| Background and aim:Helicobacter pylorus (H.pylori) is one of the most critical factors in the pathogenesis of chronic gastritis, peptic ulcers, gastric cancer and mucosa-associated lymphoid tissue lymphoma. The treatment guidelines recommended for H pylori are a standard triple therapy consisting of clarithromycin and amoxicillin or metronidazole in combination with a proton pump inhibitor (PPI). H.pylori is becoming increasingly resistant to antibiotics, and the effectiveness and eradication rate of H.pylori eradication regimen are descending. It is very important now to explore the new treatment solutions for the H.pylori eradication. Some researches indicate that the triple therapy based amoxicillin and levofloxacin can be more effective and better tolerated than the standard triple therapy and usual quadruple therapy. The triple therapy based amoxicillin and levofloxacin was recommended to widely use in the first-line H.pylori alternative or rescue therapy. In the stomach, H.pylori lives in the mucus layer and also adheres to gastric epithelial cells. Therefore, the antibiotics used to eliminate H.pylori must achieve inhibitory or bactericidal concentrations at the site of infection. The antibiotic successfully eradicating H.pylori depends heavily on the antibiotic concentration in the stomach, and it is very significant to research the distribution of antibiotics in the stomach. In the experiment, a rat mode for the study of the distribution of antibiotics in the stomach was established and the aim was to explore the gastric transport and distribution of antibiotics and the effect of rabeprazole following rabeprazole, amoxicillin and levofloxacin triple therapy. The further purpose was to research the characteristics of distribution of the antibiotics in the human stomach. This will provide experimental evidence for optimization matching of the antibiotics for eradicating H.pylori and lay an experimental base and technology platform for the study of antibiotics distribution in the stomach.Methods:1) Animal experiment(1) Preparation of model for exploring the antibiotics distribution in rat stomach:The stomach and duodenum of male Wistar rats were exposed under anesthesia at laparotomy. The duodenum was incised, and a soft rubber cannula was cannulated through the duodenal incision into the gastric antrum via pylorus. The stomach was gently lavaged, and then the cannula was pulled out and pylorus was ligated. Drugs were given by administering intravenous boluses via a tail vein. Blood sample was withdrawn from the abdominal aorta every 15 minutes in two hours after administration. Rats were sacrificed, the stomach was excised, and gastric juice was collected, and its volume and pH value were measured and recorded. The gastric mucosa was separated by blistering with injector, and the mucosa from different regions was weighed and homogenized. All the samples were stored immediately in a-80℃freezer until analysis.(2) Groups:Rats were distributed randomly into 8 groups (48 rats per group,6 rats in each sampling point) and were as follows:control group (Control:physiological saline group); amoxicillin groups (amoxicillin 50 mg/kg,100 mg/kg,200 mg/kg,200 mg/kg and rabeprazole 2 mg/kg); Levofloxacin groups (levofloxacin 50 mg/kg,100 mg/kg, 100 mg/kg and rabeprazole 2 mg/kg). (3) The HPLC method for antibiotic concentration analysis in rat samples: Concentrations of amoxicillin and levofloxacin in plasma, gastric juice and gastric mucosa samples were determined by high performance liquid chromatography (HPLC) in Waters 515 HPLC system.2) Investigation in human beings(1) Clinical trials:Patients with gastritis or peptic ulcer who were positive for H.pylori infection confirmed by gastroscopy and 14C urea test received triple therapy (rabeprazole 10mg+amoxicillin 1000mg bid+levofloxacin 500mg qd) po for 10 days. The adverse reactions of the medicine were recorded by telephone inquiry and when patients reexamined. The blood sample, gastric juice and gastric mucosa tissue samples were collected during endoscopy at either 2 (n= 13,2h group) or 4 (n= 12,4h group) hours after the morning dose administration on day 10. The gastric juice pH value was measured and gastric mucosa was weighed and homogenized. All the samples were stored immediately in a-80℃freezer until analysis.14C urea test was performanced after 4 weeks in the end of clinical trials.