Methylene blue inhibits the replication of SARS-Cov-2 in vitro
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Highlights
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Methylene blue 50% cytotoxicity concentration (CC50) > 100 µM in Vero E6 cells.
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Methylene blue EC50 of 0.3 ± 0.03 µM and EC90 of 0.75 ± 0.21 µM at MOI of 0.25 against Vero E6 cells infected with SARS-CoV-2 strain (IHUMI-3).
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In comparison, EC50 and EC90 of 1.5 and 3.0 µM for hydroxychloroquine and 20.1 and 41.9 µM for azithromycin.
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Cmax/EC50 and Cmax/EC90 ratios in blood for methylene blue after oral administration were estimated at 10.1 and 4.0, respectively, and 33.3 and 13.3 after intravenous administration.
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Methylene blue EC50 and EC90 consistent with concentrations observed in human blood.
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Methylene blue inhibited SARS-CoV-2 replication in Vero E6 cells.
Abstract
In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China. Currently, there is no antiviral treatment recommended against SARS-CoV-2. Identifying effective antiviral drugs is urgently needed. Methylene blue already demonstrated in vitro antiviral activity in photodynamic therapy, and antibacterial, antifungal or antiparasitic activity in nonphotodynamic assays. Non-photoactivated methylene blue showed in vitro activity at very low-micromolar range with EC50 of 0.3 ± 0.03 µM and EC90 of 0.75 ± 0.21 µM at MOI of 0.25 against SARS-CoV-2. The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine (1.5 and 3.0 µM) and azithromycin (20.1 and 41.9 µM). The ratios Cmax/EC50 and Cmax/EC90 in blood for methylene blue after oral administration were estimated at 10.1 and 4.0, respectively, and 33.3 and 13.3 after intravenous administration. Methylene blue EC50 and EC90 are consistent with concentrations observed in human blood. We propose that methylene blue is a promising drug for COVID-19 treatment. In vivo evaluation in animal experimental models is now required to confirm its antiviral effects on SARS-CoV-2. The potential interest of methylene blue to treat COVID-19 needs to be confirmed by prospective comparative clinical studies.
Keywords
SARS-CoV-2
COVID-19
Antiviral
Methylene blue
in vitro
1. Introduction
In December 2019, a new severe acute respiratory syndrome coronavirus (SARS-CoV-2) causing coronavirus diseases 2019 (COVID-19) emerged in Wuhan, China [1]. Despite containment measures, SARS-CoV-2 spread in Asia, Southern Europe, then in America and currently in Africa. Currently, there is no antiviral treatment recommended against SARS-CoV-2. Different drugs or combination have been evaluated worldwide. Identifying effective low cost antiviral drugs with limited side effects affordable immediately is urgently needed, especially for emerging countries.
Plasma products can transmit a wide range of pathogens in transfusion. Methylene blue, a synthesized thiazine dye, was known to be effective in photodynamic therapy against microbes and more especially virus. Methylene blue is able to intercalate into viral nucleic acid when illuminated with visible light and prevents transmission of pathogens. The illumination of methylene blue inactivated Zika, yellow fever, dengue, chikungunya, Ebola viruses and Middle East respiratory syndrome coronavirus in plasma [2], [3], [4], [5]. Methylene blue can also demonstrate antimicrobial activities without photoactivation. Methylene blue inhibited in vitro colistin-resistant strains of Acinetobacter baumannii, Mycobacterium ulcerans, Mycobacterium spp. and Candida albicans [6], [7], [8]. Methylene blue was also effective in vivo against Buruli ulcer in experimental Mycobacterium ulcerans infection in mice [7]. Additionally, methylene blue inactivated hepatis C virus in transplant organ perfused with methylene blue [9]. The most studied effects are those on malaria.
In 1891, methylene blue was first used to treat effectively two patients with uncomplicated malaria [10]. In the 2010s, methylene blue showed effective in vitro activity in the nanomolar range against Plasmodium falciparum strains and isolates [11], [12], [13], [14]. Methylene blue showed a protective effect against cerebral malaria in a murine model infected with P. berghei [15], [16], [17]. Methylene blue showed several benefits when used as partner in triple combination with artemisinin-based combination therapy in uncomplicated falciparum malaria in children [18].
Taken together, these reports suggest that methylene blue may have antiviral effects against SARS-CoV-2. Therefore the activity of methylene blue was assessed in vitro against a clinically isolated SARS-CoV-2 strain and compared with the activity of hydroxychloroquine and azithromycin, which have been already evaluated in vitro and in vivo in human [19], [20], [21], [22].
2. Material and methods
2.1. Antimalarial drugs, virus and cells
Methylene blue (methylthioninium chloride; Proveblue®) was provided from Provepharm SAS (Marseille, France). Stocks solutions of hydroxychloroquine (Sigma, Saint Louis, MO, USA) and methylene blue were prepared in water and azithromycin (Sigma) in methanol. All the stock solutions were then diluted in Minimum Essential Media (MEM, Gibco, ThermoFischer) in order to have 7 final concentrations ranging from 0.1 µM to 100 µM. The clinically isolated SARS-CoV-2 strain (IHUMI-3) [23] was maintained in production in Vero E6 cells (American type culture collection ATCC® CRL-1586™) in MEM with 4% of fetal bovine serum and 1% glutamine (complete medium).
2.2. Cytotoxicity assay
In vitro cell viability evaluation on the VERO E6 cell line was performed according to the method described by Mosmann with slight modifications [24]. Briefly, 105 cells in 200 µl of complete medium were added to each well of 96-well plates and incubated at 37°C in a humidified 5% CO2. After 24 h incubation, 25 µl of complete medium and 25 µl of each concentration of methylene blue, hydroxychloroquine or azithromycin were added and the plates were incubated 48h at 37°C. After removal of the surpernatant, 100 µL of MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide, Sigma Aldrich, France) solution (0.5 mg/ml in MEM without FBS) were then added to each well. Cells were incubated for 2 h at 37°C. After incubation, the MTT solution was removed and 100 µl of dimethyl sulfoxide (DMSO) was added to dissolve the formazan crystals. Then, plates were shaken at 700 rpm for 10 min at 37°C. The absorbance was measured at 570 nm using a TECAN Infinite F200 Microplate Reader. DMSO was used as blank. The 50% cytotoxicity concentration (CC50) was calculated with the inhibitory sigmoid Emax model, which estimated the CC50 through nonlinear regression by using a standard function of the R software (ICEstimator version 1.2, http://www.antimalarial-icestimator.net). CC50 value resulted in the mean of 6 different experimentations.
2.3. Antiviral activity assay
Briefly, 96-well plates were prepared with 5.105 cells/mL of Vero E6 (200µL per well), as previously described [20]. Methylene blue, hydroxychloroquine or azithromycin concentrations were added 4 h before infection. Vero E Cells were infected with IHUMI-3 strain at an MOI of 0.25. After 48h post-infection, the replication was estimated by RT-PCR using the Superscrit III platinum one step with Rox kit (Invitrogene) after extraction with the BIoExtract SuperBall kit (Biosellal, Dardilly, France). The primers used were previously described [25]. EC50 (median effective concentration) and EC90 (90% effective concentration) were calculated with the inhibitory sigmoid Emax model, which estimated the EC50 and EC90 through nonlinear regression by using a standard function of the R software (ICEstimator version 1.2). EC50 and EC90 values resulted in the mean of 6 different experimentations.
2.4. Data analysis and interpretation
Selectivity index (SI) as ratio of CC50/EC50 was estimated for each drug. The expected maximum blood concentration (Cmax) was estimated from literature for each drug at doses commonly administered in oral malaria treatment, and for methylene blue at intravenous doses used for FDA and MEA approved methemoglobinemia treatment. The ratios Cmax/EC50 and Cmax/EC90 were estimated to find out if the effective concentration in plasma to cure SARS-CoV-2 is achievable in human. If data on drug accumulation into lung was available, the ratios Clung/EC50 and Clung/EC90 were calculated.
3. Results
CC50, EC50, EC90 and SI for each drug are presented in Table 1. Methylene blue and hydroxychloroquine showed EC50 and EC90 at low micromolar range (Table 1). The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine and azithromycin. The ratios Cmax/EC50 and Cmax/EC90 in blood for methylene blue were estimated at 10.1 and 4.0, respectively after oral administration and at 33.3 and 13.3 after intravenous administration (Figure 1).
Table 1. Median effective concentration (EC50), 90% effective concentration (EC90) against SARS-CoV-2, 50% cytotoxicity concentration (CC50) and selectivity index (SI) for methylene blue, hydroxychloroquine and azithromycin
| Drug | EC50 in µM | EC90 in µM | CC50 in µM | SI |
|---|---|---|---|---|
| Methylene blue | 0.30 ± 0.03 | 0.75 ± 0.21 | > 100 | > 333 |
| Hydroxychloroquine | 1.5 ± 0.3 | 3.0 ±1.9 | 20.4 ± 1.4 | 13.6 |
| Azithromycin | 20.1 ± 4.5 | 41.9 ± 18.0 | > 100 | > 5 |
Figure 1. Bar chart displaying Cmax/EC50 (in black) and Cmax/EC90 (in grey) for methylene blue, hydroxychloroquine and azithromycin for in vitro activity against SARS-CoV-2.
4. Discussion
Methylene blue showed in vitro activity at very low-micromolar range with EC50 of 0.3 ± 0.03 µM and EC90 of 0.75 ± 0.21 µM at MOI of 0.25 (SI > 333) (Table 1). The EC50 and EC90 values for methylene blue are lower than those obtained for hydroxychloroquine and azithromycin. Azithromycin demonstrated low in vitro efficacy against SARS-CoV-2 used alone but potentiated the effects of hydroxychloroquine in combination [20]. An oral uptake of 325 mg of methylene blue led to a Cmax (maximum blood concentration) value of 0.97 µg/ml (around 3 µM) and t1/2 (elimination half-life) of 14.9 h [26]. Methylene blue dose of 2 mg/kg intravenous showed a Cmax of 2.917 µg/ml (around 10 µM) [27]. The ratios Cmax/EC50 and Cmax/EC90 for methylene blue were estimated at 10.1 and 4.0 for oral route and 33.3 and 13.3 for IV, respectively. Methylene blue EC50 and EC90 are consistent with concentrations observed in human blood. Around 3 to 5% of methylene blue per g of lung was found after intravenous methylene blue injection but methylene blue concentration decreased rapidly under 0.1% after 10 h [28]. In comparison, an oral uptake of 400 mg of hydroxychloroquine led to a Cmax of 1.22 µM [29]. Hydroxychloroquine accumulated 30 times more in lungs than in blood [30]. The azithromycin Cmax ranged from 0.18 to 0.4 µg/ml of blood (around 0.22 to 0.51 µM) after the last dose of oral administration of 500 mg once daily for 3 days or after a single dose of 500 mg [31], [32], [33]. These doses led to Cmax in lung ranging from 8 to 9 µg/g (around 10 to 12 µM) [31,32]. The Cmax expected in lung was below the EC50 and EC90. However, due to potentiation of the antiviral effects when azithromycin is combined with hydroxychloroquine, azithromycin can be used in vitro at lower concentrations (5 and 10 µM) [20]. These concentrations are compatible with expected concentrations in lungs.
Methylene blue showed a low cytotoxicity in vitro against Vero E6 cells with CC50 > 100 µM. The selectivity index (SI) as ratio of CC50/EC50 was estimated above 333. The present CC50 of hydroxychloroquine with SI around 13 against Vero E6 cells was higher than previous reported CC50, ranging from > 50 µM to 250 µM against Vero E6 cells [19,34] or above 500µM in Felis catus whole fetus-4 cells [35]. Azithromycin showed also a low cytotoxicity against Vero E6 cells with CC50 > 100 µM and SI > 5. CC50 for azithromycin was consistent with previous data (> 130) [34]. Methylene blue showed a low cytotoxicity but predominatingly the higher SI.
Although methylene blue is on the list of drugs potentially dangerous for patients with glucose-6-phosphate dehydrogenase (G6PD), no association between methylene blue and severe hemolysis has been detected after oral administration [36]. Additionally, IV route methylene blue has been granted a Market authorization in Europe in 2011 and in the US in 2016, for the treatment of acquired methemoglobinemia, based upon a confirmed positive benefit/risk ratio in this pathology.
5. Conclusion
Methylene blue showed high in vitro antiviral effective activity against SARS-CoV-2 with IC50 (0.3 µM) and IC90 (0.75 µM) compatible with oral uptake and intravenous administrations. This in vitro activity is higher than those obtained with drugs which are evaluated in clinical trials worldwide like hydroxychloroquine (1.5 µM), azithromycin (20.1 µM), remdisivir (23 µM), lopinavir (26.6 µM) or ritonavir (> 100 µM) [37]. We propose that methylene blue is a promising drug for COVID-19 treatment. In vivo evaluation in animal experimental models is now required to confirm its antiviral effects on SARS-CoV-2. The potential interest of methylene blue to treat COVID-19 needs to be confirmed by prospective comparative clinical studies.
Funding
This study was supported by the Institut Hospitalo-Universitaire (IHU) Méditerranée Infection, the National Research Agency under the program « Investissements d'avenir », reference ANR-10-IAHU-03.
Competiing Interests
Bernard La Scola and Bruno Pradines are associated as co-inventors with ProvePharm in the patent EP 20305425.9 (30/04/2020) but have no financial interest with the subject matter. Manon Boxberger received a PhD grant supported by L'Occitane Society. ProvePharm or funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.The other authors have no conflict of interest to declare.
Ethical approval
Not required.
Acknowledgments
The authors thank Provepharm for providing the methylene blue (Proveblue®)
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Authors contributed equally to this work.
Present address: Unité Parasitologie et Entomologie, Institut de Recherche Biomédicale des Armées, IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France
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