A multicenter, double-blind, randomized, parallel-group, placebo-controlled study to evaluate the efficacy and safety of camostat mesilate in patients with COVID-19 (CANDLE study).

BACKGROUND: In vitro drug screening studies have indicated that camostat mesilate (FOY-305) may prevent SARS-CoV-2 infection into human airway epithelial cells. This study was conducted to investigate whether camostat mesilate is an effective treatment for SARS-CoV-2 infection (COVID-19). METHODS: This was a multicenter, double-blind, randomized, parallel-group, placebo-controlled study. Patients were enrolled if they were admitted to a hospital within 5 days of onset of COVID-19 symptoms or within 5 days of a positive test for asymptomatic patients. Severe cases (e.g., those requiring oxygenation/ventilation) were excluded. Patients were enrolled, randomized, and allocated to each group using an interactive web response system. Randomization was performed using a minimization method with the factors medical institution, age, and underlying diseases (chronic respiratory disease, chronic kidney disease, diabetes mellitus, hypertension, cardiovascular diseases, and obesity). The patients, investigators/subinvestigators, study coordinators, and other study personnel were blinded throughout the study. Patients were administered camostat mesilate (600 mg qid; four to eight times higher than the clinical doses in Japan) or placebo for up to 14 days. The primary efficacy endpoint was the time to the first two consecutive negative tests for SARS-CoV-2. RESULTS: One-hundred fifty-five patients were randomized to receive camostat mesilate (n = 78) or placebo (n = 77). The median time to the first test was 11.0 days (95% confidence interval [CI]: 9.0-12.0) in the camostat mesilate group and 11.0 days (95% CI: 10.0-13.0) in the placebo group. Conversion to negative viral status by day 14 was observed in 45 of 74 patients (60.8%) in the camostat mesilate group and 47 of 74 patients (63.5%) in the placebo group. The primary (Bayesian) and secondary (frequentist) analyses found no significant differences in the primary endpoint between the two groups. No additional safety concerns beyond those already known for camostat mesilate were identified. CONCLUSIONS: Camostat mesilate did not substantially reduce the time to viral clearance, based on upper airway viral loads, compared with placebo for treating patients with mild to moderate SARS-CoV-2 infection with or without symptoms. TRIAL REGISTRATION: ClinicalTrials.gov, NCT04657497. Japan Registry for Clinical Trials, jRCT2031200198.

Randomised controlled trial of intravenous nafamostat mesylate in COVID pneumonitis: Phase 1b/2a experimental study to investigate safety, Pharmacokinetics and Pharmacodynamics.

BACKGROUND: Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials in COVID19 without characterisation of Pharmacokinetics /Pharmacodynamics including safety data. One such drug is nafamostat mesylate. METHODS: We present the findings of a phase Ib/IIa open label, platform randomised controlled trial of intravenous nafamostat in hospitalised patients with confirmed COVID-19 pneumonitis. Patients were assigned randomly to standard of care (SoC), nafamostat or an alternative therapy. Nafamostat was administered as an intravenous infusion at a dose of 0.2 mg/kg/h for a maximum of seven days. The analysis population included those who received any dose of the trial drug and all patients randomised to SoC. The primary outcomes of our trial were the safety and tolerability of intravenous nafamostat as an add on therapy for patients hospitalised with COVID-19 pneumonitis. FINDINGS: Data is reported from 42 patients, 21 of which were randomly assigned to receive intravenous nafamostat. 86% of nafamostat-treated patients experienced at least one AE compared to 57% of the SoC group. The nafamostat group were significantly more likely to experience at least one AE (posterior mean odds ratio 5.17, 95% credible interval (CI) 1.10 - 26.05) and developed significantly higher plasma creatinine levels (posterior mean difference 10.57 micromol/L, 95% CI 2.43-18.92). An average longer hospital stay was observed in nafamostat patients, alongside a lower rate of oxygen free days (rate ratio 0.55-95% CI 0.31-0.99, respectively). There were no other statistically significant differences in endpoints between nafamostat and SoC. PK data demonstrated that intravenous nafamostat was rapidly broken down to inactive metabolites. We observed no significant anticoagulant effects in thromboelastometry. INTERPRETATION: In hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous nafamostat, and there were additional adverse events. FUNDING: DEFINE was funded by LifeArc (an independent medical research charity) under the STOPCOVID award to the University of Edinburgh. We also thank the Oxford University COVID-19 Research Response Fund (BRD00230).

