Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist

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Supplemental material to this article can be found at: http://jpet.aspetjournals.org/content/suppl/2016/12/13/jpet.116.237313.DC1

1521-0103/360/2/346–355$25.00 http://dx.doi.org/10.1124/jpet.116.237313 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS J Pharmacol Exp Ther 360:346–355, February 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics

Pharmacological Characterization of a Novel Beta 3 Adrenergic Agonist, Vibegron: Evaluation of Antimuscarinic Receptor Selectivity for Combination Therapy for Overactive Bladder s

J. Di Salvo, H. Nagabukuro, L. A. Wickham, C. Abbadie, J. A. DeMartino, A. Fitzmaurice, L. Gichuru, A. Kulick, M. J. Donnelly, N. Jochnowitz, A. L. Hurley, A. Pereira, A. Sanfiz, G. Veronin, K. Villa, J. Woods, B. Zamlynny, E. Zycband, G.M. Salituro, T. Frenkl, A. E. Weber, S. D. Edmondson, and M. Struthers

Discovery and Preclinical Sciences, Merck Research Laboratories, Kenilworth, New Jersey Downloaded from Received August 17, 2016; accepted December 5, 2016

ABSTRACT Although the physiologic role of muscarinic receptors in bladder a nonselective antagonist, compared with coadministration with

function and the therapeutic efficacy of muscarinic antagonists darifenacin, a selective M3 antagonist. Furthermore, a synergistic jpet.aspetjournals.org for the treatment of overactive bladder are well established, the effect for bladder strip relaxation was observed with the combi- role of b3-adrenergic receptors (b3ARs) and their potential as nation of a b3AR agonist and tolterodine in contrast to simple therapeutics is just emerging. In this manuscript, we character- additivity with darifenacin. To determine expression in rhesus 3 ized the pharmacology of a novel b3AR agonist vibegron (MK- bladder, we employed a novel b3AR agonist probe, [ H]MRL- 4618, KRP-114V) and explored mechanistic interactions of b3AR 037, that selectively labels b3 receptors in both urothelium and agonism and muscarinic antagonism in urinary bladder function. detrusor smooth muscle. Vibegron administration caused a Vibegron is a potent, selective full b3AR agonist across species, dose-dependent increase in circulating glycerol and fatty acid and it dose dependently increased bladder capacity, decreased levels in rhesus and rat in vivo, suggesting these circulating at ASPET Journals on September 24, 2021 micturition pressure, and increased bladder compliance in lipids can be surrogate biomarkers.Thetranslationofourobser- rhesus monkeys. The relaxation effect of vibegron was enhanced vation to the clinic has yet to be determined, but the combination of when combined with muscarinic antagonists, but differentially b3AR agonists with M2/M3 antimuscarinics has the potential to influenced by muscarinic receptor subtype selectivity. The effect redefine the standard of care for the pharmacological treatment of was greater when vibegron was co-administered with tolterodine, overactive bladder.

Introduction The urinary bladder functions to collect and store urine excreted by the kidneys until voided. Alternating phases of b The beta-adrenergic receptor ( AR) family was first de- continence and micturition are controlled by the interplay of scribed more than 60 years ago (Ahlquist, 1948) and has been the central and peripheral nervous system with the local b b b since divided into three subtypes: 1, 2, and 3 (Lands et al., release of regulatory agents (Andersson and Wein, 2004; b 1967; Emorine et al., 1989). For the 3AR subtype, tissue Beckel and Holstege, 2011). Bladder filling occurs by relaxa- b b expression is more restricted compared with 1 and 2, with tion of the detrusor muscle (via parasympathetic inhibition) adipose, heart/vasculature, urinary bladder (mRNA) and with simultaneous contraction of the urethral sphincters to ovary (protein) believed to have the highest expression prevent involuntary emptying. It is also thought that release (Thomas and Liggett, 1993; Berkowitz et al., 1995; Uhlén of norepinephrine from the sympathetic hypogastric nerve et al., 2015). In the urinary bladder detrusor muscle of resulting in activation of primarily bAR receptors enhances b mammals, all three AR subtypes mRNA are expressed bladder compliance via detrusor relaxation, although only (Nomiya and Yamaguchi, 2003). Additionally, significant sparse sympathetic innervation is observed in the bladder b expression of 3AR protein is observed in bladder urothelium dome (Fowler et al., 2008). Bladder emptying (so-called (Limberg et al., 2010), the luminal epithelial lining of the micturition) on the other hand occurs via a switch in efferent urinary bladder (Birder and de Groat, 2007). signaling from sympathetic to parasympathetic, resulting in a release of acetylcholine and to a lesser extent ATP from the pelvic nerve causing detrusor contraction and interruption of release of norepinephrine from the hypogastric nerve, causing J.D.S and H.N. contributed equally to this work. dx.doi.org/10.1124/jpet.116.237313. urethral sphincter relaxation. Acetylcholine primarily acts on s This article has supplemental material available at jpet.aspetjournals.org. the detrusor via muscarinic M2 and M3 subtypes, whereas

ABBREVIATIONS: AE, adverse effects; bAR, beta-adrenergic receptor; b3AR, b3-adrenergic receptor; CIx, combination index; CI, confidence interval; EFS, electrical field stimulation; FFA, free fatty acid; OAB, overactive bladder.

346 Pharmacological Characterization of Vibegron 347

ATP acts on P2X-purine receptors, particularly under patho- A total of 16 adult female rhesus monkeys (Macaca mulatta) weighing logic conditions (Andersson, 2015), in the detrusor to initiate 5 to 7 kg and 4–7 years of age were used. Animals were either paired or bladder contraction. individually housed in temperature/humidity controlled rooms on a Disruption of this coordinated communication can result in 12-hour light/12-hour dark cycle and fed standard laboratory chow a symptom complex characterized by urinary urgency, with or (Tekland, Harlan Laboratories, Indianapolis, IN) supplemented with fresh fruit and vegetables with ad libitum water. A total of 81 male without urgency-associated urinary incontinence, referred to adult Sprague-Dawley rats weighing 250 to 350 g (Charles River, as overactive bladder (OAB) (Abrams et al., 2002). It is Wilmington, MA) were housed in temperature and light-controlled estimated that 12–23% of the general population exhibits (12-hour light/12-hour dark cycle) cages, fed standard laboratory chow symptoms of OAB, with a significant degradation in quality of (Diet 7012, Harlan) and water ad libitum. life (Irwin et al., 2006; Coyne et al., 2011). Management of Reagents. Vibegron ((6S)-N-[4-({(2S,5R)-5-[(R)-hydroxy(phenyl) OAB is typically a multimodal approach, employing both methyl]pyrrolidin-2-yl}-methyl)phenyl]-4-oxo-4,6,7,8-tetrahydropyrrolo[1,2- nonpharmacological and pharmacological treatment para- a]pyrimidine-6-carboxamide), MRL-037 ((R)-2-amino-N-(4-(((2S,5R)-5-((R)- digms. When nonpharmacological approaches (i.e., lifestyle hydroxy(phenyl)methyl)pyrrolidin-2-yl)methyl)phenyl)-5,6-dihydro- 3 and dietary modification) are ineffective, antimuscarinics are 4H-cyclopenta[d]thiazole-4-carboxamide), and [ H]MRL-037 ((R)-2- the first-line pharmacological treatment, representing the amino-N-(4-(((2S,5R)-5-((R)-hydroxy(phenyl-3,5-t2)methyl)pyrrolidin- 2-yl)methyl)phenyl-2,6-t )-5,6-dihydro-4H-cyclopenta[d]thiazole-4- most commonly prescribed drug therapy (Abrams and 2 carboxamide) were synthesized by the Discovery Chemistry Department Andersson, 2007). Although antimuscarinics have been

