Optimal Dose of Omeprazole for CYP2C19 Extensive Metabolizers in Anti-Helicobacter pylori Therapy: Pharmacokinetic Considerations

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1 July 2002 Notes Biol. Pharm. Bull. 25(7) (2002) 923 Optimal Dose of Omeprazole for CYP2C19 Extensive Metabolizers in Anti-Helicobacter pylori Therapy: Pharmacokinetic Considerations Tomoko KITA, a Toshiyuki SAKAEDA, a Nobuo AOYAMA, b Toshiyuki SAKAI, c Yuko KAWAHARA, a Masato KASUGA, c and Katsuhiko OKUMURA*,a a Department of Hospital Pharmacy, School of Medicine, Kobe University; b Department of Endoscopy, School of Medicine, Kobe University; and c Division of Diabetes, Digestive and Kidney Diseases, Department of Clinical Molecular Medicine, Graduate School of Medicine, Kobe University; Chuo-ku, Kobe , Japan. Received January 28, 2002; accepted April 2, 2002 In anti-helicobacter pylori therapy using omeprazole and antimicrobials, the efficacy can be related to the CYP2C19 genotype groups; the eradication rates were 83% in extensive metabolizers and 100% in poor metabolizers. The present study was undertaken to help predict the optimal dosage of omeprazole for extensive metabolizers in this therapy. Seven healthy Japanese subjects, classified based on the CYP2C19 genotype into extensive metabolizers (n 4) and poor metabolizers (n 3), participated in this study. Each subject received a single oral dose of omeprazole 20, 40, and 80 mg, with at least a 1-week washout period between each dose. Plasma concentrations of omeprazole and its two metabolites were monitored for 12 h after each dose of medication. After each dose was administered, the pharmacokinetic profiles of omeprazole and its two metabolites were significantly different between extensive metabolizers and poor metabolizers. The area under the plasma concentration time curve (AUC) of omeprazole in extensive metabolizers was disproportionally increased 3.2- or 19.2-fold with dose escalation from 20 to 40 or 80 mg omeprazole, respectively. In contrast, the AUC of omeprazole was proportionally increased with the higher dose in poor metabolizers. The AUC of omeprazole after 20 mg administration to poor metabolizers was almost equal to the AUC in extensive metabolizers after 80 mg administration. In anti-h. pylori therapy, the recommended dose of omeprazole for extensive metabolizers is suggested to be a maximum of 80 mg 2/d based on pharmacokinetic considerations. Key words CYP2C19 genotype; nonlinear kinetics; omeprazole Proton pump inhibitors are occasionally used in therapy for eradicating Helicobacter pylori in the combination with antimicrobials, 1 5) for the prevention of gastric and duodenal ulcer diseases. 1,2) Triple therapy using omeprazole, amoxicillin, and clarithromycin is now recommended for anti-h. pylori therapy because of its high eradication rate (greater than 90%), 3 5) although problems with this therapy have arisen including the resistance of H. pylori to clarithromycin by failure. 2,3) Omeprazole is extensively metabolized into the primary metabolites of 5 -hydroxyomeprazole and omeprazole sulfone (Fig. 1). Formation of the 5 -hydroxyomeprazole is mediated mainly by cytochrome P450 2C19 (CYP2C19) with only a minor contribution of CYP3A4 and others. 6) CYP2C19, often referred to as S-mephenytoin 4 -hydroxylase, shows genetically determined polymorphism of enzyme activity with bimodal distributions. 7) In 1994, two mutant alleles, CYP2C19*2 and CYP2C19*3, were found in addition to a wild-type allele, CYP2C19*1, and six different genotypic patterns were theoretically defined. 8 10) In our previous studies, the pharmacokinetics of omeprazole and gastric ph after omeprazole administration in healthy subjects were also defined by bimodal distribution, and two groups were genetically assigned based on CYP2C19 genotypes: extensive metabolizers and poor metabolizers. 11,12) In anti-h. pylori therapy using omeprazole (20 mg 2/d for 1 week), amoxicillin (500 mg 4/d for 1 week), and clarithromycin (200 mg 4/d for 1 week), patients who were poor metabolizers all achieved eradication, while 17% of extensive metabolizers failed. 13,14) Several studies have shown that this therapeutic inefficacy might be overcome by high-dose omeprazole. 