Pharmacogenomics: Using Genetic Testing to Guide Warfarin Therapy

1. Pharmacogenomics:Using Genetic Testing to Guide Warfarin Therapy Dan Jonas, MD, MPH Noon Conference October 29, 2007 The Right Dose of Warfarin for Every PatientThe Right Dose of Warfarin for Every Patient

2. Genetic PolymorphismsA Key to Human Individuality Polymorphisms are subtle differences in our genome Polymorphisms are common We are 99.9% identical at the DNA level But this still leaves ~3,000,000 specific DNA differences between you and others Such differences affect our appearance, our behavior, our susceptibility to disease and our response to medications

3. Single Nucleotide Polymorphisms (SNPs)A key to human variability Single nucleotide variation==Ex)10 nucleotide sequence of DNA fragment; two Fragments might differ b/c they may have a C or a TSingle nucleotide variation==Ex)10 nucleotide sequence of DNA fragment; two Fragments might differ b/c they may have a C or a T

4. What is Pharmacogenomics (PGx)? The study of how variations in the human genome affect the response to medications Tailoring treatments to unique genetic profiles “personalized” or “individualized” medicine Some use terms interchangeably with PGx But, PGx is just one aspect of PM From www.medterms.com PGx FDA definition: The investigation of variations of DNA and RNA characteristics as related to drug response. Other terms: molecular medicine, individualized therapy From www.medterms.com PGx FDA definition: The investigation of variations of DNA and RNA characteristics as related to drug response. Other terms: molecular medicine, individualized therapy

5. Individualized Medicine Current drug therapy in medicine: Efficacy may vary widely Resulting in wasted resources and time Adverse effects are common and unpredictable Complications and deaths Genetically guided therapy Direct treatment in an individualized manner To better target those most likely to benefit and least likely to be harmed Determine who to treat at all (e.g. prostate cancer?) “That background understanding and the sequencing of the human genome have lead to the promise of Individualized Medicine (or PM)”—how much is hype or reality?“That background understanding and the sequencing of the human genome have lead to the promise of Individualized Medicine (or PM)”—how much is hype or reality?

6. Pharmacogenomics The variable efficacy and unpredictability of adverse effects likely has a significant genetic component Secondary to polymorphisms Drug target polymorphisms Polymorphisms in metabolic / drug excretion pathways Cytochrome P450 Implications for drug development / discovery Ex of drug target) Angiotensin II Receptor---SNPs may encode AIIRs that are functionally different.Ex of drug target) Angiotensin II Receptor---SNPs may encode AIIRs that are functionally different.

9. Commonly prescribed Narrow therapeutic window Great hazard if outside of therapeutic window Significant variability in individual response to standard dosages No good alternative The Perfect Drug for PGx Intervention Prescribed for serious indicationsPrescribed for serious indications

10. Warfarin Commonly prescribed (2 million per year in the US) High rate of adverse events Warfarin maintenance doses are characterized by large interindividual variability Maintenance doses can range 50-fold (eg, daily dose requirements range from 0.5 to 25 mg) Warfarin is THE example of a narrow therapeutic index There have been several efforts to define this interindividual variability using genetic and non-genetic factors

11. Factors that Correlate w/ Warfarin Dose Age Body surface area (BSA) or weight Amiodarone dose Other drugs (e.g. HMG CoA Reductase inhibitors) Target INR Race Sex Plasma vitamin K level Decompensated CHF or post-operative state The patient’s genetic status with regard to polymorphisms Pie chart adapted from AMA brochurePie chart adapted from AMA brochure

12. Genes important for Warfarin Pharmacogenetics CYP2C9 Metabolizes >90% of active Warfarin Variant alleles associated with increased sensitivity to Warfarin (CYP2C9*2, *3) Vitamin K epoxide reductase (VKOR) Inhibited by Warfarin Important for replenishment of vitamin K Variant alleles of VKORC1 gene associated with altered response to Warfarin

13. In the liver, Reduced Vitamin K is essential for gamma-carboxylation of factors 2, 7, 9, and 10 (and prot C and S) to make them functional coagulation factors Warfarin (manufactured as mixture of R and S-enantiomers). S warfarin is 3-5 X more effective at inhibiting warfarin. Metabolized by CYP2C9 Warfarin produces anticoagulant effect by inhibiting VKOR (vitaminK epoxide reductase). VKOR is a multicomponent enzyme system which regenerates reduced Vitamin KIn the liver, Reduced Vitamin K is essential for gamma-carboxylation of factors 2, 7, 9, and 10 (and prot C and S) to make them functional coagulation factors Warfarin (manufactured as mixture of R and S-enantiomers). S warfarin is 3-5 X more effective at inhibiting warfarin. Metabolized by CYP2C9 Warfarin produces anticoagulant effect by inhibiting VKOR (vitaminK epoxide reductase). VKOR is a multicomponent enzyme system which regenerates reduced Vitamin K

