Molecular & Pharmacological Approach for the Optimisation of Combination Therapy of a Long-acting β2 Agonist and a Corticosteroid in Chronic Obstructive Pulmonary Disease

Combination inhalers of long-acting β2 agonists (LABAs) with inhaled corticosteroids (ICSs) are now widely used for the treatment of asthma. However, corticosteroid (CS) insensitivity can be a major barrier for the treatment of chronic obstructive pulmonary disease (COPD). Existing ICSs are less beneficial on the decline of lung function in the long term. Even at the cellular level, CSs are less effective in primary cells, peripheral blood mononuclear cells (PBMCs) obtained from COPD patients compared to PBMCs from healthy volunteers. High levels of oxidative stress in COPD have been reported to be involved in CS insensitivity. In addition, oxidative stress has been reported to affect the β2 adrenergic receptor (β2AR) signalling itself. Here I hypothesise that oxidative stress affects the function of CSs and LABAs and/or their combination. Clarifying the molecular mechanism of this defect, will give new insights for the optimisation of the ideal CS/LABA combination therapy for the treatment of COPD. Primarily, pharmacological effects of fluticasone furoate (FF), a novel CS, were evaluated in various inflammation systems in respiratory cells and compared with those of currently available fluticasone propionate (FP) and budesonide (Bud). In the course of this study, FF was found to be a potent and long-acting CS. More importantly, the anti-inflammatory effects of FP were reduced under oxidative stress, whereas the effects of FF were not affected by oxidative stress. FF was also more effective on inhibiting cytokine release in PBMCs from COPD patients. Hence, this study demonstrated that FF is an oxidative stress insensitive CS. Furthermore, effects of oxidative stress on the function or signalling of LABAs were investigated. Salmeterol (SM)-induced cyclic AMP (cAMP) production was reduced under H2O2 treatment, while the effects of formoterol (FM) were not affected by H2O2. Under conditions of oxidative stress, SM also induced β2AR internalisation, whereas FM did not. Cell signalling analysis showed that FM partially inhibited H2O2-induced Akt phosphorylation, a footprint of phosphoinositide-3 kinase (PI3K) activation, whereas SM did not. Furthermore, add-on treatment with FM could improve sensitivity of either Bud or FP in U937 cells, under simulative conditions of oxidative stress or PBMCs from COPD patients, but SM did not improve it. Moreover, vilanterol trifenatate (VT), also known as GW642444, a novel ultra LABA showed similar effects with FM, under oxidative stress. Thus, FM (and VT) is more robust than SM under oxidative stress conditions. Finally, the outcome of H2O2 on the anti-inflammatory effects of CS/LABA combination was evaluated. As concluded from the results above, FF and FM (or VT) were oxidative stress insensitive compounds. In fact, the combination of FF and VT, as well as a combination of mometasone furoate (MF) with FM showed the most potent anti-inflammatory effects under oxidative stress and in PBMCs from COPD patients. Consequently, the findings from this thesis might give new insights and support the development of new combination therapies for the treatment of chronic inflammatory airway diseases, where high levels of oxidative stress are seen

Leucine and perindopril to improve physical performance in people over 70 years with sarcopenia : the LACE factorial RCT

Funding This project was funded by the Efficacy and Mechanism Evaluation (EME) programme, a MRC and National Institute for Health and Care Research (NIHR) partnership. This will be published in full in Efficacy and Mechanism Evaluation; Vol. 9, No. 8. See the NIHR Journals Library website for further project information.Peer reviewedPublisher PD

Activin type I receptor polymorphisms and body composition in older individuals with sarcopenia—Analyses from the LACE randomised controlled trial

