Equine cytochrome P450 2B6-Genomic identification, expression and functional characterization with ketamine
Equine cytochrome P450 2B6-Genomic identification, expression and functional characterization with ketamine
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DOI：
10.1016/j.taap.2012.10.028
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发表时间：
2013-01-01
影响因子：
3.8
通讯作者：
Schmitz, A.
中科院分区：
文献类型：
--
作者：
Peters, L. M.;Demmel, S.;Schmitz, A.
氯胺酮是一种麻醉剂和止痛剂，经常用于兽医患者。由于氯胺酮几乎总是与其他药物联合给药，因此氯胺酮与其他药物之间的相互作用存在不良反应或疗效降低的风险。由于细胞色素P450酶（CYP）在大多数上市药物的I相代谢中起着关键作用，因此药物间相互作用通常发生在这些酶的活性位点。CYP已在人类和实验室动物中进行了彻底检查，但对马的CYP知之甚少。马CYP的表征对于更好地理解马的药物代谢至关重要。本文报道了马CYP 2B 6基因的注释、克隆和在V79中国仓鼠成纤维细胞中的异源表达，对所有CYP 2B基因进行计算机注释后，用RT-PCR从马肝总RNA中扩增出马CYP 2B 6的编码序列（CDS），其氨基酸序列与人的同源性为77%，相似性为93.7%。在97匹马中检测到CYP 2B 6基因外显子2的非同义变异c.226G>A。突变的A等位基因显示等位基因频率为82%。在该组的一匹和两匹马中分别检测到外显子3的两个进一步的变异体。将转染的V79细胞与作为探针底物的外消旋氯胺酮和去甲氯胺酮一起孵育以测定代谢活性。重组马CYP 2B 6将氯胺酮N-去甲基化为去甲氯胺酮，并产生去甲氯胺酮的代谢产物，如羟基化去甲氯胺酮和5，6-脱氢去甲氯胺酮。S-/和R-去甲氯胺酮形成的Vmax分别为0.49和0.45 nmol/h/mg细胞蛋白，Km分别为3.41和2.66 μ M。氯吡格雷浓度依赖性地抑制S-/R-氯胺酮的N-去甲基化，其IC 50分别为5.63和6.26 μ M。记录的遗传变异体的功能重要性仍有待探讨。确定马CYP 2B 6是一种参与氯胺酮和去甲氯胺酮代谢的β-内酰胺酶，从而证实了马肝微粒体抑制研究的结果。氯吡格雷似乎是一种可行的马CYP 2B 6抑制剂。特异性仍需用其他单一马CYP确定。单一马源性β-内酰胺酶的异源表达为实质性地提高对马的药物代谢和药物相互作用的理解开辟了新的可能性。(c)2012 Elsevier Inc. All rights reserved.
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 0.45 nmol/h/mg cellular protein and K-m was 3.41 and 2.66 mu M, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC50 of 5.63 and 6.26 mu M, 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. (c) 2012 Elsevier Inc. All rights reserved.