WO1989012390A1 - Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques - Google Patents

Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques Download PDF

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Publication number
WO1989012390A1
WO1989012390A1 PCT/US1988/002067 US8802067W WO8912390A1 WO 1989012390 A1 WO1989012390 A1 WO 1989012390A1 US 8802067 W US8802067 W US 8802067W WO 8912390 A1 WO8912390 A1 WO 8912390A1
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water
substance
insoluble reagent
acid
reagent
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PCT/US1988/002067
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English (en)
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Charles Thayer White
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E.I. Du Pont De Nemours And Company
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Priority to PCT/US1988/002067 priority Critical patent/WO1989012390A1/fr
Priority to JP63506401A priority patent/JPH03505287A/ja
Priority to EP88906570A priority patent/EP0427715A1/fr
Publication of WO1989012390A1 publication Critical patent/WO1989012390A1/fr
Priority to DK301190A priority patent/DK301190A/da
Priority to NO90905496A priority patent/NO905496L/no

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3204—Inorganic carriers, supports or substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/321—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3202—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the carrier, support or substrate used for impregnation or coating
    • B01J20/3206—Organic carriers, supports or substrates
    • B01J20/3208—Polymeric carriers, supports or substrates
    • B01J20/3212—Polymeric carriers, supports or substrates consisting of a polymer obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3214—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the method for obtaining this coating or impregnating
    • B01J20/3217—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond
    • B01J20/3219—Resulting in a chemical bond between the coating or impregnating layer and the carrier, support or substrate, e.g. a covalent bond involving a particular spacer or linking group, e.g. for attaching an active group
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/30—Processes for preparing, regenerating, or reactivating
    • B01J20/32—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating
    • B01J20/3231—Impregnating or coating ; Solid sorbent compositions obtained from processes involving impregnating or coating characterised by the coating or impregnating layer
    • B01J20/3242—Layers with a functional group, e.g. an affinity material, a ligand, a reactant or a complexing group
    • B01J20/3268—Macromolecular compounds
    • B01J20/3272—Polymers obtained by reactions otherwise than involving only carbon to carbon unsaturated bonds
    • B01J20/3274—Proteins, nucleic acids, polysaccharides, antibodies or antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans

