Surgical Oncology Research Center, Mashhad University of Medical Sciences, Iran
Department of Internal Medicine, Shariati Hospital, Mashhad University of Medical Sciences, Iran
Clinical Research Development Unit, Imam Reza Hospital, Faculty of Medicine, Mashhad University of Medical Sciences, Iran
Institute of Medical Sciences, University of Aberdeen, UK
Departments of Biochemistry, Medical School, Aristotle University of Thessaloniki, Greece
Covenant Medical Center and University Medical Center, Texas Tech University Health Science Center (TTUHSC), USA
Published Date: 15/03/2021.
*Corresponding author: Dr. Daryoush Hamidi Alamdari, Surgical Oncology Research Center, Mashhad University of Medical Sciences, Mashhad, Iran, Hamidiad@mums.ac.ir, Phone: 00989151017650
Background: Methylene blue (MB) possesses all the required properties for clinical management of COVID-19. We have coined methylene blue as an anti-hypoxemia and anti-respiratory distress agent and witnessed promising results in phase 1 & 2 clinical trials.
Methods: In the phase 3 clinical trial, the efficacy of MB has been evaluated as adjunct therapy along with standard care protocols in the treatment of COVID-19 patients. A randomized, controlled, open-label clinical trial was conducted from 20 September through 20 November 2020 and one hundred- twenty hospitalized patients with confirmed severe case of COVID-19 were recruited. Patients were randomly assigned to receive either oral MB (the reduced form: 1mg/kg T.I.D. for 2-days, followed by 1mg/kg B.I.D. for the next 12 days) along with standard-care (MB-group: 106) or standard-care alone (SC-group: 117). The outcomes were duration of hospital stay and mortality.
Results: The hospital stay, measured in days, was significantly shortened in the MB-group (6.2 ± 3.1) in comparison to the SC-group (10.6 ± 9.2, p<0.001), and a marginal significant decrease of mortality was seen in the MB-group (12.2%) in comparison to SC-group (21.7%, p= 0.07).
Conclusion: We conclude that MB, as an adjunct therapy, can be used along with standard care protocols for the treatment of COVID-19. Larger studies in other centers are needed to confirm these findings. MB is a low cost and FDA approved drug for methemoglobinemia.
Trial registration: ClinicalTrials.gov: NCT04370288; IRCT20191228045924N1
Keywords: COVID-19; Treatment; leuco-Methylene Blue; Mortality
In the clinical management of COVID-19 patients, oxygen support, anti-viral agents, antibiotics, anticoagulants, immunomodulatory drugs, antioxidants and fluid therapy are applied [1,2]. Methylene blue (MB) possesses all the required medicinal characteristics along with anti-hypoxemia and anti-respiratory properties, making it a candidate drug for the treatment of COVID-19 [3-5]. In our phase 1  and 2  clinical trials, we determined the effectiveness of MB in the treatment of COVID-19 patients and coined MB as an anti-hypoxemia drug (reduced MB converts Fe3+ in methemoglobin to Fe2+ so that the oxygen bound to Fe2+ and can be transported) and an anti-respiratory distress agent . The addition of MB to the treatment protocols for severe COVID-19 patients was associated with significant clinical benefits which resulted in decreased duration of hospital stay and mortality . In continuation of our clinical trials, phase 3 clinical trial was designed to verify the efficacy of MB (the reduced form) for treating hospitalized patients with severe COVID-19 by determining the duration of hospital stay and mortality.
This study was performed at Mashhad University of Medical Sciences, Mashhad, Iran, after ethics committee approval (IR.MUMS.REC.1399.122; IRCT20191228045924N120, September 20, 2020; ClinicalTrials.gov Identifier: NCT04370288; April 19, 2020) and taking written informed consent from patients. Enrollment for the clinical trial began on September 20, 2020, and ended on November 20, 2020. The authors were responsible for designing the trial and for collecting and analyzing the data. The clinical trial has been conducted according to the principles expressed in the Declaration of Helsinki.
This study was a randomized, controlled, parallel, open-label trial. The authors could not perform the blind study because of the blue discoloration of the urine in the intervention group. Neither statistician, nor investigators, nor patients were masked to the treatment assignment. No drugs were masked and a placebo was not used. Inclusion criteria were severe patients with age above 18 years, respiratory distress (≧26 breaths/min), oxygen saturation ≤93% at rest in the room air, a confirmed case of COVID-19 (by RT-PCR on the collected nasopharyngeal swab or clinical and typical HRCT features). Exclusion criteria were the history of G6PD deficiency, severe renal failure, cirrhosis, active chronic hepatitis, patients with a history of an allergic reaction to MB, treatment with immunosuppressive agents, pregnancy, breastfeeding, and the presence of any condition that would not allow the protocol to be followed safely such as cognitive impairments or poor mental status. Eligible patients were randomly assigned in a 1:1 ratio to either the MB-group (106 patients) or the SC-group (117 patients).
Methylene blue syrup formulation
The compositions of the syrup were MB, vitamin C, dextrose, N-acetyl cysteine. The special formulation for MB (the reduced form) was patented according to the pathology of COVID-19 disease (IR-139950140003002083) (on June 1, 2020, PCT).
For the MB-group, along with standard care, MB syrup was administered orally to patients (1 mg/kg every 8 hours for two days, followed by 1 mg/kg every 12 hours for the next 12 days). For the SC-group, the standard care protocol was continued. The standard care protocols were applied according to WHO guidelines. In the standard care protocols, severe patients receive supplemental oxygen, antiviral agents, intravenous fluids, antibiotics, anticoagulants, corticosteroids [7,8]. In the standard care protocols antiviral: Remdesivir (200 mg on the first day and 100 mg for 4 days), and, IFN-β (44µg/sc, daily for 3 doses); antibiotics: Azithromycin (500mg/day, 5 days), Meropenem (1 gr, TDS, 7-10 days), Ceftriaxone (1 gr, BID, 7 days), and Vancomycin (1 gr, BID, 7-10 days); and corticosteroids: Dexamethasone (8 mg/day for 10 days), anticoagulants (up to discharge).
The measured outcomes were duration of hospital stay and mortality rate within 28 days. It should be noted the hospital stay was counted from the day following MB treatment.
Continuous variables were compared by the t-test and Mann-Whitney test based on data distribution. The paired t-test were used to compare the mean difference of these variables (hospital stay and mortality rate) in each study group. The Significant level was less than 0.05 in all statistical analyses. SPSS version 23 was used for statistical analysis.
Role of the Funding source
The funders did not have any role in the design, collection, management, analysis, interpretation of data, writing of the report, or the decision to submit the report for publication.
Table 1: Demographic and clinical characteristics of SC-group and MB-group.
Comorbidities: Hypertension, Cardiovascular disease, Diabetes, Malignancy, Cerebrovascular disease, Asthma and COPD. *: a marginal significant difference.
Demographic characterizations of patients in the MB-group and the SC-group are presented in Table 1. Comorbidities in both group were hypertension, cardiovascular disease, diabetes, malignancy, cerebrovascular disease, asthma and COPD.
After MB therapy, the hospital stay (days) was significantly shortened in the MB-group (6.2 ± 3.1) in comparison to the SC-group (10.6 ± 9.2, p<0.001) and a marginal significant decrease of mortality was seen in the MB-group (12.2%) in comparison to SC-group (21.7%, p= 0.07). No serious adverse effects were observed in the MB-group except the color of patients' urine turned to green or blue.
This trial showed that MB, as a supplementary therapy to the standard care protocols, leads to significant decrease of hospital stay and mortality rate. Severe COVID-19 patients presented with a chief complaint of dyspnea. After 1 day of the administration of MB, 90 percent of patients expressed dyspnea relief. This finding was very important for the care of COVID-19 patients suffering from respiratory distress. In our previous trials, we explained in detail, the biochemical processes in the pathogenesis of the disease [4,5]. The rationale for considering MB for treatment was due the following mechanisms: 1) Anti-viral activity against the SARS-CoV-2 virus [9-11], 2) Anti-hypoxemia activity by converting iron from the ferric (Fe3+) state to the ferrous (Fe2+) state (an approved medicine for methemoglobinemia) , 3) Anti-respiratory distress activity (authors' observation) 4) Inhibitor of nitrite production (nitrite converts ferrous iron to ferric iron in hemoglobin) by inhibiting nitric oxide synthase and guanylate cyclase in activated macrophages , 5) Antimicrobial agent , 6) Inhibitor of reactive oxygen species (superoxide anion and hydrogen peroxide scavenger) , 7) Inhibitor of xanthine oxidase (which produces superoxide anion) , 8) Anti-platelet aggregation drug , 9) Antifungal agent  10) Anti-inflammatory agent .
MB therapy along with standard care could be very efficacious in the treatment of COVID-19. Since MB is an inexpensive, ubiquitously accessible, and FDA-approved drug for methemoglobinemia, this drug is an excellent supplementary choice for the treatment of hypoxemia in COVID-19 patients. We suggest that the golden time of MB administration should be upon diagnosis and at least before the severe stage of the disease, where patients present with multi-organ involvement and failure. MB can significantly reduce hospital stay and mortality.
Limitation of the Study
Limitations were the conducting of the trial in one university center with a small number of patients.
Ethics approval and consent to participate: Consent informed was obtained from all patients.
Ethics committee approval: IR.MUMS.REC.1399.122
1- Phase 1: Clinical Trials.gov Identifier: NCT04370288. Registered: April 19, 2020, https://clinicaltrials.gov/ct2/show/NCT04370288
2- Phase 2&3: IRCT registration number: IRCT20191228045924N1. Registered: September 20, 2020, https://en.irct.ir/trial/49767
Availability of data and materials
Data available on request through the authors and permission of the ethical committee of the university. Any physician desire to run a randomized clinical trial as a multicenter trial, the authors keen to share their experiences and the last update of their information.
All the authors do not have any possible conflicts of interest.
This work was supported by a grant from Mashhad University of Medical Sciences (Grant number: 990096, 990845).
Author Contributions Statement
Daryoush Hamidi Alamdari, Saied Hafizi Lotfabadi, Behzad Mavaji Darban, Marzieh Agheli-Rad, Shahin Saadatian, Seyed Hadi Hashemi, Fatemeh Barazandeh Ahmadabadi, Negar Morovatdar: (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Supervision, Validation, Visualization, Writing – original draft, Writing – review & editing); (Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration); Bharat Bhushan, Mohammad Arastoo, George Koliakos (Conceptualization, Data curation, Formal analysis, Validation, Visualization, Writing – original draft, Writing – review & editing).
The authors gratefully acknowledge the nurses in Iamm Reza Hospital, Shariati Hospital, Hasheminejad Hospital for their excellent cooperation.