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目的通过小鼠急性毒性试验确定异常毒性剂量范围,采用3个批次依诺肝素钠原料药给予3组小鼠,观察药后48 h内小鼠死亡情况,验证异常毒性剂量设计的合理性,从而建立依诺肝素钠原料药异常毒性检查方法。方法 80只健康ICR小鼠,雌雄各半,给药开始时体重:雄性:20.3～22.0 g,雌性:18.7～21.2 g,根据体重分层随机分组,共分8组,每组10只。给药各组剂量按等比级数0.65(714,1099,1690,2600,4000,6154,9468 mg·kg-1)设计,给药容积为20 m.lkg-1,单次皮下注射依诺肝素钠原料药溶液,对照组同时给予等给药容积的氯化钠注射液。观察药后14天内小鼠死亡情况,采用SPSS13.0统计软件包将给药剂量经对数转换的机率单位回归法(Probit过程)求出ICR小鼠单次皮下注射依诺肝素钠原料药的LD50和LD1值及95%的可信区间。根据小鼠急性毒性实验结果,综合考虑确定以急性毒性LD1值的1/6～1/3范围内的100～118 mg·kg-1剂量作为检测依诺肝素钠原料药的异常毒性剂量,参照2010年版《中国药典》二部附录XIC《异常毒性检查法》设计依诺肝素钠原料药异常毒性检查方法:采用健康雌性ICR小鼠5只,给药时体重范围17～20 g,每只小鼠皮下注射依诺肝素钠原料药溶液0.5 ml(2 mg/只),观察小鼠药后48 h内死亡情况。按照上述方法,另取2个批次依诺肝素钠原料药给予2组雌性小鼠(做过本实验的小鼠不得重复使用),观察各组小鼠药后48 h内死亡情况,以验证上述异常毒性检查方法是否可行。结果小鼠单次皮下注射依诺肝素钠原料药的LD50(95%可信限)=2982.8 mg·kg-1(2167.9～4221.5 mg·kg-1),LD1(95%可信限)=388.2 mg·kg-1(90.0～731.7 mg·kg-1),LD50下限的1/8～1/4剂量范围为:270.99～541.98mg·kg-1,LD1下限的1/6～1/3剂量范围为15.0～30.0 mg·kg-1。综合考虑确定以急性毒性LD1值的1/6～1/3范围内的100～118 mg·kg-1剂量作为依诺肝素钠原料药的异常毒性检测剂量。结果 3个批次依诺肝素钠原料药100～118 mg·kg-1剂量范围内给药,给药后48 h均未引起受试动物死亡。结论以100～118 mg·kg-1(0.5 ml,2 mg/只)作为依诺肝素钠原料药的异常毒性检测剂量,检测3个批次依诺肝素钠原料药,全部小鼠48 h内未见死亡,结果提示上述依诺肝素钠原料药异常毒性检查方法是可行的。
OBJECTIVE To determine the range of abnormal toxic dose by acute toxicity test in mice, three batches of enoxaparin sodium were given to three groups of mice to observe the death of mice within 48 h after drug administration, to verify the rationality of designing abnormal toxicity dose, Thus establishing an assay for the abnormal toxicity of enoxaparin sodium APIs. Methods Totally 80 healthy ICR mice were divided into two groups according to their body weight. They were divided into 8 groups (n = 10 in each group), male and female, weighing 20.3-22.0 g at males and 18.7-21.2 g at females. The dosage of each group was designed according to 0.65 (714, 1099, 1690, 2600, 4000, 6154, and 9468 mg · kg-1) Heparin sodium drug solution, while the control group given equal volume of sodium chloride injection. Observe the death of mice within 14 days after drug use SPSS13.0 statistical package will be administered dose logarithmic conversion probability unit regression method (Probit process) ICR mouse single subcutaneous injection of enoxaparin sodium API LD50 and LD1 values and a 95% confidence interval. According to the results of acute toxicity test in mice, the dose of 100 ~ 118 mg · kg-1 in the range of 1/6 ~ 1/3 of the acute LD1 value was determined as the abnormal toxic dose for the detection of enoxaparin sodium. 2010 edition of the “Chinese Pharmacopoeia” two appendix XIC “abnormal toxicity test” design enoxaparin sodium raw materials abnormal toxicity check method: healthy female ICR mice 5, when administered weight range 17 ~ 20 g, each small Subcutaneous injection of enoxaparin sodium drug solution 0.5 ml (2 mg / only) to observe the mice within 48 h after death. According to the above method, another two batches of enoxaparin sodium were given to two groups of female mice (the mice used in the experiment should not be reused), and the death within 48 hours after the drug in each group was observed to verify The above abnormal toxicity check method is feasible. Results LD50 (95% confidence limit) = 2982.8 mg · kg-1 (2167.9-4221.5 mg · kg-1), LD1 (95% confidence interval) = 388.2 for single subcutaneous injection of enoxaparin sodium in mice The dose range of 1/8 to 1/4 of the lower limit of LD50 ranged from 270.99 to 541.98 mg · kg-1, and the lower limit of LD1 from 1/6 to 1/3 dose (mg · kg-1, 90.0-731.7 mg · kg-1) The range of 15.0 ~ 30.0 mg · kg-1. Considering the determination of the dose of 100-118 mg · kg-1 in the range of 1 / 6-1 / 3 of the LD1 value of acute toxicity as the abnormal toxicity test of enoxaparin sodium. Results Three batches of enoxaparin sodium drug were administered within the dose range of 100-118 mg · kg-1. No death of the test animals was induced at 48 h after administration. CONCLUSION: The three batches of enoxaparin sodium were tested at the dose of 100 ~ 118 mg · kg-1 (0.5 ml, 2 mg / kg) for the detection of abnormal toxicity of enoxaparin sodium. No deaths, the results suggest that the above method of detecting toxicity of enoxaparin sodium raw material is feasible.