3. Clinical evidence of immunomodulatory and anti-inflammatory agents
Since the beginning of the COVID-19 pandemic, the determining role of inflammatory cytokines in the worsening of clinical conditions has been identified. Indeed, one of the reasons underlying the occurrence of serious symptoms (figure 1) associated with the clinical progression of SARS-CoV-2 infection is represented by the increase in levels of pro-inflammatory cytokines, mainly interleukins, granulocyte-colony stimulating factor and TNF-α. These molecules are responsible for the so-called cytokine storm that, in turn, induces ARDS, organ failure and sepsis (Guo et al., 2020b). Many drugs, acting through different mechanisms, are able to block or reduce the effects mediated by the cytokines storm (figure 2).
Tocilizumab was one of the most studied drugs able to reduce the hyperinflammatory state associated with COVID-19. It is a monoclonal antibody (figure 2), authorized for the treatment of many diseases, including rheumatoid arthritis, which is active against the interleukin-6 receptor, one of the key mediators of the inflammatory process (Scott, 2017). At the beginning of the pandemic, the drug was tested in China to reduce lung complications in 20 patients with severe SARS-CoV-2 infection, being associated with a reduction of oxygen requirement, resolution of CT lesions, normalization of lymphocyte count, reduction of C-reactive protein levels and hospital discharge (Xu et al., 2020). Few months ago, many trials evaluating the effects of tocilizumab started in Italy, after the approval of the Italian Medicine Agency and the Spallanzani Ethics Committee. However, despite the encouraging preliminary results, the availability of new evidence from other ongoing clinical trials has marked a change of course. Indeed, the study carried out by the Local Health Unit-IRCCS of Reggio Emilia (which evaluated the efficacy of tocilizumab at an early stage in patients with COVID-19 pneumonia who did not require invasive or semi-invasive mechanical ventilation procedures invasive) was earlier stopped, after the enrolment of 126 patients. The reason for the early conclusion lies in the absence of proof of benefit in treated patients either in terms of worsening (access to the ICU) or survival (Italian Medicine Agency, 2020a). Similarly, the results of the COVACTA trial, who was ongoing when we published our first paper (Scavone et al., 2020), did not reveal a benefit of tocilizumab over placebo in patients with severe COVID-19 pneumonia. Indeed, as reported in a press document released by the pharmaceutical company, no difference in clinical status between patients treated with tocilizumab and placebo were found (primary endpoint). In addition, no differences between groups were found regarding to the percentage of patients who died by week four and in ventilator-free days (Roche, 2020). Despite the negative results of these trials, tocilizumab was associated with positive outcome in other clinical studies, both interventional and observational. For instance, the single arm trial TOVICID-19 evaluated the effects of tocilizumab in hospitalized patients with SARS-CoV-2 infection, oxygen saturation at rest in ambient air ≤ 93% or required oxygen support or mechanical ventilation either non-invasive or invasive. One hundred and eighty patients received a dose of 8 mg/kg up to a maximum of 800 mg per dose and a second administration 12 h after the first one if respiratory function did not recover. The results revealed that the treatment with tocilizumab seems to reduce lethality at 30 days, although its impact at 14 days seems less relevant (Perrone et al., 2020). Lewis TC et al. reported the results of an observational cohort study of patients hospitalized at 3 hospitals within the NYU Langone Health system in New York who received tocilizumab 400-mg IV once in addition to standard of care or standard of care alone. Out of 3,580 patients, 497 were treated with tocilizumab. The propensity score analysis showed that a lower number of death occurred in tocilizumab-treated patients and that tocilizumab was associated with improved survival compared to controls (p>0.001). No difference were found between groups in term of adjusted time to hospital discharge, while patients treated with tocilizumab had longer adjusted ICU length of stay and a higher adjusted infection rate (both p>0.001) than controls (Lewis et al., 2020). Tleyjeh IM et al. carried out a systematic review and meta-analysis of 24 studies, including 5 randomized controlled trials and 19 observational studies (enrolling 1,325 and 10,021 