|Year : 2016 | Volume
| Issue : 3 | Page : 248-251
Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping
Balkrishna D Swar1, Shital R Bendkhale1, Abbas Rupawala1, Kannan Sridharan2, Nithya J Gogtay1, Urmila M Thatte1, Nilima A Kshirsagar1
1 Department of Clinical Pharmacology, Seth GS Medical College and KEM Hospital, Mumbai, Maharashtra, India
2 Department of Health Sciences, Fiji National University, Suva, Fiji
|Date of Submission||05-Jan-2016|
|Date of Decision||22-Mar-2016|
|Date of Acceptance||27-Apr-2016|
|Date of Web Publication||23-May-2016|
Dr. Nithya J Gogtay
Department of Clinical Pharmacology, Seth GS Medical College and KEM Hospital, Mumbai, Maharashtra
Source of Support: None, Conflict of Interest: None
Objectives: Cytochrome P450 2C9 (CYP2C9) is a member of cytochrome P450 (CYP) family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs. Considering the paucity of data on the polymorphisms of CYP2C9 in Western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using flurbiprofen (FLB) as a probe drug.
Materials and Methods: A 100 mg FLB capsule was administered to 298 healthy adult participants. Venous blood samples were analyzed at 2 h postdose for the estimation of FLB and 4-hydroxy FLB. Metabolic ratio (MR) was calculated to determine the extent of poor metabolizer (PM) and rapid metabolizer status using probit plot. Genotyping of CYP2C9 polymorphism was performed using polymerase chain reaction-restriction fragment length polymorphism technique.
Results: Of the total 298 participants, phenotype was assessable in 288 and genotype was performed in 289 participants. The median (range) MR of the study population was 6.6 (1.65–66.05). Five participants were found to be PMs by phenotype. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. A significant association between phenotype and genotype was observed.
Conclusion: To conclude, the present study found significant association of CYP2C9 activity by both phenotype and genotype and these findings have to be corroborated in different kinds of patients.
Keywords: Flurbiprofen, genotyping, phenotyping, polymorphism, probe drug
|How to cite this article:|
Swar BD, Bendkhale SR, Rupawala A, Sridharan K, Gogtay NJ, Thatte UM, Kshirsagar NA. Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping. Indian J Pharmacol 2016;48:248-51
|How to cite this URL:|
Swar BD, Bendkhale SR, Rupawala A, Sridharan K, Gogtay NJ, Thatte UM, Kshirsagar NA. Evaluation of cytochrome P450 2C9 activity in normal, healthy, adult Western Indian population by both phenotyping and genotyping. Indian J Pharmacol [serial online] 2016 [cited 2020 Jan 22];48:248-51. Available from: http://www.ijp-online.com/text.asp?2016/48/3/248/182885
| » Introduction|| |
Genetic polymorphisms in the drug metabolizing enzymes are one of the factors implicated in interindividual variability of the response to a drug.,, Cytochrome P450 (CYP) group of enzymes are the most widely studied and cytochrome P450 2C9 (CYP2C9) is a member of this family that accounts for nearly 18% of the total CYP protein content in the human liver microsomes and catalyzes almost 15–20% of the drugs in current clinical use, namely, warfarin, phenytoin, glipizide, tolbutamide, losartan, aspirin, diclofenac, naproxen, and flurbiprofen (FLB).
Several variant alleles have been identified for CYP2C9 enzyme (CYP2C9*1 to CYP2C9*11) of which the common ones that are reported include CYP2C9*1, CYP2C9*2 and CYP2C9*3., Of these, CYP2C9*1 is the wild type allele and the other two variants have been shown to exhibit a relatively less enzymatic activity. A recent systematic review  concluded that in comparison to the patients with CYP2C9*1/*1 genotype, the CYP2C9*1/*2, CYP2C9*1/*3, CYP2C9*2/*2, CYP2C9*2/*3, and CYP2C9*3/*3 patients required warfarin doses that were 19.6%, 33.7%, 36.0%, 56.7%, and 78.1% lower, respectively. A study  done in South and North Indian population has shown a prevalence of 0.6%, 9% and 5%, 11% for CYP2C9*2 and *3 alleles, respectively. One of the ways to assess the activity of an enzyme is by using a probe drug and studies have shown that FLB is exclusively metabolized by CYP2C9. Considering the paucity of data on the polymorphisms of CYP2C9 in western Indian population, the present study was conducted to evaluate the prevalence of CYP2C9 polymorphisms (*1, *2 and *3) and correlate it with the activity using FLB as a probe drug.
| » Materials and Methods|| |
Ethics and Study Participants
The study was conducted between April 2009 and June 2010 after obtaining approval from the Institutional Ethics Committee and written informed consent from all the study participants. Healthy participants (of either gender between 18 and 45 years) assessed as being healthy based on history and physical examination and who had at least two previous generations permanently residing in Western India (Maharashtra, Gujarat, and Rajasthan) were screened for this study. They refrained from consuming either caffeine or alcohol 48 h prior to the study entry. Those with history of any chronic disease or on any nutritional/herbal supplements or having consumed any drug in the last 7 days, nicotine and any history of or suffering from gastrointestinal, renal, liver, cardiovascular, respiratory, central nervous system and endocrine diseases were excluded from the study.
