Identification of impurities in nafamostat mesylate using HPLC-IT-TOF/MS: A series of double-charged ions / 药物分析学报

Nafamostat mesylate is a serine protease inhibitor used in the treatment of acute pancreatitis. The im-purities in nafamostat mesylate, the active pharmaceutical ingredient (API), were profiled via high performance liquid chromatography tandem ion trap coupled with time-of-flight mass spectrometer (HPLC-IT-TOF/MS). The chromatography was performed on an ACE-3 C18 column (200 mm × 4.6 mm, 3μm) using methanol and 0.1％ formic acid in purified water as mobile phase at a flow rate of 1.0 mL/min. The ions were detected by IT-TOF/MS with a full-scan mass analysis from m/z 100 to 800. In total, eleven impurities were detected in nafamostat mesylate API. The impurity profile was estimated based on the HPLC-IT-TOF/MS data, including accurate masses, MSn fingerprints of fragmentation pathways and a series of double-charged ions. Finally, seven impurities were identified and reported for the first time. The results will provide technical support for the quality control and clinical safety of nafamostat mesylate.

Validation of Nafamostat Mesilate as an Anticoagulant in Extracorporeal Membrane Oxygenation: A Large-Animal Experiment

BACKGROUND: Unfractionated heparin is commonly used for anticoagulation in extracorporeal membrane oxygenation (ECMO). Several studies have shown that nafamostat mesilate (NM) has comparable clinical outcomes to unfractionated heparin. This study compared anticoagulation with NM and heparin in a large-animal model. METHODS: Beagle dogs (n=8; weight, 6.5–9 kg) were placed on venovenous ECMO. Blood samples were taken every hour and the following parameters were compared: hemoglobin level, activated partial thromboplastin time (aPTT), thromboelastography (TEG) data, platelet function, and inflammatory cytokine levels. RESULTS: In both groups, the aPTT was longer than the baseline value. Although the aPTT in the NM group was shorter than in the heparin group, the TEG parameters were similar between the 2 groups. Hemoglobin levels decreased in both groups, but the decrease was less with NM than with heparin (p=0.049). Interleukin (IL)-1β levels significantly decreased in the NM group (p=0.01), but there was no difference in the levels of tumor necrosis factor alpha or IL-10 between the 2 groups. CONCLUSION: NM showed a similar anticoagulant effect to that of unfractionated heparin, with fewer bleeding complications. NM also had anti-inflammatory properties during ECMO. Based on this preclinical study, NM may be a good alternative candidate for anticoagulation in ECMO.

A Surgery Case of Heparin-Induced Thrombocytopenia as a Complication of Ventricular Septal Perforation after Acute Myocardial Infarction / 日本心臓血管外科学会雑誌

<p>A 66-year-old man with an unknown medical history developed chest pain and a diagnosis of acute myocardial infarction (AMI) was given by his physician. Percutaneous coronary intervention was performed in the left anterior descending artery. Echocardiography revealed ventricular septal perforation (VSP) ; therefore, the patient was transferred to our hospital. After admission, his platelet count dropped rapidly during heparin administration, and left ventricular thrombosis and deep vein thrombosis were noted, raising a suspicion of heparin-induced thrombocytopenia (HIT). To establish cardiopulmonary bypass, argatroban alone was insufficient to prolong the Powered by Editorial Manager^® and ProduXion Manager^® from the Aries Systems Corporation activated clotting time (ACT) ; thus, nafamostat mesilate was also used for coronary artery bypass grafting and surgical repair of VSP. It took many hours to normalize the ACT, requiring re-exploration for excessive bleeding. On the 37th postoperative day, the patient was transferred to another hospital. We performed cardiac surgical procedures using argatroban in a patient who developed HIT during the course of VSP following AMI ; however, we had difficulty in controlling the ACT. Since, to the best of our knowledge, there are no previous studies reporting surgical case of VSP complicated by HIT, we present this case with a review of the relevant literature.</p>

