key: cord-0906043-fod0eyuj authors: Malone, Robert W.; Tisdall, Philip; Fremont-Smith, Philip; Liu, Yongfeng; Huang, Xi-Ping; White, Kris M.; Miorin, Lisa; Olmo, Elena Moreno Del; Alon, Assaf; Delaforge, Elise; Hennecker, Christopher D.; Wang, Guanyu; Pottel, Joshua; Bona, Robert; Smith, Nora; Hall, Julie M.; Shapiro, Gideon; Clark, Howard; Mittermaier, Anthony; Kruse, Andrew C.; García-Sastre, Adolfo; Roth, Bryan L.; Glasspool-Malone, Jill; Francone, Victor; Hertzog, Norbert; Fremont-Smith, Maurice; Ricke, Darrell O. title: COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms date: 2020-06-22 journal: Res Sq DOI: 10.21203/rs.3.rs-30934/v2 sha: 38254edb4b458e5e61a0d75802dd3b439b5de990 doc_id: 906043 cord_uid: fod0eyuj SARS-CoV-2 infection is required for COVID-19, but many signs and symptoms of COVID-19 differ from common acute viral diseases. Currently, there are no pre- or post-exposure prophylactic COVID-19 medical countermeasures. Clinical data suggest that famotidine may mitigate COVID-19 disease, but both mechanism of action and rationale for dose selection remain obscure. We explore several plausible avenues of activity including antiviral and host-mediated actions. We propose that the principal famotidine mechanism of action for COVID-19 involves on-target histamine receptor H (2) activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release. during December 2019 [1] [2] [3] . COVID-19 is a disease spectrum causally associated with infection by SARS-CoV-2. De nitive COVID-19 diagnosis requires the presence of the virus, which can be isolated, grown, or otherwise detected as unique SARS-CoV-2 viral nucleic acid sequences. There are SARS-CoV-2 virus shedding or nucleic acid positive patients that do not manifest clinical COVID-19 [4] [5] [6] [7] [8] [9] . 13-20% of patients with symptoms develop severe respiratory compromise requiring oxygenation, with radiological ndings of ground glass opacities and consolidation [10] [11] [12] . Between 5 and 46% of SARS-CoV-2 positive patients are asymptomatic and do not appear to progress to COVID-19 [13] [14] [15] [16] . Therefore, SARS-CoV-2 infection is necessary but not su cient for development of clinical COVID-19 disease. Patients with COVID-19 disease can present with a range of mild to severe non-speci c clinical signs and symptoms which develop two to fourteen days after exposure to SARS-CoV-2. These symptoms include cough or shortness of breath, and at least two of the following; fever, chills, repeated rigor, myalgia, headache, oropharyngitis, anosmia and ageusia 17, 18 . More severe symptoms warranting hospital admission include di culty breathing, a persistent sense of chest pain or pressure, confusion or di culty to arouse, and central cyanosis. Of hospitalized patients, 20-42% develop ARDS, the most common cause for admission to the ICU. 39-72% of patients admitted to the ICU will die 19 . Early clinical data from a variety of sources indicate that famotidine treatment may reduce morbidity and mortality associated with COVID-19. A retrospective cohort study of 1,620 hospitalized COVID-19 patients indicates that 84 propensity score matched patients receiving famotidine during hospitalization (oral or IV, 20mg or 40mg daily) had a statistically signi cant reduced risk for death or intubation (adjusted hazard ratio (aHR) 0.42, 95% CI 0.21-0.85) and also a reduced risk for death alone (aHR 0.30, 95% CI 0. 11-0.80) 20 . In contrast, proton pump inhibitor use was not associated with reduced risk for these outcomes. A preceding anecdotal report from Wuhan, China is purported to have indicated that famotidine may be partially protective for COVID-19, but that neither cimetidine nor proton pump inhibitors were protective 21 . Together, these data have been interpreted as indicating that this increased survival pattern is due to an off-target, non-histamine receptor-mediated property of famotidine that is not shared with cimetidine. Famotidine is currently being tested under an IND waiver for treating COVID-19 in a double blind randomized clinical trial at high intravenous doses in combination with either hydroxychloroquine or remdesivir (ClinicalTrials.gov Identi er: NCT04370262). Herein we aim to investigate how famotidine may act to relieve early phase COVID-19 clinical symptoms. The most likely mechanisms of actions include: via antiviral activity, via novel human targets, or via the on-target mechanism described in the current FDA market authorization-famotidine is a histamine receptor H 2 antagonist (and inverse agonist). The idea to test the usefulness of famotidine as a medical countermeasure for COVID-19 emerged from a computational molecular docking effort aimed at identifying inhibitors of the papain-like protease (PLpro) of SARS-CoV-2 22, 23 . In addition to processing the viral polyprotein, the papain-like protease from coronaviruses (PLpro) is known to remove the cellular substrates ubiquitin and the interferon stimulated gene 15 (ISG15) from host cell proteins by cleaving the C-terminal end of the consensus sequence LXGG, a process termed deISGylation 24, 25 . Here, we used the enzymatic reaction of SARS-CoV-2 PLpro on ISG15 to assess the potential inhibition of PLpro by famotidine. The cleavage of the 8 C-terminal amino acids of ISG15 by PLpro is clearly detected by SDS-PAGE ( Figure 1, lanes 2 and 3) . However, the addition of 1 to 100 