key: cord-0862418-kt5t1gxp authors: Bernstein, Lawrence R; Zhang, Leike title: Gallium maltolate has in vitro antiviral activity against SARS-CoV-2 and is a potential treatment for COVID-19 date: 2020-12-22 journal: Antivir Chem Chemother DOI: 10.1177/2040206620983780 sha: 9f9efd230a096822726f3dfeca1cec9664c4a556 doc_id: 862418 cord_uid: kt5t1gxp BACKGROUND: Gallium has demonstrated strong anti-inflammatory activity in numerous animal studies, and has also demonstrated direct antiviral activity against the influenza A H1N1 virus and the human immunodeficiency virus (HIV). Gallium maltolate (GaM), a small metal-organic coordination complex, has been tested in several Phase 1 clinical trials, in which no dose-limiting or other serious toxicity was reported, even at high daily oral doses for several months at a time. For these reasons, GaM may be considered a potential candidate to treat coronavirus disease 2019 (COVID-19), which is caused by the SARS-CoV-2 virus and can result in severe, sometimes lethal, inflammatory reactions. In this study, we assessed the ability of GaM to inhibit the replication of SARS-CoV-2 in a culture of Vero E6 cells. METHODS: The efficacy of GaM in inhibiting the replication of SARS-CoV-2 was determined in a screening assay using cultured Vero E6 cells. The cytotoxicity of GaM in uninfected cells was determined using the Cell Counting Kit-8 (CCK-8) colorimetric assay. RESULTS: The results showed that GaM inhibits viral replication in a dose-dependent manner, with the concentration that inhibits replication by 50% (EC(50)) being about 14 µM. No cytotoxicity was observed at concentrations up to at least 200 µM. CONCLUSION: The in vitro activity of GaM against SARS-CoV-2, together with GaM’s known anti-inflammatory activity, provide justification for testing GaM in COVID-19 patients. Coronavirus disease 2019 (COVID-19) can produce severe, life-threatening symptoms due to hyperinflammatory reactions. 1 Treatments that suppress such excessive inflammatory responses, and that also act against the causative virus (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2), would be particularly useful. Gallium, a metallic element with chemical similarities to ferric iron (Fe 3þ ) and to zinc, is known to have both anti-inflammatory and antiviral activity. Its antiinflammatory activity has been recently reviewed. 2 Most relevant to COVID-19, gallium was found effective in a murine model of septic shock, in which it attenuated lipopolysaccharide-induced hepatitis and suppressed the production of nitric oxide, but had no effect on TNF-a production. 3 The hyperinflammatory reactions observed in severe cases of COVID-19 have many characteristics in common with septic shock. 4 Limited data have demonstrated antiviral activity of gallium against influenza A virus 5 and human immunodeficiency virus (HIV). 6 Further antiviral activity of gallium can be hypothesized due to gallium's chemical similarity to zinc. Zinc has efficacy against some RNA viruses, including coronaviruses and hepatitis E virus, possibly in part by acting against the RNA-dependent RNA polymerase. 7,8 SARS-CoV-2 is dependent on the host enzyme furin, 9 which can be inhibited by Zn. 10 On the other hand, Zn is essential for entry into cells by RNA viruses, including coronaviruses 11, 12 and it is probable that Ga would interfere with Zn-dependent cell entry. Furthermore, iron deprivation has been proposed as an effective means to inhibit the replication of SARS-CoV-2 13 Because Ga is a potent competitor of ferric iron, 14, 15 this is another mechanism by which Ga may act against the virus. It is noted that Ga does not enter heme, 14 accounting in part for the low observed clinical toxicity of GaM. Gallium is therefore hypothesized to be an effective treatment for COVID-19, because it could reduce inflammation, act directly against the causative virus, and deprive the virus of iron by competing against Fe 3þ in the host. In this study, a screening assay was used to determine whether gallium maltolate (GaM) is effective at inhibiting SARS-CoV-2 replication in cell culture. GaM was selected as the gallium compound because it has already completed several Phase 1 clinical trials, in a total of 94 subjects, in which it demonstrated a high level of safety and tolerability, even when administered at high daily doses for many weeks at a time. 16, 17 No dose-limiting or other serious adverse effects were reported at doses of up to 3500 mg/day for repeated 28-day cycles. 