Publications by authors named "Edith M Hessel"

45 Publications

Rhinovirus-induced Human Lung Tissue Responses Mimic COPD and Asthma Gene Signatures.

Am J Respir Cell Mol Biol 2021 Jun 28. Epub 2021 Jun 28.

Fraunhofer Institute for Toxicology and Experimental Medicine, Member of Fraunhofer international Consortium for Anti-Infective Research (iCAIR), Member of Fraunhofer Cluster of Excellence Immune-Mediated Diseases CIMD, Hannover, Germany.

Human rhinovirus (RV) is a major risk factor for COPD and asthma exacerbations. Exploration of RV pathogenesis has been hampered by a lack of disease relevant model systems. We performed a detailed characterization of host responses to RV infection in human lung tissue ex vivo and investigated whether these responses are disease relevant for patients with COPD and asthma. In addition, impact of the viral replication inhibitor rupintrivir was evaluated. Human precision-cut lung slices (PCLS) were infected with RV1B with or without rupintrivir. At day 1 and 3 post-infection, RV tissue localization, tissue viability and viral load were determined. To characterize host responses to infection, mediator and whole genome analyses were performed. RV successfully replicated in PCLS airway epithelial cells and induced both anti-viral and pro-inflammatory cytokines such as IFNα2a, CXCL10, CXCL11, IFN-γ, TNFα and CCL5. Genomic analyses revealed that RV not only induced anti-viral immune responses but also triggered changes in epithelial cell-associated pathways. Strikingly, the RV response in PCLS was reflective of gene expression changes described in COPD and asthma patients. While RV-induced host immune responses were abrogated by rupintrivir, RV-triggered epithelial processes were largely refractory to antiviral treatment. Detailed analysis of RV-infected human PCLS and comparison with COPD and asthma patient gene signatures revealed that the human RV PCLS model represents disease relevant biological mechanisms that can be partially inhibited by a well-known antiviral compound and provide an outstanding opportunity to evaluate novel therapeutics. This article is open access and distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives License 4.0 (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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http://dx.doi.org/10.1165/rcmb.2020-0337OCDOI Listing
June 2021

Nemiralisib in Patients with an Acute Exacerbation of COPD: Placebo-Controlled, Dose-Ranging Study.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1637-1646. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Background: Management of acute exacerbations of chronic obstructive pulmonary disease (COPD) is sometimes inadequate leading to either prolonged duration and/or an increased risk of recurrent exacerbations in the period following the initial event.

Objective: To evaluate the safety and efficacy of inhaled nemiralisib, a phosphoinositide 3-kinase δ inhibitor, in patients experiencing an acute exacerbation of COPD.

Patients And Methods: In this double-blind, placebo-controlled study, COPD patients (40-80 years, ≥10 pack-year smoking history, current moderate/severe acute exacerbation of COPD requiring standard-of-care treatment) were randomized to placebo or nemiralisib 12.5 µg, 50 µg, 100 µg, 250 µg, 500 µg, or 750 µg (ratio of 3:1:1:1:1:1:3; N=938) for 12 weeks with an exploratory 12-week follow-up period. The primary endpoint was change from baseline in post-bronchodilator FEV at week 12. Key secondary endpoints were rate of re-exacerbations, patient-reported outcomes (Exacerbations of Chronic Pulmonary Disease Tool, COPD Assessment Test, St George's Respiratory Questionnaire-COPD), plasma pharmacokinetics (PK) and safety/tolerability.

Results: There was no difference in change from baseline FEV at week 12 between the nemiralisib and placebo treatment groups (posterior adjusted median difference, nemiralisib 750 µg and placebo: -0.004L (95% CrI: -0.051L to 0.042L)). Overall, there were also no differences between nemiralisib and placebo in secondary endpoints, including re-exacerbations. Plasma PK increased in a dose proportional manner. The most common adverse event for nemiralisib was post-inhalation cough which appeared to be dose-related.

Conclusion: The addition of nemiralisib to standard-of-care treatment for 12 weeks did not improve lung function or re-exacerbations in patients with, and following an acute exacerbation of COPD. However, this study demonstrated that large clinical trials recruiting acutely exacerbating patients can successfully be conducted.
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http://dx.doi.org/10.2147/COPD.S309320DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184152PMC
June 2021

Exploring PI3Kδ Molecular Pathways in Stable COPD and Following an Acute Exacerbation, Two Randomized Controlled Trials.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1621-1636. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Background: Inhibition of phosphoinositide 3-kinase δ (PI3Kδ) exerts corrective effects on the dysregulated migration characteristics of neutrophils isolated from patients with chronic obstructive pulmonary disease (COPD).

Objective: To develop novel, induced sputum endpoints to demonstrate changes in neutrophil phenotype in the lung by administering nemiralisib, a potent and selective inhaled PI3Kδ inhibitor, to patients with stable COPD or patients with acute exacerbation (AE) of COPD.

Methods: In two randomized, double-blind, placebo-controlled clinical trials patients with A) stable COPD (N=28, randomized 3:1) or B) AECOPD (N=44, randomized 1:1) received treatment with inhaled nemiralisib (1mg). Endpoints included induced sputum at various time points before and during treatment for the measurement of transcriptomics (primary endpoint), inflammatory mediators, functional respiratory imaging (FRI), and spirometry.

Results: In stable COPD patients, the use of nemiralisib was associated with alterations in sputum neutrophil transcriptomics suggestive of an improvement in migration phenotype; however, the same nemiralisib-evoked effects were not observed in AECOPD. Inhibition of sputum inflammatory mediators was also observed in stable but not AECOPD patients. In contrast, a placebo-corrected improvement in forced expiratory volume in 1 sec of 136 mL (95% Credible Intervals -46, 315mL) with a probability that the true treatment ratio was >0% (Pr(θ>0)) of 93% was observed in AECOPD. However, FRI endpoints remained unchanged.

Conclusion: We provide evidence for nemiralisib-evoked changes in neutrophil migration phenotype in stable COPD but not AECOPD, despite improving lung function in the latter group. We conclude that induced sputum can be used for measuring evidence of alteration of neutrophil phenotype in stable patients, and our study provides a data set of the sputum transcriptomic changes during recovery from AECOPD.
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http://dx.doi.org/10.2147/COPD.S309303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184158PMC
June 2021

An Inhaled PI3Kδ Inhibitor Improves Recovery in Acutely Exacerbating COPD Patients: A Randomized Trial.

Int J Chron Obstruct Pulmon Dis 2021 3;16:1607-1619. Epub 2021 Jun 3.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, UK.

Purpose: This study evaluated the safety and efficacy of inhaled nemiralisib, a phosphoinositide 3-kinase δ (PI3Kδ) inhibitor, in patients with an acute exacerbation of chronic obstructive pulmonary disease (COPD).

Methods: In this double-blind, placebo-controlled study, 126 patients (40-80 years with a post-bronchodilator forced expiratory volume in 1 sec (FEV) ≤80% of predicted (previously documented)) were randomized 1:1 to once daily inhaled nemiralisib (1 mg) or placebo for 84 days, added to standard of care. The primary endpoint was specific imaging airway volume (siVaw) after 28 treatment days and was analyzed using a Bayesian repeated measures model (clintrials.gov: NCT02294734).

