Blood treatment device comprising alkaline phosphatase

Abstract

The present invention relates to a blood treatment device comprising an alkaline phosphatase, and specifically to a blood treatment device configured to dephosphorylate one or more organic compounds in the blood of a patient in need thereof in an extracorporeal blood circuit wherein the device comprises a matrix on which an alkaline phosphatase (AP) is immobilized. The blood treatment device containing alkaline phosphatase increases the stability of the immobilized AP, and the AP is not cleared by the body.

Description

[0001] This application is a divisional application of a Chinese patent application filed on March 6, 2020, with application number 202080018609.5 and invention name “Blood treatment device containing alkaline phosphatase”. Technical Field

[0002] The present invention relates to the field of extracorporeal treatment of patient blood and related hemofilter and adsorber cartridge devices. In particular, the present invention relates to alkaline phosphatase (AP)-assisted continuous renal replacement therapy (APA-CRRT) for the treatment of sepsis or sepsis-related acute kidney injury (AKI).

[0003] The present invention thus relates to a blood treatment device configured to dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof in an extracorporeal blood circuit, wherein the device comprises a matrix on which alkaline phosphatase (AP) is immobilized. The present invention also relates to an extracorporeal blood circuit comprising a blood treatment device of the present invention and to the use of a blood treatment device as a medicament or a method for treating an infection, preferably a blood or systemic infection such as sepsis, and / or for treating sepsis-related acute kidney injury (AKI). Background Art

[0004] Despite the use of modern antibiotics and resuscitation therapies, sepsis remains one of the leading causes of morbidity and mortality in critically ill patients. The outcomes of sepsis have improved overall, but the mortality rate remains unacceptably high. Sepsis is the most expensive healthcare problem in the United States, costing more than $20 billion (Martin GS et al. 2000. N Engl J Med 2003; 348: 1546-54). Sepsis is one of the most common causes of death in intensive care units (ICUs), and its incidence has more than doubled in the past 10 years (Kumar G et al., Chest 2011; 140: 1223-31). According to the Surviving Sepsis Campaign, the mortality rate from sepsis is 41% in Europe and 28.3% in the United States (Levy MM et al., Lancet Infect Dis 2012; 12: 919-24).

[0005] Acute kidney injury (AKI) or acute renal failure (ARF) is a rapid loss of the filtering function of the kidneys, characterized by metabolic acidosis, high potassium levels, and fluid imbalance. AKI occurs in 55-60% of critically ill patients, and sepsis is the most common underlying cause. The overall mortality rate of AKI / ARF is about 45%, however, the mortality rate of ARF caused by sepsis is about 70% (Acute Blood Purification, Suzuki and Hirasawa; Septic Acute Renal Failure, Mori et al., Contrib Nephrol. Basel, Karger, 2010, vol 166, pp 40-46).

[0006] Blood purification therapy for septic KI typically includes elimination of pathogens such as endotoxins or mediators that cause AKI, as well as renal replacement therapy (RRT). Adsorption of endotoxins by direct hemoperfusion using polymyxin-B immobilized fibers (PMX-DHP) can increase urine output while improving renal function (Mori et al., 2010). However, there is currently no established drug therapy for the treatment of sepsis-associated acute kidney injury (SA-AKI); only supportive renal replacement therapy (RRT) is available. AKI is associated with a complex cascade of microvascular dysregulation and cellular damage that occurs through inflammation, immune dysregulation, and oxidative damage. A range of novel therapies targeting specific enzymes or molecules involved in these pathways are at different stages of development (Bajwa A. et al., Curr Drug Targets 2009:10:1196-1204). Despite these advances, there is still an urgent need for treatments for AKI.

[0007] A new drug candidate in clinical development for the treatment or prevention of sepsis-associated acute kidney injury (SA-AKI) is alkaline phosphatase (AP). AP is a dephosphorylating, membrane-bound, endogenous enzyme that plays a detoxifying role by dephosphorylating endotoxins, which are implicated in the pathogenesis of sepsis, and other proinflammatory compounds, including extracellular ATP. When phosphorylated, lipid A molecules confer renal protection during septic AKI by dephosphorylating circulating lipopolysaccharides, which activate inflammatory pathways. In addition, AP converts ATP to adenosine, which has a protective effect on renal tubular cells. In sepsis, mitochondria release large amounts of ATP in response to cytokines and hypoxia.

[0008] Previously, clinical trials in healthy volunteers and patients with sepsis with or without AKI have established the tolerability and potential efficacy of purified bovine intestinal AP (Pickkers P et al., Eur J Clin Pharmacol 2009;65:393-402; Heemskerk S et al., Crit Care Med 2009;37:417–23); Pickkers P et al., CritCare 2012;16:R14). In patients with SA-AKI, bovine intestinal AP significantly improved renal function based on a composite endpoint of endogenous creatinine clearance, need for renal replacement therapy (RRT), and duration of RRT. Based on these results, human recombinant AP has been developed as a pharmaceutically acceptable alternative to bovine AP. To improve enzyme stability while maintaining catalytic function, the crown domain of human intestinal AP was replaced with the crown domain of human placental AP (Kifer-Moreira T et al., PLoS ONE 2014;9:e89374).

[0009] US 6290952 also discloses pharmaceutical compositions comprising AP, which are suitable for (systemic) treatment of clinical complications caused by infection, including sepsis.

[0010] Despite the progress and clinical potential of AP administration in the treatment of AKI, AP exhibits a number of disadvantages in its pharmacokinetics when administered as a pharmaceutical agent to a subject. AP is rapidly cleared from the blood, resulting in only about 10% of the initial administered activity being present after 4 hours. As a result, AP must be administered repeatedly and levels fluctuate widely, reaching suboptimal levels throughout the course of treatment. In view of the above difficulties, there is a great need in the art to develop improved means of treating AKI that provide consistent physiological improvements without the difficulties evident in the pharmacological treatment of this condition.

[0011] The present invention therefore relates to a blood treatment device comprising a matrix having alkaline phosphatase (AP) immobilized thereon. AP immobilized on a solid phase has been described previously (EP3110977, WO2013012924, WO9955828 and Chelpanova et al., Applied Biochemistry and Microbiology, vol. 52, no. 1, p. 36-42, 2016). However, immobilized AP has not been described or suggested in the context of an extracorporeal blood treatment device suitable for treating sepsis or AKI. Summary of the invention

[0012] In view of the prior art, the technical problem underlying the present invention is to provide an alternative and / or improved mechanism for treating infections such as sepsis and / or renal failure associated therewith. An object of the present invention is to provide an improved or alternative mechanism for the treatment of infections or renal failure, which provides a stable physiological improvement without the difficulties evident in the pharmacological treatment of these conditions. An object of the present invention is to provide a means for dephosphorylating extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof.

[0013] The present invention seeks to provide such a mechanism while avoiding the disadvantages known in the prior art.

[0014] This problem is solved by the features of the independent claim. Preferred embodiments of the invention are provided by the dependent claims.

[0015] The present invention therefore relates to a blood treatment device configured to dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof in an extracorporeal blood circuit, wherein the device comprises a matrix having alkaline phosphatase (AP) immobilized thereon.

[0016] In one embodiment, the blood treatment device is located in an extracorporeal blood circuit, through which the patient's blood passes, and the extracorporeal blood circuit includes means for conveying blood from the patient's vascular system to the blood treatment device at a defined flow rate and for returning the treated blood to the patient.

[0017] In another aspect, the present invention therefore relates to an extracorporeal blood circuit through which a patient's blood passes, comprising a blood treatment device as described herein and means for conveying blood from the patient's vascular system to the blood treatment device at a defined flow rate and then returning the treated blood to the patient.

[0018] The major advantage of incorporating the AP directly into a blood treatment device such as a dialyzer or hemofilter (membrane) is the increased stability of the immobilized AP and the lack of clearance of the AP by the body. The current kinetics of AP as a pharmacological agent involve rapid clearance and / or inactivation of the AP, resulting in AP activity decreasing to only 10% of the initial administered activity after 4 hours. By immobilizing the AP within the extracorporeal blood treatment device, clearance of the active agent is limited or eliminated, with no or very minimal activity loss over time.

[0019] Another significant advantage of the methods described herein is that no additional treatment or administration is required. By adding the AP to a blood treatment device suitable for extracellular dephosphorylation of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the patient's blood, the treatment is automatically incorporated once RRT or other dialysis is initiated. Patients with renal failure often begin dialysis treatment immediately to compensate for the failing renal function. This occurs in a large number of sepsis cases, where speed of treatment is critical to reducing the risk of mortality. Therefore, the present invention allows the active agent to be incorporated into a potentially already established RRT treatment that has already been initiated for the subject.

[0020] The present invention can significantly reduce costs compared to injection of AP because the amount of AP required for incorporation into the extracorporeal blood treatment apparatus is much less than that of intravenous injection due to the rapid clearance of intravenously administered AP and the requirement for continuous multiple dosing.

[0021] Thus, a matrix with AP immobilized in a blood processing device offers several advantages, including: increased duration of contact of toxic substrates with the AP, avoidance of rapid clearance of the AP from the blood when administered systemically, avoidance of off-target effects of the AP in other organs of the body (since the AP is administered only to the blood using an ex vivo system), avoidance of the need for additional treatment (since AP treatment can be concurrent with the initiation of RRT or other dialysis), simultaneous dephosphorylation of toxic substrates and removal of uremic toxins, excess ions, and water from the blood of, for example, a patient with renal failure, and faster treatment, which is particularly important in the setting of sepsis (since speed of treatment is critical to avoiding mortality).

[0022] As shown in the examples below, AP immobilization has been achieved for a variety of matrix types, including, but not limited to: PES / PVP membranes, PES / PVP / maleic anhydride membranes, PES / PVP / chitosan membranes, epoxy-functionalized adsorbent resins, amino-functionalized adsorbent resins, and polyacrylonitrile AN69 membranes. Unexpectedly, the AP immobilized on the matrix showed good activity, no or negligible leaching after prolonged rinsing, and excellent stability over extended timeframes relevant to clinical dialysis procedures, both in buffer and in human plasma. The combination of the advantages of using an in vitro approach and the unexpectedly good enzymatic properties of the immobilized AP represents a beneficial and surprisingly effective development over the prior art.

[0023] In one embodiment, the blood treatment device is a hemofilter or a dialyzer.

[0024] Such an embodiment provides a combination of two functions and can be advantageously used as a device for dephosphorylating a target protein in the blood or plasma of a person in need by an extracorporeal blood circuit as described herein, because the device simultaneously dephosphorylates the target protein and removes uremic toxins, excess ions and water in the blood of, for example, a patient with renal failure. Therefore, only one device is needed to treat a patient suffering from an infection or renal failure disease. Therefore, the extracorporeal circuit is not fundamentally different from a standard extracorporeal circuit for hemodialysis in the treatment of renal failure. Therefore, the treatment of such patients suffering from renal failure and infection (such as sepsis) is significantly simplified, and it may help to reduce the cost of cumulative treatment of the patient and increase the treatment options of the patient and the attending physician.

[0025] In one embodiment, the blood processing device is a hemofilter comprising a bundle of hollow fibers, and the alkaline phosphatase (AP) is immobilized at least on the lumen and, optionally, on the pores of the hollow fibers of the bundle.

[0026] In one embodiment, the blood treatment device as described herein is characterized in that the matrix of the device is a hollow fiber membrane, fiber mat or flat membrane composed of at least one polysaccharide derivative or synthetic polymer (examples of which are provided below) to which the AP is immobilized.

[0027] In some embodiments, APs are also immobilized to the pore surface, especially in the case of larger pore membranes such as plasma separation membranes or HCO membranes.

[0028] In some embodiments, the blood treatment device can be generally designed as a hollow fiber membrane filter (hemofilter) or a dialyzer, wherein the membrane constitutes a support, and the AP is (at least) bound to the support on the luminal side of the hollow fiber in contact with the blood. The membrane can be: a hemodialysis membrane for the treatment of renal failure, which is additionally functionalized with the AP on its luminal side; or a plasma separation membrane, which is additionally functionalized with AP on its luminal side or optionally on the outside of the hollow fiber and / or its pores.

[0029] One object of the present invention is to provide a hemofilter or hemodialyzer for blood purification that can be used to simultaneously treat patients suffering from infection (such as sepsis) or renal failure (such as SA-AKI). Due to this combined approach, the hemofilter function is combined with AP therapy and may work synergistically, thereby producing the RRT that the patient urgently needs and simultaneously treating the causes related to infectious diseases such as LPS.

[0030] In one embodiment, the device can be a blood filter for treating renal failure and infection, wherein the filter further comprises a resin (e.g. in the form of a sponge) or a non-woven fabric (functionalized with AP) in at least one end cap to dephosphorylate a target of interest. The target of interest is preferably extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP) and / or lipopolysaccharide (LPS).

[0031] In one embodiment, the blood processing device is a sorbent cartridge.

[0032] In one embodiment, the blood treatment device is located in an extracorporeal blood circuit as described herein, wherein the circuit further comprises a (separate) hemofilter located upstream or downstream of the sorbent cartridge.

[0033] In one embodiment, the sorbent cartridge includes a matrix having alkaline phosphatase (AP) immobilized thereon.

[0034] In one embodiment, the blood treatment device as described herein is characterized in that the matrix includes a support to which the AP is bonded, wherein the support includes or consists of a material selected from the group consisting of hollow fiber membranes, flat membranes, fiber mats, resins, non-woven fabrics, and open-cell foams (such as polyurethane (PU) foams).

[0035] In one embodiment, the blood treatment device described herein is characterized in that the support is a resin, and the resin is composed of the following materials: at least one polymer selected from the group consisting of alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin and agarose; an inorganic material selected from the group consisting of zeolite, ceramic, diatomaceous earth, silica, glass, activated carbon and charcoal; or a synthetic polymer, examples of which are provided below.

[0036] In one embodiment, the blood treatment device as described herein is characterized in that the support is a resin and that the resin consists of at least one synthetic polymer, examples of which are provided below.

[0037] In one embodiment, the blood treatment device as described herein is characterized in that: the support is a non-woven fabric, and the non-woven fabric is composed of the following materials: at least one biopolymer selected from the group consisting of polysaccharides, polylactic acid (PLA), polycaprolactone (PCL) and proteins, or selected from the group consisting of TiO 2 、SiO 2 or Al 2 O 3 The group consisting of at least one inorganic material, or at least one synthetic polymer, examples of which are provided below.

[0038] In one embodiment, a blood treatment device as described herein is characterized in that it is an adsorption cartridge comprising a matrix on which the AP is immobilized and which is perfused with whole blood.

[0039] Thus, the device may be or include an adsorption cartridge comprising a matrix selected from a resin or a nonwoven material, wherein any one of the functionalized with AP is configured to dephosphorylate a target of interest (extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP) and / or lipopolysaccharide (LPS), also referred to as a target protein). Such a device may be a component of an extracorporeal circuit for blood treatment, configured to provide hemodialysis, hemodiafiltration, hemofiltration or plasma exchange.

[0040] The device may be the sole blood treatment device within a blood circuit, or may be located upstream or downstream of an additional hemofilter or dialyzer in, for example, a hemodialysis, hemodiafiltration or hemofiltration circuit, or may alternatively be connected immediately to a dialyzer at a blood inlet or outlet, wherein the device is configured to be perfused with whole blood. The device may also be a component of an extracorporeal plasmapheresis circuit, wherein the device is perfused with plasma or a component thereof.

[0041] The device may also be a hybrid filtration device combining a hollow fiber membrane and a matrix in the filtrate space of the filter (WO 2014 / 079680 A1), wherein the matrix preferably comprises a resin functionalized with AP. Such a filter may be a component of an extracorporeal circuit configured for hemodialysis, hemodiafiltration or hemofiltration, wherein the filter is located upstream or downstream of a hemofilter or dialyzer for hemodialysis, hemodiafiltration or hemofiltration, or it may be used as the sole filtration device within the circuit in the absence of such a dialyzer. Such a device may be used with whole blood.

[0042] In one embodiment, the blood treatment device as described herein is characterized by an extracorporeal blood circuit, the blood treatment device located therein further comprising a plasma dialyzer or a centrifuge-based plasma separation system, which allows separation of a plasma fraction from the blood, and wherein the blood treatment device is located downstream of a plasma outlet of the plasma dialyzer. In another aspect, the present invention relates to an extracorporeal blood circuit comprising a blood treatment device as described herein, wherein the extracorporeal blood circuit comprises means for conveying blood or plasma from the patient's vascular system to the blood treatment device at a defined flow rate and means for returning the treated blood or plasma to the patient.

[0043] The active agent of the present invention is alkaline phosphatase (AP).

[0044] AP is known in various forms, each of which is not limiting of the present invention. The device described herein is characterized in that it is capable of demonstrating the function of an AP, namely, dephosphorylating extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof. The specific type of AP is limited only to the extent that it can achieve the desired function. AP has a broad specificity for phosphates of alcohols, amines, pyrophosphates, and phenols. It is commonly used to dephosphorylate proteins and nucleic acids, and is known to dephosphorylate LPS, ATP, and ADP.

