HK1230116B - Method for removing cytokines from blood with surface immobilized polysaccharides - Google Patents

Description

Method for removing cytokines from blood using surface-immobilized polysaccharides

The present application is a divisional application of Chinese patent application No. 201080062777.0 filed by the applicant on December 1, 2010, entitled “Method for removing cytokines from blood using polysaccharides fixed on a surface”.

Field of the Invention

The present invention relates to a method for removing cytokines and/or pathogens from blood or serum (blood) by contacting the blood with a solid, substantially non-microporous matrix whose surface has been treated with a polysaccharide adsorbent, such as heparin, heparan sulfate, and/or other molecules or chemical groups having binding affinity for the cytokines or pathogens to be removed (adsorbate) (adsorbent medium or medium), wherein the size of the interstitial channels within the medium is balanced with the amount of surface area of the medium and the surface concentration of binding sites on the medium to provide sufficient adsorption capacity while also allowing a relatively high flow rate of blood through the adsorbent medium. As a result, transport of the adsorbate to the binding sites on the medium occurs primarily by forced convection. (Forced) convection means flow in the patient to be treated, for example, by a pressure gradient generated by a pump, by the application of external pressure to a flexible container (or internal pressure to a rigid container), by gravity head (elevation difference), or by differences in arterial and venous pressure. The present invention provides clinically relevant adsorption capacity within a safe flow rate range typically used in clinical extracorporeal blood circuits, such as dialysis, cardiopulmonary bypass, and extracorporeal membrane oxygenation of blood. The method is in direct contrast to the much slower diffusional transport of adsorbates that typically requires microporous adsorption media, which requires the adsorbate to diffuse through the microporous membrane and/or into the micropores before binding to adsorption sites on, behind, or within the media, and thus requires very low flow rates to achieve effective separation during each blood pass. The present invention also provides methods for treating diseases by removing cytokines and/or pathogens from blood, and apparatus for implementing the methods and treatments, the methods being described by contacting blood with a substantially non-porous matrix that has been coated with a polysaccharide adsorbent, such as heparin, heparan sulfate, and/or other adsorbent materials.

background

Many disease conditions are characterized by the presence of elevated concentrations of cytokines and/or pathogens in the bloodstream. Some such conditions are treated by therapies designed to kill the pathogens, such as by administering drugs, such as anti-infective drugs. Some other conditions are treated by therapies that attempt to reduce the concentration of blood-borne cytokines or pathogens in the patient. Other diseases are treated by therapies that attempt to remove only specific components directly from the patient's blood.

For example, Guillian-Barre syndrome is currently believed to be an autoimmune disease caused by a viral infection that stimulates the body's immune system to overproduce antibodies or other proteins that can attack the patient's nervous system, resulting in increasing levels of paralysis. Most patients recover over time, but these patients appear to be prone to relapses of the disease after subsequent viral infections. One method of treating Guillian-Barre syndrome involves plasmapheresis, which "cleans" the patient's blood by removing the antibodies believed to be attacking the patient's nervous system.

Certain bioactive carbohydrates and polysaccharides can remove harmful substances from blood and biological fluids.

Heparin is a polysaccharide that can be isolated from mammalian tissue. It has a very specific distribution within mammalian tissue; it is found only in the basophilic granules of mast cells. Since its discovery by American scientist McLean in 1916, heparin's ability to prevent blood clotting and its relatively short half-life in vivo have been recognized. Systemic heparin, administered via injection as a free-form drug, has been used clinically for over 50 years as a safe and effective anticoagulant and antithrombotic agent. Heparin's effect on blood coagulation/clotting decreases very rapidly after cessation of administration, making it effective and safe for use during surgery and other procedures. Specifically, heparin's anticoagulant and antithrombotic properties are useful in many medical procedures, for example, to minimize undesirable interactions between blood and artificial surfaces in extracorporeal circulation. Once the procedure is concluded, heparin administration can be discontinued. Due to its short half-life in vivo, heparin concentrations in the patient's blood decrease to safe levels within a few hours. This is particularly important after surgery, where healing depends on the blood's ability to clot at the surgical site to avoid hemorrhagic complications. Heparin is a kind of anticoagulant that is used for the treatment of thromboembolic disease and the prevention of thrombosis. It ...rombosis. It is a kind of anticoagulant that is used for the treatment of thrombosis. It is a kind of anticoagulant that is used for the treatment of thrombosis. It is a kind of anticoagulant that is used for the treatment of thrombosis. It is a kind of anticoagulant that is used for the treatment of thrombosis.

Others have considered using the selective adsorption properties of systemic free heparin to hinder infection by introducing heparin fragments and/or so-called sialic acid-containing fragments directly into the vascular system. This therapy is based on the assumption that these fragments will bind to lectins on microorganisms and block them, so that they cannot bind to receptors on the surface of mammalian cells. Although many scientists have studied this approach, only a few successes have been reported to date. The most common problem is the bleeding complications associated with the introduction (e.g., by injection) of large amounts of free heparin into the bloodstream in order to achieve a clinically effective reduction in pathogenic microorganisms. The present invention does not require the use of any free, systemic heparin to produce therapeutic effects, and therefore can eliminate bleeding complications. This is done by permanently binding heparin or heparan sulfate to a solid matrix with a high surface area and exposing the matrix to the blood within a cartridge or filter containing this adsorption medium.

The following references relate to the issues discussed above:

Weber et al. (Weber V, Linsberger I, Ettenauer M, Loth F, et al. Development of specific adsorbents for human tumor necros is factor-α: influence of antibody immobilization on performance and biocompatibility. Biomacromolecules 2005; 6: 1864-1870) reported significant in vitro binding of TNF using cellulose microparticles coated with monoclonal anti-TNF antibodies, while Haase et al. (Haase M, Bellomo R, Baldwin I, Haase-Fielitz A, et al. The effect of three different miniaturized blood purification devices on plasma cytokine concentration in an ex vivo model of endotoxinemia. Int J Artif Organs 2008; 31: 722-729) reported significant reduction of IL-1ra, but not IL-6, using an ex vivo method similar to ours, but using a porous adsorption device. In vivo, Mariano et al. (Mariano F, Fonsato V, Lanfranco G, Pohlmeier R, et al. Tailoring high-cut-off membranes and feasible application in sepsis-associated acute renal failure: in vitro studies. Nephrol Dial Transplant 2005; 20: 1116-1126) were able to significantly reduce several circulating cytokines using hemoperfusion and high-cutoff polysulfone membranes, but also reported a reduction in serum albumin. The putative clinical relevance of these findings was shown by Schefold et al. (Schefold JC, von Haehling S, Corsepius M, Pohle C, et al. A novel selective extracorporeal intervention insepsis: immunoadsorption of endotoxin, interleukin 6, and complement-activating product 5a. Shock 2007; 28: 418-425) who were able to simultaneously reduce circulating endotoxin, IL-6, and C5a levels by selective immunoadsorption, resulting in improved organ function in a randomized study of 33 patients with septic shock.

