1. Introduction
Clopidogrel is a commonly used antiplatelet drug; medical society consensus guidelines broadly recommend its use [
1,
2,
3] for prevention of stent thrombosis in individuals treated with percutaneous intervention (PCI) [
4,
5], and in some patients medically managed after non ST elevation myocardial infarction. In PCI, a metal stent is inserted into a coronary artery to improve blood flow in patients presenting with ST elevation myocardial infarction, acute coronary syndrome (ACS), or refractory stable angina [
6,
7,
8]. Clopidogrel is an oral second-generation thienopyridine with 50% bioavailability; its concentration peaks at 1 to 2 h following administration of an initial or “loading” dose (often 300 mg) [
9,
10,
11]. Clopidogrel’s half-life ranges from 7 to 8 h [
12]. Clopidogrel is excreted almost equally by the kidney and the gastrointestinal system [
13] (
Figure 1). Based on in vitro data, approximately 85% of the orally administered drug is hydrolyzed by endogenous esterases into an inactive metabolite; the remaining drug is bioactivated in a two-step oxidative process by the cytochrome P450 (CYP450) enzyme in the liver to yield an intermediate metabolite (2-oxo-clopidogrel), which is then transformed to four isomeric thiol metabolites (H1–H4); clopi-H4 is the only active clopidogrel metabolite in vivo [
14,
15,
16].
The active clopi-H4 metabolite irreversibly binds the purinergic adenosine diphosphate (ADP) platelet receptor P2Y12 [
15], preventing platelet stimulation by ADP [
11], subsequent platelet aggregation [
7], and consequent thrombogenesis [
8]. Under normal circumstances, P2Y12 and other platelet receptors bind to one arm of the fibrinogen molecule and link the fibrinogen molecule to receptors on other uninhibited platelets that bind the other arm of fibrinogen, thereby initiating aggregation. Translational studies consistently indicate the key roles of platelet aggregation, adhesion, and activation in the pathophysiology of thrombus formation and vessel occlusion that underlie coronary atherosclerosis [
5,
17,
18,
19,
20,
21,
22]. Resultantly, rapid, reliable, and potent inhibition of platelets by drugs that bind the P2Y12 receptors on the surface of platelets is critical to decrease the frequency of ischemic events and thereby reduce cardiovascular morbidity and mortality [
4,
5].
Clopidogrel is most frequently paired with aspirin for dual antiplatelet therapy (DAPT) as an established standard of care for individuals with ischemic heart disease who have undergone PCI to reduce thrombotic risk [
7,
8]. Usually, postprocedural DAPT has a duration of up to 12 months, which depends on the type of stent placed during PCI and overall risk assessment [
3], as well as shared decision-making with each individual patient. This follows a loading dose of clopidogrel given shortly before PCI. The preprocedural loading dose and the postprocedural DAPT therapy combination generally prevents stent thrombosis and limits ischemic events recurring after PCI [
11]. Clopidogrel is in general well tolerated by individuals, with side effects mainly consisting of bleeding, gastrointestinal upset, rash, and other more rare adverse events such as hepatotoxicity or thrombotic thrombocytopenic purpura [
11].
Unfortunately, some patients demonstrate limited response to the anti-platelet effect of clopidogrel. Platelet inhibition is attenuated in such patients, who exhibit “high on treatment platelet reactivity” (HTPR), which hinders optimal DAPT efficacy [
11]. Platelets taken from patients that are tested ex vivo and found to be hyporesponsive to clopidogrel administration associate with a higher risk of ischemic coronary events, while platelets found to be hyperresponsive may associate with a higher risk of bleeding [
16]. There is therefore interindividual variability in the observed response of individuals to clopidogrel therapy [
23,
24]. In fact, up to 30% of individuals presenting to health care facilities with ACS demonstrate poor response to clopidogrel therapy, in terms of HTPR and adverse cardiovascular endpoints [
8,
25].
Various clinical factors are thought to contribute to poor response to clopidogrel therapy as described in other sections of this text, while heritability appears to be responsible for up to 70% of interindividual variability [
8,
26]. As a result, genetic polymorphisms implicated in clopidogrel pharmacodynamics and pharmacokinetics, particularly absorption and metabolism, are considered key determinants of variable interindividual response to clopidogrel therapy [
16].
2. Pharmacogenomics of Clopidogrel Response Variation
Approximately one fourth of individuals treated with clopidogrel exhibit a subtherapeutic response [
27], and variable platelet reactivity in response to clopidogrel has been found to be highly heritable [
11,
26]. In fact, the expansive pharmacodynamics and pharmacokinetics of clopidogrel involving absorption in the intestine, bioactivation by CYP450 enzymes in the liver, and deactivation by esterases, provide various avenues for considering the basis of this heritability [
11]. Such consideration could allow for discovery of differential metabolism of clopidogrel according to each individual’s genomic profile, with prediction of pharmacokinetic and pharmacodynamic responses and subsequent personalization of clopidogrel dosing in precision medicine [
28]. This has led to a search for underlying genetic variants responsible for lack of efficacy, in order to apply pharmacogenomics to this clinical problem. Pharmacogenomics studies the impact of variations in the genome on response to therapeutics, with the goal of adjusting medication dosing to improve efficacy and minimize toxicity [
28].
