The present invention relates to improvements in removal of components from whole blood or a body fluid. More specifically, the invention relates to a method, wherein blood or body fluid is allowed to pass through a rigid integral separation matrix without being excluded therefrom.
Inflammatoric processes, such as sepsis, are a major cause of morbidity and mortality in humans. It is estimated that, yearly, 400 000 to 500 000 episodes of sepsis results in 100 000 to 175 000 human deaths in the U.S. alone. In Germany, sepsis rates of up to 19% of patients stationed at Intensive Care Units have been noted. Sepsis has also become the leading cause of death in intensive care units among patients with non-traumatic illnesses. Despite the major advances of the past decades in the treatment of serious infections, the incidence and mortality due to sepsis continues to rise.
There are three major types of sepsis characterized by the type of infecting organism. Gram-negative sepsis is the most common. The majority of these infections are caused by Esherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa. Gram-positive pathogens, such as the staphylococci and the streptococci, are the second major cause of sepsis. The third major group includes the fungi. Fungal infections constitute a relatively small percentage of the sepsis cases, but they result in a high mortality rate.
A well-established mechanism in sepsis is related to the toxic components of gram-negative bacteria, i.e. the lipopolysaccharide cell wall structure (LPS, endotoxin), which is composed of a fatty acid group, a phosphate group, and a carbohydrate chain.
Several of the host responses to endotoxins have been identified, such as release of cytokines, which are produced locally. In case of an extensive stimulation, however, there is a spill over to the peripheral blood and potential harmful effects are obtained, such as induced organ dysfunction.
The key mediators of septic shock are Tumor Necrosis Factor (TNF-α), Interleukine 1 (Il-1) and Interleukine 17 (Il-17), which are released by monocytes and macrophages. They act synergistically causing a cascade of physiological changes leading to circulation collapse and multi organ failure. Indeed, high concentrations of TNF-α can mimic the symptoms and outcome of sepsis.
Normally, endotoxins are kept within the lumen of the intestine. For example, during cardiopulmonary bypass the presence of splanchic ischemia or dysoxia causes disruption of the mucosal barrier and translocation (i.e. the transport of endotoxins from the intestine to the circulation system) of endotoxins from the gut lumen to the portal circulation.
Antibiotics of varying types are widely used to prevent and treat infections. However, for many commonly used antibiotics an antibiotic resistance is developed among various species of bacteria. This is particularly true for the microbial flora resident in hospitals, where organisms are under a constant selective pressure to develop resistance. Furthermore, in the hospital the high density of potentially infected patients and the extent of staff-to-staff and staff-to-patient contact facilitate the spreading of antibiotic resistant organisms. The antibiotics used are the most economical, the safest and the most easy to administer and may not have a broad enough spectrum to suppress certain infections. Antibiotics can be toxic to varying degrees by causing allergy, interactions with other drugs, and causing direct damage to major organs (e.g. liver, kidney). Many antibiotics also change the normal intestinal flora, which can cause diarrhea and nutritional malabsorption.
Certain antibiotics are known to neutralize the action of endotoxins, such as polymyxin B. This antibiotic binds to the lipid A part of endotoxin and neutralizes its activity. Polymyxin is not used routinely due to its toxicity. It is only given to patients under constant supervision and monitoring of the renal function.
Furthermore, in order to overcome some of the limitations inherent to active immunization against bacterial components, various techniques have been used to produce endotoxin-binding antibodies. A large number of antibodies have been prepared by immunization of humans with bacteria. In order to develop more consistent preparations of therapeutic antibodies, numerous LPS-reactive monoclonal antibodies have been developed. Unfortunately, the clinical studies have not resulted in benefits. However, it should be noted that these trials were performed in humans after onset of symptoms of sepsis. It is widely believed that an anti-endotoxin antibody treatment, administered after sepsis, may yield little benefit because these antibodies cannot reverse the inflammatory cascade initiated by the endotoxin.
In JP 06022633, an adsorbent for anti-lipid antibodies is shown, which comprises a compound with an anionic functional group immobilized onto a water-insoluble porous material. The porous material can be agarose, cellulose, dextran, polyacrylamide, glass, silica gel, or a hard polymer made of a styrene-divinylbenzene copolymer, and the porous material is packed as a bed of separate particles in a separation device.
