DD221367A5 - PROCESS FOR PREPARING A BIOSPECIFIC POLYMER - Google Patents

Abstract

The present invention relates to a process for producing a biospecific polymer having immobilized reactive biological functions, having reactivity for binding specific pathological effectors or specific groups of pathological effectors thereto, characterized in that it comprises treating a biocompatible polymer carrier with an activating agent and binding a biological function having at least one functional group reactive with the activated biocompatible polymer carrier, the reaction being conducted for a time sufficient to covalently bind the biological functions to the biocompatible polymeric carrier, and wherein the biological functions have a size such that they does not penetrate into the matrix of the biocompatible polymer carrier.

Description

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Process for the preparation of a Toi ο specific polymer

Field of application of the invention

The present invention relates to biocompatible polymers having immobilized reactive biological punctures (biologicals) that bind selected specific pathological effectors or specific groups of pathological effectors associated with diseased body fluids.

Characteristic of the known technical solutions

The course of many craniofacial states is often reflected by increased levels of specific blood proteins and other molecules. This phenomenon is typically used as a diagnostic tool to define disease progression and to follow the course of clinical treatment. In many cases, these specific blood components are directly or indirectly responsible for the primary and secondary manifestations of the craniofacial process. "Autoimmune" diseases can be characterized as diseases caused by the circulation of antibodies to endogenous sub-substrates and tissue proteins that the body needs for normal growth and maintenance. "Neoplastic" diseases are typically characterized by uncontrolled growth of an undifferentiated transformed cell line that escapes or counteracts the body's natural defense mechanism.

Production of immunosuppressive blocking factors that

Antigen-surface masking components and / or growth regulator components. The compartmentalization of these pathological effectors on a biocompatible substrate is associated with the restoration of "normal" body functions by removing the pathological effectors of the disease process.

The basic function of the organs, cells and molecules that make up the immune system is to recognize and eliminate foreign substances from the body. These foreign substances are eliminated by reaction between the foreign substance and antibodies formed in response to the substance. In general, this function is performed effectively and without harm to the host. However, in certain cases, disorders may occur that lead to pathogenic disorders such as. B. An Uncontrolled Response (Allergic Disorders) or an Abnormal Response (Autoimmune Disease) The pathogenesis of both disorders is directly or indirectly associated with the production of antibodies with cross-reactivity to either the surrounding antigens (allergens) or self-antigens.

An autoimmune disease is a pathological condition that occurs when a host antibody immunologically responds by producing antibodies reactive with a self-antigen. Autoimmunity can seize almost any part of the body and generally causes a reaction between an eigenantigen and an immunoglobulin (IgM or IgG) antibody. Representative autoimmune diseases can include the thyroid, kidney, pancreas, neurons, gastric mucosa, adrenals, skin, red cells and synovial membranes, as well as thyroglobulin, insulin, deoxyribonucleic acids, and immunoglobulins.

For some types of autoimmune and neoplastic diseases, nonspecific immunosuppressive treatments such as whole body x-ray or the administration of cytotoxic drugs have been used with limited success. The disadvantages of such treatment include the toxicity of the used agents and the increased incidence of various cancers, especially lymphoma and reticulo-sarcomas, following such therapy. In addition, the use of nonspecific chronic cellular suppression agents greatly increases the susceptibility of the patient to serious infections by environmental fungi, bacteria and viruses that would not cause problems under normal circumstances. The invention disclosed herein is specific in that it removes only the pathological effectors or the groups of pathological effectors related to and responsible for the manifestations of a single disease.

In reviewing the prior art, it has recently been found that two major approaches have been taken for the therapeutic treatment of autoimmune and / or neoplastic diseases, the first being the introduction of a material into the patient that has a specific type of immunological tolerance This suppression of the antibody response would then cause tolerance to the attacking antigen A typical example of this type of procedure is described in U.S. Patent No. 4,222,907, Katz, Sep. 16, 1981. In this reference a diseased patient is given a therapeutic treatment consisting of introducing conjugates of an antigen bound to a D-aminoglutaric acid; D-lysine copolymer.

The second approach is the extracorporeal route. The methods of treatment generally involve the removal of the whole blood, the separation of cellular and soluble blood substances, the substitution or treatment of the blood plasma and the recombination infusion of the treated whole blood. The first example of this approach would be plasma substitution or exchange with salt, sugar and / or protein solutions and is described in McCullough et al., Therapeutic Plasma Exchange, Lab. Med. 12 (12), p. 745 (1981). Plasma exchange is a fairly crude technique that requires a large volume of replacement solution. A second example of this procedure requires physical and / or biochemical modifications of the plasma portion of the whole blood. Typical of the state of the art for this therapeutic treatment are, for. See, for example, Terman et al. Article "Extracorporeal Immunoadsorption: Initial Experience in Human Systemic Lupus Erythematosus", The Lancet, October 20, 1979, p. 824-826. This article describes a hemodialysis system that uses two mechanical filters with a DNA collodion charcoal filter between these two mechanical filters. However, the adsorbent column is typically only a semi-specific for immune components in this prior art because the carbon substrate does not specifically remove many vital low molecular constituents from the treated plasma. A second application of this approach can be demonstrated by the Terman et al. Article "Specific Removal of Circulated Antigens by Means of Immunoadsorption", FEBS Letters, Vol. 1, January, 1976, p. 59-62. This reference teaches the specific removal of radioactively labeled antigen by antibody treated cellulosic membranes. However, the author shows that control membranes have a significant capacity to adsorb proteins nonspecifically.

A third application of this approach is shown in the Bansal et al. Article "Ex Vivo Removal of Serum IgG in a Patient With Colon Carcinoma", Cancer, 42 (1), pp. 1-18 (1978). This report teaches semispecific adsorption of immunoglobulin by ex vivo treatment of plasma with formalin and heat killed Staphylococcus aureus. The biological activity of certain strains of S. aureus is attributed to a molecule that is on the cell wall and is called protein A, which interacts and binds with the Fc portion of mammalian IgG. This treatment does not distinguish between normal and pathological IgG components because it interacts with the Fc portion and experiments have shown the possibility of significant side effects.

A fourth application of this approach can be demonstrated by the Malchesky et al article "On-line Separation of Macromolecules by Membrane Filtration With Cryogelation", Artif. Organs 4: 205, 1980. This publication teaches the semi-specific removal of cryoglobulin substances from the plasma by the combination of filtration and cold treatment chambers. The appearance and composition of cryoglobular precipitates is not necessarily consistent with or indicative of many autoimmune or neoplastic diseases.

Another problem associated with the current state of the art is that, without systems using mechanical filtration, the specific pathological effectors to be removed could not be removed in sufficiently large amounts to condition the diseased patient because the columns do not specifically specifically absorb only the desired specific pathological effectors.

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Object of the invention

It is the object of the invention to provide a way to treat blood or plasma in an economical way * to remove with high specificity pathological effectors »

Explanation of the essence of the invention

The invention is based on the object »pathological effect factors bind to activated biocompatible polymers with biological functions.

