Methods and compositions for anticoagulation

Abstract

Compositions and methods for regional anticoagulation of an extracorporeal circuit are provided. The compositions and methods generally entail administration of a nafamostat anticoagulant agent to an extracorporeal circuit that includes either a filter comprising a polyacrylonitrile material (a PAN filter) or a filter that does not comprise a polyacrylonitrile material (a non-PAN filter) in a safe and efficient manner to provide regional anti coagulation within the circuit without concomitant systemic anti coagulation effect.

Description

METHODS AND COMPOSITIONS FOR ANTICOAGULATIONRELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application Nos. 63 / 558707 filed February 28, 2024; and 63 / 627439, filed January 31, 2024, each of which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure generally relates to methods and compositions for use in extracorporeal circuits, such as those used in renal replacement therapy, extracorporeal membrane oxygenation, plasmapheresis, and cardiopulmonary bypass.INTRODUCTION

[0003] Blood treatment procedures, such as renal replacement therapy (RRT), extracorporeal membrane oxygenation (ECMO), plasmapheresis, and cardiopulmonary bypass (CPB) procedures, provide essential interventions for use in the intensive / critical healthcare setting. For example, up to about 40% of patients in intensive care units (ICUs) present with acute kidney injury (AKI) (see, e.g., Nisula et al. (2013) Intensive Care Med. 39:420-428 and Hoste et al. (2C\S) Intensive Care Med. 41: 1411-1423) and RRT remains a mainstay of supportive measures for such critically ill patients. Continuous renal replacement therapy (CRRT) procedures are performed with lower blood flow and slower ultrafiltration rates and run continuously over approximately a week-long treatment period. CRRT is more likely to be used in the ICU than intermittent hemodialysis (IHD), particularly in those patients who are unable to tolerate rapid solute removal since it preserves hemodynamic equilibrium by gradually removing fluid and urea (Manns et al. (1999) “Continuous renal replacement therapies: an update” Am J Kidney Dis 32(2): 185-207). However, the features of CRRT that provide for lowered hemodynamic impact can cause problems within the extracorporeal circuit due to a higher potential for circuit filter fouling and occlusion. These problems arise primarily from blood clotting (coagulation), that may cause increased therapy downtime, potential patient undertreatment or loss of treatment efficacy, patient loss of clotted blood and other potential thrombogenic effects including the potential increase in catheter-related bloodstream infection. Hence, CRRT requires a level of anti coagulation that is sufficient to prevent clotting within the extracorporeal circuit (regionally) while avoiding over-anti coagulation (systemically) that can lead to complications in the patient, such as excessive bleeding.

[0004] During CRRT, blood flows from the patient typically through a double-lumen catheter and into the extracorporeal circuit and then through a dialysis filter that is driven by aperistaltic pump, allowing for blood purification and enabling fluid removal. Various anti coagulation approaches can be employed to prevent filter clotting and thus increase filter life, including regional anti coagulation therapies, selection of procedure modalities including blood flow rates and vascular access, selection of catheter types and materials, and selection of filter membrane types and materials (see, e.g., Schetz, M. (2001) “Anticoagulation in Continuous Renal Replacement Therapy”, Contrib Nephrol. 132:283-303). An international consensus guideline exists that is known as the Kidney Disease Improving Global Outcome (KDIGO) Clinical Practice Guidelines for Acute Kidney Injury (KDIGO, (2012) Kidney Inter., Suppl. 2: 1- 138). The KDIGO guideline recommends “using anti coagulation during RRT in AKI if a patient does not have an increased bleeding risk or impaired coagulation and is not already receiving anti coagulation.” (Grade IB, strong recommendation, moderate quality evidence). For patients with an increased risk of bleeding, the KDIGO guideline suggests using regional citrate anti coagulation (approved under an Emergency Use Authorization (EUA) in the US), rather than no anti coagulation, during RRT in a patient without contraindications for citrate (Grade 2C, weak recommendation, low quality evidence). However, there are currently no FDA-approved regional anticoagulants in the US.SUMMARY

[0005] CRRT is a readily available and widely employed renal replacement method in the ICU and includes intermittent hemodialysis and peritoneal dialysis. Such methodology is intended to be applied for 24 hours or longer through a continuous, slower dialysis using pump- driven extracorporeal circuits and acts as renal support through blood purification to allow solute and fluid homeostasis (Macedo et al. (2016) Am J Kidney Dis 68(4):645-657). The slower rate of fluid removal using CRRT theoretically causes less hypotension than other dialysis methods and provides advantages for hemodynamically unstable patients that often also require large volume fluid administration including medications and parenteral nutrition. However, CRRT is commonly associated with premature clotting when blood passes through the circuit filter membranes (Morabito et al. (2003) JNeph 16(4): 566-571) as a result of a coagulation cascade initiated by complex interactions between the patient’s blood and foreign surfaces of the extracorporeal circuit (Levi et al. (2006) Critical Care 10(4): 222). Such premature clotting generally leads to interruption of CRRT procedures and can result in patient blood loss. Excessive blood loss in turn causes anemia and increases the need for blood transfusion in patients, leading to a concomitant increase in morbidity and mortality in ICU patients (See, e.g., Levi et al. (supra) and Vincent et al. (2002) 4A74 288(12): 1499-1507). These considerations make improved prevention of blood clotting within the CRRT circuit, that is, regionalanti coagulation of the extracorporeal circuit, an important target for improvement in the quality and effectiveness of current critical care dialysis practice.

[0006] The effective selection of an anticoagulant agent for use in any particular CRRT procedure is dependent upon a significant number of patient / treatment variables. First and foremost, the proper selection is determined by the patient’s underlying illness or condition, the availability and regulatory status of the selected anticoagulant agent, and the technical expertise in the proper use of the anticoagulant generally available to the ordinarily skilled person. Current pharmacological CRRT anticoagulant options include standard, or “unfractionated” heparin (UFH); low molecular weight heparin (LMWH); prostacyclin; direct thrombin inhibitors such as hirudin, bivalirudin and argatroban; antiplatelet agents such as prostaglandin 12 inhibitors (epoprostenol and iloprost), prostaglandin El inhibitors such as alprostadil, and glycoprotein Ilb / IIa antagonists such as tirofiban; and serine protease inhibitors such as nafamostat. In the United States, regional citrate anti coagulation and systemic UFH anti coagulation are currently the most common pharmacological approaches to maintain patency of the extracorporeal circuit during CRRT. While both agents are administered into the CRRT circuit, the long half-life of UFH (1-2 hours) results in circulation back into the patient, hence the term “systemic” anticoagulant. A significant adverse side effect of, and general contraindication against UFH includes an increased risk of patient bleeding. Critically ill patients in general are already at a higher risk of excessive bleeding due to coagulation abnormalities such as thrombocytopenia, prolonged prothrombin time (PPT) and activated partial thromboplastin time (aPTT). A significant risk of regional citrate anti coagulation is hypocalcemia due to citrate chelation of calcium. Accordingly, for some ICU patients, clinicians may in fact choose to perform CRRT without the use of any anticoagulants for filter clotting prevention, particularly if the bleeding risk or hypocalcemia risk in the patient may exceed the benefit of providing an extended filter life in the CRRT circuit.

[0007] As stated, the main limitation of heparin (including use of UFH) is the long halflife of the heparin agent (1-2 hours), which results in systemic exposure that can lead to hemorrhage, heparin resistance, and the development of heparin-induced thrombocytopenia (Tolwani et al. (2009) “Anticoagulation for Continuous Renal Replacement Therapy” Seminars in Dialysis 22(2): 141-145). UFH can also inhibit the anti-inflammatory effects of antithrombin and trigger the release of inflammatory mediators from blood and endothelial cells (Oudemans- van Straaten (2012) “Bench-to-bedside Review: Citrate for Continuous Renal Replacement Therapy, from Science to Practice”, Critical Care 16:249). UFH can also result in heparin- induced thrombocytopenia. LMWH can also be used in dialysis but, since it is cleared by the kidney, the systemic anti coagulation effect is unacceptably long and therefore is uncommonlyused. In addition, the American College of Chest Physicians (ACCP) guideline for antithrombotic and thrombolytic therapy suggest using UFH in place of LMWH in patients with severe renal insufficiency for therapeutic anticoagulation, or to reduce the dose of LMWH (Hirsh et al. (2008) “Parenteral Anticoagulants” in American College of Chest Physicians Evidence- Based Clinical Practice Guidelines (8th Ed) 133(6 Suppl): 141S-159S). Other alternatives include heparin anti coagulation accompanied with protamine reversal, however protoamine use is complicated and often difficult to titrate and therefore is rarely used.

