【作者】 张莉蓉；

【导师】 王永铭；

【作者基本信息】 复旦大学 ， 药理学， 2003， 博士

【摘要】 氟喹诺酮类 (fluoroquinolones, FQs) 药物临床上治疗各种感染性疾病具有良好的疗效，但随着其广泛应用，不良反应（adverse drug reaction, ADR）报告日渐增多。尤其引起人们关注的是近年来因连续发生严重ADR从市场撤除、停止申报注册和限制使用的FQs，如替马沙星、曲伐沙星、格帕沙星和克林沙星。因此，为指导临床合理选择该类药物和新品种的开发，亟待对FQs进行安全性评价。本研究通过对FQs的ADR进行医院内集中监测，确定临床常用FQs品种在我国人群中的ADR发生率及风险因素（着重观察中枢神经系统反应）；研究FQs在清醒大鼠的神经毒性和毒代动力学；利用分子生物学和膜片钳技术探讨FQs 中枢神经毒性机制。主要结果如下： 1. 通过多中心的医院内集中监测对临床常用的FQs抗菌药物进行安全性评价。监测期（2001年5月~2002年6月）有2003例住院患者入选，其中262人发生ADR 279次，发生率为13.93 %。82次为中、重度ADR，222次属A型不良反应。按卫生部ADR监测中心颁发的标准进行因果关系评价，肯定的ADR有22次，占24.01 %。以左氧氟沙星、环丙沙星和氟罗沙星最为常用，ADR发生率分别为9.96 % (110/1104)，13.94 % (74/531) 和22.45 % (66/294)。累及胃肠道及中枢神经系统的ADR比例较高，分别占38.71 % (108/2003)和28.67 % (80/2003)；其次为皮肤反应, 占20.43 % (57/2003)；不同品种ADR的发生有明显差异，环丙沙星以皮肤反应最多见 (P<0.05)，氟罗沙星、左氧氟沙星以胃肠道反应较多见 (P<0.01)，而6CNS反应明显多见于氟罗沙星 (P<0.01)。ADR的发生与年龄、性别、用药种数、原发疾病的严重程度、既往过敏史及肾功能有关。这些风险因素对临床上预测FQs潜在毒性具有重要作用。2. 研究诺氟沙星在清醒大鼠的神经毒性和毒代动力学。将大鼠随机分为4组，<WP=5>每组6~10只，分别i.v. NS，诺氟沙星50，100 和200 mg·kg-1。连续记录自由活动大鼠的脑电图（EEG），并同步用微生物法测定血清药物浓度， 检测菌为大肠杆菌441102。结果显示：(1) 诺氟沙星各组大鼠EEG均出现痫样放电，并伴有局部抽搐和全身强直痉挛发作等行为学改变，脑电功率谱分析主要表现为脑电相对总功率增加 (P < 0.05)，呈剂量依赖性。 (2) 诺氟沙星的药时曲线符合二室模型，CL，Vc 和 T1/2β 与给药剂量无关，Cmax 和AUC0→∞呈剂量依赖性。 （3）以脑电相对总功率的增加为效应的定量指标，与剂量，Cmax 和AUC0→∞进行非线性拟合呈Sigmoid-Emax模型 (R2分别为0.92, 0.94, 0.95 )。 提示AUC0→∞和脑电相对总功率的变化可作为FQs中枢毒性效应判定和预测的客观指标。 3. 研究诺氟沙星与芬布芬（fenbufen）的活性代谢物对苯丙氨基苯乙酸(BPAA)合用诱发癫痫样发作与IL-1β、NO的关系。诺氟沙星 (25 mg/kg, i.p.) 与BPAA（100 mg/kg, i.g.）合用可诱发大鼠癫痫样发作。连续记录自由活动大鼠的EEG分析痫样放电，用Racine’s标准观察行为学改变，RT-PCR测定给药后大鼠额前区、海马不同时间IL-1β、iNOS mRNA的表达，免疫组化法观察IL-1β蛋白的表达。结果：诺氟沙星合用BPAA可诱导大鼠额前区、海马 IL-1β mRNA快速、暂时性上调。给药后30 min即可在额叶皮层、海马测到IL-1β mRNA 高表达，6 h后基本恢复正常。IL-1β免疫反应阳性细胞染色变浓, 密集程度亦增加。iNOS mRNA 在额叶皮层和海马的表达仅在给药后30 min明显增高（P<0.05），而在其他时间点虽有表达增加，但与对照组相比无显著差异（P>0.05）。生理盐水对照组、诺氟沙星或BPAA单用组不诱发大鼠痫样发作及IL-1β表达、iNOS mRNA表达。结果提示：IL-1β、NO可能参与诺氟沙星合用BPAA所致的痫样发作。4. 研究FQs抗菌药物（司帕沙星、氟罗沙星、氧氟沙星和左氧氟沙星）对大鼠海马锥体细胞电压门控钾通道的作用。应用全细胞式膜片钳技术在急性打散的海马锥体细胞上分离到电压门控钾电流，主要包括快速失活的IA电流和失活比较缓慢的延迟整流钾电流IK。结果显示：司帕沙星、氟罗沙星、氧氟沙星和左氧氟沙星对电压门控通道IK均有迅速可逆的阻断作用，IC50分别为6.44×10-4 M, 7.09×10-3 M, 8.42×10-3 M和 1.10×10-2 M。司帕沙星、氟罗沙星对电压门控通道IA也有迅速可逆的阻断作用，IC50分别为2.86×10-3 M，4.38×10-3 M，而氧氟沙星、左氧氟沙星在1 mM对IA无明显阻断作用。司帕沙星对IK和IA的抑制具有电压依赖性 (-20 mV~60 mV)。结果表明，FQs抗菌药物抑制海马锥体细胞的两种电压门控性钾通道，可能与其中枢毒性有关。<WP=6>更多还原

