This was a randomized, multiple-dose, placebo- and positive-controlled, parallel-group study conducted at a single clinical research center in the US. The study design was in compliance with the recommendation of the ICH E14 guideline  for thorough QT studies. The study was approved by the institutional review board (Independent Investigational Review Board; Sea View Research, Inc.) of the participating study center and was conducted in accordance with the ethical principles embodied in the Declaration of Helsinki (1989).
The study population consisted of healthy, nonsmoking male and female subjects aged between 18 and 55 years (inclusive), with a body mass index within the range of 18.5-32.0 kg/m2 and weight ≥50 kg at screening. Individuals with a history of or current ECG abnormalities (PR >240 ms; QRS complex >110 ms; QTc interval [Fridericia's; QTcF] >430 ms for males and >440 ms for females; or any significant morphological changes other than nonspecific T-wave changes) were excluded from the study. In addition, those with serum potassium <3.5 mmol/L or magnesium <0.8 mmol/L; recent blood donation or blood loss; or a history of diabetes mellitus, hyperthyroidism, impaired renal function, drug or alcohol abuse, or any significant illness within 2 weeks of dosing were also excluded. All subjects provided written informed consent prior to taking part in the study.
Study design and treatments
The study comprised a screening period (up to 21 days), a baseline visit (Day -1), a 14-day randomized treatment period (Days 1-14), and an end-of-study evaluation at 7 days after the last dose. At the start of the randomized treatment period, eligible subjects were randomized to one of the following five treatment groups in a 4:4:2:4:1 ratio: indacaterol 150 and 300 μg (therapeutic doses), indacaterol 600 μg (supratherapeutic dose), placebo, and placebo/moxifloxacin. Moxifloxacin, a fluoroquinolone antimicrobial agent, was used as a positive calibrator to determine the assay sensitivity in this study. This dose of 400 mg represents the daily therapeutic dose of moxifloxacin, single doses of which have been consistently associated with an increase in mean QT interval vs placebo in excess of 5 ms in healthy volunteers .
Indacaterol and matching placebo were administered once daily for 14 days via a single-dose dry powder inhaler (SDDPI) under double-blind conditions. Subjects randomized to the placebo/moxifloxacin arm received double-blind placebo via SDDPI for 14 days, with a single open-label oral dose of moxifloxacin 400 mg on Day 14.
Except for paracetamol and any medication needed to treat adverse events (AEs), no medication other than study drug was allowed from the start of screening until the completion of all evaluations in the study.
Serial 12-lead surface ECGs were obtained using digital ECG equipment (ELI 250™, Mortara Instrument Inc., Milwaukee, WI, USA) at baseline (Day -1) at the equivalent of predose and 1 and 12 h post-dose. During the treatment period, three ECG recordings were collected at each of the following times on Days 1 and 14: at predose, and at 10, 20, and 40 min, and 1, 2, 3, 4, 6, 12, and 24 h post-dose. All ECGs were recorded after subjects had been resting in the supine position for at least 15 min (or 8 min for the 10 min post-dose reading). The digital ECG recordings were transmitted electronically to a central facility for blinded manual interpretation and analysis. All ECG readers were blinded to all elements of the study, including treatment assignments and subject demographics.
Pharmacokinetic samples were taken to determine the relationship between indacaterol concentration and QTc interval. Serial blood samples were collected on Days 1 and 14 at predose and at 10, 20, and 40 min, and 1, 2, 3, 4, 6, 12, and 24 h post-dose. Serum was obtained from all blood samples by centrifugation and kept frozen at ≤ -20°C until analysis. Serum indacaterol was measured using a liquid chromatography/mass spectrometry/mass spectrometry method; the lower limit of quantification for indacaterol was 10 pg/mL. The maximum (peak) serum concentration (Cmax) and the area under the concentration-time curve from 0 to 24 h post-dose (AUC0-24h) were determined on Days 1 and 14, and were presented as the mean and standard deviation (SD).
Safety assessments included recording of AEs and serious adverse events (SAEs) with severity and relationship to study drug; collection of clinical laboratory data for urinalysis, hematology, and blood chemistry (including serum potassium and plasma glucose); and regular assessments of vital signs, ECGs, and body weight.
Sample size calculation and statistical analysis
The primary endpoint for the study was the QTcF interval (QT interval corrected for heart rate using Fridericia's formula, QT/RR0.33). This formula is recommended for investigating the effect of drugs that may affect pulse rate [9, 12, 13]. For each of three ECGs taken at each nominal time, the QT and RR intervals were measured from three consecutive QRST complexes from lead II. The mean of the triplicate ECG value was then calculated.
The primary objective of this study was to compare indacaterol 150, 300 and 600 μg with placebo in terms of the change in QTcF interval from baseline at each of the 10 post-dose time points on Day 14. Baseline QTcF interval was defined as the average of the pre-treatment measurements taken on Day -1 and the predose measurement on Day 1.
The sample size calculation was based on a residual standard deviation of 10.95 ms for the primary endpoint observed in a previous indacaterol study (data on file). Assuming a 5 ms difference between indacaterol 150 μg or 300 μg and placebo in terms of change from baseline in QTcF interval at the first four post-dose assessment time points on Day 14 and no difference at the next six time points, it was calculated that a sample size of 102 subjects was required in each treatment group to demonstrate that all upper one-sided 95% confidence intervals were below 10 ms.
For the comparison of indacaterol 600 μg versus placebo, 52 subjects in each treatment arm were required in order to show that all upper one-sided 95% confidence intervals were <20 ms. This calculation assumed an estimated difference of 13 ms between indacaterol 600 μg and placebo in terms of change from baseline in QTcF interval at the first four assessment time points and no difference between the treatments at the next six time points.
For the comparison between moxifloxacin and placebo, 26 subjects per treatment arm were required to conclude an assay sensitivity with 95% power based on the assumption that the difference between moxifloxacin and placebo for QTcF interval change from baseline was at least 5 ms and that this difference could be detected at least at 4 of the 10 post-dose assessment time points on Day 14. Assuming a dropout rate of approximately 10%, the number of subjects to be randomized was 116, 116, 58, 116, and 29, for indacaterol 150, 300, 600 μg, placebo, and placebo/moxifloxacin, respectively.
The absence of any meaningful effect of indacaterol 150 and 300 μg on cardiac repolarization was to be concluded if the upper limit of all the 90% two-sided CIs (equivalent to the upper limit of a 95% one-sided CI) for the mean difference in the change in QTcF interval from baseline between indacaterol (150 and 300 μg) and placebo at each time point was <10 ms. A similar analysis was performed for the supratherapeutic dose of indacaterol (600 μg) with the noninferiority margin set at a prespecified constant of 20 ms for the upper limit of the 90% two-sided CIs. Confidence intervals were calculated from pairwise determination of the pooled standard error and the 10th and 90th percentiles of the t-distribution with appropriate degrees of freedom.
The average QTcF intervals (average of all scheduled post-dose QTcF measurements up to 24 h post-dose) and the peak QTcF intervals (highest QTcF interval value observed over the period of 10 min to 24 h post-dose) were calculated for all treatment groups on both Days 1 and 14. In addition, the incidences of absolute QTcF interval values >450, >480, and >500 ms were summarized for each treatment group, as were the incidences of changes from baseline in QTcF interval >30 and >60 ms.
To determine the relationship between steady-state serum concentration of indacaterol and QTcF interval, change from baseline in QTcF interval was plotted against the corresponding indacaterol concentration for each time point on Day 14 (including all observations under placebo and all indacaterol doses). A linear relationship between the two was established by plotting a regression line, with the upper 95% simultaneous confidence band also presented.