Cardiovascular Toxicity Induced by Kinase Inhibitors: Mechanisms and Preclinical Approaches.

Kinase inhibitors have transformed the treatment of many cancers and are showing the same promise for other indications including inflammatory diseases. This class of drugs is one of the most predominant in the pharmaceutical industry, but development and clinical utility is often limited by a broad spectrum of cardiovascular (CV) toxicities including QT prolongation and arrhythmia, left ventricular dysfunction, congestive heart failure, ischemia and myocardial infarction, and hypertension. In this review article, we provide a broad overview of the spectrum of CV events detected in clinical trials of kinase inhibitors and the known and proposed on- and off-target links between kinase inhibitor targets and these specific cardiotoxicities. We also examine the unique features of kinase inhibitors that have impeded complete mechanistic understanding of kinase inhibitor-associated cardiotoxicities and contributed to the disconnect between preclinical predictions and clinical findings. We then discuss various in vitro models currently in use that are amenable for high-throughput screening as well as lower throughput models that are valuable for mechanistic insight. These physiologically relevant models, together with newer "omic"-wide approaches will help to increase our understanding of the mechanisms underlying kinase inhibitor-associated cardiotoxicity and enable rational design of kinase inhibitors in the future.

Nonclinical safety assessment of epigenetic modulatory drugs: Current status and industry perspective.

Pharmaceutic products designed to perturb the function of epigenetic modulators have been approved by regulatory authorities for treatment of advanced cancer. While the predominant effort in epigenetic drug development continues to be in oncology, non-oncology indications are also garnering interest. A survey of pharmaceutical companies was conducted to assess the interest and concerns for developing small molecule direct epigenetic effectors (EEs) as medicines. Survey themes addressed (1) general levels of interest and activity with EEs as therapeutic agents, (2) potential safety concerns, and (3) possible future efforts to develop targeted strategies for nonclinical safety assessment of EEs. Thirteen companies contributed data to the survey. Overall, the survey data indicate the consensus opinion that existing ICH guidelines are effective and appropriate for nonclinical safety assessment activities with EEs. Attention in the framework of study design should, on a case by case basis, be considered for delayed or latent toxicities, carcinogenicity, reproductive toxicity, and the theoretical potential for transgenerational effects. While current guidelines have been appropriate for the nonclinical safety assessments of epigenetic targets, broader experience with a wide range of epigenetic targets will provide information to assess the potential need for new or revised risk assessment strategies for EE drugs.

Stem cells and stem cell-derived tissues and their use in safety assessment.

Toxicology has long relied on animal models in a tedious approach to understanding risk of exposure to an uncharacterized molecule. Stem cell-derived tissues can be made in high purity, quality, and quantity to enable a new approach to this problem. Currently, stem cell-derived tissues are primarily "generic" genetic backgrounds; the future will see the integration of various genetic backgrounds and complex three-dimensional models to create truly unique in vitro organoids. This minireview focuses on the state of the art of a number of stem cell-derived tissues and details their application in toxicology.

Stem Cell Research.

Stem cells have great potential in basic research and are being slowly integrated into toxicological research. This symposium provided an overview of the state of the field, stem cell models, described allogenic stem cell treatments and issues of immunogenicity associated with protein therapeutics, and tehn concentrated on stem cell uses in regenerative medicine focusing on lung and testing strategies on engineered tissues from a pathologist's perspective.

Immunogenicity Risk Assessment of Process-Related Impurities in An Engineered T Cell Receptor Cellular Product.

Cell therapies such as genetically modified T cells have emerged as a promising and viable treatment for hematologic cancers and are being aggressively pursued for a wide range of diseases and conditions that were previously difficult to treat or had no cure. The process development requires genetic modifications to T cells to express a receptor (engineered T cell receptor (eTCR)) of specific binding qualities to the desired target. Protein reagents utilized during the cell therapy manufacturing process, to facilitate these genetic modifications, are often present as process-related impurities at residual levels in the final drug product and can represent a potential immunogenicity risk upon infusion. This manuscript presents a framework for the qualification of an assay for assessing the immunogenicity risk of AA6 and Cas9 residuals. The same framework applies for other residuals; however, AAV6 and Cas9 were selected as they were residuals from the manufacturing of an engineered T cell receptor cellular product in development. The manuscript: 1) elucidates theoretical risks, 2) summarizes analytical data collected during process development, 3) describes the qualification of an in vitro human PBMC cytokine release assay to assess immunogenicity risk from cellular product associated process residuals; 4) identifies a multiplexed inflammatory innate and adaptive cytokine panel with pre-defined criteria using relevant positive controls; and 5) discusses qualification challenges and potential solutions for establishing meaningful thresholds. The assessment is not only relevant to establishing safe exposure levels of these residuals but also in guiding risk assessment and CMC strategy during the conduct of clinical trials.

