Preclinical efficacy of THRX-200495, a dual pharmacology muscarinic receptor antagonist and β2-adrenoceptor agonist (MABA)

Pulmonary Pharmacology & Therapeutics 25 (2012) 357e363

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Preclinical efficacy of THRX-200495, a dual pharmacology muscarinic receptor antagonist and b2-adrenoceptor agonist (MABA) Alexander McNamara a, *, Tod Steinfeld b, Maria Teresa Pulido-Rios a, Eric Stangeland c, Sharath S. Hegde a, Mathai Mammen d, William J. Martin a a

Department of Pharmacology, Theravance Inc., 901 Gateway Blvd, South San Francisco, CA 94080, USA Department of Molecular and Cellular Biology, Theravance Inc., 901 Gateway Blvd, South San Francisco, CA 94080, USA Department of Medicinal Chemistry, Theravance Inc., 901 Gateway Blvd, South San Francisco, CA 94080, USA d Department of Research and Early Clinical Development, Theravance Inc, 901 Gateway Boulevard, South San Francisco, CA 94080, USA b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 March 2012 Received in revised form 12 June 2012 Accepted 25 June 2012

Combinations of a muscarinic receptor antagonist (MA) and a b2-adrenoceptor agonist (BA) improve bronchodilation in COPD patients to a greater extent than drugs with either mechanism alone. Here, using an in vivo model of bronchoprotection in guinea pigs, we characterize a single agent with dualacting MA and BA activity, THRX-200495 (MABA). THRX-200495 was compared to a fixed-dose combination of a short-acting muscarinic receptor antagonist (SAMA) and a b2-adrenoceptor agonist (SABA). The SAMA/SABA combination consisted of a 1:5.7 ratio of ipratropium and albuterol (the components of CombiventÒ). Conscious guinea pigs received aqueous nebulized solutions of vehicle or test compound by aerosol exposure. Bronchoprotective potency was estimated in anesthetized, tracheotomized and ventilated guinea pigs at predetermined time points after aerosol exposure by measuring changes in ventilation pressure. The individual (MA, BA) and composite (MABA) pharmacologies were assessed by determining protection against bronchoconstrictor responses induced by methacholine in the presence of propranolol (for MA activity), histamine (for BA activity) or methacholine (MABA activity). Bronchoprotection was calculated as percent inhibition of methacholine or histamine response relative to the vehicle group. THRX-200495 exhibited matched MA (ID50 ¼ 11.4 mg/ mL) and BA (ID50 ¼ 11.2 mg/mL) potency and potent dual pharmacology (MABA ID50 ¼ 3.5 mg/mL) that persisted for over 24 h. The combination of ipratropium/albuterol exhibited bronchoprotective activity that was 2.6-fold more potent as a BA (ID50 ¼ 5.7 mg/mL) than as an MA (ID50 ¼ 14.6 mg/mL) at 0.5 h postdose and 37-fold more potent as an MA (ID50 ¼ 4.3 mg/mL) than a BA (ID50 ¼ 159 mg/mL) at 1.5 h post aerosol exposure. Under MABA pharmacological conditions, ipratropium/albuterol produced potent bronchoprotective activity (ID50 ¼ 2.0/11.4 mg/mL) and an apparent additive effect of the two pharmacologies. In conclusion, a dual-acting prototypical MABA, THRX-200495, demonstrated potent, balanced and long-lasting bronchodilation in a guinea pig model of bronchoprotection that was greater than either the MA or BA mechanisms alone. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: COPD Muscarinic b2-Adrenoceptor Ipratropium Albuterol MABA

1. Introduction

Abbreviations: MA, Muscarinic receptor antagonist; BA, b2-adrenoceptor agonist; SAMA, short-acting muscarinic receptor antagonist; SABA, short-acting b2adrenoceptor agonist; LAMA, long-acting muscarinic receptor antagonist; LABA, long-acting b2-adrenoceptor agonist; MABA, muscarinic receptor antagonist plus b2-adrenoceptor agonist; NMS, N-methyl-scopolamine methyl chloride; DHA 4, 6propyl-dihydroalprenolol; CHO, Chinese hamster ovary; HEK, Human embryonic kidney; PBS, phosphate-buffered saline; GTPgS, guanosine 50 -O-(3-thio)triphosphate; BSA, bovine serum albumin; BEAS-2B, human bronchial epithelial cells. * Corresponding author. Tel.: þ1 650 808 6091; fax: þ1 650 808 6441. E-mail address: [email protected] (A. McNamara). 1094-5539/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.pupt.2012.06.007

