Lymphatic fatty acids in canines dosed with pharmaceutical formulations containing structured triacylglycerols

René Holma,*Trine Porsgaardb

Christopher J. H. Porterc

Carl-Erik Høyb,**Glenn A. Edwardsd

Anette Müllertza

Henning G. Kristensena

William N. Charmanc

a Department of Pharmaceuticsand Analytical Chemistry,The Danish University ofPharmaceutical Sciences,Copenhagen, Denmark

b Biochemistry and NutritionGroup, BioCentrum-DTU,Technical University of Denmark,Lyngby, Denmark

c Department of Pharmaceutics,Victorian College of Pharmacy,Monash University, Melbourne,Victoria, Australia

d Department of VeterinaryScience,University of Melbourne,Werribee, Victoria, Australia

Lymphatic fatty acids in canines dosed withpharmaceutical formulations containingstructured triacylglycerols

The intramolecular structure of dietary triacylglycerols (TAG) influences absorption. Inthis study, two different pharmaceutical formulations were compared containing TAGdiffering in fatty acid profiles and intramolecular structures: LML and MLM, where Mrepresented medium-chain fatty acids (MCFA; 8:0) and L represented long-chain fattyacids (LCFA). Lymph was collected from thoracic duct-cannulated canines for 12 h andthe fatty acid composition was determined. The lymphatic transport of total fatty acidswas significantly higher than the amount dosed; hence, the small exogenously dosedlipid recruited a large pool of endogenous fatty acids. The LML vehicle led to a signifi-cantly higher total fatty acid transport than the MLM vehicle. The amount of 8:0recovered in lymph was almost similar and low for both groups. The amount of LCFArecovered from the animals dosed with the LML vehicle was generally higher than fromthe animals dosed with the MLM vehicle; however, statistically significant differenceswere only found for 18:0 and 18:3n-3. In conclusion, these results indicated that thefatty acid profile and intramolecular structure of administered TAG influenced theabsorption of fatty acids in canines, also when the TAG was incorporated into a phar-maceutical formulation in low amounts.

Keywords: Canine, lymphatic transport, pharmaceutical formulation, structuredtriacylglycerols.

1 Introduction

Co-administration with lipid-containing food has shownto improve the bioavailability of lipophilic drugs, whichhas led the pharmaceutical industry to develop variousformulation systems based on lipids in order to increasethe clinical value and efficacy of these drugs. One verypopular approach to increase the bioavailability for thesecompounds, with respect to lipid-based pharmaceuticalformulations, is the incorporation of the active lipophiliccomponent into a self-microemulsifying drug deliverysystem (SMEDDS) [1, 2].

SMEDDS are defined as isotropic mixtures of oil, a sur-factant, and possibly one or more hydrophilic solvents orco-surfactants, which form fine oil-in-water emulsions ormicroemulsions when exposed to aqueous media underconditions of gentle agitation [3]. SMEDDS typically pro-duce emulsions with a particle size below 100 nm, prop-erties that make SMEDDS a good formulation alternativefor the oral delivery of lipophilic drugs with adequate sol-

ubility in the oil or oil/surfactant blends; however, themechanistic aspects of the bioavailability enhancementare incompletely understood. These systems have gainedconsiderable interest after it became apparent that theclinical success of the cyclosporine A formulation (Neoral)was related to the lipid surfactants [2].

The absorption of medium-chain fatty acids (MCFA) andlong-chain fatty acids (LCFA) in the intestine is different,and their ability to influence the absorption of a pharma-ceutical substance was found to be affected by this dif-ference in rats [4]. Ingested triacylglycerols (TAG) aredegraded to sn-2 monoacylglycerols (sn-2 MAG) and tofree fatty acids in the small intestine under the action ofthe pancreatic lipase [5, 6]. The hydrolysis rate by thepancreatic lipase is affected by chain length and degreeof unsaturation of the fatty acids in the sn-1,3 positions[6–8], with MCFA being hydrolyzed faster than LCFA [9,10].

The sn-2 MAG and the fatty acids are absorbed into theenterocytes. Within the enterocytes, the LCFA are rees-terified with sn-2 MAG of exogenous and endogenous

Correspondence: René Holm, Pharmaceutical R&D, H. Lund-beck A/S, Ottiliavej 9, 2500 Valby, Denmark. Phone: 14536301311, Fax: 145 36438272, e-mail: [email protected]

714 DOI 10.1002/ejlt.200600073 Eur. J. Lipid Sci. Technol. 108 (2006) 714–722

* Current address: H. Lundbeck A/S, 2500 Valby, Denmark** Professor Carl-Erik Høy passed away on February 8, 2003.

