1 Department of Drug Metabolism, Research Center, Taisho Pharmaceutical Co., Ltd, 403 Yoshino-cho 1-chome, Saitama-shi, Saitama 330-8530; 2 Department of Clinical Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan
Received 22 October 2001; returned 7 February 2002; revised 14 March 2002; accepted 30 April 2002
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Abstract |
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Introduction |
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Materials and methods |
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[6-O-methyl-14C]Clarithromycin, unlabelled clarithromycin, amoxicillin, lansoprazole and commercially available reagents were used, as described previously.2
Animals
All experiments were carried out in accordance with the Guidelines for Animal Experimentation and the Regulations for Animal Ethics in Taisho Pharmaceutical Co., Ltd. Male Wistar rats purchased from Nihon SLC Co., Ltd (Shizuoka, Japan) were acclimatized and 8-week-old rats, weighing 176225 g, were used. 2
Dosage and administration of drugs
Rats were given [14C]clarithromycin 5 mg/kg body weight, lansoprazole and amoxicillin 10 mg/kg. These drugs were made up immediately before use, as described previously.2 Rats were allocated randomly into four groups and were given the drugs listed in Table 1.
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At 15, 30, 60 and 240 min after oral (groups 1 and 2) and 15 and 60 min after intravenous (groups 3 and 4) administration of [14C]clarithromycin, the rats were exsanguinated following ether anaesthetization. The stomach was excised immediately and washed thoroughly in 20 mL of saline. The glandular stomach was separated and subdivided into mucosal and muscular layers using the stripping method, as described previously.3 Radioactivity in each biological sample was measured using a liquid scintillation counter, as described previously.3 The distribution of radioactivity in mucosa/muscle was determined.
Measurement of the composition of unchanged [14C]clarithromycin in gastric contents
At 15, 30 and 60 min after oral administration of [14C]clarithromycin to rats (groups 1 and 2), gastric contents including mucus were collected, as described above. Radioactivity in the gastric contents was measured in the same manner as for gastric tissue. Part of the gastric contents was concentrated and filtered using Ultrafree-MC (0.45 µm; Millipore, Tokyo, Japan), and was then injected into the HPLC system. For HPLC, a TSK GEL ODS-120A column (4.6 mm inner diameter x 250 mm length; Tosoh, Tokyo, Japan) was used, the mobile phase was 80% methanol/water containing 0.04% ethanolamine, the column temperature was 40°C and the flow rate was 1 mL/min. The composition of unchanged [14C]clarithromycin was calculated on the basis of the peak area of radioactivity.
Microautoradiography of gastric mucosa
Sixty minutes after oral (groups 1 and 2) and 15 min after intravenous (groups 3 and 4) administration of [14C]clarithromycin, the rats were exsanguinated, following anaesthetization with ether. The stomach was excised and the frozen sections of stomach corpus were cut, and slices were mounted on a glass slide dipped in nuclear emulsion, as described previously.3 After exposure at 4°C for 3 weeks, the glass slide was developed to observe the silver grains indicated by black coloration corresponding to the existence of radioactivity. The slide was stained with Methylene BlueFuchsin basic and observed under a light microscope.
Statistics
Results are expressed as mean ± S.D. The significance of differences was evaluated by variance analysis, using the SAS/STAT package. A significance level of 0.01 was used for all tests.
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Results and discussion |
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The concentration of radioactivity in the glandular stomach and the gastric mucosa/muscle distribution of radioactivity after administration of [14C]clarithromycin are shown in Table 2. After oral administration of [14C]clarithromycin, the concentration of radioactivity was several times higher when it was administered together with lansoprazole and amoxicillin (group 2) than when it was administered alone (group 1). After intravenous administration of [14C]clarithromycin (groups 3 and 4), there was no effect of co-administration, with regard to the concentration of radioactivity. With both oral and intravenous administration, c. 6070% of the radioactivity, which contains both unchanged [14C]clarithromycin and metabolites, was distributed in the mucosal layer. There was no change in the distribution pattern of radioactivity at any time. Thus, clarithromycin would play an important role in eradication of H. pylori at the target site. No synergic effects on the mucosa/muscle distribution were detected when [14C]clarithromycin was co-administered with lansoprazole and amoxicillin.
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The visual localization of [14C]clarithromycin in gastric mucosa was investigated microautoradiographically. Sixty minutes after oral administration of [14C]clarithromycin (group 2), silver grains indicating the existence of radioactivity were extensive in both the mucous layer and in surface epithelial cells in the corpus of the stomach (Figure 1a). Homogeneous radioactivity was observed in the fundic glands. After oral administration of [14C]clarithromycin, it was incorporated into the mucous layer and distributed to surface epithelial cells, then it could penetrate the mucosal layer of the stomach. Fifteen minutes after intravenous administration (group 4), silver grains were observed in all of the fundic glands, particularly in the gland base (Figure 1b). A magnified microautoradiogram (x400) of the gland base of Figure 1(b) is shown in Figure 1(c). Silver grains were present both in secreting cells that constituted the gland base and in the gland lumen. The gland base of the corpus has mainly zymogenic cells, which produce pepsinogen and parietal cells, which secrete acid. [14C]Clarithromycin secreted by these cells may be transported through the gland lumen to the mucous gel layer. Many silver grains were also detected in surface epithelial cells, which indicates that [14C]clarithromycin may be secreted by surface epithelial cells.
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Footnotes |
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References |
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2
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Endo, H., Yoshida, H., Ohmi, N. & Higuchi, S. (2001). Effects of lansoprazole and amoxicillin on uptake of [14C]clarithromycin into gastric tissue in rats. Antimicrobial Agents and Chemotherapy 45, 34515.
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Endo, H., Yoshida, H., Ohmi, N., Ohta, K. & Higuchi, S. (2001). Localization of [14C]amoxicillin in rat gastric tissue when administered with lansoprazole and clarithromycin. Journal of Antimicrobial Chemotherapy 48, 9236.
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