Cytochrome P450 2C19 Gene Research Articles

Clopidogrel resistance and its relevance: Current concepts

ABSTRACT Clopidogrel is the most widely used P2Y12 receptor inhibitor (P2Y12i) as a part of dual antiplatelet therapy along with aspirin. Clopidogrel is a pro-drug and is metabolized to its active metabolite by the hepatic enzyme cytochrome P4502C19 (CYP2C19). This active metabolite is responsible for the antiplatelet action of clopidogrel. Recent studies have demonstrated that single nucleotide polymorphisms in the CYP2C19 gene, including CYP2C19*2,*3,*4, and *5 alleles, result in reduced production of the active metabolite of clopidogrel, and hence reduced inhibition of platelet aggregation. This in turn enhances the incidence of stent thrombosis and recurrent cardiovascular (CV) events. We report a case of coronary stent thrombosis due to clopidogrel resistance proven by CYP2C19 genotyping. We then review the literature on clopidogrel resistance and its impact on CV outcomes. Subsequently, we discuss the methods of diagnosis of resistance, evidence from clinical trials for tailoring clopidogrel therapy, the role of potent P2Y12 inhibitors, the current guidelines, and future directions.

Genetic variation of CYP2C9 gene and its correlation with cardiovascular disease risk factors.

The major enzyme that is responsible for Sulfonylureas (SUs) metabolism is hepatic cytochrome P-450 2C9 (CYP2C9). It is encoded by the polymorphic gene CYP2C9, which has many allelic variants, among those the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. People with diabetes mellitus type 2 (T2DM) are more likely to develop cardiovascular disease (CVD), and their risk of dying from it is more than two times higher than that of people without the condition. The purpose of this study was to evaluate the association of genetic variations in the CYP2C9 gene with cardiovascular risk factors by investigating CYP2C9*1, *2, *3, *5, *11, and *13 allelic variants. A total of 226 participants were enrolled in the current case-control study. Allele-specific amplification- PCR (ASA-PCR) was used to determine the allele of different variations and the results were confirmed by sequencing. The findings of this study showed the presence of the CYP2C9*2 allele in the T2DM group does not differ from its percentage in the control group. Also, CYP2C9*3 allele frequencies identified by Hardy-Weinberg equilibrium (HWE) analysis law were not significant, p = 0.6593 and 0.5828 in T2DM and control groups. There is no statistically significant difference between the control and diabetes groups involving the distribution of CYP2C9 alleles and CYP2C9*5, *11, and *13 polymorphisms were absent in the Iraqi population. No carrier for the CYP2C9*3 homozygous state was found in both groups. According to these results T2DM patients with the CYP2C9*2 and *3 variants have an increased risk of developing hypertension.

Pharmacogenetics: Influence of CYP2C9*2 and *3 alleles polymorphisms on Iraqi type 2 diabetic patients

Abstract Background: Sulfonylureas (SUs) are the most prescribed anti-diabetic drugs. The enzyme responsible for metabolizing of SUs is hepatic cytochrome P4502C9 (CYP2C9). The CYP2C9 gene has numerous allelic variations; among those, the CYP2C9*2 and CYP2C9*3 are the most common and clinically significant allelic variations. The pharmacokinetics of SUs are dramatically impacted by CYP2C9 genotype. Objective: Evaluation of the association of genetic polymorphisms in CYP2C9 gene with the efficacy of glibenclamide (GB), second-generation SUs, by investigating two CYP2C9 allelic variants. Materials and Methods: Blood samples were collected from 113 type 2 diabetes patients. Allele specific amplification-polymerase chain reaction was used to genotype the CYP2C9 gene. Fasting serum glucose, fasting insulin, and glycated hemoglobin (HbA1c) levels were measured as part of the biochemical analysis. Results: The CYP2C9 gene variants were analyzed in a study group. The results exposed that 75 patients carried the wild (CYP2C9*1/*1) genotype, 25 were heterozygote allele (CYP2C9*1/*2) for CYP2C9*2 gene, 4 were homozygous for the variant CYP2C9*2 allele (CYP2C9*2/*2), and 9 were heterozygous for the variant CYP2C9*3 allele (CYP2C9*1/*3). Statistically significant difference was found in mean HbA1c between the mutant and wild alleles group (P = 0.044). The mean HbA1c for those carrying the CYP2C9*2 and*3 alleles (n = 38) was 8.4750 compared to 9.3177 for those carrying the CYP2C9*1 allele (n = 75), which indicate better glycemic control. Conclusion: The accordance of CYP2C9*2 and*3 was found to be associated with severe hypoglycemia (odd ratio [OR] = 2.045). The OR suggests a strong association between CYP2C9*2 and*3 alleles and hypoglycemia. Our findings imply that the diabetic patients with CYP2C9 polymorphism are more likely to suffer hypoglycemia than those with wild type alleles when treated with GB.

