key: cord-0953989-9wbq9qo2
authors: de Oliveira, Pedro Gonçalves; Termini, Lara; Durigon, Edison Luiz; Lepique, Ana Paula; Sposito, Andrei C; Pierulivo, Enrique Mario Boccardo
title: Diacerein: a potential multi-target therapeutic drug for COVID-19
date: 2020-06-01
journal: Med Hypotheses
DOI: 10.1016/j.mehy.2020.109920
sha: 09d99db3eb91040a13410fe5ba22d46b4369960c
doc_id: 953989
cord_uid: 9wbq9qo2

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 19 (COVID-19), was declared pandemic by the World Health Organization in March 2020. SARS-CoV-2 binds its host cell receptor, angiotensin-converting enzyme 2 (ACE2), through the viral spike (S) protein. The mortality related to severe acute respiratory distress syndrome (ARDS) and multi-organ failure in COVID-19 patients has been suggested to be connected with cytokine storm syndrome (CSS), an excessive immune response that severely damages healthy lung tissue. In addition, cardiac symptoms, including fulminant myocarditis, are frequent in patients in a severe state of illness. Diacerein (DAR) is an anthraquinone derivative drug whose active metabolite is rhein. Different studies have shown that this compound inhibits the IL-1, IL-2, IL-6, IL-8, IL-12, IL-18, TNF-α, NF-κB and NALP3 inflammasome pathways. The antiviral activity of rhein has also been documented. This metabolite prevents hepatitis B virus (HBV) replication and influenza A virus (IAV) adsorption and replication through mechanisms involving regulation of oxidative stress and alterations of the TLR4, Akt, MAPK, and NF-κB signalling pathways. Importantly, rhein inhibits the interaction between the SARS-CoV S protein and ACE2 in a dose-dependent manner, suggesting rhein as a potential therapeutic agent for the treatment of SARS-CoV infection. Based on these findings, we hypothesize that DAR is a multi-target drug useful for COVID-19 treatment. This anthraquinone may control hyperinflammatory conditions by multi-faceted cytokine inhibition and by reducing viral infection.

The first case of coronavirus disease 19 (COVID-19), a condition caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in the city of Wuhan in Hubei Province, China, on December 2019. Due to its incubation time (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) days) and because it is highly contagious over a short time, infection with SARS-CoV-2 has traversed global borders and caused thousands of deaths, mainly in elderly patients. On March 11th, 2020, the World Health Organization declared COVID-19 a pandemic disease. Even though some infected persons are asymptomatic, SARS-Cov-2 infection can cause severe symptoms, including high fever, severe cough and shortness of breath, which often indicates pneumonia. Severe acute respiratory distress syndrome (ARDS) has been identified as the leading cause of COVID-19-associated mortality. In addition, sepsis, acute cardiac injury, and fulminant myocarditis are common critical complications (1, 2) .

The mortality related to ARDS as well as multi-organ failure in COVID-19infected patients may be connected with cytokine storm syndrome (CSS), an excessive immune response that severely damages healthy lung tissue (3). This response may lead to macrophage activation syndrome (MAS) (4,5) or secondary haemophagocytic lymphohistiocytosis (sHLH) with fulminant and fatal hypercytokinaemia (3,6). In this scenario, a dilemma emerges due to the potential deleterious effects of immunosuppressive agents used to treat hyperinflammation, such as corticosteroids and Janus kinase (JAK) inhibitors (3), on antimicrobial immunity (7). Diacerein (DAR), also called diacetylrhein, is an anthraquinone derivative used as a symptomatic slow-acting drug for the management of osteoarthritis (SYSADOA) (8-12) licensed in countries of the European Union, Latin America and Asia for up to 20 years. The drug is administered orally and entirely converted to its active metabolite, rhein, before reaching the systemic circulation (8,12). The main mechanism of action of DAR is inhibition of the interleukin-1 (IL-1) signalling pathway (8-10,12-20). In addition, several studies have described the inhibitory effect of this compound on IL-6 (16, (20) (21) (22) (23) (24) and TNF-α (16, 21, 25, 26) . However, no reported studies have addressed the potential therapeutic use of DAR in patients with COVID-19. Considering the current global health crisis caused by COVID-19, the search for therapeutic alternatives is mandatory.

