Upload
others
View
4
Download
0
Embed Size (px)
Citation preview
i
Supersaturation as a driving force for drug absorption from
colloidal lipid species in the intestine
A thesis submitted for the degree of
DOCTOR OF PHILOSOPHY
from
Monash Institute of Pharmaceutical Sciences
Monash University (Parkville Campus)
by
YAN YAN YEAP
BPharm (Hons) Monash University
March 2013
Drug Delivery, Disposition and Dynamics
Monash Institute of Pharmaceutical Sciences
Monash University (Parkville Campus)
381 Royal Parade, Parkville,
Victoria 3052, Australia
Saturday, 7 September 2013 To: Prof Shinji Yamashita, Setsunan University Re: PhD thesis by Yan Yan Yeap Dear Prof Yamashita, Please find below the response to questions raised for the thesis entitled: Supersaturation as a driving force for drug absorption from colloidal lipid species in the intestine
by Yan Yan Yeap
The comments and feedback by Prof Yamashita are highly appreciated. I am honored that the Professor took his time to read the thesis thoroughly, and was interested enough to ask questions. It is my pleasure to provide the answers to the questions here. 1. In Chapter-3, author clearly demonstrated that receptor mediated lipid uptake does not play important roles to promote the absorption of model PWSD, cinnarizine (CIN). However, in Chapter 5, it was concluded that lipid absorption stimulates drug supersaturation and enhance the absorption. It might be possible to consider that the receptor mediated lipid uptake also promotes drug supersaturation by reducing the solibilisation capacity of LBF. Please make some comments on this possibility. The Professor raises an excellent point where receptor-mediated lipid absorption is potentially an inherent mechanism where drug supersaturation and absorption is promoted. I very much agree with the point raised, and add that the phenomenon will most likely apply for lipids that contribute significantly to colloidal drug solubilization capacity when incorporated into mixed micelles e.g. long-chain fatty acid (LCFA) more so than cholesterol. The experiments in Chapter 3 were early experiments that were carried out before the possibility of lipid absorption-induced drug supersaturation was hypothesized and tested. Therefore, in those experiments, we only tracked the systemic availability of lipids whose absorption was most likely facilitated by the receptors investigated (i.e. cholesterol for SR-BI and NPC1L1, oleic acid for CD36). For cholesterol, while its presence in vesicles is thought to stabilize the bilayer, it generally is not thought to contribute significantly to the drug solubilization capacity of colloids. Thus, cholesterol absorption is not expected to be a significant trigger for drug supersaturation and absorption. This is reflected in Fig 3.2A & Fig 3.2B of the thesis where cholesterol absorption is almost completely abolished by ezetimibe co-administration, but drug
absorption remains unchanged. On the other hand, we now know that the presence of oleic acid in bile micelles contributes significantly to the colloidal solubilization capacity for a range of lipophilic drugs. Thus, receptor-mediated absorption of oleic acid will likely stimulate drug supersaturation and absorption too. Some evidence that support this notion may be found in Fig 3.2A & Fig 3.2C of the thesis, where the absorption of oleic acid and cinnarizine appeared to be concomitantly reduced (albeit insignificantly) by CD36 inhibitor co-administration. At the time of the experiments, we did not think that the difference was significant enough to warrant further investigation. After the findings in Chapter 5 of the thesis however (i.e. LCFA absorption is a significant trigger for drug supersaturation and absorption), it seems entirely plausible that receptor-mediated LCFA uptake could also enhance drug absorption via the stimulation of drug supersaturation. At present, the absorption of LCFA is thought to occur mainly via facilitated diffusion at low concentrations, and passive diffusion at high concentrations [1]. Thus, at high LCFA load (as is typically found in the small intestine after lipid ingestion), the physiological significance of active LCFA absorption is questionable. In addition, it is still unclear if the receptors indicated to be involved in LCFA absorption (e.g. CD36 [2], FATP [3], SR-BI [4]) are indeed transporters which facilitate uptake across the apical membrane, or proteins that facilitate intracellular lipid trafficking/modify signalling processes that mediate lipid absorption [5-6]. Given these uncertainties and complexities, we were not able to adequately probe the role of receptor-mediated lipid absorption in drug supersaturation/absorption. However, in light of the comment made by the professor, it may be interesting to note the data in Fig 5.2A of the thesis, where the attenuation of the UWL acidic microclimate did not completely abolish the absorption of oleic acid, and significant amounts of oleic acid was still absorbed. This could be due to the receptor-mediated absorption component for oleic acid, and it remains highly possible that if this component of lipid absorption was also inhibited, drug absorption may be reduced further. 2. In Fig. 3.7, CIN supersaturation terminated at 1-20min (in the case of Vesicle) or >20 min (in the case of Micelles) after bile addition, however in both cases, decreased concentration of CIN becomes plateau at significantly higher level than equilibrium solubility. Generally, this phenomenon is understood as drug precipitated as amorphous or unstable polymorph. However, using polarized light microscopy, author clearly showed that the CIN precipitated in the crystalline form (Fig. 3.11). Could author explain the reason of the higher plateau concentration of CIN than the equilibrium solubility? On several occasions, the remaining mixture at the end of the precipitation kinetics experiments (colloids + bile + precipitated drug) was collected, and daily samples (after centrifugation) were taken to track the rate and extent of drug precipitation. We found that while the majority of drug precipitation occurred within the first day of mixing (as shown in Fig 3.7 of thesis), drug concentration in the supernatant took a few days to decrease to equilibrium solubility. At present I do not have an explanation for this phenomenon (where non-instantaneous equilibration to the equilibrium solubility is observed), but speculate that intestinal colloids such as mixed micelles and vesicles may have supersaturation-stabilizing properties. This would be consistent with recent studies from our group that showed that during simulated lipid digestion experiments, stabilization of drug supersaturation was typically possible at and below a supersaturation ratio (SS ratio) of 3, while rapid precipitation was typically observed at SS ratios > 3 [7]. In the experiments depicted in Fig 3.7 of thesis, it is likely that the high degree of drug supersaturation induced immediately after the addition of bile (SS ratio of 6 and 12 for micelles and vesicles,
respectively) was not sustained by the colloidal phases, leading to rapid and significant precipitation. The lower degree of drug supersaturation (SS ratio < 2 for both micelles and vesicles) that resulted after the majority of the precipitation occured, however, was able to be stabilized for longer periods of time by the colloidal mixture, giving rise to the observation where drug concentration appears to ‘plateau’ at a concentration higher than the equilibrium solubility. 3. Please speculate the possibility of LCFA colloids to interact with the components of mucin layer. I wonder if the LCFA colloids interact with mucin, which might prevent the diffusion of colloids in UWL and affect the absorption of drugs included. The possibility of LCFA colloids interacting with mucus components is an interesting one and is an area that might be usefully explored in detail. The ability of mucus glycoprotein to bind with a variety of lipids does suggest the possibility that the diffusion of LCFA colloids may be impeded by mucus-LCFA interaction. It follows that the diffusion of colloid-solubilized drugs may be slower in the mucin layer as well, although this will be in contradiction to previous modeling efforts that report enhanced solubilizate diffusion across the UWL when solubilized in bile micelles [8-10] (the UWL is often thought to be indistinguishable from the mucus layer). In the interest of discussion however, if we assume that LCFA colloid-mucin interactions do lead to slower diffusion of solubilized drug, whether or not that is beneficial or detrimental to drug absorption remains unknown. On one hand, if the diffusion of poorly water-soluble drugs through the aqueous mucin layer is indeed the rate-limiting step to drug absorption, mucin-LCFA interaction may then be expected to reduce drug absorption. However mechanistically we understand very little what happens at the mucus/UWL boundary and so it is too early to make predictions. In the presence of mucin-LCFA interaction, it is plausible that enhanced retention time in close proximity to the absorptive membrane could serve to enhance the absorption of somewhat permeability-limited drugs, or, increase the likelihood of acidic microclimate-triggered lipid absorption (which, as shown in Chapter 5 of thesis, is an important driver for drug supersaturation and absorption). In addition, analogous to lipid absorption-induced drug supersaturation, it is also possible that LCFA-mucin interaction serves to deplete LCFA from mixed micelles, and stimulate drug supersaturation as a result. The net effect of LCFA colloid-mucin interaction on drug absorption is likely difficult to predict and may be expected to vary for different drug types. Nonetheless, available literature and practical experience seem to suggest that co-administration of a lipophilic drug with precursors of LCFA often lead to enhanced drug absorption. I am therefore inclined to think that LCFA colloid-mucin interactions, if present, are in fact beneficial to the absorption of lipophilic drugs, although this judgment is made with reservations due to the lack of available scientific data. 4. Since LBFs performance in the GI tract to produce supersaturation and enhance the absorption of PWSD profoundly depends on the endogenous lipid processing events, conversely, it might become a factor to cause inter- and/or intra individual differences in drug absorption. Please make comments on the possibility of such risks of LBF system. I agree that it is possible the reliance on endogenous events to produce the necessary enhancement in drug thermodynamic activity may be a contributing factor to intra/inter-individual variability in drug absorption. However, it is well-known that the body is extremely efficient and robust in its ability to process, digest and absorb dietary lipids. Therefore, it is debatable that the
dependence of LBF on endogenous lipid processing events to promote drug absorption is any more variable than other processes that may underpin drug absorption (e.g. dose form disintegration, drug dissolution etc), or any more variable than other formulation approaches. It is worth remembering that first and foremost, LBF increases drug absorption by circumventing the need for drug dissolution in the GI tract, by increasing the mass of drug that may be present in solution in the small intestine, and by increasing the efficiency of drug presentation to the absorptive membrane (via micellar solubilization). These processes likely vary little between healthy individuals. However, in the interest of discussion, if LBF’s dependence on endogenous lipid processing events to produce drug supersaturation was indeed a significant cause for intra/inter-individual variability, the susceptibility to variability is likely different for different drug types, since different drug types may have different propensity for supersaturation to be induced by endogenous lipid processing events (one of the conclusions in Chapter 4 is that endogenously triggered supersaturation is more apparent for weak bases than neutral compounds). Therefore, for compounds where high variability in absorption may be expected, LBF may be more usefully directed to increase the bioavailability of drugs with broad therapeutic windows. References
1. Chow, S. L.; Hollander, D. A dual, concentration-dependent absorption mechanism of linoleic acid by rat jejunum in vitro. Journal of Lipid Research 1979, 20, (3), 349-56.
2. Nassir, F.; Wilson, B.; Han, X.; Gross, R. W.; Abumrad, N. A. CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine. Journal of Biological Chemistry 2007, 282, (27), 19493-19501.
3. Stahl, A.; Hirsch, D. J.; Gimeno, R. E.; Punreddy, S.; Ge, P.; Watson, N.; Patel, S.;
Kotler, M.; Raimondi, A.; Tartaglia, L. A.; Lodish, H. F. Identification of the major intestinal fatty acid transport protein. Molecular Cell 1999, 4, (3), 299-308.
4. Bietrix, F.; Yan, D.; Nauze, M.; Rolland, C.; Bertrand-Michel, J.; Coméra, C.; Schaak, S.;
Barbaras, R.; Groen, A. K.; Perret, B.; Tercé, F.; Collet, X. Accelerated lipid absorption in mice overexpressing intestinal SR-BI. Journal of Biological Chemistry 2006, 281, (11), 7214-7219.
5. Nauli, A. M.; Nassir, F.; Zheng, S.; Yang, Q.; Lo, C. M.; Von Lehmden, S. B.; Lee, D.; Jandacek, R. J.; Abumrad, N. A.; Tso, P. CD36 Is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine. Gastroenterology 2006, 131, (4), 1197-1207.
6. Tran, T. T. T.; Poirier, H.; Clément, L.; Nassir, F.; Pelsers, M. M. A. L.; Petit, V.; Degrace, P.; Monnot, M.-C.; Glatz, J. F. C.; Abumrad, N. A.; Besnard, P.; Niot, I. Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis. Journal of Biological Chemistry 2011, 286, (28), 25201-25210.
7. Williams, H. D.; Sassene, P.; Kleberg, K.; Calderone, M.; Igonin, A.; Jule, E.;
Vertommen, J.; Blundell, R.; Benameur, H.; Müllertz, A.; Pouton, C. W.; Porter, C. J. H.
Toward the establishment of standardized in vitro tests for lipid-based formulations, Part 3: Understanding supersaturation versus precipitation potential during the in vitro digestion of Type I, II, IIIA, IIIB and IV lipid-based formulations. Pharmaceutical Research 2013, DOI 10.1007/s11095-013-1038-z.
8. Westergaard, H.; Dietschy, J. M. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. Journal of Clinical Investigation 1976, 58, (1), 97-108.
9. Miller, J. M.; Beig, A.; Krieg, B. J.; Carr, R. A.; Borchardt, T. B.; Amidon, G. E.; Amidon,
G. L.; Dahan, A. The solubility–permeability interplay: Mechanistic modeling and predictive application of the impact of micellar solubilization on intestinal permeation. Molecular Pharmaceutics 2011, 8, (5), 1848-1856.
10. Sugano, K. Estimation of effective intestinal membrane permeability considering bile
micelle solubilisation. International Journal of Pharmaceutics 2009, 368, (1–2), 116-122.
I hope my response to the questions have been satisfactory, but please do not hesitate to contact me if I can provide further clarification. Sincerely,
Yan Yan Yeap, BPharm (Hons)
Current address Department of Chemical Engineering Northeastern University, Boston, Massachusetts, USA Previous address Drug Delivery Disposition and Dynamics Monash Institute of Pharmaceutical Sciences Monash University (Parkville campus), Melbourne, Victoria, Australia
ii
Notice 1
Under the Copyright Act 1968, this thesis must be used only under the normal conditions of
scholarly fair dealing. In particular no results or conclusions should be extracted from it, nor
should it be copied or closely paraphrased in whole or in part without the written consent of the
author. Proper written acknowledgement should be made for any assistance obtained from this
thesis.
Notice 2
I certify that I have made all reasonable efforts to secure copyright permissions for third-party
content included in this thesis and have note knowingly added copyright content to my work
without the owner’s permission.
iii
昨夜西风凋碧树。独上高楼,望尽天涯路。
衣带渐宽终不悔,为伊消得人憔悴。
众里寻他千百度,蓦然回首,那人却在灯火阑珊处。
- 王国维《人间词话》“三种境界”
iv
TABLE OF CONTENTS
ABSTRACT .............................................................................................................................................. ix
GENERAL DECLARATION ................................................................................................................... xi
ACKNOWLEDGEMENTS ...................................................................................................................... xii
PUBLICATIONS .....................................................................................................................................xiv
COMMUNICATIONS .............................................................................................................................. xv
LIST OF ABBREVIATIONS ...................................................................................................................xvi
CHAPTER 1 : GENERAL INTRODUCTION ................................................................... 20
1.1 STATEMENT OF THE PROBLEM ............................................................................................... 21
1.2 LIPIDS FOR THE ENHANCEMENT OF ORAL DRUG ABSORPTION .................................... 21
1.2.1 Poorly water-soluble drugs and the Biopharmaceutics Classification System ................... 22
1.2.2 Lipid-based formulations used in oral drug delivery .......................................................... 23
1.3 OVERVIEW OF LIPID DIGESTION AND ABSORPTION ......................................................... 30
1.3.1 Lipid digestion in the oral cavity and stomach ................................................................... 30
1.3.2 Lipid digestion and solubilisation in the small intestine ..................................................... 31
1.3.3 Lipid absorption across the enterocyte apical membrane ................................................... 37
1.3.4 Lipid transport into the systemic circulation ...................................................................... 38
1.4 MECHANISMS BY WHICH LIPIDS ENHANCE THE ORAL BIOAVAILABILITY OF
DRUGS ........................................................................................................................................... 40
1.4.1 Enhancement of drug dissolution in gastrointestinal fluids ................................................ 42
1.4.2 Enhancement of apparent drug solubility in intestinal fluids ............................................. 43
1.4.3 Enhancement of intestinal permeability and inhibition of enterocyte-based efflux
transporters and metabolism ............................................................................................... 44
1.4.4 Promotion of lymphatic drug transport ............................................................................... 47
1.5 PROPOSED MECHANISMS OF DRUG ABSORPTION FROM INTESTINAL MIXED
MICELLES AND VESICLES ........................................................................................................ 48
1.5.1 Current model of drug absorption and its limitations ......................................................... 48
1.5.2 Collisional uptake of solubilised drug ................................................................................ 53
1.5.3 Supersaturation-enhanced drug absorption ......................................................................... 57
1.6 STRUCTURE OF THIS THESIS ................................................................................................... 59
CHAPTER 2 : GENERAL METHODS .............................................................................. 60
2.1 INTRODUCTION ........................................................................................................................... 61
v
2.2 AUTOPERFUSED RAT JEJUNUM .............................................................................................. 61
2.2.1 Materials ............................................................................................................................. 61
2.2.2 Methods .............................................................................................................................. 62
2.2.3 Data analysis ....................................................................................................................... 68
2.2.4 Validation of the autoperfused rat jejunum ........................................................................ 70
2.2.5 Adsorption of cinnarizine onto the recirculating perfusion apparatus ................................ 78
2.3 IN VIVO BIOAVAILABILITY STUDIES .................................................................................... 81
2.3.1 Materials ............................................................................................................................. 82
2.3.2 Methods .............................................................................................................................. 82
2.4 VALIDATION OF ANALYTICAL METHODS ........................................................................... 85
2.4.1 HPLC assays for quantification of cinnarizine, halofantrine, fenofibrate, danazol and
meclofenamic acid in intestinal lipid colloidal phases ....................................................... 85
2.4.2 HPLC assay for quantification of cinnarizine in rat plasma ............................................... 89
2.4.3 LC-MS assay for quantification of danazol in rat plasma .................................................. 90
2.4.4 Enzymatic colorimetric assay for quantification of total bile salt in whole rat bile ........... 92
CHAPTER 3 : INTESTINAL BILE SECRETION PROMOTES DRUG ABSORPTION
FROM LIPID COLLOIDAL PHASES VIA INDUCTION OF SUPERSATURATION96
3.1 ABSTRACT .................................................................................................................................... 97
3.2 INTRODUCTION ........................................................................................................................... 98
3.3 METHODS .................................................................................................................................... 102
3.3.1 Materials ........................................................................................................................... 102
3.3.2 Experimental outline ......................................................................................................... 103
3.3.3 Formulation preparation ................................................................................................... 105
3.3.4 Particle sizing ................................................................................................................... 108
3.3.5 Equilibrium solubility of cinnarizine in the model micelles and vesicles ........................ 109
3.3.6 Kinetics of cinnarizine precipitation ................................................................................. 109
3.3.7 Solid-state analysis of the cinnarizine precipitate ............................................................. 109
3.3.8 Animals ............................................................................................................................. 110
3.3.9 Surgical procedures .......................................................................................................... 110
3.3.10 Cinnarizine bioavailability studies ................................................................................... 111
3.3.11 In situ single-pass rat jejunum perfusion .......................................................................... 113
3.3.12 Analytical procedures ....................................................................................................... 114
3.3.13 Statistical analysis ............................................................................................................. 117
3.4 RESULTS ...................................................................................................................................... 117
vi
3.4.1 SR-BI, CD36, NPC1L1 and endocytosis have little impact on drug absorption from
intestinal colloidal phases ................................................................................................. 117
3.4.2 Drug absorption from micelles and vesicles is determined by Cfree and not colloidal
structure ............................................................................................................................ 122
3.4.3 Bile-mediated dilution of cinnarizine-loaded micelles and vesicles generates drug
supersaturation .................................................................................................................. 126
3.4.4 Bile-induced drug supersaturation increases jejunal absorptive flux for micelles but not
vesicles .............................................................................................................................. 129
3.4.5 Bile-induced drug supersaturation increases in vivo cinnarizine exposure after
intraduodenal infusion ...................................................................................................... 132
3.5 DISCUSSION ................................................................................................................................ 132
3.6 CONCLUSION ............................................................................................................................. 141
CHAPTER 4 : THE POTENTIAL FOR DRUG SUPERSATURATION DURING
INTESTINAL PROCESSING OF LIPID-BASED FORMULATIONS IS ENHANCED FOR
BASIC DRUGS .................................................................................................................... 145
4.1 ABSTRACT .................................................................................................................................. 146
4.2 INTRODUCTION ......................................................................................................................... 147
4.3 METHODS .................................................................................................................................... 149
4.3.1 Materials ........................................................................................................................... 149
4.3.2 Experimental outline ......................................................................................................... 149
4.3.3 Preparation of model intestinal colloidal phases containing long-chain lipids ................. 151
4.3.4 Equilibrium solubility of drugs in oleic acid and soybean oil .......................................... 153
4.3.5 Equilibrium solubility of drugs in long-chain colloids ..................................................... 155
4.3.6 Kinetics of cinnarizine precipitation ................................................................................. 155
4.3.7 Solid-state analysis of the cinnarizine precipitate ............................................................. 156
4.3.8 Animals ............................................................................................................................. 156
4.3.9 Surgical procedures .......................................................................................................... 156
4.3.10 In situ single-pass rat jejunum perfusion .......................................................................... 157
4.3.11 Cinnarizine bioavailability after intraduodenal infusion .................................................. 159
4.3.12 Analytical procedures ....................................................................................................... 160
4.3.13 Statistical analysis ............................................................................................................. 162
4.4 RESULTS ...................................................................................................................................... 162
4.4.1 Trends in drug solubility in long chain lipid-based colloids as a function of bile and lipid
concentration are different for basic, neutral and acidic drugs ......................................... 162
vii
4.4.2 The addition of rat bile to drug-loaded model colloids reduces cinnarizine solubility and
promotes supersaturation, but increases danazol solubilisation ....................................... 168
4.4.3 Co-perfusion of bile with model colloids increases cinnarizine absorption to a greater
extent than danazol ........................................................................................................... 170
4.4.4 Supersaturation increases in vivo cinnarizine exposure after intraduodenal infusion of
drug-loaded model colloids .............................................................................................. 175
4.5 DISCUSSION ................................................................................................................................ 177
4.6 CONCLUSION ............................................................................................................................. 186
CHAPTER 5 : LIPID ABSORPTION TRIGGERS DRUG SUPERSATURATION AT THE
INTESTINAL UNSTIRRED WATER LAYER AND PROMOTES DRUG ABSORPTION
FROM MIXED MICELLES .............................................................................................. 190
5.1 ABSTRACT .................................................................................................................................. 191
5.2 INTRODUCTION ......................................................................................................................... 192
5.3 METHODS .................................................................................................................................... 197
5.3.1 Materials ........................................................................................................................... 197
5.3.2 Experimental outline ......................................................................................................... 197
5.3.3 Preparation of LCFA-containing intestinal colloids ......................................................... 199
5.3.4 Preparation of Brij 97 colloids .......................................................................................... 200
5.3.5 Equilibrium solubility studies of cinnarizine in colloids .................................................. 200
5.3.6 Animals ............................................................................................................................. 201
5.3.7 Surgical procedures .......................................................................................................... 201
5.3.8 In situ single-pass rat jejunum perfusion .......................................................................... 202
5.3.9 Analytical procedures ....................................................................................................... 204
5.3.10 Statistical analysis ............................................................................................................. 205
5.4 RESULTS ...................................................................................................................................... 206
5.4.1 Attenuation of the acidic microclimate using amiloride reduces oleic acid and cinnarizine
absorption from model LCFA colloids; but has no effect on the absorption of cinnarizine
from fatty acid-free Brij 97 colloids ................................................................................. 206
5.4.2 Exposure of model LCFA colloids to the acidic microclimate, and absorption of lipid
components, leads to cinnarizine supersaturation and enhanced thermodynamic activity
.......................................................................................................................................... 211
5.4.3 Attenuation of the acidic microclimate using amiloride abolishes bile-induced,
supersaturation-enhanced, cinnarizine absorption from model LCFA colloids ............... 217
5.5 DISCUSSION ................................................................................................................................ 221
viii
5.6 CONCLUSION ............................................................................................................................. 229
CHAPTER 6 : SUMMARY AND PERSPECTIVES ....................................................... 231
REFERENCES ........................................................................................................................................ 241
APPENDIX 1 ........................................................................................................................................... 261
ix
ABSTRACT
This thesis seeks to elucidate the mechanism of drug absorption from the colloidal micellar and
vesicular species that form in the gastrointestinal (GI) tract during lipid digestion. In addition to
absorption from the free fraction of drug molecules that exist in equilibrium with drug solubilised
in the colloidal reservoir, two alternative models of drug absorption were explored: (i) collisional
drug absorption where lipid colloidal phases interact directly with the absorptive membrane, and
(ii) supersaturation-enhanced absorption where transient changes to colloid structure and content
in situ lead to drug supersaturation, thereby boosting drug thermodynamic activity and rendering
solubilised drug more available for absorption via the free fraction. Collisional drug absorption
was explored by comparing the intestinal absorptive flux of cinnarizine (CIN) from two distinctly
different colloids (micelles vs. vesicles) that were matched for CIN solubilisation capacity and
drug loading (and therefore thermodynamic activity). In these systems the number of micellar
particles was substantially higher than that of vesicles, and as such collisional absorption of CIN
was expected to be higher from micelles. The absorption of CIN from micelles and vesicles,
however, was not statistically different, suggesting little collisional involvement in drug
absorption. Receptor-mediated collisional absorption was examined by assessing CIN
bioavailability from a lipid emulsion in the absence and presence of inhibitors of common lipid
uptake transporters (e.g. SR-BI, CD36, NPC1L1). CIN bioavailability was unchanged by inhibitor
co-administration. Collectively, the data suggest that collision-mediated uptake is not a significant
driver for drug absorption from intestinal lipid colloidal phases, and that drug absorption occurs
largely from the free fraction. Subsequently, attention turned to the possibility that drug
supersaturation might be stimulated during endogenous processing of intestinal lipid colloidal
phases. Two mechanisms were investigated: (i) interaction of lipid colloidal phases with bile
secretions, where bile-induced changes to colloid microstructure may lead to reductions in drug
solubilisation capacity and (ii) lipid absorption from intestinal colloids, where reductions in
colloidal lipid content may reduce drug solubilisation capacity at the intestinal unstirred water
layer (UWL). The addition of donor rat bile to CIN-loaded colloids (CIN was loaded at sub-
x
saturated concentrations) resulted in a decrease in CIN solubilisation and the generation of CIN
supersaturation. Bile-induced supersaturation was subsequently shown to increase the intestinal
absorptive flux and systemic exposure of CIN from both medium-chain and long-chain lipid
containing colloids. To assess the potential for lipid absorption to induce drug supersaturation, the
intestinal absorptive flux of CIN from oleic acid-containing colloids was assessed under
conditions of normal lipid absorption vs. inhibited lipid absorption (oleic acid absorption was
inhibited by co-administration of amiloride, an inhibitor of UWL acidity). When oleic acid
absorption was suppressed, the absorption of CIN was dramatically attenuated. Assessment of
CIN solubilisation behaviour under conditions that simulate lipid absorption at the UWL
subsequently indicated that supersaturation was likely to be responsible for the enhanced CIN
absorption observed during normal lipid absorption. In summary, supersaturation appears to be an
important driving force for drug absorption from lipid-based intestinal colloids. Two novel
mechanisms have been identified by which drug supersaturation may be naturally triggered in the
small intestine (bile dilution and lipid absorption). The findings enhance mechanistic
understanding of the effects of lipids in food or formulations on drug absorption and are expected
to inform the development of more rational design criteria for LBF.
xi
Monash University Declaration for thesis based or partially based on conjointly published or unpublished work
GENERAL DECLARATION
In accordance with Monash University Doctorate Regulation 17 Doctor of Philosophy and Research Master’s regulations the following declarations are made: I hereby declare that this thesis contains no material which has been accepted for the award of any other degree or diploma at any university or equivalent institution and that, to the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference is made in the text of the thesis. This thesis includes one original paper published in peer reviewed journals and two submitted publications. The core theme of the thesis is an understanding of the mechanism of drug absorption from intestinal colloidal species following oral administration of lipids. The ideas, development and writing up of all the papers in the thesis were the principal responsibility of myself, the candidate, working within the Drug Delivery, Disposition and Dynamics Theme of the Monash Institute of Pharmaceutical Sciences under the supervision of Prof Christopher J. H. Porter and Dr Natalie L. Trevaskis. The inclusion of co-authors reflects the fact that the work came from active collaboration between researchers and acknowledges input into team-based research. In the case of Chapter 3 my contribution to the work included the majority of the experimental work, all data analysis and interpretation, the concept and design of all studies, the preparation of initial drafts of all manuscripts and the subsequent revision and formulation of conclusions and hypotheses resulting from the relevant studies. In the case of Chapters 4 and 5 my contribution to the work included all the experimental work, data analysis and interpretation, the concept and design of all studies, the preparation of initial drafts of all manuscripts and the subsequent revision and formulation of conclusions and hypotheses resulting from the relevant studies. Thesis chapter
Publication title Publication status* Nature and extent of candidate’s contribution
3 Intestinal bile secretion promotes drug absorption from lipid colloidal phases via induction of supersaturation
In press
Planning and execution of experimental work, data evaluation, drafting and revision of manuscript
4 The potential for drug supersaturation during intestinal processing of lipid-based formulations is enhanced for basic drugs
In submission
Planning and execution of experimental work, data evaluation, drafting and revision of manuscript
5 Lipid absorption triggers drug supersaturation at the intestinal unstirred water layer and promotes drug absorption from mixed micelles
In submission
Planning and execution of experimental work, data evaluation, drafting and revision of manuscript
I have renumbered sections of submitted or published papers in order to generate a consistent presentation within the thesis. Signed: ………………………………………… Date: ………………………………
xii
ACKNOWLEDGEMENTS
I’d first like to thank my supervisors Prof Chris Porter and Dr Natalie Trevaskis for their guidance
and support. They are outstanding scientists who still believe in the personal development of their
students, for that I could not have asked for two better people to see me through my PhD. Thank
you for consistently being there, for the times when you have shown great insight, care,
enthusiasm and encouragement. Thank you also for having the foresight and courage to ask an
excellent research question that led to the conception of this project – I have had a ball, and these
years spent under your supervision will be some of the most profound and memorable experience
in my life.
To my parents, I feel truly lucky to be your daughter. You are such resilient individuals, and have
strived hard to give us a good life, and importantly, good and free minds. Thank you for always
listening and entertaining my thoughts and ideas, even if I was and still am, a little dreamy. I think
it has given me the confidence to just be myself. Thank you also for your unconditional love. I
didn’t realise the importance of having such unwavering support behind me, but I know now that
it’s the reason I do not think twice before undertaking a difficult endeavour. Thank you especially
to my mum who, in retrospect, really made the effort to ensure that our upbringing was a well-
considered one. To my siblings YXY, YYH (and Alicia and baby Xuan Xuan) and YYY, thank
you for always looking out for me and showing an interest in what I do, I hope I have outgrown
little sister status over the years.
To Hywel, in the past two years or so I have found it difficult in my mind to disentangle you from
the wonderful turn in PhD journey I have had, maybe good things do come in pairs. You have
been an absolute inspiration to me, and I am still astounded by the depth of support you continue
to provide. Thank you for so many things, from making me dinner to keeping me warm on top of
a mountain to showing me how best to import powerpoint figures to always being enthusiastic and
insightful when discussing ideas and concepts. I look forward to our future together.
To Shalini and Sifei, how will I complete my PhD a sane person without you two? Shalini you
have been a great PhD buddy. Throughout the years you have provided me with lots of laughs,
endless conversation, and some serious emotional support through the personally challenging
times. I will treasure the friendship and good times we’ve had. Sifei your wise words are worthy
of compilation, and your convincing me that breakthrough is near at my lowest point is one of the
most important things anyone has said to me. Thank you for always pointing me in the right
direction. I am grateful to have known such kind-hearted individuals full of integrity as you two.
xiii
The financial support by Australian Postgraduate Award is also gratefully acknowledged.
Lastly, I would like to thank everyone who cared and shared good times with me. Tri and Kathy
for their ‘special’ way of caring, LJ for her friendship and wisdom especially in the last few
months, Lisa for showing me how ‘easy’ it is to write a thesis/paper (in a week!), Jenny for giving
me a job with a handsome pay during my PhD years, Joe and Ian and Danielle and Khay for
lunchtime friendship during my write-up, Rachelle for yummy Thursday night Chinese takeaways,
my sister Ying, Ginny, Kary and Tim for their hospitality in this last month when I don’t have a
regular place to call home, and a special mention to Alice and Tom for being so interested in my
work. These are the things I will remember.
xiv
PUBLICATIONS
This thesis is a compilation of the following manuscripts:
Chapter 3:
Yeap YY, Trevaskis NL, Quach T, Tso P, Charman WN, Porter CJH. Intestinal bile secretion
promotes drug absorption from lipid colloidal phases via induction of supersaturation. Molecular
Pharmaceutics, 2013, in press. (Proofs appended at the end of this thesis)
Chapter 4:
Yeap YY, Trevaskis NL, Porter CJH. The potential for drug supersaturation during intestinal
processing of lipid-based formulations is enhanced for basic drugs. Manuscript in submission.
Chapter 5:
Yeap YY, Trevaskis NL, Porter CJH. Lipid absorption triggers drug supersaturation at the
intestinal unstirred water layer and promotes drug absorption from mixed micelles. Manuscript in
submission.
xv
COMMUNICATIONS
Yeap YY. Rethinking drug absorption from post-digestion lipid colloidal phases: A
supersaturation-based model of absorption. Podium presentation, AAPS Annual Meeting and
Exposition 2012, Chicago, IL, USA. (Award winning)
Yeap YY, Trevaskis NL and Porter CJH. The acidic microclimate of the intestinal unstirred water
layer can promote drug absorption from long-chain mixed micelles via induction of
supersaturation at the absorptive site. Poster presentation, AAPS Annual Meeting and Exposition
2012, Chicago, IL, USA.
Yeap YY, Trevaskis NL and Porter CJH. Bile secretion increases drug absorption from intestinal
mixed micelles via induction of supersaturation. Poster presentation, AAPS Annual Meeting and
Exposition 2011, Washington, DC, USA.
Yeap YY, Trevaskis NL and Porter CJH. The role of small intestine lipid uptake transporters in
the oral absorption of lipids and poorly water soluble drugs. Poster presentation, APSA Annual
Conference 2009, Hobart, Tasmania, Australia.
xvi
LIST OF ABBREVIATIONS
% CV % coefficient of variation
AUC area under the curve
BA bioavailability
BBMV brush border membrane vesicles
BCRP breast cancer resistant protein
BCS Biopharmaceutics Classification System
BLT-1 Block Lipid Transport-1
BS bile salts
CD36 Cluster of Differentiation 36
Ch cholesterol
CIN cinnarizine
cm centimetre
CMC critical micellar concentration
DAN danazol
DG diglyceride
DMSO dimethyl sulfoxide
FA fatty acids
FABP fatty acid binding protein
FABPpm membrane-associated fatty acid binding protein
FAT Fatty Acid Translocase
FATP fatty acid transport protein
FF fenofibrate
g gram
GI gastrointestinal
h hour
xvii
HDL high density lipoprotein
HF halofantrine
HLB Hydrophilic-Lipophilic Balance
HPLC high performance liquid chromatography
kg kilogram
L litre
LBF lipid-based formulations
LC long-chain
LCFA long-chain fatty acids
LC-MS liquid chromatography-mass spectrometry
LCT long-chain triglycerides
LDL low density lipoprotein
LFCS Lipid Formulation Classification System
Log D logarithm of the octanol-water distribution coefficient
LPC lysophosphatidylcholine
LPL lysophospholipids
m metre
mg milligram
mL millilitre
mm millimetre
mM millimolar
min minute
M molar
MC medium-chain
MCT medium-chain triglycerides
MFA meclofenamic acid
MG monoglyceride
xviii
µCi microCurie
µg microgram
µL microlitre
µm micrometre
µM micromolar
MRP multidrug resistance-associated protein
nm nanometre
ng nanogram
NMR nuclear magnetic resonance
NPC1L1 Niemann-Pick C1 Like 1
OA oleic acid
o/w oil in water
Papp apparent permeability coefficient
PC phosphatidylcholine
PEG polyethylene glycol
P-gp P-glycoprotein
PL phospholipids
PWSD poorly water-soluble drugs
rpm revolutions per minute
SAXS small-angle X-ray scattering
SDS sodium dodecyl sulphate
sec second
SEDDS self-emulsifying drug delivery systems
SEIF simulated endogenous intestinal fluid
SEM standard error of the mean
SI small intestine
SMEDDS self-microemulsifying drug delivery systems
xix
SR-BI Scavenger Receptor Class B Type 1
S-SEDDS supersaturable self-emulsifying drug delivery systems
SSO sulfo-N-succinimidyl oleate
SS ratio supersaturation ratio
TBME tert-butyl methyl ether
TG triglyceride
TRL triglyceride-rich lipoproteins
UV ultraviolet
UWL unstirred water layer
VLDL very low density lipoprotein
v/v volume in volume
w/o water in oil
w/v weight in volume
w/w weight in weight
x g relative centrifugal force
20
CHAPTER 1 : GENERAL INTRODUCTION
Chapter 1: General Introduction
21
1.1 STATEMENT OF THE PROBLEM
Lipid-based formulations (LBF) are commonly employed to increase the oral bioavailability of
poorly water-soluble drugs (PWSD). Confident and widespread application of LBF, however, is
limited by the lack of a holistic understanding of the mechanisms by which lipids enhance the
absorption of PWSD. Currently, the design and evaluation of LBF is largely predicated on the
ability of formulations to maintain drug solubilisation during in vitro simulations of formulation
dispersion and digestion in the gastrointestinal (GI) tract. This approach, while generally useful, is
largely empirical and does not always adequately predict formulation performance in vivo. In
contrast, considerably less attention has been directed towards an understanding of the
mechanism(s) of drug absorption from the colloidal phases (e.g. micellar and vesicular species)
that form in the small intestine (SI) following lipid ingestion. Improved understanding of the
fundamental processes that govern drug absorption from intestinal lipid colloidal phases has the
potential to provide enhanced design parameters for LBF, and is the main aim of this PhD thesis.
1.2 LIPIDS FOR THE ENHANCEMENT OF ORAL DRUG ABSORPTION
Growing application of high throughput activity screens and the use of complex chemical
scaffolds in drug discovery has led to increasingly frequent identification of poorly water-soluble
compounds as prospective drug candidates. Indeed, recent estimates suggest that at least 40% of
new chemical entities in development are classified as ‘poorly water-soluble’1. While these
compounds may possess high intrinsic potency at target sites, the development and clinical utility
of poorly water-soluble drug candidates are often limited by low systemic exposure after oral
administration. This reflects the need for drugs to be molecularly dispersed (i.e. in solution) in
order to pass across the absorptive cells that line the GI tract, and for poorly water-soluble
Chapter 1: General Introduction
22
compounds, this prerequisite is limited by slow dissolution rates and low solubility in the aqueous
GI milieu.
Several approaches have been employed to improve the dissolution and solubilisation
characteristics of poorly-water soluble compounds in the GI tract. These include the isolation of
drug candidates in alternate salt forms, polymorphs or co-crystals with improved dissolution and
solubility profiles; particle size reduction strategies such as milling or nanomilling; and
formulation strategies such as the use of solid dispersions, cyclodextrins, and surfactant or lipid-
based drug delivery systems. The common strategies to enhance the dissolution and solubility of
poorly water-soluble compounds has recently been reviewed in detail by Williams et al.2. The
focus of the studies in this thesis, however, is the use of LBF to enhance the absorption of PWSD.
1.2.1 Poorly water-soluble drugs and the Biopharmaceutics Classification
System
The Biopharmaceutics Classification System (BCS) categorises drugs into four classes according
to their water solubility and membrane permeability properties (Table 1.1), and allows for the
broad prediction of the rate-limiting steps in drug absorption following oral administration3.
Table 1.1: The biopharmaceutical classification system (BCS) for drugs according to their
solubility and permeability properties.
Class Solubility Permeability Examples
I High High ketoprofen, propranolol, midazolam, paracetamol
II Low High cinnarizine, danazol, phenytoin, griseofulvin
III High Low metformin, digoxin, cefotaxamine, cimetidine
IV Low Low cyclosporin A, lovostatin, amphotericin, paclitaxel
Chapter 1: General Introduction
23
PWSD are contained within Class II and IV of the BCS. In general, the absorption of Class II
drugs is limited by solubility alone, whereas the absorption of Class IV drugs is limited by both
solubility and permeability. PWSD include examples that have been likened to ‘grease balls’ that
are both hydrophobic (i.e. solubility is limited by hydration and interaction with aqueous solvents)
and lipophilic (i.e. where lipid solubility is high and melting point is typically low); and
compounds that have been likened to ‘brick dust’ that are hydrophobic but not lipophilic (i.e.
where solubility is limited by strong intermolecular bonds within the crystal lattice thereby
restricting the solubility of compounds in both water and lipids). ‘Grease ball’ like compounds
(typically found in Class II) are well-suited to formulation in LBF since the higher lipophilicity of
these compounds dictates that they have higher solubility in the formulation when compared to
‘brick dust’ like molecules. As new molecular entities become increasingly lipophilic and poorly
water-soluble1, LBF are becoming an increasingly effective and popular strategy to improve oral
absorption. However, a thorough understanding of the mechanisms by which lipids exert their
absorption-enhancing effects must first be obtained before LBF technologies can be confidently
applied.
1.2.2 Lipid-based formulations used in oral drug delivery
‘Lipid-based formulation’ is an umbrella term that encompasses a diverse group of formulations
including lipid solutions, suspensions, emulsions, microemulsions, nanoemulsions, lipid
nanoparticles, self-emulsifying drug delivery systems (SEDDS), liquid crystalline materials such
as lamellar, hexagonal, and cubic liquid crystals, and lipid complexes such as cochleates. LBF
may be employed for oral or parenteral administration. In oral drug delivery, LBF comprising
simple isotropic liquids that may be filled into gelatin capsules are the most widely used4. LBF
typically comprise a mixture of excipients including triglycerides, mixed mono- and diglycerides,
non-ionic surfactants, and cosolvents5. The choice of excipients is usually based on the need to
Chapter 1: General Introduction
24
solubilise the drug dose in the formulation, the ability to promote rapid initial emulsification of the
formulation in the GI fluids, the capacity to maintain drug solubilisation during dispersion and
digestion of the formulation in the GI tract, and the potential to alter metabolic processing
pathways such as the stimulation of lymphatic transport or the inhibition of enterocyte-based
efflux transporters and metabolism. Some examples of LBF that have been shown to enhance the
oral bioavailability of PWSD are listed in Table 1.2.
Chapter 1: General Introduction
25
Table 1.2: Examples of LBF used to enhance the oral bioavailability (BA) of PWSD.
Compound Formulation Study Observation Ref.
Cinnarizine Oleic acid solution Oral BA (beagle dogs)
4 fold increase in relative BA c.f. tablets
6
Penclomedine Trioctanoin, triolein, soybean oil, mineral oil and tributyrin o/w emulsion
Intraduodenal dosing (rats)
3-7 fold increase in absolute BA when dosed in digestible and non-digestible lipids c.f. aqueous suspension
7
Danazol Monoolein o/w emulsion Oral BA (humans)
4 fold increase in relative BA c.f. capsules
8
Griseofulvin
Dexamethasone
Peanut oil, Captex 355 and triacetin oil suspensions
Oral BA (rats) Griseofulvin: 2-3 fold increase in absolute BA c.f. aqueous suspension
Dexamethasone: 8-9 fold increase in absolute BA c.f. aqueous suspension
9
Griseofulvin Corn oil o/w emulsion Oral BA (humans)
2 fold increase in relative BA c.f. aqueous suspension
10
Ontazolast Soybean oil o/w emulsion
Oral BA (rats) 15 fold increase in absolute BA c.f. aqueous suspension
11
Halofantrine Peanut oil, Captex 355 and tributyrin solutions
Oral BA (rats) 2-3 fold increase in absolute BA c.f. aqueous suspension
12
Carvedilol SEDDS (MCT) Oral BA (beagle dogs)
4 fold increase in relative BA c.f. tablets
13
Cyclosporin Sandimmune® (SEDDS), Sandimmune Neoral® (SMEDDS)
Oral BA (humans)
Up to 2.4 fold increase in relative BA from SMEDDS c.f. SEDDS
14
Itraconazole SMEDDS (PEG) Oral BA (humans)
1.9 and 1.5 fold increase in BA in fasted and fed states, respectively
15
Silymarin SMEDDS (ethyl linoleate)
Oral BA (rabbits)
49 fold increase in relative BA c.f. suspension
16
Paclitaxel S-SEDDS Oral BA (rats) 5-fold increase in relative BA c.f. intravenous solution (Taxol®)
17
o/w = oil in water; MCT = medium-chain triglycerides; LCT = long-chain triglycerides; SEDDS = self-emulsifying
drug delivery systems; SMEDDS = self-microemulsifying drug delivery systems; S-SEDDS = supersaturable self-
emulsifying drug delivery systems
Chapter 1: General Introduction
26
To assist in the description and comparison of oral LBF, Pouton proposed a classification scheme
that originally grouped LBF into three categories18 with a fourth recently added19. The Lipid
Formulation Classification System (LFCS) allows formulations to be described according to their
composition and general behaviour during in vitro dispersion and digestion tests (Table 1.3).
Table 1.3: Typical composition of LFCS types I, II, IIIA, IIIB, and IV LBF and their
solubilisation capacity for drug before and after dispersion and digestion in the GI tract.
Type I Type II Type IIIA Type IIIB Type IV
Hydrophobic composition high
low
Lipid: TG, DG, MG (% w/w) 100 40-80 40-80 <20 -
Water-insoluble surfactants HLB < 12 (% w/w)
- 20-60 - - 0-20
Water-soluble surfactants HLB > 12 (% w/w)
- - 20-40 20-50 30-80
Hydrophilic cosolvents (% w/w) - - 0-40 20-50 0-50
Solubilisation capacity prior to dispersion
low
high
Particle size on dispersion (nm) coarse 250-2000 100-250 50-100 very fine
Loss of drug solubilisation capacity upon dispersion
limited
high
Importance of digestion crucial
not required
Loss of drug solubilisation capacity upon digestion
Possible Possible Possible Possible Possible
TG, DG, MG = triglycerides, diglycerides, monoglycerides; HLB = Hydrophilic-Lipophilic Balance
Type I formulations are comprised of a single lipid vehicle or a blend of lipids typically consisting
common plant oils or fractionated glycerides20. Type I formulations disperse poorly upon contact
with aqueous media, but typically maintain drug solubilisation during dilution by GI fluids.
Chapter 1: General Introduction
27
Digestion processes are required to increase the amphiphilicity of formulation lipids, thereby
improving dispersion properties and promoting the formation of micellar and vesicular species
that are required to maintain PWSD solubilisation and facilitate transfer to the absorptive
membrane (see Section 1.3.2 and Section 1.4.2). The requirement for digestion however, may not
limit absorption as many studies suggest that formulations containing digestible lipids have
superior performance in vivo when compared to formulations that contain poorly or non-digestible
lipids. For example, Yoshiya et al. showed that the systemic exposure of the lipophilic drug SL-
512 in rats was significantly higher after oral administration of a 2 mg/kg dose in MCT (readily
digested) when compared to administration in N-α-methylbenzyllinoleamide (poorly digested)21.
Myers et al. also noted higher bioavailability of penclomedine when administered intraduodenally
in soybean oil, triolein and trioctanoin, when compared to mineral oil7. The major drawback of
Type I formulations is their low solvent capacity for many drugs (except for drugs that possess
very high lipophilicity).
Type II formulations differ from Type I formulations in that they have improved emulsification
properties on dispersion due to the inclusion of lipophilic surfactants (HLB < 12). Type II LBF
exhibit typical ‘self-emulsifying’ drug delivery system (SEDDS) behaviour, and self-emulsify on
contact with aqueous media to form emulsions with particle sizes of 250-2000 nm. Compared to
Type III and IV systems (discussed below), Type II LBF contain limited quantities of hydrophilic
excipients and as such drug precipitation out of the formulation is unlikely during formulation
dispersion (since most formulation components are water immiscible). The use of Type II LBF,
however, has been largely superseded by Type III and IV formulations that have higher drug
solubilisation capacities and result in smaller particle sizes on initial dispersion.
Chapter 1: General Introduction
28
Type III formulations include lipids, water-soluble surfactants (HLB > 12) and cosolvents. They
form fine emulsions (particle size of 50-250 nm) on contact with aqueous media, and are often
described as self-microemulsifying drug delivery systems (SMEDDS). Type III formulations are
arbitrarily divided into IIIA and IIIB to distinguish between formulations that contain a significant
proportion of oils (Type IIIA) and those that contain greater quantities of hydrophilic components
(Type IIIB). Type III formulations are expected to have widely varying performance
characteristics due to the diverse group of formulations that fall under this category. Many
marketed LBF are Type III formulations.
Type IV formulations were most recently added to the LFCS19. They contain no classical lipids,
and are comprised predominantly of hydrophilic surfactants and cosolvents. The impact of
digestion on the performance of Type IV systems is therefore expected to be less pronounced than
those containing digestible lipids, although surfactant digestion is still possible in some cases. The
advantage of Type IV systems lies in the high solvent capacity of the formulations, which allows
for higher drug loading when compared to other types of LBF. However, since the formulations
consist of mainly water-miscible excipients, drug precipitation is likely on dilution with GI fluids.
Some toxicity concerns have also been raised regarding the chronic use of Type IV formulations
containing high quantities of surfactant and cosolvent, however there are many examples of
marketed products (e.g. the HIV protease inhibitors) where daily excipient ‘doses’ are high, but
seemingly well tolerated.
The selection of excipients for LBF is currently guided primarily by the capacity for the excipients
(or combinations of excipients) to solubilise the required drug dose, and to maintain drug
solubilisation during in vitro dispersion and digestion tests. Other factors that may influence
excipient selection include mutual miscibility, toxicity, irritancy, capsule compatibility, purity,
Chapter 1: General Introduction
29
chemical stability and cost. A summary of the common excipients used in LBF is listed in Table
1.4.
Table 1.4: Excipients commonly used in oral LBF. The table is adapted with permission from
Williams et al.2.
Excipient Class Examples
Medium-chain triglycerides Coconut or palm seed oil, Miglyol, Captex
Long-chain triglycerides Soybean, sesame, safflower, sunflower, corn, cottonseed,
olive, palm, peanut (arachis), rapeseed oils; hydrogenated
vegetable and soybean oils
Medium- and/or long-chain monoglycerides,
diglycerides, or mixed glycerides
Medium-chain: Capmul, Imwitor, Labrafac, Capmul GMO
Long-chain: Maisine 35-1, Peceol, Geleol
Low HLB surfactants (HLB < 8) Lipophilic sorbitan fatty acid esters (e.g. Span 85, Span 80);
esters of propylene glycol and fatty alcohols (Lauroglycol,
Capryol)
Water-insoluble, lipophilic surfactants (HLB 8-12) Predominantly oleate esters and include polysorbate 85
(Tween 85) and Tagat TO
Water-soluble surfactants (HLB > 12) Include products synthesized by reacting alcohols with
ethylene oxide to produce alkyl ether ethoxylates (e.g., Brij,
cetomacrogol); sorbitan esters with ethylene oxide to form
sorbitan ester ethoxylates (e.g. Tween); ethylene oxide with
castor oil to produce castor oil ethoxylates (e.g. Cremophor
EL, RH40); vegetable oils with polyethylene glycol (PEG)
to produce mixtures of monoglycerides, diglycerides,
triglycerides and PEG esters of fatty acid (Labrasol,
Labrafil, Gelucire)
Cosolvents PEG 400, propylene glycol, propylene carbonate, ethanol
HLB = Hydrophilic-Lipophilic Balance
Chapter 1: General Introduction
30
1.3 OVERVIEW OF LIPID DIGESTION AND ABSORPTION
Ingested dietary or formulation-derived lipids are subjected to a series of digestion and
solubilisation processes in the GI tract. These facilitate presentation of poorly water-soluble lipids
to the absorptive membrane of the SI at high concentration and in a molecularly dispersed,
solubilised form. The main stages of lipid processing from ingestion to uptake into the systemic
circulation are described below:
1.3.1 Lipid digestion in the oral cavity and stomach
The digestion of lipids is initiated in the oral cavity by lingual lipase, prior to the passage of food
into the stomach via the oesophagus. The presence of lipids in the GI tract leads to the secretion of
gastric lipase from gastric mucosa chief cells22, 23. Gastric lipase is acid-resistant, and partially
hydrolyses triglycerides (TG) to diglycerides (DG) and fatty acids (FA)24, 25 with specific
preference for digestion at the sn-3 position on the glycerol backbone26, 27. Gastric lipid digestion
is thought to constitute a small portion of total lipid digestion. Indeed, FA concentrations (as an
indicator of the extent of lipid digestion) are up to 7-fold higher in the duodenum than in the
stomach of humans28, and 600-fold higher in pigs29. Nonetheless, the amphiphilic nature of lipid
digestion products (DG, FA), coupled with the shear force exerted on lipid droplets by gastric
propulsion, grinding and retropulsion, aid the emulsification of lipids in the stomach and increases
the surface area available for digestion. As a result, lipids enter the duodenum via the pyloric
sphincter as crude emulsion droplets with diameters less than 0.5 µm30. The rate of gastric
emptying is thought to be regulated (slowed) by a feedback mechanism stimulated by the presence
of FA in the lower SI31, 32, which further facilitates efficient lipid digestion in the duodenum.
Chapter 1: General Introduction
31
1.3.2 Lipid digestion and solubilisation in the small intestine
The presence of lipids in the duodenum stimulates the secretion of bile from the gall bladder and
pancreatic fluid from the pancreas33, 34. Bile contains bile salts (BS), phospholipid (PL),
cholesterol (Ch), bile pigments, organic wastes, and bicarbonate ions whereas pancreatic fluid
contains enzymes that catalyse the hydrolysis of lipids (lipase and co-lipase), proteins and peptides
(trypsin, chymotrypsin and carboxypeptidase) and carbohydrates (amylases). pH change from the
stomach environment to the small intestinal environment leads to ionisation of FA, which imparts
further amphiphilicity and results in improved emulsification of lipid droplets35.
Pancreatic lipase acts on the oil/water interface of lipid droplets to hydrolyse TG and DG to 2-
monoglyceride (2-MG) and FA as the final digestion products while amphiphilic bile constituents
such as BS and PL further aid the emulsification process to produce smaller lipid droplets,
increasing the effective surface area for lipid digestion. Co-lipase is required to anchor lipase to
the oil/water interface of oil droplets in the presence of bile salts36. In addition to the digestion of
TG and DG, PL (predominantly phosphatidylcholine (PC)) is hydrolysed to lysophospholipids
(LPL) (predominantly lysophosphatidylcholine (LPC)) and FA by pancreatic phospholipase A237;
and cholesterol esters (which comprises 10-15% of dietary cholesterol) are hydrolysed to free
cholesterol by pancreatic cholesterol esterase38.
Although the aqueous solubility of lipid digestion products is inherently low, the apparent
solubility of these molecules in the aqueous environment of the SI may be increased many fold via
solubilisation within bile-derived micellar species. In effect, the amphiphilic nature of the biliary
lipids (BS, PL, LPL) and the lipid digestion products (MG, FA) enable self-association of
endogenous and exogenous sources of lipids to form a series of colloidal structures that maintain
lipid digestion products in a solubilised state.
Chapter 1: General Introduction
32
Digesting lipids in the SI display complex phase behaviour depending on the types and quantities
of lipid digestion products and biliary components present. Several studies have examined the
changes in phase behaviour that occur as lipids are diluted and digested in intestinal fluids and
provide insights into the expected phase changes that occur in vivo as lipid digestion products are
formed on the surface of a lipid droplet and are progressively diluted (i.e. solubilised) by intestinal
fluid39-41. The physical changes to dietary lipids that occur during lipid processing in the SI are
summarised in Figure 1.1.
Chapter 1: General Introduction
33
Figure 1.1: Schematic diagram showing the physical changes to dietary lipids during lipid
processing in the SI. Crudely emulsified oil droplets enter the duodenum from the stomach and
stimulate the secretion of biliary and pancreatic fluids. BS, PL and Ch further emulsify the lipid
droplets and increase the surface area available for digestion. Pancreatic lipase and co-lipase
catalyse the hydrolysis of one TG molecule to 2 FA molecules and 1 MG molecule. Lipid
digestion products accumulate on the surface of digesting oil droplets, where they become
increasingly hydrated and eventually slough off as liquid crystalline lamellar, cubic or hexagonal
phases. In the presence of raised BS, PL, Ch concentrations in the SI lumen, lipid digestion
products are incorporated into bile micelles to form a solubilised system often referred to as the
intestinal mixed micellar phase. Continued dispersion of lipid colloidal phases by secreted bile
results in the generation of progressively smaller species. Under conditions of low lipid dispersion
(i.e. high lipid, low bile), larger multilamellar vesicles are thought to predominate; whereas
smaller unilamellar vesicles and mixed micelles co-exist under conditions of high lipid dispersion
(i.e. low lipid, high bile). Absorption of MG and FA is thought to occur from mixed micelles and
unilamellar vesicles, species with smaller particle sizes (< 500 nm) that are capable of permeating
the intestinal unstirred water layer and accessing the absorptive membrane.
Chapter 1: General Introduction
34
During lipid digestion, more polar (but still poorly-water soluble) digestion products accumulate
at the surface of digesting oil droplets, where they are thought to ultimately swell (due to
hydration) and slough off as liquid crystalline phases ranging from lamellar to cubic and
hexagonal phases39-41. The presence of lamellar, inverse hexagonal and micellar/bicontinuous
cubic phases during lipid digestion has recently been confirmed by the use of a coupled in vitro
lipid digestion-synchrotron SAXS (small-angle X-ray scattering) model to study real time
evolution of liquid crystalline structures40-42. Currently, there is increasing research effort to
understand the impact that these structures have on drug solubilisation and absorption, and the
potential for the provision of modified release characteristics43, 44. In the SI lumen, the liquid
crystalline phases are diluted by (or solubilised by) intestinal fluids and biliary secretions, leading
to the generation of smaller and less lipid-rich colloidal vesicular and micellar phases. Under
conditions of low dilution, that is, when the concentration ratio of biliary components:lipid
digestion products is low, larger multilamellar vesicles are thought to predominate (an exception
to this is seen in the presence of lipids with lower hydrocarbon chain lengths (C8) where micelles
are the only species observed, presumably due to the ability of C8 FA to self-aggregate and form
micelles at high concentration39). Further dilution of the phases (i.e. increasing the concentration
ratio of biliary components:lipid digestion products) leads to colloidal properties that reflect co-
existence of smaller mixed micelles and unilamellar vesicles39. Importantly, these colloids (i.e.
mixed micelles and unilamellar vesicles) that persist under conditions of high dilution likely
represent the phases that are responsible for the presentation of lipid digestion products to the
absorptive membrane. Despite the co-existence of vesicular structures with mixed micelles in the
aqueous phase of the SI lumen during lipid digestion, the solubilised phase is frequently referred
to as the ‘mixed micellar phase’. In reality the intestinal fluids may consist of a range of highly
dispersed lipid colloidal species. The properties of different types of intestinal colloidal aggregates
are summarised in Table 1.5.
Chapter 1: General Introduction
35
Solubilisation of lipid digestion products in small colloidal species in the mixed micellar phase is
also important in the promotion of lipid transport across the intestinal unstirred water layer (UWL).
The UWL is an aqueous diffusion barrier for poorly water-soluble compounds that separates bulk
intestinal fluid from the absorptive surface of enterocytes. The UWL is estimated to be 500-800
µm wide45, 46, has a lower (acidic) pH compared with the bulk intestinal fluid, and is
indistinguishable from a viscous mucus layer consisting water (~ 95%), glycoproteins, lipids,
mineral salts and free proteins45, 47, 48. Since nano-scale particles are able to permeate intestinal
mucus layers49, 50, small colloidal phases that persist under conditions of high dilution (such as
mixed micelles and unilamellar vesicles) are expected to diffuse more readily across the UWL.
Although micellar and vesicular species are larger and slower to diffuse when compared to lipid
monomers, colloidal solubilisation enhances the net diffusion rate of lipids across the UWL, due
to their ability to shuttle more lipid molecules across the UWL in a given time, when compared to
the diffusion of lipid molecules as monomers33, 51.
Chapter 1: General Introduction
36
Table 1.5: Properties of lipid colloidal phases found in the SI during lipid digestion.
Colloidal species Properties
Simple micelles Simple micelles are small aggregates of 2-4 bile salt molecules that form spontaneously at concentrations above the critical micellar concentration of bile salts52. The bile salt molecules orientate in such a way that the aromatic hydrocarbon moieties form the core of the micelle, with the polar head groups facing the aqueous environment. Lipid digestion products are incorporated into the hydrophobic core of micelles, thus are ‘solubilised’ in the aqueous SI environment.
Mixed micelles Mixed micelles are micelles that are composed of more than one lipidic species. Simple micelles containing solubilised lipids are mixed micelles by definition. The hydrodynamic radii of mixed micelles are ~ 3–10 nm52. Some properties of simple micelles may change upon solubilisation of lipids. For example, bile salt-lecithin and bile salt-monoglyceride micelles are swollen (larger) and have greater solubilisation capacity for cholesterol and fatty acids respectively, when compared to simple bile salt micelles52.
Vesicles Vesicles are composed of a single lipid bilayer (unilamellar), or alternating lipid bilayers and layers of water (multilamellar). Simple vesicles are typically formed by phospholipids (phospholipids are capable of forming lamellar liquid crystals in water); while the inclusion of cholesterol in vesicles has been suggested to stabilise the bilayer53. Phospholipids and cholesterol in bile are thought to be secreted into the duodenum partly as unilamellar vesicles54. The hydrocarbon chains of the lipid bilayer form the hydrophobic sites in which hydrophobic regions of lipid digestion products can be solubilised in, while the hydrophilic polar groups of the lipid bilayer orientate towards the aqueous layers of water.
Two types of vesicle may be found in the SI during lipid digestion: larger multilamellar vesicles (hydrodynamic radii ~ 50–250 nm54) under conditions of low bile salt and smaller unilamellar vesicles (hydrodynamic radii ~ 40–60 nm52) at higher bile salt concentrations. Vesicles are typically able to solubilise more lipid molecules per particle when compared to micelles due to their larger size.
Liquid crystal phases
PL, FA and MG are insoluble swelling amphiphiles that may form liquid crystal phases when dispersed in aqueous media52. Liquid crystals exhibit both the long range order of crystalline materials and the disorder of liquid systems, and may include lamellar, cubic and hexagonal phases. The relationship between lipid structure and liquid crystal phase formation is defined by the critical packing parameter (p), which is calculated from the effective hydrocarbon volume (v), the fully extended hydrocarbon chain length (l) and the area of the hydrophilic headgroup (a), where p = v/al55. Factors that may affect the packing parameter include hydration, pH and temperature. For example, as water is added to anhydrous amphiphilic lipid (thus decreasing the packing parameter), structural changes from inverse cubic and hexagonal phases (w/o systems, v/al > 1) to lamellar phase (v/al = 1) to normal cubic and hexagonal phases (o/w systems, v/al < 1) may be observed55. During lipid digestion, FA and MG are thought to accumulate at the surface of digesting oil droplets, where they are increasingly hydrated and ultimately sloughed off as liquid crystal phases.
Chapter 1: General Introduction
37
1.3.3 Lipid absorption across the enterocyte apical membrane
Realising that micellar structures act as carriers of lipids across the UWL, Westergaard and
Dietschy suggested a model of absorption where micelle-solubilised lipids served as a reservoir to
replenish the passively absorbed free fraction via an equilibrium relationship51. Later, Shiau and
colleagues postulated that absorption of FA (specifically LCFA) was further facilitated by the
acidic microclimate of the intestinal UWL. They suggested that exposure of LCFA to pH values
below their pKa promoted LCFA protonation and led to an attenuation in LCFA amphiphilicity.
This in turn was suggested to enhance LCFA absorption via (i) enhanced LCFA partitioning
across the lipophilic absorptive membrane, and (ii) enhanced LCFA thermodynamic activity (due
to reduced LCFA solubility in bile micelles)56, 57. In both cases, however, uptake of FA across
enterocyte brush border membrane was thought to occur via passive diffusion.
In contrast, in a seminal paper by Chow and Hollander, linoleate (a LCFA) uptake across the
absorptive membrane was shown to be concentration dependent, and facilitated diffusion was
suggested to be the main mechanism of absorption at low lipid concentrations, whereas simple
passive diffusion was thought to dominate at high concentrations58. In subsequent years, findings
from several authors have supported the concept of dual, concentration-dependent mechanism of
absorption for LCFA59, 60, and efforts have been made to identify the plasma membrane lipid
transporters responsible for facilitated LCFA absorption. Transporters that have been identified
include CD3661, FATP462, SR-BI63 and FABPpm64, 65
, although conclusive evidence to support the
role of these receptors in the intestinal absorption of LCFA remains elusive. A number of
transporters including CD3661, SR-BI66, 67 and NPC1L168 have also been implicated in cholesterol
uptake across intestinal brush border membrane. The mechanisms of lipid absorption into
enterocytes are summarised in Figure 1.2.
Chapter 1: General Introduction
38
1.3.4 Lipid transport into the systemic circulation
The GI tract is supplied by an extensive network of blood and lymphatic vessels. Absorbed FA
and MG may be transported to the systemic circulation via either the portal blood, or the intestinal
lymph (Figure 1.2 (iv)). Lipids that are destined for transport into the portal vein diffuse directly
across enterocytes and gain access to the blood capillaries via the lamina propria. Lipids that are
destined for transport into the lymphatic vessels are trafficked to the endoplasmic reticulum where
they are re-synthesised to TG via either the 2-monoglyceride pathway (which predominates in the
fed state)69, 70 or glycerol-3-phosphate pathway (which predominates in the fasted state)71, 72. Re-
synthesised TG, together with cholesterol esters, constitutes the primary core lipids of intestinal
lipoproteins. Intestinal lipoproteins are large colloidal particles with a hydrophobic core and a
hydrophilic surface (primarily consisting phospholipid, free cholesterol and apolipoproteins). In
the fasted state, very-low-density lipoproteins (VLDL) are preferentially produced by the
enterocytes whereas after the ingestion of lipids, both VLDL and chylomicrons (larger but less
dense lipoproteins) are produced. Intestinal lipoproteins that are assembled in the endoplasmic
reticulum of enterocytes are subsequently exocytosed into the lamina propria where they
preferentially access the lymphatic vessels rather than the blood capillaries. Preferential lymphatic
access occurs because the diffusion barrier of the vascular endothelium to large lipoprotein is
significant (resulting from the presence of tight junctions between adjacent endothelial cells, and
an underlying basement membrane), whereas the lymphatic endothelia barrier is considerably
more permeable (due to the presence of wider intercellular spaces between lymphatic endothelial
cells (Figure 1.2 (iv)). In general, FA and MG with hydrocarbon chain lengths of 14 and above are
primarily transported into the intestinal lymph whereas the more water-soluble short-chain and
medium-chain FA and MG are primarily transported by the portal blood73-76. Absorbed LPC may
be re-synthesised to PC77-79 (and incorporated into the hydrophilic surface of lipoproteins) or
hydrolysed to glycerol-3-phosphorylcholine that is transported via the portal blood80, 81. In the fed
Chapter 1: General Introduction
39
state, absorbed cholesterol is esterified to cholesterol esters82-84, prior to incorporation into the
hydrophobic core of lipoproteins.
Figure 1.2: Schematic diagram outlining the mechanisms of lipid absorption into enterocytes (i, ii,
iii) and lipid transport into the systemic circulation (iv). (i) Solubilisation of lipid digestion
products within mixed micelles or unilamellar vesicles enhances the net diffusion rate of lipids
across the UWL and maximises the concentration of lipids adjacent to the absorptive membrane.
Lipid absorption occurs via the free fraction, which is in equilibrium with the solubilised fraction.
(ii) Diffusion of colloids across the acidic UWL leads to protonation of LCFA (depicted as a loss
of head groups on lipid molecules), which reduces LCFA solubility in bile micelles (thus swelling
micelles), and enhances LCFA absorption via enhanced partitioning across the absorptive
membrane and enhanced thermodynamic activity. (iii) LCFA absorption at low concentration is
thought to be mediated by a saturable, facilitated process. (iv) Absorbed lipids are transported to
the systemic circulation either via the portal blood, or the intestinal lymph. The intestinal lymph
drains via the thoracic lymph directly into the systemic circulation at the junction of the left
subclavian vein and left jugular vein, therefore avoiding first-pass through the liver.
Blood vessel
Lymph vessel
Unstirred water layerBulk lumen
lipoprotein
carrier-mediated uptake
Lipid digestion products
BS, LPC, Ch(i)
(ii)
(iv)
(iii)
Enterocyte
lamina propria
Chapter 1: General Introduction
40
1.4 MECHANISMS BY WHICH LIPIDS ENHANCE THE ORAL
BIOAVAILABILITY OF DRUGS
In the absence of carrier-mediated uptake, flux per unit area (F) across the intestinal absorptive
membrane can be represented by the following equation:
F P. C Equation 1.1
where P is the permeability coefficient of drug across the apical membrane; and C is the
concentration of drug on the abluminal side of the apical membrane.
For solubilised drug, and assuming that drug absorption is driven only by the free concentration in
the GI lumen, the maximum flux (per unit area) that can be obtained across the apical membrane
is a function of the permeability coefficient and the maximum free concentration. In the absence
of supersaturation, the maximum free concentration that can be obtained in equilibrium with a
solubilised reservoir is the equilibrium solubility (Cs) of a drug in the inter-micellar phase of the
SI content (a value that approximates the solubility of drug in water buffered to intestinal pH). The
rate of drug absorption is therefore limited by permeability across the absorptive membrane and/or
its solubility in the SI lumen. Furthermore, although drug solubilisation increases the total mass of
drug in solution, the free drug concentration is not expected to be raised above that of drug in
simple solution. Based on Equation 1.1, therefore, drug solubilisation would not be expected to
increase drug absorption. In contrast, a large number of studies detail the utility of solubilising
formulations in enhancing the oral bioavailability of PWSD85-87. This apparent anomaly may be
explained by the impact of drug solubilisation on dissolution (as described in Section 1.4.1 below)
and the potential for formulations to promote supersaturation, where the free concentration can be
Chapter 1: General Introduction
41
raised above the equilibrium aqueous solubility (see Section 1.5.3). The latter is the major topic of
this thesis.
