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.