Prevalence of CYP2C19 polymorphisms in the Lebanese population

Clopidogrel is one of the most commonly prescribed drugs, as its combination with low-dose aspirin is the recommended oral anti-platelet therapy, to prevent ischaemic events following coronary syndromes or stent placement. Numerous recent studies have shown that polymorphisms in the gene encoding the cytochrome P450 (CYP450) 2C19 enzyme (CYP2C19) contribute to variability in response to clopidogrel; patients with certain common genetic variants of CYP2C19 (*2, *3) have a reduced metabolism of clopidogrel and have a higher rate of cardiovascular events or stent thrombosis compared to patients with the CYP2C19 (*1) allele. CYP2C19*2 is most common in Caucasians, Africans and Asians while CYP2C19*3 has been found mostly in Asians. Since the prevalence of these variants in the Lebanese population has not yet been reported, our aim was to determine the genotypes of CYP2C19 in our population. CYP2C19 (*1/*2/*3) variants were assessed by Polymerase Chain Reaction-Restriction Length Polymorphism (PCR–RFLP) assays in a representative sample of 161 unrelated healthy Lebanese volunteers. The allele frequencies of CYP2C19 *2 and *3 were 0.13 and 0.03. Carriers of the CYP2C19 *2 or *3 represented 24.2% of the subjects. Our data show no significant difference in the frequency of CYP2C19 allelic variants when compared to Caucasian populations and demonstrate that the application of the recent FDA recommendations would also be beneficial in Lebanon, allowing physicians to identify patients at high risk for atherothrombotic events, and eventually advising them to consider other antiplatelet medications or alternative dosing strategies in poor metabolizers.     

Cytochrome P450 2C19 polymorphism is associated with poor clinical outcomes in coronary artery disease patients treated with clopidogrel

Patients with lesser degrees of platelet inhibition in response to clopidogrel appear to be at increased risk for recurrent ischemic events. Cytochrome P450 (CYP) polymorphisms have been proposed as possible mechanisms for nonresponsiveness to clopidogrel. Published data on the association between CYP2C19*2 polymorphism and atherothrombotic events are inconclusive. To derive a more precise estimation of the relationship, a meta-analysis was performed. A total of eight prospective cohort studies including 2,345 patients carrying CYP2C19*2 variant allele and 5,935 cases with the wild-type genotype were included in this meta-analysis. Overall, borderline statistically significantly elevated risk of adverse clinical events was associated with genotyping 681G>A polymorphism (for AA + GA vs. GG: OR, 1.46; 95% CI, 1.01 to 2.13; P = 0.05). The summary odds ratio showed a significant association between the CYP2C19*2 polymorphism and an increased risk of cardiac mortality in the follow-up period (OR, 2.07; 95% CI, 1.22 to 3.52; P = 0.007). When studies evaluating myocadial infarction, stent thrombosis, and ischemic stroke, the presence of the variant allele was associated with significantly increased risks of recurrent atherothrombotic events. In summary, this meta-analysis indicated that CYP2C19*2 carrier status is significantly associated with an increased risk of adverse cardiovascular events.     

Cytochrome allelic variants and clopidogrel metabolism in cardiovascular diseases therapy

Clopidogrel and aspirin are among the most prescribed dual antiplatelet therapies to treat the acute coronary syndrome and heart attacks. However, their potential clinical impacts are a subject of intense debates. The therapeutic efficiency of clopidogrel is controlled by the actions of hepatic cytochrome P450 (CYPs) enzymes and impacted by individual genetic variations. Inter-individual polymorphisms in CYPs enzymes affect the metabolism of clopidogrel into its active metabolites and, therefore, modify its turnover and clinical outcome. So far, clinical trials fail to confirm higher or lower adverse cardiovascular effects in patients treated with combinations of clopidogrel and proton pump inhibitors, compared with clopidogrel alone. Such inconclusive findings may be due to genetic variations in the cytochromes CYP2C19 and CYP3A4/5. To investigate potential interactions/effects of these cytochromes and their allele variants on the treatment of acute coronary syndrome with clopidogrel alone or in combination with proton pump inhibitors, we analyze recent literature and discuss the potential impact of the cytochrome allelic variants on cardiovascular events and stent thrombosis treated with clopidogrel. The diversity of CYP2C19 polymorphisms and prevalence span within various ethnic groups, subpopulations and demographic areas are also debated.     

