“3.5. Brilacidin in Combination with Other Antiviral Treatments: Synergistic Activity against SARS-CoV-2 in Combination with Remdesivir in Calu-3 Cells
As brilacidin appears to act primarily by disrupting viral integrity and inhibiting viral entry, combining the drug with antiviral treatments that have a different mechanism of action may result in synergistic inhibition when administered in combination. The potential for brilacidin to exert a synergistic inhibition of SARS-CoV-2 when combined with current frontline COVID-19 antiviral treatments, namely, remdesivir and favipiravir, was assessed. Potential toxicity of combinations of remdesivir or favipiravir with brilacidin were initially assessed in the Calu-3 cell line at 24 h post-treatment. No apparent toxicity could be detected up to a 10 µM concentration of each of the drugs in the combination regimen. To evaluate the efficacy of combining remdesivir or favipiravir with brilacidin, the cells were pretreated with brilacidin for 2 h. The virus inoculum was also independently preincubated with brilacidin for 1 h, and then the treated inoculum was overlaid on cells and the infection allowed to proceed for 1 h in the presence of brilacidin. Postinfection, the inoculum was removed and media containing both brilacidin and remdesivir or favipiravir or each drug alone for efficacy comparison were added to the infected cells. Supernatants were obtained at 24 h postinfection and infectious titer was quantified by plaque assay. The data revealed that brilacidin and favipiravir independently exerted up to 90% and 80% inhibition, respectively, and the extent of inhibition did not increase over that exerted by brilacidin alone when the two drugs were used in combination (Figure 5A). In contrast, combination of brilacidin with remdesivir at 10 and 2.5 µM concentrations, respectively, reduced the viral infectious titer by >99%, thus providing a highly effective inhibition profile (Figure 5B) and achieving greater inhibition than with either compound alone. This synergistic inhibition continued to remain higher than 99% when the concentrations of both compounds were equal (2.5 µM each) (Figure 5C).
Viruses 13 00271 g005 550
Figure 5. Efficacy of brilacidin as a combinatorial strategy as assessed in Calu-3 cells. Calu-3 cells were pretreated for 2 h with media alone or media containing brilacidin at 10 µM (for synergy treatments). (A) Cells were infected with SARS-CoV-2 at MOI 0.05 directly with brilacidin at 10 µM (for synergy treatments). or SARS-CoV-2 incubated in media alone (for favipiravir treatment alone). After 1 h, post-treatment with favipiravir alone or mixed with 10 µM brilacidin were added to cells at 1 or 2.5 µM concentrations. At 24 hpi, viral supernatants were evaluated by plaque assay as described in Materials and Methods. Calu-3 cells were pretreated for 2 h with media alone or media containing brilacidin at 10 or 2.5 µM (for synergy treatments). Cells were infected with SARS-CoV-2 at MOI 0.05 directly with brilacidin at 10 or 2.5 µM (for synergy treatments) or SARS-CoV-2 incubated in media alone (for remdesivir treatment alone). After 1 h, post-treatment with remdesivir alone, or mixed with 10 µM (B) or 2.5 µM (C) brilacidin, were added to cells at 1 or. 2.5 µM concentrations. At 24 hpi, viral supernatants were evaluated by plaque assay as described in Materials and Methods. Graphs are representative of one independent experiment performed in technical triplicates (n = 3). Brl indicates brilacidin, Rem indicates remdesivir. Statistical analyses for synergy vs. individual control treatments were determined using Unpaired Two-Tailed Student’s t test. Significance against DMSO was determined as described in Materials and Methods. * p < 0.0332, ** p < 0.0021, **** p < 0.0001.
4. Discussion
The ongoing global COVID-19 pandemic powerfully reinforces the need for therapeutic strategies that can safely and effectively address virus- and host-based events elicited during SARS-CoV-2 infection.
In multiple studies, we have attempted to evaluate the capability of brilacidin to decrease viral load in the context of the SARS-CoV-2 infection. Our experiments in the Vero cell line model demonstrate brilacidin decreases viral load in a robust manner when the virus is preincubated with brilacidin (Figure 2D), suggesting brilacidin impacts virus integrity. Brilacidin’s ability to decrease viral load in an ACE2-positive cell line is demonstrated in Figure 3, Figure 4 and Figure 5, in which Calu-3 cells were used.
All experiments conducted in Vero and Calu-3 cell line models were supportive of an early inhibition exerted by brilacidin on SARS-CoV-2, indicating the drug’s impact on viral integrity. The idea that brilacidin directly interferes with the integrity of the virion is further supported by the observation that when drug treatment was limited to the virus alone (Figure 2E), with no treatment of host cells, a robust decrease of viral load was still observed in both the Washington strain and the Italian strain of SARS-CoV-2. This mechanism of inhibition may be akin to that achieved by neutralizing antibodies that may interact with specific exposed epitopes on the surface of virions. It remains to be determined if the impact of brilacidin on viral membranes is driven by specific viral membrane compositions.