(Merck Research Laboratories) as previously described (Moyes et al., Downloaded from demonstrated to improve urgency and decrease frequency of 2014; Edmondson et al., 2016; Supplemental Fig. S1). CL316,243 micturition and urge incontinence, blockade of the M3 (disodium 5-[(2R)-2-[[(2R)-2-(3-chlorophenyl)-2-hydroxyethyl]amino]- muscarinic receptor can lead to adverse effects (AEs) such propyl]-1,3-benzodioxole-2,2-dicarboxylate hydrate), oxybutynin chlo- as dry mouth and constipation (Abrams and Andersson, ride, and methoctramine hydrate were purchased from Sigma-Aldrich 2007). These AEs contribute to a high discontinuation rate, (St. Louis, MO). Tolterodine L-tartrate and darifenacin hydrobromide resulting in significant unmet medical need for a pharmaco- were purchased from Toronto Research Chemicals (North York, logical treatment of OAB with an improved AE profile while Canada) and AK Scientific (Union City, CA), respectively. jpet.aspetjournals.org 3 — maintaining or exceeding the efficacy of antimuscarinics Autoradiography of Bladder Sections with [ H]MRL-037 Ex (Chapple et al., 2008). Vivo Receptor Occupancy Assay. Rhesus monkeys were anesthetized with an intramuscular injection of 5 mg/kg Telazol b AR activation in the bladder represents the most relevant 3 (Zoetis, Parsippany, NJ) or ketamine HCl (20 mg/kg) followed by a mechanism to increase bladder capacity without affecting lethal dose of a euthanasia drug (i.e., Euthasol; Virbac AH Inc., Fort bladder contraction. Initial studies in isolated human bladder Worth, TX). The bladder was then surgically removed and cut into using nonselective bAR agonists such as isoproterenol dem- 20-mm-thick sections, collected on superfrost/plus slides using a onstrated a pronounced bladder relaxation (Anderson, 1993). rapid sectioning cryostat (Leica CM1900), and then air dried for at ASPET Journals on September 24, 2021 – Subsequent studies using selective b3AR agonists in isolated 30 60 minutes at room temperature. After marking boundaries around human detrusor muscle strips determined that the observed the section with pan-pep, the tissue was covered with 600–1000 mlof relaxation was due to activation of b AR (Rouget et al., 2014; binding buffer (50 mM Tris-HCl, 2 mM Mg2Cl, 1 mM CaCl2,5mMKCl, 3 3 Gillespie et al., 2015; Michel and Korstanje, 2016). Given 0.1% bovine serum albumin) containing 50 nM of [ H]MRL-037 (specific increasing evidence for b AR activation as a treatment of activity of 30-50 Ci/mmol; Merck Research Laboratories) without or 3 with 10 mM of cold MRL-037 for nonspecific binding for 25 minutes. OAB, efforts to discover or repurpose potent and selective After incubation, the slides were washed for 4 minutes with ice-cold b 3AR agonists were initiated in recent years (Furuta et al., binding buffer containing 0.05% TritonX-100 for total of six times and b 2006; Drake, 2008). Subsequently, four selective 3AR ago- then soaked twice into in 30 ml of ice-cold buffer with 0.05% TritonX-100 nists, mirabegron (Tyagi and Tyagi, 2010), ritobegron in a staining jar for 4 minutes each wash, followed by dipping the slides (Maruyama et al., 2012), solabegron (Ohlstein et al., 2012), three times in ice-cold water. Slide were dried and exposed to Kodak and vibegron (Edmondson et al., 2016) entered clinical trials MR-2 895 2855 Sigma Kodak BioMax MR film (Cat # Z350400-50EA) for for treatment of OAB, with published clinical data available 5 days. Adjacent bladder sections were stained with hematoxylin and for both mirabegron and solabegron. In 2012, mirabegron eosin for histology following standard protocol (Longnecker, 1966). b demonstrated significant efficacy in reducing micturition In Vitro Potency and Selectivity of Vibegron on -Adrenergic frequency, urgency incontinence, and increasing mean volume Receptors. The ability of vibegron to activate the human, rhesus, rat, and dog b , b , and b AR was measured using CHO cell lines per micturition in patients with OAB (Chapple et al., 2014) 1 2 3 stably expressing the appropriate adrenergic receptor (Candelore and subsequently received regulatory approval. et al., 1999). For b3AR, the human cell line used expressed b3 at Herein, we describe the pharmacological characterization of levels similar to those observed in human detrusor muscle (Supple- b the novel 3AR agonist vibegron (MK-4618, KRP-114V) mental Fig. S2). To quantify the amount of released cAMP after b-AR (Edmondson et al., 2016) currently under clinical development activation, the LANCE cAMP kit (Perkin Elmer, Shelton, CT), a time- for OAB. In addition, we describe the mechanistic interaction resolved fluorescence resonance energy transfer immunoassay, was of b3AR agonists and antimuscarinics in bladder function and used. Compounds were serially diluted in DMSO and an aliquot added we propose an optimal combination of these mechanisms in to either 384-well or 96-well micro titer plates in assay buffer (5 mM ’ the treatment of OAB. HEPES, 0.1% bovine serum albumin in Hank s balanced salt solution). The reaction was initiated by the addition of 6000 cells per well in assay buffer that also contained a cAMP-specific antibody labeled with Materials and Methods Alexa Fluor 647 and a phosphodiesterase inhibitor (IBMX, Sigma). To examine serum-shifted potency, efficacy was evaluated using an assay Subjects. All procedures related to the use of animals were buffer containing 40% pooled human, rhesus, or rat serum. Because of approved by the Institutional Animal Care and Use Committee at a smaller assay window with serum compared with the buffer assay, Merck Research Laboratories (Rahway, NJ) and conform to the NIH the number of cells was increased to 10,000 per well. After 30-minute Guide for the Care and Use of Laboratory Animals (8th edition, 2011). incubation at room temperature, the cells were lysed by the addition of 348 Di Salvo et al.