A range of 30–90% of variability noted in the pharmacokinetics and pharmacodynamics of therapeutics is considered attributable to pharmacogenomic variation [
28]. Most pharmacogenomic studies over the past 20 years have involved enzymes that carry out oxidation, reduction, or hydrolysis of substrates (Phase I reactions) or enzymes that acetylate, glucuronidate, sulphate, or methylate their substrates (Phase II reactions) [
28]. This includes human cytochrome P450 enzymes (CYP450) (mixed Phase I and Phase II reactions) [
28], which have 57 active genes and 58 pseudogenes [
11,
29]. Genetic variants in
CYP2D6, CYP2C19, CYP2C9 and
CYP3A4/5 are the most extensively studied [
28,
30]. The CY2D6 enzyme metabolizes several CYP450 substrates used in clinical medicine today, but is not a primary enzyme responsible for clopidogrel metabolism [
28,
30].
The CYP2C family (CYP2C8, CYP2C9, CYP2C18 and CYP2C19) metabolizes 20% of the substrates of CYP450 [
31,
32]. Genetic variants specifically in
CYP2C9 and
CYP2C19 associate with differential metabolism of various CYP450 substrates [
28], such as clopidogrel [
11,
26,
33,
34]. Genetic variants in other CYP450 genes such as
CYP1A2 and
CYP2B6 can also influence the pharmacodynamics and pharmacokinetics of either the first or second step of clopidogrel metabolism [
11,
16,
35,
36,
37]. Thus, for clopidogrel, investigations have largely focused on genomic variants associated with metabolism to its active metabolite by these major cytochrome P450 enzymes [
11,
27,
28]. Clopidogrel metabolism can also be influenced by genetic variants in other proteins, such as the ATP-Binding Cassette Subfamily B Member 1 (
ABCB1), Paraoxonase-1 (
PON1), Carboxyl Esterase 1 (
CES1), and P2Y12 receptors [
11,
16,
35,
36,
37]. These genetic variants can interfere with regulation of gene transcription, protein translation, or substrate affinity [
38,
39].
The primary cytochrome P450 enzyme involved in the two-step bioactivation of clopidogrel has also arisen as the most highly associated with the variable response to clopidogrel:
CYP2C19 [
1,
11], which is therefore the focus of this review. The CYP2C19 enzyme is responsible for 45% of the first step and 20% of the second step in hepatic biotransformation of clopidogrel [
40,
41]. The enzymes CYP1A2 and CYP2B6 also assist in the first step, and CYP2B6, CYP2C9, CYP3A4 and CYP3A5 assist in the second step [
11,
40,
42,
43]. The CYP2C19 enzyme therefore plays an important role in both steps of this oxidative process. In a linear mixed-effects model using the AUC
0–24 as a primary outcome based on active metabolite measurements in 162 normal subjects compiled from 6 separate studies, carriage of
CYP2C19*2 or
*3 was associated with the most significant reduction (32%,
p < 0.001) in AUC
0–24, compared to genetic variation in the other cytochrome P450 enzymes involved in clopidogrel metabolism, such as CYP2C9, CYP2B6, CYP3A5 and CYP1A2 [
44]. Variants in
CYP2C19, which encodes the enzyme involved in both the first and second steps have been linked to impaired bio-activation of clopidogrel, decreased platelet inhibition, and higher risk of adverse cardiovascular events following PCI [
27].
2.1. CYP2C19 Variation
Interindividual variability in response to clopidogrel administration consists of clinical factors, and is also in part explained by genetic variants in
CYP2C19 [
25]. Polymorphisms in
CYP2C19 at least in part account for variable production of the active clopidogrel metabolite (clop-H4). As a result, CYP2C19 has arisen as a key focus of investigation in clopidogrel pharmacogenetics and pharmacogenomics [
23,
45].
The enzyme cytochrome P450 2C19 (CYP2C19) is a 55.93 kDa (490 aa) protein encoded by the
CYP2C19 gene (90.21 kb) [
28]. The gene is composed of 9 exons, which are mapped to the chromosomal location 10q24.1q24.3 [
28]. The protein is expressed in liver cells, via 1901 bp, 2395 bp, and 1417 bp RNA transcripts [
28]. CYP2C19 metabolizes a variety of drugs, including clopidogrel [
46]. Heritable variation in CYP2C19 function was first noted 45 years ago [
47]. Since then, more than 50 single nucleotide polymorphisms (SNPs) partially underlying variation in enzyme activity have been elucidated [
48,
49,
50,
51,
52,
53,
54,
55,
56,
57,
58,
59,
60,
61,
62,
63,
64,
65,
66,
67] (
Table 1). The normal or wild type allele is usually denoted
*1 (e.g., homozygous wild type
*1/
*1), with pathophysiological polymorphisms following similar nomenclature [
11]. Enzyme activity is often assessed by its classical reaction, which produces (S)-mephenytoin 4′-hydroxylation [
51].
