Study of Pharmacokinetics of Omeprazole and Its Metabolites in Iranian Volunteers Using High Performance Liquid Chromatography
M. Motevalian PhD,* F. Keyhanfar PhD,* G. Saeedi PharmD,**
M. Mahmoudian PhD*
*Department of Pharmacology, Iran University of Medical Sciences, and **Biopharmacy Research Lab, Tehran, Iran
Background-Omeprazole is widely used in various gastric, acid-related disorders and undergoes rapid and extensive metabolism in the liver.
Methods-The pharmacokinetics of omeprazole and its metabolites were studied in nine young healthy volunteers following oral administration of two 20 mg capsules. The concentration of omeprazole and its metabolites, omeprazole sulfone and hydroxy-omeprazole, were determined in plasma by a rapid and selective method using solid-phase extraction followed by HPLC analysis.
Results-The maximum concentration of omeprazole was reached at least 1 hour after oral dosing. Significant inter-subject variability was observed within pharmacokinetic parameters of omeprazole and its metabolites in volunteers. Four subjects exhibited much higher plasma levels of omeprazole compared to the others. Analysis of serum showed the existence of another metabolite of omeprazole which has not been previously reported.
Conclusion-1) The inter-individual variations in omeprazole metabolism reported in different populations was also observed in Iranian volunteers. 2) Another metabolite of omeprazole appears to exist which requires further characterization.
Introduction
Omeprazole, a selective H/K ATPase inhibitor in gastric parietal cell, is widely used in various gastric acid-related disorders.1-3 Omeprazole undergoes rapid and extensive metabolism in the liver, the oxidative processes being predominant.4-7 Two major primary metabolites of omeprazole are hydroxy-omeprazole and omeprazole sulfone. Metabolism of omeprazole is under regulatory control of S-mephenytoin 4'- hydroxylase (CYP2C19).8-12,21 Isoenzymes of hepatic cytochrome P450 such as CYP2C19 are involved in drug metabolism. CYP2C19 is polymorphically expressed in the population and the mutant allele constitutes the recessive trait.18,19 Homozygotes completely lack CYP2C19 and present higher values of plasma concentration of omeprazole, in comparison with extensive metabolisers.10,11,20,21
It has been shown that omeprazole inhibits some isoenzymes of hepatic cytochrome P450 such as CYP2C19 resulting in increased half lives of certain pharmacologic and endogenous compounds.13-15 It may also increase the activity of other isoenzymes such as CYP1A under certain conditions.16,17
Since the 4-hydroxylation polymorphism of S-mephenytoin, catalyzed by CYP2C19, demonstrates marked inter-ethnic differences in the incidence of the PM phenotypes,22-24 the present study was carried out to study the kinetics of omeprazole and its metabolites in a group of healthy Iranian volunteers to clarify further the ethnic differences in omeprazole metabolism.
Materials and Methods
Apparatus and chromatographic conditions
For quantification of omeprazole and its metabolites, a new HPLC method developed in our laboratory25 was used. A Waters' liquid chromatograph system composed of pump model 600, UV detector model 486, integrator model 746, a 4.6 ? 250 mm m bondapak C18 column (125A0-10m m) and a m bondapak C18 precolumn were used. The mobile phase was containing acetonitrile and phosphate buffer of 0.05 M, pH 7.5 (25/75, v/v). The flow rate was 0.8 ml min-1 and injection volume was 50 m l. The column effluent was monitored by a UV-absorption measurement at 302 nm, and chromatograms were traced on an integrator to determine peak heights. The procedure were performed at room temperature. Quantification was based on peak height ratio using phenacetin as internal standard.
Standard solutions
Standard solutions for plasma determination of omeprazole and metabolites were prepared by dissolving omeprazole in acetonitrile (1mg/mI), omeprazole sulfone and hydroxy-omeprazole in methanol (1mg/mI). Serial dilutions were then made in fresh double distilled water. Blank plasma (1 ml) was spiked with different amounts of standard solutions in order to prepare plasma standards at the time of slat analysis.
Subjects and sample collection
Nine healthy male volunteers, aged 28-37 years (mean 33?0.72 years), 55-85 kg body weight, from whom informed consent was obtained, participated in this study. The protocol of the study was approved by an institutional ethical committee. Each subject was given two 20 mg capsules of omeprazole (Losec) after overnight fasting and blood samples were collected at 0.0, 0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 4.0, 6.0 and 8.0 hours after drug administration. All volunteers were non-smokers and had not taken any form of medication from one week prior to and during the experiment. Volunteers were also requested not to consume coffee or tea 24 hours before or during the trial. A light breakfast was given 2 hours after drug administration and a standard lunch 4 hours after administration.