应用HRM技术对CYP2C19*2和CYP2C19*3进行双重SNP分型

氯吡格雷是一种广泛用于预防静脉血栓形成的抗血小板药物。研究表明, 携带有CYP2C19基因功能缺失型等位基因CYP2C19*2、CYP2C19*3的病人, 其体内代谢氯吡格雷成为其活性形式的能力降低, 导致氯吡格雷抑制血小板聚集功能减弱。文章旨在建立一种利用高分辨率熔解曲线分析(High-resolution melting curve analysis,HRM)技术在闭合单管中同时对CYP2C19*2、CYP2C19*3两个多态性位点进行简便、准确分型的方法。本实验针对两个SNP位点分别设计特异性的HRM引物, 并在两个位点引物的5′端分别加上富含AT和GC的序列, 保证两个位点的扩增产物熔解峰无重叠。利用HRM技术, 快速、灵敏地对64例随机DNA样本的CYP2C19*2 、CYP2C19*3两个多态性位点进行了基因分型, 且HRM方法的分型结果与测序验证结果完全一致。因此, 利用HRM技术可以实现在闭合单管中简便、准确地对CYP2C19*2 、CYP2C19*3两个多态性位点同时进行基因分型。该方法有望应用于临床, 指导氯吡格雷的个体化用药。     

Interplay between Genetic and Clinical Variables Affecting Platelet Reactivity and Cardiac Adverse Events in Patients Undergoing Percutaneous Coronary Intervention

Several clinical and genetic variables are associated with influencing high on treatment platelet reactivity (HTPR). The aim of the study was to propose a path model explaining a concurrent impact among variables influencing HTPR and ischemic events. In this prospective cohort study polymorphisms of CYP2C19*2, CYP2C19*17, ABCB1, PON1 alleles and platelet function assessed by Multiple Electrode Aggregometry were assessed in 416 patients undergoing percutaneous coronary intervention treated with clopidogrel and aspirin. The rates of major adverse cardiac events (MACE) were recorded during a 12-month follow up. The path model was calculated by a structural equation modelling. Paths from two clinical characteristics (diabetes mellitus and acute coronary syndrome (ACS)) and two genetic variants (CYP2C19*2 and CYP2C19*17) independently predicted HTPR (path coefficients: 0.11 0.10, 0.17, and -0.10, respectively; p<0.05 for all). By use of those four variables a novel score for prediction of HTPR was built: in a factor-weighted model the risk for HTPR was calculated with an OR of 3.8 (95%CI: 3.1–6.8, p<0.001) for a score level of ≥1 compared with a score of <1. While MACE was independently predicted by HTPR and age in the multivariate model (path coefficient: 0.14 and 0.13, respectively; p<0.05), the coexistence of HTPR and age ≥75 years emerged as the strongest predictor of MACE. Our study suggests a pathway, which might explain indirect and direct impact of variables on clinical outcome: ACS, diabetes mellitus, CYP2C19*2 and CYP2C19*17 genetic variants independently predicted HTPR. In turn, age ≥75 years and HTPR were the strongest predictors of MACE.     