Definitions

  • This invention relates to the preparation and use of biocompatible water-insoluble reagents for removal of substances from physiological fluids comprising specific binding ligands and biocompatibility agents immobilized on the surface of a carrier substrate.
  • LDL low density lipoprotein
  • citrate is due to calcium chelation interferring with normal nerve conduction.
  • the usefulness of extracorporeal devices containing highly selective adsorbing materials is therefore dependent not only on the specificity and affinity of the material, but also its biocompatibility with body tissues and biochemical systems.
  • Koren et al. [Biochimica et Biophysica Acta. 876, 202-207 (1986)] disclose the use of a mouse monoclonal anti-human-LDL antibody coupled to an agarose support to remove LDL from plasma samples. No apheresis was performed on any organism.
  • European Patent Application 83112042.3 published June 6, 1984 discloses the use of immobilized heparin or dextran sulfate to remove LDL from plasma.
  • the adsorbent utilized comprised a water-insoluble porous hard gel to which a binding ligand was attached.
  • Ligands were attached through reaction with epoxy groups introduced into polymer gels by reaction with epichlorohydrin or a polyoxirane compound, or in the case of inorganic gels, with epoxy groups introduced by reaction with epoxy silane coupling agents. Gels with surface hydroxyl groups were therefore preferred.
  • Japanese Patent Application JP60087854 discloses a blood purifying adsorber for removal of malignant substances or cells from blood or plasma.
  • the adsorbent comprises a carrier such as glass, charcoal. cellulose triacetate, and various polymers to which a negatively charged compound and an organic ligand have been attached. The attachment requires the use of strong electrophilic reagents, however, which cannot be utilized in concomitant or subsequent reactions incorporating complex biological binding ligands.
  • EP-103184A discloses biospecific polymers carrying immobilized biological agents.
  • the biocompatible polymer support is generally a hydrogel or other wettable polymer to which a biological agent has been covalently bounded. No biocompatibility agents are disclosed which would generally provide protection against adverse reactions in vivo.
  • Japanese patent application J602239425 discloses the use of solid carriers with attached monoclonal antiapolipoprotein B antibodies to remove LDL from human plasma. No biocompatibility agent was utilized to help protect against adverse reactions with tissues or systems in vivo.
  • Japanese patent application JP59200655 discloses the use of an adsorbent consisting of an insoluble carrier, preferably hydrophilic, with an attached organic compound containing a negatively charged group. The immobilization of the binding ligand is through the negatively charged group. The application of the adsorbent to remove malignant cells is discussed. At this time there exists a clear need for an adsorbent reagent which incorporates specific binding ligands and biocompatibility agents to remove pernicious substances from physiological fluids. Such reagents should exhibit both high selectivity and protection against activation of the complement system or blood coagulation. Enhanced biocompatibility of the reagent would enable a reduction in the amounts of anticoagulants administered.
  • the activity of complex biological ligands is highly sensitive to the immobilization technique.
  • a method of preparing such reagents is also required which is mild enough to avoid denaturation. yet flexible enough to permit concomitant or sequential immobilization of binding ligands and biocompatibility agents.
  • one aspect of this invention is a water-insoluble reagent for removal of a substance from physiological fluids comprising (a) a carrier substrate, and immobilized on the surface of said carrier substrate, (b) a binding ligand specific for said substance, and (c) a biocompatibility agent comprising an organic molecule with a nucleophilic moiety and a negatively-charged moiety separated by a spacer arm, said biocompatibility agent immobilized on said surface through reaction with said nucleophilic moiety.
  • Another aspect of this invention involves a process for preparing such a water-insoluble reagent for removal of a substance from physiological fluids comprising the step of contacting and reacting a carrier substrate with an aqueous solution of a binding ligand specific for said substance and biocompatibility agent comprising an organic molecule with a nucleophilic moiety and a negatively-charged moiety separated by a spacer arm.
  • This process can comprise both simultaneous or sequential contacting and reacting of the carrier substrate with the aqueous solutions of binding ligand and biocompatibility agent.
  • Yet another aspect of the invention involves a method of treating a physiological fluid to remove a substance comprising contacting said fluid with a water-insoluble reagent as described above.
  • the instant invention provides an apparatus for the extracorporeal treatment of physiological fluids to remove a specific substance comprising means for withdrawing whole blood from a mammal, means for separating plasma from the whole blood, means for treating said plasma including a chamber containing a water-insoluble reagent as described above which will interact with and deplete said substance from the plasma, and means for recombining the substantially substance depleted plasma with the remainder of the whole blood and for returning the recombined whole blood to the mammal.
  • preferred reagent and processes would utilize an activated neutral polymer carrier substrate, polyclonal anti-LDL antibody, and a biocompatibility agent with immobilization carried out in two steps at a pH of from about 4 to about 10 controlled by a non-nucleophilic buffer.
  • the reagent comprising the immobilization of monoclonal anti-LDL antibody and sulfamic acid on tresyl-sepharose in a one-step concomitant reaction at about pH 8 in potassium phosphate buffer.
  • Figure 1 is a bar graph representing LDL and HDL plasma concentrations pre- and post-treatment for two experiments utilizing human plasma in vitro.
  • carrier substrate is a solid supporting matrix "activated" by well-known coupling chemistries to introduce reactive surface moieties susceptible to nucleophilic attack and displacement by binding ligands or biocompatibility agents to be immobilized on the carrier substrate.
