Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing.

The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor. Technical error rates (∼1 × 10-3) of NGS obscure the true abundance of somatic mutations, which can exist at per-nucleotide frequencies ≤1 × 10-7 Using duplex sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by three carcinogens in five tissues of two strains of mice within 31 d following exposure. We observed a strong correlation between mutation induction measured by duplex sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified a primordial marker of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex sequencing can be broadly applied to basic mutational research, regulatory safety testing, and emerging clinical applications.

Nonclinical Safety Profile of Etelcalcetide, a Novel Peptide Calcimimetic for the Treatment of Secondary Hyperparathyroidism.

Etelcalcetide is a novel d-amino acid peptide that functions as an allosteric activator of the calcium-sensing receptor and is being developed as an intravenous calcimimetic for the treatment of secondary hyperparathyroidism in patients with chronic kidney disease on hemodialysis. To support clinical development and marketing authorization, a comprehensive nonclinical safety package was generated. Primary adverse effects included hypocalcemia, tremoring, and convulsions. Other adverse effects were considered sequelae of stress associated with hypocalcemia. Cardiovascular safety evaluations in the dog revealed an anticipated prolongation of the corrected QT interval that was related to reductions in serum calcium. Etelcalcetide did not affect the human ether-a-go-go gene ion channel current. Etelcalcetide was mutagenic in some strains of Salmonella, however, based on the negative results in 2 in vitro and 2 in vivo mammalian genotoxicity assays, including a 28-day Muta mouse study, etelcalcetide is considered nongenotoxic. Further support for a lack of genotoxicity was provided due to the fact that etelcalcetide was not carcinogenic in a 6-month transgenic rasH2 mouse model or a 2-year study in rats. There were no effects on fertility, embryo-fetal development, and prenatal and postnatal development. All of the adverse effects observed in both rat and dog were considered directly or secondarily related to the pharmacologic activity of etelcalcetide and the expected sequelae associated with dose-related reductions in serum calcium due to suppression of parathyroid hormone secretion. These nonclinical data indicate no safety signal of concern for human risk beyond that associated with hypocalcemia and associated QT prolongation.

Retinal Toxicity Induced by a Novel ß-secretase Inhibitor in the Sprague-Dawley Rat.

ß-Secretase 1 (BACE1) represents an attractive target for the treatment of Alzheimer's disease. In the course of development of a novel small molecule BACE1 inhibitor (AMG-8718), retinal thinning was observed in a 1-month toxicity study in the rat. To further understand the lesion, an investigational study was conducted whereby rats were treated daily with AMG-8718 for 1 month followed by a 2-month treatment-free phase. The earliest detectable change in the retina was an increase in autofluorescent granules in the retinal pigment epithelium (RPE) on day 5; however, there were no treatment-related light microscopic changes observed in the neuroretina and no changes observed by fundus autofluorescence or routine ophthalmoscopic examination after 28 days of dosing. Following 2 months of recovery, there was significant retinal thinning attributed to loss of photoreceptor nuclei from the outer nuclear layer. Electroretinographic changes were observed as early as day 14, before any microscopic evidence of photoreceptor loss. BACE1 knockout rats were generated and found to have normal retinal morphology indicating that the retinal toxicity induced by AMG-8718 was likely off-target. These results suggest that AMG-8718 impairs phagolysosomal function in the rat RPE, which leads to photoreceptor dysfunction and ultimately loss of photoreceptors.

Evaluation of genotoxicity testing of FDA approved large molecule therapeutics.

Large molecule therapeutics (MW>1000daltons) are not expected to enter the cell and thus have reduced potential to interact directly with DNA or related physiological processes. Genotoxicity studies are therefore not relevant and typically not required for large molecule therapeutic candidates. Regulatory guidance supports this approach; however there are examples of marketed large molecule therapeutics where sponsors have conducted genotoxicity studies. A retrospective analysis was performed on genotoxicity studies of United States FDA approved large molecule therapeutics since 1998 identified through the Drugs@FDA website. This information was used to provide a data-driven rationale for genotoxicity evaluations of large molecule therapeutics. Fifty-three of the 99 therapeutics identified were tested for genotoxic potential. None of the therapeutics tested showed a positive outcome in any study except the peptide glucagon (GlucaGen®) showing equivocal in vitro results, as stated in the product labeling. Scientific rationale and data from this review indicate that testing of a majority of large molecule modalities do not add value to risk assessment and support current regulatory guidance. Similarly, the data do not support testing of peptides containing only natural amino acids. Peptides containing non-natural amino acids and small molecules in conjugated products may need to be tested.

