key: cord-1027258-vipnp7df
authors: Oronsky, Bryan; Knox, Susan; Oronsky, Arnold; Reid, Tony R.
title: Desperate Times, Desperate Measures: The Case for RRx-001 in the Treatment of COVID-19
date: 2020-07-07
journal: Semin Oncol
DOI: 10.1053/j.seminoncol.2020.07.002
sha: 4715885a589d5f96e4f7c8be85feca46bac50b06
doc_id: 1027258
cord_uid: vipnp7df

This article summarizes the likely attenuation properties of RRx-001 in COVID-19 based on its mechanism of action and the putative pathogenesis of the disease, which appears to activate inflammatory, oxidative and immune cascades with the potential to culminate in acute respiratory distress syndrome (ARDS), cytokine storm and death. An ongoing pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 appears to present with three major patterns of clinical symptomatology: 1) mild upper respiratory tract infection, 2) non-life-threatening pneumonia and 3) severe pneumonia and ARDS that initially manifest as a mild prodrome lasting for 7-8 days before rapid clinical and radiological deterioration requiring ICU transfer. RRx-001 is a targeted nitric oxide (NO) donor. This small molecule, which has been evaluated in multiple Phase 1 – 2 clinical trials for cancer as well as a Phase 3 clinical trial for the treatment of small cell lung cancer called REPLATINUM (NCT03699956), is minimally toxic and demonstrates clear evidence of anti-tumor activity. During the course of these clinical trials it was noted that the rate of chronic obstructive pulmonary disease (COPD) exacerbation and pneumonia in actively smoking SCLC patients treated with RRx-001 is less than 1%. Due to extensive history of tobacco use, 40-70% of patients with lung cancer have COPD(1) and the expected rate of pulmonary infection in this population is 50-70%,(2) which was not observed in RRx-001 clinical trials. Moreover, in preclinical studies of pulmonary hypertension, RRx-001 was found to be comparable with or more effective than the FDA approved agent, Bosentan. The potential pulmonary protective effects of RRx-001 in patients with recurrent lung infections coupled with preclinical models demonstrating RRx-001-mediated reversal of pulmonary hypertension suggests therapeutic activity in patients with acute respiratory symptoms due to COVID 19. Clinical trials have been initiated to confirm the hypothesis that RRx-001 may be repurposed to treat SARS-CoV-2 infection.

and we have kept all the suggested changes in this version that we are now resubmitting. The only change we made was to insert a new equivalent Figure 3 to avoid having to pay a copyright fee for the other image.

As a sidenote, during the original submission process we listed co-author Pedro Cabrales as an author in the submission system, however the first manuscript version uploaded (Word document) was missing his name in error. We have added his name is this revised word version of the manuscript and hope that is acceptable since we did list him as an author on the author portal at the point of initial submission.

We once more wish to thank you for your wonderful editorial contributions to this manuscript.

Sincerely, Bryan Oronsky, corresponding author on behalf of all authors Given the ongoing global COVID-19 pandemic and a public call for the repurposing of existing drugs, this perspective discusses the mechanistic rationale for the experimental use of first-in-class small molecule RRx-001 for the treatment of SARS-CoV-2. RRx-001 is currently in a Phase 3 trial for lung cancer and is entering initial clinical development as an acute therapeutic for COVID-19 with a planned Phase 1 trial.

This article summarizes the likely attenuation properties of RRx-001 in COVID-19 based on its mechanism of action and the putative pathogenesis of the disease, which appears to activate inflammatory, oxidative and immune cascades with the potential to culminate in acute respiratory distress syndrome (ARDS), cytokine storm and death.

The first-in-man safety results of RRx-001 have been published in Lancet Oncology and the results of other Phase 1 and 2 trials have been published in multiple reputable journals.

We believe this short communication would be of pertinent interest and of educational value to Seminars in Oncology's readership. We thank you for considering this manuscript for publication and we look forward to your response. We are very grateful for your valuable time spent reviewing and editing this manuscript and we have kept all the suggested changes in this version that we are now re-submitting. The only change we made was to insert a new equivalent Figure 3 to avoid having to pay a copyright fee for the other image.

As a sidenote, during the original submission process we listed co-author Pedro Cabrales as an author in the submission system, however the first manuscript version uploaded (Word document) was missing his name in error. We have added his name is this revised word version of the manuscript and hope that is acceptable since we did list him as an author on the author portal at the point of initial submission.

We once more wish to thank you for your wonderful editorial contributions to this manuscript.

