Efficacy and Safety of Aviron Rapid ® in Adolescents and Children with Viral Acute Upper Respiratory Tract Infection: a Multi-Center, Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Efficacy and safety of Aviron Rapid


AIM
The main objective of this trial was to evaluate the efficacy and safety of Aviron Rapid in patients with a viral AURTI clinically diagnosed by their general practitioner (GP) and treated according to standard medical practice.

MATERIAlS AnD METhODS
This was a multi-center, Phase 4, randomized, double-blind, placebo-controlled clinical trial with Aviron Rapid in patients with a viral AURTI, recruited from 85 GP practices in Bulgaria between January 27 and March 9, 2020. The trial included 3 age cohorts: adults (18-60 years), adolescents (13)(14)(15)(16)(17) years; also referred to as 'the adolescent cohort'), and children (5)(6)(7)(8)(9)(10)(11)(12) years; also referred to as 'the pediatric cohort'). Informed consent was obtained from all participants participating in the study. Efficacy and safety data of the adult cohort in this trial have been reported previously. [10] Here, we report efficacy and safety data from the adolescent and pediatric cohorts.

Trial population
Male and female adolescents (13-17 years, both inclusive) and children (5-12 years, both inclusive) with a clinical diagnosis of a viral AURTI were enrolled in the trial. Diagnosis by the GP was based on 1) an axillary temperature of more than 37.0°C and 2) presence of one or more of the InTRODUCTIOn Acute upper respiratory tract infections (AURTIs) are associated with a significant burden of disease. Easy airdrop transmission, short incubation time, and short-lived specific immunity contribute to more than 17 billion incident cases worldwide in 2019, resulting in a considerable non-fatal burden as the symptoms may significantly affect quality of life and productivity. [1] Healthcare costs are estimated to exceed 40 billion dollars per year in the United States [2] , with an even higher economic burden attributable to loss of productivity. Children are particularly susceptible to suffering from AURTIs [3] , with an average of 6 to 8 episodes per year, compared with 2 to 4 episodes in adults [4,5] . The majority of AURTIs are caused by viruses, most commonly rhinoviruses. Most treatments focus on symptom relief, including over-the-counter analgesics, zinc, nasal decongestants, and ipratropium for cough. The few safe and effective antiviral treatments available for clinical practice are not routinely used. These include M2 channel blockers [6] (amantadine and rimantadine) with demonstrated effectiveness against influenza A virus, and neuraminidase inhibitors [7] (oseltamivir and zanamivir) with activity against influenza A and B viruses. However, considering that AUR-TIs are mostly self-limiting and mild in severity, the use of antivirals is limited to immunocompromised patients who are more likely to develop severe infections or complications. In addition, oseltamivir-resistant viral strains have been isolated from patients, particularly immunocompromised patients. [8] Frequent mutations of influenza viruses have also led to considerable resistance to M2 channel blockers. [9] Therefore, novel therapeutic options with broad clinical antiviral efficacy and safety are an unmet medical need in the management of AURTIs.
Aviron Rapid ® is a dietary supplement consisting of andrographolide, proprietary spirulina extract, and humic acid racemic mixture [10] and is indicated in adults and chil- following symptoms with onset of the first symptom within 24 hours before screening: nasal congestion, cough, sore throat, headache, fatigue, and sleep disturbance. Patients with clinical symptoms of severe flu or AURTI requiring hospitalization, or with symptoms similar to AURTI but related to other underlying diseases (infectious diseases, flu-like syndrome in systemic connective tissue disease, onco-hematologic, and other diseases) were excluded. Other exclusion criteria focused on past or current use of medications or medical disorders that could potentially confound trial results or interfere with safe completion of the trial.