2.1.1. Loss-of-Function CYP2C19 Variants
The most extensively studied variants are
CYP2C19*2 (primarily rs4244285) and
CYP2C19*3 (rs4986893), which both involve replacement of guanine (G) with adenine (A) [
11]. The
CYP2C19*2 variant is present at nucleotide 681 (i.e., c.681G > A) found in exon 5 [
11], and synonymously affects the amino acid proline at position 227;
CYP2C19*3 is present at nucleotide 636 (i.e., c.636G > A) found in exon 3, and substitution of the tryptophan codon TGG with TGA [
15,
51,
56]. Both variants result in a stop codon, indirectly via a cryptic aberrant splice site in the case of
CYP2C19*2 and directly coded by TGA in the case of
CYP2C19*3, and ultimately produce a truncated, nonfunctional, catalytically inactive CYP2C19 protein fragment [
51,
56]. Individuals who are homozygous for either
CYP2C19*2 (i.e.,
*2/*2) or
CYP2C19*3 (i.e.,
*3/*3) poorly metabolize CYP2C19 substrates [
47], such as clopidogrel, contributing to inter-individual variability in therapeutic response [
32]. Other alleles have also been associated with
CYP2C19 loss-of-function (LOF) (
CYP2C19*4,
*5,
*6,
*7 and
*8); however, these variants are rare (<1% allelic frequency) and less studied [
68,
69].
2.1.2. Gain-of-Function CYP2C19 Variant
A gain-of-function variant has also been identified [
25,
70]: the variant
CYP2C19*17 (rs12248560) involves replacement of cystine (C) with thymine (T) [
64]. The
CYP2C19*17 variant (i.e., g.−806C>T or g.−3402C>T) is present in a promotor region of the gene at the 5′-flanking region of exon 5 [
25,
71]. The genetic variant in the promoter region is thought to modify the interaction of transcription factors with the promotor region in
CYP2C19, thereby altering the extent of transcription of the gene [
71]. Thus, besides variation in the coding regions of the
CYP2C19 gene, variations in the promoter region of the gene also appears to influence variability [
47]. The
CYP2C19*17 allelic variant associates with higher levels of catalytic activity of the corresponding CYP2C19 enzyme, presumably due to increased transcription of the
CYP2C19 gene [
11,
25,
64]. The activity of CYP2C19 depends on its level of expression in the liver, which is in part determined by regulation of gene transcription [
46]. Thus, bioactivation of clopidogrel and other prodrugs to their therapeutic metabolites is increased in individuals carrying the
CYP2C19*17 allele [
15,
72]. Consequently, individuals carrying the
CYP2C19*17 allele have a higher risk of bleeding when treated with clopidogrel in the PLATO (Platelet Inhibition and Patient Outcomes) trial [
25,
73,
74]. However, other studies have not demonstrated increased platelet inhibition or altered clinical outcomes with the
CYP2C19*17 allele [
26,
75,
76,
77]. Interestingly, CYP2C19 enzyme activity levels in individuals homozygous for
CYP2C19*17 overlaps the heterogeneous enzymatic activity noted in some wild type individuals [
78]. This high enzyme activity in some individuals with wild type alleles could be due to other underlying causes of increased enzyme activity, such as response regulators described elsewhere in this article, or yet to be discovered gain of function alleles [
47]. Therefore additional studies assessing the 5′-up-stream promotor region of the
CYP2C19 gene could also help clarify the observed heterogeneity of enzyme activity in some wild type individuals [
47]. Nevertheless, in a study of livers from individuals heterozygous and homozygous for
CYP2C19*17, transcribed mRNA levels were 1.8-fold and 2.9-fold higher, respectively, than livers with the homozygous wild type allele (
*1/
*1) [
46]. Such studies may be consistent with the
*17 allelic variant serving as a regulatory polymorphism that can enhance the expression of
CYP2C19 expression [
46]. However, taken together, it is unclear how patients with the
CYP2C19 *17 allele would respond to clopidogrel, and the combined effect of
*2 and
*17 on
CYP2C19 function and response to clopidogrel is uncertain.
2.1.3. Linkage Disequilibrium
Of course, it is interesting to consider that in future studies the
*17 gain-of-function variant could potentially compensate for the
*2 or
*3 loss-of-function variant [
46]. Indeed, the
CYP2C19*17 and CYP2C19*2 variants have been found to be in linkage disequilibrium, which could explain inconsistent accounts of the effect of the
CYP2C19*17 variant on platelet aggregation and measured levels of the active clopidogrel metabolite (clopi-H4) [
15,
79,
80]. Linkage disequilibrium has also been noted between the
CYP2C19*2 and
CYP2C19*35 variants. The
CYP2C19*35 (rs12769205) involves alteration of adenine (A) at an intron 2 branch point, which results in an alternative CYP2C19 mRNA transcript in human liver that includes the entire intron 2 (exon 2B) [
81]. The reading frame on the mRNA transcript is therefore altered, with the introduction of 87 additional amino acids and a premature termination or stop codon [
81]. The
CYP2C19*35 variant in isolation can decrease
CYP2C19 canonical mRNA transcript levels and subsequent translation of the CYP2C19 protein [
81].