(2) The HPLC method for antibiotic concentration analysis:Concentrations of amoxicillin and levofloxacin in human plasma, gastric juice and gastric mucosa samples were determined by high performance liquid chromatography (HPLC) in Waters 515 HPLC system.Results:(1) The T1/2 of amoxicillin in rat blood plasma are about 1 hour. Amoxicillin concentration in the gastric juice and gastric mucosa increased accompanied with amoxicillin dosage stepping up at the same sampling point. Amoxicillin concentration in the gastric juice was higher than that in the gastric mucosa, that in the forestomach mucosa was lower than that in the gastric antrum mucosa and gastric corpus mucosa, and the concentration between in the gastric antrum mucosa and gastric corpus mucosa were not significant deviation. Amoxicillin concentration in the gastric mucosa and juice was only 1% to 2% of that in the blood plasma. The gastric transfer fraction of amoxicillin was 0.458% to 1.512%. Amoxicillin pharmacokinetic parameters in rat plasma were not affected by rabeprazole. Rabeprazole increased amoxicillin concentration in the gastric juice by increasing gastric juice pH from 2.55±0.38 to 5.66±0.48 and decreased the gastric juice volume.(2) Levofloxacin concentration in the gastric juice were far higher than that in the blood plasma, and the highest multiple was 8.21.times, and its transfer fraction was 1.672% to 2.653%. Levofloxacin concentration in the gastric antrum mucosa and gastric corpus mucosa was higher than that in the forestomach mucosa, and the concentration between in the gastric antrum mucosa and gastric corpus mucosa were not significant deviation. Rabeprazole lengthened the T1/2 of levofloxacin (mean 2.719 vs 3.645 hours), and increased the AUC0—2h, AUC0—∞, Cmax, and increased gastric juice pH,decreased the gastric juice volume, and not significantly affected the transfer fraction.(3) Antibiotics concentration in human gastric juice and gastric mucosa was higher than that in the plasma at 2 hours after administration. Amoxicillin concentration in the gastric juice was 2.46 times of that in the plasma at 4 hours after administration and that in gastric mucosa was lower than that in the gastric juice, and all were higher than that minimal inhibitory concentration on H.pylori. Levofloxacin concentration in stomach was as follow:35.165μ/ml in the gastric juice at 2 hours after administration, 8.441μg/ml at 4 hours after administration, and levofloxacin concentration between in the gastric mucosa and in the plasma were not significant deviation at 4 hours after administration, and all were higher than that minimal inhibitory concentration on H.pylori. The advantages of the triple therapy with rabeprazole, amoxicillin and levofloxacin used against Helicobacter pylori were high eradication rate and little adverse reaction.Conclusion:(1) Amoxicillin can be transported from blood into the stomach across the gastric mucosa. Amoxicillin concentration in the different region of the stomach is not the same. Amoxicillin concentration in the gastric juice is higher than that in the gastric mucosa and that in the gastric antrum mucosa and gastric corpus mucosa was higher than that in the forestomach mucosa. Increasing amoxicillin dosage could step up its concentration in stomach.(2) Amoxicillin pharmacokinetic parameters in rat plasma were not significantly affected by rabeprazole. Rabeprazole increased amoxicillin concentration in the gastric juice by increasing gastric juice pH and decreasing the gastric juice volume.(3) The amount of amoxicillin transported from blood into the stomach is very little. Amoxicillin concentration could be maintained in the high level when the stomach is infected by Helicobacter pylori.(4) Levofloxacin can penetrate the gastric mucosa into the stomach. Levofloxacin concentrations in the gastric juice and gastric mucosa are far higher than that in the blood plasma, and this maybe indicate that levofloxacin can transferred into stomach by active transport. The descending order of levofloxacin concentration is gastric juice than gastric mucosa than the plasma, and glandular stomach mucosa than the forestomach mucosa.(5) Levofloxacin pharmacokinetic parameters in rat plasma were significantly affected by rabeprazole.(6) Levofloxacin concentration in human gastric juice and gastric mucosa could be maintained in the higher level than minimal inhibitory concentration for a long time.(7) The triple therapy with rabeprazole, amoxicillin and levofloxacin will be effective to eradicate H.pylori, and cause fewer adverse reactions. The regimen can be used as first-line therapy and as second-line therapy.
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