Kikuchi_2010_Br.J.Pharmacol_159_1732

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-[(14)C]methylpiperidin-4-yl acetate ([(14)C]MP4A) and (R)-N- [(14)C]methylpiperidin-3-yl butyrate ([(14)C]MP3B_R), for AChE and butyrylcholinesterase (BChE) respectively. EXPERIMENTAL APPROACH: We investigated the kinetics of hydrolysis of [(14)C]-MP4A and [(14)C]-MP3B_R by cholinesterases, and evaluated the selectivity of [(14)C]MP4A and [(14)C]MP3B_R for human AChE and BChE, respectively, compared with traditional substrates. Then, IC(50) values of cholinesterase inhibitors in minimally diluted and highly diluted tissues were measured with [(14)C]MP4A and [(14)C]MP3B_R. KEY RESULTS: AChE and BChE activities were selectively measured as the first-order hydrolysis rates of [(14)C]-MP4A and [(14)C]MP3B_R respectively. The AChE selectivity of [(14)C]MP4A was an order of magnitude higher than traditional substrates used for the AChE assay. The IC(50) 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 IC(50) 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.