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Phytocannabinoid‐rich hemp extracts containing cannabidiol (CBD) and cannabidiolic acid (CBDA) are increasingly being used to treat various disorders in dogs. The objectives of this study were to obtain preliminary information regarding the in vitro metabolism of these compounds and their capacity to inhibit canine cytochrome P450 (CYP)‐mediated drug metabolism and canine P‐glycoprotein‐mediated transport. Pure CBD and CBDA, and hemp extracts enriched for CBD and for CBDA were evaluated. Substrate depletion assays using pooled dog liver microsomes showed CYP cofactor‐dependent depletion of CBD (but not CBDA) and UDP‐glucuronosytransferase cofactor‐dependent depletion of CBDA (but not CBD) indicating major roles for CYP and UDP‐glucuronosytransferase in the metabolism of these phytocannabinoids, respectively. Further studies using recombinant canine CYPs demonstrated substantial CBD depletion by the major hepatic P450 enzymes CYP1A2 and CYP2C21. These results were confirmed by showing increased CBD depletion by liver microsomes from dogs treated with a known CYP1A2 inducer (β‐naphthoflavone) and with a known CYP2C21 inducer (phenobarbital). Cannabinoid‐drug inhibition experiments showed inhibition (IC50 = 4.6–8.1 μM) of tramadol metabolism via CYP2B11‐mediated N‐demethylation (CBD and CBDA) and CYP2D15‐mediated O‐demethylation (CBDA only) by dog liver microsomes. CBD and CBDA did not inhibit CYP3A12‐mediated midazolam 1′‐hydroxylation (IC50 > 10 μM). CBD and CBDA were not substrates or competitive inhibitors of canine P‐glycoprotein. Results for cannabinoid‐enriched hemp extracts were identical to those for pure cannabinoids. These in vitro studies indicate the potential for cannabinoid–drug interactions involving certain CYPs (but not P‐glycoprotein). Confirmatory in vivo studies are warranted. [ABSTRACT FROM AUTHOR]

CYP2D15 is a major drug metabolizing P450 in canine liver. Like the human orthologue (CYP2D6), this enzyme is highly polymorphic with at least five common nonsynonymous variants reported that result in amino acid changes, including p.Ile109Val, p.Leu115Phe, p.Gly186Ser, p.Ile250Phe and p.Ile307Val. Furthermore, a mRNA splice variant of CYP2D15 has been found in canine liver that lacks the exon 3 gene region resulting in an inactive enzyme. The objective of this study was to evaluate whether any of these amino acid variants or the exon 3 deletion mRNA variant (exon3‐delta) was associated with differences in CYP2D15‐selective activities or protein content in a bank of canine livers. Livers were obtained from 25 Beagles and 34 dogs of various other breeds. CYP2D15‐selective activities measured included dextromethorphan o‐demethylation and tramadol o‐demethylation. Reverse transcription PCR showed that 76% of livers (44/58) expressed both exon3‐delta and normally spliced CYP2D15 RNA, while the remaining 24% (14/58) expressed only normally spliced RNA. The presence of exon3‐delta was not correlated with CYP2D15 activities or protein content. Compared with wild‐type livers, Beagle dog livers heterozygous for the p.Ile109Val and p.Gly186Ser variants showed from 40 to 50% reductions in median enzyme activities, while heterozygous p.Gly186Ser livers were associated with a 41% reduction in median CYP2D15 protein content (p <.05; Dunn's test). In the entire liver bank, livers homozygous for p.Ile109Val were also associated with a 40% reduction in median dextromethorphan O‐demethylation activities versus wild‐type livers (p <.05). These results identify several nonsynonymous CYP2D15 gene variants associated with variable CYP2D15 metabolism in canine liver. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]