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Effects of Vamicamide on Urinary Bladder Functions in Conscious Dog and Rat Models of Urinary Frequency
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Vol 154.2174-2178, December 1995 Printed in U S.A
T f i E JOURNAL OF UROLOGY
Copyright 0 1995 by AMERICANUROUX~ICAL ASSOCIATION, INC.
EFFECTS OF VAMICAMIDE ON URINARY BLADDER FUNCTIONS IN CONSCIOUS DOG AND RAT MODELS OF URINARY FREQUENCY TAKA0 YAMAMOTO, YASUSHI KOIBUCHI,* SHINTARO MIURA, TADASHI SAWADA, REIKO OZAKI, KIM10 ESUMI AND MINORU OHTSUKA From the Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan
ABSTRACT
Purpose: To investigate the usefulness of vamicamide, ( 5 )-(2R*, 4R*k4-dimethylamino-2phenyl-2-(2-pyridyl)valeramide,as a novel drug for the treatment of urinary frequency and incontinence. Materials and Methods: Urinary frequency was evaluated in specially devised conscious dog and rat models by investigating the effects of the drug on urinary bladder function of these animals by cystometrography. Results: In the dog model with transected hypogastric nerves, the bladder volume at micturition (bladder capacity) was less than 50% that of the sham-operated dog, and in the rat model with bilateral lesioning of nuclei basalis, a part of the brain, by ibotenic acid injection, bladder capacity was about 50% that of the sham-operated rat. Other bladder functions in both models were unchanged. In the dog model, orally administered vamicamide a t 0.32 and 1.0 mg./kg. significantly increased bladder capacity and did not change residual urine volume or micturition pressure. Oxybutynin 0.10 mg./kg., one of the most popular drugs for the treatment of urinary frequency and incontinence, or atropine 0.10 mg./kg. induced significant increases in bladder capacity similarly to vamicamide at 0.32mg./kg. In the rat model, oral vamicamide 0.32 mg./kg. also significantly increased bladder capacity and did not change micturition pressure or threshold pressure. Again, oxybutynin 0.10 mg./kg. or atropine 0.32 mg./kg. had almost the same effects as vamicamide 0.32 mg./kg. Conclusions: These findings suggest that vamicamide should be useful for the treatment of urinary frequency. KEYWORDS:bladder, animals, parasympatholytics Recently, the number of patients with urinary frequency and/or urinary incontinence attributed to neurogenic or unstable bladder has been increasing. In particular, the number of patients with neurogenic bladder that results from functional disturbance of the brain (for example dementia and cerebrovascular disease), injury of the spinal cord and peripheral neuropathy (for example, diabetic neuropathy) is expected to increase concomitantly with the aging of society and the increase in traffic accidents. As urinary bladder contraction is controlled largely by the parasympathetic nerve, anticholinergic (antimuscarinic) drugs, for example oxybutynin and propantheline, are generally used for the treatment of urinary frequency and incontinence, but are not necessarily adequate. Therefore, a new drug for the treatment of these diseases is eagerly awaited. To investigate the effects of drugs for the treatment of urinary frequency on urinary bladder function, distensioninduced rhythmic bladder contraction,' bladder contraction induced by pelvic nerve stimulation2 and cystometrography1.3.4 are routinely used as in vivo methods. Among these methods, cystometrography approximates clinical tests most closely, but the animals used in experimental cystometrographies generally have normal bladder function. Moreover, the drugs are administered intravenously instead of orally, as in clinical treatment, because the animals tested are usually under anesthesia or decerebration. Therefore, we devised 2 conscious animal models which had smaller bladder capacities than normal and for whom oral administration was available.
Vamicamide is a new drug for the treatment of urinary frequency and incontinence which has selective antimuscarink action to the m3 receptor subtype and longer inhibitory action on the urinary bladder than on the salivary gland and ~ t o m a c hWe . ~ investigated the effects of vamicamide on urinary bladder function in the animal model by means of cystometrography. MATERIALS AND METHODS
Dog model. Male beagle dogs (weighing 7 . 5 to 11.5 kg.) were anesthetized with sodium pentobarbital ( 3 0 mg./kg. intravenously). The abdomen was opened through a midline incision, and 2 tine polyethylene tubes (0.d. 2.0 mm.) were implanted into the bladder. The hypogastric nerves on both sides were ligated and cut at a level about 2.5 cm. distal to the caudal mesenteric ganglia. The nerves of the sham-operated dogs were not ligated or cut. More than 6 days after implantation, the dogs were placed in metabolic cages, which permitted measurement of urine volume a t micturition (micturition volume) by graduated cylinder, and cystometrography was performed. One tube was connected to a pump for saline infusion, and the other was connected to a pressure transducer (PlOEZ-1,Nihon Kohden, Tokyo, Japan) to measure intravesical pressure. The whole system was filled with saline. Saline ( 3 7 C )was infused into the bladder a t a rate of 5 ml. per minute just after the intravesical urine was drained through an infusion tube, and intravesical pressure was recorded continuously on a polygraph system (RM-6000,Nihon Kohden 1. When micturition occurred. saline infusion was Accepted for puhlication J u n e 22, 1995. * Requests for repnnts: Pharmacological Research Laboratones, stopped and the time from the beginning to the end of infuFujisawa Pharmaceutical Co.. Ltd., 2-1-6 Kashima. Yodogawa-ku, sion = micturition interval I ~ n . smwsured to cnlculatt. the Osaka 532. Japan. volume of infus;cd saline. 'rhv li)llovb iiig cyston~etricparame217.4
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