LANCE detection buffer containing a europium-labeled cAMP tracer. transducer and an infusion pump for intravesical pressure measure- Fluorescence was measured after 1-hour incubation at room temper- ment and intravesical saline infusion, respectively. The intravesical ature using a PE Envision reader, exciting at 340 nm and measuring pressure was recorded using a multiple channel data acquisition emission at 615 and 665 nm. For each assay, a cAMP standard curve system (Power Laboratory 4/30, AD Instruments) at a sampling rate of was included and used to convert fluorescence readings directly to 20 Hz. After confirming bladder emptiness by ultrasonography, saline cAMP amounts. Values were normalized to a known full agonist was intravesically infused at 15 ml/min. Saline infusion was discon-

(isoproterenol), and the EC50 and percent maximum activation was tinued when a rapid increase in the intravesical pressure due to the then determined. micturition reflex was observed. After two baseline cystometry Isometric Detrusor Muscle Tension Recordings. Rats were readings, a compound was intravenously dosed using a rising dose euthanized with CO2 and the entire bladder was surgically removed and paradigm, with a cystometry measurement performed 10 minutes the mucosa was left intact. The bladder was then placed in a bath of after each dose. Blood samples were taken for measurements of

Krebs solution (113 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2,1.2mM plasma compound levels and serum glycerol/free fatty acid (FFA) MgSO4,25mMNaHCO3,1.2mMKH2PO4, and 1.5 mM glucose). The levels right after each cystometry. The following cystometric param- bladder dome was dissected in four approximately equal pieces of about eters were obtained from each cystometry: bladder capacity (duration 6mmÂ 3 mm. Each strip was placed in a warmed (37°C) organ bath of bladder filling multiplied by intravesical infusion rate), maximum

(25 ml) containing oxygenated (95% O2 1 5% CO2) Krebs solution. The micturition pressure (the pressure reading of the first peak in intra- strips were tied at one end to the organ bath and connected at the other vesical pressure driven by the micturition reflex), and bladder end to an isometric force transducer (AD Instruments, Colorado compliance (inverse of average slope of intravesical pressure during

Springs, CO) under a resting tension of 10 mN. The responses of the filling phase). The changes from baseline were calculated and Downloaded from preparations were recorded by a multiple channel data acquisition compared with the values in the vehicle-treated group or baseline system (PowerLab, AD Instruments), and measured with an analysis values. Compounds were dissolved in sterile 60% PEG400-20% software (Chart 5, AD Instruments). After the equilibration period for at ethanol-20% saline in a volume of 0.2 ml/kg. Serum glycerol levels least 60 minutes, each tissue strip was challenged to electrical field were determined using commercially available kits (Sigma). Plasma stimulation (EFS) at 60 Hz; duration, 0.3 ms; 3 seconds; 90 V to induce concentrations of compounds were determined by liquid chromatography- contractions. Upon obtaining stable contractions with EFS, i.e., base- tandem mass spectrometry on an Applied Biosystems API 4000 mass

line, compound solution (25 ml) was applied into the organ bath in a spectrometer. Plasma glycerol and FFA levels were determined using jpet.aspetjournals.org cumulative manner, followed by another EFS (consecutive 3 stimula- commercially available kits (FG0100, Sigma and NEFA-HR2, Wako, tions with 2-minute intervals) 15 minutes after compound treatment. Richmond, VA; respectively) which rely on a series of enzyme coupled Average amplitude of three contractions at baseline and each concen- reactions. tration, changes from baseline, and then ratio to corresponding vehicle Statistical Analysis. Data are presented as arithmetic mean 6 group (% of control) were calculated. S.D., S.E.M., or geometric mean with confidence intervals (CI) except Isobologram Analysis for Assessment of Combined Drug where indicated. The mean values were compared with two-way Effects. Isobologram analysis (Tallarida, 2001) was used to deter- analysis of variance with Bonferroni’s post hoc test or one-way analysis

’ at ASPET Journals on September 24, 2021 mine whether the interactions between a selective b3AR agonist of variance with Dunnett s multiple comparison test. A probability value CL316,243 and antimuscarinic drugs were additive, synergistic, or less than or equal to 0.05 was considered significant. antagonistic. CL316,243 was used in this study, because this compound was known to be potent at rat b3AR. Dose-dependent effects were determined for each compound and for combinations at fixed constant ratios. Data points on the isobologram were evaluated Results according to their positions relative to the diagonal. Data points in Potency of Vibegron at b-Adrenergic Receptors the lower left region indicated synergism, falling on the diagonal line indicated additive effects, and the upper right region indicated across Species. Vibegron (Fig. 1A) potently activates human b antagonism. The combination index (CI) provided a means to analyze 3AR and increases cAMP levels, with an EC50 of 1.1 nM and the combined effects with a median-effect plot analysis. The CI was 87% activation relative to isoproterenol (Edmondson et al., b b calculated according to the following formula: CI 5 (dCL/IC25CL) 1 2016). Vibegron is also highly selective over 1AR and 2AR (dA/IC25A), where IC25CL is the concentration of CL316,243 required versus b3AR across multiple species, demonstrating .9000-fold to produce 25% inhibition versus control, and dCL is the concentration selectivity for activation of b3AR over b1AR or b2AR in cell of CL316,243 required to produce 25% inhibition when combined with based in vitro functional assays (Table 1). A small serum shift an antimuscarinic drug. Similarly, IC25A is the concentration of an was observed in the presence of 40% human serum (EC50 antimuscarinic required to produce 25% inhibition, and dA is the 1.7 nM; Fig. 1B). The small effect of serum was also observed for concentration of an antimuscarinic required to produce 25% of the rat and rhesus b AR, consistent with the low noncovalent effect when combined with CL316,243. The CI values were defined as 3 follows: ,0.8 5 synergism, from 0.8 to 1.2 5 additive effect, and .1.2 plasma protein binding (Edmondson et al., 2016). Localization of b AR in Rhesus Bladder with a 5 antagonism. The IC25 values and their 95% confidence intervals was 3 interpolated from the nonlinear fit curve (Prism 5, GraphPad, La Labeled b3AR Agonist. To determine expression of b3AR Jolla, CA). in rhesus tissue, we used a radiolabeled agonist of b3AR, Cystometry in Rhesus Monkeys. Cystometry was performed as [3H]MRL-037 (Fig. 2A; Supplemental Fig. S1), a pan-species described previously (Nagabukuro et al., 2011). In brief, rhesus potent and selective b3AR agonist (Moyes et al., 2014), in monkeys were anesthetized with an intramuscular injection of Telazol tissue autoradiography experiments. This compound is a (5 mg/kg) or ketamine HCl (20 mg/kg) followed by intravenous potent b AR agonist across multiple species with no activity – 3 constant rate infusion with ketamine HCl (0.2 0.3 mg/kg/min). at b or b and with physiochemical properties that make it a Animals were then placed in a supine position, and two catheters 1 2 suitable tracer for localizing b AR expression in tissue. (20 gauge) were inserted into a saphenous and/or a brachial cephalic 3 vein for compound administration and ketamine infusion/blood Similar to the staining pattern in human tissue observed sampling, respectively. A triple lumen balloon transurethral catheter with immunohistochemistry (Limberg et al., 2010), clear (7.4 Fr, Cook Medical, Bloomington, IN) was inserted into the bladder, staining of the urothelium was observed in rhesus bladder 3 and the balloon was inflated with 1 ml of saline to secure the catheter using [ H]MRL-037 (Fig. 2, B and C). Staining of human at the bladder base. Another two lumens were connected to a pressure bladder tissue with 3H-MRL-037 produced a similar pattern, Pharmacological Characterization of Vibegron 349