CYP2C19 基因多态性与冠心病危险因素对氯吡格雷抵抗的影响

目的:观察细胞色素P450系统药物代谢酶CYP2C19基因多态性以及相关临床因素对氯吡格雷抵抗的影响。方法:选择2010年11月至2011年5月我科拟行PCI术治疗的冠心病患者共145例,均给予氯吡格雷300mg负荷剂量,75mg维持剂量。①通过流式细胞仪检测血管舒张因子刺激酸磷蛋白血小板反应性指数VASP PRI(以VASP PRI≥50%,定义为氯吡格雷抵抗)分为氯吡格雷抵抗组和氯吡格雷反应组。②检测入选患者的药物代谢酶CYP2C19的基因型;根据不同等位基因功能缺失,分为快代谢基因型(*1/*1)、中间代谢基因型(*1/*2、*1/*3)和慢代谢基因型(*2/*2、*2/*3、*3/*3)。③观察CYP2C19基因型及相关临床危险因素对氯吡格雷反应性的影响,④观察氯吡格雷抵抗与临床不良终点事件主要临床不良终点事件[心源性死亡、再发心肌梗死、靶病变再次血运重建术(TLR)]和次要临床终点事件(支架内血栓形成、脑血管意外、大出血)之间的相关性。结果:检测出氯吡格雷抵抗的患者31例,其发生率为20.67%;检测出CYP2C19慢代谢基因型携带患者19例,所占比例为12.67%。慢代谢基因型患者与(快代谢基因型+中间代谢基因型患者)之间VASP PRI比为(49.20±8.45)%VS(44.17±5.41)%,P<0.05,氯吡格雷抵抗发生率之比为35.49%(n=11)VS16.81%(n=20),P<0.05。多元回归分析提示CYP2C19慢代谢基因型(OR:4.43;95%CI:3.28-8.37,P<0.05)和2型糖尿病(OR:2.76;95%CI:2.13-6.14;P<0.05)是氯吡格雷抵抗的两种危险因素。临床随访结果显示氯吡格雷抵抗组与氯吡格雷反应组主要临床不良终点事件的发生率比为6.45%(n=2)vs2.63%(n=3),P<0.05。结论:携带CPY2C19慢代谢基因型和患有2型糖尿病是导致氯吡格雷抵抗的两种重要的危险因素,氯吡格雷抵抗的发生增加了临床不良终点事件的风险。     

The effect of the CYP 2C19*2 polymorphism on stroke care

Clopidogrel is an inhibitor of platelet-aggregation used in the prevention of secondary stroke. The molecule is activated by the cytochrome P450 2C19 (CYP2C19) enzyme. The frequent CYP2C19*2 point mutation causes loss of enzyme function, a decreased (heterozygous form) or blocked (homozygous form) formation of the active molecule. Thus, for a patient harboring a mutated allele, clopidogrel does not provide effective protection against stroke. Multiple drugs inhibit the CYP2C19 enzyme and their simultaneous use with clopidogrel is especially hazardous for patients with genetically decreased enzyme activity. Frequency of the CYP2C19*2 is variable in different populations, highest rates were detected in some Asian groups. In our study the CYP2C19 genotype was determined in one Hungarian sample of 354 stroke patients and 221 healthy controls. Frequency of the minor allele was found to be 12.87% (12.85% in stroke patients, 12.89% in healthy controls). The proportion of the homozygous CYP2C19*2 variant causing total loss of gene function was 1.74%, rate of the heterozygous allele causing reduced enzyme activity was 22.26% in the total population. Our results for the allele frequencies of the CYP2C19*2 gene are similar to those found in other Caucasian populations. In conclusion, the homozygous mutation, causing ineffectiveness of clopidogrel is relatively rare. However, the heterozygous form in which interaction of CYP2C19 inhibitors causes further decrease in the genetically impaired enzyme activity is present in every fifth drug-taking patient. Based on our findings, we would like to emphasize that it is important to adjust individually antiplatelet treatment in ischemic stroke patients and to take into consideration genetic factors as well as drugs taken for comorbid conditions.     

