Quantitation of salbutamol using micro-volume blood sampling - applications to exacerbations of pediatric asthma.

OBJECTIVES: A novel gas chromatography-mass spectrometry (GC-MS) method has been developed to quantify salbutamol in micro-volumes (10 µL) of blood. A potential application is paediatric therapeutic dose monitoring (TDM) in acute severe asthma. METHODS: At presentation, the children receive multiple doses of salbutamol (inhaled, nebulised and occasionally intravenous) but it is difficult to distinguish children who do not respond to treatment because of inadequate concentrations from those with toxicity, as symptoms are similar. A comparison was made between traditional dried blood spots (DBS) and the newly developed technique volumetric absorptive micro-sampling (VAMS), with specific investigation into the effect of drying time on analyte recovery. RESULTS: For both sampling techniques, the final assay demonstrated good precision and accuracy across the concentration range tested (3-100 ng/mL), including both the normal therapeutic and toxic range. The method was developed to comply with FDA guidelines with precision and accuracy ≤15% for all concentrations, except the limit of quantification (5 ng/mL) where they were ≤20%. VAMS offered advantages in sampling ease and reduced GC-MS interference. The assay was successfully applied to the quantification of blood salbutamol concentrations in three healthy volunteers dosed with 1 mg salbutamol by inhalation. CONCLUSIONS: This demonstrated its potential for use in paediatric TDM studies, where in the acute situation considerably higher doses of salbutamol will have been administered. This is the first time that a TDM method for salbutamol has been carried out using VAMS and offers all the advantages provided by DBS, whilst eliminating the inherent sampling volume inaccuracies of traditional DBS collection.

Moderate hyperoxia induces extracellular matrix remodeling by human fetal airway smooth muscle cells.

BACKGROUND: Premature infants are at increased risk for airway diseases, such as wheezing and asthma, because of early exposure to risk factors including hyperoxia. As in adult asthma, airway remodeling and increased extracellular matrix (ECM) deposition is involved. METHODS: We assessed the impact of 24-72 h of moderate hyperoxia (50%) on human fetal airway smooth muscle (fASM) ECM deposition through western blot, modified in-cell western, and zymography techniques. RESULTS: Hyperoxia exposure significantly increased collagen I and collagen III deposition, increased pro- and cleaved matrix metalloproteinase 9 (MMP9) activity, and decreased endogenous MMP inhibitor, TIMP1, expression. Hyperoxia-induced change in caveolin-1 (CAV1) expression was assessed as a potential mechanism for the changes in ECM deposition. CAV1 expression was decreased following hyperoxia. Supplementation of CAV1 activity with caveolar scaffolding domain (CSD) peptide abrogated the hyperoxia-mediated ECM changes. CONCLUSION: These results demonstrate that moderate hyperoxia enhances ECM deposition in developing airways by altering the balance between MMPs and their inhibitors (TIMPs), and by increasing collagen deposition. These effects are partly mediated by a hyperoxia-induced decrease in CAV1 expression. In conjunction with prior data demonstrating increased fASM proliferation with hyperoxia, these data further demonstrate that hyperoxia is an important instigator of remodeling in developing airways.

cAMP-mediated secretion of brain-derived neurotrophic factor in developing airway smooth muscle.

