1. New Insights on Warfarin: How CYP 2C9 & VKORC1 Information May Improve Benefit-Risk Ratio Brian F. Gage, MD, MSc Associate Professor of Medicine, Washington University in St. Louis Blood Thinner Service Medical Director, Barnes-Jewish Hospital

2. Overview Cytochrome P450 (CYP) 2C9 Vitamin K Epoxide Reductase, Complex 1 (VKORC1) Derivation of pharmacogenetics-based warfarin dosing Validation of pharmacogenetics-based warfarin dosing

5. Effect of CYP2C9*2 on Warfarin Dose S. Sanderson et al. Genet Med. 2005 Feb;7

6. Effect of CYP2C9*3 on Warfarin Dose S. Sanderson et al. Genet Med. 2005 Feb;7

7. Vitamin K epoxide reductase, complex 1 (VKORC1) Mutations in VKORC1 Cause Warfarin Resistance and Multiple Coagulation Factor Deficiency Type 2 VKORC1 synthesizes vitamin K epoxide reductase (VKOR), which resides in the endoplasmic reticulum of the hepatocyte and other cells –VKOR is inhibited by warfarin, especially S-warfarin S-warfarin is metabolized by CYP2C9 –VKOR activity is required for post-translational modification (γ- glutamyl carboxylation) of Glu residues on clotting factors II, VII, IX, X and proteins C, S, and Z VKORC1 may or may not be part of a complex

8. VKORC1 SNPs and Warfarin Dose Hypothesis: informative SNPs in VKORC1 correlate with warfarin dose We collaborated with Mark Rieder and Allan Rettie at University of Washington –To sequence this gene in archived DNA (N = 47) from CEPH families and from the Coriell depository –To correlate informative SNPs (inferred haplotypes) in 186 patients To correlate the 4 tagSNPs and inferred haplotypes in a larger cross-sectional study

9. Effect of VKORC1 Inferred Haplotype on Warfarin Dose Rieder MJ, Reiner AP, Gage BF et al. Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose. N Engl J Med. Jun 2005. 3673 = rs9923231 aka -1639, a promoter SNP (M. Wadelius; H. Yuan; E. Sconce) 6484 = rs9934438 aka C1173T, (M. Wadelius; H. Yuan; L. Bodin; D’Andrea); 6853 = rs17886369 7566 = rs2359612

10. VKORC1 Distributions Stratified by Race Group B haplotypes had larger doses, more mRNA transcript for VKORC1, and were more frequent in white and African-American pts. Group A haplotypes had smaller doses and were more frequent in Asian pts.

11. Pyrosequencing in Dr. Eby’s and McLeod’s Labs: Thanks to Sharon, Christi, Rhonda Pyrogram of VKORC1 6853 heterozygote subject. The sequence for nucleotides is: G/C G A G C G. Frequency of VKORC1-6853C allele: 37% in white and 24% in black pts.

12. Effect of VKORC1 Haplotype on Warfarin Dose Stratified by VKORC1 haplotype and CYP2C9 status Primary cohort: UW (N=185); Replication cohort: Wash U (N=368). All participants were Caucasian.

13. D’Andrea et al. and VKORC1, Blood Genotyped 147 warfarin-treated patients for common SNPs in VKORC1 Found that 1173 did not affect mRNA processing. VKORC1 1173 Genotype Warfarin Dose (mg/d) CC (37%)6.2 CT (47%)4.8 TT (16%)3.5 P < 0.002

14. M. Wadelius et al. The Pharmacogenomics Journal (2005) Genotyped 200 warfarin-treated patients for common SNPs in VKORC1. found VKORC1 C1173T, which explained 29% of the variability in warfarin dose Combined VKORC1 SNP, the CYP2C9*2 and CYP2C9*3 SNPs, and clinical factors, to derive a regression model that accounted for 56% of the variability in the warfarin dose.

