2017 Volume 40 Issue 7 Pages 1092-1100
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 on the time courses of their ChE inhibitory effects. The potentiating effect of distigmine (10−6 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 significantly 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.
Distigmine bromide (distigmine) has a chemical structure consisting of two molecules of pyridostigmine connected by hexamethylene bonds and is a reversible carbamate cholinesterase (ChE) inhibitor (Fig. 1). The most important clinical indication of distigmine since its development in the 1950 s is myasthenia gravis,1,2) and its clinical effect is thought to be attributed to the increased acetylcholine (ACh) concentrations in neuromuscular junctions. In Japan, distigmine has also been used in the treatment of glaucoma3) and detrusor underactivity associated with spinal cord injury or chronic diseases, including diabetes.4–8) However, distigmine has not been intensively examined for its pharmacological effects or underlying mechanisms in urinary bladder (UB) smooth muscle (UBSM) preparations. Our previous pharmacological studies on distigmine using UBSM preparations indicated that this ChE inhibitor strongly potentiates ACh-induced contraction of guinea pig UBSM in vitro.9) Furthermore, distigmine produces a sustained increase in maximal intravesical pressure for at least 4 h after intravenous injection in guinea pigs,10) indicating its potential long-lasting effects.
To date, the sustainable characteristics of distigmine have been studied in both animals and humans. In d-tubocurarine-treated rats, the anticurare action of intraperitoneal distigmine was detectable for up to 24 h after injection.11) The acetylcholinesterase (AChE) activity in red blood cells, colon, UB, and submaxillary gland of rats remained suppressed 12 h after oral administration of distigmine.12) In clinical use, a myasthenia gravis patient who received an oral administration of distigmine exhibited symptomatic relief for 36 h,1) while the half-time for AChE inhibition recovery in red blood cells after distigmine administration was estimated to be approximately 40 h.13) However, the duration of distigmine’s effect on UB motility remains unclear. We have recently attempted to clarify this question in relation to the blood concentration of distigmine in guinea pigs and determined that an intravenous bolus injection of distigmine maintained an increased maximal intravesical pressure for 12 h after injection and that this effect persisted even after the elimination of distigmine from the blood.14)
This study was carried out to investigate whether distigmine exerts sustained pharmacological actions in UB tissues, despite its removal. We therefore investigated the duration of distigmine’s potentiating effects on ACh-induced contraction and its inhibitory effects on ChE activity in guinea pig UB tissue following washout, compared with those of other carbamate ChE inhibitors.
Male Hartley guinea pigs (4–13 weeks old; weight 305–580 g, Sankyo Labo Service Corporation, Tokyo, Japan) were housed under controlled conditions (21–22°C, relative air humidity 50±5%) and fixed 12 h light–dark cycle (08:00–20:00) with food and water available ad libitum. This study was approved by the Toho University Animal Care and User Committee (approval number: 15-51-294, accredited on May 22, 2015; approval number: 16-52-294, accredited on May 16, 2016) and conducted in accordance with the User’s Guideline to the Laboratory Animal Center of Faculty of Pharmaceutical Sciences, Toho University.
Drugs and MaterialsThe following drugs were used: ACh chloride (Daiichi-Sankyo Co., Ltd., Tokyo, Japan); distigmine bromide, 3-hydroxy-1-methylpyridin-1-ium bromide (compound A), N1,N6-dimethylhexane-1,6-diaminium chloride (compound B), 1-methyl-3-((methyl(6-(methylammonio)hexyl)carbamoyl)oxy)pyridine-1-ium bromide (compound C) (Torii Pharmaceutical Co., Ltd., Tokyo, Japan); ATP disodium salt hydrate, neostigmine bromide, atropine sulfate hydrate (Sigma-Aldrich, St. Louis, MO, U.S.A.); acetylthiocholine (ATCh) iodide, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan); pyridostigmine bromide (MP Biomedicals, Santa Ana, CA, U.S.A.); and ambenonium dichloride (Tocris Bioscience, Bristol, U.K.).
