European Journal of Pharmacology 605 (2009) 145–152

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European Journal of Pharmacology

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Pulmonary, Gastrointestinal and Urogenital Pharmacology

Pharmacological properties of TD-6301, a novel bladder selectivemuscarinic receptor antagonist

Alexander McNamara a,⁎, Maria Teresa Pulido-Rios a, Shelley Sweazey a, Glenmar P. Obedencio a,Harold Thibodeaux a, Travis Renner a, Scott R. Armstrong a, Tod Steinfeld a, Adam D. Hughes a,Richard D. Wilson a, Jeffrey R. Jasper b, Mathai Mammen a, Sharath S. Hegde a

a Theravance, Inc., 901 Gateway Boulevard, South San Francisco, California, 94080 USAb ARYx Therapeutics, 6300 Dumbarton Circle, Fremont, California, 94555 USA

⁎ Corresponding author. Tel.: +1 650 808 6091; fax: +E-mail address: amcnamara@theravance.com (A. Mc

0014-2999/$ – see front matter © 2009 Elsevier B.V. Aldoi:10.1016/j.ejphar.2008.12.043

a b s t r a c t

a r t i c l e i n f o

Article history:

Existing antimuscarinic dru Received 6 September 2008Received in revised form 9 December 2008Accepted 23 December 2008Available online 10 January 2009

Keywords:Overactive bladderMuscarinic receptorVolume induced bladder contraction

gs for overactive bladder have high affinity for M3/M1 muscarinic receptors andconsequently produce M3/M1-mediated adverse effects including dry mouth, constipation, mydriasis andsomnolence. TD-6301 is a M2/4 muscarinic receptor-selective antagonist developed for the treatment ofoveractive bladder. The present studies characterize the in vitro and in vivo pharmacological properties ofthis molecule in comparison to other marketed antimuscarinics agents. In radioligand binding studies, TD-6301 was found to possess high affinity for human M2 muscarinic receptor (Ki =0.36 nM) and was 31, 36, 2and 128-fold selective for the human M2 muscarinic receptor compared to the M1, M3, M4 and M5 muscarinicreceptors, respectively. The in vivo bladder selectivity of TD-6301 in rats was determined to be 26, 28, N100,16 and 0.4-fold, respectively, assessed by comparing its potency for inhibition of volume-induced bladdercontractions to that for inhibition of oxotremorine-induced salivation, inhibition of small-intestinal transit,decreases in locomotor activity, increases in pupil diameter and increases in heart rate. TD-6301 was morepotent in inhibiting volume-induced bladder contractions (ID50=0.075 mg/kg) compared to oxotremorine-induced salivation (ID50=1.0 mg/kg) resulting in a bladder/salivary gland selectivity ratio greater than thatobserved for tolterodine, oxybutynin, darifenacin and solifenacin. The preclinical properties of TD-6301suggest that this molecule is likely to be efficacious in overactive bladder patients with a lower propensity tocause M3 muscarinic receptor mediated adverse effects.

© 2009 Elsevier B.V. All rights reserved.

1. Introduction

Overactive bladder, which is characterized by symptoms of urinaryfrequency, urgency and urge incontinence, is a chronic and debilitat-ing disease (Abrams et al., 2003). The symptoms of overactive bladderare often attributed to involuntary contractions of the detrusormuscleduring bladder filling, a condition known as detrusor instability oroveractivity (de Groat, 1997). Although the precise etiology ofoveractive bladder is unknown, it is generally accepted that activationof muscarinic receptors in the detrusor via acetylcholine released frompostganglionic parasympathetic nerves is the ultimate mechanismdriving detrusor overactivity in the diseased bladder (de Groat, 1993).Thus, it is not surprising that muscarinic antagonists serve as thecornerstone in the pharmacotherapy of overactive bladder. Althoughthese antimuscarinic drugs have adequate efficacy, they fail toselectively inhibit abnormal detrusor contractions. Furthermore,existing drugs havemarginal therapeutic indices owing tomechanism

1 650 808 6441.Namara).

l rights reserved.

based dose-limiting adverse effects such as dry mouth, constipationand blurred vision. This results in poor patient compliance and limitsthe therapeutic dose. More than 80% of patients who initiate therapydiscontinue within 3 to 6 months because they do not tolerate theside effects (Kelleher et al., 1997; Chui et al., 2004). The four leadingantimuscarinic drugs for overactive bladder (tolterodine, oxybutynin,darifenacin and solifenacin) all have high affinity for M3 and/or M1

muscarinic receptors (Hegde et al., 2004). It is not surprising thatthese drugs have a poor tolerability profile given the key role playedby the M3/M1 muscarinic receptors in the salivary gland, gastro-intestinal, ocular and behavioral function (Anagnostaras et al., 2003;Caulfield, 1993; Eglen et al., 1996; Wess, 2004).

