Biol. Pharm. Bull. 40(7): 1092-1100 (2017)

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) contractility. 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 inhibitors (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 washout. 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 contraction; 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

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). UB strips were preloaded with a tension of 1 g and allowed Effects of ChE Inhibitors on ATP Concentration–Response 1094 Biol. Pharm. Bull. Vol. 40, No. 7 (2017)

Curves HBSS at 37±1.0°C, bubbled with a mixture of 95% O2 and 5% Basic experimental procedures followed those for the CO2, and allowed to equilibrate for 60 min. effects on ACh-induced contractions in previous section. After After 60-min incubation, the glass rod with the prepara- two successive contractions of approximately equal magnitude tion was immersed in a test tube filled with 3 mL of HBSS −4 −3 in response to ACh (10 M) were obtained, UBSM tissues containing acetylthiocholine (ATCh, 10 M) at 37±1.0°C, and −4 were contracted by applying 10 M ATP. This procedure was allowed to equilibrate for exactly 5 min. The glass rod with repeated until two successive contractions of approximately the preparation was then transferred to a bath containing equal magnitude had been obtained. After a 30-min equilibra- 20 mL HBSS and medium was replaced three times. Reac- −8 −4 tion period, ATP (10 –3×10 M) was cumulatively applied tion medium (2.4 mL; ATCh-containing HBSS) was then to the bath medium until a maximum response was obtained. transferred to another test tube and mixed immediately with −1 This procedure was repeated, and the second concentration– 0.5 mL phosphate buffered solution (10 M, pH=7.4) contain- −4 response curve for ATP was regarded as the control response. ing 10 M distigmine. Distigmine was added to exclude the −6 When ChE inhibitors (10 M for each) were tested, they were effects of ChE in any tissue fragments and plasma that might applied to the bath solution 30 min before the cumulative ap- have been present in the reaction medium. Next, 100 µL of −2 plication of ATP. DTNB (10 M) was added to this mixture and mixed well. Contraction was expressed as a percentage of the maximal After incubating the mixture for 1 min, absorbance at 412 nm −4 response produced by 10 M ATP applied as a single adminis- was measured using a UV-visible spectrophotometer (model tration before the cumulative application of ATP, and analyzed UV-150-02; Shimadzu, Kyoto, Japan). according to the procedures described in previous section. The glass rod with the preparation was subsequently al- −6 Potentiating Effects of ChE Inhibitors on ACh (3×10 M)- lowed to equilibrate for 30 min in 20 mL HBSS and the proce- induced Contraction and Sustainability after Washout dures were repeated twice. The third absorbance measurement UBSM preparations were preloaded with a tension of was used as the control. 0.5 g and allowed to equilibrate for 60 min, exchanging the After the third measurement, the glass rod with the prepa- bath medium every 20 min. After equilibration for 60 min, ration was incubated for 30 min with the test ChE inhibitor −6 the strips were contracted with 80 mM KCl Locke–Ringer (10 M) or potential candidate degradation products of dis- −3 solution. After washout, the strips were contracted with ACh tigmine (10 M) (Fig. 7). After this incubation period, the −4 (3×10 M) and allowed to equilibrate for a further 20 min. procedures were repeated and absorbance was measured. The This procedure was repeated until two successive contractions glass rod with the preparation was next subjected to washout −4 of approximately equal magnitude to 3×10 M ACh had been 10 times in an organ bath (20 mL) to remove residual drug. −6 obtained. After a 30-min equilibration period, ACh (3×10 M) The time of first washout was defined as 0 h, and ChE was applied to the bath medium, and this procedure was re- activity was measured every 30 min for 3 h and then every peated until three successive contractions of approximately 60 min from 3–12 h. The absorbance of an experiment without equal magnitude had been obtained. Thereafter, ChE inhibi- the addition of any drug was regarded as the blank (Ablank), −6 tors (10 M) were applied to the bath medium and allowed to and the ChE activity (%) and ChE inhibition rate (%) were equilibrate for 30 min. This equilibration period of 30 min calculated from the following equations: with distigmine was confirmed to produce an approximately AAt − blank maximum potentiating effect on the contraction induced by ChE activity (%)=×100 −6 AAcontrol− blank ACh (3×10 M) (Supplementary Fig. 1). = − After this incubation period, the contractile response ChE inhibition rate (IR) (%) 100 ChE activity −6 to 3×10 M ACh was recorded, and the bath medium was where At is absorbance at t-h after washout, Acontrol is absor- replaced with fresh medium 10 times. Next, contractile re- bance for control, and Ablank is absorbance for blank. −6 sponses to ACh (3×10 M) were recorded every 30 min for Dissociation rate constants (k) were calculated as described 12 h after the first washout procedure, which was regarded as by Perola et al.16) and Bartolini et al.,17) with some modifica- 0 h. All tension changes were recorded isometrically. Contrac- tions as described below. −6 tions in responses to 3×10 M ACh in the absence or presence Briefly, any ChE inhibitor (AB) first binds to its cor- of ChE inhibitors were expressed as a percentage of the maxi- responding enzyme (EH: ChE) to form an enzyme–drug −4 mum contraction to 3×10 M ACh which was applied before complex (EA) through its precursor intermediate (EH---AB) application of ChE inhibitors. (reaction 1). Thereafter, the ChE inhibitor bound to its enzyme Assessment of the Effects of ChE Inhibitors on Isolated (ChE) begins to dissociate from the EA by decarbamoylation Guinea Pig UBSM Tissue ChE Activities ChE activities with water (reaction 2). of the UBSM preparations were measured according to the method reported by Elman et al.,15) with minor modifications. EH ++AB EH---AB → EA HB (1) Briefly, UB was isolated from male Hartley guinea pigs and k placed in modified HBSS of the following composition in mM: EA++ H2 O →EH AOH (2)

