Effect of bromhexine in hospitalized patients with COVID-19 - Journal of Investigative Medicine
Significance of this study
What is already known about this subject?
The COVID-19 pandemic remains one of the major public health issues, despite preventive measures being implemented worldwide.
Bromhexine is a potent inhibitor of transmembrane serine protease 2 and has an antiviral effect.
One study has shown the clinical benefit of this inexpensive medicine in patients with COVID-19 pneumonia.
What are the new findings?
Bromhexine is not an effective treatment in hospitalized patients with COVID-19.
Presence of renal disease is the strongest predictor of mortality in hospitalized patients with COVID-19 in our multivariate analysis.
Bromhexine as a transmembrane serine protease 2 inhibitor could not reduce duration of hospitalization.
Bromhexine as a transmembrane serine protease 2 inhibitor could not reduce the need for mechanical ventilation compared to the control.
How might these results change the focus of research or clinical practice?
Introduction
The COVID-19 pandemic remains one of the major public health issues, despite preventive measures such as wearing mask and social distancing, being implemented worldwide.1 The search for finding the effective treatment to prevent or treat the viral infection is ongoing but, so far, has had limited success.
Bromhexine is an inexpensive and widely available medication with a low side-effect profile and has been used as mucolytic in different respiratory conditions since 1963.2 Bromhexine is a potent inhibitor of transmembrane serine protease 2 (TMPRSS2) and seems to have an antiviral effect. It has been shown that the presence of TMPRSS2 is very essential for influenza virus infection and propagation. Bromhexine has been shown to be effective in controlling influenza infection by blocking the cleavage of the surface glycoprotein hemagglutinin of the influenza virus.3 4
Researchers have proposed that bromhexine may be an effective option to reduce primary transmission, viral load, dissemination and secondary replication of SARS-CoV-2.5–8 COVID-19, like SARS-CoV, binds to human ACE 2 via its spike glycoprotein (S-protein) expressed on its envelope for entering the target cell. S protein is composed of one amino-terminal (S1) and one carboxy-terminal (S2). Cleavage at the S1–S2 junction by protease (TMPRSS2) is essential to prime the virus spikes and activate membrane fusion. It has also been proposed that bromhexine, by blocking non-endosomal pathways via serine protease 2 (TMPRSS2), theoretically blocks the priming of the spikes and virus entry into the host cell.9 10
In a small open-label trial, the clinical benefit of bromhexine administration in patients with SARS-CoV-2 pneumonia has been reported.11 This study tested whether bromhexine hydrochloride was an effective medication to improve clinical outcomes and mortality in hospitalized patients with COVID-19.
Materials and methods
This clinical trial was designed as a randomized, single center, open-label study. From 156 patients who were screened, in Masih Daneshvari Hospital, a tertiary and referral center for COVID-19, 111 patients with a diagnosis of COVID-19 pneumonia were enrolled. The study began on May 6, 2020, and enrollment of patients was completed on June 20, 2020. Written informed consent from all the study subjects was obtained.
Patients were randomized at a 1:1 ratio to receive either oral bromhexine in addition to standard therapy or standard therapy alone. Subjects who were enrolled received a trial number. Every single trial number was randomized to either arm of the study through computer randomization. The study was randomized, controlled, and open-labeled, and the trial was monitored by the data monitoring committee. Trial recruitment stopped after the target study population had been reached and was closed when all of the patients had completed their follow-up visit.
Inclusion criteria
The inclusion criteria were as follows: hospital admission, 18 years old or greater at the time of signing the informed consent, chest imaging and clinical symptoms consistent with COVID-19 pneumonia, laboratory (reverse transcription polymerase chain reaction (RT-PCR)) confirmed infection with 2019-nCoV, willingness to participate in the study, and no concurrent participation in other clinical trials.
Exclusion criteria
The following exclusion criteria were used: pregnancy or lactation, severe liver disease (eg, aspartate aminotransferase (AST)>5 times upper limit), undergoing dialysis or transferred to another hospital within 72 hours and a history of allergy to bromhexine.