[0045] Lipopolysaccharide (LPS) is part of the outer membrane of Gram-negative bacteria and plays a major role in the inflammatory cascade observed during severe infections such as sepsis. LPS induces systemic and renal inflammation through signaling through its pattern recognition receptor Toll-like receptor 4 (TLR4), which is localized to immune cells and proximal tubule epithelial cells (PTEC), resulting in epithelial inflammation, endothelial inflammation, and hypoxia. Due to the complex pathophysiology of sepsis, which involves systemic and renal inflammation as well as renal hypoxia, to be effective, treatments should aim to target all of these processes or a combination of these processes. Alkaline phosphatase (AP), a dephosphorylating enzyme, is a molecule that exerts this dual mechanism of action.

[0046] AP detoxifies LPS, which consists of an oligosaccharide core component, a polysaccharide chain, and a toxic lipid A portion with two phosphate groups. Removal of one of these two phosphate groups by AP results in a non-toxic LPS molecule that can still bind to TLR4 but then act as an antagonist.

[0047] Another protective mechanism of AP is the dephosphorylation of ATP, an endogenous signaling molecule released during cellular stress induced by, for example, inflammation and hypoxia. Excreted ATP attracts phagocytes and activates platelets and the NLRP3 inflammasome, further exacerbating inflammation and tissue damage. After being dephosphorylated by exogenous AP, ATP produces ADP, AMP, and adenosine, the latter of which exerts renal protective and anti-inflammatory effects by binding to one of the four adenosine receptors A1, A2A, A2B, and A3. AP may promote the conversion of ATP to ADP, AMP, and ultimately to adenosine, thereby increasing adenosine levels and exerting its anti-inflammatory and tissue protective effects. Therefore, the action of AP on ADP is beneficial because the same enzyme catalyzes the conversion of ATP to ADP and further catalyzes the conversion of ADP to AMP and adenosine.

[0048] As is evident from the above, dephosphorylation of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of patients in need thereof represents an effective means to combat the negative effects of these pathological signaling and bacterial molecules.

[0049] In one embodiment, the alkaline phosphatase (AP) is calf intestinal alkaline AP.

[0050] Bovine intestinal AP has been shown to be well tolerated by subjects and has shown potential efficacy in various clinical settings. In patients with SA-AKI, bovine intestinal AP significantly improved renal function as measured by a composite endpoint of endogenous creatinine clearance, need for renal replacement therapy (RRT), and duration of RRT.

[0051] In one embodiment, the alkaline phosphatase (AP) is a recombinant and / or chimeric form of AP.

[0052] In some embodiments, recombinant AP as described herein is employed in the present invention. Preferred AP variants exhibit enhanced stability and good selectivity for LPS (Kifer-Moreira T et al., PLoS ONE 2014; 9: e89374).

[0053] Early studies of the structure of placental AP (PLAP) revealed two active Zn 2+ The active sites of the residues (Zn1 and Zn2) and the Mg 2+ The third metal ion site is occupied by Zn2. While Zn2 is buried within the molecule, Zn1 is easily accessible and its reactivity can be regulated. Both Zn1 and Zn2 are coordinated to highly conserved amino acids, and mutagenesis studies revealed a major role for residue E429 in determining the affinity of Zn1, the stability and catalytic properties of the PLAP active site, and in determining the overall thermal properties of the enzyme. Introduction of the well-characterized crown domain of PLAP with the critical E429 residue into the human enteric AP construct improved enzyme stability while retaining or enhancing the catalytic function of the resulting chimeric enzyme. The resulting recombinant AP displayed greatly increased thermal stability, increased Zn 2+ Binding affinity, increased transphosphorylation, higher turnover number and narrower substrate specificity with considerable selectivity for bacterial LPS (Kifer-Moreira T. et al., PLoS ONE 2014;9:e89374).

[0054] The matrix material on the device of the present invention can be essentially any material that is suitable for contact with human blood and can immobilize AP.

[0055] In one embodiment, the substrate, preferably a hollow fiber membrane, to which alkaline phosphatase (AP) is immobilized comprises or consists of polysulfone, poly(ether)sulfone (PES) and / or polyaryl(ether)sulfone (PAES), and polyvinylpyrrolidone (PVP).

[0056] In one embodiment, the substrate, preferably a hollow fiber membrane, to which alkaline phosphatase (AP) is immobilized comprises or consists of polysulfone, PES and / or PAES, and PVP with an additive selected from chitosan and / or maleic anhydride-alt-1-octadecene alternating copolymers.

[0057] Thus, the device is a hollow fiber membrane dialyzer designed for hemodialysis and wherein the lumen side of the hollow fibers of the hemodialyzer is functionalized with alkaline phosphatase (AP).

[0058] According to one aspect of the invention, the hemodialyser is based on membranes made from polymers and polymer blends. ) are predominantly hydrophobic and are therefore combined with hydrophilic additives such as polyvinylpyrrolidone (PVP) or PEG or treated with hydrophilic copolymers such as methyl allyl sulfonate.

[0059] Examples of commercially available materials and dialyzers that can be used according to the present invention include, for example, those listed in the table below. Membranes prepared from hydrophobic / hydrophilic polymer blends are the main types of synthetic polymer membranes. Their hydrophobic base materials are polysulfones or polyethersulfones (polyarylethersulfones), and they also contain a hydrophilic component, which is typically PVP. Polyethersulfone and polysulfone-based membranes contain a small amount of water and do not contain pore stabilizers compared to other membranes. According to the present invention, these membranes are particularly suitable, as are membranes containing polyacrylonitrile.

[0060] Table: Hemofilter membrane materials and manufacturers

[0061]

[0062] According to one aspect of the invention, a polysulfone / PVP membrane is used. All polysulfone-based dialysis membranes have a foam-like support structure, which is intended to achieve specific separation characteristics. The increased hydraulic resistance of the foam-like support structure is partially compensated by a reduction in wall thickness. Examples of this type of membrane are, for example, those mentioned in the table above, such as the Helixone membrane from Fresenius Medical Care.

[0063] According to another aspect of the invention, a polyethersulfone / PVP / polyamide membrane is used. The so-called Polyamix membrane has a unique asymmetric three-layer structure, wherein the outer layer (called the support layer) is characterized by a very open finger-like morphology. The actual inner separation layer of the membrane consists of an extremely thin endothelium supported by an intermediate layer. This intermediate layer forms a very permeable foam-like structure. Thus, low resistance to convection and diffusion is ensured. The outer layer provides high mechanical stability.

[0064] According to another aspect of the invention, polyethersulfone / PVP or poly(aryl)ethersulfone membranes are used. Most membranes made of polyethersulfone and polyvinylpyrrolidone are characterized by an asymmetric structure, a dense selective endothelium in contact with blood, and a supportive porous outer layer. By appropriately adjusting the membrane making parameters and using PVP of different molecular weights, the physicochemical properties, morphological structure, solute rejection behavior and filtration performance of the underlying membrane can be improved.

[0065] The manufacture of polysulfone / PVP-based membranes (including membranes made of PES or PAES) is comparable to the manufacture of other hollow fiber membranes and is essentially known in the art (see, for example, Boschetti-de-Fierro et al., Membrane Innovation in Dialysis, Ronco (ed): Expanded Hemodialysis-Innovative Clinical Approach in Dialysis. Contrib Nephrol. Basel, Karger, 2017, vol 191, pp 100-114). For optimized applications, the inner diameter of the hollow fiber membrane ranges from 170 to 220 μm. Synthetic polymer membranes have a wall thickness of 25 to 55 μm. The effective membrane surface area of ​​the dialyzer selected for treating a patient must be adjusted according to the patient's body size. Pediatric filters (suitable for small patients) have a diameter of 0.01 to 0.6 μm. 2 The surface area of ​​the standard filter for adults is 1.0 to 2.4 m 2 surface area.

[0066] In one embodiment, the substrate, preferably a hollow fiber membrane, to which alkaline phosphatase (AP) is immobilized comprises or consists of a copolymer of acrylonitrile and sodium methallyl sulfonate.

[0067] Therefore, the blood purification device preferably includes a polyacrylonitrile (PAN) membrane, which contains i) a copolymer of acrylonitrile and sodium methallyl sulfonate, ii) polyethyleneimine, and iii) heparin on which AP has been fixed. Polyacrylonitrile is the first synthetic material developed for dialysis membranes. The material of the old AN69 membrane developed by Hospal is a copolymer of acrylonitrile and sodium methallyl sulfonate. This combination is crucial and includes the addition of sodium methallyl sulfonate that affects the membrane structure. WO 2007 / 148147 A1 describes the use of a solution of a polymer with anionic or anionizable groups in colloidal form on a membrane preferably based on a copolymer of acrylonitrile and sodium methallyl sulfonate, particularly by mixing a solution of a polymer with anionic or anionizable groups with an organic polyacid solution in a specific ratio relative to the polymer, which results in an increase in the number of polymers grafted to the membrane surface and the availability of free cations or cationizable groups on the membrane coating surface. The membrane allows the combination of a large number of compounds with anionic or anionizable groups.

[0068] In one embodiment, the matrix, preferably a hollow fiber membrane, to which alkaline phosphatase (AP) is immobilized comprises or consists of polymethyl methacrylate.

[0069] According to this embodiment, polymethyl methacrylate hollow fiber membranes are preferably used. Such high flux membranes are made of modified polymethyl methacrylate (PMMA). PMMA-based hemofilters are commercially available and have been used to treat sepsis-induced AKI. Known hemofilters comprising PMMA membranes are, for example, those provided by Toray (Sakai, in: High Performance Membrane Dialyzers. Saito et al., eds. Contributions to Nephrology, Vol 173. Basel: Karger; 2011: 137-147). NF(Nf) and traditional BG(BG).

[0070] According to the present invention, the AP is fixed to the matrix of the device of the present invention. The present invention contemplates essentially any means for fixing to the matrix. In some embodiments, the AP is fixed to the matrix so that it remains stably attached during perfusion with blood in the context of an extracorporeal blood circuit, as would be employed in the treatment of renal failure. Any form of fixation that provides a stable connection between the AP and the matrix is ​​suitable, preferably a form of fixation that results in no or negligible loss of the AP in the matrix during use of the device described herein in the blood circuit.

[0071] In further embodiments of the invention, both covalent (eg, cross-linking) and non-covalent (eg, ionic interactions) forms of attachment are contemplated.

[0072] The number and chemistry of these can be further varied. These fixing means depend on the specific matrix material used in the device and the specific location of the device in the extracorporeal blood treatment circuit.

[0073] In one embodiment, alkaline phosphatase (AP) is immobilized on a substrate via an ester bond produced by the reaction between the OH groups of polysulfone, poly(ether)sulfone (PES) and / or polyaryl(ether)sulfone (PAES) and the COOH groups of AP, preferably using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) treatment.

[0074] In one embodiment, by NH 2 Alkaline phosphatase (AP) is immobilized to the matrix, preferably using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) treatment, by reacting the peptide bond produced between the phosphodiesterase group and the COOH group of AP.

[0075] In one embodiment, the COOH groups of the maleic anhydride-1-octadecene alternating copolymer are reacted with the NH 2 The reaction between the groups generates a peptide bond, and alkaline phosphatase (AP) is immobilized to the matrix, preferably using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) treatment.

[0076] 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is known in the art to be useful for generating bonds between OH and COOH groups or between NH 2 A cross-linking agent between COOH groups. 2 and COOH groups, EDC chemistry represents an effective means of immobilizing APs onto substrates. The skilled artisan will adjust the immobilization conditions and methods depending on the specific substrate (membrane) material used in the device.

[0077] Carboxylic acids (-COOH) are present at the C-terminus of each polypeptide chain and in the side chains of aspartic acid (Asp, D) and glutamic acid (Glu, E). Like primary amines, carboxyl groups are usually located on the surface of protein structures. Carboxylic acids are reactive with carbodiimides such as EDC.

[0078] EDC and other carbodiimides are zero-length crosslinkers. They usually result in carboxylate (-COOH) groups bonding to primary amines (-NH 2) without becoming part of the final amide bond crosslink between target molecules. Since peptides and proteins contain multiple carboxyl and amine groups, direct EDC-mediated crosslinking results in random aggregation of peptides. However, this reaction chemistry is widely used in immobilization procedures.

[0079] EDC reacts with carboxylic acid groups to form active O-acylisourea intermediates, which are easily replaced by nucleophilic attacks of primary amino groups in the reaction mixture. Primary amines form amide bonds with the original carboxyl groups, and EDC byproducts are released as soluble urea derivatives. O-acylisourea intermediates are unstable in aqueous solution. Not reacting with amines can lead to intermediate hydrolysis, carboxyl regeneration and N-unsubstituted urea release. EDC cross-linking is effective under acidic (pH 4.5) conditions and is generally used in buffers that do not contain exogenous carboxyl and amine. MES buffer (4-morpholinoethanesulfonic acid) is a suitable carbodiimide reaction buffer. Phosphate buffer and neutral pH (up to 7.2) conditions are compatible with chemical reactions, but are less efficient. Increasing the amount of EDC in the reaction solution can compensate for reduced efficiency.

[0080] In some embodiments, immobilization involves cross-linking OH groups from the matrix material or surface to the COOH groups of the AP. In another embodiment, such immobilization involves cross-linking the NH 2 In another embodiment, such immobilization involves cross-linking the COOH groups generated by the maleic anhydride-1-octadecene alternating copolymer with the NH 2 Group.

[0081] Alternative means of cross-linking the AP to the device matrix are available to the skilled person and can be used without undue effort. Depending on the matrix, suitable chemicals can be used. Some matrix materials can be activated to couple directly to the AP. Other supports are made of nucleophiles or other functional groups that can be attached to proteins using cross-linkers. Carbodiimides (such as DCC and EDC) are very useful for coupling proteins to carboxyl and amine-activated glass, plastic, and polysaccharide supports. The carbodiimide procedure is usually a one-step method; however, if the reaction is carried out in an organic solvent, or if NHS or Sulfo-NHS chemistry is used to enhance the reaction, a two-step method is possible. In some embodiments, a spacer can be used. Useful spacers are diaminodipropylamine (DADPA), ethylenediamine, hexamethylenediamine, 6-amino-hexanoic acid, and any of several amino acids or peptides. When the AP is fixed too close to the matrix to allow the AP to approach its target, the spacer arm helps to overcome the steric effect.

[0082] Another aspect of the present invention relates to a method for manufacturing a blood processing device as described herein, wherein the device is configured to dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof, the method comprising immobilizing alkaline phosphatase (AP) to a matrix.

[0083] In one embodiment of the method, the blood processing device is a hemofilter comprising a membrane of a hollow fiber bundle, and the alkaline phosphatase (AP) is immobilized at least on the lumen and, optionally, on the pores of the hollow fibers of said bundle.

[0084] In one embodiment of the method, the alkaline phosphatase (AP) is covalently bound to the substrate and the method comprises treating the substrate and / or AP with a carbodiimide compound, preferably 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Suitable methods using specific EDC chemicals and protocols are disclosed in more detail in the examples below.

[0085] The method of the invention is very advantageous because production of the device does not require any fundamental changes to the structure, function or usability of standard blood treatment devices such as adsorbents or hemofilters. Existing blood treatment adsorbents or hemofilters can also be subsequently (after manufacture) treated with AP to immobilize the active agent and prepare the device for use. Since the dialyzer does not change structurally, but is only modified after manufacture, the need for investment in additional manufacturing is limited.

[0086] In some embodiments, the device for in vitro treatment containing immobilized AP produced using the methods described herein may preferably be filled or stored in a buffer or other solution, wherein the matrix containing the immobilized AP is immersed in the buffer or solution, and Mg 2+ The concentration of Zn is 0.1 to 2, preferably 0.1 to 1 mmol / L, and Zn 2+ The concentration of AP is between 5 and 150, preferably between 10 and 100 μmol / L. Depending on the AP employed, some variation of the values ​​is possible while maintaining the desired AP activity.

[0087] In some embodiments, the method for manufacturing a blood processing device as described herein comprises: a PEI coating process of a membrane (preferably a polyacrylonitrile membrane, more preferably an AN69 membrane or a hollow fiber bundle), preferably using an acidic solution, more preferably a citric acid solution, followed by glutaraldehyde treatment and AP fixation.

[0088] As shown in the examples below, a PEI coating process has been developed that is particularly effective in subsequent GA / AP functionalization and AP activity. In a preferred embodiment, a polyacrylonitrile membrane, preferably an AN69 membrane or a hollow fiber bundle, is treated with PEI in an acidic solution (preferably PEI at 100 to 300 or about 200 mg / kg, preferably 10 to 300, preferably 200 mg / g, in a citric acid solution).

[0089] In one embodiment, an optimal PEI density of 0.5 to 5, preferably 1 to 3 μmol / g of the hollow fiber membrane (before GA and AP treatment) is used.

[0090] In one embodiment, the method for manufacturing a blood treatment device as described herein comprises: subjecting a PEI-treated membrane to GA treatment, wherein the GA is applied to the membrane at an alkaline pH, preferably 7.1-11 pH, more preferably 7.3-10.5 pH, more preferably about 7.4 or 9.9 pH. These pH values ​​result in effective GA treatment and subsequent unexpectedly stable and active immobilization of AP.

