Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.
Proc Natl Acad Sci U S A. 2011 May 31;108(22):9032-7. doi: 10.1073/pnas.1100285108. Epub 2011 May 16.
Exogenous enzymes are administered orally to treat several diseases, such as pancreatic insufficiency and lactose intolerance. Due to the proteinaceous nature of enzymes, they are subject to inactivation and/or digestion in the gastrointestinal (GI) tract. Here we describe a convenient fluorescence-based assay to monitor the activity of therapeutic enzymes in real time in vivo in the GI tract. To establish the proof of principle, the assay was applied to proline-specific endopeptidases (PEPs), a group of enzymes recently proposed as adjuvant therapy for celiac disease (a highly prevalent immunogenetic enteropathy). A short PEP-specific peptide sequence which is part of larger immunotoxic sequences of gluten was labeled with a fluorescent dye and a corresponding quencher. Upon enzymatic cleavage, the fluorescence emission was dequenched and detected with an in vivo imaging system. PEPs originating from Flavobacterium meningosepticum (FM) and Myxococcus xanthus (MX) were evaluated after oral administration in rats. While MX PEP could not cleave the peptide in the stomach, FM PEP showed significant gastric activity reaching 40-60% of the maximal in vivo signal intensity. However, both enzymes produced comparable fluorescence signals in the small intestine. Coadministration of an antacid drug significantly enhanced MX PEP's gastric activity due to increased pH and/or inhibition of stomach proteases. With this simple procedure, differences in the in vivo performance of PEPs, which could not be identified under in vitro conditions, were detected. This imaging assay could be used to study other oral enzymes in vivo and therefore be instrumental in improving their therapeutic efficiency.
外源性酶经口服给药用于治疗多种疾病，如胰腺功能不全和乳糖不耐受。由于酶的蛋白质性质，它们在胃肠道（GI）中易失活和/或被消化。在这里，我们描述了一种方便的基于荧光的测定法，用于实时监测 GI 道中治疗性酶的活性。为了建立原理验证，该测定法应用于脯氨酸特异性内切酶（PEP），这是一组最近被提议作为乳糜泻（一种高度流行的免疫遗传肠病）辅助治疗的酶。将一段短的 PEP 特异性肽序列标记上荧光染料和相应的猝灭剂，该序列是谷蛋白中更大免疫毒性序列的一部分。酶切后，荧光发射被去猝灭，并通过体内成像系统检测。经口给予脑膜炎败血性黄杆菌（FM）和粘球菌（MX）来源的 PEP 后，在大鼠中进行评估。虽然 MX PEP 不能在胃中切割该肽，但 FM PEP 显示出显著的胃活性，达到体内最大信号强度的 40-60%。然而，两种酶在小肠中均产生可比的荧光信号。由于 pH 值升高和/或胃蛋白酶抑制，同时给予抗酸药物可显著增强 MX PEP 的胃活性。通过这种简单的程序，可以检测到在体外条件下无法识别的 PEP 在体内性能的差异。这种成像测定法可用于研究其他口服酶的体内性能，从而有助于提高其治疗效率。