  • Binding ligand refers to any substance, or group of substances, which has specific binding affinity for the substance to be removed from the physiological fluid to the exclusion of other substances.
  • the binding ligand may consist of a polyclonal or monoclonal antibody in the form of whole antiserum or ascites fluid, an IgG fraction or as active fragment of such materials.
  • Other specific binding proteins for example lectins, antigens, or protein A. may be utilized.
  • biocompatibility moiety refers to a substance which renders the carrier substrate more biocompatible vis-a-vis complement activation.
  • nucleophilic moiety refers to chemical groups, for example NH 2 , SH or OH, which carry an unshared pair of electrons and generally attack another molecule at a site where the atomic nucleus is poorly shielded by outer electrons.
  • negatively charged moiety refers to chemical groups, for example OSO- 3 , RSO- 3 . RSO- 2 . COO-, which carry a net negative formal charge.
  • the nucleophilic moiety and negatively charged moiety are contained within the same molecular entity which constitutes a "spacer arm” separating the two functional moieties.
  • neutral polymer refers to a water insoluble material, either natural or synthetic, that consists of repeating subunits and can be formed into a variety of possible configurations such as flat sheets, fibers, beads, porous beads, membranes, foams, etc.
  • Non-nucleophilic buffer refers to any water buffering substance that does not contain a nucleophilic moiety such as R-OH, R-SH, R-NH 2 ,
  • Polyclonal antibody refers to any collection of antibodies directed against a substance where the collection of antibodies directed against a substance arise from many different antibody producing cells.
  • “Monoclonal antibody” refers to any antibody that comes from a single antibody producing cell line.
  • the water-insoluble reagents of this invention involve the immobilization of binding ligands and biocompatibility agents on activated solid supporting matrices (carrier substrates) utilizing such mild coupling chemistry that sensitive biological ligands and biocompatibility agents can be reacted concomitantly.
  • the immobilization process is schematically represented in Equation 1 indicating
  • a variety of materials are suitable as solid supporting materials for the present invention. These materials generally possess the following characteristics: (1) sufficient surface area to effectively trap and remove the target substance due to either the existence of pores or availability in a finely divided state. (2) allow for the flow of physiological fluid over or through the material when constrained in an extracorporeal treatment apparatus, (3) surface chemical groups which can be activated for subsequent reaction with binding ligand and biocompatibility agent, and (4) stable to biological milieu. Materials fulfilling these criteria include synthetic polymers, natural polymers. silica, alumina and zirconia. The physical geometry of the solid supporting matrix can be as a particle, bead, fiber, hollow fiber, thin sheet, foams, or any other permitting fluid flow over or through the material. Table I lists a variety of commercially available carrier substrates which can be utilized to practice the instant invention. The list is representative since other materials and activations chemistries meeting the above four criteria will be known to those skilled in the art.
  • Affigel 10 (NHS activated agagrose, BioRad Co, Richmond CA)
  • Affigel 15 (NHS activated agagrose, BioRad Co, Richmond CA)
  • Tresyl-Sepharose (tresyl activated agarose, Pharmacia Chemical, Upsala, Sweden)
  • Eupergit C (allylglycidyl ether containing terpolymer, Rohn Pharma, Darmstadt Germany)
  • Chloromethylpolystyrene Activation of supporting matrix materials to produce suitable carrier substrates with surface groups susceptible to nucleophilic attack is carried out using well-known chemical techniques and process reagents. See Scouten, W.H., Affinity
  • activation reagents may include cyanogen halides. epichlorohydrins, polyoxiranes, periodates. polyethyleneimines, or glutaraldehyde.
  • Sepharose®4B (Pharmacia Fine Chemicals, Uppsala, Sweden; beaded agarose gel) possessing surface hydroxyl groups is reacted with tresyl chloride (2,2,2-trifluoroethanesulfonyl chloride) to produce a surface sulfonate ester (Tresyl-activated Sepharose®4B, Pharmacia Fine Chemicals) as depicted schematically in Equation 2.
  • activation may be achieved by reaction with an epoxysilane such as gamma-glycidoxypropyltrimethoxysilane, an aminosilane such as gamma-aminopropyltriethoxysilane, or polyethyleneimine-glutaraldehyde,
  • an epoxysilane such as gamma-glycidoxypropyltrimethoxysilane
  • an aminosilane such as gamma-aminopropyltriethoxysilane
  • polyethyleneimine-glutaraldehyde polyethyleneimine-glutaraldehyde
  • binding ligands may be utilized with the instant invention depending upon the specific substance to be removed from the physiological fluid. Binding ligands possessing great specificity permit the selective removal of the substance with the least overall impact on the composition of the physiological fluid being treated.
  • a representative list of binding ligands. conjugate substance to be removed, and the disease/disorder to be treated is provided in Table II.
  • any antigen such as Corresponding antibodies Factor VIII or IX Corresponding antibodies Factor resistant Hemophilia HLA antigen Corresponding antibodies Transplant rejection DNA, RNA, nucleotides Corresponding antibodies SLE, or other ANA related disease e.g. Sjogren's, scleroderma, etc
  • Nervous tissue antigens Corresponding antibodies Cuillan-Barre Syndrome, MS, etc Glomerular Basement membrane Corresponding antibodies Coodpasture's disease
  • Anti-LDL antibodies LDL Hypercholesterolemia. Other lipid disorders, i.e. Fabry disease
  • Anti Idiotypic Antibodies Other antibodies Autoimmune disease, hyperglobulinemia, transplant rejection, any deleterious antibodies
  • Protein A Protein A, protein G, or
  • Immunoglobulins Autoimmune disease, hyperglobulinemia, transplant rejection, any deleterious antibodies
  • Anti-killer cell antibodies or Functional white cells
  • Adoptive immunotherapy cell specific ligands eg IL-2
  • binding ligands of the present invention are not limited to those set forth in Table II which are meant to be illustrative. These binding ligands may be used alone or in mixtures if appropriate.
