Defibrotide: potential for treating endothelial dysfunction related to viral and post-infectious syndromes

ABSTRACT

Introduction

Defibrotide (DF) is a polyribonucleotide with antithrombotic, pro-fibrinolytic, and anti-inflammatory effects on endothelium. These effects and the established safety of DF present DF as a strong candidate to treat viral and post-infectious syndromes involving endothelial dysfunction.

Areas Covered

We discuss DF and other therapeutic agents that have the potential to target endothelial components of pathogenesis in viral and post-infectious syndromes. We introduce defibrotide (DF), describe its mechanisms of action, and explore its established pleiotropic effects on the endothelium. We describe the established pathophysiology of Coronavirus Disease 2019 (COVID-19) and highlight the processes specific to COVID-19 potentially modulated by DF. We also present influenza A and viral hemorrhagic fevers, especially those caused by hantavirus, Ebola virus, and dengue virus, as viral syndromes in which DF might serve therapeutic benefit. Finally, we offer our opinion on novel treatment strategies targeting endothelial dysfunction in viral infections and their severe manifestations.

Expert Opinion

Given the critical role of endothelial dysfunction in numerous infectious syndromes, in particular COVID-19, therapeutic pharmacology for these conditions should increasingly prioritize endothelial stabilization. Several agents with endothelial protective properties should be further studied as treatments for severe viral infections and vasculitides, especially where other therapeutic modalities have failed.

KEYWORDS:

• Defibrotide
• endothelial dysfunction
• COVID-19
• influenza A
• viral hemorrhagic fever
• Ebola
• coronavirus
• complement cascade

1. Defibrotide (DF): current uses and mechanisms of action

Defibrotide (DF) is a mixture of 90% single-stranded phosphodiester oligonucleotides (length, 9–80mer; average, 50mer; average molecular mass, 16.5 ± 2.5 KDa) and 10% double-stranded phosphodiester oligonucleotides extracted through controlled depolymerization of porcine gut mucosa [1]. DF has pleiotropic properties, including anti-thrombotic, pro-fibrinolytic, anti-inflammatory, and protective effects on vascular endothelia [2].

DF is approved and utilized with marked benefit in the treatment of sinusoidal obstruction syndrome (SOS; formerly known as hepatic veno-occlusive disease (VOD)) in adults and children [3,4]. The terminology VOD/SOS is now used to describe the syndrome. VOD/SOS is believed to occur secondary to endothelial cell damage, apoptosis, and extrusion of endothelial cells (ECs) into sinusoids. As an endothelial protective agent, DF is believed to limit endothelial damage and thereby inhibit the progression of VOD/SOS [5].

In the treatment of VOD/SOS, the recommended dose of DF is 6.25 mg/kg body weight every 6 hours (25 mg/kg/day), given as a 2-h infusion. The treatment should be administered for a minimum of 21 days and continued until the symptoms and signs of severe VOD/SOS resolve [6]. Pharmacokinetic studies of Defibrotide in 52 healthy volunteers, at 6.25 mg/kg dose of given as a 2-h infusion, show that the maximum plasma concentrations peaked at the end of the infusion period (t_max = 2 h) and declined thereafter with a rapid clearance and most of the samples were undetectable 3.5 hours after the start of the infusion (t_1/2 = 0.71 ± 0.35 h). Pharmacokinetic modeling simulation analysis showed that defibrotide plasma concentrations do not accumulate upon multiple-dose administration, with doses up to fourfold the therapeutic dose. The volume of distribution ranges from 8.1 to 9.1 l, consistent with the intravascular compartment. In vitro studies have demonstrated that 93% of Defibrotide is bound to plasma proteins [1].

In patients with VOD/SOS, DF has proven safety and efficacy, with significant effects demonstrated in the reversal of endothelial dysfunction and multiorgan failure [2]. DF has also demonstrated efficacy in animal models of acute graft-versus-host disease (GvHD) incorporating lung injury and has therapeutic promise in conditions underpinned by cytokine release syndrome and endotheliitis [4,5,7–12]. In clinical studies of patients with VOD/SOS treated with DF, the most frequent adverse events were hemorrhage and hypotension. Given that hemorrhagic events and hypotension are likely after underlying disease processes in these patients, supported by the greater frequency of such events in control groups, these events do not appear to represent a signal of DF intolerability or dose-limiting toxicity. DF should not be initiated in patients with active bleeding; the coadministration of DF with systemic anticoagulant or fibrinolytic therapy is also generally contraindicated, although contraindicated administration has been pursued in the context of controlled studies [1].

Non-clinical data reveal no special hazard for humans based on conventional studies of safety pharmacology, repeated dose toxicity, genotoxicity, or carcinogenicity. In animal studies, the main findings were accumulation of vacuolated macrophages in the liver of dogs and in the liver, kidneys, and lymph nodes of rats. Studies to evaluate the effects on immune functions have been performed in mice treated orally or intraperitoneally with daily dosing for 4 weeks. A number of functional assays were performed following treatment, including assays for NK cell activity, anti-SRBC antibody response, macrophage cytotoxicity, and lymphocyte proliferation: no clear alteration in immune function was observed [1].