Sincerely, Bryan Oronsky, M.D., Ph.D. corresponding author on behalf of all authors sponse to reviewers RRx-001 is a targeted nitric oxide (NO) donor. This small molecule, which has been evaluated in multiple Phase 1 -2 clinical trials for cancer as well as a Phase 3 clinical trial for the treatment of small cell lung cancer called REPLATINUM (NCT03699956), is minimally toxic and demonstrates clear evidence of antitumor activity. During the course of these clinical trials it was noted that the rate of chronic obstructive pulmonary disease (COPD) exacerbation and pneumonia in actively smoking SCLC patients treated with RRx-001 is less than 1%. Due to extensive history of tobacco use, 40-70% of patients with lung cancer have COPD 1 and the expected rate of pulmonary infection in this population is 50-70%, 2 which was not observed in RRx-001 clinical trials. Moreover, in preclinical studies of pulmonary hypertension, RRx-001 was found to be comparable with or more effective than the FDA approved agent, Bosentan. The potential pulmonary protective effects of RRx-001 in patients with recurrent lung infections coupled with preclinical models demonstrating RRx-001-mediated reversal of pulmonary hypertension suggests therapeutic activity in patients with acute respiratory symptoms due to COVID 19. Clinical trials have been initiated to confirm the hypothesis that RRx-001 may be repurposed to treat SARS-CoV-2 infection. 

This article summarizes the likely attenuation properties of RRx-001 in COVID-19 based on its mechanism of action and the putative pathogenesis of the disease, which appears to activate inflammatory, oxidative and immune cascades with the potential to culminate in acute respiratory distress syndrome (ARDS), cytokine storm and death. An ongoing pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 appears to present with three major patterns of clinical symptomatology: 1) mild upper respiratory tract infection, 2) non-life-threatening pneumonia and 3) severe pneumonia and ARDS that initially manifest as a mild prodrome lasting for 7-8 days before rapid clinical and radiological deterioration requiring ICU transfer.

RRx-001 is a targeted nitric oxide (NO) donor. This small molecule, which has been evaluated in multiple Phase 1 -2 clinical trials for cancer as well as a Phase 3 clinical trial for the treatment of small cell lung cancer called REPLATINUM (NCT03699956), is minimally toxic and demonstrates clear evidence of antitumor activity. During the course of these clinical trials it was noted that the rate of chronic obstructive pulmonary disease (COPD) exacerbation and pneumonia in actively smoking SCLC patients treated with RRx-001 is less than 1%. Due to extensive history of tobacco use, 40-70% of patients with lung cancer have COPD 1 and the expected rate of pulmonary infection in this population is 50-70%, 2 which was not observed in RRx-001 clinical trials. Moreover, in preclinical studies of pulmonary hypertension, RRx-001 was found to be comparable with or more effective than the FDA approved agent, Bosentan. The potential pulmonary protective effects of RRx-001 in patients with recurrent lung infections coupled with preclinical models demonstrating RRx-001-mediated reversal of pulmonary hypertension suggests therapeutic activity in patients with acute respiratory symptoms due to COVID 19. Clinical trials have been initiated to confirm the hypothesis that RRx-001 may be repurposed to treat SARS-CoV-2 infection.

In 2019, an enveloped positive-sense, single-stranded bat-borne 3 highly pathogenic RNA virus, SARS-CoV-2 jumped to humans (Figure 1 ). The pandemicity of the virus 4 and the ensuing economic, social, and political disruption from widespread morbidity and mortality 5 has galvanized the clinical community and regulatory authorities to accelerate the pace of vaccine and treatment trials.

RRx-001 (N-(bromoacetyl)-3,3-dinitroazetidine) is a targeted, small-molecule NO donor with anticancer activity. This agent is being studied in a Phase 3 clinical trial to treat advanced small cell lung cancer (SCLC) 6 . Like chloroquine 7 and hydroxychloroquine, RRx-001 is coupled to red blood cells 8 (RBCs), which mostly mediate the normal tissue protective, anti-tumor and antipathogenic properties of RRx-001. In preclinical testing, RRx-001 has demonstrated activity against malaria 9 , Ebola, tuberculosis and leishmaniasis. Importantly, RRx-001 is a nitric oxide (NO) donor under hypoxic conditions, resulting in targeted NO delivery to hypoxic tissues such as tumors. NO improves oxygenation through pulmonary vasodilation. 10 Consequently, targeted NO delivery to hypoxic lung tissue may lead to reversal of hypoxic pulmonary vasoconstriction caused by infection, inflammation and edema and treat or help prevent ARDS 11 . RRx-001 has been demonstrated to reduce pulmonary artery hypertension (PAH) in preclinical models (manuscript in preparation). We review the evidence that RRx-001 potentially alleviates pulmonary pathology including pneumonia, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and the potential for use of this agent to manage pulmonary symptoms caused by COVID-19.