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The present invention provides a biospecific polymer having immobilized reactive biological functions, which biological functions have a high specific activity for the binding of complements, which are pathological effectors, consisting of a biocompatible polymeric carrier, with or without a spacer, attached to the biocompatible Polymer support having a physical dimension that forces this spacer to extend from the surface of the biocompatible polymeric support and from a biological function or biological functions immobilized to the biocompatible polymeric support or spacer via a chemical bond and characterized in that said biological function or functions retain their reactivity to bind specific pathological effectors or specific groups of pathological effectors *

The invention also relates to a method for the therapeutic treatment of autoimmune diseases, consisting of the passage of the blood, plasma or other body fluid of a diseased patient via a biospecific polymer having immobilized reactive biological functions, wherein the desired pathological effectors are removed from the blood or plasma of the patient and then returning the blood to the patient,

Furthermore, this invention, generally speaking, relates to a process for the preparation of these biospecific polymers having immobilized reactive biological functions with high specific activity for the binding of complements which are pathological effectors.

Furthermore, this invention relates to a method for producing biospecific polymers on a mechanical support to provide excellent mechanical integrity.

These and other objects of the present invention are disclosed and described in the detailed description and claims.

The biocompatible polymeric carriers useful in the present invention are materials which are not prone to cause deleterious effects in contact with body fluids, such as body fluids. Plasma or all of the blood, while at the same time maintaining a reactive but immobilized biological function oriented so that the biological function protrudes from the surface of the polymeric carrier. The materials that are suitable are those that can be cast into films or other physical forms while at the same time being receptive to chemically binding the biological functions without harming neither themselves nor the functions attached thereto. The types of materials that are generally considered suitable are known in the art as hydrogels and may be either copolymers or homopolymers.

Modified cellulose and cellulose derivatives, especially cellulose acetates, have found application as biocompatible carriers useful in the present invention. Modified cellulose derivatives are understood as meaning cellulose polymers whose surface is modified by covalently bonding corresponding biocompatible surface groups to the cellulosic substrate polymer, thereby becoming more biocompatible. Such surface groups are well known and need not be described here, however, for the purposes of the present invention, albumin has particular utility as a modifying group. Methods to attach such groups are described below.

Homopolymers may also be used as suitable biocompatible polymeric carriers in the present invention. It is understood, however, that when homopolymers are mentioned, these include materials which may also be identified as slightly crosslinked homopolymers. That is, they contain a relatively small amount of a second component, which is associated with the production of the monomer or intentionally added to ensure sufficient cross-linking, to protect the homopolymers from being slowly dissolved in an aqueous solution, such as , B. blood, are dissolved. An example of this type of homopolymer, which is often lightly crosslinked, is hydroxyethyl methacrylate (HEMA).

With respect to the hydrogels, suitable polymers may be either regular homopolymers containing essentially no other material in their matrices, or they may be copolymers prepared from two or more monomers, such as e.g. As styrene and vinyl acetate. In certain cases, this type of

Tailoring the copolymers with various monomers to enhance the desirable properties of the biocompatible polymeric carrier material. Examples of suitable monomers that may be copolymerized include, for example: As hydroxyethyl methacrylate and glycidyl methacrylate.

Also suitable are terpolymers which are a subclass of copolymers containing three monomers that are polymerized. An example of a suitable terpolymer is glycidyl methacrylate / N-vinyl pyrrolidone / hydroxyethyl methacrylate (GMA / NVP / HEMA).

In addition to the specific copolymers and homopolymers listed above, copolymers prepared with or without various additional monomers and homopolymers useful in the present invention can be polymerized from the following monomers: hydroxyalkyl acrylates and hydroxyalkyl methacrylates, ζ , Hydroxyethyl acrylate, hydroxypropyl acrylate and hydroxybutyl methacrylate; Epoxy acrylates and epoxy methacrylates such. For example, glycidyl methacrylate, aminoalkyl acrylate and aminoalkyl methacrylates, N-vinyl compounds such. N-vinylpyrrolidone, N-vinylcarbazole, N-vinylacetamide and N-vinylsuccinimide; aminostyrenes; Polyvinyl alcohols and. Polyvinylamines which must be prepared from suitable polymeric precursors; Polyacrylamide and variously substituted polyacrylamides; Vinylpyridine; "vinylsulfonates and polyvinyl sulfates; vinylene carbonates; vinylic and vinylic crotonic acid; allylamines and allyl alcohol; vinyl glycidyl ethers and allyl glycidyl ethers." Processes and methods of preparation for the preparation of copolymers

and / or homopolymers of the above monomers are well known in this particular art. These parameters are not critical to the present invention, with the proviso that the final copolymer and / or homopolymer is non-toxic to animals, including humans.

The process of casting these materials into a form suitable for use in the present invention is not critical. A currently preferred method is spin casting and is carried out in Examples 2, 3 and 4.

In the context of the present invention, biological functions may be defined as chemical compounds that have the ability to covalently bind to biocompatible polymeric carriers or spacers, as defined below, while retaining activity to bind to the desired pathologically-acting component. In addition, the biological functions used must be of such size that they covalently bind to the surface of the polymer support and must not be so small as to penetrate the porous matrix of the P.I. polymer support and thereby become chemically bound within or within the support material. In view of this, a spacer must be employed to ensure that the reactive site of the biological function which is preserved and which is susceptible to binding with the desired pathological component, can actually be offered to this component, i. H. that it is kept away from the wearer by the wearer, so that it comes into contact with the body fluid flowing over the wearer. It is obvious from the

above, that, of course, the reactivity for the binding of the desired pathological component is actually maintained after the immobilization of the biological function on the biocompatible polymeric carrier. Examples of materials that may be used as biological functions include e.g. Acetylcholine, receptor proteins, tissue-compatible antigens, ribonucleic acids, basement membrane proteins, immunoglobulin classes and subclasses, myeloma protein receptors, complement components, myelin proteins and various hormones, vitamins and their receptor components. Specific examples are the attachment of insulin to a biocompatible polymeric carrier to remove anti-insulin antibodies associated with the autoimmune disease insulin resistance; Attaching Anti-Clq and / or CIq to a biocompatible polymeric carrier to remove immune complexes associated with connective tissue and proliferative diseases such as: B. Rheumatoid arthritis and carcinomas.

