Culture density contributes to hepatic functions of fresh human hepatocytes isolated from chimeric mice with humanized livers: Novel, long-term, functional two-dimensional in vitro tool for developing new drugs.

Chimeric mice with humanized livers are considered a useful animal model for predicting human (h-) drug metabolism and toxicity. In this study, the characteristics of fresh h-hepatocytes (cFHHs, PXB-cells®) isolated from chimeric mice (PXB-mice®) were evaluated in vitro to confirm their utility for drug development. cFHHs cultured at high density (2.13 × 105 cells/cm2) displayed stable production of h-albumin and cytochrome P450 (CYP) 3A activities for at least 21 days. The mRNA expression levels of 10 of 13 CYP, UDP-glucuronosyltransferase (UGT), and transporters were maintained at >10% of the levels of freshly isolated cFHHs after 21 days. From 1 week, many bile canaliculi were observed between cFHHs, and the accumulation of the multidrug resistance-associated protein and bile salt export pump substrates in these bile canaliculi was clearly inhibited by cyclosporin A. Microarray analysis of cFHHs cultured at high density and at low density (0.53 × 105 cells/cm2) revealed that high density culture maintained high expressions of some transcription factors (HNF4α, PXR, and FXR) perhaps involved in the high CYP, UGT and transporter gene expressions of cFHHs. These results strongly suggest that cFHHs could be a novel in vitro tool for drug development studies.

Organic Anion-Transporting Polypeptide (OATP)-Mediated Drug-Drug Interaction Study between Rosuvastatin and Cyclosporine A in Chimeric Mice with Humanized Liver.

The influence of transporters on the pharmacokinetics of drugs is being increasingly recognized, and DDIs via transporters may be a risk factor for adverse events. Cyclosporine A, a strong OATP inhibitor, has been reported to increase the systemic exposure of rosuvastatin, an OATP substrate, by 7.1-fold in clinical studies. PXB mice are chimeric mice with humanized livers that are highly repopulated with human hepatocytes and have been widely used for drug discovery in drug metabolism and pharmacokinetics studies. In the present study, we examined in vivo and in vitro DDIs between rosuvastatin and cyclosporine A in PXB mice and fresh human hepatocytes (PXB cells) obtained from PXB mice. We initially investigated the active transport of rosuvastatin into PXB cells, and found concentration-dependent uptake with a Michaelis-Menten constant value of 4.0 µmol/l and a Vmax value of 4.63 pmol/min per 106 cells. Cyclosporine A inhibited the uptake of rosuvastatin with an IC50 value of 0.21 µmol/l. We then examined in vivo DDIs, and the exposure of orally administered rosuvastatin increased by 3.3-fold and 11-fold in PXB mice pretreated with 10 and 50 mg/kg cyclosporine A, whereas it increased by 2.5-fold and 6.2-fold when rosuvastatin was administered intravenously, in studies that were conducted for considering gastrointestinal DDIs. The liver-to-blood concentration ratio of rosuvastatin was dose-dependently decreased by pretreatment with cyclosporine A in PXB mice and SCID mice. Observed DDIs in vivo were considered to be reasonable based on the estimated concentrations of cyclosporine A at the inlet to the liver and in the liver tissues of both mice. In conclusion, our results indicate that PXB mice might be a useful tool for predicting human OATP-mediated DDIs in drug discovery, and its limitation due to the differences of gastrointestinal condition from human should also be considered.