Periodic Reporting for period 4 - MaeBAia (Mechanisms of adverse effects of Beta-Agonists in Asthma)
Reporting period: 2023-04-01 to 2024-09-30
Summary of the context and overall objectives of the project
β2-agonists are a mainstay in the treatment of asthma. They promote bronchodilatation by inducing cyclic adenosine monophosphate (cAMP) production to promote airway smooth muscle relaxation and thereby provide symptomatic relief from bronchoconstriction. However, they are associated with adverse respiratory effects when overused or inappropriately used in the absence of inhaled corticosteroids (ICS) in patients with asthma, resulting in increased morbidity and mortality. The mechanisms underlying their adverse effects in asthma remain poorly understood. Developing an understanding of such mechanisms will be critical to improve current prescribing practices, change the way in which these medications are administered and thus improve clinical outcomes in patients with asthma.
Previous work has demonstrated that β2-agonists may induce the production of asthma relevant pro-inflammatory mediators in bronchial epithelial cells (BECs) via a cAMP response element (CRE) in the promoter region of the mediators’ genes. Many mediators implicated in asthma pathogenesis are inducible via cAMP and are therefore likely to be induced by β2-agonists. It is therefore hypothesised that β2-agonists cause adverse effects in people with asthma through induction of cAMP-induced pro-inflammatory cytokines, chemokines, remodelling associated factors, mucins and other mediators relevant to asthma.
The subsequent increase in localised inflammation in the lungs worsens disease control and increases asthma-associated morbidity. Concomitant administration of ICS abolishes this proinflammatory effect of β2-agonists by blocking the cAMP/CRE-mediated adverse effects of β2-agonists, thus attenuating local airway inflammation and improving clinical outcomes.
Overall objectives:
This research seeks to investigate these mechanisms through: in vitro studies in which BECs are stimulated with various β2-agonists to measure the subsequent induction of pro-inflammatory mediators; through ex vivo studies in which primary BECs derived from patients with asthma are stimulated with β2-agonist prior to RNA-sequencing to identify upregulation of asthma-relevant cAMP-responsive RNAs; and through a clinical study in which patients with asthma are treated with salmeterol followed by salmeterol/fluticasone to identify the effects of β2-agonists on inflammatory mediator induction and asthma control in vivo.
The in vitro studies have been completed and have demonstrated that a range of clinically relevant β2-agonists significantly induce interleukin(IL)-6, IL-11 and brain-derived neurotrophic factor (BDNF) in BECs and that they further potentiate rhinovirus-induction of these mediators. The addition of corticosteroid abolishes these effects.
These data lend support to the current hypothesis but the clinical significance of these effects in vivo remains to be seen and this forms the basis of the ongoing ex vivo studies and clinical trial (the MAELABA study) being executed at the moment to determine if these effects are seen in the human lung.
The MAELABA study has the following aims:
(1) To determine whether long-acting β2-agonist (LABA) monotherapy with salmeterol induces disease-relevant inflammatory mediators in the airways of asthma patients in vivo
(2) To determine whether combined LABA/inhaled corticosteroid (ICS) therapy with salmeterol/fluticasone abolishes the induction of disease-relevant inflammatory mediators in the airways of asthma patients in vivo
OUTCOME MEASURES Primary outcomes:
(1) Change from baseline in gene expression of disease-relevant pro-inflammatory mediators in bronchial epithelial cells (BECs) and bronchoalveolar lavage (BAL) cells and change from baseline in protein levels of disease-relevant pro-inflammatory mediators in BAL fluid and airway lining fluid following 2 weeks of salmeterol monotherapy
(2) Change from baseline in gene expression of disease-relevant pro-inflammatory mediators in BECs and BAL cells and change from baseline in protein levels of disease-relevant pro-inflammatory mediators in BAL fluid and airway lining fluid following 2 weeks of salmeterol/fluticasone combination therapy
Secondary outcomes:
Changes from baseline in the following parameters at the end of 14 days of salmeterol monotherapy and at the end of 14 days of salmeterol/fluticasone combination therapy: lung function; exhaled nitric oxide (FeNO) level; airway hyperresponsiveness (AHR); asthma symptom severity; serum BDNF and platelet BDNF concentration
POPULATION 24 adult participants with asthma
Hypothesis:
This trial seeks to test the following hypotheses:
1) The administration of 2 weeks of the LABA salmeterol in asthmatic patients will result in an increase in cAMP-mediated induction of disease-relevant pro-inflammatory cytokines, chemokines, remodelling associated factors and mucins in BECs and/or BAL cells, which in turn will cause enhanced mucosal inflammation, airway obstruction and AHR.
2) The administration of 2 weeks of salmeterol in combination with the ICS fluticasone will abolish the induction of the cAMP-mediated inflammatory markers in BECs and/or airway macrophages, resulting in reduced mucosal inflammation, airway obstruction and AHR.
