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Pharmaco-immunomodulatory interventions for averting cytokine storm-linked disease severity in SARS-CoV-2 infection

The year 2020 is characterised by the COVID-19 pandemic that has quelled more than half a million lives in recent months. We are still coping with the negative repercussions of COVID-19 pandemic in 2021, in which the 2nd wave in India resulted in a high fatality rate. Regardless of emergency vaccine approvals and subsequent meteoric global vaccination drives in some countries, hospitalisations for COVID-19 will continue to occur due to the propensity of mutation in SARS-CoV-2 virus. The immune response plays a vital role in the control and resolution of infectious diseases. However, an impaired immune response is responsible for the severity of the respiratory distress in many diseases. The severe COVID-19 infection persuaded cytokine storm that has been linked with acute respiratory distress syndrome (ARDS), culminates into vital organ failures and eventual death. Thus, safe and effective therapeutics to treat hospitalised patients remains a significant unmet clinical need. In that state, any clue of possible treatments, which save patients life, can be treasured for this time point. Many cohorts and clinical trial studies demonstrated that timely administration of immunomodulatory drugs on severe COVID-19 patients may mitigate the disease severity, hospital stay and mortality. This article addresses the severity and risk factors of hypercytokinemia in COVID-19 patients, with special emphasis on prospective immunomodulatory therapies.


Introduction
In March 2020, the World Health Organisation (WHO) declared the COVID-19 outbreak a global public health emergency. Consequently, COVID-19 waves further deteriorated situation causing persistent state of fear. This catastrophic situation created immediate need of therapeutics or vaccines resulting in emergency approval for prophylaxis and treatment of COVID-19. However, emergence of SARS-CoV-2 variants prompted concerns about the efficacy 1 3 and sensitivity of approved medicines and vaccines, which are, regrettably, still hazy. In March-April, 2021, India faced a massive surge of COVID-19 cases and deaths, and was the world's leading country in terms of infection rate. Since inception of pandemic, many suggestions and speculations were made to spot possible cure for this disease. Various drug entities showed promising clinical results, but only a small percentage of these are approved and moved forward to phase trials (Rohilla 2020;Kumar et al., 2021). Majority of these drugs were targeted to counteract inflammatory aggravation during pathologic progression of COVID-19. Hypercytokinemia is an uncontrolled hyper-inflammatory reaction that occurs when a localised inflammatory response to a viral or bacterial infection spreads across the body. Endothelial dysfunction, vascular damage, and paracrine/metabolic dysregulation are all caused by dysregulated high cytokine levels, which eventually harm many vital organ systems (Leisman et al., 2020;Ye et al., 2020;Bhaskar et al., 2020;Fodor et al., 2021). The innate immune response is the first host anti-viral response against invading viruses, which triggers the release of type I/III interferon (IFNs), pro-inflammatory cytokines like TNF-α and interleukins like IL-1, IL-6, and IL-18. These cytokines and interleukins induce collective effect on the target cells and potentiate the adaptive immune response. However, dysregulated immune response is one of the cogent reasons of mortality associated with COVID-19, known as cytokines explosion or storm, which is represented by excessive secretion of cytokines such as, IL-2, IL-6, IL-7, IL-10, IP-10 (interferon-gamma-inducible protein), MCP-1 (monocyte chemoattractant protein), TNF-α and MIP-1α (macrophage-inflammatory protein), GM-CSF in human body and such patients are in strict need of ICUs (intensive care units) (Costela-Ruiz et al., 2020;Ragab et al., 2020;Khadke et al., 2020). Despite many critics raising concern about the excessive immune response or cytokine storm during the pathogenesis of COVID-19, there are several studies highlighting that lymphopenia and pneumonia are the critical immune dysregulation developed during the severe cases of COVID-19, which characterises the higher plasma levels of pro-inflammatory cytokines (Bhaskar et al., 2020;Ye et al., 2020;Fara et al., 2020). Two different autopsy reports demonstrated that the damage to vital organs was not associated with viral inclusion of the respective organs. It could be linked to severe immune injury, caused by cytokine storm, instead of direct viral damage (Xu et al., 2020a, b;Yao et al., 2021). In severe cases, cytokine storms harm healthy cells, first the lungs, and potentially spreading to other vital organs like the heart, brain, and kidney. Some studies highlighted that increased amount of pro-inflammatory cytokines in serum (e.g. IL-1, IL-6, IL-12, IFN-, IP-10, MCP-1, MIP-1, TNF-α, and GCSF), was correlated with pulmonary inflammation and acute respiratory distress syndrome in affected patients .
The extent of the severity of the disease is directly proportional to the cytokine storm , which often leads to multi-organ failure and death. Numerous retrospective studies of cytokine blockade such as IL-6, IL1-, JAK, NF-ƙB, GM-CSF inhibitors have shown promising results (Fara et al., 2020;Ragab et al., 2020;Ye et al., 2020). Several clinical trials are underway or recently completed, and the results are eagerly expected. Regardless of speedy vaccination drives and uptake in many countries, hospitalisations with clinical severity of COVID-19 are still being reported. It might be due to the propensity of mutation and virus evasion from protection that occurs through natural immunological selection (Thompson et al., 2021). Timely therapeutic intervention in patients with hyper-inflammation could prevent the illness from progressing to ARDS and eliminate the need for invasive ventilation. To our knowledge, there have not been any updated reviews shedding light on the effectiveness of immunomodulatory therapy to combat COVID-19 severity. This review enlightened the implications and risk factor of hypercytokinemia/cytokine storm for COVID-19 patients, with special emphasis on potential therapeutic strategies used to mitigate COVID-19 severity.

