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: 8396315
• 负责人: MARIA INES MORANO
• 金额: $ 44.23万
• 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 2014-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8396315
• 关键词: Award; Biological Assay; CCAAT-Enhancer-Binding Proteins; CYP1A1 gene; CYP1A2 gene; CYP2B6 gene; CYP2C19 gene; CYP2C9 gene; CYP3A4 gene; Caymans; Cell Culture Techniques; Cell Line; Cell Survival; Cells; Chemicals; Clinical; Code; Color; Complement; Coupling; Culture Media; Cytochrome P450; Cytochromes; Data; Development; Drug Interactions; Drug Prescriptions; Enzyme Induction; Enzymes; Evaluation; Exhibits; Frequencies; Genetic Variation; Genomics; Glucocorticoid Receptor; Goals; Gold; Growth; Health; Hepatic; Hepatocyte; Housing; Human; In Vitro; Individual; Kinetics; Lead; Legal patent; Libraries; Life; Marketing; Measurement; Measures; Mediating; Messenger RNA; Metabolism; Molecular; Monitor; Nuclear; Nuclear Receptors; Patients; Pharmaceutical Preparations; Phase; Physiological; Population; Preclinical Drug Evaluation; Production; Promoter Regions; Protein Isoforms; Protocols documentation; RXR; Regulation; Regulatory Element; Reporter; Reporter Genes; Reproducibility; Risk; Safety; Sampling; Scientist; Screening procedure; Shadowing (Histology); Shipping; Ships; Small Business Innovation Research Grant; Surface; System; Technology; Testing; Time; Toxicology; Transfection; Up-Regulation; Validation; Variant; Work; Xenobiotics; base; cost effective; cytotoxicity; drug candidate; drug discovery; drug mechanism; drug metabolism; enzyme activity; flexibility; hepatoma cell; high throughput screening; improved; interest; large scale production; method development; novel; phase 1 study; promoter; prototype; receptor binding; research clinical testing; response; small molecule libraries; success; transcription factor; uptake; user-friendly

DESCRIPTION (provided by applicant): Many prescribed drugs exhibit drug-drug interactions due to up-regulation or inhibition of specific P450 enzymes. According to a recent guidance from the FDA, CYP1A2, CYP2B6, and CYP3A4 induction should be evaluated in at least an in vitro system. Enzymatic activity in freshly isolated hepatocytes is the "Gold Standard" for studying P450 induction; however, its application is hindered by variation among donors and limited supply. In addition, the exact mechanism of P450 induction cannot be revealed, and induction may be over shadowed by simultaneous inhibition of enzyme activity. Within the last few years, the mechanisms for the induction of individual P450 isoforms have been extensively explored using reporter gene systems. Many CYPs are induced by similar mechanisms through PXR, CAR, or AHR, and yet differential responses are observed among different CYPs due to preferential nuclear receptor binding or interactions with other nuclear receptors or transcription factors. During Phase I of this study, we established SEAP reporter constructs for CYP1A2, CYP2B6, and CYP3A4 each containing human genomic sequence carrying all known and putative regulatory elements required to mediate induction to xenobiotics as in liver cells. Using Originus' patented STEP (Surface Transfection and Expression Protocol) technology, these reporters were co-introduced with necessary transcription factors and nuclear receptors into human hepatoma cell lines to produce responses comparable to human hepatocytes. The CYP-SEAP reporter assay protocol is much simpler than measuring enzyme activity or mRNA, and media samples can be collected at multiple time points to monitor the kinetics of induction. Prototypes of these STEP plates were tested in house and validated externally with excellent robustness and reproducibility. During Phase II, we will further optimize the induction assays by supplementing additional nuclear receptors and transcription factors critical for the modulation of CYP promoter activities. In addition, we will expand the spectrum of reporters to include P450 CYP2C9 and CYP2C19, which are not commercially available. Further, we will transfer the reporter system into a more physiological relevant immortalized human hepatocyte cell line, Fa2N-4, to mimic the metabolism and physiological response of hepatocytes, and yet provide specific P450 induction information in a simple and reliable assay format. These assays will be applied in a high throughput screening facility on a representative drug library. Finally, we will standardize the manufacturing, packaging, and storage protocol for extended shelf life and reproducibility. The assay protocol will also be streamlined to be more user friendly and readily compatible with other assays (such as for cytotoxicity or enzyme activity measurement). Ultimately, the Phase II will lead to two lines of assay systems: one for studying individual induction mechanisms of CYP P450 in hepatoma cells, and the second for assaying the physicological CYP induction response using Fa2N-4 cells. 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(由申请人提供)：许多处方药表现出药物与药物的相互作用，这是由于特定的P450酶上调或抑制。根据FDA最近的一项指导意见，至少应该在体外系统中评估细胞色素P1A2、细胞色素P4B6和细胞色素P3A4的诱导。新鲜分离的肝细胞的酶活性是研究P450诱导的“金标准”；然而，供体之间的差异和有限的供应阻碍了它的应用。此外，P450诱导的确切机制还不能被揭示，并且诱导可能被同时抑制酶活性所掩盖。在过去的几年里，利用报告基因系统已经广泛地探索了诱导单个P450亚型的机制。许多细胞周期蛋白通过PXR、CAR或AHR被相似的机制诱导，但由于优先与核受体结合或与其他核受体或转录相互作用，不同的细胞周期蛋白之间有不同的反应 各种因素。在这项研究的第一阶段，我们建立了CYP1A2、CYP2B6和CYP3A4的SEAP报告结构，每个都包含人类基因组序列，携带所有已知和假定的调节元件，所需的中介诱导外源物质，如在肝细胞中。利用Originus的专利STEP(表面转染和表达协议)技术，这些记者与必要的转录因子和核受体共同导入人类肝癌细胞系，以产生与人类肝细胞类似的反应。CYP-SEAP报告分析方法比测定酶活性或mRNA简单得多，而且可以在多个时间点收集介质样本来监测诱导的动力学。这些阶梯板的原型在室内进行了测试，并在外部进行了验证，具有良好的健壮性和重复性。在第二阶段，我们将通过补充额外的核受体和转录因子来进一步优化诱导分析，这些核受体和转录因子对调节 CYP启动子活性。此外，我们还将扩大记者的范围，将尚未商业化的P450 CYP2C9和CYP2C19包括在内。此外，我们将把报告系统转移到更具生理学相关性的永生化人肝细胞系Fa2N-4中，以模拟肝细胞的代谢和生理反应，并以简单可靠的检测形式提供特异性的P450诱导信息。这些分析将在一个具有代表性的药物文库的高通量筛选设施中应用。最后，我们将标准化制造、包装和储存协议，以延长保质期和重现性。化验方案也将得到简化，使其更加用户友好，并易于与其他化验方法兼容(例如用于细胞毒性或酶活性测定)。最终，第二阶段将导致两个检测系统：一个用于研究CYP P450在肝癌细胞中的单独诱导机制，第二个用于分析使用Fa2N-4细胞的生理CYP诱导反应。 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。