14. CYP2C9 variant alleles CYP2C9*2, CYP2C9*3 – most common variants Seen in 20-40% of Caucasians, <10% Asians and African Americans Associated with reduced CYP2C9 enzyme activity Variant alleles associated with lower mean doses of Warfarin longer times to stabilization of INR higher risk for bleeding events CYP2C9*1 is the “normal allele”CYP2C9*1 is the “normal allele”

17. The VKOR Gene Vitamin K Epoxide Reductase Cloned in 2004 Stafford et. al (Nature 427: 541 – 544; 2004) Johannes Oldenburg, Wurzburg, Germany Resides on human chromosome 16p11.2 The target protein for warfarin’s action Encodes a protein of 163 amino acids---Mass of 18.2 kDa Consists of 3 exons (2 introns) and a 5’ and 3’ UT Interferes with ?-carboxylation of Factors II, VII, IX & X Mutations in VKORC1 coding region: Vit K clotting factor deficiency type 2 (VKCF2) Autosomal dominant Warfarin resistance Encodes a protein of 163 amino acids---Mass of 18.2 kDa Consists of 3 exons (2 introns) and a 5’ and 3’ UT Interferes with ?-carboxylation of Factors II, VII, IX & X Mutations in VKORC1 coding region: Vit K clotting factor deficiency type 2 (VKCF2) Autosomal dominant Warfarin resistance

18. Effect of VKORC1 Haplotype A or B on Warfarin dosage Determining haplotype: must look at multiple SNPs (minimum of 4?)Determining haplotype: must look at multiple SNPs (minimum of 4?)

19. AmyAmy

21. Warfarin & the FDA Changed package insert for warfarin Aug 2007 Label now provides information regarding altered metabolism in CYP2C9 and VKORC1 genetic variants Concerns regarding Provider knowledge Patient demand Potential for influencing litigation

22. What is the clinical evidence? FDA working group selected relevant studies A number found strong associations cross-sectional studies in many populations Lower dose requirements with CYP2C9 3 prospective studies Caraco 2007; Millican 2007; Limdi 2007 There were 9 (?) “solid population-based studies” Lesko (SMDM 10/2007)There were 9 (?) “solid population-based studies” Lesko (SMDM 10/2007)

23. Caraco et al. Controlled trial, prospective (“randomized” by MRN); N=283 enrolled, 191 analyzed Control group: all started on 5mg and adjust dose based on pre-established protocol Required daily monitoring of INR for initial 8 days Intervention group: CYP2C9 genotype-adjusted protocol Altered recommended dose by a set % for each of 6 different genotypes (*1/*1, *1/*2, *1/*3, etc.) Results: stable anticoagulation 18.1 days earlier TTR 80.4% vs 63.4% (P < 0.001) Discuss Limitations: 1) not practical, daily INRs for 8 days. 2) Report randomization, but not truly random---used last digit of patient’s identity number (control if uneven, study group if even) and therefore not blinded. 3) Only amiodorone was considered as an additional factor to adjust dose recommendation (no other meds or clinical factors 4) 283 enrolled and “randomized”; 191 analyzed (?selection bias—about 100 randomized and not analyzed); why the difference?: most b/c warfarin was not initiated, 8 days of warfarin therapy not completed, or “protocol violations” such as missing too many INR visits. “Stable anticoagulation”=2 consecutive therapeutic INRs 7 days apart “TTR”=calculated by linear interpolationDiscuss Limitations: 1) not practical, daily INRs for 8 days. 2) Report randomization, but not truly random---used last digit of patient’s identity number (control if uneven, study group if even) and therefore not blinded. 3) Only amiodorone was considered as an additional factor to adjust dose recommendation (no other meds or clinical factors 4) 283 enrolled and “randomized”; 191 analyzed (?selection bias—about 100 randomized and not analyzed); why the difference?: most b/c warfarin was not initiated, 8 days of warfarin therapy not completed, or “protocol violations” such as missing too many INR visits. “Stable anticoagulation”=2 consecutive therapeutic INRs 7 days apart “TTR”=calculated by linear interpolation

