Journal of Antimicrobial Chemotherapy (2001) 47, 405-410
© 2001 The British Society for Antimicrobial Chemotherapy
Effects of lansoprazole, clarithromycin and pH gradient on uptake of [14C]amoxycillin into rat gastric tissue
Department of Drug Metabolism, Research Center, Taisho Pharmaceutical Co., Ltd, 403, Yoshino-cho 1-chome, Omiya-shi, Saitama 330-8530, Japan
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Abstract |
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The effect of lansoprazole and clarithromycin on the uptake of [14C]amoxycillin into rat gastric tissue was investigated. After oral administration of [14C]amoxycillin, the levels of radioactivity in gastrointestinal tissue were two to 15 times higher than those in plasma. The level of radioactivity in glandular stomach was significantly higher when lansoprazole and [14C]amoxycillin were administered together. After intravenous administration of [14C]amoxycillin, there was less radioactivity in gastric tissue than after oral administration, and co-administration of lansoprazole and clarithromycin had no obvious effect. The gastric emptying rate of [14C]amoxycillin was not apparently affected by the co-administration of lansoprazole and clarithromycin. In vitro uptake of [14C]amoxycillin into gastric tissue depended on the pH, with uptake at pH 7.4 being four times greater than that at pH 4.0. The apparent synergic effects of lansoprazole are due to enhanced penetration of amoxycillin in gastric mucus and tissue by increasing intragastric pH and play an important role in the eradication of H. pylori.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Helicobacter pylori infection plays a pivotal role in causing peptic ulcer and is seroepidemiologically associated with the risk of gastric cancer. Several therapeutic regimens involving the use of antibiotics for the eradication of this bacterium have been tested. The macrolide antibiotic, clarithromycin,1 and the penicillin antibiotic, amoxycillin,2 show strong in vitro activity against H. pylori, but eradication rates under single-treatment regimens in vivo have been unsatisfactory.2,3 Triple therapy with amoxycillin, clarithromycin and a proton pump inhibitor, such as lansoprazole,4 achieves eradication rates of 84–95%.5–7 The pharmacokinetic interaction between amoxycillin or clarithromycin and the proton pump inhibitor, omeprazole, has been studied in humans.8 The concentration of antibiotics in gastric tissue or gastric juice is increased by the co-administration of omeprazole and the efficiency of antimicrobial agents is enhanced. However, the exact mechanism of this synergy is not completely understood.8
The purpose of our study was to investigate the effects of lansoprazole and clarithromycin on the distribution of [14C]amoxycillin in rats, focusing on the potential for amoxycillin to penetrate gastric tissue.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Chemicals
[Carbonyl-14C]amoxycillin was obtained from Daiichi Pure Chemicals Co., Ltd (Tokyo, Japan). The specific activity of the labelled compound was 5.59 MBq/mg and the radiochemical purity was 
97%, as judged by HPLC analysis. 3H-Labelled water (sp. act. 0.925 MBq/mg) was purchased from Daiichi Pure Chemicals Co., Ltd. Unlabelled amoxycillin was purchased from Sigma Chemical Co. (St Louis, MO, USA). Lansoprazole was obtained from Takeda Chemical Industries, Ltd (Osaka, Japan). Clarithromycin was synthesized at the Research Center, Taisho Pharmaceutical Co., Ltd (Saitama, Japan). All other commercially available reagents and solvents were of analytical or HPLC grade.
Animals
Animal experiments were carried out in accordance with the Guidelines for Animal Experimentation and the Regulations for Animal Ethics of Taisho Pharmaceutical Co., Ltd. Seven-week old male Wistar rats purchased from Nihon SLC Co., Ltd (Shizuoka, Japan) were housed in a temperature (23 ± 3°C) and humidity (50 ± 20%) controlled room with a 12 h light–12 h dark cycle. They were provided with commercial food [MF; Oriental Yeast Co., Ltd (Tokyo, Japan)] and water freely throughout acclimatization and the study period, except on the day before the labelled compounds were administered. The rats, weighing 174–211 g, were used at 8 weeks of age.