Muscarinic receptor antagonists (MA) and b2-adrenoceptor agonists (BA) administered either as monotherapy or in combination improve airway function in patients with chronic obstructive pulmonary disease (COPD) [1e3]. Although MA and BA produce bronchodilation by different cellular mechanisms [4] evidence suggests that these mechanisms are complementary and that biochemical cross-talk exists between M2/M3 muscarinic receptors and b2-adrenoceptors in the lung [4e7]. Indeed, the combination of bronchodilators from these two classes yields greater efficacy in moderate-to-severe COPD patients than either mechanism alone

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[8e10]. As such, the Global Initiative for Chronic Obstructive Lung Disease (GOLD) Expert Panel recommends combination therapy for the treatment of moderate-to-severe COPD [11]. Despite the potential advantages of combination bronchodilator regimens, mismatched potency and pharmacokinetic profiles, coupled with pharmaceutical incompatibility, limit the potential to develop combination bronchodilator therapies, particularly triple pharmacology therapies that include inhaled corticosteroids (ICS). Many patients currently are using both inhaled muscarinic receptor antagonists (MA) and inhaled b2-adrenoceptor agonists (BA) in either two separate inhalers [12] or via the product CombiventÒ [13], which combines the short-acting muscarinic receptor antagonist (SAMA), ipratropium and the short-acting b2adrenoceptor agonist (SABA), albuterol. Products combined in a single delivery device simplify treatment, however, CombiventÒ, which is the only marketed combination of MA and BA, bronchodilators requires dosing four times per day [14]. The need for frequent dosing lowers patient compliance which, in turn, can increase the rate of hospitalization [15]. Thus, considerable effort has been directed towards identifying long-acting muscarinic receptor antagonists (LAMA) and b2-adrenoceptor agonists (LABA) that are suitable for concomitant delivery, to reduce dosing frequency and to simplify dosing regimens. Recent clinical trials have demonstrated that combining the LAMA, tiotropium, with LABAs, salmeterol or formoterol, in COPD patients enhances clinical efficacy without increasing side effects [16e18]. However, the mismatched pharmacokinetics of tiotropium, which is administered once a day, and the twice daily b2adrenoceptor agonists suggests that both bronchodilatory mechanisms may not be optimally engaged over 24 h. Several fixed-dose combinations of long-acting bronchodilators (LAMA þ LABA), have entered clinical trials and show improved and sustained 24 h bronchodilation [19,20]. The ability to add an inhaled corticosteroid (ICS) to a fixed-dose combination of LAMA þ LABA is currently unproven; however, ‘triple therapy’ including anti-inflammatory activity may offer optimal control of symptoms associated with moderate and severe COPD [21]. The co-formulation of two bronchodilators with an ICS in a single device represents an immense technical challenge [22]. One option to overcome these difficulties is to design a single molecule with dual pharmacology (MA and BA) that could be more readily co-formulated with an ICS. The attractiveness of the MABA concept has led to the development of several MABA candidates, the most advanced being GSK961081 (TD-5959) [23]. In this study we characterized a prototype MABA molecule, THRX-200495, designed with the same multivalent approach [24,25] we used in the discovery of our lead MABA molecule GSK961081 (TD-5959). The dual pharmacological activity of THRX200495 is attributed to the covalent linkage of a BA moiety, with a carbostyril core [26,27] to a MA moiety, a biphenyl carbamic acid [28]. Using an in vivo guinea pig model of pharmacologicallyinduced bronchoconstriction [29], we characterized the bronchoprotective activity of inhaled THRX-200495 under MA, BA and MABA pharmacological conditions. 2. Material and methods 2.1. Material THRX-200495 (Fig. 1) was synthesized at Theravance, Inc. Ipratropium and albuterol, methacholine, histamine, atropine and propranolol were purchased from the SigmaeAldrich Co., St. Louis, MO. All test compounds were dissolved in sterile water (in vivo) or DMSO (in vitro) and formulated according to the base weight of the compound.

