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
基本信息
- 批准号:8531242
- 负责人:
- 金额:$ 42.82万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2011
- 资助国家:美国
- 起止时间:2011-01-25 至 2015-07-31
- 项目状态:已结题
- 来源:
- 关键词:AwardBiological AssayCCAAT-Enhancer-Binding ProteinsCYP1A1 geneCYP1A2 geneCYP2B6 geneCYP2C19 geneCYP2C9 geneCYP3A4 geneCaymansCell Culture TechniquesCell LineCell SurvivalCellsChemicalsClinicalCodeColorComplementCouplingCulture MediaCytochrome P450CytochromesDataDevelopmentDrug InteractionsDrug PrescriptionsEnzyme InductionEnzymesEvaluationExhibitsFrequenciesGenetic VariationGenomicsGlucocorticoid ReceptorGoalsGoldGrowthHealthHepaticHepatocyteHousingHumanIn VitroIndividualKineticsLeadLegal patentLibrariesLifeMarketingMeasurementMeasuresMediatingMessenger RNAMetabolismMolecularMonitorNuclearNuclear ReceptorsPatientsPharmaceutical PreparationsPhasePhysiologicalPopulationPreclinical Drug EvaluationProductionPromoter RegionsProtein IsoformsProtocols documentationRXRRegulationRegulatory ElementReporterReporter GenesReproducibilityRiskSafetySamplingScientistShadowing (Histology)ShippingShipsSmall Business Innovation Research GrantSurfaceSystemTechnologyTestingTimeToxicologyTransfectionUp-RegulationValidationVariantWorkXenobioticsbasecost effectivecytotoxicitydrug candidatedrug discoverydrug mechanismdrug metabolismenzyme activityflexibilityhepatoma cellhigh throughput screeningimprovedinterestlarge scale productionmethod developmentnovelphase 1 studypromoterprototypepublic health relevancereceptor bindingresearch clinical testingresponsescreeningsmall molecule librariessuccesstranscription factoruptakeuser-friendly
项目摘要
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.
描述(由申请人提供):由于特定P450酶的上调或抑制,许多处方药表现出药物间相互作用。根据FDA的最新指南,应至少在体外系统中评价CYP1A2、CYP2B6和CYP3A4诱导。新鲜分离的肝细胞中的酶活性是研究P450诱导的"金标准";然而,其应用受到供体之间的差异和有限供应的阻碍。此外,P450诱导的确切机制不能被揭示,并且诱导可能被同时抑制酶活性所掩盖。在过去的几年中,个别P450亚型的诱导机制已被广泛探索使用报告基因系统。许多CYP通过类似的机制通过PXR、CAR或AHR诱导,但由于优先的核受体结合或与其他核受体或转录的相互作用,在不同的CYP中观察到不同的反应
因素在本研究的I期期间,我们建立了CYP1A2、CYP2B6和CYP3A4的SEAP报告基因构建体,每个构建体均含有人类基因组序列,携带所有已知和推定的调节元件,这些元件是介导如在肝细胞中诱导外源性物质所需的。使用Originus的专利STEP(表面转染和表达协议)技术,这些报告基因与必要的转录因子和核受体共同引入人肝癌细胞系,产生与人肝细胞相当的反应。CYP-SEAP报告基因测定方案比测量酶活性或mRNA简单得多,并且可以在多个时间点收集培养基样品以监测诱导的动力学。这些STEP平板的原型在内部进行了测试,并在外部进行了验证,具有良好的耐用性和重现性。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试验,这些受体和转录因子对调节细胞凋亡至关重要。
促进活动。此外,我们将扩大报告基因的范围,以包括尚未上市的P450 CYP2C9和CYP2C19。此外,我们将报告系统转移到一个更生理相关的永生化人肝细胞系,Fa2N-4,模拟肝细胞的代谢和生理反应,但提供了一个简单可靠的分析格式的特定P450诱导信息。这些试验将在代表性药物库的高通量筛选设施中应用。最后,我们将标准化生产、包装和储存方案,以延长保质期和重现性。还将简化试验方案,使其更加用户友好,并易于与其他试验(如细胞毒性或酶活性测量)兼容。最终,第二阶段将导致两个线的测定系统:一个用于研究肝癌细胞中TP450的个体诱导机制,第二个用于测定使用Fa2N-4细胞的生理学诱导应答。
项目成果
期刊论文数量(0)
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MARIA INES MORANO其他文献
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