Research on the metabolites and key metabolic enzymes of allocryptopine in chicken liver microsomes via stable isotope tracing technology

Allocryptopine (ALL), a principal active component of the novel veterinary medicine Bopu Powder®, has gained widespread application in the poultry farming sector for the effective management of Escherichia coli (E. coli) diarrhea. In order to explore the metabolites and the pivotal enzymes associated with ALL, this study was conducted employing an in vitro chicken liver microsomal incubation. The metabolites of ALL were analyzed and identified by combining isotope tracing technology with the application of mass spectrometry fragmentation patterns. The key metabolic enzymes involved in the biotransformation of ALL were explored using the CYP450 recombinant enzyme method, which facilitated the identification of the enzymes contributing to ALL's metabolic pathway. The liver microsomal metabolism investigation revealed a total of five metabolites, with the predominant being M2 (harmol or 3-hydroxy-4-methoxy-6-methyl-5,7,8,15-tetrahydro-[1,3]dioxolo[4′,5′:4,5]benzo[1,2-g]benzo[c]azecin-14(6 H)-one). The recombinant enzyme analysis conclusively identified CYP2D6 as the pivotal CYP450 isoenzyme that plays a central role in the metabolic pathway of the principal ALL metabolite, M2. This research not only expands our comprehension of the biotransformation process of ALL but also provides significant scientific evidence for the clinical safety of ALL, which was of great importance for guiding the application of ALL in the field of veterinary medicine.