Simon Stephanie, Schlingemann Jörg, Johnson George, Brenneis Christian, Guessregen Brunhilde, Kostal Jakub, Dieckhoff Jessica
Merck Healthcare KGaA, Darmstadt, Germany.
Swansea University Medical School, Swansea, SA8PP, UK.
Arch Toxicol. 2025 Jun 11. doi: 10.1007/s00204-025-04103-2.
N-nitroso-bisoprolol (NBP) is a nitrosamine drug substance-related impurity (NDSRI) of bisoprolol, which is used to treat cardiac diseases since decades. To investigate the mutagenic potential of NBP, in vitro methods such as Enhanced Ames Test (EAT) and a mammalian cell gene mutation (HPRT) assay were used. To assess the in vivo mutagenicity, a 28-day repeat-dose study was conducted in wild-type NMRI mice, and liver and bone marrow samples were subjected to error-corrected next-generation sequencing (i.e., duplex sequencing) followed by benchmark dose analysis (BMD). NBP did not show mutagenic effects in Ames tests using 10 % and 30 % induced rat or 30 % uninduced hamster S9. However, relevant increases in mutation frequencies were observed in an EAT in the presence of 30 % induced hamster S9 in strains TA100 and TA1535, confirming that the most stringent conditions of the EAT are appropriate to detect the mutagenic activity of weak mutagens, such as NBP. In the HPRT assay conducted in V79 cells, nitroso-diethylamine (NDEA) relevantly induced the mutation frequency, but not NBP. The highly sensitive error-corrected Next-Generation Sequencing (ecNGS) method to detect mutations across the genome represents an appropriate in vivo mutagenicity investigation equally suitable as a TGR assay to assess the mutagenic potential of nitrosamines. A weak induction of mutation frequencies was detected by ecNGS in the liver and the bone marrow of mice. Using BMD analysis, new safe limits were calculated for NBP, which are higher than the published AI of 1.5 µg/person/day. Using the approach to calculate Permissible Daily Exposure (PDE) limits according to ICH Q3C, a lifetime PDE of 400 µg/person/day was derived. Based on the ICH M7 framework for derivation of Acceptable Intake (AI) limits, an AI of 64 µg/person/day was established. Consistent with regulatory emphasis on mechanistic interpretation, in vivo modeling was further supported by in silico calculations. Specifically, the validated Computer-Aided Discovery and RE-design (CADRE) tool was used to predict the potency of NBP and further differentiate its metabolic activity from the anchor nitrosamine NDEA via quantum mechanics (QM) calculations and CYP-binding predictions. Outcomes of this analysis were consistent with in vivo studies, while offering a deeper understanding of the fundamental biochemistry using a physics-led method. The integrated in vivo-in silico investigation provides a data-based determination of safe limits, suggesting that the AI based on structural considerations solely might be over-conservative and should not be capped at the TTC.
N-亚硝基比索洛尔（NBP）是比索洛尔的一种亚硝胺类药物相关杂质（NDSRI），比索洛尔已用于治疗心脏疾病数十年。为研究NBP的致突变潜力，采用了体外方法，如增强型艾姆斯试验（EAT）和哺乳动物细胞基因突变（HPRT）试验。为评估体内致突变性，在野生型NMRI小鼠中进行了为期28天的重复给药研究，并对肝脏和骨髓样本进行了纠错下一代测序（即双链测序），随后进行基准剂量分析（BMD）。在使用10%和30%诱导大鼠或30%未诱导仓鼠S9的艾姆斯试验中，NBP未显示出致突变作用。然而，在菌株TA100和TA1535中，在30%诱导仓鼠S9存在的情况下，EAT中观察到突变频率有相关增加，证实EAT的最严格条件适合检测弱诱变剂（如NBP）的诱变活性。在V79细胞中进行的HPRT试验中，亚硝基二乙胺（NDEA）显著诱导了突变频率，但NBP没有。用于检测全基因组突变的高灵敏度纠错下一代测序（ecNGS）方法是一种合适的体内致突变性研究方法，同样适用于作为评估亚硝胺致突变潜力的TGR试验。通过ecNGS在小鼠肝脏和骨髓中检测到了微弱的突变频率诱导。使用BMD分析，计算出了NBP的新安全限值，该限值高于已公布的每人每天1.5μg的AI值。根据ICH Q3C计算每日允许暴露量（PDE）限值的方法，得出每人每天400μg的终生PDE值。基于ICH M7框架推导可接受摄入量（AI）限值，确定了每人每天64μg的AI值。与监管机构对机理解释的重视一致，体内建模得到了计算机模拟计算的进一步支持。具体而言，使用经过验证的计算机辅助发现和重新设计（CADRE）工具，通过量子力学（QM）计算和CYP结合预测来预测NBP的效力，并进一步将其代谢活性与锚定亚硝胺NDEA区分开来。该分析结果与体内研究一致，同时使用基于物理学的方法对基本生物化学有了更深入的理解。体内-计算机模拟综合研究提供了基于数据的安全限值测定，表明仅基于结构考虑的AI值可能过于保守，不应以TTC为上限。
