A novel pyrrole derivative, NS-8, suppresses the rat micturition reflex by inhibiting afferent pelvic nerve activity

Authors


M. Tanaka, Discovery Research Laboratories, Nippon Shinyaku Co. Ltd. 14, Nishinosho-Monguchi-Cho, Kisshoin, Minami-ku, Kyoto, 601-8550, Japan.
e-mail: m.tanaka@po.nippon-shinyaku.co.jp

Abstract

OBJECTIVE

To investigate the suppressive effect of a newly synthesized compound, 2-amino-3-cyano-5-(2-fluorophenyl)-4-methylpyrrole (NS-8), on micturition, and its mode and sites of action in rats.

MATERIALS AND METHODS

Female rats were anaesthetized with urethane, and isovolumetric bladder contractions and cystometrograms recorded. The pelvic afferent discharges from the bladder were also monitored.

RESULTS

In the cystometric study, NS-8 increased the bladder capacity without affecting the maximum bladder contraction pressure, an effect unlike that of currently used anticholinergic drugs for the overactive bladder, which commonly decrease the maximum bladder contraction pressure. Intravesical and intravenous injection of NS-8 inhibited isovolumetric bladder contractions in a dose-dependent manner without affecting their amplitude, whereas intracerebroventricular injection with NS-8 had no such effect. During the urine storage phase of the cystometrogram, NS-8 decreased the discharge rate of the afferent pelvic nerve from the bladder, in association with a decrease in the increase in intravesical pressure.

CONCLUSION

NS-8 suppressed the micturition reflex by decreasing afferent pelvic nerve activity. Activation of calcium-sensitive potassium channel of the bladder may be responsible for such an effect. This agent has the potential to treat patients with urinary frequency and incontinence.

Abbreviations
UUI

urge urinary incontinence

MBCP

maximum bladder contraction pressure

IVBC

isovolumetric bladder contraction.

INTRODUCTION

Urinary dysfunction is an increasingly common problem in an ageing population; the troublesome symptoms are frequency, urgency and incontinence. Urge urinary incontinence (UUI) is often accompanied by abnormal spontaneous detrusor contractions unrelated to bladder urine volume, which may cause urgency and involuntary urine loss. Anticholinergic drugs, including oxybutynin and propiverine, are most commonly used to treat UUI, but drugs with anticholinergic activity can cause urinary retention and overflow incontinence, resulting from suppression of the contractility of the detrusor muscle, in addition to adverse reactions such as dry mouth, blurred vision and increased heart rate [1]. Especially in patients with UUI accompanying an impaired ability to generate a sustained bladder contraction [2], anticholinergics worsen the symptoms of urinary retention and cause overflow incontinence [3].

There is a clinical need for an effective drug for UUI which normalises voiding by suppressing abnormal detrusor activity but without affecting voiding pressure. Inhibiting the micturition reflex is one suitable method for treating UUI, and suppressing the afferent pathway from the mechanoreceptors of the bladder may be one of the best mechanisms for suppressing the micturition reflex while allowing complete emptying of the bladder [4–6]. NS-8 (2-amino-3-cyano-5-(2-fluorophenyl)-4-methylpyrrole; Fig. 1) is a newly synthesized compound designed to satisfy such needs, having calcium-sensitive potassium-channel opening activity, as a treatment for urinary frequency and incontinence. The present study was undertaken to evaluate the effect of NS-8 on lower urinary tract function in rats, investigating also its mode and sites of action.

Figure 1.

Structure of NS-8 (2-amino-3-cyano-5-(2-fluorophenyl)-4-methylpyrrole).

MATERIALS AND METHODS

All animal studies were approved by the animal research committee at the authors institution; female Sprague-Dawley rats (Japan SLC, Shizuoka, Japan; 160–250 g) were housed in groups in a room maintained at 21–25 °C and 45–65% relative humidity, in an alternating 12-h light/dark cycle, and given food and water ad libitum; in studies requiring intraduodenal administration of test compounds, animals were fasted from the day before testing.

NS-8, cromakalim, oxybutynin (hydrochloride) and propiverine were synthesized at the authors’ research laboratories. Verapamil (hydrochloride), atropine (sulphate) and gallamine (triethiodide) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan), Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan), and May & Baker (Dagenham, UK), respectively. These chemicals were suspended or dissolved in 0.5% methyl cellulose solution for intraduodenal administration, dissolved in saline solution containing 1% Tween 80 for intravenous administration, and dissolved in 2% lecithin solution for intravesical and intracerebroventricular administration.