Nafamostat mesilate promotes endothelium-dependent vasorelaxation via the Akt-eNOS dependent pathway

Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has anticoagulant and anti-inflammatory properties. The intracellular mediator and external anti-inflammatory external signal in the vascular wall have been reported to protect endothelial cells, in part due to nitric oxide (NO) production. This study was designed to examine whether NM exhibit endothelium dependent vascular relaxation through Akt/endothelial nitric oxide synthase (eNOS) activation and generation of NO. NM enhanced Akt/eNOS phosphorylation and NO production in a dose- and time-dependent manner in human umbilical vein endothelial cells (HUVECs) and aorta tissues obtained from rats treated with various concentrations of NM. NM concomitantly decreased arginase activity, which could increase the available arginine substrate for NO production. Moreover, we investigated whether NM increased NO bioavailability and decreased aortic relaxation response to an eNOS inhibitor in the aorta. These results suggest that NM increases NO generation via the Akt/eNOS signaling pathway, leading to endothelium-dependent vascular relaxation. Therefore, the vasorelaxing action of NM may contribute to the regulation of cardiovascular function.

Determination of the Genotoxicity Impurity in Mesylate Nafamostat Raw Materia by GC-MS / 中国药房

OBJECTIVE:To establish a method for determination the genotoxicity impurities (methyl methanesulfonate,ethyl methanesulfonate and isopropyl methanesulfonate) in mesylate nafamostat raw materia. METHODS:GC-MS was conducted,and the genotoxicity impurities were extracted by dichloromethane. The column was DB-5 capillary column by programmed tempera-ture,the inlet temperature was 240 ℃,column flow was 3.0 ml/min,purge flow was 6.0 ml/min,sample mode splitless injection, carrier gas was high purity helium,detector is a mass spectrometer detector,ion source temperature was 230 ℃,the interface tem-perature was 230 ℃,the delay time of solvent was 2.5 min,ionization mode was electron impact,detector voltage was respect to the tuning results,scanning(detection)method was selective ion monitoring,electron energy was 70 eV,and the injection volume was 1.0μl. RESULTS:The separation degree of 3 impurities were greater than 2.0;the linear range of 3 impurities were 0.10-20μg/ml (r≥0.999 5);RSDs of precision,stability and reproducibility tests were lower than 2%;recoveries were 97.7%-104.8%(RSD=2.8%, n=9),102.5%-110.7%(RSD=2.6%,n=9)and 103.0%-107.6%(RSD=1.6%,n=9). CONCLUSIONS:The method is simple, accurate,sensitive and rapid,and can be used for the genotoxicity impurities in mesylate nafamostat raw materia.

Cardiac arrest caused by nafamostat mesilate

A 65-year-old man was transferred from the Department of Vascular Surgery to Nephrology because of cardiac arrest during hemodialysis. He underwent incision and drainage for treatment of a buttock abscess. Nafamostat mesilate was used as an anticoagulant for hemodialysis to address bleeding from the incision and drainage site. Sudden cardiac arrest occurred after 15 minutes of dialysis. The patient was treated in the intensive care unit for 5 days. Continuous veno-venous hemodiafiltration was started without any anticoagulant in the intensive care unit. Conventional hemodialysis was reinitiated, and nafamostat mesilate was used again because of a small amount of continued bleeding. Ten minutes after hemodialysis, the patient complained of anaphylactic signs and symptoms such as dyspnea, hypotension, and facial swelling. Epinephrine, dexamethasone, and pheniramin were injected under the suspicion of anaphylactic shock, and the patient recovered. Total immunoglobulin E titer was high, and skin prick test revealed weak positivity for nafamostat mesilate. We first report a case of anaphylactic shock caused by nafamostat mesilate in Korea.

