JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 JPETThis Fast article Forward. has not been Published copyedited andon formatted. July 12, The 2007 final asversion DOI:10.1124/jpet.107.125757 may differ from this version.

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Title Page

GW427353 (solabegron) a novel selective β3 receptor agonist, evokes

bladder relaxation and increases micturition reflex threshold in the dog

Alexandra Hicks, Gerald P McCafferty, Erin Riedel, Nambi Aiyar, Mark Pullen, Downloaded from Christopher Evans, Trudy D Luce, Robert W Coatney, Gian C Rivera, Timothy D

Westfall and J. Paul Hieble

jpet.aspetjournals.org

Departments of Cardiovascular and Urogenital Biology (AH, GPM, ER, NA, MP,

TDW, JPH), Drug Metabolism and Pharmacokinetics (CE, TDL), Animal

Modeling and Imaging and Laboratory Animal Sciences (RWC, GCR) at ASPET Journals on October 3, 2021

Cardiovascular and Urogenital Center of Excellence for Drug Discovery,

GlaxoSmithKline Pharmaceuticals,

709 Swedeland Road,

P.O. Box 1539, King of Prussia, PA

19406 USA

1

Copyright 2007 by the American Society for Pharmacology and Experimental Therapeutics. JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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Running Title Page

Running Title: β3-adrenoceptor agonist in the dog

Corresponding Author:

Timothy D Westfall, PhD

Urogenital Biology, Downloaded from Cardiovascular and Urogenital Center of Excellence for Drug Discovery,

GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road,

P.O. Box 1539, jpet.aspetjournals.org

King of Prussia, PA 19406,

U.S.A.

Telephone: (001) 610 270 4320 at ASPET Journals on October 3, 2021

Fax: (001) 610 270 5681

E-mail: [email protected]

Number of text pages: 30

Number of tables: 5

Number of figures: 6

Number of references: 30

Abstract: 215 words

Introduction: 293 words

Discussion: 911 words

Abbreviations: β3-AR, β3-; CHO, Chinese Hamster Ovary

Recommended section assignment: Gastrointestinal, Hepatic, Pulmonary and Renal

2 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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Abstract

Functional studies have demonstrated that adrenoceptor agonist-evoked relaxation is mediated primarily by β3-adrenergic receptors (β3-ARs) in human bladder.

Thus, the use of selective β3-AR agonists in the pharmacological treatment of is being explored. The present studies investigated the effects of a novel selective β3-AR agonist, GW427353 (solabegron) on bladder function Downloaded from in the dog using in vitro and in vivo techniques. GW427353 stimulated cAMP accumulation in CHO cells expressing the human β3-AR with an EC50 value of 22

+ 6 nM and an intrinsic activity 95% of isoproterenol. At concentrations of jpet.aspetjournals.org

10,000 nM, GW427353 produced a minimal response in cells expressing either

β1-ARs or β2-ARs (maximum response < 10% of that to isoproterenol). In dog at ASPET Journals on October 3, 2021 isolated bladder strips, GW427353 evoked relaxation that was attenuated by the non-selective β-AR antagonist and SR59230A (reported to have β3-

AR antagonist activity). The relaxation was unaffected by , a selective

β1-AR antagonist, or ICI 118,551, a selective β2-AR antagonist. GW427353 increased the volume required to evoke micturition in the anesthetized dog following acetic acid-evoked bladder irritation, without affecting the ability of the bladder to void. GW427353-evoked effects on bladder parameters in vivo were inhibited by bupranolol. The present studies demonstrate that selective activation of β3-AR with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog.

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Introduction

Overactive bladder is a syndrome characterised by symptoms of urinary frequency, urgency, nocturia and urgency incontinence, in which there is a need for alternative therapies with novel mechanisms of action (Andersson and Wein,

2004).

The activities of the urinary bladder during urine storage and voiding are Downloaded from governed by a complex neural control system, involving both sympathetic and parasympathetic input. During bladder filling, sympathetic nerve activity to

bladder smooth muscle results in β-adrenergic receptor (β-AR) mediated jpet.aspetjournals.org relaxation (Andersson and Wein, 2004). Several subtypes of β-ARs have been identified, namely, β1, β2 and β3-ARs (Bylund et al., 1994). Evidence suggests at ASPET Journals on October 3, 2021 that species differences exist in the distributions and roles of β-ARs in the bladder

(for comprehensive review see Michel and Vrydag, 2006). Bladder relaxation evoked by β-AR agonists is mediated mainly via β1-ARs in cats (Nergardh et al.,

1977) and guinea pigs (Li et al., 1992), β2-AR in rabbits, by both β2- and β3-ARs in rats (Yamazaki et al., 1998) and pigs (Yamanishi et al., 2002) and β3-ARs in the ferret (Takeda et al., 2000), dog (Yamazaki et al., 1998) and primate (Takeda et al., 2002a). Based on mRNA expression, at least 95% of the adrenoceptor message in human bladder comprises the β3-AR subtype (Nomiya and

Yamaguchi, 2003). Further, functional studies have demonstrated that the relaxant effects induced by adrenergic receptor stimulation in human detrusor are mediated via β3-AR activation (Yamaguchi, 2002; Yamanishi et al., 2006). Thus,

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selective β3-AR stimulation of human bladder would be expected to cause bladder relaxation and as such may represent a novel strategy in the treatment of overactive bladder. The present studies investigated the effects of a novel, selective β3-AR agonist, GW427353 (solabegron; Uehling et al., 2006) on bladder function in the dog using in vitro and in vivo techniques.

Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021

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Methods

All experiments were reviewed and approved by the Institutional Animal Care and Use Committee of GlaxoSmithKline. All procedures on animals were performed in compliance with ILAR Guide for the Care and Use of Laboratory

Animals (National Research Council, 1996). Downloaded from

Radioligand binding assay

Membranes from CHO cells expressing β1, β2 or β3-ARs were prepared as jpet.aspetjournals.org described (Elshourbagy et al., 1993). Saturation binding experiments were performed as described (Schnabel et al., 2000) except a 50 µl volume was used and incubation was for 30 min at 370 C. A range of final concentrations of [125I]- at ASPET Journals on October 3, 2021

(-) (ICYP), 10 pM – 600 pM for β1 and β2 or 0.1 nM – 2.3 nM for β3. Non-specific binding was measured in the presence of SR59230A (10

µM). Protein was measured with a Biorad protein assay kit. Receptor affinity and density for the various receptors were calculated by global fit of total and non-specific binding curves using GraphPad Prism V4.0 (San Diego, CA).

Measurement of cyclic AMP

Chinese hamster ovary cells - K1 (CHO-K1) stably expressing β-ARs were grown in 50:50 Dulbecco’s modified Eagle’s medium (DMEM) plus F12 K with geneticin and hygromycin in 24 well plates (50,000 cells per well) for 48 h. On the day of experiment, medium was replaced with phosphate buffered saline

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(PBS, [Gibco cat No#21300-025]) containing 1 mM 3-isobutyl-1-methylxanthine

(IBMX). Cells were then treated with vehicle (DMSO, 2.5%), GW427353 or isoproterenol in DMSO (40X concn) in 200 µl of PBS containing 1 mM IBMX and incubated at 37° C for 30 min. After incubation cells were treated with lysis buffer and cAMP level was determined using commercial kit Amersham SPA as per package instructions by the manufacturer. The SPA measures cAMP via Downloaded from competition between cAMP and adenosine 3',5'-cyclic phosphoric acid 2'-O- succinyl-3-[125I]iodotyrosine methyl ester for an antibody/scintillant microbead system. Samples were processed under the nonacetylation protocols for the kit. jpet.aspetjournals.org

The range of sensitivity for the nonacetylation protocols of the kit was 0.2-12.8 pmol/ assay tube. Aliquots (50 µl) of the samples were transferred to 96 well at ASPET Journals on October 3, 2021 optiplate for determining cAMP level. The plates were counted on a Packard Top

Count Scintillation Counter. All experiments were performed in duplicate, with n referring to the number of independent experiments. Values are expressed as mean ± standard error of the mean (SEM) throughout the manuscript.

In vitro urinary bladder tissue assay

Mongrel and Beagle dogs (9 – 12 kg; Marshall Farms, NY, U.S.A.) were euthanized with an injection of 390 mg/ml sodium pentobarbitol into the cephalic vein followed by rapid exsanguination. The bladder was exposed and removed, fat and connective tissue cleared and the bladder body cut into longitudinal strips approximately 3 x 6 mm in size. The strips were suspended in tissue baths containing Krebs solution (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.5 mM

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MgSO4, 1.2 mM K2HPO4, 25 mM NaCO3 and 11 mM glucose). Tissues were

o maintained at 37 C and constantly aerated with 95% oxygen and 5% CO2.

Tissues were mounted to a force-displacement transducer (Biopac Inc, CA, USA) and changes in muscle tension were digitally recorded using AcqKnowledge 3.8.1 software (Biopac Inc, CA, USA).

Tissues were equilibrated for 60 min, during which the tissues were Downloaded from maintained at 9.8 mN resting tension. Concentration-response curves for

GW427353 were obtained by sequential addition (30 min interval between doses)

directly into the bath 20 min following stimulation with 15 mM KCl. jpet.aspetjournals.org

(100 µM) was added following the construction of concentration-response curves.

In a separate group of experiments, the effect of β-AR antagonists (bupranolol, at ASPET Journals on October 3, 2021 atenolol, ICI 118,551 and SR59230A) were assessed on a single dose of

GW427353 (10 nM). Antagonist or vehicle was added 5 min after 15 mM KCl stimulation and left to incubate for 15 min before addition of GW427353.

Following a 30 min incubation of GW427353, papaverine (100 µM) was added.

Stock solutions were prepared as follows: papaverine, ICI 118,551 and

SR59230A were dissolved in water, GW427353 and bupranolol in DMSO and atenolol in ethanol and diluted to desired concentrations in water. The reported concentrations are the calculated final bath concentrations.