  • an anti-LDL antibody was prepared using a standard monoclonal antibody immunization and fusion protocol [Vernon T. Oi and Leonard A. Herzenberg. Selected Methods in Cellular Immunology. Eds. B. Mishell and S. Shiigi. 1980, pp 351-372]
  • Balb/CJ mice were immunized with purified human LDL with and without complete Freund's adjuvant. The mice were boosted at regular intervals and bled at various time points for several weeks following the immunizations. Serum samples were tested in an ELISA to determine the amount of anti-LDL antibodies present in the immunized mice versus the non-immunized Balb/CJ.
  • mice were boosted with antigen and the first fusion was performed.
  • a spleen cell suspension from a primed mouse was fused with SP2/0 myeloma cells, mixed with an irradiated feeder spleen cell suspension, and plated out.
  • the clones were fed at various intervals and observed for growth.
  • Clones were selected and assayed to determine the amount of IgG being produced, and each clone was isotyped. Clones were tested regularly to monitor IgG production. At a later date, cell supernates from selected clones were tested in a LDL ELISA to determine specific activity of each selected clone.
  • a number of bifunctional chemical entities incorporating both a nucleophilic moiety, for reaction with the carrier substrate, and a negatively charged moiety separated by a spacer arm are contemplated as biocompatibility agents of the instant invention.
  • the negatively charged moiety is tethered at the surface following reaction of the nucleophilic moiety with the carrier substrate to immobilize the biocompatibility agent.
  • nucleophilic moiety and negatively charged moiety are contemplated by the present invention.
  • Nucleophilic moieties including for example RNH .
  • Negatively charged moieties including OSO- 3 , RSO- 3 , RSO 2 -, CO- 2 , are suitable.
  • a representative list of biocompatibility agents is provided in Table III. TABLE III
  • Amino Carboxylate All amino acids e.g. glycine, aspartic acid, cysteine
  • Binding ligands and biocompatibility agents are immobilized on the surface of the carrier substrate by nucleophilic displacement reactions involving a nucleophile of the binding ligand or biocompatibility agent and the activated moiety on the carrier substrate.
  • nucleophile of the binding ligand or biocompatibility agent In the case of protein binding ligands such as antibodies, the numerous amino groups within the antibody serve as nucleophiles for convenient reaction.
  • Equation 1 The processes of the instant invention are schematically represented by Equation 1.
  • the water-soluble reagent is prepared by adding the carrier substrate to a reaction solution containing the biocompatibility agent and binding ligand or vice versa.
  • the carrier substrate such as those listed in Table I, can be added as a dry material, or wetted by a suitable buffer.
  • the reaction solution contains binding ligand at a concentration of from about 10 mg/ml to about 100 mg/ml and biocompatibility agent at a concentration of from about 0.01 M to about 5 M.
  • the reaction solution can also be buffered with any non-nucleophilic buffer, such as phosphate, at a buffer concentration of from about 0.01 M to about 5 M.
  • the pH range of the reaction solution can vary from about 4 to about 10.
  • the reaction solution is separated from the water-soluble reagent and rinsed to remove unreacted biocompatibility agent and binding ligand.
  • the resultant derivatized carrier substrate comprises the water-insoluble reagent.
  • the water-insoluble reagent is prepared by first reacting the carrier substrate with binding ligand and then adding biocompatibility agent to the reaction solution in a second step. This is the preferred method in cases where the binding ligand and biocompatibility agent are not compatible in the same solution, or if the reaction conditions for immobilization of binding ligand and biocompatibility agent are sufficiently different.
  • the ranges for the reaction parameters in stepwise reactions are the same as for the simultaneous reactions described above.
  • EXAMPLE 1 Anti-LDL Water-Insoluble Reagent preparation using Simultaneous Attachment of Anti-LDL Monoclonal Antibody CLLl (binding ligand) and Sulfamic Acid (biocompatibility agent): Tresyl-Sepharose (carrier substrate; 35 gm) was sterilized by washing with acetone and filtering through a sterile sintered glass funnel. The acetone washed bead was then transferred to a sterile roller bottle (to aid transfer it is recommended that the bead be slightly wetted with acetone). To the roller bottle was added 105 ml of the reaction solution containing monoclonal antibody
  • Tresyl-Sepharose carrier substrate; 0.5 gm
  • carrier substrate 0.5 gm
  • the acetone washed bead was then transferred to a sterile centrifuge tube (to aid transfer it is recommended that the bead be slightly wetted with acetone).
  • To the centrifuge tube was added 5 mL of the reaction solution containing monoclonal antibody CLLl (3.0 rag/mL), and sodium bicarbonate (0.10 M) at pH 8.0. The slurry was gently agitated for 4 hours at ambient temperature.
  • the antibody solution was separated from the carrier substrate by centrifugation at 1000 ⁇ g for 10 min and decanting the unreacted antibody.
  • Sulfamic acid (0.20 M) in sodium carbonate buffer (0.050 M). pH 9.0, was then added and the mixture agitated overnight (16 hr) at ambient temperature. After overnight agitation, the slurry was filtered using a sintered glass funnel and the anti-LDL water-insoluble reagent washed with 1 L of citric acid (pH 3.0). followed by 1 L of buffered saline (pH 7.1). The decanted supernatant and filtrate was collected for reprocessing and reuse. The sterile anti-LDL water-insoluble reagent slurry was transferred to a sterile container where merthiolate was added (final concentration 0.1%) as a preservative of sterility until further use.
  • the water-insoluble reagent described above can be used to selectively remove substances from physiological fluids which are bound by the binding ligand.
  • the water-insoluble reagent would be confined in a container housing that would allow the flow of the physiological fluid through the water-insoluble reagent. If the physiological fluid were blood or blood plasma. the water-insoluble reagent would be used to remove from the blood or plasma a substance that is associated with a disease or disease process. This would be the case in a number of diseases where it is known or suspected that the removal of the disease associated substance would be beneficial. These include auto-immune diseases, metabolic disorders, and others.
