WO1993014127A1 - Procede et composition pour la fixation d'heparine a l'aide de polyaminocations immobilises par covalence sur des surfaces polymeres avec des segments intermediaires d'oxyde de polyethylene - Google Patents

Procede et composition pour la fixation d'heparine a l'aide de polyaminocations immobilises par covalence sur des surfaces polymeres avec des segments intermediaires d'oxyde de polyethylene Download PDF

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WO1993014127A1
WO1993014127A1 PCT/US1993/000678 US9300678W WO9314127A1 WO 1993014127 A1 WO1993014127 A1 WO 1993014127A1 US 9300678 W US9300678 W US 9300678W WO 9314127 A1 WO9314127 A1 WO 9314127A1
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pala
peo
heparin
molecular weight
pll
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PCT/US1993/000678
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Syed Fazal Mohammad
Xing Hang Ma
Sung Wan Kim
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University Of Utah
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3672—Means preventing coagulation
    • A61M1/3675—Deactivation
    • 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
    • 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
    • B01J20/327—Polymers 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
    • B01J20/3231—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

Definitions

  • compositions and methods for the removal of heparin from blood to minimize the risk of hemorrhagic complications More particularly, this invention relates to compositions consisting of a polymeric substrate which has been modified to contain polycation ligands having primary amino groups which are covalently bonded to the substrate through a polyethyleneoxide type spacer and to methods of removing heparin from blood or other solutions by contacting heparinized solution with said compositions.
  • Heparin is a powerful anticoagulant which is often administered during surgical or other procedures to prevent the formation of clots.
  • AT III Antithrombin III
  • AT III is a plasma glycoprotein with molecular weight approximately 58,000.
  • AT III binds with thrombin very tightly at a 1:1 stoichiometric ratio, which blocks the active site on thrombin and prevents it from interacting with fibrinogen.
  • the inhibition rate of thrombin with AT III is low in absence of heparin.
  • Heparin dramatically accelerates the rate of thrombin inactivation up to 2000-fold.
  • Clinically used heparin can be separated into two distinct fractions according to their affinity for AT III.
  • heparin Approximately 33% of heparin has a high affinity for AT III, which has potent anticoagulant activity (up to 90% of the activity of the unfractionated heparin). A low-affinity heparin binds to the same site on AT III, but with approximately 1000 times lower affinity.
  • Heparin Although anticoagulation is the major pharmacological activity, heparin has many other functions. Heparin inhibits the proliferation of vascular smooth muscle cells and renal mesengial cells, suppresses the delayed-type hypersensitivity, and inhibits angiogenesis. Other pharmacological functions of heparin include antithrombotic effect, antibacterial, antivirus, and antitumor angiogenesis, particularly in combination with cortisone. Although it has been clinically observed that heparin may induce thrombocytopenia, in vitro studies have shown that normal heparin enhances the release of platelets. Moreover, various heparin-binding growth factors can be purified with heparin affinity chromatography.
  • Heparin has been extensively used in many clinical applications, including cardiac surgery, peripheral vascular surgery, dialysis, autotransfusion, transplantation, the treatment of pulmonary embolism, disseminated intravascular coagulation, and venous thrombosis. The dosage is dependent on the type of application. Heparin has also been used as a prophylactic agent against deep vein thrombosis. Heparin is also of value in the treatment of thromboembolic disorders, such as pulmonary embolism and arterial thrombosis.
  • heparin is immobilized onto polymers to form nonthrombogenic surfaces to achieve a localized anticoagulant effect at the polymer blood interface.
  • heparin has been fixed onto polymers with positive charges by forming a stable complex or by being covalently bound thereto via a spacer grouping.
  • the immobilized heparin on the surface may then be released into blood by ion exchange, subsequently, the released free heparin interacts with AT III and thus exerts its anticoagulant effect.
  • heparin increases the blood compatibility of the polymer for long-term biomedical applications, particularly when bound through an appropriate spacer grouping.
  • Polymers bearing positive charges have been used to anchor heparin onto surfaces and have shown good long term blood compatibility.
