Interindividual variability of drug response has been well documented. In addition to drug-drug and drug-environment interactions, genetic factors have emerged as contributors to variability in response to several cardiovascular medications. Pharmacogenetics is the study of genetic determinants of drug response. Polymorphisms of genes encoding for proteins involved in the drug pathway, from absorption to activation (if administered as a prodrug) to action, and to elimination, may contribute to drug response ( Table 4-1 ). Genetic variants can contribute to altered pharmacokinetics and pharmacodynamics and subsequently affect a drug’s efficacy and safety profile ( Table 4-2 ).
| CLOPIDOGREL | WARFARIN | STATIN | |
|---|---|---|---|
| Absorption | ABCB1 | ABCB1 | |
| Metabolism |
| CYP2C9 |
|
| Action | P2RY12 | VKORC1 |
|
| Genetic Implications | ||
|---|---|---|
| OF DRUG EFFICACY | OF DRUG-RELATED ADVERSE EVENTS | |
| Clopidogrel | ↓ Efficacy: CYP2C19 *2 | ↑ Adverse events (↑ bleed): CYP2C19 *17 |
| Warfarin | Time to therapeutic threshold: VKORC1 haplotype A | ↑ Bleed: CYP2C9 *2 and *3, especially *3 |
| Statin | LDL cholesterol lowering: APOE, PCSK9, ?CETP | Myopathy: SLCO1B1 |
The study of pharmacogenetics may be particularly illuminating when 1) compromised medication efficacy can be serious or fatal; 2) the therapeutic window is very narrow; or 3) the predictive value of specific genetic variants for serious or fatal drug-associated side effects has been firmly established, and timely testing may prevent development of serious side effects by therapeutic modifications. These considerations have driven explorations into the pharmacogenetics of clopidogrel, warfarin, and statin therapy.
Incorporation of pharmacogenetic testing more broadly into cardiovascular therapeutics will require the availability of cost-effective pharmacogenetic assays, evidence that pharmacogenetic-guided therapy improves care, and effective evidence-based therapeutic modifications based on pharmacogenetic testing.
Clopidogrel
Drug, Indications, Mechanism of Action, and Pharmacology
Clopidogrel is an irreversible oral thienopyridine. As part of dual antiplatelet therapy in combination with aspirin, clopidogrel is indicated in the management of acute coronary syndromes (ACS) with or without percutaneous coronary interventions (PCI). The optimal dosage and duration of dual antiplatelet therapy is an area of active research.
Clopidogrel is ingested as a prodrug. Its absorption limited by intestinal efflux transporter P-glycoprotein. Upon absorption, 85% of the prodrug is hydrolyzed by esterases into an inactive carboxylic acid derivative. The remaining 15% of the prodrug is metabolized by the hepatic cytochrome P450 (CYP450) system, especially the CYP2C19 enzyme, into active thiol metabolites. Peak plasma concentrations of the active metabolites are reached within several hours with increased peak concentrations after administration of 600 mg versus 300 mg of clopidogrel as a loading dose. The active thiol metabolites irreversibly bind the P2Y 12 component of the adenosine diphosphate (ADP) receptors on the platelet surface, inducing inhibition of ADP-dependent platelet activation and aggregation that persists for the lifetime of the platelet.
Drug Interactions
Variable platelet inhibition with clopidogrel therapy has been observed and approximates a bell-shaped distribution ( Figure 4-1 ). Drug-drug interactions have been investigated as potential contributors to variable clopidogrel response, and interactions of clopidogrel with statins and proton-pump inhibitors (PPIs) have been particularly explored. Concomitant statin therapy has been shown to attenuate platelet inhibition by clopidogrel in a dose-dependent manner without an increase in clinical cardiovascular events.
