Vivo Profile of Aclidinium Bromide in Comparison With

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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. 98 34 and ipratropium. The potency, onset of action and duration of ac- 99 35 tion of each antagonist in in vitro and in vivo bronchoconstriction 2.3. Radioligand binding studies 100 36 models were also assessed. Additional studies were conducted to 101 37 investigate the potential of all four antagonists to cause systemic 2.3.1. Affinity for the human M1 to M5 muscarinic receptors 102 38 side effects in the rat pilocarpine-induced sialorrhea model. Finally, The affinity (equilibrium antagonist dissociation constant [Ki] 103 39 the stability of the four antagonists in guinea pig, rat and human values) of muscarinic antagonists at recombinant human musca- 104 40 plasma was compared. rinic M1eM5 receptors was determined as described previously 105 41 [20]. Briefly, human M1,M2,M3,M4 and M5 receptor membrane 106 42 preparations (protein concentrations 8.1, 10.0, 4.9, 4.5 and 5.0 mg/ 107 43 2. Materials and methods well, respectively) were incubated at room temperature for 2 or 6 h 108 3 44 (M1eM4 and M5, respectively) with [ H]-NMS concentrations 109 45 2.1. Chemicals and reagents approximately equal to the radioligand equilibrium dissociation 110 46 constant (Kd) for each receptor subtype (0.3 nM for M1 and M4; and 111 47 Aclidinium, glycopyrronium and tiotropium were synthesised 1 nM for M2,M3 and M5). Non-specific binding to membranes was 112 48 by the Department of Medicinal Chemistry (Almirall R&D Centre, determined in the presence of atropine 1 mM. Antagonist concen- 113 49 Barcelona, Spain). Acetylcholine hydrochloride, atropine sulfate, trations (10 5 to 10 14 M) were tested in duplicate. Incubation 114 50 carbachol chloride, ipratropium and pilocarpine were obtained times were selected to ensure equilibrium binding was achieved. 115 51 from SigmaeAldrich (Madrid, Spain); ketamine chlorhydrate Bound and free [3H]-NMS were separated by rapid vacuum filtra- 116 52 (Imalgene) was from Merial (Barcelona, Spain); xylazine (Rompun tion of GF/C filter plates (Millipore, Barcelona, Spain), and radio- 117 53 2%) from Bayer (Barcelona, Spain); acepromazine maleate (Calm- activity was quantified using a MicroBeta Trilux microplate 118 54 oneosan) from Pfizer Salud Animal (Alcobendas, Spain); propofol scintillation counter (Perkin Elmer Life and Analytical Sciences). Ki 119 55 (Lipuro) from B. Braun (Rubí, Spain); acetonitrile from Scharlau values were calculated as described by Cheng and Prusoff for 120 56 (Barcelona, Spain); Milli-Q water from Millipore S.A. (Madrid, competitive inhibitors [23]. All binding studies were performed in 121 57 Spain); and formic acid, ammonia and hydrochloric acid from non-physiological assay binding buffer containing 25 mM TRIS pH: 122 58 Merck (Madrid, Spain). 7.4. 123 59 Membrane preparations expressing recombinant human M1, 124 60 M2,M3,M4 and M5 receptors (prepared from transfected CHO-K1 2.3.2. Dissociation from M2 and M3 muscarinic receptors 125 61 cells) were obtained from Membrane Target Systems (Perkin Dissociation of radiolabelled muscarinic antagonists was 126 62 Elmer Life and Analytical Sciences, Boston, MA, USA). 1-[N-meth- assessed as described previously [20]. Association of radioligands, 127 63 yl-3H] scopolamine methyl chloride ([3H]-NMS) was obtained from with approximately 90% binding-site occupancy, was achieved by 128 3 64 Perkin Elmer Life and Analytical Sciences; [ H]aclidinium (2.89 incubating membranes expressing human M2 and M3 receptors 129 65 TBq/mmol), [3H]glycopyrronium (2.59 TBq/mmol), [3H]tiotropium (final protein concentration 15 mg/mL) at room temperature with 130

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3 3 1 [ H]aclidinium (2.5 nM), [ H]glycopyrronium (15 and 5 nM for M2 concentration that produced sub-maximal (80e90%) inhibition of 66 3 3 2 and M3 receptors, respectively), [ H]tiotropium (2.5 nM) or [ H] electrically stimulated contractions. Antagonists were then washed 67 3 ipratropium (10 nM) for 135 min. Dissociation from the receptor out and t½ offset calculated using non-linear regression analysis. 