Data analysis Integrals over a two minute period were measured before and following addition of test substances. The effects of GW427353 were expressed as percentages of the maximal relaxation induced by 100 µM papaverine. In

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cumulative concentration-response experiments, GW427353-evoked responses were compared with vehicle treated tissues using Two-way ANOVA followed by

Bonferroni post test. In single concentration experiments, GW427353-evoked responses in the absence or presence of antagonist were compared using unpaired t-test. P < 0.05 was considered statistically significant.

Downloaded from In vivo acetic acid-evoked bladder irritation in the dog

Experiments were performed on 29 adult female beagle dogs (6 – 10 kg; Marshall

Farms, NY, U.S.A.) fasted overnight with free access to water. Cannulae were jpet.aspetjournals.org inserted into both cephalic veins for intravenous (i.v.) administration of anaesthetic and test substances. Animals were then administered propofol (6 mg/kg i.v.) for anaesthetic induction. Anaesthesia was maintained with propofol at ASPET Journals on October 3, 2021 infusion (5 mg/min i.v.). The trachea was intubated to maintain a patent airway.

The left femoral artery was cannulated with a heparinized cannula (20 u ml-1 heparin in 0.9% w/v saline; PE 50) for the measurement of mean arterial blood pressure (MAP) and heart rate (HR) and for sampling arterial blood for blood gas analysis. Body temperature was maintained between 36-38° C using a blanket system (Gaymar TP-500). Blood pressure was measured using a pressure transducer (Gould Statham P23XL), and the HR derived electronically on-line from the blood pressure signal using Spike version 5.0 software (Cambridge

Electronic Design, Cambridge, U.K.). The urinary bladder was exposed by a midline abdominal incision and a cut made in the bladder dome and a cannula

(PE 60) was inserted into the bladder via a 14 gauge needle. The needle was

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removed and the cannula secured with a purse string suture. The cannula, via a

3-way tube connector (Small Parts Inc., FL, U.S.A), was connected to a pressure transducer (Gould Statham P23XL) to record intravesical bladder pressure, and to a syringe pump (Harvard Apparatus Infusion Pump, model 975) for the intravesical infusion of saline or acetic acid. Draining through the cannula allowed the bladder to be emptied of saline. Void volume was measured by Downloaded from collecting and weighing fluid released from the bladder during micturition. At the end of all surgical procedures, propofol anesthesia was discontinued and

α-chloralose administered (100 mg/kg, i.v.). Depth of anesthesia was assessed by jpet.aspetjournals.org the stability of MAP and HR, and by an absence of hind limb withdrawal in response to paw pinch. Anaesthesia was maintained via α-chloralose infusion (1 at ASPET Journals on October 3, 2021 mg/kg/min i.v.). Animals were artificially ventilated with room air by use of a positive pressure pump (Large Animal Ventilator, Harvard, MA, USA). Blood gases were monitored and maintained between 90-130 mmHg Po2, 20-35 mmHg

Pco2 and pH 7.3-7.4 with a Corning pH/blood gas analyzer (Model 248).

Adjustments of the respiratory pump rate and volume were made as necessary to maintain blood gas and pH balance. Following completion of studies, dogs were euthanized with sodium pentobarbital (120 mg/kg i.v.) without recovery.

Following surgery, animals were allowed to stabilize for 30 min during which blood gases were monitored and maintained. Warmed saline (0.9% w/v) was then infused into the bladder (3 ml/min) until voiding (the micturition reflex) occurred, characterized by a large amplitude bladder contraction with the concomitant release of fluid from the urethral opening. This fluid was measured

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and termed the void volume (mls). Micturition volume threshold (mls) was taken as the total volume of fluid required to be infused into the bladder to evoke voiding. Voiding efficiency (%) was calculated from voided volume divided by micturition volume threshold and multiplied by 100. Fluid was emptied from the bladder following voiding for a 10 min period (until the bladder was as empty as possible), after which the bladder was re-infused with saline to evoke a further Downloaded from micturition reflex. This was continued until 4 control voids had been evoked.

GW427353 or vehicle (PEG 400) was then administered as a bolus i.v. (0.15

ml/kg) followed by a 5 min period to allow stabilization of any change in baseline jpet.aspetjournals.org parameters. The saline infusion into the bladder was then replaced with 0.7 % acetic acid in 0.9 % saline (pH 3.0) and 3 acetic acid voids were evoked as outlined in the protocol above. This time period was chosen as preliminary at ASPET Journals on October 3, 2021 studies showed a reproducible hyperactive bladder response to acetic acid could be evoked using this protocol. Blood samples (200 µl) were taken following each void for plasma sample analysis of drug levels.