[0091] In one embodiment, the method for manufacturing a blood treatment device as described herein comprises: subjecting a membrane treated with PEI and GA to an AP treatment, wherein the GA is applied to the membrane in a solution at an alkaline pH, preferably 7.1 to 11 pH, more preferably 7.3 to 10.5 pH, more preferably about 7.4 or 9.9 pH. These pH values ​​result in unexpectedly stable and active immobilized AP.

[0092] In a preferred embodiment, PEI treatment is performed, preferably using a polyacrylonitrile membrane, more preferably an AN69 membrane or a hollow fiber bundle, at a pH of 7.1 to 7.8, more preferably about 7.4, and then AP treatment is performed in a solution of pH 8.5 to 10.5, more preferably about 9.9.

[0093] In some embodiments, the matrix with immobilized AP is subsequently sterilized. Surprisingly, sterilization of the matrix tested below does not result in a significant reduction in AP activity, which is problematic for clinical applications. In one embodiment, gamma sterilization is used. In one embodiment, EtO gas sterilization is used. In one embodiment, heat sterilization (steam sterilization) is not used.

[0094] In another aspect, the present invention relates to the use of a blood treatment device as described herein as a medicament for treating an infection, preferably a blood or systemic infection.

[0095] In another aspect, the present invention relates to the use of a blood treatment device as described herein as a medicament for treating any disease associated with pathological levels of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP) and / or lipopolysaccharide (LPS). LPS is generally known as a pathological molecule associated with a variety of infectious diseases. Dephosphorylation of LPS and detoxification of the molecule using the present invention can effectively counteract the pathological effects of LPS.

[0096] In one embodiment, the invention relates to the use of a blood treatment device as described herein as a medicament for treating sepsis or septic shock.

[0097] In one embodiment, the invention relates to the use of a blood treatment device as described herein as a medicament for treating infection-related renal dysfunction. In one embodiment, the infection-related renal dysfunction is sepsis-related acute kidney injury (AKI or SA-AKI).

[0098] In one embodiment, the treatment comprises continuous renal replacement therapy in an acute setting, preferably in an intensive care unit (ICU).

[0099] According to one aspect, the invention provides a method of treating or ameliorating at least one symptom of such a disease.

[0100] Therefore, another object of the present invention is to provide a method for treating or ameliorating at least one symptom of a disease associated with pathological levels of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in a patient's body, wherein the method comprises: a step of dephosphorylating extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the patient's blood in vitro by passing the patient's blood or plasma through a matrix configured as described herein and then returning the treated blood to the patient.

[0101] Therefore, one object of the present invention is to provide a device, an extracorporeal circuit and a method for treating or preventing infection, preferably blood or systemic infection, sepsis or septic shock, infection-related renal dysfunction or sepsis-related acute kidney injury (AKI), wherein the device is placed in the extracorporeal blood treatment circuit and is configured to dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP) and / or lipopolysaccharide (LPS). DETAILED DESCRIPTION

[0102] All citations of patent and non-patent literature are incorporated herein by reference in their entirety. Unless otherwise specified herein, all terms will be given their ordinary technical meanings.

[0103] The present invention is based on the in vitro dephosphorylation of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the patient's blood using alkaline phosphatase (AP) fixed to a matrix in a blood treatment device (such as a blood filter or sorbent cartridge with AP fixed thereto), and then returning the treated blood to the patient.

[0104] Extracorporeal circulation and therapy:

[0105] Extracorporeal systems into which the novel blood treatment apparatus of the present invention can be integrated are known in the prior art. An extracorporeal procedure or system is a medical procedure or system that is performed or located outside the body. This relates to a common procedure in which blood is removed from the patient's circulation and any given process is performed on it before the blood is returned to the circulation. The equipment that carries blood outside the body is called an extracorporeal blood circuit. Suitable equipment components, such as tubing, connectors, pumps, etc., are available to the skilled person.

[0106] According to the present invention, the expression "extracorporeal blood purification" preferably refers to the process of removing substances from body fluids (in vitro) by removing them from the flowing blood in a shunt circuit outside the patient's body. The substances may include endogenous toxins (i.e., uremic toxins), exogenous toxins (i.e., ethylene glycol or mycotoxins), administered drugs, viruses, bacteria, antibodies and proteins (i.e., IMHA, myasthenia gravis), abnormal cells (i.e., leukemia), and excess water. Therapeutic procedures include hemodialysis, including intermittent hemodialysis (HD, HDF, HF) and continuous renal replacement therapy (CRRT); hemoperfusion; and therapeutic apheresis.

[0107] As used herein, the expression "blood" refers to whole blood containing all components of the blood of an organism, including red blood cells, white blood cells and platelets suspended in plasma. The expression "plasma" refers to a fluid composed of about 92% water, 7% proteins (e.g. albumin, gamma globulin, fibrinogen, complement factors, coagulation factors) and 1% mineral salts, sugars, fats, electrolytes, hormones and vitamins, which plasma constitutes a part of whole blood but no longer contains red blood cells, white blood cells and platelets. In the context of the present invention, the expression "plasma" also refers to a specific part of the plasma defined above in its standard meaning, such as serum.

[0108] According to one aspect, the blood flow rate in the extracorporeal blood purification circuit is 20 ml to 700 ml / min. In addition to the case of the blood treatment device according to the present invention or in the case of a hemodialyzer configured to fix the target protein, a typical dialysate flow rate in the extracorporeal circuit including a hemodialyzer for treating renal failure is 0.5 l / h to 800 ml / min.

[0109] In hemodialysis, blood circulates in an extracorporeal circuit and its composition is altered by mass transfer of solutes and water across an interfacial semipermeable membrane by diffusion and / or convection. The magnitude and spectrum of solute transfer depends on the nature of the forces applied across the membrane, the chemical and physical properties of the solutes, and especially the size and structural properties of the membrane. Hemodialysis is the standard treatment for patients with renal failure.

[0110] Hemoperfusion is an extracorporeal adsorption therapy used to treat endogenous and exogenous poisonings that cannot be effectively removed by hemodialysis. Adsorption is the principle by which solutes, such as protein molecules, attach to the surface (matrix) of a material. In contrast to the physical separation between blood and dialysate that occurs during hemodialysis, during hemoperfusion the blood is directly exposed to an adsorbent that has the ability to selectively or non-selectively bind solutes within the blood pathway.

[0111] In therapeutic apheresis, blood is separated into its component parts, for example by centrifugation or by passing through a plasma membrane or filter, and the fraction containing the solutes to be removed (usually the plasma fraction) is specially treated before being returned to the patient. The present invention provides an apheresis treatment in which plasma (containing the target protein) is removed from the patient's flowing blood and returned to the patient after contact with a device or matrix according to the present invention ( Figure 5 Typical plasma flow rates in an extracorporeal circuit (in which a blood treatment device is perfused with plasma) are in the range of 7 ml / min to 250 ml / min.

[0112] According to one aspect, the extracorporeal blood circuit according to the present invention is configured to perform hemodialysis. In this case, the device according to the present invention is, for example, a hemodialyzer, which is additionally configured to fix the target protein according to the present invention. The circuit can be operated under different treatment modes (including hemodialysis, hemodiafiltration, hemofiltration mode) according to medical needs.

[0113] According to one aspect, the device is configured to perform "renal replacement therapy" (RRT), which refers to a therapy used to replace the normal blood filtering function of the kidneys. Hemodialysis, hemofiltration, and hemodiafiltration can be continuous or intermittent, and can use an arteriovenous approach (blood leaves from an artery and returns through a vein) or a venous approach (blood leaves from a vein and returns through a vein). This leads to various types of RRT.

[0114] For example, renal replacement therapy can be selected from, but is not limited to, the following group of therapies: continuous renal replacement therapy (CRRT), continuous hemodialysis (CHD), continuous arteriovenous hemodialysis (CAVHD), continuous veno-venous hemodialysis (CVVHD), continuous hemofiltration (CHF), continuous arteriovenous hemofiltration (CAVH or CAVHF), continuous venovenous hemofiltration (CVVH or CVVHF), continuous hemodiafiltration (CHDF), continuous arteriovenous hemodiafiltration (CAVHDF), continuous venovenous hemodiafiltration (CVVHDF), intermittent renal replacement therapy (IRRT), intermittent hemodialysis (IHD), intermittent venovenous hemodialysis (IVVHD), intermittent venovenous hemofiltration (IHF), intermittent venovenous hemofiltration (IVVH or IVVHF), intermittent hemodiafiltration (IHDF), and intermittent venovenous hemodiafiltration (IVVHDF).

[0115] According to a preferred embodiment, the extracorporeal blood circuit according to the present invention is configured to provide continuous renal replacement therapy (CCRT). Continuous renal replacement therapy (CRRT) is a slow dialysis treatment that provides continuous therapy 24 hours a day, mainly for critically ill patients in an ICU environment. Like intermittent HD in patients with chronic renal failure, solute removal in CRRT is achieved by convection (hemofiltration), diffusion (hemodialysis), or a combination of these two methods (hemodiafiltration). This process requires the use of replacement fluids to prevent iatrogenic acidosis and electrolyte depletion and excessive fluid removal. CRRT and how to use it are known in the art.

[0116] According to another aspect, the extracorporeal blood circuit according to the invention is configured for blood perfusion. Thus, the blood treatment device according to the invention is perfused with whole blood and is located in the extracorporeal circuit ( Figure 3 According to one aspect of the invention, the device is a cartridge comprising a membrane, nonwoven fabric or resin to which AP has been bonded. According to one aspect, when the matrix of the cartridge comprises a hollow fiber bundle membrane bonded to AP, the treatment mode can be hemoperfusion with a closed dialysate / filtrate port. According to yet another aspect, the cartridge can be located downstream or upstream of a hemodialyzer configured to perform hemodialysis on a patient's blood ( Figure 4 A and 4B) and can be operated in different treatment modes selected from hemodialysis, hemodiafiltration and hemodialysis.

[0117] According to one aspect, the blood treatment device is a filter comprising hollow fibers and resin in the filtrate space of the filter as described above. The filter can be operated in a blood perfusion mode, or if combined with a hemodialyzer that can be located upstream or downstream of the device according to the present invention, the treatment mode can be hemodialysis, hemodiafiltration or hemofiltration. According to another embodiment of the present invention, the device according to the present invention can be regenerated between treatments.

[0118] According to one aspect, the hollow fiber membrane of the device is a plasma separation membrane that allows plasma, together with the target contained therein, to pass through the membrane and interact with the matrix in the filtrate space, thereby allowing the target to be dephosphorylated by the AP. The purified plasma will re-enter the hollow fiber membrane within the same device, and the blood can be returned to the patient. Such a device can be located in an extracorporeal circuit upstream or downstream of a hemodialyzer, as described in WO 2014 / 079681 A2, or it can be used as the only blood perfusion device within the circuit. In another aspect, the AP can also or specifically be bound to a plasma separation membrane as described above, for example to the outside and / or pores of the membrane. In one aspect, the resin in the filtrate space can be configured to remove the same or different target proteins.

[0119] Typically, the device according to the invention is designed as a cylinder with a cylindrical housing having at least one inlet and at least one outlet for the blood or plasma treated therewith. When the device is a hemodialyzer, in addition to removing at least one target protein, it is also used for the treatment of renal failure in HD, HDF or HF, the device also comprises an inlet and an outlet for the dialysate. The device configurations that can be used according to the invention are well known and fall within the scope of the invention.

[0120] According to another embodiment, as described in US2018 / 0369783, the extracorporeal blood circuit includes a hemocompatible adsorbent for separating protein-bound uremic toxins contained in the patient's blood or plasma. The hemocompatible adsorbent is suitable for separating protein-bound uremic toxins with a molecular weight of less than 500g / mol (relative to its carrier protein) so that the uremic toxins can be dialyzed by hemodialysis, wherein the hemocompatible material includes a polymer based on cyclic oligosaccharides or a derivative thereof, which is arranged on a solid carrier component in at least one layer. Therefore, this hemocompatible adsorbent is suitable for selectively and effectively removing albumin-bound toxins with hydrophobic and / or charged domains, with a molecular weight of up to about 500Da (e.g., indoxyl sulfate, p-cresol, hippuric acid or phenylacetic acid) to support common extracorporeal blood treatments (e.g., hemodialysis or liver dialysis).

[0121] Alkaline Phosphatase:

[0122] As used herein, "alkaline phosphatase" (also known as AP, ALP, ALKP, ALPase, Alk Phos), or basic phosphatase is a homodimeric protease of about 86 kilodaltons, as described in more detail herein. Each monomer typically contains five cysteine ​​residues, two zinc atoms, and one magnesium atom that are important for its catalytic function, and it has optimal activity in an alkaline pH environment. AP has the physiological role of dephosphorylating compounds. The enzyme is found in a variety of organisms, prokaryotes and eukaryotes alike, with the same general function but in different structural forms suitable for the environment in which they function. The enzyme is generally thermostable and insensitive to the immobilization reaction chemistry disclosed herein.

[0123] One preferred embodiment of the AP involves calf intestinal alkaline phosphatase, which is commercially available (eg, from Sigma-Aldrich; P0114) and can be immobilized to the device matrix using established techniques and those described herein.

[0124] Calf intestinal alkaline phosphatase is a dimeric membrane-derived glycoprotein. At least three isoforms exist, with each monomer typically having two N-linked glycans and one or more O-linked glycans. Commercial products are available in solutions containing 50% glycerol at pH 7.0, such as 5 mM Tris, 5 mM MgCl 2 and 0.1 mM ZnCl 2 One DEA unit biAP from Sigma-Aldrich typically hydrolyzes 1 μmol of 4-nitrophenyl phosphate per minute at 37°C and pH 9.8.

[0125] An exemplary protein sequence of intestinal alkaline phosphatase (Bos Taurus) is according to GenBank: AAA30571.1, as shown below as SEQ ID NO 1. Isozymes of bovine intestinal alkaline phosphatase, as described by Besman and Coleman (J Biol Chem. 1985 Sep 15; 260(20): 11190-3), are also contemplated for use in the present invention.

[0126] SEQ ID NO 1:

[0127] MQGACVLLLLGLHLQLSLGLVPVEEEDPAFWNRQAAQALDVAKKLQPIQTAAKNVILFLGDGMGVPTVTATRILKGQMNGKLGPETPLAMDQFPYVALSKTYNVDRQVPDSAGTATAYLCGVKGNYRTIGVSA AARYNQCKTTRGNEVTSVMNRAKKAGKSVGVVTTTRVQHASPAGAYAHTVNRNWYSDADLPADAQMNGCQDIAAQLVNNMDIDVILGGGRKYMFPVGTPDPEYPDDASVNGVRKRKQNLVQAWQAKHQGAQYV WNRTALLQAADDSSVTHLMGLFEPADMKYNVQQDHTKDPTLQEMTEVALRVVSRNPRGFYLFVEGGRIDHGHHDDKAYMALTEAGMFDNAIAKANELTSELDTLILVTADHSHVFSFGGYTLRGTSIFGLAPS KALDSKSYTSILYGNGPGYALGGGSRPDVNDSTSEDPSYQQQAAVPQASETHGGEDVAVFARGPQAHLVHGVEEETFVAHIMAFAGCVEPYTDCNLPAPTTATSIPDAAHLAASPPPLALLAGAMLLLLAPTLY

[0128] An exemplary sequence of alkaline phosphatase is the human precursor protein according to GenBank: AAA51700.1, as shown in SEQ ID NO 2 below.

[0129] SEQ ID NO 2:

[0130] MQGPWVLLLLGLRLQLSLGIIPVEEENPDFWNRQAAEALGAAKKLQPAQTAAKNLIIFLGDGMGVSTVTAARILKGQKKDKLGPETFLAMDRFPYVALSKTYSVDKHVPDSGATATAYLCGVKGNFQTIGLSA AARFNQCNTTRGNEVISVMNRAKKAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADVPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPDDYSQGGTRLDGKNLVQEWLAKHQGARYV WNRTELLQASLDPSVTHLMGLFEPGDMKYEIHRDSTLDPSLMEMTEAALLLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTSEEDTLSLVTADHSHLFSFGGYPLRGSSIFGLAPG KARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDGETHAGEDVAVFARGPQAHLVHGVQEQTFIAHVMAFAACLEPYTACDLAPRAGTTDAAHPGPSVVPALLPLLAGTLLLLGTATAP

[0131] An exemplary protein sequence of alkaline phosphatase is the human protein according to GenBank: AAB59378.1, as shown below in SEQ ID NO 3. Alternative exemplary human AP sequences are also known by GenBank Accession Nos. AAC97139.1, AAA98616.1, CAA39425.1, AAA98617.1.

