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Review ArticleThe Personalization of Clopidogrel AntiplateletTherapy The Role of Integrative Pharmacogeneticsand Pharmacometabolomics

ArwaM Amin1 Lim Sheau Chin1 Dzul Azri Mohamed Noor1

Muhamad Ali SK Abdul Kader2 Yuen Kah Hay1 and Baharudin Ibrahim1

1School of Pharmaceutical Sciences Universiti Sains Malaysia Penang Malaysia2Cardiology Department Hospital Pulau Pinang Penang Malaysia

Correspondence should be addressed to Baharudin Ibrahim baharudinibrahimusmmy

Received 3 December 2016 Accepted 14 February 2017 Published 21 March 2017

Academic Editor Terrence D Ruddy

Copyright copy 2017 Arwa M Amin et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Dual antiplatelet therapy of aspirin and clopidogrel is pivotal for patients undergoing percutaneous coronary interventionHowever the variable platelets reactivity response to clopidogrel may lead to outcome failure and recurrence of cardiovascularevents Although many genetic and nongenetic factors are known great portion of clopidogrel variable platelets reactivityremain unexplained which challenges the personalization of clopidogrel therapy Current methods for clopidogrel personalizationinclude CYP2C19 genotyping pharmacokinetics and platelets function testing However these methods lack precise predictionof clopidogrel outcome often leading to insufficient prediction Pharmacometabolomics which is an approach to identifynovel biomarkers of drug response or toxicity in biofluids has been investigated to predict drug response The advantage ofpharmacometabolomics is that it does not only predict the response but also provide extensive information on the metabolicpathways implicated with the response Integrating pharmacogenetics with pharmacometabolomics can give insight on unknowngenetic and nongenetic factors associated with the response This review aimed to review the literature on factors associated withthe variable platelets reactivity response to clopidogrel as well as appraising current methods for the personalization of clopidogreltherapy We also aimed to review the literature on using pharmacometabolomics approach to predict drug response as well asdiscussing the plausibility of using it to predict clopidogrel outcome

1 Introduction

Clopidogrel is a second-generation thienopyridine antiplate-let drugwhich exerts its effect by the inhibition of the plateletrsquospurinergic receptor P2Y12 preventing adenosine diphosphate(ADP) from stimulating it Clopidogrel is crucial drug forpatients enduring high platelets reactivity such as coronaryartery disease (CAD) acute coronary syndrome (ACS) andstroke Some patients may require invasive therapy such aspercutaneous coronary intervention (PCI) with stent placedin the occluded artery to ensure enough blood flow through it[1] PCI patients have to take loading dose of clopidogrel priorto procedure followed by postprocedure dual antiplatelettherapy (DAPT) of low dose aspirin and clopidogrel for dura-tion up to 12monthsbased onstent typeandriskassessment [2]

This DAPT therapy is pivotal to prevent stent thrombosis(ST) and recurrence of ischemic events after PCI Howeversome patients may suffer from attenuated platelets inhibitionto clopidogrel or clopidogrel high on treatment plateletsreactivity (HTPR) which hinders achieving the optimumoutcome of DAPT There are genetic and nongenetic factorscontributing to clopidogrel HTPR however often challeng-ing therapeutic outcome prediction [3] Current methodsof predicting clopidogrel response do not predict clopido-grel therapeutic outcome adequatelyTherefore investigatingnew approaches to assess clopidogrel response can helpto achieve the desired outcome after PCI In this reviewwe aimed to review the literature on clopidogrel variableplatelets reactivity and appraise currentmethods to assess theclopidogrel therapeutic outcomeWe also aimed to review the

HindawiCardiology Research and PracticeVolume 2017 Article ID 8062796 17 pageshttpsdoiorg10115520178062796

2 Cardiology Research and Practice

literature on new approaches such as pharmacometabolomicsand integrative pharmacometabolomics-pharmacogeneticsin assessing clopidogrel therapeutic outcome

2 Clopidogrel Bioactivation andClopidogrel HTPR

Clopidogrel is an oral drug which has oral bioavailability of50 and the maximum peak concentration will be observedwithin 1 to 2 hours after the administration of the loadingdose (600mg) [4 5]The half-life of clopidogrel is from 7 to 8hours [6] Almost 50 of clopidogrel dose is excreted in theurine and 46 in the faeces [7] Of the oral dose approxi-mately 85 is hydrolysed by esterases into inactivemetabolitewhile the remaining 15 will be activated by the hepaticcytochrome P450 (CYP

450) enzymes to the active metabolite

through two steps of bioactivation [8] The hepatic CYP450

enzymes which are involved in the bioactivation process ofclopidogrel include the CYP1A2 CYP2B6 and CYP2C19 inthe first step and the CYP2B6 CYP2C9 CYP3A45 andCYP2C19 in the second step [9ndash11] The CYP2C19 enzymeplays vital role in the two bioactivation steps of clopidogrelby participating with 449 in the first step and 206 in thesecond step [9 12] The CYP3A4 has an essential role in thesecond step by participating with 398 [9] Clopidogrel hasminimum neutropenic side effect compared to ticlopidine(first-generation thienopyridine) [13] The main side effectsof clopidogrel are bleeding gastrointestinal disorders andrash as well as other side effects such as hepatotoxicity andthrombotic thrombocytopenic purpura albeit they are rareTherefore it is well tolerated by patients

Patients variable platelets inhibition while on clopidogrelwas first reported by Jaremo et al in 2002 [14] In thatstudy five out of the eighteen PCI patients had weak plateletsinhibition in response to clopidogrel loading dose of 300mgSince it was first reported clopidogrel HTPR has been largelydocumented It was found to be affecting 15ndash40 of thepatients [4 15 16] Clopidogrel HTPR is associated with pooroutcome after PCI Matetzky and colleagues indicated anassociation between clopidogrel HTPR and the risk of cardiaceventsrsquo recurrence among 60 ACS patients undergoing PCIwho had taken loading dose of 300mg followed by daily doseof 75mg for three months [17] Substantiating these findingsGeisler et al (2006) indicated that the primary end point ofmyocardial infarction stroke and death were significantlyincreased in clopidogrel HTPR patients who were followedup for three months after the PCI [18]

3 Genetic Factors Contributingto Clopidogrel HTPR

There are several identified genetic variabilities which con-tribute to clopidogrel HTPR In fact clopidogrel variableplatelets reactivity is highly heritable [19] As clopidogrelundergoes intestinal absorption bioactivation by the hepaticCYP450

enzymes and deactivation by esterases this processmight be affected by several genetic variabilities Genetic

variabilities which may interfere with clopidogrel vari-able platelets reactivity include the polymorphisms of theCYP2C19 [19] theCYP3A45 [20] theCYP2C9 [21] the ATP-Binding Cassette Subfamily B Member 1 (ABCB1) [22] theParaoxonase-1 (PON1) the Carboxyl Esterase 1 (CES1) [23]and the genetic polymorphism of the P2Y12 receptors [24]

There are 57 active genes of the CYP450

enzymes and58 pseudogenes in the human genome [25] For most ofthe clinically relevant CYP

450enzymes they are highly

affected by genetic polymorphisms [26] The CYP450

geneticpolymorphisms could interfere with gene transcription geneexpression regulation protein translation and affinity tosubstrate [26 27] Among the CYP

450reported genetic poly-

morphisms the CYP2D6 CYP2C9 and the CYP2C19 were-extensively studied and reported respectively [28] Of allstudied CYP

450genetic polymorphisms the CYP2C19 poly-

morphisms were found to be highly associated with clopido-grel variable response particularly that the enzyme is invol-ved in the two bioactivation steps

31 CYP2C19 Polymorphisms The CYP2C19 enzyme playssubstantial role in the metabolism and bioactivation ofdrugs such as proton pump inhibitors (PPIs) (omeprazolelansoprazole and pantoprazole) selective serotonin reuptakeinhibitors (citalopram and sertraline) tricyclic antidepres-sant (imipramine and amitriptyline) phenytoin and clopido-grel The CYP2C19 enzyme is known by its classical reactionthe (S)-mephenytoin 41015840-hydroxylation [29] The gene of theCYP2C19 is highly polymorphic with more than 25 alleleshaving variable enzymatic activity levels [30] The star oneallele (lowast1) is the normal or ldquowild typerdquo a homozygous formof this allelersquos (lowast1lowast1) phenotypes for the gained full functionof the enzyme Of all polymorphic alleles of the CYP2C19two reduced functions alleles were identified as (lowast2) and(lowast3) alleles have been extensively studied De Morais et al(1994) had found that they cause poor metabolism of (S)-mephenytoin metabolism among Japanese [29] Both theCYP2C19lowast2 and CYP2C19lowast3 alleles are single nucleotidepolymorphism (SNP) of guanine (G) to adenine (A) presentat nucleotide 681 in exon 5 and at nucleotide 636 in exon4 respectively [29 31] The CYP2C19lowast2 causes aberrantsplice site leading to the formation of stop codon whichproduces truncated nonfunctional protein which is cat-alytically inactive [29] The CYP2C19lowast3 causes prematuretermination codonwhich leads to the formation of truncatednonfunctioning protein which is also catalytically inactive[31] The CYP2C19lowast17 is another allele of the CYP2C19which is associated with increased gene transcription thatleads to increased activity of the enzyme [32] Clinicallyindividuals are classified into extensive metabolizers (EM)intermediate metabolizers (IM) poor metabolizers (PM)and ultrametabolizer (UM) of drugs based on their CYP2C19genotype [10] Table 1 shows subjectsrsquo CYP2C19 genotypeand their CYP2C19 phenotype The presence of at least oneallele of the CYP2C19lowast2 is observed in 15ndash30 33ndash40and 40ndash50 of the Caucasians African Americans andAsians respectively [33ndash35] However the CYP2C19lowast3 alleleis present in less than 1 of both Caucasians and AfricanAmericans and 7 of Asians

Cardiology Research and Practice 3

Table 1 Phenotype metabolising classes based on CYP2C19genotypelowast

Phenotype Examples ofthe genotype Enzyme activity

Ultrarapid metabolizer(UM)

lowast1lowast17 Normal or increasedenzyme activitylowast17lowast17

Extensive metabolizer(EM) lowast1lowast1 Normal enzyme activity

Intermediate metabolizer(IM)

lowast1lowast2Intermediate enzyme

activitylowast1lowast3lowast2lowast17

Poor metabolizer (PM)lowast2lowast2

Low or absent enzymeactivitylowast3lowast3

lowast2lowast3lowastThis table is adapted and modified with permission from the publisherOriginal source Scott et al (2013) [10] Copyrightcopy (2013) Published byWiley All rights reserved

The importance of the CYP2C19 loss of function (LoF)alleles CYP2C19lowast2 and CYP2C19lowast3 effect on clopidogrelresponse stems not only from their association with clopi-dogrel HTPR but also from their association with poor ther-apeutic outcome [36] Poor metabolizers (based on CYP2C19genotype) had lower plasma concentration of clopidogrelactive metabolite [21] Shuldiner and colleagues had foundsignificant association between the CYP2C19lowast2 genotypeand both the low plateletsrsquo inhibition to clopidogrel and theincrease of cardiovascular events among PCI patients [19]Mega and colleagues reported low level of the active metabo-lite of clopidogrel reduced platelets inhibition to clopidogreland higher risk of ischemic events among patients who arecarriers of theCYP2C19 LoF alleles [34]These results as wellas other findings encouraged the United States (US) FoodandDrug Administration (FDA) to issue labelled warning onclopidogrel box recommending the genotyping of CYP2C19in order to prevent recurrence of cardiovascular events [12]

32 Other Genetic Polymorphisms Apart from the CYP2C19polymorphisms the current literature is consistent aboutthe limited effect of other CYP genetic polymorphisms onthe exposure to clopidogrel active metabolite and plateletsinhibition Nevertheless some controversy exists about somegenetic variants Brandt and colleagues investigated the effectof CYP polymorphisms on the exposure to clopidogrel activemetabolite and platelets reactivity [21] They indicated thatthe presence of the CYP2C19 or the CYP2C9 LoF variantswas associated with reduced exposure to clopidogrel activemetabolite and decreased platelets inhibition However theCYP1A2 theCYP2B6 theCYP3A4 andCYP3A5 did not haveany effect Park and colleagues indicated similar findingshowever they did not investigate the effect of CYP2C9 vari-ants [37] Although several studies showed that the CYP2B6polymorphism did not have an effect a study indicated thatcarriers of the CYP2B6 had lower exposure to clopidogrelactive metabolite and lower platelets inhibition comparedto noncarriers [34] Although several studies suggested

that the association between the CYP3A4 and the CYP3A5polymorphisms and clopidogrel HTPR is not significant[34 37ndash39] others indicated significant association betweenthem [11 20 40] This might be due to the major role ofthe CYP3A4 and CYP3A5 enzymes in the second step ofclopidogrel bioactivation whichmay substantiate the effect oftheir genetic polymorphisms on clopidogrel exposure

