Transfer of lipophilic drugs between liposomal membranes and biological interfaces: Consequences for drug delivery

European Journal of Pharmaceutical Sciences 26 (2005) 251–265

Transfer of lipophilic drugs between liposomal membranes and biological interfaces: Consequences for drug delivery Alfred Fahr a,∗, Peter van Hoogevest c,d, Sylvio May b, Nill Bergstrand c, Mathew L. S. Leigh c,∗∗ a

d

Department of Pharmaceutical Technology, Friedrich-Schiller-Universit¨at Jena, Lessingstrasse 8, D-07743 Jena, Germany b Department of Physics, North Dakota State University, Fargo, ND 58105-5566, USA c Phares Drug Delivery AG, Kl¨ unenfeldstrasse 30, P.O. Box 637, CH-4132 Muttenz, Switzerland Department of Pharmaceutical Sciences, Institute of Pharmaceutical Technology, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland Received 5 October 2004; received in revised form 30 March 2005; accepted 23 May 2005 Available online 22 August 2005

Abstract This review paper describes the present knowledge on the interaction of lipophilic, poorly water soluble, drugs with liposomal membranes and the reversibility of this interaction. This interaction is discussed in the context of equilibrium and spontaneous transfer kinetics of the drug, when the liposomes are brought in co-dispersion with other artificial or natural phospholipid membranes in an aqueous medium. The focus is on drugs, which have the potential to partition (dissolve) in a lipid membrane but do not perturb membranes. The degree of interaction is described as solubility of a drug in phospholipid membranes and the kinetics of transfer of a lipophilic drug between membranes. Finally, the consequences of these two factors on the design of lipid based carriers for oral, as well as parenteral use, for lipophilic drugs and lead selection of oral lipophilic drugs is described. Since liposomes serve as model-membranes for natural membranes, the assessment of lipid solubility and transfer kinetics of lipophilic drug using liposome formulations may additionally have predictive value for bioavailability and biodistribution and the pharmacokinetics of lipophilic drugs after parenteral as well as oral administration. © 2005 Elsevier B.V. All rights reserved. Keywords: Lipophilic drugs; Phospholipid; Liposomes; Inter-membrane; Transfer kinetics; Membrane solubility; Pharmacokinetics; Bioavailability

1. Introduction The parenteral and oral administration of lipophilic drugs is often problematic because of their low water solubility. In order to administer a therapeutic dose of these drugs, formulaAbbreviations: BPD-MA, benzoporphyrin-mono acid derivative; CyA, cyclosporin A; DCP, dicetylphosphate; DMPC, 1,2 dimyristoylphosphatidylcholine; DMPG, 1,2 dimyristoylphosphatidylglycerol; DOPC, 1,2 dioleoylphosphatidylcholine; DOPS, 1,2 dioleoylphosphatidylserine; DPPC, 1,2 dipalmitoylphosphatidylcholine; HDL, high density lipoproteins; LDL, low density lipoproteins; POPC, 1-palmitoyl; 2oleoylphosphatidylcholine; MPS, mononuclear phagocytotic system; PC, phosphatidylcholine; PG, phosphatidylglycerol; ZnPc, zinc phthalocyanine ∗ Corresponding author. Tel.: +49 3641 94 9900; fax: +49 3641 949902. ∗∗ Corresponding author. Tel.: +41 61 317 9040; fax: +41 61 317 9050. E-mail addresses: [email protected] (A. Fahr), [email protected] (M.L. S. Leigh). 0928-0987/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.ejps.2005.05.012

tions containing solubilisers and/or formulations with a high dissolution rate are an absolute necessity to deliver the drug. Phospholipids can be used to solubilise such drugs. When diacyl-phospholipids with a cylindrical shape are dispersed in water, lipid vesicles comprising a phospholipid bilayer, which surrounds an aqueous compartment are formed spontaneously. Therefore, they can encapsulate hydrophilic and bind amphipathic as well as lipophilic drugs. Since the introduction of liposomes into the world of intravenous drug delivery research (Olson et al., 1982; Stamp and Juliano, 1979; Gabizon et al., 1982), liposomal formulations for lipophilic drugs have been developed and successfully, introduced on the market (Banerjee, 2001; Gregoriadis, 1995). Specific examples of such drugs are amphothericin B (Albelcet® , AmBisome® ) and benzoporphyrin (Visudyne® , Verteporfin for injection).

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The literature suggests that liposomal formulations composed of either modified, e.g. pegylated lipids or even standard natural lecithins, may alter the pharmacokinetic or pharmacodynamic behaviour of encapsulated drugs. These alterations may be related to a modified distribution of the liposomal drug in tissues. In combination with an increase of the half-life of the drug, active targeting is achieved (Van Etten et al., 2000; Hunt, 1982). This may also result in the diversion of the drug from organs where toxic effects could be induced. (Olson et al., 1982; Gabizon et al., 1982). Also direct targeting of the carrier to the site of interest in the body could be explored. (Sapra and Allen, 2003; Gotfredsen et al., 1983). Finally, the half-life of liposomes in the blood circulation can be decreased by passive targeting (i.e. spontaneous or opsonin-mediated uptake) to cells of the Mononuclear Phagocyte System (MPS) (Killion and Fidler, 1994). These approaches are directed to increase the therapeutic index of the liposomal drug. Successful examples are the marketed liposomal products for human use with the hydrophilic drug doxorubicin (Cattel et al., 2003) and the lipophilic membrane-active drug amphotericin B and in preclinical animal models, e.g. valinomycin (Juliano et al., 1987a), vincristine (Mayer et al., 1990) and lipophilic 1beta-d-arabinofuranosyl cytosine derivatives (Schwendener and Schott, 1996). Liposomes can also be used as solubilizers of poorly water soluble drugs for intravenous administration. In such situations, liposomes may behave in a comparable way to solvent formulations and do not influence the pharmacokinetics and pharmacodynamics of the drug. In this context great interest has arisen in the last decades to develop intravenous formulations of sparingly water soluble, lipophilic, drugs like cyclosporin (Fahr et al., 1995) or paclitaxel (Wenk et al., 1996) to avoid or reduce the use of solvents or detergents like Cremophor® with detrimental toxicological side effects. Also the stability of the new generation of lipophilic peptide or protein drugs can be greatly increased by liposomes and may become an issue for future drug formulations (Balasubramanian et al., 2000). Phospholipids are also employed as solubilisers for oral formulations of lipophilic drugs. The formation of the liposomal structure, upon hydration of such formulation in vivo, is not a prerequisite to keep the drug solubilised. It is assumed that after interaction with phospholipases and mixing with bile salts, the phospholipids in the formulation form, (phospho)lipid domains, including mixedmicellar/vesicular structures (see below) in which the drug is associated mono-molecularly. The potential of such formulations to enhance the oral absorption is underscored by the oral product Sirolimus® of Wyeth-Ayerst Pharmaceuticals Inc., using Phosal® , a phospholipid dispersion, as main solubiliser for the lipophilic immuno-modulator rapamycin (Carlson et al., 1998). Other examples are phospholipid based formulations for the lipophilic drug cyclosporin A comprising diacyl (Guo et al., 2001) or mixtures of diacyl- and monoacyl-phospholipids, which have been recently proposed