After oral administration, the dissolution rate of a drug in the GI fluids (i.e. the rate at which drug
passes into solution in the GI tract) may also be an important determinant of drug absorption.
Since transit time along the GI tract is finite, the rate of drug dissolution must be sufficiently rapid
to allow drug to pass into solution, and be available for absorption prior to transit past the
absorptive site. The dissolution rate of a drug is related to its solubility according to the Noyes-
Whitney equation88:
. C C Equation 1.2
where dc/dt is the dissolution rate of the drug; D is the diffusion rate of the drug in water; A is the
surface area of contact of the solid with the dissolution fluid; h is the width of the diffusion layer;
Cs is maximum drug concentration at the surface of the dissolving fluid i.e. drug solubility; and C
is the concentration of drug in the well-stirred bulk.
The low aqueous solubility of PWSD (i.e. low Cs) dictates that the dissolution rate is also low and
that the absorption of many PWSD is limited by low solubility and/or slow dissolution in the GI
fluids. In general, co-administration with lipids provides PWSD with the opportunity to intercalate
into endogenous lipid digestion and absorption pathways (stimulated by the ingestion of lipids),
ultimately leading to enhancements in drug dissolution, solubilisation, absorption and systemic
exposure as described below.
Chapter 1: General Introduction
42
1.4.1 Enhancement of drug dissolution in gastrointestinal fluids
Dosing PWSD in LBF offers significant dissolution advantages when compared to a conventional
solid dose form. LBF typically comprise lipid solutions or emulsion-preconcentrates that are filled
into soft or hard gelatin capsules prior to administration. In this cases, drug is pre-solubilised
within the formulation, thus obviating the need for traditional solid-liquid drug dissolution76.
Ideally, as these lipidic formulations are dispersed, digested and solubilised in the intestinal
content, drug remains solubilised in a monomolecular form and in equilibrium with drug free in
solution. In effect therefore, dissolution is replaced with a (rapid) partitioning process between
solubilised drug and drug in free solution. Bearing in mind the intrinsically slow rate of
dissolution of PWSD from solid dose forms, drug solubilisation in LBF therefore provides
significant dissolution rate advantages.
However, drug precipitation frequently occurs during formulation dispersion and digestion. Slow
drug re-dissolution from the precipitated solids into GI fluids may therefore limit drug absorption
even though the dose is initially solubilised in the formulation. Nonetheless, the co-administration
of lipids may also enhance the dissolution rate of solid drug in the SI via improvements in wetting,
and enhancements in drug solubilisation capacity. Specifically, raised concentrations of
amphiphilic biliary lipids (BS, PL) in the SI after lipid ingestion reduces the contact angle between
solid drug and intestinal fluid, thereby increasing the surface area available for dissolution. This
mechanism has been documented to increase the dissolution rate of phenylbutazone and
hydrocortisone89, 90. Solubilisation of drugs within fed state intestinal mixed micelles (which may
consist BS, PL, FA, MG) also significantly increases the apparent solubility of many PWSD in
intestinal fluids when compared to the fasted state39, 91, thereby increasing dissolution via
increases in solubility according to the Noyes-Whitney equation (Equation 1.2)92. Increases in
dissolution afforded by enhanced solubilisation however, may be off-set by the larger sizes of
Chapter 1: General Introduction
43
micellar species which have slower diffusion rates (hence lower D value in Equation 1.2), which
explains the less-than-proportional increase in dissolution rate (in relation to solubility) observed
in many studies93, 94.
1.4.2 Enhancement of apparent drug solubility in intestinal fluids
After the ingestion of lipids, biliary secretions combine with lipid digestion products to generate a
series of intestinal lipid colloidal phases including emulsion droplets, vesicles and mixed micelles
that have markedly increased solubilisation capacities for PWSD. Although increases in the
concentrations of BS, PL, Ch in the fed state GI lumen alone results in significantly increased
solubilisation capacity of a wide range of PWSD95-98, incorporation of digested lipids into bile salt
micelles has been shown to afford further enhancements in drug solubilisation capacity39, 91,
presumably due to the ability of lipid digestion products to swell endogenous bile micelles and to
generate a range of lipid colloidal phases with increased solubilisation capacity (discussed in
Section 1.3.2). The solubilisation enhancement afforded by the incorporation of exogenous lipids
is dependent on the nature of the digestion products and the structures of the colloidal species
formed. For example, medium-chain FA and MG are more amphiphilic than long-chain FA and
MG and more readily combine with endogenous BS, PL and Ch. Thus, at the same lipid load,
medium-chain lipids typically form smaller, more optically clear micellar dispersions while long-
chain lipids form slightly turbid systems containing both vesicular and micellar species. Drug
solubilisation enhancement afforded by the two systems at similar lipid loads are therefore
significantly different, with a less than 3-fold enhancement (relative to drug solubility in
endogenous BS, PL, Ch species) observed in the case of medium-chain lipids and up to 20-fold
enhancement observed in the case of long-chain lipids39, 99. Thus, the mass of drug that may be
solubilised in fed state GI fluids is markedly higher than that in the absence of lipid co-
administration.
Chapter 1: General Introduction
44
Analogous to the mechanism by which micellar solubilisation increases FA transport across the
intestinal UWL (outlined in Section 1.3.2), micellar solubilisation may also increase drug
transport across the UWL, thereby increasing the mass of solubilised drug that is presented to the
absorptive membrane. This concept has been applied to good effect in mathematical models
simulating the absorption of progesterone100 and danazol101 from bile micelles. Thus, current
understanding suggests that drug absorption across the apical membrane of enterocytes occurs via
the free drug fraction that is in equilibrium with the solubilised fraction; and that upon drug
absorption from the free fraction, drug rapidly partitions out of the solubilised reservoir to
replenish the free concentration and maintain solubilisation equilibrium2. In turn, the presence of
drug-rich micelles at the absorptive membrane is likely maintained by the continuous diffusion of
micelles from the lumen where lipid digestion, dispersion and drug solubilisation is on-going.
Therefore, although the concentration of drug in the free fraction (the fraction available for
absorption) is not raised by micellar solubilisation (the maximum attainable inter-micellar free
concentration remains at the solubility limit of drug in water), micellar solubilisation overcomes
the solubility limitations of PWSD on the abluminal side of the apical membrane via the provision
of a solubilised reservoir which replenishes free drug concentration during on-going absorption.
1.4.3 Enhancement of intestinal permeability and inhibition of enterocyte-
based efflux transporters and metabolism
Many LBF components (pre- or post-digestion) have been suggested to enhance passive
paracellular (i.e. between cells) and transcellular (i.e. across cells) membrane diffusion102, 103.
Excipients that enhance transcellular permeability are of relevance for PWSD, as PWSD are likely
to be absorbed via the transcellular route rather than the paracellular route due to their
hydrophobicity. Medium-chain FA and glycerides104, 105, long-chain FA and MG103, and a variety
of surfactants106, 107 have been shown to increase the transcellular permeability of the intestinal
Chapter 1: General Introduction
45
epithelial membrane in vitro as well as in Caco-2 cells. The ability of excipients to increase
membrane permeability has been attributed to their amphiphilic/surfactant properties, which
disrupt the organisation of the lipid bilayer. Generally, surfactants that have large, charged or
hydrophilic head groups and a relatively small hydrophobic tail are most likely to increase
transcellular permeability as they are taken up rapidly by the outer membrane leaflet but are slow
to translocate to the inner membrane leaflet2. The increased residence time of surfactant molecules
between the bilayers, coupled with the inability of the hydrophobic tail to fill the void between
adjacent membrane lipids, leads to monolayer/bilayer curvature strain, and the generation of a
more permeable membrane. In extreme cases, membrane solubilisation is possible where the
curvature strain is sufficiently high and membrane lipids are expelled. Bile salts are strong
membrane solubilisers103, however, the presence of phospholipid in endogenous bile is expected to
reduce the permeability enhancement effects of bile salts by decreasing bile salt thermodynamic
activity in mixed bile salt-phospholipid micelles108. Although many drugs that are formulated in
LBF have high membrane permeability (e.g. typical BCS Class II compounds), the permeability-
enhancing effects of lipid excipients may be of relevance to BCS Class IV drugs where absorption
is likely limited by both poor solubility and membrane permeability.
An increasing number of LBF excipients have recently been described to enhance drug
permeability via inhibition of intestinal efflux transporters such as P-glycoprotein (P-gp), breast
cancer resistant protein (BCRP) and multidrug resistance-associated protein (MRP)2, 109-112.
Intestinal efflux transporters are present on the apical membrane of enterocytes, and actively pump
absorbed drugs back to the intestinal lumen, thus limiting the absorption of their substrates. Well-
known substrates of efflux transporters include digoxin113, doxorubicin114, vincristine114,
cyclosporine A115, verapamil116 and the HIV protease inhibitors117. Examples of common LBF
excipients that inhibit P-gp, BCRP and MRP have been compiled by Williams et al..2 The
Chapter 1: General Introduction
46
mechanisms of efflux inhibition are thought to include competitive or allosteric inhibition of drug
binding sites111, 118-120, changes to membrane fluidity leading to protein destabilisation121-123, and
reductions in transporter expression124-126.
The CYP3A family of metabolic enzymes (also present in enterocytes) share similar substrate
specificity to P-gp127, 128, and have been suggested to be functionally linked with P-gp to limit the
systemic exposure of their substrates129, 130. Although the mechanism of interaction between P-gp
and CYP3A is not well-understood, it is postulated to be due to the function of the efflux
transporter in increasing substrate exposure to the metabolising enzyme in a given time (via
repeated cycles of drug efflux and reabsorption131, 132 which may also modulate the degree of
enzyme saturation), or increased affinity of CYP3A-generated metabolites for P-gp when
compared to the parent compound131. Thus, inhibition of intestinal efflux might also be expected
to reduce enterocyte-based first-pass metabolism, and increase the oral bioavailability of drugs.
Dietary lipids and LBF components may also affect pre-systemic metabolism via direct effects on
enzyme activity and indirect effects on enzyme expression levels133. Examples of lipids or
solubilising excipients that increase drug exposure by altering pre-systemic metabolism may be
found in reviews by Patel et al.133 and Buggins et al.134, respectively. Systemic metabolism may
be altered by lipid co-administration via the alteration of systemic lipoprotein levels (therefore
changing the extent of drug binding in the blood which may decrease or increase uptake into
metabolising compartments)133, 135, 136, although this mechanism is less likely for formulation
lipids due to the small lipid doses contained.
Although the use of LBF excipients as permeability enhancers and metabolic and efflux inhibitors
is receiving increasing interest, relatively few studies, to this point, have shown significant in vivo
Chapter 1: General Introduction
47
effects for transcellularly transported drugs. As such the major effects on bioavailability
enhancement appear to be driven by differences in solubility and dissolution.
1.4.4 Promotion of lymphatic drug transport
As described in Section 1.3.4, ingestion of lipids promotes the assembly of triglyceride-rich
lipoproteins (TRL) in the enterocyte and these are transported from the enterocyte to the systemic
circulation via the intestinal lymph. Drug transport into the systemic circulation may occur via
either the portal blood, or the intestinal lymph. Since the blood flow of the portal vein is
approximately 500-fold higher than the lymph flow in the mesenteric lymph duct137, the majority
of absorbed drugs are transported to the systemic circulation via the portal blood76. However, for
highly lipophilic (log P > 5) and highly lipid soluble (LCT solubility > 50 mg/g) drugs, the
potential for lymphatic transport is increased due to the increased likelihood of drug association
with developing TRL in the enterocyte138. Since intestinal lymph drains directly into the systemic
circulation (by-passing the liver), co-administration with lipids may increase the bioavailability of
lymphatically transported drugs via the avoidance of first-pass metabolism in the liver.
Enhancements in lymphatic drug transport have been observed for many drugs after co-
administration with dietary or formulation lipids137. Significantly, studies with halofantrine have
shown that even small amounts of long-chain lipid (e.g. quantities available within a single
capsule) are capable of supporting substantial lymphatic drug transport in the fasted state. In this
way, LBF have the potential to increase oral drug bioavailability for compounds that are highly
first-pass metabolised and that are lymphatically transported139. In general, lymphatic drug
transport increases with administered lipid load138-140, and is more effectively promoted by long-
chain and monounsaturated glycerides than their medium-chain/short-chain and saturated
counterparts12, 138, 139.
Chapter 1: General Introduction
48
1.5 PROPOSED MECHANISMS OF DRUG ABSORPTION FROM
INTESTINAL MIXED MICELLES AND VESICLES
1.5.1 Current model of drug absorption and its limitations
In contrast to the wealth of data describing lipid absorption from intestinal mixed micelles
(discussed in Section 1.3.3), the mechanism of absorption of PWSD from mixed micelles remains
poorly understood. Currently, the absorption of PWSD is believed to occur via passive diffusion
of drug monomers across the enterocyte apical membrane (see Figure 1.3(A), solid black arrow),
and drug in the solubilised fraction serves as a reservoir to replenish absorbed drug via re-
establishment of the solubilisation equilibrium:
Ccolloid ↔ Cfree
where Cfree and Ccolloid represent the free concentration and the concentration solubilised in
micelles, respectively. The total solubilised drug concentration (Ctotal) is the sum of Cfree and
Ccolloid:
Ctotal = Cfree + Ccolloid Equation 1.3
In addition to passive diffusion, some drugs may be actively absorbed by specific transporters
present on the apical membrane (see Figure 1.3 (A), dotted black arrow), usually as a result of
structural similarities of the drug to endogenous substrates of the transporter141, 142. Some
examples of active transporters in the intestine and their drug substrates are listed in Table 1.6.
Chapter 1: General Introduction
49
Table 1.6: Examples of active transporters in the intestine with specific reference to drug
substrates141.
Transporter Family and Example Substrates
amino acid – large amino acid transporter L-dopa, baclofen, gabapentin
oligopeptide – PepT1 lisinopril, cefadroxil, bestatin, cephradine
Na+ dependent phosphate transporter foscarnet, fosfomycin
monocarboxylic acid – MCT1 pravastatin, salicylic acid, carindacillin
reduced folate transporter (RFT) methotrexate
monosaccharide transporters – SGLT1 glucose, p-nitrophenyl-β-D-glucopyranoside
apical Na+ dependent bile acid transporter (ASBT) S3744 (investigational compound)
nicotinic acid transporter valproic acid, penicillins
Importantly, regardless of active or passive transport, the maximum driving concentration for drug
absorption (i.e. maximum Cfree) is not typically increased by micellar solubilisation and is defined
by drug solubility in the inter-micellar fluid (essentially the aqueous solubility). From the
equilibrium shown above, the relationship between the free fraction (f) and the solubilised fraction
(s) may be described by the following equations143:
s = (Cm – Cs)/Cm Equation 1.4
f = 1 - s Equation 1.5
where Cm is the solubility of drug in the solution with micelles and Cs is the solubility of drug in
the same medium without micelles (essentially the aqueous solubility). It is assumed that s is
Chapter 1: General Introduction
50
constant and independent of the drug concentration for a particular concentration of micelles, and
that only one type of micelles exist. Thus,
Ctotal = f.Ctotal + s.Ctotal Equation 1.6
Since the solubilised and free drug concentration are in equilibrium, the degree of drug saturation
in the free fraction and solubilised fraction are expected to be equal. For example, when a micellar
system is loaded with drug at its full solubilisation capacity, Cfree will be the saturated solubility of
the drug in the colloid-free medium (i.e. Cs), while Cmicelle will be the saturated solubility of the
drug in the colloids (i.e. Cm – Cs).
Therefore, even if micelles are efficient in promoting drug transport across the intestinal UWL and
replenishing the absorbed free fraction, the inter-micellar drug concentration at the absorptive
membrane is at best maintained at the solubility limit of free drug in water, and is very low. Since
solubilisation does not increase (and may reduce) Cfree, and drug flux across an absorptive
membrane is the product of the free drug concentration and the drug permeability across the
membrane, lipid co-administration may not be expected to enhance the absorption of drugs that
are solubility- (but not dissolution rate) limited. Indeed, many authors have shown that increasing
the total concentration of solubilised drug within bile salt or synthetic micelles does not
necessarily lead to proportional enhancements in intestinal absorptive flux100, 144, 145. These
observations recently led Miller et al. and Dahan et al. to suggest that formulations that enhance
apparent drug solubility via solubilisation may do so at the expense of overall drug absorption due
to decreases in the free fraction100, 146, 147.
Chapter 1: General Introduction
51
Co-administration with lipids, however, remains an effective means to enhance the oral absorption
of PWSD, as exemplified by the myriad drugs that demonstrate positive food effect and
bioavailability enhancement when administered in a LBF. In addition, mathematical models of
intestinal drug absorption that utilise Cfree as the driving concentration frequently underestimate
the absorption of BCS Class II compounds101, 148, 149. Indeed, the use of total solubilised
concentrations (i.e. Cfree plus Cmicelle) often led to better predictions101, 148. These observations
suggest that the absorption of lipophilic, poorly water-soluble compounds from lipid-based
colloids may not be solely dictated by membrane permeability and free solubility, and that instead
additional mechanisms may contribute to overall flux.
Therefore, the principle hypothesis in this thesis is that after lipid co-administration, the
solubilised fraction (i.e. the fraction where the majority of drug in solution resides) is not simply
an inert reservoir that replenishes Cfree, but may contribute to drug flux across absorptive
membrane via enhancements in apparent drug permeability or apparent drug solubility. The
proposed mechanisms where the solubilised fraction may contribute to drug absorption are
discussed below:
Chapter 1: General Introduction
52
Figure 1.3: Schematic diagram outlining the mechanisms of drug absorption from intestinal mixed
micelles or vesicles. (A) Current understanding of drug absorption where uptake across the enterocyte
apical membrane is driven by the free fraction (black solid arrow) while the solubilised reservoir
replenishes absorbed free drug via an equilibrium relationship. Additionally, the absorption of drugs that
possess structural similarities to endogenous substrates may be facilitated by specific transporters present
on the apical membrane of enterocytes (black dotted arrow). (B) Proposed mechanism of drug absorption
where in addition to passive diffusion of free drug, absorption may also occur via collisional uptake (red
dotted arrows), where micelles or vesicles interact directly with the apical membrane to mediate selective
transfer of solubilised drug or endocytosis of colloid-receptor complex. Collisional uptake may or may not
be receptor-mediated. (C) Proposed mechanism of drug absorption where the stimulation of drug
supersaturation in a previously stable drug-containing colloid leads to transient increases in the
thermodynamic activity (free concentration) of drug, thereby increasing the number of free drug molecules
that are available for passive diffusion across the apical membrane (red dotted arrows). These alternative
mechanisms of drug absorption from colloidal species are examined in detail in this thesis.
D DSSMixed micelle / vesicle Drug Supersaturated drug
Drug transporters Lipid uptake transporters
DDD
DDD
(A)
DD
DD DD
(B)
DDSS
DSSD DSSDSS
(C)
DD
DDD
Supersaturation stimulus
Chapter 1: General Introduction
53
1.5.2 Collisional uptake of solubilised drug
The transfer of FA between model cell membranes, proteins (receptors) and intestinal lipid
colloidal phases via collisional transfer has been extensively investigated by Storch and
colleagues150-153. Collisional transfer refers to the transfer of solubilised content from donor to
acceptor compartments via direct interactions (collisions). For example, Narayanan et al. showed
that FA transfer between mixed micelles occurred as a consequence of micelle collisions rather
than a diffusion process where FA first dissociate from donor micelles into the aqueous phase
prior to association with acceptor micelles152. Similar findings have also been demonstrated for the
transfer of LCFA from phospholipid vesicles to proteins such as brain-FABP151.
In the SI, since lipid digestion products and PWSD are also compounds with limited free solubility
in water, it is possible that the absorption of these compounds could occur via direct collisional
transfer from mixed micelles or vesicles to the absorptive membrane (see Figure 1.3(B), red dotted
arrows). Collisional transfer may or may not be receptor-mediated, however recent interest in the
role of intestinal lipid uptake transporters such as CD3661, 154, 155, SR-BI63, 67, 155, 156, NPC1L1157,
caveolin-1158-160, FATP462 and FABPpm64, 65 in lipid absorption, and reports of direct interactions
of colloidal or macromolecular species in the blood (e.g. albumin, HDL) with one or more of these
receptors have led to the hypothesis that absorption of solubilised lipids and drugs may also occur
via a similar mechanism. Thus colloid-receptor interactions between mixed micelles or vesicles
and lipid uptake transporters in the brush border membrane of the SI may facilitate lipid and/or
drug absorption (Figure 1.3(B), red dotted arrows). In this model, after initial collision,
monomeric lipids and PWSD may be selectively transferred into cells, or the entire colloid-
receptor complex may be endocytosed. A brief description of the relevant receptors and their role
in lipid uptake is given below:
Chapter 1: General Introduction
54
1.5.2.1 SR-BI
SR-BI (Scavenger Receptor Class B Type 1) is an 83 kDa glycoprotein that can bind to a variety
of ligands such as anionic phospholipids, apoptotic cells and modified/native lipoproteins in
vitro161. In vivo, SR-BI is a physiologically relevant receptor for HDL and plays a key role in
cholesterol transport into and out of cells. SR-BI acts as a docking receptor for plasma HDL (high
density lipoprotein), and mediates the selective uptake of cholesterol esters into hepatocytes and
steroidogenic tissues161. In the GI tract, SR-BI is expressed on the apical surface of intestinal villi
in the proximal small intestine67. However, the physiological ligand(s) and mechanisms of action
of SR-BI at this site remain unclear. Whilst a study by Hauser et al. reported inhibition of sterol
collisional transfer into brush border membrane vesicles (BBMV) and Caco-2 cells by anti-human
SR-BI IgG67, and there has been evidence of SR-BI receptor endocytosis in pig enterocytes156
during fat absorption, the role of SR-BI in lipid absorption is yet to be fully elucidated.
1.5.2.2 CD36
CD36 (Cluster of Differentiation 36), also known as Fatty Acid Translocase (FAT), is also a class
B scavenger receptor (88 kDa) that shares many ligands (such as native and modified lipoproteins,
anionic phospholipids and serum HDL) with SR-BI154, 155, 161. The distribution of CD36, however,
favours tissues with high metabolic needs for FA e.g. adipose tissue, heart muscle, skeletal muscle
and the intestine162, consistent with its established role as a FA transporter. The role of CD36 in
native LCFA uptake into cells has been well-studied especially in muscle162 and adipose tissues163.
For example, CD36 expression and FA transport are increased during chronic muscle
stimulation162, and CD36 knockout mice show deficits in cellular FA uptake i.e. elevated plasma
FA levels and reduced FA accumulation in muscle and adipose tissue163. Even though the role of
CD36 as a FA transporter is well-known, the exact mechanism of transport under normal
physiological conditions i.e. where FA are mostly bound to albumin, is less clear. Indeed, whether
Chapter 1: General Introduction
55
CD36 interacts with free FA or the entire FA-albumin complex in the plasma, and whether it acts
as a docking receptor or an endocytotic receptor to facilitate cellular uptake is not yet known. In
the SI, CD36 is most highly expressed on the apical side of intestinal villi of the duodenum and
jejunum163, and is believed to facilitate FA and cholesterol absorption. For example, FA and/or
cholesterol absorption is lower in vitro and in vivo in CD36 knockout mice when compared to
wild type mice61, and one study which investigated the inhibitory effect of CD36 antibodies on
radiolabelled sterol uptake into human BBMV concluded that CD36 is important in free
cholesterol uptake in the small intestine155. In contrast, in an investigation of FA uptake into the SI
of CD36 knockout mice, CD36 was found to be unimportant in FA uptake but instead played a
role in chylomicron formation and secretion in the enterocytes154. A potential role for CD36 in
intestinal lipid absorption is therefore well established. However, the mechanism whereby CD36
facilitates lipid uptake into enterocytes is not yet known.
1.5.2.3 NPC1L1
Ezetimibe, a cholesterol absorption inhibitor, is widely used in practice and has been shown to
have modest efficacy in lowering plasma cholesterol164. Although the exact mechanism of action
of ezetimibe remains unknown165, pursuit of potential sites of ezetimibe activity helped to reveal
the role of Niemann-Pick C1 Like 1 (NPC1L1) protein in cholesterol absorption. NPC1L1 is a 145
kDa165 membrane protein that is highly expressed in the SI, with detectable levels in tissues such
as the liver, gallbladder, testis, and stomach165. In the SI, NPC1L1 is predominantly expressed on
the apical side of the proximal small intestine, with the highest levels found in the jejunum164.
Unlike other lipid transporters, little is known about NPC1L1, especially its role outside the
intestine, in large part reflecting the relatively recent (2000) identification of NPC1L1 as a
potential lipid transporter166. What is known however, is the seemingly important role of NPC1L1
in cholesterol absorption and involvement in the ezetimibe-sensitive cholesterol absorption
Chapter 1: General Introduction
56
pathway. Thus, cholesterol absorption is reduced by more than 70% in NPC1L1 knockout mice
when compared to wild type mice167, and ezetimibe treatment does not reduce cholesterol
absorption further in the same knockout animals167. The available data therefore lend strong
support to a potential role of NPC1L1 in cholesterol absorption, however, the mechanism by
which NPC1L1 facilitates cholesterol absorption is still not established165. Data suggesting that
NPC1L1 aids cholesterol absorption by facilitating intracellular cholesterol transport167 (rather
than absorption directly) further complicate the issue of whether NPC1L1 is an authentic
cholesterol transporter, or a protein that facilitates intracellular trafficking, or both.
1.5.2.3 Endocytosis
Endocytosis can be mediated by several pathways including clathrin coated pits, caveolae-
mediated pathways, and non-clathrin, non-caveolae mediated endocytosis168. Briefly, clathrin-
mediated endocytosis occurs ubiquitously and involves the deformation of the plasma membrane
into a coated pit, a process mediated by surface proteins such as clathrin and adaptor protein-2.
Lipid rafts are domains of the plasma membrane that have a higher degree of order due to
enrichment by cholesterol, sphingolipids and phospholipids. Due to their less fluid (more rigid)
structure, lipid rafts are able to spatially segregate membrane lipids and proteins such that
membrane processes are also functionally separated169. Lipid rafts have received increasing recent
attention as plasma membrane platforms with involvement in the binding and uptake of LCFA160.
Caveolae are subsets of lipid rafts and are characterised by flask-like invaginations in the plasma
membrane that resemble vesicles that are poised to bud170. The expression of the scaffolding
protein, caveolin, is essential to the formation of caveolae170. Evidence also exists to suggest a role
for endocytotic pathways mediated by clathrin coated pits and caveolae in lipid uptake. For
example, the uptake of LCFA by human microvascular endothelial cells has been shown to
involve endocytosis mediated by clathrin-coated vesicles and caveolae171, and the lipid uptake
Chapter 1: General Introduction
57
transporters CD36 and SR-BI are found preferentially located in caveolae on the apical membrane
of enterocytes161, 172. Caveolin-1, the scaffolding protein of caveolae, binds FA with strong
affinity169, and may be a potential lipid uptake transporter itself. These observations suggest that
an endocytotic component to lipid uptake in the SI is possible.
1.5.3 Supersaturation-enhanced drug absorption
Supersaturation is a transient solution state that precedes precipitation, where the concentration of
solute in a system is higher than the equilibrium solubility of the solute in the same system.
Supersaturation results in an increase in thermodynamic activity, and therefore a potential means
by which drug absorption can be enhanced.
The potential for supersaturation to enhance the oral bioavailability of PWSD has received
increasing recent interest173, 174, and delivery systems that aim to generate supersaturated drug
concentrations in the GI lumen, and to stabilise the metastable supersaturated state (i.e. delaying
drug precipitation) are increasingly common. The attainment of intraluminal drug concentrations
in excessive of its equilibrium solubility may be achieved by delivering drugs in a solubilised
form that lose solubilisation capacity in situ (e.g. cosolvent systems, LBF), and the delivery of
high-energy crystal forms or amorphous materials that provide accelerated dissolution and/or
higher solubility than their most stable form173, and where precipitation does not occur
immediately.
In the context of LBF, Gao and Morozowich first reported on “supersaturable SEDDS” (S-
SEDDS), where dispersion of the formulation in the GI fluids led to drug precipitation, but where
addition of a precipitation inhibitor resulted in transient stabilisation of a supersaturated state17.
Chapter 1: General Introduction
58
They subsequently showed that the oral bioavailability of a number of drugs was significantly
improved after dosing of S-SEDDS when compared to a conventional suspension or SEDDS17, 175.
Recent data, however, suggests that many LBF, whether they are labelled as ‘supersaturable’ or
not, frequently lose drug solubilisation capacity during GI processing and have the potential to
trigger drug supersaturation. Solubilisation capacity can be lost via initiation of the digestion of
glyceride lipids91, 176 and/or surfactants177, and the dilution of co-solvents and surfactants5, 178. LBF
that lose solubilisation capacity in vivo may therefore benefit from supersaturation-enhanced drug
absorption, provided that precipitation is sufficiently delayed. In this regard, a range of polymers
have been explored for their utility in supersaturation stabilisation during GI processing of LBF,
with cellulose-based polymers appearing to be the most useful to date179.
In contrast, the possibility that supersaturation-enhanced absorption may play a role in the
absorption of solubilised drug from intestinal micellar and vesicular species has not been explored
in detail. Importantly, for a solubilised system, supersaturation is expected to increase Cfree above
drug aqueous solubility. This may provide a means to reverse the reduction in drug
thermodynamic activity resulting from solubilisation, and explain the paradox that is the ability of
solubilising LBF to increase the absorption of many PWSD despite apparent reductions in drug
thermodynamic activity (see Figure 1.3(C), red dotted arrows). In essence, the stimulation of
supersaturation in stable colloidal systems may provide a means to mobilise the solubilised drug
reservoir such that solubilised drug is more readily available for absorption. Since lipid-based
micelles and vesicles are dynamic structures that are in constant interaction with lipid dispersion,
digestion and absorption processes in the GI environment, changes to solubilisation capacity (and
therefore supersaturation opportunities) are likely to exist within endogenous lipid processing
pathways. This thesis has examined these possibilities in detail.
Chapter 1: General Introduction
59
1.6 STRUCTURE OF THIS THESIS
Chapters 3, 4 and 5 of this thesis have been directly reproduced from published or submitted
manuscripts. The constraints of journal publication dictate that these chapters focus on the results,
discussions, and conclusions of the experiments, but include relatively limited detail of the
methods employed and validation studies. A general methods chapter (Chapter 2) has therefore
been added to further describe the method development and validation studies conducted. The
thesis concludes with a final summary and perspectives chapter (Chapter 6).
60
CHAPTER 2 : GENERAL METHODS
Chapter 2: General Methods
61
2.1 INTRODUCTION
This general methods chapter contains further information on the in situ autoperfused rat jejunum
preparation, brief outline of the surgical procedures required for the conduct of in vivo
bioavailability studies, and a detailed description and validation of the analytical methods used
throughout this thesis. Other experimental methods have been included in the experimental
chapters (3 through 5) which are reproduced from published or submitted manuscripts.
2.2 AUTOPERFUSED RAT JEJUNUM
2.2.1 Materials
Sodium chloride (NaCl), D-mannitol, antipyrine, D-mannitol, [1-14C] (46.6 mCi/mmol),
antipyrine, [3-14C] (5.4 mCi/mmol) (Sigma-Aldrich, Australia), sodium hydroxide pellets (NaOH)
and ethanol 96% v/v (Merck, Australia), disodium hydrogen orthophosphate (Na2HPO4) and
sodium dihydrogen orthophosphate (NaH2PO4.2H2O) (Ajax Finechem, Australia), 0.9% Sodium
Chloride Intravenous Infusion BP (Baxter, Australia), polyethylene glycol, [1,2-3H] (1.50 mCi/g),
Irga-Safe PlusTM, SOLVABLETM (Perkin Elmer Life Sciences, MA, USA), oleic acid, [9,10-
3H(N)] (60 Ci/mmol) (American Radiolabeled Chemicals, MO, USA), heparin sodium injection
BP (1000 I.U./mL, Hospira, Australia), xylazine (100 mg/mL, Troy Laboratories, Australia),
acepromazine (10 mg/mL, Ceva Delvet, Australia), ketamine (100 mg/mL, Provet, Australia) and
pentobarbitone sodium (325 mg/mL, Virbac, Australia) were obtained from listed suppliers. Water
was obtained from a Millipore milliQ Gradient A10 water purification system (Millipore, MA,
USA).
Chapter 2: General Methods
62
2.2.2 Methods
2.2.2.1 Surgical equipment and accessories
The surgical instruments required for the setup of the autoperfused rat jejunum include a scalpel, a
pair of sharp straight scissors, a pair of iridectomy scissors, two curved anatomical forceps and
one fine tip curved forceps. A portable, multi-head, flexible cold fibre-optic illuminating system
(Microlight 150®, Fibre optic light guides, Australia) was used to obtain adequate lighting for
surgery. A heated surgical board (Ratek Instruments, Australia) was used to maintain body
temperature of the animals throughout surgery and experiments.
2.2.2.2 Animal care and anaesthesia
All rat studies were approved by the institutional animal ethics committee, and were conducted in
accordance with the guidelines of the Australian and New Zealand Council for the Care of
Animals in Research and Teaching. Male Sprague-Dawley rats (280–330 g) were used in all
experiments, and were allowed to acclimatize in the institutional animal housing facility for at
least 7 days with free access to standard chow and water. All animals were fasted overnight (12–
18 h) prior to surgery. Anaesthesia was induced in rats by subcutaneous injection of 1.0 mL/kg of
‘Cocktail I’ (37.3 mg/mL ketamine, 9.8 mg/mL xylazine, 0.4 mg/mL acepromazine in saline), and
maintained throughout the study with subcutaneous doses of 0.44 mL/kg of ‘Cocktail II’ (90.9
mg/mL ketamine, 0.9 mg/mL acepromazine) when required. To prepare for surgery, rats were
shaved and cleaned aseptically with 70% v/v ethanol on both the abdomen and the skin overlaying
the trachea, and placed in dorsal recumbency. Throughout surgery and experiment, the body
temperature of rats was maintained at 37 ºC.
Chapter 2: General Methods
63
2.2.2.3 Surgical technique
The autoperfused rat jejunum model involves the perfusion of an isolated jejunal segment and
simultaneous blood collection from the corresponding mesenteric vein branch (see Figure 2.1).
The surgical procedures for the perfusion studies are similar to those described by Singhal et al.
with slight modifications180. Firstly, the right jugular vein was cannulated to enable infusion of
donor rat blood (the surgical procedures for jugular vein cannulation is detailed in Section 2.3.2.5).
The small intestine was then exposed by a longitudinal midline incision to the abdomen, and a
piece of jejunal segment (~ 10 cm) carefully isolated and externalised. Access to the lumen was
made at the proximal and distal ends by electrocautery, and jejunal contents were flushed out with
warm perfusion buffer (150 mM Na+, 18 mM H2PO4-, 12 mM HPO42-, 108 mM Cl-, adjusted to
pH 6.30 ± 0.01 with NaOH solution). The segment was then cannulated at the proximal and distal
ends with sections of Teflon tubing (0.03-inch i.d. proximal/inlet, Upchurch Scientific, Oak
Harbor, WA, Australia; 0.0625-inch i.d. distal/outlet, Shimadzu, Kyoto, Japan) which were
secured with surgical sutures. The mesenteric vein that drains the cannulated jejunal segment was
then identified, and prepared for catheterisation by careful blunt dissection of the surrounding
connective tissue under a dissecting microscope. The rat was then heparinised (90 I.U./kg) via the
jugular vein using an appropriate volume of a 100 I.U./mL heparinised saline solution, and the
mesenteric vein immediately catheterised under the dissecting microscope with a 24G intravenous
catheter (AngiocathTM, BD, Australia), which had the top 1.5 cm tip cut so that the tip remained in
the mesenteric vein after the guide needle was withdrawn. A drop of instant cyanoacrylate glue
was placed over the site of catheterisation, the guide needle removed, and a 30 cm piece of
silicone tubing (0.025-inch i.d., Helix Medical, CA, USA) attached to the catheter tip for the
collection of venous blood. Immediately following catheterisation of the mesenteric vein and for
the remainder of the experiment, the jugular vein cannula was connected to a peristaltic pump
(Adelab Scientific, SA, Australia), and rats were infused with heparinised donor rat blood (5
Chapter 2: General Methods
64
I.U./mL) at a rate of 0.3 mL/min (this rate was selected to match the outflow of the mesenteric
blood). Donor rat blood was collected from donor rats via cardiac puncture under isoflurane
anaesthesia (IsoFlo®, Abbott Laboratories, IL, USA) immediately prior to surgery. The
temperature of the animal and preparation were maintained by a heated surgical board and lamp,
and the exposed jejunal segment was covered by saline-soaked gauze.
Figure 2.1: Autoperfused rat jejunum preparation depicting mesenteric venous blood collection
from the perfused jejunal segment (~ 10 cm). The model allows for simultaneous perfusion of the
jejunal segment and collection of mesenteric venous blood, which enables analysis of compound
absorption in a closed system.
2.2.2.4 Variations in the setup of the autoperfused rat jejunum
Various perfusion techniques may be used to conduct the autoperfused rat jejunum experiments,
including single-pass, recirculating, oscillating and closed loop perfusion181. Among the
techniques mentioned, single-pass and recirculating perfusion are most commonly employed182, 183,
and were established to determine their suitability as model systems to collect the data required for
this thesis. During single-pass perfusion, perfusate from a reservoir is continuously flowed
Chapter 2: General Methods
65
through the jejunal segment and outflowing perfusate continuously collected (Figure 2.2A). Thus,
the jejunal segment is exposed to fresh perfusate containing a constant concentration of compound
at all times (although the concentration of compound decreases during intestinal transit due to
absorption). On the other hand, during recirculating perfusion, outflowing perfusate is recirculated
back to the perfusate reservoir, and re-perfused into the jejunal segment (Figure 2.2B). Thus,
assuming that the compound of interest is absorbed, the intestine is exposed to a gradually
declining concentration of compound. As a result, the mathematical modelling for single-pass and
recirculating techniques are slightly different. Both techniques, however, have been satisfactorily
employed to study drug absorption kinetics in the small intestine182.
2.2.2.5 Experimental protocol for single-pass jejunal perfusion
The animals were equilibrated for 30 min after surgery. During this time, blood that was collected
from the cannulated mesenteric vein (~ 0.3 mL/min) was mixed with donor rat blood and re-
infused via the jugular vein. The inlet jejunal cannula was connected to a second peristaltic pump
and perfusion buffer was pumped through the jejunal segment at a rate of 0.1 mL/min. Outflowing
buffer during the equilibration period was discarded to waste. After the equilibration period and
immediately before the experiment was commenced (i.e. immediately before the solution of
interest was perfused), air was pumped through the jejunal segment for 10 sec at 0.1 mL/min, to
create an air pocket in the outflowing perfusate which differentiated outflowing perfusion buffer
and outflowing solution of interest during the first perfusate sampling interval. Outflowing
perfusate was continuously collected into 1.5 mL polypropylene tubes at 10-min intervals whilst
venous blood draining the perfused jejunal segment was collected into pre-weighed 1.5 mL
polypropylene tubes at 5-min intervals and weighed. The setup of the single-pass autoperfused rat
jejunum preparation is shown in Figure 2.3.
Chapter 2: General Methods
66
At the end of the experiment, the animal was euthanised via an intravenous injection of 100 mg
sodium pentobarbitone into the jugular vein. The jejunal segment was flushed with 10 mL warm
perfusion buffer followed by 10 mL air, excised, and dissected longitudinally for accurate
determination of surface area (calculated by multiplying the diameter by the length of the perfused
intestinal segment). The jejunal segment was then weighed and stored at -20 ⁰C for future analysis
of compound content.
2.2.2.6 Experimental protocol for recirculating jejunal perfusion
The experimental protocol for recirculating jejunal perfusion was similar to single-pass perfusion
(described in Section 2.2.2.5), except that perfusate was passed through the jejunal segment at a
rate of 0.6 mL/min, and outflowing perfusate was recirculated back to a reservoir (typically 10 mL)
and re-perfused into the jejunal segment (see Figure 2.2B). During the 30-min equilibration period,
perfusion buffer was recirculated through the jejunal segment at a rate of 0.6 mL/min.
Immediately before the experiment was commenced (i.e. immediately before the solution of
interest was perfused), air was pumped through the jejunal segment for 5 sec at 0.6 mL/min. The
perfusate reservoir was sampled at 10-min intervals (typically 100 µL aliquots) whilst venous
blood draining the perfused jejunal segment was collected into pre-weighed 1.5 mL polypropylene
tubes at 5-min intervals and weighed.
Chapter 2: General Methods
67
(A)
(B)
Figure 2.2: Schematic of autoperfused rat jejunum with (A) single-pass perfusion, and (B)
recirculating perfusion. In single-pass perfusion, perfusate is flowed through the isolated jejunal
segment once and collected. In recirculating perfusion, outflowing perfusate is redirected back to
the perfusate reservoir and re-perfused into the isolated jejunal segment. Diagrams are adapted
from Cummins et al.184.
Chapter 2: General Methods
68
Figure 2.3: Setup of the single-pass autoperfused rat jejunum preparation.
2.2.3 Data analysis
2.2.3.1 Permeability calculations for single-pass perfusion
In the single-pass perfusion model, permeability coefficients were calculated after attainment of
steady state drug absorption, and using steady state compound concentrations in perfusate and
plasma. Two permeability coefficients were calculated to describe compound absorption from the
jejunal lumen into the mesenteric venous blood as described previously184 and as follows:
′Disappearance′P . ln Equation 2.1
′Appearance′P∆
∆.
Equation 2.2
Chapter 2: General Methods
69
where ‘Disappearance’ Papp is the apparent permeability coefficient calculated from compound
loss from the perfusate (cm/sec); ‘Appearance’ Papp is the apparent permeability coefficient
calculated from compound appearance in the mesenteric blood (cm/sec); Q is the perfusate flow
rate (mL/sec); A is the surface area of the perfused jejunal segment (cm2), which is calculated by
multiplying the diameter and the length of the perfused intestinal segment; C1 is the average
steady state compound concentration exiting the perfused jejunal segment (ng/mL); C0 is the
compound concentration entering the jejunal segment (ng/mL); ∆MB/∆t is the average rate of
compound mass appearance in mesenteric venous blood at steady state (ng/sec); and <C> is the
logarithmic mean compound concentration in the lumen (ng/mL), where <C> = (C1 – C0) / (ln C1
– ln C0).
2.2.3.2 Permeability calculations for recirculating perfusion
Similar to single-pass perfusion, two permeability coefficients were calculated in the recirculating
perfusion model to describe compound absorption from the jejunal lumen into the mesenteric
venous blood. The disappearance permeability coefficient was assessed from the rate of
compound loss from the perfusate reservoir185; while the appearance permeability coefficient was
calculated from cumulative compound appearance data in the mesenteric venous blood:
′Disappearance′P . k Equation 2.3
′Appearance′P .. Equation 2.4
where ‘Disappearance’ Papp is the apparent permeability coefficient calculated from compound
loss from the perfusate (cm/sec); ‘Appearance’ Papp is the apparent permeability coefficient
calculated from compound appearance in the mesenteric venous blood (cm/sec); V is the effective
Chapter 2: General Methods
70
volume of recirculating perfusate (mL); A is the surface area of the perfused jejunal segment
(cm2); ku is the first order disappearance rate constant (sec-1), calculated from the slope of a loge
perfusate concentration vs. time plot; Co is the initial perfusate concentration (ng/mL); and dM/dt
(ng/sec) is the appearance flux of compound in the mesenteric venous blood, calculated from the
slope of a cumulative mass transported vs. time plot.
2.2.4 Validation of the autoperfused rat jejunum
2.2.4.1 Passive permeability markers
The autoperfused rat jejunum was validated via comparison of calculated disappearance and
appearance permeability coefficients for the model compounds antipyrine and mannitol to
published data using the recirculating perfusion technique. Antipyrine is passively absorbed via
the transcellular route (i.e. diffusion across the apical and basolateral membranes of enterocytes)
whereas mannitol is passively absorbed via the paracellular route (i.e. diffusion through the tight
and gap junctions between enterocytes). Antipyrine and mannitol thus act as markers for passive
permeability via the transcellular and paracellular route. Since paracellular transport comprises ~
0.01% of the total surface area available for absorption in the small intestine186, 187, the intestinal
permeability of mannitol is expected to be significantly lower than antipyrine. Comparison of the
disappearance and appearance permeability coefficients of antipyrine and mannitol to literature
values enables assessment of the reproducibility of the autoperfused rat jejunum model and the
integrity of the intestinal segments during perfusion experiments180, 182, 188.
In the validation experiments, the perfusate reservoir contained 10 mL of 1 mM mannitol in
perfusion buffer spiked with 1 µCi 14C-mannitol or 10 mL of 1 mM antipyrine in perfusion buffer
spiked with 1 µCi 14C-antipyrine. The experiments were conducted over 60 min, during which
Chapter 2: General Methods
71
time 100 µL aliquots of perfusate were sampled from the reservoir at t = 0 and at 10 min intervals
whilst blood draining the jejunal segment was collected every 5 min and plasma separated by
centrifugation (10,000 xg, 5 min). At the end of the experiment, the perfused jejunal segment was
removed and longitudinally dissected for accurate determination of surface area and quantification
of radiolabel. For quantification of antipyrine or mannitol in perfusate and plasma, 100 µL of
perfusate samples and 500 µL of plasma samples were placed in liquid scintillation (LSC) vials
containing 2 mL Irga-safe Plus scintillation fluid, and vortexed for 10 sec. For quantification of
antipyrine or mannitol in the perfused jejunal segment, the segment was placed in a 20 mL glass
vial, 2 mL of SOLVABLETM added, and the vial was incubated at 40 ºC for 48 h. The dissolved
tissue solution was divided into two equal portions where 12 mL of Irga-safe Plus was added to
each vial followed by a 1-min vortex. Radioactivity of the perfusate, plasma, and intestine samples
were quantified via scintillation counting on a Packard Tri-Carb 2000CA liquid scintillation
analyser (Packard, Meriden, CT, USA). Blood concentrations were calculated by multiplying the
plasma concentrations of antipyrine and mannitol and their respective blood:plasma concentration
ratios, which are determined in Section 2.2.4.2.
The perfusate disappearance and blood appearance profiles for antipyrine and mannitol are shown
in Figure 2.4, and calculated permeability coefficients are in Table 2.1. The apparent permeability
coefficient of antipyrine based on disappearance from the perfusate (~ 45 x 10-6 cm/sec) and the
apparent permeability coefficient of mannitol based on appearance in the mesenteric venous blood
(~ 1.9 x 10-6 cm/sec) were comparable to reported values in the literature (47-73 x 10-6 cm/sec182,
183 and 1 x 10-6 cm/sec180, respectively). The permeability coefficient of antipyrine based on
appearance in the mesenteric blood is not available in the literature to date, and the permeability
coefficient of mannitol based on disappearance from the perfusate was not calculated due to its
low disappearance from the perfusate (see Figure 2.4A) which precluded accurate assessment of
Chapter 2: General Methods
72
the permeability coefficient. The intestinal permeability of antipyrine was therefore significantly
higher than mannitol, consistent with their respective mode of absorption as discussed earlier. The
total recoveries (i.e. the sum of recovery percentages in the perfusate, blood, and the perfused
jejunal segment) of antipyrine and mannitol in the autoperfused rat jejunum preparation were
93.37% ± 8.25% and 97.02% ± 0.20% (mean ± SEM, n =3), respectively. A summary of the mass
balance for mannitol and antipyrine is shown in Table 2.2.
Thus, the permeability data obtained using mannitol and antipyrine validate the reproducibility of
the autoperfused rat jejunum and the integrity of the intestinal segments during perfusion
experiments. Although the validation experiments were conducted using the recirculating
perfusion model, the data also apply for the single-pass perfusion model, since previous studies
that compared the permeability of antipyrine using both techniques reported statistically equal
apparent permeability coefficients3, and the apparent permeability coefficient of mannitol obtained
in this thesis (using the recirculating model) was comparable to literature value that employed the
single-pass model180.
Chapter 2: General Methods
73
(A)
(B)
Figure 2.4: (A) Disappearance profiles of antipyrine (filled circles) and mannitol (open circles)
from the perfusate and (B) Appearance profiles of antipyrine (filled circles) and mannitol (open
circles) in mesenteric venous blood during recirculating perfusion of an isolated ~ 10 cm segment
of rat jejunum. Data represent mean ± SEM of n = 3 experiments, linear regression lines utilised in
calculation of permeability coefficients are shown as solid (antipyrine) or dotted (mannitol) lines.
Time (min)
0 10 20 30 40 50 60
Per
cent
initi
al
perf
usat
e co
ncen
trat
ion
(%)
50
60
70
80
90
100
AntipyrineMannitol
Time (min)
0 10 20 30 40 50 60
Cu
mu
lativ
e tr
ansp
ort
into
mes
ente
ric b
lood
(%
dos
e/10
cm
2 )
0
5
10
15
20
AntipyrineMannitol
Chapter 2: General Methods
74
Table 2.1: Permeability coefficients of antipyrine and mannitol based on disappearance from the
perfusate and appearance in mesenteric venous blood during recirculating perfusion of an isolated
rat jejunal segment (~ 10 cm). Data represent mean ± SEM of n = 3 experiments.
Disappearance Papp
(x 106 cm/sec)
Appearance Papp
(x 106 cm/sec)
Antipyrine 45.49 ± 7.86 38.27 ± 5.56
Mannitol n/a a 1.94 ± 0.42
a The disappearance Papp for mannitol could not be determined accurately due to low disappearance from
the perfusate
Table 2.2: Mass balance for mannitol and antipyrine in the autoperfused rat jejunum preparation.
Total mass balance (as a %) is the sum of percentages recovered in the perfusate, mesenteric
venous blood and perfused jejunal segment at the end of the perfusion experiments. Data represent
mean ± SEM values for n = 3 experiments.
Antipyrine Mannitol
% Recovery in perfusate 75.24 ± 6.89 94.38 ± 0.43
% Recovery in blood 16.78 ± 2.32 1.13 ± 0.20
% Recovery in jejunal segment 1.35 ± 0.14 1.50 ± 0.16
Total Mass Balance 93.37 ± 8.25 97.02 ± 0.20
Chapter 2: General Methods
75
2.2.4.2 Blood:plasma ratio determination
Calculation of appearance permeability coefficients requires accurate quantitation of total
compound transport into mesenteric venous blood. Since compound concentrations were assayed
in plasma (and not whole blood), the partitioning behaviour of compounds between whole blood
and plasma was determined such that concentrations of compound in whole blood could be back
calculated. Blood:plasma concentration ratios were therefore determined for mannitol and
antipyrine (for validation experiments), and for cinnarizine, danazol and oleic acid (for
experiments described in Chapters 3, 4 and 5), to enable conversion of plasma concentrations into
blood concentrations.
Briefly, in triplicate, 0.5 mL of blank blood was spiked with known amounts of compound to
achieve low, medium, and high concentrations (the concentrations were chosen based on the likely
amount of compound transported into mesenteric venous blood during permeability experiments)
and gently mixed. The final blood concentrations were 1, 10, 20 µM for mannitol and antipyrine;
20, 100, 500 ng/mL for cinnarizine and danazol; and 1, 5, 10 µg/mL for oleic acid. Traces of
radiolabelled mannitol, antipyrine and oleic acid were also spiked into blank blood to facilitate
quantitation. Blood samples were centrifuged at 10,000 xg for 5 min, and plasma concentrations
of compound were assayed by scintillation counting (mannitol, antipyrine and oleic acid), HPLC
(cinnarizine), or LC-MS (danazol). The blood:plasma ratio was calculated from the ratio of known
concentration in spiked blood to the concentration measured in plasma separated from spiked
blood. The blood:plasma ratios for mannitol, antipyrine, cinnarizine, danazol and oleic acid were
0.48 ± 0.02, 1.23 ± 0.03, 0.68 ± 0.03, 0.65 ± 0.01, and 1.24 ± 0.00 (mean ± SEM, n = 3),
respectively, and were not concentration-dependent within the tested range.
Chapter 2: General Methods
76
2.2.4.3 Assessment of net water flux
Changes in perfusate volume (due to water absorption from or water secretion into the lumen of
the perfused jejunal segment) affect the concentration of compound in the perfusate, and therefore
impact accurate calculation of disappearance permeability coefficients. For example, water
secretion into the lumen increases perfusate volume and lowers the concentration of compound in
the perfusate. If correction factors were not applied to the measured concentrations, compound
loss from the perfusate (i.e. mass of compound assumed to be absorbed) would be overestimated,
leading to overestimation of disappearance permeability. Therefore, net water flux across the
perfused jejunal segment is often determined during individual experiments via the inclusion of a
non-absorbable marker, such as PEG 4000189, in the perfusate. The concentration of the non-
absorbable marker is only expected to change when there is net water flux into or out of the
perfused jejunal segment. Thus, changes in the concentration of a non-absorbable marker directly
reflect volume changes in the perfused jejunal segment, and correction factors may be
subsequently calculated to adjust the concentrations of compound of interest in the perfusate at
each sampling point.
In the experiments conducted in this thesis however, the routine inclusion of a radiolabelled
marker of water flux (e.g. 14C- or 3H-PEG 4000) in each experiment was impractical since other
radiolabelled compounds (e.g. 14C-cholesterol and 3H-oleic acid) were also often included in the
perfusate. Net water flux was therefore determined in separate validation experiments, where 14C-
PEG 4000 was perfused through isolated jejunal segments using the recirculating perfusion model
(a total of five experiments were conducted). The recirculating perfusate reservoir consisted of 10
mL perfusion buffer spiked with 0.25 µCi 14C-PEG 4000. The experiments were conducted over
60 min, and 100 µL aliquots of perfusate were sampled from the reservoir at t = 0 and at 10 min
Chapter 2: General Methods
77
intervals. The perfusate samples were analysed for radiolabelled PEG 4000 content as described in
Section 2.2.4.1.
A plot of mean calculated perfusate volume vs. time is shown in Figure 2.5. The plot shows that
significant volume gain mainly occurred in the first 10 min of the experiment, and the perfusate
volume was relatively constant for the remainder of the experiment (relative to the calculated
volume at 10 min, percent volume changes from 10-60 min were < 2%, a value below
experimental error limits). The volume gain in the first 10 min likely reflects the incorporation of
small amounts of perfusion buffer that remained in the jejunal segment after the equilibration
period. The PEG 4000 experiments therefore suggested that no significant water flux occurred
between 10 and 60 min. As such, rather than applying correction factors at each sampling point,
the analysis of perfusate compound disappearance was modified slightly such that calculations of
disappearance permeability coefficients were performed using compound concentrations obtained
between 10 and 60 min (instead of between 0 and 60 min) for recirculating perfusion experiments.
For single-pass perfusion, since the analysis of perfusate compound disappearance was performed
after drug loss from perfusate reached steady state (a process that typically takes > 30 min as
exemplified by perfusate disappearance profiles in the experimental chapters), no modifications to
the method of analysis was required.
Chapter 2: General Methods
78
Figure 2.5: Mean plot of calculated perfusate volume vs. sampling time points for five perfusion
experiments where 0.25 µCi of 14C-PEG 4000, a non-absorbable marker, was included in the
perfusate and recirculated through ~ 10 cm segments of rat jejunum. Broken line represents
theoretical volume of perfusate in the absence of net water flux. A trend of water gain was evident
at all sampling time points. Data represent mean ± SEM values.
2.2.5 Adsorption of cinnarizine onto the recirculating perfusion apparatus
Validation of the autoperfused rat jejunum preparation was performed using the recirculating
perfusion model (see Section 2.2.4) since the model provides better simulations of the absorption
process in vivo, where compound absorption is continuous along the length of the small intestine.
However, in subsequent experiments where drug-containing lipid colloidal phases were perfused,
the model poorly water-soluble drug (PWSD), cinnarizine, was found to adsorb extensively to the
recirculating perfusion apparatus. Specifically, in sham experiments where cinnarizine was
solubilised in intestinal lipid colloidal phases (composition shown in Figure 2.6) and pumped
through the recirculating perfusion apparatus only (i.e. through the perfusion tubing but not
through the intestinal segment of an animal) at a rate of 0.6 mL/min, cinnarizine concentration in
Time (min)
0 10 20 30 40 50 60
Cal
cula
ted
perf
usat
e vo
lum
e (m
L)
9.0
9.5
10.0
10.5
11.0
Chapter 2: General Methods
79
the perfusate declined appreciably, with > 60% loss over 2 h. Since the stability of cinnarizine in
the phases was validated for a period of 24 h (data not shown), the decrease in perfusate
concentration of cinnarizine was attributed to adsorption onto the recirculating apparatus,
presumably to the relatively lengthy silicone tubing that was required for the recirculating model
setup.
To overcome the problem of cinnarizine adsorption onto the recirculating apparatus, the duration
of the sham experiment was increased to 6 h in an attempt to saturate the tubing with cinnarizine,
so that a constant cinnarizine perfusate concentration could be achieved before perfusate was
passed through the rat jejunal segment. This approach, however, did not prove viable as the
concentration of cinnarizine decreased to ~ 10% of initial after 5 h (Figure 2.6A), and was too low
to be used in the experiments. Thus, the recirculating model was deemed unsuitable for the jejunal
perfusion of cinnarizine in intestinal lipid colloidal phases, and the single-pass model was trialled
as an alternate method instead. The single-pass perfusion setup required shorter lengths of
polyethylene tubing, and perfusate is only passed through the tubing once. Therefore, cinnarizine
adsorption was expected to occur to a lesser extent than with the recirculating perfusion setup.
Indeed, a repeat sham experiment using the single-pass perfusion setup where cinnarizine was
solubilised in the same lipid colloidal phase and pumped through the perfusion apparatus at a rate
of 0.1 mL/min revealed negligible cinnarizine adsorption, and the concentration of cinnarizine in
the perfusate was within ± 10% of initial throughout the experiment (Figure 2.6B). Similar
outcomes were later obtained for another model PWSD, danazol, where no adsorption was
observed during the sham perfusion of danazol containing-intestinal lipid colloidal phase
(composition shown in Figure 2.7) through the perfusion apparatus at a rate of 0.1 mL/min (Figure
2.7).
Chapter 2: General Methods
80
Therefore, while validation of the animal model was carried out using the recirculating perfusion
setup, the single-pass perfusion setup was the method employed to investigate the absorption
kinetics of cinnarizine and danazol from intestinal lipid colloidal phases.
(A)
Time (mins)
0 100 200 300
Per
cent
initi
al
cinn
ariz
ine
conc
entr
atio
n (%
)
0
20
40
60
80
100
(B)
Time (min)
0 20 40 60 80 100 120
Per
cent
initi
alci
nnar
izin
e co
ncen
trat
ion
(%)
50
60
70
80
90
100
110
Figure 2.6: Plot of percent initial cinnarizine concentration in perfusate vs. time during an
adsorption test experiment where an intestinal lipid colloidal phase* containing 10 µg/mL
cinnarizine was flowed through (A) the recirculating perfusion apparatus for 6 h, and (B) the
single-pass perfusion apparatus for 2 h. In the recirculating perfusion setup, samples were taken at
t = 0, at hourly intervals for the first 5 h, and at 10 min intervals thereafter to reflect the approach
of attempting to saturate the adsorption of cinnarizine onto the perfusion apparatus for 5 h prior to
commencement of a 1 h jejunal perfusion experiment. Even though steady state (i.e. constant)
concentrations appeared to have been reached after 5 h, the concentration (~ 10% of initial) was
deemed too low for the perfusion experiments. In the single-pass perfusion setup, samples were
taken at t = 0 and at 10 min intervals for 2 h, and negligible cinnarizine adsorption was observed
during the sham experiment.
* Intestinal lipid colloidal phase consisted of 0.025 %w/v oleic acid and 0.016 %w/v monoolein solubilised
in 4 mM sodium taurocholate, 1 mM lysophosphatidylcholine and 0.25 mM cholesterol at pH 6.50.
Chapter 2: General Methods
81
Time (min)
0 10 20 30 40 50 60 70
Per
cent
initi
alda
nazo
l con
cent
ratio
n (%
)50
60
70
80
90
100
110
Figure 2.7: Plot of percent initial danazol concentration in perfusate vs. time during an adsorption
test experiment where an intestinal lipid colloidal phase** containing 14.4 µg/mL danazol was
flowed through the single-pass perfusion apparatus for 70 min. Samples were taken at t = 0 and at
10 min intervals, and negligible danazol adsorption was observed during the sham experiment.
** Intestinal lipid colloidal phase consisted of 0.10 %w/v oleic acid and 0.064 %w/v monoolein solubilised
in 4 mM sodium taurocholate, 1 mM lysophosphatidylcholine and 0.25 mM cholesterol at pH 6.30.
2.3 IN VIVO BIOAVAILABILITY STUDIES
To determine whether absorption trends observed using the in situ autoperfused rat jejunum
preparation could be extended to the in vivo setting, the bioavailability of cinnarizine and danazol
was assessed in rats following intraduodenal administration in intestinal lipid colloidal phases. In
the case of danazol, intraduodenal administration did not lead to detectable systemic plasma
concentrations (limit of quantification for danazol plasma assay was 5 ng/mL) despite significant
absorption into the mesenteric venous blood during perfusion experiments (suggesting significant
hepatic first-pass metabolism). Thus, bioavailability data for danazol is not included in this thesis.
The experimental protocol for the bioavailability studies is described in detail in Chapters 3 and 4.
This section contains details of the surgical procedures required for the setup of the animal model.
Chapter 2: General Methods
82
2.3.1 Materials
Heparin, saline, ketamine xylazine, acepromazine were sourced from suppliers listed in Section
2.2.1.
2.3.2 Methods
The right carotid artery, right jugular vein, common bile duct and duodenum were cannulated for
the purpose of: blood sample withdrawal, intravenous administration of formulation or saline,
pancreatobiliary fluid diversion and intraduodenal administration of formulation, respectively.
2.3.2.1 Conscious state
Studies that assess the bioavailability of drugs in oral formulations are most often conducted using
conscious animals since the rate of gastric emptying, intestinal mixing and digestion are likely to
be reduced in anaesthetised animals. The formulations used in the experiments throughout this
thesis, however, were highly dispersed intestinal lipid colloidal phases assembled from fully
digested lipids, and as such, drug absorption from the formulations is less likely to be affected by
the conscious state of the animals. In addition, the protocol for the bioavailability studies
necessitated abdominal incisions to be made to the animals (for the diversion of pancreatobiliary
fluids as well as the intraduodenal administration of formulations - to avoid the effects of gastric
mixing since the formulations were designed to simulate colloidal phases found in the small
intestine). These surgical interventions were expected to significantly enhance the degree of post-
operative pain and stress in the animals. Therefore, bioavailability studies were conducted using
anaesthetised rats to reduce the logistical and ethical burden of the experiments, and to match the
conditions used in the in situ jejunal perfusion experiments.
Chapter 2: General Methods
83
2.3.2.2 Surgical equipment and accessories
The surgical instruments required for the cannulation of the carotid artery, jugular vein, common
bile duct and duodenum include a scalpel, a pair of sharp straight scissors, two curved anatomical
forceps, one fine tip curved forceps, one fine tip straight forceps, a pair of iridectomy scissors, one
fine tip haemostat and one small ‘alligator’ clamp. A portable, multi-head, flexible cold fibre-optic
illuminating system (Microlight 150®, Fibre optic light guides, Australia) was used to obtain
adequate lighting for surgery. A heated surgical board (Ratek Instruments, Australia) was used to
maintain body temperature of the animals during surgery.
2.3.2.3 Animal care and anaesthesia
The care and anesthesia of animals have been described in Section 2.2.2.2.
2.3.2.4 Cannulation of the right carotid artery
A longitudinal incision (1.5 cm) was made to the right of the central neck muscle, starting from
above the right breast bone towards the head. The carotid artery (visualised as a red, pulsating
vessel running parallel to the white vagus nerve ~ 1 cm below the skin surface) was isolated by
blunt dissection of surrounding connective tissue and muscle layers. Particular care was taken in
separating the carotid artery from the vagus nerve. Blood flow to the artery from the heart was
temporarily halted by the placement of a pair of fine tip straight forceps (bridged over a 10 mL
syringe to provide leverage) underneath the vessel. A small incision was made on the top surface
of the vessel using iridectomy scissors. A bevelled 30 cm piece of polyethylene tubing (0.50 mm
i.d., 0.80 mm o.d., Microtube Extrusions, Australia; attached to a 25G syringe containing 2
I.U/mL heparinised saline) was then inserted into the incision, advanced 2.5 cm into the vessel,
and secured in place with surgical sutures (with the aid of a haemostat if needed). To ensure
Chapter 2: General Methods
84
cannula patency, the cannula was flushed with 0.2 mL heparinised saline and sealed using a naked
flame. The neck incision was closed with surgical suture.
2.3.2.5 Cannulation of the right jugular vein
A longitudinal incision (1.5 cm) was made to the right of the central neck muscle, starting from
above the right breast bone towards the head. The jugular vein (visualised as a bluish-grey vessel
close to the skin surface) was isolated by blunt dissection of surrounding connective tissue and
muscle layers. A ligature was placed around the vessel (using surgical suture), anterior to the site
of cannulation, to halt blood return from the brain via the right jugular vein. A small incision was
made on the top surface of the vessel using iridectomy scissors. A bevelled 30 cm piece of
polyethylene tubing (0.50 mm i.d., 0.80 mm o.d., Microtube Extrusions, Australia; attached to a
25G syringe containing 2 I.U/mL heparinised saline) was then inserted into the incision, advanced
2 cm into the vessel, and secured in place with surgical suture. To ensure cannula patency, the
cannula was flushed with 0.2 mL heparinised saline and sealed using a naked flame. The neck
incision was closed with surgical suture.
2.3.2.6 Cannulation of the common bile duct
The abdominal muscle wall was opened by a latitudinal incision (~ 3 cm) 1-2 cm below the
ribcage, approximately 0.5 cm to the left side of the animal from the midline. The duodenal loop
was isolated and exteriorised to expose the common bile duct, which may be visualised as a small,
yellow vessel originating from the liver and draining into the duodenum. The duct was cleared of
surrounding connective tissue using forceps, and a small puncture hole made on the top surface,
close to the duodenum, with a 25G needle. A bevelled 15 cm piece of polyethylene tubing (0.28
mm i.d., 0.68 mm o.d., Microtube Extrusions, Australia) was then inserted into the duct and
secured in place with surgical sutures. The position of the cannula was selected to be below the
Chapter 2: General Methods
85
entry points of all exocrine branches from the liver and pancreas. The cannula was externalised
through the abdominal incision which was subsequently closed with surgical suture.
2.3.2.7 Cannulation of the duodenum
The abdomen muscle wall was opened by a latitudinal incision (~ 2 cm) 1-2 cm below the ribcage,
approximately 0.5 cm to the left side of the animal from the midline. The duodenum (identified as
the brighter pink section of the small intestine, which upon gentle downward pulling reveals the
stomach) was isolated and a small puncture hole made in the duodenum, 1 cm below the pylorus,
with a 23G needle. A bevelled, J-shaped (previously heat-moulded with a cigarette lighter) piece
of polyethylene tubing (0.58 mm i.d., 0.96 mm o.d., Microtube Extrusions, Australia) was then
inserted into the hole and glued in place using instant cyanoacrylate adhesive. The cannula was
externalised through the abdominal incision which was subsequently closed with surgical sutures.
2.4 VALIDATION OF ANALYTICAL METHODS
2.4.1 HPLC assays for quantification of cinnarizine, halofantrine,
fenofibrate, danazol and meclofenamic acid in intestinal lipid colloidal
phases
The concentration of cinnarizine, halofantrine, fenofibrate, danazol and meclofenamic acid in
intestinal lipid colloidal phases was determined by High Performance Liquid Chromatography
(HPLC). The HPLC system used for the quantification of cinnarizine consisted of a Waters 610
fluid unit, Waters 717 autosampler, Model 600 fluid controller (Waters, Milford, MA, USA) and a
RF-10A XL fluorescence detector supplied by Shimadzu (Shimadzu Corp., Kyoto, Japan). Data
was recorded and integrated using Empower 2 personal chromatography data software (Waters,
Chapter 2: General Methods
86
Milford, MA, USA). The HPLC system (Shimadzu Corp., Kyoto, Japan) used for the
quantification of halofantrine, fenofibrate, danazol and meclofenamic acid consisted of a LC‐
20AD binary pump, a SIL‐20A HT autosampler, and a temperature‐controlled column
compartment (CTO‐20A) coupled with a RF-10A XL fluorescence detector and a SPD-20A
UV/Vis detector. The column compartment was maintained at 40 °C. The chromatographic data
were recorded and integrated using LabSolutions software package (Shimadzu). The injection
volume for all samples was 50 µL. The HPLC assay conditions for the five compounds are
described in Table 2.3.
The assays were validated on three separate days by analysing drug standards (prepared at low,
medium and high concentrations) in quadruplicate and comparing against a linear standard curve.
The assays for all five compounds were found to be accurate (to within ± 10% of target
concentration except for the lower limit of quantitation which was ± 15%) and precise (% CV <
10 for all concentrations except the lower limit of quantification which was < 15) for the
concentration ranges reported in Table 2.3. The validation results are summarised in Table 2.4.
Chapter 2: General Methods
87
Table 2.3: HPLC assay conditions for cinnarizine (CIN), halofantrine (HF), fenofibrate (FF),
danazol (DAN) and meclofenamic acid (MFA) in intestinal lipid colloidal phases.