hyperactivity model, a relationship between indices of bladder function and target engagement markers of acute b3AR stimulation in adipose tissue, i.e., circulating glycerol increase in rhesus monkeys, was also examined. Baseline values of urodynamic parameters in vehicle- and vibegron-treated animals are as follows: bladder capacity, 156.8 6 21.6 ml (vehicle), 123.7 6 10.7 ml (vibegron); micturition pressure, 36.7 6 1.6 cm H2O (vehicle), 35.5 6 2.8 cm H2O (vibegron); bladder compliance, 15.0 6 2.2 ml/cm H2O (vehicle), 16.6 6 2.8 ml/cm H2O (vibegron). As in the previous study (Nagabukuro et al., 2011), vehicle had no statistically significant effect on any of parameters. Vibegron increased bladder capacity in a dose-dependent manner (Fig. 3A). The maximum bladder capacity increase induced by vibegron was 156% of baseline value, which is comparable to maximum effect with antimuscarinics (Nagabukuro et al., 2011). Micturition pressure was signifi- cantly decreased at 0.3 and 3 mg/kg (Fig. 3B). Bladder compliance was increased at doses greater than 0.1 mg/kg Downloaded from Fig. 1. (A) Structure of vibegron; (B) activation of recombinant human b3AR by vibegron in the absence (s) or the presence (d) of 40% human (Fig. 3C). Vibegron also increased serum glycerol and FFA serum assayed in a recombinant CHO cell line that stably expresses levels in a dose-dependent manner (Fig. 3, D and E). Concen- b human 3AR. Percent activity is an average of N = 3 determinations; error tration response curves for bladder capacity and serum glycerol bars represent standard deviation. Potency of vibegron (1.3 nM) is only slightly less potent (1.7 nM) when assayed in the presence of 40% human closely overlapped (see Table 2 for vibegron plasma levels), serum. where the EC50 value in increasing bladder capacity was 2.9 nM in total plasma and 1.5 nM at unbound levels; for increasing jpet.aspetjournals.org albeit with weaker signal, than was observed in rhesus serum glycerol, the EC50 value was 9.9 nM in total plasma and (Supplemental Fig. S3). 5.0 nM at unbound levels (Edmondson et al., 2016). Dose-dependent Effects of Vibegron in Urodynamic Effect of Combined Treatments of a b3AR Agonist Parameters in Rhesus Monkeys. We previously reported with Antimuscarinics in Isolated Detrusor Muscle. the activity of vibegron in a rat bladder hyperactivity model CL316,243 and all antimuscarinics oxybutynin, tolterodine (Edmondson et al., 2016), measured by cystometry, and the and darifenacin, inhibited the EFS-induced isolated detrusor comparison with activity in adipose tissue as measured by muscle contractions in a concentration-dependent manner at ASPET Journals on September 24, 2021 lipolysis readouts. We sought to extend the evaluation of (Fig. 4, A and B). Unlike oxybutynin, vibegron also depressed vibegron to nonhuman primates. A nonhuman primate phar- the spontaneous contractile activity. Concentrations that macodynamic model offers advantages for pharmacological induced 25% inhibition (IC25) were used to determine evaluation of vibegron because of physiologic and anatomic combination ratios and for isobologram analyses (Table 3). similarities of the lower urinary tract to humans. Additionally, CL316,243 was cotreated with tolterodine, oxybutynin, or vibegron activates both human and rhesus monkey b3AR at a darifenacin at a fixed combination ratio. Isobologram anal- similar potency (Table 1). Accordingly, vibegron was evaluated yses are shown in Fig. 5. Based on the CI criteria (see across concentrations up to 10 mg/kg i.v. in the rhesus Materials and Methods), all combinations were synergistic, cystometry model. As investigated in the rat bladder but with different degrees of synergism: tolterodine .

TABLE 1

Potency and selectivity of vibegron at human, rhesus, rat, and dog b1, b2,andb3AR

Potency was measured using CHO cell lines stably expressing the appropriate receptor (Bmax 50–100 fmol/mg for b3AR cell lines, 300–500 fmol/mg for b1 and b2). To determine potency in the presence of serum, compounds were assayed in the presence of 40% autologous serum (pooled donors). EC50 for vibegron in the presence of 40% dog serum was not determined. Activation was determined relative to the known full agonist isoproterenol (1 mM). EC50 of isoproterenol at the human, rhesus monkey, rat, and dog b3AR receptors was 28, 12, 124, and 264 nM, respectively. Values represent geometric mean with confidence intervals (CI); N $ 6 for all values. b3AR potency has been previously described (Edmondson et al., 2016).

bAR Subtype Species EC50 (CI) EC50, nM w/ 40% Serum (CI)

nM % activation

b3 Human 1.0 (1.5,0.71); 84% 1.5 (2.3, 1.0); 102% b1 Human .10,000; 5% b2 Human .10,000; 7% b3 Rhesus Monkey 0.52 (0.81, 0.30); 108% 5.5 (12, 2.6); 98% b1 Rhesus Monkey .10,000; 4% b2 Rhesus Monkey .10,000; 0% b3 Rat 81 (119,55); 83% 118 (161,85); 89% b1 Rat .10,000; 0% b2 Rat .10,000; 1% b3 Dog 10 (15, 7.1); 82% N.D. b1 Dog .10000; 2% b2 Dog .10,000; 1%

N.D., not determined. 350 Di Salvo et al.

3 Fig. 2. MRL b3AR Agonist [ H]MRL-037 (A) is a potent full agonist at the human (EC50 = 0.12 nM), rhesus (EC50 = 0.12 nM), rat (EC50 = 1.0 nM), and dog (EC50 = 0.19 nM) b3ARs with no affinity for the b1 and b2 subtypes (EC50 and IC50 . 10 mM all species). Comparison of binding of [3H]MRL-037 to histologic staining in rhesus bladder tissue: (B) autoradiography after incubation of bladder with 50 nM [3H]MRL-037; (C) hematoxylin and eosin staining; (D) to determine nonspecific binding, incubation of bladder with 50 nM [3H]MRL-037 and an excess (10 mM) of unlabeled MRL-037 was performed followed by autoradiography. Arrows indicate regions of intact Downloaded from urothelium. Adjacent sections of bladder are shown. Data are representative of three studies. jpet.aspetjournals.org

