Efficacy of BREATHOX® Device Inhalation on Acute Symptoms Associated with COVID-19 (BREATH Study): A Randomized Pilot Clinical Trial

(1) Background: A high concentration of sodium chloride on in vitro cell culture leads to reduced SARS-CoV-2 replication. Therefore, our aim was to evaluate the effects of inhaling hypertonic NaCl particles (BREATHOX®) on the duration of COVID-19-induced acute symptoms. (2) Methods: A prospective, open label, randomized, standard of care-controlled group (SOC) pilot trial compared inhaled oral and nasal administered BREATHOX® (2.0 mg NaCl, particles size between 1–10 μm), with five or ten inhalations per day for ten days. The primary endpoint was the time to resolve COVID-19-related symptoms. Safety outcomes included adverse clinical and laboratory events. (3) Results: A total of 101 individuals were screened and 98 were randomly assigned to BREATHOX® ten sessions per day (Group 1; 33 patients), BREATHOX® five sessions per day (Group 2; 32 patients), or SOC (33 patients), and followed up for 28 days. There was an association with cough frequency after 10 days BREATHOX® compared to SOC [Group 1: hazard ratio (HR) 2.01, 95% confidence interval (CI) 1.06–3.81; Group 2: HR 2.17, 95% CI 1.17–4.04]. No differences between the groups for the reported symptoms’ resolution time were seen after 28 days. After combining both BREATHOX® groups, the period to cough resolution 10 days after randomization was significantly lower than in SOC (HR 2.10, 95% CI 1.20–3.67). An adverse event occurred in 30% of Group 1, 36% of Group 2, and 9% in SOC individuals. One patient from SOC had a serious adverse event. Nasal burning, sore or itchy nose, and dry mouth were considered related to BREATHOX® use and resolved after stopping inhalations. (4) Conclusion: BREATHOX® inhalation is safe and may be effective in reducing the duration of COVID-19-induced coughing.


Introduction
The first cases of coronavirus disease-2019 (COVID-19) were reported in December 2019.It was caused by the new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).By the first weeks of January 2023, there had been approximately 666.8 million cases, which had varied from mild to very serious symptoms with 6.7 million deaths worldwide reported in the same period [1].The most frequent symptoms in the mild disease were fever and cough [2][3][4][5].
Several anti-inflammatory treatments were investigated during the pandemic; they included cytokine modulation drugs to control the adverse effects of COVID-19.However, current guidelines for the mild disease recommend specific intervention to modify the life cycle of the virus in patients at high risk of disease progression [6].
The virus adheres to the mucosa of the upper respiratory epithelium and enters the target cell [7,8].After replication in the nucleus, the virus is released from the cell by budding.This process occurs with the greatest intensity in Type I and II respiratory epithelial cells.The new viral particles have easy access to the bloodstream and become capable of infecting any other susceptible cells, such as cells of the kidney, heart, skeletal muscle, and endocrine glands, providing the peak of viremia [7][8][9][10].
A previous in vitro study showed an 88% inhibition of virus replication in SARS-CoV-2-infected monkey lung cells exposed to a 1.1% concentration hypertonic saline solution [11].The inhibition mechanism is not known; one hypothesis is that the hyperosmotic environment triggered by the high extracellular sodium concentration changes the function of different membrane channels and prevents or attenuates virus entry into the cell.Another hypothesis is that the higher salinity increases activity of membrane Na + /K + channels, leading to increased energy expenditure, and thereby reduced energy availability for virus replication [11,12].
Thus, we hypothesized that the use of inhaled NaCl particles, through BREATHOX ® , would reduce respiratory symptoms in patients with mild COVID-19.Therefore, the primary aim of this study was to evaluate the effects of oral and nasal inhalation of hypertonic NaCl particles (BREATHOX ® ) on the duration of COVID-19-induced acute symptoms compared to the standard of care (SOC).Secondarily, we analyzed the occurrence of adverse events related to the device use.

Study Design
The BREATH study was a prospective, open label, randomized, pilot trial using inhaled oral and nasally administered BREATHOX ® (2.0 mg of NaCl-particle sizes 1-10 µm) for ten (Group 1) or five (Group 2) sessions per day for ten days.The results were compared to individuals subjected to SOC, antipyretic and/or analgesic, (Group 3) at a 1:1:1 ratio.The trial protocol was designed by the study steering committee.Use of any investigational or unregistered product within the previous 3-month or the 5-half-life period before baseline, whichever is longer; • Baseline biochemistry showing hemoglobin < 9.0 g/dL, absolute neutrophil count ≤ 1000/mm 3 , platelets ≥ 100,000/mm 3 , or creatinine clearance ≤ 30 mL/min (using the Cockcroft-Gault formula).