Standard arm
Patients received treatment based on the hospital COVID-19 treatment protocol and best practice guidelines in place at that time. (lopinavir/ritonavir) (Kaletra) 400/100 two times per day for 7 days or discharge from hospital and interferon (IFN) beta-1a (Rebif) 44 μg subcutaneous every other day for five doses in addition to supportive and symptomatic therapy.
Treatment arm
The treatment arm received oral bromhexine hydrochloride 8 mg four times a day for 2 weeks in addition of standard therapy.
Outcome measures
The primary outcome was clinical improvement within 28 days. Clinical improvement was defined as the time (in days) from initiation of the study treatment (active or placebo) until a decline of two categories on a clinical status scale occurred. The six-category ordinal scale of clinical status which ranged from hospital discharge to death and is itemized as follows: (1) hospital discharge or meeting discharge criteria (discharge criteria are defined as clinical recovery, ie, fever, respiratory rate, oxygen saturation returning to normal, and cough relief); (2) non-intensive care unit (ICU) hospitalization, not requiring supplemental oxygen; (3) non-ICU hospitalization, requiring supplemental oxygen (but not noninvasive ventilation/high-flow nasal cannula); (4) ICU/non-ICU hospitalization, requiring noninvasive ventilation/high-flow nasal cannula therapy; (5) ICU hospitalization, requiring invasive mechanical ventilation; and (6) death.
The criteria for ICU admission were worsening of respiratory distress assessed by the physician, hemodynamic instability requiring vasopressors, and oxygen desaturation of <85% that was not responsive to low-flow oxygen therapy.
Secondary outcome measures included time to hospital discharge, all-cause mortality, duration of mechanical ventilation, time to 2019-nCoV RT-PCR negativity and frequency of serious adverse drug events, within 28 days from the start of medication.
Statistical analysis
Data were analyzed using SAS V.9.4. The distribution of the demographic and clinical characteristics of the sample was summarized by treatment status. Number and percent were reported for binary outcomes. Means and SD were calculated for continuous outcomes such as time to hospital discharge. Associations between the treatment status and patient characteristics were tested for statistical significance using χ2 or Fisher's exact test, as appropriate, for categorical variables, and two-sample t-tests for continuous variables. An alpha of 0.05 was used for all significance testing.
Study subjects were tested for COVID-19 using a polymerase chain reaction (PCR) test on days 1, 7, and 28. The prevalence of PCR test positivity was plotted by time. A longitudinal data analysis using generalized estimating equations was attempted. However, there was an error in the estimation routine when fitting the generalized estimating equations logistic regression model and the convergence was questionable. Standard errors could not be generated.
Kaplan-Meier curves were created for time to improvement. Wilcoxon tests (rather than log-rank tests) were performed to determine if the survival curves for the treatment groups differed from one another in the population. The assumption of proportional hazards was violated for multiple predictors for both outcomes, time to improvement and time to death. Given these violations, HRs from Cox (proportional hazards) regression models were not calculated. Instead, ORs for improvement and mortality were calculated from logistic regression models and reported with 95% CIs and p values. Given the imbalanced distribution of several factors of clinical significance between the two study arms, ORs were adjusted for obesity (defined as a Body Mass Index (BMI) of ≥30 kg/m2), smoking, and renal disease (defined as an estimated glomerular filtration rate between 16 and 60 mL/min). Sparse data bias was a possibility, given the small number of patients who did not improve and the small number of deaths. To minimize the risk of triggering sparse data bias, Firth's penalized maximum likelihood estimation was used when estimating the unadjusted and adjusted ORs for both death and improvement.12
Results
A total of 156 patients with proven COVID-19 pneumonia were screened. Forty-five of them were excluded (33 patients were enrolled in another experimental trial, 7 were on hemodialysis, 3 had severe liver disease and 2 were transferred to another hospital). A total of 111 patients were enrolled in this randomized clinical trial. They were assigned to either the treatment with bromhexine group or the standard treatment group in a 1:1 ratio with 59 patients in the treatment arm and 52 patients in the standard/control arm. Eleven patients were lost to follow-up in the treatment arm. No attrition occurred in the control arm. Data from the total of 100 patients (48 patients in the treatment arm and 52 patients in the control arm) were analyzed (figure 1).