Sample preparation
Heparinized blood samples from various volunteers were centrifuged and the plasma collected and stored at ?20? C until the day of study. The frozen plasma was allowed to thaw at room temperature just before the extraction procedure, it was then mixed and centrifuged at 3000 rpm for 15 minutes. Then 1 ml of the sample was transferred to a clean glass tube, 100 m l of internal standard was added and mixed (final internal standard concentration was 5 m g/mI).
Whatman C18 cartridges (3 ml syringes containing 500 mg packing material) were placed on top of a vacuum manifold model A1 6000 (Analytichem International, USA), pre-washed with 1 ml methanol to elute any impurities from the cartridges and to wet the C18 packing prior to the introduction of plasma-samples. Then the mixture of plasma and internal standard was passed through the cartridge followed by 2 ml filtered double distilled water and 1 ml of phosphate buffer (0.125 M, pH-8). The effluent was discarded. Then omeprazole and metabolites were eluted with 500 m l of acetonitrile. This fraction was finally centrifuged and aliquots of each sample (50 m l) were injected to HPLC system at ambient temperature as described previously. Column eluate was monitored at 302 nm.
Materials
Omeprazole standard and omeprazole sulfone were generously donated by Union Quimico, Farmaceutica (Mallora, Barcelona) and hydroxy-omeprazole was a kind gift from Astra hassle (Molndal, Sweden). Acetonitrile of HPLC grade was purchased from Merck (Germany) and Methanol of HPLC grade from Daroupakhsh (Tehran, Iran). All other reagents were of analytical grade.
Results
The applied extraction procedure and HPLC method25 is capable of simultaneous determination of omeprazole and its metabolites in serum samples. Representative chromatograms are presented in Fig. 1. The results of plasma samples of all volunteers after an oral dose of 40 mg of omeprazole are presented in Fig. 2.
Omeprazole
The pharmacokinetic values of omeprazole are shown in Tables 1,2. The plasma concentration-time curves for each subject are shown in Fig. 2A. There was a marked inter-subject variation in Cmax values of omeprazole following 40 mg oral dose (Fig. 2A). The mean Cmax for subjects with high and low maximum concentrations were 2556.2?1100 and 322.16?93 ng/ml respectively (Table 2). The mean tmax value was 1.89?0.5 hours (Table 1). The half-life of omeprazole in the terminal phase of the plasma concentration-time curve was relatively consistent in all subjects, with the mean?SEM of 2.3?0.9 hours.
Omeprazole sulfone
Omeprazole sulfone was found to a varying extent in plasma from all individuals. Fig. 2B shows the plasma concentration-time profile of omeprazole sulfone, the profile being similar to the parent drug. The omeprazole sulfone concentration approached the peak level within three hours in most subjects, with the mean value of 2.15?0.7 hours. The mean Cmax values for subjects with high and low maximum concentrations were 585?258 and 70.02?34 ng/ml respectively (Table 2). The half-life of the omeprazole sulfone was found to be two times longer than that of omeprazole, the mean t2 being 3.16?0.9 hours (Table 1).
Hydroxy-omeprazole
Hydroxy-omeprazole was found in the plasma of all subjects studied. The results indicate that there were similar Cmax values of hydroxy-omeprazole in our subjects. The mean Cmax values was 378.5?65 ng/ml in 7 subjects and only one individual had a much lower Cmax value of 86 ng/ml (Table 2). The elimination half life of hydroxy-omeprazole is three times longer than that of omeprazole. The mean t2 was 4.498?2.2 hours (Table 1). Fig. 2C shows individual concentration-time curves for hydroxy-omeprazole after an oral dose of 40 mg of omeprazole.
Other metabolites
Another metabolite of omeprazole was found in the plasma of most volunteers with a retention time less than that of omeprazole (Fig. 1D). Plasma levels of this metabolite (X), which has not been reported previously, is shown in Fig. 2D. Following oral administration of 40 mg omeprazole, the X metabolite appeared in the plasma of three of our subjects (Table 1), who displayed a level four times higher compared to other volunteers. Those subjects with higher plasma concentrations of X metabolite could be divided to two groups, with two distinct and different ranges of tmax values. The mean tmax values were 1.5?0.3 and 6.0?0.0 hours. Fig. 2D shows the plasma level versus time curve of X metabolite for each individual separately.
