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Bladder Physiology

ffects of Combined Use ofrospium Chloride and Melatonin onn Vitro Contractility of Rat Urinary Bladderahmi Onur, Mete Ozcan, Ugur Tuygun, Tunç Ozan, Ahmet Ayar, and Irfan Orhan

BJECTIVES To examine the effects of combined use of trospium chloride and melatonin on in vitrocontractility of rat urinary bladder.

ETHODS Isolated bladder strips from 20 male Wistar rats were examined in an organ bath. Contractionswere evoked by acetylcholine (ACh). Initially, effective concentrations of trospium and mela-tonin to inhibit the contractions were determined. We subsequently tested the effects ofcombined use of melatonin and trospium on agonist-induced contractions.

ESULTS Exposure of bladder strips to 10 �M ACh significantly increased the contractions. Aftercumulative administration of increasing concentrations of trospium (1, 3, and 5 �M), agonist-induced contractions decreased significantly (P �.05). Similarly, the mean peak amplitude ofcontractions evoked by ACh was significantly inhibited by melatonin in a concentration-dependent manner (100, 200, and 300 �M) (P �.05). Further evaluation of the effects ofcombined use of trospium and melatonin on ACh-induced contractions of bladder strips revealedthat one-tenth of initially applied dose of trospium (500 nM) in addition to the lowest inhibitorydose of melatonin (100 �M) significantly lowered both the peak amplitude and area under thecontractility curve of contractions (P �.05).

ONCLUSIONS Our results showed that combined use of low dose trospium and melatonin had strong in vitroinhibitory capability on agonist-induced contractions of rat bladder strips. The present findings mayoffer an insight into lowering the dose of an antimuscarinic by combining it with an antioxidant and

endogenous hormone, melatonin. UROLOGY 75: 873–878, 2010. © 2010 Elsevier Inc.

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veractive bladder (OAB) syndrome is usuallycharacterized by urinary urgency, and/or urgeincontinence with frequency and nocturia.1 The

yndrome may be accompanied by detrusor overactivityDO), which may be either neurogenic or myogenic intiology. Involuntary bladder contractions may also occurn the presence of partial bladder outlet obstruction.2 Itas shown that the initial cholinergic denervation of theetrusor and supersensitivity of muscarinic receptorso acetylcholine (ACh) can be a contributing factorn DO.3

Current pharmacologic management of OAB consistsf use of different antimuscarinic drugs. Since ACh is therimary excitatory neurotransmitter in the human detru-or, patients with OAB and/or DO are usually treated bynticholinergic agents to block the effects of ACh onhese receptors.1,4 However, the lack of specificity andelectivity both for organ and receptor may limit their

rom the Department of Urology, Firat University, Elazig, Turkey; Department ofiophysics, Firat University, Elazig, Turkey; and Department of Physiology, Firatniversity, Elazig, TurkeyReprint requests: Rahmi Onur, M.D., Firat Universitesi, Hastanesi Uroloji Klinigi,

r3200 Elazig, Turkey. E-mail: rahmionur@yahoo.comSubmitted: May 12, 2009, accepted (with revisions): August 7, 2009

2010 Elsevier Inc.ll Rights Reserved

linical usefulness in the long term.5,6 Trospium chlorideTCl) and propiverine were reported to be least selective;owever TCl showed highest affinity for each of theuscarinic receptor subtypes.1,7 In several randomized

rials, TCl has shown improvements both in urodynamicnd symptomatic parameters in patients with OAB.7,8 Incontrolled, double-blind, multicenter clinical trial, the

ate of adverse events occurred after the use of TCl andxybutinin was 64.8% and 76.7%, respectively, and ap-roximately one-fourth of the patients terminated thetudy prematurely.8

The limiting role of side-effects of antimuscarinic drugsnd incomplete response in some patients led researcherso develop new drugs or combination of currently usedrugs in the treatment of OAB. These drugs includeotassium-channel openers, calcium channel blockers,timulation of different subtypes of alpha adrenoceptors,eta-agonist use, use of melatonin, and selective phos-hodiesterase inhibitors are among those.6,9-11 Consider-ng the challenges involved in the development of a newethod for treating OAB and efforts to decrease the

ncidence of adverse events during anticholinergic use,e hypothesized that combining an existing antimusca-

inic agent at a very low dose with melatonin may inhibit

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O. We therefore examined the effects of combined usef TCl and melatonin on in vitro contractility of ratrinary bladder.