  • charged polymers such as poly-4-vinylpyridine, Fourt et al., Adv. Chem . Ser. 87, 187 (1968); poly(amidoamine) grafted polyurethane (PUPA), Azzuuoli et al., Biomaterials 8, 61 (1987); and polyvinylchloride grafted with both polyethyleneglycol monomethacrylate and quarternized dimethylaminoethyl methacrylate (Anthron), Nagaoka et al., J. Biomater. Appl . 4 , 3 (1989), have been reported to form a stable complex with heparin.
  • the second means where binding of negatively charged heparin onto polymeric surfaces has been applied in the biomedical field in the area of heparin neutralization where heparin is removed from blood onto the polymer surface by binding.
  • heparin can be dangerous since less than desired concentrations of heparin in blood increases the likelihood of unwanted coagulation after surgery or when blood comes into contact with biopolymeric surfaces thus forming unwanted blood clots in the arteries and veins. It is well documented that most synthetic polymers activate blood coagulation when contact is made between blood and the polymer surface. Therefore, administered heparin is necessary to minimize clotting. This heparin may be present in the blood at relatively high concentrations during contact with polymeric surfaces such as during extracorporeal circulation to prevent clotting in the extracorporeal circuit. Excessive heparin in blood needs to be neutralized at the end of extracorporeal circulation due to the risk of hemorrhagic complications.
  • protamine a cationic protein
  • Anido et al. Am . J. Clin . Pathol . 76, 410 (1981).
  • protamine also has undesirable side effects, including the risk of anaphylactic reactions in some patients.
  • protamine immobilized cellulose hollow fiber Kim et al., Trans . Am. Soc . Artif. Intern.
  • Cationic polyelectrolyte surfaces proposed for binding and removal of heparin, include the use of triethylaminoethyl cellulose powder (Heparsorb, Organon Teknika, Durham, NC) and poly-L-lysine bound agarose (Sepharose 4B), Mohammad et al., Thromb . Res . , 20, 599 (1980)
  • prior art methods have demonstrated the degree of heparin removal that is desired is not sufficient or takes too long to accomplish.
  • One of the reasons for polycationic surfaces not being as effective as might be desired has to do with variables affecting heparin binding onto polycationic surfaces.
  • electrostatic attractions exist between the positively charged polycations and the negatively charged heparin.
  • steric accessibility of heparin onto polycationic interfaces is also an important consideration.
  • heparin binding onto polycationic surfaces may be controlled by the balance between the electrostatic attraction and steric accessibility factors.
  • An additional object of this invention is the formation of a polymeric substrate containing polycationic ligands having primary amino groups and preferably those selected from the group consisting of polylysine (PLL), polyallylamine (PALA) or polyvinylamine (PVA) bonded through a polyethylene oxide (PEO) spacer grouping to a biocompatible polymeric substrate.
  • PLL polylysine
  • PVA polyallylamine
  • PVA polyvinylamine
  • Another object of this invention is to provide a method for the binding and/or removal of heparin in blood by contacting the blood with a polymeric substrate containing polycationic ligands selected from the group consisting of polylysine (PLL), polyallylamine (PALA) or polyvinylamine (PVA) bonded through a polyethylene oxide (PEO) spacer grouping to a biocompatible polymeric substrate.
  • PLL polylysine
  • PVA polyallylamine
  • PVA polyvinylamine
  • An additional object to provide a method of removing heparin from blood with a minimum loss of the plasma proteins and other components of blood.
  • compositions consisting of a polymeric substrate which has been modified to contain polycations such as polyallylamine (PALA), poly- L-lysine (PLL) or polyvinylamine (PVA) covalently bonded to the substrate through a polyethyleneoxide type spacer effectively removes heparin from blood with little or no loss of plasma proteins or other blood components.
  • polycations such as polyallylamine (PALA), poly- L-lysine (PLL) or polyvinylamine (PVA) covalently bonded to the substrate through a polyethyleneoxide type spacer.
  • PALA or PLL is most preferable with PALA being the polycation determined to be best suited for purposes of this invention.
  • the polycationic ligand when covalently bound to a suitable polymeric substrate via a polyethylene oxide (PEO) spacer, provides a solution like polycationic interfacial environment.
  • the solution like conditions are due to the increased mobility of the polycation ligand as a result of the PEO spacer.
  • the flexibility of the polycation attributed to the presence of the spacer at the interface of the polymer minimizes the steric inaccessibility of the heparin thereby improving the binding characteristics.