TheABCB1 gene is the coding gene for the P-glycoproteinmultidrug resistance-1 (MDR1) intestinal transporter a mod-ulator of clopidogrel absorption [41] A genetic polymor-phism of theABCB1 causes reduced absorption of clopidogrel[22] Simon et al (2009) found that ACS patients who weretreated with clopidogrel and were carriers of the two variantsalleles of the ABCB1 had higher rate of cardiovascular eventsat one year of follow-up [35] Similarly Mega et al (2010)indicated that the ABCB1 polymorphism is significantlyassociated with the risk of deaths due to cardiovascularevents or stroke among ACS patients treated with clopidogrelin the ldquoTrial to Assess Improvement in the TherapeuticOutcomes by Optimizing Platelet Inhibition with Prasugrel-Thrombolysis in Myocardial Infarctionrdquo (TRITON-TIMI 38)trial [42] However other studies failed to indicate the samefindings [19 41]

As the majority of clopidogrel dose is being metabolizedby the CES1 enzyme a reduced function genetic polymor-phism of this enzyme can lead to variable response to clopi-dogrel [23 43] Zhu et al (2013) found that the inhibition ofCES1 enzyme and the CES1 reduced function genetic poly-morphisms are associated with the reduced hydrolyticmetabolism of clopidogrel and the increased concentrationsof clopidogrel active metabolite in an in vitro model [43]Lewis et al (2013) studied the effect of the CSE1 reducedfunction genetic variant on clopidogrel exposure and plateletsinhibition among 566 healthy volunteers from the AmishldquoPharmacogenomics of Anti-Platelet Interventionrdquo (PAPI)study The study revealed that the level of clopidogrel activemetabolite is significantly higher among carriers of thereduced function allele [23] In addition the platelets inhi-bition measured by an ADP-stimulated platelet aggregationwas higher in those whowere carriers of the reduced functionallele Such findings suggest that the CES1 reduced functiongenetic polymorphisms may play vital role in the variableplatelets inhibitory effect of clopidogrel Thus it would bevaluable for future investigations to test this role on patientsrsquooutcome

The PON1 is an esterase enzyme which is synthesized inthe liver [44]The PON1 full enzymatic activity is associatedwith atheroprotective effect which can be due to its role inenhancing an increased level of the high-density-lipoprotein(HDL) [45ndash47] Therefore patients who were carriers of thePON1 lower activity genetic polymorphism were found tobe having an increased oxidative stress and higher risk ofcardiovascular events [45] A presumed role of the PON1enzyme in the metabolism of clopidogrel and its responsive-ness stemmed controversy [46] Bouman et al (2011) usedan in vitro metabolomics profiling which indicated that thePON1 is imperative enzyme in the hydrolytic cleavage of theintermediatemetabolite of clopidogrel (2-oxo-clopidogrel) tothe active metabolite of clopidogrel [44] Accordingly the


PON1 genetic variant of lower enzymatic activity causes lowerlevel of clopidogrel active metabolite Based on their find-ings they conducted a case-cohort study on CAD patientsundergoing PCI and a further prospective replication studyon another independent sample of 1982 patients with ACS[44] Both the case-cohort and the prospective replicationstudies concluded that there was an association betweenthe genetic variant of PON1 and the pharmacokinetics thepharmacodynamics and therapeutic outcome of clopidogrel[44] These findings were challenged by other studies whichindicated that this association is not significant [46 47] Infact the effect of PON1 genetic variant on the therapeuticoutcome was attributed to the association between thisvariant and the risk of developing cardiac events [46 47]

The data on the effect of the P2Y12 genetic variants onclopidogrel response is dialectical Some studies supportedan effect of the P2Y12 polymorphism on platelets activationand clopidogrel response [24 48 49] while others refutedthis effect [50 51] or indicated a synergistic effect of it ifcoexisted with other genetic variabilities such as CYP2C19and MDR1 [52 53] There are two haplotypes of the P2Y12gene (H1 and H2) where the H2 represents a gain-of-function haplotype of the receptor which might cause anincrease in atherothrombosis and interfere with the responseto clopidogrel [48] The homozygous haplotype of H2 wasfound to be associated with clopidogrel HTPR [24]

Although the data are conflicting it could be inferredfrom current literature that except for the CYP2C19 mostof the investigated genetic polymorphisms have weak orinsignificant effect on the clopidogrel exposure and plateletsinhibition However the coexistence of polymorphisms mayhave significant effect on platelets inhibition In fact thepossession of the CYP2C19 polymorphism and other poly-morphisms can lead to synergistic effect For instance Megaand colleagues concluded that PCI patients who have bothABCB1 and CYP2C19 genetic variants may be vulnerable tothe recurrence of cardiac events while they are on clopidogreltreatment [42] Similarly some studies indicated that coexis-tence ofCYP2C19 andP2Y12 receptor genetic polymorphismshas higher effect on clopidogrel responsiveness and theclinical outcome than single polymorphism [52 53] Fur-thermore the use of a drug with enzymatic inhibitory effectin patients who have polymorphism of reduced enzymaticactivity for the same enzyme may lower platelets inhibitionto clopidogrel Park et al (2013) indicated that patients oncalcium channel blockers (CCBs) who were carriers of theCYP3A4 (IVS 10 + 12) A allele (GA and AA) had higherplatelets reactivity while on clopidogrel compared to thenoncarriers-wilt type (GG) [54]

4 Nongenetic Factors Contributingto Clopidogrel HTPR

Other multiple nongenetic factors interfere with clopidogrelresponse Clinical factors such as concomitant diseases drug-drug interaction patientrsquos compliance obesity and age areknown to influence clopidogrel response In addition lifestylefactors such as smoking and diet can also affect the response

The presence of comorbid condition such as type 2 dia-betes mellitus (T2-DM) increases platelets reactivity in CADpatients which may cause poor therapeutic outcome [55]In fact T2-DM patients were found to endure clopidogrelHTPR while on DAPT [56] However the interindividualclopidogrel variable platelets reactivity among CAD patientswith T2-DM might be due to the concomitance of geneticor nongenetic factors [55 57] It had been suggested thatthe administration of high maintenance dose of clopidogrelor more potent P2Y12 antagonists such as prasugrel can beused to overcome the clopidogrel HTPR in T2-DM [58]Similar to T2-DM patients suffering from chronic kidneydisease (CKD) had reduced platelets inhibition to clopidogrel[59] The decreased platelets inhibition in CKD patients wasindicated for both clopidogrel and aspirin in patients onDAPT [60] This reduced response is suggested to be due tothe increased baseline platelets reactivity inCKDpatients [6061] The association between renal insufficiency and the riskof death and cardiac events after PCI is well established [62]However inconsistent data is available in terms of the risk ofbleeding among PCI patients with underlying CKD [63 64]Noteworthily the concomitance of comorbidities such as T2-DMwithmoderate to severe CKDwas found to be associatedwith high platelets reactivity inCADpatients onDAPTwhichmight lead to high frequency of poor outcome in this groupof patients [65] Recently in Japanese population it has beenfound that CKD is an independent predictor of PCI outcomeregardless of theCYP2C19 genetic polymorphism status of thepatient [66]

As clopidogrel is bioactivated by the CYP450

enzymes itcan be prone to drug-drug interactions particularly drugsinterfering with the CYP

450systemThe PPIs are metabolized

by the CYP450

enzymes therefore they might interact withclopidogrel [67] However the data on the extent of PPIsinteract with clopidogrel remain controversial which suggestsfurther investigation In fact the PPIs inhibit the CYP2C19enzyme which might reduce the bioactivation of clopidogrelto its active metabolite [67 68] Accordingly this coulddecrease the antiplatelet effect of clopidogrel which mayincrease cardiovascular events [68]ThePPIsmaynot interactto the same extentwith clopidogrel [69] In otherwords somestudies concluded that omeprazole was associated with thereduced antiplatelet effect of clopidogrel while esomeprazoleand pantoprazole were not [68 69] Similarly lipophilicstatins antihyperlipidemic drugs such as atorvastatin andsimvastatin were reported to inhibit the antiplatelet effect ofclopidogrel [70ndash72] These statins interfere with the CYP3A4enzyme [71] Thus it can mitigate the antiplatelet effect ofclopidogrel However the preventive effect of statins by red-ucing lipid levels can halt cardiovascular events which mighthappen consequently to the reduced antiplatelet effect ofclopidogrel [70] Similar to the effect of statins albeit withsome controversy in the literature some drugs such assome CCBs erythromycin troleandomycin and ketocona-zole inhibit the CYP3A4 enzyme which will reduce the levelof the clopidogrel activemetabolite which can lead to reducedantiplatelet effect of clopidogrel [68 73] However the datafrom some studies did not support this suggestion [74 75]For instance the ldquoClopidogrel for the Reduction of Events


During Observationrdquo (CREDO) trial indicated that the con-comitance use of the CCBs does not reduce the efficacy ofclopidogrel and there was no evidence of interaction betweenthem [75] Anticoagulant drugs such as coumarin derivativesmay also interfere with platelets inhibition by clopidogrelSibbing et al (2010) indicated a significant reduction inthe plateletsrsquo inhibitory effect of clopidogrel among patientsconcomitantly taking phenprocoumon when compared tothose who are not taking phenprocoumon This could bebecause of the interference with the capacities of the CYP3A4and the CYP2C9 enzymes which are the main metabolizingenzymes of the phenprocoumon [76] Sulfonylureas oralhypoglycaemic drugs may also reduce platelets inhibitoryeffect of clopidogrel [77]This could be due to the interferencewith CYP2C9 enzyme as well [77 78] On the other handthere are also drugswhich can increase or decrease the level ofclopidogrel activemetabolite by the inhibition or induction ofthe CES1 enzyme [43]Thismight lead to clopidogrel variableplatelets reactivity [79] For instance the phenobarbital thedexamethasone and the polycyclic aromatic hydrocarbonsare drugswhich can interferewith the activity of CES1 leadingto variable levels of clopidogrel active metabolite [79]

Patientrsquos compliance is crucial for achieving optimumtherapeutic outcome It has been found that noncompliancecontributes significantly to clopidogrel HTPR [80] Age lipidlevels and body mass index (BMI) have been found to definenearly 22 of clopidogrel HTPR [19] An age of more than 65years old is associated with reduced platelets inhibitory effectof clopidogrel [81] Furthermore food drinks and smok-ing may interfere with clopidogrel response For examplegrapefruit can lower platelets inhibitory effect of clopidogrelwhich could be due to the interference with CYP

450isoforms

[82] In contrast caffeinated drinks can increase plateletsrsquoinhibitory effect of clopidogrel [83] Similarly smoking wasfound to increase plateletsrsquo inhibitory effect of clopidogrel[84] Smoking increases the bioactivation of clopidogrel toits active metabolite by the induction of the CYP1A2 andthe CYP2B6 enzymes which are involved in the first step ofthe bioactivation of clopidogrel This will increase plateletsinhibition effect of clopidogrel

5 Personalization of Clopidogrel Therapy

It could be clearly understood that there are multifactorialgenetic and nongenetic factors which interfere with clopi-dogrel variable platelets reactivity as depicted in Figure 1[85] However these known factors failed to explain greatportion of the variable platelets reactivity of clopidogrel [86]For instance Frelinger et al (2013) assessed the pharma-cokinetics and the pharmacodynamics of 75mg daily doseof clopidogrel administered by 160 healthy subjects for 9days The 160 subjects were free from nicotine for 6 weeksprescription drugs for 4 weeks over-the-counter drugs for 2weeks and caffeine and alcohol for 72 hours All participantswere homogenous CYP2C19 EM genotype (lowast1lowast1) Theywere genotyped for the ABCB1 PON1 and CYP3A5 poly-morphisms Of all participants 45 had clopidogrel HTPRDespite controlling the aforementioned factors altogetherfactors such as age weight sex platelet count haematocrit

and genetic polymorphisms of ABCB1 PON1 and CYP3A5explained only 18 of the clopidogrel active metabolite peakplasma concentration (119862max) and the area under the plasmaconcentration-time curve (AUC

119905) variabilities When 119862max

and AUC119905were added to these factors they explained 48

of pharmacodynamics variability Noteworthily the resultsshowed that there was no significant association betweenthe polymorphisms of ABCB1 PON1 and CYP3A5 and thepharmacokinetics and pharmacodynamics of clopidogrelThese findings indicated the burden of the unexplainedvariability of clopidogrel response which consequently affectsthe therapeutic outcome of patients This unsolved mysterycan be due to unknown genetic polymorphisms or an inter-action between several genetic polymorphisms and othernongenetic factors

Personalized therapy is the use of personrsquos geneticsproteomics and environmental information to prevent diag-nose and treat disease [87] It aims to tailor patients therapybased on their genetic and nongenetic information ratherthan treating the whole population of a disease with thesame treatment [88] Since the discovery of clopidogrelvariable platelets reactivity it has been challenging to achieveoptimum personalization of DAPT therapy However thisoptimum antiplatelet therapy could not be achieved withoutperfect prediction of the therapeutic outcome