Patients with COVID-19 due to SARS-CoV-2 infection present with a spectrum of symptomatology ranging from mild (upper respiratory infection and non-life-threatening pneumonia) to critical (hypoxemic pneumonia and ARDS); severity is related to advanced age and/or medical comorbidities such as cardiovascular disease, diabetes mellitus, morbid obesity, chronic lung disease, hypertension, and cancer. 12 Major complications of COVID-19 13 include acute lung injury (ALI)/acute respiratory disease syndrome (ARDS), sepsis, respiratory failure, heart failure, cardiomyopathies, sudden cardiac arrest and cytokine storm, which is associated with an overproduction of IL-1β, IL-2, IL-6, IL-8, both IFN-α/β, tumor necrosis factor (TNF), C-C motif chemokine 3 (CCL3), CCL5, CCL2, and IP-10. 14 A preexisting pro-inflammatory state, which is common to old age, obesity, tobacco abuse, infections, malignancies, autoimmune and cardiopulmonary disease, may be the common denominator that underlies susceptibility to cytokine storm and adverse outcomes in COVID-19 15 ; hence, the rationale to treat with immune/cytokine modulators such as chloroquine/hydroxychloroquine +/-azithromycin, 16 tocilizumab, an IL-6 inhibitor, 17 anakinra, an IL-1β antagonist, and certolizumab pegol, an anti-TNF blocker. The preexisting proinflammatory response is intensified at sites of SARS-CoV-2-tissue injury due to the release of chemokines and growth factors, which initiate an influx of neutrophils and monocytes that lead to more tissue injury with endothelial cell damage, vascular permeability, edema generation and increased inflammatory cell accumulation, constituting a vicious circle. 18 The mechanisms by which RRx-001, which has been evaluated extensively in lung cancer, both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) may attenuate the severity of SARS-Co-V-2 infection include nitric oxide release under hypoxia, anti-oxidation through Nrf2 generation and NFB inhibition.

A short-lived free radical gas, NO binds to soluble guanylyl cyclase, and increases the formation of guanosine 3′,5′-monophosphate (cGMP) from guanosine triphosphate (GTP), which is responsible for a range of effects including blood vessel relaxation and increased blood flow, bronchodilation, and antiviral activity including on SARS coronavirus. 19 20 Three distinct isoforms of the enzyme nitric oxide synthase (NOS) generate nitric oxide. These are neuronal nNOS (or NOS I), inducible iNOS (or NOS II), and endothelial eNOS (or NOS III). 21 All NOS isoforms depend on the presence of molecular oxygen to produce NO from L-arginine. 22 In ischemic/hypoxic conditions, when the endogenous NOS pathway is inactivated, the main source of NO is from deoxyhemoglobin-mediated nitrite reduction. Therefore, red blood cell (RBC) improve perfusion and oxygen supply to hypoxic tissues through nitric oxide generation. 23 RRx-001 binds to a ubiquitous residue on hemoglobin called beta cysteine 93, which enhances the enzymatic conversion of nitrite to nitric oxide and leads to its overproduction and release under hypoxic conditions only. 24 25 Unlike approved NO donors such as nitroglycerin, isosorbide dinitrate and nitroprusside, where systemic-wide NO release occurs, RRx-001-mediated nitric oxide production is selectively targeted to ischemic tissues. Consequently, side effects such as methemoglobinemia, hypotension, dizziness or headache are not observed. 26 Clinical 27 and preclinical data has shown that RRx-001 administration increases blood flow to profoundly hypoxic tumors 28 in an NO dependent manner. 29 Studies, have also demonstrated that RRx-001 improves tissue blood flow in other ischemic disorders such as sickle cell disease, hypoxic pulmonary hypertension (manuscript in preparation), doxorubicin-induced heart failure 30 , cerebral malaria-induced vasospasm 31 , ischemia-reperfusion injury 32 and hemorrhagic shock 33 while additional experiments are planned or ongoing in peripheral artery disease, critical limb ischemia, and vasculitis.

Potentially beneficial characteristics of nitric oxide for the management of COVID-19 include:

 Treatment of pulmonary arterial hypertension in ARDS  Direct antiviral potential of NO since S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, prevented SARS coronavirus (SARS-CoV-1) viral replication in vitro. 34 Pulmonary arterial hypertension (PAH) is a ubiquitous complication of acute respiratory distress syndrome (ARDS). 35 The main consequence of PAH is right heart failure with reduced systemic oxygen delivery. RRx-001 was tested in an animal model of PAH against the FDA approved endothelial receptor antagonist, Bosentan. The results demonstrated that, similar to Bosentan, RRx-001 attenuated pulmonary artery and right ventricular remodeling.

