The Bronchoprotective Effects of Dual Pharmacology, Muscarinic Receptor Antagonist and β2 Adrenergic Receptor Agonist Navafenterol in Human Small Airways

Bronchodilators and anti-inflammatory agents are the mainstream treatments in chronic obstructive and pulmonary disease (COPD) and asthma. The combination of β2 adrenergic receptor (β2AR) agonists and muscarinic antagonists shows superior bronchoprotective effects compared to these agents individually. Navafenterol (AZD8871) is a single-molecule, dual pharmacology agent combining muscarinic antagonist and β2AR agonist functions, currently in development as a COPD therapeutic. In precision-cut human lung slices (hPCLS), we investigated the bronchoprotective effect of navafenterol against two non-muscarinic contractile agonists, histamine and thromboxane A2 (TxA2) analog (U46619). Navafenterol pre-treatment significantly attenuated histamine-induced bronchoconstriction and β2AR antagonist propranolol reversed this inhibitory effect. TxA2 analog-induced bronchoconstriction was attenuated by navafenterol pre-treatment, albeit to a lesser magnitude than that of histamine-induced bronchoconstriction. Propranolol completely reversed the inhibitory effect of navafenterol on TxA2 analog-induced bronchoconstriction. In the presence of histamine or TxA2 analog, navafenterol exhibits bronchoprotective effect in human airways and it is primarily mediated by β2AR agonism of navafenterol.


Introduction
Asthma and COPD are chronic airway disorders characterized by airway hyperresponsiveness, inflammation and remodeling. Bronchodilators are the critical components of the therapeutic management of asthma and COPD. In COPD, the combination of a long-acting muscarinic antagonist (LAMA) and a long-acting β 2 adrenergic agonist (LABA) elicited bronchodilation superior to that by monotherapy with either of these agents [1,2]. Several small, single molecules with dual action-muscarinic antagonism and β 2 adrenergic agonism (MABA)-have been developed in the past [3][4][5]. Navafenterol (AZD8871, LAS191351) is an inhaled MABA currently in development for the treatment of COPD. In a phase IIa clinical trial (NCT02971293), once daily administration of navafenterol showed significant and clinically meaningful improvements in lung function-related end points in moderate to severe COPD patients compared with placebo [6]. In a further phase IIa clinical trial (NCT03645434), navafenterol demonstrated improved lung function and a reduction in COPD-related symptoms, similar to the established LAMA/LABA fixed dose combination umeclidinium/vilanterol in patients with moderate to severe COPD. Previous ex vivo studies used human bronchi and guinea pig trachea stimulated by electrical filed stimulation (EFS) to functionally dissect the muscarinic antagonism from the β 2 adrenergic agonism of navafenterol [7]. In this study, we used precision-cut human lung slices (hPCLS) to functionally dissect the β 2 adrenergic agonism of navafenterol. Compared to in vivo animal models and ex vivo studies using animal lung tissue, hPCLS yield physiologically relevant findings with translational significance. Instead of EFS stimulation, we used two physiologically relevant contractile agonists (histamine and a thromboxane A 2 analog) in the presence of the β adrenergic blocker propranolol to functionally isolate the β 2 adrenergic agonism of navafenterol. Since navafenterol is a known muscarinic antagonist, we used non-muscarinic contractile agonists to examine the β 2 AR agonism of this drug. With a focus on β 2 AR agonism, our findings advance the pharmacological characterization of navafenterol using a physiologically relevant ex vivo platform and supplement other preclinical studies on this investigative drug.

Human Precision-Cut Lung Slices (hPCLS)
Precision-cut lung slices were prepared from normal human donor lungs (n = 6) as previously described [8]. These samples are exempt from the IRB approval requirement since they are de-identified human tissue. Donor demographics are listed in Table 1.