[0132] SEQ ID NO 3:

[0133] MISPFLVLAIGTCLTNSLVPEKEKDPKYWRDQAQETLKYALELQKLNTNVAKNVIMFLGDGMGVSTVTAARILKGQLHHNPGEETRLEMDKFPFVALSKTYNTNAQVPDSAGTATAYLCGVKANEGTVGVS AATERSRCNTTQGNEVTSILRWAKDAGKSVGIVTTTRVNHATPSAAYAHSADRDWYSDNEMPPEALSQGCKDIAYQLMHNIRDIDVIMGGGRKYMYPKNKTDVEYESDEKARGTRLDGLDLVDTWKSFKPR YKHSHFIWNRTELLTLDPHNVDYLLGLFEPGDMQYELNRNNVTDPSLSEMVVVAIQILRKNPKGFFLLVEGGRIDHGHHEGKAKQALHEAVEMDRAIGQAGSLTSSEDTLTVVTADHSHVFTFGGYTPRGN SIFGLAPMLSDTDKKPFTAILYGNGPGYKVVGGERENVSMVDYAHNNYQAQSAVPLRHETHGGEDVAVFSKGPMAHLLHGVHEQNYVPHVMAYAACIGANLGHCAPASSAGSLAAGPLLLALALYPLSVLF

[0134] Also included as an exemplary embodiment is the human intestinal alkaline phosphatase precursor according to the GenBank reference sequence NP_001622.2, as shown in SEQ ID NO 4.

[0135] SEQ ID NO 4:

[0136] MQGPWVLLLLGLRLQLSLGVIPAEEENPAFWNRQAAEALDAAKKLQPIQKVAKNLILFLGDGLGVPTVTATRILKGQKNGKLGPETPLAMDRFPYLALSKTYNVDRQVPDSAATATAYLCGVKANFQTIGLSAAARFNQCNTTRGNEVISVMNRAKQAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADMPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPADASQNGIRLDGKNLVQEWLAKHQGAWYVWNRTELMQASLDQSVTHLMGLFEPGDTKYEIHRDPTLDPSLMEMTEAALRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQALTEAVMFDDAIERAGQLTSEEDTLTLVTADHSHVFSFGGYTLRGSSIFGLAPSKAQDSKAYTSILYGNGPGYVFNSGVRPDVNESESGSPDYQQQAAVPLSSETHGGEDVAVFARGPQAHLVHGVQEQSFVAHVMAFAACLEPYTACDLAPPACTTDAAHPVAASLPLLAGTLLLLGASAAP

[0137] Shorter versions are also envisioned, such as that shown in SEQ ID 5:

[0138] SEQ ID NO 5:

[0139] VIPAEEENPAFWNRQAAEALDAAKKLQPIQKVAKNLILFLGDGLGVPTVTATRILKGQKNGKLGPETPLAMDRFPYLALSKTYNVDRQVPDSAATATAYLCGVKANFQTIGLSAAARFNQCNTTRGN EVISVMRAKQAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADMPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPADASQNGIRLDGKNLVQEWLAKHQGAWYVWNRTELM QASLDQSVTHLMGLFEPGDTKYEIHRDPTLDPSLMEMTEAALRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQALTEAVMFDDAIERAGQLTSEEDTLTLVTADHSHVFSFGGYTLRGSSIFGLAPSK AQDSKAYTSILYGNGPGYVFNSGVRPDVNESESGSPDYQQQAAVPLSSETHGGEDVAVFARGPQAHLVHGVQEQSFVAHVMAFAACLEPYTACDLAPPACTTDAAHPVAASLPLLAGTLLLLGASAAP

[0140] In some embodiments, a recombinant AP as described herein is employed in the present invention. The AP variant exhibits enhanced stability and good selectivity for LPS (Kifer-Moreira T et al., PLoS ONE 2014; 9: e89374). The sequence is listed in SEQ ID NO 6.

[0141] SEQ ID NO 6:

[0142] VIPAEEENPAFWNRQAAEALDAAKKLQPIQKVAKNLILFLGDGLGVPTVTATRILKGQKNGKLGPETPLAMDRFPYLALSKTYNVDRQVPDSAATAYLCGVKANFQTIGLSAAARFNQC NTTRGNEVISVMNRAKQAGKSVGVVTTTRVQHASPAGTYAHTVNRNWYSDADMPASARQEGCQDIATQLISNMDIDVILGGGRKYMFPMGTPDPEYPADASQNGIRLDGKNLVQEWLAKHQ GAWYVWNRTELMQASLDQSVTHLMGLFEPGDTKYEIHRDPTLDPSLMEMTEAALRLLSRNPRGFYLFVEGGRIDHGHHEGVAYQALTEAVMFDDDAIERAGQLTSEEDTLTLVTADHSHVFS FGGYPLRGSSIFGLAPGKARDRKAYTVLLYGNGPGYVLKDGARPDVTESESGSPEYRQQSAVPLDEETHGGEDVAVFARGPQAHLVHGVQEQSFVAHVMAFAACLEPYTACDLAPPACTTD

[0143] In some embodiments, the AP sequence employed is functionally equivalent to a human, recombinant or bovine AP protein as described herein, in other words, such functional equivalents are defined by their ability to dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the blood of a patient in need thereof.

[0144] The present invention also includes variations in the length of the amino acid sequences described herein. A skilled artisan is able to provide amino acid sequence variants that are longer or shorter than SEQ ID NO 1, 2, 3, 4, 5 or 6 that will still exhibit sufficient similarity to the AP described herein to provide the desired results. For example, as described herein, shorter variants of SEQ ID NO 1, 2, 3, 4, 5 or 6 that contain 10, 20, 30, 40, 50 or up to 100 amino acids less than the full-length form may also achieve effective target dephosphorylation. Active fragments of AP are therefore also contemplated. In addition, as described herein, longer variants of SEQ ID NO 1, 2, 3, 4, 5 or 6 that contain any given additional sequence that is 10, 20, 30, 40, 50 or up to 100 amino acids more than the AP sequence may also achieve effective results.

[0145] In other embodiments of the present invention, the AP protein used may comprise or consist of an amino acid sequence having at least 50%, 60%, 70%, 80%, 90% or 95% sequence identity with SEQ ID NO 1, 2, 3, 4, 5 or 6. Preferably, the sequence variant comprises at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO 1, 2, 3, 4, 5 or 6, and preferably exhibits a function similar to that of the AP described herein. In a preferred embodiment, the AP protein used has at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO 1, 2, 3, 4, 5 or 6.

[0146] In some embodiments, the present invention encompasses any AP that is capable of dephosphorylating extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in the patient's blood when immobilized on a membrane, support or other matrix as described herein.

[0147] As used herein, "dephosphorylation" refers to the removal of phosphoric acid (PO) from an organic compound, such as by hydrolysis. 4 3- ) group. Phosphorylation is a reversible post-translational modification that can be removed by dephosphorylation. Dephosphorylation and its counterpart phosphorylation usually activate and inactivate enzymes, or regulate signal transduction, by separating or linking phosphate esters and anhydrides. The significant occurrence of dephosphorylation is the conversion of ATP to ADP and inorganic phosphate. Dephosphorylation uses a type of hydrolase or hydrolase that cleaves ester bonds. The main subclass of hydrolases used for dephosphorylation is phosphatases, such as AP. Phosphatases remove phosphate groups by hydrolyzing phosphate monoesters into phosphate ions and molecules with free hydroxyl (-OH) groups.

[0148] Membrane materials and hemofilters:

[0149] According to one aspect, the device is a hollow fiber membrane dialyzer designed for hemodialysis, and wherein the lumen side of the hollow fibers of the hemodialyzer is functionalized with alkaline phosphatase (AP). According to one embodiment of the present invention, the device according to the present invention comprises a hollow fiber membrane selected from the group consisting of a hemodialysis hollow fiber membrane prepared from polysulfone, polyethersulfone or polyarylethersulfone and polyvinylpyrrolidone.

[0150] The hollow fiber membranes which can be advantageously used to provide the device according to the invention preferably have an inner diameter of 100 to 500 μm. According to one embodiment of the invention, the hollow fiber membranes have a wall thickness of 20 to 150 μm. Lower wall thicknesses are disadvantageous because the mechanical properties of the fiber are reduced during production and during its use in the device according to the invention. Higher wall thicknesses are disadvantageous because they require an increased period of time for the phase inversion process, resulting in unstable process conditions and unstable membranes.

[0151] Polysulfone / PVP-based membranes that can be used according to the invention, including membranes made from PES or PAES, can be divided into "high flux" (HF), "medium cut-off" (MCO) and "high cut-off" (HCO) membranes. All these membranes have been described in the literature.

[0152] Even if the same polymer is involved, their production is associated with the defining properties that these membranes have. Today, diffusion-induced phase separation processes are mainly used, which allow fine-tuning of polymer combinations as well as pore size and diffusion transport properties. The polymer is dissolved in a suitable solvent and precipitation occurs in a non-solvent bath, preferably water. The concentration of the polymer in the polymer solution is about 20 wt%, depending on the specific recipe. The polymer solution is pumped through an annular die (spinneret) to form a hollow fiber. The internal void of the hollow fiber is formed by a pore liquid (a mixture of solvent and non-solvent), which is introduced into the interior of the spinneret. In the third step, the hollow fiber is guided through a non-solvent bath. The non-solvent bath and the pore liquid need to convert the homogeneous liquid polymer solution into a two-phase system by diffusion solvent / non-solvent exchange (impregnation precipitation). The demixing process stops at the glass transition point of the polymer-rich phase. A rigid membrane structure is formed during the polymer-rich phase, while membrane pores are formed during the liquid polymer-poor phase. The main influences on the membrane properties during the manufacturing process are the composition, viscosity and temperature of the polymer solution, the use of additives, the ability to crystallize or aggregate, the nozzle design, the composition of the coagulation bath, the conditions between the nozzle and the coagulation-bath inlet, in particular the temperature and humidity in the spinning shaft, and possible finishing treatments, such as drying and / or sterilizing the membrane by heating or radiation (e.g., see Zweigart et al. (2017) 4.11 Progress in the Development of Membranes for Kidney-Replacement Therapy. In: Drioli, E., Giorno, L. and Fontananova, E. (eds.), Comprehensive Membrane Science and Engineering, second edition. vol. 4, pp. 214-247. Oxford: Elsevier). As described in EP3010629, the performance of the final fiber bundle and filter is additionally affected by the fiber fluctuations, thereby providing them with a wavy geometry. The final assembly of the filter is described in Zweigart et al., 2017. The membranes and filters according to the present invention can be sterilized by several well-known means. It would be advantageous to sterilize the device according to the invention by irradiation with gamma rays or electron beams. Both are standard techniques. The radiation dose for gamma ray sterilization is 5 to 40 kGy. Steam sterilization can also be used and is the method of choice in terms of environmental impact and patient application. Despite the various membrane production methods available, the skilled person will have no difficulty in selecting and manufacturing a suitable membrane.

[0153] According to one aspect of the present invention, a blood purification device includes a high-flux membrane having an AP fixed thereon. The high-flux membrane used in the device, e.g. 170H (Baxter), (Baxter), EMIC2 (Fresenius Medical Care) or F180NR (Fresenius Medical Care) has been on the market for several years. Their production methods have been described in, for example, US 5,891,338 and EP2113298. Another known membrane is used in the UP series. These products are based on polyphenylene. In the polysulfone or polyethersulfone based membranes mentioned in this application, the polymer solution generally contains 10 to 20% by weight of polyethersulfone or polysulfone as hydrophobic polymer and 2 to 11% by weight of a hydrophilic polymer, in most cases PVP, wherein the PVP generally consists of low-molecular and high-molecular PVP components. The resulting high-flux membrane generally consists of 80 to 99% by weight of the hydrophobic polymer and 1 to 20% by weight of the hydrophilic polymer. During the production of the membrane, the temperature of the spinneret is generally 25 to 55°C. The polymer combinations, process parameters and performance data can be retrieved from the references mentioned or from publicly available data sheets. As used herein, the expression "high flux membrane" further refers to a membrane having a MWRO of 5 kDa to 10 kDa and a MWCO of 25 kDa to 65 kDa, as determined by dextran sieving measurements according to Boschetti-de-Fierro A et al. (Extended characterization of a new class of membranes for blood purification: The high cut-off membranes. Int J Artif Organs 2013; 36 (7), 455-463). The average pore radius before blood contact is in the range of 3.5 to 5.5 nm, wherein the pore size is determined by MWCO based on the dextran sieving coefficient according to Boschetti-de-Fierro et al. (2013).

[0154] According to one aspect of the present invention, a blood purification device includes a high cut-off membrane on which AP is fixed. This type of membrane was first disclosed in WO 2004 / 056460, in which some early high cut-off membranes were described, which have been mainly used to treat sepsis by eliminating inflammatory mediators associated with sepsis. Advanced dialyzers using high cut-off membranes on the current market are, for example, septeX and Theralite (Baxter). Known uses of the advanced high cut-off membranes include treating sepsis (EP2281625), chronic inflammation (EP2161072), amyloidosis and rhabdomyolysis, and treating anemia (US2012 / 0305487), and the most explored therapy to date is treating myeloma kidney patients (US 7,875,183B2). Therefore, these membranes and filters are already well adapted to acute environments, and are therefore particularly suitable for treatment according to the present invention. As used herein, the expression "high cut-off membrane" or "multiple high cut-off membranes" refers to membranes having a MWRO of 15-20 kDa and a MWCO of 170-320 kDa. The membrane is further characterized by a pore radius on the surface of the selective layer of the membrane of 8-12 nm before blood contact. For the avoidance of doubt, the determination of the MWRO and MWCO for a given membrane and used herein is carried out according to the method of Boschetti-de-Fierro et al. (2013). For example, methods for producing high cut-off membranes have been described in the above-mentioned references. As already disclosed in WO2004 / 056460, a key element in their production is the careful control of the temperature of the spinning process, i.e. the spinneret temperature, the spinning shaft temperature and the coagulation bath temperature relative to the spinning conditions, for the production of high flux membranes with approximately the same polymer composition. In addition, for the production of the latest high cut-off membranes such as the Theralite membrane, the ratio of water to solvent (H 2 O / solvent) also changes slightly to lower values, while the polymer content in this solution can be similar or identical for the production of high flux membranes, for example, Revaclear membranes. For the avoidance of doubt, the MWCO and MWRO values ​​used to describe the prior art membranes and the membranes according to the present invention have been measured before contact with blood or plasma, as the sieving properties of the synthetic membranes may change upon such contact.

[0155] According to one aspect of the present invention, a blood purification device includes a medium cut-off membrane on which AP is fixed. WO 2015 / 118045A1, WO 2015 / 118046A1 and, for example, Zweigart et al. (2017) describe in detail the MCO membrane and the filter comprising it and its production method. The first dialyzer on the market to include such an MCO membrane is Theranova (Baxter). The medium cut-off membrane and dialyzer fill the performance gap between high-flux and high-cut-off dialyzers. As used herein, the expression "medium cut-off membrane" refers to a membrane having an initial molecular weight retention (MWRO) of 9.0kDa to 14.0kDa and a molecular weight cut-off (MWCO) of 5kDa to 130kDa determined by a dextran sieving curve before the membrane contacts blood or blood products. Due to this very unique sieving property, the membrane greatly expands the performance of current high-flux membranes and dialyzers because they allow the removal of medium and large uremic solutes that cannot be resolved by current high-flux membranes. Therefore, they are also referred to as "membranes with increased permeability". At the same time, such membranes are able to process such higher molecular weight compounds without having to face unacceptable albumin losses during the process. Therefore, these membrane types can be used in both acute and chronic settings. For the avoidance of doubt, the expression "membrane with increased (or "extended") permeability" as used herein is equivalent to the expression "medium cut-off membrane".

[0156] According to another embodiment of the present invention, the blood purification device includes a polyacrylonitrile (PAN) membrane, which contains i) a copolymer of acrylonitrile and sodium methallyl sulfonate, ii) polyethyleneimine, and iii) heparin on which AP has been fixed. Polyacrylonitrile is the first synthetic material developed for dialysis membranes. The material of the old AN69 membrane developed by Hospal is a copolymer of acrylonitrile and sodium methallyl sulfonate. This combination is very important and includes the addition of sodium methallyl sulfonate, which seriously affects the membrane structure. These membranes are particularly suitable for treating sepsis or sepsis-induced acute renal failure, in particular for adsorbing endotoxins contained in biological fluids, for purifying certain molecules contained in blood or plasma (i.e., by dephosphorylation of target molecules described herein, and / or by extracorporeal circulation), and for reducing the systemic anticoagulation of patients during extracorporeal blood or plasma treatment.

[0157] WO 2007 / 148147 A1 also describes a method for preparing a suitable membrane. In one embodiment of the extracorporeal blood circuit, the membrane of the blood treatment device comprises 10 to 100 mg (e.g. 25 to 50 mg) of polyethyleneimine per square meter of membrane surface area and 1,500 to 10,000 UI (e.g. 3,000 to 6,000 UI, e.g. 4,500 ± 1,500 UI) of heparin per square meter of membrane surface area. In one embodiment, the blood treatment device comprises a hollow fiber membrane having an inner diameter of 180 to 260 μm (e.g. 210 μm or 240 μm). In one embodiment, the wall strength of the hollow fiber membrane is 30 to 60 μm, e.g. 40 to 50 μm. In one embodiment, the total surface area of ​​the membrane in the diffusion and / or filtration device is 1 to 3 m 2 , for example, 1.0~2.2m 2 . In one embodiment, the hollow fiber membrane in the diffusion and / or filtration device exhibits a sieving coefficient measured in bovine plasma with a protein content of 60 g / l at 37°C, wherein inulin is greater than 0.95, myoglobin is greater than 0.55 and albumin is less than 0.01. The membrane in the diffusion and / or filtration device has the ability to fix inflammatory mediators and endotoxins on its surface by adsorption. In one embodiment, the clearance of IL-6 in HDF mode with QB=400 ml / min, QD=700 ml / min and UF=100 ml / min is 20-40 ml / min. PAN membranes have a high adsorption capacity for proteins (including β2-microglobulin, cytokines and endotoxins), which makes them particularly suitable for acute treatments such as CRRT, especially for the treatment of sepsis-induced AKI, although there is a great need to improve the clinical outcomes of sepsis treatment with this type of membrane.