as oral drug delivery systems (Leigh et al., 2001) and an oral formulation comprising a phospholipid complex (silipide) with silymarin. Silymarin is an unique flavonoid complexcontaining silybin, silydianin and silychrisin, derived from the milk thistle plant (Gatti and Perucca, 1994). The question arises, what type of mechanisms are involved with the binding of lipophilic drugs to the liposomal membrane. Is this binding really irreversible and just caused by their lipophilicity? Do other factors play a role in determining the degree of binding of the lipophilic drugs to liposomal membrane and release kinetics from the liposomal bilayer membrane after contact, e.g. with the biological milieu or other lipid membranes? The appreciation of the observed reversible character of the binding of lipophilic drug to liposomes is of paramount importance, since the term “encapsulated” is often over interpreted as being “irreversibly encapsulated”. This review paper is describing the present knowledge on the interaction of poorly water soluble, lipophilic and amphipatic drugs with liposomal membranes and the reversibility of this interaction. The interaction is described in terms of the maximum solubility of such drugs in membranes and the transfer kinetics with other artificial or natural (phospho)lipid membranes in an aqueous medium. The focus is on drugs, which have the potential to partition (dissolve) in a lipid membrane. Drugs, which have the potential to perturb significantly membranes at low concentrations, like, e.g. melittin (La Rocca et al., 1999) and limonoids (Castelli et al., 2000) are not considered. Finally, the consequences of the solubility of lipophilic drugs in membranes and the transfer kinetics of lipophilic drugs between membranes, with respect to design of lipid drug carriers and bioavailability and biodistribution of lipophilic drugs are discussed.

2. Partitioning of lipophilic and amphipathic drugs into liposomes In the following section, a few examples of lipophilic and amphipathic drugs are provided to illustrate varying drug solubilities of insoluble compounds in liposomal membranes. 2.1. Cyclosporin Partitioning of lipophilic drugs into phospholipid membranes has been estimated for a variety of substances. Cyclosporin A (CyA), a very lipophilic cyclic undecapeptide with a solubility of 9.29 ␮g/mL in water (Schote et al., 2002) showed concentration-dependent liposome–water partitioning coefficients (Fig. 1) (Fahr et al., 1994). The convexly shaped curve in Fig. 1 could be interpreted as limited binding capacity of CyA to the lipid liposomal bilayer. A Scatchard plot (Fig. 2) was extrapolated and showed surprisingly that one CyA molecule could be associated with 19 phospholipid molecules, which was proven in other studies

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water molecules close to apolar solutes lose part of the rotational degree of freedom due to the lack of hydrogen bonding towards the apolar solutes). This is in contrast to many other peptide–lipid binding reactions, which involve enthalpy driven binding. Addition of cholesterol to the lipid membrane decreased the binding capacity of the liposomes for CyA, which may compete for the same lipophilic binding sites. 2.2. Paclitaxel

Fig. 1. Cyclosporin A (CyA) binding to liposomes at different concentrations of CyA. Centrifugation tubes were equilibrated overnight with a corresponding aqueous concentration of CyA to avoid excessive adsorption of CyA to the tube. The dispersion was centrifuged in a Beckman Ultracentrifuge TL-100 at 198,000 × g (ϑ = 25 ◦ C) for 6 h. Supernatant and pellet was collected. Lipid content was measured enzymatically, CyA concentration in supernatant and pellet was measured by RIA (limit of reporting: 15 ng/ml (redrawn from Fahr et al. (1994)).

(Schote et al., 2002) to be the upper limit of incorporation (i.e. solubility) of CyA in liposomal membranes. Recent studies using high sensitivity titration calorimetry, where POPC phospholipid vesicles were injected into a CyA aqueous solution revealed an average heat of reaction of H = +7.3 kcal/mol. The free energy of binding was found to be G = −7.54 kcal/mol (Schote et al., 2002). These studies revealed that the association of CyA with phospholipid membranes is accompanied by a positive enthalpy change, which must hence be overcompensated by a positive entropy change. Binding of CyA to lipid membranes thus follows the classical hydrophobic effect (the “hydrophobic effect” refers to the idea of Charles Tanford (Tanford, 1980) that

Paclitaxel (Taxol® ), another highly lipophilic substance used in cancer therapy, often formulated in liposomal carrier systems, was also thoroughly investigated concerning the binding behaviour to liposomal membranes using high sensitivity titration calorimetry (Wenk et al., 1996). An astonishingly high liposome–water partition coefficient of 9500 was observed. A partition enthalpy of H = −25 ± 3 kcal mol−1 and a free energy of binding of G = −7.9 kcal mol−1 was calculated from the binding studies. The binding reaction is enthalpy-driven, which is explained by van der Waals interactions between the hydrophobic drug and the hydrophobic region of the lipid bilayer. Noteworthy, the large H value leads to a strong temperature dependence of the partition equilibrium. A temperature increase of 10 ◦ C reduces the paclitaxel solubility in the lipid phase by a factor of 4 (Wenk et al., 1996). 2.3. Amphotericin B Amphotericin B, a polyene antibiotic used in antimycotic therapy, binds to liposomal membranes at variable amounts, depending on the liposomal membrane composition. It is well described that amphotericin B interacts more strongly with cholesterol or fungal sterols than with phospholipids (Bolard, 1986). The drug, after binding with cholesterol, forms aqueous channels increasing specifically the ion permeability of the membrane without disrupting completely the membrane (Bolard, 1986). In Albelcet® , liposomal amphotericin B for intravenous administration comprises the phospholipids DMPC/DMPG in a molar ratio of 7:3, and has a drug:lipid molar ratio of 1:1. AmBisome® , another liposomal product, comprises hydrogenated soy phosphatidylcholine, distearoylphosphatidylglycerol, cholesterol and alpha tocopherol and has a drug/lipid 1:6 (w/w) ratio. These high drug contents underscore the high solubility of the amphipathic amphotericin in lipid membranes. 2.4. Other drugs

Fig. 2. CyA binding to liposomes displayed as Scatchard plot. Measurements were done after ultracentrifugation and are described in (Fahr et al., 1994). m denotes the mean number of bound CyA to 1 liposome molecule. cL denotes the concentration of CyA. Number of liposomes in the sample was estimated by measuring lipid concentration and liposome size (180 nm) (redrawn from Fahr et al., (1994)).

Go and Ngiam (1997) found negative values of H and S for the transfer for the anti-malarial drug mefloquine from the aqueous to the gel phase of phospholipid bilayers. The partitioning is enthalpy controlled, which suggests that mefloquine interacts strongly with the ordered phospholipid domains. In contrast, the partitioning of mefloquine

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into the liquid crystalline phase of DMPC is entropy controlled which is typical of a hydrophobic interaction between mefloquine and the aqueous phase. The partitioning of mefloquine into the bulk solvents octanol and hexane were found to be enthalpy and entropy controlled, respectively. The enthalpy dominated partitioning of mefloquine into gel phase DMPC and octanol is attributed to the occurrence of hydrogen bonding and van der Waals interactions between solute and solvent. The flat shape of mefloquine may further aid its interaction with the orderly domains of the lipidic/organic phase. Also other lipophilic drugs appear to have a high solubility in liposomal membranes. For instance diazepam and triclabendazole can be dissolved in liposomes at a lipid to drug weight ratio of 14:1 and 10:1, respectively (Bergstrand and Van Hoogevest, 2004, unpublished results). These examples illustrate that the maximum solubility of a lipophilic drug in a phospholipid membrane may vary considerably from 1:1 to at least 100:1 lipid to drug weight ratio.