6 Adoption of PharmacogeneticsBiomarkers in Clinical Practice

The biomarker is a biological indicator of disease physi-ological state clinical status response to drug therapy orpathogenic process which can be estimated and appraised forits indicative accuracy [89] Accordingly genetic variabilitywhich is associated with a biological status can be used asan indicative biomarker of that status Pharmacogeneticsbiomarkers have been used to predict drug therapeutic out-come and avoid adverse drug reaction (ADR) prior to druguse In 2008 the FDA issued table of valid pharmacogeneticsbiomarkers which contains list of drugs that had FDA labelwarning of pharmacogenetics testing prior to drug use andthis list is frequently updated [90] As there are several geneticfactors whichmay interfere with clopidogrel variable plateletsreactivity genotyping of these polymorphisms was evaluatedfor clopidogrel outcome prediction [19 34] Apparently theliterature was consistent in this regard The CYP2C19 poly-morphism predominates the effect of other genetic variantsThus the FDA considered the CYP2C19 polymorphism validpharmacogenetics biomarker of clopidogrel efficacy [90]

Although there are consistent literature asserting theassociation between clopidogrel HTPR and the CYP2C19lowast2and lowast3 LoF alleles in depth analysis of the data indicatedthat this association is strong in the PM who are carriers ofthe homozygous genotypes of the CYP2C19 (lowast2lowast2lowast3lowast3)but not for the same extent with the IM who are carriers ofthe heterozygous genotypes (lowast1lowast2lowast1lowast3) [4 91ndash93] Fur-thermore patientswho are EMbut suffering from clopidogrelHTPRwould bemisclassified as responsive (having optimumclopidogrel platelets inhibition) based on their CYP2C19genotype [94] Nasyuhana Sani and colleagues studied the


Clopidogrel variable platelets reactivity

Clinical factors(i) Poor compliance

(ii) Underdosing(iii) Poor absorption(iv) Drug-drug interaction(v) Acute coronary syndrome(vi) Diabetes mellitusinsulin

resistance(vii) Elevated body mass Index

(viii) Smoking

Genetic factors(i) Polymorphism of

(a) CYP isoforms

(d)GPIIBIIIa(e) CES1(f) PON1

(b) P2Y12 receptor(c) ABCB1

Cellular factors(i) Accelerated platelet turnover

(ii) Reduced CYP metabolic activity(iii) Increased ADP exposure(iv) Upregulation of

(c) P2Y-independent pathways (collagen epinephrine thromboxane A2 thrombin)

(a) P2Y12 pathway(b) P2Y1 pathway

Figure 1 The genetic and nongenetic factors which may contribute to clopidogrel variable platelets reactivity The figure presents the knowngenetic and nongenetic factors (cellular and clinical factors) whichmay contribute to clopidogrel variable platelets reactivityCYP cytochromeP450 ADP adenosine diphosphate GPIIBIIIa Glycoprotein IIBIIIa ABCB1 ATP-Binding Cassette Subfamily B Member 1 This figure isadapted and modified with permission from the publisher Original source Angiolillo and Ferreiro (2010) [85] Copyrightcopy (2010) SociedadEspanola de Cardiologıa Published by Elsevier Espana SL All rights reserved

effect ofCYP2C19 genotype on clopidogrel platelets reactivityamong healthy volunteers from three East Asian ethnicities[92] They had found that although carriers of one or twoalleles of the CYP2C19lowast2 had significantly low plateletsinhibition while on clopidogrel low platelets inhibition wasfound in wild type homozygous carriers CYP2C19 (lowast1lowast1) aswell Similarly Mejin and colleagues had found that therewas no significant association between clopidogrel HTPRand CYP2C19 genotype among Malaysian CAD patients[93] A systematic review and meta-analysis had concludedthat with the exception of stent thrombosis the CYP2C19genotype is not significantly associated with cardiovascularevents despite its association with platelets aggregation [95]Simply put this can be merely justified by several factsThe CYP2C19lowast2 allele contributes to 12 of clopidogrelvariable platelets reactivity which means great portion ofthis variability remains unpredicted by the genotype [19 96]The total activity of the CYP2C19 enzyme is affected notonly by the genotype but also by the interacting inducer andinhibitor drugs [97] In other words some EM subjects mayexperience poor metabolizing activity of the CYP2C19 dueto concomitant administration of inhibitor drugs and viceversa the PM subjects taking concomitant inducer drugsmayexperience normal metabolizing activity of the CYP2C19Besides although it might be with considerably minimaleffect the synergetic effect of other genetic variabilities suchas the PON1 the CYP3A45 the ABCB1 the P2Y12 receptorsand the CES1 cannot be neglected [20 22ndash24 40] Indeedit is expensive and less practical to genotype all the sus-pected clopidogrel related genetic variabilities In additionthe limited role of genetic testing could be due to the factthat most of genetic mutations have inadequate predictionof the outcome [98] As early mentioned in the study ofFrelinger et al (2013) the known nongenetic and genetic

factors explained 18 of the clopidogrel pharmacokinetics(PK) and 32ndash65 of clopidogrel pharmacodynamics (PD)This means great portion of clopidogrel HTPR cannot beidentified by CYP2C19 genotyping Therefore the ClinicalPharmacogenetics Implementation Consortium Guidelinesfor CYP2C19 genotype and Clopidogrel Therapy (CPIC) inits update (2013) did not recommend the CYP2C19 guidedtherapy for all patients [10]

7 Pharmacokinetics Assessmentof Clopidogrel Response

Several studies had evaluated different pharmacokinetics(PK)methods to determine the level of the parent compoundclopidogrel and its metabolites as an indicator of clopidogrelplatelets inhibition [5 99 100] It was found that the level ofclopidogrel active metabolite is correlated with clopidogrelplatelets inhibition [86 101] and the PK of clopidogrelcould be predicted by the CYP2C19 genotype [102] Howeverapplying PK to assess clopidogrel response is limited byseveral factors The active thiol metabolite of clopidogrel isnot stable which requires adding stabilizing agent within 30seconds of blood sampling [101] Some studies used indirectquantification of the active metabolite by quantifying theparent compound and the inactive metabolite [99 103]This means an accurate quantification of the active thiolmetabolite in blood is difficult Furthermore the plasma levelof the active metabolite of clopidogrel may not reflect theresponse in subjects affected by P2Y12 receptor polymor-phism receptor density fibrinogen platelets concentrationand accelerated turnover of platelets variabilities [86 104]Therefore the PK assessment can estimate the level of theparent compound and the active metabolite but it may notbe optimum method to personalize clopidogrel antiplatelet


therapy unless it is done together with other approaches suchas pharmacogenetics or platelets function testing (PFT) toprovide further information that can help to individualize thetherapy

8 Implementation of PlateletsFunction Testing (PFT) to PersonalizeClopidogrel Therapy

Due to the limited prediction power of the pharmacogeneticsbiomarkers of clopidogrel platelets inhibition the suggestionto personalize antiplatelet therapy based on platelets functiontesting (PFT) gained attraction This had increased thedemand to evaluate the use of PFT assays in the assessment ofclopidogrel therapeutic outcome PFT is one of the importantmethods to assess the effect of antiplatelet drugs and plateletsdefectsThe PFT assessment of antiplatelet drugs relies on themeasurement of platelets inhibition which happened due tothe effect of the drug There are different PFT instrumentswhich are widely used to assess antiplatelet drugs such asthe optical light transmission aggregometry (LTA) [105]the VerifyNow system (VN) (Accumetrics Inc San DiegoCalifornia) [106] the vasodilator stimulated phosphoproteinphosphorylation assay (VASP-P) [107 108] the multipleelectrode platelet aggregometry (MEA) [105] the plateletsfunction analyser (PFA-100) [109ndash111] the Impact-R assay[110] and the platelet-works (PW) [110] The PFT instru-ments have different techniques agonist reagents and testingkits to test different classes of antiplatelet drugs [105 109]ThePFT agonist reagent induces platelets activation in the samplethrough stimulation of the drug targetThe aggregation of theplatelets will be measured by the instrument and it indicatesthe status of platelets activity after the effect of the drugTable 2 compares some of the available PFT instrumentsFor each PFT instrument there is a specified kit or agonistreagent to test clopidogrel response it mainly contains ADPas stimulant of P2Y12 receptors The LTA is the gold stan-dard PFT assay [109] However the VASP-P assay is highlyspecific to P2Y12 receptor Therefore it is often consideredthe gold standard PFT assay for P2Y12 inhibitors Severalstudies evaluated the ability of PFT assays to predict majorcardiac events after PCI [112 113] However the ldquoDo PlateletFunction Assays Predict Clinical Outcome in Clopidogrel-Pretreated PatientsUndergoing Elective PCIrdquo (Popular) studyhad compared the predictability of five different PFT assays[114]The PFT assays were the LTA VN IMPACT-R PW andPFA-100The study concluded that LTA VN and PW are thebest PFT assessment predictors of atherothrombotic eventsincluding stent thrombosis despite the limitation of lowpower in predicting bleeding risk Generally the use of PFT totailor clopidogrel therapy has its limitations The researchersfrom the ldquoDouble Randomization of a Monitoring AdjustedAntiplatelet Treatment versus a Common Antiplatelet Treat-ment for DES Implantation and Interruption versus Con-tinuation of Double AntiplateletTherapyrdquo (ARCTIC) clinicaltrial had concluded that tailoring antiplatelet therapy basedonPFTdid not improve outcomes [115]This can be explainedby several confounding factors For many of PFT assays

the results are interfered by age gender ethnicities dietplatelets count and haematocrit which lowers their accuracy[109 110 116ndash118] The results of PFT instruments whichutilize platelets aggregation technique for measurement arehighly affected by concomitant use of other antiplatelet drugssuch as GPIIb-IIIa antagonists [119] In fact the simulationnature of PFT by using agonist in an in vitro setting tostimulate platelets activation does not perfectly resemble thephysiological process of platelets activation which is affectedby many internal pathways [108] This may lead to PFTresults that do not reflect the actual platelets function of thepatients Although the LTAandVASP-P assays are consideredgold standard methods to measure platelets function theyare not advocated in clinical practice because they are timeconsuming and require lab facility and expert handling [120]On the other hand the VN-P2Y12 assay and other automatedpoint of care (POC) PFT assays are fast and easy but theyare considerably expensive which limits the routine use ofthem Platelets reactivity changes overtime in clopidogreltreated patients which necessitates frequent measurementsfor precise PFT based personalized therapy [121] Moreoverthere is inconsistency in the identification of clopidogrelresponsiveness when the prespecified cut-off points of PFTassays were compared In a study aimed to evaluate theagreement on clopidogrel HTPR identification between threePFT assays the LTA the VASP-P and the VN-P2Y12 areduced agreement was reported between the three assaysHowever when the PFT results were tested for correlationas continuous variables there was correlation between theresults [122] This implied that identifying clopidogrel HTPRbased on one PFT assay is not enough and may lead toimperfect therapeutic decision This might be one of thefactors that hinders tailoring antiplatelet therapy based onPFT

9 The Role of Systems Biology inPersonalized Medicine

Systems biology is the study of the biochemical molecularand supramolecular networks along with their connectionsand interactions with the environmental factors in orderto investigate an intricate biological perturbation of theliving organism [123] This term is an integrative approachwhich investigates not only the biological ldquoOmicsrdquo such asgenomics transcriptomics proteomics and metabolomicsbut also the environmental factors such as nutrition gutmicrobiota infections other drugs disease state rest andsleep which interfere with them In fact the conventionalbiology describes the biological processes in the living sys-tem without considering the connections and interactionsbetween them and their environment however systemsbiology is comprehensive in terms of explaining the biologicalprocesses their pathways networks and other environmentalfactors which all together contribute to a phenotype such asdisease or clinical trait [124 125] This will give an extensiveunderstanding of that phenotype by using the multidisci-plinary ldquoOmicsrdquo data as biomarkers of the phenotype [125]While the study of the genes and their association with thephenotype is the main interest of genomics the study of


Table 2 Platelets function testing devices

PFT device Specimen PFT technique Laboratory requirementpoint ofcare (POC)

AgonistreagentsCartridges

Light transmissionaggregometry (LTA) [105]

Platelet richplasma (PRP)

Optical light transmissionaggregometry

Requiring trained lab staff andlaboratory facility to do the test

ADP arachidonicacid epinephrine

Multiple electrode plateletaggregometry (MEA) [105] Whole blood Impedance aggregometry Semiautomated ADP arachidonic

acid collagen

VerifyNow system (VN)[106] Whole blood Optical light transmission

aggregometry Fully automated POCP2Y12 kitAspirin kit

GP IIbIIIa kit

Platelets function analyser(PFA-100) [109ndash111] Whole blood High shear force dynamic flow

system Semiautomated POCCollagen with ADP

Collagen withepinephrine

Impact-R [110] Whole blood Shear stress platelet adhesion

Requiring trained lab staff andlaboratory facility to do the testExtensive sample handling isneeded

ADP

Vasodilator-stimulatedphosphoproteinphosphorylation assayusing flow cytometry(VASP-P) [107 108]

