Beclomethasone dipropionate, formoterol fumarate and glycopyrronium bromide: Synergy of triple combination therapy on human airway smooth muscle ex vivo

Abstract

Background and purpose: Combining inhaled corticosteroids (ICSs), long-acting β2 -adrenoceptor agonists (LABAs) and long-acting muscarinic antagonists (LAMAs) is recommended to treat severe forms of asthma and chronic obstructive pulmonary disease (COPD). Clinical benefits have been demonstrated for ICS/LABA/LAMA combinations. This study characterized the interaction between the ICS beclomethasone dipropionate, the LABA formoterol fumarate and the LAMA glycopyrronium bromide in human airways.

Experimental approach: Human passively sensitized airways and bronchi from COPD donors were stimulated with histamine or carbachol. Tissues were incubated overnight with beclomethasone and then treated with formoterol and glycopyrronium, alone or in triple combination. The interaction was assessed by using Bliss Independence and Unified Theory theorems.

Key results: Beclomethasone/formoterol/glycopyrronium combination synergistically relaxed medium bronchi and small airways. Beclomethasone/formoterol/glycopyrronium combination at 100:6:12.5 combination ratio was a balanced drug mixture leading to very strong synergistic effect on relaxation of medium bronchi (Combination Index: from 0.042 to 0.96) and middle to very strong synergy in small airways (Combination Index: from 0.018 to 0.310). The synergy was related with the activation of intracellular glucocorticoid receptors and Gsα subunit G-protein of β2 -adrenoceptors, leading to the modulation of cyclic AMP-dependent PKA pathway.

Conclusion: Triple beclomethasone/formoterol/glycopyrronium combination induces synergistic bronchorelaxant effect in medium and small human airways, at least in ex vivo experiments. Further research is needed to confirm these findings in clinical studies in patients with asthma or COPD.

Conflict of interest statement

P.R. participated as a lecturer, speaker and advisor in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, Mundipharma, and Novartis. Her department was funded by Almirall, Boehringer Ingelheim, Novartis, Zambon, and Chiesi Farmaceutici. M.G.M. has participated as a lecturer, speaker and advisor in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, and Novartis and has been a consultant to Chiesi Farmaceutici. F.F. has no conflict of interest to declare. C.P. has acted as a Consultant for Recipharma, ImmunoRegulation, PrEP Biopharma, Ockham Biosciences and Eurodrug. C.P. also owns equity in Verona Pharma who are developing novel drugs for the treatment of respiratory diseases. M.C. has participated as a lecturer, speaker and advisor in scientific meetings and courses under the sponsorship of Almirall, AstraZeneca, Biofutura, Boehringer Ingelheim, Chiesi Farmaceutici, GlaxoSmithKline, Menarini Group, Lallemand, Mundipharma, Novartis, Pfizer, Verona Pharma, and Zambon and has been a consultant to ABC Farmaceutici, Recipharm, Chiesi Farmaceutici, Lallemand, Novartis, Verona Pharma, and Zambon. His department was funded by Almirall, Boehringer Ingelheim, Novartis, and Zambon. L.C. has participated as advisor in scientific meetings under the sponsorship of Boehringer Ingelheim and Novartis, received non‐financial support by AstraZeneca, received a research grant partially funded by Chiesi Farmaceutici, Boehringer Ingelheim, Novartis and Almirall and is or has been a consultant to ABC Farmaceutici, Recipharm, Zambon, Verona Pharma, and Ockham Biotech. His department was funded by Almirall, Boehringer Ingelheim, Novartis, Zambon, and Chiesi Farmaceutici.

© 2019 The British Pharmacological Society.

