Long-Lasting Inhibitory Effects of Distigmine on Recombinant Human Acetylcholinesterase Activity

Abstract

To elucidate the mechanism whereby distigmine, an underactive bladder remedy, potentiates urinary bladder contractions long-lastingly, the inhibition of recombinant human acetylcholinesterase (rhAChE) by distigmine was investigated. A centrifugal ultrafiltration device, Nanosep ® 10K, was used to separate rhAChE and a bound inhibitor from an unbound inhibitor, reaction substrate, and reaction product. This allowed the same aliquot of rhAChE to be repeatedly assayed for up to 48 h to confirm the long-lasting binding of an inhibitor. Cholinesterase (ChE) inhibitors, distigmine, pyridostigmine, neostigmine, and ambenonium, were tested. The dissociation rate constant (k diss ) and dissociation half-life (t 1/2 ) of each inhibitor were determined based on the changes in rhAChE activity. Within 2-4 h after removing pyridostigmine, neostigmine, or ambenonium, the rhAChE activity was restored to the control levels. The k diss values for pyridostigmine, neostigmine, and ambenonium were calculated to be 0.51 0.05, 0.66 0.03, and 1.41 0.08 h 1 , and the t 1/2 values were calculated to be 1.36, 1.05, and 0.49 h, respectively. With distigmine, the rhAChE activity initially dropped to 17% of that in the control and then slowly recovered to only 50% by 48 h after drug removal. The k diss and t 1/2 values of distigmine were calculated to be 0.012 0.001 h 1 and 57.8 h, respectively. Based on the t 1/2 values, distigmine was judged to dissociate from acetylcholinesterase (AChE) 40-120-fold slower than the other ChE inhibitors did. This may explain the long-lasting potentiation of urinary bladder contractions and motility by distigmine as a treatment for an underactive bladder. Key words distigmine bromide; acetylcholinesterase; cholinesterase inhibitorDistigmine is a reversible cholinesterase (ChE) inhibitor from the class of carbamates. It was chemically synthesized by Schmid 1) and has a chemical structure consisting of two molecules of pyridostigmine connected by hexamethylene bonds (Fig. 1). Distigmine is used clinically in some Asian and European countries, including Japan and Germany, 2-8) and the main clinical indication for distigmine is myasthenia gravis.2,9) However, in Japan, distigmine has also been used for glaucoma 3) and an underactive bladder. 4-8)The effectiveness and usefulness of distigmine for underactive bladder treatment have been shown in many clinical studies. [4][5][6][7][8] However, the experimental evidence to support these clinical reports was not convincing. Therefore, our laboratory started pharmacological studies on the effects of distigmine on urinary bladder (UB) motility and has previously reported results supporting the clinical effectiveness and usefulness of distigmine. [10][11][12][13][14][15][16][17][18] The most remarkable pharmacological characteristic of distigmine was the persistence of its effect. In particular, distigmine was found to potentiate the UB internal pressure via the micturition reflex for more than 12 h. 17)Interestingly, the long-lasting potentiating effect of distigmine o...

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