Fractional Exhaled Nitric Oxide

The study set‐up (ClinicalTrials.gov: NCT01866306) is represented schematically in Figure 1. Twenty (out of 23) stable adult mild to moderate asthma patients on inhaled corticosteroids (≤500 µg/day fluticasone propionate or equivalent), all RV16‐seronegative (<1:4), were included in this analysis. The three excluded patients were either not infected (n = 1), infected with another virus at the time of inoculation (n = 1) or the sequence reads did not match with the non‐stranded sequenced samples during the stranded library preparation step (n = 1). Nasal lavage and brushed nasal epithelial cells (NECs) were collected 7 days prior to low dose rhinovirus16 (RV16UB) inoculation (100 TICD₅₀) as baseline and at days 3, 6 and 14 after inoculation.18 RV16UB is a GMP RV16 stock prepared under auspices of U‐BIOPRED, where 100 TICD₅₀ was found to be the lowest optimal dose for effective infections in healthy individuals and asthma patients (manuscript in preparation). Blood was obtained 4 days prior, as baseline, and at day 6 post‐RV16 exposure. Viral load was measured in nasal lavage fluid at days 3, 6 and 14. In addition, a PCR screening for respiratory viruses was performed in throat swabs 1 day before RV16 challenge to ensure no other viral infections. Fractional exhaled nitric oxide (FeNO) was measured, at the same days as nasal lavage was obtained. Cold Symptom Scores and FEV₁% predicted (based on morning values) were measured every day from 7 days before until 14 days after RV16 challenge. The study was approved by the internal review boards of the participating centres, and written informed consent was obtained from all participating patients. Patient baseline characteristics are provided in Table 1. The inclusion and exclusion criteria for these patients are provided in the online data supplement.

We assessed chronic bronchitis as the combination of the symptoms of cough and mucus production in the morning during winter (defined as “CB”), which required positive answers to the following two binary-choice questions: 1) “In the winter, do you usually cough as soon as you wake up in the morning?” and 2) “In the winter, do you usually bring up mucus as soon as you wake up in the morning?” [23 (link)].
Childhood asthma at ages 1–16 years was defined if at least two of the following three criteria were fulfilled: doctor's diagnosis of asthma ever; wheezing in the past 12 months; and/or use of asthma medication during the past 12 months [24 (link)].
Current asthma was defined as a positive answer to doctor diagnosis of asthma, and at least one of the following: wheezing in the past 12 months; or use of asthma medication during the past 12 months.
Lung function was tested according to American Thoracic Society (ATS)/European Respiratory Society (ERS) spirometry criteria [25 (link)] using the Jaeger MasterScreen-IOS system (Carefusion Technologies, San Diego, CA, USA) and post-bronchodilator lung function was tested 15 min after the administration of 400 μg salbutamol. The highest values of pre- and post-forced expiratory volume in 1 s (FEV₁) and forced vital capacity (FVC) were recorded [19 (link), 20 (link)]. Predicted values and z-scores of FEV₁, FVC and FEV₁/FVC ratios were calculated for each patient using equations from the Global Lung Function Initiative (GLI) [26 (link)] according to age, sex, height and ethnicity. The lower limit of normal (LLN) was defined as the bottom fifth percentile of the predicted value and calculated by GLI equations for every participant.
Fractional exhaled nitric oxide (F_eNO) was measured using a chemiluminescence analyser (EcoMedics Exhalyzer, Duernten, Switzerland) according to the ATS/ERS guidelines [27 (link)].

