The COVID-19 pandemic has taken a massive toll on human life, with the virus spreading to 188 countries and territories and infecting nearly 17 million individuals. A very significant source of uncertainty in dealing with the pandemic has been the lack of any effective antiviral or vaccine against the causative agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) despite strenuous scientific efforts.
Novel Coronavirus SARS-CoV-2 Transmission electron micrograph of SARS-CoV-2 virus particles, isolated from a patient. Image captured and color-enhanced at the NIAID Integrated Research Facility (IRF) in Fort Detrick, Maryland. Credit: NIAID
Now, a new study published in the preprint server bioRxiv* in July 2020 reports on the antiviral activity of two drugs found in an antimalarial drug currently in phase II trials in South Korea, called Pyramax. This combination was reportedly effective in inhibiting the growth of both SARS-CoV-2 and the influenza virus, which may well circulate together in the coming winter months in the northern hemisphere, exacerbating the mortality and morbidity of the pandemic.
Among the various drugs shown to have antiviral activity in COVID-19 research are the antimalarial drugs hydroxychloroquine (HCQ) and chloroquine. These are also active against other DNA and RNA viruses, but in clinical trials on COVID-19 patients, the results have not been impressive. The current study focuses on the anti-SARS-CoV-2 activity of Pyramax.
Pyramax consists of two drugs, artesunate, and pyronaridine, in a fixed-dose combination. The two drugs are known to have a broad spectrum of antiviral activity, particularly the second. Compared to other antimalarial drugs that are trafficked in lysosomes, pyronaridine showed a higher degree of protection and produced a more marked decrease in viral load in mice and guinea pigs challenged with the Ebola virus. Artesunate, on the other hand, produced a 20-fold reduction in viral load in rats infected with cytomegalovirus.
Artesunate is a drug that is converted in the body into dihydroartemisinin (DHA). Both these molecules act against DNA viruses via the inflammatory mediators NF-kB and Sp1, both of which are components of the type-1 interferon (IFN) pathway. Artesunate is also described as protecting against lung injury in sepsis by directly inhibiting the Toll-like receptor 4 (TLR4), which is an inflammatory pathway in mice.
The Study: Antiviral Properties of Pyramax Constituents
This prompted the current study into the potential antiviral activity of SARS-CoV-2 and the influenza virus. The researchers explored the following parameters: the viral RNA loads, the cell viability, the growth patterns of the virus, and assays to measure the effect based on the time of drug addition.
They found that pyronaridine inhibited the replication of the virus, and its half-maximal inhibitory concentration, IC50, was just over 1 μM. At this concentration, half the virus cultures failed to replicate. The half-maximal cytotoxic concentration (CC50) was ~37 μM; at this level, half the cells showed toxic effects. The selectivity index (SI) was found to be ~34 at 24 hours post-infection (hpi); at this ratio, the antiviral activity occurred at a level 34 times less than the level that caused cytotoxicity.
With artesunate, the IC50 was 53 μM, with a CC50 of over 100 μM (> 100 μM) and an SI of > 1.9. The combination failed to show markedly superior inhibition of the viral activity in Vero cell cultures compared to HCQ at 24 and 48 hpi. The researchers observed, however, that in Calu-3 cells, which are derived from human lung epithelium, HCQ had no significant inhibitory effect on SARS-CoV-2 at concentrations below 100 μM, but pyronaridine and artesunate showed marked effectiveness.
Artesunate inhibited the virus in Calu-3 cells at a concentration of <2 μM at 24 hpi, compared to 53 μM in Vero cells, while the CC50 was >100 μM, and SI > 56.82. At 48 hpi the IC50 was <0.5 μM, CC50 > 100 μM, and SI > 220.8. Thus, these results are far superior to those seen in Vero cells.
In addition, they outshine the performance of pyronaridine in Calu-3 cells, with its three times higher IC50 of >6 μM, CC50 ~43 μM, and SI of about 7. At 48 hpi, its IC50 is 8.5 μM, CC50 > 100 μM, and SI > 11.7.
Viral Growth Trajectory
The researchers looked at the effect of the drugs on the replication of the virus relative to the dose. They found at a dose of 1.56-50 mM HCQ failed to inhibit the virus, but both these drugs reduced viral replication.
The time-of-addition assay produced results, which might mean that artesunate acts after the virus has accomplished host cell entry. The combination of both drugs was effective in inhibiting viral replication. Of the two, artesunate was superior in inhibiting the growth of the seasonal flu virus A, or H1N1, in Calu-3 cells.
The researchers feel that this could mean that this drug can act against both COVID-19 and the influenza virus.
Mode of Action of Artesunate
The current study shows that artesunate probably does act as described above since Vero cells are known to lack the type 1 IFN pathway, and artesunate shows a correspondingly significant lack of antiviral activity in these cells. However, it does act with significant efficacy in Calu-3 cells. The researchers also point out that artesunate does not have efficacy against the virus even as well as HCQ in Vero cells, but is far better than the latter in Calu-3 cells. This may be, they explain, because of the human airway epithelial origin of the Calu-3 cells, enabling them to represent the cells that are susceptible to the infection in reality.
Implications and Future Directions
The researchers say, “Our data demonstrate a new possibility of repurposing antimalarial drugs against both SARS-CoV-2 and influenza A viruses.” They point out that artesunate is already approved for malaria and is not known to produce toxicity as of now. It could, therefore, potentially be used as the drug of choice to combat COVID-19 if it breaks out during the winter flu season.
They also call for “an in vivo study to be conducted to evaluate the efficacy of artesunate against SARS-CoV-2 and influenza viruses.” If found effective, this combination could be used to fight the combination of flu with COVID-19 in humans, as may be seen in the upcoming winter of 2020-21.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.