  • a volume or amount of a reagent as described above would be contained in a housing that would allow for the flow of blood or plasma.
  • Anticoagulated blood or plasma from a patient would be pumped through the housing containing the water-insoluble reagent and then returned to the patient.
  • the blood or plasma treated in this way would thus be unaffected except for the depletion of the desired substance bound by the binding ligand in the water-insoluble reagent.
  • the effectiveness of the binding ligand can be determined by measuring the amount of a substance in the physiological fluid before and after passage over the water-insoluble reagent.
  • the amount of this substance depleted is a measure of the performance of the reagent.
  • the assay system for determining the amount of the substance removed is, of course, dependent upon the substance. To further illustrate how this would be accomplished, in the case of LDL, plasma that has been anticoagulated would be perfused over an anti-LDL water-insoluble reagent. Samples of the plasma, before and after perfusion over the combined sorbent. would be collected and assayed. Plasma samples would then assayed for total cholesterol and HDL. The LDL levels are easily calculated from the HDL and total cholesterol-values as the total cholesterol level of plasma is presumed to be the sum of HDL and LDL.
  • the total cholesterol determinations would be performed by standard clinical laboratory procedures. This procedure is a quantitative enzymatic reaction using cholesterol oxidase following chemical saponification of the cholesterol esters.
  • the by-product of the cholesterol oxidase (H 2 O 2 ) is coupled with peroxidase and a colorimetric substrate. This reaction leads to a colored end product which can be measured using a spectrophotometer [Stein, E. A., In. Textbook of Clinical Chemistry. Tietz, N. W., Ed., WB Saunders. Philadelphia, 1986, pp 879-886. 1818. and 1829].
  • the HDL determinations are performed by first precipitating the LDL, by use of a precipitating reagent, and assaying the supernate via the above total cholesterol procedure.
  • the effectiveness of the biocompatibility agent can be determined by measuring the amount of complement activation in the plasma as a result of exposure to the combined sorbent. Attenuation of the complement activation as compared to the untreated polymer carrier is the demonstration of performance of the biocompatibility agent. Activation of the complement cascade by a foreign substance occurs via activation of the alternate pathway.
  • the components of the complement cascade that are quantitated to determine the amount of activation are C 3a and
  • C 5a is determined by a known RIA technique [see Chenoweth. D.E. and Hugli, T.E.,
  • C 3a (or C 5a ) in a plasma sample can then be calculated from the measured amount of radioactivity.
  • the water-insoluble reagent can be contained in various shaped housings depending upon the physical form of the solid support material or the application.
  • the shape of the apparatus employed is not critical to this disclosure, any apparatus that restrains the water-insoluble reagent and allows for flow of the physiological fluid can be used.
  • the effectiveness of the water-insoluble reagent to remove LDL from plasma can be demonstrated by measuring the LDL levels before and after perfusion of a plasma sample over the water-insoluble reagent.
  • the water-insoluble reagent of Example 1 comprising anti-LDL monoclonal antibody CLLl (binding ligand) and sulfamic acid (biocompatibility agent) immobilized on tresyl Sepharose
  • demonstration of LDL binding to the immunosorbent and depletion of LDL from plasma is illustrated by the two experiments shown in Figure 1.
  • the values for LDL cholesterol (solid bars) and HDL cholesterol (hatched bars; total cholesterol is the sum of HDL and LDL) before and after plasma treatment by the immunosorbent are presented.
  • the immunosorbent effectively depletes LDL from plasma whether a high cholesterol (>300 rag/dl) or a low cholesterol (155 mg/dl) plasma is used. Also note that HDL levels are unaffected by the treatment with the LDL immunosorbent, attesting to the specificity of the water-insoluble reagents.
  • total complement activation is the sum of C 3a and C 5a and is expressed as ⁇ g/ml in the test plasma.
  • Sulfanilic and sulfamic treatment result in the use of a biocompatibility agent from Table III and a significant reduction in the complement activation of that material is seen.
  • Treatment with Tris (tris(hydroxymethyl)aminomethane, Sigma Chemical Company)) or water hydrolyzed tresyl-Sepharose does not incorporate the negatively charged moiety onto the surface and does not confer the protection obtained from the use of the biocompatibility agent.
  • a combination sorbent prepared with a binding ligand and a biocompatibility agent confers both binding and biocompatibility properties to the water-insoluble reagent.
  • the water-insoluble reagent prepared using the anti-LDL antibody CLLl and sulfamic acid demonstrate the combined effect; the ability of the biocompatibility agent to attenuate complement, and the ability of the binding ligand to bind and remove
  • the anti-LDL reagent as described above, (1.5 mL) was packed into a 1.0 ⁇ 10 cm column containing a fritted disc at the bottom to retain the water-insoluble reagent material.
  • the packed reagent was washed with several bed volumes of phosphate buffered saline at pH 7.4 to remove the merthiolate. Citrated plasma (9.0 mL) was then recirculated through the column for 1 hr. Samples of the plasma before and after recirculation were assayed to determine the amount of removal of LDL or other constituents from the plasma. The removal of LDL from the plasma is illustrated in Figure 1.
  • LDL and LDL Lipoprotein levels
  • LDL cholesterol solid bars
  • HDL cholesterol hatchched bars; total cholesterol is the sum of HDL and LDL
  • two plasma samples were perfused over the reagent. Note that the reagent removes LDL effectively whether a high cholesterol (>300 mg/dl) or a low cholesterol (155 mg/dl) plasma is used.
  • Table VI shows the effect of the plasma recirculation over the water-soluble reagent on the other plasma components before and after recirculation. There were no significant changes in the levels of the other plasma constituents.

