NOVEL HIGH THROUGHPUT PLATFORM FOR SCREENING CYTOCHROME P450 INDUCTION

描述（由申请人提供）：给药的代谢是由药物代谢酶的表达和活性决定的，如细胞色素（CYP） P450酶。这些p450受到抑制，有时被外源药物诱导，导致可能的药代动力学药物-药物相互作用。认识到这一问题，FDA发布了行业药物相互作用研究指南草案，建议至少对CYP1A2和CYP3A4进行体外诱导评估。评估CYP2B6诱导的重要性最近也被认识到。P450体外诱导试验的“金标准”是测定新鲜分离的人肝细胞中的酶活性。然而，人类肝细胞的可用性和个体可变性，以及在该系统中无法区分P450抑制和诱导，使评估复杂化。外源性药物诱导p450是由配体激活的核受体介导的，包括PXR、AhR和CAR。因此，核受体介导p450诱导的报告基因检测被认为是体外药物评价的有效方法。许多制药行业和一些cro使用PXR转激活试验，很少评估AhR活性。目前还没有市售的CAR检测方法。然而，需要几种核受体和其他转录因子的联合才能通过所有已知的诱导剂诱导特异性p450的表达。该提案的目标包括设计和实施Originus的STEP（表面转染和表达协议）技术的新应用，用于新型候选药物的安全性测试。在第一阶段，我们将开发先进的STEP平台，同时评估CYP1A2， CYP3A4和CYP2B6在人肝细胞系中使用报告基因检测的转录调控。分泌性碱性磷酸酶（SEAP）基因将在人类CYP上游调控序列的控制下作为报告基因。STEP共转染不同的CYP-SEAP报告基因和单一或相关的外源激活受体的组合将被优化，以在96孔微孔板上的多个时间点测试一组诱导剂对SEAP的反应。第一阶段的里程碑是STEP平台原型的生产和beta测试（单独或组合），具有适当的分析稳健性和可重复性，井对井，板对板和批对批。这些CYP P450诱导平台将可用于工业和学术界的高通量筛选实验室在药物发现过程的早期进行药物毒理学筛选，或作为小型实验室使用的简单检测试剂盒，成本适中。在II期，我们将扩展研究以评估其他可诱导的CYP p450，扩大测试的核受体的光谱，并进一步开发基于细胞的外源激活受体相关等位基因变异的检测。此外，我们计划开发用于CYP诱导途径分析的STEP检测平台。

DESCRIPTION (provided by applicant): Metabolism of administered drugs is determined by expression and activity of drug-metabolizing enzymes, such as cytochrome (CYP) P450 enzymes. These P450s are subject to inhibition and sometimes induction by xenobiotics, leading to possible pharmacokinetic drug-drug interactions between co-administrated drugs. Recognizing the problem, the FDA has issued a Draft Guidance for Industry Drug Interaction Studies, which advice at a minimum in vitro induction evaluation of CYP1A2 and CYP3A4. The importance of evaluating CYP2B6 induction has recently been recognized as well. The "gold standard" of P450 induction in vitro testing is the determination of enzymatic activity in freshly isolated human hepatocytes. However, availability and individual variability of human hepatocytes as well as inability to discriminate between P450 inhibition and induction in this system complicates the evaluation. The induction of P450s by xenobiotics is mediated by ligand-activated nuclear receptors, including PXR, AhR and CAR. Thus, reporter gene assays for nuclear receptors mediating induction of P450s are considered valid methods of drug evaluation in vitro. Many pharmaceutical industries and some CROs use PXR transactivation assays and very few evaluate AhR activity. There is no commercially available assay for CAR. However, combinations of several nuclear receptors and other transcription factors are needed to induce the expression of specific P450s by all known inducers. The goal of this proposal includes the design and implementation of a novel application of Originus' STEP (Surface Transfection and Expression Protocol) technology for safety testing of novel drug candidates. During Phase I, we will develop sophisticated STEP platforms for simultaneous evaluation of CYP1A2, CYP3A4 and CYP2B6 transcirptional regulation using reporter gene assays in human hepatic cell lines. Secreted alkaline phosphatase (SEAP) gene will be used as a reporter under the control of each human CYP upstream regulatory sequences. STEP co-transfection of the different CYP-SEAP reporters and single or relevant combinations of xenobiotic-activated receptors will be optimized to test SEAP response by a set of inducers at multiple time points in 96-well microplates. Milestones for Phase I are the production and beta-testing of the prototypes of STEP platforms (individually or combined) with appropriate assay robustness and reproducibility well-to-well, plate-to-plate and batch-to-batch. These CYP P450 induction platforms will be available for toxicological screening of drugs early in the drug discovery process by high throughput screening laboratories in industry and academia, or as simple assay kits for small laboratory use at a modest cost. During Phase II, we will extend the studies to evaluate other inducible CYP P450s, expand the spectrum of nuclear receptors tested, and to further develop cell-based assays of relevant allelic variants of the xenobiotic-activated receptors. In addition, we plan to develop STEP assay platforms for pathway profiling of CYP induction. PUBLIC HEALTH RELEVANCE: Drug safety is one of the major factors for compound attrition during clinical development. Simple assays for fast evaluation of metabolism and toxicology of compounds are urgently needed to minimize patient risks and improve the success rate of new molecular entities early in the drug discovery pipeline. The goal of this proposal is to use Originus' proprietary technology, STEP, to develop platforms for in vitro evaluation of drug candidates that may induce certain hepatic enzymes (cytochrome P450s) triggering drug-drug interactions and compromising the health of the patient.