Outcomes
The primary endpoint was the reported time to resolution of COVID-19-related symptoms within 10 and 28 days: fever, headache, sore throat, nasal obstruction, cough, fatigue, chest pain, dyspnea, myalgia, anosmia, dysgeusia, or gastrointestinal changes.

Safety
Safety outcomes included adverse clinical and laboratory events.

Trial Procedures
Randomization was performed through an online web-based system (REDCap ® ) using computer-generated random numbers and was age-stratified to allow equal numbers of patients > 40 years old in each group.The randomization procedure was blinded to the investigators.Data was entered directly into REDCap by the study staff.Physicians, patients, and individuals who assessed outcomes were not blinded for the assigned treatment.The REDcap system was also used as the electronic case report form (eCRF).

Clinical and Laboratory Data
Data on the demographic characteristics and physiological variables were collected.Participants were requested to report their health status according to the Ordinal Scale for Clinical Improvement [13].Laboratory analyses (complete blood count, urea, and creatinine) were performed at baseline or when necessary for complementary assessment in unscheduled visits.

Intervention
Group 1 was instructed to use BREATHOX ® ten sessions per day (two oral inhalations and one nasal inhalation in each nostril, 2 mg per inhalation) at hourly intervals during the daytime.Group 2 was instructed to use BREATHOX ® five sessions per day (two oral inhalations and one nasal inhalation in each nostril, 2 mg per inhalation), at three-hour intervals during the daytime.The treatment period was 10 days.Protocol adherence related to the medication use was assessed daily until day 10.Protocol adherence was considered acceptable at 80%.The SOC group was allowed to use antipyretics and/or analgesics; Groups 1 and 2 were allowed to use SOC at their own discretion.
All participants completed a daily symptom diary.Patients remained in the study for 28 days.The number of days with symptoms was counted from the day of inclusion.Phone interviews were carried out on day 10, and on-site visit or phone interviews were carried out on day 28.During the interviews, symptom evaluation and occurrence of endpoints or adverse events were evaluated.

Statistical Analysis
As a pilot study, we decided to include a study population of 100 patients.Statistical analysis was performed by intention to treat (ITT).Therefore, all randomized patients were analyzed according to their allocated group, regardless of adherence to treatment.The per protocol (PP) population consisted of all subjects with good compliance and without a major protocol deviation (including but not limited to violation of inclusion and exclusion criteria).The PP and ITT analyses were used to evaluate the primary endpoints.All subjects who received at least one dose of BREATHOX ® or were included in the SOC group made up the safety analysis set.
Descriptive statistics for the endpoints are presented according to the treatment group and number of visits (if applicable).Continuous data are presented as the number of subjects and reported as mean and standard deviation (SD).The mean symptom resolution time reported was calculated for each group.The safety data are presented using descriptive statistics.Kaplan-Meier curves were used to demonstrate symptom resolution time.Proportional Cox regression models were performed and a p-value of ≤ 0.003 was considered statistically significant for multiple comparisons adjusted for the Bonferroni method.The test considered the null hypothesis as the outcome being the same in all groups.The alternate hypothesis means at least one pair of groups is different.Statistical analyses were carried out using Stata Software, version 16.0 (Statcorp, College Station, TX, USA).Statistical oversight and analyses were performed at Pelotas Federal University, Brazil.

Results
One hundred and one individuals were screened, and 98 patients were included from 1 December 2021, to 3 March 2022.In total, 33 patients were assigned to Group 1, 32 patients to Group 2, and 33 to SOC.Primary endpoints were available for all individuals (Figure 1).
Descriptive statistics for the endpoints are presented according group and number of visits (if applicable).Continuous data are presen of subjects and reported as mean and standard deviation (SD).The me lution time reported was calculated for each group.The safety data a descriptive statistics.Kaplan-Meier curves were used to demonstrate s time.Proportional Cox regression models were performed and a p-va considered statistically significant for multiple comparisons adjusted method.The test considered the null hypothesis as the outcome bei groups.The alternate hypothesis means at least one pair of groups is d analyses were carried out using Stata Software, version 16.0 (Statcorp, C USA).Statistical oversight and analyses were performed at Pelotas F Brazil.