Risk factor associated with COVID-CSS (cytokine storm syndrome)
During a pandemic, COVID-19 disease is diagnosed through testing and most infections are self-limiting or mild forms, which can be recovered through standard operating procedures, but as the disease progresses and develops severe pneumonia, it becomes more difficult for clinicians to operate. Establishing ARDS during disease progression could be one of the leading causes of death. As estimated, 50 and 65% deaths were associated with ARDS and severe COVID-19 during early phase of the pandemic (Bhatraju et al., 2020). The probability of fatality or death with COVID-19 is greatly impacted by age and prior comorbidities. Older patients over 65, as well as those with chronic comorbidities such as cardiovascular disease, diabetes, hypertension, and respiratory illness, are far more vulnerable to severe or fatal disease outcomes. But, it is uncertain to determine the time of a patient's death in COVID-19, which is linked with preexisting health conditions (Streeck et al., 2020;Bartlett et al., 2020). Low arterial oxygen concentrations, alveolar damage, and a dysregulated inflammatory response in the lungs are all symptoms of COVID-19 ARDS (Matthay et al., 2019). An earlier report stated that COVID-19 ARDS was caused by a "cytokine storm," which refers to severely ill COVID-19 patients having significantly higher levels of circulating inflammatory cytokine levels than those with mild disease or healthy controls (Mehta et al., 2020a, b;Blanco-Melo, 2020;Giamarellos-Bourboulis et al., 2020). Hypercytokinemia, better described as a cytokine storm, massive release of many inflammatory cytokines in serum was determined in COVID-CSS (cytokine storm syndrome). Surprisingly, in many patients with severe COVID-19 disease, the serum IL-6 level was markedly elevated at around 100-1000 pg/ ml (Blanco-Melo et al., 2020;Herold et al., 2020;Laing et al., 2020). This magnitude of elevated IL-6 levels in COVID-CSS is comparable to severe CART-cell CRS and higher than that of other hyper-IL-6 syndromes, including multicentric Castleman disease, where elevated IL-6 levels are noted beyond 100 pg/ml (England et al., 2021;van Rhee et al., 2014). In COVID-CSS, the foundation of immune dysregulation was correlated with the common terminal hyperinflammatory pathway that is characterised by markedly elevated IL-6, severe T-cell lymphopenia, and respiratory failure (Laing et al., 2020;Giamarellos-Bourboulis et al., 2020;Lucas et al., 2020;Coomes and Haghbayan, 2020). Among the elevated cytokines, IL-6 is primarily involved in pro-inflammatory activation during cytokine-mediated organ dysfunction and tissue damage (Crayne et al., 2019) and IL-6-directed therapy acts as the cornerstone of cytokine-based therapy Kotch et al., 2019;Chen et al., 2019). Some studies demonstrated that IL-6 > 80 pg/m1 along with high C-reactive protein > 97 mg/l point towards probable respiratory failure and are specific in predicting death (Herold et al., 2020;Laguna-Goya et al., 2020). Stukas et al., 2020 reported IL-6 to be inversely proportional to the ratio of arterial oxygen tension and fraction of inspired oxygen (PaO2/FiO2) and static lung compliance . IL-6 blockade with tocilizumab resulted in partial restoration of these immune defects (Giamarellos-Bourboulis et al., 2020;Mazzoni et al., 2020). In addition to IL-6, IL-12 (p40), MIP-1a, and MIP-1b are critical chemokines, accountable for the recruitment of activated immune cells to the site of infection (Schulz et al., 2016;Coper et al., 2007;Menten et al., 2002). The hematopoietic growth factor, GM-CSF (Granulocyte-macrophage colony-stimulating factor), is also a key immunological modulator. Upon receiving immunological stimuli, it is released by many cell types such as T cells, macrophages, endothelial cells, and fibroblasts, and it can control key cytokines that can drive both innate and adaptive immune responses (Hamilton, 2000). Other elevated inflammatory indicators such as C-reactive protein are substantially linked to IL-6 plasma levels in CSS with COVID-19 . Various studies have demonstrated high levels of high-sensitivity C-reactive protein (Hs-CRP) in the serum levels of severe COVID-19 patients. Persistently high levels of CRP are a sign of ongoing chronic inflammation and it is a key inflammatory indicator linked to increased risks of death and organ damage (Clyne & Olshaker, 1999;Schmidt-Arras & Rose-John, 2016;Sharifpour et al., 2020). Elevated CRP levels have been linked to disease severity and predictions in many viral respiratory infections such as SARS, MERS-CoV, and H1N1 (Ko et al., 2016;Vasileva & Badawi, 2019;Wang et al., 2004). CRP level assessment has recently been one of the major indicators for detecting COVID-19 disease severity. The major complication associated with COVID-19 disease is cytokine storm, and it is a leading cause of multiple organ failure and ARDS. As a result, effectively repressing the cytokine storm is critical for preventing severe COVID-19 consequences and lowering mortality (Fig. 1). Numerous efforts have been made throughout the world to assess the severity of the situation in real time and apply potential therapeutics to save patients' lives. Many immunomodulatory medicines have been used off-label as COVID-19 treatments in hospitalised patients based on preclinical results, but further evidence in the context of clinical practise is needed. Many repurposed drugs have been utilised and suggested for clinical studies, and a few drugs are showing promising outcomes.

