Rapid in vitro substrate assay for the multi-drug resistance p-glycoprotein

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DESCRIPTION (provided by applicant): The ability of a drug to reach and penetrate its intended target within the body is critical to its success in treating disease. However, drug efflux proteins such as p-glycoprotein (pgp) actively pump hydrophobic drugs away from target tissues and are linked to low oral absorption and multidrug resistance in chemotherapy. Protein pumps are of increasing interest to the pharmaceutical industry, most importantly based on new draft FDA guidelines requiring knowledge of whether a drug candidate is a substrate or inhibitor of pgp. Current pgp assays are cumbersome, expensive and unreliable. Our overall goal is to prepare and validate a novel assay reagent - Fluorosome-trans-pgp - which capitalizes on the Fluorosome Technology that we have developed for measuring passive permeability of small molecules through lipid membranes. Fluorosome-trans-pgp will provide a direct, reliable, simple, rapid and inexpensive assay to determine if a drug is a pgp substrate or inhibitor. Toward this goal, in phase I of this project we have: cloned and expressed the human pgp construct containing a 10His tag in HEK293 cells. isolated and purified the protein "pgp 10His" in lipid micelles. demonstrated verapamil-stimulated ATPase activity in the purified pgp 10His lipid micelles. reconstituted the purified pgp 10His into liposome membranes. developed the methodology for the manufacture and assay of Fluorosome-trans-pgp. validated the test systems that will be used to screen potential pgp substrates and inhibitors. Phase II of this project will bring the Fluorosome-trans-pgp assay to the state of an important commercial product. To complete this development, the Specific Aims of phase II are to: 1. complete Fluorosome-trans-pgp production and validation. 2. scale up Fluorosome-trans-pgp production to commercial levels. 3. demonstrate the utility of Fluorosome-trans-pgp with representative pgp substrates and inhibitors. 4. optimize the properties of the Fluorosome-trans-pgp system. 5. develop data analysis software. Our overriding goal in Phase II is to bring Fluorosome-trans-pgp and accompanying software to market. Successful completion of phase II will satisfyi the increasing need for a reliable and economical method to measure a drug's susceptibility to efflux by the pgp pump or to act as a pgp inhibitor. This project also lays the foundation for the design of Fluorosome-based systems to test for the susceptibility of drug candidates to other drug transport proteins. Markets include pharmaceutical and biotechnology companies, contract research organizations, and in-house fee-for service assays. This project completes the development of a test which determines if drugs will be extruded from their target tissue by biological pumps. The test thereby allows the pharmaceutical industry to evaluate, at an early stage, the suitability of drug candidates for continued development. The test is reliable, simple, rapid, inexpensive and amenable to robotics.