key: cord-0996449-hlrqf7dk
authors: Bartoli, Alessandra; Gabrielli, Filippo; Alicandro, Tatiana; Nascimbeni, Fabio; Andreone, Pietro
title: COVID-19 treatment options: a difficult journey between failed attempts and experimental drugs
date: 2021-01-04
journal: Intern Emerg Med
DOI: 10.1007/s11739-020-02569-9
sha: 851139dce05a92d5a90789bd1effc2e84d7d3709
doc_id: 996449
cord_uid: hlrqf7dk

Since its outbreak in China in December 2019 a novel Coronavirus, named SARS-CoV-2, has spread worldwide causing many cases of severe pneumonia, referred to as COVID-19 disease, leading the World Health Organization to declare a pandemic emergency in March 2020. Up to now, no specific therapy against COVID-19 disease exists. This paper aims to review COVID-19 treatment options currently under investigation. We divided the studied drugs into three categories (antiviral, immunomodulatory and other drugs). For each molecule, we discussed the putative mechanisms by which the drug may act against SARS-CoV-2 or may affect COVID-19 pathogenesis and the main clinical studies performed so far. The published clinical studies suffer from methodological limitations due to the emergency setting in which they have been conducted. Nevertheless, it seems that the timing of administration of the diverse categories of drugs is crucial in determining clinical efficacy. Antiviral drugs, in particular Remdesivir, should be administered soon after symptoms onset, in the viraemic phase of the disease; whereas, immunomodulatory agents, such as tocilizumab, anakinra and steroids, may have better results if administered in pneumonia/hyperinflammatory phases. Low-molecular-weight heparin may also have a role when facing COVID-19-related coagulopathy. Up to now, treatment choices have been inferred from the experience with other coronaviruses or viral infection outbreaks. Hopefully, in the near future, new treatment strategies will be available thanks to increased knowledge on SARS-CoV2 virus and COVID-19 pathogenesis. In the meanwhile, further well-designed clinical trials are urgently needed to establish a standard of care in COVID-19 disease.

In December 2019, an outbreak of pneumonia cases of unknown origin, epidemiologically linked to the attendance of Wuhan Central Marketplace in Hubei province in China, was reported for the first time. The aetiological agent of this novel pneumonia has been named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and the name 2019 coronavirus disease (COVID-19) has been coined for the disease itself. Due to the rapid spread of the infection across all the continents, a pandemic emergency has been declared by the World Health Organization (WHO) in March 2020.

SARS-CoV-2 is an enveloped, non-segmented, positivesense single-stranded ribonucleic-acid (RNA) β-coronavirus, which shares 80% sequence homology with SARS-CoV-1 and 50% sequence homology with Middle East Respiratory Syndrome (MERS)-CoV. Like SARS-CoV-1, SARS-CoV-2 cell entry depends on binding of the viral spike protein to Angiotensin-Converting Enzyme-2 (ACE-2) receptor of lower respiratory tract cells and on spike protein priming by the trans-membrane serine protease 2 (TMPRSS2).

Even if the majority of infected people develops smooth symptoms, a considerable number progresses to pneumonia with severe respiratory failure requiring hospitalization, and a minority develops life-threatening complications with poor prognosis. Older age and comorbidities (mainly arterial hypertension, followed by diabetes and ischemic heart disease) are the most significant risk factors associated with the worst outcomes [1] . To date, no specific therapy against COVID-19 disease exists; many trials are ongoing with different treatment options but further time will be necessary to develop an effective therapy.

COVID-19 disease's clinical course can be divided into three phases. The first one, the "viraemic phase", is characterized by fast viral replication and symptoms are caused by direct cytopathic effect. This phase lasts 7-8 days and is accompanied by mild and often non-specific symptoms, such as fever, malaise, myalgia, headache, dry cough, conjunctival congestion, anosmia and ageusia, abdominal pain and diarrhoea. In patients who are able to control the infection, prognosis and recovery are excellent. The second phase, named "acute or pneumonia phase", generally occurs after 7-8 days from the symptoms onset and is characterized by high fever, cough and dyspnoea, requiring close observation and management. At this stage, patients develop a frank viral pneumonia, with imaging showing bilateral infiltrates or ground-glass opacities, and blood tests revealing lymphocytopenia and increased lactate dehydrogenase (LDH) and C-reactive protein (CRP) levels. The patient's immune system begins to develop a reaction that, if balanced, leads to the final healing, if exaggerated, brings to the "phase of complications". This third phase, which is experienced by a minority of COVID-19 patients, is characterized by extrapulmonary systemic hyperinflammatory syndrome, also referred to as cytokine storm, due to an over-reaction of the immune system with over-production of pro-inflammatory cytokines and other mediators [1, 2] . Acute respiratory distress syndrome (ARDS) is the most frequent complication (20% of the patients) followed by acute heart failure, acute kidney injury (AKI), liver failure, shock, secondary bacterial infections, coagulopathies and disseminated intravascular coagulation (DIC). Notably, 20% of COVID-19 patients, and nearly 100% of the critical ones, develop coagulation disorders, in particular a hypercoagulable state which predisposes to DIC. Indeed, micro-and macro-thrombi in lungs and extrapulmonary districts have been described in several autopsy series, suggesting that thrombosis and inflammation are two processes that reinforce each other in the pathogenesis of COVID-19 complications [3] .