Implications of this study:
If the hypotheses are proven correct, it will provide clear evidence of the mechanisms underlying the adverse effects of salmeterol in asthmatic patients and provide further justification for why salmeterol should not be used in the treatment of asthma unless it is combined with ICS in the same inhaler. This will have a significant impact on current clinical practice and will have the potential to reduce the significant morbidity and mortality that occurs with salmeterol monotherapy in asthmatics. The findings will also be of relevance to other airway diseases such as chronic obstructive pulmonary disease (COPD), where LABA therapy in the absence of ICS in the same inhaler also occurs.
This in vitro and in vivo work combined will lead to better understanding of adverse effects of the most common asthma treatments, and therefore better clinical outcomes for asthma patients.
Previous work has demonstrated that β2-agonists may induce the production of asthma relevant pro-inflammatory mediators in bronchial epithelial cells (BECs) via a cAMP response element (CRE) in the promoter region of the mediators’ genes. Many mediators implicated in asthma pathogenesis are inducible via cAMP and are therefore likely to be induced by β2-agonists. It is therefore hypothesised that β2-agonists cause adverse effects in people with asthma through induction of cAMP-induced pro-inflammatory cytokines, chemokines, remodelling associated factors, mucins and other mediators relevant to asthma.
The subsequent increase in localised inflammation in the lungs worsens disease control and increases asthma-associated morbidity. Concomitant administration of ICS abolishes this proinflammatory effect of β2-agonists by blocking the cAMP/CRE-mediated adverse effects of β2-agonists, thus attenuating local airway inflammation and improving clinical outcomes.
Overall objectives:
This research seeks to investigate these mechanisms through: in vitro studies in which BECs are stimulated with various β2-agonists to measure the subsequent induction of pro-inflammatory mediators; through ex vivo studies in which primary BECs derived from patients with asthma are stimulated with β2-agonist prior to RNA-sequencing to identify upregulation of asthma-relevant cAMP-responsive RNAs; and through a clinical study in which patients with asthma are treated with salmeterol followed by salmeterol/fluticasone to identify the effects of β2-agonists on inflammatory mediator induction and asthma control in vivo.
The in vitro studies have been completed and have demonstrated that a range of clinically relevant β2-agonists significantly induce interleukin(IL)-6, IL-11 and brain-derived neurotrophic factor (BDNF) in BECs and that they further potentiate rhinovirus-induction of these mediators. The addition of corticosteroid abolishes these effects.
These data lend support to the current hypothesis but the clinical significance of these effects in vivo remains to be seen and this forms the basis of the ongoing ex vivo studies and clinical trial (the MAELABA study) being executed at the moment to determine if these effects are seen in the human lung.
The MAELABA study has the following aims:
(1) To determine whether long-acting β2-agonist (LABA) monotherapy with salmeterol induces disease-relevant inflammatory mediators in the airways of asthma patients in vivo
(2) To determine whether combined LABA/inhaled corticosteroid (ICS) therapy with salmeterol/fluticasone abolishes the induction of disease-relevant inflammatory mediators in the airways of asthma patients in vivo
OUTCOME MEASURES Primary outcomes:
(1) Change from baseline in gene expression of disease-relevant pro-inflammatory mediators in bronchial epithelial cells (BECs) and bronchoalveolar lavage (BAL) cells and change from baseline in protein levels of disease-relevant pro-inflammatory mediators in BAL fluid and airway lining fluid following 2 weeks of salmeterol monotherapy
(2) Change from baseline in gene expression of disease-relevant pro-inflammatory mediators in BECs and BAL cells and change from baseline in protein levels of disease-relevant pro-inflammatory mediators in BAL fluid and airway lining fluid following 2 weeks of salmeterol/fluticasone combination therapy
Secondary outcomes:
Changes from baseline in the following parameters at the end of 14 days of salmeterol monotherapy and at the end of 14 days of salmeterol/fluticasone combination therapy: lung function; exhaled nitric oxide (FeNO) level; airway hyperresponsiveness (AHR); asthma symptom severity; serum BDNF and platelet BDNF concentration
POPULATION 24 adult participants with asthma
Hypothesis:
This trial seeks to test the following hypotheses:
1) The administration of 2 weeks of the LABA salmeterol in asthmatic patients will result in an increase in cAMP-mediated induction of disease-relevant pro-inflammatory cytokines, chemokines, remodelling associated factors and mucins in BECs and/or BAL cells, which in turn will cause enhanced mucosal inflammation, airway obstruction and AHR.
2) The administration of 2 weeks of salmeterol in combination with the ICS fluticasone will abolish the induction of the cAMP-mediated inflammatory markers in BECs and/or airway macrophages, resulting in reduced mucosal inflammation, airway obstruction and AHR.
