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European Journal of Pharmaceutical Sciences 42 (2011) 416–422

Contents lists available at ScienceDirect

European Journal of Pharmaceutical Sciences

journa l homepage: www.e lsev ier .com/ locate /e jps

novel excipient, 1-perfluorohexyloctane shows limited utility for the oralelivery of poorly water-soluble drugs

ené Holma,∗, Erling Bonne Jørgensena, Michael Harborga,b, Rune Larsena,b, Per Holmc,nette Müllertzb,d, Jette Jacobsenb

Preformulation, H.Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, DenmarkDepartment of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, DenmarkProduct Development and Lifecycle, H.Lundbeck A/S, Ottiliavej 9, DK-2500 Valby, DenmarkBioneer:FARMA, Danish Drug Development Center, Department of Pharmaceutics and Analytical Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen,niversitetsparken 2, DK-2100 Copenhagen, Denmark

r t i c l e i n f o

rticle history:eceived 6 November 2010eceived in revised form 13 January 2011ccepted 17 January 2011vailable online 21 January 2011

eywords:ipid based formulationemi fluorinated alkaneioavailability-Perfluorohexyloctaneoorly water soluble drugs

a b s t r a c t

The applicability of the semi-fluorinated alkane 1-perfluorohexyloctane (F6H8) as a novel excipientin lipid based drug delivery systems was studied. Solubility studies of 11 poorly water soluble drugs(cinnarizine, danazol, estradiol, fenofibrate, griseofulvin, halofantrine, lidocaine, prednisolone, probucol,rolipram and siramesine) showed significantly lower equilibrium solubility in F6H8 compared to soybean oil (long chain triglyceride). F6H8 was miscible with medium chain triglycerides (MCT) but notmiscible with long chain triglycerides, neither was pure F6H8 nor the mixture F6H8:MCT (1:1) misciblewith 7 commonly used surfactants (Cremophor EL, Span 20, Span 80, Labrasol, Softigen 767 and Gelu-cire 44/14, polysorbate 80). In vitro lipolysis studies confirmed that F6H8 was non-digestible. F6H8:MCT(1:1) showed initially faster lipolysis compared to pure MCT. Thus, final phase lipolysis was lower indi-cating that F6H8 may affect the lipolysis of MCT. However, in vivo bioavailability studies in rats showedthe same plasma concentration-time profiles when dosing 10 mg/kg halofantrine at two dose levels ofF6H8, MCT or F6H8:MCT (1:1) (AUC ranged from 3058 to 3447 h ng/ml, Tmax ∼ 6.0 h, Cmax ranged from 168

to 265 mg/ml). Generally, the addition of polysorbate 80 shortened the time to reach Cmax (Tmax ranged1.3–4.5 h), but had limited effect on the bioavailability from F6H8 or MCT in combination with polysorbate80 (4:1) (AUC ranged from 3807 to 4403 (h ng/ml)). Although a synergistic effect was obtained with halo-fantrine in F6H8:MCT:polysorbate 80 (2:2:1) (AUC 5574 ± 675 h ng/ml; mean ± SEM), it was not superiorto dosing halofantrine in pure polysorbarte 80 (AUC 7370 ± 579 h ng/ml; mean ± SEM). The applicabil-ity of F6H8 as an excipient for future use in lipid based formulations for poorly water soluble drugs istherefore considered to be very limited.

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. Introduction

A high proportion of the drug compounds in the innovativeharmaceutical companies has poor aqueous solubility (Fahr andiu, 2007; Hauss, 2007; Lipinski et al., 1997), which may limit theirioavailability after oral administration. This provides the phar-aceutical scientist with a general challenge to identify enabling

echnologies which ensures sufficient exposure in order to marketew, safer, and more effective drugs to benefit the patients. Thesenabling technologies include micronisation, stabilised nanopar-icles, solid solutions, and lipid based formulations, which either

∗ Corresponding author. Tel.: +45 3643 3596; fax: +45 3643 8242.E-mail address: rhol@lundbeck.com (R. Holm).

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928-0987/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ejps.2011.01.007

© 2011 Elsevier B.V. All rights reserved.

ncrease the dissolution rate or present the compound in a solu-ilised form, so the dissolution step is circumvented (Fahr and Liu,007).