oxybutynin . darifenacin (Fig. 5). The M2 selective antagonist radioligand, MRL-037—to label b3AR expressing cells in the methoctramine caused only marginal inhibition of the EFS- urinary bladder. From our in vitro characterization of MRL- induced detrusor contraction (0.003–10 mM; Supplemental 037, this compound is extremely selective for b3 across several Fig. S4). Given the potency of methoctramine at muscarinic species, including rhesus monkeys. Using autoradiography of 3 receptor subtypes other than M2 (Ki, ∼1 mM), significant bladder sections incubated with [ H]MRL-037, b3AR expres- inhibition at the higher concentrations was expected. But at sion was detected in urothelium. Although less intense, b3AR at ASPET Journals on September 24, 2021 least in the current experimental setting, there was less than expression was detected in detrusor as well, similar to what 25% inhibition of EFS-induced isolated detrusor contraction. was previously observed in human bladder, where b3AR In addition, pretreatment of methoctramine (1 mM) did not expression appears to be more intense in the urothelium affect CL316,243-induced inhibition of detrusor contraction compared with the detrusor (Limberg et al., 2010). The (data not shown). However, methoctramine caused a signifi- potency and selectivity of MRL-037 across several species cant change in isobologram for the darifenacin and CL316,243 provides for a unique means to directly compare and quantify combination. As shown in Fig. 5D, combination with b3AR expression across multiple species using a single tool. In CL316,243 and darifenacin exhibited much more robust addition, MRL-037 could be employed to determine ex vivo synergism with a pretreatment of methoctramine (CI, 0.16) receptor occupancy, providing a means to directly correlate compared with the same 1 mM). b3AR target engagement in the bladder to efficacy. Effect of Combined Treatments of Vibegron and We observed staining for b3AR in the urothelium of human Antimuscarinics in Rhesus Monkeys. To extend our and rhesus monkey. Given our data, along with previously findings of combination therapy with vibegron, cotreatments published observations (Limberg et al., 2010; Kullmann et al., of vibegron and two antimuscarinic agents tolterodine and 2011; Otsuka et al., 2013), b3AR is expressed in both the darifenacin were evaluated in the cystometry model in rhesus detrusor muscle and bladder urothelium across multiple monkeys. We used monotherapy data for tolterodine and species. The exact role of b3AR in the urothelium and its darifenacin from our previous publication (Nagabukuro contribution to the observed efficacy of b3AR agonists in et al., 2011) as we carried out the study with exactly the same treating OAB is unknown, but there is mounting evidence methods and within similar timeframe. All dose combinations that b3ARs in the urothelium likely contribute either directly of vibegron and tolterodine showed a greater bladder capacity or indirectly to the observed clinical effects of b3AR agonists. increase compared with each compound alone, with effects Our demonstration of agonist binding to urothelium and the that are greater than additive at low doses (Table 4). In previously reported b3AR agonist-induced functionality in an contrast, addition of darifenacin to vibegron induced greater immortalized human urothelium cell line (Harmon et al., bladder relaxation in rhesus only when used at high doses. 2005) infer that b3AR agonists can have a direct effect on activating cells within the urothelium. However, the func- b Discussion tional role of 3ARs in the urothelium has not been well documented. Masunaga et al. (2010) and Kullmann et al. Given the questionable selectivity of antibodies for staining (2011) showed that in porcine and rat bladder strips re- of b3AR in rhesus monkeys, we turned to a more novel laxation of the detrusor muscle by nonselective bAR and approach of identifying b3AR expression in the urinary selective b3AR stimulation occurs to the same extent with or bladder of this species—use of a selective b3 agonist without urothelium. Alternatively, others reported that the Pharmacological Characterization of Vibegron 351 Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021

Fig. 3. Effect of vibegron on the bladder capacity (A), maximum intravesical pressure (B), bladder compliance (C), serum glycerol (D), and nonesterified free fatty acids (NEFFA) (E) in anesthetized rhesus monkeys. Values represent mean 6 S.D. of value or of percentage of baseline values as indicated. N =6. *P , 0.05, **P , 0.01 versus baseline values, repeated-measures analysis of variance; #P , 0.05, ##P , 0.01, versus vehicle-treated group, Dunnett’s multiple comparison test. a, Not applicable for statistical analyses because of lack of data from one animal (N = 5).

presence of urothelium diminishes nonselective bAR-induced urothelial effect, whereas b3AR directly mediates the relaxa- relaxation of isolated human detrusor muscle (Otsuka et al., tion of human detrusor, and its involvement did not differ with 2008; Propping et al., 2013). By using subtype selective bAR or without urothelium (Propping et al., 2013). antagonists, b2AR was suggested to be involved in this Medicinal chemistry efforts at optimizing the potency, selectivity, and pharmacokinetic properties of a series of pyrrolidine derived amides led to the discovery of vibegron, which is currently in late stage clinical trials for OAB TABLE 2 (Edmondson et al., 2016). In two different preclinical species, Plasma levels of vibegron in anesthetized rhesus monkeys Values represent mean 6 S.D. (N =4–6). vibegron causes dose-dependent relaxation of urinary bladder, resulting in an increase in bladder capacity and a decrease in Plasma Vibegron Level micturition pressure. These pharmacodynamic effects in the nM lower urinary tract accompany an increase in circulating 6 0.003 mg/kg 0.5 0.4 glycerol and FFA, indicating the potential use of b3AR 0.01 mg/kg 1.2 6 0.1 activation in adipose tissue as a surrogate pharmacodynamic/ 0.03 mg/kg 3.0 6 0.9 0.1 mg/kg 13.1 6 3.6 target engagement readout for urinary bladder. Interpretation 0.3 mg/kg 43.4 6 15.4 of the relative pharmacodynamics effects of b3AR activation in 1 mg/kg 128.5 6 27.9 adipose tissue to bladder must be done with care when trans- 6 3 mg/kg 553.6 144.0 lating across organs and species. In rhesus cystometry studies, 352 Di Salvo et al.

observed in human adipose, and this was noted in the early clinical programs looking at b3AR agonists for obesity (Ursino et al., 2009). Chronic administration of b3AR agonists in obese rats results in weight loss without changes in food intake due to increased energy expenditures (Ursino et al., 2009; Cernecka et al., 2014), but no such effect is observed in humans (Arch, 2008). b3AR is the predominant subtype in both white and brown adipose tissue in rats, whereas in humans and rhesus monkeys only in brown adipose does b3AR predominate, with little b3AR expression in white adipose (Candelore et al., 1999). This increased expression of b3AR in rat adipose and the more pronounced effect on metabolism may explain our observation of differential sensitivity to b3AR agonists in rat adipose versus rat bladder. Alternatively, the difference may not be in tissue sensitivity but may reflect physiologic differences in the rodent versus primate lower urinary tract, such that b3AR agonists may play more active of a role in adipose tissue in rats versus primates. Downloaded from Our studies combining a b3AR agonist with antimuscarinics resulted in enhanced bladder relaxation in both rats and rhesus monkeys, with greater synergism when both muscarinic M2 and M3 subtypes were blocked compared with selective blockade of the M3 subtype. Although M3 appears to be the predominant muscarinic subtype in mediating jpet.aspetjournals.org contractile responses in bladder detrusor muscle, the role of M2 receptors in bladder is less certain despite it being the more highly expressed subtype (.90% in rat bladder) (Wang et al., 1995). It has been suggested that the M2 subtype contributes to bladder relaxation but in a more indirect role through the enhancement of M3-mediated contractions and

through inhibition of bladder relaxation (Ehlert et al., 2005; at ASPET Journals on September 24, 2021 Matsumoto et al., 2012). The inhibition of bladder relaxation mediated by M2 activation is thought to occur via an inhibition of adenylyl cyclase (the M2 receptor subtype is Gi-coupled),