SUMMARY OF THE INVENTION

An object of the present invention is to provide a method for removing cytokines and/or pathogens from mammalian blood by contacting blood with a solid, substantially non-porous matrix coated with selective adsorption molecules, biomolecules or chemical groups. Such selective adsorption molecules can include polysaccharides, such as heparin, heparan sulfate, polyethyleneimine (PEI), sialic acid, hyaluronic acid and carbohydrates with mannose sequences. When used prophylactically, such as during the collection or transfusion of stored blood, or in direct patient-to-patient transfusions, the use of the present invention can also be used to reduce or eliminate the spread of disease. Therefore, the present invention can also be used for preventing diseases and helping to cure diseases in previously infected patients.

It is an object of the present invention to provide a therapy for treating an existing disease by removing cytokines and/or pathogens from mammalian blood by contacting the mammalian blood with a solid, substantially non-porous matrix coated with heparin and/or other adsorbent molecules and returning the blood to a patient suffering from the disease.

The above objects are not intended to limit the scope of the present invention in any way.

Detailed Description of the Invention

1. Removal of cytokines or pathogens from the blood

A first aspect of the invention provides a method for removing cytokines and/or pathogens from blood, such as mammalian blood, by contacting the blood with a solid matrix, such as a solid matrix coated with heparin and/or other adsorptive carbohydrates and/or polysaccharides.

In one embodiment of this method, heparin is fixed on the matrix surface. The inventors have found that solidified heparin bound to the surface can be effectively used to remove large amounts of cytokines and pathogens from the blood. However, the general flow rate of extracorporeal blood circulation requires that the adsorption "bed" be designed to allow relatively high flow rates for safe operation. This is partly due to the general tendency of slow-moving or stagnant blood to form dangerous blood clots. In the present invention, the matrix is designed to have a sufficiently large interstitial dimension to allow blood to pass through the matrix at a high flow rate without a large pressure drop. That is, after blood is taken out from a mammalian patient, it flows through the matrix at a certain flow rate, wherein the delivery of the adsorbate to the adsorption bed surface is completed mainly by forced convection. This is in contrast to much slower molecular diffusion, which occurs when using highly porous adsorption media (such as porous silica, dextran gel, cross-linked polystyrene and other size exclusion media) and many other microporous media. When a selective permeability barrier membrane is used together with an adsorption medium, molecular diffusion is required, for example, during affinity therapy to avoid the adsorption medium from contacting blood cells and/or high molecular weight solutes.

Heparin and/or other adsorbed molecules are particularly effective in binding cytokines and pathogens in convective transport under relatively high flow rate conditions typically used for (safe) operation of extracorporeal blood circulation, for example, ≥8 cm/min, preferably about ≥24 cm/min, and more preferably about 24-329 cm/min when measured by linear flow rate, or, when measured by flow rate, about >50 mL/min and preferably >150 mL/min but less than about 2000 mL/min. In contrast, adsorption within the pores of a microporous medium may require much lower flow rates through a practically sized adsorbent bed to achieve adequate separation or purification, i.e., <50 mL/min to as low as <1 mL/min.

Strictly speaking, it is generally accepted that for media requiring diffusional transport of the adsorbate to the adsorption sites within the media, a much longer "residence time" is required on the adsorption column, compared to the shorter residence time required to transport the adsorbate to the binding sites (on the substantially non-porous media) by forced convection. However, there are practical limits on the size of safe and effective adsorption cartridges, columns, filters, etc., particularly with respect to the maximum hold-up volume of blood they can accommodate and the flow rate of blood or serum through the adsorption media. For this reason, the average flow rate through the adsorption device is considered an important design variable.

Convective and diffusive kinetics can be compared when removing cytokines or pathogens from flowing blood: Adsorbent media that rely on diffusive transport generally use very porous materials with extremely high internal surface area due to the presence of microscopic pores. On the other hand, media suitable for convective transport generally rely on macroscopic "channels" or visible spaces between solid, essentially non-porous materials, such as particles, beads, fibers, reticulated foams, or dense membranes that are optionally spirally wound.

Media that rely on forced convection transport are generally more suitable for high flow rates, while media that rely on much slower diffusion transport are much less efficient when high flow rates and shorter residence times are required. For this reason, in extracorporeal blood purification devices, adsorption media that do not require the adsorbate to slowly diffuse into the pores within the adsorption medium are more preferred. When blood is pumped through a circulation system made of artificial materials, the general practice is to use relatively high blood flow rates to avoid stagnation and reduce the risk of coagulation. On the other hand, extremely high flow rates must be avoided because they can expose blood cells to high shear rates and impact damage that can destroy or injure blood cells. Therefore, the present invention provides methods and devices for removing cytokines and/or pathogens from blood, using the preferred characteristics of convective transport and its ideal, faster kinetics. This is done by passing blood through a substantially non-microporous matrix whose surface has been treated with adsorbent molecules, such as heparin, so that it can bind to the desired cytokines or pathogens, thereby removing them from the blood. If the surface treatment effectively renders the matrix non-porous, it is also possible to use a microporous matrix in the present invention. This can occur intentionally or unintentionally (surface treatment blocks the pores when the medium is manufactured). This transforms the microporous matrix into one that does not require the adsorbate to diffuse into the pores in order to bind to the medium.

The claimed methods are intended to be used primarily for extracorporeal therapies or procedures, although they may also be used with implantable devices. "Extracorporeal therapy" means a procedure performed outside the body, such as therapy in which a desired product, such as oxygen, anticoagulants, anesthetics, etc., is added to body fluids. Conversely, undesirable products, such as naturally occurring toxins or poisons, may also be removed from body fluids using certain types of extracorporeal circulation. Examples are hemodialysis and hemofiltration, which represent technologies for reducing waste products in the blood. Adsorption on activated carbon has been used to remove blood-borne poisons, among others.

Whole blood and serum from mammals can be used in the present invention. There is no intended limitation on the amount of blood or serum that can be used in the claimed methods. It can range from less than 1 mL to more than 1 L, up to and including the patient's entire blood volume (when continuous recirculation back to the patient is employed). If desired, one or more "passes" through the adsorption bed can be used. The blood can be human or animal blood.

