Donna M. Dambach

New technologies and screening strategies for hepatotoxicity: Use of in vitro models

Hepatotoxicity remains a significant cause for drug failures during clinical trials. This is due, in part, to the idiosyncratic nature of toxicity in humans and inherent physiological differences between humans and preclinical species leading to limited correct prediction of adverse responses in humans. To address this issue, robust screening assays are being developed, which have heightened predictive capacity for human hepatotoxicity, and may be utilized throughout the discovery and development phases in conjunction with traditional in vivo methods, for decision making during drug selection and risk assessment. This manuscript describes an example application of in vitro-based strategies using human hepatocyte cultures in lead optimization screening in conjunction with ADME profiling, for evaluation of compound-associated CYP450 induction potential, and the identification of potentially useful biomarkers as predictors of hepatotoxicity for use in vitro, and in preclinical species and humans.

Exaggerated hepatotoxicity of acetaminophen in mice lacking tumor necrosis factor receptor-1. Potential role of inflammatory mediators.

Transgenic mice with a targeted disruption of the tumor necrosis factor receptor 1 (TNFR1) gene were used to analyze the role of TNF-alpha in pro- and anti-inflammatory mediator production and liver injury induced by acetaminophen. Treatment of wild-type mice with acetaminophen (300 mg/kg) resulted in centrilobular hepatic necrosis. This was correlated with expression of inducible nitric oxide synthase (NOS II) and nitrotyrosine staining of the liver. Expression of macrophage chemotactic protein-1 (MCP-1), KC/gro, interleukin-1beta (IL-1beta), matrix metalloproteinase-9 (MMP-9), and connective tissue growth factor (CTGF), inflammatory mediators known to participate in tissue repair, as well as the anti-inflammatory cytokine, interleukin-10 (IL-10), also increased in the liver following acetaminophen administration. TNFR1(-/-) mice were found to be significantly more sensitive to the hepatotoxic effects of acetaminophen than wild-type mice. This was correlated with more rapid and prolonged induction of NOS II in the liver and changes in the pattern of nitrotyrosine staining. Acetaminophen-induced expression of MCP-1, IL-1beta, CTGF, and MMP-9 mRNA was also delayed or reduced in TNFR1(-/-) mice relative to wild-type mice. In contrast, increases in IL-10 were more rapid and more pronounced. These data demonstrate that signaling through TNFR1 is important in inflammatory mediator production and toxicity induced by acetaminophen.

A Retrospective Analysis of Toxicogenomics in the Safety Assessment of Drug Candidates

Toxicogenomics is considered a valuable tool for reducing pharmaceutical candidate attrition by facilitating earlier identification, prediction and understanding of toxicities. A retrospective evaluation of 3 years of routine transcriptional profiling in non-clinical safety studies was undertaken to assess the utility of toxicogenomics in drug safety assessment. Based on the analysis of studies with 33 compounds, marked global transcriptional changes (>4% transcripts at p < 0.01) were shown to be a robust biomarker for dosages considered to be toxic. In general, there was an inconsistent correlation between transcription and histopathology, most likely due to differences in sensitivity to focal microscopic lesions, to secondary effects, and to events that precede structural tissue changes. For 60% of toxicities investigated with multiple time-point data, transcriptional changes were observed prior to changes in traditional study endpoints. Candidate transcriptional markers of pharmacologic effects were detected in 40% of targets profiled. Mechanistic classification of toxicity was obtained for 30% of targets. Furthermore, data comparison to compendia of transcriptional changes provided assessments of the specificity of transcriptional responses. Overall, our experience suggests that toxicogenomics has contributed to a greater understanding of mechanisms of toxicity and to reducing drug attrition by empiric analysis where safety assessment combines toxicogenomic and traditional evaluations.

A murine model of cigarette smoke-induced pulmonary inflammation using intranasally administered smoke-conditioned medium.