17 At the highest doses (>2000 mg/day) there was an increase in diarrhea, likely due to osmotic effects in the gut, and transient, mild iron-deficiency anemia, due to the competition of Ga 3þ with Fe 3þ . GaM can be administered orally once per day, is readily synthesized in large quantities at low cost, and is stable over a wide temperature range, making it suitable for widespread distribution and use. Gallium maltolate, manufactured under current FDA Good Manufacturing Practice (cGMP) protocols, was obtained from Gallixa LLC (Menlo Park, California, USA). The purity was >99%, based on HPLC, UV spectroscopy, FTIR, ICP-MS, and powder x-ray diffraction results. A culture of African green monkey kidney Vero E6 cells was obtained from the American Type Culture Collection (ATCC, CRL-1586), with authentication performed by a microscopic morphology examination and growth curve analysis. The cells, which tested mycoplasma-negative, were maintained in minimum Eagle's medium (MEM) supplemented with 10% fetal bovine serum (FBS) at 37 C in a humidified atmosphere containing 5% CO 2 . A clinical isolate of SARS-CoV-2 (hCoV-19/ Wuhan/WIV04/2019; GISAID EPI_ISL_402124) was propagated in the Vero E6 cells, and viral titer was determined by 50% tissue culture infective dose (TCID 50 ) using immunofluorescence assay. All the infection experiments were performed in a biosafety level-3 (BSL-3) laboratory. Pre-seeded Vero E6 cells (5 X 10 4 cells per well) were pre-treated with different concentrations of GaM for 1 h, and the virus was then added at a multiplicity of infection (MOI) of 0.01, with 1 h allowed for infection. Next, the supernatant virus-drug mixture was removed and the cells were further cultured with fresh GaMcontaining medium. At 24 h post infection, a volume of 80 mL cell supernatant was collected from each well for viral RNA measurement by quantitative real-time reverse-transcriptase polymerase chain reaction analysis (qRT-PCR). All experiments were performed in triplicate. Statistical analysis was performed using Prism software. The EC50 value was calculated using an inhibitor vs. normalized response, variable slope equation: 10exp((LogEC50-X) x HillSlope))). The cytotoxicity of GaM to uninfected Vero E6 cells was determined in triplicate with the Cell Counting Kit-8 (CCK-8) colorimetric assay (Beyotime, China), using WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt). The Vero E6 cells were suspended in growth medium in 96-well plates, and on the next day, different concentrations of GaM were added to the medium. After 24 h, the relative numbers of surviving cells were measured with the CCK8 assay in accordance with the manufacturer's instructions. GaM produced dose-dependent inhibition of viral replication, with an EC 50 (concentration producing 50% inhibition of viral replication) of about 14 mM (CI 95% 8.9-22.8 mM), Hill Slope of 0.6 (CI 95% 0.42-0.77), and r 2 ¼ 0.88 for Goodness of Fit (Figure 1 ). The gallium maltolate was not toxic to the cells at concentrations up to at least 200 mM (cell viability at 200 mM was 109(AE3)% relative to untreated cells) (Figure 2 ). Replication of SARS-CoV-2 in Vero E6 cells was strongly inhibited by GaM. Clinical studies have shown that the in vitro gallium concentrations required to inhibit SARS-CoV-2 replication are attainable with oral dosing 18 (Titan Pharmaceuticals, unpublished clinical trial data, 2005). We therefore suggest that orally administered GaM enter randomized, placebocontrolled, double-blinded clinical trials in COVID-19 patients, due to its activity against SARS-CoV-2, its anti-inflammatory activity, its Fe 3þ -competitive activity, and its reported clinical safety and tolerability. COVID-19 diagnosis and management: a comprehensive review A systematic review of the antiinflammatory effects of gallium compounds Gallium nitrate suppresses the production of nitric acid and liver damage in a murine model of LPS-induced septic shock COVID-19 gone bad: a new character in the spectrum of the hyperferritinemic syndrome? Identification and in vivo efficacy assessment of approved orally bioavailable human host protein-targeting drugs with broad anti-influenza A activity Gallium nitrate: a potent inhibitor of HIV-1 infection in vitro Zn (2þ) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture Zinc salts block hepatitis E virus replication by inhibiting the activity of viral RNA-Dependent RNA polymerase Cell entry mechanisms of SARS-CoV-2 Furin inhibition by compounds of copper and zinc Neurovirulent murine coronavirus JHM.SD uses cellular zinc metalloproteases for virus entry and Cell-Cell fusion A novel zinc-binding domain is essential for formation of the functional Jun ın virus envelope glycoprotein complex Depriving iron supply to the virus represents a promising adjuvant therapeutic against viral survival Mechanisms of therapeutic activity for gallium Gallium and its competing roles with iron in biological systems Chemistry and pharmacokinetics of gallium maltolate, a compound with high oral gallium bioavailability Hepatocellular carcinoma detection by gallium scan and subsequent treatment by gallium maltolate: rationale and case study Metallotherapeutic drugs and metal-based diagnostic agents: the use of metals in medicine We are very grateful to Haitao Yang and Zhenming Jin of the Shanghai Institute for Advanced Immunochemical Studies and School of Life Science and Technology, ShanghaiTech University, Shanghai, China, for facilitating the antiviral replication assay.