24. Millican et al. Retrospective analysis of 2 prospective cohorts to compare 2 approaches to PGx-guided warfarin initiation N=118 (46 and 72) patients scheduled for primary or revision total knee or hip arthroplasty 1st cohort: warfarin initiated and refined (target range 2-3) based on clinical factors and CYP2C9 genotype 2nd cohort: warfarin initiated (target range 1.7-2.7) based on these factors plus VKORC1 genotype; dose refinements after the 3rd dose were gene-guided 4-6 week follow up Clinical factors=age, BSA, amiodorone, target INR, statin, race, sex (?liver dz, smoking…) Limitations: 1) comparison group problem: group 1 Aug 2003-March 2004; group 2 Feb 2006-July2006 2) Orthopedic surgery patients only 3) Comparing groups with different target INR 4) Comparing with historical group--?may be several other differences in anticoag mgmt several yrs apart. 5) Composite endpoint: The primary endpoint was a composite: major hemorrhage, INR > 4 (in first cohort) or > 3.7 (in second cohort), or symptomatic venous thromboembolism within 30 days of warfarin initiation. 6) Just 4-6 weeks of anticoagulation Conclusion: A new pharmacogenetic algorithm incorporating VKORC1 genotype and a lower target INR range of 1.7-2.7 was associated with a 64% reduction in laboratory and/or clinical adverse eventsClinical factors=age, BSA, amiodorone, target INR, statin, race, sex (?liver dz, smoking…) Limitations: 1) comparison group problem: group 1 Aug 2003-March 2004; group 2 Feb 2006-July2006 2) Orthopedic surgery patients only 3) Comparing groups with different target INR 4) Comparing with historical group--?may be several other differences in anticoag mgmt several yrs apart. 5) Composite endpoint: The primary endpoint was a composite: major hemorrhage, INR > 4 (in first cohort) or > 3.7 (in second cohort), or symptomatic venous thromboembolism within 30 days of warfarin initiation. 6) Just 4-6 weeks of anticoagulation Conclusion: A new pharmacogenetic algorithm incorporating VKORC1 genotype and a lower target INR range of 1.7-2.7 was associated with a 64% reduction in laboratory and/or clinical adverse events

25. Limdi et al. Large prospective cohort study (N=490) with 2 year follow up All patients treated with standardized approach to warfarin dose adjustments Results: Variant CYP2C9 genotype Increased risk for major hemorrhage (HR 3.0; 95% CI 1.1-8.0)a, but not minor hemorrhage Variant VKORC1 genotype (1173C/T) Did not confer an increase in risk for major or minor --No comparison group; these are basically subgroup analyses --The risk of major hem for CYP2C9 was 5.3 fold greater before stabilization of therapy, but was still 2.2-2.4 fold greater after stabilization (increased risk persists after therapy is stabilized) LIMITATIONS: Limit to studying these—they’re rare events—just 25 events /358 patients in CYP wild type group had major vs 19 events/88 with variant CYP2C9 genotype. --No comparison group; these are basically subgroup analyses --The risk of major hem for CYP2C9 was 5.3 fold greater before stabilization of therapy, but was still 2.2-2.4 fold greater after stabilization (increased risk persists after therapy is stabilized) LIMITATIONS: Limit to studying these—they’re rare events—just 25 events /358 patients in CYP wild type group had major vs 19 events/88 with variant CYP2C9 genotype.

26. Pharmacogenomics at UNC to Guide Warfarin Therapy Incorporate PGx guidance in warfarin dosing at UNC through implementation of a randomized trial Integrated effort--Genetics, clinical labs, pharmacy, and providers PGx has the most to offer in choosing the initial dosing of warfarin Subsequent dosage adjustments will still be primarily guided by following INRs Thus, we need rapid identification of patients placed on warfarin We started meeting about 6 months ago to consider how to further develop the use of PGx at UNC for warfarin…We started meeting about 6 months ago to consider how to further develop the use of PGx at UNC for warfarin…

27. Structure of the UNC Warfarin PGx Study Inclusion criteria: Adults (= 18) newly starting warfarin Planned = 3 months of anticoagulation with target INR = 2. Following up at UNC (ACC or Family Practice) Exclsuion criteria: History of treatment with warfarin and know dose requirement unable to complete the study materials (questionnaires) with or without assistance (e.g. dementia), including non-English speaking patients Pregnancy Treating physician opposed to enrolling Question: For patients initiating warfarin therapy, does using genetic information to inform dosing improve outcomes compared to dosing based on clinical information only?Question: For patients initiating warfarin therapy, does using genetic information to inform dosing improve outcomes compared to dosing based on clinical information only?

