Species-Dependent Uptake of Glycylsarcosine but Not Oseltamivir in Pichia pastoris Expressing the Rat, Mouse, and Human Intestinal Peptide Transporter PEPT1
Species-Dependent Uptake of Glycylsarcosine but Not Oseltamivir in Pichia pastoris Expressing the Rat, Mouse, and Human Intestinal Peptide Transporter PEPT1
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DOI：
10.1124/dmd.111.044263
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发表时间：
2012-07-01
影响因子：
3.9
通讯作者：
Smith, David E.
中科院分区：
文献类型：
--
作者：
Hu, Yongjun;Chen, Xiaomei;Smith, David E.
本研究的目的是确定甘氨酰肌氨酸（一种模型二肽）和奥司他韦（一种抗病毒前药）是否在表达大鼠、小鼠和人类PEPT 1同系物的酵母毕赤酵母中表现出物种依赖性摄取。在表达人、小鼠和大鼠肽转运蛋白1（PEPT 1）的酵母巴斯德毕赤酵母中用[H-3]甘氨酰肌氨酸（GlySar）进行实验，其中检查了摄取与时间、浓度、潜在抑制剂以及奥司他韦对GlySar的剂量-反应抑制的函数关系。还在相同的实验条件下进行了[C-14]奥司他韦的研究。我们发现GlySar在所有三种物种中均表现出可饱和摄取，其中人（0.86 mM）>小鼠（0.30 mM）>大鼠（0.16 mM）的Km值。酵母转化体中的GlySar摄取对肽（甘氨酰脯氨酸）和肽样药物（头孢羟氨苄、头孢拉定和伐昔洛韦）具有特异性，但不受甘氨酸、L-组氨酸、头孢唑林、头孢噻吩、头孢匹林、阿昔洛韦、4-乙酰氨基-4 '-异硫氰基芪-2，2'-二磺酸、四乙基铵和依克立达的影响。尽管奥司他韦引起GlySar摄取的剂量依赖性抑制[人（27.4 mM）>大鼠（18.3 mM）>小鼠（10.7 mM）的IC 50值]，但这种相互作用在人体中的临床相关性非常低。重要的是，奥司他韦不是表达三种测试哺乳动物物种的酵母中肠PEPT 1转运蛋白的底物。相反，前药表现出与酵母载体和PEPT 1转化体的非特异性结合。最后，小鼠似乎是一个更好的动物模型，比大鼠为探索肠道吸收和药物动力学的肽和肽类药物在人体内。
The purpose of this study was to determine whether glycylsarcosine (a model dipeptide) and oseltamivir (an antiviral prodrug) exhibited a species-dependent uptake in yeast Pichia pastoris expressing the rat, mouse, and human homologs of PEPT1. Experiments were performed with [H-3]glycylsarcosine (GlySar) in yeast P. pastoris expressing human, mouse, and rat peptide transporter 1 (PEPT1), in which uptake was examined as a function of time, concentration, potential inhibitors, and the dose-response inhibition of GlySar by oseltamivir. Studies with [C-14]oseltamivir were also performed under identical experimental conditions. We found that GlySar exhibited saturable uptake in all three species, with K-m values for human (0.86 mM) > mouse (0.30 mM) > rat (0.16 mM). GlySar uptake in the yeast transformants was specific for peptides (glycylproline) and peptide-like drugs (cefadroxil, cephradine, and valacyclovir), but was unaffected by glycine, L-histidine, cefazolin, cephalothin, cephapirin, acyclovir, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, tetraethylammonium, and elacridar. Although oseltamivir caused a dose-dependent inhibition of GlySar uptake [IC50 values for human (27.4 mM) > rat (18.3 mM) > mouse (10.7 mM)], the clinical relevance of this interaction would be very low in humans. Of importance, oseltamivir was not a substrate for the intestinal PEPT1 transporter in yeast expressing the three mammalian species tested. Instead, the prodrug exhibited nonspecific binding to the yeast vector and PEPT1 transformants. Finally, the mouse appeared to be a better animal model than the rat for exploring the intestinal absorption and pharmacokinetics of peptides and peptide-like drugs in human.