Permeability-pH profiles

Wohnsland, F., Faller, B. High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J. Med. 

Permeability-pH profiles, log Pe - pH curves in arhficial membrane models (log Pjpp - pH in cehular models), generally have sigmoidal shape, similar to that of log Dod - pH cf. Fig. 3.1). However, one feature is unique to permeabihty profiles the upper horizontal part of the sigmoidal curves may be verhcally depressed, due to the drug transport resistance arising from the aqueous boundary layer (ABL) adjacent to the two sides of the membrane barrier. Hence, the true membrane contribution to transport may be obscured when water is the rate-limiting resistance to transport. This is especially true if sparingly soluble molecules are considered and if the solutions on either or both sides of the membrane barrier are poorly stirred (often a problem with 96-well microhter plate formats). 

The pH-partition theory or nonionic permeability hypothesis was first described by Jacobs in 1940 [66]. According to this concept, only neutral, preferably nonpolar compounds are able to cross biological membranes. The transcellular permeability pH-profile is then essentially characterized by the membrane partition coefficient and the pKa of the compound. The simplest quantitative description of membrane permeation is given by ... 

The effective permeability of ionizable molecules depends on pH, and the shapes of the permeability-pH profiles can be theoretically predicted when the pKa of the molecule is known, the pH partition hypothesis are valid, and the resistance of... 

Intrinsic Permeability, Permeability-pH Profiles, Unstirred Water Layers (UWL), and the pH Partition Hypothesis... 

The shapes of permeability-pH profiles mirror those of solubility-pH (see, Figs. 6.1a, 6.2a, and 6.3a), with slopes of opposite signs. In solutions saturated with an insoluble compound, the product of solubility and permeability ( flux, as described in Chapter 2) is pH-independent This is indicated in Fig. 2.2 as the maximum flux portions of the curves. 

Figure 7.34 Permeability-pH profiles of ketoprofen (a) log-log plot solid curve represents effective permeability, and the dashed curve is the membrane permeability, calculated by Eq. (7.53). The latter curve levels off at the intrinsic permeability, Pq. The effective curve levels off to approximately the unstirred water layer permeability, Pu. (b) Direct plot the inset curve for the fraction neutral substance levels of at 100% (scale not shown). [Avdeef, A., Curr. Topics Med. Chem., 1, 277-351 (2001). Reproduced with permission from Bentham Science Publishers, Ltd.]...

Figure 7.35 Permeability-pH profile of phenazopyridine under iso-pH conditions. [Based on data in Ref. 558.]...

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.]...

The effective permeability of ionizable molecules depends on pH, and the shapes of the permeability-pH profiles can be theoretically predicted when the pKa of the molecule is known [7]. The pH dependency of ionizable molecules is illustrated in Fig. 3.3 for a series of weak acids (A. Avdeef, not published). It is clear that if the wrong pH is used in screening the permeabilities of molecules, then highly promising molecules (e.g., probenecid Fig. 3.3) may be characterized as false negatives. The ideal pH to use for in vitro screening ought to reflect the in vivo pH conditions. 

Fig. 3.6. Log permeability-pH profiles of ketoprofen in 2% wt/vol DOPC in dodecane (open circles) and 20% wt/vol soy lecithin in dodecane (filled circles). The weak acid is about 17 times more permeable in soy than in DOPC, as indicated by the calculated intrinsic...

Wohnsland, E. and Ealler, B. (2001) High-Throughput Permeability pH Profile and High-Throughput Alkane/Water log P with Artificial Membranes. Journal of Medicinal Chemistry, 44, 923—930. 

In the same way, o-nitrophenyl octyl ether (o-NPOE) was immobilized on polycarbonate (PC) filters and the apparent permeability measured after 5 h incubation time was correlated to log Pnpoe for a series of reference compounds (log Pnpoe ranging from —1 to 3.6) [90]. Lipophilicity values in the alkane/water system were also determined using PAMPA with hexadecane-PC coated filters [89]. In this case, a correlation was found between intrinsic permeability (log Pq, permeability corrected for ionization and for unstirred water layer contribution, which particularly affects permeability of lipophilic compounds) and log P ik. However, log Pq is obtained from the knowledge of the pJC, value(s) and the permeability pH profile and therefore requires the full permeability pH profile to be measured for each compound, which negatively impacts the assay throughput. 