Effect of cytochrome P450 2C19 (CYP2C19) gene polymorphism and clopidogrel reactivity on long term prognosis of patients with coronary heart disease after PCI.

To investigate the impact of CYP2C19 gene polymorphism on clopidogrel reactivity and its association with long-term clinical outcome in patients with coronary heart disease (CHD) undergoing percutaneous coronary intervention (PCI). In total, 675 patients were enrolled. Based on the platelet inhibition rate, patients were categorized into two groups: clopidogrel low responsiveness (CLR) and normal clopidogrel responsiveness (NCR). The CLR group was divided into ticagrelor and clopidogrel group based on the antiplatelet drugs used in the follow-up treatment. Patients were classified into three groups (normal metabolizer, intermediate metabolizer, and poor metabolizer) based on the CYP2C19 genotype. We aimed to evaluate the impact of CYP2C19 gene polymorphism on clopidogrel reactivity. The cumulative rates of 12-month all-cause deaths, major adverse cardiovascular events (MACCEs), and bleeding events were calculated. CLR was observed in 44.4% of the overall population. Significant differences were observed in the platelet inhibition rate of clopidogrel among the three metabolic genotypes (P < 0.05). At the 12-month follow-up, 13 patients (1.9%) died and 96 patients (14.2%) experienced MACCEs. Patients with CLR (9.6% vs. 11.7% vs. 22.1%, P < 0.05) or poor metabolizer (10.7% vs. 16.4% vs. 22.6%, P = 0.026) experienced a higher rate of MACCEs. A MACCEs risk score between zero and two was calculated. The highest incidence of MACCEs significantly increased with the 2-positive results, and the area under the curve (AUC) was 0.712 (95% CI: 0.650-0.774, P < 0.05). There was no significant difference between the group with a score of one and the occurrence of MACCEs (P > 0.05). Low response to clopidogrel in CHD patients is correlated with CYP2C19 gene polymorphism. CYP2C19 genotyping combined with platelet reactivity is an independent predictor of 12-months MACCEs in patients with clopidogrel treatment after PCI, which is better than either test alone.

The Association between CYP2C19 Genetic Polymorphism and Prognosis in Patients Receiving Endovascular Therapy.

Potentially substantial impacts on the prognosis have been observed in individuals undergoing endovascular treatment due to cytochrome P450 2c19 (CYP2C19) polymorphism. In an attempt to improve prognosis and lower the recurrence rate, this study investigated the CYP2C19 polymorphism in acute ischemic stroke patients. A retrospective analysis was performed on 292 patients with cerebral infarction who had acute endovascular recanalization at the Department of Neurology of Chongqing Hospital of Traditional Chinese Medicine between May 2017 and 2019. The patients were categorized into rapid-, medium-, and slow-metabolism groups based on CYP2C19 gene polymorphism, and their prognosis was monitored. In addition, the prognosis of 188 patients selectively receiving carotid artery stenting at a selected time was also observed. Among the 292 cerebral infarction cases receiving acute endovascular recanalization, the patients in the CYP2C19 rapid-metabolism group regularly took clopidogrel and aspirin combined with antiplatelet therapy and suffered from reoccurrence of apoplexy and cerebral hemorrhage; the 90-day good prognosis had a statistical difference (P < 0.05, prognostic assessment includes hospitalization and 6 months after discharge) and the other adverse events had no statistical difference (including mortality). The 188 patients selectively receiving carotid artery stenting had a recurrence of apoplexy, cerebral hemorrhage, and restenosis rate with a statistical difference (P < 0.05), and the other adverse events had no statistical difference. In conclusion, the findings of the current study indicate that irrespective of whether patients are undergoing selective carotid artery stenting or acute endovascular recanalization, those with rapid CYP2C19 metabolism have a significantly lower likelihood of experiencing adverse prognostic events compared to those with intermediate and slow metabolism. Furthermore, this group also has a more favorable prognosis than the other two groups.

