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Drug-induced liver injury (DILI) is of concern to the pharmaceutical industry, and reliable preclinical screens are required. Previously, we established an in vitro bile acid-dependent hepatotoxicity assay that mimics cholestatic DILI in vivo. Here, we confirmed that this assay can predict cholestatic DILI in clinical situations by comparing in vitro cytotoxicity data with in vivo risk. For 38 drugs, the frequencies of abnormal increases in serum alkaline phosphatase (ALP), transaminases, gamma glutamyltranspeptidase (γGT), and bilirubin were collected from interview forms. Drugs with frequencies of serum marker increases higher than 1% were classified as high DILI risk compounds. In vitro cytotoxicity was assessed by monitoring lactate dehydrogenase release from rat and human sandwich-cultured hepatocytes (SCRHs and SCHHs) incubated with the test drugs (50 μM) for 24 hours in the absence or presence of a bile acids mixture. Receiver operating characteristic analyses gave optimal cutoff toxicity values of 19.5% and 9.2% for ALP and transaminases in SCRHs, respectively. Using this cutoff, high- and low-risk drugs were separated with 65.4-78.6% sensitivity and 66.7-79.2% specificity. Good separation was also achieved using SCHHs. In conclusion, cholestatic DILI risk can be successfully predicted using a sandwich-cultured hepatocyte-based assay.

Medication use during lactation is a matter of concern due to unnecessary exposure of infants to drugs. Although some studies have predicted the extent of drug transfer into milk from physicochemical parameters, drug concentration-time profiles in milk have not been predicted or even analyzed yet. In the present study, a drug transfer model was constructed by defining secretion and reuptake clearances (CL(sec) and CL(re), respectively) between milk and plasma based on unbound drug concentrations. Through the use of this model, drug concentration-time profiles were analyzed in human milk and plasma based on data collected from the literature. CL(sec) and CL(re) values were obtained successfully for 49 drugs. Because the CL(sec) and CL(re) values were in general similar for each drug, transport across the mammary epithelia was mediated by passive diffusion in most cases. This study demonstrated that the logarithmically transformed values of CL(sec) and CL(re) can be predicted from physicochemical parameters with adjusted R(2) values of 0.705 and 0.472, respectively. Moreover, 66.7 and 77.8% of predicted CL(sec) and CL(re) values were within 3-fold error ranges of the observed values for 45 and 27 drugs, respectively. Finally, time profiles of drug concentrations in milk were simulated from physicochemical parameters. The milk-to-plasma area under the concentration-time curve ratios also were predicted successfully within 3-fold error ranges of the observed values for 71.9% of the drugs analyzed. The method described herein therefore may be useful in predicting drug concentration-time profiles in human milk for newly developed drugs.

In a previous report, we identified the receptor for activated C-kinase 1 (RACK1) as a positive regulator of the cellular localization and expression of ATP-binding cassette B4, a phosphatidylcholine translocator expressed on the bile canalicular membrane. In the present study, we focused on the role of RACK1 on ATP-binding cassette G2 (ABCG2), which is responsible for the cellular extrusion of compounds including antitumor drugs. Protein expression of ABCG2 was up-regulated by RACK1 overexpression, although mRNA expression of ABCG2 was not dependent on RACK1. The effect of RACK1 on the expression of ABCG2 on the cell surface was confirmed by the uptake of [(3)H]estrone sulfate, an ABCG2 substrate, into isolated membrane vesicles. The expression of RACK1 affected cellular resistance to mitoxantrone, an anticancer drug excreted by ABCG2, and this effect of RACK1 was abolished in the presence of fumitremorgin C, a selective ABCG2 inhibitor. These results suggest that RACK1 has functional significance as a regulatory cofactor of ABCG2 and is indispensable for the cell surface expression and excretion function of ABCG2. The precise mechanism for RACK1-dependent expression of ABCG2 remains to be clarified, because the results of N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and chloroquine treatment and those of metabolic labeling experiments did not give us clear evidence whether the reduction of ABCG2 expression in RACK1-knocked down cells may be caused by the suppression of ABCG2 protein synthesis or by acceleration of its degradation.

The multidrug resistance-associated protein 2/ATP-binding cassette transporter family C2 (Mrp2/Abcc2) is an ATP-dependent export pump that mediates the transport of a variety of organic anions. Abcc2 is mainly expressed on the canalicular membrane of hepatocytes and also the brush-border membrane of intestinal epithelial cells. We have previously reported that Abcc2 is rapidly internalized from the canalicular membrane during acute oxidative stress, which induces protein kinase C (PKC) activation in rat liver. However, it has not been elucidated whether PKC is involved in the regulation of Abcc2 localization in other tissues. In this study, we investigated this issue in rat intestinal epithelia. Exposure to thymeleatoxin, a conventional PKC (cPKC) activator, for 20 min reduced the cumulative glutathione S-bimane efflux for 40 min via Abcc2 from 30.3 +/- 2.1 nmol/cm to 18.1 +/- 1.6 nmol/cm. Likewise, the Abcc2 expression in the brush-border membrane of the small intestine was reduced to half that of the control without changing the total amount of Abcc2 present in the homogenate. Immunoprecipitation analysis suggested an interaction between Abcc2 and ezrin, a scaffolding protein that is dominantly expressed in the intestine. Thymeleatoxin treatment decreased the amount of the active form (C-terminally phosphorylated form) of ezrin and the amount of Abcc2 that coimmunoprecipitated with ezrin. These results indicate that cPKC activation diminishes the protein-protein interaction between ezrin and Abcc2. In conclusion, the phosphorylation status of ezrin correlates with the cell surface expression of Abcc2 in the rat small intestine, which may be regulated by cPKC.

Ezetimibe (EZE) selectively blocks intestinal cholesterol absorption by interacting with Niemann-Pick C1 Like 1 (NPC1L1). After administration, EZE is extensively metabolized in liver and intestine to its phenolic glucuronide form (EZE-G) by uridine diphosphate glucuronosyltransferases (UGTs), among which UGT1A1 and 1A3 exhibit highest activity. EZE-G is excreted into bile and undergoes extensive enterohepatic recirculation. Considering the pharmacokinetic properties of EZE and an in vitro binding study showing the high affinity binding of EZE-G to NPC1L1, glucuronidation by UGTs has been believed to be essential for the pharmacological efficacy of EZE. To study the role of glucuronidation by UGTs for the cholesterol-lowering effect of EZE, in vitro and in vivo studies were performed using Gunn rats, which hereditarily lack the expression of UGT1A enzymes. The biliary excreted amount of EZE-G was reduced by 73% up to 3 h after administration of EZE (0.3 mg/kg) in Gunn rats, which is consistent with the reduction of in vitro EZE glucuronidation activity found in liver and intestinal microsome from Gunn rats. These results indicate that the formation of EZE-G in Gunn rats is much lower than that in Wistar rats. However, in vivo study showed that 0.3 mg/kg EZE, which is the clinically relevant dose, reduced cholesterol absorption in both Wistar and Gunn rats to nearly the same degree and the dose dependence was not significantly different between Wistar and Gunn rats at the range 0.001 approximately 0.3 mg/kg. These results indicate that a deficiency of UGT1A activity does not necessarily alter the cholesterol-lowering effect of EZE in rats at therapeutic doses.