  • any suitable polycation ligand rich in NH 2 groups can theoretically be utilized to bind heparin, it has been found preferable to use either polyallylamine (PALA) or poly-L-lysine (PLL) as the ligand.
  • PEO polyallylamine
  • PLL poly-L-lysine
  • a polymer substrate having free hydroxy groups such as cellulose or cellulose diacetate.
  • the PEO molecule may be derivatized by functional groups, as will be discussed more in detail below, to maximize the ability of the PEO to covalently link the ligand with the polymeric substrate.
  • Heparin is removed from blood or other fluids by bringing the fluids in contact with the polymeric substrate material containing immobilized polycations with PEO spacers where heparin is adsorbed by the material. Heparin can be released from the immobilized polycations by treatment with a basic solution, thereby allowing the material to be reused when practical.
  • the substrate containing the immobilized polycationic ligands connected via the PEO spacers must present a large surface area to the blood or other fluid in order to maximize the opportunity for the heparin to come into contact with the positively charged ligands.
  • the substrates can be in the form of films, beads, honeycombs, coated surfaces, strands, filaments, and the like.
  • the rate of heparin binding is somewhat dependant on controlling parameters such as blood flow rates, concentration of heparin in blood and exposure time of heparinized blood to the binding substrate.
  • the binding of heparin onto the polycationic surfaces grafted to the polymer surface via PEO spacers of molecular weight of between about 600 and 8,000 seems to depend on the balance of several factors such as the size of the polycation ligand, the molecular weight of the PEO spacer, the mobility of the PEO and the chain length of the PEO-polycation at the interface.
  • PALA having molecular weights ranging from about 8,500 to about 65,000 is suitable for use in the present invention.
  • PALA is categorized as PALA(L) for a low molecular weight PALA having a molecular weight of between about 8,500 and 11,000 and PALA(H) for a high molecular weight PALA having a molecular weight range of about 50,000 to 65,000.
  • a composition consisting of low molecular weight PALA (M.W. 8500) immobilized onto a biopolymer surface with a PEO spacer (M.W. 3400) is believed to provide optimal heparin binding capacity with decreased protein adsorption.
  • heparin removal from polycationic containing substrates is primarily due to the charge interaction between the polyanionic heparin and the polycations. No hemolysis or clotting is observed in circulating blood, suggesting the such a removal system will not cause adverse effects on blood.
  • the system is also effective in removing heparin from aqueous solutions other than blood.
  • substrate any suitable biopolymer having hydroxy groups to which the polycation may be covalently bonded, either directly or through a PEO type spacer.
  • the substrate may be represented by the following formula:
  • Biomedical polymers include, but are not limited to, such hydroxy containing materials as cellulose and derivatives such as cellulose diacetate (CA), poly (ethylene-vinyl alcohol) copolymers
  • PEVAL polyethylene glycol
  • agarose agarose
  • biomedical polymers such as polycarbonates, silicones, acetals, polyesters, polytetrafluroethylene, polyurethane, pyrolytic carbon, nylon or other polyamides, polyacrylonitrile, polyethersulfone and the like by devising reaction sequences and linkages which provide for covalent attachment of polycations to these surfaces.
  • polycation is meant a polymeric molecule having a repeating segment containing primary amino groups on the side chain and which is positively charged at neutral pH. These may be generically referred to as polyaminocations (PAC). As stated, polyallylamine (PALA), poly-L-lysine (PLL)) and polyvinylamine (PVA) are preferred with PALA and PLL being most preferred.
  • PAC polyaminocations
  • PLL is a polypeptide of the formula:
  • n is an integer of between about 200 and 240 and which becomes cationic when the e-amino gruups on the side chain become fully protonated. Its ionization depends upon the pH of the medium in which it is contained but is known to form electrostatic interactions with heparin at a neutral pH.
  • the positive charges of PLL per molecule can be adjusted by selecting different molecular PLL weights. The molecular weight range of PLL will generally vary between about 40,000 and 50,000.
  • PALA is a recently commercialized polymer with primary amino groups as pendant side chains having the formula:
  • m is an integer of between about 78 to 500, having a molecular weight of between about 8,500 and 65,000 and is prepared from polymerization of a monoallylammonium salt initiated by an azo compound.