The role of CYP2C9 gene polymorphism in rheumatoid arthritis

Background/Aim: The inflammatory disorder rheumatoid arthritis (RA) affects quality of life and worsens with symptoms in the extra-articular tissues and systemic joints. The most significant member of the Cytochrome P450 enzyme family, Cytochrome P450 2C9 (CYP2C9), plays an essential role in the alkylation, demethylation, and hydroxylation of a variety of substances. Insufficient studies as to whether the susceptibility to rheumatoid arthritis is genetic exists. Therefore, our study presents new information on whether CYPC9 is a genetic risk factor. In this study, we sought to determine whether rheumatoid arthritis and the CYP2C9 gene polymorphism are related. Methods: This study was conducted as a prospective case-control study. Fifty patients with RA and 50 healthy individuals were included in our study group. Blood from the controls and patients was drawn into ethylenediaminetetraacetic acid (EDTA)-containing tubes, and using a DNA isolation kit, DNA was isolated from leukocytes. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the genotypes of CYPC9*2 and CYP2C9*3 with the LightCycler-CYP2C9 mutation detection kit. Results: The heterozygous CYP2C9*2 genotype was found to carry a 2.85-fold risk when compared with the controls (odds ratio [OR]=2.85, 95% confidence interval [CI]: 0.52–15.50; P=0.22); however, this risk was not statistically significant. It was found that people with the CYP2C9*3 heterozygous genotype had a statistically significant 2.79-fold higher risk compared to the controls (OR=2.79, 95% CI: 1.13–7.00 P=0.04). Conclusion: The heterozygous genotype of CYP2C9*3 may contribute to the onset of RA.

Differences in the Proportion of CYP2C19 Loss-of-Function Between Cerebral Infarction and Coronary Artery Disease Patients.

Cytochrome P450 2C19 (CYP2C19) genotypes and metabolic phenotypes (extensive metabolizer (EM), intermediate metabolizer (IM), and poor metabolizer (PM)) are related to the metabolism of therapeutic drugs for cardiovascular and cerebrovascular diseases. This study aimed to investigate the differences of CYP2C19 gene polymorphism distribution between coronary artery disease (CAD) patients and cerebral infarction (CI) patients. We identified 413 CI patients, 509 CAD patients, and 241 CI+CAD patients from 2016 to 2020 and studied genotypes of CYP2C19 rs4986893 (636G>A) and rs4244285 (681G>A) polymorphisms using PCR-gene chip detection method. Differences in CYP2C19 genotypes and metabolic phenotypes between the groups were compared. To analyze the efficacy of CYP2C19 metabolic phenotypes in discriminating between cerebral infarction and coronary artery disease, multiple logistic regression analysis was conducted after adjusting for gender, age, smoking history, drinking history, hypertension, and diabetes. There were significant differences in the distribution of CYP2C19 genotypes and metabolic phenotypes between CI and CAD patients. The results of multivariate logistic regression (adjusted for sex, age, smoking, drinking, hypertension, and diabetes) indicated that CYP2C19 IM phenotype (IM vs EM: OR 1.443, 95% CI: 1.086-1.918, P=0.011) and CYP2C19 IM+PM phenotype (IM or PM vs EM: OR 1.440, 95% CI: 1.100-1.885, P=0.008) may be indicators of CI from CAD. CYP2C19 EM metabolic phenotype was dominant in CAD patients, and CYP2C19 IM metabolic phenotype was dominant in CI patients. After adjusting for other confounding factors, patients with the CYP2C19 IM metabolic phenotype were more likely to develop CI than CAD.

The relationship between genetic polymorphism of CYP2C19 and the efficacy of Helicobacter pylori eradication therapy in children

