No abstract

ABSTRACT:A reliable and practical CYP3A induction assay with cryopreserved human hepatocytes in a 96-well format was developed. Various 96-well plates with different basement membrane were evaluated using prototypical inducers, rifampicin, phenytoin, and carbamazepine. Thin-layer (TL) Matrigel was found to yield the highest basal and induced levels of CYP3A activity as determined by testosterone 6␤-hydroxylation. Concentration-dependent CYP3A induction of rifampicin was reproducible with the EC 50 values of 0.36 ؎ 0.28 M from four batches of human hepatocytes using the 96-well plate with TL Matrigel. The rank order of induction potency for nine inducers or noninducers at a concentration of 10 M were well comparable among the multiple donors, by expressing the results as percentage of change compared with the positive control, 10 M rifampicin. Cotreatment of avasimibe or efavirenz with 10 M rifampicin was found to reduce CYP3A activities induced by rifampicin at a lower rate than treatment with rifampicin alone, whereas treatment with phenobarbital and carbamazepine had no effect. From a comparison of induced CYP3A activities and gene expression levels, there were compounds that would cause induction of CYP3A4 mRNA but not activity, presumably due to their inhibitory effect on CYP3A activity. The cotreatment assay of test compound with rifampicin allows us to exclude the false-negative results caused by the cytotoxicity and/or the mechanism-based inactivation, when the drug candidate's ability for CYP3A induction is evaluating the enzyme activity. This 96-well plate assay, which is robust, reproducible, and convenient, has demonstrated the paramount applicability to the early drug discovery stage. IntroductionCYP3A is regarded as one of the predominant cytochrome P450 (P450) isoforms among drug-metabolizing enzymes expressed in the human liver. The induction of CYP3A can result in clinically significant drug-drug interactions (DDIs) or autoinductions. The CYP3A inducers may cause reduction in therapeutic efficacy of comedication or an increase in risk of metabolite-induced toxicity. Therefore, it is important to evaluate the induction potential of new chemical entities in the early discovery stage (Dickins, 2004;Luo et al., 2004;Lin, 2006).Several in vitro models have been developed to assess the potential of CYP3A induction, including nuclear receptor-based assays (Moore et al., 2000;El Sankary et al., 2001;Luo et al., 2002;Sinz et al., 2006), immortalized cell lines (Mills et al., 2004;Hariparsad et al., 2008), and primary hepatocytes. Among these models, the primary human hepatocytes have been considered as the most predictive model for assessing in vitro induction of P450 enzymes. Although fresh human hepatocytes are the standard for evaluating induction potential, attachable cryopreserved hepatocytes have been used more often due to increased availability. The enzymes in cryopreserved hepatocytes were shown to be inducible by standard P450 inducers, with the increased levels of mRNA, protein, and/or function...

Introduction: TAK-733 is a novel, potent, selective, non-ATP competitive, allosteric inhibitor of MEK kinase, which has a potential to inhibit cancer proliferation and survival. [18F]Fluorodeoxyglucose ([18F]FDG) is an imaging biomarker for glucose metabolism detectable using positron emission tomography (PET) and widely used in clinical cancer diagnosis and is increasingly used to assess response to anti-cancer therapy. The aim of this study was to evaluate the therapeutic efficacy of TAK-733 using [18F]FDG-PET in nude rats bearing A549 (human lung carcinoma) xenografts. Methods: A549 tumor fragments were subcutaneously implanted in female RNU nude rats. Treatments began when the mean estimated tumor mass for all groups was 250mg (Day 1). TAK-733 was orally administered at 0 (vehicle), 1, 3 and 10 mg/kg daily for two weeks (n = 8 / group). PET scans were performed before treatment (Day 0) and on Days 2, 4, 7, 10 and 14. PET image acquisition was performed for 13 min at 1.5 hr after [18F]FDG injection (400 μCi). Tracer accumulations in tumor tissue were quantified as mean standard uptake value (SUVmean) and percentage of injected dose (%ID). Results: TAK-733 showed dose-dependent inhibition of tumor growth and [18F]FDG uptake in tumor tissue. TAK-733, at 10mg/kg, produced a statistically significant difference in tumor mass (Day 14) compared to the vehicle group (31% T/C) and in SUVmean (Day 2) compared to the vehicle group. The statistical significance for the 10mg/kg did not persist however, and was only observed at a single time point for tumor mass and SUVmean. However, a significant response in SUVmean (Day 2) occurred before significant tumor growth inhibition (Day 14). In this treatment group, the SUVmean value on Day 2 (1.38 ± 0.26) was lower than that observed at pre-treatment, Day 0 (1.60 ± 0.30), and statistically lower compared to the SUVmean values in the vehicle (1.54 ± 0.46) and 1mg/kg groups (1.53 ± 0.29) on Day 2. The SUVmean value in the vehicle and 1 and 3 mg/kg groups showed an increase over time. On Day 14, the SUVmean value at 10 mg/kg (1.45 ± 0.33) was statistically significantly lower compared to the vehicle group (2.16 ± 0.33). TAK-733 produced a dose-dependent %ID response. 10mg/kg TAK-733 produced the lowest %ID for every imaging time point, while the vehicle group had the highest %ID at every imaging time point. There were no statistically significant differences in %ID response. However, %ID did not show statistically significant decreased values compared to the vehicle group, suggesting that SUVmean may be a more sensitive parameter for [18F]FDG uptake than %ID. Conclusion: [18F]FDG-PET enabled an early indication of later tumor growth in response to TAK-733 treatment in this model. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5217.