【作者】 鲍峻峻；

【导师】 许建明； 胡咏梅； 梅俏；

【作者基本信息】 安徽医科大学 ， 老年医学， 2009， 博士

【摘要】 背景应用抗菌药有效根除HP具有十分重要的临床意义。由于HP特异性定植于胃粘液及其下方黏膜表面,因此,Hp根除成功与否理应取决于治疗部位局部抗菌药的浓度。只有抗菌药在其定植部位浓度达到所需杀菌浓度方可起效。抗菌药口服后由血转运至胃,这是抗菌药胃内局部浓度升高的决定性因素,是HP根除成功的关键问题。抗菌药胃内转运具有特殊的药代动力学特点,存在胃内直接分布及肠道吸收后由血转运至胃组织的跨膜转运。研究抗菌药胃内转运、分布特点可为根除HP方案中抗菌药的选择提供试验数据。近年来HP耐药情况日益严重,经典的三联疗法根除失败率逐年升高。临床研究发现,含左氧氟沙星为基础的治疗方案是目前根除率较为理想的治疗方案。但针对左氧氟沙星胃内转运分布情况的基础研究尚未见报道,缺乏左氧氟沙星胃内区域药动学资料。另外,胃内pH值可影响抗菌药的胃内转运、分布。因此,联用PPI是否影响左氧氟沙星胃内转运、分布尚不明确。由于解剖结构的毗邻关系,胆总管直接开口于十二指肠,胆汁内抗菌药也可随胆汁返流入胃,从而影响胃内局部抗菌药浓度。研究胆汁内左氧氟沙星的药动学过程,有助于阐明左氧氟沙星抗Hp作用。目的本课题拟在建立研究抗菌药胃内和胆汁转运、分布的动物模型基础上,初步探讨左氧氟沙星在大鼠胃内和胆汁转运、分布特点;观察联合应用雷贝拉唑对上述胃内过程的影响;同时,结合动物实验研究结果,初步探讨左氧氟沙星在人体胃内、胆汁内浓度情况,从而为临床应用左氧氟沙星根除HP过程中的临床药理学优势提供实验依据。方法⑴建立抗菌药胃内转运、分布的动物模型:Wistar大鼠术前禁食,麻醉开腹行胆总管置管。在十二指肠距幽门2cm处,经切口橡胶软管通过幽门插入胃内冲洗,以备胃液收集。试验结束时,采用水泡法剥离胃黏膜,拭干、称重后研磨匀浆,用于测定药物浓度;胃粘液层厚度采用冰冻切片PAS/AB染色法进行测量。⑵建立不同生物样本中左氧氟沙星检测的HPLC方法:大鼠和人血清、胃液、胃黏膜和胆汁样本采用三氯乙酸或甲醇沉淀蛋白,取上清液测定左氧氟沙星浓度。HPLC条件:流动相乙腈-50mmol/l柠檬酸-1mol/l醋酸铵(19:80:1;v/v),Kromosil C18分析柱,柱温50℃,流速1ml/min,检测波长295nm。评价选择性、线性、准确度、精密度、稳定性和灵敏度;⑶大鼠左氧氟沙星胃内转运、分布研究:Wistar大鼠随机分为左氧氟沙星50mg/kg组、左氧氟沙星50mg/kg+雷贝拉唑组、左氧氟沙星100mg/kg组、左氧氟沙星100mg/kg+雷贝拉唑组共4组。静脉给药后2小时内按15min间隔取血处死动物,收集不同时点胃液、胃黏膜样本,检测左氧氟沙星浓度,了解大鼠左氧氟沙星胃内转运、分布特点;⑷大鼠左氧氟沙星胆汁分布研究:Wistar大鼠随机分为左氧氟沙星20mg/kg组, 50mg/kg组,100mg/kg组共3个剂量组。