SBIR-STTR Award

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 Recognizing the problem, the FDA has issued a Draft Guidance for Industry Drug Interaction Studies, which advice at a minimum in vitro induction evaluation of CYP1A2 and CYP3A4.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

Thesaurus Terms:
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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.