key: cord-342873-eu7f0zjm
authors: Yeleswaram, Swamy; Smith, Paul; Burn, Timothy; Covington, Maryanne; Juvekar, Ashish; Li, Yanlong; Squier, Peg; Langmuir, Peter
title: Inhibition of cytokine signaling by ruxolitinib and implications for COVID-19 treatment
date: 2020-06-23
journal: Clin Immunol
DOI: 10.1016/j.clim.2020.108517
sha: 
doc_id: 342873
cord_uid: eu7f0zjm

Approximately 15% of patients with coronavirus disease 2019 (COVID-19) experience severe disease, and 5% progress to critical stage that can result in rapid death. No vaccines or antiviral treatments have yet proven effective against COVID-19. Patients with severe COVID-19 experience elevated plasma levels of pro-inflammatory cytokines, which can result in cytokine storm, followed by massive immune cell infiltration into the lungs leading to alveolar damage, decreased lung function, and rapid progression to death. As many of the elevated cytokines signal through Janus kinase (JAK)1/JAK2, inhibition of these pathways with ruxolitinib has the potential to mitigate the COVID-19–associated cytokine storm and reduce mortality. This is supported by preclinical and clinical data from other diseases with hyperinflammatory states, where ruxolitinib has been shown to reduce cytokine levels and improve outcomes. The urgent need for treatments for patients with severe disease support expedited investigation of ruxolitinib for patients with COVID-19.

Coronaviruses are common human and mammalian positive-strand RNA viruses [1] . In December 2019 a new strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was identified as the pathogenic cause of coronavirus disease 2019 (COVID-19). As of April 21, 2020, there were 2,397,217 confirmed cases of and 162,956 deaths from COVID-19 worldwide [2] .

Although most patients with COVID-19 experience only mild-to-moderate disease, approximately 15% progress to severe pneumonia, and 5% develop acute respiratory distress syndrome (ARDS), septic shock, and/or multiple organ failure, which can rapidly lead to death [3] . No vaccines or specific antiviral treatments have yet proven effective against COVID-19; current clinical management consists of palliative treatments with organ support to moribund patients. Understanding the immunopathologic mechanism and appropriately targeting the key pathways involved has the potential to minimize pulmonary immune injury and mortality.

Following infection, SARS-CoV-2 binds to alveolar epithelial cells and activates innate and adaptive immune responses [1] . CD4+ and CD8+ T cells play an important role in balancing the adaptive immune response against pathogens and the potential development of autoimmunity or excessive inflammation [4] . Activation of cytotoxic CD8+ T cells is vital for clearing virus from infected cells but also induces immune injury in tissues [5] . On the other hand, rapidly activated CD4+ T cells become pathogenic T helper 1 cells that generate proinflammatory cytokines and chemokines [6] . The marked production of cytokines and chemokines leads to recruitment of lymphocytes and leukocytes to the site of infection; however, a massive release of cytokines can occur as part of a positive feedback loop associated with immune response amplification, resulting in cytokine release syndrome, or a "cytokine storm" [1] .

J o u r n a l P r e -p r o o f Journal Pre-proof Cytokine storm appears to be a common manifestation in severe COVID-19. Compared   with healthy controls, patients with COVID-19 experienced elevated plasma levels of interleukin   (IL)-1β, IL-1Rα, IL-2, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-13, IL-17, granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10/CXCL10), monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, platelet-derived growth factor-BB, MIP-1β, basic fibroblast growth factor, tumor necrosis factor alpha (TNF-α), and vascular endothelial growth factor [7] . Furthermore, patients admitted to the intensive care unit had higher plasma levels of IL-2, IL-7, IL-10, G-CSF, IP-10, MCP-1, MIP-1α, and TNF-α compared with patients who did not require critical care. The high levels of pro-inflammatory cytokines lead to massive immune cell infiltration of the lungs in patients with COVID-19, resulting in alveolar damage, decreased lung function, and rapid progression to death [7, 8] .

Indeed, respiratory failure from ARDS is the leading cause of mortality associated with COVID-19 [9,10].

Among the cytokines implicated in COVID-19-associated cytokine storm, several signal predominantly via the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. IL-2, IL-6, IL-7, IL-10, IFN-γ, G-CSF, and GM-CSF are dependent on JAK1, JAK2, or both; furthermore, IP-10, MCP-1, and MIP-1α are IFN-γ dependent [11, 12] . TNF-α has been shown to activate JAK/STAT signaling in a TNF receptor 1-dependent manner [13, 14] .

These data suggest that JAK inhibition could ameliorate the hyperinflammatory state associated with severe COVID-19.

