Background
Combining an inhaled corticosteroid (ICS) with a long-acting β
2-adrenoceptor (β
2-AR) agonist (LABA) is the cornerstone for the treatment of adult patients with asthma symptoms when a medium dose of ICS alone fails to achieve control of asthma [
1].The scientific rationale for inhaled combination therapy with β
2-AR agonists and corticosteroids has been debated for a long time [
2]. Already in the early 2000s, it was widely recognized that the addition of a LABA to an ICS provides the optimal control of asthma in most patients, and ICS/LABA fixed-dose combinations (FDCs) represent effective controllers in patients with persistent asthma [
3]. There is clear evidence that ICS/LABA FDC is superior to either drug given as monotherapy in the clinical management of moderate to severe asthma [
1,
4].
Previous works elucidated some mechanisms behind the additive ant-inflammatory effect of adding a LABA to a corticosteroid in the treatment of asthma [
5‐
9]. Nevertheless, to date there is still a large gap in the knowledge on how these two agents delivered in combination lead to superior clinical efficacy. In this regard, recent advances in multiscale modelling by using quasi-3D model may provide the opportunity of investigating how ICSs and LABAs interact each other with respect to their absorption, transport and retention into the lung at the level of lining liquid, epithelium, interstitium, airway smooth muscle (ASM), immune cells, and endothelium [
10].
Overall, β
2-AR agonists reduce the contractile tone of ASM, prevent plasma exudation, and inhibit the release of mediators from inflammatory cells and activation of sensory nerves. Conversely, corticosteroids reduce chronic inflammation and bronchial hyperresponsiveness (BHR) [
3]. Taken together, these effects allow achieving an adequate asthma control. However, the intimate interaction between the activation of membrane β
2-AR and intracellular glucocorticoid receptor (GR) is complex and not fully understood. Unquestionably, corticosteroids enhance the expression of β
2-AR and protect these receptors against down-regulation in response to chronic activation at the level of ASM cells, whereas β
2-AR agonists may increase the anti-inflammatory effects of corticosteroids at the level of inflammatory cells [
11].
Although each class of drug enhances beneficial actions induced by the other class, the doubts raised by Barnes and Giembycz [
3,
4] more than a decade ago on whether adding a LABA to an ICS results in an additive effect, or there is true synergy, are still current. In fact, little information is available on the real pharmacological characterization of ICS/LABA combination [
4].
Indeed, the concept of synergy is appealing and extensively used, as confirmed by the last update of the understanding how LABAs enhance the clinical efficacy of ICS in asthma [
12]. The increasing recognition that asthma and chronic obstructive pulmonary disease (COPD) are heterogeneous disorders has lead the attention to the needs of a group of patients with clinical features of both asthma and COPD, the so called asthma–COPD overlap syndrome (ACOS) [
13]. Nevertheless, to date it is widely recognized that it is premature to recommend the designation of ACOS as a disease entity in both primary and specialist care [
14]. In fact, more research is needed to adequately characterize patients and to obtain a validated definition of ACOS that would be based on markers that best predict treatment response in individual patients [
15]. Thus, in agreement with the current international recommendations for the diagnosis and treatment of asthma and COPD [
1,
16], ICS/LABA FDCs remain the cornerstone therapy for most asthmatic patients rather then COPD patients.
Nevertheless, to date only two studies [
5,
17] have assessed the interaction between a β
2-AR agonist and a corticosteroid by applying correct pharmacological models. These original researches [
5,
17] were carried out in murine models of allergic lung inflammation, and not in human airways. While the synergism between bronchodilator agents has been confirmed in clinical studies [
18,
19] by using specific pharmacological modeling of drug interaction, to date no clinical trials aimed to assess the potential clinical synergy between ICSs and LABAs have been carried out by using these models, namely the Bliss Independence equation and the Unified Theory [
20].
Therefore, the aim of this study was to pharmacologically characterize the impact of beclomethasone dipropionate (BDP) on the bronchorelaxant effect of formoterol fumarate (FF), administered at the concentration-ratio delivered by the currently available FDC in the marked, in human medium bronchi and small airways by using a validated ex vivo model of bronchial asthma.