[0158] Some of the disadvantages of the membranes that existed in the early stages of development are now overcome by coating the membrane surface with polycationic polyethyleneimine, which has the additional benefit of binding heparin, thereby allowing lower heparin doses to be given during dialysis, an important advantage in critically ill patients. Today, this membrane is available as AN69ST and is used in commercial dialyzers such as Evodial and oXiris, which are well known for their use in CRRT. The AN69 membrane is very suitable for covalently binding any type of protein, such as a ligand or enzyme, which can be used to change the activity or composition of a biological fluid (see, for example, US 6,260,715 B1). This property of the membrane can be advantageously used to prepare a device according to the invention by fixing AP on the membrane.

[0159] According to another aspect, wherein the membrane has a pore size that allows larger proteins to pass through, for example, a plasma separation membrane as described in WO 2008 / 003610A1, the outer surface and / or pores of the hollow fiber membrane are functionalized with AP. Alternatively, only the lumen side of such a plasma separation hollow fiber membrane is functionalized with AP.

[0160] According to yet another aspect, the device may be a hemodialyzer for treating renal failure, wherein the filter further comprises a resin (eg in the form of a sponge) or a nonwoven fabric (functionalized with AP) in at least one end cap.

[0161] Patients can be treated intermittently or continuously with the device according to the invention. Continuous treatment is used exclusively for acute renal failure (ARF). Advantages compared to intermittent therapy include improved tolerability due to slower removal of solutes and water. Treatment is performed in hemodialysis mode with a blood flow of 200-500 mL / min.

[0162] In one embodiment of the invention, wherein the membrane used to provide the device according to the invention is a plasma separation membrane or is otherwise formulated to allow the target according to the invention to pass through in significant amounts with a sieving coefficient above 0.5 and preferably above 0.7 or above 0.9, the inner layer or lumen of the hollow fiber (typically the blood contact layer) is not functionalized and does not carry any AP. The AP is instead coupled to the outer layer of the hollow fiber via a connector and optionally also to at least a portion of the layer connecting the inner layer to the outer layer, i.e., the pores of the membrane. Thus, functionalization with the AP is present only on the outer filtrate layer and optionally on at least a portion of the pore surface structure connecting the outer and inner layers of the membrane. For example, this configuration can be used for the dephosphorylation of targets in whole blood, which can be transferred from the inner layer to the outer layer due to their size, while larger blood proteins are retained on the luminal side of the membrane. As blood components, including the target, pass to the outer layer of the membrane, they are modified (dephosphorylated) by the AP.

[0163] Adsorbent Materials and Adsorbent Cartridges:

[0164] The device can also be an adsorption cartridge comprising a matrix selected from resin or nonwoven material, which is functionalized with AP. This device can be the parts of the extracorporeal circuit for blood treatment, and it is configured to provide hemodialysis, hemodiafiltration, hemofiltration or plasma exchange. The device can be the unique blood treatment device in the blood circuit, or can be located at the upstream or downstream of the dialyzer in, for example, hemodialysis, hemodiafiltration or hemofiltration circuit, or can be alternatively connected to the dialyzer immediately at the blood inlet or outlet, wherein, the device is configured to perfuse with whole blood. The device can also be the parts of the extracorporeal plasma exchange circuit, wherein, the device is perfused with blood plasma or its composition. The device can also be used for therapeutic plasma exchange (TPE) treatment, wherein, blood plasma is taken out from the patient, and then replaced by a substitute, such as fresh frozen plasma.

[0165] The device may also be a hybrid filtration device that combines a hollow fiber membrane and a matrix in the filter filtrate space, as described in (WO 2014 / 079680 A1). In the present invention, the matrix consists of a resin functionalized with AP for dephosphorylating the targets described herein. Such a filter may be a component of an extracorporeal circuit configured for hemodialysis, hemodiafiltration or hemofiltration, wherein the filter is located upstream or downstream of a dialyzer for hemodialysis, hemodiafiltration or hemofiltration, or it may be used as the sole filtration device within the circuit in the absence of such a dialyzer. Such a device may be used for whole blood.

[0166] In vitro devices and methods for removing target components from a patient's blood have been described previously. For example, WO2013 / 020967 A1 discloses the use of a device and matrix for immobilizing and removing a patient's blood group antibodies. US 8,969,322 B2 describes an extracorporeal apheresis procedure for removing soluble Flt-1 receptor from a patient's blood by a device comprising dextran sulfate. Similar to these methods, the present invention employs the fixation of AP to a blood processing device and its applicability in treating infections (such as sepsis) and renal failure (preferably SA-AKI).

[0167] Thus, in one aspect, the present invention discloses a device comprising a matrix designed to specifically dephosphorylate extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP) and / or lipopolysaccharide (LPS) in a patient's blood. According to another aspect, the present invention discloses an extracorporeal circuit comprising the device and describes how such a circuit should be configured to effectively treat the blood of a patient in need. According to yet another aspect, the present invention provides a method for reducing the level of extracellular adenosine triphosphate (ATP), adenosine diphosphate (ADP) and / or phosphorylated lipopolysaccharide (LPS) in a patient's blood, comprising the step of removing a target protein from outside the patient's body by passing the patient's blood or plasma through a device according to the present invention.

[0168] According to one aspect, a device according to the invention comprising an adsorbent, for example in the form of beads, has a relative humidity of 0.5 to 50,000 m2 per device when used for whole blood perfusion (hemoperfusion). 2 According to another aspect, when used for whole plasma perfusion (therapeutic apheresis), the device according to the invention has an active surface of 0.5 to 50,000 m 2 According to another aspect, the device for blood perfusion and / or whole plasma perfusion has a surface area of ​​0.5 to 10000 m 2 active surface area.

[0169] Thus, as used herein, the expression "matrix" refers to a material that can be used to immobilize an AP according to the present invention. Such a matrix used in the context of the present invention may in some embodiments comprise a support to which the AP is bound. Thus, the support acts as a carrier for the AP, even if it must also fulfil other functions.

[0170] According to one embodiment of the present invention, the AP includes an affinity tag for fixing it to a support. Affinity tags can be used to purify proteins during protein production and / or to fix them to a support of the matrix of the present invention. The affinity tag can be a short polypeptide sequence or a complete protein, which is co-expressed as a fusion partner with the target protein. In addition to facilitating purification and rapid fixation, fusion tags are sometimes also beneficial for improving the expression and solubility of recombinant proteins. Affinity tags can be used to ensure the correct orientation of the protein, or various orientations can be used so that functional domains can be used for interaction. Different types of affinity tags are well known in the art (Terpe, Appl Microbiol Biotechnol (2003) 60: 523-533), among which polyhistidine or His 6- Tags are described in particular detail and are an option for binding the AP according to the invention to a support material. Affinity tags that can additionally be used for binding the AP to a support may be selected from the group consisting of C-myc-tags, FLAG-tags and hemagglutinin (HA)-tags.

[0171] According to another embodiment of the present invention, the AP protein can also be immobilized on a support by adsorbing the AP using a secondary ligand. This can be achieved by using an AP that has been reacted or biotinylated with biotin and then adsorbed to a support containing immobilized avidin or streptavidin. One possible biotinylation technique is to incubate the AP with N-hydroxysuccinimide-D-biotin at pH 9. These proteins can then be immobilized using non-covalent linkages of biotin to streptavidin or avidin. These linkages have a 10 13 ~10 15 M -1 The association equilibrium constant.

[0172] According to another embodiment of the present invention, as further described below and / or as described in the prior art (Cuatrecasas, J Biol Chem (1970) 245: 3059-3065; Nisnevitch et al., J Biochem Biophys Methods (2001) 49: 467-480), the AP protein is covalently linked to the support. Covalent coupling generally includes covalent non-site-specific attachment of proteins based on the use of functional groups on the support and / or protein (Nisnevitch et al., J Biochem Biophys Methods (2001) 49: 467-480, Section 2.3). According to another embodiment of the present invention, the covalent attachment of the protein is a site-specific attachment of the protein (Nisnevitch et al., J Biochem Biophys Methods (2001) 49: 467-480, Section 2.4; Makaraviciute et al., Biosensors and Bioelectronics (2013) 50: 460-471). In essence, the technical guidance for attaching antibodies to a matrix (usually performed in immunoaffinity methods) can be used to immobilize the AP protein on a support. The references disclosed herein regarding the immobilization of antibodies to a support or matrix are also considered to be potentially relevant to the immobilization of monomeric AP variants.

[0173] As used herein, the expression "support" refers to a matrix part used as a "substrate" or "support material" to which the AP according to the present invention is bound. Such supports or support materials are sometimes also referred to as "adsorbent materials" or "adsorbents", and such expressions should be included in the expression "support" used herein.

[0174] In other embodiments, references to a "support," such as when referring to a fixation method for fixating an AP, also apply to membranes, such as the membranes of the hemofilters disclosed herein.

[0175] Suitable supports according to the present invention should be homogeneous, hydrophilic, mechanically and chemically stable over the relevant pH range and temperature, have no or negligible AP leaching during use, be selective, exhibit minimal non-specific absorption, and should otherwise be blood compatible, i.e., not cause adverse reactions (including activation of the complement system or other immune pathways), have good flow properties with whole blood and / or plasma, and provide a large surface area for AP attachment.

[0176] The support can be a resin, a film or a nonwoven fabric. As used herein, the expression "resin" refers to an insoluble material that can be in the form of a translucent gel or gel beads or in the form of microporous beads with pores and opaque appearance or in the form of a sponge. This resin can be a natural or biopolymer, a synthetic polymer and an inorganic material. Agarose, glucose and cellulose beads are commonly used natural supports. Synthetic polymers or organic supports are mainly based on acrylamide, polystyrene and polymethacrylate derivatives, and porous silica and glass are some commonly used inorganic supports. Other materials that can be used according to the present invention are described as follows.

[0177] According to one embodiment of the present invention, the resin is composed of the following materials: a polymer selected from the group consisting of alginate, chitosan, chitin, collagen, carrageenan, gelatin, cellulose, starch, pectin and agarose; an inorganic material selected from the group consisting of zeolite, ceramic, diatomaceous earth, silica, glass, activated carbon and charcoal; or a synthetic polymer selected from the group consisting of polyethylene (PE), polyoxymethylene (POM), polypropylene (PP), polyvinyl chloride (PVC), polyvinyl acetate (PVA), polyvinylidene chloride (PVDC), polystyrene (PS), polytetrafluoroethylene (PTFE), polyacrylate (PAA), polymethyl methacrylate (PMMA), polyacrylamide, polyglycidyl methacrylate (PGMA), acrylonitrile butadiene styrene (ABS), polyacrylonitrile (PAN), polyester, polycarbonate, polyethylene terephthalate (PET), polyamide, polyaramid, polyethylene glycol (PEG), polyvinyl pyrrolidone (PVP), polysulfone (PS), polyether sulfone (PES), polyarylethersulfone (PEAS), ethylene vinyl acetate (EVA), ethylene vinyl alcohol (EVOH), polyamide-imide, polyaryletherketone (PAEK), polybutadiene (PBD), polybutylene (PB), polybutylene terephthalate (PBT), polycaprolactone (PCL), polyhydroxyalkanoate, polyetheretherketone (PEEK), polyetherketoneketone (PEKK), polyetherimide (PEI), polyimide, polylactic acid (PLA), polymethylpentene (PMP), poly(p-phenylene ether) (PPE), polyurethane (P The invention relates to a group consisting of polyvinyl alcohol (PVA), ...

[0178] According to another embodiment of the present invention, the support is a nonwoven fabric. As used herein, the expression "nonwoven fabric" refers to a material that is broadly defined as a sheet, fabric or mesh structure that is combined together by mechanical, thermal or chemical means but not by weaving or knitting to entangle fibers or filaments (and by perforated membranes). They form a porous structure and can be effectively used as a support material in a device according to the present invention due to their high filtration efficiency, high surface area and high permeability. Nonwoven fabrics and their production methods (including meltblown nonwoven fabrics and spunbonded nonwoven fabrics, and devices comprising such nonwoven fabrics) are known in the art and are described in, for example, EP 1 922 097 A1, WO 2007 / 025738 A2 and Zhao et al., J Mem Sci (2011), 369: 5-12. Nonwoven fabrics can be composed of the following materials: biopolymers, selected from the group consisting of polysaccharides, polylactic acid (PLA), polycaprolactone (PCL) and proteins; inorganic materials, selected from TiO 2 、SiO 2 or AlO 2 or a synthetic polymer selected from the group consisting of polypropylene (PP), polyethylene (PE), polyacrylonitrile (PAN), poly(vinyl alcohol) (PVA), polyamideimide (PAI), polyurethane (PUR), polyethersulfone (PES), polyacrylic acid (PAA), polyethylene oxide (PEO), polystyrene (PS) and polyvinylidene fluoride (PVDF).

[0179] According to another aspect, the device according to the present invention can be a filtration device as disclosed in WO 2014 / 07680A1, comprising a hollow fiber bundle membrane and a resin in the filtrate space of the device, wherein the resin is preferably composed of beads. By selecting a membrane that allows at least the relevant target to pass through the membrane wall, such a device can be configured to be used as a device for dephosphorylating a target according to the present invention. The resin in the filtrate space of the device is used as a matrix and includes a resin support, as disclosed herein or in WO2014 / 07680A1, and the AP is bound to the resin support by the method disclosed herein or other methods known in the art.

[0180] AP Fixing:

[0181] As mentioned above, the AP according to the invention may be covalently bound to a support or a membrane. In this respect, references to the support of a matrix may also apply to the membrane of the hemofilter, depending on the specific material of the membrane and the suitability for the immobilization strategy employed.

[0182] The support that forms the basis for the generation of the matrix to which the AP can be covalently attached must provide or facilitate chemical activation, thereby allowing chemical coupling of the AP. Many coupling methods used to immobilize other ligands such as antibodies or fragments thereof are well known in the art and can be used to immobilize the AP. In general, the activation chemical should remain stable over a wide range of pH values, buffer conditions and temperatures, resulting in negligible leaching of the bound protein or ligand (AP in this case). The coupling method should avoid improper orientation, multi-site attachment or steric hindrance of the AP, which may lead to masking of active enzyme sites or pockets, resulting in loss of activity. Site-specific attachment and / or spacers can be considered to fix the AP to the support. The density of APs per volume of matrix can be optimized to promote target accessibility and activity.

[0183] Coupling can be carried out through common functional groups including amines, alcohols, carboxylic acids, aldehydes, and epoxides ( Figure 6 A and Figure 6 B). Methods for preparing supports according to the invention are known in the art and are described, for example, in US 8,142,844 B2, US 2015 / 0111194 A1 and US 2014 / 0166580 A1. These references also describe spacer groups (or "linker" groups) that can be used to produce matrices according to the invention.

[0184] According to one embodiment of the present invention, AP is coupled directly or through an amine functional group with the addition of a spacer. In the first step, the amine group is introduced onto the support. Many methods can be used to introduce the amine group into the substrate according to the present invention. For example, adding an aminated polymer (e.g., aminated polyvinyl alcohol) to the polymer solution before the membrane is precipitated, or using APTMS ((3-aminopropyl) trimethoxysilane-tetramethoxysilane), simply adsorbing PEI (poly(ethyleneimine)) or other polymers onto the membrane surface, or plasma treating the membrane with ammonium or other organic amine vapors can effectively introduce amine groups onto the membrane. In the second step, a carbodiimide compound can be used to activate the carboxyl group of the protein to directly bind to the primary amine on the membrane surface through an amide bond. The most commonly used carbodiimides are water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) for aqueous crosslinking and water-insoluble DCC (N',N'-dicyclohexylcarbodiimide) for non-aqueous organic matter synthesis methods. According to another embodiment of the present invention, hydroxyl groups can be introduced into the support. Polysaccharide-based substrates, such as cellulose or cellulose derivatives, already carry OH groups on their surfaces. Hydroxyl groups can also be introduced into the substrate, for example by plasma treatment with oxygen or air. After acylation of the hydroxyl groups with succinic anhydride, the resulting O-succinate can react with the amine of the protein in the presence of carbodiimide or other coupling agents to form an amide bond. According to another embodiment of the present invention, carboxylic acid groups can be introduced into the support. By plasma treatment with carbon dioxide, carboxyl groups can be introduced onto the substrate. For protein immobilization, carbodiimide / succinimide coupling chemistry can be used for surface attachment via the amine group of AP. According to another embodiment of the present invention, carbonyl groups (aldehydes, ketones) can be introduced for subsequent AP coupling. Aldehydes can be generated on polysaccharide-based solid substrates by oxidizing OH groups with periodic acid. Primary amines of proteins (N-termini of polypeptides and side chains of lysine) can react with aldehydes by reductive amination and formation of Schiff bases. The Schiff base formed is then hydrolyzed in aqueous solution and must be reduced to an alkylamine bond for stability. Sodium cyanoborohydride is a mild reducing agent that can induce the reaction without reducing other chemical groups of the protein. According to another embodiment of the present invention, epoxy groups can be introduced into the support. Several pre-activated resins with high-density epoxy functional groups on the surface are commercially available, see below. For example, the introduction of epoxy groups on the membrane is described in WO 2005 / 026224 A1. The epoxy groups that react with nucleophiles in the ring-opening process react with the primary amines, thiols or hydroxyls of the protein to form stable secondary amines, thioesters and ether bonds. Epoxy groups react easily with thiol groups and require a buffer system close to physiological pH (pH 7.5-8.5). Epoxy groups require high pH conditions (pH 11-12) to react with hydroxyls, and require moderate alkaline conditions (pH>9) to react with amine groups.In each case, spacers of different chain lengths can be introduced between the support and the AP.