NaCl, 136.9; KCl, 5.37; CaCl2, 1.26; MgCl2, 0.81; Na2HPO4, 0.77; KH2PO4, 0.44; NaHCO3, 4.17; glucose, 5.55. After re- If this dissociation reaction formula (reaction 2) proceeds moving the surrounding adipose and connective tissues and mathematically, the following Eq. 3 is derived: bladder trigone but not the urinary epithelium, rectangle- [EA] shaped strips (about 8 mm in width×15 mm in length) were ln t = −kt (3) prepared. The preparation was attached to a glass rod with [EA]0 a mandolin line and immersed in a bath containing 20 mL of where [EA]t is the concentration of EA at t-h after washout, Vol. 40, No. 7 (2017) Biol. Pharm. Bull. 1095

Fig. 2. Potentiating Effects of Distigmine (A), Neostigmine (B), Pyridostigmine (C), and Ambenonium (D) on Concentration–Response Curves for Acetylcholine (ACh)-Induced Contraction of Guinea Pig Urinary Bladder Smooth Muscle −7 −5 −8 −6 −7 −5 ACh was applied cumulatively to the bath solution in the absence or presence of distigmine (10 –10 M; A), neostigmine (10 –10 M; B), pyridostigmine (10 –10 M; −7 −5 C), or ambenonium (10 –10 M; D); or 1 h after washout (w/o) of each drug. Contraction is expressed as a percentage of the contraction induced by a single administra- −4 −6 tion of 10 M ACh before the experiment (100% contraction). Data are presented as means±S.E.M. Numbers of experiments are n=6 for pyridostigmine (10 M) and am- −5 −7 −5 −7 benonium (10 M); n=5 for pyridostigmine (10 and 10 M) and ambenonium (10 M); and n=4 for the others. Dis: distigmine; Neo: neostigmine; Pyr: pyridostigmine; Amb: ambenonium.