patients, respectively). The results of this study demonstrated that tocilizumab is able to reduce the risk of mechanical ventilation in hospitalized COVID-19 patients but not the short-term mortality, at least according to the results of randomized controlled trials. On the contrary, data from observational studies suggested an association between tocilizumab and lower mortality. Authors suggested that many reasons could explain differences found for the effect on the mortality, including the enrolment of different patient populations, a different inflammatory status, and timing/dosing of tocilizumab. Furthermore, in order to be sufficiently powered to detect a difference in mortality between tocilizumab and control groups, the sample size of clinical trials should have been substantially higher. In terms of safety profile, no higher risk of infections or any other adverse events was found with tocilizumab use (Tleyjeh et al., 2020). Given the evidence currently available on the effects of tocilizumab in COVID-19 patients no firm conclusion can be drawn. We still have to wait for the results of other studies, such those from the RECOVERY trial, which is currently evaluating the efficacy of tocilizumab and other drugs, including low-dose dexamethasone, colchicine, convalescent plasma, REGN-COV2 and aspirin (University of Oxford, 2020b). Furthermore, biochemical parameters like serum IL6 levels and TH17 cells may contribute to select subgroups of COVID-19 patients that would specifically benefit from the treatment with Tocilizumab, according to the view of a personalized therapy in such patients (Cacciapuoti et al., 2020). Conflicting results were also obtained for sarilumab (figure 2), a drug belonging to the same drug class of tocilizumab. In our first document (Scavone et al., 2020), we stated that three randomized controlled trials were underway. Nowadays, the results of one trial investigating the effects of sarilumab in severely or critically ill patients hospitalized with COVID-19 did not meet its primary and secondary endpoints (Sanofi, 2020b). Other studies, mainly observational, that investigated the effects of sarilumab were carried out on a very limited number of patients. For instance, Benucci M et al. evaluated the efficacy of sarilumab in 8 patients (mean age: 62 years) hospitalized at the San Giovanni di Dio Hospital (Florence, Italy). The drug was administered in a dose of 400 mg in combination with hydroxychloroquine, azithromycin, darunavir, cobicistat and enoxaparin. Seven patients had an improvement of the Horovitz index (oxygenation expressed by an increased SpO2/FiO2 ratio). A progressive reduction in the serum amyloid A and CRP inflammation parameters was observed and patients were discharge within 14 days of hospitalization (Benucci et al., 2020). Another study, carried out at the Fondazione Policlinico Universitario A. Gemelli-IRCCS (Italy), evaluated the effects of sarilumab in 53 patients (median age: 66 years) with severe COVID-19. Almost 70% of patients were also treated with darunavir/ritonavir, 24% with lopinavir/ritonavir, while the remaining patients did not receive antiviral treatment. Hydroxychloroquine, heparin and azithromycin were administered in 94,3%, 74,9% and 54,7% of patients, respectively. Thirty-nine patients were treated in medical wards (the majority with a single infusion), while 14 in ICU (the majority with two infusions). Among patients who received the drug in the medical wards, at 19 days median follow-up, almost 90% significantly improved. Among patients who received sarilumab in ICU, almost 36% were still alive at the last follow-up. The overall mortality rate was 5,7% (Gremese et al., 2020). A further retrospective study was carried out at the Azienda Ospedaliera dei Colli – Cotugno Hospital (South of Italy) on 15 patients (12 treated with a single dose and 3 with two infusions). All patients received hydroxychloroquine, lopinavir/ritonavir and heparin, while 11 patients were also treated with methylprednisolone. Improvements in respiratory parameters were observed in 10 patients after sarilumab administration. Five patients who received sarilumab died (Montesarchio et al., 2020). Overall, the majority of clinical studies carried out on sarilumab were observational and limited by a low number of patients. In addition, many other drugs were co-administered. Therefore, it is not easy to fully appreciate the positive effect deriving from sarilumab administration. Thus, also for this drug further clinical data are strongly needed.Chloroquine and hydroxychloroquine (figure 2) are noteworthy too. These are antimalarial