Benralizumab versus Mepolizumab for Eosinophilic Granulomatosis with Polyangiitis.

BACKGROUND: Eosinophilic granulomatosis with polyangiitis (EGPA) is a vasculitis characterized by eosinophilic inflammation. Benralizumab, a monoclonal antibody against the interleukin-5α receptor expressed on eosinophils, may be an option for treating EGPA. METHODS: We conducted a multicenter, double-blind, phase 3, randomized, active-controlled noninferiority trial to evaluate the efficacy and safety of benralizumab as compared with mepolizumab. Adults with relapsing or refractory EGPA who were receiving standard care were randomly assigned in a 1:1 ratio to receive benralizumab (30 mg) or mepolizumab (300 mg) subcutaneously every 4 weeks for 52 weeks. The primary end point was remission at weeks 36 and 48 (prespecified noninferiority margin, -25 percentage points). Secondary end points included the accrued duration of remission, time to first relapse, oral glucocorticoid use, eosinophil count, and safety. RESULTS: A total of 140 patients underwent randomization (70 assigned to each group). The adjusted percentage of patients with remission at weeks 36 and 48 was 59% in the benralizumab group and 56% in the mepolizumab group (difference, 3 percentage points; 95% confidence interval [CI], -13 to 18; P = 0.73 for superiority), showing noninferiority but not superiority of benralizumab to mepolizumab. The accrued duration of remission and the time to first relapse were similar in the two groups. Complete withdrawal of oral glucocorticoids during weeks 48 through 52 was achieved in 41% of the patients who received benralizumab and 26% of those who received mepolizumab. The mean (±SD) blood eosinophil count at baseline was 306.0±225.0 per microliter in the benralizumab group and 384.9±563.6 per microliter in the mepolizumab group, decreasing to 32.4±40.8 and 71.8±54.4 per microliter, respectively, at week 52. Adverse events were reported in 90% of the patients in the benralizumab group and 96% of those in the mepolizumab group; serious adverse events were reported in 6% and 13%, respectively. CONCLUSIONS: Benralizumab was noninferior to mepolizumab for the induction of remission in patients with relapsing or refractory EGPA. (Funded by AstraZeneca; MANDARA ClinicalTrials.gov number, NCT04157348.).

Eosinophil Depletion with Benralizumab for Eosinophilic Esophagitis.

BACKGROUND: Benralizumab is an eosinophil-depleting anti-interleukin-5 receptor α monoclonal antibody. The efficacy and safety of benralizumab in patients with eosinophilic esophagitis are unclear. METHODS: In a phase 3, multicenter, double-blind, randomized, placebo-controlled trial, we assigned patients 12 to 65 years of age with symptomatic and histologically active eosinophilic esophagitis in a 1:1 ratio to receive subcutaneous benralizumab (30 mg) or placebo every 4 weeks. The two primary efficacy end points were histologic response (≤6 eosinophils per high-power field) and the change from baseline in the score on the Dysphagia Symptom Questionnaire (DSQ; range, 0 to 84, with higher scores indicating more frequent or severe dysphagia) at week 24. RESULTS: A total of 211 patients underwent randomization: 104 were assigned to receive benralizumab, and 107 were assigned to receive placebo. At week 24, more patients had a histologic response with benralizumab than with placebo (87.4% vs. 6.5%; difference, 80.8 percentage points; 95% confidence interval [CI], 72.9 to 88.8; P<0.001). However, the change from baseline in the DSQ score did not differ significantly between the two groups (difference in least-squares means, 3.0 points; 95% CI, -1.4 to 7.4; P = 0.18). There was no substantial between-group difference in the change from baseline in the Eosinophilic Esophagitis Endoscopic Reference Score, which reflects endoscopic abnormalities. Adverse events were reported in 64.1% of the patients in the benralizumab group and in 61.7% of those in the placebo group. No patients discontinued the trial because of adverse events. CONCLUSIONS: In this trial involving patients 12 to 65 years of age with eosinophilic esophagitis, a histologic response (≤6 eosinophils per high-power field) occurred in significantly more patients in the benralizumab group than in the placebo group. However, treatment with benralizumab did not result in fewer or less severe dysphagia symptoms than placebo. (Funded by AstraZeneca; MESSINA ClinicalTrials.gov number, NCT04543409.).

