ABSTRACT
Many compounds that appear promising in preclinical species, fail in human clinical trials due to safety concerns. The FDA has strongly encouraged the application of modeling in drug development to improve product safety. This study illustrates how DILIsym, a computational representation of liver injury, was able to reproduce species differences in liver toxicity due to PF-04895162 (ICA-105665). PF-04895162, a drug in development for the treatment of epilepsy, was terminated after transaminase elevations were observed in healthy volunteers (NCT01691274). Liver safety concerns had not been raised in preclinical safety studies. DILIsym, which integrates in vitro data on mechanisms of hepatotoxicity with predicted in vivo liver exposure, reproduced clinical hepatotoxicity and the absence of hepatotoxicity observed in the rat. Simulated differences were multifactorial. Simulated liver exposure was greater in humans than rats. The simulated human hepatotoxicity was demonstrated to be due to the interaction between mitochondrial toxicity and bile acid transporter inhibition; elimination of either mechanism from the simulations abrogated injury. The bile acid contribution occurred despite the fact that the IC50 for bile salt export pump (BSEP) inhibition by PF-04895162 was higher (311 µmol/L) than that has been generally thought to contribute to hepatotoxicity. Modeling even higher PF-04895162 liver exposures than were measured in the rat safety studies aggravated mitochondrial toxicity but did not result in rat hepatotoxicity due to insufficient accumulation of cytotoxic bile acid species. This investigative study highlights the potential for combined in vitro and computational screening methods to identify latent hepatotoxic risks and paves the way for similar and prospective studies.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 11/antagonists & inhibitors , Anticonvulsants/toxicity , Chemical and Drug Induced Liver Injury/pathology , Models, Biological , Quinazolines/toxicity , ATP Binding Cassette Transporter, Subfamily B, Member 11/metabolism , Administration, Oral , Adolescent , Adult , Animals , Anticonvulsants/administration & dosage , Chemical and Drug Induced Liver Injury/etiology , Computer Simulation , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/standards , Epilepsy/drug therapy , HEK293 Cells , Healthy Volunteers , Hepatocytes , Humans , Inhibitory Concentration 50 , Liver/drug effects , Liver/pathology , Male , Middle Aged , Mitochondria/drug effects , Quinazolines/administration & dosage , Rats , Species Specificity , Taurocholic Acid/metabolism , Young AdultABSTRACT
To reduce costly late-stage compound attrition, there has been an increased focus on assessing compounds in in vitro assays that predict attributes of human safety liabilities, before preclinical in vivo studies are done. Relevant questions when choosing a panel of assays for predicting toxicity are (a) whether there is general concordance in the data among the assays, and (b) whether, in a retrospective analysis, the rank order of toxicity of compounds in the assays correlates with the known safety profile of the drugs in humans. The aim of our study was to answer these questions using nonsteroidal anti-inflammatory drugs (NSAIDs) as a test set since NSAIDs are generally associated with gastrointestinal injury, hepatotoxicity, and/or cardiovascular risk, with mitochondrial impairment and endoplasmic reticulum stress being possible contributing factors. Eleven NSAIDs, flufenamic acid, tolfenamic acid, mefenamic acid, diclofenac, meloxicam, sudoxicam, piroxicam, diflunisal, acetylsalicylic acid, nimesulide, and sulindac (and its two metabolites, sulindac sulfide and sulindac sulfone), were tested for their effects on (a) the respiration of rat liver mitochondria, (b) a panel of mechanistic endpoints in rat hepatocytes, and (c) the viability and organ morphology of zebrafish. We show good concordance for distinguishing among/between NSAID chemical classes in the observations among the three approaches. Furthermore, the assays were complementary and able to correctly identify "toxic" and "non-toxic" drugs in accordance with their human safety profile, with emphasis on hepatic and gastrointestinal safety. We recommend implementing our multi-assay approach in the drug discovery process to reduce compound attrition.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/toxicity , Drug Evaluation, Preclinical/methods , Hepatocytes/drug effects , Mitochondria, Liver/drug effects , Toxicity Tests/methods , Zebrafish , Animals , Cell Survival/drug effects , Cells, Cultured , Embryo, Nonmammalian/drug effects , Embryo, Nonmammalian/pathology , Hepatocytes/enzymology , Hepatocytes/metabolism , Hepatocytes/pathology , Male , Membrane Potential, Mitochondrial/drug effects , Mitochondria, Liver/enzymology , Mitochondria, Liver/metabolism , Mitochondria, Liver/pathology , No-Observed-Adverse-Effect Level , Oxygen Consumption/drug effects , Primary Cell Culture , Rats , Zebrafish/embryologyABSTRACT
Drug-induced mitochondrial dysfunction is known to contribute to late stage compound attrition. Recently, assays that identify mitochondrial dysfunction have been developed but many require expensive reagents, specialized equipment, or specialized expertise such as isolation of mitochondria. Here, we validate a new 384-well format cell-based dual parameter assay that uses commonly available detection methods to measure both mitochondrial toxicity and cytotoxicity. In our initial evaluation, antimycin A, CCCP, nefazodone, flutamide, and digitonin were tested in K562 cells in both glucose- and galactose-supplemented media with a 2h incubation. The assay was able to correctly differentiate these compounds into mitochondrial toxicants and non-mitochondrial toxicants, and had excellent reproducibility. We next tested 74 compounds in K562 cells in both types of media and show that the assay was able to correctly identify some of the compounds as mitochondrial toxicants. Moreover, the assay could be simplified, without loss of information, by using K562 cells in galactose-containing medium alone. This simple, robust assay can be positioned as a rapid, early readout of mitochondrial and cellular toxicity. However, since the assay fails to identify some mitochondrial toxicants, further assays may be required to detect mitochondrial toxicity once lead compounds have been selected.
