In vitro inhibition of the bile salt export pump correlates with risk of cholestatic drug-induced liver injury in humans.

Inhibition of the activity of the human bile salt export pump (BSEP: ABCB11) has been proposed to play a role in drug-induced liver injury (DILI). To enhance understanding of the relationship between BSEP inhibition and DILI, inhibition of human BSEP (hBSEP) and its rat ortholog (rBsep) by 85 pharmaceuticals was investigated in vitro. This was explored using assays that quantified inhibition of ATP-dependent [(3)H]taurocholate uptake into inverted plasma membrane vesicles from Sf21 insect cells, which expressed the proteins. Of the pharmaceuticals, 40 exhibited evidence of in vitro transporter inhibition and overall a close correlation was observed between potency values for inhibition of hBSEP and rBsep activity (r(2) = 0.94), although 12 drugs exhibited >2-fold more potent inhibition of hBSEP than rBsep. The median potency of hBSEP inhibition was higher among drugs that caused cholestatic/mixed DILI than among drugs that caused hepatocellular or no DILI, as was the incidence of hBSEP inhibition with IC(50) <300 µM. All drugs with hBSEP IC(50) <300 µM had molecular weight >250, ClogP >1.5, and nonpolar surface area >180Å. A clear distinction was not evident between hBSEP IC(50) or unbound plasma concentration (C(max, u)) of the drugs in humans and whether the drugs caused DILI. However, all 17 of the drugs with hBSEP IC(50) <100 µM and C(max, u) >0.002 µM caused DILI. Overall, these data indicate that inhibition of hBSEP/rBsep correlates with the propensity of numerous pharmaceuticals to cause cholestatic DILI in humans and is associated with several of their physicochemical properties.

Identification of the thiophene ring of methapyrilene as a novel bioactivation-dependent hepatic toxicophore.

Methapyrilene (MP), a 2-thiophene H(1)-receptor antagonist, is a model toxicant in the genomic and proteomic analyses of hepatotoxicity. In rats, it causes an unusual periportal necrosis that is hypothetically attributed to chemically reactive and cytotoxic metabolites. We have characterized the bioactivation of MP by hepatic microsomes and primary rat hepatocytes, and we established a possible causal linkage with cytotoxicity. Methapyrilene tritiated at C-2 of the diaminoethane moiety ([3H]MP) was metabolized via an NADPH-dependent pathway to intermediates that combined irreversibly with microsomes (rat > mouse approximately human). This binding was attenuated by the cytochrome P450 (P450) inhibitor 1-aminobenzotriazole and thiols but not by trapping agents for iminium ions and aldehydes. Reactive intermediates were trapped as thioether adducts of monooxygenated MP. Mass spectrometric and hydrogen/deuterium exchange analysis of the glutathione adduct produced by rat liver microsomes indicated that the metabolite was most probably a thioether of MP S-oxide substituted in the thiophene ring. The glutathione adduct was formed by rat hepatocytes and eliminated in bile by rats administered [3H]MP intravenously. MP produced concentration- and time-dependent cytotoxicity, depleted glutathione, and underwent irreversible binding to the hepatocytes before a significant increase in cell damage was observed. P450 inhibitors reduced turnover of the drug, production of the glutathione adduct, irreversible binding, and cytotoxicity but inhibited glutathione depletion selectively. MP underwent lesser turnover and bioactivation in mouse hepatocytes and was not cytotoxic. Analogs with phenyl and p-methoxyphenyl rings were much less hepatocytotoxic than MP. Hepatotoxicity in rats was diminished by predosing with 1-aminobenzotriazole. For the first time, a thiophene ring substituent is identified as a bioactivation-dependent toxicophore in hepatocytes.

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.

Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

Hepatic adducts, circulating antibodies, and cytokine polymorphisms in patients with diclofenac hepatotoxicity.

Diclofenac is a nonsteroidal anti-inflammatory drug that causes rare but serious hepatotoxicity, the mechanism of which is unclear. The purpose of the present study was to explore the potential role played by the immune processes. Antibodies to diclofenac metabolite-modified liver protein adducts were detected in the sera of seven out of seven patients with diclofenac-induced hepatotoxicity, 12 of 20 subjects on diclofenac without hepatotoxicity, and none of four healthy controls. The antibodies recognized adducts expressed in livers from rats treated with multiple doses of diclofenac, but not in those given single doses. In addition, several potential diclofenac adducts were identified in the liver of a patient with diclofenac-induced hepatic failure, but not from a normal human donor liver, by immunoblotting with an adduct-selective rabbit antiserum. To determine whether or not polymorphisms in genes encoding cytokine-related proteins influence susceptibility to hepatotoxicity, genotyping for the polymorphisms -627 in the interleukin (IL)-10 gene, -590 in the IL-4 gene, and codon 551 in the IL-4 receptor (IL-4R) were performed on DNA from 24 patients on diclofenac with hepatotoxicity, 48 subjects on diclofenac without hepatotoxicity, and healthy controls. The frequencies of the variant alleles for IL-10 and IL-4 were higher in patients (OR [odds ratio]: 2.8 for IL-10; 2.6 for IL-4; 5.3 for IL-10 + IL-4) compared with healthy controls and subjects on diclofenac without hepatotoxicity (OR: 2.8 for IL-10; 1.2 for IL-4; 5.0 for IL-10 + IL-4). In conclusion, the observed polymorphisms, resulting in low IL-10 and high IL-4 gene transcription, could favor a T helper (Th)-2 mediated antibody response to neoantigenic stimulation associated with disease susceptibility.