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.