Synergistic anti-SARS-CoV-2 activity of repurposed anti-parasitic drug combinations

Abstract

Background: COVID-19 pandemic has claimed millions of lives and devastated the health service system, livelihood, and economy in many countries worldwide. Despite the vaccination programs in many countries, the spread of the pandemic continues, and effective treatment is still urgently needed. Although some antiviral drugs have been shown to be effective, they are not widely available. Repurposing of anti-parasitic drugs with in vitro anti-SARS-CoV-2 activity is a promising approach being tested in many clinical trials. Combination of these drugs is a plausible way to enhance their effectiveness.

Methods: The in vitro anti-SARS-CoV-2 activity of combinations of niclosamide, ivermectin and chloroquine were evaluated in Vero E6 and lung epithelial cells, Calu-3.

Results: All the two-drug combinations showed higher potency resulting in up to 4-fold reduction in the half maximal inhibitory concentration (IC₅₀) values compared to individual drugs. Among these combinations, niclosamide-ivermectin achieved the highest inhibitory level of over 99%. Combination synergy analysis showed niclosamide-ivermectin combination to have the best synergy score with a mean Loewe synergy score of 4.28 and a peak synergy score of 24.6 in Vero E6 cells and a mean Loewe synergy score of 3.82 and a peak synergy score of 10.86 in Calu-3 cells.

Conclusions: The present study demonstrated the benefit of drug combinations on anti-SARS-CoV-2 activity. Niclosamide and ivermectin showed the best synergistic profile and should be further tested in clinical trials.

Keywords: Anti-parasitic drugs; Chloroquine; Ivermectin; Niclosamide; Repurposed drug; SARS-CoV-2.

Fig. 1

Evaluation of antiviral activity of drug candidates against SARS-CoV-2 in vitro. The dose-response curves of single drug treatments against SARS-CoV-2 are shown; (A) niclosamide, (B) ivermectin, and (C) chloroquine. Vero E6 cells were treated with various concentrations of drug for 1 hour and followed by SARS-CoV-2 infection at MOI of 0.01. After removing of virus, the cells were maintained in the medium containing drugs or 0.5%DMSO for 2 days. The virus supernatants were collected for titration using the plaque assay and one step-qRT-PCR. The dose-response curves were expressed as the percent inhibition in relative to DMSO-treated cell control. The effect of drug treatment on the cell viability was determined using MTT assay and is expressed in relative to the DMSO-treated cell control. The experiments were repeated at least three times, and data are shown as mean ± SD

Fig. 2

Niclosamide-ivermectin combination treatments against SARS-CoV-2 in Vero E6 cells. Vero E6 cells were treated for 1 hour with 16 different pairwise combinations of niclosamide and ivermectin. After that, the cells were infected with SARS-CoV-2 at MOI 0.01 for 1 hour. The virus inoculum was discarded, and the cells were further maintained in the medium containing drugs for 2 days. The viral RNA was quantitated using one-step qRT-PCR. The dose-response curves of two-drug combination treatments against SARS-CoV-2 are shown; (A) serial dilutions of niclosamide in the presence of different fixed concentrations of ivermectin, (B) serial dilutions of ivermectin in the presence of different fixed concentrations of niclosamide. The synergy scores of two-drug combinations were calculated using SynergyFinderPlus. The dose-response matrix (C) and the Loewe synergy score map of two-drug combination treatment (D) are shown. The synergy scores less than − 10 accounted for the antagonistic effect; from − 10 to 10 accounted for the additive effect; and larger than 10 accounted for the synergistic effect between two drugs. The experiments were repeated at least three times, and data are shown as mean ± SD in A, B and C or mean [95% confidence intervals (CI)] in D