Results: A total of 126 patients were randomized to treatment; 55 on active treatment and 49 on placebo completed the study. When comparing nemiralisib and placebo-treated patients, an 18% placebo-corrected increase from baseline in distal siVaw (95% credible intervals (Cr I) (-1%, 42%)) was observed on Day 28. The probability that the true treatment ratio was >0% (Pr(θ>0)) was 96%, suggestive of a real treatment effect. Improvements were observed across all lung lobes. Patients treated with nemiralisib experienced a 107.3 mL improvement in posterior median FEV (change from baseline, 95% Cr I (-2.1, 215.5)) at day 84, compared with placebo. Adverse events were reported by 41 patients on placebo and 49 on nemiralisib, the most common being post-inhalation cough on nemiralisib (35%) vs placebo (3%).

Conclusion: These data show that addition of nemiralisib to usual care delivers more effective recovery from an acute exacerbation and improves lung function parameters including siVaw and FEV. Although post-inhalation cough was identified, nemiralisib was otherwise well tolerated, providing a promising novel therapy for this acutely ill patient group.
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http://dx.doi.org/10.2147/COPD.S309129DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8184151PMC
June 2021

Predicting Re-Exacerbation Timing and Understanding Prolonged Exacerbations: An Analysis of Patients with COPD in the ECLIPSE Cohort.

Int J Chron Obstruct Pulmon Dis 2021;16:225-244. Epub 2021 Feb 5.

GlaxoSmithKline plc., Research and Development, Middlesex, UK.

Purpose: Understanding risk factors for an acute exacerbation of chronic obstructive pulmonary disease (AECOPD) is important for optimizing patient care. We re-analyzed data from the Evaluation of COPD Longitudinally to Identify Predictive Surrogate Endpoints (ECLIPSE) study (NCT00292552) to identify factors predictive of re-exacerbations and associated with prolonged AECOPDs.

Methods: Patients with COPD from ECLIPSE with moderate/severe AECOPDs were included. The end of the first exacerbation was the index date. Timing of re-exacerbation risk was assessed in patients with 180 days' post-index-date follow-up data. Factors predictive of early (1-90 days) vs late (91-180 days) vs no re-exacerbation were identified using a multivariable partial-proportional-odds-predictive model. Explanatory logistic-regression modeling identified factors associated with prolonged AECOPDs.

Results: Of the 1,554 eligible patients from ECLIPSE, 1,420 had 180 days' follow-up data: more patients experienced early (30.9%) than late (18.7%) re-exacerbations; 50.4% had no re-exacerbation within 180 days. Lower post-bronchodilator FEV (=0.0019), a higher number of moderate/severe exacerbations on/before index date (<0.0001), higher St. George's Respiratory Questionnaire total score (=0.0036), and season of index exacerbation (autumn vs winter, =0.00164) were identified as predictors of early (vs late/none) re-exacerbation risk within 180 days. Similarly, these were all predictors of any (vs none) re-exacerbation risk within 180 days. Median moderate/severe AECOPD duration was 12 days; 22.7% of patients experienced a prolonged AECOPD. The odds of experiencing a prolonged AECOPD were greater for severe vs moderate AECOPDs (adjusted odds ratio=1.917, =0.002) and lower for spring vs winter AECOPDs (adjusted odds ratio=0.578, =0.017).

Conclusion: Prior exacerbation history, reduced lung function, poorer respiratory-related quality-of-life (greater disease burden), and season may help identify patients who will re-exacerbate within 90 days of an AECOPD. Severe AECOPDs and winter AECOPDs are likely to be prolonged and may require close monitoring.
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http://dx.doi.org/10.2147/COPD.S279315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7872897PMC
June 2021

Corrected Super-Resolution Microscopy Enables Nanoscale Imaging of Autofluorescent Lung Macrophages.

Biophys J 2020 12 18;119(12):2403-2417. Epub 2020 Nov 18.

The Lydia Becker Institute of Immunology and Inflammation, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, United Kingdom. Electronic address:

Observing the cell surface and underlying cytoskeleton at nanoscale resolution using super-resolution microscopy has enabled many insights into cell signaling and function. However, the nanoscale dynamics of tissue-specific immune cells have been relatively little studied. Tissue macrophages, for example, are highly autofluorescent, severely limiting the utility of light microscopy. Here, we report a correction technique to remove autofluorescent noise from stochastic optical reconstruction microscopy (STORM) data sets. Simulations and analysis of experimental data identified a moving median filter as an accurate and robust correction technique, which is widely applicable across challenging biological samples. Here, we used this method to visualize lung macrophages activated through Fc receptors by antibody-coated glass slides. Accurate, nanoscale quantification of macrophage morphology revealed that activation induced the formation of cellular protrusions tipped with MHC class I protein. These data are consistent with a role for lung macrophage protrusions in antigen presentation. Moreover, the tetraspanin protein CD81, known to mark extracellular vesicles, appeared in ring-shaped structures (mean diameter 93 ± 50 nm) at the surface of activated lung macrophages. Thus, a moving median filter correction technique allowed us to quantitatively analyze extracellular secretions and membrane structure in tissue-derived immune cells.
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http://dx.doi.org/10.1016/j.bpj.2020.10.041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822748PMC
December 2020

Intranasal Bifidobacterium longum protects against viral-induced lung inflammation and injury in a murine model of lethal influenza infection.

EBioMedicine 2020 Oct 11;60:102981. Epub 2020 Sep 11.

Department of Medicine and School of Microbiology, APC Microbiome Ireland, National University of Ireland, Cork, Ireland.

Background: Prophylactic strategies are urgently needed for prevention of severe inflammatory responses to respiratory viral infections. Bacterial-host interactions may modify the immune response to viral infections.

Methods: We examined the contribution of Intranasal administration of two different Bifidobacterium longum strains or its isolated cell wall in controlling viral induced inflammation using a murine model of influenza infection. We monitored mortality and morbidity over a 10-day period and viral load, differential broncho alveolar lavage (BAL) fluid inflammatory cell counts, Lung tissue histology, BAL and serum cytokines, markers of vascular damage and cell death were quantified.

Findings: Intranasal administration of Bifidobacterium longum35624® or its isolated cell wall prior to virus inoculation significantly reduced viral load within the lungs and significantly improved survival. Reduced viral load was associated with reduced lung injury as suggested by cell death and vascular leakage markers, a shift from neutrophil to macrophage recruitment, reduced inflammatory cytokine levels (including IL-6), reduced type 1 and 2 interferon levels, but increased levels of interferon-λ and surfactant protein D. These protective effects were maintained when the bifidobacterial cell wall preparation was administered 24 h after viral inoculation. The protective effects were also observed for the Bifidobacterium longumPB-VIR™ strain.

Interpretation: Exposure to these bifidobacterial strains protect against the inflammatory sequelae and damage associated with uncontrolled viral replication within the lung.

