Search

Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics. [ABSTRACT FROM AUTHOR]

ABSTRACT: Background: Nafamostat, a serine protease inhibitor, has been used for the treatment of disseminated intravascular coagulation and pancreatitis. In vitro studies and clinical reports suggest its beneficial effect in the treatment of COVID-19 pneumonia. Methods: This phase 2 open-label, randomised, multicentre, controlled trial evaluated nafamostat (4.8 mg/kg/day) plus standard-of-care (SOC) in hospitalised patients with COVID-19 pneumonia (i.e., those requiring nasal high-flow oxygen therapy and/or non-invasive mechanical ventilation). The primary outcome was the time to clinical improvement. Key secondary outcomes included the time to recovery, rates of recovery and National Early Warning Score (NEWS). The trial is registered with ClinicalTrials.gov Identifier: NCT04623021. Findings: A total of 104 patients, mean age 58.6 years were enrolled in 13 clinical centres in Russia between 25/9/2020 and 14/11/2020 and randomised to nafamostat plus SOC (n=53) or SOC alone (n=51). There was no significant difference in time to clinical improvement (primary endpoint) between the nafamostat and SOC groups (median 11 [interquartile range (IQR) 9 to 14) vs 11 [IQR 9 to 14] days; Rate Ratio [RR; the ratio for clinical improvement], 1.00; 95% CI, 0.65 to 1.57; p=0.953). In 36 patients with baseline NEWS ≥7, nafamostat was superior to SOC alone in median time to clinical improvement (11 vs 14 days; RR, 2.89; 95% CI, 1.17 to 7.14; p=0.012). Patients receiving nafamostat in this subgroup had a significantly higher recovery rate compared with SOC alone (61.1% (11/18) vs 11.1 % (2/18) by Day 11, p=0.002). The 28-day mortality was 1.9% (1/52) for nafamostat and 8.0% (4/50) for SOC (95% CI, -17.0 to 3.4; p=0.155). No case of COVID-19 related serious adverse events leading to death was recorded in the patients receiving nafamostat. Interpretation: Our study found no significant difference in time to clinical improvement between the nafamostat and SOC groups, but a shorter median time to clinical improvement in a small group of high-risk COVID-19 patients requiring oxygen treatment. To assess the efficacy further, a larger Phase 3 clinical trial is warranted. Funding: Korea Research Institute of Bioscience and Biotechnology [2020M3A9H5108928] and Chong Kun Dang (CKD) Pharm (Seoul, Korea).

Nafamostat, a serine proteinase inhibitor with various actions including antithrombin, antiplasmin, and antitrypsin effects, has been used in clinical practice to treat disseminated intravascular coagulation (DIC) and pancreatitis. This case report describes the clinical course of a patient with COVID-19 pneumonia whose severe hypoxemia, probably caused by DIC and pulmonary embolism, showed remarkable improvement with combination heparin and nafamostat therapy. In addition, beneficial mechanisms of nafamostat against COVID-19 and the necessity of attention to hyperkalemia as an adverse effect are discussed.

ABSTRACT The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis. IMPORTANCE The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.