PPIs are inhibitors of the CYP2C19 enzyme, an important enzyme involved in clopidogrel metabolism. Initial observational data raised concerns about the potential association of concurrent PPI (especially omeprazole) and clopidogrel therapy with increased cardiovascular events and mortality. However, a subgroup analysis of Trial to Assess Improvement in Therapeutic Outcomes by Optimizing Platelet Inhibition with Prasugrel–Thrombolysis in Myocardial Infarction (TRITON-TIMI) 38 and Prasugrel in Comparison to Clopidogrel for Inhibition of Platelet Activation and Aggregation–Thrombolysis in Myocardial Infarction (PRINCIPLE-TIMI) 44 found no association of concurrent PPI and thienopyridine therapy with adverse clinical outcomes, although an attenuation in in vitro platelet inhibition was noted in patients on concomitant PPI therapy. The prospective, randomized controlled Clopidogrel and the Optimization of Gastrointestinal Events (COGENT) trial reported no difference in the primary cardiovascular safety endpoint—defined as the composite of death from cardiovascular causes, nonfatal myocardial infraction (MI) coronary revascularization, or ischemic stroke—and a benefit in reduction of gastrointestinal (GI) bleeding in patients concomitantly taking a PPI and clopidogrel compared with those taking clopidogrel alone. The totality of data led to a 2010 Expert Consensus Document that recommended that “PPIs are appropriate in patients with multiple risk factors for GI bleeding who require antiplatelet therapy. Routine use of either a PPI or an histamine 2–receptor antagonist (H2RA) is not recommended for patients at lower risk of upper GI bleeding.” A subsequent analysis of the French Registry of Acute ST-Elevation and Non–ST-Elevation Myocardial Infarction (FAST-MI) registry, published after the release of the Expert Consensus Document, also found no association between concurrent PPI therapy and increased cardiovascular events and mortality. Further studies focusing on PPI use will continue to help guide decisions about PPI therapy among clopidogrel-treated patients, weighing the GI bleeding versus the cardiovascular risks.
Pharmacogenetics of Clopidogrel Therapy
Polymorphisms of genes involved in clopidogrel’s absorption ( ABCB1), metabolism ( CYP2C19) , and action ( P2RY12 ) have been investigated for potential association with clopidogrel response ( Figure 4-2 , Table 4-3 ). Of these, the association between polymorphisms in the CYP2C19 gene and clopidogrel response has been most consistently replicated, and variants in the CYP2C19 gene were the only significant polymorphisms noted in a genome-wide association study (GWAS) that specifically examined the genetic influence of clopidogrel pharmacologic response.
| GENE (LOCATION) | ENCODES FOR | VARIANT STUDIED (EFFECT ON PROTEIN) | FREQUENCY OF ALLELE * | ASSOCIATED DISEASE PHENOTYPE AND RELATIVE RISK |
|---|---|---|---|---|
| ABCB1 (7q21.1) | P-glycoprotein drug efflux transporter, involved in intestinal absorption of clopidogrel | C3435T (rs1045642) | T allele:
| Biochemical: lower levels of the active metabolite with TT genotype Clinical: higher rate of cardiovascular events with TT genotype FAST-MI and TRITON-TIMI 38 genetic studies but with CC genotype in PLATO genetic study 36 |
| CYP2C19 (10q24.1-q24.3) | Eponymous protein essential in the two-step CYP450 hepatic metabolism of clopidogrel prodrug | * 2 (rs4244285, G681A) (reduced function) | A allele:
|
|
| * 3 (rs4986893) (reduced function) | A allele:
| |||
| * 17 (rs12248560, C806T ) (enhanced function) | T allele:
|
|
* Allele frequency is per HapMap phase 2 except otherwise noted.
CYP2C19
The CYP2C19 gene is highly polymorphic, with known reduced-function and enhanced-function variants. The CYP2C19 gene encodes for the CYP450 2C19 enzyme involved in both steps of hepatic activation of clopidogrel to its active metabolite (see Figure 4-2 ). Among the reduced-function variants—such as the *2, *3, *4, *5, *6, *7, and *8 variants—the *2 variant is the most common. The *2 variant (rs4244285) involves a single base-pair mutation of G→A at position 681, which creates an aberrant splice site, resulting in downstream synthesis of a truncated, nonfunctional CYP2C19 protein.
