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WO1999032080A2 - Procede d'immobilisation ciblee de l'inhibiteur de thrombogenese hirudine sur des surfaces en polymere - Google Patents

Procede d'immobilisation ciblee de l'inhibiteur de thrombogenese hirudine sur des surfaces en polymere Download PDF

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Publication number
WO1999032080A2
WO1999032080A2 PCT/DE1998/003727 DE9803727W WO9932080A2 WO 1999032080 A2 WO1999032080 A2 WO 1999032080A2 DE 9803727 W DE9803727 W DE 9803727W WO 9932080 A2 WO9932080 A2 WO 9932080A2
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WO
WIPO (PCT)
Prior art keywords
immobilization
chemical
hirudin
spatial environment
functional groups
Prior art date
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Ceased
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PCT/DE1998/003727
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German (de)
English (en)
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WO1999032080A3 (fr
Inventor
Hartwig Höcker
Jörg LAHANN
Wilhelm PLÜSTER
Doris Klee
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Individual
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Individual
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Priority claimed from DE19857904A external-priority patent/DE19857904A1/de
Application filed by Individual filed Critical Individual
Priority to DE19881935T priority Critical patent/DE19881935D2/de
Priority to AU26086/99A priority patent/AU2608699A/en
Publication of WO1999032080A2 publication Critical patent/WO1999032080A2/fr
Publication of WO1999032080A3 publication Critical patent/WO1999032080A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • A61L33/12—Polypeptides, proteins or derivatives thereof, e.g. degradation products thereof
    • A61L33/128—Other specific proteins or polypeptides not covered by A61L33/122 - A61L33/126
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/55—Protease inhibitors
    • A61K38/57—Protease inhibitors from animals; from humans
    • A61K38/58—Protease inhibitors from animals; from humans from leeches, e.g. hirudin, eglin