1) Recently, Furuta et al. 15) empirically reported that an extra-high dose of omeprazole (40 mg 3/d) achieved eradication in an extensive metabolizer. In the present study, a single oral dose of omeprazole 20, 40, and 80 mg was administered to 7 healthy Japanese subjects with the CYP2C19 genotype. The recommended dose of omeprazole for extensive metabolizers was estimated in anti-h. pylori therapy based on pharmacokinetic considerations. MATERIALS AND METHODS Chemicals Omeprazole and its two metabolites, 5 -hydroxyomeprazole and omeprazole sulfone, were obtained Fig. 1. Metabolic Pathways of Omeprazole The thickness of arrows indicates the approximate contribution of CYP isoforms to each of the metabolic pathways. To whom correspondence should be addressed. okumurak@kobe-u.ac.jp 2002 Pharmaceutical Society of Japan

2 924 Vol. 25, No. 7 from AstraZeneca Ltd. (London, U.K.). All other chemicals were of reagent grade and obtained commercially. Subjects Seven unrelated H. pylori-negative males agreed to participate in the study. The CYP2C19 genotype was determined using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 13,14) They were classified into two groups: 4 of the subjects were extensive metabolizers (CYP2C19*1/CYP2C19*1); and the remaining 3 subjects were poor metabolizers (CYP2C19*2/ CYP2C19*2 (n 1) and CYP2C19*2/CYP2C19*3 (n 2)). The demographics of age and weight for extensive metabolizers and poor metabolizers were not different and were and years (mean S.D.) and and kg, respectively. None of the subjects had hepatic or renal dysfunction or had taken any medication, including alcohol or over-the-counter drugs, for at least 1 week before and during the study. Written informed consent was obtained from all subjects before the study commenced. The protocol for the study was approved in advance by the Institutional Review Board of the Kobe University School of Medicine in Kobe, Japan. Study Protocol Each subject received a single oral dose of omeprazole (Omepral tablet, AstraZeneca Ltd.) 20, 40, and 80 mg, with at least a 1-week washout period between each dose. Each dose was taken more than 3 h after breakfast, and lunch was served 3 h after drug ingestion. Venous blood samples were collected prior to and at 1, 2, 3, 4, 6, and 12 h after taking the medication. Plasma samples were separated after centrifugation at 900 g for 10 min and were stored at 20 C until analysis. It was confirmed that there was no alteration in the concentrations of omeprazole and its two metabolites during storage. Concentration Measurements Plasma concentrations of omeprazole and its two metabolites, 5 -hydroxyomeprazole and omeprazole sulfone, were measured as described previously. 11,12) Briefly, 100 ml of 0.1 mg phenacetin/ml methanol (internal standard) and 2 ml of diethyl etherdichloromethane (7 : 3, v/v) were added to 0.5 ml of each plasma sample. They were extracted twice by shaking for 10 min and the mixture was centrifuged at 900 g for 10 min. Then, 0.5 ml of propylene glycol was added to the supernatant, and the solvent was evaporated under a nitrogen stream at 40 C. The residue was then dissolved in 300 ml of the mobile phase and filtered using a 0.2-mm filter (HPLC Sample Prep LCR4-LG, Nihon Millipore Ltd., Tokyo, Japan). Fifty-microliter aliquots were injected into the HPLC system. Pharmacokinetic Analysis The maximum plasma concentration (C max ) was obtained graphically. The areas under the plasma concentration time curve (AUC) of omeprazole and its two metabolites were calculated using the linear trapezoidal rule from 0 to 12 h. According to the rational pharmacokinetic notation, the AUC of 5 -hydroxyomeprazole and omeprazole sulfone depended on the omeprazole, and the AUC of each metabolite was corrected by dividing by that of the omeprazole to consider the metabolic processes more appropriately. Statistical Analysis Equivalence of variance was tested using the F-test. Statistical comparisons were performed using the unpaired t-test. p values of less than 0.05 were considered significant. RESULTS Extensive Metabolizers Plasma concentration time curves of omeprazole and its two metabolites in extensive metabolizers after each dose administration are shown in Fig. 2 (left panel). Plasma concentrations of omeprazole were disproportionally increased with higher