Are Antimuscarinic Drugs Effective Against Urinary Frequency Mediated by Atropine-Resistant Contractions?

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J Pharmacol Sci 115, 364 – 373 (2011) Journal of Pharmacological Sciences © The Japanese Pharmacological Society Full Paper Are Antimuscarinic Drugs Effective Against Urinary Frequency Mediated by Atropine-Resistant Contractions?

Fukumitsu Sakakibara1, Mamoru Kiniwa1, and Masato Nanri1,* 1Department of Applied Pharmacology, Tokushima Research Center, Taiho Pharmaceutical Co., Ltd., 2-224 Ebisuno, Hiraishi, Kawauchi-cho, Tokushima 771-0194, Japan

Received August 2, 2010; Accepted January 11, 2011

Abstract. In the disease states of urinary frequency and urgency, atropine-resistant contractions are known to be involved, in addition to contractions mediated by cholinergic nerves. This study was undertaken to investigate the mechanism underlying the development of atropine-resistant contractions using the representative antimuscarinic drugs solifenacin and tolterodine and also propiverine that has Ca2+ channel–antagonizing activity in addition to antimuscarinic activity. Rat models of urinary frequency were established by intravesical infusion of acetylcholine (ACh) (cholinergic nerve–mediated urinary frequency model), acetic acid (AcOH) [non-adrenergic non-

cholinergic nerve (NANC)-mediated urinary frequency model], or CaCl2 (atropine-resistant contractions-mediated urinary frequency model). Cystometrograms were obtained to measure the micturition parameters following oral administration of the aforementioned drugs. Propiverine increased the micturition weight in all the urinary frequency models. Solifenacin and tolterodine increased the micturition weight in the ACh-induced urinary frequency model but neither had any

effect in the AcOH- or CaCl2-induced urinary frequency models. While antimuscarinic drugs are, in general, effective for the control of urinary frequency and incontinence, use of drugs possessing inhibitory effects on contractions mediated by cholinergic as well as NANC nerve transmission or Ca2+ influx into smooth muscles is recommended for management of the symptoms in disease states in which atropine-resistant contractions, such as Ca2+- and capsaicin-sensitive sensory nerves, are involved.

Keywords: urinary frequency, overactive bladder, propiverine, antimuscarinic, atropine-resistant contraction

Introduction In the disease states of urinary frequency and urgency, nevertheless, not only contractions mediated by cholin- In recent years, drugs with high selectivity for musca- ergic nerves, but also atropine-resistant contractions, in- rinic receptors have been developed and demonstrated to cluding contractions mediated by non-adrenergic non- yield gratifying clinical results in the management of cholinergic (NANC) nerves, are now known to be urinary frequency and incontinence. Over time, antimus- involved, and contractions that cannot be inhibited by carinic agents have come to be used for the treatment of atropine have been demonstrated in a previous study urinary frequency and urgency in patients with overac- conducted using electrical field stimulation of isolated tive bladder (OAB) or neurogenic bladder all over the detrusor strips (2). As for contractions mediated by world. The underlying mechanism of action of these NANC nerves, contractile responses medicated by puri- drugs consists of inhibition of detrusor overactivity by nergic receptors and those mediated by capsaicin-sensi- blocking the binding of acetylcholine to muscarinic re- tive nerves are known to exist (2, 3). Moreover, involve- ceptors (1). ment of Ca2+ is also generally recognized, and it has been described that Ca2+ influx has a greater influence than *Corresponding author. [email protected] cholinergic nerves upon detrusor contractions in which Published online in J-STAGE on February 22, 2011 (in advance) NANC nerves are involved (4). Irrespective of whether doi: 10.1254/jphs.10199FP the contractions are mediated by cholinergic or NANC

