Drug-induced liver injury is the most common cause of market withdrawal of pharmaceuticals, and thus, there is considerable need for better prediction models for DILI early in drug discovery. We present a study involving 223 marketed drugs (51% associated with clinical hepatotoxicity; 49% non-hepatotoxic) to assess the concordance of in vitro bioactivation data with clinical hepatotoxicity and have used these data to develop a decision tree to help reduce late-stage candidate attrition. Data to assess P450 metabolism-dependent inhibition (MDI) for all common drug-metabolizing P450 enzymes were generated for 179 of these compounds, GSH adduct data generated for 190 compounds, covalent binding data obtained for 53 compounds, and clinical dose data obtained for all compounds. Individual data for all 223 compounds are presented here and interrogated to determine what level of an alert to consider termination of a compound. The analysis showed that 76% of drugs with a daily dose of <100 mg were non-hepatotoxic (p < 0.0001). Drugs with a daily dose of ≥100 mg or with GSH adduct formation, marked P450 MDI, or covalent binding ≥200 pmol eq/mg protein tended to be hepatotoxic (∼ 65% in each case). Combining dose with each bioactivation assay increased this association significantly (80-100%, p < 0.0001). These analyses were then used to develop the decision tree and the tree tested using 196 of the compounds with sufficient data (49% hepatotoxic; 51% non-hepatotoxic). The results of these outcome analyses demonstrated the utility of the tree in selectively terminating hepatotoxic compounds early; 45% of the hepatotoxic compounds evaluated using the tree were recommended for termination before candidate selection, whereas only 10% of the non-hepatotoxic compounds were recommended for termination. An independent set of 10 GSK compounds with known clinical hepatotoxicity status were also assessed using the tree, with similar results.
Bile salt export pump (BSEP) inhibition has been proposed to be an important mechanism for drug-induced liver injury (DILI). Modeling can prioritize knowledge gaps concerning bile acid (BA) homeostasis and thus help guide experimentation. A submodel of BA homeostasis in rats and humans was constructed within DILIsym, a mechanistic model of DILI. In vivo experiments in rats with glibenclamide were conducted, and data from these experiments were used to validate the model. The behavior of DILIsym was analyzed in the presence of a simulated theoretical BSEP inhibitor. BSEP inhibition in humans is predicted to increase liver concentrations of conjugated chenodeoxycholic acid (CDCA) and sulfate-conjugated lithocholic acid (LCA) while the concentration of other liver BAs remains constant or decreases. On the basis of a sensitivity analysis, the most important unknowns are the level of BSEP expression, the amount of intestinal synthesis of LCA, and the magnitude of farnesoid-X nuclear receptor (FXR)-mediated regulation.
The apical sodium-dependent bile acid transporter (ASBT) transports bile salts from the lumen of the gastrointestinal (GI) tract to the liver via the portal vein. Multiple pharmaceutical companies have exploited the physiological link between ASBT and hepatic cholesterol metabolism, which led to the clinical investigation of ASBT inhibitors as lipid-lowering agents. While modest lipid effects were demonstrated, the potential utility of ASBT inhibitors for treatment of type 2 diabetes has been relatively unexplored. We initiated a lead optimization effort that focused on the identification of a potent, nonabsorbable ASBT inhibitor starting from the first-generation inhibitor 264W94 (1). Extensive SAR studies culminated in the discovery of GSK2330672 (56) as a highly potent, nonabsorbable ASBT inhibitor which lowers glucose in an animal model of type 2 diabetes and shows excellent developability properties for evaluating the potential therapeutic utility of a nonabsorbable ASBT inhibitor for treatment of patients with type 2 diabetes.
This study makes use of whole embryo culture to investigate the potential embryotoxicity of benzyloxycarbonyl-phenylalanine-alanine-diazomethane (Z-Phe-Ala-CHN2) and benzyloxycarbonyl-phenylalanine-phenylalanine-diazomethane (Z-Phe-Phe-CHN2), two low molecular weight, active site-directed and irreversible inhibitors of the lysosomal cysteine proteinases. Peptidyl diazomethanes are the most specific inhibitors available for lysosomal cysteine proteinases and can be hypothesized to interrupt visceral yolk sac (VYS)-mediated nutrition during early organogenesis. When added directly to the culture medium of gestational day 10-11 rat conceptuses, both compounds inhibited lysosomal cysteine proteinase activity in the VYS in a concentration-dependent fashion that correlated with the degree of embryotoxicity observed. Z-Phe-Ala-CHN2 and Z-Phe-Phe-CHN2 were also found to increase the protein content of the VYS, even though all other conceptual growth parameters decreased. This effect was dependent on the serum content of the culture medium and the exposure time. Histological examination of Z-Phe-Ala-CHN2-treated conceptuses revealed a dramatic increase in the size and number of vacuoles in the VYS endoderm epithelium, suggestive of inhibition of VYS proteolysis. At the same time, excessive cell death was observed throughout the neuroepithelium and in specific regions of the mesenchyme of the corresponding embryos. This cell death manifested morphological characteristics of apoptosis and could be detected by supravital staining with Nile Blue Sulphate. These findings provide additional evidence in support of the hypothesis that lysosomal cysteine proteinases play a critical role in VYS-mediated histiotrophic nutrition and suggest that peptidyl diazomethanes may be useful in further characterization of these enzymes. The possible direct effects of these inhibitors on embryonic cells and the relationships between interruption of VYS-mediated nutritional processes and embryonic cell death are discussed.
Current evidence suggests that drug-induced liver disease can be caused by an allergic response (drug-induced allergic hepatitis, DIAH) induced by hepatic drug-protein adducts. The relatively low incidence of these reactions has led us to hypothesize that tolerogenic mechanisms prevent DIAH from occurring in most people. Here, we present evidence for the existence of one of these regulatory pathways. Following a hepatotoxic dose of acetaminophen in C57Bl/6 mice, lymphocyte loss that appeared to be due at least in part to apoptosis was noted in the spleen, thymus, and draining lymph nodes of the liver. There was no observable lymphocyte loss in the absence of hepatotoxicity. Acetaminophen-induced liver injury (AILI) also led to a functional suppression of the immune system as determined by the inhibition of a delayed-type hypersensitivity response to dinitrochlorobenzene. Further studies with adrenalectomized mice suggested a role for corticosterone in the depletion of lymphocytes following APAP-induced liver injury. In conclusion, these findings suggest that lymphocyte loss and immunosuppression following AILI may prevent subsequent occurrences of allergic hepatitis and possibly other forms of APAP-induced allergies induced by hepatic drug-protein adducts. Similar regulatory pathways may inhibit other hepatotoxic drugs from causing allergic reactions.
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