[0185] Several types of supports mentioned above and below can be advantageously used for coupling, for example, proteins for affinity chromatography. Affinity supports can be based on materials such as polysaccharides. Suitable polysaccharides are, for example, cellulose, nitrocellulose, chitosan, collagen, starch and cross-linked polysaccharide gels, such as agarose, Sephadex or Sepharose. Methods for preparing polysaccharide matrix derivatives are already known and are described, for example, in US 4,411,832 or US 3,947,352. The support can also be based on a synthetic organic support. Synthetic polymer matrices include hydrophilic and hydrophobic synthetic polymers and combinations thereof. Synthetic supports include supports selected from the group of supports composed, for example, of polyacrylamide supports or derivatives thereof; polymethacrylate supports or derivatives thereof; polystyrene supports or derivatives thereof; or polyethersulfone supports or derivatives thereof. In addition, derivatized silica, glass or azlactone beads can be used in the device according to the present invention. Such a device preferably uses an organic support. In the context of the present invention, the use of beads may be advantageous.

[0186] According to one embodiment of the present invention, the support material should be porous, wherein the pore size is 10 to 200 nm. For affinity applications, it has been found that pore sizes in the range of 30 to 200 nm or 60 to 200 nm are optimal. However, other pore sizes may also be advantageous, depending on the coupling chemistry, spacer and AP type used, and also on the target for dephosphorylation. If the support is used in the form of beads, the diameter of such beads may vary within a certain range. It may be advantageous to use beads with a diameter of 50 to 1000 μm. It may be further advantageous to use beads with a diameter of 60 to 800 μm, 100 to 700 μm, 120 to 800 μm.

[0187] According to one aspect of the invention, the support carries specific functional groups required for coupling the linker and / or AP. For example, many functionalized resins are commercially available and known to those skilled in the art. Pre-activated resin supports with or without spacers that already carry reactive groups for coupling the AP are commercially available and eliminate many steps of chemically activating the support prior to use (i.e., prior to coupling the AP). Such supports are typically resins as defined above, while for membrane and / or nonwoven supports an activation step typically must be performed prior to coupling. A variety of coupling chemistries including primary amines, sulfhydryls, aldehydes, hydroxyls and carboxylic acids are available in the commercial supports for covalent attachment of the AP. Examples of commercially available activated resins are UltraLink iodoacetyl resins, CarboLink coupling resins, Profinity TMEpoxy resin, Affi-Gel 10 and 15, Epoxy-activated Sepharose TM 6B, trifluoroethanesulfonyl chloride activated agarose, Tosoh AF amino or epoxy resin 650-M, ChiralVision Immobead TM 350、Resindion ReliZyme TM EXE 135 or SEPABEADS TM and epoxy functionalized Lifetech TM Methacrylate polymer.

[0188] According to one embodiment of the present invention, the support used for coupling AP is epoxy-functionalized because epoxy groups bind to different protein groups, such as -NH in lysine or nucleophiles (amino, thiol, phenolic). 2 Very stable covalent bonds are formed and immobilization can be achieved under mild pH and temperature conditions.

[0189] According to another embodiment of the present invention, the support is in the form of beads. According to another embodiment of the present invention, the support is an epoxy-functionalized methacrylate polymer. According to another embodiment of the present invention, the support is selected from the group consisting of Tosoh Epoxy 650-M, ChiralVision Immobead TM 350、ResindionReliZyme TM EXE 135, Resindion SEPABEADS TM and Lifetech TM According to one aspect, using Lifetech TM ECR8209FF epoxy methacrylate beads have epoxy groups as functional groups to which AP can bind. They have and a particle size of 150 to 300 μm. According to another aspect, using Lifetech TM ECR8215M epoxy methacrylate beads have epoxy groups as functional groups to which AP can bind. They have and a particle size of 300 to 710 μm. According to another aspect, using Lifetech TMECR8215F epoxy methacrylate beads have epoxy groups as functional groups to which AP can bind. They have The average pore size and particle size of 150-300μm.

[0190] According to another embodiment of the invention, the AP can also be non-covalently fixed to the support, for example ionically or by complexation. However, covalent binding is preferred to avoid the risk of leaching of the AP from the matrix into the patient's blood or plasma.

[0191] According to another embodiment of the present invention, the support is a membrane. According to one embodiment of the present invention, the support membrane is a hollow fiber membrane. According to another embodiment of the present invention, a plurality of hollow fiber membranes are formed into a hollow fiber bundle and embedded in a housing to form a filter or a filtering device. According to one embodiment, the support comprises a hemodialysis hollow fiber membrane dialyzer, wherein the filter is a hemodialyzer or a hemofilter.

[0192] The hollow fiber or flat membrane used as a support in the device according to the present invention can be composed of the following polymers, combinations of said polymers and any of these polymers modified by the introduction of functional groups, the polymer being cellulose, cellulose esters (cellulose acetate and cellulose triacetate), poly(methyl methacrylate) (PMMA), polyamide (PA), other nitrogen-containing polymers (polybenzimidazole, polyacrylonitrile (PAN), polyglycidyl methacrylate (PGMA), polyvinylpyrrolidone (PVP), polysulfone (PS), polyethersulfone (PES), polyarylethersulfone (PAES). According to one embodiment of the present invention, the membrane support according to the present invention comprises the following polymers, combinations of said polymers and any of these polymers modified by the introduction of functional groups, the polymer being selected from poly(methyl methacrylate) (PMMA), polyamide (PA), polyacrylonitrile (PAN), polyvinyl pyrrolidone (PVP), polysulfone (PS), polyether sulfone (PES), polyarylether sulfone (PAES) group. According to another embodiment of the present invention, the membrane support according to the present invention includes the following polymers, combinations of the polymers and any one of these polymers modified by introducing functional groups, the polymer is selected from the group consisting of polyamide (PA), polyacrylonitrile (PAN), polyvinyl pyrrolidone (PVP), polysulfone (PS), polyether sulfone (PES), polyarylether sulfone (PAES). According to yet another embodiment of the present invention, the membrane support according to the present invention includes the following polymers, combinations of the polymers and any one of these polymers modified by introducing functional groups, the polymer is selected from the group consisting of polyvinyl pyrrolidone (PVP), polysulfone (PS), polyether sulfone (PES) and polyarylether sulfone (PAES).

[0193] In order to carry out the coupling reaction for the subsequent binding of AP on the membrane surface, a polymer functionalization step is required. Known methods, such as those described in US2015 / 0111194 A1 and US2014 / 0166580 A1, can be used. For example, synthetic materials made of alkane chains such as polyethylene do not contain suitable functional groups for coupling molecules thereto. Therefore, suitable functional groups must be chemically introduced after polymer synthesis. One possibility for modifying polymers is the known plasma functionalization method, which allows functional groups to be introduced into polymers by selecting suitable gas plasmas. The method includes, for example, the use of ammonia plasma, wherein amino functional groups are formed on the surface of the treated polymer. Thus, treating, for example, polyethylene with ammonia plasma produces a polyethylene matrix with a certain amount of amino functional groups. These amino groups can then react with suitable functional groups of the linker (e.g., carboxyl groups). Alternatively, the matrix polymer can be functionalized by plasma activation to obtain carboxyl groups. For example, a method for functionalizing hollow fiber membranes in a continuous manner is further described in US2007 / 0296105 A1. In the method, the functional group introduced by the precursor gas can be an amino group, a carboxyl group, an aldehyde group, an ester group, an epoxy group, a hydroxyl group or a sulfonic acid group. The membranes that can be used as supports of the present invention include, for example, plasma separation membranes and hemodialysis membranes known in the art, including but not limited to: well-known high-flux membranes, high-retention membranes or medium-retention membranes. The goal of plasma separation is to have total plasma proteins containing blood in the separated plasma portion, while larger particulate components in the blood (such as blood cells and cell fragments) are retained by the membrane. In addition, such a plasma separation membrane should exhibit high surface porosity and total porosity of the membrane to achieve high filtration performance. It should also have hydrophilicity, a spontaneously wettable membrane structure, low fouling characteristics for long-term stable filtration, and low protein adsorption. Such a plasma separation membrane preferably has a smooth surface in contact with blood, thereby avoiding or minimizing hemolysis during blood treatment. During the entire treatment period, the membrane should show constant sieving characteristics and filtration behavior. It should also exhibit high biocompatibility, low complement activation or no complement activation and low thrombogenicity. Membranes suitable for plasma separation that can be used to provide a device according to the invention are known in the art and are described, for example, in EP 1 875 956 A1 or EP 1 875 957 A1. Other membranes that can be modified and used as a support in a device according to the invention, such as high flux membranes used in dialyzers, have been described in EP 2113 298 B1. Medium cut-off membranes for use in dialyzers, and EP 1 572 330 describes, for example, High cut-off membrane A1 used in dialyzers.

[0194] Medical Uses, Infections and Sepsis:

[0195] In some embodiments, the present invention relates to the use of a blood treatment device as described herein as a medicament for treating an infection, preferably a blood or systemic infection. In some embodiments, any infection associated with phosphorylated LPS or ATP or ADP can be treated. Corresponding treatment methods are also contemplated.

[0196] As used herein, "infection" within the scope of the present invention refers to a pathological process caused by pathogens or potential pathogens, organisms and / or microorganisms invading usually sterile tissues or body fluids, and preferably relates to infections caused by bacteria, viruses, fungi and / or parasites. Therefore, infection can be bacterial infection, viral infection and / or fungal infection. Infection can be local or systemic infection. For purposes of the present invention, viral infection can be considered as microbial infection.

[0197] In addition, the subject with infection may suffer from infection from more than one source at the same time. For example, the subject with infection may suffer from: bacterial infection and viral infection; viral infection and fungal infection; bacterial infection and fungal infection, and bacterial infection, fungal infection and viral infection, or suffer from a mixed infection comprising one or more infections listed herein, including potential repeated infections, such as one or more bacterial infections, and one or more viral infections and / or one or more fungal infections.

[0198] As used herein, "infectious diseases" include all diseases or conditions associated with bacterial and / or viral and / or fungal infections. In some embodiments, the diseases to be treated include all infectious diseases associated with phosphorylated LPS, or ATP or ADP, or all infectious diseases that would benefit from treatment with the immobilized AP described herein.

[0199] Subjects receiving treatment according to the present invention may also receive additional infection treatments. The medical treatment of the present invention may be an antibiotic treatment, wherein one or more "antibiotics" or "antibiotic agents" may be administered if an infection has been diagnosed or symptoms of an infectious disease have been determined. Other antibiotic agents / therapeutics or treatment strategies for infection or prevention of new infections include the use of antiseptics, decontamination products, antitoxins (such as liposomes); hygiene; wound care; surgery. Several of the above-mentioned antibiotic agents or treatment strategies may also be combined with fluid therapy, platelet transfusions, or blood product transfusions.

[0200] In a preferred embodiment of the present invention, the patient is diagnosed with sepsis. In some embodiments, the patient may have been diagnosed with severe sepsis and / or septic shock.

[0201] A large number of patients in intensive care units die from complications commonly referred to as "sepsis" or "septic shock". Sepsis is an overwhelming and life-threatening response of the body to infection that can lead to tissue damage, organ failure and death. General parameters of sepsis are fever (core temperature >38.3°C), hypothermia (core temperature <36°C), heart rate >90bpm or >2SD above normal for a specific age, tachypnea: >30bpm and altered mental status, as well as significant edema or positive fluid balance (>20mL kg-1 for more than 24 hours) and hyperglycemia (plasma glucose >110mg dL-1 or 7.7mM L-1) in the absence of diabetes. Sepsis progresses to severe sepsis when, in addition to the signs of sepsis, there are signs of organ dysfunction such as difficulty breathing (lung problems), low or no urine output (kidneys), abnormal liver tests (liver) and changes in mental status (brain). Almost all patients with severe sepsis require treatment in an intensive care unit (ICU).

[0202] Septic shock is the most severe level and is diagnosed when blood pressure drops to dangerous levels. In addition, trauma, burns, pancreatitis and ischemia-reperfusion injury belong to non-infectious causes of SIRS, systemic inflammatory response syndrome, multiple organ dysfunction syndrome ("MODS") and multiple organ system failure ("MOSF"). The mechanism of SIRS is the excessive release of host-derived inflammatory mediators, referred to herein as toxic mediators ("TM"). TM includes various cytokines (tumor necrosis factor, TNF; interleukins; interferons), various prostaglandins, various coagulation factors (platelet activating factor, PAF), various peptidases, reactive oxygen metabolites, and various poorly understood peptides that cause organ dysfunction (myocardial depressor factor, MDF). If the inflammatory response is excessive, damage or destruction of important organ tissues may lead to multiple organ dysfunction syndrome ("MODS"), including acute renal failure.

[0203] Sepsis can also be defined as “life-threatening organ dysfunction caused by a dysregulated host response to infection”. Septic shock is defined as an increase in lactate levels above 2 mmol L-1 in the absence of hypovolemia and initiation of vasopressor therapy to maintain mean arterial pressure above 65 mmHg. Organ dysfunction is defined as an increase of two or more points in the Sequential Organ Failure Assessment (SOFA) scoring system (Gül et al., Turk J Anaesthesiol Reanim. 2017 Jun;45(3):129–138). According to the qSOFA scoring system, sepsis is indicated if two of the following three clinical criteria are positive: 1. altered mental status (GCS score <15); 2. systolic blood pressure <100 mmHg; 3. respiratory rate >22 / min.

[0204] As used herein, the expression "sepsis" also applies to any condition that satisfies the aforementioned post-infectious sepsis parameters or the aforementioned qSOFA screening criteria, wherein an increase of two or more points in the Sequential Organ Failure Assessment (SOFA) scoring system indicates sepsis and / or qSOFA clinical criteria. For clinical operationalization, organ dysfunction may preferably be manifested as an increase of 2 or more points in the Sequential Organ Failure Assessment (SOFA) score, which is associated with an in-hospital mortality rate greater than 10%. Septic shock may be defined as a subset of sepsis in which particularly severe circulatory, cellular, and metabolic abnormalities are associated with a greater risk of mortality than sepsis alone. Patients with septic shock may be clinically identified by the need for vasopressors to maintain a mean arterial pressure of 65 mmHg or higher and a serum lactate level greater than 2 mmol / L (>18 mg / dL) in the absence of hypovolemia.

[0205] As used herein, the "Sequential Organ Failure Assessment Score" or "SOFA score" is a score used to track the status of a patient during hospitalization in an intensive care unit (ICU). The SOFA score is a scoring system used to determine the degree of organ function or failure rate of a person. The score is based on six different scores, with one score each for the respiratory system, cardiovascular system, liver, coagulation, kidneys, and nervous system. Both the average and highest SOFA scores are predictors of outcome. The increase in SOFA scores within the first 24 to 48 hours of the ICU predicts a mortality rate of at least 50% to 95%. A score below 9 predicts a mortality rate of 33%, while a score above 14 may be close to or above 95%. According to the SEPSIS-2 definition, the term "sepsis" also includes severe sepsis or septic shock. The term "sepsis" also includes subjects who meet the SEPSIS-3 definition. The term "sepsis" used herein relates to all possible stages of sepsis development.

[0206] It is therefore an object of the present invention to provide a device for the extracorporeal purification and treatment of blood that can be used to treat patients suffering from sepsis, especially in acute situations and when drugs are not indicated or effective. Another object of the present invention is to provide a device for the extracorporeal purification and treatment of blood that can be used to treat patients suffering from organ failure, especially renal failure and sepsis.

[0207] Acute renal failure (ARF) and sepsis-associated acute kidney injury (SA-AKI):

[0208] Acute renal failure (ARF), also interchangeably referred to as acute kidney injury (AKI), is a rapid loss of the filtering function of the kidneys characterized by metabolic acidosis, high potassium levels, and fluid imbalance. This condition is usually marked by an increase in serum creatinine concentration or blood urea nitrogen (BUN) concentration. As used herein, the term "acute kidney injury (AKI)" or "acute renal failure" involves a sudden and sustained decline in kidney function.