Background: Heparin-induced thrombocytopenia (HIT) involves platelet activation and aggregation caused by heparin or HIT antibodies associated with poor survival outcomes. We report a case of HIT that occurred after hemodialysis was started for rapidly progressive glomerulonephritis (RPGN), which was caused by anti-neutrophil cytoplasmic antibody-associated vasculitis (AAV), and ultimately resulted in asymptomatic cerebral infarction.Case Presentation: A 76-year-old Japanese man was urgently admitted to our hospital for weight loss and acute kidney injury (serum creatinine: 12 mg/dL). Hemodialysis therapy was started using heparin for anticoagulation. Blood testing revealed elevated titers of myeloperoxidase anti-neutrophil cytoplasmic antibodies, and renal biopsy revealed crescentic glomerulonephritis with broad hyalinization of most of the glomeruli and a pauci-immune staining pattern. These findings fulfilled the diagnostic criteria for microscopic polyangiitis, and the patient was diagnosed with RPGN caused by AAV. Steroid pulse therapy, intermittent pulse intravenous cyclophosphamide, and oral steroid therapy failed to improve the patient's renal function, and maintenance dialysis was started. However, on day 15, his platelet count had decreased to 47,000/µL, with clotting observed in the hemodialysis catheter. Magnetic resonance imaging of the head identified acute asymptomatic brain infarction in the left occipital lobe, and a positive HIT antibody test result supported a diagnosis of type II HIT. During hemodialysis, the anticoagulant treatment was changed from heparin to argatroban. Platelet counts subsequently normalized, and the patient was discharged. A negative HIT antibody test result was observed on day 622.Conclusions: There have been several similar reports of AAV and HIT co-existence. However, this is a rare case report on cerebral infarction with AAV and HIT co-existence. Autoimmune diseases are considered risk factors for HIT, and AAV may overlap with other systemic autoimmune diseases. To confirm the relationship between these two diseases, it is necessary to accumulate more information from future cases with AAV and HIT co-existence. If acute thrombocytopenia and clotting events are observed when heparin is used as an anticoagulant, type II HIT should always be considered in any patient due to its potentially fatal thrombotic complications. [ABSTRACT FROM AUTHOR]

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.

Coronavirus Disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is frequently complicated by thrombosis. In some cases of severe COVID-19, fibrinolysis may be markedly enhanced within a few days, resulting in fatal bleeding. In the treatment of COVID-19, attention should be paid to both coagulation activation and fibrinolytic activation. Various thromboses are known to occur after vaccination with SARS-CoV-2 vaccines. Vaccine-induced immune thrombotic thrombocytopenia (VITT) can occur after adenovirus-vectored vaccination, and is characterized by the detection of anti-platelet factor 4 antibodies by enzyme-linked immunosorbent assay and thrombosis in unusual locations such as cerebral venous sinuses and visceral veins. Treatment comprises high-dose immunoglobulin, argatroban, and fondaparinux. Some VITT cases show marked decreases in fibrinogen and platelets and marked increases in D-dimer, suggesting the presence of enhanced-fibrinolytic-type disseminated intravascular coagulation with a high risk of bleeding. In the treatment of VITT, evaluation of both coagulation activation and fibrinolytic activation is important, adjusting treatments accordingly to improve outcomes.

Nafamostat, a synthetic serine protease inhibitor, has been used for the treatment of inflammatory diseases such as pancreatitis. Recently, an increasing number of studies have shown the promising antiviral effects of nafamostat for the treatment of coronavirus disease-19 (COVID-19). This study aimed to develop a novel liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis and to characterize the pharmacokinetics of nafamostat in rats. Nafamostat in the rat plasma was extracted by solid phase extraction, and 13C6-nafamostat was used as an internal standard. The quantification limit of nafamostat in the rat plasma was 0.5 ng/mL. The LC-MS/MS method was fully validated and applied to characterize the pharmacokinetics of nafamostat in rats. Following intravenous injection (2 mg/kg), nafamostat in the plasma showed a multiexponential decline with an average elimination half-life (t1/2) of 1.39 h. Following oral administration of nafamostat solutions (20 mg/kg) in 10% dimethyl sulfoxide (DMSO) and in 10% DMSO with 10% Tween 80, nafamostat was rapidly absorbed, and the average oral bioavailability was 0.95% and 1.59%, respectively. The LC-MS/MS method and the pharmacokinetic information of nafamostat could be helpful for the further preclinical and clinical studies of nafamostat.