364 Antimuscarinics in Pollakiuria Models 365 nerves, Ca2+ influx is involved in smooth muscle contrac- passed subcutaneously and led out of the occipital region tions. Therefore, it is important for the control of such to be passed into a spring. symptoms as urinary frequency and urgency to also in- The measurements were carried out on Day 7 after the hibit atropine-resistant contractions; however, the com- operation. A swivel (22 ga single channel stainless steel monly used muscarinic receptor–specific drugs do not swivel; Instech Laboratories, Inc., Plymouth Meeting, inhibit these contractions. PA, USA) was coupled to the catheter connected to the Under these circumstances, we conducted this study bladder of the rat and the other end of the catheter lumen using an atropine-resistant contractions-mediated urinary was divided into two branches with a three-way stopcock. frequency model to verify whether antimuscarinic agents One branch was connected via a pressure transducer currently in use in the clinical setting might be useful for (Disposal Blood Pressure Monitoring Life Kit DX-360; the management of urinary frequency and urgency in Nihon Kohden Corporation, Tokyo) to a pressure ampli- which atropine-resistant contractions are also involved. fier (Blood Pressure Amplifier AP-641G combined with The drugs assessed included solifenacin and tolterodine, the main unit case RMP-6008M, Nihon Kohden Corpo- both of which exhibit high specificity for the muscarinic ration) to measure the intravesical pressure. The intra- receptor, and propiverine that is endowed with an anti- vesical pressure was recorded with a PowderLab 16/30 muscarinic effect as well as Ca2+ channel–antagonizing System (Chart v5.2.2 for Windows; ADInstruments Pty activity (5). To confirm the involvement of NANC nerves Ltd., Sydney, Australia). The urine weight was recorded in the development of urinary frequency, an acetylcholine with the PowerLab 16/30 System simultaneously in (ACh)-induced urinary frequency model as a cholinergic terms of changes in the urine weight measured using an system–mediated urinary frequency model and an acetic electronic balance (HF200; A and D Company Co., Ltd., acid (AcOH)-induced urinary frequency model as an Tokyo) placed directly underneath the cage. The other NANC nerve-mediated urinary frequency model were branch was connected to a syringe fitted to a continuous used (3, 6). In addition, a CaCl2-induced urinary fre- injector (Micro syringe pump KDS200; KD Scientific, quency model was also established to explore the effects Colorado Springs, CO, USA). on urinary frequency mediated by Ca2+ channels encoun- Intravesical infusion of physiological saline was per- tered in various disease states. The effects of atropine, formed at the rate of 3.0 mL/h. The test drugs were ad- verapamil, and desensitization with capsaicin were also ministered orally after achieving stabilization of the in- investigated in all of these models. travesical pressure, and at the same time, the physiological saline infused intravesically was switched to physiologi- Materials and Methods cal saline containing 30 mM ACh (Wako Pure Chemical Industries, Ltd., Osaka), 0.3% AcOH (Wako Pure