Preparation of dosage form and administration of drug
Rats were given [14C]amoxycillin 10 mg, lansoprazole 10 mg and clarithromycin 5 mg per kg body weight. For oral administration these drugs were suspended in 5% gum arabic adjusted to pH 7.0 with 1 M KOH. When given intravenously, [14C]amoxycillin was dissolved in 2.33% KH2PO4–1.44% NaHCO3 isotonic buffer (pH 7.4). Intravenous clarithromycin was given in a solution of saline with equimolar HCl used to dissolve the drug. All preparations were made up immediately before use. Rats were distributed randomly into six groups (three rats per group) and were given the drugs as shown in Table I
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Effect of lansoprazole and clarithromycin on the distribution of [14C]amoxycillin
Fifteen, 30, 60 and 240 min after oral administration (groups 1–3) and 15 and 60 min after intravenous administration (groups 4–6) of [14C]amoxycillin, the rats were anaesthetized with ether and whole blood was withdrawn from the inferior aorta into heparinized containers. Plasma was obtained by centrifugation of the blood at 1600g for 10 min at 4°C. Immediately after blood had been collected, the liver, kidney, heart, lung, stomach and intestine (duodenum–ileum) were excised. Gastrointestinal tissues were washed thoroughly in 20–50 mL of saline. Aliquots (500 mg) of gastrointestinal contents were radioassayed. The stomach was separated into the fore-stomach (stratified squamous epithelium, a characteristic tissue for rats, not found in humans) and the glandular stomach, which is similar to human gastric tissue. The duodenum was separated from the intestine. All tissues were weighed and a 100 mg sample was taken for measurement of radioactivity.
Radioactivity in each biological sample was dissolved with 0.5–1 mL of Soluene-350 (Packard Instrument Co., Meriden, CT, USA) and was decoloured by adding 0.4–0.5 mL of 30% hydrogen peroxide. Then, 10 mL of Insta-gel Plus scintillator (Packard Instrument Co.) was added to the sample after neutralization with 0.5–1 mL of 1 M HCl. Radioactivity was measured using an LS6000TA liquid scintillation counter (Beckman Coulter, Fullerton, CA, USA).
Preparation of drug solution for in vitro studies
[14C]Amoxycillin was dissolved in an isotonic buffer of pH 2.3 (containing, per litre, 6.1 mL of 12 M HCl, citric acid 7.7 g, NaOH 2.9 g, NaCl 3.6 g), pH 4.0 (containing, per litre, 3.8 mL of 12 M HCl, citric acid 12.6 g, NaOH 4.8 g, NaCl 2.1 g), pH 5.5 (containing, per litre, NaCl 5.0 g, Na2HPO4•12H2O 1.2 g, KH2PO4 8.6 g) or pH 7.4 (containing, per litre, NaCl 4.0 g, Na2HPO4•12H2O 19.1 g, KH2PO4 1.8 g), to a final concentration of 200 mg/L. When drug interactions were being investigated, lansoprazole to give a final concentration of 200 mg/L or lansoprazole and clarithromycin at final concentrations of 200 and 100 mg/L, respectively, were added to the solution of [14C]amoxycillin. 3H-Labelled water was added to each isotonic buffer above as a control.
Penetration of [14C]amoxycillin and [3H]water into gastric tissue in vitro
Rats were fasted for 16 h, anaesthetized with ether and then killed by bleeding. The inside of the stomach was washed with 10 mL of saline, the cardia and pylorus were tied and the entire stomach was immediately excised. A solution of 1 mL of [14C]amoxycillin or [3H]water in an isotonic buffer of pH 2.3, 4.0, 5.5 or 7.4 was injected through a needle into the excised stomach sac, followed by incubation at 37°C for 1 h in 5 mL of isotonic buffer, pH 7.4. After incubation, the sac was opened and washed in 20 mL of the same pH isotonic buffer, and aliquots (500 mg) of inside and outside buffers were radioassayed. The gastric tissue was separated into the fore-stomach and the glandular stomach. They were weighed and 100 mg samples were cut to measure radioactivity. The radioactivity in each biological sample was determined in the same manner as for the in vivo study.
In order to clarify the effect of lansoprazole and clarithromycin on the penetration of [14C]amoxycillin, rats were given drugs as shown in Table I
. One hour after the final administration, their stomachs were washed with saline and excised, then the isotonic buffer plus [14C]amoxycillin and lansoprazole or [14C]amoxycillin, lansoprazole and clarithromycin were injected into the sacs, and experiments were continued, as above.
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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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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We evaluated the levels of radioactivity in blood, plasma and main organs after oral or intravenous administration of [14C]amoxycillin to rats. The synergic effect of lansoprazole and clarithromycin on the uptake of [14C]amoxycillin into gastric tissue comprising both gastric mucus and mucosa as target sites of H. pylori9 was also studied.
The levels of radioactivity in blood, plasma and main organs after administration of [14C]amoxycillin are shown in Tables II and III. With both oral and intravenous administration of [14C]amoxycillin, there was 10 times more radioactivity in the liver and kidney than in plasma, but the levels in blood, lung and heart were low. This distribution pattern was similar to the data of previous investigators, who used the bioassay method to determine amoxycillin concentrations.10,11 As the level of radioactivity in the plasma was twice as high as that in the blood, it is likely that [14C]amoxycillin in blood was present mainly in plasma and not in erythrocytes. Lansoprazole and clarithromycin had no apparent influence on the level of radioactivity in blood, plasma and main organs after either oral or intravenous administration of [14C]amoxycillin.