Fig. 1. Chemical structure of THRX-200495, a muscarinic receptor antagonist pharmacophore (biphenyl carbamate piperidine) linked to a b2- adrenoceptor agonist pharmacophore (carbostyryl group). The linkage is a 4,4-propyl ethyl biphenyl ether. The chemical name is biphenyl-2-ylcarbamic acid 1-{3-[4-(4-{2-[(R)-2-hydroxy-2-(8hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]ethyl}-phenoxy)phenyl]propyl} piperidin-4-yl ester.

[3H]-N methyl scopolamine ([3H]NMS) was purchased from GE Healthcare (Piscataway, NJ). [3H]-dihydroalprenolol ([3H]DHA), [125I]-cyanopindolol ([125I]CYP) and [35S]-Guanosine 50 -(gammathio)triphosphate ([35S]GTPgS) were purchased from PerkinElmer (Waltham, MA).

2.2. In vitro pharmacological characterization of THRX-200495 at human muscarinic acetylcholine receptors and beta adrenoceptors: displacement of radioligand binding Radioligand binding studies were conducted using membranes prepared from CHO-K1 cells stably expressing human recombinant muscarinic M1, M2, M3, M4 or M5 acetylcholine receptors or HEK293 cells stably expressing human recombinant b1- or b2-adrenoceptors. Assays were conducted with 1 nM [3H]NMS in a 10 mM HEPES buffer containing 100 mM NaCl, 10 mM MgCl2, and 0.025% BSA, pH 7.4 at 20  C (muscarinic receptors) or with 1 nM [3H]DHA in a 75 mM Tris/HCl buffer containing 12.5 mM MgCl2, 1 mM EDTA, 0.025% BSA, pH 7.4 at 37  C (beta adrenoceptors). Nonspecific binding was defined in the presence of 10 mM atropine (muscarinic receptors) or 10 mM propranolol (b-adrenoceptors). Membrane fractions were incubated with radioligand and unlabeled test compounds for up to 6 h at 37  C to achieve equilibrium. Following separation by vacuum filtration onto GF/B filter plates presoaked with 0.3% polyethyleneimine, the quantity of membrane bound radioligand was measured by scintillation counting using a TopCount scintillation counter (PerkinElmer, Waltham, MA).

2.3. In vitro pharmacological characterization of THRX-200495 at human beta adrenoceptors: agonist Stimulated cAMP accumulation To determine beta adrenoceptor agonist potencies, cAMP accumulation in HEK-293 cells expressing human recombinant b1- or b2-adrenoceptors was measured by a homogeneous [125I]cAMP radioimmunoassay (Flashplate, PerkinElmer NEN). Agonists were incubated with cells for 10 min at 37  C in vendor supplied stimulation buffer. Assays were terminated with the addition of ice-cold stop solution, provided by the vendor. Scintillation counting was used to quantify antibody-captured radiolabeled cAMP. To measure intrinsic activity of b2-adrenoceptor agonists, the cAMP assay mentioned above was performed using human bronchial epithelial cells (BEAS-2B, ATCC, licensed from NIH) expressing low endogenous levels of b2-adrenoceptors [30]. Cells were grown to 75e90% confluency in complete, serum free medium (LHC 9 MEDIUM containing epinephrine and retinoic acid, Biosource International, Camarillo, CA). The day before the assay, medium was switched to LHC 8 containing no epinephrine or retinoic acid.