  • PALA is obtained commercially as a HCl salt wherein the HCl is removed by increasing the pH with NaOH.
  • PALA has a higher positive charge density than PLL.
  • PEG poly(ethylene glycol)
  • PEO poly (ethylene oxide)
  • n is an integer of between about 13 and 180.
  • PEO is extremely water soluble.
  • Low molecular weight PEO (less than 600) is a viscous fluid at room temperature. As the molecular weight increases, PEO becomes a waxy solid and morphology changes from amorphous to crystalline. PEO-water interaction is found to increase sharply with increasing molecular weight until a weight of about 3400 is reached and then remains constant within the 3400-20000 molecular weight range.
  • spacer grouping it has been found that the chain mobility of PEO increases with increasing molecular weight up to about 4,000.
  • PALA and PLL were both immobilized on PEVAL copolymer covalently using cyanogen bromide as a coupling agent.
  • Cellulose diacetate and cellulose hollow fibers could also be used because each of these materials have hydroxyl groups available after proper treatment which can be activated by BrCN. Therefore, the amino groups of PALA and PLL can be coupled directly with the hydroxyl groups on these polymeric supports.
  • the imidocarbonate of Formula 4 is then reacted with either PLL or PALA in an aqueous solution to covalently link a primary amino group from a pendant PLL or PALA chain with the polymer surface as follows in
  • the PLL or PALA polycation When the PLL or PALA polycation is covalently immobilized on the polymer surface, it is relatively rigid and not laterally movable due to the intramolecular and intermolecular charge repulsions.
  • the relatively low binding of heparin on polycations rigidly bound may be ascribed to the steric inaccessibility of heparin into the root part of the relatively rigid polycationic interface. Therefore, heparin may not bind efficiently on polycationic directly immobilized on to polymeric surfaces.
  • PALA or PLL are coupled directly onto a poly(ethylene-vinyl alcohol) (PEVAL) surface using BrCN as a coupling reagent under exactly the same experimental conditions
  • PALA immobilized surface (Formula 6) demonstrates a heparin binding capacity which is about 1.3 times that of PLL (Formula 5) which is believed to be due to the higher charge density of PALA per molecule when compared with PLL.
  • Heparin binding of PALA was 0.71 ⁇ 0.08 ⁇ g/cm 2 and of PLL was 0.52 ⁇ 0.19 ⁇ g/cm 2 .
  • the primary factors affecting heparin binding onto polycationic surfaces are electrostatic attractions between the positively charged surfaces and the negatively charged heparin, and steric accessibility of heparin into polycationic interfaces.
  • the polycation When a polycation is covalently immobilized directly onto a polymer surface, the polycation is relatively rigid and not laterally movable due to the intramolecular and intermolecular charge repulsions.
  • heparin binding onto polycationic surfaces may be enhanced by the balancing of the electrostatic binding and steric accessibility factors. It has been found that the heparin binding efficiency on polycation- immobilized surfaces is increased if the effect of the steric inaccessibility is minimized and a solution like polycationic interfacial environment is provided.
  • Polyethylene glycol also referred to herein as polyethylene oxide "PEO"
  • PEO polyethylene oxide
  • the PEO provides a larger intermolecular gap between the polycations at the interface of the polymer surface and therefore minimizes the steric inaccessibility. Therefore, the binding properties of heparin are increased when PEO spacer molecules are inserted between the surface and the polycations.
  • the heparin removal system is formed by reacting a polymeric surface containing free hydroxy groups, preferably with a PEO molecule which has been derivatized to form a diacid thereby creating an ester linkage between the hydroxy groups of the surface and the carboxylic acid group of the diacid.
  • PEO may be derivatized to form diacids by : 1) the reaction of polyethylene oxide with succinic anhydride as shown in Formula 7; 2) the reaction of an alkoxide of polyethylene oxide with 2-halo-alkyl acetate as shown in Formula 8; and 3) the reaction of polyethylene oxide with an alkylene diacid such as azelaic acid as shown in Formula 9.
