A Novel Cleavage Pattern of Complement C5 Induced by Chlamydia trachomatis Infection via the Chlamydial Protease CPAF

Urogenital Chlamydia trachomatis infection is one of the most common bacterial sexually transmitted diseases globally. Untreated C. trachomatis infections can ascend to the upper genital tract and establish a series of severe complications. Previous studies using C3−/− and C5−/− mice models demonstrated that C3-independent activation of C5 occurred during C. trachomatis infection. However, the mechanism of how chlamydial infection activates C5 in the absence of C3 has yet to be elucidated. To delineate interactions between C5 and chlamydial infection, cleavage products in a co-incubation system containing purified human C5 and C. trachomatis-HeLa229 cell lysates were analyzed, and a novel cleavage pattern of C5 activation induced by C. trachomatis infection was identified. C5 was cleaved efficiently at the previously unidentified site K970, but was cleaved poorly at site R751. C5b was modified to C5bCt, which later formed C5bCt-9, which had enhanced lytic ability compared with C5b-9. The chlamydial serine protease CPAF contributed to C3-independent C5 activation during C. trachomatis infection. Nafamostat mesylate, a serine protease inhibitor with a good safety profile, had a strong inhibitory effect on C5 activation induced by chlamydial infection. These discoveries reveal the mechanism of C3-independent C5 activation induced by chlamydial infection, and furthermore provide a potential therapeutic target and drug for preventing tubal fibrosis caused by chlamydial infection.

Anaphylactic Reactions Caused by Nafamostat Mesylate during Hemodialysis before Surgery for Carpal Tunnel Syndrome

Nafamostat mesylate (NM) has been used to treat pancreatitis and disseminated intravascular coagulation during hemodialysis (HD). However, there have been some reports of adverse effects related to anaphylactic reactions. We present a case in which anaphylactic reactions caused by NM during preoperative HD caused repeated postponement of surgery for carpal tunnel syndrome. Symptoms including fever, shivering, chills, low blood pressure, tachycardia, nausea, and vomiting appeared during preoperative HD, and surgery was postponed thrice. Initially, the patient was misdiagnosed with sepsis because of elevated C-reactive protein and procalcitonin levels. However, since the symptoms appeared only when NM was administered and disappeared quickly after the administration of NM was terminated, the condition was diagnosed as anaphylactic reactions caused by NM. Therefore, it is essential to consider anaphylactic reactions caused by NM as differential diagnoses, when symptoms, such as fever, are observed during perioperative HD.

Association between Nafamostat Mesylate and In-Hospital Mortality in Patients with Coronavirus Disease 2019: A Multicenter Observational Study

Nafamostat mesylate may be effective against coronavirus disease 2019 (COVID-19). However, it is not known whether its use is associated with reduced in-hospital mortality in clinical practice. We conducted a retrospective observational study to evaluate the effect of nafamostat mesylate in patients with COVID-19 using the Medical Data Vision Co. Ltd. hospital-based database in Japan. We compared patients with COVID-19 who were (n = 121) and were not (n = 15,738) administered nafamostat mesylate within 2 days of admission between January and December 2020. We conducted a 1:4 propensity score matching with multiple imputations for smoking status and body mass index and combined the 20 imputed propensity score-matched datasets to obtain the adjusted odds ratio for in-hospital mortality. Crude in-hospital mortality was 13.2% (16/121) and 5.0% (790/15,738), respectively. In the propensity score-matched analysis with multiple imputations, the adjusted odds ratio (use vs. no use of nafamostat mesylate) for in-hospital mortality was 1.27 (95% confidence interval: 0.61–2.64; p = 0.52). Sensitivity analyses showed similar results. The results of this retrospective observational study did not support an association between nafamostat mesylate and improved in-hospital outcomes in patients with COVID-19, although further studies with larger sample sizes are warranted to assess the generalizability of our findings.