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2.4. Animals Male Duncan-Hartley guinea pigs (Harlan, Indianapolis, IN), weighing between 250 and 400 g were housed in a climate controlled environment (12 h light, 12 h dark cycle) and given unrestricted access to food and water. All studies were conducted at Theravance, Inc., an AAALAC accredited facility, in accordance with Institutional Animal Care and Use Committee policies. 2.5. Dosing Test compound or vehicle (sterile deionized water) was dosed by aerosol exposure for 10 min in a pie shaped whole body inhalation chamber (R þ S Molds, San Carlos, CA) using 5 mL of dosing solution. The total volume nebulized was approximately 2 mL. Animals were exposed to an aerosol, generated by a LC Star Nebulizer Set (Model22F51, PARI Respiratory Equipment, Inc. Midlothian, VA) driven by Bioblend (5% CO2/95% atmospheric air) at a pressure of 22 psi. Doses of THRX-200495 and the ipratropium/albuterol fixeddose combination are reported as the concentration of dosing solution nebulized. Ipratropium/albuterol doses for testing MA, BA and MABA bronchoprotection were co-formulated at a concentration ratio of 1:5.7 similar to that used in CombiventÒ [31]. 2.6. Bronchoprotection: animal preparation and experimental protocol Bronchoprotection measurements for THRX-200495 were made at 1.5 h, 8 h and 24 h after aerosol exposure, for determination of potency and duration of action. Bronchoprotection measurements for the ipratropium/albuterol combination were made 0.5 h and 1.5 h after aerosol exposure for determination of potency. Prior to evaluating pulmonary function, animals were anesthetized with an intramuscular injection of a cocktail consisting of ketamine (43.7 mg/kg), xylazine (3.5 mg/kg), acepromazine (1.05 mg/kg). Supplemental anesthesia (50% of initial dose) was administered if animals responded to a toe pinch. The jugular vein was isolated and cannulated with a saline filled polyethylene catheter (PE 50) to allow intravenous (i.v.) injection of either methacholine or histamine. The trachea was then dissected free and cannulated with a 14G needle (#NE 014, Small Parts, Miami Lakes, FL). The guinea pigs were then ventilated using a respirator (Model 683, Harvard Apparatus, Inc., MA) set at a stroke volume of 1 mL/100 g body weight but not exceeding 2.5 mL volume, and at a rate of 100 strokes per min. A T-connector was attached to the respirator expiratory tubing to measure changes in ventilation pressure using a Biopac transducer that was connected to a Biopac (TSD 137C) pre-amplifier. Body temperature was maintained at 37  C using a heating pad. Prior to initiating data collection, pentobarbital (25 mg/kg, i.p.) was administered to suppress spontaneous breathing and obtain a stable baseline. The changes in ventilation pressure were recorded on a Biopac Windows data collection interface. Baseline values were collected for at least 5 min prior to initiating pharmacologically-induced airway obstruction. We implemented test protocols that enabled us to isolate the bronchoprotection conferred by muscarinic receptor antagonist (MA) or b2-adrenoreptor agonist (BA) properties of either a single molecule or two distinct molecules. The MA, BA and MABA bronchoprotective potencies were estimated by obtaining bronchoconstrictor doseeresponse curves to i.v. bronchoconstricting agents under the following conditions. Under all conditions, guinea pigs were challenged, non-cumulatively, with 2-fold incremental i.v. doses of bronchoconstricting agents. To measure only the MA effects of THRX-200495 or the ipratropium/albuterol combination,

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methacholine was administered in the presence of a 5 mg/kg, i.v. dose of propranolol, a non-selective beta-adrenoceptor antagonist that blocks the BA contribution of MABA bronchoprotection, and thus isolates the MA activity. Histamine was used to determine the contribution of BA to the bronchoprotection produced by THRX200495 or the ipratropium/albuterol combination. Lastly, administration of methacholine, in the absence of propranolol, was used to assess the dual pharmacology (MABA) effects of either THRX200495 or the ipratropium/albuterol combination. After the completion of study, the animals were euthanized by CO2 asphyxiation followed with a thoracotomy. 3. Data analysis 3.1. In vitro Radioligand binding data were normalized to percent specific binding and analyzed using a four-parameter logistic equation in GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). Hill coefficients did not differ significantly from unity, and therefore, IC50 values were determined with slopes fixed to unity. Inhibitory binding constants (KI) were calculated from the IC50 values [32]. The amount of cAMP produced per mL of reaction was calculated from the counts observed for each sample and the cAMP standard curve, as described in the manufacturer’s user manual. Data were analyzed by nonlinear regression analysis with GraphPad Prism using the four-parameter model for sigmoidal dose response. Potency data are reported as EC50 values. Compound intrinsic activity (I.A.) was calculated from the ratio of the maximal response for the test molecule to the maximal response for 10 mM isoproterenol. 3.2. Guinea pig Einthoven model Change in ventilation pressure was measured in cm of H2O. Change in ventilation pressure (cm H20) ¼ peak pressure (after bronchoconstrictor challenge) -peak baseline pressure. The doseeresponse curve to methacholine or histamine was fitted to a four parameter logistic equation using GraphPad Prism, version 3.00 for Windows (GraphPad Software, San Diego, California). The mean bronchoconstrictor response at submaximal doses of methacholine (16 mg/kg, i.v.) or histamine (32 mg/kg, i.v.) in vehicle and drug-treated animals were compared to calculate percent inhibition of methacholine/histamine -induced bronchoconstriction. Inhibition curves were fitted using the four parameter logistic equation from GraphPad software. The ID50 was defined as the dose producing 50% inhibition of the bronchoconstrictor response. All in vivo results reported as mean  SEM. Student’s ttest was used for statistical analysis. The criterion for significant differences between groups was p < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001). 4. Results 4.1. In vitro results In competition binding studies, THRX-200495 and ipratropium displayed subnanomolar affinity for all muscarinic receptor subtypes, including for the receptors most relevant to bronchodilatory effects: hM2 and hM3. (THRX-200495; M2 pKI ¼ 9.21  0.04; M3 pKI ¼ 9.53  0.13; and ipratropium; M2 pKI ¼ 9.03  0.16; M3 pKI ¼ 8.97  0.15; Table 1). THRX-200495 exhibited subnanomolar affinity for hb2 adrenoceptors, (pKI ¼ 9.37  0.09) compared to (pKI ¼ 5.40  0.46) hb2 adrenoceptor affinity measured for albuterol (Table 2). THRX-

