Drug-induced liver injury (DILI) is the most common adverse event causing drug nonapprovals and drug withdrawals. Using drugs as test agents and measuring a panel of cellular phenotypes that are ...directly linked to key mechanisms of hepatotoxicity, we have developed an in vitro testing strategy that is predictive of many clinical outcomes of DILI. Mitochondrial damage, oxidative stress, and intracellular glutathione, all measured by high content cellular imaging in primary human hepatocyte cultures, are the three most important features contributing to the hepatotoxicity prediction. When applied to over 300 drugs and chemicals including many that caused rare and idiosyncratic liver toxicity in humans, our testing strategy has a true-positive rate of 50–60% and an exceptionally low false-positive rate of 0–5%. These in vitro predictions can augment the performance of the combined traditional preclinical animal tests by identifying idiosyncratic human hepatotoxicants such as nimesulide, telithromycin, nefazodone, troglitazone, tetracycline, sulindac, zileuton, labetalol, diclofenac, chlorzoxazone, dantrolene, and many others. Our findings provide insight to key DILI mechanisms, and suggest a new approach in hepatotoxicity testing of pharmaceuticals.
Drug-induced liver injury (DILI) is one of the most important reasons for drug development failure at both preapproval and postapproval stages. There has been increased interest in developing ...predictive in vivo, in vitro, and in silico models to identify compounds that cause idiosyncratic hepatotoxicity. In the current study, we applied machine learning, a Bayesian modeling method with extended connectivity fingerprints and other interpretable descriptors. The model that was developed and internally validated (using a training set of 295 compounds) was then applied to a large test set relative to the training set (237 compounds) for external validation. The resulting concordance of 60%, sensitivity of 56%, and specificity of 67% were comparable to results for internal validation. The Bayesian model with extended connectivity functional class fingerprints of maximum diameter 6 (ECFC_6) and interpretable descriptors suggested several substructures that are chemically reactive and may also be important for DILI-causing compounds, e.g., ketones, diols, and α-methyl styrene type structures. Using Smiles Arbitrary Target Specification (SMARTS) filters published by several pharmaceutical companies, we evaluated whether such reactive substructures could be readily detected by any of the published filters. It was apparent that the most stringent filters used in this study, such as the Abbott alerts, which captures thiol traps and other compounds, may be of use in identifying DILI-causing compounds (sensitivity 67%). A significant outcome of the present study is that we provide predictions for many compounds that cause DILI by using the knowledge we have available from previous studies. These computational models may represent cost-effective selection criteria before in vitro or in vivo experimental studies.
Mitochondrial toxicity is increasingly implicated in a host of drug-induced organ toxicities, including hepatotoxicity. Nefazodone was withdrawn from the U.S. market in 2004 due to hepatotoxicity. ...Accordingly, we evaluated nefazodone, another triazolopyridine trazodone, plus the azaspirodecanedione buspirone, for cytotoxicity and effects on mitochondrial function. In accord with its clinical disposition, nefazodone was the most toxic compound of the three, trazodone had relatively modest effects, whereas buspirone showed the least toxicity. Nefazodone profoundly inhibited mitochondrial respiration in isolated rat liver mitochondria and in intact HepG2 cells where this was accompanied by simultaneous acceleration of glycolysis. Using immunocaptured oxidative phosphorylation (OXPHOS) complexes, we identified Complex 1, and to a lesser amount Complex IV, as the targets of nefazodone toxicity. No inhibition was found for trazodone, and buspirone showed 3.4-fold less inhibition of OXPHOS Complex 1 than nefazodone. In human hepatocytes that express cytochrome P450, isoform 3A4, after 24 h exposure, nefazodone and trazodone collapsed mitochondrial membrane potential, and imposed oxidative stress, as detected via glutathione depletion, leading to cell death. Our results suggest that the mitochondrial impairment imposed by nefazodone is profound and likely contributes to its hepatotoxicity, especially in patients cotreated with other drugs with mitochondrial liabilities.