Definitions

  • the invention relates to a method for immobilizing the thrombogenesis inhibitor hirudin on polymer surfaces of medical objects to be used extracorporeally and / or intracorporeally by interaction between functional groups of hirudin with corresponding functional groups of the polymers directly or via suitable spacer systems.
  • the invention also relates to a thrombogenesis inhibitor for improving the hemocompatibility of surgical suture material or other medical objects, consisting of a hirudin suitable for the immobilization of the surface thereof.
  • Functional groups are to be understood as meaning those chemical groups which are suitable for binding hirudin directly, after activation or by means of spacer systems, ie. H. for example amino, hydroxyl, thiol or carboxyl groups.
  • polymers as biomaterials for medical applications. If these materials come into direct contact with blood, e.g. B. as stents, vascular prostheses, catheters, dialysis membranes, artificial heart valves or hearts or as surgical sutures, the problem of thrombus formation occurs. It is also known that the immobilization of biologically active, antithrombogenic substances on the surface can improve the hemocompatibility. Such a thromobogenesis inhibitor, heparin, has long been used in clinical practice for the treatment of diseases of the coagulation system. Therapeutic use is often accompanied by side effects such as excessively prolonged clotting times.
  • Heparin and heparin derivatives have often been immobilized on polymer surfaces in order to improve the hemocompatibility of the medical objects and to reduce the systemically administered amount of heparin. It is known that such a thrombogenesis inhibitor can be immobilized using a wide variety of methods. In addition to methods based primarily on adsorptive immobilization, methods which aim at covalent attachment of the thrombogenesis inhibitor to the polymer surface have become increasingly popular. Such methods are e.g. B. from US Patent 4,613,665. In addition to the widespread use of spacer systems as a bivalent connector between the surface and thrombogenesis inhibitor, z. B. in EP-PS 081 853 proposed the use of heparin-albumin conjugates for immobilization.
  • Hirudin is the strongest known thrombin inhibitor with a complex formation constant of approx. 10 "12 mol / 1 and has a number of advantages compared to heparin.
  • the antithrombogenic effect of heparin on the presence of antithrombin III or of heparin cofactor II is one example Heparin is also inhibited in its antithrombogenic effect by platelet factor 4.
  • Hirudin In addition to prolonged bleeding, heparin not infrequently causes thrombocytopenia, hirudin shows none of these effects and, moreover, has a more antithrombogenic effect than heparm platelets and platelet activation by thrombin.Hirudin is also able to block thrombin in its action, mitogenic effects on fibroblasts, activating endothelial cells and chemotactic effects on monocytes and leucocytes, the latter being closely related to the inflammatory effects at the implant ion of biomeater materials often are to be seen to be seen. Hirudin thus has a number of favorable properties which make it appear to be a potentially superior substance to immobilization on biomaterial surfaces for blood contact, which is potentially superior to heparin.
  • the invention is therefore based on the object of providing a method for immobilizing hirudin in which the antiplatelet and / or thrombogenesis-inhibiting effect is largely retained, and also to provide a suitable hirudin-based thrombogenesis inhibitor.
  • the object is achieved in that in a treatment step preceding the immobilization, the chemical and / or spatial environment of functional groups of hirudin is changed with regard to their immobilization selectivity and that after the immobilization, the change in the chemical and / or spatial environment again will be annulled.
  • the invention assumes that one of the functional groups is essential for the thrombogenesis-inhibiting effect of hirudin. At the same time, the same functional group reacts very selectively with the usual coupling reagents that can be used to immobilize the hirudin on a surface. Without changing the chemical and / or spatial environment of functional groups of hirudin, hirudin cannot be immobilized while maintaining its thrombogenesis-inhibiting activity.
  • hirudin by changing the chemical and / or spatial environment of functional groups of hirudin, ie by changing their electronic and / or energy getisch state, a metastable hirudin derivative is generated, which shows a changed immobilization rank selectivity of the functional groups in a subsequent immobilization compared to hirudin.
  • immobilization rank selectivity is the ratio in which the available functional groups of hirudin govern during immobilization.
  • the chemical and / or spatial environment is changed while keeping part of the functional grapple free. This free part of the grappa is then available for binding the hirudin to the thrombin. It has been found that it is particularly advantageous if the chemical and / or spatial environment is changed while freeing more than 50% of the functional grapples, which are preferably amino groups.
  • An essential feature of the invention is therefore that the change in the chemical and / or spatial environment explained is reversible, ie that the original environment of the functional groups of hirudin not required for connection to the polymer surface can be restored after immobilization.
  • the proposed procedure thus ensures that functional groups necessary for thrombin activity are not bound during immobilization and are therefore available for interactions with thrombin after immobilization. This causes an increased antithrombogenic and / or platelet-repellent effect of the surface-bound hirudin.
  • the invention according to the method thus shows a way that functional grappes of hirudin, which due to their chemical state are reactive both in the immobilization and in the reaction with thrombin, are also available after the immobilization of hiradin on polymer surfaces for the interactions with thrombin.
  • the chemical environment of the functional grapes of hirudin can be changed by a chemical reaction of the hirudin with substances to form a covalent, non-valent or ionogenic bond.
  • a change in the chemical environment of the functional groups of hirudin under the influence of energy-donating fields or by thermal or photochemical treatment or reaction corresponds to the essence of the invention.
  • the change in the spatial environment of the functional grappes of the hirudin e.g. B. by changing the surrounding medium, by oxidation or reduction reactions, by thermal or photochemical treatment or by the influence of energy-giving fields.
  • a change in the spatial environment of the functional grappes of the Hiradm in connection with a change in their chemical environment expressly corresponds to the essence of the invention.
  • both the immobilization on a polymer which has free functional groups or on which those have been generated by surface modification, and the immobilization on a metal, a ceramic or some other non-polymeric substrate as a result a polymer coating has free functional grapples, possible.
  • the reaction of the hirudin with 2- (methylsulfonyl) ethyl-N-succinimidyl carbonate at pH 3 to 8 is particularly suitable for changing the chemical environment of the functional groups of the hirudin in such a way that an optimal immobilization rank selectivity is achieved with regard to a later availability of the functional grappes necessary for the interactions of the brain arm with thrombin.
  • any treatment which cancels out the change in the chemical and / or spatial environment carried out before the immobilization is suitable for restoring the thrombin activity of hirudin after immobilization.
  • all substances are suitable for changing the chemical and / or spatial environment of the functional groups of hirudin, which change the immobilization range selectivity of hirudin and in which the change in the chemical and / or spatial environment caused by them can be restored by immobilization.