Benralizumab for allergic asthma: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Benralizumab induces rapid and near-complete depletion of eosinophils from blood and lung tissue. We investigated whether benralizumab could attenuate the allergen-induced late asthmatic response (LAR) in participants with allergic asthma. METHODS: Participants with allergic asthma who demonstrated increased sputum eosinophils and LAR at screening were randomised to benralizumab 30 mg or matched placebo given every 4 weeks for 8 weeks (3 doses). Allergen challenges were performed at weeks 9 and 12 when blood, sputum, bone marrow and bronchial tissue eosinophils and LAR were assessed. RESULTS: 46 participants (mean age 30.9 years) were randomised to benralizumab (n=23) or placebo (n=23). Eosinophils were significantly reduced in the benralizumab group compared with placebo in blood at 4 weeks and sputum and bone marrow at 9 weeks after treatment initiation. At 7 h after an allergen challenge at week 9, sputum eosinophilia was significantly attenuated in the benralizumab group compared to placebo (least squares mean difference -5.81%, 95% CI -10.69- -0.94%; p=0.021); however, the LAR was not significantly different (least squares mean difference 2.54%, 95% CI 3.05-8.12%; p=0.363). Adverse events were reported for seven (30.4%) and 14 (60.9%) participants in the benralizumab and placebo groups, respectively. CONCLUSION: Benralizumab administration over 8 weeks resulted in a significant attenuation of blood, bone marrow and sputum eosinophilia in participants with mild allergic asthma; however, there was no change in the LAR, suggesting that eosinophils alone are not a key component of allergen-induced bronchoconstriction.

Pharmacokinetics/pharmacodynamics of benralizumab in chronic rhinosinusitis with nasal polyps: Phase III, randomized, placebo-controlled OSTRO trial.

AIMS: Benralizumab, a humanized, afucosylated monoclonal antibody against the interleukin 5 receptor, α subunit, causes rapid depletion of eosinophils by antibody-dependent cellular cytotoxicity. We investigated the pharmacokinetic and pharmacodynamic effects of benralizumab in patients with chronic rhinosinusitis with nasal polyps (CRSwNP) from the phase III OSTRO trial. METHODS: Patients received a placebo or 30 mg of benralizumab by subcutaneous injection every 8 weeks (first three doses every 4 weeks) to week 48; a subset of patients continued in an extended follow-up period to assess treatment durability to week 80. Serum benralizumab concentrations and blood eosinophil and basophil counts were assessed to week 80. Biomarker assessments were performed on nasal polyp tissue biopsies at week 56 and nasal lining fluid at weeks 24 and 56 to examine changes in immune cells and inflammatory mediators. RESULTS: Among 185 patients in this analysis, 93 received benralizumab. Serum benralizumab concentrations reached a steady state by week 24 (median concentration 385.52 ng mL-1); blood eosinophils were almost fully depleted and blood basophils were reduced between weeks 16 and 56. Nasal polyp tissue eosinophils decreased with benralizumab from 57.6 cells mm-2 at baseline to 0 cells mm-2 at week 56 (P < .001 vs placebo), and tissue mast cells were numerically reduced. In nasal lining fluid, eosinophil-derived neurotoxin was significantly reduced at weeks 24 and 56 (P < .001) and interleukin-17 at week 56 (P < .05) with benralizumab. CONCLUSION: Benralizumab treatment led to rapid, sustained, nearly complete depletion of eosinophils from blood and nasal polyp tissue in patients with CRSwNP.

INEXAS: A Phase 2 Randomized Trial of On-demand Inhaled Interferon Beta-1a in Severe Asthmatics.

