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Interaction of Cimetidine and Ranitidine, the H2 Receptor Blockers, with Mexazolam, a Benzodiazepinooxazole-Anxiolytic in Rats

Toshihiko IKEDA, Izumi MORI, Toru KoMAI and Minoru TANAKA

Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd., 1-2-58, Hiromachi, Shinagawa-ku, Tokyo 140, Japan

Key words : drug interaction ; H2-receptor antagonist ; anxiolytics ; metabolism ; rat ; microsome ; plasma concentration

Summary Mexazolam, a benzodiazepinooxazole-anxiolytic, was first N-dealkylated to M, and then hydroxylated to in rat liver microsomes. The production of MI in vitro was inhibited by SKF-525 A, carbon monoxide and in the atmosphere of . The heat-treated microsomes, the omission of an NADPH-generating system and the substitution of NADPH with NADH showed practically no activity. The microsomes obtained from the treated rats showed increased activity toward both the production of Ml and lorazepam from mexazolam, but the clofibrate and 3-methylcholanthrene treatments failed to induce this activity. Cimetidine inhibited non-competitively both steps in the metabolism of mexazolam : mexazolam to M, (Kt : 375,aM) and Ml to lorazepam (Kt : 390 uM). Ranitidine did not inhibit in vitro metabolism of mexazolam at the concentrations so far investigated ('400 ,uM). The pretreatment of rats with cimetidine (200 mg/kg, i.p.) 30 min prior to the administration of mexazolam (50 mg/kg, i.p.), increased AUC and the plasma half-life of mexazolam 8.8-fold and 7.7-fold, respectively. On the other hand, the co-administration of ranitidine did not change the pharmacokinetic para meters of mexazolam. Introduction

The H2-receptor antagonists, cimetidine and ranitidine, are extensively used in the treatment of peptic ulcers and other gastrointestinal disorders. Cimetidine is known to prolong the half-life of warfarin, , propranolol, antipyrine, , valproate, and other drugs.'-") On the other hand, an increasing number of papers demonstrate that raniti dine is much less effective than cimetidine in terms of interaction with other drugs.10"'S' Along with the H2-receptor antagonists, minor tranquilizers such as diazepam are commonly co-administered with other anti-ulcer agents in patients. From clinical point of view, the drug interaction between tranquilizers and H2-receptor antagonists is an important subject that should be thoroughly investigated. Mexazolam is a newly developed minor tranquilizer (anxiolytic) which has a benzodia zepinooxazole structure."-") As shown in Chart 1, it is metabolized through two separate pathways, i.e., ring-scission to M3 via M2 (pathway 1) and oxidation to lorazepam via MI (pathway 2).19' Pathway 1 represents an inactivating process of mexazolam, while the meta bolites formed in pathway 2 still retain their pharmacological activity. After incubation with rat liver microsomes, we have found that the major metabolites of mexazolam are MI and lorazepam, the pharmacologically active metabolites. In this paper, we have demonstrated that cimetidine inhibits the in vitro metabolism of mexazolam by a non competitive mode and prolongs the plasma half-life of mexazolam in vivo. Ranitidine showed

Chart 1. Metabolic pathway of mexazolam no significant effects on the metabolism of mexazolam both in vivo and in vitro.

