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In silico pharmacogenetics of warfarin metabolism

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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Figure 1: R-warfarin metabolism in males of 13 inbred mouse strains.
Figure 2: Analysis of R-warfarin metabolites.
Figure 3: Haplotype-based genetic analysis of warfarin metabolites.
Figure 4: Haplotype map and expression of Cyp2c genes.
Figure 5: Cyp2c29 in R-warfarin biotransformation.
Figure 6: Immunoblot analysis of Cyp2c29 protein in liver extracts prepared from seven inbred strains.

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References

  1. Evans, W.E. & Relling, M.V. Pharmacogenomics: translating functional genomics into rational therapeutics. Science 286, 487–491 (1999).

    Article  CAS  Google Scholar 

  2. Wang, J. & Peltz, G. Haplotype-based computational genetic analysis in mice. in Computational Genetics and Genomics: New Tools for Understanding Disease (ed. Peltz, G.) 51–70 (Humana Press Inc., Totowa, NJ, 2005).

    Chapter  Google Scholar 

  3. Wang, J., Liao, G., Usuka, J. & Peltz, G. Computational genetics: from mouse to human? Trends Genet. 21, 526–532 (2005).

    Article  CAS  Google Scholar 

  4. Liao, G. et al. In silico genetics: identification of a functional element regulating H2-Ealpha gene expression. Science 306, 690–695 (2004).

    Article  CAS  Google Scholar 

  5. Wang, J. et al. Haplotypic structure of the mouse genome. in Computational Genetics and Genomics: New Tools for Disease Biology (ed. Peltz, G.) 71–83 (Humana Press Inc., Totowa, NJ, 2005).

    Chapter  Google Scholar 

  6. Phillips, K.A., Veenstra, D.L., Oren, E., Lee, J.K. & Sadee, W. Potential role of pharmacogenomics in reducing adverse drug reactions: a systematic review. J. Am. Med. Assoc. 286, 2270–2279 (2001).

    Article  CAS  Google Scholar 

  7. Suttie, J.W. The biochemical basis of warfarin therapy. Adv. Exp. Med. Biol. 214, 3–16 (1987).

    CAS  PubMed  Google Scholar 

  8. Nelsestuen, G.L., Zytkovicz, T.H. & Howard, J.B. The mode of action of vitamin K. Identification of gamma-carboxyglutamic acid as a component of prothrombin. J. Biol. Chem. 249, 6347–6350 (1974).

    CAS  PubMed  Google Scholar 

  9. Stenflo, J., Fernlund, P., Egan, W. & Roepstorff, P. Vitamin K dependent modifications of glutamic acid residues in prothrombin. Proc. Natl. Acad. Sci. USA 71, 2730–2733 (1974).

    Article  CAS  Google Scholar 

  10. James, A.H., Britt, R.P., Raskino, C.L. & Thompson, S.G. Factors affecting the maintenance dose of warfarin. J. Clin. Pathol. 45, 704–706 (1992).

    Article  CAS  Google Scholar 

  11. Hallak, H.O. et al. High clearance of (S)-warfarin in a warfarin-resistant subject. Br. J. Clin. Pharmacol. 35, 327–330 (1993).

    Article  CAS  Google Scholar 

  12. Kaminsky, L.S. & Zhang, Z.Y. Human P450 metabolism of warfarin. Pharmacol. Ther. 73, 67–74 (1997).

    Article  CAS  Google Scholar 

  13. Rettie, A.E., Wienkers, L.C., Gonzalez, F.J., Trager, W.F. & Korzekwa, K.R. Impaired (S)-warfarin metabolism catalysed by the R144C allelic variant of CYP2C9. Pharmacogenetics 4, 39–42 (1994).

    Article  CAS  Google Scholar 

  14. Daly, A.K. & King, B.P. Pharmacogenetics of oral anticoagulants. Pharmacogenetics 13, 247–252 (2003).

    Article  CAS  Google Scholar 

  15. Rieder, M.J. et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N. Engl. J. Med. 352, 2285–2293 (2005).

    Article  CAS  Google Scholar 

  16. Jansing, R.L., Chao, E.S. & Kaminsky, L.S. Phase II metabolism of warfarin in primary culture of adult rat hepatocytes. Mol. Pharmacol. 41, 209–215 (1992).