Objective: To investigate the relationship between genetic polymorphisms of cytochrome P450 2C19 (CYP2C19) and the efficacy of Helicobacter pylori (Hp) eradication therapy in children. Methods: The retrospective cohort study was conducted on 125 children with gastroscopy and positive rapid urease test (RUT) from September 2016 to December 2018 who presented to the Children's Hospital of Zhejiang University School of Medicine due to gastrointestinal symptoms including nausea, vomiting, abdominal pain, bloating, acid reflux, heartburn, chest pain, vomiting blood and melena. Hp culture and drug susceptibility test were carried out with gastric antrum mucosa before treatment. All the patients completed 2 weeks of standardized Hp eradication therapy and had 13C urea breath test 1 month after that, which was used to evaluate the curative effect. The DNA of gastric mucosa after RUT was analyzed and CYP2C19 gene polymorphism was detected. Children were grouped according to metabolic type. Combined with the results of Hp culture and drug susceptibility, the relationship between CYP2C19 gene polymorphism and the efficacy of Hp eradicative treatment was analyzed in children. Chi square test was used for row and column variables, and Fisher exact test was used for comparison between groups. Results: One hundred and twenty five children were enrolled in the study, of whom 76 were males and 49 females. The genetic polymorphism of CYP2C19 in these children found poor metabolizer (PM) of 30.4% (38/125), intermediate metabolizer (IM) of 20.8% (26/125), normal metabolizer (NM) of 47.2% (59/125), rapid metabolizer (RM) of 1.6% (2/125), and ultrarapid metabolizer (UM) of 0. There were statistically significant in positive rate of Hp culture among these groups (χ2=124.00, P<0.001). In addition, the successful rates of Hp eradication in PM, IM, NM and RM genotypes were 84.2% (32/38), 53.8% (14/26), 67.8% (40/59), and 0, respectively, with significant differences (χ2=11.35, P=0.010); those in IM genotype was significantly lower than that in PM genotype (P=0.011). With the same standard triple Hp eradicative regimen, the successful rate of Hp eradication for IM type was 8/19, which was lower than that of PM (80.0%, 24/30) and NM type (77.3%, 34/44) (P=0.007 and 0.007, respectively). There was a significant difference in the efficacy of Hp eradication treatment among different genotypes (χ2=9.72, P=0.008). According to the clarithromycin susceptibility result, the successful rate of Hp eradication treatment for IM genotype was 4/15 in the sensitive group and 4/4 in the drug-resistant group (χ2=6.97, P=0.018). Conclusions: The genetic polymorphism of CYP2C19 in children is closely related to the efficacy of Hp eradication treatment. PM has a higher successful rate of eradication treatment than the other genotypes.

Identification and in vitro functional assessment of 10 CYP2C9 variants found in Chinese Han subjects.

Cytochrome P450 2C9 (CYP2C9) participates in about 15% of clinical drug metabolism, and its polymorphism is associated with individual drug metabolism differences, which may lead to the adverse drug reactions (ADRs). In this study, 1163 Chinese Han individuals were recruited to investigate their distribution pattern of CYP2C9 gene and find out the variants that may affect their drug metabolic activities. We successfully developed a multiplex PCR amplicon sequencing method and used it for the genetic screening of CYP2C9 in a large scale. Besides the wild type CYP2C9*1, totally 26 allelic variants of CYP2C9 were detected, which included 16 previously reported alleles and 10 new non-synonymous variants that had not been listed on the PharmVar website. The characteristics of these newly detected CYP2C9 variants were then evaluated after co-expressing them with CYPOR in S. cerevisiae microsomes. Immunoblot analysis revealed that except for Pro163Ser, Glu326Lys, Gly431Arg and Ile488Phe, most of newly detected variants showed comparable protein expression levels to wild type in yeast cells. Two typical CYP2C9 probe drugs, losartan and glimepiride, were then used for the evaluation of metabolic activities of variants. As a result, 3 variants Thr301Met, Glu326Lys, and Gly431Arg almost lost their catalytic activities and most of other variants exhibited significantly elevated activities for drug metabolism. Our data not only enriches the knowledge of naturally occurring CYP2C9 variants in the Chinese Han population, but also provides the fundamental evidence for its potential clinical usage for personalized medicine in the clinic.

Analysis of CYP2C19 gene polymorphism and influencing factors of pharmacological response of clopidogrel in patients with cerebral infarction in Zhejiang, China.