BACKGROUND: Upper respiratory tract infections (URTIs) are important triggers for asthma exacerbations. We hypothesized that inhalation of the anti-viral cytokine, interferon (IFN)-ß, during URTI, could prevent these exacerbations. OBJECTIVE: To evaluate the efficacy of on-demand inhaled IFN-ß1a (AZD9412) to prevent severe asthma exacerbations following symptomatic URTI. METHODS: This was a randomized, double-blind, placebo-controlled trial in which patients with severe asthma (GINA 4-5; n = 121) reporting URTI symptoms were randomized to 14 days of once-daily nebulized AZD9412 or placebo. The primary endpoint was severe exacerbations during treatment. Secondary endpoints included 6-item asthma control questionnaire (ACQ-6) and lung function. Exploratory biomarkers included IFN-response markers in serum and sputum, blood leucocyte counts and serum inflammatory cytokines. RESULTS: Following a pre-planned interim analysis, the trial was terminated early due to an unexpectedly low exacerbation rate. Asthma worsenings were generally mild and tended to peak at randomization, possibly contributing to the lack of benefit of AZD9412 on other asthma endpoints. Numerically, AZD9412 did not reduce severe exacerbation rate, ACQ-6, asthma symptom scores or reliever medication use. AZD9412 improved lung function (morning peak expiratory flow; mPEF) by 19.7 L/min. Exploratory post hoc analyses indicated a greater mPEF improvement by AZD9412 in patients with high blood eosinophils (>0.3 × 109 /L) at screening and low serum interleukin-18 relative change at pre-treatment baseline. Pharmacodynamic effect of AZD9412 was confirmed using IFN-response markers. CONCLUSIONS & CLINICAL RELEVANCE: Colds did not have the impact on asthma patients that was expected and, due to the low exacerbation rate, the trial was stopped early. On-demand AZD9412 treatment did not numerically reduce the number of exacerbations, but did attenuate URTI-induced worsening of mPEF. Severe asthma patients with high blood eosinophils or low serum interleukin-18 response are potential subgroups for further investigation of inhaled IFN-ß1a.

Upper Airway Cell Transcriptomics Identify a Major New Immunological Phenotype with Strong Clinical Correlates in Young Children with Acute Wheezing.

Asthma exacerbations are triggered by rhinovirus infections. We employed a systems biology approach to delineate upper-airway gene network patterns underlying asthma exacerbation phenotypes in children. Cluster analysis unveiled distinct IRF7hi versus IRF7lo molecular phenotypes, the former exhibiting robust upregulation of Th1/type I IFN responses and the latter an alternative signature marked by upregulation of cytokine and growth factor signaling and downregulation of IFN-γ. The two phenotypes also produced distinct clinical phenotypes. For IRF7lo children, symptom duration prior to hospital presentation was more than twice as long from initial symptoms (p = 0.011) and nearly three times as long for cough (p < 0.001), the odds ratio of admission to hospital was increased more than 4-fold (p = 0.018), and time to recurrence was shorter (p = 0.015). In summary, our findings demonstrate that asthma exacerbations in children can be divided into IRF7hi versus IRF7lo phenotypes with associated differences in clinical phenotypes.

Dynamics of IFN-ß Responses during Respiratory Viral Infection. Insights for Therapeutic Strategies.

Rationale: Viral infections are major drivers of exacerbations and clinical burden in patients with asthma and chronic obstructive pulmonary disease (COPD). IFN-ß is a key component of the innate immune response to viral infection. To date, studies of inhaled IFN-ß treatment have not demonstrated a significant effect on asthma exacerbations.Objectives: The dynamics of exogenous IFN-ß activity were investigated to inform on future clinical indications for this potential antiviral therapy.Methods: Monocyte-derived macrophages (MDMs), alveolar macrophages, and primary bronchial epithelial cells (PBECs) were isolated from healthy control subjects and patients with COPD and infected with influenza virus either prior to or after IFN-ß stimulation. Infection levels were measured by the percentage of nucleoprotein 1-positive cells using flow cytometry. Viral RNA shedding and IFN-stimulated gene expression were measured by quantitative PCR. Production of inflammatory cytokines was measured using MSD.Measurements and Main Results: Adding IFN-ß to MDMs, alveolar macrophages, and PBECs prior to, but not after, infection reduced the percentage of nucleoprotein 1-positive cells by 85, 56, and 66%, respectively (P < 0.05). Inhibition of infection lasted for 24 hours after removal of IFN-ß and was maintained albeit reduced up to 1 week in MDMs and 72 hours in PBECs; this was similar between healthy control subjects and patients with COPD. IFN-ß did not induce inflammatory cytokine production by MDMs or PBECs but reduced influenza-induced IL-1ß production by PBECs.Conclusions:In vitro modeling of IFN-ß dynamics highlights the potential for intermittent prophylactic doses of exogenous IFN-ß to modulate viral infection. This provides important insights to aid the future design of clinical trials of IFN-ß in asthma and COPD.