  • HO-(-CH 2 CH 2 O-)- n H + - O - n 50-100
  • diacids may be generically represented by a single formula as seen in Formula 10 as follows:
  • the diacid PEO intermediate as represented by Formula 10 is first grafted onto the surface of a polymer, preferably one containing OH groups (Formula 1), using an agent such as 1,3-dicyclohexylcarcodiimide (DCC) to esterify the carboxylic acid groups of the diacid derivatized PEO with the hydroxyl group of the polymer surface according to following sequence:
  • DCC 1,3-dicyclohexylcarcodiimide
  • the esterified PEO grafted onto the polymer surface as represented by Formula 11, then has polycations, such as PLL or PALA, immobilized to the polymer by reacting the -COOH at the opposing end of the PEO with the polycation using an agent such as (3- dimethlyamino-propyl)carbodimide (EDC).
  • EDC (3- dimethlyamino-propyl)carbodimide
  • the amount of polycation in the reaction solution will be in excess resulting in only one amino group of PLL or PALA per molecule reacting with each COOH group on the surface.
  • This reaction sequence is represented as follows resulting in PLL or PALA immobilized compositions represents by Formulas 12 and 13 as follows:
  • the final product formed contains polylysine (PLL) or polyallylamine (PAPA) separated from the surface by a spacer molecule of PEO.
  • PLL polylysine
  • PAPA polyallylamine
  • any polycation having free amino groups which can be covalently bonded to the PEO spacer may be a suitable agent and is within the scope of this invention. Therefore, the compositions of the invention may be broadly defined by the following formula 14:
  • PAC stands for any suitable polyaminocation and preferably one selected from the group consisting of polyallyl amine (PALA), poly-L-lysine (PLL) or polyvinlyamine.
  • PVA polyallyl amine
  • PLL poly-L-lysine
  • polyvinlyamine polyvinlyamine
  • the material upon which the spacer molecule is bonded can be any substance that has free hydroxy groups to which the PEO diacid intermediate can be esterified, for example, cellulose, cellulose acetate or a poly(ethylene vinyl alcohol) copolymer.
  • the formula representing the compositions of this invention may be further simplified for purposes of claiming.
  • the structures of the PEO spacer, and the PALA and PLL ligands are given in detail above. All have certain repeating units within their respective molecular structures and hence, with the formulas established, it is the molecular weights of these units which determines their functionality in the binding of heparin. Therefore the following formula will be referred to in the claims:
  • Substrate-OC-PEO-CNH-PAC where Substrate is the same as Formula 1, -OC(O)-PEO- C(O)- has the same meaning as the PEO diacid of Formula 10 and -NH-PAC is the polyaminocation ligand and is preferably the PLL of Formula 2 or the PALA of Formula 3.
  • -NH-PAC is the polyaminocation ligand and is preferably the PLL of Formula 2 or the PALA of Formula 3.
  • This examples describes the derivatization of PEO with succinic anhydride to prepare PEO derivatives having carboxyl end groups according to the reaction sequence shown in Formula 7.
  • the hydroxyl end groups of PEO (having molecular weights of 600, 2000, 3400, and 8000) were reacted with succinic anhydride in dioxane using 4-Dimethylaminopyridine (DMAP) and triethylamine (TEA) as catalysts according to the above reaction sequence.
  • DMAP 4-Dimethylaminopyridine
  • TAA triethylamine
  • Succinic anhydride (20 mmol), 4- Dimethylaminopyridine (10 mmol) and triethylamine (TEA) (10 mmol) were dissolved in 20 ml of dioxane and added to a solution of 5 mmol of polyethylene oxide (PEO) (10 mmmol of OH groups) in 50 ml of dioxane. The mixture was stirred for 12 hr at 60° C. Most of the dioxane was then removed by a rotavapor. The residue was dissolved by dichloromethane and precipitated by ether. The products were dissolved and precipitated twice from dichloromethane/ether and then vacuum dried.
  • PEO polyethylene oxide
  • the terminal carboxylic acid groups of polyethylene oxide (PEO) diacids was determined by non-aqueous titration. PEO diacids (5 x 10 -4 mol) were dissolved in dioxane and titrated by sodium methoxide (0.1 N) in methanol-benzene (2.8) using 0.2% thymol blue as an indicator. The pure polyethylene oxide (PEO) in dioxane was used as a blank. The amount of sodium methoxide consumed by PEO diacid gave the information on the amount of carboxylic acid groups at two ends of polyethylene oxide (PEO).
