The 2% DOPC in dodecane (model 1.0, Table 7.3) was the first PAMPA model explored by the pION group [25-28,556-558]. The lipid is commercially available in a highly purified preparation (in flame-sealed ampules packed under nitrogen), and is most like that used in the original BLM experiments [516,518,519, 523,532,542]. The lipid is completely charge neutral. It shows relatively low membrane retention for most molecules in Table 7.5, with the exception of chlorpromazine, phenazopyridine, primaquine, and progesterone. Our experience has been that as long as R < 90%, most drug molecules have sufficient UV absorptivity to be adequately characterized when the initial concentrations are —50 mm (a typical concentration in high-throughput applications). Lipid systems based on 10% or higher lecithin content can show very high membrane retention, in some cases preventing the assessment of permeability by UV spectro-photometry.

A few molecules have unexpectedly low permeability in 2% DOPC, not consistent with their octanol-water partition coefficients. Notably, metoprolol has a Pe value —10 times lower in 2% DOPC, compared to 10% egg lecithin. Also, prazosin Pe appears to be significantly lower in DOPC, compared to other lipids.

The quality of the data collected from 2% DOPC membranes is unmatched by any other system we have explored. It's not uncommon to see interplate reproducibility <5% and intraplate even better than that (1-3% SD). As will be seen later, lipid model 1.0 does not predict GIT absorption as well as some of the newer pION models. However, this may not be the case when BBB models are explored in detail.

Figure 7.23 Relative acceptor compartment concentrations versus octanol-water apparent partition coefficients [550]. [Reprinted from Kansy, M.; Fischer, H.; Kratzat, K.; Senner, F.; Wagner, B.; Parrilla, I., in Testa, B.; van de Waterbeemd, H.; Folkers, G.; Guy, R. (Eds.). Pharmacokinetic Optimization in Drug Research, Verlag Helvetica Chimica Acta: Zürich and Wiley-VCH, Weinheim, 2001, pp. 447-464, with permission from Verlag Helvetica Chimica Acta AG.]

Olive oil was the ''original'' model lipid for partition studies, and was used by Overton in his pioneering research [518,524]. It fell out of favor since the 1960s, over concerns about standardizing olive oil from different sources. At that time, octanol replaced olive oil as the standard for partition coefficient measurements. However, from time to time, literature articles on the use of olive oil appear. For example, Poulin et al. [264] were able to demonstrate that partition coefficients based on olive oil-water better predict the in vivo adipose-tissue distribution of drugs, compared to those from octanol-water. The correlation between in vivo log Kp (adipose tissue-plasma) and log Kp (olive oil-water) was 0.98 (r2), compared to 0.11 (r2) in the case of octanol. Adipose tissue is white fat, composed mostly of triglycerides. The improved predictive performance of olive oil may be due to its triglyceride content.

It was thus interesting for us to examine the permeability and membrane retention properties of olive oil. As Table 7.5 shows, most of the Pe values for olive oil are less than or equal to those of 2% DOPC, with notable exceptions; for instance, quinine is 4 times more permeable and progesterone is 16 times less permeable in olive oil than in DOPC. Both lipids show progesterone retention to be >80%, but quinine retention in olive oil is substantially greater than in DOPC. Octanol

Octanol permeability is important to explore, since it is the principal basis for the lipophilicity scale in pharmaceutical research. Most interesting to us, in this light, is to address the question of ion pair partitioning and its meaning in the prediction of absorption of charged drugs. It has been discussed in the literature that quaternary ammonium drugs, when matched with lipophilic anions, show considerably increased octanol-water partition coefficients [291]. It has been hypothesized that with the right counterion, even charged drugs could be partly absorbed in the GIT. Given the structure of wet octanol, it could be argued that the 25 mol% water in octanol may be an environment that can support highly charged species, if lipophi-lic counterions are added. Unexpectedly high partition coefficients can be measured for ion pair forming drugs. But does this mean that ion pair transport takes place in vivo? This was addressed by the pION group by comparing permeability coefficients derived from DOPC and octanol lipid membrane models. For molecules showing very low permeabilities in DOPC (model 1.0) and very high permeabilities in octanol-impregnated membranes (Model 3.0), one could hypothesize that the water clusters in wet octanol act like ''ion pair shuttles,'' an interesting effect, but perhaps with uncertain physiological interpretation [560].

Figure 7.22b shows that hydrophilic molecules, those with log < 1, are much more permeable in octanol than in olive oil. The same may be said in comparison to 2% DOPC and dodecane. Octanol appears to enhance the permeability of hydrophi-lic molecules, compared to that of DOPC, dodecane, and olive oil. This is dramatically evident in Fig. 7.7, and is confirmed in Figs. 7.8c and 7.22b. The mechanism is not precisely known, but it is reasonable to suspect a ''shuttle'' service may be provided by the water clusters in octanol-based PAMPA (perhaps like an inverted micelle equivalent of endocytosis). Thus, it appears that charged molecules can be substantially permeable in the octanol PAMPA. However, do charged molecules permeate phospholipid bilayers to any appreciable extent? We will return to this question later, and will cite evidence at least for a partial answer.

Membrane retention of lipophilic molecules is significantly increased in octanol, compared to 2% DOPC. Chlorpromazine and progesterone show R > 90% in octanol. Phenazopyridine, verapamil, promethazine, and imipramine show R > 70%. Dodecane

Dodecane-coated filters were studied to determine what role hydrogen-bonding and electrostatic effects play in the 2% DOPC system. Measuring the differences between Pe deduced from 2% DOPC in dodecane and 0% DOPC in dodecane might indicate the extent of H-bonding and/or electrostatic interactions for specific probe molecules. Table 7.5 indicates that some molecules are retarded by the presence of DOPC (e.g., phenazopyridine, verapamil, metoprolol, theophylline, terbutaline, antipyrine), while most molecules are accelerated by DOPC (e.g., chlorpromazine, imipramine, diltiazem, prazosin, progesterone). The quantitative structure-permeability relationships for a much larger set of drug-like molecules are currently investigated in our laboratory (see Section 7.7.8).

It is also quite interesting that lipid model 4.0 may be used to obtain alkane partition coefficients at high-throughput speeds, as suggested by Faller and Wohnsland [509,554]. It is also interesting to note that since our Pe are corrected for membrane retention, the slope in Fig. 7.11 corresponding to the dashed line (our data) is 1.0, whereas the data not corrected for retention (solid line) show a lesser slope. This may not matter if the objective is to obtain alkane-water log Kp values at high speeds.

7.7.2 Membrane Retention (under Iso-pH and in the Absence of Sink Condition)

The membrane retention R is often stated as a mole percentage of the sample lost to the membrane. Its value can at times be very high, as high as 85% for chlorproma-zine and 70% for phenazopyridine, with membranes made of 2% DOPC dissolved in dodecane. Regression analysis of log %R versus log Kd(74), the octanol-water apparent partition coefficient, produces r2 0.59. For DOPC-free dodecane, such analysis yields a higher r2 (0.67). Olive oil and octanol further improve, with r2 of 0.80 and 0.90, respectively. As far as %R representing lipophilicity as indicated by octanol-water partition coefficients is concerned, the order of ''octanol-like-ness'' is octanol > olive oil > dodecane > DOPC in dodecane. Figure 7.24 shows the log %R/log Kd plot for octanol-impregnated membranes, at pH 7.4. It's clear that retention is due to the lipophilicity of molecules.

Culture-cell assays are also subject to sample retention by the monolayer. Sawada et al. [574] studied the transport of chlorpromazine across MDCK cell

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