DF has been shown to interact directly with ECs, after which DF may be internalized by macropinocytosis. This process, classically considered the bulk and nonselective uptake of extracellular fluid, has received special attention in the last decade as an entry route for genetic material and drug delivery. Unlike receptor-mediated endocytosis and phagocytosis, macropinocytosis is not regulated through cargo–receptor interactions coordinating the recruitment and activity of specific effector molecules to sites at the plasma membrane [13]. With inhibition of macropinocytosis, however, the effects of DF on intracellular signaling are preserved, suggesting that DF can exert effects on the endothelial cell from the cell surface [7]. This interaction impacts multiple-signaling cascades within ECs, including reductions of both the p38/MAPK pathway and the PI3K/Akt pathway [7,8].

DF protects endothelial cells via modulation of several molecular pathways. Downregulation of histone deacetylases (HDACs) is thought to be a key mechanism by which defibrotide exerts its endothelial protective effects. Treatment with DF normalized the overexpression of HDAC1 in a dose-dependent manner in human umbilical cord vein endothelial cells exposed to sera of patients with chronic kidney disease, a condition associated with endothelial dysfunction; in these conditions, defibrotide inhibited the expression of ROS, ICAM-1, vWF, and Toll-like receptor 4 to levels consistent with normal endothelial function [14]. DF also decreases the expression of endothelial cell adhesion molecules critical to leukocyte transmigration and platelet activation, such as P-selectin, E-selectin, VCAM-1, and ICAM-1 [9]. DF reduces serum levels of cytokines and proinflammatory molecules including IL-6, IL-12, TNF-α, IFN-γ, VEGF, thromboxane A2, leukotriene B4, and ROS, furthering DF’s anti-inflammatory properties relevant in the treatment of numerous infectious syndromes [8,9]. DF potently inhibits heparanase expression and activity in a variety of settings [10]. Moreover, DF competes with heparan sulfate to diminish heparanase-mediated degradation, thereby preserving heparan sulfate on the glycocalyx and subendothelial basement membrane [15].

DF has demonstrated long-lasting, dose-dependent anti-thrombotic and thrombolytic activities in a variety of experimental approaches without causing a hemorrhagic state. Treatment with DF directly increases cyclic adenosine monophosphate production in platelets [16]. Furthermore, DF increases the expression of thrombomodulin, tissue plasminogen activator (t-PA), tissue factor pathway inhibitor, prostaglandin E2, prostaglandin I₂, and protein C plasmin activity, while simultaneously decreasing thromboxane A2, leukotriene B4, von Willebrand factor (VWF), plasminogen activator inhibitor-1 (PAI-1), platelet activating factor, and thrombin [7,8,17]. Notably, DF has been shown to counteract lipopolysaccharide-induced increases in PAI-1 levels and decreases in t-PA expression by ECs [18]. DF also decreases leukocyte and platelet aggregates in thrombi and attenuates the complement activation [19,20]. The sum of these multiple effects constitutes DF’s antithrombotic mechanism. The safety profile of DF is very favorable in this mechanism, as DF carries a low risk for treatment-associated thrombocytopenia or significant systemic bleeding [21].

The demonstrated long-term safety of DF, confirmed even in a high-risk pediatric population [22], and the pleiotropic effects of DF establish the drug as a strong candidate to treat the vascular complications of various post-infectious viral syndromes involving endothelial dysfunction, as well as other diseases driving vascular inflammation. For the purposes of this review article, PubMed/Medline™ databases were accessed at https://pubmed.ncbi.nlm.nih.gov from 12^th November 2020 to 12^th May 2021, in order to assess the latest pathophysiology of COVID-19 and other virology literature describing pathogens with endothelial effects.

2. Defibrotide and COVID-19

2.1. COVID-19 pathophysiology: components of endothelial dysfunction and coagulopathy

Coronavirus Disease 2019 (COVID-19), the infectious syndrome caused by the highly transmissible and pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in 2019 and remains a major threat to human health and public safety today. As of May 12^th, 2021, 159,896,332 cases of COVID-19 were recorded, with 3,321,888 deaths [23]. The infectious syndrome caused by SARS-CoV-2 includes a wide range of symptoms and potentially severe, life-threatening manifestations. Severe COVID-19 can manifest as an acute respiratory distress syndrome (ARDS), characterized by acute hypoxemic respiratory failure presentation, within 1 week of initial respiratory symptoms, and bilateral airspace disease on imaging, where cardiac failure is not the primary cause of respiratory failure [24].

In the early disease stages, severe hypoxemia may occur secondary to impaired regulation of pulmonary blood flow in the context of preserved alveolar compliance [25]; in late stages of disease, however, low compliance has been linked with increased mortality [26]. Some patients with COVID-19-related ARDS exhibit hyperperfusion of gasless tissue, demonstrative of impaired recruitment of pulmonary vasculature, with responses to prone positioning due to gravitational forces rather than dynamic changes in vascular tone [25]. Poor recruitment of oxygenated lung parenchyma may be attributed to endothelial cell dysfunction, loss of vascular tone, and diffuse formation of microthrombi throughout pulmonary microvasculature [27]. Compared to influenza-affected lungs, histopathological specimens of SARS-CoV-2 affected tissue demonstrate increased vascular neutrophil infiltration and neutrophil extracellular trap formation [28]. These findings point toward an innate immune axis as a potential driver of vascular inflammation and microthrombosis in COVID-19.