The eligible participants were administered a single capsule of 100 mg FLB orally with 100 ml of water after an overnight fast. Ten milliliters of blood sample (5 ml in heparin for FLB analysis and 5 ml in ethylenediaminetetraacetic acid for genotyping) was collected in a heparinized tube 2 h postdose for the estimation of FLB and its metabolite, 4-hydroxy FLB (4-OH-FLB). The plasma was separated and stored at −20°C pending analysis.
Phenotyping was done by the analysis of FLB and 4-OH-FLB by reverse phase high-performance liquid chromatography using minor modification of the method as described by Tracy et al . The metabolic ratio (MR) was calculated by FLB/4-OH-FLB (2 h concentration), which gave the extent of hydroxylation by the CYP2C9 enzyme. Genotyping was done by a method as described by Gu et al .
Descriptive statistics were used for demographic data and normality of MR assessed by Kolmogorov–Smirnov test. Logarithmic values were obtained for each MR and a frequency histogram plotted. Probit plot was prepared with log MR on x -axis and probit values on y -axis. Trendlines were added to the plot to get the best linear fit. Based on the selected trendline, a polynomial equation of regression was obtained. Intercept at the x -axis was considered as the antimode. Individuals with log MR equal to or higher than the antimode were classified as poor metabolizers (PMs) and those with values less than antimode were categorized as rapid metabolizers. These are expressed in proportions with 95% confidence intervals (CIs).
The allelic frequencies of CYP2C9*1, *2 and *3 were represented using percentages and 95% CI. Chi-square test for independence was used to assess whether they follow Hardy–Weinberg equilibrium. Association of genotype and phenotype was performed using Chi-square test for association and the trend analysis of the MR with genotype was done with Jonckheere Terpstra test. A P < 0.05 was considered statistically significant. All the statistical tests were performed using SPSS version 17.0 (SPSS Inc., released 2008, SPSS statistics for Windows, version 17.0, SPSS Inc., Chicago, IL, USA). As no previous data were available on the prevalence of variant genotypes in western India, no formal sample size calculations were performed.
| » Results|| |
A total of 300 participants were screened and 298 were enrolled. Of these, DNA could not be extracted for nine participants and phenotyping could not be done for ten participants and therefore, the final analysis consisted of 289 participants for genotyping and 288 for phenotyping. The demographic details of the study participants are shown in [Table 1]. Seven participants have reported mild gastritis following the drug administration.
The median (range) MR of the study population was 6.6 (1.65–66.05). The frequency histogram and probit plot of the MR were plotted and it showed a bimodal distribution as shown in [Figure 1]. The probit values were obtained and the probit plot [Figure 2] gave an antimode of 29.51 (log MR = 1.47). Those participants with an MR > 29.51 were labeled as PMs (n = 5 [1.7%]). All these PMs were males; two were from Gujarat while three were from Rajasthan.
|Figure 2: Probit plot after single dose of flurbiprofen. MR: Metabolic ratio|
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The ladder pattern of different genotypes is represented in [Figure 3]a and [Figure 3]b. Of the total 289 participants, 209 (72.3%) (66.7, 77.2) had CYP2C9*1/*1, 25 (8.7%) (5.8, 12.7) with CYP2C9*1/*2, 55 (19%) (14.8, 24.1) had CYP2C9*1/*3, 3 (1%) (0.3, 3.3) had CYP2C9*2/*3 genotype. There were no individuals with homozygous *2 or *3. The allelic frequencies (%) for *1, *2 and *3 were 86.2 (83, 88.8), 4.3 (3.3, 7) and 9.5 (7.8, 12.8), respectively, and were found to follow Hardy–Weinberg equilibrium (P > 0.05).
