C

Tt

u

Basic and Translational Science

Inhibitory Effect ofStandardized Cannabis sativaExtract and Its Ingredient Cannabidiolon Rat and Human Bladder ContractilityRaffaele Capasso, Gabriella Aviello, Francesca Borrelli, Barbara Romano, Matteo Ferro,Luigi Castaldo, Vittorino Montanaro, Vincenzo Altieri, and Angelo A. Izzo

OBJECTIVES To evaluate the effect of a Cannabis sativa extract enriched in cannabidiol (CBD) botanic drugsubstance (BDS) and pure CBD, on bladder contractility in vitro. Cannabis based-medicines,including CBD-enriched extracts, have been shown to reduce urinary urgency, incontinenceepisodes, frequency, and nocturia in patients with multiple sclerosis.

METHODS Strips were cut from male Wistar rats and the human bladder body and placed in organ bathscontaining Krebs solution. Contractions were induced by electrical field stimulation, acetylcho-line, KCl, and �,�-methylene adenosine triphosphate.

RESULTS CBD BDS significantly reduced the contractions induced by acetylcholine, but not those inducedwith electrical field stimulation, KCl, or �,�-methylene adenosine triphosphate in the isolatedrat bladder. The inhibitory effect of CBD BDS was not significantly modified by the cannabinoidor opioid receptor antagonists or by modulators of calcium levels, but it was increased byruthenium red and capsazepine, 2 transient receptor potential vanilloid type-1 blockers. Inhumans, CBD BDS and pure CBD significantly reduced acetylcholine-induced contractions, aneffect that was not changed by the transient receptor potential vanilloid type-1 blockers.

ONCLUSIONS Our data have suggested that CBD BDS reduces cholinergic-mediated contractility and that thiseffect is modulated by transient receptor potential vanilloid type-1 in rats but not in humans.CBD is the chemical ingredient of CBD BDS responsible for such activity. If confirmed invivo, such results could provide a pharmacologic basis to explain, at least in part, the effi-cacy of Cannabis medicines in reducing incontinence episodes in patients with multiple

sclerosis. UROLOGY 77: 1006.e9 –1006.e15, 2011. © 2011 Elsevier Inc.

Anecdotal reports from patients with multiplesclerosis have suggested that preparations fromthe plant Cannabis sativa might have a beneficial

effect on lower urinary tract symptoms. The main activeingredient of the plant is �9-tetraidrocannabinol (�9-

HC), which binds specific G-protein-coupled receptors,ermed cannabinoid (CB1 and CB2) receptors.1 CB1 and

CB2 receptors have been identified in rodent and humanrothelium and nerve fibers.2-4 Cannabinoids inhibit

neurally mediated bladder contractions in vitro by CB1

activation5,6 and increase micturition in vivo by CB2

receptor activation.4

In addition to �9-THC, the marihuana plant Cannabissativa also contains nonpsychotropic cannabinoids,

From the Departments of Experimental Pharmacology and Endocannabinoid ResearchGroup, and Urology, University of Naples Federico II, Naples, Italy

Reprint requests: Angelo A. Izzo, Ph.D., Department of Experimental Pharmacol-ogy, University of Naples Federico II, Via D, Montesano 49, Naples 80131 Italy.

E-mail: aaizzo@unina.it

Submitted: September 30, 2010, received (with revisions): December 5, 2010

© 2011 Elsevier Inc.All Rights Reserved

which exert pharmacologic actions without binding tocannabinoid receptors. The most studied among thesecompounds has been cannabidiol (CBD), which exerts aplethora of pharmacologic effects (eg, analgesic, anti-inflammatory, antioxidant, and antitumoral) that are me-diated by multiple pharmacologic and biochemical mech-anisms.7,8 CBD has an extremely safe profile in humans,and it has been clinically evaluated (albeit in a prelimi-nary fashion) for the treatment of anxiety, psychosis, andmovement disorders.8 CBD is a main ingredient of aCannabis-derived medicine (sold under the brand nameSativex) used for the treatment of pain and spasticityassociated with multiple sclerosis. Sativex is composedprimarily of a 1:1 ratio of 2 Cannabis sativa extracts,Cannabis sativa extract enriched with �9-THC and aCannabis sativa enriched with CBD (that we have termed“CBD botanic drug substance” [BDS]). Clinical studieshave shown that Sativex reduces urgency, the numberand volume of incontinence episodes, and frequency and

nocturia in patients with multiple sclerosis.9,10 Another

0090-4295/11/$36.00 1006.e9doi:10.1016/j.urology.2010.12.006

a

caAapfI1d

3aei1mti

te

ii

o

tBrtsCttii1etoo

hcAGS

ncwsTi

au

1

randomized placebo-controlled study showed that orallyadministered CBD-enriched extract reduced urge incon-tinence in patients with multiple sclerosis.11

Given the reported clinical efficacy of standardizedCannabis sativa-derived medicines in relieving the blad-der hyperactivity associated with multiple sclerosis, inthe present study, we evaluated the effect of CBD BDSand its main active ingredient CBD on rat and humanbladder contractility.