Several innate immune signalling proteins are targeted by SARS-CoV-2 viral proteins, among all, Interferon (IFN) and Nf-Kb pathways. In particular, SARS-CoV-2 seems to be able to activate NLRP3 (NOD like receptor pyrin domaincontaining 3) inflammasome causing the abnormal production of pro-inflammatory cytokines. The cytokine storm is induced by the activation of large numbers of white blood cells, including B cells, T cells, natural killer (NK) cells, macrophages, dendritic cells, neutrophils, monocytes, and resident tissue cells, such as epithelial and endothelial cells, which release high amounts of pro-inflammatory cytokines. Among the numerous molecules that increase in serum in a cytokine storm, complements, IFN-γ, interleukin (IL)-1β, IL-6, IL-12, and IL-17, and tumor necrosis factor-α (TNFα) are of crucial importance. An Italian study found an increased capability of cluster of differentiation (CD)4 + or CD8 + T cells from COVID-19 patients with pneumonia to produce in vitro IL-17, that is able to strengthen the inflammation response and to activate neutrophils [4] . Pathway analysis of peripheral blood mononuclear cells transcriptome revealed that COVID-19 patients lymphopenia may be associated with activation of apoptosis and P53 signalling pathway in lymphocytes, in which SARS-CoV-2 seems to enter through the CD147 receptor. Virus-activated "cytokine storm syndrome" is a common feature of severe COVID-19 cases, a major reason for ARDS and multiorgan failure, and the main cause of mortality in COVID-19 disease [5] .

Understanding COVID-19 disease pathogenesis is important to make the best treatment choices. For instance, antiviral drugs are likely more useful in the phases ruled by the direct cytopathic effect of SARS-CoV-2; whereas, at the later stages of COVID-19 disease, the treatment options to be theoretically considered more appropriate would be the ones which affect the immune response, such as corticosteroids and immunosuppressive/immunomodulatory agents [1, 2] . However, the pathogenic features of COVID-19 disease are yet far from being completely elucidated and no validated specific therapeutic options exist.

This paper intends to review COVID-19 treatment options which are currently under investigation. For each drug deemed to be potentially effective on COVID-19 disease, firstly we discuss the putative mechanisms by which the drug may act against SARS-CoV-2 or may affect COVID-19 pathogenesis, then we comment on the major clinical studies among COVID-19 patients involving the particular drug.

All the relevant studies were independently retrieved by two researchers by interrogating Pubmed and Google Scholar databases, using the following search strategies: "COVID-19 Treatment OR Therapy" and each single drug under investigation for COVID-19 treatment; attention has been paid to "cytokine storm in COVID-19" and "coagulation and COVID-19". The bibliographic research has been conducted until July 24, 2020.

The most important antiviral drugs tested in the COVID-19 pandemic are detailed below. Table 1 describes the main clinical studies on antiviral drugs for treating COVID-19 patients. [6] . On this basis, LPV/r has been tested since the beginning of the pandemic. As shown in Table 1 -LPN/r section, the studies on LPV/r carried out up to now present several criticisms, notably including small samples sizes and poor study design. Nevertheless, it is interesting to observe that in the single available randomized controlled clinical trial LPV/r therapy has not proven effectiveness in patients with severe disease and was not superior to standard therapy when started 12 days or later after symptoms onset. In the few studies evaluating LPN/r in association with other drugs, such as Umifenovir, Ribavirin and Interferons, combination therapies seem to perform better than LPN/r alone [6] [7] [8] [9] . Further studies with a better study design on larger populations are urgently needed to establish if LPN/r could be an effective therapeutic option for COVID-19 disease and to evaluate the appropriate timing of administration of this drug.

Darunavir is a second-generation protease inhibitor used for HIV therapy. It is associated with cobicistat, which increases its plasma concentration by inhibiting cytochrome P450 3A4 isoform. DAR/COB is an alternative to treatment with LPV/r when the latter is not tolerated because of intestinal side effects [10] . In Table 1 -DAR/COB section, we reported the design of a clinical trial testing DAR/COB in China.

Remdesivir is an adenosine analogue, which is included in nascent viral RNA chains resulting in premature termination. Formerly evaluated for the treatment of Ebola Virus infection, Remdesivir is a promising broad-spectrum antiviral drug active against a wide range of RNA viruses, including SARS-CoV and MERS-CoV in cultured cells, mice and non-human primate models. The drug seems to reduce viral load in lung tissue in SARS-CoV pneumonia in mice, leading to improvement in ventilatory function and healing of the damaged tissue [11] . It is worthy to acknowledge, however, that when the drug was administered after peak viral replication with airway epithelium damage already occurred, it did not improve survival and healing. This indicates that Remdesivir should be administered at the early stages of the disease [12] . Accordingly, Remdesivir reduced clinical signs, pulmonary lesions and viral replication if administered early after infection in a non-human primate model infected by SARS-CoV2 [11] . Since SARS-CoV and SARS-CoV2 RNA-dependent RNA polymerase (RdRp) share 96% sequence identity, it could be hypothesized that drugs targeting viral RdRp proteins of SARS-CoV can be effective on SARS-CoV2 too [13] . Indeed, Wang et al. demonstrated that, compared to other antiviral drugs, Remdesivir contrasts and controls SARS-CoV2 infection in vitro at lower micromolar concentrations with a very high selectivity index [14] . The Food and Drug Administration has authorized Remdesivir compassionate use for the treatment of adults and children with severe COVID-19 disease who do not respond to other treatments [15] . Holshue was first in reporting the case of a young man with COVID-19 disease, in which the treatment with Remdesivir started 7 days after symptoms onset, was effective in reducing radiological involvement and improving symptoms [16] . A case series of 61 patients treated with Remdesivir seemed to show drug effectiveness by improving oxygen support class and symptoms even when administered later after symptoms onset (9-12 days) in severe disease [17] . However, when tested in larger populations, the results seemed to be more conflicting.

