Abstract
A substantial burden of cardiovascular disease results from thrombosis that manifests clinically as acute coronary syndrome, stroke, and venous thromboembolism. Although antithrombotic drugs that inhibit platelets and coagulation factors have been shown to reduce thrombotic events, there exists considerable variability in response to many of these drugs that affect the relative efficacy and safety in individual patients. Pharmacogenomics offers the opportunity for personalized medicine through the identification of medically actionable variants, such as predicting the efficacy of a specific drug ( CYP2C19 genotyping for selecting antiplatelet therapy) or determining optimal dosing regimens and minimizing adverse events ( CYP2C9 and VKORC1 genotyping for warfarin).
Keywords
Antithrombotic, acute coronary syndrome (ACS), anticoagulant, antiplatelet, pharmacogenetic
Introduction
A substantial burden of cardiovascular disease results from thrombosis that manifests clinically as acute coronary syndrome (ACS), stroke, and venous thromboembolism. Clot formation results from a disruption in the complex hemostatic interactions involving the vascular endothelium, platelets, and coagulation factors. Although antithrombotic drugs that inhibit platelets and coagulation factors have been shown to reduce thrombotic events, there exists considerable variability in response to many of the cardiovascular drugs routinely used in clinical practice that affects the relative efficacy and safety in individual patients. Clinical, environmental, and genetic factors all contribute to variability in drug response. This chapter will focus on the major pharmacogenetic variants associated with antiplatelet agents (aspirin and clopidogrel) and vitamin K anticoagulants (warfarin). In addition, we highlight the therapeutic implications of these variants and review the potential role of pharmacogentic testing in the practice of “personalized” or “precision” medicine.
Antiplatelet Agents
Platelets are integral to the development of the pathological thrombus in coronary arteries that are responsible for ACS . Platelets contribute to clot formation through a process of adhesion, activation, and aggregation. Disruption of the endothelium exposes platelets to the adhesive proteins of the subendothelial matrix. Platelet adhesion is dependent on the interactions between the matrix proteins and platelet-receptor glycoproteins. Platelet adhesion results in the release of activators such as adenosine diphosphate (ADP), adrenaline, serotonin, thrombin, and thromboxane A2 . These agonists bind to G-protein-coupled receptors, which further potentiate platelet activation. Finally, platelet aggregation occurs when glycoprotein IIb/IIIa complexes on platelets bind to fibrinogen. Pharmacologic targeting of platelet activating factors and their receptors is a cornerstone of antithrombotic therapy ( Fig. 9.1 ).
Aspirin
Aspirin (acetylsalicylic acid) blocks the conversion of arachidonic acid to thromboxane A2 by irreversibly inhibiting cyclooxygenase (COX)-1 by acetylating a serine residue near the narrow catalytic site of the COX-1 channel . Thromboxane A2 inhibition is cumulative with repeated low doses of aspirin because of the permanent and irreversible enzyme inactivation throughout the 7–10-day lifetime of anucleated platelets, which allows for aspirin to be given once daily despite a half-life of 15–20 minutes .
Peak plasma concentration of aspirin is obtained within 30 minutes after ingestion of regular aspirin and up to 4 hours after ingestion of the enteric-coated formulation . For patients with ACS, a recommended 150–325 mg of oral aspirin is chewed to achieve rapid inhibition of thromboxane A2 . For chronic therapy, low-dose aspirin (e.g., 81 mg/d) is recommended.
The issue of aspirin “resistance” is controversial. Since aspirin completely inhibits COX-1 in almost all patients, investigation has focused on genetic loci that determine platelet function. Several genes ( PEAR1 , ITGB3 , VAV3 , GPVI , F2R , GP1BA , and LPA ) have been associated with platelet function in response to aspirin; however, most studies have failed to demonstrate a clear link between these variants and clinical outcomes .
Therapeutic Implications
The initial genetic association studies focused on only on impaired laboratory response to aspirin, and many of the clinical outcomes studies were underpowered case–control studies that did not account for concurrent medications. The largest prospective cohort study (nearly 3500 patients and 700 events) did not find any association between variants associated with laboratory aspirin resistance and was well relatively well powered (80%–88% power to detect of hazard ratio of ≥1.3) . Given the lack of clear association of variants related to platelet function in response to aspirin with clinical outcomes, there is currently no established role for pharmacogenetic testing. Variability in platelet inhibition with aspirin in clinical settings is likely not primarily determined by genetic factors but rather secondary to proinflammatory conditions (ACS or diabetes) characterized by a faster rate of platelet turnover or hyperreactivity .
