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.
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.