According to the present invention, an adsorption medium for removing cytokines or pathogens from blood is optimized for use in conventional extracorporeal blood circulation with flow rates >50 mL/min, and preferably between about 150-2000 mL/min. If measured by linear flow rate, it is ≥8 cm/min, preferably about ≥24 cm/min and more preferably about 24-329 cm/min. Such high flow rates produce short residence times in the adsorption column, and convective transport dominates over Brownian diffusion transport. This is particularly important for binding large molecular weight proteins or cytokines, such as TNF-α, and larger particles, such as viruses, bacteria, and parasites, because they diffuse very, very slowly. In the present invention, the primary adsorption sites for removing cytokines and pathogens are located on the surface within the interstitial space of the media bed, through which blood flows or is delivered by forced convection. In order to process blood, the interstitial channel needs to be large enough to allow the transport of red blood cells, which have an average diameter of 6 microns. In order to allow the placement of a filled adsorption cartridge in an extracorporeal circulation with high blood flow rates, the interstitial channel must be several times larger than the diameter of the red blood cells. This avoids high shear rates that can lead to hemolysis while minimizing the pressure drop in the blood flowing through the packed bed or cartridge. Additionally, the media is preferably rigid to minimize deformation due to compaction that could clog the filter cartridge. Based on these preferences, an optimized rigid media balances interstitial channel size and total surface area, for example, for the effective removal of pathogens and/or cytokines in high-flow extracorporeal blood circulation.

2. The matrix used in the present invention.

The material of various shapes and compositions can be used as the matrix in the present invention.All suitable matrices provide larger surface area, and simultaneously (mainly) promote adsorbate to be transported to the adsorption site in conjunction with it by forced convection transport.Generally provide filling in container, for example the medium in column, container is designed to hold medium, makes it not be taken away by flowing blood (also known as medium migration) and allows blood to flow through substantially all medium surfaces.The useful matrix of manufacturing adsorption medium comprises non-porous rigid beads, particles or the reticulated foam of filling, rigid integral bed (monolithic bed) (for example formed by sintered beads or particles), the column of filling braided or non-woven fabric, the column of filling yarn or solid or hollow dense (non-microporous) monofilament fiber, the spiral wound tube formed by planar film or dense membrane, or the combination of medium, for example mixed beads/fabric tube.A kind of suitable matrix for the present invention is microporous initially but before (for example by the heparin connected at the end) produces adsorption site, during or after the surface becomes substantially non-porous matrix after treatment.

The column has a macroporous structure that presents a large surface area to the blood or serum while avoiding large pressure drops and high shear rates. In addition to potentially damaging the blood through hemolysis, high pressure drops should be avoided because they can shut down the extracorporeal circulation equipped with an automatic shutoff switch that responds to the pressure drop.

Matrix also can adopt dense membrane, also known as the form of diaphragm.In this embodiment, by heparin, heparan sulfate or another adsorptive polysaccharide and optionally not derived from heparin, heparan sulfate, or the adsorbent component of adsorptive polysaccharide are bonded to the surface of membrane surface modification non-porous film together.Alternatively, can be by using substantially non-porous material, for example polymer fills hole before, during or after connecting binding site and makes microporous membrane become non-porous or " dense ".The film of thin sheet or (hollow) fiber form can be arranged in shell to present the large surface area for the blood contact that is applicable in the practice of the present invention.

2.1. Beads as a matrix

A useful matrix is in the form of solid beads or particles. The "beads" can be made of materials that are rigid enough to resist deformation/compaction at the flow rate to be tolerated. Resistance to deformation is necessary to maintain free volume and the subsequent small pressure drop of the packed bed "switch". The lack of accessible pores in the matrix block eliminates the need for the adsorbate to diffuse into the pores before adsorption. The adsorption sites of the present invention are mainly on the surface of the medium, so that they are easily accessible to the adsorbate in the blood delivered to the surface mainly by convection. Suitable matrices do not need to be completely smooth on their surface, because roughness leads to an increase in the surface area desired for connecting binding sites (for example, by covalent or ionic binding of heparin). On the other hand, accessible internal pores of molecular size are largely avoided to eliminate the need for the adsorbate to diffuse into the pores before connecting to the binding sites. Various types of beads can be used in the present invention. Useful beads should have sufficient size and rigidity to avoid deformation/compaction during use of the method and have sufficient surface area that can be coated with heparin for use in the method.

Evidence of adequate matrix stiffness is the absence of a significant increase in the pressure drop across the absorbent bed during approximately one hour of water or saline flow at rates typical of clinical use: for example, <10-50% increase in the relative initial pressure drop (measured within the first minute of flow) when measured with, for example, saline at similar flow rates.

Beads or other high surface area matrices can be made of several different biocompatible materials, such as natural or synthetic polymers or non-polymeric materials that are substantially free of leachable impurities, including glass, ceramics, and metals. Some representative polymers include polyurethane, polymethyl methacrylate, copolymers of polyethylene or ethylene and other monomers, polyethyleneimine, polypropylene, and polyisobutylene. Useful matrix examples include non-porous ultra-high molecular weight polyethylene (UHMWPE). Other suitable beads are polystyrene, high-density and low-density polyethylene, silica, polyurethane, and chitosan.

Methods for making these beads are known in the art. Polyethylene and other polyolefin beads are produced directly during the synthesis process and are generally used without further size reduction. Other polymers may require grinding or spray drying and classification, or processing to produce beads of the desired size distribution and properties.

As mentioned above, in order to be used for the method for the present invention, the size of the passage or interstitial space between the individual beads for extracorporeal blood filtration should be optimized to avoid the significant pressure reduction between the inlet and outlet of the tube, allowing blood cells to pass safely between the individual beads in a high flow rate environment, and providing suitable interstitial surface area for making the polysaccharide adsorbent bind cytokines or pathogens in the blood. In a bed tightly packed with 300 microns, roughly spherical beads, the diameter of a suitable interstitial pore size is about 68 microns. Useful beads have a size ranging from about 100 to greater than 500 micron diameters. The average size of beads can be from 150-450 microns. For example, polyethylene beads with a 0.3 mm average diameter from Polymer Technology Group (Berkeley, USA) are suitable. Interstitial pores are a function of bead size.

For use, suitable beads are packaged in a container, such as a column.

Other suitable forms of matrices are described below.

Reticulated foam has open cells and can be made from, for example, polyurethane and polyethylene. Control of pore size can be achieved by controlling the manufacturing process. Generally, reticulated foam can have 3-100 pores/inch and can exhibit a surface area of ≥66 ^cm2 / ^cm3 .

The beads can be sintered into a monolithic porous structure by chemical or physical methods. Polyethylene beads can be sintered by heating the beads above their melting temperature in a drum and applying pressure. The resulting interstitial pore size is slightly reduced compared to that of a packed bed of non-sintered beads of the same size. This reduction can be determined experimentally and used to produce the desired final interstitial pore size.