Moderate hyperoxic exposure in preterm infants contributes to subsequent airway dysfunction and to risk of developing recurrent wheeze and asthma. The regulatory mechanisms that can contribute to hyperoxia-induced airway dysfunction are still under investigation. Recent studies in mice show that hyperoxia increases brain-derived neurotrophic factor (BDNF), a growth factor that increases airway smooth muscle (ASM) proliferation and contractility. We assessed the mechanisms underlying effects of moderate hyperoxia (50% O2) on BDNF expression and secretion in developing human ASM. Hyperoxia increased BDNF secretion, but did not alter endogenous BDNF mRNA or intracellular protein levels. Exposure to hyperoxia significantly increased [Ca2+]i responses to histamine, an effect blunted by the BDNF chelator TrkB-Fc. Hyperoxia also increased ASM cAMP levels, associated with reduced PDE4 activity, but did not alter protein kinase A (PKA) activity or adenylyl cyclase mRNA levels. However, 50% O2 increased expression of Epac2, which is activated by cAMP and can regulate protein secretion. Silencing RNA studies indicated that Epac2, but not Epac1, is important for hyperoxia-induced BDNF secretion, while PKA inhibition did not influence BDNF secretion. In turn, BDNF had autocrine effects of enhancing ASM cAMP levels, an effect inhibited by TrkB and BDNF siRNAs. Together, these novel studies suggest that hyperoxia can modulate BDNF secretion, via cAMP-mediated Epac2 activation in ASM, resulting in a positive feedback effect of BDNF-mediated elevation in cAMP levels. The potential functional role of this pathway is to sustain BDNF secretion following hyperoxic stimulus, leading to enhanced ASM contractility and proliferation.

TLR3 activation increases chemokine expression in human fetal airway smooth muscle cells.

Viral infections, such as respiratory syncytial virus and rhinovirus, adversely affect neonatal and pediatric populations, resulting in significant lung morbidity, including acute asthma exacerbation. Studies in adults have demonstrated that human airway smooth muscle (ASM) cells modulate inflammation through their ability to secrete inflammatory cytokines and chemokines. The role of ASM in the developing airway during infection remains undefined. In our study, we used human fetal ASM cells as an in vitro model to examine the effect of Toll-like receptor (TLR) agonists on chemokine secretion. We found that fetal ASM express multiple TLRs, including TLR3 and TLR4, which are implicated in the pathogenesis of respiratory syncytial virus and rhinovirus infection. Cells were treated with TLR agonists, polyinosinic-polycytidylic acid [poly(I:C)] (TLR3 agonist), lipopolysaccharide (TLR4 agonist), or R848 (TLR7/8 agonist), and IL-8 and chemokine (C-C motif) ligand 5 (CCL5) secretion were evaluated. Interestingly, poly(I:C), but neither lipopolysaccharide nor R848, increased IL-8 and chemokine (C-C motif) ligand 5 secretion. Examination of signaling pathways suggested that the poly(I:C) effects in fetal ASM involve TLR and ERK signaling, in addition to another major inflammatory pathway, NF-κB. Moreover, there are variations between fetal and adult ASM with respect to poly(I:C) effects on signaling pathways. Pharmacological inhibition suggested that ERK pathways mediate poly(I:C) effects. Overall, our data show that poly(I:C) initiates activation of proinflammatory pathways in developing ASM, which may contribute to immune responses to infection and exacerbation of asthma.

Perfluorocarbon emulsion therapy attenuates pneumococcal infection in sickle cell mice.

Impaired immunity and tissue hypoxia-ischemia are strongly linked with Streptococcus pneumoniae pathogenesis in patients with sickle cell anemia. Perfluorocarbon emulsions (PFCEs) have high O2-dissolving capacity and can alleviate tissue hypoxia. Here, we evaluate the effects of intravenous PFCE therapy in transgenic sickle cell (HbSS) mice infected with S. pneumoniae. HbSS and C57BL/6 (control) mice intravenously infected with S. pneumoniae were treated intravenously with PFCE or phosphate-buffered saline (PBS) and then managed in either air/O2 (FiO2 proportion, 50%; hereafter referred to as the PFCE-O2 and PBS-O2 groups) or air only (hereafter, the PFCE-air and PBS-air groups) gas mixtures. Lungs were processed for leukocyte and bacterial counts and cytokine measurements. HbSS mice developed severe pneumococcal infection significantly faster than C57BL/6 mice (Kaplan-Maier analysis, P < .05). PFCE-O2-treated HbSS mice had significantly better survival at 72 hours than HBSS mice treated with PFCE-air, PBS-O2, or PBS-air (P < .05). PFCE-O2-treated HbSS mice also had significantly lower pulmonary leukocyte counts, lower interleukin 1ß and interferon γ levels, and higher interleukin 10 levels than PFCE-air-treated HbSS mice. Clearance of S. pneumoniae from lungs of HbSS mice or C57BL/6 mice was not altered by PFCE treatment. Improved survival of PFCE-O2-treated HbSS mice infected with S. pneumoniae is associated with altered pulmonary inflammation but not enhanced bacterial clearance.