MATERIAL AND METHODS

Bladder Preparations and Drug AdministrationMale Wistar rats (200-240 g) were purchased from HarlanLaboratories (San Pietro al Natisone (UD), Italy) and main-tained under controlled temperature (24° � 2°C) and humidity(60%) conditions until used. The rats had free access to waterand food. All experiments complied with the Italian D.L. No.116 of January 27, 1992 and the associated guidelines in theEuropean Communities Council Directive of November 24,1986 (86/609/ECC). The rats were killed by asphyxiation withcarbon dioxide. The urinary bladder was removed and placed inKrebs solution (composition NaCl 119 mM, KCl 4.75 mM,KH2PO4 1.2 mM, NaHCO3 25 mM, MgSO4 1.5 mM, CaCl22.5 mM, and glucose 11 mM). Strips of approximately 2 �10 mm were cut from the bladder body (2 strips from 1 rat) andplaced in 20-mL organ baths containing Krebs solution equili-brated with 95% oxygen and 5% carbon dioxide at 37°C. Thetissues were connected to an isometric transducer (tension9.81 mN). The contractions were recorded using a PowerLabsystem (Ugo Basile, Comerio, Italy). After a minimal 1-hourequilibration period, the strips were subjected to electrical fieldstimulation (EFS, 0.5 Hz for 1 second, 500 mA, 0.25-ms pulseduration, 20-minute interval between stimulations), deliveredby electrodes placed around the tissue or stimulated with exog-enous agonists, such as acetylcholine (10�6 M), �,�-methylenedenosine triphosphate (�,�-methylene-ATP, 10�5 M), or KCl

(8 � 10�2 M). The conditions of EFS were selected on the basisof our previous work,12 in which we have shown that suchontractions could be modulated by drugs acting at the presyn-ptic level. The contact time for acetylcholine, �,�-methylene-TP, and KCl was 1, 3, and 4 minutes, respectively. These

gonists gave a contractile responses that were similar in am-litude to those of EFS. Stable and reproducible contractionsor 3 hours were obtained with stimulation every 20 minutes.n preliminary experiments, the effect of tetrodotoxin (3 �0�7 M, contact time 10 minutes), atropine (10�6 M), oresensitization with �,�-methylene-ATP (5 � 10�5 M; 3 suc-

cessive applications of �,�-methylene-ATP for 5 minutes at-minute intervals) on EFS-induced contractions was evalu-ted. After the stable control contractions evoked by EFS or byxogenous agonists were recorded, the responses were observedn the presence of increasing CBD BDS concentrations (0.1-00 �g/mL). The contact time for each concentration was 20inutes. Preliminary experiments showed that this contact

ime was sufficient for CBD BDS to achieve the maximalnhibitory effect.

In different sets of experiments, the effect of CBD BDS onhe acetylcholine contractions was also evaluated in the pres-nce of rimonabant plus SR144528 (10�6 and 10�7 M, respec-

tively, to block cannabinoid receptors), naloxone (10�6 M, to

block opioid receptors), ruthenium red and capsazepine (both at T

006.e10

10�5 M, to block transient receptor potential vanilloid type 1[TRPV1]), nifedipine (10�7 M, to block L-type calcium chan-nels), and cyclopiazonic acid (10�5 M, to block the sarcoplas-mic reticulum calcium-ATPase). These concentrations wereselected on the basis of previous published studies.13,14 Thenhibitory response curve to CBD BDS against acetylcholine-nduced contractions was repeated for each set of experiments.

In some experiments, the effect of increasing concentrationsf pure CBD (0.1-100 �g/mL) on the acetylcholine (10�6

M)-induced contractions was also evaluated (contact time 20minutes per concentration of CBD). In these experiments, theeffect of CBD was also evaluated in the presence of rutheniumred (10�5 M) or capsazepine (10�5 M).