Physical Compatibility of Nafamostat with Analgesics, Sedatives, and Muscle Relaxants for Treatment of Coronavirus Disease 2019.

BACKGROUND: Severe coronavirus disease 2019 (COVID-19) may require continuous administration of analgesics, sedatives, and muscle relaxants. Nafamostat has recently been reported as a therapeutic agent for COVID-19. However, there is a lack of information on the compatibility of nafamostat with the aforementioned drug classes. This study evaluated the physical compatibility of nafamostat with these drug classes. METHODS: Nafamostat was combined with 1-3 target drugs (fentanyl, morphine, midazolam, dexmedetomidine, and rocuronium). Fifteen physical compatibility tests were conducted. Nafamostat was dissolved in 5% glucose solution; the final concentration was 10 mg/mL. All other medications were diluted in 0.9% sodium chloride to obtain clinically relevant concentrations. The power of hydrogen (pH) of all medications was measured during each test. Compatibility tests were conducted with 4 test solutions in which nafamostat and the target drugs were compounded at equal volume ratios (1:1, 1:1:1, or 1:1:1:1). Visual appearance, turbidity, and pH were evaluated immediately after mixing and at 1 and 3 hours. Physical incompatibilities were defined as gross precipitation, cloudiness, appearance of the Tyndall effect, or a turbidity change of ≥0.5 nephelometric turbidity units (NTU) based on nafamostat. RESULTS: The mean pH of nafamostat was 3.13 ± 0.03. The combination of nafamostat, fentanyl, and dexmedetomidine had the highest pH (3.39 ± 0.01; 3 hours after mixing). All drugs were compatible with nafamostat until 3 hours after admixture, with a mean turbidity value of ≤0.03 NTU. CONCLUSIONS: Infusions combining nafamostat with the tested sedatives, analgesics, and muscle relaxants could be safely administered.

Nafamostat reduces systemic inflammation in TLR7-mediated virus-like illness.

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.

Nafamostat-Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

A Novel Computational Approach for the Discovery of Drug Delivery System Candidates for COVID-19.

In order to treat Coronavirus Disease 2019 (COVID-19), we predicted and implemented a drug delivery system (DDS) that can provide stable drug delivery through a computational approach including a clustering algorithm and the Schrödinger software. Six carrier candidates were derived by the proposed method that could find molecules meeting the predefined conditions using the molecular structure and its functional group positional information. Then, just one compound named glycyrrhizin was selected as a candidate for drug delivery through the Schrödinger software. Using glycyrrhizin, nafamostat mesilate (NM), which is known for its efficacy, was converted into micelle nanoparticles (NPs) to improve drug stability and to effectively treat COVID-19. The spherical particle morphology was confirmed by transmission electron microscopy (TEM), and the particle size and stability of 300-400 nm were evaluated by measuring DLSand the zeta potential. The loading of NM was confirmed to be more than 90% efficient using the UV spectrum.

COVID-19-associated coagulopathy and disseminated intravascular coagulation.

The pathology of coronavirus disease 2019 (COVID-19) is exacerbated by the progression of thrombosis, and disseminated intravascular coagulation (DIC), and cytokine storms. The most frequently reported coagulation/fibrinolytic abnormality in COVID-19 is the increase in D-dimer, and its relationship with prognosis has been discussed. However, limits exist to the utility of evaluation by D-dimer alone. In addition, since the coagulation/fibrinolytic condition sometimes fluctuates within a short period of time, regular examinations in recognition of the significance of the examination are desirable. The pathophysiology of disseminated intravascular coagulation (DIC) associated with COVID-19 is very different from that of septic DIC, and both thrombotic and hemorrhagic pathologies should be noted. COVID-19 thrombosis includes macro- and microthrombosis, with diagnosis of the latter depending on markers of coagulation and fibrinolysis. Treatment of COVID-19 is classified into antiviral treatment, cytokine storm treatment, and thrombosis treatment. Rather than providing uniform treatment, the treatment method most suitable for the severity and stage should be selected. Combination therapy with heparin and nafamostat is expected to develop in the future. Fibrinolytic therapy and adsorption therapy require further study.

