Totsuka S, Watanabe T, Koyanagi F, Tanaka K, Yasuda M, Manabe S
Laboratory Animal Science and Toxicology Laboratories, Sankyo Co., Ltd., Fukuroi, Shizuoka, Japan.
Arch Toxicol. 1999 Jun-Jul;73(4-5):203-7. doi: 10.1007/s002040050607.
The ratio of urinary 6beta-hydroxycortisol (6beta-OHF) to free cortisol (F), i.e., the 6beta-OHF/F ratio, has been reported to be a specific marker for human CYP3A induction by in vivo studies of human subjects. In the development of drugs, it is quite beneficial to predict human CYP3A induction in preclinical safety studies using urine samples from experimental animals. We examined the 6beta-OHF/F ratio in urine of common marmosets administered with rifampicin, a potent inducer of CYP3A, to evaluate the usefulness of common marmosets for the prediction of CYP3A induction. Rifampicin was orally administered to three groups of four male common marmosets at doses of 0, 10, and 20 mg/kg per day for 4 days. Amounts of 6beta-OHF and F in urine samples were determined by means of high-performance liquid chromatography (HPLC) during the experimental period. One day after the 4th dosing, animals were killed, and P450 contents and P450-catalyzed. 7-alkoxycoumarin O-dealkylase (ACD) activities in the liver were measured. Western blot analysis of liver microsomes was also performed using anti-rat P450 (CYP1A1, 2B1/2, 3A, and 4A) antibodies. The results indicated elevations in the 6beta-OHF/F ratios that were dependent on both the dosing period and dose levels adopted. The ratios on day 4 reached 4.7- and 5.3-fold the pre-administration values in the 10 and 20 mg/kg per day groups, respectively. P450 contents and ACD activities were also elevated in all of the groups. Western blot analysis showed specific increases in the protein which cross-reacts with anti-rat CYP3A antibody in all of the groups. Furthermore, the 6beta-OHF/F ratio was well correlated with the CYP3A contents in liver (r = 0.906). These results indicated that increase in urinary excretion of 6beta-OHF is a specific marker of the induction of hepatic CYP3A in common marmosets just as in humans. Consequently, the present study suggested that human CYP3A induction elicited by chemical agents can be predicted in common marmosets by measuring the urinary excretion of 6beta-OHF.
据体内人体研究报道，尿中6β-羟基皮质醇（6β-OHF）与游离皮质醇（F）的比值，即6β-OHF/F比值，是人体CYP3A诱导的特异性标志物。在药物研发过程中，利用实验动物的尿液样本在临床前安全性研究中预测人体CYP3A诱导情况是非常有益的。我们检测了给予CYP3A强效诱导剂利福平的普通狨猴尿液中的6β-OHF/F比值，以评估普通狨猴在预测CYP3A诱导方面的实用性。将三组每组四只雄性普通狨猴分别按每天0、10和20mg/kg的剂量口服利福平，持续4天。在实验期间，通过高效液相色谱法（HPLC）测定尿液样本中6β-OHF和F的含量。第4次给药后一天，处死动物，测定肝脏中的P450含量和P450催化的7-烷氧基香豆素O-脱烷基酶（ACD）活性。还使用抗大鼠P450（CYP1A1、2B1/2、3A和4A）抗体对肝脏微粒体进行了蛋白质印迹分析。结果表明，6β-OHF/F比值的升高取决于给药时间和所采用的剂量水平。第4天，每天10mg/kg和20mg/kg组的比值分别达到给药前值的4.7倍和5.3倍。所有组的P450含量和ACD活性也均升高。蛋白质印迹分析显示，所有组中与抗大鼠CYP3A抗体发生交叉反应的蛋白质均有特异性增加。此外，6β-OHF/F比值与肝脏中CYP3A含量高度相关（r = 0.906）。这些结果表明，6β-OHF尿排泄增加是普通狨猴肝脏CYP3A诱导的特异性标志物，与人类情况相同。因此，本研究表明，通过测量6β-OHF的尿排泄情况，可以在普通狨猴中预测化学物质引起的人体CYP3A诱导。