Implications of this study:
If the hypotheses are proven correct, it will provide clear evidence of the mechanisms underlying the adverse effects of salmeterol in asthmatic patients and provide further justification for why salmeterol should not be used in the treatment of asthma unless it is combined with ICS in the same inhaler. This will have a significant impact on current clinical practice and will have the potential to reduce the significant morbidity and mortality that occurs with salmeterol monotherapy in asthmatics. The findings will also be of relevance to other airway diseases such as chronic obstructive pulmonary disease (COPD), where LABA therapy in the absence of ICS in the same inhaler also occurs.
This in vitro and in vivo work combined will lead to better understanding of adverse effects of the most common asthma treatments, and therefore better clinical outcomes for asthma patients.
Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far
In-vitro studies of the adverse effects of beta agonist have been completed and an abstract reporting these results was submitted to the American Thoracic Society meetings in 2020, however this meeting was cancelled due to the COVID-19 pandemic.
The abstract is as follows:
Title:
Clinically Relevant β2-agonists Induce and Augment Rhinovirus-induction of Asthma-relevant Pro-inflammatory Mediators in Human Bronchial Epithelial Cells
Authors:
Kartik Kumar, Tatiana Kebadze, Francesca Losa, Patrick Mallia, Aran Singanayagam, Michael R Edwards, Sebastian L Johnston
Institution:
National Heart and Lung Institute, Imperial College London, Norfolk Place, London, W2 1PG, UK
Rationale:
Short-acting β2-agonist (SABA) overuse and long-acting β2-agonist (LABA) use without inhaled corticosteroids are associated with increased mortality in asthma. Molecular mechanisms underlying these adverse effects are poorly understood. Salmeterol and formoterol induce asthma-relevant pro-inflammatory mediators in bronchial epithelial cells (BECs). We hypothesised these effects are common to other β2-agonists and questioned whether muscarinic antagonists, the other major class of bronchodilators, exhibit similar effects.
Methods:
BEAS-2B BECs were stimulated with SABAs (fenoterol, salbutamol), LABAs (formoterol, salmeterol), ultra-LABAs (indacaterol, olodaterol, vilanterol), short-acting (ipratropium) or long-acting (tiotropium, aclidinium, umeclidinium) muscarinic antagonists, at concentrations 0.1-100nM or with vehicle control. Additionally, we assessed the effect of β2-agonists with rhinovirus(RV)A-16 infection to mimic overuse during exacerbations; and the effect of adding the corticosteroid fluticasone (propionate for all except vilanterol [furoate]) to β2-agonists, both alone or combined with RV infection. Levels of IL-6, IL-11 and brain-derived neurotrophic factor (BDNF) – asthma-relevant mediators induced by β2-agonists1 – were quantified in supernatants by ELISA.
Results:
IL-6 and IL-11 were induced by all β2-agonists in a dose- and time-dependent manner (all P<0.05) with peak inductions in the hundreds of pg/mL and of the order of 2-4-fold relative to vehicle control. Salbutamol mostly induced at 100nM; LABAs induced at concentrations as low as 0.1nM. LABAs also induced BDNF (~1.6-1.85-fold P<0.05).
β2-agonists significantly augmented RV-induction of all three mediators by 1.3-1.7-fold for salbutamol, mostly at 100nM; and by 2-3-fold for LABAs, at lower concentrations (P<0.05). Adding fluticasone to β2-agonist alone and to β2-agonist/RV infection abolished induction of all three mediators by 72-100% (P<0.05).
Fenoterol gave inductions similar to or greater than salbutamol; formoterol similar to salmeterol; and indacaterol and olodaterol similar to vilanterol.
Ipratropium, tiotropium, aclidinium and umeclidinium did not significantly induce any of the three mediators at any drug concentration up to 100nM (all P>0.05).
Conclusion:
β2-agonists upregulate BEC production of asthma-relevant pro-inflammatory mediators, while muscarinic antagonists do not. β2-agonists also augment RV-induction of disease-relevant mediators. Corticosteroid co-administration abolishes these pro-inflammatory effects. Pro-inflammatory mediator induction by β2-agonists may explain the adverse effects associated with their inappropriate use. In vivo studies need to determine the clinical significance of these findings.
Funding:
European Research Council Advanced Grant; NIHR Imperial Biomedical Research Centre
Development of the MAELABA protocol and participant documents has been completed, as well as amending these documents and submitting them for Ethics and local authority approvals.
We initially developed a paper case record form (CRF) for this study, but were subsequently informed by Imperial College that we were obliged to develop an electronic CRF (eCRF) instead.
We engaged an eCRF provider to develop this, but were then forced into changing eCRF provider due to severe delays incurred by the original eCRF provider. The new eCRF provider OpenClinica has now completed eCRF development the eCRF is now fully operational.