Whole bloodFlow cytometric fluorescentquantification of intraplatelet

VASP

Requiring trained lab staffintensive lab work samplepreparation

VASP-P2Y12 assay

Platelet works (PW) [110] Whole blood Single platelet count Semiautomated ADPPOC point of care ADP adenosine diphosphate GP IIbIIIa glycoprotein IIbIIIa and VASP-P vasodilator-stimulated phosphoprotein phosphorylationP2Y12 kit VerifyNow kit designed to test the P2Y12 receptor blockade to assess the response to P2Y12 inhibitors Aspirin kit VerifyNow kit designed to test theresponse to aspirin via arachidonic acid initiated reaction GP IIbIIIa kit VerifyNow kit designed to test the response to GP IIbIIIa inhibitors VASPP2Y12assay VASP phosphorylation correlates with the P2Y12 receptor inhibition

the messenger ribonucleic acid (mRNA) and transcriptionprocess is the aim of transcriptomics [126] The proteomicsfocuses on peptides and proteins expression [127] Eventuallyas all the substrates in the organism will be subjected tometabolism the metabolomics focuses on the study of themetabolites [128] The role of systems biology in the predic-tion of the phenotype and the personalization of therapy isshown in Figure 2

Apart from pharmacogenomics biomarkers which hadbeen extensively studied there is scant data on the use ofother systems biology approaches such as transcriptomicsproteomics and metabolomics to find novel biomarkers ofclopidogrel variable platelets reactivity and cardiovascularoutcome In a proteomics approach Caruso et al 2015were the first to report an investigation of platelets pro-teomes after 24 hours of clopidogrel loading dose in 12ACS patients (6 responders and 6 nonresponders) Theyindicated significant upregulation of the platelet adhesionmolecule cluster differentiation-226 (CD226) downregula-tion of peroxiredoxin-4 and increased expression of trans-ferrin in patients suffering from clopidogrel HTPR [129] Tothe best of our literature review there is no study which hadinvestigated the use of metabolomics to assess clopidogrelresponse

10 Disease and Drug ResponsePhenotyping Using Metabolomics

Metabolomics (metabonomics in some literature) is the studyof the metabolites of the living organismsrsquo metabolome [130]

Themetabolites are the ultimate small molecules compoundswhich are outcomes of the metabolism of the systems biologycomponents and the environmental factors interfering withit [131] The metabolome of each living system varies withthe variations in the systems biology components and thevarious environmental factors affecting it [130] Accordinglythe metabolomes of subjects having particular phenotype(disease condition or drug response) cluster in discrimina-tory way from their controls upon analysing the metabolomeof their biological samples [132] This differentiation usuallyhappens due to the presence of discriminating metabolites inthe metabolome which is termed the metabolic fingerprintor metabotype [133] The identified metabotype can be usedas a biomarker of the phenotype The metabolomics analysisinvolves analysing biological samples such as urine serumplasma exhaled breath sputum cerebrospinal fluids (CSF)and cell culture using spectroscopic instruments such asnuclear magnetic resonance (NMR) or mass spectroscopy(MS) to identify and quantify the metabolites in the sam-ple [134ndash136] Metabolomics approach has been used inseveral studies to find novel biomarkers of diseases Itwas used to identify new biomarkers of asthma chronicobstructive pulmonary disease (COPD) cancer and otherdiseases [135 137 138] The use of metabolomics approachto explore and understand different cardiac conditions hasbeen evolved In an animal model of rats harbouring humanrenin and angiotensinogen genes Mervaala and colleaguesused metabolomics to test whether angiotensin II alters themetabolic profile of the heart [139] They concluded thatangiotensin II plays key role in the metabolic profile of the


Patient medical historyand current clinical data

Biomarkers

Personalized medicine

GenomicsDNA

TranscriptomicsRNA

ProteomicsProteins

MetabolomicsMetabolites

Envi

ronm

enta

l fac

tors

Phenotypedisease or drug response

Syste

ms b

iolo

gy

Figure 2 The role of Systems Biology in Personalized Medicine The figure elucidates how all ldquoOmicsrdquo disciplines can provide phenotypebiomarkers (disease or drug response biomarkers) Together with patient medical history and current clinical data these biomarkers can helpto personalize patientrsquos therapyThemetabolomics is the ultimate ldquoOmicsrdquo that is close to the phenotype as well as reflecting the perturbationin other ldquoOmicsrdquo This figure is adapted and modified with permission from the publisher Original source Louridas and Lourida (2012)Published by Hippokratia All rights reserved [125]

heart Tenori and colleagues investigated the use of NMRprofiling of serum and urine to find a metabolic finger printassociated with heart failure (HF) and its relationship withNew York Heart Association (NYHA) functional classes inhumans [140] The NMR analysis of serum and urine sig-nificantly discriminated HF patients from their healthy con-trols with high accuracy (greater than 86) However it wasnot able to discriminate patients among the NYHA classes ofHF

11 Pharmacometabolomics

Pharmacometabolomics or pharmacometabonomics in someliterature is a metabolomics analysis which aims to discovernovel biomarkers in the metabolome that predict drug res-ponse or toxicity [141 142] These novel biomarkers can beused as classifying tool to classify patients to responsiveand nonresponsive to drug or may develop and may notdevelop drug toxicity [143] Drug response metabotype notonly predicts patientrsquos response but also explains responserelated pathways and monitor patientrsquos outcome duringdisease management which will improve the personalizationof therapy [136 143 144] The term was first proposedby Clayton et al 2006 They analysed urine samples ofrat pre-dose and postdose of 600mg of paracetamol usingproton nuclear magnetic resonance (1H-NMR) spectroscopyto find metabotype which is associated with paracetamolinduced hepatotoxicity [145] It was revealed that high levelof predose taurine was associated with low liver damage

assessed by mean histology score (MHS) [145] Howeverpredose low levels of trimethylamine-N-oxide (TMAO) andbetaine were associated with increased paracetamol inducedliver damage A repeat of the study in healthy volunteersindicated that high level of predose urinary p-cresol sul-fate was associated with low postdose urinary ratios ofacetaminophen sulfate to acetaminophen glucoronate (SG)which is due to the competence between acetaminophen andp-cresol on sulfotransferase enzyme [144] Thus the highlevel of endogenous p-cresol causes an increase in liver amen-ability for acetaminophen hepatotoxicity and its urine sulfateform can be used as predose predictive biomarker of it Thepharmacometabolomics studies have been evolved in usingdifferent biofluids samples to find metabotypes of drugefficacy and toxicity [136 146ndash149] In Table 3 the findingsof some pharmacometabolomics studies in humans are sum-marized

111 Pharmacometabolomics Spectroscopic Techniques Thereare many spectroscopic methods commonly used in meta-bolomicspharmacometabolomics studies such as nuclearmagnetic resonance (NMR) or 1H-NMR MS combined withgas or liquid chromatographic extraction (GC LC) andFourier transform infrared spectroscopy (FT-IR) [150ndash152]Among them 1H-NMR and MS are widely used [153] Eachof them has its advantages and limitations The selection ofthe suitable spectroscopic method in a study depends on thefeatures of each method and the compatibility of the methodwith the studyrsquos aim The GC or LC combined MS method is


Table 3 Examples of pharmacometabolomics studies in humans

Study Drug Analytical method Specimen Main findings

Holmes et al 2006[136]

Antipsychoticdrugslowast

1HNMR CSF

A metabotype of schizophrenia which discriminated antipsychoticdrug naive patients from healthy control This metabotype wasalleviated in half of the patients to normal after short term treatmentwith antipsychotic drugs

Kim et al 2010[146]

Cyclosporine A(CsA) amp

Tacrolimus(TAC)

1HNMR Serum

Time dependent metabolites changes in response to differenttreatment The difference in the level of trimethylamine-N-oxide(TMAO) which is associated with graft dysfunction between 2groups of immunosuppressant drugs treatment CsA amp TAC were notsignificant Lipid metabolites were higher in CsA group increasing therisk of cardiovascular diseases

Wang et al 2012[147] Methotrexate 1HNMR Serum

Identification of 11 metabolites which can discriminate betweenmethotrexate efficacy groups in patients with early rheumatoidarthritis

Kaddurah-Daouket al 2013 [148] Sertraline GC-TOF-MS Serum

Discriminating metabotypes of symptoms reduction betweensertraline and placebo in MDD patients on one week and 4 weeks oftreatment Symptoms reduction after one week of sertraline treatmentwas associated with the reduction of 5-methoxytryptamine levelswhile it was associated with lower levels of branched chain aminoacids at four weeks of sertraline treatment

Yerges-Armstronget al 2013 [164] Aspirin GC-MS Serum

Identification of aspirin exposure metabotype of 18 metabolites inhealthy volunteers who were on aspirin 81mg for 14 days (HAPIstudy) Aspirin exposure metabotype was significantly associatedwith purine metabolic pathway Inosine and adenosine weresignificantly higher after aspirin in aspirin HTPR group

Villasenor et al2014 [149] Ketamine LC-QTOF-MS Plasma

Identification of discriminating metabolites between responders andnonresponders of ketamine among bipolar depression patients Thediscriminating metabolites were related to mitochondrial 120573-oxidationof fatty acid

Ellero-Simatos etal 2014 [166] Aspirin LC-MS Serum

Elevated level of baseline serum serotonin is associated with aspirinHTPR based on collagen induced PFT assessment of healthy subjectswho were on aspirin 81mg for 14 days (HAPI study)

1HNMR proton nuclear magnetic resonance GC gas chromatography TOF time of flight MS mass spectroscopy QTOF quadrupole time of flight LCliquid chromatography CSF cerebrospinal fluid CVDs cardiovascular diseases CsA Cyclosporine A TAC Tacrolimus TMAO trimethylamine-N-oxideMDD major depressive disorder HAPI Heredity and Phenotype Intervention and HTPR high on treatment platelets reactivitylowastAntipsychotic drugs atypical antipsychotic drug such as amisulpride clozapine olanzapine risperidone quetiapine and ziprasidone

more sensitive than 1H-NMR thus it is highly recommendedfor metabolomics studies where particular group of com-pounds is being targeted such as lipid compounds [152 154]However as it requires prior separation using LC or GCit is time consuming and necessitates more lab work [145]The 1H-NMR method is fast and does not require extensivesample processing [155] Besides prepared 1H-NMR samplescan be run in large quantities with automated bulk run ofsamples [156 157] Samples prepared for 1H-NMR analysiscan be stored and rerun for further analysis Therefore itis preferred choice for untargeted metabolomics analysis oflarge number of patientsrsquo samples

112 Pharmacometabolomics Data Analysis Pharmacometa-bolomics data analysis relies on prediction modelling usingchemometrics analysis [158]This includes multivariate anal-ysis through the use of unsupervised and supervisedmachinelearning methods [159] The commonly used unsupervised

method inmetabolomics is the principal component analysis(PCA) while the supervised methods include methods suchas partial least square (PLS) discriminant function analysis(DFA) and artificial neural networks (ANNs) The PCAprovides an overview screening for the systematic variationof the data regardless of prior classification Thus it isexploratory analysis and usually followed by the supervisedanalysis where a prior input classification is introduced Theidentification and pathway interpretation of the metabo-lites include combining knowledge in the pathophysiol-ogy of the phenotype with the analytical chemistry of thepotential compound [160] There are available databaseswhich can be used to identify the putative biomarkers inmetabolomics analysis such asHumanMetabolomeDatabase(HMDB) the Chenomx (Chenomx Inc Canada) theMetaboLights the Birmingham Metabolite Library (BML-NMR) and the Biological Magnetic Resonance Data Bank(BMRB-metabolomics)


12 Pharmacometabolomics InformsPharmacogenomics

The ldquopharmacometabolomics informs pharmacogenomicsrdquoapproach implies that pharmacometabolomics can be usedto identify genetic variation which is associated with thevariation of drug response or toxicity [161] Simply put thisconcept is based on the fact that the variation in genes orgenesrsquo expression may lead to variations in proteins andeventually the metabolites levels which are associated withthese pathways will change [162] Accordingly a metabotypewhich is associated with drug response may contain somemetabolites associated with the variations in genes or genesexpression which are implicated in the variable response Jiand colleagues were the first to elucidate this concept on thevariable response to the selective serotonin reuptake inhibitor(SSRI) escitalopram [163] From major depressive disorder(MDD) patients on escitalopram whom were included in theMyo-PGRN SSRI study 20 responders and 20 nonresponderswere recruited for plasma pharmacometabolomics analysisBaseline glycine levels were significantly different (119875 = 0005)between the two groups and had negative associationwith thetreatment outcome (remission) [163]The researchers studiedthe glycine biosynthesis and metabolism to assign SNPswhich are possibly related to these pathways The selectedSNPs were genotyped using the DNA of 512 patients fromthe same study (Myo-PGRN SSRI) Of the 135 assigned SNPsthe rs10975641 SNP from the glycine dehydrogenase genewas associated with response and remission Similarly usingthe same approach novel genetic variability associated withaspirin HTPR was discovered [164 165] Yerges-Armstronget al (2013) identified aspirin response metabotype in serumsamples of healthy volunteers from the Heredity and Pheno-type Intervention (HAPI) Heart study [164] Aspirin HTPRgroup had significantly higher postaspirin levels of purinemetabolites inosine and adenosine Association analysis ofSNPs in the purine genes and aspirin HTPR led to the identi-fication of the rs16931294 SNP in the adenosine kinase (ADK)gene region Aspirin HTPR was significantly associated withthe less common G allele rather than the more common Aallele of the rs16931294 (120573 = 08 119875 = 000034) Furthermorethe G allele was significantly associated with preaspirin levelsof adenosine monophosphate hypoxanthine and xanthineand postaspirin levels of inosine and guanosine Thereforepharmacometabolomics gained informationmay help to dis-cover unknown genetic variations and response metabotypescan be used as an economical tool to predict both theresponse and the possible genetic cause of the response