Sepsis is a life-threatening syndrome resulting from a dysregulated host response to infection. It is the primary cause of death in the intensive care unit, posing a substantial challenge to human health and medical resource allocation. The pathogenesis and pathophysiology of sepsis are complex. During its onset, pro-inflammatory and anti-inflammatory mechanisms engage in intricate interactions, possibly leading to hyperinflammation, immunosuppression, and long-term immune disease. Of all critical outcomes, hyperinflammation is the main cause of early death among patients with sepsis. Therefore, early suppression of hyperinflammation may improve the prognosis of these patients. Nafamostat mesilate is a serine protease inhibitor, which can inhibit the activation of the complement system, coagulation system, and contact system. In this review, we discuss the pathophysiological changes occurring in these systems during sepsis, and describe the possible targets of the serine protease inhibitor nafamostat mesilate in the treatment of this condition., (© 2024 The Authors.)

Inhibition of transmembrane serine protease 2 (TMPRSS2) is expected to block the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nafamostat, a potent TMPRSS2 inhibitor as well as a candidate for anti-SARS-CoV-2 drug, possesses the same acyl substructure as camostat, but is known to have a greater antiviral effect. A unique aspect of the molecular binding of nafamostat has been recently reported to be the formation of a covalent bond between its acyl substructure and Ser441 in TMPRSS2. In this study, we investigated crucial elements that cause the difference in anti-SARS-CoV-2 activity of nafamostat and camostat. In silico analysis showed that Asp435 significantly contributes to the binding of nafamostat and camostat to TMPRSS2, while Glu299 interacts strongly only with nafamostat. The estimated binding affinity for each compound with TMPRSS2 was actually consistent with the higher activity of nafamostat; however, the evaluation of the newly synthesized nafamostat derivatives revealed that the predicted binding affinity did not correlate with their anti-SARS-CoV-2 activity measured by the cytopathic effect (CPE) inhibition assay. It was further shown that the substitution of the ester bond with amide bond in nafamostat resulted in significantly weakened anti-SARS-CoV-2 activity. These results strongly indicate that the ease of covalent bond formation with Ser441 in TMPRSS2 possibly plays a major role in the anti-SARS-CoV-2 effect of nafamostat and its derivatives.

Aortic aneurysms are sometimes associated with enhanced-fibrinolytic-type disseminated intravascular coagulation (DIC). In enhanced-fibrinolytic-type DIC, both coagulation and fibrinolysis are markedly activated. Typical cases show decreased platelet counts and fibrinogen levels, increased concentrations of fibrin/fibrinogen degradation products (FDP) and D-dimer, and increased FDP/D-dimer ratios. Thrombin-antithrombin complex or prothrombin fragment 1 + 2, as markers of coagulation activation, and plasmin-α2 plasmin inhibitor complex, a marker of fibrinolytic activation, are all markedly increased. Prolongation of prothrombin time (PT) is not so obvious, and the activated partial thromboplastin time (APTT) is rather shortened in some cases. As a result, DIC can be neither diagnosed nor excluded based on PT and APTT alone. Many of the factors involved in coagulation and fibrinolysis activation are serine proteases. Treatment of enhanced-fibrinolytic-type DIC requires consideration of how to control the function of these serine proteases. The cornerstone of DIC treatment is treatment of the underlying pathology. However, in some cases surgery is either not possible or exacerbates the DIC associated with aortic aneurysm. In such cases, pharmacotherapy becomes even more important. Unfractionated heparin, other heparins, synthetic protease inhibitors, recombinant thrombomodulin, and direct oral anticoagulants (DOACs) are agents that inhibit serine proteases, and all are effective against DIC. Inhibition of activated coagulation factors by anticoagulants is key to the treatment of DIC. Among them, DOACs can be taken orally and is useful for outpatient treatment. Combination therapy of heparin and nafamostat allows fine-adjustment of anticoagulant and antifibrinolytic effects. While warfarin is an anticoagulant, this agent is ineffective in the treatment of DIC because it inhibits the production of coagulation factors as substrates without inhibiting activated coagulation factors. In addition, monotherapy using tranexamic acid in cases of enhanced-fibrinolytic-type DIC may induce fatal thrombosis. If tranexamic acid is needed for DIC, combination with anticoagulant therapy is of critical importance.