Discussion
Our data in the present study show some peculiar features of the pharmacokinetics of omeprazole in this small group of Iranian volunteers. After administration of an enteric coated formulation of omeprazole, there is a lag time in absorption of the drug, the peak occurring after 1 hour in most individuals. After oral administration, four subjects (No.s 5,6,8&9, Table 3) showed significantly higher concentrations of omeprazole than the rest of the subjects. The pronounced inter-individual variability in the pharmacokinetic profile of omeprazole observed in this study, has also been reported previously by others.4,27-29 The observed higher concentration in the above mentioned subjects may be due to a greater bio-availability of omeprazole in these four individuals compared to others, and might have resulted from a higher degree of absorption of omeprazole in the gastrointestinal tract. Known to be unstable in acid conditions, the enteric coating of omeprazole would be expected to confer a substantial degree of protection from acid degradation. This is however, unlikely to be complete and may show inter-individual variations. Since omeprazole is metabolized completely and its urinary recovery in the unchanged form is negligible,3,4 it seems likely that hepatic enzyme activity could be an alternative reason for the existence of inter-individual differences observed in the pharmacokinetic profile of omeprazole in our subjects.
The plasma concentration-time profile and kinetic variable of omeprazole sulfone showed a behavior similar to that of the parent drug (Fig. 2A,B) which is in agreement with previous reports.10,30 The Cmax and tmax values of hydroxy-metabolite seems to be less variable between our volunteers.
A new finding in this study is the appearance of an extra peak in the chromatogram of the plasma samples of most of our volunteers (7 out of 9) and does not exist in blank samples. Considering the preliminary mass spectrometric data31 and the time course of its concentration, this peak may be an unknown metabolite of omeprazole (X metabolite). This X metabolite has not been previously reported. The reason could be the liquid-liquid extraction procedure used by other workers. Alternatively, its peak might have been covered by the peak of internal standard in HPLC chromatograms. H 168/24 which has been used as an internal standard in most other studies26 has a similar retention time to this metabolite. It was found that those volunteers who had low levels of omeprazole sulfone in plasma did not produce X-metabolite in a detectable level (Table 3). This may indicate that the X-metabolite might be a secondary metabolite of omeprazole sulfone. Further work is under way to identify and characterize this metabolite.
Acknowledgement
The Authors would like to thank Dr. F. Salek-Moghadam, Dr. L. Tayebi and Dr. M. Amini for their assistance during the course of this study. The financial support of Dr. Abidi Pharmaceutical Co. is gratefully acknowledged.
References
1 Holt S, Howden CW. Omeprazole: overview and opinion. Dig Dis Sci 1991; 36:385-93.
2 McTavish D, Buckley MMT, Heel RC. Omeprazole: an updated review of its pharmacology and therapeutic use in acid-related disorders. Drugs 1991; 42:138-70.
3 Maton PN. Omeprazole. N Eng J Med 1991; 324:965-75.
4 Regardh CG, Andersson T, Lagerstrom P, et al. The pharmacokinetics of omeprazole in humans. A study of single intravenous and oral doses. Ther Drug Monit 1990; 12:163-72.
5 Regardh CG, Gabrielsson M, Hoffman KJ, et al. Pharmacokinetics and metabolism of omeprazole in animals and man. Scan J Gastroenterol Suppl 1985; 108:79-94.
6 Regardh CG. Pharmacokinetics and metabolism of omeprazole in man. Scan J Gastroenterol Suppl 1986; 118:99-104.
7 Cederberg C, Andersson T, Skanberg I. Omeprazole pharmacokinetics and metabolism in man. Scan J Gastroenterol Suppl 1989; 166:33-40.
8 Yasuda S, Horai Y, Tomono Y, et al. Comparison of the kinetic disposition and metabolism of E3810, a new proton pump inhibitor, and omeprazole in relation to S-mephenytoin-4'-hydroxylation status. Clin Pharm Ther 1995; 58(2):143-54.
9 Rost KL, Roots I. Nonlinear kinetics after high-dose omeprazole caused by saturation of genetically variable cyp 2C19. Hepatology 1996; 23(6):1491-6.
10 Sohn DR, Kobauashi K, Chiba K, et al. Disposition kinetics and metabolism of omeprazole in extensive and poor metabolizers of S-mephenytin-4'-hydroxylation recruited from an oriental population. J Pharm Exper Ther 1992; 262(3):1195-202.