© 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com

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Eur. J. Lipid Sci. Technol. 108 (2006) 714–722 Lymphatic fatty acid profiles in canines 715

origin to form a new population of TAG [8, 11], which aresubsequently packed into chylomicrons and excretedinto the lymph [12, 13]. MCFA are, however, preferentiallytransported via the portal vein to the liver for oxidation [14,15]; the shorter the chain length, the lower the transportvia the lymphatics [16]. MCFA and LCFA can be combinedin structured TAG where the glycerol backbone is ester-ified with different fatty acids, giving special functionaland nutritional properties to the TAG.

Lipophilic compounds like halofantrine [17], mepitionstan[18], ontazolast [19] and penclomedine [20] are all exam-ples of pharmaceutically active compounds transportedfrom the intestine via the lymphatic system, in associationwith the lipid core of lipoproteins. The pharmaceuticaladvantages associated with the intestinal lymphatictransport comprise the possibility of enhanced bioavail-ability of some very lipophilic compounds, the avoidanceof hepatic first-pass metabolism and the potential toselectively target drugs to the lymphatic system.

Holm et al. [21] reported that in canines the intestinallymphatic transport and the portal absorption of halofan-trine, a highly lipophilic antimalaria drug (clogP = 8.5),was affected when co-administered in a SMEDDS basedon a structured TAG, when compared to a SMEDDS witha long-chain TAG (LCT). Lipids, unlike many other usedpharmaceutical excipients, are processed both chemi-cally and physically within the gastrointestinal tract beforeabsorption. Administered lipids in pharmaceutical for-mulations have their own right in terms of formulationproperties, but many of their effects are mediated aftertransformation by the natural biochemical processes inthe gastrointestinal tract.

The objective of the present study was therefore toinvestigate the impact of two different structured TAGvarying in fatty acid compositions and intramolecularstructures (MLM and LML, where M represented MCFAand L represented LCFA) and incorporated into a humanrelevant pharmaceutical formulation, both in terms ofcomposition and volume dosed, on the intestinal lym-phatic transport of various fatty acids in conscious lymph-cannulated canines.

2 Materials and methods

2.1 Pharmaceutical formulation

The composition of the emulsions was (in wt/wt):the active pharmaceutical ingredient halofantrine/TAG/Maisine 35–1/Cremophor EL/absolute ethanol(5 : 29 : 29 : 30 : 7), a formulation optimized for halofan-trine and previously described by Khoo et al. [22]. In the

present study, TAG was one of two structured TAG, MLMor LML, produced by enzymatic interesterifications at theBioCentrum-DTU (Technical University of Denmark), aspreviously described [23]. In the case of MLM, sunflowerseed oil (Róco, Copenhagen, Denmark) was used as TAGand caprylic acid (Sigma, St. Louis, MO, USA) as free fattyacid, whereas in the case of LML, tricaprylin (Sigma) wasused as TAG and linoleic acid (Sigma) as free fatty acid.Halofantrine was a gift from GlaxoSmithKline (Mysore,India). Cremophor EL was donated by BASF (Ludwig-shafen, Germany) and Maisine 35–1 by Gattéfosse (Saint-Priest, France).

Approximately 4 g of each formulation was prepared theday before use, by firstly weighing halofantrine into a12-mL Teflon-lined screw-capped glass conical tube,followed by addition of Cremophor EL, Maisine 35–1and MLM or LML. The components were mixed bygentle stirring, and heated in a 50 7C water bath until alldrug compounds had dissolved. The mixture was thencooled to ambient temperature, absolute ethanol wasadded, and the mixture was stirred to ensure uniformity.Of the formulation, 1.00 g was filled into a soft gelatinecapsule using a syringe and a needle. A disintegrationexperiment was performed each day to assess the effi-cacy of the self-microemulsification using a standarddissolution apparatus (Erweka, Heusenstamm, Ger-many). The droplet size of the emulsions was deter-mined by photo correlation spectroscopy using a Zeta-sizer 3000 (Malvern Instruments Ltd., Malvern, UK) toensure that the mixed formulations did not differ in par-ticle size distribution.