Trial design
Adolescents and children presenting to a GP practice with symptoms suggestive of viral AURTIs were clinically examined (including measurement of axillary temperature) by the GP on day 1 and, if eligible, were enrolled and randomly assigned (1:1) to receive active treatment or placebo in a double-blinded fashion. Patients in the active group (Group 1) received standard symptomatic therapy + Aviron Rapid, patients in the placebo group (Group 2) received standard symptomatic therapy + placebo. Randomization of patients was done using software designed by an external, independent vendor and was stratified by center. The active and placebo treatments were packed in blisters and delivered to the GP practices in white paper boxes, labeled with unique treatment codes. Manufacturing, packaging, and labeling were performed by the sponsor of the trial. Patients, GPs, and the sponsor's project team were blinded to treatment group assignments throughout the trial and until database lock.
On day 1, patients or their parents were given a diary to record axillary temperature, antipyretics intake, symptom severity, and recovery status twice daily with 12-hour intervals (once in the morning, once in the evening) during the treatment period, i.e., days 1 to 5 for adolescents and days 1 to 7 for children. Adolescents recorded data in the diary under supervision of their parents. For children, data was recorded by the parents. After the end of treatment, on day 6 (adolescents) or day 8 (children), patients returned to the GP practice for a closing visit. During this visit, the GP clinically examined (including measurement of axillary temperature) the patient and verified completeness of the data recorded in the diary. In addition, on day 1, symptom severity was assessed and on day 6, a general evaluation of the patient's condition (recorded as 'healthy' or 'ill') was performed.

Trial treatment
Aviron Rapid was provided as 647-mg tablets for oral use containing 10 mg andrographolide, 100 mg proprietary spirulina extract, and 250 mg proprietary humic acid racemic mixture. Placebo was provided as tablets visually matching the Aviron Rapid tablets. Patients were treated according to the following schedule: adolescents: 3 tablets 3 times daily on day 1, 2 tablets 3 times daily on day 2, and 1 tablet 3 times daily on days 3 to 5; children: 2 tablets 3 times daily on day 1 and 1 tablet 3 times daily on days 2 to 7. Tablets were taken with water, in the morning, at noon, and in the evening. Standard symptomatic therapy included non-steroidal anti-inflammatory drugs, decongestants, bronchodilators, mucolytics, antitussives, and other drugs for treatment of chronic diseases. Use of other antiviral remedies, antihistamines, antibiotics, and interferons was not allowed.

Efficacy outcome measures and assessments
The primary endpoints of this trial were the number (and percentage) of clinically recovered patients and the mean disease duration. Patients were considered clinically recovered if the following 3 criteria were met: 1) persistent improvement of every symptom to "very mild" or "lack of symptoms" (severity score <2 as measured on a visual analogue scale [VAS] for adolescents or a modified face pain scale [FPS-R] for children) until the end of follow-up and a total severity score for all symptoms [i.e. the sum of the 6 individual symptom severity scores] <12 points; 2) persistent decrease of the axillary temperature to <37.0°C, i.e. temperature <37.0°C at 2 consecutive measurements with a 12-hour interval and no new increase >37°C (i.e., fever) until the end of follow-up; 3) the decrease in axillary temperature <37°C was achieved without the use of antipyretics. The severity of the symptoms nasal congestion, cough, sore throat, headache, fatigue, and sleep disturbance was measured using a VAS (for adolescents) or FPS-R (for children) ranging from 0 to 10 with 0 = lack of symptoms and 10 = very severe symptoms. Disease duration was defined as the interval between treatment initiation and the time point at which the patient met the criteria for clinically recovery. The primary endpoints were assessed twice daily with 12-hour intervals during the treatment period (days 1 to 5 for adolescents or days 1 to 7 for children) and recorded in the patient's diary.
Secondary efficacy endpoints included number (and percentage) of patients with persistent decrease of the axillary temperature <37.0°C without the use of antipyretics, mean time to persistent decrease of the axillary temperature <37.0°C, number (and percentage) of patients taking antipyretics, mean duration of antipyretic treatment, mean total severity score for all symptoms, number (and percentage) of patients with persistent improvement of a particular symptom (severity score <2), and number (and percentage) of patients considered fully recovered. Patients were considered fully recovered if at 2 consecutive measurements with a 12-hour interval they scored 2 or 3 on a scale ranging from 1 to 3 with 1 = I still feel ill; 2 = I feel better; 3 = I feel healthy. Secondary efficacy endpoints were assessed twice daily at 12-hour intervals and recorded in the patient's diary from day 1 to the end of treatment (day 5

Safety outcome measures and assessments
Safety-related endpoints included the incidence of adverse events (AEs), as recorded in the patient's diary during the treatment period, and physical examinations by the GP on days 1 and 6 (adolescents) or 8 (children).