µM famotidine to the reaction does not signi cantly reduce the amount of ISG15 cleaved during the assay ( Figure 1 , lanes 4 to 6), thus suggesting that famotidine does not inhibit SARS-CoV-2 PLpro. A previous virtual screening report 26 suggested that famotidine might bind to the 3 chymotrypsinlike protease (3CLpro), more commonly referred to as the main protease (Mpro), however this mechanism was recently discounted 27 . To assess the possibility that famotidine may inhibit SARS-CoV-2 infection by other routes, a Vero E6 cell-based assay was performed to compare median tissue culture infectious doses (TCID50/mL) of famotidine, remdesivir, and hydroxychloroquine ( Figure 2 ). While both remdesivir and hydroxychloroquine demonstrated antiviral activity, no inhibition of SARS-CoV-2 infection was observed with famotidine. Famotidine does not act via sigma-1 or -2 receptor binding A wide-ranging study recently presented a map of interactions between viral and host proteins 28 . It was shown that regulation of the sigma-1 and sigma-2 receptors had antiviral effects. Sigma and histamine receptors share several ligands in common, like the antipsychotic haloperidol, the antihistamines astemizole and clemastine, the antidepressive clomipramine, and many more. As such, we tested for possible interaction between famotidine and sigma-1 or sigma-2 receptors (Figure 3 ). We performed radioligand competition binding experiments using cloned sigma receptors, following established procedures 29 30 . In these assays, famotidine showed no detectable displacement of radioligand probes for either sigma-1 or sigma-2 receptors at famotidine concentrations up to 10 μM. Hence, famotidine's binding to sigma-1 and sigma-2 receptors is likely negligible at physiologically relevant concentrations. As is well-known 31 , famotidine is a selective blocker of the histamine H 2 receptor with a nity of approximately 14 nM, substantially more active than the 590 nM cimetidine ( Figure 4A ). Here we nd it to have highly e cacious inverse agonist activity (reducing basal activity by 75%) with a potency of 33 nM ( Figure 4C ). Intriguingly, and unlike cimetidine, while famotidine acts to block G s protein signaling it actually acts as a partial agonist of arrestin recruitment, with an e cacy of about 15% that of histamine, and an EC 50 of 105 nM ( Figure 4D ), suggesting that the molecule promotes arrestin-scaffolded signaling -such as through the ERK pathway, 32 and promotes internalization of the receptor and further noncanonical signaling once internalized 33, 34 through an arrestin-biased mechanism. These features distinguish famotidine certainly from cimetidine, and potentially from other H 2 blockers, as such biased activation of arrrestin recruitment for GPCR antagonists, while not unprecedented, is not common. Finally, we note that a screen for activation of 318 receptors of the GPCR-ome reveals only seven receptors with an average fold of basal increase above 3.0, including H 2 ( Figure 5 ). In all cases, the quadruplicate replicates were not in agreement and require follow-up studies. Chief among these are the CCR2L and CXCR3 chemokine receptors [35] [36] [37] [38] . Such activity would be intriguing because these receptors would be expected to activate immune cell mobilization and may plausibly have a role in famotidine's bene cial activities, especially at the high systemic concentrations it is expected to reach in the clinical studies. This would also be consistent with famotidine's lack of direct anti-viral activity in the Vero cell direct infectivity assays, where immune cells are not present. Famotidine reaches functionally relevant systemic concentrations, whereas cimetidine does not We calculated predicted steady state concentrations of famotidine and cimetidine at different doses based on published pharmacokinetic and biodistribution data [39] [40] [41] . This modeling demonstrated that the different clinical outcomes exhibited by COVID-19 patients taking famotidine vs. cimetidine could be readily explained by the distinctive pharmacokinetic and pharmacodistribution properties of the two agents. Therapeutic e cacy of a pharmacological antagonist requires that it achieves a steady-state concentration that substantially exceeds the half maximal inhibitory concentration (IC 50 ) for its target. Thus, in order to evaluate the relative systemic effects of famotidine and cimetidine, the IC 50 higher, and these data were used for the current analyses 39, 41 . In these reports, the IC 50 for the H 2 receptor were reported as 13 g/L (0.039 M) for famotidine and 400-780 g/L (1.59-3.09 M) for cimetidine. Css values were calculated using pharmacokinetic data for dosing, clearance, bioavailability, and volume of distribution as summarized previously 41 . Table 1 lists the Css values for both famotidine and cimetidine. In primary human neutrophils and eosinophils, H 2 activation by histamine inhibits neutrophil effector functions including O 2 release 42,43 , platelet-activating-factor induced chemotaxis 44 and leukotriene biosynthesis 45 . Eosinophil functions are also inhibited by H 2 activation; histamine binding diminishes eosinophil peroxidase release 46 and, at high concentrations, inhibits eosinophil chemotaxis 47, 48 . Famotidine is one of the most effective antagonists of these H 2 -mediated histamine effects on neutrophils and