Measuring the Concentration of Serum Syndecan-1 to Assess Vascular Endothelial Glycocalyx Injury During Hemodialysis

Glycocalyx is present on the surface of healthy endothelium, and the concentration of serum syndecan-1 can serve as an injury marker. This study aimed to assess endothelial injury using serum syndecan-1 as a marker of endothelial glycocalyx injury in patients who underwent hemodialysis. In this single-center, retrospective, observational study, 145 patients who underwent hemodialysis at the Gifu University Hospital between March 2017 and December 2019 were enrolled. The median dialysis period and time were 63 months and 3.7 h, respectively. The serum syndecan-1 concentration significantly increased from 124.6 ± 107.8 ng/ml before hemodialysis to 229.0 ± 138.1 ng/ml after hemodialysis (P < 0.001). Treatment with anticoagulant nafamostat mesylate inhibited hemodialysis-induced increase in the levels of serum syndecan-1 in comparison to unfractionated heparin. Dialysis time and the change in the syndecan-1 concentration were positively correlated. Conversely, the amount of body fluid removed and the changes in the syndecan-1 concentration were not significantly correlated. The reduction in the amount of body fluid removed and dialysis time inhibited the change in the syndecan-1 levels before and after hemodialysis. In conclusion, quantitative assessment of the endothelial glycocalyx injury during hemodialysis can be performed by measuring the serum syndecan-1 concentration, which may aid in the selection of appropriate anticoagulants, reduction of hemodialysis time, and the amount of body fluid removed.

Delayed Administration of Nafamostat Mesylate Inhibits Thrombin-Mediated Blood-Spinal Cord Barrier Breakdown During Acute Spinal Cord Injury in Rats

Background: Nafamostat mesylate (NM), an FDA-approved serine protease inhibitor, exerts anti-neuroinflammation and neuroprotective effect on rat spinal cord injury (SCI). However, the time window for NM administration after SCI as well as its underlying mechanism remains unclear. Methods: A series of different first administration time points of NM was tested on rat contusive SCI model. The optimal time window of NM was screened by evaluating hindlimb locomotion and electrophysiology. We performed western blot and immunofluorescence to evaluate the drug target thrombin as well as its downstream Protease activated receptor 1 (PAR-1), and matrix metalloproteinase-9 (MMP9). Enzyme activity assay was used to test thrombin activity. The permeability of blood-spinal cord barrier (BSCB) was assessed by Evans Blue leakage. The infiltration of peripheral inflammatory cell was observed by immunofluorescence.Results: The optimal administration time window of NM was 2-12 h. The thrombin specific inhibitor, Argatroban, had similar pattern. The temporal expression pattern of thrombin peaked at 12 hours and returned to normal level at 7 days post SCI. PAR-1, the thrombin receptor, was observed a significant upregulation after SCI. MMP9, downstream of PAR-1, was also increased along with thrombin and PAR1. The most significant increase of thrombin expression was detected in vascular endothelial cells (ECs). NM significantly downregulated the thrombin and MMP9 expression as well as thrombin activity in the spinal cord, especially in ECs. NM administration at 2-12 h after SCI could inhibit the leakage of Evans blue in the epicenter and upregulate tight junction proteins (TJPs) expression. 8 h administration of NM effectively inhibited the infiltration of peripheral macrophages in the acute SCI. Conclusions: Our study provided preclinical data of NM administration time window in SCI model, which is clinically relevant in the acute SCI. We elucidated the protective mechanism of NM through BSCB protection and anti-neuroinflammation via thrombin intervention.

Multifocal Epithelioid Hemangioendothelioma Complicated with Disseminated Intravascular Coagulation

Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular neoplasm that requires long-term management. Several reports describe disseminated intravascular coagulation (DIC) associated with angiosarcoma, but no association with EHE has been reported. We encountered a patient with DIC complicated by multifocal EHE. The patient was an 83-year-old woman with spinal lesions, small lung nodules, and a soft tissue mass in the right buttock. The tumor was biopsied and diagnosed as EHE. The patient received pain control therapy without antitumor therapy. One month later, DIC developed with tumor progression. DIC subsided with nafamostat mesylate infusion, and oral apixaban was administered. DIC was managed for 5 months until the patient died of brain metastases. This is the first report of a patient with DIC complicated by EHE. It should be noted that progression of EHE can cause DIC. We were able to manage DIC using anticoagulant agents.