Discussion
This study investigated the effects of a new intervention aimed at controlling respiratory symptoms in mild COVID-19.We showed that the period to reported cough resolution was lower in both BREATHOX ® groups than the SOC group.The effect of BREATHOX ® on coughing may hypothetically be related to a hyperosmotic response influencing the function of different membrane channels consequently preventing virus entry into the cells.Moreover, the hypertonic solution may increase the mucociliary clearance and reduce the destructive inflammatory process in the airways with a decrease in respiratory symptoms.Hypertonic saline solution has been widely used in patients with acute respiratory diseases as an adjuvant to other therapies for improving respiratory symptoms [14][15][16][17].
In the three years of the COVID-19 pandemic, scientists have developed strategies to recommend the best evidence for treating SARS-CoV-2 infections.Pharmacological guidelines are prepared in accordance with the disease severity [6,18].The main goal of intervention is to reduce the risk of death and the need for mechanical ventilation in moderate-severe COVID-19 cases.Therefore, the use of antiviral drugs, corticosteroids, and immunosuppressive interventions is recommended for moderate to severe COVID-19 cases [6,18].Indeed, monoclonal antibodies are not recommended in SARS-CoV-2 preexposure or to control the progression of disease since the omicron sub-lineages escape the monoclonal antibodies [6].
Most of the pharmacological guidelines for COVID-19 treatment were designed for previous sub-lineages of SARS-CoV-2, such as Alpha, Beta, Gama, and Delta which cause lower respiratory tract infections [1].Currently, the Omicron variant represents the most divergent mutation across the world, which has modified the disease approach due to its higher transmission rate and milder clinical picture.Most of the patients with the Omicron variant present a mild illness with fever, cough, sore throat, malaise, headache, muscle pain, ageusia, and anosmia.Therefore, treatment is conducted at home and often through telemedicine.In this case, antiviral drugs are recommended to specific populations with a high probability of developing severe COVID-19.On the other hand, patients without comorbidities or risk factors may use symptomatic treatment to relieve symptoms.However, the use of drugs commonly prescribed for the common cold may not relieve symptoms and BREATHOX ® can be considered in this population.
Mild COVID-19 may be associated with mild respiratory symptoms such as coughing.NICE guidelines recommend the use of non-pharmacological treatments as the first line, such as honey, to manage COVID-19-induced coughing.This should be followed by codeine linctus, codeine phosphate, or morphine sulfate [18].In this randomized controlled trial, we showed positive results for cough resolution with BREATHOX ® in mild COVID-19.
Concerning the administration method, in addition to direct application in the upper airways, the inhalation of hypertonic saline has been shown to improve quality of life for COPD patients.Therefore, the availability of inhaler and nasal devices with hypertonic saline may facilitate the care of patients with chronic respiratory diseases [16,17].We did not observe an increased adverse event severity related to BREATHOX ® use; most of the mild adverse events were nasal burning which did not require modification of treatment.This demonstrates the opportunity to conduct a novel study on coughing caused by mild COVID-19 by using a symptomatic relief intervention.
There are limitations to this study.As a pilot trial, it is possible that the low sample size prevented statistical differences from being found between the BREATHOX ® and SOC groups in COVID-19-related symptoms other than coughing.Additionally, we did not conduct an analysis of the virus cycle threshold using BREATHOX ® , which represents a gap in our investigation.Large double-blinded, placebo-controlled randomized clinical trials are therefore needed to confirm the efficacy of BREATHOX ® on cough resolution following COVID-19 and to further investigate BREATHOX ® efficacy on other COVID-19 symptoms.Also, the results may be biased by the lack of a placebo-controlled group.

Conclusions
The inhaled use of BREATHOX ® was safe and effective in reducing the duration of COVID-19-induced coughing in the studied population.Funding: This research was funded by LIITA Care.

Institutional Review Board Statement:
The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Ethics Committee (Approval number 5.627.523).The study protocol was registered on the clinical trials registry (NCT05196581).All study activities, including project coordination, data management, and site monitoring were performed at the Botucatu Medical School, São Paulo State University, Brazil.The trial was overseen by a data-safety monitoring board of independent experts.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Table 1 .
Patient demographics and baseline clinical data.

Table 1 .
Patient demographics and baseline clinical data.

Table 4 .
Number of adverse events.