Data analysis Arterial blood and bladder pressures were continuously displayed on a chart recorder (Grass Instruments) and captured (200 samples per s) by a

CED 1401 interface (Cambridge Electronic Design, U.K.) to allow data to be acquired and analyzed off-line using Spike version 5 software (Cambridge

Electronic Design, U.K.). HRs were derived electronically on-line from the blood pressure signal using this software. All baseline values were the mean values over a 30 s period and were measured 1 min before infusion of saline or acetic

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acid into the bladder. The amplitude (mmHg) and duration(s) of each reflex-evoked bladder contraction was measured. The micturition pressure thresholds were taken as the bladder pressure (mmHg) at which voiding occurred.

The control values for various parameters were calculated from the mean of the last two pre-acetic acid voids (micturition parameters) or the mean of the last three voids (MAP and HR). The mean of the next 3 voids following treatment Downloaded from with vehicle or GW427353 were used to measure the change in void parameters,

MAP and HR. Changes were compared to control values and treatment values

using a paired t or student t test as appropriate. jpet.aspetjournals.org

Plasma analysis

Analysis of female dog plasma samples for GW427353 was performed using at ASPET Journals on October 3, 2021 liquid chromatography/tandem mass spectrometric (LC/MS/MS) detection.

Analytical standards for GW427353 were prepared in 50 µl of female dog plasma.

Dog plasma samples were thawed, plasma proteins were precipitated with 200 µl of 95/5 acetonitrile/10 mM aqueous ammonium formate (pH 3.0), containing 200 ng/ml of a mass spectral internal standard, and the resulting mixture was vortex- mixed for two min followed by centrifugation for 30 min at > 2000 x g. 2.0 µl of the resulting supernatant was injected onto the LC/MS/MS system using an HTS

PAL autosampler (CTC Analytics, Zwingen, Switzerland). The mobile phase consisted of a 2.1 min linear gradient using aqueous 10 mM ammonium formate

(pH 3.0) and acetonitrile mobile phases (500 ul/min flow rate). A 2 x 50 mm, 3 µ,

Polaris C8 Ether analytical column (Varian, Lake Forest, CA) was used. The

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eluent flowed into a Sciex API4000 triple-quadrupole mass spectrometer (Applied

Biosystems, Foster City, California) using positive-ion electrospray multiple- reaction monitoring. GW427353 was characterized by the transition of the m/z

411 parent (M+H)+ precursor ion to its m/z 240 product ion, generated at an optimized collision energy.

Downloaded from Data analysis Data were reported as quantitative concentrations as determined by standard calibration curve analysis, using quadratic fitting of a 1/x-weighted plot

of the GW427353/mass spectral internal standard peak area ratios versus jpet.aspetjournals.org

GW427353 concentration, where the analytical range of quantitation was 50 to

10,000 ng/ml (r=0.9944), LLQ to HLQ, respectively.

at ASPET Journals on October 3, 2021

Plasma protein binding

2 male beagle dogs (8.5 and 14.5 kg) were fasted overnight with free access to water. Cannulas were inserted into both cephalic veins and animals were dosed with GW427353 i.v. (over a 5 min infusion period, in 0.025 M methanesulfonic acid in 5% mannitol) with a total dose of 0.2 mg/kg via one of the catheters. 5 min following dosing, blood samples (2 ml) were collected via the other cephalic vein. The plasma was separated from red blood cells by centrifugation and frozen at -20º C. The extracts were analysed for GW427353 using similar analytical methodologies as described above. Protein plasma binding was determined by ultrafiltration at 37º C using an Amicon Centrifree disposable ultrafiltration device (Millipore Corp., Bedford, MA, U.S.A.). An absorption test indicated that

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GW427353 did not bind to the membrane or plastic components of the ultrafiltration device.

Materials

(R)-3'-[[2-[[2-(3-chlorophenyl)-2-hydroxyethyl]amino]ethyl]amino]-[1,1'- biphenyl]-3-carboxylic acid (GW427353; Uheling et al., 2006) and bupranolol Downloaded from hydrochloride (1-(2-chloro-5-methylphenoxy)-3-[(1,1-dimethylethyl)amino]-2- propanol) were synthesised at GlaxoSmithKline Pharmaceuticals, King of Prussia,

PA, U.S.A. Potassium chloride, papaverine hydrocholoride and atenolol ((±)-4- jpet.aspetjournals.org

[2-Hydroxy-3-[(1- methylethyl)amino]propoxy]benzeneacetamide) were purchased from Sigma Aldrich Fine Chemicals, St. Louis, MO, U.S.A. ICI

118,551 hydrochloride ((±)-1-[2,3-(Dihydro-7-methyl-1H-inden-4-yl)oxy]-3-[(1- at ASPET Journals on October 3, 2021 methylethyl)amino]-2-butanol hydrochloride) and SR59230A hydrochloride (1-

(2-Ethylphenoxy)-3-[[(1S)-1,2,3,4-tetrahydro-1-naphthalenyl]amino]-(2S)-2- propanol hydrochloride) were purchased from Tocris Cookson, Ltd, Bristol, U.K.

[125I]-(-)Iodocyanopindolol (2200 Ci/mmol) was purchased from PerkinElmer,

Boston, MA, U.S.A.