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k r s were investigated: bladder capacity (volume of infused Comparison test following the randomized block designed saline until micturition), micturition pressure (maximum in- alnalysis of variance. A probability level c0.05 was accepted travesical pressure at micturition) and residual volume aIS significant. (bladder capacity minus micturition volume, residual volume of saline in the bladder just after micturition). Test RESULTS drugs were administered orally after the cystometrogram had become stable. The number of dogs in each group was 4. In conscious dogs, slow saline infusion into the urinary bladCystometrography was performed 1hour after drug admin- ler increasedthe inhvesical pressure and induced micturition istration, and the effects on cystometric parameters (= blad- tccompanied with transient bladder contraction (fig. 1). The der function) were assessed by comparing with the averages yatometrogrampattern of the hypogastric denervated dog was of 2 cystometrograms just before drug administration. The h o s t the same as that of the sham-operated dog, but the pupillary diameter of the dog was measured before and 1 )ladder capacity (volume of infused saline until micturition) hour after drug administration. uas significantly smaller (table 1).Micturition pressure (maxRat model. Male Wistar rats (7 to 8 weeks old) under mum intravesical pressure at micturition)and residual volume sodium pentobarbital anesthesia (45 mg./kg. intraperitone- residual volume of saline in the bladder just aRer micturition) ally) were operated on according to the methods of Dubois et were not changed by the denemation, and reaidual volume was al.6 and Hara et al.? Rats were placed in a stereotaxic appannall in this modeL We also tried to measurethreaholdpresratus (Kopf Instruments, Tujunga, California)and subjected 3ure (intravesicalpressure immediately before micturition)but to bilateral lesion of the brain by ibotenic acid injection. The muld not always do it w d y because, during the cy% coordinates of the cannula placements used were: anterior, tomehgraphy, the dogs were often in a sitting position until 7.7 mm.; lateral, 2.4 mm.; horizontal, 2.9 mm. according to Jjust before micturition although they were always in standing Paxinos and Watson.8 Ibeotenic acid was infused through a position at micturition. 0.4 mm. stainless steel cannula connected via Teflon tubing In the hypogastric denervated dogs, vamicamide (0.32and to a Hamilton microsyringe. Ibotenic acid (Research Bio- 1.0 mg./kg.) significantly increased bladder capacity and did chemicals,Inc., Natick, Massachusetts) was dissolved in Dul- not change residual volume or micturition pressure 1hour becco's phosphate buffered saline (D-PBS, pH 7.4) at a con- aRer oral administration (fig. 2). At only 1.0 mglkg. mild centration of 10 mg./ml. and infused in a volume of 0.75 4. mydriasis was observed (fig. 3). At the highest dose of vamifor 3 minutes by means of a syringe pump. The sham-oper- camide (3.2mglkg.), a decrease in micturition pressure with ated rats were infused with only D-PBS. One to 3 weeks after severe mydriasis was observed, and the effect on bladder surgery, rats were subjected to cystometrography. Under capacity was attenuated. Oxybutynin (0.10mgJkg.1 or atroether anesthesia, the abdomen was opened through a midline pine (0.10 mglkg.) induced significant increases in bladder incision, and a fine stainless steel cannula (0.d. 1.0 mm.)was capacity, and their potencies were almost the same as that of implanted into the bladder. The cannula was connected to a vamimmide (fig. 2). These drugs also did not change residual double lumen tube (outer tube: 0.d. 1.7 mm., inner tube: 0.d. volume or micturition pressure. At a higher dose (0.32mgJ 0.61 mm.) to infuse saline (370and to measure intravesical kg.), however, they did not induce an increase in bladder pressure simultaneously. A polyethylene tube (0.d. 0.97 mm.) capacity, and mild (oxybutynin) or severe (atropine) mydriawas placed into the stomach via the esophagus to administer sis was observed. Moreover, oxybutynin (1.0 mglkg.) showed drugs orally. Rats were placed in Bollman's cages and al- a tendency to decrease micturition pressure with severe mylowed to recover from anesthesia for about 3 hours. The free driasis, and atropine induced a decrease in micturition prestips of the bladder tubing were connectedto a pressure trans- sure even at 0.32 mgJkg. Cystometrograms of the conscious rats were almost the ducer (TP400T.Nihon Kohden: outer tube) and to a syringe pump (inner tube). Saline (37C)was infused into the bladder same as those of the conscious dogs except that bladder by means of a syringe pump at a rate of 61.5 4. per minute, capacity and micturition pressure were smaller in rats (data and intravesical pressure was recorded continuously on a not shown). Rats with bilateral lesions on the nuclei basalis polygraph system (RM-6000, Nihon Kohden). The micturi- by ibotenic acid injection had significantly smaller bladder tion interval was measured to calculate the volume of infused capacity than the sham-operated rats, but micturition pre% saline at each cystometrogram. The following cystometric sure and threshold pressure did not differ (table 2). parameters were investigated: bladder capacity, micturition pressure and threshold pressure (intravesid pressure hmediately prior to micturition). Test drugs were adminisSham Operation tered orally via preplaced polyethylene tube after the cystometrogram had become stable. The number of rats in each group was 5 with only a few exceptions (4 or 7).Cystometrography was performed 30 minutes (or 2 or 3 hours) after drug administration, and the effects on cystometric parameters (the averages of 2 or 3 cystometrograms) were assessed by comparing with the averages of 3 cystometrogramsjust be5mk 10 mm S M of (smw fore drug administration. Saline infusion (Drugs.Vamicamide (synthesized by Fujisawa PharmaceuTransection of Hypogastric Nerve tical Co. La.,Osaka, Japan) was dissolved in quimolar hydrochloric acid, neutralized by sodium bicarbonate and diluted with water. Oxybutynin (oxybutynin hydrochloride, synthesized by Fujisawa Pharmaceutical Co., La.) and atropine (atropine sulfate monohydrate, Wako Pure Chem. Inc., La.,Osaka, Japan) were dissolved in water. stut 01 5Statistical analysis. The results are given as mean values Saline Infusion (2 the standard error of the mean. Unpaired comparisons between operated and sham-operated groups were performed FIG.1. Typical cystometrogramtracing in conscious dog. Ivp:inby Student's or Aspin-Welch t test, and paired comparisons travesical reasure; $ :start of micturition ( = end of saline infusion); between treatments before and after drug administration HorizonJaxis: time from beginning of iuiimon; ( ): volume of were performed by Student's t test or Dunnett's multiple infused ealine.
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
2176
TABLE1. Cvstometric parameters of conscious dogs Sham-operated Hypogastic denervated
61.5 2 Bladder capacity (rnl.) Micturition pressure (rnm.Hg) 25.3 2 3.6 5 Residual volume (rnl.) Mean values 2 the standard error of the *: p (0.05 versus sharn-operated group.
8.5 5.4 1.3 mean ( n
26.3 f 1.2* 33.5 z 4.7 2.3 2 0.6 4).
=
9
6
6
=
5 .
0.1
40
1
ml 30 Residual Volume
**
I*
3 2 mgha
I
0.32
0012
0.1
mmHg 80
I nUh4
0032 0.1 0.32 Dmc
I mgh'
FIG.3. Effects of vamicamide and other drugs on pupillary diameter in conscious hypogastric denervated dogs. Open column: predosing. Closed column: postdosing (1 hour after administration). Mean values 2 standard error of mean (n = 4). *, **: p 10.05 or 0.01 versus predosing.
25
% .
