Screening of OATP1B1/3 and OCT1 inhibitors in cryopreserved hepatocytes in suspension

Abstract

Drug–drug interactions involving hepatic drug transporters may have clinical consequences and jeopardize development of promising drug candidates. Organic anion transporting polypeptides (OATP/Oatp) and the organic cation transporters (OCT/Oct) are among the most important transporters involved in xenobiotic uptake in the liver. In the present study, 179 molecules have been tested as inhibitors of the uptake of estradiol-17βD-glucuronide (E₂17βG), substrate of OATP1B1/3 (rOatp), or 1-methyl-4-phenylpyridinium (MPP+), substrate of OCT1 (rOct1), into suspended cryopreserved hepatocytes from humans and rats. Uptake was assessed in 96-well plates by measuring intracellular accumulation of radioactive substrate in hepatocytes in presence or absence of inhibitor.

In rat hepatocytes 140 compounds were identified as inhibitors (inhibition at 20 μM ≥ 30%) of E₂17βG uptake and 77 compounds inhibitors of MPP+ uptake. The most potent inhibitors of rOatp and rOct1 were dantrolene sodium (K_i = 2 ± 9 μM) and bepridil (K_i = 14 ± 2 μM), respectively. In human hepatocytes, the most potent inhibitors of E₂17βG and MPP+ uptake were capsazepine (K_i = 14 ± 5 μM) and cyproheptadine (K_i = 19 ± 3 μM), respectively.

Structure–activity relationship (SAR) analysis of all tested compounds suggested that lipophilicity, polarity, pK_a and the number of hydrogen bond donors and acceptors play a role in their interaction with the transporters investigated.

The method used here is a simple tool to screen large number of compounds as inhibitors of the uptake of substrates into suspended hepatocytes.

Introduction

Drug metabolism in liver is the most important pathway for xenobiotic biotransformation. In hepatocytes, drugs are metabolised by intracellular enzymes (phases I and II) to more polar compounds readily excreted through the bile or the urine. Although most lipophilic drugs pass the cell membrane passively, leading to equilibrium between extracellular and intracellular concentrations of the free drug, carrier-mediated transport of more polar drugs into hepatocytes may be the rate-limiting step for their metabolism by intracellular enzymes.

Uptake transporters accumulate drugs in hepatocytes at concentrations higher than in the extracellular space (Soars et al., 2007). By doing so, these transporters expose intracellular enzymes and canalicular transporters to higher concentrations of drugs.

Although the role of drug transport has now been established for a number of marketed drugs (Li et al., 2009, Shitara et al., 2006), there is still a lack of selective tool compounds (substrates and inhibitors) to further assess the importance of hepatic drug transport in drug pharmacokinetics or to assess the risk of drug–drug interactions on a specific transport protein.

For drugs where hepatic uptake is the rate-limiting step for disposition, interactions may occur when these drugs are co-administered with inhibitors of transport proteins (Soars et al., 2009, Endres et al., 2006). Assessing the potential of a new drug candidate to inhibit a specific uptake transporter, may be important to fully characterise the risk of drug–drug interactions (Treiber et al., 2007).

There are a number of uptake transporters in hepatocytes, some of the most important being the organic anion transporting polypeptides (OATP/Oatp) and the organic cation transporters (OCT/Oct). OATP and OCT belongs to the solute carrier superfamily. They are distributed widely throughout the body (liver, kidney, intestine, lung, brain, etc.) (Koepsell et al., 2007).

OATP and OCT are located in the basolateral membrane, transporting drugs from the sinusoidal space into hepatocytes. Intracellular free drugs are either metabolised and/or excreted to the bile by canalicular efflux transporters. The drug or its metabolites can also be excreted by basolateral efflux transporters, the multidrug resistance-associated protein 2 and 6 (MRP2, 6) back to the sinusoidal space (Diaz, 2000).

From a drug industry perspective, developing drugs that interact with hepatic uptake or efflux proteins is a factor susceptible to increase the risk of inter-individual variabilities in systemic exposures. Inter-individual differences in hepatic transport proteins (consequence of phenotype of genotype differences) (Choi and Song, 2008, Maeda and Sugiyama, 2008, Shu et al., 2007) in addition to inter-individual differences in hepatic metabolism (differences in enzymes activity) may increase inter-individual differences of the pharmacokinetics parameters. Co-administration of drugs interacting with the same transport proteins may increase the complexity of the pharmacokinetic profile leading to unpredictable drug–drug interactions or adverse drug reactions.

In the present study, we have used a simple protocol previously described by us (Jørgensen et al., 2007) to assess the effect of 179 molecules on the uptake of estradiol-17βD-glucuronide (E₂17βG), substrate of OATP1B1/3 (rOatp) or 1-methyl-4-phenylpyridinium (MPP+), substrate of OCT1 (rOct1), in rat hepatocytes and the effect of 84 compounds were tested in human hepatocytes.