Subject(s)
Drug-Related Side Effects and Adverse Reactions , High-Throughput Screening Assays , Mitochondria/drug effects , Biological Assay , Cell Death , Cell Line , Drug Evaluation, Preclinical , Hepatocytes , Humans , K562 Cells , Myocytes, Cardiac , Reproducibility of Results , Stem Cells/cytologyABSTRACT
Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/EiJ, with the latter two being sequenced for the first time. The bioenergetic profile of the four strains of MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial function were tested for their effect on the ATP content of the four strains of MEFs in both glucose- and galactose-containing media. Our results showed that there were strain-dependent differences in the response to some of the drugs. We propose that this model is a useful starting point to study compounds that may cause mitochondrial off-target toxicity in early stages of drug development, thus decreasing the number of experimental animals used.
Subject(s)
DNA, Mitochondrial/metabolism , Embryo, Mammalian/metabolism , Energy Metabolism/drug effects , Energy Metabolism/genetics , Fibroblasts/metabolism , Mitochondria/metabolism , Polymorphism, Single Nucleotide/genetics , Polymorphism, Single Nucleotide/physiology , Adenosine Triphosphate/metabolism , Animals , Cell Proliferation/drug effects , Cell Survival/drug effects , Citrate (si)-Synthase/metabolism , DNA, Mitochondrial/drug effects , Electron Transport Complex I/drug effects , Electron Transport Complex I/metabolism , Electron Transport Complex IV/drug effects , Electron Transport Complex IV/metabolism , Embryo, Mammalian/drug effects , Fibroblasts/drug effects , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Oxygen Consumption/drug effects , Rats, Inbred Strains , Rotenone/pharmacology , Species Specificity , Uncoupling Agents/pharmacologyABSTRACT
High-throughput applicable screens for identifying drug-induced mitochondrial impairment are necessary in the pharmaceutical industry. Hence, we evaluated the XF96 Extracellular Flux Analyzer, a 96-well platform that measures changes in the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of cells. The sensitivity of the platform was bench-marked with known modulators of oxidative phosphorylation and glycolysis. Sixteen therapeutic agents were screened in HepG2 cells for mitochondrial effects. Four of these compounds, thiazolidinediones, were also tested in primary feline cardiomyocytes for cell-type specific effects. We show that the XF96 platform is a robust, sensitive system for analyzing drug-induced mitochondrial impairment in whole cells. We identified changes in cellular respiration and acidification upon addition of therapeutic agents reported to have a mitochondrial effect. Furthermore, we show that respiration and acidification changes upon addition of the thiazoldinediones were cell-type specific, with the rank order of mitochondrial impairment in whole cells being in accord with the known adverse effects of these drugs.
Subject(s)
Mitochondria, Heart/metabolism , Myocytes, Cardiac/metabolism , Oxygen Consumption/drug effects , Thiazolidinediones/pharmacology , Animals , Cats , Drug Evaluation, Preclinical/methods , Female , Glycolysis/drug effects , Hep G2 Cells , Humans , Male , Mitochondria, Heart/pathology , Myocytes, Cardiac/pathology , Oxidative Phosphorylation/drug effectsABSTRACT
Mitochondrial dysfunction has been shown to be a pharmacotoxicological response to a variety of currently-marketed drugs. In order to reduce attrition due to mitochondrial toxicity, high throughput-applicable screens are needed for early stage drug discovery. We describe, here, a set of immunocapture based assays to identify compounds that directly inhibit four of the oxidative phosphorylation (OXPHOS) complexes: I, II, IV, and V. Intra- and inter-assay variation were determined and specificity tested by using classical mitochondrial inhibitors. Twenty drugs, some with known mitochondrial toxicity and others with no known mitochondrial liability, were studied. Direct inhibition of one or more of the OXPHOS complexes was identified for many of the drugs. Novel information was obtained for several drugs including ones with previously unknown effects on oxidative phosphorylation. A major advantage of the immunocapture approach is that it can be used throughout drug screening from early compound evaluation to clinical trials.