Subgroup analyses of clinical trials and registry databases, as well as a GWA study and a meta-analysis, have identified reduced-function CYP2C19 variants to be independently associated with diminished inhibition of ADP-induced platelet aggregation and with increased risk of death and ischemic events in the setting of clopidogrel therapy. Compared with noncarriers, carriers of at least one copy of a reduced-function CYP2C19 allele have approximately 30% lower levels of active clopidogrel metabolite and approximately 25% relatively less platelet inhibition with clopidogrel. Carriers of both one and two CYP2C19 reduced-function alleles appear to be at increased risk for adverse cardiovascular outcomes: meta-analyses of patients treated with clopidogrel predominantly for PCI found carriers of one and two reduced-function CYP2C19 alleles to have about a 1.5-fold increase in the risk of cardiovascular death, MI, or stroke and a threefold increase in the risk for stent thrombosis compared with noncarriers.
Genetic studies of patients receiving clopidogrel predominantly not for PCI yielded different results. The Clopidogrel for High Atherothrombotic Risk and Ischemic Stabilization, Management, and Avoidance (CHARISMA) genomics substudy, which involved patients with stable atherothrombotic diseases, found patients homozygous but not heterozygous for CYP2C19 *2 to have an increased risk of ischemic events and decreased risk of bleeding with dual antiplatelet therapy. Genetic analysis of the Clopidogrel in Unstable Angina to Prevent Recurrent Events (CURE) trial, which studied patients with non–ST-elevation ACS managed predominantly without PCI, found that carrier status of CYP2C19 reduced-function alleles did not affect clopidogrel efficacy. Differences in the patient populations (stable atherothrombotic disease vs. acute coronary syndrome) and exposure to PCI may in part explain the differential findings of the CHARISMA and CURE genetic studies versus those based on trials predominantly involving patients with ACS for PCI.
The enhanced-function CYP2C19 *17 variant has also been reported to influence clopidogrel therapy and involves a single base-pair mutation of C→T at position 808. It has been associated with increased transcriptional activity of the CYP2C19 enzyme, extensive clopidogrel metabolism with enhanced production of active clopidogrel metabolites, greater inhibition of ADP-induced platelet aggregation, and increased risk of bleeding in a gene–dose-dependent fashion without significant impact on stent thrombosis, combined 30-day ischemic endpoint of death or MI, or urgent target vessel revascularization. Genetic analysis of the CURE study found carrier status of the CYP2C19 *17 allele to be associated with more pronounced reductions of cardiovascular events with clopidogrel therapy.
ABCB1
The ABCB1 gene, also known as MDR1, encodes for the xenobiotic efflux P-glycoprotein pump involved in the intestinal absorption of clopidogrel. The C3435T polymorphism has been variably associated with clopidogrel response, and the 3435TT genotype has been associated with decreased peak plasma concentrations of clopidogrel and its active metabolites. In the FAST-MI registry, carriers of TT and CT genotypes, compared with carriers of the wild-type genotype, had an increase in cardiovascular events in the setting of treatment with clopidogrel therapy after an acute MI. Likewise, in the setting of treatment with clopidogrel in TRITON–TIMI 38, ABCB1 3435TT homozygotes experienced a 72% increased risk of adverse cardiovascular events compared with CT/CC individuals. Data from the Platelet Inhibition and Patient Outcomes (PLATO) trial provided contrasting results with an association between 3435CC genotype and higher rates of ischemic events.
PON1
The PON1 gene encodes for paraoxonase-1, an esterase synthesized in the liver and associated with high-density lipoprotein (HDL) in the blood. Using in vitro metabolomic profiling followed by subsequent analyses of a case-cohort study and an independent replication study, another study identified PON1 Q192R polymorphism to affect variability in clopidogrel efficacy and to confer increased risks for definite stent thrombosis. The impact of the PON1 Q192R polymorphism on clopidogrel treatment effect was thought to be mediated by the role of paraoxonase-1 in bioactivation of clopidogrel from the intermediate product formed by the cytochromes in the liver to the active metabolite in the bloodstream. However, PON1 Q192R was not associated with clopidogrel treatment effect in a previously published GWAS of clopidogrel pharmacogenomics, and the study that identified this novel PON1 Q192R polymorphism was unable to reproduce the well-replicated effect of CYP2C19 loss-of-function alleles on clopidogrel therapy. A larger study, which specifically investigated the impact of PON1 Q192R genotype in parallel to that of CYP2C19 *2 on clopidogrel response and stent thrombosis, found no association of PON1 Q192R with platelet response to clopidogrel and risk of stent thrombosis, although it confirmed the effect of CYP2C19 *2 on clopidogrel antiplatelet response and risks for stent thrombosis.