SARS-CoV-2 enters the cell through multiple mechanisms involving the viral spike protein. Known mechanisms include binding of furin-cleaved spike protein fragments to neuropilin-1 and binding of primed spike proteins to angiotensin-converting enzyme 2 (ACE2). NRP1 is expressed at high levels in the respiratory and olfactory epithelium, especially by epithelial cells and ECs [29]. ACE2 is expressed at high levels by type II pneumocytes of the lung and enterocytes of the small intestine, as well as ECs, pericytes, and vascular smooth muscle cells across numerous organ systems [27,30,31]. The distribution of these target molecules on cellular surfaces corresponds with the observed tropism of SARS-CoV-2, especially respiratory epithelium and endothelium; of note, SARS-CoV-2 infection of ECs in multiple organs has been substantiated by pathological findings from autopsies [27,32]. This histological evidence, along with biomarker data, indicates that direct infection of ECs is a likely contributor to the widespread endothelial barrier dysfunction and endothelitis observed in COVID-19 [33].

The entry of SARS-CoV-2 through binding of ACE2 disables a key homeostatic process employed by the endothelium to regulate Angiotensin II (Ang II) signaling. The ACE2 on the cell surface converts Ang II into Angiotensin 1–7, which binds the Mas receptor and downregulates NADPH oxidase, the generation of reactive oxygen species, and the vasoconstrictive and proinflammatory effects driven by Ang II through p38/MAPK activation. Binding of SARS-CoV-2 to ACE2 leads to endocytosis and proteolytic cleavage, resulting in decreased ACE2 expression and functionality, thereby diminishing the counterbalancing effect served by ACE2 [30]. The effects of Ang II are amplified at the endothelial cell, as the ratio of Ang II to Angiotensin 1–7 increases. Results of this phenomenon include upregulation of p38/MAPK, which activates the transcription of proinflammatory cytokines such as IL-6, TNF-α, and IL-1β, all of which are detected at high levels in patients with COVID-19 in comparison with controls. Unchecked p38/MAPK activity can promote endotheliitis and vasoconstriction, while also augmenting the viral life cycle by enhancing viral replication [34].

Other consequences of acute increases in Ang II include autocrine vasoconstriction, death of ECs, destruction of EC connections to pericytes, disruption of tight junctions between ECs, and increased production of reactive oxygen species, all of which drives adverse cardiovascular remodeling, increasing vascular permeability, and a procoagulant state that increases arterial and microvascular thrombosis [35,36]. These observations point toward Ang II as a potential mediator of sequelae observed during the infectious syndrome associated with SARS-CoV-2 and afterward. Ischemic stroke, for instance, has been observed at an alarmingly high rate in patients with COVID-19, with one study reporting a 7.6-fold increase in the risk of stroke of patients with COVID-19 compared to influenza [37]. Throughout the pandemic, many centers have reported an increased incidence of large vessels and cryptogenic strokes [38]. These thrombotic events may be linked to alterations in Ang II signaling associated with SARS-CoV-2 infection, and modulation of Ang II signaling is being investigated as a therapeutic strategy during the infectious syndrome and subsequent infection. A prominent retrospective study demonstrated that, among hospitalized patients with COVID-19 and hypertension, those using angiotensin-converting enzyme inhibitors (ACE inhibitors) and angiotensin-II receptor blockers (ARBs) demonstrated a lower risk of all-cause mortality as compared to non-users [39].

COVID-19-related endotheliitis is also characterized by elevations of numerous endothelial-derived proinflammatory and procoagulant factors. These include integrin ligands ICAM-1, and VCAM-1, which are upregulated in COVID-19, with dramatic elevations seen in severe cases [40]. Overexpression of these molecules promotes increased leukocyte activation, adhesion, leukocyte extravasation, and subsequent tissue inflammation, contributing to damage of both endothelium and proximal lung parenchyma. Furthermore, endothelial barrier dysfunction and rupture permit interstitial and alveolar edema leading to ARDS [26,27,33]. A cross-sectional study of patients with confirmed COVID-19 demonstrated significant elevation of VWF antigen, factor VIII, soluble P-selectin, and soluble thrombomodulin [41]. Increased expression and release of these hemostatic factors from damaged ECs spark a widespread activation of platelets and the coagulation cascade, driving the profound prothrombotic state observed in COVID-19. This hypercoagulable state leads to dramatically increased rates of thromboembolism, elevated levels of D-dimer and fibrinogen, and prolonged prothrombin time and activated partial thromboplastin time [42]. Recent findings indicate that serum from critically ill COVID-19 patients activates platelets in an immune complex-mediated reaction through crosslinking of the Fcγ receptor IIA (FcγRIIA) [43]; this evidence supports a role for platelets as one of the key drivers involved in the increased thromboembolic risk in severe COVID-19 patients [44].

