Rodrigues A D, Mulford D J, Lee R D, Surber B W, Kukulka M J, Ferrero J L, Thomas S B, Shet M S, Estabrook R W
Department 46V, Abbott Laboratories, Abbott Park, IL 60064, USA.
Drug Metab Dispos. 1995 Jul;23(7):765-75.
The metabolism of terfenadine was studied with a cDNA-expressed/purified recombinant fusion protein containing human liver microsomal cytochrome P4503A4 (CYP3A4) linked to rat NADPH-P450 reductase (rF450[mHum3A4/mRatOR]L1) and was compared with that observed in the presence of human liver microsomes and precision-cut human liver tissue slices. In all three cases, [3H]terfenadine was metabolized to at least three major metabolites. LC/MS (electrospray) analysis confirmed that these metabolites were alpha, alpha-diphenyl-4-piperidinomethanol (M5), t-butyl hydroxy terfenadine (M4), and t-butyl carboxy terfenadine (M3), although the level of M5 detected in the presence of fusion protein was greater than that found with microsomes or tissue slices. Two additional metabolites, M1 (microsomes and tissue slices) and M2 (fusion protein), were also detected, but remain uncharacterized. Consumption of parent drug (microsomes: KM = 9.58 +/- 2.79 microM, Vmax = 801 +/- 78.3 pmol/min/nmol CYP; fusion protein: KM = 14.1 +/- 1.13 microM, Vmax = 1670 +/- 170 pmol/min/nmol CYP) and t-butyl hydroxylation to M4 (microsomes: KM = 12.9 +/-3.74 microM, Vmax = 643 +/- 62.5 pmol/min/nmol CYP, ; fusion protein: KM = 30.0 +/- 2.55 microM, Vmax = 1050 +/- 141 pmol/min/nmol CYP) obeyed Michaelis-Menten kinetics over the terfenadine concentration range of 1-200 microM. Ketoconazole, a well-documented CYP3A inhibitor, effectively inhibited terfenadine metabolism in all three models. The conversion of M4 to M3, studied with human liver microsomes and fusion protein, was NADPH-dependent and inhibited by ketoconazole. It is concluded that cDNA-expressed CYP3A4, in the form of a NADPH-P450 reductase-linked fusion protein, may also serve as a model for studying the metabolism of terfenadine in vitro and many other drugs.
用一种含有与大鼠NADPH - P450还原酶相连的人肝微粒体细胞色素P4503A4（CYP3A4）的cDNA表达/纯化重组融合蛋白（rF450[mHum3A4/mRatOR]L1）研究了特非那定的代谢，并与在人肝微粒体和精密切割的人肝组织切片存在下观察到的代谢情况进行了比较。在所有这三种情况下，[3H]特非那定都代谢为至少三种主要代谢物。液相色谱/质谱（电喷雾）分析证实，这些代谢物是α，α - 二苯基 - 4 - 哌啶甲醇（M5）、叔丁基羟基特非那定（M4）和叔丁基羧基特非那定（M3），尽管在融合蛋白存在下检测到的M5水平高于在微粒体或组织切片中发现的水平。还检测到另外两种代谢物，M1（微粒体和组织切片）和M2（融合蛋白），但仍未鉴定。母体药物的消耗（微粒体：KM = 9.58±2.79 microM，Vmax = 801±78.3 pmol/min/nmol CYP；融合蛋白：KM = 14.1±1.13 microM，Vmax = 1670±170 pmol/min/nmol CYP）以及向M4的叔丁基羟基化（微粒体：KM = 12.9±3.74 microM，Vmax = 643±62.5 pmol/min/nmol CYP；融合蛋白：KM = 30.0±2.55 microM，Vmax = 1050±141 pmol/min/nmol CYP）在1 - 200 microM的特非那定浓度范围内符合米氏动力学。酮康唑是一种有充分文献记载的CYP3A抑制剂，在所有三种模型中均有效抑制特非那定的代谢。用人肝微粒体和融合蛋白研究的M4向M3的转化是NADPH依赖性的，并受酮康唑抑制。结论是，以NADPH - P450还原酶连接的融合蛋白形式的cDNA表达的CYP3A4也可作为体外研究特非那定和许多其他药物代谢的模型。