Aim: We aimed to describe the clinical characteristics, treatment and outcomes of patients with COVID‐19 pneumonia, in particular older patients, admitted to tertiary and partner hospitals in Saitama, Japan. Methods: We retrospectively reviewed the medical records of patients with COVID‐19 pneumonia admitted to tertiary and partner hospitals in Saitama, Japan. Twenty‐six patients with COVID‐19 were categorized into two groups, i.e., older (≥75 years) and younger adults (≤74 years). We evaluated the clinical characteristics, comorbidities, symptoms, laboratory test results, treatments and outcomes of the patients. Results: The majority of the older patients had comorbidities, such as dementia, cardiovascular disease and bone fractures. Comorbidities were significantly more frequent in older patients than younger patients. No association was found between age and body temperature or the incidence of respiratory failure. White blood cell count was significantly lower in older patients (P = 0.018) and the decrease in lymphocytes was greater in younger patients (P = 0.009). Computed tomography (CT) of all patients showed non‐segmental, peripherally dominant ground‐glass opacities consistent with COVID‐19 pneumonia. In older patients, antiviral drugs, anticoagulants and anti‐inflammatory drugs were administered on a compassionate use basis. The difference in mortality between the older and the younger patients was not statistically significant. Conclusions: In older patients, typical clinical symptoms and blood test changes were often absent; however, CT always contained typical findings of COVID‐19, suggesting that CT may be a useful diagnostic tool. Our report illustrates that appropriate treatment, taking patient background into consideration, may improve their condition regardless of age. Geriatr Gerontol Int 2020; 20: 1044–1049. [ABSTRACT FROM AUTHOR]

Patients who undergo renal replacement therapy often exhibit a high plasma linezolid concentration. Linezolid is metabolized via oxidation. Nafamostat mesilate has antioxidant effects and is frequently used as an anticoagulant during renal replacement therapy. We aimed to investigate the effect of nafamostat mesilate on plasma linezolid concentration. We examined whether the co‐administration of linezolid and nafamostat had any effect on plasma linezolid concentration. Mice were randomly allocated to two groups (n = 18/group): linezolid (100 mg kg−1, subcutaneous injection) + nafamostat (30 mg kg−1, intraperitoneal injection) and linezolid + saline. At 5 hours, the linezolid concentration was significantly higher in the linezolid + nafamostat co‐administration group than that in the linezolid + saline group (20.6 ± 9.8 vs 3.6 ± 1.2 μg/mL, respectively P <.001). The antioxidant effects of nafamostat may inhibit linezolid metabolism, resulting in the adverse event of high linezolid concentration if both are administered concurrently during renal replacement therapy. [ABSTRACT FROM AUTHOR]