Lipid based formulations are particularly effective for some ofhe most difficult compounds (Fahr and Liu, 2007; Gursoy andenita, 2004; Hauss, 2007). A low aqueous solubility does notecessarily lead to a high solubility in excipients used in lipidased formulations (Hauss, 2007). Therefore, new solvents or lipidxcipients are constantly sought to solubilise the drugs beforedministration. Semi fluorinated alkanes (SFAs) could be consid-

red as such a lipid excipient. SFAs are diblock molecules, inhich a hydrocarbon segment and a fluorocarbon segment are

ovalently bound. The general formula of SFAs can be writtens CnF2n+1CmH2m+1 (FnHm) (Krafft and Riess, 2009; Riess, 2002).he fluorine atom is significantly larger than hydrogen, as seen

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R. Holm et al. / European Journal of Ph

y their van der Waals radius of 1.47 A and 1.20 A respectivelyBondi, 1964). This makes fluorocarbon chains more bulky thanheir corresponding hydrocarbon chains. The volume of CH2 andH3 groups is estimated at 27 A3 and 54 A3 respectively, whereasF2 and CF3 groups are 38 A3 and 92.5 A3 (Tiddy, 1985). Fluorocar-on chains are stiffer than their hydrocarbon counterparts (Eatonnd Smart, 1990), and in order to minimize steric hindrance, fluoro-arbon chains adopt a helical conformation whereas hydrocarbonhains adopt the classical planar trans zigzag-conformation (Krafftnd Riess, 2009). Fluorocarbon chains combine two characteris-ics that are usually considered to be conflicting, as they are bothydrophobic and lipophobic at the same time (Broniatowski andynarowicz-Latka, 2008; Krafft, 2001; Krafft and Riess, 2009; Loostro, 2003; Sabín et al., 2006; Turbgerg and Brady, 1988) andre therefore described as being fluorophilic moieties (Krafft andiess, 2009). SFAs are generally considered to be chemically andiologically inert (Krafft and Riess, 2009). These properties makehem a potential new solvent or surfactant for soft gelatine capsuleormulations.

The biological properties of SFAs have recently been extensivelyeviewed by Krafft and Riess (2009). The reported toxicity of SFAs isery low (Cook and Pierce, 1973; Riess et al., 1991, 1994; Tsai, 2005;arif et al., 1994) and no overt toxicity or negative effects on theurvival and growth of mice have been noted after intraperitonealnjection of 30 g/kg of selected SFAs (Riess et al., 1994). In additionFAs have a wide array of medical applications and suggested appli-ations include; stabilizers in blood substitutes (reviewed by Krafftt al., 2003; Krafft and Riess, 2009; Riess, 2001); a component in thereatment of retinal detachment (Broniatowski and Dynarowicz-atka, 2008; Colthurst et al., 2000; Hoerauf et al., 2001; Peymant al., 1995); a solvent for pulmonary drug delivery (Krafft, 2001;iess, 2002; Sadtler et al., 1996); and in topical gels (Krafft andiess, 1994). This makes it of interest to investigate if SFAs could betilized in oral drug delivery. The overall aim of the present workas, therefore, to examine if F6H8 is useful as an excipient in oralrug formulation for poorly water-soluble drugs, using both in vitrond in vivo methods.

. Materials and methods

.1. Materials

1-Perfluorohexyloctane (F6H8) was kindly donated byovaliq (Heidelberg, Germany). Halofantrine free base and

nternal standard (IS) 2,4-dichloro-6-trifluoromethyl-9[1-[2-dibutylamino)ethyl]]-phenathrenemethanol hydrochloride wereindly donated by GlaxoSmithKline (West Sussex, UK). Dana-ol and glycerol were purchased from Unikem (Copenhagen,enmark) and lecithin (Lipoid E80 and Lipoid S PC, phos-hatidylcholine 98% pure) from Lipoid GmbH (Ludwigshafen,ermany). Cinnarizine, estradiol, lidocaine, fenofibrate, griseoful-in, prednisolone, probucol, rolipram, soybean oil, crude porcineancreas extract, crude porcine bile extract, tris-maleate, Span0, Span 80, polysorbate 80, glycerol, PEG400 and propylenelycol were all obtained from Sigma–Aldrich (St. Louis, MO,SA) and used as received. Ethanol (96%) was obtained from Deanske Spritfabrikker (Aalborg, Denmark) and Siramesine was

rom H. Lundbeck A/S (Valby, Denmark). Viscoleo, Ph.Eur gradeedium chain triglyceride, C8/C10 (MCT) was purchased from

elios V (Illertissen, Germany) and Softigen 767 from Sasol Ltd.