For cystometry under urethane anaesthesia (900 mg/kg, subcutaneously) rats were placed supine. The bladder was exposed through a midline incision in the abdomen, and a polyethylene catheter (PE50, Clay Adams, Parsippany, NJ, USA) inserted into the apex of the bladder dome and connected to a three-way stopcock. One outlet was connected to a pressure transducer (TP-200T, Nihon Kohden, Tokyo, Japan) for recording the intravesical pressure with a pen recorder (RJG-4124, Nihon Kohden). The other outlet was connected to an infusion pump (STC-521, Terumo, Tokyo, Japan) for infusing physiological saline, and the abdominal incision sutured. After the bladder was emptied, reflex micturition was induced by filling the bladder with warm saline at 2.8 mL/h with the infusion pump. The flow of saline was terminated when reflex micturition occurred. The bladder capacity and maximum bladder contraction pressure (MBCP) were read from the cystometrogram. Cystometry was applied several times until the bladder capacity and MCBP had stabilized. Test compounds were then injected into the duodenum and their effects on bladder capacity and MBCP evaluated by cystometry again.

For isovolumetric bladder contractions (IVBCs), under urethane anaesthesia (900 mg/kg) the abdominal cavity of rats placed supine was opened by a midline incision and both ureters cannulated by passing polyethylene catheters (PE10) to drain urine from the kidneys. The urinary bladder was cannulated with a polyethylene catheter (PE60) through the urethra, secured by a ligature at the urethral orifice and connected to a three-way stopcock. One outlet was connected to a TP-200T pressure transducer for recording the intravesical pressure (pen recorder), and the other connected to an STC-521 infusion pump to fill the bladder by 0.1-mL increments of warm physiological saline until rhythmic IVBCs occurred. When the contractions were at constant intervals, test compounds were injected intravenously and cumulatively at 15-min intervals, or the bladder was emptied and then filled with the same volume of warm physiological saline containing the test compound.

For intracerebroventricular injections, IVBCs were induced as described and the rat then placed in a stereotaxic instrument, a small craniotomy made, a guide cannula for drug administration introduced into the lateral ventricle and fixed to the skull with dental cement. When IVBCs occurred at constant intervals an injection cannula was introduced into the lateral ventricle through the guide cannula and test compounds injected with the cannula connected to a micro-injection pump (CMA/100, Carnegie Medicine, Stockholm, Sweden) operated at 4 µL/min.

The method of Hotta et al.[7] was used to measure afferent neural activities. Under urethane anaesthesia (900 mg/kg) rats were placed supine and a PE60 catheter inserted into the bladder through the urethra, secured by a ligature at the urethral orifice, and connected to a three-way stopcock. One outlet was connected to a TP-200T pressure transducer for recording the intravesical pressure and the other connected to an STC-521 infusion pump for infusing physiological saline. The bilateral hypogastric and pelvic nerves were exposed through a midline incision in the abdomen, sectioned 10 mm proximal to the pelvic nerve plexus, and one distal stump of the pelvic nerve from the bladder placed in contact with bipolar platinum electrodes. Skin flaps were tied to a metal frame with thread to retain an abdominal pool of warm mineral oil to cover the nerves and organs. Afferent neural activity from the bladder was amplified with a preamplifier (MEG-1200, Nihon Kohden) and displayed on an oscilloscope. Spikes were transformed into unit square-wave pulses, and those of amplitude above the background noise were selected with a window discriminator (MET-1100, Nihon Kohden). Gallamine (20 mg/kg, intravenously) was given before the start of nerve stimulation; additional doses were given as appropriate, after checking the depth of anaesthesia. After the bladder was emptied it was filled with warm saline at a constant rate of 0.15 or 0.2 mL/min with the infusion pump. The flow of saline was terminated within 5–10 min, when the infused volume had reached 1.2 mL. The discharges were accumulated over 5-s intervals and recorded on a pen recorder simultaneously with the intravesical pressure.

The results are expressed as the mean (sem) and analysed statistically using one-way anova followed by Dunnett's test for multiple comparisons in the cystometric study and in the study of IVBCs when test compounds were administered intravesically, Student's t-test in the latter study when test compounds were administered intravenously, and a paired t-test in the study of pelvic afferent discharges; P < 0.05 was considered to indicate statistical significance.