Animals Chemical Industries, Ltd.), or CaCl2 (30, 50, 80 mM; Female 11-week-old Sprague-Dawley rats were used. Wako Pure Chemical Industries, Ltd.) (3, 6). The cysto- The animals, purchased from Charles River Japan, Inc. metric measurements were initiated 30 min after the (Yokohama), were accommodated in cages in an envi- dosing and continued for over 2.5 h with the animals in ronmentally controlled room at an ambient temperature a conscious state without restraint. and relative humidity of 20°C – 26°C and 30% – 70%, respectively, under a 12-h light/dark cycle (lights from Drugs and administration 6:00 to 18:00 h). Water and food were provided ad libi- Atropine sulfate (1 mg/kg; Sigma-Aldrich, St. Louis, tum. All experiments were conducted in accordance with MO, USA), verapamil hydrochloride (3 mg/kg; Wako the Laboratory Animal Care Code of Ethics of Taiho Pure Chemical Industries, Ltd.), propiverine hydrochlo- Pharmaceutical Co., Ltd. ride (1 – 30 mg/kg, Taiho Pharmaceutical Co., Ltd., Tokyo), solifenacin succinate (0.3 – 10 mg/kg; Taiho Cystometry Pharmaceutical Co., Ltd.), and tolterodine tartrate (0.1 – After induction of anesthesia, the body temperature of 3 mg/kg; Taiho Pharmaceutical Co., Ltd.) were dissolved the animals was maintained at 37°C using a heating in purified water to prepare the dosing solutions at the board (PS-53; Sakura Finetek Japan, Tokyo) during the time of switching of the intravesical infusion fluid. Cap- operation. The abdomen was opened and the bladder was saicin (125 mg/kg, Sigma-Aldrich) was dissolved with exposed from the abdominal cavity, followed by a small ethanol at a concentration of 200 mg/mL and mixed with incision of the bladder vertex. A polyethylene tube (PE- an equal volume of Tween 80 (Sigma- Aldrich). This 50; Becton Dickinson, Franklin Lakes, NJ, USA) was mixture was mixed with 8 times its volume of physiologi- inserted into the urinary bladder and fixed therein, fol- cal saline to prepare the dosing solution. For desensitiza- lowed by suture of the incised area. The catheter was tion with capsaicin, the prepared injection fluid was ad- 366 F Sakakibara et al ministered subcutaneously at 50 mg/kg in the morning 5 linear regression analysis. For the statistical analyses, days before the measurements, at 25 mg/kg about 12 h EXSAS (EXSAS 7.10; Arm Systex, Osaka) was used, afterwards, and at 50 mg/kg in the morning 4 days before with the P-value for significant difference set at <0.05 in the measurements. All injections were performed while the Dunnett’s test and Student’s t-test and at <0.025 in the animals were under halothane anesthesia according the Williams’ test. to the method of Chuang et al., and the anesthesia was maintained for approximately an hour after the capsaicin Results injection attenuated the pain (3). Effects of the agents examined in the normal rat Phenylalkylamine (PAA) sensitive L type Ca2+ channel The experiment was conducted on rats that had been affinity operated on 7 days previously. Cystometric measure- The rat cerebral cortex was used for PAA-sensitive ments in the animals were performed in the usual manner L-type Ca2+ channels. After the tissue was excised, it was to record the voiding behavior. The effects of the drugs homogenized and suspended in 19 volumes of 50 mM were assessed during the course of continuous intravesi- Tris-HCl, pH 7.4 (Wako Pure Chemical Industries, Ltd.) cal infusion of saline following administration of distilled and used as the receptor preparation. [3H]-Des- water or one of the drugs. Thus, the effects of purified methoxyverapamil (Amersham Biosciences, Little water, atropine (1.0 mg/kg), verapamil (10 mg/kg), de- Chalfont, UK) was used as the tracer substance, and sensitization (capsaicin, 125 mg/kg), propiverine (30 mg/ methoxyverapamil (from 1 × 10−8 M to 3 × 10−6 M, kg), solifenacin (10 mg/kg), or tolterodine (3 mg/kg) Sigma-Aldrich) was used as the positive control. The were evaluated. The micturition weights in the groups effects of atropine, propiverine, tolterodine, and solifena- given purified water, atropine, verapamil, capsaicin, cin were assessed in the concentration range of 3 × 10−7 propiverine, solifenacin, and tolterodine were 1.09 ± 0.13, M to 1 × 10−4 M. After the receptor preparations, mea- 1.15 ± 0.06, 1.17 ± 0.06, 1.37 ± 0.10, 1.34 ± 0.12, 1.37 ± surement compounds, and tracers were mixed and incu- 0.14, and 1.40 ± 0.11 g, respectively; and the maximal bated at 25°C for 60 min, the incubated mixture was fil- voiding pressures were 14.5 ± 2.2, 11.6 ± 1.6, 17.2 ± 1.5, tered through a cell harvester (cell harvester M-30R; 19.3 ± 2.6, 15.6 ± 1.0, 14.9 ± 1.2, and 15.7 ± 0.4 mmHg, BRANDEL, Gaithersburg, MD, USA) and then washed respectively. with 50 mM Tris-HCl. The radioactivity was measured with a scintillation counter (scintillation counter 1500 Effects of antimuscarinic agents in the ACh-induced and 2500; PerkinElmer Life & Analytical Science, urinary frequency model Boston, MA, USA). The Kd values were calculated by a The effects of the drugs were assessed during the Scatchard analysis of the results of the binding tests, and course of continuous intravesical infusion of ACh fol- the Ki values were computed using the formula: lowing administration of distilled water or each drug.