Tocilizumab (IL-6 inhibitor)
Tocilizumab is a recombinant humanised monoclonal antibody that has been approved for use in the treatment of cytokine release syndrome. It is particularly effective in inhibiting IL-6 production. The dysregulated host immune response is reported in the severe case of COVID-19, which has a propensity to release excessive amounts of cytokines, resulting in a cytokine storm or pulmonary inflammation (Bhaskar et al. 2020;Ye et al., 2020). Among all the cytokines, IL-6 is predominately produced and can be an ideal target to circumvent COVID-CSS. Based on that, Tocilizumab, a monoclonal antibody inhibitor of IL-6, can be used to ameliorate the intense inflammatory response associated with severe COVID-19, and thus improve clinical outcomes. Various clinical studies have been showing promising results in COVID-19 treatment (Kumar et al., 2021) (Table 1). Xu et al. 2020a, b were the first to test tocilizumab's efficacy on 21 severe or critical COVID-19 patients. Clinical data indicated that symptoms, hypooxygenemia, and CT opacity changes improved quickly after tocilizumab treatment in the majority of patients, indicating that tocilizumab might be an effective COVID-19 treatment. This observation has prompted many clinical trial studies on tocilizumab. A few studies contradicted the efficacy of tocilizumab, and no significant clinical improvements were achieved through the administration of tocilizumab. But these trials have limitations like the inclusion of small numbers of patients, less severely ill patients, and the exclusion of critically ill patients (Stone et al., 2020;Hermine et al., 2021). The study by many research groups suggested that TCZ may lower the frequency of ICU hospitalisations and/or fatalities in patients with severe COVID-19 (Alattar et al., 2020;Commes and Haghbayan, 2020). Gupta et al., 2021, conducted a multicentre cohort study, enrolling 4485 critically ill COVID-19 patients. After 27 days of therapy, individuals who received tocilizumab had a reduced risk of mortality than those who did not receive tocilizumab. The anticipated 30-day mortality was 27.5% in the tocilizumabtreated patients and 37.1% in the control groups (risk difference, 9.6%). In this investigation, patients who received tocilizumab in the first two days of ICU admission had a decreased risk of in-hospital mortality than patients who did not get tocilizumab in the first two days of ICU admission (Gupta et al., 2021). Gordon et al., 2021 conducted a large randomised, embedded, multifactorial adaptive platform trial for community acquired pneumonia (REMAP-CAP) and included a total of 803 patients. Among them, 353 patients were assigned to tocilizumab to examine the efficacy of tocilizumab on survival and organ support in critically ill patients with COVID-19, and the outcome showed a survival benefit over the current standard of care, which included corticosteroids (Gordon et al., 2021). Guillén et al., 2020 investigated the efficacy of tocilizumab in 64 hospitalised COVID-19 patients. After receiving tocilizumab, 49 patients (76.6%) demonstrated an early positive response. There were no deaths or recurrences of illness. There were no more bacterial infections as a result of the treatment during the hospitalisation. However, patients who responded well to TCZ were younger in age (Gullein et al., 2020).

Sarilumab
Sarilumab is completely a human MCA (monoclonal antibody) responsible for inhibiting the binding of IL-6 and its own α receptor. Sarilumab is approved by FDA (Food and drug administration) for its use against the treatment of ankylosing spondylitis and arthritis rheumatoid (severe and moderate) (Khiali et al., 2020). As it inhibits both membrane bound and soluble form of IL-6 and possibly suppresses the pro-inflammatory signalling by immune cells as well as pulmonary-epithelial cells; its use can be possibly predicted in reducing the pulmonary complications raised due to COVID-19, along with respiratory failure (Lescure et al., 2021). Sarilumab suppresses the growth of Calu3 (human lung cancer cell line) and binds with high affinity to epitope of IL-6R, thereby blocks both the activation (trans and cis) of IL-6 signalling (Yousefi et al., 2021). It is considered to be a safe and well-tolerated drug with recommended dosage not exceeding 200 mg every fortnight (Khiali et al., 2020;León López et al., 2020).
Various studies have been conducted by various researchers to check the efficacy and safety of sarilumab in patients suffering from rheumatoid arthritis ( Table 2). A study done by (Bae and Lee, 2017) with four randomised controlled trials with 2667 patients suffering from rheumatoid arthritis   Salvarani et al., 2021 showed that Sarilumab at 150 mg and 200 mg is effective as well as tolerated among the patients. The adverse event rate of Sarilumab is very low, which is around 6.7-9.4 per 100 patients per year (Khiali et al., 2020). In a study conducted by (Benucci et al., 2020) over 8 patients (6 males and 2 females, mean age 62 years) with confirmed positive PCR (polymerase chain reaction) reports, the patients were given sarilumab (400 mg) along with standard therapy of hydroxychloroquinone (400 mg), darunavir (800 mg), enoxaparin (100 U/kg), and azithromycin (500 mg). The study revealed that there was improvement of oxygenation and the oxygen requirement was reduced by 30%. Seven patients showed progressive and significant reduction in echo score and improvement of Horovitz index was observed significantly (Benucci et al., 2020). Within 14 days, 7 of the 8 patients were tested negative and discharged from the hospital, while an 83-year-old patient showed no improvement.