P2Y 12 Receptor Antagonists
Clopidogrel
The use of aspirin in combination with a P2Y 12 receptor antagonist, known as dual antiplatelet therapy, is the primary antithrombotic regimen in patients with ACS and those undergoing coronary stenting. The P2Y 12 ADP receptor inhibitor clopidogrel is one of the most widely prescribed medications in patients with cardiovascular disease. Among patients presenting with ACS, the addition of clopidogrel to aspirin reduces the risk of death and ischemic complications by approximately 20% .
Clopidogrel, a thienopyridine, is a prodrug. After intestinal absorption, 85% of clopidogrel is hydrolyzed rapidly by esterases to an inactive carboxylic metabolite . The remaining 15% undergoes two sequential hepatic oxidation steps involving the cytochrome P450 isoenzymes, mainly CYP2C19 , to form the active thiol metabolite that irreversibly binds to the platelet P2Y 12 ADP receptor, inhibiting ADP-dependent platelet activation and hence aggregation.
There exists substantial variability in response to clopidogrel . The mechanisms causing this variability are multifactorial and include drug, environmental, and genetic interactions, in addition to clinical features such as diabetes and obesity. Several studies have demonstrated that patients treated with clopidogrel who have high residual on-treatment platelet reactivity have an increased rate of ischemic complications, such as myocardial infarction and stent thrombosis . A pharmacogenetic explanation of clopidogrel’s variable response has focused on polymorphisms in genes involved with clopidogrel’s absorption ( ABCB1 ) and metabolism ( CYP2C19 and PON1 ) ( Table 9.1 ).
| Gene | Variant | Active Clopidogrel Metabolite Concentration | Platelet Inhibition | Clinical Outcomes |
|---|---|---|---|---|
| CYP2C19 | *2 (rs4244285, c.681 G>A) | Decreased | Decreased | MACE
|
| CYP2C19 | *17 (rs12248560, c.-806C>T) | Increased | Increased | MACE
|
| ABCB1 | (rs1045642, c.343C>T) | Decreased | Decreased | MACE
|
CYP2C19
The CYP2C19 gene encodes for the cytochrome 450 2C19 enzyme, which is involved in both steps of hepatic activation of clopidogrel to its active metabolite. The CYP2C19 gene is polymorphic. CYP2C19*1 is the wild-type allele, but more than 25 variants are recognized, with the majority of variants associated with reduced or absent enzymatic function . The most common of these loss-of-function alleles is the *2 variant, with ~25% to 30% of those of European and African ancestry and up to 70% of those with Asian ancestry carrying at least one copy. 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.
Both candidate gene and genomewide association studies (GWAS) have demonstrated an association with loss-of-function CYP2C19 variants with reduced high on-treatment platelet reactivity . Compared to noncarriers, individuals with at least one copy of loss-of-function allele have approximately 30% lower levels of active clopidogrel metabolite and approximately 25% relative less platelet inhibition with clopidogrel . Although the CYP2C19*2 allele has a stronger independent effect on ex vivo platelet function than any clinical risk factor, it still accounts only for up to ~12% of the observed variability in clopidogrel platelet response .
The impact of CYP2C19 loss-of-function alleles on adverse cardiovascular events is more controversial. In a cohort of patients treated with clopidogrel predominantly for percutaneous coronary intervention (PCI), carriers of both one and two CYP2C19 loss-of-function alleles were at increased risk for major adverse cardiovascular outcomes, with a 1.5-fold increase in the risk of cardiovascular death, myocardial infarction (MI), or stroke as well as a threefold increase in risk for stent thrombosis compared to noncarriers . In contrast, genetic association studies of patients receiving clopidogrel who were predominantly medically managed (i.e., did not undergo PCI) did not show an association between CYP2C19 loss-of-function variants and outcomes . The most plausible explanation for discordant findings is that the magnitude of the benefit of clopidogrel in a specific patient population influences the risk associated with CYP2C19 loss-of-function variants. If the magnitude of the benefit of clopidogrel in that population is small, the impact of genotype on clopidogrel efficacy will be minimal. Therefore, the risk associated with loss-of-function CYP2C19 variants appears greatest among patients for whom clopidogrel has the greatest efficacy—those undergoing PCI. To support this hypothesis, the odds ratio for major adverse cardiovascular events (MACE) in CYP2C19 loss-of-function allele carriers as compared with that in noncarriers is 1.57 (95% CI: 1.13–2.16) in one meta-analysis, in which >90% of included patients had undergone PCI , as compared with 1.18 (95% CI: 1.09–1.28) in another meta-analysis, which included a broader range of lower risk patients .