Strong Binding of Leupeptin with TMPRSS2 Protease May Be an Alternative to Camostat and Nafamostat for SARS-CoV-2 Repurposed Drug: Evaluation from Molecular Docking and Molecular Dynamics Simulations.

The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial.

Camostat mesylate against SARS-CoV-2 and COVID-19-Rationale, dosing and safety.

The coronavirus responsible for COVID-19, SARS-CoV-2, utilizes a viral membrane spike protein for host cell entry. For the virus to engage in host membrane fusion, SARS-CoV-2 utilizes the human transmembrane surface protease, TMPRSS2, to cleave and activate the spike protein. Camostat mesylate, an orally available well-known serine protease inhibitor, is a potent inhibitor of TMPRSS2 and has been hypothesized as a potential antiviral drug against COVID-19. In vitro human cell and animal studies have shown that camostat mesylate inhibits virus-cell membrane fusion and hence viral replication. In mice, camostat mesylate treatment during acute infection with influenza, also dependent on TMPRSS2, leads to a reduced viral load. The decreased viral load may be associated with an improved patient outcome. Because camostat mesylate is administered as an oral drug, it may be used in outpatients as well as inpatients at all disease stages of SARS-CoV-2 infection if it is shown to be an effective antiviral agent. Clinical trials are currently ongoing to test whether this well-known drug could be repurposed and utilized to combat the current pandemic. In the following, we will review current knowledge on camostat mesylate mode of action, potential benefits as an antiviral agent and ongoing clinical trials.

A comparative analysis of remdesivir and other repurposed antivirals against SARS-CoV-2.

The ongoing SARS-CoV-2 pandemic stresses the need for effective antiviral drugs that can quickly be applied in order to reduce morbidity, mortality, and ideally viral transmission. By repurposing of broadly active antiviral drugs and compounds that are known to inhibit viral replication of related viruses, several advances could be made in the development of treatment strategies against COVID-19. The nucleoside analog remdesivir, which is known for its potent in vitro activity against Ebolavirus and other RNA viruses, was recently shown to reduce the time to recovery in patients with severe COVID-19. It is to date the only approved antiviral for treating COVID-19. Here, we provide a mechanism and evidence-based comparative review of remdesivir and other repurposed drugs with proven in vitro activity against SARS-CoV-2.

Dynamic changes in fibrinogen and D-dimer levels in COVID-19 patients on nafamostat mesylate.

Critical illnesses associated with coronavirus disease 2019 (COVID-19) are attributable to a hypercoagulable status. There is limited knowledge regarding the dynamic changes in coagulation factors among COVID-19 patients on nafamostat mesylate, a potential therapeutic anticoagulant for COVID-19. First, we retrospectively conducted a cluster analysis based on clinical characteristics on admission to identify latent subgroups among fifteen patients with COVID-19 on nafamostat mesylate at the University of Tokyo Hospital, Japan, between April 6 and May 31, 2020. Next, we delineated the characteristics of all patients as well as COVID-19-patient subgroups and compared dynamic changes in coagulation factors among each subgroup. The subsequent dynamic changes in fibrinogen and D-dimer levels were presented graphically. All COVID-19 patients　were classified into three subgroups: clusters A, B, and C, representing low, intermediate, and high risk of poor outcomes, respectively. All patients were alive 30 days from symptom onset. No patient in cluster A required mechanical ventilation; however, all patients in cluster C required mechanical ventilation, and half of them were treated with venovenous extracorporeal membrane oxygenation. All patients in cluster A maintained low D-dimer levels, but some critical patients in clusters B and C showed dynamic changes in fibrinogen and D-dimer levels. Although the potential of nafamostat mesylate needs to be evaluated in randomized clinical trials, admission characteristics of patients with COVID-19 could predict subsequent coagulopathy.

