Synthetic and natural guanidine derivatives as antitumor and antimicrobial agents: A review.

Guanidines are fascinating small nitrogen-rich organic compounds, which have been frequently associated with a wide range of biological activities. This is mainly due to their interesting chemical features. For these reasons, for the past decades, researchers have been synthesizing and evaluating guanidine derivatives. In fact, there are currently on the market several guanidine-bearing drugs. Given the broad panoply of pharmacological activities displayed by guanidine compounds, in this review, we chose to focus on antitumor, antibacterial, antiviral, antifungal, and antiprotozoal activities presented by several natural and synthetic guanidine derivatives, which are undergoing preclinical and clinical studies from January 2010 to January 2023. Moreover, we also present guanidine-containing drugs currently in the market for the treatment of cancer and several infectious diseases. In the preclinical and clinical setting, most of the synthesized and natural guanidine derivatives are being evaluated as antitumor and antibacterial agents. Even though DNA is the most known target of this type of compounds, their cytotoxicity also involves several other different mechanisms, such as interference with bacterial cell membranes, reactive oxygen species (ROS) formation, mitochondrial-mediated apoptosis, mediated-Rac1 inhibition, among others. As for the compounds already used as pharmacological drugs, their main application is in the treatment of different types of cancer, such as breast, lung, prostate, and leukemia. Guanidine-containing drugs are also being used for the treatment of bacterial, antiprotozoal, antiviral infections and, recently, have been proposed for the treatment of COVID-19. To conclude, the guanidine group is a privileged scaffold in drug design. Its remarkable cytotoxic activities, especially in the field of oncology, still make it suitable for a deeper investigation to afford more efficient and target-specific drugs.

Comparison of disinfectants-induced gene expression profile: Potential adverse effects.

Despite their importance in combating the spread of the COVID-19 pandemic, adverse effects of disinfectants on human health, especially the respiratory system, have been of continuing concern to researchers. Considering that bronchi are the main target of sprayed disinfectants, we here treated the seven major active ingredients in disinfectant products accepted by the US EPA to human bronchial epithelial cells and determined the subtoxic levels. Then, we performed microarray analysis using total RNA obtained at the subtoxic level and designed a network representing disinfectant-induced cellular response using the KEGG pathway analysis technique. Polyhexamethylguanidine phosphate, a lung fibrosis inducer, was used as a reference material to verify the relationship between cell death and pathology. The derived results reveal potential adverse effects along with the need for an effective application strategy for each chemical.

Comprehensive analysis of disinfectants on the horizontal transfer of antibiotic resistance genes.

The propagation of antimicrobial resistance (AMR) is constantly paralyzing our healthcare systems. In addition to the pressure of antibiotic selection, the roles of non-antibiotic compounds in disseminating antibiotic resistance genes (ARGs) are a matter of great concerns. This study aimed to explore the impact of different disinfectants on the horizontal transfer of ARGs and their underlying mechanisms. First, the effects of different kinds of disinfectants on the conjugative transfer of RP4-7 plasmid were evaluated. Results showed that quaternary ammonium salt, organic halogen, alcohol and guanidine disinfectants significantly facilitated the conjugative transfer. Conversely, heavy-metals, peroxides and phenols otherwise displayed an inhibitory effect. Furthermore, we deciphered the mechanism by which guanidine disinfectants promoted conjugation, which includes increased cell membrane permeability, over-production of ROS, enhanced SOS response, and altered expression of conjugative transfer-related genes. More critically, we also revealed that guanidine disinfectants promoted bacterial energy metabolism by enhancing the activity of electron transport chain (ETC) and proton force motive (PMF), thus promoting ATP synthesis and flagellum motility. Overall, our findings reveal the promotive effects of disinfectants on the transmission of ARGs and highlight the potential risks caused by the massive use of guanidine disinfectants, especially during the COVID-19 pandemic.

A Double-Blind, Randomized, Placebo-Controlled, Phase II Clinical Study To Evaluate the Efficacy and Safety of Camostat Mesylate (DWJ1248) in Adult Patients with Mild to Moderate COVID-19.