Drug Stationary phase
Mobile phase Flow rate (mL/min)
Detection Retention time (min)
Validated concentration
range
CIN Waters Symmetry®
C18, 5 µm, 3.9 x 150 mm
50% v/v Acetonitrile : 50%
v/v 20 mM NH4H2PO4
1.0
Fluorescence
λ = 249/311 nm
5.7 20–1000
ng/mL
HF Phenomenex Luna® C8(2), 5 µm, 4.6 x 250
mm
75% v/v Acetonitrile : 25%
v/v H2O (with 0.2% w/v SDSa & 0.2% v/v
acetic acid)
1.5 UV
λ = 254 nm
4.2 0.5–25
µg/mL
FF Waters XbridgeTM C18, 5 µm, 4.6 x 150
mm
80% v/v Acetonitrile : 20%
v/v H2O (total 0.01% v/v formic acid)
1.0 UV
λ = 286 nm
3.9 0.5–25
µg/mL
DAN Waters XbridgeTM C18, 5 µm, 4.6 x 150
mm
75% v/v Methanol : 25% v/v H2O
1.0 UV
λ = 286 nm
4.4
0.25–50
µg/mL
MFA Phenomenex Luna® C8(2), 5 µm, 4.6 x 250
mm
80% v/v Acetonitrile : 20% v/v 10 mM H3PO4
1.0 UV
λ = 285 nm
4.8 0.5–25
µg/mL
a SDS is sodium dodecyl sulphate
Chapter 2: General Methods
88
Table 2.4: Precision (% coefficient of variation) and accuracy (% of the target value) of the HPLC
assays for cinnarizine (CIN), halofantrine (HF), fenofibrate (FF), danazol (DAN) and
meclofenamic acid (MFA) in intestinal lipid colloidal phases.
Drug Target concentration
Day 1 Day 2 Day 3
Precision Accuracy Precision Accuracy Precision Accuracy
CIN 50 ng/mL 1.9 107.7 1.5 109.3 0.8 104.1
200 ng/mL 1.0 95.1 0.5 93.4 0.8 92.6
1000 ng/mL 0.2 101.8 0.2 104.5 1.2 106.1
HF 0.5 µg/mL 0.5 102.4 0.7 103.6 0.4 103.3
5 µg/mL 0.1 100.4 0.2 100.1 0.4 100.5
25 µg/mL 0.2 100.1 0.2 100.4 0.5 101.2
FF 0.5 µg/mL 0.2 94.4 0.4 86.1 0.5 86.2
5 µg/mL 0.2 100.0 0.0 97.5 0.0 97.8
25 µg/mL 0.2 100.4 0.0 99.5 0.1 99.7
DAN 0.25 µg/mL 0.3 110.0 0.2 108.6 0.3 92.3
5 µg/mL 0.2 99.4 0.2 98.9 0.1 97.8
50 µg/mL 0.1 100.0 0.1 100.1 0.8 100.4
MFA 0.5 µg/mL 0.5 99.7 0.3 96.8 0.3 113.1
5 µg/mL 0.6 98.1 1.6 95.8 1.0 97.8
25 µg/mL 0.6 101.0 0.1 99.7 0.1 100.2
Chapter 2: General Methods
89
2.4.2 HPLC assay for quantification of cinnarizine in rat plasma
Cinnarizine plasma samples were prepared for HPLC analysis using a validated extraction
procedure. Standards for the assay were prepared by spiking 100 μL aliquots of blank plasma (in 4
mL polypropylene tubes) with 20 μL of 50, 100, 200, 400, 800, 1600 ng/mL cinnarizine in mobile
phase solution, which provided spiked plasma concentrations in the range of 10‐320 ng/mL
cinnarizine. 20 µL of an internal standard solution (500 ng/mL flunarizine in mobile phase) was
also added, and tubes briefly vortexed. 20 µL of trichloroacetic acid was added to precipitate
plasma protein, and the tubes vortexed for 1 min. 2 mL of tert-butyl methyl ether (TBME) was
added to extract cinnarizine and flunarizine into the organic phase. The tubes were vortexed for 1
min, allowed to equilibrate for 10 min, and vortexed for a further minute before being centrifuged
at 10,000 xg for 15 min. 1.6 mL aliquots of the supernatant were then transferred into new
polypropylene tubes, and TBME evaporated using a nitrogen evaporator at 40 ºC. Cinnarizine and
flunarizine in the tubes were reconstituted with 150 µL mobile phase and assayed via HPLC.
The HPLC assay conditions for the quantification of cinnarizine in rat plasma were similar to that
for the quantification of cinnarizine in intestinal lipid colloidal phases described in Section 2.4.1,
with slight modifications. The mobile phase used in the plasma assay consisted 45% v/v
acetonitrile : 55% v/v 20 mM NH4H2PO4, i.e. the organic solvent content was lowered to enable
better separation of cinnarizine and flunarizine peaks. The injection volume was 100 µL, and the
retention time for cinnarizine and flunarizine were 8.9 min and 11.3 min, respectively. Unknown
concentrations were determined by comparing the unknown cinnarizine:flunarizine area under the
curve (AUC) ratio against a standard curve of cinnarizine:flunarizine AUC ratio vs. cinnarizine
concentration. The assay was validated on three separate days by analysing drug standards
(prepared at low, medium and high concentrations) in quadruplicates and comparing against a
linear standard curve. The assay was found to be accurate (to within ± 10 % of target
Chapter 2: General Methods
90
concentration except for the lower limit of quantitation which was ± 15%) and precise (% CV <
10 for all concentrations except the lower limit of quantification which was < 15) for cinnarizine
plasma concentrations between 10-320 ng/mL. The validation results are shown in Table 2.5.
Table 2.5: Precision (% coefficient of variation) and accuracy (% of the target value) of the
plasma cinnarizine HPLC assay.
[Cinnarizine]
(ng/mL)
Day 1 Day 2 Day 3
Precision Accuracy Precision Accuracy Precision Accuracy
10 11.5 91.0 6.6 93.2 1.3 92.2
80 2.2 91.4 6.3 110.0 2.8 98.8
320 9.0 100.9 2.5 106.2 2.0 99.3
2.4.3 LC-MS assay for quantification of danazol in rat plasma
Danazol plasma samples were prepared for LC-MS analysis using a validated precipitation
procedure. Standards for the assay were prepared by spiking 125 μL aliquots of blank plasma with
5 μL of 125, 250, 1250, 2500 and 6250 ng/mL danazol in acetonitrile, which provided spiked
plasma concentrations in the range of 5‐250 ng/mL danazol. 5 µL of an internal standard solution
(2000 ng/mL progesterone in acetonitrile) was also added to each sample, and tubes vortexed for
30 sec. Following that, 62.5 μL of saturated ammonium sulphate solution was added, tubes
vortexed for 30 sec; 125 µL ACN was added, and the tubes vortexed again for 30 sec. The tubes
were left to stand for 20 min at room temperature prior to centrifugation at 35,000 xg for 5 min.
100 μL aliquots of the organic phase (top layer of supernatant) were then transferred into
autosampler vials and 10 μL injected onto the LC‐MS. Unknown concentrations were determined
Chapter 2: General Methods
91
by comparing the unknown danazol:progesterone peak height ratio against a standard curve of
danazol:progesterone peak height ratio vs. danazol concentration.
The LC-MS system (LCMS 2020, Shimadzu, Japan) consisted a LC‐20AD binary pump, a SiL‐
20AC Ht refrigerated autosampler, a mobile phase vacuum degassing unit (DGU‐20A3), and a
temperature‐controlled column compartment (CTO‐20A) coupled with a single‐quadrupole mass
spectrometric (MS) detector (Shimadzu LCMS 2020) equipped with an electrospray ionisation
source. The autosampler was maintained at 4 °C and the column at 40 °C. The stationary phase
was a Phenomenex Gemini® C6 phenyl column (50 × 2.0 mm, 3 µm). Samples were eluted using
a gradient at a total flow rate of 0.3 mL/min. The mobile phases consisted a mixture of solvent A
(95% v/v water : 5% v/v methanol ) and solvent B (5% v/v water : 95% v/v methanol) both
containing 1 mM ammonium formate and 0.1% v/v formic acid. The initial percentage of solvent
B was 60% and was linearly increased to 100% over 6.5 min and was held at 100% for 7 min.
After 14.25 min, the gradient was returned to 60% solvent B which was held until the end of the
run (17 min). The MS conditions were as follows: drying gas flow 20 L/min; nebulising gas flow
1.5 L/min; drying gas temperature 200 °C; interface voltage 3.5 kV; detector voltage 1.0 kV.
Selected‐ion monitoring was accomplished at m/z +338.2 for danazol [M+H]+ and m/z +314.9 for
progesterone [M+H]+. The chromatographic data were acquired and analysed using LabSolutions
software package (Shimadzu).
The assay was validated on three separate days by analysing drug standards (prepared at low,
medium and high concentrations) in quadruplicates and comparing against a linear standard curve.
The assay was found to be accurate (to within ± 10 % of target concentration except for the lower
limit of quantitation which was ± 15%) and precise (% CV < 10 for all concentrations except the
Chapter 2: General Methods
92
lower limit of quantification which was < 15) for cinnarizine plasma concentrations between 5-
250 ng/mL. The validation results are shown in Table 2.6.
Table 2.6: Precision (% coefficient of variation) and accuracy (% of the target value) of the
plasma danazol LC-MS assay.
[Danazol]
(ng/mL)
Day 1 Day 2 Day 3
Precision Accuracy Precision Accuracy Precision Accuracy
5 8.2 96.0 7.8 103.5 5.3 87.6
50 4.5 103.2 1.1 108.5 2.0 104.4
250 6.4 97.6 4.4 106.7 2.0 93.0
2.4.4 Enzymatic colorimetric assay for quantification of total bile salt in
whole rat bile
Total bile salt concentration in whole rat bile was analysed using an enzymatic colorimetric assay
(Total Bile Acids kit # 431-15001; Wako Pure Chemical Industries, Osaka, Japan). Standards for
the assay were freshly prepared 50, 100, 200, 400, 500 µM sodium glycocholate in water. Rat bile
samples were diluted 1:100 with water. Two 20 µL aliquots of sample were added to two wells of
a 96-well plate. 50 µL of ‘enzyme reagent’ and 50 µL of ‘enzyme reagent for blank test’ were
added to the first and second sample, respectively. The plate was then incubated at 37 ⁰C for 10
min. At the end of the incubation period, 50 µL of ‘stopper solution’ was added to both samples.
Sample absorbance was then measured at a wavelength of 540 nm on plate reader (Fluostar
Optima plate reader, BMG Labtechnologies, Germany). The difference between the specific
absorption of the sample treated with ‘enzyme reagent’ and the sample treated with ‘enzyme
reagent for blank test’ was used in the determination of total bile salt concentration. The assay was
Chapter 2: General Methods
93
validated on three separate days by analysing three standards (prepared at low, medium and high
concentrations) in quadruplicates and comparing against the linear standard curve. The assay was
found to be accurate (to within ± 10 % of target concentration except for the lower limit of
quantitation which was ± 15%) and precise (% CV < 10 for all concentrations except the lower
limit of quantification which was < 15) for total bile salt concentrations between 50-500 µM. The
validation results are shown in Table 2.7.
Table 2.7: Precision (% coefficient of variation) and accuracy (% of the target value) of the total
bile salt enzymatic colorimetric assay.
[Glycocholate] (µM)
Day 1 Day 2 Day 3
Precision Accuracy Precision Accuracy Precision Accuracy
50 13.2 85.9 14.6 93.1 11.6 87.3
200 4.5 100.6 1.2 102.8 4.8 107.3
500 4.0 101.9 2.8 101.8 0.7 103.6
94
Monash University
Declaration for Thesis Chapter 3 Declaration by candidate In the case of Chapter 3, the nature and extent of my contribution to the work was the following:
Nature of contribution Extent of contribution (%)
Concept and design of studies, planning and execution of experimental work, data analysis and interpretation, formulation of conclusions and hypotheses resulting from the relevant studies, drafting and revision of manuscript
60%
The following co-authors contributed to the work. Co-authors who are students at Monash University must also indicate the extent of their contribution in percentage terms:
Name Nature of contribution Extent of contribution (%)
C.J.H. Porter Project supervisor, data and manuscript review NA
N.L. Trevaskis Project co-supervisor, data and manuscript review NA
T. Quach Synthesis of sulfo-N-succinimidyl oleate NA
W.N. Charman Manuscript review NA
P. Tso Manuscript review NA
Candidate’s Signature
Declaration by co-authors The undersigned hereby certify that:
(1) the above declaration correctly reflects the nature and extent of the candidate’s contribution to this work, and the nature of the contribution of each of the co-authors.
(2) they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise;
(3) they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication;
(4) there are no other authors of the publication according to these criteria; (5) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or
publisher of journals or other publications, and (c) the head of the responsible academic unit; and
(6) the original data are stored at the following location(s) and will be held for at least five years from the date indicated below:
Location(s) Monash Institute of Pharmaceutical Sciences
Department of Pathology and Laboratory Medicine, University of Cincinnati
95
Signature 1
Signature 2
Signature 3
Signature 4
Signature 5
96
CHAPTER 3 : INTESTINAL BILE
SECRETION PROMOTES DRUG
ABSORPTION FROM LIPID COLLOIDAL
PHASES VIA INDUCTION OF
SUPERSATURATION
Yan Yan Yeap1, Natalie L. Trevaskis1, Tim Quach2, Patrick Tso3, William N. Charman1,
Christopher J. H. Porter1
1 Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia
2 Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia
3 Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45221, USA
Molecular Pharmaceutics (2013); In press.
Chapter 3: Bile-induced supersaturation
97
3.1 ABSTRACT
The oral bioavailability of poorly water-soluble drugs (PWSD) is often significantly enhanced by
co-administration with lipids in food or lipid-based oral formulations. Co-administration with
lipids promotes drug solubilisation in intestinal mixed micelles and vesicles, however, the
mechanism(s) by which PWSD are absorbed from these dispersed phases remain poorly
understood. Classically, drug absorption is believed to be a product of the drug concentration in
free solution and the apparent permeability across the absorptive membrane. Solubilisation in
colloidal phases such as mixed micelles increases dissolution rate and total solubilised drug
concentrations, but does not directly enhance (and may reduce) the free drug concentration. In the
absence of changes to cellular permeability (which is often high for lipophilic, PWSD), significant
changes to membrane flux are therefore unexpected. Realising that increases in effective
dissolution rate may be a significant driver of increases in drug absorption for PWSD, we explore
here two alternate mechanisms by which membrane flux might also be enhanced: (1) collisional
drug absorption where drug is directly transferred from lipid colloidal phases to the absorptive
membrane, and (2) supersaturation-enhanced drug absorption where bile mediated dilution of lipid
colloidal phases leads to a transient increase in supersaturation, thermodynamic activity and
absorption. In the current study, collisional uptake mechanisms did not play a significant role in
the absorption of a model PWSD, cinnarizine, from lipid colloidal phases. In contrast, bile-
mediated dilution of model intestinal mixed micelles and vesicles led to drug supersaturation. For
colloids that were principally micellar, supersaturation was maintained for a period sufficient to
promote absorption. In contrast, for primarily vesicular systems, supersaturation resulted in rapid
drug precipitation and no increase in drug absorption. This work suggests that on-going dilution
by bile in the gastrointestinal tract may invoke supersaturation in intestinal colloids and promote
absorption, and thus presents a new mechanism by which lipids may enhance the oral absorption
of PWSD.
Chapter 3: Bile-induced supersaturation
98
3.2 INTRODUCTION
The potential for lipid-based formulations (LBF) to enhance the oral bioavailability of poorly
water-soluble drugs (PWSD) has been recognised for over 40 years2. Lipid co-administration is
thought to enhance the oral absorption of PWSD by providing mechanisms to overcome both slow
dissolution and low solubility of PWSD in the aqueous gastrointestinal (GI) milieu. Firstly, LBF
present drug to the GI tract in a molecularly dispersed form (i.e. in solution in the formulation),
thereby circumventing the need for dissolution from the solid to the liquid state. Subsequently, the
intercalation of formulation lipids into endogenous lipid digestion pathways results in the
generation of intestinal lipid colloidal phases (such as vesicular and micellar species) that enhance
the solubilisation capacity of the small intestine, promote drug solubilisation and reduce the risk of
drug precipitation.
Solubilisation within lipid colloidal phases therefore increases the apparent solubility of PWSD in
the intestinal fluids and promotes dissolution. However, in the absence of solid drug, solubilisation
also results in a reduction in thermodynamic activity143. In simple micellar systems this reduction
in thermodynamic activity is manifest in a decrease in the free concentration of drug. Where
solubilised drug exists in equilibrium between the free concentration (Cfree) and the concentration
solubilised in intestinal colloids (Ccolloid), the total solubilised drug concentration (Ctotal) is the sum
of Cfree and Ccolloid,
Ctotal = Cfree + Ccolloid Equation 3.1
Under these circumstances, the solubility of drug in the inter-micellar phase (effectively the
aqueous solubility of the drug) provides the upper limit for Cfree and the presence of solubilising
colloidal species typically increase the total drug concentration but do not increase, and often
Chapter 3: Bile-induced supersaturation
99
reduce, Cfree. Classical models of passive drug absorption suggest that drug flux across an
absorptive membrane is the product of the free drug concentration and the drug permeability
across the membrane. Therefore, where solubilisation reduces Cfree (but does not alter
permeability), absorption is expected to be reduced. Indeed, in solubilised systems, even at
saturation, Cfree does not exceed the equilibrium solubility of drug in (non-micellar) aqueous
solution. Solubilisation in intestinal colloidal phases therefore provides no practical advantage in
free drug concentration, and in the absence of changes to permeability, is unlikely to lead to
appreciable increases in membrane flux when compared to an aqueous solution containing drug at
close to saturated solubility. In support of this suggestion, many authors have shown that
increasing the total concentration of a PWSD by solubilisation does not necessarily result in
proportional increases in absorptive flux100, 143-146. Dahan et al. and Miller et al. recently proposed
a model to quantify this phenomena and referred to the existence of a ‘solubility-permeability
interplay’ where potential increases in membrane flux due to increases in solubilised drug
concentration were off-set by a reduction in the apparent permeability, the latter being, in large
part, a function of decreases in free fraction100, 146.
The dispersion and digestion of LBF therefore creates a solubilised reservoir that is in rapid
equilibrium with drug in free solution and provides significant advantage in the effective rate of
dissolution of a PWSD when compared to traditional dissolution from a solid dose form. In
contrast, the inherent solubility limitations to flux (rather than dissolution-rate limitations) are
seemingly unchanged when viewed from the perspective of the free concentration or may be made
worse. This appears at odds with the wealth of experimental and practical observations that
suggest the ability of lipids (either formulation- or dietary-derived) to enhance the oral absorption
of a range of PWSD2. A possible explanation for this anomaly is that the traditional view of drug
absorption from colloidal dispersions may not adequately describe the dynamic manner in which
Chapter 3: Bile-induced supersaturation
100
LBF exert absorption-enhancing effects in the GI tract. Two alternative potential mechanisms of
drug absorption are therefore examined here (Figure 3.1).
Figure 3.1: Alternative mechanisms of drug absorption from intestinal lipid colloidal phases. In
collisional drug absorption (left panel), in addition to the diffusion of free drug molecules (a), lipid
colloidal phases collide with the absorptive membrane and facilitate direct transfer of solubilised
drug into absorptive cells (b). Collisional transfer may or may not be receptor-mediated. In
supersaturation-enhanced drug absorption (right panel), the interaction between secreted bile and
lipid colloidal phases leads to drug supersaturation (possibly via stimulation of phase changes to
less lipid-rich colloids with lowered drug solubilisation capacity). The increase in thermodynamic
activity manifests in increases in free drug concentration, and enhanced diffusional flux (a) across
the absorptive membrane.
The first mechanism evaluated was the potential for drug absorption to occur via direct collisional
transfer from lipid colloidal phases to the absorptive membrane, and thus to be mediated not only
Vesicles Mixed micelles Receptors
D Drug Dss Supersaturated drug
Bile components
DssD (a)
(a)Bile
secretion
Dss
DssD Dss
DDss
Diffusion Collision(a) (b)
Collisional drug absorption Supersaturation-enhanced drug absorption
D
D
(b)
(a)
(a)
(b)
D
DD
D
DD
D
DD
D
DD
DD
DD
(b)
(b)D
DD
D
DDDD
DssD
Chapter 3: Bile-induced supersaturation
101
by Cfree but also by the solubilised fraction, Ccolloid. Previously, studies by Storch and colleagues
have shown that the transfer of poorly water-soluble fatty acids between model cell membranes
and proteins may occur via collisional transfer150, 151. More recently, the possibility of drug
absorption via collisional uptake has been suggested by Yano et al. and Gao et al.190, 191.
Collisional uptake may or may not be receptor-mediated192, however, lipid uptake receptors such
as CD3661, SR-BI67 and NPC1L168 have been suggested to facilitate the absorption of cholesterol
and fatty acids; poorly water-soluble molecules that are also solubilised in intestinal colloidal
phases. In the case of SR-BI and CD36, direct interaction of the receptor with colloidal structures
such as HDL (high-density lipoprotein), bile salt micelles and phospholipid vesicles66, 67, has also
been suggested, raising the possibility that lipid uptake receptors may interact directly with
intestinal lipid colloidal phases to facilitate collisional absorption of solubilised contents,
including PWSD.
The second mechanism evaluated was the potential for endogenous lipid processing pathways to
lead to drug supersaturation in lipid colloidal phases. Supersaturation increases the
thermodynamic activity of solubilised drug, and in the solubilisation model described by Equation
3.1, will increase Cfree above the equilibrium aqueous drug solubility. This in turn is expected to
enhance drug flux. The potential for supersaturation to enhance the oral bioavailability of PWSD
has received increasing recent interest173, 191. For LBF, supersaturation may be generated by the
loss of drug solubilisation capacity resulting from the digestion of triglycerides91, 176, 193 and/or
surfactants177, and the dilution of co-solvents5 during GI processing176. In contrast, the possibility
that supersaturation may result directly from interactions between lipid colloidal phases and
biliary fluids has been almost entirely ignored. Although traditional micellar solubilisation models
suggest that increases in bile salt concentrations increase drug solubilisation, previous studies have
also shown that dilution of lipid colloidal phases with model intestinal fluids (containing bile salts,
Chapter 3: Bile-induced supersaturation
102
phospholipid and cholesterol) may lead to the generation of less lipid-rich colloidal phases with
lowered solubilisation capacities194. This provides a plausible mechanism for supersaturation
generation in the small intestine and has been examined in detail here.
The data suggest that under the conditions explored, collisional drug absorption has little impact
on drug absorption from intestinal colloidal species. In contrast, supersaturation-enhanced drug
absorption, mediated by the interaction between secreted bile and lipid colloidal phases, may
provide an endogenous mechanism to promote supersaturation and to facilitate drug absorption
from lipid colloidal phases.
3.3 METHODS
3.3.1 Materials
Cinnarizine, flunarizine dihydrochloride, monensin sodium, sodium taurocholate, sodium
taurodeoxycholate, sodium glycocholate, sodium glycochenodeoxycholate, cholesterol, L-α-
lysophosphatidylcholine (LPC, from egg yolk), L-α-phosphatidylcholine (PC, from dried egg
yolk), oleic acid, caprylic acid, monocaprylin, N-hydroxysulfosuccinimide sodium,
dicyclohexylcarbodiimide solution (60%w/v in xylenes), N,N-dimethylformamide, TWEEN® 80,
potassium dihydrogen phosphate (KH2PO4) and sodium chloride (NaCl) were obtained from
Sigma-Aldrich, Australia. Sodium taurochenodeoxycholate, sodium glycodeoxycholate, ortho-
phosphoric acid 85% (H3PO4), sodium hydroxide pellets (NaOH), tert-butyl methyl ether (TBME),
dimethyl sulfoxide (DMSO), glacial acetic acid and absolute ethanol were from Merck, Australia.
Disodium hydrogen orthophosphate (Na2HPO4), sodium dihydrogen orthophosphate
(NaH2PO4.2H2O) and ammonium dihydrogen orthophosphate (NH4H2PO4) (Ajax Finechem,
Chapter 3: Bile-induced supersaturation
103
Australia), cholesterol, [4-14C]- (49.8 mCi/mmol) and Irga-Safe PlusTM (Perkin Elmer Life
Sciences, MA, USA), oleic acid, [9,10-3H(N)] (60 Ci/mmol) (American Radiolabelled Chemicals,
MO, USA), Block Lipid Transport-1 (BLT-1, i.e. 2-hexyl-1-cyclopentanone thiosemicarbazone)
(Chembridge, CA, USA), ezetimibe (Jai Radhe Sales, AMD, India), heparin sodium injection BP
(1000 I.U./mL, Hospira, Australia), xylazine (100 mg/mL, Troy Laboratories, Australia),
acepromazine (10 mg/mL, Ceva Delvet, Australia), ketamine (100 mg/mL, Provet, Australia) and
pentobarbitone sodium (325 mg/mL, Virbac, Australia) were obtained from listed suppliers.
Acetonitrile, methanol, and chloroform used were analytical reagent grade. Water was obtained
from a Millipore milliQ Gradient A10 water purification system (Millipore, MA, USA).
3.3.1.1 Sulfo-N-succinimidyl oleate (SSO) synthesis
SSO synthesis was adapted from the procedure of Harmon et al.195. Briefly,
dicyclohexylcarbodiimide (1.26 mmol) and N-hydroxysulfosuccinimide (sodium salt, 1.20
mmol) were added to a solution of oleic acid (1.20 mmol) dissolved in N,N-dimethylformamide (2
mL) and the reaction stirred at room temperature overnight. The precipitated dicyclohexylurea
was removed by filtration and ethyl acetate (2 mL) added to the filtrate, which was left to stand at
4° C overnight. SSO (precipitate) was then collected by filtration and dried under vacuum (1
mmHg). SSO identity was confirmed by NMR and mass spectrometry.
3.3.2 Experimental outline
To assess the role of receptor-mediated collisional drug absorption, cinnarizine bioavailability
was assessed after administration of a lipid emulsion formulation in the absence and presence of
BLT-1, SSO, and ezetimibe. BLT-1, SSO and ezetimibe are chemical inhibitors of SR-BI196,
CD36197, and NPC1L1198, respectively. The possibility of endocytosis-mediated uptake was also
investigated by the use of a general endocytosis inhibitor, monensin.
Chapter 3: Bile-induced supersaturation
104
The role of collisional drug absorption was assessed more generically using an in situ rat jejunum
perfusion model to compare the absorptive flux of cinnarizine from two distinctly different lipid
colloidal phases (micelles vs. vesicles) with matched drug solubilisation capacities. Colloidal
systems with the same total solubilisation capacity, loaded with drug at the same concentration
have the same thermodynamic activity and therefore Cfree is the same in both cases. Under these
circumstances, comparison of the flux profiles obtained from two structurally different colloids,
but with identical Cfree, provides a means of determining whether the nature of the colloid, or Cfree,
is the principal determinant of absorption. Generation of identical flux profiles from both systems
would therefore confirm the dependence of flux on thermodynamic activity and free concentration,
whereas a significant difference in flux would indicate a role for factors beyond Cfree in
determining flux. These include the potential for collisional drug absorption mechanisms since
collision rates are a statistical function of particle number and are expected to be markedly higher
for micelles (where the smaller particle size results in higher particle numbers) when compared to
vesicles.
To assess the potential for intestinal fluids to enhance drug absorption from lipid colloidal phases
via the induction of drug supersaturation, whole bile was collected from fasted rats, and mixed
with model micelles and vesicles to simulate the process of interaction with bile in vivo. The
potential for bile to generate drug supersaturation was evaluated in vitro by assessing changes in
cinnarizine solubility, and by monitoring the kinetics of cinnarizine solubilisation and
precipitation, following bile addition to cinnarizine-loaded micelles and vesicles. Subsequently,
the impact of drug supersaturation on the intestinal absorptive flux of cinnarizine from micelles
and vesicles was assessed in an in situ rat jejunum perfusion model, with and without co-perfusion
of donor bile. Finally, the relevance of bile-induced drug supersaturation in vivo was assessed via
Chapter 3: Bile-induced supersaturation
105
examination of changes to cinnarizine bioavailability after administration of drug-loaded micelles
and vesicles (with matched thermodynamic activity) in bile-intact vs. bile-diverted rats.
3.3.3 Formulation preparation
3.3.3.1 Lipid emulsion
The lipid emulsion (3 mL per dose) consisted of 1 mg cinnarizine and 49 mg oleic acid solubilised
in 8 mM sodium taurocholate, 2 mM phosphatidylcholine, 2 mM cholesterol and trace amounts of
14C-cholesterol (1 µCi/3 mL) and/or 3H-oleic acid (3 µCi/3 mL). The emulsion was prepared in
7.5 mL batches by weighing appropriate masses of cinnarizine in oleic acid stock solution (20
mg/g), phosphatidylcholine and cholesterol into a glass vial, and the mixture made up to volume
with a buffered sodium taurocholate solution (buffer consisted 18 mM Na2H2PO4.2H2O and 12
mM Na2HPO4). Appropriate volumes of 14C-cholesterol, 3H-oleic acid, 5 mg/mL BLT-1 in
ethanol (for SR-BI inhibition experiments only), 25 mg/mL SSO in DMSO (for CD36 inhibition
experiments only), and 10 mg/mL monensin in ethanol (for endocytosis inhibition experiments
only) were spiked into the vial, and vortexed for 1 min. The formulation was emulsified by
ultrasonification with a Misonix XL 2020 ultrasonic processor (Misonix, Farmingdale, NY, USA)
equipped with a 3.2-mm microprobe tip running at an amplitude of 240 µm and a frequency of 20
kHz for 1.5 min. The total solvent concentration in the emulsion was ≤ 2.5% v/v. The emulsion
was used within 4 h of preparation, and the concentration of drug and labelled cholesterol and/or
oleic acid assayed before dosing (in duplicate) to confirm compound content in the emulsion and
to allow for dose normalisation between rats.
Chapter 3: Bile-induced supersaturation
106
3.3.3.2 Intravenous formulation
The formulation (1 mL per dose) used for intravenous administration of cinnarizine and 14C-
cholesterol comprised 0.5% w/v Tween® 80 in buffer (36 mM Na2HPO4 and 22 mM KH2PO4,
adjusted to pH 4 with acetic acid). Cinnarizine and cholesterol were added to the formulation by
spiking 5% v/v DMSO (containing 10 mg/mL cinnarizine) and 5% v/v ethanol (containing 5
mg/mL cholesterol and 40 µCi/mL 14C-cholesterol) into the micellar solution. The formulation
was mixed by vortexing, and the concentration of drug and labelled cholesterol assayed before
dosing to confirm compound content in the formulation and to allow for dose normalisation
between rats. The formulation was used within 1 h of preparation.
3.3.3.3 Model micelles and vesicles
The preparation of model micelles and vesicles was guided by the methods and phase diagram
published by Kossena et al.39. Medium-chain lipid containing colloids were chosen over long-
chain systems since the former have previously been shown to generate monophasic micellar and
vesicular systems39. Relatively high lipid concentrations were chosen to reflect the species that are
expected to initially form during the digestion of medium-chain triglycerides91. The model
colloids consisted of tricaprylin digestion products (caprylic acid and monocaprylin) solubilised in
simulated endogenous intestinal fluid (SEIF). SEIF comprised the six most prevalent bile salts in
human bile199, lysophosphatidylcholine (LPC), and cholesterol. The total bile salt:LPC:cholesterol
molar ratio was maintained at 16:4:1, reflecting known ratios within fasted human bile200, 201. The
combination of bile salts used here comprised 25 mol% sodium glycocholate, 17.5 mol% sodium
glycodeoxycholate, 25 mol% sodium glycochenodeoxycholate, 12.5 mol% sodium taurocholate,
7.5 mol% sodium taurodeoxycholate and 12.5 mol% sodium taurochenodeoxycholate. The
concentration ratios of the bile salts were chosen based on average concentrations of the six most
prevalent bile salts found in human bile199. The caprylic acid:monocaprylin molar ratio was kept at
Chapter 3: Bile-induced supersaturation
107
2:1, reflecting the ratio of digestion products expected on digestion of 1 mole of triglyceride. The
concentration of micellar and vesicular components was varied by trial and error (but maintaining
the ratios described above) to identify systems with similar drug solubilisation capacities. It has
previously been shown that the thermodynamic activity (i.e. free concentration) of drug in a
solubilised system may be estimated via assessment of solubility behaviour, such that different
colloidal solutions containing drug at a fixed proportion of the saturated solubility, results in
matched free concentrations202. Thus, drug was loaded into either micellar or vesicular systems at
the same concentration (and the same proportion of saturated solubility) and was therefore present
at the same thermodynamic activity (i.e. Ctotal, Ccolloid and Cfree were the same in both micellar and
vesicular systems). The compositions of the identified micellar and vesicular systems are shown in
Table 3.1.
Table 3.1: Composition of model micelles and vesicles.^
Total bile salt# (mM)
LPC$ (mM)
Cholesterol (mM)
Caprylic acid (mM)
Monocaprylin (mM)
Micelles 8 2 0.5 69.3 34.7
Vesicles 2 0.5 0.125 52.0 26.0
^ Micelles and vesicles also consist of 18 mM NaH2PO4.2H2O and 12 mM Na2HPO4. Sodium strength was adjusted to 150 mM with NaCl. Final pH of phases was adjusted to 6.30 ± 0.01
# Total bile salt consist of 25 mol% sodium glycocholate, 17.5 mol% sodium glycodeoxycholate, 25 mol% sodium glycochenodeoxycholate, 12.5 mol% sodium taurocholate, 7.5 mol% sodium taurodeoxycholate, 12.5 mol% sodium taurochenodeoxycholate
$ LPC is lysophosphatidylcholine
SEIF (8 mM total bile salt:2 mM LPC:0.5 mM cholesterol) was prepared in 50 mL batches.
Briefly, LPC and cholesterol were dissolved in 1 mL chloroform in a round bottom flask, followed
Chapter 3: Bile-induced supersaturation
108
by solvent evaporation under vacuum. The thin film formed by solvent evaporation was
reconstituted with buffered bile salts solution (8 mM total bile salt, 18 mM NaH2PO4.2H2O and 12
mM Na2HPO4, 100 mM NaCl), vortexed for 1 min, and allowed to equilibrate at room
temperature overnight. When vesicles were prepared, a similar procedure was adopted, but in this
case SEIF was diluted 4-fold with buffer (18 mM NaH2PO4.2H2O and 12 mM Na2HPO4, 108 mM
NaCl) to reduce the bile salt:lipid concentration ratio. Micelles and vesicles were prepared in 10
mL batches by adding caprylic acid and monocaprylin (quantities in Table 3.1) to SEIF, followed
by pH adjustment to 6.30 with solid NaOH and vortexing for 1 min. The phases were then
ultrasonicated as described earlier (30 sec continuous ultrasonication followed by pulsatile, 1 sec-
on/1 sec-off ultrasonication for 5 min). When included in the colloids, cinnarizine was pre-
dissolved in caprylic acid, and the drug/fatty acid solution allowed to equilibrate overnight prior to
micelle/vesicle preparation.
3.3.4 Particle sizing
The particle size of the model micelles and vesicles was determined by photon correlation
spectroscopy (Malvern Instruments Nano-ZS Zetasizer, Malvern, UK) at 37 °C. Light scattering
cells were cleaned by rinsing with ultrapure laboratory grade water (Milli-Q, Millipore, MA,
USA), and allowed to dry while inverted on lint-free wipes (Kimwipes®, Kimberly-Clark,
Australia). Samples were transferred into the cells using sterile 1 mL syringes, and analysed
without dilution to prevent structural changes to the phases. CONTIN analysis method was used to
analyse the autocorrelation functions and to provide a calculated polydispersity index. Micelles
had a mean particle size of 9 ± 1 nm [polydispersity index 0.128 ± 0.040], and vesicles had a mean
particle size of 443 ± 32 nm [polydispersity index 0.456 ± 0.038]. Data reported are mean ± SEM
of n = 3 determinations.
Chapter 3: Bile-induced supersaturation
109
3.3.5 Equilibrium solubility of cinnarizine in the model micelles and vesicles
Excess solid cinnarizine was added to 2 mL micelles or vesicles in glass vials. Vials were briefly
vortexed, incubated at 37 °C, and samples taken every 24 h over a period of 120 h. During
sampling, vials were centrifuged (2,200 xg, 10 min, 37 °C), 50 µL of supernatant sampled, and
vials re-vortexed. Equilibrium solubility was defined when drug concentrations in consecutive
samples varied by ≤ 5%, and was determined on three separate occasions. Equilibrium solubility
of cinnarizine was also determined after 1:1 addition of bile/bile pH 6.30/buffer pH 6.30 to the
different colloidal phases. The pH of fresh bile was adjusted to 6.30 with H3PO4. Buffer pH 6.30
consisted of 18 mM NaH2PO4.2H2O, 12 mM Na2HPO4, and 108 mM NaCl.
3.3.6 Kinetics of cinnarizine precipitation
The kinetics of cinnarizine precipitation was monitored after addition of bile to the micellar and
vesicular phases, to determine whether a period of drug supersaturation existed prior to drug
precipitation. In a temperature and stirring rate-controlled vessel, 2.5 mL of bile was added to 2.5
mL micelles or vesicles containing 0.2 mg/mL cinnarizine (~ 80% saturated solubility). Samples
(100 µL) were taken before the addition of bile, and at 1, 10, 20, 30, 40, 50, 60, 80, 100, 120 min
after bile addition. Samples were immediately centrifuged (2,200 xg, 5 min, 37 °C) to separate
precipitated drug, and 50 µL of supernatant assayed for drug content. The proportion of the initial
solubilised cinnarizine concentration, that remained solubilised after bile addition, was assessed as
the percent of the drug mass remaining in solution (i.e. concentration in the supernatant multiplied
by the volume remaining in vessel) relative to the total drug mass in the vessel at each time point.
3.3.7 Solid-state analysis of the cinnarizine precipitate
Selected cinnarizine pellets from the precipitation kinetics experiments were analysed using a
Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with crossed polarising
Chapter 3: Bile-induced supersaturation
110
filters. At the end of the precipitation kinetics experiments, 1.5 mL of remaining bile + colloid
mixture were centrifuged (2,200 xg, 10 min, 37 °C), the supernatant was discarded, and a small
amount of pellet was carefully placed on a microscope slide. Samples were analysed under cross-
polarised light, and images were recorded using a Canon PowerShot A70 digital camera (Canon,
Tokyo, Japan).
3.3.8 Animals
Animal care and anaesthesia have been described in Section 2.2.2.2. All animals were fasted
overnight (12–18 h) prior to surgery. At the end of all experiments, rats were euthanized via an
intravenous or intracardiac injection of 100 mg sodium pentobarbitone.
3.3.9 Surgical procedures
3.3.9.1 Cinnarizine bioavailability studies following intraduodenal
administration
The surgical procedures for the conduct of bioavailability studies included cannulations of the
right carotid artery, right jugular vein, duodenum (1 cm below pylorus), and common bile duct
(only for bile-diverted rats). The surgical procedures for the cannulations have been described in
Section 2.3.2.
3.3.9.2 Fasted rat bile collection
The bile duct was cannulated near the hilum of the liver (where the duct is free of pancreatic
tissue) in order to facilitate the collection of bile fluid without contamination by exocrine
pancreatic secretions203. Rats were rehydrated via saline infusion (1.5 mL/h) into a cannula
inserted into the right jugular vein, and bile continuously collected for 5 h. The concentration of
Chapter 3: Bile-induced supersaturation
111
total bile salt in collected bile was assayed using a validated enzymatic colorimetric assay (Total
Bile Acids kit #431-15001; Wako Pure Chemical Industries, Osaka, Japan) on a plate reader
(Fluostar Optima plate reader, BMG Labtechnologies, Germany) measuring absorbance at a
wavelength of 540 nm. In all subsequent experiments, bile was used within 24 h of collection.
3.3.9.3 Single-pass rat jejunum perfusion
The model employed to assess flux across rat jejunum involved in situ perfusion (single-pass) of
an isolated jejunal segment and simultaneous blood collection from the corresponding mesenteric
vein branch. The surgical procedures for the setup of the single-pass rat jejunum perfusion model
have been described in Section 2.2.2.3.
3.3.10 Cinnarizine bioavailability studies
A 30-min equilibration period was allowed between the end of surgery and drug dosing. To
examine the impact of lipid uptake receptors on drug absorption, studies were conducted in the
presence or absence of lipid uptake inhibitors. To examine the impact of bile-induced drug
supersaturation on drug absorption, studies were conducted in bile-intact or bile-diverted rats.
Colloidal systems (lipid emulsion, micelles, vesicles) containing cinnarizine were infused into the
duodenum of rats at a rate of 1.5 mL/h for 2 h. When the dose infusion was complete, saline was
infused at a rate of 1.5 mL/h for 10 min to flush any remaining formulation in the tubing into the
duodenum. Blood samples (0.3 mL) were collected via the carotid artery cannula up to 8 h after
infusion initiation into tubes containing 3 I.U. heparin. The sampling intervals were: t = 0, 1, 2, 2.5,
3, 4, 6, 8 h for the receptor inhibition studies; and t = 0, 1, 1.5, 2, 3, 4, 6, 8 h for the bile-induced
drug supersaturation studies. After each blood sample was taken, the cannula was flushed with 0.3
mL of 2 I.U./mL heparinised saline to ensure cannula patency, and to replace the volume of blood
Chapter 3: Bile-induced supersaturation
112
removed. Plasma was separated by centrifugation (10,000 xg, 5 min) to enable analysis of drug
and labelled lipid content.
3.3.10.1 Administration of lipid uptake inhibitors (BLT-1, SSO, ezetimibe) and
endocytosis inhibitor (monensin)
Ezetimibe has previously been dosed at 0.3 mg/kg intravenously into rats, and has been shown to
inhibit cholesterol absorption (from an intraduodenally dosed lipid emulsion) without reports of
toxicity204. Therefore, in our study, intravenous administration of ezetimibe (0.3 mg/kg via the
jugular vein) was selected as the route to administer the inhibitor at the beginning of the 30-min
equilibration period. An appropriate volume of 5 mg/mL ezetimibe in ethanol was spiked into
blank rat plasma, and 0.8 mL of resultant plasma dosed into rats as an intravenous bolus. The total
ethanol concentration was less than 2.5% v/v. In the case of BLT-1 and SSO, the inhibitors had
not been previously administered intravenously, therefore local (i.e. intestinal) administration was
selected to limit the systemic effects of the inhibitors. Monensin was also administered directly
into the intestine to minimise systemic endocytosis inhibition. Here, 100 µM BLT-1, 1 mM SSO
and 100 µM monensin were pre-infused intraduodenally (in saline) at a rate of 1.5 mL/h during
the 30-min equilibration period, and subsequently co-infused at the same concentration as part of
the lipid emulsion. BLT-1 and SSO have previously been shown to inhibit lipid uptake in cell-
based studies at concentrations of 1-10 µM196 and 400 µM195, respectively. Monensin has
previously been shown to inhibit endocytosis in cultured cells at 10 µM205.
Intravenous administration studies were also conducted in control rats and monensin-treated rats
to assess the effect of endocytosis inhibition on the systemic distribution and clearance of
cinnarizine and cholesterol. In these studies, blank (i.e. not containing cinnarizine and 14C-
cholesterol) lipid emulsion (with or without 100 µM monensin) was infused intraduodenally as
Chapter 3: Bile-induced supersaturation
113
described above, and intravenous infusion of the cinnarizine and 14C-cholesterol containing
intravenous formulation was commenced at the same time. The intravenous formulation (1 mL)
was infused into the right jugular vein at a rate of 0.05 mL/15 sec (total infusion period < 5 min).
Blood samples (0.3 mL) were collected via the carotid artery cannula at t = 5, 15, 30, 60, 120, 180,
240, 360, 480 min after infusion initiation into tubes containing 3 I.U. heparin. After each blood
sample was taken, the cannula was flushed with 0.3 mL of 2 I.U./mL heparinised saline. Plasma
was separated by centrifugation (10,000 xg, 5 min) to enable analysis of drug and labelled
cholesterol content.
3.3.11 In situ single-pass rat jejunum perfusion
After surgery, animals were equilibrated for 30-min, during which time heparinised donor rat
blood was infused via the jugular vein as described above. During re-equilibration, blood from the
cannulated mesenteric vein (~ 0.3 mL/min) was collected for re-infusion. Perfusion buffer was
pumped through the jejunal segment at a rate of 0.1 mL/min and outflowing buffer discarded to
waste. The exposed jejunal segment was kept moist by covering with saline-soaked gauze
throughout the experiment.
In all experiments, the concentration of cinnarizine in the perfusate was held at 0.1 mg/mL (~ 40%
saturated solubility). Therefore, in experiments where micelles or vesicles were perfused alone,
cinnarizine was loaded into the perfusate at 0.1 mg/mL. In experiments where micelles or vesicles
were co-perfused in a 1:1 v/v ratio with a secondary perfusate of bile/bile pH 6.30/buffer pH 6.30,
cinnarizine was loaded into the primary perfusate at 0.2 mg/mL, such that 1:1 v/v dilution led to a
final perfusate concentration of 0.1 mg/mL.
Chapter 3: Bile-induced supersaturation
114
Perfusate flow was maintained at 0.1 mL/min in all experiments to minimise variations in the
thickness of the unstirred water layer206. For experiments where 1:1 v/v co-perfusion of the phases
with bile/bile pH 6.30/buffer pH 6.30 was required, micelles/vesicles and bile/buffer were pumped
at 0.05 mL/min, and mixed via a three-way “T” connector immediately prior to entry into the
jejunal segment, providing a total perfusate flow of 0.1 mL/min. Perfusate was sampled at t = 0 to
confirm lipid and drug concentrations. After this time, the outflowing perfusate was continuously
collected at 10-min intervals, and briefly vortexed before samples were taken for analysis of drug
and lipid content. For experiments where drug supersaturation was generated, perfusate samples
were taken before and after centrifugation (2,200 xg, 2 min), to assess the degree of drug
precipitation within the jejunal segment. Blood draining the perfused jejunal segment was
collected at 5-min intervals, plasma separated by centrifugation (10,000 xg, 5 min), and samples
taken for analysis of drug content by HPLC as described below.
3.3.12 Analytical procedures
3.3.12.1 Sample preparation and HPLC assay conditions for cinnarizine
Samples of lipid emulsion were prepared for HPLC assay by an initial 80-fold dilution with
chloroform:methanol (2:1 v/v), followed by a 10-fold dilution with mobile phase (50% v/v
acetonitrile:50% v/v 20 mM NH4H2PO4). Samples of IV formulation and micelles/vesicles were
prepared for HPLC assay by a 900-fold, and a 400-fold dilution with mobile phase (50% v/v
acetonitrile:50% v/v 20 mM NH4H2PO4), respectively. Plasma samples were prepared for HPLC
using a validated extraction procedure, with flunarizine as an internal standard, as reported
previously43.
Chapter 3: Bile-induced supersaturation
115
Cinnarizine HPLC assay conditions were as described previously43, with slight modification to the
mobile phase employed for cinnarizine quantification in plasma, to 45% v/v acetonitrile:55% v/v
20 mM NH4H2PO4 in this study. Replicate analysis of n = 4 quality control samples revealed
acceptable accuracy and precision (± 10%, ± 15% at the limit of quantification) for concentrations
between 20–1000 ng/mL for IV formulation, emulsion, micelles and vesicles, and 10–320 ng/mL
for plasma.
3.3.12.2 Scintillation counting
Quantification of 14C-cholesterol and 3H-oleic acid in the plasma was performed via scintillation
counting on a Packard Tri-Carb 2000CA liquid scintillation analyser (Packard, Meriden,
Connecticut, USA). Plasma samples (50 µL) were added to 2 mL Irga-safe Plus scintillation fluid
followed by a 10-sec vortex. Samples were corrected for background radioactivity by the inclusion
of a blank plasma sample in each run.
3.3.12.3 Blood:plasma ratio determination for cinnarizine
The blood:plasma ratio for cinnarizine was determined by spiking 0.5 mL blank blood with known
amounts of cinnarizine to achieve low, medium, and high concentrations (in triplicate). Plasma
was separated by centrifugation (10,000 xg, 5 min) and plasma drug concentration assayed by
HPLC. The blood:plasma ratio was calculated from the ratio of known concentration in spiked
blood to the concentration measured in plasma separated from spiked blood. The mean
blood:plasma ratio was subsequently used to convert plasma concentrations to blood
concentrations in perfusion experiments, enabling quantification of total drug transport into
mesenteric blood.
Chapter 3: Bile-induced supersaturation
116
3.3.12.4 Calculations
In the single-pass rat jejunum perfusion model, permeability coefficients were calculated using
steady state drug concentrations in perfusate and blood. Two apparent permeability coefficients
(Papp) were calculated as described previously184:
′Disappearance P . ln Equation 3.2
′Appearance′P∆
∆.
Equation 3.3
where ‘Disappearance’ Papp is the apparent permeability coefficient calculated from drug loss from
the perfusate (cm/s); ‘Appearance’ Papp is the apparent permeability coefficient calculated from
drug appearance in the mesenteric blood (cm/s); Q is the perfusate flow rate (mL/s); A is the
surface area of the perfused jejunal segment (cm2), which is calculated by multiplying the
diameter by the length of the perfused intestinal segment as described previously207; C1 is the
average steady state drug concentration exiting the perfused jejunal segment (ng/mL); C0 is the
drug concentration entering the jejunal segment (ng/mL); ∆MB/∆t is the average rate of drug mass
appearance in mesenteric blood at steady state (ng/s); and <C> is the logarithmic mean drug
concentration in the lumen (ng/mL), where <C> = (C1 – C0)/(ln C1 – ln C0).
3.3.12.5 Non-compartmental pharmacokinetic analysis
The maximum plasma concentration (Cmax), time to reach Cmax (Tmax), area under the plasma
concentration-time curve from time zero to the last measured concentration (AUC0-8 h), area under
the plasma concentration-time curve extrapolated to infinity (AUC0-inf), elimination rate constant
Chapter 3: Bile-induced supersaturation
117
(Ke), volume of distribution (Vd), and clearance (Cl) were calculated using WINONLIN version
5.3 (Pharsight Inc., Apex, NC, USA).
3.3.13 Statistical analysis
Results were analysed using Student’s t test. A P value of < 0.05 was considered to be a
significant difference. Analyses were performed using SPSS v19 for Windows (SPSS Inc.,
Chicago, IL, USA).
3.4 RESULTS
3.4.1 SR-BI, CD36, NPC1L1 and endocytosis have little impact on drug
absorption from intestinal colloidal phases
Inhibition of the lipid uptake receptors SR-BI (by co-infusion of 100 µM BLT-1), CD36 (by co-
infusion of 1 mM SSO), and NPC1L1 (by intravenous administration of 0.3 mg/kg ezetimibe) did
not result in significant changes to the systemic plasma concentration-time profiles or systemic
exposure of cinnarizine (Figure 3.2A, Table 3.2). Inhibition of endocytosis (by co-infusion of 100
µM monensin) unexpectedly led to significant increases in cinnarizine systemic plasma
concentration at t = 2.5 and 4 h (Figure 3.2A), and increased the AUC0-8 h 1.5-fold (Table 3.2).
However, subsequent intravenous dosing studies revealed significantly lower Vd and Cl values in
the monensin-treated rats when compared to control (Table 3.3), suggesting that the increase in
cinnarizine systemic exposure was due to a decrease in cinnarizine systemic distribution and
clearance, rather than changes to intestinal absorption. The data suggest a limited role for SR-BI,
CD36, NPC1L1, and endocytosis generally, in the absorption of cinnarizine from micelles and
vesicles.
Chapter 3: Bile-induced supersaturation
118
In contrast, inhibition of NPC1L1 did lead to significantly lower systemic plasma concentrations
of exogenously dosed cholesterol at t = 2, 2.5, 3, 4, 6, 8 h (Figure 3.2B). Plasma concentrations of
exogenously dosed oleic acid also appeared lower in rats following inhibition of CD36 although
differences were not significantly different (Figure 3.2C). While inhibition of SR-BI and CD36
did not lead to significant changes to the systemic plasma concentration-time profile of
exogenously dosed cholesterol, inhibition of endocytosis significantly increased the systemic
plasma concentrations of exogenously dosed cholesterol at t = 4, 8 h (Figure 3.2B). This increase
in exposure is likely explained by a decrease in systemic distribution of 14C-cholesterol in the
monensin-treated rats, as plasma 14C-cholesterol concentration at early sample time points (which
reflect the distribution phase) in intravenous studies were significantly higher in the monensin-
treated rats (Figure 3.3B) when compared to controls (Vd and Cl could not be calculated for
cholesterol as a typical elimination phase was not evident in the systemic plasma concentration vs.
time profiles of cholesterol - Figure 3.2B and Figure 3.3B).
Chapter 3: Bile-induced supersaturation
119
Figure 3.2: Systemic plasma concentration-time profiles of (A) cinnarizine (CIN), (B) 14C-
labelled cholesterol (Ch), and (C) 3H-labelled oleic acid (OA) following intraduodenal infusion of
a 3 mL lipid emulsion consisting 1 mg cinnarizine emulsified in 59 mM oleic acid, 8 mM sodium
taurocholate, 2 mM phosphatidylcholine, 2 mM cholesterol, 1 µCi 14C-cholesterol and/or 3 µCi 3H-oleic acid. Experiments were performed in control rats (filled circle); rats treated with 100 µM
BLT-1 (open circle); rats treated with 1 mM SSO (open triangle), rats treated with 0.3 mg/kg
ezetimibe (open square), and rats treated with 100 µM monensin (open diamond), to inhibit the
lipid uptake receptors SR-BI, CD36, NPC1L1, and endocytosis, respectively. BLT-1, SSO, and
monensin were co-infused as part of the lipid emulsion; ezetimibe was administered intravenously.
Data represent mean ± SEM of n = 4 rats for (A) and (B); and n = 3 rats for (C). Statistically
significant difference with respect to control rats (p < 0.05) is denoted by the symbol *.
Time (h)
0 2 4 6 8
Pla
sma
CIN
con
c.
(ng
/mL)
0
100
200
300
400
500 ControlBLT-1SSO Ezetimibe Monensin
A
*
*
Time (h)
0 2 4 6 8
Pla
sma
14C
-Ch
con
c.(n
g/m
L)
0
20
40
60
ControlBLT-1SSOEzetimibeMonensin
B
* ** * * *
* *
Time (h)
0 2 4 6 8
Pla
sma
3H
-OA
co
nc.
(ng
/mL)
0.00
0.01
0.02
0.03
0.04
0.05
0.06ControlSSO
C
Chapter 3: Bile-induced supersaturation
120
Table 3.2: Pharmacokinetic parameters for cinnarizine after intraduodenal administration of a 3
mL lipid emulsion in rats. Experiments were performed in control rats; rats treated with 100 µM
BLT-1; rats treated with 1 mM SSO, rats treated with 0.3 mg/kg ezetimibe, and rats treated with
100 µM monensin, to inhibit the lipid uptake receptors SR-BI, CD36, NPC1L1, and endocytosis,
respectively. Values represent mean ± SEM of n = 4 rats.
Experimental group Formulation type
CIN dose
(mg/kg)
AUC0-8 h
(ng h/mL)
Cmax
(ng/mL)
Tmax
(h)
Control Emulsion 3.33 978 ± 122 288.1 ± 26.6 2.4 ± 0.1
BLT-1 treated Emulsion 3.33 883 ± 21 219.0 ± 20.0 2.6 ± 0.5
SSO-treated Emulsion 3.33 924 ± 88 195.5 ± 33.6 2.4 ± 0.2
Ezetimibe-treated Emulsion 3.33 1048 ± 92 333.9 ± 13.6 2.3 ± 0.1
Monensin-treated Emulsion 3.33 1487 ± 103a 432.8 ± 45.9a 2.3 ± 0.1
a Significant difference when compared to control group
Chapter 3: Bile-induced supersaturation
121
Time (min)
0 100 200 300 400 500
Pla
sma
CIN
co
nc.
(ng
/mL
)
10
100
1000ControlMonensin
** * *
(A)
Time (min)
0 100 200 300 400 500
Pla
sma
14
C-C
h c
on
c.
(ng
/mL
)
100
1000ControlMonensin*
**
(B)
Figure 3.3: Systemic plasma concentration-time profiles of (A) cinnarizine (CIN), and (B) 14C-
labelled cholesterol (Ch) following intravenous infusion over 5 min of 1 mL formulation
containing 0.5 mg cinnarizine and 1 µCi 14C-Ch. Experiments were performed in control rats
(filled circle) and rats treated with 100 µM monensin (open diamond). Intraduodenal infusion of
blank lipid emulsion was also commenced at t = 0 (to match experimental conditions in Figure
3.2). Monensin, an endocytosis inhibitor, was pre-infused intraduodenally 30 min prior to t = 0,
and subsequently co-infused as part of the lipid emulsion. Data represent mean ± SEM of n = 4
rats for (A); and n = 3 rats for (B). Statistically significant difference with respect to control rats (p
< 0.05) is denoted by the symbol *.
Table 3.3: Pharmacokinetic parameters for cinnarizine after intravenous administration in rats.
Values represent mean ± SEM of n = 3-4 rats
Experimental group
CIN dose (mg/kg)
AUC0-inf
(ng h/mL) Ke
(1/h) Vd
(L/kg) Cl
(L/h/kg)
Control 1.67 821 ± 24 0.25 ± 0.03 5.8 ± 0.2 2.03 ± 0.06
Monensin-treated 1.67 1077 ± 28a 0.28 ± 0.04 4.0 ± 0.4a 1.55 ± 0.04a
a Significant difference when compared to control group
Chapter 3: Bile-induced supersaturation
122
3.4.2 Drug absorption from micelles and vesicles is determined by Cfree and
not colloidal structure
The intestinal perfusion of colloidal media with markedly different compositional profiles
(micelles and vesicles) but with comparable Cfree and thermodynamic activity did not result in
significant differences in steady-state absorptive flux, disappearance Papp or appearance Papp of
cinnarizine (Table 3.4, Figure 3.4A – filled symbols). Similarly, in spite of large differences in
particle size and composition, the intraduodenal infusion of the same micellar or vesicular systems
to bile-diverted rats did not result in significant differences in systemic plasma concentration-time
profiles (Figure 3.5 – filled symbols) and pharmacokinetic parameters of cinnarizine (Table 3.5 -
micelles vs. vesicles in bile-diverted rats). The results suggest that cinnarizine absorption from
lipid colloidal phases is relatively insensitive to the physical nature of the colloidal milieu, and is
instead controlled largely by Cfree.
Chapter 3: Bile-induced supersaturation
123
Figure 3.4: (A) Absorptive flux of cinnarizine (CIN) into mesenteric blood (ng/5 min/10 cm2) and
(B) & (C) CIN disappearance from intestinal perfusate (% drug dose passing through jejunum)
when micelles/vesicles were perfused through an isolated rat jejunal segment (~ 10 cm2), with
(open symbols) and without (filled symbols) 1:1 v/v co-perfusion with rat bile. Co-perfusion of
micelles and vesicles with donor bile generates drug supersaturation in situ within the perfused
jejunal segment. SS denotes experiments where drug is supersaturated in the perfusate. The degree
of drug precipitation within the perfusate is illustrated in (B) and (C) as the difference in perfusate
concentration between pre- and post-centrifugation data. Precipitation was significant in the
vesicle, but not micellar groups. Experiments were performed using an in situ single-pass rat
jejunum perfusion model. In all experiments, the concentration of cinnarizine in perfusate and the
total perfusate flow rate were kept constant at 0.1 mg/mL and 0.1 mL/min, respectively. Data
represent mean ± SEM of n = 3-4 experiments.
Time (min)
0 10 20 30 40 50 60
CIN
flu
x in
to m
ese
nte
ric
blo
od
(n
g/5
min
/10
cm
2)
0200400600800
100012001400
Micelles
Micelles + Bile ss
Vesicles
Vesicles + Bile ss
A
Time (min)
0 20 30 40 50 60
% D
ose
pas
sin
g
thro
ug
h je
jun
um
0
20
40
60
80
100
MicellesMicelles + Bile SS (pre-centrifugation)Micelles + Bile SS (post-centrifugation)
B
Time (min)
0 20 30 40 50 60
% D
ose
pa
ssin
g
thro
ug
h je
jun
um
0
20
40
60
80
100
VesiclesVesicles + Bile SS (pre-centrifugation)Vesicles + Bile SS (post-centrifugation)
C
Chapter 3: Bile-induced supersaturation
124
Table 3.4: Cinnarizine disappearance Papp (x 106 cm/s) from the intestinal perfusate, appearance
Papp (x 106 cm/s) in the mesenteric blood, and steady state absorptive flux into mesenteric blood
(ng/5 min/10 cm2) after 60 min of single-pass perfusion of ~ 10 cm2 segments of rat jejunum with
model micelles and vesicles, with and without 1:1 v/v co-perfusion with rat bile, rat bile pH 6.30
or buffer pH 6.30. Values calculated using data obtained after steady state attainment (t = 40-60
min). In all experiments, cinnarizine concentration in perfusate and total perfusate flow rate were
kept constant at 0.1 mg/mL and 0.1 mL/min, respectively. Data represent mean ± SEM of n = 3-4
experiments.
SS ratio #
Disappearance Papp
(x 106 cm/s)
Appearance Papp
(x 106 cm/s)
Flux into mesenteric blood (ng/5 min/10 cm2)
Micelles 0.4 19.4 ± 3.0 1.1 ± 0.2 310 ± 50
Micelles + Bile SS 6.1 29.3 ± 3.0 a 3.7 ± 0.1 a 979 ± 30 a
Micelles + Bile pH 6.30 SS 3.0 39.5 ± 5.6 a 4.2 ± 1.0 a 1099 ± 279 a
Micelles + Buffer pH 6.30 SS 2.1 38.9 ± 4.1 a 4.4 ± 0.4 a 1180 ± 100 a
Vesicles 0.4 22.0 ± 2.2 1.2 ± 0.1 340 ± 29
Vesicles + Bile SS 11.6 66.0 ± 18.3b 2.0 ± 0.7 499 ± 155
a Significant increase from micelles alone b Significant increase from vesicles alone SS denotes drug supersaturation in perfusate # SS ratio = Supersaturation ratio = (Supersaturated) concentration of drug in perfusate / Equilibrium
solubility of drug in perfusate
Chapter 3: Bile-induced supersaturation
125
(A)
(B)
Time (h)
0 2 4 6 8
Pla
sma
CIN
con
c.
(ng/
mL)
020406080
100120140160
Micelles (Bile-diverted)Micelles (Bile-intact)
* * *
Time (h)
0 2 4 6 8
Pla
sma
CIN
con
c.
(ng/
mL)
020406080
100120140160
Vesicles (Bile-diverted)Vesicles (Bile-intact)
Figure 3.5: Systemic plasma concentration-time profiles of cinnarizine (CIN) following a 2 h
intraduodenal infusion of cinnarizine-loaded (0.2 mg/mL) (A) micelles and (B) vesicles to bile-
intact and bile-diverted rats. Consistent with observations in rat jejunum perfusion studies, bile-
induced supersaturation translated into increased in vivo exposure during the absorption phase in
the case of micelles but not vesicles. Data represent mean ± SEM of n = 4 rats. Statistical
significance (p < 0.05) is denoted by the symbol *.
Table 3.5: Pharmacokinetic parameters for cinnarizine after intraduodenal administration of
cinnarizine-loaded (0.2 mg/mL) micelles and vesicles to bile-intact and bile-diverted rats. Values
represent mean ± SEM of n = 4 rats.
Experimental group Formulation type
CIN dose (mg/kg)
AUC0-8 h (ng h/mL)
Cmax (ng/mL)
Tmax
(h)
Micelles (bile-intact) Micelles 2 502 ± 59a 138.8 ± 11.6a 1.6 ± 0.2
Micelles (bile-diverted) Micelles 2 362 ± 39 85.7 ± 14.3 2.0 ± 0.4
Vesicles (bile-intact) Vesicles 2 411 ± 74 109.4 ± 10.6 2.3 ± 0.3
Vesicles (bile-diverted) Vesicles 2 393 ± 82 97.6 ± 12.8 2.0 ± 0.4
a Significant increase when compared to micelles (bile-diverted) group
Chapter 3: Bile-induced supersaturation
126
3.4.3 Bile-mediated dilution of cinnarizine-loaded micelles and vesicles
generates drug supersaturation
The equilibrium solubility and change in cinnarizine solubilisation capacity of model micelles and
vesicles before and after 1:1 v/v dilution with bile, bile pH 6.30 or buffer pH 6.30 are tabulated in
Table 3.6, and shown graphically in Figure 3.6.
Table 3.6: Equilibrium solubility (37 °C) and percent original solubilisation capacity values of
cinnarizine in model micelles and vesicles, before and after 1:1 v/v addition of rat bile, rat bile pH
6.30 or buffer pH 6.30. Data represent mean ± SEM of n = 3-4 determinations.
Equilibrium solubility (µg/mL)
Percent original solubilisation capacity (%) #
Micelles Micelles alone 236 ± 9.3 100
+ Bile (1:1) 16.5 ± 0.8 14.0 ± 0.7
+ Bile pH 6.30 (1:1) 33.1 ± 1.0 28.0 ± 0.9
+ Buffer pH 6.30 (1:1) 48.7 ± 0.8 41.2 ± 0.6
Vesicles Vesicles alone 232 ± 3.3 100
+ Bile (1:1) 8.60 ± 0.5 7.4 ± 0.4
+ Bile pH 6.30 (1:1) 17.4 ± 0.4 15.0 ± 0.3
+ Buffer pH 6.30 (1:1) 24.9 ± 0.3 21.4 ± 0.2
Percentoriginalsolubilisationcapacity# Solubility ∗ VolumeSolubility ∗ Volume
x100%
Chapter 3: Bile-induced supersaturation
127
% o
rigin
al C
IN
solu
bilis
atio
n ca
paci
ty
0
20
40
60
80
100
120
140Colloid onlyColloid + Bile (1:1)Colloid + Bile pH 6.30 (1:1)Colloid + Buffer pH 6.30 (1:1)
Micelles Vesicles
**
*
* * *
Figure 3.6: Percent original cinnarizine (CIN) solubilisation capacity of model colloids (micelles
or vesicles), before and after a 1:1 v/v addition of rat bile, rat bile pH 6.30 or buffer pH 6.30. Data
represent mean ± SEM of n = 3-4 determinations. Statistical significant difference (p < 0.05) to
colloid only is denoted by the symbol *.
Dilution of the micellar or vesicular systems in a 1:1 v/v ratio with bile obtained from donor
animals led to significant decreases in cinnarizine solubilisation capacity. As a proportion of initial,
the solubilisation capacity of the micellar system dropped significantly from 100% to 14%, and
for vesicles the decrease was even greater from 100% to 7%. This was in spite of the fact that the
bile salt concentration in donor bile was higher than that in the micellar or vesicular system
(average total bile salt concentration in donor bile was 14.7 ± 0.9 mM; mean ± SEM, n = 13), and
therefore ‘dilution’ with bile did not reduce bile salt concentrations below the critical micellar
concentration (CMC) and instead increased the overall bile salt concentration.
The pH of donor bile was higher than intestinal pH (average pH of donor bile was 8.02 ± 0.02;
mean ± SEM, n = 5). As such additional studies were performed to examine whether the effects on
solubility reflected a pH effect. 1:1 v/v dilution of the phases with pH adjusted bile (pH of bile
adjusted to 6.30 to match the pH of the micelles and vesicles) also resulted in significant decreases
Chapter 3: Bile-induced supersaturation
128
in solubilisation capacity (to 28% and 15% of initial for micelles and vesicles, respectively),
although the decrease was slightly attenuated when compared to non-pH adjusted bile. Finally, the
micellar and vesicular systems were diluted 1:1 v/v with buffer at pH 6.30 in an attempt to
uncouple simple dilution effects from pH effects and bile-mediated effects. Dilution with pH 6.30
buffer also decreased cinnarizine solubilisation capacity significantly, although to a lesser extent
(41% and 21% in micelles and vesicles, respectively).
Analysis of the kinetics of drug precipitation (Figure 3.7) demonstrated that bile addition to
cinnarizine-loaded micelles and vesicles (cinnarizine present in phases at 0.2 mg/mL, ~ 80%
saturated solubility) did not result in immediate drug precipitation, and was preceded by a period
of supersaturation. The time taken for cinnarizine precipitation to occur was variable, however
supersaturation was maintained for longer periods in micelles (> 20 min in all cases) when
compared to vesicles (1-20 min).
Time (min)
0 20 40 60 80 100 120
% o
rigin
al C
IN in
sol
utio
n
0
20
40
60
80
100
120
Micelles + BileVesicles + Bile
Equilibrium solubility: Micelles + Bile Equilibrium solubility: Vesicles + Bile
Add bile
Figure 3.7: Kinetics of cinnarizine (CIN) precipitation from model micelles (filled circles, n = 5)
and model vesicles (open circles, n = 4) upon addition of rat bile (in a 1:1 v/v ratio) at t = 0.
Addition of bile reduces the equilibrium cinnarizine solubilisation capacity of micelles and
vesicles to 14% and 7% of initial, respectively (see Table 3.6, and shown here as the lines denoted
Chapter 3: Bile-induced supersaturation
129
equilibrium solubility). Cinnarizine supersaturation appeared to be maintained for longer in
micelles than in vesicles. Cinnarizine was loaded into micelles and vesicles at 80% saturation (~
0.2 mg/mL). Each line represents individual experiments.
3.4.4 Bile-induced drug supersaturation increases jejunal absorptive flux for
micelles but not vesicles
The absorptive flux vs. time profiles for cinnarizine under supersaturated conditions (i.e. when bile
was co-perfused with the phases) and under non-supersaturated conditions (when bile was not co-
perfused with the phases) are shown in Figure 3.4A. Steady state-absorptive flux, disappearance
Papp, and appearance Papp of cinnarizine in all perfusion experiments are reported in Table 3.4.
Co-perfusion of micelles with bile in a 1:1 v/v ratio increased the absorptive flux, disappearance
Papp, and appearance Papp of cinnarizine from micelles 3.2-fold, 1.5-fold, and 3.4-fold, respectively.
In contrast, 1:1 v/v co-perfusion of vesicles with bile did not lead to significant changes in
cinnarizine absorptive flux or appearance Papp. Disappearance Papp for cinnarizine did increase 3.0-
fold when vesicles were co-perfused with bile, however the drop in perfusate drug concentration
was largely a result of rapid drug precipitation in the perfusate (see below).
For the micellar preparation, bile-induced supersaturation was relatively stable throughout the
experimental period. Thus the cinnarizine concentration in the perfusate was essentially the same
before or after centrifugation (Figure 3.4B). In contrast, when vesicles were co-perfused with bile,
significant drug precipitation was observed during the time required for perfusate to transit the
jejunal segment (as indicated by the difference between the pre- and post-centrifugation data in
Figure 3.4C). This was consistent with the in vitro dilution profiles in Figure 3.7.
