Science Pool

Prediction of drug-induced liver injury (DILI) is challenging. Translation from animals to humans is poor and manifestation of DILI can be complex mechanistically. 

In this poster, we focus on:

• transcriptomics analysis of 128 compounds form the FDA Liver Toxicity Knowledge Base (68 associated with DILI and 60 not associated with DILI)
• the use of machine learning in accurately predicting DILI and providing an insight into the mechanism of toxicity

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Cardiotoxicity is one of the most reported adverse effects that leads to pre-clinical and clinical drug failure. To tackle this, the International Conference of Harmonization (ICH) S7B guideline in 2005, proposed a non-clinical assessment of new drug entities using in vitro electrophysiology studies (typically hERG ion channel) and in vivo telemetry in animal models. Although these are very sensitive approaches, they may have also led to unwarranted drug attrition of many potentially valuable therapeutics due to the low specificity nature of the assays.

More recently, the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative was proposed by experts in the field and was established to move safety pharmacology towards in silico and in vitro approaches utilising new and emerging technologies such as stem-cell-derived cardiomyocytes. Building on this new safety paradigm, colleagues from Evotec and Cyprotex have worked collaboratively to develop a non-clinical model using cutting-edge techniques with improved predictive power to de-risk cardiotoxicity in early drug discovery. We have presented the output of this work in a research article titled “In-depth mechanistic analysis including high-throughput RNA sequencing in the prediction of functional and structural cardiotoxicants using hiPSC cardiomyocytes” published in a recent edition of Expert Opinion on Drug Metabolism and Toxicology.

In this article, we describe the use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as an in vitro model system together with three high-throughput technologies incorporating structural assays (high-content imaging, HCI), functional assays (Ca²⁺ transience, CaT) and high-throughput RNA-sequencing (ScreenSeq) for the pre-clinical risk assessment of novel compounds. The transcriptional responses of hiPSC-CMs to 24 h treatment with 33 cardiotoxicants (12 structural cardiotoxicants, 14 functional cardiotoxicants, 7 structural/functional cardiotoxicants) and 9 non-cardiotoxicants of mixed therapeutic indications were investigated and compound-induced differential gene expression (DEG) was calculated in comparison with vehicle treated controls. Likewise, the hiPSC-CMs responses to six structural readouts (cell count, cellular ATP, mitochondrial mass, mitochondrial membrane potential, calcium content, DNA structure and nuclear size) and four functional readouts (amplitude, frequency, peak width and decay time) were analysed. In summary, hiPSC-CMs recapitulated expected structural and functional toxicity mechanisms, validating their use as in vitro model system to detect and characterize modes of toxicity. ScreenSeq identified several molecular mechanisms of toxicity such as alterations in cardiac pathways, genotoxicity, ER stress and mitochondrial toxicity. Together, HCI, CaT and ScreenSeq provided the best cardiotoxicity prediction metrics (10x C_max: 100% specificity, 82% sensitivity, 86% accuracy; 25x C_max: 89% specificity, 91% sensi-tivity, 90% accuracy).

This study not only provides invaluable cardiotoxic mechanistic information of the drugs tested, but it also demonstrates the potential of this mechanism-driven risk assessment approach in predicting drug-induced cardiotoxicity in hiPSC-CMs.

Read the paper.

Read more about our Cardiotox Screen assay consisting of both a functional assay (examining the mechanical function of the cardiomyocytes) and a structural assay (assessing morphological changes and loss of viability).

In addition, discover more about our transcriptomics offerings here.

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Cardiotoxicity remains one of the most reported adverse drug reactions that lead to drug attrition during pre-clinical and clinical drug development. Drug-induced cardiotoxicity may develop as a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or as a structural change, resulting in loss of viability and morphological damage to cardiac tissue.

Our latest work, published in Expert Opinion on Drug Metabolism & Toxicology, is now available to download.

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• The genotoxic potential is one of the major risks associated to potential pitfalls in drug development.
• True positive results in the genotoxicity screening observed during the Lead Optimization phase not always represent a showstopper, but need to be carefully characterized.
• In this scenario, the toxicologist could provide a strategy supporting the next IND enabling phase by adopting the most effective risk management to allow the highest success rate possible.
• The process proposed can be adapted to any scenario, and provides data supporting the risk assessment ensuring the lowest attrition and the ethical use of animals.