Figures

Figure 1

Bronchorelaxant effect of monocomponents and triple combination therapy in passively sensitized human airways submaximally contracted by histamine: The yellow bars refer to the effect of beclomethasone alone, the blue bars refer to effect of formoterol alone, the orange bars refer to the effect of GB alone, the green bars refer to the effect of beclomethasone/formoterol/glycopyrronium administered in combination at 100:6:12.5 combination ratio, and the grey bars refer to the expected effect accordingly with the Bliss Independence model (a and b). Delta effect between the observed and expected relaxant response (c and d) and graphical representation of Unified Theory analysis via logarithmic Combination Index plot (e and f) for beclomethasone/formoterol/glycopyrronium administered at 100:6:12.5 combination ratio in medium (a, c, e) and small (b, d, f) passively sensitized human airways submaximally contracted by histamine. *P < .05 versus the expected additive relaxant effect as predicted by Bliss Independence equation (statistical significance assessed by unpaired Student's t‐test); #P < .05 versus the expected additive relaxant effect as predicted by Bliss Independence equation (statistical significance assessed by two‐way ANOVA). Points represent the mean ± SEM of n = 5 subsegmental bronchi from different subjects. BDP, beclomethasone dipropionate; CI, combination index; EC70, concentration inducing 70% Emax; Emax, maximal effect; Fa, fraction affected; FF, formoterol fumarate; GB, glycopyrronium bromide; His, histamine

Figure 2

Bronchorelaxant effect of monocomponents and triple combination therapy in human airways collected from COPD donors submaximally contracted by carbachol: The yellow bars refer to the effect of beclomethasone alone, the blue bars refer to effect of formoterol alone, the orange bars refer to the effect of glycopyrronium alone, the green bars refer to the effect of beclomethasone/formoterol/glycopyrronium administered in combination at 100:6:12.5 combination ratio, and the grey bars refer to the expected effect accordingly with the Bliss Independence model (a and b). Delta effect between the observed and expected relaxant response (c and d) and graphical representation of Unified Theory analysis via logarithmic Combination Index plot (e and f) for beclomethasone/formoterol/glycopyrronium administered at 100:6:12.5 combination ratio in medium (a, c, e) and small (b, d, f) human airways collected from COPD donors and submaximally contracted by carbachol. *P < .05 versus the expected additive relaxant effect as predicted by Bliss Independence equation (statistical significance assessed by unpaired Student's t‐test); #P < 0.05 versus the expected additive relaxant effect as predicted by Bliss Independence equation (statistical significance assessed by two‐way ANOVA). Points represent the mean ± SEM of n = 5 subsegmental bronchi from different subjects. BDP, beclomethasone dipropionate; CCh, carbachol; CI, combination index; EC70, concentration inducing 70% Emax; Emax, maximal effect; Fa, fraction affected; FF, formoterol fumarate; GB, glycopyrronium bromide

Figure 3

Effect of adding beclomethasone 10 ng·ml−1 to the formoterol/glycopyrronium combination administered at 0.6:1.25 ng·ml−1 in passively sensitized human airways submaximally contracted by histamine (a) and airways collected from COPD donors submaximally contracted by carbachol. *P < .05, versus formoterol/glycopyrronium combination (statistical significance assessed by unpaired Student's t‐test). Bars represent the mean ± SEM of n = 5 subsegmental bronchi from different subjects. BDP, beclomethasone dipropionate; CCh, carbachol; EC70, concentration inducing 70% Emax; Emax, maximal effect; FF, formoterol fumarate; GB, glycopyrronium bromide; His, histamine

Figure 4

Impact of inhibition of intracellular glucocorticoid receptors and β2‐AR pathways, by RU486 1 μM and NF449 20 μM, respectively, on the bronchorelaxant synergistic interaction elicited by beclomethasone/formoterol/glycopyrronium combination administered at 10/0.6/1.25 ng·ml−1 in passively sensitized human bronchi stimulated by histamine (a, asthma model) and at 3/0.18/0.375 ng·ml−1 in human airways collected from COPD donors stimulated by carbachol (b, stable COPD model). *P < .05 versus beclomethasone/glycopyrronium expected additive effect as predicted by Bliss Independence equation (statistical significance assessed by unpaired Student's t‐test). Bars represent the mean ± SEM of n = 5 subsegmental bronchi from different subjects. β2‐AR, β2‐adrenoceptor; BDP, beclomethasone dipropionate; CCh, carbachol; EC70, concentration inducing 70% Emax; Emax, maximal effect; FF, formoterol fumarate; GB, glycopyrronium bromide; His, histamine; NF449, selective Gsα subunit G‐protein antagonist; RU486, high affinity antagonist of the intracellular glucocorticoid receptors