ATERIAL AND METHODS

he protocol of this study has been reviewed and approved byhe Institutional Ethics Committee on animal experimentalesearch. A total of 20 male Wistar rats weighing 200-220 gere used in these experiments.

xperimental Proceduresll rats were killed by cervical dislocation and urinary bladdersere removed. Whole bladder was immediately placed in phys-

ological saline solution (PSS) containing (in mmol/L): NaCl20, KCl 5.9, CaCl2 2.5, MgCl2 1.1, NaHCO3 15, NaH2PO4

.2, glucose 11, and HEPES 10. A total of 48 strips, 2 to 3 stripsrom each rat bladder, (� 10 mm long and 2 mm wide) wererepared from the longitudinally incised bladder of each rat andlaced in a 10-mL jacketed tissue bath containing PSS at 37°C,H 7.4. The organ bath was continuously bubbled with 95% O2

nd 5% CO2. The lower end of the strip was tied to a stationaryetal hook and the upper end was attached to an isometric

orce-displacement transducer (FDT 10, May IOBS 99, Com-at Co., Ankara, Turkey) with surgical silk on each side. The

ransducer signals were recorded through transducer data acqui-ition system (May, TDA95, Commat Ltd., Ankara, Turkey)ith the help of software (Commat Co., Ankara, Turkey).The amplitude and area under the contractility curve (AUC)

uring the 10-minute period after equilibration and before anyreatment were termed “basal.” All data were normalized withespect to the basal amplitude and AUC of contraction andxpressed as percent of basal values.

The amplitude of bladder contractility was defined as thealue of the distance between the highest point and the initialaseline of the evoked contractility in the test periods. The areander the AUC was determined by measuring the integral ofontractile force as the area under the curve using the software.elaxation responses of trospium and melatonin on ACh-in-uced responses are expressed as a percentage decrease of theasal contractile response.

gonist Used to Induce Contractions and Effectsf Trospium and Melatonin on Rat Urinary Bladdersfter equilibration under an initial load of 1 g for 60 minute,

trip contractions were evoked by 10 �M ACh. All constituentsf PSS solution, ACh, and melatonin were obtained fromigma (Deisenhofen, Germany).We performed preliminary experiments to determine the

ffective concentration of trospium required to inhibit theontractions induced by ACh (10 �M). Trospium was providedy the manufacturer (Dr. R. Pfleger GmbH, Bamberg, Ger-any). Similar steps were taken to determine the concentration

f melatoninthat lowers the amplitude to the basal level. Theffects of individually administered trospium and melatonin ongonist-induced contractions in all strips were assessed. In theext step, we examined combined use of melatonin and tro-pium. To determine the combination dose, we tested half dose,ne-fourth, and one-tenth of initially applied dose of trospiumy combining it with the lowest inhibitory dose of melatonin100 �M). Similarly, we evaluated the level of inhibition in

ean amplitude and AUCs for combination effect. m

74

tatistical Analysisll values were indicated as mean � standard error of the mean

SEM). Differences between groups were calculated by means ofne-way analysis of variance followed by a post-hoc Tukey HSDest to determine significant differences among data groups. Forll analyses, P �.05 was accepted as evidence of significance.

ESULTSxposure of bladder strips to ACh (10 �M) caused aignificant stimulation of contraction compared to theasal (Fig. 1A) levels. Mean peak amplitude of contrac-ions after ACh stimulation was 1199 g (SEM, 151).fter cumulative administration of increasing concentra-

ions of trospium (1, 3, and 5 �M), the mean peakmplitudes were 693, 403, and 70 g, respectively (SEM,17, 62, 38, respectively). The decrease in amplitudes ofontractions after each TCl administration was statisti-ally significant (P �.05). Similarly, the mean peak am-litude of contractions evoked by ACh was significantlynhibited by melatonin in a concentration-dependent

igure 1. The effects of cumulatively administered (adm)rospium and melatonin decreased both (A) the mean am-litude of contractions induced by acetylcholine (ACh) andB) the area under curve.

anner (P �.05) (Fig. 1). A mean peak amplitude of

UROLOGY 75 (4), 2010

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362 g (SEM, 109) was detected after ACh stimulation,nd 3 doses of melatonin administration (100, 200, and00 �M) significantly reduced the peak amplitudes ofontractions to 793, 308, and 89 g, respectively (SEM,8, 103, 21, respectively). Normalized data with respecto basal amplitude and percentage of inhibition for eachrug are shown in Fig. 1A. The inhibitory effects ofumulative administration of TCl and melatonin werelso significant on AUC (Fig. 1B).