Vitamin D attenuates cytokine-induced remodeling in human fetal airway smooth muscle cells.

Asthma in the pediatric population remains a significant contributor to morbidity and increasing healthcare costs. Vitamin D3 insufficiency and deficiency have been associated with development of asthma. Recent studies in models of adult airway diseases suggest that the bioactive Vitamin D3 metabolite, calcitriol (1,25-dihydroxyvitamin D3 ; 1,25(OH)2 D3 ), modulates responses to inflammation; however, this concept has not been explored in developing airways in the context of pediatric asthma. We used human fetal airway smooth muscle (ASM) cells as a model of the early postnatal airway to explore how calcitriol modulates remodeling induced by pro-inflammatory cytokines. Cells were pre-treated with calcitriol and then exposed to TNFα or TGFß for up to 72 h. Matrix metalloproteinase (MMP) activity, production of extracellular matrix (ECM), and cell proliferation were assessed. Calcitriol attenuated TNFα enhancement of MMP-9 expression and activity. Additionally, calcitriol attenuated TNFα and TGFß-induced collagen III expression and deposition, and separately, inhibited proliferation of fetal ASM cells induced by either inflammatory mediator. Analysis of signaling pathways suggested that calcitriol effects in fetal ASM involve ERK signaling, but not other major inflammatory pathways. Overall, our data demonstrate that calcitriol can blunt multiple effects of TNFα and TGFß in developing airway, and point to a potentially novel approach to alleviating structural changes in inflammatory airway diseases of childhood.

Hyperoxia-induced changes in estradiol metabolism in postnatal airway smooth muscle.

Supplemental oxygen, used to treat hypoxia in preterm and term neonates, increases the risk of neonatal lung diseases, such as bronchopulmonary dysplasia (BPD) and asthma. There is a known sex predilection for BPD, but the underlying mechanisms are not clear. We tested the hypothesis that altered, local estradiol following hyperoxia contributes to pathophysiological changes observed in immature lung. In human fetal airway smooth muscle (fASM) cells exposed to normoxia or hyperoxia, we measured the expression of proteins involved in estrogen metabolism and cell proliferation responses to estradiol. In fASM cells, CYP1a1 expression was increased by hyperoxia, whereas hyperoxia-induced enhancement of cell proliferation was blunted by estradiol. Pharmacological studies indicated that these effects were attributable to upregulation of CYP1a1 and subsequent increased metabolism of estradiol to a downstream intermediate 2-methoxyestradiol. Microarray analysis of mouse lung exposed to 14 days of hyperoxia showed the most significant alteration in CYP1a1 expression, with minimal changes in expression of five other genes related to estrogen receptors, synthesis, and metabolism. Our novel results on estradiol metabolism in fetal and early postnatal lung in the context of hyperoxia indicate CYP1a1 as a potential mechanism for the protective effect of estradiol in hyperoxia-exposed immature lung, which may help explain the sex difference in neonatal lung diseases.

Fetal human airway smooth muscle cell production of leukocyte chemoattractants is differentially regulated by fluticasone.

BACKGROUND: Adult human airway smooth muscle (ASM) produce cytokines involved in recruitment and survival of leukocytes within airway walls. Cytokine generation by adult ASM is glucocorticoid-sensitive. Whether developing lung ASM produces cytokines in a glucocorticoid-sensitive fashion is unknown. METHODS: Cultured fetal human ASM cells stimulated with TNF-α (0-20 ng/ml) were incubated with TNF-α receptor-blocking antibodies, fluticasone (1 and 100 nm), or vehicle. Supernatants and cells were assayed for the production of CCL5, CXCL10, and CXCL8 mRNA and protein and glucocorticoid receptor phosphorylation. RESULTS: CCL5, CXCL10, and CXCL8 mRNA and protein production by fetal ASM cell was significantly and dose-dependently following TNF-α treatment. Cytokine mRNA and protein production were effectively blocked by TNF-α R1 and R2 receptor neutralizing antibodies but variably inhibited by fluticasone. TNF-α-induced TNF-R1 and R2 receptor mRNA expression was only partially attenuated by fluticasone. Glucocorticoid receptor phosphorylation at serine (Ser) 211 but not at Ser 226 was enhanced by fluticasone. CONCLUSION: Production of CCL5, CXCL10, and CXCL8 by fetal ASM appears to involve pathways that are both qualitatively and mechanistically distinct to those described for adult ASM. The findings imply developing ASM has potential to recruit leukocyte into airways and, therefore, of relevance to childhood airway diseases.

