Laboratory of Toxicology, Department of Bioanalysis, Faculty of Pharmaceutical Sciences, Ghent University , Ottergemsesteenweg 460, 9000 Ghent, Belgium.
Institute of Forensic Medicine, Forensic Toxicology, Medical Center - University of Freiburg, Faculty of Medicine, University of Freiburg , Albertstr. 9, 79104 Freiburg, Germany.
Anal Chem. 2016 Dec 6;88(23):11476-11485. doi: 10.1021/acs.analchem.6b02600. Epub 2016 Nov 7.
Synthetic cannabinoids (SCs) are the largest group of compounds currently monitored in Europe by the EU Early Warning System on new psychoactive substances. Emerging recreational use of these products has led to multiple cases of adverse health effects and even death. In contrast to marijuana, where Δ-tetrahydrocannabinol (ΔTHC) is metabolized to only one major active metabolite, it has been reported that several major phase I metabolites of SCs remain biologically active, exerting cannabinoid (CB) receptor affinity, potency, and efficacy greater than those of ΔTHC. It is therefore reasonable that more SCs can also be biotransformed into molecules with various levels of CB activity. Here, we developed and applied a new G-protein coupled receptor (GPCR) activation assay based on NanoLuc binary technology (Promega). More specifically, by demonstrating CB1 and CB2 receptor activation by JWH-018 and a selection of its metabolites, we are the first to show the suitability of the newly developed bioassay for monitoring GPCR-mediated activity. We also successfully applied this reporter system to evaluate the in vitro activity of JWH-122, JWH-210, and PB-22, their 5-fluoro analogues (MAM-2201, EAM-2201, and 5F-PB-22, respectively), and their main phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at cannabinoid receptors. All of these active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to its toxicity profile. We also demonstrate a proof of concept of the applicability of the newly developed bioassay for screening urine for CB receptor activity exerted by SCs.
合成大麻素 (SCs) 是欧盟新精神活性物质早期预警系统目前在欧洲监测的最大化合物群体。这些产品的新兴娱乐用途导致了多种不良健康影响，甚至死亡。与大麻不同，Δ-四氢大麻酚 (ΔTHC) 仅代谢为一种主要的活性代谢物，据报道，SCs 的几个主要的 I 相代谢物仍然具有生物活性，发挥大麻素 (CB) 受体亲和力、效力和功效大于 ΔTHC。因此，更多的 SCs 也可以被生物转化为具有不同 CB 活性水平的分子是合理的。在这里，我们开发并应用了一种基于 NanoLuc 双技术（Promega）的新型 G 蛋白偶联受体 (GPCR) 激活测定法。更具体地说，通过证明 JWH-018 及其一些代谢物对 CB1 和 CB2 受体的激活，我们首次表明新开发的生物测定法适合监测 GPCR 介导的活性。我们还成功地将该报告系统应用于评估 JWH-122、JWH-210 和 PB-22 及其 5-氟类似物 (MAM-2201、EAM-2201 和 5F-PB-22) 及其主要的 I 相代谢物的体外活性。通过这样做，我们证明了这些 SCs 的几种主要代谢物在大麻素受体上保持其活性。所有这些活性代谢物都可能延长母体化合物的精神和生理作用，并可能有助于其毒性特征。我们还证明了新开发的生物测定法用于筛选尿液中 SC 对 CB 受体活性的适用性的概念验证。
