Manier Sascha K, Richter Lilian H J, Schäper Jan, Maurer Hans H, Meyer Markus R
Department of Experimental and Clinical Toxicology, Institute of Experimental and Clinical Pharmacology and Toxicology, Saarland University, Center for Molecular Signaling (PZMS), Homburg, Germany.
State Bureau of Criminal Investigation Bavaria, München, Germany.
Drug Test Anal. 2018 Jan 4. doi: 10.1002/dta.2355.
In vitro and in vivo experiments are widely used for studying the metabolism of new psychoactive substances (NPS). The availability of such data is required for toxicological risk assessments and development of urine screening approaches. This study investigated the in vitro metabolism of the 5 pyrrolidinophenone-derived NPS alpha-pyrrolidinobutyrophenone (alpha-PBP), alpha-pyrrolidinopentiothiophenone (alpha-PVT), alpha-pyrrolidinohexanophenone (alpha-PHP), alpha-pyrrolidinoenanthophenone (alpha-PEP, PV8), and alpha-pyrrolidinooctanophenone (alpha-POP, PV9). First, they were incubated with pooled human liver microsomes (pHLM) or pooled human liver S9 fraction (pS9) for identification of the main phase I and II metabolites. All substances formed hydroxy metabolites and lactams. Longer alkyl chains resulted in keto group and carboxylic acid formation. Comparing these results with published data obtained using pHLM, primary human hepatocytes (PHH), and authentic human urine samples, PHH provided the most extensive metabolism. Second, enzyme kinetic studies showed that the initial metabolic steps were formed by cytochrome P450 isoforms (CYP) CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 resulting in pyrrolidine, thiophene or alkyl hydroxy metabolites depending on the length of the alkyl chain. The kinetic parameters indicated an increasing affinity of the CYP enzymes with increase of the length of the alkyl chain. These parameters were then used to calculate the contribution of a single CYP enzyme to the in vivo hepatic clearance. CYP2C19 and CYP2D6 were mainly involved in the case of alpha-PBP and CYP1A2, CYP2C9 and CYP2C19 in the case of alpha-PVT, alpha-PHP, alpha-PEP, and alpha-POP.
体外和体内实验被广泛用于研究新型精神活性物质（NPS）的代谢。毒理学风险评估和尿液筛查方法的开发需要此类数据。本研究调查了5种吡咯烷酮衍生的NPSα-吡咯烷基丁酰苯（α-PBP）、α-吡咯烷基戊硫酮（α-PVT）、α-吡咯烷基己酰苯（α-PHP）、α-吡咯烷基庚酰苯（α-PEP，PV8）和α-吡咯烷基辛酰苯（α-POP，PV9）的体外代谢。首先，将它们与混合人肝微粒体（pHLM）或混合人肝S9组分（pS9）孵育，以鉴定主要的I相和II相代谢物。所有物质均形成羟基代谢物和内酰胺。较长的烷基链导致酮基和羧酸的形成。将这些结果与使用pHLM、原代人肝细胞（PHH）和真实人尿液样本获得的已发表数据进行比较，PHH提供了最广泛的代谢。其次，酶动力学研究表明，初始代谢步骤由细胞色素P450同工酶（CYP）CYP1A2、CYP2B6、CYP2C9、CYP2C19、CYP2D6和CYP3A4形成，根据烷基链的长度产生吡咯烷、噻吩或烷基羟基代谢物。动力学参数表明，CYP酶的亲和力随着烷基链长度的增加而增加。然后使用这些参数计算单一CYP酶对体内肝脏清除率的贡献。对于α-PBP，主要涉及CYP2C19和CYP2D6；对于α-PVT、α-PHP、α-PEP和α-POP，主要涉及CYP1A2、CYP2C9和CYP2C19。