Nafamostat Mesilate Inhibits TNF-alpha-Induced Vascular Endothelial Cell Dysfunction by Inhibiting Reactive Oxygen Species Production

Nafamostat mesilate (NM) is a serine protease inhibitor with anticoagulant and anti-inflammatory effects. NM has been used in Asia for anticoagulation during extracorporeal circulation in patients undergoing continuous renal replacement therapy and extra corporeal membrane oxygenation. Oxidative stress is an independent risk factor for atherosclerotic vascular disease and is associated with vascular endothelial function. We investigated whether NM could inhibit endothelial dysfunction induced by tumor necrosis factor-alpha (TNF-alpha). Human umbilical vein endothelial cells (HUVECs) were treated with TNF-alpha for 24 h. The effects of NM on monocyte adhesion, vascular cell adhesion molecule-1 (VCAM-1) and intracellular adhesion molecule-1 (ICAM-1) protein expression, p38 mitogen-activated protein kinase (MAPK) activation, and intracellular superoxide production were then examined. NM (0.01~100 microg/mL) did not affect HUVEC viability; however, it inhibited the increases in reactive oxygen species (ROS) production and p66shc expression elicited by TNF-alpha (3 ng/mL), and it dose dependently prevented the TNF-alpha-induced upregulation of endothelial VCAM-1 and ICAM-1. In addition, it mitigated TNF-alpha-induced p38 MAPK phosphorylation and the adhesion of U937 monocytes. These data suggest that NM mitigates TNF-alpha-induced monocyte adhesion and the expression of endothelial cell adhesion molecules, and that the anti-adhesive effect of NM is mediated through the inhibition of p66shc, ROS production, and p38 MAPK activation.

The Use of Nafamostat Mesilate as an Anticoagulant during Continuous Renal Replacement Therapy for Children with a High Risk of Bleeding

PURPOSE: Nafamostat mesilate (NM), a synthetic serine protease inhibitor, has been investigated as an anticoagulant for adult patients with a high risk of bleeding, who need chronic renal replacement therapy (CRRT). However, little is known about the use of NM as an anticoagulant in pediatric CRRT. The aim of this study was to evaluate the ideal dosage, efficacy, and safety of NM in pediatric CRRT. METHODS: We conducted a retrospective study of 40 pediatric patients who had undergone at least 24 h of venovenous CRRTs between January 2011 and October 2013. We divided the patients according to risk of bleeding. Those at high risk received no anticoagulation (group 1) or NM as an anticoagulant (group 2), while those at low risk received heparin (group 3). RESULTS: Forty patients (25 male and 15 female; mean age, 8.2+/-6.6 years) were enrolled. The mean duration of CRRT was 13.0 days, and the survival rate was 57.5%. The mean hemofilter lifespan was 39.3 h in group 1 and 11.3 h in group 3. In group 2, hemofilter lifespan was extended from 7.5 h to 27.4 h after the use of NM (P=0.001). The mean hemofilter lifespan with NM was greater than with heparin (P=0.018). No patient experienced a major bleeding event during treatment with NM. CONCLUSION: NM may be a good alternative anticoagulant in pediatric patients with a high risk of bleeding requiring CRRT, and is not associated with bleeding complications.

Could Nafamostat or Gabexate Prevent the Post Endoscopic Retrograde Cholangiopancreatography Pancreatitis? / 대한소화기학회지

BACKGROUND/AIMS: ERCP is the most common procedure for the diagnosis and treatment of bile duct and pancreatic disease, but Post-ERCP pancreatitis makes poor outcome in some cases. The protease inhibitors, nafamostat and gabexate, have been used to prevent pancreatitis related to ERCP, but there is some debate. We tried to evaluate the efficacy of gabexate and nafamostat for the prevention of post-ERCP pancreatitis. METHODS: Two hundred forty two patients (73 patients in the gabexate group, 88 patients in the nafamostat group and 81 patients in the placebo group) were included in the study after selective exclusion. The incidence of pancreatitis after ERCP was compared among groups. RESULTS: The incidence of pancreatitis were 6.8% in the gabexate group, 5.7% in the nafamostat group and 6.2% in the placebo group (p=0.954). CONCLUSIONS: There was no meaningful difference among the gabexate, nafamostat and placebo group.

