manuscript doi: 10.5599/admet.4.1.277 54 ADMET & DMPK 4(1) (2016) 54-59; doi: 10.5599/admet.4.1.277 Open Access : ISSN : 1848-7718 http://www.pub.iapchem.org/ojs/index.php/admet/index Original scientific paper Reversed phase parallel artificial membrane permeation assay for log P measurement Zihao Song, Katsuhide Terada, Kiyohiko Sugano* Department of pharmaceutics, Faculty of Pharmaceutical Sciences, Toho University, 2-2-1, Miyama, Funabashi, Chiba, 274-8510, Japan *Corresponding Author: E-mail: kiyohiko.sugano@phar.toho-u.ac.jp; Tel.: +81 47 472 1494 Received: March 17, 2016; Published: March 31, 2016 Abstract 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. Keywords octanol, partition, parallel artificial membrane permeation assay, pKa, drug 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 pKa 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 (Papp) across the octanol impregnated filter membrane was found to correlate with logP. However, the Papp – 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 bell- shaped relationship between Papp 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 pKa measurement, especially for low solubility compounds. http://www.pub.iapchem.org/ojs/index.php/admet/index ADMET & DMPK 4(1) (2016) 54-59 Reversed phase PAMPA doi: 10.5599/admet.4.1.277 55 Figure 1. Schematic configuration of RP-PAMPA Materials and Methods Materials Octanol, pentoxifylline, 1-naphthol, dipyridamole, and acid blue 9 were purchased from Tokyo chemical industry (Tokyo, Japan). Agar powder, agarose S, agarose H, prednisone, sulfamethoxazole, carbamazepine, caffeine, chlormphenicol, ethanol, trisodium citrate, sodium dihydrogenphosphate, disodium hydrogenphosphate, sodium hydroxide solution, propranolol hydrochloride, warfarin sodium, piroxicam, and ketoprofen were purchased from Wako pure chemicals (Tokyo, Japan). Phenacetin was purchased from Yamamoto Corporation (Osaka, Japan). The other reagents were of analytical grade. Papp 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 pKa measurement sodium - phosphate and sodium - citric acid buffers (100 mM of anion species) were used to construct the water membrane. Papp was calculated as previously reported [6]: C C P A V V t A equilibrium app D A ln(1 / ) (1/ 1/ )       (1)  C C V C V V Vequilibrium D D A A D A/( )     (2) where CA and CD are the drug concentrations in the donor and acceptor phases at time t, respectively. VD and VA are the volumes of the donor and acceptor phases, respectively. A is the membrane surface area (0.28 cm 2 ). Results Construction of RP-PAMPA We first investigated the stability of the water phase (water membrane) constructed on the hydrophilic Z. Song et al. ADMET & DMPK 4(1) (2016) 54-59 56 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 Papp 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 Papp – log P relationship The log Papp and log P data are summarized in Table 1 [1,7,8]. Figure 2 shows the correlation between log Papp 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. Table 1. Papp and log P Drug log P (literature) a log Papp (cm s -1 , mean ± S.D., N = 6) phenacetin 1.58 -6.38 ± 0.04 caffeine 0.10 -5.53 ± 0.02 carbamazepine 2.1 -6.52 ± 0.04 prednisone 1.56 -6.25 ± 0.01 chloramphenicol 1.14 -6.13 ± 0.01 sulfamethoxazole 0.70 -5.96 ± 0.02 1-naphthol 2.85 -6.95 ± 0.03 pentoxifylline 0.38 -5.78 ± 0.02 a Refs. [1,7,8] Figure 2. Log P – log Papp relationship. pH - Papp profile The pH - Papp profiles were shown in Figure 3 and Table 2. The pKa values were obtained as the intersection of the slope and the horizontal lines. Estimated and literature pKa values are shown in Table 3 [1,9]. y = -0.4835x - 5.5584 R² = 0.9842 -7.0 -6.8 -6.6 -6.4 -6.2 -6.0 -5.8 -5.6 -5.4 -5.2 -5.0 0 1 2 3 lo g P a p p ( cm /s e c) logP ADMET & DMPK 