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指导原则编号:【Z】GPT4-1中药、天然药物刺激性和溶血性研究的技术指导原则二○○五年三月1目录一、概述····················································································································································1二、基本内容·········································································································································2（一）基本原则····························································································································2（二）刺激性试验·······················································································································3（三）光毒性试验·······················································································································4（四）溶血性试验······················································································································5（五）结果分析及评价············································································································6（六）常见问题及处理············································································································7（七）不同剂型的中药、天然药物试验项目的选择············································9三、参考文献·······································································································································10四、附录··················································································································································11（一）刺激性试验方法·········································································································111、血管刺激性试验···············································································································112、肌肉刺激性试验···············································································································123、皮肤刺激性试验···············································································································124、粘膜刺激性试验···············································································································15（二）光毒性试验方法·········································································································20（三）溶血性试验方法·········································································································221、常规体外试管法（肉眼观察法）·········································································222、改进的体外溶血性试验法（分光光度法）·····················································243、体外红细胞计数法··········································································································254、体内溶血试验法（红细胞计数法）····································································25五、著者·················································································································································252中药、天然药物刺激性和溶血性研究的技术指导原则一、概述刺激性是指中药、天然药物制剂（包括活性成分和赋形剂）经皮肤、粘膜、腔道、肌肉、血管等非口服途径给药，经局部吸收或注射后对给药部位以及全身产生的毒性作用，包括血管、肌肉、粘膜等刺激性。溶血性是指中药、天然药物制剂（包括活性成分和赋形剂）产生的溶血或红细胞凝聚等反应。它是临床前安全性评价的组成部分。中药、天然药物制剂，包括活性成分或组分、配伍后产生的新成分、体内代谢物、制备过程中的杂质、辅料及制剂的理化性质（如pH、渗透压等）等均是可能导致给药部位毒性或溶血性反应产生的因素。因此，为了指导临床合理用药，提示临床应用时可能出现的毒性反应和程度、安全剂量或浓度、安全范围、临床研究监测指标、解毒或解救措施等，应进行中药、天然药物制剂局部应用的毒性试验。本指导原则旨在阐明进行临床前非口服途径给药的毒性研究的目的、意义和重要性，阐明进行中药新药安全性评价的基本原则、相关技术要求及该领域存在的问题，阐明应利用具体问题具体分析的方法，针对药物的自身特点，结合药学、药效学、其他毒理学及临床应用信息，通过合理、客观的试验设计和研究，探索、揭示或发现中药、天然药物非口服途径给药的毒性反应，预测临床应用中可能存在的潜在毒性，指导临床用药。本指导原则适用于非口服给药途径的中药、天然药物，包括中药注射剂及局部用药制剂。主要内容包括研究和评价的基本原则、刺激性试验、1光毒性试验和溶血性试验的相关内容。二、基本内容（一）基本原则根据《中华人民共和国药品管理法》，药物的免疫毒性试验必须执行“药物非临床研究质量管理规范”。实验设计应符合随机、对照、重复的原则。应在遵循安全性评价普遍规律的基础上，运用具体问题具体分析的方法，结合受试物的自身特点，充分考虑和结合药学、药效学、其他毒理学（长毒、急毒）及拟临床应用情况等信息，体现整体性、综合性的原则，在阐明其研究方法或手段科学、合理的前提下进行规范性试验，对试验结果应进行全面分析，对药物的安全性做出综合评价。（二）刺激性试验刺激性是指非口服给药制剂给药后对给药部位产生的可逆性炎症反应，若给药部位产生了不可逆性的组织损伤则称为腐蚀性。刺激性试验是考察动物的血管、肌肉、皮肤、粘膜等部位接触受试物后是否引起红肿、充血、渗出、变性或坏死等局部反应。1．受试物受试物应同临床应用制剂相一致，即采用中试或中试以上规模的、制备工艺稳定、符合临床试用质量标准规定的样品。否则，应有充分的理由。同时，应注明受试物的名称、来源、批号、含量（或规格）、保存条件及配制方法等。试验中所用辅料、溶剂或赋形剂等应标明批号、规格和生产厂家，并符合试验要求。22．对照可用溶媒和/或赋形剂和/或基质作为阴性对照。必要时可设阳性对照。因局部刺激性与受试物制剂密切相关，相同活性成分不同制剂的受试物局部刺激性可能明显不同，若出现毒性反应，应与已上市品种进行比较性研究，以保证药物临床应用的安全性。3．动物应选择与人类皮肤、粘膜反应比较相近的动物，如家兔、豚鼠和小型猪等。动物种属依据要观察的指标和模型的合理性进行选择。通常一个试验类型选择一种动物进行评价。动物应符合国家有关规定的等级要求，来源、遗传背景清楚，并具有试验动物质量合格证。刺激性试验结果从动物外推到人的可靠性有限。若用二种动物进行试验得到类似结果，则会增加从动物外推到人的可靠性。4．剂量可选择几种不同浓度的制剂进行试验，其中包括拟用于临床研究的浓度，为临床研究的安全性提供选择依据。可以通过改变给药频次进行剂量的调整。对于局部用膏状制剂，在给药面积不变的情况下，不应通过增加厚度来满足增加给药量的目的。5．给药途径一般应与临床用药途径一致，否则应加以说明。6．给药部位一般应选择与临床应用相似的部位。并注意对可能接触到受试物的周围组织的影响。设计剂量和给药频次时，应考虑受试动物给药部位的解剖3和生理特点，保证受试物在给药部位的有效暴露时间。7．给药频率和时间给药频率和时间应依据拟定临床用药方案来决定，多次给药一般不超过4周。8．观察指标8.1肉眼观察应详细描述动物局部反应，包括红斑、水肿、充血程度及范围，并以计分的方式表示。同时观察动物的一般状态、行为、体征等。8.2组织病理学检查组织病理学检查应详细描述给药部位的病理变化，并进行半定量分析、判断。提供相应的组织病理学照片。8.3可逆性观察若出现刺激性反应，应进行停药后的恢复期观察，以明确毒性反应的恢复情况。9．试验方法具体试验方法可参照附录（一）。附录中所列方法仅为推荐的方法，若有新的、更灵敏或适合的方法，可结合不同试验进行选择。（三）光毒性（光刺激性）试验光敏反应是用药后皮肤对光线产生的不良反应，包括光毒性反应和光过敏反应两类，均由受试物所含的感光物质引起，但两者机制不同，实验方法、临床表现及意义亦不同。光毒性是由光诱导的非免疫性的皮肤对光的反应，是指药物吸收的紫外光能量在皮肤中释放导致皮肤损伤的作用，可通过直接作用或通过血循环间接作用，即皮肤或全身接触或应用药物后，继而暴露于紫外线照射下所引起的一种皮肤毒性反应。光毒性反应是光敏反应中最常见的一种反应。4具有剂量依赖性，其临床表现与晒伤相似，表现为红斑、水肿、皮肤瘙痒和色素沉着。严重者可产生局部坏死、溃烂