High purity human-induced pluripotent stem cell-derived cardiomyocytes: electrophysiological properties of action potentials and ionic currents.

Human-induced pluripotent stem cells (hiPSCs) can differentiate into functional cardiomyocytes; however, the electrophysiological properties of hiPSC-derived cardiomyocytes have yet to be fully characterized. We performed detailed electrophysiological characterization of highly pure hiPSC-derived cardiomyocytes. Action potentials (APs) were recorded from spontaneously beating cardiomyocytes using a perforated patch method and had atrial-, nodal-, and ventricular-like properties. Ventricular-like APs were more common and had maximum diastolic potentials close to those of human cardiac myocytes, AP durations were within the range of the normal human electrocardiographic QT interval, and APs showed expected sensitivity to multiple drugs (tetrodotoxin, nifedipine, and E4031). Early afterdepolarizations (EADs) were induced with E4031 and were bradycardia dependent, and EAD peak voltage varied inversely with the EAD take-off potential. Gating properties of seven ionic currents were studied including sodium (I(Na)), L-type calcium (I(Ca)), hyperpolarization-activated pacemaker (I(f)), transient outward potassium (I(to)), inward rectifier potassium (I(K1)), and the rapidly and slowly activating components of delayed rectifier potassium (I(Kr) and I(Ks), respectively) current. The high purity and large cell numbers also enabled automated patch-clamp analysis. We conclude that these hiPSC-derived cardiomyocytes have ionic currents and channel gating properties underlying their APs and EADs that are quantitatively similar to those reported for human cardiac myocytes. These hiPSC-derived cardiomyocytes have the added advantage that they can be used in high-throughput assays, and they have the potential to impact multiple areas of cardiovascular research and therapeutic applications.

The electrophysiological effects of cardiac glycosides in human iPSC-derived cardiomyocytes and in guinea pig isolated hearts.

BACKGROUND/AIMS: Monitoring changes in the field potential (FP) of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) following compound administration has been proposed as a novel screening tool to evaluate cardiac ion channel interactions and QT liability. Here we extended the use of FP to evaluate the pharmacological and toxicological properties of cardiac glycosides. METHODS: FPs were recorded using microelectrode arrays (MEAs) in spontaneously beating hiPSC-CMs. The in vitro effects of ouabain and digoxin on FPs were compared with data generated on hemodynamic and ECG parameters in guinea pig Langendorff hearts. RESULTS: In hiPSC-CMs, ouabain and digoxin reduced Na(+)-spike amplitude, shortened FP duration (FPD), increased Ca(2+)-wave amplitude, and dose-dependently induced arrhythmic beats. The ouabain-induced changes observed in hiPSC-CMs correlated well with the effects seen in isolated hearts which revealed QT shortening, enhancement of contractility, and arrhythmogenesis. Nifedipine, an L-type Ca(2+) channel blocker, reduced Ca(2+)-wave amplitude and FPD in hiPSC-CMs, and led to parallel effects of decreased ventricular contractility and shortened QT interval in isolated hearts. Further, nifedipine attenuated the Ca(2+)-peak amplitude and proarrhythmic effect of both glycosides. These results suggested that FPD and Ca(2+)-wave amplitude are comparable surrogates of QT interval and contractility of intact hearts, respectively. CONCLUSION: hiPSC-CMs reflect similar cardiac pharmacology as seen in isolated cardiac preparations and thus are a suitable model in study of the pharmacology and toxicology of cardioactive ion channel and transporter modulators.

Characterization of xenobiotic-induced hepatocellular enzyme induction in rats: anticipated thyroid effects and unique pituitary gland findings.