[EA]0 is the concentration of EA at the start of the dissocia- to the ratio of ChE inhibition rate (ChE IR), and the following tion reaction, and k is the dissociation rate constant. Here, the Eq. 4 is derived: ratio of EA concentrations ([EA]t/[EA]0) can be approximated 1096 Biol. Pharm. Bull. Vol. 40, No. 7 (2017)

Fig. 3. Effects of Distigmine (A), Neostigmine (B), Pyridostigmine (C), and Ambenonium (D) on Concentration–Response Curves for ATP-Induced Contraction of Guinea Pig Urinary Bladder Smooth Muscle −6 ATP was applied cumulatively to the bath solution in the absence or presence of each inhibitor (10 M). Experiments with neostigmine were carried out in the presence −6 −4 of atropine (10 M). Contraction is expressed as the percentage of the contraction induced by a single administration of 10 M ATP before the experiment (100% contraction). Data are presented as means±S.E.M. (n=4). Dis: distigmine; Neo: neostigmine; Pyr: pyridostigmine; Amb: ambenonium.

Table 1. Effects of ChE Inhibitors on pD2 Values (Shown in Fig. 2)

ChE inhibitor −log M Control Inhibitor (+) 1 h after washout

Distigmine 7 4.79±0.35 5.57±0.40 5.48±0.10 6 4.74±0.07 6.28±0.10** 6.13±0.07** 5 4.52±0.45 6.31±0.18* 6.27±0.19* Neostigmine 8 4.58±0.54 5.75±0.17 5.07±0.18 7 4.98±0.12 6.50±0.11** 5.20±0.09## 6 5.03±0.32 6.96±0.09** 5.31±0.35# Pyridostigmine 7 4.78±0.26 5.07±0.24* 5.05±0.18 6 4.70±0.17 6.28±0.11** 5.14±0.16*## 5 4.59±0.17 6.75±0.07** 5.51±0.19**# Ambenonium 7 4.45±0.38 6.08±0.09* 4.40±0.65 6 5.04±0.16 6.19±0.07** 5.74±0.16** 5 4.40±0.31 5.44±0.17 6.43±0.54

−6 −5 −7 −5 Data are presented as means±S.E.M. Numbers of experiments are n=6 for pyridostigmine (10 M) and ambenonium (10 M); n=5 for pyridostigmine (10 and 10 M) and −7 # ## ambenonium (10 M); and n=4 for the others. * p<0.05, ** p<0.01 vs. control. p<0.05, p<0.01 vs. each inhibitor.

preparations. The significance of differences between mean ChE IR ln t = −kt (4) values was evaluated by Student’s paired t-test or one-way ChE IR 0 ANOVA followed by Dunnett’s multiple comparison test or where ChE IRt is ChE IR at t-h after washout and ChE IR0 is Tukey’s multiple comparison test using GraphPad Prism™. p ChE IR at the start of the dissociation reaction. values of less than 0.05 were considered statistically signifi- In accordance with Eq. 4, a linear regression line was ob- cant. tained from the ratio of ChE IR, and the dissociation rate constant (k) was determined from the slope of the regression line, RESULTS obtained using GraphPad Prism™. Statistical Analysis All values in the text and illustra- Effects of Repeated ACh and ATP Application on Iso- tions are presented as means±standard error of the mean lated Guinea Pig UBSM We first determined whether −8 −4 (S.E.M.) of the data obtained from different numbers (n) of repeated cumulative application of ACh (10 –3×10 M) or Vol. 40, No. 7 (2017) Biol. Pharm. Bull. 1097

−6 Fig. 4. Representative Traces of the Enhancing Effects of Distigmine (A), Neostigmine (B), Pyridostigmine (C), and Ambenonium (D) on 3×10 M ACh-Induced Contraction in Guinea Pig Urinary Bladder Smooth Muscle Up to 12 h after Washout −6 Qualitatively, the same results were obtained from four experiments for each inhibitor. Arrow: administration of 3×10 M ACh; w: washout; Dis: distigmine; Neo: neostigmine; Pyr: pyridostigmine; Amb: ambenonium.