68 4 was initiated by the addition of atropine 10 mM(final concentra- 69 5 tion). The amount of bound radioligand remaining over time was 70 2.5. In vivo potency, onset of action and duration of action in 6 assessed by separating bound and free radioligand as described in 71 anaesthetised guinea pigs 7 section 2.3.1. Dissociation half-lives (t½) were calculated using one- 72 8 phase exponential decay. All binding studies were performed in 73 The in vivo potency, onset and duration of bronchodilation were 9 non-physiological assay binding buffer containing 25 mM TRIS pH: 74 assessed in an anaesthetised guinea pig bronchoconstriction 10 7.4. 75 model. Conscious guinea pigs were placed in a methacrylate box 11 76 and exposed to a nebulised aerosol of antagonist solution. Antag- 12 2.4. In vitro potency and duration of action at native M and M 77 2 3 onists were administered for 1 min at a flow rate of 3 L/min and 13 muscarinic receptors 78 animals were allowed to breathe freely for a 5-min period. This 14 79 procedure was then repeated. Muscarinic antagonists (1e1000 mg/ 15 2.4.1. M receptors 80 2 mL) or vehicle were administered to guinea pigs (n ¼ 4e9 by dose 16 Potency and duration of action at M receptors were assessed in 81 2 and time point) as nebulised aerosols via an ultrasonic nebuliser 17 the isolated guinea pig left-atria preparation. Briefly, left atria 82 (DeVilbiss UltraNeb 2000; Sunrise Medical, Somerset, PA). This 18 (n ¼ 3e13) were dissected and suspended in an organ bath con- 83 nebulisation was driven by a mixture of 5% CO , 21% O , 74% N at a 19 taining oxygenated Krebs-Henseleit solution at 32 C. Isolated tis- 2 2 2 84 flow of 3L/min as previously described [20]. At various time points 20 sues were connected to a force transducer (Letica TRI201, 85 (1, 2, 4, 6, 18 and 24 h) after antagonist exposure, guinea pigs were 21 Barcelona, Spain) and isometric changes recorded using PowerLab 86 anaesthetised with an intramuscular injection of ketamine 22 software (AD instruments, Panlab, Barcelona, Spain). A stable 87 (43.8 mg/kg), xylazine (3.5 mg/kg) and acrepromazine (1.1 mg/kg); 23 resting tone was achieved by applying a pre-load of 1 g prior to 88 additional anaesthetic was administered as necessary. Animals 24 electrical stimulation (1 Hz, 8 V 5 ms); baseline contractions were 89 were adequately anesthetised during surgical procedure and dur- 25 assessed during a 60-min stabilization period. Inhibition of elec- 90 ing all study time points. Airflow, transpulmonary pressure and 26 trically induced contractions via the M receptor was achieved by 91 2 blood pressure were monitored throughout the procedure as pre- 27 the addition of carbachol 1 mM. Increasing cumulative concentra- 92 viously described [20]. During experiments, guinea pigs were 28 tions of antagonists (0.01e1000 nM) were added every 5e10 min to 93 artificially ventilated as previously described [20]; body tempera- 29 assess the potency of each compound to reverse carbachol-induced 94 ture was maintained at 37 C with a homeothermic blanket. Pul- 30 relaxation of electrically stimulated contractions. The EC (con- 95 50 monary airway resistance (Raw) was assessed as a measure of 31 centration required to produce 50% inhibition of the maximum 96 bronchoconstriction. Raw was calculated as the quotient of the 32 carbachol-induced relaxation) was determined for each compound 97 changes in flow and pressure between isovolumetric points on 33 using non-linear regression. 98 inspiration and expiration. Measurements were initiated once 34 Duration of action was assessed as time to 50% recovery of the 99 baseline Raw values were in the range 0.1e0.2 cM H2O/mL per s. 35 maximum carbachol-induced relaxation (t½ offset). Following the 100 Bronchoconstriction was induced by intravenous administration 36 addition of carbachol 10 mM, antagonists were added at a concen- 101 of a single bolus dose of acetylcholine (30 mg/kg), and the inhibitory 37 tration that inhibited 80% of the maximum carbachol-induced 102 effect of each antagonist was assessed relative to vehicle. Potency 38 relaxation; inhibition of tone was then allowed to stabilise for 103 was defined as the concentration required to produce 50% inhibi- 39 20e30 min. The antagonists were washed out and the atria re- 104 tion of acetylcholine-induced bronchoconstriction (EC50), deter- 40 incubated with carbachol 10 mM for 4 h. The t½ offset was calcu- 105 mined from a sigmoidal doseeresponse curve constructed using 41 lated using one-phase (aclidinium, glycopyrronium and tio- 106 the inhibition values at each of the time points studied. Onset of 42 tropium) or two-phase (ipratropium) exponential decay. 