Studies on the tissue distribution of the new synthetic opioid U-47700 and its main metabolite N-desmethyl-U-47700 revealed about sixfold higher metabolite concentrations in pig brain as compared with the parent compound. To better assess the toxic potential of this drug, the aim of this study was to assess the in vitro μ-opioid receptor (MOR) activation potential of the main metabolites of U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, using a live cell-based reporter assay based on NanoLuc Binary Technology®. Cells stably expressing human MOR and β-arrestin 2 (βarr2), each fused via a flexible linker to two complementary inactive subunits of the nanoluciferase, were seeded on poly-d-lysine-coated 96-well plates and treated with N-desmethyl-U-47700, N,N-bisdesmethyl-U-47700, U-47700, or hydromorphone as reference standard. MOR activation results in functional complementation of the nanoluciferase, which can be assessed via luminescence monitoring. The potency of the metabolites is lower than that of U-47700 (EC₅₀ of 186 nM for U-47700, 3770 nM for N-desmethyl-U-47700, and >5 μM for N,N-bisdesmethyl-U-47700). The maximal efficacy (E_max) observed (relative to hydromorphone, set arbitrarily at 100%) decreased from 183% to 127% and 39.2% for U-47700, N-desmethyl-U-47700, and N,N-bisdesmethyl-U-47700, respectively. Thus, the loss of one or two methyl groups reduced the MOR activation potential, which was more pronounced if both methyl groups were removed. It is thus anticipated that the impact on MOR exerted by the higher metabolite concentration in brain has only little—if any relevance for the strong toxic effects of U-47700.

中文翻译：

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U-47700 的 N-去甲基化代谢物是否比其母体化合物更活跃？N-去甲基-U-47700和N,N-双去甲基-U-47700的体外μ-阿片受体活化

对新型合成阿片类药物 U-47700 及其主要代谢物N -desmethyl-U-47700 的组织分布研究表明，与母体化合物相比，猪脑中的代谢物浓度高出约六倍。为了更好地评估该药物的毒性潜力，本研究的目的是评估 U-47700、 N-去甲基-U-47700 和N的主要代谢物的体外 μ-阿片受体 (MOR) 活化潜力，N -bisdesmethyl-U-47700，使用基于 NanoLuc Binary Technology® 的基于活细胞的报告基因分析。将稳定表达人 MOR 和 β-arrestin 2 (βarr2) 的细胞接种到 poly -d-赖氨酸包被的 96 孔板，用N-去甲基-U-47700、N、N-双去甲基-U-47700、U-47700 或氢吗啡酮作为参考标准处理。MOR 激活导致纳米荧光素酶的功能互补，这可以通过发光监测进行评估。代谢物的效力低于 U-47700（U-47700 的 EC ₅₀为 186 nM，N-desmethyl-U-47700 的 EC 50 为 3770 nM，N , N - bisdesmethyl - U-47700 的 EC 50 >5 μM ） . 对于 U- 47700 、N - _{desmethyl -U-} 47700和N、N-双去甲基-U-47700。因此，一个或两个甲基基团的损失降低了 MOR 的活化电位，如果两个甲基基团都被去除，这一点会更加明显。因此，预计大脑中较高的代谢物浓度对 MOR 的影响与 U-47700 的强毒性作用几乎没有相关性。