Post or other shell shapes can be filled with weaving or non-woven heparinized fabric, or after shell has been filled with matrix medium, heparin, heparan sulfate or optional non-heparin adsorption site can be connected, for example, by covalent bond, ionic bond or other chemical or physical bond.By controlling fiber fineness (denier) and the density of fabric during weaving or knitting or during manufacturing non-woven net, the interstitial aperture can be controlled.Useful non-woven fabric can be the form of the net that felting (fel ts), melt-blown or electrostatically formed, has the random orientation that is held together by the entanglement of fiber and/or the adhesion of cross-fiber or cohesion.Available braided fabric has more definite and non-random structure.

The column can be filled with fibers or yarns made from fibers. Polyethylene and other fibers can be drawn into thin hollow fibers or solid monofilament fibers or multifilament yarns, which can be packed into the cartridge in the same manner as hollow fiber membranes are installed in conventional hemodialysis cartridges or blood oxygenators. In the present invention, the originally porous hollow fibers are made dense or non-porous before, during, or after heparin or other adsorbents are attached to the outer and/or inner surfaces. Dyneema from Royal DSM is a high-strength solid fiber made of UHMWPE. Dyneema can be heparinized and packed into cartridges to provide a high surface area support for removing cytokines and pathogens.

The spiral wound drum comprises a film or membrane tightly wound together with an optional spacer material to avoid contact between adjacent surfaces. The membrane can be made of polymers such as polyurethane, polyethylene, polypropylene, polysulfone, polycarbonate, PET, PBT, etc.

2.2. Linkage of Adsorbent Polysaccharides

The adsorptive polysaccharides of the present invention can be bound to the surface of a solid substrate by a variety of methods including covalent or ionic attachment.

The adsorption medium of the present invention may comprise heparin covalently linked to the surface of a solid substrate. Heparin can be linked to the desired substrate using a variety of known methods, such as those described in the review article by Wendel and Ziemer (H.P. Wendel and G. Ziemer, European Journal of Cardio-thoracic Surgery 16 (1999) 342-350). In one embodiment, heparin is linked to the solid substrate via covalent end-linking. This method improves the safety of the device by reducing or eliminating the release of heparin from the substrate surface that may enter the bloodstream. It is important to avoid heparin being "filtered out" of the blood and entering the bloodstream because this can increase the risk of bleeding and heparin-induced thrombocytopenia.

Covalent attachment of a polysaccharide, such as heparin, to a solid matrix provides better control over parameters such as surface density and orientation of the immobilized molecule compared to non-covalent attachment. The inventors have demonstrated that these parameters are important for providing optimal binding of cytokines or pathogens to solidified carbohydrate molecules. The surface concentration of heparin on the solid matrix can be in the range of 1-10 μg/cm ^2. Covalent end attachment means that a polysaccharide, such as heparin, is covalently attached to the solid matrix via the terminal residues of the heparin molecule. Heparin can also be attached at multiple points. End attachment is preferred.

If beads are used, they may be hydrophilized prior to attachment to a polysaccharide such as heparin, or other compound.Possible methods for preparing beads include acid etching, plasma treatment, and exposure to strong oxidizing agents such as potassium permanganate.

2.3. Amount of polysaccharide per gram of matrix

The amount of polysaccharide adsorbent per gram of substrate can vary. In a specific embodiment, if beads are used, the amount of polysaccharide, e.g., heparin, per gram of beads is determined by the number of layers used and the size of the beads. The larger the beads, the less polysaccharide, e.g., heparin, is obtained per gram of beads. A preferred amount is 2.0 ± 0.5 mg heparin/g beads per MBTH procedure.

The molecular weight of the polysaccharide used in the claimed method can vary. For example, native heparin has an average molecular weight of 22 kDa. Nitric acid-decomposed heparin has a molecular weight of 8 kDa.

3. Mixture of beads with different surface functionalities

Heparin is a bioactive carbohydrate that binds to cytokines, pathogens, and other proteins. However, heparin's best-known and most widespread use is as an anticoagulant to prevent blood clotting. It has been safely used clinically for 50 years as an injectable, systemic anticoagulant. Additionally, it has been used by manufacturers for many years as a coating or surface treatment for medical devices, with the sole purpose of improving the safety of medical devices in blood-contact applications. This is particularly important in devices that must expose large surface areas to blood for large-volume transport. Examples include dialyzers and blood oxygenators. The surface of the adsorption media used in the present invention contains heparin for two purposes: 1) heparin is able to bind pathogens, cytokines, and other blood-borne substances that contribute to disease, and 2) heparin is able to prevent blood clotting and related reactions after contact with foreign, e.g., artificially manufactured, surfaces. Heparin is therefore a key component of the adsorption media of the present invention, as it provides both effectiveness and safety during the removal of harmful substances from blood and other biological fluids.

In addition to heparin and heparan sulfate, other bioactive chemical moieties, including other carbohydrates, can remove harmful substances from blood and other biological fluids that cannot be effectively removed by solidified heparin alone. For example, chitosan, a highly cationic, positively charged carbohydrate, binds endotoxins. Other positively charged molecules, such as polyethyleneimine (PEI), can also bind endotoxins. However, cationic surfaces are less compatible with blood than heparinized surfaces and can lead to increased procoagulant properties, a hazard in blood-contacting devices. (See Sagnella S., and Mai-Ngam K. 2005, Colloids and Surfaces B: Biointerfaces, Vol. 42, pp. 147-155, Chitosan based surfactant polymers designed to improve blood compatibility on biomaterials and Keuren J.F.W., Wielders S.J.H., Willems G.M., Morra M., Cahalan L., Cahalan P., and Lindhout T. 2003, Biomaterials, Vol. 24, pp. 1917-1924. Thrombogenity of polysaccharide-coated surfaces.) Although adsorption cartridges containing PEI, chitosan, or other inherently thrombogenic surfaces as bioactive adsorbents can be used to remove LPS or endotoxins from blood, patients will require high doses of systemic anticoagulants due to the serious risk of clotting. In the case of systemic heparin, this may lead to bleeding risks and possible thrombocytopenia.

The method of the present invention prepares an adsorption bed using a mixture of a heparinized medium and an inherently thrombogenic medium. By assembling an adsorption cartridge having a heparinized surface and, for example, a cationic surface (or other inherently thrombogenic surface), cytokines, pathogens, and endotoxins can be safely removed from blood or biological fluids.

Another bioactive, but thrombogenic carbohydrate is sialic acid. Sialic acid is known to bind to viral lectins, including influenza (C, Mandal. 1990, Experientia, Vol. 46, pp. 433-439, Sialic acid binding lectins). Mixed cartridges of heparinized and sialic acid-coated beads can be used to treat patients, for example, during influenza epidemics.