During routine safety evaluation of RO2910, a non-nucleoside reverse transcriptase inhibitor for HIV infection, histopathology findings concurrent with robust hepatocellular induction occurred in multiple organs, including a unique, albeit related, finding in the pituitary gland. For fourteen days, male and female rats were administered, by oral gavage vehicle, 100, 300, or 1000 mg/kg/day of RO2910. Treated groups had elevated serum thyroid-stimulating hormone and decreased total thyroxine, and hypertrophy in the liver, thyroid gland, and pituitary pars distalis. These were considered consequences of hepatocellular induction and often were dose dependent and more pronounced in males than in females. Hepatocellular centrilobular hypertrophy corresponded with increased expression of cytochrome P450s 2B1/2, 3A1, and 3A2 and UGT 2B1. Bilateral thyroid follicular cell hypertrophy occurred concurrent to increased mitotic activity and sometimes colloid depletion, which were attributed to changes in thyroid hormone levels. Males had hypertrophy of thyroid-stimulating hormone-producing cells (thyrotrophs) in the pituitary pars distalis. All findings were consistent with the well-established adaptive physiologic response of rodents to xenobiotic-induced hepatocellular microsomal enzyme induction. Although the effects on the pituitary gland following hepatic enzyme induction-mediated hypothyroidism have not been reported previously, other models of stress and thyroid depletion leading to pituitary stimulation support such a shared pathogenesis.

A functional three-dimensional microphysiological human model of myeloma bone disease.

Human myeloma bone disease (MBD) occurs when malignant plasma cells migrate to the bone marrow and commence inimical interactions with stromal cells, disrupting the skeletal remodeling process. The myeloma cells simultaneously suppress osteoblastic bone formation while promoting excessive osteoclastic resorption. This bone metabolism imbalance produces osteolytic lesions that cause chronic bone pain and reduce trabecular and cortical bone structural integrity, and often culminate in pathological fractures. Few bone models exist that enable scientists to study MBD and the effect therapies have on restoring the bone metabolism imbalance. The purpose of this research was to develop a well characterized three-dimensional (3D) bone organoid that could be used to study MBD and current or potential treatment options. First, bone marrow stromal cell-derived osteoblasts (OBs) mineralized an endosteal-like extracellular matrix (ECM) over 21 days. Multiple analyses confirmed the generation of hydroxyapatite (HA)-rich bone-like tissue fragments that were abundant in alkaline phosphatase, calcium, and markers of osteoblastic gene expression. On day 22, bone marrow macrophage (BMM)-derived osteoclasts (OCs) were introduced to enhance the resorptive capability of the model and recapitulate the balanced homeostatic nature of skeletal remodeling. Tartrate-resistant acid phosphatase 5b (TRAcP-5b), type I collagen C-telopeptide (CTX-1), and gene expression analysis confirmed OC activity in the normal 3D organoid (3D in vitro model of normal bonelike fragments [3D-NBF]). On day 30, a human multiple myeloma (MM)-derived plasmacytoma cell line was introduced to the 3D-NBF to generate the 3D-myeloma bone disease organoid (3D-MBD). After 12 days, the 3D-MBD had significantly reduced total HA, increased TRAcP-5b levels, increases levels of CTX-1, and decreased expression of osteoblastic genes. Therapeutic intervention with pharmaceutical agents including an immunomodulatory drug, a bisphosphonate, and monoclonal restored HA content and reduced free CTX-1 in a dose-dependent manner. This osteogenically functional model of MBD provides a novel tool to study biological mechanisms guiding the disease and to screen potential therapeutics. © 2021 American Society for Bone and Mineral Research (ASBMR).

Investigation Into the Role of ERK in Tyrosine Kinase Inhibitor-Induced Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common and debilitating adverse event that can alter patient treatment options and halt candidate drug development. A case study is presented here describing the preclinical and clinical development of CC-90003, a small molecule extracellular signal-regulated kinase (ERK)1/2 inhibitor investigated as an oncology therapy. In a Phase Ia clinical trial, CC-90003 elicited adverse drug-related neuropathy and neurotoxicity that contributed to discontinued development of CC-90003 for oncology therapy. Preclinical evaluation of CC-90003 in dogs revealed clinical signs and electrophysiological changes consistent with peripheral neuropathy that was reversible. Mice did not exhibit signs of neuropathy upon daily dosing with CC-90003, supporting that rodents generally poorly predict CIPN. We sought to investigate the mechanism of CC-90003-induced peripheral neuropathy using a phenotypic in vitro assay. Translating preclinical neuropathy findings to humans proves challenging as no robust in vitro models of CIPN exist. An approach was taken to examine the influence of CIPN-associated drugs on human-induced pluripotent stem cell-derived peripheral neuron (hiPSC-PN) electrophysiology on multielectrode arrays (MEAs). The MEA assay with hiPSC-PNs was sensitive to CIPN-associated drugs cisplatin, sunitinib, colchicine, and importantly, to CC-90003 in concordance with clinical neuropathy incidence. Biochemical data together with in vitro MEA data for CC-90003 and 12 of its structural analogs, all having similar ERK inhibitory activity, revealed that CC-90003 disrupted in vitro neuronal electrophysiology likely via on-target ERK inhibition combined with off-target kinase inhibition and translocator protein inhibition. This approach could prove useful for assessing CIPN risk and interrogating mechanisms of drug-induced neuropathy.