Any well known chemical attachment method is sufficient to attach the biological functions to the biocompatible polymeric support, with the limitation that the biological function still has at least one active site for the single autoimmune disease related component. In general, the methods of chemical bonding fall into three classes or ways of tying. These three ways are

1. spontaneous attachment,

2. chemical activation of terminal functional groups and

3. Bonding with coupling agents.

Spontaneous covalent attachment of biological functions to polymer support surfaces occurs via chemically reactive groups that depart from the polymer support. To couple z. For example, reactive groups such as aldehyde and epoxy leaving the polymer carrier immediately contain the available hydroxyl, amino or thiol groups with biological functions. Likewise couple z. B. free aldehyde groups on the polymer support via acetal bonds with hydroxyl-containing biological functions and imide bonds with amino-containing molecules. In addition, z. B. free oxime groups via alkylamine, ether and Thioetherbrücken with biological functions containing amine, hydroxyl and thio groups, accordingly. For simplicity, all of these bonds and couplings are defined as immobilizations. Further discussions of these reactions may, for. In Chemical Procedures for Enzyme Immobilization of Porous Cellulose Beads, Chen, L.F. et al, Biotechnology and Bioengineering, Vol. XIX, pp. 1463-1473 (1977) and "Epoxy Activated Sepharose", 6B, Pharmacia Fine Chemicals, Affinity Chromatography, pp. 27-32 (1979).

Chemical activation of terminal functional groups can be achieved by activation of the polymer surface functional groups by chemical modification of their terminal components. This process can be illustrated by the oxidation of terminal epoxy functions with periodic acid to form active aldehyde groups. This method will be further explained z. In "Immobilization of Amyloglucosidosis on Poly (glycidyl methacrylate) -Co- (Ethylene Dimethacrylate) -Carrier and Its Derivatives", Svec, F. et al., Biotechnology and Bioengineering, Vol. XX, pp. 1319-1328 (1978). Immobilization of biological functions

happens as described above. Condensation reactions can be carried out between free carboxyl and amino groups via carbodiimide activation of the carboxy groups as described e.g. In New Approaches to Non-Thrombogenic Materials, Hoffman et al., Coagulation - Current Research and Clinical Applications, Academic Press, N.Y. (1973). The immobilization of the biological functions is performed by carbodiimide activation of either the polymer or carboxyl groups of the biological function and condensation with a free amine to form a stable peptide bond. The final orientation of the biological function generally determines whether to use an amine or a carboxyl-containing polymer.

Bonding via coupling reagents can be performed using a variety of coupling agents to form covalent bridges between the polymeric and biological functions. Here, free hydroxyl and / or amine group-containing polymers and biological functions are covalently coupled by reagents such. As cyanogen bromide, diisocyanates, dialdehydes and trichloro-s-triazines. A more exhaustive discussion of this technique may e.g. found in the Chen et al article cited above.

The preferred method of immobilizing reactive biological functions on a biocompatible polymeric substrate in a given case is generally dictated by the molecular location of the reactive binding portion of the biological function and by the biological functional groups

Function and the polymer substrate, which can be covalently combined. For example, it is presently preferred to activate polymer substrates having terminal hydroxy functions by treatment with an alkaline solution of cyanogen bromide (10 to 20% w / v). Typically, the reaction mixture is held at room temperature (20 to 25 ° C) for about 30 minutes. The pH of the solution is maintained in a range of about 10 to 12 by addition of an alkaline material, e.g. B. KOH or NaOH. The polymer is washed extensively with physiological saline (0.9 g%) and incubated with solutions of a purified biological function dissolved in a slightly alkaline buffer solution for about 12 to 16 hours at 2 to 8 ^° C. The polymer is rinsed extensively with physiological saline to remove unbound or non-specifically bound biological components.

Biological functions are immobilized on polymers containing glycidyl groups via ethers, thioethers or alkylamine bonds. Epoxy-activated polymer substrates are rinsed and swollen with aqueous neutral buffer solutions at room temperature. Purified biological functions, dissolved borates, carbonates or phosphate buffer solutions are incubated with the glycidyl polymer substrate for 12 to 20 hours at 4 to 30 ° C. Excess and non-specifically bound biological functions are removed by rinsing the polymer with saline, acetic acid (0.2 to 1.0 M) and phosphate buffered (pH = 7.2 + 0.2) saline solutions. Activation of amine and carboxyl group containing polymer matrices is accomplished by treatment with purified biological functions dissolved in slightly acidic ones

(pH 4.5 to 6.5) buffer solutions of a water-soluble carbodiimide. Biological functions are covalently coupled to polymeric carrier substrates by incubation of polymer support, the biological function and carbodiimide reactants 12 to 16 hours at 2 to 8 ⁰ C. The polymer-biological function conjugates are washed alternately in acid and Alkalispüllösungen until the rinse solutions free from biological functions and carbodiimide reactants.

To determine the specific binding properties of the polymer-immobilized biological functions, physiological serum solutions of complementary biomolecules were treated with activated membranes. The amounts of biomolecules were measured radiochemically. The significant reduction of specific biomolecules gave the following short comments on the biologically modified polymer substrates.

In the present invention, a spacer may be defined as a molecule or compound capable of binding to the surface of a biospecific polymeric carrier and which is large enough to protrude from the surface of this carrier and which is capable of functioning as a biological agent and / or or biological functions to immobilize. The spacer ensures that the biological function active site is held away from the wearer so that it more effectively contacts the body fluid. It is obvious from the above that, of course, the reactivity for binding with the desired disease complex is maintained even after the immobilization of the biological functions on the spacer and thereby on the biocompatible polymeric carrier.

The spacers are derived from organic molecules having at least two reactive functional groups, which are generally at opposite ends of the molecule. Such groups serve as binding agents capable of coupling the spacer to the polymer support and to the biological function. The reactive functional groups on the spacer may be the same or different except that they react with the functional groups along the surface of the polymeric support and the functional groups that protrude from the biological functions to form covalent bonds. Any known method for carrying out such coupling reactions is sufficient. For example, the methods described herein and the coupling pathways can be used to bind biological functions directly to a polymer support.

Suitable examples of spacers that may be used in the present invention where the reactive functional groups are the same include, for example, U.S. Pat. For example, 1,6-diaminohexane, glutaraldehyde, 1,4-cyclohexane-dicarboxylic acid, ethylenediamine-tetraacetic acid, triethylene glycol, 1,4-butanediol diglycidyl ether, methylene-p-phenyl diisocyanate and succinic anhydride. Examples of spacers in which the reactive functional groups are not the same include e.g. A: 6-aminocaproic acid, p-nitrobenzoyl chloride, 1,2-epoxy-3- (p-nitrophenoxy) -propane, aminopropyltriethoxy-silane and homocysteine thiolactone.

Also polypeptides and proteins can be used as spacers in the present invention. Albumin, a low affinity protein, has e.g. B. successfully applied as a spacer. In addition, albumin and other natural proteins serve to make the polymer carrier more biocompatible.

Finally, it is understood that certain materials serve simultaneously as a spacer and as an activator in the reaction used to join the spacer and the biocompatible carrier. Examples of this type of compounds include, for example: As a: glutaraldehyde and 1, 4-Butandioldiglycidylether.