Statistical analysis
The statistical analyses were done by an external blinded statistician. In both cohorts, a total number of 320 patients (160 per treatment group) were required to detect a difference of 5% between the two treatment groups with 80% power.
All patients who completed the treatment and all planned trial visits per protocol and did not have protocol deviations were included in the efficacy analyses. For the primary endpoint analysis, the 2-proportion Z-test was used to compare the percentages of clinically recovered patients between the active and placebo group. The mean disease duration in both groups was compared using an independent samples t-test. The secondary endpoints were analyzed using the same statistical methods as used for the primary endpoint analyses.
The safety analysis included all patients who received at least one dose of Aviron Rapid or a placebo. The incidence of AEs was summarized descriptively. All statistical tests were two-sided except for the 2-proportion Z-test. Statistical significance was set at p<0.05. All statistical analyses were performed using SPSS 17.

Patient disposition and baseline demographics
The trial started on 27 January 2020 (i.e., enrolment of the first patient in the trial) and was completed on 14 March 2020 (i.e., end of follow-up for the last patient in the trial). In total, 380 adolescents and 401 children were enrolled in the respective cohorts and randomly assigned to one of the two treatment groups. Safety analyses included all randomized patients. Efficacy analyses comprised 329 adolescents and 319 children. The reasons for excluding patients from the efficacy analysis are shown in Fig. 1.
Overall, in the adolescent cohort, the mean age was 14.6 years and 54% of patients were male. In the pediatric cohort, the mean age was 8.6 years and 51% of patients were male. Patient demographics were comparable in the two treatment groups in both cohorts. The total symptom severity score at baseline was slightly higher in the adolescent cohort (25.6 and 24.9 in the active and placebo group, respectively) compared with the pediatric cohort (24.6 and 23.5, respectively). The mean axillary temperature was 38°C in the active and placebo groups in both cohorts. The percentages of patients reporting any symptom at baseline were generally lower in the pediatric cohort, compared with the adolescent cohort. Nasal congestion was the most common symptom in both cohorts (reported in >91% of adolescents and >88% of children). There were no significant differences between the active and placebo group in both cohorts (Table 1).
For nasal congestion, cough, sore throat, headache, fatigue, and sleep disturbance, n (%) refers to the number (and percentage) of patients with a particular symptom at baseline; the mean values refer to the mean severity of a particular symptom at baseline.

Primary Endpoint
In the adolescent cohort, the percentages of patients meeting the criteria for clinical recovery were consistently higher in the active group compared with the placebo group from 24 hours after initiation of treatment (i.e., morning of day 2: 8.1% versus 1.9%, respectively; p=0.0055) until the end of treatment (i.e., evening of day 5: 90.7% versus 80.9%; p=0.0052). Differences were statistically significant (p<0.05) as of the morning of day 2 onwards until end of treatment, except for the morning of day 4 (p=0.0786) (Fig. 2 (Fig. 3).
Similar results were obtained in the pediatric cohort with consistently higher percentages of clinically recovered patients in the active group compared with the placebo group from 24 hours after initiation of treatment (i.e., morning of day 2: 5.6% versus 4.4%, respectively; p=0.31) until end of treatment (i.e., evening of day 7: 98.8% versus 89.2%; p=0.001). Differences were statistically significant (p<0.05) as of the morning of day 3 and throughout the treatment period ( Fig. 2

Persistent decrease of temperature
From the morning of day 2 onwards until the end of treatment, the percentage of patients in the adolescent and pediatric cohorts with a persistent temperature decrease of <37°C was consistently higher in the active group than that in the placebo group, with the biggest difference between the two treatment groups being noticed on the morning  Fig. 5).

