【作者】 初乃惠；

【导师】 朱莉贞；

【作者基本信息】 北京市结核病胸部肿瘤研究所 ， 结核病学， 2008， 博士

【摘要】 研究背景氯法齐明(Clofazimine,Cfz),是1944年在开发抗结核新药时被发现的,但在当时的豚鼠及猴子结核病模型中未显示活性,此后一直被忽视对其抗结核作用的进一步研究和应用。所幸的是在抗麻风杆菌的研究中发现了它具有很好的抗分枝杆菌作用,至今已用于治疗麻风病及鸟型分枝杆菌病多年,是一种较为有效的抗麻风病药物。对于氯法齐明抗结核活性的研究仅有个别报道,在世界范围内尤其是我国的耐多药以及严重耐多药结核病治疗十分困难的情况下,进行氯法齐明抗结核作用的基础与临床研究,评价其抗结核活性显然是十分必要的,将为临床应用提供依据,为肺结核特别是耐药肺结核患者提供可能的治疗药物打下基础。研究目的通过评价氯法齐明对结核分枝杆菌标准株及耐药结核分枝杆菌临床株的体外活性、小鼠结核病模型的体内治疗作用观察;以及氯法齐明的组织分布和异烟肼、吡嗪酰胺对其组织分布的影响的研究,并采用自身对照的方法评价氯法齐明联合其他抗结核药治疗耐多药肺结核的有效性和安全性,为氯法齐明治疗耐药结核病的临床应用提供帮助。研究方法1.应用微孔板指示剂法测定氯法齐明对结核分枝杆菌标准株及对30株临床分离耐多药结核分枝杆菌的最低抑菌浓度;通过建立小鼠静脉感染H37Rv 1×105CFU/只的结核病模型,按照氯法齐明20mg/kg(Cfz-1)每周给药5次;10 mg/kg(Cfz-2)每周给药5次及20 mg/kg (Cfz-3)每周给药2次的方案给予治疗;30天时进行活菌计数评价氯法齐明在小鼠体内的抗结核活性;并将耐药结核分枝杆菌临床分离株感染小鼠,应用氯法齐明和联合化疗方案治疗60天,评价氯法齐明对耐药结核病模型小鼠的效果。2.采用昆明种小鼠和BALB/C小鼠,单次给药研究氯法齐明的组织分布并在小鼠体内研究异烟肼、吡嗪酰胺对氯法齐明组织分布的影响:灌胃给药,于给药后1h、5h、24h,眶下取血或摘眼球取血。解剖取小鼠肝、肾、脾、肺、脂肪等,测量组织浓度。3.氯法齐明治疗耐多药结核病的初步研究。选择耐多药结核病患者,自愿签署知情同意书;年龄18-65岁;采用为期24个月自身对照的临床试验。根据药敏试验结果,选择含氯法齐明(Cfm)、丙硫异烟胺(Pto)、对氨基水杨酸钠(PAS)、左旋氧氟沙星(Lfx)、克拉霉素(Clr)、丁胺卡那霉素(Am)以及敏感药组成的5-7种药的强化期为6个月,巩固期为24个月的化疗方案,观察治疗耐多药结核病的有效性和安全性。结果:1.氯法齐明对结核分枝杆菌H37Rv的MIC为0.12ug/ml,对耐多药结核分枝杆菌MIC范围是0.12-1.96μg/ml。2.在标准株感染小鼠的结核病模型中显示:氯法齐明虽然抗结核的活性不如异烟肼,但可以将小鼠肺脏中的结核分枝杆菌降低1.8～2.9Log10 CFU,脾脏中降低1.5～2.5 Log10 CFU,表明具有较好的体内抗结核作用。3.在耐药株感染小鼠的结核病模型中显示:治疗2月后氯法齐明可以将肺中的活菌数降低2.32 Log10 CFU,而5药联合化疗后可以降低3.9 Log10 CFU。4.氯法齐明在小鼠体内的组织分布顺序依次为脂肪、脾、肾、肝、肺等组织,异烟肼和吡嗪酰胺均可提高氯法齐明在肺组织的浓度,氯法齐明在给药2月停药后的2月仍不能彻底消除。5.氯法齐明在治疗的9例耐多药结核病患者中,疗程从3个月到10个月不等,有3例患者在3个月时痰菌阴转,同时病灶有吸收,在治疗的9例病人中,临床症状均有不同程度的改善。血药浓度的测定显示个体差异比较大,且与疗效关系不密切。主要的副作用为面部、头发和四肢暴露皮肤的色素沉着。研究结论1.氯法齐明具有较好的体外及体内抗结核分支杆菌活性,对敏感菌及耐药菌均有效。2.氯法齐明在脂肪、脾、肾、肝、肺等组织分布浓度比较高,异烟肼和吡嗪酰胺均可提高氯法齐明在肺组织的浓度。氯法齐明在组织中消除所需时间较长,这也是皮肤红染的主要原因。3.氯法齐明对耐药结核病可能有效,但由于例数较少,仍需要进行大样本的临床对照研究。更多还原

