Abstract
Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.
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Acknowledgements
Y.G. was supported by a grant (1 R01 GM068885-01A1) from the National Institute of General Medical Sciences awarded to G.P. We would like to thank David Shaw, Ezra Tai, Witold Woroniecki, Lisa Lohr, Will Tao, Grace Lam and Larry Bowen for help with this manuscript.
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Several of the authors are employees of Roche Palo Alto. However, we have no direct financial interest in the results presented in this manuscript.
Supplementary information
Supplementary Fig. 1
β–glucuronidase hydrolysis of M8 (putative warfarin conjugates). (PDF 69 kb)
Supplementary Fig. 2
A logarithmic plot comparing the plasma concentrations of R-warfarin, 7-hydroxywarfarin and M8 in strains with a high rate (Balb/cbyJ) and a low rate (B.10.D2-H2/oSnJ) of generating 7-hydroxywarfarin metabolites. (PDF 46 kb)
Supplementary Fig. 3
The Area Under Concentration-time Curve (AUC) for 7-hydroxywarfarin metabolites (7-OH +M8) within 8 hr after a 10 mg/kg IP dose of 14C-R-warfarin was administered to males of 13 inbred mouse strains. (PDF 55 kb)
Supplementary Table 1
R-warfarin metabolic profiles determined by radiometric methods in pooled plasma samples (1-8 h postdose) after an IP dose 10 mg/kg of 14C-R-warfarin to males of 13 inbred mouse strains. (PDF 60 kb)
Supplementary Table 2
The calculated AUC 0-8 h forR-warfarin and its metabolites following a single IP dose of 10 mg/kg of 14C-R-warfarin to males of 13 inbred mouse strains. (PDF 68 kb)
Supplementary Table 3
All genomic regions where the C57B/6J and B.10.D2-H2/oSnJ share a unique haplotype that differs from the other 11 strains. (PDF 77 kb)
Supplementary Table 4
Kinetic parameters for in vitro biotransformation of R-warfarin to 6-, 7-and 8-hydroxywarfarin by recombinant Cyp2c29 and CD-1 mouse liver microsomes. (PDF 68 kb)
Supplementary Table 5
MRM parameters for the detection of warfarin, 4’-, 6-, 7-, 8-, 10-hydroxywarfarin, deuterium-labeled 7-hydroxywarfarin (IS) and 7-hydroxywarfarin-4-glucuronide. (PDF 67 kb)
Supplementary Table 6
PCR primers used in the cDNA cloning and RT-PCR experiments. (PDF 36 kb)
Supplementary Note
Analysis of R-warfarin metabolites in inbred mouse strains. (DOC 24 kb)
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Guo, Y., Weller, P., Farrell, E. et al. In silico pharmacogenetics of warfarin metabolism. Nat Biotechnol 24, 531–536 (2006). https://doi.org/10.1038/nbt1195
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DOI: https://doi.org/10.1038/nbt1195
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