Effect of chronic mucus hypersecretion on treatment responses to inhaled therapies in patients with chronic obstructive pulmonary disease: Post hoc analysis of the IMPACT trial.

This is the final version. Available from Wiley via the DOI in this record. DATA AVAILABILITY STATEMENT: Anonymized individual participant data and study documents can be requested for further research from www.clinicalstudydatarequest.com.BACKGROUND AND OBJECTIVE: Chronic mucus hypersecretion (CMH) is a clinical phenotype of COPD. This exploratory post hoc analysis assessed relationship between CMH status and treatment response in IMPACT. METHODS: Patients were randomized to once-daily fluticasone furoate/umeclidinium/vilanterol (FF/UMEC/VI) 100/62.5/25 μg, FF/VI 100/25 μg or UMEC/VI 62.5/25 μg and designated CMH+ if they scored 1/2 in St George's Respiratory Questionnaire (SGRQ) questions 1 and 2. Endpoints assessed by baseline CMH status included on-treatment exacerbation rates, change from baseline in trough forced expiratory volume in 1 second, SGRQ total score, COPD Assessment Test (CAT) score, proportion of SGRQ and CAT responders at Week 52 and safety. RESULTS: Of 10,355 patients in the intent-to-treat population, 10,250 reported baseline SGRQ data (CMH+: 62% [n = 6383]). FF/UMEC/VI significantly (p < 0.001) reduced on-treatment moderate/severe exacerbation rates versus FF/VI and UMEC/VI in CMH+ (rate ratio: 0.87 and 0.72) and CMH- patients (0.82 and 0.80). FF/UMEC/VI significantly (p < 0.05) reduced on-treatment severe exacerbation rates versus UMEC/VI in CMH+ (0.62) and CMH- (0.74) subgroups. Similar improvements in health status and lung function with FF/UMEC/VI were observed, regardless of CMH status. In CMH+ patients, FF/VI significantly (p < 0.001) reduced on-treatment moderate/severe and severe exacerbation rates versus UMEC/VI (0.83 and 0.70). CONCLUSION: FF/UMEC/VI had a favourable benefit: risk profile versus dual therapies irrespective of CMH status. The presence of CMH did not influence treatment response or exacerbations, lung function and/or health status. However, CMH did generate differences when dual therapies were compared and the impact of CMH should be considered in future trial design.GlaxoSmithKleinNational Institute for Health Research (NIHR) Manchester Biomedical Research Centre (BRC

Methods for detection of NOX-derived superoxide radical anion and hydrogen peroxide in cells

NADPH oxidases (NOX) are transmembrane enzymes, which catalyze the formation of reactive oxygen species (ROS). In humans and most mammals, the NOX family comprises seven members, namely, NOX1-5 and the dual oxidases DUOX1 and 2. The primary product of most NOX isoforms is the superoxide radical anion O2ċ-, which is rapidly dismutated in hydrogen peroxide (H2O2), while NOX4 and DUOX mostly generate H2O2. ROS are multifunctional molecules in tissues, and NOX-derived ROS cellular functions are as diverse as microbial killing (NOX2), thyroid hormone synthesis (DUOX2), or otoconia formation in the inner ear (NOX3). NOX are potential pharmacological targets in numerous diseases such as diabetes, fibrosis, and brain ischemia, and NOX inhibitors are currently under development. Here we describe two cellular assays to detect extracellular O2ċ- and H2O2 in cells overexpressing specific NOX isoforms and their subunits

Singlet molecular oxygen regulates vascular tone and blood pressure in inflammation

