Date of Defense

Summer 8-20-1997

Department

Biological Sciences

First Advisor

Zhiyang Zhao, Pharmacia & Upjohn

Second Advisor

Christine Byrd, Biological Sciences

Third Advisor

John Spitsbergen, Biological Sciences

Abstract

Compound 1-cyclopropyl-4-phenyl-1,2,3,6-tetrahydropyridine (CPTP) is a structural analog of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which becomes a neurotoxin after bioactivation, causing Parkinson's syndrome. The cyclopropyl ring on CPTP gives it a unique structure that allows it to inhibit MAO-B, the enzyme responsible for the activation of MPTP and the toxicity. Therefore, CPTP is currently being studied to determine the metabolic pathways and enzymes involved in its metabolism. In this project, CPTP metabolism was studied in vitro in human liver microsomes, and it was found that the process was NADPH dependent, suggesting the involvement of P450 enzymes. Kinetic parameters (Km and Vmax) were measured for metabolites M2 (4-phenyl-1,2,3,6-tetrahydropyridine) and M3 (1-cyclopropyl-4-phenylpyridinium). M2, which is of primary importance because it involves the decyclopropylation from CPTP, seemed to have at least two enzymes involved in its formation, with the following kinetic parameters: Kml=82.3±8.05 µM; Vmax1=1.91±0.098 nmol/mg/min; Km2=275±44.0 µM, Vmax2=4.33±0.32 nmmol/mg/min. Correlation, inhibition, and cloned P450 isoform studies were used to determine the specific cytochrome P450 isoforms involved in the metabolism of CPTP. CYP2D6 and 2A6 showed involvement in the formation fm2, with minor contributions from CYP3A4 and possibly 2B6, 2C8, and 2C9. Alternate metabolic mechanism studies, involving hydrogen peroxide, peroxidase, and superoxide dismutase (SOD), were also conducted, in an effort to examine the involvement of other enzymes. It was found that the presence of reductase had no effect on the matbolite formation. SOD did have some effect on the metabolism, but further studies need to be conducted in this area. CYP3A4 is found to be involved in the formation of M3, and contributions from other enzymes need to be further evaluated.

Access Setting

Honors Thesis-Campus Only

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