13 Conclusion

Clopidogrel had been well established as essential antiplatelettherapy paired with aspirin in DAPT therapy Due to its highmargin of safety in terms of bleeding and side effects andits price clopidogrel remains the preferred P2Y12 antagonistsin clinical practice However some patients fail to achievetherapeutic outcome while on clopidogrel Finding precisemethod for predicting clopidogrel outcome is crucial toguide antiplatelet therapy Current methods the CYP2C19

genotyping PK and PFT have their drawbacks Investigat-ing other systems biology approaches such as proteomicsand metabolomics may be the preeminence for clopidogrelpersonalization Pharmacometabolomics approach has beenused in assessing drug response as well as identifyingnovel genetic variations associated with the response As theperturbation in the metabolome is close to the phenotypeit can be highly reflective of drugrsquos variable response Todate pharmacometabolomics had never been evaluated inpersonalizing clopidogrel therapy An integration of phar-macometabolomics and pharmacogenomics may not onlytailor the therapy but also provide better understanding of themystery behind clopidogrel HTPR

Conflicts of Interest

The authors declare that they have no conflicts of interest

References

[1] J M Torpy C Lynm and R M Glass ldquoPercutaneous coronaryinterventionrdquoThe Journal of the American Medical Associationvol 291 no 6 p 778 2004

[2] GN Levine E R Bates J A Bittl et al ldquo2016ACCAHAguide-line focused update on duration of dual antiplatelet therapy inpatients with coronary artery disease a report of the AmericanCollege of CardiologyAmerican Heart Association Task ForceonClinical Practice Guidelinesrdquo Journal of the American Collegeof Cardiology vol 68 no 10 pp 1082ndash1115 2016

[3] S Goswami A Cheng-Lai and J Nawarskas ldquoClopidogrel andgenetic testing is it necessary for everyonerdquo Cardiology inReview vol 20 no 2 pp 96ndash100 2012

[4] A D Oprea and W M Popescu ldquoP2Y12receptor inhibitors in

acute coronary syndromes what is new on the horizonrdquo Car-diology Research and Practice vol 2013 Article ID 195456 15pages 2013

[5] D Taubert A Kastrati S Harlfinger et al ldquoPharmacokineticsof clopidogrel after administration of a high loading doserdquoThrombosis and Haemostasis vol 92 no 2 pp 311ndash316 2004

[6] K Schror ldquoClinical pharmacology of the adenosine dipho-sphate (ADP) receptor antagonist clopidogrelrdquo Vascular Medi-cine vol 3 no 3 pp 247ndash251 1998

[7] R W Shepard ldquoPharmacology antiplatelet and antithrombintherapy in acute coronary syndromesrdquoThe Journal of cardiovas-cular nursing vol 15 no 1 pp 54ndash61 2000

[8] R Shameem M S Hamid A Randhawa C Spaccavento andK Garatt ldquoP2Y12 Antagonists pharmacology efficacy andpatient considerationsrdquo Journal of Cardiovascular Disease vol2 pp 91ndash100 2014

[9] M Kazui Y Nishiya T Ishizuka et al ldquoIdentification of thehuman cytochromeP450 enzymes involved in the two oxidativesteps in the bioactivation of clopidogrel to its pharmacologicallyactivemetaboliterdquoDrugMetabolism andDisposition vol 38 no1 pp 92ndash99 2010

[10] S A Scott K Sangkuhl C M Stein et al ldquoClinical pharma-cogenetics implementation consortiumguidelines forCYP2C19genotype and clopidogrel therapy 2013 updaterdquo Clinical Phar-macology andTherapeutics vol 94 no 3 pp 317ndash323 2013

[11] R Priyadharsini D G Shewade K Subraja et al ldquoSinglenucleotide polymorphism of CYP3A5lowast3 contributes to clopi-dogrel resistance in coronary artery disease patients among


Tamilian populationrdquoMolecular Biology Reports vol 41 no 11pp 7265ndash7271 2014

[12] D R Holmes Jr G J Dehmer S Kaul D Leifer P TOrsquoGara and C M Stein ldquoACCFAHA clopidogrel clinical alertapproaches to the FDA lsquoboxed warningrsquo a report of the Amer-ican College of Cardiology Foundation Task Force on clinicalexpert consensus documents and the American Heart Associ-ation endorsed by the Society for Cardiovascular Angiographyand Interventions and the Society of Thoracic Surgeonsrdquo Jou-rnal of the American College of Cardiology vol 56 no 4 pp321ndash341 2010

[13] S Maseneni M Donzelli A B Taegtmeyer K Brecht and SKrahenbuhl ldquoToxicity of clopidogrel and ticlopidine on humanmyeloid progenitor cells importance of metabolitesrdquo Toxi-cology vol 299 no 2-3 pp 139ndash145 2012

[14] P Jaremo T L Lindahl S G Fransson and A Richter ldquoIndi-vidual variations of platelet inhibition after loading doses ofclopidogrelrdquo Journal of Internal Medicine vol 252 no 3 pp233ndash238 2002

[15] P A Gurbel and U S Tantry ldquoDrug insight clopidogrel nonre-sponsivenessrdquoNature Clinical Practice CardiovascularMedicinevol 3 no 7 pp 387ndash395 2006

[16] P A Gurbel and U S Tantry ldquoClopidogrel resistancerdquoThrom-bosis Research vol 120 no 3 pp 311ndash321 2007

[17] S Matetzky B Shenkman V Guetta et al ldquoClopidogrel resis-tance is associated with increased risk of recurrent athero-thrombotic events in patients with acutemyocardial infarctionrdquoCirculation vol 109 no 25 pp 3171ndash3175 2004

[18] T Geisler H Langer M Wydymus et al ldquoLow response toclopidogrel is associated with cardiovascular outcome aftercoronary stent implantationrdquo European Heart Journal vol 27no 20 pp 2420ndash2425 2006

[19] A R Shuldiner J R OrsquoConnell K P Bliden et al ldquoAssociationof cytochrome P450 2C19 genotype with the antiplatelet effectand clinical efficacy of clopidogrel therapyrdquo The Journal of theAmericanMedical Association vol 302 no 8 pp 849ndash857 2009

[20] J-W Suh B-K Koo S-Y Zhang et al ldquoIncreased risk ofatherothrombotic events associated with cytochrome P4503A5 polymorphism in patients taking clopidogrelrdquo CanadianMedical Association Journal vol 174 no 12 pp 1715ndash1722 2006

[21] J T Brandt S L Close S J Iturria et al ldquoCommon polymor-phisms of CYP2C19 and CYP2C9 affect the pharmacokineticand pharmacodynamic response to clopidogrel but not prasug-relrdquo Journal of Thrombosis and Haemostasis vol 5 no 12 pp2429ndash2436 2007

[22] J Su J Xu X Li et al ldquoABCB1 C3435T polymorphism andresponse to clopidogrel treatment in coronary artery disease(CAD) patients a meta-analysisrdquo PLoS ONE vol 7 no 10Article ID e46366 2012

[23] J P Lewis R B Horenstein K Ryan et al ldquoThe functionalG143E variant of carboxylesterase 1 is associated with increasedclopidogrel active metabolite levels and greater clopidogrelresponserdquo Pharmacogenetics and Genomics vol 23 no 1 pp 1ndash8 2013

[24] P Staritz K Kurz M Stoll E Giannitsis H A Katus andB T Ivandic ldquoPlatelet reactivity and clopidogrel resistance areassociated with the H2 haplotype of the P2Y12-ADP receptorgenerdquo International Journal of Cardiology vol 133 no 3 pp 341ndash345 2009

[25] M Ingelman-Sundberg ldquoPharmacogenetics of cytochromeP450 and its applications in drug therapy the past present and

futurerdquo Trends in Pharmacological Sciences vol 25 no 4 pp193ndash200 2004

[26] R K Bains ldquoAfrican variation at Cytochrome P450 genes evo-lutionary aspects and the implications for the treatment ofinfectious diseasesrdquo Evolution Medicine and Public Health vol2013 no 1 pp 118ndash134 2013

[27] I Lee and D Kim ldquoPolymorphic metabolism by functional alt-erations of human cytochrome P450 enzymesrdquoArchives of Pha-rmacal Research vol 34 no 11 pp 1799ndash1816 2011

[28] M Ingelman-Sundberg S C Sim A Gomez and C Rod-riguez-Antona ldquoInfluence of cytochrome P450 polymorphismson drug therapies pharmacogenetic pharmacoepigenetic andclinical aspectsrdquo Pharmacology amp Therapeutics vol 116 no 3pp 496ndash526 2007

[29] S M F De Morais G R Wilkinson J Blaisdell K NakamuraU A Meyer and J A Goldstein ldquoThe major genetic defectresponsible for the polymorphism of S-mephenytoin meta-bolism in humansrdquo Journal of Biological Chemistry vol 269 no22 pp 15419ndash15422 1994

[30] S-J Lee W-Y Kim H Kim J-H Shon S L Sang and J-G Shin ldquoIdentification of new CYP2C19 variants exhibitingdecreased enzyme activity in themetabolism of S-mephenytoinand omeprazolerdquo Drug Metabolism and Disposition vol 37 no11 pp 2262ndash2269 2009

[31] S M F DeMorais G RWilkinson J Blaisdell U A Meyer KNakamura and J A Goldstein ldquoIdentification of a new geneticdefect responsible for the polymorphism of (S)-mephenytoinmetabolism in Japaneserdquo Molecular Pharmacology vol 46 no4 pp 594ndash598 1994

[32] S C Sim C Risinger M-L Dahl et al ldquoA common novelCYP2C19 gene variant causes ultrarapid drug metabolismrelevant for the drug response to proton pump inhibitors andantidepressantsrdquo Clinical Pharmacology and Therapeutics vol79 no 1 pp 103ndash113 2006

[33] H-R Luo R E Poland K-M Lin and Y-J Y Wan ldquoGeneticpolymorphism of cytochrome P450 2C19 in Mexican Ameri-cans a cross-ethnic comparative studyrdquo Clinical PharmacologyandTherapeutics vol 80 no 1 pp 33ndash40 2006

[34] J L Mega S L Close S D Wiviott et al ldquoCytochrome P-450 polymorphisms and response to clopidogrelrdquoNew EnglandJournal of Medicine vol 360 no 4 pp 354ndash362 2009

[35] T Simon C Verstuyft M Mary-Krause et al ldquoGenetic deter-minants of response to clopidogrel and cardiovascular eventsrdquoNew England Journal of Medicine vol 360 no 4 pp 363ndash3752009

[36] J L Mega T Simon J-P Collet et al ldquoReduced-functionCYP2C19 genotype and risk of adverse clinical outcomes amongpatients treated with clopidogrel predominantly for PCI ameta-analysisrdquoThe Journal of the AmericanMedical Associationvol 304 no 16 pp 1821ndash1830 2010

[37] J J Park K W Park J Kang et al ldquoGenetic determinantsof clopidogrel responsiveness in Koreans treated with drug-eluting stentsrdquo International Journal of Cardiology vol 163 no1 pp 79ndash86 2013

[38] SMG SmithHM Judge G Peters et al ldquoCommon sequencevariations in the P2Y12 and CYP3A5 genes do not explainthe variability in the inhibitory effects of clopidogrel therapyrdquoPlatelets vol 17 no 4 pp 250ndash258 2006

[39] P Fontana J-S Hulot P De Moerloose and P GaussemldquoInfluence of CYP2C19 and CYP3A4 gene polymorphismson clopidogrel responsiveness in healthy subjectsrdquo Journal ofThrombosis and Haemostasis vol 5 no 10 pp 2153ndash2155 2007


[40] D J Angiolillo A Fernandez-Ortiz E Bernardo et al ldquoContri-bution of gene sequence variations of the hepatic cytochromeP450 3A4 enzyme to variability in individual responsiveness toclopidogrelrdquo Arteriosclerosis Thrombosis and Vascular Biologyvol 26 no 8 pp 1895ndash1900 2006

