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.
We tested in vitro anti-SARS-CoV-2 activity of combinations of Niclosamide, Ivermectin, and Chloroquine; and show here that these combinations resulted in more than 10-fold reduction in the half maximal inhibitory concentration (IC50) as compared to individual drugs.
In vitro screenings of FDA-approved drugs have identified a number of anti-parasitic drugs with anti-SARS-CoV-2 activity and potential for drug repurposing for treatment of COVID-19 patients [4, 20]. The early hope to get an effective treatment using these drugs was let down by the failure to show clinical benefit of Chloroquine in clinical trials .
On the other hand, Ivermectin has shown promising results in many clinical trials [1, 6, 10–12, 17, 25, 40]. Ivermectin has been shown to cause up to 5000-fold reduction in SARS-CoV-2 replication in vitro [9, 13, 19].
The drug has been widely used to treat various parasitic diseases in humans and animals for four decades with little safety concern. It was also used in the mass treatment campaign against river blindness (Onchocerciasis) with good safety record . It is therefore, an attractive option for drug repurposing for COVID-19 treatment.
The drug has been shown to exhibit broad antiviral activity against a wide range of viruses . These anti-parasitic drugs with potent in vitro anti-SARS-CoV-2 activity are widely available, inexpensive, and considered relatively safe for short-term usage. They were therefore selected for synergistic testing in order to find combination regimens with good potential for drug repurposing in COVID-19 treatment.
Our study shows that the repurposed anti-parasitic drugs, Niclosamide, Ivermectin and Chloroquine possess high in vitro activity against SARS-CoV-2 as the IC50 values are in the low micromolar range. These results on the IC50 against SARS-CoV-2 of these single drugs are in agreement with previous studies [9, 20, 28, 30].
Previous in vitro studies suggested that Ivermectin inhibits host importin alpha/beta-1 nuclear transport proteins, thus preventing the viruses from suppressing the host antiviral response . Recently, it was found that Ivermectin may interfere with the attachment of SARS-CoV-2 spike protein to the ACE2 receptor on human cell membrane .
Several studies also reported antiviral activity of Ivermectin on other viruses such as Zika virus , Dengue virus  and Human immunodeficiency virus type 1(HIV-1) . And with its board spectrum antiviral activity, Ivermectin is thought to act on host cells for its antiviral activity.
Niclosamide showed broad antiviral activity against a wide range of viruses such as SARS-CoV [43–45], MERS-CoV , Zika virus , HCV , Ebola virus  and HIV-1 . Several evidences found in other viruses suggested the plausible mechanisms of Niclosamide in SARS-CoV-2 inhibition by blocking of viral entry via altering endosomal pH and the prevention of autophagy that lead to the inhibition of virus replication [15, 21, 37].
Although Niclosamide was originally thought to act on parasitic worms in the gut lumen and is barely absorbed to the blood stream, it was tested for various systemic repurposed treatments, and a maximal plasma concentration ranged from 35.7 to 182 ng ml−1 (corresponding to 0.11-0.56 µM) was observed in a pharmacokinetic study [3, 8, 24, 34].
Chloroquine inhibits a broad range of viruses by blocking viral entry via inhibition of endosomal acidification . It was recently shown that Chloroquine could not inhibit SARS-CoV-2 in human lung cells because of the expression of TMPRSS2 . This may at least partially explain the lack of clinical efficacy of this drug.
Despite these in vitro anti-SARS-CoV-2 activities, clinical application of these drugs to COVID-19 treatment has not yet been successful. While some clinical trials of Ivermectin on COVID-19 treatment have shown promising results [1, 6, 11, 12, 17, 25, 40], clinical trials for Chloroquine mostly showed negative results  and there have been little clinical data on Niclosamide.
The lack of obvious clinical efficacy suggests that either these in vitro activities could not take effect in vivo or the activities may not be sufficiently potent. An obvious strategy to enhance the potency is drug combination. While combining direct acting antivirals with different targets almost always results in additive or synergistic effect, combining drugs that act on host machineries does not always cause a synergistic effect and can even result in an antagonistic effect [7, 30].
Selecting proper drug combinations with synergistic effect is therefore crucial for development of efficacious regimens. Our data may be useful in guiding the design of clinical trials that may generate a badly needed efficacious regimen for COVID-19 treatment and prevention.
In conclusion, our study demonstrated the benefit of combining Ivermectin, Niclosamide and Chloroquine on their anti-SAR-CoV-2 activities.
Among the combinations, Ivermectin and Niclosamide showed the best synergistic profile. This combination should be further tested in clinical trials.