注射用灯盏花素豚鼠全身主动过敏性试验评价方法的比较

Comparison of methods for active systemic anaphylactic reaction of guinea pigs induced by breviscapine injection

目的通过《中国药典》2010年版一部附G中药注射液安全性检查法应用指导原则中"过敏反应检查法"(以下简称为"药典法")和《中药、天然药物免疫毒性(过敏性、光过敏反应)研究的技术指导原则》2005版中载明的方法(以下简称为"指导原则法")分别评价并比较灯盏花素对豚鼠全身过敏反应,为药物临床前安全评价提供参考。方法将豚鼠分为阴性对照组、阳性对照组、注射用灯盏花素5和50 mg·kg-1组,分别按照"药典法"和"指导原则法"观察豚鼠对注射用灯盏花素全身主动过敏反应。致敏阶段,豚鼠ip给予注射用灯盏花素溶液每只0.5 m L,剂量分别为5和50 mg·kg-1,隔日1次,共3次。"药典法"在首次致敏后第14和21天ip给予注射用灯盏花素每只1 m L激发;"指导原则法"在末次注射后第12天激发1次,观察豚鼠的过敏症状和过敏时间。结果 "药典法":第1次激发(首次致敏后第14天)注射用灯盏花素5 mg·kg-1组在不同时间点出现喷嚏1声和(或)搔鼻1次的现象,注射用灯盏花素50 mg·kg-1组不同时间点出现喷嚏1声或2声和(或)搔鼻1次等现象,判定过敏反应阴性。第2次激发(首次致敏后第21天)灯盏花素5 mg·kg-1组给药后分别在不同时间点出现发抖、喷嚏1声或2声和(或)搔鼻1次的现象,判定过敏反应阴性;注射用灯盏花素剂量50 mg·kg-1组4只豚鼠(4/4)给药后在不同时间点分别出现喷嚏1声和3声、咳嗽1声和2声、搔鼻1次以及排尿等现象;1只豚鼠(1/4)出现连续喷嚏3声和发抖,判定过敏反应阳性。"指导原则法":末次注射后第12天激发,注射用灯盏花素5 mg·kg-1组激发后10 min内均出现了不同程度的过敏反应症状,其中3只豚鼠出现搔鼻症状,为弱阳性;3只豚鼠出现喷嚏和(或)排尿症状,为阳性。注射用灯盏花素50 mg·kg-1组过敏症状包括搔鼻、喷嚏、咳嗽和(或)排尿症状,为阳性。结论在进行临床前安全性评价豚鼠全身过敏试验过程中,从动物数量、评定标准、观察时间和剂量设置等多方面要采取2种方法的优势,利用"药典法"对供试品进行初筛,一旦有疑似反应,最好采用"指导原则法"进行更加细致的观察。

OBJECTIVE To compare the difference of methods for active systemic anaphylactic reaction induced by breviscapine injection between ″Pharmacopoeia″ 2010 edition, an Attached ⅫG in Traditional Chinese Medicine lnjection Safety Test Application Guidelines ″ Anaphylactic Reaction Test″（thereafter referred to as the method of ″Pharmacopoeia″） and the Traditional Chinese Medicine, Natural Medicine lmmune Toxicity （Anaphylaxis, Anaphylactic Reaction of Light） Technology Guiding Principles in the 2005 Version （thereafter referred to as the method of ″Guiding Principle″） and provide reference for non-clinical safety evaluation of drugs. METHODS According to the methods of ″Pharmacopoeia″and ″Guiding Principle″, respectively, the effect of breviscapine injection on active systemic anaphylactic reaction of guinea pigs was investigated. The guinea pigs were divided into four groups, negative control group, positive control group, breviscapine injection 5 and 50 mg.kg-1 groups. ln the sensitization phase, the guinea pigs were ip administrered with breviscapine injection 0.5 mL each every other day for 3 times. The dose was 5 and 50 mg.kg-1 , respectively. For the method of ″Pharmacopoeia″, on the 14th and 21st days after the first sensitization, the guinea pigs were iv administrered with breviscapine injec-tion 1 mL. For the method of ″Guiding Principle″, the guinea pigs were provocated on the 12th day after the first sensitization. Each group was studied by observing the symptom of anaphylactic reaction and immune time. RESULTS For the method of ″Pharmacopoeia″, on the 14th day after the first sensitization, there was 1 guinea pig with sneezing and （or） the nose-scratching at different time in the 5 mg.kg-1 group. ln the 50 mg.kg-1 group, there was one or two cases of sneezing and （or） 1 case of nose-scratc-hing. The 5 and 50 mg.kg-1 dose groups conformed to the regulations. On the 21st day after the first sensitization, trembling occurred in the 5 mg.kg-1 group, with 1 or 2 guinea pigs sneezing and （ or） scratching the nose. There were 4 guinea pigs （4/ 4） with sneezing 1 and 3 times, cough once or twice, 1 scratching nose and urination at different time, and 1 guinea pigs （1/ 4） appeared 3 times consecutive sneezing and shivering in 50 mg.kg-1 group. The 5 mg.kg-1 group conformed to the regulations, while the 50 mg.kg-1 group did not. For the method of ″Guiding Principle″, the 5 mg.kg-1 group was weak positive or positive, with different degrees of symptoms of an anaphylactic reaction, including 3 guinea pigs scratched nasal symptoms. And the 50 mg.kg-1 group of anaphylactic symptoms including scratc-hing nose, sneezing, coughing and （or） urination, showed positive. CONCLUSION During the active systemic anaphylactic reaction of drugs non-clinical safety evaluation of drugs the advantage of either method should be brought into play. The method of ″ Pharmacopoeia″ may be used for preliminary screening of test samples. ln case pf suspected reactions, the method of ″Guiding Principle″ should be used for more detailed observations.