We further evaluated the combined use of trospiumnd melatonin on ACh-induced contractions of bladdertrips. To determine the combination dose, we againested cumulative doses of trospium by combining 100M melatonin. Because of strong inhibitory pattern atach dose, we finally diluted the original trospium dose to/10 and examined the effects at 3 different combinationoses (100 �M melatonin � 100, 300, and 500 nM ofrospium, respectively). Combined use of decreased dosesf melatonin and trospium significantly lowered both theormalized mean amplitude and AUC of contractionsP �.05) (Table 1). Considering the effects on AUCs, its clear that the last 2 trospium doses combined with

elatonin (100 �M) caused significant inhibition ofontractions (Fig. 2).

OMMENTrinary incontinence is a highly prevalent and distress-

Table 1. Effects of melatonin and melatonin plus reduced dafter ACh induced contractions in rat urinary bladder strips

Drug(s)Norm

ACh (10 �M)MelatoninMelatonin � 100 nM trospiumMelatonin � 300 nM trospiumMelatonin � 500 nM trospium

ACh indicates acetylcholine; SEM, standard error of mean.* P �.05.

igure 2. Contractile activity showing that combined use ofnhibited the agonist induced contractions (P �.05) (ACh: a

ng health problem both in women and men.11-13 Cur- H

ROLOGY 75 (4), 2010

ently, muscarinic receptor antagonists are the most com-only used medications in the pharmacotherapy forAB related symptoms. Different antimuscarinics mar-

eted worldwide were shown to have similar efficaciesith different pharmacokinetic and adverse event pro-les.1 TCl is a quaternary ammonium derivative, and ineveral randomized trials, it has proved effective in im-roving different urodynamic parameters and symp-oms.7,14 In the present study, we showed that trospiumas able to block muscarinic receptors even at very lowoses. It was previously shown that trospium has poorlood-brain barrier passage. Additionally, it is mainlyxcreted unchanged and has no pharmacologic interac-ions at the level of the hepatic cytochrome P450 sys-em.14,15

Large-scale, randomized and controlled studies havehown that patients receiving any type of oral antimus-arinics report significant improvement in OAB symp-oms.1 However, despite their good initial responses,hese antimuscarinics may fall short because of substan-ial side-effects, insufficient effect on continuing incon-inence, and long-term compliance problems.16 The mostrequently reported adverse events are dry mouth, con-tipation, headache, and blurred vision.1,5,7 Recently, aore serious concern related to adverse events of anti-uscarinics is increase in heart rate, QT prolongation,

nd induction of polymorphic ventricular tachycardia.1,17

of trospium on normalized amplitude and normalized area

d Amplitude (%)n � SEM

Normalized Area (%)Mean � SEM

� 0 100 � 0� 7 54 � 6� 10 50 � 6.6� 7* 29 � 6.1�� 0.6* 10 � 2.7�

red doses of melatonin (100 �M) and trospium significantlylcholine).

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uscarinics increases the risk of cardiac events whendministered at recommended therapeutic doses.1 Nev-rtheless, the lack of specificity and selectivity of anti-uscarinics for end organ limits their usefulness, and

dverse events in some patients along with long-termompliance problems still direct researchers to developew therapeutic drugs that have potential usefulness inreatment of OAB.