Therapeutic Implications
In March 2010, the U.S. Food and Drug Administration (FDA) approved a new label for clopidogrel with the addition of a boxed warning regarding pharmacogenetics, noting diminished effectiveness of therapy in poor metabolizers (defined as having two loss-of-function CYP2C19 alleles). The boxed warning further states that “tests are available to identify a patient’s CYP2C19 genotype and can be used as an aid in determining therapeutic safety [and to] consider alternative treatment or treatment strategies in patients identified as CYP2C19 poor metabolizers.”
Pharmacogenetic Testing in Clopidogrel Therapy
Although tests to identify a patient’s CYP2C19 genotype are available, clopidogrel pharmacogenetic testing is not routinely performed. Local “point of care” assays have been piloted at specific institutions. The role and predictive value of clopidogrel pharmacogenetic testing are being actively studied. Key questions under investigations include 1) determining the best assays to use to guide clopidogrel therapy (e.g., which platelet function assays, if any, CYP2C19 genotyping, or a combination of the two); 2) assessing whether testing and therapies guided by testing results improve outcomes; and 3) estimating the cost effectiveness of testing to tailor clopidogrel therapy (to be generic in the United States in 2012) versus treating with nongeneric therapies with less interpatient variability.
Therapeutic modifications
Potential therapeutic modifications for individuals found to carry the CYP2C19 *2 allele include escalation of clopidogrel dosage or switching to an alternate agent. Earlier smaller studies suggested that tailoring the clopidogrel loading dose to platelet reactivity may improve cardiovascular outcome and that increased loading and maintenance doses of clopidogrel (up to 1200 mg loading dose and up to 150 mg daily maintenance dose), or repeated reloading with 600 mg clopidogrel based on serial vasodilator-stimulated phosphoprotein phosphorylation (VASP) measurements, may improve platelet inhibition in carriers of a reduced-function CYP2C19 *2 allele. However, results from the randomized clinical trial Gauging Responsiveness with a Verify-Now Assay–Impact on Thrombosis and Safety (GRAVITAS) study, which enrolled 2796 mostly stable angina patients for elective PCI, did not suggest a benefit in cardiovascular outcomes or stent thrombosis with doubling the dose of clopidogrel (from 75 mg to 150 mg after reloading with another 600 mg) in clopidogrel nonresponders identified by high residual platelet activity. Nonetheless, findings from the GRAVITAS study did not definitively rule out the use of tailoring thienopyridine therapy based on platelet function testing; further studies are warranted. The ELEVATE-TIMI 56 study found that among patients with stable cardiovascular disease, tripling the maintenance dose of clopidogrel to 225 mg/day in CYP2C19 *2 heterozygotes achieved levels of platelet reactivity similar to those seen with the standard 75-mg dose in noncarriers. However, doses as high as 300 mg/day did not result in comparable degrees of platelet inhibition in CYP2C19* 2 homozygotes.
