Douglas A. Keller

An Analysis of Pharmaceutical Experience with Decades of Rat Carcinogenicity Testing: Support for a Proposal to Modify Current Regulatory Guidelines

Data collected from 182 marketed and nonmarketed pharmaceuticals demonstrate that there is little value gained in conducting a rat two-year carcinogenicity study for compounds that lack: (1) histopathologic risk factors for rat neoplasia in chronic toxicology studies, (2) evidence of hormonal perturbation, and (3) positive genetic toxicology results. Using a single positive result among these three criteria as a test for outcome in the two-year study, fifty-two of sixty-six rat tumorigens were correctly identified, yielding 79% test sensitivity. When all three criteria were negative, sixty-two of seventy-six pharmaceuticals (82%) were correctly predicted to be rat noncarcinogens. The fourteen rat false negatives had two-year study findings of questionable human relevance. Applying these criteria to eighty-six additional chemicals identified by the International Agency for Research on Cancer as likely human carcinogens and to drugs withdrawn from the market for carcinogenicity concerns confirmed their sensitivity for predicting rat carcinogenicity outcome. These analyses support a proposal to refine regulatory criteria for conducting a two-year rat study to be based on assessment of histopathologic findings from a rat six-month study, evidence of hormonal perturbation, genetic toxicology results, and the findings of a six-month transgenic mouse carcinogenicity study. This proposed decision paradigm has the potential to eliminate over 40% of rat two-year testing on new pharmaceuticals without compromise to patient safety.

HISTOCHEMICAL LOCALIZATION OF FORMALDEHYDE DEHYDROGENASE IN THE RAT

Formaldehyde dehydrogenase (FDH) activity has been demonstrated biochemically in the olfactory and respiratory mucosae and in the liver of the rat, but the cellular localization of this enzyme has not been investigated. A histochemical procedure was developed to permit cellular localization of FDH. This allowed us to examine the relationship between distribution of FDH and formaldehyde-induced toxicity. Cold-processed glycol methacrylate embedded tissues were used to localize FDH activity in the rat respiratory tract, kidney, liver, and brain. Five- or ten-micrometer tissue sections were incubated in a reaction mixture containing formaldehyde (HCHO), glutathione (GSH), NAD+, nitroblue tetrazolium, pyrazole, and disulfiram. A blue formazan precipitate was formed at the site of FDH activity. Epithelial cell cytoplasm of both the respiratory and the olfactory mucosae of the nose stained for FDH, and olfactory sensory cell nuclei were also positive. Underlying Bowmans and seromucous glands were weakly positive. The lung had FDH activity located mainly in the Clara cells of the airways, with only diffuse weak activity in the lung parenchyma. Liver had activity in the cytoplasm of the hepatocytes, while in the kidney FDH was most prominent in the brush border of the P2 segment of the proximal tubules. Brain white matter stained strongly for FDH, while in gray matter only the neuropil exhibited weak activity. Corresponding tissue sections were stained for sulfhydryls; these sections indicated that GSH is likely to be present in all cells with FDH activity. For the respiratory tract these results demonstrate distinct differences between the location of FDH activity and previously reported nonspecific aldehyde dehydrogenase activity in the nose (M. S. Bogdanffy, H. W. Randall, and K. T. Morgan, 1986, Toxicol. Appl. Pharmacol. 82, 560-567). While high aldehyde dehydrogenase activities were found in tissues with low toxicities due to acetaldehyde exposure and vice versa, FDH activity was observed in tissues whether or not they exhibited a toxic response to inhaled HCHO. While not able to account for the localized toxicity of HCHO, the presence of FDH and glutathione in the epithelial layer of the nasal cavity presents a barrier to inhaled formaldehyde at low concentrations and may partially explain the observed nonlinearity of HCHO toxicity.

FutureTox II: In vitro Data and In Silico Models for Predictive Toxicology

FutureTox II, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in January, 2014. The meeting goals were to review and discuss the state of the science in toxicology in the context of implementing the NRC 21st century vision of predicting in vivo responses from in vitro and in silico data, and to define the goals for the future. Presentations and discussions were held on priority concerns such as predicting and modeling of metabolism, cell growth and differentiation, effects on sensitive subpopulations, and integrating data into risk assessment. Emerging trends in technologies such as stem cell-derived human cells, 3D organotypic culture models, mathematical modeling of cellular processes and morphogenesis, adverse outcome pathway development, and high-content imaging of in vivo systems were discussed. Although advances in moving towards an in vitro/in silico based risk assessment paradigm were apparent, knowledge gaps in these areas and limitations of technologies were identified. Specific recommendations were made for future directions and research needs in the areas of hepatotoxicity, cancer prediction, developmental toxicity, and regulatory toxicology.