Inhaled nitric oxide has been shown to decrease V/Q mismatch and shunt 36 and to improve arterial oxygenation in patients with ARDS 37 since the vasodilatory effects are limited to well-ventilated lung regions. However, with RRx-001, oxygenation is expected to improve due to improved cardiac output and reduced pulmonary venous remodeling.

In the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), with which COVID-19 is closely associated. Oxidative stress and inflammation 38 are two key interrelated factors 39 . RRx-001 has previously been demonstrated to activate Nrf2 40 and inhibit NF-κB. Inflammation induces oxidative stress, which, in turn, feeds forward and amplifies inflammation through the activation of the pro-inflammatory mediator, nuclear factor kappa B (NF-κB). In ALI/ARDS, excessive ROS mediates epithelial and endothelial injury, lung edema, and protein leakage. Nrf2, a master regulator of antioxidative responses, induces genes that reduce overall reactive oxygen species (ROS) levels. Consequently, RRx-001 is expected to help protect pulmonary parenchyma by induction of Nrf2.

At the other end of the spectrum, NF-κB, a p50/p65 heterodimer is one of the main transcription factors, which produce proinflammatory cytokines and enzymes such as IL-1β, TNF-, cyclooxygenase-2 (COX-2), and IL-6 and induce lung injury. Thus, as a dual Nrf2 inducer and NF-κB inhibitor, RRx-001 may provide antioxidant protection against COVID-19-induced lung injury, as illustrated in Figure 2 .

RRx-001 has been evaluated and well tolerated in over 300 patients treated in clinical trials for patients with cancer. The main RRx-001-related adverse event is localized pain or discomfort at the site of infusion, which is mitigated with corticosteroid premedication. No maximally tolerated dose (MTD) was ever reached with RRx-001 and no dose-limiting toxicities (DLTs) have been reported to date either as monotherapy or in combination with chemotherapy.

In the face of the dire global threat presented by the SARS-CoV-2 virus, more than 20 medicines 41 , including human immunoglobulin from convalescent sera, interferons, inhaled nitric oxide, chloroquine, hydroxychloroquine, arbidol, remdesivir, favipiravir, lopinavir, ritonavir, oseltamivir, methylprednisolone, bevacizumab, and traditional Chinese medicines have been therapeutically repurposed or repositioned and used to treat patients. Hopefully, like with the Ebola virus, a fully protective vaccine candidate will emerge. 42 However, in the meantime, new treatment strategies are urgently needed given the high mortality rate and the economic and social upheaval, which has ensued.

Evidence in both animal models and cancer clinical trials has demonstrated that RRx-001 is protective for the lungs against the toxicities of chemotherapy and radiation potentially through Nrf2 activation and NF-κB inhibition. Furthermore, in animal models of pulmonary hypertension, RRx-001 has been shown to blunt adverse pulmonary vascular remodeling and reduce right ventricular hypertrophy. In clinical studies involving RRx-001, the incidence of COPD exacerbations and pneumonia in a heavily smoking SCLC population has been significantly less-than-expected, suggesting protection of lung parenchyma.

On the basis of its targeted production of NO in hypoxic tissue, RRx-001 has the potential to reduce the pulmonary toxicity associated with the COVID-19 infection. RRx-001 has demonstrated a favorable safety profile in clinical studies of over 300 cancer patients and will be evaluated for safety and activity in clinical trials for patients experiencing infection with COVID-19. As a red blood cell based therapeutic, RRx-001 may serve as a less toxic alternative to chloroquine for the treatment of COVID-19. Finally, because RRx-001 experimentally ameliorated ischemia-reperfusion injury, it may also have the potential to reduce the morbidity of oxygen toxicity and ventilator-induced lung injury.

In summary, while protease inhibitors may decrease viral burden, inhaled nitric oxide may reduce V/Q mismatch, anticytokine therapy may treat cytokine storm or prevent its progression, and immunomodulators like chloroquine/hydroxychloroquine may inhibit viral replication, RRx-001 may serve as an all-in-one therapy, which improves cardiopulmonary function, decreases viral burden and reduces the systemic inflammatory response. Results from pending clinical trials of RRx-001 in COVID-19 disease are eagerly awaited.