In attempts to remove components from blood, different adsorbent materials have been prepared. An endotoxin removal adsorbent comprising a ligand immobilized on a solid phase support medium is shown in WO 01/23413. A preferred support medium is in the form of beads. When packed in a separation device, the solid phase support medium is porous enough to allow passage of blood cells between the beads.
In WO 00/62836, the adsorbent material has a size and a structure adapted to remove β-2 microglobulin from blood. The adsorbent material of this document can be a macroporous synthetic polymer with a surface of beads and of pores modified as to prevent adsorption of proteins and plateletes. However, individual spherical beads of the polymer were mechanically destroyed at a loading of about 500 g, which is obtained in for example a column packed with the beads. Such a loading results in a considerable pressure drop over of the column.
In order to reduce the pressure drop, an absorbent has been prepared in EP 464872, which comprises waterinsoluble porous hard gel particles having an exclusion limit of 106-109 Dalton. The gel bed is used for selective removal of lipoproteins from blood or plasma in extracorporeal circulation treatment.
Likewise, in WO 01/23413 the porous support material for endotoxin removal is beads, which can be filled into a container, the beads having a size sufficient to provide the requisite space between the beads when packed into a column or filter bed. The porous support material can also be microfiltration hollow-fibers or flat sheet membranes in order to minimize pressure drops.
In EP 424698 an adsorbent for eliminating biomacromolecules is shown, which consists of a carrier of porous spherical particles having a particle size of 50-150 microns and an exclusion limit of at least 105 Dalton. Polymyxin B is coupled to the particles, which are subsequently filled in a cartridge to be used in a system for extracorporeal endotoxin removal from whole blood.
In these traditional systems for extracorporeal removal of toxic components from blood, a container or cartridge is first filled with a liquid and the adsorbing porous beads are introduced afterwards. In U.S. Pat. No. 6,408,894 a method is shown, which provides a more uniform distribution and denser packing of the beads. The method involves forcedly supplying a mixture of liquid and beads into a container in such a manner that the liquid is squeezed out of the mixture and out of the container.
Thus, an elimination of blood cells facilitates the removal of compounds present in plasma as described above, e.g. in WO 00/62836 or WO 01/23413. However, such a technique involves two separation steps which both could contribute to an enhanced risk of adverse cellular activation due to bioincompatability.
The purpose of the present invention is to provide a new method for selective binding and separating at least one component from whole blood or body fluids, whereby the above mentioned problems in connection with inflammatoric processes are eliminated.
Another purpose is to provide such a method, whereby the selective binding and separation can be accomplished on whole blood without the need of separating blood into plasma and blood cells.
A further purpose of the invention is to provide such a method, which is not size-dependent, i.e. the blood components are not separated by means of exclusion.
Still another object of the invention is to provide such a method, whereby high flow rates can be obtained in a separation device without significant pressure drop with time.
Yet a further purpose is to provide such a method without subjecting the blood to shear forces in a separation device even at very high flow rates while maintaining a low line pressure in order to avoid damage to bloodvessels.
These objects are achieved by the present invention having the characteristic features of claim 1. Other advantages of the invention will become apparent from claim 21 and the subclaims.
According to the invention, a method is provided for selectively binding and separating at least one component from whole blood or a body fluid. The blood or body fluid is allowed to pass through a rigid integral separation matrix without being excluded therefrom, the matrix having a porous structure with a pore size ranging from 5 micron to 500 micron and an active surface ranging from 0.5 cm2 to 10 m2, which is able to bind one or several components.
In a preferred embodiment of the invention the matrix further comprises at least one functional group which has been introduced by means of coating and/or surface modification of the porous structure. This results in that the active surface obtained, alone or in combination with non-functionalized regions of the same, is able to selectively bind at least one component of whole blood or a body fluid. The components to be removed can be natural as well as non-natural, i.e. a specific ligand, such as an antibody, is attached to the component.
Furthermore, the functional groups, obtained by means of direct or indirect selective conversion of the surface of the porous structure, have been further used for immobilization of ligands. However, the functional groups of the porous structure can be utilized as they are in the inventive method.