Most, if not all, of the suitable biocompatible polymer carriers have very little mechanical stability. Most of these materials are indeed gels or gellike unlike materials with high mechanical stability such B. polypropylene films. Therefore, in most embodiments of the present invention, a mechanically stable support member is necessary. This support allows large surface areas to be used to ensure rapid, both medically and commercially acceptable, removal of immune-related components. The support member, in addition to being mechanically stable, should also not be expensive and must be sterilizable to render it compatible for use in a system wherein the blood of a diseased patient must be treated according to the present invention. Examples of materials suitable for the present invention as support members include e.g. A: filter paper,

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Polyester fiber, polycarbonates, reticulated polyurethanes * NORYL, a polyphenylene oxide polymer, microporous polyolefins such as polypropylene and cotton fabric *

Many methods for binding the activated or biocompatible polymeric carrier that has chemically bound biological functions can be used; so z * B, methods such as spin coating, horizontal casting, vacuum impregnation, dip coating, dip coating with subsequent crosslinking and solution copolymerization. Specific examples of these methods can be found in the examples below. "

Generally speaking, the therapeutic aspect of the present invention contemplated herein encompasses the therapeutic . see treating autoimmune diseases and other diseases that exposes the blood or plasma of a diseased patient to a biospecific polymer having immobilized reactive biological functions, removing specific pathological effectors from the patient's blood or plasma, and then returning that blood to the patient , This therapeutic treatment may or may not require the use of blood separation techniques. Therefore, it is intended to perform the treatment in a manner similar to dialysis treatment with the benefit that all blood separation may be unnecessary and that little, if any, physical damage to the normal Blood components occurs.

It is of course also possible to use the present invention and the method of the present invention for the treatment of plasma. The plasma can be obtained from whole blood by any of the currently known and performed methods. Therefore, z. For example, the plasma may be separated from the blood of a patient by known methods, then treated according to the present invention and then recombined with the other blood components and returned to the patient using currently known procedures. In addition, plasma used in known medical treatments may be used in the present invention to treat the plasma before it is administered to a patient in need of plasma from a blood bank, e.g. B., is administered. Of course, all the blood of a blood bank can also be treated according to the present invention.

It is also to be understood that the present invention may also be used with other body fluids to effect the removal of pathological effectors.

Because of the advantages of the present invention, as well as those listed above, as well as others known to those skilled in the art, it is believed that many types of disease states will be directed to the present invention when used in a therapeutic manner. Generally speaking, six groups of disease states could be advantageously treated. These six disease categories are disorders of immune components, drug excesses, poison intake, body substance imbalances, infections and neoplastic conditions. Many diseases are currently being treated using

Plasmapheresis and cytopheresis, where the desired result is the removal of a specific substance. The present invention and method of the invention would be applicable to the diseases currently being treated by plasmapheresis and cytopheresis.

Examples of immune complex diseases that can be treated are e.g. For example, any disease state that elicits antibodies, antigen, antibody antigen, antigen-antigen and antibody-antibody interactions, cell surface complexes, cytoplasmic complexes, etc.

Examples of drug overdoses that can be treated are e.g. Overdoses of iron, dioxin, aspirin, tylenol, methotrexate and other tricyclics.

Examples of poisons and toxins for which the present invention is suitable are ζ. Lead, aluminum, fungi (anatoxin) and organic phosphates.

Body substances that are present in excess can lead to the disease. Examples of these substances which can be removed using the present invention include e.g. Cholesterol, uric acid, immunoglobulins, sickle cells, uremic toxins, bilirubin, porphyrin, cortisone and prostaglandins.

Some examples of infectious agents that can be treated are e.g. B. viral disorders such as cytomegalovirus; Protozoal disorders such as malaria, trypanosomes and Leishmania; bacterial infections such as streptococci,

Fungal infections such as tinea versicolor, mycoplasmas such as pleuro-pneumonia-like organisms; Rickettsia, diseases such as typhus and typhus; Spirochetes such as syphilis and Chlamydia in the psittacosis-lympho-granulotrachoma disease group.

Neoplasms that can be treated using the present invention include, for example, As lymphomas, sarcomas, carcinomas and leukemias. These can be removed by the specific removal of a cell line, inhibitors, initiators of the disease and combinations.

Further examples of disease states that can be treated using the present invention include, for example, For example, the following:

Infections like Poststreptococcale Glomerulonephritis, Subacute Bacterial Endocarditis, Secondary Syphilis, Pneumococcale Sepsis, Lepomatous Leprosy, Ventricular Shunt Infection, Infectious Mononucleosis, Typhoid Fever, Subacute Sclerosing Encephalitis, Landry-Guillain-Barre Syndrome, Hepatitis B Infection, Quartanmalaria, Schistosomiasis and trypanosomiasis.

Neoplasms such as hepatoma, lymphoma and hodgkin disease, acute leukemia, hypernephroma, colon carcinoma, bronchogenic carcinoma and Burkitts lymphoma.

Connective tissue disorders such as periarteritis nodosa, chronic glomerulonephritis, acute or subacute thyroiditis, vinyl chloride overgrowth, chronic liver disease, mixed cryoglobulinemia, Berger's disease or IgA nephropathy, rapidly progressive glomerulonephritis and sickle cell anemia.

Hematologic disorders such as thrombotic thrombocytopenic purpura, autoimmune hemolytic anemia, idiopathic thrombocytopenic purpura, idiopathic neutropenia, cold-hemoglobin, paroxysmal cold hemoglobinuria, circulating anticoagulants, acquired hemophilia, leukemia, lymphomas, fetal erythroblastosis, pernicious anemia, and Rh disorders.

Neurological diseases such as acute demyelinating encephalitis, multiple sclerosis, Landry paralysis, Guillain-Barre syndrome, peripheral neuritis and myasthenia gravis.

Collagen diseases such as Raynaud's, lupus erythematosus, polyarteritis nodosa, scleroderma, dermatomyositis, Sjogren syndrome, rheumatoid arthritis, rheumatic fever and erythema nodosa.

Endocrine diseases such. Cushing's Syndrome and Cushing's Disease, Thyroiditis, Thyrotoxicosis, Addison's Disease and Aspermatogenesis.

Gastrointestinal disorders such as portal cirrhosis, acute hepatitis, chronic active hepatitis, lupoid hepatitis, billiard cirrhosis, ulcerative colitis, regional enteritis and pancreatitis.

Varied diseases such. B-. Hypercholesterolemia, glomerulonephritis, basement membrane diseases, psychogenic conditions - drugs, postaortic valve prostheses - hemolytic anemia, exfoliative dermatitis, Id reaction, psoriasis, Behcet's syndrome, carcinomas, subacute bacterial endocarditis, hypertension, asthma, hereditary angioneurotic edema, meningococcemia, Crohn's disease, hepatitis Encephalopathy and Raynaud's disease.

Furthermore, diseases characterized by antibodies to nuclear antigens, cytoplasmic antigens, cell surface antigens and subclasses can be treated by the present invention. Suitable examples include, for. B. antibodies against native DNA (double strand) or single and double strand, antibodies against SS-DNA, antibodies against deoxyribonucleoprotein, antibodies against histones, antibodies against Sm, antibodies against RNP, antibodies against Sc 1-1 - Scleroderma, antibodies against SS-A Sjogrens syndrome, sicca complex, antibodies against RAP rheumatoid arthritis, Sjogrens syndrome, antibodies against PM-I - polymyositis dermatomyositis and antibodies against nucleolar systemic sclerosis, Sjogren syndrome.