Cigarette smoke enhances proliferation and extracellular matrix deposition by human fetal airway smooth muscle.

Cigarette smoke is a common environmental insult associated with increased risk of developing airway diseases such as wheezing and asthma in neonates and children. In adults, asthma involves airway remodeling characterized by increased airway smooth muscle (ASM) cell proliferation and increased extracellular matrix (ECM) deposition, as well as airway hyperreactivity. The effects of cigarette smoke on remodeling and contractility in the developing airway are not well-elucidated. In this study, we used canalicular-stage (18-20 wk gestational age) human fetal airway smooth muscle (fASM) cells as an in vitro model of the immature airway. fASM cells were exposed to cigarette smoke extract (CSE; 0.5-1.5% for 24-72 h), and cell proliferation, ECM deposition, and intracellular calcium ([Ca(2+)]i) responses to agonist (histamine 10 µM) were used to evaluate effects on remodeling and hyperreactivity. CSE significantly increased cell proliferation and deposition of ECM molecules collagen I, collagen III, and fibronectin. In contrast, [Ca(2+)]i responses were not significantly affected by CSE. Analysis of key signaling pathways demonstrated significant increase in extracellular signal-related kinase (ERK) and p38 activation with CSE. Inhibition of ERK or p38 signaling prevented CSE-mediated changes in proliferation, whereas only ERK inhibition attenuated the CSE-mediated increase in ECM deposition. Overall, these results demonstrate that cigarette smoke may enhance remodeling in developing human ASM through hyperplasia and ECM production, thus contributing to development of neonatal and pediatric airway disease.

A Phase 1, Randomized, Double-Blind, Placebo-Controlled, Single Ascending Dose Study to Evaluate the Pharmacokinetics, Immunogenicity, Safety, and Tolerability After Subcutaneous Administration of Tozorakimab in Healthy Chinese Participants.

Tozorakimab is a high-affinity human immunoglobulin G1 monoclonal antibody that neutralizes interleukin (IL)-33, an IL-1 family cytokine. This phase 1, single-center, randomized, double-blind, placebo-controlled, single ascending dose study (NCT05070312) evaluated tozorakimab in a healthy Chinese population. Outcomes included the characterization of the pharmacokinetic (PK) profile and immunogenicity of tozorakimab. Safety outcomes included treatment-emergent adverse events (TEAEs) and clinical laboratory, electrocardiogram, and vital sign parameters. Healthy, non-smoking, male, and female Chinese participants aged 18-45 years with a body mass index 19-24 kg/m2 were enrolled. In total, 36 participants across 2 cohorts of 18 participants were randomized 2:1 to receive a single subcutaneous dose of tozorakimab (300 mg [2 mL] or 600 mg [4 mL]) or matching placebo (2 or 4 mL). Tozorakimab showed dose-dependent serum PK concentrations with an approximate monophasic distribution in serum over time and a maximum observed peak concentration of 20.1 and 33.7 µg/mL in the 300- and 600-mg cohorts, respectively. No treatment-emergent anti-drug antibodies for tozorakimab were observed in any of the participants. There were no clinically relevant trends in the occurrence of TEAEs across the treatment groups. There were no clinically relevant trends over time in clinical laboratory (hematology, clinical chemistry, and urinalysis), electrocardiogram, or vital sign parameters in any treatment group. Overall, tozorakimab demonstrated dose-dependent systemic exposure in healthy Chinese participants and was well tolerated, with no safety concerns identified in this study.