[0008] In the United States, only UFH is approved for CRRT anti coagulation (which recirculates systemically as previously mentioned), therefore there are no FDA-approved agents for regional anticoagulation. Alternatives to heparin include regional citrate anti coagulation (RCA). One type of RCA is Regiocit ®, which has only an emergency use authorization (EUA) by the FDA. Other types of RCA are used off-label and are discussed below. In RCA, the citrate agent chelates calcium (which is needed to form a clot), acting as a local anticoagulant when regionally administered (prefilter). Calcium is then infused post-filter (of systemically into the patient) to reverse the hypocalcemic effect of citrate. However, use of the citrate agent creates a risk of hypocalcemia, metabolic acidosis or metabolic alkalosis if partially metabolized and accumulated and citrate toxicity giving rise to fulminant hepatic failure or liver shock, and clinical errors that may lead to overdose. Accordingly, RCA requires advanced training, monitoring and frequent blood draws to ensure that patients do not develop life-threatening hypocalcemia or alkalosis. In addition, the FDA recently issued an urgent warning against the use of concentrated citrate following the cardiac arrest of a hemodialysis patient, and most centers that use RCA use tri-sodium citrate solutions approved for apheresis: either 4% sodium citrate, anticoagulant citrate dextrose solution (ACD-A), or compounded citrate solutions. These approaches carry non-trivial hazards. The use of ACD-A or 4% sodium citrate requires large volumes (typically 1-2 liters per day during CRRT) of ACD-A or 4% sodium citrate infused into the system via an intravenous pump. Since these solutions require an infusion pump that is not part of the dialysis system, this pump does not automatically stop if the CRRT pump stops, thereby risking citrate back-flow into the patient’s systemic circulation, which can cause acute hypocalcemia and fluid overload. Furthermore, since all patients requiring CRRT have renal impairment and may also have liver impairment, off-label use of RCA can also result in citrate toxicity. In the largest randomized controlled trial of RCA, nearly 6% of patients experienced a critically low level of ionized calcium of less than 0.9 mmol / L (Oudemans-van Straaten (2009) Crit Care Med 37(2):545-552). Critically low calcium levels can lead to life-threatening ventricular tachycardia and ventricular fibrillation (Cecchi (2015) Clin Cases Miner Bone Metab 12(3):265-8).

[0009] The third most common anti coagulation option, that is the option of the use of no anti coagulation agent in CRRT, is typically directed to patients with coagulopathies, hepatic failure or thrombocytopenia, and may be indicated in those patients with a platelet count <50,0000, and international normalized ratio >2.0, an aPTT > 60 seconds or who are actively bleeding or have had a hemorrhagic event within the previous 24 hours. Such procedures may include an extracorporeal circuit priming step using saline solution or heparin, wherein intermittent saline flushes may also be employed.

[0010] Given the limitations of the various currently available pharmacological UFH or regional anti coagulation approaches for use in CRRT, there remains a significant unmet medical need for a safe and effective regional anticoagulation option in CRRT that does not suffer from the aforementioned adverse events. One such agent is nafamostat, a small molecule, broadspectrum protease inhibitor that inhibits prothrombin conversion to thrombin, as well as inhibiting thrombin at the platelet thrombin receptor, PARI (Fuse et al. (1999) Platelets 10:212- 218). Nafamostat also inhibits various enzyme systems, such as the coagulation-fibrinolysis system (Xlla, Xa, Vila, and plasmin), the kallikrein-kinin system, the complement system, and pancreatic proteases (see, e.g., Hitomi et al. (1985) Haemostasis 15(3): 164-168, Fujii et al.(1981) Biochem Biophys Acta 661:342-345, and Aoyama et al. (1984) Japan J Pharmacol 35:203-227).

[0011] Nafamostat has a small molecular weight of 539.59 Da and an ultra-short systemic circulation half-life (~8 minutes), making it ideally suited as a regional anticoagulant in extracorporeal circuits. When nafamostat is administered into the afferent limb of the CRRT circuit, blood becomes anti coagulated because of nafamostat’ s rapid inhibition of thrombin. As blood is transported into the hemodialyzer, nafamostat’ s small molecular weight allows a significant proportion of the agent to be removed via filtration. Thus, the amount of nafamostat that is returned to the patient via the efferent limb is limited and will be rapidly metabolized. Accordingly, the anti coagulation activity of nafamostat is primarily limited to a regional effect within the extracorporeal circuit. An illustration of the chemical structure of the mesylate salt of nafamostat is depicted in Figure 1.

[0012] Nafamostat is currently approved and has been marketed as Futhan® in Japan and South Korea for regional anti coagulation where it has no contraindications as an anticoagulant for use in CRRT, nor does it have any limitation on the treatment / administration period (FUTHAN Package Insert, August 2009, Version 7. Japan). Nafamostat can be used in patients who are at risk of bleeding and in patients for whom the use of heparin is contraindicated (see, e.g., Hwang et al. (2013) Int J Artif Organs 36(3):208-216, Kubota et al. (1997) Can J Anaesth 44(11): 1182-1186 and Baek et al. (2012) Renal Failure 34(3):279-285) since the agent exhibits asignificantly reduced incidence of bleeding during continuous hemofiltration and / or continuous hemodiafiltration as compared against UFH and LMWH in critically ill patients (see, e.g., Ohtake et al. (1991) Contrib Nephrol 93:215-217).

[0013] Accordingly, selection of nafamostat as the regional anticoagulant agent for use in CRRT forms an integral part in the practice of the present disclosure. Nafamostat is a potent regional anticoagulant agent option that avoids the systemic anticoagulant effect of heparin-based options and does not give rise to citrate’s adverse systemic side effect profile that requires continual clinical monitoring and / or additional medical prevention measures or pharmaceutical / therapeutic intervention. However, the effective use of nafamostat in CRRT must also take into consideration the particular attributes of the extracorporeal circuit filter that has been selected for use in a particular CRRT process. In this regard, a CRRT filter membrane can be formed from a polyacrylonitrile (PAN) polymeric material such as AN69 or oXiris, from a non-PAN polymeric material such as polysulfone, polyethersulfone or polyarylethersulfone, or the filter membrane may be formed from a blended material containing PAN and non-PAN polymer or copolymer components including, for example, a filter membrane material that is coated with a PAN component (e.g., AN69ST). The CRRT filter membrane acts as both a screentype filter to collect and remove particles from the fluid stream passing through the extracorporeal circuit by direct (size) exclusion and / or by diffusional interception (adsorption) of smaller particulates via membrane surface interactions that cause such smaller particles to adhere to the surface of the internal pores or fibers of the filter medium and then attract similar particles. Protein complexes of coagulated blood are thus separated from the dialysis fluid filtrate by exclusion or diffusion and become trapped on the surface of the CRRT filter membrane. The accumulation of such adhered particles on the membrane surface can form a so-called “protein cake” that eventually results in occlusion (or “fouling”) of the filter membrane, leading to reduced filter efficiency and lifespan.

[0014] The significant performance differences between various combinations of nafamostat and selected CRRT filter membrane materials are manifest. For example, whereas the Futhan label addresses and confirms the use of nafamostat with PAN surface-treated membrane materials (e.g., AN69ST) and non-PAN membrane materials such as polysulfone (PS) and polyarylethersulfone (PAES), the use of nafamostat with PAN membrane materials such as AN69 and non-PAN materials such as polyethersulfone (PES) are contraindicated (FUTHAN Package Insert, supra). In recognition of such differences, the inventors of International Patent Publication Number WO 2022 / 225723 disclose a number of strategies for priming and then operating an extracorporeal circuit to provide regional anti coagulation with nafamostat in combination with PAN filter membranes or non-PAN filter membranes depending upon thediffering absorptive potentials that such membrane materials have to the nafamostat anticoagulant agent. However, to date there are no existing criteria governing the use of nafamostat to provide safe and effective regional anti coagulation in an extracorporeal circuit for service in patient populations that are at high risk of bleeding without affecting the efficiency of the procedure, decreasing the effective life of circuit filters and / or causing undue and unwanted systemic anti coagulation as well as increasing the potential for causing significant adverse events in such patients.

[0015] There accordingly remains an urgent and long-felt need in the art to develop novel methods and compositions suitable for providing safe and effective regional anti coagulation in extracorporeal circuits as generally employed and operated worldwide, using commonly available critical circuit components such as traditional circuit filter membranes as well as emerging new generation circuit filter membranes. In answer to this urgent and long-felt need, the present disclosure is directed to a set of readily employable guidelines for operating extracorporeal circuits in a safe and effective manner in patients at high risk of bleeding by establishing and maintaining regional anticoagulation within the circuit during a blood treatment procedure such as CRRT while avoiding potential systemic anti coagulation or other significant safety concerns.

[0016] Accordingly, it is a primary aspect of the present disclosure to provide a method for providing regional anti coagulation in an extracorporeal circuit during a blood treatment procedure, where the circuit has a filter component that comprises a polyacrylonitrile (PAN) material (a PAN filter). The method entails initiating the procedure in a subject whose bloodstream is connected to the circuit without first performing an initial anticoagulant priming step as normally directed. An anticoagulant agent is introduced into the circuit upon initiation of the procedure to provide an anticoagulant effect within the circuit, that is, to provide regional anti coagulation. The anticoagulant agent employed in the practice of the method includes nafamostat, and the agent is introduced into the circuit at a higher infusion rate than with other filters. Avoidance of performing a standard initial priming step and then initiating and performing the blood treatment procedure using a higher rate of infusion with the nafamostat anticoagulant helps overcome the high filter binding to PAN filters. Priming the PAN circuits is of no use as the filter has an almost limitless ability to bind nafamostat. Therefore, higher rates of infusion allow the binding to occur while still providing adequate circuit anticoagulation. The high rate of removal of nafamostat from the circuit by filter binding to PAN filters as well as the ultra-short half-life of nafamostat prevents or reduces any significant systemic concentration of the anticoagulant.

[0017] In certain preferred aspects of the present disclosure, the blood treatment procedure comprises a continuous renal replacement therapy (CRRT), wherein the CRRT procedure can further comprise hemofiltration, hemodialysis or hemodiafiltration. In certain further preferred aspects of the present disclosure, the nafamostat anticoagulant agent is administered at an initial infusion rate sufficient to provide about 15 mg of nafamostat per hour to the circuit. In other aspects, the nafamostat anticoagulant agent is administered at an initial infusion rate sufficient to provide at least about 20, 25, 30, 35, 40 or 50 mg of nafamostat per hour to the circuit. In addition, the nafamostat anticoagulant agent can be infused over a period of about 1 hour, or about 24, 30, 48, 78, 96 hours or longer.

