Assessing the genotoxicity of N-nitrosodiethylamine with three in vivo endpoints in male Big Blue® transgenic and wild-type C57BL/6N mice.

The detection of N-nitrosamines in drug products has raised global regulatory interest in recent years due to the carcinogenic potential of some nitrosamines in animals and a need to identify a testing strategy has emerged. Ideally, methods used would allow for the use of quantitative analysis of dose-response data from in vivo genotoxicity assays to determine a compound-specific acceptable intake for novel nitrosamines without sufficient carcinogenicity data. In a previous study we compared the dose-response relationships of N-nitrosodiethylamine (NDEA) in three in vivo genotoxicity endpoints in rats. Here we report a comparison of NDEA's genotoxicity profile in mice. Big Blue® mice were administered NDEA at doses of 0.001, 0.01, 0.1, 1 and 3 mg/kg/day by oral gavage for 28 days followed by 3 days of expression. Statistically significant increases in the NDEA induced mutations were detected by both the transgenic rodent mutation assay (TGR) using the cII endpoint and by duplex sequencing in the liver but not bone marrow of mice. In addition, administration of NDEA for two consecutive days in male C57BL/6N mice caused elevated DNA damage levels in the liver as measured by % tail DNA in comet assay. The benchmark dose (BMD) analysis shows a BMDL50 of 0.03, 0.04 and 0.72 mg/kg/day for TGR, duplex sequencing and comet endpoints, respectively. Overall, this study demonstrated a similar genotoxicity profile of NDEA between mice and rats and provides a reference that can be used to compare the potential potency of other novel nitrosamines for the induction of gene mutations.

Experiments in the EpiDerm 3D Skin In Vitro Model and Minipigs In Vivo Indicate Comparatively Lower In Vivo Skin Sensitivity of Topically Applied Aneugenic Compounds.

Risk management of in vitro aneugens for topically applied compounds is not clearly defined because there is no validated methodology to accurately measure compound concentration in proliferating stratum basale keratinocytes of the skin. Here, we experimentally tested several known aneugens in the EpiDerm reconstructed human skin in vitro micronucleus assay and compared the results to flow cytometric mechanistic biomarkers (phospho-H3; MPM2, DNA content). We then evaluated similar biomarkers (Ki-67, nuclear area) using immunohistochemistry in skin sections of minipigs following topical exposure the potent aneugens, colchicine, and hesperadin. Data from the EpiDerm model showed positive micronucleus responses for all aneugens tested following topical or direct media dosing with similar sensitivity when adjusted for applied dose. Quantitative benchmark dose-response analysis exhibited increases in the mitotic index biomarkers phospho-H3 and MPM2 for tubulin binders and polyploidy for aurora kinase inhibitors are at least as sensitive as the micronucleus endpoint. By comparison, the aneugens tested did not induce histopathological changes, increases in Ki-67 immunolabeling or nuclear area in skin sections from the in vivo minipig study at doses in significant excess of those eliciting a response in vitro. Results indicate the EpiDerm in vitro micronucleus assay is suitable for the hazard identification of aneugens. The lack of response in the minipig studies indicates that the barrier function of the minipig skin, which is comparable to human skin, protects from the effects of aneugens in vivo. These results provide a basis for conducting additional studies in the future to further refine this understanding.

2-Hydroxypyridine N-Oxide is not genotoxic in vivo.