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Table 1 Binding affinities of THRX-200495 and ipratropium at the five human recombinant muscarinic receptors. Negative logarithm equilibrium inhibition binding constants (pKI) were measured in competition studies with [3H]NMS and human recombinant receptors expressed in CHO-K1 cell membranes. Data are expressed as mean pKI  S.D. Compound

M1

M2

M3

M4

M5

THRX-200495 9.19  0.02 9.21  0.04 9.53  0.13 9.38  0.08 8.62  0.07 Ipratropium 8.75  0.30 9.03  0.16 8.97  0.15 8.87  0.16 8.27  0.16

200495 displayed approximately 70-fold higher affinity for hb2 over hb1-adrenoceptors in these assays, while albuterol was relatively non-selective between the two subtypes. In recombinant b-adrenoceptors cAMP assays, THRX-200495 was a potent b-adrenoceptor agonist (b2 pEC50 ¼ 9.69  0.10), with approximately 40-fold selectivity for b2-adrenoceptors over b1-adrenoceptors. THRX-200495 was 140-fold more potent at the b2-adrenoceptor than albuterol. The rank order of potencies measured for these compounds was maintained in cells expressing low levels of endogenous b2-adrenoceptors (BEAS-2B cells). Relative to isoproterenol, the intrinsic activities of THRX-200495 and albuterol were 0.76 and 0.43, respectively. 4.2. In vivo results The potency of THRX-200495 (3, 10, 30 and 100 mg/mL) was determined 1.5 h after aerosol exposure. THRX-200495 produced dose-dependent dextral shifts of the methacholine and histamine dose response curves (Fig. 2). THRX-200495 also inhibited the bronchoconstrictive response elicited by single submaximal bronchoconstrictive doses of methacholine (16 mg/kg) in the presence of propranolol, MA pharmacological conditions; histamine (32 mg/kg), BA pharmacological conditions; and of methacholine (16 mg/kg), MABA pharmacological conditions (Fig. 3). THRX-200495 exhibited bronchoprotective potency (estimated ID50 values [95% CI]) as a MA and a BA of 11.4 [7.8e16.7] and 11.2 [7.2e18.6] mg/mL, respectively and an MABA potency of 3.5 [2.9e4.2] mg/mL (Table 3). Based on non-overlapping confidence intervals, the MABA potency of THRX200495 was significantly greater than either mechanism alone. To determine the duration of bronchoprotection, THRX-200495 was tested at a nebulized concentration (30 mg/mL) that produced 96% MABA-mediated bronchoprotection at 1.5 h post aerosol exposure. Under these conditions, all three mechanisms (MA, BA and MABA) of THRX-200495 contributed to the bronchoprotection 8 h and 24 h post aerosol exposure. At these time points, the MA and BA ratio remained unchanged and the MABA bronchoprotection was greater than that achieved through either MA or BA activity alone (Fig. 4).

