Reversed phase parallel artificial membrane permeation assay for log P measurement

A reversed phase parallel artificial membrane permeation assay (RP-PAMPA) was newly invented for log P measurement. An oil/water/oil sandwich was constructed using a conventional PAMPA instrument. 1 % agarose was used to improve the physical stability of the water phase. A linear correlation between log P and the apparent permeability was observed in the -0.24 < log P < 2.85 region (R 2 = 0.98). RP-PAMPA was also applied to pKa measurement.


Introduction
High throughput physicochemical profiling of a drug is still challenging in early drug discovery.Various methods have been proposed for octanol -water partition coefficient (log P), solubility, and pK a measurements [1].The parallel artificial membrane permeation assay (PAMPA) has been widely used in drug discovery as PAMPA is compatible with high throughput screening (HTS) [2,3].In normal phase (NP-) PAMPA methods, a lipid phase is immobilized on a filter (usually a 96 well filter plate) and the permeability of a drug across the lipid membrane is measured.Previously, Faller et al. applied NP-PAMPA to log P measurement [4].The apparent permeability (P app ) across the octanol impregnated filter membrane was found to correlate with logP.However, the P app -log P curve showed a bell-shaped relationship with a plateau around log P = 1.Therefore, it was impossible to estimate the log P values around log P = 1.A bellshaped relationship between P app and lipophilicity of drugs is usually observed in NP-PAMPA [5].
The purpose of the present study was to overcome the drawback of NP-PAMPA for log P measurement.A reversed phase PAMPA (RP-PAMPA) method for log P measurement was newly invented.In RP-PAMPA, an oil/water/oil sandwich was constructed using a conventional PAMPA instrument (Figure 1).In addition, RP-PAMPA was applied for pK a measurement, especially for low solubility compounds.

P app measurement
The schematic configuration of RP-PAMPA is shown in Figure 1.In RP-PAMPA, the water phase (water membrane) was immobilized on the hydrophilic filter with the aid of agarose.Agarose S was dissolved in hot water or a buffer at 1.0 % and then poured into a hydrophilic filter (Multi Screen-HV, pore size 0.48 μm, low protein binding, Millipore).A model drug was dissolved in octanol at 10 mM and added to the donor plate (downside, 300 μL).The filter plate was then put on the donor plate.The filter plate was filled with 200 μL of octanol.After 16 hour incubation at room temperature (25 ± 1 °C), both octanol phases were diluted tenfold by ethanol and the drug concentrations in the donor and accepter sides were measured by UV spectroscopy.For pK a measurement sodium -phosphate and sodium -citric acid buffers (100 mM of anion species) were used to construct the water membrane.P app was calculated as previously reported [6]: where C A and C D are the drug concentrations in the donor and acceptor phases at time t, respectively.V D and V A are the volumes of the donor and acceptor phases, respectively.A is the membrane surface area (0.28 cm 2 ).

Construction of RP-PAMPA
We first investigated the stability of the water phase (water membrane) constructed on the hydrophilic filter with the aid of agarose.1.0 % concentration was selected to enable pipetting of the hot sol phase while maintaining the physical strength of the agarose gel.It was found that at least 30 μL of 1 % agarose was required to provide sufficient physical strength for P app measurement.The ager powder was found to form a less stable water phase compared to Agarose S and H.In a preliminary study, it was found that more than 10 hours were required to achieve a steady -state flux across the water phase (data not shown).Therefore, the Agarose S 30 μL water membrane and 16 hour incubation time were employed in the following studies.

Log P app -log P relationship
The log P app and log P data are summarized in Table 1 [1,7,8].Figure 2 shows the correlation between log P app and log P. A linear correlation was observed in the -0.24 < log P < 2.85 region (R 2 = 0.98).The slope of the log-log plot was -0.48.

pH -P app profile
The pH -P app profiles were shown in Figure 3 and Table 2.The pK a values were obtained as the intersection of the slope and the horizontal lines.Estimated and literature pK a values are shown in    a Measured at 25 °C.The buffer concentration was 100 mM.b Refs.[1], [9].

Discussion
In this study, RP-PAMPA for log P measurement was investigated for the first time.1.0 % agarose was used to improve the physical stability of the water membrane.The mesh size of agarose is significantly larger than the size of drug molecules so that it does not affect the diffusion coefficient of drugs [10].By using RP-PAMPA, log P in the -0.24 < log P < 2.85 range can be accurately measured.The measurable range can be expanded by using a more sensitive quantitation method such as LC-MS.The slope of the P app -log P relationship was 0.45, which is significantly smaller than 1.If P app follows the solubility -partition theory for membrane permeation, i.e., P app = PD/h where D is the diffusion coefficient and h is the thickness of the membrane, the slope of the log-log plot should be unity [11].The reason for this deviation is not clear.
Previously, Kwon et al. reported a poly(dimethylsiloxane)(PDMS) permeation assay, which might be regarded as a kind of reversed phase membrane permeation assay [12].However, the configuration of the PDMS permeation assay was largely different from the one used in the present study that is usually referred as PAMPA.In the PDMS permeation assay, a side-by-side single diffusion chamber was employed.A PDMS membrane was put between two chambers filled with aqueous bulk fluids.In addition, PDMS disks were added to both the donor and acceptor sides as dosing and sampling (extracting) phases, respectively.The aqueous phases were stirred by magnetic stirrers.In the PDMS permeation assay, a good correlation was observed between log P app and log P in the range of log P > 3 even though PDMS was used instead of octanol as the oil phase.
For low solubility drugs, it has been difficult to measure pK a by using conventional methods such as pH titration.The pH -solubility profile can be used to estimate pK a for low solubility drugs [13,14].However, this method may not be accurate due to aggregate formation, low detection limit, etc.In RP-PAMPA, a drug is solubilized in the organic solvent phase.Therefore, it would become possible to measure pK a for low solubility drugs by using RP-PAMPA.As the pH of the water membrane was changed in RP-PAMPA, the pH -P app relationship should become a mirror image of that for NP-PAMPA.However, the pH -P app relationship deviated from the Henderson -Hasselbalch equation.Therefore, the pK a values of the model drugs were estimated as the intersection of the slope and the horizontal lines.The pK a values of acidic drugs were underestimated by the RP-PAMPA method.The reason for this deviation is not clear.One possible reason may be that the incubation time of 16 hours might not be sufficient to achieve a steady state at pH > pK a for acids.The pK a of diclofenac has been reported to be ca.4.0 in most cases in the literature.However, pK a of 5.7 was obtained from the pH-solubility profile [15].The P app value at a pH where a drug molecule is undissociable (intrinsic water permeability, P w,int ) also correlated with log P.However, P w,int deviated from the log P app -log P line for the undissociable drugs about 0.3 log unit.The difference of the water membrane (pure water vs. a buffer) could be a reason for the discrepancy.The present RP-PAMPA method

Table 3 .
pK a values