Funding: This work has been funded, in part, by a research grant from GlaxoSmithKline, PrecisionBiotics Group Ltd., Swiss National Science Foundation grants (project numbers CRSII3_154488, 310030_144219, 310030_127356 and 310030_144219) and Christine Kühne - Center for Allergy Research and Education (CK-CARE).
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http://dx.doi.org/10.1016/j.ebiom.2020.102981DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7495089PMC
October 2020

Drug Interactions for Low-Dose Inhaled Nemiralisib: A Case Study Integrating Modeling, In Vitro, and Clinical Investigations.

Drug Metab Dispos 2020 04 2;48(4):307-316. Epub 2020 Feb 2.

Drug Metabolism and Pharmacokinetics (A.P., A.W.H., K.S.T., M.T., H.T.) and Bioanalysis, Immunogenicity and Biomarkers (A.G.), GlaxoSmithKline R&D, Ware, United Kingdom; RD Projects Clinical Platforms & Sciences, GlaxoSmithKline R&D, Stevenage, United Kingdom (R.W.); Global Clinical and Data Operations, GlaxoSmithKline R&D, Ermington, Australia (K.R.); Discovery Medicine, GlaxoSmithKline, Stevenage, United Kingdom (A.P.C.); Safety and Medical Governance, GlaxoSmithKline R&D, Stockley Park, Uxbridge, United Kingdom (M.M.); and Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom (E.M.H.).

In vitro data for low-dose inhaled phosphoinositide 3-kinase delta inhibitor nemiralisib revealed that it was a substrate and a potent metabolism-dependent inhibitor of cytochrome P450 (P450) 3A4 and a P-glycoprotein (P-gp) substrate. An integrated in silico, in vitro, and clinical approach including a clinical drug interaction study as well as a bespoke physiologically based pharmacokinetic (PBPK) model was used to assess the drug-drug interaction (DDI) risk. Inhaled nemiralisib (100 µg, single dose) was coadministered with itraconazole, a potent P4503A4/P-gp inhibitor, following 200 mg daily administrations for 10 days in 20 male healthy subjects. Systemic exposure to nemiralisib (AUC) increased by 2.01-fold versus nemiralisib alone. To extrapolate the clinical data to other P4503A4 inhibitors, an inhaled PBPK model was developed using Simcyp software. Retrospective simulation of the victim risk showed good agreement between simulated and observed data (AUC ratio 2.3 vs. 2.01, respectively). Prospective DDI simulations predicted a weak but manageable drug interaction when nemiralisib was coadministered with other P4503A4 inhibitors, such as the macrolides clarithromycin and erythromycin (simulated AUC ratio of 1.7), both common comedications in the intended patient populations. PBPK and static mechanistic models were also used to predict a negligible perpetrator DDI effect for nemiralisib on other P4503A4 substrates, including midazolam (a sensitive probe substrate of P4503A4) and theophylline (a narrow therapeutic index drug and another common comedication). In summary, an integrated in silico, in vitro, and clinical approach including an inhalation PBPK model has successfully discharged any potential patient DDI risks in future nemiralisib clinical trials. SIGNIFICANCE STATEMENT: This paper describes the integration of in silico, in vitro, and clinical data to successfully discharge potential drug-drug interaction risks for a low-dose inhaled drug. This work featured assessment of victim and perpetrator risks of drug transporters and cytochrome P450 enzymes, utilizing empirical and mechanistic approaches combined with clinical data (drug interaction and human absorption, metabolism, and pharmacokinetics) and physiologically based pharmacokinetic modeling approaches to facilitate bespoke risk assessment in target patient populations.
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http://dx.doi.org/10.1124/dmd.119.089003DOI Listing
April 2020

Influenza-induced monocyte-derived alveolar macrophages confer prolonged antibacterial protection.

Nat Immunol 2020 02 13;21(2):145-157. Epub 2020 Jan 13.

Immunoregulation Laboratory, Francis Crick Institute, London, UK.

Despite the prevalence and clinical importance of influenza, its long-term effect on lung immunity is unclear. Here we describe that following viral clearance and clinical recovery, at 1 month after infection with influenza, mice are better protected from Streptococcus pneumoniae infection due to a population of monocyte-derived alveolar macrophages (AMs) that produce increased interleukin-6. Influenza-induced monocyte-derived AMs have a surface phenotype similar to resident AMs but display a unique functional, transcriptional and epigenetic profile that is distinct from resident AMs. In contrast, influenza-experienced resident AMs remain largely similar to naive AMs. Thus, influenza changes the composition of the AM population to provide prolonged antibacterial protection. Monocyte-derived AMs persist over time but lose their protective profile. Our results help to understand how transient respiratory infections, a common occurrence in human life, can constantly alter lung immunity by contributing monocyte-derived, recruited cells to the AM population.
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http://dx.doi.org/10.1038/s41590-019-0568-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983324PMC
February 2020

Optimization of Orally Bioavailable PI3Kδ Inhibitors and Identification of Vps34 as a Key Selectivity Target.

J Med Chem 2020 01 8;63(2):638-655. Epub 2020 Jan 8.

Cellzome GmbH , GlaxoSmithKline , Meyerhofstrasse 1 , 69117 Heidelberg , Germany.

Optimization of a lead series of PI3Kδ inhibitors based on a dihydroisobenzofuran core led to the identification of potent, orally bioavailable compound . Selectivity profiling of compound showed similar potency for class III PI3K, Vps34, and PI3Kδ, and compound was not well-tolerated in a 7-day rat toxicity study. Structure-based design led to an improvement in selectivity for PI3Kδ over Vps34 and, a focus on oral phramacokinetics properties resulted in the discovery of compound , which showed improved toxicological outcomes at similar exposure levels to compound .
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http://dx.doi.org/10.1021/acs.jmedchem.9b01585DOI Listing
January 2020

Converging TLR9 and PI3Kgamma signaling induces sterile inflammation and organ damage.

Sci Rep 2019 12 13;9(1):19085. Epub 2019 Dec 13.

Department of Biochemistry and Immunology, Institute of Biological Sciences, Feredal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.

Toll-like receptor 9 (TLR9) and Phosphatidylinositol-3-kinase gamma (PI3Kγ) are very important effectors of the immune response, however, the importance of such crosstalk for disease development is still a matter of discussion. Here we show that PI3Kγ is required for immune responses in which TLR9 is a relevant trigger. We demonstrate the requirement of PI3Kγ for TLR9-induced inflammation in a model of CpG-induced pleurisy. Such requirement was further observed in inflammatory models where DNA sensing via TLR9 contributes to disease, such as silicosis and drug-induced liver injury. Using adoptive transfer, we demonstrate that PI3Kγ is important not only in leukocytes but also in parenchymal cells for the progression of inflammation. We demonstrate this crosstalk between TLR9 and PI3Kγ in vitro using human PBMCs. The inhibition of PI3Kγ in CpG-stimulated PBMCs resulted in reduction of both cytokine production and phosphorylated Akt. Therefore, drugs that target PI3Kγ have the potential to treat diseases mediated by excessive TLR9 signalling.
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http://dx.doi.org/10.1038/s41598-019-55504-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6910931PMC
December 2019

Obesity and disease severity magnify disturbed microbiome-immune interactions in asthma patients.