Certain genetic and non-genetic factors may cause damaged platelet inhibition by clopidogrel. We aimed to determine the effect of cytochrome P4502C19 (CYP2C19) polymorphism, along with other clinical factors, on the platelet response to clopidogrel in patients with acute ischemic stroke (AIS). A total of 214 patients with AIS receiving clopidogrel at a maintenance dose of 75 mg daily admitted to the Ningbo First Hospital between 1 January 2020, and 31 December 2021, were enrolled. Platelet aggregation analysis was performed to determine clopidogrel resistance. Quantitative real-time polymerase chain reaction (QRT-PCR) was used to determine CYP2C19 genotype. Other laboratory data on complete blood count and biochemical parameters were taken from patient medical files. Among the 214 AIS patients treated with clopidogrel in the Ningbo population, the incidence of clopidogrel resistance was approximately 43.9%, and the distribution of CYP2C19 genotypes was highest for CYP2C19(*1/*2) (43.0%), followed by CYP2C19 (*1/*1) (38.8%). The distribution of alleles *1, *2, *3, and *17 was 62.1, 32.5, 4.9, and 0.5%, respectively. A chi-squared test showed that the gene frequencies of alleles *2 and *3 were significantly higher in the clopidogrel-resistant group than in the clopidogrel-sensitive group (p < 0.001), and a Mann-Whitney U-test showed that high HCY levels were significantly correlated with clopidogrel resistance (p < 0.001). Multi-factor logistic regression analysis demonstrated that mutant heterozygous genotype [OR 2.893; 95% confidence interval (CI) 1.456-5.748; p = 0.002], mutant homozygous genotype (OR 4.741; 95% CI 1.828-12.298; p = 0.001), and high HCY levels (OR 1.209; 95% CI 1.072-1.362; p = 0.002) were significantly associated with clopidogrel resistance. According to our results, carrying the CYP2C19*2/*3 allele and high HCY levels are independent risk factors for clopidogrel resistance after clopidogrel therapy in patients with AIS. These two factors should be considered prior to clopidogrel administration.

Efficacy and Adverse Events of Clopidogrel and Ticagrelor in Patients with Cytochrome P450 2C19 Gene Deletion

To compare the safety and efficacy of clopidogrel and ticagrelor among patients with cytochrome P450 2C19*2 and *3 mutations, and to assess the necessity of pharmacogenetic testing is the objective of the study.The study is a two-center retrospective, observational cohort study of 425 patients. Compare the differences of patients with different genotypes in the rehospitalization owing to original disease, major adverse cardiovascular events, initial readmission duration, frequency and all-cause mortality, dyspnea, minor hemorrhagic incidents, as well as significant hemorrhagic incidents.No significant differences were observed between the two groups in terms of rehospitalization incidence and duration due to the primary disease, recurrent myocardial infarction, revascularization, stent thrombosis, as well as ischemic stroke (p>0.05). Nevertheless, ticagrelor-treated patients experienced a higher incidence of dyspnea events compared to those treated with clopidogrel (43.1 % vs. 31.7 %, p=0.039). Furthermore, the incidence of minor bleeding was similar between both groups (6.6 % vs. 2.8 %, p=0.184) and no major bleeding events were reported. In patients with cytochrome P450 2C19 gene deletion, clopidogrel and ticagrelor have similar efficacy, but clopidogrel has less dyspnea. Therefore, there is no more benefit from replacing clopidogrel with ticagrelor.

Editor's Summaries of the Articles Published in This Issue of Precision Medicine Communications

Editor's Summaries of the Articles Published in This Issue of Precision Medicine Communications

Antiplatelet response to clopidogrel is associated with a haplotype in CYP2C19 gene in Pakistani patients

Clopidogrel, an antiplatelet drug, is frequently prescribed to patients diagnosed with ischemic diseases such as those suffering from acute coronary syndromes or ischemic stroke. Despite the drug being effective in majority of the patients, some still experience ischemic events early in the treatment which might be due to poor platelet inhibition. This study aims to investigate the association of cytochrome P450 2C19 (CYP2C19) loss-of-function polymorphisms, haplotypes as well as a wide range of clinical and demographic variables with platelet aggregation phenotypes to clopidogrel in a Pakistani cohort. The study comprised of a total of 120 patients diagnosed with cardiovascular diseases and were treated with clopidogrel. Antiplatelet response to clopidogrel was monitored by Helena AggRAM (HL-2-1785P) and patients with maximal platelet aggregation more than 50% were categorized as low responders and those with less than 50% as high responders. Our results show that 56.6% of patients were homozygous for the CYP2C19 wild-type allele, 38.3% of patients possessed one copy of the CYP2C19*2 allele and 5% of patients possessed both CYP2C19*2 alleles. No CYP2C19*3 allele was found in our patient cohort. There was no statistically significant difference between the high and low responder groups to clopidogrel in terms of extensive, intermediate and poor metabolizer genotypes. However, haplotype (H1), leukocyte count, random blood glucose, and history of diabetes mellitus was associated with the antiplatelet response to clopidogrel. The prevalence of clopidogrel resistance in our population was in line with that reported for other regional and global populations.

