Mark R. Dowling

In Vitro and in Vivo Pharmacological Characterization of 5-[(R)-2-(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (Indacaterol), a Novel Inhaled β2 Adrenoceptor Agonist with a 24-h Duration of Action

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled β2 adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human β2 adrenoceptor (Emax = 73 ± 1% of the maximal effect of isoprenaline; pEC50 = 8.06 ± 0.02), whereas salmeterol displays only partial efficacy (38 ± 1%). The functional selectivity profile of indacaterol over β1 human adrenoceptors is similar to that of formoterol, whereas its β3 adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 ± 4 min) similar to formoterol and salbutamol, and a long duration of action (529 ± 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled β2 adrenoceptor agonists.

Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor

1 Slow receptor dissociation kinetics has been implicated in the long clinical duration of action of the muscarinic receptor antagonist tiotropium. However, despite the potential benefits of new drugs with slow dissociation kinetics, the rate parameters of new compounds are seldom measured due to technical difficulties and financial implications associated with radiolabeling multiple ligands. Here we describe the development and optimisation of a medium throughput assay which is capable of measuring the kinetic parameters of novel, unlabelled compounds. 2 Radioligand binding studies were performed with membranes derived from CHO cells recombinantly expressing the human M3 muscarinic receptor. 3 Initial characterisation of the radioligand [3H]‐NMS yielded on and off rates of 4.1±0.2 × 108 M−1 min−1 and 0.015±0.0005 min−1, respectively. 4 The specific binding of [3H]‐NMS was measured over time in the presence and absence of several concentrations of unlabelled competitor compounds. These data were analysed using a competition kinetic model to provide on and off rates for the unlabelled competitor. Comparison of the kinetically derived Kd (koff/kon) with Ki values generated at equilibrium showed an excellent correlation (r2=0.99), providing good validation of the method. 5 The on and off rates were also used in theoretical computer simulations to successfully predict the effect of incubation time on apparent IC50 values. 6 This study demonstrates that a medium‐throughput competition kinetic binding assay can be used to determine accurate on and off rates of unlabelled compounds, providing the opportunity to optimise for kinetic parameters early in the drug discovery process.

Exploring the mechanism of agonist efficacy: a relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor.

Although there are several empirical approaches that enable the comparison of relative agonist efficacy, the molecular basis that underlies differences in the ability of G protein-coupled receptor agonists to elicit a response is still largely unexplained. Several models have been described that incorporate the kinetics of receptor-mediated initiation of the G protein cycle, but these have not directly addressed the influence of agonist-binding kinetics. To test this, we investigated the relationship between the efficacy of seven M3 muscarinic receptor agonists and their rate of dissociation (koff) from the M3 receptor. The association and dissociation rate constants of the agonists were determined using a l-[N-methyl]-[3H]scopolamine methyl chloride competition binding assay in the presence of GTP. The agonists displayed a range of association and dissociation rates. Relative agonist efficacy was measured at two points after M3 receptor activation: the stimulation of guanosine 5′-O-(3-[35S]thio)triphosphate binding to Gα subunits, and the subsequent increase in intracellular calcium levels. These experiments revealed a range of intrinsic efficacy, from the low-efficacy pilocarpine and oxotremorine to high-efficacy acetylcholine. There was no relationship between agonist efficacy and the equilibrium binding affinity of each agonist (Kd). When efficacy was compared with the dissociation rate constant, however, the two were highly correlated, suggesting a relationship between the duration of agonist binding at the receptor and the intrinsic efficacy. These data suggest that kinetic models incorporating the mean lifetime of specific complexes will be required to fully explain the nature of agonist efficacy.

The Influence of Receptor Kinetics on the Onset and Duration of Action and the Therapeutic Index of NVA237 and Tiotropium

Studies under nonphysiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. Our aim was to determine how clinically relevant dissociation rates under more physiological conditions influence the differences in onset of action between tiotropium and 3-[(cyclopentylhydroxyphenylacetyl oxy]-1,1-dimethyl-pyrrolidinium bromide (NVA237), a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease. In addition, we have investigated kinetic selectivity at each of the muscarinic receptor subtypes to determine whether the improved cardiovascular therapeutic index obtained with NVA237 in animal models is attributable to differences in kinetic rate constants. The binding of radioligand [3H]N-methyl-scopolamine was measured in the presence/absence of several concentrations of unlabeled competitors, and data were analyzed using a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus nonphysiological conditions (173 and 462 min, respectively). NVA237 had a more rapid onset of action (3–4.8 times) versus tiotropium, determined in an vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had greater equilibrium binding and kinetic selectivity for muscarinic type 3 (M3) versus muscarinic type 2 (M2) receptors, with a faster off rate from M2 versus M3 receptors than tiotropium, potentially affording it a more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.

Investigating the molecular mechanisms through which FTY720-P causes persistent S1P1 receptor internalization.

The molecular mechanism underlying the clinical efficacy of FTY720‐P is thought to involve persistent internalization and enhanced degradation of the S1P1 receptor subtype (S1P1R). We have investigated whether receptor binding kinetics and β‐arrestin recruitment could play a role in the persistent internalization of the S1P1R by FTY720‐P.

The pharmacological rationale for combining muscarinic receptor antagonists and β-adrenoceptor agonists in the treatment of airway and bladder disease

Highlights • Muscarinic receptors increase smooth muscle tone in airways and urinary bladder.• β-Adrenoceptors relax smooth muscle tone and oppose muscarinic contraction.• Opposition involves transmitter release, signal transduction and receptor expression.• This supports the combined use of muscarinic antagonists and β-adrenoceptor agonists.

A single point mutation (N514Y) in the human M3 muscarinic acetylcholine receptor reveals differences in the properties of antagonists: evidence for differential inverse agonism.

A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (∼300%) in agonist-independent [3H]inositol phosphate ([3H]IPx) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M3 and N514YM3 mACh receptor-mediated basal [3H]IPx accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the N514YM3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate N514YM3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the N514YM3 mACh receptor was approximately 100% greater than that seen for the WT-M3 receptor. The ability of antagonists to decrease basal N514YM3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [3H]IPx accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

Measuring Receptor Target Coverage: A Radioligand Competition Binding Protocol for Assessing the Association and Dissociation Rates of Unlabeled Compounds

The kinetics of the ligand‐receptor interaction is an important feature in lead optimization for new drug candidates. The protocol described in this unit is a kinetic radioligand competition binding assay that makes possible the determination of both the association and dissociation rates of unlabeled receptor ligands. Curr. Protoc. Pharmacol. 50:9.14.1‐9.14.30.

Selective inhibition of histamine-evoked Ca2+ signals by compartmentalized cAMP in human bronchial airway smooth muscle cells

Graphical abstract

Endogenous lysophosphatidic acid (LPA1 ) receptor agonists demonstrate ligand bias between calcium and ERK signalling pathways in human lung fibroblasts.

Human lung fibroblasts (HLF) express high levels of the LPA1 receptor, a GPCR that responds to the endogenous lipid mediator, lysophosphatidic acid (LPA). Several molecular species or analogues of LPA exist and have been detected in biological fluids such as serum and plasma. The most widely expressed of the LPA receptor family is the LPA1 receptor, which predominantly couples to Gq/11, Gi/o and G12/13 proteins. This promiscuity of coupling raises the possibility that some of the LPA analogues may bias the LPA1 receptor towards one signalling pathway over another.