032
Dor
DLU
Vamicamidt ml 45 Bladder Capacity
60 40
TABLE2 . Cystometric parameters
10
20
Dose
0.1 0.32 1 3.2 mgkg Dose
mgkg
0.1 0.32 1 Dose
3.2 mgkg
oxybutynin ml
45 1 Bladder Capacity
ml
30 1 Residual Volume
Bladder Capacity ( ~ 1 . ) 843 2 71 443 i El** 18.9 5 1.6 17.5 2 1.5 Micturition Pressure (mm.Hg) 4.1 2 0.8 Threshold Pressure (rnm.Hg) 4.2 2 0.7 Mean values 2 the standard error of the mean (n = 5 to 6) **: p <0.01versus sham-operated group.
mmHg 80 1 Micturition Pressure Vamicamide
40 35 30
:$
60
Threshdd Rrrurr
Bladder Capacity
Micturition R r r u r c
mmHg
mmHg
40
10
25
20
5
20 15
of conscious rats
Sham-operated Ibotenic acid treated
0
0.032 0.1 0.32 I m a g Dose
0.0320.1 0.32 1 m a g Dose
Ampine ml 45 Bladder Capacity
ml 30 Residual Volume
M .-
25 -_
I .
0
0.032 0.1 0.32 1 mgkg Dose
0.1
I
mmHg
I
':L 60
25
10
0.032 0.10.32 I m a g Dose
" 0.032
- 0.032 0.1
.=
r
0.032 0.1
O.32mehg
-
0 0.032 0.1 0 . 3 2 m 0 g
Dore
Rcrw~
:"ifll
200
0.032 0.1
Dw
0.32mgkg
DOU
Atropine
n
0.1 0.32 I m%lrg Dose
5
Micturilion
mmHg
"Add
20
5
20
Threshold Rswrc 20
8001:
40
30
Dose
mmHg
IOM
O32mOg
01
DOX
PI
80 Micturition Pressure
0 3 2 mgkg
01
Bladder Capaciiy
35
IC ._
O32mOg
Dw Oxybulynin
Threshold Rsrurc
Bladder Capacity PI
0.32 I mg/Lg Dose.
FIG.2. Effects of vamicamide and other drugs on urinary bladder function in conscious hypogastric denervated dogs. Open column: predosing. Closed column: postdosing (1 hour after administration). Mean values 2 standard error of mean (n = 4). *, **: p <0.05 or 0.01 versus predosing.
Vamicamide (0.32 mg./kg.) induced significant increases in bladder capacity a n d did not change micturition or threshold pressure (intravesical pressure immediately before micturition) in t h e conscious rats with ibotenic lesions 0.5 to 1 hour after oral administration (fig. 4). Almost t h e same effects were observed with oxybutynin 0.10 mg.kg. or atropine 0.32 mg./kg., but a high dose of oxybutynin (0.32mg./kg.) failed to increase bladder capacity (fig. 4). Atropine 0.10 mg./kg. showed a tendency to increase bladder capacity. We also investigated t h e duration of t h e effects of vamicamide and oxybutynin on bladder capacity in this model and found a significant effect of vamicamide 0.32 mg./kg. lasting for 3 to 3.5 hours, while the effect of oxybutynin 0.10 mg./kg. disappeared within 2 hours (fig. 5).
Micturition R r r u m
Y2
%?
r
800
200
0 32 mehg
0.1 Dose
rln
0.32 m a g
0.1
Dose
0.1
0 . 3 2 mgkg
Dose
FIG. 4. Effects of vamicamide and other drugs on urinary bladder function in conscious rats with ibotenic lesion of nuclei basalis. Open column: predosing; closed column: postdosing (0.5 to 1.0 hour after administration). Mean values 2 standard error of mean ( n = 4 to 5 ) . *: p 10.05 versus predosing. DISCUSSION
Urinary bladder contraction is induced by activation of t h e parasympathetic pathway (pelvic nerves), a n d stimulation of t h e hypogastric nerves (sympathetic pathway) elicits inhibitory responses in t h e bladder due to direct inhibitory action on smooth muscle cells and suppression of t h e parasympathetic ganglia on t h e surface of t h e bladder, too.:'."-11 Fukudal" reported that bilateral hypogastric nerve transection of t h e decerebrate dog induced a decrease of about 20% in bladder capacity and Morikawn et aI.1:' reported t h a t the same operation in conscious rats induced a decrease of about 50%.Similarly, bladder capacity ot'thc conscious hypogastric
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
2177
antimwcarinic action to the m3 receptor subtype, which seemed to be responsible for contraction of the bladder in vitro, and that it had longer antimwcarinic action on the bladder than on the salivary gland and stomach in vivo.6 Judging from such a pharmacological profile, vamicamide ** * seemed to be suitable for the treatment of urinary firesuency and incontinence because antimusuuinic drugs are generally used for the treatment of these diseases, but both efficacy and elimination of side effects (dry mouth, mydriasie) are not necessarily adequate. A clinical trial of vamicamide for the treatment of urinary frequency and incontinenceis presently underway. In the dog model of urinary fresuency, orally administered vamicamide (0.32 and 1.0 mg./kg.) induced a significant increase in bladder capacity without changing residual urine volume and micturition pressure (fig. 2). In the rat model of urinary frequency, vamicamide 0.32 mg-. induced a significant increase in bladder capacity without " 0.5-1.0 2.0-2.5 3 . 0 - 3 . 5 hr changing micturition pressure and threshold preesure (fig. 4). Oxybutynin 0.10 mgJkg., one of the most popular drugs Time after administration for the treatment of urinary frequency and incontinence, showed almost the same effects as vamicamide 0.32 mg./kg. in both animal models. These findings suggested that vami0 VUnicMlide camide, like oxybutynin, could improve urinary fresuency Post (0.32mgkgp.o.) without influencing other bladder functione. Oxybutynin was FtG. 5. Duration of effect of vamicamide or o but on bladder said to inhibit bladder contraction both by an antimmcarinic capacity in conscious rate with ibotenic lesion 3 n u c E a e a l i e . re: effect and by other mechanisms, such as a direct muscle fredosing. Post: pstdosing. Mean values % standard error of mean relaxant effect, but the latter was observed at 100-foldhigher II = 5 to 7). *, * . p <0.05 or 0.01 versus predosing. concentration than the former in our previous study.6 Judging from our previous data, the pMcipal mechanism, not only of vamicamide but also of oxybutynin, for increasing denervated dogs was less than 50% that of the sham-oper- bladder capacity in both models seemed to be blockade of ated dogs in our study (table 1).These facts strongly suggest mwcarinic receptors on the bladder smooth muscle cells that the hypogastric nerves play an important role in bladder connecting to the nerve endings. In the dog model, both function and that the dog whose bladder capacity is de- vamicamide and oxybutynin induced mild mydriasis at 3.2 creased by meana of hypogastric denervation is a useful times higher doses than the minimum effective doses on the model of urinary frequency caused by peripheral nervous bladder (fig. 3). On the other hand, atropine, one of the most system disorder, which causes superiority of the parasympa- typical antimusearinic drugs, also induced a significant inthetic nerve over the sympathetic nerve in the bladder. We crease in bladder capacity at 0.10mghg., but induced severe therefore performed cystometrography on the hypogastric mydriasis and decrease in micturition pressure at 0.32 mgJ denervated dog to investigate the effects of vamicamide. kg.in the dog model (figs. 3,4). Judging from these findings, Moreover, as we used conscious dogs, this test had other the antimucarink side effects of atropine might happen advantages over the cystometrographiesperformed formerly. more fresuently than those of oxybutynin or vamicamide, if These were ability to administer the drugs orally, eliminate atropine should be administered for the treatment of urinary the effects of anesthesia and investigate the pupillary