Cardiovascular microphysiological systems (CVMPS) for safety studies - a pharma perspective.

The integrative responses of the cardiovascular (CV) system are essential for maintaining blood flow to provide oxygenation, nutrients, and waste removal for the entire body. Progress has been made in independently developing simple in vitro models of two primary components of the CV system, namely the heart (using induced pluripotent stem-cell derived cardiomyocytes) and the vasculature (using endothelial cells and smooth muscle cells). These two in vitro biomimics are often described as immature and simplistic, and typically lack the structural complexity of native tissues. Despite these limitations, they have proven useful for specific "fit for purpose" applications, including early safety screening. More complex in vitro models offer the tantalizing prospect of greater refinement in risk assessments. To this end, efforts to physically link cardiac and vascular components to mimic a true CV microphysiological system (CVMPS) are ongoing, with the goal of providing a more holistic and integrated CV response model. The challenges of building and implementing CVMPS in future pharmacological safety studies are many, and include a) the need for more complex (and hence mature) cell types and tissues, b) the need for more realistic vasculature (within and across co-modeled tissues), and c) the need to meaningfully couple these two components to allow for integrated CV responses. Initial success will likely come with simple, bioengineered tissue models coupled with fluidics intended to mirror a vascular component. While the development of more complex integrated CVMPS models that are capable of differentiating safe compounds and providing mechanistic evaluations of CV liabilities may be feasible, adoption by pharma will ultimately hinge on model efficiency, experimental reproducibility, and added value above current strategies.

Identification of a kinase profile that predicts chromosome damage induced by small molecule kinase inhibitors.

Kinases are heavily pursued pharmaceutical targets because of their mechanistic role in many diseases. Small molecule kinase inhibitors (SMKIs) are a compound class that includes marketed drugs and compounds in various stages of drug development. While effective, many SMKIs have been associated with toxicity including chromosomal damage. Screening for kinase-mediated toxicity as early as possible is crucial, as is a better understanding of how off-target kinase inhibition may give rise to chromosomal damage. To that end, we employed a competitive binding assay and an analytical method to predict the toxicity of SMKIs. Specifically, we developed a model based on the binding affinity of SMKIs to a panel of kinases to predict whether a compound tests positive for chromosome damage. As training data, we used the binding affinity of 113 SMKIs against a representative subset of all kinases (290 kinases), yielding a 113x290 data matrix. Additionally, these 113 SMKIs were tested for genotoxicity in an in vitro micronucleus test (MNT). Among a variety of models from our analytical toolbox, we selected using cross-validation a combination of feature selection and pattern recognition techniques: Kolmogorov-Smirnov/T-test hybrid as a univariate filter, followed by Random Forests for feature selection and Support Vector Machines (SVM) for pattern recognition. Feature selection identified 21 kinases predictive of MNT. Using the corresponding binding affinities, the SVM could accurately predict MNT results with 85% accuracy (68% sensitivity, 91% specificity). This indicates that kinase inhibition profiles are predictive of SMKI genotoxicity. While in vitro testing is required for regulatory review, our analysis identified a fast and cost-efficient method for screening out compounds earlier in drug development. Equally important, by identifying a panel of kinases predictive of genotoxicity, we provide medicinal chemists a set of kinases to avoid when designing compounds, thereby providing a basis for rational drug design away from genotoxicity.

Thalidomide Inhibits Human iPSC Mesendoderm Differentiation by Modulating CRBN-dependent Degradation of SALL4.