107 action for each compound was defined as the time to maximal 43 108 inhibition of bronchoconstriction (t ) taken from EC values. The 44 2.4.2. M receptors max 50 109 3 duration of action, defined as the time to 50% recovery of the 45 The potency and duration of action of antagonists at M3 re- 110 maximal inhibitory effect achieved by the antagonist (t½ offset), 46 ceptors were assessed in the isolated guinea pig trachea prepara- 111 was derived from time-course bronchoconstriction inhibition 47 tion, as described previously [20,24]. Briefly, trachea were excised 112 curves using one-phase exponential decay. 48 and mounted in a superfusion chamber containing oxygenated 113 49 Krebs-Henseleit solution, supplemented with propranolol 1 mMat 114 50 37 C. Trachea strips (n ¼ 3e13) were connected to a force trans- 2.6. Salivation in conscious rats 115 51 ducer and isometric changes recorded as in Section 2.4.1. A pre-load 116 52 of 1 g was applied to obtain a stable resting tone prior to the in- The effect of aclidinium, glycopyrronium and tiotropium on 117 53 duction of M3 receptor-mediated contractions (10 s trains of square salivation in conscious rats was assessed as follows: rats (n ¼ 6e24) 118 54 wave pulses of 5 Hz and 0.1 ms every 2 min). Baseline was estab- were fasted for 18 h (with water ad libitum) prior to administration 119 55 lished by stimulating trachea strips for 20 min at a voltage of of aclidinium (0.1e1000 mg/kg), glycopyrronium (0.1e10 mg/kg), 120 56 10e15% above that required for a maximal response. Increasing tiotropium (0.1e100 mg/kg) or vehicle subcutaneously in the 121 57 concentrations of antagonists (0.01e1000 nM) were then added intercapular area. After 30 min, pilocarpine (0.5 mg/kg) was 122 58 every 30 min to assess the potency of each compound to inhibit administered via the caudal vein. The presence of any sialorrhea 123 59 electrically stimulated contractions. A cumulative concen- (excess saliva) was recorded during the first 15-min post- 124 60 trationeresponse curve for inhibition of electrically stimulated pilocarpine administration by gently pressing filter paper on the 125 61 contractions was constructed and the EC50 determined using non- animal's snout. Animals were considered positive for sialorrhea if 126 62 linear regression. the filter paper was spotted. The proportions of animals showing 127 63 Duration of action at M3 receptors was assessed as t½ offset salivation following antagonist treatment were compared with 128 64 (time to 50% recovery of electrically-stimulated contractions). Tis- vehicle-treated animals using Fisher's exact test. The ED50 values 129 65 sues were incubated for 45 min in antagonist solution, at a (dose required to inhibit pilocarpine-induced salivation in 50% of 130

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1 Table 1 3.2. Dissociation from human M2 and M3 muscarinic receptors 66 2 Binding affinity of aclidinium, glycopyrronium, tiotropium and ipratropium for 67 human M ,M ,M,M and M receptors. 3 1 2 3 4 5 The dissociation half-lives of [3H]aclidinium, [3H]glyco- 68 3 3 4 Ki (nM) pyrronium, [ H]tiotropium and [ H]ipratropium were determined 69 3 5 M1 M2 M3 M4 M5 (Table 2). [ H]aclidinium dissociated more slowly from both M2 and 70 6 M receptors than [3H]glycopyrronium; [3H]tiotropium dissociated 71 Aclidinium 0.10 ± 0.00 0.14 ± 0.04 0.14 ± 0.02 0.21 ± 0.04 0.16 ± 0.01 3 7 Glycopyrronium 0.42 ± 0.02 1.77 ± 0.06 0.52 ± 0.04 0.78 ± 0.04 1.29 ± 0.09 the most slowly from both receptors (approximately 2- to 3-fold 72 3 3 8 Tiotropium 0.13 ± 0.00 0.13 ± 0.04 0.19 ± 0.04 0.30 ± 0.09 0.18 ± 0.06 slower than [ H]aclidinium) and [ H]ipratropium dissociated 73 9 Ipratropium 1.31 ± 0.15 1.12 ± 0.13 1.24 ± 0.08 1.92 ± 0.18 3.22 ± 0.15 most rapidly (Fig. 1a and b; Table 2). All the antagonists displayed a 74 10 Data are reported as mean ± standard error of the mean of three independent ex- similar magnitude of kinetic selectivity for M3 over M2 receptors (as 75 11 periments. determined by the t½ M3/M2 ratio; Table 2). 76 12 Ki, antagonist dissociation constant. 77 13 78 3.3. Potency and duration of action at endogenous M2 and M3 14 79 rats) were calculated by non-linear regression (sigmoidal dos- muscarinic receptors 15 80 eeresponse curve fit). 