.
Chapter 3: Bile-induced supersaturation
130
To distinguish between bile-induced increases in cinnarizine absorptive flux resulting from drug
supersaturation, pH increases (since an increase in pH might be expected to increase the
permeability of a weak base), and non-specific effects of bile components on membrane
permeability, micelles were also co-perfused with pH-adjusted bile (pH 6.30) and buffer (pH 6.30).
1:1 v/v co-perfusion of micelles with bile pH 6.30 or buffer pH 6.30 increased cinnarizine
absorptive flux 3.5-fold and 3.8-fold, respectively, when compared to the perfusion of micelles
alone (Figure 3.8, Table 3.4). Stable supersaturation was also generated within the perfused
jejunal segment in these experiments (Figure 3.9 - cinnarizine concentration in the perfusate was
essentially the same before and after centrifugation).
Time (min)
0 10 20 30 40 50 60
CIN
flu
x in
to m
esen
teric
blo
od (
ng/5
min
/10
cm2 )
0
500
1000
1500
2000MicellesMicelles + Bile SS
Micelles + Bile pH 6.30 SS
Micelles + Buffer pH 6.30 SS
Figure 3.8: Absorptive flux-time profiles of cinnarizine (CIN) when micelles were perfused
through an isolated rat jejunal segment (~ 10 cm2), with and without 1:1 v/v co-perfusion with rat
bile, rat bile pH 6.30 or buffer pH 6.30. SS denotes experiments where drug is supersaturated in
perfusate. Co-perfusion of rat bile, rat bile pH 6.30 or buffer pH 6.30 with micelles generates drug
supersaturation in situ within the perfused jejunal segment in all cases, and increased cinnarizine
absorptive flux by 3.2-fold, 3.5-fold and 3.8-fold, respectively. Experiments were performed using
an in situ single-pass rat jejunum perfusion model. In all experiments, the concentration of
cinnarizine in perfusate and the total perfusate flow rate were kept constant at 0.1 mg/mL and 0.1
mL/min, respectively. This series of experiments provides further support for the suggestion that
Chapter 3: Bile-induced supersaturation
131
supersaturation was responsible for the increase in absorptive flux seen in Figure 3.4A. Data
represent mean ± SEM of n = 3-4 experiments.
Time (min)
0 20 30 40 50 60
% D
ose
pass
ing
thro
ugh
jeju
num
0
20
40
60
80
100
Micelles + Bile pH 6.30 (pre-centrifugation)Micelles + Bile pH 6.30 (post-centrifugation)
(A)
Time (min)
0 20 30 40 50 60
% D
ose
pass
ing
thro
ugh
jeju
num
0
20
40
60
80
100
Micelles + Buffer pH 6.30 (pre-centrifugation) Micelles + Buffer pH 6.30 (post-centrifugation)
(B)
Figure 3.9: Perfusate disappearance (% drug dose passing through jejunum) profiles of
cinnarizine (CIN) when micelles were perfused through an isolated rat jejunal segment (~ 10 cm2),
with and without 1:1 v/v co-perfusion with (A) rat bile pH 6.30 or (B) buffer pH 6.30. Co-
perfusion of micelles with rat bile pH 6.30 or buffer pH 6.30 generates drug supersaturation in situ
within the perfused jejunal segment. The degree of drug precipitation within the perfusate is
represented by the difference in perfusate concentration between pre- and post-centrifugation data.
Experiments were performed using an in situ single-pass rat jejunum perfusion model. In all
experiments, the concentration of cinnarizine in perfusate and the total perfusate flow rate were
kept constant at 0.1 mg/mL and 0.1 mL/min, respectively. Data represent mean ± SEM of n = 3-4
experiments.
Since 1:1 v/v co-perfusion of micelles with bile, bile pH 6.30 and buffer pH 6.30 are all expected
to generate cinnarizine supersaturation within the perfused jejunal segment (according to the
solubility data in Table 3.6, and the lack of drug precipitation in outflow perfusate in all cases), the
observation that absorptive flux enhancement was similar in all groups (Figure 3.8) suggest that
the enhancement was attributable to drug supersaturation, and not an increase in system pH (by
comparing micelles + bile group with micelles + bile pH 6.30 group), or non-specific effects of
bile on membrane permeability (by comparing micelles + bile pH 6.30 group with micelles +
Chapter 3: Bile-induced supersaturation
132
buffer pH 6.30 group). However, the degree of flux enhancement did not correlate with the degree
of supersaturation, as flux enhancement was similar in all groups despite the significantly higher
supersaturation ratio generated in the micelles + bile group, when compared to the micelles + bile
pH 6.30 group and micelles + buffer pH 6.30 group (supersaturation ratio of 6 vs. 2-3) (Table 3.4).
Previous studies have shown that for lipophilic drugs such as cinnarizine, the drug fraction
extracted into octanol (and analogous to the permeable fraction) as a function of pH is shifted to
lower pHs than would be expected based on the unionised fraction208, 209. This left shift would
limit pH effects on permeability over the range of pH 6-8, consistent with the observations here.
3.4.5 Bile-induced drug supersaturation increases in vivo cinnarizine
exposure after intraduodenal infusion
The systemic plasma concentration-time profiles of cinnarizine following intraduodenal infusion
of cinnarizine-loaded micelles and vesicles (0.2 mg/mL; ~ 80% saturated solubility) to bile-intact
and bile-diverted rats are shown in Figure 3.5. The pharmacokinetic parameters of cinnarizine for
the bioavailability studies are reported in Table 3.5. Consistent with the results from the rat jejunal
permeability studies, intraduodenal administration of micelles to bile-intact rats resulted in
significantly higher systemic plasma concentrations of cinnarizine at t = 1, 1.5 and 2 h (AUC0-8 h
increased 1.4-fold) when compared to bile-diverted rats. In contrast, when vesicles were dosed to
bile-intact rats, the systemic plasma concentration-time profiles and AUC0-8 h of cinnarizine were
not different to that observed in bile-diverted rats.
3.5 DISCUSSION
After oral administration, the absorption of poorly water-soluble drugs (PWSD) is often limited by
slow dissolution and low solubility in the GI tract. LBF overcome many of the dissolution
limitations of PWSD (by providing a mechanism to circumvent dissolution from the solid to liquid
Chapter 3: Bile-induced supersaturation
133
state), however, the solubility limitations of PWSD are seemingly unaddressed, since
solubilisation in the lipid-based colloidal phases that result from the digestion of LBF does not
typically enhance free drug concentrations101, 143. Nonetheless, co-administration of lipids (either
formulation lipids or via co-administration with lipid-rich foods) remains a highly effective means
to promote the absorption of PWSD. This suggests the potential for alternative mechanisms by
which LBF enhance drug absorption. In the current communication, two possible alternatives to
the traditional model of drug absorption from LBF have been explored. Firstly, that drug
absorption from lipid colloidal phases may involve a collisional uptake component (i.e. drug
absorption directly from the solubilised phase), and secondly that flux across the absorptive
membrane may be enhanced by a transient increase in the thermodynamic activity of drug in
intestinal colloidal phases due to supersaturation.
The data describing drug absorption from micellar and vesicular colloidal phases suggest that
direct interactions between colloids (or at least the systems examined here) and the absorptive
membrane do not play an important role in cinnarizine absorption. Thus, comparable absorptive
flux (Figure 3.4A – filled symbols) and systemic plasma concentration-time profiles (Figure 3.5 –
bile diverted rats) were observed following jejunal perfusion and intraduodenal infusion of
micelles and vesicles. The thermodynamic activity and Cfree of both systems were held constant,
but the large difference in hydrodynamic radius (9 nm of micelles vs. 443 nm of vesicles) and
higher bile salt, LPC and lipid concentrations in the micellar system (Table 3.1) suggest that the
number of administered micellar particles was substantially higher than that of vesicular particles.
Collision-mediated absorption is highly sensitive to increased particle number, since this increases
the statistical likelihood of collisions and collisional transfer150. As such collisional interactions
did not appear to dictate the degree of drug absorption from micelles and vesicles and instead,
absorption was seemingly controlled by thermodynamic activity (or Cfree). Differences in particle
Chapter 3: Bile-induced supersaturation
134
size might also be expected to alter colloid diffusion across the unstirred water layer (UWL). The
similarity in absorption profiles from micelles and vesicles (Figure 3.4A – filled symbols)
therefore suggests that diffusion across the UWL was either non-limiting, or was not affected by
particle size in this experimental model. Similarly, the rates of replenishment of Cfree (i.e. the rate
of re-establishment of the equilibrium between solubilised and free drug) might be expected to be
different between the two different particle size colloids, however this was presumably
sufficiently fast in both cases to have little impact on drug absorption in the current model.
Data obtained from the lipid uptake receptor inhibition studies further support the notion that
collisional uptake mechanisms have a limited role in drug absorption from these systems. SR-BI,
CD36, and NPC1L1 were examined since they have previously been shown to mediate the cellular
uptake of fatty acids and/or cholesterol61, 66, 210; the absorption of which is also facilitated by
micellar solubilisation. Whether SR-BI, CD36, and NPC1L1 function as authentic transporters
that directly mediate lipid absorption61, 66-68, 210, or whether they act to facilitate intracellular lipid
trafficking or to modify signalling processes that mediate lipid absorption154, 211, 212, or both,
remains contentious but nonetheless all merit examination here. Recent reports also suggest the
involvement of SR-BI, CD36, and NPC1L1 in the absorption of fat-soluble nutrients (such as
carotenoids66, 213, vitamin D214, and vitamin E215), providing further support for a role in drug
absorption. In the current study, consistent with previous reports in vivo, inhibition of NPC1L1
reduced cholesterol absorption (Figure 3.2B)68, and inhibition of CD36 reduced (albeit non-
significantly) the absorption of oleic acid (Figure 3.2C)61. In contrast, inhibition of SR-BI and
CD36 had little impact on cholesterol absorption (Figure 3.2B). Inhibition of SR-BI and CD36
was expected to reduce cholesterol absorption based on previous in vitro studies61, 66, 67. However,
in vivo evidence of a role of SR-BI and CD36 in cholesterol absorption is less clear and, for
example, no significant differences in intestinal cholesterol absorption were reported in SR-BI
Chapter 3: Bile-induced supersaturation
135
knockout vs. wild-type mice66, 157. The lack of effect of SR-BI and CD36 inhibition on cholesterol
absorption in the current study therefore runs contrary to previous in vitro studies but is in
agreement with some previous in vivo data. Inhibition of endocytosis pathways via administration
of monensin, also failed to reduce cholesterol and cinnarizine absorption. Rather, the volume of
distribution and clearance of cholesterol and cinnarizine appeared to be reduced, resulting in
increases in plasma exposure. Since exogenously dosed 14C-cholesterol and cinnarizine are likely
to be present within lipoproteins in the systemic circulation, the changes in systemic disposition of
14C-cholesterol and cinnarizine in the monensin-treated rats may reflect inhibition of receptor-
mediated endocytosis of LDL216.
Inhibition of SR-BI, CD36 and NPC1L1 did not result in significant changes to the systemic
exposure of cinnarizine following intraduodenal infusion of a lipid emulsion formulation in rats
(Figure 3.2A). The data suggest that while lipid colloidal phases may be capable of direct
interaction with lipid receptors (as suggested previously), the transfer of solubilised content into
absorptive cells is likely selective and more applicable to solubilised lipids and nutrients rather
than drugs. Together with the data describing cinnarizine absorption from micelles and vesicles,
the results indicate that cinnarizine absorption from lipid colloidal phases is largely independent of
the physical nature of the infused colloid (realising that in these first experiments cinnarizine was
present at concentration well below the solubilisation limit and therefore under conditions where
solubility/precipitation-mediated events were avoided), is not influenced significantly by common
lipid uptake receptors, and instead appears to be primarily dependent on the free drug
concentration in equilibrium with the solubilised reservoir.
Subsequent studies addressed the possibility that bile secretion may enhance drug absorption via
the induction of supersaturation during the intestinal processing of dietary or formulation-related
Chapter 3: Bile-induced supersaturation
136
lipids. The addition of bile to cinnarizine-loaded micelles led to sustained drug supersaturation
that ultimately led to increased intestinal drug absorption and systemic drug exposure. In contrast,
although addition of bile to cinnarizine-loaded vesicles also led to supersaturation, the metastable
supersaturated state was less stable than that generated by bile addition to micelles, resulting in
more rapid precipitation of solubilised drug and therefore a lack of increase in drug absorption and
systemic drug exposure. The data indicate that bile-mediated dilution of lipid colloidal phases may
represent an endogenous mechanism of supersaturation generation during lipid processing in the
small intestine; and that the transient increase in thermodynamic activity may lead to enhanced
drug absorption. The observations also highlight both the potential for supersaturation to enhance
drug absorption, and the need to achieve an optimal balance between drug supersaturation and
drug precipitation.
Interaction of intestinal colloidal phases with bile leads to dilution, an increase in pH, and an
increase in the concentrations of bile components (bile salts, phosphatidylcholine and cholesterol)
associated with the colloidal species. For a solubilised system above the critical micellar
concentration (CMC), simple 1:1 v/v dilution is expected to reduce the solubilised concentration,
but to maintain total solubilisation capacity (i.e. for the drug concentration to drop by 50% but the
volume to double and therefore for solubilisation capacity to remain unchanged). However, the
data in Table 3.6 show that dilution of the micelles or vesicles with bile or buffer results in a drop
in total solubilisation capacity to only 7-41% of initial. Greater proportional decreases were
apparent for dilution of vesicles (in all cases when compared to micelles), and after dilution with
bile rather than buffer (Table 3.6). The loss of solubilisation capacity on dilution suggests the
likelihood of a phase transition to structures with reduced solubilisation capacity. Although a
complete explanation for these phase transition is not apparent at this time, for the micellar
systems, it may be related to the ability of ionised caprylic acid to self-associate and form fatty
Chapter 3: Bile-induced supersaturation
137
acid micelles at high lipid concentration217. Thus, dilution of medium-chain colloidal phases may
reduce the concentration of caprylic acid below the CMC, leading to a loss of cinnarizine
solubilisation capacity in the micellar phase. For vesicles, previous studies have suggested that
increasing bile concentrations facilitate a vesicular to micellar transition218, 219. Since micelles are
expected to have lower solubilisation capacities for lipophilic drugs than vesicles (micelles are
smaller and less lipid-rich)92, 194, a reduction in cinnarizine solubilisation capacity might therefore
be anticipated when bile is added to model vesicles. In the case of the micelles, therefore, it seems
likely that the addition of bile disrupted the structure of swollen, mixed micelles leading to lower
colloidal lipid content and lower solubilisation capacity. Unfortunately, attempts to quantify
changes to particle size on bile addition were unsuccessful due to high polydispersity. However,
the broad trends observed were consistent with the suggestions above and decreases in particle
size were apparent for vesicles (consistent with initiation of a vesicle to micelle transition) and
increases in particle size for the mixed micelles (consistent with micellar destabilisation and
transformation to less dispersed structures) (data not shown). An additional complexity in these
dilution studies was the realisation that the pH of bile (pH 8.02) is higher than that normally
employed for simulated intestinal fluids39, 220, and therefore incubation of the colloidal phases
(prepared at pH 6.30) with bile increases system pH. Cinnarizine is a weak base with a pKa of
7.47221 and is therefore expected to be less ionised, less soluble and potentially more permeable at
higher pHs. The impact of pH in the current studies was therefore examined by dilution of
micelles and vesicles with pH-corrected bile at pH 6.30. Comparison to the data obtained with bile
at pH 8.02 suggests that the higher pH of bile provides an additional driver for drug
precipitation/supersaturation since the solubility drop was greater after incubation with bile at pH
8.02 vs. bile at pH 6.30.
Chapter 3: Bile-induced supersaturation
138
While dilution of micelles and vesicles with bile resulted in decreases in drug solubilisation
capacity, cinnarizine did not precipitate immediately. A period of drug supersaturation was
evident for both micellar and vesicular systems, although drug precipitation from vesicles was
much more rapid than from micelles. This in turn translated into increases in drug absorption
(Figure 3.4A) and systemic exposure (Figure 3.5A) for the micellar systems. The difference in the
capacity of micelles and vesicles to maintain drug supersaturation (Figure 3.7) may be explained
by the difference in the degree of drug supersaturation induced by dilution with bile. The degree
of supersaturation is described by the supersaturation ratio, which is the ratio of the
(supersaturated) concentration of drug in solution relative to the equilibrium solubility of the drug
in the same system173. Crystallization theory suggests that the thermodynamic drivers of
precipitation from supersaturated solutions increase with increasing supersaturation ratios, as the
likelihood of nucleation and crystal growth increases with increases in (metastable) drug
concentration in solution173, 174. Here, 1:1 v/v addition of bile resulted in the attainment of
cinnarizine supersaturation ratios of 6 and 12, in micelles and vesicles, respectively. Therefore, the
faster rate of drug precipitation from vesicles (when compared to micelles) may be explained by
the higher supersaturation ratio induced by bile addition. It is also possible that micellar structures
are more effective in stabilising supersaturation when compared to vesicular structures, although
this has not been examined explicitly here. Notably, cinnarizine was found to precipitate in the
crystalline form in these experiments (Figure 3.10), precluding the possibility that the enhanced
cinnarizine absorption from micelles (when bile was co-perfused) observed in Figure 3.4A was
due to accelerated cinnarizine dissolution from precipitated amorphous forms.
Chapter 3: Bile-induced supersaturation
139
(A)
(B)
Figure 3.10: Analysis of the cinnarizine precipitate using polarised light microscopy following
precipitation kinetics experiments where fasted rat bile was added in a 1:1 v/v ratio to cinnarizine-
loaded (A) micelles or (B) vesicles. Cinnarizine was loaded into micelles and vesicles at 80%
saturation (~ 0.2 mg/mL). Addition of bile reduced the equilibrium cinnarizine solubilisation
capacity of micelles and vesicles to 14% and 7% of initial, respectively, and triggered cinnarizine
supersaturation and precipitation (see Figure 3.7). The polarised light microscopy images show
that cinnarizine precipitated in the crystalline form after the addition of bile. The crystals obtained
from the vesicles + bile group were significantly smaller than those obtained from the micelles +
bile group, an observation that likely reflects the higher degree of supersaturation stimulated in the
vesicular system (SS ratio = 12) when compared to the micellar system (SS ratio = 6).
The role of bile in enhancing drug absorption from LBF has been reported previously222-224. In the
majority of cases, bile-mediated bioavailability enhancement has been suggested to stem from the
ability of bile to expand the solubilisation reservoir for PWSD in the GI tract. This is typically
assumed to occur via PWSD solubilisation in simple bile micelles, or via the ability of bile to
solubilise lipid digestion products and to generate more complex lipid colloidal phases with
enhanced solubilisation capacities. It seems likely that the ability to solubilise lipid digestion
products and to promote colloid formation remains an integral part of the role of bile in supporting
drug (and lipid) absorption. However, the data described here suggest that continued dilution of
lipid colloidal phases with bile in the small intestinal lumen may also lead to physical changes that
Chapter 3: Bile-induced supersaturation
140
promote drug supersaturation, and ultimately promote drug absorption. In doing so,
supersaturation induction may be a means by which the decrease in thermodynamic activity
inherent in solubilisation is reversed, such that the free concentration of drug available for
absorption is maximised. Thus, a dual role of bile in facilitating drug absorption from LBF may be
conceived (see Figure 3.11). First, bile-mediated solubilisation of lipid digestion products at the
interface of a digesting lipid droplet results in the generation of lipid colloidal phases such as
vesicles and micelles that promote drug solubilisation during lipid digestion. Second, continued
bile-mediated dilution of existing lipid colloidal phases promotes drug supersaturation, and
enhances drug absorption by significantly increasing drug thermodynamic activity in colloidal
phases. The combination of these two highly kinetic events likely contributes to the effective drug
absorption often observed with lipid co-administration, as it affords a means to simultaneously
increase solubilisation capacity and promote thermodynamic activity of co-administered PWSD in
the small intestine.
Supersaturation induction via interaction with bile also provides a means of overcoming the
recently described solubility-permeability interplay observed in studies where PWSD are co-
administered with cosolvents, cyclodextrins or surfactant systems100, 146, 225. In these studies the
authors describe the reduction in thermodynamic activity common to most solubilisation
technologies and show that this off-sets the potential increases in membrane flux that might be
expected by an increase in solubilised drug concentration100, 146. More recent studies by the same
authors have shown that this solubility-permeability interplay can be addressed via the use of
amorphous solid dispersion formulations that stimulate supersaturation, but do not promote
solubilisation147, 226. Here we report that essentially similar outcomes are also possible with
solubilising formulations, when the solubilising formulations contain lipids and when the kinetic
changes that occur in the GI lumen in the presence of bile secretion promote supersaturation. The
Chapter 3: Bile-induced supersaturation
141
current data therefore suggest that endogenous lipid processing pathways provide an exquisitely
sensitive and triggerable supersaturation mechanism that allows drug to remain in a solubilised
state during initial lipid digestion and at high lipid:bile concentration ratios, but that on-going bile
secretion subsequently provides a boost to thermodynamic activity and in doing so supports
enhanced absorption. This is in contrast to other common solubilisation strategies that may not
interact with the dynamic GI environment, or for which interaction with bile typically reduces
thermodynamic activity by increasing solubilisation capacity. Conversely, the ability of LBF to
promote drug solubilisation until drug supersaturation is triggered in the small intestine may
confer an advantage over formulation approaches that only utilise supersaturating strategies, as the
risk of drug precipitation in the GI tract may be reduced by solubilisation within lipid colloidal
phases.
3.6 CONCLUSION
Improved understanding of the mechanism of drug absorption from lipid colloidal phases such as
micelles and vesicles is required to provide a platform for more rational design of lipid-based
formulations. Using medium-chain lipids, we have demonstrated that the absorption of cinnarizine
(a lipophilic, poorly water-soluble drug) may be enhanced when drug supersaturation is generated
during bile-mediated dilution of lipid colloidal phases. Previous studies suggest that a similar
induction of supersaturation may occur as a result of initiation of digestion of some microemulsion
based LBFs21. These observations indicate that supersaturation, and its associated benefits in
enhancing drug absorption, may occur intrinsically during LBF incorporation into endogenous
lipid processing pathways in the small intestine. Future work will be directed toward assessing the
impact of bile dilution on supersaturation tendency, and thus absorption, for an extended range of
PWSD and in a series of different micellar and vesicular colloidal systems.
Chapter 3: Bile-induced supersaturation
142
Figure 3.11: The dual role of bile during lipid digestion and dispersion. (i) Bile-mediated
solubilisation of lipid digestion products at the interface of a digesting oil droplet results in the
generation of lipid colloidal phases such as vesicles and micelles that maintain drug solubilisation
in the small intestine. (ii) The continuing interaction of secreted bile with existing lipid colloidal
phases in the lumen results in progressively less lipid-rich phases with lowered solubilisation
capacity. Thus, on-going bile-mediated dilution of lipid colloidal phases promotes drug
supersaturation and enhances drug absorption by increasing drug thermodynamic activity in
colloidal phases. The combination of the two highly kinetic, bile-mediated events affords a means
to simultaneously increase solubilisation capacity and promote thermodynamic activity of co-
administered drug in the small intestine, and may contribute to the increase in drug absorption
often observed with lipid co-administration. D represents the free concentration of drug available
for absorption. Dss is used to signify the increase in free concentration resulting from bile-
mediated supersaturation that drives increases in drug absorption.
Oildroplet
Common bile duct
DD
D D
D
DD
Multilamellarvesicles
Mixed micelles
Unilamellarvesicles
Simple micelles
(i) (ii)
D
DDD D
DD D
D
D
DDss DssDDss Dss
Absorption Absorption
Pancreatic lipase/co-lipase
Bile micelles
Lipid digestion products
Drug
Supersaturateddrug
Small intestine
Increasing bile dispersion
(ii) Bile-mediated supersaturation(i) Bile-mediated solubilisation
143
Monash University
Declaration for Thesis Chapter 4 Declaration by candidate In the case of Chapter 4, the nature and extent of my contribution to the work was the following:
Nature of contribution Extent of contribution (%)
Concept and design of studies, planning and execution of experimental work, data analysis and interpretation, formulation of conclusions and hypotheses resulting from the relevant studies, drafting and revision of manuscript
70%
The following co-authors contributed to the work. Co-authors who are students at Monash University must also indicate the extent of their contribution in percentage terms:
Name Nature of contribution Extent of contribution (%)
C.J.H. Porter Project supervisor, data and manuscript review NA
N.L. Trevaskis Project co-supervisor, data and manuscript review NA
Candidate’s Signature
Declaration by co-authors The undersigned hereby certify that:
(7) the above declaration correctly reflects the nature and extent of the candidate’s contribution to this work, and the nature of the contribution of each of the co-authors.
(8) they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise;
(9) they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication;
(10) there are no other authors of the publication according to these criteria; (11) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or
publisher of journals or other publications, and (c) the head of the responsible academic unit; and
(12) the original data are stored at the following location(s) and will be held for at least five years from the date indicated below:
Location(s) Monash Institute of Pharmaceutical Sciences
144
Signature 1
Signature 2
145
CHAPTER 4 : THE POTENTIAL FOR
DRUG SUPERSATURATION DURING
INTESTINAL PROCESSING OF LIPID-
BASED FORMULATIONS IS ENHANCED
FOR BASIC DRUGS
Yan Yan Yeap, Natalie L. Trevaskis, Christopher J. H. Porter
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia
Manuscript in submission.
Chapter 4: Supersaturation potential is enhanced for basic drugs
146
4.1 ABSTRACT
Co-administration of poorly water-soluble drugs (PWSD) with dietary or formulation lipids
stimulates the formation of lipid colloidal phases such as vesicular and micellar species, and
significantly expands the drug solubilisation capacity of the small intestine. The mechanism of
drug absorption from the solubilising phases, however, has not been fully elucidated. Recently, we
observed that drug supersaturation may be triggered during endogenous processing of lipid
colloidal phases containing medium-chain lipid digestion products, and that this may represent a
mechanism to enhance absorption and to reverse the reduction in thermodynamic activity inherent
in drug solubilisation. The current studies expand these preliminary findings and explore the
supersaturation tendency of five model PWSD during endogenous processing of intestinal
colloidal phases containing long-chain lipid digestion products. Bile:lipid concentration ratios
progressively increase during colloid transit through the gastrointestinal tract due to biliary
dispersion of lipid digestion products and lipid absorption. Supersaturation potential was therefore
evaluated under conditions of increasing bile and decreasing lipid concentrations and was found to
be greater for basic drugs (cinnarizine and halofantrine) than neutral (fenofibrate and danazol) or
acidic drugs (meclofenamic acid). Assessment of intestinal absorptive flux using rat jejunal
perfusion experiments subsequently showed that the absorption enhancement afforded by bile
dilution was greater for cinnarizine than danazol. The results confirm that bile may play a
significantly greater role in the absorption of weak bases such as cinnarizine from long-chain
intestinal colloids when compared to uncharged molecules such as danazol, and that this
difference reflects a greater propensity for supersaturation as intestinal colloids are dispersed and
diluted by bile. The data also suggest that co-administered digestible lipids may have a particular
utility in enhancing the absorption of poorly water-soluble weak bases.
Chapter 4: Supersaturation potential is enhanced for basic drugs
147
4.2 INTRODUCTION
The oral bioavailability of many poorly water-soluble drugs (PWSD) is low and variable due to
slow dissolution and low solubility in the aqueous gastrointestinal (GI) contents. Lipid-based
formulations (LBF) have proven to be an effective approach in improving the oral bioavailability
of lipophilic PWSD. LBF overcome the dissolution and solubility limitations of PWSD by
presenting drug to the GI tract in a molecularly dispersed form (i.e. in solution in the formulation),
and by stimulating the generation of lipid colloidal phases that promote drug solubilisation and
reduce the risk of drug precipitation2, 76. Classically, therefore, absorption enhancement by LBF
has been considered to result from mechanisms that enhance drug dissolution and expand the
solubilisation capacity of the GI tract.
Recently, interest has increased in the potential for LBF to both promote solubilisation, and
simultaneously to stimulate supersaturation as a means of enhancing drug absorption175, 191. The
dispersion and digestion of LBF typically leads to a reduction in drug solubilisation capacity5, 91,
176, 177, 193, and where precipitation is not immediate, a period of supersaturation results where
solubilised drug concentrations are higher than the equilibrium drug solubility in an identical
colloidal milieu176, 227. Supersaturation has the potential to enhance drug absorption via transient
increases in thermodynamic activity. Conversely, however, supersaturation is also a driver of drug
nucleation and precipitation, processes that break the continuity of solubilisation and commonly
reduce drug absorption. Balancing supersaturation and precipitation is therefore a key design
element of LBF.
The digestion of triglycerides or partial glycerides in LBF, or dietary lipids, liberates amphiphilic
digestion products including fatty acids and monoglycerides. Digestion products so formed
combine with biliary components to generate a range of lipid colloidal phases in which co-
Chapter 4: Supersaturation potential is enhanced for basic drugs
148
administered PWSD may be solubilised. At the surface of a digesting oil droplet, digestion
products accumulate and ultimately slough off to form lipid-rich dispersed liquid crystalline
phases such as lamellar, cubic and hexagonal phases with relatively large particle sizes and high
drug solubilisation capacities39-41. On dilution with intestinal fluids these colloidal structures
become smaller and less lipid-rich and typically have lower drug solubilisation capacities39. Drug
absorption is thought to occur from these smaller, more dilute lipid colloidal phases such as
unilamellar vesicles and mixed micelles, although the mechanisms involved in drug absorption
from these species have not been fully elucidated2, 76, 101.
Recently we described the ability of bile to promote drug absorption from lipid colloidal phases in
the small intestine. Contrary to traditional solubilisation paradigms, the addition of bile to
intestinal colloidal phases containing digestion products of medium-chain (MC) triglycerides
enhanced drug absorption in vivo by reducing the solubilisation capacity of the colloidal phases
for a model PWSD (cinnarizine), thereby generating a transiently supersaturated state (Chapter 3).
These initial findings were suggested to provide a means to reverse the reduction in drug
thermodynamic activity, and therefore absorptive drug flux that is inherent in solubilisation-
mediated formulation approaches100, 146.
The current study expands this preliminary evaluation of supersaturation events during the LBF
processing and has examined the impact of bile on the potential for drug supersaturation in
intestinal colloidal phases containing digestion products of long-chain (LC) triglycerides.
Importantly the work also delineates the relative sensitivities to supersaturation for weak bases,
acids and neutral compounds and shows that weak bases may be particularly suited to
supersaturation-mediated absorption enhancement from LBF.
Chapter 4: Supersaturation potential is enhanced for basic drugs
149
4.3 METHODS
4.3.1 Materials
Cinnarizine, flunarizine dihydrochloride, fenofibrate, meclofenamic acid, progesterone,
ammonium sulphate, sodium taurocholate, sodium taurodeoxycholate, sodium glycocholate,
sodium glycochenodeoxycholate, cholesterol, L-α-lysophosphatidylcholine (LPC, from egg yolk),
oleic acid, monoolein, sodium chloride (NaCl), sodium dodecyl sulphate (SDS), formic acid and
ammonium formate were obtained from Sigma-Aldrich, Australia. Sodium
taurochenodeoxycholate, sodium glycodeoxycholate, ortho-phosphoric acid 85% (H3PO4), sodium
hydroxide pellets (NaOH), tert-butyl methyl ether (TBME), glacial acetic acid and absolute
ethanol were from Merck, Australia. Disodium hydrogen orthophosphate (Na2HPO4), sodium
dihydrogen orthophosphate (NaH2PO4.2H2O) and ammonium dihydrogen orthophosphate
(NH4H2PO4) (Ajax Finechem, Australia), oleic acid, [9,10-3H(N)] (60 Ci/mmol) (American
Radiolabeled Chemicals, MO, USA), danazol (Sterling Pharmaceuticals, Australia), halofantrine
base (GlaxoSmithKline, King of Prussia, PA, USA), heparin sodium injection BP (1000 I.U./mL,
Hospira, Australia), xylazine (100 mg/mL, Troy Laboratories, Australia), acepromazine (10
mg/mL, Ceva Delvet, Australia), ketamine (100 mg/mL, Provet, Australia) and pentobarbitone
sodium (325 mg/mL, Virbac, Australia) were obtained from listed suppliers. Acetonitrile,
methanol and chloroform used were analytical reagent grade. Water was obtained from a
Millipore milliQ Gradient A10 water purification system (Millipore, MA, USA).
4.3.2 Experimental outline
Nine colloidal systems designed to mimic the colloidal phases that form in the small intestine
during the digestion of LC triglycerides were prepared. The systems consisted of triolein digestion
products (oleic acid and monoolein) solubilised in bile components (bile salts,
Chapter 4: Supersaturation potential is enhanced for basic drugs
150
lysophosphatidylcholine and cholesterol). The concentrations of lipid and bile components in the
systems were varied systematically, as shown in Table 4.2. The equilibrium solubility of five
lipophilic PWSD (two weak bases and two neutral drugs with differing triglyceride solubilities,
and one weak acid) were determined in each of the colloidal systems, to assess drug solubility
trends as a function of colloidal bile salt concentration [bile] and colloidal lipid concentration
[lipid]. The melting points, octanol-water distribution coefficients, pKa, molecular weights,
soybean oil and oleic acid solubilities of the five drugs are shown in Table 4.3.
During bile-mediated dilution of lipid colloidal phases in the small intestine, increases in the
concentration of biliary components and decreases in lipid concentrations occur in tandem. Under
these circumstances, drug solubilisation capacity may increase (decreasing thermodynamic
activity at constant drug concentration) or decrease (invoking the potential for precipitation or
supersaturation where precipitation is not immediate). The net effect is dictated by the magnitude
of the change in solubility expected by increasing [bile] and decreasing [lipid]. Under conditions
of lipid absorption, lipids from intestinal colloids are also removed via absorption, further
decreasing lipid concentrations. In the second part of the current studies, two model drugs with
markedly different solubility relationships with [bile] and [lipid] were selected for further study.
For these compounds, absorption was assessed from a model long-chain colloidal system in situ
and in vivo, to determine the potential for endogenous lipid processing pathways to generate drug
supersaturation, and to enhance drug absorption, from intestinal colloidal phases containing LC
lipid digestion products.
Chapter 4: Supersaturation potential is enhanced for basic drugs
151
4.3.3 Preparation of model intestinal colloidal phases containing long-chain
lipids
Colloidal systems containing triolein digestion products (oleic acid and monoolein) solubilised in
simulated endogenous intestinal fluid (SEIF) were prepared. SEIF comprised the six most
prevalent bile salts in human bile199, lysophosphatidylcholine (LPC), and cholesterol. The total
bile salt:LPC:cholesterol molar ratio was kept constant at 16:4:1, reflecting known ratios within
fasted human bile200, 201. The bile salts employed were glycocholate, glycodeoxycholate,
glycochenodeoxycholate, taurocholate, taurodeoxycholate, and taurochenodeoxycholate, at a
molar ratio of 4.0:2.8:4.0:2.0:1.2:2.0. The concentration ratios of the bile salts were chosen based
on average concentrations of the six most prevalent bile salts found in human bile199. The oleic
acid:monoolein molar ratio was kept at 2:1, reflecting the ratio of digestion products expected
from digestion of 1 mole of triolein. The concentrations of lipid and bile components in the
systems were varied systematically, according to the compositions shown in Table 4.2. Bile salt
concentrations of 4–16 mM were selected to span the typical concentration range seen in vivo76
and previously used in simulated intestinal fluids220; lipid concentrations were chosen to reflect
the solubilised concentrations that might be attained in the intestine following the digestion of LC
triglycerides91. Based on physical examination and the maintenance of consistent drug solubilising
capacities, colloids were stable for 5 days after preparation.
SEIF 16 (16 mM total bile salt:4 mM LPC:1 mM cholesterol) was prepared in 50 mL batches.
Briefly, LPC and cholesterol were dissolved in 2 mL chloroform in a round bottom flask, followed
by solvent evaporation under vacuum. The thin film formed was reconstituted with buffered bile
salt solution (consisting 16 mM total bile salt, 18 mM NaH2PO4.2H2O, 12 mM Na2HPO4, 92 mM
NaCl), vortexed for 1 min, and allowed to equilibrate at room temperature overnight. When SEIF
8 (8 mM total bile salt:2 mM LPC:0.5 mM cholesterol) and SEIF 4 (4 mM total bile salt:1 mM
Chapter 4: Supersaturation potential is enhanced for basic drugs
152
LPC:0.25 mM cholesterol) were required, SEIF 16 was diluted 2-fold and 4-fold, respectively,
with buffer (18 mM NaH2PO4.2H2O and 12 mM Na2HPO4, 108 mM NaCl). The colloidal systems
were prepared in 10 mL batches by adding appropriate masses of oleic acid and monoolein into
SEIF, followed by a 1-min vortex. The mixture was then ultrasonicated (30 sec continuous
ultrasonication followed by pulsatile, 1 sec-on/1 sec-off ultrasonication for 5 min) with a Misonix
XL 2020 ultrasonic processor (Misonix, Farmingdale, NY, USA) equipped with a 3.2-mm
microprobe tip running at an amplitude of 240 µm and a frequency of 20 kHz. The colloids were
subsequently adjusted to pH 6.30 with H3PO4 solution and/or NaOH solution.
For the preparation of drug-loaded model colloids (for in situ jejunal perfusion and in vivo
bioavailability studies), cinnarizine was first dissolved in oleic acid at a concentration of 61 mg/g
and 115 mg/g prior to generating colloidal systems as described above, resulting in colloids
containing 65 µg/mL cinnarizine (representing ~ 40% of cinnarizine saturated solubility in the
colloidal system, solubility determined as described below) and 130 µg/mL (~ 80% saturated
solubility), respectively. Due to the lower solubility of danazol in oleic acid, colloids containing
danazol were drug-loaded by spiking 50 µL of 1.4 mg/mL or 2.8 mg/mL danazol in ethanol
solution into 10 mL blank model colloids, followed by vortexing for 1 min, and equilibration for 1
h to prepare colloids containing 7 µg/mL (~ 40% saturated solubility) and 14 µg/mL (~ 80%
saturated solubility) danazol, respectively. As a final step, trace amounts of 3H-oleic acid (0.25
µCi/mL in jejunal perfusion studies; 1 µCi/mL in bioavailability studies) were also added to the
drug-loaded colloids, followed by a 1-min vortex. The total ethanol concentration in the colloids
was ≤ 1% v/v. Drug and oleic acid concentrations in the colloidal systems were confirmed by
HPLC and scintillation counting immediately prior to use.
Chapter 4: Supersaturation potential is enhanced for basic drugs
153
4.3.4 Equilibrium solubility of drugs in oleic acid and soybean oil
Excess solid drug (cinnarizine (CIN), halofantrine (HF), fenofibrate (FF), danazol (DAN),
meclofenamic acid (MFA)) was added to 2 mL glass vials containing approximately 0.5 g oleic
acid or soybean oil. Vials were briefly vortexed, incubated at 37 °C, and samples taken every 24 h
over a period of 120 h. During sampling, vials were centrifuged (2,200 xg, 10 min, 37 °C), and 1-2
drops of supernatant accurately weighed into 5 mL volumetric flasks using glass Pasteur pipettes.
After sampling, vials were re-vortexed such that undissolved drug particles were re-suspended in
the lipid slurry. The 5 mL volumetric flasks containing accurately weighed supernatant samples
were made up to volume with chloroform-methanol (2:1, v/v) and briefly vortexed. 50 µL aliquots
were then diluted > 10-fold with respective mobile phases (Table 4.1) and analysed for drug
content via HPLC (HPLC assay conditions for CIN, HF, FF, DAN and MFA in LC colloids are
reported in Table 4.1). Equilibrium solubility was defined when drug concentrations in
consecutive samples varied by ≤ 5%, and was determined on three separate occasions.
Chapter 4: Supersaturation potential is enhanced for basic drugs
154
Table 4.1: HPLC assay conditions for cinnarizine (CIN), halofantrine (HF), fenofibrate (FF), danazol
(DAN) and meclofenamic acid (MFA) in long-chain colloidal systems.
Drug Stationary phase
Mobile phase Flow rate (mL/min)
Detection Retention time (min)
Validated concentration
range
CIN^ Waters Symmetry®
C18, 5 µm, 3.9 x 150 mm
50% v/v Acetonitrile : 50%
v/v 20 mM NH4H2PO4
1.0
Fluorescence
λ = 249/311 nm
5.7 20–1000 ng/mL
HF Phenomenex Luna® C8(2), 5 µm, 4.6 x 250
mm
75% v/v Acetonitrile : 25% v/v H2O (0.2% w/v SDS & 0.2% v/v
acetic acid)
1.5 UV
λ = 254 nm
4.2 0.5–25 µg/mL
FF Waters XbridgeTM C18, 5 µm, 4.6 x 150
mm
80% v/v Acetonitrile : 20%
v/v H2O (total 0.01% v/v formic acid)
1.0 UV
λ = 286 nm
3.9 0.5–25 µg/mL
DAN# Waters XbridgeTM C18, 5 µm, 4.6 x 150
mm
75% v/v Methanol : 25% v/v H2O
1.0 UV
λ = 286 nm
4.4
0.25–50 µg/mL
MFA Phenomenex Luna® C8(2), 5 µm, 4.6 x 250
mm
80% v/v Acetonitrile : 20% v/v 10 mM H3PO4
1.0 UV
λ = 285 nm
4.8 0.5–25 µg/mL
^ CIN in long-chain colloidal systems was assayed via HPLC as described above; while CIN in plasma was assayed via HPLC using a validated extraction procedure (with flunarizine as an internal standard) and slightly modified HPLC conditions as reported previously43.
# DAN in long-chain colloidal systems was assayed via HPLC as described above; while DAN in plasma was assayed via LC-MS using a validated precipitation procedure (with progesterone as an internal standard) and slightly modified HPLC conditions as reported previously228.
Chapter 4: Supersaturation potential is enhanced for basic drugs
155
4.3.5 Equilibrium solubility of drugs in long-chain colloids
Excess solid drug (CIN, HF, FF, DAN, MFA) was added to 2 mL of long-chain colloids (prepared
as above) in glass vials. Vials were briefly vortexed, incubated at 37 °C, and samples taken every
24 h over a period of 120 h. During sampling, vials were centrifuged (2,200 xg, 10 min, 37 °C), 50
µL of supernatant removed, and vials re-vortexed. 50 µL aliquots were then diluted > 10-fold with
respective mobile phases (see Table 4.1) and analysed for drug content via HPLC (HPLC assay
conditions for CIN, HF, FF, DAN and MFA in LC colloids are reported in Table 4.1). Equilibrium
solubility was defined when drug concentrations in consecutive samples varied by ≤ 5%, and was
determined on three separate occasions. Equilibrium solubilities of CIN and DAN were also
determined after 1:1 v/v addition of fasted rat bile (obtained from donor animals as described
below) or buffer (18 mM NaH2PO4.2H2O, 12 mM Na2HPO4, 108 mM NaCl; adjusted to pH 6.30)
to the colloidal phases.
4.3.6 Kinetics of cinnarizine precipitation
The kinetics of cinnarizine precipitation was monitored after addition of fasted rat bile (obtained
from donor animals as described below) to model colloids, to determine whether a period of drug
supersaturation preceded drug precipitation. In a temperature (37 ºC) and stirring rate-controlled
vessel, 2.5 mL of bile was added to 2.5 mL model colloids containing 130 µg/mL cinnarizine (~
80% saturated solubility). A 1:1 v/v addition ratio of bile to colloids was used based on the known
rates of bile flow in rats in vivo (~ 1.2 ± 0.05 mL/h; mean ± SEM; n = 22) and the volume of a
typical oral dose in rats (~ 1 mL/h). Samples (100 µL) were taken before the addition of bile, and
at 1, 10, 20, 30, 40, 50, 60, 80, 100, 120 min after bile addition. Samples were immediately
centrifuged (2,200 xg, 5 min, 37 °C) to separate precipitated drug, and 50 µL of supernatant
assayed for drug content. The proportion of the initial solubilised cinnarizine concentration that
remained solubilised after bile addition was assessed as the percent of the drug mass remaining in
Chapter 4: Supersaturation potential is enhanced for basic drugs
156
solution (i.e. the concentration in the supernatant multiplied by the volume remaining in vessel
after sampling) relative to the total drug mass in the vessel at each time point.
4.3.7 Solid-state analysis of the cinnarizine precipitate
Selected cinnarizine pellets from the precipitation kinetics experiments were analysed using a
Zeiss Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with crossed polarising
filters. At the end of the precipitation kinetics experiments, 1.5 mL of remaining bile + colloid
mixture were centrifuged (2,200 xg, 10 min, 37 °C), the supernatant was discarded, and a small
amount of pellet was carefully placed on a microscope slide. Samples were analysed under cross-
polarised light, and images were recorded using a Canon PowerShot A70 digital camera (Canon,
Tokyo, Japan).
4.3.8 Animals
Animal care and anaesthesia have been described in Section 2.2.2.2. All animals were fasted
overnight (12–18 h) prior to surgery. At the end of all experiments, rats were euthanized via an
intravenous or intracardiac injection of 100 mg sodium pentobarbitone.
4.3.9 Surgical procedures
4.3.9.1 Fasted rat bile collection
The bile duct was cannulated near the hilum of the liver (where the duct is free of pancreatic tissue)
in order to facilitate the collection of bile fluid without contamination with exocrine pancreatic
secretions203. The bile duct and jugular vein were cannulated as described previously12, 229. Rats
were rehydrated via a continuous infusion of saline (1.5 mL/h) into a cannula inserted in the right
jugular vein, and bile continuously collected for 5 h. The concentration of total bile salt in
Chapter 4: Supersaturation potential is enhanced for basic drugs
157
collected bile was assayed using a validated enzymatic colorimetric assay (Total Bile Acids kit
#431-15001; Wako Pure Chemical Industries, Osaka, Japan) on a plate reader (Fluostar Optima
plate reader, BMG Lab technologies, Germany) measuring absorbance at a wavelength of 540 nm.
In all subsequent experiments, bile was stored at 4°C (and allowed to warm up to ambient
temperature before use) and used within 24 h of collection.
4.3.9.2 Single-pass rat jejunal perfusion
The model employed to assess flux across rat jejunum involved in situ perfusion (single-pass) of
an isolated jejunal segment and simultaneous blood collection from the corresponding mesenteric
vein branch. The surgical procedures for the setup of the single-pass rat jejunum perfusion model
have been described in Section 2.2.2.3.
4.3.9.3 Cinnarizine bioavailability studies following intraduodenal
administration
The surgical procedures for the conduct of bioavailability studies included cannulations of the
right carotid artery, right jugular vein, duodenum (1 cm below pylorus), and common bile duct
(only for bile-diverted rats). The surgical procedures for the cannulations have been described in
Section 2.3.2.
4.3.10 In situ single-pass rat jejunum perfusion
After surgery, animals were equilibrated for 30 min, during which time heparinised donor rat
blood was infused via the jugular vein as described previously12, 229. During the equilibration
period, blood from the cannulated mesenteric vein (~ 0.3 mL/min) was collected to enable re-
infusion via the jugular vein. Perfusion buffer was pumped through the jejunal segment at a rate of
Chapter 4: Supersaturation potential is enhanced for basic drugs
158
0.1 mL/min and outflowing buffer discarded to waste. The exposed jejunal segment was kept
moist by covering with saline-soaked gauze throughout the experiment.
In all experiments to assess cinnarizine intestinal absorptive flux, the inflow concentration of
cinnarizine was constant at 65 µg/mL. Therefore, in experiments where model colloids were
perfused alone, cinnarizine was loaded into the perfusate at 65 µg/mL (~ 40% saturated solubility).
In experiments where model colloids were co-perfused in a 1:1 v/v ratio with a secondary
perfusate of bile, cinnarizine was loaded into the primary perfusate at 130 µg/mL (~ 80% saturated
solubility), such that 1:1 v/v dilution led to a final inflow perfusate concentration of 65 µg/mL. In
this way the concentration of cinnarizine on entry into the intestine was corrected for biliary
dilution effects, realising that the intent of the current studies was to evaluate whether bile addition
resulted in additional changes to drug solubilisation capacity and thermodynamic activity beyond
simple dilution.
Similarly, in all experiments that assessed danazol intestinal absorptive flux, the concentration of
danazol flowing into the perfused jejunum segment was held at 7.2 µg/mL. Therefore, in
experiments where model colloids were perfused alone, danazol was loaded into the perfusate at
7.2 µg/mL (~ 40% saturated solubility). In experiments where model colloids were co-perfused in
a 1:1 v/v ratio with a secondary perfusate of bile, danazol was loaded into the primary perfusate at
14.4 µg/mL (~ 80% saturated solubility), such that 1:1 v/v dilution led to a final inflow perfusate
concentration of 7.2 µg/mL.
Perfusate flow was maintained at 0.1 mL/min in all experiments to minimise variations in the
thickness of the unstirred water layer that may influence drug flux206. For experiments where 1:1
v/v co-perfusion was required, model colloids and bile were individually pumped at 0.05 mL/min,
Chapter 4: Supersaturation potential is enhanced for basic drugs
159
and mixed via a three-way “T” connector immediately prior to entry into the jejunal segment,
providing a total perfusate flow of 0.1 mL/min. Perfusate was sampled at t = 0 to confirm drug and
3H-oleic acid concentrations. After this time, the outflowing perfusate was continuously collected
for 10 min intervals, and briefly vortexed before samples were taken for analysis of drug and 3H-
oleic acid content. For experiments where drug supersaturation was generated (i.e. cinnarizine in
model colloids + bile experiments), perfusate samples were briefly vortexed, and samples taken
before and after centrifugation (2,200 xg, 2 min) to obtain an indication of the degree of drug
precipitation within the jejunal segment. Blood draining the perfused jejunal segment was
collected at 5 min intervals, plasma separated by centrifugation (10,000 xg, 5 min), and samples
taken for analysis of drug and 3H-oleic acid concentrations.
4.3.11 Cinnarizine bioavailability after intraduodenal infusion
A 30 min-equilibration period was allowed between the end of surgery and drug dosing. To
examine the impact of bile-induced drug supersaturation on cinnarizine absorption, studies were
conducted in bile-intact or bile-diverted rats. Model colloids containing 130 µg/mL cinnarizine (~
80% saturated solubility) were infused into the duodenum of rats at a rate of 1.5 ml/h for 2 h.
Following infusion of the model colloids, saline was infused at a rate of 1.5 mL/h for 10 min to
flush remaining formulation in the tubing into the duodenum. Blood samples (0.3 mL) were
collected via the carotid artery cannula up to 8 h after infusion initiation into tubes containing 3
I.U. heparin. The sampling intervals were t = 0, 1, 1.5, 2, 3, 4, 6, 8 h. After each blood sample was
taken, the cannula was flushed with 0.3 mL of 2 I.U./mL heparinised saline to ensure cannula
patency, and to replace the volume of blood removed. Plasma was separated by centrifugation
(10,000 xg, 5 min) to enable analysis of plasma drug and 3H-oleic acid concentrations as below.
Chapter 4: Supersaturation potential is enhanced for basic drugs
160
4.3.12 Analytical procedures
4.3.12.1 Sample preparation and HPLC assay conditions for CIN, HF, FF, DAN,
MFA
The HPLC assay conditions for CIN, HF, FF, DAN and MFA in LC colloids are reported in Table
4.1. LC colloid samples were prepared for HPLC assay by a minimum of 5-fold dilution with the
respective mobile phases (see Table 4.1). The injection volume for all samples was 50 µL.
Replicate analysis of n = 4 quality control samples revealed acceptable accuracy and precision (±
10%, ± 15% at the limit of quantification) for the reported concentration ranges.
CIN plasma samples were prepared for HPLC using a validated extraction procedure, with
flunarizine as an internal standard, as reported previously43. DAN plasma samples were prepared
for LC-MS using a validated precipitation procedure, with progesterone as an internal standard, as
reported previously228. The assay conditions for CIN and DAN in plasma are described
elsewhere227, 228. Replicate analysis of n = 4 quality control samples revealed acceptable accuracy
and precision (± 10%, ± 15% at the limit of quantification) for CIN plasma concentrations
between 10–320 ng/mL, and DAN plasma concentrations between 5–250 ng/mL.
4.3.12.2 Scintillation counting
Quantification of 3H-oleic acid in the perfusate and plasma was performed via scintillation
counting on a Packard Tri-Carb 2000CA liquid scintillation analyser (Packard, Meriden, CT,
USA). Perfusate samples (100 µL) and plasma samples (50 µL for in vivo studies, 200 µL for in
situ studies) were added to 2 mL Irga-safe Plus scintillation fluid followed by a 10 sec vortex.
Samples were corrected for background radioactivity by the inclusion of a blank sample in each
run.
Chapter 4: Supersaturation potential is enhanced for basic drugs
161
4.3.12.3 Blood:plasma ratio determination for CIN, DAN, oleic acid
The blood:plasma ratio for cinnarizine, danazol, and oleic acid were determined by spiking 0.5
mL blank blood with known amounts of compound to achieve low, medium, and high
concentrations (in triplicate). Plasma was separated by centrifugation (10,000 xg, 5 min) and
plasma drug concentration assayed by HPLC (cinnarizine), LC-MS (danazol), or scintillation
counting (oleic acid). The blood:plasma ratio was calculated from the ratio of known
concentration in spiked blood to the concentration measured in plasma separated from spiked
blood. The mean blood:plasma ratio was subsequently used to convert plasma concentrations to
blood concentrations in perfusion experiments, enabling quantification of total transport into
mesenteric blood.
4.3.12.4 Calculations
In the single-pass rat jejunum perfusion model, permeability coefficients were calculated from the
flux data obtained after attainment of steady state drug transport into mesenteric blood. Two
apparent permeability coefficients (Papp) were calculated as described previously184:
′Disappearance′P . ln Equation 4.1
′Appearance′P∆
∆.
Equation 4.2
where ‘Disappearance’ Papp is the apparent permeability coefficient calculated from drug loss
from the perfusate (cm/sec); ‘Appearance’ Papp is the apparent permeability coefficient calculated
from drug appearance in the mesenteric blood (cm/sec); Q is the perfusate flow rate (mL/sec); A is
the surface area of the perfused jejunal segment (cm2), which is calculated by multiplying the
Chapter 4: Supersaturation potential is enhanced for basic drugs
162
diameter by the length of the perfused intestinal segment as described previously207; C1 is the
average steady state drug concentration exiting the perfused jejunal segment (ng/mL); C0 is the
drug concentration entering the jejunal segment (ng/mL); ∆MB/∆t is the average rate of drug mass
appearance in mesenteric blood at steady state (ng/s); and <C> is the logarithmic mean drug
concentration in the lumen (ng/mL), where <C> = (C1 – C0) / (ln C1 – ln C0).
4.3.12.5 Pharmacokinetic analysis
The maximum plasma concentration (Cmax), the time to reach Cmax (Tmax) and the area under the
plasma concentration-time curve from time zero to the last measured concentration (AUC0-8 h)
were calculated using WinNonLin version 5.3 (Pharsight Inc., Apex, NC, USA).
4.3.13 Statistical analysis
Results were analysed using Student’s t test. A P value of < 0.05 was considered to be a
significant difference. Analyses were performed using SPSS v19 for Windows (SPSS Inc.,
Chicago, IL, USA).
4.4 RESULTS
4.4.1 Trends in drug solubility in long chain lipid-based colloids as a
function of bile and lipid concentration are different for basic, neutral and
acidic drugs
The solubilities of cinnarizine (CIN), halofantrine (HF), fenofibrate (FF), danazol (DAN) and
meclofenamic acid (MFA) in the lipid colloidal systems examined are presented in Table 4.2. The
solubility enhancement (or reduction) resulting from increasing bile concentrations (holding lipid
Chapter 4: Supersaturation potential is enhanced for basic drugs
163
concentration constant), and solubility reduction caused by decreasing lipid concentration (holding
bile concentration constant), are shown in Figure 4.1 and Figure 4.2, respectively. Percent changes
in solubility were calculated relative to low bile concentration (i.e. 4 mM) and high lipid
concentration (i.e. 7.08 mM) in Figure 4.1 and Figure 4.2 respectively, to assess likely changes in
drug solubilisation during bile dilution (where bile concentration increases and lipid concentration
decreases in tandem) and lipid absorption (where lipid concentration decreases).
Table 4.2: Equilibrium solubility (37 °C) of cinnarizine (CIN), halofantrine (HF), fenofibrate
(FF), danazol (DAN) and meclofenamic acid (MFA) in long-chain colloidal systems.
[Lipid] #
(mM) ##
[Bile] ^
(mM) ^^
Saturated solubility (µg/mL)
CIN HF FF DAN MFA
1.77 4 49 330 13 12 85
8 19 267 15 18 90
16 13 250 21 29 130
3.54 * 4 * 163 704 37 18 62
8 84 610 32 24 86
16 35 471 37 35 157
7.08 4 357 1270 84 29 84
8 261 1276 71 36 107
16 144 1023 63 48 268
# Lipid refers to oleic acid and 1-monoolein in 2:1 molar ratio ## Concentration refers to concentration of oleic acid ^ Bile contained bile salts, lysophosphatidylcholine, cholesterol in 16:4:1 molar ratio. The bile salt mixture comprised 25 mol% sodium glycocholate, 17.5 mol% sodium glycodeoxycholate, 25 mol% sodium glycochenodeoxycholate, 12.5 mol% sodium tauroocholate, 7.5 mol% sodium taurodeoxycholate and 12.5 mol% sodium taurochenodeoxycholate
^^ Concentration refers to concentration of total bile salt
* Model long-chain colloidal system used in in situ and in vivo experiments is highlighted
Chapter 4: Supersaturation potential is enhanced for basic drugs
164
CIN
HF
FF
DAN
MFA
Figure 4.1: Percent change in cinnarizine (CIN), halofantrine (HF), fenofibrate (FF), danazol
(DAN) and meclofenamic acid (MFA) solubility stimulated by increasing concentrations of bile at
varying lipid concentrations. Values calculated using raw data in Table 4.2. Solubility change was
calculated relative to the solubility in 4 mM bile at each lipid concentration.
The different drugs were found to have very different solubility behaviours with respect to bile
concentration in the prepared colloidal systems (Table 4.2, Figure 4.1). The solubilisation of the
weak bases (CIN and HF) was significantly reduced by increasing bile concentration (i.e. an
inverse relationship was apparent between solubility and bile concentration – as exemplified by
negative slopes in Figure 4.1); whilst the solubilisation of the neutral drugs (DAN and FF) and the
weak acid (MFA) increased in most cases in the presence of increasing bile concentrations (i.e. a
direct relationship between solubility and bile concentration - as exemplified by positive slopes in
Figure 4.1). The rank order by which bile ‘enhanced’ the solubilisation of drugs was CIN < HF <
[Bile] (mM)4 8 164 8 164 8 164 8 164 8 16
Sol
ubili
tyen
hanc
emen
t (%
)
020406080
100
4 8 164 8 164 8 164 8 164 8 160
20406080
100
4 8 164 8 164 8 164 8 164 8 160
100
200
300
4 8 164 8 164 8 164 8 164 8 160
100
200
300
4 8 164 8 164 8 164 8 164 8 160
100
200
300
Chapter 4: Supersaturation potential is enhanced for basic drugs
165
FF < DAN < MFA, and appeared to be inversely correlated to drug solubility in oleic acid, such
that drugs that were most soluble in oleic acid (see Table 4.3) had their solubility reduced the most
by increases in bile concentration, whereas those with lower oleic acid solubility had their
solubilities increased by increases in bile concentration. The magnitude of the change was
seemingly greater for drugs with lower triglyceride (TG) solubility, for example, CIN
solubilisation was reduced to a greater extent when compared to HF; and the solubilisation of
DAN was increased to a greater extent when compared to FF.
In contrast, the solubility relationship between the drugs and colloidal lipid concentrations were
more predictable (Figure 4.2). With the exception of MFA at lower bile concentrations, the
solubility of all the drugs in the model colloids decreased with decreasing lipid concentration (i.e.
all drugs demonstrated a direct relationship between solubility and lipid concentration). The
proportional decrease in drug solubility varied significantly across the drug types, however, and
was greatest for CIN (which reduced to ~ 7% of the solubility value in colloids containing 7.08
mM lipid when the lipid content was dropped to 1.77 mM), followed by FF (which reduced to
15%), HF (21%), DAN (41%), and MFA (49%). The rank order by which decreasing lipid
concentration reduced the solubilisation of drugs was therefore CIN > HF ≈ FF > DAN > MFA,
and reflected drug solubility in oleic acid, such that drugs with the highest solubility in oleic acid
(see Table 4.3) had their solubility reduced the most by decreasing lipid (oleic acid and monoolein)
concentration.
Chapter 4: Supersaturation potential is enhanced for basic drugs
166
CIN
HF
FF
DAN
MFA
Figure 4.2: Percent solubility reduction of cinnarizine (CIN), halofantrine (HF), fenofibrate (FF),
danazol (DAN) and meclofenamic acid (MFA) by decreasing lipid concentration, at various bile
concentrations. Values calculated using raw data in Table 4.2. Solubility reduction was relative to
the solubility in colloids containing 7.08 mM oleic acid at each bile concentration. Blue solid lines
(without symbols) represent theoretical changes in solubility upon simple dilution.
[Lipid] (mM)7.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.77
Sol
ubili
tyre
duct
ion
(%)
020406080
100
7.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.770
20406080
100
7.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.770
20406080
100
7.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.770
20406080
100
7.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.777.08 3.54 1.770
20406080
100
Chapter 4: Supersaturation potential is enhanced for basic drugs
167
Table 4.3: Melting points, Log DpH7.5 (octanol-water distribution coefficients at pH 7.5), pKa,
molecular weights, and lipid solubilities of cinnarizine (CIN), halofantrine (HF), fenofibrate (FF),
danazol (DAN) and meclofenamic acid (MFA).
Drug Drug type Tm* (°C)
Log DpH7.5 pKa Molecular weight
Solubility in soybean oil (mmol/mol)
Solubility in oleic acid
(mmol/mol)
CIN Weak base 120$ 5.48** 1.95^^
, 7.47§§ 368.52 63.9** 264.0
HF Weak base 83# 8.85** 5.58*** 536.88 82.4** 111.9
FF Neutral 81^ 5.24$$ n/a 360.83 197.7 81.6
DAN Neutral 229$ 4.53** n/a 337.46 10.1** 4.8
MFA Weak acid 257-260§ 3.61##
3.76$$$ 296.15 19.4 7.8
n/a – not applicable
* Tm denotes melting point $ Data from Bergström et al.230 # Data from McIntosh et al.231 ^ Data from Van Speybroeck et al.232 § Data from Sanphui et al.233
** Data from Kaukonen et al.91 $$ Data quoted in Guichard et al.234 ## Calculated using Advanced Chemistry Development (ACD/Labs) Software Solaris V9.12 (1994–2006
ACD/Labs) ^^ Data quoted in Gu et al.235 §§ Data from Peeters236
*** Data from Khoo et al.237 $$$ Data quoted in Marriner et al.238
Chapter 4: Supersaturation potential is enhanced for basic drugs
168
4.4.2 The addition of rat bile to drug-loaded model colloids reduces
cinnarizine solubility and promotes supersaturation, but increases danazol
solubilisation
Cinnarizine (CIN) and danazol (DAN) were selected as model drugs for more detailed assessment
of the impact of solubilisation properties on in situ and in vivo absorption. These compounds were
chosen since bile had opposing effects on their colloidal solubility properties (Figure 4.1,Table
4.2), and lipid concentration had a greater effect on the solubility of CIN when compared to DAN
(Figure 4.2,Table 4.2). To explore the impact of dilution with bile on CIN and DAN solubilisation
(rather than dilution with model intestinal fluids in Figure 4.1), donor bile was collected from rats,
and the change in CIN and DAN solubilisation capacity in model colloids assessed before and
after 1:1 v/v addition of rat bile (Figure 4.3A). 1:1 v/v addition of donor bile decreased CIN
solubility from 157.7 µg/mL to 9.2 µg/mL and therefore, as a proportion of initial, reduced CIN
solubilisation capacity from 100% to 12% (taking into account the 2-fold increase in volume after
the addition of bile). In contrast, for DAN, combination of the model colloids with bile in a 1:1 v/v
ratio increased DAN solubility from 17.6 µg/mL to 22.6 µg/mL, and therefore increased DAN
solubilisation capacity from 100% to 257% (again taking into account a doubling in volume on
bile addition). Dilution of the colloids with the same volume of buffer did not result in significant
changes in the solubilisation capacity of either DAN or CIN (Figure 4.3A). As such, the changes
in solubilisation capacity observed on addition of bile were not simply due to a volume/dilution
effect (i.e. decreasing colloidal lipid concentration), but instead appear to reflect increases in bile
concentration, since the average total bile salt concentration in donor bile was 15.0 ± 1.0 mM
(mean ± SEM, n = 4) whereas the bile concentration in the model colloidal system was 4 mM.
The solubility of CIN therefore decreased with increasing bile concentration, whereas the
solubility of DAN increased with increasing bile concentration. This trend with donor rat bile was
Chapter 4: Supersaturation potential is enhanced for basic drugs
169
consistent with the in vitro data obtained using SEIF (Figure 4.1), and suggests that CIN may
supersaturate when drug-loaded model colloids interact with secreted bile in vivo.
To assess the kinetics of drug precipitation on interaction with bile, CIN-loaded model colloids
(CIN loaded at 130 µg/mL, ~ 80% saturated solubility) were incubated 1:1 v/v with donor rat bile
in a temperature (37 °C) and stirring rate-controlled vessel, and samples taken over time to
evaluate whether precipitation occurred. Figure 4.3B shows that bile addition did not result in
immediate drug precipitation, and instead was preceded by a period of supersaturation that lasted
for at least 80 min in all cases. Since DAN solubility increased rather than decreased on bile
addition, supersaturation/precipitation did not occur and therefore precipitation kinetics were not
evaluated.
(A)
% o
rigin
al d
rug
solu
bilis
atio
n ca
paci
ty
0
100
200
300
400
Model colloidModel colloid + Buffer (1:1)Model colloid + Bile (1:1)
Cinnarizine Danazol
100 100 100
12
257
94
*
*
(B)
Figure 4.3: (A) Percent original solubilisation capacity of cinnarizine (CIN) and danazol (DAN),
before and after a 1:1 v/v addition of buffer or rat bile to model colloids. After the addition of rat
bile, solubility of CIN decreased from 157.7 ± 3.0 µg/mL to 9.2 ± 0.7 µg/mL, while solubility of
DAN increased slightly from 17.6 ± 0.5 µg/mL to 22.6 ± 2.5 µg/mL. After the 1:1 v/v addition of
buffer, solubility of CIN and DAN was approximately halved, from 157.7 ± 3.0 µg/mL to 73.8 ±
1.0 µg/mL, and 17.6 ± 0.5 µg/mL to 8.8 ± 0.2 µg/mL, respectively. Percent original solubilisation
capacity is the solubilisation capacity of the colloids after bile addition (taking into account
Time (min)0 20 40 60 80 100 120%
orig
inal
CIN
in s
olut
ion
0
20
40
60
80
100
120
Add bile
Equilibrium solubility: Model colloid + Bile (1:1)
Chapter 4: Supersaturation potential is enhanced for basic drugs
170
volume increases as a result of dilution) relative to the solubilisation capacity of the colloids prior
to bile addition. Data represent mean ± SEM of n = 3 determinations. Significant difference (p <
0.05) to colloid only is denoted by the symbol *. (B) Kinetics of CIN precipitation from model
colloids following 1:1 v/v addition of rat bile at t = 0. CIN was loaded into colloids at 80%
saturation (~130 µg/mL). Each line represents individual experiments. The addition of bile to
drug-loaded colloids resulted in CIN supersaturation that was maintained for at least 80 min in all
experiments.
4.4.3 Co-perfusion of bile with model colloids increases cinnarizine
absorption to a greater extent than danazol
The perfusate disappearance and mesenteric blood appearance profiles of cinnarizine (CIN),
danazol (DAN) and oleic acid after perfusion of drug in LC lipid-loaded model colloids through
an isolated jejunal segment (~ 10 cm2), with and without 1:1 v/v co-perfusion with donor rat bile,
are shown in Figure 4.4. Steady state-absorptive flux, disappearance Papp, and appearance Papp
from all perfusion experiments are reported in Table 4.4.
Co-perfusion of model colloids with bile increased the absorptive flux, disappearance Papp, and
appearance Papp of CIN 3.2-fold, 3.0-fold, and 4.2-fold, respectively. Throughout the perfusion
experiments, bile-induced supersaturation appeared to be stable, consistent with the in vitro
studies (Figure 4.3B), since the CIN perfusate concentration was unchanged before or after
centrifugation, therefore indicating a lack of CIN precipitation during co-perfusion with bile
(Figure 4.4AI).
Co-perfusion of model colloids with bile was expected to lead to an increase in DAN
solubilisation capacity within the perfused jejunal segment and therefore to not stimulate DAN
supersaturation or an increase in thermodynamic activity. In spite of this, however, co-perfusion of
Chapter 4: Supersaturation potential is enhanced for basic drugs
171
model colloids with bile led to a moderate (1.7-fold) increase in both the absorptive flux and
appearance Papp of DAN (disappearance Papp was unchanged) (Figure 4.4II and Table 4.4). The
increase in absorptive flux in the presence of bile was, however, notably higher for CIN than DAN
(3.2-fold increase in CIN vs.1.7-fold increase in DAN).
In assessing the permeability of CIN and DAN, the concentration of the drug in the perfusate was
kept constant (65 µg/mL and 7 µg/mL, respectively), allowing direct comparisons of absorptive
flux in the absence and presence of bile. For oleic acid, however, the concentration in the
perfusate was reduced by 50% due to bile co-perfusion. Therefore, direct comparisons of absolute
oleic acid absorptive flux was not possible, and transport into the mesenteric blood is represented
instead as % dose transported (Figure 4.4BIII). Nonetheless, bile co-perfusion led to an increase in
the proportion of oleic acid absorbed (Figure 4.4BIII), and an increase in the appearance Papp of
oleic acid (Table 4.4).
