Exogenous glial cell line-derived neurotrophic factor (GDNF) exhibits potent survival-promoting effects on dopaminergic neurons of the nigrostriatal pathway that is implicated in Parkinson's disease and also protects neurons in forebrain ischemia of animal models. However, a role for endogenous GDNF in brain function has not been established. Although mice homozygous for a targeted deletion of the GDNF gene have been generated, these mice die within hours of birth because of deficits in kidney morphogenesis, and, thus, the effect of the absence of GDNF on brain function could not be studied. Herein, we sought to determine whether adult mice, heterozygous for a GDNF mutation on two different genetic backgrounds, demonstrate alterations in the nigrostriatal dopaminergic system or in cognitive function. While both neurochemical and behavioural measures suggested that reduction of GDNF gene expression in the mutant mice does not alter the nigrostriatal dopaminergic system, it led to a significant and selective impairment of performance in the spatial version of the Morris water maze. A standard panel of blood chemistry tests and basic pathological analyses did not reveal alterations in the mutants that could account for the observed performance deficit. These results suggest that endogenous GDNF may not be critical for the development and functioning of the nigrostriatal dopaminergic system but it plays an important role in cognitive abilities.

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We tested the hypothesis that the regular inhalation of a beta 2-adrenergic receptor (beta 2AR) agonist increases airway responsiveness in guinea pigs. A potent beta 2AR agonist, fenoterol hydrobromide, in sublaryngeal doses equivalent to maximal doses used in the treatment of asthma on a weight basis (5.28 micrograms/kg), was administered by nebulizer three times a day for 6 weeks to normal adolescent guinea pigs (FEN, n = 10) and to ovalbuminsensitized guinea pigs challenged twice weekly with ovalbumin (OA + FEN, n = 20), although not in the 12 h prior to or 4 h after antigen challenge. Controls included saline-treated normal animals (CON, n = 10) and ovalbumin-sensitized animals treated with repeated antigen challenge and saline (OA, n = 20). At 72 h after the last administration of saline, fenoterol, and ovalbumin, the dose-response relationship between pulmonary resistance (RL) and nebulized acetylcholine (ACh) was measured. RLmax increased 2-fold and the ACh concentration causing a 10-fold increase in RL (PC10) decreased 4-fold in the FEN, OA, and OA + FEN groups as compared to the CON group. In the FEN, OA, and OA + FEN groups, in vitro tracheal smooth-muscle contractile responses to maximal concentrations of acetylcholine increased 2-fold, and this increase was not due to increased smooth-muscle mass. There was no evidence for beta 2AR desensitization as judged by in vitro tracheal smooth-muscle relaxant responses to fenoterol. These results suggest that chronic beta 2AR stimulation increases airway smooth-muscle contractility and in vivo airways responsiveness to a degree similar to that induced by chronic antigen exposure. A similar effect in human asthmatics may explain the adverse effects observed during prolonged treatment with these drugs.

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).

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 [1,19-biphenyl]-2-ylcarbamate], a novel first-in-class inhaled bifunctional compound possessing both muscarinic antagonist (MA) and b 2 -adrenoceptor agonist (BA) properties (MABA). In competition radioligand binding studies at human recombinant receptors, GSK-961081 displayed high affinity for hM 2 (K i 5 1.4 nM), hM 3 muscarinic receptors (K i 5 1.3 nM) and hb 2 -adrenoceptors (K i 5 3.7 nM). GSK-961081 behaved as a potent hb 2 -adrenoceptor agonist (EC 50 5 0.29 nM for stimulation of cAMP levels) with 440-and 320-fold functional selectivity over hb 1 -and hb 3 -adrenoceptors, respectively. In guinea pig isolated tracheal tissues, GSK-961081 produced smooth muscle relaxation through MA (EC 50 5 50.2 nM), BA (EC 50 524.6 nM), and MABA (EC 50 5 11 nM) mechanisms. In the guinea pig bronchoprotection assay, inhaled GSK-961081 produced potent, dose-dependent inhibition of bronchoconstrictor responses via MA (ED 50 5 33.9 mg/ml), BA (ED 50 5 14.1 mg/ml), and MABA (ED 50 5 6.4 mg/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 b 2 -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.