16 81 3.3.1. M2 receptors 17 82 To evaluate the potency and duration of action at endogenous 18 2.7. In vitro rat, guinea pig and human plasma stability 83 M2 receptors, the ability of muscarinic antagonists to inhibit the 19 84 effects of carbachol in electrically stimulated guinea pig left-atria 20 In vitro plasma stability was assessed as described previously 85 fl ¼ preparations was assessed. Each of the antagonists reversed 21 [22]. Brie y, guinea pig (n 10) plasma samples were prepared 86 carbachol-mediated inhibition of electrically stimulated contrac- 22 using sodium heparin as anticoagulant (25 units/mL; 2000 g at 87 ¼ tions in a concentration-dependent manner. Tiotropium displayed 23 4 C). Plasma samples from human volunteers (n 6) were ob- 88 the greatest potency at endogenous M2 receptors, whereas the 24 tained in a similar manner. Rat plasma was commercially available. 89 potencies of aclidinium, glycopyrronium and ipratropium were 25 Triplicate plasma samples were pre-incubated at 37 C for 5 min 90 comparable (Table 3). Glycopyrronium had a t½ offset time 26 prior to the addition of aclidinium, glycopyrronium, tiotropium or 91 approximately 3-fold shorter than that of aclidinium and 6-fold 27 ipratropium at a final concentration of 83 nM, 126 nM, 102 nM and 92 shorter than that of tiotropium. By contrast, the t½ offset of glyco- 28 120 nM, respectively (40 ng/mL, expressed as cation) to initiate the 93 pyrronium was 8-fold longer than that of ipratropium (Table 3). 29 reaction. Following incubation for predetermined time points up to 94 m m 30 1h,100 L aliquots of each reaction were combined with 300 L 95 31 ice-cold acetonitrile:1 N HCl (90/10, v/v). Samples were centrifuged 3.3.2. M3 receptors 96 32 at 2500 g for approximately 10 min at 4 C. Control plasma in- The ability of antagonists to inhibit cholinergic tone in isolated 97 33 cubations in the absence of antagonist were also performed. Sam- guinea pig trachea was investigated to determine the potency and 98 34 ples were analysed by ultra performance liquid chromatography duration of action of each compound at endogenous M3 receptors. 99 35 (Acquity Ultra Performance LC, Waters, Milford, MA, USA) with All four antagonists exhibited comparable, low nanomolar, potency 100 e 36 mass spectrometry detection (Quattro Premier, Micromass Tech- (3.0 5.3 nM) at endogenous M3 receptors (Table 3). The duration of 101 37 nologies, Waters). For each time point, the percentage of remaining action of the three LAMAs at M3 receptors was comparable, 102 38 unaltered compound was calculated. The dissociation half-life (t½) whereas the duration of action of the SAMA, ipratropium, was 103 39 in plasma was calculated using WinNonlin software (version 5.0.1., much shorter (>480 min versus 42 min, respectively; Table 3). 104 40 Pharsight Corporation, USA). 105 41 3.4. In vivo onset of action, potency and duration of action in 106 42 3. Results anaesthetised guinea pigs 107 43 108 44 3.1. Determination of affinity of muscarinic antagonist for the All of the antagonists produced concentration-dependent inhi- 109 45 human M1 to M5 muscarinic receptors bition of acetylcholine-induced bronchoconstriction in vivo; 110 46 maximal inhibition was 97e99% with all four antagonists. With 111 47 The affinity of aclidinium, glycopyrronium, tiotropium and regard to onset of action, aclidinium, glycopyrronium and ipra- 112 e 48 ipratropium for the human M1 M5 receptors was assessed in tropium achieved tmax 2 h post-administration compared with 4 h 113 49 competitive binding experiments using membranes from trans- with tiotropium. At the onset of action for each compound, EC50 114 50 fected CHO-K1 cells, stably expressing each of the recombinant values were comparable across all four antagonists, ranging from 115 51 receptors. Prior to determining ligand affinity, the amount of drug 1.4 to 3.8 mg/mL (Table 4). 116 52 required to saturate a population of receptors and the Kd value for 117 53 each receptor were established in saturation (equilibrium binding) 118 54 experiments using [3H]-NMS; these data have been reported pre- Table 2 119 3 3 3 fi Dissociation half-lives of [ H]aclidinium, [ H]glycopyrronium, [ H]tiotropium and 55 viously [20]. All of the antagonists tested inhibited the speci c 3 120 3 [ H]ipratropium from human M2 and M3 receptors. 