drugs that have also been used for the treatment of different rheumatic diseases (Mascolo et al., 2018). Both drugs show many pharmacological effects, including the stabilization of the lysosomal membranes, the inhibition of prostaglandin synthesis, polymorphonuclear chemotaxis and phagocytosis and a possible interference with the production of interleukin 1 by monocytes and inhibition of the release of superoxide by neutrophils (Italian Medicine Agency). These drugs have demonstrated to be effective in inhibiting the replication of SARS-CoV-2 in experimental models (figure 2) (Italian Medicine Agency) and have been increasingly tested for COVID-19 in many clinical studies. Specifically, many studies, which are currently ongoing, are evaluating the effects of hydroxychloroquine as prophylactic agent (Lother et al., 2020; Niriella et al., 2020; Tirupakuzhi Vijayaraghavan et al., 2020). At the moment, only one randomized, double-blind, placebo-controlled study on the prophylactic role of hydroxychloroquine was concluded. The trial enrolled 821 subjects who had had family or occupational exposure with a subject with COVID-19. According to the study results, there was no difference in the incidence of new onset of COVID-19 between participants treated with hydroxychloroquine (11.8%) and those who received placebo (14.3%). Adverse events were more commonly observed with hydroxychloroquine than with placebo, although no serious adverse reactions were reported (Boulware et al., 2020). Many clinical trials evaluated the efficacy and safety of hydroxychloroquine, alone or in combination with azithromycin, as therapeutic agent. Gautret P et al. carried out a single-arm interventional study, which evaluated the effects of hydroxychloroquine (plus azithromycin) in 36 hospitalized patients with COVID-19. Authors reported that a significant reduction in viral load was found on day 6 in patients treated with hydroxychloroquine compared to controls. The addition of azithromycin to hydroxychloroquine therapy significantly contributed to the reduction of viral load (Gautret et al., 2020). Another study enrolled 11 hospitalized patients treated with hydroxychloroquine and azithromycin. After 5-6 days of starting treatment, the nasopharyngeal swab gave a positive result in 8/10 patients. These virological results are in contrast with those reported by Gautret et al. and raise doubts about the effectiveness of the drugs combination (Molina et al., 2020). A further study found no difference in viral load reduction and clinical outcomes between hydroxychloroquine and standard of care (CHEN et al., 2020). In addition, the results of the randomized clinical study by Tang et al., which was carried out on 150 hospitalized patients with COVID-19 (148 with mild to moderate disease and 2 with severe disease), did not show any difference on the viral clearance at 28 days. The only positive result came from a post hoc analysis in a subgroup of patients who did not take antivirals in which a greater effect on symptoms was observed (Tang et al., 2020). Similarly, the results of a randomized, double-blind, placebo-controlled clinical trial (carried out in 491 outpatients at an early stage of COVID-19) found no differences in symptom severity over 14 days between hydroxychloroquine and placebo (relative difference 12%; p=0.117). (Skipper et al., 2020). Huang et al. conducted a randomized study that showed no significant differences up to 14 days, in terms of viral clearance and improvement on CT scan between chloroquine and lopinavir/ritonavir, but on day 14, 100% of patients treated with chloroquine were discharged from the hospital, compared with 50% in the lopinavir / ritonavir group. However, it should be underlined that patients in the chloroquine group were younger and they had started the treatment earlier (Huang et al., 2020). Lastly, the preliminary results of the SOLIDARITY study seem to suggest that hydroxychloroquine, the combination lopinavir / ritonavir and IFN-based regimens have little or no effect on mortality at 28 days or on hospital course (World Health Organization, 2020c). Alongside with data on the efficacy profile of chloroquine and hydroxychloroquine, many data on the safety profile of both drugs were collected as well. For example, a recent warning discouraged the friendly use of chloroquine in the COVID-19 disease due to the possible side effects (like a severe haemolytic crisis) in G6PD deficient males and in unaware carriers of the disease, that are estimated to be about 400 million worldwide (Capoluongo et al., 2020). Based on these, the main regulatory agencies