WO1991015252A1 - Copolymeres non thrombogenes au glycosaminoglycan - Google Patents

Copolymeres non thrombogenes au glycosaminoglycan Download PDF

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Publication number
WO1991015252A1
WO1991015252A1 PCT/CA1991/000120 CA9100120W WO9115252A1 WO 1991015252 A1 WO1991015252 A1 WO 1991015252A1 CA 9100120 W CA9100120 W CA 9100120W WO 9115252 A1 WO9115252 A1 WO 9115252A1
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Prior art keywords
copolymer
heparin
glycosaminoglycan
fragment
oco
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PCT/CA1991/000120
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English (en)
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M. Abdul Mazid
Frank M. Unger
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Chembiomed, Ltd.
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Priority to JP91506427A priority Critical patent/JPH05507298A/ja
Publication of WO1991015252A1 publication Critical patent/WO1991015252A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/64—Macromolecular compounds not provided for by groups C08G18/42 - C08G18/63
    • C08G18/6484—Polysaccharides and derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/0005—Use of materials characterised by their function or physical properties
    • A61L33/0011—Anticoagulant, e.g. heparin, platelet aggregation inhibitor, fibrinolytic agent, other than enzymes, attached to the substrate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L33/00—Antithrombogenic treatment of surgical articles, e.g. sutures, catheters, prostheses, or of articles for the manipulation or conditioning of blood; Materials for such treatment
    • A61L33/06—Use of macromolecular materials
    • A61L33/08—Polysaccharides
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0078—Degradation products
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • C08G18/06—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen
    • C08G18/28—Polymeric products of isocyanates or isothiocyanates with compounds having active hydrogen characterised by the compounds used containing active hydrogen
    • C08G18/40—High-molecular-weight compounds
    • C08G18/64—Macromolecular compounds not provided for by groups C08G18/42 - C08G18/63
    • C08G18/6453—Macromolecular compounds not provided for by groups C08G18/42 - C08G18/63 having sulfur

Definitions

  • the present invention relates to synthesis of glycosaminoglycan copolymers which are biocompatible and nonthrombogenic in vitro and thus can be used in biomedical devices.
  • biomedical devices The importance of the clinical application of devices or materials which contact blood is well recognized in the practice of modern medicine. Generally speaking, all materials used in biomedical devices need to be biocompatible. The use of biomedical devices has frequently been limited due to adverse reactions between the foreign surface and one or more components of blood, a major restriction being the propensity of foreign surfaces to support thrombosis.
  • heparin In a completely different approach, a chemical or radiochemical treatment of heparin induces the generation of a macro-radical which in conjunction with a monomer results in a covalently incorporated heparin copolymer.
  • these may or may not have an anticoagulant activity and are rather poor compared to intact heparin (Boffa, M.C, et al., Thromb Haemostas (1979) 1:346; Salzman, E. , et al., in Chemistry and Biolocrv of Heparin (R. Lundblad et al., Eds.) Elsevier, New York, 1980, p. 435).
  • the antithrombin-mediated inhibition of thrombin requires a segment of at least four (trisulfated) disaccharide units in addition to the pentasaccharide sequence of the active-site for thrombin (Lane, D.A. , et al., Biochem J (1984) 21S.: 25-732) .
  • the sizes of these heparin fragments are far from those of conventional heparin which may contain a considerable proportion of higher molecular weight fractions, the latter being responsible for inducing platelet adhesion/aggregation as well as binding to other plasma components. Therefore, incorporation of smaller heparin segments with anti-Xa activity into a polymeric * backbone would provide a permanently antithrombotic and chronically nonthrombogenic (thromboresistant) or antithrombogenic biomaterial.
  • the advantage of using lower amounts of heparin segments would be to prevent thrombosis while reducing or eliminating the hemorrhagic risks as ⁇ ociated with high anticoagulant activity of conventional heparin.
  • a biomaterial incorporating such heparin fragments into a polymeric backbone would be useful for the prevention of surface thrombosis in a more efficient manner since the surface would be les ⁇ susceptible to competing interactions with various heparin-neutralizing or degrading species including lipoproteins, fibrinogen, etc. , which are more abundant than the antithrombin in plasma. This surface would also be less prone to platelet adhesion and promotion of their aggregation or interaction with such cell surface components as fibronectin and laminin.
  • the material would provide a negatively-charged nonthrombogenic surface, somewhat similar to that of a vascular endothelium, and a pseudo-affinity for antithrombin by mimicking the array of essential sulfate groups in the binding site for antithrombin, yet inappropriate for thrombogenic interaction with fibrinogen or platelets.
  • the present invention solve ⁇ the ⁇ e problems by including anticoagulant and antithrombotic glycosamino ⁇ glycan fragments as monomeric units in copolymers with non-glycosaminoglycan monomeric units that confer the physical properties required for construction of the device.
  • the devices may be constructed, in those portions which contact the blood, with the copolymer which itself has anticoagulant, nonthrombogenic, and antithrombotic properties.
  • these copolymers may be used to coat surfaces of devices intended for blood or other biological contact.
  • water soluble copolymers are useful as pharmacological agents for the treatment of coagulation disorders or of certain immunological, cardiovascular and viral diseases.
  • copolymers of the invention can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers to provide forms of the copolymers with more rigid physical properties, if desired.
  • the copolymers can also be synthesized in the presence of crosslinkers
  • SUBSTITUTE SHEET further derivatized to additional moietie ⁇ by employing monomeric units which contain functional groups that permit further derivatization.
  • This derivatization can include derivatization to, for example, affinity ligands, label, or additional glycosaminoglycan units to enhance the anticoagulation effect.
  • the invention is directed to a biologically compatible copolymer suitable for the construction and/or coating of biomedical devices which contact the blood.
  • the copolymer is constructed by copolymerization of at least one monomeric unit which is a glycosaminoglycan fragment capable of conferring the desirable antithrombotic, nonthrombogenic, and anticoaculant propertie ⁇ along with at lea ⁇ t one additional non-glycosa inoglycan monomeric unit which confers the suitable physical properties required for the construction or coating of the device.