The use of drugs with established mechanisms of action has emerged as a valid tool to identify compounds that can contribute to managing this disease.

SARS-CoV-2 recognizes and binds its host receptor, angiotensin-converting enzyme 2 (ACE2), through the viral spike (S) protein. This binding occurs through the S1 domain of the viral protein, while the S2 domain is responsible for fusion of the viral envelope to the cell membrane (27) (28) (29) . Therefore, the level and pattern of human ACE2 expression in different tissues might be critical for differences in susceptibility, symptoms and outcome between different individuals (30) . In addition, it is important to consider that the expression of ACE2, including its related polymorphisms, can differ in the population (30) (31) (32) . This enzyme is mainly expressed in endothelial cells of the blood vessels but is also present in other organs, including the heart, lungs and kidneys.

This may explain the extra-pulmonary manifestations of the disease as well as some focus on specific targets. On the other hand, other approaches, such as the use of corticoids, present a more nonspecific mechanism of action. It is clear from the data described above that several therapeutic alternatives are under examination. However, at present, no drug for COVID-19 management has been explored from a multi-target perspective.

The active metabolite of DAR, an anthraquinone, is rhein (8, 12) . Different studies have demonstrated that the main mechanism of action of DAR is the inhibition of IL-1β production (15, 17) . In addition, this drug reduced the number IL-1 receptor (IL-1R) in chondrocytes (15) , induced the upregulation of IL-1 receptor agonist (IL-1ra) in cartilage culture (17), and prevented IL-1-induced Nuclear Factor-B (NF-B) activation by inhibiting the degradation of its main inhibitor, IκBα (18). Moreover, rhein was shown to reduce the production of IL-1 converting enzyme (ICE) in cartilage, leading to a reduction in activation of IL-1β from its inactive (pro-IL-1) form (19). Recently, Chang et al. (60) demonstrated that rhein suppressed caspase-1 protease activity and IL-1β production by interfering with formation of the NLRP3 multi-protein complex. In addition, it may exert its anti-inflammatory action through inhibition of the NF-B and NALP3 inflammasome pathways (61, 62) . Based on these mechanisms of action, rhein could suppress lung inflammatory injury induced by human respiratory syncytial virus in mice (63) . Finally, the inhibitory effect of this molecule on IL-6 (20-24,64,65) and TNF-α 

Monolayer cultures of lung, cardiac, endothelial and epidermal cells will be maintained under standard cell culture conditions. The different cell lines will be seeded in 24-well plates and infected with SARS-CoV-2 at different multiplicity of infection (MOI) values. After 48 hours, cell cultures will be treated with rhein at different concentrations (1 M to 400 M) for 3, 6, 12, 24, 48 and 72 hours. Production of the pro-inflammatory and anti-inflammatory cytokines IL-1b, IL-2, IL-6, IL-7, IL-8, IL-10, IL-12, IL-18, IL-37, IL-38 and TNF-α in cell extracts and the supernatants and whole-cell extracts of the cell cultures described will be determined by Western blotting, flow cytometry, Luminex assay and enzyme-linked immunosorbent assay (ELISA). Additionally, release of the damage-associated molecules APT and HMGB-1 in the cell culture supernatants will be measured by ELISA. The expression and function of the NACHT, LRR and PYD domains-containing protein (NALP3) inflammasome in the cell lines treated with rhein will be analysed by Western blotting and RT-polymerase chain reaction (PCR). All the experiments will be performed in triplicate. Moreover, the levels of activated caspase-1 and HMGB1 will be tested.

The effect of rhein-mediated inhibition of the interaction between the S protein and ACE2 on SARS-CoV-2 will be evaluated as described by Ho et al. (73) . The method will consist of purification and biotinylation of recombinant SARS-CoV-2 S protein, biotinylated ELISA, immunofluorescence assay (IFA), and the infection of Vero cells and lung-, cardiac-, endothelial-and epidermal-derived cell lines with S proteinpseudotyped retrovirus. Cell viability will be evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The study will be performed in the presence of rhein at varying concentrations (1 M-400 M).