2.1.4. Multiple Variants
Most genes with variants that associate with poor clopidogrel response exert a weak on platelets inhibition [
11]. However, several authors have suggested additive or synergistic effects on clopidogrel response in the context of coexistence of
CYP2C19 variants with polymorphisms in other genes [
11,
82,
83,
84]. For example, synergistic effects may be noted with coexistence of
CYP2C19 polymorphisms and
ABCB1 genetic variants in patients undergoing PCI [
82]. Other studies suggest that coexistence of
CYP2C19 and
P2Y12 variants leads to a greater impact on clopidogrel response and clinical outcomes than either polymorphism alone [
11,
83,
84]. As such, models to predict clopidogrel response should account for the effect of multiple variants in different proteins. Similar multimarker models have been developed in other areas of medicine with effective prediction of outcomes [
85].
4. Regulators of Response
Careful examination of a myriad of studies reveals inconsistency of the association of
CY2C19 loss-of-function or gain-of-function allelic variants with reduced antiplatelet activity, plasma clopidogrel active metabolite concentration, and ischemic cardiovascular outcomes, or the converse, respectively [
15,
23,
73,
76,
103,
118]. Some studies suggest that
CYP2C19 genetic polymorphisms are responsible for a sizeable genetic defect of up to 12% of the interindividual variability of platelet aggregation noted in individuals who have undergone PCI, and that >80% of the variability can be explained by other factors collectively [
119]. Among reasons for such variability are the inability of various genotyping assays to detect all variants that possibly could affect CYP2C19 activity (although only a handful of variants potently impact enzyme activity), differential drug absorption among individuals, and drug–drug interactions [
15]. The observation of genotype-phenotype discordance in some individuals can also imply differential regulation of the
CYP2C19 gene leading to an acquired deficiency in enzyme activity [
47]. Indeed, antiplatelet response to clopidogrel administration may depend on genetic, cellular, environmental, and clinical factors [
100].
Platelet function and cardiovascular outcomes are affected not only by medical therapy and genetic variation, but also by non-genetic factors, such as concomitant diseases, patient compliance, obesity, age, race, sex, ethnicities, diet, smoking, platelet count, and hematocrit [
11,
23,
32,
92,
120,
121,
122,
123] (
Figure 3). It should be noted that platelet reactivity levels and plasma concentration of active metabolites may vary with time and in different clinical settings, such as diabetes mellitus, chronic kidney disease, type of coronary lesions, stent-related factors, presentation with stable ischemic heart disease versus ACS, and inflammatory state (e.g., CRP or interleukin levels post-PCI [
107,
124,
125,
126]). Some studies suggest that high-sensitivity CRP and interleukin levels can predict response to clopidogrel [
127], while other studies suggest that clopidogrel and other antiplatelet agents might be anti-inflammatory. Further, concomitant polymorphisms, differences in P2Y12 receptor density, concentration of fibrinogen and platelets, and accelerated turnover of platelets can also play a role [
45,
108]. Taken together, these observations elucidate the complex and nonlinear nature of the relationships among allelic variation, platelet reactivity, plasma concentration of active metabolite, and cardiovascular outcomes in the pharmacogenomics of CYP2C19 [
16].
4.1. Variation by Ethnicity
Homozygosity for LOF
CYP2C19 variants is noted in approximately 2% of whites, 4% of blacks, and 14% of Chinese individuals [
129] (
Table 3). Heterozygosity for
CYP2C19*2 is noted in up to 30% of Caucasians, up to 40% in African Americans, and up to 50% in East Asians [
23,
44,
75,
130]. The
CYP2C19*3 allele is less common, and is found in less than 1% of self-reported Caucasians and African Americans and in 7–9% of Asians [
11,
15]. The CYP2C19*2 and *
3 alleles are the most common and account for ≥99% LOF in a multi-ethnic population [
68,
69]. Of note, a high prevalence of LOF variants has been discovered in Vanuatu and Papua New Guinea, where allele frequencies are approximately 70% (
*2) and 13% (
*3) of
CYP2C19 alleles in Vanuatu, and approximately 40% and 30%, respectively, in Papua New Guinea, several orders of magnitude higher than in other ethnic populations [
47,
131,
132]. The
CYP2C19*
17 allele also exhibits variation by ethnicity, with a low incidence of <5% in Japanese and Chinese populations, and higher a frequency of up to 30% in European and African populations [
133].