Nat Commun 2019 12 13;10(1):5711. Epub 2019 Dec 13.

Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland.

In order to improve targeted therapeutic approaches for asthma patients, insights into the molecular mechanisms that differentially contribute to disease phenotypes, such as obese asthmatics or severe asthmatics, are required. Here we report immunological and microbiome alterations in obese asthmatics (n = 50, mean age = 45), non-obese asthmatics (n = 53, mean age = 40), obese non-asthmatics (n = 51, mean age = 44) and their healthy counterparts (n = 48, mean age = 39). Obesity is associated with elevated proinflammatory signatures, which are enhanced in the presence of asthma. Similarly, obesity or asthma induced changes in the composition of the microbiota, while an additive effect is observed in obese asthma patients. Asthma disease severity is negatively correlated with fecal Akkermansia muciniphila levels. Administration of A. muciniphila to murine models significantly reduces airway hyper-reactivity and airway inflammation. Changes in immunological processes and microbiota composition are accentuated in obese asthma patients due to the additive effects of both disease states, while A. muciniphila may play a non-redundant role in patients with a severe asthma phenotype.
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http://dx.doi.org/10.1038/s41467-019-13751-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6911092PMC
December 2019

An Innovative Approach to Characterize Clinical ADME and Pharmacokinetics of the Inhaled Drug Nemiralisib Using an Intravenous Microtracer Combined with an Inhaled Dose and an Oral Radiolabel Dose in Healthy Male Subjects.

Drug Metab Dispos 2019 12 24;47(12):1457-1468. Epub 2019 Oct 24.

Drug Metabolism and Pharmacokinetics (A.W.H., G.Y., R.C., D.K.) and Bioanalysis, Immunogenicity and Biomarkers (A.G., A.P.), GlaxoSmithKline R&D, Ware, United Kingdom; RD Projects Clinical Platforms & Sciences (R.W.), Drug Metabolism and Pharmacokinetics (C.B.), Discovery Medicine (Y.L.M.), Biostatistics (E.J.), GlaxoSmithKline R&D and Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom (E.M.H.); Safety and Medical Governance (M.M.) and Discovery Medicine (W.A.F.), GlaxoSmithKline R&D, Stockley Park, Uxbridge, United Kingdom; Global Clinical and Data Operations, GlaxoSmithKline R&D, Ermington, Australia (K.R.); Covance Laboratories, Harrogate, United Kingdom (L.C.); and Hammersmith Medicines Research, London, United Kingdom (D.W.).

An innovative open-label, crossover clinical study was used to investigate the excretion balance, pharmacokinetics, and metabolism of nemiralisib-an inhaled phosphoinositide 3-kinase delta inhibitor being developed for respiratory diseases. Six healthy men received a single intravenous microtracer of 10 g [C]nemiralisib with a concomitant inhaled nonradiolabeled 1000 g dose followed by an oral 800 g dose of [C]nemiralisib 14 days later. Complementary methods including accelerator mass spectrometry allowed characterization of a range of parameters including oral absorption (F), proportion of nemiralisib escaping gut wall metabolism (F), hepatic extraction (E), fraction of dose absorbed from inhaled dose (F), and renal clearance. Intravenous pharmacokinetics of nemiralisib were characterized by low blood clearance (10.0 l/h), long terminal half-life (55 hours), and high volume of distribution at steady state (728 l). Nemiralisib exhibited moderate inhaled and oral bioavailability (38% and 35%) while F was 29%. Absorption and first-pass parameters were corrected for blood renal clearance and compared with values without correction. Any swallowed nemiralisib was relatively well absorbed (F, 0.48) with a high fraction escaping gut wall metabolism and low extraction by the liver (F and E being 0.83 and 0.10, respectively). There were no major human plasma metabolites requiring further qualification in animal studies. Both unchanged nemiralisib and its oxidative/conjugative metabolites were secreted in bile, with nemiralisib likely subject to further metabolism through enterohepatic recirculation. Direct renal clearance and metabolism followed by renal clearance were lesser routes of elimination. SIGNIFICANCE STATEMENT: A number of innovative features have been combined into one small clinical study enabling a comprehensive description of the human pharmacokinetics and metabolism of an inhaled molecule. Design elements included an intravenous C tracer administration concomitant with an inhalation dose that enabled derivation of parameters such as fraction absorbed (F), the proportion of drug escaping first-pass extraction through the gut wall and liver (F and F) and hepatic extraction (E). Entero-test bile sampling enabled characterization of biliary elimination pathways.
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http://dx.doi.org/10.1124/dmd.119.088344DOI Listing
December 2019

Airway host-microbiome interactions in chronic obstructive pulmonary disease.

Respir Res 2019 Jun 6;20(1):113. Epub 2019 Jun 6.

University of Manchester and University Hospital of South Manchester, Manchester, M23 9QZ, UK.

Background: Little is known about the interactions between the lung microbiome and host response in chronic obstructive pulmonary disease (COPD).

Methods: We performed a longitudinal 16S ribosomal RNA gene-based microbiome survey on 101 sputum samples from 16 healthy subjects and 43 COPD patients, along with characterization of host sputum transcriptome and proteome in COPD patients.

Results: Dysbiosis of sputum microbiome was observed with significantly increased relative abundance of Moraxella in COPD versus healthy subjects and during COPD exacerbations, and Haemophilus in COPD ex-smokers versus current smokers. Multivariate modeling on sputum microbiome, host transcriptome and proteome profiles revealed that significant associations between Moraxella and Haemophilus, host interferon and pro-inflammatory signaling pathways and neutrophilic inflammation predominated among airway host-microbiome interactions in COPD. While neutrophilia was positively correlated with Haemophilus, interferon signaling was more strongly linked to Moraxella. Moreover, while Haemophilus was significantly associated with host factors both in stable state and during exacerbations, Moraxella-associated host responses were primarily related to exacerbations.

Conclusions: Our study highlights a significant airway host-microbiome interplay associated with COPD inflammation and exacerbations. These findings indicate that Haemophilus and Moraxella influence different components of host immune response in COPD, and that novel therapeutic strategies should consider targeting these bacteria and their associated host pathways in COPD.
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http://dx.doi.org/10.1186/s12931-019-1085-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6555748PMC
June 2019

Safety, Tolerability, and Pharmacokinetics of a New Formulation of Nemiralisib Administered via a Dry Powder Inhaler to Healthy Individuals.

Clin Ther 2019 06 7;41(6):1214-1220. Epub 2019 May 7.

GlaxoSmithKline, Stevenage, United Kingdom. Electronic address:

Purpose: Nemiralisib, a phosphoinositide 3-kinase δ inhibitor, is being investigated as an immunomodulatory agent with anti-inflammatory properties in chronic obstructive pulmonary disease. This study evaluated the pharmacokinetic (PK) properties and safety of a new formulation of nemiralisib that contains 0.4% magnesium stearate.