Background: Ageing is associated with changes in body composition including an overall reduction in muscle mass and a proportionate increase in fat mass. Sarcopenia is characterised by losses in both muscle mass and strength. Body composition and muscle strength are at least in part genetically determined, consequently polymorphisms in pathways important in muscle biology (e.g., the activin/myostatin signalling pathway) are hypothesised to contribute to the development of sarcopenia.Methods: We compared regional body composition measured by DXA with genotypes for two polymorphisms (rs10783486, minor allele frequency (MAF) =0.26 and rs2854464, MAF =0.26) in the activin 1B receptor (ACVR1B) determined by PCR in a cross-sectional analysis of DNA from 110 older individuals with sarcopenia from the LACE trial.Results: Neither muscle mass nor strength showed any significant associations with either genotype in this cohort. Initial analysis of rs10783486 showed that males with the AA/AG genotype were taller than GG males (174±7cm vs 170±5cm, p=0.023) and had higher arm fat mass, (median higher by 15%, p=0.008), and leg fat mass (median higher by 14%, p=0.042). After correcting for height, arm fat mass remained significantly higher (median higher by 4% padj=0.024). No associations (adjusted or unadjusted) were seen in females.Similar analysis of the rs2854464 allele showed a similar pattern with the presence of the minor allele (GG/AG) being associated with greater height (GG/AG = 174±7 cm vs AA = 170 ±5cm, p=0.017) and greater arm fat mass (median higher by 16%, p=0.023). Again, the difference in arm fat remained after correction for height. No similar associations were seen in females analysed alone.Conclusion: These data suggest that polymorphic variation in the ACVR1B locus could be associated with body composition in older males. The activin/myostatin pathway might offer a novel potential target to prevent fat accumulation in older individuals

ACE I/D genotype associates with strength in sarcopenic men but not with response to ACE inhibitor therapy in older adults with sarcopenia:Results from the LACE trial

BACKGROUND: Angiotensin II (AII), has been suggested to promote muscle loss. Reducing AII synthesis, by inhibiting angiotensin converting enzyme (ACE) activity has been proposed as a method to inhibit muscle loss. The LACE clinical trial was designed to determine whether ACE inhibition would reduce further muscle loss in individuals with sarcopenia but suffered from low recruitment and returned a negative result. Polymorphic variation in the ACE promoter (I/D alleles) has been associated with differences in ACE activity and muscle physiology in a range of clinical conditions. This aim of this analysis was to determine whether I/D polymorphic variation is associated with muscle mass, strength, in sarcopenia or contributed to the lack of response to treatment in the LACE study.METHODS: Sarcopenic individuals were recruited into a 2x2 factorial multicentre double-blind study of the effects of perindopril and/or leucine versus placebo on physical performance and muscle mass. DNA extracted from blood samples (n = 130 72 women and 58 men) was genotyped by PCR for the ACE I/D polymorphism. Genotypes were then compared with body composition measured by DXA, hand grip and quadriceps strength before and after 12 months' treatment with leucine and/or perindopril in a cross-sectional analysis of the influence of genotype on these variables.RESULTS: Allele frequencies for the normal UK population were extracted from 13 previous studies (I = 0.473, D = 0.527). In the LACE cohort the D allele was over-represented (I = 0.412, D = 0.588, p = 0.046). This over-representation was present in men (I = 0.353, D = 0.647, p = 0.010) but not women (I = 0.458, D = 0.532, p = 0.708). In men but not women, individuals with the I allele had greater leg strength (II/ID = 18.00 kg (14.50, 21.60) vs DD = 13.20 kg (10.50, 15.90), p = 0.028). Over the 12 months individuals with the DD genotype increased in quadriceps strength but those with the II or ID genotype did not. Perindopril did not increase muscle strength or mass in any polymorphism group relative to placebo.CONCLUSION: Our results suggest that although ACE genotype was not associated with response to ACE inhibitor therapy in the LACE trial population, sarcopenic men with the ACE DD genotype may be weaker than those with the ACE I/D or II genotype.</p

Activin type I receptor polymorphisms and body composition in older individuals with sarcopenia-Analyses from the LACE randomised controlled trial

