The invention relates novel wound coverings with which it is possible to remove interfering factors of the wound healing process from the wound fluid of chronic wounds in a targeted manner and in this way to initiate or promote the normal healing process.
The treatment of wounds which heal poorly or not at all has always been a problem which it has not hitherto been possible to solve satisfactorily. In the past, attempts have been made to promote wound healing by application of the most diverse substances and substance mixtures. The range of topical applications has extended from blood, serum or excrement via sulphur, pitch or heavy metal salts to milk, sugar or honey. Active compounds currently being developed for topical wound treatment are often substances endogenous to the body which are prepared by genetic engineering and are assumed to be present in the chronic wound in only a reduced amount, if at all. In some cases, it has been possible in all instances to detect a reduced concentration compared with healthy tissue. The proliferation-stimulating growth factors are an example (W. Meyer-Ingold, TIBTECH 1993, 11:387). The reasoning behind these modern set-ups is the concept that the absence or inadequate presence of such stimuli can be compensated by supplementing them and a normal wound healing which proceeds unimpeded therefore results. A large number of animal studies in recent years confirm this assumption. In spite of intensive efforts, the clinical breakthrough—apart from one exception (PDGF)—nevertheless has not yet been achieved. Even active compounds such as peptides or sugar polymers, which are derived from the natural structures of connective tissue and showed promising results in animal studies, have so far largely failed to achieve clinical success.
The disappointing results from clinical studies prompted an intensive world-wide analysis of the target of topical wound therapeutics, the chronic wound itself. Attention is mainly focused here on the exudate from the chronic wound, which is called wound fluid. Although these studies are still in their infancy, they nevertheless already show differences between chronic and acute, i.e. “normal” wound fluid, especially the observation, supported by determination of inflammatory markers (such as e.g. interleukin-1-alpha and collagenase (E. J. Barone et al., Wound Rep. Reg. 1995; 3:374), that the inflammatory character of chronic wound fluid is increased significantly compared with acute wound fluid, and furthermore is also persistent.
The current requirements of the function of wound coverings for chronic wounds are attributed to G. Winter (Nature 1962; 193:293) and have recently been reformulated by T. D. Turner (Wound Rep. Reg. 1994; 2:202). The main task is to provide a damp wound healing medium which, in contrast to traditional dry wound treatment with e.g. gauze compresses, offer physiological and therefore better conditions to the natural processes of wound healing. The wound covering here must absorb most of the exudate, but at the same time leave a film of fluid on the wound itself, in which fluid the actual moist wound healing takes place. These requirements are achieved by gel- or sponge-like structures and/or additional swellable or water-binding substances in the wound covering. On the outside, a breathable film ensures permeability to oxygen and water vapour and at the same time a barrier function against germs possibly penetrating from the environment.
In this connection, it is remarkable that by varying the properties of the wound coverings in respect of absorption of liquid or permeability to water vapour, in some cases opposite effects can be observed, e.g. a general concentration of protein (cf e.g. V. Achterberg, J. Wound Care, 1996; 5:79). The selective properties of Sorbact®, a cellulose wound covering rendered hydrophobic by coupling with stearic acid (T. Wadström et al., Acta Pathol. Microbiol. Immunol. Scand. 1985; 93B:359), are indeed in principle suitable for preferentially binding bacteria via hydrophobic interactions, as described in EP 0 162 026 and a clinical study by G. Friman (Current Therapeutic Research 1987; 42:88), but a differentiation between different strains of bacteria is not possible with this.
Disadvantages of the wound coverings known in the prior art are, inter alia, the inadequate healing-promoting action on chronic wounds and the abovementioned, in some cases opposite effects which are achieved by different properties in respect of liquid absorption or permeability to water vapour of the wound coverings.
The object of the invention is therefore to provide wound coverings which are particularly suitable for initiating or promoting healing of chronic wounds, i.e. wounds which heal poorly or not at all.
The object is achieved according to the invention in that additional substances which interact with interfering factors present in wounds exudate, i.e. with factors which impede the Rewound healing process, are used in the production of the wound coverings, these additional substances being covalently bonded to a carrier material
In this connection, the term “interfering factors” is understood generally as materials or substances which impede or slow down the healing process of wounds and therefore lead to the development of chronic wounds. These include suspended cells (e.g. inflammatory cells, such as leukocytes and macrophages or bacteria) and cell fragments or dissolved constituents, such as antigens, free radicals (such as e.g. reactive oxygen species, ROS), ions, (such as e.g. iron ions), proteins, peptides, lipids and free fatty acids, present in the wound exudate.