ATP was dissolved in 2.5×10−2 M Tris–HCl buffer (pH=8.0) to give a stock solution of 10−2 M and diluted further with 2.5×10−2 M Tris–HCl buffer (pH=7.4) to the desired concentration. ATCh was dissolved in Hank’s Balanced Salt Solution (HBSS) to give a stock solution of 10−3 M. DTNB was dissolved in 10−1 M phosphate buffer (pH 7.4) to give a stock solution of 10−2 M. All other drugs were prepared as aqueous solutions and diluted with distilled water.
Assessment of the Effects of ChE Inhibitors on Isolated Guinea Pig UBSM Tissue ContractionPreparation of Guinea Pig UBSM TissuesGuinea pigs were euthanized by cervical dislocation and UBs were immediately removed and placed in Locke–Ringer 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. After removing surrounding adipose and connective tissues and bladder trigone, the bladder was opened with a longitudinal incision. UB strips (approximately 2 mm in width×15 mm in length) were prepared, suspended in a 20 mL organ bath filled with Locke–Ringer solution at 32±1.0°C, and bubbled with a mixture of 95% O2 and 5% CO2.
Effects of ChE Inhibitors on ACh Concentration–Response CurvesUB strips were preloaded with a tension of 1 g and allowed to equilibrate for 20 min. After equilibration, the strips were contracted with 80 mM KCl Locke–Ringer solution of the following composition in mM: NaCl, 79.6; KCl, 80; CaCl2, 2.2; MgCl2, 2.1; NaHCO3, 5.9; and glucose, 2.8. After washout, the strips were contracted with ACh (10−4 M) and allowed to equilibrate for a further 20 min. This procedure was repeated until two successive contractions of approximately equal magnitude had been obtained. After a 30-min equilibration period, ACh (10−8–3×10−4 M) was cumulatively applied to the bath medium until a maximum response was obtained. This procedure was repeated, and the second concentration–response curve for ACh was regarded as the control response. When ChE inhibitors (distigmine, neostigmine, pyridostigmine, and ambenonium) were tested, they were applied to the bath solution 30 min before the cumulative application of ACh. After obtaining a concentration–response curve for ACh in the presence of the tested ChE inhibitor, bath medium was replaced with fresh solution 10 times, and allowed to equilibrate for 60 min. Following this equilibration period, ACh was again cumulatively applied to construct its concentration–response curve in the absence of ChE inhibitor to determine the washout effect. All tension changes in the study were recorded isotonically.
Concentration–response curves for ACh were constructed, expressing the contraction as a percentage of the maximum contractile response to 10−4 M ACh, which was administered as a single application before the cumulative application of ACh. Data were plotted as a function of drug concentration and fitted to the following equation:
 |
Basic experimental procedures followed those for the effects on ACh-induced contractions in previous section. After two successive contractions of approximately equal magnitude in response to ACh (10−4 M) were obtained, UBSM tissues were contracted by applying 10−4 M ATP. This procedure was repeated until two successive contractions of approximately equal magnitude had been obtained. After a 30-min equilibration period, ATP (10−8–3×10−4 M) was cumulatively applied to the bath medium until a maximum response was obtained. This procedure was repeated, and the second concentration–response curve for ATP was regarded as the control response. When ChE inhibitors (10−6 M for each) were tested, they were applied to the bath solution 30 min before the cumulative application of ATP.
Contraction was expressed as a percentage of the maximal response produced by 10−4 M ATP applied as a single administration before the cumulative application of ATP, and analyzed according to the procedures described in previous section.
Potentiating Effects of ChE Inhibitors on ACh (3×10−6 M)-induced Contraction and Sustainability after WashoutUBSM preparations were preloaded with a tension of 0.5 g and allowed to equilibrate for 60 min, exchanging the bath medium every 20 min. After equilibration for 60 min, the strips were contracted with 80 mM KCl Locke–Ringer solution. After washout, the strips were contracted with ACh (3×10−4 M) and allowed to equilibrate for a further 20 min. This procedure was repeated until two successive contractions of approximately equal magnitude to 3×10−4 M ACh had been obtained. After a 30-min equilibration period, ACh (3×10−6 M) was applied to the bath medium, and this procedure was repeated until three successive contractions of approximately equal magnitude had been obtained. Thereafter, ChE inhibitors (10−6 M) were applied to the bath medium and allowed to equilibrate for 30 min. This equilibration period of 30 min with distigmine was confirmed to produce an approximately maximum potentiating effect on the contraction induced by ACh (3×10−6 M) (Supplementary Fig. 1).
