Floby E, Briem S, Terelius Y, Sohlenius-Sternbeck A K
Research DMPK and Biomarkers, AstraZeneca R&D Södertälje, S-151 85 Södertälje, Sweden.
Xenobiotica. 2004 Nov-Dec;34(11-12):949-59. doi: 10.1080/00498250400017257.
A cocktail of the following probe substrates for human drug-metabolizing enzymes was used to characterize hepatocyte preparations: phenacetin (for CYP1A2), diclofenac (CYP2C9), diazepam (CYP2C19), bufuralol (CYP2D6), midazolam (CYP3A4/5) and 7-hydroxycoumarin (for glucuronidation and sulphation). The cocktail was incubated with cryopreserved human, dog or minipig hepatocytes or with freshly prepared rat hepatocytes. Sample analysis was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an Open Access environment that allowed less experienced MS operators to login, submit and analyse sample sets using predefined settings without the immediate attendance of an experienced analyst. Intrinsic clearances (CLint) were calculated from the disappearance of the compounds from the incubations. Initially, the cocktail used for human, rat and dog hepatocyte incubations contained 7-ethoxycoumarin instead of 7-hydroxycoumarin. However, 7-ethoxycoumarin had an inhibitory effect on the metabolism of phenacetin. The highest CLint estimated with human and dog hepatocytes was observed for 7-hydroxycoumarin. For rat and minipig hepatocytes, the highest CLint was observed for bufuralol. In incubations with dog and minipig hepatocytes, the lowest CLint was seen with diclofenac, whereas for human and rat hepatocytes, the lowest value was observed with diazepam and phenacetin, respectively. When the cocktail was incubated together with human hepatocytes and 1 microM ketoconazole, the CLint of midazolam was decreased to about 7.5% of the control value, whereas the metabolism of the other cocktail compounds was virtually unaffected by this CYP3A inhibitor. It is suggested that a cocktail of specific human probe substrates for drug-metabolizing enzymes can be used routinely for the determination of the metabolic capacity of hepatocyte preparations in order to ensure the quality and reproducibility of experiments. Moreover, a cocktail of specific probe substrates can also be a useful tool for studies on enzyme inhibition.
非那西丁（用于CYP1A2）、双氯芬酸（CYP2C9）、地西泮（CYP2C19）、布非洛尔（CYP2D6）、咪达唑仑（CYP3A4/5）和7-羟基香豆素（用于葡糖醛酸化和硫酸化）。该混合物与冷冻保存的人、狗或小型猪肝细胞或新鲜制备的大鼠肝细胞一起孵育。样品分析通过液相色谱-串联质谱法（LC-MS/MS）在开放获取环境中进行，该环境允许经验较少的质谱操作人员登录、提交和使用预定义设置分析样品集，而无需经验丰富的分析师立即在场。根据孵育中化合物的消失情况计算内在清除率（CLint）。最初，用于人、大鼠和狗肝细胞孵育的混合物含有7-乙氧基香豆素而非7-羟基香豆素。然而，7-乙氧基香豆素对非那西丁的代谢有抑制作用。用7-羟基香豆素观察到人与狗肝细胞的最高CLint估计值。对于大鼠和小型猪肝细胞，布非洛尔的CLint最高。在与狗和小型猪肝细胞的孵育中，双氯芬酸的CLint最低，而对于人和大鼠肝细胞，分别观察到地西泮和非那西丁的最低值。当该混合物与人肝细胞和1 microM酮康唑一起孵育时，咪达唑仑的CLint降至对照值的约7.5%，而其他混合物化合物的代谢几乎不受这种CYP3A抑制剂的影响。建议将用于药物代谢酶的特定人类探针底物混合物常规用于测定肝细胞制剂的代谢能力，以确保实验的质量和可重复性。此外，特定探针底物混合物也可以是酶抑制研究的有用工具。