have issued many recommendations for clinicians. For instance, the EMA drew the attention on risks of serious adverse reactions (including heart rhythm disturbances) (European Medicine Agency, 2020a), highlighting the need for prescribers to closely monitor patients treated with both drugs (European Medicine Agency, 2020b). In addition, the European agency recommended the use of these medicines only in clinical trials or in national emergency management programs in hospitalized patients under close monitoring (European Medicine agency, 2020). The FDA has also highlighted the need for patients’ monitoring when treated with hydroxychloroquine, especially regarding to the risk of severe changes in heart rhythm (tachycardia, atrial fibrillation and torsades de pointes), observed in patients treated with these drugs often in combination with azithromycin and other drugs able to prolong the QT (US Food and Drug Administration, 2020a). The American agency also recommended the use of chloroquine and hydroxychloroquine only in the context of clinical trials and revoked, in June 2020, the previously granted Emergency Use Authorization (US Food and Drug Administration, 2020a). In May 2020, the World Health Organization (WHO) suspended the hydroxychloroquine treatment arm in the multicentre clinical trial SOLIDARITY (World Health Organization, 2020e). In June 2020, the hydroxychloroquine treatment arm was reopened for enrolments, only to be permanently closed in the same month (Italian Medicine Agency, 2020b). Lastly, on November 2020, the PRAC of the EMA recommended updating the product information for chloroquine or hydroxychloroquine due to the risk of psychiatric disorders and suicidal behaviour (European Medicine Agency, 2020d). In conclusion, data related to the efficacy and safety profile of antimalarial drugs are quite conflicting. We should wait for the results of clinical trials currently ongoing in COVID-19 patients (www.clinicaltrials.gov). At the time of the publication of our first paper (Scavone et al., 2020), the evidence on the efficacy of corticosteroids in patients with COVID-19 was quite conflicting. Nowadays, we know that these drugs, especially dexamethasone and methylprednisolone, could be considered one of the mainstays in the treatment of COVID-19, especially for patients with severe disease, to prevent its worsening and complications and to resolve its serious symptoms. As reported in figure 2, corticosteroids are associated with multiple mechanisms, including the increase in the gene transcription of anti-inflammatory cytokines, the decrease in gene transcription of pro-inflammatory cytokines, chemokines, and adhesion molecules and a reduction in inflammatory response. All these effects seem to be responsible of the counteraction of the cytokine storm caused by SARS-CoV-2 infection. First and foremost, the preliminary results of the RECOVERY trial, which were recently published on the NEJM, provided the most significant evidence supporting the use of dexamethasone. In this controlled, open-label trial, patients were randomized to receive oral or intravenous dexamethasone 6 mg/day for up to 10 days (n=2,104) or the usual care alone (n=4,321). Within 28 days after randomization, 22.9% of patients treated with dexamethasone and 25.7% of patients in the usual care group died (age-adjusted rate ratio, 0.83; 95% confidence interval [CI], 0.75 to 0.93; P<0.001). According to the level of respiratory support, the incidence of death was lower in dexamethasone group among patients receiving invasive mechanical ventilation or oxygen without invasive mechanical ventilation (Horby et al., 2020a). These results, together with those obtained from other randomized clinical studies (Angus et al., 2020; Dequin et al., 2020; Jeronimo et al., 2020; Tomazini et al., 2020), showed that dexamethasone could be considered a treatment option in hospitalized subjects with severe COVID-19 infection who require oxygen therapy, whether or not they have mechanical ventilation (invasive or not invasive). Based on the preliminary results of RECOVERY trial, on September 2020 the CHMP of the EMA concluded that dexamethasone can be considered a treatment option for patients who require oxygen therapy and the EMA is endorsing the use of this drug in adults and adolescents (from 12 years of age and weighing at least 40 kg) who require supplemental oxygen therapy at a dose of 6 milligrams once a day for up to 10 days (European Medicine Agency, 2020c). Recently the WHO REACT Working Group carried out a meta-analysis of seven randomized clinical trials that evaluated the efficacy