Pharmacogenomics:Using Genetic Testing to Guide Warfarin Therapy

Dan Jonas, MD, MPH

Noon Conference

October 29, 2007

                           

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

Single Nucleotide Polymorphisms (SNPs)A key to human variability

DNA sequence variation at a single nucleotide that DNA sequence variation at a single nucleotide that may alter the function of the encoded protein may alter the function of the encoded protein

Functional but Functional but alteredaltered protein proteinFunctional proteinFunctional protein

Polymorphisms are common and contribute to common diseases and influence our response to medications

*

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

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?)

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

Goldstein DB, et al. Nature Reviews 2003;4: 937-947Genetic variants found to be significantly associated with drug response in

at least two studies

Nature Reviews 2003;4:937-947

Goldstein DB, et al. Nature Reviews 2003;4: 937-947Genetic variants found to be significantly associated with drug response in

at least two studies

Nature Reviews 2003;4:937-947

• Commonly prescribed

• Narrow therapeutic window

• Great hazard if outside of therapeutic window

• Significant variability in individual response to standard dosages

• No good alternative

Warfarin (Coumadin)

The Perfect Drug for PGx Intervention

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

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

CYP2C9 (up to 15%)

VKORC1 (up to 25%)

Other factors (up to 40%)

Age, sex,

weight (10-20%)

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

CYP2C9CYP2C9

CYP1A1CYP1A1CYP1A2CYP1A2CYP3A4CYP3A4

RR--warfarin

warfarin

SS--warfarin

warfarin

Oxidized Vitamin KOxidized Vitamin K Reduced Vitamin KReduced Vitamin K

OO22

HypofunctionalHypofunctionalF. V, VII, IX, XF. V, VII, IX, X

Functional Functional F. II, VII, IX, XF. II, VII, IX, X

Vitamin K Vitamin K dependent dependent

carboxylasecarboxylase

Vitamin K Vitamin K ReductaseReductase

COCO22

WarfarinWarfarin

RR--war

farin

war

farin

SS--warfarin

warfarin

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

C y to c h ro m e P 4 5 0 G e n o ty p e a n d W a rfa r in D o s e

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N = 3 6 9

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Time to Event for Anticoagulation-Related Outcomes

HigashiHigashi MK, et al. MK, et al. JAMAJAMA. 2002;287:1690. 2002;287:1690--1698.1698.

Pro

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No. at RiskNo. at RiskVariantVariant 5858 3333 1717 66 66 33 22 22 22Wild TypeWild Type 127127 3939 1919 1010 66 33 33 22 22

5858 2323 1616 99 99 66 44 33127127 7171 5454 3434 2222 1010 66 00

1.01.0

0.80.8

0.60.6

0.40.4

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0.00.000 100100 200200 300300 400400 500500 600600 700700 800800 900900 10001000

FollowFollow--up, dup, d

=8.30; =8.30; PP=.004=.0042211

Time to Stable DosingTime to Stable Dosing1.01.0

0.80.8

0.60.6

0.40.4

0.20.2

0.00.000 400400 36003600

FollowFollow--up, dup, d

=6.21; =6.21; PP=.01=.012211

Time to First Serious orTime to First Serious orLifeLife--Threatening BleedThreatening Bleed

Ble

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CYP2C9 variant

CYP2C9 variant

Wild type

Wild type

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

Effect of VKORC1 Haplotype A or B on Warfarin dosage

Rieder et al. New England Journal of Medicine 2005

Individual Variability in Warfarin Dose

Warfarin maintenance dose (mg/day)

SENSITIVITYSENSITIVITY

CYP2C9 coding SNPs

RESISTANCERESISTANCE

VKORC1 coding SNPs

0.5 5 15

Fre

qu

ency

Common Common VKORC1VKORC1 non- non-coding SNPscoding SNPs

Adapted from Rettie and Tai, Molecular Interventions 2006

(*3/*3)

Warfarin dosing algorithm in UNC patientswww.warfarindosing.org

R2 = 0.4828

0

2

4

6

8

10

12

0 2 4 6 8 10 12Predicted dose

Act

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ean

dose

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

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

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)