Chapter 4: Supersaturation potential is enhanced for basic drugs
172
(AI)
Time (min)
0 30 40 50 60 70
% C
IN d
ose
pass
ing
thro
ugh
jeju
num
40
60
80
100
Model colloids Model colloids + Bile (pre-centrifugation)SS
Model colloids + Bile (post-centrifugation)SS
(BI)
Time (min)
0 10 20 30 40 50 60 70
CIN
flu
x in
to m
esen
teric
bl
ood
(ng/
5 m
in/1
0 cm
2 )
0
100
200
300
400
500
600Model colloids (CIN 65 µg/mL)Model colloids + Bile (CIN 65 µg/mL)SS
(AII)
(BII)
Time (min)
0 10 20 30 40 50 60 70
DA
N f
lux
into
mes
ente
ricbl
ood
(ng/
5 m
in/1
0 cm
2 )
0
50
100
150
200
250
300Model colloids (DAN 7 µg/mL)Model colloids + Bile (DAN 7 µg/mL)
(AIII)
(BIII)
Time (min)
0 10 20 30 40 50 60 70% O
A d
ose
into
mes
ente
ric
bloo
d (%
/5 m
in/1
0 cm
2 )
0
1
2
3
4
5Model colloids (OA 1 µg/mL)Model colloids + Bile (OA 0.5 µg/mL)
Figure 4.4: (A) Perfusate disappearance (% dose passing through jejunum) and (B) mesenteric
blood appearance of (I) cinnarizine (CIN), (II) danazol (DAN), and (III) oleic acid (OA) when
model colloids were perfused through an isolated rat jejunal segment (~ 10 cm2), with (open
symbols) and without (closed symbols) 1:1 v/v co-perfusion with donor rat bile. Appearance
profiles are plotted as absorptive flux (ng/5 min/10 cm2) for CIN and DAN, and % dose passing
Time (min)
0 30 40 50 60 70
% D
AN
do
se p
ass
ing
thro
ug
h je
jun
um
40
60
80
100
Model colloids Model colloids + Bile
Time (min)
0 30 40 50 60 70
% 3
H-O
A d
ose
pa
ssin
gth
rou
gh
jeju
nu
m
40
60
80
100
Model colloidsModel colloids + Bile (pre-centrifugation)Model colloids + Bile (post-centrifugation)
Chapter 4: Supersaturation potential is enhanced for basic drugs
173
through jejunum for OA. Co-perfusion of model colloids with bile generates supersaturation in
situ within the perfused jejunal segment for CIN but not DAN. It is not known if co-perfusion of
colloids with bile generates OA supersaturation within the perfused jejunal segment. SS denotes
experiments where drug is supersaturated in perfusate. Degree of CIN and OA (if any)
precipitation within the perfusate is illustrated in (AI) and (AIII) as the difference in perfusate
concentration pre- and post-centrifugation. Experiments were performed using an in situ single-
pass rat jejunum perfusion model. In all experiments, the total perfusate flow rate was kept
constant at 0.1 mL/min. Data represent mean ± SEM of n = 3-4 rats.
Chapter 4: Supersaturation potential is enhanced for basic drugs
174
Table 4.4: Cinnarizine (CIN), danazol (DAN) and oleic acid (OA) disappearance Papp (x 106 cm/s)
from the intestinal perfusate, appearance Papp (x 106 cm/s) in the mesenteric blood, and steady
state absorptive flux into mesenteric blood (ng/5 min/10 cm2) after 70 min of single-pass perfusion
of ~ 10 cm2 segments of rat jejunum with model colloids (composition reported in Table 4.2), with
and without 1:1 v/v co-perfusion with donor rat bile. Values calculated using data obtained after
steady state attainment (t = 55-70 min). In all experiments, total perfusate flow rate was kept
constant at 0.1 mL/min. Data represent mean ± SEM of n = 3-4 rats.
Perfusate Conc. perfused (µg/mL)
SS ratio #^
Disappearance Papp (x
106 cm/s)
Appearance Papp
(x 106 cm/s) Flux into
mesenteric blood (ng/5 min/10 cm2)
CIN Model colloid only
65.0 0.4 12.2 ± 2.8 0.6 ± 0.2 138 ± 28
Model colloid + Bile SS
65.0 7.1 36.9 ± 5.5 a 2.5 ± 0.2 a 443 ± 34 a
DAN Model colloid only
7.2 0.4 27.7 ± 6.8 3.8 ± 0.1 75 ± 2
Model colloid + Bile
7.2 0.3 29.4 ± 3.0 6.6 ± 0.8 a 131 ± 15 a
OA Model colloid only
1.0 n/a 18.0 ± 5.9 2.0 ± 0.3 6 ± 1
Model colloid + Bile
0.5 n/a 26.1 ± 3.8 6.1 ± 0.8 a 9 ± 1
a Significant difference from model colloid only SS denotes drug supersaturation in perfusate # SS ratio = Supersaturation ratio = (Supersaturated) concentration of drug in perfusate / Equilibrium
solubility of drug in perfusate ^ SS ratio not reported for OA because equilibrium solubility of OA in perfusate was not determined
Chapter 4: Supersaturation potential is enhanced for basic drugs
175
4.4.4 Supersaturation increases in vivo cinnarizine exposure after
intraduodenal infusion of drug-loaded model colloids
The systemic plasma concentration-time profiles for cinnarizine (CIN) and 3H-oleic acid
following intraduodenal infusion of CIN-loaded model colloids (130 µg/mL CIN; ~ 80% saturated
solubility) to bile-intact and bile-diverted rats are shown in Figure 4.5, and the pharmacokinetic
parameters reported in Table 4.5. Consistent with the results from the rat jejunal permeability
studies, intraduodenal administration of drug-loaded model colloids to bile-intact rats resulted in
significantly higher systemic plasma concentrations of CIN at t = 1.5, 2, 3 and 4 h, and
significantly higher AUC0-8 h (2.0-fold increase) when compared to bile-diverted rats. The
systemic plasma concentrations of 3H-oleic acid were also significantly higher in bile-intact rats at
all sample time points.
Bioavailability studies for danazol (DAN) are not reported here because the dose administered
(0.14 mg/kg – corresponding to 80% saturated solubility) did not lead to detectable systemic
plasma concentrations in both bile-intact and bile-diverted rats at all sample time points (limit of
quantification for DAN plasma assay was 5 ng/mL).
Chapter 4: Supersaturation potential is enhanced for basic drugs
176
(A)
Time (h)
0 2 4 6 8
Pla
sma
CIN
con
c.
(ng/
mL)
020406080
100120140
Bile-divertedBile-intact*
*
*
*
(B)
Figure 4.5: Systemic plasma concentration-time profiles of (A) cinnarizine (CIN) and (B) oleic
acid (OA) following a 2-hour intraduodenal infusion of 3 mL model colloids containing 130
µg/mL CIN (~ 80% saturated solubility) and 1 µCi/mL OA to bile-intact and bile-diverted rats.
Consistent with observations in rat jejunum perfusion studies, bile-induced supersaturation
translated into increased in vivo CIN exposure. Data represent mean ± SEM of n = 3 rats.
Statistical significance (p < 0.05) is denoted by the symbol *.
Table 4.5: Pharmacokinetic parameters for cinnarizine (CIN) after intraduodenal administration
of model colloids (composition reported in Table 4.2) loaded with 1.3 mg/kg CIN (~ 80%
saturated solubility) to bile-intact and bile-diverted rats. The interaction between the colloids and
secreted bile in the duodenum is expected to generate CIN supersaturation in bile-intact animals,
whilst CIN is expected to be sub-saturated in bile-diverted animals. Values represent mean ± SEM
of n = 3 rats.
Compound Experimental group
AUC0-8 h
(ng h/mL) Cmax
(ng/mL) Tmax
(h)
Cinnarizine Bile-intact 403 ± 3 a 107.1 ± 10.8 a 1.8 ± 0.2
Bile-diverted 190 ± 17 53.0 ± 8.8 1.7 ± 0.3
a Significant difference when compared to bile-diverted group
Time (h)
0 2 4 6 8
Pla
sma
3H
-OA
con
c.(d
pm/m
L)
0
20000
40000
60000Bile-divertedBile-intact
**
**
** *
Chapter 4: Supersaturation potential is enhanced for basic drugs
177
4.5 DISCUSSION
Previously, we described the ability of bile secretion to trigger drug supersaturation and to
enhance the absorption of a model PWSD (cinnarizine) from intestinal colloidal phases containing
digestion products of medium-chain triglycerides (Chapter 3). In the current study, we aimed to
extend these initial observations to include assessment of colloidal phases containing long-chain
lipids (to evaluate the dependence on lipid chain length) and PWSD with differing
physicochemical properties (to assess the importance of acid/base functionality, lipid solubility
and octanol-water distribution coefficient). The supersaturation tendency of the different PWSD
during endogenous processing of post-digestion lipid colloidal phases was predicted by assessing
drug solubility relationships with increasing bile concentration and decreasing lipid concentration;
and the absorption of drugs with different supersaturation tendencies was evaluated in situ and in
vivo. The data suggest that under the conditions explored, bile dilution of, and lipid
removal/absorption from, LC colloids may induce supersaturation and enhance the absorption of
PWSD, and that the potential for drug supersaturation during lipid processing is more pronounced
for basic drugs.
The solubility of lipophilic PWSD in lipid-containing intestinal colloids is a complex function of
the solid state properties of the drug (usually captured by melting point), and drug affinity for core
lipids, the colloid interface and water, and is not easily predicted de novo. For example, the lipid
solubility of FF far exceeds that of the similarly lipophilic (by log P) DAN (Table 4.3),
presumably due to differences in melting point. However, both drugs had comparable solubility in
the colloidal systems examined here (Table 4.2). In contrast, although CIN and HF have similar
solubility properties in lipids, the solubility of HF in the model colloids was up to an order of
magnitude higher than CIN, suggesting specific affinity of HF for the colloidal structures rather
than their component parts. MFA has low solubility in lipids but had moderate solubility in the
Chapter 4: Supersaturation potential is enhanced for basic drugs
178
prepared colloids, presumably due to ionisation (and higher water solubility) at the pH of model
intestinal fluids.
Examination of the impact of bile concentration on the drug solubilisation capacities of the long-
chain colloidal systems revealed widely varying solubility trends for the drugs evaluated (Figure
4.1 and Table 4.2). Traditionally it has been assumed that bile secretion promotes drug
solubilisation (and in this way promotes drug absorption), and indeed increases in drug solubility
were evident with increasing bile concentration for the neutral (DAN, FF) and acidic (MFA) drugs.
In contrast, however, and in large part counter-intuitively, the solubility of the basic drugs (CIN,
HF) in the long-chain colloids decreased with increasing bile concentration. The different
relationships between the colloid solubility of the basic, neutral and acidic drugs and bile
concentration was not expected, but may reflect changes in the structure of the colloidal phases on
bile addition, as well as differences in the preferred drug solubilisation site. Under digesting
conditions in the small intestine, many amphiphilic molecules (e.g. bile salts, lyso-phospholipids,
fatty acids, monoglycerides) co-exist, and the colloidal species formed display complex phase
behaviour that is dependent on the type and concentration of the components present199, 239, 240. For
example, raising the concentration of endogenous biliary components (e.g. bile salts) relative to
the concentration of lipid digestion products has been shown to stimulate phase changes from
larger lipid rich species such as cubic, hexagonal and multilamellar liquid crystals to smaller, more
highly dispersed species such as unilamellar vesicles and mixed micelles39, 40. Continued phase
changes from unilamellar vesicles to mixed micelles on addition of higher concentrations of bile
salts are also well-documented218, 241, 242. In view of the dynamic nature of the structure of
intestinal lipid colloidal species, drug solubilisation capacity may also change with the addition or
removal of amphiphilic components, since different phases possess different solubilisation
capacities for PWSD194, 243.
Chapter 4: Supersaturation potential is enhanced for basic drugs
179
Solubility changes as a function of microstructure have been described for many poorly water-
soluble compounds in larger, microemulsion-based colloidal systems244-246. The dependency of
compound solubility on microstructure is thought to be related to the preferred solubilisation locus
within the microstructure, and drugs that preferentially reside at an interface may have increased
solubility when the interfacial area of colloidal systems is increased247. It is thus possible that in
the colloidal systems examined here, differences in the solubility behaviour of the
basic/neutral/acidic drugs reflect differences in preferred solubilisation sites within the colloids.
CIN and HF have higher solubilities in oleic acid (presumably due to favourable electrostatic
interactions in lipid solution), and are therefore more likely to co-localise with oleic acid in the
hydrophobic core of a micelle or in the lipid bilayer of a vesicle when compared to DAN and
MFA, which in turn may be more highly solubilised at the amphiphilic interface. Under these
circumstances, the addition of bile constituents to the colloids (which disperses the lipids,
decreasing the proportional volume of the lipid core and increasing the colloid interfacial area)
may lead to increased DAN and MFA solubilisation, but reduced solubilisation of CIN and HF.
It is also possible that within the lipophilic sub-compartments of the dispersed colloidal structures,
molecules of oleic acid and the weak bases orientate in such a way that the hydrophobic aspects of
each molecule remain dissolved in the lipid core, whereas the charged species orientate towards
the interface where the cationic weak bases and the carboxylic acid head groups of oleic acid are
free to favourably interact. Under these circumstances increased concentrations of bile acids may
also disrupt intermolecular interactions between oleic acid and weak bases at the colloidal
interface, further lowering drug solubilisation capacity.
Reduced solubilisation of the weak bases with increasing bile concentration may also occur via the
formation of insoluble drug-bile salt complexes between the cationic weak bases and anionic bile
Chapter 4: Supersaturation potential is enhanced for basic drugs
180
salts. To investigate the potential importance of the effects of bile on interactions between the
weak bases and oleic acid vs. drug interaction or complexation with bile salt, a series of colloidal
systems analogous to those in Table 4.2 (i.e. containing bile components and monoolein) but
without the inclusion of oleic acid were prepared, and CIN solubility assessed. As expected, the
solubility of CIN in the oleic acid-free colloidal systems (Table 4.6) was substantially lower than
the corresponding oleic acid-containing colloidal systems (Table 4.2), illustrating the high
dependency of the colloid solubility of the weak bases on the presence of oleic acid. More
significantly, in the oleic acid-free systems, CIN solubility increased (rather than decreased) with
increasing bile concentration (Table 4.6). The stark difference in CIN solubility behaviour in oleic
acid-free vs. oleic acid-containing colloidal systems suggests that the effect of bile in reducing
CIN solubilisation in bile salt mixed micelles is oleic acid dependent, and that the reduction in
CIN solubility was not due to bile salt-drug complexation, but rather bile-mediated disruption of
intermolecular interactions between oleic acid and the weak bases.
Chapter 4: Supersaturation potential is enhanced for basic drugs
181
Table 4.6: Equilibrium solubility (37 °C) of cinnarizine (CIN) in colloidal systems consisting 1-
monoolein solubilised in bile components. Colloidal systems presented in this table correspond to
those in Table 4.2, but without the inclusion of oleic acid.
[1-Monoolein] (mM) [Bile]^ (mM)^^ CIN saturated solubility (µg/mL)
0.88 4 5
8 7
16 13
1.77 4 8
8 10
16 16
3.54 4 16
8 16
16 20
^ Bile refers to total bile salt, lysophosphatidylcholine, cholesterol in 16:4:1 molar ratio. Total bile salt consisted of 25 mol% sodium glycocholate, 17.5 mol% sodium glycodeoxycholate, 25 mol% sodium glycochenodeoxycholate, 12.5 mol% sodium tauroocholate, 7.5 mol% sodium taurodeoxycholate, 12.5 mol% sodium taurochenodeoxycholate
^^ Concentration refers to concentration of total bile salt
Endogenous lipid processing pathways that lead to decreases in the drug solubilisation capacity of
the intestinal colloidal phases may lead to drug supersaturation (provided that precipitation is not
instantaneous). Since the bile:lipid concentration ratio progressively increases on passage down
the GI tract, the tendency for a drug to supersaturate may be predicted by the relationship between
drug solubility and increasing bile/decreasing lipid concentrations. Thus, during bile dilution of
lipid colloidal phases, drugs that have an inverse solubility relationship with bile concentration are
expected to supersaturate, and the higher the proportional decrease in solubilisation (caused by
increasing bile concentration), the higher the supersaturation tendency. Although bile dilution also
Chapter 4: Supersaturation potential is enhanced for basic drugs
182
leads to a decrease in colloidal lipid concentration, the percent solubility reduction vs. [lipid] plots
(Figure 4.2) suggest that drug solubility decreases no more than, and in most cases less than, the
dilution factor (solubility plots mostly overlap or are above the dilution line). As such, the overall
solubilisation capacity of the intestinal colloidal phases (with respect to a decrease in lipid
concentration) is likely to remain unchanged or even increase. Supersaturation tendency due
simply to bile dilution is therefore expected to be less dependent on changes in lipid concentration
and more dependent on changes in bile concentration. During lipid absorption, however, colloidal
structures are further depleted of lipid content (in the absence of volume changes). In this case
solubilisation capacity is expected to drop (and therefore supersaturation tendency to increase) and
this would be greatest for drugs with the highest solubility dependency on lipid concentration.
Taking into consideration the proportional changes in drug solubility caused by increasing bile
concentration and decreasing lipid concentration, the overall drug supersaturation tendency
resulting from both bile dilution of and lipid absorption from lipid colloidal phases follows the
rank order CIN > HF > FF > DAN > MFA, i.e. supersaturation tendency was greatest for basic
drugs followed by neutral drugs and acidic drugs.
Indeed, when CIN-loaded colloids were mixed with endogenous rat bile (to simulate the process
of bile dilution), CIN solubilisation capacity was reduced significantly, and the period of drug
supersaturation that preceded precipitation translated into enhanced drug absorption (Figure 4.4BI)
and enhanced systemic exposure (Figure 4.5A). Notably, cinnarizine was found to precipitate in
the crystalline form in these experiments (Figure 4.6), precluding the possibility that the enhanced
cinnarizine absorption from model colloids (when bile was co-perfused) observed in Figure 4.4BI
was due to accelerated cinnarizine dissolution from precipitated amorphous forms. In addition,
during bile co-perfusion, the proportion of oleic acid absorbed was increased (Figure 4.4BIII).
Since CIN solubilisation capacity is directly related to colloidal lipid content, the increased
Chapter 4: Supersaturation potential is enhanced for basic drugs
183
absorption of oleic acid (when bile was co-perfused) was expected to magnify the decrease in drug
solubilisation capacity, and further increase CIN supersaturation potential and absorption.
Figure 4.6: Analysis of the cinnarizine precipitate using polarised light microscopy following
precipitation kinetics experiments where fasted rat bile was added in a 1:1 v/v ratio to cinnarizine-
loaded model colloids (cinnarizine was loaded into model colloids at 80% saturation (~ 130
µg/mL). Addition of bile reduced the equilibrium cinnarizine solubilisation capacity of model
colloids to 12% of initial, and triggered cinnarizine supersaturation and precipitation (see Figure
4.3). The polarised light microscopy image show that cinnarizine precipitated in the crystalline
form after the addition of bile.
When similar studies were repeated with DAN (which has significantly lower absolute solubility
in lipid digestion products and lower supersaturation potential based on the impact of increases in
bile concentration and decreases in lipid concentration on DAN solubility; Table 4.3, Figure 4.1,
Figure 4.2), absorptive flux also increased when model colloids were co-perfused with bile, but to
a much lower extent than CIN (1.7-fold, compared to 3.2-fold for CIN). The effect of bile in
enhancing DAN absorption most likely reflected differences in oleic acid absorption in the
presence of bile (Figure 4.4BIII). Thus, bile increased oleic acid absorption, and in turn decreased
DAN solubilisation capacity in the lipid depleted mixed micelles, resulting in an increase in DAN
Chapter 4: Supersaturation potential is enhanced for basic drugs
184
thermodynamic activity at the absorptive membrane. This occurred despite a slight decrease in
DAN thermodynamic activity in the bulk lumen due to bile addition (via an increase in drug
solubilisation capacity due to bile). Other potential explanations for the change in DAN absorption
include an increase in flux resulting from an increase in permeability due to bile-induced
membrane damage248, 249 or bile-induced changes to pH (the average pH of donor bile used was
8.02 ± 0.02 (mean ± SEM, n = 5)). These factors were, however, excluded in previous studies
using similar experimental methods and colloidal systems (Chapter 3). The presence of
phospholipids in endogenous bile is also expected to reduce the toxicity (and permeability
enhancement effects) of bile salts by decreasing bile salt thermodynamic activity in mixed bile
salt-phospholipid micelles108. The permeability of DAN, a non-ionisable drug, is not expected to
be affected by changes to pH.
Differences in the extent to which bile enhanced the absorption of CIN and DAN may therefore be
attributed to differences in the supersaturation tendencies of the two drugs during bile dilution and
lipid absorption. In the case of CIN, increases in thermodynamic activity were triggered by both
bile dilution and lipid absorption; whereas in the case of DAN, the increase in thermodynamic
activity was triggered only during lipid absorption. These observations suggest that bile-induced
supersaturation may be a phenomenon that is specific to basic drugs; while lipid absorption-
induced supersaturation is expected to apply to all lipophilic drugs, and the degree of
supersaturation induced is likely to be greatest for basic drugs (which had higher solubility
dependency on the concentration of lipid digestion products within bile micelles).
Thus, the in vitro and in vivo observations in this study suggest that drugs with high solubility in
lipid digestion products (such as weak bases in fatty acid-containing colloids) may better harness
the supersaturation-generating potential of endogenous lipid processing pathways. This reflects
three separate, but sequential, processes (see Figure 4.7). First, strong affinity for solubilised lipid
Chapter 4: Supersaturation potential is enhanced for basic drugs
185
digestion products may enable favourable drug partitioning from digesting triglyceride droplets
into post-digestion lipid colloidal phases250. Subsequently, drug is preferentially solubilised in the
core of lipid colloidal phases at high concentration and is therefore predisposed to bile-induced
supersaturation in the event of a drop in solubilisation capacity. Finally, lipid absorption (i.e. lipid
removal from colloids) exaggerates the loss of drug solubilisation capacity and therefore
encourages supersaturation if precipitation is not immediate. Drug affinity for lipid digestion
products may therefore be a useful indicator of supersaturation potential during LBF processing in
the GI tract.
In summary, we previously described a dual role for bile in facilitating drug absorption after co-
administration with medium-chain formulation lipids, where bile-mediated solubilisation of lipid
digestion products and bile-mediated dilution of existing lipid colloidal phases provides a means
to simultaneously increase solubilisation capacity and promote the thermodynamic activity of a
co-administered drug in the small intestine (Chapter 3). The data described here further establish
that endogenous processing of post-digestion lipid colloidal phases (via bile dilution and lipid
absorption) may generate drug supersaturation after co-administration of long-chain lipids; and
that the potential for supersaturation generation and ensuing absorption enhancement is greatest
for basic drugs. The unique manner by which lipids promote both drug solubilisation and drug
supersaturation likely contributes to effective drug absorption after lipid co-administration, and
has implication both for LBF design and better understanding the effects of lipids in food on drug
absorption. The sequential manner (see Figure 4.7) by which supersaturation is triggered during
endogenous lipid processing (e.g. during lipid dispersion and digestion176, 227, as a result of on-
going bile dilution, and in response to lipid absorption), combined with the presence of drug
solubilising lipid colloidal phases in the small intestine that ‘buffer’ against the generation of
excessive supersaturation, dictates that the rate and extent of supersaturation generated by LBF in
Chapter 4: Supersaturation potential is enhanced for basic drugs
186
vivo is highly controlled, and has the potential to maximise absorption-enhancement and reduce
the risk of drug precipitation.
4.6 CONCLUSION
Supersaturation-triggered enhancements in thermodynamic activity may represent an important
mechanism by which solubilised drug is made available for absorption from the lipid colloidal
phases formed in the intestine following digestion of dietary or formulation lipids. In this study,
we investigated drug supersaturation during bile-mediated dilution of intestinal colloid phases
containing digestion products of long-chain triglycerides. The increase in colloidal concentration
of biliary solubilisers upon dilution with bile was a highly effective trigger for supersaturation for
basic drugs, whereas for acidic or neutral drugs, bile-mediated enhancements in lipid absorption
and therefore decreases in colloidal lipid content appeared to provide the required reduction in
solubilisation capacity to promote supersaturation. The data suggest that supersaturation tendency
during endogenous processing of intestinal colloids is greatest for basic when compared to
neutral/acidic drugs and likely reflects the high affinity of weak bases for colloidal systems
containing oppositely charged fatty acids. Studies to evaluate the importance of intestinal colloids
containing natural glyceride digestion products (i.e. fatty acids rather than systems based on
uncharged lipids or synthetic solubilisers) in maximising the absorption of co-administered drugs
are on-going.
Chapter 4: Supersaturation potential is enhanced for basic drugs
187
Figure 4.7: Endogenous lipid processing pathways that facilitate both drug solubilisation and drug
supersaturation in the small intestine. (i) Bile-mediated solubilisation of lipid digestion products at the
interface of a digesting oil droplet results in the generation of lipid colloidal phases such as vesicles and
micelles that promote drug solubilisation (ii) Digestion and dispersion of lipid based formulations (LBF)
may reduce drug solubilisation capacity and trigger drug supersaturation176, 227 (iii) The continuing
interaction of secreted bile and existing lipid colloidal phases in the lumen may reduce the solubilisation
capacity of basic drugs and trigger drug supersaturation (iv) The removal of colloidal lipid components via
lipid absorption reduces drug solubilisation capacity and also triggers drug supersaturation at the absorption
site. Drugs that have high affinity for lipid digestion products (e.g. basic drugs such as cinnarizine and
halofantrine) may be more prone to supersaturation events as they preferentially partition into post-
digestion phases (instead of remaining in a digesting TG droplet), are predisposed to bile-induced
supersaturation, and show exaggerated losses in drug solubilisation capacity (i.e. greater supersaturation
potential) during lipid absorption. The sequential manner by which supersaturation is triggered during
endogenous LBF processing (ii, iii, iv), combined with the supersaturation ‘buffering’ properties of lipid
colloidal phases (via increases in intestinal solubilisation capacity), dictate that the rate and extent of
supersaturation generated by LBF in vivo is intrinsically controlled, thus maximising absorption-
enhancement potential of supersaturation and reducing the risk of drug precipitation. The unique ability of
LBF components to simultaneously increase solubilisation capacity and promote thermodynamic activity of
co-administered drug in the small intestine may contribute to effective drug absorption often observed with
LBF co-administration.
Oildroplet
Common bile duct
DD
D D
D
DD
Multilamellarvesicles
Mixed micelles
Unilamellarvesicles
DD
D
DD
AbsorptionAbsorption
Pancreatic lipase/co‐lipase
Bile micelles
Lipid digestion products
Drugmolecule
Small intestine
(i) Bile‐mediated solubilisation(ii) LBF digestion and dispersion‐induced supersaturation(iii) Bile dilution‐induced supersaturation(iv) Lipid absorption‐induced supersaturation
DD
DSS DSS
Absorption
Enterocytes
Supersaturated drug
(ii)
(i) (iii)(iv)
DDSS DSS
(iii) (iii)
DDSS DSS DDSS DSS
DLipid absorption
Lipid absorption
DD
DD
DDD
Chapter 4: Supersaturation potential is enhanced for basic drugs
188
Monash University
Declaration for Thesis Chapter 5 Declaration by candidate In the case of Chapter 5, the nature and extent of my contribution to the work was the following:
Nature of contribution Extent of contribution (%)
Concept and design of studies, planning and execution of experimental work, data analysis and interpretation, formulation of conclusions and hypotheses resulting from the relevant studies, drafting and revision of manuscript
70%
The following co-authors contributed to the work. Co-authors who are students at Monash University must also indicate the extent of their contribution in percentage terms:
Name Nature of contribution Extent of contribution (%)
C.J.H. Porter Project supervisor, data and manuscript review NA
N.L. Trevaskis Project co-supervisor, data and manuscript review NA
Candidate’s Signature
Declaration by co-authors The undersigned hereby certify that:
(13) the above declaration correctly reflects the nature and extent of the candidate’s contribution to this work, and the nature of the contribution of each of the co-authors.
(14) they meet the criteria for authorship in that they have participated in the conception, execution, or interpretation, of at least that part of the publication in their field of expertise;
(15) they take public responsibility for their part of the publication, except for the responsible author who accepts overall responsibility for the publication;
(16) there are no other authors of the publication according to these criteria; (17) potential conflicts of interest have been disclosed to (a) granting bodies, (b) the editor or
publisher of journals or other publications, and (c) the head of the responsible academic unit; and
(18) the original data are stored at the following location(s) and will be held for at least five years from the date indicated below:
Location(s) Monash Institute of Pharmaceutical Sciences
Chapter 4: Supersaturation potential is enhanced for basic drugs
189
Signature 1
Signature 2
190
CHAPTER 5 : LIPID ABSORPTION
TRIGGERS DRUG SUPERSATURATION
AT THE INTESTINAL UNSTIRRED WATER
LAYER AND PROMOTES DRUG
ABSORPTION FROM MIXED MICELLES
Yan Yan Yeap, Natalie L. Trevaskis, Christopher J. H. Porter
Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia
Manuscript in submission.
Chapter 5: Lipid absorption triggers drug supersaturation
191
5.1 ABSTRACT
Purpose. To evaluate the potential for the acidic intestinal unstirred water layer (UWL) to induce
drug supersaturation and enhance drug absorption from intestinal mixed micelles, via the
promotion of fatty acid absorption. Methods. Using a single-pass rat jejunal perfusion model, the
absorptive-flux of cinnarizine and 3H-oleic acid from oleic acid-containing intestinal mixed
micelles was assessed under normal acidic microclimate conditions and conditions where the
acidic microclimate was attenuated via the co-administration of amiloride. As a control, the
absorptive-flux of cinnarizine from micelles of Brij® 97 (a non-ionisable, non-absorbable
surfactant) was assessed in the absence and presence of amiloride. Cinnarizine solubility was
evaluated under conditions of decreasing pH and decreasing micellar lipid content to assess
changes in solubilisation and thermodynamic activity during micellar passage across the UWL.
Results. In the presence of amiloride, the absorptive-flux of cinnarizine and 3H-oleic acid from
mixed micelles decreased 6.5-fold and 3.0-fold, respectively. In contrast, the absorptive-flux of
cinnarizine from Brij® 97 micelles remained unchanged by amiloride, and was significantly lower
than from the long-chain micelles. Cinnarizine solubility in long-chain micelles decreased under
conditions where pH and micellar lipid content decreased simultaneously. Conclusion. The acidic
microclimate of the intestinal UWL promotes drug absorption from intestinal mixed micelles via
the promotion of fatty acid absorption which subsequently stimulates drug supersaturation. The
observations suggest that formulations containing absorbable lipids (or their pre-digestive
precursors) may outperform formulations that lack absorbable components due to the benefits
associated with lipid absorption-induced drug supersaturation.
Chapter 5: Lipid absorption triggers drug supersaturation
192
5.2 INTRODUCTION
Co-administration of poorly water-soluble drugs (PWSD) with lipids often leads to a significant
enhancement in oral bioavailability2. In the small intestine, the digestion of formulation or dietary-
derived di/triglycerides liberates fatty acids and monoglycerides that are solubilised by biliary
components (bile salts, phosphatidylcholine, cholesterol) to generate a series of lipid colloidal
structures including vesicles and mixed micelles. These colloidal phases in turn provide dispersed
lipidic microenvironments for the solubilisation of co-administered PWSD, thereby increasing the
drug solubilisation capacity of the small intestine when compared to conditions in the fasted
state76.
Although solubilisation increases the apparent solubility of PWSD in the small intestine, the total
concentration of drug in solution (Ctotal) exists in equilibrium between the concentration
solubilised in the colloidal fraction (Ccolloid) and the concentration in the free fraction (Cfree):
Ctotal = Cfree + Ccolloid Equation 5.1
In the absence of solid drug, solubilisation in colloidal structures such as micelles and vesicles is
expected to result in a reduction in drug thermodynamic activity143. In simple micellar systems a
reduction in thermodynamic activity manifests as a decrease in Cfree. Thus, solubilisation in
colloids does not increase (and may reduce) free drug concentrations. Whether increases in total
solubilisation capacity translate into enhancements in drug absorption is therefore difficult to
predict with certainty. Indeed, recent studies suggest that in the absence of an increase in free drug
concentrations, solubilisation may not result in enhanced drug absorption despite increases in total
solubilised drug concentrations100, 145, 146.
Chapter 5: Lipid absorption triggers drug supersaturation
193
Recently, however, we have observed that formulations containing lipids may provide unique
absorption benefits for solubilising formulation, since drug supersaturation appears to be triggered
during lipid processing in the gastrointestinal (GI) tract4, 176, 227, 251. Under these circumstances, the
induction and maintenance of supersaturation has the potential to reverse (or at least attenuate) the
reduction in drug thermodynamic activity inherent in solubilisation, and may significantly enhance
free drug concentrations above the aqueous solubility. For lipid-based formulations (LBF), drug
supersaturation may be generated by several processes. Firstly, when the formulation loses
solubilisation capacity during the dilution of water miscible co-solvents or surfactants17, 178, 191,
secondly, as a result of the digestion of triglycerides and/or surfactants within the formulation4, 91,
176, 251, and thirdly as lipid-rich colloidal species are diluted by biliary secretions (Chapters 3 and
4).
The ability for drug supersaturation in lipid-based colloids to translate into enhanced drug
absorption has been shown using systems that model the species likely to form during the
digestion of glyceride lipids (i.e. micelles and vesicles containing bile salts, lysophospholipid,
cholesterol, fatty acid and monoglyceride) (Chapters 3 and 4). In these studies, interaction of
secreted bile with lipid colloidal phases reduced the solubilisation capacity of the colloids for
poorly water-soluble weak bases, but since drug precipitation was not immediate, supersaturation
was induced. The period of drug supersaturation that preceded drug precipitation coincided with
significant enhancements in the absorption of cinnarizine across rat jejunum (Chapters 3 and 4).
Interestingly, in the same studies, some increase in absorption in the presence of bile was also
apparent for danazol, even though interaction with bile did not reduce micellar solubilisation
capacity in vitro (and therefore did not stimulate supersaturation). Indeed, addition of bile
increased drug solubility in a fashion more consistent with traditional models for micellar
Chapter 5: Lipid absorption triggers drug supersaturation
194
solubilisation (where the addition of solubilising species such as bile typically increases
solubilisation capacity). For danazol, the driver for increased drug absorption was suggested to be
the potential for lipid absorption (i.e. the removal of micellar lipid content) to reduce micellar drug
solubilisation capacity and to trigger drug supersaturation at the absorptive site (assuming lipid
absorption was faster than drug absorption). These preliminary data provide the background for
the current chapter that has examined in detail the role of lipid absorption from intestinal micellar
species as a driver of drug supersaturation, and therefore increased drug absorption.
The absorption of long-chain fatty acids (LCFA) is extremely efficient and facilitated by the acidic
microclimate (pH 5.3–6.245, 46, 57, 252) that is present within the unstirred water layer (UWL) at the
absorptive surface of the small intestine56, 57. The UWL (see Figure 5.1) separates bulk intestinal
fluid from the surface of intestinal absorptive cells, and is estimated to be 500-800 µm wide45, 46.
The UWL exists coincident with, and is indistinguishable from, a viscous mucus layer consisting
of water (~ 95%), glycoproteins, lipids, mineral salts and free proteins45, 47, 48. The acidic
microclimate of the UWL is maintained by the action of the Na+/H+ antiporter present at the brush
border membrane of enterocytes46, as well as the mucus coating which retards H+ diffusion into
bulk luminal fluid45, 46. Shiau and colleagues were the first to describe the facilitatory role of the
acidic microclimate in dietary LCFA absorption from intestinal mixed micelles56. These studies
showed that LCFA absorption was higher in the presence of the low pH microclimate of the UWL.
The authors postulated that the exposure of micelles to the UWL acidic microclimate led to the
protonation of ionised LCFA and an increase in lipid absorption via two mechanisms (depicted in
Figure 5.1(i)). Firstly, protonated LCFA were expected to preferentially partition into and across
the absorptive membrane in accord with classical pH-partition theory56, 57. Secondly, the
protonation of fatty acids was suggested to reduce LCFA amphiphilicity and thereby reduce
LCFA solubility in bile salt micelles. The decrease in micellar LCFA solubility was subsequently
Chapter 5: Lipid absorption triggers drug supersaturation
195
suggested to stimulate micellar dissociation, resulting in increased LCFA thermodynamic activity
and increased LCFA absorption. A decrease in pH at the UWL is therefore expected to lead to a
reduction in the lipid content of intestinal mixed micelles via promotion of LCFA micellar
dissociation and absorption (Figure 5.1(i)).
Since the presence of lipid digestion products within mixed micelles contributes significantly to
drug solubilisation capacity39, 91, in the current chapter we have explored the hypothesis that in
promoting LCFA micellar dissociation and absorption, the acidic microclimate also promotes a
reduction in the drug solubilisation capacity of LCFA-containing intestinal mixed micelles. This
in turn is expected to facilitate drug absorption via the induction of drug supersaturation at the
UWL.
The importance of the presence of absorbable lipids in intestinal mixed micelles as a driver of
drug absorption has been studied in two ways. Firstly by examining drug absorption from LCFA-
containing intestinal mixed micelles in the absence and presence of amiloride. Amiloride is an
inhibitor of the Na+/H+ antiporter that is responsible for acidifying the UWL and is therefore an
inhibitor of fatty acid absorption. Secondly, comparison has been made between drug absorption
from LCFA-containing micelles and from micellar systems comprising non-ionic surfactant (Brij
97), where the micellar components are not expected to be absorbed and therefore where changes
to drug solubilisation capacity are not expected.
The data suggest that lipid absorption is a significant trigger for the induction of drug
supersaturation, and that the combination of fatty acid-containing solubilising species and the
acidic intestinal unstirred water layer may be a particularly powerful driver for drug
supersaturation and absorption. The results provide an improved mechanistic understanding of the
Chapter 5: Lipid absorption triggers drug supersaturation
196
enhancements in drug absorption often observed from lipid-based systems containing digestible
lipids, and also serve to exemplify the beneficial effects of digestible lipids in food on drug
absorption.
Figure 5.1: Schematic of the proposed mechanisms by which the UWL acidic microclimate
facilitates the absorption of micellar solubilised (i) long-chain fatty acids (LCFA) and (ii) poorly
water-soluble drug (PWSD). (i) Exposure of mixed micelles to the acidic microclimate leads to
protonation of LCFA, attenuating their amphiphilic character and reduces LCFA solubility in
mixed micelles. Increased LCFA thermodynamic activity subsequently promotes LCFA
dissociation from mixed micelles and absorption across apical membrane. (ii) At the UWL,
removal of LCFA from mixed micelle via dissociation and absorption decreases the solubilisation
capacity for PWSD, therefore triggering drug supersaturation in close proximity to the absorptive
site, and enhances drug absorption via increases in thermodynamic potential. D represents drug.
Drug is either free and available for absorption or associated with micelles. Dss is used to signify
acidic microclimate-induced drug supersaturation that drives increases in drug absorption.
Unstirred water layer(acidic microclimate)
(mucus layer)
H+
H+
D
H+
Na+
Bile saltsFatty acid
Bulk lumen
(i) Lipid
(ii) Drug
--
--
----
-
-
-
-
-
-
--
----
-
-D DD DD
-
-
DD DD DD
-
-D
Dss
Dss
Dss
D D
H+
Small Intestine
Chapter 5: Lipid absorption triggers drug supersaturation
197
5.3 METHODS
5.3.1 Materials
Cinnarizine, flunarizine dihydrochloride, amiloride hydrochloride hydrate, sodium taurocholate,
sodium taurodeoxycholate, sodium glycocholate, sodium glycochenodeoxycholate, cholesterol, L-
α-lysophosphatidylcholine (LPC, from egg yolk), oleic acid, sodium chloride (NaCl) and Brij® 97
were obtained from Sigma-Aldrich, Australia. Sodium taurochenodeoxycholate, sodium
glycodeoxycholate, ortho-phosphoric acid 85% (H3PO4), sodium hydroxide pellets (NaOH) and
tert-butyl methyl ether (TBME) were from Merck, Australia. Disodium hydrogen orthophosphate
(Na2HPO4), sodium dihydrogen orthophosphate (NaH2PO4.2H2O) and ammonium dihydrogen
orthophosphate (NH4H2PO4) (Ajax Finechem, Australia), Irga-Safe PlusTM (Perkin Elmer Life
Sciences, MA, USA), oleic acid, [9,10-3H(N)] (60 Ci/mmol) (American Radiolabelled Chemicals,
MO, USA), transcutol HP (Gattefossé, France), heparin sodium injection BP (1000 I.U./mL,
Hospira, Australia), xylazine (100 mg/mL, Troy Laboratories, Australia), acepromazine (10
mg/mL, Ceva Delvet, Australia), ketamine (100 mg/mL, Provet, Australia) and pentobarbitone
sodium (325 mg/mL, Virbac, Australia) were obtained from listed suppliers. Acetonitrile and
chloroform used were analytical reagent grade. Water was obtained from a Millipore milliQ
Gradient A10 water purification system (Millipore, MA, USA).
5.3.2 Experimental outline
In situ rat jejunal perfusion experiments were conducted to assess the role of the acidic
microclimate in LCFA and drug absorption from LCFA-containing intestinal colloids. Specifically,
the intestinal absorptive flux of oleic acid and cinnarizine from a model LCFA-containing colloid
(“model LCFA colloids”) was assessed in the absence and presence of 2 mM amiloride, a
competitive inhibitor (with respect to Na+) of the plasma membrane Na+/H+ exchanger253 that has
Chapter 5: Lipid absorption triggers drug supersaturation
198
previously been shown to attenuate the acidic microclimate on the cell surface of the rat jejunum46.
As a control, the absorption of cinnarizine from “model Brij 97 colloids” (Brij 97 is a non-
ionisable and non-absorbable surfactant) in the absence and presence of 2 mM amiloride was also
assessed. The total cinnarizine concentration (130 µg/mL) and cinnarizine thermodynamic activity
(~ 80% saturated solubility) were matched in both colloidal systems. Cinnarizine (a weak base)
was selected as a model PWSD in this proof-of-concept study, as the solubility of cinnarizine in
LCFA-containing intestinal colloids was previously found to be highly dependent on oleic acid
content (Chapter 4), and therefore may be more amenable to enhancement in drug thermodynamic
activity induced by fatty acid absorption. The model LCFA colloids used in this study were
representative of the diluted post-digestion lipid colloidal phases likely responsible for the
presentation of solubilised drug to the absorptive membrane39, 76.
In vitro solubility studies were conducted to evaluate expected changes in cinnarizine
solubilisation when model LCFA or Brij 97 colloids were exposed to the acidic microclimate in
vivo. The equilibrium solubility of cinnarizine was assessed in a series of LCFA colloids with
decreasing system pH and decreasing lipid concentration (to simulate exposure to the acidic
microclimate and lipid absorption); as well as in a series of Brij 97 colloids with decreasing
system pH (to simulate exposure to the acidic microclimate only, as Brij 97 is not absorbed).
The role of the acidic microclimate in the absorption of cinnarizine from supersaturated, LCFA-
containing colloids was also assessed in rat jejunal perfusion studies via co-perfusion of donor bile
with cinnarizine-loaded LCFA colloids (conditions previously shown to induce cinnarizine
supersaturation in situ and to promote intestinal drug absorption – Chapter 4), in the absence and
presence of 2 mM amiloride.
Chapter 5: Lipid absorption triggers drug supersaturation
199
5.3.3 Preparation of LCFA-containing intestinal colloids
Model LCFA colloids used in in situ rat perfusion studies consisted 0.1% w/v oleic acid and 0.06%
w/v monoolein solubilised in simulated endogenous intestinal fluid (SEIF)39 at pH 6.30 ± 0.01.
SEIF comprised of 4 mM total bile salt (25 mol% glycocholate, 17.5 mol% glycodeoxycholate, 25
mol% glycochenodeoxycholate, 12.5 mol% taurocholate, 7.5 mol% taurodeoxycholate, 12.5 mol%
taurochenodeoxycholate), 1 mM LPC and 0.25 mM cholesterol. The oleic acid:monoolein molar
ratio was kept at 2:1, reflecting the ratio of digestion products expected from digestion of 1 mole
of triolein. To model the effect of colloid interaction with the acidic microclimate and the
absorption of lipid components on cinnarizine solubility, systems were prepared at decreasing pH
(pH 6.3, 5.8, 5.3, 4.8) and containing decreasing quantities of lipids (0.1, 0.05, 0.025, 0% w/v
oleic acid, with a proportional decrease in monoolein concentrations) for cinnarizine equilibrium
solubility determination studies.
The preparation of SEIF and LCFA colloids were as described in Chapter 4. pH adjustment of
colloids to 6.30, 5.80, 5.30, 4.80 was achieved by drop wise addition of H3PO4 solution. For the
preparation of drug-loaded LCFA colloids (for in situ jejunal perfusion studies), cinnarizine was
pre-dissolved in oleic acid and allowed to equilibrate overnight at a concentration of 61 mg/g and
115 mg/g, prior to preparation of colloids, such that the final concentration of cinnarizine in the
colloids was 65 µg/mL (~ 40% saturated solubility) and 130 µg/mL (~ 80% saturated solubility),
respectively. As a final step, trace quantities of 3H-oleic acid (to achieve 0.25 µCi/mL) were added
to the drug-loaded colloids, followed by a 1-min vortex. When amiloride was included in the
LCFA colloids, the appropriate mass of amiloride was dissolved in the prepared colloids at 37 °C,
and used within 30 min of preparation. The total sodium strength in all the prepared colloids was
kept constant at 150 mM.
Chapter 5: Lipid absorption triggers drug supersaturation
200
5.3.4 Preparation of Brij 97 colloids
Brij 97 (a liquid at 37 °C) was weighed into a volumetric flask and made to volume with
phosphate buffer (18 mM NaH2PO4.2H2O, 12 mM Na2HPO4, 108 mM NaCl), followed by pH
adjustment to 6.30 ± 0.01 with H3PO4 solution. From a plot of cinnarizine solubility vs. Brij 97
concentration, 3.09% w/v Brij 97 was identified as the concentration required to provide equal
cinnarizine solubilisation capacity as the model LCFA colloid (i.e. 157.7 µg/mL). This
concentration (3.09% w/v) was therefore used to form the model Brij 97 colloids used in jejunal
perfusion experiments. Solutions of 3.09% w/v Brij 97 were also prepared at pH 5.80, 5.30, 4.80
(pH adjustment via drop wise addition of H3PO4 solution) for cinnarizine equilibrium solubility
determination studies. For the preparation of drug-loaded Brij 97 colloids (for in situ jejunal
perfusion studies), 100 µL of a 130 mg/mL cinnarizine in transcutol stock solution was spiked into
10 mL of model Brij colloids to achieve a final concentration of 130 µg/mL cinnarizine (~ 80%
saturated solubility). When amiloride was included in the Brij 97 colloids, the appropriate mass of
amiloride was dissolved in the prepared colloids at 37 °C, and used within 30 min of preparation.
The total sodium concentration in all the prepared colloids was kept constant at 150 mM.
5.3.5 Equilibrium solubility studies of cinnarizine in colloids
To determine the equilibrium solubility of cinnarizine in LCFA colloids and Brij 97 colloids,
excess solid drug was added to 2 mL of the colloids in glass vials. Vials were briefly vortexed,
incubated at 37 °C, and samples taken every 24 h over a period of 120 h. During sampling, vials
were centrifuged (2,200 xg, 10 min, 37 °C), 50 µL of supernatant sampled, and vials re-vortexed.
Cinnarizine concentration in the supernatant was determined via HPLC. Equilibrium solubility
was defined when cinnarizine concentrations in consecutive supernatant samples varied by ≤ 5%
on three separate occasions. The equilibrium solubility of cinnarizine was also determined when 2
mM amiloride was included in model LCFA colloids, model Brij 97 colloids, and 1:1 v/v mixture
Chapter 5: Lipid absorption triggers drug supersaturation
201
of model LCFA colloids and fasted rat bile, to confirm that solubilisation capacity for cinnarizine
was unaltered by the inclusion of amiloride. Based on physical examination and the maintenance
of consistent drug solubilisation capacity, model LCFA and Brij 97 colloids were stable for 5 days
after preparation.
For some of the LCFA colloids prepared at system pHs < 5.8, phase separation into a highly
dispersed aqueous phase and an undispersed oil phase was evident. Since an oil phase is not likely
to be retained at the UWL in vivo (unionised LCFA is expected to be readily absorbed in close
proximity to the absorptive membrane), the drug solubilisation capacity of the aqueous micellar
phase rather than the oil phase was deemed relevant in assessing expected changes to drug
solubilisation and thermodynamic activity in the UWL. Therefore, to accurately determine the
equilibrium solubility of cinnarizine in the aqueous phase for all LCFA colloids, solubility
samples were left to equilibrate at 37 ºC for 120 h (as equilibrium solubility was attained by this
time), and ultracentrifuged for 30 min at 37 ºC and 400,000 xg (Optima xL-100 K centrifuge, SW-
60 rotor, Beckman, Palo Alto, CA, USA) to separate samples into an aqueous phase, an
undispersed oil phase (if any), and a pellet phase (containing excess solid drug) as described
previously254. Samples from the aqueous phase were assayed for cinnarizine content by HPLC.
5.3.6 Animals
Animal care and anaesthesia have been described in Section 2.2.2.2. All animals were fasted
overnight (12–18 h) prior to surgery. At the end of all experiments, rats were euthanized via an
intravenous or intracardiac injection of 100 mg sodium pentobarbitone.
5.3.7 Surgical procedures
Chapter 5: Lipid absorption triggers drug supersaturation
202
5.3.7.1 Single-pass rat jejunum perfusion
The model employed to assess flux across rat jejunum involved in situ perfusion (single-pass) of
an isolated jejunal segment and simultaneous blood collection from the corresponding mesenteric
vein branch. The surgical procedures for the setup of the single-pass rat jejunum perfusion model
have been described in Section 2.2.2.3.
5.3.7.2 Fasted rat bile collection
Surgical procedures for the collection of fasted bile from donor rats have been described in
Section 4.3.9.1.
5.3.8 In situ single-pass rat jejunum perfusion
After surgery, animals were equilibrated for 30-min, during which time blood was collected from
the cannulated mesenteric vein (~ 0.3 mL/min) to enable re-infusion via the jugular vein.
Perfusion buffer (150 mM Na+, 18 mM H2PO4-, 12 mM HPO42-, 108 mM Cl-, adjusted to pH 6.30
± 0.01) was pumped through the jejunal segment at a rate of 0.1 mL/min and outflowing buffer
discarded to waste. For experiments where amiloride was administered, perfusion buffer
containing 2 mM amiloride was perfused during the equilibration period. The exposed jejunal
segment was kept moist by covering with saline-soaked gauze throughout the experiment.
Perfusate flow through the jejunal segment was maintained at 0.1 mL/min in all experiments to
minimise variations in the thickness of the unstirred water layer which may influence drug flux206.
Following the equilibration period, perfusion buffer was replaced with model colloids. The
perfused colloids were sampled at t = 0 to confirm cinnarizine and/or 3H-oleic acid concentrations.
After this time, the outflowing perfusate was continuously collected at 10-min intervals, and
briefly vortexed before samples were taken for analysis of drug and/or 3H-oleic acid content.
Chapter 5: Lipid absorption triggers drug supersaturation
203
Blood draining the perfused jejunal segment was also collected at 5-min intervals, plasma
separated by centrifugation (10,000 xg, 5 min), and samples taken for analysis of cinnarizine
and/or 3H-oleic acid concentrations.
The concentration of cinnarizine flowing into the perfused jejunal segment was held at 130 µg/mL
in studies assessing the role of the acidic microclimate in the absorption of cinnarizine from model
LCFA-containing or Brij 97 colloids. When amiloride was included in these experiments, it was
pre-dissolved in the model colloids at 37 ºC at a concentration of 2 mM amiloride. In studies that
assessed the role of the acidic microclimate in the absorption of cinnarizine from supersaturated
LCFA-containing colloids (i.e. where co-perfusion of donor rat bile triggered drug supersaturation
in the model LCFA colloids), the concentration of cinnarizine flowing into the perfused jejunal
segment was held at 65 µg/mL. Therefore, in experiments where model colloids were perfused
alone, cinnarizine was loaded into the perfusate at 65 µg/mL (~ 40% saturated solubility). In
experiments where model colloids were co-perfused in a 1:1 v/v ratio with a secondary perfusate
of bile, cinnarizine was loaded into the primary perfusate at 130 µg/mL (~ 80% saturated
solubility), such that 1:1 v/v dilution led to a final perfusate concentration of 65 µg/mL cinnarizine.
When amiloride was included, it was pre-dissolved in bile at 37 ºC at 4 mM, such that 1:1 v/v
dilution led to a final perfusate concentration of 2 mM amiloride. Both model LCFA colloids and
bile were pumped at 0.05 mL/min, and mixed via a three-way “T” connector immediately prior to
entry into the jejunal segment, such that total perfusate flow was maintained at 0.1 mL/min. Since
luminal cinnarizine supersaturation was generated in these experiments, outflowing perfusate
samples were taken before and after centrifugation (2,200 xg, 2 min), to obtain an indication of the
degree of drug precipitation within the jejunal segment.
Chapter 5: Lipid absorption triggers drug supersaturation
204
5.3.9 Analytical procedures
5.3.9.1 Sample preparation and HPLC assay conditions for cinnarizine
Samples of LCFA colloids and Brij 97 colloids were prepared for HPLC assay by a minimum 20-
fold dilution with mobile phase (50% v/v acetonitrile:50% v/v 20 mM NH4H2PO4). Plasma
samples were prepared for HPLC using a validated extraction procedure, with flunarizine as an
internal standard, as reported previously227. Replicate analysis of n = 4 quality control samples
revealed acceptable accuracy and precision (± 10%, ± 15% at the limit of quantification) for
cinnarizine concentrations between 20–1000 ng/mL for colloids, and 10–320 ng/mL for plasma.
5.3.9.2 Scintillation counting
Quantification of 3H-oleic acid in perfusate and collected plasma was performed via scintillation
counting on a Packard Tri-Carb 2000CA liquid scintillation analyser (Packard, Meriden,
Connecticut, USA). Perfusate samples (100 µL) and plasma samples (200 µL) were added to 2 mL
Irga-safe Plus scintillation fluid followed by a 10-sec vortex. Samples were corrected for
background radioactivity by the inclusion of a blank sample in each run.
5.3.9.3 Blood:plasma ratio determination for cinnarizine, oleic acid
The blood:plasma ratios for cinnarizine and oleic acid have been determined as described in
Chapter 2. The mean blood:plasma ratio was used to convert plasma concentrations to blood
concentrations in perfusion experiments, enabling quantification of total compound transport into
mesenteric blood.
Chapter 5: Lipid absorption triggers drug supersaturation
205
5.3.9.4 Calculations
In the single-pass rat jejunum perfusion model, permeability coefficients were calculated from the
flux data obtained after attainment of steady state drug transport into mesenteric blood. Two
apparent permeability coefficients (Papp) were calculated as described previously184:
′Disappearance′P . ln Equation 5.2
′Appearance′P∆
∆.
Equation 5.3
where ‘Disappearance’ Papp is the apparent permeability coefficient calculated from drug loss from
the perfusate (cm/sec); ‘Appearance’ Papp is the apparent permeability coefficient calculated from
drug appearance in the mesenteric blood (cm/sec); Q is the perfusate flow rate (mL/sec); A is the
surface area of the perfused jejunal segment (cm2), which is calculated by multiplying the
diameter and the length of the perfused intestinal segment as described previously207; C1 is the
average steady state drug concentration exiting the perfused jejunal segment (ng/mL); C0 is the
drug concentration entering the jejunal segment (ng/mL); ∆MB/∆t is the average rate of drug mass
appearance in mesenteric blood at steady state (ng/sec); and <C> is the logarithmic mean drug
concentration in the lumen (ng/mL), where <C> = (C1 – C0) / (ln C1 – ln C0).
5.3.10 Statistical analysis
Statistically significant differences were determined by ANOVA followed by Tukey's test for
multiple comparisons at a significance level of α = 0.05 using SPSS v19 for Windows (SPSS Inc.,
Chicago, IL, USA).
Chapter 5: Lipid absorption triggers drug supersaturation
206
5.4 RESULTS
5.4.1 Attenuation of the acidic microclimate using amiloride reduces oleic
acid and cinnarizine absorption from model LCFA colloids; but has no effect
on the absorption of cinnarizine from fatty acid-free Brij 97 colloids
Figure 5.2 shows the intestinal absorptive flux vs. time profiles of oleic acid (from LCFA
containing colloids), and cinnarizine (from both LCFA and Brij 97 colloids) in the absence and
presence of 2 mM amiloride during in situ rat jejunum perfusion experiments. Corresponding
steady state-absorptive flux, disappearance Papp, and appearance Papp data are reported in Table 5.1
Perfusate disappearance profiles are shown in Figure 5.3.
Administration of amiloride reduced the absorption of oleic acid, resulting in a significant (α <
0.05) 3.0-fold, 1.4-fold, and 3.0-fold reduction in absorptive flux, disappearance Papp, and
appearance Papp of oleic acid, respectively (Figure 5.2A, Table 5.1). Amiloride administration also
led to a significant reduction in cinnarizine absorption from LCFA colloids, resulting in 6.5-fold,
2.8-fold, and 5.7-fold reductions (α < 0.05) in the absorptive flux, disappearance Papp, and
appearance Papp of cinnarizine, respectively (Figure 5.2B, Table 5.1). In contrast, co-
administration of amiloride with Brij 97 colloids did not lead to significant changes in the
absorptive flux, disappearance Papp, and appearance Papp of cinnarizine (Figure 5.2C, Table 5.1).
The model colloids were chosen such that the cinnarizine solubilisation capacity in each was the
same (157.7 ± 3.0 µg/mL and 153.2 ± 2.5 µg/mL for LCFA colloids and Brij 97 colloids
respectively, mean ± SEM of n = 3 determinations), and drug was loaded at the same
concentration (130 µg/mL). Cinnarizine thermodynamic activity was therefore also matched and
cinnarizine was dissolved at ~ 80% saturated solubility in both colloidal solutions. In the absence
Chapter 5: Lipid absorption triggers drug supersaturation
207
of amiloride (i.e. in the presence of an intact acidic microclimate), the absorptive flux of
cinnarizine from model LCFA colloids was 4.9-fold higher than from Brij 97 colloids (Figure
5.2B & C, Table 5.1). This difference was abolished in the presence of amiloride (i.e. under
conditions where the acidic microclimate was attenuated and lipid absorption inhibited). The data
suggest that cinnarizine absorption is significantly more efficient from LCFA-containing intestinal
colloids than from Brij 97 colloids, in spite of matched initial thermodynamic activity in both
colloids, and that this is dependent on the presence of an acidic microclimate at the intestinal
UWL. For the model LCFA colloids, the increase in cinnarizine absorption in the absence of
amiloride also occurred coincidentally with an increase in oleic acid absorption (Figure 5.2A & B,
Table 5.1).
Chapter 5: Lipid absorption triggers drug supersaturation
208
(A)
(B)
(C)
Figure 5.2: Mesenteric blood appearance profiles of (A) oleic acid (OA) from model LCFA
colloids, (B) cinnarizine (CIN) from model LCFA colloids, and (C) cinnarizine (CIN) from model
Brij 97 colloids, after 70 min single-pass perfusion of ~ 10 cm2 segments of rat jejunum with and
without co-administration of 2 mM amiloride, which attenuates the acidic microclimate of the
intestinal unstirred water layer. Model LCFA colloids and model Brij 97 colloids had equal
solubilisation capacity for CIN (157.7 ± 3.0 µg/mL and 153.2 ± 2.5 µg/mL, respectively; average
± SEM of n = 3 determinations). CIN was loaded into both colloids at constant concentration (130
µg/mL) and thermodynamic activity (~ 80% saturated solubility). Data represent mean ± SEM of
n = 3-4 experiments. Steady state-absorptive flux, disappearance Papp, and appearance Papp of CIN
and OA from this series of experiments are tabulated in Table 5.1.
Time (min)
0 10 20 30 40 50 60 70
OA
flux
into
mes
ent
eric
b
loo
d (
ng
/5 m
in/1
0 c
m2
)0
2000400060008000
100001200014000 LCFA colloids
LCFA colloids + amiloride
Time (min)
0 10 20 30 40 50 60 70
CIN
flu
x in
to m
esen
teric
bl
ood
(ng/
5 m
in/1
0 cm
2 )
0
200
400
600
800
1000LCFA colloidsLCFA colloids + amiloride
Time (min)
0 10 20 30 40 50 60 70
CIN
flu
x in
to m
esen
teric
bl
ood
(ng/
5 m
in/1
0 cm
2 )
0
200
400
600
800
1000 Brij 97 colloids Brij 97 colloids + amiloride
Chapter 5: Lipid absorption triggers drug supersaturation
209
Table 5.1: Cinnarizine (CIN) and oleic acid (OA) disappearance Papp (x 106 cm/sec) from the
intestinal perfusate, appearance Papp (x 106 cm/sec) in the mesenteric blood, and steady state
absorptive flux into mesenteric blood (ng/5 min/10 cm2) after 70 min of single-pass perfusion of ~
10 cm2 segments of rat jejunum with model long chain fatty acid (LCFA) colloids or model Brij
97 colloids, with and without co-administration of 2 mM amiloride. Values calculated using data
obtained after steady state attainment (t = 55-70 min). Data represent mean ± SEM of n = 3-4
experiments.
Perfusate CIN conc.
(µg/mL)
OA conc.
(µg/mL)
Disappearance Papp (x
106 cm/sec)
Appearance Papp
(x 106 cm/sec) Mesenteric blood
flux (ng/5min/10cm2)
CIN LCFA colloid 130 - 35.5 ± 2.2 1.7 ± 0.4 600 ± 129
LCFA colloid + amiloride
130 - 12.7 ± 4.1 a 0.3 ± 0.1 a 93 ± 25 a
Brij 97 colloid 130 - 14.0 ± 6.7 a 0.3 ± 0.0 a 123 ± 14 a
Brij 97 colloid + amiloride
130 - 5.4 ± 1.2 a 0.2 ± 0.0 a 90 ± 3 a
OA LCFA colloid - 1000 22.1 ± 2.0 3.3 ± 0.4 9144 ± 991
LCFA colloid + amiloride
- 1000 15.3 ± 0.9 b 1.1 ± 0.1 b 3045 ± 304 b
a Significant difference (α < 0.05) from model LCFA colloid group (containing 130 µg/mL CIN) in the absence of amiloride
b Significant difference (α < 0.05) from model LCFA colloid group (containing 1000 µg/mL OA) in the absence of amiloride
Chapter 5: Lipid absorption triggers drug supersaturation
210
(A)
(B)
(C)
Figure 5.3: Perfusate disappearance profiles of (A) oleic acid (OA) from model LCFA colloids,
(B) cinnarizine (CIN) from model LCFA colloids, and (C) cinnarizine (CIN) from model Brij 97
colloids, after 70 min single-pass perfusion of ~ 10 cm2 segments of rat jejunum with and without
co-administration of 2 mM amiloride, which attenuates the acidic microclimate of the intestinal
unstirred water layer. Model LCFA colloids and model Brij 97 colloids had equal solubilisation
capacity for cinnarizine (157.7 ± 3.0 µg/mL and 153.2 ± 2.5 µg/mL, respectively; average ± SEM
of n = 3 determinations). CIN was loaded into both colloids at constant concentration (130 µg/mL)
and thermodynamic activity (~ 80% saturated solubility). Data represent mean ± SEM of n = 3-4
experiments.
Time (min)
0 30 40 50 60 70
% O
A d
ose
pass
ing
thro
ugh
jeju
num
60
70
80
90
100
LCFA colloids LCFA colloids + amiloride
Time (min)
0 30 40 50 60 70
% C
IN d
ose
pass
ing
thro
ugh
jeju
num
60
70
80
90
100
LCFA colloids LCFA colloids + amiloride
Time (min)
0 30 40 50 60 70
% C
IN d
ose
pass
ing
thro
ugh
jeju
num
60
70
80
90
100
Brij 97 colloids Brij 97 colloids + amiloride
Chapter 5: Lipid absorption triggers drug supersaturation
211
5.4.2 Exposure of model LCFA colloids to the acidic microclimate, and
absorption of lipid components, leads to cinnarizine supersaturation and
enhanced thermodynamic activity
To examine the possible mechanisms by which pH changes at the UWL (due to the acidic
microclimate), and the process of lipid absorption, result in changes to cinnarizine absorption, the
equilibrium solubility of cinnarizine in LCFA colloids was assessed in systems with decreasing
pH and decreasing lipid concentration (to simulate exposure to the acidic microclimate and lipid
absorption, Figure 5.4A). Equivalent data were also generated for Brij 97 colloids with decreasing
system pH, in this case to simulate exposure to the acidic microclimate only, as Brij 97 is not
absorbed (Figure 5.4B).
As system pH decreased, the LCFA colloids became increasingly turbid, ultimately leading to
phase separation into an aqueous phase and an undispersed oil phase (Figure 5.5). Phase
separation occurred earlier (i.e. at higher pHs) in systems containing higher concentrations of lipid.
Systems that phase separated are asterisked in Figure 5.4A. Phase separation occurred at pH 4.8
for all LCFA-based colloids and at and below pH 5.3 for colloids containing the highest lipid load
(i.e. 0.1% w/v oleic acid). Even where phase separation did not occur, the turbidity of the LCFA
colloidal solutions increased with increasing lipid concentration. Development of turbidity was not
evident in SEIF (i.e. 0% oleic acid) or the Brij 97 colloids (Figure 5.5).
Changes in turbidity of the LCFA-containing colloids appeared to correlate with changes to
solubilisation capacity, such that drug solubility was higher in systems with increasing turbidity.
As depicted in Figure 5.4A, cinnarizine solubility in LCFA colloids increased significantly with
increasing lipid concentration and also increased (albeit more moderately) with decreasing pH.
These trends continued until phase separation occurred, at which point cinnarizine solubility in the
Chapter 5: Lipid absorption triggers drug supersaturation
212
micellar phase was reduced due to partitioning of lipids from the micellar phase into a poorly
dispersed lipid phase. At 0% incorporated lipid (i.e. SEIF only), cinnarizine solubility also
increased slightly with decreasing pH. However, this increase was small when compared to the
increase in solubility afforded by increasing lipid concentration or decreasing pH in the LFCA-
containing colloids.
In vivo, exposure of LCFA colloids to the intestinal acidic microclimate results in a decrease in pH
and an increase in LCFA absorption. Changes to cinnarizine solubility under the same
circumstances are therefore expected to be predicted by assessment of solubility changes under
conditions where system pH and lipid concentration are decreased simultaneously. ‘O’ in Figure
5.4 identifies the maximum cinnarizine solubilisation capacity of the LCFA and Brij 97 colloids.
Cinnarizine was loaded into model colloids at 80% saturated solubility, i.e. 130 µg/mL – blue
dotted line. At the highest lipid load (0.1% w/v OA), exposure of the LCFA containing colloids to
decreases in pH that are consistent with conditions in the acidic microclimate, initially increased
cinnarizine solubility (at pH 5.8), but ultimately reduced cinnarizine solubility at pH 5.3 (the
lowest reported microclimate pH45) (Figure 5.4). The solubility of cinnarizine was also highly
dependent on the concentration of incorporated lipids, and a reduction in oleic acid content
(consistent with the reductions expected on lipid absorption) significantly reduced cinnarizine
solubility. Indeed, the drop in cinnarizine solubility seen at pH 5.3 most likely reflects a loss of
micellar lipid content due to phase separation rather than an effect of pH alone. A theoretical line
in Figure 5.4A (red broken arrow) depicts the changes in cinnarizine solubility expected under
conditions where pH is reduced to pH 5.30 and lipids are fully absorbed. Under these
circumstances, cinnarizine solubility is reduced dramatically and in the absence of precipitation, is
expected to lead to supersaturation, enhanced thermodynamic activity and improved absorption.
Chapter 5: Lipid absorption triggers drug supersaturation
213
Conversely, in Brij 97 colloids, a decrease in system pH led to increased cinnarizine solubility
(Figure 5.4B), presumably due to increased cinnarizine ionisation at lower pH. Since Brij 97 is not
absorbed, the concentration of micellar surfactant is expected to remain constant during passage
across the UWL, and the only driver of changes to drug solubility at the UWL is the reduction in
pH due to the acidic microclimate. As depicted by the red broken arrow in Figure 5.4B,
cinnarizine solubilisation is therefore expected to increase during passage of Brij 97 colloids
across the UWL, leading to reduced thermodynamic activity.
Chapter 5: Lipid absorption triggers drug supersaturation
214
(A)
LCFA colloids
(B)
3.09% w/v Brij 97 colloids
Figure 5.4: (A) Three-dimensional plot of cinnarizine (CIN) solubility (37 °C) in the aqueous
phase of LCFA-containing intestinal colloids as a function of oleic acid and monoolein
concentration in bile micelles (concentration on y-axis refers to oleic acid concentration) and
system pH (B) CIN solubility (37 °C) in 3.09% w/v Brij 97 colloids as a function of system pH.
Concentration of bile components in (A) was kept constant. Black solid arrows depict the
expected change in CIN solubilisation capacity when (1) colloids are exposed to the acidic
microclimate of the UWL, and (2) as oleic acid and monoolein are absorbed and removed from
colloids (absorption of colloidal components only applies to LCFA colloids as Brij 97 is non-
absorbable). * in (A) denotes colloidal systems where phase separation into an aqueous phase and
undispersed oil phase was evident during the 120 h equilibrium solubility determination study (see
Figure 5.5). ‘O’ denotes the solubilisation capacity of the model LCFA and Brij 97 colloids used
in Figure 5.2, and the blue dotted line shows the concentration of CIN (i.e. 130 µg/mL – 80%
saturated solubility) in the model colloids used in Figure 5.2 and Table 5.1. The red broken arrows
depict (A) the theoretical solubility trend when pH of system is reduced by 1 unit to pH 5.3
(lowest pH reported in the literature45) and lipids are completely removed, and (B) when pH of
system is reduced by 1 unit to pH 5.3 such as on entry into the UWL. The passage of model LCFA
colloids across the UWL and subsequent absorption of lipid digestion products is therefore
expected to reduce cinnarizine solubility, leading to supersaturation, enhanced thermodynamic
potential and absorption; while the passage of model Brij 97 colloids across the UWL leads to a
small increase in CIN solubility and reduced thermodynamic potential.