The substances which interact with these interfering factors are substances which remove the interfering factors from the wound exudate. In the following, the substances which interact with the interfering factors are also called “trapper molecules”, but this name is not understood as a limitation. The removal or elimination can take place in various ways, depending on the nature of the interfering factor, i.e. by a physical, chemical or immunological route, such as, for example, by binding, complexing or chelating. In an individual case, however, the interfering factor can also be eliminated by reacting chemically with the substance (the “trapper molecule”) and being converted into a compound which no longer impedes wound healing or impedes it to only a reduced extent. In the context of the present invention, the “trapper molecules” can be chosen, for example, from the group consisting of antibodies, chelators, enzyme inhibitors, enzymes, enzyme mimics, peptides and other proteins. In each case, it is particularly advantageous if the substance bonded to the carrier has a high specificity for the interfering factor to be eliminated or to be removed from the wound fluid, since removal also of substances which are important for the wound healing process and the result of an adverse effect can be prevented in this manner.
The invention accordingly relates to a wound covering, which is characterized in that substances which interact with interfering factors present in the wound exudate which impede the wound healing process are covalently bonded to a carrier material
The advantage of the invention lies in the fact that a selective removal of the interfering factors is possible by the substances covalently bonded to the carrier material, the “trapper molecules” also being removed on removal of the wound covering, without remaining in the wound or in the wound fluid. In this manner, on the one hand introduction of further substances which, where appropriate, could impede the wound healing process into the wound with the “trapper molecules” is prevented. On the other hand, for example in the case of substances which chelate or bond in the form of a complex or in another manner the interfering factors, such as e.g. ions, enzymes or other proteins, as it were “withdraw” the interfering factors from the wound exudate on removal of the wound covering. According to the invention, the interaction can also include a conversion of interfering factors in to substances which no longer impede wound healing. The substances used for the interaction which are covalently bonded to the wound covering are thus introduced into the wound only temporarily and, after they have performed their task as intended (i.e. have undergone the above-mentioned interaction), are removed from the wound region again. According to a preferred embodiment of the invention, the complexed interfering factors or such bonded to the trapper molecules are removed here at the same time. The healing process of chronic wounds, i.e. wounds which heal poorly or not at all, is improved or initiated by this selective removal elimination of the interfering factors.
In the context of the present invention, it is therefore not only possible to generate a moist wound medium or to apply growth-promoting substances in order to improve the healing process of chronic wounds, according to the invention the healing process can be accelerated and promoted further in that interfering factors present in the wound fluid are removed or eliminated specifically, e.g. by selective bonding to a wound covering or by the abovementioned conversion processes.
In the inflammatory phase of wound healing, which follows blood clotting and blood platelet aggregation after injury and trauma, neutrophiles and monocytes preferentially migrate into the damaged tissue. They start the phagocytosis of germs and the breakdown of destroyed tissue and foreign antigens there. Activated by chemical messenger substances and microorganisms, a greatly increased production of ROS, also called “oxidative burst”, occurs. These ROS are stored in granula and, on further stimulation, are released into the extracellular tissue in high local concentrations for combating microorganisms.
Another weapon of neutrophiles for combating substances foreign to the body are proteolytic enzymes, such as elastase, cathepsin G (J. Travies, American Journal of Med., 1988; 84:37) and collagenases, in particular MNLP-8 (M. Weckroth et al., J. Invest. Dermatol., 1996; 106:1119) They are usually stored in cytoplasmic granula and enter the extracellular medium only in a controlled manner. Rapid dissolution and sudden leakage of the cell membrane and therefore uncontrolled secretion of the proteolytic enzymes occurs by spontaneous cell death (necrosis) of the neutrophiles (C. Haslett and P. Henson, in: The Molecular and Cellular Biology of Wound Repair, 1996; Ed. R. Clark, Plenum Press, New York & London, p. 143). The secretion takes place in the form of latent proenzymes, and, after the N-terminal propeptides have been split off, leads to breakdown of the connective tissue proteins elastin, collagen, proteoglycan (J. Travies, American Journal of Med., 1988; 84:37), fibronectin (F. Grinell and M.