  • a glycosaminoglycan fragments and/or mixtures of copolymerizing monomeric units can be u ⁇ ed to form the copolymer ⁇ .
  • the phy ⁇ ical characteristics desired in the resulting copolymer will determine the choice of the nonglycosaminoglycan monomeric unit.
  • the physical characteristic ⁇ are also affected by the type and amount of crosslinking employed.
  • Water-soluble forms of the copolymers are also included in the invention, as are derivatized forms of these copolymers.
  • the invention is directed to a method to prepare the copolymer of the invention which method comprises mixing the individual monomeric units or their components under condition ⁇ wherein copolymeri ⁇ zation takes place.
  • the invention is directed to medical devices con ⁇ tructed and/or coated with the copolymer of the invention.
  • Figure 1 shows the degradation of heparin and typical products from (A) nitrous acid cleavage and (B) heparinase digestion.
  • Figure 2 shows copolymerization schemes for acrylamide with typical (A) heparin fragment generated by heparinase digestion (Example l) , (B) allyl glycoside derivation of product from heparinase digestion, and (C) aminoethylmethacrylate derivative of nitrous acid cleavage product (Example 2).
  • Figure 3 shows the proton NMR spectrum in D 2 0 of a "putative copolymer" of acrylamide with heparin decasaccharide obtained from heparinase digestion (Preparation A).
  • the copolymer (designated CH5-139, prepared by Example 1) contains 7.9 weight % heparin fragment after purification by desalting on a Sephadex G-25 column, followed by dialy ⁇ i ⁇ again ⁇ t 50 mM EDTA and finally against Type I water.
  • Inset show ⁇ proton NMR spectra of corresponding heparin deca ⁇ accharide.
  • Figure 4 show ⁇ a typical proton NMR spectrum in 0,0 of a copolymer of acrylamide with aminoethylmeth ⁇ acrylate derivative of heparin octasaccharide produced by limited deaminative cleavage with nitrou ⁇ acid (Preparation B) .
  • the copolymer (CH8-32B, prepared by Example 2) contains 28 wt % heparin fragment, following purification on a Sepharose 4B column eluted with 0.2 M NaCl and dialysis against pure water.
  • Figure 5 shows the proton NMR spectrum in deuterated DMSO of a polyurethane-type polymer prepared by copolymerization of tolylene diisocyanate and ethylene glycol with low molecular weight heparin produced from nitrous acid cleavage.
  • the copolymer (CF3-22C, prepared by Example 6) contains 0.7 wt % heparin after extensive washing with chloroform, ethanol and finally with water until free from unreacted heparin and other monomers.
  • Figure 6 shows the proton NMR spectrum in deuterated DMSO of the copolymer of acrylonitrile with an allyl derivative of heparin hexadecasaccharide (from heparinase digestion) containing 8% (by weight) of heparin, prepared in Example 11.
  • Figure 7 shows a typical FTIR spectrum of a polyurethane-type copolymer, as prepared in Example 18, of tolylene-2, -diisocyanate and Tris-(hydroxymethyl) amino ethane-derivatized, low molecular weight heparin (4,000-6,000 Da).
  • the copolymer contains 5.24 ⁇ g heparin/mg of solid.
  • Figure 8 shows copolymerization schemes for the synthesis of copolyurethanes with low molecular weight heparin fragments.
  • the invention is directed to copolymers which are useful in construction and/or coating of medical devices which contact blood. These copolymers are advantageous because their biocompatibility properties are such that they can be used to contact blood without the thrombotic effects usually evidenced when blood contacts nonbiocompatible surfaces.
  • nonthrombogenic means that the material does not itself cause the formation of thrombi or the coagulation of blood.
  • Antithrombotic and anticoagulant mean that the materials not only do not themselves cause clotting but actively prevent the formation of blood clots encouraged by other stimuli.
  • the copolymers of the invention are both antithrombotic and nonthrombogenic.
  • the fragment is generally prepared by depolymerization of a suitable glycosaminoglycan, but the possibility of synthetic preparation of these fragments is by no means excluded. At the present time, preparation through depolymerization is more economic.
  • GAGs are biological polymers, generally sulfated, which are repeating disaccharide units of uronic acid-glycosamine disac- charides.
  • Heparin is perhaps the best known GAG, and consists mostly of repeating iduronic acid-glucosamine subunits which are sulfated at some of the available hydroxyl and amino positions.
  • Dermatan sulfate is comprised of disaccharide units which are either iduronic or glucuronic acid coupled to a galactosamine residue, also sulfated at some of the amino and hydroxyl positions.
  • Other GAGs useful in the invention include hyaluronic acid, heparan sul ⁇ fate, and chondroitin sulfate, especially types A and C
  • the GAGs are susceptible to depolymerization using appropriate enzymes or by the use of chemical degradation.
  • heparin is conveniently degraded by heparinase digestion; chondroitin sulfate by chondroitinase.
  • Chemical degradation is most commonly conducted using nitrous acid or periodate.
  • the nature of the degradation products will depend on the mode of degradation. Furthermore, the nature of the degradation scheme will al ⁇ o determine the availability of functional group ⁇ for further polymerization in the product low molecular weight GAG.
  • the reducing terminal aldehyde is retained, this can be derivatized after the degradation to obtain an allyl derivative which can participate in addition polymerization with un ⁇ aturated non-GAG monomer ⁇ .
  • Additional carbonyl moietie ⁇ may also be generated by certain degradation schemes, and these can be further derivatized or used directly in the polymerization reaction.
  • Available carbonyl groups can also be reduced to the alcohols which can participate in polymerization reactions with isothiocyanates. Some degradation schemes directly generate carbon-carbon unsaturation in the sugar residues.
  • the available functional groups in the degradation products can also, if desired, be provided with linkers which attach suitable functional groups for polymerization reactions.
  • FIG. l An illustrative scheme for the degradation of GAGs is shown for heparin in Figure l.