−8 −4 −8 −6 −7 −5 ATP (10 –3×10 M) generated reproducible concentration– (10 –10 M), pyridostigmine (10 –10 M), and ambenonium −7 −5 response curves in isolated guinea pig UBSM preparations. (10 –10 M) on UBSM tissue contractions in response to −8 −4 −7 −5 pD2 values for ACh and Emax values of the contractions ACh (10 –3×10 M). Distigmine (10 –10 M) potentiated were as follows, and were not signiﬁcantly different. pD2 ACh-induced contractions in a concentration-dependent man- values were 4.46±0.38 (n=4, 1st response), 4.47±0.42 (n=4, ner, which was clearly demonstrated by the leftward shift of

2nd response), and 4.49±0.27 (n=4, 3rd response). Emax values the concentration–response curves for ACh in the presence of −4 −7 −5 for ACh (3×10 M)-induced contractions were 120.2±8.9% distigmine (Fig. 2A, Table 1). All concentrations (10 –10 M)

(n=4, 1st response), 121.6±11.6% (n=4, 2nd response), and of distigmine increased the pD2 value of ACh, and statistical 134.7±9.7% (n=4, 3rd response). ATP-induced contractions significances were particularly apparent at concentrations of −6 −5 were also shown to be reproducible for at least two repetitions, 10 –10 M (Table 1). Interestingly, the potentiating effects of judging from the insignificant differences between the val- distigmine were preserved even 1 h after washout. −8 −6 ues: pD2 values for ATP were 5.06±0.44 (n=4, 1st response) Neostigmine (10 –10 M) also potentiated ACh-induced and 4.84±0.35 (n=4, 2nd response); and Emax values for ATP contractions (Fig. 2B), and a significant increase in pD2 was −4 −7 −6 (3×10 M)-induced contractions were 105.3±10.6% (n=4, 1st obtained at concentrations of 10 –10 M (Table 1). However, response) and 104.1±10.3% (n=4, 2nd response). 1 h after washout, the potentiating effects of neostigmine dis-

Effects of ChE Inhibitors on UBSM Tissue Contrac- appeared, as shown by the signiﬁcant decrease in pD2 values tions in Response to ACh and ATP Figure 2 shows for ACh (Table 1). −7 −5 −7 −5 the effects of distigmine (10 –10 M), neostigmine Similarly, pyridostigmine (10 –10 M) shifted the concen- 1098 Biol. Pharm. Bull. Vol. 40, No. 7 (2017)