3. Typical examples of lipophilic drug release from liposomal formulations The in vitro or in vivo (if available) release characteristics of a few selected liposomal lipophilic drugs are given below to illustrate the variety of release kinetics. 3.1. Cyclosporin A CyA was investigated thoroughly for encapsulation in liposomes. Several studies were concerned with the binding and release of CyA to liposomal membranes and the pharmacokinetic impact (Fahr et al., 1995; Fahr and Reiter, 1999; Fahr and Seelig, 2001). An in vitro system for measuring the transfer of lipophilic drug molecules from the liposomal carrier system to model membranes mimicking other membranous binding places in the body (erythrocyte membranes, endothelial cell membranes, LDL, etc.) was developed, analogous to the assay proposed by McLean and Phillips (1981). It consists of the insertion of the drug into negatively charged liposomes using standard techniques. Neutral liposomes (mostly PCliposomes) were used as acceptor medium at an excess of 10 in relation to the donor liposomes. After mixing the two liposome populations together, samples were processed at defined time points over an ion-exchange column, which allows only the neutral (acceptor) liposomes to be eluted (Fahr and Seelig, 2001). Analysis of the amount of drug in the acceptor liposomes is either done by HPLC, RIA or most easily by radioactive tracers, which were available in the case of CyA. Fig. 3 shows the results of the experiment. It can easily be seen that despite its high lipophilicity, CyA exchanges between its binding sites very rapidly. In agreement with previous studies on the spontaneous cholesterol transfer between

Fig. 3. Transfer of CyA and Cholesterol between liposomes. Donor liposomes (egg-PC:DCP:Chol = 7:1:2) were loaded with 14 C-CyA (phospholipid:CyA = 300:1) or 14 C-cholesterol. Acceptor liposomes, composed of POPC:Chol = 8:2, were mixed with donor liposomes in a relation of 10:1 (acceptor:donor liposomes) at a temperature of 37 ◦ C. At specified time points after mixing, samples were applied to an ion-exchange column. Only acceptor liposomes were eluted and counted for radioactivity (for details, Fahr and Seelig (2001)).

liposomal membranes (Bar et al., 1987, 1986), cholesterol exchanges more slowly compared with CyA. As a control for the assay, the radioactive lipid marker, cholesteryloleoylether exchanges even more slowly, as one would expect from a lipid marker, which is claimed to be a non-exchangeable marker. A parallel experiment using radiolabelled lecithin showed that under the used experimental conditions, the lecithin did not co-migrate with CyA. These in vitro experiments were supported by pharmacokinetic experiments, which showed that liposomal CyA intravenous pharmacokinetics are not significantly different from a classical detergent based (Cremophor® ) formulation (Fahr et al., 1995). An exception was found for very high dosages of liposomes, which may act as a competing binding reservoir for CyA with physiological binding places in the blood (lipoproteins, erythrocytes, etc.). Alternatively, the cells of the MPS in liver and spleen may be saturated and the elimination of the liposomes from the circulation by these cells is delayed. 3.2. Paclitaxel Paclitaxel is a natural product with antitumor activity. Natural paclitaxel is extracted from Taxus chinensis and purified. Liposomal formulations are described to lower toxicity and to change body distribution (Cabanes et al., 1998). Paclitaxel transfer studies were also performed similar to the one described for CyA above (Fahr, Reszka, Schaufelberger, unpublished results). The findings were also similar to those using CyA. In a time frame of minutes, paclitaxel seems to exchange between membrane binding sites.

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3.3. Amphotericin B Amphotericin B is known to transfer rapidly between membranes (van Hoogevest and de Kruijff, 1978; Bolard, 1986). This transfer depends on the physical state (i.e., gel state or liquid crystalline state) of the donor membrane and the acceptor membrane. Transfer is rapid between, gel state, DPPC-containing liposomes and also between, liquid crystalline, egg-PC-liposomes, however, no transfer was observed at room temperature from DPPC liposomes to egg-PC liposomes. However, when the temperature was increased to above the phase transition temperature of DPPC to 48 ◦ C (at which the DPPC is in the liquid crystalline state), transfer occurred rapidly. The role of liposome composition (i.e. the influence of presence of negatively charged phospholipids like DMPG) and temperature in the distribution of amphotericin B with serum lipoproteins were determined by Wasan et al. (1993). The authors conclude that lipid charge and temperature play a role in amphotericin B distribution into serum lipoproteins. Furthermore, amphotericin B and DMPG may co-transfer as an intact drug–lipid complex to serum lipoproteins. 3.4. Other drugs RS-93522 is a dihydropyridine Ca2+ channel blocker (Fig. 4) with a solubility of 0.012 g/l and is, therefore, characterized as a very insoluble drug. An i.v. formulation, was developed using DOPC and DOPG as lipids for a liposomal formulation (Lidgate et al., 1988). The authors observed that a maximum loading of 1 mol of drug for 3.5 mol of lipids is achievable. By studying the influence of the liposome size on the incorporation degree of the drug, the authors concluded that the drug distributes preferably into the smaller sized liposomes as shown by size exclusion chromatography, as the ratio of lipid:drug becomes smaller at high fraction numbers (corresponding to smaller liposomes). A possible explanation for this behaviour could be the higher curvature of the lipid membrane, which may offer more space for drug molecules. Incubation of liposomal RS-93522 with fresh human serum at 37 ◦ C showed an almost complete recovery of the lipophilic drug in the albumin peak (Fig. 5). The authors write: “thus, upon exposure to serum, the drug moved rapidly

Fig. 4. RS-93522, a diydropyridine Ca2+ channel blocker (redrawn from Lidgate et al. (1988)).