Johannesburg, South Africa). Cremophor EL was a gift from BASFLudwigshafen, Germany) and Gelucire 44/14, and Labrasol a giftrom Gattefossé (Saint-Priest, France). All other reagents were ofnalytical or HPLC grade. Purified water was obtained from a Mil-

bTwTi

ceutical Sciences 42 (2011) 416–422 417

ipore Milli-Q Ultrapure water purification system (Billerica, MA,SA).

.2. Solubility studies

The solubility of a number of poorly water soluble drugs (cin-arizine, danazol, estradiol, fenofibrate, griseofulvin, halofantrine,

idocaine, prednisolone, probucol, rolipram and siramesine) in6H8 and soybean oil was determined by adding an excess of eachompound to each of the two vehicles into glass vials with Teflon-ined caps. The mixture was sonicated in a Covaris S2 acousticransducer (Covaris Inc., Woburn, MA, USA) for 100 s using the fol-owing settings: duty cycle 20, intensity 10 and cycles/bursts 1000Nixon et al., 2009). The glass vials were then placed on a horizontalod and rotated (30 rpm) for 24 h at ambient temperature. After 24 hhe samples were filtered through a 0.20 �m filter (Millex-FG, Milli-ore Corporation, Billerica, MA, USA), diluted with tetrahydrofurano an appropriate concentration and the drug content determinedy reverse phase HPLC with a flow rate of 1 ml/min and a columnemperature at 40 ◦C. The HPLC system comprised an L-7300 col-mn oven, L-7400 UV detector, L-7200 autosampler, L-7110 pumpnd D-7000 interface, all from Merck (Darmstadt, Germany). Theolumn was an XBridge 3.5 �m C18, 4.6 mm × 150 mm (Watersorporation, Milford, MA, USA). The different compounds werenalysed with different mobile phases and detection at differentavelengths (see Table 1).

.3. Miscibility of F6H8 with lipid excipients

The miscibility of lipids or surfactants was studied visually inure F6H8 and a 1:1 mixture of F6H8 and MCT (v:v) at ambi-nt temperature employing MCT, long chain tri/mono-glycerides,olysorbate 80, Cremophor EL, Span 20, Span 80, Labrasol, Softigen67 and Gelucire 44/14, ethanol, glycerol, PEG400 and propylenelycol. The miscibility was conducted using the titrimetic methodescribed by Gao et al. (1998). F6H8 or F6H8:MCT mixtures wereeighed directly into a 12 ml vial with a teflon lined cap. Aliquots of

ach investigated excipient were then mixed into the F6H8 phasend left for 30 min at ambient temperature before assessed visu-lly. Miscibility was investigated in the range from 9 to 91% (v/v)or all investigated mixtures.

.4. In vitro lipolysis

The procedure for the dynamic in vitro lipolysis experimentas based – with minor modifications – on a previously devel-

ped lipolysis model (Zangenberg et al., 2001a,b; Christensen et al.,004; Larsen et al., 2008). Bile acids, phosphatidylcholine, NaCl andris-maleate were mixed in a thermostatically controlled vessel37 ± 0.5 ◦C) at concentrations of 5, 1.25, 150 and 2 mM, respec-ively. The formulation, 0.5 ml of either F6H8, MCT or F6H8:MCT1:1, v/v) was added to the bile salt medium, and the pH was imme-iately adjusted to 6.5 with 1.00 M NaOH. In parallel with this a

ipase suspension was prepared by accurately weighing pancreatinn centrifuge tubes, adding water, centrifuged at 4000 rpm for 7 mint 37 ◦C (Labofuge 400R, Heraeus Instruments, Osterode, Germany)nd adjusting pH to 6.3 with 1.00 M NaOH. The supernatant waseparated and heated to 37 ◦C before use. The lipase suspensionas used within 15 min after centrifugation to avoid precipita-

ion. After an equilibration time of 3 min., lipolysis was initiated

y the addition of lipase, producing a volume of 300 ml in total.he hydrolysis was followed by potentiostatic titration at pH 6.5ith 1.00 M NaOH. The equipment for the titration consists of 824

itrando controller, 800 Dosino drive, 842 Titrando pump, 807 dos-ng unit, 804 Titrando stander and an 802 rod stirrer with a 96 mm