Another study done by (Benucci et al., 2020) on COVIDpositive hospitalised patients (n = 8, t = 14 days), three doses were given viz. first dose (400 mg) after the first day of hospitalisation; second dose (200 mg) after 2 days and third dose (200 mg) after 4 days. Other standard treatments were also carried out alongside such as azithromycin, cobicistat, HCQ, darunavir and enoxaparin. The result showed significant improvement in the count of lymphocytes, CRP and echo score. Gremese et al., 2020 conducted open-label observational study on 53 patients suffering from COVID-19 with severe pneumonia. The patients were given 1-2 doses of sarilumab (400 mg) in 1-11-day intervals along with LPV/r, HCQ, DRV/r, GC and heparin azithromycin. The result showed significant improvement in 89.7% of the patients who were not admitted in ICU. After the treatment, oxygen therapy was not required by 85.7% of the patients, while 64.2% of the patients admitted in ICU no longer require the intensive care and was shifted to normal wards.
There are limited data available addressing the use of sarilumab in the case of COVID-19 and various clinical trials are still in progress. Overall, it can be concluded from the limited published evidences that sarilumab can be used in treatment of COVID-19 (moderate to severe cases). However, further research is still required to explore the efficacy and safety of Sarilumab in treatment of COVID-19 (Khiali et al., 2020). Della-Torre et al., in 2020, conducted an open-label cohort study and check the efficacy of sarilumab in severe COVID-19 pneumonia patients. Fifty-six patients (PaO2/FiO2 < 300 mm Hg with hyper-inflammation) were enrolled and given sarilumab therapy. Twenty-eight patients received 400 mg sarilumab intravenously in addition to standard of care, while rest 28 patients treated with standard of care alone. Sarilumab treatment resulted in clinical improvements in patients and they showed faster recovery. In patients with lung consolidation < 17 percent at CT scan, the

Anakinra
Anakinra, a well-known IL-1 receptor antagonist, is used for the treatment of various hyper-inflammatory conditions like Still's disease, juvenile idiopathic arthritis, familial Mediterranean fever, etc. Anakinra is found to be super effective in treating cytokine storm syndrome including cytokine release syndrome as well as macrophage activation syndrome (Filocamo et al., 2020;Cavalli et al., 2020). It is approved by US-FDA (United States Food and Drug administration) for its use in the treatment of arthritis rheumatoid and multiinflammatory diseases (Navarro et al., 2020). It is known to possess the ability to block the activity of pro-inflammatory cytokines (IL-1β and IL-1α). As previously discussed, patients with COVID-19 disease exhibit hyper-inflammatory symptoms similar to cytokine storm and the release of interferon, IL-18, IL-6 and IL-1β in patients with macrophage activation syndrome. These disorders can be prevented up to great extent using agents that block the cytokines. Anakinra is one such blocking agent that is found to be effective in the treatment of these diseases. The use of anakinra permits the prompt discontinuation due to its short half-life, and thus considered suitable to be used in the case of critical patients.
Also, the anakinra is considered much safer as compared to other cytokine-blocking agents . Higher dosage of anakinra is recommended for patients suffering from heavy load of viral infection as in case of H1N1, Ebola and EBV (Filocamo et al., 2020). Anakinra tends to lower the death rate in patients having sepsis, liver dysfunction and disseminated-intravascular coagulation. Due to its short halflife and its property of inhibiting both the IL-1 subtypes, it could possibly be a promising treatment to cure severe COVID-19. A number of studies have been reported showing the beneficial effects of anakinra during initial treatment in patients suffering from COVID-19 disease (Aomar-Millán et al., 2021; Navarro-Millán et al., 2020) ( Table 3). A study was conducted by (Aomar-Millán et al., 2021) on 143 patients suffering from moderate hyper-inflammation and severe COVID pneumonia with the aim to depict the effect of anakinra on COVID patients. The patients were given standard therapy and were divided into 3 groups. Group 1 received pulses of methyl prednisolone, group 2 received tocilizumab in addition to methyl prednisolone and group 3 received anakinra as per the protocol. The first dose of anakinra was decided as per the weight of the patients. Patients with weight ranging from 50 to 60 kg were given dosage of 100 mg/12 h; 60 to 75 kg were given 100 mg/ 8 h and those above 75 kg were given 100 mg/ 6 h. All patients received a dosage of 100 mg/12 h from the second day onwards till day 6. The study lasted for 60 days. The results indicated that patients from group 3 who were receiving the dose of anakinra were associated with a lower risk of death rate. Also, the mean CD-4 count was reduced in group 3 after 3 days of the treatment. It was observed that none of the patients developed any complications after treatment with anakinra, and it was tolerated well among all the patients.