CONSORT flow diagram. Patient enrollment and treatment assignment.
The distributions of most of the demographic and disease characteristics were similar in the treatment and standard groups (table 1).
Demographic and clinical characteristics of the 100 study subjects*
The mean age±SD was 50.7±16.4 years among the treated arm and 53.1±15.2 in the standard arm. In terms of gender, the percentage of men in both the treatment and standard groups was approximately 46%. There was a significant difference (p<0.0001) in the mean BMI between the treatment group (26.2±1.8) and the standard treatment group (33.2±4.5). The distribution of other comorbidities such as asthma, hypertension, diabetes, chronic obstructive pulmonary disease, cancer and cerebrovascular accident were almost identical between the study arms.
Primary clinical outcome
There was no significant difference in the primary outcome of this study, which was time to clinical improvement. The median time to improvement in the bromhexine arm was 7 days, while that in the control arm it was 6 days. The p value from the Wilcoxon test for equality of the survival curves in the population was 0.61 (figure 2).
Kaplan-Meier estimates of cumulative clinical improvement. The median time to clinical improvement was 7.0 days (95% CI 6.0 to 7.0) in the bromhexine arm and 6.0 days (95% CI 6.0 to 7.0) in the placebo arm.
The unadjusted OR for clinical improvement comparing patients in the bromhexine arm with those in the standard treatment arm was 0.92. After adjusting for obesity, smoking, and renal disease, this OR was 4.15 (95% CI 0.13 to 138.25, p=0.43) (table 2).
Unadjusted and adjusted ORs* for clinical improvement in 100 patients with COVID-19: 96 improved vs 4 did not improve
Patients with no renal disease had 25 times the odds of improving compared to patients with renal disease (1/0.04=25): adjusted OR=0.04, 95% CI 0.004 to 0.42, p=0.007.
Unadjusted and adjusted ORs for death are listed in table 3.
Unadjusted and adjusted ORs* for time to death in 100 patients with COVID-19: 4 died vs 96 survived
The unadjusted OR for death comparing patients in the bromhexine group with those in the control arm was 1.09. After adjustment for obesity, smoking, and renal disease, the bromhexine OR was 0.24. Given this result, it appears, at first, that there was a 76% reduction in the odds of dying (bromhexine vs standard treatment); however, this result was not statistically significant: 95% CI:0.007 to 8.03, p=0.43. In a similar fashion, the obesity OR was also affected by strong joint confounding by the remaining three variables found in the multiple logistic regression model. The confounding was severe enough to reverse the direction of the association: unadjusted obesity, OR=1.13, and adjusted obesity, OR=0.48. Neither of the obesity ORs were statistically significant. In contrast, both the unadjusted and adjusted ORs for mortality for the presence of renal disease were above 1 and statistically significant: adjusted renal disease, OR=24.98, 95% CI 2.40 to 259.71, p=0.007.
Secondary clinical outcomes
There was no significant difference in the mean (average) time to hospital discharge between the two arms. There were also no differences observed in the mean ICU stay, frequency of intermittent mandatory ventilation, duration of supplemental oxygenation and risk of death by day 28 noted between the two arms. The prevalence of the use of high-flow nasal oxygenation in the bromhexine group was significantly higher than the prevalence in the standard arm (56.3% vs 23.1%, p=0.001) (table 4).
Outcomes of the 100 study subjects
The temporal trend in the probability of being PCR positive was assessed. On days 1, 7, and 28, 100%, 60.4%, and 0%, respectively, of the patients in the bromhexine arm were PCR positive. Among patients in the standard arm, the prevalence of PCR positivity on days 1, 7, and 28 were 100%, 34.6%, and 0%, respectively.
Adverse events
No major adverse events were noted.
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