Ki = IC50 / (1 + L / Kd). Typical charts are presented in Fig. 1. In regard to the control of the urinary frequency in the ACh-induced Statistical analyses urinary frequency model, atropine caused a 76.0% in- The waveforms of the micturition weight and intra- crease of the micturition weight (P = 0.002) and vera- vesical pressure measurement data were analyzed by pamil produced a 46.0% increase, but the difference was using PowerLab Chart v5.2.2. For all the parameters as- not statistically significant (P = 0.061). Desensitization sessed, the mean and standard error (S.E.M.) of the with capsaicin was ineffective (P = 0.193). None of these measurements from the first micturition to the last mic- treatments had any effects on the intravesical pressure turition were calculated. The paired t-test was used for (Fig. 1). Then, the effects of propiverine (1 – 10 mg/kg), comparison of the pre and post model responses. Com- solifenacin (0.3 – 3 mg/kg), and tolterodine (0.1 – 1 mg/ parison between the results in the normal group infused kg) were explored. In the ACh-induced urinary frequency with physiological saline and those in each of the ACh-, model, propiverine given at the dose of 10 mg/kg, solif-

AcOH-, and CaCl2-induced urinary frequency models enacin at the dose of 3 mg/kg, or tolterodine at the dose was performed by Student’s t-test. Comparison between of 1 mg/kg caused a significant increase in the micturi- the results in each model group and each of the antimus- tion weight without affecting the intravesical pressure carinic agent–treated groups was carried out using Wil- (Fig. 2). The ED50 of propiverine, solifenacin, and tol- liams’ test. Increase of the micturition weight as com- terodine were 3.05, 0.56, and 0.35 mg/kg, respectively. pared with that in the control group was observed in the antimuscarinic agent–related group, and the percent effective ratio was calculated. The ED50 was determined by Antimuscarinics in Pollakiuria Models 367

A : Vehicle B : Atropine

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Fig. 1. Effects of intravesical instillation of 30 mM ACh on the intravesical pressure and micturition weight in conscious freely- moving normal rats and desensitized rats. Upper panel shows the intravesical pressures, and the lower panel shows the micturition weight. The dots represent the micturition points. A: vehicle, B: atropine (1 mg/kg), C: verapamil (10 mg/kg), D: desensitized rat.

1.5 * ** model of urinary frequency evoked by stimulation of capsaicin-sensitive nerves. Desensitization with capsai-

1 cin abolished AcOH-induced urinary frequency without ## affecting the intravesical pressure. Atropine and verapamil were ineffective (P = 0.981, P = 0.948) (Fig. 3). 0.5 Then, the effects of propiverine (3 – 30 mg/kg), solifena-

Micturition weight (g) cin (1 – 10 mg/kg), and tolterodine (0.3 – 3 mg/kg) were

0 assessed. Propiverine given at the dose of 30 mg/kg N C 1 3 10 0.3 1 3 0.1 0.3 1 (mg/kg) caused an increase in the micturition weight without af- Propiverine Solifenacin Tolterodine fecting the intravesical pressure. Solifenacin and tol- ACh 30 mM terodine had no appreciable effect on the micturition Fig. 2. Effects of propiverine (1, 3, and 10 mg/kg), solifenacin (0.3, parameters (Fig. 4). 1, and 3 mg/kg, p.o.), and tolterodine (0.1, 0.3, and 1 mg/kg, p.o.) on the micturition weight in a rat model of ACh (30 mM)-induced urinary Assessments in the CaCl2-induced urinary frequency frequency. N: Normal group (saline infusion + vehicle administra- model tion), C: ACh group (ACh infusion + vehicle administration). Each Rats receiving continuous intravesical infusion of ## value is the mean ± S.E.M. of 6 – 11 animals/group. P < 0.01, sig- CaCl at various concentrations were observed for nificant difference from the saline group (Student’s t-test). *P < 0.025, 2 significant difference from the ACh group (William’s test). changes in their voiding behavior. Continuous infusion of 30 mM CaCl2 did not produce any change in the voiding behavior as compared with the observations in the rats administered intravesical infusion of physiological