The pore size as well as the surface of the skeletal-like porous structure has been adapted to be used in the separation matrix of the inventive method in connection with whole blood purification. However, the method according to the invention can also be used for the removal of components from other body fluids as well as aqueous solutions. It is an important aspect of the invention that neither any component nor any solvent is excluded from the matrix during a separation procedure.
According to the invention, the rigid integral matrix should have an available surface from 0.5 cm2 to 10 m2, and the density of the matrix structure is not limiting for performing the inventive method.
In this connection the term “rigid” means that the matrix is not flexible, not bending or yielding, but able to withstand a pressure of at least 0.5 bar. The term “integral” means that the matrix with high surface area is an entire entity.
The porous structure of the matrix in the inventive method is made of metal, inorganic oxide, carbon, glass, ceramic, synthetic polymer, and/or natural polymer, or mixtures thereof. Porous solid metal structures with well-defined pore sizes and high surface areas can be manufactured by using strictly controlled sintering processes that produces uniformly-sized pores.
Different polymers have been produced as a moulded or extruded porous material with a porous structure, having the desired pore size as well a high surface area for the matrix. They have also been produced as foam. For example, polyurethanes prepared from isocyanates and various other organic compounds have active hydrogen atoms, which have been used for producing poly-addition products. This active hydrogen can come from bifunctional or polyfunctional compounds, such as polyalcohols, polyamines. Reactions with water gives rise to primary amines which have been used for covalent immobilization of specific ligands.
A wide variety of metals and alloys have been used, such as stainless steel, nickel, titanium, monel, inconel, hastelloy and other special metal materials. High surface area inorganic oxides, especially alumina and zirconia, have also been utilized with the same techniques to produce ceramic materials with defined pore structures. Likewise, such ceramics as well as sintered glass can be purchased, which have adequate pore sizes.
Other natural rigid materials, such as amorphous silica, e.g. zeolites, and lava rock, have also been used.
Natural materials and hybrides thereof, which can be used as a matrix material in the inventive method, are polysaccharides, such as cellulose, and other polymeric carbohydrate materials. Other suitable natural polymeric materials are polyamino acids, also those involving synthetic amino acids, polylactic acid, polyglycolic acid and its copolymers with lactic acid.
In this connection the term hybrid encompasses derivatives of such natural materials, for example cellulose diacetate, which is a preferred polysaccharide derivative.
Suitable synthetic polymers for the matrix to be used in the present invention are polyolefines, such as polyethylene, polypropylene, polybutylene, polymetylpentene, and ethylene vinyl acetate copolymers; vinylic polymers, such as polyvinyl alcohol, polyvinyl acetals, and polyvinylpyrrolidone; fluorine containing polymers, such as polytetrafluoroethylene, fluorinated ethylene-propylene copolymer, polychloroflouroethylene, polyvinylfluoride, and polyvinylidene fluoride; polyacrylates, such as polymethylmethacrylate, cyanoacrylate, polyacrylonitrile, and polymetacrylates; polyamides, such as polyacrylamide; polyimides, such as polyethylenimines; polystyrene and its copolymers, such as polystyrene and acrylonitrile-butadiene-styrene-polymers; silicone rubbers; polyesters/ethers; polycarbonates; polyurethanes; polysulfonates; polyglycols; polyalkydeoxides such as polyehtyleneoxide, polypropyleneoxide; and copolymers or hybrids thereof.
In the preferred embodiment, at least one functional group has been introduced onto a porous structure of the rigid integral separation matrix. The functional groups can be of different kinds, i.e. of the anionic, cationic or nonionic type. The functional groups of the porous structure have been used to covalent bind substances like peptides/proteins and bile acids (e.g. deoxycholic acid), antibodies and fragments thereof as well as other biomolecules and substances having the ability to selectively bind endotoxins and/or proinflammatory mediators.