Zhu, J. Invest. Dermatol. 1996; 106:335; S. Herrick et al., Laboratory Investigations 1997; 77:281) and also plasma factors, such as fibrin and antithrombin III (W. Bode et al., EMBO J 1986; 5:2453), and therefore damage to the tissue and a delay in wound healing. ROS and plasmin as mediators of inflammatory cells can activate the pro-matrix metalloproteinases (pro-MMPs) (A. Docherty et al., TIBTECH 1992; 10:200) and contribute here to a further increase in the protease concentration.
In wound healing which progresses normally, after successful elimination of the triggering stimuli the inflammatory phase ends and the reconstruction of the tissue can start. However, if these stimuli persist, further leukocytes subsequently migrate into the tissue and are activated again, which leads to a permanently inflamed or chronic wound. The associated increased secretion of ROS (O. Senel et al., Annals of Plastic Surgery 1997; 39:516) and proteolytic enzymes can lead to a release of iron from damaged tissue, the plasma proteins transferrin and ferritin (C. Thomas et al., J. Mol. Biol. 1985; 260:3275; P. Biemond et al., Free Radio. Biol. Med 1988; 4:185), iron-containing enzymes, such as aconitase (P. R. Gardner et al., J. Mol. Biol. 1995; 270:13399) and other iron-sulphur proteins (J. Fridovich, Annu. Rev. Biochem. 1995; 64:97). The breakdown of ferritin and transferrin into the acid autophagocytic vacuoles of macrophages by lysosomal hydrolases leads to a further increase in the concentration of non-bonded iron (S. Sakaida et al., Mol. Pharmacol. 1990; 37:435). This transient, extracellular pool of released iron reacts with oxygen dissolved in the wound exudate to give iron(III) ions and superoxide, which dismutates rapidly into hydrogen peroxide. This non-charged molecule can penetrate through biological membranes, in contrast to superoxide, and react further at specific points in the cytosol. It dissociates into highly reactive hydroxyl radicals, under catalysis by iron(II) ions, in the reaction called the Fenton reaction. In the presence of physiological reducing agents, cyclic redox reactions occur, ROS being generated continuously (B. Halliwell and J. Gutteridge, Free Radicals in Biology and Medicine 1989; 2nd edition, Clarendon Press, Oxford). Under non-pathological conditions, iron metabolism is under strict control, and iron-mediated free radical reactions of the cell occur to only a limited extent.
The important role of iron ions and also copper ions in a cyclic redox reaction to form ROS (superoxide anions, peroxide dianions, singlet oxygen and hydroxyl radicals) and therefore the oxidative damage to biomolecules and cells has been adequately documented. Free, mobile iron and weakly complexed iron bonded to phosphoric acid esters, organic acids and membrane lipids (J. Mutanté et al., Agents Actions 1991; 32:167; D. A. Rowley et al., Clin. Sci. 1984; 66:691; B. Halliwell et al., FEBS Lett 1988; 66:691) cause e.g. damage to lysosomal membranes by lipid peroxidation with accompanying secretion of hydrolytic enzymes, DNA and RNA modification and strand breakages, degradation of polysaccharides, oxidation of amino acids and cleavage of enzymes and other proteins. Reactive oxygen species are therefore held responsible for many further processes in the body, such as ageing, rheumatoid arthritis, arteriosclerosis, muscular dystrophy, Parkinson's disease or autoimmune reactions, in addition to tissue damage. Studies on leg ulcers in which an increased iron content and a reduced amount of the endogenous growth factor transforming growth factor β (TGF-β) have been found also indicate the relationship between iron ions, ROS and tissue damage. Macrophages showed the greatest iron-specific staining here, followed by fibroblasts (Y. J. Francillon et al., Wound Rep. and Regeneration 1996; 4:240). By phagocytosls of senescent erythrocytes and iron-containing proteins, the iron is deposited in intracellular depots in the form of the iron storage proteins ferritin and haemosiderin.
