High performance liquid chromatographic assays with UV-detection for evaluation of inhibitors of acetylcholinesterase and butyrylcholinesterase

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.