doses. The C max and AUC of omeprazole were increased 3.2- and 19.2-fold with dose escalation from 20 to 40 or 80 mg, respectively (Table 1). Plasma concentrations of 5 -hydroxyomeprazole in extensive metabolizers were proportionally increased with the higher dose. The C max and AUC of 5 -hydroxyomeprazole were proportionally increased 1.5- and 4.0-fold and 1.9- and 4.5-fold with dose escalation from 20 to 40 or 80 mg. In contrast, the plasma profiles and pharmacokinetic parameters of omeprazole sulfone corresponded to those of omeprazole. The significant decrease in the ratio of the AUC for 5 -hydroxyomeprazole to the AUC for omeprazole (AUC H-OPZ / AUC OPZ ) was seen at the dose of 80 mg, although the ratio of the AUC for omeprazole sulfone to the AUC for omeprazole (AUC OPZ-SFN /AUC OPZ ) did not differ among the three doses. Poor Metabolizers Plasma concentrations and AUC of omeprazole in poor metabolizers were proportionally increased with higher doses, and the C max and AUC were increased 3.0- and 4.2-fold and 3.0- and 4.5-fold with dose escalation from 20 to 40 or 80 mg, respectively (Fig. 2, Table 1). The plasma concentrations and AUC of 5 -hydroxyomeprazole in poor metabolizers did not show dose dependency. Plasma profiles and pharmacokinetic parameters of omeprazole sulfone corresponded to those of omeprazole. Neither AUC H-OPZ /AUC OPZ nor AUC OPZ-SFN /AUC OPZ differed among the three doses. Comparison between Extensive Metabolizers and Poor Metabolizers Compared with extensive metabolizers, the plasma concentrations, AUC of omeprazole and omeprazole sulfone, and AUC OPZ-SFN /AUC OPZ at the same dose in poor metabolizers were much higher (Fig. 2, Table 1). In contrast, the plasma concentrations and AUC of 5 -hydroxyomeprazole in poor metabolizers were relatively lower than extensive. Figure 3 shows the nonlinear and linear kinetics of omeprazole in extensive and poor metabolizers. The AUC of omeprazole 20 mg in poor metabolizers was similar to the AUC in extensive metabolizers after 80 mg administration. DISCUSSION Two mutant alleles, CYP2C19*2 and CYP2C19*3, result in deficient metabolic activity, and CYP2C19 activity is classified into three groups: homozygous extensive metabolizers (CYP2C19*1/CYP2C19*1); heterozygous extensive metabolizers (CYP2C19*1/CYP2C19*2 and CYP2C19*1/CYP2C19*3); and poor metabolizers (CYP2C19*2/CYP2C19*2, CYP2C19*2/ CYP2C19*3, and CYP2C19*3/CYP2C19*3). Subjects have been classified into these three genotype groups, and the relation between the CYP2C19 genotypes and phenotypes have been studied. 16,17) An in vitro human liver microsome study indicated that the intrinsic clearance rates (V max /K m ) of the high-affinity component for heterozygous extensive metabolizers were only 2-fold lower than those for homozygous extensive metabolizers at the therapeutic concentration, and no difference in V max was found between homozygous extensive

3 July Fig. 2. Plasma Concentration Time Curves (Mean S.E.) of Omeprazole (A), 5 -Hydroxyomeprazole (B), and Omeprazole Sulfone (C) after a Single Oral Dose of Omeprazole 20 (Circle), 40 (Triangle) and 80 mg (Square) in Extensive Metabolizers (Open Symbols in Left Panel, n 4) and Poor Metabolizers (Closed Symbols in Right Panel, n 3) Classified Using the CYP2C19 Genotype metabolizers and heterozygous extensive metabolizers. 18) In our previous studies, the pharmacokinetics of omeprazole and gastric ph after omeprazole administration in healthy subjects were well correlated with the CYP2C19 genotype, 11,12) although heterozygous extensive metabolizers can be included with homozygous extensive metabolizers as extensive metabolizers. Omeprazole is a key drug in anti-h. pylori therapy. 2) In a previous study, the dosage regimen in the triple therapy consisting of omeprazole (20 mg 2/d for 1 week), amoxicillin (500 mg 4/d for 1 week) and clarithromycin (200 mg 4/d for 1 week) was suitable for poor metabolizers, but 100% eradication was not attained for extensive metabolizers. 13,14) In the present study, the AUC of omeprazole 20 mg in poor