Effects of antimuscarinic agents in the AcOH-induced saline. Infusion of 50 mM CaCl2 caused a reduction in urinary frequency model the micturition interval, that is, induced urinary fre- The effects of the antimuscarinic agents on AcOH- quency, without affecting the intravesical pressure. Infu- induced urinary frequency were explored in this animal sion of 80 mM of CaCl2 further shortened the micturition 368 F Sakakibara et al

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Fig. 3. Effects of intravesical instillation of 0.3% AcOH on the intravesical pressure and micturition weight in conscious freely- moving normal rats and desensitized rats. Upper panel shows the intravesical pressures, and the lower panel shows the micturition weight. The dots represent the micturition points. A: vehicle, B: atropine (1 mg/kg), C: verapamil (10 mg/kg), D: desensitized rat.

1.5 urinary frequency model. Verapamil produced a 72.3% increase (P = 0.004) of the micturition weight without * exerting any influence on the intravesical pressure in the 1 rats with urinary frequency induced by 50 mM CaCl2.

## Neither atropine nor desensitization with capsaicin had 0.5 produce any effect (Fig. 6). Micturition weight (g)

0 Effects of antimuscarinic agents in the CaCl2-induced NNC 31030 1 310 0.313(mg/kg) urinary frequency model Propiverine Solifenacin Tolterodine The effects of propiverine (1 – 10 mg/kg), solifenacin 0.3% AcOH (0.3 – 3 mg/kg), and tolterodine (0.1 – 1 mg/kg) on Fig. 4. Effects of propiverine (3, 10, and 30 mg/kg), solifenacin (1, CaCl2-induced urinary frequency were explored. As 3, and 10 mg/kg, p.o.), and tolterodine (0.3, 1, and 3 mg/kg, p.o.) on compared with the observations in the control group, the micturition weight in a rat model of AcOH (0.3%)-induced urinary propiverine at 10 mg/kg significantly increased the mic- frequency. N: Normal group (saline infusion + vehicle administra- turition weight without exerting any effects on the intra- tion), C: AcOH group (AcOH infusion + vehicle administration). vesical pressure. The micturition weight tended to in- ## Each value is the mean ± S.E.M. of 18 animals/group. P < 0.01, crease in response to administration of solifenacin or significant difference from the saline group (Student’s t-test). *P < 0.025, significant difference from the AcOH group (William’s test). tolterodine at the doses tested (Fig. 7). In view of this finding, we further assessed the effects of these two drugs at higher dose levels in a preliminary study, but neither drug produced any significant improvements (data not interval, so that it was difficult to identify micturition at shown). The ED50 of propiverine was 3.29 mg/kg and the all in occasional animals (Fig. 5). Based on these results, values for the other antimuscarinic agents could not be a CaCl2 concentration of 50 mM was adopted for this calculated. Antimuscarinics in Pollakiuria Models 369

A : 0 mM B: 30 mM

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Micturition -1 -1 06309000090 06330900 Time (min) Time (min)

Fig. 5. Effects of each concentration of intravesical CaCl2 on the intravesical pressure and micturition weight in conscious freely-moving rats. Upper panel shows the intravesical pressures, and the lower panel shows the micturition weight. The dots represent the micturition points. A: 0 mM, B: 30 mM, C: 50 mM, D: 80 mM.