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Results

In vitro cellular assays

GW427353 stimulated cAMP accumulation in CHO cells expressing the recombinant human β3-AR with an EC50 value of 22 ± 6.0 nM and producing a maximum response 95 ± 5% of that produced by the nonselective β-adrenoceptor Downloaded from agonist isoproterenol. At concentrations of 10,000 nM, GW427353 produced a minimal response in cells expressing either β1-ARs or β2-ARs (maximum response < 10% of that to isoproterenol) (Table 1). GW427353 did not produce jpet.aspetjournals.org antagonism at any of the β-ARs (not shown). Receptor density was determined in membranes from CHO cells expressing the recombinant β-ARs. β3-AR at ASPET Journals on October 3, 2021 expression was found to be lower than that of both β1- and β2-AR expression

(Table 2).

In vitro urinary bladder tissue assay

Cumulative addition of GW427353 evoked concentration-dependent relaxation of

KCl pre-contracted dog bladder strips attaining a significant effect at 100 nM (n =

18; Fig. 1). However, significant decreases in the tension of strips was observed over time in vehicle-treated tissues and thus a single concentration experimental protocol was used to evaluate the effects of antagonists. In the single concentration experiments, decreases in the presence of vehicle were much less

(12 ± 3.3%). A single concentration of 10 nM GW427353 produced 82 ± 2.5%

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relaxation (n = 6; Fig. 2A). Higher concentrations of GW427353 (30 and 100 nM) produced a degree of relaxation that was not statistically different from 10 nM (77 ± 6.9% and 89 ± 4.7%, respectively, n = 8, data not shown), thus 10 nM

GW427353 was used as the agonist concentration in antagonist studies. The non- selective β-AR antagonist bupranolol (10 µM) significantly attenuated the relaxation evoked by 10 nM GW427353 by approximately 64%, without having Downloaded from significant activity alone (n = 4, Fig. 2A). Neither the selective β1-AR antagonist, atenolol (1 µM) nor the selective β2-AR antagonist ICI 118,551 (1 µM) had any effects of bladder responses evoked by GW427353 (n = 8; Fig. 2B & 2C). jpet.aspetjournals.org

SR59230A, reported to have antagonistic properties at the β3-AR (Manara et al.,

1996), evoked a significant relaxation of isolated canine bladder strips alone, at ASPET Journals on October 3, 2021 evoking maximal responses of 47 ± 17% and 38 ± 5.8% at 1 and 3 µM, respectively (n = 4; Fig. 2D). In the presence of 1 µM and 3 µM SR59230A,

GW427353-evoked relaxations were significantly attenuated by 21% and 30% respectively (n = 4; Fig. 2D).

In vivo acetic acid-evoked bladder irritation in the dog

Control micturition reflexes

Infusion of saline into the bladder evoked the micturition reflex, characterised by the appearance of a large amplitude (56 ± 5.8 mmHg; n = 22) bladder contraction lasting 34 ± 7.2 (n = 23) seconds, with the concomitant release of 27 ± 5.3 ml of

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fluid from the urethral opening (void volume; n = 23). The mean volume infused to evoke the micturition reflex (micturition volume threshold) was 38 ± 4.3 ml (n

= 23) with a calculated voiding efficiency (voided volume as a percentage of micturition reflex threshold) of 84 ± 13% (n = 23). This parameter was sometimes calculated to be greater than 100% as not all the residual volume could be drained from the bladder before the start of the next bladder infusion. Manual expression Downloaded from of residual urine from the bladder was not performed to prevent potential sensitisation of bladder nociceptive afferent nerves during the control period. The mean bladder pressure threshold to evoke the micturition reflex was 38 ± 3.9 jpet.aspetjournals.org mmHg (micturition pressure threshold; n = 22). Baseline MAP and HR were 123

± 5.6 mmHg and 116 ± 7.3 beats per min, respectively (n = 22). The mean at ASPET Journals on October 3, 2021 baseline data for bladder and cardiovascular parameters are shown in Table 3.

Effects of GW427353 on acetic acid-evoked bladder irritation

In vehicle pre-treated dogs, intravesical infusion of acetic acid evoked a decrease in the micturition volume threshold of 10 ± 2.3 ml or 29% (n = 7; Table 3; Fig. 3).

Intravesical acetic acid facilitated voiding, evoking a concomitant increase in void volume, voiding efficiency and bladder contraction amplitude, likely as a result of residual volumes that were not emptied during the control period now being evacuated (Table 3). Acetic acid had no effect on micturition pressure threshold, bladder contraction duration, MAP and HR (Table 3). GW427353 (3 mg/kg i.v.) evoked an increase in micturition volume threshold of 6.5 ± 4.4 ml or 22% and

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thus prevented the acetic-acid-evoked decreases in this parameter (Table 3; Fig.

