NOVEL HIGH THROUGHPUT PLATFORM FOR SCREENING CYTOCHROME P450 INDUCTION

点击翻译按钮获取中文摘要

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. 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.