A : Atropine B : Verapamil

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Intravesical Intravesical 0 0630900 56 45 Fig. 6. Effects of intravesical instillation of 50 mM CaCl2 stimula- 34 3 tion on intravesical pressure and micturition weight in conscious 2 (g) 1 freely-moving normal rats and desensitized rat. Upper panel shows weight 0

Micturition -1-1 the intravesical pressure, and the lower panel shows the micturition 063309000090 weight. The dots represent the micturition points. A: atropine (1 mg/ Time (min) kg), B: verapamil (10 mg/kg), C: desensitized rat. 370 F Sakakibara et al

1.5 * to improve the symptom of urinary frequency in rats with ibotenic acid–induced destruction of the nucleus basalis magnocellularis (13), and verapamil at the dose of 10 1 ## mg/kg, p.o. was reported to produce a significant decrease of the blood pressure in normal rats (14); therefore, these 0.5 doses of atropine and verapamil were selected for this

Micturition weight (g) study. Desensitization with capsaicin was performed in the usual manner (3). The administration of each drug 0 N C 1 3 10 0.3 1 3 0.1 0.3 1 (mg/kg) and the desensitization had no significant effect on the Propiverine Solifenacin Tolterodine micturition weight or intravesical pressure in normal

CaCl2 50 mM animals. In the evaluation of antimuscarinic agents, atropine Fig. 7. Effect of propiverine (1, 3, and 10 mg/kg), solifenacin (0.3, exerted a conspicuous ameliorative effect in the ACh- 1, and 3 mg/kg, p.o.), and tolterodine (0.1, 0.3, and 1 mg/kg, p.o.) on induced urinary frequency model, whereas the Ca2+ an- the micturition weight in a rat model of CaCl2 (50 mM)-induced urinary frequency. N: Normal group (saline infusion + vehicle adminis- tagonist verapamil produced some improvement, but not tration), C: CaCl2 group (CaCl2 infusion + vehicle administration). statistically significant, of urinary frequency in this Each value is the mean ± S.E.M. of 6 or 7 animals/group. ##P < 0.01, model. Verapamil was demonstrated to reduce the maxi- significant difference from the saline group (Student’s t-test). *P < mal contractile response to carbachol (pD2′ = 6.98) in a 0.025, significant difference from the CaCl group (William’s test). 2 study on isolated urinary bladder (15), and this effect may underlie the improvement of the urinary frequency in the ACh-induced urinary frequency mode. It was thus Affinity of antimuscarinic agents for PAA-sensitive L- inferred that in addition to the antimuscarinic activity, type Ca2+ channel the Ca2+ channel–antagonizing activity might also con-

In order to corroborate their efficacy in the CaCl2- tribute to the control of ACh-induced urinary frequency. induced urinary frequency model, we investigated the In the ACh-induced urinary frequency model, the anti- binding affinity of the anticholinergic drugs for PAA- muscarinic agents used in this study were demonstrated sensitive L-type Ca2+ channels. The positive control to cause a dose-dependent increase in the micturition methoxyverapamil inhibited [3H]-desmethoxyverapamil weight; this finding was consistent with previously re- binding in a concentration-dependent manner, and the Ki ported data obtained in a rat model of cerebral infarction value for the PAA-sensitive Ca2+ channels was 4.97 × 10−8 (16). M. It was confirmed that all of the anticholinergic agents We also conducted assessments in the AcOH-induced also inhibited [3H]-desmethoxyverapamil binding in a urinary frequency model established via stimulation of concentration-dependent manner under these conditions. capsaicin-sensitive nerves (3). None of the drugs, includ- The Ki values of propiverine, solifenacin, tolterodine, ing atropine, the other two specific antimuscarinic drugs and atropine for PAA-sensitive Ca2+ channels were examined (solifenacin and tolterodine) and verapamil, 1.61 × 10−6, 2.91 × 10−6, 6.38 × 10−7, and 4.18 × 10−5 M, improved urinary frequency, whereas significant im- respectively. Based on these results it was concluded that provement of the urinary frequency was observed in the propiverine, solifenacin, and tolterodine possess affinity propiverine-treated group and the group that was sub- for the PAA-sensitive Ca2+ channels and exhibit antago- jected to capsaicin desensitization. It has been demon- nist activity. strated that propiverine directly binds to transient receptor potential vanilloid subtype 1 (TRPV1) and thereby inhib- Discussion its capsaicin-stimulated Ca2+ influx (17). The inhibition of AcOH-stimulated urinary frequency observed in the The present study represents an attempt to examine present study was considered to be consequent to inhibi- whether antimuscarinic agents currently in use in the tion of C-fiber hyperfunction via inhibition of Ca2+ influx clinical setting might be useful in the management of caused by propiverine binding to TRPV1. It was inferred urinary frequency mediated by atropine resistant contrac- from these findings that antimuscarinic and Ca2+ channel– tions, by comparing the drugs in three different urinary antagonizing activities are ineffective for the control of frequency animal models. The doses of the antimusca- urinary frequency caused by C-fiber stimulation, whereas rinic agents employed in this study were determined by propiverine alleviates the urinary frequency in this model reference to human and rat plasma concentrations of via TRPV1. these drugs (7 – 12). Of the drugs used as positive con- A CaCl2-induced urinary frequency model in which trols, atropine at the dose of 0.32 mg/kg p.o. was reported concentration-dependent urinary frequency can be Antimuscarinics in Pollakiuria Models 371

