Different in vitro and in vivo tools for elucidating the human metabolism of alpha‐cathinone‐derived drugs of abuse

Abstract

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.

Toxicometabolomics of the new psychoactive substances α-PBP and α-PEP studied in HepaRG cell incubates by means of untargeted metabolomics revealed unexpected amino acid adducts

“…α-PBP was described to be transformed to five metabolites, one after reduction of the cathinone carbonyl group resulting in a dihydro metabolite and several others after oxidation of the pyrrolidine ring, resulting in a mono and dihydroxylated metabolite, as well as an oxo and a ring-opened hydroxy metabolite. HepaRG did only form the dihydro metabolite, one of the main metabolites of α-PBP (Manier et al 2018). Oxidation of the pyrrolidine ring was not observed, although, for example, the metabolite found after lactam formation was also described as the second main metabolite.…”

“…The metabolism of α-PBP and α-PEP has been thoroughly investigated in previous publications using either pHLM (Manier et al 2018(Manier et al , 2019a, primary human hepatocytes (Swortwood et al 2016), or authentic human urine (Matsuta et al 2015). α-PBP was described to be transformed to five metabolites, one after reduction of the cathinone carbonyl group resulting in a dihydro metabolite and several others after oxidation of the pyrrolidine ring, resulting in a mono and dihydroxylated metabolite, as well as an oxo and a ring-opened hydroxy metabolite.…”

“…This is most probably due to fact that the HepaRG cell line was derived from a donor known to be a CYP2D6 poor metabolizer (Guillouzo et al 2007). Investigations of the CYP isoforms involved in the metabolism of α-PBP revealed that lactam formation of α-PBP was only catalyzed by CYP2D6 (Manier et al 2018). Additionally, pyrrolidine ring hydroxylation was catalyzed by CYP2B6, CYP2C19, and CYP2D6, of which CYP2D6 is expressed to a reduced extent due to the cells being derived from a poor metabolizing patient and CYP2B6 has a low contribution to the hepatic total net clearance of α-PBP.…”

“…Several in vitro models have been developed and used for investigating the metabolism of drugs of abuse (DOA) and new psychoactive substances (NPS), and also for xenobiotics in general (Manier et al 2018;Richter et al 2017a, b;Sinz 2012;Sinz and Kim 2006). They are based on subcellular fractions such as pooled human liver microsomes (pHLM), pooled human liver cytosol, or pooled human liver S9 fraction (pHLS9), as well as hepatic cell lines such as HepG2 and HepaRG.…”

“…Consumption and seizure of these stimulants has been reported in Europe as well as Japan (EMCDDA 2015;Kudo et al 2015;Odoardi et al 2016;Swortwood et al 2016). The general metabolism of the two NPS in, e.g., pHLM has so far been investigated (Manier et al 2018(Manier et al , 2019aMatsuta et al 2015;Swortwood et al 2016). The results shall be compared with those studies.…”