[41] J Jaitner T Morath R A Byrne et al ldquoNo association ofABCB1 C3435T genotype with clopidogrel response or risk ofstent thrombosis in patients undergoing coronary stentingrdquoCirculation Cardiovascular Interventions vol 5 no 1 pp 82ndash88 2012

[42] J L Mega S L Close S D Wiviott et al ldquoGenetic variantsin ABCB1 and CYP2C19 and cardiovascular outcomes aftertreatment with clopidogrel and prasugrel in the TRITON-TIMI38 trial a pharmacogenetic analysisrdquo The Lancet vol 376 no9749 pp 1312ndash1319 2010

[43] H-J Zhu X Wang B E Gawronski B J Brinda D JAngiolillo and J S Markowitz ldquoCarboxylesterase 1 as a deter-minant of clopidogrel metabolism and activationsrdquo Journal ofPharmacology and Experimental Therapeutics vol 344 no 3pp 665ndash672 2013

[44] H J Bouman E Schomig J W van Werkum et al ldquoParaoxo-nase-1 is a major determinant of clopidogrel efficacyrdquo NatureMedicine vol 17 no 1 pp 110ndash116 2011

[45] T Bhattacharyya S J Nicholls E J Topol et al ldquoRelationshipof paraoxonase 1 (PON1) gene polymorphisms and functionalactivity with systemic oxidative stress and cardiovascular riskrdquoThe Journal of the AmericanMedical Association vol 299 no 11pp 1265ndash1276 2008

[46] J P Lewis and A R Shuldiner ldquoParaoxonase 1 Q192R variantand clopidogrel efficacy fact or fictionrdquo Circulation Cardiovas-cular Genetics vol 5 no 2 pp 153ndash155 2012

[47] K W Park J J Park J Kang et al ldquoParaoxonase 1 gene poly-morphism does not affect clopidogrel response variability butis associated with clinical outcome after PCIrdquo PLoS ONE vol 8no 2 Article ID e52779 2013

[48] P Fontana A Dupont S Gandrille et al ldquoAdenosine dipho-sphate-induced platelet aggregation is associated with P2Y12gene sequence variations in healthy subjectsrdquo Circulation vol108 no 8 pp 989ndash995 2003

[49] N Zoheir S A Elhamid N Abulata M E Sobky D Khafagyand A Mostafa ldquoP2Y12 receptor gene polymorphism andantiplatelet effect of clopidogrel in patients with coronaryartery disease after coronary stentingrdquo Blood Coagulation andFibrinolysis vol 24 no 5 pp 525ndash531 2013

[50] L Bonello N Bonello-Palot S Armero et al ldquoImpact of P2Y12-ADP receptor polymorphism on the efficacy of clopidogreldose-adjustment according to platelet reactivity monitoring incoronary artery disease patientsrdquoThrombosis Research vol 125no 4 pp e167ndashe170 2010

[51] N Von Beckerath O Von Beckerath W Koch M EichingerA Schomig and A Kastrati ldquoP2Y12 gene H2 haplotype isnot associated with increased adenosine diphosphate-inducedplatelet aggregation after initiation of clopidogrel therapy witha high loading doserdquo Blood Coagulation and Fibrinolysis vol 16no 3 pp 199ndash204 2005

[52] X-F Tang J-H Zhang J Wang et al ldquoEffects of coexist-ing polymorphisms of CYP2C19 and P2Y12 on clopidogrelresponsiveness and clinical outcome in patients with acutecoronary syndromes undergoing stent-based coronary inter-ventionrdquo Chinese Medical Journal vol 126 no 6 pp 1069ndash10752013

[53] K K Shalia V K Shah P Pawar S S Divekar and S Pay-annavar ldquoPolymorphisms of MDR1 CYP2C19 and P2Y12 genesin Indian population effects on clopidogrel responserdquo IndianHeart Journal vol 65 no 2 pp 158ndash167 2013

[54] J J Park K W Park J Kang et al ldquoCYP3A4 genetic statusmay be associated with increased vulnerability to the inhibitoryeffect of calcium-channel blockers on clopidogrelrdquo CirculationJournal vol 77 no 5 pp 1289ndash1296 2013

[55] D J Angiolillo E Bernardo M Zanoni et al ldquoImpact ofinsulin receptor substrate-1 genotypes on platelet reactivityand cardiovascular outcomes in patients with type 2 diabetesmellitus and coronary artery diseaserdquo Journal of the AmericanCollege of Cardiology vol 58 no 1 pp 30ndash39 2011

[56] D J Angiolillo A Fernandez-Ortiz E Bernardo et al ldquoPlateletfunction profiles in patients with type 2 diabetes and coronaryartery disease on combined aspirin and clopidogrel treatmentrdquoDiabetes vol 54 no 8 pp 2430ndash2435 2005

[57] E T Carreras W Hochholzer A L Frelinger et al ldquoDiabetesmellitus CYP2C19 genotype and pharmacodynamic responseto clopidogrel dosing insights from the ELEVATE-TIMI 56trialrdquo Circulation vol 128 Article ID A12325 2013

[58] S D Wiviott E Braunwald D J Angiolillo et al ldquoGreater cli-nical benefit of more intensive oral antiplatelet therapy withprasugrel in patients with diabetes mellitus in the trial toassess improvement in therapeutic outcomes by optimizingplatelet inhibition with prasugrelndashthrombolysis in myocardialinfarction 38rdquo Circulation vol 118 no 16 pp 1626ndash1636 2008

[59] C Muller S Caillard L Jesel et al ldquoAssociation of estimatedGFR with platelet inhibition in patients treated with clopido-grelrdquo American Journal of Kidney Diseases vol 59 no 6 pp777ndash785 2012

[60] T Gremmel M Muller S Steiner et al ldquoChronic kidneydisease is associated with increased platelet activation andpoor response to antiplatelet therapyrdquo Nephrology DialysisTransplantation vol 28 no 8 pp 2116ndash2122 2013

[61] T Geisler D Graszlig B Bigalke et al ldquoThe residual platelet aggre-gation after deployment of intracoronary stent (PREDICT)scorerdquo Journal of Thrombosis and Haemostasis vol 6 no 1 pp54ndash61 2008

[62] P J M Best R Lennon H H Ting et al ldquoThe impact ofrenal insufficiency on clinical outcomes in patients undergoingpercutaneous coronary interventionsrdquo Journal of the AmericanCollege of Cardiology vol 39 no 7 pp 1113ndash1119 2002

[63] F Latif N S Kleiman D J Cohen et al ldquoIn-hospital and 1-yearoutcomes among percutaneous coronary intervention patientswith chronic kidney disease in the era of drug-eluting stents areport from the EVENT (evaluation of drug eluting stents andischemic events) registryrdquo JACC Cardiovascular Interventionsvol 2 no 1 pp 37ndash45 2009

[64] P J M Best S R Steinhubl P B Berger et al ldquoThe efficacyand safety of short- and long-term dual antiplatelet therapyin patients with mild or moderate chronic kidney diseaseresults from theClopidogrel for the Reduction of EventsDuringObservation (CREDO) Trialrdquo American Heart Journal vol 155no 4 pp 687ndash693 2008

[65] D J Angiolillo E Bernardo D Capodanno et al ldquoImpactof chronic kidney disease on platelet function profiles indiabetes mellitus patients with coronary artery disease takingdual antiplatelet therapyrdquo Journal of the American College ofCardiology vol 55 no 11 pp 1139ndash1146 2010


[66] N Tabata S Hokimoto T Akasaka et al ldquoChronic kidneydisease status modifies the association of CYP2C19 polymor-phism in predicting clinical outcomes following coronary stentimplantationrdquoThrombosis Research vol 134 no 5 pp 939ndash9442014

[67] N S Abraham M A Hlatky E M Antman et al ldquoACCFACGAHA 2010 expert consensus document on the concomi-tant use of proton pump inhibitors and thienopyridines afocused update of the ACCFACGAHA 2008 expert consensusdocument on reducing the gastrointestinal risks of antiplatelettherapy and NSAID userdquo Journal of the American College ofCardiology vol 56 no 24 pp 2051ndash2066 2010

[68] E R Bates W C Lau and D J Angiolillo ldquoClopidogrel-druginteractionsrdquo Journal of the American College of Cardiology vol57 no 11 pp 1251ndash1263 2011

[69] J M Siller-Matula A O Spiel I M Lang G Kreiner G Christand B Jilma ldquoEffects of pantoprazole and esomeprazole onplatelet inhibition by clopidogrelrdquo American Heart Journal vol157 no 1 pp 148e1ndash148e5 2009

[70] M Schmidt M B Johansen M Maeng et al ldquoConcomitantuse of clopidogrel and statins and risk of major adversecardiovascular events following coronary stent implantationrdquoBritish Journal of Clinical Pharmacology vol 74 no 1 pp 161ndash170 2012

[71] W C Lau L A Waskell P B Watkins et al ldquoAtorvastatinreduces the ability of clopidogrel to inhibit platelet aggregationa new drug-drug interactionrdquo Circulation vol 107 no 1 pp 32ndash37 2003

[72] H Neubauer B Gunesdogan C Hanefeld M Spiecker and AMugge ldquoLipophilic statins interfere with the inhibitory effectsof clopidogrel on platelet functionmdasha flow cytometry studyrdquoEuropean Heart Journal vol 24 no 19 pp 1744ndash1749 2003

[73] N A Farid C D Payne D S Small et al ldquoCytochrome P4503A inhibition by ketoconazole affects prasugrel and clopidogrelpharmacokinetics and pharmacodynamics differentlyrdquo ClinicalPharmacology andTherapeutics vol 81 no 5 pp 735ndash741 2007

[74] J B Olesen G H Gislason M G Charlot et al ldquoCalcium-channel blockers do not alter the clinical efficacy of clopidogrelafter myocardial infarction a nationwide cohort studyrdquo Journalof the American College of Cardiology vol 57 no 4 pp 409ndash4172011

[75] C W Good S R Steinhubl D M Brennan A M LincoffE J Topol and P B Berger ldquoIs there a clinically significantinteraction between calcium channel antagonists and clopido-grel Results from the clopidogrel for the reduction of eventsduring observation (CREDO) trialrdquoCirculation CardiovascularInterventions vol 5 no 1 pp 77ndash81 2012

[76] D Sibbing N Von Beckerath T Morath et al ldquoOral antico-agulation with coumarin derivatives and antiplatelet effects ofclopidogrelrdquo European Heart Journal vol 31 no 10 pp 1205ndash1211 2010

[77] A M Harmsze J W Van Werkum F Moral et al ldquoSulfony-lureas and on-clopidogrel platelet reactivity in type 2 diabetesmellitus patientsrdquo Platelets vol 22 no 2 pp 98ndash102 2011

[78] D S H Bell ldquoPractical considerations and guidelines for dosingsulfonylureas as monotherapy or combination therapyrdquoClinicalTherapeutics vol 26 no 11 pp 1714ndash1727 2004

[79] J M Siller-Matula D Trenk S Krahenbuhl A D Michelsonand G Delle-Karth ldquoClinical implications of drug-drug inter-actions with P2Y12 receptor inhibitorsrdquo Journal of Thrombosisand Haemostasis vol 12 no 1 pp 2ndash13 2014

[80] V Serebruany G Cherala C Williams et al ldquoAssociation ofplatelet responsiveness with clopidogrel metabolism Role ofcompliance in the assessment of ldquoresistancerdquordquo American HeartJournal vol 158 no 6 pp 925ndash932 2009

[81] T Geisler E Schaeffeler J Dippon et al ldquoCYP2C19 andnongenetic factors predict poor responsiveness to clopidogrelloading dose after coronary stent implantationrdquo Pharmacoge-nomics vol 9 no 9 pp 1251ndash1259 2008

[82] M T Holmberg A Tornio M Neuvonen P J Neuvonen J TBackman and M Niemi ldquoGrapefruit juice inhibits the meta-bolic activation of clopidogrelrdquo Clinical Pharmacology andTherapeutics vol 95 no 3 pp 307ndash313 2014

[83] E I Lev M E Arikan M Vaduganathan et al ldquoEffect ofcaffeine on platelet inhibition by clopidogrel in healthy subjectsand patients with coronary artery diseaserdquo American HeartJournal vol 154 no 4 pp 694e1ndash694e7 2007

[84] K P Bliden J DiChiara L Lawal et al ldquoThe association ofcigarette smoking with enhanced platelet inhibition by clopi-dogrelrdquo Journal of the American College of Cardiology vol 52no 7 pp 531ndash533 2008

[85] D J Angiolillo and J L Ferreiro ldquoPlatelet adenosine diphos-phate P2Y12 receptor antagonism benefits and limitations ofcurrent treatment strategies and future directionsrdquo RevistaEspanola de Cardiologia vol 63 no 1 pp 60ndash76 2010

[86] A L Frelinger III D L Bhatt R D Lee et al ldquoClopidogrelpharmacokinetics and pharmacodynamics vary widely des-pite exclusion or control of polymorphisms (CYP2C19 ABCB1PON1) noncompliance diet smoking co-medications (Inclu-ding Proton Pump Inhibitors) and pre-existent variability inplatelet functionrdquo Journal of the American College of Cardiologyvol 61 no 8 pp 872ndash879 2013