Using human genetics to improve safety assessment of therapeutics.

Human genetics research has discovered thousands of proteins associated with complex and rare diseases. Genome-wide association studies (GWAS) and studies of Mendelian disease have resulted in an increased understanding of the role of gene function and regulation in human conditions. Although the application of human genetics has been explored primarily as a method to identify potential drug targets and support their relevance to disease in humans, there is increasing interest in using genetic data to identify potential safety liabilities of modulating a given target. Human genetic variants can be used as a model to anticipate the effect of lifelong modulation of therapeutic targets and identify the potential risk for on-target adverse events. This approach is particularly useful for non-clinical safety evaluation of novel therapeutics that lack pharmacologically relevant animal models and can contribute to the intrinsic safety profile of a drug target. This Review illustrates applications of human genetics to safety studies during drug discovery and development, including assessing the potential for on- and off-target associated adverse events, carcinogenicity risk assessment, and guiding translational safety study designs and monitoring strategies. A summary of available human genetic resources and recommended best practices is provided. The challenges and future perspectives of translating human genetic information to identify risks for potential drug effects in preclinical and clinical development are discussed.

Raf inhibition causes extensive multiple tissue hyperplasia and urinary bladder neoplasia in the rat.

Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney. A seven-day time course study in rats showed morphologic evidence of epithelial proliferation in the nonglandular stomach within four to five hours after a single dose of C-1. Similar indications of cellular proliferation were observed in the urinary bladder by day 2 and in the heart, kidney, and liver by day 3. Transcriptional evidence of proliferation in the urinary bladder was detected within four to five hours after a single dose consistent with activation of the PI3K/AKT and ERK/MAPK pathways. In a twenty-eight-day rat toxicity study of C-1, hyperplasia was observed in the esophagus, nonglandular stomach, skin, urinary bladder, kidney, and heart. Hyperplasia of transitional epithelium of the urinary bladder was particularly severe and in one female rat was accompanied by the presence of a transitional cell carcinoma. These results suggest that these Raf inhibitors induce early transcriptional changes driving unchecked cell proliferation, resulting in marked tissue hyperplasia that can progress to carcinoma within a short time frame.

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.

Biomarkers of genome instability in normal mammalian genomes following drug-induced replication stress.

Genome instability is a hallmark of most human cancers and is exacerbated following replication stress. However, the effects that drugs/xenobiotics have in promoting genome instability including chromosomal structural rearrangements in normal cells are not currently assessed in the genetic toxicology battery. Here, we show that drug-induced replication stress leads to increased genome instability in vitro using proliferating primary human cells as well as in vivo in rat bone marrow (BM) and duodenum (DD). p53-binding protein 1 (53BP1, biomarker of DNA damage repair) nuclear bodies were increased in a dose-dependent manner in normal proliferating human mammary epithelial fibroblasts following treatment with compounds traditionally classified as either genotoxic (hydralazine) and nongenotoxic (low-dose aphidicolin, duvelisib, idelalisib, and amiodarone). Comparatively, no increases in 53BP1 nuclear bodies were observed in nonproliferating cells. Negative control compounds (mannitol, alosteron, diclofenac, and zonisamide) not associated with cancer risk did not induce 53BP1 nuclear bodies in any cell type. Finally, we studied the in vivo genomic consequences of drug-induced replication stress in rats treated with 10 mg/kg of cyclophosphamide for up to 14 days followed by polymerase chain reaction-free whole genome sequencing (30X coverage) of BM and DD cells. Cyclophosphamide induced chromosomal structural rearrangements at an average of 90 genes, including 40 interchromosomal/intrachromosomal translocations, within 2 days of treatment. Collectively, these data demonstrate that this drug-induced genome instability test (DiGIT) can reveal potential adverse effects of drugs not otherwise informed by standard genetic toxicology testing batteries. These efforts are aligned with the food and drug administration's (FDA's) predictive toxicology roadmap initiative.