A Randomized Phase I Study of the Anti-Interleukin-33 Antibody Tozorakimab in Healthy Adults and Patients With Chronic Obstructive Pulmonary Disease.

Tozorakimab is a human monoclonal antibody that neutralizes interleukin (IL)-33. IL-33 is a broad-acting epithelial "alarmin" cytokine upregulated in lung tissue of patients with chronic obstructive pulmonary disease (COPD). This first-in-human, phase I, randomized, double-blind, placebo-controlled study (NCT03096795) evaluated the safety, tolerability, pharmacokinetics (PKs), immunogenicity, target engagement, and pharmacodynamics (PDs) of tozorakimab. This was a 3-part study. In part 1, 56 healthy participants with a history of mild atopy received single escalating doses of either intravenous or subcutaneous tozorakimab or placebo. In part 2, 24 patients with mild COPD received multiple ascending doses of subcutaneous tozorakimab or placebo. In part 3, 8 healthy Japanese participants received a single intravenous dose of tozorakimab or placebo. The safety data collected included treatment-emergent adverse events (TEAEs), vital signs, and clinical laboratory parameters. Biological samples for PKs, immunogenicity, target engagement, and PD biomarker analyses were collected. No meaningful differences in the frequencies of TEAEs were observed between the tozorakimab and placebo arms. Three tozorakimab-treated participants with COPD experienced treatment-emergent serious adverse events. Subcutaneous or intravenous tozorakimab demonstrated linear, time-independent PKs with a mean half-life of 11.7-17.3 days. Treatment-emergent anti-drug antibody frequency was low. Engagement of tozorakimab with endogenous IL-33 in serum and nasal airways was demonstrated. Tozorakimab significantly reduced serum IL-5 and IL-13 levels in patients with COPD compared with placebo. Overall, tozorakimab was well tolerated, with a linear, time-independent serum PK profile. Additionally, biomarker studies demonstrated proof of mechanism. Overall, these data support the further clinical development of tozorakimab in COPD and other inflammatory diseases.

Dried blood spots and sparse sampling: a practical approach to estimating pharmacokinetic parameters of caffeine in preterm infants.

AIMS: Dried blood spots (DBS) alongside micro-analytical techniques are a potential solution to the challenges of performing pharmacokinetic (PK) studies in children. However, DBS methods have received little formal evaluation in clinical settings relevant to children. The aim of the present study was to determine a PK model for caffeine using a 'DBS/microvolume platform' in preterm infants. METHODS: DBS samples were collected prospectively from premature babies receiving caffeine for treatment of apnoea of prematurity. A non-linear mixed effects approach was used to develop a population PK model from measured DBS caffeine concentrations. Caffeine PK parameter estimates based on DBS data were then compared with plasma estimates for agreement. RESULTS: Three hundred and thirty-eight DBS cards for caffeine measurement were collected from 67 preterm infants (birth weight 0.6-2.11 kg). 88% of cards obtained were of acceptable quality and no child had more than 10 DBS samples or more than 0.5 ml of blood taken over the study period. There was good agreement between PK parameters estimated using caffeine concentrations from DBS samples (CL = 7.3 ml h⁻¹ kg⁻¹; V = 593 ml kg⁻¹; t(½) = 57 h) and historical caffeine PK parameter estimates based on plasma samples (CL = 4.9-7.9 ml h⁻¹ kg⁻¹; V = 640-970 ml kg⁻¹; t(½) = 101-144 h). We also found that changes in blood haematocrit may significantly confound estimates of caffeine PK parameters based on DBS data. CONCLUSIONS: This study demonstrates that DBS methods can be applied to PK studies in a vulnerable population group and are a practical alternative to wet matrix sampling techniques.

GC-MS analysis of ethanol and other volatile compounds in micro-volume blood samples--quantifying neonatal exposure.