The number of patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly increased, although the WHO declared a pandemic. However, drugs that function against SARS-CoV-2 have not been established. SARS-CoV-2 has been suggested to bind angiotensin-converting enzyme 2, the receptor of the SARS coronavirus. SARS coronavirus and coronavirus 229E, the cause of the common cold, replicate through cell-surface and endosomal pathways using a protease, the type II transmembrane protease. To examine the effects of protease inhibitors on the replication of coronavirus 229E, we pretreated primary cultures of human nasal epithelial (HNE) cells with camostat or nafamostat, each of which has been used for the treatment of pancreatitis and/or disseminated intravascular coagulation. HNE cells were then infected with coronavirus 229E, and viral titers in the airway surface liquid of the cells were examined. Pretreatment with camostat (0.1-10 µ g/mL) or nafamostat (0.01-1 µ g/mL) reduced the titers of coronavirus 229E. Furthermore, a significant amount of type II transmembrane protease protein was detected in the airway surface liquid of HNE cells. Additionally, interferons have been reported to have antiviral effects against SARS coronavirus. The additive effects of interferons on the inhibitory effects of other candidate drugs to treat SARS-CoV-2 infection, such as lopinavir, ritonavir and favipiravir, have also been studied. These findings suggest that protease inhibitors of this type may inhibit coronavirus 229E replication in human airway epithelial cells at clinical concentrations. Protease inhibitors, interferons or the combination of these drugs may become candidate drugs to inhibit the replication of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The coronavirus, a subfamily of the coronavirinae family, is an RNA virus with over 40 variations that can infect humans, non-human mammals and birds. There are seven types of human coronaviruses, including SARS-CoV-2, is responsible for the recent COVID-19 pandemic. The current study is focused on the identification of drug molecules for the treatment of COVID-19 by targeting human proteases like transmembrane serine protease 2 (TMPRSS2), furin, cathepsin B, and a nuclear receptor named farnesoid X receptor (FXR). TMPRSS2 and furin help in cleaving the spike protein of the SARS-CoV-2 virus, while cathepsin B plays a critical role in the entry and pathogenesis. FXR, on the other hand, regulates the expression of ACE2, and its inhibition can reduce SARS-CoV-2 infection. By inhibiting these four protein targets with non-toxic inhibitors, the entry of the infectious agent into host cells and its pathogenesis can be obstructed. We have used the BioSolveIT suite for pharmacophore-based computational drug designing. A total of 1611 ligands from the ligand library were docked with the target proteins to obtain potent inhibitors on the basis of pharmacophore. Following the ADMET analysis and protein ligand interactions, potent and druggable inhibitors of the target proteins were obtained. Additionally, toxic substructures and the less toxic route of administration of the most potent inhibitors in rodents were also determined computationally. Compounds namely N -(diaminomethylene)-2-((3-((1 R ,3 R)-3-(2-(methoxy(methyl)amino)-2-oxoethyl)cyclopentyl)propyl)amino)-2-oxoethan-1-aminium (26), (1 R ,3 R)-3-(((2-ammonioethyl)ammonio)methyl)-1-((4-propyl-1 H -imidazol-2-yl)methyl)piperidin-1-ium (29) and (1 R ,3 R)-3-(((2-ammonioethyl)ammonio)methyl)-1-((1-propyl-1 H -pyrazol-4-yl)methyl)piperidin-1-ium (30) were found as the potent inhibitors of TMPRSS2, whereas, 1-(1-(1-(1 H -tetrazol-1-yl)cyclopropane-1‑carbonyl)piperidin-4-yl)azepan-2-one (6), (2 R)-4-methyl-1-oxo-1-((7 R ,11 S)-4-oxo-6,7,8,9,10,11-hexahydro-4 H -7,11-methanopyrido[1,2- a ]azocin-9-yl)pentan-2-aminium (12), 4-((1-(3-(3,5-dimethylisoxazol-4-yl)propanoyl)piperidin-4-yl)methyl)morpholin-4-ium (13), 1-(4,6-dimethylpyrimidin-2-yl)- N -(3-oxocyclohex-1-en-1-yl)piperidine-4-carboxamide (14), 1-(4-(1,5-dimethyl-1 H -1,2,4-triazol-3-yl)piperidin-1-yl)-3-(3,5-dimethylisoxazol-4-yl)propan-1-one (25) and N,N -dimethyl-4-oxo-4-((1 S ,5 R)-8-oxo-5,6-dihydro-1 H -1,5-methanopyrido[1,2- a ][1,5]diazocin-3(2 H ,4 H ,8 H)-yl)butanamide (31) inhibited the FXR preferentially. In case of cathepsin B, N -((5-benzoylthiophen-2-yl)methyl)-2-hydrazineyl-2-oxoacetamide (2) and N -([2,2′-bifuran]-5-ylmethyl)-2-hydrazineyl-2-oxoacetamide (7) were identified as the most druggable inhibitors whereas 1-amino-2,7-diethyl-3,8-dioxo-6-(p -tolyl)-2,3,7,8-tetrahydro-2,7-naphthyridine-4‑carbonitrile (5) and (R)-6-amino-2-(2,3-dihydroxypropyl)-1 H -benzo[ de ]isoquinoline-1,3(2 H)-dione (20) were active against furin. [Display omitted] • Identification of drug molecules for the treatment of COVID-19. • TMPRSS2, furin, cathepsin B, FXR were used as target proteins. • BioSolveIT suite was used for the pharmacophore-based computational drug designing. • Several potent druggable inhibitors were identified. [ABSTRACT FROM AUTHOR]