[0018] The nafamostat anticoagulant agent can comprise any therapeutically effective form of nafamostat, such as the mesylate salt of nafamostat, and the filter used in the circuit can consist entirely of a polyacrylonitrile filter material or merely just contain a polyacrylonitrile component such as in a copolymer. Successful practice of the methods of the present disclosure can be assessed by monitoring a patient’s post-filter activated clotting time (ACT) after initiation of the blood treatment procedure to determine that there is a significant increase from that patient’s baseline ACT, for example as measured after about 24 or 48 hours of treatment. Postfilter ACT can also be compared against the patient’s systemic ACT to determine successful practice of the methods, for example wherein the 24-, 48- and / or 72-hour post-filter and systemic ACT are measured, and the post-filter ACT is higher than the systemic ACT at one or more of each such time points after initiation of the blood treatment procedure.

[0019] In addition, successful practice of the methods of the present disclosure can be assessed by determining increased lifespan of the extracorporeal circuit filter, such as where there is about a 10 or 15 percent increase of effective filter life as compared against the same procedure performed without the use of an anticoagulant. Increased lifespan of the extracorporeal circuit filter can also be determined by noting the number of filter changes required during any portion of the procedure up to the amount of filter changes required during conduct of the entire procedure.

[0020] In certain aspects of the present disclosure wherein the blood treatment procedure comprises dialysis, successful practice of the disclosed methods can be assessed by determining enhanced dialysis efficiency of the procedure as measured using a time-averaged concentration of blood urea nitrogen (TACurea) test, wherein the patient’s time-averaged concentration of blood nitrogen measured at about 24 hours from initiation of the procedure is lower as compared against the same procedure when performed without the use of an anticoagulant. Alternatively, in those aspects of the present disclosure wherein the blood treatment procedure comprises dialysis, successful practice of the disclosed methods can be assessed by determining enhanced dialysisefficiency of the procedure as measured using a time-averaged concentration of plasma 132 microglobulin test, wherein the patient’s time-averaged concentration of plasma 132 microglobulin measured at about 24 hours from initiation of the procedure is lower as compared against the same procedure when performed without the use of an anticoagulant.

[0021] It is a further primary aspect of the present disclosure to provide a method for providing regional anti coagulation in an extracorporeal circuit, where the circuit has a filter component that does not comprise a polyacrylonitrile material (a non-PAN filter). Non-PAN filters bind nafamostat less than PAN filters and therefore can benefit from being primed with an anticoagulant to saturate the binding and allow a lower infusion rate. For an ultra-short acting anticoagulant agent such as nafamostat, this technique is more appropriate to allow for a rapid anti coagulation effect on the circuit filter while not over-anticoagulating some patients. The method thus entails an initial bolus priming of the non-PAN filter prior to initiation of a blood treatment procedure by administering a bolus of an anticoagulant agent comprising nafamostat to the circuit to establish a regional anti coagulation effect in the circuit. The priming bolus that contains at least about 25, 30, 35, 40, 45, 50, 55 or 60 mg or more of nafamostat is introduced into the circuit to initiate regional anti coagulation. After the bolus priming step is carried out, the regional anti coagulation is maintained in the circuit by infusing the nafamostat anticoagulant agent to the circuit at a low infusion rate. In certain preferred aspects of the present disclosure, the anti coagulation maintenance infusion rate is sufficient to provide about 18, 15, 10 or less mg of nafamostat per hour to the circuit. The maintenance infusion of the nafamostat anticoagulant agent can be carried out for at least about 1 hour, 24, 48 hours or more during the procedure.

[0022] In certain preferred aspects of the present disclosure, the blood treatment procedure comprises a continuous renal replacement therapy (CRRT), wherein the CRRT procedure can further comprise hemofiltration, hemodialysis or hemodiafiltration.

[0023] The nafamostat anticoagulant agent used in the methods of the present disclsoure can comprise any therapeutically effective form of nafamostat, such as the mesylate salt of nafamostat.

[0024] Successful practice of the methods of the present disclosure can be assessed by monitoring a patient’s post-filter ACT after initiation of the blood treatment procedure to determine that there is a significant increase from that patient’s baseline ACT, for example as measured after about 24 or 48 hours of treatment. Post-filter ACT can also be compared against the patient’s systemic ACT to determine successful practice of the methods, for example wherein the 24-, 48- and / or 72-hour post-filter and systemic ACT are measured, and the post-filter ACT is higher than the systemic ACT at one or more of each such time points after initiation of the blood treatment procedure.

[0025] In addition, successful practice of the methods of the present disclosure can be assessed by determining increased lifespan of the extracorporeal circuit filter, such as where there is about a 10 or 15 percent increase of effective filter life as compared against the same procedure performed without the use of an anticoagulant. Increased lifespan of the extracorporeal circuit filter can also be determined by noting the number of filter changes required during any portion of the procedure up to the amount of filter changes required during conduct of the entire procedure.

[0026] In certain aspects of the present disclosure wherein the blood treatment procedure comprises dialysis, successful practice of the disclosed methods can be assessed by determining enhanced dialysis efficiency of the procedure as measured using a TACurea test, wherein the patient’s time-averaged concentration of blood nitrogen measured at about 24 hours from initiation of the procedure is lower as compared against the same procedure when performed without the use of an anticoagulant. Alternatively, in those aspects of the present disclosure wherein the blood treatment procedure comprises dialysis, successful practice of the disclosed methods can be assessed by determining enhanced dialysis efficiency of the procedure as measured using a time-averaged concentration of plasma B2 microglobulin test, wherein the patient’s time-averaged concentration of plasma B2 microglobulin measured at about 24 hours from initiation of the procedure is lower as compared against the same procedure when performed without the use of an anticoagulant.

[0027] These aspects of the present disclosure, as well as others, are described in detail in the following sections of this application and expressly set forth in the appended claims.BRIEF DESCRIPTION OF THE FIGURES

[0028] Figure 1 depicts a chemical structure of the mesylate salt of nafamostat.

[0029] Figure 2 depicts a proposed benefit of using a higher prime dose of nafamostat coupled with a lower infusion dose in a blood treatment procedure using a non-PAN filter in order to quickly achieve and then maintain a post-filter activated clotting time within a narrow regional anti coagulation window during the procedure. In the figure, the top curve represents the Futhan® product administered as a 20 mg bolus followed by a 20 mg / hr infusion rate. The bottom curve represents nafamostat mesylate administered in accordance with the present disclosure using a 50 mg bolus followed by a 15 mg / hr infusion rate.DETAILED DESCRIPTION

[0030] For convenience, certain terms employed in this entire application (including the specification, figures and appended claims) are expressly defined throughout. Unless expressly defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.Definitions

[0031] As used throughout this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a PAN membrane material” includes a mixture of two or more such materials, and the like.

[0032] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” therefore indicates inclusion rather than limitation. The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the aspect. As used herein the term “consisting essentially of’ refers to those elements required for a given aspect. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that aspect of the disclosure.

[0033] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, protein, and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002); Harlow and Lan, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Principles of Neural Science, 4th ed., Eric R. Kandel, James H. Schwart, Thomas M. Jessell eds. McGraw-Hill / Appleton & Lange: New York, N.Y. (2000); The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Research Laboratories (2006) (ISBN 0-911910-19-0), Robert S. Porter et al. eds., The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd. (1994) (ISBN 0-632-02182-9); and Current Protocols in Protein Sciences (2009) Wiley Intersciences, Coligan et al., eds.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0035] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about” whether or not expressly indicated as such. The term “about” when used in connection with percentages, hours, days or dosages or other amounts can mean + / - 10%.

[0036] The terms “anti coagulated” and “anti coagulation” as used herein refer to the prevention or reduction of blood coagulation (clotting). An “anticoagulant” agent is a pharmacologically or therapeutically active agent that can anticoagulate blood such that the anti coagulated blood has a longer clotting time in the presence of such agent as compared to in its absence.

[0037] The term “bolus” as used herein refers to the administration of a discrete amount of therapeutically active agent, such as an anticoagulant, within a specific, short period of time generally from about 1 to about 45 minutes, wherein such bolus administration is intended to quickly raise the concentration of the administered agent to a therapeutically or pharmacologically effective level.

[0038] The terms “lower”, “decrease”, “reduce”, “reduced”, “reduction”, “decrease”, and “inhibit” are all used interchangeably herein generally to mean a decrease by a statistically significant amount relative to a reference. However, for avoidance of doubt, such terms typically mean a decrease by at least 1% as compared to a reference level and can include, for example, a decrease by at least about 5%, or at least about 10%, or at least about 20%, or at least about 50%, or at least about 60%, at least about 65%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 99%, up to and including, for example, the complete absence of the given entity or parameter as compared to the reference level, or any decrease between 1-99% as compared to the absence of a given treatment or procedure.

[0039] The terms “increased”, “increase” or “enhanced” are all used interchangeably herein generally to mean an increase by a statically significant amount; for the avoidance of any doubt, the terms denote an increase of at least 1% as compared to a reference level, for example an increase of at least about 5%, or at least about 10%, or at least about 20%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase or any increase between 1-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or more as compared to a reference level.

[0040] The term “pharmaceutically acceptable” refers to a material or agent that has been approved or is approvable for pharmaceutical use by a regulatory agency of a relevant federal or state government and / or is listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animal subjects, and more particularly in humans.

[0041] A “pharmaceutically acceptable salt” refers to a salt of a therapeutically active molecule or compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent molecule or compound. Pharmaceutically acceptable salts of the therapeutically active anticoagulant agents described herein include those salts derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts include acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate, glucoheptanoate, glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, malonate, methanesulfonate, mesylate, 2- naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate, tartrate, thiocyanate, tosylate and undecanoate salts. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining pharmaceutically acceptable acid addition salts. Salts derived from appropriate bases include alkali metal (e.g., sodium and potassium), alkaline earth metal (e.g., magnesium), ammonium and salts.