Neonatal Mouse Model: Review of Methods and Results

The neonatal mouse model, in various forms, has been used experimentally since 1959 and a large number of chemicals have been tested. The neonatal model is known to be very sensitive for the detection of carcinogens that operate via a genotoxic mode of action. In contrast, it is known not to respond to chemicals that act via epigenetic mechanisms, commonly observed in the two-year carcinogenicity studies. As such, the model has a high sensitivity and specifi city in its response. Dose selection for the neonatal model is based on the maximum tolerated or feasible dose. Traditionally, compounds have been tested via the IP route of administration in this model. In some cases, this has limited the amount of material that can be administered because of the low dosing volumes (10 to 20 μL) that can be administered IP. For the ILSI project, the neonatal model was adapted for oral administration, which has the advantages of being the same route for which most pharmaceuticals are administered. In addition, a 10-fold increase in the volume of administration (100 to 200 μL) and the ability to dose drugs in suspension, permits much higher doses to be used as compared to the IP route of administration. The spontaneous tumors in the neonatal model occurred mainly in the liver of male mice and lung of male and female mice with a few tumors observed in the Harderian gland. The positive control, DEN produced a robust, uniform, and reproducible tumor response with the target organs essentially limited to liver and lung. A total of 13 compounds out of the 21 ILSI ACT compounds were evaluated in the neonatal model involving 18 studies with duplicate studies for some compounds. The genotoxic carcinogens including those used as positive controls were clearly positive (cyclophosphamide, diethylnitrosamine, 6-nitrochrysene). The non-genotoxicrodent carcinogens were clearly negative (chlorpromazine, sulfi soxazole, sulfamethoxazole, clofi brate, DEHP, haloperidol, metaproteranol, and phenobarbital). The non-genotoxic human carcinogen (cyclosporin) was clearly negative. The two other human carcinogens phenacetin and DES were negative and interestingly estradiol was negative in one of the two oral studies, but was clearly positive in the other. Considering the mode of action for three of the human carcinogens (DES, cyclosporin and phenacetin), which were negative in this model, the mode of action in humans is likely to be epigenetic. Overall, for the 3 clearly genotoxic chemicals, all were positive. For the 9 clearly non-genotoxic chemicals, all 9 were negative. The two human carcinogens for which genotoxicity may or may not play a role (DES and phenacetin) were negative and estradiol was positive in 1 of the two oral studies. Overall, the extensive database for compounds tested in the neonatal mouse model would support its use as an alternative model for the assessment of the carcinogenic potential of a chemical. The model responds to chemicals that act via a genotoxic mode of action that represent a greater concern for human cancer risk.

Mechanistic studies on chloral toxicity: Relationship to trichloroethylene carcinogenesis

Chloral (trichloroacetaldehyde), the major metabolite of trichloroethylene (TCE), was investigated for its potential to form DNA-protein cross-links (DPX), a lesion produced by other aldehydes. Chloral did not form DPX in rat liver nuclei at concentrations up to 250 mM for 30 min at 37 degrees C, while chloroacetaldehyde (47 mM) and acetaldehyde (200 mM) did form cross-links. Experiments with the aldehyde-trapping reagents thiosemicarbazide and semicarbazide showed that chloral did not react, in contrast with aldehydes that form DPX. This indicates a very strong hydration of chloral. Mice given 800 mg/kg [14C]chloral after pretreatment with 1500 mg/kg TCE for 10 days had no detectable covalent binding of 14C to DNA in the liver. These results do not support a genotoxic theory of carcinogenesis for TCE mediated through chloral.

