Abstract

Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are considered as viable targets in the treatment of Alzheimer’s disease and other dementias. However, since clinically used AChE or dual AChE/BuChE inhibitors only provide some symptomatic treatment and lose their therapeutic effectiveness over time, in the last years an intensive search for multi-target compounds has started, which address additional targets besides AChE and BuChE, like endocannabinoid degrading enzymes. For the evaluation of the AChE and BuChE inhibitory potency of the test compounds in such drug development projects, usually the traditional Ellman’s method is used. Since this method has some analytical drawbacks, we have developed alternative robust HPLC/UV based assays using pyridin-2-ylmethyl acetate as substrate for AChE and benzoylcholine for BuChE. The enzyme products pyridin-2-ylmethanol and benzoic acid, respectively, were determined by reversed phase HPLC adding the ion pair reagent sodium octane-1-sulfonate to the mobile phase in case to the polar pyridine derivative. AChE from electric eel, human recombinant AChE and BuChE from equine serum were employed as enzymes. For method validation, the IC₅₀-values of the known AChE and BuChE inhibitors physostigmine, rivastigmine and tacrine were measured. The obtained data were within the very wide range of the IC₅₀-values given in the literature for these compounds.

摘要

乙酰胆碱酯酶 (AChE) 和丁酰胆碱酯酶 (BuChE) 被认为是治疗阿尔茨海默病和其他痴呆症的可行靶点。然而，由于临床上使用的 AChE 或双 AChE/BuChE 抑制剂只能提供一些对症治疗，随着时间的推移会失去治疗效果，因此在过去几年中，已经开始对多靶点化合物进行深入研究，以解决除 AChE 和 BuChE 之外的其他靶点，例如内源性大麻素降解酶。对于此类药物开发项目中测试化合物的 AChE 和 BuChE 抑制效力的评估，通常使用传统的 Ellman 方法。由于这种方法有一些分析缺陷，我们开发了替代的基于 HPLC/UV 的检测，使用 pyridin-2-ylmethyl acetate 作为 AChE 的底物和 BuChE 的苯甲酰胆碱。酶产物吡啶-2-基甲醇和苯甲酸分别通过反相HPLC测定，向流动相中加入离子对试剂辛烷-1-磺酸钠，以防极性吡啶衍生物。来自电鳗的AChE、来自马血清的人重组AChE和BuChE被用作酶。对于方法验证，IC_测量了已知的 AChE 和 BuChE 抑制剂毒扁豆碱、利凡斯的明和他克林的 50 值。获得的数据在这些化合物的文献中给出的IC ₅₀值的非常宽的范围内。