Evaluation of the activity of CYP2C19 in Gujrati and Marwadi subjects living in Mumbai (Bombay)
© Panchabhai et al; licensee BioMed Central Ltd. 2006
Received: 08 May 2006
Accepted: 24 October 2006
Published: 24 October 2006
Inherited differences in the metabolism and disposition of drugs, and genetic polymorphisms in the targets of drug therapy (e.g., receptors), can greatly influence efficacy and toxicity of medications. Marked interethnic differences in CYP2C19 (a member of the cytochrome P-450 enzyme superfamily catalyzing phase I drug metabolism) which affects the metabolism of a number of clinically important drugs have been documented. The present study evaluated the activity of CYP2C19 in normal, healthy Gujrati and Marwadi subjects by phenotyping (a western Indian population).
All subjects received 20 mg of omeprazole, which was followed by blood collection at 3 hrs to estimate the metabolic ratio of omeprazole to 5-hydroxyomeprazole. The analysis was done by HPLC.
It was seen that 10.36% of this population were poor metabolizers(PM) whereas 89.63% were extensive metabolizers(EM).
A genotyping evaluation would better help in identifying population specific genotypes and thus help individualize drug therapy.
The differences among individuals in the way they respond to medications  can be attributed to inherited differences in the metabolism and disposition of drugs, and genetic polymorphisms in the targets of drug therapy (e.g., receptors) [2–4] other than the conventional factors like individual's age and race, organ function, concomitant therapy, drug interactions, and concomitant illnesses . CYP2C19, a member of the cytochrome P-450 enzyme superfamily(catalyzing phase I drug metabolism)  affects the metabolism of a number of clinically important drugs, such as proton pump inhibitors (omeprazole , lanzoprazole, rabeprazole), tricyclic antidepressants (imipramine, amitryptiline), phenytoin, propranolol and benzodiazepines (diazepam) . Marked interethnic differences in the polymorphism frequency [6, 9] have led to 21 variant alleles (CYP2C19*2 to CYP2C19*8) being documented; that predict poor metabolizers (PMs); of which the most commonly encountered ones contributing to a PM phenotype were CYP2C19*2 and CYP2C19*3 genotypes. The prevalence of PMs has been reported to be 2–5% in Caucasians [10, 11], 4–8 % in Africans  and 11–23 % in Orientals .
Polymorphisms can be determined by phenotyping and genotyping methodology. The phenotyping method employs the use of "Probe Drugs". These are drugs which are characteristically metabolized by a single enzyme system and hence can be used to classify individuals as extensive metabolizers (EMs) or poor metabolizers (PMs). The disadvantages in using older probe drugs like mephenytoin has prompted the use of others like omeprazole  and proguanil . The fact that omeprazole is almost exclusively metabolized by CYP2C19 to 5-hydroxy omeprazole and to a lesser extent by CYP3A4 to omeprazole sulphone makes it a valuable probe drug for establishing the genotype-phenotype correlation for CYP2C19.
Omeprazole is used in combination regimens for the treatment of gastric as well as duodenal ulcers, gastroesophageal reflux disease and for eradication of Helicobacter pylori infection. There exist significant differences in intragastric pH  and differences in cure rates for H. pylori infection  between extensive metabolizers (EMs) and poor metabolizers (PMs) who are treated using omeprazole. Gastric acid suppression and eradication of H. pylori infection are important determinants in the management of the pathologies mentioned vide supra. It has been shown that a higher concentration of omeprazole in PMs results in greater gastric acid suppression as compared with extensive metabolizers . Whereas the frequency of these polymorphisms in North  and South  Indians (who respectively belong to Aryan and Dravidian races) has been documented, the variations in CYP2C19 activity in Western Indian population has not been determined so far. The present study thus evaluated the activity of CYP2C19 in normal, healthy, Gujrati and Marwadi subjects by phenotyping using omeprazole as the probe drug.
The study was conducted after approval from the Institutional Review Board, and in accordance with Ethical Guidelines for Biomedical Research in Human subjects of ICMR (2000) . Written, informed consent was obtained from all participating subjects.