4 926 Vol. 25, No. 7 Table 1. Pharmacokinetic Parameters of Omeprazole and Its Two Metabolites in Extensive Metabolizers and Poor Metabolizers a) 20 mg 40 mg 80 mg CYP2C19 genotype group Extensive Poor Extensive Poor Extensive Poor metabolizers metabolizers metabolizers metabolizers metabolizers metabolizers (n 4) (n 3) (n 4) (n 3) (n 4) (n 3) Omeprazole C max (ng/ml) (1.0) b) (1.0) (2.4) (3.0) (13.7) (4.2) AUC 0 12 h (ng h/ml) ** **,## (1.0) (1.0) (3.2) (3.0) (19.2) (4.5) 5-Hydroxyomeprazole C max (ng/ml) ## * (1.0) (1.0) (1.5) (2.3) (4.0) (2.0) AUC 0 12 h (ng h/ml) ## ** (1.0) (1.0) (1.9) (1.6) (4.5) (1.5) AUC H-OPZ /AUC OPZ * # Omeprazole sulfone C max (ng/ml) ** **,## (1.0) (1.0) (2.7) (1.9) (6.0) (4.1) AUC 0 12 h (ng h/ml) **,## (1.0) (1.0) (3.3) (2.0) (11.1) (3.5) AUC OPZ-SFN /AUC OPZ AUC 0 12 h, area under the plasma concentration time curve up to 12 h; C max, maximum plasma concentration; AUC H-OPZ /AUC OPZ, ratio of AUC 0 12 h for 5 -hydroxyomeprazole to AUC 0 12 h for omeprazole; AUC OPZ-SFN /AUC OPZ, ratio of AUC 0 12 h for omeprazole sulfone to AUC 0 12 h for omeprazole. a) Each value represents the mean S.E. b) The ratio of C max or AUC 0 12 h to those with omeprazole 20 mg are given in parentheses to examine the linearity. p 0.05, p 0.01 as compared with extensive metabolizers. # p 0.05, ## p 0.01 as compared with omeprazole 20 mg for each group classified using the CYP2C19 genotype. Fig. 3. AUC of Omeprazole after a Single Oral Dose of 20, 40, and 80 mg in Extensive Metabolizers (, n 4) and Poor Metabolizers (, n 3) Classified Using the CYP2C19 Genotype metabolizers was similar to the AUC in extensive metabolizers after administration of 80 mg (Fig. 3). Omeprazole inhibits H /K -ATPase irreversibly, and the correlation between this inhibition and the AUC for omeprazole was well described with a sigmoid E max model. 19) Although there were no serious adverse events after single dose of omeprazole 80 mg in extensive metabolizers, multiple doses of 80 mg 2/d for 1 week might be much enough in anti-h. pylori therapy. 20) Further studies on efficacy and safety should be undertaken to estimate the optimal dose of omeprazole. The nonlinear kinetics of omeprazole derived from the saturation of CYP2C19 activity have been known, 21,22) although little information is available on the CYP2C19 genotype groups of extensive metabolizers and poor metabolizers. 23) In the present study, nonlinear and linear kinetics of omeprazole were seen in extensive metabolizers and poor metabolizers, respectively (Fig. 3). CYP2C19-dependent reactions were significantly slowed and kinetic parameters would be shifted toward values observed in poor metabolizers with higher doses (80 mg) of omeprazole in extensive metabolizers (Fig. 2, Table 1). These results were explained by measurements using recombinant human CYP2C19 and CYP3A4 enzymes, which had activities for omeprazole 5 -hydroxylation, with low K m (2280 or 4214 ng/ml) and high V max for the CYP2C19, and high K m (17271 or ng/ml) and low V max for the CYP3A4. 24,25) Maximum plasma concentrations of omeprazole in extensive metabolizers ( ng/ml) and poor metabolizers ( ng/ml) with the 80 mg dose were much lower than K m for the CYP2C19 and CYP3A4, respectively. Based on pharmacokinetic considerations, the recommended dose of omeprazole for extensive metabolizers was suggested to be a maximum of 80 mg 2/d in anti-h. pylori therapy. REFERENCES 1) Bayerdorffer E., Miehlke S., Mannes G. A., Sommer A., Hochter W., Weingart J., Heldwein W., Klann H., Simon T., Schmitt W., Gastroenterology, 108, (1995). 2) Lind T., Megraud F., Unge P., Bayerdorffer E., O morain C., Spiller R., Veldhuyzen van Z. S., Bardhan K. D., Hellblom M., Wrangstadh M., Zeijlon L., Cederberg C., Gastroenterology, 116, (1999). 3) Tytgat G. N. J., Aliment. Pharmacol. Ther., 8, (1994). 4) Labenz J., Gyenes E., Ruhl G. H., Borsch G., Gut, 34, (1993). 5) Lind T., Veldhuyzen van Z. S., Unge R., Bayerdorffer E., O Morain C., Bardhan K. D., Bradette M., Chiba N., Wrangstadh M., Cederberg C., Idstrom J. P., Helicobacter, 1, (1996). 6) Sohn D. R., Kobayashi K., Chiba K., Lee K. H., Shin S. G., Ishizaki T., J. Pharmacol. Exp. Ther., 262, (1992). 7) Nakamura K., Goto F., Ray W. A., McAllister C. B., Jacqz E., Wilkinson G. R., Branch R. A., Clin. Pharmacol. Ther., 38, (1985). 8) de Morais S. M. F., Wilkinson G. R., Blaisdell J., Nakamura K., Meyer U. A., Goldstein J. A., J. Biol. Chem., 269, (1994).

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