  • Figure *1A degradation in nitrous acid result ⁇ in deamination and ring contraction of the reducing terminal glucosamine residue to obtain a 2,5-anhydromannose.
  • the re ⁇ ulting free aldehyde at the reducing terminu ⁇ i ⁇ a convenient reactive group for derivatization to material ⁇ capable of forming copolymers.
  • Periodate degradation would also generate aldehyde functionalities. This free aldehyde may also be reduced to obtain the corresponding anhydromannitol.
  • Figure 2 illustrates some of the modalities for the copolymerization of the GAG fragments with additional monomeric units. As shown in Figure 2A, for example, the
  • SUBSTITUTE SHEET 4,5-uronate resulting from heparinase digestion can be reacted with acrylamide to obtain an addition polymer having a heparin fragment integrated into the re ⁇ ulting copolymer.
  • multiple acrylamide residues may (and typically will) form the "monomeric unit" with which the glycosaminoglycan fragment is copolymerized.
  • "monomeric unit” refers respectively to the glycosaminoglycan and non-glycosaminoglycan subunits of the copolymer which will be, of course, nonuniform, in a typical copolymerization.
  • heparinase fragments could dimerize or trimerize by virtue of the unsaturation at the nonreducing terminus; it is extremely likely that oligomer formation of the acrylamide components will take place.
  • component is used to refer to an individual monomer; “monomeric unit” is used to refer to an individual GAG or non-GAG region of the copolymer.
  • Figure IB show ⁇ a different approach for providing unsaturation in the GAG fragment for participation in addition polymerization.
  • the reducing terminus can be conveniently derivatized to an unsaturated moiety, such as here shown in the formation of an allyl glycoside. Formation of allyl derivatives is standard in the art. These then can copolymerize with a non-GAG monomeric unit as illustrated here, with acrylamide. Again, formation of oligomeric units as the defined "monomeric unit" from the acrylamide occurs. While the illustration shows involvement of the allyl group in the polymer, it is of course possible that polymerization will take place at the nonreducing terminus as well, in a manner analogous to that shown in Figure 2A.
  • Figure 2C illustrate ⁇ ⁇ till another approach involving derivatization of a nitrou ⁇ acid degradation product of heparin through amination of the aldehyde to provide the desired unsaturation, in this case to the aminoethyl ethacrylate.
  • the unsaturation in the methacrylate is then a convenient site for copolymerization with the additional monomeric units supplied by acrylamide.
  • the functionality appropriate for inclusion of the GAG fragment in an addition copolymer can be supplied by derivatization of a convenient functional group on the fragment.
  • a C-C double bond can be derivatized to the reducing terminus of the fragment by virtue of the reactivity of the aldehyde or hemiacetal.
  • An allyl group can be derivatized directly as de ⁇ cribed in the figure, or a C-C double bond containing residue, such as acrylic acid or a derivative thereof, can be coupled with the free aldehyde or the open chain form at the reducing end.
  • C-C double bonds residing in the GAG per se as a result of degradation at the nonreducing terminus can be used directly in the copolymerization.
  • addition polymers can be formed using diisocyanates as shown in Figure 8.
  • the appropriate reactive group i ⁇ hydroxy or amino. Addition is to the N-C double bond of the isocyanate.
  • a ⁇ the GAG fragment will have, in many cases, a multiplicity of hydroxyl groups, this method is most advantageous when a primary alcohol can be supplied to react preferentially in comparison to the mostly secondary hydroxyl ⁇ in the fragment.
  • the products of nitrous acid depolymerization provide an aldehyde which is readily reduced to a primary alcohol using, for example, a borohydride.
  • the foregoing copolymerization may also be modified by including in the non-GAG "monomeric unit" a diol which polymerizes initially with the diisocyanate.
  • a diol which polymerizes initially with the diisocyanate.
  • ethylene glycol, propylene glycol, 1,3-propanediol and the like can be included in the copolymerization mixture, resulting in copolymers of the diol with the diisocyanate as well as inclusion of the GAG fragment.
  • the non-GAG "monomeric unit" used in the copolymer can be composed of a variety of monomers, dimer ⁇ , trimers, and oligomer ⁇ wherein the ⁇ e monomeric units may be homogeneous with regard to the monomer ⁇ which comprise them or which may be heterogeneous.
  • Candidates for components which comprise the non-GAG monomeric units in the copolymers of the invention include acrylic and methacrylic acid and their carboxyl derivatives such as the amide ⁇ and e ⁇ ters thereof.
  • the amides may be the primary amide as in the illustration of Figure 2 or may be, for example, an alkyl amide of 1-6C
  • the esters may be the alkyl esters of 1-6C or the alkyl groups of the ester may be substituted, preferably with hydrophilic substituents such as hydroxyl.
  • suitable as components of the non-GAG monomeric unit are the hydroxyalkyl ester ⁇ , the aminoalkyl ester ⁇ , and the glyceryl e ⁇ ter ⁇ of acrylic or methacrylic acid.
  • hydrophilic alkylenes such as ester ⁇ of vinyl alcohol, allyl alcohol, acrylonitrile, and the like.
  • the e ⁇ ter groups of the vinyl alcohol esters can be hydrolyzed, if desired, after polymerization. In some instances, less
  • BSTITUTE SHEET hydrophilic non-GAG component ⁇ ⁇ uch as styrene, styrene sulfonic acid, or propylene can be used.
  • the non-GAG "monomeric unit” may comprise copolymers of a multiplicity of these components.
  • a copolymer of hydroxyethyl methacrylate and a vinyl alcohol ester could be used.
  • multiplicity means that two or more different GAG or non-GAG components are u ⁇ ed either with re ⁇ pect to a monomeric unit, the copolymer a ⁇ a whole, or both.
  • the non-GAG fragment can also be a urethane type of monomeric unit.
  • Urethane-type unit ⁇ are obtained by conden ⁇ ation of a diol with a diisocyanate.
  • Useful diisocyanates include tolylene- diisocyanate, and diisocyanate derivatives of the various alkylene diamines.