References: 1 38 Sarma JV, Ward PA. Oxidants and redox signaling in acute lung injury. Compr Physiol (2011) 3:1365-81. 39 Baetz D, Shaw J, Kirshenbaum LA. Nuclear factor-kappaB decoys suppress endotoxin-induced lung injury. Mol Pharmacol (2005) 4:977-9. 40 

This article summarizes the likely attenuation properties of RRx-001 in COVID-19 based on its mechanism of action and the putative pathogenesis of the disease, which appears to activate inflammatory, oxidative and immune cascades with the potential to culminate in acute respiratory distress syndrome (ARDS), cytokine storm and death. An ongoing pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), COVID-19 appears to present with three major patterns of clinical symptomatology: 1) mild upper respiratory tract infection, 2) non-life-threatening pneumonia and 3) severe pneumonia and ARDS that initially manifest as a mild prodrome lasting for 7-8 days before rapid clinical and radiological deterioration requiring ICU transfer.

RRx-001 is a targeted nitric oxide (NO) donor. This small molecule, which has been evaluated in multiple Phase 1 -2 clinical trials for cancer as well as a Phase 3 clinical trial for the treatment of small cell lung cancer called REPLATINUM (NCT03699956), is minimally toxic and demonstrates clear evidence of antitumor activity. During the course of these clinical trials it was noted that the rate of chronic obstructive pulmonary disease (COPD) exacerbation and pneumonia in actively smoking SCLC patients treated with RRx-001 is less than 1%. Due to extensive history of tobacco use, 40-70% of patients with lung cancer have COPD 1 and the expected rate of pulmonary infection in this population is 50-70%, 2 which was not observed in RRx-001 clinical trials. Moreover, in preclinical studies of pulmonary hypertension, RRx-001 was found to be comparable with or more effective than the FDA approved agent, Bosentan. The potential pulmonary protective effects of RRx-001 in patients with recurrent lung infections coupled with preclinical models demonstrating RRx-001-mediated reversal of pulmonary hypertension suggests RRx-001 may have therapeutic activity in patients with acute respiratory symptoms due to COVID 19. Clinical trials have been initiated to confirm the hypothesis that RRx-001 may be repurposed to treat SARS-CoV-2 infection.

In 2019, an enveloped positive-sense, single-stranded bat-borne 3 highly pathogenic RNA virus, SARS-CoV-2 jumped to humans (Figure 1) . The pandemicity of the virus 4 and the ensuing economic, social, and political disruption from widespread morbidity and mortality 5 has galvanized the clinical community and regulatory authorities to accelerate the pace of vaccine and treatment trials.

RRx-001 (N-(bromoacetyl)-3,3-dinitroazetidine) (Figure 2) is a targeted, small-molecule NO donor with anticancer activity. This agent is being studied in a Phase 3 clinical trial to treat advanced small cell lung cancer (SCLC) 6 . Like chloroquine 7 and hydroxychloroquine, RRx-001 is coupled to red blood cells 8 (RBCs), which mostly mediate the normal tissue protective, antitumor and anti-pathogenic properties of RRx-001 (Figure 3) . In preclinical testing, RRx-001 has demonstrated activity against malaria 9 , Ebola, tuberculosis and leishmaniasis. Importantly, under hypoxic conditions, RRx-001 is a nitric oxide (NO) donor under hypoxic conditions, resulting in targeted NO delivery to hypoxic tissues such as tumors. NO improves oxygenation through pulmonary vasodilation. 10 Consequently, targeted NO delivery to hypoxic lung tissue may lead to reversal of hypoxic pulmonary vasoconstriction caused by infection, inflammation and edema and might treat or help prevent ARDS 11 . In preclinical models, RRx-001 has been demonstrated to reduce pulmonary artery hypertension (PAH) in preclinical models (manuscript in preparation). We review the evidence that RRx-001 potentially alleviates pulmonary pathology including pneumonia, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) and the potential for use of this agent to manage pulmonary symptoms caused by COVID-19.

Patients with COVID-19 due to SARS-CoV-2 infection present with a spectrum of symptomatology ranging from mild (upper respiratory infection and non-life-threatening pneumonia) to critical (hypoxemic pneumonia and ARDS); severity is related to advanced age and/or medical comorbidities such as cardiovascular disease, diabetes mellitus, morbid obesity, chronic lung disease, hypertension, and cancer. 12 Major complications of COVID-19 13 include acute lung injury (ALI)/acute respiratory disease syndrome (ARDS), sepsis, respiratory failure, heart failure, cardiomyopathies, sudden cardiac arrest and cytokine storm, which is associated with an overproduction of IL-1β, IL-2, IL-6, IL-8, both IFN-α/β, tumor necrosis factor (TNF), C-C motif chemokine 3 (CCL3), CCL5, CCL2, and IP-10. 14 A preexisting pro-inflammatory state, which is common to old age, obesity, tobacco abuse, infections, malignancies, autoimmune and cardiopulmonary disease, may be the common denominator that underlies susceptibility to cytokine storm and adverse outcomes in COVID-19 15 ; hence, the rationale to treat with immune/cytokine modulators such as chloroquine/hydroxychloroquine ± azithromycin, 16 tocilizumab, an IL-6 inhibitor, 17 anakinra, an IL-1β antagonist, and certolizumab pegol, an anti-TNF blocker. The preexisting proinflammatory response is intensified at sites of SARS-CoV-2-tissue injury due to the release of chemokines and growth factors, which initiate an influx of neutrophils and monocytes that lead to more tissue injury with endothelial cell damage, vascular permeability, edema generation and increased inflammatory cell accumulation, constituting a vicious circle. 18 

