Effect Of Phospholipid

Figure 7.25 Membrane retention in 2%DOPC/dodecane-soaked filters versus dodecane-soaked filters.

Figure 7.25 Membrane retention in 2%DOPC/dodecane-soaked filters versus dodecane-soaked filters.

(0.08-0.12 mL). The choice rests on the presumed structure of the membrane lipids (and where the drug preferentially partitions), which is not absolutely certain at present (see Section 7.3.6). It may be best to treat r as an empirical parameter, determined by regression against some lipophilicity model.

Figure 7.25 is a plot of %R (2%DOPC in dodecane) versus %R (100% dode-cane). It shows that even 2% DOPC in dodecane can influence membrane retention to a considerable extent, compared to retentions observed in the absence of DOPC. Many molecules show retentions exceeding 70% in DOPC, under conditions where the retentions in dodecane are below 10%. However, it cannot be assumed that retention is always very low in dodecane, since several points in Fig. 7.25 are below the diagonal line, with values as high as 90% (chlorpromazine).

7.7.3 Two-Component Anionic Lipid Models with Sink Condition in the Acceptor Compartment

The use of simple single-component neutral lipids has played a valuable role in development of the PAMPA technique. Since it was an early objective of such work to predict GIT absorption, it became necessary to test the effect of phospho-lipid mixtures, where variable amounts of negative lipid could be introduced. Table 7.1 indicates that brush-border membrane (BBM) lipid mixture contains one negative phospholipid for every 3.5 zwitterionic lipids, and the blood-brain barrier (BBB) lipid has even a higher negative lipid content. The simplest model to simulate the BBM mixture could consist of two components: DOPC plus a negatively charged phospholipid: for example, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol, phosphatidic acid, or cardiolipin (see Fig. 7.4). Even a fatty acid, such as dodecylcarboxylic acid (DA), could play the role of introducing negative charge to the mixture. Our design criterion was to begin with 2% DOPC and add the additional negatively charged lipid in the proportion consistent with BBM (0.6% added lipid) or BBB (1.1% added lipid) negative-zwitterionic proportions (Table 7.1).

Since there would be increased overall lipid concentration in the dodecane solution, we decided to create a sink condition in the acceptor wells, to lower the membrane retention. We discovered that the pH 7.4 buffer saturated with sodium laurel sulfate serves as an excellent artificial sink-forming medium. Since the new PAMPA membranes would possess substantial negative charge, the negatively charged micellar system was not expected to act as an aggressive detergent to the two-component artificial membrane infused in the microfilter.

Six two-component models were tested under sink conditions (models 5.1-10.1 in Table 7.3), employing three negatively charged lipids (dodecylcarboxylic acid, phosphatidic acid, and phosphatidylglycerol). These models were also tested in the absence of the sink condition (models 5.0-10.0 in Table 7.3).

Tables 7.6-7.8 list the Pe, SD, and %R of the 32 probe molecules in the thirteen new PAMPA lipid models, one of which is 2% DOPC assayed under sink conditions (model 1.1). The latter model served as a benchmark for assessing the effects of negative membrane charge.

TABLE 7.6 Two-Component Anionic Lipid PAMPA Models (with Sink), pH 7.4a
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