evoked by continuous intravesical infusion of CaCl2 has than 100,000-fold difference in the binding affinity of 2+ been newly established. The CaCl2-induced urinary fre- atropine for Ca channels and muscarinic receptors was quency model was considered as a non-cholinergic uri- found (23). Among the anticholinergic drugs, the Ki nary frequency model in as much as improvement of the value of propiverine for the PAA sensitive L-type Ca2+ urinary frequency in this model was obtained by admin- channels was 4.6-fold higher than for the M3 muscarinic istration of atropine. The Ca2+-channel antagonist vera- receptor (24), which is thought to be the reason for the pamil, on the other hand, also improved the urinary fre- close ED50 values obtained between the ACh-induced quency, indicating that the model represents a frequency model and CaCl2-induced frequency model. Ca2+-induced urinary frequency model as well. Desensi- Thus, propiverine was confirmed as a drug capable of tization with capsaicin had no effect on the urinary fre- improving urinary frequency at clinical dose levels both quency in this model, suggesting the absence of signifi- via its antimuscarinic effect and its effect of inhibiting cant involvement of capsaicin-sensitive sensory nerves. Ca2+ influx into the urinary bladder myocytes. A more Zhang et al. (18) obtained evidence showing that ATP than 100-fold difference was observed in the binding could also be an NANC mediator in the bladder because affinity of tolterodine and solifenacin between the M3 ATP-induced urinary frequency was not inhibited by muscarinic receptors and the PAA sensitive L-type Ca2+ resiniferatoxin desensitization. The results would be channels (24), which was inferred to be the reason why strengthened by investigating the involvement of puri- effect of these drugs in ameliorating urinary frequency nergic nerves in the CaCl2-induced urinary frequency. was inadequate at the doses used in this study. The effi- However, it was difficult to prove that the CaCl2-induced cacy of administering solifenacin at 100 mg/kg and tol- urinary frequency model was equivalent to the only pu- terodine at 30 mg/kg was investigated in a preliminary rinergic nerve-mediated urinary frequency model based study; however, the increase in amount of the micturition on only this study. weight decreased. Moreover, the effect of solifenacin This model was considered to reproduce a disease state and tolterodine, although weak, in ameliorating urinary in which Ca2+ homeostasis plays a significant role, in that frequency is likely to be attributable to their anticholin- there have been reports demonstrating the effectiveness ergic actions. of Ca2+ antagonists in the management of urinary ur- In the present experiments, the urinary frequency gency, frequency, and incontinence (19). Meanwhile, the symptom due to cholinergic nerve stimulation was found Ca2+ channel antagonist verapamil has been documented to be responsive to solifenacin, tolterodine, and propi- to increase the bladder capacity and at the same time, verine. Urinary frequency due to stimulation with AcOH induce arrhythmia when administered intravenously in or Ca2+, and hence not mediated by the cholinergic ner- rats with bladder outlet obstruction (20). On account of vous system, on the other hand, was not responsive to the the noticeable influence of the clinically used Ca2+ an- highly selective muscarinergic receptor antagonists, tagonists on the cardiovascular system, we considered whereas propiverine, endowed with both antimuscarinic that Ca2+ antagonists with high selectivity for the urinary and Ca2+ channel–antagonizing activities, produced bladder relative to that for the cardiovascular system marked improvement. would be preferable for the management of urinary fre- It has been documented in contraction experiments on quency and urgency associated with abnormalities of detrusor strips that ATP- and CaCl2-induced contractions Ca2+ homeostasis. Of the antimuscarinic drugs used in were conspicuously enhanced in the bladder of patients this study, propiverine produced attenuation of the uri- with neurogenic bladder, whereas ACh-stimulated detru- nary frequency in the CaCl2-induced urinary frequency, sor contractions were essentially comparable between while the other agents proved less effective. Propiverine patients with neurogenic bladder and healthy subjects is known to have an inhibitory effect on ACh-, KCl-, or (25). According to a report by Yoshida et al., ATP-