Melatonin, which is an endogenous hormone and aery potent antioxidant, has recently been used for itsnhibitory effect on smooth muscle cells of different or-ans.6,18 Ayar et al19 described that the inhibitory actionf melatonin on smooth muscle cells may result from thenhibitory action on voltage-dependent calcium entry inultured rat dorsal root ganglia neurons. Considering therogenital organs, Ellis and Buhrley20 examined the ef-ects of melatonin on contractility of rat seminiferousubules and concluded that the effect of melatonin onubular contractility might be due to change in cellulara�� levels. Similarly, melatonin was shown to produce

n inhibition on electrical field stimulation evoked con-ractions in the ipsilateral and contralateral rat vasaeferentia.21 Melatonin was reported to be decreasingontractility by binding to Ca��-activated calmodulinith high affinity and preventing it from activating my-sin light-chain kinase.19,22 In the present study, we alsohowed that melatonin significantly reduced the peakmplitudes of contractions induced by ACh in isolatedat urinary bladders. In addition to its well known anti-xidant effects, melatonin has more unique features thatere not shared by other antioxidants. These features ofelatonin, apart from antioxidative effects, have been

ocumented in chemical-induced hyperglycemia and inther circumstances, such as colitis, liver and lung dam-ge, and alkylating-agent toxicity.23 Moreover, melato-in was reported to be a safe and effective molecule with

ack of side effects. It has been administered in bothhysiological and pharmacologic amounts to humans andnimals, and there is widespread agreement that it is aontoxic molecule.24 It is easily synthesized in a phar-acologically pure form and is inexpensive and afford-

ble.25 Another advantage of melatonin over classicalntioxidants is its lack of prooxidative actions. All clas-ical antioxidants are potential electron donors, and theyxhibit both reduced and oxidized forms. Melatonin notnly does not consume cellular GSH; it also preserves orven increases the content of GSH in tissues. Thus,elatonin is a unique and strong antioxidant.26

Uninhibited detrusor contractions are not solely de-endent on release of Ach, and involuntary contractionsay arise because of different interactions between neu-

onal and nonneuronal sources.6 In the present study, weimed to evaluate the effects of combined use of trospiumnd melatonin, thereby bringing a synergy to potentiatehe degree of inhibition. Since these agents have 2 dif-erent mechanisms of actions, we proposed that combin-

ng the melatonin to a lower dose of trospium may

76

roduce the same effect. Thus, using trospium at a lowerose, less side effects may occur in the clinical setting.ur results showed that using one-third of melatonin

ose (100 �M) and combining it with a much lower dosef trospium (500 nM) showed the same efficacy comparedo the efficacies obtained by each drug separately.

In conclusion, there is continuous effort to developew therapeutic applications to treat the condition ofAB. Recently, synergistic activation of different mus-

arinic receptors, use of alpha3-adrenoceptor agonists,urinergic receptor agonists, phosphodiesterase inhibi-ors, neurokinin-1 receptor antagonists, opioids, andho-kinase inhibitors were examined in preliminary ex-erimental studies.1,9,11 Currently, we believe that one ofhe aims in the treatment of OAB symptoms should be toower the incidence of adverse events of antimuscarinics.hus, our study is the first to offeran insight into lowering

he dose of an antimuscarinic by combining it with anndogenous hormone, melatonin. Since we administeredrospium at a dose of one-tenth of its initial dose, weelieve that using this dose in combination with mela-onin would induce little or no side effects and may helpo decrease the rate of withdrawals in therapeutic trials.owever, these findings need to be confirmed in large

andomized and controlled series for possible in vivo use.

eferences1. Abrams P, Andersson KE. Muscarinic receptor antagonists for

overactive bladder. BJU Int. 2007;100:987-1006.2. Su X, Stein R, Stanton MC, et al. Effect of partial outlet obstruc-

tion on rabbit urinary bladder smooth muscle function. Am J PhysiolRenal Physiol. 2003;284:644-652.

3. Sibley GN. Developments in our understanding of detrusor insta-bility. Br J Urol. 1997;80:54-61.

4. Wein AJ. Diagnosis and treatment for the overactive bladder.Urology. 2003;62:20-27.

5. Chapple C, Khullar V, Gabriel Z, et al. The effects of antimusca-rinic treatments in overactive bladder: a systematic review andmeta-analysis. Eur Urol. 2005;48:5-26.

6. Semercioz A, Onur R, Ayar A, et al. The inhibitory role ofmelatonin isolated guinea-pig urinary bladder: an endogenous hor-mone effect. BJU Int. 2004;94:1373-1376.