COVID-19-related endotheliitis is also associated with the upregulation of heparanase activity [45]. Heparanase-1 (Hpa1) is an endo-β-glucuronidase that degrades the heparan sulfate scaffold of the glycocalyx and subendothelial basement membrane, increasing endothelial dysfunction and allowing extravasation of activated immune cells into the extravascular compartment [15,46]. Heparanase-2 (Hpa2), by contrast, lacks endoglycosidase activity and inhibits activity of Hpa1 [47]. Studies have demonstrated that COVID-19 is associated with an acquired Hpa2 deficiency and a subsequent increase in the ratio of Hpa1 to Hpa2, precipitating hyperactivity of Hpa1 and degradation of the endothelial glycocalyx [48]. This degradation renders the endothelium increasingly vulnerable to inflammatory damage and subsequent dysfunction, making heparanase an important target in the treatment of COVID-19.

SARS-CoV-2 infection has also been associated with increased complement cascade activation, supported by both preclinical and clinical studies. Preclinical work has demonstrated that SARS-CoV-2 spike proteins bind directly to heparan sulfate to activate the alternative complement pathway on cell surfaces [49]. In humans, immunohistochemistry analyses of COVID-19-associated cutaneous lesions have revealed the colocalization of SARS-CoV-2 spike proteins with C4d and C5b-9 in the microvasculature [50]. In sera from 46 patients with severe COVID-19, proteomic, and metabolomic characterizations revealed upregulation of membrane attack complex proteins including C5, C6, and C8 [51]. These findings suggest a role for complement activation in endothelial injury and coagulopathy, highlighting the complement cascade as an additional target for therapeutic intervention in COVID-19.

In addition, recent studies have identified neutrophil extracellular traps (NETs) as a significant contributor in COVID-19 pathophysiology. NETs consist of extracellular chromatin webs, microbicidal proteins, and oxidant enzymes released by neutrophils as a component of the response to infections [52]. When produced in an unregulated manner, as seen in some instances of ARDS secondary to viral infection, NETs have the capacity to induce thrombosis and widespread inflammation [53]. Sera from patients with severe COVID-19 has significantly elevated myeloperoxidase DNA and citrullinated histone H3, both of which are specific markers of NETs [54]. Together with clinical data from these patients, these findings suggest that NETs also contribute to the pathogenesis of thrombosis and endothelial damage in COVID-19, establishing NETs as yet another therapeutic target COVID-19.

2.2. Defibrotide in the treatment of COVID-19

DF stands as an important candidate for the treatment of COVID-19, based on the pathophysiology of COVID-19 and DF’s pleiotropic mechanisms of action (Figure 1) [55]. As DF can interact directly with ECs to influence cell signaling, DF may reduce activation of the p38/MAPK pathway associated with COVID-19-related endotheliitis, thereby regulating endothelial dysfunction driven by the loss of ACE2 and potentially limiting viral replication within infected ECs [34]. In this manner, DF may also stand to counterbalance the deleterious effects of excessive Ang II signaling, achieving a similar therapeutic purpose to ACE inhibitors and ARBs in this respect.

Figure 1. Pathophysiologic mechanisms of COVID-19 and proposed mechanisms of defibrotide in treatment of COVID-19. SARS-CoV-2 directly infects endothelial cells by binding to ACE2, leading to endocytosis and degradation of ACE2, and loss of ACE2 permits dysregulation of the Angiotensin II signaling axis [34]. Angiotensin-II levels are increased in patients with SARS-CoV-2, likely secondary to decreased conversion to Angiotensin 1–7; increased Angiotensin-II levels lead to activation of p38/MAPK, which contributes to endotheliitis, vasoconstriction, production of inflammatory cytokines, expression of adhesion molecules VCAM-1 and ICAM-1, release of Weibel Palade bodies, and up-regulation of Heparanase-1 [36]. Defibrotide inhibits activity of p38/MAPK, reduces production of proinflammatory cytokines, decreases expression of adhesion molecules, diminishes Weibel Palade body release, and limits expression and activity of Heparanase-1 [2,7–12]. (Created with BioRender.com)

As DF decreases the expression of endothelial cell adhesion molecules including P-selectin, E-selectin, VCAM-1, and ICAM-1, allow which are elevated in COVID-19, DF may serve to decrease inflammation and the procoagulant state driven by endothelial dysfunction in COVID-19 [9]. Treatment with DF also reduces IL-6, IL-12, TNF-α, IFN-γ, VEGF, thromboxane A2, leukotriene B4, and ROS, furthering DF’s anti-inflammatory properties relevant in the treatment of COVID-19 [7–9].

DF also stands to reduce thrombosis and enhance fibrinolysis in COVID-19 by increasing t-PA and thrombomodulin expression, and simultaneously decreasing VWF and PAI-1, which are notably elevated in the context of severe disease [2,7,9,13,15]. Importantly, studies of patients with severe COVID-19 have demonstrated decreased fibrinolysis in response to elevated fibrinolytic inhibitors [56], contributing to an increased risk for thrombosis. DF stands to directly counteract this phenomenon through inhibition of PAI-1.

