Identification of canine cytochrome P‐450s ( CYP s) 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 CYP 2D15 and to (+)‐M2 by multiple CYP s, but primarily CYP 2B11. 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 CYP s involved in metabolizing (+)‐M1 and (+)‐M2 using recombinant enzymes, untreated dog liver microsomes ( DLM s), inhibitor‐treated DLM s, and DLM s 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 CYP 2C21 with minor contributions from CYP 2C41 and CYP 2B11. (+)‐M5 formation in DLM s from (+)‐M1 was potently inhibited by sulfaphenazole ( CYP 2C inhibitor) and chloramphenicol ( CYP 2B11 inhibitor) and was greatly increased in DLM s from phenobarbital‐treated dogs. (+)‐M5 was formed from (+)‐M2 predominantly by CYP 2D15. (+)‐M5 formation from (+)‐M1 in DLM s was potently inhibited by quinidine ( CYP 2D 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 DLM s. 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 CYP s in the complex metabolism of tramadol in dogs.