In a randomized double-blind trial no differences were noted in 28 days mortality and clinical improvement in patients treated with Remdesivir compared with subjects treated with placebo; it should be acknowledged that this study was performed on patients with advanced disease and didn't reach its enrolment target [13] . Conversely, a larger study on a population of 1059 hospitalized COVID-19 patients demonstrated that Remdesivir started at an advanced disease stage was superior compared to standard therapy in time to recovery and mortality; however, albeit statistically significant, the results of Remdesivir were only slightly better than placebo. Notably, since at the time of article submission a large amount of patients was still hospitalized, the data of this study were incomplete [17] . Finally, a study that compared 5-day versus 10-day Remdesivir administration failed to demonstrate better clinical outcomes depending on Remdesivir therapy duration; moreover, a higher burden of side effects was observed among patients in the 10-day therapy group [18] . These studies, taken together, seem to suggest that Remdesivir is the most effective antiviral drug available so far; for these reasons the Italian drug regulatory agency (AIFA) authorized Remdesivir administration in adult patients affected by severe COVID-19 disease. Since all the studies testing Remdesivir in COVID-19 disease up to now are based on patients with severe disease in an advanced stage, it would be useful to evaluate the drug activity on moderate COVID-19 patients, administering the molecule soon after symptoms onset to observe if, in these categories of subjects, better results and an higher efficacy could be reached. The main studies are reported in Table 1 -Remdesivir section.

Favipiravir is a new generation RdRp inhibitor active against a wide range of viruses. Its use, which is hampered by significant side effects (teratogenicity and suicide induction), is authorized in Japan, but not in Europe and USA, for influenza treatment when other antivirals are not effective [19] . Several studies with Favipiravir among COVID-19 patients have been authorized by regulatory agencies, many of which are still ongoing [20] . The available clinical trials are based on small populations, are not randomized and not doubleblinded. Moreover, groups features were heterogeneous, making it hard to draw conclusions on its effect on COVID-19 disease. In the two studies reported in Table 1 -Favipiravir section, it seemed slightly more effective than LPN/r and Umifenovir in improving imaging, viral clearance and clinical signs [19, 21] .

Umifenovir is a small indole-derived molecule developed in Russia and approved in Russia and China for prophylaxis and treatment of influenza and other respiratory viral infections. It is able to block viral fusion with the cell membrane in Influenza A and B viruses. Umifenovir and its derivate Umifenovir mesylate had been reported to have antiviral activity against SARS-CoV in cell cultures [22] . A retrospective study based on a small population of patients affected by moderate and severe COVID-19 disease failed to observe significant differences in viral clearance and symptoms resolution between Umifenovir and standard therapy [23] . The limited evidence regarding the use of this drug on COVID-19 patients makes it impossible to draw conclusions.

The main immunomodulatory drugs are reported below and the major studies on these molecules are detailed in Table 2 .