As Hpa1 activity becomes unchecked through acquired Hpa2 deficiency in COVID-19, DF stands to attenuate heparanase hyperactivity and promote restoration of endothelial integrity and function. DF may also interfere with SARS-CoV-2 interaction with heparan sulfate to diminish viral adherence and complement activation. In patients with post-transplant thrombotic microangiopathy, presenting as atypical hemolytic uremic syndrome, treatment with DF reduced complement activation, and associated vascular damage [11]. DF may similarly regulate the complement hyperactivation associated with severe COVID-19. In fact, two patients with pediatric inflammatory multisystem syndrome temporally associated with SARS-CoV-2 infection received DF, leading to symptomatic resolution and normalization of complement activation and fibrinolysis [12]. Furthermore, as NETs have been identified as a contributor to COVID-19-related thrombosis, DF may be beneficial in its capacity to neutralize NETs and reduce associated endothelial death; in vitro studies have demonstrated that DF directly engages cationic components of NETs, including histones, to counteract endothelial cell activation and cell death driven by these components [57].

This demonstrates the myriad of targets for DF specific to the pathophysiology of COVID-19, and this scientific background is the rationale for several studies including the Spanish DEFACOVID phase IIb randomized, double-blind, placebo-controlled, clinical trial (clinicaltrials.gov: NCT04348383), as well as studies in Italy, Ireland, Germany, and the United Kingdom, with over 220 patients enrolled and treated to date across these various protocols. Clinical trials in the United States are also underway at the University of Michigan in Ann Arbor, MI, USA (clinicaltrials.gov: NCT04530604) and Brigham and Women’s Hospital in Boston, MA, USA (clinicaltrials.gov: NCT04652115). Patients receiving DF (and being administered with either prophylactic or therapeutic dosing of low molecular weight or unfractionated heparins) are being comprehensively assessed for inflammatory markers, including heparanase, inflammatory cytokines, immune subpopulations, and prothrombotic/antifibrinolytic markers including VWF and soluble thrombomodulin. Preliminary clinical results of safety and efficacy, as well as biomarker studies, have been encouraging, in patients treated in Spain, the United States, and Italy; in aggregate, these data may confirm the effects of DF on multiple targets that are fundamental components of pathobiology in COVID-19.

3. Defibrotide and other viral infectious syndromes driving endothelial dysfunction

3.1. Defibrotide in the treatment of severe influenza A virus

Influenza A viruses infect the upper respiratory tract and commonly result in a self-limited infection with a mild course. In severe cases, however, influenza A may cause viral pneumonia, progressing to pulmonary edema, hypoxemia, and respiratory failure. Endothelial dysfunction of pulmonary microvasculature likely underlies these phenomena [58]. Additional therapies are required for these cases as antiviral drugs have limited effect on mortality at late stages of influenza A infection [59]. In 2019, a meta-analysis of disease burden estimated an average of 389,000 deaths (with an uncertainty range from 294,000 to 518,000) from influenza globally per year during the study period [60].

Endothelial dysfunction in severe influenza may result from elevated cytokines and chemokines, including TNF-α, IL-6, and IL-1β, which can upregulate trypsin and thereby enhance degradation of endothelial tight junction proteins, altering barrier function, and driving increased vascular permeability [58]. ECs also become activated in the context of influenza A infection, with increased levels of circulating VWF, increased coagulation, and necrosis observed in the context of infection [61]. These sequelae of infection can have critical effects on pulmonary and extrapulmonary tissues, driving the high morbidity and mortality observed in severe influenza A infection.

These features of influenza A infection establishes this stage of disease as a candidate for DF treatment. As DF stands to reduce levels of TNF-α, IL-6, and IL-1β, decrease circulating VWF, decrease thrombosis, and enhance fibrinolysis, DF might play a role in ameliorating symptoms associated with pulmonary vascular compromise. Given that type A influenza viruses have caused numerous pandemics with massive death tolls, we propose that investigation of DF in the treatment of severe infections is warranted to mitigate future mortality.

3.2. Defibrotide in the treatment of viral hemorrhagic fever

Viral hemorrhagic fevers are caused by four families of RNA viruses: arenaviruses, bunyaviruses, filoviruses, and flaviviruses. These viruses have diverse pathogenic mechanisms, resulting in varying disease severity and presentation [62]. The Ebola virus, a filovirus, accounts for few infections annually but stands among the most lethal viruses known, with reported fatality rates ranging from 24% to 100% in different outbreaks [63]. Dengue virus, a flavivirus, accounts for an estimated 390 million infections per year, of which only 96 million manifests with disease symptoms [64]. Hantavirus, a bunyavirus, manifests itself as two hemorrhagic febrile diseases: hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) [65]. While these diseases differ in severity, incidence, and presentation, their severe manifestations share features of vascular instability, hemorrhage, and cytokine storm [62]. These viruses are noteworthy, furthermore, for their interaction with ECs, such as Ebola virus, dengue virus, and Hantavirus each directly infect ECs in the course of disease pathogenesis [66–68].

