Identification of canine cytochrome P‐450s (CYPs) metabolizing the tramadol (+)‐M1 and (+)‐M2 metabolites to the tramadol (+)‐M5 metabolite in dog liver microsomes

We previously showed that (+)‐tramadol is metabolized in dog liver to (+)‐M1 exclusively by CYP2D15 and to (+)‐M2 by multiple CYPs, but primarily CYP2B11. However, (+)‐M1 and (+)‐M2 are further metabolized in dogs to (+)‐M5, which is the major metabolite found in dog plasma and urine. In this study, we identified canine CYPs involved in metabolizing (+)‐M1 and (+)‐M2 using recombinant enzymes, untreated dog liver microsomes (DLMs), inhibitor‐treated DLMs, and DLMs from CYP inducer‐treated dogs. A canine P‐glycoprotein expressing cell line was also used to evaluate whether (+)‐tramadol, (+)‐M1, (+)‐M2, or (+)‐M5 are substrates of canine P‐glycoprotein, thereby limiting their distribution into the central nervous system. (+)‐M5 was largely formed from (+)‐M1 by recombinant CYP2C21 with minor contributions from CYP2C41 and CYP2B11. (+)‐M5 formation in DLMs from (+)‐M1 was potently inhibited by sulfaphenazole (CYP2C inhibitor) and chloramphenicol (CYP2B11 inhibitor) and was greatly increased in DLMs from phenobarbital‐treated dogs. (+)‐M5 was formed from (+)‐M2 predominantly by CYP2D15. (+)‐M5 formation from (+)‐M1 in DLMs was potently inhibited by quinidine (CYP2D inhibitor) but had only a minor impact from all CYP inducers tested. Intrinsic clearance estimates showed over 50 times higher values for (+)‐M5 formation from (+)‐M2 compared with (+)‐M1 in DLMs. This was largely attributed to the higher enzyme affinity (lower Km) for (+)‐M2 compared with (+)‐M1 as substrate. (+)‐tramadol, (+)‐M1, (+)‐M2, or (+)‐M5 were not p‐glycoprotein substrates. This study provides a clearer picture of the role of individual CYPs in the complex metabolism of tramadol in dogs.