Relationship between recurrent ischemic events in cerebrovascular disease and cytochrome P450 2C19 gene polymorphism on the basis of thrombelastography.

Recurrent ischemic events in cerebrovascular disease present a difficult problem in clinical practice. The predictive value of cytochrome P450 2C19 (CYP2C19) gene polymorphism and high platelet reactivity for recurrent ischemic events in cerebrovascular disease is not clear. A total of 295 patients with acute ischemic cerebrovascular disease admitted to the cerebrovascular disease center of Northern Theater General Hospital between January 1, 2020 and February 2, 2021 were enrolled in this study. Thrombelastography (TEG) was used to detect platelet reactivity and CYP2C19 gene polymorphism. Among the 118 noncarriers, 97 had normal platelet reactivity and 21 had high platelet reactivity. Of the 177 carriers, 120 showed normal platelet reactivity and 57 showed high platelet reactivity. The area under the curve (AUC) of CYP2C19 gene polymorphism in predicting recurrent ischemic events was 0.66. The regression coefficients of hypertension, stroke history, carriers, and high platelet reactivity with recurrent ischemic events were 0.341, 0.402, 0.358, and 0.281, respectively, with significant positive correlation (P<0.05). Hypertension, stroke history, carriers, and high platelet reactivity are all independent risk factors for recurrent ischemic events. CYP2C19 gene polymorphism and high platelet reactivity can be used as effective predictors of recurrent ischemic events in clinical cerebrovascular disease.

Incidence of Device-Related Thrombosisin WatchmanPatients Undergoing a Genotype-Guided Antithrombotic Strategy.

This study sought to report the incidence of device-related thrombosis (DRT) and thromboembolic (TE) events when an alternative to clopidogrel is prescribed in loss-of-function (LOF) allele carriers of the cytochrome P450 2C19 (CYP2C19) gene. LOF polymorphisms of the CYP2C19 gene are associated with reduced hepatic bioactivation of clopidogrel. A total of 1,002 Watchman patients were included. Six hundred forty-five patients underwent CYP2C19 genetic testing; among patients with clopidogrel resistance, clopidogrel was replaced by either prasugrel (pilot cohort) or half dose direct oral anticoagulant ([DOAC]/Group 1), both in combination with aspirin. We compared the incidence of DRT/TE events among genotyped patients and a control group which received standard dual antiplatelet therapy (DAPT) (Group 2; n=357). All reported events occurred during a timeframe between 45- and 180-day follow-up transesophageal echocardiograms, when the 2 different antithrombotic strategies (genotype-guided vs standard DAPT) were adopted. In the pilot cohort (n=244), bleeding events occurred in 10.2% of patients who received aspirin plus prasugrel, leading to early discontinuation of the prasugrel-based protocol. DOAC Group 1 patients (n=401), 25.7% were reduced metabolizers, and clopidogrel was replaced by half dose direct oral anticoagulant. DRT was documented in 1 (0.2%) patient of Group 1 and 7 (1.96%) patients of Group 2 (log-rank P =0.021). The composite endpoint of DRT/TE events was significantly loweramong patients receiving a genotype-guided antithrombotic strategy (0.75% vs 3.10%; log-rank P =0.017). In Watchman patients, a genotype-based antithrombotic strategy with aspirin plus half dose DOAC in reduced clopidogrel metabolizers was superior to standard DAPT with respect to DRT/TE events.

Influence of CYP2C9 Polymorphisms on Plasma Concentration of Warfarin and 7-Hydroxy Warfarin in South Indian Patients.