Lanosterol Synthase Regulates Human Rhinovirus Replication in Human Bronchial Epithelial Cells.

Human rhinovirus (RV) infections are a significant risk factor for exacerbations of asthma and chronic obstructive pulmonary disease. Thus, approaches to prevent RV infection in such patients would give significant benefit. Through RNA interference library screening, we identified lanosterol synthase (LSS), a component of the cholesterol biosynthetic pathway, as a novel regulator of RV replication in primary normal human bronchial epithelial cells. Selective knock down of LSS mRNA with short interfering RNA inhibited RV2 replication in normal human bronchial epithelial cells. Small molecule inhibitors of LSS mimicked the effect of LSS mRNA knockdown in a concentration-dependent manner. We further demonstrated that the antiviral effect is not dependent on a reduction in total cellular cholesterol but requires a 24-hour preincubation with the LSS inhibitor. The rank order of antiviral potency of the LSS inhibitors used was consistent with LSS inhibition potency; however, all compounds showed remarkably higher potency against RV compared with the LSS enzyme potency. We showed that LSS inhibition led to an induction of 24(S),25 epoxycholesterol, an important regulator of the sterol pathway. We also demonstrated that LSS inhibition led to a profound increase in expression of the innate antiviral defense protein, IFN-ß. We found LSS to be a novel regulator of RV replication and innate antiviral immunity and identified a potential molecular mechanism for this effect, via induction of 24(S),25 epoxycholesterol. Inhibition of LSS could therefore be a novel therapeutic target for prevention of RV-induced exacerbations.

Seasonal and geographic variations in the incidence of asthma exacerbations in the United States.

BACKGROUND: Exacerbations drive the burden of asthma and lead to significant morbidity and consumption of health care resources. Many prior studies of the epidemiology of asthma exacerbations have relied upon data from hospital care. OBJECTIVE: The objective of this study was to determine US patterns of geographic and seasonal variations of asthma exacerbations being defined as asthma episodes requiring hospital care and/or a prescription for oral steroid. METHODS: The study was a retrospective observational cohort study using administrative claims data for insured individuals from the HealthCore Integrated Research Database, including around 43 million members in the United States. Analyses examined 3 age groups, 6-17, 18-64, and ≥65 years and four US regions, Northeast, Southeast, Central, and Western. RESULTS: Monthly rates of asthma exacerbations showed the greatest variation over the year in children, less so in adults and in the elderly. Clinically important differences in rates of asthma exacerbation were observed between regions with the Western Region having the lowest in all three age groups followed by the Northeast, Central, and Southeast regions. Peaks in children occurred in the early fall following troughs in the summer months, and peaks at year-end occurred in adults, particularly in those over 65 years. CONCLUSIONS: There is a striking seasonal variation in asthma exacerbations in the United States. Substantial differences between regions of the United States in asthma exacerbation rates cannot readily be explained and invite further investigation.

A comparative study of blood cell count in four automated hematology analyzers: An evaluation of the impact of preanalytical factors.