11 Caraco Y, Wilinson GR, Wood AJ. Differences between white subjects and chinese subjects in the in vivo inhibition of cyp2C19, cyp2D6 and cyp3A by omeprazole. Clin Pharm Ther 1996; 60(4):396-404.
12 Balian JD, Sukhova N, Harris JW, et al. The hydroxylation of omeprazole correlates with S-mephenytoin metabollism: A population study. Hepatology 1995; 23(3):268-77.
13 Lampen A, Christians U, Guengerich FP, et al. Cytochrome p450 drug interaction and interindividual variability. Drug Metabol Dispos 1995; 23(12):1315-24.
14 Andersson T, Andren K, Cederberg C. Effect of omeprazole and cimetidine on plasma diazepam levels. Eur J Clin Pharmacol 1990; 47:79-85.
15 Diaz D, Fabre I, Danjat M, et al. Omeprazole is an aryl hydrocarbon-like inducer of human cytochrome p450. Gastroenterology 1990; 99:737-47.
16 Rost KL, Roots I. Accelerated caffeine metabolism after omeprazole treatment is indicated by urinary metabolite ratios. Clin Pharm Ther 1994; 55(4):402-11.
17 Rost KL, Brosiche H, Brockmoller J, et al. Increase of cytochrome p450IA2 activity by omeprazole. Clin Pharmacol Ther 1992; 52:170-80.
18 Inaba T, Jurima M, Kalow W. Family studies of mephenytoin hydroxylation deficiency. Am J Hum Genet 1986; 38:768-72.
19 Ward SA, Goto F, Nakamura K, et al. S-mephenytin-4'-hydroxylation is inherited as an autosomal trait in Japanese families. Clin Pharm Ther 1987; 42:96-9.
20 Andersson T, Regardh CG, Lou YC. Polymorphic hydroxylation of S-mephenytoin and omeprazole metabolism in caucasian and chinese subjects. Pharmacogenetics 1992; 2:25-31.
21 Chang M, Tybring G, Dahl ML, et al. Interphenotype differences in disposition and effect on gastrin levels of omeprazole-Suitability of omeprazole as a probe for cyp2C19. Br J Clin Pharmacol 1995; 39:511-8.
22 Bertilsson L, Lou YQ, Du YL. Pronounced differences between native Chinese and Swedish populations in the polymorphic hydroxylations of debrisoquine and S-mephenytoin. Clin Pharm Ther 1992; 51:388-97.
23 Sohn DR, Kusaka M, Ishizaki T, et al. Incidence of S-mephenytoin hydroxylation deficiency in Korean population and the interphenotipic differences in diazepam pharmacokinetics. Clin Pharm Ther 1992; 52:160-9.
24 Bertilsson L, Kalow W. Why are diazepam metabolism and polymorphic S-mephenytoin hydroxylation associated with each other in white and Korean populations, but not in chinese population? Clin Pharm Ther 1993; 53:608-10.
25 Saeedi G, Motevalian M, Tayebi L, et al. A new method for the measurement of omeprazole in plasma by HPLC. 13th Iranian Congress of Pharmacology and Physiology. Isfahan, 1997:627-8.
26 Amantea MA, Narang PK. Improved procedure for quantitation of omeprazole and metabolites using reversed-phase HPLC. J Chroma 1988; 426:216-22.
27 Larson C, Cavuto NJ, Flochhart DA, et al. Bioavailability and efficacy of omeprazole given orally and by nasogastric tube. Dig Dis Sci 1996; 41(3):475-9.
28 Ishizaki T, Sohn DR, Kobayashi K, et al. Interethnic differences in omeprazole metabolism in the two S-mephenytoin hydroxylation phenotypes studied in caucasians and orientals. Ther Drug Monit 1994; 16:214-5.
29 Andersson T, Regardh CG, Dahl-Puustinen ML, et al. Slow omeprazole metabolizers are also poor S-mephenytoin hydroxylators. Ther Drug Monit 1990; 12: 415-6.
30 Andersson T, Cederberg C, Regardh CG, Skanberg I. Pharmacokinetics of various single intravenous and oral doses of omeprazole. Eur J Pharmacol 1990; 39:195-7.
31 Motevalian M, Keyhanfar F, Amanlou M, Mahmoudian M. Study of pharmacokinetics of omeprazole and its metabolites in human by HPLC using extraction. 13th IUPHAR Congress. Munich, 1998.