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.

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.).

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.

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.

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.

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.

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 Mucociliary Clearance in Smokers with Mild-to-Moderate Chronic Obstructive Pulmonary Disease and Chronic Bronchitis from Planar Radionuclide Imaging Using the Change in Penetration Index.

Background: Mucociliary clearance (MCC) rate from the lung has been shown to be reduced in chronic obstructive pulmonary disease (COPD). This study compared the use of change in penetration index (PI) with conventional whole lung clearance in assessing MCC in mild-to-moderate disease. Methods: Measurement of lung MCC using planar gamma camera imaging was performed in three groups: (1) healthy nonsmoking controls (n = 9), (2) smoking controls who were current smokers with normal lung function (n = 10), and (3) current smokers with mild-to-moderate COPD and bronchitis (n = 15). The mean (±standard deviation) forced expiratory volume at 1 second (FEV1) for the three groups was 109 (±18), 94 (±5), and 78 (±12), respectively. Following inhalation of a technetium-99m labeled aerosol, planar imaging was performed over 4 hours and then at 24 hours. Total lung clearance and tracheobronchial clearance (TBC; normalized to 24-hour clearance) were calculated. A novel parameter, the normalized change in PI (NOCHIP), was also evaluated. PI is the ratio of counts between outer and inner lung zones normalized to lung volume. Results: More aerosol was deposited in central airways in COPD compared to nonsmoking controls, using 24-hour clearance measurements (p < 0.001). Smoking controls had intermediate values. The optimal endpoint for MCC assessment was chosen to be 3 hours, when intersubject variability was minimal, while preserving a measure of early clearance. There was no statistical difference between the three groups in mean total lung clearance, or TBC, at 3 hours. NOCHIP at 3 hours was reduced significantly, compared to nonsmoking controls, in both smoking controls (p = 0.007) and COPD (p < 0.0001). It also correlated with FEV1 (p = 0.003). A higher proportion of smoking control subjects had NOCHIP values in the nonsmoking control range than in the COPD group. Conclusions: NOCHIP was a more sensitive measure of MCC than whole lung clearance and TBC in mild-to-moderate COPD.

Attached stratified mucus separates bacteria from the epithelial cells in COPD lungs.

The respiratory tract is normally kept essentially free of bacteria by cilia-mediated mucus transport, but in chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), bacteria and mucus accumulates instead. To address the mechanisms behind the mucus accumulation, the proteome of bronchoalveolar lavages from COPD patients and mucus collected in an elastase-induced mouse model of COPD was analyzed, revealing similarities with each other and with the protein content in colonic mucus. Moreover, stratified laminated sheets of mucus were observed in airways from patients with CF and COPD and in elastase-exposed mice. On the other hand, the mucus accumulation in the elastase model was reduced in Muc5b-KO mice. While mucus plugs were removed from airways by washing with hypertonic saline in the elastase model, mucus remained adherent to epithelial cells. Bacteria were trapped on this mucus, whereas, in non-elastase-treated mice, bacteria were found on the epithelial cells. We propose that the adherence of mucus to epithelial cells observed in CF, COPD, and the elastase-induced mouse model of COPD separates bacteria from the surface cells and, thus, protects the respiratory epithelium.

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.

A Novel Class of Small Molecule Agonists with Preference for Human over Mouse TLR4 Activation.

The best-characterized Toll-like receptor 4 (TLR4) ligands are lipopolysaccharide (LPS) and its chemically modified and detoxified variant, monophosphoryl lipid A (MPL). Although both molecules are active for human TLR4, they demonstrate a potency preference for mouse TLR4 based on data from transfected cell lines and primary cells of both species. After a high throughput screening process of small molecule libraries, we have discovered a new class of TLR4 agonist with a species preference profile differing from MPL. Products of the 4-component Ugi synthesis reaction were demonstrated to potently trigger human TLR4-transfected HEK cells but not mouse TLR4, although inclusion of the human MD2 with mTLR4 was able to partially recover activity. Co-expression of CD14 was not required for optimal activity of Ugi compounds on transfected cells, as it is for LPS. The species preference profile for the panel of Ugi compounds was found to be strongly active for human and cynomolgus monkey primary cells, with reduced but still substantial activity for most Ugi compounds on guinea pig cells. Mouse, rat, rabbit, ferret, and cotton rat cells displayed little or no activity when exposed to Ugi compounds. However, engineering the human versions of TLR4 and MD2 to be expressed in mTLR4/MD2 deficient mice allowed for robust activity by Ugi compounds both in vitro and in vivo. These findings extend the range of compounds available for development as agonists of TLR4 and identify novel molecules which reverse the TLR4 triggering preference of MPL for mouse TLR4 over human TLR4. Such compounds may be amenable to formulation as more potent human-specific TLR4L-based adjuvants than typical MPL-based adjuvants.

Study on risk factors and phenotypes of acute exacerbations of chronic obstructive pulmonary disease in Guangzhou, China-design and baseline characteristics.

BACKGROUND: To describe a study design that focuses on risk factors and patterns of chronic obstructive pulmonary disease (COPD) exacerbations. METHODS: A 2-year, single centre, observational study was conducted in Guangzhou in China. The study enrolled 318 subjects with COPD aged 40-79 years, stratified into different but equally sized groups according to global initiative for chronic obstructive lung disease (GOLD) stage (including Stage 0) and 86 lung healthy controls. An assessment each year was scheduled including questionnaires, lung function testing, Chest X-ray and blood collection. A sub-group, called sub-group X, consisting of 203 subjects with COPD and 51 lung healthy controls, was selected to answer a symptom questionnaire daily (EXACT-PRO) via a BlackBerry Personal Digital Assistant (PDA) device. Upon an alert that indicated a change in daily symptom pattern, the patients were contacted by the clinic to decide whether they had experienced an exacerbation and should have an extra visit within 24-48 hours. At an extra visit, nasal and throat swabs, induced sputum and blood were collected. Air pollution, temperature and humidity were also monitored daily. A subset of sub-group X, called sub-group M that consisted of 52 COPD patients and 15 healthy controls was dedicated to measure muscle strength and a dexa scan. RESULTS: More than 78% of the enrolled patients completed the study successfully. There appeared a difference between the patient groups and the controls in gender, age, body mass index (BMI), forced expiratory volume in 1 second (FEV1), FEV1/FVC and smoking at baseline. In sub-group X 90 out of 203 (44.4%) selected COPD patients developed one or more exacerbations in the 2-year observation period. They were more severe COPD patients according to GOLD stage at study start. On average most exacerbations occurred in the month March and the least number of exacerbations occurred in October. CONCLUSIONS: This study with the obtained patient dataset will allow a better insight in many aspects of exacerbations in COPD (e.g., the identification, the risk factors, phenotypes and the biomarkers).

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.