Fig. 5. Elution profile obtained by gel chromatograpy of liposomally solubilized RS-93522, after 5 min of incubation in serum. (. . .) 14 C-lipid-marker (liposome distribution), (- - -) RS-93522 concentration (␮g/ml). The first peak represents liposomes, the second peak represents albumin particles in the serum.

into the same serum pool as it would if it were solubilized by the conventional formulation” (Lidgate et al., 1988). The authors also made an in vivo evaluation of their liposomal formulation and a formulation of the lipophilic substance, which employed ethanol/PEG 400. The half-life (0.65 h), volume of distribution and other relevant pharmacokinetic parameters in rats were not significantly different between the liposomal and the classical formulation. Zinc phthalocyanine (ZnPc) is an experimental photosensitizer, which is virtually insoluble in water. The drug can be incorporated in small liposomes, composed of POPC and DOPS (9:1, w/w), at a 100:1 phospholipid to ZnPc weight ratio (Isele et al., 1994). The monomeric state of the ZnPc in the liposomal membrane appeared essential for an efficient body and tumor distribution (Isele et al., 1995). In contrast to monomeric ZnPc, aggregated ZnPc was only eliminated from the body slowly and showed poor tumor loading compared to monomeric ZnPc. Rensen et al., (1994) studied the interaction of small unilamellar liposomes containing Zn-Pc with purified human plasma lipoproteins. The bulk of Zn-Pc was incorporated into HDL and LDL; very little 14 C-labelled POPC, the most abundant phospholipid in the formulation, was associated with lipoproteins. The enrichment of the lipoprotein fraction by the lipophilic photosensitizer is believed to be a key property for obtaining efficient tumor targeting and treatment. This is because these drug–lipoprotein complexes are internalized by tumor cells via low-density (LDL) receptors, which are upregulated in rapidly dividing tissues undergoing rapid growth or repair. Pre-association of such drugs with LDLs has been shown to result in better delivery to target tissues and improved efficacy in vivo (Allison et al., 1991, 1990). Benzoporphyrin (BPD-MA, Visudyne® , Verteporfin for injection) is a lipophilic photosensitizer intravenously administered in liposomes, for the photodynamic treatment of macular degeneration. The liposomes are composed of egg phosphatidylglycerol, dimyristoyl phosphatidylcholine,

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ascorbyl palmitate and butylated hydroxytoluene, with a drug/total phospholipid weight ratio of 1 to 7.5–15. The distribution of liposomal BPD-MA after incubation with human blood for varying time periods was analyzed to determine the partitioning behavior into various blood compartments. Additionally, the analysis enabled the physical association of the BPD-MA with liposomes to be compared with a DMSO solution of the drug (Desai et al., 2000). In general, it was shown that the drug equilibrated rapidly and to a greater extent with lipoproteins when administered with liposomes. By applying a fluorescence assay, the transfer out of the liposomes of the drug was found to be almost instantaneous (less than 2 min) (Chowdhary et al., 2003b). Gel electrophoresis showed that both the drug and phospholipid components were transferred to the lipoprotein fraction concurrently. As a result the lipid-based structures were readily destabilized in the presence of relatively low concentrations of plasma, and for that reason the authors conclude that liposomes of this lipid composition (DMPC, egg-PG) were highly unlikely to be found intact in the circulation following intravenous injection. The explanation of this concurrent transfer of drug and phospholipid by Chowdhary et al. (2003b) was that “as biological transporters of lipids in the circulation, it is perhaps inevitable that lipoproteins should readily and completely acquire phospholipids following injection”. This process is greatly accelerated by the presence of phospholipid transferases in the circulation. The majority of interactions are likely to be with HDL, which can acquire up to 25% its own weight in lipids without displaying an increase in particle size, and reaching saturation at 0.25 mg phosphatidylcholine/mg (Lasic, 1993). Blood concentrations of these lipoproteins are 10 mg/ml, so it is not surprising that intact liposomes or other lipid phases were not detectable in the presence of plasma concentrations, even at excessively high lipid-based BPD-MA concentrations. The correlation of delivery of benzoporphyrin (BPD-MA) and its derivatives in lipid-based, Pluronic® P123 and solvent based formulations to lipoproteins and the efficacy in tumor and arthritis mouse models has been described (Chowdhary et al., 2003a). It was found that the formulations in which the drug was in a monomeric form were better able to transfer drug to lipoproteins, which in turn led to superior photodynamic therapy in vivo. After density centrifugation of animal plasma samples, the solvent formulation with pure DMSO showed a significant increase of aggregated and sedimented lipophilic photosensitizer. There was also a corresponding reduction in the amount of photosensitizer present in the lipoprotein fraction. These findings were consistent for all of the benzoporphyrin derivatives studied. These examples clearly show that lipophilic drugs can be reversibly bound to liposomes and that the binding can be highly dynamic. Depending on the size of the liposomes and lipid composition and the properties of the drug, the spontaneous transfer half-life may be in the range from milliseconds to several days. The transfer target may be lipoproteins and/or

albumin, dependent on the affinity of the drug for these blood components. For tumor treatment transfer of the anti-tumor drug from formulations to specific lipoprotein classes may be desired to obtain optimal efficacy. Care should be taken to formulate the drug in liposomal formulations in such a way that the drug is present in the monomeric state. At high intravenous liposomal doses, the total liposomal lipid pool may compete with the acceptor lipid pool and the fraction of liposomal drug able to transfer to, e.g. lipoproteins is reduced. Drug transfer from liposomes, either in vitro or in vivo to lipoproteins may be accompanied by co-migration of phospholipids, because of the high affinity of the lipoproteins for phospholipids. For these reasons, solvent formulations of a lipophilic drug, free from phospholipids, may direct the drug to other lipoproteins than compared to drug in a liposomal formulation.

4. Models for membrane-partitioning/transfer of lipophilic drugs Liposomes offer several possibilities to entrap drugs, either within the membrane-enclosed aqueous compartment or by direct association with the lipid bilayer. The latter case requires a favourable drug-membrane association free energy, which can be provided by various interactions. A proper understanding of these interactions and their implications are a prerequisite not only for drug immobilization, but also for the controlled drug release after parenteral administration of a liposome formulation. Electrostatic interactions of cationic, water soluble drugs with charged lipid head groups lead to binding onto the membrane surface without a notable structural perturbation of the lipophilic domain of the membrane. In contrast, hydrophobic interactions induce the penetration of the drug into the hydrocarbon core of the membrane. This case is of particular pharmaceutical importance, because it provides an efficient way to administer highly lipophilic drugs through liposomal/phospholipid based formulations. 4.1. Membrane-partitioning of lipophilic drugs Liposomes have been and are being used to study the inter-membrane transfer rates of natural membrane components like cholesterol (Bar et al., 1986, 1987; Fugler et al., 1985; Jezek et al., 1997; Lund-Katz et al., 1988; McLean and Phillips, 1981; Nemecz et al., 1988), fatty acids (Hamilton and Cistola, 1986; Kamp et al., 1993, 1995; Kleinfeld and Storch, 1993; Kleinfeld et al., 1997a,b; Pohl et al., 2000; Storch and Kleinfeld, 1986; Zhang et al., 1996) and phospholipids (Bai and Pagano, 1997; McLean and Phillips, 1981, 1984). Most lipophilic drugs do not have a structural similarity with phospholipids and do not orientate in a bilayer configuration like cylindrically shaped phospholipids. However, the models describing the spontaneous transfer from membrane to membrane of these natural membrane components need