56 binding of [ H]-NMS to human M1eM5 receptors in a 121 57 concentration-dependent manner. Aclidinium and tiotropium had M2 M3 Relative half-life t½ 122 a t½ (h) t½ (h) at M receptor M /M ratio 58 comparable affinity for all of the receptor subtypes and higher af- 3 3 2 123 59 finity compared with glycopyrronium and ipratropium (Table 1). Aclidinium 4.69 ± 0.29 29.24 ± 0.61 62 6.2 124 ± ± 60 The affinity of glycopyrronium was 4- to 13-fold lower than that of Glycopyrronium 1.07 0.20 8.10 0.45 17 7.3 125 ± ± e Tiotropium 15.11 1.57 62.19 2.96 132 4.1 61 aclidinium across the M1 M5 receptors (Table 1). Ipratropium had Ipratropium 0.08 ± 0.01 0.47 ± 0.02 1 5.9 126 62 the lowest affinity for all receptor subtypes with the exception of 127 Data are reported as mean ± standard error of the mean from three independent 63 M (Table 1). Glycopyrronium was the only antagonist that 128 2 experiments. fi 64 exhibited some degree of preference in terms of af nity for M3 t½, dissociation half-life. 129 a 3 65 versus M2 receptors (approximately 3-fold; Table 1). Half-lives expressed relative to [ H]ipratropium. 130

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1 antagonists produced equi-effective (97e98%) inhibition of 66 2 acetylcholine-induced bronchoconstriction at the selected doses 67 3 (Fig. 2). The in vivo duration of bronchodilator action of aclidinium 68 4 was more than 2-fold that of glycopyrronium (t½ offset ¼ 29 h 69 5 versus 13 h, respectively; Fig. 2). Tiotropium had the longest 70 6 duration of action (t½ offset ¼ 64 h) and ipratropium the shortest 71 7 (t½ offset ¼ 8h)(Fig. 2). 72 8 73 9 3.5. Salivation in conscious rats 74 10 75 11 The ability of aclidinium, glycopyrronium and tiotropium to 76 12 inhibit salivation was assessed in the rat pilocarpine-induced sia- 77 13 lorrhea model. All three compounds inhibited sialorrhea in a dose- 78 14 dependent manner (Fig. 3). However, the doseeresponse curve for 79 15 aclidinium demonstrated a rightward shift compared with that of 80 16 tiotropium. Consistent with this, the dose of aclidinium required to 81 17 produce a 50% inhibition of pilocarpine-induced salivation (ED50) 82 18 was 43e51-fold lower than that for tiotropium and glycopyrronium 83 19 (ED50 [mg/kg] ¼ 38, 0.88 and 0.74 for aclidinium, tiotropium and 84 20 glycopyrronium respectively; Fig. 3). 85 21 86 22 3.6. In vitro guinea pig and rat plasma stability 87 23 88 24 Fig. 4 shows the stability of all four antagonists in rat (Fig. 4a), 89 25 guinea pig (Fig. 4b) and human (Fig. 4c) plasma. Plasma stability 90 26 data for aclidinium, tiotropium and ipratropium in rat and guinea 91 27 pig have been reported previously [22]. In rat plasma, aclidinium 92 28 was rapidly hydrolysed with t½ ¼ 0.19 h (Table 5) whereas glyco- 93 29 pyrronium was hydrolysed more slowly (t½ ¼ 12 h; Table 5). Tio- 94 30 tropium was hydrolysed more slowly than aclidinium, but more 95 31 quickly than either glycopyrronium or ipratropium. Aclidinium was 96 32 more stable in guinea pig plasma compared with rat (Table 5), 97 33 whereas glycopyrronium was less stable. Aclidinium was least 98 34 stable in human plasma (t½ ¼ 0.04 h), whereas the stability of the 99 35 other three antagonists in human plasma was intermediate to that 100 36 observed in rat and guinea pig plasma. The rank order of plasma 101 37 stability was the same in all three species, with 102 38 aclidinium < tiotropium < glycopyrronium < ipratropium (Table 5, 103 39 Fig. 4aec). 104 40 105 41 4. Discussion 106 42 107

43 Fig. 1. Dissociation of [3H]aclidinium, [3H]glycopyrronium, [3H]tiotropium and [3H] The use of a LAMA in the maintenance treatment of stable COPD 108

44 ipratropium from (a) human M2 receptors, and (b) human M3 receptors. is well established [1]. However, until recently, tiotropium was the 109 45 only LAMA approved for the treatment of COPD. The recent 110 46 approval of aclidinium and glycopyrronium for use as maintenance 111 47 The duration of bronchodilator action was assessed using a bronchodilator treatment in patients with COPD expands the 112 m 48 single concentration of each inhaled antagonist (aclidinium 100 g/ therapeutic options for these patients. Here, we compared the 113 m m 49 mL, glycopyrronium 100 g/mL, tiotropium 10 g/mL and ipra- in vitro and in vivo profiles of aclidinium and glycopyrronium with 114 m 50 tropium 30 g/mL). At 1 h post-administration, all of the those of tiotropium and the SAMA, ipratropium. Our results 115 51 demonstrate that while all four muscarinic receptor antagonists 116 Table 3 52 have high affinity for M1 to M5 receptors and demonstrate similar 117 Potency and duration of action of aclidinium, glycopyrronium, tiotropium and 53 kinetic selectivity for M3 versus M2 receptors, they have unique 118 ipratropium at native M2 receptors (isolated guinea pig left atria) and M3 receptors 54 (isolated guinea pig trachea). profiles with respect to dissociation from the therapeutic target (M3 119 55 receptors), and in vitro and in vivo onset and duration of action. 