In another series of experiments, the effect of both CBD BDS(0.1-100 �g/mL) and CBD (0.1-100 �g/mL) was evaluated onhe contractions evoked by acetylcholine in human tissues.ladder tissue was taken from patients who had undergone

adical cystectomy because of carcinoma of the bladder orransvescical adenomectomy because of adenomatous hyperpla-ia of the prostate. No differences in the effect of CBD BDS andBD against the acetylcholine-induced contractions evoked in

he bladder from patients with adenomatous hyperplasia orhose with cancer were observed. The ethics committee at ournstitution approved the present study, and each subject providednformed consent before study entry. Bladder strips (approximately0 mm) were set up under a resting tension of 9.81 mN. Thexperimental protocol, including drug administration and contactime, was identical to that described for the rat bladder. The effectf CBD BDS and pure CBD was evaluated alone or in the presencef ruthenium red (10�5 M) or capsazepine (10�5 M) using the

same protocol as was used for the rat tissue.

DrugsAcetylcholine hydrochloride, �,�-methylene-ATP, naloxone

ydrochloride, tetrodotoxin, nifedipine, cyclopiazonic acid,apsazepine, and ruthenium red were purchased from Sigmaldrich (Milan, Italy) and KCl from Merck KGaA (Damstadt,ermany) and rimonabant and SR144528 were a gift fromanofi-Aventis (Montpellier, France).The standardized Cannabis sativa extract enriched with can-

abidiol (CBD BDS) and pure cannabidiol (CBD, purity ac-ording to high-performance liquid chromatography 99.76%)ere supplied by GW Pharmaceutical (Porton Down, Wilt-

hire, UK). CBD BDS was standardized to contain 65.6% CBD.he concentrations of CBD BDS reported in the present report

ndicated the amount of CBD contained in the extract (eg, 1 �gCBD BDS contained 1 �g CBD).

Rimonabant, SR144528 (N-[-1S-endo-1,3,3-trimethyl bicy-clo (2.2.1) heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide), capsazepine, nifedi-pine, CBD BDS, and cannabidiol were dissolved in dimethylsulphoxide. The other drugs were dissolved in distilled water.Dimethyl sulphoxide (�0.01%) did not modify bladder con-tractility.

Statistical AnalysisThe results are expressed as the mean � standard error of themean. Comparisons between 2 sets of data were made usingStudent’s t test for paired data. When multiple comparisonsgainst a single control were made, analysis of variance wassed, followed by the Tukey-Kramer multiple comparisons test.

he concentrations of CBD BDS (or CBD) that produced 50%

UROLOGY 77 (4), 2011

mpw

o

tr

(an

13

sd

opF2lFoetram

1af

oCdS32

tccbet

c

ir*

inhibition of acetylcholine-induced contractions (IC50) or theaximal inhibitory effect (Emax) were used to characterize its

otency and efficacy, respectively. The IC50 and Emax valuesere calculated with the aid of a computer program.

RESULTS

Rat BladderAcetylcholine, at a concentration of 10�6 M, causedsubmaximal contractions (magnitude 10.3 � 0.5 mN,n � 117) of the rat urinary bladder strips that weresimilar in amplitude to those obtained with electricalstimulation (magnitude 9.0 � 1.5 mN, n � 9). EFS-induced contractions were abolished by tetrodotoxin(3 � 10�7 M). A combination of desensitization with�,�-methylene-ATP (10�4 M), followed by applicationf atropine (10�6 M), greatly reduced the electrically

evoked contractions. Thus, the electrically induced con-tractions were predominately due to the release of ace-tylcholine and purines from the bladder neurons. Incontrast, acetylcholine-induced contractions were com-pletely abolished by atropine (10�6 M) but were leftunchanged by tetrodotoxin (3 � 10�7 M). In addition,he contractions due to acetylcholine were unaffected byimonabant (10�6 M) plus SR1442558 (10�7 M), nalox-

one (10�6 M), ruthenium red (10�5 M), or capsazepine10�5 M; data not shown), increased by cyclopiazoniccid (10�5 M, 27.16% increase, n � 8), and decreased byifedipine (10�7 M, 49.56% inhibition, n � 8).CBD BDS, at a concentration of 1-100 �g/mL, de-

creased the contractions induced by acetylcholine, with-out significantly modifying the contractions induced byelectrical stimulation, KCl, or �,�-methylene-ATP (Fig.). Statistical significance was achieved starting with the-�g/mL concentration. The IC50 and Emax was 2.14