diam- frequency. Moreover, the fact that, in the rat model, vamiceter simultaneously as a parameter of antimusuuinic side amide had a longer lasting effect on bladder capacity than oxybutynin suggested that the efficacy of vamicamide on effects. than that of 0xybutyn.h The conscious rats with ibotenic lesions of the nuclei basa- urinary frequency might last longer . lis also had smaller bladder capacity than the sham-operated (fig. 5j. In the dog model, the increasing effect of vamicamide on rats, as was the case in the dog model (table 2). Generally, this operation (ibotenic acid treatment) is used to cause se- bladder capacity was attenuated at a dose that induced severe neuronal damage to the nucleus basalis magnocellularis vere mydriasis (3.2mgJkg.) and that of oxybutynin or atro(NBM) in the basal forebrain as a model of senile dementia of pine disappeared at the minimum dose inducing mydriasis the Alzheimer type, and it was reported that the decrease of (0.32mgJkg.) (fig. 2).Also, the increasing effect of oxybutycholine-acetyltransferase (ChAT)activity in the prefrontal nin on bladder capacity in the rat model disappeared at a and sensorimotor cortices was evidence of such damage.6 higher dose (0.32mg.). The reasons for these phenomena are Recently, Hara et al.7 reported that rats with NBM lesions by not clear, but there have been suggestions in other reports. ibotenic acid injection showed urinary incontinence 2 to 3 All 3 drugs are considered to increase bladder capacity days aRer the omration. They attributed this finding to chiefly by blocking muscarinic receptors on the smooth musdamage in the adjacent lateralhypothalamw. judging f;om cle cells connected to nerve endinas, but Gallaaher et al.14 the fact that decreases not only of cortical ChAT activity but and de Groat et al.16 reported that there were sts synaptic also of plasma antidiuretic hormone (ADH)were observed. In muscarinic receptors in the parasympathetic ganglia on the the present study, the conscious rats with ibotenic lesions surface of the bladder as well. The musCarinic receptors in had smaller bladder capacity than the sham-operated rats the parasympathetic ganglia mediated inhibitory postsynap(table 2),and it suggested that their symptom was not merely tic potential although the nicotinic receptors in the ganglia due to polyuria induced by a decrease in plasma ADH but mediated major excitatory postsynaptic potential. Judging rather to the impairment of regulation of micturition from from these reports, a n t i m d c drugs might block, not the central nervous system. Although the exact cause of only contractile muscarinic receptors on the bladder smooth these symptoms is unknown, rats with ibotenic lesions of the muscle cells, but also inhibitory muscarinic receptors in the nuclei basalis would be a useful model of pollakiuria caused ganglia when the dose was high enough to induce antimuscarinic side effects and their effect to increase bladder capacby central nerve system disorder. Previously, we reported that vamicamide had a selective ity was attenuated or disappeared. Indeed, Morikawa et al.l
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ANIMAL. MODELS OF URINARY FREQUENCY AND VAMICAMIDE
reported that high doses of atropine (0.01 and 0.1 mg.lkg., intravenously) suppressed the amplitude of distensioninduced rhythmic bladder contractions but increased their frequency in treated rats. In the same paper, the same doses of atropine, 0.01 and 0.1 mg.kg. intravenously, were reported not to induce an increase in bladder capacity in the cystometrographiesof anesthetized rats and dogs. Moreover, Guarneri et a1.16 reported that oxybutynin administered intravenously induced a significant increase in bladder capacity only at 0.03 mg./kg., but not at higher doses, in the eystometrography of conscious rats. These reports seem to support our hypothesis. In conclusion, our results of cystometrographies in 2 conscious animal models of urinary frequency strongly suggested that vamicamide should be useful for the treatment of urinary frequency caused by neurogenic or unstable bladder. REFERENCES
1. Morikawa, K., Kakiuchi, M., Yamauchi, T., Hashimoto, S.,
Miyashita, N., Sawada, Y., Kato, H. and Ito, Y.: Pharmacological studies on the micturition reflex. 11. Effects of various drugs on bladder and urethral functions in rats and dogs. Oyo Yakuflharmacometrics, 37: 27, 1989. 2. Nishizawa, 0.. Moriya, I., Fukuda, T., Matsuzaki, A. and Tsuchida, S.: Effects of the autonomic antagonists on canine urethral responses to autonomic nerve stimulation. Urol. Int., 40:314, 1985. 3. De Groat, W. C. and Saum, W. R.: Sympathetic inhibition of the urinary bladder and of pelvic ganglionic transmission in the cat. J. Physiol., 220 297, 1972. 4. Tsuchida, S., Nishizawa, 0. and Noto, H.: A comparative study of the effects of propiverine, terodiline, oxybutynin on the lower urinary tract function in the decerebrate dog. Acta Urol. Jpn., 3 6 915, 1990. 5. Yamemoto, T., Matsuo, M., Yamazaki, S., Ueshima, K., Sawada, T., Furuichi, A., Ozaki, R., Nishii, M., Miura, S., Kusunoki, T., Sato, N., Koibuchi, Y., Esumi, K. and Ohtsuka, M.: Antimus-
carinic properties of vamicamide, a novel compound for the treatment of pollakiuria. Drug Dev. Res., 34:9, 1995. 6. Dubois, B., Mayo, W., k d , Y., Le Moal, M. and Simon, H.: Profound disturbances of spontaneous and learned behaviors following lesions of the nucleus basalis magnocellularis in the rat. Brain Res., 338 249, 1985. 7. Hara, C., Kasahara, N. and Ogawa, N.: Characteristics of behavioral and physical disturbances of rats with bilateral lesions of basal forebrain by ibotenic acid. Jpn. J . Pharmacol., suppl. 1, 58: 58P,1992. 8. Paxinos, G. and Watson, C.: The rat brain in stereotaxic coordinates. Sydney, Australia: Academic Press, 1982. 9. De Groat, W. C. and Saum, W. R.: Adrenergic inhibition in mammalian parasympathetic ganglia. Nature New Biol., 231: 188, 1971. 10. Ohtsuka, M., Mori, J., Tsujioka, K. and Kumada, S.: Pharmacological study on sympathetic inhibition of the urinary bladder in dogs. Jpn. J . Pharmacol., 3 0 187, 1980. 11. Ohtsuka, M., Mori, J., Mukumoto, S. and Kumada, S.: Morphological evidence for sympathetic mechanism in extra-bladder pelvic plexus in dogs. Jpn. J . Pharmacol., 3 0 922, 1980. 12. Fukuda, T.: Effects of pelvic and hypogastric nerve transection on the micturition cycle in decerebrate dogs. Acta Urol. Jpn., 3 3 1608, 1987. 13. Morikawa, K., Kakiuchi, M., Fukuoka, M., Kato, H., Ito, Y. and Gomi, Y.: Effects of various drugs on bladder function in conscious restrained-denervated rats placed in a restraining cage and produced by transection of the hypogastric nerve.: Jpn. J. Pharmacol., 5 2 405, 1990. 14. Gallagher, J. P., Griffith, W. H. and Shinnick-Gallagher, P.: Cholinergic transmission in cat parasympathetic ganglia. J. Physiol., 332 473, 1982. 15. De Groat, W. C. and Booth, A. M.: Inhibition and facilitation in parasympathetic ganglia of the urinary bladder. Fed. Proc., 39:2990, 1980. 16. Guarneri, L., Cova, R., Angelico, P., Colli, E. and Testa, R.: Effects of different drugs on the cystometrogram in conscious rats. Pharmacol. Res., 24: 175, 1991.