Abdominal septic shock is often caused by Escherichia coli (E. coli), a Gram-negative bacterium. Gram-negative bacteria generally do not bind heparin, so the use of a versatile adsorption column that binds these bacteria in addition to cytokines and/or endotoxins would be useful. Carbohydrates with mannose sequences, such as methyl α-D-mannopyranoside, are known to bind to E. coli, K. pneumoniae, P. aeruginosa, and Salmonella. (Ofek I., and Beachey E.H.1, 1978, Infection and Immunity, Vol.22, pp.247-254, Mannose Binding and Epithelial Cell Adherence of Escherichia coli and Sharon, N.2, 1987, FEBS letters, Vol.217, pp.145-157Bacterial lectins, cell-cellrecognition and infectious disease.)

The use of this embodiment is based on the concept that close contact or close proximity of the antithrombotic surface and the thrombogenic surface can avoid the formation of large clinical thrombi (which would occur if the inherent thrombogenic surface was used alone). In the case of adsorption media in the form of beads or particles, a preferred application of the present invention is to mix the different adsorption media together before filling them into a cartridge or other housing. This provides close contact of the various surface chemistries on adjacent beads while allowing efficient manufacture of adsorption cartridges or filters. A related approach is to layer the different media in a "parfait-type" arrangement within the housing so that blood contacts the different media sequentially or in parallel flow. One arrangement of the different media within the cartridge is to place unmixed antithrombotic media at the inlet and/or outlet of the cartridge, with an optionally mixed area containing a more thrombogenic medium inserted between the inlet and outlet regions. In the case of media in the form of fibers, mixed woven, knitted or non-woven structures can be prepared by methods well known in the textile industry for forming fabrics from mixed fibers. Alternatively, yarns can be made from thinner multifilament yarns or monofilaments made from two or more fibers with different surface chemistries, as long as one fiber type contains a surface that effectively prevents blood from clotting upon contact. Mixed fiber yarns can then be used to make fabrics for blood contact. Hollow fiber or solid fiber adsorption media can be mixed and used to make cartridges similar to hollow fiber dialyzers or oxygenators. For membrane or film-type adsorption media used in spirally wound adsorption cartridges, two or more surface chemistries can be used in close proximity to each other so that blood must (almost) simultaneously contact both surface chemistries. This can be achieved by a regular or random array of multiple binding components within the membrane or film surface layer, or by forming a blood flow channel between two closely spaced thin or film-like films, one of which is anti-thrombotic.

4. Equipment used in the method of the present invention

Another aspect of the present invention provides the use of a device comprising a solid substrate modified with an adsorbent having a binding affinity for the cytokine or pathogen for the in vitro removal of cytokines or pathogens from mammalian blood.

The devices mentioned in the uses and methods according to the invention may comprise conventional devices for the extracorporeal treatment of blood and serum of patients (eg suffering from renal failure).

It is known that local blood flow patterns in blood-contacting medical devices used for extracorporeal circulation influence clot formation through shear activation and platelet aggregation in stagnant zones. Therefore, the devices used in various aspects of the present invention should be designed in a manner that does not produce these problems.

The apparatus used in some embodiments of the present invention may, for example, have the following properties:

Blood flow in the range of 150-2000 ml/min, or ≥8 cm/min if measured by linear flow velocity.

Low flow resistance.

A large surface area of the matrix having carbohydrates immobilized thereon, for example about 0.1-1 ^m2 .

Stable coating (no clinically significant leakage of carbohydrates into the blood following contact with blood).

Suitable hemodynamic performance in the device (no stagnant zones).

Optimal biocompatibility.

A non-limiting example of such a device that can be used in accordance with the purposes or methods of the present invention is a pediatric blood flow dialyzer, which is an extracorporeal blood filtration device for removing cytokine molecules and is compatible with high flow rates. One such device is available from Exthera Medical. Other models or types of devices for extracorporeal treatment of blood or serum may also be used, such as the Prisma M10 blood filter/dialyzer from Gambro AB, Sweden.

High flow conditions can be defined as blood flow that exceeds the diffusion limit.

5. Cytokines

As used herein, the term "cytokine" means a protein released, for example, in association with microbial infection or immunity, selected from the group consisting of interleukins, interferons, chemokines, and tumor necrosis factors. Examples of cytokines are vascular cell adhesion molecule (VCAM), antithrombin, activation-regulated normal T-cell expressed and secreted protein (RANTES), interferons, tumor necrosis factor alpha (TNF-α), tumor necrosis factor beta (TNF-β), interleukin-1 (IL-1), IL-8, GRO-α, and interleukin-6 (IL-6).

The method also provides for selective removal of cytokines from blood by strongly adsorbing heparin-binding molecules. Some molecules have a higher binding affinity for heparin than others. For example, TNF-α has a high affinity for heparin.

6. Pathogens

Another aspect of the present invention provides a method for treating a disease by removing cytokines and/or pathogens from mammalian blood by contacting the mammalian blood with a solid matrix as disclosed in the above method. Examples of pathogens that can be removed from blood using the heparinized matrix according to the present invention include:

Viruses - Adenovirus, Coronavirus, Dengue Virus, Hepatitis B, Hepatitis C, HIV, HPV, CMV, etc.

Bacteria - Bacillus anthracis, Chlamydia pneumoniaem, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, MRSA, Streptococcus pyrogenes, Yersinia enterocolitica, etc.

Parasites - Giardia lambitia, Plasmodium spp., etc.

See also, Chen, Y. Gotte M., Liu J., and Park P. W., Mol. Cells, 26, 415-426.

An example of a disease treatable according to the present invention is sepsis. Sepsis is generally considered a systemic response to infection that can lead to organ failure and often death. This condition can be caused by bacterial, viral, parasitic or fungal infections. It is known that this condition is particularly dangerous in hospitals where patients may already be immunocompromised. During sepsis, patients experience a so-called cytokine storm, and the body's immune system attacks healthy tissue, leading to multiple organ failure in highly perfused organs. Reducing TNF-α and other inflammatory molecules will modulate the immune response and can serve as an organ protection strategy. In addition, any heparin-bound pathogens in the blood can be removed, which will help reduce their further colonization and can reduce the amount of antibiotics needed to treat the infection. This can improve patient safety by reducing the risk of side effects associated with antibiotic treatment.

The methods of the present invention can be applied before or after other conventional treatments, such as administration of antibiotics.

7. Combining the present invention with other filtration/separation steps

In one embodiment of the method of treatment according to the invention, the removal and reintroduction of blood may be performed in a continuous circuit comprising a portion of the subject's blood stream.

In another aspect, the above methods can be combined with other methods for filtering or treating mammalian blood. For example, a convection-based cartridge can be used in tandem with conventional extracorporeal circulation, such as CPB, hemodialysis, and oxygenation.