Protease Inhibitor Treatment of Disseminated Intravascular Coagulation Associated with Aortic Dissection / 대한내과학회지

Disseminated intravascular coagulation (DIC) is a rare complication of aortic dissection. We report an unusual case of a 64-year-old woman with DIC associated with chronic aortic dissection who developed catastrophic intracranial hemorrhage. Computed tomography (CT) revealed partially thrombosed false lumen in the chronic dissected aneurysm of the thoracoabdominal aorta, which remained after surgery for acute type A aortic dissection. The laboratory profile showed features of DIC, including thrombocytopenia, hypofibrinogenemia, and increased D-dimer levels. Bleeding diathesis, including ecchymosis and coagulopathy, showed improvement following treatment with protease inhibitors (nafamostat and camostat).

A Case of Re-operation for Paravalvular Leakage after Mitral Valve Replacement Complicated by Heparin-Induced Thrombocytopenia / 日本心臓血管外科学会雑誌

Anticoagulation management in cardiac surgery can be difficult in patients with heparin-induced thrombocytopenia (HIT). We report a patient who underwent reoperation of cardiopulmonary bypass (CPB) using argatroban in combination with nafamostat mesilate. A bolus of 0.25 mg/kg argatroban was administered, followed by continuous infusion of 5-10 μg/kg/min argatroban and 100 mg/h nafamostat mesilate. No complications such as thrombosis were observed during either CPB or the perioperative period. Although we used argatroban and nafamostat mesilate, which has a shorter half-life than argatroban, the anticoagulant effect was prolonged, and the patient had an uneventful postoperative course despite requiring substantial blood transfusion.

Low-Dose Nafamostat Mesilate in Hemodialysis Patients at High Bleeding Risk / 대한신장학회지

PURPOSE: Systemic anticoagulation, usually with heparin, is required to prevent thrombosis in the blood circuit of hemodialysis. In patients at high bleeding risk, strategies to minimize the bleeding risk include heparin-free or regional anticoagulation methods. Nafamostat mesilate with conventional dose (35 mg/hr) has been used for this purpose. But it is an expensive anticoagulant to use conveniently for the dialysis therapy. Application of low-dose nafamostat mesilate has almost never been tried yet on hemodiaysis management. In this study, we examined the effect of low-dose nafamostat mesilate compared to heparin-free in hemodialysis patients with high risk of bleeding. METHODS: The current study was conducted on 35 hemodialysis patients with high bleeding risk (on-going bleeding, hemorrhage, surgery or severe thrombocytopenia). In the low-dose nafamostat group (n=17, mean age: 59+/-15 years), 238 sessions were performed with continuous infusion of nafamostat mesilate (12.5 mg/hr). In the control group with saline-flushing no heparin methods (n=18, mean age: 57+/-17 years), 247 sessions were analyzed. RESULTS: No significant differences were found in baseline characteristics between the low-dose nafamostat group and the saline group. In the progress of bleeding complications, there were no significant differences between the two groups (11.8% vs. 11.1%). In saline group, however, massive clotting occurred in 44.5 per 1000 sessions, while it occurred in 4.2 per 1000 sessions in the low-dose nafamostat group (p=0.006). CONCLUSION: In patients at high bleeding risk, low-dose nafamostat mesilat can be used as an inexpensive, effective, and safe anticoagulant for hemodialysis.

Use of Nafamostat Mesilate as an Anticoagulant during Extracorporeal Membrane Oxygenation

