COVID-19 Treatments: Taiwanese Study Finds That Zinc Supplements Enhances Certain Repurposed Drugs’ Efficacy Against SARS-CoV-2

COVID-19 Treatments: A new study by researchers from National Taiwan University Hospital and National Taiwan University College of Medicine has found that zinc supplementation enhances the efficacy of certain repurposed drugs such as triclabendazole and emetine.

The SARS-CoV-2 has caused vast number of infections and fatalities worldwide with almost 104 million infections and more than 2.25 million deaths so far.  As the development and safety validation of effective vaccines are ongoing, drug repurposing is most efficient approach to search FDA approved agents against the COVID-19 disease.
 
In this study,  the study team found that endogenous ACE2 expressions could be detected in H322M and Calu-3 cell lines, as well as their ACE2 mRNA and protein expressions were suppressed by pyrrolidine dithiocarbamate (PDTC), a NF-kappa B inhibitor, in dose- and time-dependent manners. Moreover, N-acetyl-cysteine (NAC) pretreatment reversed PDTC-induced ACE2 suppression, as well as the combined treatment of hydrogen peroxide and knockdown of p50 subunit of NF-kappa B by siRNA reduced ACE2 expression in H322M cells. In addition, the anthelmintic drug triclabendazole and antiprotozoal drug emetine as well as repurposed drugs of NF-kappa B inhibitor also inhibited ACE2 mRNA and protein expressions in H322M cells.
 
Importantly, zinc supplementation augmented the suppressive effects of triclabendazole and emetine on ACE2 suppression in H322M and Calu-3 cells. Taken together, these results indicate that ACE2 expression is modulated by reactive oxygen species (ROS) and NF-kappa B signal in human lung cell lines, and zinc combination with triclabendazole or emetine has the clinical potential for the prevention and treatment of COVID-19.
 
The study findings were published on a preprint server and are currently being peer reviewed. https://www.biorxiv.org/content/10.1101/2021.01.27.428372v1
 
It has been found that the angiotensin-converting enzyme 2 (ACE2) receptor is widely expressed in the human body, and plays a role in the functioning of the renin-angiotensin system (RAS). SARS-CoV-2 binds to the receptor by its spike protein, which has two subunits, S1 and S2.

Importantly the S1 subunit mediates the initial attachment of the virus, leading to the cleavage of the spike at the S1/S2 interface. Following the spike-ACE2 binding, S2 is cleaved by the host protease TMPRSS2, an essential step for endocytosis and internalization of SARS-CoV-2 into the host cell.
 
The critical role and importance of the ACE2 receptor has led to the exploration of many compounds which may prevent the virus-ACE2 interactions.
 
Some includes soluble ACE2 molecules, used as a decoy for the virus, ACE2 inhibitors, and TMPRSS2 inhibitors. The hope is that these will entrap the virus and inhibit spike-ACE2 binding. However, no effective and safe drugs have yet been identified.
 
Also it is important to note that the viral spike or the double-stranded genetic material of the virus may activate the NF-κB pathway in the cells of the alveolar epithelium or the vascular endothelium. The triggering of this inflammatory pathway may finally re sult in a cytokine storm, characteristic of severe COVID-19.
 
Hence the inhibition of this pathway is, therefore, a therapeutic target to prevent disease progression and adverse outcomes in COVID-19. The glucocorticoid dexamethasone has been found to reduce mortality among patients with severe COVID-19 who are on invasive mechanical ventilation or supplemental oxygen, but not on those with less severe disease.
 
To date it has been suggested to many researchers that this beneficial activity of dexamethasone is in part at least mediated by its suppression of the pro-inflammatory NF-κB activation in patients with severe disease.
 
The study team found that the NF-κB inhibitor, ammonium pyrrolidine dithiocarbamate (PDTC), as well as the drugs triclabendazole and emetine, were capable of suppressing ACE2 expression. Both the mRNA encoding ACE2 and ACE2 protein expression were found to be increased in cells treated with PDTC, in a time-dependent manner.
 
This finding indicates that the expression of ACE2 is regulated by the NF-κB pathway, such that inhibitors of NF-κB could potentially be helpful in reducing the severity of COVID-19.
 
The study team also found that inhibition of the p50 subunit of NF-κB by PDTC was not responsible for inhibiting ACE2 expression. Instead, PDTC is a chelator with both pro-oxidant and antioxidant effects.
 
