TAK-994 Mechanistic Investigation into Drug-Induced Liver Injury.

The frequency of drug-induced liver injury (DILI) in clinical trials remains a challenge for drug developers despite advances in human hepatotoxicity models and improvements in reducing liver-related attrition in preclinical species. TAK-994, an oral orexin receptor 2 agonist, was withdrawn from phase II clinical trials due to the appearance of severe DILI. Here, we investigate the likely mechanism of TAK-994 DILI in hepatic cell culture systems examined cytotoxicity, mitochondrial toxicity, impact on drug transporter proteins, and covalent binding.

Current Therapeutic Landscape and Safety Roadmap for Targeting the Aryl Hydrocarbon Receptor in Inflammatory Gastrointestinal Indications.

Target modulation of the AhR for inflammatory gastrointestinal (GI) conditions holds great promise but also the potential for safety liabilities both within and beyond the GI tract. The ubiquitous expression of the AhR across mammalian tissues coupled with its role in diverse signaling pathways makes development of a "clean" AhR therapeutically challenging. Ligand promiscuity and diversity in context-specific AhR activation further complicates targeting the AhR for drug development due to limitations surrounding clinical translatability.

Assessment of a 3D neural spheroid model to detect pharmaceutical-induced neurotoxicity.

Drug-induced neurotoxicity is a leading cause of safety-related attrition for therapeutics in clinical trials, often driven by poor predictivity of preclinical in vitro and in vivo models of neurotoxicity. Over a dozen different iPSC-derived 3D spheroids have been described in recent years, but their ability to predict neurotoxicity in patients has not been evaluated nor compared with the predictive power of nonclinical species. To assess the predictive capabilities of human iPSC-derived neural spheroids (microBrains), we used 84 structurally diverse pharmaceuticals with robust clinical and pre-clinical datasets with varying degrees of seizurogenic and neurodegenerative liability.

Human-relevant mechanisms and risk factors for TAK-875-Induced liver injury identified via a gene pathway-based approach in Collaborative Cross mice.

Development of TAK-875 was discontinued when a small number of serious drug-induced liver injury (DILI) cases were observed in Phase 3 clinical trials. Subsequent studies have identified hepatocellular oxidative stress, mitochondrial dysfunction, altered bile acid homeostasis, and immune response as mechanisms of TAK-875 DILI and the contribution of genetic risk factors in oxidative response and mitochondrial pathways to the toxicity susceptibility observed in patients. We tested the hypothesis that a novel preclinical approach based on gene pathway analysis in the livers of Collaborative Cross mice could be used to identify human-relevant mechanisms of toxicity and genetic risk factors at the level of the hepatocyte as reported in a human genome-wide association study.

Polygenic architecture informs potential vulnerability to drug-induced liver injury.

Drug-induced liver injury (DILI) is a leading cause of termination in drug development programs and removal of drugs from the market; this is partially due to the inability to identify patients who are at risk. In this study, we developed a polygenic risk score (PRS) for DILI by aggregating effects of numerous genome-wide loci identified from previous large-scale genome-wide association studies. The PRS predicted the susceptibility to DILI in patients treated with fasiglifam, amoxicillin-clavulanate or flucloxacillin and in primary hepatocytes and stem cell-derived organoids from multiple donors treated with over ten different drugs.

New phenotypic cytotoxicity assay for ROS-inducing compounds using rat renal epithelial cells.

An important mechanism of chemical toxicity is the induction of oxidative stress through the production of excess reactive oxygen species (ROS). In this study, we show that the level of drug-induced ROS production between NRK52E and HepG2 cells is significantly different for several marketed drugs and a number of Takeda's internal proprietary compounds. Nifedipine, a calcium channel blocker and the initial focus of the study, was demonstrated to promote in vitro ROS production and a decrease in cell viability in NRK52E cells but not HepG2 cells.

An Integrated Multi-Omics Analysis Defines Key Pathway Alterations in a Diet-Induced Obesity Mouse Model.

Obesity is a multifactorial disease with many complications and related diseases and has become a global epidemic. To thoroughly understand the impact of obesity on whole organism homeostasis, it is helpful to utilize a systems biological approach combining gene expression and metabolomics across tissues and biofluids together with metagenomics of gut microbial diversity. Here, we present a multi-omics study on liver, muscle, adipose tissue, urine, plasma, and feces on mice fed a high-fat diet (HFD).

