The arginine-vasopressin (AVP) neuropeptide system is involved in complex social behaviors and emotional states across species. Within the brain, the vasopressin 1A receptor (V1AR) is the predominant subtype localized in circuits underlying emotion (e.g., anxiety, aggression) and social behaviors (e.g., pair bonding) with particularly high expression in the limbic system, hypothalamus, and brainstem. Given that social emotional processing deficits are present in many neurobehavioral disorders, modulation of the AVP system through the V1AR provides a tractable approach for the treatment of these disorders.

Revefenacin (TD-4208) is a novel, long-acting, and lung-selective muscarinic cholinergic receptor (mAChR) antagonist in development as a nebulized inhalation solution for the treatment of chronic obstructive pulmonary disease (COPD) patients. This study evaluated the pharmacology of revefenacin at human recombinant mAChRs and in airway tissues from rats, guinea pigs, and humans. At human recombinant mAChRs, revefenacin displayed high affinity (pK(I)=8.2-9.8) and behaved as a competitive antagonist (pKI, apparent=9.4-10.9) at the five human recombinant mAChRs. Kinetic studies demonstrated that revefenacin dissociated significantly slower from the hM(3) (t(1/2)=82minutes) compared to the hM(2) (t(1/2)=6.9minutes) mAChR at 37 degrees C, thereby making it kinetically selective for the former subtype. Similarly, in functional studies, revefenacin-mediated antagonism of acetylcholine (ACh)-evoked calcium mobilization responses were reversed less rapidly at hM(3) compared to the hM(2) mAChR. In isolated tracheal tissues from rat and guinea pig and isolated bronchial tissues from humans, revefenacin potently antagonized mAChR-mediated contractile responses. Furthermore, the antagonistic effects of revefenacin in rat, guinea pig, and human airway tissues were slowly reversible (t(1/2) of 13.3, >16, and >10hours, respectively). These data demonstrate that revefenacin is a potent, high affinity, and selective functional mAChR antagonist with kinetic selectivity for the hM(3) receptor and produces potent and long-lasting antagonism of mAChR-mediated contractile responses in rat, guinea pig, and human airway tissue. These data suggest that revefenacin has the potential to be a potent once-daily dosed inhaled bronchodilator in COPD patients.

A series of novel carbazole-containing amides and ureas were synthesized. A structure-activity relationship study of these compounds led to the identification of potent cryptochrome modulators. Based on the desired pharmacokinetic/pharmacodynamic parameters and the results of efficacy studies in db/db mice, compound 50 was selected for further profiling.

Through application of our multivalent approach to drug discovery we previously reported the first discovery of dual pharmacology MABA bronchodilators, exemplified by 1. Herein we describe the subsequent lead optimization of both muscarinic antagonist and β2 agonist activities, through modification of the linker motif, to achieve 24 h duration of action in a guinea pig bronchoprotection model. Concomitantly we targeted high lung selectivities, low systemic exposures and identified crystalline forms suitable for inhalation devices. This article culminates with the discovery of our first clinical candidate 12f (TD-5959, GSK961081, batefenterol). In a phase 2b trial, batefenterol produced statistical and clinically significant differences compared to placebo and numerically greater improvements in the primary end point of trough FEV1 compared to salmeterol after 4 weeks of dosing in patients with moderate to severe chronic obstructive pulmonary disease (COPD).

A series of novel carbazole-containing sulfonamides and sulfamides were synthesized. A structure–activity relationship study of these compounds led to the identification of potent cryptochrome modulators. Based on the results of efficacy studies in diet-induced obese (DIO) mice, and the desired pharmacokinetic parameters, compound 41 was selected for further profiling.

