Permeability PAMPA

Many organizations use colon adenocarcinoma (Caco-2) for detailed study of permeability however, this method can be resource intensive. Parallel artificial-membrane permeability (PAMPA) [19] has proven to be a reliable predictor of passive transcellular permeability for intestinal absorption prediction. It is also useful to interpret results of cell-based discovery assays, in which cell-membrane permeability is limiting. Finally, pTf provides insight into the pH dependence of solubility and permeability. It can be measured [20] or calculated to get an understanding of the regions of the intestine in which the compound will be best absorbed, as well as to anticipate the effect of pH on solubility and pemieability. Permeability at the blood-brain barrier (BBB) also can be rapidly profiled [21]. 

The experimental Pjpp data have been used to build predictive models. However, since PAMPA is already a model, an in silica model based on this is a model of a model. The predictability for in vivo permeability or absorption of such in silica PAMPA model can be queshoned (see Eq. 11), since it is two steps from reality ... 

The evaluation of the apparent ionization constants (i) can indicate in partition experiments the extent to which a charged form of the drug partitions into the octanol or liposome bilayer domains, (ii) can indicate in solubility measurements, the presence of aggregates in saturated solutions and whether the aggregates are ionized or neutral and the extent to which salts of dmgs form, and (iii) can indicate in permeability measurements, whether the aqueous boundary layer adjacent to the membrane barrier, Umits the transport of drugs across artificial phospholipid membranes [parallel artificial membrane permeation assay (PAMPA)] or across monolayers of cultured cells [Caco-2, Madin-Darby canine kidney (MDCK), etc.]. 

Fig. 3.4 Permeability profiles for (a) warfarin (acid), (b) propranolol (base) and (c) morphine (ampholyte) based on a BBB PAMPA model (plON) composed of animal brain extract of lipids. The data (unpublished) were analyzed with the pCEL-X program (plON), with the refined parameters indicated in the three frames. In all three cases, there was evidence for the permeation of charged...

In PAMPA, the effective permeability coefficient, Pe, is related to the membrane and ABL permeability coefficients, P and Pabl. respectively, as... 

Avdeef A. HT solubility and permeability MAD-PAMPA analysis. In Pharmacokinetic Profiling in Drug Research, Testa, B., Kramer, S. D., Wunderli-Allenspach, H., Eolkers, G. 

PAMPA is typically used to make a prediction of the passive, transcellular absorption of a compound. Compounds which may be absorbed by a paracellular mechanism or may be substrates for active transport (uptake or efflux) are usually better assessed in a cell based system. A combination of assays can be applied to gain a greater understanding of the permeability and transport properties of a compound. 

Figure 8 shows PAMPA data for a subset of compounds from the two series in Fig. 7. The potential concern about low permeability for Series D is confirmed between the MDCK and PAMPA data. Series C, with the exception of one compound, also demonstrates good correlation between MDCK and PAMPA permeability the compound with low PAMPA permeability should be further analyzed for relevant structure-permeability information.

BBB PAMPA Blood brain barrier parallel artificial membrane permeability assay... 

During the characterization process, hits are typically tested for kinetic solubility and permeability in a model of passive diffusion such as PAMPA [22]. As new compounds are synthesized, additional parameters also need to be considered, such as pZa, chemical and plasma stability, and protein binding. Calculated properties such as MW, clogP, and PSA should also be tracked. 

The coverage of permeability in this book is more comprehensive than that of solubility, lipophilicity, and ionization. This decision was made because permeability is not as thoroughly treated in the pharmaceutical literature as the other topics, and also because much emphasis is placed on the PAMPA in this book, which is indeed a very new technique [547] in need of elaboration. 

PARALLEL ARTIFICIAL-MEMBRANE PERMEABILITY ASSAY (PAMPA)... 

Figure 7.11 Intrinsic permeabilities versus alkane-water partition coefficients for drugs PAMPA filters soaked with alkane [509].

Permeability-Retention-Gradient-Sink PAMPA Models (plON Models)... 

Figure 7.12 Chugai model PAMPA permeabilities as a function of pH for several drug...

These general observations have been confirmed in PAMPA measurements in our laboratory, using the 2% DOPC-dodecane lipid. With very lipophilic molecules, glycocholic acid added to the donor solution slightly reduced permeabilities, taurocholic acid increased permeabilities, but SLS arrested membrane transport altogether in several cases (especially cationic, surface-active drugs such as CPZ). 

In PAMPA measurements each well is usually a one-point-in-time (single-timepoint) sample. By contrast, in the conventional multitimepoint Caco-2 assay, the acceptor solution is frequently replaced with fresh buffer solution so that the solution in contact with the membrane contains no more than a few percent of the total sample concentration at any time. This condition can be called a physically maintained sink. Under pseudo-steady state (when a practically linear solute concentration gradient is established in the membrane phase see Chapter 2), lipophilic molecules will distribute into the cell monolayer in accordance with the effective membrane-buffer partition coefficient, even when the acceptor solution contains nearly zero sample concentration (due to the physical sink). If the physical sink is maintained indefinitely, then eventually, all of the sample will be depleted from both the donor and membrane compartments, as the flux approaches zero (Chapter 2). In conventional Caco-2 data analysis, a very simple equation [Eq. (7.10) or (7.11)] is used to calculate the permeability coefficient. But when combinatorial (i.e., lipophilic) compounds are screened, this equation is often invalid, since a considerable portion of the molecules partitions into the membrane phase during the multitimepoint measurements. 

Figure 7.22b shows that hydrophilic molecules, those with log Kj < 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 hydrophilic 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. 

Figure 7.35 shows the characteristic log Pe-pH curve for a weak base, phenazo-pyridine (pKa 5.15). With bases, the maximum permeability is realized at high pH values. As in Fig. 7.34, the PAMPA assays were performed under iso-pH conditions (same pH in donor and acceptor wells), using the 2% DOPC in dodecane lipid... 

The above iso-pH measurements are based on the 2% DOPC/dodecane system (model 1.0 over pH 3-10 range). Another membrane model was also explored by us. Table 7.16 lists iso-pH effective permeability measurements using the soy lecithin (20% wt/vol in dodecane) membrane PAMPA (models 17.1, 24.1, and 25.1) The negative membrane charge, the multicomponent phospholipid mixture, and the acceptor sink condition (Table 7.1) result in different intrinsic permeabilities for the probe molecules. Figure 7.40 shows the relationship between the 2% DOPC and the 20% soy iso-pH PAMPA systems for ketoprofen. Since the intrinsic permeability of ketoprofen in the soy lecithin membrane is about 20 times greater than in DOPC membrane, the flat diffusion-limited transport region of the log Pe... 

Figure 7.40 Permeability-pH profiles for ketoprofen under iso-pH conditions for two different PAMPA models unfilled circles = 2% DOPC/dodecane, filled circles = 20% soy lecithin/dodecane. [Reprinted from Avdeef, A., in van de Waterbeemd, H. Lennemas, H. Artursson, P. (Eds.). Drug Bioavailability. Estimation of Solubility, Permeability, Absorption and Bioavailability. Wiley-VCH Weinheim, 2003 (in press), with permission from Wiley-VCH Verlag GmbH.]...

Figure 7.44 Collander relationship between intrinsic permeabilities of 20% soy lecithin versus 2% DOPC PAMPA models.

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