Vol 154.2174-2178, December 1995 Printed in U S.A
T f i E JOURNAL OF UROLOGY
Copyright 0 1995 by AMERICANUROUX~ICAL ASSOCIATION, INC.
EFFECTS OF VAMICAMIDE ON URINARY BLADDER FUNCTIONS IN CONSCIOUS DOG AND RAT MODELS OF URINARY FREQUENCY TAKA0 YAMAMOTO, YASUSHI KOIBUCHI,* SHINTARO MIURA, TADASHI SAWADA, REIKO OZAKI, KIM10 ESUMI AND MINORU OHTSUKA From the Pharmacological Research Laboratories, Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan
ABSTRACT
Purpose: To investigate the usefulness of vamicamide, ( 5 )-(2R*, 4R*k4-dimethylamino-2phenyl-2-(2-pyridyl)valeramide,as a novel drug for the treatment of urinary frequency and incontinence. Materials and Methods: Urinary frequency was evaluated in specially devised conscious dog and rat models by investigating the effects of the drug on urinary bladder function of these animals by cystometrography. Results: In the dog model with transected hypogastric nerves, the bladder volume at micturition (bladder capacity) was less than 50% that of the sham-operated dog, and in the rat model with bilateral lesioning of nuclei basalis, a part of the brain, by ibotenic acid injection, bladder capacity was about 50% that of the sham-operated rat. Other bladder functions in both models were unchanged. In the dog model, orally administered vamicamide a t 0.32 and 1.0 mg./kg. significantly increased bladder capacity and did not change residual urine volume or micturition pressure. Oxybutynin 0.10 mg./kg., one of the most popular drugs for the treatment of urinary frequency and incontinence, or atropine 0.10 mg./kg. induced significant increases in bladder capacity similarly to vamicamide at 0.32mg./kg. In the rat model, oral vamicamide 0.32 mg./kg. also significantly increased bladder capacity and did not change micturition pressure or threshold pressure. Again, oxybutynin 0.10 mg./kg. or atropine 0.32 mg./kg. had almost the same effects as vamicamide 0.32 mg./kg. Conclusions: These findings suggest that vamicamide should be useful for the treatment of urinary frequency. KEYWORDS:bladder, animals, parasympatholytics Recently, the number of patients with urinary frequency and/or urinary incontinence attributed to neurogenic or unstable bladder has been increasing. In particular, the number of patients with neurogenic bladder that results from functional disturbance of the brain (for example dementia and cerebrovascular disease), injury of the spinal cord and peripheral neuropathy (for example, diabetic neuropathy) is expected to increase concomitantly with the aging of society and the increase in traffic accidents. As urinary bladder contraction is controlled largely by the parasympathetic nerve, anticholinergic (antimuscarinic) drugs, for example oxybutynin and propantheline, are generally used for the treatment of urinary frequency and incontinence, but are not necessarily adequate. Therefore, a new drug for the treatment of these diseases is eagerly awaited. To investigate the effects of drugs for the treatment of urinary frequency on urinary bladder function, distensioninduced rhythmic bladder contraction,' bladder contraction induced by pelvic nerve stimulation2 and cystometrography1.3.4 are routinely used as in vivo methods. Among these methods, cystometrography approximates clinical tests most closely, but the animals used in experimental cystometrographies generally have normal bladder function. Moreover, the drugs are administered intravenously instead of orally, as in clinical treatment, because the animals tested are usually under anesthesia or decerebration. Therefore, we devised 2 conscious animal models which had smaller bladder capacities than normal and for whom oral administration was available.