eosinophils 49 . IC 50 for two measures that relate to these phenotypes are also listed in coincided with onset of fever (102 o F), night sweats, shortness of breath and a feeling of chest pressure. was initiated upon receiving the PCR diagnosis due to symptoms meeting FDA criteria for severe COVID-19, combined with high risk preexisting conditions. The famotidine drug regime was continued for 30 days. After initiating famotidine in the evening, the patient was able to sleep through the night and reported complete relief from the chest pressure sensation, reduction in cough, but continued to be febrile (101.6 o F). On day 10, he presented to the emergency room (ER) with continuing complaints of diarrhea, abdominal cramping, eructation, low energy, dry cough, arthralgia, myalgia, anosmia and ageusia and shortness of breath on exertion. Day 10 ER physical examination, including the chest, was unremarkable and vital signs were normal. The patient BMI was 36 (Du Bois BSA 26.78 ft 2 ). SpO2 was 93%, rising to 97% and 99% on 3 L/min by nasal cannula over the next 30 minutes. An intranasal sample was obtained for SARS-CoV-2 rtPCR diagnostic analysis. Comprehensive metabolic panel showed a mild decrease in serum sodium and chloride with hyperglycemia (260 mg/dL). Complete blood count (CBC) was normal, speci cally including the lymphocyte count. Urinalysis showed a speci c gravity of 1.025 but was otherwise normal. A portable chest X-ray had poor inspiration but was interpreted as showing "bibasilar areas of airspace disease" consistent with COVID-19 ( Figure 6 , CXR day 10). The patient was diagnosed as dehydrated, given ondansetron IV, 1 L IV of normal saline and discharged home with a hospital pulse oximeter. At the time of departure, he had an SpO2 of 94% on room air that did not drop with ambulation. The patient again presented to the emergency room on day 15 after experiencing near-syncope during showering. Physical examination was unremarkable. Vital signs were normal. SpO2 showed values of 98%, 93% and 97% on room air over the 2 hour period. Basic metabolic panel showed only hyperglycemia (266 mg/dL). CBC was normal except for a mild lymphopenia (0.96; reference range 1.00-3.00 X10 3 /μL) and mild monocytosis (0.87; 0.20-0.80 X10 3 /μL). Chest X-ray was interpreted as showing "Faint patchy consolidation of lung bases bilaterally, similar to perhaps minimally improved at the lower left lung base compared to prior" ( Figure 6 CXR day 15). The patient was placed on azithromycin and discharged to home. On days 27 and 28 after initial symptoms, he tested negative (2x, successive days) for SARS-CoV-2 nucleic acid by PCR (intranasal swab) and returned to his work at the local hospital 31 days after initial symptoms. 47 days after rst developing COVID-19 symptoms he continues to note a lack of ability to taste or smell, but otherwise considers himself largely recovered from COVID-19 ( Figure 6 timeline). Use of famotidine in this patient was recommended due to meeting FDA criteria for severe COVID-19 and his COVID-19 risk factors: male, 47yo, hypertension, obesity and diabetes mellitus Type 2. Although this is an anecdotal example, the patient experienced relief of symptoms overnight upon initiating use of famotidine. While not su cient to demonstrate proof of cause and effect, this case does provide context for typical COVID-19 presentation and symptoms, as well as support for additional well-controlled famotidine therapeutic clinical trials in an outpatient setting. Famotidine is an off-patent drug available as either branded ("PEPCID ® ") or generic medicines in tablet, capsule or intravenous forms. The general pharmacology of famotidine is well-characterized, with an excellent absorption, distribution, metabolism, excretion and toxicology pro le 53 20 . In contrast, proton pump inhibitor use was not associated with reduced risk for these outcomes. Anecdotal reports and undisclosed data indicating that famotidine provided protection from COVID-19 mortality while neither cimetidine nor proton pump inhibitors were similarly protective lead to an initial inference that the bene cial effects of famotidine were not related to the known on-target activity of the drug 21 . Studies detailed in this report and others, however, indicate that famotidine does not act by directly inhibiting either of the principal SARS-CoV-2 proteases (PLpro or Mpro) 27 . Vero E6-based cell assays also indicate that famotidine has no direct antiviral activity in this cell line, although antiviral activity in cells that express H 2 has not been tested. Additional hypotheses that famotidine may act via binding either the sigma-1 or -2 receptors have not been supported by the studies summarized herein. The most straightforward explanation of the apparent famotidine activity as a COVID-19 therapy is that the drug acts via its antagonism or inverse-agonism of histamine signaling and via its arrestin biased activation-all a result of its binding to histamine receptor H 2 . If true, then it is reasonable to infer that a The data presented herein provides a rationale for famotidine dose selection to maintain a steady state concentration at a reasonable multiple of the IC 50 for systemic antagonism of H 2 and indicate that oral tablet dosages of between 40mg every eight hours to 60mg every eight hours should be su cient to insure maximal H 2 target effects. As