Although several antiviral agents have become available for coronavirus disease 2019 (COVID-19) treatment, oral drugs are still limited. Camostat mesylate, an orally bioavailable serine protease inhibitor, has been used to treat chronic pancreatitis in South Korea, and it has an in vitro inhibitory potential against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study was a double-blind, randomized, placebo-controlled, multicenter, phase 2 clinical trial in mild to moderate COVID-19 patients. We randomly assigned patients to receive either camostat mesylate (DWJ1248) or placebo orally for 14 days. The primary endpoint was time to clinical improvement of subject symptoms within 14 days, measured using a subjective 4-point Likert scale. Three hundred forty-two patients were randomized. The primary endpoint was nonsignificant, where the median times to clinical improvement were 7 and 8 days in the camostat mesylate group and the placebo group, respectively (hazard ratio [HR] = 1.09; 95% confidence interval [CI], 0.84 to 1.43; P = 0.50). A post hoc analysis showed that the difference was greatest at day 7, without reaching significance. In the high-risk group, the proportions of patients with clinical improvement up to 7 days were 45.8% (50/109) in the camostat group and 38.4% (40/104) in the placebo group (odds ratio [OR] = 1.33; 95% CI, 0.77 to 2.31; P = 0.31); the ordinal scale score at day 7 improved in 20.0% (18/90) of the camostat group and 13.3% (12/90) of the placebo group (OR = 1.68; 95% CI, 0.75 to 3.78; P = 0.21). Adverse events were similar in the two groups. Camostat mesylate was safe in the treatment of COVID-19. Although this study did not show clinical benefit in patients with mild to moderate COVID-19, further clinical studies for high-risk patients are needed. (This trial was registered with ClinicalTrials.gov under registration no. NCT04521296).

Efficacy and safety of nafamostat mesilate anticoagulation in blood purification treatment of critically ill patients: a systematic review and meta-analysis.

BACKGROUND: Nafamostat mesilate (NM), a broad-spectrum and potent serine protease inhibitor, can be used as an anticoagulant during extracorporeal circulation, as well as a promising drug effective against coronavirus disease 2019 (COVID-19). We conducted a systematic meta-analysis to evaluate the safety and efficacy of NM administration in critically ill patients who underwent blood purification therapy (BPT). METHODS: The Cochrane Library, Web of Science and PubMed were comprehensively searched from inception to August 20, 2021, for potential studies. RESULTS: Four randomized controlled trials (RCTs) and seven observational studies with 2723 patients met the inclusion criteria. The meta-analysis demonstrated that conventional therapy (CT) significantly increased hospital mortality compared with NM administration (RR = 1.25, p = 0.0007). In subgroup analyses, the in-hospital mortality of the NM group was significantly lower than that of the anticoagulant-free (NA) group (RR = 1.31, p = 0.002). The CT interventions markedly elevated the risk ratio of bleeding complications by 45% (RR = 1.45, p = 0.010) compared with NM interventions. In another subgroup analysis, NM used exhibited a significantly lower risk of bleeding complications than those of the low-molecular-weight heparin (LMWH) used (RR = 4.58, p = 0.020). The filter lifespan was decreased significantly (MD = -10.59, p < 0.0001) in the NA groups compared with the NM groups. Due to the poor quality of the included RCTs, these results should be interpreted with caution. CONCLUSION: Given the better survival outcomes, lower risk of bleeding, NM anticoagulation seems to be a safe and efficient approach for BPT patients and could yield a favorable filter lifespan. More multi-center RCTs with large samples are required for further validation of this study.

Nafamostat Mesylate for Treatment of COVID-19 in Hospitalised Patients: A Structured, Narrative Review.

The search for clinically effective antivirals against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is ongoing. Repurposing of drugs licensed for non-coronavirus disease 2019 (COVID-19) indications has been extensively investigated in laboratory models and in clinical studies with mixed results. Nafamostat mesylate (nafamostat) is a drug licensed in Japan and Korea for indications including acute pancreatitis and disseminated intravascular coagulation. It is available only for continuous intravenous infusion. In vitro human lung cell line studies with nafamostat demonstrate high antiviral potency against SARS-CoV-2 (half maximal inhibitory concentration [IC50] of 0.0022 µM [compared to remdesivir 1.3 µM]), ostensibly via inhibition of the cellular enzyme transmembrane protease serine 2 (TMPRSS2) preventing viral entry into human cells. In addition, the established antithrombotic activity is hypothesised to be advantageous given thrombosis-associated sequelae of COVID-19. Clinical reports to date are limited, but indicate a potential benefit of nafamostat in patients with moderate to severe COVID-19. In this review, we will explore the pre-clinical, pharmacokinetic and clinical outcome data presently available for nafamostat as a treatment for COVID-19. The recruitment to ongoing clinical trials is a priority to provide more robust data on the safety and efficacy of nafamostat as a treatment for COVID-19.

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.

