Permeability of lipophilic compounds in drug discovery using in-vitro human absorption model, Caco-2

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Abstract

Highly lipophilic compounds are often encountered in the early stages of drug discovery. The apparent permeability (Papp) of these compounds in Caco-2 cell could be underestimated because of considerable retention by the Caco-2 monolayer and non-specific binding to transwell surface. We have utilized a general approach for the determination of permeability of these compounds, which includes the addition of 1–5% DMSO in the apical (AP) and 4% bovine serum albumin (BSA) in the basolateral (BA) side. Two highly lipophilic and highly protein bound Schering compounds, SCH-A and SCH-B, exhibited poor recovery and low Papp in the conventional Caco-2 system that included 1% DMSO in the AP and BA sides. In contrast, both compounds were well absorbed in cynomolgus monkeys. Inclusion of BSA (up to 4%) in the BA side provided necessary absorptive driving force similar to in vivo sink conditions improving both recovery and Papp of these compounds as well as progesterone, a model highly lipophilic and highly protein bound compound. Whereas, the recovery and Papp of mannitol (high recovery, low permeability) and propranolol (high recovery, high permeability) remained unaffected. The presence of 4% BSA increased Papp of SCH-A, SCH-B, and progesterone by five-, four-, and three-fold, respectively. We also compared this approach with a second, based on the disappearance of the compound from the AP side, which resulted in a reasonable estimate of the permeability (23.3×10−6 cm/s) for SCH-A. The results demonstrated that the reliable estimates of permeability of highly lipophilic compounds that are subjected to considerable retention by the cell monolayer and exhibit non-specific binding are obtained by the addition of BSA to the BA side.

Introduction

The majority of new molecular entities (NME's) generated in drug discovery in the pharmaceutical industry are intended for oral administration because of convenience, safety, and better patient compliance. Thus, oral absorption potential, if not assessed early in drug discovery, could seriously jeopardize the development of a lead compound mainly due to pharmacokinetic deficiencies (Barr et al., 1996). Among various in vitro systems to predict oral absorption in humans, Caco-2 monolayers seem to be the best available model in terms of throughput and reliability. Accordingly, the permeability evaluation in Caco-2 monolayers is becoming the most widely used screen for oral absorption potential in a drug discovery setting in the pharmaceutical industry (Yee, 1996). This system is amenable to higher throughput, which can meet the challenge of the vast increase in the number of NME's emanating as a result of combinatorial chemistry and high throughput screening (HTS).

Studies have shown a good correlation between permeability in the Caco-2 monolayers and the oral absorption in humans (Bailey et al., 1996, Artursson et al., 1996, Rubas et al., 1993). However, these correlations, for the most part, were made with compounds that were well characterized. Unfortunately, at a very early stage of drug discovery, it is not possible to fully characterize compounds in terms of physicochemical properties. Moreover, the in vitro pharmacological screens such as receptor binding assays tend to be selective toward more lipophilic and poorly water-soluble compounds. These compounds pose a major challenge in terms of estimating permeability in the Caco-2 monolayers because of their poor solubility and high lipophilicity, which may result in considerable retention of compounds by the cell monolayer as well as non-specific binding or adsorption to in vitro systems.

Systematic studies addressing permeability as it is affected by the recovery of the compound are lacking. The pre- or post- experimental approaches reported earlier do not address the problems associated with considerable retention of compounds in the Caco-2 monolayers. Additionally, approaches such as post-experimental incubation with organic solvents, (e.g. dimethylsulfoxide and methanol) are tedious and not suitable for higher-throughput (Augustinjns et al., 1993, Hosoya et al., 1996). Other approaches such as the pre-treatment with human serum albumin (HSA) may not work for some compounds because of the extensive adsorption of compounds to the system despite the pre-treatment with HSA (Chan et al., 1996) or considerable uptake by the cell monolayers (Wils et al., 1994). The use of surfactants such as Tween 80, or cremophore EL to minimize non-specific binding seems promising. However, surfactants may interfere with p-glycoprotein (p-gp) which may confound the results (Nerurkar et al., 1996).

Therefore, a universal rather than a compound specific approach is needed to improve recovery so that a reasonable estimate of permeability could be attained. This has an added importance in drug discovery, since the quantities of compounds are very limited, precluding reiterative studies. In this report, we have systematically investigated the approach that might be useful in determining permeability of compounds that could otherwise be underestimated because of significant retention by cell monolayers and non-specific binding to in vitro set-up. Next, this approach was compared with another that is based on the disappearance of the compound from the apical side.

Section snippets

Materials

3H-propranolol, and 3H-progesterone were purchased from Sigma Chemical Co (St. Louis, MO). 14C-mannitol was from Moravek Biochemicals (Brea, CA). Corresponding unlabeled chemicals were from Sigma Chemical Co. (St. Louis, MO). SCH-A, SCH-B, and SCH-E were from the Chemical Research Division, Schering-Plough Research Institute, Kenilworth, NJ. The radiochemical purity of the compounds used was>95%.

Cell and Transwell culture

Caco-2 cells were obtained from American Type Culture Collection (ATCC) at passage #17. The cells

Results

The recovery and permeability of propranolol, progesterone, mannitol, and three NME's (SCH-A, SCH-B, and SCH-E) that were structurally unrelated except SCH-A and SCH-B were first evaluated. The recovery values without the methanol wash were about 80 and 100% for propranolol and mannitol, respectively. The recovery was<70% for a relatively more lipophilic compound, progesterone.

SCH-A and progesterone, lipophilic and consequently high protein bound (>99 and 96–99%, respectively,) compound with

Discussion

SCH-A, SCH-B, and SCH-E are all moderate to well absorbed compounds in cynomolgus monkeys (unpublished data); however, SCH-A and SCH-B exhibited low permeability in conventional Caco-2 system with 1% DMSO or 1% CH3OH as a co-solvent in the AP and BA sides. The log P values for SCH-A and SCH-B, were higher than that of progesterone, with a rank order of SCH-A>SCH-B (Table 3). The log P value for SCH-E was lower than that of propranolol (Table 3). Consistent with high lipophilicity, protein binding

Conclusion

Reliable estimates of permeability were attained for compounds that exhibited extensive retention by the Caco-2 monolayers and non-specific binding to in vitro apparatus by including bovine serum albumin in the basolateral side. The inclusion of the BSA in the basolateral side resulted in a considerable improvement in recovery and consequently permeability values for highly protein bound lipophilic compounds, SCH-A, SCH-B, and progesterone, which exhibited poor recovery. The permeability for

Acknowledgements

The authors would like to thank Mitchell N. Cayen, Ph.D. and Ronald E White, Ph.D. for a critical review of the manuscript and valuable suggestions. The authors would also like to thank Laura Norton for excellent technical assistance.

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A part of this work was presented as an abstract and poster at First AAPS Frontier Symposium, ‘From Good Ligands to Good Drugs: Optimizing Pharmaceutical Properties by Accelerated Screening’, February 19–21, 1998, Bethesda, MA.

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