Table 2 In vitro pharmacological characterization of THRX-200495 and albuterol b-adrenoceptor agonist activity. The negative logarithm of agonist concentrations eliciting 50% of the maximum cAMP response (pEC50) were measured in HEK-293 cells expressing human recombinant b-adrenoceptors (b1 or b2) or in BEAS-2B cells expressing low levels of endogenous b2-adrenoceptors. Intrinsic activities (I.A.) were measured in BEAS-2B cells relative to isoproterenol. Negative logarithm equilibrium inhibition binding constants (pKI) at the b1- or b2-adrenoceptors were measured in competition studies with [3H]DHA and human recombinant receptors expressed in HEK-293 cell membranes. Data are expressed as mean  S.D. pEC50

b1

b2

pEC50 (I.A.)

pKI

BEAS-2B

b1

b2

THRX-200495 8.09  0.17 9.69  0.10 8.92  0.19 7.53  0.25 9.37  0.09 (0.76  0.08) Albuterol 6.04  0.22 7.55  0.21 6.60  0.31 5.12  0.15 5.40  0.46 (0.43  0.08)

Fig. 2. Bronchoprotective effects of inhaled THRX-200495 (MA, BA, MABA) in the guinea pig Einthoven model 1.5 h after drug aerosol exposure. Abscissa represents the i.v. bolus dose of the bronchoconstrictor agent on a logarithmic scale. Ordinate represents the change in ventilation pressure (cm H20). All data are expressed as mean  SEM (n ¼ 4e11 per group). Data points without error bars represent an SEM less than the symbol size.

Next, to enable a comparison with THRX-200495, we assessed the bronchoprotective potency (ID50 values [95% CI]) of the ipratropium/albuterol combination (concentration ratio of 1:5.7) under MA, BA and MABA pharmacological conditions, at 0.5 h and 1.5 h post aerosol exposure. Previously, we have demonstrated that albuterol exhibits a fast onset of activity in this guinea pig model of bronchoprotection and, consequently, was 19-fold more potent at 0.5 h than at 1.5 h [29]. We therefore examined the potency of the ipratropium/albuterol combination at 0.5 h and 1.5 h post aerosol exposure to preclude an underestimate of the MABA potency. At 0.5 h and 1.5 h post aerosol exposure, the estimated MA potencies

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Fig. 3. Bronchoprotective effects of inhaled THRX-200495 (MA, BA, MABA) in the guinea pig Einthoven model 1.5 h after drug aerosol exposure. Abscissa represents the aerosol exposure dose of the test drug on a logarithmic scale. Ordinate represent percent inhibition of the bronchoconstrictor responses to methacholine (16 mg/kg, i.v.) in the presence of propranolol, MA pharmacological conditions; histamine (32 mg/kg, i.v.), BA pharmacological conditions; or methacholine (16 mg/kg, i.v.), MABA pharmacological conditions. All data are expressed as mean  SEM (n ¼ 6 per group). Data points without error bars represent an SEM less than the symbol size. *P < 0.05, **P < 0.01, ***P < 0.001 compared to MABA bronchoprotection.

Fig. 4. Duration of bronchoprotective activity of inhaled THRX-200495 (30 mg/mL) in anesthetized guinea gigs. All data are expressed as mean  SEM (n ¼ 4e6 per group). Abscissa represents the time (in hours) following aerosol exposure of the test compound. Ordinate represent percent inhibition of the bronchoconstrictor responses to methacholine (16 mg/kg, i.v.) in the presence of propranolol, MA pharmacological conditions; histamine (32 mg/kg, i.v.), BA pharmacological conditions; or methacholine (16 mg/kg, i.v.), MABA pharmacological conditions. Data points without error bars represent an SEM less than the symbol size. *P < 0.05, **P < 0.01, ***P < 0.001 compared to MABA bronchoprotection.

of the ipratropium/albuterol combination were 14.6 (9.2e23.3) and 4.3 (2.4e7.8) mg/mL respectively (Fig. 5, Table 3); At 0.5 h and 1.5 h post aerosol exposure the estimated BA potencies of the ipratropium/albuterol combination were 5.7 (1.9e2.4) and 159 mg/mL respectively (Fig. 5, Table 3). At 0.5 h and 1.5 h post aerosol exposure the estimated MABA potencies of the ipratropium/albuterol combination were 1.7 (1.1e2.7)/9.8 (6.3e15.4) mg/mL and 2.0 (0.8e5.0)/11.4 (4.6e28.5), respectively (Fig. 5, Table 3).