Another study conducted by (Navarro-Millán et al., 2020), on patients suffering from SARS CoV-2 with high fever and acute-hypoxemic respiratory failure (AHR failure). Out of 14 patients, 11 received anakinra treatment (100 mg every 6 h, subcutaneously) for maximum 19 days. Seven patients with AHR failure did not require mechanical ventilation anymore after anakinra treatment of ≤ 36 h and were discharged from the hospital. While the other 3 patients who did not received the anakinra treatment needed mechanical ventilation. The study suggested that the use of anakinra could prove to be beneficial for treating patients suffering from COVID-19 with cytokine storm syndrome. Various other studies are available and are still in queue to evaluate the efficacy and safety of anakinra in COVID-19 patients. , they have administrated high dose (5 mg/kg, twice daily, intravenously) and observed the positive outcome, which showed improved respiratory function among the patients. No adverse events were observed. Baricitinib Baricitinib (C16H17N7O2S), a reversible JAK inhibitor, i.e. Janus-associated-kinase inhibitor (JAK 1/ JAK 2) is approved in more than 65 countries to treat patients suffering from rheumatoid arthritis (moderate to severe) (Jorgensen et al., 2020). This small molecule not only inhibits the release of cytokines but also reduces the chances of viral entry, and thus seems to be an important drug possessing anti-inflammatory activity (Cantini et al., 2020). Using benevolent algorithms of artificial intelligence and due to the dual effective nature of baricitinib against viral entry and release of cytokines, its use can be predicted in COVID-19 infection. The anti-viral efficacy using baricitinib was demonstrated in liver spheroids of humans and showed a good safety profile when administered to patients suffering from COVID-19. Administration of baricitinib to patients reduced the mortality rate, their chances of admission to the intensive care unit, and a drop in the count of viral load was observed when detected through nasopharyngeal swabs (Petrone et al., 2021;Stebbing et al., 2020). Various adaptive treatment trials of COVID-19 showed that baricitinib when used in combination with remdesivir claimed better results in terms of reducing the recovery time and speeding up the status of improvement among COVID-19 patients ( Table 4). Various in vitro studies and machine learning algorithms suggested that clathrin-mediated endocytosis in case of SARS CoV-2 can be inhibited using baricitinib due to its affinity towards AP2-associated protein AAK1, which is responsible for reducing the SARS-CoV-2 endocytosis (Cantini et al., 2020). A study conducted by (Cantini et al., 2020) for duration of 14 days on 12 patients (2 females and 10 males) suffering from mild-to-moderate corona disease (CoV-19), the patients were given treatment of baricitinib (4 mg per day) along with ritonavir-lopinavir therapy. The treatment was tolerated normally without any adverse event. However, 1 patient was withdrawn from the study on 10th day due to elevation of transaminases (ALT: 298 U/L; AST: 267 U/L), which might be due to anti-viral therapy and not that of baricitinib treatment. Despite this, no other opportunistic or bacterial infection, haematologic toxicity or thrombophlebitis were observed. Significant improvement in parameters of respiratory function and all other clinical characteristic were observed in weeks 1 and 2 in comparison with baseline. The major depiction of the study involved the admission of 33% (i.e. 4 out of 12) of control group to ICU, which was 0 in case of baricitinib-treated group. Also, discharge rate of the patients in week 2 was 58% (i.e. 7 out of 12) in case of baricitinib-treated group, which was only 8% (i.e. 1 out of 12) in case of controls.
Another in vitro study was conducted by (Petrone et al., 2021) to evaluate the effect of baricitinib on the whole blood of 39 COVID-positive patients. Usually, low magnitude of cytokines (Th-2), predominance of Th-1 and high IFN-γ response are associated with immune response of COVID-19 patients. The result of the study depicts that there was significant drop in various immune factors such as IFN-γ; growth factors (FGF, GM-CSF); pro-inflammatory cytokines such as TNF-α, IL-6 and IL-1 β; Th-2 cytokines including IL-4 and 13, IL-10; chemokines (MIP-1 β, MCP-1 and IP-10) and other antigens upon baricitinib addition. All these factors are produced by innate immunity cells and by T and B cells and are considerably increased during the infection of COVID-19. The staphylococcal enterotoxin B-induced response was also dropped significantly upon baricitinib addition. Overall, the study concluded that baricitinib addition is responsible for lowering the SARS-CoV-2-specific response, including in patients suffering from mild or moderate coronavirus disease or in patients having a lymphocyte count of ≥ 1 × 10 3 /μl.
Many other trials and studies have been done and are still in queue by various institutions in different countries to evaluate the effect of baricitinib on COVID-19 patients. Majority of the results confirms the safety of baricitinib therapy in number of clinical trials where no hematological or cardiovascular adverse event or infection took place after two weeks treatment (Cantini et al., 2020). This two-week duration is similar to that of duration in ongoing COVID-19 studies involving baricitinib therapy which lasts from 7 to 14 days (Jorgensen et al., 2020). Carpio-Orantes et al., in 2021 enrolled 30 patients, and 90% of them had moderate and 10% had severe ARDS. Patients receiving 4 mg/ day baricitinib for 14 days demonstrated clinical improvement, with improved oxygen saturation and SAFI after 7 and 14 days of therapy. Ninety percent of patients healed, while ten percent died while undergoing therapy (Carpio-Orantes et al., in 2021). In 2021, Kalil et al. conducted a doubleblind, randomised, placebo-controlled study in which they enrolled 1033 hospitalised COVID-19 patients and treated 515 of them with baricitinib in combination with remdesivir. When compared to remdesivir alone and the placebo group, treatment with combination medication resulted in better clinical results. Patients who received combo treatment healed more quickly than the control group (7 days vs. 8 days). Patients on mechanical ventilation respond better to combination treatment, recovering in 10 days compared to 18 days for placebo. The use of combination treatment resulted in fewer side effects (Kalil et al., 2021;Goletti and Cantini 2021). Marconi et al., 2021 conducted a randomised, double-blind, parallel-group, placebo-controlled phase 3 trial, and 1525 participants were enrolled from 101 centres across 12 countries in Asia, Europe, North America, and South America. 764 of them received 4 mg/day baricitinib for 14 days with systemic corticosteroids or 761 patients assigned to placebo group who received only systemic corticosteroids. The baricitinib-treated group had a  Marconi et al., 2021 38.2 percent lower death rate on day 28. On days 28 and 60, the estimated mortality rate of the baricitinib-treated group was 8% (n = 62) and 10% (n = 79), respectively, compared to placebo, which was 13% (n = 100) and 15% (n = 110). In comparison to the placebo, the baricitinib-treated group demonstrated fewer serious adverse reactions like serious infections and venous thromboembolic events (Marconi et al., 2021).