【Abstract】 Background:Clofazimine, which was found in 1944 during the developing of new anti-tuberculosis drugs, no more research and application on tuberculosis because it has no activity against Mycobacterium tuberculosis in the guinea pigs and monkeys models. Fortunately, it was effect on the research of anti-leprosy bacilli, used in treating leprosy and bird-Mycobacterium disease for many years. There was few report on the research of anti-tuberculosis activity with Clofazimine. Because it is very difficult to treat the MDR-TB and XDR-TB in the world especially in China, we carried out the basic and clinical studies on the tuberculosis of Clofazimine, the object is to estimate the activity of anti-tuberculosis and supply the base to clinical application, which will be an anti-tuberculosis drug especially to the drug-resistant TB.Object:We observed the in vitro activity against Mycobacterium tuberculosis standard strain and Mycobacterium tuberculosis clinical resistant strains, and the effect in vivo in mice TB models treated with Clofazimine. The tissue distribution of Clofazimine and the effect of Isoniazid、Pyrazinamide to the tissue distribution of it. We adopted the method of self-control to estimate the efficacy and safety of Clofazimine combined with other anti-tuberculosis drugs in treating the MDR-TB, and help the clinical application of Clofazimine to treat the drug resistant TB.Method:1. Apply the microplate Alamar Blue method to determine the MIC of Clofazimine to standard strains of mycobacterium tuberculosis and 30 clinical isolates of MDR mycobacterium tuberculosis. Design tuberculosis mice model infected with H37RV 1×105CFU through vein, administer Clofazimine 20mg/kg(CLF-1) 5 times a week; 10 mg/kg(CLF-2) 5 times a\week and 20 mg/kg (CLF-3) 2 times a week; after 30 days we achieved the viable count to evaluate the anti-TB activity of Clofazimine in mice, and estimate the effect on the tuberculosis model mice which infected the clinical isolates of drug resistant mycobacterium tuberculosis were treated with Clofazimine and combined chemotherapy for 60 days.2. Study the tissue distribution of Clofazimine and the effect by isoniazid、pyrazinamide which administration one time in BALB/C mice, after administration 1h,5h,24h, blood from infraorbital or eyeball extraction. Determined the concentration of liver, kidney, spleen, lungs, fat, etc using by HPLC.3. Preliminary study on MDR-TB treated with Clofazimine. We choose the multi-drug resistant TB patients, signed informed consent voluntarily, aged from 18 to 65 years, and adopt the 24-month own control clinical trials. Based on the sensitivity test results, We select 5-7 kinds of drugs to strengthen period for 6 months and consolidation period for 24 months including Clofazimine (Cfm), Protionamide (Pto), Sodium Aminosalicylate (PAS), Levofloxacin (Lfx), Clarithromycin (Clr ), Amikacin (Am) and sensitive drugs , to observe the efficacy and safety of the treatment to MDR-TB .Results:1.The MIC of Clofazimine to Mycobacterium tuberculosis H37Rv was 0.12 ug / ml, The range of MIC to multi-drug resistant Mycobacterium TB was 0.12-1.96μg / ml.2.In the TB mice model infected with standard strains showed: Clofazimine has less anti-TB activity than Isoniazid, but it can reduce 1.8 ~ 2.9 Log 10 CFU of Mycobacterium tuberculosis in lungs, 1.5 ~ 2.5 Log 10 CFU in spleen, which has good anti-tuberculosis activity in vivo.3. In the mice model infected with resistant strain showed: after 2 months treatment, Clofazimine can reduce 2.32 Log 10 CFU of the viable count in lung, and 3.9 Log 10 CFU with 5 kinds of drugs combined chemotherapy.4.The order of tissue distribution in mice about Clofazimine is fat, spleen, kidney, liver, lung, and other organizations, Isoniazid and Pyrazinamide can improve the concentration of Clofazimine in lung tissue, after 2 months treatment with Clofazimine it is still not complete elimination in the followed 2 months. 5.In the 9th multi-drug resistant TB patients treated with Clofazimine, the course of treatment is from 3 months to 10 months, 3 of them has sputum negative after 3 months, while focus of infection were absorbed. All of them have different degrees of improvement in the clinical symptoms. Determination of plasma display larger differences individually, which has no close relationship with the effect. The main side effect is pigmentation in the face, hair and exposed skin of limbs.Conclusion:1.Clofazimine has good anti- Mycobacterium TB activity in vitro and in vivo, against the sensitive and drug-resistance TB.2.The distribution of Clofazimine is higher in fat, spleen, kidney, liver, lungs and other organization, Isoniazid and Pyrazinamide can improve the concentration of Clofazimine in lung tissue. The main reason of skin pigmentation is Clofazimine eliminated from the organization after a longer period of time since withdrawal.3.Clofazimine may be effective to the drug-resistant TB, but there is few case, we will need a large samples of clinical control study.更多还原