  • the copolymerization of the GAG and non-GAG subunits will be conducted under conditions suitable for the particular components chosen.
  • the non-GAG fragment may be preoligomerized or, more typically and preferably, the components of the fragment may be supplied as the monomers in the copolymerization mixture.
  • T conditions for such copolymerizations are known to those in the art.
  • GAG component Gl may be copolymerized with the non-GAG component NG1 to obtain copolymers, e.g., of the form:
  • GAG components e.g., Gl and G2
  • Gl and G2 may be u ⁇ ed to obtain, for example:
  • Typical molecular weights of the resulting copolymer are sufficiently high that the appropriate physical characteristic ⁇ will be conferred to allow the construction or coating of the desired medical devices.
  • Such devices include catheters, delivery lines for extracorporeal treatment, endoscopic instruments, and the like.
  • water-soluble copolymers in particular can be used as pharmacological agent ⁇ for the treatment of coagulation disorders or certain immunological, cardiovascular and viral diseases.
  • the copolymer may also be formed in the presence of a crosslinker to obtain a product with physical properties which may be more desirable for the particular use intended.
  • Typical crosslinker ⁇ for addition-type polymers include monomer components with two centers of un ⁇ aturation, such as bis forms of acrylamide or of the hydroxyalkyl acrylate ester ⁇ .
  • the crosslinking moiety is included in the polymerization mixture a weight percent of about 5-15% of the non-GAG ⁇ onomer component, preferably about 10% of the non-GAG monomer component.
  • the copolymer ⁇ can further be derivatized to other u ⁇ eful moieties, including, for example, affinity ligands, label or, in particular, additional low molecular weight GAG.
  • additional functional groups unreactive under the conditions of polymerization, but capable of reacting thereafter with functional groups in the moiety to be derivatized to the copolymer.
  • use of aminoethyl methacrylate in the non-GAG portion provides free amino groups, which can derivatize with the aldehyde groups of low molecular weight heparin. Any convenient coupling strategy can be employed depending on the nature of the substance to be derivatized and the functionality of the resulting copolymer.
  • the backbone copolymer contains only non-GAG components and includes low molecular weight GAG as an intact derivatized ligand.
  • functional groups inert to the polymerization are included in the monomeric component ⁇ and then used to bind selectively at only one functional group, for example, the reducing terminus, of the GAG. This form of the invention is illustrated below in Examples 13-15, 17 and 19-21.
  • Preparation A Degradation of Heparin with Heparinase from Flavobacteriu ⁇ i Heparinum and Derivatization of Heparin Fragments into Allvl Glvcosides
  • One gram of commercial porcine mucosal heparin (Sigma) is dissolved in 5.0 mL of 25 mM sodium acetate, 0.25 mM calcium acetate, pH 7.0.
  • 1250 units of commercial heparinase from Flavobacterium heparinum (also, from Sigma) is dissolved in 5.0 mL of 50 mM sodium phosphate, pH 7.4. These two solutions are combined, mixed well and allowed to incubate overnight in a 30•C water-bath.
  • the enzymatic degradation product is monitored periodically by measuring the absorbance of 20 ⁇ l of digestion mixture in 2.0 mL of 30 mM HC1 at 232 nm.
  • the filtrate and wa ⁇ hings are pooled, concentrated to a email volume, and loaded onto a rever ⁇ e pha ⁇ e silica gel column (20 cm x 1.5 cm).
  • the column is washed sequentially with 50 mL water, 50 mL methanol:H 2 0 fraction containing the allyl glycoside of the heparin fragment is then lyophilized.
  • heparin Depolymerization of heparin is carried out according to the procedure of B. Casu et al. fBiochem J. (1981) 197:599-609) .
  • One gram of commercial procine mucosal heparin is dissolved -in 400 mL of Type I water to make a 0.25% solution.
  • This ⁇ olution i ⁇ brought to 0.1 M H 2 S0 4 by adding to it 2.14 mL of concentrated H 2 S0 4 .
  • the mixture is cooled to 10*C in an ice-bath and then made 0.2 M in NaN0 2 by dissolving 5.55 g NaN0 2 in it. This i ⁇ allowed to react for 2 min at 10 ⁇ C.
  • the solution is then neutralized by adding 10 M NaOH until the pH stabilized at 7.0-7.2.
  • the mixture is concentrated and partially desalted u ⁇ ing a 500 molecular-weight-cut-off Amicon membrane, and lyophilized.
  • the dried heparin fragments are then redissolved in a small volume of Type I water and resolved on Sephadex G-50 superfine columns, as described in preparation A. Fractions are assayed for heparin content using the uronic acid assay of T. Bitter and H.M. Muir (Anal Biochem (1962) 1:330-334).
  • the heparin fragments of different size (disaccharides to hexadecasaccharides) present in the column fractions are dialyzed separately against Type I water and lyophilized.
  • a typical polymerization mixture contains 10 mg of a heparin fragment (hexa- to hexadecasaccharide) with the ,5-unsaturated bond (prepared as described in paragraph A of preparation A), 11.6 mg of acrylamide, and 0.725 ⁇ L TEMED (N,N,N' fN'-tetramethylethylenediamine) , in 180 ⁇ L water. This mixture is degassed for 30 min before addition of 0.18 ⁇ g ammonium persulfate in 5 ⁇ L water.
  • LMWH low molecular weight heparin
  • ASM 2-aminoethyl methacrylate
  • Low molecular weight heparin (Sigma, H5640, average molecular weight 4,000-6,000 Daltons) is derivatized with aminoethylmethacrylate (AEM) .
  • AEM aminoethylmethacrylate
  • AEM-derivatized heparin is dissolved in 240 mL of formamide by warming to 70 ⁇ C
  • the resulting heparin solution is made 0.1% in sodium dodecyl sulfate (SDS) by adding 0.27 mg SDS to the reaction mixture.
  • SDS sodium dodecyl sulfate
  • This solution is degassed by bubbling N 2 through it for 20 minutes.
  • AIBN 2,2'-azo-bis-isobutyronitrile
  • 5 ⁇ L of a 5 mg/mL potassium persulfate solution are then added.