Antipyretics use
The percentage of adolescent patients taking antipyretics was consistently lower in the active group compared with the placebo group from the evening of day 1 onwards until the end of treatment. In the pediatric cohort, statistically significantly lower percentages of patients on antipyretics were observed as of the morning of day 3 until the end of treatment (Fig. 6) (Supplementary Table 3) (see Appendix). The mean duration of antipyretics intake was signifi-        Fig. 7).

Symptom severity
The mean total severity of clinical symptoms in the adolescents in the active group was consistently lower than that in the placebo group from the evening of day 1 until the end of treatment, with day 3 showing the biggest difference between the two groups (Supplementary Table 4) (see Appendix). For nasal congestion, cough, and sleep disturbance, the percentages of adolescents with persistent improvement were consistently higher in the active group compared with the placebo group throughout the entire treatment period. As of the morning of day 2 until the end of treatment, a higher percentage of patients in the active group had persistent improvement of sore throat compared with the placebo group. No relevant differences between the two groups were observed for headache and fatigue (Supplementary Tables 5-10) (see Appendix). Among children in the active group, the mean total severity of clinical symptoms was consistently lower than that of the placebo group as of the morning of day 3 until the end of treatment. Before that time point, the mean total score was higher in the placebo group (Supplementary Table 4) (see Appendix). A similar pattern was observed for the individual symptoms nasal of congestion and cough, for which statistically significantly higher percentages of children with persistent improvement in the active group were only observed from day 5 (for nasal congestion) or day 6 (for cough) onwards. For fatigue, sore throat, and sleep disturbance, the percentages of children with persistent improvement were consistently higher in the active group compared with the placebo group as of the evening of day 1 or the morning of day 2 and throughout the entire treatment period. The largest difference between the two groups was observed on days 3 and 4 for fatigue and sleep disturbance, and on days 4 and 5 for sore throat. For headache, similar percentages were observed in both groups throughout the treatment period until the morning of day 6. As of the evening of day 6 until the end of treatment, significantly higher percentages of patients in the active group had persistent improvement of headache compared with the placebo group (Supplementary Tables 5-10) (see Appendix).
The percentage of adolescents and children being considered fully recovered was consistently higher in the active group as of the evening of day 2 or the evening of day 1, respectively, until nearly all patients in both groups reached full recovery by the end of treatment (Supplementary Table 11) (see Appendix).

Safety endpoints
In both cohorts, the incidence of AEs was similar in the active and placebo groups. No serious AEs were reported. In the adolescent cohort, 14 (7.3%) patients in the active group and 23 (12.2%) in the placebo group reported AEs, the most common being antibiotic treatment for unknown reason (reported in 13 [6.8%] and 17 [9.0%] patients, respectively). In the pediatric cohort, 30 (14.8%) and 27 (13.6%) patients in the active and placebo group, respectively, reported AEs, the most common also being antibiotic treatment for unknown reason (in 20 [9.9%] and 18 [9.1%] patients, respectively) ( Table 2). Only one child in the placebo group experienced an AE of allergic reaction, and one adolescent (in the active group) and four children (2 in each treatment group) were hospitalized. No clinically relevant findings were reported for physical examinations.