Reagents
Navafenterol was provided by AstraZeneca. Propranolol HCl, diluent DMSO, histamine dihydrochloride and thromboxane A 2 analog (U46619) were obtained from Sigma Aldrich (St. Louis, MO, USA). HAM/F-12 cell culture medium, PBS and media supplements were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

Reconstitution of Reagents and Exposure Protocol
Navafenterol (10 mM) was reconstituted in DMSO and stored at −20 • C. Propranolol HCl (10 mM) and histamine dihydrochloride (10 mM) were freshly prepared in sterile HAM/F-12 cell culture medium without serum. Thromboxane A 2 analog (U46619, 10 mg/mL, 28.5 mM) was supplied in methyl acetate and stored at −20 • C. Ten-fold serial dilutions of U46619 or histamine were prepared in HAM/F-12 cell culture medium without serum. Slices were treated with 0.1% DMSO or navafenterol (3, 10, 30, 100 and 300 nM) for 1 h.

Generation of Concentration Response Curves
Following exposure to navafenterol or vehicle, slices were exposed to incremental concentrations of histamine (10 −10 M to 10 −4 M) or U46619 (10 −10 M to 10 −5 M). Slices were incubated in each concentration for 5 min in the continued presence of navafenterol or vehicle. In a subset, 10 µM propranolol was co-incubated with each concentration of the contractile agonist to block β 2 AR. The airway lumens were captured and analyzed using Image J as previously described [8]. Briefly, the airway lumen images were captured after incubation with each concentration using an inverted light microscope-linked camera (40× magnification). The luminal areas were measured in each airway with Image J. The change in airway lumen area was calculated as the percentage of baseline area of each airway (percentage bronchoconstriction. Figure S1 shows representative images of histamine-induced bronchoconstriction from a single donor).

Sample Size and Data Analysis
Human PCLS from at least 5 independent lung donors (donor characteristics are provided in Table 1) were used in each experiment. From each donor, 3 slices (technical replicates) were used for each treatment. The mean or mean± SEM of each experimental condition are presented in the graph. The means were statistically compared using GraphPad Prism 9.0, with one-way ANOVA and Dunnett's test for multigroup comparisons or unpaired, two-tailed Student's t-test for two-group comparisons. The means were considered significantly different if p < 0.05.

Effect of Navafenterol on Histamine-Induced Bronchoconstriction
Navafenterol (3-300 nM) attenuated histamine-induced bronchoconstriction in a concentration-dependent manner ( Figure 1A,B). In the presence of the β 2 AR blocker propranolol (10 µM), navafenterol had little effect on histamine-induced bronchoconstriction at lower (30 and 100 nM) concentrations ( Figure 1C,D). However, the highest concentration (300 nM) of navafenterol still attenuated histamine-induced bronchoconstriction in the presence of propranolol. The potency of histamine (p[EC] 50 ) was not significantly different in the presence or absence of propranolol ( Figure 1E). In slices treated with navafenterol (30 and 100 nM), propranolol increased the potency of histamine compared to the slices not treated with propranolol ( Figure 1E). With or without propranolol, histamine-induced maximal contraction (E max ) was not significantly different in the presence of navafenterol ( Figure 1F).

Effect of Navafenterol on Thromboxane-Induced Bronchoconstriction
Our findings show that navafenterol had H 1 receptor antagonism at the highest concentration. To functionally isolate the β 2 AR agonism of navafenterol, we measured the effect of navafenterol on thromboxane A 2 analog-induced bronchoconstriction. Navafenterol attenuated thromboxane-induced bronchoconstriction only at the highest concentration (300 nM) (Figure 2A,). Blocking of β 2 AR by propranolol completely reversed the inhibitory effect of navafenterol ( Figure 2C,D). Thromboxane A 2 analog-induced bronchoconstriction at the baseline conditions was not affected by propranolol. However, in the presence of navafenterol, propranolol slightly enhanced the potency of thromboxane ( Figure 2E). Navafenterol had little effect on thromboxane-induced maximal bronchoconstriction (E max ) in the presence or absence of propranolol ( Figure 2F).