RESULTS

Representative cystometric recordings before and 30 min after the intraduodenal administration of NS-8 are shown in Fig. 2. NS-8 at 3 and 10 mg/kg increased the bladder capacity without affecting the MBCP. The time course for the effect of NS-8 on bladder capacity is shown in Fig. 3; there were statistically significant increases in bladder capacity at both doses, with the maximum effect at 30 min after dosing and a duration of action of > 3 h.

Figure 2.

Representative urodynamic recordings of anaesthetized rats before and after the intraduodenal administration of NS-8 (upper traces, 3 mg/kg; lower traces, 10 mg/kg. BC, bladder capacity; arrow, start of saline infusion; M, micturition.

Figure 3.

Changes in bladder capacity after intraduodenal NS-8 in anaesthetized rats. The baseline bladder capacity values were 0.38 (0.01), 0.35 (0.02), 0.36 (0.01) and 0.37 (0.02) mL in the control (green open circles), NS-8 1 (red closed square), 3 (light green closed circle) and 10 mg/kg (light red open square) groups, respectively. Each point represents the mean (sem) (six rats). *P < 0.01, significantly different from vehicle control (Dunnett's test).

The effect of NS-8 and reference drugs on bladder capacity and MBCP is summarized in Fig. 4. Oxybutynin decreased the MBCP at doses of > 30 mg/kg, but caused no changes in bladder capacity. Propiverine increased the bladder capacity at 100 mg/kg but decreased the MBCP at 30 mg/kg, a dose insufficient to significantly increase the bladder capacity. Cromakalim had no effect on either variable at 0.1–1 mg/kg.

Figure 4.

Effect of intraduodenal administration of NS-8 and reference compounds on the bladder capacity and MBCP in anaesthetized rats. Baseline values: bladder capacity, 0.35 (0.02) to 0.41 (0.04) mL; MBCP 18.0 (1.5) to 24.6 (1.6) mmHg. Each column represents the mean (sem) at the maximum change after administration (five or six rats). C, control group; *P < 0.05; **P < 0.01, significantly different from vehicle control at the corresponding time (Dunnett's test).

Representative recordings of IVBCs before and after intravenous NS-8 and reference compounds are shown in Fig. 5. The effects of NS-8 and reference compounds on the contraction pressure and contraction interval are shown in Fig. 6. NS-8 inhibited the IVBCs in a dose-dependent manner without affecting their amplitude. There was significant inhibition at doses of > 0.03 mg/kg. Verapamil inhibited the IVBCs similarly to NS-8 at doses of > 1 mg/kg. Atropine reduced the amplitude, but not the frequency, of the IVBCs at doses of > 0.01 mg/kg. Cromakalim did not affect either variable at 0.01–1 mg/kg.

Figure 5.

Representative recordings of IVBCs in anaesthetized rats. Test compounds were administered intravenously and cumulatively.

Figure 6.

The effect of intravenous NS-8 (A), verapamil (B), atropine (C) and cromakalim (D) on contraction pressure and duration of disappearance of IVBCs in anaesthetized rats. Open bars, control group; shaded bars, treated group. Each column represents the mean (sem) of 6 rats.

Representative recordings of IVBCs after intravesical administration of NS-8 are shown in Fig. 7, and the effect of NS-8 on the contraction interval in Fig. 8. NS-8 inhibited IVBCs without affecting the amplitude at concentrations of > 30 µg/mL.

Figure 7.

Representative recordings of IVBCs after intravesical administration (arrow) of NS-8 in anaesthetized rats.

Figure 8.

The effect of an intravesical injection with NS-8 on IVBCs in anaesthetized rats. Each column represents the mean (sem) of three or four rats. *P < 0.05, **P < 0.01, significantly different from the control (Dunnett's test).

Representative recordings of IVBCs after intracerebroventricular NS-8 and morphine are shown in Fig. 9. The contractions were unaffected by NS-8 at 3–30 µg (six rats), and by the same volume of vehicle (six). In contrast, morphine inhibited the IVBCs at 0.3 µg (four).

Figure 9.

Representative recordings showing the effect of intracerebroventricular vehicle (A), NS-8 (B) and morphine (C) on IVBCs in anaesthetized rats. Drug solutions were injected over the periods indicated by the horizontal bars.