The mechanisms by which RRx-001, which has been evaluated extensively in lung cancer, both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) may attenuate the severity of SARS-Co-V-2 infection include nitric oxide release under hypoxia, antioxidation through Nrf2 generation and NFB inhibition.

NO, aA short-lived free radical gas, NO binds to soluble guanylyl cyclase, and increases the formation of guanosine 3′,5′-monophosphate (cGMP) from guanosine triphosphate (GTP), an increase which is responsible for a range of effects including blood vessel relaxation and increased blood flow, bronchodilation, and antiviral activity including on against SARS coronavirus. 19 20 Three distinct isoforms of the enzyme nitric oxide synthase (NOS) generate nitric oxide. These are neuronal nNOS (or NOS I), inducible iNOS (or NOS II), and endothelial eNOS (or NOS III). 21 All NOS isoforms depend on the presence of molecular oxygen to produce NO from L-arginine. 22 In ischemic/hypoxic conditions, when the endogenous NOS pathway is inactivated, the main source of NO is from deoxyhemoglobin-mediated nitrite reduction. Therefore, red blood cells (RBCs) improve perfusion and oxygen supply to hypoxic tissues through nitric oxide generation. 23 RRx-001 binds to a ubiquitous residue on hemoglobin called beta cysteine 93, which enhances the enzymatic conversion of nitrite to nitric oxide and leads to its overproduction and release only under hypoxic conditions. 24, 25 (Figure 3 ) Unlike approved NO donors such as nitroglycerin, isosorbide dinitrate and nitroprusside, where systemic-wide NO release occurs, RRx-001-mediated nitric oxide production is selectively targeted to ischemic tissues. Consequently, side effects such as methemoglobinemia, hypotension, dizziness or headache are not observed. 26 Clinical 27 and preclinical data has shown that RRx-001 administration increases blood flow to profoundly hypoxic tumors 28 in an NO dependent manner. 29 Studies, have also demonstrated that RRx-001 improves tissue blood flow in other ischemic disorders such as sickle cell disease, hypoxic pulmonary hypertension (manuscript in preparation), doxorubicin-induced heart failure 30 , cerebral malaria-induced vasospasm 31 , ischemia-reperfusion injury 32 and hemorrhagic shock 33 while additional experiments are planned or ongoing in peripheral artery disease, critical limb ischemia, and vasculitis.

Potentially beneficial characteristics of nitric oxide for the management of COVID-19 include:  Treatment of pulmonary arterial hypertension in ARDS  Direct antiviral potential of NO since S-nitroso-N-acetylpenicillamine (SNAP), an NO donor, prevented SARS coronavirus (SARS-CoV-1) viral replication in vitro. 34 Pulmonary arterial hypertension (PAH) is a ubiquitous complication of acute respiratory distress syndrome (ARDS). 35 The main consequence of PAH is right heart failure with reduced systemic oxygen delivery. RRx-001 was tested in an animal model of PAH against the FDA approved endothelial receptor antagonist, Bosentan. The results demonstrated that, similar to Bosentan, RRx-001 attenuated pulmonary artery and right ventricular remodeling.

Inhaled nitric oxide has been shown to decrease V/Q mismatch and shunt 36 and to improve arterial oxygenation in patients with ARDS 37 since the vasodilatory effects are limited to well-ventilated lung regions. However, with RRx-001, oxygenation is expected to improve due to improved cardiac output and reduced pulmonary venous remodeling.