2-Hydroxypyridine N-oxide (HOPO) is an important coupling reagent used in pharmaceutical synthesis. Our laboratory previously reported HOPO as equivocal in the Ames assay following extensive testing of multiple lots of material. Given the lack of reproducibility between lots of material and the weak increase in revertants observed, it was concluded that it would be highly unlikely that HOPO would pose a mutagenic risk in vivo. The purpose of the current investigation was to assess experimentally in rats the mutagenic (Pig-a mutation induction) and more broadly genotoxic (micronucleus and comet induction) potential of HOPO. Rats were administered HOPO (0, 50, 150, 300, and 500 mg/kg/day) by oral gavage for 28 days. At the end of study, the following parameters were assessed: frequency of Pig-a mutant red blood cells and reticulocytes, frequency of peripheral blood micronuclei, and the incidence of comet formation in liver. Toxicokinetic data collected on study Days 1 and 28 demonstrated systemic exposure to HOPO. Although there were no overt clinical signs, animals treated with HOPO showed a dose-related decrease in body weight gain. There were no increases observed in any of the genotoxicity endpoints assessed. The results from this study further support the conclusion that in the context of pharmaceutical synthesis, HOPO should not be considered a mutagenic impurity but rather controlled as a normal process-related impurity. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Development and validation of an in vitro micronucleus assay platform in TK6 cells.

The Organization for Economic Co-operation and Development (OECD) has recently adopted Test Guideline 487 (TG487) for conducting the in vitro micronucleus (MNvit) assay. The purpose of this study is to evaluate and validate treatment conditions for the use of p53 competent TK6 human lymphoblastoid cells in a TG487 compliant MNvit assay. The ten reference compounds suggested in TG487 (mitomycin C, cytosine arabinoside, cyclophosphamide, benzo-a-pyrene, vinblastine sulphate, colchicine, sodium chloride, nalidixic acid and di(2-ethylhexyl)phthalate and pyrene) and noscapine hydrochloride were chosen for this study. In order to optimize the micronucleus response after treatment with some positive substances, we extended the recovery time after pulse treatment from 2 cell cycles recommended in TG487 to 3 cell cycles for untreated cells (40h). Each compound was tested in at least one of four exposure conditions: a 4h exposure followed by a 40h recovery, a 4h exposure followed by a 24h recovery, a 4h exposure in the presence of an exogenous metabolic activation system followed by a 40h recovery period, and a 27h continuous direct treatment. Results show that the direct acting clastogens, clastogens requiring metabolic activation and aneugens caused a robust increase in micronuclei in at least one test condition whereas the negative compounds did not induce micronuclei. The negative control cultures exhibited reproducibly low and consistent micronucleus frequencies ranging from 0.4 to 1.8% (0.8±0.3% average and standard deviation). Furthermore, extending the recovery period from 24h to 40h produced a 2-fold higher micronucleus frequency after a 4h pulse treatment with mitomycin C. In summary, the protocol described in this study in TK6 cells produced the expected result with model compounds and should be suitable for performing the MNvit assay in accordance with guideline TG487.

Discovery of two clinical histamine H(3) receptor antagonists: trans-N-ethyl-3-fluoro-3-[3-fluoro-4-(pyrrolidinylmethyl)phenyl]cyclobutanecarboxamide (PF-03654746) and trans-3-fluoro-3-[3-fluoro-4-(pyrrolidin-1-ylmethyl)phenyl]-N-(2-methylpropyl)cyclobutanecarboxamide (PF-03654764).

The discovery of two histamine H(3) antagonist clinical candidates is disclosed. The pathway to identification of the two clinical candidates, 6 (PF-03654746) and 7 (PF-03654764) required five hypothesis driven design cycles. The key to success in identifying these clinical candidates was the development of a compound design strategy that leveraged medicinal chemistry knowledge and traditional assays in conjunction with computational and in vitro safety tools. Overall, clinical compounds 6 and 7 exceeded conservative safety margins and possessed optimal pharmacological and pharmacokinetic profiles, thus achieving our initial goal of identifying compounds with fully aligned oral drug attributes, "best-in-class" molecules.

Concordance analysis of an in vitro micronucleus screening assay and the regulatory chromosome aberration assay using pharmaceutical drug candidates.