Ruxolitinib
Early hypercytokinemia causes an increase in IL-6 levels, which activates the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, perpetuating the inflammatory processes. IL-6 forms a complex with either surface or soluble receptors (mIL-6R or sIL-6R) that activates gp130, which interacts with Janus kinases and regulates levels of cytokines. Blocking signalling events related to or at the level of cytokine production, such as JAK-STAT signalling in macrophages to alleviate IL-1 and IL-6 production, offers a promising therapeutic strategy for overcoming cytokine storm. Ruxolitinib is a JAK 1/2 inhibitor, licensed for use for the treatment of polycythemia vera and myelofibrosis (Ajayi et al., 2018). It has also been reported to be effective in the treatment of secondary hemophagocytic lymphohistiocytosis (Ahmed et al., 2019) by targeting the lethal effects of an aberrant host inflammatory response. Ruxolitinib alleviates the level of pro-inflammatory cytokines (IL-6, TNF-α, GM-CSF, MCP-1, and MIP-1α) and proliferation of cytotoxic T cells through the activation of regulatory T cells. Ruxolitinib can be effective against the consequences of the CSS in patients with COVID-19 (Table 5). Cao et al., in 2020 conducted a randomised, multicenter, placebo-controlled, single-blind phase II trial in hospitalised patients with severe COVID-19 to evaluate the efficacy and safety of ruxolitinib. In terms of clinical improvement, however, no significant differences were found, despite the fact that individuals treated with ruxolitinib had a numerically shorter median time to clinical improvement [IQR, 10-19] days vs 15 [IQR,[10][11][12][13][14][15][16][17][18] days, compared to placebo. In secondary outcomes, administration of ruxolitinib significantly mitigated the exuberant cytokine storm featured in severe COVID-19, alleviated the elevated levels of 48 cytokines, among which the levels of IL-6, NGF, IL-12 (p40), MIF, MIP-1a, MIP-1b, and VEGF were significantly lower in the ruxolitinib group compared to control group. The finding depicts that ruxolitinib may exert its inhibitory impact on multiple critical cytokines rather than any particular cytokine, and these cytokines could be exploited as surrogate biomarkers in future ruxolitinib trials. Gozzetti et al., 2020 enrolled fourteen patients in their study to check the efficacy of ruxolitinib and administrated it at a dose of 7.5 mg/day for 14 days with a COVID-19 Inflammation Score (CIS) ≥ 10 out of 16 points. The authors reported that 12/14 patients achieved a significant reduction of CIS by ≥ 25% on day 7 with a sustained clinical improvement in 11/14 patients. D'Alessio et al., in 2021 conducted a non-randomised clinical study to assess the effect of ruxolitinib in patients with severe COVID-19 pneumonia, without mechanical ventilation. Five mg of ruxolitinib was given orally twice a day for 7 days and then tapered to a single dose per day for up to 10 days. In addition to ruxolitinib, all patients received methylprednisolone 1 mg/kg intravenously for three days followed by 0.5 mg/kg for five days and then oral prednisone, which was slowly tapered in the course of 2 weeks. The results depicted that the mortality rate of the treated patients was significantly reduced and there were no significant side effects compared to the control group. Secondary outcomes were decreased inflammatory response, defined as afebrile and 30% decreased CRP levels at the second clinical observation, i.e. 3-10 days after hospital admission . In a study by Capochiani et al., 2020, 16 out of 18 enrolled patients exhibited a significant improvement in respiratory responsiveness during the first 48 h. After 7 days of ruxolitinib treatment, 11/18 patients had completely regained respiratory function (pO2 > 98 percent in spontaneous breathing), 4/18 patients had minimal oxygen requirements (2-4 L/m), 1/18 patients had stable disease, and 2/18 patients had advancing disease.16/18 patients had perfect respiratory function on day 14 of ruxolitinib therapy. 16/18 patients had perfect respiratory function on day 14 of ruxolitinib therapy. IL-6 levels dropped quickly in responsive patients (16/18). However, the non-responsive patients (2/18) showed a substantial rise in IL-6 plasma levels (Capochiani et al., 2020). In patients on ruxolitinib, anaemia is the most prevalent side effect. Longterm ruxolitinib treatment, on the other hand, was associated with a low rate of non-hematological side effects (Kiladjian et al., 2020).