Although the incidence of bleeding complications during extracorporeal membrane oxygenator (ECMO) support has decreased in various trials, bleeding is still the most fatal complication. We investigated the ideal dosage and efficacy of nafamostat mesilate for use with ECMO in patients with acute cardiac or respiratory failure. We assessed 73 consecutive patients who received ECMO due to acute cardiac or respiratory failure between January 2006 and December 2009. To evaluate the efficacy of nafamostat mesilate, we divided the patients into 2 groups according to the anticoagulants used during ECMO support. All patients of nafamostat mesilate group were male with a mean age of 49.2 yr. Six, 3, 5, and 3 patients were diagnosed with acute myocardial infarction, cardiac arrest, septic shock, and acute respiratory distress syndrome, respectively. The mean dosage of nafamostat mesilate was 0.64 mg/kg/hr, and the mean duration of ECMO was 270.7 hr. The daily volume of transfused packed red blood cells, fresh frozen plasma, and cryoprecipitate and the number of complications related to hemorrhage and thrombosis was lower in the nafamostat mesilate group than in the heparin group. Nafamostat mesilate should be considered as an alternative anticoagulant to heparin to reduce bleeding complications during ECMO.

Saline Versus Nafamostat Mesilate Anticoagulation for Continuous Veno-venous Hemofiltration (CVVH) in Patients at High Risk of Bleeding: A Prospective Study / 대한신장학회지

PURPOSE:In patients with a higherrisk of bleeding, performing CVVH with heparin or saline anticoagulation is associated with increased bleeding or thrombotic risk. Nafamostat mesilate (NM), a serine proteinase inhibitor, while inhibiting various clotting factors in filter circuit, is characterized by short half life resulting in little systemic anticoagulation effect. Accordingly, we prospectively evaluated the anticoagulant effect and safety of NM in patients with a higher risk of bleeding who underwent CVVH. METHODS:Among 43 patients with high risk of bleeding [defined by (1) INR>2, aPTT>20 sec, platelet2, aPTT>20 sec, platelet<50,000/mm3), the positive effect of NM on circuit lifespan persisted irrespective of the coagulation status. CONCLUSION:As compared with saline bolus, nafamostat mesilate infusion was associated with higher CVVH filter life. In patients with high risk of bleeding, nafamostat mesilate can be used as a safe and effective anticoagulant for CVVH with acceptable filter life

Nafamostat for Prophylaxis against Post-Endoscopic Retrograde Cholangiopancreatography Pancreatitis Compared with Gabexate

BACKGROUND/AIMS: The 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. CONCLUSIONS: Administration of nafamostat before ERCP was not inferior to gabexate in protecting against the development of pancreatitis.

Risk Factors for Post-ERCP Pancreatitis in Patients Pretreated with Nafamostat Mesilate / 대한소화기내시경학회지

BACKGOUND/AIMS: Pancreatitis is the most common and important complication of an endoscopic retrograde cholangiopancreatography (ERCP). The aim of this study was to identify risk factors for post ERCP-pancreatitis in patients pretreated with nafamostat mesilate, a synthetic protease inhibitor. METHODS: A total of 247 patients who underwent an ERCP were evaluated prospectively. Potential risk factors of post-ERCP pancreatitis in patients pretreated with nafamostat mesilate were evaluated. RESULTS: Twenty-four patients (9.7%) and nine patients (3.6%) developed post-ERCP hyperamylasemia and pancreatitis, respectively. As determined by univariate analysis among the potential risk factors, we found a procedure time over 20 minutes, pancreatic duct cannulation over four times, prior post-ERCP pancreatitis and the absence of a common bile duct (CBD) stone as risk factors for post-ERCP hyperamylasemia. We also found a patient age under 60 years, a procedure time over 20 minutes, pancreatic duct cannulation over four times and the absence of a CBD stone as risk factors for post-ERCP pancreatitis (p<0.05). As determined by multivariate analysis, pancreatic cannulation over four times is independently associated with post-ERCP hyperamylasemia (p=0.038; OR, 5.165; 95% CI, 1.093~24.412) and post-ERCP pancreatitis (p=0.002; OR, 33.122; 95% CI, 3.526~311.138). CONCLUSIONS: A repeated pancreatic duct cannulation is the most important risk factor for post-ERCP pancreatitis in patients pretreated with nafamostat mesilate.