Differential white blood cell counts are frequently used in diagnosis, patient stratification, and treatment selection to optimize therapy responses. Referral laboratories are often used but challenged with use of different hematology platforms, variable blood shipping times and storage conditions, and the different sensitivities of specific cell types. To extend the scientific literature and knowledge on the temporal commutability of blood samples between hematology analyzers, we performed a comparative ex-vivo study using four of the most utilized commercial platforms, focusing on the assessment of eosinophils given its importance in asthma management. Whole blood from healthy volunteers with and without atopy (n = 6+6) and participants with eosinophilic asthma (n = 6) were stored under different conditions (at 4, 20, 30, and 37°C, with or without agitation) and analyzed at different time points (3, 6, 24, 48 and 72h post-sampling) in parallel on the Abbott CELL-DYN Sapphire, Beckman Coulter DxH900, Siemens ADVIA 2120i and Sysmex XN-1000V. In the same blood samples, eosinophil-derived neurotoxin (EDN), eosinophil activation and death markers were analyzed. All platforms gave comparable measurements of cell differentials on fresh blood within the same day of sampling. However, by 24 hours, significant temporal and temperature-dependent differences were observed, most markedly for eosinophils. None of the platforms performed perfectly across all temperatures tested during the 72 hours, showing that handling conditions should be optimized depending on the cell type of interest and the hematology analyzer. Neither disease status (healthy vs. asthma) nor agitation of the sample affected the cell quantification result or EDN release. The eosinophil activation markers measured by flow cytometry increased with time, were influenced by temperature, and were higher in those with asthma versus healthy participants. In conclusion, hematology analyzer, time window from sampling until analysis, and temperature conditions must be considered when analyzing blood cell differentials, particularly for eosinophils, via central labs to obtain counts comparable to the values obtained in freshly sampled blood.

Budesonide and formoterol effects on rhinovirus replication and epithelial cell cytokine responses.

BACKGROUND: Combination therapy with budesonide and formoterol reduces exacerbations of asthma, which are closely associated with human rhinovirus (RV) infections in both children and adults. These data suggest that budesonide and formoterol inhibit virus-induced inflammatory responses of airway epithelial cells. METHODS: To test this hypothesis, bronchial epithelial (BE) cells were obtained from airway brushings of 8 subjects with moderate-to-severe allergic asthma and 9 with neither asthma nor respiratory allergies. Cultured BE cells were incubated for 24 hours with budesonide (1.77 µM), formoterol (0.1 µM), both, or neither, and then inoculated with RV-16 (5×10(6) plaque forming units [PFU]/mL). After 24 hours, viral replication (RV RNA), cytokine secretion (CXCL8, CXCL10, TNFa, IFN-ß, IL-28) and mRNA expression (CXCL8, CXCL10, TNF, IFNB1, IL-28) were analyzed. RESULTS: RV infection induced CXCL10 protein secretion and IFNB1 and IL28 mRNA expression. Drug treatments significantly inhibited secretion of CXCL10 in mock-infected, but not RV-infected, BE cells, and inhibited secretion of TNFa under both conditions. Neither budesonide nor formoterol, alone or in combination, significantly affected viral replication, nor did they inhibit RV-induced upregulation of IFNB1 and IL28 mRNA. Overall, RV replication was positively related to CXCL10 secretion and induction of IFNB1 and IL28 mRNA, but the positive relationship between RV RNA and CXCL10 secretion was stronger in normal subjects than in subjects with asthma. CONCLUSIONS: Budesonide and formoterol can inhibit BE cell inflammatory responses in vitro without interfering with viral replication or production of interferons. These effects could potentially contribute to beneficial effects of budesonide/formoterol combination therapy in preventing RV-induced asthma exacerbations.

BAL Fluid Eosinophilia Associates With Chronic Lung Allograft Dysfunction Risk: A Multicenter Study.