Methods: In this randomized, double-blind, parallel-group study, healthy individuals received a single dose of 500 or 750 μg of nemiralisib administered via the Ellipta dry powder inhaler (DPI) (n = 6 in each treatment group). Aerodynamic particle size distribution (APSD) data comparing previous and new formulations were available before the study. Serial PK analyses for plasma exposure and safety assessments were performed during the first 24 h after dosing, with follow-up measurements on days 3 and 6 in clinic.

Findings: APSD had increases of approximately 6-fold and 2-fold in very fine particle mass and fine particle mass over the previous (Diskus) formulation. In humans, systemic exposure (AUC) was greater after inhalation of 750 versus 500 μg of nemiralisib (AUC: 17,200 h∙pg/mL; 95% CI, 10,900-27,200 h∙pg/mL and 13,100; 95% CI, 8130-21,000 h∙pg/mL, respectively). A low frequency of individual adverse events and no serious adverse events were reported after both doses.

Implications: After single-dose inhalation of 500 and 750 μg of nemiralisib from the Ellipta DPI in healthy individuals, plasma PK data were well defined, and as predicted based on previous PK and APSD data, exposure was increased with the new formulation. Nemiralisib was well tolerated with no new safety issues identified. These data supported progression of nemiralisib to a Phase IIb study in patients with chronic obstructive pulmonary disease. ClinicalTrials.gov identifier: NCT03189589.
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http://dx.doi.org/10.1016/j.clinthera.2019.04.008DOI Listing
June 2019

Translation of Inhaled Drug Optimization Strategies into Clinical Pharmacokinetics and Pharmacodynamics Using GSK2292767A, a Novel Inhaled Phosphoinositide 3-Kinase Inhibitor.

J Pharmacol Exp Ther 2019 06 2;369(3):443-453. Epub 2019 Apr 2.

Refractory Respiratory Inflammation Discovery Performance Unit, GlaxoSmithKline, Stevenage, United Kingdom (M.B., C.D.E., J.N.H., E.M.H.); Clinical Pharmacology & Model Stimulation, (E.P., R.W.), Drug Metabolism & Pharmacokinetics, (G.V., D.M., J.M.), Refractory Respiratory Inflammation DPU, (S.U.), In Vitro In Vivo Translation, (C.F.N.), Discovery Medicine, (A.C) GlaxoSmithKline, Stevenage, United Kingdom; Global Clinical Science & Delivery, GlaxoSmithKline, Stockley Park, Uxbridge, United Kingdom (M.M., J.G.); In Vitro In Vivo Translation, (A.H.), Drug Product Design & Development, (M.I.H.), GlaxoSmithKline, Ware, United Kingdom (M.I.H.); Department of Biological Chemistry, Babraham Institute, Cambridge, United Kingdom (J.C.); and Clinical Unit Cambridge, Addenbrookes Hospital, Cambridge, United Kingdom (F.E., D.F.)

This study describes the pharmacokinetic (PK) and pharmaco-dynamic (PD) profile of -(5-(4-(5-(((2,6)-2,6-dimethylmorpholino)methyl)oxazol-2-yl)-1-indazol-6-yl)-2-methoxypyridin-3-yl)methanesulfonamide (GSK2292767A), a novel low-solubility inhaled phosphoinositide 3-kinase delta (PI3K) inhibitor developed as an alternative to 2-(6-(1H-indol-4-yl)-1H-indazol-4-yl)-5-((4-isopropylpiperazin-1-yl)methyl)oxazole (nemiralisib), which is a highly soluble inhaled inhibitor of PI3K with a lung profile consistent with once-daily dosing. GSK2292767A has a similar in vitro cellular profile to nemiralisib and reduces eosinophilia in a murine PD model by 63% ( = 5, < 0.05). To explore whether a low-soluble compound results in effective PI3K inhibition in humans, a first time in human study was conducted with GSK2292767A in healthy volunteers who smoke. GSK2292767A was generally well tolerated, with headache being the most common reported adverse event. PD changes in induced sputum were measured in combination with drug concentrations in plasma from single (0.05-2 mg, = 37), and 14-day repeat (2 mg, = 12) doses of GSK2292767A. Trough bronchoalveolar lavage (BAL) for PK was taken after 14 days of repeat dosing. GSK2292767A displayed a linear increase in plasma exposure with dose, with marginal accumulation after 14 days. Induced sputum showed a 27% (90% confidence interval 15%, 37%) reduction in phosphatidylinositol-trisphosphate (the product of phosphoinositide 3-kinase activation) 3 hours after a single dose. Reduction was not maintained 24 hours after single or repeat dosing. BAL analysis confirmed the presence of GSK2292767A in lung at 24 hours, consistent with the preclinical lung retention profile. Despite good lung retention, target engagement was only present at 3 hours. This exposure-response disconnect is an important observation for future inhaled drug design strategies considering low solubility to drive lung retention.
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http://dx.doi.org/10.1124/jpet.119.257311DOI Listing
June 2019

Use of autologous Technetium-labelled neutrophils to quantify lung neutrophil clearance in COPD.

Thorax 2019 07 23;74(7):659-666. Epub 2019 Jan 23.

Department of Medicine, University of Cambridge, Cambridge, UK.

Rationale: There is a need to develop imaging protocols which assess neutrophilic inflammation in the lung.

Aim: To quantify whole lung neutrophil accumulation in (1) healthy volunteers (HV) following inhaled lipopolysaccharide (LPS) or saline and (2) patients with COPD using radiolabelled autologous neutrophils and single-photon emission computed tomography/CT (SPECT/CT).

Methods: 20 patients with COPD (Global initiative for chronic obstructive lung disease (GOLD) stages 2-3) and 18 HVs were studied. HVs received inhaled saline (n=6) or LPS (50 µg, n=12) prior to the injection of radiolabelled cells. Neutrophils were isolated using dextran sedimentation and Percoll plasma gradients and labelled with Technetium (Tc)-hexamethylpropyleneamine oxime. SPECT was performed over the thorax/upper abdomen at 45 min, 2 hours, 4 hours and 6 hours. Circulating biomarkers were measured prechallenge and post challenge. Blood neutrophil clearance in the lung was determined using Patlak-Rutland graphical analysis.

Results: There was increased accumulation of Tc-neutrophils in the lungs of patients with COPD and LPS-challenged subjects compared with saline-challenged subjects (saline: 0.0006±0.0003 mL/min/mL lung blood distribution volume [mean ±1 SD]; COPD: 0.0022±0.0010 mL/min/mL [p<0.001]; LPS: 0.0025±0.0008 mL/min/mL [p<0.001]). The accumulation of labelled neutrophils in 10 patients with COPD who underwent repeat radiolabelling/imaging 7-10 days later was highly reproducible (0.0022±0.0010 mL/min/mL vs 0.0023±0.0009 mL/min/mL). Baseline interleukin (IL)-6 levels in patients with COPD were elevated compared with HVs (1.5±1.06 pg/mL [mean ±1 SD] vs 0.4±0.24 pg/mL). LPS challenge increased the circulating IL-6 levels (7.5±2.72 pg/mL) 9 hours post challenge.