Ruxolitinib (INCB018424) is a selective inhibitor of JAK1 and JAK2 that is approved for the treatment of myelofibrosis (MF), polycythemia vera, and steroid-refractory acute graft-versus-J o u r n a l P r e -p r o o f Journal Pre-proof 6 host disease (SR-aGVHD) [15] . The in vitro pharmacology of ruxolitinib has been studied using enzymes and cell-based assays. In biochemical assays, ruxolitinib has demonstrated potent inhibition of JAK1 and JAK2, with half maximal inhibitory concentration (IC50) values in the single digit nanomolar range (Table 1 ) [16] . Ruxolitinib has consistently demonstrated robust inhibition of JAK/STAT signaling in cell-based assays. In whole blood assays for the inhibition of phosphorylated STAT3 following stimulation with IL-6 (a prototype cytokine that signals through The effect of ruxolitinib was also evaluated in a major histocompatibility complexmismatch mouse model of aGVHD characterized by significant upregulation of inflammatory cytokines (IFN-γ, TNF-α, and IL-6) in peripheral blood ( Fig. 1 ) [18] . Ruxolitinib (60 mg/kg BID) treatment significantly reduced the inflammatory cytokine milieu in circulation. No differences J o u r n a l P r e -p r o o f Journal Pre-proof 7 were observed in the proportion of peripheral CD4+ or CD8+ T cells in groups treated with ruxolitinib ( Fig. 2) , and there were no detrimental effects on donor engraftment. These alloreactive GVHD data are consistent with previous reports suggesting that ruxolitinib has immunomodulatory but not immune-depleting effects [17, 19] .

Data supporting reduction of the cytokine burden has emerged from multiple clinical studies with ruxolitinib. MF is a type of myeloproliferative neoplasm with progressive cytopenias, bone marrow fibrosis, and splenomegaly, driven by a hyper-inflammatory state [20] . Plasma levels of pro-inflammatory cytokines, including IFN-α, IL-6, IL-8, IL-16, IL-18, as well as Creactive protein, intracellular adhesion molecule 1, vascular adhesion molecule 1, and matrix metalloproteinase 2, were significantly higher at baseline in patients with MF compared with healthy controls (Fig. 3A) . After one cycle of therapy with ruxolitinib (28 days), levels of these pro-inflammatory biomarkers decreased (Fig. 3B ). These changes were not related to JAK2 mutational status or disease subtype, indicating that the effects of ruxolitinib in patients with MF are reflective of a broad anti-inflammatory effect. In addition, constitutive phosphorylation of STAT3 and/or STAT5 was observed at baseline in patients with MF, and a dose-and timedependent reduction of phosphorylated STAT3 was observed after treatment with ruxolitinib. high-risk MF [20, 21] . Anemia and thrombocytopenia were the most frequent any-grade and grade 3-4 adverse events experienced.

SR-aGVHD is a condition characterized by an allogeneic hyperinflammatory response that can lead to organ damage and death [22] . Ruxolitinib was approved for SR-aGVHD based on the results of the phase 2 REACH1 trial [23] . Ruxolitinib Proteomics analysis revealed robust changes in the expression of inflammatory mediators after treatment with ruxolitinib and corticosteroids, with IL-2-receptor alpha among the most significantly downregulated proteins [24] .

Hemophagocytic lymphohistiocytosis (HLH) is another disease with elevation of many pro-inflammatory cytokines (eg, IFN-γ, IL-2, IL-6, IL-10, IL-18, IP-10, MIP-1α, and TNF-α) that frequently results in cytokine storm [25, 26] . Ruxolitinib (5-20 mg BID) has demonstrated improvement in symptoms and inflammatory markers in the treatment of patients with HLH [26] [27] [28] . In two consecutive patients treated with ruxolitinib, rapid reduction in fever was observed [28] . In a study of 34 patients with HLH, the overall response rate was 73.5% with a complete response rate of 14.7% [26] . In the 25 patients who responded, there was a significant reduction in the levels of IFN-γ, IL-18, MIP-1α, and IP-10. In another study of five patients with secondary HLH and two additional patients treated off-protocol, 100% achieved a response at the time of the first assessment (day 14), with three patients achieving a complete response [27] . Furthermore, hematologic parameters including platelet, red blood cell, and neutrophil counts improved within the first week of ruxolitinib treatment. All patients treated on-protocol also experienced substantial improvements in ferritin and soluble IL-2 receptor concentrations.

At 15 mg BID, ruxolitinib was generally well tolerated in this population.