7. Cardozo L, Chapple CR, Toozs-Hobson P, et al. Efficacy of tro-spium chloride in patients with detrusor instability: a placebo-controlled, randomized, double-blind, multicentre clinical trial.BJU Int. 2000;85:659-664.

8. Halaska M, Ralph G, Wiedemann A, et al. Controlled, double-blind, multicentre clinical trial to investigate long-term tolerabilityand efficacy of trospium chloride in patients with detrusor insta-bility. World J Urol. 2003;20:392-399.

9. Amend B, Hennenlotter J, Schäfer T, et al. Effective treatment ofneurogenic detrusor dysfunction by combined high-dosed antimus-carinics without increased side-effects. Eur Urol. 2008;53:1021-1028.

0. Hassouna M, Nishizawa O, Miyagawa I, et al. Role of calcium ionantagonists of the bladder detrusor muscle: in vitro and in vivostudy. J Urol. 1986;135:1327-1335.

1. Andersson KE, Wein AJ. Pharmacology of the lower urinary tract:basis for current and future treatments of urinary incontinence.Pharmacol Rev. 2004;56:581-631.

2. Abrams P, Blaivas JG, Stanton SL, et al. Standardization of termi-nology of lower urinary tract function. Neurourol Urodyn. 1988;7:

403-427.

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3. Wagner TH, Hu T. Economic costs of urinary incontinence in1995. Urology. 1998;51:355-361.

4. Alloussi S, Laval KU, Eckert R, et al. Trospium chloride (Spasmo-lyt) in patients with motor urge syndrome (detrusor instability): adouble-blind, randomised, multicentre, placebo-controlled study.J Clin Res. 1998;1:439-451.

5. Beckmann-Knopp S, Rietbrock S, Weyhenmeyer R, et al. Inhibi-tory effects of trospium chloride on cytochrome P450 in humanliver microsomes. Pharmacol Toxicol. 1999;85:299-304.

6. Drake MJ. Emerging drugs for treatment of overactive bladder anddetrusor overactivity. Expert Opin Emerg Drugs. 2008;13:431-446.

7. Sanguinetti MC, Tristani-Firouzi M. hERG potassium channelsand cardiac arrhythmia. Nature. 2006;440:463-469.

8. Satake N, Shibata S, Takagi T. The inhibitory action of melatoninon the contractile response to 5-hydroxytryptamine in variousisolated vascular smooth muscles. Gen Pharmacol. 1986;17:553-558.

9. Ayar A, Martin DJ, Ozcan M, et al. Melatonin inhibits highvoltage activated calcium currents in cultured rat dorsal root gan-glion neurones. Neurosci Lett. 2001;313:73-77.

0. Ellis LC, Buhrley LE. Inhibitory effects of melatonin, prostaglandinE1, cyclic AMP, dibutyrylcyclic AMP and theophylline on ratseminiferous tubular contractility in vitro. Biol Reprod. 1978;19:217-222.

1. Barun S, Ekingen G, Vural IM, et al. The effects of melatonin onelectrical field stimulation-evoked biphasic twitch responses in theipsilateral and contralateral rat vasa deferentia after unilateraltesticular torsion/detorsion. Naunyn Schmiedebergs Arch Pharmacol.2005;371:351-358.

2. Ouyang H, Vogel HJ. Melatonin and serotonin interactions withcalmodulin: NMR, spectroscopic and biochemical studies. BiochimBiophys Acta. 1998;1383:37-47.

3. Korkmaz A, Reiter RJ, Topal T, et al. Melatonin: an establishedantioxidant worthy of use in clinical trials. Mol Med. 2009;15:43-50.

4. Seabra ML, Bignotto M, Pinto LR Jr, et al. Randomized, double-blind clinical trial, controlled with placebo, of the toxicology ofchronic melatonin treatment. J Pineal Res. 2000;29:193-200.

5. Reiter R, Gultekin F, Flores LJ, et al. Melatonin: potential utilityfor improving public health. TAF Prev. Med Bull. 2006;5:131-158.

6. Tan DX, Reiter RJ, Manchester LC, et al. Chemical and physicalproperties and potential mechanisms: melatonin as a broad spec-trum antioxidant and free radical scavenger. Curr Top Med Chem.2002;2:181-197.