2.2 Animals and surgery

All surgical and experimental procedures were reviewedand approved by the local Institutional Animal Experi-mentation Ethics Committee (University of Melbourne,Australia). Studies were conducted in male greyhounddogs (28–35 kg), and their health status was verified by aveterinarian prior to the study. The dogs received a pre-anesthetic by subcutaneous injection of acetylpromazinemaleate (0.5 mg/kg; Delvet Pty. Ltd., Taren Point, Aus-tralia) and were subsequently anesthetized with an intra-venous injection of propofol (3–6 mg/kg; Schering-Plough, Baulkham Hills, Australia). The surgical anesthe-sia was maintained by delivery of halothane and oxygen.Following induction of surgical anesthesia, the thoraciclymph duct was cannulated as previously described byKhoo et al. [24]. Briefly, each dog was fed a small lipidmeal prior to surgery to facilitate subsequent identifica-tion of the thoracic lymph duct. The dogs received intra-venous infusion of saline during surgery, and post-opera-


716 R. Holm et al. Eur. J. Lipid Sci. Technol. 108 (2006) 714–722

tive injection of antibiotic (cephazolin 20 mg/kg; SigmaPharmaceuticals, South Croydon, Australia) and analge-sic (carprofen 4 mg/kg; Pfizer, West Ryde, Australia).

Following surgery, the dogs were allowed to recoverunrestrained in a closed run for 16–20 h. In the initial peri-od of recovery, saline was administered intravenously toensure adequate hydration and to prevent hypoproteine-mia. The dogs were allowed to return to normal ambula-tory movement prior to administration of the lipid-basedformulation.

2.3 Administration of oil and collection of lymph

The dogs remained fasted throughout the recovery andstudy period. A single capsule at 1.00 g with the SMEDDS(290 mg MLM or LML) was administered with 50 mLwater. To prevent dehydration, 25 mL Hartmann’s solutionwas administered at hourly intervals. Water was availablead libitum.

Lymph was collected in hourly fractions into 50-mL tubescontaining 75 mg dipotassium EDTA (Merck, Darmstadt,Germany) for 12 h post-dosing. The amount of lymphcollected in each interval was weighed, and from thehourly samples, four 1-mL aliquots were obtained andplaced into individual Eppendorf tubes and stored at220 7C before analysis. At the end of the experiment, theanimals were sacrificed by an overdose of sodium pen-tobarbitone given intravenously.

2.4 Analysis of structured TAG and lymph lipids

The fatty acid profile of total TAG in MLM and LML wasdetermined by gas-liquid chromatography (GLC) aftermethylation with KOH in methanol [25]. The structureof MLM and LML represented by fatty acids in the sn-2position of the TAG was determined by Grignard deg-radation with allyl magnesium bromide followed byisolation of the sn-2 MAG fraction [26] and methylationwith KOH in methanol. The resulting fatty acid methylesters were analyzed using a Hewlett-Packard 5890chromatograph with a fused-silica capillary column(SP-2380, 60 m, i.d. 0.25 mm, 0.2 mm film thickness;Supelco Inc., Bellefonte, PA, USA), flame ionizationdetection (FID; Hewlett-Packard GmbH, Ingelheim,Germany), and helium as carrier gas. The oven tem-perature was 70 7C followed by temperature program-ming: 15 7C/min until 160 7C, followed by 1.5 7C/minuntil 200 7C, which was maintained for 15 min, andfinally the temperature was raised to 225 7C and main-tained for 5 min. Peak areas were calculated using aHewlett-Packard computing integrator and were used

to calculate the mol-% of fatty acids following correc-tion for response factors based on calibrated stand-ards (Nu-Chek-Prep, Elysian, MN, USA).

Total lipid was extracted from lymph fractions accordingto the method by Folch et al. [27] after addition of internalstandard (TAG 15:0). After methylation with KOH in meth-anol, the fatty acids were analyzed by GLC as describedabove. The internal standard was used to calculate theamounts of fatty acids transported in the lymph.

