【作者】 李国庆；

【导师】 蒋建东； 游雪甫；

【作者基本信息】 北京协和医学院 ， 微生物与生化药学， 2011， 博士

【摘要】 喹诺酮类抗生素作为目前我国临床治疗感染性疾病的重要药物之一,具有抗菌谱广、抗菌活性强、药代动力学特性好等特点,尤其氟喹诺酮类抗生素具有更大的优势。从喹诺酮类抗生素发现至今,已有数十种新喹诺酮类化合物进入临床,当前广泛使用的环丙沙星、氧氟沙星、左氧氟沙星、莫西沙星等是临床抗感染不可或缺的药物。昌欣沙星是由我国自主研发的1.1类新氟喹诺酮类抗生素,是由莫西沙星进行结构改造,将8位二氟甲氧基取代甲氧基而得,具有良好的体内外广谱抗菌活性。本论文第一部分研究了昌欣沙星的体外药效学,包括对1754株临床分离菌的MIC分布、MBC、KCs、抗菌活性影响因素、细胞毒性和对DNA回旋酶和拓扑异构酶Ⅳ的抑制试验。结果表明,昌欣沙星对革兰氏阳性菌和革兰氏阴性菌均具有较强的广谱抗菌活性,对肺炎链球菌、流感嗜血杆菌具有非常强的抗菌活性,MIC90分别为0.25μg/mL、0.03μg/mL,与莫西沙星相当或稍强,对金葡菌、表葡菌、化脓链球菌、粪肠球菌、肺炎克雷伯杆菌的MIC5o为0.06～0.5μg/mL之间,显示出良好的活性。昌欣沙星对临床耐药菌仍有较好的抗菌活性,对MRSA、CR-MRSA、MRSE、CR-MRSE、PISP、PRSP等的MICso为0.125～4μg/mL之间,对产ESBL的大肠埃希菌和肺炎克雷伯杆菌的活性稍弱,但仍与莫西沙星相当。昌欣沙星对金葡菌、肺炎链球菌、流感嗜血杆菌、副流感嗜血杆菌的敏感率较高,分别为52.3%、97.8%、100%和86.5%,具有较好的临床使用前景。MBC试验和杀菌曲线试验结果表明,昌欣沙星对金葡菌、化脓链球菌、肺炎链球菌、大肠埃希菌、肺炎克雷伯杆菌是一种有效的杀菌剂,对金葡菌、肺炎链球菌、大肠埃希菌、肺炎克雷伯杆菌的杀菌效果呈现明显的浓度依赖关系,对化脓链球菌呈现时间依赖关系,有待于进一步研究。影响因素试验表明,培养基pH值、细菌接种量、血清浓度等对昌欣沙星的抗菌活性无明显影响。细胞毒性试验结果表明,昌欣沙星对人肺腺癌A549细胞毒性较小,IC5o为551.04μg/mL(大于1000μM),与莫西沙星等其他临床常用喹诺酮类化合物相当。昌欣沙星对喹诺酮类靶点DNA回旋酶和拓扑异构酶Ⅳ的抑制试验表明,昌欣沙星对DNA回旋酶的促旋活性抑制的IC5o为1.05μM,稍弱于莫西沙星,但与环丙沙星相当；对拓扑异构酶Ⅳ的解离活性抑制的IC5o为2.25μM,弱于莫西沙星和环丙沙星；松弛活性抑制的IC5o为0.79μM,强于莫西沙星和环丙沙星。此外,为更深入的研究昌欣沙星的药效学,克隆表达了金葡菌中喹诺酮类抗生素的靶点之一的拓扑异构酶Ⅳ,为进一步研究昌欣沙星的作用机制打下了基础。本论文第二部分研究了昌欣沙星的体内药效学。在小鼠全身感染模型中,昌欣沙星口服给药与莫西沙星口服给药治疗效果相当,对革兰氏阳性菌优于左氧氟沙星,对革兰氏阴性菌弱于左氧氟沙星。其中对金葡菌、肺炎链球菌、大肠埃希菌、肺炎克雷伯杆菌杆菌引起的全身感染具有较好的治疗效果,ED5o为1.25 mg/kg至8.28 mg/kg;对粪肠球菌引起的全身感染治疗效果稍差,EDso为25.02 mg/kg o同时,研究了昌欣沙星在小鼠体内的药代动力学特点,通过计算昌欣沙星5 mg/kg口服给药后小鼠体内的药代动力学参数,得到昌欣沙星在小鼠体内的Cmax为1085ng/mL,半衰期为1.425h,AUC0-24h为1101.31h*ng/mL。根据体外药效学MIC数据计算获得金葡菌、肺炎链球菌、肺炎克雷伯杆菌、流感嗜血杆菌、副流感嗜血杆菌的Cmax/MIC50分别为18.08、8.68、4.34、135.625、31.17,化脓链球菌的T>MIC为2.03h。试验结果说明昌欣沙星具有良好的药代动力学特征,进一步说明昌欣沙星具有良好的体内抗菌能力。更多还原

