缬沙坦中亚硝胺药物成分相关杂质 (Q)SAR评价及Ames试验研究

Study on (Q)SAR Evaluation and Ames Test of Nitrosamine Drug Substance-related Impurities in Valsartan

关键词:  缬沙坦  亚硝胺药物成分相关杂质(NDSRI)  (定量)构效关系  致突变性  Ames试验  失活特征

Key words:  valsartan  nitrosamine drug substance-related impurities (NDSRI)  (quantitative) structure activity relationship  mutagenicity  Ames test  deactivating feature

摘要:

目的 结合基因毒性软件预测和细菌回复突变试验(Ames试验)评价缬沙坦中亚硝胺药物成分相关杂质(nitrosamine drug substance-related impurities, NDSRI)V6-NO的基因突变风险。方法 采用CASE Ultra软件对V6-NO进行初步预测和分类,采用标准平板掺入法Ames试验(±S9：10%大鼠肝S9混合液)和预培养法Mini-Ames试验(±S9：30%大鼠肝S9混合液,37℃预培养1 h)评价V6-NO的致突变性。结果 因V6-NO具有N-亚硝基类警示结构,基于统计学规则模型和专家知识规则模型的预测结果均为阳性,ICH M7分类为第3类,但基于统计学规则的预测结果显示该杂质结构中具有6个失活特征。Ames试验结果显示,在±S9条件下,在标准平板掺入法Ames试验中,V6-NO在62 μg/皿~5000 μg/皿剂量下回复突变菌落数均未超过溶媒对照组的2倍,在预培养法Mini-Ames试验中,V6-NO在62.5 μg/孔~1000 μg/孔剂量下回复突变菌落数也未超过溶媒对照组的2倍,Ames试验和Mini-Ames试验结果均为致突变阴性。结论 当NDSRI的软件预测结果为阳性,且与Ames试验结果不一致时,要综合分析和进一步的验证,主要从其结构中存在的活化和失活特征入手,并通过改进Ames试验条件进行验证,全面分析和了解NDSRI的致突变性风险,为药物研发提供有价值的参考信息。

Abstract:

ABSTRACT: OBJECTIVE To evaluate the risk of gene mutation of nitrosamine drug substance-related impurities (NDSRI) V6-NO in valsartan by combining genotoxicity software prediction and bacterial reverse mutation test (Ames test). METHODS CASE Ultra software was used for the preliminary prediction and classification of V6-NO, and the mutagenicity of V6-NO was evaluated by the Ames test (±S9: 10% rat liver S9 mixture) using the standard plate incorporation method, and by the Mini-Ames test using the pre-incubation method (±S9: 30% rat liver S9 mixture, pre-incubated at 37℃ for 1h). RESULTS Because V6-NO had an N-nitroso alert structure, the prediction results of statistical-based model and expert rule-based model are positive, and the ICH M7 was classified as category 3, but the prediction results of statistical-based model showed that the impurity structure had 6 deactivating features. The results of Ames test showed that in the presence and absence of S9 conditions, the number of revertant colonies of V6-NO at the dose of 62 μg/plate ~ 5000 μg/plate did not exceed 2 times that of the vehicle control group in the Ames test of standard plate incorporation method, and the number of revertant colonies of V6-NO at the dose of 62.5 μg/well ~ 1000 μg/well did not exceed 2 times that of the vehicle control group in the pre-incubation method Mini-Ames test. Both the Ames test and the Mini-Ames test are non-mutagenic. CONCLUSION When the prediction result of NDSRI software is positive and inconsistent with the Ames test results, it is necessary to comprehensively analyze and further verify, mainly starting from the activating and deactivating feature existing in its structure, and verify it by improving the Ames test conditions, so as to comprehensively analyze and understand the mutagenicity risk of NDSRI, and provide valuable reference information for drug development.