A surface modification, i.e. a surface functionalization in an indirect way, was accomplished by means of electro-deposition, electro-evaporation, plasma chemical deposition, deposition from an ion plasma flow, or chemical vapor deposition (e.g. plasma polymerization, plasma enhanced surface polymer deposition). The surface modification methods are known per se and found in “Plasma surface modification and plasma polymerization” by N. Inagaki, 1996, Technomic Publishing, Lancaster, USA. Different three-dimensional matrix structures have been treated by means of these methods, a very homogeneous modification of the active surface being achieved.
Polymerization of bifunctional monomers of acrylic or allylic double bonds with polar groups as OH, NH2, CN and COOH have been used to produce plasma polymers with high density of the functional groups. For example, surface functionalization of the inorganic and organic surfaces have been carried out in a plasma environment of allyl compounds, such as allylamine.
It has also been possible to organic polymeric surfaces in NH3, O2, or CO2 plasma environments, which give rise to either of the functional groups ═NH, —NH2, ═CN, —OH, or —COOH. Other examples of gases used are well-known within the art.
A plasmachemical processing have also been combined with classic chemical synthesis, the selectivity of surface modifications for polymers being significantly enhanced. One approach has been to apply a specific plasma gas surface functionalization immediately followed by a chemical unification of the coexisting plasma functional groups.
Another way of introducing the functional groups is by means of a direct functionalization, i.e. coating the surface with a polymeric material. In this connection the synthetic or natural polymer has been coated onto the high surface metal, inorganic oxide, carbon, glass, ceramic, as well as another suitable synthetic polymer, and/or a natural polymer, or mixtures thereof.
Many of the above-mentioned polymers, especially those without functional groups, such as polyethylene, polypropylene, polytetrafluoroethylene etc., need a further treatment in order to alter their surface properties. Thus, a plasma or corona treatment, as mentioned above, of the polymer surface will generate a very unique functional group, like hydroxyl, carbonyl, carboxyl, amino, and imino groups etc, which are covalently attached to the surface.
The coating has also been accomplished by means of adhesion or adsorption of a polymeric substance having functional groups. Examples of such substances are polylysine, polyarginine, and polyethyleneimine.
By for example using a plasma technique, polyethyleneimine-like substances was obtained on the porous surface. When a separation matrix is used in the method according to the invention for selectively binding and separating at least one component from whole blood or a body fluid, the hydrophilic as well as the hydrophobic regions of proteineous blood components can interact with the processed surface in order to remove the desired components. After functionalization, when the matrix surface for selective binding and separation comprises a polyolefine, e.g. a polyethylene or polypropylene, the positive charges of the amino groups are likewise used for electrostatic interactions and the hydrophobic regions are used for hydrophobic interactions. This approach is used in the inventive method for the selective binding of different regions of for example lipopolysaccharides.
Polymers and metals, having for example reactive hydroxyls, can also be functionilized by means of silanization.
Accordingly, various different functional groups have been covalently coupled to the high surface porous matrix structure. After a direct and/or indirect functionalization, the porous structure can have hydrophilic as well as hydrophobic regions, which can interact the different blood components. Thus, the characteristic properties of a substance of interest are utilized when preparing the surface to be used in the method according to the invention.
Preferably, the functional groups of the active surface are sulfhydryls, carboxylates, amines, aldehydes, ketones, hydroxyls, halogens, hydrazides, and active hydrogen.
In another preferred embodiment, a ligand has been coupled to the at least one functional group of the high surface porous structure in a covalent way. In this connection, a ligand is a substance with high affinity for the component to be removed from whole blood or a body fluid. Thus, the ligand is used to enhance the adsorption properties and the efficacy of binding.
The ligand can be a protein, preferably a recombinant protein, a peptide, an antibody or a fragment thereof, a carbohydrate, e.g. a polysaccharide, a hormone, an antioxidant, a glycoprotein, a lipoprotein, a lipid, a fat soluble vitamin, e.g. vitamin E, a bile acid, a reactive dye, allantoin, uric acid, or polymyxin, or combinations thereof.
A suitable bile acid is deoxycholic acid, which is an endogenous hydrophobic substance. Such a bile acid can be coupled either directly to the functional groups, via a spacer, or coupled via a large molecule, and is then used for removing endotoxins from blood, body fluids and aqueous solutions as in the method according to the invention.
In this connection a spacer is a molecule, large or small, which connects the ligand to the surface of the porous structure.