Also antibodies that are associated with specific autoimmune disorders such as antibodies against smooth muscle - chronic hepatitis, antibodies against acetylcholine receptors - myasthenia gravis, antibodies against basement membrane at the dermal-epidermal junction

- Bullus pemphigus, antibody to mucopolysaccharide-protein complex or intracellular binder substance

- Pemphigus, antibodies to immunoglobulins rheumatoid arthritis, antibodies to glomerular basement membrane - glomerulonephritis, Goodpasture's syndrome, idiopathic primary hemasiderosis, antibodies to erythrocytes - autoimmune hemolytic anemia, antibodies to the thyroid - Hashimoto, antibodies to the intrinsic factor - pernicious anemia, antibodies to plut platelets - idiopathic thrombocytopenic purpura, alloimmunization, antibodies against mitochondria - primary biliary cirrhosis, antibodies against salivary gland - Sjogren's syndrome,

Adrenal gland antibodies - iodiopathic adrenal atropathy, antibodies to thyroid microsomes - Grave's disease, antibodies to thyroglobulin - Addison's disease and antibodies to islet diabetes mellitus.

Paraproteinemias such as multiple myeloma, macroglobulinemia, cryoglobulinemia and L-chain disease.

Hyperlipidemia such as primary biliary cirrhosis and familial hypercholesterolemia.

Endocrinopathies such as Grave's disease and diabetes mellitus.

Alloimmunization such as neonatal hemolytic disease and renal homograft rejection.

Furthermore, suitable for the treatment using the present invention z. Post-transfusion purpura and autoantibody diseases such as Goodpasture syndrome, myasthenia gravis, pemphigus vulgaris, hematologic diseases, idiopathic (autoimmune) thrombocytopenic purpura, autoimmune hemolytic anemia, factor VIII inhibitor, and polyradiculopathy / Guillain-Barre syndrome.

Immune complex diseases can also be treated and include: Systemic lupus erythematosus, polyarteritis nodosa, cutaneous vasculitis, rheumatoid arthritis, glomerulonephritis and dermatomyositis.

While one theory may not take precedence over another, a review of the likely course of autoimmune disease images indicates that the pathological consequence is most likely elicited by rejection of a free antigen followed by antibody development and is completed and terminated by antibody excess and complement fixation formed complexes. Therefore, for proper selection of biospecific polymer formulation and provision for proper efficacy, preliminary diagnostic procedures would be necessary to determine the predominant form of the autoimmune effector. Ice vivid example of this is described below for the treatment of rheumatoid diseases. In short, rheumatoid diseases can be characterized as following the course of

a) free RF antigen (atypical Ig) (rheumatic condition),

b) free RF antibody formation and RF complexation, and finally

c) Antibody excess and complement-activated RF complex fixation.

Therefore, the treatment of rheumatoid diseases at their early stage could be determined by the detection of atypical immunoglobulins by monoclonal rheumatoid factor (m-RF) antibodies. Treatment at this stage would be most efficiently performed by m-RF activated biospecific polymers to remove the stimulating antigen and thereby prevent the development of endogenous RF (e-RF) antibodies. Diagnostic evidence of e-RF would be the Indicate use of biospecific polymers containing both m-RF and gamma-globulin bound biological

tions (RF antigen). Alternatively, two biospecific polymers could be used in series, each carrying some sort of active biological function. In one case, this combination of m-RF and bound gamma globulin would adsorb the stimulating antigen and antibody molecules to inhibit the progression of the disease. In the case where significant levels of RF antigen-antibody complexes are discovered, biospecific polymers containing CIq and / or collagen effector molecules would be required. Finally, if the disease process has progressed to the stage of complement fixation of formed imunocal complexes, an effective biospecific polymer would contain one or more anti-complement antibodies, e.g. Anti-Clq, anti-C, or anti-C. The biological functions may, if more than one is desired, be re-immobilized on a single biocompatible carrier or each may be on a separate carrier and with respect to the Blood or plasma flow in series.

As suggested above, the effective use of the present invention is realized by careful definition of the dynamics and stage of the immune response for effective disease control.

Today, plasmapheresis and cytophoresis are the treatments for diseases by removing harmful substances or cells from the blood. It is presently believed that any disease treated by plasmapheresis and / or cytopheresis where the desired result is the removal of a specific substance can be advantageously treated with the product and method of the present invention.

More specifically, a currently contemplated therapeutic measure for the entire blood may be carried out as follows:

• a) 'A vascular access is provided which is suitable for

b) a blood flow of about 30 ml / min to about 200 ml / min,

c) an anticoagulant is administered into the blood and

d) a pump is provided;

e) the blood is passed, with contact to the present invention;

f) depending on the anticoagulant used, additional medication may be necessary or desirable to neutralize the anticoagulant effect in the blood thus treated;

g) the treated blood is returned to the patient.

The duration currently contemplated for the above procedure is approaching within a range of about 2 hours to about 4 hours. It is, of course, taken into account that, depending on the situation, such a time frame may be either shortened or extended.

A currently contemplated therapeutic use for plasma can be described as follows:

a) a vascular access is created, which is calculated for

b) a blood flow of about 30 ml / min to about 200 ml / min,

c) an anticoagulant is administered into the blood; and

d) a pump is provided;

e) separation means for the separation of plasma and formed blood component are provided;

f) the plasma is slid, with contact to the present invention;

g) filtration through a 0.2 μπι filter to remove any microemboli, bacteria and / or fungi;

h) the treated plasma and the blood components formed are recombined;

i) depending on the anticoagulant used, additional medication may be necessary or desired to neutralize the anticoagulant effect in the treated blood;

j) the treated blood is returned to the patient. ,

Vascular access can be created using "well-known techniques and procedures in the medical arts, for example, an inherent large cannula can be used intravenously or arterially." Examples of suitable veins or arteries include the anticubital vein, subclavian veins, and brachial or It is further understood that an arterial-venous shunt or fistulae (AV shunt) may also be used, in which case the heart is the pump, unless the AV shunt or fistulae is used, the preferred pump is during of venous access, a delivery peristaltic pump capable of providing a flow rate of about 30 ml / min to about 200 ml / min.

Suitable anticoagulants suitable in the method of the present invention include e.g. For example, acidic citrate dextrose (approximately 1 ml per 8 ml of total blood), heparin, heparin / acid citrate-dextrose mixtures (eg, 1250 IU of heparin in 125 ml of acidic

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Citrate dextrose / L), and prostaglandin. It is generally understood that when anticoagulants such as heparin and prostaglandin are used, the treated blood or plasma should be given an opposite medication before it is returned or before that blood or plasma is given to a patient.