[0042] The term “prime” or “priming” as used herein refers to an initial infusion of a solution into an extracorporeal circuit or catheter, whether or not the circuit is dry or previously filled with another solution or blood.

[0043] The terms “regional” or “regionally” as used herein mean, with respect to an anti coagulation effect, that such event takes place outside of the body of a subject, for example within an extracorporeal circuit.

[0044] The term “subject” is used interchangeably in this specification and the appended claims with the term “patient” and is used to refer to a human individual, for example a human subject undergoing a blood treatment procedure that employs an extracorporeal circuit apparatus.

[0045] The terms “systemic” or “systemically” as used herein mean, with respect to an anti coagulation effect or an anticoagulant agent, that such event or agent is detectable at a biologically significant level in the blood plasma of a subject.

[0046] It should be understood that this disclosure is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. Theterminology used herein is for the purpose of describing particular aspects only and is not intended to limit the scope of the present disclosure, which is defined solely by the claims.Methods of Treatment, Compositions:

[0047] Extracorporeal procedures are medical procedures conducted outside of the body using an extracorporeal device or apparatus, such as an extracorporeal circuit, where blood is taken from a subject’s circulation for treatment or processing prior to being returned to the subject’s systemic circulation. Such blood treatments include renal replacement therapy (RRT), continuous renal replacement therapy (CRRT), intermittent hemodialysis (IHD), extracorporeal membrane oxygenation (ECMO), plasmapheresis, and cardiopulmonary bypass (CPB) procedures.

[0048] In all extracorporeal circuits there necessarily is significant contact between the internal surfaces of the circuit and the surfaces of circuit components with the blood that is being treated. Contact of blood flowing through the circuit with such “foreign” surfaces of the circuit causes a natural cascade of biochemical reactions within the circulating blood that results in clotting. Blood clotting activity within the circuit during a blood treatment procedure reduces the quality and efficiency of the procedure since such clotting causes physical obstruction within the circuit, leading to increased therapy downtime, potential patient undertreatment or loss of treatment efficacy, patient loss of clotted blood and other potential thrombogenic effects including the potential increase in catheter-related bloodstream infection. Excessive coagulation within the circuit requires disruption of the blood treatment procedure in order to remove and clogged circuit components, and then subsequent downtime while re-initiating the procedure.

[0049] A number of non-pharmacological approaches to addressing the issue of clotting in extracorporeal circuits have been directed to providing alterations in the rate of blood flow through the circuit, the use of longer or shorter catheters and / or surface-modified catheters and other similar strategies. However, the filter membrane in the circuit has by far the most surface area contact with the circulating blood. Accordingly, new generation circuit filters have sought to improve non-clotting behavior through providing higher membrane fiber density, reduced membrane fiber length, and altered filter structures (such as flat plate formats versus cylindrical structures), as well as using filter surface modifications such as anticoagulant coatings. Although such approaches may provide improvements to the performance of extracorporeal circuits, the most common approaches to filter clotting performance issues must employ pharmacological anticoagulant agents that are administered usually within the circuit in order to reduce or eliminate blood clotting activity. In the United States and many other countries, the only available anticoagulant agents are either heparin-based or citrate. However, both of these agentshave significant safety and efficacy issues that limit their utility and applicability in addressing the issue of undue blood clotting in extracorporeal circuits.

[0050] Accordingly, there remains a significant need to develop novel methods and compositions suitable for providing safe and effective regional anti coagulation in extracorporeal circuits using commonly available circuit components including circuit filter membranes combined with a safe and effective anticoagulant agent. The present disclosure is thus directed to such novel methods and compositions and allow ready operation of extracorporeal circuits in a safe and effective manner, particularly in patients that may be at high risk of bleeding. These methods and compositions can be used to establish and maintain regional anti coagulation within an extracorporeal circuit during a blood treatment procedure while avoiding potential systemic anti coagulation or other significant safety concerns, such as hypocalcemia, in the patient.

[0051] Initially, nafamostat has been selected as the regional anticoagulant agent for use in the practice of the methods of the present disclosure. As discussed herein above, nafamostat is a broad-spectrum protease inhibitor that inhibits prothrombin conversion to thrombin, as well as inhibiting thrombin at the platelet thrombin receptor, PARI. Nafamostat also inhibits various important enzyme systems, such as the coagulation-fibrinolysis system (Xlla, Xa, Vila, and plasmin), the kallikrein-kinin system, the complement system, and pancreatic proteases. The nafamostat agent can be used in patients who are at risk of bleeding as well as in patients for whom the use of a heparin anticoagulant is contraindicated. The small molecular weight and short systemic circulation half-life of nafamostat makes it ideally suited as a regional anticoagulant in extracorporeal circuits. When nafamostat is added to an extracorporeal circuit, the circulating blood becomes anti coagulated because of nafamostat’ s rapid inhibition of thrombin. Nafamostat’ s small molecular weight allows a significant proportion of the agent to then be removed via filtration and the reduced amount of nafamostat that is returned to the patient’s circulation will be rapidly metabolized. Accordingly, when used as a regional anticoagulant, the anti coagulation activity of nafamostat is primarily limited to a regional effect within the extracorporeal circuit.

[0052] The anti coagulation effect provided by the nafamostat anti coagulation agent can be readily assessed using standard methods to measure blood coagulation parameters in a blood sample, such as by measuring one or any combination of the activated clotting time (ACT), the activated partial thromboplastin time (aPTT), the prothrombin time (PT), the international normalized ratio (PT / INR), or the thromboelastography (TEG) of that sample.

[0053] The nafamostat regional anticoagulant agent is then used in combination with a selected extracorporeal circuit filter membrane. In the practice of the methods of the present disclosure, nafamostat can be used with a membrane that is formed from a polyacrylonitrile(PAN) polymeric material such as AN69 or oXiris, referred to herein as a “PAN filter”, or a membrane formed from a non-PAN polymeric material such as polysulfone, polyethersulfone or polyarylethersulfone, referred to herein as a “non-PAN filter”. Alternatively, the filter membrane may be formed from a blended material containing PAN and a non-PAN polymer or copolymer component including, for example, a non-PAN filter membrane material that is coated with a PAN component (e.g., AN69ST). For clarity, if the final membrane material contains any PAN component, such filter constitutes a PAN filter. Examples of non-PAN filters that may be used in the conduct of the present methods include the Fresenius F50-F80, NxStage CAR-125 / 502 / 505 polyethersulfone (PES) filters, and Baxter HF 1000 / 1400 non-PAN filters. Examples of PAN filters that may be used in the conduct of the present methods include the Baxter M60, Ml 00 and Ml 50 PAN filters.

[0054] The selection of either a PAN filter or a non-PAN filter then requires the selection of novel operational parameters for the extracorporeal circuit as set forth in the present methods that increase both the safety and efficiency of the blood treatment procedure being employed in such methods. Not being bound by any particular theory, the methods of the present disclosure are designed to quickly establish and then maintain a narrow window of regional anti coagulation with the nafamostat agent in the extracorporeal circuit during the blood treatment procedure, wherein such anti coagulation window is effective to prevent or reduce clotting in the filter membrane while at the same time not giving rise to significant systemic anti coagulation effects in the patient. In the present methods that employ a PAN filter, the PAN filter is primed with a standard priming solution (a priming solution that does not contain any nafamostat anticoagulant agent). Upon initiation of the blood treatment procedure, the nafamostat agent is then infused at a higher infusion rate as compared to methods employing a non-PAN filter. This is because PAN filters adsorb a high amount of nafamostat, almost to the point where PAN filters are considered unsaturable by nafamostat. Accordingly, there is no benefit to priming the filter with nafamostat but rather by selection of a higher initial infusion rate than with non-PAN filters, there will be sufficient nafamostat anticoagulant agent in the blood running through the filter during the treatment procedure to avoid clotting, even though a significant amount of that agent will be adsorbed to the filter. This technique represents a significant departure from the prior art, wherein the use of PAN filters is contra-indicated on the Futhan® label, the only approved nafamostat anti coagulation agent (approved in Japan and South Korea). In the present methods that employ a non-PAN filter, the non-PAN filter is bolus primed with a high dose of the nafamostat agent to quickly establish regional anti coagulation within the extracorporeal circuit prior to initiation of the blood treatment procedure. After the bolus priming step has been completed, the blood treatment procedure is initiated using a low initial infusion rate in order to maintain the regionalanti coagulation within the prescribed narrow anti coagulation window. Here again, this technique represents a significant departure from the prior art, wherein the Futhan® label directs that priming and the initial infusion rate should be established and then maintained using identical infusion rates (20 mg / hr priming infusion rate, followed by 20 mg / hr initial infusion rate). A representation of the differences between the Futhan® priming / infusion technique and the technique of the present methods is set forth in Figure 2. As can be seen, use of the high bolus prime / low initial infusion rate technique of the present methods quickly establishes an effective steady state regional anti coagulation that then remains within the narrow anti coagulation window depicted between the dashed lines during the blood treatment procedure, whereas practice of the Futhan® technique gives rise to a slow onset of anti coagulation with a low priming dose and a constantly increasing degree of anti coagulation with the higher intital rate that can exceed the narrow anti coagulation window during conduct of the blood treatment procedure.