Nafamostat Mesylate Monotherapy in Patients with Moderate COVID-19: a Single-Center, Retrospective Study.

Coronavirus disease (COVID-19) has spread dramatically worldwide. Nafamostat mesylate inhibits intracellular entry of the novel severe acute respiratory syndrome coronavirus 2 and is believed to have therapeutic potential for treating patients with COVID-19. In this study, patients with moderate COVID-19 who were admitted to our hospital were retrospectively analyzed. Thirty-one patients received monotherapy with nafamostat mesylate, and 33 patients were treated conservatively. Nafamostat mesylate was administered with continuous intravenous infusion for an average of 4.5 days. Compared with the conservative treatment, nafamostat mesylate did not improve outcomes or laboratory data 5 days after admission. In addition, no significant differences in laboratory data 5 days after admission and outcomes in high-risk patients were observed. The incidence of hyperkalemia was significantly higher in the nafamostat mesylate group; however, none of the patients required additional treatment. In conclusion, monotherapy with nafamostat mesylate did not improve clinical outcomes in patients with moderate COVID-19. This study did not examine the therapeutic potential of combining nafamostat mesylate with other antiviral agents, and further investigation is required. Because of the high incidence of hyperkalemia, regular laboratory monitoring is required during the use of nafamostat mesylate.

SARS-CoV-2 Omicron BA.5: Evolving tropism and evasion of potent humoral responses and resistance to clinical immunotherapeutics relative to viral variants of concern.

BACKGROUND: Genetically distinct viral variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been recorded since January 2020. The introduction of global vaccine programs has contributed to lower COVID-19 hospitalisation and mortality rates, particularly in developed countries. In late 2021, Omicron BA.1 emerged, with substantially altered genetic differences and clinical effects from other variants of concern. Shortly after dominating global spread in early 2022, BA.1 was supplanted by the genetically distinct Omicron lineage BA.2. A sub-lineage of BA.2, designated BA.5, presently has an outgrowth advantage over BA.2 and other BA.2 sub-lineages. Here we study the neutralisation of Omicron BA.1, BA.2 and BA.5 and pre-Omicron variants using a range of vaccine and convalescent sera and therapeutic monoclonal antibodies using a live virus neutralisation assay. Using primary nasopharyngeal swabs, we also tested the relative fitness of BA.5 compared to pre-Omicron and Omicron viral lineages in their ability to use the ACE2-TMPRSS2 pathway. METHODS: Using low passage clinical isolates of Clade A.2.2, Beta, Delta, BA.1, BA.2 and BA.5, we determined humoral neutralisation in vitro in vaccinated and convalescent cohorts, using concentrated human IgG pooled from thousands of plasma donors, and licensed monoclonal antibody therapies. We then determined infectivity to particle ratios in primary nasopharyngeal samples and expanded low passage isolates in a genetically engineered ACE2/TMPRSS2 cell line in the presence and absence of the TMPRSS2 inhibitor Nafamostat. FINDINGS: Peak responses to 3 doses of BNT162b2 vaccine were associated with a 9-fold reduction in neutralisation for Omicron lineages BA.1, BA.2 and BA.5. Concentrated pooled human IgG from convalescent and vaccinated donors and BNT162b2 vaccination with BA.1 breakthrough infections were associated with greater breadth of neutralisation, although the potency was still reduced 7-fold across all Omicron lineages. Testing of clinical grade antibodies revealed a 14.3-fold reduction using Evusheld and 16.8-fold reduction using Sotrovimab for the BA.5. Whilst the infectivity of BA.1 and BA.2 was attenuated in ACE2/TMPRSS2 entry, BA.5 was observed to be equivalent to that of an early 2020 circulating clade and had greater sensitivity to the TMPRSS2 inhibitor Nafamostat. INTERPRETATION: Observations support all Omicron variants to significantly escape neutralising antibodies across a range of vaccination and/or convalescent responses. Potency of therapeutic monoclonal antibodies is also reduced and differs across Omicron lineages. The key difference of BA.5 from other Omicron sub-variants is the reversion in tropism back to using the well-known ACE2-TMPRSS2 pathway, utilised efficiently by pre-Omicron lineages. Monitoring if these changes influence transmission and/or disease severity will be key for ongoing tracking and management of Omicron waves globally. FUNDING: This work was primarily supported by Australian Medical Foundation research grants MRF2005760 (ST, GM & WDR), MRF2001684 (ADK and ST) and Medical Research Future Fund Antiviral Development Call grant (WDR), Medical Research Future Fund COVID-19 grant (MRFF2001684, ADK & SGT) and the New South Wales Health COVID-19 Research Grants Round 2 (SGT).