The objective of the present studies was to characterize the pharmacologic properties of GSK-961081 [TD-5959; (R)-1-(3-((2-chloro-4-(((2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethyl)amino)methyl)-5-methoxyphenyl)amino)-3-oxopropyl) piperidin-4-yl [1,1′-biphenyl]-2-ylcarbamate], a novel first-in-class inhaled bifunctional compound possessing both muscarinic antagonist (MA) and β2-adrenoceptor agonist (BA) properties (MABA). In competition radioligand binding studies at human recombinant receptors, GSK-961081 displayed high affinity for hM2 (Ki = 1.4 nM), hM3 muscarinic receptors (Ki = 1.3 nM) and hβ2-adrenoceptors (Ki = 3.7 nM). GSK-961081 behaved as a potent hβ2-adrenoceptor agonist (EC50 = 0.29 nM for stimulation of cAMP levels) with 440- and 320-fold functional selectivity over hβ1- and hβ3-adrenoceptors, respectively. In guinea pig isolated tracheal tissues, GSK-961081 produced smooth muscle relaxation through MA (EC50 = 50.2 nM), BA (EC50=24.6 nM), and MABA (EC50 = 11 nM) mechanisms. In the guinea pig bronchoprotection assay, inhaled GSK-961081 produced potent, dose-dependent inhibition of bronchoconstrictor responses via MA (ED50 = 33.9 µg/ml), BA (ED50 = 14.1 µg/ml), and MABA (ED50 = 6.4 µg/ml) mechanisms. Significant bronchoprotective effects of GSK-961081 were evident in guinea pigs via MA, BA, and MABA mechanisms for up to 7 days after dosing. The lung selectivity index of GSK-961081 in guinea pigs was 55- to 110-fold greater than that of tiotropium with respect to systemic antimuscarinic antisialagogue effects and was 10-fold greater than that of salmeterol with respect to systemic β2-adrenoceptor hypotensive effects. These preclinical findings studies suggest that GSK-961081 has the potential to be a promising next-generation inhaled lung-selective bronchodilator for the treatment of airway diseases, including chronic obstructive pulmonary disease.

Lowering of intra-ocular pressure is the primary pharmacologic approach for the treatment of glaucoma and a number of distinct mechanisms of action have been clinically validated. Targeting of multiple mechanisms in combination therapies has proven effective both clinically and commercially although potential improvements with regards to efficacy, tolerability and dosing frequency remain. Application of Theravance’s multivalent approach to drug discovery towards linked dual-pharmacology prostaglandin F receptor (FP) agonist/carbonic anhydrase (CA)-II inhibitor compounds is described. Compound 29 exhibits weak potency (pEC50=5.7, IA>1.0) as an FP agonist with high binding affinity (pKi=8.1) to the CA-II enzyme, and has comparable corneal permeability to the CA-II inhibitor dorzolamide.

Combinations of a muscarinic receptor antagonist (MA) and a β(2)-adrenoceptor agonist (BA) improve bronchodilation in COPD patients to a greater extent than drugs with either mechanism alone. Here, using an in vivo model of bronchoprotection in guinea pigs, we characterize a single agent with dual-acting MA and BA activity, THRX-200495 (MABA). THRX-200495 was compared to a fixed-dose combination of a short-acting muscarinic receptor antagonist (SAMA) and a β(2)-adrenoceptor agonist (SABA). The SAMA/SABA combination consisted of a 1:5.7 ratio of ipratropium and albuterol (the components of Combivent®). Conscious guinea pigs received aqueous nebulized solutions of vehicle or test compound by aerosol exposure. Bronchoprotective potency was estimated in anesthetized, tracheotomized and ventilated guinea pigs at predetermined time points after aerosol exposure by measuring changes in ventilation pressure. The individual (MA, BA) and composite (MABA) pharmacologies were assessed by determining protection against bronchoconstrictor responses induced by methacholine in the presence of propranolol (for MA activity), histamine (for BA activity) or methacholine (MABA activity). Bronchoprotection was calculated as percent inhibition of methacholine or histamine response relative to the vehicle group. THRX-200495 exhibited matched MA (ID(50) = 11.4 μg/mL) and BA (ID(50) = 11.2 μg/mL) potency and potent dual pharmacology (MABA ID(50) = 3.5 μg/mL) that persisted for over 24 h. The combination of ipratropium/albuterol exhibited bronchoprotective activity that was 2.6-fold more potent as a BA (ID(50) = 5.7 μg/mL) than as an MA (ID(50) = 14.6 μg/mL) at 0.5 h post-dose and 37-fold more potent as an MA (ID(50) = 4.3 μg/mL) than a BA (ID(50) = 159 μg/mL) at 1.5 h post aerosol exposure. Under MABA pharmacological conditions, ipratropium/albuterol produced potent bronchoprotective activity (ID(50) = 2.0/11.4 μg/mL) and an apparent additive effect of the two pharmacologies. In conclusion, a dual-acting prototypical MABA, THRX-200495, demonstrated potent, balanced and long-lasting bronchodilation in a guinea pig model of bronchoprotection that was greater than either the MA or BA mechanisms alone.