M2 andM3 are the predominantmuscarinic receptor subtypes foundwithin the bladder and are present in a ratio of about 4 to 1 (M2:M3)(HegdeandEglen,1999;Hegde, 2006).M3muscarinic receptorsmediatethe direct contractile responses necessary for normal bladder functionthrough a mechanism that depends on entry of extracellular calciumthrough L-type calcium channels and activation of a rho kinase(Schneider et al., 2004). The functional role of the M2 muscarinicreceptor in detrusor contraction remains unclear. Studies suggest thatM2muscarinic receptor activation alsomediate detrusor contraction but

Fig. 1. Chemical structure of TD-6301 is composed of a benzhydryl motif substitutedwith a carboxamide and a pyrrolidine. The pyrrolidine is linked to a 4-amino piperidinethrough a heptyl alkyl chain. The exocyclic tertiary amine contains an isopropylsubstituent while the piperidine has a 4-methoxy pyridine methyl group. The chemicalname is proposed to be 1-(4-methoxypyridin-3-ylmethyl)-4-{N-[7-(3-(S)-1-carba-moyl-1,1-diphenylmethyl)pyrrolidin-1-yl) hept-1-yl]-N-(isopropyl) amino} piperidine.

146 A. McNamara et al. / European Journal of Pharmacology 605 (2009) 145–152

through an indirect mechanism involving inhibition of sympatheticallymediated (β3 adrenoceptor evoked) relaxation via a cAMP-dependentmechanism (Hegde et al., 1997; Yamanishi et al., 2002; Ehlert et al.,2005; Ehlert et al., 2007). More importantly, since M2 muscarinicreceptors do not appear to be functionally important in the salivarygland, gastrointestinal tract and eye (Bymaster et al., 2003; Eglen et al.,1996; Eglen andHarris,1993;Gil et al.,1997; Ishizaka et al.,1998;Messeret al., 1990), antagonists which selectively target this receptor may beexpected to possess a better tolerability profile. However, the potentialeffects of an M2 muscarinic receptor selective antagonist on heart rateand in the central nervous system (CNS) needs to be considered giventhe known importance ofM2muscarinic receptors in regulation of heartrate (Caulfield, 1993) and autoregulation of acetylcholine release in thebrain (Zhang et al., 2002).

TD-6301 (Fig. 1) is a selective M2/M4 muscarinic receptorantagonist developed for the treatment of overactive bladder. Theobjective of the present studies was to characterize the in vitro and invivo pharmacological properties of this molecule in comparison tomarketed antimuscarinics agents. The in vivo bladder selectivity of themolecule was assessed in a range of animal models that evaluated theantimuscarinic potency in the bladder, salivary gland, eye, gastro-intestinal tract, heart and CNS.

2. Materials and methods

2.1. Material

TD-6301 was synthesized at Theravance, Inc. Tolterodine (tartratesalt), oxybutynin (hydrochloride salt), darifenacin (trifluoroacetatesalt) and solifenacin (hydrochloride salt) were all prepared usingpublished methods. All test compounds were dissolved in 5% dextrosein water and formulated with respect to the base weight of thecompound.

2.2. In vitro pharmacological characterization of TD-6301 in humanmuscarinic receptor subtypes; displacement of [3H]-N-methylscopolamine

Membranes were prepared from CHO-K1 cells stably expressingthe human muscarinic receptor subtypes, hM1, hM2, hM3 or hM4.Membrane fractions expressing the human M5 muscarinic receptorwere purchased from Perkin Elmer (Waltham, MA). Radioliganddisplacement binding assays were performed using [3H] N-methylscopolamine (1 nM final concentration) in a buffer consisting of10 mM HEPES, 100 mM NaCl, 10 mM MgCl2 and 0.025% bovine serumalbumin. Following a 60 min incubation at 37 °C, membranes werecollected on glass fiber filtermats pre-treated with 1% BSA using aPerkinElmer 96 well harvester. Bound radioactivity was measured byscintillation counting.

2.3. In vivo pharmacological characterization of TD-6301

All studies were conducted in accordance with the TheravanceInstitutional Animal Care and Use Committee.

2.3.1. AnimalsMale and female Sprague Dawley rats (Harlan, San Diego, CA)

weighing 90–350 g were used in these studies. The animal colonyholding rooms were temperature controlled and kept on a 12 h light/dark cycle. Rats were housed in pairs in plastic cages with corncobchip bedding. Food and water was available ad libitum.

2.4. M2/M3 muscarinic receptor selectivity in pithed rats

The objective of this study was to determine theM2/M3muscarinicreceptor selectivity of TD-6301 in an in vivo setting. This was achievedby determining the relative potency of TD-6301 in antagonizingmethacholine evoked M2 muscarinic receptor mediated decreases inheart rate (bradycardia response) compared toM3muscarinic receptormediated decreases in mean arterial pressure (depressor response) inangiotensin-supported pithed rats (Armstrong et al., 2008).

