key: cord-0858640-y74jbnb2
authors: Ky, Bonnie; Mann, Douglas L.
title: COVID-19 Clinical Trials: A Primer for the Cardiovascular and Cardio-Oncology Communities
date: 2020-04-16
journal: JACC. Basic to translational science
DOI: 10.1016/j.jacbts.2020.04.003
sha: 70da11a74fc5161cabba03f4def1b01c65a7eb36
doc_id: 858640
cord_uid: y74jbnb2

Abstract The COVID-19 pandemic has resulted in a proliferation of clinical trials that are designed to slow the spread of SARS-CoV-2, the virus that causes COVID-19. The overwhelming majority of cardiovascular and cancer patients are at increased risk for SARS-CoV-2 infection; accordingly, the cardiovascular and cardio-oncology communities are playing a major role in caring for COVID-19 patients. Many of the therapeutic agents that are being used to treat patients with COVID-19 are repurposed treatments for influenza, drugs that were not effective in Ebola patients, or treatments for malaria that were developed decades ago, and are unlikely to be familiar to the cardiovascular and cardio-oncology communities. Here we have provided a foundation for cardiovascular and cardio-oncology physicians who are on the frontline providing care to COVID-19 patients, so that they can better understand the emerging cardiovascular epidemiology of COVID-19, as well as the biological rationale for the clinical trials that are ongoing for the treatment of COVID-19 patients.

The COVID-19 pandemic has resulted in a proliferation of clinical trials that are designed to slow the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. These therapies range from vaccines, to repurposed treatments for influenza, to drugs that were not effective in Ebola patients, to treatments for malaria that were developed decades ago. Recognizing that patients with underlying cardiovascular risk factors, cardiovascular disease, or cancer have an increased risk for adverse outcomes with COVID-19, and recognizing that these vulnerable populations may be enrolled in COVID-19 clinical trials, here we present a critical review of the rationale for the different therapeutics that are currently being employed. As background, we first review the epidemiology of COVID-19, followed by the biology of coronavirus. We then briefly define the complex interplay between the coronavirus and the renin-angiotensin system (RAS), which is directly relevant to the care of the majority of patients with cardiovascular disease or cancer who are receiving drugs that modulate this system. Finally, we review the mechanisms of action the multiple therapies that are currently being studied in clinical trials. Given the breadth of information that is emerging, we will not discuss the role of vaccines.

The current impact of the novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unquantifiable. The number of confirmed cases and deaths from the global COVID-19 pandemic increase daily (https://www.statnews.com/2020/03/26/covid-19tracker/ https://coronavirus.jhu.edu/map.html). While there is a great deal that still remains to be 4 understood, initial reports from 552 hospitals in China describing 1,099 of the 7,736 COVID-19 infected patients provide some insight into the disease (1) . In this multi-center retrospective analysis, the majority were Wuhan residents or had contact with Wuhan residents, although 25 .9% were neither. The median age of patients was 47 years (Interquartile range [IQR] 35 to 58), and 41.9% were female. Patients with more severe disease tended to be older, and tended to suffer from at least one comorbidity, compared to those with non-severe disease. In this retrospective analyses, patients commonly received intravenous antibiotics (58.0%). Oseltamivir was administered in 35.8%, systemic steroids in 18.6%, and oxygen in 41.3%. The median duration of hospitalization was 12.0 days, interquartile range 10.0-14.0 days); however, the majority of the patients (93.6%) remained hospitalized at the time of data analyses and as such, the clinical course still largely remains to be defined.

Epidemiologic data thus far suggest that patients with cardiovascular risk factors, including older age; cardiovascular disease; or cancer are more susceptible to infection and suffer from worse clinical outcomes (2) . COVID-19 can also directly result in a number of cardiovascular complications, including fulminant myocarditis, myocardial injury, heart failure, and arrhythmia (1, 3, 4) . There have been a number of published case reports of clinically suspected myocarditis with markedly elevated troponin levels, ST-segment elevations on electrocardiogram without obstructive coronary disease in the presence of severely decreased left ventricular systolic function and shock (5) , with cardiac magnetic resonance imaging evidence of diffuse myocardial edema and gadolinium enhancement (6) . However, in another isolated autopsy report from a patient who suffered from SARS-CoV-2related pneumonia and cardiac arrest, no obvious histological changes in the myocardium were observed with the exception of few interstitial mononuclear inflammatory infiltrates (7). 5 Elevated troponin levels have also been observed in those with worse clinical outcomes.