2.5 Calculations and statistical analyses

Results are expressed as means 6 SEM (n = 4). Therecovery of fatty acids was calculated as the amount of afatty acid found in the lymph divided by the amount of thesame fatty acid in the structured TAG and in Maisine 35–1,multiplied by 100. The recovery calculations included acontribution of endogenous fatty acids transported in thelymph; thus, recoveries could exceed 100%. Differencesbetween lymphatic transport at different time points wereevaluated statistically by one-way ANOVA using Sigma-Stat for Windows version 2.0 (Systat Software GmbH,Erkrath, Germany), and the Student-Newman-Keuls mul-tiple comparison test was applied for analysing potentialdifferences between the formulations. The results wereconsidered significant if p ,0.05.

3 Results

3.1 Fatty acid composition of structured TAG

The major fatty acids (in mol-%) both in total TAG and insn-2 MAG of the two structured TAG, and the fatty acidsin total TAG of Maisine 35–1, are presented in Tab. 1(Cremophor EL is based on castor oil and will hence notbe absorbed). The data confirm the accuracy of the pro-posed structure with respect to the presence of MCFAand LCFA in the sn-2 position of LML and MLM, respec-tively. The differences observed in contents of 8:0, 18:1n-9, and 18:3n-3 in the two structured TAG originated fromthe differences in the raw material used during synthesisof the TAG, but both TAG were enriched in 8:0 and 18:2n-6 as intended.

3.2 Lymph flow

The total 12-h lymph flow was 535.1 6 270.5 and648.0 6 74.5 g in the MLM and LML groups, respectively.There were no differences in flow rate between the twogroups (p .0.05).



Tab. 1. Fatty acid composition (mol-%) of total TAG ofthe structured TAG and Maisine 35–1 and of sn-2 MAG ofthe structured TAG{.

Fattyacid

Maisine35-1TAG

MLM LML

TAG sn-2 TAG sn-2

8:0 0.0 48.7 1.9 36.9 88.516:0 7.2 2.4 0.8 1.8 0.518:0 4.3 0.7 0.3 0.8 0.218:1n-9 22.2 6.9 13.2 16.8 3.218:1n-7 0.9 0.1 0.1 1.1 0.218:2n-6 63.6 40.0 82.0 37.3 6.518:3n-3 1.2 0.1 0.0 4.7 0.8Others{ 0.6 1.1 1.7 0.6 0.1

{ Values represent the means of three determinations.{ Others represent fatty acids that contributed less than0.5 mol-% of total fatty acids.

3.3 Lymphatic transport of MCFA

A portion of the MCFA was transported through the thor-acic lymph duct. The maximum lymphatic transport of 8:0was obtained after 2 h (Fig. 1), but no significant differ-ence between the transported levels of 8:0 from the twoformulations was observed (p .0.05).

The accumulated lymphatic transport of 8:0 did not differbetween the two vehicles (Tab. 2), and neither did the

recovery of 8:0 when dosed in the MLM vehicle(6.2 6 0.1%) when compared with the LML vehicle(7.8 6 2.0%, p .0.05).

3.4 Lymphatic transport of LCFA

The maximum lymphatic transport of 18:2n-6 wasobserved after 2 h when administered in the MLM vehicleand after 1 h in the LML vehicle (Fig. 2); however, no sig-nificant difference between the maximum transport wasobserved (p .0.05). Similar absorption profiles were seenfor 18:0, 18:1n-9 and 18:3n-3 (data not shown), and asignificant difference between the maximum transport for18:3n-3 (p = 0.029) was found.

The accumulated lymphatic transport of both total andindividual fatty acids at selected time points (4, 8, and12 h) after administration of the lipid vehicle is shown inTab. 2. Higher accumulated transport of all fatty acids,except 8:0, was observed after administration of the LMLvehicle when compared with the MLM vehicle; the differ-ence was, however, only statistically significant for 18:0and 18:3n-3 (p ,0.05). This difference was visible already4 h after lipid administration. The 12-h recoveries of theindividual fatty acids are also presented in Tab. 2. Therecovery of 16:0 and 18:0 was statistically highest in theanimals dosed with the LML vehicles, whereas no statis-tically significant differences were observed between therecoveries of the other individual fatty acids, when dosed

Fig. 1. The lymphatic transport of 8:0after oral administration of a pharma-ceutical formulation containing 290 mgstructured TAG; MLM (circle) or LML (tri-angle) expressed as mg transportedper h (mean 6 SEM of four canines).



Tab. 2. Accumulated lymphatic transport (mg) of selected fatty acids at 4, 8, and 12 h in lymph-can-nulated canines administered a lipid-based pharmaceutical formulation{.