[0209] The diagnostic criteria are usually, but not limited to, as follows: serum creatinine rises > 0.3 mg / dl (26.5 μmol / L) within 48 hours; or serum creatinine increases to 1.5 times the baseline, known or presumed to occur within 7 days; or urine output < 0.5 ml / (kg·h) for 6 to 12 hours. Depending on the severity, the condition is divided into stages 1, 2, and 3. The Kidney Disease: Improving Global Outcomes (KDIGO) group defines these stages as: 1: serum creatinine increases 1.5-1.9 times from baseline or increases >26.4umol / L (0.3mg / dl) or urine output <0.5ml / kg / h during a 6-hour block, 2: serum creatinine increases 2.0-2.9 times from baseline or urine output <0.5ml / kg / h during two 6-hour blocks, and, 3: serum creatinine increases >3 times from baseline or increases to >353umol / L (4mg / dl) or initiation of renal replacement therapy or urine output <0.3ml / kg / h during more than 24 hours or anuria for more than 12 hours. As used herein, the term "infection-related renal dysfunction" refers to any loss of renal function in a patient with an infection.

[0210] Renal dysfunction has been known to be associated with a variety of infectious pathogens since the 1970s (Zech et al., Adv Nephrol Necker Hosp. 1971; 1: 231-58.). Without limitation, renal damage is known to occur in acute nephropathy following septicemic Staphylococcus, Leptospira, and Rickettsial infections. Gram-negative sepsis, possibly through septic shock, is more likely to cause tubulopathy. Hemolytic sepsis is known to cause tubular necrosis. In severe cases, pulmonary or urinary tract infections may also lead to renal dysfunction.

[0211] As used herein, the term "sepsis-associated acute kidney injury (SA-AKI)" refers to AKI in patients with sepsis. Sepsis is the most common cause of ARF. When blood cultures are positive, about 19% of patients with moderate sepsis, 23% of patients with severe sepsis, and 51% of patients with septic shock will develop ARF. Sepsis is different from a single mechanism of its etiology. It is associated with a whole cellular mechanism (adaptive and maladaptive) that reinforces each other and ultimately leads to clinical SA-AKI. The microcirculation is an important physiological compartment where these mechanisms come together and exert their comprehensive and harmful effects. These mechanisms include endothelial dysfunction, inflammation, coagulopathy, and adaptive cellular responses to injury. This ultimately leads to renal failure, and the key event in the early dysfunction of the kidney during sepsis appears to be bioenergetic stress of tubular epithelial cells in response to amplified inflammatory signals generated by peritubular microvascular dysfunction.

[0212] Thus, the present invention addresses the negative effects of renal failure and any pathology caused by, for example, LPS. Lipopolysaccharide (LPS) can cause a variety of acute pathophysiological effects, such as fever, lethality, macrophage and B lymphocyte activation, especially during sepsis. Thus, the present invention provides a method of ameliorating the negative effects of LPS while providing RRT. BRIEF DESCRIPTION OF THE DRAWINGS

[0213] The present invention is further described by the following figures. These are intended to represent a more detailed description of various preferred non-limiting embodiments or aspects of the present invention, without limiting the scope of the invention described herein.

[0214] Figure 1 : Schematic diagram of a preferred embodiment of the present invention, showing an AP coupled to the lumen of a hollow dialysis membrane, connected to a blood treatment device in a device for an extracorporeal blood treatment circuit.

[0215] Figure 2 : Results of immobilized alkaline phosphatase activity assay.

[0216] (A) Figure 2 A shows a standard curve of the absorbance (405 nm) of dephosphorylated pNPP used to determine the activity of AP on the modified membrane. The measured activity of the membrane samples with immobilized AP is shown in the table in μmol of pNPP min-1 together with the weight of each sample. The activity was extrapolated to the activity of the entire dialyzer using the sample membrane weight and the membrane weight of the entire dialyzer.

[0217] (B) Figure 2B shows the absorbance measured when a pNPP solution reacts with AP immobilized on epoxy resin (ECR8209F) and NH2 (ECR8409F) beads. The development of absorbance over time was measured after mixing the resin with the commercial pNPP solution in a ratio of 1:4. The absorbance was corrected for the signal observed when the unmodified resin does not contain AP.

[0218] Figure 3 : Schematic diagram of an extracorporeal treatment circuit comprising a blood treatment device. The device can be a cartridge or filter comprising a membrane, resin or nonwoven based support to which an AP has been incorporated. The circuit can be operated in a hemoperfusion mode. In the case where the blood treatment device is a hollow fiber membrane filtration device, the treatment mode can be hemodialysis, hemodiafiltration, hemofiltration or hemoperfusion with a filter having a closed dialysate / filtrate port.

[0219] Figure 4 : Schematic diagram of an extracorporeal treatment circuit including a blood treatment device. The device may be an adsorption cartridge comprising a resin or a nonwoven fabric or a filter comprising a membrane to which the AP has been respectively bonded. The blood treatment device may be located upstream of a separate hemodialyzer (arranged before the dialyzer, Figure 4 A) or downstream of the hemodialyzer (post-dialyzer setting, Figure 4 B). The non-functionalized hemodialyzer in the circuit can be operated in different treatment modes (including hemodialysis, hemodiafiltration or hemofiltration mode) according to medical needs.

[0220] Figure 5 : Schematic diagram of an extracorporeal processing circuit including a blood processing device. The device is perfused with plasma. In the embodiment shown, a plasma separation filter is used to separate plasma from whole blood. The plasma filter produces a plasma fraction containing a target protein through a pore size of 0.03 μm to 2 μm. The plasma is perfused through a blood processing device that includes a matrix based on a nonwoven fabric, resin or membrane support to which the AP has been bound.

[0221] Figure 6 : Schematic diagram of covalent coupling of target protein to epoxy activated or amino support. The support can be a resin, a membrane, including a hollow fiber membrane, a flat membrane or a fiber mat or a non-woven fabric. (A) shows the direct coupling of the protein to the support through the amino group of the protein (Example 6). (B) shows that the covalent fixation of the enzyme is based on the use of amino resins. The amino resin can be preactivated with glutaraldehyde and then used for covalent fixation of the enzyme. The aldehyde group reacts quickly with the amino group of the target protein and forms a Schiff base (imine), thereby forming a stable multi-point covalent bond between the enzyme and the support. The imine double bond can be further reduced with borohydride.

[0222] Figure 7: A scanning electron microscope (SEM) image of an AN69 hollow fiber is shown together with a schematic setup of how such hollow fibers can be used for dialysis when used in bundles.

[0223] Figure 8 : Results of AP activity testing of micromodules after gamma sterilization.

[0224] Example

[0225] The present invention is further described by the following examples. These are intended to provide support for the operability of many preferred non-limiting embodiments or aspects of the present invention, but not to limit the scope of the invention described herein.

[0226] Example 1: Immobilization of AP on hemofilter membrane

[0227] Membrane preparation:

[0228] In a proof of principle method of the invention, alkaline phosphatase (AP) was immobilized on various membranes. The table describes the different components of the membranes used as a percentage of the solution used to cast the membrane (weights of 50 g total solution weight are given in brackets).

[0229] Table: Membrane components

[0230]

[0231] *Dissolve the citric acid in the water first, then dissolve the chitosan overnight before adding to the rest of the mixture.

[0232] Under continuous stirring, the components are dissolved at 60°C overnight. After all components are completely dissolved, the solution is removed from the oil bath and cooled for 1 hour. A flat film is cast on an electronic casting machine using a 100μm knife. The knife is placed on a clean glass plate and about 10mL of polymer solution is applied. The engine is then started at a speed of 25mm / s until the polymer film covers the entire glass plate. The engine returns to its starting position, the knife is removed, and the coated glass is placed in a 500mL softened water bath. After 5 minutes, the water is replaced with clean water, and the process is repeated after 2 hours. The film is placed in the water bath for another 4 hours and then placed in vacuum at 60°C overnight to completely remove all NMP.

[0233] Alkaline phosphatase coupled to membrane:

[0234] The membrane was cut into small pieces (approximately 1 x 1 cm) and its exact weight was determined (usually about 3-7 mg). A total of 5 mg of alkaline phosphatase (Santa Cruz Biotechnology) with an activity of 4000 U / mg and a protein content of 24.5 mg / mL was divided into 5 μL aliquots, each containing 100 U / μL. For each fixation experiment, a 5 μL aliquot was dissolved in 1 mL of 1 mM CaCl 2 0.5 M TRIS, pH 8.5, to obtain a 500 U / mL solution. The membrane was placed in a 900 μL Millipore HO 2 O, 100 μL of 500U / mLAP and 2.5mg / mL EDC in an EP (Eppendorf) tube. The membrane was then kept on a drum shaker for 3-4 hours at room temperature and placed overnight at 4°C. After 18 hours, the membrane was placed in 50mL of 0.5M NaCl solution and placed on a tumbler shaker to remove unbound AP. After 4 hours, the wash solution was replaced with 1x PBS and placed on a drum shaker for another 2 hours. Alternatively, the membrane was placed in an EP tube containing 1mL of MES buffer containing 2.5mg / mL EDC for 30 minutes, removed and washed, and then placed in 1mL of 50U / mLAP overnight.

[0235] Activity determination of immobilized alkaline phosphatase:

[0236] After the last washing step, the membrane was placed in 1 mL of p-nitrophenyl phosphate (pNPP) liquid substrate system (Sigma Aldrich). To determine the activity, 75 μL of pNPP solution was removed and the absorbance was measured at t = 10 min, 20 min, 30 min and 60 min. At each time point, the absorbance of the previous time point was determined again, and the change in absorbance over time was used to assess the release of alkaline phosphatase from the membrane (because the release causes continuous color development and the readings at subsequent time points will show an increase in absorbance). The absorbance was then compared with the absorbance of a standard concentration of dephosphorylated pNPP solution, and the activity of alkaline phosphatase on the membrane was measured in μmol of pNPP converted per minute.

[0237] result:

[0238] The results for the two different membranes showed that the measured activities were between 0.06 and 0.10 μmol min -1 To clarify the results and compensate for the weight of the different samples of membrane, the activity was extrapolated to the activity of the entire dialyzer (containing 20 g of membrane), which indicated an activity of approximately 560 μmol min -1The release of alkaline phosphatase was minimal in all cases and, with one exception, did not exceed the standard deviation of the mean activity. The results of these experiments are shown in Figure 2 As shown in A.

[0239] Example 2a: Immobilization of AP onto epoxy-functionalized adsorbent resin:

[0240] First, equilibrate the resin. The resin is washed with fixation buffer and filtered. A resin / buffer ratio of 1 / 1 (w / v) is preferred. A fixation buffer that is compatible with the AP is selected. This process is repeated 2 to 4 times. The AP solution is prepared by dissolving the protein in the fixation buffer. For example, 100 to 200 mg of AP can be loaded per gram of wet resin. The protein concentration can be determined by using a standard protein content assay. Dissolve the AP in a sufficient amount of buffer to obtain a resin / buffer ratio of 1:4 (w / v). This ratio can be optimized depending on the protein used (the range can vary between 1:1 and 1:4). Immobilization begins with transferring the fixation buffer containing the AP protein to the immobilization vessel. Then an epoxy-functionalized resin, such as the one described herein, is added. Lifetech TM Resin. The slurry is gently mixed at 70-80 rpm for 18 hours and then allowed to stand for an additional 20 hours. Magnetic stirring should be avoided during protein immobilization as this will damage the beads. Depending on the stability of the protein, immobilization can be performed at a temperature of 20°C to 30°C. Immobilization should not be performed at high temperatures as this will cause degradation (hydrolysis) of the epoxy resin ring and promote microbial growth. Finally, the liquid phase is filtered out and collected. The protein content in the liquid is determined and the immobilization yield is calculated. The resin is then washed with wash buffer. The process is repeated 2 to 4 times with gentle stirring or column washing. Additional washing steps can be performed using a buffer containing 0.5 M NaCl to completely desorb non-covalently bound proteins. Excess water is removed by filtration. The immobilized AP protein can then be characterized based on moisture content and specific binding activity.

[0241] Example 2b: Immobilization of AP on amino-functionalized adsorbent resin:

[0242] First, the resin is equilibrated. The resin is washed with the fixation buffer and filtered. A resin / buffer ratio of 1:1 (w / v) is preferred. A fixation buffer that is compatible with the AP protein is selected. In the second step, a 2% glutaraldehyde buffer is prepared starting from a solution of 25% (w / v) glutaraldehyde. A 2% glutaraldehyde (v / v) solution is prepared using the fixation buffer. In the third step, the amino resin is activated by adding the 2% glutaraldehyde buffer prepared in step 2 to the resin. The optimal volume of the 2% glutaraldehyde buffer should be in the range of a resin / buffer ratio of 1:4 (w / v). The slurry is mixed at 20°C to 25°C for 60 minutes. The beads are then filtered and washed with the fixation buffer at a resin / buffer ratio of 1:4 (w / v). Storing the preactivated resin for more than 48 hours should be avoided. The beads are then ready for the fixation step. In the fourth step, an AP protein solution is prepared. To this end, the protein is dissolved in the fixation buffer. For example, 1 mg to 100 mg of AP protein can be loaded per gram of wet resin. Protein concentration can be determined by using a standard protein assay.

[0243] Dissolve the protein in buffer to obtain a 1:4 (w / v) resin / buffer ratio. This ratio can be optimized in further experiments (the range can vary from 1:1–1:4). In the fifth step, the protein is immobilized. Transfer the immobilization buffer to the immobilization vessel and add the preactivated amino resin prepared in step 3 (e.g. from Lifetech TM ). The slurry is gently mixed at 70-80 rpm for 18 hours. Magnetic stirring should be avoided during the immobilization process as this will damage the beads. Depending on the stability of the AP protein, immobilization can be performed at 20°C to 30°C. Immobilization should not be performed at high temperatures as this may cause side reactions of the aldehyde groups on the resin formed in step 3. Finally, the liquid phase is filtered out and collected. The protein content in the liquid is determined and the immobilization yield is calculated. The resin is then washed with wash buffer. This process is repeated 2-4 times with gentle stirring or column washing. Additional washing steps can be performed using a buffer containing 0.5 M NaCl to completely desorb non-covalently bound proteins. Excess water is removed by filtration. The immobilized AP protein can then be characterized based on moisture content and specific enzyme activity.

[0244] Activity determination of immobilized alkaline phosphatase:

[0245] The epoxy resin and amino-functionalized resin treated to immobilize AP as described above were then placed in 1 mL of p-nitrophenylphosphonic acid (pNPP) liquid substrate system (Sigma Aldrich).

[0246] To determine activity, 75 μL of the pNPP solution was removed and the absorbance was measured over time as described above. The absorbance was then corrected for any background signal obtained when the unmodified resin was not treated with AP.

[0247] result:

[0248] Results from epoxy and amino-functionalized resins indicate that APs were effectively immobilized and active based on the commercial pNPP solution used in this assay. Figure 2 As shown in B.

[0249] Example 3: Immobilization of AP onto membrane under different pH conditions:

[0250] Polyacrylonitrile AN69 membranes were used to determine the optimal pH conditions for AP binding to the membrane and were validated by subsequent AP activity testing. The pH optimization tests were performed using "M-Filter" membranes (MF150) prepared as "micromodules", i.e. small filters that are commonly used to test membrane properties. The tests described were performed on AN69 micromodules, each of which contained fiber bundles, wherein each bundle contained approximately 40 hollow fibers.

[0251] AN69 is an established polymer for dialysis applications and is a copolymer of acrylonitrile and sodium methallyl sulfonate. The sulfonate groups form a negatively charged hydrophilic membrane which forms a hydrogel structure by retaining water. This composition of the membrane allows the adsorption of medium-sized proteins. Figure 7 A scanning electron microscope (SEM) image of an AN69 hollow fiber and a schematic setup of how such a fiber bundle can be used in dialysis are shown. The inner surface of the AN69 micromodule is approximately 1 cm per fiber. 2 This corresponds to 40 cm per micromodule. 2 inner surface.

[0252] The micromodule was connected using common hoses (Promedt) to a pump station, by means of which the liquid (pump from Ismatec) could be pumped at a controlled rate through the micromodule.

[0253] To prepare the immobilized AP on the AN69 micromodule, the micromodule was coated with polyetherimide (PEI) before linking glutaraldehyde (GA) to the AP. Due to the presence of negative charges on the AN69 membrane, positively charged polymers can be linked via ionic interactions. In this case, PEI carrying positively charged amino groups was used. PEI linkage is usually performed in an acidic medium, so the amino groups are protonated and the charge density increases. By using citric acid in the process, more PEI can be bound to the membrane compared to alkaline media. The optimization of PEI processing will be discussed in more detail below.

[0254] After PEI treatment, the AN69 membrane was treated with glutaraldehyde (GA) before immobilization of AP. Glutaraldehyde acts as a bifunctional spacer, generating a pre-activated polymer surface to bind the enzyme. After surface pre-activation, the free aldehyde groups of glutaraldehyde are available to enable binding of another component via the same reaction. In this case, AP is subsequently covalently bound via the free amino groups in a second modification step.