While drug toxicity (especially hepatotoxicity) is the most frequent reason cited for withdrawal of an approved drug, no simple solution exists to adequately predict such adverse events. Simple ...cytotoxicity assays in HepG2 cells are relatively insensitive to human hepatotoxic drugs in a retrospective analysis of marketed pharmaceuticals. In comparison, a panel of pre-lethal mechanistic cellular assays hold the promise to deliver a more sensitive approach to detect endpoint-specific drug toxicities. The panel of assays covered by this review includes steatosis, cholestasis, phospholipidosis, reactive intermediates, mitochondria membrane function, oxidative stress, and drug interactions. In addition, the use of metabolically competent cells or the introduction of major human hepatocytes in these in vitro studies allow a more complete picture of potential drug side effect. Since inter-individual therapeutic index (TI) may differ from patient to patient, the rational use of one or more of these cellular assay and targeted in vivo exposure data may allow pharmaceutical scientists to select drug candidates with a higher TI potential in the drug discovery phase.
Idiosyncratic drug hepatotoxicity represents a major problem in drug development due to inadequacy of current preclinical screening assays, but recently established rodent models utilizing bacterial ...LPS co-administration to induce an inflammatory background have successfully reproduced idiosyncratic hepatotoxicity signatures for certain drugs. However, the low-throughput nature of these models renders them problematic for employment as preclinical screening assays. Here, we present an analogous, but high-throughput,
in vitro approach in which drugs are administered to a variety of cell types (primary human and rat hepatocytes and the human HepG2 cell line) across a landscape of inflammatory contexts containing LPS and cytokines TNF, IFNγ, IL-1α, and IL-6. Using this assay, we observed drug–cytokine hepatotoxicity synergies for multiple idiosyncratic hepatotoxicants (ranitidine, trovafloxacin, nefazodone, nimesulide, clarithromycin, and telithromycin) but not for their corresponding non-toxic control compounds (famotidine, levofloxacin, buspirone, and aspirin). A larger compendium of drug–cytokine mix hepatotoxicity data demonstrated that hepatotoxicity synergies were largely potentiated by TNF, IL-1α, and LPS within the context of multi-cytokine mixes. Then, we screened 90 drugs for cytokine synergy in human hepatocytes and found that a significantly larger fraction of the idiosyncratic hepatotoxicants (19%) synergized with a single cytokine mix than did the non-hepatotoxic drugs (3%). Finally, we used an information theoretic approach to ascertain especially informative subsets of cytokine treatments for most highly effective construction of regression models for drug- and cytokine mix-induced hepatotoxicities across these cell systems. Our results suggest that this drug–cytokine co-treatment approach could provide a useful preclinical tool for investigating inflammation-associated idiosyncratic drug hepatotoxicity.
The present study examined the interaction of four 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (atorvastatin, lovastatin, and simvastatin in acid and lactone forms, and pravastatin in ...acid form only) with multidrug resistance gene 1 (MDR1, ABCB1) P-glycoprotein, multidrug resistance-associated protein 2 (MRP2, ABCC2), and organic anion-transporting polypeptide 1B1 (OATP1B1, SLCO21A6). P-glycoprotein substrate assays were performed using Madin-Darby canine kidney (MDCK) cells expressing MDR1, and the efflux ratios the ratio of the ratio of basolateral-to-apical apparent permeability and apical-to-basolateral permeability between MDR1 and MDCK were 1.87, 2.32/4.46, 2.17/3.17, and 0.93/2.00 for pravastatin, atorvastatin (lactone/acid), lovastatin (lactone/acid), and simvastatin (lactone/acid), respectively, indicating that these compounds are weak or moderate substrates of P-glycoprotein. In the inhibition assays (MDR1, MRP2, Mrp2, and OATP1B1), the IC50 values for efflux transporters (MDR1, MRP2, and Mrp2) were >100 microM for all statins in acid form except lovastatin acid (>33 microM), and the IC50 values were up to 10-fold lower for the corresponding lactone forms. In contrast, the IC50 values for the uptake transporter OATP1B1 were 3- to 7-fold lower for statins in the acid form compared with the corresponding lactone form. These data demonstrate that lactone and acid forms of statins exhibit differential substrate and inhibitor activities toward efflux and uptake transporters. The interconversion between the lactone and acid forms of most statins exists in the body and will potentially influence drug-transporter interactions, and may ultimately contribute to the differences in pharmacokinetic profiles observed between statins.