�g/mL (95% confidence interval [CI] 0.95-4.87) and76.7% (95% CI 58.0%-95.3%), respectively. CBD BDS,at the 0.1-�g/mg and 0.3-�g/mL concentration caused amall, nonsignificant, increase in the acetylcholine-in-uced contractions.Figure 2 shows the effect of CBD BDS in the presence

f drugs that block cannabinoid receptors (rimonabantlus SR144528; Fig. 2A), opioid receptors (naloxone;ig. 2B), or TRPV1 (capsazepine and ruthenium red; Fig.C) and in the presence of drugs that influence intracel-ular calcium levels (nifedipine and cyclopiazonic acid;ig. 2D). Capsazepine and ruthenium red, but not thether drugs used, significantly modified the inhibitoryffect of CBD BDS on the acetylcholine-induced con-ractions. In the presence of both capsazepine anduthenium red, the inhibitory effect of CBD BDS oncetylcholine-induced contractions was significantly aug-ented (Fig. 2C).Similar to CBD BDS, pure CBD, at concentrations of

-100 �g/mL, decreased the contractions induced bycetylcholine (Fig. 3). The effect was significant startingrom 30 �g/mL. The IC50 and Emax was 4.96 �g/mL (95%

CI 0.72-21.22) and 46.7% (95% CI 9.3%-84.1%), re-

UROLOGY 77 (4), 2011

spectively. The inhibitory effect of pure CBD on theacetylcholine-induced contractions was significantly in-creased in the presence of both ruthenium red and cap-sazepine (Fig. 3).

Neither CBD-BDS nor CBD affected the spontaneousmotility of the rat urinary bladder.

Human BladderFigure 4 shows the effect of CBD BDS (Fig. 4A) andCBD (Fig. 4B), both at concentrations of 0.1-100 �g/mL,on the contractions induced by acetylcholine (10�6 M)n the human bladder. The addition of CBD BDS orBD to the organ bath produced a concentration-depen-ent inhibition of acetylcholine-induced contractions.ignificant inhibitory effects were achieved starting with0 �g/mL. The IC50 for CBD BDS and pure CBD was.84 �g/mL (95% CI 0.77-12.70) and 6.15 �g/mL (95%

CI 0.85-22.25), respectively. The Emax for CBD BDS andCBD was 49.40% (95% CI 30.0%-73.3%) and 33.8%(95% CI 21.9%-45.6%), respectively. Neither CBD BDSnor CBD affected the spontaneous motility of the humanurinary bladder.

COMMENTBladder dysfunction is a common problem for patientswith multiple sclerosis. Confirming anecdotal reports, aquestionnaire study of patients with multiple sclerosisregularly using Cannabis for symptom relief found that, ofhose subjects with urinary problems, more than one halflaimed improvement in urgency.15 Clinical studies haveonsistently shown that a standardized Cannabis sativa-ased medicine (ie, Sativex), as well as a cannabidiol-nriched extract, reduced incontinence episodes in pa-ients with multiple sclerosis.9-11 A recent multicenter

Figure 1. Effect of standardized Cannabis sativa extractenriched with cannabidiol (CBD BDS, 0.1-100 �g/mL) onontractile response produced by EFS, acetylcholine (10�6

M), �,�-methylene ATP (10�5 M), or KCl (8 � 10�2 M) insolated rat bladder. Each point represented mean of 8-10ats. Vertical lines show standard error of mean. *P � .05,*P � .01, and ***P � .001 vs corresponding control.

double-blind randomized clinical study has shown that

1006.e11

ib

(

Vr( BDS

((

Sativex reduced the number of episodes of nicturia, thenumber of voids daily, and the number of daytime voidsin subjects with overactive bladder due to multiple scle-

Figure 2. Acetylcholine-induced contractions in isolated ratwith cannabidiol (CBD BDS, 0.1-100 �g/mL) alone (vehicl10�7 M; to block cannabinoid receptors) (CB1 � CB2 acapsazepine (10�5 M), (D) or nifedipine (10�7 M) and cyclopertical lines show standard error of mean. No statistical siepresenting inhibitory effect of CBD BDS on acetylcholineP � .01) from curves representing inhibitory effect of CBD

