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University of Wyoming

UW Technologies Available for Licensing


Technology Disclosure: Technology Disclosure: 05-075 Novel Substrates for Cytochrome P450 Mediated N-Dealkylation

 

New drug development is a major area of significance to healthcare in America, and our system of new drug approval is among the most rigorous in the world. For a new drug to be approved by the FDA, it has to be analyzed for its metabolic behavior. A new drug’s metabolic pathways must be studied to understand its disposition from the body and the potential for interactions with other drugs.

To date, testing a drug’s metabolic behavior has been very difficult. The main biological system involved in drug metabolism is the cytochrome P450 system. Cytochromes P450 are the principal enzymes for phase I oxidative metabolism of drugs, and there are not many direct, facile assays available to measure the N-dealkylating power of these enzymes. To test a new drug against these N-dealkylating P450 enzymes, specific substrates must be available. There are very few substrates commercially available for N-dealkylase activities and none available where the dealkylase activity can be measured directly in real time. Available substrates are expensive, require time consuming procedures for quantitation, or are not true measures of N-dealkylase activity but rather O-dealkylase activity.

Researchers at the University of Wyoming School of Pharmacy and the USDA’s Arthropod-Borne Animal Diseases Research Laboratory have developed inexpensive fluorescent substrates that are specific to the cytochromes P450 most commonly responsible for the N-dealkylation metabolism of drugs. This novel technology is based on conversion of low fluorescing substrates by cytochromes P450 into highly fluorescent product. The substrates can be quickly incorporated into metabolic assay tests, are inexpensive, can be measured quantitatively by fluorescence, and are suitable for high throughput screening. These substrates have the potential to be widely used in drug metabolism testing.

Cytochrome P450 isozyme CYP2D6 is a well-documented N-dealkylase. Figure 1 depicts a standard assay using novel “UW Substrate A” and its metabolism by cDNA expressed human CYP2D6 (and reductase) into the highly fluorescent metabolite.

CYP2D6 is apparently reactive only with three of the substrate derivatives (A, B, and C) as shown in Figure 2. Further, these substrates are highly specific for N-dealkylases CYP2D6 and CYP1A1 (Figure 3). No activity was observed with CYP3A5, CYP 1A2, CYP3A4, and CYP2C19.

Further evidence that these substrates are true N-dealkylase substrates comes from the use of known inhibitors of cytochrome P450 N-dealkylases. Figure 4 shows the results of additions of varying amounts of quinidine, imipramine, and amitriptyline to reaction mixtures containing “UW Substrate A” and CYP2D6. The calculated I50’s are comparable to those reported in the literature for other substrates metabolized by CYP2D6.

Besides being suitable substrates for expressed CYP’s, the substrates also work with microsomes (human, monkey, and rat) and cell suspensions. Figure 5 shows fluorescence trace of a reaction with human hepatocellular carcinoma cells and “UW Substrate A”. The lag in the trace is presumably due to the amount of time for the substrate to transport across the cell membrane.

The development of sensitive and specific assays is critical to the study of drug metabolism and interactions. Just as one example, phosphonates are an evolving group of drugs that have a great potential for use as protease inhibitors (cancer, AIDS, West Nile virus infections), insecticides, or fungicides; however, little is known about their metabolic fate. The novel substrates developed at the University of Wyoming will give insight into which P450 enzyme systems are involved and whether this group of drugs could have some potentially dangerous interactions with other drugs.

These substrates are also applicable to the growing field of pharmacogenomics that is defined as the study of the association between genetics and drug response. Not every individual reacts to a drug in the same way. This variable drug responsiveness is caused by polymorphisms within multiple genes responsible for metabolism of the drug. These substrates can be used in a clinical laboratory to assess the metabolic behavior of one person towards a specific drug to help avoid dosing errors and improve efficacy. They will also aid in the development of customized, genotype-based therapies to target groups of patients.

If you would like to learn more about these novel substrates and how your company may apply them in commercial situations, please contact the director of the University of Wyoming Research Products Center, Davona Douglass. We would be pleased to share further details.