  • This example describes the preparation of a cellulose acetate substrate to which is attached the PEO-diacid spacer prepared according to Example 1.
  • Cellulose acetate was dissolved in acetone to make a 10% solution.
  • a casting knife was used to make a film with 0.8 mm thickness on a glass plate and allowed to air dry.
  • the dried cellulose film was peeled off by rinsing the plate with distilled water.
  • the cellulose acetate (CA) film (1000 cm 2 ) was immersed in dichloromethane:toluene solvent (5:5) overnight, then 9x10 -4 mol of PEO diacid of Example 1 and 9X10 -1 mol of 4-dimethylaminopyridine (DMAP) were added into the solution and stirred at 4°C for one hour.
  • a 5 ml aliquot of 9X10 -4 mole of DCC in dichloromethane:toluene (5:5) was added dropwise to the reaction mixture and stirred for an additional 1 hour at 4°C. The whole solution was moved to 22 °C and allowed to stand for 48 hr.
  • the amount of PEO coupled onto CA film surfaces was determined by acid-base back titration of its free end of carboxylic acid.
  • the coupled film was thoroughly washed with distilled water and then immersed in distilled water (10 ml).
  • Sodium hydroxide (0.01 N) was added to adjust the pH above 7.0.
  • the solution and film were titrated potentiometrically with 0.01 N hydrochloric acid and the pH was monitored by a Corning pH Meter.
  • CA film without PEO was used as blank.
  • the amount of HCl consumed by PEO grafted film between the second equivalent point subtracted from the amount of HCl used by the blank at the same pH gave the amount of carboxylic acid groups, hence the PEO content on CA film.
  • the results are shown in the table in following Example 3.
  • This example describes the reaction of polyallyl amine with the CA-PEO film formed in Example 2.
  • CA-PEO-COOH film prepared as described above (1000 cm 2 ) was immersed in water.
  • PALA (2 x10 -4 mol) was added into the film suspension and stirred at 4°C for 1 hr.
  • EDC 2.5x10 -4 mol
  • the solution was then stirred for an additional 1 hr. at 4°C.
  • the whole solution was moved to 22°C and allowed to stand for 24 hr.
  • Two additional volumes of EDC were added to the solution at 3 and 6 hr during the reaction. After the reaction was completed, the CA-PEO-PALA films were washed with distilled water.
  • the CA film contained immobilized PALA connected to the film surface via PEO spacers as shown in Formula 13. Both low molecular weight PALA(L) and high molecular weight PALA(H) were immobilized with varying PEO molecular weight spacers.
  • the content of amino groups from PALA immobilized on the surface was determined by acid-base potentiometric back titration and also by spectroscopy analysis. At pH ⁇ 7, all the -NH 2 groups are changed to - NH 3 + which can be potentiometrically titrated by NaOH. The difference in the amounts of NaOH used between the CA-PEO-PALA and a blank CA gives the amounts of the amino groups on the surface.
  • the results, measured in surface concentrations of 10 -8 mole/cm 2 are shown in the following table:
  • the amount of amino groups on PALA(H) immobilized surfaces is about 2-3 times higher than that on PALA(L) surfaces.
  • a similar procedure to that described above can be used to immobilize PLL onto a CA-PEO film to prepare a product as shown in Formula 12.
  • CA-PEO-PALA films (240 cm 2 ) were placed in 10 ml of 40 ⁇ g/ml heparin solution (0.1 N Sodium carbonate buffer solution or saline) with shaking for 1 hr. The difference of heparin amount before and after binding gave the net amount heparin bound on the surfaces.
  • CA-glutaric acid-PALA films was also evaluated as a control and compared with CA-PEG-PALA surfaces. The glutaric acid spacer did not provide sufficient linkage space to have any affect on the binding capacity of the PALA.
  • the amount of heparin on PALA(L) (molecular weight 8,500) immobilized with PEO spacers of varying molecular weight is shown in the following table with a concentration of immobilized PALA on the cellulose acetate surface being given in concentrations of 10 -10 mole/cm 2 :
  • the amount of heparin on PALA(H) (molecular weight 50,000) immobilized with PEO spacers of varying molecular weight is shown in the following table with a concentration of immobilized PALA on the cellulose acetate surface being given in concentrations of 10 -11 mole/cm 2 :
  • the PEO-PALA(L) interface was more solution like and less steric restriction than the PEO- PALA(H) interface.