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  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Biophysics (AREA)
  • Inorganic Chemistry (AREA)
  • Microbiology (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Endocrinology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Developmental Biology & Embryology (AREA)
  • Food Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Genetics & Genomics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • External Artificial Organs (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention décrit la préparation et le procédé d'utilisation de réactifs biocompatibles insolubles dans l'eau, qui sont destinés à extraire des substances de fluides physiologiques. Le réactif, qui comprend un substrat véhicule avec une couche d'agent de liaison immobilisé et d'agent de biocompatibilité, est produit par contact concomitant ou séquentiel d'un substrat véhicule activé avec l'agent de liaison et avec l'agent de biocompatibilité. L'agent de biocompatibilité, qui contient une molécule organique ayant une fraction nucléophile et une fraction chargée négativement séparées par un bras d'espacement, réagit avec la surface du substrat par l'intermédiaire de sa fraction nucléophile. Le réactif peut être incorporé dans des systèmes extracorporels traditionnels de perfusion de sang ou de plasma pour l'extraction de substances telles que des lipoprotéines basse densité (LBD).
PCT/US1988/002067 1988-06-20 1988-06-20 Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques WO1989012390A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
PCT/US1988/002067 WO1989012390A1 (fr) 1988-06-20 1988-06-20 Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques
JP63506401A JPH03505287A (ja) 1988-06-20 1988-06-20 生理学的流体を処理するための生物適合性、物質特異的試薬
EP88906570A EP0427715A1 (fr) 1988-06-20 1988-06-20 Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques
DK301190A DK301190A (da) 1988-06-20 1990-12-19 Bioforenelige, stof-specifikke reagenser til behandling af fysiologiske vaesker
NO90905496A NO905496L (no) 1988-06-20 1990-12-19 Biologisk forenelige, stoff-spesifikke reagenser til behandling av fysiologiske vaesker.