Discussion
The results of this study confirmed that FF is a potent and effective bronchorelaxant agent at the level of medium bronchi, as previously reported by studies carried out in 1990s [
59,
60]. Although in a previous study we documented that FF is able to abolish the contraction of small airways induced by acetylcholine [
47], in the present research FF did not completely relax bronchioles submaximally pre-contracted by histamine. However, we have provided for the first time the evidence that FF is ≃0.5 logarithm more potent in passively sensitized than in non-sensitized airways and completely abolishes the histaminergic tone of passively sensitized PCLS. These evidences indicate that FF has a beneficial bronchorelaxant impact especially in human hyperresponsive airways. On the contrary, the results indicated that the overnight incubation with BDP did not modulate the bronchial contractility induced by histamine in both medium and small airways, even after passive sensitization procedure.
Conversely, combining BDP with FF at 100:6 concentration-ratio not only improved the effectiveness of the β2-AR agonist, but elicited a synergistic bronchorelaxant effect in both medium and small airways, either non-sensitized or passively sensitized.
The BI analysis showed that the in non-sensitized tissues the concentration of drugs mixture necessary to induce the maximal synergistic interaction in bronchioles was ≃2 logarithms higher than that necessary to elicit the greatest synergism in medium bronchi. The total concentration of BDP/FF combination required to induce submaximal relaxation of non-sensitized medium bronchi was in the order of magnitude of ng/ml, whereas in small airways the order of magnitude to elicit the same effect was in the range of μg/ml. On the other hand, in passively sensitized tissues low concentrations of BDP/FF combination, in the order of magnitude of ng/ml, were sufficient to submaximally relax both medium and small airways. These findings prove that passively sensitized small airways are more sensitive to the beneficial synergistic interaction induced by adding BDP to FF, compared to non-sensitized bronchioles.
The BI approach showed that the BDP/FF 100:6 combination-ratio induced statistically significant synergism, and permitted to quantify the overall extent of bronchodilation and compare the observed synergism with the expected additive effect. Nevertheless, the BI equation did not allow assessing what is the real magnitude of synergism occurring between BDP and FF. At concentrations of drugs mixture corresponding to EC
50 or below it is not difficult to detect synergism, whereas a synergistic interaction may be more difficult to be detected at higher concentrations producing a submaximal effect. Therefore, after have performed the statistical analysis of the synergism between BDP and FF by using the BI model, we have carried out a further evaluation by applying the Unified Theory in order to quantify the magnitude of the interaction [
57].
The analysis of the logarithmic Combination Index plots and isobolograms evidenced that in medium bronchi, either non-sensitized or passively sensitized, BDP/FF 100:6 combination-ratio elicited a certain level of bronchorelaxant synergism, even at high concentrations. In non-sensitized small airways low concentrations induced additive effect, and very high concentrations were necessary to elicit synergistic interaction. Surprisingly, in passively sensitized small airways, even very small total concentrations of BDP/FF combination, ranging between 1.06 ng/ml and 10.6 ng/ml, were effective in producing a marked synergistic relaxation of ASM. Certainly the increased acute bronchorelaxant effect of FF in isolated airways incubated overnight with BDP can be prevalently due to the genomic effect of the corticosteroid [
61,
62], although investigating this matter was beyond the scope of our study.
The findings of this study are undoubtedly interesting, considering the potential translational implications. In fact, we have previously demonstrated that the relaxant effect of bronchodilator agents detected ex vivo in human medium isolated airways was related with their impact in vivo on the changes in forced expiratory volume in 1 s (FEV
1) [
63]. Furthermore, the luminal area of small airways studied by using PCLS with an internal diameter < 2 mm is related with the flow in small airways that, in turn, seems to be associated with the forced expiratory flow between 25% and 75% of vital capacity (FEF 25–75) [
64,
65]. Overall, the flow resistance in medium and small bronchi contributes to the total airway resistance in asthmatic patients, which may influence also the alveolar moiety as demonstrated by the measurement of nitric oxide at different flow rates [
66‐
68].
Lower airways significantly contribute to the severity of chronic obstructive pulmonary disorders, such as asthma and COPD [
69]. The dysfunction of bronchioles has been also demonstrated in specific asthma phenotypes, namely nocturnal asthma, exercise-induced asthma, and allergic asthma [
70,
71]. Thus, delivering an adequate amount of inhaled ICS/LABA combination to distal airways represents a central target to treat asthmatic patients. However, although the new generation of inhaler devices emitting extrafine formulations have lead to an improved lung deposition of drugs mixture, and a more effective aerosol penetration into the lung periphery [
70,
72], in asthmatic patients approximately two-thirds of extrafine formulation of BDP/FF FDC are deposited in the central lung region, and only one-third reaches the peripheral lung [
73,
74]. Therefore, it is crucial that the amount of drugs mixture that reaches small airways is effective at concentrations lower than those detectable in larger airways, and that the extent of effectiveness remains sustained also at the low concentrations that are present in the airways immediately before the next dose is inhaled.