A static headspace gas chromatography coupled mass spectrometry (GC-MS) method was developed and fully validated for the quantitative measurement of acetaldehyde, acetone, methanol, ethanol and acetic acid in the headspace of micro-volumes of blood using n-propanol as an internal standard. The linearity of the method was established over the range 0.2-100 mg/L (R(2) > 0.99) and the limits of detection were 0.1-0.2 mg/L and lower limits quantification 0.5-1 mg/L. Precision and accuracies fell within acceptable limits (20 % for LLOQ and 15 %) for both intra- and inter-day analyses for all compounds except acetaldehyde which had inter-day variability of ≤25 %. The method was applied to analyse blood samples from neonatal patients receiving courses of ethanol excipient containing medications. Baseline levels of acetaldehyde, acetone, methanol and ethanol could be measured in patients before dosing commenced and an increase in levels of some volatiles were observed in several neonates after receiving ethanol-containing medications.

A randomised phase 2a study to investigate the effects of blocking interleukin-33 with tozorakimab in patients hospitalised with COVID-19: ACCORD-2.

Background: Increased serum interleukin (IL)-33 predicts poor outcomes in patients hospitalised with coronavirus disease 2019 (COVID-19). We examined the efficacy and safety of tozorakimab, a monoclonal antibody that neutralises IL-33, in improving outcomes in ACCORD-2 (EudraCT: 2020-001736-95). Methods: ACCORD-2 was an open-label, phase 2a study in adults hospitalised with COVID-19. Patients were randomised 1:1 to tozorakimab 300 mg plus standard of care (SoC) or SoC alone. The primary end-point was time to clinical response (sustained clinical improvement of ≥2 points on the World Health Organization ordinal scale, discharge from hospital or fit for discharge) by day 29. Other end-points included death or respiratory failure, mortality and intensive care unit admission by day 29, and safety. Serum IL-33/soluble stimulated-2 (sST2) complex levels were measured by high-sensitivity immunoassay. Results: Efficacy analyses included 97 patients (tozorakimab+SoC, n=53; SoC, n=44). Median time to clinical response did not differ between the tozorakimab and SoC arms (8.0 and 9.5 days, respectively; HR 0.96, 80% CI 0.70-1.31; one-sided p=0.33). Tozorakimab was well tolerated and the OR for risk of death or respiratory failure with treatment versus SoC was 0.55 (80% CI 0.27-1.12; p=0.26), while the OR was 0.31 (80% CI 0.09-1.06) in patents with high baseline serum IL-33/sST2 complex levels. Conclusions: Overall, ACCORD-2 results suggest that tozorakimab could be a novel therapy for patients hospitalised with COVID-19, warranting further investigation in confirmatory phase 3 studies.

Oxygen dose responsiveness of human fetal airway smooth muscle cells.

Maintenance of blood oxygen saturation dictates supplemental oxygen administration to premature infants, but hyperoxia predisposes survivors to respiratory diseases such as asthma. Although much research has focused on oxygen effects on alveoli in the setting of bronchopulmonary dysplasia, the mechanisms by which oxygen affects airway structure or function relevant to asthma are still under investigation. We used isolated human fetal airway smooth muscle (fASM) cells from 18-20 postconceptual age lungs (canalicular stage) to examine oxygen effects on intracellular Ca(2+) ([Ca(2+)](i)) and cellular proliferation. fASM cells expressed substantial smooth muscle actin and myosin and several Ca(2+) regulatory proteins but not fibroblast or epithelial markers, profiles qualitatively comparable to adult human ASM. Fluorescence Ca(2+) imaging showed robust [Ca(2+)](i) responses to 1 µM acetylcholine (ACh) and 10 µM histamine (albeit smaller and slower than adult ASM), partly sensitive to zero extracellular Ca(2+). Compared with adult, fASM showed greater baseline proliferation. Based on this validation, we assessed fASM responses to 10% hypoxia through 90% hyperoxia and found enhanced proliferation at <60% oxygen but increased apoptosis at >60%, effects accompanied by appropriate changes in proliferative vs. apoptotic markers and enhanced mitochondrial fission at >60% oxygen. [Ca(2+)](i) responses to ACh were enhanced for <60% but blunted at >60% oxygen. These results suggest that hyperoxia has dose-dependent effects on structure and function of developing ASM, which could have consequences for airway diseases of childhood. Thus detrimental effects on ASM should be an additional consideration in assessing risks of supplemental oxygen in prematurity.