Materials and Methods

Materials Glucose-6-phosphate, glucose-6-phosphate dehydrogenase and NADP+ were purchased from Sigma Chemicals Co., St. Louis, USA. 3-Methylcholanthrene and phenobarbital (sodium salt) were purchased from Tokyo Kasei Kogyo Co., Ltd., Tokyo, Japan. Clofibrate was a generous gift from Ayerst Laboratories Inc., New York, USA. The assay kit for protein determination using the dye-complex method was purchased from Bio-Rad Lab ., Richmond, USA. Mexazolam, M1, M2, M3, lorazepam, cimetidine and ranitidine were prepared in the Chemical Research Laboratories of Sankyo Co., Ltd. The other chemicals used were all of the reagent grade. Preparation of the Liver Microsomal Fractions Male rats of the Sprague-Dawley strain were sacrificed by decapitation after brief anesthesia with ether. The were excised and homogenized in a Potter-Elvehjem glass homogenizer with a Teflon pestle after the addition of a 3-fold volume of a 50 mM potas sium phosphate buffer containing 1. 15 % potassium chloride (pH 7. 0). The homogenate was centrifuged for 20 min at 9, 000 g in a refrigerated centrifuge (Model RS-18 GL, Tomy Seiko Co., Ltd., Tokyo, Japan). The supernatant fraction at 9, 000 g was centrifuged for 1 hr at 105, 000 g in a ultracentrifuge (Model L 5-65, Beckman Instruments Inc ., Palo Alto, USA) to sediment the microsomal fraction. The pellet at 105, 000 g (microsomal fractions) was washed once with the same buffer. The microsomal fraction was resuspended in a 50 mM phosphate buffer containing 30 % glycerol (pH 7. 0) and stored at -80°C. To obtain the liver microsomes with the induced activities of the different drug-metabolizing enzymes , a group of three male rats was treated with clofibrate, phenobarbital and 3-methylcholanthrene , respectively. Clofi brate was mixed with powdered laboratory chow (Clea Japan Inc., Tokyo , Japan) at concentration of 0. 2 %. The rats were given the 0. 2 % clofibrate chow for 2 weeks . Phenobar bital (80 mg/ml-water) was administered once a day intraperitoneally at a dose of 80 mg/kg for 3 days. 3-Methylcholanthrene (80 mg/ml-corn oil) was given once intraperitoneally at a dose of 150 mg/kg. The content of cytochrome P-450 was measured according to Omura and Sato.20 The Effect of Cimetidine and Ranitidine on Mexazolam Metabolism in vitro Mexazolam (0-300 ttM) was incubated with the rat liver microsomes (2 mg protein/ml) and an NADPH-generating system for 15 min at 37°C in a final volume of 0. 5 ml. Glucose 6-phosphate dehydrogenase (2 Sigma units), glucose-6-phosphate (25 MM), MgCl2 (10 MM) and NADP (2. 5 mM) were used as the NADPH-generating system. The reaction was started by the addition of an ethanol solution of mexazolam (15 pl). An aqueous solution of cime tidine and ranitidine was added to the mixture at the concentrations of 0, 100, 200 and 400 µM. After the reaction was stopped by the addition of 2 ml of , the incubation mixture was centrifuged in an Eppendorf centrifuge for 2 min. Ten p1 of the supernatant were analyzed for mexazolam and its metabolites by high-performance liquid chromatography (HPLC), as described at the end of this section. M2 was dissolved in methanol and used as the internal standard for the HPLC assay. The Effect of Cimetidine and Ranitidine on the of Mexazolam Three male rats of the Sprague-Dawley strain weighing 250 g were used. Cimetidine and ranitidine were dissolved in distilled water (200 mg/ml) and administered intraperitoneally to the rats at a dose of 200 mg/kg. Thirty min after the administration of H2-receptor antagonists, mexazolam in dimethylsulfoxide (50 mg/ml) was administered intraperitoneally at a dose of 50 mg/kg. Blood samples (0. 25 ml) were taken from the cannulated femoral artery, 15, 30, 45, 60, 90, 120, 180, 240, 360 and 480 min after the administration of mexa zolam. Before blood sampling, the rats were heparinized with i.v. injection of heparin solution (10, 000 units). The tubes were centrifuged in the Eppendorf centrifuge for 1 min and the 50 pl-aliquots of the plasma samples were put into another set of Eppendorf tubes (0. 5 ml content). Fifty lcl of acetonitrile containing monochlorobenzene (7,"1/50 ml) were added to the plasma samples as the internal standard for the HPLC-assay and mixed vigorously by a