Effects of Cytochrome P450 2C19 and Paraoxonase 1 Polymorphisms on Antiplatelet Response to Clopidogrel Therapy in Patients with Coronary Artery Disease

Clopidogrel is an antiplatelet prodrug that is recommended to reduce the risk of recurrent thrombosis in coronary artery disease (CAD) patients. Paraoxonase 1 (PON1) is suggested to be a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to active thiol metabolite with inconsistent results. Here, we sought to determine the associations of CYP2C19 and PON1 gene polymorphisms with clopidogrel response and their role in ADP-induced platelet aggregation. Clopidogrel response and platelet aggregation were determined using Multiplate aggregometer in 211 patients with established CAD who received 75 mg clopidogrel and 75–325 mg aspirin daily for at least 14 days. Polymorphisms in CYP2C19 and PON1 were genotyped and tested for association with clopidogrel resistance. Linkage disequilibrium (LD) and their epistatic interaction effects on ADP-induced platelet aggregation were analysed. The prevalence of clopidogrel resistance in this population was approximately 33.2% (n = 70). The frequencies of CYP2C19*2 and *3 were significantly higher in non-responder than those in responders. After adjusting for established risk factors, CYP2C19*2 and *3 alleles independently increased the risk of clopidogrel resistance with adjusted ORs 2.94 (95%CI, 1.65–5.26; p<0.001) and 11.26 (95%CI, 2.47–51.41; p = 0.002, respectively). Patients with *2 or *3 allele and combined with smoking, diabetes and increased platelet count had markedly increased risk of clopidogrel resistance. No association was observed between PON1 Q192R and clopidogrel resistance (adjusted OR = 1.13, 95%CI, 0.70–1.82; p = 0.622). Significantly higher platelet aggregation values were found in CYP2C19*2 and *3 patients when compared with *1/*1 allele carriers (p = 1.98×10⁻⁶). For PON1 Q192R genotypes, aggregation values were similar across all genotype groups (p = 0.359). There was no evidence of gene-gene interaction or LD between CYP2C19 and PON1 polymorphisms on ADP-induced platelet aggregation. Our findings indicated that only CYP2C19*2 and *3 alleles had an influence on clopidogrel resistance. The risk of clopidogrel resistance increased further with smoking, diabetes, and increased platelet count.     

Effect of genetic and coexisting polymorphisms on platelet response to clopidogrel in Chinese Han patients with acute coronary syndrome

Polymorphisms of CYP2C19 are associated with platelet response to clopidogrel. This study was conducted to evaluate the contribution of the previously identified polymorphisms to the response of clopidogrel in a cohort of Chinese Han patients. A total of 222 acute coronary syndrome patients undergoing percutaneous coronary intervention treated with clopidogrel were enrolled from September 2012 to June 2013. Residual platelet aggregations for all patients were measured by the VerifyNow P2Y12 system. Sixteen single-nucleotide polymorphisms among nine genes were genotyped including CYP2C19, ABCB1 and PON1. In this study, CYP2C19*2 and CYP2C19*17 were strongly associated with higher platelet aggregation and lower platelet aggregation to clopidogrel treatment, respectively (P<0.001). Patients with CYP2C19*2 allele had a higher risk of high on-treatment platelet reactivity than non carriers (adjusted OR, 5.434; 95% CI, 1.918–15.399, P=0.01). The coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and the coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were also associated with poor response to clopidogrel. No significant relation of CYP2C19*3 and other polymorphisms to the platelet aggregation was found. In conclusion, CYP2C19*2, CYP2C19*17 coexistence of CYP2B6*9 (rs8192719) and P2Y12 (rs2046934) and coexistence of CYP2B6*1B (rs7254579) and P2Y12 (rs2046934) were identified to be associated with response to clopidogrel treatment in Chinese Han patients.     

Comparison between MassARRAY and pyrosequencing for CYP2C19 and ABCB1 gene variants of clopidogrel efficiency genotyping