CQ and HCQ are used as antimalarial drugs. However, they have a very broad spectrum of action being effective against bacteria, fungi, protozoa and viruses. HCQ safety profile is better than that of CQ, therefore the former should be preferred as it gives the possibility to maintain higher doses for a longer time. CQ and HCQ are effective in vitro against diverse RNA and DNA viruses and inhibit in vitro replication of SARS-CoV229E in lung epithelial cells. CQ and HCQ have multiple mechanisms of action. Firstly, they are able to interfere with cell surface receptors. Specifically, HCQ inhibits sialic acid synthesis. Sialic acid is a monosaccharide located at the end of the sugar chains bound to transmembrane signaling proteins and is a critical component for ligand recognition. Avoiding this fundamental path, the drug interferes with the virus entry. Moreover, these drugs affect cell receptor glycosylation. In SARS-CoV in vitro studies, HCQ glycosylates ACE-2 receptor resulting in failure of recognition, binding and entry of the virus in the cell. HCQ also alkalizes the lysosomal pH. To facilitate the fusion of the virion with the cell, the lysosomal pH should be acid; an alkaline pH inhibits this path. Finally, HCQ can increase the intracellular pH and inhibit lysosomal activity in antigenpresenting cells preventing antigen processing and major histocompatibility complex (MHC) class II autoantigen presentation to T cells. As a consequence, the drug reduces T cell's activation, differentiation and expression of co-stimulatory proteins e.g. CD154 on CD4 + T cells and cytokines production from T and B cells (IL-1, IL-6 and TNF). Even the production of type I Interferons is attenuated [24] . Several studies have been performed to assess the efficacy of these molecules on COVID-19 patients and many other studies are still ongoing (see Table 2 -CQ/HCQ section). Notably, CQ and HCQ have been included in the Guidelines for the Prevention, Diagnosis, and Treatment of Pneumonia Caused by SARS-CoV2 in China [25] . However, the results of CQ and HCQ for COVID-19 treatment are contradictory; some studies showed a clinically significant improvement in patients treated with CQ or HCQ with respect to untreated patients, while others did not show differences between the groups [25] [26] [27] [28] [29] [30] [31] [32] [33] . A group of French investigators tested the efficacy of HCQ in association with Azithromycin in mild-moderate COVID-19 patients soon after symptoms onset, in two different studies, showing a clear effectiveness of the above-cited molecules compared with standard therapy [26, 27] . The first study was severely criticized due to design faults and incomplete data [26] ; the second one, even if followed by a detailed analysis, still had a small sample size [27] . A larger French clinical trial on a population of 1061 COVID-19 patients confirmed clinical improvement and faster viral clearance in patients treated with HCQ and Azithromycin. It should be highlighted that this study only enrolled patients with mild/moderate disease or asymptomatic subjects with a positive swab; moreover, treatment was started very early after symptoms onset [28] . After these studies, many others showed no clear benefits of HCQ and CQ therapy in COVID-19 patients. Some trials on small populations of severe hospitalized COVID-19 patients even showed an increased mortality in HCQ and CQ treated patients compared to untreated groups [29] [30] [31] [32] [33] . However, it is very difficult to draw definite conclusions since study design and heterogeneous populations were some of the many drawbacks of these studies. Recently, a multinational registry analysis on 96,032 severe/critical hospitalized COVID-19 patients was very welcomed by the scientific community since it was thought that a study on such a large population could be diriment and conclusive in understanding the real effectiveness of these two drugs. This analysis not only showed that HCQ and CQ alone and associated with macrolides were not effective in COVID-19 disease, but also that they determined an increased rate of cardiac side effects and an increased mortality rate, depicting an unsettling scenario on these drugs related to toxicity and danger [33] . The publication of this analysis was rapidly followed by the discontinuation of ongoing trials and by the prohibition of HCQ and CQ administration in COVID-19 disease patients by national regulatory agencies and WHO [34] . In a little time, many criticisms were raised against this trial, due to conflicting and missing data and doubts about veracity and accuracy of data collection; as a result, an investigation has been initiated and the study has been withdrawn [35] . Up to now, little can be affirmed with certainty from studies on HCQ and CQ; nevertheless, it seems that these molecules could be effective if administered early after symptoms onset. Further studies with a strong design and a large population need to be performed to understand the real effectiveness of these drugs in early disease stages. Recently, a clinical trial on HCQ used as prophylaxis of COVID-19 disease did not show differences between HCQ and placebo groups in preventing COVID-19 disease after a high-risk exposure [36] . Further studies are needed to understand if HCQ and CQ could be useful as pre-exposure prophylaxis in subjects at high risk (hospital staff, partners of COVID-19 positive patients, etc.).

Corticosteroids were largely used during SARS-CoV and MERS-CoV epidemics. During SARS-CoV epidemic in 2003, systemic glucocorticoids, even at a very high dosage (> 500 mg of methylprednisolone (MTP)/day), were widely used in infected patients who developed severe respiratory disease. A cohort study during SARS-CoV outbreak showed that the use of pulsed high-dose MTP was associated with clinical improvement; however, a previous retrospective study found that the use of pulsed steroids in severe SARS-CoV pneumonia patients was associated with higher 30-day mortality. A systematic review and meta-analysis on the use of corticosteroids in hospitalized patients with communityacquired pneumonia observed a reduction in mortality and need for mechanical ventilation in treated patients. However, a study on MERS-CoV pneumonia stated that the use of steroids was associated with a delayed viral clearance [37] . Against this background, WHO and the Center for Disease Control and prevention (CDC) did not recommend the administration of steroids in COVID-19 disease, at least not in the first disease phases [38] . A Chinese consensus conference proposed to use a low-medium steroid dosage (0.5-1 mg/kg/day of MTP) for 7 days, in patients with acute inflammatory response and dyspnoea [37] . Another Chinese group recommended MTP dosage of 40-80 mg/day (to be gradually reduced) in patients with severe COVID-19 pneumonia and 80-160 mg/day (to be gradually reduced) in critical patients [39] . On the one hand, steroids have an immunosuppressive activity that determines decreased viral clearance velocity, but on the other hand they show a potent anti-inflammatory activity. The latter action is now thought to be very useful in COVID-19 since the immunesystem inflammatory response and cytokine storm drive the intermediate and advanced phases of the disease [40] . Pathological findings in COVID-19 pneumonia lungs consisted of pulmonary oedema with proteinaceous exudate and hyaline membrane formation, implying that the use of corticosteroids could be useful in severe patients [39] . Since trials on the use of corticosteroids among patients with respiratory complications from other infections yielded conflicting results, it is necessary to wait for the results of new ad hoc clinical trials to draw some conclusions on the use of systemic glucocorticoids for the treatment of COVID-19 disease (see Table 2 -Corticosteroids section). Up to now, studies on the use of corticosteroids in COVID-19 disease were all conducted on very small populations of severe or critical patients and showed faster time in fever resolution, faster improvement in imaging and oxygen saturation and a better survival rate in ARDS patients treated with steroids compared to untreated patients without a delay in viral clearance [41] [42] [43] . In a very recent communication, Oxford University disclosed the preliminary results of the RECOV-ERY trial on the use of dexamethasone in severe and moderate COVID-19 disease. The study was conducted on a large population and showed the efficacy of dexamethasone in reducing deaths especially among ventilated patients and among those receiving oxygen supplementation; no benefit was found among patients not requiring respiratory support [44] . The results of this trial emphasize the importance of steroids as a potent weapon to be used without fear in intermediate and advanced COVID-19 disease phases. More studies are certainly needed to understand which molecule is more suitable, the dosage and the timing that may contribute to achieve the best results.