In a retrospective, single-center analysis of 416 hospitalized patients with confirmed COVID-19, 19 .7% displayed evidence of cardiac injury, as defined by elevated high sensitivity Troponin I levels greater than the 99 th percentile upper limit. Those with confirmed cardiac injury tended to be older (median age of 74 versus 60 years) and suffer from hypertension (59.8% versus 23.4%), diabetes (24.4% versus 12.0%), coronary heart disease (29.3% versus 6.0%), heart failure (14.6% versus 1.5%), or cancer (8.5% versus 0.6%) (8).

Patients with cardiovascular risk factors or disease are at increased risk for suffering from worse clinical outcomes with COVID-19. In an analysis of 2 cohorts from Jinyintan Hospital and Wuhan Hospital of 191 patients, patients with hypertension, diabetes, coronary heart disease were each at increased risk of mortality upon hospital admission (9) . The prevalence of hypertension amongst non-survivors was 48% as compared to 30% in survivors; 31% versus 19% for diabetes, and 13% versus 8% for cardiovascular disease. These comorbidities were also more likely to be present in patients who required intensive care unit admission (2 (13) . In contrast, the estimated cumulative incidence of all COVID-19 cases in Wuhan was 0.37%. As a result, the odds of infection in cancer patients were estimated to be 2.31 greater (95% CI 1.89-3.02). Cancer patients who were infected had a median age of 66 years and were more likely to have non-small cell lung cancer (58.3%). Five of these patients were being treated with chemotherapy, immunotherapy, or radiation therapy. Three deaths were recorded.

In a multicenter, prospective cohort study of 2,007 cases from 575 hospitals, 1% of the 1590 COVID-19 cases had a history of cancer (13) . This in contrast to an incidence of cancer in the Chinese population of 0.29% per 100,000 people. Again, amongst those infected, lung cancer was most common and patients tended to be older. When compared to patients without cancer, patients with cancer also suffered from an increased risk of adverse events that tended to occur earlier, including admission to the intensive care unit (ICU), need for invasive ventilation, or death, which occurred in 7 of 18 patients (39%), compared to 124 of 1,572 patients without 7 cancer (8%). Cancer patients who were recently treated with chemotherapy or surgery were also more likely to suffer from clinically severe adverse events.

Coronaviruses (CoVs) represent a large family of hundreds of enveloped, single- . Identification and sequencing of the virus responsible for COVID-19 established that it was a novel CoV (2019-nCoV) that shared 88% sequence identity with two bat-derived SARS-like CoVs (14) .

Subsequently, 2019-nCoV was shown to share a 79.5% sequence homology with SARS-CoV, and was subsequently renamed SARS-CoV-2 (14) . The genome of the coronaviruses encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein (Central Illustration). The S protein is responsible for facilitating entry of the CoV into the target cell (14) (15) , and is comprised of a short intracellular tail, a transmembrane anchor, and a large ectodomain that consists of a receptor binding S1 subunit and a membrane-fusing S2 subunit (14) . 8 

Given that far more is known with respect to the virology of SARS-CoV than SARS-CoV-2, and given that these two coronaviruses appear to have some overlapping biology and clinical presentations, we will discuss these two viruses together, with an emphasis on the most recent studies that have revealed unique aspects biological aspects of SARS-CoV-2. We will review viral attachment, entry and replication of SARS-CoV and SARS-CoV-2 in host cells. This discussion will be integrated with a review of the ongoing clinical trials that target these different aspects of the biology of SARS-CoV-2 (see Tables 1-5 ).