120 M receptors M receptors 56 2 3 Furthermore, their propensity to inhibit salivation in a rodent 121 a a 57 EC50 (nM) t1/2 offset (min) EC50 (nM) t1/2 offset (min) model varies, which may be related, in part, to differences in the 122 58 Aclidinium 17.4 ± 1.1 102 5.3 ± 1.6 >480 plasma stability of each compound. 123 59 Glycopyrronium 17.3 ± 1.2 30 4.2 ± 0.3 >480 Aclidinium exhibited sub-nanomolar affinity for all five receptor 124 ± ± > 60 Tiotropium 11.8 1.1 184 3.0 0.6 480 subtypes with no selectivity in terms of binding affinity at any of 125 Ipratropium 19.9 ± 1.1 4 3.0 ± 0.4 42 61 the receptors. Consistent with previous reports [21,25], glyco- 126 62 EC50, concentration required to produce 50% inhibition of the maximum carbachol- pyrronium also had high affinity for each of the five receptor sub- 127 induced relaxation (M ) or 50% inhibition of electrically stimulated contractions 63 2 types; however, its affinity was 4- to 13-fold lower than that of 128 (M3); t1/2 offset, time to 50% recovery of the maximum carbachol-induced relaxation 64 129 (M2) or to 50% recovery of electrically-stimulated contractions (M3). aclidinium across the receptors. Whilst glycopyrronium exhibited a 65 Data reported as mean ± standard error of the mean; n ¼ 3e13. some degree of selectivity for M3 versus M2 receptors 130

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1 Table 4 66 2 Onset of action and potency of aclidinium, glycopyrronium, tiotropium and ipratropium in reversing acetylcholine-induced bronchoconstriction in guinea pigs (n ¼ 4e9by 67 dose and time point). 3 68 m 4 Onset time (h) EC50, g/mL (95% CI) 69 5 1h 2h 4h 18h 24h 70 6 71 Aclidinium 2 5.9 (3.7, 9.4) 2.5 (1.7, 3.5) 2.9 (1.8, 4.7) 12.4 (4.1, 37.6) 23.1 (9.3, 57.3) 7 Glycopyrronium 2 7.2 (4.1, 12.8) 3.8 (2.5, 5.7) 8.8 (5.2, 14.8) 68.7 (39.6, 119.2) 242.3 (162.0, 362.2) 72 8 Tiotropium 4 2.4 (1.4, 3.8) 3.9 (2.0, 7.6) 1.4 (0.7, 2.5) 1.4 (0.7, 2.9) 3.3 (2.0, 5.2) 73 9 Ipratropium 2 6.9 (4.0, 11.7) 3.4 (1.9, 5.9) 7.3 (4.0, 13.4) 689.7 (337.1, 1411.0) NA 74

10 CI, confidence interval; Onset time, time to maximal inhibition of bronchoconstriction (h); EC50, concentration required to produce 50% inhibition of bronchoconstriction 75 11 induced by acetylcholine (30 mg/kg i.v.); h, hour; i.v., intravenous; NA, not available. 76 12 77 13 78 fi 14 (approximately 3-fold), binding af nities at each receptor were still inotropic effects of acetylycholine in the heart [9,35]. All four 79 fi 15 in the low nanomolar range. As previously reported, the af nity of muscarinic receptor antagonists have been shown previously to 80 16 aclidinium for M1 to M5 receptors was comparable to that of tio- dissociate faster from M2 receptors compared with M3, conferring 81 17 tropium; ipratropium was the least potent of the four compounds some degree of kinetic selectivity [20,21,26]. In this study, aclidi- 82 18 overall [20,21]. nium and glycopyrronium had comparable kinetic selectivity for 83 19 The difference in duration of action of LAMAs and SAMAs is M3 versus M2 receptors, whereas tiotropium exhibited the lowest 84 20 thought to be primarily due to their longer residence times at hu- kinetic selectivity. 85 21 man M3 receptors [21,26]. Aclidinium, glycopyrronium and tio- Aclidinium and glycopyrronium had similar relative potencies at 86 fi 22 tropium have been reported to have a residence half-life at native M3 and M2 receptors, suggesting that the higher af nity of 87 23 recombinant human M3 receptors of between 6.1 h and 62.2 h aclidinium versus glycopyrronium for M3 and M2 receptors in 88 e 24 (compared with 13.2 28.2 min for ipratropium) [20,21,26], making binding experiments does not necessarily translate into improved 89 25 them suitable for once- or twice-daily dosing in the clinical setting potency at native M3 and M2 receptors. Both antagonists were 3- to 90 26 compared with four times a day for ipratropium. In this study, the 4-fold more potent at native M3 receptors compared with native