|Figure 3: (a) Ladder pattern of CYP2C9*2 genotype. Well 1 - 100 base pair ladder, well 2 - uncut, Well 3 - heterozygote mutant (positive control, well 4, 5, 6, 7, 8 - wild type, well 9 and 10 - heterozygote mutants. (b) Gel picture of CYP2C9*3 genotype. Well 1 - 100 base pair ladder, well 2 - uncut, well 3 - heterozygote mutant (positive control), well 4, 5, 6, 8, 10 - wild type, well 7 and 9 - heterozygote mutants. CYP2C9: Cytochrome P450 2C9|
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Genotype and Phenotype Correlation
Both the genotype and phenotype data were available for 281 participants. A significant association and trend was observed in the MR for different genotypes [Table 2].
|Table 2: Association of genotype and phenotype in normal, healthy, adult Western Indian population (n=281)|
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| » Discussion|| |
The present study evaluated the CYP2C9 activity both by phenotype (FLB as a probe drug) and by genotype of CYP2C9*1, *2, and *3 polymorphisms in adult healthy Western Indian participants. We found 1.7% of the participants to be PMs by phenotype and 27.7% participants harboring any of the *2 or *3 alleles. We also found a significant association between the phenotype and genotype of the study participants.
FLB is an orally administered, almost completely bioavailable, nonsteroidal analgesic primarily metabolized by oxidation through CYP2C9 in liver. Studies have shown that FLB is a good probe drug for evaluating CYP2C9 activity ,, including even when administered as a cocktail with other probe drugs. The T max(time taken for maximum concentration to be achieved in blood) of FLB has been reported to be 2.0 ± 1.0 h. Hence, we collected the blood sample at 2 h for the estimation of parent and its metabolite. To the best of our knowledge, there is no published literature on the evaluation of CYP2C9 activity in any of the populations using FLB and its estimation from the blood sample. However, studies , done with phenytoin as a probe drug have shown similar results of CYP2C9 activity as like that of the present study.
Majority (72.3%) of the individuals in the present study were found to have wild type of CYP2C9 gene. The study population consists of Maharashtrians, Marwaris (living in Rajasthan), and Gujaratis which are classified as Caucasoid. Since these three states share boundaries and have sociocultural and linguistic similarities, they were together taken as one population. The genetic polymorphisms of CYP2C9 in the present are found to be similar to other populations including North and South Indians.,, CYP2C9 enzyme is mainly involved in the metabolism of narrow therapeutic indexed drugs such as warfarin and phenytoin. Studies have shown that individuals with mutant CYP2C9 alleles require lesser doses of warfarin and phenytoin for their appropriate therapeutic effect.,, A recent randomized study also suggests that genotype-based titration of warfarin dose is associated with more time to be spent on the therapeutic range of international normalized ratio and lesser incidence of over anticoagulation as compared to routine clinical practice. Also, one of our previous studies has shown a significant association of CYP2C9 polymorphisms with toxic concentrations of phenytoin in the serum. We also found a significant association of metabolizing status with CYP2C9 genotype with an increased chance of being a PM with the presence of mutant allele/s. However, one out of 204 (0.005%) individual with wild genotype was found to be a PM. This reiterates the fact that the presence of a wild genotype increases the chances of an individual to have normal activity of the enzyme but not necessarily. Hence, the evaluation of CYP2C9 polymorphism becomes important in a patient before prescribing any of these drugs. However, cost-effectiveness studies evaluating the pre-prescription genotyping-based treatment initiation need to be undertaken before firm recommendation of the same. The present study and a study done in Caucasians  have shown that FLB predicts CYP2C9 genotype especially the presence of *3 allele. Evaluation of the CYP2C9 polymorphism by using FLB is also relatively cheap as compared to genotyping. However, it is hampered by a complicated protocol of testing, risks of adverse drug reactions, problem with incorrect phenotype assignment due to co-administration of drugs, and confounding effect of disease.
Over thirty polymorphic alleles have been identified in CYP2C9. Our study was limited with the fact that we looked only two of these alleles, *2 and *3, which are the common ones along with *1. The restriction fragment length polymorphism methodology itself is limited by the fact that an allele which was neither a *2 or *3 is assumed to be wild. Although only a single 2 h sampling was performed in the present study, considering the variability of T max, multiple time point assessment would serve as the best strategy. Additionally, we did not carry out any a subgroup analysis specific to gender.
| » Conclusion|| |
To conclude, the present study found a significant association of CYP2C9 activity by both phenotype and genotype. These findings have to be corroborated in different kinds of patients.
We thank all the volunteers for participating in the study. The authors also wish to thank Dr. Tim Tracy for sharing the metabolite with us, FDC limited for providing the pure powder of flurbiprofen and Dr. Sanjay Oak, Dean, Seth GS Medical College, and KEM Hospital for allowing us to use the institutional facilities for the purpose of the study.
Financial Support and Sponsorship
Conflicts of Interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2]