of corticosteroids (dexamethasone, hydrocortisone, or methylprednisolone) in 1,703 critically ill patients with COVID-19 on all-cause mortality at 28 days and the occurrence of serious adverse events. The results showed that, compared with usual care or placebo, the OR for the association with mortality was 0.64 (p<0.001) for dexamethasone, 0.69 (p=0.13) for hydrocortisone and 0.91 (p=0.87) for methylprednisolone. Sixty-four serious adverse events were observed among patients randomized to corticosteroids and eighty serious adverse events among patients randomized to usual care or placebo. Authors concluded that the administration of systemic corticosteroids, compared with usual care or placebo, was associated with lower 28-day all-cause mortality (Sterne et al., 2020). Lastly, several clinical trials are currently underway or in development to evaluate corticosteroids for the treatment of COVID-19 (www.clinicaltrials.gov). Similarly to corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs) (figure 2) and paracetamol represent valuable therapeutic options in patients with COVID-19, especially for outpatients at early stage (not severe form), to treat fever and muscle pain. At the beginning of the outbreak, the role of NSAIDs was misjudged due to some concerns that arose from few studies (Capuano et al., 2020b). Specifically, in 2009 a group of researchers from the University of Rochester highlighted that NSAIDs might lower host defence following infection or vaccination (Bancos et al., 2009). On April 2019 the French regulatory agency reported the results of a survey carried out by the regional pharmacovigilance centres of Tours and Marseille in which serious infectious complications occurred in patients receiving NSAIDs, mainly ibuprofen and ketoprofen, used in the treatment of fever and pain (ANSM, 2019). On March 2020, the French Minister of Health recommended the use of paracetamol instead of ibuprofen or oral cortisone for the treatment of fever in patients with COVID-19. This statement was supported also by the French Authorities, which announced that NSAIDs might worsen clinical conditions of patients with COVID-19 (Le monde, 2020; Ministère de la Santé, 2020). Recently few clinical data have become available, mainly from observational studies, regarding to the use of NSAIDs in patients with COVID-19. For instance, Rinott E et al. carried out a retrospective cohort study on 403 patients with COVID-19. The authors evaluated the association between the use of ibuprofen, administered from a week before diagnosis of COVID-19 throughout the disease, and the mortality and the need for respiratory support. Of the entire cohort, 87 patients received ibuprofen and 316 did not. No significant difference was found between groups in terms of mortality and respiratory support. The comparison of ibuprofen users with paracetamol users did not reveal any difference in mortality rates or the need for respiratory support (Rinott et al., 2020). A prospective cohort study enrolled patients with COVID-19, who were interviewed about NSAID use and infection outcomes (such as death, admission, severity, time to clinical improvement, oxygen requirement and length of stay) through a telephone questionnaire. According to study’s results, neither the acute use of ibuprofen nor chronic NSAID use were associated with a greater risk of mortality compared to non-use (adjusted HR: 0.632 [95% CI 0.073-5.441; P = 0.6758 and adjusted HR 0.492 [95% CI 0.178-1.362; P = 0.1721], respectively) (Abu Esba et al., 2020). Lastly, a further multicentre, observational study, which collected data related to 1222 patients with COVID-19 admitted to eight UK hospitals (of whom 54 were routinely prescribed NSAIDs prior to admission), found no evidence that routine NSAID use was associated with higher COVID-19 mortality (Bruce et al., 2020). Other drugs able to reduce the hyperinflammation are currently under evaluation in patients with COVID-19, including baricitinib, ruxolitinib, aviptadil and eculizumab. For instance, the effects ofbaricitinib , a Janus kinase (JAK) inhibitor authorized for the treatment of rheumatoid arthritis and able to suppress the cytokine storm and to interrupt the passage and intracellular assembly of SARS-CoV-2 into the target cells (Zhang et al., 2020b), were evaluated in 12 patients with mild-moderate COVID-19 pneumonia. At week 1 and week 2, the drug demonstrated to improve the clinical and laboratory parameters and none of the patients required ICU support. Even though encouraging, this study enrolled a limited number of patients, had no randomization and no control