−6 Fig. 5. Summarized Data for the Effects of Distigmine (A), Neostigmine (B), Pyridostigmine (C), and Ambenonium (D) on 3×10 M ACh-Induced Contraction in Guinea Pig Urinary Bladder Smooth Muscle Up to 12 h after Washout (w/o) of Each Inhibitor, Following the Protocol Shown in Fig. 4 −4 Contraction is expressed as the percentage of the contraction induced by a single administration of 3×10 M ACh before the experiment (100% contraction). Data are presented as means±S.E.M. (n=4). * p<0.05, ** p<0.01 vs. before treatment with each inhibitor. tration–response curves for ACh to the left (Fig. 2C), an ob- servation clearly supported by the significant increase in pD2 values for ACh (Table 1). Washout of pyridostigmine strongly decreased its potentiating effect, although the effect was still significant after 1 h (Fig. 2C, Table 1). −7 −5 Ambenonium (10 –10 M) also potentiated ACh-induced contractions (Fig. 2D) although increases in pD2 were signifi- −7 −6 cant at 10 –10 M in limited numbers of experimental trials (Table 1). One hour after washout, the potentiating effect of −7 ambenonium at 10 M disappeared. However, it remained sig- −6 −5 nificant at 1 h for a concentration of 10 –10 M. −6 No ChE inhibitor (10 M) had a significant effect on ATP −8 −4 (10 –3×10 M)-induced contractions (Fig. 3), despite exerting Fig. 6. Inhibiting Effects of Distigmine, Neostigmine, Pyridostigmine, a significant potentiating effect on ACh-induced contractions and Ambenonium on ChE Activity in Guinea Pig Urinary Bladder at these concentrations. Smooth Muscle Up to 12 h after Washout (w/o) of Each Drug Potentiating Effects of ChE Inhibitors on UBSM Tissue A: ChE activity in guinea pig urinary bladder smooth muscle up to 12 h after −6 Contractions in Response to 3×10 M ACh, and the Preser- washout of each inhibitor. ChE activity is expressed as the percentage of the activity prior to treatment with each inhibitor (100% activity). B: Plots for analysis of vation of These Effects after Washout Figure 4 represents the dissociation rate constant (k). Data are presented as means±S.E.M. (n=4). w/o: typical recordings of the effects of ChE inhibitors on UBSM washout of each inhibitor. −6 tissue contractions in response to 3×10 M ACh and the preservation of this effect up to 12 h after washout. All ChE inhib- pyridostigmine, and 0.61±0.04 h−1 for ambenonium, as indi- itors potentiated ACh-induced contractions although, with the cated by the slopes of the plots shown in Fig. 6B. exception of distigmine, these effects disappeared over time However, inhibition of ChE activity by distigmine was pre- within 2–3 h after washout (Figs. 4B–D, 5B–D). However, the served until 12 h after washout (Fig. 6A), and thus, the k value potentiating effect of distigmine remained significantly pre- for distigmine could not be calculated (Fig. 6B). served until 12 h after washout (Figs. 4A, 5A). Effects of Distigmine Degradation Product Candidates Inhibitory Effects of ChE Inhibitors on UB ChE Activ- on UB ChE Activity Compounds A–C in Fig. 7 are can- ity and the Preservation of These Effects after Washout didates for degradation products of distigmine, as estimated Figure 6 shows the effects of ChE inhibitors on UB ChE using the general rules for binding and dissociation reactions activity and their preservation of these effects up to 12 h after between ChE and carbamate ChE inhibitors. None of the com- washout. ChE activity was restored in a time-dependent man- pounds significantly inhibited ChE at 12 h after washout, even −3 ner after washout of all ChE inhibitors except distigmine (i.e., at a concentration of 10 M (1000 times more concentrated −6 neostigmine, pyridostigmine, and ambenonium) (Fig. 6A). The than distigmine 10 M) (Fig. 7). k values were 0.40±0.02 h−1 for neostigmine, 0.50±0.04 h−1 for Vol. 40, No. 7 (2017) Biol. Pharm. Bull. 1099

Fig. 7. Effects of Distigmine Degradation Products (Compounds A–C) on ChE Activity in Guinea Pig Urinary Bladder Smooth Muscle Up to 12 h after Washout ChE activity is expressed as the percentage of activity before the administration of inhibitor (100% activity). Data are presented as means±S.E.M. (n=3).