418 R. Holm et al. / European Journal of Pharmaceutical Sciences 42 (2011) 416–422

Table 1Mobile phase and wavelength used to analyse the investigated drugs and equilibrium solubility of different drugs in 1-perfluorohexyloctane (F6H8) and soy bean oil. Allvalues are mean ± SD (n = 3).

Compound Mobile phase (%, v/v) Wavelength (nm) Solubility (mg/ml)

ACNa MeOHb Bufferc F6H8 Soy bean oil

Cinnarizine 70 – 30 254 0.9 ± 0.1 39.6 ± 2.0Danazol – 65 35 290 0.1 ± 0.0 2.9 ± 0.1Estradiol – 50 50 220 LOQd 0.7 ± 0.1Fenofibrate – 65 35 290 7.7 ± 1.1 46.1 ± 1.2Griseofulvin – 50 50 296 LOQd 0.3 ± 0.0Halofantrine 70 – 30 250 5.0 ± 0.4 44.5 ± 5.9Lidocaine – 50 50 225 48.5 ± 0.9 148.3 ± 4.8Prednisolone 20 – 80 245 LOQd 0.1 ± 0.0Probucol 70 – 30 223 6.1 ± 0.1 56.2 ± 0.5Rolipram 20 – 80 234 LOQd 0.8 ± 0.1Siramesinee – 65 35 255 0.6 ± 0.1 32.5 ± 1.6

a ACN: acetonitrile.

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b MeOH: methanol.c Buffer: 25 mM phosphate buffer, pH 6.d Solubility below detection limit of the analytical method.e log P: 8.5; pKa: 8.7; solubility in 50 mM phosphate buffer, pH 7.4: 0.5 �g/ml.

tirring propeller at a speed ensuring fast and continuous mix-ng during the lipolysis and a 602 Combined Metrosensor glass pHlectrode and temperature sensor. Anti diffusion burette tips weresed and the software used to control the titration was Tiamo ver-ion 2.0. All equipment was from Metrohm (Herisau, Switzerland).he background level of lipolysis was determined from a lipolysisxperiment without adding formulation. CaCl2 (0.5 M) was dis-ended at a speed of 0.09 ml/min giving a final concentration ofbout 13 mM. The lipase activity was 675 tributyrin units/ml in thenal reaction medium. Calculation of 100% released free fatty acidsas based upon molecular weight of the MCT and the belief that

wo moles of free fatty acids could be released per mole of triglyc-ride. The actual released amount was based upon the assumptionhat one mole of NaOH is needed to neutralise one mole of freeatty acid (Zangenberg et al., 2001a,b). F6H8 was not assumed toeutralise any NaOH.

.5. Oral formulations for in vivo study

Oral formulations for the in vivo study were prepared by weigh-ng halofantrine and adding the relevant amount of F6H8, MCT,olysorbate 80 or mixture hereof. Subsequently, the content was

ixed by mechanical stirring until all halofantrine was dissolved.The formulations were prepared to contain 10 mg halo-

antrine/kg in either 1.5 or 3.0 ml/kg of vehicle (see Table 2).ormulation A was prepared and stored at an elevated tempera-ure (37 ◦C) to ensure solubilisation of halofantrine, whereas all the

able 2omposition of investigated oral formulations and volumes administered to rats.ll formulations contained halofantrine at a dose of 10 mg/kg.

Formulation Content (%, w/w) Dosed volume(ml/kg)

F6H8a MCTb PS 80c

A 100 – – 1.5B 100 – – 3.0C – 100 – 1.5D 50 50 – 3.0E 80 – 20 1.5F – 80 20 1.5G 40 40 20 1.5H – – 100 1.5

a 1-Perfluorohexyloctane.b Medium chain triglyceride.c Polysorbate 80.