Inhaled beta-2-adrenergic receptor (β2) agonists and inhaled muscarinic acetylcholine antagonists are the most frequently used bronchodilators in the treatment of chronic obstructive pulmonary disease. While short-acting agents (4–6 h) serve as ‘rescue’ therapy, long-acting (12–24 h) bronchodilators can reduce the incidence and number of exacerbations as well as improve lung function. Due to the complementary nature of the two mechanisms, combinations of the two classes provide even greater improvement in lung function than either mechanism alone. This review focuses on the multivalent origins of dual pharmacology MABA bronchodilators and describes the supporting in vitro characterization and reported animal studies. Topics discussed include approaches taken to identify novel MABA molecules, highlighting preferred muscarinic and β2-binding groups and how these two entities are linked together; challenges in designing MABA molecules; and the potential benefits of enhanced bronchoprotection and opportunity for ‘triple therapy’ with an inhaled corticosteroid.

We sought to design dual pharmacology bronchodilators targeting both the M(3) muscarinic acetylcholine and beta-2 adrenergic (β(2)) receptors by applying our multivalent approach to drug discovery. Herein, we describe our initial discovery and the SAR of the first such compounds with matched potencies at both receptors.

Biphenyl-2-yl-carbamic acid 1-{9-[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydro-quinolin-5-yl)-ethylamino]-nonyl}-piperidin-4-yl ester (THRX-198321) is a single molecule composed of a muscarinic acetylcholine receptor (mAChR) antagonist moiety, represented by the fragment MA, linked by a C9 polymethylene chain to a β(2)-adrenoceptor (β(2)AR) agonist moiety, represented by the fragment 8-hydroxy-5-((R)-1-hydroxy-2-methylamino-ethyl)-1H-quinolin-2-one (BA). THRX-198321 exhibited high affinity for mAChR (M(2) pK(I,App) = 10.57 ± 0.09; M(3) pK(I,App) = 10.07 ± 0.11) and β(2)AR (pK(I,App) = 9.54 ± 0.15), with potent mAChR antagonist (M(2) pK(I,Fn) = 9.69 ± 0.23; M(3) pK(I,Fn) = 10.05 ± 0.17) and β(2)AR agonist (pEC(50) = 9.25 ± 0.02) activities. Consistent with multivalent interactions, THRX-198321 binding affinity was >300-fold higher at mAChR and 29-fold higher at β(2)AR relative to its monovalent fragments biphenyl carbamic acid piperidinyl ester (MA) and BA, respectively. THRX-198321 was a competitive antagonist at mAChR (M(2) pK(B) = 9.98 ± 0.13; M(3) pK(B) = 10.31 ± 0.89), whereas THRX-198321 agonist activity at β(2)AR was competitively inhibited by propranolol. Interactions of THRX-198321 with an allosteric site on mAChR and a novel extracellular allosteric site on β(2)AR, respectively, were detected by measuring THRX-198321-evoked changes in the dissociation rates for the orthosteric radioligands, [N-methyl-(3)H]scopolamine methyl chloride (M(2) pEC(50,diss) = 6.73 ± 0.10; M(3) pEC(50,diss) = 5.02 ± 0.14) and [4,6-propyl-(3)H]dihydroalprenolol (β(2)AR pEC(50,diss) = 3.82 ± 0.25). The carbostyril-linker fragment (BA-L) binds to the allosteric site of mAChR (M(2) pEC(50,diss) = 5.06 ± 0.03; M(3) pEC(50,diss) = 4.15 ± 0.25), whereas the MA fragment binds to the allosteric site of β(2)AR (pEC(50,diss) = 3.60 ± 0.18). Collectively, these observations suggest that THRX-198321 exhibits a multivalent bimodal orientation in the orthosteric and allosteric binding pockets of mAChR and β(2)AR, a phenomenon that may be unique to this class of molecule.