0
50
100
150
200
0.1000.1000.1000.100
0.0500.0500.0500.0500.0250.0250.0250.025
0.0000.0000.0000.0004.8
5.35.8
6.3
* **
*
O
(1)(2)
pH
6.3 5.8 5.3 4.8
CIN
so
lub
ility
(µ
g/m
L)
0
50
100
150
200
250
300
O
(1)
Chapter 5: Lipid absorption triggers drug supersaturation
215
(A)
0% OA 0% MO
(B)
0.025% w/v OA 0.016% w/v MO
(C)
0.050% w/v OA 0.032% w/v MO
(D)
0.10% w/v OA 0.06% w/v MO
(E)
3.09% w/v Brij 97
Figure 5.5: The appearance of LCFA-containing intestinal colloids (panel A-D) and 3.09% w/v
Brij 97 colloids (panel E) 120 h into cinnarizine equilibrium solubility determination studies, after
vials were centrifuged at 2,200 xg for 10 min at 37 °C. LCFA-containing colloids consisted oleic
acid (OA) and monoolein (MO) (concentrations as labelled) solubilised in simulated endogenous
intestinal fluid (SEIF) containing 4 mM total bile salt, 1 mM lysophosphatidylcholine and 0.25
mM cholesterol. In each panel, the system pH of colloids was, from left to right, 6.30, 5.80, 5.30
Chapter 5: Lipid absorption triggers drug supersaturation
216
and 4.80, respectively. In LCFA-containing systems, the turbidity of samples was observed to
increase when system pH was decreased, up to a point where colloids were destabilised and
underwent phase separation into an aqueous phase and an undispersed oil phase (undispersed oil
phase floats on top of aqueous phase and may not be seen clearly in pictures), by which point the
samples appeared clear again. * denotes colloidal systems where phase separation into an aqueous
phase and undispersed oil phase was evident. Since the concentration of bile components was held
constant, phase separation of colloidal system with 0.10% w/v OA (Panel D) occurred at a higher
pH than systems containing 0.05 and 0.025% w/v OA (Panel B and C). Turbidity did not develop
in SEIF containing 0% OA (Panel A) or 3.09% w/v Brij 97 colloids (Panel E). White mass at the
bottom of vials is excess solid cinnarizine added in the solubility determination studies. The
pictures illustrate the impact of the acidic microclimate on the microstructure of LCFA-containing
intestinal colloids, where exposure of LCFA colloids to the microclimate pH leads to protonation
of LCFA, increasing the ratio of unionised LCFA:ionised LCFA and resulting in the formation of
larger colloids with reduced thermodynamic stability in vitro. In vivo, the increase in LCFA
thermodynamic activity is expected to lead to enhanced micellar dissociation and absorption.
Chapter 5: Lipid absorption triggers drug supersaturation
217
5.4.3 Attenuation of the acidic microclimate using amiloride abolishes bile-
induced, supersaturation-enhanced, cinnarizine absorption from model
LCFA colloids
In Chapter 4, co-perfusion of fasted rat bile with cinnarizine-loaded model LCFA colloids
triggered cinnarizine supersaturation in the GI lumen and enhanced cinnarizine absorptive flux by
3.2-fold in an in situ rat jejunal perfusion model (Figure 5.6B, Table 5.2). In the current chapter,
inclusion of 2 mM amiloride in the same perfusate (i.e. 1:1 v/v mixture of model LCFA colloids
and fasted rat bile) reduced the absorptive flux and appearance Papp of oleic acid by 4.6-fold and
4.4-fold, respectively (Table 5.2, Figure 5.6A). A coincident decrease in the absorptive flux and
appearance Papp of cinnarizine (a decrease of 5.2-fold and 5.0-fold, respectively) was also
observed. Cinnarizine appearance permeability in the presence of bile and amiloride was therefore
not significantly different to that in the absence of bile (and therefore in the absence of
supersaturation) (Figure 5.6B, Table 5.2). Analysis of the outflowing perfusate in the permeability
experiment suggested that cinnarizine supersaturation was maintained in the bulk GI fluids, in
both the absence and presence of amiloride, (i.e. precipitation did not occur, Figure 5.7B).
Amiloride co-administration therefore negated the increase in cinnarizine absorption stimulated by
bile-mediated supersaturation. Amiloride did not directly influence cinnarizine solubility in model
LC colloids in the presence or absence of bile (cinnarizine solubility in LC colloids + amiloride
was 151.6 ± 17.0 µg/mL, cinnarizine solubility in LC colloids + bile + amiloride was 10.3 ± 0.3
µg/mL respectively; mean ± SEM; n =3).
Chapter 5: Lipid absorption triggers drug supersaturation
218
(A) (B)
Figure 5.6: Absorptive flux-time profiles of (A) oleic acid (OA) and (B) cinnarizine (CIN) when
model LCFA colloids were perfused via single-pass through an isolated rat jejunal segment (~ 10
cm2), with and without 1:1 v/v co-perfusion with donor rat bile, in the absence and presence of 2
mM amiloride. * Data in the absence of amiloride are reproduced from Chapter 4. Co-perfusion of
rat bile generates CIN supersaturation in situ within the perfused jejunal segment, and increased
cinnarizine absorptive flux by 3.2-fold. Inclusion of 2 mM amiloride in the perfusate, however,
abolished the absorption-enhancing effects of bile-induced supersaturation. SS denotes drug
supersaturation in perfusate. Data represent mean ± SEM of n = 3-4 rats. Steady state-absorptive
flux, disappearance Papp, and appearance Papp of CIN and OA from this series of experiment are
tabulated in Table 5.2.
Time (min)
0 10 20 30 40 50 60 70% O
A d
ose
into
me
sen
teric
b
loo
d (
%/5
min
/10
cm
2)
0
1
2
3
4
5
Time (min)
0 10 20 30 40 50 60 70
CIN
flu
x in
to m
esen
teric
bl
ood
(ng/
5 m
in/1
0 cm
2 )
0
100
200
300
400
500
600
Chapter 5: Lipid absorption triggers drug supersaturation
219
Table 5.2: Cinnarizine (CIN) and oleic acid (OA) disappearance Papp (x 106 cm/sec) from the
intestinal perfusate, appearance Papp (x 106 cm/sec) in the mesenteric blood, and steady state
absorptive flux into mesenteric blood (ng/5 min/10 cm2) after 70 min of single-pass perfusion of ~
10 cm2 segments of rat jejunum with model LCFA colloids, with and without 1:1 v/v co-perfusion
with donor rat bile, in the absence and presence of 2 mM amiloride. Data in the absence of
amiloride are obtained from Chapter 4. Values calculated using data obtained after steady state
attainment (t = 55-70 min). Data represent mean ± SEM of n = 3-4 experiments.
Perfusate CIN conc.
(µg/mL)
OA conc.
(µg/mL)
DisappearancePapp
(x 106 cm/sec)
Appearance Papp
(x 106 cm/sec)
Mesenteric blood flux
(ng/5min/10cm2)
CIN LCFA colloid * 65 - 12.2 ± 2.8 0.6 ± 0.2 138 ± 28
LCFA colloid + Bile SS * 65 - 36.9 ± 5.5 a 2.5 ± 0.2 a 443 ± 34 a
LCFA colloid + Bile + amiloride SS 65 - 52.9 ± 6.3 a 0.5 ± 0.0 b 85 ± 4 b
OA LCFA colloid * - 1000 18.0 ± 5.9 2.0 ± 0.3 5607 ± 863
LCFA colloid + Bile * - 500 26.1 ± 3.8 6.2 ± 0.8 a 8557 ± 1205
LCFA colloid + Bile + amiloride - 500 35.0 ± 4.1 a 1.4 ± 0.4 b 1853 ± 441 b
a Significant difference (α<0.05) from model LCFA colloid group in the absence of amiloride b Significant difference (α<0.05) from model LCFA colloid + Bile group in the absence of amiloride
* Papp and flux data reproduced from Chapter 4 SS denotes drug supersaturation in perfusate, induced by bile dilution of model LCFA colloids
Chapter 5: Lipid absorption triggers drug supersaturation
220
(A) (B)
Figure 5.7: Perfusate disappearance (% drug dose passing through jejunum) profiles of (A) oleic
acid (OA) and (B) cinnarizine (CIN) when model LCFA colloids were perfused via single-pass
through an isolated rat jejunal segment (~ 10 cm2), with and without 1:1 v/v co-perfusion with
donor rat bile, in the absence and presence of 2 mM amiloride. * Data in the absence of amiloride
are reproduced from Chapter 4. Co-perfusion of model LCFA colloids with rat bile generates drug
supersaturation in situ within the perfused jejunal segment. The degree of drug precipitation
within the perfusate is represented by the difference in CIN perfusate concentration pre- and post-
centrifugation. SS denotes drug supersaturation in perfusate. Data represent mean ± SEM of n = 3-4
rats.
Time (min)
0 30 40 50 60 70
% 3
H-O
A d
ose
pa
ssin
gth
rou
gh
jeju
nu
m
50
60
70
80
90
100
Time (min)
0 30 40 50 60 70
% C
IN d
ose
pas
sin
g
thro
ug
h je
jun
um
50
60
70
80
90
100
Chapter 5: Lipid absorption triggers drug supersaturation
221
5.5 DISCUSSION
The mechanism of absorption of long-chain fatty acid (LCFA) from intestinal mixed micelles is
well described in the literature. Westergaard and Dietschy’s seminal studies initially proposed that
solubilisation of LCFA within bile salt micelles increased LCFA diffusion across the intestinal
unstirred water layer (UWL), and increased the concentration of LCFA presented to the absorptive
membrane51. Shiau and colleagues later suggested that the acidic microclimate within the UWL
further enhanced LCFA absorption by protonating ionised LCFA, leading to enhanced micellar
dissociation and absorption56. These initial studies have also been followed by several studies
describing the role of active transport systems such as CD3661, FATP62 and SR-BI63 in lipid
uptake across the apical absorptive membrane, although the quantitative importance of active vs.
passive transport as a means of lipid absorption under differing lipid loads remains contentious58-60,
64, 255.
In contrast to lipid absorption, the mechanism of absorption of poorly water-soluble drugs (PWSD)
from intestinal mixed micelles has been less clearly defined. We recently described the potential
for drug supersaturation to be induced during the interactions of lipid colloidal phases (mixed
micelles and vesicles) with secreted bile (Chapter 3 and 4), and suggested that supersaturation-
enhanced absorption may be an endogenous mechanism to reverse the reduction in
thermodynamic activity inherent in drug solubilisation within colloidal phases. During these
studies, however, it became apparent that multiple mechanisms may underpin the generation of
drug supersaturation in intestinal mixed micelles. For weak bases with high solubility in LCFA
containing micelles, interaction with bile appeared to decrease micellar drug solubility with
increasing bile concentration. This in turn resulted in transient supersaturation and absorption
promotion. However, interaction with bile also promoted (albeit to a lesser extent) the absorption
of a non-ionic drug (danazol), where micellar solubility increased rather than decreased with
Chapter 5: Lipid absorption triggers drug supersaturation
222
increasing bile concentrations. This occurred simultaneously with an increase in lipid absorption.
These previous studies suggest that lipid absorption and drug absorption may be linked, and
stimulated the current hypothesis, i.e. that lipid absorption from intestinal mixed micelles reduces
drug solubility in micellar structures, thereby inducing supersaturation and promoting drug
absorption.
The possibility that protonation and absorption of LCFA from intestinal mixed micelles during
passage across the UWL can trigger drug supersaturation and enhance drug absorption has been
studied in two ways. Firstly, via an examination of changes to cinnarizine solubility in mixed
micelles as a function of changes to pH and lipid concentration; and secondly by modifying the
conditions within the UWL such that LCFA protonation and absorption are suppressed, and
examining the effects of suppressed lipid absorption on cinnarizine absorption. The latter was
achieved by inhibiting the action of the Na+/H+ antiporter at the enterocyte brush border, thereby
reversing the acidity of the microclimate adjacent to the absorptive surface46. The acidic
microclimate has long been suggested to be important in the absorption of LCFA56. The pKa of
oleic acid, a common LCFA component (or digestion product of dietary or formulation-derived
lipids), is 9.85256. When solubilised in bile salt micelles, however, the pKa of oleic acid decreases
to 6.3–6.5257. As such pH changes from 6.5 in the GI lumen, to 5.3 in the UWL45, significantly
increase the unionised:ionised fraction for LCFA. A decrease in ionisation is expected to increase
LCFA absorption by 1) increasing cellular partitioning due to the pH-partition effect and 2)
increasing thermodynamic activity due to a reduction in amphiphilicity and micellar solubility,
and stimulation of micellar dissociation (as described by Shiau and coworkers56, 57). In contrast,
conjugated bile salts are not absorbed in the upper GI tract. The pKa values of taurine and glycine
conjugated bile salts are <1 and ~3.8, respectively258. Bile salts therefore remain ionised during
passage across the UWL, and in the absence of absorption, bile salt micellar structures are
Chapter 5: Lipid absorption triggers drug supersaturation
223
expected to persist. The lipid content of the micelles however, is expected to reduce (due to
enhanced LCFA micellar dissociation and absorption), reducing drug solubilisation capacity and
generating the conditions required for drug supersaturation. In the current studies cinnarizine was
employed as a model PWSD to examine the impact of the acidic UWL on drug absorption from
mixed micelles. Cinnarizine was chosen for two reasons. Firstly, previous studies have shown that
cinnarizine is highly soluble in bile salt-fatty acid mixed micelles; and solubility is highly
dependent on micellar oleic acid content (Chapter 4). Thus, cinnarizine is likely to be amenable to
enhancements in drug thermodynamic activity induced by oleic acid absorption. Secondly, as a
base, exposure to the UWL microenvironment acidity is expected to increase drug ionisation, and
therefore decrease drug absorption based on pH partition relationships. As such, any increase in
drug absorption that occurs due to microenvironment acidity is expected to reflect mechanisms
unrelated to drug ionisation/partitioning.
Under pH conditions reflective of the acidic UWL, the LCFA containing colloids examined here
became increasingly turbid, suggesting increases in colloid particle size consistent with the
observations of Shiau et al.57. The increase in turbidity with decreasing pH only occurred in
systems containing LCFA, and developed instantly (see Figure 5.8 for the appearance of model
LCFA colloids after the addition of one drop of 20% v/v H3PO4). Reduced LCFA solubility in bile
salt micelles manifests as an increase in system turbidity or phase separation in vitro. However, in
vivo where an absorptive sink is present directly adjacent to the UWL, increased LCFA
thermodynamic activity and an increase in unionised:ionised LCFA is expected to contribute to
enhanced absorption56, 57. Consistent with this suggestion, the absorptive flux of oleic acid was
significantly reduced when the acidic microclimate of the UWL was attenuated by co-
administration with amiloride (Figure 5.2A). This is consistent with previous data that have shown
reduced oleic acid uptake into rat jejunal brush border vesicles in the presence of amiloride259.
Chapter 5: Lipid absorption triggers drug supersaturation
224
(A)
Model LCFA colloids
(B)
Model Brij 97 colloids
Figure 5.8: Appearance of cinnarizine-loaded (A) model LCFA colloids and (B) model Brij 97
colloids immediately after the addition of one drop of 20% v/v H3PO4. Turbidity instantly
developed in model LCFA colloids while model Brij 97 colloids remained optically clear. The
addition of H3PO4 reduced the system pH of model LCFA colloids and Brij 97 colloids from 6.30
to 5.92, and 6.30 to 6.01, respectively. Magnetic stirrers are present at the bottom of vials. This
figure visually depicts the effect of protonation of oleic acid (OA) when model LCFA colloids are
exposed to acidic conditions. An increase in the ratio of unionised OA:ionised OA is thought to
enhance the thermodynamic potential of OA, and induce a micelle to emulsion transition in the
colloids.
In light of the significant reduction in lipid absorption in the presence of amiloride, this
experimental system was utilised to explore the potential link between lipid absorption and drug
absorption. The data in Figure 5.2B show a very clear reduction in cinnarizine absorption in the
presence of amiloride. The reduction in drug absorption occurred coincidently with the reduction
in LCFA absorption (Figure 5.2A). In contrast, perfusion of Brij 97 colloids containing the same
concentrations of cinnarizine at the same thermodynamic activity resulted in drug flux that was
significantly lower than that from LCFA-containing colloids in the absence of amiloride, and was
independent of the co-administration of amiloride. The data suggest that drug absorption from
micellar systems containing LCFA is inherently more effective than from solubilising systems that
Chapter 5: Lipid absorption triggers drug supersaturation
225
lack fatty acids, and that fatty acid absorption is critical in mobilising the solubilised fraction to
maximise drug absorption. These findings are significant in the context of the design of lipid-
based formulations, and suggest that formulations containing absorbable lipids may have inherent
advantages over systems containing non-absorbable surfactants and co-solvents. Consistent with
this suggestion, previous studies comparing danazol absorption from LBF comprising surfactants
and cosolvents alone (i.e. LFCS Type IV formulations) and formulations where the same
surfactants and cosolvents were combined with glyceride lipids (i.e. a more traditional LFCS
Type III self-emulsifying formulation), suggest increased drug absorption from the lipid
containing formulations260. Thus, despite similar solubilisation properties in in vitro dispersion
and digestion tests, a glyceride lipid-containing formulation (55% w/w Cremophor RH 40, 7.5%
w/w ethanol, 37.5% w/w Soybean oil:Maisine (1:1 w/w)) outperformed (~ 2-fold difference in in
vivo exposure of danazol) a similar surfactant/cosolvent formulation (55% w/w Cremophor RH 40,
7.5% w/w ethanol, 37.5% w/w Pluronic L121). We were previously unable to explain these
observations, but the current studies provide some justification for the apparent advantage of
formulations containing absorbable lipids.
To better understand the impact of microclimate pH and lipid absorption on drug absorption from
LCFA-containing intestinal colloids, a series of studies was undertaken to detail changes in drug
solubility under conditions of both reduced pH and decreased lipid content. As a weak base with a
pKa of 7.47, cinnarizine is increasingly ionised at lower pH, and this was reflected in increases in
solubility when the pH of blank SEIF (i.e. bile micelles with 0% solubilised lipids) and model Brij
97 colloids was decreased from 6.3 to 4.8 (Figure 5.4). In LCFA-containing colloids, decreasing
pH initially resulted in the formation of swollen colloidal particles with increased turbidity and
higher cinnarizine solubilisation capacity (Figure 5.4), consistent with previous studies that
suggest increases in drug solubility in larger colloidal particles39. The expected changes to
Chapter 5: Lipid absorption triggers drug supersaturation
226
cinnarizine solubility resulting from pH changes in the acidic UWL alone (i.e. an increase in
ionisation and solubility, leading to a decrease in thermodynamic activity), are therefore unlikely
to promote drug absorption and are unable to explain the in vivo data in Figure 5.2B.
However, a second major change to colloidal structure was expected on entry into the UWL in
vivo, namely a significant reduction in micellar lipid content due to the promotion of LCFA
micellar dissociation and absorption. At lower pHs in vitro, decreases in micellar lipid content
were also stimulated by phase separation out of the micellar phase (as indicated by asterisks in
Figure 5.4A, Figure 5.5). Thus, during passage of LCFA colloids across the acidic UWL in vivo,
the lipid content of the micellar phase is expected to drop due to lipid absorption and potentially
due to lipid phase separation, although in vivo, in the presence of a large absorption sink, phase
separation may be less prevalent.
Since cinnarizine solubility in intestinal colloids is highly dependent on the concentration of
incorporated lipids (cinnarizine solubility in LCFA colloids decreased dramatically with
decreasing lipid load at each pH studied - Figure 5.4A), decreasing lipid concentration is likely to
be the predominant factor in determining cinnarizine solubilisation at the UWL. Therefore, in
predicting the impact of both a decrease in pH and a coincident decrease in micellar lipid
concentration in the UWL on drug solubilisation (Figure 5.4A), the net change to the solubility of
cinnarizine in the aqueous phase is likely to be a reduction due to the loss in lipid content. In the
absence of precipitation, this will drive supersaturation and enhance drug thermodynamic activity,
consistent with the significant increase in drug flux seen in vivo under conditions where lipid
absorption was enhanced (Figure 5.2A & B).
Chapter 5: Lipid absorption triggers drug supersaturation
227
The ability of the LCFA colloids to harness acidic microclimate-induced drug supersaturation
therefore stems from the ability of LCFA to respond to a decrease in pH at the UWL, leading to
enhanced LCFA absorption, subsequently decreasing colloid solubilisation capacity for PWSD.
That is, the presence of oleic acid confers pH sensitivity to LCFA-containing colloids and serves
as a trigger for acidic microclimate-induced drug supersaturation, ultimately enabling the
absorption of solubilised drug via enhancements in thermodynamic activity. Similar events might
also be expected for non-ionisable, but absorbable micellar components such as monoglycerides,
although in this case, lipid absorption is not stimulated by changes in pH and therefore the effects
of acidic microclimate attenuation on drug absorption may not be as significant. Conversely, for
colloids that lack an absorbable (and/or pH-responsive) component, potential increases in drug
thermodynamic activity due to entry into the acidic microclimate are unlikely, and thermodynamic
activity (and absorption) of drug is expected to remain constant (or to decrease as drug is absorbed)
during colloidal passage across the UWL. The data obtained here for cinnarizine absorption from
Brij 97 colloids (a non-ionisable, non-absorbable surfactant) is consistent with this suggestion
(Figure 5.2C). Indeed, when the acidic microclimate was attenuated, and when lipid absorption
was effectively inhibited, cinnarizine absorption from LCFA and Brij 97 micelles was comparable
and low (Figure 5.2B & C).
The intercalation of lipid digestion products into bile-derived intestinal colloids has previously
been shown to increase solubilisation capacity for a range of PWSD39, 91, 99. Therefore, although
cinnarizine was used as a model PWSD in the current study, acidic microclimate-induced drug
supersaturation is likely to be a common endogenous mechanism of absorption enhancement for
lipophilic PWSD after co-administration with absorbable lipids. The degree of supersaturation that
is induced by entry into the acidic microclimate will be dictated by the sensitivity of drug
solubility to micellar lipid content, and the efficiency of lipid absorption. Under these
Chapter 5: Lipid absorption triggers drug supersaturation
228
circumstances the combination of weak bases, with LBF comprising fatty acids or glyceride that
generate fatty acids in situ, may be particularly beneficial since the colloid solubility of weak
bases in structures containing fatty acids is high (refer Chapter 4), and the efficiency of fatty acid
absorption is also high.
Previously, we have shown that the interaction of biliary components (i.e. bile salts, phospholipid,
cholesterol) with similar lipid colloidal phases to those examined here leads to changes in colloid
microstructure that dramatically reduce cinnarizine solubilisation capacity (Chapters 3 and 4).
Similar to the events described in the UWL here, precipitation in the previous studies was delayed
sufficiently that supersaturation occurred in the bulk luminal fluids and ultimately enhanced the
intestinal absorptive flux of cinnarizine. In an attempt to link the current studies at the UWL, with
these previous studies that describe luminal supersaturation, a final experiment was performed to
explore the importance of a functioning acidic microclimate on increases in drug absorption
resulting from supersaturation in the bulk luminal fluids. From the data in Figure 5.6B it is
apparent that the increase in cinnarizine absorptive flux that occurred due to interaction of LCFA-
containing colloids with bile (and the period of drug supersaturation in the luminal fluids that
ensued), were abolished when the acidic microclimate was inhibited with amiloride. It seems
likely therefore that even where supersaturation is generated in the intestinal lumen by, for
example, initiation of lipid digestion4, 91, 176 or interaction with bile, micellar structures are still
required to promote transport across the UWL, and the acidity of the UWL is critical to allowing
efficient absorption of LCFA and PWSD from these micellar structures. Thus, on approach to the
absorptive surface, LFCA protonation and absorption is required to translate luminal
supersaturation into enhanced drug absorption, presumably via enhancements in thermodynamic
activity that enable drug absorption from the supersaturated solubilised reservoir.
Chapter 5: Lipid absorption triggers drug supersaturation
229
As a final caveat, the generation of drug supersaturation during the processing of LBF is not
always expected to be beneficial. Indeed, supersaturation is also a precursor to crystal nucleation
and drug precipitation, events that are likely to reduce drug absorption for PWSD with
dissolution-rate limited absorption. The benefits of supersaturation generation must therefore be
weighed against the potential for precipitation. However, supersaturation at the UWL may be
particularly effective in enhancing drug absorption from intestinal colloids, as proximity to the
absorptive membrane is likely to provide an effective sink for effective removal (i.e. absorption)
of supersaturated drug, thereby minimising the risk of drug precipitation. Similar concepts in non-
lipid based systems have recently been elegantly exemplified by the Augustijns group where the
likelihood of drug precipitation from supersaturated solutions (formed in this case by solvent shift)
was dramatically reduced by the presence of an absorptive membrane261. The viscous, mucus layer
that is present at the UWL may also play a role in stabilising drug supersaturation, as increased
viscosity has been suggested to delay nucleation and crystal growth of solutes from supersaturated
solutions262, 263.
5.6 CONCLUSION
Lipid absorption is an endogenous mechanism that triggers drug supersaturation and facilitates
effective drug absorption from intestinal mixed micelles. Formulations containing absorbable
lipids or the pre-digestive precursors to absorbable lipids may therefore be more effective in
enhancing PWSD absorption when compared to formulations that lack absorbable components.
The degree of drug supersaturation (and therefore absorption enhancement potential) generated by
lipid absorption is likely maximised under conditions where the dependency of drug solubility on
micellar lipid content is high, and where lipid absorption is highly efficient. Combinations of drug
and excipients that are formulated according to the above paradigm (e.g. weak bases and lipid-
Chapter 5: Lipid absorption triggers drug supersaturation
230
based formulations comprising precursors of fatty acids) may therefore maximise the absorptive
benefits associated with lipid absorption-induced drug supersaturation. These data reinforce
previous suggestions that lipid-based formulations are able to interact with the dynamic GI
environment, and in doing so promote both drug solubilisation and thermodynamic activity. This
provides unique benefits for drug absorption when compared to formulations that promote
solubilisation or supersaturation alone.
231
CHAPTER 6 : SUMMARY AND
PERSPECTIVES
Chapter 6: Summary and perspectives
232
The ability of lipids in food or lipid-based formulations (LBF) to enhance the oral bioavailability
of poorly water-soluble drugs (PWSD) has long been recognised. However, confident application
of LBF, and accurate simulation or prediction of oral drug absorption from LBF, is limited by the
lack of a holistic understanding of the mechanisms by which lipids enhance the absorption of
PWSD. The studies described in this thesis aimed to address this gap in understanding, and
focussed on elucidating the mechanism of drug absorption from the colloidal micellar and
vesicular species that are formed in the gastrointestinal (GI) tract during lipid digestion.
Co-administration with lipids promotes drug solubilisation within intestinal lipid colloidal phases
and increases the apparent solubility of PWSD in the small intestine (SI). The mechanism of drug
absorption from these solubilising species, however, remains poorly-defined. Current
understanding suggests that drug flux across absorptive membrane is the product of the free drug
concentration and drug permeability across the membrane. For solubilised systems, therefore, drug
solubilised within colloidal phases serves to rapidly replenish the free drug fraction via re-
establishment of the equilibrium between solubilised and free drug. This results in a significant
increase in dissolution rate when compared to dissolution from a typical solid dose form. Since
solubilisation does not directly enhance the inter-micellar free drug concentration, however, lipid
co-administration may not increase membrane flux for drugs where absorption is solubility-
limited (rather than dissolution-limited). Indeed, it has recently been suggested that solubilising
formulation approaches result in a ‘solubility-permeability interplay’ where potential
enhancements in drug absorption due to solubilisation are compromised by decreases in free drug
fraction100, 146. This suggestion, however, runs counter to a body of evidence that demonstrates that
solubilising formulations such as LBF commonly increase the oral bioavailability of PWSD87.
Whilst the advantages of LBF may stem from increases in dissolution rate alone, the available data
also suggest that drug flux in excess of that predicted by the free drug concentration is likely. This
Chapter 6: Summary and perspectives
233
apparent contradiction between robust drug absorption in the presence of LBF or food, and the
realisation that this occurs even though the free drug concentration is often extremely low,
stimulated the work described in this thesis. The principle hypothesis that underpins the work is
that drug absorption from intestinal colloidal species is not simply a function of the free drug
concentration in equilibrium with the solubilised reservoir. The alternative hypotheses that have
been explored include the possibility that drug absorption occurs directly from the solubilised
fraction and/or that transient changes to colloidal structure in situ may lead to colloidal
supersaturation and an increase in the free drug concentration that subsequently increases drug
absorption.
In Chapter 3 the prospect for the solubilised fraction to contribute directly to drug absorption has
been explored via examination of the potential role of collisional drug absorption. This was
investigated by comparing the intestinal absorption of a model PWSD, cinnarizine (CIN), from
micelles and vesicles that had equal CIN solubilisation capacities and were loaded with drug at the
same concentration and therefore that had the same thermodynamic activity/free concentration.
The micelles and vesicles employed were assembled using differing quantities of medium-chain
fatty acid and monoglyceride solubilised in simulated intestinal fluid. Whilst both species had
identical solubilising properties, the structure of the two colloids was notably different and the
particle size of the vesicles was significantly larger than that of the micelles (443 nm vs. 9 nm
respectively). Since the rate of collisional transfer is directly related to the number of particles,
collisional absorption of CIN from micelles was expected to be higher since the number of
micellar particles was substantially higher than that of vesicular particles. In contrast, the data in
Chapter 3 show that intestinal absorption of CIN from vesicles and micelles was the same when
the free concentration was matched, suggesting limited collisional transfer and instead reliance on
the free concentration.
Chapter 6: Summary and perspectives
234
Receptor-mediated collisional absorption was also investigated by assessing CIN bioavailability in
the absence and presence of inhibitors of common lipid uptake transporters (e.g. SR-BI, CD36,
NPC1L1). These transporters were hypothesised to interact directly with intestinal colloids to
mediate the absorption of solubilised lipid and drug since they have similar interactions with other
colloidal particles (e.g. HDL) in the blood161. Consistent with the micelle/vesicle data, however,
no significant differences in CIN absorption were seen in the presence and absence of the
inhibitors, providing further support for the suggestion that collision-mediated uptake was not a
significant driver for drug absorption from intestinal lipid colloidal phases, and that drug
absorption occurred largely from the free fraction.
Subsequently, attention turned to the possibility that drug absorption from intestinal colloidal
phases may be promoted, not by direct absorption from the solubilised phase, but by boosting
thermodynamic activity, thereby rendering solubilised drug more available for absorption via the
free fraction. In the first instance, the potential for increases in thermodynamic activity based on
the generation of supersaturation due to interaction with biliary fluids was explored. Bile dilution
was hypothesised to stimulate drug supersaturation since the addition of bile to intestinal lipid
colloidal phases has previously been shown to generate colloids that are less lipid-rich with lower
solubilisation capacities39, 194. To investigate the impact of biliary dilution on drug absorption from
lipid colloidal phases, fasted bile was collected from donor rats and added to CIN-loaded (CIN
was loaded at sub-saturated concentrations) micelles and vesicles in a 1:1 v/v ratio. Bile addition
resulted in CIN supersaturation, and supersaturation was maintained for longer time periods in
micellar systems when compared to vesicles. The ability of bile-induced supersaturation to
enhance drug absorption was subsequently evaluated in situ and in vivo via the assessment of CIN
absorptive flux from colloids with and without the co-perfusion of donor bile in rat jejunal
perfusion studies, and the assessment of CIN bioavailability in bile diverted vs. bile-intact rats.
Chapter 6: Summary and perspectives
235
The results showed that bile-induced supersaturation led to enhanced intestinal absorption and
systemic exposure of CIN from micelles, but failed to enhance the absorption of CIN from
vesicles due to rapid precipitation. Thus, bile-induced supersaturation was identified as a novel
mechanism to increase drug thermodynamic activity and to better mobilise the solubilised drug
fraction for absorption.
The ability of bile to enhance the absorption of PWSD is well-described in the literature, however
the mechanism by which this occurs, has previously been assumed to reflect increases in drug
solubilisation. The novelty of the data in chapter 3 therefore lies in showing that bile may also
enhance drug absorption via promotion of drug supersaturation. The difference in the absorption
enhancement of CIN from micelles and vesicles also highlights the need to achieve an optimal
balance between drug supersaturation and precipitation.
Chapter 4 extended the findings in Chapter 3 to include micellar systems containing long-chain
lipids (which have the added advantage of increasing relevance beyond LBF to also include
typical food-related lipids), and to a range of drugs with differing physicochemical properties.
Initially, the sensitivity of the solubility of five PWSD (2 basic drugs, 2 neutral drugs, 1 acidic
drug) to colloidal bile or long-chain lipid content was evaluated. Consistent with the data obtained
with CIN and the medium-chain lipid containing colloids in Chapter 3, the solubility of the basic
drugs in long-chain lipid containing micelles was found to decrease with increasing bile
concentration. In contrast, and more consistent with typical solubilisation behaviour, the solubility
of the neutral and acidic drugs increased with increasing bile (surfactant) concentration. The data
suggest that the potential for drug supersaturation to be triggered during bile dilution is highest for
basic drugs. The basis for supersaturation stimulation was suggested to be related to drug affinity
for the core lipids within the colloids. Thus, high affinity for colloidal lipids resulted in high initial
Chapter 6: Summary and perspectives
236
solubility, and the addition of bile served to disrupt intermolecular interactions between drug and
lipid, leading to reductions in solubilisation capacity. Consistent with this suggestion, in situ
evaluation of the absorption enhancement afforded by 1:1 v/v co-perfusion of bile with drug-
containing colloids showed that bile enhanced the absorption of cinnarizine (a basic drug) to a
greater extent than danazol (a neutral drug).
Interestingly, however, bile did stimulate some increase in danazol absorption, in spite of a
reduction in thermodynamic activity (bile addition increased danazol solubility in colloidal species
thereby decreasing thermodynamic activity). Since oleic acid absorption was also increased by
bile co-perfusion, the increase in danazol absorption was suggested to be due to the increase in
lipid absorption in the presence of bile. Thus lipid absorption was hypothesised to reduce micellar
drug solubilisation capacity, and therefore to increase thermodynamic activity and absorption
(assuming that absorption enhancement precedes drug precipitation).
In Chapter 5, the potential for lipid absorption (i.e. a reduction in colloidal lipid content) to
stimulate drug supersaturation and absorption was examined directly. The acidic microclimate of
the intestinal unstirred water layer (UWL) facilitates the absorption of long-chain fatty acids
(LCFA) by protonating LCFA and increasing thermodynamic activity and membrane
partitioning56, 57. In this series of experiments, therefore, the acidity of the UWL was attenuated
using amiloride in order to inhibit oleic acid absorption. This enabled a comparison of CIN
absorption from oleic acid-containing colloids under conditions of normal lipid absorption vs.
conditions of inhibited lipid absorption. In the rat jejunal perfusion model the absorptive flux of
CIN was dramatically attenuated (6.5-fold) when oleic acid absorption was suppressed. In vitro
assessment of CIN solubilisation behaviour under conditions that simulate lipid absorption at the
UWL subsequently indicated that supersaturation was likely to be responsible for the enhancement
Chapter 6: Summary and perspectives
237
in CIN absorption during normal lipid absorption. Importantly, in this chapter, it was shown that
the intrinsic solubilisation capacity of colloids did not dictate the efficiency of drug absorption.
Instead, the ability of colloids to lose solubilisation capacity and to generate drug supersaturation
at the UWL was seemingly more important. Thus, head-to-head comparison of CIN absorption
from two colloidal systems with equal solubilisation capacity (colloids were loaded with CIN at
the same concentration such that total, solubilised, and free concentrations were equal) showed
that drug absorption was dramatically more efficient when micelles comprised bile salt and oleic
acid, when compared to micelles comprising Brij 97 (a non-ionisable and non-absorbable
surfactant). These differences were abolished when oleic acid absorption was inhibited. The data
support the importance of lipid absorption-induced supersaturation as a means to mobilise the
solubilised drug fraction and to enable effective drug absorption from intestinal mixed micellar
species.
The major findings from this thesis therefore support a consensus view that LBF inherently
generate drug supersaturation when incorporated into endogenous lipid processing pathways.
Specifically, supersaturation has previously been shown to be generated when solubilisation
capacity is lost during LBF dispersion and digestion5, 91, 176, 177, 193. The current studies further
suggest that supersaturation is generated during biliary dispersion of lipid digestion products, and
during the absorption of lipid digestion products. The role of bile and the acidic UWL in
promoting drug supersaturation and absorption has not been described previously, and represent
interesting new avenues for further research. Thus, LBF have traditionally been regarded as
formulations that promote the absorption of PWSD by increasing solubilisation. The current data,
however, suggest that in vivo, LBF are capable of promoting both drug solubilisation and drug
supersaturation.
Chapter 6: Summary and perspectives
238
The unique ability of LBF to simultaneously increase the solubilised reservoir and to also enhance
the thermodynamic activity of co-administered drug may distinguish LBF from other enabling
formulations and help explain why lipids are often very effective in enhancing the oral absorption
of a range of lipophilic PWSD. Firstly, the formation of intestinal mixed micelles and vesicles
during lipid digestion increases the solubilisation capacity of the SI, and allows for higher
amounts of drug to be maintained in solution. Secondly, although drug thermodynamic activity is
initially lowered by solubilisation, the ensuing interaction between drug-containing colloids and
endogenous lipid processing pathways triggers drug supersaturation, and provides a means to
reverse the reduction in thermodynamic activity and to mobilise the solubilised drug fraction for
absorption. Lastly, the multi-step, sequential manner by which drug supersaturation is triggered in
the SI, coupled with the increased drug solubilisation capacity of the SI after lipid co-
administration, ensures that the supersaturation generated during LBF processing is rate and
extent-controlled, therefore providing a balance between solubilisation and supersaturation that
maximises the potential for drug absorption while minimising the risk of drug precipitation. In this
way, LBF possess inherent advantages when compared to other enabling formulations that either
promote drug solubilisation only (e.g. cyclodextrins) or drug supersaturation only (e.g. solid
dispersions), and may provide a means to finely balance the need to solubilise the drug dose, and
to maintain or increase drug thermodynamic activity.
The findings from this thesis provide significant new insights into the fundamental mechanisms by
which lipids enhance the oral absorption of PWSD. The studies show that supersaturation is an
important driving force for drug absorption after co-administration with lipids, and identify two
novel mechanisms (i.e. bile dilution and lipid absorption) by which drug supersaturation may be
triggered during intestinal lipid processing. Better understanding of the mechanisms of
supersaturation generation is expected to contribute to an increasingly rational basis for the
Chapter 6: Summary and perspectives
239
selection of lipid excipients. Although more work is needed, practical applications related to the
findings of this thesis are conceivable, and relate to the opportunity to generate colloidal
assemblies with built-in sensitivity to bile dilution and lipid absorption mediated supersaturation.
For example, the studies in Chapter 4 suggest that the use of lipid-drug combinations where drug
possesses high affinity for lipid digestion products may predispose post-digestion colloids to lose
solubilisation capacity during bile dilution via the disruption of drug-lipid intermolecular
interactions. Studies in Chapter 5 further suggest that the utilisation of absorbable lipids (or
precursors of absorbable lipids) may lead to potent stimulation of drug supersaturation at the UWL,
especially where the drug has high affinity for the lipids and where lipid absorption is efficient.
Collectively, these studies suggest that combinations of drug and excipients that are formulated
according to the above principles (e.g. weak bases with LBF comprising precursors of fatty acids)
may maximally harness the absorptive benefits associated with bile and lipid absorption-induced
drug supersaturation. The in vitro studies in Chapter 4 that delineate drug solubility relationships
with bile components or lipid digestion products also serve to demonstrate that the likelihood of
supersaturation generation in vivo may be predicted in vitro, and may be amenable to high
throughput screening.
Finally, the data in Chapter 5 demonstrate a crucial role of the UWL acidic microclimate in the
translation of luminal supersaturation into enhanced absorption (inhibition of UWL acidity
blocked increases in drug absorption due to bile-mediated promotion of luminal supersaturation).
This suggests that supersaturated luminal colloids alone are not sufficient to promote drug
absorption and that micellar dissociation via a decrease in UWL pH is an important mechanism to
mobilise supersaturated solubilised drug for absorption. This may be an important consideration
for solubilising formulations that lack pH-sensitivity.
Chapter 6: Summary and perspectives
240
In conclusion, this thesis has demonstrated for the first time that effective drug absorption from
intestinal colloidal species is a result of dynamic interactions between the solubilised drug
reservoir and the GI environment that lead to multiple opportunities for supersaturation-enhanced
absorption. This improved understanding of the mechanism of drug absorption is ultimately
expected to inform rational design criteria for LBF and to improve oral absorption simulation
models. Future work might be usefully directed towards further characterising the parameters that
dictate the propensity of LBF to generate drug supersaturation during formulation processing, and
in particular an assessment of the balance between drug supersaturation and drug precipitation.
241
REFERENCES
1. Lipinski, C. A. Drug-like properties and the causes of poor solubility and poor permeability. Journal of Pharmacological and Toxicological Methods 2000, 44, (1), 235-249.
2. Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.; Charman, W. N.; Pouton, C. W.; Porter, C. J. H. Strategies to address low drug solubility in discovery and development. Pharmacological Reviews 2013, 65, (1), 315-499.
3. Amidon, G. L.; Lennernäs, H.; Shah, V. P.; Crison, J. R. A theoretical basis for a biopharmaceutic drug classification: The correlation of in vitro drug product dissolution and in vivo bioavailability. Pharmaceutical Research 1995, 12, (3), 413-420.
4. Williams, H. D.; Anby, M. U.; Sassene, P.; Kleberg, K.; Bakala-N’Goma, J.-C.; Calderone, M.; Jannin, V.; Igonin, A.; Partheil, A.; Marchaud, D.; Jule, E.; Vertommen, J.; Maio, M.; Blundell, R.; Benameur, H.; Carrière, F.; Müllertz, A.; Pouton, C. W.; Porter, C. J. H. Toward the establishment of standardized in vitro tests for lipid-based formulations. 2. The effect of bile salt concentration and drug loading on the performance of Type I, II, IIIA, IIIB, and IV formulations during in vitro digestion. Molecular Pharmaceutics 2012, 9, (11), 3286-3300.
5. Pouton, C. W.; Porter, C. J. H. Formulation of lipid-based delivery systems for oral administration: Materials, methods and strategies. Advanced Drug Delivery Reviews 2008, 60, (6), 625-637.
6. Tokumura, T.; Tsushima, Y.; Tatsuishi, K.; Kayano, M.; Machida, Y.; Nagai, T. Enhancement of the oral bioavailability of cinnarizine in oleic acid in beagle dogs. Journal of Pharmaceutical Sciences 1987, 76, (4), 286-288.
7. Myers, R. A.; Stella, V. J. Systemic bioavailability of penclomedine (NSC-338720) from oil-in-water emulsions administered intraduodenally to rats. International Journal of Pharmaceutics 1992, 78, (1–3), 217-226.
8. Charman, W. N.; Rogge, M. C.; Boddy, A. W.; Berger, B. M. Effect of food and a monoglyceride emulsion formulation on danazol bioavailability. The Journal of Clinical Pharmacology 1993, 33, (4), 381-386.
9. Dahan, A.; Hoffman, A. The effect of different lipid based formulations on the oral absorption of lipophilic drugs: The ability of in vitro lipolysis and consecutive ex vivo intestinal permeability data to predict in vivo bioavailability in rats. European Journal of Pharmaceutics and Biopharmaceutics 2007, 67, (1), 96-105.
10. Bates, T. R.; Sequeira, J. A. Bioavailability of micronized griseofulvin from corn oil-in-water emulsion, aqueous suspension, and commercial tablet dosage forms in humans. Journal of Pharmaceutical Sciences 1975, 64, (5), 793-797.
11. Hauss, D. J.; Fogal, S. E.; Ficorilli, J. V.; Price, C. A.; Roy, T.; Jayaraj, A. A.; Keirns, J. J. Lipid-based delivery systems for improving the bioavailability and lymphatic transport of a poorly water-soluble LTB4 inhibitor. Journal of Pharmaceutical Sciences 1998, 87, (2), 164-169.
12. Caliph, S. M.; Charman, W. N.; Porter, C. J. H. Effect of short-, medium-, and long-chain fatty acid-based vehicles on the absolute oral bioavailability and intestinal lymphatic transport of halofantrine and assessment of mass balance in lymph-cannulated and non-cannulated rats. Journal of Pharmaceutical Sciences 2000, 89, (8), 1073-1084.
242
13. Wei, L.; Sun, P.; Nie, S.; Pan, W. Preparation and evaluation of SEDDS and SMEDDS containing carvedilol. Drug Development & Industrial Pharmacy 2005, 31, (8), 785-794.
14. Mueller, E. A.; Kovarik, J. M.; van Bree, J. B.; Tetzloff, W.; Grevel, J.; Kutz, K. Improved dose linearity of cyclosporine pharmacokinetics from a microemulsion formulation. Pharmaceutical Research 1994, 11, (2), 301-304.
15. Woo, J. S.; Song, Y.-K.; Hong, J.-Y.; Lim, S.-J.; Kim, C.-K. Reduced food-effect and enhanced bioavailability of a self-microemulsifying formulation of itraconazole in healthy volunteers. European Journal of Pharmaceutical Sciences 2008, 33, (2), 159-165.
16. Wu, W.; Wang, Y.; Que, L. Enhanced bioavailability of silymarin by self-microemulsifying drug delivery system. European Journal of Pharmaceutics and Biopharmaceutics 2006, 63, (3), 288-294.
17. Gao, P.; Rush, B. D.; Pfund, W. P.; Huang, T.; Bauer, J. M.; Morozowich, W.; Kuo, M.-S.; Hageman, M. J. Development of a supersaturable SEDDS (S-SEDDS) formulation of paclitaxel with improved oral bioavailability. Journal of Pharmaceutical Sciences 2003, 92, (12), 2386-2398.
18. Pouton, C. W. Lipid formulations for oral administration of drugs: non-emulsifying, self-emulsifying and ‘self-microemulsifying’ drug delivery systems. European Journal of Pharmaceutical Sciences 2000, 11, Supplement 2, (0), S93-S98.
19. Pouton, C. W. Formulation of poorly water-soluble drugs for oral administration: Physicochemical and physiological issues and the lipid formulation classification system. European Journal of Pharmaceutical Sciences 2006, 29, (3–4), 278-287.
20. Hauss, D. J. Oral lipid-based formulations. Advanced Drug Delivery Reviews 2007, 59, (7), 667-676.
21. Yoshiya, Y.; Takeshi, N.; Hiroshi, T.; Tadao, M. Biopharmaceutical studies of lipid-containing oral dosage forms: Relationship between drug absorption rate and digestibility of vehicles. International Journal of Pharmaceutics 1979, 3, (1), 23-31.
22. Borovicka, J.; Schwizer, W.; Mettraux, C.; Kreiss, C.; Remy, B.; Asal, K.; Jansen, J. B.; Douchet, I.; Verger, R.; Fried, M. Regulation of gastric and pancreatic lipase secretion by CCK and cholinergic mechanisms in humans. American Journal of Physiology - Gastrointestinal and Liver Physiology 1997, 273, (2), G374-G380.
23. Moreau, H.; Bernadac, A.; Gargouri, Y.; Benkouka, F.; Laugier, R.; Verger, R. Immunocytolocalization of human gastric lipase in chief cells of the fundic mucosa. Histochemistry 1989, 91, (5), 419-423.
24. Hamosh, M.; Scanlon, J. W.; Ganot, D.; Likel, M.; Scanlon, K. B.; Hamosh, P. Fat digestion in the newborn. Characterization of lipase in gastric aspirates of premature and term infants. Journal of Clinical Investigation 1981, 67, (3), 838-846.
25. Abrams, C. K.; Hamosh, M.; Lee, T. C.; Ansher, A. F.; Collen, M. J.; Lewis, J. H.; Benjamin, S. B.; Hamosh, P. Gastric lipase: localization in the human stomach. Gastroenterology 1988, 95, (6), 1460-1464.
26. Ransac, S.; Rogalska, E.; Gargouri, Y.; Deveer, A. M.; Paltauf, F.; de Haas, G. H.; Verger, R. Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic
243
lipases, a kinetic study using the monomolecular film technique. Journal of Biological Chemistry 1990, 265, (33), 20263-70.
27. Canaan, S.; Roussel, A.; Verger, R.; Cambillau, C. Gastric lipase: Crystal structure and activity. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1999, 1441, (2–3), 197-204.
28. Armand, M.; Borel, P.; Pasquier, B.; Dubois, C.; Senft, M.; Andre, M.; Peyrot, J.; Salducci, J.; Lairon, D. Physicochemical characteristics of emulsions during fat digestion in human stomach and duodenum. American Journal of Physiology - Gastrointestinal and Liver Physiology 1996, 271, (1), G172-G183.
29. Jensen, M. S.; Jensen, S. K.; Jakobsen, K. Development of digestive enzymes in pigs with emphasis on lipolytic activity in the stomach and pancreas. Journal of Animal Science 1997, 75, (2), 437-45.
30. Carey, M. C.; Small, D. M.; Bliss, C. M. Lipid digestion and absorption. Annual Review of Physiology 1983, 45, (1), 651-677.
31. Ohtani, N.; Sasaki, I.; Naito, H.; Shibata, C.; Matsuno, S. Mediators for fat-induced ileal brake are different between stomach and proximal small intestine in conscious dogs. J Gastrointest Surg 2001, 5, (4), 377-382.
32. Van Citters, G. W.; Lin, H. C. The ileal brake: A fifteen-year progress report. Curr Gastroenterol Rep 1999, 1, (5), 404-409.
33. Nordskog, B. K.; Phan, C. T.; Nutting, D. F.; Tso, P. An examination of the factors affecting intestinal lymphatic transport of dietary lipids. Advanced Drug Delivery Reviews 2001, 50, (1–2), 21-44.
34. Mu, H.; Høy, C.-E. The digestion of dietary triacylglycerols. Progress in Lipid Research 2004, 43, (2), 105-133.
35. Ros, E. Intestinal absorption of triglyceride and cholesterol. Dietary and pharmacological inhibition to reduce cardiovascular risk. Atherosclerosis 2000, 151, (2), 357-379.
36. Lowe, M. E. Structure and function of pancreatic lipase and colipase. Annual review of nutrition 1997, 17, 141-158.
37. Borgström, B.; Dahlqvist, A.; Lundh, G.; Sjövall, J. Studies of intestinal digestion and absorption in the human. Journal of Clinical Investigation 1957, 36, (10), 1521-1536.
38. Meyer, J. G. Lipolytic enzymes of the human pancreas. II. Purification and properties of cholesterol ester hydrolase. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1989, 1002, (1), 89-92.
39. Kossena, G. A.; Charman, W. N.; Boyd, B. J.; Dunstan, D. E.; Porter, C. J. H. Probing drug solubilization patterns in the gastrointestinal tract after administration of lipid-based delivery systems: A phase diagram approach. Journal of Pharmaceutical Sciences 2004, 93, (2), 332-348.
40. Salentinig, S.; Sagalowicz, L.; Leser, M. E.; Tedeschi, C.; Glatter, O. Transitions in the internal structure of lipid droplets during fat digestion. Soft Matter 2011, 7, (2), 650-661.
244
41. Warren, D. B.; Anby, M. U.; Hawley, A.; Boyd, B. J. Real time evolution of liquid crystalline nanostructure during the digestion of formulation lipids Using synchrotron small-angle X-ray scattering. Langmuir 2011, 27, (15), 9528-9534.
42. Fatouros, D. G.; Deen, G. R.; Arleth, L.; Bergenstahl, B.; Nielsen, F. S.; Pedersen, J. S.; Mullertz, A. Structural development of self nano emulsifying drug delivery systems (SNEDDS) during in vitro lipid digestion monitored by small-angle X-ray scattering. Pharmaceutical Research 2007, 24, (10), 1844-1853.
43. Kossena, G. A.; Charman, W. N.; Boyd, B. J.; Porter, C. J. H. A novel cubic phase of medium chain lipid origin for the delivery of poorly water soluble drugs. Journal of Controlled Release 2004, 99, (2), 217-229.
44. Nguyen, T.-H.; Hanley, T.; Porter, C. J. H.; Boyd, B. J. Nanostructured liquid crystalline particles provide long duration sustained-release effect for a poorly water soluble drug after oral administration. Journal of Controlled Release 2011, 153, (2), 180-186.
45. Shiau, Y. F.; Fernandez, P.; Jackson, M. J.; McMonagle, S. Mechanisms maintaining a low-pH microclimate in the intestine. American Journal of Physiology - Gastrointestinal and Liver Physiology 1985, 248, (6), G608-G617.
46. Ikuma, M.; Hanai, H.; Kaneko, E.; Hayashi, H.; Hoshi, T. Effects of aging on the microclimate pH of the rat jejunum. Biochimica et Biophysica Acta (BBA) - Biomembranes 1996, 1280, (1), 19-26.
47. Li, C.-Y.; Zimmerman, C. L.; Wiedmann, T. S. Diffusivity of bile salt/phospholipid aggregates in mucin. Pharmaceutical Research 1996, 13, (4), 535-541.
48. Khanvilkar, K.; Donovan, M. D.; Flanagan, D. R. Drug transfer through mucus. Advanced Drug Delivery Reviews 2001, 48, (2–3), 173-193.
49. Norris, D. A.; Sinko, P. J. Effect of size, surface charge, and hydrophobicity on the translocation of polystyrene microspheres through gastrointestinal mucin. Journal of Applied Polymer Science 1997, 63, (11), 1481-1492.
50. Sanders, N. N.; De Smedt, S. C.; Demeester, J. The physical properties of biogels and their permeability for macromolecular drugs and colloidal drug carriers. Journal of Pharmaceutical Sciences 2000, 89, (7), 835-849.
51. Westergaard, H.; Dietschy, J. M. The mechanism whereby bile acid micelles increase the rate of fatty acid and cholesterol uptake into the intestinal mucosal cell. Journal of Clinical Investigation 1976, 58, (1), 97-108.
52. Carey, M. C.; Small, D. M. The characteristics of mixed micellar solutions with particular reference to bile. The American Journal of Medicine 1970, 49, (5), 590-608.
53. Garcıa, R. A.; Pantazatos, S. P.; Pantazatos, D. P.; MacDonald, R. C. Cholesterol stabilizes hemifused phospholipid bilayer vesicles. Biochimica et Biophysica Acta (BBA) - Biomembranes 2001, 1511, (2), 264-270.
54. Mansbach II, C. M.; Tso, P.; Kuksis, A.; eds, Intestinal lipid metabolism. Springer: 2000.
55. Israelachvili, J. N., Intermolecular and surface forces: Revised third edition. Academic press: 2011.
245
56. Shiau, Y. F. Mechanism of intestinal fatty acid uptake in the rat: the role of an acidic microclimate. The Journal of Physiology 1990, 421, (1), 463-474.
57. Shiau, Y. F.; Kelemen, R. J.; Reed, M. A. Acidic mucin layer facilitates micelle dissociation and fatty acid diffusion. American Journal of Physiology - Gastrointestinal and Liver Physiology 1990, 259, (4), G671-G675.
58. Chow, S. L.; Hollander, D. A dual, concentration-dependent absorption mechanism of linoleic acid by rat jejunum in vitro. Journal of Lipid Research 1979, 20, (3), 349-56.
59. Ling, K.-Y.; Lee, H.-Y.; Hollander, D. Mechanisms of linoleic acid uptake by rabbit small intestinal brush border membrane vesicles. Lipids 1989, 24, (1), 51-55.
60. Goré, J.; Hoinard, C.; Couet, C. Linoleic acid uptake by isolated enterocytes: Influence of α-linolenic acid on absorption. Lipids 1994, 29, (10), 701-706.
61. Nassir, F.; Wilson, B.; Han, X.; Gross, R. W.; Abumrad, N. A. CD36 is important for fatty acid and cholesterol uptake by the proximal but not distal intestine. Journal of Biological Chemistry 2007, 282, (27), 19493-19501.
62. Stahl, A.; Hirsch, D. J.; Gimeno, R. E.; Punreddy, S.; Ge, P.; Watson, N.; Patel, S.; Kotler, M.; Raimondi, A.; Tartaglia, L. A.; Lodish, H. F. Identification of the major intestinal fatty acid transport protein. Molecular Cell 1999, 4, (3), 299-308.
63. Bietrix, F.; Yan, D.; Nauze, M.; Rolland, C.; Bertrand-Michel, J.; Coméra, C.; Schaak, S.; Barbaras, R.; Groen, A. K.; Perret, B.; Tercé, F.; Collet, X. Accelerated lipid absorption in mice overexpressing intestinal SR-BI. Journal of Biological Chemistry 2006, 281, (11), 7214-7219.
64. Stremmel, W. Uptake of fatty acids by jejunal mucosal cells is mediated by a fatty acid binding membrane protein. Journal of Clinical Investigation 1988, 82, (6), 2001-2010.
65. Schoeller, C.; Keelan, M.; Mulvey, G.; Stremmel, W.; Thomson, A. B. R. Role of a brush border membrane fatty acid binding protein in oleic acid uptake into rat and rabbit jejunal brush border membrane. Clinical and Investigative Medicine 1995, 18, (5), 380-380.
66. Van Bennekum, A.; Werder, M.; Thuahnai, S. T.; Han, C.-H.; Duong, P.; Williams, D. L.; Wettstein, P.; Schulthess, G.; Phillips, M. C.; Hauser, H. Class B Scavenger Receptor-mediated intestinal absorption of dietary β-carotene and cholesterol. Biochemistry 2005, 44, (11), 4517-4525.
67. Hauser, H.; Dyer, J. H.; Nandy, A.; Vega, M. A.; Werder, M.; Bieliauskaite, E.; Weber, F. E.; Compassi, S.; Gemperli, A.; Boffelli, D.; Wehrli, E.; Schulthess, G.; Phillips, M. C. Identification of a receptor mediating absorption of dietary cholesterol in the intestine. Biochemistry 1998, 37, (51), 17843-17850.
68. Altmann, S. W.; Davis, H. R.; Zhu, L.; Yao, X.; Hoos, L. M.; Tetzloff, G.; Iyer, S. P. N.; Maguire, M.; Golovko, A.; Zeng, M.; Wang, L.; Murgolo, N.; Graziano, M. P. Niemann-Pick C1 Like 1 protein is critical for intestinal cholesterol absorption. Science 2004, 303, (5661), 1201-1204.
69. Niot, I.; Poirier, H.; Tran, T. T. T.; Besnard, P. Intestinal absorption of long-chain fatty acids: Evidence and uncertainties. Progress in Lipid Research 2009, 48, (2), 101-115.
246
70. Mansbach, C. M.; Siddiqi, S. A. The biogenesis of chylomicrons. Annual Review of Physiology 2010, 72, (1), 315-333.
71. Johnston, J. M.; Rao, G. A. Triglyceride biosynthesis in the intestinal mucosa. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1965, 106, (1), 1-9.
72. Tso, P.; Karlstad, M. D.; Bistrian, B. R.; DeMichele, S. J. Intestinal digestion, absorption, and transport of structured triglycerides and cholesterol in rats. American Journal of Physiology - Gastrointestinal and Liver Physiology 1995, 268, (4), G568-G577.
73. Bloom, B.; Chaikoff, I. L.; Reinhardt, W. O. Intestinal lymph as pathway for transport of absorbed fatty acids of different chain lengths. American Journal of Physiology -- Legacy Content 1951, 166, (2), 451-455.
74. Chaikoff, I. L.; Bloom, B.; Stevens, B. P.; Reinhardt, W. O.; Dauben, W. G. Pentadecanoic acid-5-C14; its absorption and lymphatic transport. Journal of Biological Chemistry 1951, 190, (1), 431-435.
75. Kiyasu, J. Y.; Bloom, B.; Chaikoff, I. L. The portal transport of absorbed fatty acids. Journal of Biological Chemistry 1952, 199, (1), 415-419.
76. Porter, C. J. H.; Trevaskis, N. L.; Charman, W. N. Lipids and lipid-based formulations: optimizing the oral delivery of lipophilic drugs. Nat Rev Drug Discov 2007, 6, (3), 231-248.
77. Nilsson, Å. Intestinal absorption of lecithin and lysolecithin by lymph fistula rats. Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism 1968, 152, (2), 379-390.
78. Sato, Y. The metabolic fate of lysolecithin administered into rat duodenal lumen. The Tohoku Journal of Experimental Medicine 1970, 100, (3), 277-287.
79. Scow, R. O.; Stein, Y.; Stein, O. Incorporation of dietary lecithin and lysolecithin into lymph chylomicrons in the rat. Journal of Biological Chemistry 1967, 242, (21), 4919-4924.
80. Le Kim, D.; Betzing, H. Intestinal absorption of polyunsaturated phosphatidylcholine in the rat. Hoppe Seylers Z Physiol Chem 1976, 357, (9), 1321-1331.
81. Ottolenghi, A. Estimation and subcellular distribution of lecithinase activity in rat intestinal mucosa. Journal of Lipid Research 1964, 5, (4), 532-537.
82. Clark, S. B.; Tercyak, A. M. Reduced cholesterol transmucosal transport in rats with inhibited mucosal acyl CoA: Cholesterol acyltransferase and normal pancreatic function. Journal of Lipid Research 1984, 25, (2), 148-159.
83. Gallo, L. L.; Newbill, T.; Hyun, J.; Vahouny, G. V.; Treadwell, C. R. Role of pancreatic cholesterol esterase in the uptake and esterification of cholesterol by isolated intestinal cells. Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.) 1977, 156, (2), 277-281.
84. Borja, C. R.; Vahouny, G. V.; Treadwell, C. R. Role of bile and pancreatic juice in cholesterol absorption and esterification. American Journal of Physiology -- Legacy Content 1964, 206, (1), 223-228.
247
85. Gao, P.; Guyton, M. E.; Huang, T.; Bauer, J. M.; Stefanski, K. J.; Lu, Q. Enhanced oral bioavailability of a poorly water soluble drug PNU-91325 by supersaturatable formulations. Drug Development and Industrial Pharmacy 2004, 30, (2), 221-229.
86. Carrier, R. L.; Miller, L. A.; Ahmed, I. The utility of cyclodextrins for enhancing oral bioavailability. Journal of Controlled Release 2007, 123, (2), 78-99.
87. Porter, C. J. H.; Pouton, C. W.; Cuine, J. F.; Charman, W. N. Enhancing intestinal drug solubilisation using lipid-based delivery systems. Advanced Drug Delivery Reviews 2008, 60, (6), 673-691.
88. Noyes, A. A.; Whitney, W. R. The rate of solution of solid substances in their own solutions. Journal of the American Chemical Society 1897, 19, (12), 930-934.
89. Naylor, L. J.; Bakatselou, V.; Dressman, J. B. Comparison of the mechanism of dissolution of hydrocortisone in simple and mixed micelle systems. Pharmaceutical Research 1993, 10, (6), 865-870.
90. Miyazaki, S.; Inoue, H.; Yamahira, T.; Nadai, T. Interaction of drugs with bile components. I. Effects of bile salts on the dissolution behavior of indomethacin and phenylbutazone. Chemical & pharmaceutical bulletin 1979, 27, (10), 2468-2472.
91. Kaukonen, A. M.; Boyd, B.; Porter, C.; Charman, W. Drug solubilization behavior during in vitro digestion of simple triglyceride lipid solution formulations. Pharmaceutical Research 2004, 21, (2), 245-253.
92. Hörter, D.; Dressman, J. B. Influence of physicochemical properties on dissolution of drugs in the gastrointestinal tract. Advanced Drug Delivery Reviews 1997, 25, (1), 3-14.
93. Crison, J. R.; Shah, V. P.; Skelly, J. P.; Amidon, G. L. Drug dissolution into micellar solutions: Development of a convective diffusion model and comparison to the film equilibrium model with application to surfactant-facilitated dissolution of carbamazepine. Journal of Pharmaceutical Sciences 1996, 85, (9), 1005-1011.
94. Balakrishnan, A.; Rege, B. D.; Amidon, G. L.; Polli, J. E. Surfactant-mediated dissolution: Contributions of solubility enhancement and relatively low micelle diffusivity. Journal of Pharmaceutical Sciences 2004, 93, (8), 2064-2075.
95. Bates, T. R.; Gibaldi, M.; Kanig, J. L. Solubilizing properties of bile salt solutions I: Effect of temperature and bile salt concentration on solubilization of glutethimide, griseofulvin, and hexestrol. Journal of Pharmaceutical Sciences 1966, 55, (2), 191-199.
96. Kassem, M. A.; Mattha, A. G.; El-Nimr, A. E. M.; Omar, S. M. Study of the influence of sodium taurocholate (STC) and sodium glycocholate (SGC) on the mass transfer of certain drugs. Digoxin. International Journal of Pharmaceutics 1982, 12, (1), 1-9.
97. Kararli, T. T.; Gupta, V. W. Solubilization and dissolution properties of a leucotriene-D4 antagonist in micellar solutions. Journal of Pharmaceutical Sciences 1992, 81, (5), 483-485.
98. Luner, P. E.; Babu, S. R.; Radebaugh, G. W. The effects of bile salts and lipids on the physicochemical behavior of gemfibrozil. Pharmaceutical Research 1994, 11, (12), 1755-1760.
248
99. Kossena, G. A.; Charman, W. N.; Boyd, B. J.; Porter, C. J. H. Influence of the intermediate digestion phases of common formulation lipids on the absorption of a poorly water-soluble drug. Journal of Pharmaceutical Sciences 2005, 94, (3), 481-492.
100. Miller, J. M.; Beig, A.; Krieg, B. J.; Carr, R. A.; Borchardt, T. B.; Amidon, G. E.; Amidon, G. L.; Dahan, A. The solubility–permeability interplay: Mechanistic modeling and predictive application of the impact of micellar solubilization on intestinal permeation. Molecular Pharmaceutics 2011, 8, (5), 1848-1856.
101. Sugano, K. Estimation of effective intestinal membrane permeability considering bile micelle solubilisation. International Journal of Pharmaceutics 2009, 368, (1–2), 116-122.
102. Heerklotz, H. Interactions of surfactants with lipid membranes. Quarterly reviews of biophysics 2008, 41, (3-4), 205-264.
103. Aungst, B. J. Intestinal permeation enhancers. Journal of Pharmaceutical Sciences 2000, 89, (4), 429-442.
104. Sakai, M.; Imai, T.; Ohtake, H.; Azuma, H.; Otagiri, M. Effects of absorption enhancers on the transport of model compounds in Caco-2 cell monolayers: Assessment by confocal laser scanning microscopy. Journal of Pharmaceutical Sciences 1997, 86, (7), 779-785.
105. Tomita, M.; Sawada, T.; Ogawa, T.; Ouchi, H.; Hayashi, M.; Awazu, S. Differences in the enhancing effects of sodium caprate on colonic and jejunal drug absorption. Pharmaceutical Research 1992, 9, (5), 648-653.
106. Chao, A. C.; Nguyen, J. V.; Broughall, M.; Recchia, J.; Kensil, C. R.; Daddona, P. E.; Fix, J. A. Enhancement of intestinal model compound transport by DS-1, a modified Quillaja saponin. Journal of Pharmaceutical Sciences 1998, 87, (11), 1395-1399.
107. Rege, B. D.; Kao, J. P. Y.; Polli, J. E. Effects of nonionic surfactants on membrane transporters in Caco-2 cell monolayers. European Journal of Pharmaceutical Sciences 2002, 16, (4–5), 237-246.
108. Martin, G. P.; Marriott, C. Membrane damage by bile salts: The protective function of phospholipids. Journal of Pharmacy and Pharmacology 1981, 33, (1), 754-759.
109. Seelig, A.; Gerebtzoff, G. Enhancement of drug absorption by noncharged detergents through membrane and P-glycoprotein binding. Expert Opinion on Drug Metabolism & Toxicology 2006, 2, (5), 733-752.
110. Constantinides, P. P.; Wasan, K. M. Lipid formulation strategies for enhancing intestinal transport and absorption of P-glycoprotein (P-gp) substrate drugs: In vitro/In vivo case studies. Journal of Pharmaceutical Sciences 2007, 96, (2), 235-248.
111. Werle, M. Polymeric and low molecular mass efflux pump inhibitors for oral drug delivery. Journal of Pharmaceutical Sciences 2008, 97, (1), 60-70.
112. Goole, J.; Lindley, D. J.; Roth, W.; Carl, S. M.; Amighi, K.; Kauffmann, J.-M.; Knipp, G. T. The effects of excipients on transporter mediated absorption. International Journal of Pharmaceutics 2010, 393, (1–2), 17-31.
249
113. de Lannoy, I. A. M.; Silverman, M. The MDR1 gene product, P-glycoprotein, mediates the transport of the cardiac glycoside, digoxin. Biochemical and Biophysical Research Communications 1992, 189, (1), 551-557.
114. Gottesman, M. M.; Pastan, I. Biochemistry of multidrug resistance mediated by the multidrug transporter. Annual Review of Biochemistry 1993, 62, (1), 385-427.
115. Augustijns, P. F.; Bradshaw, T. P.; Gan, L. S. L.; Hendren, R. W.; Thakker, D. R. Evidence for a polarized efflux system in Caco-2 cells capable of modulating cyclosporine A transport. Biochemical and Biophysical Research Communications 1993, 197, (2), 360-365.
116. Saeki, T.; Ueda, K.; Tanigawara, Y.; Hori, R.; Komano, T. P-glycoprotein-mediated transcellular transport of MDR-reversing agents. FEBS Letters 1993, 324, (1), 99-102.
117. Williams, G. C.; Sinko, P. J. Oral absorption of the HIV protease inhibitors: A current update. Advanced Drug Delivery Reviews 1999, 39, (1–3), 211-238.
118. Friche, E.; Jensen, P. B.; Sehested, M.; Demant, E. J.; Nissen, N. N. The solvents Cremophor EL and Tween 80 modulate daunorubicin resistance in the multidrug resistant Ehrlich ascites tumor. Cancer communications 1990, 2, (9), 297-303.