famotidine is primarily cleared by the kidney, adequate renal function is required for higher dosages 53 . In addition to H 2 antagonism, famotidine may also act as an inverse agonist thereby lowering the concentration of cyclic-Adenosine Monophosphate (c-AMP) 32 . Endothelial cell permeability has been attributed to histamine H 2 activation and is blunted by famotidine pretreatment 54 . Histamine, bradykinin and des-arg-bradykinin receptor engagements can lead to increased endothelial permeability through a common pathway that results in AKT-1 activation 55 . The H2 receptor also signals through Gq/11 proteins, resulting in inositol phosphate formation and increases in cytosolic Ca2+ concentrations which may account for the increased endothelial cell uid permeability 56 . One alternative hypothesis is that famotidine may not only inhibit signaling through the H 2 receptor but may also engage in cross talk with the kinin B1 receptor, which moderates the response of endothelial cells to DABK and DAKD ligands. Data provided here in are not consistent with this hypothesis; no activation of bradykinin receptor B1 or B2 were observed in quadruplicate replicate TANGO assay. While COVID-19 symptoms affect multiple organ systems, respiratory failure due to acute respiratory distress syndrome (ARDS) is the most common cause of death. Examination of RNA expression pro les of the cells which contribute to lung anatomy and function demonstrate the presence of multiple ACE2/TMPRSS2 positive cell types susceptible to SARS-CoV-2 infection in the lung. In addition, these and other associated lung cells that are positive for histamine receptors H 1 and H 2 could respond to local histamine release following mast cell degranulation 57 , and therefore those cells positive for H 2 may be responsive to famotidine effects. To understand how famotidine may act to reduce pulmonary COVID-19 symptoms requires an understanding of COVID-19 lung pathophysiology, which appears to have two principal disease phases. In turn, this requires an appreciation of pulmonary tissue and cell types. Pulmonary edema results from loss of a regulation of uid transfer that occurs at several levels in the alveolus, as diagrammed in Figure 7 . shortness of breath 62 . The image in Figure 9 panel B does not permit evaluation for microvascular thrombi. These ndings are supported in a separate autopsy case report of a patient dying 5 days after onset of COVID-19 symptoms. In this case, photomicrographs also show a non-in ammatory transudative-type edema 63 . In both of these studies, the observed non-in ammatory edema in early-stage COVID-19 pulmonary disease is consistent with histamine release by mast cells. Mast cell degranulation correlates with the COVID-19 natural history that progresses through functionally and clinically different early and later phases. Most SARS-CoV-2 infections follow the typical early phase pattern of any lower respiratory virus, in which a majority of patients have asymptomatic or minimal disease, while a minority go on to later phase acute respiratory distress syndrome (ARDS). Within this spectrum typical of any severe viral disease, COVID-19 has a number of distinctive features. In the out-patient setting, early COVID-19 is usually indistinguishable from other "in uenza-like illnesses", presenting with various non-speci c symptoms ranging from sore throat, headache and diarrhea to fever, cough, and myalgias. In these rst few days however, COVID-2 may also be associated with anosmia, a unique feature 64 . It is towards the end of the rst week of symptoms that COVID-19 patients develop shortness of breath (SOB). This follows cough and fever by several days, a feature not typical of other viruses 65 . On physical examination of COVID-19 patients with SOB, the oxygen saturation drops dramatically on exertion. CT scan will usually show bilateral bibasilar ground glass opaci cations consistent with pulmonary edema. Nasopharyngeal swabs test positive for SARS-CoV-19. This SOB correlates with a distinctive clinical phenotype of hypoxia with near normal compliance (i.e. >50 mLcmH2O). Some authors attribute this to a loss of pulmonary vasoconstriction, one cause of which could be histamine effect on the H 2 receptors of pericytes and/or vascular smooth muscle. H 1 -related edema and microthrombosis of lung vessels could also be causes. These are the patients that PEEP ventilation will not help, as there are no recruitable alveoli. These patients are helped by lying prone 66 . It is at this stage that the patient is at greatest risk to progress onto the serious complications of later disease, especially ARDS with its 60-80% mortality if ventilation is required. Patients may also present with additional neurological symptoms and complications including ischemic stroke [67] [68] [69] . Cardiac complications of later COVID-19 include myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and venous thromboembolic events 70, 71 . Multiple studies have demonstrated a hypercoagulable state in COVID-19 patients requiring hospitalization. Results from a recent large autopsy study suggests that there is also a novel lung-centric coagulopathy that manifests as a small vessel microthrombosis. Based on this study, there are indications that over 50% of patients dying of COVID-19 have pulmonary microthrombosis 72 . This thrombosis is not only in