Vehicle Fattyacid

Time Recovery (%){

4 h 8 h 12 h

MLM Total 1329.0 6 319.3 1864.4 6 458.0 2010.2 6 425.0* 305 6 64*LML Total 2441.9 6 398.9 3518.1 6 531.7 4064.2 6 697.6* 616 6 106*MLM 8:0 8.2 6 0.5 8.7 6 0.1 8.7 6 0.1 6.2 6 0.1LML 8:0 7.9 6 2.4 8.4 6 2.2 8.4 6 2.2 7.8 6 2.0MLM 16:0 383.6 6 70.3 355.9 6 101.1 383.6 6 95.5 1223 6 304*LML 16:0 460.7 6 70.1 671.2 6 97.1 779.5 6 131.5 3020 6 510*MLM 18:0 160.4 6 21.5* 241.2 6 62.1* 271.6 6 30.7* 1646 6 186*LML 18:0 262.1 6 29.7* 402.0 6 49.9* 480.4 6 74.2* 3248 6 501*MLM 18:1n-9 361.3 6 129.7 494.6 6 185.6 517.1 6 181.6 521 6 183LML 18:1n-9 734.3 6 125.8 1041.2 6 164.2 1182.3 6 213.3 1045 6 189MLM 18:2n-6 364.8 6 65.0 482.8 6 93.5 515.6 6 85.3 172 6 28LML 18:2n-6 635.0 6 132.6 882.7 6 161.9 1001.6 6 194.5 253 6 46MLM 18:3n-3 7.8 6 3.2* 11.2 6 5.2* 11.6 6 5.2* 307 6 138LML 18:3n-3 27.8 6 5.0* 38.5 6 6.4* 43.3 6 7.9* 342 6 67

{ Data represent means 6 SEM of four canines.{ Calculated after 12 h as described in Materials and methods.* LML significantly higher (p ,0.05) than MLM for the specific fatty acid at the same time.

Fig. 2. The lymphatic transport of18:2n-6 after administration of a phar-maceutical formulation containing290 mg structured TAG; MLM (circle) orLML (triangle) expressed as mg trans-ported per h (mean 6 SEM of fourcanines).

in the two vehicles. However, a tendency for a higherrecovery of 18:2n-6 from the LML vehicle compared withthe MLM vehicle (p = 0.056) was observed.

In both groups, 20:4n-6 was observed in the lymph.Totally 87.6 6 10.4 mg was found over the 12-h collectionperiod for the animals dosed with the MLM vehicle and123 6 38.7 mg for the animals dosed with the LML vehi-cle. This difference was not statistically significant(p = 0.41).

3.5 Lymphatic transport of total fatty acids

The maximum lymphatic transport of total fatty acids wasseen after 2 h when administered in the MLM vehicle andafter 1 h in the LML vehicle (not shown). The amounttransported at the peak was statistically significantlydifferent (p = 0.048) for the two vehicles, where dosingof the LML vehicle led to the highest amount(1058.9 6 331.4 mg/h compared with 670.5 6 303.2 mg/h). The accumulated lymphatic transport of total fatty



acids was statistically higher (p = 0.046) at 12 h for theLML vehicle than for the MLM vehicle (Tab. 2). Therecovery of total fatty acids was 305 6 64% for MLM and616 6 106% for LML after 12 h, which was statisticallydifferent (p = 0.046).

4 Discussion

The lymphatic transport of fatty acids was examined afteroral administration to thoracic duct-cannulated caninesof two pharmaceutical formulations containing structuredTAG differing in intramolecular structure. As the accept-able volume of a capsule or a tablet for a pharmaceuticaldosage form is relatively small, the amount of lipid dosedin the present study is limited, when compared to thedoses normally seen in nutritional studies. The presentstudy, however, demonstrated that even 580 mg ofabsorbable lipid was sufficient to trigger the biochemicalpathways leading to lymphatic transport with a high con-tribution of endogenous fatty acids, consistent with aprevious study [28]. Khoo et al. [28] administered a for-mulation with a similar overall composition as the oneused in this study; however, Khoo et al. used either medi-um-chain TAG (MCT) or LCT as the lipid components.When dosed to lymph-cannulated canines, this induced atotal TAG transport over 12 h of 3.4 6 2.2 g for the LCTgroup, 0.9 6 0.2 g for the MCT group and 0.5 6 0.4 g for anon-lipid group, clearly demonstrating the impact of thelipid. In the present study, differences were observed inaccumulated transport of total fatty acids between theMLM and LML vehicles, with higher amounts beingtransported after ingestion of the LML vehicle. Signifi-cantly higher transport of total fatty acids was observed inthis study when compared to the fasted group publishedby Khoo et al. [28] for both the MLM and the LML vehicle(p = 0.040 and p = 0.007). This finding is consistent withthe MCFA having little impact on endogenous lipid turn-over in lymph, most likely because its digestion productsare absorbed primarily via the portal vein for oxidation inthe liver. The total fatty acid recovery of the LML vehiclewas at the same level as the values reported by Khoo etal. [28] for the LCT group, whereas the MLM vehicle led toan intermediate between Khoo et al.’s MCT group and theLML vehicle. These results suggest that the intramole-cular structure of TAG ingested together with a lipophilicpharmaceutical component might influence the absorp-tion of this component and thereby influence the effi-ciency with which the component will work.