TCZ is a humanized monoclonal antibody against IL-6 receptor approved in Europe for the treatment of rheumatoid arthritis and Takayasu arteritis. It is also effectively used in Chimeric Antigen Receptor (CAR)-T-cell cytokine release syndrome. TCZ acts as a cytokine storm blocker, reducing the systemic inflammatory response and macrophage activation. TCZ has been considered as one of the most promising drugs for COVID-19 disease based on the results of Chinese case reports showing a fast improvement in oxygen saturation, imaging and clinical conditions shortly after the molecule administration [45] . Nevertheless, results from Italian studies are more controversial. The partial results of a first Italian study seem to disprove this efficacy rate showing no differences in ICU admission and 7 days mortality rate among treated and untreated groups [46] . A recent press release of AIFA on the results of an Italian multicentre randomized clinical trial on the early administration of TCZ in non-severe COVID-19 disease patients reported that the study was interrupted due to futility since no differences in ICU admission, respiratory failure and mortality were seen among the treated and untreated patients [47] . Conversely, a retrospective observational Italian cohort study suggested that TCZ administration in severe COVID-19 disease resulted in a significantly reduced risk of death and mechanical ventilation [48] . These studies demonstrated that TCZ is probably not useful in preventing severe complications in patients with non-severe COVID-19 disease but may have beneficial effects in subjects with severe COVID-19 disease [47, 48] . Further randomized studies are needed to assess whether the drug could be useful in specific categories of patients. The main studies on the use of TCZ in COVID-19 disease are reported in Table 2 -TCZ section.

Sarilumab is another IL-6 receptor antagonist. Several trials evaluating Sarilumab as a potential therapeutic option for COVID-19 treatment are ongoing [49] . Of note, AIFA authorized a clinical trial to evaluate the efficacy and safety of Sarilumab in severe/advanced disease (see Table 2 -Sarilumab section).

Anakinra is a recombinant antagonist of IL-1 receptor. IL-1 is another important cytokine involved in cytokine storm. Some studies have shown that MERS-CoV virus causes pyroptosis, a highly inflammatory form of programmed cell death, with a massive release of IL-1β. Since it seems that the same phenomenon happens in SARS-CoV2 infection, blocking this cytokine could be useful. Some case reports documented the efficacy of this drug in moderate/severe COVID-19 pneumonia [50] . Moreover, a retrospective, nonrandomized, small sample size cohort study on a population of severe COVID-19 patients with ARDS, also showed Anakinra's efficacy in improving respiratory function and in reducing 21-day mortality [51] . Considering the encouraging results of these preliminary studies together with the drug mechanism of action and its remarkable safety profile, anakinra could theoretically be extremely useful in fighting COVID-19 disease. As such, many clinical trials testing this molecule are now ongoing (see Table 2 -Anakinra section).

A group of approved drugs that can inhibit clathrin-mediated endocytosis and consequently virus entry into target cells was discovered through artificial intelligence. The selected drugs are inhibitors of members of the Numb-Associated-Kinase (NAK) family, including AP2 associated kinase 1 (AAK1) and cyclin G associated kinase (GAK), the inhibition of which has been shown to reduce viral infection in vitro. Baricitinib was identified as a NAK inhibitor, in particular, it showed high affinity for AAK. Also Fedratinib and Ruxolitinib have been found as potential therapeutic weapons belonging to this class. All these three drugs are potent Janus Kinase (JAK)-inhibitors approved for rheumatoid arthritis and myelofibrosis treatment and have powerful anti-inflammatory properties. A dosage study showed that the best option could be Baricitinib, due to the once a day oral administration, the acceptable safety profile and the double mechanism of action targeting JAK inhibition and clathrin-mediated endocytosis (i.e. inflammation and virus entry) at the tolerated dosage. Conversely, Fedratinib and Ruxolitinib are able to inhibit JAK but not the virus endocytosis at tolerated dosage. In addition, Baricitinib could be used in combination with other therapies due to its low plasma protein binding and minimal cytochrome interactions. However, there are also some concerns about Baricitinib use because the simultaneous inhibition of AAK and JAK can reduce Interferon-α levels determining a worsening of immune response with subsequent clinical deterioration [52] . For this reason, other authors suggested Fedratinib as the best drug option among this group because of its selective inhibition of JAK2. Fedratinib decreases IL-17 and IL-22 expression by T helper (Th)17 lymphocytes, suppresses GM-CSF function, but it does not compromise IL-21 mediated B cells function. As a consequence, JAK inhibitors would reduce the cytokine storm via multiple mechanisms [53] . More laboratory and clinical data are needed to clarify the use of these therapeutic options in COVID-19 disease. 