In the pathogenesis of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), with which occurs in COVID-19, is closely associatedo. Oxidative stress and inflammation 38 are two key interrelated factors 39 . RRx-001 has previously been demonstrated to activate Nrf2 40 and inhibit NF-κB. Inflammation induces oxidative stress, which, in turn, feeds forward and amplifies inflammation through the activation of the pro-inflammatory mediator, nuclear factor kappa B (NF-κB). In ALI/ARDS, excessive ROS mediates epithelial and endothelial injury, lung edema, and protein leakage. Nrf2, a master regulator of antioxidative responses, induces genes that reduce overall reactive oxygen species (ROS) levels. Consequently, RRx-001 is expected to help protect pulmonary parenchyma by induction of Nrf2.

At the other end of the spectrum, NF-κB, a p50/p65 heterodimer is one of the main transcription factors, which produce proinflammatory cytokines and enzymes such as IL-1β, TNF-, cyclooxygenase-2 (COX-2), and IL-6 and induce lung injury. Thus, as a dual Nrf2 inducer and NF-κB inhibitor, RRx-001 may provide antioxidant protection against COVID-19induced lung injury, as illustrated in Figure 42 .

RRx-001 has been evaluated and well tolerated in over 300 patients treated in clinical trials for that have enrolled patients with cancer ( Table 1) . The main RRx-001-related adverse event is localized pain or discomfort at the site of infusion, which is mitigated with corticosteroid premedication. No maximally tolerated dose (MTD) was ever reached with RRx-001 and no dose-limiting toxicities (DLTs) have been reported to date either as monotherapy or in combination with chemotherapy.

In the face of the dire global threat presented by the SARS-CoV-2 virus, more than 20 medicines 41 , including human immunoglobulin from convalescent sera, interferons, inhaled nitric oxide, chloroquine, hydroxychloroquine, arbidol, remdesivir, favipiravir, lopinavir, ritonavir, oseltamivir, methylprednisolone, bevacizumab, and traditional Chinese medicines have been therapeutically repurposed or repositioned and used to treat patients with COVID-19. Hopefully, like with the Ebola virus, a fully protective vaccine candidate will emerge. 42 However, in the meantime, new treatment strategies are urgently needed given the high mortality rate and the economic and social upheaval, which has ensued.

Evidence in both animal models and cancer clinical trials has demonstrated that RRx-001 is protective for the lungs against the toxicities of chemotherapy and radiation potentially through Nrf2 activation and NF-κB inhibition. Furthermore, in animal models of pulmonary hypertension, RRx-001 has been shown to blunt adverse pulmonary vascular remodeling and reduce right ventricular hypertrophy. In clinical studies involving RRx-001, the incidence of COPD exacerbations and pneumonia in a heavily smoking SCLC population has been significantly less-than-expected, suggesting protection of lung parenchyma.

On the basis of its targeted production of NO in hypoxic tissue, RRx-001 has the potential to reduce the pulmonary toxicity associated with the COVID-19 infection. RRx-001 has demonstrated a favorable safety profile in clinical studies of over 300 cancer patients and will be evaluated for safety and activity in clinical trials for patients experiencing infection with COVID-19. As a red blood cell based therapeutic, RRx-001 may serve as a less toxic alternative to chloroquine for the treatment of COVID-19. Finally, because RRx-001 experimentally ameliorated ischemia-reperfusion injury, it may also have the potential to reduce the morbidity of oxygen toxicity and ventilator-induced lung injury.

In summary, while protease inhibitors may decrease viral burden, inhaled nitric oxide may reduce V/Q mismatch, anticytokine therapy may treat cytokine storm or prevent its progression, and immunomodulators like chloroquine/hydroxychloroquine may inhibit viral replication, RRx-001 may serve as an all-in-one therapy, which improves cardiopulmonary function, decreases viral burden and reduces the systemic inflammatory response. Results from pending clinical trials of RRx-001 in COVID-19 disease are eagerly awaited. RRx-001 may provide support for patients with ALI/ARDS and related complications. It is associated with Nrf2/antioxidant response element signaling, which effectively reduces ROS levels, which are elevated in acute lung injury (ALI), and inhibition of the proinflammatory NF-κB pathway ALI: Acute lung injury; ARDS: Acute respiratory distress syndrome. 

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Characteristics and Outcomes of 21 Critically Ill Patients With COVID-19 in Washington State

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

COVID-19 infection: the perspectives on immune responses

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

China approves use of Roche drug in battle against coronavirus complications

Neutrophil clearance: when the party is over, clean-up begins

Is Nitric Oxide (NO) the Last Word in Radiosensitization? A Review

Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus

Nitric oxide synthases: regulation and function

Nitric oxide synthase structure and mechanism

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

NO to cancer: The complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001

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Safety and activity of RRx-001 in patients with advanced cancer: a first-inhuman, open-label, dose-escalation phase 1 study