Nafamostat-Mediated Inhibition of SARS-CoV-2 Ribosomal Frameshifting Is Insufficient to Impair Viral Replication in Vero Cells. Comment on Munshi et al. Identifying Inhibitors of -1 Programmed Ribosomal Frameshifting in a Broad Spectrum of Coronaviruses. Viruses 2022, 14, 177.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, which has been reported to have caused 18 [...].

Efficacy and safety of camostat mesylate in early COVID-19 disease in an ambulatory setting: a randomized placebo-controlled phase II trial.

OBJECTIVES: This study aimed to assess the efficacy and safety of 300 mg camostat mesylate three times daily in a fasted state to treat early phase COVID-19 in an ambulatory setting. METHODS: We conducted a phase II randomized controlled trial in symptomatic (maximum 5 days) and asymptomatic patients with confirmed COVID-19 infection. Patients were randomly assigned in a 2:1 ratio to receive either camostat mesylate or a placebo. Outcomes included change in nasopharyngeal viral load, time to clinical improvement, the presence of neutralizing antibodies, and safety. RESULTS: Of 96 participants randomized between November 2020 and June 2021, analyses were performed on the data of 90 participants who completed treatment (N = 61 camostat mesylate, N = 29 placebo). The estimated mean change in cycle threshold between day 1 and day 5 between the camostat and placebo group was 1.183 (P = 0.511). The unadjusted hazard ratio for clinical improvement in the camostat group was 0.965 (95% confidence interval, 0.480-1.942, P = 0.921 by Cox regression). The percentage distribution of the 50% neutralizing antibody titer at day 28 visit and frequency of adverse events were similar between the two groups. CONCLUSION: Under this protocol, camostat mesylate was not found to be effective as an antiviral drug against SARS-CoV-2. TRIAL REGISTRATION: ClinicalTrials.gov NCT04625114; November 12, 2020.

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).

Peramivir, an Anti-Influenza Virus Drug, Exhibits Potential Anti-Cytokine Storm Effects.

Coronavirus Disease 2019 (COVID-19) infected by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has been declared a public health emergency of international concerns. Cytokine storm syndrome (CSS) is a critical clinical symptom of severe COVID-19 patients, and the macrophage is recognized as the direct host cell of SARS-CoV-2 and potential drivers of CSS. In the present study, peramivir was identified to reduce TNF-α by partly intervention of NF-κB activity in LPS-induced macrophage model. In vivo, peramivir reduced the multi-cytokines in serum and bronchoalveolar lavage fluid (BALF), alleviated the acute lung injury and prolonged the survival time in mice. In human peripheral blood mononuclear cells (hPBMCs), peramivir could also inhibit the release of TNF-α. Collectively, we proposed that peramivir might be a candidate for the treatment of COVID-19 and other infections related CSS.

Protease inhibitor Camostat Mesyalte blocks wild type SARS-CoV-2 and D614G viral entry in human engineered miniature lungs.

The catastrophic global effects of the SARS-CoV-2 pandemic highlight the need to develop novel therapeutics strategies to prevent and treat viral infections of the respiratory tract. To enable this work, we need scalable, affordable, and physiologically relevant models of the human lung, the primary organ involved in the pathogenesis of COVID-19. To date, most COVID-19 in vitro models rely on platforms such as cell lines and organoids. While 2D and 3D models have provided important insights, human distal lung models that can model epithelial viral uptake have yet to be established. We hypothesized that by leveraging techniques of whole organ engineering and directed differentiation of induced pluripotent stem cells (iPSC) we could model human distal lung epithelium, examine viral infection at the tissue level in real time, and establish a platform for COVID-19 related research ex vivo. In the present study, we used type 2 alveolar epithelial cells (AT2) derived from human iPSCs to repopulate whole rat lung acellular scaffolds and maintained them in extended biomimetic organ culture for 30 days to induce the maturation of distal lung epithelium. We observed emergence of a mixed type 1 and type 2 alveolar epithelial phenotype during tissue formation. When exposing our system to a pseudotyped lentivirus containing the spike of wildtype SARS-CoV-2 and the more virulent D614G, we observed progression of the infection in real time. We then found that the protease inhibitor Camostat Mesyalte significantly reduced viral transfection in distal lung epithelium. In summary, our data show that a mature human distal lung epithelium can serve as a novel moderate throughput research platform to examine viral infection and to evaluate novel therapeutics ex vivo.