Fig. 4. Effects of concentrations of a b3AR agonist CL316,243 and which opposes the increase in cAMP elicited by activation of muscarinic antagonists on EFS-induced contraction of rat bladder strips. b3AR (Matsui et al., 2003; Ehlert et al., 2007). In the absence of (A) Representative recordings of isometric tension of bladder strips during an M2 response, forskolin and isoproterenol exhibit a greater the course of cumulative treatment of vehicle, CL316,243, and oxybutynin. (B) Dose titration of CL316,243, tolterodine, oxybutynin, and darifenacin. relaxant activity compared with conditions under which M2 is N =7or8. active (Matsui et al., 2003; Ehlert et al., 2007). Inhibition of M2 activity would have a direct impact on the effects of b3AR elevated levels of serum glycerol correlated with b3AR- activation by increasing cAMP levels and would therefore mediated increases in bladder capacity, with the two readouts provide greater relaxation mediated through b3AR activation, of b3AR activation closely overlapping each other. In rats, which is in accordance with our findings in this study. although decreases in micturition pressure are accompanied by Recently, Furuta et al. (2016) reported that in conscious an increase in circulating glycerol levels, we observed elevated female rats, the combination therapy of a b3AR agonist and glycerol levels in the absence of any effect on bladder compli- muscarinic M3 antagonists was more effective in increasing ance, suggesting that rat adipose is the more sensitive tissue to bladder capacity than monotherapy and that M2 antagonism b3AR activation relative to bladder (Edmondson et al., 2016). had no impact on the effect of a b3AR agonist. We also Although b3AR is also involved in lipolysis in human adipose confirmed that simple M2 antagonism did not influence (Bordicchia et al., 2014), the effects of b3AR activation in rat b3AR agonist-induced relaxation of rat bladder strips. Addi- adipose appear to be much more profound compared with that tionally, muscarinic receptor subtype contribution to bladder

TABLE 3 Potency of CL316,243, tolterodine, oxybutynin, and darifenacin in inhibiting EFS-induced contraction of rat bladder strips and combination index of each antimuscarinic with CL316,243

Drug IC25 Upper 95% CI Lower 95% CI Combination Index with CL316,243

nM CL316,243 2.86 5.91 1.18 — Tolterodine 4.71 11.68 1.89 0.21 Oxybutynin 22.28 68.45 8.86 0.50 Darifenacin 5.84 11.29 3.07 0.71 Pharmacological Characterization of Vibegron 353

Fig. 5. Isobologram analysis of the effects of combined treatments with a b3AR agonist CL316,234 and three different muscarinic antagonists in isolated rat detrusor muscle: tolterodine (A), oxybutynin (B), and darifenacin (C). An additional

analysis was done with darifenacin with a Downloaded from pretreatment of methoctramine (D). Com- bination ratio of two drugs is shown in parentheses. Each represents mean 6 95% confidence interval. jpet.aspetjournals.org at ASPET Journals on September 24, 2021

function may differ between species. In nonhuman primates, In conclusion, we described the pharmacology of a new muscarinic subtypes other than M3 appear to contribute more potent and selective b3AR agonist, vibegron, and mechanistic to bladder storage functions than rodents (Nagabukuro et al., interplay between b3AR agonists and muscarinic antagonists 2011). Multiple clinical studies have demonstrated additional in urinary bladder. We also demonstrated that circulating benefits by combining solifenacin and mirabegron (Abrams glycerol and free fatty acid levels could potentially be used as et al., 2015; De Nunzio, et al., 2015; Kosilov et al., 2015). surrogate pharmacodynamic readouts to predict bladder Because solifenacin is more M3 selective compared with effects of vibegron, although care must be exercised when tolterodine and oxybutynin (Ohtake et al., 2007), it may be comparing pharmacodynamic effects in different tissue beds possible to improve further efficacy at bladder relaxation (Morgan et al., 2012; Cook et al., 2014). The potential for b3AR based on our data, suggesting a b3AR agonist combined with agonists as monotherapy to effectively treat OAB has been a nonselective M2/M3 antagonist as the optimal combination validated with the recent approval of mirabegron, but given therapy in humans. that both muscarinic and b3ARs play a critical role in bladder

TABLE 4 Bladder capacity increases with combined treatments with vibegron and tolterodine and darifenacin in rhesus monkeys Values in table represent Mean 6 SEM of % increase over baseline (N=4).

Vibegron Vehicle 0.003 0.01 0.03 0.1 0.3 1

mg/kg Vehicle 4.1 6 3.5 18.0 6 5.7 27.3 6 5.1 33.9 6 9.5 31.3 6 5.0 55.9 6 18.5 Tolterodine 0.01 10.7 6 5.0a 26.7 6 4.9 31.0 6 6.3 0.03 16.8 6 13.9a 35.5 6 9.3 36.4 6 4.0 43.4 6 6.8 0.1 40.3 6 9.8a 62.9 6 8.3 56.8 6 8.1 69.6 6 14.6 Darifenacin 0.01 8.9 6 7.1a 13.7 6 4.0 23.7 6 3.3 0.03 18.3 6 8.6a 37.1 6 9.4 43.3 6 12.6 0.1 29.4 6 8.9a 58.0 6 19.5 68.7 6 20.9

aData from previous publication (Nagabukuro et al., 2011) 354 Di Salvo et al. function, additional efficacy (with potential for an improved Coyne KS, Sexton CC, Vats V, Thompson C, Kopp ZS, and Milsom I (2011) National community prevalence of overactive bladder in the United States stratified by sex adverse effects profile) may be achieved by combining stan- and age. Urology 77:1081–1087. dard of care antimuscarinics with b3AR agonists. Our obser- De Nunzio C, Presicce F, Pirozzi L, Castellan P, Schips L, Cindolo L, Lombardo R, b and Tubaro A (2015) The current indications and the benefits of combining a b3-agonist vations indicate that combination of 3AR agonists with dual with an anticholinergic for the treatment of OAB. Curr Drug Targets 16:1198–1206. M2 and M3 antagonists rather than selective M3 antagonists Drake MJ (2008) Emerging drugs for treatment of overactive bladder and detrusor provides optimal efficacy in the treatment of OAB. Our overactivity. Expert Opin Emerg Drugs 13:431–446. Edmondson SD, Zhu C, Kar NF, Di Salvo J, Nagabukuro H, Sacre-Salem B, Dingley hypothetical translation to the clinic has yet to be determined, K, Berger R, Goble SD, Morriello G, et al. (2016) Discovery of vibegron: a potent but we propose this potential therapeutic approach to redefine and selective b3 adrenergic receptor agonist for the treatment of overactive bladder. J Med Chem 59:609–623. the standard of care for the pharmacological treatment of Ehlert FJ, Ahn S, Pak KJ, Park GJ, Sangnil MS, Tran JA, and Matsui M (2007) OAB. Neuronally released acetylcholine acts on the M2 muscarinic receptor to oppose the relaxant effect of isoproterenol on cholinergic contractions in mouse urinary bladder. J Pharmacol Exp Ther 322:631–637. Authorship Contributions Ehlert FJ, Griffin MT, Abe DM, Vo TH, Taketo MM, Manabe T, and Matsui M (2005) Participated in research design: Di Salvo, Nagabukuro, Wickham, The M2 muscarinic receptor mediates contraction through indirect mechanisms in mouse urinary bladder. J Pharmacol Exp Ther 313:368–378. Abbadie, DeMartino, Fitzmaurice, Gichuru, Donnelly, Jochnowitz, Emorine LJ, Marullo S, Briend-Sutren MM, Patey G, Tate K, Delavier-Klutchko C, Hurley, Pereira, Sanfiz, Voronin, Villa, Woods, Zamlynny, Zycband, and Strosberg AD (1989) Molecular characterization of the human beta Salituro, Frenkl, Weber, Edmondson, and Struthers. 3-adrenergic receptor. Science 245:1118–1121. Conducted experiments: Di Salvo, Nagabukuro, Wickham, Abbadie, Fowler CJ, Griffiths D, and de Groat WC (2008) The neural control of micturition. Nat Rev Neurosci 9:453–466.