Since wound healing is an extremely complex biological process, within which many individual steps pass through a coordinated cascade, the possibilities of interference are also manifold. Cellular interfering factors, such as bacteria or an excess of endogenous inflammatory cells, such as leukocytes or macrophages, can slow down wound healing just as much as imbalanced regulatory proteins or peptides of the cytokine type, such as catabolic enzymes from the classes of proteases, lipases, phospholipases or glycosidases, and such as enzymes which are active in signal transduction, such as, for example, kinases Small molecules or ions are also changed in their concentration in chronic wound fluid, for example reactive oxygen species or iron ions are increased. It is therefore obvious that the removal of such interfering factors helps the normal wound healing process.
Substances which interact with interfering factors of the type mentioned e.g. to form stable complexes can also be manifold, according to the diversity of the potential interfering factors. In the context of the present invention, the “trapper molecules” are preferably proteins or organic molecules.
According to a particular embodiment of the invention, specific antibodies against a cytokine, against an enzyme or against a regulatory peptide are suitable as the “trapper molecule”. Antibodies are therefore primarily suitable for the removal of the abovementioned inflammatory markers, which as a rule are proteins. For as quantitative as possible a removal, for example of a defined cytokine, antibodies of high affinity (if possible greater than 103 mol−1; E. Harlow and D. Lane: Antibodies—A Laboratory Manual, 1988; p. 514, Cold Spring Harbor Laboratory) must be chosen, since otherwise complete bonding of the interfering factor cannot take place, in spite of the use of high antibody concentrations.
Further interacting substances which are to be used according to the invention are integrins (R. O. Hynes, Cell 1992; 69:11) or generally RGD peptides (i.e. peptides which contain the tripeptide sequence arginine-glycine-aspartate), which can be used in an immobilized form as specific receptors for cell-cell and cell-matrix interactions in order to bind a certain cell population in a controlled manner.
Analogously, adhesins can be employed for specific bonding of bacterial surface structures (C. Heilmann et al., Mol. Microbiology 1996; 20:1083) and therefore for specific bonding of bacteria (J. M. Higashi et al., J. Biomed. Mater. Res. 1998; 39:341).
Furthermore, it is possible to use inhibitors for enzymes if the formation of the corresponding enzyme-inhibitor complex proceeds irreversibly (so-called “dead-end complex”). Inhibitors which are particularly suitable according to the invention are synthetic low molecular weight inhibitors for proteolytic enzymes, such as e.g. antipain, leupeptin, cystain, pepstatin, diisopropyl fluorophosphate, 4-(2-aminoethyl)-phenylsulphonyl fluoride or phenylmethanesulphonvyl fluoride. Naturally occurring, proteinogenic protease inhibitors, such as e.g. those of the class of tissue inhibitors of matrix metalloproteinases or aprotinin, anti-2-antiplasmin, alpha-2-macroglobulin, alpha-1-antichymotrypsin, soya bean trypsin inhibitor and/or alpha-1-protease inhibitor, are also suitable.
In the context of the present invention, enzymes or enzyme mimics can also be employed as the binding principle as substances which interact with the interfering factors if a corresponding substrate is the interfering factor and can be rendered harmless by reaction with the enzyme covalently bonded to the carrier. This can take place, for example, in the case of ROS.
The use of other known biological binding phenomenon, such as, for example, the property of the blood protein albumin to add on to free fatty acids, can lead to a reduction in the content of free fatty acids in wound fluid if required.
Specific chelators, such as e.g. diethylenetriaminepentaacetic acid, N,N′-bis-(o-hydroxybenzyl)-ethylenediamine-N,N′-diacetic acid, 1,2-dimethyl-3-hydroxypyrid-4-on, 1,2-dimethyl-3-hydroxy-3-hydroxypyridin-4-on (B. Porter, Acta Haematol. 1996; 95:13) and deferrioxamine (deferoxamine, 30-amino-3,14,25-trihydroxy-3,9,14,20,25-pentaaza-triacontane-2,10,13,21,24-1g pentaone, DFO) (G. J. Kontoghiorghes, Toxicol. Lett. 1995; 80:1; J. B. Galey et al., Free Radic. Res. 1995; 22:67), which form complexes with very high bonding constants, are suitable foe selective removal of interfering ions such as e.g. iron ions. DFO, a bacterial siderophor isolated from Streptomyces pilosus (H. Bickel et al., Helv. Chim. Acta 1963; 46:1385), and its mesylate (Desferal®; DFOM) are complexing agents with a high bonding affinity in a molar ratio of 1:1 for aluminium and iron ions. DFO is built up from one molecule of acetic acid, two molecules of succinic acid and three molecules of 1-amino-5-hydroxylaminopentane, which form with iron(III) ions, via their hydroxamic acid groups, an intensely orange-coloured complex (K=10−31), which is called ferrioxamine (H. Keberle, Ann. N.Y. Acad. Sci., 1964; 119:758). A drastically reduced complexing occurs with iron(II) ions (K=10−10). The protection which DFO forms against the formation of hydroxyl radicals lies in the complete enclosure of the bonded iron ions, in contrast to the complexing agent ethylenediaminetetraacetate, which still allows addition of an easily replaceable water molecule (E. Graf et al., J. Biol. Chem. 1984; 259:3620). As a result, the participation of the iron ion in the reaction cycle called the Fenton reaction (equation 1) with the formation of reactive hydroxyl radicals is suppressed.