Conclusions: This study shows the ability to quantify 'whole lung' neutrophil accumulation in HVs following LPS inhalation and in subjects with COPD using autologous radiolabelled neutrophils and SPECT/CT imaging. Moreover, the reproducibility observed supports the feasibility of using this approach to determine the efficacy of therapeutic agents aimed at altering neutrophil migration to the lungs.
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http://dx.doi.org/10.1136/thoraxjnl-2018-212509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6585304PMC
July 2019

A Multicentre, Randomized, Double-Blind, Placebo-Controlled, Crossover Study To Investigate the Efficacy, Safety, Tolerability, and Pharmacokinetics of Repeat Doses of Inhaled Nemiralisib in Adults with Persistent, Uncontrolled Asthma.

J Pharmacol Exp Ther 2018 12 14;367(3):405-413. Epub 2018 Sep 14.

Respiratory Therapy Area Unit (S.K.), Clinical Pharmacology Science and Study Operations (M.M.), Global Clinical Safety and Pharmacovigilance (Y.C.), and Clinical Pharmacology Modelling and Simulation, Quantitative Sciences, RD Projects Clinical Platforms and Sciences (S.Y.), GSK, Stockley Park, Uxbridge, Middlesex, United Kingdom; Discovery Medicine (A.C.), Refractory Respiratory Inflammation Discovery Performance Unit (M.B., H.W., J.N.H., E.M.H.), Clinical Pharmacology Science and Study Operations (A.H.), and Clinical Statistics (J.R.), GSK, Stevenage, Hertfordshire, United Kingdom; Clinical Operations Department, GSK, Zeist, The Netherlands (C.L.); KLB Gesundheitsforschung Lübeck, Lübeck, Germany (A.L.-S.); and IKF Pneumologie Frankfurt, Clinical Research Centre Respiratory Diseases, Frankfurt, Germany (O.K.)

Phosphoinositide 3-kinase (PI3K) is a lipid kinase involved in leukocyte recruitment and activation. Activation of PI3K has been linked to airway inflammation and asthma pathogenesis. This randomized, double-blind, placebo-controlled, crossover study investigated the efficacy, safety, tolerability, and pharmacokinetics of a PI3K inhibitor, nemiralisib (GSK2269557), in patients with persistent, uncontrolled asthma. Patients ( = 50) received once-daily inhaled nemiralisib (1000 g) or placebo for 28 days, with a crossover to the alternative treatment following a 4-week washout period. Spirometry demonstrated no discernible difference in trough forced expiratory volume in 1 second (FEV) from baseline (adjusted posterior median 7 ml; 95% credible interval -83, 102 ml) between nemiralisib and placebo treatment at day 28 (primary endpoint). These results were supported by most secondary endpoints, including weighted mean FEV (0-4 hours) and change in trough forced vital capacity at day 28. Nemiralisib was generally well-tolerated, with few side effects except for post-inhalation cough (nemiralisib: 35%; placebo: 9%). At day 14, sputum interleukin (IL)-5, IL-13, IL-6, and IL-8 levels were reduced by a median of 17%, 7%, 15%, and 8%, respectively, when comparing nemiralisib with placebo [ = 15 (IL-5, IL-8) or 16 (IL-6, IL-13); posterior probability of a true ratio >0%: 78%, 64%, 76%, and 63%, respectively]. These results suggest that nemiralisib inhibited PI3K locally; however, this did not translate into meaningful clinical improvement. Further studies will investigate the potential efficacy of nemiralisib in patients with asthma with other specific more severe phenotypes, including those who are colonized with bacteria and frequently exacerbate.
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http://dx.doi.org/10.1124/jpet.118.249516DOI Listing
December 2018

PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner.

Nat Commun 2018 08 9;9(1):3174. Epub 2018 Aug 9.

Laboratory of Lymphocyte Signalling and Development, Babraham Institute, Cambridge, CB21 3AT, UK.

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can confer protection against repeated exposure to S. pneumoniae, yet vaccines offer only partial protection. Patients with Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knock-in mouse model of this disease and identify a CD19B220 B cell subset that is induced by PI3Kδ signaling, resides in the lungs, and is correlated with increased susceptibility to S. pneumoniae during early phases of infection via an antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a potential therapeutic target.
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http://dx.doi.org/10.1038/s41467-018-05674-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085315PMC
August 2018

Safety, Tolerability, and Pharmacokinetics of Single and Repeat Doses of Nemiralisib Administered via the Ellipta Dry Powder Inhaler to Healthy Subjects.

Clin Ther 2018 08 25;40(8):1410-1417. Epub 2018 Jul 25.

GlaxoSmithKline, Stevenage, United Kingdom. Electronic address:

Purpose: Novel therapies to treat chronic obstructive pulmonary disease are highly desirable. The safety, tolerability, and pharmacokinetic (PK) parameters of nemiralisib, a phosphoinositide 3-kinase δ inhibitor, administered via the Ellipta dry powder inhaler (GlaxoSmithKline, Research Triangle Park, North Carolina) was evaluated, including an assessment of oral bioavailability.

Methods: This single-center, 3-part, placebo-controlled trial in 22 healthy subjects evaluated single (100 and 200 μg) and repeat (200 μg for 10 days) doses of inhaled nemiralisib in parts A (n = 12) and B (n = 12) (double-blind) and single doses of inhaled nemiralisib (200 µg) with and without charcoal block in Part C (n = 6) (open-label, 2-period, crossover). There was a minimum 14-day washout period between dosing days.

Findings: 21 subjects completed the study, mean age was similar in the three parts (A: 49 years; B: 44 years; C: 55 years). After single doses of nemiralisib, observed plasma C dropped rapidly, followed by a slower elimination phase. Near-dose proportionality was observed: mean (95% CI) plasma C and AUC values were 174.3 pg/mL (96.9-313.3) and 694.6 pg·h/mL (503.5-958.2) for 100 μg and 398.9 pg/mL (318.3-500.1) and 1699.6 pg·h/mL (1273.3-2268.7) for 200 μg, respectively. Repeat dosing for 10 days showed exposures ∼2- to 4-fold higher than on the single dose (peak, trough, and AUC levels), achieving steady-state by day 6. Mean AUC was 2193.6 pg·h/mL and 1645.3 pg·h/mL in the absence/presence of charcoal. Two non-drug-related adverse events were observed; neither was serious or resulted in withdrawal.

Implications: Inhalation of nemiralisib was well tolerated in these healthy subjects. Plasma pharmacokinetic variables were well defined, and charcoal block data indicate that ∼23% of the total systemic exposure after inhalation from Ellipta was attributable to orally absorbed drug. ClinicalTrials.gov identifier: NCT02691325.
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http://dx.doi.org/10.1016/j.clinthera.2018.06.011DOI Listing
August 2018

The effects of repeated Toll-like receptors 2 and 4 stimulation in COPD alveolar macrophages.

Int J Chron Obstruct Pulmon Dis 2018 2;13:771-780. Epub 2018 Mar 2.

Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, The University of Manchester, Manchester University NHS Foundation Trust, Manchester.