Lenzilumab/mavrilimumab/otilimab (GM-CSF inhibitors)
GM-CSF is an immunoregulatory cytokine that plays an important role in the onset and progression of inflammatory disorders. GM-CSF levels have recently been found to be elevated in COVID-19 patients (Mehta et al., 2020a, b). Although GM-CSF is considered an appropriate response under some circumstances, in this case, the inflammatory response is observed to be disproportionate. The lung damage caused by COVID-19-induced hyper-inflammation can be improved by blocking the GM-CSF signalling, which can be done by opposition of the GM-CSF receptor or direct binding of circulating GM-CSF (Table 6). COVID-19 patients treated with a single dose of mavrilimumab, a monoclonal antibody which binds to the GM-CSF receptor, showed improvement in oxygenation and resulted in shorter hospitalisations (Bonaventura et al., 2020). Cremer et al., 2021, conducted multicentre, double-blind, randomised trial on 40 hospitalised COVID-19 patients with pneumonia, hypoxaemia and a C-reactive protein > 5 mg/dL. The primary efficacy outcome at day 14 for checking the patients free from mechanical ventilation was found to be 57% with mavrilimumab as compared to 47% with placebo; whereas, secondary outcome at day 28 to check the survival ratio was that 95% of the patients in mavrilimumab group were alive compared to 79% in untreated. However, the observation was not statistically significant, but this finding hypothesises the need for larger clinical trials to check their efficacy (Cremer et al., 2021). Lenzilumab is a novel Humaneered® anti-human GM-CSF monoclonal antibody that binds directly to the GM-CSF and blocks further signalling through its receptor. It also showed strong binding affinity (25 pM) and a slow offrate for glycosylated human GM-CSF (Padron et al., 2013). Temesgen et al., in 2020a, conducted the first clinical trial of Lenzilumab in neutralising the GM-CSF in patients with severe COVID-19-associated pneumonia. In their study, lenzilumab treatment resulted in clinical improvement in 11 out of 12 (92%), with a median time to discharge of 5 days. On day 3, mean CRP and IL-6 levels improved significantly (137.3 mg/l vs 51.2 mg/l, p = 0.040; 26.8 pg/ml vs 16.1 pg/ ml, p = 0.035, respectively). After two days of lenzilumab therapy, cytokine analysis revealed a decrease in inflammatory myeloid cells. Temesgen et al., 2020b conducted a cohort study to evaluate the efficacy of lenzilumab in the treatment of patients hospitalised with COVID-19 pneumonia. Intravenous treatment with three doses of 600 mg of lenzilumab in 12 patients versus 27 patients comprising the matched control cohort (untreated), led to a significantly shorter time for clinical improvement in lenzilumab-treated patients, with a median of 5 days versus 11 days (P = 0.006). Compared to the untreated, the proportion of patients with acute respiratory distress syndrome (oxygen saturation/ fraction of inspired oxygen < 315 mm Hg) was considerably reduced over time when given lenzilumab (P < 0.001). Inflammatory markers (C-reactive protein and interleukin 6) and disease severity markers (absolute lymphocyte count) improved significantly in lenzilumab-treated individuals compared to untreated patients. No treatment-emergent adverse events were associated with lenzilumab. Recently, Temesgen et al., 2021, conducted phase-3 randomised, double-blind, placebo-controlled study in 520 hospitalised severe COVID-19 patients across 29 sites in the United States and Brazil, and investigated the efficacy and safety of lenzilumab in improving the likelihood of ventilator-free survival without ventilation (SWOV). The likelihood of SWOV through day 28 was reduced by 54% in COVID-19 hospitalised patients with early treatment with lenzilumab. Although the patients were hypoxic, they did not require mechanical ventilation. The severity of COVID-19 disease is linked to a high level of CRP. In this trial, 77% of subjects with an evaluable CRP, had a baseline value < 150 mg/l and 74% had a baseline CRP value < 150 mg/l and age < 85. This study revealed that a CRP level of less than 150 mg/l was related with obtaining SWOV and distinguished developing hyper-inflammation from full-fledged CSS during the course of treatment. This study also demonstrated that early intervention with lenzilumab led to neutralisation of GM-CSF, which is a key factor responsible for initiation of CS in hypoxic patients. This also improved the probability of survival without the usage of mechanical ventilation and provided the greatest benefit over the usage of steroids and/ or remdesivir.

Heparin
Heparin is a member of a family of polyanionic polysaccharides called glycosaminoglycans (Young, 2008). It is still one of the most commonly prescribed anticoagulants in clinical practice, for the prevention of thrombosis. Heparin has been shown to have various functions in inhibiting SARS-CoV-2 in the treatment of COVID-19 (Hippensteel et al., 2020). It is used as an anticoagulant in treating coagulopathy associated with severe SARS-CoV-2 infection. Tang et al., 2020 were the first to conduct a retrospective, cohort study, including 449 severe COVID-19 patients, 99 of whom had taken low-dose preventive doses of heparin for 7 days or longer. Those who received prophylactic heparin with sepsis-induced coagulopathy (SIC) score ≥ 4 had ~ 24.0 percent lowered mortality than non-users patients (40.0 percent vs 64.2 percent, P = 0.029) (Tang et al., 2020).