8. Examples

Various aspects of the present invention are further described in the following examples. These examples are not intended to be limiting. For example, heparin is used in this example. However, other carbohydrate and polysaccharide adsorbents may be used alone or in addition to the heparin-coated matrices exemplified below.

Example 1

Preparation of heparin column

Polyethylene (PE) beads (lot 180153) with an average diameter of 0.3 mm were provided by Polymer Technology Group (Berkeley, USA), and columns (Mobicol, 1 mL) were obtained from MoBiTec (Germany). Heparin and polyethyleneimine (PEI) were purchased from Scientific Protein Laboratories (Waunakee, Wisconsin, USA) and BASF (Ludwigshafen, Germany), respectively. All chemicals used were of analytical grade or higher.

Immobilization of heparin on beads was performed as described by Larm et al. (Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Devices Artif Organs 1983; 11: 161-173).

The polymer surfaces were heparinized using the following general procedure.

The polymer surface is etched with an oxidizing agent (potassium permanganate, ammonium persulfate) to introduce hydrophilic properties and some reactive functional groups ( _-SO3H , -OH, -C=O, -C=C-). The surface can also be etched with plasma or corona. For example, PE beads were etched with an oxidizing agent (potassium permanganate in sulfuric acid). These hydrophilized beads, which contain particularly OH groups and double bonds, were used later as a control.

Reactive amino functional groups are introduced by treatment with polyamines, polyethyleneimine (PEI) or chitosan. For some purposes, polyamines can be stabilized on the surface by crosslinking with bifunctional reagents such as crotonaldehyde or glutaraldehyde.

By further stabilizing coating with sulfopolysaccharide (dextran sulfate or heparin) ion crosslinking. If necessary, repeat these steps and set up sandwich structure. Between each step, should carry out careful rinsing (water, suitable buffer).After finally adding PEI or chitosan, utilize the aldehyde functional group in the natural heparin reducing terminal residue, carry out with the end connection (EPA) of natural heparin amination surface by reductive amination.

Partial nitrous degradation of heparin can yield more reactive aldehyde functional groups in the reducing terminal residues. This shortens the reaction time, but the immobilized heparin will have a lower molecular weight. Attachment is performed by reductive amination (cyanoborohydride, CNBH ₃ ⁻ ) in aqueous solution.

In this alternative method, the aminated medium is suspended in acetate buffer (800 ml, 0.1 M, pH 4.0) and 4.0 g of nitrous acid-decomposed heparin (heparin from Pharmacia, Sweden) is added. After shaking for 0.5 h, NaBH ₃ CN (0.4 g) is added. The reaction mixture is shaken for 24 h and then processed as above to obtain the heparinized medium.

1-10 μg/cm ² heparin can be attached to all hydrophilic surfaces such as glass, cellulose, chitin, etc., and almost all hydrophobic polymers such as polyvinyl chloride, polyethylene, polycarbonate, polystyrene, PTFE, etc.

The resulting PE beads with covalently end-linked heparin were sterilized with ethylene oxide (ETO) and rinsed with 0.9% sodium chloride and ultrapure water. The amount of heparin measured using the MBTH method was 2.0 mg heparin/g bead. (Larm O, Larsson R, Olsson P. A new non-thrombogenic surface prepared by selective covalent binding of heparin via a modified reducing terminal residue. Biomater Med Devices Artif Organs 1983; 11: 161-173 and Riesenfeld J, Roden L. Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides. Anal Biochem 1990; 188: 383-389).

The polyethylene beads used have an average diameter of 0.3mm, and use the technology that guarantees that the covalent end of heparin molecule is connected to surface to carry out heparinization, therefore makes the protein that heparin/heparan sulfate has affinity more accessible carbohydrate chain.The average molecular weight of the heparin of solidification is about 8kDa, and every gram of beads connects 2mg (equal to about 360IU).By removing as expected and flowing through heparinization, rather than 75% antithrombin (AT) concentration in the blood of non-heparinized beads has verified the integrity of this surface.

These data agree well with previous observations published by Bindslev et al. on extracorporeal lung assistance in septic patients using a surface heparinized oxygenator. (Bindslev L, Eklund J, Norlander O, Swedenborg J, et al., Treatment of acute respiratory failure by extracorporeal carbon dioxide elimination performed with a surface heparinized artificial lung. Anesthesiology 1987;67:117-120.)

8.2. Example 2

patient

The study protocol was approved by the local ethics committee of Karolinska University Hospital, and signed informed consent was obtained from each patient. Arterial blood was drawn from the hemodialyzers of three patients (2M/1F, aged 43, 56, and 68 years; Table 1) with sepsis (fever >38°C, chills, and white blood cell count >12 x ¹⁰⁹ cells/L).

Table 1. Clinical manifestations of blood donation patients

Patients were previously treated with broad-spectrum antibiotics (ceftazidime or cefuroxime, with aminoglycosides; 1 dose each) and heparin (200 IU/kg body weight at the start of dialysis). Blood was collected in EDTA vacuum tubes and immediately transferred to an adjacent room. 1 mL of blood was applied to a previously prepared column and passed through the column at a flow rate of 1, 5, and 10 mL/min using a roller pump. Blood was immediately collected at the other end of the column and refrigerated centrifuged (4500 G). The supernatant was then collected and frozen at -80°C for later analysis.

8.3. Example 3

Quantitative analysis of cytokines

The amount of cytokines was measured using a plate reader (Multiskan Ascent) using photoluminescence. Each sample was measured in 3 wells, and the geometric mean was used for analysis. The intra-assay coefficient of variation for all kits was less than 8%. We used the Coamatic Antithrombin Kit (Haemochrom, Catalog No. 8211991), Quantikine Human IL-6 (R&D Systems, Catalog No. D6050), Quantikine Human IL-10 (R&D Systems, Catalog No. D1000B), Protein C Antigen Detection 96 (HLScandinavia AB, Catalog No. H5285), Human CCL5/RANTES Quantikine (R&D Systems, Catalog No. DRN00B), Quantikine Human sVCAM-1 (CD106) (R&D Systems, Catalog No. DVC00), Quantikine Human IFN-γ (R&D Systems, Catalog No. DIF50), Quantikine HS TNF-α/TNFSF1A (R&D Systems, Catalog No. HSTA00D), and BD OptEIA Human C5a ELISA Kit II (BD Biosciences, Catalog No. 557965).

Statistical evaluation

The blood concentrations of each cytokine before and after column passage were compared using a paired Kruskal-Wallis test, with a two-sided p-value less than 0.05 indicating significance. The results are summarized in Table 2.