Clopidogrel is one of the most frequently used drugs in patients to reduce cardiovascular events. Since patients with different genetic variations respond quite differently to clopidogrel therapy, the related genetic testing plays a vital role in its dosage and genetic testing related to clopidogrel therapy is currently considered as routine test worldwide. In this study, we aim to use two different methods MALDI-TOF mass spectrometry and pyrosequencing to detect gene variant of CYP2C19 and ABCB1. Six single nucleotides polymorphisms (SNP) within CYP2C19 (*2, *3, *4, *5, *17) and ABCB1 C3435T in 458 Chinese Han patients were determined using both MassARRAY and Pyrosequencing. Sanger sequencing was used for verification. Results of both methods were analyzed and compared. Allele frequencies of each SNP and distribution of different genotypes were calculated based on the MassARRAY and Sanger sequencing results. Both methods provided 100% call rates for gene variants, while results of six samples were different with two methods. With Sanger sequencing as the reference results, MassARRAY generated all the same results. The minor allele frequencies of the above six SNPs were 27.1% (CYP2C19*), 5.9% (CYP2C19*3), 0% (CYP2C19*4), 0% (CYP2C19*5), 1.1% (CYP2C19*17), 40.9% (ABCB1), respectively. MassARRAY provides accurate clopidogrel related genotyping with relatively high cost-efficiency, throughput and short time when compared with pyrosequencing.     

Paraoxonase-1 is a major determinant of clopidogrel efficacy

Clinical efficacy of the antiplatelet drug clopidogrel is hampered by its variable biotransformation into the active metabolite. The variability in the clinical response to clopidogrel treatment has been attributed to genetic factors, but the specific genes and mechanisms underlying clopidogrel bioactivation remain unclear. Using in vitro metabolomic profiling techniques, we identified paraoxonase-1 (PON1) as the crucial enzyme for clopidogrel bioactivation, with its common Q192R polymorphism determining the rate of active metabolite formation. We tested the clinical relevance of the PON1 Q192R genotype in a population of individuals with coronary artery disease who underwent stent implantation and received clopidogrel therapy. PON1 QQ192 homozygous individuals showed a considerably higher risk than RR192 homozygous individuals of stent thrombosis, lower PON1 plasma activity, lower plasma concentrations of active metabolite and lower platelet inhibition. Thus, we identified PON1 as a key factor for the bioactivation and clinical activity of clopidogrel. These findings have therapeutic implications and may be exploited to prospectively assess the clinical efficacy of clopidogrel.     

Inhibitory Potency of 8-Methoxypsoralen on Cytochrome P450 2A6 (CYP2A6) Allelic Variants CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22: Differential Susceptibility Due to Different Sequence Locations of the Mutations

Human cytochrome P450 2A6 (CYP2A6) is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6*15, CYP2A6*16, CYP2A6*21 and CYP2A6*22) have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP), in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC₅₀ values. In general, a similar trend of change in the IC₅₀ and K_m values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6*16, differences in IC₅₀ values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6*16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.     

Association of poor metabolizers of cytochrome P450 2C19 with head and neck cancer and poor treatment response

A case-control study consisting of 300 patients and an equal number of healthy controls was carried out to investigate the association of polymorphism in cytochrome P450 2C19 (CYP2C19), which results in poor and extensive metabolizers (PMs and EMs) genotypes, with squamous cell carcinoma of head and neck (HNSCC) and treatment response in patients receiving combination of chemo-radiotherapy. A higher frequency of CYP2C19*2 variants was observed in the cases resulting in significantly higher risk to HNSCC (Ad OR 3.36, 95% CI 1.94-5.82, p-value<0.05). The PM genotype of CYP2C19*3 was also found to be slightly increased in the cases, though the increase in risk was not significant when analyzed by multivariate logistic regression model. Tobacco chewing amongst the cases resulted in almost 13-fold increase in the risk with CYP2C19*2 (OR: 12.39) and 3-fold with CYP2C19*3 genotype (OR: 2.90) when compared to the tobacco chewers amongst the controls. Likewise, cigarette smoking in the cases increased the risk approximately 9-fold and 3-fold with CYP2C19*2 (OR: 8.93) and CYP2C19*3 (OR: 2.18) genotypes respectively when compared to smokers amongst the controls. Similar increase in risk was associated with alcohol use amongst the cases carrying variant genotypes of CYP2C19*2 (OR: 7.75) or CYP2C19*3 (OR: 2.60), demonstrating the importance of gene-environment interaction in modifying susceptibility to HNSCC. Interestingly, patients with PMs of CYP2C19 (CYP2C19*2 and CYP2C19*3) exhibited little response to the respective chemotherapy than the patients carrying wild-type genotype demonstrating that functional enzyme deficiencies due to polymorphism in CYPs may not only be important in modifying the susceptibility to HNSCC but also in determining chemotherapeutic response.     