Background: COPD is a progressive inflammatory airway disease characterized by increased numbers of alveolar macrophages in the lungs. Bacterial colonization of the lungs is a common feature in COPD and can promote inflammation through continual and repeated Toll-like receptor (TLR) stimulation. We have studied the response of COPD alveolar macrophages to repetitive stimulation with TLR2 and TLR4 ligands. We investigated the effect of sequential stimulation with different ligands to determine whether this results in tolerance or amplification of the immune response.

Methods: We stimulated alveolar macrophages from COPD patients (n=9) and smokers (n=8) with the TLR4 agonist lipopolysaccharide (LPS) or the TLR2 agonist Pam3CSK4 for 24 hours before restimulating again for 24 hours. Cytokine protein release and gene expression were investigated.

Results: Repetitive stimulation of COPD and smokers macrophages with LPS for both 24-hour periods caused a reduction in tumor necrosis factor α, CCL5, and IL-10 production compared to cells that were not exposed initially to LPS. IL-6 and CXCL8 production were not significantly altered following repetitive LPS stimulation. The same pattern was observed for repeated stimulation with Pam3CSK4. Using COPD macrophages, LPS followed by Pam3CSK4 stimulation increased the levels of all cytokines compared to media followed by Pam3CSK4.

Conclusion: TLR tolerance in COPD alveolar macrophages occurs after repetitive stimulation with the same TLR ligand, but this only occurs for selected cytokines. CXCL8 production is not reduced after repetitive TLR stimulation with the same ligand; this may be an important mechanism for the increased CXCL8 levels that have been observed in COPD. We showed that TLR4 stimulation followed by TLR2 stimulation does not cause tolerance, but enhances cytokine production. This may be a relevant mechanism by which bacteria cause excessive inflammation in COPD patients.
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http://dx.doi.org/10.2147/COPD.S97071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5841324PMC
September 2018

First-in-Human Study With the Inhaled TLR9 Oligonucleotide Agonist AZD1419 Results in Interferon Responses in the Lung, and Is Safe and Well-Tolerated.

Clin Pharmacol Ther 2018 08 6;104(2):335-345. Epub 2017 Dec 6.

Dynavax Technologies, Berkeley, California, USA.

Current asthma treatments address symptoms rather than the underlying disease pathophysiology, a better understanding of which has led to the identification of the Th2 high endotype. The activation of Toll-like receptors to induce Type I interferons directly in the lungs represents a novel therapeutic approach to reset this underlying Th2 pathophysiology with the potential to provide long-term disease modification. We present the nonclinical data and phase I clinical profile of an inhaled TLR9 agonist, AZD1419, a C-type CpG designed to induce interferon in the lung. In healthy volunteers, AZD1419 was found to be safe and well-tolerated. Target engagement in the lung was demonstrated at all dose levels tested. No evidence of tolerization or amplification of responses was evident on repeated dosing and 15.4 mg was defined as the maximum tolerated dose. AZD1419 clinical data supports its continued development as a potentially disease-modifying therapeutic in asthma.
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http://dx.doi.org/10.1002/cpt.938DOI Listing
August 2018

Ampicillin resistance in from COPD patients in the UK.

Int J Chron Obstruct Pulmon Dis 2017 22;12:1507-1518. Epub 2017 May 22.

Division of Infection, Immunity and Respiratory Medicine, Medicines Evaluation Unit, University Hospital of South Manchester Foundation Trust, University of Manchester.

Background: is commonly isolated from the airways of COPD patients. Antibiotic treatment may cause the emergence of resistant strains, particularly ampicillin-resistant strains, including β-lactamase-negative ampicillin resistance (BLNAR) strains. Genetic identification using sequencing is the optimum method for identifying mutations within BLNAR strains. The prevalence of BLNAR in COPD patients during the stable state has not been reported. We investigated the antibiotic resistance patterns of present in the sputum of stable COPD patients, focusing on ampicillin resistance; the prevalence of enzyme and non-enzyme-mediated ampicillin resistance was determined. A subset of patients was followed up longitudinally to study strain switching and antibiotic sensitivity changes.

Patients And Methods: Sputum sampling was performed in 61 COPD patients, with 42 samples obtained at baseline; was detected by polymerase chain reaction in 28 samples. In all, 45 patients completed the follow-up for 2 years; 24 isolates were obtained.

Results: Disk diffusion showed the highest antibiotic resistance in the penicillin antibiotic group (eg, 67% for ampicillin) and macrolides (eg, 46% for erythromycin), whereas all isolates were susceptible to quinolones. Of the 16 isolates resistant to ampicillin, 9 (56%) were β-lactamase positive. The β-lactamase-negative isolates were further investigated; none of these fulfilled the phenotypic BLNAR classification criteria of ampicillin minimum inhibitory concentration >1 µg/mL, and only one demonstrated an mutation. Frequent strain switching was confirmed using multilocus sequence typing and was associated with changes in the antibiotic sensitivity pattern.

Conclusion: We observed an overidentification of ampicillin resistance by disk diffusion. The majority of ampicillin resistance was due to enzyme production. strain changes during the stable state may be associated with a change in antibiotic sensitivity; this has implications for empirical antibiotic prescribing.
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http://dx.doi.org/10.2147/COPD.S135338DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446963PMC
March 2018

An investigation of the anti-inflammatory effects and a potential biomarker of PI3Kδ inhibition in COPD T cells.

Clin Exp Pharmacol Physiol 2017 Sep;44(9):932-940

The University of Manchester, Division of Infection, Immunity and Respiratory Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University Hospital of South Manchester NHS Foundation Trust, The University of Manchester, Manchester, UK.

Lymphocyte numbers are increased in the lungs of chronic obstructive pulmonary disease (COPD) patients. Phosphatidylinositol-3-kinase delta (PI3Kδ) is involved in lymphocyte activation. We investigated the effect of PI3Kδ inhibition on cytokine release from COPD lymphocytes. We also evaluated phosphorylated ribosomal S6 protein (rS6) as a potential biomarker of PI3Kδ activation. Peripheral blood mononuclear cells (PBMCs) and bronchoalveolar lavage (BAL) cells isolated from healthy never smokers (HNS), smokers (S) and COPD patients were stimulated to induce a T cell receptor response. The effects of a PI3Kδ specific inhibitor (GSK045) on cytokine release and rS6 phosphorylation were measured by Luminex and flow cytometry respectively. The effects of GSK045 on cytokine production from PHA stimulated chopped lung samples were investigated. GSK045 reduced cytokine release from PBMCs, BAL cells and chopped lung. Inhibition was greatest in the chopped lung model, with approximately 80% inhibition of interferon (IFN) γ, interleukin (IL)-2, IL-17 and IL-10. PI3Kδ inhibition suppressed rS6 phosphorylation in unstimulated airway T-lymphocytes by up to 60%. Inhibition of PI3Kδ suppressed T cell cytokine production in COPD patients. rS6 phosphorylation shows potential as a biomarker to assess PI3Kδ activity.
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http://dx.doi.org/10.1111/1440-1681.12784DOI Listing
September 2017

Membrane nanoclusters of FcγRI segregate from inhibitory SIRPα upon activation of human macrophages.

J Cell Biol 2017 04 13;216(4):1123-1141. Epub 2017 Mar 13.