CoV-2. Viruses enter cells by binding to host cell-encoded proteins that facilitate the entry of the virus into the cell, as well as allow the virus to survive and replicate within the cell. Some viruses, including certain strains of CoVs are capable of down-modulating the entry receptor once they gain access to the cell. Receptor down-modulation is a strategy broadly used by many viruses to escape the immune system, as well as establish the best environment for viral replication and spread (16) . Receptor down-modulation may also disrupt many of the natural physiologic functions of the host cell, resulting in cell death leading to organ level dysfunction.

The entry receptor utilized by both SARS-CoV and SARS-CoV-2 is the Angiotensin-Converting Enzyme 2 (ACE2) (Central Illustration), which is type I transmembrane carboxypeptidase with 40% homology ACE. ACE plays a critical role in activation of the RAS, by processing Angiotensin I (Angiotensin 1-10) to Angiotensin II (Angiotensin 1-8), the major effector peptide of RAS , which mediates its effects through selective interactions with G-protein coupled Angiotensin II type 1 (AT1) and type 2 (AT2) receptors (17) . ACE, however, has not been implicated in the entry of human coronaviruses into cells. 9 ACE2 is highly expressed in the mouth, tongue, and type I and II alveolar epithelial cells in the lungs. ACE2 is also abundantly expressed by cardiovascular endothelium, cardiac myocytes, cardiac fibroblasts, as well as epithelial cells of the kidney and testis. The major substrate of ACE2 is angiotensin II, which is cleaved to Ang 1-7 (Figure 1) , and functions through association with the G-protein-coupled receptor Mas receptor. The ACE2-Ang (1-7)-Mas receptor axis is regarded as the counter-regulatory arm of the RAS by opposing the effects of the ACE-Angiotensin II axis-AT1. Although the precise role of ACE2 is still being evaluated, studies been shown that ACE2 exerts protective effects in the pulmonary and the cardiovascular systems, where it serves to oppose the deleterious effects of RAS activation (18) (19) (20) .

protein on the surface of the coronavirus to ACE2 that is expressed on the cell surface. The receptor binding domain of the S protein of SARS-CoV-2 is located on the S1 subunit, which undergoes a conformational change when it binds to ACE2, which facilitates viral attachment to the surface of target cells (15) . Binding of SARS-CoV-2 to ACE2 can result in uptake of virions into endosomes (Central Illustration). Viral entry into the cell requires priming of the S protein by the serine protease transmembrane protease serine 2 (TMPRSS2), which cleaves the viral S protein at the S1/S2 and the S2' site, and allows fusion of viral and cellular membranes (21) . The S proteins of SARS-CoV-2 can also use pH sensitive endosomal proteases (cathepsin B and L) for priming and entry into cells. Interestingly, the binding affinity the SARS-CoV-2 S ectodomain to ACE2 is 10-to 20-fold higher than the binding of the SARS-CoV ectodomain to ACE2 (15) . The increase in stickiness of the SARS-CoV-2 capsid S protein makes disease transmission more likely, and might explain the increased person-to-person transmission with SARS-CoV-2 compared to SARS-CoV. Insofar as the viral S proteins are the part of the virus that interacts with the immune system, they may serve as a promising target for vaccines.

Relevant to this discussion, convalescent sera from SARS patients have been shown to block the entry of SARS-CoV-2 entry into cultured cells, albeit with less efficiency that SARS-CoV (21) .

However, monoclonal antibodies raised against the receptor binding domain of the S1 protein of SARS-CoV do not bind to receptor binding domain of the S1 protein of SARS-CoV-2, suggesting that SARS-directed antibodies are not cross reactive, and that SARS-CoV-2 proteins are necessary to develop effective antibodies. Although ACE inhibitors do not inhibit ACE2, Hoffman and colleagues demonstrated that anti-ACE2 antibody prevented entry of viral vectors into cell lines expressing the SARS-CoV-2 S protein (21) .

System. An additional layer of complexity to understanding the pathophysiology of the SARS-CoV-2 in humans stems from the complexity of the interactions of CoVs with the RAS (Figure 1) , as well as the widespread use of drugs that interfere with the RAS, including angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, or angiotensin receptor neprilysin inhibitors. Each of these drugs has different effects on the expression of the various components of the RAS in different tissue beds. Here we will briefly discuss these important interactions, as well as their implications for the treatment of COVID-19 patients.