Figure 3. Acetylcholine-induced contractions in isolated ratbladder. Effect of pure cannabidiol (0.1-100 �g/mL) alonevehicle) or with ruthenium red (10�5 M) or capsazepine10�5 M). Each point represented mean of 8-9 rats. Verticallines showed standard error of mean. *P � .05 vs corre-sponding control. Note, curve representing inhibitory effectof cannabidiol on acetylcholine-induced contractions (vehi-cle curve) was significantly different (P � .01) from curvesrepresenting inhibitory effect of cannabidiol in presence ofruthenium red or in presence of capsazepine.

rosis.16

1006.e12

Previous investigators have found that a number ofcannabinoid receptor agonists, including �9-THC, inhib-ted nerve-mediated contractions of the rodent bladdery activation of the CB1 receptors.5 Another study

showed that the ability of cannabinoid agonists to inhibitneurogenic contractions of the rodent bladder was notconserved across all mammalian species. Cannabinoidreceptor activation was found to be ineffective againstelectrically evoked contractions of the human bladder.6

Our results have provided evidence that CBD BDS, aswell as pure CBD, both devoid of psychotropic activity,inhibit rat and human contractility.

We have shown that CBD BDS inhibited the contrac-tions induced by acetylcholine (which contracts thebladder through direct activation of the smooth muscles),but it had no significant effects on the contractile re-sponse due to EFS (which is mostly mediated by therelease of ATP and acetylcholine from the nerves) in therat bladder. These results suggest a postsynaptic site ofaction. In addition, the postsynaptic inhibitory action ofCBD BDS was not one of a general depressant, becausethe responses of the tissue to KCl or �,�-methylene-ATPwere not significantly affected by CBD BDS. Collec-tively, such results suggest that CBD BDS selectivelyinhibits cholinergic contractions by acting at a postsyn-aptic site on the bladder smooth muscle. In accordancewith our results, it has previously been demonstrated that

er: effect of standardized Cannabis sativa extract enrichedin presence of (A) rimonabant (10�6 M) and SR144528B) naloxone (10�6 M), (C) ruthenium red (10�5 M) andnic acid (10�5 M). Each point represented mean of 7-9 rats.ance found among curves, except for Fig. C, in which curveced contractions (vehicle curve) was significantly different

in presence of ruthenium red or capsazepine.

bladde) ornt), (iazo

gnific-indu

CBD inhibited acetylcholine- but not EFS-induced con-

UROLOGY 77 (4), 2011

bb

t

g

tractions in the guinea pig ileum.17 The ability of CBDBDS (or CBD) to reduce acetylcholine-induced contrac-tions, but not EFS-induced contractions, could indicatethat CBD BDS (or CBD) activates receptors or releaseendogenous molecules that have an excitatory effect onneuronal transmission that counterbalances the inhibi-tory effect exerted at the postjunctional level. Althoughthe reasons for the selective inhibition of CBD BDS onacetylcholine-induced contractions remain to be ex-plained, CBD has been shown to selectively inhibit theadrenergic �1-mediated contractile response in themouse isolated vas deferens.18 In contrast, it has recentlyeen shown that CBD inhibited the contractions evokedy different agonists in the isolated mouse ileum.19 Fi-

nally, it should be highlighted that the inhibitory effectof CBD BDS (or CBD) on rat bladder contractility differsfrom that of �9-THC, which, in contrast to CBD, is thedirect-acting cannabinoid CB1 receptor agonist.7,8 CB1

Figure 4. Acetylcholine-induced contractions in isolatedhuman bladder. Effect of standardized Cannabis sativa ex-ract enriched with (A) cannabidiol (CBD BDS, 0.1-100

�g/mL) or (B) pure cannabidiol (0.1-100 �g/mL). Drugsiven alone (vehicle) or in presence of ruthenium red (10�5

M) or capsazepine (10�5 M). Each point represented meanof 8-9 experiments. Vertical lines show standard error ofmean. *P � .05 vs corresponding control. No statisticallysignificant differences noted among curves.

receptor agonists, such as �9-THC, inhibit electrically

UROLOGY 77 (4), 2011

evoked contractions by activating presynaptic CB1 recep-tors and thus reducing contractile transmitter releasefrom bladder nerves.5,6 In contrast, CBD has been shownto activate indirectly cannabinoid receptors by inhibitionof endocannabinoid inactivation, only in some—but notall—experimental conditions7,8 (see also the followingparagraphs).