Female Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 225 and 275 g were used in this study. The animal wasanesthetized with pentobarbital (60 mg/kg, i.p.). The trachea wascannulated with PE-240 tubing. The right jugular vein (PE-10 fused toPE-50 tubing) and both femoral veins (micro-renethane tubing) werecatheterized for i.v. drug administration. Next, the femoral artery wascatheterized (PE-10 fused to PE-50 tubing) for measurement of arterialpressure and heart rate. Both vagus nerveswere isolated adjacent to thecarotid artery and transected. The animal was then pithed through theorbit of the eye with a stainless steel pithing rod (1.5 mm in diameter)and ventilated artificially (60 cycles/min with a volume of 1.5 ml/kg ofbodyweight) using a small animal ventilator (Harvard ApparatusModel683, Hollistan, MA). Following a stabilization period, angiotensin-II(0.3 µg/kg/min, i.v.) was infused via one of the femoral veins to elevatearterial pressure to normotensive levels (between 100 and 125mmHg).The angiotensin-II supported arterial pressure was allowed to stabilizefor approximately 15 min. Thereafter, a non-cumulative dose–responsecurve to methacholine (i.v.) was constructed. TD-6301 or vehicle wasdosed as a continuous i.v. infusion, via one of the femoral veins, to studyantagonistic effects under steady-state drug exposure conditions.A loading infusion dose and a maintenance dose was deliveredover 30 min, based on previously obtained pharmacokinetic data, toachieve appropriate steady-state drug levels. Thereafter, a non-cumulative dose–response curve to methacholine was constructed.After eachmethacholine dose, the depressor and bradycardic responseswere allowed to recover to baseline before administration of thesubsequent dose before administration of TD-6301.

2.5. Volume-induced bladder contraction model

Female Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 180 and 300 g were used in this study. Animals wereanesthetized with urethane (1.5 g/kg, s.c.) and the level of anesthesiawas maintained with additional supplemental doses (0.25 g/kg, s.c.) ifrequired. Following cannulation of the trachea (PE-205), the femoralvein was catheterized (micro-renathane tubing, 0.3 mm ID×0.64 mmOD) for drug administration. The lower abdominal cavity was exposedand the ureters were ligated and severed proximal to the ligation.The bladder was cannulated via the urethra to measure intra-vesicalpressure. The bladder cannula was secured in place around theexternal urethral orifice using a purse string suture. After surgery,saline was infused (200 µl/min for ∼5 min followed by a maintenanceinfusion of ∼5 µl/min) into the bladder to evoke rhythmic volume-induced bladder contractions which were manifested as rhythmicchanges in intravesical pressure. After a stabilization period, thevehicle was dosed i.v. and changes in volume-induced bladdercontraction amplitude were recorded for 15 min. Thereafter, theappropriate dose of the antagonist was administered and changes involume-induced bladder contraction amplitude recorded for 15 minafter which atropine (0.1 mg/kg, i.v.) was dosed and datawas recorded

147A. McNamara et al. / European Journal of Pharmacology 605 (2009) 145–152

for an additional 15 min. In this model, the atropine-sensitivemuscarinic component accounts for 50–55% of the volume-inducedbladder contraction amplitude (Hegde et al., 1997).

2.6. Anti-sialagogue model

Anti-sialagogue activity was evaluated by determining the effectsof TD-6301 or standards on subcutaneously dosed oxotremorine(muscarinic agonist)-induced salivation (Sanchez and Lembol, 1994).Female Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 180 and 350 g were used in this study. Animals wereanesthetized with urethane (1.5 g/kg, s.c.) with additional supple-mental doses (0.25 g/kg, s.c.) if required. Following induction ofanesthesia and a stabilization period, either vehicle or the appropriatedose of the antagonist was dosed i.v. via the tail vein. The animal wasplaced head down on its ventral side at a 20° angle plane. A pre-weighed sponge was placed inside the animal's mouth. Five min afteradministration of vehicle/antagonist, the animal was challenged withoxotremorine (1 mg/kg, s.c.). Ten min later, the sponge was removedand re-weighed to determine the output of saliva.

2.7. Pupil dilation model

Effects on ocular function were evaluated by determining theinfluence of test drugs on pupil diameter in conscious rats (Parry andHeathcote, 1982). Conscious male Sprague–Dawley rats (Harlan,Indianapolis, IN) weighing between 90 and 150 g were held under amicroscope (Jena03, magnification 0.8×, objective GF/Pw 12.5× (20)),the right eyepiece of which had a graduated eyepiece for measure-ment of pupil diameter. A fiber optic light (Fibre-Lite, Dolah-JennerIndustries, Boxborough, MA) was directed towards the left eye of theanimal. Baseline pupil diameter was recorded. One min later, theanimal was dosed i.v. via the tail vein with either vehicle or theappropriate dose of the antagonist. Ten min later, measurements ofpupil diameter were repeated.

2.8. Locomotor activity model

Effects on behavior were evaluated by determining the influence ofTD-6301 or standards on locomotor activity in conscious rats. In thismodel, CNS-penetrant muscarinic antagonists produce increases inlocomotor activity due to antagonismof inhibitorymuscarinic receptors(M4) in the nigro-striatal pathway (Shannon and Peters, 1990).

Male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 200 and 300 g were used in this study. Locomotor activitywas measured by placing the animals in a cage that is positionedbetween infrared photocell beams (Digiscan DMicro Animal ActivityMonitor with MicroPro V 1.30i software, AccuScan Instruments,Columbus, OH). Movement of animals interferes with the light beamthat allows for objective measurement of locomotor activity. On theday of the study, baseline locomotor activity was measured in rats for60 min. The animals were then dosed i.v. via the tail vein with eithervehicle or the appropriate dose of the antagonist. Locomotor activitywasmeasured and recorded for 60min post-dose. Atropine (10mg/kg,i.v.) was used as the positive control.