Inflammation and cardiovascular status impact midazolam pharmacokinetics in critically ill children: An observational, prospective, controlled study.

Altered physiology caused by critical illness may change midazolam pharmacokinetics and thereby result in adverse reactions and outcomes in this vulnerable patient population. This study set out to determine which critical illness-related factors impact midazolam pharmacokinetics in children using population modeling. This was an observational, prospective, controlled study of children receiving IV midazolam as part of routine care. Children recruited into the study were either critically-ill receiving continuous infusions of midazolam or otherwise well, admitted for elective day-case surgery (control) who received a single IV bolus dose of midazolam. The primary outcome was to determine the population pharmacokinetics and identify covariates that influence midazolam disposition during critical illness. Thirty-five patients were recruited into the critically ill arm of the study, and 54 children into the control arm. Blood samples for assessing midazolam and 1-OH-midazolam concentrations were collected opportunistically (critically ill arm) and in pre-set time windows (control arm). Pharmacokinetic modeling demonstrated a significant change in midazolam clearance with acute inflammation (measured using C-Reactive Protein), cardio-vascular status, and weight. Simulations predict that elevated C-Reactive Protein and compromised cardiovascular function in critically ill children result in midazolam concentrations up to 10-fold higher than in healthy children. The extremely high concentrations of midazolam observed in some critically-ill children indicate that the current therapeutic dosing regimen for midazolam can lead to over-dosing. Clinicians should be aware of this risk and intensify monitoring for oversedation in such patients.

Visualization of exhaled breath metabolites reveals distinct diagnostic signatures for acute cardiorespiratory breathlessness.

Acute cardiorespiratory breathlessness accounts for one in eight of all emergency hospitalizations. Early, noninvasive diagnostic testing is a clinical priority that allows rapid triage and treatment. Here, we sought to find and replicate diagnostic breath volatile organic compound (VOC) biomarkers of acute cardiorespiratory disease and understand breath metabolite network enrichment in acute disease, with a view to gaining mechanistic insight of breath biochemical derangements. We collected and analyzed exhaled breath samples from 277 participants presenting acute cardiorespiratory exacerbations and aged-matched healthy volunteers. Topological data analysis phenotypes differentiated acute disease from health and acute cardiorespiratory exacerbation subtypes (acute heart failure, acute asthma, acute chronic obstructive pulmonary disease, and community-acquired pneumonia). A multibiomarker score (101 breath biomarkers) demonstrated good diagnostic sensitivity and specificity (≥80%) in both discovery and replication sets and was associated with all-cause mortality at 2 years. In addition, VOC biomarker scores differentiated metabolic subgroups of cardiorespiratory exacerbation. Louvain clustering of VOCs coupled with metabolite enrichment and similarity assessment revealed highly specific enrichment patterns in all acute disease subgroups, for example, selective enrichment of correlated C5-7 hydrocarbons and C3-5 carbonyls in heart failure and selective depletion of correlated aldehydes in acute asthma. This study identified breath VOCs that differentiate acute cardiorespiratory exacerbations and associated subtypes and metabolic clusters of disease-associated VOCs.

Extracorporeal membrane oxygenation and term neonatal respiratory failure deaths in the United Kingdom compared with the United States: 1999 to 2005.