M2 91 27 residence half-life of aclidinium at human M3 receptors was receptors, in contrast to the slight preference of glycopyrronium 92 28 approximately four times longer than that of glycopyrronium. versus aclidinium for human recombinant M3 compared with M2 93 29 Tiotropium had the longest residency time, consistent with its use receptors. The differences in results between the binding studies 94 30 as a once-daily treatment [27,28]. By contrast, ipratropium had a and the in vitro potency studies may be due, in part, to the differ- 95 < fi 31 dissociation half-life at M3 receptors of 1 h. The longer residency ence in receptor af nities between species. Consistent with their 96 32 time of aclidinium versus glycopyrronium at M3 receptors suggests long residency time at recombinant human M3 receptors, aclidi- 97 33 a longer duration of action in vivo for aclidinium than glyco- nium, glycopyrronium and tiotropium had a long duration of action 98 > 34 pyrronium. Interestingly, in the clinical setting, aclidinium is ( 8 h) in isolated guinea pig trachea. 99 35 administered twice daily [29,30] versus once daily for glyco- The faster onset of action of aclidinium and glycopyrronium 100 e 36 pyrronium [31 33]. A recent study by Sykes et al., comparing gly- compared with tiotropium in this study is consistent with the 101 fi 37 copyrronium and tiotropium, demonstrated that receptor-binding clinical pro le of these compounds [36,37]. At the time of maximal 102 fi 38 properties, including af nity and dissociation rates, can be over- effect, aclidinium, glycopyrronium, tiotropium and ipratropium 103 39 estimated under non-physiological assay conditions suggesting were equipotent inhibitors of bronchoconstriction in vivo. The 104 40 that other factors, in particular drug rebinding, may play an duration of bronchodilator action of each antagonist in vivo 105 ' 41 important role in determining duration of action in vivo [34]. mirrored that for M3 receptors residency times seen in the binding 106 42 Kinetic selectivity for M3 versus M2 receptors is considered 107 43 desirable because: (i) inhibition of presynaptic M2 receptors may 108 44 facilitate cholinergic signalling in the airway by blocking negative 109 45 feedback mechanisms regulating acetylcholine release from para- 110 46 sympathetic nerves [7,9]; and (ii) the characteristic tachycardia 111 47 seen with anticholinergics is a consequence of inhibition of cardiac 112 48 M2 receptors which mediate the negative chronotropic and 113 49 114 50 115 51 116 52 117 53 118 54 119 55 120 56 121 57 122 58 123 59 124 60 125 61 126 62 127 63 128 64 Fig. 2. Duration of action of aclidinium, glycopyrronium, tiotropium and ipratropium Fig. 3. Effects of aclidinium, glycopyrronium and tiotropium on pilocarpine-induced 129 65 in reversing acetylcholine-induced bronchoconstriction in guinea pigs. salivation in conscious male Wistar rats. 130

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1 Table 5 66 2 Estimated stability of aclidinium, glycopyrronium, tiotropium and ipratropium in rat 67 and guinea pig plasma. 3 68 4 Rat plasma Guinea pig Human 69 5 t½ (h) plasma t½ (h) plasma t½ (h) 70 6 Aclidinium 0.19 0.64 0.04 71 7 a) Glycopyrronium 11.6 5.5 6 72 8 Tiotropium 1.2 1.9 1.6 73 Ipratropium 23.6 73.4 33 9 74 10 h, hour; t½, hydrolysis half-life. 75 11 76 12 77 studies, with aclidinium having a longer duration of effect in 13 78 anaesthetised guinea pigs than glycopyrronium and ipratropium, 14 79 and a shorter duration of effect than tiotropium. 15 80 Anticholinergic compounds, including tiotropium and ipra- 16 81 tropium, are typically associated with systemic side effects such as 17 82 dry mouth and tachycardia [12,13,15,16]. In a previous study, acli- 18 83 dinium was shown to produce a transient increase in heart rate in 19 84 conscious dogs that was resolved 2.5 h post-administration, 20 85 whereas tiotropium caused a significant increase that persisted 21 86 for at least 6 h post-administration [20]. In this study, aclidinium 22 87 was a much less potent inhibitor of salivation than either glyco- 23 88 pyrronium or tiotropium, suggesting a lower propensity for acli- 24 89 dinium to cause dry mouth in the clinical setting. These preclinical 25 b) 90 observations are supported by results from Phase III clinical trials 26 91 which have demonstrated that the incidence of dry mouth and 27 92 cardiovascular side effects with