NOVEL HIGH THROUGHPUT PLATFORM FOR SCREENING CYTOCHROME P450 INDUCTION

用于筛选细胞色素 P450 诱导的新型高通量平台

• 批准号: 8058570
• 负责人: MARIA INES MORANO
• 金额: $ 22.24万
• 依托单位: ORIGINUS, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-25 至 2012-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8058570
• 关键词: Academia; Alkaline Phosphatase; Americas; Aryl Hydrocarbon Receptor; Award; Binding; Biological Assay; CYP1A1 gene; CYP1A2 gene; CYP2B6 gene; CYP3A4 gene; Cell Line; Cells; Clinical; Cytochrome P450; Cytochromes; DNA; DNA receptor; Development; Drug Evaluation; Drug Industry; Drug Interactions; Drug Kinetics; Elements; Enzyme Induction; Enzymes; Evaluation; Family member; Genes; Genetic Variation; Goals; Gold; Health; Hepatic; Hepatocyte; Human; In Vitro; Individual; Industry; Laboratories; Lead; Ligands; Manufacturer Name; Mediating; Medicine; Metabolism; Methods; Molecular; Nuclear; Nuclear Receptors; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacologic Substance; Phase; Plasmids; Population; Preclinical Drug Evaluation; Process; Production; Productivity; Protocols documentation; Receptor Activation; Regulation; Reporter; Reporter Genes; Reproducibility; Research; Risk; Safety; Screening procedure; Small Business Innovation Research Grant; Surface; Surveys; System; Technology; Testing; Time; Toxicology; Transactivation; Transfection; United States Food and Drug Administration; Validation; Variant; Work; Xenobiotics; base; constitutive androstane receptor; cost; design; drug candidate; drug discovery; drug metabolism; enzyme activity; high throughput screening; human AHR protein; improved; in vitro Assay; in vitro testing; novel; pregnane X receptor; promoter; prototype; receptor; response; safety testing; success; transcription factor

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 Recognizing the problem, the FDA has issued a Draft Guidance for Industry Drug Interaction Studies, which advice at a minimum in vitro induction evaluation of CYP1A2 and CYP3A4.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

描述（申请人提供）：所给药物的代谢取决于药物代谢酶（例如细胞色素（CYP）P450酶）的表达和活性。这些P450受到外源性物质的抑制和有时诱导，导致共同给药药物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发布了一份行业药物相互作用研究指南草案，建议至少对CYP 1A 2和CYP 3A 4进行体外诱导评价。最近也认识到评价CYP 2B 6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中的酶活性。然而，人肝细胞的可用性和个体变异性以及在该系统中无法区分P450抑制和诱导使评价复杂化。外源性物质对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，介导P450诱导的核受体的报告基因测定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，很少评估AhR活性。目前还没有市售的CAR检测试剂盒。然而，需要几种核受体和其他转录因子的组合来诱导特异性P450的表达。 该提案的目标包括设计和实施Originus STEP（表面转染和表达协议）技术的新应用，用于新药候选物的安全性测试。在第一阶段，我们将开发复杂的STEP平台，用于在人肝细胞系中使用报告基因测定法同时评价CYP 1A 2、CYP 3A 4和CYP 2B 6的转录调控。分泌型碱性磷酸酶（SEAP）基因将被用作报告基因，受每个人源性前列腺素上游调控序列的控制。将优化不同CYP-SEAP报告基因和外源性激活受体的单一或相关组合的STEP共转染，以在96孔微孔板中在多个时间点通过一组诱导剂测试SEAP反应。I期的关键是STEP平台原型的生产和β测试（单独或组合），具有适当的试验耐用性和孔间、板间和批间重现性。这些CMP 450诱导平台将可用于工业和学术界高通量筛选实验室在药物发现过程早期进行药物毒理学筛选，或作为简单的测定试剂盒以适度的成本供小型实验室使用。在第二阶段，我们将扩展研究，以评估其他诱导型cMP 450，扩大测试的核受体谱，并进一步开发基于细胞的异源激活受体相关等位基因变体的测定。此外，我们计划开发STEP检测平台，用于分析β-内酰胺酶诱导的途径。 公共卫生相关性：药物安全性是临床开发期间化合物损耗的主要因素之一。迫切需要快速评估化合物代谢和毒理学的简单测定，以最大限度地降低患者风险并提高药物发现管道早期新分子实体的成功率。该提案的目标是使用Originus的专有技术STEP开发用于体外评价候选药物的平台，这些候选药物可能诱导某些肝酶（细胞色素P450），从而引发药物相互作用并损害患者的健康。