DITORIAL COMMENTntimuscarinic drugs have been for many years the most effec-

ive first-line pharmacologic treatment for urgency, frequency,nd urge incontinence, all typical symptoms for the overactiveladder syndrome (OAB). Unfortunately, antimuscarinic treat-ent is not always helpful, and is associated with bothersome

ide effects, which limit its clinical use. Development of otherrugs, such as calcium-channel blockers, potassium-channelpeners, and phosphodiesterase inhibitors, has been of promise,ut did not yet receive widespread clinical application.1 Thedea of synergistic combined use of existing antimuscarinicrugs with a second compound might be an attractive andimple alternative. The ultimate goal of such a combinationould be optimal effectiveness and persistence of use without

ignificant side effects in the treatment of OAB.The present article describes the synergistic effects of mela-

onin combined with the antimuscarinic trospium chloride.cetylcholine-evoked contractions of rat bladder strips were

nhibited by different concentrations of melatonin, trospiumhloride, or a combination of melatonin and trospium chlo-

ide. The optimal combination of melatonin and trospium l

ROLOGY 75 (4), 2010

hloride resulted in a 10-fold decrease in the use of trospiumhloride alone.

The idea of combining trospium chloride with the antioxi-ant melatonin in the treatment of OAB is very elegant andppealing. Potentially, it unlocks an array of possible combina-ions of existing drugs to optimize treatment of OAB. However,any issues have to be resolved before the described combina-

ion can be tested in clinical practice. For instance, the poten-ial mechanism of action of the synergistic effects should belucidated and tested using in vivo experiments. Furthermore,he article describes only muscle effects of the combined drugs,hereas symptoms typical for OAB originate probably fromoth sensory urothelial and motor muscle dysfunction. Sensoryignaling of the bladder and its urothelium is modulated byctivation of muscarinic receptors,2 and antimuscarinics inhibithese muscarinic receptors.3

In conclusion, the combined use of melatonin and trospiumhloride shows a promising synergistic inhibitory effect oncetylcholine-evoked contractions in rat urinary bladder strips.he importance of this observation needs to be confirmed in

ntact animal experiments before it can be used in clinicalractice.

ertil F.M. Blok, M.D., Ph.D., Department of Urology,rasmus Medical Center, Rotterdam, the Netherlands

eferences. Andersson KE, Chapple CR, Cardozo L, et al. Pharmacological

treatment of overactive bladder: report from the International Con-sultation on Incontinence. Curr Opin Urol. 2009;19:380-394.

. Kullman FA, Artim DE, Birder LA, et al. Activation of muscarinicreceptors in rat bladder sensory pathways alters reflex bladder activ-ity. J Neurosci. 2008;28:1977-1987.

. Kim Y, Yoshimura N, Masuda H, et al. Antimuscarinic agentsexhibit local inhibitory effects on muscarinic receptors in bladderafferent pathways. Urology. 2005;65:238-242.

oi:10.1016/j.urology.2009.09.072ROLOGY 75: 877, 2010. © 2010 Published by Elsevier

nc.

EPLYhe idea of combining 2 different pharmacologic agents in the

reatment of overactive bladder syndrome (OAB) came fromhe complexity of the pathogenesis and from the recent ad-ances in the role of urothelium. Abnormal detrusor contrac-ions are not only dependent on the cholinergic system but alsoesult from intrinsic malfunction of the detrusor smooth muscle,alfunction of neuronal input into the detrusor, and/or fromalfunction of sensory signals originating in the bladder.1 Thus,

y targeting only 1 type of receptor, it may not be possible tolock all uninhibited contractions in every OAB patient. For aetter response, increasing the dose or long-term use may bessociated with more side effects and withdrawals of the drug. Inhe present study, our primary aim was to define a new strategynd assess the synergism of different molecules in the inhibitionf detrusor contractions. For this purpose, melatonin was espe-ially selected for combination with an anticholinergic as it is aotent antioxidant, an endogenous hormone, and a smoothuscle relaxator, which is free of most of side effects. We were

ble to obtain the same efficacy in the inhibition using much

ower doses of each drug when they were combined. Consider-

877