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to be considered to understand the drug migration between liposomes. In the case of compounds, which show structural similarities with natural membrane components, like, e.g. steroidal drugs and cholesterol, the proposed mechanisms of transfer for cholesterol may apply. The transfer process of natural membrane lipids in aqueous liposomal dispersions (when passage through the water phase between membranes is the main transfer route) can be divided into the following sequential steps: (1) flip-flop movement of the membrane component from the inner to the outer leaflet (monolayer) of the donor membrane (spontaneous and/or catalysed by proteins), (2) departure of the membrane component from the membrane into the aqueous phase, (3) association of the membrane component in the aqueous phase with the acceptor membrane, followed by (4) flip flop to the inner membrane leaflet. Lipophilic drugs, which do not have membrane lipid like structures, probably are not subject to flip-flop. The transfer steps are therefore: (1) drug dissolved in the lipid domain of the membrane, (2) departure of the drug from the membrane into the aqueous phase, (3) association of the drug component in the aqueous phase with the acceptor membrane, followed by (4) dissolving of the drug in the acceptor membrane. As explained below these steps may differ at high phospholipid concentrations at which it is believed that collision between the lipid vesicles is the main transferring mechanism. Given that the lipophilicity of a drug is a measure of its ability to intrude into the hydrophobic region of the lipid membrane, one consequently expects a highly lipophilic drug to be buried deeply into the hydrocarbon core of the host membrane. At this fully membrane-inserted state, a drug is supposed to be highly immobilized with respect to leaving the membrane and exchanging with the aqueous environment (step 2, above). In other words, there should be a substantial energetic barrier for a membrane-buried drug to partition into the water phase compared to the other transfer steps described above. These equilibrium considerations are, however, not at all correlated with the experimentally observed kinetics of the transfer process. For example, despite its high lipophilicity cyclosporin A (CyA) exhibits remarkably high exchange rates between different lipid layers (Fahr and Seelig, 2001) (see Fig. 3) and paclitaxel (A. Fahr et al., unpublished data) behaves in a similar manner. When the passage through the water phase between membranes is the main transfer route, the question arises what energetic driving force could be behind the apparently fast transfer of highly lipophilic drugs through the water phase? 4.2. Thermodynamic considerations The lipophilicity of a drug determines the partition equilibrium between an aqueous and oily phase. The more lipophilic the drug is, the further the partition equilibrium is shifted to the oily phase. The oily phase can be an alkane phase or – in order to better represent the amphipathic nature of a lipid

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bilayer – consist of an alcohol such as octanol. Regarding its lipophilicity, the lipid chain region of a lipid bilayer is comparable to an oily phase. There is, however, a significant difference: the structural properties of the oil phase are uniform, isotropic and homogeneous. In contrast, a lipid bilayer is a thin, self-assembled, film with strongly inhomogeneous properties. The lipid chains within a membrane – despite being in the fluid-like state – are orientated in an ordered manner and thus render the bilayer anisotropic. The packing properties of the hydrocarbon chains in a lipid bilayer are significantly different from those in an alkane phase. The free energy of the transfer process related to this difference in packing properties is described in Eq. (1): F = Fsol + Flip

(1)

F is the difference of the free energy of a drug when it transfers from the water phase into the lipid bilayer or with the opposite sign for F, when it transfers from lipid layer to the water phase. The equation is composed of two major contributions (for a discussion of other relevant contributions see Ben-Tal et al. (1996) and for a detailed discussion of the so-called cratic entropy see Chan and Dill (1997)). The first contribution is the solvation free energy, Fsol , which accounts for changes in electrostatic and hydrophobic interactions of membrane-drug associations and constitutes the classical hydrophobic effect. The second contribution, Flip , is characteristic for a lipid bilayer and arises from the drug-induced structural membrane perturbation. The presence of the contribution Flip is a consequence of the abovementioned anisotropic orientation of the lipid chains within the lipid bilayer. Based on these theoretical thermodynamic considerations, in spite of high solubility in an (isotropic) oily phase, it may be speculated that drugs may be repelled/excluded from a (anisotropic) lipid bilayer, because of packing defects caused by the incorporation of the drug into the fatty acid chain region. The magnitude of this tendency is governed by the individual properties of the drug molecule such as its size, shape, orientation and hydrophobic moment. As a result, an equilibrium exists between the drug dissolved in the membrane and drug dissolved (to a much lower extent) in the water phase. Upon addition of an acceptor membrane, a flux of the drug, through the water phase, from the donor to the acceptor membrane will be initiated. Alternatively, when the transfer occurs through collision of vesicles (see below), the drug moves directly along its concentration gradient from the donor lipid domain to the acceptor lipid domain (possibly through an aqueous boundary layer) at the moment of collision. We shall illustrate the influence of the characteristic packing properties of the hydrocarbon chains in a lipid bilayer on the location and orientation of a drug in a membrane using a simple generic model. In this model we represent a rigid, bulky, entirely lipophilic drug as a stiff cylinder. We choose the cylinder length, L ≈ 1.6 nm, to correspond roughly to the

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Fig. 6. Schematic illustration of two different hypothetical model drug-membrane association states. The model drug (black regions) is represented as a rigid ˚ and length L ∼ ˚ associated with a lipid membrane (some lipids are displayed schematically) of equilibrium impenetrable cylinder of radius R ∼ =6A = 16 A, ˚ of its hydrophobic core. In (A) the cylinder is fully buried into the membrane, with its long axis parallel to the membrane midplane. Case (B) thickness D ∼ = 30 A shows the cylinder orientation in normal direction to the membrane. The cylinder is inserted into only one monolayer, inducing a weak structural perturbation of this monolayer and leaving the opposite monolayer essentially unperturbed.

length of a lipid tail, and its radius, R ≈ 0.6 nm, to account for the volume of a typical drug. CyA is an example whose spatial dimensions roughly fit with our choice of L and R (Fahr and Reiter, 1999). In the following example, it is argued that our simple lipophilic model drug is interacting with the membrane in a way that does not agree with the simplified assumption of the drug being buried deeply within the membrane hydrocarbon core and being safely shielded by the lipid headgroups from any contact with the aqueous phase. Let us compare two different drug-membrane association states: consider first the long axis of the cylindrical model drug being parallel to the bilayer midplane as illustrated schematically in Fig. 6A. For this drug orientation, we expect the solvation free energy, Fsol to be strongly negative because after transfer from water into the hydrocarbon core of the membrane, no part of the drug is exposed to the polar aqueous milieu. However, the presence of the drug induces a substantial structural membrane perturbation. This perturbation results from the rigidity of the cylinder, which prevents the penetration of the lipid hydrocarbon chains into the drug interior and thus restricts the number of potentially accessible chain conformations. In order to get an impression of the corresponding cost of free energy, we refer to the results of a recently presented statistical-thermodynamic mean-field theory of chain packing (Zemel et al., 2004) which resulted in ˚ Flip /L = 3.2 kJ/(mol A). ˚ the perturbation free With the cylinder length, L = 16 A, energy of the membrane amounts to Flip ≈ 51.2 kJ/mol. In contrast to this, the same chain packing theory predicts Flip = 12.4 kJ/mol (Zemel et al., 2005), if the cylinder is oriented in parallel to the fatty acid chains of the lipid bilayer; see Fig. 6(B). The different magnitudes for Flip in (A) and (B) reflect a different loss of chain conformational freedom. The membrane-buried cylinder in (A) restricts mainly the terminal region of the hydrocarbon chains in both lipid monolayers. In the absence of the cylinder, this region is particularly “fluid” as indicated by measurements of the lipid chain order parameter (Lafleur et al., 1990). Inserting a rigid lipophilic inclusion (i.e. drug) within the hydrocarbon region