Colchicine is an old drug used in auto-inflammatory disorders and in gout. It counteracts the assembly of the NLRP3 inflammasome (the leading component in the development of ARDS), thereby reducing the release of IL-1b and an array of other interleukins, including IL-6, that are produced in response to danger signals. Colchicine also inhibits microtubule dynamics by binding to unpolymerized tubulin heterodimers. As such, it seems that colchicine could directly inhibit the virus entry phase in human cells due to interference with clathrin-mediated endocytosis via microtubules inhibition [54] . Indeed, in mice studies colchicine showed efficacy in inhibition of respiratory syncytial virus (RSV) replication and suppression of RSV-induced airway inflammation [55] . Moreover, colchicine was thought to be potentially useful for its double action on the heart and pericardium and on lungs due to a decrease in cytokine levels and interstitial cells activation [56] . Many trials in the early use of colchicine for COVID-19 are ongoing all over the world. A brief overview can be seen in Table 2 -Colchicine section.

Interferons are a group of cytokines used for the communication between cells to trigger the defense mechanisms against pathogens. In particular, they have a critical role in the innate immune response, such as the activation of natural killer lymphocytes and macrophages. They also have several antiviral activities, including the induction of viral degradation, the alteration of RNA transcription and protein synthesis, and the promotion of cellular apoptosis. Interferons family is composed of type I, II and III interferons. As a member of type I interferons, Interferon-α is quickly produced during viral infections as part of the innate immune response. Its action leads to the inhibition of viral replication even in Coronaviruses infecting humans and animals. Interferon-β inhibits SARS-CoV virus replication in vitro while γ-Interferon doesn't. Interferon-α, and to a lesser extent Interferon β-1, were widely used in SARS-CoV and MERS-CoV epidemics, due to promising results demonstrated by in vitro and in animal studies. SARS-CoV virus produces proteins able to inhibit type I Interferon release causing a delayed production of type I Interferons and a delayed immune response. The rise in serum levels of type I Interferons at the advanced disease stage contributes to the development of cytokine storm with systemic hyperinflammation and accumulation of macrophages and monocytes in lung tissue. It seems that this mechanism also occurs in COVID-19 disease. In particular, SARS-CoV2 virus antagonizes STAT1, a key protein in the Interferon-mediated immune response. The importance of T cell-mediated immune response in respiratory Coronaviruses is well established. Type I Interferon response is crucial in T cell survival and T cells reduce cytokine storm by modulating the innate immune response. A combination of type I Interferon and γ-Interferon or λ-Interferon was shown to synergistically inhibit the virus replication in vitro [57] . Combining interferons gives the opportunity of lowering the dosage of each one to decrease side effects. The timing of Interferon administration is crucial. Type I Interferons effects are protective against virus infections in the first phases; in advanced disease high titers of α and β-Interferons are associated with worst outcomes because they sustain inflammatory response [58] . Up to date, evidence on the use of Interferons in COVID-19 pneumonia is lacking. Chinese national health service recommends α-Interferon 5 million IU twice a day nebulization as a coadjutant treatment option in COVID-19 infection; however, nebulization is a risky procedure due to the aerosol spread of the virus in the environment [59] . Some studies about the administration of Interferons in COVID-19 are reported in Table 2 -Interferons section. These studies suggest better results in groups of COVID-19 patients treated with the association of antiviral drugs and Interferons (nebulized or sub-cutaneous administration) or Interferon alone compared to antiviral drugs alone [9, 60, 61] . However, up to date, well-designed studies on large populations are lacking.

Emapalumab is a human monoclonal antibody against Interferon-γ. It is used in the treatment of refractory primary hemophagocytic lymphohistiocytosis (HLH) [62]. The cytokine storm developed during advanced hyperinflammatory COVID-19 disease phase may resemble HLH [40] . Moreover, Interferon-γ levels are associated with an amplification of inflammation and generalized activation of immune response with subsequent hyper-inflammatory state and damage [57] . On this basis, researchers decided to begin clinical trials with Emapalumab as a potential therapeutic option for COVID-19 disease (see Table 2 -Emapalumab section).

In this section diverse drugs belonging to different classes with different mechanisms of action which may have potential therapeutic implications for COVID-19 disease are treated. The main studies on these molecules are reported in Table 3 . 

IVIGs are human immunoglobulin preparations derived from plasma, indicated for the treatment of diverse diseases such as autoimmune, inflammatory disorders and various immunodeficiencies [63] . During the SARS-CoV epidemic, many observational studies and case reports described IVIGs use in combination with anti-viral drugs for the treatment of critically ill patients. In a clinical review of different treatment protocols for SARS-CoV, the use of IVIGs combined with Interferon was described as ineffective [60] . Since the beginning of SARS-CoV2 infection in Wuhan, clinicians have used IVIGs in patients affected by COVID-19 pneumonia. Some authors, on the basis of the experience acquired with SARS-CoV pneumonia, proposed the use of IVIGs at the dosage of 0.3-0.5 g/kg/day for 5 days in the treatment of COVID-19 pneumonia. According to the authors' opinion, this treatment should be started as soon as possible in patients who present these features: leukopenia and lymphopenia (< 1000/ μL), D-dimer elevation above 4 times the upper limit of normal value (ULN) and cytokine increased levels (in particular IL-6). The rationale behind this approach is to try to reduce the cytokine storm developed in the most rapidly evolving patients [1] . Only a few case reports are available on the use of IVIGs in COVID-19 disease patients [64] . Because of the lack of evidence on IVIGs treatment in COVID-19 disease, physicians have proposed a number of trials to assess the efficacy of IVIGs compared to the standard care in severe patients (ClinicalTrials. gov Identifier: NCT04381858) (see Table 3 -Intravenous immunoglobulins section).