15. M. Wadelius et al. The Pharmacogenomics Journal (‘05) rs2359612

16. L. Bodin et al. Measured FVII & INRs in 222 Patients after 1 dose of Acenocoumarol:

17. E. Sconce et al. ‘05 Derived (N = 297) & Validated (N=38) a Dosing Algorithm using VKORC1, CYP2C9, Age, Height R 2 = 54%-64%

18. 6 (6%)30 (3%) Takes amiodarone, N (%) 4.8 (1.6) Therapeutic warfarin dose mean (SD) 2.4 (0.4) Target INR mean (SD) 60 (60%)584 (65%) Men, N (%) 84 (84%)743 (83%) Caucasian, N (%) 2.0 (0.24)2.0 (0.26) Body surface area, mean (SD), m2 67 (13)65 (14) Age, mean (SD), y Variables Validation (N = 100) Derivation (N = 900) Development and Validation of a Warfarin Dosing Algorithm at Washington University Gage BF, Eby C, Johnson JA, Rieder MJ, Ridker PM…McLeod H. [ASH abstract]

19. Warfarin Dosing Equation in Derivation Cohort, N= 900 52%-5% (0 to –10%)-0.053Simvastatin or fluvastatin11 52%9% (2% to 16%)0.085Smokes10 51%-12% (–1% to –17%)-0.126African American9 51%-24% (–15% to –32%)-0.277Amiodarone8 49%8% (5% to 12%)0.161Target INR, per 0.5 increase7 48%-33% (–19% to –44%)-0.395African American * VKOR5808 6 47%-7% (–6% to –9%)-0.007Age, per decade5 43%-20% (–16% to –23%)-0.2182C9*24 39%-33% (–29% to –38%)-0.4082C9*33 32%12% (10% to 14%)0.454BSA, per 0.25 m22 22%-27% (–25% to –30%)-0.319Caucasian * VKOR6853C1 R 2 after entry Effect on Warfarin Dose Coefficient VariableEntry into Model

20. R 2 = 26% for clinical model. Accuracy in Validation Cohort (N = 100) (P<0.0001 vs. standard dose; P = 0.02 vs. clinical dose) R 2 = 56% for pharmacogenetics model, but lower in African- Americans By reducing the dosing error from 1.79 to 1.31 mg, pharmacogenetics should be able to increase the time in range in month one by 2%-4% and should decrease adverse events in patients w/ usual genotypes

21. Future Studies SNP discovery in targeted genes: APOE, calumenin, Factors II, VII, IX, X, γ-GCx Quantifying the relationship between new SNPs and warfarin dose –VKORC1 SNPs specific to African-American populations. –Factor VII G-402A & G-401T –Factor II 165Thr>Met –APOE –γ-GCx Prospective validation of a pharmacogenetics- dosing model –Would benefit from a platform that could quickly and economically genotype individuals

22. Conclusions The maintenance warfarin dose can be estimated from clinical and pharmacogenetic factors that can be obtained at the time of warfarin initiation –> 50% of the variability in the warfarin dose can be predicted from regression model using 2 genes: CYP2C9 and VKORC1 Although no dosing algorithm has been prospectively validated, the relationship between SNPs in these genes and the therapeutic warfarin dose is biologically and statistically compelling –For patients initiating warfarin therapy, we estimate a 2%-4% increase in time in the therapeutic INR range in month one with less benefit thereafter After month 1, pharmacogenetic knowledge may allow for more cautious dose escalation patients with CYP2C9*2 or CYP2C9*3

23. Methods for this Prospective, Open- labeled Study Patients are scheduled for elective orthopedic surgery Screen for exclusion criteria, obtain consent for clinical trial, gather sample for genotyping, and obtain non genetic data Determine CYP2C9 genotype (in commercial lab run by Mark Linder) and estimate pharmacogenetic dose 5- or 10-mg dose of warfarin taken the day before surgery To OR Inpatient (daily INR) and outpatient pharmacogenetics-based warfarin doses (2-3x/week INR) to be adjusted and monitored by anticoagulation service for 6 weeks Time D. Voora et al. Thromb Haemost 2005

24. Results

25. Time Until INR > 4 or Major Bleed