For example, if in the inventive method the porous structure of the separation matrix comprises a polyolefine having an added amine-group, this group can have an albumin coupled thereto and in turn at least one a bile acid moiety coupled to this large molecule.
Thus, the invention also refers to a new use of a bile acid moiety immobilized on a support for eliminating a component from an aqueous solution comprising the same. Preferably, the bile acid moiety is a deoxycholic acid moiety.
Accordingly, a suitable solid support for immobilization of the bile acid moiety is a rigid integral separation matrix having a porous structure with a pore size ranging from 5 micron to 500 micron, preferably from 70 micron to 170 micron, and an active surface ranging from 0.5 cm2 to 10 m2.
It is also preferred that the ligand of the matrix in the inventive method is albumin or an albumin produced by means of recombinant technology, which can be used instead of serum albumin, polymyxin B (i.e. charged groups on a hydrophobic structure), or deoxycholic acid.
Thus, a ligand can also act as a spacer in the method according to the invention. For example, it has also been possible to first covalently attach a human recombinant protein or another large molecule (e.g. hyaluronic acid) to the porous structure, which allows for a subsequent binding of the ligand specific for the component to be removed.
If necessary, a crosslinker is coupled between the at least one functional group and the ligand in a covalent way. In this connection, a cross-linker is an element that covalently bonds the ligand to the supportive porous structure, the element being a spacer when linking the ligand at a distance from the porous structure itself. Such molecular spacers are known within the art. They have been introduced in order to increase the affinity for the component to be bound and separated from whole blood or body fluids by providing a better availability to the ligands. The biocompatibility of the surface of the porous matrix structure is also increased by the introduction of these molecular spacers.
A crosslinker/spacer can comprise a zero-length cross-linker alone or in a combination with an intervening crosslinker, the final complex obtained being bound together by virtue of chemical substances that add structures to the crosslinked substance. These intervening crosslinkers can be of type homobifunctional (e.g. dialdehydes), heterobifuntional (e.g. amino acids) or trifunctional crosslinking type.
The main purpose of the spacer is to increase the bioavailability of the specific ligand used.
The spacer can for example be a silane, a diisocyanate, a glycolate, a polyethyleneglycol, a succinimidyl reagent, a dihydrazine, adipidic acid, a diamine, an amino acid, a poly or oligo amino acid, a polyamino acid, a peptide, or a protein. Preferably, the protein is a human recombinant protein.
The functional groups of the cross-linker are designed to react with amino groups (Lys, Arg), with sulfhydryls (Cys), or with carboxyls (Asp, Glu), to cite a few examples.
In connection with the chemistry of reactive groups, reference is made to Bioconjugate Techniques, Greg T Hermanson, Academic Press, USA 1996.
Thus, the active porous matrix surface is in the inventive method capable of removing for example endotoxins, alone or in combination with non-functionilized regions of the available surface of the porous structure. The active surface can also be used as a tool for covalent immobilization of chemicals, such as biomolecules like amino acids, polypeptides and antibodies in order to selectively enhance the elimination of such specific components.
A separation matrix, which is intended for selective removal of at least one component from whole blood or body fluids, can be produced with a porous structure of a certain pore size and/or a certain pore size range in dependence on the intended application. Preferably, the porous structure should permit passage of blood cells. Accordingly, certain types of blood cells can also be removed from whole blood by means of the inventive method. Such cells sick cells or cells with specific surface receptors, for example activated phagocyting cells.
The metal structure can for especial applications be magnetic. A magnetic matrix can for example be obtained by coating sintered magnetite with a polymer, e.g. polyethylene. An efficient removal of cells can then be performed allowing antibodies, having a magnetic dextran iron label, to attach to specific cells in the blood.
The pore size should be within the range from 5 micron to 500 micron, preferably from 70 micron to 170 micron, most preferred from 80 micron to 100 micron, so that high flow rates can be maintained without cellular damage or cellular exclusion. Thus, the separation accomplished with the method according to the invention is not based on any size distribution of components. Virtually all components of whole blood or a body fluid might be eliminated by means of the inventive method.