Romiplostim (Nplate®) as an effective radiation countermeasure to improve survival and platelet recovery in mice.

Purpose: Rapid depletion of white blood cells, platelets, and reticulocytes are hallmarks of hematopoietic injury of acute radiation syndrome (H-ARS) and, if left untreated, can lead to severe health consequences including death. While the granulocyte colony stimulating factors (G-CSF) filgrastim (Neupogen®), pegfilgrastim (Neulasta®), and sargramostim (Leukine®) are approved to increase survival in patients exposed to a myelosuppressive dose of radiation, no medical countermeasure is currently available for treatment of the thrombocytopenia that also results following radiation exposure. Romiplostim (Nplate®), a thrombopoietin receptor agonist, is the first FDA-approved thrombopoiesis-stimulating protein for the treatment of low platelet (PLT) counts in adults with chronic immune thrombocytopenia. Herein, we present the results of an analysis in mice of romiplostim as a medical countermeasure to improve survival and PLT recovery following acute radiation.Materials and methods: Male and female C57BL/6J mice (11 - 12 weeks of age, n = 21/sex/group) were total body irradiated (TBI) with 6.8 Gy X-rays that reduces 30-day survival to 30% (LD70/30). Vehicle, romiplostim, and/or pegfilgrastim were administered subcutaneously beginning 24 h after TBI for 1-5 days. Evaluation parameters included 30-day survival, pharmacokinetics, and hematology.Results: Full or maximal efficacy with an ∼40% increase in survival was achieved after a single 30 µg/kg dose of romiplostim. No further survival benefit was seen with higher (100 µg/kg) or more frequent dosing (3 or 5 once daily doses at 30 µg/kg) of romiplostim or combined treatment with pegfilgrastim. Pharmacodynamic analysis revealed that the platelet nadir was not as low and recovery was faster in the irradiated mice treated with romiplostim when compared with irradiated control animals (Day 8 versus 10 nadir; Day 22 versus 29 recovery to near baseline). Platelet volume also increased more rapidly after romiplostim injection. Kinetic profiles of other hematology parameters were similar between TBI romiplostim-treated and control mice. Peak serum levels of romiplostim in TBI mice occurred 4 - 24 h (Tmax) after injection with a t1/2 of ∼24 h. Cmax values were at ∼6 ng/ml after 30 µg/kg ± TBI and ∼200 ng/ml after 300 µg/kg. A 10-fold higher romiplostim dose increased the AUClast values by ∼35-fold.Conclusion: A single injection of romiplostim administered 24 h after TBI is a promising radiation medical countermeasure that dramatically increased survival, with or without pegfilgrastim, and hastened PLT recovery in mice.

Pharmacodynamics of romiplostim alone and in combination with pegfilgrastim on acute radiation-induced thrombocytopenia and neutropenia in non-human primates.

Purpose: Evaluation of the pharmacodynamics (PD) and pharmacokinetics (PK) of romiplostim alone and in combination with pegfilgrastim in a non-human primate (NHP) model of acute radiation syndrome (ARS).Materials and methods: Male and female rhesus macaques were subjected to Cobalt-60 γ irradiation, at a dose of 550 cGy 24 h prior to subcutaneous administration of either romiplostim alone as a single (2.5 or 5.0 mg/kg on Day 1) or repeat dose (5.0 mg/kg on Days 1 and 8), pegfilgrastim alone as a repeat dose (0.3 µg/kg on Day 1 and 8), or a combination of both agents (romiplostim 5.0 mg/kg on Day 1; pegfilgrastim 0.3 µg/kg on Days 1 and 8). Clinical outcome, hematological parameters and PK were assessed throughout the 45 d study period post-irradiation.Results: Administration of romiplostim, pegfilgrastim or the combination of both resulted in significant improvements in hematological parameters, notably prevention of severe thrombocytopenia, compared with irradiated, vehicle control-treated NHPs. The largest hematologic benefit was observed when romiplostim and pegfilgrastim were administered as a combination therapy with much greater effects on both platelet and neutrophil recovery following irradiation compared to single agents alone.Conclusions: These results indicate that romiplostim alone or in combination with pegfilgrastim is effective at improving hematological parameters in an NHP model of ARS. This study supports further study of romiplostim as a medical countermeasure to improve primary hemostasis and survival in ARS.