DISCUSSION almost the same rate. In contrast, the k value could not be calculated for distigmine within the timeframe of this study The objective of this study was to clarify the mechanism by (12 h) (Fig. 6B), as AChE activity had not sufficiently recov- which distigmine produces long-lasting characteristic potentia- ered at 12 h after distigmine washout (Fig. 6A). This finding tion of UBSM contraction. In this study, in order to determine strongly supports the hypothesis that the dissociation of dis- whether the potentiating effects of distigmine on UBSM tigmine from AChE occurs slowly after the stable complex contraction require the continued presence of distigmine near of distigmine and AChE has formed. However, the possibility UBSM tissue, we evaluated the duration of contraction en- that distigmine molecules accumulate in the plasma membrane hancement of isolated guinea pig UBSM by distigmine and adjacent to UBSM, and are thus gradually exposed to AChE, inhibition of ChE activity after washout of distigmine from cannot be overlooked. To rule out this possibility and to sub- the tissue bath solution. In addition, the effects of distigmine stantiate the hypothesis that distigmine itself strongly binds were compared with those of other ChE inhibitors. We first to AChE to generate a stable complex, further studies using showed that the potentiating effect of distigmine on UBSM AChE isolated from UBSM cells are needed. We are currently contraction was exerted against ACh- but not against ATP conducting experiments to demonstrate that distigmine resists (a co-neurotransmitter of the parasympathetic nerve)-induced dissociation from AChE using recombinant human AChE contraction18) (Fig. 3). Next, we determined that the potentiat- purified from cells, and to calculate the k value of distigmine ing effects of distigmine on ACh-induced contraction and its from this AChE. inhibitory effects on ChE activity persisted for 12 h, even after The sustained potentiation by distigmine of UBSM contrac- its removal from the bath solution (Figs. 4–6). In contrast, tility and its inhibitory effects on AChE were shown in vitro both effects were strongly attenuated for other ChE inhibitors using isolated UBSM tissues. These findings suggest that the after washout. These in vitro results are consistent with the re- inhibitory effect on AChE is produced by distigmine itself. sults of our previous in vivo study, which showed that the po- However, there is a possibility that these inhibitory effects tentiating effect of distigmine on UB motility persisted for at are instead mediated via decomposed products of distigmine. least 6 h after the elimination of distigmine from the blood.14) Therefore, we further explored this possibility. Based on these findings, the sustained potentiation of UBSM Distigmine has a chemical structure of two pyridostigmine contractile activity by distigmine appears independent of the molecules bonded via a hexamethylene group and thus has continued presence of distigmine in the UBSM tissue. two carbamoyl groups available to bind to AChE (Fig. 1). One possible explanation for the sustained potentiation by Furthermore, some ChE inhibitors without carbamoyl groups distigmine of ACh-induced UBSM contractility irrespective are reported to exert their ChE inhibitory effects by acting on of the presence of distigmine is that distigmine strongly binds sites other than the ChE active site serine residue.19) There- to UBSM AChE to form a stable complex, and thus inhibits fore, it is plausible that the degradation products of distigmine the decomposition of ACh over a prolonged time. Therefore, released from AChE bind again to AChE and thus inhibit the k value of the distigmine and AChE complex was calcu- its activity over a long duration. However, none of the three lated to determine the rate at which distigmine dissociates types of candidate degradation products that are presumed to from AChE. As shown in Fig. 6B, the k values for ChE in- be formed from distigmine showed inhibitory activity against hibitors other than distigmine were approximately 0.5 h−1, and AChE (Fig. 7). Therefore, the sustained inhibition of AChE thus they were estimated to dissociate from UBSM AChE at by distigmine was unlikely caused by its degradation products 1100 Biol. Pharm. Bull. Vol. 40, No. 7 (2017) but was likely produced by distigmine itself. Ubretid in the handling of glaucoma, myasthenia gravis and esotro- Among the three types of candidate degradation products of pia. Ophthalmologica, 159, 339–362 (1969). distigmine evaluated in this study, compound C (Fig. 7) con- 4) Inada T, Ebisuta K, Harada T. Use of “Ubretid” in the field of urol- tains one carbamoyl group that can bind to AChE, although ogy. Hinyokika Kiyo, 13, 429–432 (1967). 