After the removal of one or more primary toxic effectors, i.e. an endotoxin, further secondary toxic effectors can be removed. The secondary effectors can be cytokines (e.g. TNF-α), interleukines (e.g. Il-1), reactive oxygen and nitrogen radicals, etc.
When performing the method according to the invention, one or several separation matrixes are protected within a housing, which can have various shapes and varying and/or different in- and outlets depending on the application. Such a device can then be used for endotoxin removal and/or cytokine removal and/or cytokine neutralization. This is accomplished by passing blood or other body fluids through the device, applied intra, para, or extracorporally, without the liquid being excluded from the rigid integral separation matrix therein. The active surface of the porous structure, the functional groups and/or specific ligands thereon then selectively binds and separates at least one component from the liquid. The device can advantageously also be used for removal of endotoxins from aqueous solutions.
An important feature of the inventive method is that all aspects of septic shock can be provided for, i.e. primary as well as secondary toxic effectors can be removed by means of the inventive method.
Reference is made to FIG. 1 in connection with performing the method according to the invention. A device 1 comprises a housing 2, the housing (or cartridge) of the device being integrated into a closed circulation, in which whole blood or body fluids is circulated by means of a pump. In the housing 2 at least one separation matrix 5 a, 5 b, . . . is arranged, each intended to selectively remove one component from whole blood or body fluids. The housing 2 is provided with an inlet 3 and an outlet 4, the sites of which are of no importance as long as an adequate flow is obtained within the separation matrix(es) and the housing. Preferably, the pump is arranged upstream the inlet 3.
In this way a device is obtained which can maintain flow rates from 5 ml/h to 6 000 ml/min without a significant pressure drop. When applied extracorporeally, a line pressure of not more than 300 mm Hg from pump to cannula is obtained even at very high flow rates.
The rigid integral separation matrix can be produced in different shapes to be used in the inventive method. It can for example be designed as a disk, a rod, a cylinder, a ring, a sphere, a tube, a hollow tube, a flat sheet, or other moulded shapes.
Since the flow within each separation matrix is dependent on its porosity, the contact time of the components in blood or a body fluid with the active surface can be controlled. Furthermore, a desired flow gradient can be created within a separation device by changing the porosity and configuration of the individual separation matrixes therein.
In FIG. 2 and FIG. 3 different schematic embodiments of devices are shown, which can be used when performing the method according to the invention. Arrows indicate the flow of blood or body fluid within the individual separation matrixes and the housings therefor, large arrows indicating a higher flow rate than small arrows. In these examples of different configurations the separation matrixes can have the same or different porosities with or without the same or various types of functional groups or ligands in order to remove one or several components from blood or a body fluid.
The separation matrixes are preferably integrated with the housings (each having an inlet 3 and an outlet 4) in order to ensure that no liquid or components therein are prevented from entering the matrix or matrixes, i.e. being excluded therefrom. In FIGS. 2(a) and (b) examples of one separation matrix 5 within a housing 2 are given, the matrix being of different configurations. Examples of two separation matrixes 5 a, 5 b within a housing 2 are shown in FIGS. 2(c) and (d). In the device of FIG. 2(c) an impermeable coating 6, such as an applied skin, on the outside periphery of the separation matrix 5 a ensures that all the material supplied to the device will pass this entire matrix. In the device of FIG. 2(d), on the other hand, some of the material supplied will have a shorter residence time in the separation matrix 5 a than in the separation matrix 5 b, and vice versa.
In FIG. 3 each device comprises several separation matrixes 5 a-5 g. In FIG. 3(a) a partition wall 7 ensures a flow through all matrixes. The separation matrixes can be positioned laterally or transversally relative to their longitudinal directions, as in FIGS. 3(b) and (c), respectively. In FIG. 3(d) the device comprises separation matrixes of different sizes.
In conclusion, the inventive method can be used with an intra, para, or extracorporeally applied or stand alone device, which is thereby capable of reducing circulating endotoxins and potential harmful pro inflammatory mediators, especially TNF-α, IL-1 and IL-17, preferably in blood. It is also possible to selective remove endotoxins from other aqueous solutions. The components are considered to bind to the active surface of the rigid integral separation matrix by means of adhesion.