[0055] Throughout conduct of the methods of the present disclosure, the degree of regional anti coagulation provided by the nafamostat agent can be monitored by measuring the post-filter activated clotting time (ACT) from blood samples taken immediately downstream of the circuit filter. Such post-filter samples represent the regional anticoagulation in the filter itself. The infusion rate of the nafamostat agent can then be easily titrated down or up in order to maintain regional anti coagulation within a preselected narrow anti coagulation window. In certain aspects of the present disclosure, the anticoagulation window can extend between about 175 to about 225 seconds. In addition, the safety of the methods of the present disclosure can be monitored by comparing the measured post-filter ACT against the patient’s systemic ACT to see if there has been any significant increase in such clotting time, which would indicate that there is an unwanted systemic anti coagulation effect provided by the nafamostat anti coagulation agent.

[0056] Accordingly, it is a primary aspect of the present disclosure to provide a method for providing regional anti coagulation in an extracorporeal circuit during a blood treatment procedure, wherein the circuit has a filter component that comprises a polyacrylonitrile (PAN) material, i.e., a PAN filter. The method entails initiating the procedure in a subject whose bloodstream is connected to the circuit without first performing an initial anticoagulant priming step as normally directed. The nafamostat anticoagulant agent is then introduced into the circuit at a high initial infusion rate upon initiation of the procedure to quickly provide an anticoagulant effect within the circuit, that is, to provide regional anti coagulation. In certain preferred aspects of the disclosure, the initial infusion rate is about 35 mg nafamostat per hour. In other preferred aspects, the initial infusion rate is about 15 mg nafamostat per hour, about 20 mg / hr, about 25 mg / hr or about 30 mg / hr. In other aspects, the initial infusion rate is about 40 mg nafamostat per hour, about 45 mg / hr or about 50 mg / hour or greater.

[0057] The nafamostat anti coagulation agent used in the practice of the present methods can be provided in any pharmaceutically acceptable form including as a free base or in the form of any pharmaceutically acceptable salt of nafamostat such as the mesylate salt of nafamostat. The blood treatment procedure can be carried out over any medically significant duration, for example wherein the nafamostat agent is infused into the extracorporeal circuit for at least about 1 hour, or at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours or longer. The PAN filter used in the practice of the present methods can consist entirely of a polyacrylonitrile filter material or can merely just comprise a polyacrylonitrile filter material component.

[0058] In certain preferred aspects of the present disclosure, the blood treatment procedure is a dialysis procedure. In other aspects, the dialysis procedure is a continuous renal replacement therapy (CRRT), such as a CRRT procedure that comprises hemofiltration, hemodialysis or hemodiafiltration.

[0059] During conduct of the blood treatment pursuant to the present methods, effective regional anti coagulation can be assessed by measuring post-filter activated clotting time (ACT) from a blood sample taken immediately downstream of the circuit filter. Effective anti coagulation can then be determined by comparing the measured clotting time against the patient’s baseline systemic ACT to determine if there has been an increase in clotting time. Such post-filter sampling can be carried out over any selected frequency, for example with samples taken at about 15 minutes after initiation of the infusion period, at about 1 hour or about 4, 16, 24, 48, 72, 96 hours or greater after initiation of the infusion period, or any combination or derivation thereof. In addition, effective regional anti coagulation can be assessed by also measuring the patient’s systemic ACT and then comparing the measured systemic clotting time against the measured post-filter clotting time at various selected timepoints, wherein a post-filter ACT that is higher than the subject’s systemic ACT represents effective regional anti coagulation without concomitant excessive systemic anti coagulation. Such measurement timepoints can comprise measurements taken at about 24, 48, 72, 96 hours or greater after initiation of the infusion period, or any combination or derivation thereof.

[0060] In addition to the above-described procedure assessments conducted during the practice of the present methods, the overall efficacy of regional anti coagulation provided during the blood treatment procedure can be monitored using indirect measures such as monitoring the effective lifespan of the extracorporeal circuit filter as compared against the same procedure conducted without use of the anticoagulant agent. Such indicia include, but are not limited to, tracking the filter life during conduct of the blood treatment procedure, wherein an increased lifespan of at least about 10% or 15% would demonstrate increased efficiency due to theeffective regional anti coagulation provided by the nafamostat agent. In addition, in those methods wherein the blood treatment procedure is a dialysis procedure, the methods could further entail the step of measuring dialysis efficiency using a time-averaged concentration of blood urea nitrogen (TACurea) test, wherein a lowered time-averaged concentration of blood urea nitrogen at about 24 hours after initiation of the procedure would demonstrate an enhanced dialysis efficiency as compared against the same procedure performed without the use of the nafamostat anticoagulant agent. Alternatively or additionally, dialysis efficiency can be assessed using a further step of measuring a time-averaged concentration of plasma B2 microglobulin, wherein a lowered time-averaged concentration of plasma B2 microglobulin at about 24 hours after initiation of the procedure would also demonstrate an enhanced dialysis efficiency as compared against the same procedure performed without the use of the nafamostat anticoagulant agent.

[0061] It is a further primary aspect of the present disclosure to provide a method for providing regional anti coagulation in an extracorporeal circuit, where the circuit has a filter component that does not comprise a polyacrylonitrile material (a non-PAN filter). The method entails an initial high bolus to prime the non-PAN filter prior to initiation of a blood treatment procedure by administering a large bolus of an anticoagulant agent comprising nafamostat to the circuit to quickly establish a regional anti coagulation effect in the circuit. After the bolus priming step is carried out, the regional anti coagulation is maintained in the circuit by infusing the nafamostat anticoagulant agent to the circuit at a lower infusion rate. In certain preferred aspects of the disclosure, the amount of nafamostat in the priming bolus is at least about 50 mg. In other preferred aspects, the amount of nafamostat in the priming bolus is at least about 25 mg, 30 mg, 35 mg, 40 mg or 45 mg. In other aspects, the amount of nafamostat in the priming bolus is at least about 55 mg or 60 mg. In certain other preferred aspects of the disclosure, the nafamostat anti coagulation agent is infused at a low initial infusion rate of about 15 mg or less, or about 14 mg, 13 mg, 12 mg, 11 mg or 10 mg or less in order to maintain regional anticoagulation. The maintenance infusion of the nafamostat anticoagulant agent can be carried out for at least about 1 hour, 24, 48 hours or more during the procedure.

[0062] The nafamostat anti coagulation agent used in the practice of the present methods can be provided in any pharmaceutically acceptable form including as a free base or in the form of any pharmaceutically acceptable salt of nafamostat such as the mesylate salt of nafamostat. The blood treatment procedure can be carried out over any medically significant duration, for example wherein the nafamostat agent is infused into the extracorporeal circuit to maintain regional anti coagulation for at least about 1 hour, or at least about 24 hours, at least about 48 hours, at least about 72 hours, at least about 96 hours or longer. The non-PAN filter used in the practice of the present methods does not contain any polyacrylonitrile filter material.

[0063] In certain preferred aspects of the present disclosure, the blood treatment procedure is a dialysis procedure. In other aspects, the dialysis procedure is a continuous renal replacement therapy (CRRT), such as a CRRT procedure that comprises hemofiltration, hemodialysis or hemodiafiltration.

[0064] During conduct of the blood treatment pursuant to the present methods, effective regional anti coagulation can be assessed by measuring post-filter activated clotting time (ACT) from a blood sample taken immediately downstream of the circuit filter. Effective anti coagulation can then be determined by comparing the measured clotting time against the patient’s baseline systemic ACT to determine if there has been an increase in clotting time. Such post-filter sampling can be carried out over any selected frequency, for example with samples taken at about 15 minutes after initiation of the infusion period, at about 1 hour or about 4, 16, 24, 48, 72, 96 hours or greater after initiation of the infusion period, or any combination or derivation thereof. In addition, effective regional anti coagulation can be assessed by also measuring the patient’s systemic ACT and then comparing the measured systemic clotting time against the measured post-filter clotting time at various selected timepoints, wherein a post-filter ACT that is higher than the subject’s systemic ACT represents effective regional anti coagulation without concomitant excessive systemic anti coagulation. Such measurement timepoints can comprise measurements taken at about 24, 48, 72, 96 hours or greater after initiation of the infusion period.

[0065] In addition to the above-described procedure assessments conducted during the practice of the present methods, the overall efficacy of regional anti coagulation provided during the blood treatment procedure can be monitored using indirect measures such as monitoring the effective lifespan of the extracorporeal circuit filter as compared against the same procedure conducted without use of the anticoagulant agent. Such indicia include, but are not limited to, tracking the filter life during conduct of the blood treatment procedure, wherein an increased lifespan of at least about 10% or 15% would demonstrate increased efficiency due to the effective regional anti coagulation provided by the nafamostat agent. In addition, in those methods wherein the blood treatment procedure is a dialysis procedure, the methods could further entail the step of measuring dialysis efficiency using a time-averaged concentration of blood urea nitrogen (TACurea) test, wherein a lowered time-averaged concentration of blood urea nitrogen at about 24 hours after initiation of the procedure would demonstrate an enhanced dialysis efficiency as compared against the same procedure performed without the use of the nafamostat anticoagulant agent. Alternatively or additionally, dialysis efficiency can be assessed using a further step of measuring a time-averaged concentration of plasma 132 microglobulin, wherein a lowered time-averaged concentration of plasma 132 microglobulin at about 24 hours afterinitiation of the procedure would also demonstrate an enhanced dialysis efficiency as compared against the same procedure performed without the use of the nafamostat anticoagulant agent.