Early double stent thrombosis associated with clopidogrel hyporesponsivenesss

A 57-year-old male patient without cardiovascular history suffered an acute myocardial infarction and underwent drug-eluting stent implantation in the left anterior descending artery. A few days later, the right coronary artery was also stented (drug-eluting stent). Three days later, he was re-admitted to our hospital in cardiogenic shock. Emergent coronary angiography showed total occlusion of both stents. Platelet function analysis (PFA) showed attenuated platelet inhibition in response to clopidogrel treatment. The patient was the carrier of a loss-of-function polymorphism in the CYP2C19 gene, which has been associated with increased incidence of adverse thrombotic events. Antiplatelet therapy was switched to prasugrel and PFA revealed an adequate antiplatelet effect.     

CYP2C19 Phenotype,Stent Thrombosis,Myocardial Infarction,and Mortality in Patients with Coronary Stent Placement in a Chinese Population

Background

Several studies have indicated that CYP2C19 loss-of-function polymorphisms have a higher risk of stent thrombosis (ST) after percutaneous coronary interventions (PCIs). However, this association has not been investigated thoroughly in a Chinese population. In this study, we aimed to determine the effect of CYP2C19*2 and CYP2C19*3 loss-of-function polymorphisms on the occurrence of ST and other adverse clinical events in a Chinese population.

Methods

We designed a cohort study among 1068 consecutive patients undergoing intracoronary stent implantation after preloading with 600 mg of clopidogrel. CYP2C19*2 and CYP2C19*3 were genotyped by using polymerase chain reaction-restriction fragment length polymorphism analysis. The adverse clinical events recorded were ST, death, myocardial infarction (MI), and bleeding events. The primary end point of the study was the incidence of cumulative ST within 1 year after PCI. The secondary end point was other adverse clinical outcomes 1 year after the procedure.

Results

The cumulative 1-year incidence of ST was 0.88% in patients with extensive metabolizers (EMs) (CYP2C19*1/*1 genotype), 4.67% in patients with intermediate metabolizers (IMs) (CYP2C19*1/*2 or *1/*3 genotype), and 10.0% in patients with poor metabolizers (PMs) (CYP2C19*2/*2, *2/*3, or *3/*3 genotype) (P<0.001). The one-year event-free survival was 97.8% in patients with EMs, 96.5% in patients with IMs, and 92.0% in patients with PMs (P = 0.014). Multivariate analysis confirmed the independent association of CYP2C19 loss-of-function allele carriage with ST (P = 0.009) and total mortality (P<0.05).

Conclusion

PM patients had an increased risk of ST, death, and MI after coronary stent placement in a Chinese population.     

Role of CYP2C9 and CYP2C19 polymorphisms in patients with atherosclerosis

The arachidonic acid metabolizing CYP enzymes with prominent roles in vascular regulation are epoxygenases of the two gene family which generate epoxyeicosatrienoic acids. Carriers of CYP2C9 mutant alleles exhibit a diminished CYP2C9 metabolic capacity leading to decreased endothelium-derived hyperpolarizing factors (EDHF) synthesis and an increased risk for atherosclerosis. We investigated whether the polymorphisms of CYP2C9/19 are related with atherosclerosis. We examined 108 patients having angioraphically > or =70 coronary artery narrowing and 90 healthy controls. CYPC2C9/19*2 and CYP2C9/19*3 alleles were investigated in both patients and controls by a real time PCR instrument. There was no significant difference in the distribution of the CYP2C9*2/*3 alleles between cases and the controls. We found that smoker patients having CYP2C9*2 heterozygote genotype have 3.7-fold risk of developing atherosclerosis. CYP2C19*3 heterozygote alleles are more frequent in patients than in controls (10.2%, 5.6% respectively) and it is related with a three-fold risk of atherosclerosis (odds ratio (OR) = 3.75, confidence interval (CI) = 0.75-18.65). It becomes clear that cigarette smoking can cause almost all major diseases prevalent today, such as cancer or heart disease. This inter-subject variability in cigarette-induced pathologies is partly mediated by genetic variants of genes that may participate in detoxification processes, e.g., cytochrome P450 (CYP), cellular susceptibility to toxins, such as p53, or disease development such as atherosclerosis.     