  • 24 ⁇ L of inhibitor-free styrene is added and * the mixture i ⁇ stirred vigorously at 60°C for 24 hours.
  • Example 6 Copolymerization of Tolvlene Diisocyanate and Ethvlene Glvcol With Heparin Fragments From Deaminative Cleavage With Nitrous Acid A.
  • Low molecular weight heparin (Sigma H5640, average molecular weight 4,000-6,000 Daltons) is reduced with sodium borohydride. 110 mg of the heparin is dissolved in 2 mL of water and a solution of 76 mg of NaBH 4 in 1 mL of ice-cooled water is added to it. Reduction of the anhydromannosyl moiety of heparin is carried out overnight (approximately 20 hrs) at room temperature. The reaction mixture is acidified with
  • the precipitate is washed well with chloroform, ethanol, and finally with water until the supernatant i ⁇ free from unreacted heparin, the latter being determined by the carbazole method for uronic acid.
  • the copolymer (designated, CF3-22C) is insoluble in water but dissolves readily in dimethylsulfoxide, and the NMR spectra relate ⁇ closely to aromatic residues in the polyurethane structure as well as some new bands assigned to heparin, as shown in Figure 5.
  • Copolymers are evaluated for anticoagulant and antithrombotic activity by the APTT (Activated Partial Thromboplastin Time, Sigma Procedure No. A7668) and anti- Factor Xa (Sigma Technical Bulletin No. 870, Heparin in Plasma) assays, the latter being based on the neutralization of activated factor X (Xa) by its pla ⁇ ma inhibitor (antithrombin III) .
  • APTT Active Partial Thromboplastin Time
  • anti- Factor Xa Sigma Technical Bulletin No. 870, Heparin in Plasma
  • the ratio of anti-Xa activity to APTT is important in determining a substance's antithrombotic potential with respect to it ⁇ anticoagulant activity. Substances with a high ratio appear to be more antithrombotic and carry with them less risk of hemorrhage. This is because the anti-Xa as ⁇ ay measures the ability of a substance to block the formation of thrombin at an early stage in the coagulation cascade, whereas the APTT measures the ability of a substance to stop coagulation at many steps along the coagulation sequence. Thus, anti-Xa activity specifically relates to the local antithrombotic potential, while APTT is a measure of the global anticoagulant activity. Commercial heparin has an anti-Xa/APTT ratio of approx. l and the low molecular weight heparin from Sigma ha ⁇ a ratio of approximately 2.
  • Table I shows re ⁇ ult ⁇ from analyses of copolymers .prepared as described in Examples l and 2. These copolymers are soluble in water, and can thus be assayed directly by the APTT and anti-Xa methods. Analyses of these copolymers prepared with small heparin fragments show that they generally retain the high anti- Xa/APTT ratios characteristic of the short oligosaccharides used to prepare the copolymers. For example, the anti-Xa/APTT ratios for heparin fragments of size octa-, deca- and dodecasaccharide by preparation A are 4.3, 7.4 and 6.5, respectively.
  • anti- Xa/APTT ratios for heparin fragments of size octa-and decasaccharide by preparation B are 10.4 and 10.0, re ⁇ pectively.
  • One of the copolymer ⁇ , namely the one designated CH8-36A, show ⁇ higher anti-Xa/APTT compared to the corresponding fragments, perhap ⁇ due to a variation in the inherent activity of the fragment ⁇ re ⁇ ulting from different preparation ⁇ or due to a better utilization of the active fragment ⁇ which are randomly incorporated at a distance from each other and as pendant moieties into the copolymer backbone.
  • the monomer phase contains 60 mg heparin fragments of size ranging from octasaccharide to hexadecasaccharide, 240 mg acrylamide, 60 mg polyvinylpyrrolidone, and 20 mg N,N -methylene-bis- acryla ide (Bis) dissolved in 1.6 mL type I water, while the organic phase consists of a mixture of 11 mL toluene, 5 mL chloroform and 80 ⁇ L Span 85.
  • the monomer phase is cooled in an ice-bath to about 0°C and is mixed with 160 ⁇ L of a 50 mg/mL ammonium persulfate solution and 20 ⁇ L TEMED (N, N, N 1 , N - tetramethylethylenediamine) .
  • This solution is pipetted into the organic phase contained in a three-neck round-bottomed flask which is being stirred rapidly with a paddle-stirrer. Nitrogen gas is gently blown through the flask until all of the monomer phase has been added. The mixture is allowed to react for 30 min at 25°C while stirring at high speed and then at 65°c for 60 min at low speed.
  • the reaction mixture is transferred to a sintered glass funnel and washed with type I water, acetone and finally methanol.
  • the copolymer beads are suspended in water and allowed to soak for five days to remove the polyvinylpyrrolidone and any unreacted components.
  • the beads are washed again on a sintered gla ⁇ funnel with a large volume of type I water and methanol.
  • the ⁇ olid copolymer is then vacuum- dried at 60°C for 4-5 h. This is assayed for heparin content using the toluidine blue dye binding method of Smith et al. (1980) , indicating a content of 2 mg of heparin/g of solid beads (0.2% heparin by weight).
  • heparin Low molecular wight heparin (4,000 - 6,000 Da), obtained from Sigma Chemical Co. , is derivatized with aminoethylmethacrylate (AEM) following the procedure described in Example 2 (paragraph A) and also in Example 3.
  • AEM aminoethylmethacrylate
  • the heparin derivative is purified further by dialysis in 3,500 MWCO Spectrapore tubing (to remove excess of aminoethylmethacrylate) and the inner dialysate is lyophilized.
  • a typical copolymerization mix contains 30 mg acrylamide, 30 mg AEM-derivatized low molecular weight heparin (AEM-LMWH) and 2.5 mg bis- acrylamide, dissolved in 1.0 L type I water and cooled to about 0°C in an ice-bath. To this is added 160 ⁇ L of a 50 mg/mL ammonium persulfate solution and 20 ⁇ L TEMED. The solution is mixed well and nitrogen gas is gently bubbled through the reaction mixture. The mixture is left to polymerize at room temperature overnight. The reaction mixture is dialyzed in 3,500 MWCO tubing against 50 mM EDTA and subsequently against type I water.