CaCl2-induced contractile responses of human and rat induced detrusor contractions in humans increased pro- detrusor muscles (2, 21). Furthermore, propiverine has gressively with advancing age in humans (26). It has also been demonstrated to inhibit Ca2+ uptake in experiments been demonstrated in contraction inhibition tests with conducted with isolated guinea-pig urinary bladder and atropine and α,β-methylene ATP on detrusor contractions murine urinary bladder myocytes and is known to be evoked by electrical field stimulation in humans and rats endowed with both antimuscarinic activity and Ca2+ an- that the cholinergic nerve system–mediated contractile tagonistic activity (5, 22). elements diminished and the purinergic nerve system– When we calculated the Ki values of atropine and the mediated contractile elements increased progressively other antimuscarinic agents for the PAA sensitive L-type with advancing age in humans (26). Therefore, it is Ca2+ channels in order to corroborate efficacy of these generally thought that the effects of antimuscarinic drugs agents in the CaCl2-induced frequency model, a more are weak in patients with neurogenic bladder and elderly 372 F Sakakibara et al subjects and that inhibition of atropine-resistant contrac- 9 Olsson B, Brynne N, Johansson C, Arnberg H. Food increases tions including the NANC nervous system-mediated the bioavailability of tolterodine but not effective exposure. J contractions is important for improving the urinary fre- Clin Pharmacol. 2001;41:298–304. quency and urgency associated with an OAB neurogenic 10 Påhlman I, Kankaanranta S, Palmér L. Pharmacokinetics of tolterodine, a muscarinic receptor antagonist, in mouse, rat and dog. bladder in elderly patients. Attention is being focused Arzneimittelforschung. 2001;51:134–144. increasingly on the role of atropine-resistant contractions 11 Tanaka T, Yamaguchi O. Phase I clinical study of solifenacin in the treatment of urinary frequency and urgency due to succinate. –Single oral administration study–. Jpn Pharmacol OAB or neurogenic bladder and numerous studies have Ther. 2006;34:S5–S13. been conducted (27, 28). In the clinical setting, intravesi- 12 Yanagihara T, Aoki T, Soeishi Y, Iwatsubo T, Kamimura H. De- cal resiniferatoxin or botulinum toxin A–injection therapy termination of solifenacin succinate, a novel muscarinic receptor has been shown to be successful for the control of abnor- antagonist, and its major metabolite in rat plasma by semi-micro high performance liquid chromatography. J Chromatogr B. 2007; mal excitation of atropine-resistant contractions in pa- 859:241–245. tients with symptoms refractory to antimuscarinic drugs 13 Yamamoto T, Koibuchi Y, Miura S, Sawada T, Ozaki R, Esumi (29). K, et al. 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