3). A lower dose of GW427353 (1 mg/kg i.v.) appeared to block the reduction in micturition threshold produced by acetic acid, but this effect was not significant

(Fig. 3). Neither dose of GW427353 had any effect on void volume, voiding efficiency and bladder contraction amplitude and duration (Table 3). Both doses of GW427353 evoked a significant decrease in MAP (25 ± 3.0 mmHg or 22% and Downloaded from 35 ± 5.3 mmHg or 30%) and increase in HR (83 ± 16 mmHg or 105% and 97 ±

14 mmHg or 82%) following administration of 1 and 3 mg/kg, respectively (Table

3; Fig. 4). Flushing of the dogs was also observed but not measured. jpet.aspetjournals.org

In vivo antagonist experiments were performed with the higher dose of

GW427353 (3 mg/kg i.v.). Bupranolol (1 mg/kg i.v.) alone had no significant at ASPET Journals on October 3, 2021 effects on micturition threshold volume (Fig. 5) or cardiovascular parameters when compared to vehicle treatment (not shown). Pre-treatment with bupranolol prevented the GW427353-evoked increases in micturition threshold volume (Fig.

5). In addition, bupranolol significantly attenuated the GW427353-evoked increase in HR, but not the GW427353-evoked decrease in MAP (Fig. 6).

Plasma analysis and protein binding

Plasma samples were taken after each acetic acid void following i.v. dosing with

GW427353 in the above experiments and plasma concentrations determined

(Table 4). Average plasma concentrations were 2130 ng/ml (4.8 µM) and 3343 ng/ml (7.5 µM) following doses of 1 mg/kg and 3 mg/kg respectively. In separate

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experiments, dog plasma protein binding of GW427353 was determined to be

99.3% (Table 5).

Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021

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Discussion

The present studies demonstrate that stimulation of β3-ARs with GW427353 evokes bladder relaxation and facilitates bladder storage mechanisms in the dog, suggesting that β3-AR agonists may represent a novel approach in the treatment of overactive bladder. In human bladder, at least 95% of the adrenoceptor message comprises the β3-AR subtype and adrenoceptor agonist-evoked relaxation is Downloaded from mediated principally by these receptors (Yamaguchi, 2002; Nomiya and

Yamaguchi, 2003; Yamanishi et al., 2006). GW427353 has been found to relax isolated human bladder strips and reduce spontaneous contractile activity (Biers et jpet.aspetjournals.org al., 2006; Westfall, unpublished observations). In rat models, β3-AR agonists have been shown to increase micturition interval in bladder instability evoked by at ASPET Journals on October 3, 2021 cerebral infarction (Kaidoh et al., 2002), intravesical acetic acid or PGE2 (Takeda et al., 2002b; Woods et al., 2001), inhibit spontaneous inter-micturition contractions in obstruction-induced bladder hyperplasia (Woods et al., 2001) and decrease the frequency of rhythmic bladder contractions (Takasu et al., 2007). β3-

AR mediated bladder relaxation is also observed in the anaesthetized ferret

(Takeda et al., 2000).

GW427353 is a highly selective β3-AR agonist as evidenced by selective stimulation of cAMP accumulation in CHO cells expressing the human β3-AR, with an intrinsic activity nearly equivalent to that of isoproterenol. GW427353 did not prossess antagonistic activity at any of the β-ARs (binding experiments with GW427353 were not included as they would not add to the functional data).

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In an in vitro functional bladder assay, GW427353 evoked potent relaxation of isolated canine bladder strips. Further, the non-selective β-AR antagonist bupranolol (Pietri-Rouxel and Strosberg, 1995) attenuated GW427353-evoked relaxations, demonstrating the involvement of β-AR stimulation in mediating these effects. Atenolol and ICI 118,551, at concentrations shown to be effective in selectively blocking β1- and β2-ARs, respectively (Baker, 2005), were without Downloaded from effect. The present findings are in agreement with the studies by Takeda et al

(2003) who showed potent relaxation of dog bladder strips with selective β3-, but jpet.aspetjournals.org not β1- or β2-AR agonists. Yamazaki et al (1998) provided additional in vitro functional evidence using selective β-AR agonists and antagonists to show that

β3-ARs mediate the bladder relaxation induced by β-AR agonists in the dog. at ASPET Journals on October 3, 2021 GW427353 also demonstrated the ability to relax canine detrusor in vivo as measured by a significant increase in the micturition volume threshold, i.e. an increase in bladder capacity, under conditions of acetic acid-evoked bladder irritation. No effects on voiding efficiency were observed, demonstrating that

GW427353 did not detrimentally affect the ability of the bladder to void.

Consistent with the in vitro findings in canine isolated detrusor strips, bupranolol blocked the GW427353-evoked increases in micturition volume threshold, suggesting the involvement of β-AR stimulation. However the in vivo results must be interpreted with caution as β1-AR or β2-AR selective antagonists were not tested in vivo and GW427353 was not tested for selectivity at canine recombinant β-ARs, therefore a contribution of β1-AR and or β2-AR activity can

21 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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not be ruled out. Based on plasma exposures of GW427353 following administration, the high plasma protein binding and low volume of distribution

(not shown) of this compound in the dog, drug levels may not have been sufficient at the 1 mg/kg dose, especially at the level of the bladder tissue, to evoke a similar pharmacological effect to 3 mg/kg i.v. on bladder function.