119. Spoelstra, E. C.; Dekker, H.; Schuurhuis, G. J.; Broxterman, H. J.; Lankelma, J. P-glycoprotein drug efflux pump involved in the mechanisms of intrinsic drug resistance in various colon cancer cell lines: Evidence for a saturation of active daunorubicin transport. Biochemical Pharmacology 1991, 41, (3), 349-359.
120. Zordan-Nudo, T.; Ling, V.; Liu, Z.; Georges, E. Effects of nonionic detergents on P-glycoprotein drug binding and reversal of multidrug resistance. Cancer Research 1993, 53, (24), 5994-6000.
121. Woodcock, D. M.; Linsenmeyer, M. E.; Chojnowski, G.; Kriegler, A. B.; Nink, V.; Webster, L. K.; Sawyer, W. H. Reversal of multidrug resistance by surfactants. Br J Cancer 1992, 66, (1), 62-68.
122. Loe, D. W.; Sharom, F. J. Interaction of multidrug-resistant Chinese hamster ovary cells with amphiphiles. Br J Cancer 1993, 68, (2), 342-351.
123. Regev, R.; Assaraf, Y. G.; Eytan, G. D. Membrane fluidization by ether, other anesthetics, and certain agents abolishes P-glycoprotein ATPase activity and modulates efflux from multidrug-resistant cells. European Journal of Biochemistry 1999, 259, (1-2), 18-24.
124. Risovic, V.; Sachs-Barrable, K.; Boyd, M.; Wasan, K. M. Potential mechanisms by which Peceol® increases the gastrointestinal absorption of amphotericin B. Drug Development & Industrial Pharmacy 2004, 30, (7), 767-774.
125. Sachs-Barrable, K.; Thamboo, A.; Lee, S. D.; Wasan, K. M. Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells. J Pharm Pharmaceut Sci 2007, 10, (3), 319-331.
126. Barta, C. A.; Sachs-Barrable, K.; Feng, F.; Wasan, K. M. Effects of monoglycerides on P-glycoprotein: Modulation of the activity and expression in Caco-2 cell monolayers. Molecular Pharmaceutics 2008, 5, (5), 863-875.
250
127. Kivisto, K. T.; Kroemer, H. K.; Eichelbaum, M. The role of human cytochrome P450 enzymes in the metabolism of anticancer agents: Implications for drug interactions. British Journal of Clinical Pharmacology 1995, 40, (6), 523-530.
128. Wacher, V. J.; Wu, C.-Y.; Benet, L. Z. Overlapping substrate specificities and tissue distribution of cytochrome P450 3A and P-glycoprotein: Implications for drug delivery and activity in cancer chemotherapy. Molecular Carcinogenesis 1995, 13, (3), 129-134.
129. Benet, L. Z.; Wu, C.-Y.; Hebert, M. F.; Wacher, V. J. Intestinal drug metabolism and antitransport processes: A potential paradigm shift in oral drug delivery. Journal of Controlled Release 1996, 39, (2–3), 139-143.
130. Benet, L. Z.; Cummins, C. L. The drug efflux–metabolism alliance: Biochemical aspects. Advanced Drug Delivery Reviews 2001, 50, Supplement 1, (0), S3-S11.
131. Watkins, P. B. The barrier function of CYP3A4 and P-glycoprotein in the small bowel. Advanced Drug Delivery Reviews 1997, 27, (2–3), 161-170.
132. Benet, L. Z.; Izumi, T.; Zhang, Y.; Silverman, J. A.; Wacher, V. J. Intestinal MDR transport proteins and P-450 enzymes as barriers to oral drug delivery. Journal of Controlled Release 1999, 62, (1–2), 25-31.
133. Patel, J. P.; Brocks, D. R. The effect of oral lipids and circulating lipoproteins on the metabolism of drugs. Expert Opinion on Drug Metabolism & Toxicology 2009, 5, (11), 1385-1398.
134. Buggins, T. R.; Dickinson, P. A.; Taylor, G. The effects of pharmaceutical excipients on drug disposition. Advanced Drug Delivery Reviews 2007, 59, (15), 1482-1503.
135. Wasan, K. M.; Brocks, D. R.; Lee, S. D.; Sachs-Barrable, K.; Thornton, S. J. Impact of lipoproteins on the biological activity and disposition of hydrophobic drugs: Implications for drug discovery. Nat Rev Drug Discov 2008, 7, (1), 84-99.
136. Humberstone, A. J.; Porter, C. J. H.; Edwards, G. A.; Charman, W. N. Association of halofantrine with postprandially derived plasma lipoproteins decreases its clearance relative to administration in the fasted state. Journal of Pharmaceutical Sciences 1998, 87, (8), 936-942.
137. Trevaskis, N. L.; Charman, W. N.; Porter, C. J. H. Lipid-based delivery systems and intestinal lymphatic drug transport: A mechanistic update. Advanced Drug Delivery Reviews 2008, 60, (6), 702-716.
138. Charman, W. N. A.; Stella, V. J. Effects of lipid class and lipid vehicle volume on the intestinal lymphatic transport of DDT. International Journal of Pharmaceutics 1986, 33, (1–3), 165-172.
139. Khoo, S.-M.; Shackleford, D. M.; Porter, C. J. H.; Edwards, G. A.; Charman, W. N. Intestinal lymphatic transport of halofantrine occurs after oral administration of a unit-dose lipid-based formulation to fasted dogs. Pharmaceutical Research 2003, 20, (9), 1460-1465.
140. White, K. L.; Nguyen, G.; Charman, W. N.; Edwards, G. A.; Faassen, W. A.; Porter, C. J. H. Lymphatic transport of methylnortestosterone undecanoate (MU) and the bioavailability of methylnortestosterone are highly sensitive to the mass of coadministered lipid after oral administration of MU. Journal of Pharmacology and Experimental Therapeutics 2009, 331, (2), 700-709.
251
141. Tsuji, A.; Tamai, I. Carrier-mediated intestinal transport of drugs. Pharmaceutical Research 1996, 13, (7), 963-977.
142. Lee, V. H. L. Membrane transporters. European Journal of Pharmaceutical Sciences 2000, 11, Supplement 2, S41-S50.
143. Poelma, F. G. J.; Breäs, R.; Tukker, J. J.; Crommelin, D. J. A. Intestinal absorption of drugs. The influence of mixed micelles on the disappearance kinetics of drugs from the small intestine of the rat. Journal of Pharmacy and Pharmacology 1991, 43, (5), 317-324.
144. Katneni, K.; Charman, S. A.; Porter, C. J. H. Permeability assessment of poorly water-soluble compounds under solubilizing conditions: The reciprocal permeability approach. Journal of Pharmaceutical Sciences 2006, 95, (10), 2170-2185.
145. Amidon, G. E.; Higuchi, W. I.; Ho, N. F. H. Theoretical and experimental studies of transport of micelle-solubilized solutes. Journal of Pharmaceutical Sciences 1982, 71, (1), 77-84.
146. Dahan, A.; Miller, J. M.; Hoffman, A.; Amidon, G. E.; Amidon, G. L. The solubility–permeability interplay in using cyclodextrins as pharmaceutical solubilizers: Mechanistic modeling and application to progesterone. Journal of Pharmaceutical Sciences 2010, 99, (6), 2739-2749.
147. Miller, J. M.; Beig, A.; Carr, R. A.; Spence, J. K.; Dahan, A. A win–win solution in oral delivery of lipophilic drugs: Supersaturation via amorphous solid dispersions increases apparent solubility without sacrifice of intestinal membrane permeability. Molecular Pharmaceutics 2012, 9, (7), 2009-2016.
148. Parrott, N.; Lave, T. Applications of physiologically based absorption models in drug discovery and development. Molecular Pharmaceutics 2008, 5, (5), 760-775.
149. Hilgers, A. R.; Smith, D. P.; Biermacher, J. J.; Day, J. S.; Jensen, J. L.; Sims, S. M.; Adams, W. J.; Friis, J. M.; Palandra, J.; Hosley, J. D.; Shobe, E. M.; Burton, P. S. Predicting oral absorption of drugs: A case study with a novel class of antimicrobial agents. Pharmaceutical Research 2003, 20, (8), 1149-1155.
150. Wootan, M. G.; Bernlohr, D. A.; Storch, J. Mechanism of fluorescent fatty acid transfer from adipocyte fatty acid binding protein to membranes. Biochemistry 1993, 32, (33), 8622-8627.
151. Thumser, A.; Tsai, J.; Storch, J. Collision-mediated transfer of long-chain fatty acids by neural tissue fatty acid-binding proteins (FABP). Journal of Molecular Neuroscience 2001, 16, (2), 143-150.
152. Narayanan, V. S.; Storch, J. Fatty acid transfer in taurodeoxycholate mixed micelles. Biochemistry 1996, 35, (23), 7466-7473.
153. Storch, J.; Kleinfeld, A. M. Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles. Biochemistry 1986, 25, (7), 1717-1726.
154. Nauli, A. M.; Nassir, F.; Zheng, S.; Yang, Q.; Lo, C. M.; VonLehmden, S. B.; Lee, D.; Jandacek, R. J.; Abumrad, N. A.; Tso, P. CD36 Is important for chylomicron formation and secretion and may mediate cholesterol uptake in the proximal intestine. Gastroenterology 2006, 131, (4), 1197-1207.
155. Werder, M.; Han, C.-H.; Wehrli, E.; Bimmler, D.; Schulthess, G.; Hauser, H. Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine. Biochemistry 2001, 40, (38), 11643-11650.
252
156. Hansen, G. H.; Niels-Christiansen, L.-L.; Immerdal, L.; Danielsen, E. M. Scavenger receptor class B type I (SR-BI) in pig enterocytes: Trafficking from the brush border to lipid droplets during fat absorption. Gut 2003, 52, (10), 1424-1431.
157. Altmann, S. W.; Davis Jr, H. R.; Yao, X.; Laverty, M.; Compton, D. S.; Zhu, L.-j.; Crona, J. H.; Caplen, M. A.; Hoos, L. M.; Tetzloff, G.; Priestley, T.; Burnett, D. A.; Strader, C. D.; Graziano, M. P. The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 2002, 1580, (1), 77-93.
158. Smart, E. J.; De Rose, R. A.; Farber, S. A. Annexin 2–caveolin 1 complex is a target of ezetimibe and regulates intestinal cholesterol transport. Proceedings of the National Academy of Sciences of the United States of America 2004, 101, (10), 3450-3455.
159. Valasek, M. A.; Weng, J.; Shaul, P. W.; Anderson, R. G. W.; Repa, J. J. Caveolin-1 is not required for murine intestinal cholesterol transport. Journal of Biological Chemistry 2005, 280, (30), 28103-28109.
160. Pohl, J.; Ring, A.; Ehehalt, R.; Schulze-Bergkamen, H.; Schad, A.; Verkade, P.; Stremmel, W. Long-chain fatty acid uptake into adipocytes depends on lipid raft function. Biochemistry 2004, 43, (14), 4179-4187.
161. Krieger, M. Scavenger receptor class B type I is a multiligand HDL receptor that influences diverse physiologic systems. Journal of Clinical Investigation 2001, 108, (6), 793-797.
162. Abumrad, N.; Coburn, C.; Ibrahimi, A. Membrane proteins implicated in long-chain fatty acid uptake by mammalian cells: CD36, FATP and FABPm. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1999, 1441, (1), 4-13.
163. Drover, V. A.; Abumrad, N. A. CD36-dependent fatty acid uptake regulates expression of peroxisome proliferator activated receptors. Biochemical Society Transactions 2005, 33, 311-315.
164. Sudhop, T.; Lütjohann, D.; von Bergmann, K. Sterol transporters: Targets of natural sterols and new lipid lowering drugs. Pharmacology & Therapeutics 2005, 105, (3), 333-341.
165. Hui, D. Y.; Howles, P. N. Molecular mechanisms of cholesterol absorption and transport in the intestine. Seminars in Cell & Developmental Biology 2005, 16, (2), 183-192.
166. Davies, J. P.; Levy, B.; Ioannou, Y. A. Evidence for a Niemann–Pick C (NPC) gene family: Identification and characterization of NPC1L1. Genomics 2000, 65, (2), 137-145.
167. Iyer, S. P.; Yao, X.; Crona, J. H.; Hoos, L. M.; Tetzloff, G.; Davis, H. R.; Graziano, M. P.; Altmann, S. W. Characterization of the putative native and recombinant rat sterol transporter Niemann-Pick C1 Like 1 (NPC1L1) protein. Biochimica et biophysica acta 2005, 1722, (3), 282-292.
168. Kirkham, M.; Parton, R. G. Clathrin-independent endocytosis: New insights into caveolae and non-caveolar lipid raft carriers. Biochim Biophys Acta 2005, 1745, (3), 273-286.
169. Ehehalt, R.; Füllekrug, J.; Pohl, J.; Ring, A.; Herrmann, T.; Stremmel, W. Translocation of long chain fatty acids across the plasma membrane – lipid rafts and fatty acid transport proteins. Molecular and Cellular Biochemistry 2006, 284, (1-2), 135-140.
253
170. Patel, H. H.; Murray, F.; Insel, P. A. Caveolae as organizers of pharmacologically relevant signal transduction molecules. Annual review of pharmacology and toxicology 2008, 48, 359-391.
171. Ring, A.; Pohl, J.; Völkl, A.; Stremmel, W. Evidence for vesicles that mediate long-chain fatty acid uptake by human microvascular endothelial cells. Journal of Lipid Research 2002, 43, (12), 2095-2104.
172. Babitt, J.; Trigatti, B.; Rigotti, A.; Smart, E. J.; Anderson, R. G. W.; Xu, S.; Krieger, M. Murine SR-BI, a high density lipoprotein receptor that mediates selective lipid uptake, is N-glycosylated and fatty acylated and colocalizes with plasma membrane caveolae. Journal of Biological Chemistry 1997, 272, (20), 13242-13249.
173. Brouwers, J.; Brewster, M. E.; Augustijns, P. Supersaturating drug delivery systems: The answer to solubility-limited oral bioavailability? Journal of Pharmaceutical Sciences 2009, 98, (8), 2549-2572.
174. Augustijns, P.; Brewster, M. E. Supersaturating drug delivery systems: Fast is not necessarily good enough. Journal of Pharmaceutical Sciences 2012, 101, (1), 7-9.
175. Gao, P.; Akrami, A.; Alvarez, F.; Hu, J.; Li, L.; Ma, C.; Surapaneni, S. Characterization and optimization of AMG 517 supersaturatable self-emulsifying drug delivery system (S-SEDDS) for improved oral absorption. Journal of Pharmaceutical Sciences 2009, 98, (2), 516-528.
176. Anby, M. U.; Williams, H. D.; McIntosh, M.; Benameur, H.; Edwards, G. A.; Pouton, C. W.; Porter, C. J. H. Lipid digestion as a trigger for supersaturation: Evaluation of the impact of supersaturation stabilization on the in vitro and in vivo performance of self-emulsifying drug delivery systems. Molecular Pharmaceutics 2012, 9, (7), 2063-2079.
177. Cuiné, J.; Charman, W.; Pouton, C.; Edwards, G.; Porter, C. Increasing the proportional content of surfactant (Cremophor EL) relative to lipid in self-emulsifying lipid-based formulations of danazol reduces oral bioavailability in beagle dogs. Pharmaceutical Research 2007, 24, (4), 748-757.
178. Mohsin, K.; Long, M. A.; Pouton, C. W. Design of lipid-based formulations for oral administration of poorly water-soluble drugs: Precipitation of drug after dispersion of formulations in aqueous solution. Journal of Pharmaceutical Sciences 2009, 98, (10), 3582-3595.
179. Warren, D. B.; Benameur, H.; Porter, C. J. H.; Pouton, C. W. Using polymeric precipitation inhibitors to improve the absorption of poorly water-soluble drugs: A mechanistic basis for utility. Journal of Drug Targeting 2010, 18, (10), 704-731.
180. Singhal, D.; Ho, N. F. H.; Anderson, B. D. Absorption and intestinal metabolism of purine dideoxynucleosides and an adenosine deaminase-activated prodrug of 2′ ,3′ -dideoxyinosine in the mesenteric vein cannulated rat ileum. Journal of Pharmaceutical Sciences 1998, 87, (5), 569-577.
181. Schurgers, N.; Bijdendijk, J.; Tukker, J. J.; Crommelin, D. J. A. Comparison of four experimental techniques for studying drug absorption kinetics in the anesthetized rat in situ. Journal of Pharmaceutical Sciences 1986, 75, (2), 117-119.
182. Grassi, M.; Cadelli, G. Theoretical considerations on the in vivo intestinal permeability determination by means of the single pass and recirculating techniques. International Journal of Pharmaceutics 2001, 229, (1–2), 95-105.
254
183. Salphati, L.; Childers, K.; Pan, L.; Tsutsui, K.; Takahashi, L. Evaluation of a single-pass intestinal-perfusion method in rat for the prediction of absorption in man. Journal of Pharmacy and Pharmacology 2001, 53, (7), 1007-1013.
184. Cummins, C. L.; Salphati, L.; Reid, M. J.; Benet, L. Z. In vivo modulation of intestinal CYP3A metabolism by P-glycoprotein: Studies using the rat single-pass intestinal perfusion model. Journal of Pharmacology and Experimental Therapeutics 2003, 305, (1), 306-314.
185. Ho, N. F. H.; Higuchi, W. I. Theoretical model studies of intestinal drug absorption IV: Bile acid transport at premicellar concentrations across diffusion layer—membrane barrier. Journal of Pharmaceutical Sciences 1974, 63, (5), 686-690.
186. Madara, J. L.; Pappenheimer, J. R. Structural basis for physiological regulation of paracellular pathways in intestinal epithelia. J. Membrain Biol. 1987, 100, (1), 149-164.
187. Fagerholm, U.; Nilsson, D.; Knutson, L.; Lennernäs, H. Jejunal permeability in humans in vivo and rats in situ: Investigation of molecular size selectivity and solvent drag. Acta Physiologica Scandinavica 1999, 165, (3), 315-324.
188. Lindahl, A.; Krondahl, E.; Grudén, A.-C.; Ungell, A.-L.; Lennernäs, H. Is the jejunal permeability in rats age-dependent? Pharmaceutical Research 1997, 14, (9), 1278-1281.
189. Pappenheimer, J. R.; Reiss, K. Z. Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat. J. Membrain Biol. 1987, 100, (1), 123-136.
190. Yano, K.; Masaoka, Y.; Kataoka, M.; Sakuma, S.; Yamashita, S. Mechanisms of membrane transport of poorly soluble drugs: Role of micelles in oral absorption processes. Journal of Pharmaceutical Sciences 2010, 99, (3), 1336-1345.
191. Gao, P.; Morozowich, W. Development of supersaturatable self-emulsifying drug delivery system formulations for improving the oral absorption of poorly soluble drugs. Expert Opinion on Drug Delivery 2006, 3, (1), 97-110.
192. Gu, X.; Trigatti, B.; Xu, S.; Acton, S.; Babitt, J.; Krieger, M. The efficient cellular uptake of high density lipoprotein lipids via Scavenger Receptor Class B Type I requires not only receptor-mediated surface binding but also receptor-specific lipid transfer mediated by its extracellular domain. Journal of Biological Chemistry 1998, 273, (41), 26338-26348.
193. Porter, C. J. H.; Kaukonen, A. M.; Boyd, B. J.; Edwards, G. A.; Charman, W. N. Susceptibility to lipase-mediated digestion reduces the oral bioavailability of danazol after administration as a medium-chain lipid-based microemulsion formulation. Pharmaceutical Research 2004, 21, (8), 1405-1412.
194. Kossena, G. A.; Boyd, B. J.; Porter, C. J. H.; Charman, W. N. Separation and characterization of the colloidal phases produced on digestion of common formulation lipids and assessment of their impact on the apparent solubility of selected poorly water-soluble drugs. Journal of Pharmaceutical Sciences 2003, 92, (3), 634-648.
195. Harmon, C. M.; Luce, P.; Beth, A. H.; Abumrad, N. A. Labeling of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-chain fatty acids: Inhibition of fatty acid transport. Journal of Membrane Biology 1991, 121, (3), 261-268.
255
196. Nieland, T. J. F.; Penman, M.; Dori, L.; Krieger, M.; Kirchhausen, T. Discovery of chemical inhibitors of the selective transfer of lipids mediated by the HDL receptor SR-BI. PNAS 2002, 99, (24), 15422-15427.
197. Coort, S. L. M.; Willems, J.; Coumans, W. A.; van der Vusse, G. J.; Bonen, A.; Glatz, J. F. C.; Luiken, J. J. F. P. Sulfo-N-succinimidyl esters of long chain fatty acids specifically inhibit fatty acid translocase (FAT/CD36)-mediated cellular fatty acid uptake. Molecular and Cellular Biochemistry 2002, 239, (1), 213-219.
198. Garcia-Calvo, M.; Lisnock, J.; Bull, H. G.; Hawes, B. E.; Burnett, D. A.; Braun, M. P.; Crona, J. H.; Davis, H. R.; Dean, D. C.; Detmers, P. A.; Graziano, M. P.; Hughes, M.; MacIntyre, D. E.; Ogawa, A.; O'Neill, K. A.; Iyer, S. P. N.; Shevell, D. E.; Smith, M. M.; Tang, Y. S.; Makarewicz, A. M.; Ujjainwalla, F.; Altmann, S. W.; Chapman, K. T.; Thornberry, N. A. The target of ezetimibe is Niemann-Pick C1-Like 1 (NPC1L1). Proceedings of the National Academy of Sciences of the United States of America 2005, 102, (23), 8132-8137.
199. Staggers, J. E.; Hernell, O.; Stafford, R. J.; Carey, M. C. Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 1. Phase behavior and aggregation states of model lipid systems patterned after aqueous duodenal contents of healthy adult human beings. Biochemistry 1990, 29, (8), 2028-2040.
200. Hay, D. W.; Cahalane, M. J.; Timofeyeva, N.; Carey, M. C. Molecular species of lecithins in human gallbladder bile. Journal of Lipid Research 1993, 34, (5), 759-68.
201. Duane, W. C.; Ginsberg, R. L.; Bennion, L. J. Effects of fasting on bile acid metabolism and biliary lipid composition in man. Journal of Lipid Research 1976, 17, (3), 211-9.
202. Boyd, B. J.; Porter, C. J. H.; Charman, W. N. Using the polymer partitioning method to probe the thermodynamic activity of poorly water-soluble drugs solubilized in model lipid digestion products. Journal of Pharmaceutical Sciences 2003, 92, (6), 1262-1271.
203. Waynforth, H. B.; Flecknell, P. A. Experimental and surgical technique in rat. Academic Press 1992.
204. Van Heek, M.; Farley, C.; Compton, D. S.; Hoos, L.; Alton, K. B.; Sybertz, E. J.; Davis, H. R. Comparison of the activity and disposition of the novel cholesterol absorption inhibitor, SCH58235, and its glucuronide, SCH60663. British Journal of Pharmacology 2000, 129, (8), 1748-1754.
205. Kyle, M. E.; Nakae, D.; Sakaida, I.; Miccadei, S.; Farber, J. L. Endocytosis of superoxide dismutase is required in order for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide. Journal of Biological Chemistry 1988, 263, (8), 3784-3789.
206. Winne, D. Rat jejunum perfused in situ: Effect of perfusion rate and intraluminal radius on absorption rate and effective unstirred layer thickness. Naunyn-Schmiedeberg's Archives of Pharmacology 1979, 307, (3), 265-274.
207. Johnson, B. M.; Chen, W.; Borchardt, R. T.; Charman, W. N.; Porter, C. J. H. A kinetic evaluation of the absorption, efflux, and metabolism of verapamil in the autoperfused rat jejunum. Journal of Pharmacology and Experimental Therapeutics 2003, 305, (1), 151-158.
256
208. Wagner, J. G.; Sedman, A. J. Quantitation of rate of gastrointestinal and buccal absorption of acidic and basic drugs based on extraction theory. Journal of Pharmacokinetics and Biopharmaceutics 1973, 1, (1), 23-50.
209. Dahan, A.; Miller, J. M.; Hilfinger, J. M.; Yamashita, S.; Yu, L. X.; Lennernäs, H.; Amidon, G. L. High-permeability criterion for BCS classification: Segmental/pH dependent permeability considerations. Molecular Pharmaceutics 2010, 7, (5), 1827-1834.
210. Haikal, Z.; Play, B.; Landrier, J.-F.; Giraud, A.; Ghiringhelli, O.; Lairon, D.; Jourdheuil-Rahmani, D. NPC1L1 and SR-BI are involved in intestinal cholesterol absorption from small-size lipid donors. Lipids 2008, 43, (5), 401-408.
211. Tran, T. T. T.; Poirier, H.; Clément, L.; Nassir, F.; Pelsers, M. M. A. L.; Petit, V.; Degrace, P.; Monnot, M.-C.; Glatz, J. F. C.; Abumrad, N. A.; Besnard, P.; Niot, I. Luminal lipid regulates CD36 levels and downstream signaling to stimulate chylomicron synthesis. Journal of Biological Chemistry 2011, 286, (28), 25201-25210.
212. Yu, L.; Bharadwaj, S.; Brown, J. M.; Ma, Y.; Du, W.; Davis, M. A.; Michaely, P.; Liu, P.; Willingham, M. C.; Rudel, L. L. Cholesterol-regulated Translocation of NPC1L1 to the Cell Surface Facilitates Free Cholesterol Uptake. Journal of Biological Chemistry 2006, 281, (10), 6616-6624.
213. Moussa, M.; Landrier, J.-F.; Reboul, E.; Ghiringhelli, O.; Coméra, C.; Collet, X.; Fröhlich, K.; Böhm, V.; Borel, P. Lycopene absorption in human intestinal cells and in mice involves Scavenger Receptor Class B Type I but not Niemann-Pick C1-Like 1. The Journal of Nutrition 2008, 138, (8), 1432-1436.
214. Reboul, E.; Goncalves, A.; Comera, C.; Bott, R.; Nowicki, M.; Landrier, J.-F.; Jourdheuil-Rahmani, D.; Dufour, C.; Collet, X.; Borel, P. Vitamin D intestinal absorption is not a simple passive diffusion: Evidences for involvement of cholesterol transporters. Molecular Nutrition & Food Research 2011, 55, (5), 691-702.
215. Reboul, E.; Klein, A.; Bietrix, F.; Gleize, B.; Malezet-Desmoulins, C.; Schneider, M.; Margotat, A.; Lagrost, L.; Collet, X.; Borel, P. Scavenger Receptor Class B Type I (SR-BI) Is involved in vitamin E transport across the enterocyte. Journal of Biological Chemistry 2006, 281, (8), 4739-4745.
216. Goldstein, J. L.; Brown, M. S.; Anderson, R. G. W.; Russell, D. W.; Schneider, W. J. Receptor-mediated endocytosis: Concepts emerging from the LDL receptor system. Annual Review of Cell Biology 1985, 1, (1), 1-39.
217. Mukerjee, P.; Mysels, K. J. Critical micelle concentrations of aqueous surfactant systems. Washington, DC: National Bureau of Standards 1971, 88-170.
218. Schubert, R.; Schmidt, K. H. Structural changes in vesicle membranes and mixed micelles of various lipid compositions after binding of different bile salts. Biochemistry 1988, 27, (24), 8787-8794.
219. Hofmann, A. F. Bile acids: The good, the bad, and the ugly. Physiology 1999, 14, (1), 24-29.
220. Vertzoni, M.; Fotaki, N.; Nicolaides, E.; Reppas, C.; Kostewicz, E. S.; Stippler, E.; Leuner, C.; Dressman, J. Dissolution media simulating the intralumenal composition of the small intestine: physiological issues and practical aspects. Journal of Pharmacy and Pharmacology 2004, 56, (4), 453-462.
257
221. Järvinen, T.; Järvinen, K.; Schwarting, N.; Stella, V. J. β-cyclodextrin derivatives, SBE4-β-CD and HP–β-CD, increase the oral bioavailability of cinnarizine in beagle dogs. Journal of Pharmaceutical Sciences 1995, 84, (3), 295-299.
222. Tønsberg, H.; Holm, R.; Mu, H.; Boll, J. B.; Jacobsen, J.; Müllertz, A. Effect of bile on the oral absorption of halofantrine in polyethylene glycol 400 and polysorbate 80 formulations dosed to bile duct cannulated rats. Journal of Pharmacy and Pharmacology 2011, 63, (6), 817-824.
223. Ericzon, B. G.; Todo, S.; Lynch, S.; Kam, I.; Ptachcinski, R. J.; Burckart, G. J.; Van Thiel, D. H.; Starzl, T. E.; Venkataramanan, R. Role of bile and bile salts on cyclosporine absorption in dogs. Transplant Proc. 1987, 19, (1 Pt 2), 1248-1249.
224. Behrens, D.; Fricker, R.; Bodoky, A.; Drewe, J.; Harder, F.; Heberer, M. Comparison of cyclosporin A absorption from LCT and MCT solutions following intrajejunal administration in conscious dogs. Journal of Pharmaceutical Sciences 1996, 85, (6), 666-668.
225. Miller, J. M.; Beig, A.; Carr, R. A.; Webster, G. K.; Dahan, A. The solubility–permeability interplay when using cosolvents for solubilization: Revising the way we use solubility-enabling formulations. Molecular Pharmaceutics 2012, 9, (3), 581-590.
226. Dahan, A.; Beig, A.; Ioffe-Dahan, V.; Agbaria, R.; Miller, J. M. The twofold advantage of the amorphous form as an oral drug delivery practice for lipophilic compounds: Increased apparent solubility and drug flux through the intestinal membrane. The AAPS Journal 2012, In press.
227. Yeap, Y. Y.; Trevaskis, N. L.; Quach, T.; Tso, P.; Charman, W. N.; Porter, C. J. H. Bile secretion promotes drug absorption from lipid colloidal phases via induction of supersaturation. Submitted.
228. Van Speybroeck, M.; Williams, H. D.; Nguyen, T.-H.; Anby, M. U.; Porter, C. J. H.; Augustijns, P. Incomplete desorption of liquid excipients reduces the in vitro and in vivo performance of self-emulsifying drug delivery systems solidified by adsorption onto an inorganic mesoporous carrier. Molecular Pharmaceutics 2012, 9, (9), 2750-2760.
229. Trevaskis, N. L.; Porter, C. J. H.; Charman, W. N. Bile increases intestinal lymphatic drug transport in the fasted rat. Pharmaceutical Research 2005, 22, (11), 1863-1870.
230. Bergström, C. A. S.; Wassvik, C. M.; Johansson, K.; Hubatsch, I. Poorly soluble marketed drugs display solvation limited solubility. Journal of Medicinal Chemistry 2007, 50, (23), 5858-5862.
231. McIntosh, M. P.; Charman, W. N.; Campbell, M.; Porter, C. J. H. Influence of physicochemical properties on the patterns of association of a series of aliphatic esters of halofantrine with plasma lipoproteins. Journal of Controlled Release 2004, 95, (2), 275-289.
232. Van Speybroeck, M.; Mellaerts, R.; Mols, R.; Thi, T. D.; Martens, J. A.; Van Humbeeck, J.; Annaert, P.; Van den Mooter, G.; Augustijns, P. Enhanced absorption of the poorly soluble drug fenofibrate by tuning its release rate from ordered mesoporous silica. European Journal of Pharmaceutical Sciences 2010, 41, (5), 623-630.
233. Sanphui, P.; Bolla, G.; Nangia, A. High solubility piperazine salts of the nonsteroidal anti-Inflammatory drug (NSAID) meclofenamic acid. Crystal Growth & Design 2012, 12, (4), 2023-2036.
258
234. Guichard, J. P.; Blouquin, P.; Qing, Y. A new formulation of fenofibrate: Suprabioavailable tablets. Current Medical Research and Opinion 2000, 16, (2), 134-138.
235. Gu, C.-H.; Rao, D.; Gandhi, R. B.; Hilden, J.; Raghavan, K. Using a novel multicompartment dissolution system to predict the effect of gastric pH on the oral absorption of weak bases with poor intrinsic solubility. Journal of Pharmaceutical Sciences 2005, 94, (1), 199-208.
236. Peeters, J. Determination of ionization constants in mixed aqueous solvents of varying composition by a single titration. Journal of Pharmaceutical Sciences 1978, 67, (1), 127-129.
237. Khoo, S.-M.; Prankerd, R. J.; Edwards, G. A.; Porter, C. J. H.; Charman, W. N. A physicochemical basis for the extensive intestinal lymphatic transport of a poorly lipid soluble antimalarial, halofantrine hydrochloride, after postprandial administration to dogs. Journal of Pharmaceutical Sciences 2002, 91, (3), 647-659.
238. Marriner, S.; Bogan, J. A. The influence of the rumen on the absorption of drugs: studies using meclofenamic acid administered by various routes to sheep and cattle. Journal of Veterinary Pharmacology and Therapeutics 1979, 2, (2), 109-115.
239. Hernell, O.; Staggers, J. E.; Carey, M. C. Physical-chemical behavior of dietary and biliary lipids during intestinal digestion and absorption. 2. Phase analysis and aggregation states of luminal lipids during duodenal fat digestion in healthy adult human beings. Biochemistry 1990, 29, (8), 2041-2056.
240. Humberstone, A. J.; Charman, W. N. Lipid-based vehicles for the oral delivery of poorly water soluble drugs. Advanced Drug Delivery Reviews 1997, 25, (1), 103-128.
241. O'Connor, C. J.; Wallace, R. G.; Iwamoto, K.; Taguchi, T.; Sunamoto, J. Bile salt damage of egg phosphatidylcholine liposomes. Biochimica et Biophysica Acta (BBA) - Biomembranes 1985, 817, (1), 95-102.
242. Schubert, R.; Beyer, K.; Wolburg, H.; Schmidt, K. H. Structural changes in membranes of large unilamellar vesicles after binding of sodium cholate. Biochemistry 1986, 25, (18), 5263-5269.
243. Rosoff, M.; Serajuddin, A. T. M. Solubilization of diazepam in bile salts and in sodium cholate-lecithin-water phases. International Journal of Pharmaceutics 1980, 6, (2), 137-146.
244. Spernath, A.; Yaghmur, A.; Aserin, A.; Hoffman, R. E.; Garti, N. Food-grade microemulsions based on nonionic emulsifiers: Media to enhance lycopene solubilization. Journal of Agricultural and Food Chemistry 2002, 50, (23), 6917-6922.
245. Spernath, A.; Yaghmur, A.; Aserin, A.; Hoffman, R. E.; Garti, N. Self-diffusion nuclear magnetic resonance, microstructure transitions, and solubilization capacity of phytosterols and cholesterol in Winsor IV food-grade microemulsions. Journal of Agricultural and Food Chemistry 2003, 51, (8), 2359-2364.
246. Garti, N.; Amar, I.; Yaghmur, A.; Spernath, A.; Aserin, A. Interfacial modification and structural transitions induced by guest molecules solubilized in U-type nonionic microemulsions. Journal of Dispersion Science and Technology 2003, 24, (3-4), 397-410.
247. Rane, S. S.; Anderson, B. D. What determines drug solubility in lipid vehicles: Is it predictable? Advanced Drug Delivery Reviews 2008, 60, (6), 638-656.
259
248. Feldman, S.; Gibaldi, M. Bile salt-induced permeability changes in the isolated rat intestine. Experimental Biology and Medicine 1969, 132, (3), 1031-1033.
249. Feldman, S.; Reinhard, M.; Willson, C. Effect of sodium taurodeoxycholate on biological membranes: Release of phosphorus, phospholipid, and protein from everted rat small intestine. Journal of Pharmaceutical Sciences 1973, 62, (12), 1961-1964.
250. Day, J. P. R.; Rago, G.; Domke, K. F.; Velikov, K. P.; Bonn, M. Label-free imaging of lipophilic bioactive molecules during lipid digestion by multiplex coherent anti-stokes raman scattering microspectroscopy. Journal of the American Chemical Society 2010, 132, (24), 8433-8439.
251. Williams, H. D.; Sassene, P.; Kleberg, K.; Calderone, M.; Igonin, A.; Jule, E.; Vertommen, J.; Blundell, R.; Benameur, H.; Mullertz, A.; Pouton, C. W.; Porter, C. J. H. Toward the establishment of standardized in vitro tests for lipid-based formulations: 3) Understanding supersaturation versus precipitation potential during the in vitro digestion of Type I, II, IIIA, IIIB and IV lipid-based formulations. Pharmaceutical Research 2013, This issue.
252. Lucas, M. L.; Schneider, W.; Haberich, F. J.; Blair, J. A. Direct measurement by pH-microelectrode of the pH microclimate in rat proximal jejunum. Proceedings of the Royal Society of London. Series B. Biological Sciences 1975, 192, (1106), 39-48.
253. Mahnensmith, R. L.; Aronson, P. S. The plasma membrane sodium-hydrogen exchanger and its role in physiological and pathophysiological processes. Circulation Research 1985, 56, (6), 773-88.
254. Williams, H. D.; Sassene, P.; Kleberg, K.; Bakala-N'Goma, J.-C.; Calderone, M.; Jannin, V.; Igonin, A.; Partheil, A.; Marchaud, D.; Jule, E.; Vertommen, J.; Maio, M.; Blundell, R.; Benameur, H.; Carrière, F.; Müllertz, A.; Porter, C. J. H.; Pouton, C. W. Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: Method parameterization and comparison of in vitro digestion profiles across a range of representative formulations. Journal of Pharmaceutical Sciences 2012, 101, (9), 3360-3380.
255. Chow, S. L.; Hollander, D. Linoleic acid absorption in the unaesthetized rat: Mechanism of transport and influence of luminal factors on absorption. Lipids 1979, 14, (4), 378-385.
256. Kanicky, J. R.; Shah, D. O. Effect of degree, type, and position of unsaturation on the pKa of long-chain fatty acids. Journal of Colloid and Interface Science 2002, 256, (1), 201-207.
257. Hofmann, A. F., Molecular association in fat digestion. In Molecular Association in Biological and Related Systems, AMERICAN CHEMICAL SOCIETY: 1968; Vol. 84, pp 53-66.
258. Hofmann, A. F.; Mysels, K. J. Bile salts as biological surfactants. Colloids and Surfaces 1987, 30, (1), 145-173.
259. Schoeller, C.; Keelan, M.; Mulvey, G.; Stremmel, W.; Thomson, A. B. R. Oleic acid uptake into rat and rabbit jejunal brush border membrane. Biochimica et Biophysica Acta (BBA) - Biomembranes 1995, 1236, (1), 51-64.
260. Cuiné, J. F.; McEvoy, C. L.; Charman, W. N.; Pouton, C. W.; Edwards, G. A.; Benameur, H.; Porter, C. J. H. Evaluation of the impact of surfactant digestion on the bioavailability of danazol after oral administration of lipidic self-emulsifying formulations to dogs. Journal of Pharmaceutical Sciences 2008, 97, (2), 995-1012.
260
261. Bevernage, J.; Brouwers, J.; Annaert, P.; Augustijns, P. Drug precipitation–permeation interplay: Supersaturation in an absorptive environment. European Journal of Pharmaceutics and Biopharmaceutics 2012, 82, (2), 424-428.
262. Raghavan, S. L.; Trividic, A.; Davis, A. F.; Hadgraft, J. Crystallization of hydrocortisone acetate: Influence of polymers. International Journal of Pharmaceutics 2001, 212, (2), 213-221.
263. Machefer, S.; Huddar, M. M.; Schnitzlein, K. Effect of polymer admixtures on the growth habit of ionic crystals. Study on crystal growth kinetics of potassium dihydrogen phosphate in water/polyol mixtures. Journal of Crystal Growth 2008, 310, (24), 5347-5356.
261
APPENDIX 1
Re-printed with permission from Yeap YY, Trevaskis NL, Quach T, Tso P, Charman WN,
Porter CJH. Intestinal bile secretion promotes drug absorption from lipid colloidal phases
via induction of supersaturation. Molecular Pharmaceutics, 2013, in press.
1 Intestinal Bile Secretion Promotes Drug Absorption from Lipid2 Colloidal Phases via Induction of Supersaturation3 Yan Yan Yeap,† Natalie L. Trevaskis,*,† Tim Quach,‡ Patrick Tso,§ William N. Charman,†
4 and Christopher J. H. Porter*,†
5†Drug Delivery, Disposition and Dynamics and ‡Medicinal Chemistry and Drug Action, Monash Institute of Pharmaceutical Sciences,
6 Monash University, 381 Royal Parade, Parkville, Victoria, 3052, Australia
7§Department of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, Ohio 45221, United States
8 *S Supporting Information
9 ABSTRACT: The oral bioavailability of poorly water-soluble10 drugs (PWSD) is often significantly enhanced by coadminis-11 tration with lipids in food or lipid-based oral formulations.12 Coadministration with lipids promotes drug solubilization in13 intestinal mixed micelles and vesicles, however, the mecha-14 nism(s) by which PWSD are absorbed from these dispersed15 phases remain poorly understood. Classically, drug absorption16 is believed to be a product of the drug concentration in free17 solution and the apparent permeability across the absorptive18 membrane. Solubilization in colloidal phases such as mixed19 micelles increases dissolution rate and total solubilized drug20 concentrations, but does not directly enhance (and may21 reduce) the free drug concentration. In the absence of changes to cellular permeability (which is often high for lipophilic,22 PWSD), significant changes to membrane flux are therefore unexpected. Realizing that increases in effective dissolution rate may23 be a significant driver of increases in drug absorption for PWSD, we explore here two alternate mechanisms by which membrane24 flux might also be enhanced: (1) collisional drug absorption where drug is directly transferred from lipid colloidal phases to the25 absorptive membrane, and (2) supersaturation-enhanced drug absorption where bile mediated dilution of lipid colloidal phases26 leads to a transient increase in supersaturation, thermodynamic activity and absorption. In the current study, collisional uptake27 mechanisms did not play a significant role in the absorption of a model PWSD, cinnarizine, from lipid colloidal phases. In28 contrast, bile-mediated dilution of model intestinal mixed micelles and vesicles led to drug supersaturation. For colloids that were29 principally micellar, supersaturation was maintained for a period sufficient to promote absorption. In contrast, for primarily30 vesicular systems, supersaturation resulted in rapid drug precipitation and no increase in drug absorption. This work suggests that31 ongoing dilution by bile in the gastrointestinal tract may invoke supersaturation in intestinal colloids and promote absorption,32 and thus presents a new mechanism by which lipids may enhance the oral absorption of PWSD.
33 KEYWORDS: absorption, poorly water-soluble drug, lipid-based formulation, supersaturation, bile, micelles, food effect,34 membrane permeability, cinnarizine
35 ■ INTRODUCTION
36 The potential for lipid-based formulations (LBF) to enhance
37 the oral bioavailability of poorly water-soluble drugs (PWSD)
38 has been recognized for over 40 years.1 Lipid coadministration
39 is thought to enhance the oral absorption of PWSD by
40 providing mechanisms to overcome both slow dissolution and
41 low solubility of PWSD in the aqueous gastrointestinal (GI)
42 milieu. First, LBF present drug to the GI tract in a molecularly
43 dispersed form (i.e., in solution in the formulation), thereby
44 circumventing the need for dissolution from the solid to the
45 liquid state. Subsequently, the intercalation of formulation
46 lipids into endogenous lipid digestion pathways results in the
47 generation of intestinal lipid colloidal phases (such as vesicular48 and micellar species) that enhance the solubilization capacity of
49the small intestine, promote drug solubilization and reduce the50risk of drug precipitation.51Solubilization within lipid colloidal phases therefore increases52the apparent solubility of PWSD in the intestinal fluids and53promotes dissolution. However, in the absence of solid drug,54solubilization also results in a reduction in thermodynamic55activity.2 In simple micellar systems this reduction in56thermodynamic activity is manifest in a decrease in the free57concentration of drug. Where solubilized drug exists in58equilibrium between the free concentration (Cfree) and the
Received: November 15, 2012Revised: March 9, 2013Accepted: March 12, 2013
Article
pubs.acs.org/molecularpharmaceutics
© XXXX American Chemical Society A dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXX
tms00 | ACSJCA | JCA10.0.1465/W Unicode | research.3f (R3.5.i1:3915 | 2.0 alpha 39) 2012/12/04 10:21:00 | PROD-JCAVA | rq_2253907 | 3/20/2013 16:18:31 | 15
59 concentration solubilized in intestinal colloids (Ccolloid), the60 total solubilized drug concentration (Ctotal) is the sum of Cfree61 and Ccolloid,
= +C C Ctotal free colloid62 (1)
63 Under these circumstances, the solubility of drug in the64 intermicellar phase (effectively the aqueous solubility of the65 drug) provides the upper limit for Cfree and the presence of66 solubilizing colloidal species typically increases the total drug67 concentration but does not increase, and often reduces, Cfree.68 Classical models of passive drug absorption suggest that drug69 flux across an absorptive membrane is the product of the free70 drug concentration and the drug permeability across the71 membrane. Therefore, where solubilization reduces Cfree (but72 does not alter permeability), absorption is expected to be73 reduced. Indeed, in solubilized systems, even at saturation, Cfree74 does not exceed the equilibrium solubility of drug in75 (nonmicellar) aqueous solution. Solubilization in intestinal76 colloidal phases therefore provides no practical advantage in77 free drug concentration and, in the absence of changes to78 permeability, is unlikely to lead to appreciable increases in79 membrane flux when compared to an aqueous solution80 containing drug at close to saturated solubility. In support of81 this suggestion, many authors have shown that increasing the82 total concentration of a PWSD by solubilization does not83 necessarily result in proportional increases in absorptive flux.2−6
84 Dahan et al. and Miller et al. recently proposed a model to85 quantify this phenomenon and referred to the existence of a86 “solubility-permeability interplay” where potential increases in87 membrane flux due to increases in solubilized drug88 concentration were offset by a reduction in the apparent89 permeability, the latter being, in large part, a function of90 decreases in free fraction.5,6
91 The dispersion and digestion of LBF therefore creates a92 solubilized reservoir that is in rapid equilibrium with drug in93 free solution and provides significant advantage in the effective94 rate of dissolution of a PWSD when compared to traditional95 dissolution from a solid dose form. In contrast, the inherent96 solubility limitations to flux (rather than dissolution-rate97 limitations) are seemingly unchanged when viewed from the98 perspective of the free concentration or may be made worse.99 This appears at odds with the wealth of experimental and100 practical observations that suggest the ability of lipids (either101 formulation- or dietary-derived) to enhance the oral absorption102 of a range of PWSD.1 A possible explanation for this anomaly is103 that the traditional view of drug absorption from colloidal104 dispersions may not adequately describe the dynamic manner105 in which LBF exert absorption-enhancing effects in the GI tract.106 Two alternative potential mechanisms of drug absorption are
f1 107 therefore examined here (Figure 1).108 The first mechanism evaluated was the potential for drug109 absorption to occur via direct collisional transfer from lipid110 colloidal phases to the absorptive membrane, and thus to be111 mediated not only by Cfree but also by the solubilized fraction,112 Ccolloid. Previously, studies by Storch and colleagues have shown113 that the transfer of poorly water-soluble fatty acids between114 model cell membranes and proteins may occur via collisional115 transfer.7,8 More recently, the possibility of drug absorption via116 collisional uptake has been suggested by Yano et al. and Gao et117 al.9,10 Collisional uptake may or may not be receptor-118 mediated,11 however, lipid uptake receptors such as CD36,12
119 SR-BI13 and NPC1L114 have been suggested to facilitate the120 absorption of cholesterol and fatty acids, poorly water-soluble
121molecules that are also solubilized in intestinal colloidal phases.122In the case of SR-BI and CD36, direct interaction of the123receptor with colloidal structures such as HDL (high-density124lipoprotein), bile salt micelles and phospholipid vesicles13,15 has125also been suggested, raising the possibility that lipid uptake126receptors may interact directly with intestinal lipid colloidal127phases to facilitate collisional absorption of solubilized contents,128including PWSD.129The second mechanism evaluated was the potential for130endogenous lipid processing pathways to lead to drug131supersaturation in lipid colloidal phases. Supersaturation132increases the thermodynamic activity of solubilized drug and,133in the solubilization model described by eq 1, will increase Cfree134above the equilibrium aqueous drug solubility. This in turn is135expected to enhance drug flux. The potential for super-136saturation to enhance the oral bioavailability of PWSD has137received increasing recent interest.10,16 For LBF, supersatura-138tion may be generated by the loss of drug solubilization139capacity resulting from the digestion of triglycerides17−19 and/140or surfactants,20 and the dilution of cosolvents21 during GI141processing.19 In contrast, the possibility that supersaturation142may result directly from interactions between lipid colloidal143phases and biliary fluids has been almost entirely ignored.144Although traditional micellar solubilization models suggest that145increases in bile salt concentrations increase drug solubilization,146previous studies have also shown that dilution of lipid colloidal147phases with model intestinal fluids (containing bile salts,148phospholipid and cholesterol) may lead to the generation of149less lipid-rich colloidal phases with lowered solubilization150capacities.22 This provides a plausible mechanism for super-151saturation generation in the small intestine and has been152examined in detail here.
Figure 1. Alternative mechanisms of drug absorption from intestinallipid colloidal phases. In collisional drug absorption (left panel), inaddition to the diffusion of free drug molecules (a), lipid colloidalphases collide with the absorptive membrane and facilitate directtransfer of solubilized drug into absorptive cells (b). Collisionaltransfer may or may not be receptor-mediated. In supersaturation-enhanced drug absorption (right panel), the interaction betweensecreted bile and lipid colloidal phases leads to drug supersaturation(possibly via stimulation of phase changes to less lipid-rich colloidswith lowered drug solubilization capacity). The increase inthermodynamic activity manifests in increases in free drugconcentration, and enhanced diffusional flux (a) across the absorptivemembrane.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXB
153 The data suggest, that under the conditions explored,154 collisional drug absorption has little impact on drug absorption155 from intestinal colloidal species. In contrast, supersaturation-156 enhanced drug absorption, mediated by the interaction between157 secreted bile and lipid colloidal phases, may provide an158 endogenous mechanism to promote supersaturation and to159 facilitate drug absorption from lipid colloidal phases.
160 ■ EXPERIMENTAL SECTION161 Materials. Cinnarizine, flunarizine dihydrochloride, mon-162 ensin sodium, sodium taurocholate, sodium taurodeoxycholate,163 sodium glycocholate, sodium glycochenodeoxycholate, choles-164 terol, L-α-lysophosphatidylcholine (LPC, from egg yolk), L-α-165 phosphatidylcholine (PC, from dried egg yolk), oleic acid,166 caprylic acid, monocaprylin, N-hydroxysulfosuccinimide so-167 dium, dicyclohexylcarbodiimide solution (60% w/v in xylenes),168 N,N-dimethylformamide, TWEEN 80, potassium dihydrogen169 phosphate (KH2PO4) and sodium chloride (NaCl) were170 obtained from Sigma-Aldrich, Australia. Sodium taurocheno-171 deoxycholate, sodium glycodeoxycholate, ortho-phosphoric172 acid 85% (H3PO4), sodium hydroxide pellets (NaOH), tert-173 butyl methyl ether (TBME), dimethyl sulfoxide (DMSO),174 glacial acetic acid and absolute ethanol were from Merck,175 Australia. Disodium hydrogen orthophosphate (Na2HPO4),176 sodium dihydrogen orthophosphate (NaH2PO4·2H2O) and177 ammonium dihydrogen orthophosphate (NH4H2PO4) (Ajax178 Finechem, Australia), cholesterol, [4-14C] (49.8 mCi/mmol),179 and Irga-Safe Plus (Perkin-Elmer Life Sciences, Waltham, MA),180 oleic acid, [9,10-3H(N)] (60 Ci/mmol) (American Radio-181 labeled Chemicals, St. Louis, MO), Block Lipid Transport-1182 (BLT-1, i.e., 2-hexyl-1-cyclopentanone thiosemicarbazone)183 (Chembridge, San Diego, CA), ezetimibe (Jai Radhe Sales,184 AMD, India), heparin sodium injection BP (1000 IU/mL,185 Hospira, Australia), xylazine (100 mg/mL, Troy Laboratories,186 Australia), acepromazine (10 mg/mL, Ceva Delvet, Australia),187 ketamine (100 mg/mL, Provet, Australia) and pentobarbitone188 sodium (325 mg/mL, Virbac, Australia) were obtained from189 listed suppliers. Acetonitrile, methanol and chloroform used190 were analytical reagent grade. Water was obtained from a191 Millipore Milli-Q Gradient A10 water purification system192 (Millipore, Billerica, MA).193 Sulfo-N-succinimidyl Oleate (SSO) Synthesis. SSO syn-194 thesis was adapted from the procedure of Harmon et al.23
195 Briefly, dicyclohexylcarbodiimide (1.26 mmol) and N-hydrox-196 ysulfosuccinimide (sodium salt, 1.20 mmol) were added to a197 solution of oleic acid (1.20 mmol) dissolved in N,N-198 dimethylformamide (2 mL), and the reaction was stirred at199 room temperature overnight. The precipitated dicyclohexylurea200 was removed by filtration and ethyl acetate (2 mL) added to201 the filtrate, which was left to stand at 4 °C overnight. SSO202 (precipitate) was then collected by filtration and dried under203 vacuum (1 mmHg). SSO identity was confirmed by NMR and204 mass spectrometry.205 Experimental outline. To assess the role of receptor-206 mediated collisional drug absorption, cinnarizine bioavailability207 was assessed after administration of a lipid emulsion208 formulation in the absence and presence of BLT-1, SSO and209 ezetimibe. BLT-1, SSO and ezetimibe are chemical inhibitors of210 SR-BI,24 CD3625 and NPC1L1,26 respectively. The possibility211 of endocytosis-mediated uptake was also investigated by the use212 of a general endocytosis inhibitor, monensin.213 The role of collisional drug absorption was assessed more214 generically using an in situ rat jejunum perfusion model to
215compare the absorptive flux of cinnarizine from two distinctly216different lipid colloidal phases (micelles vs vesicles) with217matched drug solubilization capacities. Colloidal systems with218the same total solubilization capacity, loaded with drug at the219same concentration, have the same thermodynamic activity and220therefore Cfree is the same in both cases. Under these221circumstances, comparison of the flux profiles obtained from222two structurally different colloids, but with identical Cfree,223provides a means of determining whether the nature of the224colloid, or Cfree, is the principal determinant of absorption.225Generation of identical flux profiles from both systems would226therefore confirm the dependence of flux on thermodynamic227activity and free concentration, whereas a significant difference228in flux would indicate a role for factors beyond Cfree in229determining flux. These include the potential for collisional230drug absorption mechanisms since collision rates are a231statistical function of particle number and are expected to be232markedly higher for micelles (where the smaller particle size233results in higher particle numbers) when compared to vesicles.234To assess the potential for intestinal fluids to enhance drug235absorption from lipid colloidal phases via the induction of drug236supersaturation, whole bile was collected from fasted rats and237mixed with model micelles and vesicles to simulate the process238of interaction with bile in vivo. The potential for bile to generate239drug supersaturation was evaluated in vitro by assessing changes240in cinnarizine solubility, and by monitoring the kinetics of241cinnarizine solubilization and precipitation, following bile242addition to cinnarizine-loaded micelles and vesicles. Sub-243sequently, the impact of drug supersaturation on the intestinal244absorptive flux of cinnarizine from micelles and vesicles was245assessed in an in situ rat jejunum perfusion model, with and246without coperfusion of donor bile. Finally, the relevance of bile-247induced drug supersaturation in vivo was assessed via248examination of changes to cinnarizine bioavailability after249administration of drug-loaded micelles and vesicles (with250matched thermodynamic activity) in bile-intact vs bile-diverted251rats.252Formulation Preparation. Lipid Emulsion. The lipid253emulsion (3 mL per dose) consisted of 1 mg of cinnarizine254and 49 mg of oleic acid solubilized in 8 mM sodium255taurocholate, 2 mM phosphatidylcholine, 2 mM cholesterol256and trace amounts of 14C-cholesterol (1 μCi/3 mL) and/or 3H-257oleic acid (3 μCi/3 mL). The emulsion was prepared in 7.5 mL258batches by weighing appropriate masses of cinnarizine in oleic259acid stock solution (20 mg/g), phosphatidylcholine and260cholesterol into a glass vial, and the mixture made up to261volume with a buffered sodium taurocholate solution (buffer262consisted 18 mM Na2H2PO4·2H2O and 12 mM Na2HPO4).263Appropriate volumes of 14C-cholesterol, 3H-oleic acid, 5 mg/264mL BLT-1 in ethanol (for SR-BI inhibition experiments only),26525 mg/mL SSO in DMSO (for CD36 inhibition experiments266only) and 10 mg/mL monensin in ethanol (for endocytosis267inhibition experiments only) were spiked into the vial and268vortexed for 1 min. The formulation was emulsified by269ultrasonification with a Misonix XL 2020 ultrasonic processor270(Misonix, Farmingdale, NY) equipped with a 3.2 mm271microprobe tip running at an amplitude of 240 μm and a272frequency of 20 kHz for 1.5 min. The total solvent273concentration in the emulsion was ≤2.5% v/v. The emulsion274was used within 4 h of preparation, and the concentration of275drug and labeled cholesterol and/or oleic acid was assayed276before dosing (in duplicate) to confirm compound content in277the emulsion and to allow for dose normalization between rats.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXC
278 Intravenous Formulation. The formulation (1 mL per279 dose) used for intravenous administration of cinnarizine and280
14C-cholesterol comprised 0.5% w/v Tween 80 in buffer (36281 mM Na2HPO4 and 22 mM KH2PO4, adjusted to pH 4 with282 acetic acid). Cinnarizine and cholesterol were added to the283 formulation by spiking 5% v/v DMSO (containing 10 mg/mL284 cinnarizine) and 5% v/v ethanol (containing 5 mg/mL285 cholesterol and 40 μCi/mL 14C-cholesterol) into the micellar286 solution. The formulation was mixed by vortexing, and the287 concentration of drug and labeled cholesterol was assayed288 before dosing to confirm compound content in the formulation289 and to allow for dose normalization between rats. The290 formulation was used within 1 h of preparation.291 Model Micelles and Vesicles. The preparation of model292 micelles and vesicles was guided by the methods and phase293 diagram published by Kossena et al.27 Medium-chain lipid294 containing colloids were chosen over long-chain systems since295 the former have previously been shown to generate mono-296 phasic micellar and vesicular systems.27 Relatively high lipid297 concentrations were chosen to reflect the species that are298 expected to initially form during the digestion of medium-chain299 triglycerides.18 The model colloids consisted of tricaprylin300 digestion products (caprylic acid and monocaprylin) solubilized301 in simulated endogenous intestinal fluid (SEIF). SEIF302 comprised the six most prevalent bile salts in human bile,28
303 lysophosphatidylcholine (LPC) and cholesterol. The total bile304 salt:LPC:cholesterol molar ratio was maintained at 16:4:1,305 reflecting known ratios within fasted human bile.29,30 The306 combination of bile salts used here comprised 25 mol % sodium307 glycocholate, 17.5 mol % sodium glycodeoxycholate, 25 mol %308 sodium glycochenodeoxycholate, 12.5 mol % sodium taur-309 ocholate, 7.5 mol % sodium taurodeoxycholate and 12.5 mol %310 sodium taurochenodeoxycholate. The concentration ratios of311 the bile salts were chosen based on average concentrations of312 the six most prevalent bile salts found in human bile.28 The313 caprylic acid:monocaprylin molar ratio was kept at 2:1,314 reflecting the ratio of digestion products expected on digestion315 of 1 mol of triglyceride. The concentration of micellar and316 vesicular components was varied by trial and error (but317 maintaining the ratios described above) to identify systems with318 similar drug solubilization capacities. It has previously been319 shown that the thermodynamic activity (i.e., free concen-320 tration) of drug in a solubilized system may be estimated via321 assessment of solubility behavior, such that having different322 colloidal solutions containing drug at a fixed proportion of the323 saturated solubility results in matched free concentrations.31
324 Thus, drug was loaded into either micellar or vesicular systems325 at the same concentration (and the same proportion of326 saturated solubility) and was therefore present at the same327 thermodynamic activity (i.e., Ctotal, Ccolloid and Cfree were the328 same in both micellar and vesicular systems). The compositions329 of the identified micellar and vesicular systems are shown in
t1 330 Table 1.331 SEIF (8 mM total bile salt:2 mM LPC:0.5 mM cholesterol)332 was prepared in 50 mL batches. Briefly, LPC and cholesterol333 were dissolved in 1 mL of chloroform in a round-bottom flask,334 followed by solvent evaporation under vacuum. The thin film335 formed by solvent evaporation was reconstituted with buffered336 bile salts solution (8 mM total bile salt, 18 mM337 NaH2PO4·2H2O and 12 mM Na2HPO4, 100 mM NaCl),338 vortexed for 1 min and allowed to equilibrate at room339 temperature overnight. When vesicles were prepared, a similar340 procedure was adopted, but in this case SEIF was diluted 4-fold
341with buffer (18 mM NaH2PO4·2H2O and 12 mM Na2HPO4,342108 mM NaCl) to reduce the bile salt:lipid concentration ratio.343Micelles and vesicles were prepared in 10 mL batches by adding344caprylic acid and monocaprylin (quantities in Table 1) to SEIF,345followed by pH adjustment to 6.30 with solid NaOH and346vortexing for 1 min. The phases were then ultrasonicated as347described earlier (30 s continuous ultrasonication followed by348pulsatile, 1 s on/1 s off ultrasonication for 5 min). When349included in the colloids, cinnarizine was predissolved in caprylic350acid, and the drug/fatty acid solution was allowed to equilibrate351overnight prior to micelle/vesicle preparation.352Particle Sizing. The particle size of model micelles and353vesicles was determined by photon correlation spectroscopy354(Malvern Instruments Nano-ZS Zetasizer, Malvern, U.K.).355Micelles had a mean particle size of 9 ± 1 nm [polydispersity356index 0.128 ± 0.040], and vesicles had a mean particle size of357443 ± 32 nm [polydispersity index 0.456 ± 0.038]. Data358reported are mean ± SEM of n = 3 determinations.359Equilibrium Solubility of Cinnarizine in the Model360Micelles and Vesicles. Excess solid cinnarizine was added to3612 mL micelles or vesicles in glass vials. Vials were briefly362vortexed and incubated at 37 °C and samples taken every 24 h363over a period of 120 h. During sampling, vials were centrifuged364(2200g, 10 min, 37 °C), 50 μL of supernatant was sampled, and365vials were revortexed. Equilibrium solubility was defined when366drug concentrations in consecutive samples varied by ≤5% and367was determined on three separate occasions.368Equilibrium solubility of cinnarizine was also determined369after 1:1 addition of bile/bile pH 6.30/buffer pH 6.30 to the370different colloidal phases. The pH of fresh bile was adjusted to3716.30 with H3PO4. Buffer pH 6.30 consisted of 18 mM372NaH2PO4·2H2O, 12 mM Na2HPO4 and 108 mM NaCl.373Kinetics of Cinnarizine Precipitation after the374Addition of Bile to Model Micelles and Vesicles. The375kinetics of cinnarizine precipitation was monitored after376addition of bile to the micellar and vesicular phases, to377determine whether a period of drug supersaturation existed378prior to drug precipitation. In a temperature and stirring rate-379controlled vessel, 2.5 mL of bile was added to 2.5 mL of380micelles or vesicles containing 0.2 mg/mL cinnarizine (∼80%381saturated solubility). Samples (100 μL) were taken before the382addition of bile, and at 1, 10, 20, 30, 40, 50, 60, 80, 100, 120383min after bile addition. Samples were immediately centrifuged384(2200g, 5 min, 37 °C) to separate precipitated drug, and 50 μL385of supernatant was assayed for drug content. The proportion of386the initial solubilized cinnarizine concentration that remained387solubilized after bile addition was assessed as the percent of the388drug mass remaining in solution (i.e., concentration in the
Table 1. Composition of Model Micelles and Vesiclesa
concn (mM)
total bilesaltb LPCc cholesterol
caprylicacid monocaprylin
micelles 8 2 0.5 69.3 34.7vesicles 2 0.5 0.125 52.0 26.0
aMicelles and vesicles also consist of 18 mM NaH2PO4·2H2O and 12mM Na2HPO4. Sodium strength was adjusted to 150 mM with NaCl.Final pH of phases was adjusted to 6.30 ± 0.01. bTotal bile salt consistof 25 mol % sodium glycocholate, 17.5 mol % sodium glycodeox-ycholate, 25 mol % sodium glycochenodeoxycholate, 12.5 mol %sodium taurocholate, 7.5 mol % sodium taurodeoxycholate, 12.5 mol% sodium taurochenodeoxycholate. cLPC is lysophosphatidylcholine.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXD
389 supernatant multiplied by the volume remaining in vessel)390 relative to the total drug mass in the vessel at each time point.391 Solid-State Analysis of the Cinnarizine Precipitate392 Using Polarized Light Microscopy. Selected cinnarizine393 pellets from the precipitation kinetics experiments were394 analyzed using a Zeiss Axiolab microscope (Carl Zeiss,395 Oberkochen, Germany) equipped with crossed polarizing396 filters. At the end of the precipitation kinetics experiments,397 1.5 mL of remaining bile + colloid mixture was centrifuged398 (2200g, 10 min, 37 °C), the supernatant was discarded, and a399 small amount of pellet was carefully placed on a microscope400 slide. Samples were analyzed under cross-polarized light, and401 images were recorded using a Canon PowerShot A70 digital402 camera (Canon, Tokyo, Japan).403 Animals. All rat studies were approved by the institutional404 animal ethics committee and were conducted in accordance405 with the guidelines of the Australian and New Zealand Council406 for the Care of Animals in Research and Teaching. Male407 Sprague−Dawley rats (280−330 g) were used in all experi-408 ments and were allowed to acclimatize in the institutional409 animal housing facility for at least 7 days with free access to410 standard chow and water. All animals were fasted overnight411 (12−18 h) prior to surgery.412 Surgical Procedures. Anesthesia was induced in rats by413 subcutaneous injection of 1.0 mL/kg of “Cocktail I” (37.3 mg/414 mL ketamine, 9.8 mg/mL xylazine, 0.4 mg/mL acepromazine415 in saline), and maintained throughout the study with416 subcutaneous doses of 0.44 mL/kg of “Cocktail II” (90.9417 mg/mL ketamine, 0.9 mg/mL acepromazine) when required.418 Rats were maintained on a 37 °C heated pad throughout419 surgery and experiments. At the end of all experiments, rats420 were euthanized via an intravenous or intracardiac injection of421 100 mg of sodium pentobarbitone.422 Cinnarizine Bioavailability Studies following Intraduode-423 nal Administration. The surgical procedures for the conduct of424 bioavailability studies included cannulations of the right carotid425 artery, right jugular vein, duodenum (1 cm below pylorus) and426 common bile duct (only for bile-diverted rats). The surgical427 procedures for the cannulations were as described previ-428 ously.32,33
429 Fasted Rat Bile Collection. The bile duct was cannulated430 near the hilum of the liver (where the duct is free of pancreatic431 tissue) in order to facilitate the collection of bile fluid without432 contamination by exocrine pancreatic secretions.34 Rats were433 rehydrated via saline infusion (1.5 mL/h) into a cannula434 inserted into the right jugular vein, and bile was continuously435 collected for 5 h. The concentration of total bile salt in436 collected bile was assayed using a validated enzymatic437 colorimetric assay (Total Bile Acids kit #431-15001; Wako438 Pure Chemical Industries, Osaka, Japan) on a plate reader439 (Fluostar Optima plate reader, BMG Labtechnologies,440 Germany) measuring absorbance at a wavelength of 540 nm.441 In all subsequent experiments, bile was used within 24 h of442 collection.443 Single-Pass Rat Jejunum Perfusion. The model employed444 to assess flux across rat jejunum involved in situ perfusion445 (single-pass) of an isolated jejunal segment and simultaneous446 blood collection from the corresponding mesenteric vein447 branch. The surgical procedures for the perfusion studies are448 similar to those described elsewhere, with slight modifica-449 tions.35 Briefly, the right jugular vein was cannulated to enable450 infusion of donor blood. A piece of jejunal segment (∼10 cm2)451 was isolated and cannulated at the proximal and distal ends
452with sections of Teflon tubing (0.03 in. i.d. proximal/inlet,453Upchurch Scientific, Oak Harbor, WA; 0.0625 in. i.d. distal/454outlet, Shimadzu, Kyoto, Japan). Jejunal contents were initially455flushed with warm perfusion buffer (150 mM Na+, 18 mM456H2PO4
−, 12 mM HPO42−, 108 mM Cl−, adjusted to pH 6.30 ±4570.01). The mesenteric vein draining the jejunal segment was458then isolated, the rat heparinized (90 IU/kg) via the jugular459vein and the mesenteric vein immediately catheterized. A drop460of superglue was placed over the site of catheterization, and461silicone tubing (0.025 in. i.d., Helix Medical, CA) attached to462the catheter tip for the collection of venous blood. Immediately463following catheterization of the mesenteric vein and for the464remainder of the experiment, rats were infused with heparinized465donor rat blood (5 IU/mL) via the jugular vein, and the rate466(0.3 mL/min) was adjusted based on the outflow from the467mesenteric blood.468Cinnarizine Bioavailability after Intraduodenal Infu-469sion in Anesthetized Rats. A 30 min equilibration period470was allowed between the end of surgery and drug dosing. To471examine the impact of lipid uptake receptors on drug472absorption, studies were conducted in the presence or absence473of lipid uptake inhibitors. To examine the impact of bile-474induced drug supersaturation on drug absorption, studies were475conducted in bile-intact or bile-diverted rats. Colloidal systems476(lipid emulsion, micelles, vesicles) containing cinnarizine were477infused into the duodenum of rats at a rate of 1.5 mL/h for 2 h.478When the dose infusion was complete, saline was infused at a479rate of 1.5 mL/h for 10 min to flush any remaining formulation480in the tubing into the duodenum. Blood samples (0.3 mL) were481collected via the carotid artery cannula up to 8 h after infusion482initiation into tubes containing 3 IU heparin. The sampling483intervals were as follows: t = 0, 1, 2, 2.5, 3, 4, 6, 8 h for the484receptor inhibition studies; and t = 0, 1, 1.5, 2, 3, 4, 6, 8 h for485the bile-induced drug supersaturation studies. After each blood486sample was taken, the cannula was flushed with 0.3 mL of 2 IU/487mL heparinized saline to ensure cannula patency, and to replace488the volume of blood removed. Plasma was separated by489centrifugation (10000g, 5 min) to enable analysis of drug and490labeled lipid content.491Administration of Lipid Uptake Inhibitors (BLT-1, SSO,492Ezetimibe) and Endocytosis Inhibitor (Monensin). Ezetimibe493has previously been dosed at 0.3 mg/kg intravenously into rats,494and has been shown to inhibit cholesterol absorption (from an495intraduodenally dosed lipid emulsion) without reports of496toxicity.36 Therefore, in our study, intravenous administration497of ezetimibe (0.3 mg/kg via the jugular vein) was selected as498the route to administer the inhibitor at the beginning of the 30499min equilibration period. An appropriate volume of 5 mg/mL500ezetimibe in ethanol was spiked into blank rat plasma, and 0.8501mL of resultant plasma was dosed into rats as an intravenous502bolus. The total ethanol concentration was less than 2.5% v/v.503In the case of BLT-1 and SSO, the inhibitors had not been504previously administered intravenously, therefore local (i.e.,505intestinal) administration was selected to limit the systemic506effects of the inhibitors. Monensin was also administered507directly into the intestine to minimize systemic endocytosis508inhibition. Here, 100 μM BLT-1, 1 mM SSO and 100 μM509monensin were preinfused intraduodenally (in saline) at a rate510of 1.5 mL/h during the 30 min equilibration period, and511subsequently coinfused at the same concentration as part of the512lipid emulsion. BLT-1 and SSO have previously been shown to513inhibit lipid uptake in cell-based studies at concentrations of 1−
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXE
514 10 μM24 and 400 μM,23 respectively. Monensin has previously515 been shown to inhibit endocytosis in cultured cells at 10 μM.37
516 Intravenous administration studies were also conducted in517 control rats and monensin-treated rats to assess the effect of518 endocytosis inhibition on the systemic distribution and519 clearance of cinnarizine and cholesterol. In these studies,520 blank (i.e., not containing cinnarizine and 14C-cholesterol) lipid521 emulsion (with or without 100 μM monensin) was infused522 intraduodenally as described above, and intravenous infusion of523 the cinnarizine and 14C-cholesterol containing intravenous (iv)524 formulation was commenced at the same time. The iv525 formulation (1 mL) was infused into the right jugular vein at526 a rate of 0.05 mL/15 s (total infusion period <5 min). Blood527 samples (0.3 mL) were collected via the carotid artery cannula528 at t = 5, 15, 30, 60, 120, 180, 240, 360, 480 min after infusion529 initiation into tubes containing 3 IU of heparin. After each530 blood sample was taken, the cannula was flushed with 0.3 mL of531 2 IU/mL heparinized saline. Plasma was separated by532 centrifugation (10000g, 5 min) to enable analysis of drug and533 labeled cholesterol content.534 Intestinal Absorptive Flux Assessment via in Situ535 Single-Pass Rat Jejunum Perfusion. After surgery, animals536 were equilibrated for 30 min, during which time heparinized537 donor rat blood was infused via the jugular vein as described538 above. During re-equilibration, blood from the cannulated539 mesenteric vein (∼0.3 mL/min) was collected for reinfusion.540 Perfusion buffer was pumped through the jejunal segment at a541 rate of 0.1 mL/min and outflowing buffer discarded to waste.542 The exposed jejunal segment was kept moist by covering with543 saline-soaked gauze throughout the experiment.544 In all experiments, the concentration of cinnarizine in the545 perfusate was held at 0.1 mg/mL (∼40% saturated solubility).546 Therefore, in experiments where micelles or vesicles were547 perfused alone, cinnarizine was loaded into the perfusate at 0.1548 mg/mL. In experiments where micelles or vesicles were549 coperfused in a 1:1 v/v ratio with a secondary perfusate of550 bile/bile pH 6.30/buffer pH 6.30, cinnarizine was loaded into551 the primary perfusate at 0.2 mg/mL, such that 1:1 v/v dilution552 led to a final perfusate concentration of 0.1 mg/mL.553 Perfusate flow was maintained at 0.1 mL/min in all554 experiments to minimize variations in the thickness of the555 unstirred water layer.38 For experiments where 1:1 v/v556 coperfusion of the phases with bile/bile pH 6.30/buffer pH557 6.30 was required, micelles/vesicles and bile/buffer were558 pumped at 0.05 mL/min, and mixed via a three-way “T”559 connector immediately prior to entry into the jejunal segment,560 providing a total perfusate flow of 0.1 mL/min. Perfusate was561 sampled at t = 0 to confirm lipid and drug concentrations. After562 this time, the outflowing perfusate was continuously collected563 at 10 min intervals and briefly vortexed before samples were564 taken for analysis of drug and lipid content. For experiments565 where drug supersaturation was generated, perfusate samples566 were taken before and after centrifugation (2200g, 2 min), to567 assess the degree of drug precipitation within the jejunal568 segment. Blood draining the perfused jejunal segment was569 collected at 5 min intervals, plasma was separated by570 centrifugation (10000g, 5 min) and samples were taken for571 analysis of drug content by HPLC as described below.572 Analytical Procedures. Sample Preparation and HPLC573 Assay Conditions for Cinnarizine. Samples of lipid emulsion574 were prepared for HPLC assay by an initial 80-fold dilution575 with chloroform:methanol (2:1 v/v), followed by a 10-fold576 dilution with mobile phase (50% v/v acetonitrile:50% v/v 20
577mM NH4H2PO4). Samples of iv formulation and micelles/578vesicles were prepared for HPLC assay by a 900-fold and a 400-579fold dilution with mobile phase (50% v/v acetonitrile:50% v/v58020 mM NH4H2PO4), respectively. Plasma samples were581prepared for HPLC using a validated extraction procedure,582with flunarizine as an internal standard, as reported583previously.39
584Cinnarizine HPLC assay conditions were as described585previously,39 with slight modification to the mobile phase586employed for cinnarizine quantification in plasma, to 45% v/v587acetonitrile:55% v/v 20 mM NH4H2PO4 in this study. Replicate588analysis of n = 4 quality control samples revealed acceptable589accuracy and precision (±10%, ±15% at the limit of590quantification) for concentrations between 20 and 1000 ng/591mL for iv formulation, emulsion, micelles and vesicles, and 10592and 320 ng/mL for plasma.593Scintillation Counting. Quantification of 14C-cholesterol594and 3H-oleic acid in the plasma was performed via scintillation595counting on a Packard Tri-Carb 2000CA liquid scintillation596analyzer (Packard, Meriden, CT, USA). Plasma samples (50597μL) were added to 2 mL of Irga-safe Plus scintillation fluid598followed by a 10 s vortex. Samples were corrected for599background radioactivity by the inclusion of a blank plasma600sample in each run. The counting method was validated by601spiking blank plasma with low, medium and high concen-602trations of labeled cholesterol and oleic acid (in triplicate). The603measured concentrations were within 5% of the nominal604concentration.605Blood:Plasma Ratio Determination for Cinnarizine. The606blood:plasma ratio for cinnarizine was determined by spiking6070.5 mL of blank blood with known amounts of cinnarizine to608achieve low, medium and high concentrations (in triplicate).609Plasma was separated by centrifugation (10000g, 5 min) and610plasma drug concentration assayed by HPLC. The blood:-611plasma ratio was calculated from the ratio of known612concentration in spiked blood to the concentration measured613in plasma separated from spiked blood. The mean blood:-614plasma ratio was subsequently used to convert plasma615concentrations to blood concentrations in perfusion experi-616ments, enabling quantification of total drug transport into617mesenteric blood.618Calculations. In the single-pass rat jejunum perfusion model,619permeability coefficients were calculated using steady-state drug620concentrations in perfusate and blood. Two apparent621permeability coefficients (Papp) were calculated as described622previously:35
= − ·PCC
disappearanceQA
lnapp1
0 623(2)
=·⟨ ⟩
ΔΔ( )
PA C
appearance
Mt
app
B
624(3)
625where “disappearance Papp” is the apparent permeability626coefficient calculated from drug loss from the perfusate (cm/627s); “appearance Papp” is the apparent permeability coefficient628calculated from drug appearance in the mesenteric blood (cm/629s); Q is the perfusate flow rate (mL/s); A is the surface area of630the perfused jejunal segment (cm2), which is calculated by631multiplying the diameter by the length of the perfused intestinal632segment as described previously;40 C1 is the average steady-633state drug concentration exiting the perfused jejunal segment634(ng/mL); C0 is the drug concentration entering the jejunal
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXF
635 segment (ng/mL); ΔMB/Δt is the average rate of drug mass636 appearance in mesenteric blood at steady state (ng/s); and ⟨C⟩637 is the logarithmic mean drug concentration in the lumen (ng/638 mL), where ⟨C⟩ = (C1 − C0)/(ln C1 − ln C0).639 Noncompartmental Pharmacokinetic Analysis. The max-640 imum plasma concentration (Cmax), time to reach Cmax (Tmax),641 area under the plasma concentration−time curve from time642 zero to the last measured concentration (AUC0−8h), area under643 the plasma concentration−time curve extrapolated to infinity644 (AUC0−inf), elimination rate constant (Ke), volume of645 distribution (Vd) and clearance (Cl) were calculated using646 WINONLIN version 5.3 (Pharsight Inc., Apex, NC, USA).647 Statistical Analysis. Results were analyzed using Student’s648 t test. A P value of <0.05 was considered to be a significant649 difference. Analyses were performed using SPSS v19 for650 Windows (SPSS Inc., Chicago, IL, USA).