2.9. Gastro-intestinal motility model

Effects on intestinal function were evaluated by determining theinfluence of test drugs on small intestinal transit of a charcoal meal inrats. Female Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 140 and 220 gwere used in this study. Animalswere dosed i.v.via the tail vein with either vehicle or the appropriate dose of theantagonist. Five min later, a charcoal meal (10% charcoal in 2% Arabicgum) was administered by oral gavage. Twenty min later, the animalwas sacrificed and the small intestine was removed and stretched out

on the bench. The length of the intestine from the pyloric sphincterto the ileo–caecal junction and distance traveled by the charcoalfront was measured with a graduated ruler and recorded. Atropine(1 mg/kg, i.v.) was used as a positive control and reduced smallintestinal transit by 30–50% of the vehicle control.

2.10. Cardiovascular model

Cardiovascular adverse effects, chiefly tachycardia, are a potentialconcern during antimuscarinic therapy. Tachycardia results fromantagonism of M2 muscarinic receptors in the heart leading tosuppression of the inhibitory vagal drive to the heart (Hulme et al.,1990; Caulfield, 1993). The objective of this study was to assess theeffects of TD-6301 onmean arterial pressure and heart rate in conscious,instrumented free-roaming rats.

Male Sprague–Dawley rats (Harlan, Indianapolis, IN) weighingbetween 200 and 300 g were used in this study. Animals wereanesthetized with isoflurane (5% induction and 3% maintenance) toallow cannulae implantation. The intrascapular and femoral triangleareas were shaved and cleansed with betadine and 70% alcohol. Asurgical drape was used to isolate and provide a sterile surgical field.Animals were instrumented for measurement of arterial pressure (viafemoral artery) and i.v. drug administration (via the femoral vein). Thearterial cannula was constructed by fusing PE-10 to PE-50 tubing.The venous cannula was made of micro-renathane tubing (0.033 in.ID, Braintree Scientific, Braintree, MA). The arterial and venouscannulae were tunneled subcutaneously and exteriorized throughthemidscapular region. Both incisionswere cleansed free of blood andsutured with 4–0 silk. The cannulae were “locked” with a solution of1000 U heparin and 50% sucrose, sealed with stainless steel pins andsecured into a jacketed pouch. After surgery, animals were adminis-tered buprenorphine (0.05mg/kg, i.m.) plus 3ml of saline and allowedto recover from anesthesia on a warming blanket before beingreturned to their study room in the vivarium. On the day of the study,arterial catheters were connected to pressure tranducers and baselinehemodynamic parameters were measured for 30 min after whichvehicle or TD-6301 was dosed i.v. (dosing volume of 0.3 ml/kg) withescalating doses administered every 10 min.

2.11. Data analysis

2.11.1. In vitroThe binding data were analyzed by nonlinear regression analysis

using GraphPad Prism software. Ki values for test compoundswere calculated from observed IC50 values and the KD value of theradioligand (determined from saturation binding studies performedfor each receptor subtype) according to (Cheng and Prusoff, 1973).

2.11.2. Pithed ratHeart rate was measured at pre-dose and at the peak response post

methacholine. Mean arterial pressure was measured at pre-dose andfollowing recovery of the heart rate response to within 15% of pre-methacholine values (to minimize the confounding influence of heartrate changes on mean arterial pressure). Changes from baseline (pre-dose) in mean arterial pressure and heart rate were calculated for eachdoseofmethacholine. Dose-ratios (DR)were calculated and a Schild plot(log [DR-1] vs log [antagonist])was constructed fromwhich estimates ofDR10 (dose required to produce a 10-fold dextral shift of the agonistdose response curve) were obtained for both the M2 andM3 muscarinicreceptor endpoints. TheM2/M3muscarinic receptor selectivity ratiowascalculated as the the ratio of DR10 (M3) to DR10 (M2).

2.11.3. Volume-induced bladder contractionsThe average volume-induced bladder contraction amplitude

during the 5–15 min post-drug (test antagonist/atropine) periodwas obtained and subtracted from the average volume-induced

Table 1Radioligand binding affinities (Ki in nM; standard deviation, S.D.) determined bydisplacement of [3H]N-methylscopolamine for TD-6301 for tolterodine, oxybutynin,darifenacin, and solifenacin at human M1, M2, M3, M4, and M5 muscarinic receptorsubtypes

Radioligand binding affinities (Ki in nM)

hM1 hM2 hM3 hM4 hM5

TD-6301 11 (S.D. 5.4) 0.36 (S.D. 0.10) 13 (S.D. 4.5) 0.78 (S.D. 0.26) 46 (S.D. 16)Tolterodine 8.1 (S.D. 3.2) 9.8 (S.D. 4.2) 17 (S.D. 5.4) 13 (S.D. 5.3) 21 (S.D. 7.0)Oxybutynin 5.0 (S.D. 2.3) 14.5 (S.D. 7.9) 3.7 (S.D. 2.0) 5.3 (S.D. 3.3) 40 (S.D. 14.0)Darifenacin 22.0 (S.D. 7.4) 94 (S.D. 62) 2.3 (S.D. 1.0) 49 (S.D. 29) 13.0 (S.D. 29)Solifenacin 1.9 (S.D. 0.24) 8.0 (S.D. 2.7) 2.6 (S.D. 0.9) 3.4 (S.D. 1.6) 3.3 (S.D. 1.7)

Across the five receptor subtypes, N=46–50 for TD-6301, N=192–207 for tolterodine,N=168–187 for oxybutynin, N=133–146 for darifenacin and N=10–18 for solifenacin.