Another study showed that African-Americans had a higher prevalence of
CYP2C19*2 allele carrier status and higher on-treatment platelet reactivity than Europeans [
134]. This study also suggested that African-American ethnicity and
CYP2C19*2 allele carrier status were both independent predictors of HTPR. This implies that beyond
CYP2C19*2 allele carrier status, additional factors may contribute to higher HTPR in African-Americans [
46]. These factors may be genetic variants unique to African-Americans, conventional response regulators or response regulators based on a more comprehensive precision medicine approach described in subsequent sections. In addition, the
CYP2C19*35 loss-of-function allelic variant is found in linkage disequilibrium with rs4244285 on
CYP2C19*2 in whites, but is expressed in isolation in African-Americans [
135]. On the other hand, a liver sample of African descent exhibited an approximately 12-fold increase in metabolic activity that was not accounted for by the presence of a
CYP2C19*17 allelic variant, suggesting the existence of possible additional undiscovered variants or regulatory mechanisms [
46].
One study identified lower frequency of the
CYP2C19*2 variant in North Indians of Asian descent compared with the global population [
136], while another study suggested comparable frequencies (40–47%) in Asian Indian patients with ischemic heart disease [
137]. A meta-analysis showed that carrier status for LOF genetic variants in
CYP2C19 associated with increased risk of adverse clinical events [
138]. The study also suggested higher risk of adverse clinical events in Asians compared to Western populations [
138]. Paradoxically, although Japanese and other East Asian individuals have a higher incidence of
CYP2C19 LOF allelic variants than Europeans, Asians in general are reported to have a greater frequency of bleeding than Europeans [
88,
139]. The increased bleeding tendency observed in Asians, however, does not translate to less thrombotic events in Asians with
CYP2C19 LOF alleles receiving clopidogrel. For example, major cardiovascular and cerebrovascular events occurred at a significantly higher rate in Koreans and East Asians when treated with clopidogrel who had
CYP2C19 poor metabolizer status compared to wild type individuals [
140,
141].
4.2. Drug-Drug Interactions
Bioactivation of clopidogrel by CYP2C19 can be affected by drug-drug interactions [
11]. The enzyme CYP2C19 also metabolizes several other drugs that are clinically useful, such as proton pump inhibitors (PPIs) (e.g., omeprazole, lansoprazole, and pantoprazole), selective serotonin reuptake inhibitors (e.g., citalopram and sertraline), tricyclic antidepressants (e.g., imipramine and amitriptyline), phenytoin, and clopidogrel [
11,
71]. In fact, there are almost 500 drugs that act as substrates (281), inhibitors (263), or inducers (23) of the CYP2C19 enzyme, or some combination of these.
Some studies suggest that PPIs inhibit the CYP2C19 enzyme, and that this may interfere with clopidogrel bioactivation to yield its active metabolite clopi-H4 [
23,
143,
144]. Similar to carrying a LOF
CYP2C19 allele, co-administration of clopidogrel with certain PPIs leads to HTPR, and an approximately 40% increased risk in associated adverse cardiovascular outcomes and mortality, particularly in high-risk individuals [
23,
143]. Some PPIs exhibit a greater effect than others, such that omeprazole (the most potent CYP2C19 inhibitor) and lansoprazole, for example, associated with decreased platelet inhibition by clopidogrel, while esomeprazole and pantoprazole did not [
11,
23,
143,
145,
146].
4.3. Conventional Regulators of Response
4.3.1. Adherence
As with any pharmacologic agent, patient adherence to the prescribed regimen is key to achieve optimal therapeutic effect and associated clinical outcomes. Studies indicate that nonadherence to prescribed clopidogrel therapy is a significant contributor to HTPR, but that nonadherence minimally accounts for the widely observed interindividual variability [
91,
147].
4.3.2. Ischemic Heart Disease Risk Factors
It has been demonstrated that ischemic heart disease risk factors associate with low antiplatelet response to clopidogrel therapy [
112] (
Figure 2).
Sociodemographic Characteristics
Age (>65 years) and body mass index (BMI) are reported to account for approximately 20% of HTPR [
26,
148], and may be independent risk factors for reduced antiplatelet response to clopidogrel [
112]. Individuals with effective response to clopidogrel have a BMI that is significantly less than the BMI for individuals with reduced antiplatelet response to clopidogrel therapy [
149]. Some authors suggest weight-adjusting clopidogrel dosing [
112].
Lifestyle Habits
Lifestyle habits such as diet, caffeine, and smoking can all increase platelet inhibition in response to clopidogrel, while grapefruit can interfere with platelet inhibition [
11,
150,
151,
152]. These effects are thought to be mediated by interaction with the CYP450 enzymes.
Comorbid Conditions
Type 2 Diabetes Mellitus (DM2) appears to be an independent modulator of platelet response to clopidogrel [
112]. HTPR is noted in patients with DM2 and associates with poor therapeutic outcomes [
153,
154], which may be overcome by administering a higher maintenance dose of clopidogrel such as 150 mg [
11,
155]. Some authors suggest BMI and DM2 status as more reliably predictive of HTPR and poor prognosis following PCI than other risk factors or genetic variant status alone [
112]. In fact, it has been suggested that the poor metabolizer phenotype, BMI, and DM2 be combined as a more credible triple predictor of resistance to platelet inhibition by clopidogrel, and that these three can synergistically interfere with clopidogrel’s antiplatelet activity to incite major adverse cardiovascular events after PCI [
112].