Furthermore, it is understood, in the case of the treatment of plasma, that any conventional method of removing formed blood components can be used. Suitable examples of methods for separating plasma from formed blood components include: plasmapheresis, centrifugal cell separation, and cell sedimentation in a plasma sac. It is possible that both continuous separation and discontinuous separation are suitable; the aforementioned methods of separation are independent of present invention and its use.

Finally, the shape of the present invention is generally not critical. Therefore, the present invention can be used as a biocompatible carrier containing, for example, the biological function in the form of films, hollow fibers, cylindrical fibers, reticulated network, cylindrical or rectangular channels, spheres, and combinations thereof. The use of a fluidized bed can also be advantageous in some cases his.

Working Example

The invention will be explained in more detail below with reference to some examples.

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example 1

This example describes a method of casting the biocompatible polymeric carrier and a method of chemically attaching a biological function directly to the polymeric carrier. This example is also used to describe the use of a system that is not connected to any mechanical support *

Absorption of Anti-insulin Antibodies Using Insulin-Activated Polyhydroxyethyl Methacrylate: (p~HEMA) - Membrane:

A) Polymer casting:

Solutions of the monomer are prepared by mixing 15.0 g of 2-hydroxyethyl methacrylate, 15.0 g of ethylene glycol, 0.08 g of sodium bisulfite and 0.036 g of ammonium persulfate. The solution was stirred for 15 minutes at room temperature. Approximately 5 ml of the solution was placed on a glass plate (127 mm 1 χ 127 mm w χ 9.5 mm) (5 "1 x 5" w χ 3/8 "t) in the middle of a polyethylene spacer (0.5 mm thick) cut to form a 102mm χ 102mm window sealing ring, a second glass plate was placed over the gasket and solution, clamped in place, and the whole set up at 60 ^° C overnight The clips were removed and the glass plates were slightly spread apart and placed in a deionized water bath for at least 24 hours

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carefully removed from the glass plates and rinsed «- watered for at least three days in freshly exchanged deionised water (500 ml per day),

B) Polymer activation:

Membrane disks (5 mm diameter) were cut from the polymer film for activation and analysis. A 10 to 20 g /% cyanogen bromide solution was prepared by dissolving 1.69 g of finely divided BrCN crystals in 10 ml of 0.2 M Na ₁₃ CO (pH 11; 1) with continuous stirring at 4 ⁰ C. The pH of the solution was kept above 11 by dropwise addition of 5N NaOH until the crystals were dissolved and the pH was stabilized. Four membrane discs were placed in a small sieve and rinsed with approximately 5 ml of 0.1 N HCl and incubated in the cyanogen bromide solution for 15 minutes. The disks were each rinsed at least two more times with 5 ml portions of 0.1N HCl and placed in 5.0 ml overnight

U / 100 regulately incubated ILETIN insulin injection solution adjusted to pH 8.7 by addition of 1N NaOH. The membrane discs were rinsed with 5 ml of 0.5 M NaCl, 0.1 M Na ₂ CO solution and 3 times in 5 ml portions of phosphate (0.05 M) buffered saline (0.9 g%) (pH = 7.4),

G) Evaluation of membrane adsorption of anti-insulin antibodies

A double antibody competitive radioimmunoassay was performed by incubating 560 picograms

125 I labeled porcine insulin and serial dilutions

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(980 to 15 pg) of unlabelled hepatic insulin or p.-HEMA membrane slices with 280 pg guinea pig anti-swine insulin antibody in 0.5 ml of phosphate (0.05 M) buffered saline (pH 7.4) ( 0.9 g%) containing 1 g% bovine serum albumin for two hours at room temperature. The p-HEMA slices were removed from each test solution. A 0.1 ml portion of goat anti-guinea pig gamma globulin was added to each test tube Test solutions were mixed and incubated for a further 2 hours at room temperature A 1.0 ml aliquot of cold (2 to 4 C) phosphate buffered saline (pH 7.4) was added to each tube, each test solution was mixed and centrifuged at 4 for 15 minutes ⁰ C at 7500 G, and the supernatant was decanted into 20 ml scintillation vials The supernatant was gelled with 5.0 ml of Aquasol liquid as a scintillation fluid and counted for 4 minutes in an Isocap 300 Counter Insulin-treated membrane discs adso rbierten 111 pg anti-insulin antibody

2 from the solution or 690 pg per cm surface,

Example 2

This example describes how to make a biospecific membrane without a carrier. It also describes how a 6-aminocaproic acid (with a chain of 6 carbons atoms) can be used as a spacer to attach insulin to the biocompatible polymeric carrier used to remove the insulin antibody and to adsorb anti-insulin antibody using the insulin-activated polyhydroxyethyl methacrylate membrane (p-HEMA),

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A) Polymer casting

Solutions of the monomers were prepared by mixing 15.0 g of 2-hydroxyethyl methacrylate (Polysciences Ine *, Warrington, PA) with 15.0 g of ethylene glycol, 0.08 g of sodium bisulfite, and 0.036 g of ammonium persulfate. The solution was allowed to stand at room temperature for 15 minutes touched. Approximately 5 times of the solution was placed on a glass plate (127 mm χ 127 mm x 9.5 mm) in the middle of a polyethylene <intermediate piece (0.25 mm thick) cut so as to be a sealing ring A second glass plate was placed over the sealing ring and solution, clamped in place, and the entire assembly was incubated at 60 ^° C. overnight. The clamps were removed and the glass plates were placed slightly spread apart and placed in a bath of deionized water for at least 24 hours. The swollen polymer membrane was carefully removed from the glass plates and rinsed - soaked for at least three days in freshly exchanged deionized water (50C ml) per day),

B) Polymer activation

Membrane discs were prepared as described in Example 1. A 10 to 20 g% cyanogen bromide solution was prepared by dissolving 1.69 g of finely divided cyanogen bromide crystals in 10 mL of 0.2 M Na ₂ CO (pH 11.1) by continuous stirring at 4 ° C The pH of the solution was maintained above 11 by the dropwise addition of 5 N NaOH,

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until the crystals were dissolved and the pH was stabilized. Four membrane discs were placed in a small sieve and rinsed with approximately 5 ml of 0.1 N HCl and incubated in the cyanogen bromide solution for 15 min. The disks were each rinsed at least two more times with 5 ml portions of 0.1 N HCl and incubated overnight in 10 ml of a 10 g% 6-aminocaproic acid solution (w / v) prepared in 0 _t 1 M Na ₂ CO, O, 5 M NaCl buffer solution, pH 8.6. The membrane discs were rinsed with 5 ml of O ₁ IM Na ₂ CO, 0.5 M NaCl buffer and three 5 ml aliquots of phosphate buffered (0.05 M) (pH = 7.4) saline (0.9 g%) ₀ The membrane discs were removed from the rinse solution, activated by incubation in 10 ml of a 10% (w / v) 1-cyclohexal-3- (2-morpholinoethyl) <- carbodiimide solution prepared in 0.1 M ( 2-N-morpholino-ethanesulfonic acid) (MES) buffer (pH 6.0) for 30 minutes at room temperature, and each disk was dissolved in 5 ml ka! rinsed with salt solution.