PAMPA is often used at various pH values in order to measure permeability pH profiles as the permeability of ionisable compounds depends heavily on the pH of the buffer. As the pH range of the intestinal tract varies between pH 6 and pH 8 this is the range of pH values that mostly is used. Kerns (2004) recommended to measure from pH 4 to pH 7,4 in order to predict both bases and acids correctly. Ruell (2003) used permeation pH profiles from pH 4 to pH 9 together with the pKa values of the compounds under investigation to establish the optimum pH value for a single pH PAMPA measurement. 

Also the fact that only transcellular permeation is regarded in PAMPA experiments is valuable as it allows comparing PAMPA results to cellular permeation experiments that feature all possible permeation mechanism including paracellular or active transport and active efflux. Kerns (2004) recommended this comparison to get an insight into the permeation mechanism applied by a compound under investigation. The possibility to obtain permeability pH profiles is also really helpful to identify the relevant permeability value of a compound and cannot be determined by cellular assays due to the limited pH range usable with living cells. 

Wohnsland F, Faller B (2001) High-throughput permeability pH profile and high-throughput alkane/water log P with artificial membranes. J Med Chem 44 923-930 Youdim KA, Avdeef A, Abbott N (2003) In vitro transmonolayer permeability calculations often forgotten assumptions. DDT 8 997-1003... 

PAMPA-pKa fiux optimized design (pOD)-permeabiiity Iso-pH mapping unstirred PAMPA was used to measure the effective permeability, Pe, as a function of pH from 3 to 10, of five weak monoprotic acids (ibuprofen, naproxen, ketoprofen, salicylic acid, benzoic acid), an ampholyte (piroxicam), five monoprotic weak bases (imipramine, verapamil, propranolol, phenazopyridine, metoprolol), and a diprotic weak base (quinine). The intrinsic permeability, Po, the UWL permeability, Pu, and the apparent pKa (pKa.fiux) were determined from the pH dependence of log Pg. The underlying permeability-pH equations were derived for multiprotic weak acids, weak bases, and ampholytes. The average thickness of the UWL on each side of the membrane was estimated to be nearly 2000 p, somewhat larger than that found in Caco-2 permeability assays (unstirred). As the UWL thickness in the human intestine is believed to be about forty times smaller, it is critical to correct the in vitro permeability data for the effect of the UWL. Without such correction, the in vitro permeability coefficient of lipophilic molecules would be indicative only of the property of water. In single-pH PAMPA (e.g., pH 7.4), the uncertainty of the UWL contribution can be minimized if a specially selected pH (possibly different from 7.4) were used in the assay. From the analysis of the shapes of the log Pe-pH plots, a method to improve the selection of the assay pH, called pOD-PAMPA, was described and tested. From an optimally selected assay pH, it is possible to estimate Pg, as well as the entire membrane permeability-pH profile. 

Permeability pH Profiles Ionization, Membrane Retention, and Unstirred Layers... 

Figure 15.3. Diclofenac effective permeability pH profile corrected for the unstirred layer effect. The solid line represents the effective membrane permeability, while the dotted line represents the membrane permeabiltiy, corrected by the unstirred layer effect.

Artificial membranes are a relatively low-cost assay for fast screening of transcellular passive diffusion. The system allows easy access to permeability pH profiles and unstirred water layer correction. Due to the simple composition of the buffer reagents, the analytics is much easier than with cell cultures. Straight UV detection can be used in most cases and LC-MS (low soluble samples or compounds lacking UV chromophore) is considerably simplified in comparison to Caco-2, as the system matrix is relatively simple. A reasonable correlation has been obtained between artificial membrane and reported human GIT permeability in our laboratory (Figure 15.6A). 

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