Singlet molecular oxygen (O-1(2)) has well-established roles in photosynthetic plants, bacteria and fungi(1-3), but not in mammals. Chemically generated O-1(2) oxidizes the amino acid tryptophan to precursors of a key metabolite called N-formylkynurenine(4), whereas enzymatic oxidation of tryptophan to N-formylkynurenine is catalysed by a family of dioxygenases, including indoleamine 2,3-dioxygenase 1(5). Under inflammatory conditions, this haem-containing enzyme is expressed in arterial endothelial cells, where it contributes to the regulation of blood pressure(6). However, whether indoleamine 2,3-dioxygenase 1 forms O-1(2) and whether this contributes to blood pressure control have remained unknown. Here we show that arterial indoleamine 2,3-dioxygenase 1 regulates blood pressure via formation of O-1(2). We observed that in the presence of hydrogen peroxide, the enzyme generates O-1(2) and that this is associated with the stereoselective oxidation of L-tryptophan to a tricyclic hydroperoxide via a previously unrecognized oxidative activation of the dioxygenase activity. The tryptophan-derived hydroperoxide acts in vivo as a signalling molecule, inducing arterial relaxation and decreasing blood pressure; this activity is dependent on Cys42 of protein kinase G1 alpha. Our findings demonstrate a pathophysiological role for O-1(2) in mammals through formation of an amino acid-derived hydroperoxide that regulates vascular tone and blood pressure under inflammatory conditions

NAD deficiency, congenital malformations and niacin supplementation

Background Congenital malformations can manifest as combinations of phenotypes that co-occur more often than expected by chance. In many such cases it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral and renal defects, amongst others, in four unrelated patients. Methods Genomic sequencing was used to identify potentially pathogenic gene variants in four families. The variants were functionally tested using in vitro enzyme activity assays, and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using CRISPR/Cas9. Results Variants were identified in 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU) genes encoding kynurenine pathway enzymes. Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNY proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan via the kynurenine pathway. The patients had reduced circulating NAD levels. Haao or Kynu null mouse embryos developed similar defects to the patients due to NAD deficiency. In null mice, averting NAD deficiency during gestation prevented defects. Conclusions Disruption of NAD synthesis causes a deficiency and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice

Mitochondrial CoQ deficiency is a common driver of mitochondrial oxidants and insulin resistance

© Fazakerley et al. Insulin resistance in muscle, adipocytes and liver is a gateway to a number of metabolic diseases. Here, we show a selective deficiency in mitochondrial coenzyme Q (CoQ) in insulin-resistant adipose and muscle tissue. This defect was observed in a range of in vitro insulin resistance models and adipose tissue from insulin-resistant humans and was concomitant with lower expression of mevalonate/CoQ biosynthesis pathway proteins in most models. Pharmacologic or genetic manipulations that decreased mitochondrial CoQ triggered mitochondrial oxidants and insulin resistance while CoQ supplementation in either insulin-resistant cell models or mice restored normal insulin sensitivity. Specifically, lowering of mitochondrial CoQ caused insulin resistance in adipocytes as a result of increased superoxide/hydrogen peroxide production via complex II. These data suggest that mitochondrial CoQ is a proximal driver of mitochondrial oxidants and insulin resistance, and that mechanisms that restore mitochondrial CoQ may be effective therapeutic targets for treating insulin resistance

High-Throughput Screening of NOX Inhibitors.

MEDLINE:31172487Development of new, selective inhibitors of nicotinamide adenine dinucleotide phosphate oxidase (NOX) isoforms is important both for basic studies on the role of these enzymes in cellular redox signaling, cell physiology, and proliferation and for development of new drugs for diseases carrying a component of increased NOX activity, such as several types of cancer and cardiovascular and neurodegenerative diseases. High-throughput screening (HTS) of large libraries of compounds remains the major approach for development of new NOX inhibitors. Here, we describe the protocol for the HTS campaign for NOX inhibitors using rigorous assays for superoxide radical anion and hydrogen peroxide, based on oxidation of hydropropidine, coumarin boronic acid, and Amplex Red. We propose using these three probes to screen for and identify new inhibitors, by selecting positive hits that show inhibitory effects in all three assays. Protocols for the synthesis of hydropropidine and for confirmatory assays, including oxygen consumption measurements, electron paramagnetic resonance spin trapping of superoxide, and simultaneous monitoring of superoxide and hydrogen peroxide, are also provided