BACKGROUND: Ageing is associated with changes in body composition including an overall reduction in muscle mass and a proportionate increase in fat mass. Sarcopenia is characterised by losses in both muscle mass and strength. Body composition and muscle strength are at least in part genetically determined, consequently polymorphisms in pathways important in muscle biology (e.g., the activin/myostatin signalling pathway) are hypothesised to contribute to the development of sarcopenia.METHODS: We compared regional body composition measured by DXA with genotypes for two polymorphisms (rs10783486, minor allele frequency (MAF) = 0.26 and rs2854464, MAF = 0.26) in the activin 1B receptor (ACVR1B) determined by PCR in a cross-sectional analysis of DNA from 110 older individuals with sarcopenia from the LACE trial.RESULTS: Neither muscle mass nor strength showed any significant associations with either genotype in this cohort. Initial analysis of rs10783486 showed that males with the AA/AG genotype were taller than GG males (174±7cm vs 170±5cm, p = 0.023) and had higher arm fat mass, (median higher by 15%, p = 0.008), and leg fat mass (median higher by 14%, p = 0.042). After correcting for height, arm fat mass remained significantly higher (median higher by 4% padj = 0.024). No associations (adjusted or unadjusted) were seen in females. Similar analysis of the rs2854464 allele showed a similar pattern with the presence of the minor allele (GG/AG) being associated with greater height (GG/AG = 174±7 cm vs AA = 170 ±5cm, p = 0.017) and greater arm fat mass (median higher by 16%, p = 0.023). Again, the difference in arm fat remained after correction for height. No similar associations were seen in females analysed alone.CONCLUSION: These data suggest that polymorphic variation in the ACVR1B locus could be associated with body composition in older males. The activin/myostatin pathway might offer a novel potential target to prevent fat accumulation in older individuals.</p

Clinical and transcriptomic features of persistent exacerbation-prone severe asthma in U-BIOPRED cohort

Background: Exacerbation-prone asthma is a feature of severe disease. Yet, the basis for its persistency remains unclear. Objectives: To determine the clinical and transcriptomic features of the frequent-exacerbator (FE) and of persistent FEs (PFE) in U-BIOPRED cohort. Methods: We compared features of FE (≥2 exacerbations in past year) to infrequent exacerbators (IE, <2 exacerbations) and of PFE with repeat ≥2 exacerbations during the following year to persistent IE (PIE). Transcriptomic data in blood, bronchial and nasal epithelial brushings, bronchial biopsies and sputum cells were analysed by gene set variation analysis for 103 gene signatures. Results: Of 317 patients, 62.4 % were FE of whom 63.6% were PFE, while 37.6% were IE of whom 61.3% were PIE. Using multivariate analysis, FE was associated with short-acting beta-agonist use, sinusitis and daily oral corticosteroid use, while PFE with eczema, short-acting beta-agonist use and asthma control index. CEA Cell Adhesion Molecule 5 (CEACAM5) was the only differentially-expressed transcript in bronchial biopsies between PE and IE. There were no differentially-expressed genes in the other 4 compartments. There were higher expression scores for Type 2 , T-helper type-17 and Type 1 pathway signatures together with those associated with viral infections in bronchial biopsies from FE compared to IE, while higher expression scores of Type 2, Type 1 and steroid insensitivity pathway signatures in bronchial biopsies of PFE compared to PIE. Conclusion: FE group and its PFE subgroup are associated with poor asthma control while expressing higher Type 1 and Type 2 activation pathways compared to IE and PIE, respectively

Effect of perindopril or leucine on physical performance in older people with sarcopenia: the LACE randomized controlled trial

Acknowledgements: AAS, TA and MDW acknowledge support from the NIHR Newcastle Biomedical Research Centre. AA acknowledges support from the Health Services Research Unit which is core funded by the Chief Scientist Office of the Scottish Government Health and Social Care Directorate. The authors acknowledge support from the NIHR Ageing Clinical Research Network and the NHS Scotland Support for Science programme, The authors would also thank the efforts of all the research nurses and other ants to the trial, all the participants, and all the staff of the Tayside Clinical Trials Unit for their support of the trial. Funding: The LACE trial (project reference 13/53/03) is funded by the Efficacy and Mechanism Evaluation (EME) Programme, an MRC and NIHR partnership. The views expressed in this publication are those of the authors and not necessarily those of the MRC, NIHR or the Department of Health and Social Care.Peer reviewedPublisher PD

Sputum proteomics and airway cell transcripts of current and ex-smokers with severe asthma in U-BIOPRED: an exploratory analysis