Fig. 1. Separation of mexazolam, Ml, M2 and lorazepam by HPLC Vortex mixer (Scientific Industries Inc., Bohemia, USA). After centrifugation in the Eppen dorf centrifuge for 2 min, 50 ul of the deproteinized supernatant were analyzed by HPLC. Analysis of Mexazolam and Its Metabolites by High-Performance Liquid Chroma togrpahy (HPLC) Deproteinized samples obtained from in vitro and in vivo experiments, 1011 and 50 p1, respectively, were injected into a high-performance liquid chromatograph (Waters Associates Inc., Milford, USA) with the ultraviolet detector set at 254 nm. Satisfactory separation of mexazolam and its metabolites was achieved on ODS-column (ERC-ODS-1162, Erma Co., Tokyo, Japan) with the 55% acetonitrile-45 % phosphate buffer (50 mM, pH 8.0) as a mobile phase at a flow rate of 1 ml/min as shown in Fig. 1. The calibration curve for the quantitative analysis was obtained by plotting the peak height ratio (mexazolam or metabolites/internal standard) against the concentration. M2 and monochlorobenzene were used as the internal standards for the analysis of the incubated samples in vitro and plasma samples in vivo, respectively.

Fig. 2. Incubation products of mexazolam with rat liver microsomes Results

Participation of Cytochrome P-450 in the Metabolism of Mexazolam in vitro After incubation of mexazolam with the rat liver microsomes and the NADPH-genera ting system, MI was detected by HPLC as the major metabolite along with the small amounts of lorazepam as shown in Fig. 2. M2 was not detected at all, demonstrating that the pathway leading to lorazepam is the main metabolic pathway in vitro. Thus, M2 was used as the internal standard for the HPLC assay in the experiments in vitro. The generation of Me from mexazolam was significantly reduced by the omission of microsomes (4. 2 %), the heat-treatment of the microsomes for 2 min at 100°C (4. 2 %), the omission of an NADPH generating system (3. 7 %) and the substitution of NADPH with NADH (6. 7 %), as shown in Table I. The addition of SKF-525 A, bubbling with carbon monoxide and incubation in the nitrogen atmosphere resulted in a significant inhibition of activity (89, 75 and 55 %inhibition, respectively). The formation of Ml was increased by 36 % after the induction with phenoba rbital and was accompanied by an increase of the cytochrome P-450 contents (81 % increase), as shown in Table II. The cytochrome P-450 contents were increased by 18% and 52% after treatments with clofibrate and 3-methylcholanthrene, respectively, however, the formation of M, was reduced by 42-43% after the administration of these inducers. The amount of loraze

Table I. Effects of various treatments on the metabolism of mexazolam in vitro

a) Average from the duplicated experiments. b) Mexazolam (300 pM), liver microsomes (1 mg/ml) and an NADPH-generating system .

Table II. Effects of cytochrome P-450 inducers on the metabolism of mexazolam in vitro

a) nmole/mg-protein. b) leg-M, formed/15 min/mg-protein. c) leg-lorazepam formed/15 min/mg-protein. d) Significantly different from the control, p<0.05. e) Significantly different from the control, p<0.01. Fig. 3. Effects of cimetidine on the formation of Ml from mexazolam (Dixon-plot)