Vamicamide is a new drug for the treatment of urinary frequency and incontinence which has selective antimuscarink action to the m3 receptor subtype and longer inhibitory action on the urinary bladder than on the salivary gland and ~ t o m a c hWe . ~ investigated the effects of vamicamide on urinary bladder function in the animal model by means of cystometrography. MATERIALS AND METHODS
Dog model. Male beagle dogs (weighing 7 . 5 to 11.5 kg.) were anesthetized with sodium pentobarbital ( 3 0 mg./kg. intravenously). The abdomen was opened through a midline incision, and 2 tine polyethylene tubes (0.d. 2.0 mm.) were implanted into the bladder. The hypogastric nerves on both sides were ligated and cut at a level about 2.5 cm. distal to the caudal mesenteric ganglia. The nerves of the sham-operated dogs were not ligated or cut. More than 6 days after implantation, the dogs were placed in metabolic cages, which permitted measurement of urine volume a t micturition (micturition volume) by graduated cylinder, and cystometrography was performed. One tube was connected to a pump for saline infusion, and the other was connected to a pressure transducer (PlOEZ-1,Nihon Kohden, Tokyo, Japan) to measure intravesical pressure. The whole system was filled with saline. Saline ( 3 7 C )was infused into the bladder a t a rate of 5 ml. per minute just after the intravesical urine was drained through an infusion tube, and intravesical pressure was recorded continuously on a polygraph system (RM-6000,Nihon Kohden 1. When micturition occurred. saline infusion was Accepted for puhlication J u n e 22, 1995. * Requests for repnnts: Pharmacological Research Laboratones, stopped and the time from the beginning to the end of infuFujisawa Pharmaceutical Co.. Ltd., 2-1-6 Kashima. Yodogawa-ku, sion = micturition interval I ~ n . smwsured to cnlculatt. the Osaka 532. Japan. volume of infus;cd saline. 'rhv li)llovb iiig cyston~etricparame217.4
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
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k r s were investigated: bladder capacity (volume of infused Comparison test following the randomized block designed saline until micturition), micturition pressure (maximum in- alnalysis of variance. A probability level c0.05 was accepted travesical pressure at micturition) and residual volume aIS significant. (bladder capacity minus micturition volume, residual volume of saline in the bladder just after micturition). Test RESULTS drugs were administered orally after the cystometrogram had become stable. The number of dogs in each group was 4. In conscious dogs, slow saline infusion into the urinary bladCystometrography was performed 1hour after drug admin- ler increasedthe inhvesical pressure and induced micturition istration, and the effects on cystometric parameters (= blad- tccompanied with transient bladder contraction (fig. 1). The der function) were assessed by comparing with the averages yatometrogrampattern of the hypogastric denervated dog was of 2 cystometrograms just before drug administration. The h o s t the same as that of the sham-operated dog, but the pupillary diameter of the dog was measured before and 1 )ladder capacity (volume of infused saline until micturition) hour after drug administration. uas significantly smaller (table 1).Micturition pressure (maxRat model. Male Wistar rats (7 to 8 weeks old) under mum intravesical pressure at micturition)and residual volume sodium pentobarbital anesthesia (45 mg./kg. intraperitone- residual volume of saline in the bladder just aRer micturition) ally) were operated on according to the methods of Dubois et were not changed by the denemation, and reaidual volume was al.6 and Hara et al.? Rats were placed in a stereotaxic appannall in this modeL We also tried to measurethreaholdpresratus (Kopf Instruments, Tujunga, California)and subjected 3ure (intravesicalpressure immediately before micturition)but to bilateral lesion of the brain by ibotenic acid injection. The muld not always do it w d y because, during the cy% coordinates of the cannula placements used were: anterior, tomehgraphy, the dogs were often in a sitting position until 7.7 mm.; lateral, 2.4 mm.; horizontal, 2.9 mm. according to Jjust before micturition although they were always in standing Paxinos and Watson.8 Ibeotenic acid was infused through a position at micturition. 0.4 mm. stainless steel cannula connected via Teflon tubing In the hypogastric denervated dogs, vamicamide (0.32and to a Hamilton microsyringe. Ibotenic acid (Research Bio- 1.0 mg./kg.) significantly increased bladder capacity and did chemicals,Inc., Natick, Massachusetts) was dissolved in Dul- not change residual volume or micturition pressure 1hour becco's phosphate buffered saline (D-PBS, pH 7.4) at a con- aRer oral administration (fig. 2). At only 1.0 mglkg. mild centration of 10 mg./ml. and infused in a volume of 0.75 4. mydriasis was observed (fig. 3). At the highest dose of vamifor 3 minutes by means of a syringe pump. The sham-oper- camide (3.2mglkg.), a decrease in micturition pressure with ated rats were infused with only D-PBS. One to 3 weeks after severe mydriasis was observed, and the effect on bladder surgery, rats were subjected to cystometrography. Under capacity was attenuated. Oxybutynin (0.10mgJkg.1 or atroether anesthesia, the abdomen was opened through a midline pine (0.10 mglkg.) induced significant increases in bladder incision, and a fine stainless steel cannula (0.d. 1.0 mm.)was capacity, and their potencies were almost the same as that of implanted into the bladder. The cannula was connected to a vamimmide (fig. 2). These drugs also did not change residual double lumen tube (outer tube: 0.d. 1.7 mm., inner tube: 0.d. volume or micturition pressure. At a higher dose (0.32mgJ 0.61 mm.) to infuse saline (370and to measure intravesical kg.), however, they did not induce an increase in bladder pressure simultaneously. A polyethylene tube (0.d. 0.97 mm.) capacity, and mild (oxybutynin) or severe (atropine) mydriawas placed into the stomach via the esophagus to administer sis was observed. Moreover, oxybutynin (1.0 mglkg.) showed drugs orally. Rats were placed in Bollman's cages and al- a tendency to decrease micturition pressure with severe mylowed to recover from anesthesia for about 3 hours. The free driasis, and atropine induced a decrease in micturition prestips of the bladder tubing were connectedto a pressure trans- sure even at 0.32 mgJkg. Cystometrograms of the conscious rats were almost the ducer (TP400T.Nihon Kohden: outer tube) and to a syringe pump (inner tube). Saline (37C)was infused into the bladder same as those of the conscious dogs except that bladder by means of a syringe pump at a rate of 61.5 4. per minute, capacity and micturition pressure were smaller in rats (data and intravesical pressure was recorded continuously on a not shown). Rats with bilateral lesions on the nuclei basalis polygraph system (RM-6000, Nihon Kohden). The micturi- by ibotenic acid injection had significantly smaller bladder tion interval was measured to calculate the volume of infused capacity than the sham-operated rats, but micturition pre% saline at each cystometrogram. The following cystometric sure and threshold pressure did not differ (table 2). parameters were investigated: bladder capacity, micturition pressure and threshold pressure (intravesid pressure hmediately prior to micturition). Test drugs were adminisSham Operation tered orally via preplaced polyethylene tube after the cystometrogram had become stable. The number of rats in each group was 5 with only a few exceptions (4 or 7).Cystometrography was performed 30 minutes (or 2 or 3 hours) after drug administration, and the effects on cystometric parameters (the averages of 2 or 3 cystometrograms) were assessed by comparing with the averages of 3 cystometrogramsjust be5mk 10 mm S M of (smw fore drug administration. Saline infusion (Drugs.Vamicamide (synthesized by Fujisawa PharmaceuTransection of Hypogastric Nerve tical Co. La.,Osaka, Japan) was dissolved in quimolar hydrochloric acid, neutralized by sodium bicarbonate and diluted with water. Oxybutynin (oxybutynin hydrochloride, synthesized by Fujisawa Pharmaceutical Co., La.) and atropine (atropine sulfate monohydrate, Wako Pure Chem. Inc., La.,Osaka, Japan) were dissolved in water. stut 01 5Statistical analysis. The results are given as mean values Saline Infusion (2 the standard error of the mean. Unpaired comparisons between operated and sham-operated groups were performed FIG.1. Typical cystometrogramtracing in conscious dog. Ivp:inby Student's or Aspin-Welch t test, and paired comparisons travesical reasure; $ :start of micturition ( = end of saline infusion); between treatments before and after drug administration HorizonJaxis: time from beginning of iuiimon; ( ): volume of were performed by Student's t test or Dunnett's multiple infused ealine.