Deferrioxamine has been used for than 20 years in treatment of severe ion poisoning, of disturbances in iron storage and of diseases which lead to increased iron values. Examples are idiopathic haemochromotosis, thalassaemia (hereditary disorder in globin synthesis of haemoglobin), sickle cell anaemia, frequent blood transfusions, excess iron absorption or haemolyses induced by medicaments. An accelerated wound contraction was also to be found in wound healing in animal studies after systemic administration of DFO (H. Cohly et al., Diabetes 1997; 46:295A). The low toxicity (J. B. Porter and E. R. Huens, Bailliere's Clin. Haematol. 1989; 2:257) and the low tendency to penetrate through biological membranes (J. B. Lloyd et al., Biochem. Pharmacol. 1991; 41:1361) make DFO an ideal active compound for trapping excess iron out of extracellular fluids. It is known from in vitro experiments that on incubation of dissolved DFO with ferritin and haemosiderin, iron can be dissolved out up to the maximum saturation of DFO. On incubation with iron-saturated transferrin, 10 to 15% of the iron can be dissolved out, while the iron cannot be removed from the porphyrin ring of haemoglobin and myoglobin (H. Keberle , Annu. N.Y Acad. Sci. 1964; 119:758). In vivo, DO thus bonds only depot iron and—to a small extent—transport iron. Haemoglobin and myoglobin and iron-containing enzymes of the respiratory chain are not influenced. The high specificity for iron furthermore prevents the elimination of other metal ions essential for wound healing, such as magnesium, calcium and zinc.
Colonization and infection of poorly healing wounds by microorganisms is a major and frequently occurring problem in the treatment of wounds. Bacteria can trigger an excessive immune response and thus adversely influence the course of wound healing (M. C. Robson et al., Clinics in Plastic Surgery, 1990; 17:485). Chronic wounds are affected more often than 5 acute wounds because of tissue which has already died (P. Mertz et al., Dermatologic Clinics 1993; 11:739). For example, a persistent colonization of a leg ulcer with Staphylococcus aureus led to an enlargement of the ulcer with the development of haemorrhagia and incipient necrosis (S. Munk et al., Acta Pathol. Microbiol et Immunol. Scand. 1996; 104:895; J. Danielsen et al., J. Wound Care, 1997; 6:308). The iron transport proteins transferrin and lactoferrin the latter is also secreted from leukocytes—occurring in the serum have a potent bacteriostatic action by withdrawal of iron from bacteria, and therefore contribute towards controlling infections (R. Critchton, in: Inorganic Biochemistry of Iron Metabolism, 1991; Elis Horwood, N.Y., 101). A high-affinity iron chelator immobilized on a wound covering shows an analogous action, the growth of bacteria being inhibited and recolonization being made difficult.
In respect of ROS, in aerobic biological systems the dimeric or tetrameric enzyme superoxide dismutase (SOD) plays the main role in cell defence against the oxygen-mediated toxicity of ROS and in regulating the intracellular oxygen concentration (I., Fridovich, Annu. Rev. Biochem. 1995; 64: 97). The ubiquitous enzyme catalyses the dismutation reaction of superoxide into less toxic hydrogen peroxide and oxygen (equation 2). SOD furthermore prevents reaction of superoxide with nitrogen monoxide, which is responsible for regulation of blood pressure (R. F. Furchgott, Nature 1980; 288:373). In eukaryotic cells, SOD is in the cytosol and in the mitochondria. The various enzymes with molecular weights from 32,000 to 56,000 Da have metal ions, such as copper and zinc (Cu—Zn-SOD), manganese (Mn-SOD) or iron (Fe-SOD) as co-factors in the catalytic centre.