148 A. McNamara et al. / European Journal of Pharmacology 605 (2009) 145–152

bladder contraction amplitude during the 5–15 min post-vehicleperiod to obtain the test antagonist and atropine-induced change involume-induced bladder contraction amplitude. The inhibitory effectsof the test drug were normalized to the atropine response and theresulting dose–response curves were fitted with a four parameterlogistic equation to obtain estimates of ID50 (dose required to produce50% of the maximal response).

2.11.4. Oxotremorine-induced salivationDose–response curves were fitted to obtain estimates of ID50 (dose

required to produce 50% of the maximal response) and ID25 (doserequired to produce 25% of the maximal response).

2.11.5. Pupil dilationChange in pupil diameter (post-dose — baseline) was calculated

for each treatment. Atropine (0.1 mg/kg, i.v.) was used as a positivecontrol. Dose–response curves were to obtain estimates of ED50 (doserequired to produce 50% of the atropine response).

2.11.6. LocomotorThe total number of ambulatory counts during the 30-min period

post-dosing was used as the endpoint. Dose–response curves werefitted to obtain estimates of ED50 (dose required to produce 50% of theatropine response).

2.11.7. Small intestinal transitIntestinal transit is measured as the percent of distance traveled by

the charcoal front relative to the total length of the intestine. In vehicletreated animals, distance traveled by the charcoal front was usuallyapproximately 60–70% of the total small intestinal length. Dose–response curves were fitted to obtain estimates of ID50 (dose requiredto produce 50% of the atropine response).

2.11.8. CardiovascularHeart rate and mean arterial pressure were derived from the

pressure pulse using MP100 Biopac Data Acquisition Systems (BIOPACSystems, Santa Barbara, CA). Change in heart rate and mean arterialpressure from baseline was used for analysis. Statistical analysis wasperformed using two-way ANOVA using Bonferroni post-hoc test.Pb0.05 was considered to be statistically significant.

3. Results

3.1. Binding affinities for human muscarinic subtypes

TD-6301 binds with high affinity to human M2 and M4 muscarinicreceptors (Ki =0.36 and 0.78 nM, respectively) and was 36-fold more

Fig. 2. Inhibition of [3H]NMS binding to the muscarinic M1 to M5 receptors by themuscarinic antagonist TD-6301. [3H]NMS binding to M1 (●), M2 (■), M3 (▲), M4 (▼) orM5 (♦) receptors was inhibited by various concentrations of TD-6301. CPM data werenormalized to percent specific binding of [3H] NMS and were fit to a four-parameterlogistic equation. Hill coefficients were not significantly different from unity and weretherefore, fixed to 1. Inhibition binding constants (KI) were calculated from IC50s valuesaccording to methods described by Cheng and Prusoff (1973).

selective for the human M2 muscarinic receptor compared to the M3

muscarinic receptor (Ki =13 nM). Across the five muscarinic receptorsubtypes, the rank order from highest to lowest binding affinity forTD-6301 was: hM2=hM4NhM1NhM3NhM5 (Fig. 2, Table 1). Incomparison, tolterodine, oxybutynin and solifenacin did not discri-minate between the five muscarinic subtypes; darifenacin had highaffinity and selectivity for the M3 muscarinic receptor; solifenacinhad high affinity for all five subtypes with marginal selectivity forthe M1 muscarinic receptor over the other four subtypes. Oxybutynindisplayed the highest affinity for the M1, M3 and M4 muscarinicreceptor subtypes.

3.2. M2/M3 selectivity in pithed rats

As reported previously in the pithed rat model, tolterodine andsolifenacin do not discriminate between M2 and M3 muscarinicreceptors in the pithed rat model in vivo, oxybutynin is modestlyselective forM3 receptors whereas darifenacin is highlyM3muscarinicreceptor selective (Armstrong et al., 2008). In contrast, TD-6301produced dose-dependent dextral shifts of the methacholine brady-cardia (M2 muscarinic receptor) and depressor (M3 muscarinicreceptor) dose–response curves. The DR10s (mg/kg, i.v.) for TD-6301were 0.029 for M2 muscarinic receptor antagonism and 0.6 for M3

muscarinic receptor antagonism (Fig. 3). The calculated M2/M3

muscarinic receptor selectivity ratio of TD-6301 was 21.