[0066] It is to be understood that the above description is intended to be illustrative and not restrictive. Many other aspects will be apparent to those of skill in the art upon reading the above description. It should be noted that specific aspects discussed in different portions of the description and / or referred to in the figures can be combined to form additional aspects of the present disclosure. The scope of the disclosure should, therefore, be determined with reference to the appended claims, along with the full scope of equivalents to which such claims are entitled. All publications, patents and patent documents are incorporated by reference herein, as though individually set forth herein in their entirety.EXAMPLES

[0067] The disclosure is illustrated herein by the experiments described by the following examples, which should not be construed as limiting. Those skilled in the art will understand that this disclosure may be embodied in many different forms and should not be construed as limited to the aspects set forth herein. Rather, these examples are provided so that this disclosure will fully convey the disclosure to those skilled in the art. Many modifications and other aspects of the disclosure will come to mind in one skilled in the art to which this disclosure pertains having the benefit of the teachings presented in the foregoing description. Although specific terms are employed, they are used as in the art unless otherwise indicated.Example 1:

[0068] The purpose of this Example 1 is to conduct a randomized, placebo-controlled, double-blind, multi-center clinical study to assess the efficacy of various combinations of a nafamostat regional anti coagulation agent in a CRRT circuit using either a PAN or a non-PAN circuit membrane filter element. In the study, patients undergoing CRRT who cannot tolerate heparin or are at a higher risk for bleeding are evaluated to measure the anti coagulation efficacy of each nafamostat / circuit membrane filter combination. The efficacy endpoint for the study is the mean post-filter activated clotting time (ACT) composed of measurements made at 1, 4, 16, and 24 hours post-CRRT initiation for an active anticoagulant agent (nafamostat) group as compared to the mean ACT composed of measurements made at 1, 4, 16, and 24 hours post- CRRT initiation for a placebo (saline) group. These same measurements taken at 48, 72, 96 or more hours post-CRRT initiation can further be used to assess the durability of nafamostat’ s regional anti coagulation effect in the CRRT circuit.

[0069] Study Design. This prospective, randomized, placebo-controlled clinical study will investigate the safety and efficacy of nafamostat mesylate for anti coagulation of extracorporeal blood circulating through a dialysis filter in patients undergoing CRRT who cannot tolerate heparin or are at higher risk for bleeding. The study will aim to enroll 166 patients undergoing CRRT in a 1: 1 randomization allocation to active (nafamostat) treatment or placebo (saline) for up to 7 days. Eligible patients will receive either the (active) regional anticoagulant nafamostat or a saline (placebo) infusion at the initiation of their CRRT treatment. Patients will be stratified by filter type (non-polyacrylonitrile (PAN) filter or PAN filter). Study treatment will initiate upon the first CRRT cycle for a patient or, if the patient is already undergoing CRRT, study treatment will initiate upon a filter change, provided however, that such patient began CRRT within the past 48 hours. Eligible subjects must be receiving a target effluent flow rate of at least 20 mL / kg of body weight per hour for the first 24 hours of the study and a CRRT blood flow rate of 100 - 250 mL / min. It is expected that the use of the nafamostat regional anticoagulant agent will result in increased post-filter mean activated clotting time (ACT) as compared to placebo. The overall dialysis efficacy of the CRRT procedure will also be assessed as measured by the time-averaged concentration of patient blood urea nitrogen in the first 24 hours of treatment, with samples being taken at 1, 4, 16 and 24 hours post initiation of the procedure.

[0070] Inclusion Criteria. In order to be eligible to participate in the study, an individual must meet all of the following criteria: (1) patients are 18 to 80 years of age; (2) patients require CRRT or are undergoing CRRT initiated within the prior 48 hours; (3) patients who cannot tolerate heparin or are at high-risk of bleeding due to any one of the following as assessed against normal values ranges per local laboratory: elevated activated partial thromboplastin time (aPTT), elevated international normalized ratio (INR), thrombocytopenia (less than 150,000 / pL), history of intra-cranial hemorrhage, major surgery within the prior 7 days, poly-trauma within the prior 7 days, history of gastrointestinal bleeding, and / or history of pulmonary hemorrhage; and (4) patient has a baseline ACT < 150 seconds.

[0071] Exclusion Criteria. An individual who meets any of the following criteria will be excluded from participation in the study: (1) patients weighing less than 50 kg; (2) patients receiving systemic anticoagulation; (3) patients with intra-cranial hemorrhage within the past 7 days; (4) patients with severe thrombocytopenia (less than 25,000 / pL); (5) patients with active bleeding; (6) patients with a prior allergic reaction to nafamostat mesylate; (7) patients prescribed a target effluent flow rate of less than 20 mL / kg of body weight per hour; and (8) patients receiving a prohibited medication (metformin, procainamide, topotecan, estrone sulphate, acyclovir, ganciclovir, oxaliplatin, cephalexin, cephradine, oxazolidnones, fexofenadine, cimetidine, and pyrmethamine) currently or within the 24 hours prior to screening.

[0072] Randomization and Blinding. Up to 166 eligible patients will be randomly assigned in a 1 : 1 ratio to either Active (nafamostat treatment) or Placebo (saline) over up to 10 independent clinical sites. At the time of randomization patients will be stratified by filter type (non-PAN filter versus PAN filter). The assignment to Active or Placebo will be performed randomly. The randomization schedule will be generated using a validated randomization program and verified for accuracy using strict quality control procedures. Assignment of the randomization number and treatment assignment will be centrally coordinated through the study’s interactive web randomization system (IWRS). Randomization numbers will be assigned by the IWRS. All study site personnel, with the exception of the unblinded pharmacist(s), will remain blinded through to study intervention assignment. Once prepared, all properties of the intervention and delivery system will be identical to maintain the blind. In order to enhance the blinding, only the study coordinator and site investigator will know the post-filter ACT values and make the corresponding dose adjustments. Therefore, the results of the post-filter ACT will not be charted and will only be shared with the clinical team if there is a safety concern.Assessment of the relatedness of an adverse event to the experimental treatment will be made by a blinded observer (e.g., co-investigator or attending physician) and not the site investigator who is responsible for adjusting the dose to a target ACT.

[0073] Materials and Methods. All preparation of priming and study treatment infusion solutions with either active anticoagulant agent or placebo will be performed by an unblinded pharmacist. Study personnel will then carry-out the priming of the CRRT machine and titration of study treatment infusion in a blinded manner. Baxter Viaflex 1000 mL saline bags (for priming) and 250 mL saline bags (for study treatment infusion) will be used for the infusion solutions. All CRRT devices and products used for the CRRT procedure (e.g., dialysate, replacement fluids) will be FDA cleared / approved. Priming of the filter and titration will occur in a membrane-specific manner as described below. Eligible patients will be prescribed a target effluent flow rate of at least 20 mL / kg of body weight per hour for the first 24 hours of the study and a CRRT blood flow rate of 100 - 250 mL / min. The experimental conditions for the study involve infusing an anticoagulant directly into the dialysis circuit and measuring the level of anti coagulation immediately post filter. The control arm will receive a saline placebo infusion in lieu of the anticoagulant.

[0074] The study anticoagulant agent is supplied in a 50 mg vial as a sterile lyophilized white crystalline powder composed of nafamostat mesylate with succinic acid and D-mannitol. The details for the study anticoagulant agent are as set forth in Table 1 below.Table 1 Study Anticoagulant Composition and Description.

[0075] The placebo control group consists of 0.9% sodium chloride.

[0076] The CRRT circuit will be primed according to the type of filter (PAN or non- PAN), the effective surface area of the filter and in accordance with the filter manufacturer and CRRT extracorporeal circuit manufacturer specifications. Accordingly, the priming step may be carried out over a period of from about 0 to about 45 minutes prior to initiation and titration of the treatment infusion. Priming the circuit when non-PAN filters are used will avoid premature coagulation of blood during CRRT. Non-PAN filters typically bind nafamostat in a saturable manner, therefore sufficient priming of the circuit allows these binding sites to be saturated prior to study infusion. Examples of non-PAN filters that may be used in the conduct of the study include the Fresenius F50-F80, NxStage CAR-125 / 502 / 505 polyethersulfone (PES) filters, and Baxter HF 1000 / 1400 non-PAN filters. PAN filters bind nafamostat to a much higher degree such that they are unsaturable. Therefore, PAN filters will be primed with standard priming solution (without the nafamostat anticoagulant). To account for the high degree of nafamostat binding to PAN filters, the study treatment infusion rate will be run at a higher rate than for non- PAN filters as set forth below. Examples of PAN filters used in the conduct of the study include the Baxter M60, Ml 00 and Ml 50 PAN filters.

[0077] The priming solution for non-PAN filters in patients randomized to the active treatment (nafamostat anticoagulant) group will be reconstituted in the vial using 5 mL of water for injection or 5% dextrose for injection. Following reconstitution, 5 mL of the reconstituted solution (50 mg nafamostat mesylate) will be injected into each Baxter Viaflex 0.9% sodium chloride 1000 mL bag that is needed for priming, with the priming volume (e.g., IL or 2L) being based on the specific CRRT filter size being used. The priming solution for all of the randomized PAN filter patients and for the entire randomized placebo (saline) group patients will consist of Baxter Viaflex 0.9% sodium chloride 1000 mL bags without addition of any anticoagulant into the solution.

[0078] The nafamostat anti coagulation agent infusion is prepared by first reconstituting 5 vials (using 5 mL of water for injection or 5% dextrose for injection for each vial) and then withdrawing 5 mL of the reconstituted solution from each vial for a total of 25 mL (250 mg of nafamostat mesylate). 25 mL of the saline solution is then withdrawn from a Baxter Viaflex 0.9% sodium chloride 250 mL bag and discarded, and the 25 mL of the reconstituted nafamostat that was withdrawn from the five reconstituted vials is added to the remaining 225 mL of the 0.9% saline in the 250 mL bag. The resulting solution will contain 250 mg of nafamostat mesylate in 250 mL saline for a final concentration of 1 mg / mL nafamostat mesylate. The placebo (saline) infusion will consist of a Baxter Viaflex 0.9% sodium chloride 250 mL bag.