strongly decreases the number of chain conformations the lipids in the immediate vicinity of the drug can adopt and thus entails a large entropic penalty. Hence, Flip is large. In contrast to (A) the conformational restrictions in case (B) are much smaller. Here, the perturbation of the drug-containing lipid monolayer results mainly from weak motional limitations of a few lipid chains in the immediate vicinity of the drug molecule. The opposite lipid monolayer remains nearly unperturbed. The calculated value of Flip = 12.4 kJ/mol (see Fig. 6B) can be explained by considering a simple model which has recently been found to be qualitatively in agreement with mean-field chain packing calculations (May and Ben-Shaul, 2000). This so called “director model” is based on the representation of a single hydrocarbon chain by a rigid fluctuating director. To be more specific, a director is defined as the end-to-end vector of a hydrocarbon chain, pointing from the glycerol backbone to the terminal methyl segment. Different chain conformations correspond to different director orientations. Within the director model it is assumed that all possible director orientations within the hydrocarbon core occur with equal probability; yet, the director cannot penetrate into the model drug. Based on a simple statistical model (May and Ben-Shaul, 2000), one obtains the perturbation free energy induced by a monolayer spanning cylinder (see Fig. 6): Flip = (1 − ln 2)

πDR kB T a0

(2)

˚ 2 denotes the cross-sectional area of a single, Here, a0 = 30 A fluid-like lipid chain that resides in the membrane (the typical cross-sectional area per double-chained lipid in a lipid ˚ 2 ), kB is the Boltzmann constant and T bilayer is about 60 A ˚ is the absolute temperature. Inserting furthermore D = 30 A ˚ into Eq. (2) leads to Flip = 14.9 kJ/mol which and R = 6 A is close to the value of Flip = 12.4 kJ/mol, calculated on the basis of the chain packing theory. The director model suggests that Flip results mainly from the entropic loss due to conformational restrictions of the drug-facing lipid chains. Of course, interactions between the drug and the lipid head

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groups, which are not accounted for by the director model, may further affect Flip .

5. Discussion 5.1. Examples on lipid solubility and transfer tendency of lipophilic drugs The examples cited here show a rather clear picture: lipophilic drugs are not all necessarily “caught” irreversibly in the lipid bilayer of liposomal carrier systems: there is enough evidence, that most of the described (marketed) lipophilic drugs have a high solubility in the membrane and are associated with the lipid bilayer. This has been investigated, e.g. for cyclosporin A using deuterated lipids to analyse the position of the drug inside the bilayer (Fahr and Seelig, 2001; Schote et al., 2002) and for amphotericin B, which could be incorporated up to 5 mol% in several types of liposomes, with only modest disruptive effects as observed with ESR and freeze fracture electron microscopy (Juliano et al., 1987b). Also, as studied by 1 H NMR and fluorescence techniques, the antibiotic rifampicin is deeply buried in the liposomal membrane but does not cause disruption of the membrane at the studied drug/lipid ratios (Rodrigues et al., 2003). Although these drugs (and others as well) are buried inside the liposomal membrane, they nevertheless seem to exchange often quite rapidly between artificial membranes or with other lipophilic binding places in vivo. 5.2. Transfer models The inter-membrane transfer phenomenon is first described as part of membrane biochemistry studies with liposomes as model membranes for biological membranes. In the past, two models to explain the transfer between two lipid domains (either inter-membrane transfer or transfer from membrane to, e.g. plasma components) of lipophilic membrane components were hypothesized. One model proposes a collision mechanism for, e.g. phosphatidylcholine (Jones and Thompson, 1989) and cholesterol transfer (Steck et al., 1988). The other model proposes transfer through the water phase as demonstrated by cholesterol transfer (Lange et al., 1983; McLean and Phillips, 1981) and phosphatidylcholine transfer studies (McLean and Phillips, 1981). Others claim that both mechanisms may simultaneously play a role, as demonstrated by the transfer of monoacylglycerols from SUV’s to brush border membrane vesicles (Schulthess et al., 1994). The mathematical equations describing a “First Order Model” (for lipid transfer between vesicles through the aqueous phase via desorption from the bilayer) and a “Second Order Model” (for transfer upon collision of donor and acceptor vesicles in addition to transfer to the aqueous phase) can be found in Jones and Thompson (1989).

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Which mechanism plays a role under in vitro conditions is largely dependent on the in vitro transfer assay conditions, like phospholipid concentration (Jones and Thompson, 1989), pH and membrane configuration and hydrophobicity/lipophilicity of the membrane component, which is subject of the transfer study (Yang and Huestis, 1993). Monomer transfer through the water phase predominates for less hydrophobic lipids at all values of pH and membrane concentration, and for more hydrophobic lipids at very high membrane dilutions. Transient collision transfer contributes significantly to the rate for relatively hydrophobic lipids in concentrated donor–acceptor systems. The size and surface configuration of donor and recipient membranes also alter the relative contributions of through-solution and collision transfer study (Yang and Huestis, 1993). In how far these in vitro mechanisms play a role in vivo has still to be discovered. The spontaneous transfer for phosphatidylcholine between liposomes, catalysed by non-specific lipid transfer proteins, seems also to occur both through collision and monomer diffusion through the water phase at the same time (Nichols, 1988). Lipophilic drugs may exchange between lipid domains in the same way as natural membrane components, i.e. by collision transfer or monomeric diffusion transfer. From the theoretical section, the calculation of the perturbation free energy Flip for the model drug shows that the lipid membrane provides a strong driving force towards the orientation of the cylinder in membrane normal direction. In fact, we estimated a difference in perturbation free energy of about 40 kJ/mol upon reorientation. As can be seen in Fig. 6(B), the normal orientation implies an additional exposure of the cylinder end-cap to the aqueous environment. Whether the lipid headgroups will be able to participate in shielding of the cylinder cap from the contact to water is likely to depend on molecular details of both the drug and the involved lipid headgroups. (We mention that a lipid headgroup reorientation has been observed experimentally in CyA-containing membranes (Schote et al., 2002).) However, even if such an additional shielding would be absent, we estimate that the solvation free energy Fsol = γπR2 ≈ 25 kJ/mol of the water-exposed hydrophobic end-cap (where γ ≈ 0.1, 40 mN/m measures the interfacial tension between water and oil) is still sufficiently small to allow the conformation in Fig. 6(B) to be adopted. This water-exposure orientation is conclusive for amphotericin B, which has to act as a membrane-spanning aggregate of several molecules (van Hoogevest and de Kruijff, 1978). Experimental evidence for this type of orientation was also shown for cyclosporin A (Schote et al., 2002). Note that the partial exposure of a lipophilic drug to the aqueous environment is expected to enhance both transfer mechanisms. That is, it furthers the exchange of drug molecules between membrane binding places via partitioning into the aqueous phase according to the partition coefficient. It also facilitates the direct exchange between donor and acceptor membranes through the collision mechanism.