The Key Characteristics of Carcinogens: Relationship to the Hallmarks of Cancer, Relevant Biomarkers, and Assays to Measure Them.

The key characteristics (KC) of human carcinogens provide a uniform approach to evaluating mechanistic evidence in cancer hazard identification. Refinements to the approach were requested by organizations and individuals applying the KCs. We assembled an expert committee with knowledge of carcinogenesis and experience in applying the KCs in cancer hazard identification. We leveraged this expertise and examined the literature to more clearly describe each KC, identify current and emerging assays and in vivo biomarkers that can be used to measure them, and make recommendations for future assay development. We found that the KCs are clearly distinct from the Hallmarks of Cancer, that interrelationships among the KCs can be leveraged to strengthen the KC approach (and an understanding of environmental carcinogenesis), and that the KC approach is applicable to the systematic evaluation of a broad range of potential cancer hazards in vivo and in vitro We identified gaps in coverage of the KCs by current assays. Future efforts should expand the breadth, specificity, and sensitivity of validated assays and biomarkers that can measure the 10 KCs. Refinement of the KC approach will enhance and accelerate carcinogen identification, a first step in cancer prevention.See all articles in this CEBP Focus section, "Environmental Carcinogenesis: Pathways to Prevention."

Cardiovascular response to small-molecule APJ activation.

Heart failure (HF) remains a grievous illness with poor prognosis even with optimal care. The apelin receptor (APJ) counteracts the pressor effect of angiotensin II, attenuates ischemic injury, and has the potential to be a novel target to treat HF. Intravenous administration of apelin improves cardiac function acutely in patients with HF. However, its short half-life restricts its use to infusion therapy. To identify a longer acting APJ agonist, we conducted a medicinal chemistry campaign, leading to the discovery of potent small-molecule APJ agonists with comparable activity to apelin by mimicking the C-terminal portion of apelin-13. Acute infusion increased systolic function and reduced systemic vascular resistance in 2 rat models of impaired cardiac function. Similar results were obtained in an anesthetized but not a conscious canine HF model. Chronic oral dosing in a rat myocardial infarction model reduced myocardial collagen content and improved diastolic function to a similar extent as losartan, a RAS antagonist standard-of-care therapy, but lacked additivity with coadministration. Collectively, this work demonstrates the feasibility of developing clinical, viable, potent small-molecule agonists that mimic the endogenous APJ ligand with more favorable drug-like properties and highlights potential limitations for APJ agonism for this indication.

The liver pharmacological and xenobiotic gene response repertoire.

We have used a supervised classification approach to systematically mine a large microarray database derived from livers of compound-treated rats. Thirty-four distinct signatures (classifiers) for pharmacological and toxicological end points can be identified. Just 200 genes are sufficient to classify these end points. Signatures were enriched in xenobiotic and immune response genes and contain un-annotated genes, indicating that not all key genes in the liver xenobiotic responses have been characterized. Many signatures with equal classification capabilities but with no gene in common can be derived for the same phenotypic end point. The analysis of the union of all genes present in these signatures can reveal the underlying biology of that end point as illustrated here using liver fibrosis signatures. Our approach using the whole genome and a diverse set of compounds allows a comprehensive view of most pharmacological and toxicological questions and is applicable to other situations such as disease and development.

Validation of putative genomic biomarkers of nephrotoxicity in rats.