BACKGROUND: Chronic lung allograft dysfunction (CLAD) is the leading cause of death among lung transplant recipients. Eosinophils, effector cells of type 2 immunity, are implicated in the pathobiology of many lung diseases, and prior studies suggest their presence associates with acute rejection or CLAD after lung transplantation. RESEARCH QUESTION: Does histologic allograft injury or respiratory microbiology correlate with the presence of eosinophils in BAL fluid (BALF)? Does early posttransplant BALF eosinophilia associate with future CLAD development, including after adjustment for other known risk factors? STUDY DESIGN AND METHODS: We analyzed BALF cell count, microbiology, and biopsy data from a multicenter cohort of 531 lung recipients with 2,592 bronchoscopies over the first posttransplant year. Generalized estimating equation models were used to examine the correlation of allograft histology or BALF microbiology with the presence of BALF eosinophils. Multivariable Cox regression was used to determine the association between ≥ 1% BALF eosinophils in the first posttransplant year and definite CLAD. Expression of eosinophil-relevant genes was quantified in CLAD and transplant control tissues. RESULTS: The odds of BALF eosinophils being present was significantly higher at the time of acute rejection and nonrejection lung injury histologies and during pulmonary fungal detection. Early posttransplant ≥ 1% BALF eosinophils significantly and independently increased the risk for definite CLAD development (adjusted hazard ratio, 2.04; P = .009). Tissue expression of eotaxins, IL-13-related genes, and the epithelial-derived cytokines IL-33 and thymic stromal lymphoprotein were significantly increased in CLAD. INTERPRETATION: BALF eosinophilia was an independent predictor of future CLAD risk across a multicenter lung recipient cohort. Additionally, type 2 inflammatory signals were induced in established CLAD. These data underscore the need for mechanistic and clinical studies to clarify the role of type 2 pathway-specific interventions in CLAD prevention or treatment.

Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD.

Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.

Selection of a screening panel of rhinoviral serotypes.

A novel methodology for the selection of a representative primary and secondary screening panel of rhinoviral serotypes for the purposes of identifying potential antiviral agents is presented. This methodology focuses on the active-sites of the rhinoviral proteins but does not invoke historical SAR data, thereby avoiding compound bias.

A sputum 6-gene signature predicts airway inflammation endotypes and exacerbation frequency in chronic obstructive pulmonary disease.

Aim: To validate a sputum 6-gene signature (6GS), comprising of CLC, CPA, DNASE1L3, IL-1B, ALPL and CXCR2, for identifying different endotypes in chronic obstructive pulmonary disease. Methodology & results: Sputum cell CLC, CPA3 and DNASE1L3 gene expression correlated with eosinophil percentage, while IL-1B, ALPL and CXCR2 correlated with neutrophil percentage. Hierarchical cluster analyses of IL-1B, ALPL and CXCR2, and CLC, CPA3 and DNASE1L3, identified patient groups that differed in their sputum neutrophil and eosinophil levels, respectively. Multiple logistic regressions demonstrated that the 6GS could distinguish between eosinophilHigh and eosinophilLow patients, as well as neutrophilHigh and neutrophilLow, and could also predict exacerbation history. Conclusion: The 6GS may have applications in clinical practice or for stratifying patients for clinical trials.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. COPD is made up of a number of disease subgroups, which require different treatments. It is important for clinicians to be able to identify these subgroups. We have measured the activity levels of 6 sputum cell genes and demonstrated that the levels differ in two different subgroups of COPD, which are known to respond differently to treatment. We have also shown that the amount these genes are turned on allows us to identify patients who might suffer a worsening in their symptoms in the future.

Increased mast cell activation in eosinophilic chronic obstructive pulmonary disease.

Objectives: A subset of chronic obstructive pulmonary disease (COPD) patients have increased numbers of airway eosinophils associated with elevated markers of T2 inflammation. This analysis focussed on mast cell counts and mast cell-related gene expression in COPD patients with higher vs lower eosinophil counts. Methods: We investigated gene expression of tryptase (TPSAB1), carboxypeptidase A3 (CPA3), chymase (CMA1) and two mast cell specific gene signatures; a bronchial biopsy signature (MCbb) and an IgE signature (MCIgE) using sputum cells and bronchial epithelial brushings. Gene expression analysis was conducted by RNA-sequencing. We also examined bronchial biopsy mast cell numbers by immunohistochemistry. Results: There was increased expression of TPSAB1, CPA3 and MCbb in eosinophilhigh than in eosinophillow COPD patients in sputum cells and bronchial epithelial brushings (fold change differences 1.21 and 1.28, respectively, P < 0.01). Mast cell gene expression was associated with markers of T2 and eosinophilic inflammation (IL13, CLCA1, CST1, CCL26, eosinophil counts in sputum and bronchial mucosa; rho = 0.4-0.8; P < 0.05). There was no difference in MCIgE gene expression between groups. There was no difference in the total number of bronchial biopsy mast cells between groups. Conclusion: These results demonstrate that eosinophilic inflammation is associated with altered mast cell characteristics in COPD patients, implicating mast cells as a component of T2 inflammation present in a subset of COPD patients.