Fitzmaurice, Gichuru, Kulick, Donnelly, Jochnowitz, Hurley, Pereira Furuta A, Suzuki Y, Kimura S, Koike Y, Egawa S, and Yoshimura N (2016) Combi- Downloaded from Sanfiz, Veronin, Villa Zamlynny, and Zycband. nation therapy with b3 -adrenoceptor agonists and muscarinic acetylcholine receptor – Contributed new reagents or analytic tools: Di Salvo, Nagabukuro, antagonists: Efficacy in rats with bladder overactivity. Int J Urol 23:425 430. Furuta A, Thomas CA, Higaki M, Chancellor MB, Yoshimura N, and Yamaguchi O Wickham, Abbadie, DeMartino, Fitzmaurice, Gichuru, Kulick, (2006) The promise of beta3-adrenoceptor agonists to treat the overactive bladder. Donnelly, Jochnowitz, Hurley, Pereira Sanfiz, Veronin, Villa, Woods Urol Clin North Am 33:539–543, x x. Zamlynny, Zycband, Salituro, Frenkl, Weber, Edmondson, and Gillespie JI, Rouget C, Palea S, Granato C, and Korstanje C (2015) Beta adrenergic modulation of spontaneous microcontractions and electrical field-stimulated contractions Struthers. in isolated strips of rat urinary bladder from normal animals and animals with partial – Performed data analysis: Di Salvo, Nagabukuro, Wickham, bladder outflow obstruction. Naunyn Schmiedebergs Arch Pharmacol 388:719 726. jpet.aspetjournals.org Abbadie, DeMartino, Fitzmaurice, Gichuru, Kulick, Donnelly, Harmon EB, Porter JM, and Porter JE (2005) Beta-adrenergic receptor activation in immortalized human urothelial cells stimulates inflammatory responses by PKA- Jochnowitz, Hurley, Pereira, Sanfiz, Veronin, Villa, Woods Zamlynny, independent mechanisms. Cell Commun Signal 3:10–19. Zycband, Salituro, Frenkl, Weber, Edmondson, and Struthers. Irwin DE, Milsom I, Hunskaar S, Reilly K, Kopp Z, Herschorn S, Coyne K, Kelleher Wrote or contributed to the writing of the manuscript: Di Salvo, C, Hampel C, Artibani W, et al. (2006) Population-based survey of urinary incontinence, overactive bladder, and other lower urinary tract symptoms in five Nagabukuro, Edmondson, and Struthers. countries: results of the EPIC study. Eur Urol 50:1306–1315. Kullmann FA, Downs TR, Artim DE, Limberg BJ, Shah M, Contract D, de Groat WC, References and Rosenbaum JS (2011) Urothelial beta-3 adrenergic receptors in the rat bladder. Neurourol Urodyn 30:144–150.

