| BackgroundIt has a very important clinical significance to effectively eradicate Hp by using the antibiotics. The specificity of Hp is colonized at gastric mucus and its lower gastric mucosa surface, therefore, the success of eradication Hp depends on the concentration of local antibiotic in treatment position. The antibiotic works only when the concentration of antibiotic reached the required bactericidal concentration at the colonized position. The antibiotic will be transported into stomach from blood after oral administration, which is the decisive factor that the local concentration of antibiotic in stomach is raised and the key problem to successfully eradicate Hp. The transport of antibiotic in stomach has the feature of pharmacokinetics, transmembrane transport exists from blood to stomach organization after direct distribution in stomach and intestinal absorption. The research on the transport of antibiotic in stomach and distribution features can provide the data for the selection of antibiotic in Hp eradication scheme.Recently, the drug resistance of Hp is more and more serious, and the failure rate of classical triple therapy for eradiation increases yearly. Clinical researches show that the eradication scheme contained with levofloxacin is a comparatively ideal treatment in eradication rate at present. However, the basic research aimed at the transport and distribution condition of levofloxacin in stomach has not been reported so far, and the pharmacokinetic data of levofloxacin in stomach lacks. In addition, the pH in stomach may affect the transport and distribution of antibiotics in stomach. Therefore, whether the PPI will influence the transport and distribution of levofloxacin in stomach is not clear. Due to the neighborhood relationship of anatomical structure, the common bile duct is directly opened at duodenum, and the antibiotics in bile also can back flow to stomach with bile, so as to affect the local antibiotics concentration in stomach. Studying the pharmacokinetic process of levofloxacin in bile is helpful to explain the anti- Helicobacter pylori function of levofloxacin.PurposeTo discuss the transport and distribution characteristic of levofloxacin in stomach and bile of rat, on the basis of building the animal model for the transport and distribution of antibiotics in stomach and bile; to observe the effect of treatment combined with rebeprazole on above process in stomach; at the same time, combining with the results of experiments, to preliminarily discuss the concentration condition of levofloxacin in human stomach and bile, so as to provide the experimental basis for clinical pharmacology during the process of eradication Hp by using levofloxacin.Methods⑴Build the animal model for transport and distribution of antibiotic in the stomach: carried out the fasting for Wistar rats before operation, and after anaesthesia, carried out the indwelling tube operation in bile duct. At 2cm of duodenum from pylorus, insert the rubber hose into the stomach through the pylorus and collected the gastric juice after intraoperative irrigation of stomach. Determined the concentration of drug by stripping of stomach mucosa of rat with method of blistering at the end of experiment, and then wiped off water and weighed, and finally grinded and homogenated; and the thickness of gastric mucus layer was measured by frozen-section PAS/AB staining method.⑵Establish the HPLC method for detection of levofloxacin in different biological samples: Trichloroacetic acid or methanol was used to precipitate proteins in rats/human serum, gastric juice, stomach mucosa and bile, then 20μl filtrates were injected for determining the concentration of levofloxacin. HPLC conditions: A Kromosil C18 column was used as chromatographic column, whose temperature was maintained at 50℃. The mobile phase was prepared as follow: cetonitrile-50mmol/l citric acid-1mol/l ammonium acetate (19:80:1, v/v) with the flow rate of 1.0 ml/min. The detection wavelength was 295nm. The parameters of selectivity, linearity, accuracy, precision, stability, sensitivity were evaluated for method validation.⑶Study on transport and distribution of levofloxacin in rat stomach: divided Wistar rats randomly into 4 groups: levofloxacin 50mg/kg, levofloxacin 50mg/kg + rabeprazole, levofloxacin 100mg/kg, levofloxacin 100mg/kg + rabeprazole. Within 2 hours after intravenous administration, collected the serum, gastric juice, stomach mucosa samples at an interval of 15min and detected the concentration of levofloxacin of all the samples to find out the feature of transport and distribution of levofloxacin in rat stomach.