Vivo Profile of Aclidinium Bromide in Comparison With
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YPUPT1381_proof ■ 7 June 2014 ■ 1/8 Pulmonary Pharmacology & Therapeutics xxx (2014) 1e8 55 Contents lists available at ScienceDirect 56 57 Pulmonary Pharmacology & Therapeutics 58 59 60 journal homepage: www.elsevier.com/locate/ypupt 61 62 63 64 65 1 The in vitro and in vivo profile of aclidinium bromide in comparison 66 2 67 3 with glycopyrronium bromide 68 4 69 * 5 Q4 Amadeu Gavalda , Israel Ramos, Carla Carcasona, Elena Calama, Raquel Otal, 70 6 Jose Luis Montero, Sonia Sentellas, Monica Aparici, Dolors Vilella, Joan Alberti, 71 7 Jorge Beleta, Montserrat Miralpeix 72 8 73 9 Almirall R&D Centre, Laurea Miro, 408-410, 08980 Sant Feliu de Llobregat, Barcelona, Spain 74 10 75 11 76 12 article info abstract 77 13 78 14 Article history: This study characterised the in vitro and in vivo profiles of two novel long-acting muscarinic antagonists, 79 Received 8 January 2014 15 aclidinium bromide and glycopyrronium bromide, using tiotropium bromide and ipratropium bromide 80 Received in revised form 16 as comparators. All four antagonists had high affinity for the five muscarinic receptor sub-types (M1 23 May 2014 81 eM ); aclidinium had comparable affinity to tiotropium but higher affinity than glycopyrronium and 17 Accepted 24 May 2014 5 82 ipratropium for all receptors. Glycopyrronium dissociated faster from recombinant M receptors than 18 Available online xxx 3 83 aclidinium and tiotropium but more slowly than ipratropium; all four compounds dissociated more 19 84 rapidly from M2 receptors than from M3 receptors. In vitro, aclidinium, glycopyrronium and tiotropium 20 Q5 Chemical compounds studied in this article: had a long duration of action at native M receptors (>8 h versus 42 min for ipratropium). In vivo, all 85 21 Aclidinium bromide (PubChem CID: 3 compounds were equi-potent at reversing acetylcholine-induced bronchoconstriction. Aclidinium, gly- 86 22 11519741) Glycopyrronium bromide (PubChem CID: copyrronium and ipratropium had a faster onset of bronchodilator action than tiotropium. Aclidinium 87 23 11693) had a longer duration of action than glycopyronnium (time to 50% recovery of effect [t½ offset] ¼ 29 h and 88 24 Tiotropium bromide (PubChem CID: 13 h, respectively); these compare with a t½ offset of 64 h and 8 h for tiotropium and ipratropium, 89 25 5487426) respectively. Aclidinium was less potent than glycopyrronium and tiotropium at inhibiting salivation in 90 Ipratropium bromide (PubChem CID: 26 conscious rats (dose required to produce half-maximal effect [ED50] ¼ 38, 0.74 and 0.88 mg/kg, respec- 31098) 91 27 tively) and was more rapidly hydrolysed in rat, guinea pig and human plasma compared with glyco- 92 pyrronium or tiotropium. These results indicate that while aclidinium and glycopyrronium are both 28 Keywords: 93 29 Aclidinium potent antagonists at muscarinic receptors with similar kinetic selectivity for M3 receptors versus M2, COPD 94 30 aclidinium has a longer dissociation half-life at M3 receptors and a longer duration of bronchodilator Glycopyrronium action in vivo than glycopyrronium. The rapid plasma hydrolysis of aclidinium, coupled to its kinetic 95 31 Long-acting muscarinic antagonists selectivity, may confer a reduced propensity for systemic anticholinergic side effects with aclidinium 96 32 Muscarinic receptors versus glycopyrronium and tiotropium. 97 Preclinical 33 © 2014 Published by Elsevier Ltd. 98 34 99 35 100 36 101 37 102 38 1. Introduction 103 39 104 40 Abbreviations: COPD, chronic obstructive pulmonary disease; EC50, concentra- Chronic obstructive pulmonary disease (COPD) is characterised 105 41 tion required to produce 50% effect; ED50, dose required to produce 50% effect; Kd, by persistent airflow limitation, and an enhanced chronic inflam- 106 42 equilibrium dissociation constant; Ki, antagonist dissociation constant; LAMA, long- e e 3 matory response in airways and lung to noxious particles or gases 107 43 acting muscarinic antagonist; M1 M5, muscarinic receptor subtypes 1 5; [ H]- NMS, 1-[N-methyl-3H] scopolamine methyl chloride; Raw, airway resistance; SAMA, [1]. Characteristic symptoms of COPD include airway limitation and 108 44 short-acting muscarinic antagonist; t½, dissociation half-life/hydrolysis half-life; t½ chronic coughing due to mucus hypersecretion [1]. Acetylcholine is 109 45 offset, time to 50% recovery of effect; tmax, time to maximal effect. the primary parasympathetic neurotransmitter in the airways [2] 110 46 * Corresponding author. Tel.: þ34 93 291 3818; fax: þ34 93 291 3465. 