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ther formulations were prepared and stored at ambient temper-ture. Prior to dosing the formulations were drawn into syringes;ince formulations F, G and H were biphasic systems, these for-ulations were stirred while being drawn into the syringe. The

ormulations were administered orally via gavage within 24 h ofreparation and the exact concentration of halofantrine in the for-ulation was verified by HPLC. All used formulations contained the

arget concentration within 1% of precision.

.6. Animal study

The protocol used was approved by the Animal Welfare Com-ittee appointed by the Danish Ministry of Justice and all animal

rocedures were carried out in compliance with EC Directive6/609/EEC, the Danish law regulating experiments with animalsnd the NIH guidelines on animal welfare. Male Sprague–Dawleyats (284–333 g) were purchased from Charles River DeutschlandSulzfeld, Germany). The animals were acclimatized and main-ained on standard feed with free access to apples and water forminimum of 5 days prior to the study. Before initiation of the

xperiment the animals were fasted for 16–20 h with free accesso water and randomly assigned to receive one of the treatments.

The animals were orally dosed by gavage, with halofantrine10 mg/kg) solubilised in either 1.5 or 3 ml/kg of the oral formula-ions (see Table 2). Blood samples of 200 �l were obtained from theail vein by individual vein puncture and collected into potassium-DTA tubes (Microvette 500 K3E, Sarstedt, Nümbrecht, Germany)t 1, 2, 3, 4, 6, 8, 10, 24 and 30 h after administration of the oralormulations. Plasma was harvested immediately by 10 min ofentrifugation at 4 ◦C, 2765 × g (Multifuge 1 S-R, Heraeus, Hanau,ermany) and stored at −20 ◦C until analysed. The animals werellowed access to apples 10 h after oral dosing and water at allimes. At the end of the experiment, the animals were sacrificedy gas.

.7. Quantitative analysis of plasma samples

The concentrations of halofantrine in the plasma samplesere quantified by reverse phase HPLC-UV. The plasma sam-

les were analysed by the use of a previously described methodHumberstone et al., 1995) with a few modifications. In short, thelasma samples were extracted by adding 100 �l internal standard2 mg/ml in acetonitrile) and 1 ml methanol to 100 �l plasma fol-owed by mixing. 4 ml tert-butylmethylether was added and the

R. Holm et al. / European Journal of Pharma

Table 3Some physical chemical parameters of F6H8 (Riess and Krafft, 2006).

F6H8 Water

Molecular weight (g/mol) 432 18Specific gravity, 25 ◦C (g/cm3) 1.331 0.97Refractive index, 20 ◦C 1.343 1.333Melting point (◦C) −5.2 0Boiling point (◦C) 223 100Viscosity, 25v (mPa s) 3.44 0.89Vapor pressure (kPa) <0.01 (25 ◦C) 2.3 (20 ◦C)Surface tension (nM/m) 19.65 72

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O2-solubility, 25 ◦C (vol.%) 40.3 2.8CO2-solubility, 25 ◦C (vol.%) 130 n.a.

amples mixed and centrifuged for 15 min at 4 ◦C, 4000 rpm (6-6K, Sigma Laborzentrifugen Gmbh, Osterode am Harz, Germany).he supernatant was transferred to an evaporation glass containing00 �l 7 �M HCl in acetonitrile and the mixture was evaporated toryness under a stream of nitrogen at 55 ◦C (TurboVap LV Concen-ration Workstation, Zymark, Hopkinton, MA, USA). The residueas reconstituted with 100 �l methanol and 25 �l was analysed

y reverse phase HPLC on an L-5025 column thermostat, L-4250V–VIS detector, AS-2000A auto sampler, L-6200A intelligentump and D-6000 interface, all from Merck (Darmstadt, Germany).he column was an XBridge 3.5 �m C18, 4.6 mm × 150 mm (Watersorporation, Milford, MA, USA). The mobile phase consisted of 72%v/v) methanol and 28% (v/v) phosphate buffer (25 mM, pH 3). Theow-rate was set to 1 ml/min, column temperature at 40 ◦C andhe absorbance was measured at 257 nm. Standard curves were lin-ar in the investigated concentration range 40–1000 ng/ml and theecovery of the extraction exceeded 90% over the evaluate range.