[87] National cancer Institute National Cancer Institute Person-alized Medicine Dictionary of Cancer Terms National Can-cer Institute 2016 httpwwwcancergovpublicationsdiction-ariescancer-termscdrid=561717

[88] K Offit ldquoPersonalized medicine new genomics old lessonsrdquoHuman Genetics vol 130 no 1 pp 3ndash14 2011

[89] W Colburn V G DeGruttola D L DeMets et al ldquoBiomarkersand surrogate endpoints preferred definitions and conceptualframework Biomarkers Definitions Working Grouprdquo ClinicalPharmacology ampTherapeutics vol 69 pp 89ndash95 2001

[90] Food and Drug Administration Food and Drug Administration(FDA) Table of Pharmacogenomic Biomarkers in Drug LabelingUS Food and Drug Administration 2015 httpwwwfdagovdrugsscienceresearchresearchareaspharmacogeneticsucm0-83378htm

[91] S G Ahn J Yoon J Kim et al ldquoGenotype- and phenotype-directed personalization of antiplatelet treatment in patientswith non-ST elevation acute coronary syndromes undergoingcoronary stentingrdquoKoreanCirculation Journal vol 43 no 8 pp541ndash549 2013

[92] Y Nasyuhana Sani L Sheau Chin L Luen Hui et al ldquoTheCYP2C19lowast1lowast2 genotype does not adequately predict clopi-dogrel response in healthy Malaysian volunteersrdquo CardiologyResearch and Practice vol 2013 Article ID 128795 7 pages 2013

[93] M Mejin W N Tiong L Y H Lai et al ldquoCYP2C19 genotypesand their impact on clopidogrel responsiveness in percutaneouscoronary interventionrdquo International Journal of Clinical Phar-macy vol 35 no 4 pp 621ndash628 2013

[94] P Fontana M Cattaneo C Combescure and J-L RenyldquoTailored Thienopyridine therapy no urgency for CYP2C19


genotypingrdquo Journal of the American Heart Association vol 2no 2 Article ID e000131 2013

[95] M V Holmes P Perel T Shah A D Hingorani and J P CasasldquoCYP2C19 genotype clopidogrel metabolism platelet functionand cardiovascular events a systematic review and meta-analysisrdquo JAMA - Journal of the American Medical Associationvol 306 no 24 pp 2704ndash2714 2011

[96] T Yin and T Miyata ldquoPharmacogenomics of clopidogrel evi-dence and perspectivesrdquo Thrombosis Research vol 128 no 4pp 307ndash316 2011

[97] Y B Saab R Zeenny and W H Ramadan ldquoOptimizing clopi-dogrel dose response a new clinical algorithm comprisingCYP2C19 pharmacogenetics and drug interactionsrdquo Therapeu-tics and Clinical Risk Management vol 11 pp 1421ndash1427 2015

[98] M A Hamburg and F S Collins ldquoThe path to personalizedmedicinerdquo New England Journal of Medicine vol 363 no 4 pp301ndash304 2010

[99] N K Patel G Subbaiah H Shah M Kundlik and P S Shri-vastav ldquoRapid LC-ESI-MS-MS method for the simultaneousdetermination of clopidogrel and its carboxylic acid metabolitein human plasmardquo Journal of Chromatographic Science vol 46no 10 pp 867ndash875 2008

[100] C J Peer S D Spencer D A H VanDenBerg M APacanowski R B Horenstein and W D Figg ldquoA sensitive andrapid ultra HPLC-MSMS method for the simultaneous detec-tion of clopidogrel and its derivatized active thiol metabolitein human plasmardquo Journal of Chromatography B AnalyticalTechnologies in the Biomedical and Life Sciences vol 880 no 1pp 132ndash139 2012

[101] J T Brandt C D Payne S D Wiviott et al ldquoA comparison ofprasugrel and clopidogrel loading doses on platelet functionmagnitude of platelet inhibition is related to active metaboliteformationrdquo American Heart Journal vol 153 no 1 pp 66e9ndash66e16 2007

[102] I Y Gong N Crown C M Suen et al ldquoClarifying the impor-tance of CYP2C19 and PON1 in the mechanism of clopidogrelbioactivation and in vivo antiplatelet responserdquo European HeartJournal vol 33 no 22 pp 2856ndash2864 2012

[103] H Mani S W Toennes B Linnemann et al ldquoDeterminationof clopidogrel main metabolite in plasma a useful tool formonitoring therapyrdquoTherapeutic Drug Monitoring vol 30 no1 pp 84ndash89 2008

[104] D J Angiolillo A Fernandez-Ortiz E Bernardo et al ldquoVari-ability in individual responsiveness to clopidogrel clinicalimplications management and future perspectivesrdquo Journal ofthe AmericanCollege of Cardiology vol 49 no 14 pp 1505ndash15162007

[105] R Paniccia E Antonucci N Maggini et al ldquoAssessment ofplatelet function on whole blood by multiple electrode aggre-gometry in high-risk patients with coronary artery diseasereceiving antiplatelet therapyrdquo American Journal of ClinicalPathology vol 131 no 6 pp 834ndash842 2009

[106] Accumetrics Verify Now P2Y12 Platelet Reactivity Test Instruc-tion for Use Accumetrics 2013

[107] RK SharmaD J Voelker R SharmaHK ReddyHDod andJ D Marsh ldquoEvolving role of platelet function testing in coro-nary artery interventionsrdquo Vascular Health and Risk Manage-ment vol 8 no 1 pp 65ndash75 2012

[108] F J Falcao L Carvalho M Chan C M Alves A C Car-valho and A M Caixeta ldquoReceptores plaquetarios P2Y12importancia na intervencao coronariana percutaneardquo ArquivosBrasileiros de Cardiologia vol 101 pp 277ndash282 2013

[109] C Gachet and B Aleil ldquoTesting antiplatelet therapyrdquo EuropeanHeart Journal Supplements vol 10 pp A28ndashA34 2008

[110] R Paniccia R Priora A A Liotta and R Abbate ldquoPlateletfunction tests a comparative reviewrdquo Vascular Health and RiskManagement vol 11 pp 133ndash148 2015

[111] SHd PFA-100 System US Assay Menu Edited by US SHd 2012[112] M J Price S Endemann R R Gollapudi et al ldquoPrognostic sig-

nificance of post-clopidogrel platelet reactivity assessed by apoint-of-care assay on thrombotic events after drug-elutingstent implantationrdquo European Heart Journal vol 29 no 8 pp992ndash1000 2008

[113] R Marcucci A M Gori R Paniccia et al ldquoCardiovasculardeath and nonfatal myocardial infarction in acute coronarysyndrome patients receiving coronary stenting are predicted byresidual platelet reactivity to ADP detected by a point-of-careassay a 12-month follow-uprdquoCirculation vol 119 no 2 pp 237ndash242 2009

[114] N J Breet JW VanWerkumH J Bouman et al ldquoComparisonof platelet function tests in predicting clinical outcome inpatients undergoing coronary stent implantationrdquo JAMAmdashJournal of the American Medical Association vol 303 no 8 pp754ndash762 2010

[115] J-P Collet T Cuisset G Range et al ldquoBedside monitoring toadjust antiplatelet therapy for coronary stentingrdquo New EnglandJournal of Medicine vol 367 no 22 pp 2100ndash2109 2012

[116] C H Miller A S Rice K Garrett and S F Stein ldquoGenderrace and diet affect platelet function tests in normal subjectscontributing to a high rate of abnormal resultsrdquo British Journalof Haematology vol 165 no 6 pp 842ndash853 2014

[117] YGKim J-W Suh J J Park et al ldquoDifferent influences of hem-atocrit on the results of two point-of-care platelet function teststhe verifynow assay and multiple electrode platelet aggregome-tryrdquo PLoS ONE vol 9 no 11 Article ID e114053 2014

[118] N Kakouros T S Kickler K M Laws and J J Rade ldquoHema-tocrit alters VerifyNow P2Y12 assay results independently ofintrinsic platelet reactivity and clopidogrel responsivenessrdquoJournal of Thrombosis and Haemostasis vol 11 no 10 pp 1814ndash1822 2013

[119] P A Gurbel K P Bliden K A Zaman J A Yoho K M HayesandU S Tantry ldquoClopidogrel loading with eptifibatide to arrestthe reactivity of platelets results of the Clopidogrel LoadingWith Eptifibatide to Arrest the Reactivity of Platelets (CLEARPlatelets) studyrdquo Circulation vol 111 no 9 pp 1153ndash1159 2005

[120] J A Jakubowski C Zhou B Egan et al ldquoModification of theVerifyNow P2Y12 test BASE channel to accommodate highlevels of P2Y12 antagonismrdquo Platelets vol 22 no 8 pp 619ndash6252011

[121] W Hochholzer C T Ruff R A Mesa et al ldquoVariabilityof individual platelet reactivity over time in patients treatedwith clopidogrel insights from the ELEVATEndashTIMI 56 trialrdquoJournal of the American College of Cardiology vol 64 no 4 pp361ndash368 2014

[122] G Lemesle J-B Landel A Bauters et al ldquoPoor agreementbetween light transmission aggregometry Verify Now P2Y12and vasodilatator-stimulated phosphoprotein for clopidogrellow-response assessment a potential explanation of negativeresults of recent randomized trialsrdquo Platelets vol 25 no 7 pp499ndash505 2014

[123] G E Louridas I E Kanonidis and K G Lourida ldquoSystemsbiology in heart diseasesrdquo Hippokratia vol 14 no 1 pp 10ndash162010


[124] D W D Kuster D Merkus J V D Velden A J M Verhoevenand D J Duncker ldquorsquoIntegrative Physiology 20rsquo integrationof systems biology into physiology and its application tocardiovascular homeostasisrdquo Journal of Physiology vol 589 no5 pp 1037ndash1045 2011

[125] G E Louridas and K G Lourida ldquoThe new biology a bridge toclinical cardiologyrdquoHippokratia vol 16 no 2 pp 106ndash112 2012

[126] T P Cappola andK BMargulies ldquoFunctional genomics appliedto cardiovascular medicinerdquo Circulation vol 124 no 1 pp 87ndash94 2011

[127] S Piran P Liu A Morales and R E Hershberger ldquoWheregenome meets phenome rationale for integrating genetic andprotein biomarkers in the diagnosis andmanagement of dilatedcardiomyopathy and heart failurerdquo Journal of the AmericanCollege of Cardiology vol 60 no 4 pp 283ndash289 2012

[128] K Saito and F Matsuda ldquoMetabolomics for functional geno-mics systems biology and biotechnologyrdquo Annual Review ofPlant Biology vol 61 no 1 pp 463ndash489 2010

[129] R Caruso S Rocchiccioli A M Gori et al ldquoInflammatory andantioxidant pattern unbalance in lsquoclopidogrel-resistantrsquo patientsduring acute coronary syndromerdquo Mediators of Inflammationvol 2015 Article ID 710123 12 pages 2015

[130] R Goodacre ldquoMetabolomics of a superorganismrdquo Journal ofNutrition vol 137 no 1 2007

[131] I A Gutiu A Andries CMircioiu F Radulescu A-M Georg-escu and D Cioaca ldquoPharmacometabonomics pharmacoge-nomics and personalized medicinerdquo Romanian Journal of Inter-nal Medicine vol 48 no 2 pp 187ndash191 2010

[132] P A Gladding ldquoClinical applications of pharmacogenetics pre-sent and near futurerdquo Cleveland Clinic Journal of Medicine vol80 no 8 pp 477ndash482 2013

[133] N Semmar ldquoMetabotype concept flexibility usefulness andmeaning in different biological populationsrdquo in MetabolomicsD U Roessner Ed InTech 2012

[134] J Lindon J Nicholson and E HolmesThe Handbook of Meta-bonomics and Metabolomics Elsevier BV Amsterdam TheNetherlands 2006

[135] B Ibrahim M Basanta P Cadden et al ldquoNon-invasive phe-notyping using exhaled volatile organic compounds in asthmardquoThorax vol 66 no 9 pp 804ndash809 2011

[136] E Holmes TM Tsang J T-J Huang et al ldquoMetabolic profilingof CSF evidence that early intervention may impact on diseaseprogression and outcome in schizophreniardquo PLoSMedicine vol3 no 8 pp 1420ndash1428 2006

[137] M Basanta B Ibrahim R Dockry et al ldquoExhaled volatileorganic compounds for phenotyping chronic obstructive pul-monary disease a cross-sectional studyrdquo Respiratory Researchvol 13 article 72 2012

[138] P Montuschi D Paris and D Melck ldquoMetabolomic analysisby nuclear magnetic resonance spectroscopy of exhaled breathcondensate in patient with cystic fibrosisrdquo European RespiratoryJournal vol 34 supplement 53 p 63s 2009

[139] E Mervaala A Biala S Merasto et al ldquoMetabolomics inangiotensin II-induced cardiac hypertrophyrdquoHypertension vol55 no 2 pp 508ndash515 2010