3.3. Bladder/salivary gland selectivity

TD-6301, tolterodine, oxybutynin, darifenacin and solifenacinproduced dose-dependent inhibition of volume-induced bladdercontractions and oxotremorine-induced salivation (Fig. 4). Thebladder/salivary gland selectivity ratios, calculated from ID50

Fig. 3. Schild analysis of the antagonist effects of TD-6301 on M2-mediated bradycardicand M3-mediated depressor response. Ordinate axis shows the dose ratio of the MChcurve (post antagonist/pre-antagonist infusion) on a logarithmic scale. Abscissa axisshows the dose of antagonist (i.v. infusion) on a logarithmic scale. The doses of TD-6301represent the total infused dose (mg/kg). Data are expressed as mean±SEM. N=3–4.

Fig. 4. Inhibitory dose–response curves for tolterodine, oxybutynin, solifenacin and TD-6301 against volume-induced bladder contractions (VIBC) and oxotremorine-inducedsalivation (OIS). Ordinate axis shows the % VIBC or OIS responses expressed as % of control. Abscissa axis shows the dose of antagonist. Values represent mean±SEM. N=6–9 (VIBC),N=and 13–28 (OIS).

149A. McNamara et al. / European Journal of Pharmacology 605 (2009) 145–152

estimates, of TD-6301, tolterodine, oxybutynin, darifenacin andsolifenacin were 14, 4.8, 1.4, 2.1 and 5-fold, respectively (Table 2).The bladder/salivary gland selectivity ratios, calculated from ID25

estimates, of TD-6301, tolterodine, oxybutynin, darifenacin andsolifenacin were 26, 5.3, 1.7, 3.3 and 5.9-fold, respectively (Table 3).At a dose which inhibits volume-induced bladder contractions by 80%,TD-6301, tolterodine, oxybutynin, darifenacin and solifenacin inhib-

Table 2Bladder to salivary gland selectivity, estimated at ID50, of TD-6301, tolterodine,oxybutynin, darifenacin, and solifenacin in anesthetized rats

Compound VIBC ID50 (mg/kg) OIS ID50 (mg/kg) Bladder/salivary glandselectivity ratio

TD-6301 0.075 1.01 14(0.056–0.10) (0.83–1.2) (8.3–21.4)

Tolterodine 0.025 0.12 4.8(0.021–0.029) (0.095–0.1) (3.3–4.8)

Oxybutynin 0.062 0.089 1.4(0.050–0.076) (0.072–0.11) (0.9–2.2)

Darifenacin 0.037 0.078 2.1(0.031–0.045) (0.065–0.096) (1.4–3.1)

Solifenacin 0.017 0.085 5.0(0.014–0.02) (0.071–0.10) (3.6–7.1)

VIBC=volume-induced bladder contraction, OIS=oxotremorine-induced salivation.Values represent mean with 95% confidence interval. N=6–9 (VIBC), N=13–28 (OIS)bladder/salivation selectivity ratio calculated as ID50 (OIS) to ID50 (VIBC).

ited oxotremorine-induced salivation by 5%, 44%, 75%, 75% and 33%,respectively (Fig. 4).

3.4. Bladder/pupil dilation selectivity

TD-6301, tolterodine, oxybutynin, darifenacin and solifenacinproduced dose-dependent increase in pupil dilation with ED50s

Table 3Bladder to salivarygland selectivity, estimated at ID25, of test compounds in anesthetizedrats

Compound VIBC ID25 (mg/kg) OIS ID25 (mg/kg) Bladder/salivary glandselectivity ratio

TD-6301 0.025 0.64 26(0.017–0.037) (0.45–0.92) (12.2–54.2)

Tolterodine 0.0094 0.050 5.3(0.0073–0.012) (0.037–0.069) (3.1–9.5)

Oxybutynin 0.019 0.033 1.7(0.015–0.026) (0.024–0.046) (0.92–3.1)

Darifenacin 0.012 0.040 3.3(0.0090–0.015) (0.030–0.053) (2–5.9)

Solifenacin 0.0064 0.038 5.9(0.0047–0.0086) (0.029–0.049) (3.4–10.4)

VIBC=volume-induced bladder contraction, OIS=oxotremorine-induced salivation.Values represent mean with 95% confidence interval. N=6–9 (VIBC), N=13–28 (OIS).Bladder/salivation selectivity ratio calculated as ID25 (OIS) to ID25 (VIBC).

Table 4Bladder selectivity of TD-6301, tolterodine, oxybutynin, darifenacin and solifenacin in rats

Compound VIBC ID50a

(mg/kg)ID50 (PD)b

(mg/kg)Bladder/ocularselectivity ratioc

ED50 (LMA)d

(mg/kg)Bladder/CNSselectivity ratioc

ID50 (SIT)e

(mg/kg)Bladder/gutselectivity ratioc

TD-6301 0.075 2.1(0.056–0.10) N1.2 N16 N7.5 N100 (1.0–4.2) 28

Tolterodine 0.025 0.34 14 2.5 100 0.66(0.021–0.029) (0.23–0.48) (1.3–4.9) (0.24–1.8) 26

Oxybutynin 0.062 0.29 0.52 0.22(0.050–0.076) (0.23–0.39) 4.7 (0.28–0.99) 8.4 (0.13–0.37) 3.5