Incorporating phenotypic screening as a key strategy enhances predictivity and translatability of drug discovery efforts. Cellular imaging serves as a "phenotypic anchor" to identify important ...toxicologic pathology that encompasses an array of underlying mechanisms, thus provides an effective means to reduce drug development failures due to insufficient safety. This mini-review highlights the latest advances in hepatotoxicity, cardiotoxicity, and genetic toxicity tests that utilized cellular imaging as a screening strategy, and recommends path forward for further improvement.
OATP1B1 (a.k.a. OATP-C, OATP2, LST-1, or SLC21A6) is a liver-specific organic anion uptake transporter and has been shown to be a higher affinity bilirubin uptake transporter than OATP1B3. Using ...human embryonic kidney (HEK 293) cells stably transfected with OATP1B1, we have studied the effects of indinavir, saquinavir, cyclosporin A, and rifamycin SV on human OATP1B1 transport function. These drugs are potent inhibitors of OATP1B1 transport activity in vitro. We further provide evidence that the calculated fraction of OATP1B1 inhibited at the clinical exposure level correlated very well with the observed hyperbilirubinemia outcome for these drugs in humans. Our data support the hypothesis that inhibition of OATP1B1 is an important mechanism for drug-induced unconjugated hyperbilirubinemia. Inhibition of OATPs may be an important mechanism in drug–drug and drug–endogenous substance interactions.
CP-724,714, a potent and selective orally active HER2 tyrosine kinase inhibitor, was discontinued from clinical development due to unexpected hepatotoxicity in cancer patients. Based on the clinical ...manifestation of the toxicity, CP-724,714 likely exerted its hepatotoxicity via both hepatocellular injury and hepatobiliary cholestatic mechanisms. The direct cytotoxic effect, hepatobiliary disposition of CP-724,714, and its inhibition of active canalicular transport of bile constituents were evaluated in established human hepatocyte models and in vitro transporter systems. CP-724,714 exhibited direct cytotoxicity using human hepatocyte imaging assay technology with mitochondria identified as a candidate organelle for its off-target toxicity. Additionally, CP-724,714 was rapidly taken up into human hepatocytes, partially via an active transport process, with an uptake clearance approximately fourfold higher than efflux clearance. The major human hepatic uptake transporter, OATP1B1, and efflux transporters, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein, were involved in hepatobiliary clearance of CP-724,714. Furthermore, CP-724,714 displayed a concentration-dependent inhibition of cholyl-lysyl fluorescein and taurocholate (TC) efflux into canaliculi in cryopreserved and fresh cultured human hepatocytes, respectively. Likewise, CP-724,714 inhibited TC transport in membrane vesicles expressing human bile salt export pump with an IC50 of 16μM. Finally, CP-724,714 inhibited the major efflux transporter in bile canaliculi, MDR1, with an IC50 of ∼28μM. These results suggest that inhibition of hepatic efflux transporters contributed to hepatic accumulation of drug and bile constituents leading to hepatocellular injury and hepatobiliary cholestasis. This study provides likely explanations for clinically observed adverse liver effects of CP-724,714.
Major reasons preventing many early candidates reaching market are the inappropriate ADME (absorption, distribution, metabolism and excretion) properties and drug-induced toxicity. From a commercial ...perspective, it is desirable that poorly behaved compounds are removed early in the discovery phase rather than during the more costly drug development phases. As a consequence, over the past decade, ADME and toxicity (ADMET) screening studies have been incorporated earlier in the drug discovery phase. The intent of this review is to introduce the desirable attributes of a new chemical entity (NCE) to the medicinal chemist from an ADMET perspective. Fundamental concepts, key tools, reagents and experimental approaches used by the drug metabolism scientist to aid a modern project team in predicting human pharmacokinetics and assessing the "drug-like" molecule are discussed.
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