CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide
CYP2C8 and CYP3A4 are the principal enzymes involved in the human in vitro biotransformation of the insulin secretagogue repaglinide
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DOI：
10.1046/j.0306-5251.2003.01862.x
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发表时间：
2003-09-01
影响因子：
3.4
通讯作者：
Hansen, KT
中科院分区：
文献类型：
--
作者：
Bidstrup, TB;Bjornsdottir, I;Hansen, KT
目的鉴定负责瑞格列奈人体体外生物转化的主要人体细胞色素P450（CYP）酶。以前的实验已经确定CYP 3A 4主要负责瑞格列奈的体外代谢，但临床研究的结果表明，一个以上的酶可能参与瑞格列奈biotransformation.Methods [C-14]-瑞格列奈孵育与重组人肝微粒体（HLM）从个人捐助者的抑制性抗体的存在下，对个人的酶的特异性。采用液相色谱-质谱联用技术（LC-MS）和LC-MS-核磁共振在线联用技术（LC-MS-NMR）对样品中的代谢物进行了定性和定量分析结果CYP 3A 4和CYP 2C 8参与瑞格列奈的代谢，M4（由哌啶环系统上的羟基化产生）和M1（芳族胺）。针对CYP 3A 4和CYP 2C 8的特异性抑制性单克隆抗体可显著抑制HLM中M4和M1的形成（> 71%）。在来自12个个体供体的一组薄膜中，M4和M1的形成分别在约160-880 pmol min（-1）mg（-1）蛋白和100-1110 pmol min（-1）mg（-1）蛋白之间变化。发现CYP 2C 8产生的主要代谢产物为M4。HLM中该代谢物的形成速率与紫杉醇6 α-羟基化显著相关（r（s）= 0.80; P= 0.0029）。还检测到其他两种次要代谢物。其中一个为M1，另一个为异丙基羟基化瑞格列奈（M0-OH）。在CYP 2C 8 Supersomes（TM）中，M4的形成速率为2.5 pmol min（-1）pmol（-1）β-内酰胺酶，而在CYP 3A 4 Supersomes（TM）中，M4的形成速率仅约为0.1 pmol min（-1）pmol（-1）β-内酰胺酶。CYP 3A 4产生的主要代谢产物为M1。HLM中该代谢物的形成速率与睾酮6 β-羟基化显著相关（r（s）= 0.90; P = 0.0002）。鉴别了其他三种代谢产物，即MO-OH、M2（哌啶环氧化开环形成的二羧酸）和M5。在CYP 3A 4 Supersomes（TM）中M1的形成速率为1.6 pmol min（-1）pmol（-1）β-内酰胺酶，而在CYP 2C 8 Supersomes TM中M1的形成速率仅为0.4 pmol min（-1）pmol（-1）β-内酰胺酶。如果一个β途径有足够的能力在另一个β途径被抑制时进行补偿，这种双β生物转化可能会对瑞格列奈的临床药代动力学和药物相互作用产生影响。
Aims To identify the principal human cytochrome P450 (CYP) enzyme(s) responsible for the human in vitro biotransformation of repaglinide. Previous experiments have identified CYP3A4 as being mainly responsible for the in vitro metabolism of repaglinide, but the results of clinical investigations have suggested that more than one enzyme may be involved in repaglinide biotransformation.Methods [C-14]-Repaglinide was incubated with recombinant CYP and with human liver microsomes (HLM) from individual donors in the presence of inhibitory antibodies specific for individual CYP enzymes. Metabolites, measured by high-performance liquid chromatography (HPLC) with on-line radiochemical detection, were identified by liquid chromatography-mass spectrophotometry (LC-MS) and LC-MS coupled on-line to a nuclear magnetic resonance spectrometer (LC-MS-NMR).Results CYP3A4 and CYP2C8 were found to be responsible for the conversion of repaglinide into its two primary metabolites, M4 (resulting from hydroxylation on the piperidine ring system) and M1 (an aromatic amine). Specific inhibitory monoclonal antibodies against CYP3A4 and CYP2C8 significantly inhibited (> 71%) formation of M4 and M1 in HLM. In a panel of FILM from 12 individual donors formation of M4 and M1 varied from approximately 160-880 pmol min(-1) mg(-1) protein and from 100-1110 pmol min(-1) mg(-1) protein, respectively. The major metabolite generated by CYP2C8 was found to be M4. The rate of formation of this metabolite in HLM correlated significantly with paclitaxel 6alpha-hydroxylation (r(s) = 0.80; P= 0.0029). Two other minor metabolites were also detected. One of them was M1 and the other was repaglinide hydroxylated on the isopropyl moiety (M0-OH). The rate of formation of M4 in CYP2C8 Supersomes(TM) was 2.5 pmol min(-1) pmol(-1) CYP enzyme and only about 0.1 pmol min(-1) pmol(-1) CYP enzyme in CYP3A4 Supersomes(TM). The major metabolite generated by CYP3A4 was M1. The rate of formation of this metabolite in HLM correlated significantly with testosterone 6beta-hydroxylation (r(s) = 0.90; P = 0.0002). Three other metabolites were identified, namely, MO-OH, M2 (a dicarboxylic acid formed by oxidative opening of the piperidine ring) and M5. The rate of M1 formation in CYP3A4 Supersomes(TM) was 1.6 pmol min(-1) pmol(-1) CYP enzyme but in CYP2C8 Supersomes TM it was only approximately 0.4 pmol min(-1) pmol(-1) CYP enzyme.Conclusions The results confirm an important role for both CYP3A4 and CYP2C8 in the human in vitro biotransformation of repaglinide. This dual CYP biotransformation may have consequences for the clinical pharmacokinetics and drug-drug interactions involving repaglinide if one CYP pathway has sufficient capacity to compensate if the other is inhibited.