.8. Pharmacokinetic data analysis

The pharmacokinetic parameters following oral administrationf halofantrine were obtained by a non-compartmental analysisy the use of WinNonlin Professional version 5.2 (Pharsight Cor-oration, Mountain View, CA, USA). The area under the curve foralofantrine after oral administration (AUC0-30) was calculatedsing the linear trapezoidal rule from time zero to the last measuredlasma concentration at 30 h post-dose.

.9. Statistical analysis

Sigma Stat for Windows software, version 3.0.1 from SPSSnc. (Chicago, IL, USA) was used for the statistical calculations.he statistical difference between two means was calculatedsing Student’s t-test. When more than two means were com-ared one way analysis of variance (ANOVA) followed by atudent–Neuman–Keuls test was used. Two sided p-values below% (p < 0.05) were considered statistically significant.

. Results and discussion

One of the pharmaceutical technologies, which may be usedo ensure sufficient bioavailability of low aqueous solubility com-ounds, is lipid based formulations. However, poor aqueousolubility is not equal to good solubility in traditional lipid excip-

ents, hence this work evaluates the possibility of using F6H8 as aew excipient for the hydrophobic compounds. Some of the phys-

cal chemical properties of F6H8 compared to water can be foundn Table 3.

3

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ceutical Sciences 42 (2011) 416–422 419

.1. Solubility study

The solubility capability of F6H8 relative to soybean oil, a lipidommonly used in lipid based formulations, was evaluated for aumber of compounds with very different physical chemical prop-rties. The results are presented in Table 1. For all the examinedompounds it was evident that the solubility in F6H8 was signif-cantly lower than in soybean oil. There could be several possibleeasons for this difference in solubility power. The relative stiffnessf the F6H8 when compared to the triglyceride, but also the differ-nce in surface activity could be an explanation. Lower solubilityn surfactants than in triglycerides has been reported previouslyTønsberg et al., 2010).

.2. Miscibility of F6H8 with lipid excipients

On the basis of the chemical structure of F6H8, which is closeo saturated fatty acids, it was decided to examine the feasibil-ty of using F6H8 in lipid-based formulations replacing ordinaryipids or co-solvents. The possibility of making self-microemulsionrug delivery systems containing the semifluorinated alkenesC8F17–CH2–CH = CH–C4H9) and polysorbate 80 has previouslyeen reported (Cecutti et al., 1990; Lattes and Ricolattes, 1994).6H8 was miscible with medium chain triglycerides (MCT) butot long chained triglycerides; hence the compound could replaceome oils.

Several surfactants and cosolvents were also evaluated withoth pure F6H8 and F6H8:MCT (1:1, v/v) including polysorbate0, Cremophor EL, Span 20, Span 80, Labrasol, Softigen 767, Gelu-ire 44/14, ethanol, PEG400, glycerol and propylene glycol. Of thesenly ethanol at concentrations above 40% (v/v) was miscible with6H8:MCT, whereas none of the other excipients were miscibleith either F6H8 or the F6H8:MCT mixture; hence the generalossibility of using F6H8 in lipid based formulations seems to beestricted to simple lipid solutions. The solubility of F8H2 in oliveil has been reported to be 29 mM (Le et al., 1996), the number ofydrogenated carbons, hence seems to be important for the lipidiscibility.

.3. In vitro lipolysis

F6H8 does not possess any digestible bonds; therefore the effectf F6H8 on the lipolysis of MCT was studied in the dynamic in vitroipolysis model. The lipolysis of MCT and F6H8:MCT (1:1, v/v) ishown in Fig. 1. Adding only F6H8 to the lipolysis model did asnticipated not consume any NaOH (data not shown). The lipoly-is process could be divided into three phases: a short lag-phase,fast initial phase and a slow final phase for both the MCT and

he MCT:F6H8 mixture. The initial lipolysis rate was slightly fasteror the mixture of F6H8 and MCT than the pure MCT, but the finalegree of lipolysis was significantly lower. Based on the amount ofitrated free fatty acids the percentage extent of hydrolysed MCTas 96.6% for pure MCT and 72.6% for the F6H8:MCT (1:1, v/v) mix-

ure. The pattern for the hydrolysis of MCT was in accordance withreviously published results (Christensen et al., 2004; Kaukonent al., 2004; Sek et al., 2002). The change in hydrolysis activity,.e. the shift from the fast to the slow lipolysis phase, sets in at aater time for the pure MCT compared to the mixture. This may be aeflection of the presence of more substrate in the MCT experimentr more available substrate for the lipase when F6H8 is absent.

