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米尔贝肟的非临床安全性评价及对犬蛔虫体外作用的研究
Non-Clinical Safety Evaluation and in Vitro Study Against Toxocara Canis of Milbemycin Oxime
【作者】 曲明丽；
【导师】 李继昌；
【作者基本信息】 东北农业大学 ， 基础兽医学， 2011， 硕士
【摘要】 米尔贝肟（milbemycin oxime）是从链霉菌属中分离而得到的大环内酯类抗生素,主要由米尔贝霉素A3肟和米尔贝霉素A4肟按2:8比例组成的混合物。为了探讨其安全性,本研究利用小鼠经口急性毒性试验、Ames试验、小鼠骨髓微核试验、小鼠精子畸形试验、大鼠长期毒性试验等方法对米尔贝肟进行了药物毒理学安全性评价。小鼠急性毒性试验结果表明,米尔贝肟对昆明系小白鼠毒性很低,并且有显著的性别差异,其对雄性和雌性小白鼠的LD50分别为1832 mg·kg-1和727 mg·kg-1,95%可信限为1637.57～2022.08 mg·kg-1和603.95～868.96 mg·kg-1;Ames试验结果表明,各剂量组回变菌落数均未超过阴性对照组回变菌落数的2倍,亦无剂量——反应关系。在小鼠骨髓嗜多染红细胞微核试验中,米尔贝肟未引起骨髓嗜多染红细胞微核率的增加。在精子畸形试验中,各剂量组精子畸形率差异不显著,亦无剂量——反应关系。采用Wistar大鼠进行米尔贝肟的三个月慢性毒性实验,给药后一个月,高剂量组大鼠出现精神沉郁,但给药停止后症状随之消失,其他组动物外观体征和行为活动未发生明显变化;血液学指标各给药组均无明显差异;血液生化指标均在正常范围内;高剂量组肺脏、肝脏、小肠、脾脏出现轻微病理变化,但在恢复期均恢复正常。其他组织,未见异常。为满足临床应用的需要,本文就米尔贝肟对犬蛔虫的体外作用进行了研究。从哈尔滨周边地区杂种犬小肠分离犬蛔虫成虫,观察在10-4、10-5、10-6、10-7、10-8 g·mL-1浓度下,给药后4、8、12、24、48h,米尔贝肟对犬蛔虫成虫的作用。结果表明,10-4、10-5、10-6 g·mL-1剂量组米尔贝肟对犬蛔虫虫体抑制作用的有效率均达到100%,而10-7、10-8 g·mL-1剂量组的有效率为66%,药物对虫体活力的影响程度与药物剂量有很大的关系。按照上述浓度进行药物对犬蛔虫卵作用试验,结果表明,随着药物浓度增大,虫卵孵育所需时间延长,Ⅴ期幼虫所占比例也有所下降;将与药物作用后并发育为Ⅴ期的蛔虫卵对小鼠进行感染,结果显示米尔贝肟能明显减弱虫卵的感染力。正常发育的感染性犬蛔虫卵与10-5、10-6、10-7 g·mL-1米尔贝肟作用后,灌服小鼠（约1000个虫卵）。感染3d、5d后对小鼠进行剖检,观察肺脏损害情况并进行病理观察。结果表明,感染3d和5d后,灌服低浓度药物(10-7 g·mL-1)且作用12h虫卵的小鼠,肺脏呈明显病变。而高浓度药物(10-6、10-5 g·mL-1)作用相同时间,肺脏病变轻微甚至无变化。
【Abstract】 Milbemycin oxime is one of a series of antibiotics produced through fermentation by Streptomyces hygroscopicus subspecies aureolacrimosus. The substance is a mixture of milbemycin A3 oxime and milbemycin A4 oxime at a 2:8 ratio. The objective of this study was to determine toxicological safety of milbemycin oxime by acute toxicity, Ames, micronucleus, sperm malformation and long-term toxicity tests. Kunming mice were chosen to study acute toxicity, micronucleus and sperm malformation, while chronic toxicity to Wistar rats. The results showed that the single use of milbemycin oxime did not effect the spontaneous movement, creep pole ability or express any synergy with continual in subthreshold dose. Milbemycin oxime had little toxicity to mice, and showed sex differences, the LD50 of milbemycin oxime to male and female mice were 1832 mg·kg-1 and 727 mg·kg-1, the 95% confidence intervals were 1637.57～2 022.08mg·kg-1 and 603.95～868.96mg·kg-1, respectively. In the Ames assay, four genotypic variants of the Salmonella strains （TA97, TA98, TA100 and TA102） carrying mutations in several genes were used. The average number of revertant colonies per plate treated with milbemycin oxime was less than double as compared to that of the negative control. In the micronucleus assay, the numbers of micro-nucleated PCE were counted, and the micronuclear rates were calculated. The obtained results indicated that milbemycin oxime could not induce the increase of micronuclear rates of PCE in bone marrow. In the sperm malformation test, the milbemycin oxime-treated groups showed no significant changes both in sperm abnormality and in a dose-dependent manner. In one month after administration, there were no significant changes in the appearance and movement of rats, except some rats show low spirit, but recovered when the administration stopped; and the hematology parameters were all in the normal scales; blood biochemical indicators and uronoscopy were normal before and after administration; in the high dose group there were some pathological changes in lung, liver, intestine and spleen, while others were normal.For the convenience of clinical application, the study on the pharmacodynamics of milbemycin oxime in vitro against Toxocara canis was carried out. In vitro action of milbemycin oxime against isolated adult Toxocara canis from dog intestine was observed after exposing 4, 8, 12, 24h at the concentration of 10-4, 10-5, 10-6, 10-7, 10-8 g·mL-1, the results showed that the concentrations of 10-4, 10-5, 10-6g·mL-1 were 100% efficiency to worms, while those of 10-7, 10-8 g·mL-1 were 66%, indicating that the concentration played important role on it. The above concentrations were used to study the effect of milbemycin oxime to Toxocara canis eggs, with the more dose increasing and prolonging of incubation time, the eggs need longer development time, and the number of stageⅤeggs were less; at the same time, the eggs that incubated 28d after acting with drug were used to infect mice, the results showed that milbemycin oxime could obviously decrease the infectivity of eggs. After being coacted with different concentrations (10-5、10-6、10-7 g·mL-1) of milbemycin oxime, infective Toxocara canis eggs （about 1,000 eggs） that were developed from fertilized eggs were orally infected on mice. Over 3 and 5 days of post-inoculation （p.i.）, mice were killed by cervical dislocation, respectively. The lungs were removed to record the macroscopic and histopathological injury. After 3 and 5 days of p.i., the lung of mice inoculated by infective Toxocara canis eggs with low concentration of milbecycin oxime (10-7 g·mL-1) for 12h showed obvious macroscopic lesions, while no lesions were found at high concentration of milbecycin oxime (10-5, 10-6 g·mL-1). It indicated that milbemycin oxime could make the infective Toxocara canis eggs lose the infection.
【Key words】 milbemycin oxime； toxicological safety evaluation； Toxocara canis； in vitro；