arterial vessels, but also can be found in alveolar capillaries in the absence of in ammation and ARDS, as seen in Figure 10 73 . There is widening of the alveolar septae by extensive brinous occlusion of capillaries (open black arrows). There is alveolar space edema with red blood cell extravasation. Septae show a mild mononuclear in ltrate. Alveolar edema shows neutrophils in proportion to the blood. Capillary wall disruption accompanied by brin deposition and red cell extravasation, with neutrophils in the septa and within the alveolar spaces. (Hematoxylin and eosin, 1000x) . For further discussion of microvascular coagulation associated with COVID-19, see 73 . Because small microthrombi are di cult to identify on CT scan even with iodinated contrast 74 , premortem diagnosis is di cult. Laboratory coagulation tests have typically shown normal or mildly prolonged Prothrombin time (PT) and activated partial thromboplastin time (aPTT), normal to increased or slightly decreased platelet counts, elevated brinogen levels and very elevated D-dimers 75 . Although referred to by some authors as a DIC-like state, this pulmonary microthrombosis does not appear as a typical coagulation factor consumptive bleeding condition typical of overt DIC, but instead more closely resembles hypercoagulable thrombosis. This coagulopathy appears to be a core pathophysiology of COVID-19 as rising D-dimer levels, correlate with a poor prognosis, as do rising levels of IL-6 and CRP. IL-6 levels have been correlated to brinogen levels in one study, possibly through the acute phase reactant response 76 . The pathogenesis of microthrombosis of the lung in COVID-19 is not known. There are multiple working hypotheses concerning this nding currently being assessed 77 . Damage to the vascular endothelial glycocalyx can be caused by TNF-α, ischemia and bacterial lipopolysaccharide. As well, activated mast cells release cytokines, proteases, histamine, and heparinase, which degrade the glycocalyx 78 and may thereby contribute to microthrombosis. Disruption of the glycocalyx exposes endothelial cell adhesion molecules, triggering further in ammation, rolling and adhesion of white blood cells and platelets 79 . Glycocalyx components measured in serum positively correlate with increased mortality in septic patients 80 . Other causes of hypercoagulability include direct damage to ACE2 positive endothelial cells by viral invasion or secondary damage from the in ammatory response to the infection. Mast cells release heparin which activates the contact system, producing plasmin and bradykinin. Plasmin activation could account for the singular rise in D-dimer levels. Activation of platelets also seems likely as part of the thrombo-in ammatory response but their precise role in thrombus formation remains to be elucidated 81 . A more complete understanding awaits further study. In addition to the usual features of a viral infection, early COVID-19 often presents with anosmia, ageusia, skin rashes including pruritis and urticaria, neuropsychiatric symptoms (including altered dream states), and silent hypoxia. These symptoms are all consistent with histamine signaling. Anosmia, ageusia, and other symptoms relating to cachexia are often reported in both COVID-19 and mast cell degranulation syndrome, and the potential role of histamine signaling in driving the pathophysiology of cachexia has been reviewed 82, 83 . As summarized in Figure 11 , the distinctive later ndings of abnormal coagulation, involvement of other organ systems and ARDS occur in the second week after the appearance of symptoms. This is coincidental with a rising immunoglobulin response to SARS-CoV-2 antigens. For a subset of patients, disease progress may suddenly worsen at days 7-10, and this correlates with the onset of SARS-CoV-2 spike protein neutralizing antibody titers 84 . In this study, it was shown that IgG starts to rise within 4 days post-symptoms, inconsistent with a rst antigenic exposure 84 . Rapid onset of speci c neutralizing antibody responses beginning less than seven days after exposure to SARS-CoV-2 implies a recall rather than primary B cell response, and therefore the response is being driven by a pre-existing memory cell population. These memory cells may have been educated by prior exposure to another coronavirus (e.g. circulating alphanumeric coronaviruses), raising concerns that this second phase of COVID-19 disease progression could share an immunologic basis with Dengue hemorrhagic fever 85 . Antibodies produced from this early rapid humoral response may drive further mast cell degranulation. During this phase rising D-dimer levels correlate with poor prognosis, as do measured levels of CRP and IL-6. Current reviews seek to explain COVID-19 clinical and pathologic ndings based on standard models of antiviral innate and adaptive immune responses which do not consider the potential role of mast cell activation and degranulation. Reviews emphasize the in ammatory cell response cascade associated with monocytes, macrophages 86 , and adaptive T and B cell helper and effector responses 87 . These types of immune responses are also invoked to explain the novel microvascular pulmonary intravascular coagulopathy associated with COVID-19 88 . We propose an alternative paradigm; SARS-CoV-2 infection-induced mast cell activation could account for some of the core pathologic