The sudden surge in hospitalization of patients with COVID-19 and the high mortality rate of hospitalized patients has encouraged treating physicians to look to repurpose approved drugs to lessen the burden of disease. Increased understanding of the immunopathology of severe COVID-19 [6] [7] [8] Monoclonal antibodies targeting IL-6 are likely to have an impact on the cytokine storm associated with COVID-19 given that IL-6 is among the cytokines reported to be elevated in those patients compared with healthy individuals. At the time of writing, anti-IL-6 antibody products tocilizumab and sarilumab are being evaluated in phase 3 studies [29, 30] . However, other cytokines, such as IL-2, IL-7, IL-10, IFN-γ, G-CSF, and GM-CSF, are also elevated and may be equally or more important in the inflammatory response in patients with severe COVID-19. As these cytokines signal through JAK1 and/or JAK2, it is likely that treatment with ruxolitinib will result in broader anti-inflammatory activity than targeting any one of the cytokines alone (Fig. 4) .

In addition to MF, SR-aGVHD, and HLH noted above, JAK inhibitors have shown promise in several autoimmune and inflammatory diseases such as rheumatoid arthritis, psoriasis, and ulcerative colitis [31] . Ruxolitinib was the first JAK inhibitor approved in the United States and European Union, indicated first for MF; others have since been approved. There are notable differences in selectivity profiles between approved JAK inhibitors. Ruxolitinib is a balanced JAK1/JAK2 inhibitor with good selectivity over non-Janus kinases, tofacitinib is a pan-JAK inhibitor, upadacitinib is a JAK1 inhibitor, and fedratinib is a JAK2 inhibitor with activity against FLT-kinase and other non-kinase proteins. These differences result in distinct biomarker activity profiles. Singer et al. determined gene signatures of four different JAK inhibitors in a panel of 12 human primary cell systems and concluded that only ruxolitinib has a biomarker profile that is consistent with broad anti-inflammatory activity [32] . These differences in selectivity may in turn be responsible for the differentiated safety profiles. For example, fedratinib shows a high incidence of gastrointestinal intolerance and cases of Wernicke's encephalopathy [33] , whereas tofacitinib has been associated with an increased risk for J o u r n a l P r e -p r o o f Journal Pre-proof lymphomas as well as cardiovascular events in patients 50 years of age and older with at least one cardiovascular risk factor [34] . Non-melanoma skin cancers and elevated lipid parameters have occurred in patients treated with ruxolitinib [15] . Dose-dependent and reversible cytopenias have been commonly observed with ruxolitinib treatment in patients with MF, PV, and GVHD [15, 20, 21, 35, 36] . Use of ruxolitinib has also been associated with viral reactivation, including cytomegalovirus and herpes zoster virus [15, 35] , suggesting the potential for an increase in infections with ruxolitinib treatment.

The pharmacokinetic profile of ruxolitinib is characterized by rapid oral absorption and a short terminal elimination half-life of approximately 3 hours (Fig. 5A ) as well as a concentrationdependent and reversible pharmacodynamic effect (Fig. 5B) [37]. This profile is in contrast with that of antibodies such as tocilizumab, which has a half-life of approximately 2 weeks [38] , and other JAK inhibitors such as fedratinib, which has a half-life of 62-78 hours [39] . Thus, ruxolitinib is more conducive to short-term therapy and withdrawal as needed. Based on the similarity of the reported elevation of cytokine levels in COVID-19 to HLH and MF, dose ranges of 5 to 15 mg BID may result in adequate inhibition of cytokine signaling while minimizing adverse events. Furthermore, it is anticipated that patients would receive ruxolitinib for approximately 14 days, a brief time period that should minimize the risk of long-term infection or other complications, such as severe cytopenias. Although preclinical models and clinical data show a lack of any impact on T-cell function and immune response with ruxolitinib treatment at pharmacologically relevant doses [17, 19, 40] , it would be prudent to both select patients who are likely to develop cytokine storm based on evolving clinical criteria such as H-score, and to identify the best time to initiate treatment based on onset of symptoms and other clinical indicators such as respiratory distress or the need for supplemental oxygen.