Severe manifestations of the Ebola virus including overt hemorrhage, shock, lymphopenia, and thrombocytopenia. Excessive proinflammatory cytokine production and absence of immune responses are recognized as major factors in the development of these phenomena [69]. As the Ebola virus infects monocytes, macrophages, dendritic cells, and ECs, direct infection may also contribute to disruption of endothelial homeostasis [70]. Widespread endothelial activation and dysfunction are an integral component of pathogenesis, leading to loss of vascular integrity, increased vascular permeability, and activation-dysregulation of the coagulation pathway [66]. This presents the endothelium as an attractive target in therapeutic approaches for Ebola virus infection, a disease with few established therapeutic strategies. Indeed, statins and ARBs have been utilized for their endothelial protective effects in the treatment of Ebola virus infection, with reported improvements in patient mortality [71]. As DF also protects endothelium and may similarly downregulate the Ang-II signaling axis, DF may also achieve therapeutic effects in patients with Ebola virus infection. As cytokine storm is recognized as a critical factor in Ebola virus disease; moreover, DF may stand to modulate the impact of excessive cytokine production on the endothelium. To our knowledge, DF has not been utilized in the treatment of these infections.

While the vast majority of cases are asymptomatic, dengue virus infection also has severe manifestations including hemorrhage, shock, and thrombocytopenia, secondary to widespread capillary leakage [72]. Multiple processes can contribute to vascular leakage and instability in Dengue virus infection, including direct infection of ECs and antibody-dependent enhancement, wherein poorly neutralizing antibodies drive immune hyperactivation and subsequent cytokine storm [73]. Dengue virus depends on envelope protein binding to heparan sulfate moieties for entry into target cells [68]. Dengue virus infection of ECs, furthermore, contributes to alteration of barrier functions, directs immune cell targeting of ECs, increases cytokine and chemokine responses, and augments viral load [67]. Strategies to prevent dengue virus infection of ECs stand to modulate this significant contribution to disease pathogenesis. DF, for one, might limit viral entry through inhibition of heparanase activity and subsequently decreased heparan sulfate expression on EC surfaces. DF may serve additional benefits in the treatment of dengue virus infection for its alteration of cytokine production and endothelial responses to cytokine storms, as observed in antibody-dependent enhancement. These effects may improve endothelial homeostasis to reduce hemorrhage, increase hemostasis, and promote hemodynamic stability.

The severity of the Hantavirus infection also ranges from mild to severe, with reported case fatality rates up to 35%. HCPS is marked by lung edema and acute hypoxic respiratory failure which may require supplemental oxygen, intubation with mechanical ventilation or extracorporeal membrane oxygenation. HFRS, on the other hand, presents with renal impairment requiring hemodialysis in severe cases [74]. Additional severe manifestations of Hantavirus infection include shock and diffuse hemorrhage due to increased vascular permeability. Vascular endothelial growth factor (VEGF) has been identified as a key factor in Hantavirus infection, as pathogenic hantaviruses augment endothelial permeability in response to VEGF signals [75]. Hantavirus targets beta-integrins, which normally form complexes with VEGF receptors, thereby interfering with the normal regulation of VEGF signaling at the endothelial cell surface through this mechanism [67]. The resultant increase in VEGF signaling contributes to widespread vascular leakage in hantavirus-related diseases. In vitro studies have demonstrated reduced production of VEGF by stromal cells in response to the DF treatment [10], a potentially advantageous effect in the treatment of hantavirus infection. Hantaviruses also binds VE-cadherins on the surface of ECs as one route of cell entry, disrupting inter-endothelial adherens junctions [65]. In vitro studies with DF have also shown that DF can suppress the VE-cadherin expression on ECs [8], and DF may thereby limit the infectivity of hantavirus by decreasing its binding target on ECs. In the context of hantavirus infection, however, this effect of DF may stand to further destabilize adherent junctions.

While the established mechanisms and clinical effects of DF demonstrate its indications in these syndromes, the therapeutic effects of DF may be limited by the challenging features of these diseases as the extent of vascular instability, hemorrhage, and thrombocytopenia is often profound [62]. Furthermore, hemorrhagic complications have been observed in patients treated with DF, which may impart caution in the treatment of severe hemorrhagic disease [76]. However, one study of DF in 88 patients with severe VOD, all with severe thrombocytopenia (platelets <20,000/#x1D6CD;l), reported no worsening of clinical bleeding as measured by hemodynamic instability, acute transfusion requirement, or end-organ compromise [77]. These observations reaffirm the potential efficacy of DF in patients with compromised hemostasis.

4. Conclusion

The effects of DF in reversing endothelial activation, dysfunction, and diminishing vascular inflammation present DF as a strong candidate for the treatment of numerous disease processes, including COVID-19, severe influenza A infections, and viral hemorrhagic fevers. Trials with DF in the treatment of severe COVID-19 are ongoing, with encouraging preliminary results. We hope this discussion will prompt further consideration of DF and other agents as experimental treatments for numerous diseases involving endothelial dysfunction, potentially warranting future trials in these areas.

5. Expert opinion

Endothelial dysfunction has been identified as a critical component in the pathogenesis of numerous infectious syndromesin particular, COVID-19, which remains a major and growing human health crisis today. Influenza A viruses, furthermore, have also caused global pandemics and continue to challenge public health. For these pathogens and others, therapeutic research has largely focused on vaccination strategies to enhance host immunity, antiviral agents to inhibit viral infection or replication within hosts, and anti-inflammatory molecules aimed at decreasing host cytokine production or neutralizing cytokines in circulation [78,79]. Despite innovations in these areas, a significant percentage of patients with severe viral infections progress to ARDSin particular, those infected by SARS-CoV-2, and require intensive care. Endothelial dysfunction stands as a key frontier requiring further investigation in order to improve care and the outcome of patients with these potentially devastating syndromes.