.4. In vivo study

F6H8 has capabilities to function as a surfactant (Turbgerg andrady, 1988; Krafft and Riess, 2009). Cremophor RH40 and polysor-

420 R. Holm et al. / European Journal of Pharmaceutical Sciences 42 (2011) 416–422

Fig. 1. Percentage lipid digested during in vitro lipolysis. The amount of free fattyafF(

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Table 4Pharmacokinetic parameters following oral administration of 10 mg/kg halofantrinein different formulations. All values are mean ± SEM (n = 5–6).

Formulation AUC (h ng/ml) Tmax (h) Cmax (ng/ml)

A 3058 ± 421a 5.8 ± 0.7e 168 ± 29e

B 3447 ± 455a 5.0 ± 0.6e 212 ± 17a

C 3327 ± 488a 6.0 ± 0.5e 265 ± 40a

D 3074 ± 201a 6.0 ± 0.5e 198 ± 26a

E 3807 ± 323a 1.3 ± 0.2f 329 ± 34g

F 4403 ± 494b 4.5 ± 0.5e 306 ± 44b

G 5574 ± 675c 2.7 ± 0.6f 398 ± 33h

H 7370 ± 579d 2.0 ± 0.4f 639 ± 41d

a Significantly different from formulation G and H.b Significantly different from formulation H.c Significantly different from formulation A, B, C, D, E and F.d Significantly different from formulation A, B, C, D, E, F and G.

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cid has been normalized to 100% of the theoretical amount of titratable freeatty acid when adding following formulations. (A) 0.5 ml MCT and (B) 0.25 ml6H8 + 0.25 ml MCT with the background lipolysis subtracted. All values are meann = 3). Background lipolysis (n = 3) has been subtracted from the obtained results.

ate 80 have limitations in the amount used due to toxicologicalffects (Gad et al., 2006), whereas SFAs have been reported to beery safe (Cook and Pierce, 1973; Riess et al., 1991, 1994; Tsai, 2005;arif et al., 1994), consequently F6H8 could potential replace somef these surfactants in lipid based formulations. Due to the partlyydrophilic character of surfactants lower solubilities comparedo triglycerides are not unusual for lipophilic compounds, as alsobserved for F6H8. F6H8 had an influence on the digestion pro-le of MCT in the in vitro lipolysis model, which has also previouslyeen reported for the extensively used surfactant Cremophor RH40Cuiné et al., 2008). Based upon the potential of F6H8, the excipientas investigated in vivo with halofantrine as a model compound,

requently used in the investigations of lipid based formulations.The plasma concentration time profiles for rats dosed orally

ith the simple solutions, i.e. formulations A–D, are shown in Fig. 2

nd the pharmacokinetic parameters are presented in Table 4. Tour knowledge no other studies have investigated the oral absorp-ion of a drug co-administered with any SFA. The present studyemonstrated that the groups dosed with both 1.5 and 3 ml/kg of

ig. 2. Plasma concentration versus time of halofantrine in male rats after oral dos-ng of 10 mg/kg halofantrine in; formulation (A) 0.5 ml F6H8 (�), (B) 1 ml F6H8 (�),C) 0.5 ml MCT (�), and (D) 0.5 ml F6H8 + 0.5 ml MCT (�). All values are mean + SEMn = 5–6).

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e Significantly different from formulation E, G and H.f Significantly different from formulation A, B, C, D and F.g Significantly different from formulation A and H.h Significantly different from formulation A, B, C, D, and H.

6H8 lead to the same AUC as the group dosed with both MCT andhe mixture of MCT and F6H8. No statistically significant differ-nces were observed for the simple solutions, i.e. formulations A–D,n neither AUC, Tmax nor Cmax. Though F6H8 seemed to change then vitro lipolysis of MCT, this did not translate into lower bioavail-bility in vivo. The relatively good absorption of halofantrine fromhe F6H8 containing vehicles, therefore might suggest that (i) F6H8s absorbed and thereby taking its load of halofantrine into thenterocyte, or (ii) F6H8 is capable of effectively preventing precip-tation of halofantrine in the gastro intestine during the absorptionf the compound. Dosing higher amounts of F6H8 to the animalsid not compromise or enhance the absorption when compared tohe lower dosing volume, which could support the proposal that6H8 is absorbed orally.