Drug-induced renal injury is a common finding in the early preclinical phase of drug development. But the specific genes responding to renal injury remain poorly defined. Identification of drug-induced gene changes is critical to provide insights into molecular mechanisms and detection of renal damage. To identify genes associated with the development of drug-induced nephrotoxicity, a literature survey was conducted and a panel of 48 genes was selected based on gene expression changes in multiple published studies. Male Sprague-Dawley rats were dosed daily for 1, 3 or 5 days to the known nephrotoxicants gentamicin, bacitracin, vancomycin and cisplatin, or the known hepatotoxicants ketoconazole, 1-naphthyl isothiocyanate and 4,4-diaminodiphenylmethane. Histopathological evaluation and clinical chemistry revealed renal proximal tubular necrosis in rats treated with the nephrotoxicants, but not from those treated with the hepatotoxicants. RNA was extracted from the kidney, and RT-PCR was performed to evaluate expression profiles of the selected genes. Among the genes examined, 24 genes are confirmed to be highly induced or repressed in rats treated with nephrotoxicants; further investigation identified that 5 of the 24 genes were also altered by hepatotoxicants. These data led to the identification of a set of genomic biomarker candidates whose expression in kidney is selectively regulated only by nephrotoxicants. Among those genes displaying the highest expression changes specifically in nephrotoxicant-treated rats were kidney injury molecule 1 (Kim1), lipocalin 2 (Lcn2), and osteopontin (Spp1). The establishment of such a genomic marker set offers a new tool in our ongoing quest to monitor nephrotoxicity.

Modernizing Human Cancer Risk Assessment of Therapeutics.

Cancer risk assessment of therapeutics is plagued by poor translatability of rodent models of carcinogenesis. In order to overcome this fundamental limitation, new approaches are needed that enable us to evaluate cancer risk directly in humans and human-based cellular models. Our enhanced understanding of the mechanisms of carcinogenesis and the influence of human genome sequence variation on cancer risk motivates us to re-evaluate how we assess the carcinogenic risk of therapeutics. This review will highlight new opportunities for applying this knowledge to the development of a battery of human-based in vitro models and biomarkers for assessing cancer risk of novel therapeutics.

A gene expression biomarker provides early prediction and mechanistic assessment of hepatic tumor induction by nongenotoxic chemicals.

There are currently no accurate and well-validated short-term tests to identify nongenotoxic hepatic tumorigens, thus necessitating an expensive 2-year rodent bioassay before a risk assessment can begin. Using hepatic gene expression data from rats treated for 5 days with one of 100 structurally and mechanistically diverse nongenotoxic hepatocarcinogens and nonhepatocarcinogens, a novel multigenebiomarker (i.e., signature) was derived to predict the likelihood of nongenotoxic chemicals to induce liver tumors in longer term studies. Independent validation of the signature on 47 test chemicals indicates an assay sensitivity and specificity of 86% and 81%, respectively. Alternate short-term in vivo pathological and genomic biomarkers were evaluated in parallel for comparison, including liver weight, hepatocellular hypertrophy, hepatic necrosis, serum alanine aminotransferase activity, induction of cytochrome P450 genes, and repression of Tsc-22 or alpha2-macroglobulin messenger RNA. In contrast to these biomarkers, the gene expression-based signature was more accurate. Unlike existing tests, an understanding of potential modes of action for hepatic tumorigenicity can be derived by comparison of the signature profile of test chemicals to hepatic tumorigens of known mechanism, including regenerative proliferation, proliferation associated with xenobiotic receptor activation, peroxisome proliferation, and steroid hormone-mediated mechanisms. This signature is not only more accurate than current methods, but also facilitates the identification of mode of action to aid in the early assessment of human cancer risk.

Development of 2-aminooxazoline 3-azaxanthene ß-amyloid cleaving enzyme (BACE) inhibitors with improved selectivity against Cathepsin D.

As part of an ongoing effort at Amgen to develop a disease-modifying therapy for Alzheimer's disease, we have previously used the aminooxazoline xanthene (AOX) scaffold to generate potent and orally efficacious BACE1 inhibitors. While AOX-BACE1 inhibitors demonstrated acceptable cardiovascular safety margins, a retinal pathological finding in rat toxicological studies demanded further investigation. It has been widely postulated that such retinal toxicity might be related to off-target inhibition of Cathepsin D (CatD), a closely related aspartyl protease. We report the development of AOX-BACE1 inhibitors with improved selectivity against CatD by following a structure- and property-based approach. Our efforts culminated in the discovery of a picolinamide-substituted 3-aza-AOX-BACE1 inhibitor absent of retinal effects in an early screening rat toxicology study.

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.