cascade and much of the unusual symptomatology associated with COVID-19 89 . Many of the unique clinical symptoms observed during the early phase of COVID-19 are consistent with known effects of histamine release. Histamine may act as an autocrine regulator of mast cell cytokine and TNF-a release in a PGE2-dependent fashion and based on in vitro studies the autocrine feedback appears to be mediated by H 2 and H 3 90 . This model is consistent with the histopathologic ndings seen at surgery, autopsies, and is supported by clinical pharmacologic ndings suggesting potential bene ts of histamine H2 receptor blockade using famotidine. This model is also supported by the signi cant overlap in the clinical signs and symptoms of the initial phase of COVID-19 disease and those of mast cell activation syndrome (MCAS) [91] [92] [93] [94] as well similarities to Dengue hemorrhagic fever and shock syndrome (including T cell depletion) during the later phase of COVID-19 85, 95, 96 . The cardiac events, stroke, and related outcomes associated with COVID-19 also appear consistent with the Kounis Analysis of the mechanism of action of famotidine Famotidine was originally selected by the authors for advancement as a potential repurposed drug candidate therapeutic for COVID-19 based on molecular docking data to PLpro. Based on this analysis the FDA granted an IND waiver for the subsequent double blinded randomized clinical trial currently in progress (ClinicalTrials.gov Identi er: NCT04370262). Brie y, a ranked list of licensed compounds with predicted binding activity in the PLpro catalytic site was computationally generated, and the PLpro catalytic site binding pose of each of the top compounds was examined and ranked by a team of pharmaceutical chemists. Package inserts or product monographs for the licensed compounds which generated high computational binding scores and passed inspection were then reviewed and used to rank compounds based on adverse events, FDA warnings, drug interactions on-target mechanisms, pharmacokinetic and absorption, metabolism, excretion and toxicity (ADMET), protein binding and available therapeutic window considerations. Famotidine ("PEPCID ® "), a histamine H2 antagonist widely available in tablet form over-the-counter, as well as in solution form for intravenous administration, was repeatedly computationally ranked as among the most promising of the compounds tested and was associated with the most favorable pharmacokinetic and safety pro le. Other compounds considered at this stage of docking model optimization included camostat mesylate and isoquercitrin. Camostat was rejected for further development due to US regulatory status, lack of suitability for outpatient use, and metabolism issues. Isoquercitrin was rejected due to poor oral bioavailability and lack of prior FDA authorization as a therapeutic (including lack of drug master le). A series of analogs of famotidine were generated using PubChem, and many of these scored even higher as potential candidates. Recognizing that computational docking predictions are typically associated with about a 20% success rate, we applied the method of multiple working hypotheses 77 to assess the mechanism of action of famotidine as a potential treatment for COVID-19. Hypotheses tested included 1) direct binding and action as an inhibitor of SARS-CoV-2 PLpro; 2) action as a direct acting inhibitor of SARS-CoV-2 infection or replication; 3) off-target binding of a non-histamine H2 G-coupled protein receptor 4) histamine H2 receptor inhibition. Production of recombinant SARS-CoV-2 Plpro applied at serial 10-fold dilutions ranging from 10−1 to 10−6 and, after 5 days, viral CPE was detected by staining cell monolayers with crystal violet. Median tissue culture infectious doses (TCID50)/mL were calculated using the method of Reed and Muench. Sigma-1 and sigma-2 competition binding assays terminated by ltration through a glass ber lter using a Brandel cell harvester. Glass ber lters were soaked in 0.3% polyethylenimine for at least 30 min at room temperature before harvesting. All reactions were performed in triplicate using a 96-well block. After the membranes were transferred to the lters and washed, the lters were soaked in 5 mL Cytoscint scintillation uid overnight, and radioactivity was measured using a Beckman Coulter LS 6500 scintillation counter. Data were analyzed using GraphPad Prism software. Ki values were computed by directly tting the data and using the experimentally determined probe K d to calculate a K i value, using the GraphPad Prism software. This process implicitly uses a Cheng-Prusoff correction, so no secondary correction was applied. Sigma-2 competition curves were performed in a similar manner, using Expi293 cells overexpressing the human sigma-2 (TMEM97) and using [ 3 H] DTG as the radioactive probe. The known on-target activity of famotidine considered the known primary mechanism of action is as an antagonist of the histamine H2 receptor. This hypothesis was originally rejected due to unveri ed reports that clinical researchers in PRC (Wuhan) had observed that famotidine use was associated with protection from COVID-19 mortality, while the histamine H2R antagonist cimetidine was not. Positing that this difference in clinical effectiveness for the two different H2R antagonists may re ect absorption, pharmacokinetic and pharmacodistribution