Effect of lipophilicity modulation on inhibition of human rhinovirus capsid binders.

To try and generate broad spectrum human rhinovirus VP1 inhibitors with more attractive physicochemical, DMPK and safety profiles, we explored the current SAR of known VP1 compounds. This lead to the identification of specific structural regions where reduction in polarity can be achieved, so guiding chemistry to analogues with significantly superior profiles to previously reported inhibitors.

Combined siRNA and Small-Molecule Phenotypic Screening Identifies Targets Regulating Rhinovirus Replication in Primary Human Bronchial Epithelial Cells.

Human rhinovirus (RV) is the most common cause of acute upper respiratory tract infections and has recently been shown to play a significant role in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). There is a significant unmet medical need for agents for the prevention and/or treatment of exacerbations triggered by human RV infection. Phenotypic drug discovery programs using different perturbation modalities, for example, siRNA, small-molecule compounds, and CRISPR, hold significant value for identifying novel drug targets. We have previously reported the identification of lanosterol synthase as a novel regulator of RV2 replication through a phenotypic screen of a library of siRNAs against druggable genes in normal human bronchial epithelial (NHBE) cells. Here, we describe a follow-up phenotypic screen of small-molecule compounds that are annotated to be pharmacological regulators of target genes that were identified to significantly affect RV2 replication in the siRNA primary screen of 10,500 druggable genes. Two hundred seventy small-molecule compounds selected for interacting with 122 target gene hits were screened in the primary RV2 assay in NHBE cells by quantifying viral replication via in situ hybridization followed by secondary quantitative PCR-based assays for RV2, RV14, and RV16. The described follow-up phenotypic screening allowed us to identify Fms-related tyrosine kinase 4 (FLT4) as a novel target regulating RV replication. We demonstrate that a combination of siRNA and small-molecule compound screening models is a useful phenotypic drug discovery approach for the identification of novel drug targets.

Quantitative Assessment of Regional Mucociliary Clearance in Smokers with Mild-to-Moderate Chronic Obstructive Pulmonary Disease and Chronic Bronchitis from Planar Radionuclide Imaging.

Background: Mucociliary clearance (MCC) rate from the lung has been shown to be reduced in chronic obstructive pulmonary disease (COPD). This study investigates the value of regional clearance measurements in assessing MCC in mild-to-moderate disease. Methods: Measurement of lung MCC using planar gamma camera imaging was performed in three groups: (i) healthy nonsmoking controls (NSCs) (n = 9), (ii) smoking controls (SCs) who were current smokers with normal lung function (n = 10), and (iii) current smokers with mild-to-moderate COPD and bronchitis (n = 15). The mean (±standard deviation) forced expiratory volumes at 1 second (FEV1) for the three groups were 109 (± 18), 94 (± 5), and 78 (± 12), respectively. After inhalation of a technetium-99m labeled aerosol, planar imaging was performed over 4 hours and then at 24 hours. Both lung clearance and tracheobronchial clearance (TBC) (normalized to 24 hours clearance) were calculated for inner and outer lung zones. Inner zone clearance was corrected for input from the outer zone. A novel parameter, the bronchial airways clearance index (BACI), which combined clearance data from both zones, was also evaluated. Regional results were compared with whole lung clearance in the same subjects. Results: Corrected inner zone clearance at 3 hours was not reduced compared with NSC in either SCs or COPD. Outer zone clearance was higher in COPD than in the other groups. Corrected inner zone TBC showed significant reductions in SC and COPD compared with NSC. BACI was significantly reduced in COPD compared with NSC and also correlated with FEV1. The mean BACI for SC was also reduced compared with NSC, but the distribution of results was bimodal, with a significant proportion of subjects having values in the NSC range. Conclusions: Regional MCC demonstrated differences between NSCs, SCs, and subjects with mild-to-moderate COPD, which were not apparent with whole lung measurements.