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Genetic Polymorphisms A Key to Human Individuality. Polymorphisms are subtle differences in our genomePolymorphisms are commonWe are 99.9% identical at the DNA levelBut this still leaves ~3,000,000 specific DNA differences between you and othersSuch differences affect our appearance, our behavior, our susceptibility to disease and our response to medications.
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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