We also found that CBD BDS was more active andeffective than pure CBD in reducing the acetylcholine-induced contractions. The differences in potency andefficacy between CBD BDS and pure CBD clearly indi-cated that additional compounds, such as minor phyto-cannabinoids, are present in the extract and that thesecompounds can, in a synergistic or additive way, poten-tiate the effect of the principal phytocannabinoid. Syn-ergistic actions among CBD and other phytocannabi-noids have been previously documented.20

CBD is known to exert a wide range of pharmacologicactions that are mediated by several mechanisms of ac-tion.7,8 To give insights into the mode of action of CBD,we evaluated the possible involvement of (a) the endo-cannabinoid system, (b) opioid receptors, (c) TRPV1,and (d) calcium. The rationale for investigating the pos-sible involvement of these systems/receptors was as fol-lows:

1. It is well known that CBD does not directly activateCB1 or CB2 receptors.7,8 However, CBD could indi-rectly activate cannabinoid receptors because of itsability to inhibit the fatty acid amide hydrolase (ie,the enzyme responsible for endocannabinoid inactiva-tion). CBD, possibly by indirect activation of canna-binoid receptors, has been shown to induce apoptosisin human leukemia cells, to facilitate the extinction ofcontextual fear memory in rodents, and to exert in-testinal antiprokinetic effects.7,8 In contrast, directactivation of CB1 receptors by plant and syntheticcannabinoids is known to reduce the contractility ofthe rat isolated bladder.6

2. CBD has been shown to be an allosteric modulator atthe �- and �-opioid receptors.21 Immunohistochemi-cal studies have shown the presence of opioid immu-noreactive peptides in the rat detrusor,22 and opioidshave been shown to inhibit bladder motility.23

3. The contractions of all smooth muscles, including thebladder, absolutely depend on the presence of cal-cium. The calcium ions that activate the contractileproteins are derived from intracellular stores or fromextracellular influx, mainly through L-type calciumchannels.24 In hippocampal cells, the CBD responseswere reduced by inhibitors of L-type calcium channelblockers and by depletors of the intracellular store.25

4. TRPV1 is a nonselective ion channel activated bycapsaicin, heat, protons, and endogenous ligands, suchas the endocannabinoid anandamide.26 It is largelyexpressed in the urinary tract of mammals, not only inthe sensory fibers, but also in the non-neuronal cells,

such as urothelial and smooth muscle cells.27 Patients

1006.e13

icTCrCcieth

ap

1

1

with an overactive bladder have shown increasedTRPV1 mRNA expression in the trigonal mucosa.28

TRPV1 activation is known to elicit contractions ofthe rat bladder.29 In contrast, TRPV1 antagonistscounteract bladder overactivity.13,30 Although noTRPA1 antagonist is commercially available for thetreatment of overactive bladder, such drugs are underinvestigation for the treatment of the symptoms ofoveractive bladder.28,31,32 CBD has been shown to bea TRPV1 agonist and to exert antipsychotic and an-algesic effects by TRPV1 activation.8

Experiments using selective CB1 and CB2 receptorantagonists (ie, rimonabant and SR144528), naloxone(an opioid receptor antagonist), nifedipine (which blockscalcium entry into the cell by antagonizing L-type cal-cium channels), and cyclopiazonic acid (which inhibitsthe sarcoplasmic reticulum calcium-ATPase and thus de-pletes agonist-releasable intracellular pools) suggestedthat CBD BDS does not act by activation of cannabinoidor opioid receptors or modulation of L-type calciumchannels or sarcoplasmic reticulum calcium uptake.However, we found that the inhibitory effect of CBDBDS on acetylcholine-induced contractions was furtheron augmented by both capsazepine, a competitiveTRPV1 antagonist, and ruthenium red, a noncompetitiveTRPV1 blocker.13 Both antagonists also increased thenhibitory effect of pure CBD on acetylcholine-inducedontractions. Collectively, such results suggest thatRPV1 negatively modulates the depressant effect ofBD BDS on rat bladder contractility and that CBD is

esponsible for such modulation. Thus, the ability ofBD to activate TRPV1 makes this nonpsychotropic

annabinoid (as well as CBD BDS) less active in reduc-ng bladder contractility. Others have shown that thendocannabionid anandamide, by way of TRPV1 activa-ion, contributes to the development of hyperreflexia andyperalgesia during cystitis.33