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5.3. Relevance of membrane solubility and transfer for intravenous administration Solubilisation of lipophilic drugs by surfactants depends on the molar ratio of micellar surfactant to the solubilised drug (Almog et al., 1986). As the critical micellar concentration of phospholipids is very low (<10−9 M), solubilisation by liposomes is determined by the ratio of the total molar phospholipid concentration to drug concentration. The amount of drug bound seems to vary in a large extent according to early citations in the literature (Israelachvili et al., 1977; Lidgate et al., 1988). Our own studies indicate that about 20 phospholipid molecules are minimally necessary to host one lipophilic drug molecule. This holds for Foscan® , cyclosporin A, paclitaxel and other investigated substances in our laboratory (Fahr and Reiter, 1999; Wenk et al., 1996). Drug to phospholipid ratios from 1:20 to 1:1 up are theoretically certainly possible, especially when the drug has a membrane-component like structure. This may, however, be accompanied with a slower transfer tendency. When the drug is present in excess to phospholipid then the liposomal structure tends to be distorted and may convert at the extreme end to other lipid–drug complex structures than liposomes like, e.g. amorphous colloidal drug/detergent co-precipitates (List and Sucker, 1995). It can also be assumed that addition of cholesterol to liposomal membranes reduces the capacity of the liposomal membrane for hosting lipophilic drug molecules. This could be shown for CyA, were the partition coefficient is considerably reduced (Schote et al., 2002). A plausible explanation is the tendency of cholesterol to stiffen the fatty acid chains in the liposomal membrane, making it more difficult to host guest molecules. The nature of binding of lipophilic drugs to liposomal membranes does also influence the characteristics and the possibility to associate the lipophilic drug with the liposomal formulation. Paclitaxel (Taxol® ) exhibits large negative binding enthalpies, which indicates a binding reaction with release of heat being the driving force for paclitaxel partitioning into liposomal membranes (Wenk et al., 1996). Other physico-chemical characteristics of the association of drug to membranes may also play significant roles in formulation aspects. Binding of paclitaxel to liposomes is four times stronger at 20 ◦ C than at 37 ◦ C, which might affect the rapid release of paclitaxel at body temperature (Wenk et al., 1996). For cyclosporin A, the solubility in water increases with decreasing temperature, which might cause problems in stability-related issues of cyclosporin A liposomal formulations, as CyA can partition out of the liposomal membrane and form crystals in the suspending medium (Fahr, unpublished results). It is obvious from the conclusions so far, that liposomal formulations may be used as a non-toxic solubilizing agent for lipophilic drugs. An immediate release of the drug to blood components is desirable, if liposomes are intended to act as a solubilizing agent. If the drug is not released from

the liposomes sufficiently fast then the liposomal drug may be partially taken up together with the liposomes by cells of the MPS (i.e. macrophages). Based on the fast transferring properties of lipophilic drugs, an elegant two vial system comprising a transparent placebo liposome dispersions and a transfer medium with drug in water miscible solvent has been developed recently allowing the in situ preparation of drug loaded liposomes (Van Hoogevest et al., 2004). If the drug has fast transferring properties, the drug leaves the liposome immediately after intravenous administration to bind with other lipid domains in the blood. If liposomes without cholesterol are used, the liposomes immediately disintegrate after intravenous administration (Scherphof et al., 1978) and this further stimulates the transfer of the drug to other lipid domains. If it is desirable that the drug is preferentially distributed to particular body targets, for example, in tumours, a delayed release pattern would be helpful. In the range of the described drugs, this could be supposed for the photosensitizer temoporfin used in Foscan® (Huang et al., 1992). Considering the release time in the hours range, liposomal temoporfin could be passively targeted to tumour tissue in a time range of hours, after that the cargo is released in the vicinity of the target. The improved tolerability of paclitaxel (Cabanes et al., 1998) and cyclosporin A (Fahr and Seelig, 2001) in liposomes can be explained as follows. At the beginning of the injection procedure the concentration of drug and liposome is quite high. The drug may be bound in this early stage to the vast amount of liposomal lipid in the blood, and is therefore not able to interact with other sites in a toxic manner. With time, after distribution of the liposomes to the entire body, the concentration of drug in the blood falls below an acute toxic level. This understanding of transfer kinetics of lipophilic drugs implies that experimental liposomal dosage forms for fast transferring drugs may be inter-exchangeable with other solubilising formulations principles during the development from the pre-clinical stage to the clinical stage. This interexchangeability of oil-in-water emulsions, liposomes and solutions has been recently demonstrated for the experimental lipophilic drug KW-3902, which is an adenosine A1-receptor antagonist. Formulations comprising a lipid emulsion about 130 nm in diameter composed of egg yolk lecithin:soybean oil:oleic acid = 1:1:0.048, a liposome with about 100 nm in diameter composed of egg yolk lecithin, and a saline solution containing 1% (v/v) each of dimethyl sulfoxide and 1N NaOH showed the same i.v. pharmacokinetic parameters of KW-3902 and its metabolite (M1) in rats (Hosokawa et al., 2002). From a historical perspective it should be noted that diazepam is being used in three types of intravenous formulations, organic solvent mixture, oil-inwater emulsions and mixed micelles showing similar efficacy. This strongly suggests that diazepam must be a fast transferring drug. It is very well possible that fast transferring drugs will show shorter alpha elimination kinetics compared to slower transferring drugs.

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However, when a lipophilic drug with slow transferring properties (as determined by applying a transfer assay) is formulated with liposomes, these formulations are not interexchangeable with other solubilising formulation types. Due to the longer circulation time period, the probability that the drug co-transfers with lipids or is taking up by macrophages increases. On the other hand, the chance that passive localization at, e.g. tumor sites takes place will increase as well. From a development perspective, the preclinical liposomal dosage form has to be the same as the clinical dosage form. In any case, the assessment of inter-membrane transfer properties yields valuable information on the use of liposomes as either as solubilisers or as targeting devices for the lipophilic drug. In addition, the transferring properties may be predictive to some extent for the distribution and retention kinetics of drugs in the biomembranes after parenteral administration. 5.4. Relevance of membrane solubility and transfer for oral administration The solubility of lipid drugs in biological membranes determines directly the degree of mass transport through a membrane (Collander, 1949; Overton, 1899) and from a formulation perspective determines how much drug can be maximally incorporated in a phospholipid based dosage form. In the following, the correlation between passive mass transport of a lipophilic drug through a membrane and the membrane solubility of the drug is explained. In Eq. (3), the mass transport through any biomembrane is described (Lennern¨as, 1995). The transport is composed of a paracellular component and a transcellular component. Lipophilic drugs use the transcellular route, whereas (low molecular weight) hydrophilic drugs use the paracellular route (Lennern¨as, 1995). Eq. (3) can be transformed into Eq. (4), by taking into consideration the passive transcellular diffusion and the active transcelluar diffusion caused by transporters in the apical membrane. The ± sign indicates that transporters could act in two directions. In this equation, K is the membrane-aqueous partition coefficient of the lipophilic compound, Am the surface area of permeation, λ the thickness of the membrane, Dm the diffusion coefficient and Clumen is the drug concentration in the lumen. When a lipophilic drug is only passively absorbed, then the active transcellular and the paracellular components can be eliminated in Eq. (4). The partition coefficient K, being the ratio of the drug concentration in the membrane and the lumen: Cmembrane /Clumen , can be eliminated as well. The resulting Eq. (5) then describes the passive transcellular transport component and shows that dM/dt is directly proportional to Cmembrane (Cm ).
  