After this incubation period, the contractile response to 3×10−6 M ACh was recorded, and the bath medium was replaced with fresh medium 10 times. Next, contractile responses to ACh (3×10−6 M) were recorded every 30 min for 12 h after the first washout procedure, which was regarded as 0 h. All tension changes were recorded isometrically. Contractions in responses to 3×10−6 M ACh in the absence or presence of ChE inhibitors were expressed as a percentage of the maximum contraction to 3×10−4 M ACh which was applied before application of ChE inhibitors.
Assessment of the Effects of ChE Inhibitors on Isolated Guinea Pig UBSM Tissue ChE ActivitiesChE activities of the UBSM preparations were measured according to the method reported by Elman et al.,15) with minor modifications. Briefly, UB was isolated from male Hartley guinea pigs and placed in modified HBSS of the following composition in mM: 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 removing the surrounding adipose and connective tissues and bladder trigone but not the urinary epithelium, rectangle-shaped strips (about 8 mm in width×15 mm in length) were prepared. The preparation was attached to a glass rod with a mandolin line and immersed in a bath containing 20 mL of HBSS at 37±1.0°C, bubbled with a mixture of 95% O2 and 5% CO2, and allowed to equilibrate for 60 min.
After 60-min incubation, the glass rod with the preparation was immersed in a test tube filled with 3 mL of HBSS containing acetylthiocholine (ATCh, 10−3 M) at 37±1.0°C, and allowed to equilibrate for exactly 5 min. The glass rod with the preparation was then transferred to a bath containing 20 mL HBSS and medium was replaced three times. Reaction medium (2.4 mL; ATCh-containing HBSS) was then transferred to another test tube and mixed immediately with 0.5 mL phosphate buffered solution (10−1 M, pH=7.4) containing 10−4 M distigmine. Distigmine was added to exclude the effects of ChE in any tissue fragments and plasma that might have been present in the reaction medium. Next, 100 µL of DTNB (10−2 M) was added to this mixture and mixed well. After incubating the mixture for 1 min, absorbance at 412 nm was measured using a UV-visible spectrophotometer (model UV-150-02; Shimadzu, Kyoto, Japan).
The glass rod with the preparation was subsequently allowed to equilibrate for 30 min in 20 mL HBSS and the procedures were repeated twice. The third absorbance measurement was used as the control.
After the third measurement, the glass rod with the preparation was incubated for 30 min with the test ChE inhibitor (10−6 M) or potential candidate degradation products of distigmine (10−3 M) (Fig. 7). After this incubation period, the procedures were repeated and absorbance was measured. The glass rod with the preparation was next subjected to washout 10 times in an organ bath (20 mL) to remove residual drug.
The time of first washout was defined as 0 h, and ChE activity was measured every 30 min for 3 h and then every 60 min from 3–12 h. The absorbance of an experiment without the addition of any drug was regarded as the blank (Ablank), and the ChE activity (%) and ChE inhibition rate (%) were calculated from the following equations:
 |
Dissociation rate constants (k) were calculated as described by Perola et al.16) and Bartolini et al.,17) with some modifications as described below.
Briefly, any ChE inhibitor (AB) first binds to its corresponding enzyme (EH: ChE) to form an enzyme–drug complex (EA) through its precursor intermediate (EH---AB) (reaction 1). Thereafter, the ChE inhibitor bound to its enzyme (ChE) begins to dissociate from the EA by decarbamoylation with water (reaction 2).
 | (1) |
 | (2) |
If this dissociation reaction formula (reaction 2) proceeds mathematically, the following Eq. 3 is derived:
 | (3) |
 | (4) |
In accordance with Eq. 4, a linear regression line was obtained from the ratio of ChE IR, and the dissociation rate constant (k) was determined from the slope of the regression line, obtained using GraphPad Prism™.