Fig. 3

Niclosamide-chloroquine combination treatments against SARS-CoV-2 in Vero E6 cells. Vero E6 cells were treated for 1 hour with 16 different pairwise combinations of niclosamide and chloroquine. After that, the cells were infected with SARS-CoV-2 at MOI 0.01 for 1 hour. The virus inoculum was discarded, and the cells were further maintained in the medium containing drugs for 2 days. The viral RNA was quantitated using one-step qRT-PCR. The dose-response curves of two-drug combination treatments against SARS-CoV-2 are shown; (A) serial dilutions of niclosamide in the presence of different fixed concentrations of chloroquine, (B) serial dilutions of chloroquine in the presence of different fixed concentrations of niclosamide. The synergy scores of two-drug combinations were calculated using SynergyFinderPlus. The dose-response matrix (C) and the Loewe synergy score map of two-drug combination treatment (D) are shown. The synergy scores less than − 10 accounted for the antagonistic effect; from − 10 to 10 accounted for the additive effect; and larger than 10 accounted for the synergistic effect between two drugs. The experiments were repeated at least three times, and data are shown as mean ± SD in A, B and C or mean [95% confidence intervals (CI)] in D

Fig. 4

Ivermectin-chloroquine combination treatments against SARS-CoV-2 in Vero E6 cells. Vero E6 cells were treated for 1 hour with 16 different pairwise combinations of ivermectin and chloroquine. After that, the cells were infected with SARS-CoV-2 at MOI 0.01 for 1 hour. The virus inoculum was discarded, and the cells were further maintained in the medium containing drugs for 2 days. The viral RNA was quantitated using one-step qRT-PCR. The dose-response curves of two-drug combination treatments against SARS-CoV-2 are shown; (A) serial dilutions of ivermectin in the presence of different fixed concentrations of chloroquine, (B) serial dilutions of chloroquine in the presence of different fixed concentrations of ivermectin. The synergy scores of two-drug combinations were calculated using SynergyFinderPlus. The dose-response matrix (C) and the Loewe synergy score map of two-drug combination treatment (D) are shown. The synergy scores less than − 10 accounted for the antagonistic effect; from − 10 to 10 accounted for the additive effect; and larger than 10 accounted for the synergistic effect between two drugs. The experiments were repeated at least three times, and data are shown as mean ± SD in A, B and C or mean [95% confidence intervals (CI)] in D

Fig. 5

Single drug treatment against SARS-CoV-2 in Calu-3 cells. The dose-response curves of a single drug treatment against SARS-CoV-2 in Calu-3 cells are shown; (A) niclosamide, and (B) ivermectin. Calu-3 cells were treated with twofold serial dilutions of drug for 1 hour and followed by SARS-CoV-2 infection at 500 TCID₅₀. Then the cells were maintained in the medium containing drugs or 0.5%DMSO for two days. Virus titers were determined using the plaque assay. The dose-response curves were expressed as the percent inhibition in relative to DMSO-treated cell control. The cell viability was determined using MTT assay and is expressed in relative to the DMSO-treated cell control. The experiments were repeated at least three times, and data are shown as mean ± SD

Fig. 6

Niclosamide-Ivermectin combination treatments against SARS-CoV-2 in Calu-3 cells. Calu-3 cells were treated for 1 hour with 16 different pairwise combinations of niclosamide and ivermectin. After that, the cells were infected with SARS-CoV-2 at 500 TCID₅₀ for 1 hour. The virus inoculum was discarded, and the cells were further maintained in the medium containing drugs for 2 days. The viral titers were determined using the plaque assay. The dose-response curves of two-drug combination treatments against SARS-CoV-2 are shown; (A) serial dilutions of niclosamide in the presence of different fixed concentrations of ivermectin, (B) serial dilutions of ivermectin in the presence of different fixed concentrations of niclosamide. The synergy scores of two-drug combinations were calculated using SynergyFinderPlus. The dose-response matrix (C) and the Loewe synergy score map of two-drug combination treatment (D) are shown. The synergy scores less than − 10 accounted for the antagonistic effect; from − 10 to 10 accounted for the additive effect; and larger than 10 accounted for the synergistic effect between two drugs. The experiments were repeated at least three times, and data are shown as mean ± SD in A, B and C or mean [95% confidence intervals (CI)] in D