C57 Black/6 (7 to 9 weeks old) mice will be infected with the mouse-adapted SARS-CoV-2 MA15 strain (85). For infection, mice will be intranasally inoculated with 1×10 4 PFU mouse-adapted SARS-CoV-2 in 50 l of PBS. Treatment of 3 experimental groups and equivalent control and placebo-treated groups with rhein (1 M to 400 M) will start at 2, 6 or 12 hours after viral inoculation. Body weight and pulmonary function (using a single-chamber, whole-body plethysmograph (Buxco Eletronics Inc., NC, USA)) will be assessed daily until the 5 th day post-inoculation. At day 5, the blood, lungs, heart, kidneys and peripheral lymph nodes will be harvested after euthanasia. The lungs will be scored for haemorrhage, and the inferior right lobe will be used for viral load determination by Vero E6 cell infection according to the protocol described by Sheahan et al. (86) . For lung damage measurements, the lungs will be fixed and embedded in paraffin. Scores will be given according to the American Thoracic Society scoring tool, which takes into consideration neutrophil infiltration in the alveolar space, neutrophils in the interstitial spaces, hyaline membranes, proteinaceous debris in air spaces, and alveolar septal thickening. Moreover, necrosis, haemorrhagic areas and cellular sloughing will be examined. The hearts and kidneys will also be fixed and embedded for histology to search for necrosis and haemorrhagic areas. The lymph nodes will be mechanically dissociated into single-cell suspensions. Cells will be labelled with Proliferation Cell Dye (BD Biosciences, Carlsbad, CA) and stimulated with 10 mg/ml TPA and 1 g/ml ionomycin for 4 days. After that period, cell culture supernatants will be harvested for cytokine secretion determination (Th1/Th2/Th17 CBA kit, BD Biosciences, Carlsbad, CA), and cells will be harvested and labelled with anti-CD4, anti-CD8, anti-CD19 and anti-CD25 to determine the proliferation of specific populations.

Serum isolated from the blood will be used to determine the cytokine concentration by Luminex and the lactate concentration to evaluate acidosis using a colorimetric assay (Merck/Sigma).

The effect of rhein on SARS-CoV-2 replication will be evaluated as described by Sun et al. (71) with modifications. Briefly, the different cell lines described above will be infected with SARS-CoV-2 virions and cultured in the presence of rhein at different concentrations. After 72 hours, the effect of rhein on viral replication will be determined by measuring viral load by quantitative real-time PCR. The expression levels of SARS-CoV-2 surface antigens S and E will be determined by ELISA and a virus neutralization test (VNT) after rhein treatment. Rhein will be tested under the conditions described above. The half-maximal inhibitory concentration (IC50) will be calculated.

Total extracts from monolayer cell cultures infected with SARS-CoV-2 and treated with rhein under the conditions described above will be analysed using commercially available protein arrays to determine the levels and activation state of proteins involved in the TLR-, Akt-, MAPK-, and NF-B-regulated signalling pathways.

Data will be analysed using ImageJ software. The levels of reactive oxygen species (ROS)/reactive nitrogen species (RNS) will be determined in the total cell extracts and supernatants from monolayer cell cultures infected with SARS-CoV-2 and treated with rhein under the conditions described above.

important tool to test our hypothesis in humans and, based on their results, accelerate the translational development of this potential therapeutic alternative for COVID-19.

Considering the data presented above, we hypothesize that DAR is a multi-target drug useful for COVID-19 treatment. The mechanisms of action involved include the control of hyperinflammatory conditions by multi-faceted cytokine inhibition of IL-1, IL-2, IL-6, IL-8, IL-12, IL-18 and TNF-α; anti-platelet aggregation activity; and potential effects on viral infection and replication.

Pedro Gonçalves de Oliveira is responsible for R&D activities at TRB Pharma Indústria Química e Farmacêutica Ltda. TRB Pharma is the owner of the product ARTRODAR ® , a diacerein-based product for osteoarthritis treatment.

The other authors declare no conflict of interest. 

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