SBIR-STTR Award

Cytochrome P450s are enzymes that metabolize drugs and other potentially toxic compounds in the human body. Without them, toxic chemicals may accumulate to unsafe levels and administered drugs may continue to act indefinitely. Interindividual variation in P450 constituency is one reason for an FDA mandate to pharmaceutical companies, that drug candidates be tested for their potential to induce P450 enzymes. Further, combinatorial chemical technology produces thousands of biologically active compounds daily that require screening for P450 inducibility. To address these needs, this proposal develops a high- volume in vitro assay demonstrating therapeutic-mediated induction of human P450 enzymes. This test system will employ two human P450s, CYP3A4 and 1A2. Both enzymes play major roles in the biostransformation of therapeutic agents and are inducible by a variety of xenobiotics. Regulatory regions of these P450 genes will be ligated to a reporter gene, green fluorescent protein (GFP), and transfected into human tumor cell lines. An "ELISA-like" plate format will be developed for the treatment of transfected cells with candidate drugs. Increased expression of GFP is monitored fluorimetrically and correlates to the drugs' ability to induce a particular P450 enzyme. The ability to predict the behavior of drugs in humans prior to clinical trials, thereby addressing FDA requirements, in a fast and easy format are the hallmarks of this technology.Proposed Commercial Application:Not avaliable

Thesaurus Terms:
cytochrome P450, enzyme induction /repression, gene expression, immunologic assay /test, method development, regulatory gene genetic susceptibility, green fluorescent protein, reporter gene cell line, enzyme linked immunosorbent assay, transfection

The focus of this phase II application is to develop an easy-to-use high-throughput screening (HTS) system for evaluating drug candidates for their ability to induce the drug metabolizing P450 enzymes. During phase I, vectors were developed that contained the regulatory elements of CYP1A2 and CYP3A4 which when transiently transfected were responsive to drugs causing transcriptional activation of a reporter gene. In phase II, five specific aims are proposed to initiate marketing of these HTS systems. In aim one, Puracyp will establish stable hepatomas containing CYPIA2 and CYP3A4 enhancers. The cell lines will alleviate variability and reduce production time and costs. Aim two contains experiments to compare results of inducers in primary hepatocyte cultures with those obtained in stable transformants. These studies will demonstrate the utility of Puracyp's HTS system as a surrogate for hepatocytes. In aim three, Puracyp will assemble the HTS systems and establish quality control standards. Specific aim 4 will isolate other defined response elements of human drug metabolizing P450 genes including the phenobarbital response element of CYP2B6 and the glucocorticoid responsive region of CYP3A5. Vectors will be developed in aim 5 that contain the phenobarbital response element of CYP3A4 enabling additional drugs to be tested for their inductive properties of this P450. Finally, aim 6 experiments will incorporate the enhancer elements of other inducible human drug metabolizing P450s. Collectively; Puracyp will market the first HTS for evaluating drug-mediated induction of P450 enzymes in a simplified, low cost, and timesaving system. PROPOSED COMMERCIAL APPLICATION: It is a federal mandate that all preclinical testing include studies to determine the inducibility of the drug metabolizing P450 enzymes by candidate drugs. In light of current genomic and combinatorial paradigms, it is imperative that the pharmaceutical industry adopt FITS methods for preclinical drug discovery. To date, an HTS system for assessing P450 inducibility is unavailable. Puracyp is currently developing such a system which will produce reliable, accurate and consistent results.