Higher relative proportions of 18:1n-9 and 18:3n-3 werepresent in LML than in MLM, which to some degreemakes a direct comparison between the two differentstructures less cogent, a difference that could have been

minimized by a time-consuming and expensive HPLCpurification of the structured TAG. This was, however, notconducted as the structure of the lipid and their effects insmall quantities was in focus, not the exact composition.Furthermore, the study would have gained higher power ifa control group, receiving either no lipid or a natural lipidincorporated into the formulation, had been included.However, as very limited canine data detailing intestinallymphatic transport of lipids is available in the literature,we believe the study to be of general interest, even withthese experimental limitations, but the results discussedbelow should be interpreted with these complications inmind.

The lymphatic transport of 8:0 from MLM after a singleoral bolus administration has previously been exam-ined in rats. Ikeda et al. [29] found a recovery of 8:0 at6.7 6 0.6%, which corresponded well with the findingsreported by Mu and Høy (7.3 6 0.9%) [16], by Straarupet al. (4–5%) [30], as well as with the results in thepresent canine study (6.2 6 0.1%). Most of the 8:0 wasprobably transported through the portal vein afterhydrolysis by the pancreatic lipase. In contrast to thefindings reported in other studies [29–31], the results inthe present study did not show a more efficient lym-phatic transport of MCFA located in the sn-2 positionof the structured TAG when compared to those in thesn-1,3 positions. Only 7.8 6 2.0% of the dosed 8:0 inthe LML vehicle was found in the lymph from thecanines in the present study. In the rat studies referredto above, a larger amount of structured TAG wasdosed when compared to what was dosed to caninesin the present study (270 mg TAG to ,300-g rats ver-sus 290 mg to ,30-kg canines). The requirement forsn-2 MAG for reesterification to TAG in the enterocyteswas consequently very low in the present study incomparison with the aforesaid studies and could mostlikely be supplied by the Maisine 35–1 incorporated inthe pharmaceutical formulation, as a co-surfactant,and by fatty acids from the bile, which may have led tothe relatively low incorporation of 8:0 when dosed inthe LML vehicle.

Jandacek et al. [7] reported that the in vitro hydrolysis ofTAG with MCFA in the sn-1,3 positions and an LCFA inthe sn-2 position was more rapid than the hydrolysis ofan LCT. Similarly, Nagata et al. [10] observed a faster invitro hydrolysis of MLM-type structured TAG than ofLML-type structured TAG. Ikeda et al. [29] suggestedthat this rapid hydrolysis was responsible for the higherlymphatic transport of 18:2n-6 reported in rats when theLCFA was located in the sn-2 position in comparison tothe LML structure where the LCFA are present in theouter positions of the structured TAG. In the present



study, where only a small amount of lipid was dosed toa large animal, the findings were different; a significantlyhigher mass of 18:2n-6 was found in the lymph whenthe LML vehicle was administered compared with theMLM vehicle. It is at present not clear why this differ-ence appears, but possible explanations for thesedivergences include different species and, conse-quently, different gastrointestinal environments; very lit-tle lipid was administered, hence digestion capacity wassufficient to hydrolyze the TAG quickly, and the additionof the pharmaceutical excipient Cremophor EL pro-duced an emulsion with a particle size of 45 nm [21],leading to a very efficient hydrolysis. Furthermore,Porsgaard et al. [31] did not find faster in vitro hydrolysisof MLM in comparison with LML, although the lympha-tic transport in rats was enhanced for fatty acids locat-ed in the sn-2 position of the administered structuredTAG. The purification level of the structured TAG couldalso be a factor influencing the results. In the studies byIkeda et al. and Nagata et al., highly purified TAG wereused, while the time-consuming HPLC purification wasnot applied in the study by Porsgaard et al. [31] and inthe present study.