Previous studies have shown that SARS-CoV spike proteins induce the expression of a cell surface metalloenzyme termed ADAM (A Disintegrin And Metalloproteinase)-17, which was originally described as the enzyme that cleaves membrane bound tumor necrosis factor (TNF)-α from the cell surface and allows it be circulate in soluble form of (sTNF-α) (22) . As shown in Figure 1 , activation of ADAM-17 results in the proteolytic cleavage of ACE2 (referred 11 to as shedding) from the cell surface, with the release of the catalytically active soluble ACE2 (sACE2) ectodomains into the circulation (referred to as soluble ACE2) (20, 23) . A decrease in ACE2 levels on the cell surface would be expected to result in a decrease in the levels Ang 1-7 levels (cytoprotective) and a corresponding increase in tissue levels of angiotensin-II (proinflammatory and pro-fibrotic). The importance of SARS-CoV2-induced down regulation of cell surface ACE2 was demonstrated in experimental studies, wherein administration of recombinant human ACE2 protein, genetic deletion of the AT1 receptor or administration of an AT1 receptor antagonist were shown to be protective in acute lung injury models (19, 20) . These, and other observations have suggested that the use of AT1 receptor antagonists may be beneficial in COVID-19 patients (24) , and consistent with this, losartan is currently being tested in randomized, double-blind placebo controlled studies as a potential therapy in hospitalized infected patients ( Table 1) . Relevant to this discussion, the ACE inhibitors in clinical use do not directly affect ACE2 activity (25) . The biological significance of circulating sACE2 is not known. Of note, sACE2 retains its ability to bind the S protein of SARS-CoV and was shown prevent entry of SARS-CoV into cells in vitro (26) . Thus, sACE2 may act as a decoy receptor that prevents SARS-CoV-2 from binding to ACE2 on the cell surface. APN01 is a human recombinant soluble ACE2 (hrsACE2) that has been shown to block the early stages if SARS-CoV-2 infections in cell culture and human tissue organoid cultures. (27) . APN01has already undergone safety and tolerability testing in a phase II trial I healthy volunteers (NCT00886353), but at the time of this writing is not being tested clinically in COVID-19 patients.

The recognition that many COVID-19 patients have underlying medical conditions that are treated with angiotensin converting enzyme (ACE) inhibitors and AT1 receptor antagonists (28) , coupled with the knowledge that hypertension and diabetes treated with these agents have 12 increased ACE2 levels (24) , has given rise to the concern that pharmacologic upregulation of ACE2 by RAS inhibitors may influence the infectivity of SARS-CoV-2 in patient population that is already at high risk for severe COVID-19 infection (29) . However, as noted in a recent review on this topic, the experimental and clinical data often yield conflicting results with respect the role of ACE inhibitors and AT1 receptor antagonists on ACE2 levels in different pathophysiological contexts (30) , suggesting the effects on RAS inhibitors on ACE2 are complex and nuanced, and should not be assumed to be the same for all RAS inhibitors, nor should it be assumed that changes in ACE2 levels in the heart or other tissues necessarily reflect changes in ACE2 levels in the lung, which is the portal of entry for SARS-CoV-2. Given that we have limited understanding with respect to the interaction of RAS inhibitors, ACE2 levels and SARS-CoV-2 infectivity in humans, we do not believe that it is possible to make definitive statements that go beyond the joint statement issued on March 17, 2020, by the Heart Failure Society of America/the American College of Cardiology/American Heart Association, who together recommended "continuing RAAS antagonists for those patients who are currently prescribed such agents for indications for which these agents are known to be beneficial" (31) .