Initial Clinical and Advanced Imaging Outcomes from a Multi-Institutional Phase I Dose-Escalation Trial of RRx-001 Plus Whole Brain Radiation for Patients with Brain Metastases

Dinitroazetidines are a novel class of anticancer agents and hypoxia-activated radiation sensitizers developed from highly energetic materials

Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-001: a superagonist of nitric oxide generation

Cardioprotective Effect of Phase 3 Clinical Anticancer Agent, RRx-001, in Doxorubicin-Induced Acute Cardiotoxicity in Mice

From METS to malaria: RRx-001, a multi-faceted anticancer agent with activity in cerebral malaria

The macrophage stimulating anti-cancer agent, RRx-001, protects against ischemia-reperfusion injury

Impact of hemoglobin nitrite to nitric oxide reductase on blood transfusion for resuscitation from hemorrhagic shock

Inhibition of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine, a nitric oxide donor compound

Pulmonary hypertension due to acute respiratory distress syndrome

Inhaled nitric oxide for the adult respiratory distress syndrome

Inhaled nitric oxide therapy in adults

Impact of COPD on prognosis of lung cancer: from a perspective on disease heterogeneity

Postobstructive pneumonia in lung cancer

Bat-man disease transmission: zoonotic pathogens from wildlife reservoirs to human populations

Information for Healthcare Professionals

Evaluation and Treatment Coronavirus

StatPearls

REPLATINUM Phase III randomized study: RRx-001 + platinum doublet versus platinum doublet in third-line small cell lung cancer

Uptake of chloroquine by human erythrocytes

A look inside the mechanistic black box: Are red blood cells the critical effectors of RRx-001 cytotoxicity?

From METS to malaria: RRx-001, a multi-faceted anticancer agent with activity in cerebral malaria

Role of nitric oxide in management of acute respiratory distress syndrome

Hypoxic pulmonary vasoconstriction in the adult respiratory distress syndrome

Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study

Characteristics and Outcomes of 21 Critically Ill Patients With COVID-19 in Washington State

Pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology

COVID-19 infection: the perspectives on immune responses

Hydroxychloroquine and azithromycin as a treatment of COVID-19: results of an open-label non-randomized clinical trial

China approves use of Roche drug in battle against coronavirus complications

Neutrophil clearance: when the party is over, clean-up begins

Is Nitric Oxide (NO) the Last Word in Radiosensitization? A Review

Nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus

Nitric oxide synthases: regulation and function

Nitric oxide synthase structure and mechanism

The Capacity of Red Blood Cells to Reduce Nitrite Determines Nitric Oxide Generation under Hypoxic Conditions

NO to cancer: The complex and multifaceted role of nitric oxide and the epigenetic nitric oxide donor, RRx-001

RRx-001, a hypoxia activated, nitric oxide generating cytotoxic agent: Phase 1 study results' Presentation at 3rd International Workshop on Nitric Oxide in Cancer Therapy

Safety and activity of RRx-001 in patients with advanced cancer: a first-inhuman, open-label, dose-escalation phase 1 study

Initial Clinical and Advanced Imaging Outcomes from a Multi-Institutional Phase I Dose-Escalation Trial of RRx-001 Plus Whole Brain Radiation for Patients with Brain Metastases

Dinitroazetidines are a novel class of anticancer agents and hypoxia-activated radiation sensitizers developed from highly energetic materials

Targeting tumor hypoxia with the epigenetic anticancer agent, RRx-001: a superagonist of nitric oxide generation

Cardioprotective Effect of Phase 3 Clinical Anticancer Agent, RRx-001, in Doxorubicin-Induced Acute Cardiotoxicity in Mice

From METS to malaria: RRx-001, a multi-faceted anticancer agent with activity in cerebral malaria

The macrophage stimulating anti-cancer agent, RRx-001, protects against ischemia-reperfusion injury

Impact of hemoglobin nitrite to nitric oxide reductase on blood transfusion for resuscitation from hemorrhagic shock

Inhibition of SARS-coronavirus infection in vitro by S-nitroso-N-acetylpenicillamine, a nitric oxide donor compound

Pulmonary hypertension due to acute respiratory distress syndrome

Inhaled nitric oxide for the adult respiratory distress syndrome

Inhaled nitric oxide therapy in adults

Oxidants and redox signaling in acute lung injury

Nuclear factor-kappaB decoys suppress endotoxin-induced lung injury

RRx-001 protects normal tissues but not tumors via Nrf2 induction and Bcl-2 inhibition

Efficacy and effectiveness of an rVSV-vectored vaccine in preventing Ebola virus disease: final results from the Guinea ring vaccination, open-label, cluster-randomised trial (Ebola Ça Suffit!)