【Abstract】 Fluoroquinolones (FQs) have been shown to be very effective for the treatment of various bacterial infections. However, with FQs widely used, more and more adverse drug reactions (ADRs) have been reported. Several new FQs have been withdrawn from the market in recent years because of rare or exceptional but life-threatening ADR (e.g. temafloxacin, trovafloxacin, grepafloxacin and clinafloxacin). Thus, it is necessary to evaluate fluoroquinolone safety for the guideline of clinical rational use and the development of new candidates.The present studies firstly evaluated the safety in everyday clinical usage of fluoroquinolones by hospital intensive monitoring and determining incidence rates and risk factors of ADR. Secondly, the neurotoxicity and toxicokinetics of norfloxacin in freely moving rats were studied. Finally, the underlying mechanisms of neurotoxicity were investigated using molecular biological techniques and patch-clamp electrophysiological techniques. The results are summarized as follows:1. The safety in everyday clinical usage of fluoroquinolones in P. R. China was evaluated by multicentre hospital intensive monitoring. The main outcome measures were patient background factors, including age, sex, underlying disease, complications, history, etc.; the indication for prescribing the drug being monitored; the start and stop dates of treatment and the events recorded during and after treatment. 2003 hospitalized patients who took fluoroquinolones were monitored between May 2001 and June 2002. There were 279 ADRs identified in 262 patients. The overall event rate is 13.93% (279/2003). Eighty-two ADRs were characterized as moderate or severe in degree, and 222 ADR were classified as type A reactions. Causality assessment (according to standardized algorithm of ADR Monitoring Centre， Ministry of Public Health, China) revealed that cerain causality was established in 22 (24.01%) of total ADR. The most<WP=8>commonly used FQs were levofloxacin, ciprofloxacin and fleroxacin. The ADR rates of levofloxacin, ciprofloxacin and fleroxacin were 9.96% (110/1104), 13.94% (74/531) and 22.45% (66/294), respectively. Gastrointestinal, CNS and skin manifestations accounted for 38.71% (108/2003), 28.67% (80/2003) and 20.43%(57/2003) of the ADR, respectively. We found some significant differences in the safety profiles of individual FQs: ciprofloxacin was more frequently associated with skin reactions (P<0.05), fleroxacin and levofloxacin with gastrointestinal reactions (P<0.01), and fleroxacin with CNS reactions (P<0.01). Age, sex, number of concomitant drugs, allergic history and renal function were closely related to ADR. The aforementioned risk factors may play an important role for clinician to predict the potential risk of FQs. 2. The neurotoxicity and toxicokinetics of norfloxacin were studied in freely moving rats . Rats were assigned randomly to four treatment groups that received a single iv dose of 50, 100, 200 mg/kg of norfloxacin and 0.9 % saline, respectively. Electroencephalogram (EEG) was continuously recorded with a computerized system in freely moving rats. Venous blood samples were collected for determination of the norfloxacin concentration using microbioassay method with Escherichia coli 441102 as the test strain. Toxicokinetic parameters were determined from serum concentration-time data with the 3p97 program. Result: (1) The epileptiform discharges appeared in all norfloxacin groups with different latent periods, accompanied with limb twitching and clonic-tonic seizures. The relative total power of the EEG increased. (2) Drug serum concentration-time curves of different doses conformed to two compartment model. The values of clearance, volume of distribution, and terminal half-life were dose-independent, while maximum serum concentrations （Cmax） and the areas under the concentration-time curve(AUC0→∞) of norfloxacin increased with dosage. (3) The increase in relative total power of the EEG was chosen as quantitative measurement of CNS stimulant effect of norfloxacin. Th更多还原