Polysorbate 80 is a digestible surfactant (Cuiné et al., 2008),hich has been used in combination with halofantrine previously

Tønsberg et al., 2010) and was consequently used in the presenttudy in combination with the indigestible F6H8. The plasma con-entration time profiles for rats dosed orally with formulations

ontaining polysorbate 80 (formulations D–H) are shown in Fig. 3nd the pharmacokinetic parameters are presented in Table 4.here was a clear trend, though only statistically different, for one ofhe formulations (i.e. formulation H), that addition of polysorbate

ig. 3. Plasma concentration versus time of halofantrine in male rats after oral dos-ng of 10 mg/kg halofantrine in; formulation (E) 0.4 ml F6H8 + 0.1 ml PS 80 (�), (F).4 ml MCT + 0.1 ml PS 80 (�), (G) 0.2 ml F6H8 + 0.2 ml MCT + 0.1 ml PS 80 (�) and (H).5 ml PS 80 (�). All values are mean + SEM (n = 6).

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0 increased the AUC. When F6H8 is added to water it accumu-ates in the bottom of the beaker, however when polysorbate isdded a slight white color appears indicating partly dispersion of6H8. Assuming that addition of polysorbate 80 increased the dis-ersion of all the formulations the observations are in accordanceith a number of studies (Araya et al., 2005; Balandraud-Pieri et al.,

997; de Smidt et al., 2004; Holt et al., 1994; Iwanaga et al., 2006;eier-Kriesche et al., 2000, 2001; Nielsen et al., 2008; Toguchi et al.,

990a,b). Further, addition of polysorbate 80 generally decreaseshe time to reach maximum plasma exposure. Despite an increasen the AUC when polysorbate 80 was added to the formulations,he availability of halofantrine was still highest when solubilised inure polysorbate 80, which led to an AUC similar to previous studiesLind et al., 2008; Tønsberg et al., 2010). An interesting observa-ion is that the AUC for the F6H8/MCT mixture with polysorbate0 was significantly higher than the AUC obtained when polysor-ate 80 was mixed with pure F6H8 or MCT, though the latter wasot statistically significant. This increase indicates that when dos-

ng a mixture of F6H8, MCT and polysorbate 80 a synergistic effectccurs and hence, that the excipient may have some interestingiopharmaceutical properties.

Halofantrine is described in the literature as being transportedymphatically in rats (Caliph et al., 2000; Holm et al., 2002; Portert al., 1996), and both polysorbate 80 (Lind et al., 2008; Seeballuckt al., 2004) and MCT (Caliph et al., 2000) have been demonstratedo facilitate this absorption route. The impact of F6H8 on lymphaticransport is unknown.

. Conclusions

In conclusion, the present study demonstrates that F6H8 hasimited solubilising effect on the poorly water-soluble drugs tested

hen compared to soy bean oil. In fact, the solubility of the testedrugs was higher in soy bean oil compared to F6H8 by a factor–60. F6H8 was not digested in the in vitro model, but it changedhe lypolysis profile of MCT when the two components were co-dministered in the in vitro lipolysis model. As described in thentroduction SFAs in general possess interesting biopharmaceuti-al and toxicological properties, however, the solubility data makehe use of F6H8 as an enabling excipient for low aqueous solubleompounds questionable.

The in vivo study demonstrated that F6H8 was able to providebioavailability at the same level as MCT. Furthermore, improving

he dispersion of F6H8 by the addition of polysorbate 80 had a lim-ted effect on bioavailability, but a synergistic effect was obtainedy combining F6H8, MCT and polysorbate 80. This supports theesults from the in vitro data, that the use of F6H8 as a generalxcipient for poorly aqueous soluble compounds is very unlikely.

cknowledgement

Bernhard Günther and Dieter Scherer from Novaliq GmbH arecknowledged for providing the F6H8 and for the discussions ofhe possible use of the material. The personnel in animal facilities atundbeck are gratefully acknowledged for their skilful conductancef the animal experiment.

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