  • Substances which can be assigned to the following classes of substances are particularly suitable: 1-chloroformic acid alkyl ester, preferably 1-chloroformic acid (5-trisilylpropyl) ester, 1-chloroformic acid aryl ester, preferably 9-fluorenylmethyloxycarbonyl chloride, 1-azidoformic acid alkyl ester or -aryl ester, preferably 1-azidoyl formate , Diaryl or arylalkyl carbonates, aryl or alkyl (orthonitrophenyl) carbonates, preferably isobutyl (orthonitrophenyl) carbonate or arylalkyl or dialkyl dicarbonates, preferably diisobutyl dicarbonate.
  • the restoration of the chemical and / or spatial environment can be carried out according to the invention by all methods which are sufficiently gentle on the primary, secondary and tertiary structure of hirudin, the bond between hiradin and the polymer surface and on the polymer surface used.
  • the chemical and / or spatial environment by incubating the hiradin in an aqueous mixture of an organic solvent, preferably dimethylformamide, dimethyl sulfoxide, isopropanol, ethanol, acetone, diethyl ether, hexane or tetrahydrofuran, combined with the addition of reducing agents, preferably peptides or proteins carrying mercaptoethanol, cysteine or cysteine residues, glutathione, dithiothreitol or also sodium tetrathionate, the chemical and / or spatial environment of the functional grapple of the hirudin is also changed in such a way that these show changed immobilization selectivity.
  • an organic solvent preferably dimethylformamide, dimethyl sulfoxide, isopropanol, ethanol, acetone, diethyl ether, hexane or tetrahydrofuran
  • the metastable form takes place by incubating the hiradin under reductive conditions in an aqueous mixture of an organic solvent, preferably dimethylformamide, dimethyl sulfoxide, isopropanol, ethanol, acetone, diethyl ether, hexane or tetrahydrofuran. It when a reducing agent, such as. B. sodium tetrathionate to the chemical and / or spatial environment of the functional groups of hirudin is used so that these groups show a changed immobilization rank selectivity.
  • an organic solvent preferably dimethylformamide, dimethyl sulfoxide, isopropanol, ethanol, acetone, diethyl ether, hexane or tetrahydrofuran.
  • glutathione, cystopine, peptide or protein carrying cysteine residues or dithiothereitrol is added.
  • a thrombogenesis inhibitor suitable for improving the hemocompatibility of surgical suture material or other medical objects consists of a hiradin suitable for immobilizing the surface thereof, according to the invention this thrombogenesis inhibitor is characterized by a hiradine derivative and an aftertreatment consisting of aqueous piperidine solution with pH 6 to 13 . It goes without saying that the aftertreatment is used retrospectively to cancel the immobilization.
  • Such a thrombogenesis inhibitor is available in the form of a derivative which can be brought into contact in a simple manner with the suture material to be treated or other medical objects in order to then develop the targeted and desired effect, i. H. Functional grappes are still available, which are required for thrombin activity or for the antiplatelet effect. This ensures that the hiradin has its beneficial effects.
  • a thrombogenesis inhibitor has been found to be particularly useful in which the hiradine derivative from r-hiradin, a ten-fold excess of 2- (methylsulfonyl) ethyl-N-succinimidyl carbonate and 20 ml of 0.1 M sodium acetate solution, preferably at pH 5 , 9, it being again expedient that the aftertreatment is a ten percent aqueous piperidine solution at pH 6 to 13.
  • Fig. 1 Thrombin activity after immobilization of r-hirudin or the metastable r-hirudin derivative on EDC-activated polyurethane depending on the volume concentration (n> 6)
  • Fig. 2 Thrombin activity after immobilization of r-hirudin or the metastable r-hirudin derivative on HDI-activated hydroxymethyl-ppx as a function of the volume concentration (n> 6); Samples in which no coagulation is observed are normalized to 90 s
  • Fig. 4 Fluorescence images of the adherent platelets on the hydroxymethyl-ppx-coated stainless steel foil after reaction with HDI and immobilization of the metastable r-hirudin derivative (right) or unmodified (left), bar 20 ⁇ m
  • argon plasma treatment microwave, pulsed plasma, 30 s, 2000 W
  • the polymer films activated in this way were graft-copolymerized with a 5% aqueous acrylic acid solution using UV radiation (excimer, 308 ⁇ m) over a period of 10 min. After cleaning the graft-copolymerized surfaces, the mixture was activated for 1 h at 4 ° C. with EDC (pH 4.8). The activated foils were subsequently reacted with a metastable hirudin derivative at pH 3.5 for 24 h. The surface was then washed five times with surfactant-containing phosphate buffer and incubated with ten percent piperidine solution for 2 hours. The thrombin activity of the hirudin immobilized by this method is shown in Fig. 1 in comparison to the directly immobilized hirudin.
  • Example 2 The thrombin activity of the hirudin immobilized by this method is shown in Fig. 1 in comparison to the directly immobilized hirudin.
  • Example 2 The thrombin activity of the hirudin
  • the dimer [2,2] -hydroxymethyl-paracyclophane was first cleaved at 700 ° C. and 30 Pa in a pretreatment step and then deposited on the stainless steel plate by cooling to about 120 ° C. (hydroxymethyl-ppx). Then the hirudin derivative was bound to the surface using hexamethylene diisocyanate as a spacer.
  • the coated metal plate was incubated in an ethereal hexamethylene diisocyanate solution for 12 hours at room temperature and then extracted with ether. A solution of the metastable hirudin derivative was then added to the sample at 4 ° C. for 24 h. The surface was then washed five times with surfactant-containing phosphate buffer and then incubated with ten percent aqueous piperidine solution for four hours. Finally, the surface was washed again five times with surfactant-containing phosphate buffer.
  • the TT clotting time (Fig. 2) already shows a high value of (40.5 ⁇ 7.2) s at a volume concentration of 1.05 nmol / ml.
  • An increase in the hirudin concentration in solution from 10.5 nmol / ml to 21 nmol / ml does not significantly increase the TT time.
  • At a volume concentration of 42 nmol / ml all thrombin is removed from the solution, so that coagulation can no longer be observed. All experiments in which no coagulation takes place are standardized to a coagulation time of 90 s.
  • r-Hirudin specifically complexes the coagulation enzyme thrombin; furthermore, in contrast to heparin, which, for example, can promote platelet aggregation, no further interactions with plasma or cellular blood components are known. For this reason, in v / trol l Hemocompatibility of immobilized hirudin methods important that detect biomaterial-induced thrombin generation.
  • thrombirin / antithrombin complex was determined in direct contact with blood plasma.
  • Fig. 3 shows the TAT content of r-hirudin immobilized according to the invention in comparison to the reference surfaces glass, hydroxymethyl-ppx and stainless steel. The mean value for glass is set to 100%, all other results are given relative to it. While the hydroxymethyl-ppx coating of the stainless steel surface with (22.5 ⁇ 1.4)% of the value for glass does not result in a significant change compared to the stainless steel surface ((36.2 ⁇ 6.8)%), the is reduced TAT value after immobilization of r-hirudin according to the invention to (2.9 ⁇ 0.8)% of the reference value.
  • Fig. 4 shows representative fluorescence images of the hydroxymethyl-ppx-coated stainless steel foil to which the metastable r-hirudin was immobilized (right). For comparison, the hydroxymethyl-ppx-coated stainless steel foil (left) is compared. The investigation of platelet adhesion was carried out with platelet-rich human blood plasma.
  • the hydroxymethyl-ppx shown on the left shows a relatively dense occupancy with platelets, which are partially rounded. In addition, there are also activated cells spread out flat.
  • the r-hirudin immobilized according to the invention brings about a drastic reduction in platelet adhesion. In addition to areas where there are no cells at all, there are occasionally adherent platelets that are globular and therefore not activated or only slightly activated.
  • the hemocompatibility examination of the hydroxymethyl-ppx-coated metal surfaces presented here, to which r-hirudin was bound using the protective rubber technique while maintaining its biological activity can be summarized as follows:
  • the biomaterial-induced thrombin generation can be almost completely prevented by the immobilization of r-hirudin according to the invention.

