4. Approaching therapies for COVID-19 patients.
In the attempt to identify the effective anti-SARS-CoV2 therapy some clinical trials are still ongoing. In particular, the therapeutic approach can be classified in two big branches: the antiviral, which aims to diminish virus replication, and the anti-inflammatory agents to hijack the cytokine storm that the virus is able to induce. In particular, the antiviral drug that is currently proving of efficacy in COVID-19 is remdesivir, which tightly binds and inhibits the virus RNA-dependent RNA polymerase (RdRp) (Elfiky, 2020). In a cohort of patients hospitalized for severe COVID-19 who were treated with compassionate-use, remdesivir proved of clinical improvement in 36 out of 53 patients (68%) (Grein et al. 2020). Instead, lopinavir and ritonavir, anti-HIV drugs, showed disappointing results beyond standard care in that the viral load and the mortality were not altered (Caoet al. 2020).
On the other hand, disease-modifying antirheumatic drugs (DMARDS), such as chloroquine and hydroxychloroquine, as well as immunotherapeutic agents, such as monoclonal antibodies (mAbs), are being used. In particular, chloroquine as well as hydroxychloroquine, antimalarial drugs, can interfere with lysosomal activity and autophagy, interact with membrane stability and alter signalling pathways and transcriptional activity, which can result in inhibition of cytokine production and modulation of immune co-stimulatory molecules (Schrezenmeier and Dörner, 2020). Thus, they can inhibit lysosomal activity, preventing major histocompatibility complex (MHC) class II-mediated antigen presentation. Moreover, they can accumulate in endosomes and bind to double-stranded DNA, inhibiting both Toll-like receptor (TLR) signaling (i.e. TLR7 and TLR9) (Kuznik et al.2011) and the nucleic acid sensor cyclic guanosine monophosphate–adenosine monophosphate (cGMP-AMP or cGAMP) synthase (cGAS) (Zhang et al. 2014). By preventing TLR signalling and cGAS–stimulator of interferon genes (STING) signalling, hydroxychloroquine can reduce the production of pro-inflammatory cytokines (van den Borne et al. 1997). However, the adverse effects need to be taken into consideration, especially in regards to the alteration of heart rhythm which cautiously limits their use.
Importantly, immunotherapeutic agents, such as tocilizumab or sarilumab, which are mAbs against IL-6 signalling, highly released during the interstitial pneumonia, have proved an effective treatment in severe patients of COVID-19 to calm the inflammatory storm and reduce mortality (Xu et al. 2020c; http://www.news.sanofi.us/2020-03-16-Sanofi-and-Regeneron-begin-global-Kevzara-R-sarilumab-clinical-trial-program-in-patients-with-severe-COVID-19). This encouraging clinical trial indicates that neutralizing mAbs against other pro-inflammatory cytokines may also be of use, with potential targets including IL-1, IL-17 and their respective receptors. However, tocilizumab, as well as sarilumab, can induce hepatotoxicity, neutropenia, tumorigenesis, hypersensitivity, opportunistic infections (https://www.accessdata.fda.gov/drugsatfda_docs/label/2016/125276s107_125472s018lbl.pdf;https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/761037s001lbl.pdf); therefore, mAbs targeting IL-6 signalling cannot be administered to all patients, because of co-morbidities to COVID-19 need carefully to be taken into consideration. Nevertheless, they represent the promise for blocking cytokine storm-related immunopathology of moderate to severe COVID-19.
Another clinical trial for stable COVID-19 patients is on the activity of colchicine, an anti-gout drug, which blocks the mitotic cells in metaphase, but is also able to block Nod-like receptor protein 3 (NLRP3) inflammasome inhibiting the release of IL-1-like cytokines (https://clinicaltrials.gov/ct2/show/NCT04322565), such as IL-1β, that was in the attempt to be blocked by means of anakinra in another clinical trial (https://clinicaltrials.gov/ct2/show/NCT04366232). Additionally, emapalumab, a monoclonal antibody against IFN-γ, associated to anakinra, has been proposed (https://clinicaltrials.gov/ct2/show/NCT04324021). However, the inhibition of such an important anti-viral cytokine could be on one side important to block the cytokine storm, but on the other can be hijacked by the virus due to the absence of one of the most important army against viral infections, creating further opportunistic pathologies.