4(1) (2016) 54-59 Reversed phase PAMPA doi: 10.5599/admet.4.1.277 57 Figure 3. pH – Papp relationship. Mean ± S.D. N = 3. Table 2. Papp of dissociable drugs at each pH a Compound pH Papp (10 -6 cm sec -1 ) Compound pH Papp (10 -6 cm sec -1 ) Ketoprofen 3.1 0.17 ± 0.03 Dipyridamole 3.9 0.31 ± 0.02 (log P = 3.2) 3.5 0.17 ± 0.07 (log P = 3.9) 4.5 0.30 ± 0.01 4.1 0.15 ± 0.05 5.1 0.20 ± 0.02 4.5 0.18 ± 0.04 5.5 0.11 ± 0.02 5.0 0.28 ± 0.09 5.9 0.07 ± 0.02 5.5 0.44 ± 0.10 6.5 0.05 ± 0.01 6.0 1.33 ± 0.18 7.0 0.05 ± 0.03 6.5 2.00 ± 0.23 7.5 0.04 ± 0.00 Piroxicam 3.0 0.81 ± 0.07 Propranolol 8.0 1.05 ± 0.06 (log P = 2.0) 3.5 0.81 ± 0.19 (log P = 2.9) 8.5 0.72 ± 0.08 4.0 0.77 ± 0.07 9.0 0.63 ± 0.10 4.5 0.68 ± 0.07 9.5 0.42 ± 0.04 5.0 0.81 ± 0.15 10.0 0.40 ± 0.12 5.5 0.98 ± 0.13 10.5 0.39 ± 0.04 6.0 1.62 ± 0.08 11.0 0.38 ± 0.01 6.5 2.37 ± 0.24 11.5 0.38 ± 0.03 Warfarin 3.0 0.16 ± 0.01 (log P = 3.1) 3.5 0.16 ± 0.01 4.0 0.16 ± 0.02 4.5 0.23 ± 0.02 5.0 0.22 ± 0.04 5.5 0.23 ± 0.02 6.0 0.34 ± 0.04 6.5 0.80 ± 0.05 a Mean ± S.D. N = 3. Measured at 25 °C. The buffer concentration was 100 mM. Z. Song et al. ADMET & DMPK 4(1) (2016) 54-59 58 Table 3. pKa values Drug This study a Literature b Ketoprofen 4.9 4.0 Piroxicam 4.8 4.7 Warfarin 5.7 5.0 Dipyridamole 5.9 6.1 Propranolol 9.4 9.5 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 Papp – log P relationship was 0.45, which is significantly smaller than 1. If Papp follows the solubility – partition theory for membrane permeation, i.e., Papp = 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 Papp 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 pKa by using conventional methods such as pH titration. The pH – solubility profile can be used to estimate pKa 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 pKa for low solubility drugs by using RP-PAMPA. As the pH of the water membrane was changed in RP-PAMPA, the pH – Papp relationship should become a mirror image of that for NP-PAMPA. However, the pH – Papp relationship deviated from the Henderson – Hasselbalch equation. Therefore, the pKa values of the model drugs were estimated as the intersection of the slope and the horizontal lines. The pKa 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 > pKa for acids. The pKa of diclofenac has been reported to be ca. 4.0 in most cases in the literature. However, pKa of 5.7 was obtained from the pH-solubility profile [15]. The Papp value at a pH where a drug molecule is undissociable (intrinsic water permeability, Pw,int) also correlated with log P. However, Pw,int deviated from the log Papp – 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 ADMET & DMPK 4(1) (2016) 54-59 Reversed phase PAMPA doi: 10.5599/admet.4.1.277 59 needs to be improved for pKa measurement in the future. In conclusion, in the present study, RP-PAMPA for log P measurement was constructed for the first time. 1.0 % agarose can be used to stabilize the water membrane. RP-PAMPA was applied to log P and pKa measurements. As PAMPA is compatible with the current HTS instrument, RP-PAMPA will be a useful tool in drug discovery. References [1] A. Avdeef. Absorption and Drug Development. Hoboken: Wiley-Interscience, NJ; 2003. [2] M. Kansy, F. Senner, K. Gubernator. J. Med. Chem. 41 (1998) 1007-1010. [3] A. Avdeef, S. Bendels, L. Di, B. Faller, M. Kansy, K. Sugano, Y. Yamauchi. J. Pharm. Sci. 96 (2007) 2893-2909. [4] B. Faller, H.P. Grimm, F. Loeuillet-Ritzler, S. Arnold, X. Briand. J. Med. Chem. 48 (2005) 2571-2576. [5] M. Kansy, H. Fischer, K. Kratzat, F. Senner, B. Wagner, I. Parrilla. High-throughput artifical membrane permeability studies in early lead discovery and development. In: Testa B, Van de Waterbeemd H, Folkers G, Guy R, editors. Pharmacokinetic optimization in drug research. 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