Be was in 5 ml of cold (4 ⁰ C) phosphate-buffered cooking =?

Membrane discs were in duplicate overnight in 5 ml U-IOO regular Iletin® Insulininjek tion solution either ~ or pork insulin regular Iletin® solution at 4 ⁰ C incubated _# The membrane discs were removed buffered from the protein solutions and rinsed three times in 5 ml of phosphate "brine,

C) Evaluation of membrane adsorption of anti-insulin antibody

A double antibody competitively binding radioimmunoassay

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125

was carried out by incubating 560 pg I-labeled whole insulin with serial dilutions (980 to 15 pg) of unlabeled porcine insulin or p-HEMA membrane slices with 280 pg of guinea pig anti-pig insulin antibody in 0.5 ml of phosphate (0.05 M) Buffered (pH 7.4) saline (0.9 g%) containing 1 g% bovine serum albumin for two hours at room temperature. "The p-HEMA slices were removed from each test solution. A 0.1 ml Goat anti-sera WGammai globulin was added to each test tube. The test solutions were mixed and incubated for a further two hours at room temperature. A 1.0 ml portion of cold (2 to 4 ⁰ C) phosphate buffered saline (pH 7.4) was added to each tube. Oede test solution was mixed and centrifuged for 15 min at 4 ⁰ C at 7500 G and the supernatant was decanted into 20 ml scintillation vials. The supernatant was gelled with 5.0 ml of Aquasol liquid scintillation fluid and counted for 4 minutes in an Isocap 300 Counter. Insulin-treated membrane discs adsorbed 271 pg of anti-insulin antibody from solution or 690 pg per cm surface area.

Example 3

This example describes a method of casting the compatible polymer supports both with and without mechanical support by spin casting. "There is also a second way of chemically binding the biological function to the

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biocompatible polymer carrier described »

Adsorption of Anti-Human Immunoglobulin G (IgG) Antibody (Rheumatoid Type "FACTORS") Using Immunoglobulin-Activated Polyhydroxyethyl Acrylate-Co-Glycidyl Methacrylate (p-HEGL) Membranes:

I, polymer casting

The following example describes the preparation of both supported and unsupported polymer membranes by rotary casting techniques »

A) Drafting devices

The Drehgußvorrichtung consists of a closed aluminum cylinder with 6 mm thick walls. The inner dimensions of the cylinder are 10 cm in diameter and 13 cm in length. The cylinder is connected to a mofor that rotates the cylinder, and the cylinder rpm is measured with a strobe phototachometer (Model 1891M, A heat blow gun heats the rotating cylinder, thermoelectric elements measure the internal cylinder temperature and the temperature of the air flowing out The cylinder is cleaned with nitrogen before and during the polymerization,

B) Preparation of the carrier membrane

Whatman Grade 50 tempered filter paper was used

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as a back support to give the rotary castings mechanical strength. The paper was cut into rectangular sheets (101 mm to 24 mm χ 30 mm to 11 mm) and then soaked in ethylene glycol (EG) for 30 minutes at room temperature. Excess glycol was drained from the paper after draining

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The blower (about 35 ft / min) and the heater on the heater blower were started, and the cylinder was heated to 70 to 75 ⁰ C for 90 minutes. The heat was then discontinued, but the blower was started up to cool the cylinder until the inner cylinder temperature had dropped to about 30 ° C. The cold cylinder was removed from the rotary cast iron and filled with deionized water. After soaking for one hour, the cast was removed from the cylinder,

II. Polymer activation

Membrane discs were prepared as described above in Example 1. Fourteen different discs were each incubated in 1.0 ml of 1.0 M hexanediamine solution for 72 hours at 4 ° C. Slices were removed from the hexanediamine solution and three times with 2 ml of phosphate buffered saline A 4.0 g% human gamma-globulin (HGG) solution was prepared by dissolving 4.0 g of HGG in 100 ml of 0.1 M MES buffer solution (pH 6.0) with gentle stirring at room temperature. After the protein was completely dissolved, serial dilutions were made by consecutive transfers of 1.0 ml of protein solution to 9.0 ml of MES solution to give protein concentrations of 4 mg / ml, 400 μg / ml, 40 μg / ml and 4 μg / to obtain buffer. Two different polymer disks were each incubated in 0.5 ml of the protein solutions and 0.5 ml of a 0.25 M l-ethyl-3- (3-dimethyl-aminopropyl) -carbodiimide (Sigma Chemical Co.) solution prepared in MES buffer 72 STUN

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The 4 ⁰ C _- Gede membrane disc was removed from the protein solution and rinsed three times with 2 ml cold (4 C) phosphate buffered saline.

III, Evaluation of Membrane Adsorption of Anti-IgG Antibodies from Physiological Solutions

A radioimmunoassay was carried out by incubation

125

of individual membrane slides with 10 ng of goat anti-human IgG in 1.0 ml of PBS containing 1.0 g% human serum albumin for two hours at room temperature. The radiotracer solution was removed and each membrane was rinsed three times with 2.0 ml PBS solution, the membranes were incubated overnight at 4 ⁰ C in the last rinse solution. Individual membranes were removed from the rinse solutions and counted in an Innotron Hydragamma counter for one minute each. The counts per minute were converted to decays per minute (DPM) by dividing with the detector efficiency. The amount of adsorbed antibody was approximated by dividing the average DPM by the specific activity of the radiotracer. The following results were obtained:

HGG treatment Adsorbed AntilgG

(mg / ml) (pg / cm) ⁺

20.0 2453

2,01919

0.2 1271

0.02 664

0.002 ++

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⁺ Picogram of radiotracer material per cm of membrane

Background activity

Example 4

This example demonstrates the use of aminocarboxylic acid as a spacer for gamma globulin.

Adsorption of antihuman immunoglobulin G (IgG) antibodies (rheumatoid type "Factors") using immunoglobulin-activated polyhydroxyethyl methacrylate-co-glycidyl methacrylate (p-HEGL) membranes.

A) Polymer casting

Rotary cast p-HEGL membranes were prepared as described in Example 2 »

B) Polymer derivatization and activation

Membrane discs were prepared and treated as described in Example 2 except that 1.0 M 6-aminocarboxylic acid was substituted for hexanediamine as a derivatization and spacer reagent *

C) Evaluation of the Membrane Adsorption of Anti-IgG Antibodies from Physiological Solutions »

A radioimmunoassay was performed as described in Example 3 and the following results were obtained:

HGG treatment adsorbed anti-IgG (Mg / ml) (pg cm ² ) * 20.0 2395 ' 2.0 1828 0.2 1310 0.02 732 0,002 158

* Picogram of radiotracer material per cm ² membrane.