The in vitro micronucleus assay is under consideration by regulatory agencies as a suitable alternative to the in vitro chromosome aberration (CA) assay. At Pfizer, we utilized a non-Good Laboratory practices cytokinesis-block in vitro micronucleus (CBMN) assay in CHO cells as a screen to predict the regulatory outcome of the human lymphocyte CA assay, and we have retrospectively analyzed a highly select set of 112 internal drug candidates to measure concordance. Overall, our exploratory CBMN correctly classified 97 of 112 (86.6%) compounds in the CA assay. Specificity was high with 87 of 92 (94.6%) CA negative compounds correctly classified by CBMN. Sensitivity was low at 50% with 10 of 20 CA positive compounds correctly classified by CBMN; this may be attributed to the low number of CA positives in the select set. In an attempt to improve sensitivity, we increased the number of CA positives by combining our internal set with an industrial data set previously published (Miller B et al. 1997: Mutat Res 392:45-59). When combined, concordance was 86.7% (143/165), specificity was 91.2% (114/125), and sensitivity increased to 72.5% (29/40). Because cytotoxicity is considered a confounding factor of in vitro test systems, we also examined, within the Pfizer data set, the influence of cytotoxicity in the CBMN assay, and the results indicated that seemingly low (<50%) or excessively high (>70%) levels of cytotoxicity did not significantly alter predicted CA outcome. These collective analyses contribute to growing evidence that the CBMN assay is a suitable regulatory option in the standard battery of genetic toxicology tests.

Piperidinyl-2-phenethylamino inhibitors of DPP-IV for the treatment of type 2 diabetes.

A highly ligand efficient lead molecule was rapidly developed into a DPP-IV selective candidate series using focused small library synthesis. A significant hurdle for series advancement was genetic safety since some agents in this series impaired chromosome division that was detected using the in vitro micronucleus assay. A recently developed high-throughput imaging-based in vitro micronucleus assay enabled the identification of chemical space with a low probability of micronucleus activity. Advanced profiling of a subset within this space identified a compound with a clean safety profile, an acceptable human DPP-IV inhibition profile based on the rat PK/PD model and a projected human dose that was suitable for clinical development.

Yeast DEL assay detects clastogens.

Chromosomal rearrangements, including DNA deletions are involved in carcinogenesis. The deletion (DEL) assay scoring for DNA deletions in the yeast Saccharomyces cerevisiae is able to detect a wide range of carcinogens. Among approximately 60 compounds of known carcinogenic activity, the DEL assay detected 86% correctly whereas the Ames Salmonella assay detected only 30% correctly [R.J. Brennan, R.H. Schiestl, Detecting carcinogens with the yeast DEL assay, Methods Mol. Biol. 262 (2004) 111-124]. Since the DEL assay is highly inducible by DNA double strand breaks, this study examined the utility of the DEL assay for detecting clastogens. Ten model compounds, with varied mechanisms of genotoxicity, were examined for their effect on the frequency of DNA deletions with the DEL assay. The compounds tested were: actinomycin D, camptothecin, methotrexate and 5-fluorodeoxyuridine, which are anticancer agents, noscapine and furosemide are therapeutics, acridine, methyl acrylate and resorcinol are industrial chemicals and diazinon is an insecticide. The in vitro micronucleus assay (IVMN) in CHO cells, a commonly used tool for detection of clastogens, was performed on the same compounds and the results of the two assays were compared. The results of our study show that there is 70% concordance in the presence of metabolic activation (rat liver S9) and 80% concordance in the absence of metabolic activation between the DEL assay and the standard in vitro micronucleus assay. The lack of cytotoxicity observed for four of the ten compounds examined indicates limited diffusion of lipophilic compounds across the yeast cell wall. Thus, the development of a more permeable yeast tester strain is expected to greatly improve concordance of the DEL assay with the IVMN assay. The yeast DEL assay is inexpensive, amenable to automation and requires less expertise to perform than the IVMN. Thus, it has a strong potential as a robust, fast and economical screen for detecting clastogens in vitro.