The recovery of total fatty acids was far more than100% (304 6 64% for MLM and 616 6 106% for LMLafter 12 h). These high values suggest extensive trans-port of endogenous fatty acids as well as those admin-istered exogenously, which was also evident from thehigh recoveries of 18:2n-6 and findings of 20:4n-6.Comparison to the fatty acid profile from a groupreceiving no lipid would have strengthened this study;however, these findings are in accordance with otherstudies reported in the literature. Shiau et al. [32]showed that a substantial fraction of total lymphaticTAG was derived from endogenous sources duringabsorption, and Porsgaard and Høy [33] found a 24-hrecovery of 121 6 10% in rats after administration ofolive oil. They suggested this high finding to be initi-ated through mobilization of the endogenous stores offatty acids after postprandial release of gastrointestinalhormones such as glucose-dependent insulintropicpolypeptide. This explanation could also cover theresults in the present study. The LML vehicle produceda statistically higher recovery of total fatty acids thanthe MLM vehicle. Both vehicles contained the sameweight percentage of lipid, but as the molecular weightof the two explored TAG differs, so does the molaramount of LCFA dosed. The LML vehicle, hence, con-tained a larger molar amount of LCFA (586 mmol) thanthe MLM vehicle (560 mmol), which theoretically shouldlead to a slightly higher lymphatic transport of fattyacids, but not in the range observed in this study. TheLML vehicle leads to a higher turnover of the endoge-

nous pool of fatty acids than the MLM structure, whichcannot completely be explained by the amount ofLCFA administered, and may imply a structural rela-tionship between the dosed TAG and the endogenousmobilization. It may be considered that the LML vehi-cle caused the formation of smaller, phospholipid-richer chylomicrons based on the high endogenousmobilization. The clearance of chylomicrons is de-pendent on the size [34]; hence, this hypothesis couldbe supported if changes were seen in the clearance ofthe co-administered drug; but as all the lymph is col-lected in the present study, the drug found in plasmadoes only reflect the passive portal absorption. On theother hand, a study comparing the size and number oflymph particles after administration of MLM and LMLoils to rats showed that TAG structure had no influenceon the size of lymph particles during absorption [35].From a pharmaceutical point of view, this informationis of great importance as a correlation between thetotal lymphatic TAG transport and the drug transporthas been reported by a number of authors [36, 37]. Thepharmaceutical scientist hereby has the possibility ofenhancing the absorption of poorly water-solublecompounds by utilizing an endogenous transport sys-tem. On the other hand, the scientist has the possibilityof directing the compounds more specifically into thelymphatic system in the situation where site-directeddelivery is the goal. As stated by Ikeda et al. [29], theMLM-type structured TAG can be used both as a fatenergy source for patients with insufficient supply ofpancreatic lipase and as a source of essential fattyacids for these patients. Jensen et al. [38] generallydiscussed the possibility of designing TAG with customfatty acid formulations for nutritional and pharmacolog-ical applications, but designer lipids could also findapplication in drug delivery, as the structure of the TAGcan be used to direct the compound into the absorp-tion pathway (i.e. lymph or portal).

In conclusion, this study demonstrates that the intramo-lecular structure of administered TAG influenced theabsorption of fatty acids in canines, also when the TAGwas incorporated into a pharmaceutical formulation inlow amounts.

Acknowledgments

This work was financially supported by the Danish Medi-cal Research Council (Center for Drug Discovery andTransport) and the Augustinus Foundation. The authorswould like to thank Dr. X. Xu from the Food Biotechnologyand Engineering Group at the Technical University ofDenmark for the production of structured TAG. Majella



Ryan is gratefully acknowledged for technical assistanceduring the sampling period, Grete Peitersen for technicalassistance with determination of the fatty acid profile ofthe lymph samples, and Maggie Palludan for linguisticsupport.

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[Received: March 28, 2006; accepted: July 5, 2006]


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Lymphatic fatty acids in canines dosed with pharmaceutical formulations containing structured triacylglycerols