OBJECTIVE: To compare national neonatal extracorporeal membrane oxygenation data and deaths from primary respiratory disorders of term neonates between the United Kingdom and the United States from 1999 to 2005. DESIGN: Cross-sectional study. SETTING: National data sets from the United Kingdom and the United States. PATIENTS: Neonatal extracorporeal membrane oxygenation patients submitted to the Extracorporeal Life Support Organization Registry and national birth and death registrations. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Meconium aspiration syndrome was the most common indication for extracorporeal membrane oxygenation in the United Kingdom: 50.6% vs. 25.8% in the United States (p < .001). Congenital diaphragmatic hernia was most common indication for extracorporeal membrane oxygenation in the United States: 30.7% vs. 15.4% in the United Kingdom (p < .001).Extracorporeal membrane oxygenation use was greater in the United States than the United Kingdom: rate ratio, 1.81 (95%, confidence interval, 1.64, 2.00). The extracorporeal membrane oxygenation rate decreased over time in the United States (p < .001) but was unchanged for all diagnoses in the United Kingdom (p = .49). The rates of extracorporeal membrane oxygenation use for meconium aspiration syndrome were equivalent in both countries: rate ratio, 0.92 (95% confidence interval, 0.80, 1.07) but greater in the United States for congenital diaphragmatic hernia: rate ratio, 3.60, (95% confidence interval, 2.82, 4.66) and persistent pulmonary hypertension newborn: rate ratio, 4.67 (95% confidence interval, 3.33, 6.74).National neonatal death rates included nonextracorporeal membrane oxygenation + extracorporeal membrane oxygenation death. Meconium aspiration syndrome deaths were equivalent overall between the two countries: rate ratio, 0.99 (95% confidence interval, 0.77, 1.29), but decreased in the United States (p < .001) although not in the United Kingdom (p = .17). Congenital diaphragmatic hernia deaths were more prevalent in the United Kingdom than in the United States: rate ratio, 1.57 (95% confidence interval, 1.34, 1.84). CONCLUSIONS: Extracorporeal membrane oxygenation is used more often in the United States: clinicians seem less willing to offer extracorporeal membrane oxygenation for persistent pulmonary hypertension of the newborn and congenital diaphragmatic hernia in the United Kingdom. In contrast to the United States, no reduction in either extracorporeal membrane oxygenation use or death due to meconium aspiration syndrome was observed in the United Kingdom. Early transfer to a tertiary center is recommended for term neonates with respiratory failure.

Intranasal GSK2245035, a Toll-like receptor 7 agonist, does not attenuate the allergen-induced asthmatic response in a randomized, double-blind, placebo-controlled experimental medicine study.

BACKGROUND: Allergic asthma is a heterogenous disorder predominantly driven by a type 2 inflammatory response to aeroallergens. Therapeutic modulation to rebalance these type 2 responses may offer clinical benefit for allergic respiratory inflammatory diseases, with the potential for disease modification. GSK2245035, a selective toll-like receptor-7 agonist, preferentially stimulates the induction of type 1 interferon alpha, reducing type 2 responses. OBJECTIVE: This study investigated whether intranasal GSK2245035 reduced allergen-induced bronchial reactivity in mild allergic asthma. METHODS: This double-blind, placebo-controlled, parallel-group Phase IIa trial randomized (1:1) participants with mild allergic asthma to intranasal GSK2245035 20 ng or placebo once weekly for 8 weeks; follow-up was conducted 1, 4, and 12 weeks after treatment. Allergen-induced late asthmatic response 1 week after treatment was measured as minimum and weighted mean forced expiratory volume in 1 second (FEV1) 4-10 hours following bronchial allergen challenge (primary endpoint). Pharmacodynamic and allergic biomarkers, and adverse events, were assessed. A Bayesian analysis framework was used; a posterior probability >0.7 denoted primary endpoint success. RESULTS: Thirty-six participants were randomized (GSK2245035, n = 22; placebo, n = 14). The percentage attenuation in late asthmatic response was -4.6% (posterior probability: 0.385) and -10.5% (posterior probability: 0.303) for minimum and weighted mean FEV1, respectively. Type 2 responses were confirmed by changes in lung function, eosinophils (blood and sputum), interleukin-5 (sputum) and fractional exhaled nitric oxide biomarkers pre- and post-bronchial allergen challenge. However, no treatment effect was observed. Adverse events were reported by 10/14 (71%) and 21/22 (95%) participants in the placebo and GSK2245035 groups, respectively; headache was the most common. CONCLUSIONS AND CLINICAL RELEVANCE: Although target engagement was observed, weekly intranasal GSK2245035 20 ng for 8 weeks did not substantially attenuate the late asthmatic response in participants with mild allergic asthma. Overall, treatment was well tolerated.