  • the PEO-PALA(L)-immobilized surfaces when glutaric acid was used to link PALA(L) with the CA film, no spacer effect was considered due to its short chain. Therefore, heparin binding efficiency was low due to the rigid PALA(L) at the interface.
  • PEO(0.6K) as a spacer, despite the increased mobility of PALA(L) at the interface, the short chain length of PALA(L)-PEO (0.6K) at the interface could not enhance heparin binding. This minimum chain length requirement at the interface may be ascribed to the relatively large size of the heparin macromolecule.
  • CA-PEO (2K) -PALA(L) film was immersed in 1 ml of the heparinized bovine plasma or whole blood (40 ⁇ g heparin/ml blood, 2% of 35 S-labeled heparin). After incubation for 1 hr, the film was rinsed with distilled water and 1 ml of NaOH solution was added. After 5 min shaking, CytoScint cocktail (15ml) was added. The solution was mixed well on a vortex shaker and the radioactivity was counted.
  • CA- PEO (2K)-PALA(L) film was immersed in 1:10 diluted serum solution containing heparin (40 ⁇ g heparin/ml serum solution). After incubation for 1 hr., 0.5ml of solution was taken out for Azure II colorimetric assay.
  • the amount of heparin bound on CA-PEO(2K)-PALA(L), CA-PEO(2K)-PALA(H), CA-glutaric acid-PALA(L), and CA- glutaric acid-PALA(H) membranes in bovine serum was compared with the corresponding heparin binding amount in aqueous solution. The comparison did not show significant difference between these two media.
  • the amount of heparin binding in bovine plasma or whole blood was determined by counting the radioactivity of 35 S-heparin on the film.
  • the amount of heparin bound on the CA-PEO(2K)-PAL-(L) film from plasma or whole blood was compared with the corresponding heparin bound amount in PBS using tracer 35 S-heparin, no significant differences in heparin binding amount among the three media were observed.
  • the data shows blood components do not affect heparin binding on CA-PEO-PALA surfaces.
  • Iodine-125 labeled bovine albumin was used as a model protein to study protein adsorption on CA-PEO-PALA surfaces using low molecular weight PALA and PEO spacers varying in molecular weight of from 600 (0.6K) to 8,000 (8K).
  • the concentration of albumin in serum was determined by the bromcresol green method.
  • PALA and PLL are structurally similar in having primary amino groups on the side chain and both being positively charged at neutral pH.
  • PALA has a higher charge density per molecule than PLL due to the smaller repeat unit.
  • Empirical results show that both PALA and PLL have similar heparin binding capacities.
  • Comparison of average pKa of PALA and PLL indicates that the basic strength of PALA is slightly lower than that of PLL but the difference was not significant.
  • the repulsive interactions of the intramolecular amino groups on the PALA chain is greater than that of PLL which may be attributed to the higher charge density per molecule of PALA than PLL.
  • the greater intramolecular force between functional amino groups on PALA also translates into the fact that changes of pKa value of PALA is more sensitive than to the changes of pH than with PLL.
  • Another similarity with PALA and PLL is that the degree of ionization is similar at about neutral pH.
  • the pKa of PLL is lightly higher than that of PALA inferring that he interaction between PLL and polyanions such as heparin may be somewhat stronger than that of PALA.
  • the higher positive charge density of PALA per molecule when compared with PLL suggests that PALA may actually bind more heparin than PLL.
  • the criteria for selecting a polycation as a heparin binding reagent are (a) capability of binding heparin, (b) functional groups which can be covalently grafted onto polymeric surfaces and (c) commercial availability at a reasonable price.
  • Any polycation having primary amino groups is considered to be a candidate for use in this invention due to the likelihood of various chemical reactions with the free amino groups.
  • Inclusive of polycations which are currently commercially available and have free amino groups are PLL, PALA and PVA. The high price of PLL limits its practical usage.