5) Niijima T, Asano M. Clinical appraisal of Ubretid: a new cholines- the second carbamoyl group of distigmine is absent. However, terase inhibitor. Hinyokika Kiyo, 13, 423–428 (1967). although compound C was predicted to show AChE inhibitory 6) Philp NH, Thomas DG. The effect of distigmine bromide on void- activity, no inhibition was observed (Fig. 7). Interestingly, a ing in male paraplegic patients with reflex micturition. Br. J. Urol., compound with a structure of two neostigmine (Fig. 1) mol- 52, 492–496 (1980). ecules linked with a long-chain methylene group was reported 7) Izumi K, Maolake A, Maeda Y, Shigehara K, Namiki M. Effects to exert sustained anti-myasthenia effects after oral adminis- of bethanechol chloride and distigmine bromide on postvoiding tration.1) Similarly, the introduction of a long-chain methylene residual volume in patients with underactive bladder. Minerva Urol. group or cyclic structure into physostigmine, a carbamate- Nefrol., 66, 241–247 (2014). type ChE inhibitor with a cyclic structure, increased its as- 8) Bougas DA, Mitsogiannis IC, Mitropoulos DN, Kollaitis GC, Se- sociation with AChE.16) Based on these findings, the presence rafetinides EN, Giannopoulos AM. Clinical efficacy of distigmine bromide in the treatment of patients with underactive detrusor. Int. of two pyridostigmine (aromatic) structures appears to be Urol. Nephrol., 36, 507–512 (2004). required for distigmine’s sustained AChE inhibitory action. 9) Horinouchi T, Aoki T, Akiyama R, Ono T, Shibano M, Tanaka Although the molecular basis of these mechanisms requires Y, Koike K. Effects of distigmine, a long-acting cholinesterase further investigation, the π–π interaction presumed to arise inhibitor, on urinary bladder detrusor and urethra smooth muscles from the aromatic ring structures bonded to both ends of the of guinea-pig: pharmacological analysis in vitro and in vivo. Ōyō pyridostigmine molecule might contribute to the stable bind- Yakuri/Pharmacometrics, 64, 45–52 (2003). ing of distigmine to AChE. 10) Sekiya S, Takahashi H, Seki Y, Teraoka A, Aikawa N, Tanaka Y, In summary, the potentiating effect of distigmine on UBSM Koike K. Comparison of the effects of distigmine and neostigmine contraction was shown to persist for 12 h after washout and on guinea-pig urinary bladder contractile functions assessed by in was unique to distigmine compared with other ChE inhibi- vivo and in vitro studies. Ōyō Yakuri/Pharmacometrics, 75, 85–96 (2008). tors. We also showed that the sustained potentiation by dis- 11) Herzfeld E, Kraupp O, Pateisky K, Stumpf C. Pharmacological and tigmine of the ACh-induced contractile response of UBSM is clinical effects of cholinesterase antagonist hexamethylene-bis(N- likely caused by distigmine itself rather than its degradation methylcarbaminoyl-1-methyl-3-oxypyridiniumbromide); (BC 51). products. One plausible explanation for the sustained effects Wien. Klin. Wochenschr., 69, 245–248 (1957). of distigmine is that it forms a strong and stable complex 12) Ito Y, Harada T, Fushimi K, Kagawa Y, Oka H, Nakazawa H, with AChE, which in turn produces sustainable inhibition of Homma R, Kato Y, Yamada S. Pharmacokinetic and pharmaco- UBSM AChE activity. dynamic analysis of acetylcholinesterase inhibition by distigmine bromide in rats. Drug Metab. Pharmacokinet., 25, 254–261 (2010). Acknowledgments Distigmine bromide and distigmine 13) Breuel HP, Bohn-Olszewsky W, Engelsen SJ, Samhaber EM, degradation products were a gift from Torii Pharmaceutical Niklaus H. Inhibition of acetylcholinesterase by distigmine bromide Co., Ltd. (Tokyo, Japan). The authors would like to thank Ms. (Ubretid). Int. J. Clin. Pharmacol. Ther. Toxicol., 31, 230–235 (1993). Eriko Kaneki and Ms. Aya Katayose for their expert technical 14) Obara K, Chino D, Tanaka Y. Distigmine bromide produces sus- assistance. tained potentiation of guinea-pig urinary bladder motility by inhibiting cholinesterase activity. Biol. Pharm. Bull., 40, 807–814 (2017). Conflict of Interest The authors declare no conflict of 15) Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM. A new interest. and rapid colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol., 7, 88–95 (1961). Supplementary Materials The online version of this ar- 16) Perola E, Cellai L, Lamba D, Filocamo L, Brufani M. 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