Taken together, these data suggest that ruxolitinib at pharmacologically achievable doses may be able to mitigate the hyperinflammatory state observed in patients experiencing COVID-19-J o u r n a l P r e -p r o o f Journal Pre-proof associated cytokine storm. Indeed, early clinical evidence supports this premise. A team in Northern Italy has reported on the use of ruxolitinib in four of their hospitalized patients requiring supplemental oxygen, with clinical improvement seen in all four patients [41] . Furthermore, emerging data from ongoing investigator-initiated trials suggest a potential benefit of ruxolitinib with a manageable adverse event profile. [42] [43] [44] [45] In a randomized trial of 43 patients in Wuhan, China, patients treated with ruxolitinib had a numerically shorter time to clinical improvement compared with those treated with standard of care (median 12 vs 15 days) and better outcome for survival (0 vs 3 deaths in the control arm). [42] Importantly, although higher rates of grade 1-2 anemia and thrombocytopenia were observed with ruxolitinib in this study, there was no increase in grade 3-4 cytopenias, and patients receiving ruxolitinib experienced a significantly shorter time to improvement of lymphopenia compared with standard of care (median 5 vs 8 days; hazard ratio, 3.307 [95% CI, 1.097 to 8.409]; P = 0.033). Additionally, no increase in infections was seen with ruxolitinib treatment in these patients.

These encouraging early clinical results, combined with a thorough understanding of the evidence supporting the posited mechanism of action of ruxolitinib in COVID-19-associated cytokine storm and the urgent need for treatments in patients with severe disease, support expedited investigation of ruxolitinib for patients with COVID-19 in phase 3 clinical trials. Table 1 In vitro enzymatic and functional potency of ruxolitinib [16] . 

IC50

Coronavirus infections and immune responses

World Health Organization, Coronavirus disease 2019 (COVID-19) situation report -92

Clinical characteristics of coronavirus disease 2019 in China

Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it?

Acute respiratory distress revealing antisynthetase syndrome

Pathogenic T cells and inflammatory monocytes incite inflammatory storm in severe COVID-19 patients

Clinical features of patients infected with 2019 novel coronavirus in

Pathological findings of COVID-19 associated with acute respiratory distress syndrome

Clinical predictors of mortality due to COVID-19 based on an analysis of data of 150 patients from Wuhan, China

Therapeutic targeting of Janus kinases

JAKs in pathology: role of Janus kinases in hematopoietic malignancies and immunodeficiencies

SOCS-1 suppresses TNF-αinduced apoptosis through the regulation of Jak activation

Tumor necrosis factor α (TNF-α) activates Jak1/Stat3-Stat5B signaling through TNFR-1 in human B cells

Jakafi (ruxolitinib)

Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Activity of therapeutic JAK 1/2 blockade in graft-versus-host disease

Pharmacologic blockade of JAK1/JAK2 reduces GvHD and preserves the graft-versus-leukemia effect

Safety and efficacy of INCB018424, a JAK1 and JAK2 inhibitor, in myelofibrosis

JAK inhibition with ruxolitinib versus best available therapy for myelofibrosis

Ruxolitinib: a potential treatment for corticosteroid refractory acute graft-versus-host disease

Ruxolitinib for the treatment of steroid-refractory acute GVHD (REACH1): a multicenter, open-label, phase 2 trial

Stratification of responders and non-responders in the REACH-1 trial based on serum proteomic analysis

Ruxolitinib in adult patients with secondary haemophagocytic lymphohistiocytosis: an open-label, single-centre

Resolution of secondary hemophagocytic lymphohistiocytosis after treatment with the JAK1/2 inhibitor ruxolitinib

NCT04320615) A study to evaluate the safety and efficacy of tocilizumab in patients with severe COVID-19 pneumonia (COVACTA)

Selective JAKinibs: prospects in inflammatory and autoimmune diseases

Comparative phenotypic profiling of the JAK2 inhibitors ruxolitinib, fedratinib, momelotinib, and pacritinib reveals distinct mechanistic signatures

Inrebic (fedratinib) [prescribing information

Xeljanz (tofacitinib) [prescribing information

Ruxolitinib versus Standard Therapy for the Treatment of Polycythemia Vera

The pharmacokinetics, pharmacodynamics, and safety of orally dosed INCB018424 phosphate in healthy volunteers

ACTEMRA ® (tocilizumab)

A randomized, placebo-controlled study of the pharmacokinetics, pharmacodynamics, and tolerability of the oral JAK2 inhibitor fedratinib (SAR302503) in healthy volunteers

Pharmacologic inhibition of JAK1/JAK2 signaling reduces experimental murine acute GVHD while preserving GVT effects

first positive results from 'anti-intensive care' drug

Ruxolitinib in treatment of severe coronavirus disease 2019 (COVID-19): A multicenter, single-blind, randomized controlled trial

Side effects of ruxolitinib in patients with SARS-CoV-2 infection: Two case reports

The Janus kinase 1/2 inhibitor ruxolitinib in COVID-19 with severe systemic hyperinflammation

Medical writing assistance was provided by Beth Burke, PhD, CMPP, of