Semi-Mechanistic Pharmacokinetic-Pharmacodynamic Model of Camostat Mesylate-Predicted Efficacy against SARS-CoV-2 in COVID-19.

The SARS-CoV-2 coronavirus, which causes COVID-19, uses a viral surface spike protein for host cell entry and the human cell-surface transmembrane serine protease, TMPRSS2, to process the spike protein. Camostat mesylate, an orally available and clinically used serine protease inhibitor, inhibits TMPRSS2, supporting clinical trials to investigate its use in COVID-19. A one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) model for camostat and the active metabolite FOY-251 was developed, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. The model predicts that 95% inhibition of TMPRSS2 is required for 50% inhibition of viral entry efficiency. For camostat 200 mg dosed four times daily, 90% inhibition of TMPRSS2 is predicted to occur but with only about 40% viral entry inhibition. For 3-fold higher camostat dosing, marginal improvement of viral entry rate inhibition, up to 54%, is predicted. Because respiratory tract viral load may be associated with negative outcome, even modestly reducing viral entry and respiratory tract viral load may reduce disease progression. This modeling also supports medicinal chemistry approaches to enhancing PK/PD and potency of the camostat molecule. IMPORTANCE Strategies to repurpose already-approved drugs for the treatment of COVID-19 has been attractive since the beginning of the pandemic. Camostat mesylate, a serine protease inhibitor approved in Japan for the treatment of acute exacerbations of chronic pancreatitis, inhibits TMPRSS1, a host cell surface serine protease essential for SARS-CoV-2 viral entry. In vitro experiments provided data suggesting that camostat might be effective in the treatment of COVID-19. Multiple clinical trials were planned to test the hypothesis that camostat would be beneficial for treating COVID-19 (for example, clinicaltrials.gov, NCT04353284). The present work used a one-compartment pharmacokinetic (PK)/pharmacodynamic (PD) mathematical model for camostat and the active metabolite FOY-251, incorporating TMPRSS2 reversible covalent inhibition by FOY-251, and empirical equations linking TMPRSS2 inhibition of SARS-CoV-2 cell entry. This work is valuable to guide further development of camostat mesylate and possible medicinal chemistry derivatives for the treatment of COVID-19.

Incidence and risk factors for hyperkalaemia in patients treated for COVID-19 with nafamostat mesylate.

WHAT IS KNOWN AND OBJECTIVE: Nafamostat mesylate (NM) is used clinically in combination with antiviral drugs to treat coronavirus disease (COVID-19). One of the adverse events of NM is hyperkalaemia due to inhibition of the amiloride-sensitive sodium channels (ENaC). The incidence and risk factors for hyperkalaemia due to NM have been studied in patients with pancreatitis but not in COVID-19. COVID-19 can be associated with hypokalaemia or hyperkalaemia, and SARS-CoV-2 is thought to inhibit ENaC. Therefore, frequency and risk factors for hyperkalaemia due to NM may differ between COVID-19 and pancreatitis. Hyperkalaemia may worsen the respiratory condition of patients. The objective of this study was to determine the incidence and risk factors for hyperkalaemia in COVID-19 patients treated with favipiravir, dexamethasone and NM. METHODS: This retrospective study reviewed the records of hospitalized COVID-19 patients treated with favipiravir and dexamethasone, with or without NM, between March 2020 and January 2021. Multivariable logistic regression analysis was performed to identify the risk factors for hyperkalaemia. RESULTS AND DISCUSSION: Of 45 patients who received favipiravir and dexamethasone with NM for the treatment of COVID-19, 21 (47%) experienced hyperkalaemia. The duration of NM administration was a significant predictor of hyperkalaemia (odds ratio: 1.55, 95% confidence interval: 1.04-2.31, p = 0.031). The receiver-operating characteristic curve analysis determined that the cut-off value for predicting the number of days until the onset of hyperkalaemia was 6 days and the area under the curve was 0.707. WHAT IS NEW AND CONCLUSION: This study revealed that the incidence of hyperkalaemia is high in patients treated for COVID-19 with NM, and that the duration of NM administration is a key risk factor. When NM is administered for the treatment of COVID-19, it should be discontinued within 6 days to minimize the risk of hyperkalaemia.

Pharmacokinetics of Nafamostat, a Potent Serine Protease Inhibitor, by a Novel LC-MS/MS Analysis.

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.

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.

In Silico Analysis and Synthesis of Nafamostat Derivatives and Evaluation of Their Anti-SARS-CoV-2 Activity.

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.