In this regard, the very strong synergistic interaction elicited by low concentrations of BDP/FF administered at 100:6 combination-ratio in hyperresponsive small airways may explain the superiority of BDP/FF FDC (400/24 μg daily) delivered via an extrafine formulation in improving asthma control, compared to the combination of the same drugs formulated as larger non-extrafine agents administered at equipotent doses [
75]. Furthermore, considering lung functional parameters related with peripheral airway dysfunction, extrafine BDP/FF FDC treatment was superior to an equipotent dose of the non-extrafine fluticasone propionate/salmeterol combination in improving air trapping in moderate to severe asthmatic patients [
76]. The beneficial impact of BDP/FF FDC (400/24 μg daily) on small airways in asthma was further confirmed by a pilot study that demonstrated an improvement in closing capacity after 12 weeks of treatment [
71]. Intriguingly, in this study [
71] the authors also evidenced that BDP/FF FDC was effective in decreasing the BHR of larger airways. This finding indirectly confirms the results of our study, with regard to the evidence that combining an ICS with a LABA can target both medium and small hyperresponsive airways, leading to bronchorelaxant synergistic interaction.
Although the data of the present study result from a widely validated human model of non-specific BHR typical of bronchial asthma [
23,
36,
44,
77‐
80], this research remains an ex vivo study characterized by intrinsic limitations [
27,
57]. The findings of ex vivo studies aimed to assess the pharmacological interaction between drugs characterized by different mechanisms of action [
81] have been generally confirmed by a translational approach in clinical trials [
18,
19], and vice versa [
82,
83]. Nevertheless, we must highlight that, although the responses of human isolated ASM strictly resembles those elicited in vivo, results obtained from ex vivo studies need to be confirmed by clinical trials specifically designed to detect pharmacological interaction of drugs mixture [
57,
82].
One of the major objectives of having synergistic drug combinations is to reduce the dose of the drugs used, thereby reducing the risk of adverse events, while optimizing the efficacy [
57,
58]. In this respect, our data support the delivery of BDP/FF 100:6 combination-ratio via extrafine formulation to reduce the total dose of the monocomponents, improve the distribution in the lung, and optimize the effectiveness, compared to non-extrafine formulations. This approach permits to reduce the deposition of drugs mixture in the oropharynx, resulting in decreased systemic absorption through the gastrointestinal tract and obvious beneficial consequences on the safety profile [
84].
A recent and extensive review of Newton and Giembycz [
12] attempted to explain how LABAs enhance the clinical efficacy of ICS in asthma. The authors reported that combining an ICS with a LABA might produce profound synergy at the level of genes and proteins expression involved in ASM contractility and airway inflammation [
12]. Unfortunately, the original studies [
61,
85‐
88] cited in that review [
12] to support the positive interaction between ICSs and LABAs provided arbitrary interpretation of synergy because no methods were used to adequately analyze the real drug interaction. Furthermore, the functional impact of combining a corticosteroid with a β
2-ARs was indirectly assessed by using cytosolic surrogate of ASM contractility [
86]. In any case, even assuming that an ICS/LABA combination can modulate the expression of genes and proteins in asthmatic ASM, it is not implicit that such an interaction may lead to functional synergy [
62]. Therefore, at the best of our knowledge, the present study provides for the first time the evidence-based pharmacological characterization of the synergism between an ICS and a LABA, at least with regard to the beneficial impact against BHR in human airways.
Competing interests
LC 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, Edmond Pharma, Zambon, Verona Pharma, and Ockham Biotech. His department was funded by Almirall, Boehringer Ingelheim, Novartis, Zambon and Chiesi Farmaceutici.
MGM has participated as a lecturer 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.
FF has no conflict of interest to declare.
MC has participated as a lecturer 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, Edmond Pharma, Chiesi Farmaceutici, Lallemand, Novartis, Verona Pharma, and Zambon. His department was funded by Almirall, Boehringer Ingelheim, Novartis, and Zambon.
PR participated as a lecturer 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.