The effect of nafamostat mesilate on the prevention of cerulein-induced acute pancreatitis / 대한내과학회지

BACKGROUND: Many protease inhibitors show a protective effect for acute pancreatitis as seen in animal models. In previous studies, the protease inhibitors were administered before induction of pancreatitis, and there are few published reports examining effects when these agents were administered after induction of pancreatitis. The timing of drug administration may provide an explanation for the ineffectiveness of protease inhibitors for the treatment of patients with acute pancreatitis. Herein, we assessed the protective effect of nafamostat mesilate (NM), a potent protease inhibitor, in a mouse model of cerulean-induced pancreatitis and compared the results of administering the drug before and after the induction of pancreatitis. METHODS: Cerulein, a cholecystokinin analogue, was injected into mice intraperitoneally to induce pancreatitis. The mice received intravenous NM administration before and after the induction of pancreatitis. The serum concentration of amylase and lipase was measured, histological changes were measured, and the tissue expression of myeloperoxidase was measured to assess the degree of inflammation. Expression of p38 MAPK (mitogen-activated protein kinase), phospho-p38 MAPK, and IL-6 (interleukin-6) in tissue was evaluated. RESULTS: Acute pancreatitis was induced successfully by intraperitoneal injection of cerulein. Acute pancreatitis could be prevented when NM was administered before the induction of pancreatits. However, the effect was not guaranteed when given after the induction of pancreatitis. For a group of mice with induced pancreatitis, tissue expression of phospho-p38 MAPK was prominent and there was no marked difference in the expression of IL-6 between groups with or without induced pancreatitits. CONCLUSIONS: Although the efficacy of NM for treatment of acute pancreatitis is doubtful, pretreatment with NM for an expected condition like endoscopic retrograde cholangiopancreatography (ERCP), might be helpful for the prevention of pancreatitis.

A Multi-Center Phase III Clinical Trial to Assess the Influence to Bleeding and Anticoagulant Effect of Nafamostat Mesilate (Futhan) in Hemodialysis Patients with High Bleeding-Risk / 대한신장학회잡지

BACKGROUND: Routine hemodialysis is performed with systemic anticoagulation, usually with heparin, to prevent thrombosis in the extracorporeal blood circuit. However, systemic anticoagulation can produce hemorrhagic complications in patients at high risk of bleeding. To minimize the risk of bleeding, a number of alternative regimens has been proposed, however, each of those methods has its own limitations and complication. METHODS: 58 hemodialysis patients at risk for bleeding due to previous surgery or hemorrhagic complication were treated with Futhan as regional anticoagulant and compared with that of low-dose heparin anticoagulation. There were 29 (50%) postoperative cases and 29 (50%) cases of hemorrhage from various sites. RESULTS: The exacerbation of bleeding by hemodialysis was noted in only 4% in heparin treated group and none in Futhan group. Clotting times at site A (intracorporeal circulation) were not prolonged with Futhan, whereas those of heparin were prolonged slightly, which is not statistically significant. Degrees of residual blood in the dialyzer and blood clottings in the venous drip-chamber were less in Futhan than in heparin group. Adverse reactions related to Futhan therapy were minor and the incidence of adverse reactions was comparable in both groups. CONCLUSION: Futhan is a safe and effective regional anticoagulant for hemodialysis especially for patients with high bleeding risk.

The Effect of Nafamostat Mesilate for the Treatment of Disseminated Intravascular Coagulation after Surgery Using Cardiopulmonary Bypass / 日本心臓血管外科学会雑誌

To investigate the effect of nafamostat mesilate (FUT) for disseminated intravascular coagulation (DIC) after surgery using cardiopulmonary bypass, we studied DIC scores and parameters of coagulation and fibrinolysis in the DIC cases. Although 12 patients developed DIC, the platelet counts improved by administration of FUT apart from one complicated by sepsis. The DIC scores decreased as a result of the increase of platelets and fibrinogen and improvement of FDP. Thrombin-antithrombin III complex, D-dimer and plasmin-α₂ plasmin inhibitor complex showed an even higher value at the endpoint of FUT administration. These results indicate that patients with DIC after cardiopulmonary bypass may have severe fibrinolytic acceleration and that administration of FUT can be useful in those cases.