Finally, we confirmed the main findings reported forthe rat bladder in human tissue. We found that CBDBDS and CBD inhibited acetylcholine-induced contrac-tions in the human bladder. Similar to the findings forthe rat, we found that CBD BDS was more active andeffective than pure CBD in reducing acetylcholine-in-duced contractions. However, in contrast to the findingsfrom the rat experiments, the inhibitory effect of bothCBD BDS and CBD on acetylcholine-induced contrac-tions was not significantly modified by either capsazepineor ruthenium red in the human bladder. This was notsurprising because of the observation that the well-known TRPV1 agonist capsacin contracted isolated ratbladder tissue, but not human bladder tissue.13,34,35

CONCLUSIONSWe have shown that CBD BDS reduces—through apostsynaptic site of action—cholinergic-mediated con-

tractility in the rat and human bladder. CBD is likely the

1006.e14

chemical ingredient responsible for the activity of theextract, although synergistic/additive effects have beenobserved. The effect of both CBD BDS and pure CBDwas modulated—in the rat but not in the human blad-der—by TRPV1. If confirmed in vivo, such results couldprovide a pharmacologic basis to explain, at least in part,the efficacy of Cannabis preparations in reducing urinarysymptoms associated to multiple sclerosis and open thepossibility of evaluating CBD BDS and/or pure CBD forpossible use in treating urinary incontinence. Additionalstudies are needed to verify whether CBD BDS affects thecontractility in experimental models of overactive blad-der as well as in the tissues from patients with overactivebladder.

Acknowledgment. To Enrico and Enrica Sovena Foundationnd GW Pharmaceuticals (Porton Down, Wiltshire, UK) forroviding us with CBD and CBD BDS.

References1. Pertwee RG. Emerging strategies for exploiting cannabinoid recep-

tor agonists as medicines. Br J Pharmacol. 2009;156:397-411.2. Hayn MH, Ballesteros I, de Miguel F, et al. Functional and immu-

nohistochemical characterization of CB1 and CB2 receptors in ratbladder. Urology. 2008;72:1174-1178.

3. Gratzke C, Streng T, Park A, et al. Distribution and function ofcannabinoid receptors 1 and 2 in the rat, monkey and humanbladder. J Urol. 2009;181:1939-1948.

4. Gratzke C, Streng T, Stief CG, et al. Effects of cannabinor, a novelselective cannabinoid 2 receptor agonist, on bladder function innormal rats. Eur Urol. 2010;57:1093-1100.

5. Pertwee RG, Fernando SR. Evidence for the presence of cannabi-noid CB1 receptors in mouse urinary bladder. Br J Pharmacol.1996;118:2053-2058.

6. Martin RS, Luong LA, Welsh NJ, et al. Effects of cannabinoidreceptor agonists on neuronally-evoked contractions of urinarybladder tissues isolated from rat, mouse, pig, dog, monkey andhuman. Br J Pharmacol. 2000;129:1707-1715.

7. Pertwee RG. The diverse CB1 and CB2 receptor pharmacology ofthree plant cannabinoids: delta9-tetrahydrocannabinol, cannabi-diol and delta9-tetrahydrocannabivarin. Br J Pharmacol. 2008;153:199-215.

8. Izzo AA, Borrelli F, Capasso R, et al. Non-psychotropic plantcannabinoids: new therapeutic opportunities from an ancient herb.Trends Pharmacol Sci. 2009;30:515-527.

9. Wade DT, Makela P, Robson P, et al. Do cannabis-based medicinalextracts have general or specific effects on symptoms in multiplesclerosis? A double-blind, randomized, placebo-controlled study on160 patients. Mult Scler. 2004;10:434-441.

10. Brady CM, DasGupta R, Dalton C, et al. An open-label pilot studyof cannabis-based extracts for bladder dysfunction in advancedmultiple sclerosis. Mult Scler. 2004;10:425-433.

11. Freeman RM, Adekanmi O, Waterfield MR, et al. The effect ofcannabis on urge incontinence in patients with multiple sclerosis:a multicentre, randomised placebo-controlled trial (CAMS-LUTS). Int Urogynecol J Pelvic Floor Dysfunct. 2006;17:636-641.

12. Maggi CA, Bevan S, Walpole CS, et al. A comparison of capsaz-epine and ruthenium red as capsaicin antagonists in the rat isolatedurinary bladder and vas deferens. Br J Pharmacol. 1993;108:801-805.

3. Capasso R, Borrelli F, Capasso F, et al. Inhibitory effect of theantidepressant St. John’s wort (Hypericum perforatum) on rat blad-der contractility in vitro. Urology. 2004;64:168-172.

