NOVEL HIGH THROUGHPUT PLATFORM FOR SCREENING CYTOCHROME P450 INDUCTION

说明书(申请人提供)：所用药物的代谢由药物代谢酶的表达和活性决定，如细胞色素P450酶。这些P450受到抑制，有时被外源物质诱导，导致联合给药之间可能的药代动力学药物-药物相互作用。认识到这个问题，FDA已经发布了行业药物相互作用研究指南草案，其中建议至少对CYP1A2和CYP3A4进行体外诱导评估。最近，人们也认识到了评估细胞色素P450_2B6诱导的重要性。P450体外诱导试验的“金标准”是测定新鲜分离的人肝细胞的酶活性。然而，在这个系统中，人肝细胞的可用性和个体变异性以及无法区分P450抑制和诱导使评估变得复杂。外源生物对P450的诱导是由配体激活的核受体介导的，包括PXR、AhR和CAR。因此，核受体介导的P450诱导的报告基因分析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反式激活试验，很少有人评估AhR的活性。目前还没有商业化的CAR检测方法。然而，需要几种核受体和其他转录因子的组合才能通过所有已知的诱导剂诱导特定P450的表达。这项建议的目标包括设计和实施Originus的STEP(表面转染和表达协议)技术的一种新应用，用于新药候选的安全性测试。在第一阶段，我们将开发复杂的STEP平台，使用报告基因分析在人肝细胞系中同时评估CYP1A2、CYP3A4和CYP2B6的跨循环调节。分泌型碱性磷酸酶(SEAP)基因将作为报告基因，在每个人类CYP上游调控序列的调控下进行调控。不同的CYP-SEAP报告和单个或相关的外源激活受体组合的分步共转染将被优化，以在96孔微孔板上通过一组诱导剂在多个时间点测试SEAP反应。第一阶段的里程碑是生产和测试STEP平台的原型(单独或组合)，具有适当的分析稳健性和重复性，从井到井、板到板和批次到批次。这些CYP P450诱导平台将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对药物进行毒理学筛选，或作为小型实验室使用的简单分析试剂盒，成本适中。在第二阶段，我们将扩大研究范围，以评估其他可诱导的CYP P450，扩大所测试的核受体的光谱，并进一步发展基于细胞的外源激活受体相关等位基因变体的分析。此外，我们还计划开发用于CYP诱导途径分析的STEP分析平台。 公共卫生相关性：在临床开发过程中，药物安全性是化合物磨损的主要因素之一。迫切需要快速评估化合物代谢和毒理学的简单分析方法，以最大限度地减少患者风险，并在药物发现管道的早期提高新分子实体的成功率。这项提议的目标是利用Originus的专利技术STEP开发用于体外评估候选药物的平台，这些候选药物可能会诱导某些肝酶(细胞色素P450)，从而触发药物-药物相互作用，并危及患者的健康。

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 Recognizing the problem, the FDA has issued a Draft Guidance for Industry Drug Interaction Studies, which advice at a minimum in vitro induction evaluation of CYP1A2 and CYP3A4.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.