Randomised Controlled Trial of Intravenous Nafamostat Mesylate in COVID pneumonitis: Phase 1b/2a Experimental Study to Investigate Safety, Pharmacokinetics and Pharmacodynamics

ABSTRACTDespite the success of vaccines and selected repurposed treatments, COVID-19 is likely to remain a global health problem and further chemotherapeutics are required. Many repurposed drugs have progressed rapidly to Phase 2 and 3 trials without characterisation of Pharmacokinetics (PK)/Pharmacodynamics (PD) including safety in COVID-19. One such drug is Nafamostat Mesylate (Nafamostat), a synthetic serine protease inhibitor with anticoagulant and anti-inflammatory properties. Preclinical data has demonstrated that it is has potent antiviral activity against SARS-CoV-2 by directly inhibiting the transmembrane protease serine 2 (TMPRSS2) dependent stage of host cell entry.MethodsWe present the findings of a phase Ib/II open label, platform randomised controlled trial (RCT), exploring the safety 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. Secondary endpoints included clinical endpoints such as number of oxygen free days and clinical improvement/ deterioration, PK/PD, thromboelastometry, D Dimers, cytokines, immune cell flow cytometry and viral load.ResultsData is reported from 42 patients, 21 of which were randomly assigned to receive intravenous Nafamostat. The Nafamostat group developed significantly higher plasma creatinine levels, more adverse events and a lower number of oxygen free days. There were no other statistically significant differences in the primary or secondary endpoints between Nafamostat and SoC. PK data demonstrated that intravenous Nafamostat was rapidly broken down to inactive metabolites. We observed an antifibrinolytic profile, and no significant anticoagulant effects in thromboelastometry. Participants in the Nafamostat group had higher D Dimers compared to SoC. There were no differences in cytokine profile and immune cell phenotype and viral loads between the groups.ConclusionIn hospitalised patients with COVID-19, we did not observe evidence of anti-inflammatory, anticoagulant or antiviral activity with intravenous Nafamostat. Given the number of negative trials with repurposed drugs, our experimental medicine trial highlights the value of PK/PD studies prior to selecting drugs for efficacy trials. Given the mechanism of action, further evaluation of Nafamostat delivered via a different route may be warranted. This trial demonstrates the importance of experimental trials in new disease entities such as COVID-19 prior to selecting drugs for larger trials.

Co-Spray Dried Nafamostat Mesylate with Lecithin and Mannitol as Respirable Microparticles for Targeted Pulmonary Delivery: Pharmacokinetics and Lung Distribution in Rats

Coronavirus disease 2019 (COVID-19), caused by a new strain of coronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is spreading rapidly worldwide. Nafamostat mesylate (NFM) suppresses transmembrane serine protease 2 and SARS-CoV-2 S protein-mediated fusion. In this study, pharmacokinetics and lung distribution of NFM, administered via intravenous and intratracheal routes, were determined using high performance liquid chromatography analysis of blood plasma, lung lumen using bronchoalveolar lavage fluid, and lung tissue. Intratracheal administration had higher drug delivery and longer residual time in the lung lumen and tissue, which are the main sites of action, than intravenous administration. We confirmed the effect of lecithin as a stabilizer through an ex vivo stability test. Lecithin acts as an inhibitor of carboxylesterase and delays NFM decomposition. We prepared inhalable microparticles with NFM, lecithin, and mannitol via the co-spray method. The formulation prepared using an NFM:lecithin:mannitol ratio of 1:1:100 had a small particle size and excellent aerodynamic performance. Spray dried microparticles containing NFM, lecithin, and mannitol (1:1:100) had the longest residual time in the lung tissue. In conclusion, NFM-inhalable microparticles were prepared and confirmed to be delivered into the respiratory tract, such as lung lumen and lung tissue, through in vitro and in vivo evaluations.