β-Adrenoceptors are important mediators of smooth muscle relaxation in the urinary bladder, but the concomitant presence of a muscarinic agonist, e.g., carbachol, can attenuate relaxation responses by reducing potency and/or efficacy of β-adrenoceptor agonists such as isoprenaline. Therefore, the present study was designed to explore the subtypes and signalling pathways of muscarinic receptors involved in the attenuation of isoprenaline-induced isolated rat detrusor preparations using novel subtype-selective receptor ligands. In radioligand binding studies, we characterized BZI to be a M(3)-sparing muscarinic agonist, providing selective M(2) stimulation in rat bladder, and THRX-182087 as a highly M(2)-selective antagonist. The use of BZI and of THRX-182087 in the presence of carbachol enabled experimental conditions with a selective stimulation of only M(2) or M(3) receptors, respectively. Confirming previous findings, carbachol attenuated isoprenaline-induced detrusor relaxation. M(2)-selective stimulation partly mimicked this attenuation, indicating that both M(2) and M(3) receptors are involved. During M(3)-selective stimulation, the attenuation of isoprenaline responses was reduced by the phospholipase C inhibitor U 73,122 but not by the protein kinase C inhibitor chelerythrine. We conclude that both M(2) and M(3) receptors contribute to attenuation of β-adrenoceptor-mediated relaxation of rat urinary bladder; the signal transduction pathway involved in the M(3) component of this attenuation differs from that mediating direct contractile effects of M(3) receptors.

Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables drug discovery by facilitating the iterative process of screening and optimization of chemical leads. This unit describes isolated tissue functional assays for the quantification of ligand affinity and efficacy at the M 1 , M 2 , M 3 , M 4 , and M 5 muscarinic receptor subtypes in tissues expressing the native receptor using organ bath techniques. Curr. Protoc. Pharmacol . 48:4.15.1‐4.15.29. © 2010 by John Wiley & Sons, Inc.

M(2) and M(3) muscarinic receptor subtypes are attractive drug targets for the treatment of pulmonary and urological disorders. Described in this unit is an in vivo pithed rat assay for estimating agonist and antagonist potency at M(2) and M(3) receptors. In the pithed rat, the muscarinic agonist methacholine induces reduction in heart rate (bradycardia) and blood pressure (depressor response) through interaction with M(2) and M(3) receptors, respectively. The dissociation of the peripheral and central nervous system in the pithed rat preparation permits the direct assessment of compound effects on the heart and vasculature in the absence of cardiovascular reflexes. Estimates of antagonist potency can be reliably established by quantifying the shift in the agonist dose-effect curve produced under appropriate equilibrium or non-equilibrium conditions.

G-protein-coupled muscarinic receptors (mAChRs), of which there are five subtypes (M(1)-M(5)), are attractive drug targets for a number of disorders. Described in this unit are radioligand-binding assays for defining the selectivity and affinity of chemical agents at the five mAChR subtypes. Detailed methodologies and troubleshooting strategies are provided for saturation-binding studies, to estimate K(D) and B(max) values, and for competition-binding studies to estimate K(i) values. Emphasis is placed on experimental details that are critical for executing a robust and reliable assay.

Existing antimuscarinic drugs for overactive bladder have high affinity for M3/M1 muscarinic receptors and consequently produce M3/M1-mediated adverse effects including dry mouth, constipation, mydriasis and somnolence. TD-6301 is a M2/4 muscarinic receptor-selective antagonist developed for the treatment of overactive bladder. The present studies characterize the in vitro and in vivo pharmacological properties of this 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, 2 and 128-fold selective for the human M2 muscarinic receptor compared to the M1, M3, M4 and M5 muscarinic receptors, respectively. The in vivo bladder selectivity of TD-6301 in rats was determined to be 26, 28, > 100, 16 and 0.4-fold, respectively, assessed by comparing its potency for inhibition of volume-induced bladder contractions 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 more potent 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 that observed for tolterodine, oxybutynin, darifenacin and solifenacin. The preclinical properties of TD-6301 suggest that this molecule is likely to be efficacious in overactive bladder patients with a lower propensity to cause M3 muscarinic receptor mediated adverse effects.