Individuals with chronic kidney disease (CKD) also demonstrate HTPR on clopidogrel [
156] and increased adverse cardiovascular outcomes after PCI, in part related to elevated baseline platelet reactivity [
157,
158,
159]. Furthermore, comorbid DM2 and CKD associate with HTPR on clopidogrel and poor outcomes [
160]. Lipid levels also appear to account for approximately 20% of HTPR on clopidogrel [
26]. Future studies on risk assessment should determine which and how many risk factors could be included in a multimarker predictor of clopidogrel resistance.
5. Multi-Omic Precision Medicine Approach to Response Regulation
Significant progress has been made in the diagnosis, prognosis, and therapeutics of ACS, yet initial and recurrent coronary events continue to plague some individuals especially after PCI. Pharmacogenomics has identified allelic variants in
CYP2C19 and other proteins involved in clopidogrel pharmacodynamics and pharmacokinetics that are associated with recurrent cardiovascular events. However, beyond conventional regulators of platelet response to clopidogrel therapy lie non-traditional regulators as described above that should be considered in precision cardiovascular medicine. Personalized care in precision medicine is not complete after pharmacogenomic testing and application alone. Precision medicine extends beyond genomics into multi-genomic marker models, transcriptomics, epigenomics, exposomics, miRNA regulomics, proteomics, metabolomics, microbiomics, and in silico computational and mathematical modeling, among other precision and systems medicine tools (
Figure 4). Systems medicine applies systems biology to medicine. Systems biology studies whole organisms and networks by focusing not only on the details of each component, but also on how the system works together as a whole to respond to perturbations to produce emergent properties. As a result, a true precision approach to the pharmacogenomics of clopidogrel therapy considers various “omics” and other biotechnological tools resulting from, related to, and regulating pharmacogenomics. Precision cardiovascular medicine therefore integrates genomics with various other components of the human system and accounts for connectivity and interactions within the system as it responds to perturbation (in this case clopidogrel therapy) and develops emergent characteristics (in this case the observed clinical phenotype).
The new frontier in pharmacogenomics is likely to be the multidimensional interface between pharmacogenomics and various other components integrated in precision cardiovascular medicine. These components include not only the biotechnological tools, but also clinical factors unique to each individual, in the context of their environment and the resulting exposome. Undoubtedly the exposures influence each individual’s epigenomics, behaviors, and prognosis, all interacting with other omics.
5.1. Transcriptomics
Transcriptomics looks at the complement of large-scale gene expression profiles at a particular timepoint while the organism is in its current state. In one study, analysis of specific mRNA transcripts in peripheral blood samples from a large community-based cohort identified platelet-derived inflammatory transcripts that associated with BMI; several transcripts were deemed heritable [
161]. This supports associations previously noted between BMI and platelet reactivity [
149]. Transcription can display tremendous variability in gene expression profiles in response to stimuli [
27] (i.e., perturbations such as clopidogrel therapy). These profiles can be evaluated for discovery of grouped gene expression or pathways partially underlying variability in clopidogrel response.
5.2. Epigenomics and Exposomics
Epigenomics involves modification of DNA via physical additions without change in DNA sequence, and includes methylation, acetylation, histone modification, and transcription factor binding, among other modifications. Several studies suggest that environmental characteristics or exposures such as pregnancy, old age, cancer, or congestive heart failure can modify CYP2C19 activity [
162,
163,
164,
165]. Such modifications can contribute to genotype-phenotype discordance [
47]. This could occur via CpG methylation, for example, or by influencing the expression or activity of transcription factors, or post-translational regulation mechanisms [
47]. Physical binding of transcription factors to promotor regions to initiate gene transcription can be affected by methylation of CpG motifs in the promoter region or other location in the gene to be transcribed [
47,
166]. For example, methylation status at three P2Y12 promoter sites (CpG11, CpG12, and CpG13) correlated with HTPR and an increased risk of ischemic events in patients treated with clopidogrel [
167,
168]. Epigenomic modification can silence an allele and result in preferential expression of the complementary allele. This phenomenon, known as allelic expression imbalance, may contribute to CYP2C19 expression levels [
47]. Further, DNA methylation throughout the genome and acetylation of chromatin to modify histone binding can be heritable [
27,
169].
5.3. miRNA Regulomics
MicroRNAs (or miRNAs) are small non-coding RNAs that suppress protein translation by directly binding to mRNA transcripts for the affected protein [
22]. These miRNAs can promiscuously bind various transcripts in related biological networks and pathways, thereby having profound pathophysiological effects that can fine-tune complex biological processes [
27,
170]. Thus, miRNAs can post-transcriptionally regulate CYP2C19 expression, as predicted by in silico modeling and simulation [
171] and confirmed experimentally [
172]. The miRNA combination miR-103/107 binds the 3′UTR of
CYP2C19 at 222–242 bp and 138–152 bp down-stream of a stop codon [
172]. Thus,
CYP2C19 post-transcriptional regulation may contribute to interindividual variability in response to clopidogrel therapy [
47].
