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 , Albertstrasse 9, 79104 Freiburg, Germany.
Anal Chem. 2017 Sep 5;89(17):9527-9536. doi: 10.1021/acs.analchem.7b02552. Epub 2017 Aug 18.
Synthetic cannabinoids (SCs) continue to be the largest group of new psychoactive substances (NPS) monitored by the European Monitoring Center of Drugs and Drugs of Abuse (EMCDDA). The identification and subsequent prohibition of single SCs has driven clandestine chemists to produce analogues of increasing structural diversity, intended to evade legislation. That structural diversity, combined with the mostly unknown metabolic profiles of these new SCs, poses a big challenge for the conventional targeted analytical assays, as it is difficult to screen for "unknown" compounds. Therefore, an alternative screening method, not directly based on the structure but on the activity of the SC, may offer a solution for this problem. We generated stable CB1 and CB2 receptor activation assays based on functional complementation of a split NanoLuc luciferase and used these to test an expanded set of recent SCs (UR-144, XLR-11, and their thermal degradation products; AB-CHMINACA and ADB-CHMINACA) and their major phase I metabolites. By doing so, we demonstrate that several major metabolites of these SCs retain their activity at the cannabinoid receptors. These active metabolites may prolong the parent compound's psychotropic and physiological effects and may contribute to the toxicity profile. Utility of the generated stable cell systems as a first-line screening tool for SCs in urine was also demonstrated using a relatively large set of authentic urine samples. Our data indicate that the stable CB reporter assays detect CB receptor activation by extracts of urine in which SCs (or their metabolites) are present at low- or subnanomolar (ng/mL) level. Hence, the developed assays do not only allow activity profiling of SCs and their metabolites, it may also serve as a screening tool, complementing targeted and untargeted analytical assays and preceding analytical (mass spectrometry based) confirmation.
合成大麻素（SCs）仍然是欧洲毒品和药物滥用监测中心（EMCDDA）监测的最大类新精神活性物质（NPS）。单一 SCs 的鉴定和随后的禁止促使秘密化学家生产出结构多样性不断增加的类似物，旨在逃避立法。这种结构多样性，加上这些新 SCs 的代谢概况大多未知，给常规靶向分析检测方法带来了巨大挑战，因为很难筛选“未知”化合物。因此，一种替代的筛选方法，不是直接基于结构，而是基于 SC 的活性，可能为这个问题提供解决方案。我们生成了基于 NanoLuc 荧光酶分裂功能互补的稳定 CB1 和 CB2 受体激活测定法，并使用这些测定法测试了一组新的 SC（UR-144、XLR-11 及其热降解产物；AB-CHMINACA 和 ADB-CHMINACA）及其主要的 I 相代谢物。通过这样做，我们证明了这些 SC 的几种主要代谢物在大麻素受体上保持其活性。这些活性代谢物可能延长母体化合物的精神和生理作用，并可能有助于毒性特征。使用相对较大的一组真实尿液样本，还证明了生成的稳定细胞系统作为尿液中 SC 的一线筛选工具的实用性。我们的数据表明，稳定的 CB 报告基因测定法可检测到尿液提取物中存在 SC（或其代谢物）时的 CB 受体激活，其浓度低至纳摩尔（ng/mL）或亚纳摩尔（ng/mL）水平。因此，开发的测定法不仅允许对 SC 及其代谢物进行活性分析，还可以作为筛选工具，补充靶向和非靶向分析检测方法，并在分析（基于质谱）确认之前进行。
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