This study was a multi-centre, randomized, double-blind, parallel, active controlled phase III clinical trial to evaluate the efficacy and safety of DW1601 compared to DW16011 or P. sidoides in patients with acute bronchitis. This study was conducted in six institutions in Republic of Korea between December 2018 and December 2019. This study consisted of a screening visit (visit 1), a baseline visit (visit 2), a Day 4 ± 1 day visit (visit 3), and an end of treatment visit on Day 7 ± 1 day (visit 4). The primary outcome was efficacy of DW1601 compared to that of DW16011 or P. sidoides in reducing the bronchitis severity score (BSS) at 4 days after medication in patients with acute bronchitis [20,21]. The secondary outcome was to assess the efficacy of DW1601 compared to DW16011 or P. sidoides in reducing total BSS at 7 days and in relief of each symptom through BSS assessment at 4 and 7 days post-treatment and to assess patient satisfaction with improvement of symptoms at 4 and 7 days post-treatment. This study protocol was uploaded to Clinicaltrial.gov before the beginning of the study (Number: NCT04260555). The study protocol, available in Supplementary Methods, was approved by the Institutional Review Boards of all applicable institutions (Korea University Guro Hospital, IRB number: 2018GR0369; Kangbuk Samsung Hospital, KBSMC2018-11-010). This study was conducted in accordance with the Declaration of Helsinki and the international conference on harmonization-good clinical practice. We ensured protection of patient privacy and anonymity. All patients provided written informed consent before enrollment, and the study was monitored by an independent data and safety monitoring committee.

The inclusion criteria were age ≥ 19 years, experienced acute bronchitis and persistent cough and purulent sputum production within 48 hours before randomization, and patients with total score of BSS ≥ 5 points and sputum BSS ≥ 1 point at the stage of randomization. The composite BSS was determined by the sum of severity ratings, 0 (absent), 1 (mild), 2 (moderate), 3 (severe), or 4 (very severe) for the five bronchitis-related features of cough, sputum, dyspnea, chest pain, and crackle [20,21].

The exclusion criteria were severe lung disease (e.g., bronchiectasis, lung cancer, interstitial lung disease, pneumonia, pulmonary tuberculosis, chronic obstructive pulmonary disease, asthma, chronic bronchitis, and emphysema); antibiotic use because of present infection; sleep apnea; elevated liver enzyme (≥ 3 times the normal range); severe renal dysfunction (glomerular filtration rate < 30 mL/min); uncontrolled diabetes mellitus (random plasma glucose ≥ 250 mg/dL); uncontrolled hypertension (systolic blood pressure ≥ 160 or diastolic blood pressure ≥ 100 mmHg); active peptic ulcer and gastrointestinal bleeding; coagulopathy; cataract(s); advanced malignancy; urinary obstruction due to benign prostate hypertrophy; significant heart disease (e.g., Class III/IV heart failure, pulmonary hypertension, peripheral artery disease, corrected QT interval > 450 seconds on electrocardiograph); antibiotics, antiviral agents, steroids, anticoagulants, or other medications for relieving cough and sputum during the study period; monoamine oxidase (MAO) inhibitors (e.g., antidepressant, antipsychotics, or anti-Parkinson’s drugs) within 2 weeks before randomization; smokers (≥ 15 cigarettes/day); glucose intolerance; pregnant or lactating women; drug allergy for substances associated with study drugs; loss to follow-up; use of drugs that can cause drug-drug interactions with study drugs; and failure to provide informed consent.

The Data and Safety Monitoring Board performed randomization based on an interactive web response system. Patients received placebo or drug using a double-blind, 1:1:1 ratio of DW16011, P. sidoides, and DW1601 through a random table. A stratified block randomization method was used.

Of the screened patients, those eligible were randomly assigned into one of three groups: DW16011, P. sidoides, or DW1601. Patients in each group received the assigned medication three times per day for 7 days: (1) DW1601 arm (DW1601 20 mL + DW16011 placebo 20 mL + P. sidoides placebo 9 mL); (2) DW16011 arm (DW1601 placebo 20 mL + DW16011 20 mL + P. sidoides placebo 9 mL); (3) P. sidoides arm (DW1601 placebo 20 mL + DW16011 placebo 20 mL + P. sidoides 9 mL). The efficacy and safety of each drug were evaluated at 4 and 7 days post-treatment by the Data and Safety Monitoring Board. After the end of treatment, patients were interviewed by telephone to monitor the safety of the drug for 5 days (Supplementary Fig. 1). We recommended that enrolled patients take medication at above 75% of the total drug dosage and assessed medication compliance through the return of unused medications. Patients who took the medication below 75% of the total drug dosage were excluded from the final analysis. For safety monitoring, the Data and Safety Monitoring Board investigated adverse events, adverse drug reactions, and serious adverse events in enrolled patients from the time of enrolment to the follow-up period (for 5 days after end of treatment).

The primary outcome was efficacy of DW1601 compared to that of DW16011 or P. sidoides in reducing total BSS in patients with acute bronchitis after 4 days of medication. The secondary outcomes were change in BSS on day 7 compared to baseline, change in symptom-specific BSS on day 4 and day 7, response rate (proportion of subjects whose BSS dropped below 3 points or whose BSS decreased by more than 7 points compared to baseline), and response rate reported by investigators. We also investigated self-reported patient satisfaction as very satisfied, satisfied, neutral, dissatisfied, or very dissatisfied; satisfaction rate was determined by the percentage of subjects whose treatment was rated either “very satisfied” or “satisfied.”