CYP2C19*17 (rs12248560, single base pair mutation of C → T in the promoter region of the gene) is another common variant that leads to gain-of-function as a result of an increase in trascriptional activity that has been correlated with a greater inhibition of ADP-induced platelet aggregation . Clinically, the CYP2C19*17 variant is associated with an increased risk of bleeding with clopidogrel and protection from ischemic events, although the magnitude of the effect is lower than that of loss-of-function alleles .
ABCB1
ABCB1 [adenosine triphosphate-binding cassette (ABC) subfamily B member 1] encodes for an efflux P-glycoprotein transporter that reduces the bioavailability of a broad array of substrates. The C3434T (rs1045642) polymorphism has been associated with reduced intestinal absorption of clopidogrel. Patients with ACS randomized to clopidogrel in the TRITON-TIMI 38 trial who were TT homozygous had a 72% increased risk of adverse cardiovascular events compared with CT/CC patients . In contrast, the 3435CC genotype was associated with a higher risk of ischemic events in the ACS patients randomized to clopidogrel in the PLATO trial . Meta-analyses confirm the significantly increased risk of MACE as well as a decreased risk of bleeding .
PONI
The PON1 gene encodes for paraoxonase-1, an esterase synthesized in the liver. Initial studies suggested that PON1 may play a role in the hepatic conversion of an intermediate clopidogrel metabolite (2-oxo-clopidogrel) to the active thiol metabolite and that the missense variant PONI Q12R was associated with an increased risk of ischemic events in patients treated with clopidogrel . Subsequent larger studies, however, have failed to demonstrate any association between this variant and clopidogrel efficacy and subsequent pharmacology studies have questioned whether paraoxonase-1 actually plays a role in the clopidogrel metabolism . A recent meta-analysis suggests no significant impact of PON1variants on cardiovascular outcomes .
Therapeutic Implications
In March 2010, the US Food and Drug Administration (FDA) issued a boxed warning on the clopidogrel (Plavix) product label informing clinicians of the diminished efficacy of clopidogrel in patients designated as “poor metabolizers” by virtue of having two copies of CYP2C19 loss-of-function alleles. The boxed warning does not mandate genetic testing but rather provides information on the potential risk associated with certain genotypes and has general guidance on alternative approaches. Potential strategies for patients found to carry a loss-of-function CYP2C19 variant include using higher doses of clopidogrel or using alternative antiplatelet agents.
The ELEVATE-TIMI 56 trial demonstrated that tripling the maintenance dose of clopidogrel to 225 mg daily in CYP2C19 *2 heterozygotes achieved comparable on-treatment platelet reactivity as the standard 75 mg dose in wild-type individuals. However, doses up to 300 mg did not achieve bioequivalence in patients with two loss-of-function alleles . There are no studies that have demonstrated that personalizing the dose of clopidogrel based on genetic testing improves clinical outcomes.
Alternatively, the third generation P2Y 12 inhibitors prasugrel or ticagrelor could be substituted for clopidogrel. Prasugrel, like clopidogrel, is a thienopyridine that irreversibly binds the platelet P2Y 12 receptor to inhibit ADP-induced platelet aggregation. It is also a prodrug but activation only requires a single hepatic CYP-dependent oxidative step . Ticagrelor is cyclopentyltriazolopyrimidine and is an active compound and therefore does not require CYP450 activation. Both prasugrel, in the TRITON-TIMI 38 trial, and ticagrelor, in the PLATO trial, reduced cardiovascular death, MI, and stroke in patients with ACS compared to clopidogrel, although at the expense of increasing major spontaneous bleeding . Both agents are approved for use in patients with ACS. Genetic analyses within both trials demonstrated that loss-of-function polymorphisms in CYP2C19 did not affect the clinical efficacy of prasugrel or ticagrelor .
There is considerable debate regarding the necessity and clinical utility of genetic testing to inform decisions regarding antiplatelet therapy. Guidelines have not supported routine CYP2C19 genotyping in clinical practice given the absence of randomized trial data that such a strategy improves patient outcomes. They do, however, acknowledge that genetic testing may play a role in select patients. The American College of Cardiology Foundation/American Heart Association consensus guidelines advocate consideration of CYP2C19 genotyping before initiating clopidogrel therapy in patients at high risk of adverse cardiovascular events – for example, patients undergoing multivessel PCI . Certainly if the genotype is known that information should guide therapy. The Clinical Pharmacogenetics Implementation Consortium guidelines recommend that prasugrel or ticagrelor be used in patients with ACS undergoing PCI if they are carriers of loss-of-function CYP2C19 alleles . With the superior clinical efficacy of prasugrel and ticagrelor compared to clopidogrel, it is also reasonable to consider one of these agents instead of clopiodgrel without genotyping patients first as long as they do not have a clinical contraindication.
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