Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine

Peters, Laureen; Demmel, Steffi; Pusch, G.; Buters, J. T. M.; Thormann, W.; Zielinski, Jana; Leeb, Tosso; Mevissen, Meike; Schmitz, Andrea (2013). Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine. Toxicology and Applied Pharmacology, 266(1), pp. 101-8. Elsevier 10.1016/j.taap.2012.10.028

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Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in ketamine and norketamine metabolism, thus confirming results from inhibition studies with horse liver microsomes. Clopidogrel seems to be a feasible inhibitor for equine CYP2B6. The specificity still needs to be established with other single equine CYPs. Heterologous expression of single equine CYP enzymes opens new possibilities to substantially improve the understanding of drug metabolism and drug interactions in horses.

Item Type:

Journal Article (Original Article)

Division/Institute:

05 Veterinary Medicine > Department of Clinical Research and Veterinary Public Health (DCR-VPH) > Veterinary Pharmacology and Toxicology
05 Veterinary Medicine > Department of Clinical Research and Veterinary Public Health (DCR-VPH) > Experimental Clinical Research
05 Veterinary Medicine > Department of Clinical Research and Veterinary Public Health (DCR-VPH) > Institute of Genetics

UniBE Contributor:

Peters, Laureen; Demmel, Steffi; Zielinski, Jana; Leeb, Tosso; Mevissen, Meike and Schmitz, Andrea

Subjects:

600 Technology > 630 Agriculture
500 Science > 590 Animals (Zoology)

ISSN:

0041008X

Publisher:

Elsevier

Language:

English

Submitter:

Susanne Portner

Date Deposited:

28 Jul 2014 14:58

Last Modified:

16 Apr 2018 16:44

Publisher DOI:

10.1016/j.taap.2012.10.028

PubMed ID:

23142468

Uncontrolled Keywords:

Horse, Cytochrome P450 2B6, Ketamine, Clopidogrel, Drug metabolism

URI:

https://boris.unibe.ch/id/eprint/43899

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