Baricitinib has been identified as a molecule potentially useful in COVID-19 because of a double action to down-modulate the inflammatory storm and reduce the entry of the virus into type II pneumocytes due to the blockade of the AP2-associated protein kinase 1 (AAK1), a regulator of the endocytosis of the virus (Richardson et al. 2020). Moreover, baricitinib also binds to the cyclin G-associated kinase, another regulator of endocytosis. Thus, baricitinib may be useful for both reducing inflammatory response and viral endocytosis.
It has to be pointed out that all the above ongoing clinical trials include monitoring of coagulation parameters, such as D-dimer, which is a metabolite of fibrin aggregates. Although there are no published case series reporting abnormal coagulation parameters in hospitalized severe COVID-19 patients, in a multicenter retrospective cohort study in China, elevated D-dimer levels (> 1 g/L) were strongly associated with in-hospital deaths, therefore to severe COVID-19 (Zhou et al. 2020a). To date, low molecular weight heparin (LMWH), enoxaparin, has been proposed for these patients either to avoid thromboembolism events (Tang et al. 2020a) or to inhibit the cytokine storm (Shiet al. 2020), due to non-anticoagulant fraction of enoxaparin suppresses in vitro IL-6 and IL-8 release from human pulmonary epithelial cells (Shastri et al. , 2015). Moreover both in vitro and in vivo experimental studies have shown that human coronaviruses utilize heparin sulfate proteoglycans for attachment to target cells (Milewska et al. 2014). Indeed, interaction between the SARS-CoV2 Spike S1 protein receptor binding domain (SARS-CoV-2 S1 RBD) and heparin has been recently showed, suggesting a role for heparin in the therapeutic armamentarium against COVID-19 (Mycroft-West et al. 2020).
Another immunotherapeutic agent that was suggested is eculizumab, a mAb against C5 complement. Diffuse microvascular thrombi in multiple organs in COVID-19 non-survivors have been announced and even more important, thrombotic microangiopathy (TMA) can occur in many different clinical scenarios including pathogenic complement activation (Campbell and Kahwash, 2020). Altered complement system occurs in a number of pathologic settings, leading to diffuse thrombotic microangiopathy (TMA), microangiopathic hemolytic anemia, thrombocytopenia, and acute renal failure up to organ dysfunction. If given early, eculizumab therapy can reverse both renal and cardiac dysfunction (Campbell and Kahwash, 2020). Campbell and Kahwash (2020) suggest that complement inhibition could be a promising treatment for severe COVID19 by reducing the innate immune-mediated consequences of severe coronavirus infection, and it would pair well with direct anti-viral therapy.