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
2176
TABLE1. Cvstometric parameters of conscious dogs Sham-operated Hypogastic denervated
61.5 2 Bladder capacity (rnl.) Micturition pressure (rnm.Hg) 25.3 2 3.6 5 Residual volume (rnl.) Mean values 2 the standard error of the *: p (0.05 versus sharn-operated group.
8.5 5.4 1.3 mean ( n
26.3 f 1.2* 33.5 z 4.7 2.3 2 0.6 4).
=
9
6
6
=
5 .
0.1
40
1
ml 30 Residual Volume
**
I*
3 2 mgha
I
0.32
0012
0.1
mmHg 80
I nUh4
0032 0.1 0.32 Dmc
I mgh'
FIG.3. Effects of vamicamide and other drugs on pupillary diameter in conscious hypogastric denervated dogs. Open column: predosing. Closed column: postdosing (1 hour after administration). Mean values 2 standard error of mean (n = 4). *, **: p 10.05 or 0.01 versus predosing.
25
% .
032
Dor
DLU
Vamicamidt ml 45 Bladder Capacity
60 40
TABLE2 . Cystometric parameters
10
20
Dose
0.1 0.32 1 3.2 mgkg Dose
mgkg
0.1 0.32 1 Dose
3.2 mgkg
oxybutynin ml
45 1 Bladder Capacity
ml
30 1 Residual Volume
Bladder Capacity ( ~ 1 . ) 843 2 71 443 i El** 18.9 5 1.6 17.5 2 1.5 Micturition Pressure (mm.Hg) 4.1 2 0.8 Threshold Pressure (rnm.Hg) 4.2 2 0.7 Mean values 2 the standard error of the mean (n = 5 to 6) **: p <0.01versus sham-operated group.
mmHg 80 1 Micturition Pressure Vamicamide
40 35 30
:$
60
Threshdd Rrrurr
Bladder Capacity
Micturition R r r u r c
mmHg
mmHg
40
10
25
20
5
20 15
of conscious rats
Sham-operated Ibotenic acid treated
0
0.032 0.1 0.32 I m a g Dose
0.0320.1 0.32 1 m a g Dose
Ampine ml 45 Bladder Capacity
ml 30 Residual Volume
M .-
25 -_
I .
0
0.032 0.1 0.32 1 mgkg Dose
0.1
I
mmHg
I
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25
10
0.032 0.10.32 I m a g Dose
" 0.032
- 0.032 0.1
.=
r
0.032 0.1
O.32mehg
-
0 0.032 0.1 0 . 3 2 m 0 g
Dore
Rcrw~
:"ifll
200
0.032 0.1
Dw
0.32mgkg
DOU
Atropine
n
0.1 0.32 I m%lrg Dose
5
Micturilion
mmHg
"Add
20
5
20
Threshold Rswrc 20
8001:
40
30
Dose
mmHg
IOM
O32mOg
01
DOX
PI
80 Micturition Pressure
0 3 2 mgkg
01
Bladder Capaciiy
35
IC ._
O32mOg
Dw Oxybulynin
Threshold Rsrurc
Bladder Capacity PI
0.32 I mg/Lg Dose.
FIG.2. Effects of vamicamide and other drugs on urinary bladder function in conscious hypogastric denervated dogs. Open column: predosing. Closed column: postdosing (1 hour after administration). Mean values 2 standard error of mean (n = 4). *, **: p <0.05 or 0.01 versus predosing.
Vamicamide (0.32 mg./kg.) induced significant increases in bladder capacity a n d did not change micturition or threshold pressure (intravesical pressure immediately before micturition) in t h e conscious rats with ibotenic lesions 0.5 to 1 hour after oral administration (fig. 4). Almost t h e same effects were observed with oxybutynin 0.10 mg.kg. or atropine 0.32 mg./kg., but a high dose of oxybutynin (0.32mg./kg.) failed to increase bladder capacity (fig. 4). Atropine 0.10 mg./kg. showed a tendency to increase bladder capacity. We also investigated t h e duration of t h e effects of vamicamide and oxybutynin on bladder capacity in this model and found a significant effect of vamicamide 0.32 mg./kg. lasting for 3 to 3.5 hours, while the effect of oxybutynin 0.10 mg./kg. disappeared within 2 hours (fig. 5).
Micturition R r r u m
Y2
%?