    CAS  PubMed  Google Scholar 

  17. Rowland, M. & Tozer, T.N. Clinical Pharmacokinetics: Concepts and Applications, 367–393 (Lippincott Williams & Wilkins, Philadelphia, 1995).

    Google Scholar 

  18. Riley, R.J., Hemingway, S.A., Graham, M.A. & Workman, P. Initial characterization of the major mouse cytochrome P450 enzymes involved in the reductive metabolism of the hypoxic cytotoxin 3-amino-1,2,4-benzotriazine-1,4-di-N-oxide (tirapazamine, SR 4233, WIN 59075). Biochem. Pharmacol. 45, 1065–1077 (1993).

    Article  CAS  Google Scholar 

  19. Crespi, C.L. & Miller, V.P. The use of heterologously expressed drug metabolizing enzymes—state of the art and prospects for the future. Pharmacol. Ther. 84, 121–131 (1999).

    Article  CAS  Google Scholar 

  20. Luo, G., Zeldin, D.C., Blaisdell, J.A., Hodgson, E. & Goldstein, J.A. Cloning and expression of murine CYP2Cs and their ability to metabolize arachidonic acid. Arch. Biochem. Biophys. 357, 45–57 (1998).

    Article  CAS  Google Scholar 

  21. Meyer, R.P., Hagemeyer, C.E., Knoth, R., Kurz, G. & Volk, B. Oxidative hydrolysis of scoparone by cytochrome p450 CYP2C29 reveals a novel metabolite. Biochem. Biophys. Res. Commun. 285, 32–39 (2001).

    Article  CAS  Google Scholar 

  22. Rossi, D.T. Sample preparation and handling for LC/MS in drug discovery. in Mass Spectrometry in Drug Discovery (ed. Rossi, D.T. & Sinz, M.W.) 171–214 (Marcel Dekker, Inc., New York, 2002).

    Google Scholar 

  23. Ufer, M., Kammerer, B., Kirchheiner, J., Rane, A. & Svensson, J.O. Determination of phenprocoumon, warfarin and their monohydroxylated metabolites in human plasma and urine by liquid chromatography-mass spectrometry after solid-phase extraction. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 809, 217–226 (2004).

    Article  CAS  Google Scholar 

  24. Edelbroek, P.M., van Kempen, G.M., Hessing, T.J. & de Wolff, F.A. Analysis of phenprocoumon and its hydroxylated and conjugated metabolites in human urine by high-performance liquid chromatography after solid-phase extraction. J. Chromatogr. 530, 347–358 (1990).

    Article  CAS  Google Scholar 

  25. Fitch, W.L. et al. Identification of glutathione-derived metabolites from an IP receptor antagonist. Drug Metab. Dispos. 32, 1482–1490 (2004).

    Article  CAS  Google Scholar 

  26. Patten, C.J. et al. Kinetic analysis of the activation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by heterologously expressed human P450 enzymes and the effect of P450-specific chemical inhibitors on this activation in human liver microsomes. Arch. Biochem. Biophys. 333, 127–138 (1996).

    Article  CAS  Google Scholar 

  27. Omura, T. & Sato, R. The carbon monoxide-binding pigment of liver microsomes. I. Evidence for its hemoprotein nature. J. Biol. Chem. 239, 2370–2378 (1964).

    CAS  PubMed  Google Scholar 

  28. Zhang, Z.Y., King, B.M. & Wong, Y.N. Quantitative liquid chromatography/mass spectrometry/mass spectrometry warfarin assay for in vitro cytochrome P450 studies. Anal. Biochem. 298, 40–49 (2001).

    Article  CAS  Google Scholar 

  29. Buters, J.T., Shou, M., Hardwick, J.P., Korzekwa, K.R. & Gonzalez, F.J. cDNA-directed expression of human cytochrome P450 CYP1A1 using baculovirus. Purification, dependency on NADPH-P450 oxidoreductase, and reconstitution of catalytic properties without purification. Drug Metab. Dispos. 23, 696–701 (1995).

    CAS  PubMed  Google Scholar 

  30. Zhang, Z., Fasco, M.J., Huang, Z., Guengerich, F.P. & Kaminsky, L.S. Human cytochromes P4501A1 and P4501A2: R-warfarin metabolism as a probe. Drug Metab. Dispos. 23, 1339–1346 (1995).

    CAS  PubMed  Google Scholar 

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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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Correspondence to Gary Peltz.

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Competing interests

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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