Exposure to thalidomide during a critical window of development results in limb defects in humans and non-human primates while mice and rats are refractory to these effects. Thalidomide-induced teratogenicity is dependent on its binding to cereblon (CRBN), the substrate receptor of the Cul4A-DDB1-CRBN-RBX1 E3 ubiquitin ligase complex. Thalidomide binding to CRBN elicits subsequent ubiquitination and proteasomal degradation of CRBN neosubstrates including SALL4, a transcription factor of which polymorphisms phenocopy thalidomide-induced limb defects in humans. Herein, thalidomide-induced degradation of SALL4 was examined in human induced pluripotent stem cells (hiPSCs) that were differentiated either to lateral plate mesoderm (LPM)-like cells, the developmental ontology of the limb bud, or definitive endoderm. Thalidomide and its immunomodulatory drug (IMiD) analogs, lenalidomide, and pomalidomide, dose-dependently inhibited hiPSC mesendoderm differentiation. Thalidomide- and IMiD-induced SALL4 degradation can be abrogated by CRBN V388I mutation or SALL4 G416A mutation in hiPSCs. Genetically modified hiPSCs expressing CRBN E377V/V388I mutant or SALL4 G416A mutant were insensitive to the inhibitory effects of thalidomide, lenalidomide, and pomalidomide on LPM differentiation while retaining sensitivity to another known limb teratogen, all-trans retinoic acid (atRA). Finally, disruption of LPM differentiation by atRA or thalidomide perturbed subsequent chondrogenic differentiation in vitro. The data here show that thalidomide, lenalidomide, and pomalidomide affect stem cell mesendoderm differentiation through CRBN-mediated degradation of SALL4 and highlight the utility of the LPM differentiation model for studying the teratogenicity of new CRBN modulating agents.

Human ileal organoid model recapitulates clinical incidence of diarrhea associated with small molecule drugs.

Drug-induced gastrointestinal toxicity (GIT) is a common treatment-emergent adverse event that can negatively impact dosing, thereby limiting efficacy and treatment options for patients. An in vitro assay of GIT is needed to address patient variability, mimic the microphysiology of the gut, and accurately predict drug-induced GIT. Primary human ileal organoids (termed 'enteroids') have proven useful for stimulating intestinal stem cell proliferation and differentiation to multiple cell types present in the gut epithelium. Enteroids have enabled characterization of gut biology and the signaling involved in the pathogenesis of disease. Here, enteroids were differentiated from four healthy human donors and assessed for culture duration-dependent differentiation status by immunostaining for gut epithelial markers lysozyme, chromogranin A, mucin, and sucrase isomaltase. Differentiated enteroids were evaluated with a reference set of 31 drugs exhibiting varying degrees of clinical incidence of diarrhea, a common manifestation of GIT that can be caused by drug-induced thinning of the gut epithelium. An assay examining enteroid viability in response to drug treatment demonstrated 90% accuracy for recapitulating the incidence of drug-induced diarrhea. The human enteroid viability assay developed here presents a promising in vitro model for evaluating drug-induced diarrhea.

Developing in vitro assays to transform gastrointestinal safety assessment: potential for microphysiological systems.

Drug-induced gastrointestinal toxicities (DI-GITs) are among the most common adverse events in clinical trials. High prevalence of DI-GIT has persisted among new drugs due in part to the lack of robust experimental tools to allow early detection or to guide optimization of safer molecules. Developing in vitro assays for the leading GI toxicities (nausea, vomiting, diarrhoea, constipation, and abdominal pain) will likely involve recapitulating complex physiological properties that require contributions from diverse cell/tissue types including epithelial, immune, microbiome, nerve, and muscle. While this stipulation may be beyond traditional 2D monocultures of intestinal cell lines, emerging 3D GI microtissues capture interactions between diverse cell and tissue types. These interactions give rise to microphysiologies fundamental to gut biology. For GI microtissues, organoid technology was the breakthrough that introduced intestinal stem cells with the capability of differentiating into each of the epithelial cell types and that self-organize into a multi-cellular tissue proxy with villus- and crypt-like domains. Recently, GI microtissues generated using miniaturized devices with microfluidic flow and cyclic peristaltic strain were shown to induce Caco2 cells to spontaneously differentiate into each of the principle intestinal epithelial cell types. Second generation models comprised of epithelial organoids or microtissues co-cultured with non-epithelial cell types can successfully reproduce cross-'tissue' functional interactions broadening the potential of these models to accurately study drug-induced toxicities. A new paradigm in which in vitro assays become an early part of GI safety assessment could be realized if microphysiological systems (MPS) are developed in alignment with drug-discovery needs. Herein, approaches for assessing GI toxicity of pharmaceuticals are reviewed and gaps are compared with capabilities of emerging GI microtissues (e.g., organoids, organ-on-a-chip, transwell systems) in order to provide perspective on the assay features needed for MPS models to be adopted for DI-GIT assessment.

Evaluation of the GreenScreen GADD45alpha-GFP indicator assay with non-proprietary and proprietary compounds.

