Semester

Fall

Date of Graduation

2001

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Timothy S. Tracy.

Abstract

Cytochrome P450 enzymes can exhibit non-Michaelis-Menten, or atypical kinetics. In particular, dapsone activates CYP2C9-mediated flurbiprofen 4 '-hydroxylation. However, the mechanism of activation of CYP2C9 is poorly understood. The activation of flurbiprofen, naproxen, and piroxicam metabolism by dapsone was investigated, fitting kinetic data to a two-site effector model, indicating that the CYP2C9 active site may have multiple binding regions. In addition the structural requirements for activation of CYP2C9 were explored using structural analogs of dapsone, with results suggesting that a sulfone group between two benzene rings is necessary for activation. CYP2C9 *2(R144C) and *3(I359L) allelic variants and the F114L mutant enzymes have also been used to see if these amino acid changes alter dapsone activation of CYP2C9. In this study, flurbiprofen 4'-hydroxylation was activated ∼5-fold in the F114L mutant, but only ∼2-fold for wild-type enzyme in the presence of dapsone. Also, piroxicam 5'-hydroxylation was not activated by dapsone in the F114L mutant, which had higher piroxicam hydroxylase activity than CYP2C9 wild-type, suggesting a role for F114 in activation. In addition, substrate metabolism was studied in CYP2C9/19 chimeric and CYP2C19 mutant enzymes. Results suggest that amino acids 228--292 are important for catalysis of flurbiprofen, naproxen, and piroxicam. In addition, the CYP2C19 KSN mutant has the greatest activity towards flurbiprofen 4 '-hydroxylation, indicating that changes at residues 241, 286, and 289 confer activity to CYP2C19. It has also been discovered that hydroxy-dapsone, the major phase I metabolite of dapsone activates flurbiprofen metabolism, although through a different mechanism. Spectral binding studies confirm kinetic data, indicating that dapsone directly affects the binding of flurbiprofen, whereas hydroxy-dapsone does not. Additional studies with ibuprofen indicates that dapsone alters the binding orientation of this substrate, as the ratio of 3-OH to 2-OH ibuprofen changes in the presence of dapsone. Lastly a urine and plasma assay for detection of flurbiprofen and 4'-hydroxy flurbiprofen was developed for use in analysis of samples in a clinical study investigating activation of metabolism in vivo. It was found that dapsone activates flurbiprofen 4'-hydroxylation in vivo about 10%, much less than that observed in vitro.

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