The entry of enveloped viruses into host cells occurs through two primary mechanisms: one is direct fusion of the viral membrane with the plasma membrane of the host cells, which allows the virus to directly deliver their genomic material into the cytosol; and the second is that virus hijacks the cell's endocytic machinery, by binding to a cell surface receptor, which then triggers endocytosis of the virus-receptor complex (Central Illustration). In the endocytic pathway, the endocytosed virions are subjected to an activation step within the endosome, which is typically mediated by the acidic environment of the endosome, resulting in fusion of the viral and endosomal membranes, which allows for the 13 release of the viral genome into the cytosol. Several viruses, including HIV and SARS-CoV use direct membrane fusions at the cell surface or endocytosis to enter cells. As noted above, recent studies suggest that SARS-CoV-2 binds to ACE2, which is leads to endocytosis of the receptorvirus complex (21) . What is not known at this time is whether SARS-CoV-2 is also capable of directly fusing with the lipid membrane of cells. However, based on the similarities of how SARS-CoV and SARS-CoV-2 behave, it is likely that their modes of entry into cells will be similar. Understanding these differences in cell entry has implications for developing novel therapeutics.

occur by direct fusion of the viral membranes with the plasma membrane of the host cell (Central Illustration), through a process that requires processing of the viral S protein by TMPRSS2 at or near the cell surface. Processing of the S protein exposes the fusion peptide of the S protein that inserts into the cell membrane, which brings the envelope of the viral membrane into closer approximation with the membrane of the host cell, thereby facilitating fusion (32) . At the time of this writing, the uptake of SARS-CoV-2 into cells has been shown to occur through endocytosis of the SARS-CoV-2 -ACE2 complex, which also requires priming of the S protein by TMPRSS2. It is not known whether SARS-CoV-2 also enters though direct fusion.

Based on the evidence linking TMPRSS2-mediated SARS-CoV-2 activation to SARS-CoV-2 infectivity (21, 33) , the small molecule serine protease inhibitor camostat mesylate may also be an attractive target for clinical trials with SARS-CoV-2 ( Table 2) . Camostat mesylate has already shown to inhibit replication of influenza and parainfluenza viruses and to prevent the 14 development of pneumonia and viral myocarditis in infected mice (34) . Given that the SARS-CoV-2 S protein is activated by the pH dependent cysteine protease cathepsin L, this processing step may be sensitive to inhibition with drugs that indirectly inhibit cathepsin L activity by interfering with endosomal acidification (e.g. bafilomycin A1), or by compounds which directly block the proteolytic activity of cathepsin L.

It has also been suggested that the antimalarial drugs chloroquine and hydroxychloroquine might exert a potent antiviral effect by virtue of its ability to increase endosomal pH. Inside cells, chloroquine and hydroxychloroquine are rapidly protonated and concentrated in endosomes. The positive charge of the chloroquine increases the pH of the endosome, which prevents cathepsin-induced priming of the viral S protein. Both chloroquine and hydroxychloroquine decrease SARS-CoV-2 replication in cultured cells; however, hydroxychloroquine was more potent than chloroquine (35) . In a small single-arm study of patients with confirmed COVID-19, treatment with hydroxychloroquine was associated with a significant difference in clearing of viral nasopharyngeal carriage of SARS-CoV-2 within 3 -6 to days when compared to untreated controls. Azithromycin when added to hydroxychloroquine was significantly more efficient for virus elimination (36) . However, both therapies can result in QT prolongation, and as such, caution needs to be exercised when using these therapies together.

Chloroquine and hydroxychloroquine can also manifest in cardiotoxicity, including cardiomyopathy, both systolic and diastolic, atrioventricular and bundle branch block (37) . As detailed below, Hydroxychloroqine will be used as one of the treatment arms in the World Health Organization (WHO) multi-national SOLIDARTY trial (38) , and is also currently being investigated in a number of other studies (Tables 2 and 5) . Interestingly, amiodarone, which is a cationic amphiphile, was shown to inhibit Ebola virus infection in vitro in target cells, using 15 concentrations of amiodarone that overlapped those detected in the sera of patients treated for arrhythmias. Both amiodarone and its main metabolite, monodesethyl amiodarone (MDEA), were shown to interfere with the fusion of the viral envelope with the endosomal membrane, thus blocking viral replication (39) . Amiodarone has also been shown to inhibit SARS-CoV infection and spreading in vitro, by altering the late compartments of the endocytic pathway by acting after the transit of the virus through endosomes (40) . Once the genomic RNA of SARS-CoV is released into the cytoplasm of the host cell, the positive-strand viral RNA is translated on host ribosomes into one large polypeptide termed the replicase, which undergoes proteolytic cleavage to yield proteins that are required from genome replication, including a viral RNA-dependent RNA polymerase (RdRp). The viral RdRp generates a full-length, antisense negative-strand viral RNA template, which is used for replicating positive strand viral genomic RNA, as well as shorter subgenomic negative strand RNAs that serve as templates for synthesizing mRNAs that code for structural proteins of the virus, including the spike (S), membrane (M), envelope (E), and nucleocapsid (N) proteins.