Fig. 4. Effects of ranitidine on the formation of M, from mexazolam (Dixon-plot) pam formed from mexazolam was also increased by phenobarbitalYtreatment (5.18-fold), but not by clofibrate and 3-methylcholanthrene treatments. Phenobarbital, clofibrate and 3-methyl cholanthrene are known to induce different types of cytochrome P-450 that have different drug-metabolizing activities. The results shown in Tables I and II indicate that mexazolam is a good substrate for the drug-metabolizing enzyme which can be induced by phenobarbital. Effects of Cimetidine and Ranitidine on the Metabolism of Mexazolam in vitro The effects of cimetidine and ranitidine (100, 200 and 400,UM) on the in vitro meta bolism of mexazolam were examined. Both of the two steps in the metabolism of mexazolam, that is, the N-dealkylation of mexazolam to Ml and the oxidation of Ml to lorazepam, were inhibited significantly by cimetidine. The mode of inhibition by cimetidine was non-compe titve, as shown in the Dixon-plot for the Ml-forming activity from mexazolam (Ki ; 375 pM, Fig. 3). The non-competitive inhibition by cimetidine was also demonstrated for the loraze pam-forming activity from Ml (Ki ; 390 ttM, results not shown). In contrast, the Dixon-plots for these two metabolic reactions in the presence of ranitidine resulted in parallel lines without focal point, demonstrating that there was no significant inhibition by ranitidine (typically shown for Ml formation from mexazolam in Fig. 4). Effects of Cimetidine and Ranitidine on the Metabolism of Mexazolam in vivo The effects of the pretreatment of rats with H2-receptor antagonists (200 mg/kg , i.p.) or, the plasma concentration of mexazolam were examined. Three male rats were injected with mexazolam (50 mg/kg) 30 min after the intraperitoneal injection of H2-receptor antagonists . In the control rats, the plasma level of mexazolam decreased in three phases, as shown in Fig. 5 (half-lives: 39. 0, 95. 2 and 370 min, respectively). The administration of ranitidine did

Fig. 5. Effects of cimetidine and ranitidine on the concentration of mexazolam in rat plasma Table III. Effects of cimetidine and ranitidine on the pharmacokinetic parameters of mexazolam in rats

a) Area under the time-plasma concentration curve. b) Maximum plasma concentration . c) Significantly different from the control , p<0.05. not change the time course of the plasma concentration of mexazolam, showing the same plasma level and half-life of each phase as those in the control (half-lives: 29. 9, 117 and 448 min, respectively). On the other hand, the plasma level of mexazolam was elevated significantly by the cimetidine pretreatment , as demonstrated by the fact that AUC and the maximum plasma level (Cp..) increased, in comparison to control , 8. 8-fold and 2. 4-fold, respectively (Table III). After the treatment with cimetidine, the elimination mode of mexa zolam from the plasma changed to apparently single-phase elimination, showing a half-life 7. 7-fold longer than that of the a-phase in the control.

Discussion

The data shown in this paper have confirmed a pronounced drug interaction between cimetidine and mexazolam. On the other hand, ranitidine did not show any interaction with mexazolam both in vitro and in vivo. Cimetidine is reported to bind strongly to hepatic cytochrome P-450 (Ks values for the first binding site : 0. 04-0. 07 mM).21-23) The moiety of cimetidine is considered to be the principal part that interacts with cytochrome P-450. This binding causes the inhibition of drug-metabolizing enzymes, thus resulting in the drug interaction of cimetidine with many drugs in vivo. The participation of cytochrome P 450 in the metabolism of mexazolam in vitro was demonstrated in this paper, and hence the metabolism of mexazolam was significantly inhibited by cimetidine. The binding affinity of ranitidine to hepatic cytochrome P-450, on the other hand, is considerably lower than that of cimetidine (Ks values : 1. 4-2. 8 mM), because of the substitution of the imidazole moiety with the furan ring. 141 Accordingly, most cases of interactions with ranitidine are not attribu table to the inhibition of drug-metabolizing enzymes but to the elevation of gastric pH, competition at the renal excretion, a possible reduction of the hepatic blood flow, etc.25> Although the apparent interactions with ranitidine due to the inhibition of cytochrome P-450 are known 21) ranitidine is unlikely to interact with mexazolam according to the results shown in this paper.

Acknowledgments

The authors would like to express theis sincere gratitude to Prof. S. Tsurufuji of Tohoku University for his valuable discussions. Thanks are also due to Dr. Hideyo Shindo, the director of the Analytical and Metabolic Research Laboratories, Sankyo Co., Ltd. for his continuous encouragement.

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