However, GW427353 evoked effects on cardiovascular parameters, characterized Downloaded from by flushing, decreases in MAP and a positive chronotropic effect, consistent with

β3-AR stimulation in the dog, at both doses tested (Shen et al., 1996; Tavernier et

al., 1992). Differences in the distribution of GW427353 to visceral organs and jpet.aspetjournals.org peripheral circulation or differences in β3-AR density and physiological function at these two sites may explain the differential magnitude of the effects of at ASPET Journals on October 3, 2021 GW427353 on bladder versus the cardiovascular system. GW427353-evoked cardiovascular responses were only partially attenuated by bupranolol. It has been reported that complete sinoaortic denervation or combined ganglionic, cholinergic and adrenergic (β1- and β2-AR) blockade is required to completely block β3-AR agonist-evoked increases in HR in the dog. This indicates that the

β3-AR agonist effects on cardiovascular function in this species are complex, and that the effects on MAP and HR involve a variety of direct and indirect mechanisms (Donckier et al., 2001; Shen et al., 1996; Tavernier et al., 1992). In addition, the apparent β3-AR mediated cardiovascular effects in dogs appear to be species specific as reports suggest selective β3-AR agonism in nonhuman

22 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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primates and humans does not result in similar cardiovascular effects (Shen et al.,

1996; Larsen et al., 2002).

SR59230A, which has been shown to have antagonistic properties at the

β3-AR (Manara et al., 1996), was used in the present studies in an attempt to provide further supporting evidence for the mechanism of action of GW427353.

SR59230A evoked responses alone in the isolated tissue studies suggesting β3-AR Downloaded from agonist activity. Partial β3-AR agonist activity of SR59230A has previously been reported in guinea pig gastrointestinal tissues (Horinouchi and Koike, 2001) and jpet.aspetjournals.org at the mouse β3-AR (Hutchinson et al., 2005). We also observed partial agonist activity of SR59230A on CHO cells expressing human recombinant β3-ARs (EC50

= 3.4 nM, IA= 0.28; data not shown). at ASPET Journals on October 3, 2021 In summary, the present studies have demonstrated the ability of a novel selective β3-AR agonist GW427353 to evoke bladder relaxation and facilitate bladder storage mechanisms in the dog. Since β3-ARs are postulated to play an important role in the control of bladder smooth muscle tone in both dog and man, these results support the premise that β3-AR agonists such as GW427353 represent a useful clinical strategy for the treatment of overactive bladder.

23 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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Footnotes

Current Address (AH):

Respiratory, Inflammation & Autoimmune Diseases

Roche

340 Kingsland Street, Nutley, NJ Downloaded from 07006 USA

jpet.aspetjournals.org at ASPET Journals on October 3, 2021

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Legends for Figures

Fig. 1. Effect of GW427353 on KCl pre-contracted canine bladder strips.

*P< 0.05, **P<0.01 as compared to vehicle-treated tissues.

Fig. 2. Effect of GW427353 (10 nM) on KCl pre-contracted bladder strips alone Downloaded from and in the presence of (A) 10 µM bupranolol (B) 1 µM atenolol (C) 1 µM ICI

118551 and (D) 1 µM and 3 µM SR59230A. *P < 0.05, **P < 0.01, ***P < 0.001 for comparison of effects of compound versus vehicle. ##P < 0.01, ###P < 0.001 jpet.aspetjournals.org for comparison of GW427353 effect in presence and absence of antagonist.

at ASPET Journals on October 3, 2021 Fig. 3. Effect of GW427353 on micturition threshold in the anesthetized dog treated with intravesical acetic acid. Each bar represents the mean of 7 (vehicle and GW427353 3 mg/kg) or 3 (GW427353 1 mg/kg) experiments ± SEM.

Significant difference ++P<0.01 from pre treatment and **P<0.01 from vehicle.

Fig. 4. Effect of GW427353 on HR and MAP in the anesthetized dog. Values represent the mean of 6 (vehicle and GW427353 3 mg/kg) or 3 (GW427353 1 mg/kg) experiments ± SEM. Significant difference +P<0.05, ++P<0.01 from pre dosing; *P<0.05, and ***P<0.001 from vehicle.

Fig. 5. Effects of intravenous administration of GW427353, bupranolo1, and the combination of bupranolol and GW427353 on change in micturition threshold in

31 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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anesthetized dog. Bars represent the mean of 6-7 (vehicle and GW427353) or 3

(bupranolol and bupranolol + GW427353) experiments ± SEM. Significant difference *P<0.05 from vehicle and ##P<0.01 from GW427353.