Translation of viral mRNAs occurs using the host endoplasmic reticulum. Once the viral structural proteins, S, E, and M are translated in the endoplasmic reticulum (ER), they move 16 along the secretory pathway to the endoplasmic reticulum-Golgi intermediate compartment.

There, the viral proteins become encapsulated and bud into membranes containing viral structural proteins. Following assembly and maturation, virions are transported to the cell surface in vesicles and released by exocytosis (41, 42) .

Therapeutics for Viral Replication. There are a number of anti-viral drugs that are being repurposed for the treatment of SARS-CoV2. A partial list of these antiviral drugs are discussed below.

anti-viral activity. Remdesivir is a prodrug that is metabolized to its active form GS-441524, which interferes with the action of viral RNA-dependent RNA polymerase, resulting a in decrease in viral RNA production. It is not known, however, whether Remdesivir terminates RNA chains or causes mutations in them. Remdesivir was effective against multiple types of coronaviruses in cell culture and a mouse model of SARS (43) ; however, it did not show an effect in patients with Ebola. Remdesivir is currently being tested in several clinical trials for hospitalized patients with COVID-19 and pneumonia (Tables 3 and 5) . Remdesivir is also one of the four treatment arms in the multi-national SOLIDARITY trial, which is WHO sponsored multi-national randomized, open clinical trial to evaluate the safety and comparative efficacy of Hydroxychloroquine, Remdesivir, the combination of Lopinavir and Ritonavir, and the combination Lopinavir and Ritonavir plus interferon-beta (38) . SOLIDARITY will use an adaptive design, which will allow for discontinuation of drugs that lack effectiveness, as well as adding new drugs that appear promising. This type of trial design offers flexibility and efficiency, particularly in the identification of early signals related to either efficacy or toxicity, while maintaining study validity (44) .

RNA-dependent RNA polymerase. Like Remdesivir it is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5'-triphosphate (favipiravir-RTP). Although Favipiravir has undergone phase III clinical trials for the treatment of influenza, it is not yet approved by the FDA. Japan has granted approval for Favipiravir for treating viral strains unresponsive to current antivirals. In preliminary studies Favipiravir was shown to have more potent antiviral activity than lopinavir/ritonavir (45) . (50) , and will also be evaluated in the SOLIDARITY trial (38) .

A number of additional immunomodulatory agents are also currently being evaluated, including the IL-6 inhibitor, Tocilizumab, and glucocorticosteroids (Tables 4 and 5) , given the cytokine storm syndrome which has been observed in subgroups with severe COVID-19 (51) with increased levels of interleukin (IL)-2, IL-6, IL-7, and additional inflammatory cytokines (52) . One meta-analysis suggested that the mean IL-6 levels were 2.9-fold (95% CI 1. 17, 7.19) greater in patients with complicated compared to non-complicated COVID-19 (52) . Tocilizumab 19 (ACTEMRA®) is FDA approved for the treatment of severe cytokine release syndrome in patients treated with CAR T cell therapy and also approved for the treatment of rheumatoid arthritis (53) (54) (55) (56) . Tocilizumab is a monoclonal antibody that binds the IL-6 receptor, both the membrane bound and soluble forms, thus inhibiting both classic and trans-IL-6 downstream signaling. Similarly, the IL-6 humanized murine chimeric monoclonal antibody Siltuximab, although not FDA approved for the treatment of cytokine release syndrome, has also been used in the treatment of cytokine release syndrome and is also being studied as a potential therapy in severe COVID-19 infections. Siltuximab (SYLVANT®) binds directly to IL-6 and prevents the activation of immune effector cells. Sarilumab (KEVZARA®) is a human monoclonal antibody against the IL-6 receptor that was developed for the treatment of rheumatoid arthritis, that is also being evaluated for severe COVID-19.

