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The more lipophilic molecules preferentially concentrate in the more lipophilic phase, leading to decreased permeabilities, according to the effect of the negative term in Eq. (7.44), as the concentration of solute in the lower-lipophilicity phase decreases. In the soy lecithin models, the lipid phases are systematically varied, with reference to a molecule of a particular lipophilicity. The plots in Figs. 7.31a-c are orthogonally equivalent to the Kubinyi model type plots (Fig. 7.19d), with each curve representing a particular molecule and the horizontal axis corresponding to varied lipid ratios. Eq. 7.44 applies and Figs. 7.31a-c may be interpreted as bilinear curves, for both sink and sinkless domains. For example, the maximum permeability for most molecules occurs at about 20% wt/vol lecithin in dodecane. For higher lecithin content, the negative term in Eq. (7.44) dominates, causing the Pe values to decrease. Sink Condition to Offset the Attenuation of Permeability

The preceding section treats the decrease in permeabilities with increasing lecithin content in dodecane in terms of shifting concentration distributions between a weak lipophilic domain (dodecane) and a stronger lipophilic domain (lecithin). Another view of this may be that at the molecular level, as the amount of phospholipid increases, the effects of electrostatic and H-bonding play a more prominent role in the transport process. Generally, %R of the lipophilic molecules increases with increasing lecithin content, most dramatically in the case of lipophilic bases. Such losses of compound to the membrane pose a challenge to the analysis of concentrations, which can be significantly diminished (to undetectable levels at times) in the aqueous compartments. At the same time, the permeability drops to near vanishing values in 68% soy lecithin-dodecane membranes. Under these conditions, the permeabilities of the lipophilic bases and acids converge to similar low values, significantly departing from the expected values based on the octanol-water lipo-philicity scale (Table 7.4) and the pH partition hypothesis. This excessive drug-membrane binding would not be expected under in vivo conditions in the small intestine, due to the naturally occurring sink state. There would be competing lipid environments in the receiving compartment (serum proteins, other membrane barriers, etc.), and the solute-binding membrane would release a portion of the retained lipophilic molecules, resulting in a concomitant higher effective permeability.

The transport properties of the molecules in concentrated soy lecithin, Tables 7.12-7.14, do not adequately model the in vivo permeabilities reported by Winiwarter et al. [56] (Table 7.4). The strategy to overcome this shortcoming of the model involves creating a model sink condition. However, the use of BSA or other serum proteins, although easily effected, is not practical in high-throughput screening, since the UV absorption due to the proteins would render determination of the compound concentrations in the acceptor compartments by direct UV spectropho-tometry nearly impossible in most cases. Without knowledge of the concentration of sample in the acceptor compartment, the determination of %R would not be practical. Some PAMPA practitioners, using BSA to create sink conditions, make the simplifying assumption that membrane retention is zero. It is neither reason able nor warranted to expect that membrane retention is eliminated in the presence of serum proteins or other practical substitutes in the acceptor compartment. Figures 7.32a-c clearly show that retention under sink can be substantial.

Since lipophilic molecules have affinity for both the membrane lipid and the serum proteins, membrane retention is expected to decrease, by the extent of the relative lipophilicities of the drug molecules in membrane lipid versus serum proteins, and by the relative amounts of the two competitive-binding phases [see Eqs. (7.41)-(7.43)]. Generally, the serum proteins cannot extract all of the sample molecules from the phospholipid membrane phase at equilibrium. Thus, to measure permeability under sink conditions, it is still necessary to characterize the extent of membrane retention. Generally, this has been sidestepped in the reported literature.

We found that the negatively charged surfactant, sodium laurel sulfate, can be successfully substituted for the serum proteins used previously. In low ionic strength solutions, the cmc of the surfactant is 8.1 mM [577]. We explored the use of both sub-CMC (data not shown) and micelle-level concentrations. Saturated micelle solutions are most often used at pION.

The addition of surfactant to the acceptor solution allows for the re-distribution of lipophilic permeants between the PAMPA membrane phase and the surfactant phase in the acceptor compartment, in the manner of Kubini's [23] analysis (Sec. 7.6), according to the relative lipophilicities of the two oil phases. This redistribution can be approximated. Garrone et al. [600] derived a Collander relationship for a series of substituted benzoic acids, relating their lipophilicities in 30-100 mM sodium laurel sulfate to the octanol-water system. The Collander equation comparing the drug partitioning in liposome-water to octanol-water systems (Fig. 5.6) can be combined with that of the above micellar relationship to get the approximate equation: log Kpm¿c = 1.4 log Kp,ijposome — 1.6. If it is assumed that the PAMPA membrane lipophilicity can be approximated by that of liposomes, then the strength of the surfactant-created acceptor sink can be compared to that of the PAMPA membrane, according to the latter expression. The most lipophilic molecules will favor the micellar phase when their liposome partition coefficients, log Kp,liposome, are greater than 4. (The micellar and PAMPA lipid volumes are nearly the same.) Positively charged drug molecules will favor additional binding to the negatively charged micelles, unless the PAMPA membrane lipid composition also has negative charge.

The effect of the surfactant is most dramatic for the bases and neutral molecules studied, as shown in Tables 7.13 and 7.14. Permeabilities increased by up to fourfold for the lipophilic bases and neutral molecules, and membrane retentions were decreased by 50% in most cases of bases and neutral compounds (Figs. 7.31 and 7.32).

The transport properties of the acids did not respond significantly to the presence of the sink. This may be because at pH 7.4 the acids are negatively charged, as are the phospholipid membranes and also the surfactant micelles; electrostatic repulsions balanced out the attractive forces due to increased membrane lipophilicity. Lowered surface pH may also play a balancing role [457]. Comparing Egg and Soy Lecithin Models

The negative-charge lipid content in the egg lecithins is not as high as that found in BBM and especially BBB lipids (Table 7.1). Furthermore, the negative-charge content in the egg lecithin is about one-fourth that in the soy lecithin. This is clearly evident in the membrane retention parameters for the bases at the 10% lecithin levels (models 12.0 or 14.0 in Table 7.8 vs. model 16.0 in Table 7.12), as they are ^20-30% lower for the lipophilic bases in egg, compared to soy.

For acids, the membrane retention actually increases in the case of egg lecithin, compared to soy lecithin. This may be due to decreased repulsions between the negatively charged sample and negatively charged phospholipid, allowing H-bond-ing and hydrophobic forces to more fully realize in the less negatively charged egg lecithin membranes. The neutral molecules display about the same transport properties in soy and egg lecithin, in line with the absence of direct electrostatic effects. These differences between egg and soy lecithins make soy lecithin the preferred basis for further model development. Titrating a Suspension of Soy Lecithin

Since soy lecithin (''20% extract'' from Avanti) was selected as a basis for absorption modeling, and since 37% of its content is unspecified, it is important to at least establish that there are no titratable substituents near physiological pH. Asymmetric triglycerides, the suspected unspecified components, are not expected to ionize. Suspensions of multilamellar vesicles of soy lecithin were prepared and titrated across the physiological pH range, in both directions. The versatile Bjerrum plots (Chapter 3) were used to display the titration data in Fig. 7.33. (Please note the extremely expanded scale for nH.) It is clear that there are no ionizable groups

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