In this guinea pig model of bronchoprotection, co-formulation of ipratropium and albuterol demonstrated potent, short-lasting bronchoprotection which was consistent with the frequency of administration (q.i.d.) in patients. These results were also consistent with the reported preclinical efficacy of ipratropium and albuterol [33e35]. Albuterol was 2.6-fold more potent than ipratropium when the MA and BA components of ipratropium/albuterol were pharmacologically isolated at 0.5 h post aerosol exposure. These results were consistent with the potency of either drug administered alone in this guinea pig model of bronchoprotection [29]. This result suggests that the aerosol delivery of either drug is not influenced by co-formulation. Based on comparison to our previously demonstrated MA potency of ipratropium 1.5 h post aerosol exposure in the Einthoven model [29], the selected dose of propranolol (5 mg/kg, i.v.) in this study completely inhibited the BA contribution of albuterol in the ipratropium/albuterol combination. In addition, intravenous administration of propranolol (5 mg/kg) did not increase airway sensitivity to methacholine in the Einthoven model (Fig. 2). Clinically, ipratropium plus albuterol has proven more effective than either ipratropium or albuterol alone [14]. In the guinea pig model and when in combination with ipratropium, the BA potency

5. Discussion The present study demonstrated that a single molecule, THRX200495, possesses in vitro and in vivo characteristics consistent with dual activity as a muscarinic receptor antagonist and a b2adrenoceptor agonist (MABA). The MABA activity of THRX-200495 was compared to the combination of two short-acting MA and BA bronchodilators ipratropium and albuterol in a preclinical in vivo model of bronchoconstriction. Similar to ipratropium/albuterol, THRX-200495 engaged both MA and BA mechanisms in vivo. However, unlike what is observed with ipratropium/albuterol, the MA/BA balance of THRX-220495 and the additive effects of these two mechanisms remained unchanged up to 24 h.

Table 3 The bronchoprotective effects of THRX-200495 at 1.5 h post aerosol exposure, and the ipratropium/albuterol combination (at 0.5 and 1.5 h post aerosol exposure) in the guinea pig Einthoven model (ID50). The muscarinic antagonist (MA) and b2- adrenoceptor agonist (BA) and composite (MABA) bronchoprotections calculated as inhibition of methacholine (16 mg/kg) in the presence of propranolol, of histamine (32 mg/kg) and of methacholine (16 mg/kg) in the absence of propranolol, respectively. Results estimated ID50 values (95% CI), n ¼ 5e11. Bronchoprotection 0.5 h ID50 (mg/mL)

Bronchoprotection 1.5 h ID50 (mg/mL)

Compound

MA

BA

MABA

MA

BA

MABA

THRX-200495 Ipratropium/Albuterol

n.d. 14.6 (9.2e23.3)

n.d. 5.7 (1.9e2.4)

n.d. 1.7 (1.1e2.7)/9.8 (6.3e15.4)a

11.4 (7.8e16.7) 4.3 (2.4e7.8)

11.2 (7.2e18.6) 159

3.5 (2.9e4.2) 2.0 (0.8e5.0)/11.4 (4.6e28.5)a

n.d. ¼ not dosed. a Ipratropium/Albuterol MABA ID50 calculated as the drug concentration of ipratropium plus albuterol producing 50% inhibition of the bronchoconstrictor response.

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Fig. 5. Bronchoprotective effects of an inhaled ipratropium/albuterol combination (MA, BA, MABA) in the guinea pig Einthoven model 0.5 h (A) and 1.5 h (B). Abscissa represents the aerosol dose of the test drug on a logarithmic scale. Ordinate represent percent inhibition of the bronchoconstrictor responses to methacholine (16 mg/kg, i.v.) in the presence of propranolol, MA pharmacological conditions; histamine (32 mg/kg, i.v.), BA pharmacological conditions; or methacholine (16 mg/kg, i.v.), MABA pharmacological conditions. All data are expressed as mean  SEM (n ¼ 4e12 per group). Data points without error bars represent an SEM less than the symbol size. *P < 0.05, **P < 0.01, ***P < 0.001 compared to MA þ BA bronchoprotection.