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.

• Nafamostat blocks SARS-CoV-2 entry by inhibiting TMPRSS2 activity on epithelial cells. • This exploratory multicentre RCT assessed the antiviral effects and safety of nafamostat. • Nafamostat significantly reduced the SARS-CoV-2 viral load in nasopharyngeal samples. • The most common adverse event was phlebitis due to nafamostat. This study aimed to evaluate the antiviral effects and safety of nafamostat in early-onset patients with coronavirus disease 2019 (COVID-19). In this exploratory multicentre randomized controlled trial, patients were assigned to three groups within 5 days of symptom onset, with 10 participants in each group: nafamostat at either 0.2 mg/kg/h or 0.1 mg/kg/h or a standard-of-care group. The primary endpoint was area under the curve for decrease in SARS-CoV-2 viral load in nasopharyngeal samples from baseline to day 6. Of the 30 randomized patients, 19 received nafamostat. Overall, 10 patients received low-dose nafamostat, 9 patients received high-dose nafamostat, and 10 received standard-of-care. The detected viruses were Omicron strains. The regression coefficient for area under the curve for decrease in viral load as the response variable and nafamostat dose per body weight as the explanatory variable showed a significant relationship of −40.1 (95% confidence interval, −74.1 to −6.2; P = 0.022). Serious adverse events were not observed in either group. Phlebitis occurred in ca. 50% of patients treated with nafamostat. Nafamostat exerts virus load-reducing effects in patients with early-onset COVID-19. [ABSTRACT FROM AUTHOR]

• Antiviral treatment did not show efficacy for hypoxemia and DIC in a COVID-19 patient. • Heparin and nafamostat combination therapy showed dramatic efficacy on the illness. • Mechanisms by which nafamostat is effective for COVID-19 have been fully explained. • This would be an instructional lecture on nafamostat therapy for COVID-19 patients. Nafamostat, a serine proteinase inhibitor with various actions including antithrombin, antiplasmin, and antitrypsin effects, has been used in clinical practice to treat disseminated intravascular coagulation (DIC) and pancreatitis. This case report describes the clinical course of a patient with COVID-19 pneumonia whose severe hypoxemia, probably caused by DIC and pulmonary embolism, showed remarkable improvement with combination heparin and nafamostat therapy. In addition, beneficial mechanisms of nafamostat against COVID-19 and the necessity of attention to hyperkalemia as an adverse effect are discussed. [ABSTRACT FROM AUTHOR]

• Nafamostat, known as anticoagulant, has antiviral effect to COVID-19. • Nafamostat, a serine protease inhibitor, has antiinflammatory effect. • Nafamostat is a effective and safe drug to COVID-19 pneumonia. 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. [ABSTRACT FROM AUTHOR]