We propose that agents with endothelial protective properties be further studied in the treatment of endothelial damage and endothelialitis after viral infection, especially in cases where other therapeutic modalities have failed. As a pleiotropic drug with numerous favorable effects on endothelial dysfunction, DF stands as a promising therapeutic candidate for several syndromes, including but not limited to those discussed in this review. While the approved use of DF principally targets sinusoidal endothelium of the liver as part of the treatment of VOD/SOS, the stabilizing and protective effects of DF should be considered for restoring endothelial function in numerous tissues, including the lungs and kidneys, especially as DF has demonstrated efficacy in patients with significant renal injury, pulmonary dysfunction, and related systemic endotheliitis [80].

Recent data has demonstrated a role for antibody-mediated platelet activation in the pathogenesis of COVID-19-related thrombosis, wherein platelet activating anti-platelet factor 4 and heparin antibodies engage FcγRIIA to drive platelet activation [43]. Since the FcRγ-chain is physically associated with glycoprotein VI, the central activating collagen receptor in platelets, some promising pharmacological strategies blocking this receptor have emerged to effectively inhibit thrombotic events without effect on thrombus formation [81]. DF, heparin, anti-human GPVI F(ab) fragments, or soluble GPVI-Fc fusion proteins could interfere with immune complexes on FcγRIIa receptors by blocking the collagen-binding site. This might subsequently further the effects of these agents in reducing platelet activation, with the benefit of no bleeding time prolongation.

DF should also be evaluated as a prophylactic treatment in patients at high risk for multiorgan failure due to widespread endothelial dysfunction, especially in the setting of severe COVID-19. Importantly, defibrotide prophylaxis has demonstrated efficacy in preventing VOD/SOS, especially in the context of salvaged and second-round hematopoietic stem cell transplantation [82]. As a therapeutic strategy for COVID-19, DF prophylaxis, or early intervention may offer superior outcomes to DF treatment after respiratory failure and intubation, at which stage endothelial damage may be both advanced and relatively irreversible. In addition, new schedules of DF infusions are being investigated in COVID-19 patients, such as intermittent vs. continuous intravenous infusion of the drug, as this may both decreased provider exposure to infection in the acute setting and offer potential therapeutic advantages. Studies have identified markers with a prognostic potential, including blood levels of VWF and soluble thrombomodulin [41]. Concentrations of soluble thrombomodulin may be utilized to predict severe clinical outcomes in patients with COVID-19 and so demarcate a threshold for the preemptive administration of DF.

In addition to DF, other pharmacologic agents have potential as treatments targeting endothelial dysfunction, particularly through modulation of the Ang-II and p38/MAPK signaling axis. As antagonists of the Ang-II pathway, ACE inhibitors and ARBs stand to inhibit the signaling cascade initiated by Ang-II at the EC, which leads to p38/MAPK activation. In the treatment of endotheliitis of viral etiology, ACE inhibitors and ARBs should be studied for their capacity to downregulate this axis and mitigate the contribution of Ang-II to inflammatory pathologies involving the endothelium. We also propose that ACE inhibitors and ARBs should be investigated as both pre-exposure and post-exposure prophylaxis for COVID-19, as overactivity of Ang-II may play a central role in both acute and chronic manifestations of the syndrome. Direct inhibitors of p38/MAPK may also be considered in this capacity, as several approaches for direct p38/MAPK inhibition have been developed and studied in numerous contexts [83].

Applications of these agents should be considered beyond viral and post-infectious syndromes, to include chronic inflammatory conditions affecting vasculature. Of note are the vasculitides, many of which have relapsing and remitting courses, with limited therapeutic modalities outside of corticosteroids and immunomodulators [84]. For antineutrophil cytoplasmic antibody (ANCA)-associated vasculitides, the principal therapies for induction of remission are methotrexate and cyclophosphamide, both of which carry significant toxic effects [85]. DF, on the other hand, bears a remarkable safety profile and with few adverse effects. DF might serve as a therapeutic adjuvant enhancing induction of remission by minimizing endothelial damage and reducing requirements for cytotoxic therapy, such as methotrexate and cyclophosphamide. DF might also be utilized at basal levels to prevent relapse of vasculitisfor instance, in systemic lupus erythematosus, where p38/MAPK activity may have a critical role in disease pathogenesis [86].

Kawasaki's disease is another form of vasculitis that has notably emerged in pediatric patients with COVID-19 [87]. The pathogenesis of Kawasaki disease includes activation of NF-κB and p38/MAPK as drivers of cytokine production and metalloproteinase expression, leading to vascular lesions. DF stands to downregulate this process through the inhibition of both NF-κB and p38/MAPK [88]. Complement cascade activation has also been reported to play an important role in endothelial dysfunction during complex vasculitis and SARS-CoV-2 infection, and inhibitors of complement activation drugs have been proposed as targeted therapies [89]. In this regard, DF has been shown to reduce complement activation in thrombotic microangiopathies and so decrease the levels of C5b-C9 complex in patients with the pediatric inflammatory multisystem syndrome associated with COVID-19 [12].