  • AEM-LMWH AEM-derivatized low molecular weight heparin
  • the inner dialysate is lyophilized and filtered through a column of Sephadex G-150 superfine gel u ⁇ ing 0.2 M NaCl as the running buffer. Fractions (15 min) are collected at a flow rate of approximately 0.1 mL/min and assayed for uronic acid to identify the fractions containing copolymer. These fractions are pooled, desalted and lyophilized. Uronic acid, APTT and anti-Factor Xa assays are performed on the copolymer to determine the heparin content and biological activity. Result ⁇ show a heparin content of 17%, APTT activity of 2.9 U/mg and anti-Xa activity of 6.63 U/mg of copolymer.
  • Example 10 Copolymerization of Allyl-heoarin with Acrylamide Usin ⁇ bis-acrylamide as a Cross-linker Allyl glycoside of heparin fragment obtained from enzymatic digestion is prepared as described in preparation A (paragraph B) .
  • the copolymerization follows essentially the same procedure as in Example 9 except that 30 mg of allyl-heparin (hexadecasaccharide) is used instead of AEM-hepa in.
  • the copolymer appropriately purified and lyophilized, has a heparin content of 11% and an APTT activity of only 0.62 U/mg of copolymer, while there is practically no anti-Xa activity.
  • Example 11 Copolymerization of Allyl-heparin with Acrylonitrile
  • Enzymatically produced heparin hexadecasaccharide is derivatized to allyl glycoside as before.
  • 20 mg of allyl-heparin is dissolved in 200 ⁇ L of inhibitor-free acrylonitrile and mixed with 6 mg of AIBN (2,2 1 - azo-bis-isobutyronitrile) in 150 ⁇ L of type I water.
  • AIBN 2,2 1 - azo-bis-isobutyronitrile
  • the mixture i ⁇ ⁇ tirred at 70°C in an oil-bath for 4.5 h.
  • a solid copolymer is formed which is recovered by centrifugation and washed exten ⁇ ively with type I water.
  • the copolymer i ⁇ dried and assayed for heparin content using the toluidine blue dye binding method.
  • the biological activity is determined by the APTT and anti- Factor Xa assays.
  • the results of these as ⁇ ays indicate a heparin content of 8%, while the APTT and anti-Xa activities are 0.15 U/mg and 1.0 U/mg of solid, respectively.
  • a typical NMR spectrum of the copolymer is given in Figure 6. The spectrum clearly shows the absence of the characteristic signal corresponding to the allyl group.
  • AEM-heparin prepared as described in Example 9 (paragraph A), is dissolved in water. 25 mL of AEM- heparin solution (10 mg/mL) is warmed in a 100 mL round- bottomed flask to 70°C in a thermostated oil-bath. A solution of 1.7 mg AIBN in 5 mL of inhibitor-free acrylonitrile is added to the flask and the temperature is raised to 75 ⁇ C The mixture is allowed to react for 3 h with stirring, whereupon a white precipitate is formed inside the flask. The precipitate is removed and washed extensively with type I water in a sintered glass funnel. This is resuspended in type I water and lyophilized. Heparin content is found to be 0.5%, and the copolymer shows high anticoagulant activity.
  • Molecular Weight Heparin 2.5 g of AEM is dissolved in 10 mL of dimethyl sulfoxide (DMSO) and to the solution are added l mL of inhibitor-free acrylonitrile (1:1 molar ratio of AEM to acrylonitrile) and 0.3 g of AIBN.
  • DMSO dimethyl sulfoxide
  • the solution is warmed to 70 ⁇ C while stirring and allowed to react for 4 h.
  • the reaction mixture turns into a viscous solution and the copolymer is precipitated by dropwise addition of the reaction mixture to 1 L of methylene chloride with vigorous stirring.
  • the precipitated copolymer is filtered in a sintered glass funnel and washed well with methylene chloride.
  • the copolymer is dried in a vacuum oven at 70 ⁇ C and crushed into a powder.
  • Example 16 Copolymerization of an Aminoethylmethacrylate Derivative of Heparin with 4-styrenesulfonic Acid
  • the AEM derivative of low molecular weight heparin (4,000 - 6,000 Da) is prepared as noted in Example 9 (paragraph A) .
  • 400 mg of 4-styrenesulfonic acid is dissolved in 10 mL of an AEM-heparin solution in water (10 mg/mL) .
  • 300 mg of VAZO-67 is added and the temperature is raised to 70°C
  • the polymerization reaction is carried out overnight with stirring.
  • the solution is then dialyzed in a 6,000 - 8,000 MWCO tubing against 50 mM EDTA followed by type I water, and lyophilized.
  • Heparin content of the dried copolymer is found to be 12.7% by the uronic acid assay.
  • Biological activity is determined using the APTT and anti-Factor Xa methods, which give 16 U/m
  • Example 17 Copolymerization of Aminoethylmethacrylate with Vinyl Acetate and Derivatization of the Copolvmer with Low Molecular Weight Heparin Vinyl acetate (1.75 g) and AEM (0.25 g) are dissolved in DMF (25 mL) and VAZO-67 (20 mg) is added to the solution under a nitrogen atmosphere. The solution is heated at 70°C in a constant temperature oil-bath for 20 h. The resulting solution is concentrated by distilling under vacuum and the copolymer is precipitated by suspending the viscous solution in water with vigorous ⁇ tirring. The precipitate is filtered, washed repeatedly with water, and lyophilized. The copolymer is derivatized with LMWH (Sigma) in DMF solution following the same general procedure as described in Examples 13- 15. This shows a heparin content of 0.26% (by weight) .
  • LMWH Sigma
  • tolylene-2,4-diisocyanate (large exce ⁇ ) is added from a syringe, and the solution is diluted with 5 mL dimethylsulfoxide.