Allele and genotype frequencies of the polymorphic cytochrome P450 genes (CYP1A1, CYP3A4, CYP3A5, CYP2C9 and CYP2C19) in the Jordanian population

Drug metabolizing enzymes participate in the neutralizing of xenobiotics and biotransformation of drugs. Human cytochrome P450, particularly CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, play an important role in drug metabolism. The genes encoding the CYP enzymes are polymorphic, and extensive data have shown that certain alleles confer reduced enzymatic function. The goal of this study was to determine the frequencies of important allelic variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 in the Jordanian population and compare them with the frequency in other ethnic groups. Genotyping of CYP1A1(m1 and m2), CYP2C9 (*2 and *3), CYP2C19 (*2 and *3), CYP3A4*5, CYP3A5 (*3 and *6), was carried out on Jordanian subjects. Different variants allele were determined using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). CYP1A1 allele frequencies in 290 subjects were 0.764 for CYP1A1*1, 0.165 for CYP1A1*2A and 0.071 for CYP1A1*2C. CYP2C9 allele frequencies in 263 subjects were 0.797 for CYP2C9*1, 0.135 for CYP2C9*2 and 0.068 for CYP2C9*3. For CYP2C19, the frequencies of the wild type (CYP2C19*1) and the nonfunctional (*2 and *3) alleles were 0.877, 0.123 and 0, respectively. Five subjects (3.16?%) were homozygous for *2/*2. Regarding CYP3A4*1B, only 12 subjects out of 173 subjects (6.9?%) were heterozygote with none were mutant for this polymorphism. With respect to CYP3A5, 229 were analyzed, frequencies of CYP3A5*1,*3 and *6 were 0.071, 0.925 and 0.0022, respectively. Comparing our data with that obtained in several Caucasian, African-American and Asian populations, Jordanians are most similar to Caucasians with regard to allelic frequencies of the tested variants of CYP1A1, CYP2C9, CYP2C19, CYP3A4 and CYP3A5.     

Reduced number of cardiovascular events and increased cost-effectiveness by genotype-guided antiplatelet therapy in patients undergoing percutaneous coronary interventions in the Netherlands

Aim

This study explores clinical outcome in cytochrome P450 2C19 (CYP2C19)-related poor metaboliser patients treated with either clopidogrel or prasugrel after percutaneous coronary intervention (PCI) and investigates whether this could be cost-effective.

Methods and results

This single-centre, observational study included 3260 patients scheduled for elective PCI between October 2010 and June 2013 and followed for adverse cardiovascular events until October 2014. Post PCI, CYP2C19 poor metaboliser patients were treated with clopidogrel or prasugrel, in addition to aspirin. In total, 32 poor metabolisers were treated with clopidogrel and 41 with prasugrel. The number of adverse cardiovascular events, defined as death from cardiovascular cause, myocardial infarction, stent thrombosis, every second visit to the catheterisation room for re-PCI in the same artery, or stroke, within 1.5 years of PCI, was significantly higher in the CYP2C19 poor metaboliser group treated with clopidogrel (n = 10, 31?%) compared with the poor metaboliser group treated with prasugrel (n = 2, 5?%) (p = 0.003). Costs per gained quality-adjusted life years (QALY) were estimated, showing that genotype-guided post-PCI treatment with prasugrel could be cost-effective with less than € 10,000 per gained QALY.

Conclusion

This study provides evidence that for CYP2C19-related poor metabolisers prasugrel may be more effective than clopidogrel to prevent major adverse cardiovascular events after PCI and this approach could be cost-effective.