28. UNC Warfarin PGx Study Exclusion: not starting chronic (>3 months) anticoagulation; previously on warfarin with know dose requirmentExclusion: not starting chronic (>3 months) anticoagulation; previously on warfarin with know dose requirment

29. Experimental Group

30. Outcomes Time in therapeutic range (TTR) # of visits required Complications Minor and Major bleeding INRs > 4 Process measures Genotyping turn-around-time Provider knowledge and attitudes Cost-effectiveness

31. When Starting Warfarin…consider Genotype! Warfarin genotyping panel (pertinent VKOR and CYP polymorphisms) will be available soon In the context of the study For clinical use 1) Available in the context of the study, and also for any patients you want to order it on (probably by Nov-Dec)1) Available in the context of the study, and also for any patients you want to order it on (probably by Nov-Dec)

33. Thank You! Genetics and Medicine: Jim Evans Betsy Bryant Brent Ferrell Leslie Lange Kristy Lee Kandamurugu Manickam Stephan Moll Cécile Skrzynia Marcia Van Riper Maimoona Zariwala Pharmacy and Institute for Pharmacogenomics and Individualized Therapy Howard McLeod Stephen Eckel John Valgus Laboratory Medicine Karen Weck Jessica Booker Mike Langley Family Practice Sarah Ford

36. Prevalence of genetic variations influencing warfarin maintenance dose CYP2C9 ~4% PM’s (two inactive alleles eg. *3/*3) ~35% IM’s (one inactive allele eg. *1/*3) ~60% EM’s (two active alleles eg *1/*1) VKORC1 ~37% GG, highest maintenance doses ~47% AG, intermediate maintenance doses ~16% AA, lowest maintenance doses

37. CYP2C9 Polymorphisms

38. Individualized Medicine Predisposition and Screening The current status of disease screening in medicine In spite of aggregate benefit… Relatively little benefit to a given individual Actual harm to some Tremendous waste of resources Genetically guided screening holds the promise of: Preventing disease in those susceptible Early detection Rational use of society’s limited resources

39. CYP450 Gene Nomenclature CYP 2 C 19 *1 (normal allele) Variant alleles (named in order of discovery): CYP 2 C 19 *2 CYP 2 C 19 *3 CYP 2 C 19 *4

40. Major CYP450 enzymes involved in drug metabolism CYP1A2 CYP2C9 CYP2C19 genetically variable CYP2D6 CYP2E1 CYP3A4 CYP3A5

41. CYP2C9 gene variants

42. CYP2C9 Allele frequencies

43. VKORC1 gene variants

44. Percent of warfarin dose variability explained by CYP2C9 and VKORC1

45. Warfarin dose variance in European Caucasians VKORC1 genotype CYP2C9*2,*3 Dosing algorithms (VKORC1+ CYP2C9 + age + body mass +other meds) Other factors??? 21-25% of dose variance 6-10% of dose variance 50-60% 40-50%

46. Warfarin dosing algorithm (based on age, height, CYP2C9 and VKOR)

48. Structure of the UNC Warfarin Service/Study Project manager or pharmacist will be notified of all inpatients or ED patients who are prescribed warfarin or heparin Consult provider and approach patient for consent Patients randomized to one of two arms: Dosing based on algorithm which takes genotype into account Dosing based on same algorithm, but without genetic data Blood drawn for genotyping Laboratory genotypes for VKOR and CYP polymorphisms TAT of <24 hours Pharmacist calculates recommended dose using algorithm Relays information to clinicians and orders newly adjusted dose

49. Provider Education Crucial to success of efforts to incorporate PGx into clinical practice If providers consider genotyping before giving the first dose more beneficial impact on proper optimal dosing will result Educate providers about utility of genotyping to stimulate orders at the time which warfarin is first considered Attendings House staff Nursing Pharmacy personnel We will also take this opportunity to survey attitudes and knowledge about PGx before,during and after the study Of providers Of patients

50. In the liver, Reduced Vitamin K is essential for gamma-carboxylation of factors 2, 7, 9, and 10 (and prot C and S) to make them functional coagulation factors Warfarin (manufactured as mixture of R and S-enantiomers). S warfarin is 3-5 X more effective at inhibiting warfarin. Metabolized by CYP2C9 Warfarin produces anticoagulant effect by inhibiting VKOR (vitaminK epoxide reductase). VKOR is a multicomponent enzyme system which regenerates reduced Vitamin KIn the liver, Reduced Vitamin K is essential for gamma-carboxylation of factors 2, 7, 9, and 10 (and prot C and S) to make them functional coagulation factors Warfarin (manufactured as mixture of R and S-enantiomers). S warfarin is 3-5 X more effective at inhibiting warfarin. Metabolized by CYP2C9 Warfarin produces anticoagulant effect by inhibiting VKOR (vitaminK epoxide reductase). VKOR is a multicomponent enzyme system which regenerates reduced Vitamin K