group (Cantini et al., 2020a). The same research group carried out an observational, retrospective, multicentre study in hospitalized patients with moderate COVID-19 pneumonia to compare the 2-week effectiveness and safety of baricitinib and lopinavir/ritonavir (n=113) vs. hydroxychloroquine and lopinavir/ritonavir (n=78). The results showed that the fatality rate was significantly lower among patients treated with baricitinib (0% vs. 6.4%; p= 0.010). Similarly, ICU admission was significantly lower among patients treated with baricitinib (0.88% vs. 17.9%) (Cantini et al., 2020b). The drug was also used in a patient with severe COVID-19 with insufficient response to lopinavir/ritonavir, hydroxychloroquine, azithromycin and sarilumab. Authors reported that after the administration of baricitinib, the patient experienced a prompt resolution of the respiratory function and improvement in the radiological findings (Cingolani et al., 2020). Based on data currently available, no firm conclusion can be drawn on the effects of baricitinib in COVID-19 patients. In addition, due to a possible increased risk of herpes zoster and simplex infections, a group of Italian researchers suggested that the use of baricitinib should be considered with extreme caution (Favalli et al., 2020). Ruxolitinib is a Janus kinase (JAK) 1 and JAK2 inhibitor authorized for the treatment of myeloproliferative malignancies (El Bairi et al., 2020). The effects of the drug were evaluated in the RUXCOVID study, a phase III, multicentre, randomised and placebo-controlled trial, but its results are still not available. Furthermore, the drug is currently evaluated in the RUXCOVID-DEVENT trial (NCT04377620) in COVID-19 patients with ARDS requiring mechanical ventilation.Aviptadil is a synthetic vasoactive intestinal polypeptide (VIP), which increases the activity of adenosine cyclase (Leuchte et al., 2008). By acting as a weak pulmonary vasodilator, the drug is able to improve the oxygenation in patients with chronic lung disease (Ferrara et al., 2005). Currently, many clinical studies are evaluating the effects of aviptadil as therapeutic or prophylactic agent. For instance, clinical trials NCT04311697 and NCT04360096 are investigating the effects of aviptadil on acute lung injury, ARDS, and dyspnoea associated with COVID-19, while the phase I study NCT04536350 will evaluate the prophylactic effect of aviptadil for COVID-19 in 80 participants (at this moment all studies are still recruiting participants) (www.clinicaltrials.gov). Lastly, two recent studies highlighted the benefits deriving from the treatment with eculizumab (Annane et al., 2020; Laurence et al., 2020). This is a monoclonal antibody approved for the treatment of atypical haemolytic uremic syndrome, refractory generalized myasthenia gravis and neuromyelitis spectrum disorders. The drug is able to inhibit the terminal portion of the complement cascade involved in the inflammatory response (Gralinski et al., 2018). The study carried out by Laurence J et al. reported the experience of 3 critical COVID-19 patients who experienced a marked decline in D-dimers and neutrophil counts after eculizumab treatment (Laurence et al., 2020). The results of the proof-of-concept study carried out by Annane D et al. showed that, in a population of 80 patients with severe COVID-19 admitted to ICU, the estimated survival was 82.9% with eculizumab and 62.2% without eculizumab (log-rank test, P = 0.04). Based also on the positive results on rapid decrease in lactate, blood urea nitrogen, bilirubin levels and a rapid increase in platelet count and prothrombin time observed with eculizumab, authors suggested that the drug could improve survival and reduce hypoxia in patients with severe COVID-19 (Annane et al., 2020). In conclusion, many immunomodulatory and anti-inflammatory drugs have been tested in patients with COVID-19, but today the evidence is quite conflicting for most of them. In addition, many of the published studies are observational or suffer from many limitations, including the lack of a sample size calculation or control groups and the use of surrogate endpoints (viral load instead of mortality rate). At present, the highest number of concluded clinical studies was found for tocilizumab, hydroxychloroquine and corticosteroids. Among these drugs, only the use of corticosteroids seems to be supported by robust evidence, while data related to the efficacy and safety of tocilizumab and hydroxychloroquine are quite conflicting. Therefore, further data from randomized controlled trial or well-designed observational studies are strongly needed.