Table 2. Cytokine concentrations measured before and after blood passed through different columns

Cytokine binding

The concentrations of the analyzed cytokines before and after column passage are shown in Table 2 and Figure 1. Briefly, passage through the heparinized beads resulted in significantly greater reductions in blood VCAM and TNF compared to non-heparinized beads.

Effect of bead volume

The data obtained using blood:bead volumes of 1:1 and 1:10 did not vary significantly (Table 2).

Effect of flow rate

Varying the blood flow rate from 1 to a maximum of 10 mL/min did not significantly affect the amount of individual cytokines removed, indicating that the observed binding to the immobilized heparin molecules is a very fast event, apparently independent of diffusion kinetics.

8.4. Example 4

In this example, 5 liters of platelet-poor plasma to which recombinant TNF-α was added were tested using a clinical-sized cartridge. A 300 ml cartridge was filled with heparinized PE beads as described in Example 1. The equipment was sterilized using ETO sterilization, using a 16-hour cycle and a temperature of 50 degrees. The equipment was allowed to "gas out" for an additional 12 hours before the study began.

5.1 L of frozen, unfiltered porcine heparinized platelet-poor plasma was purchased from Innovative Research and stored at -20°C until the day of use. 1 mg of recombinant human tumor necrosis factor-α was obtained in powder form from Invitrogen.

On the morning of the procedure, plasma was removed from a -20°C freezer and thawed in a warm water bath. 1 ml of sterile water was mixed with 1 mg of powdered TNF-α to reconstitute a concentration of 1 mg/ml.

A Fresenius 2008K dialysis machine was equipped with "Combiset" hemodialysis tubing (standard tubing used on Fresenius machines) in a closed system configuration, with the Fresenius Kidney replaced by a seraph hemofilter. Five liters of plasma were transferred into a 5-liter storage bag, which was connected to both arterial and venous patient lines. The dialysis machine and tubing were pre-treated with saline to ensure proper function and the absence of air in the closed loop.

The 5L blood plasma bag is placed on a plate rocker wrapped with a Bair Hugger insulation blanket, and the insulation blanket is wrapped to maintain temperature during the program. The control sample before the infusion (pre-infusion) is collected and placed in liquid nitrogen for quick freezing. The sample is then transferred to a sample storage box and placed on dry ice. After confirming all connections, 0.415ml TNF-α is injected into the port of the storage bag. Before collecting the control sample after the infusion, the blood plasma with TNF-α is allowed to mix on the rocker for 10 minutes. The sample after the infusion is collected, quick frozen in liquid nitrogen, and then moved to the sample storage box on dry ice. The dialysis system is cleaned with saline and the system clock is turned on when the blood plasma passes through the closed system. The first sample is collected from the filter upstream at 5 minutes.

During the first hour of the test run, samples were collected every 5 minutes from the ports immediately upstream and downstream of the hemofilter. The samples were snap-frozen in liquid nitrogen and placed in sample storage boxes on dry ice.

During the second and third hours of the test run, samples were collected every 10 minutes from the ports immediately upstream and downstream of the hemofilter. The samples were snap-frozen in liquid nitrogen and placed in sample storage boxes on dry ice.

During the 4th and 5th hours of the test run, samples were collected every 20 minutes from the upstream and downstream ports. These samples were also snap-frozen in liquid nitrogen and placed in sample storage boxes on dry ice.

A new needle and a new syringe were used for each sample collection at both upstream and downstream locations at each time point to avoid residual TNF-α.

An ELISA is performed to monitor the amount of TNF-α in plasma. To coat a 96-well plate, dilute 10 μL of capture antibody in 10 μL of coating buffer A and add 100 μL to each well. Cover the plate with parafilm and store at 4°C overnight or over the weekend. Remove the samples from the -80°C freezer, record them, and thaw them. Remove the plate from the 4°C freezer, aspirate the wells, rinse once with 400 μL/well assay buffer, then invert and blot dry on an absorbent paper towel. Add 300 μL of assay buffer to each well and incubate the plate for 60 minutes. During this time, prepare standards from the TNF-α standard included in the CytoSet by diluting them in assay buffer. Dilute the thawed samples in assay buffer. Once the samples are diluted, they are replaced in the appropriate locations in the freezer box and returned to the -80°C freezer, as indicated in the record. Wash the plate again with 400 μL/well assay buffer, invert, and blot dry. Place 100 μL into the wells according to the ELISA template sheet. Samples and standards were assayed in duplicate, and a standard curve was included on each plate. 8.8 μL of detection antibody was diluted in 5.49 mL of assay buffer and 50 μL was added to each well after all samples and standards were added. The plates were incubated at room temperature for 2 hours on an orbital shaker. The plates were washed and blotted 5 times as described above. Streptavidin conjugate (16 μL) was diluted in 10 mL of assay buffer and 100 mL was added to each well. The plates were incubated at room temperature on an orbital shaker for 30 minutes, then washed 5 times with assay buffer as described above. 100 μL of TMB solution was added to each well and the plates were incubated on an orbital shaker for an additional 30 minutes. 100 μL of stop solution was added to each well and the plates were read at 450 nm on a Vmax plate reader within 30 minutes of adding the stop solution.

The results are plotted in Figure 1. Within 40 minutes of continuous circulation of TNF-enriched serum, an 80% reduction in TNF was observed with no subsequent release. After ideal mixing, all of the TNF in the plasma would have passed through the adsorption column within 33 minutes. The fact that most of the TNF was captured during this period demonstrates that TNF-α was removed by adsorption based on convection kinetics.

8.5. Example 5

Quartz crystal microbalance (QCM) experiments were performed to attempt to standardize the non-competitive binding capacity of TNF-α to heparinized surfaces. QCM is a technique that measures the weight of adsorbent on specially prepared crystals. The minimum detection limit is 0.5 ng/ ^cm2 . The QCM measures the resonant frequency of the crystal. Because the mass of the crystal increases with adsorption, the change in resonant frequency is proportional to the increase in mass. The following equation describes the mass change (Δm):

n-overtone

where Δf is the change in frequency.

Heparinized polystyrene-coated QCM crystals were prepared according to the method described in Example 1. A 120 ml solution was prepared in which recombinant TNF-α was added to PBS to obtain a final concentration of 83 μg/L. The solution was then passed through a chamber containing 4 QCM crystals at a rate of 50 μl/min. Two crystals were heparinized and two crystals were control crystals (untreated polystyrene). The total experimental time was 20 hours. The results of the amount of TNF-α adsorbed on the heparinized crystals and the control crystals are shown in Figure 2. A maximum of 1234 ng/ ^cm2 of TNF-α was adsorbed on the heparinized crystals, and adsorption on the control surface was negligible.

