In vitro metabolism and metabolite identification of eutylone using rat liver microsomes

Abstract

The determination of toxicokinetic parameters allows the characterization of the toxicological profile of a wide range of substances. Several models have been employed to in vitro kinetic evaluations, in which liver microsomes stands out due to its simple handling and obtaining. Eutylone is a New Psychoactive Substance (NPS) classified as a synthetic cathinone with stimulant effects on central nervous system. This substance was initially synthesized for pharmaceutical applications but ultimately became subject to recreational use, with constant seizures worldwide. Herein, the in vitro metabolism and the production of eutylone phase I and phase II metabolites was investigated using rat liver microsomes (RLM). Eutylone presented a low metabolic stability showing an in vitro elimination half-life (t_1/2) of 2.27 min. The unbound fractions of eutylone in microsomal (f_u-mic) and plasma (f_u-p) proteins were 0.93 and 0.15, respectively. A sigmoidal profile defined by Hill equation were observed, allowing kinetic parameters calculations. The Hill coefficient (H) was 1.21, in vitro maximum velocity (V_max) was 19.40 μmol/mg/min, substrate concentration at half V_max (S₅₀) was 4.78 μM, intrinsic maximum clearance (CL_{max, in vitro}) was 3.36 mL/min/mg, in vivo intrinsic clear-ance (CL_{int, in vivo}) was 8.20 mL/min/kg, hepatic clearance (CL_H) was 1.29 mL/min/kg, and hepatic extraction rate (E_H) was 0.02. Eight eutylone metabolites were identified, four produced by phase I reactions and four by phase I followed by phase II reactions. Demethylenation and O-glucuronidation were pivotal in eutylone’s metabolism. These findings provide valuable information about the metabolism of eutylone, allowing practical implications for evaluating its safety and toxicity.