Darifenacin 0.037 0.33 3.4 0.3(0.031–0.045) (0.28–0.40) 8.7 (2.4–4.9) 89 (0.090–0.93) 7.9

Solifenacin 0.017 0.088 5.2 0.39 0.37(0.014–0.02) (0.070–0.11) (0.15–1.0) 23 (0.24–0.59) 22

Values represent mean±95% CI. N=6–9 (VIBC), N=13–28 (Ocular), N=5–10 (LMA) and N=8–16 (GI).a VIBC=volume-induced bladder contraction. ID50 estimates expressed as mean (95% confidence intervals).b PD=pupil diameter. ED50 estimates expressed as mean (95% confidence intervals).c Ratio of ED50 (PD), ED50 (LMA) or ID50 (SIT) to ID50 (VIBC).d LMA=locomotor activity. ED50 estimates expressed as mean (95% confidence intervals).e SIT=small intestinal transit. ID50 estimates expressed as mean (95% confidence intervals).

150 A. McNamara et al. / European Journal of Pharmacology 605 (2009) 145–152

(mg/kg, i.v.) of N1.2, 0.34, 0.29, 0.33 and 0.088, respectively. Thebladder-to-ocular selectivity ratio of TD-6301, tolterodine, oxybu-tynin, darifenacin and solifenacin are N16, 14, 4.7, 8.7 and 5.2-fold,respectively. The potencies (ED50s/ID50s) for pupil dilation, volume-induced bladder contractions and the calculated bladder/ocularselectivity ratios are shown in Table 4.

Fig. 5. Effects of TD-6301 on mean arterial pressure (A) and heart rate (B) in consciousrats. Ordinate shows responses expressed as peak change from baseline. Abscissa axisshows the dose of antagonist. Values represent mean±SEM. N=6 per group (Bonferronipost test following two-way ANOVA analysis).

3.5. Bladder/CNS selectivity

Tolterodine, oxybytynin, darifenacin and solifenacin produced dose-dependent increases in locomotor activitywith ED50s (mg/kg, i.v.) of 2.5,0.52, 3.4 and 0.39, respectively. TD-6301 produced no increase inlocomotor activity at doses up to 7.5 mg/kg. The calculated bladder-to-CNS selectivity ratios of TD-6301, tolterodine, oxybytynin, darifenacinand solifenacin were N100, 100, 8.4, 89 and 23, respectively. Theinhibitory potencies (ED50s/ID50s) for locomotor activity, volume-induced bladder contractions and the calculated bladder/CNS selectivityratios are shown in Table 4.

3.6. Bladder/small intestinal transit selectivity

TD-6301, tolterodine, oxybutynin, darifenacin and solifenacinproduced dose-dependent decreases in small intestinal transit withID50s (mg/kg, i.v.) of 2.1, 0.66, 0.22, 0.3 and 0.37, respectively. Thebladder/gut selectivity ratios TD-6301, tolterodine, oxybutynin, dar-ifenacin and solifenacin were 28, 26, 3.5, 7.9 and 22-fold, respectively.The inhibitory potencies (ID50s) for small intestinal transit, volume-induced bladder contractions and the calculated bladder/gut selectiv-ity ratios are shown in Table 4.

3.7. Cardiovascular effects

Baseline mean arterial pressure ranged from 115–120 mm Hg andbaseline heart rate ranged from 415–447 beats/min in the drug-treated groups. TD-6301 (0.03–1 mg/kg, i.v.) produced dose-depen-dent increases in heart rate. However these increases in heart ratewere not statistically significant in comparison to vehicle treatedanimals, perhaps because of the small sample size. The maximalincreases in heart rate were 15, 47, 55 and 49 beats/min, respectivelyat 0.03, 0.1, 0.3 and 1 mg/kg, i.v. (Fig. 5). TD-6301 had no effects onmean arterial pressure.

4. Discussion

Muscarinic antagonists currently remain the only effective ther-apeutic strategy for controlling the symptoms of overactive bladder,however the non-selective orM3muscarinic receptor selective profile ofexisting drugs inevitably limit the therapeutic index of these agents dueto adverse effects. Incidences of drymouth are particularly confoundingas patients increase fluid consumption thus marginalizing the benefitsof controlling overactive bladder with antimuscarinic therapies. Con-stipation, blurred vision and CNS (fatigue, confusion and restlessness)effects reported during treatment may also influence patient compli-ance and persistence. Ideally, antimuscarinic therapy for overactive

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bladder should minimize bladder dysfunction and have an acceptabletolerability profile. Targeting overactive bladder with an M2 muscarinicreceptor selective antagonist may be an attractive alternative to currentnon-selective or M3 muscarinic receptor selective treatment optionssince it has the potential of selectively inhibiting detrusor instabilitywithout causing the aforementioned antimuscarinic adverse effects.