⑷Study on distribution of levofloxacin in rat bile: divided Wistar rats randomly into 3 dose groups: levofloxacin 20mg/kg, 50mg/kg, 100mg/kg. Carried out the indwelling tube in carotid artery and common bile duct during operation, then respectively collected blood and bile samples at different time points every 1 hour in 12 hours after intravenous administration and detected the concentration of levofloxacin to find out the feature of distribution of levofloxacin in rat bile;⑸Study on distribution of levofloxacin in human stomach: 4 cases of Hp infected patients, treat by the scheme of oral rabeprazole 10mg bid + levofloxacin 500mg qd + amoxicillin 1.0g bid, carried out gastroscopy in the 10th day, collected gastric juice samples, take gastric mucosa samples from top to bottom at gastric antrum, gastric corpus and gastric fundus, and detected the concentration of levofloxacin, so as to find out the feature of distribution of levofloxacin in human stomach;⑹Study on distribution of levofloxacin in human bile: 17 cases of patients with biliary disease and indwelling T-tube after exploration of biliary tract, took an intravenous drip of 200mg, 400mg and 500mg levofloxacin. At 24 hours after administration, collected blood samples at 1h, 2h, 4h, 8h, 12h, 24h time points, collected the bile samples at an interval of 1h, and detected the concentration of levofloxacin, so as to find out the feature of distribution of levofloxacin in human bile.Results⑴Through verifying key experimental methods involved in the study of animal model of transport and distribution of antibiotics in the stomach, such as the gastric juice collection method, gastric mucosa stripping method and gastric secretion layer measurement method, and HPLC detection method of levofloxacin in different biological samples, it was proved that the animal model and HPLC detection method were reliable, simple and convenient for the technical requirements.⑵Transport and distribution of levofloxacin in rat stomach: from comparison of the drug concentration of levofloxacin 50mg/kg group and 100mg/kg group in each part at different time points, it was found, after administration for about 45-60 minutes, that the drug concentration of gastric juice was gradually higher than the drug concentration of serum, and the drug concentrations of gastric corpus, gastric antrum and glandular stomach at different time points were higher than the drug concentration of serum and the drug concentration of forestomach, however, there was no difference in the drug concentration of gastric corpus and the gastric antrum. Comparison of the results of group of levofloxacin and the group of levofloxacin combined with rabeprazole indicated that the drug concentrations of gastric juice, forestomach, gastric corpus, gastric antrum and glandular stomach at different time points for the group of levofloxacin combined with rabeprazole were all higher than that of the group of levofloxacin, but there was no significant difference in statistics. The levofloxacin transport fraction in the stomach for levofloxacin 50mg/kg group, levofloxacin 50mg/kg + rabeprazole group, levofloxacin 100mg/kg group, and levofloxacin 100mg/kg + rabeprazole group is respectively 2.36, 2.42, 2.52 and 2.55.(3) Distribution of levofloxacin in rat bile: the Cmax of bile for levofloxacin 20mg/kg, 50mg/kg and 100mg/kg dose group was 57.42mg/l, 88.81mg/l and 137.60mg/l separately. The statistical analysis showed that there was difference of the drug concentration of the bile for all doses at all time points.⑷Distribution of levofloxacin in human stomach: the levofloxacin concentration of human gastric juice was 2.26±1.32μg/ml, and the levofloxacin concentration of samples of gastric fundus, gastric corpus and gastric antrum was 5.51±1.46μg/ml, 9.99±4.01μg/ml and 8.52±2.45μg/ml separately, which were all higher than the levofloxacin concentration of gastric juice.⑸Distribution of levofloxacin in human bile: the Cmax for levofloxacin 200mg, 400mg and 500mg dose groups was respectively 11.92mg/l, 16.52mg/l and 20.53mg/l, and the statistics showed that there was Cmax difference in all dose groups. Besides, through comparing the drug concentration of the bile of all dose groups at each time point, it was found that the difference of drug concentration of the bile exist nearly in all time points and all dose groups.ConclusionsThrough the study of transport and distribution of levofloxacin in rat stomach and bile, study of the effect of combining with rabeprazole, as well as preliminary discussion of distribution of levofloxacin in human stomach and bile, then obtained the following conclusions:⑴In the rat body, along with the extension of administration time, the levofloxacin concentration of gastric juice gets higher than the serum concentration, indicating that there is an active transport process for the levofloxacin in the stomach.⑵The levofloxacin concentration of the gastric antrum of rats is higher than that of the forestomach, there is a rising trend partially with increase of administration dose, and combining with PPI has a slight influence on the drug concentration and distribution of levofloxacin in the stomach.⑶Higher levofloxacin concentration in the bile of both rats and human indicates that the bile is an important excretory process for levofloxacin.⑷It is preliminarily found in the human body test that the levofloxacin concentration of gastric mucosa is higher than the drug concentration of gastric juice, and partially higher concentration of gastric antrum and gastric corpus indicates that the levofloxacin may gather at Hp specificity colonization areas.
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