Abstract
The enzyme kinetics of the hydrolysis of the one-ring open metabolites of the antioxidant cardioprotective agent dexrazoxane [ICRF-187; (+)-1,2-bis(3,5-dioxopiperazin-1-yl)propane] to its active metal ion binding form ADR-925 [N,N′-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)glycine] by dihydroorotase (DHOase) has been investigated by high-performance liquid chromatography (HPLC). A spectrophotometric detection HPLC assay for dihydroorotate was also developed. Dexrazoxane is clinically used to reduce the iron-based oxygen free radical-mediated cardiotoxicity of the anticancer drug doxorubicin. DHOase was found to catalyze the ring opening of the metabolites with an apparentVmax that was 11- and 27-fold greater than its natural substrate dihydroorotate. However, the apparentKm for the metabolites was 240- and 550-fold larger than for dihydroorotate. This report is the first that DHOase might be involved in the metabolism of a drug. Furosemide inhibited DHOase, but the neutral 4-chlorobenzenesulfonamide did not. Because dihydroorotate, the one-ring open metabolites, and furosemide all have a carboxylate group, it was concluded that a negative charge on the substrate strengthened binding to the positively charged active site. The presence of DHOase in the heart may explain the cardioprotective effect of dexrazoxane. Thus, dihydropyrimidinase and DHOase acting in succession on dexrazoxane and its metabolites to form ADR-925 provide a mechanism by which dexrazoxane is activated to exert its cardioprotective effects. The ADR-925 thus formed may either remove iron from the iron-doxorubicin complex, or bind free iron, thus preventing oxygen radical formation.
Footnotes
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This work was supported by the Canadian Institutes of Health Research, the Canada Research Chairs program, and a Canada Research Chair in Drug Development for Brian Hasinoff, and National Science Foundation Grant MCB-98-08562). P.S. was supported by a Manitoba Health Research Council studentship and Canadian Institutes of Health Research studentship.
- Abbreviations used are::
- HPLC
- high-pressure liquid chromatography
- DHOase
- dihydroorotase
- DHPase
- dihydropyrimidine amidohydrolase or dihydropyrimidinase
- CAD
- trifunctional protein catalyzing the first three steps of de novo pyrimidine synthesis in higher eukaryotes
- ADR-925
- N,N′-[(1S)-1-methyl-1,2-ethanediyl]bis[N-(2-amino-2-oxoethyl)glycine
- Received June 28, 2002.
- Accepted September 3, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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