5.4. Proteomics
Once mRNA transcripts are translated, the protein products can be measured on a large scale in a proteomics approach. In a study, the proteome of platelets was interrogated 24 h after administration of the clopidogrel loading dose [
173]. Higher levels of the platelet adhesion molecule cluster differentiation-226 (CD226) and transferrin, and lower levels of peroxiredoxin-4, associated with HTPR on clopidogrel [
173]. Since proteins also may undergo chemical modifications such as phosphorylation, nitrosylation, or acetylation, to regulate structure and function, the use of proteomics also aims to identify such chemical modifications associated with some perturbation of the studied system [
27].
5.5. Metabolomics and Microbiomics
Metabolomics studies the small molecule compounds (often active metabolites) that result from biotransformation of prodrugs like clopidogrel. An organism’s metabolome varies with perturbation of the system by environmental factors, drugs, or other modalities [
174]. The metabolomic analysis can yield a metabolic fingerprint or metabotype that discriminates an individual’s response to the perturbation, such as association of metabolic biomarkers of drug response with variable platelet reactivity on clopidogrel therapy [
11,
175]. Thus, metabolomics or pharmacometabolomics can partner with pharmacogenomics to potentially predict cardiovascular outcomes on clopidogrel therapy [
11]. In the same way, microbiomics can also partner with pharmacogenomics. A poignant example involves trimethylamine
N-oxide (TMAO), which is a small and colorless amine oxide produced by microbial metabolism [
176]. The metabolite TMAO is derived from dietary phosphatidylcholine, and also from carnitine and betaine, by gut microbes [
176,
177]. Studies have now linked high levels of TMAO with platelet hyperactivity and incident thrombosis [
178], and consequently increased risk for cardiovascular events [
177]. Even after adjusting for conventional cardiovascular risk factors and risk biomarkers such as high-sensitivity C-reactive protein, TMAO levels predict all-cause mortality risk [
179]. Notably, administration of aspirin appears to attenuate the effect of TMAO on platelets [
179], likely due to observed effects of aspirin on composition of the gut microbiota [
180]. Conversely, high levels of TMAO may antagonize the antiplatelet activity of aspirin, as well as clopidogrel [
179]. This raises an important issue for both clopidogrel and aspirin in DAPT, which is independent of
CYP2C19 variation and can potentially synergize the effect of the LOF
*2 and
*3 alleles or counteract any effect of gain-of-function alleles such as possibly
*17.
5.6. Mathematical, Computational, and Molecular Modeling
Mathematical, computational, and molecular modeling can be used to simulate clopidogrel pharmacodynamics and/or pharmacokinetics in silico. In a dynamic pharmacokinetic model, developed for clopidogrel and its active metabolite clopi-H4, four CYP2C19 metabolic phenotypes are simulated: poor, intermediate, extensive, and ultra-rapid metabolizers [
181]. Each simulated phenotype virtually receives a clopidogrel loading dose of 300 mg, then a maintenance dose of 75 mg. Several approaches were used to validate the model, which accurately predicted pharmacokinetics of clopidogrel and its intermediate and final active metabolite in the four phenotypic metabolism groups [
181]. Another model simulated in silico epigenomics to evaluate and predict the function of binding sites in
CYP2C19 that could potentially regulate antiplatelet response to clopidogrel.
6. Toward Implementation in Clinical Practice
DAPT therapy remains pivotal for patients undergoing PCI [
47]. However, interindividual variability in platelet reactivity and clinical response to clopidogrel lead to therapeutic failure in some patients [
47]. These observations have led to extensive searches on a variety of causes of treatment failure [
22]. Some of the variability is determined by interacting inducer and inhibitor drugs [
182]. Minimal effects on variability are associated with genetic variants in
PON1,
CYP3A4/5,
ABCB1,
P2Y12, and
CES1 [
11,
33,
35,
36,
37,
183].
Clinical and pharmacogenomic (primarily involving
CYP2C19) factors causes have been identified, but these account for only approximately 20% of clopidogrel pharmacokinetics and up to 65% of clopidogrel pharmacodynamics [
108]. Allelic variants alone in
CYP2C19 account for only 12% of clopidogrel variability in platelet reactivity [
26,
184]. Thus, the underlying pathophysiology of a great proportion of the etiology of clopidogrel HTPR has not yet been elucidated [
47].
Pharmacogenomic testing is commercially available and often reimbursable for
CYP2C19 genotyping [
11,
27]. Of note, some commercial target arrays measure only the common SNPs, e.g.,
*2,
*3, and
*17. The absence of these SNPs reflexes the report to wild type, i.e.,
*1. This could be misleading clinically, as other LOF genotypes (however rare) such as
*4–
*7 and
*20–
*21, are misclassified as normal. In the future, next generation sequencing, which would assess the entire
CYP2C19 gene sequence, may be useful to more precisely identify CYP2C19 variants in clinical settings. Nevertheless, most physicians have not begun to order or act on such testing [
11]. Consequently, additional studies would be needed to assess cost-effectiveness and best practice methods for implementation and delivery, likely requiring specialized physician education and utilizing systematic clinical decision support tools [
11], for shared decision-making sessions with patients. Findings from some studies suggest that genotyping
CYP2C19 may be cost-effective for personalization of clopidogrel therapy [
7].
