COVID-19: Famotidine, Histamine, Mast Cells, and Mechanisms

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 H2 activity, and that development of clinical COVID-19 involves dysfunctional mast cell activation and histamine release.

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).

Famotidine does not bind to SARS-CoV-2 proteases
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 .

Famotidine does not directly inhibit SARS-CoV-2 infection
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.

Human receptors
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.

Famotidine is selective for receptor H 2
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.

Famotidine may activate other GPCRs
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 μg/L (0.039 μM) for famotidine and 400-780 μg/L (1.59-3.09 μM) 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 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.

Discussion
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 SARS-CoV-2 infection that results in COVID-19 is at least partially mediated by pathologic histamine release. The anecdotal lack of protection provided by oral administration of the H 2 antagonist cimetidine can be accounted for by insu cient systemic drug levels after oral administration and does not contradict potential bene t provided by famotidine H 2 binding. Intravenous cimetidine at su cient doses may achieve levels high enough for clinical bene t and would further support this hypothesis. Failure to achieve clinical COVID-19 responses with cimetidine may indicate that inverse agonism or other GPCRmediated effects of famotidine may play an important role in the (preliminary) observed clinical bene ts. 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. In the capillary wall, there are the glycocalyx, the endothelial cell with associated tight junctions, and the basement membrane. In the epithelium there is a surfactant layer on the alveolar lining uid, manufactured and secreted by the Type II pneumocyte, and the Type I pneumocyte itself with its tight junctions and negatively charged basement membrane which restricts albumin. The pulmonary pericytes located in the terminal conducting airway region play a critical role in synthesizing the endothelial basement membrane and regulating blood ow in the precapillary arteriole, the capillary and the postcapillary venule. Disruption of any of these cells or layers can lead to edema. This edema uid may be a transudate in milder dysfunctions or an exudate when in ammation or necrosis develop. Two possible pathologies that could result in edema of the alveolar wall and space include infection of cells by SARS-CoV-2 and mast cell degranulation with release of hundreds of compounds that can impact on cellular and basement membrane functions, glycocalyx and tight junction integrity. These compounds include histamine, bradykinin, heparin, tryptase and cytokines.
Gene expression patterns of these pulmonary cells provide insight into which cells are likely to be infected, and which express the H 2 receptor that could be directly impacted by famotidine treatment and resulting H 2 antagonism or inverse agonism ( Figure 8). These patterns suggest that epithelial cells and endothelial cells are more likely to be infected based on ACE2 and TMPRSS2 expression patterns in those cell types. The cells most likely to show a famotidine effect include Type 2 pneumocytes, smooth muscle cells, pericytes, and myeloid granulocytes (which includes mast cells, neutrophils and eosinophils).
The limited tissue pathology available from early COVID-19 cases seems to support both viral infection as well as histamine effects in the lung. In a singular study of early COVID-19, Sufang Tian et al 59 describe the viral lung pathology of early COVID-19 in tissue resected for cancer. Their photomicrographs show two different patterns of disease. As shown in Figure 9 panel B, some samples of this lung tissue demonstrate the usual mononuclear in ammatory pattern of interstitial pneumonitis and brinous exudate that one would associate with a viral infection. It is striking that no neutrophils or eosinophils are observed in the in ammatory in ltrate. One explanation is that H 2 activation of neutrophils 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 .
The reports of Tian et al 59 and Zeng et al 60 also include images in which there is interstitial and alveolar edema while the alveolar septae retain normal architecture (Figure 9 panel A). This is not a pattern typically observed in viral infection, as there is no in ammation, and the uid appears to be a transudate. It is consistent with dysregulation of the uid barrier due to the effect of histamine or other mast cell products on endothelial cells, pericytes or Type II pneumocytes. Increased endothelial permeability due to histamine is driven by H 1 receptor activation, and so if any potential famotidine treatment effect on these cells occurs it would most likely be indirect by inhibition of mast cell degranulation. Forskolin activates the enzyme adenylyl cyclase and increases intracellular levels of cAMP, and can be used to inhibit the release of histamine from human basophils and mast cells 61 . Histamine may act as an autocrine regulator of mast cell cytokine and TNF-a release in a PGE2-dependent fashion. Based on in vitro studies, this autocrine feedback appears to be mediated by H 2 and H 3 (88). Endothelial cells are also susceptible to infection by SARS-CoV-2. Mast cell degranulation-related pulmonary edema could correlate with the early phase silent hypoxia and the high compliance non-ARDS ventilation pattern associated with 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 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  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. for overnight stimulation. Plates were then counted as above.

5) Famotidine could act by blocking histamine receptor(s)
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     Lung alveolus cell interactions and gas exchange