Another suggested approach includes the off-label use of Camostat or Nafamostat mesylate, inhibitors of the host cell protease TMPRSS2, that could arrest coronavirus infections by controlling viral entry into the human cells. It has to be noted that, if on one side TMPRSS2 inhibitors could prevent SARS-CoV2 replication by blocking the fusion of the virus envelope with host cell surface membranes, they could also be effective in controlling pathological conditions correlated to COVID-19, such as coagulation and inflammation, based on their pharmacological properties. As it is well-known, Nafamostat mesylate has been used as a short-acting anticoagulant in patients with disseminative blood vessel coagulation, hemorrhagic lesions, and hemorrhagic tendencies (Maruyama et al.2011; Choi et al. 2015) due to its ability to competitively inhibit various enzyme systems, such as coagulation and fibrinolytic systems (thrombin, Xa, and XIIa), the KKS, the complement system, pancreatic proteases and activation of protease-activated receptors (PARs) (Kim et al. 2016). Similarly, Gabexate mesylate, binds and inhibits kallikrein, plasmin and thrombin (Tamura et al. 1977). Therefore, it was suggested the use of these drugs to prevent thrombosis and disseminated intravascular coagulation typical of COVID-19 patients (Tang et al. 2020b; Cui et al. 2020). Beyond their anticoagulant propriety, both Gabexate and Nafamostat mesylate show anti-inflammatory effects, which could be useful in COVID-19 uncontrolled inflammation (Tay et al. 2020). In particular, Gabexate mesylate decreases the production of inflammatory cytokines, such as TNF-α by attenuating NF-κB and JNK pathway activity, most probably through the proteolytic destruction of IκB (Yuksel et al. 2003); Nafamostat mesylate shows an anti-inflammatory effectin vitro , where it mediates the inhibition of lipopolysaccharide-induced nitric oxide production, apoptosis, IL-6 and IL-8 production in cell cultures (Kang et al. 2015; Choi et al. 2016). In this context, Camostat mesylate could be also useful to reduce the production of inflammatory cytokines due to SARS-CoV2 infection. Indeed, it was already found that Camostat mesylate reduces the release of IL-6 and TNF-α into cell supernatants infected with influenza virus (Yamaya et al. 2016).
5. Further therapeutic hypotheses.
So far, the published clinical observations of biochemical markers in COVID-19 patients include elevated LDH, D-dimer, bilirubin, high levels of pro-inflammatory cytokines that accompany interstitial pneumonia, renal and cardiac injury due to thromboembolic events, which also underlie septic shock that occurs in severe COVID-19 patients. Therefore, based on what described above and cross-linking biochemical with clinical outcomes, in this review we propose another therapeutic approach based on the inhibition of both BK receptors and HMWK. Icatibant is an antagonist of B2 receptor blocking the activity of the BK avoiding both the pro-inflammatory cytokine storm and cell proliferation; it is a drug approved by the European medical agency (EMA) for the treatment of angioedema in both children and adults (https://www.ema.europa.eu/en/documents/assessment-report/firazyr-epar-public-assessment-report_en.pdf). No specific adverse events have been reported, unless urticaria, nausea and headache, though, specific attention to be paid in patients with compromised cardiovascular system (i.e. ischemia and angina pectoris). However, preclinical studies did not show any genotoxic activity, alteration of the cardiac conduction and ischemia events or hemodynamic parameters. Nevertheless, it has been demonstrated that icatibant highly binds to B2 receptor, while the affinity to the analogous B1 receptor is at least 100 times lower. It has to be pointed out, though, that little is known about B1 receptor, which is the inducible receptor during inflammatory conditions, although several pre-clinical and phase I/II trials have been performed to evaluate possible use of agents targeting B1 receptor for inflammation-related diseases (Qadri and Bader, 2018).
In addition, another drug to point the attention on could be lanadelumab, which is a monoclonal antibody against the plasmatic kallikrein, which is important for the cleavage of HMWK into BK, and is involved in the coagulation as well as in the induction of the complement system (Figure 2). Actually, lanadelumab is used for the treatment of angioedema and has not reported adverse, severe events, other than hypersensitivity, myalgia and hepatic alteration of alanine aminotransferase (ALT) (https://www.ema.europa.eu/en/documents/assessment-report/takhzyro-epar-public-assessment-report_en.pdf). Differently from icatibant, lanadelumab could block upstream the activity of BK, avoiding the inflammatory and coagulation storm besides the complement system in SARS-CoV2 infected patients, likely preventing the exacerbation of COVID-19, in parallel with antiviral therapy.
In conclusion, we believe that the blockade of ACE2 increases not only the activity of angiotensin II on the cardiovascular system, but also the levels of DABK derived by HMWK. Therefore, the hypothesis to block the production of DABK upstream by blocking the metabolism of HMWK could be another option to face this tremendous pandemic event that affected whole world life style obliging to social limitations and stay-at-home politics.