Background: The use of continuous renal replacement therapy (CRRT) in critically ill children is rapidly increasing, but the standard of care has not yet been established and prognosis remains poor. To develop optimal CRRT strategies, we launched a research project generating the Japanese Pediatric CRRT registry, a multicenter registry of CRRT in Japanese pediatric intensive care units (PICUs), to investigate the actual status of CRRT in recent years in PICUs, where data are lacking., Methods: This manuscript presents a protocol for planning a multicenter prospective registry. As of April 2023, 15 Japanese PICUs are voluntarily participating. Patients enrolled are those <16 years of age who enter the PICUs of the collaborating institutions, require CRRT, and have the guardians' consent. CRRT is defined as anticipated to be required for >24 hours, and CRRT connected to extracorporeal membrane oxygenation is also included. The registry is an online registry system managed by the University Hospital Medical Information Network. The primary outcomes are Pediatric Cerebral Performance Category Scale at PICU discharge and 6 months post-discharge (deaths included), persistent need for dialysis, and PICU readmission within 6 months. The secondary outcomes are adverse events during and immediately after CRRT initiation, and initial circuit life span., Conclusions: This project will examine the differences in outcomes of CRRT in PICUs in specific patient and treatment groups and will be used to design future interventional studies. We will also aim to establish a platform for a multicenter registry study in Japanese PICUs, considering the current lack of such a platform., Competing Interests: The authors declare no conflicts of interest in relation to the work presented in the manuscript., (© 2023 Society for Clinical Epidemiology.)

Aim: To describe the stability of nafamostat in infusion solutions, during blood sample collection and in extracted plasma samples in the autosampler. Methods: Nafamostat infusion solutions were stored at room temperature in the light for 24 h. For sample collection stability, fresh blood spiked with nafamostat was subjected to combinations of anticoagulants, added esterase inhibitor and temperature. Nafamostat was monitored in the extracted plasma samples in the autosampler. Results: Nafamostat was stable in infusion solutions. Nafamostat in whole blood was stable for 3 h before centrifugation when collected in sodium fluoride/potassium oxalate tubes (4°C). Nafamostat in extracted plasma samples degraded at 4.7 ± 0.7% per h. Conclusion: Viable samples can be obtained using blood collection tubes with sodium fluoride, chilling and processing promptly.

We aimed to clarify the effect of nafamostat mesilate (nafamostat) on intestinal mucositis as well as the potentiation of intestinal 5-hydroxytryptamine (5-HT) dynamics induced by methotrexate, an anti-cancer drug, in rats. Rats received intraperitoneal methotrexate at 12.5 mg/kg/day for 4 days. In addition, 1, 3, or 10 mg/kg/day of nafamostat was given subcutaneously for 4 days. Ninety-six hours after the first administration of methotrexate, jejunal tissues were collected for analysis. The results showed that 1 mg/kg, but not 3 or 10 mg/kg, of nafamostat significantly ameliorated the methotrexate-induced body weight loss. Moreover, 1 mg/kg of nafamostat significantly improved methotrexate-induced mucositis, including villus atrophy. Nafamostat (1 mg/kg) significantly inhibited the methotrexate-induced mRNA expression of pro-inflammatory cytokines and cyclooxygenase-2, as well as methotrexate-induced 5-HT content and tryptophan hydroxylase (TPH) activity. In addition, it tended to inhibit the number of anti-TPH antibody-positive cells and significantly inhibited the number of anti-substance P antibody-positive cells. These findings suggest that low-dose nafamostat ameliorates tissue injury and 5-HT and substance P synthesis in methotrexate-induced mucositis. Nafamostat may be a novel therapeutic strategy for the prevention and treatment of mucositis as well as 5-HT- and/or substance P-related adverse effects in cancer chemotherapy., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background/AimsThe protease inhibitors, nafamostat and gabexate, have been used to prevent pancreatitis related to endoscopic retrograde cholangiopancreatography (ERCP). In vitro, nafamostat inhibits the pancreatic protease activities 10-100 times more potently than gabexate. We evaluated the efficacy of nafamostat for prophylaxis against post-ERCP pancreatitis in comparison with gabexate.Methods : Five hundred patients (208 patients in the nafamostat-treated group and 292 in the gabexate-treated group) were analyzed retrospectively after selective exclusion. The incidences of pancreatitis and hyperamylasemia after the ERCP were compared between the nafamostat and gabexate groups.Results : The incidences of acute pancreatitis and hyperamylasemia were 9.1% and 40.9%, respectively, in the nafamostat-treated group, and 8.6% and 39.4% in the gabexate-treated group. The frequencies of post-ERCP pancreatitis and hyperamylasemia did not differ significantly between the two groups, Post-ERCP pancreatitis in two group did not vary according to the different ERCP procedures. The mean serum amylase level at 6 h after ERCP was significantly lower in the nafamostat-treated group than in the gabexate-treated group (p=0.020). However, the difference in serum amylase level did not persist at 18 h and 36 h post-ERCP.Conclusion : sAdministration of nafamostat before ERCP was not inferior to gabexate in protecting against the development of pancreatitis.