Evidence shows that hyperimmune (or convalescent) plasma from patients who have recovered from various viral infections can be useful for the disease treatment without particular warnings. Hyperimmune (or convalescent) plasma has been already used as a last attempt in patients with critical SARS-CoV pneumonia not responding to maximal treatment [65] . Different studies on SARS-CoV pneumonia showed that hyperimmune plasma was effective in reducing hospitalization and mortality. A meta-analysis of 32 studies on SARS-CoV infection and severe influenza showed a statistically significant reduction in the pooled odds of mortality following convalescent plasma therapy compared with placebo or no therapy (OR 0.25; 95% CI 0.14-0.45) [66] . A protocol to encode the use of hyperimmune plasma also in patients affected by MERS-CoV pneumonia was established in 2015. Up to now, there are several case reports of COVID-19 patients treated with hyperimmune plasma that suggested a beneficial effect probably mediated by its antiviral activity.

These case reports showed that COVID-19 patients treated with convalescent plasma had large reductions in serum viral loads and most were virus negative 3 days after infusion [66] [67] [68] . Recently the results of a Chinese randomized clinical trial have been published. This work, based on a population of 103 severely-ill or with life-threatening COVID-19 disease subjects, did not show statistically significant benefits of convalescent plasma compared to standard therapy in 28 days clinical improvement, mortality and time to discharge. In a subgroup analysis, severely-ill but not critical patients did show a faster clinical improvement when treated with convalescent plasma (p = 0.03). Of note, the study was underpowered, not reaching its target sample size of 200 patients, because enrolment was terminated prematurely due to control of infection spreading in China. It is important to highlight that conventionally convalescent plasma has the maximum efficacy in early viraemic stages of disease; whereas, in this study, it was administered very late after symptoms onset (median of 30 days) [69] . For these reasons, it would be useful to test hyperimmune plasma efficacy in early stages of COVID-19 disease to understand if a timelier administration could be associated with better outcomes. See Table 2 -Hyperimmune plasma section.

The International Society of Thrombosis and Haemostasis (ISTH), based on the current literature, recommends measuring D-Dimer, prothrombin time and platelet count in all patients with COVID-19 disease. This strategy may help clinicians in stratifying patients who may need admission and close monitoring or not [70] . An adjunctive parameter to be considered is the serum fibrinogen, useful for the diagnosis of DIC, a condition highly prevalent in COVID-19 patients who did not survive the infection [7] . Due to the strong association between coagulopathy and mortality in COVID-19, the inhibition of thrombin generation may be beneficial. Moreover, it has been shown that heparin displays an anti-inflammatory action and various immunomodulatory properties. As such, prophylaxis dose LMWH has been proposed in all patients who require hospital admission for COVID-19 disease, in the absence of any contraindications [70] . The effectiveness of this approach is attested by Doctor Ning Tang [71] . An Italian group of ICU physicians highlighted the fact that since severe/critical COVID-19 disease is associated with an increased rate of DIC and VTE, augmented LMWH dosing could be useful in reducing the coagulopathy risk in mechanically ventilated patients.

A study conducted in a northern Italy ICU department showed no increased mortality and no major bleeding after anticoagulant therapy augmentation (anticoagulant dosage of LMWH and UFH) indicating the need for further studies comparing the different doses of heparin and outcomes with a greater interest in mortality and adverse events [72] . The main studies on this topic can be found in Table 3 -Low molecular weight heparin (LMWH) and unfractioned heparin (UFH) section [3, 72, 73] .

Azithromycin is an antibiotic belonging to the macrolide class. It inhibits bacterial synthesis but also shows in vitro activity against viruses such as Influenza A H1N1 (interference with internalization process) and Zika (upregulation of virus-induced Interferons type I and III). Moreover, azithromycin anti-inflammatory effect on lung tissue, even during viral infections, is well known. It acts suppressing T-helper 1 and 2 lymphocyte-related cytokines (IL-1, IL-6, TNFα) and INF inducible protein 10 (IP-10)/macrophage derived chemokine (MDC) in monocytic cell line. Its use has been associated with decreased major respiratory complications during viral respiratory infections in clinical studies [74] .

Doxycycline is an antibiotic belonging to the tetracycline class. It interferes with bacterial protein synthesis and demonstrated in vitro activity against viral infections (i.e. Influenza and Dengue). Moreover, doxycycline presents documented anti-inflammatory effects by acting on Nf-Kb pathway and by inhibiting the production of pro-inflammatory cytokines (IL-1b, IL-6 and TNFα). An experimental study on mice showed that the use of doxycycline during severe influenza pneumonia was associated with a better outcome with less inflammatory lung lesions and ARDS prevention [75] . Another study suggested that the administration of doxycycline in patients with Dengue haemorrhagic fever reduced cytokines (IL-6 and TNFα) blood levels, mortality and discharge time [76] . Recent studies suggested that coronaviruses induce the proliferation of mast cells within the respiratory submucosa and that chemically-modified tetracyclines can induce apoptosis of mast cells and activation of protein-kinase C, thus decreasing levels of circulating inflammatory mediators [77] . For these reasons, some studies are testing doxycycline efficacy in COVID-19 pneumonia.