Quantitative Systems Toxicology Analysis of In Vitro Mechanistic Assays Reveals Importance of Bile Acid Accumulation and Mitochondrial Dysfunction in TAK-875-Induced Liver Injury.

TAK-875 (fasiglifam), a GPR40 agonist in development for the treatment of type 2 diabetes (T2D), was voluntarily terminated in Phase III trials due to adverse liver effects. The potential mechanisms of TAK-875 toxicity were explored by combining in vitro experiments with quantitative systems toxicology (QST) using DILIsym, a mathematical representation of drug-induced liver injury. In vitro assays revealed that bile acid transporters were inhibited by both TAK-875 and its metabolite, TAK-875-Glu.

Navigating tissue chips from development to dissemination: A pharmaceutical industry perspective.

Tissue chips are poised to deliver a paradigm shift in drug discovery. By emulating human physiology, these chips have the potential to increase the predictive power of preclinical modeling, which in turn will move the pharmaceutical industry closer to its aspiration of clinically relevant and ultimately animal-free drug discovery. Despite the tremendous science and innovation invested in these tissue chips, significant challenges remain to be addressed to enable their routine adoption into the industrial laboratory.

Development of dietary soluble fibres by enzymatic synthesis and assessment of their digestibility in in vitro, animal and randomised clinical trial models.

The aim was to develop novel fibres by enzymatic synthesis, to determine their total dietary fibre by AOAC method 2009.01 and to estimate their potential digestibility and assess their digestibility in vivo using glycaemic and insulinaemic responses as markers in mice and randomised clinical trial models. We found that fibre candidates to which α-(1,2) branching was added were resistant to digestion in the mouse model, depending on the amount of branching.

Fasiglifam (TAK-875) Alters Bile Acid Homeostasis in Rats and Dogs: A Potential Cause of Drug Induced Liver Injury.

Fasiglifam (TAK-875), a Free Fatty Acid Receptor 1 (FFAR1) agonist in development for the treatment of type 2 diabetes, was voluntarily terminated in phase 3 due to adverse liver effects. A mechanistic investigation described in this manuscript focused on the inhibition of bile acid (BA) transporters as a driver of the liver findings. TAK-875 was an in vitro inhibitor of multiple influx (NTCP and OATPs) and efflux (BSEP and MRPs) hepatobiliary BA transporters at micromolar concentrations.

Doxorubicin: Comparison between 3-h continuous and bolus intravenous administration paradigms on cardio-renal axis, mitochondrial sphingolipids and pathology.

Doxorubicin (DOX) is a potent and effective broad-spectrum anthracycline antitumor agent, but its clinical usefulness is restricted by cardiotoxicity. This study compared pharmacokinetic, functional, structural and biochemical effects of single dose DOX bolus or 3-h continuous iv infusion (3-h iv) in the Han–Wistar rat to characterize possible treatment-related differences in drug safety over a 72 h observation period. Both DOX dosing paradigms significantly altered blood pressure, core body temperature and QA interval (indirect measure of cardiac contractility); however, there was no recovery observed in the bolus iv treatment group.

Early prediction of polymyxin-induced nephrotoxicity with next-generation urinary kidney injury biomarkers.

Despite six decades of clinical experience with the polymyxin class of antibiotics, their dose-limiting nephrotoxicity remains difficult to predict due to a paucity of sensitive biomarkers. Here, we evaluate the performance of standard of care and next-generation biomarkers of renal injury in the detection and monitoring of polymyxin-induced acute kidney injury in male Han Wistar rats using colistin (polymyxin E) and a polymyxin B (PMB) derivative with reduced nephrotoxicity, PMB nonapeptide (PMBN). This study provides the first histopathological and biomarker analysis of PMBN, an important test of the hypothesis that fatty acid modifications and charge reductions in polymyxins can reduce their nephrotoxicity.

Cellular impedance assays for predictive preclinical drug screening of kinase inhibitor cardiovascular toxicity.

Cardiovascular (CV) toxicity is a leading contributor to drug attrition. Implementing earlier testing has successfully reduced human Ether-à-go-go-Related Gene-related arrhythmias. How- ever, analogous assays targeting functional CV effects remain elusive.