Statistical AnalysisAll values in the text and illustrations are presented as means±standard error of the mean (S.E.M.) of the data obtained from different numbers (n) of preparations. The significance of differences between mean values was evaluated by Student’s paired t-test or one-way ANOVA followed by Dunnett’s multiple comparison test or Tukey’s multiple comparison test using GraphPad Prism™. p values of less than 0.05 were considered statistically significant.
We first determined whether repeated cumulative application of ACh (10−8–3×10−4 M) or ATP (10−8–3×10−4 M) generated reproducible concentration–response curves in isolated guinea pig UBSM preparations.
pD2 values for ACh and Emax values of the contractions were as follows, and were not significantly different. pD2 values were 4.46±0.38 (n=4, 1st response), 4.47±0.42 (n=4, 2nd response), and 4.49±0.27 (n=4, 3rd response). Emax values for ACh (3×10−4 M)-induced contractions were 120.2±8.9% (n=4, 1st response), 121.6±11.6% (n=4, 2nd response), and 134.7±9.7% (n=4, 3rd response). ATP-induced contractions were also shown to be reproducible for at least two repetitions, judging from the insignificant differences between the values: pD2 values for ATP were 5.06±0.44 (n=4, 1st response) and 4.84±0.35 (n=4, 2nd response); and Emax values for ATP (3×10−4 M)-induced contractions were 105.3±10.6% (n=4, 1st response) and 104.1±10.3% (n=4, 2nd response).
Effects of ChE Inhibitors on UBSM Tissue Contractions in Response to ACh and ATPFigure 2 shows the effects of distigmine (10−7–10−5 M), neostigmine (10−8–10−6 M), pyridostigmine (10−7–10−5 M), and ambenonium (10−7–10−5 M) on UBSM tissue contractions in response to ACh (10−8–3×10−4 M). Distigmine (10−7–10−5 M) potentiated ACh-induced contractions in a concentration-dependent manner, which was clearly demonstrated by the leftward shift of the concentration–response curves for ACh in the presence of distigmine (Fig. 2A, Table 1). All concentrations (10−7–10−5 M) of distigmine increased the pD2 value of ACh, and statistical significances were particularly apparent at concentrations of 10−6–10−5 M (Table 1). Interestingly, the potentiating effects of distigmine were preserved even 1 h after washout.
ACh was applied cumulatively to the bath solution in the absence or presence of distigmine (10−7–10−5 M; A), neostigmine (10−8–10−6 M; B), pyridostigmine (10−7–10−5 M; C), or ambenonium (10−7–10−5 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 administration of 10−4 M ACh before the experiment (100% contraction). Data are presented as means±S.E.M. Numbers of experiments are n=6 for pyridostigmine (10−6 M) and ambenonium (10−5 M); n=5 for pyridostigmine (10−7 and 10−5 M) and ambenonium (10−7 M); and n=4 for the others. Dis: distigmine; Neo: neostigmine; Pyr: pyridostigmine; Amb: ambenonium.
Neostigmine (10−8–10−6 M) also potentiated ACh-induced contractions (Fig. 2B), and a significant increase in pD2 was obtained at concentrations of 10−7–10−6 M (Table 1). However, 1 h after washout, the potentiating effects of neostigmine disappeared, as shown by the significant decrease in pD2 values for ACh (Table 1).
Similarly, pyridostigmine (10−7–10−5 M) shifted the concentration–response curves for ACh to the left (Fig. 2C), an observation 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).
| 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 |
Data are presented as means±S.E.M. Numbers of experiments are n=6 for pyridostigmine (10−6 M) and ambenonium (10−5 M); n=5 for pyridostigmine (10−7 and 10−5 M) and ambenonium (10−7 M); and n=4 for the others. * p<0.05, ** p<0.01 vs. control. # p<0.05, ## p<0.01 vs. each inhibitor.