r
800
200
0 32 mehg
0.1 Dose
rln
0.32 m a g
0.1
Dose
0.1
0 . 3 2 mgkg
Dose
FIG. 4. Effects of vamicamide and other drugs on urinary bladder function in conscious rats with ibotenic lesion of nuclei basalis. Open column: predosing; closed column: postdosing (0.5 to 1.0 hour after administration). Mean values 2 standard error of mean ( n = 4 to 5 ) . *: p 10.05 versus predosing. DISCUSSION
Urinary bladder contraction is induced by activation of t h e parasympathetic pathway (pelvic nerves), a n d stimulation of t h e hypogastric nerves (sympathetic pathway) elicits inhibitory responses in t h e bladder due to direct inhibitory action on smooth muscle cells and suppression of t h e parasympathetic ganglia on t h e surface of t h e bladder, too.:'."-11 Fukudal" reported that bilateral hypogastric nerve transection of t h e decerebrate dog induced a decrease of about 20% in bladder capacity and Morikawn et aI.1:' reported t h a t the same operation in conscious rats induced a decrease of about 50%.Similarly, bladder capacity ot'thc conscious hypogastric
ANIMAL MODELS OF URINARY FREQUENCY AND VAMICAMIDE
2177
antimwcarinic action to the m3 receptor subtype, which seemed to be responsible for contraction of the bladder in vitro, and that it had longer antimwcarinic action on the bladder than on the salivary gland and stomach in vivo.6 Judging from such a pharmacological profile, vamicamide ** * seemed to be suitable for the treatment of urinary firesuency and incontinence because antimusuuinic drugs are generally used for the treatment of these diseases, but both efficacy and elimination of side effects (dry mouth, mydriasie) are not necessarily adequate. A clinical trial of vamicamide for the treatment of urinary frequency and incontinenceis presently underway. In the dog model of urinary fresuency, orally administered vamicamide (0.32 and 1.0 mg./kg.) induced a significant increase in bladder capacity without changing residual urine volume and micturition pressure (fig. 2). In the rat model of urinary frequency, vamicamide 0.32 mg-. induced a significant increase in bladder capacity without " 0.5-1.0 2.0-2.5 3 . 0 - 3 . 5 hr changing micturition pressure and threshold preesure (fig. 4). Oxybutynin 0.10 mgJkg., one of the most popular drugs Time after administration for the treatment of urinary frequency and incontinence, showed almost the same effects as vamicamide 0.32 mg./kg. in both animal models. These findings suggested that vami0 VUnicMlide camide, like oxybutynin, could improve urinary fresuency Post (0.32mgkgp.o.) without influencing other bladder functione. Oxybutynin was FtG. 5. Duration of effect of vamicamide or o but on bladder said to inhibit bladder contraction both by an antimmcarinic capacity in conscious rate with ibotenic lesion 3 n u c E a e a l i e . re: effect and by other mechanisms, such as a direct muscle fredosing. Post: pstdosing. Mean values % standard error of mean relaxant effect, but the latter was observed at 100-foldhigher II = 5 to 7). *, * . p <0.05 or 0.01 versus predosing. concentration than the former in our previous study.6 Judging from our previous data, the pMcipal mechanism, not only of vamicamide but also of oxybutynin, for increasing denervated dogs was less than 50% that of the sham-oper- bladder capacity in both models seemed to be blockade of ated dogs in our study (table 1).These facts strongly suggest mwcarinic receptors on the bladder smooth muscle cells that the hypogastric nerves play an important role in bladder connecting to the nerve endings. In the dog model, both function and that the dog whose bladder capacity is de- vamicamide and oxybutynin induced mild mydriasis at 3.2 creased by meana of hypogastric denervation is a useful times higher doses than the minimum effective doses on the model of urinary frequency caused by peripheral nervous bladder (fig. 3). On the other hand, atropine, one of the most system disorder, which causes superiority of the parasympa- typical antimusearinic drugs, also induced a significant inthetic nerve over the sympathetic nerve in the bladder. We crease in bladder capacity at 0.10mghg., but induced severe therefore performed cystometrography on the hypogastric mydriasis and decrease in micturition pressure at 0.32 mgJ denervated dog to investigate the effects of vamicamide. kg.in the dog model (figs. 3,4). Judging from these findings, Moreover, as we used conscious dogs, this test had other the antimucarink side effects of atropine might happen advantages over the cystometrographiesperformed formerly. more fresuently than those of oxybutynin or vamicamide, if These were ability to administer the drugs orally, eliminate atropine should be administered for the treatment of urinary the effects of anesthesia and investigate the pupillary diam- frequency. Moreover, the fact that, in the rat model, vamiceter simultaneously as a parameter of antimusuuinic side amide had a longer lasting effect on bladder capacity than oxybutynin suggested that the efficacy of vamicamide on effects. than that of 0xybutyn.h The conscious rats with ibotenic lesions of the nuclei basa- urinary frequency might last longer . lis also had smaller bladder capacity than the sham-operated (fig. 5j. In the dog model, the increasing effect of vamicamide on rats, as was the case in the dog model (table 2). Generally, this operation (ibotenic acid treatment) is used to cause se- bladder capacity was attenuated at a dose that induced severe neuronal damage to the nucleus basalis magnocellularis vere mydriasis (3.2mgJkg.) and that of oxybutynin or atro(NBM) in the basal forebrain as a model of senile dementia of pine disappeared at the minimum dose inducing mydriasis the Alzheimer type, and it was reported that the decrease of (0.32mgJkg.) (fig. 2).Also, the increasing effect of oxybutycholine-acetyltransferase (ChAT)activity in the prefrontal nin on bladder capacity in the rat model disappeared at a and sensorimotor cortices was evidence of such damage.6 higher dose (0.32mg.). The reasons for these phenomena are Recently, Hara et al.7 reported that rats with NBM lesions by not clear, but there have been suggestions in other reports. ibotenic acid injection showed urinary incontinence 2 to 3 All 3 drugs are considered to increase bladder capacity days aRer the omration. They attributed this finding to chiefly by blocking muscarinic receptors on the smooth musdamage in the adjacent lateralhypothalamw. judging f;om cle cells connected to nerve endinas, but Gallaaher et al.14 the fact that decreases not only of cortical ChAT activity but and de Groat et al.16 reported that there were sts synaptic also of plasma antidiuretic hormone (ADH)were observed. In muscarinic receptors in the parasympathetic ganglia on the the present study, the conscious rats with ibotenic lesions surface of the bladder as well. The musCarinic receptors in had smaller bladder capacity than the sham-operated rats the parasympathetic ganglia mediated inhibitory postsynap(table 2),and it suggested that their symptom was not merely tic potential although the nicotinic receptors in the ganglia due to polyuria induced by a decrease in plasma ADH but mediated major excitatory postsynaptic potential. Judging rather to the impairment of regulation of micturition from from these reports, a n t i m d c drugs might block, not the central nervous system. Although the exact cause of only contractile muscarinic receptors on the bladder smooth these symptoms is unknown, rats with ibotenic lesions of the muscle cells, but also inhibitory muscarinic receptors in the nuclei basalis would be a useful model of pollakiuria caused ganglia when the dose was high enough to induce antimuscarinic side effects and their effect to increase bladder capacby central nerve system disorder. Previously, we reported that vamicamide had a selective ity was attenuated or disappeared. Indeed, Morikawa et al.l
T
L
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ANIMAL. MODELS OF URINARY FREQUENCY AND VAMICAMIDE
reported that high doses of atropine (0.01 and 0.1 mg.lkg., intravenously) suppressed the amplitude of distensioninduced rhythmic bladder contractions but increased their frequency in treated rats. In the same paper, the same doses of atropine, 0.01 and 0.1 mg.kg. intravenously, were reported not to induce an increase in bladder capacity in the cystometrographiesof anesthetized rats and dogs. Moreover, Guarneri et a1.16 reported that oxybutynin administered intravenously induced a significant increase in bladder capacity only at 0.03 mg./kg., but not at higher doses, in the eystometrography of conscious rats. These reports seem to support our hypothesis. In conclusion, our results of cystometrographies in 2 conscious animal models of urinary frequency strongly suggested that vamicamide should be useful for the treatment of urinary frequency caused by neurogenic or unstable bladder. REFERENCES
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