Semester

Spring

Date of Graduation

2008

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Physiology, Pharmacology & Neuroscience

Committee Chair

Patrick S. Callery.

Abstract

Busulfan is a bifunctional alkylating agent that is used to treat mylogenous leukemia. The major elimination pathway of busulfan is through glutathione-S-transferase (GST) catalyzed conjugation to form glutathione sulfonium conjugate. The aim of this work is to elucidate the novel metabolic pathways of busulfan that may explain its toxicity. The observed data showed that busulfan is not a substrate for CYP450. The sulfonium ion conjugate of busulfan was found to be inactive as it did not inhibit GST in human liver cytosol, did not react with 4-(4-nitrobenzyl)pyridine, did not induce apoptosis in NCI-H460 cells, and was not stable in basic conditions with a half life of 6.0 hours at pH 7.4. The degradation products were identified to be tetrahydrothiophene and dehydroglutathione. Dehydroglutathione is a glutathione analogue in which the cysteine moiety is replaced by dehydroalanine moiety, which makes it a Michael acceptor. This secondary metabolite of busulfan produced cytotoxicity against C6 glioma cells and reacted in vitro with sulfhydryl nucleophiles such as glutathione and cysteine. An alternative metabolic pathway for the sulfonium ion conjugate of busulfan is through the mercapurate pathway which will lead to the formation of the cysteine sulfonium conjugate of busulfan (THT-A). THT-A was found to undergo a non-enzymatic beta-elimination reaction at pH 7.4 and 37 °C to yield tetrahydrothiophene, pyruvate and ammonia. This reaction is accelerated by (a) rat liver, kidney and brain homogenates, (b) isolated rat liver mitochondria, and (c) pyridoxal 5'-phosphate (PLP). A PLP-dependent enzyme in rat liver cytosol that catalyzes a beta-lyase reaction with THT-A was identified as cystathionine gamma-lyase. This unusual drug metabolism pathway represents an alternate route for intermediates in the mercapturate pathway.

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