Example 5

This example demonstrates the use of albumin (67,000 MW) as a spacer for folate. The folate is used to remove folic acid binding protein.

Adsorption of folate binding proteins (FABP) by folate-albumin activated polyhydroxyethyl methacrylate (p-HEMA) membranes:

A) Polymer casting:

Filter paper reinforced by p-HEMA polymer membranes was spin cast as described in Example 2 using the following polymer formulation:

15.0 g of 2-hydroxyethyl methacrylate

15.0 g of ethylene glycol

0.08 g of sodium metabisulfite

0.036 g of ammonium persulfate

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B) Polymer derivatization and activation

A folic acid-bovine serum albumin complex was prepared by carbodiimide condensation of folate carboxyl groups with albumin-terminated amino groups. To achieve this, 200 mg of folic acid was dissolved in 8 ml of 0.1 N NaOH and 400 mg of 1-cyclohexy1-3 (2- Mo rpholinoethyl) -ca-rbodiimide-metho-p-toluenesulfonate were dissolved in 2.0 ml of 0.1 M MES buffer (pH 6.0) and 1.0 g of bovine serum albumin (BSA) was dissolved in 40.0 ml of 0.1 M MES buffer * The solutions were combined, mixed and incubated for 72 hours at 4 ° C. Unreacted folate and carbodiimide were removed from the solution by mixing 20 ml of the mixture with 20 ml of a BSA (2.5 g%) ~ carbon (1.25 g%) suspension was treated for 30 minutes at 4 ^° C. The suspension was centrifuged for 15 minutes at 3400 G at 4 ° C., decanted and filtered through a 0.22 μm filter.

Membrane discs were prepared and treated with cyanogen bromide solution as described previously in Example 1. After the discs were rinsed in cold saline, sets of eight slices were prepared in 20 ml of either physiological saline, 160 mg% BSA or the folate-albumin complex solution previously prepared was added, and incubated. The discs were incubated for 72 hours at 4 C. CJeder diaphragm assembly was removed from the solution _t blotted dry and suspended in 20 ml saline solution at 4 ⁰ C added to rinse for at least 24 hours, double membranes were treated with 8 ml of 1% glutaraldehyde solution, a minute and about Rinsed overnight in 20 ml phosphate-buffered saline »

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C) Evaluation of membrane adsorption of folic acid-binding protein (FABP) from physiological solution

A competitive protein-binding radioassay was performed by incubating 370 pg of H-pteroylglutamic acid (PGA) and standard solutions (48 to 348 pg) of non-radioactive PGA or p-HEMA membrane slices with 234 pg of binding activity on FABP (Kamen, B, A, and Caston, O.D. ₄ , "Direct Radiochemical Assay for Serum Folate: Competition between H ^ Folic Acid and 5-Methyl-Tetrahydrofolic Acid for a Folate Binder", 0, Lab * Clin, Med., 83, 164, 1974). in 1.0 ml of 0.05 M phosphate buffer (pH 7.6) containing 20 μM folate-free normal human serum and 5 mg sodium ascorbate. The radioassay tubes were mixed, incubated for 30 minutes at room temperature and incubated for 10 minutes at 4 ° C. Individual membrane discs were removed from the test solutions and 0.5 ml of a cold (4 ^° C) BSA (2.5 g%) charcoal (1.25 g%) suspension was added to each tube.

All test solutions were incubated at 4 ^° C. for 10 minutes and centrifuged at 2000 ° C. for 15 minutes at 4 ^° C. The supernatants were decanted into 20 ml scintillation vials. 12 (12.0) ml of liquid scintillation fluid was added to each vial. The samples were counted in an isocap counter for 2 minutes. The following results were obtained:

membrane treatment adsorbed FABP (pg / cm ² Saline 67 cyanogen bromide 54 Bovine Serum Albumin 39 Folate-BSA complex 758

The examples described above serve to illustrate the present invention without limiting it.

Claims (15)

62 829 18 invention sanj A process for preparing a biospecific polymer having immobilized reactive biological punctures having reactivity for binding specific pathological effectors or specific groups of pathological effectors thereto, characterized in that it comprises treating a biocompatible polymeric carrier with an activating agent and binding a biological puncture with at least one functional group that is reactive with the activated biocompatible polymeric carrier, wherein the reaction is carried out for a time sufficient to covalently bind the biological functions to the biocompatible polymeric carrier, and wherein the biological functions are sized such that _: they do not interfere the matrix of the biocompatible polymer carrier can penetrate. 2. The method according to item 1, characterized in that for binding the biological function, a spacer which has a reactive with the activated biocompatible carrier polymer function, reacting with the carrier polymer, the spacer treated on the carrier polymer with a zv / eiten activating agent and then the biological function immobilized on it. 3- (3-d: ethyl-aminopropyl) carbodiimide or 1-cyclohexal-3- (2-morli-thioloethyl) -carbodiimide. 3. The method according to item 1, characterized in that to bind the biological function, the biocompatible carrier polymer is reacted with a spacer which binds spontaneously with the biocompatible carrier polymer, treated the spacer on the carrier polymer with an activating agent and then immobilized the biological function on it. 62 329 18 4. The method according to item 1, characterized in that the Akt ivierungsmittel C] MBR. 5. The method according to item 2, characterized in that the spacer is selected from the group 6-aminocaproic acid or albumin. 6. The method according to item 2, characterized in that the first Alct ivierungsmittel GITBR. 7. The method according to item 2, characterized in that the second Alct ivierungsmittel is selected from the group 1-ethyl-3- (3-dimethyl-aminopropyl) carbodiimide or 1-cyclohexal-3- (2-morpholinoethyl) carbodiimide. 8, method according to item 3, characterized in that the spacer is 1,6-diaminohexane. 9. The method according to item 3, characterized in that the activating agent is selected from the group 1-ethyl 10, method according to item 2 or 3, characterized in that the activating agent and the biological functions react simultaneously with the polymer carrier spacer. 11. - Method according to item .1, 2 or 3 »characterized by that the biocompatible polymer carrier is a hydrogel. 12. The method according to item 1, 2, or 3, characterized in that the biocompatible polymer carrier is the slightly crosslinked homopolymer Hydroxyethy1-methacrylate. 13. The method according to item 1, 2 or 3, characterized in that the biocompatible polymer carrier is selected from the group consisting of polymerized glycidyl acrylate and polymerized glycidyl methacrylate. 14. A method according to any one of items 1, '2 or 3', characterized in that the biocompatible polymer carrier is selected from the group consisting of copolymerized N-vinyl-pyrrolidone and glycidyl methacrylate, the copolymer further containing a monomer selected from the group consisting of: A group consisting of hydroxyalkyl acrylates and hydroxyalkyl methacrylates and mixtures thereof., 15. The method according to item 1, 2 or 3, characterized in that the biospecific polymer support is bonded to a mechanically stable support member. 16 _o . Process according to item 1, 2 or 3, characterized in that the biological puncture is selected from the group consisting of insulin, gamma globulin and polate.