There are no systematically obtained clinical data that yet support a benefit to the use of steroids, and some reports have suggested a possible detriment with delayed viral clearance and increased risk of infection with MERS and SARS, although the role of steroids in COVID-19 is an area of active investigation ( Table 4 ) (57) .

The COVID-19 pandemic has presented innumerable challenges to health care organizations and health care providers. Given that the vast majority of cardiovascular patients are at high risk for SARS-CoV-2 infection, the cardiovascular and cardio-oncology community will play a major role in caring for COVID-19 patients now and for the foreseeable future. As a community, we have a long tradition of enrolling patients into clinical trials that evaluate therapeutic agents whose mechanism(s) of action is familiar, which has for clinical equipoise when enrolling patients in clinical trials. In the coming months that lie ahead our communities 20 will be asked to contribute patients to clinical trials where the mechanism of action of the therapeutic agents is less familiar and the knowledge base required for providing care for COVID-19 is accelerating at a dizzying pace. Here we have tried to provide a foundation for cardiovascular physicians who are on the frontline providing care to COVID-19 patients, so that they can better understand the emerging cardiovascular epidemiology of COVID-19, as well as the biological rationale for the plethora of clinical trials that are either being designed or are currently recruiting patients. 21 

The SARS-CoV-2 virus genome encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein. The S protein is responsible for facilitating entry of the CoV into the target cell. The routes employed by SARS-CoV include endocytosis and membrane fusion. The route employed by SARS-CoV-2 is via endocytosis; whether SARS-CoV-2 enters cells by membrane fusion is not known. Binding of the spike protein of SARS-CoV to ACE2, lead to the in uptake of the virions into endosomes, where the viral spike protein is activated by the pH dependent cysteine protease cathepsin L. Activation of the spike protein by cathepsin L can be blocked by bafilomycin A1 and ammonium chloride, which indirectly inhibit the activity of cathepsin L by interfering with endosomal acidification. Chloroquine and hydroxychloroquine are weak bases that diffuse into acidic cytoplasmic vesicles such as endosomes, lysosomes, or Golgi vesicles and thereby increases their pH. MDL28170 inhibits calpain and cathepsin L. SARS-CoV can also directly fuse with host cell membranes, after processing of the virus spike protein by TMPRSS2, a type II cell membrane serine protease. Camostat mesylate is an orally active serine protease inhibitor. , which is the major effector peptide of the renin angiotensin system. Angiotensin II mediates its effects through selective interactions with G-protein coupled Angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. Angiotensin II is degraded to Ang 1-7 by angiotensin converting enzyme 2, Ang 1-7 binds to the Mas receptor (not shown). The ACE2-Ang (1-7)-Mas receptor axis opposes the effects of ACE-Angiotensin II-AT1-axis. The binding of the SARS-CoV-2 spike protein to ACE2 induces ACE2 shedding by activating ADAM-17. A decrease in ACE2 levels would be expected to result in a decrease in the levels Ang 1-7 levels (cytoprotective) and a corresponding increase in tissue levels of angiotensin-II (pro-inflammatory and pro-fibrotic). TMPRSS2, a type II cell membrane serine protease that activates the S protein of SARS-CoV-2 and allows it to bind to ACE2. Abbreviations: RAS = renin angiotensin system; URI = upper respiratory infection; SOFA = Sequential Organ Failure Assessment 

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Abbreviations: RT -PCR = reverse transcriptase polymerase chain reaction; NEWS = National Early Warning Score; ICU = Intensive Care Unit; IL-6 = interleukin-6