The study was conducted in 170 normal healthy (by history and focused clinical examination) Gujrati and Marwadi subjects residing in the state of Maharashtra, ensuring that their native places were in the states of Gujrat and Rajasthan. The sample size was calculated assuming a 12% prevalence of PMs with 95% CI at 5% significance. The prevalence for the sample size calculation was taken as 12% based on the data of 14% and 12% prevalence in North  and South  India respectively. Subjects were admitted to the Clinical Pharmacology ward and received Omeprazole 20 mg (Lomac-20®, batch no: G57688, Cipla Ltd, Mumbai, India) after an overnight fast. The drug was administered orally under direct supervision and the blood sample (10 ml of venous blood collected from an antecubital vein into 1 tube containing 100 μL Heparin) was collected in accordance with the protocol 3 hours after ingestion of the drug. Alcohol, caffeine and citrus juices were avoided for at least 48 hours before the intake of omeprazole. The plasma was separated and stored at -20°C pending further analysis. Omeprazole and 5-hydroxy omeprazole plasma concentrations were determined by reverse phase high performance liquid chromatography. The analytes were detected at 302 nm and absorbance was set at 0.005 Aufs. The sensitivity of the method and percentage extraction was 10 ng/ml and 90.0% for both drug and metabolite. The assay was found to be linear over the concentration range from 0.1 to 10.00 μg/ml with all the concentrations giving precision and accuracy within 15%. The phenotyping was done based on minor modifications of the method of Kobayashi et al. . Statistical analysis was performed using the Graphpad Instat statistical software (Graph Pad Software Inc., San Diego, CA, USA). The Kolmogorov-Smirnov test was used to test normality in the metabolic ratios.
Of 170 samples 6 samples could not be analyzed for various reasons and phenotyping was done for the remaining 164 samples. There were 110 males (67.07%) and 54 females (32.92%), the mean age of the population being 24.79 ± 11.51 (mean ± SD)(95%CI; 26.55, 23.03). Of the 164 subjects, there were 122 Gujratis (77 males and 45 females) and 42 Marwadis (32 males and 10 females). No subject reported any adverse event to the single dose of the drug.
In the study group of 164 volunteers,147 (89.63%) were EMs with a mean MR of 3.27 ± 0.36 (mean ± SEM) and 17 (10.36%) were PMs with a mean MR of 36.85 ± 3.32 (mean ± SEM). It was further observed that the 55 females, 5(09.09%) were PMs and 50(91.91%) were EMs. Among the 109 males,12(11.00%) were PMs and 97(89.00%) were EMs. In the individual subgroup of Gujratis, 4(8.88%) of the total 45 females and 10(12.98%) of the total 77 males were PMs, the % of PMs in the subgroup as a whole being 11.47%. In the Marwadi subgroup, 1(10%) of the total 10 females and 2(6.25%) of the total 32 were PMs, the % of PMs in the subgroup as a whole being 7.14%.
Discussion and conclusion
Most of the current literature related to pharmacogenetics of CYP2C19 has studied polymorphisms in Caucasians [10, 11], African-American  and Oriental populations , while far less is known about other ethnic groups. This is the first study from the country to establish the CYP2C19 phenotype frequencies in the Gujrati and Marwadi (Western Indian) population. Indian population comprises of more than 1 billion people. The population is divided into 4 major morphological types – Caucasoid, Mongoloid, Australoid and Negrito. The states of Maharashtra (Maharashtrians), Gujrat (Gujratis) and Rajasthan (Marwadis) belong to the Caucasoid morphological type . All the subjects of the study are actually migrants to the city of Mumbai (Bombay). There were 122 Gujratis and 42 Marwadis. The states of Gujrat and Rajasthan share a common boundary and have socio cultural, and linguistic similarities and hence the total sample size was taken as 164 subjects.
Data on PMs and EMs in Gujratis and Marwadis
Gujratis n = 122
Marwadis n = 42
Total N = 164
Males n = 109
PM n = 12
EM n = 97
Females n = 55
PM n = 5
EM n = 50
CYP2C19 metabolizes many clinically important drugs, notably proton-pump inhibitors, proguanil and benzodiazepines. The clinical implications of the CYP2C19 metabolism are many and varied, ranging from marked efficacy in the treatment of Helicobacter pylori infection with omeprazole in PMs to increased risk of failure of anti-malarial prophylaxis with proguanil in the same subgroup. This study was done as a part of a project to determine the polymorphisms of CYP2C19 by phenotyping as well as genotyping. The fact that 10.36% of our study population possesses the PM phenotype makes it essential to evaluate the phenotype-genotypes correlation in this population which can be done after the genotyping data is available. This is required to circumvent adverse drug reactions associated with increased accumulation of the parent drug and would also serve to decrease cost and duration of therapy, as a lower dose may suffice in the individuals who do not metabolize the drug extensively.
We wish to thank all the study participants as well as the staff of the Department of Clinical Pharmacology for their assistance in conducting the study. We also thank Mr Abhijit Karwa who rendered valuable help in volunteer recruitment. We wish to thank Cipla Ltd for the supply of omeprazole powder and capsules. The authors are also grateful to Dr Kjell Anderson from Astra Zeneca, Sweden, for the donation of 5-hydroxy omeprazole pure powder and Dr C Adithan Professor – Director in Pharmacology at JIPMER, Pondicherry for training given in pharmacogenetics. This study was done as a short term studentship (STS) project by the second author under the Indian Council of Medical Research (ICMR) undergraduate student research scheme for the year 2006 and under the aegis of the ICMR centre for advanced research in Clinical Pharmacology. The authors are also grateful to the Department of Science and Technology, for funding the HPLC under their FIST scheme.
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