【Abstract】 Quinolone antibiotics are important kind of medicines dealing with clinical infectious diseases in China, which are broad-spectrum, highly potent and have good pharmacokinetics parameters, especially the fluoroquinolones which are more effective. Since the discovery of the quinolones, there are several scores of ones with new structure being used in clinical. Ciprofloxacin, ofloxacin, levofloxacin and moxifioxaxin are enssential in the treatment of infection. Chinfloxacin is a Class I new quinolone drug developed by our country, which is the difluoride substitution of moxifloxacin in the 8-methoxy group, and it shows broad-spectrum activities against bacteria in vitro and in vivo.In the first part of this work, the in vitro pharmacodynamics characteristics of Chinfloxacin were studied to 1756 clinical isolates and standard isolates, including the MICs, MBC, KCs, incubation conditions’effects, cytotoxic and inhibition test against DNA gyrase and topoisomerase IV. The result demonstrated that Chinfloxacin had good antibacterial activity against both Gram-positive and Gram-negative organisms, notablely the antibacterial activity against S. pneumoniae, H. influenzae were extremely good, MIC90S were 0.25μg/mL、0.03μg/mL, which were equal to or better than moxifloxacin. Besides, the MIC50S of Chinfloxacin against the other frequent clinical isolates such as S. aureus, S. epidermidis, S. pyogenes, E. faecalis and K. pneumoniae were between 0.06μg/mL to 0.5μg/mL, showed good activities too. Chinfloxacin had relatively good antibacterial activity to clinical drug-resistant isolates. The MIC50S against MRSA, CR-MRSA, MRSE, CR-MRSE, PISP and PRSP were from 0.125μg/mL to 4μg/mL. The activities of Chinfloxacin against ESBL- E. coli and ESBL-K. pneumonia were slightly week, but still equal to that of moxifloxacin. The susceptible rates of Chinfloxacin to S. aureus, S. pneumoniae, H. influenzae and H. parainfluenzae were quite high, which were 52.3%,97.8%,100% and 86.5%, showed good potential. MBC experiment and KCs experiment revealed that Chinfloxacin was an effective bactericidal agent against S. aureus, S. pyogenes, S. pneumoniae, E. coli and K. pneumoniae. The activities of Chinfloxacin against S. aureus, S. pneumoniae, E. coli and K. pneumoniae were concentration dependent, but were time dependent against S. pyogenes, which should be studied later. The pH, inoculum size and serum concentration had no apparent effects on the activity of Chinfloxacin.The cytotoxicity experiment showed that Chinfloxacin was safe to human adenocarcinoma lung cell A549, with IC50 551.04μg/mL (more than 1000μM), which was similar to the other quinolones usually used in clinical.The inhibition test of Chinfloxacin against DNA gyrase and topoisomerase IV, the target of the quinolone, revealed that the IC50 of Chinfloxacin in the supercoling inhibition test is 1.05μM, a little weak than moxifloxacin, but equal to ciprofloxacin. The IC50 of Chinfloxacin in the decatenation inhibition test is 2.25μM, which is inferior than moxifloxacin and ciprofloxacin. Then the IC50 of Chinfloxacin in the relaxation inhibition test is 0.79μM, which is superior than moxifloxacin and ciprofloxacin.Besides, to do some further research, we express the topoisomeraseⅣof S. aureus, one of the target of quinolones. We purified the two submits of the enzyme, quantitate the concentration and then determined the activity of the enzyme, to do the groundwork for the further research of the mechanism of action.In the second part of this work, the in vivo pharmacodynamics of Chinfloxacin was studied. In the mouse systemic infection model, Chinfloxacin showed the same therapeutic efficacy with moxifloxacin by oral administration, which is better than levofloxacin against Gram-positive bacteria, and is inferior than levofloxacin against Gram-negative bacteria. Chinfloxacin showed good efficacy against S. aureus, S. pneumoniae, E. coli and K. pneumoniae, with ED50s 1.25 mg/kg to 8.28 mg/kg, and showed weaker efficacy against E. faecalis in vivo, with ED50 25.02 mg/kg.Meanwhile the pharmacokinetics of Chinfloxacin in mouse was studied. The Cmax, T1/2, AUC0-24h were 1085ng/mL,1.425h,1101.31h*ng/mL respectively calculated by the pharmacokinetics parameters of mouse after oral administration of Chinfloxacin. On the basis of the MIC in vitro, we obtained the Cmax/MIC50 of S. aureus, S. pneumoniae, K. pneumoniae, H. influenzae and H. parainfluenzae which were 18.08、8.68、4.3、135.625、31.17 respectively, and the T>MIC50 of S. pyogenes was 2.03h. The results demonstrate that Chinfloxacin had good pharmacokinetics performance, and revealed the good antibacterial potential in vivo.更多还原