Use of a novel radiometric method to assess the inhibitory effect of donepezil on acetylcholinesterase activity in minimally diluted tissue samples

Abstract

Background and purpose: Cholinesterase inhibitors have been widely used for the treatment of patients with dementia. Monitoring of the cholinesterase activity in the blood is used as an indicator of the effect of the cholinesterase inhibitors in the brain. The selective measurement of cholinesterase with low tissue dilution is preferred for accurate monitoring; however, the methods have not been established. Here, we investigated the effect of tissue dilution on the action of cholinesterase inhibitors using a novel radiometric method with selective substrates, N‐[14C]methylpiperidin‐4‐yl acetate ([14C]MP4A) and (R)‐N‐[14C]methylpiperidin‐3‐yl butyrate ([14C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively.Experimental approach: We investigated the kinetics of hydrolysis of [14C]‐MP4A and [14C]‐MP3B_R by cholinesterases, and evaluated the selectivity of [14C]MP4A and [14C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC50 values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [14C]MP4A and [14C]MP3B_R.Key results: AChE and BChE activities were selectively measured as the first‐order hydrolysis rates of [14C]‐MP4A and [14C]MP3B_R respectively. The AChE selectivity of [14C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC50 values of specific AChE and BChE inhibitors, donepezil and ethopropazine, in 1.2‐fold diluted human whole blood were much higher than those in 120‐fold diluted blood. In addition, the IC50 values of donepezil in monkey brain were dramatically decreased as the tissue was diluted.Conclusions and implications: This method would effectively monitor the activity of cholinesterase inhibitors used for therapeutics, pesticides and chemical warfare agents.

Analysis of Acetylcholinesterase Activity in Cell Membrane Microarrays of Brain Areas as a Screening Tool to Identify Tissue Specific Inhibitors

“…In summary, we propose a novel platform based on CMMs obtained from a tissue collection to estimate membrane-associated AChE activity and to screen compounds that act through membrane-bound cholinesterases, in a single miniaturized assay. Several attempts have been performed to quantify AChE activity and to screen AChE ligands in serum samples, membrane homogenates or AChE-enriched solutions with high sensibility and reproducibility [4][5][6][7][8][9][10][11][12][13][14][15][16][17]. However, these methods are optimized to soluble AChE and require the addition of detergent to study the membrane-bound AChE, which can alter the lipid environment in addition to enzyme structure and function.…”

“…Several approaches have been developed to study AChE activity and to test the inhibitory capacity of AChE inhibitors including colorimetric [4,5], spectrophotometric [6,7], fluorometric [8][9][10][11], radiometric [12,13], or electrochemical techniques [14][15][16][17]. Among them, the most commonly used technologies are based on the photometric Ellman's method, which consists of measuring the increase in yellow color produced from thiocholine, generated from the hydrolysis of the acetylthiocholine by cholinesterases when it reacts with dithiobisnitrobenzoate ion [6].…”

Donepezil suppresses intracellular Ca2+ mobilization through the PI3K pathway in rodent microglia

“…Donepezil was diluted with the standard external solution to obtain the final concentration (5 μM). This donepezil concentration is sufficient to inhibit the AChE activity in both human blood cells and monkey brain samples [ 23 ] or to suppress the LPS-induced NO production in mouse primary microglial cells [ 21 ]. Drugs that were insoluble in water were first dissolved in dimethylsulfoxide (DMSO; Wako Pure Chemical Industries, Osaka, Japan) and then diluted in the standard external solution.…”

The progress in the cholinesterase quantification methods

“…These substrates were originally developed as in vivo probes of cerebral AChE and BChE by positron emission tomography. This method would effectively monitor the inhibitory properties of cholinesterase inhibitors used for therapeutics, pesticides, and chemical warfare agents [118].…”