To date, few animal models of chronic obstructive pulmonary disease (COPD) exist that are ideal for the evaluation of pathophysiology, as they typically require many months of cigarette smoke exposure in inhalation facilities. Here we show that pulmonary inflammation and some of the inflammatory hallmarks of COPD can be induced in mice by cigarette smoke-conditioned media (CS) administered by the intranasal route. Balb/c mice were challenged with CS for up to 40 days. At the end of smoke treatment, mice were sacrificed and bronchoalveolar lavage (BAL) fluid collected. Total cell counts and cell differentials were performed. Enzyme-linked immunosorbent assays (ELISAs) for KC and tumor necrosis factor alpha (TNF- α) were performed on BAL fluid. Lungs and nasal cavities were examined histologically. Intranasal CS treatment significantly increased BAL neutrophils, lymphocytes, KC, TNF- α, and mucin. Changes in pulmonary reactivity to methacholine were also observed in mice challenged with CS for 40 days. The model described above demonstrates that within 1 to 8 weeks of intranasal instillation of CS, mice develop pulmonary inflammation and cellular lung changes that are characteristic of human COPD and therefore may be a good short-term in vivo model that can be utilized to monitor intervention strategies targeted for COPD.

Dilated Cardiomyopathy in Juvenile Portuguese Water Dogs

Dilated cardiomyopathy recently has been recognized in juvenile Portuguese Water Dogs. The purpose of this study was to evaluate unaffected and affected puppies by physical examination, electrocardiogram (ECG), echocardiogram, specific biochemical assays, and ultrastructure to document disease progression and to develop a method of early detection. Results of segregation analysis were consistent with autosomal recessive inheritance. Of 124 puppies evaluated clinically and echocardiographically, 10 were affected. No significant differences were found between unaffected and affected puppies for blood and myocardial carnitine or taurine concentrations, serum chemical variables, results of ophthalmological examinations, ECGs, or measurement of urine metabolites. Ultrastructural examination of myocardium from affected dogs revealed myofibrillar atrophy and small regions of myo‐fibrillar degeneration, most prominently at the region of the intercalated discs. Only echocardiography allowed detection of affected puppies before clinical signs became evident. Echocardiography revealed a significant difference in the shortening fraction, E point to septal separation, and the end systolic and diastolic left ventricular internal diameters. Affected puppies were detected 1–4 weeks before the development of acute congestive heart failure.

Key Challenges and Opportunities Associated with the Use of In Vitro Models to Detect Human DILI: Integrated Risk Assessment and Mitigation Plans

Drug-induced liver injury (DILI) is a major cause of late-stage clinical drug attrition, market withdrawal, black-box warnings, and acute liver failure. Consequently, it has been an area of focus for toxicologists and clinicians for several decades. In spite of considerable efforts, limited improvements in DILI prediction have been made and efforts to improve existing preclinical models or develop new test systems remain a high priority. While prediction of intrinsic DILI has improved, identifying compounds with a risk for idiosyncratic DILI (iDILI) remains extremely challenging because of the lack of a clear mechanistic understanding and the multifactorial pathogenesis of idiosyncratic drug reactions. Well-defined clinical diagnostic criteria and risk factors are also missing. This paper summarizes key data interpretation challenges, practical considerations, model limitations, and the need for an integrated risk assessment. As demonstrated through selected initiatives to address other types of toxicities, opportunities exist however for improvement, especially through better concerted efforts at harmonization of current, emerging and novel in vitro systems or through the establishment of strategies for implementation of preclinical DILI models across the pharmaceutical industry. Perspectives on the incorporation of newer technologies and the value of precompetitive consortia to identify useful practices are also discussed.

Cloning, expression and characterization of monkey (Macaca fascicularis) CD137

CD137 plays an important role as a co-stimulatory molecule in activated T cells. Agonistic CD137 specific antibodies have been investigated as therapeutic agents to promote tumor-specific immune responses by direct activation of T cells. As part of the pre-clinical pharmacological evaluation of cynomolgus monkeys, monkey CD137 was cloned and characterized. The deduced amino acid sequence encoded a full-length gene of 254 amino acids 95% identical to human CD137. Sequence variants identified in monkey CD137 include four splice variants lacking the transmembrane domain. These variants were detectable in human including two previously unreported variants. Two missense single nucleotide polymorphisms were detected present in 42 and 50% of 36 monkeys tested. In both monkey and human, mRNA expression of full-length CD137 and splice variants were significantly increased in peripheral blood mononuclear cells (PBMCs) upon stimulation by anti-CD3 antibodies. Recombinant monkey CD137 protein was bound with high affinity by an agonistic anti-human CD137 antibody but not by an anti-mouse CD137 antibody. In summary, compared to human, monkey CD137 showed distinct extracellular domain amino acid sequence and sequence polymorphisms. Thus, antibodies directed against epitopes in this extracellular domain could have differences in pharmacologic activity between cynomolgus monkeys and human or across individual cynomolgus monkeys.