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Abstract

L'invention concerne un procédé permettant d'immobiliser l'inhibiteur de la thrombogénèse hirudine sur des surfaces en polymère d'objets médicaux, destinés à une utilisation extracorporelle et/ou intracorporelle, par interaction de groupes fonctionnels de l'hirudine avec des groupes fonctionnels correspondants des polymères, directement ou par l'intermédiaire de systèmes espaceurs appropriés. Selon ce procédé, il est proposé qu'au cours d'une étape de traitement à exécuter avant l'immobilisation, on modifie l'environnement chimique et/ou spatial de groupes fonctionnels de l'hirudine pour obtenir de celle-ci une sélectivité d'immobilisation, et qu'après l'immobilisation, on mette fin à la modification de l'environnement chimique et/ou spatial. Grâce à la procédure proposée, les groupes fonctionnels nécessaires à l'activité de la thrombine ne sont pas liés lors de l'immobilisation et restent donc à disposition, après l'immobilisation, pour les interactions avec la thrombine. On obtient ainsi un effet élevé antithrombogène et/ou empêchant la formation de thrombocytes de l'huridine lié auxdites surfaces.
PCT/DE1998/003727 1997-12-19 1998-12-18 Procede d'immobilisation ciblee de l'inhibiteur de thrombogenese hirudine sur des surfaces en polymere Ceased WO1999032080A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
DE19881935T DE19881935D2 (de) 1997-12-19 1998-12-18 Verfahren zur gezielten Immobilisierung des Thromobogeneseinhibitors Hirudin auf Polymeroberflächen
AU26086/99A AU2608699A (en) 1997-12-19 1998-12-18 Device for targeted immobilization of the thrombogenic inhibitor hirudine on polymer surfaces

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19756508 1997-12-19
DE19756508.5 1997-12-19
DE19857904A DE19857904A1 (de) 1997-12-19 1998-12-16 Verfahren zur gezielten Immobilisierung des Thrombogeneseinhibitors Hirudin auf Polymeroberflächen
DE19857904.7 1998-12-16

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WO1999032080A2 true WO1999032080A2 (fr) 1999-07-01
WO1999032080A3 WO1999032080A3 (fr) 1999-10-14

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DE (1) DE19881935D2 (fr)
WO (1) WO1999032080A2 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2000887A1 (fr) * 1988-11-01 1990-05-01 Cecilia S.L. Ku Materiaux thromboresistants et methode de production

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WO1999032080A3 (fr) 1999-10-14
DE19881935D2 (de) 2001-01-04
AU2608699A (en) 1999-07-12

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