Ambenonium (10−7–10−5 M) also potentiated ACh-induced contractions (Fig. 2D) although increases in pD2 were significant at 10−7–10−6 M in limited numbers of experimental trials (Table 1). One hour after washout, the potentiating effect of ambenonium at 10−7 M disappeared. However, it remained significant at 1 h for a concentration of 10−6–10−5 M.
No ChE inhibitor (10−6 M) had a significant effect on ATP (10−8–3×10−4 M)-induced contractions (Fig. 3), despite exerting a significant potentiating effect on ACh-induced contractions at these concentrations.
ATP was applied cumulatively to the bath solution in the absence or presence of each inhibitor (10−6 M). Experiments with neostigmine were carried out in the presence of atropine (10−6 M). Contraction is expressed as the percentage of the contraction induced by a single administration of 10−4 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.
Figure 4 represents typical recordings of the effects of ChE inhibitors on UBSM tissue contractions in response to 3×10−6 M ACh and the preservation of this effect up to 12 h after washout. All ChE inhibitors potentiated ACh-induced contractions although, with the exception of distigmine, these effects disappeared over time within 2–3 h after washout (Figs. 4B–D, 5B–D). However, the potentiating effect of distigmine remained significantly preserved until 12 h after washout (Figs. 4A, 5A).
Qualitatively, the same results were obtained from four experiments for each inhibitor. Arrow: administration of 3×10−6 M ACh; w: washout; Dis: distigmine; Neo: neostigmine; Pyr: pyridostigmine; Amb: ambenonium.
Figure 6 shows the effects of ChE inhibitors on UB ChE activity and their preservation of these effects up to 12 h after washout. ChE activity was restored in a time-dependent manner after washout of all ChE inhibitors except distigmine (i.e., neostigmine, pyridostigmine, and ambenonium) (Fig. 6A). The k values were 0.40±0.02 h−1 for neostigmine, 0.50±0.04 h−1 for pyridostigmine, and 0.61±0.04 h−1 for ambenonium, as indicated by the slopes of the plots shown in Fig. 6B.
However, inhibition of ChE activity by distigmine was preserved until 12 h after washout (Fig. 6A), and thus, the k value for distigmine could not be calculated (Fig. 6B).
Contraction is expressed as the percentage of the contraction induced by a single administration of 3×10−4 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.
A: ChE activity in guinea pig urinary bladder smooth muscle up to 12 h after 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 the dissociation rate constant (k). Data are presented as means±S.E.M. (n=4). w/o: washout of each inhibitor.
Compounds A–C in Fig. 7 are candidates for degradation products of distigmine, as estimated using the general rules for binding and dissociation reactions between ChE and carbamate ChE inhibitors. None of the compounds significantly inhibited ChE at 12 h after washout, even at a concentration of 10−3 M (1000 times more concentrated than distigmine 10−6 M) (Fig. 7).
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).
The objective of this study was to clarify the mechanism by which distigmine produces long-lasting characteristic potentiation of UBSM contraction. In this study, in order to determine whether the potentiating effects of distigmine on UBSM contraction require the continued presence of distigmine near UBSM tissue, we evaluated the duration of contraction enhancement of isolated guinea pig UBSM by distigmine and inhibition of ChE activity after washout of distigmine from the tissue bath solution. In addition, the effects of distigmine were compared with those of other ChE inhibitors. We first showed that the potentiating effect of distigmine on UBSM contraction was exerted against ACh- but not against ATP (a co-neurotransmitter of the parasympathetic nerve)-induced contraction18) (Fig. 3). Next, we determined that the potentiating effects of distigmine on ACh-induced contraction and its inhibitory effects on ChE activity persisted for 12 h, even after its removal from the bath solution (Figs. 4–6). In contrast, both effects were strongly attenuated for other ChE inhibitors after washout. These in vitro results are consistent with the results of our previous in vivo study, which showed that the potentiating effect of distigmine on UB motility persisted for at least 6 h after the elimination of distigmine from the blood.14) Based on these findings, the sustained potentiation of UBSM contractile activity by distigmine appears independent of the continued presence of distigmine in the UBSM tissue.