[0079] Initiation of CRRT and Titration of Study Treatment Infusion. Following priming of the CRRT circuit with the appropriate priming solution as described above, the study treatment infusion is initiated using an infusion pump to introduce the infusion solution into the circuit using a standard anticoagulant infusion site. Infusion is initiated immediately prior to the start of blood flow through the system at the flow rate as set forth in Table 2 below.Table 2. Study Treatment Initial Infusion Rate Based on Filter Type.

[0080] After initiation of the study treatment infusion into the extracorporeal pre-filter circuit, patient activated clotting time (ACT) is measured in the post-filter circuit at 15 minutes. Based upon the measured 15-minute ACT value, the infusion rate of the study treatment (Active (nafamostat) or Placebo (saline)) can be titrated up or down as needed (up to a maximum of 50 mL / hr) to achieve a post-filter ACT of between about 175 and about 225 seconds. Such titration of the study treatment infusion is based upon the titration guideline set forth in Table 3 below.Table 3. Titration of Study Treatment Infusion based on Measured ACT Values.* Maximum infusion rate is 50 mL / hr.

[0081] Following priming of the CRRT circuit and initiation of the study treatment infusion and subsequent titration (as needed) as described above, the CRRT procedure under the study will be carried out for at least 3 days (completer population) and may be continued for up to 7 days or until the end of such CRRT procedure, whichever occurs first. If the study infusion treatment is interrupted during CRRT for an arising medical procedure, the treatment can resume after such medical procedure has been adequately addressed and / or completed.

[0082] Patient Baseline Measurements. Prior to receiving any treatment under the study, patients will be screened to establish baseline values as follows. Patient systemic ACT should be measured no more than 12 hours prior to randomization. ACT at screening and pre-dose will be measured by iSTAT Celite method from systemic circulation according to the manufacturer’s specifications and instructions. The liver function screening panel should be collected no more than 24 hours prior to randomization and include measurement of total bilirubin, alanine transaminase (ALT), aspartate transaminase (AST), AST / ALT ratio, alkaline phosphatase (ALP), albumin, total protein and lactate dehydrogenase (LD). Hematology screening samples should be collected no more than 24 hours prior to randomization and include measurement of hematocrit, hemoglobin, platelet count, red blood cell count and white blood cell count with differential. The clinical serum chemistry screening panel should be collected no more than 24 hours prior to randomization and include measurement of creatinine, serum urea, chloride, bicarbonate, sodium, potassium, calcium, phosphorus, magnesium and glucose. Coagulation parameters other than ACT will include establishment of international normalized ratio (INR), prothrombin time (PT) and activated thromboplastin time (aPTT).

[0083] Study Assessments and Measurements. Patient ACT from post-filter samples will be measured by iSTAT Celite method during the infusion period at 15 minutes, 1, 4, 16 and 24 hours, and then at 48, 72, 96, 120, 144 and 168 hours (if relevant). In addition, patient systemic circulation ACT will be measured during the infusion period at 24 hours, and then daily thereafter (if relevant) to assess potential systemic effect. Patient aPTT from post-filter samples will be measured at 1, 4, 16 and 24 hours. Time-averaged concentration (TAC) of blood urea (TACurea) and TAC of plasma 132 microglobulin will be determined at 1, 4, 16 and 24 hours. Patient clinical serum chemistry will be measured daily throughout the study. CRRT filter parameters will be assessed prior to patient randomization, at initiation of the infusion period and then monitored throughout the infusion period. These parameters will include access pressures, filter pressure, effluent pressure, return pressure and transmembrane pressure (TMP). In addition, the number of filters changed due to clotting and average filter lifespan (in hours) due to clotting during the CRRT procedure will be tracked to monitor anti coagulation efficacy. Any events such as visual appearance of filter clots, visible clots in the circuit that obstruct blood flow, bloodpump inability to rotate due to obstruction by clot in the membrane or circuit, or CRRT machine system clotted off will also be tracked, and the number of blood transfusions within each group in the first 72 hours of infusion will be determined.

[0084] Study Results. The primary efficacy endpoint for the study will be the mean postfilter ACT comprised of measurements made at 1, 4, 16 and 24 hours post initiation of the treatment for the Active (nafamostat) group as compared against the mean post-filter ACT comprised of measurements made at 1, 4, 16 and 24 hours post initiation of the treatment for the Placebo (saline) group. The null and alternative hypotheses are given by:Where / zfis the mean ACT for the Active treatment groupis the mean ACT for the placebo group.

[0085] The secondary efficacy endpoints for the study will comprise: the mean post-filter ACT from measurements taken during the first 72 hours of treatment (infusion); the average number of filters changed due to clotting in each group within the first 72 hours of treatment; the average filter lifespan (in hours) due to clotting by filter type over the first 72 hours of treatment in each group; the average number of transfusions in the first 72 hours of treatment in each group; and dialysis efficacy in each group as measured by the time-averaged concentration (TAC) of blood urea nitrogen in the first 24 hours. The null and alternative hypotheses for the each of the secondary efficacy endpoints are:(1) Mean post-filter ACT composed of measurements made in the first 72 hours:where / zfis the mean ACT for the Active group andis the mean ACT for the Placebo group;(2) Average number of filters changed due to clotting in each group in the first 72 hours:where / zfis the mean number of filters changed in the first 72 hours for the Active group and is the mean number of filters changed in the first 72 hours for the Placebo group;(3) Average filter lifespan (in hours) due to clotting by filter type over first 72 hours:where / zfis the mean filter lifespan (in hours) due to clotting by filter type over first 72 hours for the Active group and is the mean filter lifespan (in hours) due to clotting by filter type overfirst 72 hours for the Placebo group;(4) Average number of transfusions in the first 72 hours:where / zfis the mean number transfusions in the first 72 hours for the Active group andis the mean number of transfusions in the first 72 hours for the Placebo group; and(5) Dialysis efficacy as measured by the time-averaged concentration (TAC) of blood urea nitrogen in the first 24 hours:where / zfis the mean TAC of urea in the first 24 hours for the Active groupis the mean TAC of urea in the first 24 hours for the Placebo group.Example 2:

[0086] The purpose of this Example 2 is to conduct a randomized, placebo-controlled, double-blind, multi-center clinical study to assess the metabolism of and systemic exposure to nafamostat in patients resulting from the use of various combinations of a nafamostat regional anti coagulation agent in a CRRT circuit paired with either a PAN or a non-PAN circuit membrane filter element, as well as any potential systemic pharmacological anti coagulation effect thereof as measured by ACT in those patients. In the study, patients undergoing CRRT who cannot tolerate heparin or are at a higher risk for bleeding are evaluated to assess the metabolism of and systemic exposure to nafamostat by measuring the concentration of nafamostat and its inactive metabolites (6-amidino-2 -naphthol (6-AN) and 4-guanidinobenzoic acid (4-GBA)) in systemic blood samples from both Active (regional nafamostat anti coagulation) and Placebo (saline) groups taken during study treatment infusion. The study will further assess the effect of the selected filter type (PAN or non-PAN) on regional dosing of the nafamostat anticoagulant that may be required to maintain a patient’s systemic ACT within a particular desired range.

[0087] In order to conduct this study, the clinical trial study of Example 1 is carried out exactly as described above.

[0088] Additional Study Assessments and Measurements. In addition to the study assessments and measurements obtained in accordance with the Example 1 study protocol above, blood samples (4 mL per sample) will be obtained from systemic circulation at 24, 48, and 72 hours after the start of study treatment (infusion) for up to 60 patients in the study (up to 30 takenfrom the Active (nafamostat regional anticoagulant) treatment group and up to 30 taken from the Placebo (saline) treatment group). A validated LC / MS / MS method will then be used to analyze the systemic blood sample for: nafamostat mesylate with lower limit of quantitation of 0.50 ng / mL; the 6-AN nafamostat metabolite with a lower limit of quantitation of 1.0 ng / mL; and the 4-GBA nafamostat metabolite with a lower limit of quantitation of 1.0 ng / mL. The summary of these measurements will then be used to determine systemic exposure to the regionally administered nafamostat anticoagulant.

[0089] Patient ACT from post-filter samples will be measured by iSTAT Celite method during the infusion period at 15 minutes, 1, 4, 16 and 24 hours, and then at 48, 72, 96, 120, 144 and 168 hours (if relevant). In addition, patient systemic circulation ACT will be measured during the infusion period at 24 hours, and then daily thereafter (if relevant) to compare postfilter ACT with systemic ACT, and these results will be further assessed to determine the effect of the selected filter type (PAN or non-PAN) of differences in the average daily dose of regional nafamostat anticoagulant as may be required to maintain a patient’s systemic ACT within a particular desired range.Example 3:

[0090] The purpose of this Example 3 is to conduct a randomized, placebo-controlled, double-blind, multi-center clinical study to assess the safety of using a regional anticoagulant versus not using any regional anticoagulant in CRRT procedures for patients as further differentiated by employing various combinations of the experimental regional anti coagulation agent, nafamostat (or placebo agent, saline) in a CRRT circuit when paired with either a PAN or a non-PAN circuit membrane filter element. In the study, patients undergoing CRRT who cannot tolerate heparin or are at a higher risk for bleeding are evaluated to assess the safety profile of using nafamostat as a regional anticoagulant in the circuit across a number of different commonly used CRRT filter materials the use of which the ordinarily skilled person would expect to encounter in ICU settings worldwide. Accordingly, the proportion of patients experiencing adverse events of special interest (AESIs) including bleeding complications, hypersensitivity events such as anaphylaxis (as assessed using Sampson’s criteria (Sampson et al. (2006) J Allergy Clin Immunol 117(2):391-397), hyponatremia (< 130 mEq / L), hyperkalemia (> 5.5 mEq / L), decreased white blood count (< 3.5 x 109 / L), hepatic function disorder (jaundice with increased aspartate aminotransferase (AST / GOT), alanine aminotransferase (ALT / SGPT) and gamma-glutamyl transpeptidase (y — GTP)), or study-related need for blood transfusions (i.e., packed red blood cell for Hgb < 7 or > 7 with evidence of acute bleeding; patient platelet level decrease below 20,000 / mL or below 50,000 / mL with evidence of acute bleeding; or prothrombin time decreases below 70% with evidence of bleeding or suspected disseminated intravascularcoagulation) are tracked and assessed in this study, and the results from the Active and Placebo groups compared.