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Blood microsampling is a less invasive and simplified alternative to traditional venipuncture for PK/TK sampling, used mainly in small-animal studies. The purpose of this work was to evaluate the possibility of using microsampling technique also to support PK/TK studies in non-human primates.
A comparison of plasma PK parameters was conducted by traditional blood collection from the femoral vein and microsampling from the tail vein of six non-naïve cynomolgus monkeys. Four drugs were selected for this comparison, based on acid-base properties and volume of distribution. 
The results obtained in this work, supported by robust statistics, demonstrated the suitability of microsampling in supporting PK/TK studies in non-human primates. 
The plasma exposures of the tested drugs are comparable for both sampling techniques and are not influenced by acid-base characteristics and volume of distribution. 
Microsampling used in non-human primates avoids the occurrence of hematomas at the animal sampling site and can also refine practices to limit pain and distress to which animals are exposed (refinement of 3Rs) and, as a result, may reduce the impact of animal stress on PK/TK readouts; moreover, it also provides significant advantages for animal technicians during in life handling.

To request a copy of the article, contact the authors. For Evotec: massimo.breda@evotec.com

Hepatic transporter inhibition, mitochondrial dysfunction, and reactive metabolite formation are some of the most common mechanisms associated with intrinsic DILI. The cytochrome P450 (CYP450) superfamily of enzymes play an important role in phase 1 metabolism within the liver. For certain chemical entities, reactive metabolites may form with increased toxicity compared to the parent. These reactive metabolites may result in hepatotoxicity through the formation of reactive oxygen species, DNA damage, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. In our research the pan-specific CYP450 inhibitor, 1-aminobenzotriazole (1-ABT), was used in combination with high-content imaging to evaluate the effects of potential reactive metabolites on cell health parameters in hepatocytes. The endpoint assessed included nuclear features, glutathione (GSH) content, mitochondrial dysfunction, and reactive oxygen species (ROS) formation, as well as cellular ATP content. A calculated fold-shift in cell health features between the plus and minus 1-ABT dosing conditions was used to determine reactive metabolite formation. A panel of known DILI reference compounds associated with the formation of reactive metabolites were assessed through this HCI bioactivation assay within metabolically competent HepaRG cells, primary human hepatocytes (PHH) and primary mouse hepatocyte (PMH).

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Peripheral neuropathy can be induced by many chemotherapeutics. Symptoms include numbness, tingling or abnormal sensations which can impact on the long-term quality of a patient’s life. Animal models used to evaluate these side effects may be difficult to interpret and are labor-intensive. In this study, we further optimized a previously developed DRG assay by comparing the neurite outgrowth responses of a group of chemotherapeutics from different classes at 24 h and 72 as an in vitro cell-based model for peripheral neuropathy. Cytotoxicity was assessed alongside neurite outgrowth. Taxanes (paclitaxel, docetaxel), microtubule interfering agents (vincristine, vinblastine, colchicine, nocodazole) and epothilones (ixabepilone) were assessed.

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The generation of anti-drug antibodies (ADA) towards a therapeutic agent can have severe implications with respect to drug safety and efficacy. Therefore, identification of this risk during the initial phase of development is imperative to improve both patient outcomes and downstream attrition rates. As preclinical species often fail to mimic the complexity of the human immune system, the immunogenic potency of therapeutic agents and their potential to elicit a human-specific proinflammatory response can be grossly underestimated during in vivo safety assessment. In our research, a high-throughput in vitro approach to immunogenicity screening was developed using two distinct models: i) peripheral blood mononuclear cells (PBMC) and ii) monocyte-derived dendritic cells (MoDC) isolated from the blood of healthy human donors. The effect of serum-free culture conditions was also evaluated. The immunogenicity of monoclonal antibodies, small molecules associated with delayed-onset hypersensitivity reactions and oligonucleotides were assessed in each model through lymphocyte proliferation and cytokine release.

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The periodic activation and inactivation of various cardiac ion channels enables the regular and synchronous contraction of the heart. Therefore, any disruption of the cardiac ion channel signaling network can result in a change in membrane potential and in the shape of cardiac action potential (AP). As a result, the cardiac excitation-contraction coupling can be modified which may lead to arrhythmias and potential patient death. Local Extracellular Action Potential (LEAP) is a technique which enables non-invasive, label-free monitoring of cardiac action potential in a high-throughput real-time format. In our research, we used the Axion LEAP MEA assay to analyse the effect of various selective and non-selective ion channel inhibitors on cardiac AP by quantification of action potential morphology, repolarization irregularities, and arrhythmic risk factors such as triangulation.

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Drug-induced cardiotoxicity may result from a functional change in cardiac electrophysiology (acute alteration of the mechanical function of the myocardium) and/or from a structural change, resulting in damage to the cardiac tissue. In our research, the effects of 42 reference compounds in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were investigated in a combined risk assessment strategy. Functional cardiotoxicity was evaluated through kinetic monitoring of calcium transients (CaT), while structural morphology changes and gross cytotoxicity were assessed using high-content imaging (HCI) and cellular ATP measurements. In addition, whole genome high-throughput RNA-sequencing (ScreenSeq) was performed in matched-sister plates. Data were analysed to determine differentially expressed genes (DEGs) and any associated perturbed pathways.

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