2O2 −+2H+→H2O2+O2tm (2)
The hydrogen peroxide formed by reaction of the SOD is then converted into water and oxygen in a multi-stage catalytic cycle by the redox enzyme catalase, which is ubiquitous in aerobic organisms (P. Gouet et al., Nature Structural Biology 1996; 3:951). In addition to catalase, the enzymes gluthathione peroxidase and myeloperoxidase are also capable of breaking down hydrogen peroxide. The most widespread form of catalase is a homotetramer (235,000 Da, bovine liver) with a porphyrinic group with one iron atom per sub-unit.
According to the invention, these abovementioned substances (“trapper molecules”) are not administered per se, but are employed in a form covalently bonded to a wound covering. By fixing the “trapper molecules” to the wound covering, the site of action is precisely defined and is limited to the wound region or the wound fluid. A systemic action, i.e. an interaction with the entire organism, is avoided in this way. The action principle thus comprises a complexing, chelating or bonding reaction of the interfering factor or a chemical reaction with the interfering factor on the wound covering.
The present invention thus relates to novel wound coverings which impart an additional healing effect by the controlled removal or elimination of interfering factors, i.e. of substances which impede healing of chronic wounds.
The interfering factors can be removed or eliminated by a physical, chemical or immunological route. In particular, bonding or complexing or chelating of interfering factors is a preferred embodiment of the invention, since the interfering factors are removed finally ,from the wound region when the dressing is changed.
For such a wound dressing to function as intended, covalent bonding of the “trapper molecules” to the wound covering is of great importance. The carrier material of the wound covering according to the invention, i.e. the polymeric material (of natural or synthetic origin) to which the “trapper molecule” is covalently bonded, must be activatable for the coupling or, if this should not be the case, rendered activatable in a prior reaction. All carriers of materials which still carry functional groups such as e.g. —OH, —SH, —NH2, —NHR, —CHO, —COCH or —COOR on the surface after production of the wound covering can in principle be activated. The coupling steps then necessary can take place chemically or enzymatically, and are described in standard works as general immobilization techniques (J. M. S. Cabral and J. F. Kennedey in: Protein Immobilization—Fundamentals and Applications, 1991; p. 73, ed. R. F. Taylor, Marcel Dekker, New York; Immobilization of Enzymes and Cells, 1997; ed. G. F. Bickerstaff, Humana Press, Totowa, N.J.). However, it is also possible for the “trapper molecules” already to be bonded to starting materials which are employed for the production of the carrier material or the wound covering.
According to a preferred embodiment of the invention, polymers with activatable hydroxyl groups are the starting substances and are used either per se as the wound covering or as one of several components of a wound covering.
According to the invention, for example, the following carrier materials are possible: naturally occurring and modified and other forms of cellulose [such as e.g. carboxymethylcellulose, cellulose acetate and viscose fibres, bacterial cellulose (U. Geyer et al., Int. J. Biol. Macromol. 1994; 16:343)], alginates, hyaluronic acid, chitin or chitosans and other polysaccharides, such as e.g. Sephadex®, Sephacryl®, Sepharose® and Superdex®, synthetic polymers, such as e.g. polyamides, polyesters, polyolefins, polyacrylates, polyvinyl alcohols, polyurethanes and silicones, including mixtures and copolymers thereof, into which various functional groups, such as e.g. hydroxyl, carbonyl, carboxyl, amino or peroxy, can be introduced on the surface by corona treatment, plasma treatment or polymer grafting (chemical grafting) (D. M. Coates and S. L. Kaplan, MRS Bulletin 1995; 8:43). The above-mentioned carrier materials can be employed as films, gels, foams, laminates and composites (mixtures).