In 2020, Paranjpe et al., enrolled 2773 hospitalised patients with COVID-19 to evaluate the efficacy of anticoagulant therapy. In-hospital mortality was found to be 29.1% in patients receiving therapy and on mechanical ventilation support, with a median survival of 21 days, compared to 62.7% in non-users with a median survival of 9 days (Paranjpe et al 2020). Furthermore, heparin also displayed anti-viral characteristics, a study by Lang et al. 2011 demonstrated that heparinase or exogenous heparin blocked spike protein from attaching to hostile cells and prevented pseudovirus SARS infection. In 2020, using a surface plasmon resonance direct binding assay, Kim et al. demonstrated that the SARS-CoV-2 spike protein formed a highly strong (and nearly irreversible) bond with heparin sulphate, and has shown a greater affinity than either SRS-CoV or MERS-CoV spike protein (Kim et al., 2020). Despite this, few studies showed that the blocking of proinflammatory signalling pathways mediated by MAPK, NF-kB, and STAT-3 was also linked to heparin's protective response in sepsis (Iba and Levy, 2017;Li et al., 2020). Heparin has been demonstrated to be easily coupled and absorbed into the cytosolic compartment, where it binds to the positively charged nuclear localisation sequence and prevents NF-kB from translocating to the nucleus (Letourneur et al., 1995;Akimoto et al., 1996;Dudas et al., 2000). The production of pro-inflammatory cytokines, chemokines, and adhesion molecules can be regulated by reducing inflammatory gene activation by blocking of NF-kB. Lower mortality was observed in COVID-19 patients who were treated with heparin, even after taking various parameters like age, gender, oxygen saturation < 90% and temperature > 37 °C (Ayerbe et al., 2020). Recently, a study in the US of COVID-19-hospitalised patients (n = 786) showed that patients who received anticoagulants had a reduced risk of mortality. Studies showed that early treatment with prophylactic anticoagulation resulted in reduced 30-day mortality, compared to no anticoagulation among hospitalised COVID-19 patients (Paranjpe et al., 2020). Amongst the 4297 COVID-19 patients admitted, 3627 (84.4%) who received prophylactic anticoagulation within 24 h of admission had a 27% reduced risk for 30-day mortality as compared to patients with no prophylactic administration (Rentsch et al., 2021). The treatments had no increased risk of serious bleeding events. Many more studies suggested that the inhaled unfractionated heparin (UFH) may provide therapeutic advantage over the course of the disease. UHF may prevent infection in upper respiratory tract, given by inhalations into the upper airways, a significant site of entrance for the virus (Haren et al., 2020;Braz-de-Melo et al., 2021).

Antifibrotics (tyrosine kinase inhibitors)
Nintedanib has been demonstrated to reduce IL-1 concentrations in bronchoalveolar lavage, and pirfenidone has been proven to diminish serum and lung IL-6 concentrations in murine models of pulmonary fibrosis, adding to the biological basis for Nintedanib and pirfenidone's usage in COVID-19. Umemura et al. (2021) conducted an interventional study for evaluation of the safety and efficacy of nintedanib for pulmonary fibrosis in COVID-19 patients with severe pneumonia. There was no significant difference in 28-day mortality in 30 patients that received nintedanib therapy in comparison to 30 control patients not receiving it (23.3% vs 20%, P = 0.834). However, the nintedanib group had considerably shorter periods of mechanical ventilation. It was hypothesised that the usage of nintedanib may offer potential benefits in minimising the COVID-19 induced lung injury (Umemura et al., 2021). Based on this observation, Nintedanib was referred for clinical trials to check its efficacy in the treatment of SARS-Cov-2 induced pulmonary fibrosis (NINTECOR) [ClinicalTrials.gov Identifier: NCT04541680]. A case report conducted by Ogata et al., in 2021, on a 78-year-old Japanese woman with no smoking history who suffered from a near-fatal COVID-19, required four-week invasive mechanical ventilation, with subsequent radiological features of pulmonary fibrosis. In parallel with systemic steroids, the patient was also given nintedanib therapy, which led to the patient being able to walk using a walking aid utilising oxygen at 4 L/min (Ogata et al., 2021). The current case suggests that nintedanib could be used to treat post COVID-19 fibrosis in a unique way. However, only one patient was examined in this study and more investigations are, thus, necessary.
Pirfenidone, also known as 5-methyl-1-phenyl-2-[1H]pyridone, is a new anti-fibrotic agent with little side effects. Pirfenidone is licenced for the treatment of patients with mild-to-moderate Idiopathic Pulmonary Fibrosis (IPF). Pirfenidone has been shown to suppress apoptosis, downregulate ACE receptor expression, reduce inflammation through a variety of pathways, and alleviate oxidative stress and, thus, protect pneumocytes and other cells from COVID-19 invasion and cytokine storm concurrently. Based on known action mechanism and pathophysiology of pirfenidone for COVID-19 (Seifirad, 2020;Singh et al., 2021), the treatment with pirfenidone for severe COVID-19-related severe ARDS is referred for the clinical trials [Clinical trials NCT04653831].

Conclusion
Numerous initiatives have been undertaken to combat the SARS-CoV-2 epidemic. Many medicines and vaccines have received emergency approval from the WHO and FDA and are being used to treat the symptoms of this disease. Despite this, outbreaks continue to be diagnosed and spread in a number of countries. The emergence of SARS-CoV-2 variants raises concerns about the efficacy of authorised medicines and vaccines, and we still do not know what the SARS-CoV-2 virus and its variants may bring. CSS and ARDS are the most common clinical problems linked to severe instances of COVID-19 illness. The increased cytokine level causes typical organ damage in the more established CSS, although many clinical trials demonstrate that the cytokineblocking yields in clinical benefit and survival. Numerous studies have shown that immunomodulatory treatments can help to reduce the severity of this disease and save lives. However, variability, limited sample sizes, and random recruitment raise concerns about the research that has been undertaken. A number of studies, both supporting and contradictory, have been published. We commended the efforts of all health authorities, supporting personnel, and scientists for identifying potential treatment options for severe instances of COVID-19 illness. Moreover, efforts on a global level are still needed, as demonstrated by the CSS phenotype on the severe end of the illness spectrum in COVID-19 patients. Studies should be undertaken with a large sample size with a dysregulated immune response to determine the real effectiveness of immunomodulatory treatments.