Background: Severe asthma patients with a significant smoking history have airflow obstruction with reported neutrophilia. We hypothesise that multi-omic analysis will enable the definition of smoking and ex-smoking severe asthma molecular phenotypes. Methods: The U-BIOPRED severe asthma patients containing current-smokers (CSA), exsmokers (ESA), non-smokers (NSA) and healthy non-smokers (NH) was examined. Blood and sputum cell counts, fractional exhaled nitric oxide and spirometry were obtained. Exploratory proteomic analysis of sputum supernatants and transcriptomic analysis of bronchial brushings, biopsies and sputum cells was performed. Results: Colony stimulating factor (CSF)2 protein levels were increased in CSA sputum supernatants with azurocidin 1, neutrophil elastase and CXCL8 upregulated in ESA. Phagocytosis and innate immune pathways were associated with neutrophilic inflammation in ESA. Gene Set Variation Analysis of bronchial epithelial cell transcriptome from CSA showed enrichment of xenobiotic metabolism, oxidative stress and endoplasmic reticulum stress compared to other groups. CXCL5 and matrix metallopeptidase 12 genes were upregulated in ESA and the epithelial protective genes, mucin 2 and cystatin SN, were downregulated. Conclusion: Despite little difference in clinical characteristics, CSA were distinguishable from ESA subjects at the sputum proteomic level with CSA having increased CSF2 expression and ESA patients showed sustained loss of epithelial barrier processes

Molecular & pharmacological approach for the optimisation of combination therapy of a long-acting β2 agonist and a corticosteroid in chronic obstructive pulmonary disease

Combination inhalers of long-acting β2 agonists (LABAs) with inhaled corticosteroids (ICSs) are now widely used for the treatment of asthma. However, corticosteroid (CS) insensitivity can be a major barrier for the treatment of chronic obstructive pulmonary disease (COPD). Existing ICSs are less beneficial on the decline of lung function in the long term. Even at the cellular level, CSs are less effective in primary cells, peripheral blood mononuclear cells (PBMCs) obtained from COPD patients compared to PBMCs from healthy volunteers. High levels of oxidative stress in COPD have been reported to be involved in CS insensitivity. In addition, oxidative stress has been reported to affect the β2 adrenergic receptor (β2AR) signalling itself. Here I hypothesise that oxidative stress affects the function of CSs and LABAs and/or their combination. Clarifying the molecular mechanism of this defect, will give new insights for the optimisation of the ideal CS/LABA combination therapy for the treatment of COPD. Primarily, pharmacological effects of fluticasone furoate (FF), a novel CS, were evaluated in various inflammation systems in respiratory cells and compared with those of currently available fluticasone propionate (FP) and budesonide (Bud). In the course of this study, FF was found to be a potent and long-acting CS. More importantly, the anti-inflammatory effects of FP were reduced under oxidative stress, whereas the effects of FF were not affected by oxidative stress. FF was also more effective on inhibiting cytokine release in PBMCs from COPD patients. Hence, this study demonstrated that FF is an oxidative stress insensitive CS. Furthermore, effects of oxidative stress on the function or signalling of LABAs were investigated. Salmeterol (SM)-induced cyclic AMP (cAMP) production was reduced under H2O2 treatment, while the effects of formoterol (FM) were not affected by H2O2. Under conditions of oxidative stress, SM also induced β2AR internalisation, whereas FM did not. Cell signalling analysis showed that FM partially inhibited H2O2-induced Akt phosphorylation, a footprint of phosphoinositide-3 kinase (PI3K) activation, whereas SM did not. Furthermore, add-on treatment with FM could improve sensitivity of either Bud or FP in U937 cells, under simulative conditions of oxidative stress or PBMCs from COPD patients, but SM did not improve it. Moreover, vilanterol trifenatate (VT), also known as GW642444, a novel ultra LABA showed similar effects with FM, under oxidative stress. Thus, FM (and VT) is more robust than SM under oxidative stress conditions. Finally, the outcome of H2O2 on the anti-inflammatory effects of CS/LABA combination was evaluated. As concluded from the results above, FF and FM (or VT) were oxidative stress insensitive compounds. In fact, the combination of FF and VT, as well as a combination of mometasone furoate (MF) with FM showed the most potent anti-inflammatory effects under oxidative stress and in PBMCs from COPD patients. Consequently, the findings from this thesis might give new insights and support the development of new combination therapies for the treatment of chronic inflammatory airway diseases, where high levels of oxidative stress are seen.EThOS - Electronic Theses Online ServiceGBUnited Kingdo