Warfarin is primarily metabolized by cytochrome P450 2C9 (CYP2C9) enzyme, which is encoded by the CYP2C9 gene. CYP2C9*2 and CYP2C9*3 variants significantly influence warfarin metabolism and subsequently the required dose of warfarin. The current retrospective study was aimed to determine the influence of CYP2C9 variants on warfarin metabolic ratio (MR, warfarin/7-hydroxy warfarin) and warfarin maintenance therapy in 210 patients (mean age 44.6±11.6 (SD) years; male to female ratio 81:129). High-performance liquid chromatography (HPLC) with UV detector was used to measure plasma concentrations of warfarin and 7-hydroxy warfarin. Plasma samples were collected 12 h after the previous dose of warfarin was administered. CYP2C9 variants (rs1799853 and rs1057910) were identified using real-time polymerase chain reaction allele-discrimination method. The mean daily maintenance dose of warfarin was 4.6±1.8 (SD) mg. The mean plasma warfarin and 7-hydroxy warfarin concentrations were 3.7±1.6 (SD) μg/mL and 1.1±0.54 (SD) μg/mL, respectively. Patients carrying other CYP2C9 variants required 39% lower warfarin maintenance dose 3.3±1.2(SD)mg than CYP2C9*1*1 carrier 4.9±1.8(SD)mg, (p<0.0001). MRs differed significantly between CYP2C9 variant carriers (8.1±5.1) and normal genotype carriers (4.8±3.9) (p<0.0001). Probit analysis identified an MR value of 7.6 as the anti-mode (sensitivity of 84% and specificity of 55%) to differentiate poor and intermediate metabolizers (carriers of any CYP2C9*2 or CYP2C9*3 variants) from normal metabolizers (CYP2C9*1*1 genotype). The present study results provide, insights on the effect of CYP2C9 genetic polymorphisms on inter-individual variability in warfarin metabolism and emphasizes utility of phenotyping in a setting of genotype-guided dosing of warfarin in South Indian population.

B-PO02-157 INCIDENCE OF DEVICE-RELATED THROMBOSIS IN WATCHMAN PATIENTS UNDERGOING A GENOTYPE-GUIDED ANTITHROMBOTIC STRATEGY

Loss of Function (LOF) polymorphisms of cytochrome P450 2C19 (CYP2C19) are associated with reduced hepatic bioactivation of clopidogrel. To report the incidence of device-related thrombosis (DRT) and thromboembolic (TE) events when an alternative to clopidogrel is prescribed in patients with a LOF allele of CYP2C19 gene. Among 1002 Watchman patients, 645 underwent CYP2C19 genetic testing; among patients with clopidogrel resistance, clopidogrel was replaced by either prasugrel (pilot cohort) or half dose novel oral anticoagulant (NOAC; Group 1) both in combination with aspirin. We compared the incidence of DRT/TE events among genotyped patients and a control group which received standard dual antiplatelet therapy (DAPT; Group 2; n=357). All reported events occurred during a timeframe between 45- and 180-day follow-up TEEs, when the genotype-guided or standard DAPT strategies were adopted. In the pilot cohort (n=244), bleeding events occurred in 10.2% of patients receiving prasugrel, leading to discontinuation of the prasugrel-based protocol. Among Group 1 patients (n=401), 25.7% were reduced metabolizers and clopidogrel was replaced with half dose NOAC. DRT was documented in 1 (0.2%) patient of Group 1 and 7 (1.96%) of Group 2 (log-rank p-value=0.021). The composite endpoint of DRT/TE events was significantly lower among patients receiving a genotype-guided strategy (0.75% vs. 3.1%; log-rank p-value=0.017). In Watchman patients, a genotype-based antithrombotic strategy with aspirin plus half dose NOAC in reduced clopidogrel metabolizers was superior to standard DAPT with respect to DRT/TE events.

Cytochrome P450 2C19 gene polymorphisms (CYP2C19*2 and CYP2C19*3) in chronic myeloid leukemia patients: in vitro and in silico studies

Polymorphisms in the CYP2C19 have a huge impact on drug processing, out of which CYP2C19*2 and CYP2C19*3 are the most common variants associated with reduced metabolism of drugs. Mechanism by which two variants contribute in poor metabolization of drugs and cancer is not well understood. Here, we hypothesized that the mutations in CYP2C19 gene might affect the risk of chronic myeloid leukemia patients (CML). Present study has two main objectives: first to investigate the allele frequencies of CYP2C19*2 and CYP2C19*3 associated gene polymorphisms in CML patients and to elucidate the structural stability, conformation and functions of protein encoded by such variants. Genotyping of CYP2C19 was performed in 103 CML patients and 103 matched healthy controls. Heterozygous genotype of CYP2C19*2 was higher in CML patients (13.59%) than the controls (4.85%). Whereas, CYP2C19*3 allele frequency was not observed in cases as well as in controls. Furthermore, molecular dynamics (MD) simulation was applied to monitor the structural and conformational effect of above mutants. MD simulation results demonstrated that these mutants formed unstable proteins with distorted conformations, altered residues network and affected drug binding site which led to malfunction of mutant proteins. Hence, the study provides the role of CYP2C19 gene polymorphisms in susceptibility to CML population and explored the molecular basis of malignancies caused which may aid in the development of precise medicine or adjusting the drug dosages so as to reduce the chemotherapeutic side effects. Communicated by Ramaswamy H. Sarma

