Institute of Legal Medicine, Saarland University, Homburg, Germany.
Laboratory of Toxicology, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium.
Drug Test Anal. 2022 Apr;14(4):713-717. doi: 10.1002/dta.3182. Epub 2021 Oct 25.
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 (EC 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 ) 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.
对新型合成阿片类药物 U-47700 及其主要代谢物 N-去甲基-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约六倍。为了更好地评估这种药物的毒性潜力，本研究旨在使用基于 NanoLuc Binary Technology®的活细胞报告测定法评估 U-47700 的主要代谢物 N-去甲基-U-47700、N,N-双去甲基-U-47700 的体外 μ 阿片受体 (MOR) 激活潜力。通过柔性接头分别与两个互补的纳米荧光素酶无活性亚基融合，稳定表达人 MOR 和β-arrestin 2 (βarr2) 的细胞被播种在聚-d-赖氨酸包被的 96 孔板上，并分别用 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，可通过发光监测进行评估。代谢物的效力低于 U-47700（U-47700 的 EC 为 186 nM，N-去甲基-U-47700 的 EC 为 3770 nM，N,N-双去甲基-U-47700 的 EC 为 >5 μM）。观察到的最大功效 (E)（相对于氢吗啡酮，任意设定为 100%）从 U-47700 的 183%下降到 127%和 39.2%，对于 N-去甲基-U-47700 和 N,N-双去甲基-U-47700。因此，一个或两个甲基的丢失降低了 MOR 激活潜力，如果两个甲基都被去除，则这种降低更为明显。因此，可以预期，在大脑中更高的代谢物浓度对 MOR 产生的影响对于 U-47700 的强烈毒性作用只有很小的影响——如果有的话。