One possible explanation for the sustained potentiation by distigmine of ACh-induced UBSM contractility irrespective of the presence of distigmine is that distigmine strongly binds to UBSM AChE to form a stable complex, and thus inhibits the decomposition of ACh over a prolonged time. Therefore, the k value of the distigmine and AChE complex was calculated to determine the rate at which distigmine dissociates from AChE. As shown in Fig. 6B, the k values for ChE inhibitors other than distigmine were approximately 0.5 h−1, and thus they were estimated to dissociate from UBSM AChE at almost the same rate. In contrast, the k value could not be calculated for distigmine within the timeframe of this study (12 h) (Fig. 6B), as AChE activity had not sufficiently recovered at 12 h after distigmine washout (Fig. 6A). This finding strongly supports the hypothesis that the dissociation of distigmine from AChE occurs slowly after the stable complex of distigmine and AChE has formed. However, the possibility that distigmine molecules accumulate in the plasma membrane adjacent to UBSM, and are thus gradually exposed to AChE, cannot be overlooked. To rule out this possibility and to substantiate the hypothesis that distigmine itself strongly binds to AChE to generate a stable complex, further studies using AChE isolated from UBSM cells are needed. We are currently conducting experiments to demonstrate that distigmine resists dissociation from AChE using recombinant human AChE purified from cells, and to calculate the k value of distigmine from this AChE.
The sustained potentiation by distigmine of UBSM contractility and its inhibitory effects on AChE were shown in vitro using isolated UBSM tissues. These findings suggest that the inhibitory effect on AChE is produced by distigmine itself. However, there is a possibility that these inhibitory effects are instead mediated via decomposed products of distigmine. Therefore, we further explored this possibility.
Distigmine has a chemical structure of two pyridostigmine molecules bonded via a hexamethylene group and thus has two carbamoyl groups available to bind to AChE (Fig. 1). Furthermore, some ChE inhibitors without carbamoyl groups are reported to exert their ChE inhibitory effects by acting on sites other than the ChE active site serine residue.19) Therefore, it is plausible that the degradation products of distigmine released from AChE bind again to AChE and thus inhibit its activity over a long duration. However, none of the three types of candidate degradation products that are presumed to be formed from distigmine showed inhibitory activity against AChE (Fig. 7). Therefore, the sustained inhibition of AChE by distigmine was unlikely caused by its degradation products but was likely produced by distigmine itself.
Among the three types of candidate degradation products of distigmine evaluated in this study, compound C (Fig. 7) contains one carbamoyl group that can bind to AChE, although the second carbamoyl group of distigmine is absent. However, although compound C was predicted to show AChE inhibitory activity, no inhibition was observed (Fig. 7). Interestingly, a compound with a structure of two neostigmine (Fig. 1) molecules linked with a long-chain methylene group was reported to exert sustained anti-myasthenia effects after oral administration.1) Similarly, the introduction of a long-chain methylene group or cyclic structure into physostigmine, a carbamate-type ChE inhibitor with a cyclic structure, increased its association with AChE.16) Based on these findings, the presence of two pyridostigmine (aromatic) structures appears to be required for distigmine’s sustained AChE inhibitory action. Although the molecular basis of these mechanisms requires further investigation, the π–π interaction presumed to arise from the aromatic ring structures bonded to both ends of the pyridostigmine molecule might contribute to the stable binding of distigmine to AChE.
In summary, the potentiating effect of distigmine on UBSM contraction was shown to persist for 12 h after washout and was unique to distigmine compared with other ChE inhibitors. We also showed that the sustained potentiation by distigmine of the ACh-induced contractile response of UBSM is likely caused by distigmine itself rather than its degradation products. One plausible explanation for the sustained effects of distigmine is that it forms a strong and stable complex with AChE, which in turn produces sustainable inhibition of UBSM AChE activity.
Distigmine bromide and distigmine degradation products were a gift from Torii Pharmaceutical Co., Ltd. (Tokyo, Japan). The authors would like to thank Ms. Eriko Kaneki and Ms. Aya Katayose for their expert technical assistance.
The authors declare no conflict of interest.
The online version of this article contains supplementary materials.