[140] L Tenori X Hu P Pantaleo et al ldquoMetabolomic fingerprintof heart failure in humans a nuclear magnetic resonancespectroscopy analysisrdquo International Journal of Cardiology vol168 no 4 pp e113ndashe115 2013

[141] G Corona F Rizzolio A Giordano and G Toffoli ldquoPharmaco-metabolomics an emerging lsquoomicsrsquo tool for the personalization

of anticancer treatments and identification of new valuabletherapeutic targetsrdquo Journal of Cellular Physiology vol 227 no7 pp 2827ndash2831 2012

[142] J K Nicholson I D Wilson and J C Lindon ldquoPharmacome-tabonomics as an effector for personalized medicinerdquo Pharma-cogenomics vol 12 no 1 pp 103ndash111 2011

[143] Z Yang and F Marotta ldquoPharmacometabolomics in drug dis-covery amp development applications and challengesrdquoMetabolo-mics vol 2 article e122 2012

[144] T A Clayton D Baker J C Lindon J R Everett and J KNicholson ldquoPharmacometabonomic identification of a signifi-cant host-microbiome metabolic interaction affecting humandrug metabolismrdquo Proceedings of the National Academy ofSciences of the United States of America vol 106 no 34 pp14728ndash14733 2009

[145] T A Clayton J C Lindon O Cloarec et al ldquoPharmaco-meta-bonomic phenotyping and personalized drug treatmentrdquoNature vol 440 no 7087 pp 1073ndash1077 2006

[146] C Kim E Kim H Yoo et al ldquoMetabonomic analysis of serummetabolites in kidney transplant recipients with cyclosporine a-or tacrolimus-based immunosuppressionrdquoTransplantation vol90 no 7 pp 748ndash756 2010

[147] Z Wang Z Chen S Yang et al ldquo1H NMR-based metabolomicanalysis for identifying serum biomarkers to evaluatemethotrexate treatment in patients with early rheumatoidarthritisrdquo Experimental and Therapeutic Medicine vol 4 no 1pp 165ndash171 2012

[148] R Kaddurah-Daouk M B Bogdanov W R Wikoff et alldquoPharmacometabolomic mapping of early biochemical changesinduced by sertraline andplacebordquoTranslational Psychiatry vol3 article no e223 2013

[149] A Villasenor A Ramamoorthy M S D Santos et al ldquoA pilotstudy of plasma metabolomic patterns from patients treatedwith ketamine for bipolar depression evidence for a response-related difference in mitochondrial networksrdquo British Journal ofPharmacology vol 171 no 8 pp 2230ndash2242 2014

[150] K Gilany R S M Pouracil and M R Sadeghi ldquoFouriertransform infrared spectroscopy a potential technique fornoninvasive detection of spermatogenesisrdquo Avicenna Journal ofMedical Biotechnology vol 6 no 1 pp 47ndash52 2014

[151] F Savorani M A Rasmussen M S Mikkelsen and S BEngelsen ldquoA primer to nutritional metabolomics by NMRspectroscopy and chemometricsrdquo Food Research Internationalvol 54 no 1 pp 1131ndash1145 2013

[152] A Scalbert L Brennan O Fiehn et al ldquoMass-spectrometry-based metabolomics limitations and recommendations forfuture progress with particular focus on nutrition researchrdquoMetabolomics vol 5 no 4 pp 435ndash458 2009

[153] A Alonso S Marsal and A JuliA ldquoAnalytical methods inuntargeted metabolomics state of the art in 2015rdquo Frontiers inBioengineering and Biotechnology vol 3 article 23 2015

[154] M V Silva Elipe ldquoAdvantages and disadvantages of nuclearmagnetic resonance spectroscopy as a hyphenated techniquerdquoAnalytica Chimica Acta vol 497 no 1-2 pp 1ndash25 2003

[155] W B Dunn N J C Bailey and H E Johnson ldquoMeasuring themetabolome current analytical technologiesrdquoThe Analyst vol130 no 5 pp 606ndash625 2005

[156] V Shulaev ldquoMetabolomics technology and bioinformaticsrdquoBriefings in Bioinformatics vol 7 no 2 pp 128ndash139 2006

[157] J K Nicholson and J C Lindon ldquoSystems biology metabo-nomicsrdquo Nature vol 455 no 7216 pp 1054ndash1056 2008


[158] D Bu Chemometric Analysis for Spectroscopy Bridging theGap between the State and Measurement of a Chemical System2007 httpwwwcamocomdownloadsresourcesapplication_notesChemometric20Analysis20for20Spectroscopypdf

[159] R Goodacre S Vaidyanathan W B Dunn G G Harriganand D B Kell ldquoMetabolomics by numbers acquiring andunderstanding global metabolite datardquo Trends in Biotechnologyvol 22 no 5 pp 245ndash252 2004

[160] G Pennazza M Santonico EMartinelli A DrsquoAmico and C DNatale ldquoInterpretation of exhaled volatile organic compoundsrdquoin Exhaled Biomarkers European Respiratory Society Mono-graph pp 115ndash129 2010

[161] R Kaddurah-Daouk and R M Weinshilboum ldquoPharma-cometabolomics implications for clinical pharmacology andsystems pharmacologyrdquo Clinical Pharmacology and Therapeu-tics vol 95 no 2 pp 154ndash167 2014

[162] LM Raamsdonk B TeusinkD Broadhurst et al ldquoA functionalgenomics strategy that uses metabolome data to reveal thephenotype of silentmutationsrdquoNature Biotechnology vol 19 no1 pp 45ndash50 2001

[163] Y Ji S Hebbring H Zhu et al ldquoGlycine and a glycine dehy-drogenase (GLDC) SNP as citalopramescitalopram responsebiomarkers in depression pharmacometabolomics-informedpharmacogenomicsrdquo Clinical Pharmacology and Therapeuticsvol 89 no 1 pp 97ndash104 2011

[164] L M Yerges-Armstrong S Ellero-Simatos A Georgiades etal ldquoPurine pathway implicated in mechanism of resistanceto aspirin therapy pharmacometabolomics-informed pharma-cogenomicsrdquo Clinical Pharmacology and Therapeutics vol 94no 4 pp 525ndash532 2013

[165] J P Lewis L M Yerges-Armstrong S Ellero-Simatos A Geor-giades R Kaddurah-Daouk and T Hankemeier ldquoIntegrationof pharmacometabolomic and pharmacogenomic approachesreveals novel insights into antiplatelet therapyrdquo Clinical Phar-macology andTherapeutics vol 94 no 5 pp 570ndash573 2013

[166] S Ellero-Simatos J P Lewis A Georgiades et al ldquoPharma-cometabolomics reveals that serotonin is implicated in aspirinresponse variabilityrdquoCPT PharmacometricsampSystems Pharma-cology vol 3 no 7 article e125 2014

Submit your manuscripts athttpswwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


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EndocrinologyInternational Journal of



Disease Markers


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Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


literature on new approaches such as pharmacometabolomicsand integrative pharmacometabolomics-pharmacogeneticsin assessing clopidogrel therapeutic outcome

2 Clopidogrel Bioactivation andClopidogrel HTPR

Clopidogrel is an oral drug which has oral bioavailability of50 and the maximum peak concentration will be observedwithin 1 to 2 hours after the administration of the loadingdose (600mg) [4 5]The half-life of clopidogrel is from 7 to 8hours [6] Almost 50 of clopidogrel dose is excreted in theurine and 46 in the faeces [7] Of the oral dose approxi-mately 85 is hydrolysed by esterases into inactivemetabolitewhile the remaining 15 will be activated by the hepaticcytochrome P450 (CYP

450) enzymes to the active metabolite

through two steps of bioactivation [8] The hepatic CYP450

enzymes which are involved in the bioactivation process ofclopidogrel include the CYP1A2 CYP2B6 and CYP2C19 inthe first step and the CYP2B6 CYP2C9 CYP3A45 andCYP2C19 in the second step [9ndash11] The CYP2C19 enzymeplays vital role in the two bioactivation steps of clopidogrelby participating with 449 in the first step and 206 in thesecond step [9 12] The CYP3A4 has an essential role in thesecond step by participating with 398 [9] Clopidogrel hasminimum neutropenic side effect compared to ticlopidine(first-generation thienopyridine) [13] The main side effectsof clopidogrel are bleeding gastrointestinal disorders andrash as well as other side effects such as hepatotoxicity andthrombotic thrombocytopenic purpura albeit they are rareTherefore it is well tolerated by patients

Patients variable platelets inhibition while on clopidogrelwas first reported by Jaremo et al in 2002 [14] In thatstudy five out of the eighteen PCI patients had weak plateletsinhibition in response to clopidogrel loading dose of 300mgSince it was first reported clopidogrel HTPR has been largelydocumented It was found to be affecting 15ndash40 of thepatients [4 15 16] Clopidogrel HTPR is associated with pooroutcome after PCI Matetzky and colleagues indicated anassociation between clopidogrel HTPR and the risk of cardiaceventsrsquo recurrence among 60 ACS patients undergoing PCIwho had taken loading dose of 300mg followed by daily doseof 75mg for three months [17] Substantiating these findingsGeisler et al (2006) indicated that the primary end point ofmyocardial infarction stroke and death were significantlyincreased in clopidogrel HTPR patients who were followedup for three months after the PCI [18]

3 Genetic Factors Contributingto Clopidogrel HTPR

There are several identified genetic variabilities which con-tribute to clopidogrel HTPR In fact clopidogrel variableplatelets reactivity is highly heritable [19] As clopidogrelundergoes intestinal absorption bioactivation by the hepaticCYP450

enzymes and deactivation by esterases this processmight be affected by several genetic variabilities Genetic

variabilities which may interfere with clopidogrel vari-able platelets reactivity include the polymorphisms of theCYP2C19 [19] theCYP3A45 [20] theCYP2C9 [21] the ATP-Binding Cassette Subfamily B Member 1 (ABCB1) [22] theParaoxonase-1 (PON1) the Carboxyl Esterase 1 (CES1) [23]and the genetic polymorphism of the P2Y12 receptors [24]

There are 57 active genes of the CYP450

enzymes and58 pseudogenes in the human genome [25] For most ofthe clinically relevant CYP

450enzymes they are highly

affected by genetic polymorphisms [26] The CYP450

geneticpolymorphisms could interfere with gene transcription geneexpression regulation protein translation and affinity tosubstrate [26 27] Among the CYP

450reported genetic poly-

morphisms the CYP2D6 CYP2C9 and the CYP2C19 were-extensively studied and reported respectively [28] Of allstudied CYP

450genetic polymorphisms the CYP2C19 poly-

morphisms were found to be highly associated with clopido-grel variable response particularly that the enzyme is invol-ved in the two bioactivation steps

31 CYP2C19 Polymorphisms The CYP2C19 enzyme playssubstantial role in the metabolism and bioactivation ofdrugs such as proton pump inhibitors (PPIs) (omeprazolelansoprazole and pantoprazole) selective serotonin reuptakeinhibitors (citalopram and sertraline) tricyclic antidepres-sant (imipramine and amitriptyline) phenytoin and clopido-grel The CYP2C19 enzyme is known by its classical reactionthe (S)-mephenytoin 41015840-hydroxylation [29] The gene of theCYP2C19 is highly polymorphic with more than 25 alleleshaving variable enzymatic activity levels [30] The star oneallele (lowast1) is the normal or ldquowild typerdquo a homozygous formof this allelersquos (lowast1lowast1) phenotypes for the gained full functionof the enzyme Of all polymorphic alleles of the CYP2C19two reduced functions alleles were identified as (lowast2) and(lowast3) alleles have been extensively studied De Morais et al(1994) had found that they cause poor metabolism of (S)-mephenytoin metabolism among Japanese [29] Both theCYP2C19lowast2 and CYP2C19lowast3 alleles are single nucleotidepolymorphism (SNP) of guanine (G) to adenine (A) presentat nucleotide 681 in exon 5 and at nucleotide 636 in exon4 respectively [29 31] The CYP2C19lowast2 causes aberrantsplice site leading to the formation of stop codon whichproduces truncated nonfunctional protein which is cat-alytically inactive [29] The CYP2C19lowast3 causes prematuretermination codonwhich leads to the formation of truncatednonfunctioning protein which is also catalytically inactive[31] The CYP2C19lowast17 is another allele of the CYP2C19which is associated with increased gene transcription thatleads to increased activity of the enzyme [32] Clinicallyindividuals are classified into extensive metabolizers (EM)intermediate metabolizers (IM) poor metabolizers (PM)and ultrametabolizer (UM) of drugs based on their CYP2C19genotype [10] Table 1 shows subjectsrsquo CYP2C19 genotypeand their CYP2C19 phenotype The presence of at least oneallele of the CYP2C19lowast2 is observed in 15ndash30 33ndash40and 40ndash50 of the Caucasians African Americans andAsians respectively [33ndash35] However the CYP2C19lowast3 alleleis present in less than 1 of both Caucasians and AfricanAmericans and 7 of Asians





























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