  • the terminal amino groups which can be covalently grafted onto polymeric surfaces

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Abstract

Compositions de formule (I) où substrat représente n'importe quel biopolymère approprié auquel des polycations peuvent être liés par covalence au moyen d'un groupement de segments intermédiaires; -OC(O)-PEO-C(O)- est un polymère d'oxyde de polyéthylène présentant un poids moléculaire compris entre environ 600 et 8000; et -NH-PAC représente n'importe quel polyaminocation approprié qui renferme une charge positive à un pH neutre et élimine efficacement l'héparine du sang avec peu ou pas de perte de protéines plasmatiques ou d'autres constituants sanguins. L'emploi d'une polyallylamine ou d'une poly-L-lysine comme polyaminocation est idéal, la polyallylamine étant le polycation convenant le mieux aux applications de la présente invention. Le ligand polycationique, lorsqu'il est lié par covalence à un substrat polymère approprié par le biais d'un segment intermédiaire d'oxyde de polyéthylène (PEO), donne un environnement interfacial polycationique analogue à une solution. Ces conditions analogues à une solution sont dues à la mobilité accrue du ligand polycationique par suite de la présence du segment intermédiaire de PEO. La flexibilité du polycation attribuée à la présence du segment intermédiaire au niveau de l'interface du polymère réduit au minimum l'inaccessibilité stérique de l'héparine, améliorant ainsi les caractéristiques de fixation. Le substrat sur lequel est liée la molécule de segment intermédiaire peut être toute substance qui possède des groupes hydroxy libres auxquels peut être estérifié l'intermédiaire diacide de PEO, et comporter de la cellulose ou de l'acétate de cellulose.
PCT/US1993/000678 1992-01-21 1993-01-19 Procede et composition pour la fixation d'heparine a l'aide de polyaminocations immobilises par covalence sur des surfaces polymeres avec des segments intermediaires d'oxyde de polyethylene WO1993014127A1 (fr)

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WO1995024437A1 (fr) * 1994-03-11 1995-09-14 Hoechst Aktiengesellschaft Derives d'acide malonique conjugues a un polymere et leur utilisation comme medicaments ou agents diagnostiques
WO1998022162A1 (fr) * 1996-11-20 1998-05-28 Biocompatibles Limited Compositions biocompatibles
WO1999001195A1 (fr) * 1997-07-01 1999-01-14 Terumo Cardiovascular Systems Corporation Elimination d'agents biologiquement actifs
US5891196A (en) * 1997-04-16 1999-04-06 Baxter International Inc. Method for actively binding heparin to crosslinked biological tissues
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US6303179B1 (en) 1999-02-08 2001-10-16 Medtronic, Inc Method for attachment of biomolecules to surfaces through amine-functional groups
EP1676581A2 (fr) * 1993-08-11 2006-07-05 Genzyme Corporation Polymère à utilisation thérapeutique

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1676581A2 (fr) * 1993-08-11 2006-07-05 Genzyme Corporation Polymère à utilisation thérapeutique
EP1676581A3 (fr) * 1993-08-11 2008-12-03 Genzyme Corporation Polymère à utilisation thérapeutique
WO1995024437A1 (fr) * 1994-03-11 1995-09-14 Hoechst Aktiengesellschaft Derives d'acide malonique conjugues a un polymere et leur utilisation comme medicaments ou agents diagnostiques
US5817742A (en) * 1994-03-11 1998-10-06 Hoechst Aktiengesellschaft Polymer-conjugated malonic acid derivatives and their use as medicaments and diagnostic agents
WO1998022162A1 (fr) * 1996-11-20 1998-05-28 Biocompatibles Limited Compositions biocompatibles
US6432314B1 (en) 1996-11-20 2002-08-13 Biocompatibles Limited Anion exchange materials and processes
US5891196A (en) * 1997-04-16 1999-04-06 Baxter International Inc. Method for actively binding heparin to crosslinked biological tissues
WO1999001195A1 (fr) * 1997-07-01 1999-01-14 Terumo Cardiovascular Systems Corporation Elimination d'agents biologiquement actifs
US6143354A (en) * 1999-02-08 2000-11-07 Medtronic Inc. One-step method for attachment of biomolecules to substrate surfaces
US6303179B1 (en) 1999-02-08 2001-10-16 Medtronic, Inc Method for attachment of biomolecules to surfaces through amine-functional groups

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