Biol. Pharm. Bull. 40(7): 1092-1100 (2017)
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1092 Biol. Pharm. Bull. 40, 1092–1100 (2017) Vol. 40, No. 7 Regular Article The Long-Lasting Enhancing Effect of Distigmine on Acetylcholine- Induced Contraction of Guinea Pig Detrusor Smooth Muscle Correlates with Its Anticholinesterase Activity Keisuke Obara, Tsukasa Ogawa, Daisuke Chino, and Yoshio Tanaka* Department of Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Toho University; 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan. Received February 27, 2017; accepted April 11, 2017 Distigmine bromide (distigmine), a reversible, long-lasting cholinesterase (ChE) inhibitor, is used for the treatment of underactive bladder in Japan and has been shown to potentiate urinary bladder (UB) con- tractility. We studied the duration of distigmine’s potentiating effects on acetylcholine (ACh)-induced UB contraction and its inhibitory effects on ChE activity, and compared that with those of other ChE inhibi- tors (neostigmine, pyridostigmine, and ambenonium). The duration of potentiating/inhibitory effects of ChE inhibitors, including distigmine, on ACh-induced guinea pig UB contraction/ChE activity was evaluated for 12 h following washout. Dissociation rate constants (k) of the inhibitors were also tentatively calculated based −6 on the time courses of their ChE inhibitory effects. The potentiating effect of distigmine (10 M) on ACh- induced UB contraction and its inhibitory effect on ChE activity were significantly sustained 12 h after wash- out. The potentiating effect of other ChE inhibitors on ACh-induced UB contraction, however, was sustained only until 3 h after washout. The ChE inhibitory effects of these inhibitors dissipated in a time-dependent manner after washout, with more than 75% of ChE activity restored by 4 h after washout. The k values of ChE inhibitors approached 0.50 h−1, except for distigmine, where k could not be determined. Compared with that of other ChE inhibitors, the potentiating effect of distigmine on UB contractile function was significant- ly more sustainable following washout, which was likely associated with its corresponding long-lasting ChE inhibitory effect. Distigmine may associate more strongly with UB ChE than other ChE inhibitors, which would partly explain its sustained effects. Key words distigmine; guinea pig urinary bladder; cholinesterase activity; acetylcholine-induced contrac- tion; dissociation rate constant; urinary underactivity Distigmine bromide (distigmine) has a chemical structure distigmine exhibited symptomatic relief for 36 h,1) while the consisting of two molecules of pyridostigmine connected by half-time for AChE inhibition recovery in red blood cells hexamethylene bonds and is a reversible carbamate cholines- after distigmine administration was estimated to be approxi- terase (ChE) inhibitor (Fig. 1). The most important clinical mately 40 h.13) However, the duration of distigmine’s effect on indication of distigmine since its development in the 1950 s is UB motility remains unclear. We have recently attempted to myasthenia gravis,1,2) and its clinical effect is thought to be at- clarify this question in relation to the blood concentration of tributed to the increased acetylcholine (ACh) concentrations in distigmine in guinea pigs and determined that an intravenous neuromuscular junctions. In Japan, distigmine has also been bolus injection of distigmine maintained an increased maxi- used in the treatment of glaucoma3) and detrusor underactiv- mal intravesical pressure for 12 h after injection and that this ity associated with spinal cord injury or chronic diseases, effect persisted even after the elimination of distigmine from including diabetes.4–8) However, distigmine has not been the blood.14) intensively examined for its pharmacological effects or un- This study was carried out to investigate whether distig- derlying mechanisms in urinary bladder (UB) smooth muscle mine exerts sustained pharmacological actions in UB tissues, (UBSM) preparations. Our previous pharmacological studies despite its removal. We therefore investigated the duration of on distigmine using UBSM preparations indicated that this distigmine’s potentiating effects on ACh-induced contraction ChE inhibitor strongly potentiates ACh-induced contraction of and its inhibitory effects on ChE activity in guinea pig UB guinea pig UBSM in vitro.9) Furthermore, distigmine produces tissue following washout, compared with those of other carba- a sustained increase in maximal intravesical pressure for at mate ChE inhibitors. least 4 h after intravenous injection in guinea pigs,10) indicat- ing its potential long-lasting effects. MATERIALS AND METHODS To date, the sustainable characteristics of distigmine have been studied in both animals and humans. In d-tubocurarine- Animals Male Hartley guinea pigs (4–13 weeks old; treated rats, the anticurare action of intraperitoneal distigmine weight 305–580 g, Sankyo Labo Service Corporation, Tokyo, was detectable for up to 24 h after injection.11) The acetylcho- Japan) were housed under controlled conditions (21–22°C, linesterase (AChE) activity in red blood cells, colon, UB, and relative air humidity 50±5%) and fixed 12 h light–dark cycle submaxillary gland of rats remained suppressed 12 h after (08:00–20:00) with food and water available ad libitum. This oral administration of distigmine.12) In clinical use, a myas- study was approved by the Toho University Animal Care and thenia gravis patient who received an oral administration of User Committee (approval number: 15-51-294, accredited on * To whom correspondence should be addressed. e-mail: [email protected] © 2017 The Pharmaceutical Society of Japan Vol. 40, No. 7 (2017) Biol. Pharm. Bull. 1093 Fig. 1. Structures of Reversible Carbamate Cholinesterase (ChE) Inhibitors (Distigmine, Neostigmine, Pyridostigmine, and Ambenonium) May 22, 2015; approval number: 16-52-294, accredited on May to equilibrate for 20 min. After equilibration, the strips were 16, 2016) and conducted in accordance with the User’s Guide- contracted with 80 mM KCl Locke–Ringer solution of the fol- line to the Laboratory Animal Center of Faculty of Pharma- lowing composition in mM: NaCl, 79.6; KCl, 80; CaCl2, 2.2; ceutical Sciences, Toho University. MgCl2, 2.1; NaHCO3, 5.9; and glucose, 2.8. After washout, the −4 Drugs and Materials The following drugs were used: strips were contracted with ACh (10 M) and allowed to equil- ACh chloride (Daiichi-Sankyo Co., Ltd., Tokyo, Japan); dis- ibrate for a further 20 min. This procedure was repeated until tigmine bromide, 3-hydroxy-1-methylpyridin-1-ium bromide two successive contractions of approximately equal magnitude (compound A), N1,N6-dimethylhexane-1,6-diaminium chloride had been obtained. After a 30-min equilibration period, ACh −8 −4 (compound B), 1-methyl-3-((methyl(6-(methylammonio) hexyl)- (10 –3×10 M) was cumulatively applied to the bath medium carbamoyl) oxy) pyridine-1-ium bromide (compound C) (Torii until a maximum response was obtained. This procedure was Pharmaceutical Co., Ltd., Tokyo, Japan); ATP disodium salt repeated, and the second concentration–response curve for hydrate, neostigmine bromide, atropine sulfate hydrate (Sig- ACh was regarded as the control response. When ChE inhibi- ma-Aldrich, St. Louis, MO, U.S.A.); acetylthiocholine (ATCh) tors (distigmine, neostigmine, pyridostigmine, and ambenoni- iodide, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Tokyo um) were tested, they were applied to the bath solution 30 min Chemical Industry Co., Ltd., Tokyo, Japan); pyridostigmine before the cumulative application of ACh. After obtaining a bromide (MP Biomedicals, Santa Ana, CA, U.S.A.); and am- concentration–response curve for ACh in the presence of the benonium dichloride (Tocris Bioscience, Bristol, U.K.). tested ChE inhibitor, bath medium was replaced with fresh −2 ATP was dissolved in 2.5×10 M Tris–HCl buffer (pH=8.0) solution 10 times, and allowed to equilibrate for 60 min. Fol- −2 to give a stock solution of 10 M and diluted further with lowing this equilibration period, ACh was again cumulatively −2 2.5×10 M Tris–HCl buffer (pH=7.4) to the desired concen- applied to construct its concentration–response curve in the tration. ATCh was dissolved in Hank’s Balanced Salt Solu- absence of ChE inhibitor to determine the washout effect. All −3 tion (HBSS) to give a stock solution of 10 M. DTNB was tension changes in the study were recorded isotonically. −1 dissolved in 10 M phosphate buffer (pH 7.4) to give a stock Concentration–response curves for ACh were constructed, −2 solution of 10 M. All other drugs were prepared as aqueous expressing the contraction as a percentage of the maximum −4 solutions and diluted with distilled water. contractile response to 10 M ACh, which was administered Assessment of the Effects of ChE Inhibitors on Isolated as a single application before the cumulative application of Guinea Pig UBSM Tissue Contraction ACh. Data were plotted as a function of drug concentration Preparation of Guinea Pig UBSM Tissues and fitted to the following equation: Guinea pigs were euthanized by cervical dislocation and nnHHn H UBs were immediately removed and placed in Locke–Ringer EE=×max A/C(E 50 + A ) solution of the following composition in mM: NaCl, 154; KCl, 5.6; CaCl2, 2.2; MgCl2, 2.1; NaHCO3, 5.9; and glucose, 2.8. where E is % response at a given drug concentration, Emax After removing surrounding adipose and connective tissues is the maximum response, A is the concentration of drug, and bladder trigone, the bladder was opened with a longitudi- nH is the slope function, and EC50 is the effective drug con- nal incision. UB strips (approximately 2 mm in width×15 mm centration that produces a 50% response. Curve fitting was in length) were prepared, suspended in a 20 mL organ bath performed using GraphPad Prism™ (version 6.04) (GraphPad filled with Locke–Ringer solution at 32±1.0°C, and bubbled Software, Inc., San Diego, CA, U.S.A.). The contractile po- with a mixture of 95% O2 and 5% CO2. tency of ACh was expressed as pD2 value (negative logarithm Effects of ChE Inhibitors on ACh Concentration–Response of the effective drug concentration that produced 50% of the Curves maximum response).