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 2 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]-8hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled 2 adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human 2 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 1 human adrenoceptors is similar to that of formoterol, whereas its 3 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 2 adrenoceptor agonists. Agents that act as agonists of the 2 adrenoceptor are effective in the management of asthma and chronic obstructive pulmonary disease (COPD), primarily through their bronchodilatating properties. These drugs induce bronchodilatation by causing direct relaxation of airway smooth muscle through activation of adenylate cyclase, which in turn increases intracellular cAMP levels. Salbutamol is an inhaled 2 adrenoceptor agonist that provides rapid bronchodilatation and has been widely used over the past 30 years. However, its major drawback is its short duration of action (4–6 h), requiring the drug to be administered several times a day. Two longer acting inhaled 2 adrenoceptor agonists, formoterol and salmeterol, are now available and are used in the management of asthma and COPD (Sutherland, 2004). These two drugs have a bronchodilating effect lasting for 12 h after a single inhalation and are therefore given twice daily. Despite the decrease in dosing frequency with the longer acting inhaled 2 adrenoceptor agonists, patient compliance is still an issue (Ying et al., 1999). In addition, the recent launch of tiotropium bromide, a once-daily inhaled muscarinic antagonist for the treatment of COPD (Gross, 2004), and the development of once-daily inhaled corticosteroids for the treatment of asthma (Dent, 2002; Wardlaw et al., 2004), would suggest that a new inhaled 2 adrenoceptor agonist with a duration of action compatible with once-daily administration is likely to become the future bronchodilator of choice, either on its own or when used with a once-daily muscarinic antagonist in COPD and when used with a once-daily inhaled corticosteroid for the treatment of asthma. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.105.098251. ABBREVIATIONS: COPD, chronic obstructive pulmonary disease; 5-HT, 5-hydroxytryptamine. 0022-3565/06/3172-762–770$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 317, No. 2 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 98251/3101434 JPET 317:762–770, 2006 Printed in U.S.A. 762 at A PE T Jornals on M ay 6, 2017 jpet.asjournals.org D ow nladed from Systemic exposure to 2 adrenoceptor agonists results in predictable adverse events such as tremor, palpitation, headache, muscle cramps, and hypokalemia. These adverse events are minimized when these drugs are given by inhalation; salbutamol, salmeterol, and formoterol are perceived to be safe at the recommended clinical doses. However, the therapeutic margin of these drugs is rather small, such that doubling the dose of, e.g., salmeterol and formoterol, can result in a significant increase in the incidence of side effects (Guhan et al., 2000; Sovani et al., 2004). Furthermore, such undesirable side effects have been linked to reduced compliance to treatment (White and Sander, 1999). Therefore, a higher therapeutic margin would be desirable for the new generation of inhaled 2 adrenoceptor agonists. 5-[(R)-2-(5,6-Diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8hydroxy-1H-quinolin-2-one (indacaterol) (Fig. 1), previously known as QAB149, is a novel, chirally pure inhaled 2 adrenoceptor agonist that was discovered in a program to identify compounds with a duration of action compatible with oncedaily dosing in human, combined with a fast onset of action and an increased therapeutic index compared with the available inhaled 2 adrenoceptor agonists. Here, we report the preclinical in vitro and in vivo pharmacological characteristics of indacaterol and compare the properties of this compound to those of the marketed inhaled 2 adrenoceptor agonists salbutamol, formoterol, and salmeterol (Fig. 1). Materials and Methods Materials. Indacaterol maleate or hydrochlorate and formoterol fumarate were synthesized by the Department of Chemistry (Novartis, Horsham, UK). Salbutamol hemisulfate and isoprenaline were purchased from Sigma Chemical (Poole, Dorset, UK). Salmeterol was synthesized or isolated from clinical dosage forms by the Department of Chemistry (Novartis) or purchased from Tocris Cookson Inc. (Bristol, UK). The xinafoate salt form for salmeterol was isolated from clinical dosage forms or prepared by the Department of Chemistry (Novartis). In Vitro Cell-Based Assays. Chinese hamster ovary cells stably transfected with human adrenoceptors ( 1, 2, or 3; Novartis) were grown adherently and maintained in Ham’s F-12 Nutrient Mix GlutaMAX-1, containing 10% fetal calf serum, 2 mM L-glutamine, and 0.5 mg/ml Geneticin (G-418) at 37°C in 5% CO2. Cells were subcultured at a ratio between 1:10 and 1:20 twice weekly. Membrane preparations were made when cells were between 70 and 90% confluence. Cells were suspended in buffer (10 mM HEPES and 10 mM EDTA, pH 7.4) at 4°C and disrupted with a homogenizer (5 times 5-s bursts with an IKA T8 Ultra Turrax using a 1to 50-ml S8N-8G dispersing element; IKA Labortechnik, Staufen, Germany). The homogenate was then centrifuged at 48,000g for 25 min. The resulting pellet was resuspended in buffer at 4°C, rehomogenized as described above, and centrifuged at 48,000g for a further 25 min. The resulting pellet was resuspended in buffer (10 mM HEPES and 0.1 mM EDTA, pH 7.4), and the protein was determined by Bio-Rad protein assay (Bio-Rad, Hercules, CA). [I]Iodo-( )-cyanopindolol (PerkinElmer Life and Analytical Sciences, Boston, MA) binding assays were performed with membrane preparations (10 g of protein/well) in a final assay volume of 250 l in a Hanks’ buffered saline solution containing 0.1% bovine serum albumin, pH 7.4 (assay buffer), in the presence of 100 M guanosine 5 -triphosphate to ensure monophasic binding curves. For saturation binding experiments, up to 600 pM [I]iodo-( )-cyanopindolol was used for 1 and 2 and up to 1600 pM for 3. Nonspecific binding was determined in the presence of 10 M alprenolol. For competition binding, [I]iodo-( )-cyanopindolol was used at 10 pM for 1 and 2 and 120 pM for 3. Membranes were incubated for 3 h at 22°C, filtered through Millipore GF/B filters [containing 0.5% (w/v) polyethylenimine per well] using ice-cold wash buffer (75 mM Tris; pH