Journal of Antimicrobial Chemotherapy (2001) 48, 923-926
© 2001 The British Society for Antimicrobial Chemotherapy
Brief report |
Localization of [14C]amoxicillin in rat gastric tissue when administered with lansoprazole and clarithromycin
Department of Drug Metabolism, Research Center, Taisho Pharmaceutical Co., Ltd, 403 Yoshino-cho 1-chome, Saitama-shi, Saitama 330-8530, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
The gastric mucosal distribution of [14C]amoxicillin when administered to rats with or without lansoprazole and clarithromycin was investigated. After oral administration, the amount found in the gastric mucosa was higher than after iv administration. Co-administration of lansoprazole and clarithromycin had no apparent effect on the distribution pattern of [14C]amoxicillin within the deeper stomach layers. About 50–60% of the radioactivity in the gastric tissue was present in the mucosal layer, irrespective of the route of administration. Microautoradiograms of the gastric mucosa indicated that [14C]amoxicillin was distributed in both the mucous layer and surface epithelial cells following oral administration. [14C]amoxicillin was secreted mainly by surface epithelial cells after iv administration, although only in small quantities.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Currently, amoxicillin is co-administered with clarithromycin and a proton pump inhibitor to eradicate Helicobacter pylori.1 H. pylori colonizes both the apical surface of surface epithelial cells2 and within the surface mucous gel layer.3 The existence of sanctuary sites, possibly intracellular, in which H. pylori can evade the effects of antimicrobial therapy, has been suggested.4 We investigated localization of [14C]amoxicillin in the rat gastric mucosa, and attempted to clarify in detail the distribution of [14C]amoxicillin in gastric cells, using microautoradiography.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Chemicals
[Carbonyl-14C]amoxicillin, unlabelled amoxicillin, lansoprazole, clarithromycin and commercially available reagents were used, as described.5
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 40 8-week-old rats, weighing 178–202 g, were used.5
Preparation of dosage and administration of drugs
Rats were given [14C]amoxicillin 10 mg/kg body weight, lansoprazole 10 mg/kg and clarithromycin 5 mg/kg. These drugs were made up immediately before use, as described.5 Rats were allocated randomly into four groups and were given the drugs listed in Table 1
.
|
Measurement of unchanged [14C]amoxicillin in gastric contents
At 15, 30 and 60 min after oral administration of [14C]amoxicillin to rats (groups 1 and 2), these animals were exsanguinated under ether anaesthesia. Gastric contents including mucus were collected, the stomach was excised and washed thoroughly in 10 mL of saline, then an equal volume of 50 mM phosphate buffer pH 7.0 was added. The gastric contents were then filtered using Ultrafree-MC (0.45 µm; Millipore, Tokyo, Japan) and 0.1 mL of filtrate was injected on to the high performance liquid chromatography (HPLC) system. For HPLC, a YMC-Pack ODS-AM column (4.6 mm internal diameter x 150 mm length; YMC Co., Ltd, Tokyo, Japan) was used, the mobile phase was 50 mM phosphate buffer pH 7.0/ acetonitrile (95:5), the column temperature was 35°C and the flow rate was 0.8 mL/min. Composition of unchanged [14C]amoxicillin was calculated on the basis of the peak area of radioactivity. Radioactivity of gastric contents was measured in a liquid scintillation counter, as described.5
Measurement of radioactivity in gastric mucosal and muscular layers
At 15, 30, 60 and 240 min after oral (groups 1 and 2) and 15 and 60 min after iv (groups 3 and 4) administration of [14C]amoxicillin, the rats were exsanguinated under ether anaesthesia. The stomach was excised immediately and washed, as described above. The glandular stomach was separated and subdivided into mucosal and muscular layers using the stripping method reported by Arakawa et al.6 Radioactivity in each biological sample was measured in the same manner as for gastric contents. Distribution of radioactivity in mucosa/muscle was then determined.
Microautoradiography of the gastric mucosa
Sixty minutes after oral (groups 1 and 2) and 15 min after iv (groups 3 and 4) administration of [14C]amoxicillin, the ether-anaesthetized rats were exsanguinated. Cardia and pylorus of the stomach were ligated and excised. The stomach was fixed with a small amount of embedding medium (OCT Compound; Sakura Finetek USA, Torrance, CA, USA) and frozen in dry ice–hexane. The stomach was cut on a microtome cryostat (HN500M; Meiwa-Shoji, Tokyo, Japan) at about -20°C. Frozen 8 µm sections of the stomach corpus were cut serially. Under dark conditions, the slice was mounted on a glass slide dipped in nuclear emulsion (K5; Ilford Imaging Ltd, UK). K5 consists of silver halide with a crystal size of 0.20 µm and is suited for optical microscopy using a high energy nuclide. After exposure at 4°C for 4 weeks, the glass slide was developed to observe the silver grains indicated by black colour corresponding to the presence of radioactivity. The slide was stained with basic methylene blue–fuchsin and observed under a light microscope, Leitz DMRBE (Leica AG, Tokyo, Japan).
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.
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
The level of radioactivity in gastric contents 15 min after oral administration of [14C]amoxicillin alone (group 1) or with lansoprazole and clarithromycin (group 2) was c. 12% of the dose given and diminished gradually with time. Since amoxicillin showed almost no degradation in the gastric contents, most of the radioactivity was in an unchanged form in both groups until 60 min after the oral administration of [14C]amoxicillin. These results showed that active [14C]amoxicillin can penetrate the gastric mucosa.
