Abstract
Doxorubicin (DOX) and daunorubicin (DAUN) are effective anticancer drugs; however, considerable interpatient variability exists in their pharmacokinetics. This may be caused by altered metabolism by nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in genes encoding aldo-keto reductases (AKRs) and carbonyl reductases. This study examined the effect of 27 ns-SNPs, in eight human genes, on the in vitro metabolism of both drugs to their major metabolites, doxorubicinol and daunorubicinol. Kinetic assays measured metabolite levels by high-performance liquid chromatography separation with fluorescence detection using purified, histidine-tagged, human wild-type, and variant enzymes. Maximal rate of activity (Vmax), substrate affinity (Km), turnover rate (kcat), and catalytic efficiency (kcat/Km) were determined. With DAUN as substrate, variants for three genes exhibited significant differences in these parameters compared with their wild-type counterparts: the A106T, R170C, and P180S variants significantly reduced metabolism compared with the AKR1C3 wild-type (Vmax, 23–47% decrease; kcat, 22–47%; kcat/Km, 38–44%); the L311V variant of AKR1C4 significantly decreased Vmax (47% lower) and kcat and kcat/Km (both 43% lower); and the A142T variant of AKR7A2 significantly affected all kinetic parameters (Vmax and kcat, 61% decrease; Km, 156% increase; kcat/Km, 85% decrease). With DOX, the R170C and P180S variants of AKR1C3 showed significantly reduced Vmax (41–44% decrease), kcat (39–45%), and kcat/Km (52–69%), whereas the A142T variant significantly altered all kinetic parameters for AKR7A2 (Vmax, 41% decrease; kcat, 44% decrease; Km, 47% increase; kcat/Km, 60% decrease). These findings suggest that ns-SNPs in human AKR1C3, AKR1C4, and AKR7A2 significantly decrease the in vitro metabolism of DOX and DAUN.
Footnotes
These studies were supported by the Canadian Institutes of Health Research [Grant MOP-68896]. O.S.B. was supported by a Canadian Institutes of Health Research Doctoral Research Award.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.173179.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- AKR
- aldo-keto reductase
- CBR
- carbonyl reductase
- DOX
- doxorubicin
- DAUN
- daunorubicin
- DOXol
- doxorubicinol
- DAUNol
- daunorubicinol
- HPLC
- high-performance liquid chromatography
- ns-SNP
- nonsynonymous single-nucleotide polymorphism
- Km
- substrate affinity
- kcat
- turnover rate
- kcat/Km
- catalytic efficiency
- Ni-NTA
- nickel-nitrilotriacetic acid affinity
- PCR
- polymerase chain reaction
- IPTG
- isopropyl β-d-1-thiogalactopyranoside
- FXa
- factor Xa.
- Received July 20, 2010.
- Accepted September 3, 2010.
- Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics
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