Radioligand displacement studies demonstrated that TD-6301possesses a high affinity to the humanM2muscarinic receptor subtypeand is highly selective forM2 over theM3muscarinic receptor subtype.The uniqueM2/M3muscarinic receptor selectivity of TD-6301was alsoreproduced in vivo in pithed rats. In contrast, themarketed comparatormolecules (tolterodine, oxybutynin, darifenacin and solifenacin) wereeither non-selective or displayed some selectivity for theM1 and/orM3

muscarinic receptor.Inhibitory effects of TD-6301 on bladder function, mediated at

least in part through M2 muscarinic receptor antagonism, wereassessed on reflex volume-induced bladder contractions in anesthe-tized rats (Lecci et al., 1995; Hegde et al., 1997; Sugaya and de Groat,2007). Thismodel is unique and highly relevant for the following threereasons: first, the reflex contractions in this model are of physiologicalrelevance since they require the operation of the entire spinal-bulbo-spinal micturition reflex (de Groat et al., 1993); second, it allows one tostudy the effects of drugs on bladder function in isolation without theconfounding influence of outlet resistance changes; third, since thebladder contractions are mediated isovolumetrically, there is con-comitant activation of both parasympathetic and sympathetic systemsallowing one to study the interaction of M2 muscarinic receptor andbeta adrenoceptors in an in vivo setting.

TD-6301, tolterodine, oxybutynin, darifenacin and solifenacin dose-dependently inhibited volume induced bladder contractions. The highbladder inhibitory potencies of all drugs which have varying potenciesand selectivities for M2 and M3 muscarinic receptors are consistentwith a functional role of both M2 and M3 muscarinic receptors inmediation of VIBC (Hegde et al., 1997). Oxybutynin and darifenacinwere equipotent in inhibiting volume-induced bladder and salivation.The discrepancy between the rank order of binding affinities and invivo bladder potency for TD-6301 and the comparatormolecules couldbe explained by three factors. First, themolecular weight of TD-6301 is1.5 to 2-fold higher than the comparator molecules. Second, inhibitionof VIBC by TD-6301 is likely to be mediated by both M2 and M3

receptors and this is reflected by the shallower inhibitory dose–response curve of TD-6301 compared to the other molecules. Third,differences in pharmacokinetic exposures of the molecules could alsocontribute to their potency differences in vivo.

Tolterodine and solifenacin had a modest degree of bladder/salivary selectivity. By contrast, TD-6301 demonstrated the highestdegree of bladder/salivary selectivity at the ID50 dose level. It isimportant to note that TD-6301, unlike other agents, was able toinhibit volume-induced bladder up to 80% with minimal inhibition ofoxotremorine-induced salivation. These data are consistent with afunctional role of M2 muscarinic receptors in volume-induced bladdercontractions but not in salivation. The bladder inhibitory effects of TD-6301 and other molecules need to be confirmed in a conscious animalmodel of cystometry. The role of M4 muscarinic receptors in theinhibitory effects of TD-6301 cannot be ruled out although thereis limited evidence for a functional role of this subtype in bladderfunction.

The bladder-to-ocular selectivity of TD-6301 (N24-fold) is equiva-lent or slightly better than that of tolterodine but greatly exceeds thatof oxybutynin, darifenacin and solifenacin. The bladder-to-gutselectivity of TD-6301, based on inhibition of transport of a charcoalmeal, is equivalent to that of tolterodine and solifenacin but exceedsthat of oxybutynin and darifenacin. These observations are inaccordance with the low affinity of TD-6301 for M3 muscarinicreceptors. The bladder/CNS selectivity of TD-6301, based on increasedlocomotor activity, is equivalent to that of tolterodine and darifenacin

but exceeds that of oxybutynin and solifenacin. These observations areconsistent with the low affinity of TD-6301 for M1 muscarinicreceptors and its limited ability to penetrate the CNS (unpublisheddata). The modest positive chronotropic effects of TD-6301 are likelythe result of M2 muscarinic receptor antagonistic activity and raise theconcern that selective M2 muscarinic receptor antagonism mightcause increases in heart rate in humans by suppression of vagal driveto the heart. It should be noted that the elderly population, whichrepresents the bulk of overactive bladder patients, exhibit diminishedcardiac vagal/muscarinic responsiveness (Brodde et al., 1998). Never-theless, it is pertinent to note that a M2 muscarinic receptor selectiveantagonist has never been clinically assessed in the target overactivebladder population and the effect on heart rate in this population isunknown at present.

5. Conclusion

In rats, the bladder-to-salivary gland selectivity of TD-6301 (14 -26-fold) and bladder-to-ocular selectivity (N24-fold) is superior to allcompetitors. Furthermore, the bladder-to-gut selectivity of TD-6301 isequivalent to that of tolterodine but exceeds oxybutynin, darifenacinand solifenacin. Lastly, the bladder-to-CNS selectivity of TD-6301 isequivalent to that of tolterodine and darifenacin but exceeds that ofoxybutynin and solifenacin. Overall, TD-6301 is a highly M2-selectiveantagonist that has the potential to be efficacious in overactivebladder patients, causing little or no drymouth, ocular adverse effects,(mydriasis and blurred vision), constipation or adverse CNS effects attherapeutic doses in overactive bladder patients. The potential of TD-6301 to cause increases in heart rate in the target population needs tobe considered during clinical development of this compound.

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