twice-daily aclidinium was low and 28 93 comparable to that with placebo [29,30]. Aclidinium was rapidly 29 94 hydrolysed in rat and guinea pig plasma with a t½ in both species 9- 30 95 to 61-fold shorter than glycopyrronium and 3- to 6-fold shorter 31 96 than tiotropium. Aclidinium was least stable in human plasma, with 32 97 a t½ 150-fold shorter than glycopyrronium and 40-fold shorter than 33 98 tiotropium. Furthermore, in healthy volunteers aclidinium has been 34 99 shown to be rapidly eliminated from plasma [38,39]. The rapid 35 100 plasma hydrolysis of aclidinium results in very low systemic 36 101 exposure which, coupled to its kinetic selectivity for M receptors 37 3 102 over M , may confer a reduced propensity for systemic side effects 38 2 103 compared with other anticholinergic compounds. 39 104 In summary, aclidinium has high affinity for muscarinic re- 40 105 ceptors that is comparable to tiotropium but higher than glyco- 41 106 pyrronium. While all four muscarinic antagonists have 42 107 comparable kinetic selectivity, aclidinium dissociates from M 43 3 108 c) receptors more slowly than glycopyrronium and has a longer 44 109 bronchodilatory action in vivo.Inaddition,aclidiniumismore 45 110 rapidly hydrolysed in plasma compared with both glyco- 46 111 pyrronium and tiotropium, which may translate into a reduced 47 112 propensity for systemic anticholinergic side effects. The avail- 48 113 ability of different LAMAs with unique pharmacological and 49 114 physical properties may be important in providing additional 50 115 therapeutic options for these patients. 51 116 52 117 53 Funding source 118 54 119 55 This study was funded by Almirall S.A., Barcelona, Spain. IR, CC, 120 56 EC, RA, JLM, SS and MA performed the study and were involved in 121 57 the acquisition and analysis of the data. AG, DV, JA, JB and MM 122 58 contributed to study conception, design and data interpretation. AG 123 59 and MM were involved in the planning and coordination of the 124 60 study and contributed to the writing of the manuscript. All authors 125 61 were involved in revising the manuscript for intellectual content 126 62 Fig. 4. Stability of aclidinium, glycopyrronium, tiotropium and ipratropium over time and approved the final version of the manuscript for submission. 127 63 in (a) rat plasma, (b) guinea pig plasma and (c) human plasma. The sponsor was involved in the design of the study, analysis of the 128 64 data, review of the data, and review and approval of the 129 65 manuscript. Q1 130

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1 [18] Novartis. Novartis receives European Commission approval for once-daily 59 Q2 Disclosures ® ® 2 Seebri Breezhaler as maintenance COPD treatment in the EU [Internet]; 60 2012 [cited 2013 March 28]. [Available from:]. Q3 3 Amadeu Gavalda, Israel Ramos, Carla Carcasona, Elena Calama, [19] Food and Drug Administration. Tudorza™ Pressair™ (aclidinium bromide) 61 4 Raquel Otal, Jose Luis Montero, Sonia Sentellas, Monica Aparici, [Internet] [cited 2013 March 28]. Available from:, http://www.accessdata.fda. 62 ¼ 5 Dolors Vilella, Joan Alberti, Jorge Beleta and Montserrat Miralpeix gov/scripts/cder/drugsatfda/index.cfm?fuseaction Search.DrugDetails; 2012. 63 [20] Gavalda A, Miralpeix M, Ramos I, Otal R, Carreno~ C, Vinals~ M, et al. Charac- 6 are employees of Almirall S.A. terization of aclidinium bromide, a novel inhaled muscarinic antagonist, with 64 7 long duration of action and a favorable pharmacological profile. J Pharmacol 65 8 Exp Ther 2009;331:740e51. 66 Acknowledgements [21] Casarosa P, Bouyssou T, Germeyer S, Schnapp A, Gantner F, Pieper M. Pre- 9 clinical evaluation of long-acting muscarinic antagonists: comparison of tio- 67 10 e 68 We would like to acknowledge the significant technical support tropium and investigational drugs. J Pharmacol Exp Ther 2009;330:660 8. 11 [22] Sentellas S, Ramos I, Albertí J, Salva M, Anton F, Miralpeix M, et al. Aclidinium 69 of Luis Boix, Mireia Verdú, Felix Muruzabal, Rosario Cerrato, Manel 12 bromide, a new, long-acting, inhaled muscarinic antagonist: in vitro plasma 70 De Luca, MaRosa Ortiz, Isabel Pagan and Núria Toran (Almirall). inactivation and pharmacological activity of its main metabolites. Eur J Pharm 13 e 71 We also thank Deborah McGregor, PhD, of Complete Medical Sci 2010;39:283 90. 14 [23] Cheng Y, Prusoff WH. 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