Manchester Collaborative Centre for Inflammation Research, University of Manchester, Manchester M13 9NT, England, UK

Signal integration between activating Fc receptors and inhibitory signal regulatory protein α (SIRPα) controls macrophage phagocytosis. Here, using dual-color direct stochastic optical reconstruction microscopy, we report that Fcγ receptor I (FcγRI), FcγRII, and SIRPα are not homogeneously distributed at macrophage surfaces but are organized in discrete nanoclusters, with a mean radius of 71 ± 11 nm, 60 ± 6 nm, and 48 ± 3 nm, respectively. Nanoclusters of FcγRI, but not FcγRII, are constitutively associated with nanoclusters of SIRPα, within 62 ± 5 nm, mediated by the actin cytoskeleton. Upon Fc receptor activation, Src-family kinase signaling leads to segregation of FcγRI and SIRPα nanoclusters to be 197 ± 3 nm apart. Co-ligation of SIRPα with CD47 abrogates nanocluster segregation. If the balance of signals favors activation, FcγRI nanoclusters reorganize into periodically spaced concentric rings. Thus, a nanometer- and micron-scale reorganization of activating and inhibitory receptors occurs at the surface of human macrophages concurrent with signal integration.
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http://dx.doi.org/10.1083/jcb.201608094DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5379948PMC
April 2017

Histamine-secreting microbes are increased in the gut of adult asthma patients.

J Allergy Clin Immunol 2016 11 27;138(5):1491-1494.e7. Epub 2016 Jul 27.

Swiss Institute of Allergy and Asthma Research (SIAF), University of Zurich, Davos, Switzerland. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2016.05.049DOI Listing
November 2016

IFNλ is a potent anti-influenza therapeutic without the inflammatory side effects of IFNα treatment.

EMBO Mol Med 2016 09 1;8(9):1099-112. Epub 2016 Sep 1.

Immunoregulation Laboratory, Mill Hill Laboratory, Francis Crick Institute, London, UK

Influenza A virus (IAV)-induced severe disease is characterized by infected lung epithelia, robust inflammatory responses and acute lung injury. Since type I interferon (IFNαβ) and type III interferon (IFNλ) are potent antiviral cytokines with immunomodulatory potential, we assessed their efficacy as IAV treatments. IFNλ treatment of IAV-infected Mx1-positive mice lowered viral load and protected from disease. IFNα treatment also restricted IAV replication but exacerbated disease. IFNα treatment increased pulmonary proinflammatory cytokine secretion, innate cell recruitment and epithelial cell death, unlike IFNλ-treatment. IFNλ lacked the direct stimulatory activity of IFNα on immune cells. In epithelia, both IFNs induced antiviral genes but no inflammatory cytokines. Similarly, human airway epithelia responded to both IFNα and IFNλ by induction of antiviral genes but not of cytokines, while hPBMCs responded only to IFNα. The restriction of both IFNλ responsiveness and productive IAV replication to pulmonary epithelia allows IFNλ to limit IAV spread through antiviral gene induction in relevant cells without overstimulating the immune system and driving immunopathology. We propose IFNλ as a non-inflammatory and hence superior treatment option for human IAV infection.
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http://dx.doi.org/10.15252/emmm.201606413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5009813PMC
September 2016

Development of a Novel Quantitative Structure-Activity Relationship Model to Accurately Predict Pulmonary Absorption and Replace Routine Use of the Isolated Perfused Respiring Rat Lung Model.

Pharm Res 2016 11 11;33(11):2604-16. Epub 2016 Jul 11.

Refractory Respiratory Inflammation DPU, GlaxoSmithKline Medicines Research Centre, Gunnels Wood Road, Stevenage, Hertfordshire, SG1 2NY, UK.

Purpose: We developed and tested a novel Quantitative Structure-Activity Relationship (QSAR) model to better understand the physicochemical drivers of pulmonary absorption, and to facilitate compound design through improved prediction of absorption. The model was tested using a large array of both existing and newly designed compounds.

Methods: Pulmonary absorption data was generated using the isolated perfused respiring rat lung (IPRLu) model for 82 drug discovery compounds and 17 marketed drugs. This dataset was used to build a novel QSAR model based on calculated physicochemical properties. A further 9 compounds were used to test the model's predictive capability.

Results: The QSAR model performed well on the 9 compounds in the "Test set" with a predicted versus observed correlation of R(2) = 0.85, and >65% of compounds correctly categorised. Calculated descriptors associated with permeability and hydrophobicity positively correlated with pulmonary absorption, whereas those associated with charge, ionisation and size negatively correlated.

Conclusions: The novel QSAR model described here can replace routine generation of IPRLu model data for ranking and classifying compounds prior to synthesis. It will also provide scientists working in the field of inhaled drug discovery with a deeper understanding of the physicochemical drivers of pulmonary absorption based on a relevant respiratory compound dataset.
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http://dx.doi.org/10.1007/s11095-016-1983-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5040732PMC
November 2016

Acute Respiratory Distress Syndrome Neutrophils Have a Distinct Phenotype and Are Resistant to Phosphoinositide 3-Kinase Inhibition.

Am J Respir Crit Care Med 2016 10;194(8):961-973

1 Department of Medicine, University of Cambridge, Cambridge, United Kingdom.

Rationale: Acute respiratory distress syndrome is refractory to pharmacological intervention. Inappropriate activation of alveolar neutrophils is believed to underpin this disease's complex pathophysiology, yet these cells have been little studied.

Objectives: To examine the functional and transcriptional profiles of patient blood and alveolar neutrophils compared with healthy volunteer cells, and to define their sensitivity to phosphoinositide 3-kinase inhibition.

Methods: Twenty-three ventilated patients underwent bronchoalveolar lavage. Alveolar and blood neutrophil apoptosis, phagocytosis, and adhesion molecules were quantified by flow cytometry, and oxidase responses were quantified by chemiluminescence. Cytokine and transcriptional profiling were used in multiplex and GeneChip arrays.

Measurements And Main Results: Patient blood and alveolar neutrophils were distinct from healthy circulating cells, with increased CD11b and reduced CD62L expression, delayed constitutive apoptosis, and primed oxidase responses. Incubating control cells with disease bronchoalveolar lavage recapitulated the aberrant functional phenotype, and this could be reversed by phosphoinositide 3-kinase inhibitors. In contrast, the prosurvival phenotype of patient cells was resistant to phosphoinositide 3-kinase inhibition. RNA transcriptomic analysis revealed modified immune, cytoskeletal, and cell death pathways in patient cells, aligning closely to sepsis and burns datasets but not to phosphoinositide 3-kinase signatures.

Conclusions: Acute respiratory distress syndrome blood and alveolar neutrophils display a distinct primed prosurvival profile and transcriptional signature. The enhanced respiratory burst was phosphoinositide 3-kinase-dependent but delayed apoptosis and the altered transcriptional profile were not. These unexpected findings cast doubt over the utility of phosphoinositide 3-kinase inhibition in acute respiratory distress syndrome and highlight the importance of evaluating novel therapeutic strategies in patient-derived cells.
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http://dx.doi.org/10.1164/rccm.201509-1818OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067816PMC
October 2016
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