一个简捷的甾体5α-还原酶抑制剂体外筛选模型

A Convenient and Rapid Model in vitro to Screen Steroid 5 alpha-reductase Inhibitors

通过模拟睾丸酮经甾体5α-还原酶催化,从NADPH(辅酶)中获得H^+还原成双氢睾丸酮(DHT)这一反应,参照文献资料以酶动力学原理,分别测定反应底物NADPH和产物[~3H]-DHT含量变化的初始速率以代表酶活性,建立了“分光光度计法”和“同位素法”体外筛药模型,通过这两种模型对比,以非那甾胺为阳性对照药物,对爱普列特进行了体外模型验证研究。两种筛选模型结果一致,确定非那甾胺和爱普列特的抑制常数:用测定OD_(340)nm值的“分光光度计法”求得爱普列特抑制常数Ki=24.9±1.3nmol/L,半数抑制浓度IC_(50)=39.0 nmol/L;非那甾胺Ki=15.3±2.3 nmol/L,IC_(50)=58.6 nmol/L。用测定[~3H」-DHT dpm值的“同位素法”求得爱普列特Ki=67.4±13.2 nmol/L,IC_(50)=49.6nmol/L;非那甾胺Ki=25.0±1.9 nmol/L,IC_(50)=5.1nmol/L。该结果与国外文献中报道的爱普列特Ki=5～23 nmol/L,非那甾胺Ki=23.6 nmol/L相接近。

A convenient and rapid model in vitro to screen steroid 5a-reductase inhibitors which were effective in the treatment of benign p'rostatic hyperplasia (BPH) was developed. In the attendance of nicotinamide adenine dinucleotide phosphare (NADPH), steroid 5a-reductase converts testosterone to dihydrotestosterone which is a major etio-logic factor of BPH. NADPH has characteristic absorbance at 340nmol/L, and the ab-sorbance spectrum may be used to identify NADPH as a kind of the substrate in this enzymatic reaction. In this paper, NADPH, steroid 5a-reductase, series concentration of testosterone and finasteride, and 4 ml Tris-HCl buffer were continuously incubated together at 37 癈 and the NADPH OD value were continually measured. The descending rate of NADPH OD340 nm value by linear regression from the beginning to the tenth minute is close to the initial velocity of the enzymatic reaction. The precise activity of the steroid 5ot-reductase was the slope after subtracted the one of the blank control. The inhibition constant (Ki) of steroid 5a-reductase inhibitors could be calculated according to the Lineweaver-Burk plots. Two drug screen models, the common isotope model and this novel model, were compared in this paper. The result showed that the latter one is more economical, quicker and more effective than the former one.