  • the copolymerization i ⁇ continued overnight (about 18 h) with ⁇ tirring under the N 2 atmo ⁇ phere at 4°C.
  • the re ⁇ ulting copolymer i ⁇ precipitated by adding a mixture of methanol and chloroform (100 mL each) . The precipitate is filtered and washed exten ⁇ ively with chloroform, methanol, methanol-water and finally pure water.
  • the copolymer is dried in a vacuum oven at 70°C, and it shows a heparin content of 5.24 ⁇ g/mg of solid by the toluidine blue dye binding method of Smith et al. (1980) .
  • the biological activity is measured by the APTT and anti- Factor Xa assay ⁇ which give 7.3 mU/mg and 8.35 mU/mg of solid, respectively.
  • a typical FTIR spectrum is given in Figure 7 which shows strong absorption bands for hydroxyl groups at
  • the copolymer is precipitated with chloroform, washed well with methanol and water, and dried in a vacuum oven at 60°C This yields 0.4 ⁇ g heparin/mg of solid with APTT and anti-Xa activities of 13 mU/mg and 14 mU/mg, respectively.
  • Example 19 Copolymerization of an Isocyanate-Terminated Prepolvmer with Tris- (hydroxymethyl) Aminomethane-Derivatized Low Molecular Weight Heparin
  • the same procedure as described in Example 18 is followed with the exception that 81 mg LMWH, 49.6 mg Tris and 5.1 mg NaBH 4 are dissolved in 3 mL formamide.
  • the derivatization of LMWH with Tris is performed overnight (19 h) at room temperature. This is followed by reaction with 2.04 g Hypol 2000 (an isocyanate- terminated prepolymer containing 1.6 mg NCO/g) , diluted with 7 mL dimethylsulfoxide and transferred into a round- bottomed flask under N 2 atmosphere.
  • the reaction is continued for about 22 h at 4 ⁇ C with the addition of 18 mL chloroform after 6 h to reduce the vi ⁇ cosity of the mixture.
  • a semi-solid mass is obtained which is disper ⁇ ed in 200 mL CHC1 3 and then precipitated from a large volume (-800 mL) of ethanol.
  • the precipitate, wa ⁇ hed exten ⁇ ively with methanol and water, is dried in a vacuum oven as before.
  • the copolymer has a heparin content of 1.05 ⁇ g/mg of solid, APTT activity of 8.4 mU/mg and anti-Xa activity of 7.2 mU/mg.
  • Example 20 Copolymerization of Tolylene Diisocyanate with Serinol Hydrochloride and Derivatization of the Polymer with Low Molecular Weight Heparin
  • One mmole serinol hydrochloride (127.4 mg) is dis ⁇ olved in 5 mL dimethylacetamide to which is added 142 ⁇ L tolylene-2,4-diisocyanate (equimolar) and 50 ⁇ L dibutyltin dilaurate (catalyst) .
  • the mixture is allowed to react in a rotary evaporator at 65°C for approximately 30 min and then at room temperature (21°C) for 18 h.
  • the copolymer is precipitated by su ⁇ pending the mixture in 200 L chloroform, washed well with methanol and water, filtered, and dried in a vacuum oven. 35.6 mg of the dried copolymer is dissolved in formamide, and the solution mixed with 61.3 mg LMWH and 8.4 mg NaBH 4 , also in formamide, for a total of 4 mL. The mixture is left overnight (- 17 h) at room temperature for derivatization of the copolymer, followed by precipitation and washing with water.
  • the precipitate is vacuum-dried and shows 0.26 ⁇ g of heparin/mg of solid by the toluidine blue dye binding method, while the APTT and anti-Xa activities are 30 mU/mg and 240 mU/mg, respectively.
  • Example 20 The same procedure as described in Example 20 is used except that 120 mg Tris (l mmole) is dissolved in 6 mL dimethylformamide. An equimolar amount of tolylene- 2,4-dii ⁇ ocyanate (142 ⁇ L) is added and the copolymerization is carried out in presence of 50 ⁇ L dibutyltin dilaurate. The copolymer is precipitated, washed, and dried in a vacuum oven as before. The dried copolymer (36 mg) is dissolved in formamide (4 mL) together with low molecular weight heparin (61.3 mg) and sodium borohydride (8.4 mg) . The derivatization of the copolymer is continued overnight at room temperature.
  • the resulting product is dialyzed extensively against pure water using 6,000 - 8,000 MWCO Spectrapore dialysis tubing.
  • the dialyzed solution is concentrated in a Rotovap and then dried in a vacuum oven at 70°C.
  • the product contains 64 ⁇ g of heparin/mg of solid and shows high anti-Xa activity.

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Abstract

L'invention concerne de nouveaux coplymères biocompatibles au glycosaminoglycan qui sont antithrombotiques et antithrombogènes et qui sont destinés à des applications biomédicales nécessitant une surveillance permanente ou à long terme des propriétés anticoagulantes. Les nouveaux copolymères de cette invention sont composés de petits fragments ou de tronçons de glycosaminoglycans comme de l'héparine, copolymérisée avec des composants monomères de synthèse. L'invention tire profit du fait que de petits tronçons d'héparine, ou d'autres glycosaminoglycans possèdent et maintiennent une activité antithrombotique qui retarde ou empêche la thrombose. Les fragments anticoagulants sont commodément produits à l'aide d'un moyen enzymatique ou chimique et copolymérisés avec les monomères de synthèse.
PCT/CA1991/000120 1990-04-10 1991-04-10 Copolymeres non thrombogenes au glycosaminoglycan WO1991015252A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0623150A1 (fr) * 1992-11-19 1994-11-09 Case Western Reserve University Surfaces d'implants non thrombogenes
EP0610441A4 (fr) * 1991-10-29 1996-01-10 Clover Cons Ltd Polysaccharides, polycations et lipides reticulables destines a l'encapsulation et la liberation de medicaments.