4. Nocerino E, Izzo AA, Borrelli F, et al. Relaxant effect of capsaz-epine in the isolated rat ileum. Naunyn Schmiedebergs Arch Phar-

macol. 2002;365:187-192.

UROLOGY 77 (4), 2011

2

2

2

2

2

3

3

3

3

3

3

15. Consroe P, Musty R, Rein J, et al. The perceived effects of smokedcannabis on patients with multiple sclerosis. Eur Neurol. 1997;38:44-48.

16. Kavia R, De Ridder D, Constantinescu C, et al. Randomizedcontrolled trial of Sativex to treat detrusor overactivity in multiplesclerosis. Mult Scler. 2010;16:1349-1359.

17. Layman JM, Milton AS. Some actions of delta-1 tetrahydrocan-nabinol and cannabidiol at cholinergic junctions. Br J Pharmacol.1971;41:379P-380P.

18. Pertwee RG, Ross RA, Craib SJ, et al. (-)-Cannabidiol antagonizescannabinoid receptor agonists and noradrenaline in the mouse vasdeferens. Eur J Pharmacol. 2002;456:99-106.

19. Capasso R, Borrelli F, Aviello G, et al. Cannabidiol, extracted fromCannabis sativa, selectively inhibits inflammatory hypermotility inmice. Br J Pharmacol. 2008;154:1001-1008.

20. Williamson EM. Synergy and other interactions in phytomedi-cines. Phytomedicine. 2001;8:401-409.

21. Kathmann M, Flau K, Redmer A, et al. Cannabidiol is an allostericmodulator at mu- and delta-opioid receptors. Naunyn SchmiedebergsArch Pharmacol. 2006;372:354-361.

22. Berggren A, Sillén U, Rubenson A. In vivo motor effects ofloperamide on the rat urinary bladder. Acta Physiol Scand. 1992;145:33-37.

23. Dhattiwala AS, Jindal MN, Kelkar VV. Effects of some drugs onthe responses of the rat isolated, innervated urinary bladder toindirect electrical stimulation. Br J Pharmacol. 1970;39:738-747.

4. Fry CH, Hussain M, McCarthy C, et al. Recent advances in detrusormuscle function. Scand J Urol Nephrol Suppl. 2004;215:20-25.

25. Drysdale AJ, Ryan D, Pertwee RG, et al. Cannabidiol-inducedintracellular Ca2� elevations in hippocampal cells. Neuropharma-

cology. 2006;50:621-631.

UROLOGY 77 (4), 2011

6. Holzer P. The pharmacological challenge to tame the transientreceptor potential vanilloid-1 (TRPV1) nocisensor. Br J Pharmacol.2008;155:1145-1162.

7. Avelino A, Cruz F. TRPV1 (vanilloid receptor) in the urinarytract: expression, function and clinical applications. NaunynSchmiedebergs Arch Pharmacol. 2006;373:287-299.

8. Everaerts W, Gevaert T, Nilius B, et al. On the origin of bladdersensing: tr(i)ps in urology. Neurourol Urodyn. 2008;27:264-273.

9. Saitoh C, Kitada C, Uchida W, et al. The differential contractileresponses to capsaicin and anandamide in muscle strips isolatedfrom the rat urinary bladder. Eur J Pharmacol. 2007;570:182-187.

0. Charrua A, Cruz D, Narayanan S, et al. GRC-6211, a new oralspecific TRPV1 antagonist, decreases bladder overactivity and nox-ious bladder input in cystitis animal models. J Urol. 2009;181:379-386.

1. Ouslander JG. Management of overactive bladder. N Engl J Med.2004;350:786-799.

2. Yoshimura N, Kaiho Y, Miyazato M, et al. Therapeutic receptortargets for lower urinary tract dysfunction. Naunyn SchmiedebergsArch Pharmacol. 2008;377:437-448.

3. Dinis P, Charrua A, Avelino A, et al. Anandamide-evoked acti-vation of vanilloid receptor 1 contributes to the development ofbladder hyperreflexia and nociceptive transmission to spinal dorsalhorn neurons in cystitis. J Neurosci. 2004;24:11253-11263.

4. Kalbfleisch RE, Daniel EE. The role of substance P in the humanurinary bladder. Arch Int Pharmacodyn Ther. 1987;285:238-248.

5. Maggi CA, Patacchini R, Santicioli P, et al. Further studies on themotor response of the human isolated urinary bladder to tachy-kinins, capsaicin and electrical field stimulation. Gen Pharmacol.

1989;20:663-669.

1006.e15