In anaesthetized rats the infusion of saline into the bladder at a constant rate increased the afferent pelvic nerve activity, concomitant with an increase in intravesical pressure. Intravenous treatment with vehicle alone did not affect either of these variables (Fig. 10A). Intravenous NS-8 suppressed the increase in the pelvic afferent discharge frequency and inhibited the increase in intravesical pressure (Fig. 10B). Verapamil also suppressed both increases, although the degree of suppression on the increase in intravesical pressure was greater than that of NS-8 (Fig. 10C).

Figure 10.

The effect of intravenous vehicle (A), NS-8 (B) and verapamil (C) on intravesical pressure and pelvic afferent neuronal discharges in anaesthetized rats, before (green circles) and after (red squares) administration. Saline was infused into the bladder at a constant rate. Each point represents the mean (sem) (six rats). *P < 0.05, **P < 0.01, significantly different from the values before drug treatment (paired t-test).

DISCUSSION

The causes of bladder overactivity are not well understood, but theoretically increased afferent activity, decreased inhibitory control in the CNS or peripheral ganglia, and increased sensitivity of the detrusor to efferent stimulation may be involved. Of several targets for pharmacological intervention, the inhibition of afferent pelvic nerve activity may be the most attractive to ‘normalise’ the diseased bladder without affecting the contractility needed for adequate bladder emptying.

In the present urodynamic study, NS-8 increased the bladder capacity without affecting the MBCP; this effect is qualitatively different from that of anticholinergic drugs used for treating urinary frequency and incontinence, e.g. oxybutynin and propiverine [8–10]. In the present study, oxybutynin and propiverine markedly decreased the MBCP, in agreement with the findings of others [11,12]. Contraction of the bladder in the voiding phase is mainly elicited by the stimulation of muscarinic acetylcholine receptors, and is inhibited by anticholinergics, resulting in a decrease in MBCP instead of an increase in bladder capacity. ATP-sensitive potassium-channel openers, e.g. cromakalim [13,14], inhibit the excitability of detrusor smooth muscle cells by hyperpolarizing the membrane potential [15], and have recently been developed for treating urinary frequency and incontinence. However, cromakalim barely increased the bladder capacity even at doses high enough to produce a marked decrease in blood pressure and increase in heart rate [16,17], an effect that is consistent with a previous report [18]. The results of the present urodynamic study with NS-8, which showed a significant increase in bladder capacity with no effect on voiding pressure, suggest that this drug has a different mechanism of action in inhibiting the micturition reflex from those of anticholinergics or cromakalim. Not only by avoiding the deficiencies, but also by improving the efficacy of anticholinergic drugs, NS-8 may be clinically preferable for treating UUI.

To investigate the sites and modes of action of NS-8, its effects on IVBCs were compared when it was administered by different routes. Intravenous NS-8 inhibited IVBCs without affecting their amplitude, similar to that of verapamil, a voltage-dependent calcium-channel antagonist. An anticholinergic, atropine, did not inhibit the initiation of contractions, although it caused a marked decrease in their amplitude. Intravesically administered NS-8 also inhibited IVBCs, but intracerebroventricular NS-8, even at high doses, did not. Together with the fact that IVBCs are neurally controlled by the vesico-spinal and brainstem pathways, these results indicate that NS-8 acts at the bladder wall, including the detrusor smooth muscle, or at mechanoreceptors or afferent neuronal pathways from the bladder that participate in the micturition reflex, rather than at the micturition centre in the brain. The idea that the site of action of NS-8 in inhibiting the micturition reflex is in the afferent pathways, including the bladder, is supported by the finding that this drug inhibited the afferent pelvic nerve activity from the bladder, concomitant with the decrease in the rise of intravesical pressure during the bladder filling phase.

NS-8 activates the calcium-sensitive potassium-channel of the bladder [19] and has a preference for large-conductance calcium-sensitive potassium (BKCa) channels, suggested by an electrophysiological and pharmacological study in isolated detrusor smooth muscle cells and tissues (unpublished observations). In the urinary bladder, BKCa channels are important in modulating cell excitability by helping to maintain the membrane potential and by regulating the duration and amplitude of the action potential [20,21]. Therefore, the activation of BKCa channel by NS-8 may contribute to the relaxation of the bladder smooth muscle which brings about the decrease in mechanoreceptor and/or afferent nerve activity.

In conclusion, NS-8 suppressed the micturition reflex associated with a marked increase in bladder capacity without affecting the maximum bladder pressure. The inhibition of afferent pelvic nerve activity may be responsible for this suppression of the micturition reflex by NS-8. This compound is expected to be a useful drug for treating urinary frequency and incontinence.

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