111 and plays an important role in regulating both airway smooth 47 E-mail addresses: [email protected] (A. Gavalda), israel.ramos@ muscle tone [3] and mucus secretion [4,5] via stimulation of airway 112 48 almirall.com (I. Ramos), [email protected] (C. Carcasona), elena. [email protected] (E. Calama), [email protected] (R. Otal), jose.luis. muscarinic receptors. The primary reversible component of airway 113 49 [email protected] (J.L. Montero), [email protected] (S. Sentellas), limitation is sensitive to muscarinic receptor antagonists [2,6].Of 114 50 [email protected] (M. Aparici), [email protected] (D. Vilella), the five muscarinic receptors identified to date (M1eM5), only the 115 51 [email protected] (J. Alberti), [email protected] (J. Beleta), e 116 [email protected] (M. Miralpeix). M1 M3 subtypes are found in the airways [7]. The M3 receptor 52 117 53 http://dx.doi.org/10.1016/j.pupt.2014.05.005 118 54 1094-5539/© 2014 Published by Elsevier Ltd. 119 Please cite this article in press as: Gavalda A, et al., The in vitro and in vivo profile of aclidinium bromide in comparison with glycopyrronium bromide, Pulmonary Pharmacology & Therapeutics (2014), http://dx.doi.org/10.1016/j.pupt.2014.05.005 YPUPT1381_proof ■ 7 June 2014 ■ 2/8 2 A. Gavalda et al. / Pulmonary Pharmacology & Therapeutics xxx (2014) 1e8 1 mediates acetylcholine-induced contraction of airway smooth (3.11 TBq/mmol) and [3H]ipratropium (2.70 TBq/mmol) were 66 2 muscle [8,9], and stimulation of M1 and M3 receptors on submu- custom synthesised by GE Healthcare UK Ltd (Slough, UK). 67 3 cosal mucus glands promotes mucus secretions in airways [5,10].By All equilibrium binding studies were performed in 96-well 68 4 contrast, M2 receptors are presynaptic autoreceptors which serve plates (NUNC; Thermo Fischer Scientific, Roskilde, Denmark). All 69 5 as a negative feedback mechanism to modulate acetylcholine assay reagents were dissolved in assay buffer (TRIS 25 mM pH: 7.4) 70 6 release from parasympathetic nerves [7]. [SigmaeAldrich, Tres Cantos, Spain]) and test compounds were 71 7 As a consequence of the central role of muscarinic receptors in dissolved in dimethyl sulfoxide. Aclidinium was prepared in 0.2% 72 8 mediating the underlying pathophysiology of COPD, anticholiner- HCl/20% polyethylene glycol for use in in vitro organ bath experi- 73 9 gics, specifically muscarinic receptor antagonists, are recom- ments and in vivo studies; carbachol, ipratropium, glycopyrronium 74 10 mended as a first-line bronchodilator treatment option in patients and tiotropium were dissolved in distilled water. Krebs-Henseleit 75 11 with COPD [1,11]. Short-acting muscarinic antagonists (SAMAs), solution was composed of: NaCl 118 nM, KCl 4.7 nM, CaCl2 76 12 such as ipratropium bromide, are recommended for use in Group-A 2.52 nM, MgSO4 1.2 nM, NaHCO3 24.9 nM, KH2PO4 1.18 nM, glucose 77 13 patients who are characterised as having few symptoms and a low 5.55 nM and sodium pyruvate 2 nM. In plasma stability studies, 78 14 risk of exacerbation [1]. By contrast, long-acting muscarinic an- stock solutions (1 mg/mL) of aclidinium, glycopyrronium, tio- 79 15 tagonists (LAMAs), such as aclidinium bromide, glycopyrronium tropium and ipratropium were prepared in 20:80, v/v 0.1 N HCl/ 80 16 bromide and tiotropium bromide, are preferred for maintenance acetonitrile; working solutions were dissolved in Milli-Q water. Rat 81 17 treatment in patients with more severe airflow limitation, more plasma was obtained from RCC Cida (Barcelona, Spain). 82 18 symptoms or a higher risk of exacerbation (Groups CeD) [1]. 83 19 However, ipratropium and tiotropium, which have been available 2.2. Animals 84 20 for many years, are associated with systemic side effects typical of 85 21 the anticholinergic class of compounds, such as dry mouth [12,13] Male Dunkin-Hartley guinea pigs (400e600 g) were obtained 86 22 and an increased risk of cardiovascular side effects [14e16]. from Harlan (Interfauna Iberica, Sant Feliu de Codines, Spain). 87 23 In 2012, two new LAMAs, aclidinium and glycopyrronium, were Guinea pigs were housed in groups of four or five, at 20e24 C 88 24 approved in Europe for maintenance bronchodilator treatment in under a 12-h light/dark cycle and fed a maintenance diet for guinea 89 25 adult patients with COPD [17,18]; aclidinium has also been pigs, supplemented with vitamin C (SAFE114, SAFE, France); water 90 26 approved in the US [19]. In preclinical studies, both aclidinium [20] was ad libitum. Guinea pigs were allowed to acclimatise for a 91 27 and glycopyrronium [21] had high affinity for all five muscarinic minimum of 5 days prior to experimental procedures. Male Wistar 92 28 receptors. Aclidinium was also shown to be rapidly hydrolysed in rats (180e260 g) were also obtained from Harlan. Rats were housed 93 29 human plasma to two inactive metabolites [22], suggesting a at 20e24 C under a 12-h light/dark cycle. Standard chow and water 94 30 reduced potential for systemic anticholinergic effects with were available ad libitum. All experiments were approved and 95 31 aclidinium. monitored by the Animal Ethical Committee of Almirall (Barcelona, 96 32 Here we compare the in vitro pharmacology of aclidinium and Spain) and in accordance with EU Directive 2010/63/EU for animal 97 33 glycopyrronium at muscarinic receptors with that of tiotropium experiments.