术中行颈动脉置管术及胆总管置管术,分别在静脉给药后12小时按1小时间隔,收集不同时点血液及胆汁样本,检测左氧氟沙星浓度,了解大鼠左氧氟沙星胆汁分布特点;⑸人体左氧氟沙星胃内分布研究:4例HP感染患者,采用口服雷贝拉唑10mg bid +左氧氟沙星500mg qd +阿莫西林1.0 bid方案治疗,第10天行胃镜检查,留取胃粘液标本后,自下而上分别留取胃窦、胃体、胃底各部位胃黏膜样本,检测左氧氟沙星浓度,了解人体左氧氟沙星胃内分布特点;⑹人体左氧氟沙星胆汁分布研究:17例因胆系疾病行胆道探查术后留置T管的患者,分别静滴左氧氟沙星200mg、400mg和500mg。给药结束后24小时,按1h, 2h, 4h, 8h, 12h, 24h时间点留取血标本,按1小时间隔留取胆汁样本,检测左氧氟沙星浓度,了解人体左氧氟沙星胆汁分布特点。结果⑴对研究抗菌药胃内和胆汁转运、分布的动物模型中所涉及的胃液收集方法、胃黏膜剥离方法以及胃粘液层测量方法等关键实验方法和不同生物样本中左氧氟沙星HPLC检测方法进行验证,证明动物模型和HPLC检测方法具有可靠、简便的技术要求。⑵大鼠左氧氟沙星胃内转运、分布:比较左氧氟沙星50mg/kg组和左氧氟沙星100mg/kg组不同时点各部位药物浓度发现,给药后45-60min左右,胃液药物浓度逐渐高于血药浓度,不同时间点胃体、胃窦、腺胃药物浓度均高于血药浓度及前胃药物浓度,但胃体和胃窦药物浓度无差异。比较单用左氧氟沙星组和左氧氟沙星+雷贝拉唑联用组结果发现,联用雷贝拉唑组的胃液、前胃、胃体、胃窦和腺胃不同时点药物浓度均高于单用左氧氟沙星组,但统计学无显著差异。左氧氟沙星50mg/kg组、左氧氟沙星50mg/kg+雷贝拉唑组、左氧氟沙星100mg/kg组、左氧氟沙星100mg/kg+雷贝拉唑组左氧氟沙星胃内转运分数分别为2.36,2.42,2.52,2.55。⑶大鼠左氧氟沙星胆汁分布:左氧氟沙星20mg/kg,50mg/kg,100mg/kg各剂量组胆汁药位浓度Cmax分别为57.42mg/l,88.81 mg/l,137.60 mg/l。经统计分析发现,各剂量组各时点胆汁药物浓度均有差异。⑷人体左氧氟沙星胃内分布:人胃液左氧氟沙星浓度为2.26±1.32μg/ml,胃底、胃体、胃窦样本左氧氟沙星浓度分别为5.51±1.46μg/ml,9.99±4.01μg/ml,8.52±2.45μg/ml,均高于胃液左氧氟沙星浓度。⑸人体左氧氟沙星胆汁分布:左氧氟沙星200mg,400mg,500mg各剂量组胆汁药物浓度Cmax分别为11.92mg/l,16.52mg/l,20.53mg/l,经统计分析发现,各剂量组Cmax有差异。比较各剂量组各时间点胆汁药浓度,发现不同剂量组几乎在不同时间点胆汁药浓度均有差异。结论本课题通过研究大鼠左氧氟沙星胃内和胆汁转运、分布情况及其联用雷贝拉唑的影响,以及初步探讨人体左氧氟沙星胃内和胆汁分布情况,得出以下结论:⑴在大鼠体内,随给药时间延长胃液左氧氟沙星浓度高于血药浓度,提示左氧氟沙星胃内存在主动转运过程。⑵大鼠胃窦左氧氟沙星浓度高于前胃,并具有随给药剂量增大而局部浓度增大趋势,联用PPI对左氧氟沙星胃内药物浓度及分布影响较小。⑶大鼠与人胆汁内左氧氟沙星浓度较高,提示胆汁是左氧氟沙星一个重要的排泄过程。⑷在人体试验中初步发现,胃黏膜中左氧氟沙星浓度高于胃液药物浓度,其中胃窦和胃体局部浓度较高,提示左氧氟沙星可聚集于Hp特异性定植部位。更多还原

【Abstract】 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.更多还原