Alternate therapeutic options for patients requiring dual antiplatelet therapy for ACS and PCI are available. Prasugrel is a third-generation thienopyridine that irreversibly binds the platelet P2Y 12 receptor to inhibit ADP-induced platelet aggregation. The TRITON-TIMI 38 trial found prasugrel to have superior efficacy compared with clopidogrel in reducing all-cause mortality or vascular complications, including stent thrombosis, but with an increased risk of bleeding. The FDA approved prasugrel for use in PCI for ACS in July 2009. A genetic analysis within the TRITON-TIMI 38 trial found that polymorphisms in several genes, including CYP2C19 , did not affect active metabolite levels, platelet aggregation inhibition, or clinical cardiovascular event rates in individuals treated with prasugrel. The impact of CYP2C19 polymorphisms on clopidogrel compared with that on prasugrel is likely mediated by differential involvement of esterases and the CYP450 system in the activation of clopidogrel and prasugrel. For clopidogrel, esterases shunt the majority of ingested clopidogrel to a dead-end inactive pathway with the remaining prodrug requiring a two-step CYP-dependent oxidation to produce active clopidogrel metabolites; for prasugrel, esterases are part of the activation pathway, and activation of prasugrel requires only a single CYP-dependent oxidative step. The ABCB1 C3435T polymorphism, which has inconsistently been reported to influence clopidogrel therapy in some studies, was also found not to affect clinical or pharmacologic outcomes in patients treated with prasugrel.
Another therapeutic option for patients is ticagrelor, an oral reversible antagonist of the platelet ADP P2Y 12 receptor, approved for use in Europe and the United States. Superior efficacy of ticagrelor to clopidogrel in ACS was established by the PLATO trial, which found ticagrelor (180 mg loading dose, 90 mg twice daily maintenance dose) to be superior to clopidogrel (300 to 600 mg loading dose, 75 mg daily maintenance dose) in reduction of vascular death, MI, or stroke but with an increase in the rate of non–procedure-related bleeding. Ticagrelor is an active compound and not a prodrug, and thus it does not require hepatic CYP450-mediated activation. Pharmacogenetic analysis of the Randomized Double-Blind Assessment of the Onset and Offset of the Antiplately Effects of Ticagrelor Versus Clopidogrel in Patients with Stable Coronary Artery Disease (ONSET/OFFSET and RESPOND) confirmed that the antiplatelet effect of ticagrelor was consistently superior to clopidogrel irrespective of CYP2C19 genotype, including the *2 poor metabolizer and *17 ultrametabolizer. A genetic analysis within the PLATO trial found ticagrelor superior to clopidogrel in treatment of ACS irrespective of CYP2C19 polymorphism, although the magnitude of benefit tended to be greater in carriers of loss-of-function alleles.
Cost Effectiveness of Clopidogrel Pharmacogenetics Testing
Universal reimbursement policies for pharmacogenetic testing or platelet function testing for clopidogrel remain to be defined. Although cost may compromise the feasibility and utility of current commercial clopidogrel pharmacogenetic testing, the availability of clopidogrel in generic form in the near future may offset the cost of testing. Furthermore, technological advances and increased availability of testing may shorten the turnaround time, making it easier for test results to be incorporated into clinical decision making. Currently, inexpensive and rapid point-of-care testing has been locally developed and pioneered at several institutions. Formal cost-effectiveness analyses of clopidogrel pharmacogenetic testing will continue to provide useful data.
Future Directions
Prospective clinical trials will be helpful to further evaluate whether genetic testing indeed improves outcome in the setting of antiplatelet therapy, whether pharmacogenetic and platelet function testing are complementary, and whether testing of all patients versus only the high-risk population is most feasible. Several clinical trials are ongoing to further investigate the impact of antiplatelet therapy for PCI guided by platelet function testing (ARCTIC, NCT00827411) or genotyping (GIANT NCT01134380; TARGET-PCI, NCT01177592; Genotype Guided Comparison of Clopidogrel and Prasugrel Outcomes Study, NCT00995514). These studies will assist in determining the optimal way to use pharmacogenetic testing to select among different antiplatelet regimens.
Warfarin
Drug, Indications, Mechanism of Action, and Pharmacology
Warfarin is an oral anticoagulant used for treatment of venous thromboembolism and for perioperative prevention of venous thromboembolism, anticoagulation for mechanical heart valves, and prevention of stroke in patients with atrial fibrillation. Warfarin exerts its anticoagulant effects by antagonizing vitamin K.