     

Hepatocellular toxicity of clopidogrel: Mechanisms and risk factors

Clopidogrel is a prodrug used widely as a platelet aggregation inhibitor. After intestinal absorption, approximately 90% is converted to inactive clopidogrel carboxylate and 10% via a two-step procedure to the active metabolite containing a mercapto group. Hepatotoxicity is a rare but potentially serious adverse reaction associated with clopidogrel. The aim of this study was to find out the mechanisms and susceptibility factors for clopidogrel-associated hepatotoxicity. In primary human hepatocytes, clopidogrel (10 and 100 μM) was cytotoxic only after cytochrome P450 (CYP) induction by rifampicin. Clopidogrel (10 and 100 μM) was also toxic for HepG2 cells expressing human CYP3A4 (HepG2/CYP3A4) and HepG2 cells co-incubated with CYP3A4 supersomes (HepG2/CYP3A4 supersome), but not for wild-type HepG2 cells (HepG2/wt). Clopidogrel (100 μM) decreased the cellular glutathione content in HepG2/CYP3A4 supersome and triggered an oxidative stress reaction (10 and 100 µM) in HepG2/CYP3A4, but not in HepG2/wt. Glutathione depletion significantly increased the cytotoxicity of clopidogrel (10 and 100 µM) in HepG2/CYP3A4 supersome. Co-incubation with 1 μM ketoconazole or 10 mM glutathione almost completely prevented the cytotoxic effect of clopidogrel in HepG2/CYP3A4 and HepG2/CYP3A4 supersome. HepG2/CYP3A4 incubated with 100 μM clopidogrel showed mitochondrial damage and cytochrome c release, eventually promoting apoptosis and/or necrosis. In contrast to clopidogrel, clopidogrel carboxylate was not toxic for HepG2/wt or HepG2/CYP3A4 up to 100 µM. In conclusion, clopidogrel incubated with CYP3A4 is associated with the formation of metabolites that are toxic for hepatocytes and can be trapped by glutathione. High CYP3A4 activity and low cellular glutathione stores may be risk factors for clopidogrel-associated hepatocellular toxicity.     

The roles of different porcine cytochrome P450 enzymes and cytochrome b5A in skatole metabolism

Boar taint is the unfavourable odour and taste from pork fat, which results in part from the accumulation of skatole (3-methylindole, 3MI). The key enzymes in skatole metabolism are thought to be cytochrome P450 2E1 (CYP2E1) and cytochrome 2A (CYP2A); however, the cytochrome P450 (CYP450) isoform responsible for the production of the metabolite 6-hydroxy-3-methylindole (6-OH-3MI, 6-hydroxyskatole), which is thought to be involved in the clearance of skatole, has not been established conclusively. The aim of this study was to characterize the role of porcine CYP450s in skatole metabolism by expressing them individually in the human embryonic kidney HEK293-FT cell line. This system eliminates the problems of the lack of specificity of antibodies, inhibitors and substrates for CYP450 isoforms in the pig, and contributions of any other CYP450s that would be present. The results show that pig CYP1A1, CYP2A19, CYP2C33v4, CYP2C49, CYP2E1 and CYP3A and human CYP2E1 (hCYP2E1) are all capable of producing the major skatole metabolite 3-methyloxyindole (3MOI), as well as indole-3-carbinol (I3C), 5-hydroxy-3-methylindole (5-OH-3MI), 6-OH-3MI, 2-aminoacetophenone (2AAP) and 3-hydroxy-3-methyloxindole. CYP2A19 produced the highest amount of the physiologically important metabolite 6-OH-3MI, followed by porcine CYP2E1 and CYP2C49; CYP2A19 also produced more 6-OH-3MI than hCYP2E1. Co-transfection with CYB5A increased the production of skatole metabolites by some of the CYP450s, suggesting that CYB5A plays an important role in the metabolism of skatole. We also show the utility of this expression system to check the specificity of selected substrates and antibodies for porcine CYP450s. Further information regarding the abundance of different CYP450 isoforms is required to fully understand their contribution to skatole metabolism in vivo in the pig.