Clin Pharmacol Ther. 2007 Sep 12; [Epub ahead of print]; Hadassah University, Israel

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

Millican et al., Blood. 2007 Sep 1;110(5):1511-5. Epub 2007 Mar 26; Wash U;Voora et al., Thromb Haemost. 2005 Apr;93(4):700-5;Grice, Gage, et al. ACCP 2007 Poster

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

Limidi et al., Clin Pharmacol Ther. 2007 Jul 25; [Epub ahead of print]; UAB

aAdjusted for age, gender, race, BMI, VKORC1, vitamin K and alcohol intake, warfarin dose, interacting drugs, number of comorbid conditions, and INR at the time of the event

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

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

PM or Clinical pharmacist notified via:

•Orders for heparin or warfarin •Physician•U/S Doppler tech

Project Manager prescreens subject for possible

inclusion via electronic medical record

PM contacts physician and approaches patient for consent

PM contacts clinical pharmacist, who orders blood draw for CYP2C9 and VKOR

Blood drawn and sent to lab along with signed consent; results reported in Webcis

Subjects are randomized to the control or experimental group

UNC Warfarin PGx Study

Experimental GroupPharmacist calculates dose using algorithm ASAP without genetic info & re-calculates

dose including genetic info as soon as available

Pharmacist communicates recommended dose to the treating physician & ensures

patient is d/c’d on that dose

Pharmacist calculates dose using algorithm ASAP without genetic info

Clinical pharmacist makes dose change

Subjects follow up for routine care in the ACC or Family Medicine Center

Anticoagulation Clinc

Control Group

Collect outcomes data over first 3 months of treatment: visits, TTR, utilization…

Pharmacist communicates recommended dose to the treating physician & ensures

patient is d/c’d on that dose

Clinical pharmacist makes dose change

Subjects follow up for routine care in the ACC or Family Medicine Center

Anticoagulation Clinc

Collect outcomes data over first 3 months of treatment: visits, TTR, utilization…

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

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

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

DEPARTMENT OF GENETICS

EXTRA SLIDES

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

CYP2C9 Polymorphisms

(*2) Arg Cys codon 144

(*3) Ile Leu codon 359

(*4) Ile Thr codon 359

(*5) Asp Glu codon 360

(*1) wild type

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

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

Family Subfamily Gene Allele Variant

Major CYP450 enzymes involved in drug metabolism

• CYP1A2

• CYP2C9

• CYP2C19genetically variable

• CYP2D6

• CYP2E1

• CYP3A4

• CYP3A5

CYP2C9 gene variants

Enzyme Activity

Normal

Reduced (50-70%)

Reduced (5-15%)

I359L*

R144C*

CYP2C9*1(wild type)

CYP2C9*2

CYP2C9*3

CYP2C9 Allele frequencies

African Americans

Caucasians Asians

*1 0.953 0.743 0.984

*2 Rare 0.10-0.16 Rare

*3 0.01 0.05-0.10 0.02-0.4

*4 0.01 Absent? Absent?

*5 0.01 Absent? Absent?

*6 (818delA) 0.01 Absent? Absent?

*11 (R335W) 0.023 Rare Absent?

VKORC1 gene variants

Enzyme ClinicalActivity Effect

spontaneous bleeding (VKCFD2)

Warfarin Warfarin resistance binding?

OR OR Warfarin dose

R98W*

SNPs

Percent of warfarin dose variability explained by CYP2C9 and VKORC1

Ref CYP2C9 VKORC1 Gx total Total*

1 55%

7 7% 25% 32% 51%

8 20% 21% 41% 63%

9 10% 34% 44% 57%

11 15% 18% 33% 45%

12 17% 13% 30% 59%

13 5% 21% 26% 39%

14 18% 34% 52% 60%

Avg 13% 24% 37% 54%

*Total variability explained by genetic, demographic and clinical variablesClinical and demographic factors alone explain 20-25% of dose variability

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%

Sconce, et al. Blood 2005

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

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

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

CYP2C9

7-hydroxywarfarin6-hydroxywarfarin8-hydroxywarfarin

10-hydroxywarfarin

CYP1A1CYP1A2CYP3A4