A NovelIn VitroHepatocyte Culture System (icHep) for Biliary Drug Excretion Analysis Using Permeation Assay

Abstract

<b>Abstract ID 15604</b> <b>Poster Board 576</b> <b>Purpose:</b> Biliary excretion via drug-metabolizing enzymes and transporters is often critical as the hepatobiliary disposition is directly linked to drug efficacy and safety. Sandwich-cultured hepatocyte (SCH) is an <i>in&nbsp;vitro</i> model widely used to assess the biliary excretion potential of drugs. However, SCH does not allow time-dependent and quantitative evaluation because drugs accumulate in the closed bile canaliculi formed between hepatocytes and their amounts cannot be measured directly. This structural problem of SCH can be overcome by inducing a formation of an open canaliculus lumen face to a culture support. The aim of this study is to establish a novel hepatocyte culture model with open-form bile canaliculi that enables to evaluate biliary excretion of drugs by a permeation assay but not by accumulation assay. <b>Methods:</b> Primary human hepatocytes and PXB-cells derived from liver-humanized mice were cultured on a permeable support coated with claudin, a tight junction protein. The formation of bile canaliculi was evaluated by fluorescent immunostaining of the MRP2 localized on the canalicular membrane. The permeation assay was performed using substrates for transporters (BSEP, MRP2 and P-gp) involved in biliary excretion. Human <i>in&nbsp;vivo</i> biliary excretion clearance of six drugs whose clearances are reported was estimated using the apparent permeability coefficient <i>P_app</i> calculated by permeation assays of each test compound. <b>Results and Discussion:</b> When human hepatocytes were cultured on a permeable support coated with specific claudin proteins, open hemi-luminal morphology as indicated by the canalicular membrane marker MRP2 was newly formed on the surface of the permeable support. Therefore, it is considered that the surface of support coated with claudin proteins functioned as a pseudo-hepatocyte membrane for bile canaliculus formation and this culture system is termed as an induced open-form bile canaliculus hepatocyte (icHep). A continuous monolayer of hepatocytes with tight cell-to-cell contact is formed using PXB-cells. In addition, the cells maintained the functions of a wide range of liver-specific drug uptake transporters and metabolizing enzymes. Permeation assay in icHep showed unidirectional transport of taurocholate (BSEP), E₂17βG (MRP2), and digoxin (P-gp) from blood to bile side. Each transport was suppressed upon treatment with the respective inhibitors. Furthermore, the estimated biliary drug excretion from the permeability obtained in icHep was well-correlated with the reported human <i>in&nbsp;vivo</i> values. <b>Conclusions:</b> icHep permeation assay system established in the present study overcomes the problems of the conventional SCH method and enables easy and rapid collection of biliary excreted drugs and estimation of their biliary excretion clearance. This approach is useful to assess pharmacokinetics and drug safety during the preclinical stage of drug discovery and development.