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The levels of radioactivity in gastrointestinal tissues after administration of [14C]amoxycillin to rats are shown in Figures 1 and 2
. During the first 30 min after oral administration of [14C]amoxycillin alone (group 1), levels of radioactivity in the fore-stomach, glandular stomach and duodenum were three to 15 times higher than those of plasma; they then decreased rapidly and, 1 h after administration, the levels were almost twice as high as those in the plasma (Figure 1). The level of radioactivity in glandular stomach was significantly different (P < 0.01) among three groups (groups 1–3), because of the synergic effect of lansoprazole. This effect was not observed in fore-stomach and duodenum. In contrast, when [14C]amoxycillin was administered intravenously (groups 4–6), the level of radioactivity in gastric tissues was not altered by co-administration of lansoprazole or clarithromycin (Figure 2).
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, 2 
and 3
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Fifteen minutes after oral administration of [14C]amoxycillin (group 1), 14% of the radioactivity was recovered in the gastric contents and 55% in the intestinal contents; these values gradually diminished, reaching 1% and 33%, respectively, after 60 min. Sixty minutes after intravenous administration (group 4), recovery of radioactivity from gastric and intestinal contents was approximately 0.1% and 11%, respectively. In both oral (groups 1–3) and intravenous (groups 4–6) administration, no significant differences were observed between single treatment and combination treatment. These findings suggest that the gastric emptying rate of [14C]amoxycillin was not affected by co-administration of lansoprazole or clarithromycin.
After oral administration, [14C]amoxycillin could be taken up into gastric tissue by ‘penetration’ from the gastric lumen or by ‘secretion’ through the blood circulation. After intravenous administration of [14C]amoxycillin, the radioactivity was homogeneously distributed in the whole stomach and there was less radioactivity than in plasma. After oral administration, however, there was more radioactivity in gastric tissue than in plasma. We suggest, therefore, that uptake of [14C]amoxycillin into gastric tissue occurs mainly by the ‘penetration’ route and that the contribution by ‘secretion’ is small. The synergic effect would be due to the ‘penetration’ of [14C]amoxycillin and would have no influence on ‘secretion’.
To clarify how co-administration of lansoprazole enhances the ‘penetration’ of [14C]amoxycillin, we studied the effects of pH and additional drugs in in vitro experiments using excised stomach sacs. After injection of [14C]amoxycillin alone into the stomach sacs, the uptake of radioactivity in glandular stomach tissue depended significantly (P < 0.01) on pH. The uptake at pH 2.3, 4.0, 5.5 and 7.4 was 0.85, 0.80, 1.48 and 3.26%, respectively (Figure 3). For all conditions of pH, a small amount of radioactivity in the fore-stomach was detected and the radioactivity in the outside buffer showed a little recovery. Lansoprazole and clarithromycin did not affect the uptake of [14C]amoxycillin into gastric tissue. The ‘penetration’ of [14C]amoxycillin into glandular stomach was more active at neutral pH than at acidic pH. No direct drug interaction seemed to exist regarding the ‘penetration’ of [14C]amoxycillin.
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), outside buffer (
).When 3H-labelled water was injected into the stomach sac, the uptake of radioactivity in the fore-stomach (approximately 1%) or glandular stomach (approximately 7%) and recovery in the outside buffer (approximately 32%) were constant under all pH conditions. Therefore, amoxycillin behaved differently from water.
It has been reported that the viscosity of the gastric mucus decreases (because of increased intragastric pH) when omeprazole is administered to healthy volunteers.12 A change in the viscosity of gastric mucus may be one factor that explains the increased uptake of [14C]amoxycillin into gastric tissue in vivo. Our study suggests that the lansoprazole-mediated change in gastric pH mainly influences the ‘penetration’ of [14C]amoxycillin into gastric mucus and tissue. Amoxycillin is an amphoteric drug, containing both a basic (–NH2) and an acidic group (–COOH) (pKa 2.4 and 7.2).13 A greater proportion of the molecule is in the un-ionized form at alkaline pH and at very acidic pH, and the lipid solubility of amoxycillin may be enhanced. The effect of lansoprazole on the uptake of amoxycillin into gastric tissue is likely to play an important role in the eradication of H. pylori.
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Notes |
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* Corresponding author. Tel: +81-48-663-1111; Fax: +81-48-652-7254; E-mail: S14952{at}ccm.taisho.co.jp
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References |
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Received 4 August 2000; returned 25 September 2000; revised 31 October 2000; accepted 17 November 2000
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