Human induced pluripotent stem cells and their use in drug discovery for toxicity testing.

Predicting human safety risks of novel xenobiotics remains a major challenge, partly due to the limited availability of human cells to evaluate tissue-specific toxicity. Recent progress in the production of human induced pluripotent stem cells (hiPSCs) may fill this gap. hiPSCs can be continuously expanded in culture in an undifferentiated state and then differentiated to form most cell types.

Evaluation of cellular impedance measures of cardiomyocyte cultures for drug screening applications.

Cardiovascular toxicity is a leading contributor to drug withdrawal and late-stage attrition. Earlier and broader screening is a validated approach to build-in cardiovascular safety as demonstrated with human Ether-à-go-go-related gene (hERG) screening to reduce drug-induced arrhythmia. There is an urgent need for novel in vitro assays to address other mechanistic aspects of cardiovascular function, including contractility, heart rate, toxicity, hypertrophy, and non-hERG arrhythmia.

What do we need to know prior to thinking about incorporating an epigenetic evaluation into safety assessments?

The International Life Sciences Institute, Health and Environmental Sciences Institute sponsored a workshop entitled "State of the Science: Evaluating Epigenetic Changes," hosted by the National Institute of Environmental Health Sciences, Research Triangle Park, NC, 28-30 October 2009. The goal was to evaluate and enhance the scientific knowledge base regarding epigenetics and its role in disease, including potential relationships between epigenetic changes and transgenerational effects. A distinguishing aspect of the workshop was the highly interactive discussion session on the final morning.

The accurate prediction of protein family from amino acid sequence by measuring features of sequence fragments.

The rapid advances in proteomic analyses coupled with the completion of multiple genomes have led to an increased demand for determining protein functions. The first step is classification or prediction into families. A method was developed for the prediction of protein family based only on protein sequence using support vector machine (SVM) models.

Evaluations of the trans-sulfuration pathway in multiple liver toxicity studies.

Drug-induced liver injury has been associated with the generation of reactive metabolites, which are primarily detoxified via glutathione conjugation. In this study, it was hypothesized that molecules involved in the synthesis of glutathione would be diminished to replenish the glutathione depleted through conjugation reactions. Since S-adenosylmethionine (SAMe) is the primary source of the sulfur atom in glutathione, UPLC/MS and NMR were used to evaluate metabolites involved with the transulfuration pathway in urine samples collected during studies of eight liver toxic compounds in Sprague-Dawley rats.

Biomarkers of carcinogenicity and their roles in drug discovery and development.

The screening of drug candidates to assess their carcinogenic potential has long been a challenge for drug development. While genotoxic compounds can be readily detected with a battery of standard tests, including short-term in vitro and in vivo assays, predicting nongenotoxic carcinogenicity remains a major challenge. The 2-year rodent bioassay has been held as the gold standard for the assessment of carcinogenic risk to humans.

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS.

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously.

Interlaboratory evaluation of genomic signatures for predicting carcinogenicity in the rat.

The Critical Path Institute recently established the Predictive Safety Testing Consortium, a collaboration between several companies and the U.S. Food and Drug Administration, aimed at evaluating and qualifying biomarkers for a variety of toxicological endpoints.

Self-self hybridization as an alternative experiment design to dye swap for two-color microarrays.

Dye-specific bias effects, commonly observed in the two-color microarray platform, are normally corrected using the dye swap design. This design, however, is relatively expensive and labor-intensive. We propose a self-self hybridization design as an alternative to the dye swap design.

Differential gene expression in mouse primary hepatocytes exposed to the peroxisome proliferator-activated receptor alpha agonists.

Background: Fibrates are a unique hypolipidemic drugs that lower plasma triglyceride and cholesterol levels through their action as peroxisome proliferator-activated receptor alpha (PPARalpha) agonists. The activation of PPARalpha leads to a cascade of events that result in the pharmacological (hypolipidemic) and adverse (carcinogenic) effects in rodent liver.

Results: To understand the molecular mechanisms responsible for the pleiotropic effects of PPARalpha agonists, we treated mouse primary hepatocytes with three PPARalpha agonists (bezafibrate, fenofibrate, and WY-14,643) at multiple concentrations (0, 10, 30, and 100 microM) for 24 hours.