of albuterol was modestly but significantly improved at the high dose. These results suggest that, given the 2.6:1 in vivo potency ratio of ipratropium:albuterol, the 1:5.7 co-formulation ratio of ipratropium/albuterol does not maximize the additive effects of the two mechanisms during the early acute phase of bronchoprotection. At 0.5 h and 1.5 h post aerosol exposure, the MABA potencies of ipratropium/albuterol were similar, however the slow onset of ipratropium and short duration of albuterol result in an MABA condition without temporal MA/BA balance. THRX-200495 displayed a pharmacological profile clearly distinct from that of ipratropium/albuterol. At 1.5 h post aerosol exposure, the molecule displayed potent and balanced MA and BA activities when the individual pharmacologies were studied in isolated fashion. As a result, the additive effects of the two mechanisms led to more potent bronchoprotection when both MA and BA pharmacologies were active and studied as an MABA. Furthermore, this prolife was time-independent and persisted for 24 h after a single aerosol exposure. Several features of this study may limit the interpretation and extrapolation of our results. First, multiple technical features of the in vitro assays as well as the intrinsic properties of the molecules could contribute to the apparent increase in b2- vs. b1-adrenoceptor selectivity for albuterol. In our opinion, explanation of the data requires consideration of i) the shift in functional potency relative to binding affinity which is frequently observed in assays using recombinant cells with high receptor density and/or a high receptor reserve and ii) the relative potencies of THRX-200495 (high) and albuterol (modest). The binding and functional assays confirmed that THRX-200495 exhibits subnanomolar affinity and potency for b2-adrenoreptor and selectivity over b1-adrenoceptor. Second, based on the intrinsically short duration of activity and with this a change in estimated potency of albuterol and ipratropium from 0.5 h to 1.5 h post aerosol exposure [29], the duration of the albuterol/ipratropium combination was not tested over the full 24 h time frame. Third, we did not directly compare THRX-200495 to a long-acting MA or BA bronchodilator. However, based on our previous studies [29], THRX-200495 is 2-fold less potent as a MA than tiotropium and 6-fold less potent as a BA than salmeterol in the Einthoven model. The long-lasting bronchoprotection of tiotropium and salmeterol in the Einthoven model was consistent

with established clinical dosing regimens [29]. Therefore, it is reasonable to assume that a combination of tiotropium and salmeterol would provide MA/BA bronchoprotection 24 h following aerosol exposure in the Einthoven model. Fourth, instead of intratracheal administration, we chose an inhalation dosing method using a whole body chamber to deliver the compounds. While nebulized delivery may better mimic the clinical setting [36,37], we did not characterize the aerosol or lung deposition in this study. Therefore, this approach does not lend itself to absolute quantitative comparisons of intratracheal instillation doses used in other preclinical studies [35,38]. However, we have previously demonstrated that nebulized delivery in the Einthoven model was predictive of the potency and duration of clinically used bronchodilators [29]. Moreover, by exploring the full dose range and by standardizing doses across compounds, we were able to accurately assess the relative potencies of the individual and combined components of the treatments. Lastly, by using a model of pharmacologically-induced bronchoconstriction we may have potentially underestimated the overall clinical benefits of a dual acting molecule, for example, specifically to reducing mucus secretion [39] and the airway inflammatory response [40e43]. Despite these limitations, this study provides confidence that MABA bronchoprotection is an improvement over MA þ BA bronchoprotection. A molecule with dual MA and BA pharmacology, like THRX200495, offers a fixed ratio of pharmacodynamics through a single compound pharmacokinetics thereby achieving temporal uniformity in efficacy at both the targets. This approach may manifest as subnanomolar potency and 24 h of bronchoprotection that is at least additive and potentially synergistic. It is worth noting that COPD patients may also require inhaled therapy with corticosteroids, and therefore patients would still be required to use multiple inhalers for maintenance therapy of their disease. An MABA molecule if co-administered with an ICS would simplify triple therapy in a single inhaler. In summary, this study provided in vivo evidence that the combination of muscarinic receptor antagonist and b2-adrenoceptor agonist activities yield additive bronchoprotection. Moreover, the current work provides insight into the potential benefits as well as limitations of fixed-dose combination therapy, specifically the necessity to optimize both

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