Partitioning Into Liposomes

The octanol-water partition model has several limitations; notably, it is not very "biological." The alternative use of liposomes (which are vesicles with walls made of a phospholipid bilayer) has become more widespread [149,162,275, 380-444]. Also, liposomes contain the main ingredients found in all biological membranes.


Figure 5.1 shows a tetrad of equilibrium reactions related to the partitioning of a drug between an aqueous environment and that of the bilayer formed from phospholipids. (Only half of the bilayer is shown in Fig. 5.1.) By now, these reaction types might be quite familiar to the reader. The subscript "mem" designates the partitioning medium to be that of a vesicle formed from a phospholipid bilayer. Equations (4.1)-(4.4) apply. The pKmem in Fig. 5.1 refers to the "membrane" pKa. Its meaning is similar to that of pK<oct; when the concentrations of the uncharged and the charged species in the membrane phase are equal, the aqueous pH at that point defines pKmem, which is described for a weak base as

Absorption and Drug Development: Solubility, Permeability, and Charge State. By Alex Avdeef ISBN 0-471-423653. Copyright © 2003 John Wiley & Sons, Inc.

Figure 5.1 Phospholipid membrane-water tetrad equilibria. Only half of a bilayer is shown. [Avdeef, A., Curr. Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]

The salt dependence of constants discussed in Section 4.2 also applies to the pKmem and log PL constants. Although they are conditional, the dependence on ionic strength is subtle [433,442]. It is thought that when a charged drug migrates into the lipid environment of a liposome, the counterion that at first accompanies it may be exchanged with the zwitterionic phosphatidylcholine head groups, as suggested in Fig. 5.1. As the nature of the ion pair may be different with liposome partitioning, the term surface ion pair (SIP) is used to denote it. We use the term diffmem to designate the difference between the neutral species partitioning and the surface ion pair partitioning [see Eq. (4.6)].


There are no convenient databases for liposome log P values. Most measured quantities need to be ferreted from original publications [149,162,376,381-387,443]. The handbook edited by Cevc [380] is a comprehensive collection of properties of phospholipids, including extensive compilations of structural data from X-ray crystallographic studies. Lipid-type distributions in various biological membranes have been reported [380,388,433].


Based on the observed nuclear Overhauser effect in a 31P{1H} nuclear magnetic resonance (NMR) study of egg phosphatidylcholine (eggPC) bilayers, Yeagle et al. [399] concluded that the N-methyl hydrogens were in close proximity to phosphate oxygens in neighboring phospholipids, suggesting that the surface of the bilayer was a ''shell'' of interlocking (intermolecular) electrostatic associations. Added cholesterol bound below the polar head groups, and did not interact with them directly. However, its presence indirectly broke up some of the surface structure, making the surface more polar and open to hydration.

Boulanger et al. [420,421] studied the interactions of the local anesthetics procaine and tetracaine with eggPC multilamellar vesicles (MLV, 52-650 mM), as a function of pH, using deuterium nmr as a structural probe. They proposed a three-site model, similar to that in Fig. 5.1, except that the membrane-bound species (both charged and uncharged) had two different locations, one a weakly bound surface site (occupied at pH 5.5), and the other a strongly bound deeper site (occupied at pH 9.5). Membrane partition coefficients were estimated for both sites. Westman et al. [422] further elaborated the model by applying the Gouy-Chapman theory. When a charged drug partitions into the bilayer, a Cl— is likely bound to the surface, to maintain charge neutrality. They found unexpected low values of diffmem of 0.77 for tetracaine and 1.64 for procaine (see Section 4.7). Kelusky and Smith [423], also using deuterium NMR, proposed that at pH 5.5, there was an electrostatic bond formed between the protonated drug and the phosphate groups, (=P—O—. . . +H3N—), and a hydrogen bond formed between the aminobenzene proton and the acyl carbonyl oxygen. At pH 9.5, the ionic bond breaks as the secondary amine moves deeper into the interior of the bilayer; however, the amino-benzene H bond, (—CO. . . H2N—), continues to be an anchoring point.

Bauerle and Seelig [395] studied the structural aspects of amlodipine (weak base, primary amine pKa 9.26 [162]) and nimodipine (nonionizable) binding to phospholipid bilayers, using NMR, microcalorimetry, and zeta-potential measurements. They were able to see evidence of interactions of amlodipine with the cis double bond in the acyl chains. They saw no clear evidence for (=P—O— . . . +H3N—) electrostatic interactions.

Herbette and co-workers [425-428,445] studied the structures of drugs bound to liposomes using a low-angle X-ray diffraction technique. Although the structural details were coarse, it was apparent that different drugs position in different locations of the bilayer. For example, amlodipine is charged when it partitions into a bilayer at physiological pH; the aromatic dihydropyridine ring is buried in the vicinity of the carbonyl groups of the acyl chains, while the —NH3+ end points toward the aqueous phase, with the positive charge located near the phosphate negativecharge oxygen atoms [426-428]. A much more lipophilic molecule, amiodarone (weak base with pKa 9.1 [pION]), positioned itself closer to the center of the hydrocarbon interior [425].


Davis et al. [394] studied the thermodynamics of the partitioning process of substituted phenols and anisoles in octanol, cyclohexane, and dimyristoylphosphatidyl-choline (DMPC) at 22°C (which is below the gel-liquid transition temperature of DMPC). Table 5.1 shows the results for 4-methylphenol. The phenol partitioned into the lipid phases in the order DMPC > octanol > cyclohexane, as indicated by AGtj. Thus, the free energy of transfer into DMPC was greater than into octanol or cyclohexane. Partitioning was generally-entropy driven, but the components of the free energy of transfer were greatly different in the three lipid systems (Table 5.1). Octanol was the only lipid to have an exothermic heat of transfer (negative enthalpy), due to H-bond stabilization of the transferred solute, not found in cyclohexane. Although AHtI in the DMPC system is a high positive number (endothermic), not favoring partitioning into the lipid phase, the entropy increase (+114.1 eu) was even greater, more than enough to offset the enthalpy destabiliza-tion, to end up an entropy-driven process. The large AHtt and AStr terms in the DMPC system are due to the disruption of the ordered gel structure, found below the transition temperature.

The partition of lipophilic drugs into lipid phases is often believed to be entropy-driven, a hydrophobic effect. Bauerle and Seelig [395] studied the thermodynamics of amlodipine and nimodipine binding to phospholipid bilayers (above the transition temperature) using highly sensitive microcalorimetry. The partitioning of the drugs into the lipid bilayer was enthalpy-driven, with AHtt —38.5 kJ mol—1 bound amlodipine. The entropy of transfer is negative, contrary to the usual interpretation

TABLE 5.1 Energy of Transfer (kJ/mol) into Lipid Phase for 4-Methylphenol


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