High levels of anti-SSA/Ro antibodies in COVID-19 patients with severe respiratory failure: a case-based review : High levels of anti-SSA/Ro antibodies in COVID-19.

We treated two patients with severe respiratory failure due to coronavirus disease 2019 (COVID-19). Case 1 was a 73-year-old woman, and Case 2 was a 65-year-old-man. Neither of them had a history of autoimmune disease. Chest computed tomography scans before the antiviral therapy showed bilateral multiple patchy ground-glass opacities (GGO) consistent with COVID-19 pneumonia. The GGO regressed over the course of the antiviral treatment; however, new non-segmental patchy consolidations emerged, which resembled those of interstitial lung disease (ILD), specifically collagen vascular disease-associated ILD. We tested the patients' sera for autoantibodies and discovered that both patients had high anti-SSA/Ro antibody titers. In Case 1, the patient recovered with antiviral therapy alone. However, in Case 2, the patient did not improve with antiviral therapy alone but responded well to corticosteroid therapy (methylprednisolone) and made a full recovery. The relationship between some immunological responses and COVID-19 pneumonia exacerbation has been discussed previously; our discovery of the elevation of anti-SSA/Ro antibodies suggests a contribution from autoimmunity functions of the immune system. Although it is unclear whether the elevation of anti-SSA/Ro antibodies was a cause or an outcome of aggravated COVID-19 pneumonia, we hypothesize that both patients developed aggravated the COVID-19 pneumonia due to an autoimmune response. In COVID-19 lung injury, there may be a presence of autoimmunity factors in addition to the known effects of cytokine storms. In patients with COVID-19, a high level of anti-SSA/Ro52 antibodies may be a surrogate marker of pneumonia severity and poor prognosis.

Case Report: Viral Shedding for 60 Days in a Woman with COVID-19.

Novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has become a public health emergency of international concern. This was first noted in Wuhan, Hubei Province, China, and since then has become widespread globally. We report a 71-year-old woman with documented viral shedding (based on reverse transcription-polymerase chain reaction (RT-PCR) testing) of SARS-CoV-2 for 60 days from the onset of symptoms (55 days from her first positive test and 36 days after complete resolution of symptoms). This is to our knowledge the longest duration of viral shedding reported to date. This case demonstrates that viral shedding after COVID-19 diagnosis can be prolonged.

Three cases of treatment with nafamostat in elderly patients with COVID-19 pneumonia who need oxygen therapy.

No effective treatment for COVID-19 has been well established yet. Nafamostat, known as anticoagulant, has potential anti-inflammatory and anti-viral activities against COVID-19. We report three cases of COVID-19 pneumonia who progressed while using antiviral drugs and needed supplementary oxygen therapy, improved after treatment with nafamostat. These preliminary findings show the possibility that Nafamostat can be considered to be used in elderly patients with COVID-19 pneumonia who need oxygen therapy. The effectiveness of nafamostat should be evaluated in further studies.

Potential therapeutic targets and promising drugs for combating SARS-CoV-2.

As of April 9, 2020, a novel coronavirus (SARS-CoV-2) had caused 89,931 deaths and 1,503,900 confirmed cases worldwide, which indicates an increasingly severe and uncontrollable situation. Initially, little was known about the virus. As research continues, we now know the genome structure, epidemiological and clinical characteristics, and pathogenic mechanisms of SARS-CoV-2. Based on this knowledge, potential targets involved in the processes of virus pathogenesis need to be identified, and the discovery or development of drugs based on these potential targets is the most pressing need. Here, we have summarized the potential therapeutic targets involved in virus pathogenesis and discuss the advances, possibilities, and significance of drugs based on these targets for treating SARS-CoV-2. This review will facilitate the identification of potential targets and provide clues for drug development that can be translated into clinical applications for combating SARS-CoV-2.