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. This article describes the main steps that need to be taken and highlights key considerations in order to transform tissue chip technology from the hands of the innovators into those of the industrial scientists. Written by scientists from 13 pharmaceutical companies and partners at the National Institutes of Health, this article uniquely captures a consensus view on the progression strategy to facilitate and accelerate the adoption of this valuable technology. It concludes that success will be delivered by a partnership approach as well as a deep understanding of the context within which these chips will actually be used. Impact statement The rapid pace of scientific innovation in the tissue chip (TC) field requires a cohesive partnership between innovators and end users. Near term uptake of these human-relevant platforms will fill gaps in current capabilities for assessing important properties of disposition, efficacy and safety liabilities. Similarly, these platforms could support mechanistic studies which aim to resolve challenges later in development (e.g. assessing the human relevance of a liability identified in animal studies). Building confidence that novel capabilities of TCs can address real world challenges while they themselves are being developed will accelerate their application in the discovery and development of innovative medicines. This article outlines a strategic roadmap to unite innovators and end users thus making implementation smooth and rapid. With the collective contributions from multiple international pharmaceutical companies and partners at National Institutes of Health, this article should serve as an invaluable resource to the multi-disciplinary field of TC development.

Current nonclinical testing paradigm enables safe entry to First-In-Human clinical trials: The IQ consortium nonclinical to clinical translational database

&NA; The contribution of animal testing in drug development has been widely debated and challenged. An industry‐wide nonclinical to clinical translational database was created to determine how safety assessments in animal models translate to First‐In‐Human clinical risk. The blinded database was composed of 182 molecules and contained animal toxicology data coupled with clinical observations from phase I human studies. Animal and clinical data were categorized by organ system and correlations determined. The 2 × 2 contingency table (true positive, false positive, true negative, false negative) was used for statistical analysis. Sensitivity was 48% with a 43% positive predictive value (PPV). The nonhuman primate had the strongest performance in predicting adverse effects, especially for gastrointestinal and nervous system categories. When the same target organ was identified in both the rodent and nonrodent, the PPV increased. Specificity was 84% with an 86% negative predictive value (NPV). The beagle dog had the strongest performance in predicting an absence of clinical adverse effects. If no target organ toxicity was observed in either test species, the NPV increased. While nonclinical studies can demonstrate great value in the PPV for certain species and organ categories, the NPV was the stronger predictive performance measure across test species and target organs indicating that an absence of toxicity in animal studies strongly predicts a similar outcome in the clinic. These results support the current regulatory paradigm of animal testing in supporting safe entry to clinical trials and provide context for emerging alternate models. HighlightsTranslational database created to determine animal to human safety concordance.182 molecules with animal and clinical data; concordance parameters determinedPositive predictive value of 43%; Negative predictive value of 86%Absence of toxicity in animal study strongly predicts similar outcome in the clinic.Database supports animal testing to ensure human safety in the clinic.

FutureTox III: Bridges for Translation

Future Tox III, a Society of Toxicology Contemporary Concepts in Toxicology workshop, was held in November 2015. Building upon Future Tox I and II, Future Tox III was focused on developing the high throughput risk assessment paradigm and taking the science of in vitro data and in silico models forward to explore the question-what progress is being made to address challenges in implementing the emerging big-data toolbox for risk assessment and regulatory decision-making. This article reports on the outcome of the workshop including 2 examples of where advancements in predictive toxicology approaches are being applied within Federal agencies, where opportunities remain within the exposome and AOP domains, and how collectively the toxicology community across multiple sectors can continue to bridge the translation from historical approaches to Tox21 implementation relative to risk assessment and regulatory decision-making.

Regulatory Forum Opinion Piece*: Use and Utility of Animal Models of Disease for Nonclinical Safety Assessment: A Pharmaceutical Industry Survey

An Innovation and Quality (IQ) Consortium focus group conducted a cross-company survey to evaluate current practices and perceptions around the use of animal models of disease (AMDs) in nonclinical safety assessment of molecules in clinical development. The IQ Consortium group is an organization of pharmaceutical and biotechnology companies with the mission of advancing science and technology. The survey queried the utilization of AMDs during drug discovery in which drug candidates are evaluated in efficacy models and limited short-duration non-Good Laboratory Practices (GLP) toxicology testing and during drug development in which drug candidates are evaluated in GLP toxicology studies. The survey determined that the majority of companies used AMDs during drug discovery primarily as a means for proactively assessing potential nonclinical safety issues prior to the conduct of toxicology studies, followed closely by the use of AMDs to better understand toxicities associated with exaggerated pharmacology in traditional toxicology models or to derisk issues when the target is only expressed in the disease state. In contrast, the survey results indicated that the use of AMDs in development is infrequent, being used primarily to investigate nonclinical safety issues associated with targets expressed only in disease states and/or in response to requests from global regulatory authorities.