Fig. 6. Effects of intravenous administration of GW427353, bupranolo1, and the combination of bupranolol and GW427353 on change in cardiovascular Downloaded from parameters in anesthetized dog. Bars represent the mean of 7 (GW427353) or 3

(bupranolol and bupranolol + GW427353) experiments ± SEM. Significant difference #P<0.05 from GW427353. jpet.aspetjournals.org at ASPET Journals on October 3, 2021

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Table 1

Effect of isoproterenol and GW427353 on cAMP accumulation in CHO cells expressing human recombinant β-adrenoceptors. Values are mean ± SEM.

Receptor Isoproterenol GW427353 % of the Isoproterenol

pEC50 pEC50 Maximum Maximum at Response to 10 µM Isoproterenol (% over basal)

β1 8.10 ± 0.26 >5 4.2 ± 1.7 401.6 ± 81.5

β2 9.46 ± 0.22 >5 8.8 ± 2.4 441.1 ± 73.1 Downloaded from

β3 8.48 ± 0.10 8.16 ± 0.19 89.1 ± 4.2 457 ± 70.7 β1 = 3.1 ± 0.3 pg/well (4); β2 = 11 ± 2.8 pg/well (7); β3 = 3.2 ± 0.2 pg/well (7) jpet.aspetjournals.org at ASPET Journals on October 3, 2021

33 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 2

Relative receptor density in CHO cell membranes expressing recombinant β-ARs and affinity for ICYP. Values are mean ± SEM.

Subtype Bmax Kd ICYP

fmol/mg pM

β1 1070 ± 117 76 ± 7.1 Downloaded from

β2 605 ± 61 42 ± 3.8

β3 164 ± 22 537 ± 48 jpet.aspetjournals.org at ASPET Journals on October 3, 2021

34 Table 3

Summary of baseline and treatment data for bladder and cardiovascular parameters in dogs treated with vehicle (n=7) or This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. GW427353 (1 mg/kg; n=3) (3 mg/kg; n=7). Baseline values represent the mean of the last two voids prior to initiation of acetic acid infusion. Vehicle and GW427353 values represent the mean of three voids following infusion of acetic acid. Values are mean JPET FastForward.PublishedonJuly12,2007asDOI:10.1124/jpet.107.125757

± SEM.

1 mg/kg 3 mg/kg

Baseline Vehicle Baseline GW427353 Baseline GW427353

+ Acetic Acid + Acetic Acid + Acetic Acid

± ± ± ± ± ± Threshold Volume 36 8.7 26 6.7 38 6.3 36 11 30 6.1 37 3.6 (ml) ± ± ± ± ± ± Voided Volume 25 7.9 27 6.5 48 17 41 12 14 2.2 27 4.2 (ml) ± ± ± ± ± ± Voiding Efficiency 77 13 142 59 140 29 116 2.2 36 14 34 8.0 (%) ± a ± a ± ± ± ± Threshold Pressure 45 2.0 44 14 20 3.0 17 0.7 39 10 34 6.1 (mmHg) ± a ± a ± ± ± ± Contraction 67 8.3 57 5.2 36 3.2 38 6.8 48 11 42 6.8 Amplitude (mm Hg) ± ± ± ± ± ± Contraction 40 12 21 3.3 55 32 31 3.6 36 14 34 8.0 Duration (sec) MAP (mmHg) 116 ± 10b 113 ± 11b 111 ± 10 87 ± 11 110 ± 6.4b 77 ± 10b

HR (bpm) 137 ± 12b 126 ± 18b 81 ± 5.0 163 ± 14 118 ± 13b 214 ± 25b

35

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on October 3, 2021 3, October on Journals ASPET at JPET #125757 aIntravesical pressures in one dog not included due to technical problem with measurement bCardiovascular parameters in one dog not included due to technical problem with measurement This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. JPET FastForward.PublishedonJuly12,2007asDOI:10.1124/jpet.107.125757

36

Downloaded from from Downloaded jpet.aspetjournals.org at ASPET Journals on October 3, 2021 3, October on Journals ASPET at JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version.

Table 4

Plasma levels of GW427353 attained following each void after initiation of acetic acid infusion in the dog following i.v. administration. Values are mean ± SEM in ng/ml.

Treatment n Void

4 5 6

2830 ± 542 1949 ± 403 1612 ± 347 3 GW427353 (1 mg/kg) Downloaded from (6.4 µM) (4.4 µM) (3.6 µM)

4598 ± 685 3123 ± 507 2307 ± 356 GW427353 (3 mg/kg) 7 (10.3 µM) (7.0 µM) (5.2 µM) jpet.aspetjournals.org

at ASPET Journals on October 3, 2021

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Table 5

Protein binding of GW427353 in the dog following i.v. administration (0.2 mg/kg).

Total Conc. Free Conc Animal % Free Mean % Free (ng/ml) (ng/ml) Dog 1 1696 10.9 0.64 0.66 Dog 2 2718 18.4 0.68

Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021

38 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021 JPET Fast Forward. Published on July 12, 2007 as DOI: 10.1124/jpet.107.125757 This article has not been copyedited and formatted. The final version may differ from this version. Downloaded from jpet.aspetjournals.org at ASPET Journals on October 3, 2021