differences between famotidine and cimetidine, steady state concentrations were calculated for both drugs when administered at standard oral doses as well as the elevated doses of famotidine which are being prescribed off-label for outpatient clinical use to treat COVID-19 or are being used in the ongoing inpatient clinical trial (NCT04370262), and these were compared to the published H2R IC50 for each drug. GloSensor cAMP assays: cAMP production was determined in transiently transfected HEK293T cells A Novel Coronavirus from Patients with Pneumonia in China Characteristics of and Important Lessons From the Coronavirus Disease 2019 (COVID-19) Outbreak in China: Summary of a Report of 72314 Cases From the Chinese Center for Disease Control and Prevention The SARS-CoV-2 outbreak: What we know SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients Cluster of coronavirus disease 2019 (Covid-19) in the French Alps Asymptomatic carrier state, acute respiratory disease, and pneumonia due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2): Facts and myths Asymptomatic cases in a family cluster with SARS-CoV-2 infection Evidence Supporting Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 While Presymptomatic or Asymptomatic Epidemiologic characteristics of early cases with 2019 novel coronavirus (2019-nCoV) disease in Korea Clinical features of patients infected with 2019 novel coronavirus in Wuhan Clinical Characteristics of 138 Hospitalized Patients With 2019 Novel Coronavirus-Infected Pneumonia in Wuhan Radiological ndings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study The clinical feature of silent infections of novel coronavirus infection (COVID-19) in Wenzhou Characteristics of COVID-19 infection in Beijing Clinical characteristics of 24 asymptomatic infections with COVID-19 screened among close contacts in Nanjing Estimating the asymptomatic proportion of coronavirus disease 2019 (COVID-19) cases on board the Diamond Princess cruise ship Self-reported olfactory and taste disorders in SARS-CoV-2 patients: a crosssectional study Olfactory and gustatory dysfunctions as a clinical presentation of mild-tomoderate forms of the coronavirus disease (COVID-19): a multicenter European study Interim Clinical Guidance for Management of Patients with Con rmed Coronavirus Disease (COVID-19) Famotidine Use is Associated with Improved Clinical Outcomes in Hospitalized COVID-19 Patients: A Retrospective Cohort Study. medRxiv New York clinical trial quietly tests heartburn remedy against coronavirus Structural Insights into the Interaction of Coronavirus Papain-Like Proteases and Interferon-Stimulated Gene Product 15 from Different Species The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds Nidovirus papain-like proteases: Multifunctional enzymes with protease, deubiquitinating and deISGylating activities Papain-like protease 2 (PLP2) from severe acute respiratory syndrome coronavirus (SARS-CoV): expression, puri cation, characterization, and inhibition Analysis of therapeutic targets for SARS-CoV-2 and discovery of potential drugs by computational methods Broad-spectrum inhibition of coronavirus main and papain-like proteases by HCV drugs A SARS-CoV-2 protein interaction map reveals targets for drug repurposing Crystal structure of the human σ1 receptor Identi cation of the gene that codes for the sigma2 receptor Pharmacology of the novel H2 antagonist famotidine: in vitro studies Physiological implications of biased signaling at histamine H2 receptors GPCR signaling along the endocytic pathway Protein-Coupled Receptor Signaling Through beta-Arrestin-Dependent Mechanisms Clinical signi cance of chemokine receptor antagonists Dual CCR5/CCR2 targeting: opportunities for the cure of complex disorders The Role of CXCR3 in Neurological Diseases Mechanisms regulating expansion of CD8+ T cells during HIV-1 infection Effects of antacids and food on absorption of famotidine Single-dose pharmacokinetics and bioavailability of famotidine in man. Results of multicenter collaborative studies Pharmacokinetic and pharmacodynamic properties of histamine H2-receptor antagonists. Relationship between intrinsic potency and effective plasma concentrations Histamine inhibits activation of human neutrophils and HL-60 leukemic cells via H2-receptors Human polymorphonuclear neutrophils. Pharmacological characterization of histamine receptors mediating the elevation of cyclic AMP Inhibition by histamine of platelet-activating-factor-induced neutrophil chemotaxis in bronchial asthma Histamine-induced inhibition of leukotriene biosynthesis in human neutrophils: involvement of the H2 receptor and cAMP Histamine H(2) receptors mediate the inhibitory effect of histamine on human eosinophil degranulation In vitro effects of histamine on eosinophil migration The selective eosinophil chemotactic activity of histamine Evidence for ligand-speci c conformations of the histamine H(2)-receptor in human eosinophils and neutrophils Human skin mast cells express H2 and H4, but not H3 receptors Clinical pharmacokinetics of cimetidine Clinical pharmacokinetics of famotidine PEPCID® (famotidine) tablets, for oral use Histamine H2 receptor activation exacerbates myocardial ischemia/reperfusion injury by disturbing mitochondrial and endothelial function Akt1 is critical for acute in ammation and histamine-mediated vascular leakage