8.6. Example 6

The following study was conducted to detect the removal of cytokines from the plasma of macaques infected with Bacillus anthracis using heparinized beads. Plasma was collected and combined when several animals died. A 1ml filter syringe was filled with 0.5 grams of PE beads heparinized according to the procedure outlined in Example 1, or untreated PE beads used as a control. A total of 3 sample syringes and 3 control syringes were used. A top porous plate was placed on top of the beads to avoid floating the beads when saline or plasma was added to the filter syringe. The filter syringe was pretreated with 2ml of Tris-buffered saline (TBS). A 5ml syringe was used to pass 2ml of saline through the filter. The piston of the 5ml syringe was then retracted and filled with 4ml of air. Air was then passed through the filter syringe to push the remaining saline out of the beads. 0.5ml of plasma was then drawn into the 5ml syringe and immediately pushed through the filter syringe. Another 4ml of air was passed through the filter syringe to remove any residual plasma. After the plasma was emptied from the syringe, aliquots were sampled and quick-frozen for cytokine concentration analysis. Multiplex assay was used to detect GRO-α, IL-8, MIP-1β, Rantes, and TNF-β. The results are summarized in Table 3.

Table 3.

8.7. Example 7

Cartridges combining cytokines and endotoxins using layered assembly

Two different adsorption media were used to make the cartridge. Heparinized beads were used to capture cytokines and PEI beads were used to capture endotoxins. Blood compatibility was maintained by controlling the ratio of heparinized beads to PEI beads. PEI beads were made according to the first two steps in Example 1.

In this embodiment, heparinized beads and polyethyleneimine (PEI) coated beads are used to make a tube. A 300ml adsorption column is fixed on an upright stand. 50ml of an average of 300 microns of heparinized beads are then added to the tube and allowed to settle. A 50:50 mixture of PEI beads and heparinized beads is then added to form the next 200ml. The tube is then filled with the last 50ml of heparinized beads. The tube is then sealed, and the tight packing of beads maintains the layered structure of the adsorption medium. In this tube, 2/3 of the beads are heparinized, while 1/3 are aminated, and therefore inherently thrombogenic.

8.8. Example 8

Cartridges that combine cytokines and endotoxin using a consistent mixture of beads

The cartridge is manufactured using two different adsorption media: heparinized beads for cytokine capture and PEI beads for endotoxin capture. By controlling the ratio of heparinized to PEI beads, the overall hemocompatibility of the device can be maintained.

In this embodiment, heparinized beads and PEI-coated beads were used to make a cartridge. A 300ml adsorption column was fixed on an upright stand. 100ml of PEI-coated beads were added to 200ml of heparinized beads and thoroughly mixed. 300ml of beads were then added to the cartridge and sealed. The tight packing of beads maintained a random mixing of beads. In this cartridge, 2/3 of the beads were heparinized, while 1/3 were aminated.

8.9. Example 9

Hierarchically assembled cartridges that bind cytokines, Gram-negative bacteria, and endotoxins

The cartridges are manufactured using three different adsorption media. Heparinized beads are used to capture cytokines, mannose-functionalized beads are used to capture Gram-negative bacteria, and PEI-coated beads are used to capture endotoxins. Hemocompatibility can be maintained by controlling the ratio of heparinized, mannose-functionalized, and PEI-coated beads.

A 300ml adsorption column was fixed on a vertical stand. 50ml of an average of 300 microns of heparinized beads were then added to the tube and allowed to settle. 200ml of beads containing equal amounts of heparinized beads, PEI beads, and mannose-functionalized beads were then added to the tube. The tube was then filled to the top with the last 50ml of heparinized beads. The tube was then sealed, and the tight packing of beads maintained the layered structure of the adsorption medium. In this tube, 55.6% of the beads were heparinized, 22.2% were PEI beads, and 22.2% were mannose-functionalized.

Embodiments of the invention are further described in the following claims.

Claims (12)

1. An apparatus for removing at least one cytokine or pathogen from blood, comprising a solid matrix in a container, the solid matrix having one or more polysaccharide adsorbents having binding affinity for the cytokine or pathogen on its surface, wherein the matrix is sufficiently rigid such that blood does not pass through pores in the solid matrix, and wherein the size of the gap channels between the portions of the solid matrix and the amount of gap surface area are such that when the blood flows through the apparatus at a linear flow rate of at least 24 cm/min and contacts the solid matrix, the cytokine or pathogen binds to the one or more polysaccharide adsorbents and is thereby separated from the blood, the gap channels being several times larger than the diameter of red blood cells, and the blood being transported through the matrix more by convection than by Brownian diffusion. 2. An apparatus for removing at least one cytokine or pathogen from blood, comprising a matrix containing a plurality of rigid polyethylene beads in a container, wherein the surface of the plurality of rigid polyethylene beads comprises one or more polysaccharide adsorbents having binding affinity for the cytokine or pathogen, wherein the plurality of rigid polyethylene beads are rigid enough that blood does not pass through pores in the beads, and wherein the size of the interstitial space between each of the plurality of rigid polyethylene beads and the amount of the interstitial surface area of the plurality of rigid polyethylene beads are such that when the blood flows in contact with the plurality of rigid polyethylene beads at a linear flow rate of at least 24 cm/min, the cytokine or pathogen binds to the one or more polysaccharide adsorbents and is thereby separated from the blood, wherein the interstitial space is several times larger than the diameter of a red blood cell, and the flow transport of the blood through the matrix is more by convection transport than by Brownian diffusion transport. 3. The device according to claim 1 or 2, wherein at least one of the one or more polysaccharide adsorbents is selected from heparin, heparan sulfate, hyaluronic acid, salicylic acid, carbohydrates having a mannose sequence, and chitosan. 4. The device according to claim 1 or 2, wherein the linear flow rate is between 24 and 328 cm/min. 5. The device of claim 2, wherein the plurality of rigid polyethylene beads have a diameter ranging from 100 to 450 micrometers. 6. The device according to claim 5, wherein the plurality of rigid polyethylene beads have an average diameter of 0.3 mm. 7. The device according to claim 2, wherein the plurality of rigid polyethylene beads are coated with 0.5-10 mg heparin/g of various rigid polyethylene beads. 8. The device according to claim 7, wherein the plurality of rigid polyethylene beads are coated with 2 ± 0.5 mg heparin/g of the plurality of rigid polyethylene beads. 9. The device of claim 3, wherein the heparin has an average molecular weight of 8 kDa. 10. The device of claim 3, wherein the heparin is covalently connected to a plurality of rigid polyethylene beads. 11. The apparatus of claim 1, wherein the solid matrix is a variety of rigid polyethylene beads with an average diameter of 150-450 micrometers. 12. The apparatus of claim 2, wherein the matrix is a variety of rigid polyethylene beads with an average diameter of 150-450 micrometers.