睾酮代谢的种属差异研究

摘要

Objective To investigate the species difference of testosterone metabolism among human and 7 animal species including mice,rats,guinea pigs,rabbits,dogs,pigs,sheep,and cattle.Methods Testosterone was incubated with liver microsomes of humans and investigated animals,together with CYP-mediated metabolic reaction factors including NADP Na,glucose-6-phosphate,and glucose-6-phosphate dehydrogenase.Incubation samples were introduced into HPLC to determinate testosterone and its metabolites.Heatmap was used to visualize the metabolic profile while principle component analysis was used to tell the difference of metabolic profile among species.High resolution mass spectrometry was used to assist the identification of metabolite structures.Results 6β-hydroxylation is not the unique metabolic pathway of testosterone.Eighteen NADP-dependent metabolites were observed in humans or other investigated animals.Although 6β-hydroxylation showed positive correlation with the substrate elimination,this specific metabolic pathway could not compensate all substrate consumption.Guinea pig generates the most metabolites while sheep showed the weakest capacity on testosterone metabolism.Mice,dogs,and rabbits showed the most similar metabolic profile to humans.Conclusion 6β-hydroxylation metabolites of testosterone was not applicable to evaluate the CYP 3A activity of all species.Mice,dogs,and rabbits are recommended in pre-clinical trial if the target is proved to be metabolized by human CYP 3A4.%目的 研究睾酮在人及小鼠、大鼠、犬、兔、猪、豚鼠、牛、羊等实验动物肝微粒体代谢中的差异.方法 睾酮分别于上述种属肝微粒体进行孵育,加入NADP Na、葡萄糖-6-磷酸、葡萄糖-6-磷酸脱氢酶等试剂启动体外P450酶反应.样品采用高效液相色谱紫外检测器及高分辨质谱进行分析.代谢轮廓用热图进行可视化,并用主成分分析进行差异研究.结果 6β-羟基化不是睾酮唯一的代谢途径.本研究在不同种属中共发现了18种睾酮的代谢产物.尽管6β-羟基化在不同种属中与睾酮的底物消除基本呈正相关,但该途径无法补偿其他途径造成的底物消除.豚鼠具有最强的睾酮代谢能力,而羊对睾酮的代谢能力最弱.小鼠、犬、兔肝微粒体中,睾酮的代谢轮廓与人最相似.结论 6β羟基化途径的定量不能用作所有物种CYP3A活性评判的精准依据.如果药物证明由CYP 3A4进行代谢,在临床前期研究中,应结合药理学模型的需要,尽量采用小鼠、犬、兔作为动物模型,以保证药物体内暴露与人体实验尽量接近.