Also the potential role of reactive oxygen species (ROS) in inducing ACE2 suppression in response to cell exposure to PDTC was explored by pretreating the cells with N-acetyl-cysteine (NAC). This thiol compound is known to inhibit ROS production induced by PDTC.
 
The study team observed an increase in ACE2 mRNA and protein levels in cells treated with NAC alone. When NAC was used for pretreatment, followed by PDTC, the expected PDTC-induced suppression of ACE2 was not observed, at transcriptional and translational levels.
 
This lead to the conclusion was that PDTC is an oxidant. It suppresses ACE2 expression by inducing oxidant activity, similar to the effects produced by treatment with hydrogen peroxide on cells lacking the p50 subunit of NF-κB. This effect of hydrogen peroxide was not observed in control cells.
 
Hence, both NF-κB inhibition and oxidant activity induced by PDTC are required for the latter to suppress ACE2 expression.
 
The study findings suggest that PDTC suppresses ACE2 expression through ROS induction and NF-κB inhibition.”
 
Typically drug repurposing is a method to rapidly identify drugs capable of treating a disease condition without the time and expense involved in conventional drug discovery protocols.
 
The team found that the drugs emetine and triclabendazole, which have anti-protozoal and anthelmintic activity, respectively, have been found to suppress this signaling pathway. The current study seeks to explore their effects on ACE2 expression.
 
The study team used non-toxic dosages of both triclabendazole and emetine on cultured cells, and observed a dose- and time-dependent suppression of ACE2 expression.

The IC50 values of inhibiting IκBα phosphorylation for emetine and triclabendazole are 0.31 mM and 25.1 mM, respectively. Administration of 100 nM emetine or 100 mM triclabendazole significantly repressed ACE2 expressions in both of mRNA and protein levels in H322M cells. It is noteworthy that the serum levels of inflammatory cytokines were significantly reduced by emetine treatment in the pulmonary hypertension rat model.
 
Moreover, triclabendazole treatment obviously reduced inflammatory cytokine levels, indicating the improvement of the immunological responses of the patients with acute or chronic stage of fasciolosis.

The study findings suggest that triclabendazole and emetine exhibit anti-SARS-CoV-2 activity via ACE2 suppression.
 
Interestingly the study also explored the potential for combination therapy, using either of these repurposed drugs with zinc. This could increase the efficacy of treatment as well as reduce the required dose and thus the adverse effects.
 
The supplement zinc is a trace mineral that enhances the clearance of virus particles through the mucociliary apparatus, augments epithelial barrier function, reduces viral replication, modulates inflammatory processes, and promotes anti-oxidant activity. Hospitalized adult COVID-19 patients have shown correlations between lower basal zinc levels and increased mortality, complication rate, and length of hospital stay.
 
The study team found that zinc was able to produce a small decrease in ACE2 mRNA and protein levels. When combined with either triclabendazole or emetine, vs. the use of each of these drugs alone, the researchers found that monotherapy with any of these three compounds failed to produce a significant drop in ACE2 levels.
 
But when zinc was combined with either of the other two drugs, ACE2 levels were suppressed, with no signs of cell toxicity.
 
To date, the exact changes in ACE2 expression following viral binding are unclear, but some studies suggest an increase on airway epithelium by about threefold. If so, viral amplification could be promoted by the increase in ACE2 levels following infection.
 
Most significantly triclabendazole or emetine plus zinc supplementation may be more effective in reducing virus amplification through decreasing ACE2 protein expression than triclabendazole or emetine monotherapy.

Combination therapy has proven successful in a variety of medical areas, such as infectious and metabolic diseases. The advantages of combination therapy include to increase treatment efficacy, to reduce the dose and side effects of toxic drugs, prevent the development of drug resistance, and to reduce the duration of treatment. Previous studies had implicated that zinc may possess antiviral activity and anti‑inflammatory activity through inhibition of SARS‑CoV RNA polymerase and disruption of NF‑κB signaling, respectively. In addition, it was noted that 57.4% of the COVID-19 patients were zinc deficient, as well as these patients had higher rates of complications, acute respiratory distress syndrome, prolonged hospital stay, and increased mortality.
 
The study findings indicate the suitability of this combination for further studies, to investigate its efficacy in alleviating the severity of COVID-19 as well as possibly prevent further infection. The researchers suggest a future focus on the development of these drugs in an inhalable form, to optimize the efficacy on airway epithelium.
 
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