Abrams P and Andersson KE (2007) Muscarinic receptor antagonists for overactive at ASPET Journals on September 24, 2021 bladder. BJU Int 100:987–1006. Kosilov K, Loparev S, Ivanovskaya M, and Kosilova L (2015) A randomized, con- Abrams P, Cardozo L, Fall M, Griffiths D, Rosier P, Ulmsten U, van Kerrebroeck P, trolled trial of effectiveness and safety of management of OAB symptoms in elderly Victor A, and Wein A; Standardisation Sub-committee of the International Con- men and women with standard-dosed combination of solifenacin and mirabegron. – tinence Society (2002) The standardisation of terminology of lower urinary tract Arch Gerontol Geriatr 61:212 216. function: report from the Standardisation Sub-committee of the International Lands AM, Arnold A, McAuliff JP, Luduena FP, and Brown TG, Jr (1967) Differentiation – Continence Society. Neurourol Urodyn 21:167–178. of receptor systems activated by sympathomimetic amines. Nature 214:597 598. Abrams P, Kelleher C, Staskin D, Rechberger T, Kay R, Martina R, Newgreen D, Limberg BJ, Andersson KE, Aura Kullmann F, Burmer G, de Groat WC, b Paireddy A, van Maanen R, and Ridder A (2015) Combination treatment with and Rosenbaum JS (2010) -Adrenergic receptor subtype expression in myocyte – mirabegron and solifenacin in patients with overactive bladder: efficacy and safety and non-myocyte cells in human female bladder. Cell Tissue Res 342:295 306. results from a randomised, double-blind, dose-ranging, phase 2 study (Symphony). Longnecker DS (1966) A program for automated hematoxylin and eosin staining. Am Eur Urol 67:577–588. J Clin Pathol 45:229. Ahlquist RP (1948) A study of the adrenotropic receptors. Am J Physiol 153:586–600. Maruyama I, Tatemichi S, Goi Y, Maruyama K, Hoyano Y, Yamazaki Y, and Kusama b Anderson KE (1993) Pharmacology of lower urinary tract smooth muscles and penile H (2012) Effects of ritobegron (KUC-7483), a novel selective 3-adrenoceptor agonist, – erectile tissues. Pharmacol Rev 45:253–308. on bladder function in cynomolgus monkey. J Pharmacol Exp Ther 342:163 168. b Andersson KE (2015) Purinergic signalling in the urinary bladder. Auton Neurosci Masunaga K, Chapple CR, McKay NG, Yoshida M, and Sellers DJ (2010) The 3- b 191:78–81. adrenoceptor mediates the inhibitory effects of -adrenoceptor agonists via the – Andersson KE and Wein AJ (2004) Pharmacology of the lower urinary tract: basis for urothelium in pig bladder dome. Neurourol Urodyn 29:1320 1325. current and future treatments of urinary incontinence. Pharmacol Rev 56:581–631. Matsui M, Griffin MT, Shehnaz D, Taketo MM, and Ehlert FJ (2003) Increased Arch JR (2008) The discovery of drugs for obesity, the metabolic effects of leptin and relaxant action of forskolin and isoproterenol against muscarinic agonist-induced variable receptor pharmacology: perspectives from beta3-adrenoceptor agonists. contractions in smooth muscle from M2 receptor knockout mice. J Pharmacol Exp – Naunyn Schmiedebergs Arch Pharmacol 378:225–240. Ther 305:106 113. Beckel JM and Holstege G (2011) Neurophysiology of the lower urinary tract. Handb Matsumoto Y, Miyazato M, Yokoyama H, Kita M, Hirao Y, Chancellor MB, Exp Pharmacol 202:149–169. and Yoshimura N (2012) Role of M2 and M3 muscarinic acetylcholine receptor Berkowitz DE, Nardone NA, Smiley RM, Price DT, Kreutter DK, Fremeau RT, subtypes in activation of bladder afferent pathways in spinal cord injured rats. and Schwinn DA (1995) Distribution of beta 3-adrenoceptor mRNA in human tis- Urology 79: 1184.e15–1184.e20. sues. Eur J Pharmacol 289:223–228. Michel MC and Korstanje C (2016) b3-Adrenoceptor agonists for overactive bladder Birder LA and de Groat WC (2007) Mechanisms of disease: involvement of the uro- syndrome: Role of translational pharmacology in a repositioning clinical drug de- thelium in bladder dysfunction. Nat Clin Pract Urol 4:46–54. velopment project. Pharmacol Ther 159:66–82. Bordicchia M, Pocognoli A, D’Anzeo M, Siquini W, Minardi D, Muzzonigro G, Morgan P, Van Der Graaf PH, Arrowsmith J, Feltner DE, Drummond KS, Wegner Dessì-Fulgheri P, and Sarzani R (2014) Nebivolol induces, via b3adrenergic CD, and Street SD (2012) Can the flow of medicines be improved? Fundamental receptor, lipolysis, uncoupling protein 1, and reduction of lipid droplet size in pharmacokinetic and pharmacological principles toward improving Phase II sur- human adipocytes. JHypertens32:389–396. vival. Drug Discov Today 17:419–424. Candelore MR, Deng L, Tota L, Guan XM, Amend A, Liu Y, Newbold R, Cascieri MA, Moyes CR, Berger R, Goble SD, Harper B, Shen DM, Wang L, Bansal A, Brown PN, and Weber AE (1999) Potent and selective human beta(3)-adrenergic receptor Chen AS, Dingley KH, et al. (2014) Design, synthesis, and evaluation of con- antagonists. J Pharmacol Exp Ther 290:649–655. formationally restricted acetanilides as potent and selective b3 adrenergic receptor Cernecka H, Sand C, and Michel MC (2014) The odd sibling: features of b3-adrenoceptor agonists for the treatment of overactive bladder. J Med Chem 57:1437–1453. pharmacology. Mol Pharmacol 86:479–484. Nagabukuro H, Villa KL, Wickham LA, Kulick AA, Gichuru L, Donnelly MJ, Voronin Chapple CR, Cardozo L, Nitti VW, Siddiqui E, and Michel MC (2014) Mirabegron in GO, Pereira T, Tong X, Nichols A, et al. (2011) Comparative analysis of the effects overactive bladder: a review of efficacy, safety, and tolerability. Neurourol Urodyn of antimuscarinic agents on bladder functions in both nonhuman primates and 33:17–30. rodents. J Pharmacol Exp Ther 338:220–227. Chapple CR, Khullar V, Gabriel Z, Muston D, Bitoun CE, and Weinstein D (2008) Nomiya M and Yamaguchi O (2003) A quantitative analysis of mRNA expression of The effects of antimuscarinic treatments in overactive bladder: an update of a alpha 1 and beta-adrenoceptor subtypes and their functional roles in human nor- systematic review and meta-analysis. Eur Urol 54:543–562. mal and obstructed bladders. J Urol 170:649–653. Cook D, Brown D, Alexander R, March R, Morgan P, Satterthwaite G, and Pangalos Ohlstein EH, von Keitz A, and Michel MC (2012) A multicenter, double-blind, ran- MN (2014) Lessons learned from the fate of AstraZeneca’s drug pipeline: a five- domized, placebo-controlled trial of the b3-adrenoceptor agonist solabegron for dimensional framework. Nat Rev Drug Discov 13:419–431. overactive bladder. Eur Urol 62:834–840. Pharmacological Characterization of Vibegron 355

Ohtake A, Saitoh C, Yuyama H, Ukai M, Okutsu H, Noguchi Y, Hatanaka T, Suzuki Thomas RF and Liggett SB (1993) Lack of beta 3-adrenergic receptor mRNA M, Sato S, Sasamata M, et al. (2007) Pharmacological characterization of a new expression in adipose and other metabolic tissues in the adult human. Mol Phar- antimuscarinic agent, solifenacin succinate, in comparison with other anti- macol 43:343–348. muscarinic agents. Biol Pharm Bull 30:54–58. Tyagi P and Tyagi V (2010) Mirabegron, a b3-adrenoceptor agonist for the potential Otsuka A, Kawasaki H, Matsumoto R, Shinbo H, Kurita Y, Iwashita T, and Ozono S treatment of urinary frequency, urinary incontinence or urgency associated with (2013) Expression of b-adrenoceptor subtypes in urothelium, interstitial cells overactive bladder. IDrugs 13:713–722. and detrusor of the human urinary bladder. Low Urin Tract Symptoms 5: Uhlén M, Fagerberg L, Hallström BM, Lindskog C, Oksvold P, Mardinoglu A, 173–180. Sivertsson Å, Kampf C, Sjöstedt E, Asplund A, et al. (2015) Proteomics. Tissue- Otsuka A, Shinbo H, Matsumoto R, Kurita Y, and Ozono S (2008) Expression and based map of the human proteome. Science 347:1260419. functional role of beta-adrenoceptors in the human urinary bladder urothelium. Ursino MG, Vasina V, Raschi E, Crema F, and De Ponti F (2009) The beta3-adrenoceptor Naunyn Schmiedebergs Arch Pharmacol 377:473–481. as a therapeutic target: current perspectives. Pharmacol Res 59:221–234. Propping S, Wuest M, Eichhorn B, Wirth MP, Kaumann AJ, and Ravens U (2013) Wang P, Luthin GR, and Ruggieri MR (1995) Muscarinic acetylcholine receptor Mucosa of human detrusor impairs contraction and b-adrenoceptor-mediated re- subtypes mediating urinary bladder contractility and coupling to GTP binding laxation. BJU Int 112:1215–1222. proteins. J Pharmacol Exp Ther 273:959–966. Rouget C, Rekik M, Camparo P, Botto H, Rischmann P, Lluel P, Palea S, and Westfall TD (2014) Modulation of nerve-evoked contractions by b3-adrenoceptor agonism in human and rat isolated urinary bladder. Pharmacol Res 80:14–20. Address correspondence to: Jerry Di Salvo, Evotec USA, 303B College Tallarida RJ (2001) Drug synergism: its detection and applications. J Pharmacol Exp Road East, Princeton, NJ 08540. E-mail: [email protected] Ther 298:865–872. Downloaded from jpet.aspetjournals.org at ASPET Journals on September 24, 2021