Levels of radioactivity in the glandular stomach, and mucosa/muscle distribution (%) after administration of [14C]amoxicillin to rats are shown in Table 2. The level of radioactivity in the glandular stomach after oral administration of [14C]amoxicillin (groups 1 and 2) was much higher than that after iv administration (groups 3 and 4). After oral administration of [14C]amoxicillin, total radioactivity in the glandular stomach was 7–14% in group 1 and 15–50% in group 2 of that in gastric contents, respectively. The level was two to three times higher when [14C]amoxicillin was given together with lansoprazole and clarithromycin (group 2) than when it was given alone (group 1). Previous work revealed that this synergy is due to the co-administration of lansoprazole.5 After iv administration of [14C]amoxicillin (groups 3 and 4), total radioactivity in the glandular stomach was the same level as in the gastric contents. There was no apparent effect on the level of radioactivity in the glandular stomach when lansoprazole and clarithromycin were co-administered. With both routes, c. 50–60% of the radioactivity was distributed in the mucosal layer, and there was no change in the ratio at any time. It seems likely that [14C]amoxicillin penetrated the muscular layer through the mucosal layer. There were no synergic effects on the mucosa/muscle distribution when [14C]amoxicillin was co-administered with lansoprazole and clarithromycin.
|
The visual distribution of [14C]amoxicillin in the mucosal layer was examined microautoradiographically. Microautoradiograms (x200) of gastric mucosal sections after oral or iv administration are shown in Figure 1
. At 60 min after oral administration of [14C]amoxicillin (Figure 1a), grains indicating the existence of radioactivity were highly visible in both mucous layer and surface epithelial cells surrounding gland pits in the corpus. [14C]Amoxicillin was incorporated into the mucous layer and surface epithelial cells; this process was followed by penetration into the mucosal layer.
|
At 15 min after iv administration of [14C]amoxicillin (group 4), the grains were observed in the mucous layer and in surface epithelial cells in the corpus (Figure 1b
). [14C]amoxicillin seems to have been secreted by surface epithelial cells after iv administration. A magnified microautoradiogram (x400) of isthmus to glandular neck of Figure 1(b) is shown in Figure 1(c). The grains were found on parietal cells but were much fewer than on surface epithelial cells, which means that parietal cells in the isthmus may secrete [14C]amoxicillin, although the secreted radioactivity was only a small amount. Therefore, compared with penetration, the secretion of [14C]amoxicillin may contribute little to the eradication of H. pylori. In the present study, we found that oral [14C]amoxicillin penetrated well into both mucous layer and surface epithelial cells. Thus, amoxicillin plays an important role in the eradication of H. pylori. The distribution pattern of [14C]amoxicillin in the gastric mucosa was little affected by the co-administration of lansoprazole and clarithromycin and the synergism was a result of a quantitative modification of [14C]amoxicillin penetration.
|
Notes |
|---|
* Corresponding author. Tel: +81-48-663-1111; Fax: +81-48-652-7254; E-mail: hiromi.endou{at}po.rd.taisho.co.jp
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
1 . Lind, T., Zanten, S. V., Unge, R., Spiller, R., Bayerdörffer, E., O'Morain, C. et al. (1996). Eradication of Helicobacter pylori using one-week triple therapies combining omeprazole with two antimicrobials: the MACH 1 study. Helicobacter 1, 138–44.[Medline]
2 . Slominary, B. L. & Slomiany, A. (1992). Mechanism of Helicobacter pylori pathogenesis: focus on mucus. Journal of Clinical Gastroenterology 14, Suppl. 1, S114–21.
3 . Akamatsu, T., Ota, H., Shimizu, T., Matsuzawa, K., Fujimori, Y., Nakamura, N. et al. (1995). Histochemical study of Helicobacter pylori and surface mucous gel layer in various gastric lesions. Acta Histochemistry and Cytochemistry 28, 181–5.
4 . Engstrand, L., Graham, D. Y., Scheynius, A., Genta, R. M. & El-Zaatari, F. (1997). Is the sanctuary where Helicobacter pylori avoids antibacterial treatment intracellular? American Journal of Clinical Pathology 108, 504–9.[Web of Science][Medline]
5
.
Endo, H., Yoshida, H., Ohmi, N. & Higuchi, S. (2001). The effects of lansoprazole and clarithromycin and pH gradient on the uptake of [14C]amoxycillin into rat gastric tissue. Journal of Antimicrobial Chemotherapy 47, 405–10.
6 . Arakawa, T., Nakamura, H., Chono, S., Yamada, H. & Kobayashi, K. (1980). Prostaglandin E2 in the rat gastric mucosa (first report)—establishment of assay procedure and effects of nonsteroidal antiinflammatory compounds. Chemotherapy 77, 1052–9.
Received 2 January 2001; returned 10 April 2001; revised 13 August 2001; accepted 4 September 2001
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  H. Endo, H. Yoshida, N. Ohmi, K. Ohta, S. Higuchi, and T. Suga Localization of [14C]clarithromycin in rat gastric tissue when administered with lansoprazole and amoxicillin J. Antimicrob. Chemother., August 1, 2002; 50(2): 285 - 288. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||