We have recruited and appointed the Trial Steering Committee (TSC), the Data and Safety Monitoring committee (DSMB) and have completed the extremely onerous Imperial College Faculty of Medicine General Data Protection Regulation (GDPR) process, as well as the even more onerous ERC Ethics approval process.
We have received ethics approval and Imperial College Healthcare NHS Trust approval to start the MAELABA study. Screening for recruitment to the MAELABA clinical trial is ongoing and the first study participant completed the first study visit today.
The abstract is as follows:
Title:
Clinically Relevant β2-agonists Induce and Augment Rhinovirus-induction of Asthma-relevant Pro-inflammatory Mediators in Human Bronchial Epithelial Cells
Authors:
Kartik Kumar, Tatiana Kebadze, Francesca Losa, Patrick Mallia, Aran Singanayagam, Michael R Edwards, Sebastian L Johnston
Institution:
National Heart and Lung Institute, Imperial College London, Norfolk Place, London, W2 1PG, UK
Rationale:
Short-acting β2-agonist (SABA) overuse and long-acting β2-agonist (LABA) use without inhaled corticosteroids are associated with increased mortality in asthma. Molecular mechanisms underlying these adverse effects are poorly understood. Salmeterol and formoterol induce asthma-relevant pro-inflammatory mediators in bronchial epithelial cells (BECs). We hypothesised these effects are common to other β2-agonists and questioned whether muscarinic antagonists, the other major class of bronchodilators, exhibit similar effects.
Methods:
BEAS-2B BECs were stimulated with SABAs (fenoterol, salbutamol), LABAs (formoterol, salmeterol), ultra-LABAs (indacaterol, olodaterol, vilanterol), short-acting (ipratropium) or long-acting (tiotropium, aclidinium, umeclidinium) muscarinic antagonists, at concentrations 0.1-100nM or with vehicle control. Additionally, we assessed the effect of β2-agonists with rhinovirus(RV)A-16 infection to mimic overuse during exacerbations; and the effect of adding the corticosteroid fluticasone (propionate for all except vilanterol [furoate]) to β2-agonists, both alone or combined with RV infection. Levels of IL-6, IL-11 and brain-derived neurotrophic factor (BDNF) – asthma-relevant mediators induced by β2-agonists1 – were quantified in supernatants by ELISA.
Results:
IL-6 and IL-11 were induced by all β2-agonists in a dose- and time-dependent manner (all P<0.05) with peak inductions in the hundreds of pg/mL and of the order of 2-4-fold relative to vehicle control. Salbutamol mostly induced at 100nM; LABAs induced at concentrations as low as 0.1nM. LABAs also induced BDNF (~1.6-1.85-fold P<0.05).
β2-agonists significantly augmented RV-induction of all three mediators by 1.3-1.7-fold for salbutamol, mostly at 100nM; and by 2-3-fold for LABAs, at lower concentrations (P<0.05). Adding fluticasone to β2-agonist alone and to β2-agonist/RV infection abolished induction of all three mediators by 72-100% (P<0.05).
Fenoterol gave inductions similar to or greater than salbutamol; formoterol similar to salmeterol; and indacaterol and olodaterol similar to vilanterol.
Ipratropium, tiotropium, aclidinium and umeclidinium did not significantly induce any of the three mediators at any drug concentration up to 100nM (all P>0.05).
Conclusion:
β2-agonists upregulate BEC production of asthma-relevant pro-inflammatory mediators, while muscarinic antagonists do not. β2-agonists also augment RV-induction of disease-relevant mediators. Corticosteroid co-administration abolishes these pro-inflammatory effects. Pro-inflammatory mediator induction by β2-agonists may explain the adverse effects associated with their inappropriate use. In vivo studies need to determine the clinical significance of these findings.
Funding:
European Research Council Advanced Grant; NIHR Imperial Biomedical Research Centre
Development of the MAELABA protocol and participant documents has been completed, as well as amending these documents and submitting them for Ethics and local authority approvals.
We initially developed a paper case record form (CRF) for this study, but were subsequently informed by Imperial College that we were obliged to develop an electronic CRF (eCRF) instead.
We engaged an eCRF provider to develop this, but were then forced into changing eCRF provider due to severe delays incurred by the original eCRF provider. The new eCRF provider OpenClinica has now completed eCRF development the eCRF is now fully operational.
We have recruited and appointed the Trial Steering Committee (TSC), the Data and Safety Monitoring committee (DSMB) and have completed the extremely onerous Imperial College Faculty of Medicine General Data Protection Regulation (GDPR) process, as well as the even more onerous ERC Ethics approval process.
We have received ethics approval and Imperial College Healthcare NHS Trust approval to start the MAELABA study. Screening for recruitment to the MAELABA clinical trial is ongoing and the first study participant completed the first study visit today.
Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)
We currently do not have any results as we are screening for participants.