ACE2 is a monocarboxypeptidase that hydrolyses multiple peptides, including angiotensin; in particular, the enzyme forms angiotensin-(1-7) by cleaving angiotensin II. The former has a vasodilator, anti-inflammatory and anti-fibrotic effect, while the latter determines vasoconstriction and enhances inflammation. Since ARB and ACE inhibitors determine an increase in ACE2 expression, at the beginning of COVID-19 pandemic, it was thought that they could be harmful by facilitating SARS-CoV2 attachment and entry. However, it has been subsequently demonstrated that SARS-CoV2, once attached to ACE2, determines a down-regulation in its expression in the lungs and as a consequence a decrease in angiotensin-(1-7) and an increase in angiotensin II with possible acute lung injury and enhanced global inflammation. As such, it is now thought that these two drug classes may be beneficial in controlling lung damage and inflammation. ARB and ACE-inhibitors may act as a double-edged sword: on the one hand, these two drugs could enhance infectivity by inducing ACE2 over-expression; on the other hand, the subjects treated with ARB or ACE-inhibitors, once infected, exhibit a more balanced equilibrium between angiotensin II and angiotensin-(1-7) which may prevent systemic and lung inflammation and damage [78] . Some recent studies suggested that patients chronically treated with ARB and ACEi not only are not at increased risk of SARS-CoV2 infection or worse COVID-19 outcomes but even may show a less severe disease course. Another possible therapeutic approach targeting this pathway consists in soluble recombinant ACE2 intravenous infusion; this strategy determines a reduction in viral load since the virus could bind the "false" receptor [79] .

TMPRSS2 is a serine protease located on the epithelial cell surface of specific tissues including the respiratory tract. This protease cleaves SARS-CoV2 spike protein and activates the virus internalization. Researchers have tried to find ways to reduce TMPRSS2 expression or to block its activity as a potential therapeutic option for COVID-19 disease. Two classes of drugs have been thought to be used so far: anti-androgens or oestrogens to reduce the TMPRSS2 expression, and Camostat Mesilate, a potent serine-protease inhibitor. In vitro studies have shown a potent inhibitory effect of Camostat Mesilate on SARS-CoV2 entry ability [80] . Five clinical studies to test Camostat Mesilate efficacy in COVID-19 disease are now ongoing.

In this review, we described the main drugs tested until now or with a putative therapeutic role against SARS-CoV2 infection; in particular, we detailed the possible mechanisms of action of the selected molecules in COVID-19 disease and, when available, we described the results of the main clinical studies in COVID-19 patients. An important issue that is worth to highlight is the emergency setting in which these studies have been conducted so far. As such, the vast majority of the studies that have been analysed suffer from several methodological limitations, notably including a lack of randomized, double-blind design, a lack of control populations, small or underpowered sample size, and even a lack of peer review evaluations before publication. Moreover, in many cases, the compared groups of patients are heterogeneous in clinical features, disease severity and concurrent pharmacological treatment. Finally, the reported outcomes include a plethora of virologic, biochemical, radiological and clinical aspects that have poor consistency across studies and are limited in time of follow-up. Taking all these considerations in mind, it is difficult to draw some valid conclusions on drugs efficacy in COVID-19 disease treatment.

For what concerns antiviral drugs, the available pathophysiological knowledge on SARS-CoV2 infection and the clinical studies performed so far suggest that the timing of administration of this class of molecules is important. Indeed, they can be effective if dispensed in the first viraemic stage of infection, early after symptoms onset. Among these drugs, the most effective seems to be Remdesivir, even if further large, randomized, double-blind studies are eagerly awaited.

Among immunomodulatory drugs, CQ and HCQ are two controversial molecules. From early studies conducted in France it seems they can be effective if administered very early, at disease presentation. However, their use in hospitalized critical/severe COVID-19 patients do not seem to be associated with improved outcomes and there are concerns about their cardiotoxicity. Importance must be attributed to immunomodulatory drugs such as corticosteroids (in particular dexamethasone) and biological molecules (Tocilizumab, Anakinra and others) which seem to be valid and useful therapeutic options in the inflammatory phase of the disease. However, further studies are necessary to better understand if they can also be active in the early phases of the disease.

Among other classes of drugs, LMWH and UFH may also have a major therapeutic role when facing COVID-19-related coagulopathy, but future studies have to establish the correct dosing, timing and length of treatment.

In conclusion, COVID-19 is a novel, life-threatening disease, that does not have a cure able to arrest its progression yet. Up to now, treatment choices have been inferred from the experience with other coronaviruses or viral infection outbreaks. New treatment strategies will hopefully be available in the near future in parallel with the increased knowledge of SARS-CoV2 virus and COVID-19 pathogenesis. In the meantime, further well-designed clinical trials on homogeneous populations are urgently needed to establish a standard of care in COVID-19 disease.

Author contributions AB retrieved and analysed the literature and wrote the main draft of the paper; FG and TA revised the paper; FN retrieved and analysed the literature and revised the paper; PA conceived the study and was in charge of overall direction and planning. All the Authors approved the final version of the manuscript.

Funding Nothing to declare concerning this study.

Conflict of interest The authors declare that they have no conflict of interest.

Informed consent Not applicable.

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