dM dM dM = + (3) dt total dt transcellular dt paracellular







dM dt

dM dt

 total

  Dm = Am × K × × Clumen passive λ   Jmax transcellular ± active Km + Clumen 
 dM transcellular + dt paracellular 

 total

Am × Dm = λ × Cm

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(4)

 (5) transcellular

For formulation scientists working with liposomes, one of the first activities to characterise an envisaged liposomal dosage form for a lipophilic drug is to measure the incorporation degree in liposomes (i.e. degree of solubility of the drug in the membrane). However, as part of predicting oral bioavailability, this determination of the solubility of the drug in the membrane, is not being performed. One of the reasons may be that the Biopharmaceutical Classification System (BCS) (Amidon et al., 1995) is based on the water solubility of the drug substance and is not focussing on the lipid solubility as criterion. In addition, the BCS also only considers the characteristics of drug substances on their own and does not consider the properties of formulated drugs. Lipidic delivery system like microemulsions (Porter and Charman, 2001) and mononacyl/diacyllipid based oral dosage forms as proposed by Leigh et al. (2001), increase the apparent solubility of the lipophilic drugs in the lumen and the lipophilic drug shifts from the low solubility class to the high solubility class of the BCS (Porter and Charman, 2001). It should be realised that log P values, often used to characterize drugs, does not replace Cm in predictive value, since log P values are determined from saturated aqueous solutions of lipophilic drugs and measurement of the concentration of the drug in the octanol phase. In this way no information on the (maximum) solubility in the lipid phase is obtained. Considering the above, we conclude that the knowledge on the solubility of lipophilic drug in artificial liposomal- or natural membranes is as valuable as the knowledge of the water solubility of water soluble drugs. Inter-membrane transfer of lipophilic drugs plays also a role after oral administration of such drugs. This becomes immediately apparent by the observations that colloidal mixtures containing bile salts, phosphatidylcholine, (medium and long chain) mono-glycerides and fatty acids mimicking the gastrointestinal content after digestion of formulation derived lipids, showed an increasing solubilisation proportion of solubilising capacity of the vesicular (i.e. liposomal) fraction for increasingly lipophilic drugs (Kossena et al., 2003). The transfer kinetics of lipophilic drugs from such lipid structures to the epithelial membrane and the solubility of the drug in such vesicles may play a role in the over all absorption kinetics. Also the transfer capability of the drug from the formulation to the epithelial membrane may play a significant role

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Fig. 7. Schematic illustration of the two penetration routes of (low MW) hydrophilic and lipophilic drugs through the GI tract epithelium and corresponding membrane permeation and transfer steps.

in the dissolution process of, for example, solid dispersions of a lipophilic drug. In a recent paper (Verreck et al., 2004), it is suggested that the lipophilic drug forms supersaturated solutions at the site of dissolution of solid dispersions in the GI tract. We, however, think that such a supersaturation is not always needed to achieve high absorption, because if the drug has fast transferring properties, an extremely low concentration of the drug in water could be already sufficient to allow a proper interaction with the receiving epithelial membrane. After dissolution of, for example, a solid dispersion or sequestration of a lipid based formulation (micro-emulsions or phospholipid complexes) in the GI tract lumen, the lipophilic drug will be absorbed along the transcellular route, whereas the paracellular route is reserved for water soluble drugs. The transcellular route is mainly described in the literature schematically as a rough arrow piercing though an epithelial cell monolayer. This indicates that the details of this passage route are not very well understood in detail. We assume that after transfer of the drug from the dosage form, the drug will find its way further through the epithelium by sequential steps of transfer through the water phase to other lipid domains and continued permeation through membranes along the concentration gradient of the drug (Fig. 7). In other words, lipid based formulations may act as a reservoir of lipophilic drug in which the drug is present at high concentrations. From this reservoir the drug transfers into the apical membrane; it is then transferred through the cytosol diffusing from one lipid domain (e.g. cell organelles, proteins) to another, along its gradient, so reaching the basolateral membrane. After passage through the basolateral membrane, the drug transfers into the interstitial fluid, and diffuses again from lipid domain to lipid domain reaching the vascular epithelial membrane. After passage through this membrane the drug transfers to

lipid domains in the blood (red blood cells, lipoproteins, albumin, etc.). These steps can be divided into membrane passage, membrane desorption and membrane absorption steps. It is obvious that because of the lipophilicity of the drug, the desorption step and entering into the hostile aqueous environment present the highest energy barrier and might be rate limiting to the overall cascade of the epithelial passage avenue. Since this desorption step is an essential step in the inter-membrane transfer assay, measurement of its kinetics may then be predictive for absorption kinetics of lipophilic drugs. Lipophilic drugs, when properly formulated (i.e. the drug should be present in a form which allows an efficient transfer, e.g. mono-molecularly dispersed in a solid dispersion with polymers or phospholipids, amorphous precipitates, micro-emulsions), may therefore show a short tmax after oral administration. In that respect, it could be concluded that for (formulated) water soluble drugs the dissolution rate in water is of importance, whereas for (formulated) lipophilic drug the transfer process is the crucial property to predict the performance of the formulation. Future work studying the correlation between formulation dissolution/transfer characteristics, in vitro inter-membrane transfer kinetics and in vivo absorption kinetics is needed to understand these complex matters.

6. Outlook In summary, phospholipid based dosage forms could be useful solubilisation carrier systems for a large class of lipophilic drugs in need of oral and parenteral administration. These dosage forms should be investigated for their inter-membrane transfer properties of the entrapped drug. Fast transferring lipophilic drugs could be administered in an intravenous liposomal form, which possesses the same

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pharmacokinetics as conventional dosage forms, e.g. organic solvent solutions. However, in contrast, drugs with a high lipophilicity that have a molecular structure like phospholipid molecules are likely to behave differently when associated with liposomes. This is because this type of drug will be encapsulated and bound to the liposomal membrane as part of the bilayer (Awiszus and Stark, 1988) and will have a similar fate as the carrying liposomes. Finally, it should be realised that phospholipid-based formulation work, including liposomes for intravenous use and lipid drug associates for oral use (Leigh et al., 2001), can simultaneously provide the pharmaceutical researcher with information on the interaction of the drug with biological membranes. Understanding this interaction may have a predictive value for the in vivo absorption and distribution of the drug. This advantage should stimulate the use of this type of colloidal carrier, either as a delivery system and/or analytical tool in the future. The in vitro assessment of transfer properties and lipid solubility may be useful predictive parameters for the in vivo pharmacokinetics of drugs. By determining the transfer and lipid solubility characteristics of marketed drug products it is apparent to note that most of them have fast transferring properties and high lipid solubility. More research is, however, needed to validate this finding by, e.g. comparison of the pharmacokinetics of fast transferring drug, with slow transferring drugs.

Acknowledgement Sylvio May thanks ND EPSCoR through NSF grant #EPS0132289.

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