US5718694A (en) * 1993-11-09 1998-02-17 The Board Of Regents Of The University Of Nebraska Inhibition of adherence of microorganisms to biomaterial surfaces by treatment with carbohydrates
US5945457A (en) * 1997-10-01 1999-08-31 A.V. Topchiev Institute Of Petrochemical Synthesis, Russian Academy Of Science Process for preparing biologically compatible polymers and their use in medical devices
EP1172386A1 (fr) * 1999-04-02 2002-01-16 Netech Inc. Polymere de glycosaminoglycane fonctionnalise et leur utilisation en medecine et en pharmacie
US7314860B2 (en) 1997-11-25 2008-01-01 Jenny Ja Antti Wilhurin Rahasto Heparin-like compounds, their preparation and use to prevent arterial thrombosis associated with vascular injury and interventions
WO2013052779A3 (fr) * 2011-10-06 2013-10-17 Bvw Holding Ag Copolymères de segments hydrophobes et hydrophiles réduisant l'adsorption de protéines
US8673333B2 (en) 2002-09-25 2014-03-18 The Johns Hopkins University Cross-linked polymer matrices, and methods of making and using same
WO2020070258A1 (fr) 2018-10-03 2020-04-09 Carmeda Ab Entités biologiques immobilisées

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1558216A4 (fr) * 2002-09-25 2007-01-03 Univ Johns Hopkins Med Matrices de polymere reticule et procede de fabrication de celles-ci
US20050186679A1 (en) * 2004-02-24 2005-08-25 Christian Viskov Method for determining specific groups constituting heparins or low molecular weight heparins
EP2682408A1 (fr) * 2012-07-05 2014-01-08 Huntsman International Llc Polysaccharide dérativisé

Citations (3)

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US4239664A (en) * 1978-10-31 1980-12-16 Research Corporation Anti-thrombogenic PVP-heparin polymer
US4521564A (en) * 1984-02-10 1985-06-04 Warner-Lambert Company Covalent bonded antithrombogenic polyurethane material
WO1987000060A1 (fr) * 1985-07-08 1987-01-15 Battelle Memorial Institute Substrat dont la surface presente une activite antithrombogenique

Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
US4239664A (en) * 1978-10-31 1980-12-16 Research Corporation Anti-thrombogenic PVP-heparin polymer
US4521564A (en) * 1984-02-10 1985-06-04 Warner-Lambert Company Covalent bonded antithrombogenic polyurethane material
WO1987000060A1 (fr) * 1985-07-08 1987-01-15 Battelle Memorial Institute Substrat dont la surface presente une activite antithrombogenique

Cited By (21)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0610441A4 (fr) * 1991-10-29 1996-01-10 Clover Cons Ltd Polysaccharides, polycations et lipides reticulables destines a l'encapsulation et la liberation de medicaments.
EP0623150A4 (en) * 1992-11-19 1999-08-25 Univ Case Western Reserve Nonthrombogenic implant surfaces.
EP0623150A1 (fr) * 1992-11-19 1994-11-09 Case Western Reserve University Surfaces d'implants non thrombogenes
US5718694A (en) * 1993-11-09 1998-02-17 The Board Of Regents Of The University Of Nebraska Inhibition of adherence of microorganisms to biomaterial surfaces by treatment with carbohydrates
US5945457A (en) * 1997-10-01 1999-08-31 A.V. Topchiev Institute Of Petrochemical Synthesis, Russian Academy Of Science Process for preparing biologically compatible polymers and their use in medical devices
US7314860B2 (en) 1997-11-25 2008-01-01 Jenny Ja Antti Wilhurin Rahasto Heparin-like compounds, their preparation and use to prevent arterial thrombosis associated with vascular injury and interventions
US8415324B2 (en) 1997-11-25 2013-04-09 Jenny Ja Antti Wihurin Rahasto Heparin-like compounds, their preparation and use to prevent arterial thrombosis associated with vascular injury and interventions
US7504113B2 (en) 1997-11-25 2009-03-17 Jenny Ja Antti Wihurin Rahasto Heparin-like compounds, their preparation and use to prevent arterial thrombosis associated with vascular injury and interventions
EP1172386A1 (fr) * 1999-04-02 2002-01-16 Netech Inc. Polymere de glycosaminoglycane fonctionnalise et leur utilisation en medecine et en pharmacie
US7005513B1 (en) * 1999-04-02 2006-02-28 Netech, Inc. Functionalized glycosaminoglycan polymer and medical instruments and drugs by using the same
AU771610B2 (en) * 1999-04-02 2004-04-01 Netech Inc. Functionalized glycosaminoglycan polymer and medical instruments and drugs by using the same
EP1172386A4 (fr) * 1999-04-02 2002-07-10 Netech Inc Polymere de glycosaminoglycane fonctionnalise et leur utilisation en medecine et en pharmacie
US8673333B2 (en) 2002-09-25 2014-03-18 The Johns Hopkins University Cross-linked polymer matrices, and methods of making and using same
WO2013052779A3 (fr) * 2011-10-06 2013-10-17 Bvw Holding Ag Copolymères de segments hydrophobes et hydrophiles réduisant l'adsorption de protéines
CN104144962A (zh) * 2011-10-06 2014-11-12 Bvw控股公司 减少蛋白吸附的疏水性和亲水性链段的共聚物
CN104144962B (zh) * 2011-10-06 2017-03-08 Bvw控股公司 减少蛋白吸附的疏水性和亲水性链段的共聚物
US10485822B2 (en) 2011-10-06 2019-11-26 Bvw Holding Ag Copolymers of hydrophobic and hydrophilic segments that reduce protein adsorption
US11524030B2 (en) 2011-10-06 2022-12-13 Bvw Holding Ag Copolymers of hydrophobic and hydrophilic segments that reduce protein adsorption
WO2020070258A1 (fr) 2018-10-03 2020-04-09 Carmeda Ab Entités biologiques immobilisées
CN113164660A (zh) * 2018-10-03 2021-07-23 卡梅达股份公司 固定化的生物实体
US11850334B2 (en) 2018-10-03 2023-12-26 Carmeda Ab Immobilised biological entities

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