The variables to be measured are listed in tables 1–3.
Data will primarily be collected via eCRFs completed yearly by the treating HCP, and PROs via patient questionnaires every 3 months (with relevant translations). These will provide a consistently collected set of variables aligned to NOVELTY objectives.
The feasibility study revealed that many data required for NOVELTY were not consistently recorded in EMRs [30 ], necessitating the study-specific eCRF.
Spirometry will be performed by trained site personnel. For sites with suitable spirometers, data will be recorded in the eCRF. For other sites, spirometers (ERT FlowScreen®, ERT, Philadelphia, PA, USA), which meet European Respiratory Society/American Thoracic Society standards [31 (link)], will be provided and data collected centrally (centralised over-read performed at baseline only). For predicted values, reference equations from the 2012 Global Lung Function Initiative [32 (link)] will be used. Reversibility of airway obstruction will be assessed following withholding of bronchodilators: pre- and post-bronchodilator forced expiratory volume over 1 s and forced vital capacity will be measured immediately before, and ≥15 min after, a bronchodilator is given. Fractional exhaled nitric oxide will be measured at baseline using Niox Vero devices (Circassia Pharmaceuticals Inc., Morrisville, NC, USA).
PROs will be recorded after the yearly clinic visits and every 3 months (tables 2 and 3). While most respiratory symptom tools are validated for only asthma or COPD, a distinctive feature of NOVELTY is that the same PROs will be administered to the whole study population, irrespective of diagnosis. The chronic airways assessment test (CAAT), a modified version of the COPD assessment test (CAT) [33 (link)], will be used with permission of the copyright holder (GlaxoSmithKline, Brentford, UK), with reference to COPD replaced with “your pulmonary disease”. As part of CAAT validation, a subset of patients with COPD will also complete the CAT. Generic information on respiratory symptoms will be collected, modelled on guidelines for asthma symptom control, but without referring to asthma. In addition to the above PROs, patients with asthma will complete the Asthma Control Test [34 (link)]. The CAPTURE tool (COPD Assessment in Primary Care to Identify Undiagnosed Respiratory Disease and Exacerbation Risk) [35 (link)] will also be evaluated in NOVELTY.
Data on medications, comorbidities, exacerbations and HCU will be recorded in the eCRF, and information on medications, exacerbations and HCU will also be collected from patients. Patient-reported adherence will be compared with prescribing data. Information collected on comorbidities includes type, duration and ongoing status.
Investigators will be asked to record in the eCRF any specialised assessments performed during routine care (e.g. diffusion capacity of the lung for carbon monoxide, fractional exhaled nitric oxide, computed tomography scans, 6-minute walk distance and blood clinical chemistry), for which results are available.
With patient consent, blood and urine samples will be collected. Blood samples will be used to measure biomarkers over time, and, with patients' specific consent, for exploratory genetic research, such as investigating genomic, transcriptomic and metabolomic variants associated with the disease phenotypes that may reveal underlying endotypes. Standardised laboratory protocols have been developed for collection, handling, storage and shipping of biosamples (see online supplement), and all site staff will be trained in sample collection and handling. Biosamples will be stored in a central repository and held for batched analysis (see online supplement). Peripheral blood differential counts will be available for baseline analysis.
In countries selected for EMR analysis, retrospective EMR data may be collected with patient consent, and will be compared with eCRF data to assess EMRs as a source for NOVELTY and future studies, and to evaluate if patients in NOVELTY are representative of nonenrolled patients with a similar diagnosis.

Ecleralimab is formulated as a PulmoSol engineered powder in hard capsules and delivered to the lungs via a Breezhaler dry powder inhaler device, all provided by Novartis (Basel, Switzerland). Based on clinical and nonclinical safety data, a starting dose of 4 mg administered once daily for 12 weeks was expected to provide adequate pulmonary exposure to assess pharmacodynamic effects against allergen-induced airway responses. Each randomised subject received a single inhaled dose of ecleralimab or placebo on day 1 at the investigational site. Subsequent daily dosing began on day 3 provided no safety concerns were identified in the 48 h following the initial dose. Dosing was self-administered during the morning at home or at the study site during scheduled visits up to and including day 84. MIC and AIC testing commenced at least 1 h after dosing. Compliance to study treatment was recorded by the subject in a diary and assessed at each visit using blister pack counts and the diary.
13 visits were scheduled during the 12-week treatment period, including two allergen challenge triads (MIC–AIC–MIC) at days 41–43 and days 83–85. Safety, pharmacokinetics and pharmacodynamics assessments were also performed. To enter the treatment period, subjects needed to demonstrate return to baseline with FEV₁ ≥70% predicted, and FEV₁ and forced vital capacity were to be within 10% and the methacholine PC₂₀ not more than 1 doubling concentration lower than the values measured at day −15 during screening.
MIC was conducted during screening (to qualify subjects for the study), pre-treatment on day 1, and 24 h pre-AIC and 24 h post-AIC, as previously described [21 (link)], until a 20% decrease in FEV₁ occurred. The methacholine PC₂₀ was calculated from the log concentration versus response curve.
AIC was conducted during screening and again at day 42 and day 84 as previously reported (see supplementary material) [22 (link)]. At screening, doubling concentrations of commercially available aero-allergen extracts (table 1) were inhaled at 12-min intervals until a ≥20% decrease in FEV₁ was reached. FEV₁ was then measured at regular intervals for 7 h to identify subjects with positive LAR. Allergen concentrations administered on days 42 and 84 were the same as those administered at screening. EAR and LAR were reported as time-adjusted area of percentage decrease in FEV₁ (EAR AUC_0–2h and LAR AUC_3–7h), maximum percentage decrease in FEV₁ (EAR% and LAR%) and minimum FEV₁ (EAR_min and LAR_min).
Sputum was induced before the start of treatment on day 1, and during each allergen challenge triad at 24 h pre-allergen and at 7 and 24 h post-allergen, and processed using a method modified from Pizzichiniet al. [23 (link)].
Fractional exhaled nitric oxide (F_ENO) was sampled before each MIC, beginning on day −1 and also at 7 h post-allergen (prior to 7 h spirometry), using a Niox VERO (Aerocrine, Stockholm, Sweden) F_ENO testing device. Peripheral blood eosinophils were also assessed.
Safety assessments included physical examinations, systems review, open-ended health inquiry, ECGs, vital signs, haematology, blood chemistry, urinalysis, and monitoring of adverse events (AEs) and serious AEs (SAEs). Subjects completed an end-of-treatment period visit on day 85 and entered a 4-week follow-up period.