Warfarin is ingested as a combination of active R- and S-warfarin enantiomers, with the S-enantiomer about three to five times more potent than the R-enantiomer. Warfarin is rapidly absorbed with near complete bioavailability. It circulates primarily bound to albumin with a mean plasma half-life of 40 hours. Warfarin interrupts hepatic vitamin K recycling by inhibiting vitamin K epoxide reductase and vitamin K quinine reductase (Braunwald, Fig. 87-13). Depletion of vitamin K reserve compromises the vitamin K–dependent gamma-carboxylation necessary for production of coagulation factors II, VII, IX, and X and anticoagulant proteins C and S. Full anticoagulation effects of warfarin are observed at least 24 to 72 hours after initiation of therapy, when clotting factors previously synthesized have been depleted. S-warfarin is inactivated by CYP2C9-mediated hydrolysis; the less active R-enantiomer is metabolized by CYP1A2 and CYP3A4 enzymes.
Patients treated with warfarin are routinely monitored for their International Normalized Ratio (INR), with an INR target of 2 to 3, except for those patients with a mechanical mitral valve, in whom the INR target range is 2.5 to 3.5. Excessive anticoagulation by warfarin and preoperative reversal of warfarin effects are managed with administration of vitamin K and, when acute reversal is indicated, with additional repletion of clotting factors via infusion of fresh frozen plasma.
Drug Interactions
Warfarin is well known for its narrow therapeutic window and extensive drug-drug and drug-diet interactions, which make the titration of warfarin dosage challenging. Increase in total-body vitamin K reserve through ingestion of vitamin K–rich foods or a decrease in vitamin K reserve during antibiotic therapy as a result of reduced GI tract production of vitamin K, respectively, attenuates or accentuates warfarin’s anticoagulant effects. Concomitant therapy with CYP2C9 inducers or inhibitors, respectively, accelerates or reduces metabolism of the active S-warfarin enantiomer. CYP1A2 and CYP3A4, which metabolize the R-warfarin enantiomer, can also be inhibited by quinolone and macrolides, respectively, and both may be inhibited by azoles. Concurrent therapy with other anticoagulants or antiplatelet agents may potentiate bleeding risk. Other cardiovascular medications, including amiodarone and statins, may also interact with warfarin. Amiodarone and its major metabolites inhibit CYP2C9, thereby potentiating the anticoagulant effects and bleeding risks of warfarin. Small retrospective studies, case series, and case reports suggest that fluvastatin, lovastatin, simvastatin, and rosuvastatin may accentuate warfarin’s effect, potentially via inhibition of CYP2C9, CYP3A4, or both.
Pharmacogenetics of Warfarin Therapy
Genetic polymorphisms along the warfarin pathway have been explored. GWASs have confirmed the association between genetic polymorphisms in VKORC1 , CYP2C9 , and CYP4F2 genetic polymorphisms and warfarin dosing variability. Polymorphisms of these three genes have been reported to account for about 20% to 30%, about 12%, and 1% to 4%, respectively, of variability in warfarin dosage.
VKORC1
The VKORC1 gene on chromosome 16 encodes for the vitamin K epoxide reductase complex 1 that is the molecular target of warfarin ( Figure 4-3 ). VKORC1 haplotypes were noted to stratify patients into groups with differing warfarin maintenance dose requirements. Specifically, haplotype A (more prevalent in Asian populations) was associated with a lower warfarin requirement, and haplotype B (more prevalent in African populations) was associated with higher warfarin maintenance dose requirement. Haplotype combinations of A/A, A/B, and B/B, respectively, confer low-, intermediate-, and high-maintenance dose requirements of warfarin. Two noncoding single-nucleotide polymorphisms (SNPs) from haplotype A rs9923231 (−1639G>A, also known as VKORC1 *2) and rs9934438 (1173C>T), which are in linkage disequilibrium (LD), have consistently been associated with a lowered warfarin dosage requirement. The minor allelic frequency of rs9923231 and rs9934438 both vary among racial groups ( Table 4-4 ). However, the presence of the rs9923231 or rs9934438 variants, irrespective of race, was consistently associated with a decrease in warfarin dosage requirement in all three of the racial groups—Asians, whites, and blacks—specifically studied in the International Warfarin Pharmacogenetics Consortium (IWPC) cohort.