While brilacidin’s mechanism of action appears primarily to be extracellular, it may also impact intracellular viral replication and is planned to be researched further. Supportive of this, an in silico quantum mechanical molecular screening study of 11,522 compounds identified brilacidin as a potential inhibitor of SARS-CoV-2 based on the potential of its physicochemical properties to interfere with the intracellular replication of SARS-CoV-2's main protease (Mpro) [69].
The high CC50 (a measure of cytotoxicity) and low IC50 (a measure of potency) values observed for brilacidin in Calu-3 cells—yielding a Selectivity Index (SI) for brilacidin of 426 (CC50 = 241 µM/IC50 = 0.565 µM)—strongly support brilacidin’s treatment potential to achieve positive antiviral outcomes in humans. A vast majority of other drugs being evaluated as potential COVID-19 treatments, including repurposed drugs, have SIs that are much lower than that achieved by brilacidin [70], with most drugs failing to show anti-SARS-CoV-2 potency in the <1 µM range [71]. Of note, the IC50 (0.565 µM) and IC90 (2.63 µM) values for brilacidin observed in the Calu-3 cell line are well below clinically achievable concentrations based on pharmacokinetics observed in Phase 2 clinical trials with brilacidin for the treatment of Acute Bacterial Skin and Skin Structure Infections (ABSSSI). Applying the in vitro IC50 and IC90 parameter targets to in vivo human plasma concentration data, simulated dose regimens for brilacidin are similar to that already tested in clinical trials for ABSSSI and even exceed such targets, thereby further supporting the progression of brilacidin to clinical testing for treatment of COVID-19.
As of January 2021, FDA Investigational New Drug approval (with FDA Fast Track designation), and a similar regulatory approval from an overseas health authority, has been obtained for conduct of a multinational Phase 2 clinical trial of intravenously administered brilacidin in hospitalized patients with COVID-19. Brilacidin has been tested in numerous human trials (a total of eight) for other clinical indications, providing established safety and efficacy data on over 460 subjects.
A desirable outcome for any potential COVID-19 therapeutic will be its ability to synergize with existing COVID-19 treatments, particularly if the mechanisms of action of the synergistic treatments can impact more than one step of the viral lifecycle. Such combinations are more likely to elicit an additive response while also reducing the likelihood of viral resistance developing. Along these lines, we conducted experiments to evaluate the potential of brilacidin to work in conjunction with remdesivir and favipiravir (Figure 5), two frontline COVID-19 treatments, which proved supportive of synergistic inhibition between brilacidin and remdesivir. Remdesivir is a SARS-CoV-2 nucleotide analog RNA polymerase inhibitor that impacts the viral RNA synthesis step of the infectious process. By that mechanism, remdesivir may help decrease progeny viral genomes in infected cells but will not be conducive to inhibiting progressive infection of naïve cells once the progeny virions have been released from infected cells.
By combining remdesivir with brilacidin, a two-pronged strategy of inhibiting viral entry and viral RNA synthesis might be successfully leveraged to most effectively control progression of SARS-CoV-2 infection. The opportunity that combination treatments with brilacidin could potentially offer in treating COVID-19 requires further exploration, and in vivo animal studies are in planning stages.
Clearly, an effective COVID-19 therapeutic (or therapeutics in combination) ideally would control both viral load and the corresponding inflammatory damage due to SARS-CoV-2 [72], and mitigate bacterial coinfections. With its HDP mimetic properties—antiviral, immuno/anti-inflammatory, and antibacterial—brilacidin may be able to address the different disease parameters of COVID-19 within the one therapeutic treatment. The results of the planned Phase 2 clinical trial, with intravenous treatment of COVID-19 in addition to standard of care, are highly anticipated.
In this manuscript, we demonstrate brilacidin exhibits robust inhibition of SARS-CoV-2 in Vero cells and Calu-3 cells, and in two strains of the virus. Likely to function as a viral entry inhibitor [73,74,75], the proposed mechanism of action for brilacidin includes affecting the integrity of the viral membrane and interfering with viral entry. Brilacidin also exhibited an excellent synergistic inhibitory profile against SARS-CoV-2 in combination with remdesivir. Destabilizing viral integrity is a desirable antiviral property, especially in relation to pan-coronavirus agents, as the viral membrane is highly conserved and similar in construct across different coronavirus strains. Further research will be conducted in the context of other lethal coronaviruses (MERS-CoV, SARS-CoV) toward assessing the potential of brilacidin as a broad-spectrum inhibitor of coronaviruses.”