Discussion
Navafenterol is a novel, single-molecule, dual pharmacology bronchodilator combining muscarinic (M 3 ) cholinergic receptor antagonist and β 2 AR agonist functions [7]. Previous pharmacological characterizations of navafenterol were performed in isolated human bronchi and guinea pig tracheal rings using electrical field stimulation (EFS). Studies also focused on guinea pig and canine models to assess the bronchoprotective and off-target effects of navafenterol. The objective of the current study is to functionally dissociate the β 2 AR agonism of navafenterol from its reported antagonism towards histamine (H 1 ) receptors.
Human PCLS is an innovative ex vivo platform to characterize investigational drugs, toxicants and infectious agents. The direct physiological measurements from small airways in hPCLS have immense physiological and translational values compared to other preclinical models. The current study is yet another piece of evidence that demonstrates the application of hPCLS in the pharmacological characterization of a drug targeting distinct receptors in human airways.
A number of investigational drugs with combined muscarinic antagonist and β 2 adrenergic agonist activities have been developed [3,5,9]. These small molecules are combined antagonist and agonist entities connected by a chemical linker with the structure of the linker influencing the balance between muscarinic antagonist and β 2 AR agonist functions. In isolated human bronchi, navafenterol showed more dominant muscarinic antagonism than β 2 AR agonism compared to batefenterol (GSK961081), another MABA [7]. The same study identified navafenterol to have moderate affinity for histamine H 1 receptors (IC 50 = 85 nM, p[IC] 50 = 7.1). Our findings support this observation by showing that 300 nM of navafenterol retains the inhibitory effect on bronchoconstriction in the presence of propranolol with a similar measure of compound affinity (pA 2 ) of 7.5 ( Table 2). Thromboxane is one of the several prostanoids with roles in airway inflammation and hyperresponsiveness (reviewed in [10]). Acting through thromboxane prostanoid (TP) receptors, TxA 2 signals through G αq/11 , mobilizing cytosolic Ca 2+ and eliciting bronchoconstriction. Thromboxane A 2 analog-induced bronchoconstriction was used to further demonstrate the β 2 AR agonism of navafenterol. In contrast to histamine-induced bronchoconstriction, the inhibitory effect of navafenterol on TxA 2 analog-induced airway narrowing was modest, showing significant inhibition only at the highest concentration of the drug. However, the complete reversal of that navafenterol inhibition in the presence of propranolol suggests that thromboxane prostanoid (TP) receptors are not antagonized by navafenterol.
In summary, we have functionally distinguished the histamine antagonism of navafenterol from its β 2 AR agonism using two distinct contractile agonists in human small airways. These findings demonstrate that the bronchoprotective effect of navafenterol in human small airways is primarily mediated through β 2 AR agonism (summarized in Figure 3). Further studies are required to determine if the H 1 antagonism seen with navafenterol may contribute to a therapeutically meaningful bronchoprotective effect in diseases where histamine may play a role.
presence of propranolol suggests that thromboxane prostanoid (TP) receptors are not antagonized by navafenterol.
In summary, we have functionally distinguished the histamine antagonism of navafenterol from its 2AR agonism using two distinct contractile agonists in human small airways. These findings demonstrate that the bronchoprotective effect of navafenterol in human small airways is primarily mediated through 2AR agonism (summarized in Figure 3). Further studies are required to determine if the H1 antagonism seen with navafenterol may contribute to a therapeutically meaningful bronchoprotective effect in diseases where histamine may play a role. Funding: This study was funded by AstraZeneca.
Institutional Review Board Statement: Ethical review and approval were waived for this study due to the use of de-identified human tissue (IRB-exempt).
Informed Consent Statement: Patient consent was waived due to the use of de-identified human tissue.
Data Availability Statement: Data is contained within the article and supplementary material. Original numerical data will be made available upon reasonable request.
Acknowledgments: This study was supported by a sponsored research agreement with Astra-Zeneca.

Conflicts of Interest:
Reynold Panettieri reports financial support was provided by Astra-Zeneca. Reynold Panettieri reports a relationship with AstraZeneca that includes consulting or advisory roles, funding grants, and speaking and lecture fees. Ian Dainty reports a relationship with Astra-Zeneca that includes employment and equity or stocks. Joseph Antony Jude, Nikhil Karmacharya and William Jester declare no conflicts of interest. Funding: This study was funded by AstraZeneca.
Institutional Review Board Statement: Ethical review and approval were waived for this study due to the use of de-identified human tissue (IRB-exempt).
Informed Consent Statement: Patient consent was waived due to the use of de-identified human tissue. Data Availability Statement: Data is contained within the article and supplementary material. Original numerical data will be made available upon reasonable request.