651 ■ RESULTS
652 SR-BI, CD36, NPC1L1 and Endocytosis Have Little653 Impact on Drug Absorption from Intestinal Colloidal654 Phases. Inhibition of the lipid uptake receptors SR-BI (by655 coinfusion of 100 μM BLT-1), CD36 (by coinfusion of 1 mM656 SSO) and NPC1L1 (by intravenous administration of 0.3 mg/657 kg ezetimibe) did not result in significant changes to the658 systemic plasma concentration−time profiles or systemic
f2t2 659 exposure of cinnarizine (Figure 2A, Table 2). Inhibition of660 endocytosis (by coinfusion of 100 μM monensin) unexpectedly
661led to significant increases in cinnarizine systemic plasma662concentration at t = 2.5 and 4 h (Figure 2A), and increased the663AUC0−8h 1.5-fold (Table 2). However, subsequent intravenous664dosing studies revealed significantly lower Vd and Cl values in665the monensin-treated rats when compared to control (Table S1666in the Supporting Information), suggesting that the increase in667cinnarizine systemic exposure was due to a decrease in668cinnarizine systemic distribution and clearance, rather than669changes to intestinal absorption. The data suggest a limited role670for SR-BI, CD36, NPC1L1 and endocytosis generally, in the671absorption of cinnarizine from micelles and vesicles.672In contrast, inhibition of NPC1L1 did lead to significantly673lower systemic plasma concentrations of exogenously dosed674cholesterol at t = 2, 2.5, 3, 4, 6, 8 h (Figure 2B). Plasma675concentrations of exogenously dosed oleic acid also appeared676lower in rats following inhibition of CD36 although differences677were not significantly different (Figure 2C). While inhibition of678SR-BI and CD36 did not lead to significant changes to the679systemic plasma concentration−time profile of exogenously680dosed cholesterol, inhibition of endocytosis significantly681increased the systemic plasma concentrations of exogenously682dosed cholesterol at t = 4, 8 h (Figure 2B). This increase in683exposure is likely explained by a decrease in systemic684distribution of 14C-cholesterol in the monensin-treated rats,685as plasma 14C-cholesterol concentrations at early sample time686points (which reflect the distribution phase) in intravenous687studies were significantly higher in the monensin-treated rats688(Figure S1B in the Supporting Information) when compared to689controls (Vd and Cl could not be calculated for cholesterol as a690typical elimination phase was not evident in the systemic691plasma concentration vs time profiles of cholesterol: Figure 2B692and Figure S1B in the Supporting Information).693Drug Absorption from Micelles and Vesicles Is694Determined by Cfree and Not Colloidal Structure. The695intestinal perfusion of colloidal media with markedly different696compositional profiles (micelles and vesicles) but with697comparable Cfree and thermodynamic activity did not result in698significant differences in steady-state absorptive flux, disappear-699 t3f3ance Papp or appearance Papp of cinnarizine (Table 3, Figure 3A:700filled symbols). Similarly, in spite of large differences in particle701size and composition, the intraduodenal infusion of the same702micellar or vesicular systems to bile-diverted rats did not result703in significant differences in systemic plasma concentration−704 f4time profiles (Figure 4: filled symbols) and pharmacokinetic705 t4t5parameters of cinnarizine (Table 5: micelles vs vesicles in bile-
Figure 2. Systemic plasma concentration−time profiles of (A)cinnarizine (CIN), (B) 14C-labeled cholesterol (Ch) and (C) 3H-labeled oleic acid (OA) following intraduodenal infusion of a 3 mLlipid emulsion consisting of 1 mg of cinnarizine emulsified in 59 mMoleic acid, 8 mM sodium taurocholate, 2 mM phosphatidylcholine, 2mM cholesterol, 1 μCi of 14C-cholesterol and/or 3 μCi of 3H-oleicacid. Experiments were performed in control rats (filled circle); ratstreated with 100 μM BLT-1 (open circle); rats treated with 1 mM SSO(open triangle); rats treated with 0.3 mg/kg ezetimibe (open square);and rats treated with 100 μM monensin (open diamond), to inhibitthe lipid uptake receptors SR-BI, CD36, NPC1L1 and endocytosis,respectively. BLT-1, SSO, and monensin were coinfused as part of thelipid emulsion; ezetimibe was administered intravenously. Datarepresent mean ± SEM of n = 4 rats for A and B; and n = 3 ratsfor C. Statistically significant difference with respect to control rats (p< 0.05) is denoted by the symbol *.
Table 2. Pharmacokinetic Parameters for Cinnarizine afterIntraduodenal Administration of a 3 mL Lipid Emulsion inRatsa
expl group AUC0−8h (ng h/mL) Cmax (ng/mL) Tmax (h)
control 978 ± 122 288.1 ± 26.6 2.4 ± 0.1BLT-1-treated 883 ± 21 219.0 ± 20.0 2.6 ± 0.5SSO-treated 924 ± 88 195.5 ± 33.6 2.4 ± 0.2ezetimibe-treated 1048 ± 92 333.9 ± 13.6 2.3 ± 0.1monensin-treated 1487 ± 103b 432.8 ± 45.9b 2.3 ± 0.1
aIn each case, the formulation type was emulsion and the CIN dosewas 3.33 mg/kg. Experiments were performed in control rats; ratstreated with 100 μM BLT-1; rats treated with 1 mM SSO; rats treatedwith 0.3 mg/kg ezetimibe; and rats treated with 100 μM monensin, toinhibit the lipid uptake receptors SR-BI, CD36, NPC1L1, andendocytosis, respectively. Values represent mean ± SEM of n = 4rats. bSignificant difference when compared to control group.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXG
706 diverted rats). The results suggest that cinnarizine absorption707 from lipid colloidal phases is relatively insensitive to the708 physical nature of the colloidal milieu, and is instead controlled709 largely by Cfree.710 Bile-Mediated Dilution of Cinnarizine-Loaded Mi-711 celles and Vesicles Generates Drug Supersaturation.712 The equilibrium solubility and change in cinnarizine solubiliza-713 tion capacity of model micelles and vesicles before and after 1:1714 v/v dilution with bile, bile pH 6.30 or buffer pH 6.30 are
f5 715 tabulated in Table 4, and shown graphically in Figure 5.716 Dilution of the micellar or vesicular systems in a 1:1 v/v ratio717 with bile obtained from donor animals led to significant718 decreases in cinnarizine solubilization capacity. As a proportion719 of initial, the solubilization capacity of the micellar system720 dropped significantly from 100% to 14%, and for vesicles the721 decrease was even greater from 100% to 7%. This was in spite722 of the fact that the bile salt concentration in donor bile was723 higher than that in the micellar or vesicular system (average724 total bile salt concentration in donor bile was 14.7 ± 0.9 mM;725 mean ± SEM, n = 13), and therefore “dilution” with bile did726 not reduce bile salt concentrations below the critical micellar727 concentration (CMC) and instead increased the overall bile salt728 concentration.729 The pH of donor bile was higher than intestinal pH (average730 pH of donor bile was 8.02 ± 0.02; mean ± SEM, n = 5). As731 such additional studies were performed to examine whether the732 effects on solubility reflected a pH effect. 1:1 v/v dilution of the733 phases with pH adjusted bile (pH of bile adjusted to 6.30 to734 match the pH of the micelles and vesicles) also resulted in735 significant decreases in solubilization capacity (to 28% and 15%736 of initial for micelles and vesicles, respectively), although the737 decrease was slightly attenuated when compared to non-pH738 adjusted bile. Finally, the micellar and vesicular systems were
739diluted 1:1 v/v with buffer at pH 6.30 in an attempt to740uncouple simple dilution effects from pH effects and bile-741mediated effects. Dilution with pH 6.30 buffer also decreased742cinnarizine solubilization capacity significantly, although to a743lesser extent (41% and 21% in micelles and vesicles,744respectively).745 f6Analysis of the kinetics of drug precipitation (Figure 6)746demonstrated that bile addition to cinnarizine-loaded micelles747and vesicles (cinnarizine present in phases at 0.2 mg/mL,748∼80% saturated solubility) did not result in immediate drug749precipitation, and was preceded by a period of supersaturation.750The time taken for cinnarizine precipitation to occur was
Table 3. Cinnarizine Disappearance Papp (×106 cm/s) fromthe Intestinal Perfusate, Appearance Papp (×106 cm/s) in theMesenteric Blood and Steady-State Absorptive Flux intoMesenteric Blood (ng/5 min/10 cm2) after 60 min of Single-Pass Perfusion of ∼10 cm2 Segments of Rat Jejunum withModel Micelles and Vesicles, with and without 1:1 v/vCoperfusion with Rat Bile, Rat Bile pH 6.30 or Buffer pH6.30a
SSratiob
disappearancePapp (×10
6
cm/s)
appearancePapp (×10
6
cm/s)
flux into mesentericblood (ng/5 min/10
cm2)
micelles 0.4 19.4 ± 3.0 1.1 ± 0.2 310 ± 50micelles +bileSSc
6.1 29.3 ± 3.0d 3.7 ± 0.1d 979 ± 30d
micelles +bile pH6.30SS
3.0 39.5 ± 5.6d 4.2 ± 1.0d 1099 ± 279d
micelles +buffer pH6.30SS
2.1 38.9 ± 4.1d 4.4 ± 0.4d 1180 ± 100d
vesicles 0.4 22.0 ± 2.2 1.2 ± 0.1 340 ± 29vesicles +bileSS
11.6 66.0 ± 18.3e 2.0 ± 0.7 499 ± 155
aValues calculated using data obtained after steady-state attainment (t= 40−60 min). In all experiments, cinnarizine concentration inperfusate and total perfusate flow rate were kept constant at 0.1 mg/mL and 0.1 mL/min, respectively. Data represent mean ± SEM of n =3−4 experiments. bSS ratio = supersaturation ratio = (supersaturated)concentration of drug in perfusate/equilibrium solubility of drug inperfusate. cSS denotes drug supersaturation in perfusate. dSignificantincrease from micelles alone. eSignificant increase from vesicles alone.
Figure 3. (A) Absorptive flux of cinnarizine (CIN) into mesentericblood (ng/5 min/10 cm2) and (B, C) CIN disappearance fromintestinal perfusate (% drug dose passing through jejunum) whenmicelles/vesicles were perfused through an isolated rat jejunal segment(∼10 cm2), with (open symbols) and without (filled symbols) 1:1 v/vcoperfusion with rat bile. Coperfusion of micelles and vesicles withdonor bile generates drug supersaturation in situ within the perfusedjejunal segment. SS denotes experiments where drug is supersaturatedin the perfusate. The degree of drug precipitation within the perfusateis illustrated in B and C as the difference in perfusate concentrationbetween pre- and postcentrifugation data. Precipitation was significantin the vesicle, but not micellar groups. Experiments were performedusing an in situ single-pass rat jejunum perfusion model. In allexperiments, the concentration of cinnarizine in perfusate and the totalperfusate flow rate were kept constant at 0.1 mg/mL and 0.1 mL/min,respectively. Data represent mean ± SEM of n = 3−4 experiments.
Figure 4. Systemic plasma concentration−time profiles of cinnarizine(CIN) following a 2 h intraduodenal infusion of cinnarizine-loaded(0.2 mg/mL) micelles (left panel) and vesicles (right panel) to bile-intact and bile-diverted rats. Consistent with observations in ratjejunum perfusion studies, bile-induced supersaturation translated intoincreased in vivo exposure during the absorption phase in the case ofmicelles but not vesicles. Data represent mean ± SEM of n = 4 rats.Statistical significance (p < 0.05) is denoted by the symbol *.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXH
751 variable, however supersaturation was maintained for longer752 periods in micelles (>20 min in all cases) when compared to753 vesicles (1−20 min).754 Bile-Induced Drug Supersaturation Increases Jejunal755 Absorptive Flux for Micelles but Not Vesicles. The756 absorptive flux vs time profiles for cinnarizine under super-757 saturated conditions (i.e., when bile was coperfused with the758 phases) and under nonsupersaturated conditions (when bile759 was not coperfused with the phases) are shown in Figure 3A.760 Steady-state absorptive flux, disappearance Papp and appearance761 Papp of cinnarizine in all perfusion experiments are reported in762 Table 3.763 Coperfusion of micelles with bile in a 1:1 v/v ratio increased764 the absorptive flux, disappearance Papp and appearance Papp of765 cinnarizine from micelles 3.2-fold, 1.5-fold and 3.4-fold,766 respectively. In contrast, 1:1 v/v coperfusion of vesicles with767 bile did not lead to significant changes in cinnarizine absorptive768 flux or appearance Papp. Disappearance Papp for cinnarizine did769 increase 3.0-fold when vesicles were coperfused with bile,770 however the drop in perfusate drug concentration was largely a771 result of rapid drug precipitation in the perfusate (see below).772 For the micellar preparation, bile-induced supersaturation773 was relatively stable throughout the experimental period. Thus774 the cinnarizine concentration in the perfusate was essentially775 the same before or after centrifugation (Figure 3B). In contrast,776 when vesicles were coperfused with bile, significant drug777 precipitation was observed during the time required for
778perfusate to transit the jejunal segment (as indicated by the779difference between the pre- and postcentrifugation data in780Figure 3C). This was consistent with the in vitro dilution781profiles in Figure 6.782To distinguish between bile-induced increases in cinnarizine783absorptive flux resulting from drug supersaturation, pH784increases (since an increase in pH might be expected to785increase the permeability of a weak base) and nonspecific786effects of bile components on membrane permeability, micelles787were also coperfused with pH-adjusted bile (pH 6.30) and788buffer (pH 6.30). 1:1 v/v coperfusion of micelles with bile pH7896.30 or buffer pH 6.30 increased cinnarizine absorptive flux 3.5-790fold and 3.8-fold, respectively, when compared to the perfusion791 f7of micelles alone (Figure 7, Table 3). Stable supersaturation
Table 4. Equilibrium Solubility (37 °C) and Percent OriginalSolubilization Capacity Values of Cinnarizine in ModelMicelles and Vesicles, before and after 1:1 v/v Addition ofRat Bile, Rat Bile pH 6.30 or Buffer pH 6.30a
equilibrium solubility(μg/mL)
percent original solubilizationcapacity (%)b
Micellesmicelles alone 236 ± 9.3 100
+ bile (1:1) 16.5 ± 0.8 14.0 ± 0.7+ bile pH6.30 (1:1)
33.1 ± 1.0 28.0 ± 0.9
+ buffer pH6.30 (1:1)
48.7 ± 0.8 41.2 ± 0.6
Vesiclesvesicles alone 232 ± 3.3 100
+ bile (1:1) 8.60 ± 0.5 7.4 ± 0.4+ bile pH6.30 (1:1)
17.4 ± 0.4 15.0 ± 0.3
+ buffer pH6.30 (1:1)
24.9 ± 0.3 21.4 ± 0.2
aData represent mean ± SEM of n = 3−4 determinations. bPercentoriginal solubilization capacity = (solubilityfinal × volumefinal/solubilityinitial × volumeinitial) × 100%.
Table 5. Pharmacokinetic Parameters for Cinnarizine after Intraduodenal Administration of Cinnarizine-Loaded (0.2 mg/mL)Micelles and Vesicles to Bile-Intact and Bile-Diverted Ratsa
exptl group formulation type CIN dose (mg/kg) AUC0−8h (ng h/mL) Cmax (ng/mL) Tmax (h)
micellesbile-intact micelles 2 502 ± 59b 138.8 ± 11.6b 1.6 ± 0.2bile-diverted micelles 2 362 ± 39 85.7 ± 14.3 2.0 ± 0.4
vesiclesbile-intact vesicles 2 411 ± 74 109.4 ± 10.6 2.3 ± 0.3bile-diverted vesicles 2 393 ± 82 97.6 ± 12.8 2.0 ± 0.4
aValues represent mean ± SEM of n = 4 rats. bSignificant increase when compared to micelles (bile-diverted) group.
Figure 5. Percent original cinnarizine (CIN) solubilization capacity ofmodel colloids (micelles or vesicles), before and after a 1:1 v/vaddition of rat bile, rat bile pH 6.30 or buffer pH 6.30. Data representmean ± SEM of n = 3−4 determinations. Statistical significantdifference (p < 0.05) to colloid only is denoted by the symbol *.
Figure 6. Kinetics of cinnarizine (CIN) precipitation from modelmicelles (filled circles, n = 5) and model vesicles (open circles, n = 4)upon addition of rat bile (in a 1:1 v/v ratio) at t = 0. Addition of bilereduces the equilibrium cinnarizine solubilization capacity of micellesand vesicles to 14% and 7% of initial, respectively (see Table 4, andshown here as the lines denoted equilibrium solubility). Cinnarizinesupersaturation appeared to be maintained for longer in micelles thanin vesicles. Cinnarizine was loaded into micelles and vesicles at 80%saturation (∼0.2 mg/mL). Each line represents individual experiments.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXI
792 was also generated within the perfused jejunal segment in these793 experiments (Figure S2 in the Supporting Information:794 cinnarizine concentration in the perfusate was essentially the795 same before and after centrifugation).796 Since 1:1 v/v coperfusion of micelles with bile, bile pH 6.30797 and buffer pH 6.30 are all expected to generate cinnarizine798 supersaturation within the perfused jejunal segment (according799 to the solubility data in Table 4, and the lack of drug800 precipitation in outflow perfusate in all cases), the observation801 that absorptive flux enhancement was similar in all groups802 (Figure 7) suggests that the enhancement was attributable to803 drug supersaturation, and not an increase in system pH (by804 comparing micelles + bile group with micelles + bile pH 6.30805 group), or nonspecific effects of bile on membrane permeability806 (by comparing micelles + bile pH 6.30 group with micelles +807 buffer pH 6.30 group). However, the degree of flux808 enhancement did not correlate with the degree of super-809 saturation, as flux enhancement was similar in all groups despite810 the significantly higher supersaturation ratio generated in the811 micelles + bile group, when compared to the micelles + bile pH812 6.30 group and micelles + buffer pH 6.30 group (super-813 saturation ratio of 6 vs 2−3) (Table 3). Previous studies have814 shown that, for lipophilic drugs such as cinnarizine, the drug815 fraction extracted into octanol (and analogous to the permeable816 fraction) as a function of pH is shifted to lower pHs than would817 be expected based on the un-ionized fraction.41,42 This left shift818 would limit pH effects on permeability over the range of pH 6−819 8, consistent with the observations here.820 Bile-Induced Drug Supersaturation Increases in Vivo821 Cinnarizine Exposure after Intraduodenal Infusion. The822 systemic plasma concentration−time profiles of cinnarizine823 following intraduodenal infusion of cinnarizine-loaded micelles824 and vesicles (0.2 mg/mL; ∼80% saturated solubility) to bile-825 intact and bile-diverted rats are shown in Figure 4. The826 pharmacokinetic parameters of cinnarizine for the bioavail-827 ability studies are reported in Table 5. Consistent with the828 results from the rat jejunal permeability studies, intraduodenal829 administration of micelles to bile-intact rats resulted in
830significantly higher systemic plasma concentrations of cinnar-831izine at t = 1, 1.5 and 2 h (AUC0−8h increased 1.4-fold) when832compared to bile-diverted rats. In contrast, when vesicles were833dosed to bile-intact rats, the systemic plasma concentration−834time profiles and AUC0−8h of cinnarizine were not different835from that observed in bile-diverted rats.
836■ DISCUSSION837After oral administration, the absorption of poorly water-838soluble drugs (PWSD) is often limited by slow dissolution and839low solubility in the GI tract. LBF overcome many of the840dissolution limitations of PWSD (by providing a mechanism to841circumvent dissolution from the solid to liquid state), however,842the solubility limitations of PWSD are seemingly unaddressed,843since solubilization in the lipid-based colloidal phases that result844from the digestion of LBF does not typically enhance free drug845concentrations.2,43 Nonetheless, coadministration of lipids846(either formulation lipids or via coadministration with lipid-847rich foods) remains a highly effective means to promote the848absorption of PWSD. This suggests the potential for alternative849mechanisms by which LBF enhance drug absorption. In the850current communication, two possible alternatives to the851traditional model of drug absorption from LBF have been852explored: first, that drug absorption from lipid colloidal phases853may involve a collisional uptake component (i.e., drug854absorption directly from the solubilized phase); and second,855that flux across the absorptive membrane may be enhanced by a856transient increase in the thermodynamic activity of drug in857intestinal colloidal phases due to supersaturation.858The data describing drug absorption from micellar and859vesicular colloidal phases suggest that direct interactions860between colloids (or at least the systems examined here) and861the absorptive membrane do not play an important role in862cinnarizine absorption. Thus, comparable absorptive flux863(Figure 3A: filled symbols) and systemic plasma concen-864tration−time profiles (Figure 4: bile-diverted rats) were865observed following jejunal perfusion and intraduodenal infusion866of micelles and vesicles. The thermodynamic activity and Cfree867of both systems were held constant, but the large difference in868hydrodynamic radius (9 nm of micelles vs 443 nm of vesicles)869and higher bile salt, LPC and lipid concentrations in the870micellar system (Table 1) suggest that the number of871administered micellar particles was substantially higher than872that of vesicular particles. Collision-mediated absorption is873highly sensitive to increased particle number, since this874increases the statistical likelihood of collisions and collisional875transfer.7 As such collisional interactions did not appear to876dictate the degree of drug absorption from micelles and vesicles877and, instead, absorption was seemingly controlled by878thermodynamic activity (or Cfree). Differences in particle size879might also be expected to alter colloid diffusion across the880unstirred water layer (UWL). The similarity in absorption881profiles from micelles and vesicles (Figure 3A: filled symbols)882therefore suggests that diffusion across the UWL either was883nonlimiting or was not affected by particle size in this884experimental model. Similarly, the rates of replenishment of885Cfree (i.e., the rate of re-establishment of the equilibrium886between solubilized and free drug) might be expected to be887different between the two different particle size colloids,888however this was presumably sufficiently fast in both cases to889have little impact on drug absorption in the current model.890Data obtained from the lipid uptake receptor inhibition891studies further support the notion that collisional uptake
Figure 7. Absorptive flux−time profiles of cinnarizine (CIN) whenmicelles were perfused through an isolated rat jejunal segment (∼10cm2), with and without 1:1 v/v coperfusion with rat bile, rat bile pH6.30 or buffer pH 6.30. SS denotes experiments where drug issupersaturated in perfusate. Coperfusion of rat bile, rat bile pH 6.30 orbuffer pH 6.30 with micelles generates drug supersaturation in situwithin the perfused jejunal segment in all cases, and increasedcinnarizine absorptive flux by 3.2-fold, 3.5-fold and 3.8-fold,respectively. Experiments were performed using an in situ single-passrat jejunum perfusion model. In all experiments, the concentration ofcinnarizine in perfusate and the total perfusate flow rate were keptconstant at 0.1 mg/mL and 0.1 mL/min, respectively. This series ofexperiments provides further support for the suggestion thatsupersaturation was responsible for the increase in absorptive fluxseen in Figure 3A. Data represent mean ± SEM of n = 3−4experiments.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXJ
892 mechanisms have a limited role in drug absorption from these893 systems. SR-BI, CD36 and NPC1L1 were examined since they894 have previously been shown to mediate the cellular uptake of895 fatty acids and/or cholesterol,12,15,44 the absorption of which is896 also facilitated by micellar solubilization. Whether SR-BI, CD36897 and NPC1L1 function as authentic transporters that directly898 mediate lipid absorption,12−15,44 or whether they act to899 facilitate intracellular lipid trafficking or to modify signaling900 processes that mediate lipid absorption,45−47 or both, remains901 contentious, but nonetheless all merit examination here. Recent902 reports also suggest the involvement of SR-BI, CD36 and903 NPC1L1 in the absorption of fat-soluble nutrients (such as904 carotenoids,15,48 vitamin D49 and vitamin E50), providing905 further support for a role in drug absorption. In the current906 study, consistent with previous reports in vivo, inhibition of907 NPC1L1 reduced cholesterol absorption (Figure 2B),14 and908 inhibition of CD36 reduced (albeit nonsignificantly) the909 absorption of oleic acid (Figure 2C).12 In contrast, inhibition910 of SR-BI and CD36 had little impact on cholesterol absorption911 (Figure 2B). Inhibition of SR-BI and CD36 was expected to912 reduce cholesterol absorption based on previous in vitro913 studies.12,13,15 However, in vivo evidence of a role of SR-BI914 and CD36 in cholesterol absorption is less clear and, for915 example, no significant differences in intestinal cholesterol916 absorption were reported in SR-BI knockout vs wild-type917 mice.15,51 The lack of effect of SR-BI and CD36 inhibition on918 cholesterol absorption in the current study therefore runs919 contrary to previous in vitro studies but is in agreement with920 some previous in vivo data. Inhibition of endocytosis pathways921 via administration of monensin also failed to reduce cholesterol922 and cinnarizine absorption. Rather, the volume of distribution923 and clearance of cholesterol and cinnarizine appeared to be924 reduced, resulting in increases in plasma exposure. Since925 exogenously dosed 14C-cholesterol and cinnarizine are likely to926 be present within lipoproteins in the systemic circulation, the927 changes in systemic disposition of 14C-cholesterol and928 cinnarizine in the monensin-treated rats may reflect inhibition929 of receptor-mediated endocytosis of LDL.52
930 Inhibition of SR-BI, CD36 and NPC1L1 did not result in931 significant changes to the systemic exposure of cinnarizine932 following intraduodenal infusion of a lipid emulsion for-933 mulation in rats (Figure 2A). The data suggest that while lipid934 colloidal phases may be capable of direct interaction with lipid935 receptors (as suggested previously), the transfer of solubilized936 content into absorptive cells is likely selective and more937 applicable to solubilized lipids and nutrients rather than drugs.938 Together with the data describing cinnarizine absorption from939 micelles and vesicles, the results indicate that cinnarizine940 absorption from lipid colloidal phases is largely independent of941 the physical nature of the infused colloid (realizing that in these942 first experiments cinnarizine was present at concentration well943 below the solubilization limit and therefore under conditions944 where solubility/precipitation-mediated events were avoided),945 is not influenced significantly by common lipid uptake946 receptors and, instead, appears to be primarily dependent on947 the free drug concentration in equilibrium with the solubilized948 reservoir.949 Subsequent studies addressed the possibility that bile950 secretion may enhance drug absorption via the induction of951 supersaturation during the intestinal processing of dietary or952 formulation-related lipids. The addition of bile to cinnarizine-953 loaded micelles led to sustained drug supersaturation that954 ultimately led to increased intestinal drug absorption and
955systemic drug exposure. In contrast, although addition of bile to956cinnarizine-loaded vesicles also led to supersaturation, the957metastable supersaturated state was less stable than that958generated by bile addition to micelles, resulting in more rapid959precipitation of solubilized drug and therefore a lack of increase960in drug absorption and systemic drug exposure. The data961indicate that bile-mediated dilution of lipid colloidal phases may962represent an endogenous mechanism of supersaturation963generation during lipid processing in the small intestine; and964that the transient increase in thermodynamic activity may lead965to enhanced drug absorption. The observations also highlight966both the potential for supersaturation to enhance drug967absorption and the need to achieve an optimal balance between968drug supersaturation and drug precipitation.969Interaction of intestinal colloidal phases with bile leads to970dilution, an increase in pH and an increase in the971concentrations of bile components (bile salts, phosphatidylcho-972line and cholesterol) associated with the colloidal species. For a973solubilized system above the critical micellar concentration974(CMC), simple 1:1 v/v dilution is expected to reduce the975solubilized concentration, but to maintain total solubilization976capacity (i.e., for the drug concentration to drop by 50% but977the volume to double and therefore for solubilization capacity978to remain unchanged). However, the data in Table 4 show that979dilution of the micelles or vesicles with bile or buffer results in a980drop in total solubilization capacity to only 7−41% of initial.981Greater proportional decreases were apparent for dilution of982vesicles (in all cases when compared to micelles), and after983dilution with bile rather than buffer (Table 4). The loss of984solubilization capacity on dilution suggests the likelihood of a985phase transition to structures with reduced solubilization986capacity. Although a complete explanation for these phase987transitions is not apparent at this time, for the micellar systems,988it may be related to the ability of ionized caprylic acid to self-989associate and form fatty acid micelles at high lipid990concentration.53 Thus, dilution of medium-chain colloidal991phases may reduce the concentration of caprylic acid below992the CMC, leading to a loss of cinnarizine solubilization capacity993in the micellar phase. For vesicles, previous studies have994suggested that increasing bile concentrations facilitate a995vesicular to micellar transition.54,55 Since micelles are expected996to have lower solubilization capacities for lipophilic drugs than997vesicles (micelles are smaller and less lipid-rich),22,56 a998reduction in cinnarizine solubilization capacity might therefore999be anticipated when bile is added to model vesicles. In the case1000of the micelles, therefore, it seems likely that the addition of1001bile disrupted the structure of swollen, mixed micelles leading1002to lower colloidal lipid content and lower solubilization1003capacity. Unfortunately, attempts to quantify changes to1004particle size on bile addition were unsuccessful due to high1005polydispersity. However, the broad trends observed were1006consistent with the suggestions above and decreases in particle1007size were apparent for vesicles (consistent with initiation of a1008vesicle to micelle transition) and increases in particle size for1009the mixed micelles (consistent with micellar destabilization and1010transformation to less dispersed structures) (data not shown).1011An additional complexity in these dilution studies was the1012realization that the pH of bile (pH 8.02) is higher than that1013normally employed for simulated intestinal fluids,27,57 and1014therefore incubation of the colloidal phases (prepared at pH10156.30) with bile increases system pH. Cinnarizine is a weak base1016with a pKa of 7.47
58 and is therefore expected to be less ionized,1017less soluble and potentially more permeable at higher pHs. The
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXK
1018 impact of pH in the current studies was therefore examined by1019 dilution of micelles and vesicles with pH-corrected bile at pH1020 6.30. Comparison to the data obtained with bile at pH 8.021021 suggests that the higher pH of bile provides an additional driver1022 for drug precipitation/supersaturation since the solubility drop1023 was greater after incubation with bile at pH 8.02 vs bile at pH1024 6.301025 While dilution of micelles and vesicles with bile resulted in1026 decreases in drug solubilization capacity, cinnarizine did not1027 precipitate immediately. A period of drug supersaturation was1028 evident for both micellar and vesicular systems, although drug1029 precipitation from vesicles was much more rapid than from1030 micelles. This in turn translated into increases in drug1031 absorption (Figure 3A) and systemic exposure (Figure 4A)1032 for the micellar systems. The difference in the capacity of1033 micelles and vesicles to maintain drug supersaturation (Figure1034 6) may be explained by the difference in the degree of drug1035 supersaturation induced by dilution with bile. The degree of1036 supersaturation is described by the supersaturation ratio, which1037 is the ratio of the (supersaturated) concentration of drug in1038 solution relative to the equilibrium solubility of the drug in the1039 same system.16 Crystallization theory suggests that the1040 thermodynamic drivers of precipitation from supersaturated1041 solutions increase with increasing supersaturation ratios, as the1042 likelihood of nucleation and crystal growth increases with1043 increases in (metastable) drug concentration in solution.16,59
1044 Here, 1:1 v/v addition of bile resulted in the attainment of1045 cinnarizine supersaturation ratios of 6 and 12, in micelles and1046 vesicles, respectively. Therefore, the faster rate of drug1047 precipitation from vesicles (when compared to micelles) may1048 be explained by the higher supersaturation ratio induced by bile1049 addition. It is also possible that micellar structures are more1050 effective in stabilizing supersaturation when compared to1051 vesicular structures, although this has not been examined1052 explicitly here. Notably, cinnarizine was found to precipitate in1053 the crystalline form in these experiments (Figure S3 in the1054 Supporting Information), precluding the possibility that the1055 enhanced cinnarizine absorption from micelles (when bile was
1056coperfused) observed in Figure 3A was due to accelerated1057cinnarizine dissolution from precipitated amorphous forms.1058The role of bile in enhancing drug absorption from LBF has1059been reported previously.60−62 In the majority of cases, bile-1060mediated bioavailability enhancement has been suggested to1061stem from the ability of bile to expand the solubilization1062reservoir for PWSD in the GI tract. This is typically assumed to1063occur via PWSD solubilization in simple bile micelles, or via the1064ability of bile to solubilize lipid digestion products and to1065generate more complex lipid colloidal phases with enhanced1066solubilization capacities. It seems likely that the ability to1067solubilize lipid digestion products and to promote colloid1068formation remains an integral part of the role of bile in1069supporting drug (and lipid) absorption. However, the data1070described here suggest that continued dilution of lipid colloidal1071phases with bile in the small intestinal lumen may also lead to1072physical changes that promote drug supersaturation, and1073ultimately promote drug absorption. In doing so, super-1074saturation induction may be a means by which the decrease1075in thermodynamic activity inherent in solubilization is reversed,1076such that the free concentration of drug available for absorption1077is maximized. Thus, a dual role of bile in facilitating drug1078 f8absorption from LBF may be conceived (see Figure 8). First,1079bile-mediated solubilization of lipid digestion products at the1080interface of a digesting lipid droplet results in the generation of1081lipid colloidal phases such as vesicles and micelles that promote1082drug solubilization during lipid digestion. Second, continued1083bile-mediated dilution of existing lipid colloidal phases1084promotes drug supersaturation, and enhances drug absorption1085by significantly increasing drug thermodynamic activity in1086colloidal phases. The combination of these two highly kinetic1087events likely contributes to the effective drug absorption often1088observed with lipid coadministration, as it affords a means to1089simultaneously increase solubilization capacity and promote1090thermodynamic activity of coadministered PWSD in the small1091intestine.1092Supersaturation induction via interaction with bile also1093provides a means of overcoming the recently described1094solubility−permeability interplay observed in studies where
Figure 8. The dual role of bile during lipid digestion and dispersion. (i) Bile-mediated solubilization of lipid digestion products at the interface of adigesting oil droplet results in the generation of lipid colloidal phases such as vesicles and micelles that maintain drug solubilization in the smallintestine. (ii) The continuing interaction of secreted bile with existing lipid colloidal phases in the lumen results in progressively less lipid-rich phaseswith lowered solubilization capacity. Thus, ongoing bile-mediated dilution of lipid colloidal phases promotes drug supersaturation and enhances drugabsorption by increasing drug thermodynamic activity in colloidal phases. The combination of the two highly kinetic, bile-mediated events affords ameans to simultaneously increase solubilization capacity and promote thermodynamic activity of coadministered drug in the small intestine, and maycontribute to the increase in drug absorption often observed with lipid coadministration. D represents the free concentration of drug available forabsorption. Dss is used to signify the increase in free concentration resulting from bile-mediated supersaturation that drives increases in drugabsorption.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXL
1095 PWSD are coadministered with cosolvents, cyclodextrins or1096 surfactant systems.5,6,63 In these studies the authors describe1097 the reduction in thermodynamic activity common to most1098 solubilization technologies and show that this offsets the1099 potential increases in membrane flux that might be expected by1100 an increase in solubilized drug concentration.5,6 More recent1101 studies by the same authors have shown that this solubility−1102 permeability interplay can be addressed via the use of1103 amorphous solid dispersion formulations that stimulate super-1104 saturation, but do not promote solubilization.64,65 Here we1105 report that essentially similar outcomes are also possible with1106 solubilizing formulations, when the solubilizing formulations1107 contain lipids and when the kinetic changes that occur in the GI1108 lumen in the presence of bile secretion promote super-1109 saturation. The current data therefore suggest that endogenous1110 lipid processing pathways provide an exquisitely sensitive and1111 triggerable supersaturation mechanism that allows drug to1112 remain in a solubilized state during initial lipid digestion and at1113 high lipid:bile concentration ratios, but that ongoing bile1114 secretion subsequently provides a boost to thermodynamic1115 activity and in doing so supports enhanced absorption. This is1116 in contrast to other common solubilization strategies that may1117 not interact with the dynamic GI environment, or for which1118 interaction with bile typically reduces thermodynamic activity1119 by increasing solubilization capacity. Conversely, the ability of1120 LBF to promote drug solubilization until drug supersaturation1121 is triggered in the small intestine may confer an advantage over1122 formulation approaches that only utilize supersaturating1123 strategies, as the risk of drug precipitation in the GI tract1124 may be reduced by solubilization within lipid colloidal phases.
1125 ■ CONCLUSION
1126 Improved understanding of the mechanism of drug absorption1127 from lipid colloidal phases such as micelles and vesicles is1128 required to provide a platform for more rational design of lipid-1129 based formulations. Using medium-chain lipids, we have1130 demonstrated that the absorption of cinnarizine (a lipophilic,1131 poorly water-soluble drug) may be enhanced when drug1132 supersaturation is generated during bile-mediated dilution of1133 lipid colloidal phases. Previous studies suggest that a similar1134 induction of supersaturation may occur as a result of initiation1135 of digestion of some microemulsion-based LBFs.21 These1136 observations indicate that supersaturation and its associated1137 benefits in enhancing drug absorption may occur intrinsically1138 during LBF incorporation into endogenous lipid processing1139 pathways in the small intestine. Future work will be directed1140 toward assessing the impact of bile dilution on supersaturation1141 tendency, and thus absorption, for an extended range of PWSD1142 and in a series of different micellar and vesicular colloidal1143 systems.
1144 ■ ASSOCIATED CONTENT
1145 *S Supporting Information1146 Table S1 showing pharmacokinetic parameters for cinnarizine1147 after intravenous administration in rats. Figure S1 showing1148 systemic plasma concentration−time profiles of cinnarizine and1149
14C-labeled cholesterol following intravenous infusion in rats.1150 Figure S2 showing perfusate disappearance profiles of1151 cinnarizine when micelles were perfused through an isolated1152 rat jejunal segment, with and without 1:1 v/v coperfusion with1153 rat bile pH 6.30 or buffer pH 6.30. Figure S3 showing polarized1154 light microscopy images of the crystalline cinnarizine
1155precipitate following precipitation kinetics experiments. This1156material is available free of charge via the Internet at http://1157pubs.acs.org.
1158■ AUTHOR INFORMATION1159Corresponding Author1160*N.L.T.: phone, +61 3 9903 9138; fax, +61 3 9903 9583; e-1161mail, [email protected]. C.J.H.P.: phone, +61 311629903 9649; fax, +61 3 9903 9583; e-mail, [email protected] authors declare no competing financial interest.
1166■ ACKNOWLEDGMENTS1167Funding support from the National Health and Medical1168Research Council (NHMRC) is gratefully acknowledged.
1169■ ABBREVIATIONS USED1170LBF, lipid-based formulations; PWSD, poorly water-soluble1171drugs; GI, gastrointestinal; LPC, L-α-lysophosphatidylcholine;1172CD36, cluster of differentiation 36; SR-BI, scavenger receptor1173class B type 1; NPC1L1, Niemann-Pick C1 like 1; BLT-1, block1174lipid transport-1; SSO, sulfo-N-succinimidyl oleate; iv, intra-1175venous; HPLC, high performance liquid chromatography;1176NMR, nuclear magnetic resonance; DMSO, dimethyl sulfoxide;1177TBME, tert-butyl methyl ether; SEIF, simulated endogenous1178intestinal fluid; SEM, standard error of the mean; CMC, critical1179micellar concentration; UWL, unstirred water layer; HDL,1180high-density lipoprotein; LDL, low-density lipoprotein; CIN,1181cinnarizine; Ch, cholesterol; OA, oleic acid; SS ratio, super-1182saturation ratio
1183■ REFERENCES(1) 1184Williams, H. D.; Trevaskis, N. L.; Charman, S. A.; Shanker, R. M.;
1185Charman, W. N.; Pouton, C. W.; Porter, C. J. H. Strategies to address1186low drug solubility in discovery and development. Pharmacol. Rev.11872013, 65 (1), 315−499.
(2) 1188Poelma, F. G. J.; Breas, R.; Tukker, J. J.; Crommelin, D. J. A.1189Intestinal absorption of drugs. The influence of mixed micelles on the1190disappearance kinetics of drugs from the small intestine of the rat. J.1191Pharm. Pharmacol. 1991, 43 (5), 317−324.
(3) 1192Katneni, K.; Charman, S. A.; Porter, C. J. H. Permeability1193assessment of poorly water-soluble compounds under solubilizing1194conditions: The reciprocal permeability approach. J. Pharm. Sci. 2006,119595 (10), 2170−2185.
(4) 1196Amidon, G. E.; Higuchi, W. I.; Ho, N. F. H. Theoretical and1197experimental studies of transport of micelle-solubilized solutes. J.1198Pharm. Sci. 1982, 71 (1), 77−84.
(5) 1199Miller, J. M.; Beig, A.; Krieg, B. J.; Carr, R. A.; Borchardt, T. B.;1200Amidon, G. E.; Amidon, G. L.; Dahan, A. The solubility−permeability1201interplay: Mechanistic modeling and predictive application of the1202impact of micellar solubilization on intestinal permeation. Mol.1203Pharmaceutics 2011, 8 (5), 1848−1856.
(6) 1204Dahan, A.; Miller, J. M.; Hoffman, A.; Amidon, G. E.; Amidon, G.1205L. The solubility−permeability interplay in using cyclodextrins as1206pharmaceutical solubilizers: Mechanistic modeling and application to1207progesterone. J. Pharm. Sci. 2010, 99 (6), 2739−2749.
(7) 1208Wootan, M. G.; Bernlohr, D. A.; Storch, J. Mechanism of1209fluorescent fatty acid transfer from adipocyte fatty acid binding protein1210to membranes. Biochemistry 1993, 32 (33), 8622−8627.
(8) 1211Thumser, A.; Tsai, J.; Storch, J. Collision-mediated transfer of1212long-chain fatty acids by neural tissue fatty acid-binding proteins1213(FABP). J. Mol. Neurosci. 2001, 16 (2), 143−150.
(9) 1214Yano, K.; Masaoka, Y.; Kataoka, M.; Sakuma, S.; Yamashita, S.1215Mechanisms of membrane transport of poorly soluble drugs: Role of
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXM
1216 micelles in oral absorption processes. J. Pharm. Sci. 2010, 99 (3),1217 1336−1345.
(10)1218 Gao, P.; Morozowich, W. Development of supersaturatable self-1219 emulsifying drug delivery system formulations for improving the oral1220 absorption of poorly soluble drugs. Expert Opin. Drug Delivery 2006, 31221 (1), 97−110.
(11)1222 Gu, X.; Trigatti, B.; Xu, S.; Acton, S.; Babitt, J.; Krieger, M. The1223 efficient cellular uptake of high density lipoprotein lipids via Scavenger1224 Receptor Class B Type I requires not only receptor-mediated surface1225 binding but also receptor-specific lipid transfer mediated by its1226 extracellular domain. J. Biol. Chem. 1998, 273 (41), 26338−26348.
(12)1227 Nassir, F.; Wilson, B.; Han, X.; Gross, R. W.; Abumrad, N. A.1228 CD36 is important for fatty acid and cholesterol uptake by the1229 proximal but not distal intestine. J. Biol. Chem. 2007, 282 (27),1230 19493−19501.
(13)1231 Hauser, H.; Dyer, J. H.; Nandy, A.; Vega, M. A.; Werder, M.;1232 Bieliauskaite, E.; Weber, F. E.; Compassi, S.; Gemperli, A.; Boffelli, D.;1233 Wehrli, E.; Schulthess, G.; Phillips, M. C. Identification of a receptor1234 mediating absorption of dietary cholesterol in the intestine.1235 Biochemistry 1998, 37 (51), 17843−17850.
(14)1236 Altmann, S. W.; Davis, H. R.; Zhu, L.; Yao, X.; Hoos, L. M.;1237 Tetzloff, G.; Iyer, S. P. N.; Maguire, M.; Golovko, A.; Zeng, M.; Wang,1238 L.; Murgolo, N.; Graziano, M. P. Niemann-Pick C1 Like 1 protein is1239 critical for intestinal cholesterol absorption. Science 2004, 303 (5661),1240 1201−1204.
(15)1241 Van Bennekum, A.; Werder, M.; Thuahnai, S. T.; Han, C.-H.;1242 Duong, P.; Williams, D. L.; Wettstein, P.; Schulthess, G.; Phillips, M.1243 C.; Hauser, H. Class B Scavenger Receptor-mediated intestinal1244 absorption of dietary β-carotene and cholesterol. Biochemistry 2005,1245 44 (11), 4517−4525.
(16)1246 Brouwers, J.; Brewster, M. E.; Augustijns, P. Supersaturating1247 drug delivery systems: The answer to solubility-limited oral1248 bioavailability? J. Pharm. Sci. 2009, 98 (8), 2549−2572.
(17)1249 Porter, C. J. H.; Kaukonen, A. M.; Boyd, B. J.; Edwards, G. A.;1250 Charman, W. N. Susceptibility to lipase-mediated digestion reduces1251 the oral bioavailability of danazol after administration as a medium-1252 chain lipid-based microemulsion formulation. Pharm. Res. 2004, 211253 (8), 1405−1412.
(18)1254 Kaukonen, A. M.; Boyd, B.; Porter, C.; Charman, W. Drug1255 solubilization behavior during in vitro digestion of simple triglyceride1256 lipid solution formulations. Pharm. Res. 2004, 21 (2), 245−253.
(19)1257 Anby, M. U.; Williams, H. D.; McIntosh, M.; Benameur, H.;1258 Edwards, G. A.; Pouton, C. W.; Porter, C. J. H. Lipid digestion as a1259 trigger for supersaturation: Evaluation of the impact of supersaturation1260 stabilization on the in vitro and in vivo performance of self-emulsifying1261 drug delivery systems. Mol. Pharmaceutics 2012, 9 (7), 2063−2079.
(20)1262 Cuine, J.; Charman, W.; Pouton, C.; Edwards, G.; Porter, C.1263 Increasing the proportional content of surfactant (Cremophor EL)1264 relative to lipid in self-emulsifying lipid-based formulations of danazol1265 reduces oral bioavailability in beagle dogs. Pharm. Res. 2007, 24 (4),1266 748−757.
(21)1267 Pouton, C. W.; Porter, C. J. H. Formulation of lipid-based1268 delivery systems for oral administration: Materials, methods and1269 strategies. Adv. Drug Delivery Rev. 2008, 60 (6), 625−637.
(22)1270 Kossena, G. A.; Boyd, B. J.; Porter, C. J. H.; Charman, W. N.1271 Separation and characterization of the colloidal phases produced on1272 digestion of common formulation lipids and assessment of their1273 impact on the apparent solubility of selected poorly water-soluble1274 drugs. J. Pharm. Sci. 2003, 92 (3), 634−648.
(23)1275 Harmon, C. M.; Luce, P.; Beth, A. H.; Abumrad, N. A. Labeling1276 of adipocyte membranes by sulfo-N-succinimidyl derivatives of long-1277 chain fatty acids: Inhibition of fatty acid transport. J. Membr. Biol.1278 1991, 121 (3), 261−268.
(24)1279 Nieland, T. J. F.; Penman, M.; Dori, L.; Krieger, M.;1280 Kirchhausen, T. Discovery of chemical inhibitors of the selective1281 transfer of lipids mediated by the HDL receptor SR-BI. Proc. Natl.1282 Acad. Sci. U.S.A. 2002, 99 (24), 15422−15427.
(25)1283 Coort, S. L. M.; Willems, J.; Coumans, W. A.; van der Vusse, G.1284 J.; Bonen, A.; Glatz, J. F. C.; Luiken, J. J. F. P. Sulfo-N-succinimidyl
1285esters of long chain fatty acids specifically inhibit fatty acid translocase1286(FAT/CD36)-mediated cellular fatty acid uptake. Mol. Cell. Biochem.12872002, 239 (1), 213−219.
(26) 1288Garcia-Calvo, M.; Lisnock, J.; Bull, H. G.; Hawes, B. E.; Burnett,1289D. A.; Braun, M. P.; Crona, J. H.; Davis, H. R.; Dean, D. C.; Detmers,1290P. A.; Graziano, M. P.; Hughes, M.; MacIntyre, D. E.; Ogawa, A.;1291O’Neill, K. A.; Iyer, S. P. N.; Shevell, D. E.; Smith, M. M.; Tang, Y. S.;1292Makarewicz, A. M.; Ujjainwalla, F.; Altmann, S. W.; Chapman, K. T.;1293Thornberry, N. A. The target of ezetimibe is Niemann-Pick C1-Like 11294(NPC1L1). Proc. Natl. Acad. Sci. U.S.A. 2005, 102 (23), 8132−8137.
(27) 1295Kossena, G. A.; Charman, W. N.; Boyd, B. J.; Dunstan, D. E.;1296Porter, C. J. H. Probing drug solubilization patterns in the1297gastrointestinal tract after administration of lipid-based delivery1298systems: A phase diagram approach. J. Pharm. Sci. 2004, 93 (2),1299332−348.
(28) 1300Staggers, J. E.; Hernell, O.; Stafford, R. J.; Carey, M. C. Physical-1301chemical behavior of dietary and biliary lipids during intestinal1302digestion and absorption. 1. Phase behavior and aggregation states of1303model lipid systems patterned after aqueous duodenal contents of1304healthy adult human beings. Biochemistry 1990, 29 (8), 2028−2040.
(29) 1305Hay, D. W.; Cahalane, M. J.; Timofeyeva, N.; Carey, M. C.1306Molecular species of lecithins in human gallbladder bile. J. Lipid Res.13071993, 34 (5), 759−68.
(30) 1308Duane, W. C.; Ginsberg, R. L.; Bennion, L. J. Effects of fasting1309on bile acid metabolism and biliary lipid composition in man. J. Lipid1310Res. 1976, 17 (3), 211−9.
(31) 1311Boyd, B. J.; Porter, C. J. H.; Charman, W. N. Using the polymer1312partitioning method to probe the thermodynamic activity of poorly1313water-soluble drugs solubilized in model lipid digestion products. J.1314Pharm. Sci. 2003, 92 (6), 1262−1271.
(32) 1315Trevaskis, N. L.; Porter, C. J. H.; Charman, W. N. Bile increases1316intestinal lymphatic drug transport in the fasted rat. Pharm. Res. 2005,131722 (11), 1863−1870.
(33) 1318Caliph, S. M.; Charman, W. N.; Porter, C. J. H. Effect of short-,1319medium-, and long-chain fatty acid-based vehicles on the absolute oral1320bioavailability and intestinal lymphatic transport of halofantrine and1321assessment of mass balance in lymph-cannulated and non-cannulated1322rats. J. Pharm. Sci. 2000, 89 (8), 1073−1084.
(34) 1323Waynforth, H. B.; Flecknell, P. A. Experimental and surgical1324technique in rat; Academic Press: 1992.
(35) 1325Cummins, C. L.; Salphati, L.; Reid, M. J.; Benet, L. Z. In vivo1326modulation of intestinal CYP3A metabolism by P-glycoprotein:1327Studies using the rat single-pass intestinal perfusion model. J.1328Pharmacol. Exp. Ther. 2003, 305 (1), 306−314.
(36) 1329Van Heek, M.; Farley, C.; Compton, D. S.; Hoos, L.; Alton, K.1330B.; Sybertz, E. J.; Davis, H. R. Comparison of the activity and1331disposition of the novel cholesterol absorption inhibitor, SCH58235,1332and its glucuronide, SCH60663. Br. J. Pharmacol. 2000, 129 (8),13331748−1754.
(37) 1334Kyle, M. E.; Nakae, D.; Sakaida, I.; Miccadei, S.; Farber, J. L.1335Endocytosis of superoxide dismutase is required in order for the1336enzyme to protect hepatocytes from the cytotoxicity of hydrogen1337peroxide. J. Biol. Chem. 1988, 263 (8), 3784−3789.
(38) 1338Winne, D. Rat jejunum perfused in situ: Effect of perfusion rate1339and intraluminal radius on absorption rate and effective unstirred layer1340thickness. Naunyn-Schmiedeberg’s Arch. Pharmacol. 1979, 307 (3),1341265−274.
(39) 1342Kossena, G. A.; Charman, W. N.; Boyd, B. J.; Porter, C. J. H. A1343novel cubic phase of medium chain lipid origin for the delivery of1344poorly water soluble drugs. J. Controlled Release 2004, 99 (2), 217−1345229.
(40) 1346Johnson, B. M.; Chen, W.; Borchardt, R. T.; Charman, W. N.;1347Porter, C. J. H. A kinetic evaluation of the absorption, efflux, and1348metabolism of verapamil in the autoperfused rat jejunum. J. Pharmacol.1349Exp. Ther. 2003, 305 (1), 151−158.
(41) 1350Wagner, J. G.; Sedman, A. J. Quantitaton of rate of1351gastrointestinal and buccal absorption of acidic and basic drugs1352based on extraction theory. J. Pharmacokinet. Biopharm. 1973, 1 (1),135323−50.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXN
(42)1354 Dahan, A.; Miller, J. M.; Hilfinger, J. M.; Yamashita, S.; Yu, L.1355 X.; Lennernas, H.; Amidon, G. L. High-permeability criterion for BCS1356 classification: Segmental/pH dependent permeability considerations.1357 Mol. Pharmaceutics 2010, 7 (5), 1827−1834.
(43)1358 Sugano, K. Estimation of effective intestinal membrane1359 permeability considering bile micelle solubilisation. Int. J. Pharm.1360 2009, 368 (1−2), 116−122.
(44)1361 Haikal, Z.; Play, B.; Landrier, J.-F.; Giraud, A.; Ghiringhelli, O.;1362 Lairon, D.; Jourdheuil-Rahmani, D. NPC1L1 and SR-BI are involved1363 in intestinal cholesterol absorption from small-size lipid donors. Lipids1364 2008, 43 (5), 401−408.
(45)1365 Tran, T. T. T.; Poirier, H.; Clement, L.; Nassir, F.; Pelsers, M.1366 M. A. L.; Petit, V.; Degrace, P.; Monnot, M.-C.; Glatz, J. F. C.;1367 Abumrad, N. A.; Besnard, P.; Niot, I. Luminal lipid regulates CD361368 levels and downstream signaling to stimulate chylomicron synthesis. J.1369 Biol. Chem. 2011, 286 (28), 25201−25210.
(46)1370 Nauli, A. M.; Nassir, F.; Zheng, S.; Yang, Q.; Lo, C. M.;1371 VonLehmden, S. B.; Lee, D.; Jandacek, R. J.; Abumrad, N. A.; Tso, P.1372 CD36 Is important for chylomicron formation and secretion and may1373 mediate cholesterol uptake in the proximal intestine. Gastroenterology1374 2006, 131 (4), 1197−1207.
(47)1375 Yu, L.; Bharadwaj, S.; Brown, J. M.; Ma, Y.; Du, W.; Davis, M.1376 A.; Michaely, P.; Liu, P.; Willingham, M. C.; Rudel, L. L. Cholesterol-1377 regulated Translocation of NPC1L1 to the Cell Surface Facilitates1378 Free Cholesterol Uptake. J. Biol. Chem. 2006, 281 (10), 6616−6624.
(48)1379 Moussa, M.; Landrier, J.-F.; Reboul, E.; Ghiringhelli, O.;1380 Comera, C.; Collet, X.; Frohlich, K.; Bohm, V.; Borel, P. Lycopene1381 absorption in human intestinal cells and in mice involves Scavenger1382 Receptor Class B Type I but not Niemann-Pick C1-Like 1. J. Nutr.1383 2008, 138 (8), 1432−1436.
(49)1384 Reboul, E.; Goncalves, A.; Comera, C.; Bott, R.; Nowicki, M.;1385 Landrier, J.-F.; Jourdheuil-Rahmani, D.; Dufour, C.; Collet, X.; Borel,1386 P. Vitamin D intestinal absorption is not a simple passive diffusion:1387 Evidences for involvement of cholesterol transporters. Mol. Nutr. Food1388 Res. 2011, 55 (5), 691−702.
(50)1389 Reboul, E.; Klein, A.; Bietrix, F.; Gleize, B.; Malezet-1390 Desmoulins, C.; Schneider, M.; Margotat, A.; Lagrost, L.; Collet, X.;1391 Borel, P. Scavenger Receptor Class B Type I (SR-BI) Is involved in1392 vitamin E transport across the enterocyte. J. Biol. Chem. 2006, 281 (8),1393 4739−4745.
(51)1394 Altmann, S. W.; Davis, H. R., Jr; Yao, X.; Laverty, M.; Compton,1395 D. S.; Zhu, L.-j.; Crona, J. H.; Caplen, M. A.; Hoos, L. M.; Tetzloff, G.;1396 Priestley, T.; Burnett, D. A.; Strader, C. D.; Graziano, M. P. The1397 identification of intestinal scavenger receptor class B, type I (SR-BI) by1398 expression cloning and its role in cholesterol absorption. Biochim.1399 Biophys. Acta, Mol. Cell Biol. Lipids 2002, 1580 (1), 77−93.
(52)1400 Goldstein, J. L.; Brown, M. S.; Anderson, R. G. W.; Russell, D.1401 W.; Schneider, W. J. Receptor-mediated endocytosis: Concepts1402 emerging from the LDL receptor system. Annu. Rev. Cell Biol. 1985,1403 1 (1), 1−39.
(53)1404 Mukerjee, P.; Mysels, K. J. Critical micelle concentrations of1405 aqueous surfactant systems; National Bureau of Standards: Washington,1406 DC, 1971; pp 88−170.
(54)1407 Schubert, R.; Schmidt, K. H. Structural changes in vesicle1408 membranes and mixed micelles of various lipid compositions after1409 binding of different bile salts. Biochemistry 1988, 27 (24), 8787−8794.
(55)1410 Hofmann, A. F. Bile acids: The good, the bad, and the ugly.1411 Physiology 1999, 14 (1), 24−29.
(56)1412 Horter, D.; Dressman, J. B. Influence of physicochemical1413 properties on dissolution of drugs in the gastrointestinal tract. Adv.1414 Drug Delivery Rev. 1997, 25 (1), 3−14.
(57)1415 Vertzoni, M.; Fotaki, N.; Nicolaides, E.; Reppas, C.; Kostewicz,1416 E. S.; Stippler, E.; Leuner, C.; Dressman, J. Dissolution media1417 simulating the intralumenal composition of the small intestine:1418 physiological issues and practical aspects. J. Pharm. Pharmacol. 2004,1419 56 (4), 453−462.
(58)1420 Jarvinen, T.; Jarvinen, K.; Schwarting, N.; Stella, V. J. β-1421 Cyclodextrin derivatives, SBE4-β-CD and HP-β-CD, increase the oral
1422bioavailability of cinnarizine in beagle dogs. J. Pharm. Sci. 1995, 84 (3),1423295−299.
(59) 1424Augustijns, P.; Brewster, M. E. Supersaturating drug delivery1425systems: Fast is not necessarily good enough. J. Pharm. Sci. 2012, 1011426(1), 7−9.
(60) 1427Tønsberg, H.; Holm, R.; Mu, H.; Boll, J. B.; Jacobsen, J.;1428Mullertz, A. Effect of bile on the oral absorption of halofantrine in1429polyethylene glycol 400 and polysorbate 80 formulations dosed to bile1430duct cannulated rats. J. Pharm. Pharmacol. 2011, 63 (6), 817−824.
(61) 1431Ericzon, B. G.; Todo, S.; Lynch, S.; Kam, I.; Ptachcinski, R. J.;1432Burckart, G. J.; Van Thiel, D. H.; Starzl, T. E.; Venkataramanan, R.1433Role of bile and bile salts on cyclosporine absorption in dogs.1434Transplant Proc. 1987, 19 (1 Part 2), 1248−1249.
(62) 1435Behrens, D.; Fricker, R.; Bodoky, A.; Drewe, J.; Harder, F.;1436Heberer, M. Comparison of cyclosporin A absorption from LCT and1437MCT solutions following intrajejunal administration in conscious1438dogs. J. Pharm. Sci. 1996, 85 (6), 666−668.
(63) 1439Miller, J. M.; Beig, A.; Carr, R. A.; Webster, G. K.; Dahan, A.1440The solubility−permeability interplay when using cosolvents for1441solubilization: Revising the way we use solubility-enabling formula-1442tions. Mol. Pharmaceutics 2012, 9 (3), 581−590.
(64) 1443Dahan, A.; Beig, A.; Ioffe-Dahan, V.; Agbaria, R.; Miller, J. M.1444The twofold advantage of the amorphous form as an oral drug delivery1445practice for lipophilic compounds: Increased apparent solubility and1446drug flux through the intestinal membrane. AAPS J. 2012,1447DOI: 10.1208/s12248-012-9445-3.
(65) 1448Miller, J. M.; Beig, A.; Carr, R. A.; Spence, J. K.; Dahan, A. A1449win−win solution in oral delivery of lipophilic drugs: Supersaturation1450via amorphous solid dispersions increases apparent solubility without1451sacrifice of intestinal membrane permeability.Mol. Pharmaceutics 2012,14529 (7), 2009−2016.
Molecular Pharmaceutics Article
dx.doi.org/10.1021/mp3006566 | Mol. Pharmaceutics XXXX, XXX, XXX−XXXO