[0091] In order to conduct this study, the clinical trial study of Example 1 is carried out exactly as described above.

[0092] Additional Study Assessments and Measurements. In addition to the study assessments and measurements obtained in accordance with the Example 1 study protocol above, incidence of AESIs will be tracked and assessed, as well as measurements taken from the laboratory values (hematology, clinical chemistry, liver function panel and coagulation parameters) obtained throughout the CRRT procedure.

Claims

CLAIMS:

1. A method of providing regional anti coagulation in an extracorporeal circuit during a blood treatment procedure, wherein the circuit comprises a filter comprising a polyacrylonitrile (PAN) material and said method comprises: initiating the procedure in a subject whose bloodstream is connected to the circuit without conducting any anticoagulant priming step in the circuit; and introducing and then administering an anticoagulant comprising nafamostat into the circuit to provide regional anti coagulation, wherein said anticoagulant is introduced and administered into the circuit at an initial infusion rate of at least about 15 mg nafamostat per hour.

2. The method of claim 1, wherein the anticoagulant is infused at an initial rate of at least about 20 mg nafamostat per hour.

3. The method of claim 2, wherein the anticoagulant is infused at an initial rate of at least about 25 mg nafamostat per hour.

4. The method of claim 3, wherein the anticoagulant is infused at an initial rate of at least about 30 mg nafamostat per hour.

5. The method of claim 4, wherein the anticoagulant is infused at an initial rate of at least about 35 mg nafamostat per hour.

6. The method of claim 5, wherein the anticoagulant is infused at an initial rate of at least about 40 mg nafamostat per hour.

7. The method of claim 6, wherein the anticoagulant is infused at an initial rate of at least about 50 mg nafamostat per hour.

8. The method of any one of claims 1-7, wherein the anticoagulant is infused into the circuit for at least about 1 hour.

9. The method of claim 8, wherein the anticoagulant is infused into the circuit for at least about 24 hours.

10. The method of claim 9, wherein the anticoagulant is infused into the circuit for at least about 48 hours.

11. The method of claim 10, wherein the anticoagulant is infused into the circuit for at least about 72 hours.

12. The method of claim 11, wherein the anticoagulant is infused into the circuit for at least about 96 hours.

13. The method of any one of claims 1 to 12, wherein the anticoagulant comprises the mesylate salt of nafamostat.

14. The method of any one of claims 1 to 13, wherein the blood treatment procedure is a continuous renal replacement therapy (CRRT).

15. The method of claim 14, wherein the CRRT procedure comprises hemofiltration, hemodialysis or hemodiafiltration.

16. The method of any one of claims 1 to 15, wherein the PAN filter consists of a polyacrylonitrile filter material.

17. The method of any one of claims 1 to 16 further comprising the step of measuring postfilter activated clotting time (ACT), wherein said post-filter ACT measured at twenty -four hours after initiation of the procedure is increased relative to the subject’s baseline systemic ACT.

18. The method of claim 17, wherein said post-filter ACT measured at seventy -two hours after initiation of the procedure is increased relative to the subject’s baseline systemic ACT.

19. The method of any one of claims 1 to 18 further comprising the step of measuring the subject’s systemic ACT, wherein the subject’s systemic ACT measured at twenty -four hours after initiation of the procedure has not significantly increased from the subject’s baseline ACT.

20. The method of any one of claims 17 to 19 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at twenty -four hours after initiation of the procedure.

21. The method of any one of claims 17 to 19 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at forty-eight hours after initiation of the procedure.

22. The method of any one of claims 17 to 19 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at seventy-two hours after initiation of the procedure.

23. The method of any one of claims 1 to 22, wherein the effective lifespan of the filter is increased by at least about 10% as compared to the lifespan of a filter used in the same procedure performed without the use of an anticoagulant.

24. The method of any one of claims 1 to 23, wherein the effective lifespan of the filter is increased by at least about 15% as compared to the lifespan of a filter used in the same procedure performed without the use of an anticoagulant.

25. The method of any one of claims 1 to 24 wherein the blood treatment procedure is a dialysis procedure and the method further comprises the step of measuring the dialysis efficiency of the procedure using a time-averaged concentration of blood urea nitrogen (TACurea) test, wherein the subject’s time-averaged concentration of blood urea nitrogen measured at twenty- four hours after initiation of the procedure is lower than when using same procedure performed without the use of an anticoagulant.

26. The method of any one of claims 1 to 25 wherein the blood treatment procedure is a dialysis procedure and the method further comprises the step of measuring the dialysis efficiency of the procedure using a time-averaged concentration of plasma 132 microglobulin test, wherein the subject’s time-averaged concentration of plasma 132 microglobulin measured at twenty -four hours after initiation of the procedure is lower than when using same procedure performed without the use of an anticoagulant.

27. A method of providing regional anti coagulation in an extracorporeal circuit wherein the circuit comprises a filter that does not comprise a polyacrylonitrile material (non-PAN), said method comprising: bolus priming the non-PAN circuit filter prior to initiating a blood treatment procedure in a subject whose bloodstream is connected to the circuit by administration of a bolus of an anticoagulant comprising at least about 25 mg of a nafamostat anticoagulant agent to the circuit; and after bolus priming the circuit, establishing regional anti coagulation in the circuit by administering the anticoagulant at a low infusion rate.

28. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 30 mg nafamostat.

29. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 35 mg nafamostat.

30. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 40 mg nafamostat.

31. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 45 mg nafamostat.

32. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 50 mg nafamostat.

33. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 55 mg nafamostat.

34. The method of claim 27, wherein the amount of anticoagulant administered during the bolus priming step comprises at least about 60 mg nafamostat.

35. The method of any one of claims 27 to 34, wherein the anticoagulant is administered during the procedure at an initial infusion rate sufficient to provide about 15 mg or less nafamostat per hour to the circuit.

36. The method of claim 42, wherein the anticoagulant is administered during the procedure at an initial infusion rate sufficient to provide about 10 mg or less nafamostat per hour to the circuit.

37. The method of any one of claims 27 to 36, wherein the anticoagulant is infused into the circuit for at least about 1 hour during the procedure.

38. The method of claim 37, wherein the anticoagulant is infused into the circuit for at least about 24 hours during the procedure.

39. The method of claim 38, wherein the anticoagulant is infused into the circuit for at least about 48 hours during the dialysate procedure.

40. The method of any one of claims 27 to 39, wherein the blood treatment procedure is a CRRT procedure.

41. The method of claim 40, wherein the CRRT procedure comprises hemofiltration, hemodialysis or hemodiafiltration.

42. The method of any one of claims 27 to 41, wherein the anticoagulant comprises the mesylate salt of nafamostat.

43. The method of any one of claims 27 to 42 further comprising the step of measuring post- ACT, wherein said post-filter ACT measured at twenty -four hours after initiation of the procedure is increased relative to the subject’s baseline systemic ACT.

44. The method of claim 43, wherein said post-filter ACT measured at seventy-two hours after initiation of the procedure is increased relative to the subject’s baseline systemic ACT.

45. The method of any one of claims 27 to 44 further comprising the step of measuring the subject’s systemic ACT, wherein the subject’s systemic ACT measured at twenty -four hours after initiation of the procedure has not significantly increased from the subject’s baseline ACT.

46. The method of any one of claims 43 to 45 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at twenty -four hours after initiation of the procedure.

47. The method of any one of claims 43 to 45 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at forty-eight hours after initiation of the procedure.

48. The method of any one of claims 43 to 45 wherein both the post-filter ACT and the subject’s systemic ACT are measured, and the post-filter ACT is higher than the subject’s systemic ACT at seventy-two hours after initiation of the procedure.

49. The method of any one of claims 27 to 48, wherein the effective lifespan of the filter is increased by at least about 10% as compared to the lifespan of a filter used in the same procedure performed without the use of an anticoagulant.

50. The method of any one of claims 27 to 49, wherein the effective lifespan of the filter is increased by at least about 15% as compared to the lifespan of a filter used in the same procedure performed without the use of an anticoagulant.

51. The method of any one of claims 27 to 50 wherein the blood treatment procedure is a dialysis procedure and the method further comprises the step of measuring the dialysis efficiency of the procedure using a TACurea test, wherein the subject’s time-averaged concentration of blood urea nitrogen measured at twenty-four hours after initiation of the procedure is lower than when using same procedure performed without the use of an anticoagulant.

52. The method of any one of claims 27 to 51 wherein the blood treatment procedure is a dialysis procedure and the method further comprises the step of measuring the dialysis efficiency of the procedure using a time-averaged concentration of plasma 132 microglobulin test, wherein the subject’s time-averaged concentration of plasma 132 microglobulin measured at twenty -four hours after initiation of the procedure is lower than when using same procedure performed without the use of an anticoagulant.

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