International Union of Basic and Clinical Pharmacology. XCVIII. Histamine Receptors LungGENS': a web-based tool for mapping single-cell gene expression in the developing lung Pulmonary Pathology of Early-Phase 2019 Novel Coronavirus (COVID-19) Pneumonia in Two Patients With Lung Cancer Pulmonary Pathology of Early Phase COVID-19 Pneumonia in a Patient with a Benign Lung Lesion Forskolin inhibits the release of histamine from human basophils and mast cells The mystery of the pandemic's 'happy hypoxia Implications for forensic death investigations from rst Swiss post-mortem CT in a case of non-hospital treatment with COVID-19 Sudden and Complete Olfactory Loss Function as a Possible Symptom of COVID-19 The Early Natural History of SARS-CoV-2 Infection: Clinical Observations From an Urban, Ambulatory COVID-19 Clinic COVID-19 pneumonia: ARDS or not? Neurologic Manifestations of Hospitalized Patients With Coronavirus Disease Management of acute ischemic stroke in patients with COVID-19 infection: Report of an international panel Cardiovascular complications in COVID-19 Management of Acute Myocardial Infarction During the COVID-19 Pandemic Pulmonary post-mortem ndings in a large series of COVID-19 cases from Northern Italy. medRxiv Complement associated microvascular injury and thrombosis in the pathogenesis of severe COVID-19 infection: a report of ve cases Diagnosis, Prevention, and Treatment of Thromboembolic Complications in COVID-19: Report of the National Institute for Public Health of the Netherlands Hypercoagulability of COVID-19 patients in Intensive Care Unit. A Report of Thromboelastography Findings and other Parameters of Hemostasis The procoagulant pattern of patients with COVID-19 acute respiratory distress syndrome The Method of Multiple Working Hypotheses The endothelial glycocalyx: a review of the vascular barrier Therapeutic strategies targeting the endothelial glycocalyx: acute de cits, but great potential Increased levels of glycosaminoglycans during septic shock: relation to mortality and the antibacterial actions of plasma Thromboin ammation: challenges of therapeutically targeting coagulation and other host defense mechanisms Is Neuronal Histamine Signaling Involved in Cancer Cachexia? Implications and Perspectives Olfactory dysfunction in seasonal and perennial allergic rhinitis Rapid generation of neutralizing antibody responses in COVID-19 patients. medRxiv Original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever Pathological in ammation in patients with COVID-19: a key role for monocytes and macrophages Immunology of COVID-19: current state of the science Immune mechanisms of pulmonary intravascular coagulopathy in COVID-19 pneumonia Mast cells contribute to coronavirus-induced in ammation: New anti-in ammatory strategy Histamine inhibits tumor necrosis factor alpha release by mast cells through H2 and H3 receptors Diagnosis of mast cell activation syndrome: a global "consensus-2 Mast Cell Activation Syndrome: Tools for Diagnosis and Differential Diagnosis Mast Cell Activation Syndrome: A Primer for the Gastroenterologist Dengue: Status of current and under-development vaccines Anaphylaxis-induced atrial brillation and anesthesia: Pathophysiologic and therapeutic considerations Kounis syndrome: an update on epidemiology, pathogenesis, diagnosis and therapeutic management Mast cell activation disorders presenting with cerebral vasospasm-related symptoms: a "Kounis-like" syndrome? As a potential treatment of COVID-19: Montelukast Recent advances in our understanding of mast cell activationor should it be mast cell mediator disorders? An expression plasmid containing the sequence for (His)6-TEVsite-SARS-CoV-2 PLpro (nsp3 from Wuhan-Hu-1 isolate, polyprotein 1ab 1564-1878) was obtained commercially from ATUM. The plasmid was The expression plasmid for proISG15 (2-165) was a gift from David Komander (Addgene plasmid # 110762 150 mM NaCl, 2 mM DTT, 0.1 mg.mL-1 BSA, with 10 µM of ISG15 in a nal volume of 20 µL for 1 h at room temperature. Control was incubated without enzyme Famotidine does not appear to directly inhibit SARS-CoV-2 infection or replication in Vero cells Viral Growth and Cytotoxicity Assays in the Presence of Inhibitors Plates were then transferred into the BSL3 facility and 100 PFU (MOI 0.025) was added in 50ul of DMEM (2% FBS), bringing the nal compound concentration to those indicated. Plates were then incubated for 48 h at 37C. After infection, supernatants were removed and cells were xed with 4% formaldehyde for 24 hours prior to being removed from the BSL3 facility. The cells were then immunostained for the viral NP protein with a DAPI counterstain Infected cells/Total cells) -Background) *100 and the DMSO control was then set to 100% infection for analysis. The IC50 and IC90 for each experiment were determined using the Prism (GraphPad Software) software. For select inhibitors Cytotoxicity was also performed using the MTT assay (Roche), according to the manufacturer's Infectious titers were quanti ed by limiting dilution titration on Vero E6 cells. Brie y, Vero E6 cells were seeded in 96-well plates at 20,000 cells/well. The next day, SARS-CoV2-containing supernatant was Surveillance (CEIRS, contract # HHSN272201400008C) and by supplements to NIAID grant U19AI135972 and DoD grant W81XWH-20-1-0270 to A. G.-S. The Natural History of COVID-19. Modi ed from Oudkerk et al 74.