Acid-sensing ion channels (ASICs) are blocked by many cationic compounds. Mechanisms of action, which may include pore block, modulation of activation and desensitization, need systematic analysis to allow predictable design of new potent and selective drugs. In this work, we studied the action of the serine protease inhibitors nafamostat, sepimostat, gabexate and camostat, on native ASICs in rat giant striatal interneurons and recombinant ASIC1a and ASIC2a channels, and compared it to that of well-known small molecule ASIC blocker diminazene. All these compounds have positively charged amidine and/or guanidine groups in their structure. Nafamostat, sepimostat and diminazene inhibited pH 6.5-induced currents in rat striatal interneurons at −80 mV holding voltage with IC 50 values of 0.78 ± 0.12 μM, 2.4 ± 0.3 μM and 0.40 ± 0.09 μM, respectively, whereas camostat and gabexate were practically ineffective. The inhibition by nafamostat, sepimostat and diminazene was voltage-dependent evidencing binding in the channel pore. They were not trapped in the closed channels, suggesting "foot-in-the-door" mechanism of action. The inhibitory activity of nafamostat, sepimostat and diminazene was similar in experiments on native ASICs and recombinant ASIC1a channels, while all of them were drastically less active against ASIC2a channels. According to our molecular modeling, three active compounds bind in the channel pore between Glu 433 and Ala 444 in a similar way. In view of the relative safety of nafamostat for clinical use in humans, it can be considered as a potential candidate for the treatment of pathophysiological conditions linked to ASICs disfunction, including inflammatory pain and ischemic stroke. [ABSTRACT FROM AUTHOR]

Introduction: Asthma is characterized by an imbalance between proteases and their inhibitors. Hence, an attractive therapeutic option could be to interfere with asthma-associated proteases. Here we exploited this option by assessing the impact of nafamostat, a serine protease inhibitor known to neutralize mast cell tryptase., Methods: Nafamostat was administered in a mouse model for asthma based on sensitization by house dust mite (HDM) extract, followed by the assessment of effects on airway hyperreactivity, inflammatory parameters and gene expression., Results: We show that nafamostat efficiently suppressed the airway hyperreactivity in HDM-sensitized mice. This was accompanied by reduced infiltration of eosinophils and lymphocytes to the airways, and by lower levels of pro-inflammatory compounds within the airway lumen. Further, nafamostat had a dampening impact on goblet cell hyperplasia and smooth muscle layer thickening in the lungs of HDM-sensitized animals. To obtain deeper insight into the underlying mechanisms, a transcriptomic analysis was conducted. This revealed, as expected, that the HDM sensitization caused an upregulated expression of numerous pro-inflammatory genes. Further, the transcriptomic analysis showed that nafamostat suppressed the levels of multiple pro-inflammatory genes, with a particular impact on genes related to asthma., Discussion: Taken together, this study provides extensive insight into the ameliorating effect of nafamostat on experimental asthma, and our findings can thereby provide a basis for the further evaluation of nafamostat as a potential therapeutic agent in human asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Allam, Waern, Taha, Akula, Wernersson and Pejler.)