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US1988/002067 WO1989012390A1 (fr) 1988-06-20 1988-06-20 Reactifs biocompatibles a reaction specifique selon les substances destines au traitement de fluides physiologiques

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WO1989012390A1 true WO1989012390A1 (fr) 1989-12-28

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EP (1) EP0427715A1 (fr)
JP (1) JPH03505287A (fr)
DK (1) DK301190A (fr)
NO (1) NO905496L (fr)
WO (1) WO1989012390A1 (fr)

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WO1991010360A1 (fr) * 1990-01-17 1991-07-25 Octapharma Ag Procede de suppression de virus dans les liquides biologiques
US5753227A (en) * 1993-07-23 1998-05-19 Strahilevitz; Meir Extracorporeal affinity adsorption methods for the treatment of atherosclerosis, cancer, degenerative and autoimmune diseases
US6582386B2 (en) 2001-03-06 2003-06-24 Baxter International Inc. Multi-purpose, automated blood and fluid processing systems and methods
US6706008B2 (en) 2001-03-06 2004-03-16 Baxter International Inc. Automated system and method for withdrawing compounds from blood
US7868124B2 (en) * 2002-08-06 2011-01-11 Commissariat A L'engergie Atomique Polyphenylene-type polymers, preparation method thereof, membranes and fuel cell device comprising said membranes
CN114160110A (zh) * 2021-11-30 2022-03-11 广州康盛生物科技股份有限公司 一种预活化的多糖微球及其制备方法、应用

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Proc. Natl. Acad. Sci. USA, Vol. 78, No. 1., issued January 1981 (Washington, D.C. USA) STOFFEL et al., Selective Removal of Apolipoprotein B-Containing Serum Lipoproteins from Blood Plasma", pp 611-615. see Abstract. *
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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991010360A1 (fr) * 1990-01-17 1991-07-25 Octapharma Ag Procede de suppression de virus dans les liquides biologiques
US5753227A (en) * 1993-07-23 1998-05-19 Strahilevitz; Meir Extracorporeal affinity adsorption methods for the treatment of atherosclerosis, cancer, degenerative and autoimmune diseases
US6039946A (en) * 1993-07-23 2000-03-21 Strahilevitz; Meir Extracorporeal affinity adsorption devices
US6569112B2 (en) 1993-07-23 2003-05-27 Meir Strahilevitz Extracorporeal affinity adsorption device
US6676622B2 (en) 1993-07-23 2004-01-13 Meir Strahilevitz Extracorporeal affinity adsorption methods for the treatment of atherosclerosis, cancer, degenerative and autoimmune diseases
US6582386B2 (en) 2001-03-06 2003-06-24 Baxter International Inc. Multi-purpose, automated blood and fluid processing systems and methods
US6706008B2 (en) 2001-03-06 2004-03-16 Baxter International Inc. Automated system and method for withdrawing compounds from blood
US7868124B2 (en) * 2002-08-06 2011-01-11 Commissariat A L'engergie Atomique Polyphenylene-type polymers, preparation method thereof, membranes and fuel cell device comprising said membranes
CN114160110A (zh) * 2021-11-30 2022-03-11 广州康盛生物科技股份有限公司 一种预活化的多糖微球及其制备方法、应用

Also Published As

Publication number Publication date
EP0427715A1 (fr) 1991-05-22
DK301190D0 (da) 1990-12-19
DK301190A (da) 1990-12-19
NO905496D0 (no) 1990-12-19
NO905496L (no) 1990-12-19
JPH03505287A (ja) 1991-11-21

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