New Insights on Warfarin: How CYP 2C9

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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

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Overview

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

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Effect of CYP2C92 on Warfarin Dose
S. Sanderson et al. Genet Med. 2005 Feb7
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Effect of CYP2C93 on Warfarin Dose
S. Sanderson et al. Genet Med. 2005 Feb7
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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 (?-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

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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

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Effect of VKORC1 Inferred Haplotype on Warfarin
Dose
3673 rs9923231 aka -1639, a promoter SNP (M.
Wadelius H. Yuan E. Sconce) 6484 rs9934438
aka C1173T, (M. Wadelius H. Yuan L. Bodin
DAndrea) 6853 rs17886369 7566 rs2359612
Rieder MJ, Reiner AP, Gage BF et al. Effect of
VKORC1 haplotypes on transcriptional regulation
and warfarin dose. N Engl J Med. Jun 2005.
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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.
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Pyrosequencing in Dr. Ebys and McLeods Labs
Thanks to Sharon, Christi, Rhonda
Frequency of VKORC1-6853C allele 37 in white
and 24 in black pts.
Pyrogram of VKORC1 6853 heterozygote subject.
The sequence for nucleotides is G/C G A G C G.
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Effect of VKORC1 Haplotype on Warfarin
Dose Stratified by VKORC1 haplotype and CYP2C9
status Primary cohort UW (N185) Replication
cohort Wash U (N368). All participants were
Caucasian.
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DAndrea 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 lt 0.002
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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 CYP2C92 and CYP2C93
  SNPs, and clinical factors, to derive a
  regression model that accounted for 56 of the
  variability in the warfarin dose.

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M. Wadelius et al. The Pharmacogenomics Journal
(05)
rs2359612
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L. Bodin et al. Measured FVII INRs in 222
Patients after 1 dose of Acenocoumarol
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E. Sconce et al. 05 Derived (N 297)
Validated (N38) a Dosing Algorithm using VKORC1,
CYP2C9, Age, Height
R2 54-64
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Development and Validation of a Warfarin Dosing
Algorithm at Washington University
Gage BF, Eby C, Johnson JA, Rieder MJ, Ridker
PMMcLeod H. ASH abstract
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Warfarin Dosing Equation in Derivation Cohort, N
900
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Accuracy in Validation Cohort (N 100)
R2 56 for pharmacogenetics model, but lower in
African-Americans
R2 26 for clinical model.
(Plt0.0001 vs. standard dose P 0.02 vs.
clinical dose)
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
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Future Studies

• SNP discovery in targeted genes APOE, calumenin,
  Factors II, VII, IX, X, ?-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 165ThrgtMet
• APOE
• ?-GCx
• Prospective validation of a pharmacogenetics-
  dosing model
• Would benefit from a platform that could quickly
  and economically genotype individuals

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Conclusions

• The maintenance warfarin dose can be estimated
  from clinical and pharmacogenetic factors that
  can be obtained at the time of warfarin
  initiation
• gt 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 CYP2C92 or CYP2C93

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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
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Results
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Time Until INR gt 4 or Major Bleed