It has been inferred that perhaps patients with
CYP2C19 LOF alleles should be warned about their probabilistic risk for adverse cardiovascular outcomes on clopidogrel following PCI [
32]. If so, a higher dose of clopidogrel has been proposed as an option in attempts to overcome clopidogrel resistance [
76]. However, in the GRAVITAS/GIFT trial, an increased clopidogrel maintenance dose of 150 mg daily (with a loading dose of 600 mg) as compared to 75 mg did not seem to overcome the risk of HTPR in
CYP2C19*2 carriers [
185] (add 2011 GRAVITAS reference also). In the ELEVATE-TIMI 56 trial demonstrated that a higher maintenance clopidogrel dose of 225 or 300 mg significantly reduced the number of
CYP2C19*2 heterozygotes who had HTPR from 52% to 10% (
p < 0.001), but homozygotes remained resistant at a dose as high as 300 mg [
186]. The long-term safety of maintenance clopidogrel doses at 225 or 300 mg (or potentially of loading doses >600 mg) is unknown and cannot yet be safely recommended. Prasugrel and ticagrelor as alternatives to clopidogrel would be reserved for poor and intermediate metabolizers [
7] (
Figure 5). This would be in keeping with guidelines from the Clinical Pharmacogenetics Implementation Consortium (CPIC), which recommend
CYP2C19 genotyping in individuals with ACS undergoing PCI, and alternative antiplatelet therapy with prasugrel or ticagrelor for individuals with 1 or 2
CYP2C19 LOF alleles (i.e., intermediate or poor metabolizers, respectively) [
15,
72]. The CPIC guidelines have been useful for standardization and consistency at various institutions involved in a network collaboration (The Translational Pharmacogenetics Program; TPP) funded by the National Institutes of Health (NIH) Pharmacogenomics Research Network [
69]. The TPP is tasked with developing strategies for real-world dissemination and implementation of pharmacogenomics and assists with interpretation of alleles such as those salient to treatment with clopidogrel.
Determining the
CYP2C19 genotype for each patient could therefore have utility [
32]. Yet, there has been marginal translation of this potential utility into clinical practice [
47]. This is in large part due to insufficient precision in prediction of response to clopidogrel and lack of prospective data demonstrating benefit in treating patients with
CYP2C19 LOF alleles with alternative anti-platelet therapy [
47,
187]. The probable value of routine pharmacogenetic testing in patient on clopidogrel therapy is still uncertain [
25]. Thus,
CYP2C19 genotyping, or phenotypic testing with platelet reactivity or clopidogrel active metabolite concentration, does not yet carry enough evidence to guide therapeutic management [
99].
The United States Food and Drug Administration issued a black box warning regarding the use of clopidogrel [
41]. The statement suggested genotype testing to identify patients who may be
CYP2C19 poor metabolizers, obviating consideration of alternative therapies. However, routine testing is not yet recommended by the American College of Cardiology (ACC), American Heart Association (AHA), or Society for Cardiovascular Angiography and Interventions (SCAI) [
2,
3,
15,
41,
188]. Adequately sized clinical trials would be needed to determine the effectiveness of routine pharmacogenetic testing for clopidogrel therapy [
11]. To address this persistent need, two separate large prospective randomized clinical trials, TAILOR-PCI and the POPular Genetics study are currently ongoing [
15,
117,
189]. Data from such large prospective randomized clinical trials could be incorporated into predictive mechanisms on the horizon. Proposed algorithms (e.g.,
Figure 5), may then shed light on pathways for patients with perplexing combinations of CYP2C19 polymorphism and platelet reactivity. This would apply for patients potentially presenting with genetic variants suggesting poor clopidogrel metabolism but with observation of low platelet reactivity, or patients with variants suggesting rapid clopidogrel metabolism but with observation of high platelet reactivity. The algorithm currently proposes a choice of either genotype testing or platelet reactivity testing, with only one type of test guiding therapy.
Indeed, determining precise methods to predict response to clopidogrel therapy is crucial to guide antiplatelet therapy in the future [
11]. Developing such precision would depend on tailoring treatment based on each individual patient’s genetic and non-genetic clinical information [
190]. Truly personalized care in precision cardiovascular medicine will necessitate integration of genomics, transcriptomics, epigenomics, exposomics (based on exposures), miRNA regulomics, proteomics, metabolomics, microbiomics, mathematical and computational modeling, to prevent, diagnose, prognosticate, and manage disease [
191]. A systems biology approach in precision medicine [
85] will likely be the next frontier of clopidogrel therapy personalization [
11] (
Figure 6).