Two-Pair Primers PCR-CTPP Optimization to Identify rs1057910 CYP2C9 Gene Variants in Surakarta

Cytochrome P450 2C9 (CYP2C9) gene variants (rs1057910) cause inter-individual drug dose variability. Polymerase chain reaction confronting two-pair primers (PCR-CTPP) is cost effective and time-saving method. Before genotyping a large number of samples, the PCR-CTPP need to be optimized. This research aimed to optimize PCR CTPP in order to identify rs1057910 CYP2C9 gene variant in Surakarta. Forty deoxyribose nucleic acid (DNA) samples were obtained from Universitas Sebelas Maret Hospital. The optimization steps were determining of two-pair primer ratio and the optimum PCR annealing temperature (Ta). The PCR-CTPP results were analyzed directly by agarose gel electrophoresis. The optimum Ta was at C using outer (Foward1-Reverse2) and inner (Foward2-Reverse1) primer ratio of 1:2. From 40 DNA samples, CYP2C9*1/*1 (92.5%) and CYP2C9*1/*3 (7.5%) were observed based on PCR-CTPP and sequencing results. The PCR-CTPP optimization method successfully identified the rs1057910 CYP2C9 gene variants. The results were consistent with validation using DNA sequencing.

Commiphora myrrha (Nees) Engl. resin extracts induce phase-I cytochrome P450 2C8, 2C9, 2C19, and 3A4 isoenzyme expressions in human hepatocellular carcinoma (HepG2) cells

Commiphora myrrha (Nees) Engl. (C. myrrha) resin is the most Middle Eastern herbal medicine used against numerous diseases. After being decocted or macerated, this resin is widely consumed among Saudi Arabian patients who are already under prescribed medication. Despite its popularity, no studies have been reported on potential modulation effects of these resin extracts on drug metabolism. Therefore, we studied C. myrrha resin extracts on the expression of cytochrome P450 (CYP) drug-metabolizing isoenzyme in human hepatocellular carcinoma cell line HepG2. The C. myrrha extracts were prepared by sonication and boiling, resembling the most popular traditional preparations of maceration and decoction, respectively. Both boiled and sonicated aqueous extracts were fingerprinted using high-performance liquid chromatography equipped with ultra-violet detector (HPLC-UVD). The viability of HepG2 cells treated with these aqueous extracts was determined using CellTiter-Glo® assay in order to select the efficient and non-toxic resin extract concentrations for phase-I metabolic CYP isoenzyme expression analysis. The isoenzyme gene and protein expression levels of CYP 2C8, 2C9, 2C19, and 3A4 were assessed using reverse transcription-quantitative polymerase chain reaction and Western blot technologies. The HPLC-UVD fingerprinting revealed different chromatograms for C. myrrha boiled and sonicated aqueous extracts. Both aqueous extracts were toxic to HepG2 cells when tested at concentrations exceeding 150 µg/ml of the dry crude extract. The CYP 2C8, 2C9, and 2C19 mRNA expression levels increased up to 4.0-fold in HepG2 cells treated with either boiled or sonicated C. myrrha aqueous extracts tested between 1 and 30 µg/ml, as compared with the untreated cells. However, CYP3A4 mRNA expression level exceeded the 2.0-fold cutoff when the cells were exposed to 30 µg/ml of C. myrrha extracts. The up-regulation of CYP mRNA expression levels induced by both boiled and sonicated C. myrrha aqueous extracts was confirmed at the CYP protein expression levels. In conclusion, both sonicated and boiled C. myrrha aqueous extracts modulate CYP 2C8, 2C9, 2C19, and 3A4 gene expression at clinically-relevant concentrations regardless of preparation methods. Further in vitro and in vivo experiments are required for CYP isoenzyme activity assessment and the establishment of herb-drug interaction profile for these traditional medicinal resin extracts.