The activation of the functional group for covalent coupling of proteins, antibodies, enzymes or low molecular weight organic substances via amino groups is described in detail in standard works, such as e.g. W. H. Scouten, Immobilized Enzymes and Cells, in: Methods Enzymol., ed. K. Mosbach, 1987; 135:30 and in: Immobilization of Enzymes and Cells, 1997; ed. G. F. Bickerstaff, Humana Press, Totowa, N.J. Functional amino groups which can be used in the case of proteins, antibodies and enzymes are the amino group of the amino terminus and/or the ε-amino groups of amino acid side chains of lysine and arginine exposed to the surface. Reagents such as, for example, 1,1′-carbonylditriazole, 1,1′-carbonyldiimidazole (CDI), cyanuric chloride, chloroformic acid esters, succinic anhydride, dicyclohexylcarbodiimide, p-nitrophenyl chloroformate, 2,4,5-trichlorophenyl chloroformate, p-toluyl chloride and tricresyl chloride can be employed for activation of hydroxyl groups which are on a soluble polymer (compare C. Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1992; 9:249).
Corresponding coupling processes are known in the prior art and are employed e.g. in the preparation of substances for preparative and analytical uses. Thus, for example, EP 0 087 786 describes a process for immobilization of the iron chelator DFO via bonding of the primary amine group, DFO being bonded to agarose-polyaldehyde gel beads. The agarose was activated beforehand by perlodate oxidation. The covalent bonding of DFO to dissolved biopolymers, such as dextran, hyaluronic acid, hydroxyethyl-starch, methylcellulose, inulin and human serum albumin, is furthermore described in EP 0 304 183. In this case, the terminal amine function of the DFO is coupled with an aldehyde group of the polysaccharide to give a Schiff's base, and is then reduced with sodium borohydride, as described by S. Margel and E. Weisel (J. Pol. Chem. Sci., 1984; 22:145) and EP 0 087 786. Other medicaments or proteins can also be coupled covalently to agarose-polyaldehyde gel beads via the reaction of primary amine or thiol groups with the aldehyde groups. WO 96/39125 describes a matrix comprising a high-viscosity cubic phase of e.g. amphiphilic glyceryl monooleate, in which horseradish peroxidase or catalase is embedded. This matrix represents a barrier between the enzyme bonded in this manner and external proteolytic enzymes or cell defence systems of the patient and thus prevents release of the enzyme. The catalytic activity of the enzyme remains accessible to the particular substrate through pores and channels in the matrix. M. M. Hossain and D. D. Do (Biotechnology & Bioengineering, 1988; 31:730) describe the immobilization of the enzyme catalase on glass particles of controlled pore size and subsequent kinetic measurements. The covalent modification of SOD (P. Pyatak et al., Res. Com. Chem. Path. and Pharmacol., 1980; 29:115) and CAT (A. Abukovski et al., J. Biol. Chem., 1976; 252:3582) and other enzymes (M .L. Nucci et al., Advanced Drug Delivery Reviews, 1991; 6:133) with polyethylene glycol by coupling of activated polyethylene glycol on to amino groups of the enzyme is described. H. Hirane et al. (J. Controlled Release, 1994; 28:203) describe the preparation of SOD polymer conjugates with activated divinyl ether and maleic anhydride. WO 95/15352 describes covalent bonding of peroxidase and catalase via amino groups into a polymer gel comprising the protein BSA and preactivated polyethylene glycol. G. Maneke and D. Polakowski (J. Chrom., 1981; 215:13) describe the immobilization of α-chymotrypsin on a polymer matrix of polyvinyl alcohol and terephthalaldehyde
The prior art has not described to date the use of substances which interact with interfering factors which are present in wound exudate and impede the wound healing process for the production of wound coverings. By analogous use of the abovementioned coupling processes, the substances which interact with the interfering factor are coupled covalently according to the invention on to carrier materials suitable for wound coverings by chemical reaction and novel wound coverings are produced in a controlled manner for selective removal of interfering factors from the wound fluid of wounds which heal poorly or not at all. The present invention provides for the first time novel wound coverings for improving the healing course of these wounds.
Possible wound coverings are, for example, wound coverings from the group consisting of dressings, dressing gauze, bandages, compresses, cotton-wool, patches, foils, films, hydrocolloid dressings, gels and the like.
In the context of the present invention, the wound coverings known in the prior art can thus be used and can be modified according to the invention by covalent bonding of “trapper molecules”. Thus, in particular, wound coverings which absorb Moisture can be used. Furthermore, it is of course possible to apply simultaneously with the wound coverings according to the invention wound healing-promoting substances, such as e.g. growth factors (e.g. PDGF), regulatory cytokines, peptides and hormones, which are suitable for promoting wound healing or accelerating the healing process, into the wound.
The present invention is explained in the following with the aid of examples and figures: