Latest Review on Antiviral Potential of Doxycycline against SARS-CoV-2
Written By : Dr. Kamal Kant Kohli
Published On 2021-02-04 07:15 GMT | Update On 2022-12-07 05:57 GMT
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Severe acute respiratory syndrome coronavirus (SARS-CoV-2), causing coronavirus disease 2019 (COVID-19) first emerged in Wuhan, China 1 and despite containment measures continues to spread across Asia, Southern Europe, America, and Africa. While the vaccine still seems a while away before it could reach the masses, and a large population getting infected calling for need for therapeutic modalities; many existing agents continue to be repurposed for treatment against SARS-CoV-2 infections. We review some of the most recent evidence shedding light on the antiviral effects of Doxycycline against SARS-CoV-2 infections which continue to emerge endurably over time.
Doxycycline - Promising Track Record against Community Infections
Doxycycline is a broad-spectrum antimicrobial (2) and additionally possesses anti-inflammatory activities (3) . Doxycycline was approved as prophylaxis against malaria by the Food and Drug Administration in 1994 and has been used since 2006 in oral form (100 mg/day) by the French military forces deployed in malaria-endemic areas (4) .
In many studies, Doxycycline also inhibited replication of the dengue virus by inhibition of NS2B-NS3 serine protease 5 and even shown inhibition of entry and replication of Chikungunya virus (6) . Additionally, doxycycline reportedly also inhibited early-stage replication of the porcine reproductive and respiratory syndrome virus, which causes respiratory disease (7) . Thus, since early weeks of the pandemic, Doxycycline seemed to be an attractive candidate for the treatment of COVID-19 infections (8)
The activity of doxycycline was assessed in vitro against a clinically isolated SARS-CoV-2 strain and was compared with the activity of chloroquine. (9)
Study Methodology Overview
Agent, Virus, and Cells: Stock solution of doxycycline hyclate (Sigma, Saint Louis, MO, USA) was prepared in methanol and diluted in Minimum Essential Media (MEM, Gibco, ThermoFischer, Waltham, MA, USA) to have 7 final concentrations ranging from 0.1 microM to 100 microM. Chloroquine diphosphate (Sigma) was used as a comparator. The clinically isolated SARS-CoV-2 strain (IHUMI-3) was maintained in production in Vero E6 cells (American type culture collection ATCC® CRL-1586TM ) in MEM with 4% of fetal bovine serum and 1% glutamine medium.
Cytotoxicity Assay: In vitro cell viability evaluation on the VERO E6 cell line was performed according to the method described by Mosmann with some modifications. The 50% cytotoxicity concentration (CC50) was calculated with the inhibitory sigmoid Emax (the maximum effect attributable to the drug) model, which estimated the CC50 through nonlinear regression by using a standard function of the ICEstimator version 1.2 software. CC50 value resulted in the mean of 5 different experimentations.
Antiviral Activity Assay: About 96 well plates were prepared with 5.10 5 cells/mL of Vero E6 (200 L per well). Doxycycline and chloroquine concentrations were added 4 hours before infection. Vero E Cells were infected with IHUMI-3 strain at the multiplicity of infection (MOI) of 0.25. After 48 h post-infection, the replication was estimated by Reverse transcription-polymerase chain reaction (RT-PCR) using the Superscrit III platinum one step with Rox kit (Invitrogene) after extraction with the BioExtract SuperBall kit (Biosellal, Dardilly, France).
Data Analysis and Interpretation: Results were estimated as a mean and standard deviation of 5 to 10 experiments. Selectivity index (SI) as a ratio of CC50/EC50 was estimated for doxycycline. The expected maximum blood concentration (Cmax) was estimated from the literature for doxycycline at. doses commonly administered in oral or intravenous treatment.
Results
The cytotoxicity evaluation of doxycycline and chloroquine showed that the CC50 values were >100 microM for 48 hours. The CC50 value of chloroquine was found to be consistent with those previously described (10) .
The antiviral effects of doxycycline against the clinically isolated SARS-CoV-2 strain (IHUMI-3) were concentration-dependent.
The median effective concentration (EC50) and 90% effective concentration (EC90) for doxycycline were 4.5 + 2.9 microM and 23.5 + 16.5 microM, respectively. The EC50 and EC90 for chloroquine were 3.2 + 1.8 microM and 13.9 + 6.4 microM, respectively. The EC50 value for chloroquine was consistent with previous results on Vero E6 cells at MOI of 0.2 to 0.25.
The ratios Cmax/EC50 and Cmax/EC90 in blood for doxycycline were estimated at 0.75 and 0.07, respectively after an oral administration of doxycycline 100 mg, at 2.21 and 0.41 after an oral administration of doxycycline 200 mg and at 1.14 and 0.21 after intravenous administration of 100 mg of doxycycline.
The results demonstrated that doxycycline interacted at both entry and post-entry stages of the SARS-CoV-2 infection in Vero E6 cells.
Practice Relevant Message from the Study
The in vitro activity of doxycycline against the SARS-CoV-2 was consistent with those reported with hydroxychloroquine, chloroquine, amodiaquine, ferroquine, or mefloquine, antiviral agents like remdesivir or lopinavir, and macrolides like azithromycin.
Doxycycline seems to be interacting at both levels - entry and post-entry stages of the SARS-CoV-2 infection.
Doxycycline seems to strongly bind the spike protein(S) of SARS-CoV-2 (11) which uses the ACE-2 receptor for its entry, thus offering a potential target against the SARS-CoV-2 virus.
Lastly, doxycycline also reportedly inhibits the RNA-directed 5'-3' polymerase activity of SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) - a key enzyme for replication of the SARS-CoV-2 (12) , which seems similar to remdesivir (13) .
Doxycycline Benefits COVID-19 Patients – Brief Overview of Testimony from Clinical Studies
Early treatment with doxycycline of 100 mg a day for 7 days in 89 high-risk patients affected with moderate to severe COVID-19 infections was associated with early clinical recovery, decreased hospitalization, and reduced mortality (14) .
Another observational study on 54 high-risk patients across 3 long term care facilities in New York, USA demonstrated that the use of a combination of 100 mg of doxycycline twice a day for 7 days and 400 mg of hydroxychloroquine twice a day on the first day and daily for next 6 days was associated with a decrease in hospital admissions and reduced mortality (15)
In Summary
Doxycycline showed a high in vitro antiviral effective activity against SARS-CoV-2 compatible with oral uptake and intravenous administrations. Doxycycline interacts with SARS-CoV-2 virus at multiple levels - on entry and in replication after its initial entry.
In addition, doxycycline has anti-inflammatory effects by decreasing the expression of various pro-inflammatory cytokines and could prevent co-infections and superinfections by virtue of its broad-spectrum antimicrobial activity. 9 We are close to 10 months on this threatening pandemic and it is indeed promising to witness a valuable role of doxycycline continuing to emerge durably in the context of COVID-19 infections, and it seems doxycycline could remain a potential partner in the management of COVID-19 infections.
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References
Adapted from
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14 Alam, M.M.; Mahmud, S.; Rahman, M.M.; Simpson, J.A.; Aggarwal, S.; Ahmed, Z. Clinical outcomes of early treatment with doxycycline for 89 high-risk COVID-19 patients in long-term care facilities in New York. Cureus 2020, 12, e9658
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