DISCUSSIOn
Acute upper respiratory tract infections are a major cause of morbidity and one of the most frequent reasons for chil-dren and adults to visit GP offices. Given that the majority of AURTIs have a viral etiology, their management is often limited to symptomatic medications. However, there is no conclusive evidence that these medications also shorten symptom duration. [16,17] Moreover, the use of over-thecounter medications for AURTIs in children is often limited given the higher risk of side effects. [18] The rapid evolvement of certain viruses to escape human immunity limits the efficacy of antivirals and complicates the development of vaccines. Given the major burden AURTIs have on society due to missed work and (unnecessary) medical care and the emergence of new viruses potentially giving rise to major outbreaks or even pandemics, novel approaches with efficacy against a broad range of common viruses are urgently needed. In this trial, an alternative approach in the management of AURTIs was evaluated, relying on the potential synergistic activity of three naturally occurring ingredients with demonstrated in vitro and in vivo antiviral effects. Aviron Rapid is a dietary supplement approved for human use containing andrographolide, proprietary spirulina extract, and humic acid, which have the potential to target viral replication at different levels, including viral cell surface attachment [19] , viral envelope fusion to the endosomal membrane [20] , viral RNA polymerase endonuclease activity [21] , and intracellular viral particle transport [22] . The clinical benefit of Aviron Rapid in the management of AURTIs in adolescents and children was supported by results obtained in the adult cohort of this trial [10] , which demonstrated that daily use of Aviron Rapid for 5 days decreases disease duration, intake of antipyretics, and symptom severity. Significant differences were observed as early as 12 or 24 hours after initiation of treatment.
This clinical trial demonstrated that daily use of Aviron Rapid for 5 (adolescents) or 7 (children) days in combination with standard symptomatic therapy resulted in a significantly shorter duration of disease compared with placebo. The percentage of clinically recovered patients was significantly higher in adolescents and children on Aviron Rapid than in those on placebo from 24 and 48 hours after start of treatment, respectively. Aviron Rapid intake Acute tonsillitis 0 (0%) 1 (0.5%) 0 (0%) 1 (0.5%) Allergic reaction 0 (0%) 0 (0%) 0 (0%) 1 (0.5%) significantly reduced the duration of fever (i.e., an axillary temperature >37°C) and the duration of antipyretics intake. As early as 24 hours after start of treatment, a significantly higher percentage of adolescents and children on Aviron Rapid achieved persistent decrease of temperature <37°C. The effect of Aviron Rapid treatment on symptom relief was generally more pronounced in adolescents than in children. Remarkably, throughout the entire treatment period, a higher percentage of adolescents on Aviron Rapid experienced relief of nasal congestion, cough, and sleep disturbance compared with placebo. In children, a similar beneficial effect on nasal congestion and cough was observed, though it became apparent later in the course of disease (i.e. by days 5 and 6, respectively). The largest difference in percentage of patients with relief of sore throat between the Aviron Rapid and placebo group was observed on days 3 and 4 for adolescents and on days 4 and 5 for children. Across both cohorts, a low number of AEs was reported and no major differences in the incidence of individual AEs were observed between the two treatment groups.
The results of this trial confirm the promising results previously obtained with Aviron Rapid in adults [10] and warrant further research into the potential synergistic effects of the three active ingredients in Aviron Rapid in the management of viral AURTIs. The therapeutic potential of Aviron Rapid in this age group is further supported by the fact that children are even more susceptible to AURTIs than adults are.
Given that the standard approach to managing AURTIs in contemporary outpatient practice does not include testing to identify the infectious agent, one limitation of this trial was the lack of confirmation of the viral etiology of the AURTIs. Moreover, it cannot be guaranteed that the trial included only patients who visited their general practitioner's office within 24 hours of the onset of their first symptom. Another limitation of the trial was the relatively small sample size.

COnClUSIOnS
The results of this trial in adolescents and children confirms previous data reporting on the efficacy of Aviron Rapid, a dietary supplement containing andrographolide, proprietary spirulina extract, and humic acid, in the management of AURTIs in adults. Aviron Rapid treatment rapidly increased the number of clinically recovered patients and reduced overall disease duration and duration of symptoms, in particular fever, while being well tolerated.

Ethics approval and consent to participate
The trial protocol and informed consent were approved by the Ethics

Consent for publication
Not applicable

Availability of data and materials
The dataset supporting the conclusions of this article is included within the article (and the Supplementary Tables).

Competing interests
The authors declare that they have no competing interests.

Funding
This trial was funded by Neopharm Bulgaria Ltd. The funder of the trial had no role in the trial design, data collection, data analysis, data interpretation, or writing of the manuscript.

Supplementary tables
Additional tables which present results of the trial not included in the body of the manuscript. Only patients experiencing this symptom at baseline were included in the analysis.
Symptom severity was measured on a VAS (for adolescents) or FPS-R (for children), both ranging from 0 to 10. Only patients experiencing this symptom at baseline were included in the analysis.
Symptom severity was measured on a VAS (for adolescents) or FPS-R (for children), both ranging from 0 to 10. Only patients experiencing this symptom at baseline were included in the analysis.
Symptom severity was measured on a VAS (for adolescents) or FPS-R (for children), both ranging from 0 to 10.