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

Perez Jimenez, Tania E., Mealey, Katrina L., Schnider, Darren, Grubb, Tamara L., Greene, Stephen A., Court, Michael H.
Journal of veterinary pharmacology and therapeutics 2018 v.41 no.6 pp. 815-824
P-glycoproteins, cell lines, central nervous system, chloramphenicol, cytochrome P-450, dogs, enzymes, liver, liver microsomes, metabolism, metabolites, quinidine, urine
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.