Seddon T, Michelle I, Chenery R J
Smith, Kline and French Ltd, The Frythe, Herts, U.K.
Biochem Pharmacol. 1989 May 15;38(10):1657-65. doi: 10.1016/0006-2952(89)90314-6.
Diazepam (DZ) was used as a substrate in drug metabolism studies to characterise the differences in metabolite profiles in hepatocytes isolated from four species: Wistar rat, cynomolgus monkey, beagle dog and man. Hepatocytes were incubated with DZ (20 microM) for 180 min at 3 hr post isolation in culture, and the disappearance of parent compound and appearance of its metabolites determined. DZ disappearance was found to be monoexponential in rat, monkey and human cells, but that DZ disappearance in dog hepatocytes was best described by a two compartment process. There were considerable differences in both the rates of formation and the profiles of metabolites produced from DZ in each species. Drug metabolism of DZ was determined in five human hepatocyte preparations. The rates of formation of both the major metabolites, temezepam (TEM) and nordiazepam (NOR) were highly variable between samples, and oxazepam (OX) was detected in only three of the preparations. There was no evidence of further metabolism of these metabolites, and the profiles were comparable with in vivo findings. In a single case, human hepatocytes were cultured for five days, and DZ was used as substrate to characterise the changes in drug metabolising activities. There was a rapid loss in the production of OX in the initial 24 hr, and a complete loss of 3-hydroxylation activities in the succeeding 120 hr. N-demethylation activities, however, were well maintained, and the appearance of NOR declined to 47% of initial rate. The hepatocytes of all species were found to produce NOR and TEM as metabolites; NOR representing the principal metabolite in the dog, monkey and human cells. In the dog, TEM was found only as a minor metabolite. OX was a significant metabolite in the monkey and a minor metabolite in the dog and human hepatocytes, but was not detected in rat cultures. The principal metabolite in rat cells was 4'-hydroxy diazepam, which was rapidly further metabolised to its glucuronide. The drug metabolising activities of the hepatocyte cultures towards DZ were comparable with the drug metabolism of DZ found in vivo in each species. These findings substantiate hepatocytes as an in vitro model of hepatic metabolism.
地西泮（DZ）在药物代谢研究中用作底物，以表征从四种物种分离的肝细胞中代谢物谱的差异：Wistar大鼠、食蟹猴、比格犬和人类。肝细胞在分离后3小时于培养物中与DZ（20微摩尔）孵育180分钟，并测定母体化合物的消失及其代谢物的出现。发现DZ在大鼠、猴和人类细胞中的消失呈单指数形式，但犬肝细胞中DZ的消失最好用双室过程来描述。每种物种中DZ产生的代谢物的形成速率和谱都存在相当大的差异。在五种人肝细胞制剂中测定了DZ的药物代谢。两种主要代谢物替马西泮（TEM）和去甲地西泮（NOR）的形成速率在样品之间高度可变，并且仅在三种制剂中检测到奥沙西泮（OX）。没有证据表明这些代谢物有进一步的代谢，并且其谱与体内研究结果相当。在一个案例中，人肝细胞培养了五天，并且使用DZ作为底物来表征药物代谢活性的变化。在最初的24小时内，OX的产生迅速丧失，并且在随后的120小时内3-羟基化活性完全丧失。然而，N-去甲基化活性保持良好，并且NOR的出现下降至初始速率的47%。发现所有物种的肝细胞都产生NOR和TEM作为代谢物；NOR是犬、猴和人类细胞中的主要代谢物。在犬中，仅发现TEM是次要代谢物。OX是猴中的重要代谢物，在犬和人肝细胞中是次要代谢物，但在大鼠培养物中未检测到。大鼠细胞中的主要代谢物是4'-羟基地西泮，其迅速进一步代谢为其葡糖醛酸苷。肝细胞培养物对DZ的药物代谢活性与每种物种体内发现的DZ药物代谢相当。这些发现证实了肝细胞作为肝脏代谢的体外模型。