[0255] Therefore, the AN69 micromodule is:

[0256] - Rinse to remove glycerol (using reverse osmosis water, inject 5 mL per micromodule at 1.5 mL / min, then inject 5-20 mL at 3 mL / min). Visually adjust the volume to remove air,

[0257] - treated with PEI (PEI in 200 mg / kg citric acid solution, 20 mL per micromodule, flow rate 3 mL / min),

[0258] - Rinse (20 mL per micromodule in PBS at a flow rate of 3 mL / min)

[0259] - Treatment with glutaraldehyde (2.5% GA solution, 40 mL per micromodule, flow rate 1.5 mL / min),

[0260] - Rinse (50 mL per micromodule in PBS at a flow rate of 1.5 mL / min)

[0261] - Treatment with AP (1000 U AP per micromodule in PBS, 20 mL recirculated at 1.5 mL / min for 16 hours), and

[0262] - Final rinse (50 mL per micromodule in PBS at a flow rate of 1.5 mL / min).

[0263] As a readout, the activity of the immobilized AP is assessed. A test solution containing the pNPP substrate (disodium p-nitrophenylphosphate hexahydrate) and magnesium chloride and zinc chloride matrices in PBS buffer is used. To quantify the AP activity on the membrane, individual fibers are cut from the minimodule. The module is opened, the fibers are washed with PBS, and each fiber is then placed in a well of a plate. The fibers are cut into pieces of appropriate size and the pNPP test solution is applied. Readings are taken at 60 minutes and 120 minutes. As usual, the samples are measured against a standard solution of the free enzyme, so the results are expressed as the activity of the free AP under ideal standard (alkaline) conditions.

[0264] To evaluate the optimal pH conditions for AP immobilization, two processes were modified, namely (1) coating the membrane with glutaraldehyde (hereinafter referred to as "GA-pH") and (2) subsequent immobilization of AP (hereinafter referred to as "AP-pH"). For each of these treatments, multiple pH values ​​of the relevant solutions were tested.

[0265] The following table shows the results of several protocols (I-IV) and the results in U / cm after 60 and 120 minutes. 2 The results are for AP activity in units. To simplify the discussion, the corresponding average values ​​were calculated and displayed.

[0266]

[0267]

[0268] Experiments under II gave 0.38-0.45 U / cm 2 Reproducible high AP activity, which is empirically estimated to be the minimum limit of therapeutically relevant activity (0.05 U / cm 2 Other pH conditions also seemed to be effective, but the reproducibility was not as good as in Experiment II.

[0269] Example 4: Immobilization of AP onto beads under different pH conditions:

[0270] Experiments similar to those in Example 3 above were performed using amino-functionalized beads (Purolite Lifetech ECR 8408F) as a matrix for immobilizing AP. The beads were pre-treated with GA at different pH values ​​and then treated with AP at different pH values.

[0271] The experiments were performed in 96-well plates using GA / AP treated beads in 900 μL of pNPP solution under constant stirring. 75 μL of the reaction was removed and analyzed at 10, 30, 60 and 120 min in separate 96-well plates using the above mentioned photometric activity measurements.

[0272] The table below summarizes the AP activity measured after 10 minutes using beads as solid matrix.

[0273]

[0274] From the above results, it can be seen that the AP activity is maintained after coupling to the beads, and the most effective immobilization conditions are GA pH 9.9 + AP pH 9.9 or GA pH 9.9 + AP pH 7.4, similar to the AN69 microassembly experiments described above. When using these beads (homobifunctional spacer glutaraldehyde), the GA step is important because by placing the beads directly together with the AP, the enzyme binding activity cannot be observed separately.

[0275] Example 5: Adjustment of cofactors of AP activity:

[0276] The AP used in the above examples is commercially available and was mixed with 1 mM MgCl 2 and 0.1 mM ZnCl 2 In the above AP activity test, the cofactor concentration was 0.5 mM MgCl 2 and 0.1 mM ZnCl 2 In the PBS simulation test, the cofactor Mg 2+ and Zn 2+ It was also added at concentrations of 0.5 mmol / L and 0.1 mmol / L, respectively (see below for simulated use stability test).

[0277] Further experiments were performed using the AN69 membrane micromodule from Example 3 to increase the Mg content in the dilution series. 2+ The concentration was titrated from 5 mM to 0.005 mM. Although a slowdown of the AP reaction was observed, no obvious differences between the kinetic curves of different magnesium concentrations were observed, suggesting that immobilized AP may exhibit suitable activity at cofactor concentrations found in human blood samples.

[0278] Plasma concentrations in healthy individuals are similar to serum concentrations, ranging from 0.7 to 1.0 mM. Therefore, when the device is used clinically, the magnesium content in the blood will be high enough to support AP activity. The same is true for zinc. The serum zinc concentration is 12.4 ± 1.4 μmol / L (9 to 22 μmol / L for women; 12 to 26 μmol / L for men).

[0279] This example also shows that the device for in vitro treatment containing immobilized AP can be preferably filled or stored in a buffer or other solution, wherein the matrix containing immobilized AP is immersed in the buffer or solution, and Mg 2+ The concentration of Zn is 0.1-2, preferably 0.1-1 mmol / L, and Zn 2+ The concentration is 5 to 150, preferably 10 to 100 μmol / L.

[0280] Example 6: Optimizing PEI density on membrane

[0281] The "TNBS" test of APA-CRRT is used to determine the optimal PEI coating and density. The TNBS test is known to the skilled person (based on 2,4,6-trinitrobenzenesulfonic acid) and can be used for the quantitative analysis of amino functional groups, for example on the membrane surface. TNBS reacts with active amine molecules to produce a highly colored (orange) product, whose absorbance at 335-345 nm can be measured using a plate reader or spectrophotometer. In this example, the amino functional groups are derived from PEI.

[0282] A 50 g / L TNBS solution was used for the test. The absorbance of the solution was measured at 340 nm on a photometer blanked with reverse osmosis water. In 15 mL of a 0.1 g / L TNBS solution, all 40 fibers were taken out of a mini-module and added to the solution in pieces of about 1 cm long. After shaking in the dark at room temperature for 30 minutes, the supernatant of each solution was removed and the absorbance was measured. The decrease in absorbance is directly related to the number of active amines. The optimal PEI density on the membrane is 1 to 3 μmol / g of hollow fiber membrane.

[0283] It was found that membranes coated using the method described in Example 3 could be AP functionalized very effectively. A citric acid based PEI coating process (see Example 3; preferably 200 mg / kg PEI in 200 mg / kg citric acid solution) was effective. Further experiments were also performed using PEI / GA / AP functionalization based on pre-coated (PEI coated) ST filters, which are the same as AN69 membranes but pre-coated with PEI in commercial production. Immobilization of AP onto pre-treated PEI membranes was also successful. However, those hollow fibers containing AP showed significantly lower enzyme activity, as described above.

[0284] Example 7: Simulated use and stability of AP activity in PBS and human plasma:

[0285] To evaluate whether the immobilized AP was stable over time, the following experiment was performed. The micromodule was prepared as described in Experiment II of Example 3 above and then connected to a pump station as in Example 3.

[0286] In all experiments, the micromodules were closed on the dialysate side and perfused with the appropriate medium on the lumen side for recirculation. Flow rates and volumes were set accordingly to simulate 72 h of dialysis, which corresponds to the maximum allowed usage time of a standard dialysis filter for acute treatment.

[0287] For standard dialysis, a flow rate of about 200 mL / min to 500 mL / min and a blood volume of about 5 L are used. The inner surface of the dialyzer used here is, for example, 1.5 m 2 For an area of ​​40cm 2 This results in a flow rate of 0.5 mL / min to 1.3 mL / min and a cell volume of 13 mL. Since the calculated values ​​are too low for laboratory testing, the flow rate is set to 1.5 mL / min and the cell volume is set to 20 mL.

[0288] Use 0.5mmol / L MgCl 2 and 0.1mmol / L ZnCl 2PBS buffer was used as the perfusion medium. The assemblies were then perfused under a heating hood at 37°C for the specified time. At certain times, samples were taken from the pool and the activity was measured to investigate possible leaching or flushing processes. Fibers from the corresponding assemblies were then subjected to the above-mentioned activity tests without further treatment.

[0289] Activity measurements showed that the activity was 0.4 U / cm before 72 hours of perfusion. 2 , and the activity after testing was 0.7~2.0U / cm 2 No activity was observed in the eluate pool, indicating that no or negligible leaching or washing of the immobilized AP occurred.

[0290] When the micromodules with AP-functionalized membranes were perfused with PBS in the presence of AP cofactor, the immobilization activity was thus reliably retained (and even reproducibly increased) for more than 72 h. Assuming that the AP before perfusion had already consumed some of the cofactor, the enzyme was subsequently supplied by the buffer and thus not only retained activity but also increased activity over time. Regardless, the AP on the hollow fiber membranes was highly stable.

[0291] This effect was even more pronounced in human plasma. The experiment was repeated using human plasma instead of PBS buffer. In essence, human plasma already contains the required AP cofactor. Activity measurements showed an activity of 0.4 U / cm in plasma before 72 h of perfusion. 2 , and the activity after testing was 1.6~2.4U / cm 2 No activity was observed in the eluate pool, indicating that no or negligible leaching or washing of the immobilized AP occurred.

[0292] Example 8: Stability of AP activity after sterilization:

[0293] Clinical products require sterilization techniques to inactivate potential microbial pathogens. Due to the essential AP activity, heat treatment is possible but not preferred as steam sterilization may damage the membrane and / or denature the enzyme.

[0294] Sterilization using gamma radiation has been evaluated as a promising method. In the gamma sterilization process, gamma radiation is generated by a radioactive radiation source and then reaches the product. Gamma rays have a high penetration depth and can therefore be used to sterilize the product in its final packaging.

[0295] The micromodules as described above were gamma sterilized using a dose of about 30 kGy. Using this sterilization method, the micromodules that were still wetted by the modification process (about 0.5 g of water / micromodule) could be closed with stoppers and did not need to be filled with glycerol. After coating (as in Example 3), the micromodules on the blood and dialysate sides were filled with reverse osmosis water and closed with stoppers and sent for gamma sterilization.

[0296] After sterilization at a dose of 30.6 kGy, the water contained in the micromodules was collected and stored. The two micromodules 91 and 92 were sterilized with MgCl 2 and ZnCl 2 The microassembly was pre-flushed with saline solution. To this end, the microassembly was connected to the hose set and filled at a flow rate of 3 mL / min. The hose was then disconnected and the microassembly was allowed to stand for 30 minutes before being evacuated and tested for activity. The results are shown in Figure 8 .

[0297] As described above, two micromodules 89 and 90 were directly tested for activity. Two rinsed micromodules 91 and 92 showed approximately 0.5 U / cm 2 The unwashed micromodule 89 showed an activity of 0.02 U / cm 2 The micromodule 90 showed a very low activity of about 0.3 U / cm 2 The same procedure was modified and the non-sterilized micromodules showed a higher activity of about 0.4 U / cm 2 Therefore, sterilization of water-filled micromodules had little effect on 3 of the 4 micromodules when evaluating AP activity.

[0298] Alternatively, ethylene oxide (EtO) gas is also used for sterilization. The microcomponents are first filled with glycerol to prevent the gel film from drying out and then processed using standard techniques. Ethylene oxide (EtO) is a common gas used for low-temperature sterilization. It is a colorless, toxic gas that attacks the cellular proteins and nucleic acids of microorganisms. It is most often used to sterilize medical devices with long lumens and materials that must be sterilized but cannot withstand higher temperatures.

[0299] As described above, the microassemblies were sterilized with EtO and subsequently assayed for AP activity. EtO sterilization resulted in a slight decrease in AP activity. Of the five microassemblies tested (two unsterilized and three sterilized with EtO), only one microassembly lost AP activity (likely due to an unknown technical error). Of the two microassemblies that retained AP activity, initial data showed that 40% of AP activity was maintained after EtO sterilization compared to the unsterilized control after 120 minutes of activity testing. These test results are preliminary and are being replicated.

[0300] In conclusion, both gamma and EtO sterilization appear to be feasible in order to maintain the activity of membrane-immobilized APs.

Claims

1. A blood treatment apparatus configured to dephosphorylate one or more organic compounds in a patient's blood in an extracorporeal blood circuit, wherein: The blood processing device includes a matrix having alkaline phosphatase (AP) immobilized thereon.

2. The blood treatment apparatus according to claim 1, wherein: The organic compound is a phosphorylated target compound in the patient's blood that is susceptible to dephosphorylation by the alkaline phosphatase (AP).

3. The blood treatment apparatus according to claim 1, wherein: The organic compound is a phosphorylated target protein or nucleic acid in the patient's blood that is susceptible to dephosphorylation by the alkaline phosphatase (AP).

4. The blood treatment apparatus according to claim 1, wherein: The organic compound is a protein for regulating signal transduction, or an enzyme activated or inactivated by phosphorylation.

5. The blood treatment apparatus according to claim 1, wherein: The blood treatment device is located in an extracorporeal blood circuit through which the patient's blood flows and which includes means for conveying blood from the patient's vascular system to the blood treatment device at a defined flow rate and for returning the treated blood to the patient.

6. The blood treatment apparatus according to claim 1, wherein: The blood treatment device is a hemofilter.

7. The blood processing apparatus according to claim 1, wherein: The blood processing device is a sorbent cartridge.

8. The blood treatment apparatus according to claim 7, wherein: The blood treatment device is located in the extracorporeal blood circuit, wherein the extracorporeal blood circuit further comprises a blood filter located upstream or downstream of the sorbent cartridge.

9. The blood treatment apparatus according to claim 1, wherein: The alkaline phosphatase (AP) is calf intestinal alkaline AP.

10. The blood treatment apparatus according to claim 1, wherein: The alkaline phosphatase (AP) is a recombinant and / or chimeric form of AP.

11. The blood treatment apparatus according to claim 6, wherein: The blood processing device is a hemofilter comprising a bundle of hollow fiber membranes, and the alkaline phosphatase (AP) is immobilized at least on the lumens of the hollow fiber membranes.

12. The blood treatment apparatus according to claim 1, wherein: The matrix on which alkaline phosphatase (AP) is immobilized comprises: a. a copolymer of acrylonitrile and sodium methallyl sulfonate, or b. polysulfone, polyethersulfone (PES) and / or polyarylethersulfone (PAES), and polyvinylpyrrolidone (PVP), or c. Polysulfone, PES and / or PAES with an additive selected from chitosan and / or maleic anhydride-1-octadecene alternating copolymer, and PVP.

13. The blood treatment apparatus according to claim 12, wherein: The alkaline phosphatase (AP) is immobilized on the matrix by: a. by an ester bond produced by the reaction between the OH groups of polysulfone, polyethersulfone (PES) and / or polyarylethersulfone (PAES) and the COOH groups of AP, or b. through a peptide bond generated by the reaction between the NH2 groups of chitosan and the COOH groups of AP, or c. Peptide bonds produced by the reaction between COOH groups produced from maleic anhydride-1-octadecene alternating copolymer and NH2 groups of AP.

14. A method for manufacturing a blood treatment apparatus according to any one of claims 1 to 13, wherein: The blood processing device is configured to dephosphorylate one or more organic compounds in the blood of a patient in need thereof, and the method for manufacturing the blood processing device includes immobilizing alkaline phosphatase (AP) to a matrix.

15. The method for manufacturing a blood treatment apparatus according to claim 14, wherein: The blood processing device is a hemofilter comprising a bundle of hollow fiber membranes, and the alkaline phosphatase (AP) is immobilized at least on the lumens of the hollow fiber membranes.

16. The method for manufacturing a blood processing apparatus according to claim 14, wherein: The alkaline phosphatase (AP) is covalently bound to the matrix, and the method for manufacturing the blood processing device includes treating the matrix and / or AP with a carbodiimide compound.

17. An extracorporeal blood circuit through which a patient's blood passes, comprising a blood treatment device according to any one of claims 1 to 13 and means for conveying blood from the patient's vascular system to the blood treatment device at a defined flow rate and then returning the treated blood to the patient.

18. Use of alkaline phosphatase (AP) in the preparation of a medicament for treating infection, the medicament comprising alkaline phosphatase (AP) immobilized on a matrix of a blood treatment device according to any one of claims 1 to 13.

19. The use according to claim 18, wherein The treatment involves dephosphorylating a phosphorylated target compound in the patient's blood.

20. The use according to claim 18, wherein The treatment involves dephosphorylating a phosphorylated target protein or nucleic acid in the patient's blood.

21. The use according to claim 18, wherein The infection is sepsis or septic shock.

22. The use according to claim 18, for treating infection-related renal dysfunction.

23. The use according to claim 22, wherein The infection-related renal dysfunction is sepsis-related acute kidney injury (AKI).

24. The use according to claim 22, wherein The treatment includes continuous renal replacement therapy in the acute setting.

25. Use of alkaline phosphatase (AP) in the manufacture of a blood treatment device according to claim 1 for treating infection.

26. Use of a substrate having alkaline phosphatase (AP) immobilized thereon for the manufacture of a blood treatment device according to claim 1 for treating an infection.

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