The GreenScreen GADD45alpha indicator assay has been assessed for its concordance with in vitro genotoxicity and rodent carcinogenicity bioassay data. To test robustness, sensitivity, and specificity of the assay, 91 compounds with known genotoxicity results were screened in a blinded manner. Fifty seven of the compounds were classified as in vitro genotoxic whereas 34 were non-genotoxic. Out of the 91 compounds, 50 had been tested in 2-year carcinogenicity assays, with 33 identified to be rodent carcinogens and 17 non-carcinogens. Gadd45alpha assay sensitivity and specificity for genotoxicity was 30% and 97%, respectively (17/57 and 33/34), whereas its sensitivity and specificity for rodent carcinogenicity was 30% and 88%, respectively (10/33 and 15/17). Gadd45alpha assay genotoxicity results from this validation study exhibited a high concordance with previously published results as well as for compound test results generated at two different sites (91%, 19/21), indicating that the assay is both robust and reproducible. In conclusion, results from this blinded and independent validation study indicate that the GreenScreen GADD45 indicator assay is reproducible and reliable with low sensitivity and high specificity for identifying genotoxic and carcinogenic compounds.

The state-of-the-art in predictive toxicogenomics.

Predictive toxicogenomics, ie, the acquisition of advanced knowledge of the safety profile of a compound using genomic biomarkers, is a technology that provides much optimism for improving early drug discovery decisions. Toxicogenomics creates an opportunity to shift attrition to earlier stages in drug development to a point where course-corrective action can be taken with relatively lower financial costs, thus improving the efficiency of the drug development process. This review will survey the current state-of-the-art in toxicogenomics for predicting toxicity, both in vivo and in vitro, with emphasis on the use of classification algorithms and the importance of toxicogenomic databases for biomarker discovery and validation.

Preclinical drug safety analysis by chemogenomic profiling in the liver.

The economic hurdles of drug development and the emergence of genomic technologies such as chemogenomics are combining to shift the existing paradigms in preclinical drug development. Today, the information gleaned from high content molecular data has begun to augment traditional approaches to the assessment of drug safety. The optimal approach is a hybrid strategy employing chemogenomic data and gene expression-based biomarkers of drug efficacy and toxicity to supplement low content and insensitive methods for risk assessment and mechanistic evaluation of drug candidates. Large reference databases of chemogenomic data are essential to the derivation and validation of accurate and predictive gene expression biomarkers. An example of the development of a predictive biomarker for hepatic bile duct hyperplasia is described herein. As gene expression technologies improve, biomarkers will achieve higher throughput, and become more cost effective and increasingly accurate. This will elevate the value of chemogenomics in drug development, shift attrition to earlier in the process, and reduce the overall cost of drug development. Over the past 2 to 3 years, the transition of chemogenomics from a research tool to a decision-making tool has begun and regulatory agencies are anxiously awaiting implementation of this technology to make faster and more informed evaluations of potential drugs.

Development of a large-scale chemogenomics database to improve drug candidate selection and to understand mechanisms of chemical toxicity and action.

Successful drug discovery requires accurate decision making in order to advance the best candidates from initial lead identification to final approval. Chemogenomics, the use of genomic tools in pharmacology and toxicology, offers a promising enhancement to traditional methods of target identification/validation, lead identification, efficacy evaluation, and toxicity assessment. To realize the value of chemogenomics information, a contextual database is needed to relate the physiological outcomes induced by diverse compounds to the gene expression patterns measured in the same animals. Massively parallel gene expression characterization coupled with traditional assessments of drug candidates provides additional, important mechanistic information, and therefore a means to increase the accuracy of critical decisions. A large-scale chemogenomics database developed from in vivo treated rats provides the context and supporting data to enhance and accelerate accurate interpretation of mechanisms of toxicity and pharmacology of chemicals and drugs. To date, approximately 600 different compounds, including more than 400 FDA approved drugs, 60 drugs approved in Europe and Japan, 25 withdrawn drugs, and 100 toxicants, have been profiled in up to 7 different tissues of rats (representing over 3200 different drug-dose-time-tissue combinations). Accomplishing this task required evaluating and improving a number of in vivo and microarray protocols, including over 80 rigorous quality control steps. The utility of pairing clinical pathology assessments with gene expression data is illustrated using three anti-neoplastic drugs: carmustine, methotrexate, and thioguanine, which had similar effects on the blood compartment, but diverse effects on hepatotoxicity. We will demonstrate that gene expression events monitored in the liver can be used to predict pathological events occurring in that tissue as well as in hematopoietic tissues.