Hence, due to its multitargeted, broadly positive effects on endothelium and its restoration of fibrinolytic balance, DF may be an ideal candidate to treat diseases underpinned by inflammatory signaling cascades, cytokine release, and thrombo-inflammation. DF is already approved in adults and children more than 1 month of age for the treatment of severe VOD/SOS with multi-organ dysfunction, and its consistently favorable safety profile is an important factor to consider in performing future clinical studies [12]. Taken together, there is substantial potential for naturally derived polydeoxyribonucleotide to treat and improve outcomes for patients with thrombo-inflammatory disorders, warranting comprehensive evaluation to include ex vivo and clinical studies.

Preclinical research using in vitro systems is difficult to envisage due to the difficulty of adequately modeling such a complex human disease. For example, in vitro studies of virus-exposed endothelial cells that are pharmacologically modulated with DF may be attractive, but this methodology is likely to provide data that do not reflect the human disease. The same considerations apply to the development of animal models: the complexity of human disease and interaction with other pathological processes would be challenging to recapitulate in mice, with further difficulties arising in the validation of such models. Correlative studies using biological samples from patients treated with DF remain a strong option to identify markers with prognostic roles. Valuable information may also be generated by bulk and single-cell RNA sequencing to dissect the expression profiles of different peripheral blood mononuclear cells in the context of COVID-19.

The aforementioned studies ongoing in Europe and the United States include one randomized placebo-controlled trial (clinicaltrials.gov: NCT04348383) and several non-randomized trials; to date, these trials have shown that DF treatment of patients with severe COVID-19 does not confer safety issues. These studies are also assessing clinically significant efficacy endpoints and are expected to provide efficacy data. The varying complexity, heterogeneity, and clinical severity of patients with COVID-19 has become increasingly apparent throughout these studies. Therefore, in the near future, the clinical development of DF will require a specific focus on its efficacy in well-defined and homogeneous cohorts of COVID-19 patients. In order to obtain robust efficacy data, homogeneous cohorts of patients should be selected on the basis of the WHO ordinal scale and stringent oxygen requirements.

We recommend that randomized phase two-thirds trials be prioritized, utilizing DF in addition to standard of care therapy. When non-randomized trials are performed, a robust contemporary control group of patients should be used. These studies should also explore the impact of DF on the long-term sequelae of SARS-CoV-2 infection, including the effects on neurological, cardiac, and pulmonary complications. Appropriate studies are also required to explore DF efficacy in onco-hematological patients experiencing SARS-CoV-2 infection, as these patients represent an especially fragile population that is likely to experience diminished protection with vaccination and vulnerability to endothelial complications [90].

Article Highlights

• Defibrotide (DF) has numerous effects on endothelium with potential benefits specific to the pathophysiologic components of several viral syndromes.

• Endothelial dysfunction is a critical component of COVID-19 pathobiology, underpinning numerous systemic phenomena in the disease.

• Other viruses that drive disruption of endothelial homeostasis include influenza A and viral hemorrhagic fevers.

• Clinical studies of DF in the treatment of COVID-19 are ongoing and should continue, with priority to randomized phase two-thirds trials.

• Agents with endothelial protective properties, such as DF, angiotensin-converting enzyme inhibitors, angiotensin-II receptor blockers, and p38/MAPK inhibitors should be further studied as treatments for severe viral infections and vasculitides.

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Declaration of interest

R Jara declares honorarium from Edwards Lifesciences and Getinge. M Palomo declares speaker’s fees from Jazz Pharmaceuticals. R Baron serves on the Advisory Boards for Merck and Genentech. G Yanik has grant support from Jazz Pharmaceuticals. M Díaz-Ricart declares research funding and speaker’s fees from Jazz Pharmaceuticals. P Richardson declares service on advisory committees for Jazz Pharmaceuticals. C Carlo-Stella has received research support from ADC Therapeutics; has served as consultant or advisor for Servier, Novartis, ADC Therapeutics, Roche, Sanofi, Karyopharm Therapeutics; and has received honoraria for speaker engagements from Bristol-Myers Squibb, Merck Sharp & Dohme, Janssen Oncology, Astra-Zeneca, Incyte, and Gilead Science. JM Moraleda reports grants and consulting honoraria from Jazz Pharmaceuticals during the conduct of the study; consulting honoraria and travel expenses from Gilead, consulting honoraria from Novartis, Sandoz, and Takeda, outside the submitted work. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership, or options, expert testimony, grants, or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers in this manuscript have no relevant financial or other relationships to disclose

Acknowledgments

We acknowledge Ashley Ford for administrative assistance during this endeavor.

Additional information

Funding

This work has been funded in part by the Instituto de Salud Carlos III (ISCIII) of the Spanish Ministry of Science and Innovation, through the project COV20/0039 and by a research grant from Jazz Pharmaceuticals, as well as the Paula, and Rodger Riney Foundation, and the RJ Corman Multiple Myeloma Research Fund.