The number of subjects was calculated with reference to the results of past clinical trials having the same indications and the same endpoints. The minimum sample size assuming two-sided α of 0.05, 90% power (total power over 80%), and drop-out rate of 20% was 204 (68 per group). For efficacy and safety evaluation, subjects were randomized into three groups in a 1:1:1 ratio by stratified block randomization using site as the stratification variable. In efficacy evaluation analysis, full analysis set was used as the main analysis, and per-protocol set was used as the sub-analysis. The descriptive statistics of the endpoints of each group are shown, and unless otherwise noted, every test was conducted with a two-sided test with a significance level of 5%. Primary endpoint analysis required comparisons between the test group and each control group by analysis of covariance (ANCOVA) with total baseline BSS as a covariate. The 95% two-sided confidence interval (CI) and p value of difference of least squares mean are shown, and the test group was superior to each control group if the upper limit of the CI was less than zero. Secondary endpoints were difference of total BSS on day 7 and difference of BSS of each symptom on day 4 and day 7 and were analysed by ANCOVA with total BSS at baseline as a covariate. Response rates at day 4 and day 7 and satisfaction rate, and frequency and percentage of each administration group are presented, and the difference among the three groups was analysed by Pearson’s chi-square test or Fisher’s exact test.

During the study period, 204 patients with acute bronchitis were randomized to the DW1601, DW16011, or P. sidoides group. We performed this study with 68 patients randomized to DW1601, 70 patients randomized to DW16011, and 66 patients randomized to P. sidoides. Of the 68 patients randomized to DW1601 and the 66 patients randomized to P. sidoides, two were excluded because of duplicate registration. In addition, of the 66 patients randomized to P. sidoides, one was excluded because of missing BSS measurements after randomization. Therefore, the data of the 67 patients randomized to DW1601, 70 patients randomized to DW16011, and 64 patients randomized to P. sidoides were included in the final analysis (Fig. 1).

The baseline characteristics of the patients are shown in Table 1. There were no differences in baseline characteristics among groups. The mean age was 40 years, and 34.3% (69/201) were male. The mean body mass index was 23 kg/m², and there were 162 (80.6%) non-smokers. The symptom duration of cough and sputum production was almost 32 hours, and 43 patients (21.4%) had comorbid diseases. Patient compliance was almost 98%, and there was no difference in compliance among the three groups.

The primary outcome was efficacy of DW1601 compared to DW16011 or P. sidoides alone in reduction of total BSS in patients with acute bronchitis at 4 days after medication. The decrease in total BSS from baseline was significantly greater in the DW1601 group (indicating lesser severity of acute bronchitis) than in the DW16011 group (−3.51 ± 0.18 vs. −2.65 ± 0.18, p = 0.001) or the P. sidoides group (−3.56 ± 0.18 vs. −2.64 ± 0.19, p < 0.001) (Fig. 2, Supplementary Table 1). The average difference in BSS change from predose to post-dose between DW1601 and DW16011 was −0.86 ± 0.26, (95% CI, −1.37 to −0.35), while that between DW1601 and P. sidoides was −0.92 ± 0.26 (95% CI, −1.44 to −0.40). The upper limits of the 95% CI prove the superiority of DW1601 over DW16011 or P. sidoides (p = 0.001, p < 0.001, respectively).

At 7 days after medication, decrease of total BSS from baseline was significantly greater in the DW1601 group than in the DW16011 group (−5.38 ± 0.14 vs. −4.46 ± 0.14, p < 0.001) or P. sidoides group (−5.44 ± 0.16 vs. −4.66 ± 0.17, p = 0.001) (Fig. 2, Supplementary Table 1). In addition, the change in BSS of cough and sputum production at day 4 and day 7 was significantly more reduced in the DW1601 group compared with the DW16011 group or P. sidoides group. However, differences in other components of the BSS (dyspnoea, chest pain, and crackle) before and after medication were not statistically significant (Fig. 3, Supplementary Table 1). Patients treated with DW1601 showed a higher response rate than those treated with DW16011 (78.8% vs. 60.3%, p = 0.02) or P. sidoides (78.8% vs. 53.1%, p = 0.002) at day 4. In addition, the response rates at day 7 were 100% for the DW1601 group, 85.7% for the DW16011 group, and 85.9% for the P. sidoides group (Fig. 4, Supplementary Table 2). DW1601-treated patients reported a significantly higher satisfaction rate than DW16011- or P. sidoides-treated patients both on day 4 and day 7 (Supplementary Table 3).

Totals of five patients (7.5%) in the DW1601 group, six patients (8.6%) in the DW16011 group, and six patients (9.2%) in the P. sidoides group reported treatment-emergent adverse events. In addition, four patients (6.0%) in the DW1601 group, six patients (8.6%) in the DW16011 group, and four patients (6.2%) in the P. sidoides group reported adverse drug reactions. The reported adverse events were somnolence, nausea, facial oedema, and dry mouth, and all resolved spontaneously. There were no severe adverse events, such as death, near-death, irreversible functional disability, or an aggravated condition requiring hospital admission (Supplementary Table 4).