Drug – bio-affecting and body treating compositions – Enzyme or coenzyme containing
Reexamination Certificate
2000-01-28
2004-08-10
Monshipouri, Maryam (Department: 1652)
Drug, bio-affecting and body treating compositions
Enzyme or coenzyme containing
C424S400000, C424S422000, C424S423000
Reexamination Certificate
active
06773703
ABSTRACT:
The healing of therapy-resistant wounds has since lime immemorial been a great challenge to medicine and science. Present-day specifications for the function of interactive dressings for chronic wounds derive from Winter (1962, Nature 193, 293) and have recently been reformulated by Turner (1994, Wound Rep. Reg. 2, 202). The emphasis in this connection is on the creation of a moist wound healing environment which, in contrast to traditional dry wound treatment with, for example, gauze compresses, provides physiological and thus better conditions for the natural processes of wound healing.
The principle of moist wound healing can at present be regarded as the state of the art in the therapy of wounds which are healing with difficulty or not at all. The dressing must absorb most of the exudate but, at the same time, leave on the wound itself a liquid film in which the actual moist wound healing takes place. Dry wounds and those with little exudation must be provided with adequate moisture to achieve rehydration of the dehydrated tissue. In the moist wounds which have been established in this way there is then proliferation of new blood vessels and reduced bacterial growth, with a suitable pH being set up. These requirements are met by sponge-like structures such as, for example, hydrogels which have an excess in the form of bound water.
Hydrogels are generally produced by hydrophilic, coherent monomers forming in a dispersant a three-dimensional network structure into whose interstices the dispersant, normally water, can infiltrate. A hydrogel should have a certain oxygen permeability and assume a barrier function towards microbes possibly penetrating in from the environment. Fundamental requirements for a hydrogel are also a simple production process and a certain storage stability.
The use and the variety of possible uses of hydrogels in wound treatment, and their composition and production are sufficiently well documented (Peppas in: Hydrogels in Medicine and Pharmacy, 1986, CRC Press, Volume II, Chapter 4). Hydrogels are preferably employed for treating dry necrotic wounds such as, for example, bums and chronic venous ulcers (Thomas, in: Wound Management and Dressings, 1990, The Pharmaceutical Press, London, p. 50).
They are made of insoluble polymeric materials able to swell in aqueous media. They should furthermore have a high water content, be inert to biological processes, permeable for cellular metabolites and, in particular, not induce any irritation on contact with living tissue.
A bioartificial or semisynthetic hydrogel can be synthesized by covalently linking the hydrophilic synthetic monomer to the surface of a protein, whereupon there is formation of a three-dimensional polymer-protein matrix inside the dispersant. This class of hydrogels made from synthetic polymers and biopolymers have recently become the subject of research, These novel biomaterials are referred to as bioartificial hydrogels (Giusti et al., 1993, Trends in Polymeric Science, 9, 261).
U.S. Pat. No. 5,804,213 reports on the production of a biologically active, water-containing gel as wound dressing. In this case, a dry, hydrocolloidal polymer is mixed with water and a biologically active substance.
The use of dehydrated, crosslinked collagen materials as drug delivery system is described in WO 98/22153. In WO 96/31551, proteins or peptides in the dry state are mixed as active agents with polyurethane-crosslinked microgels. The microgels swell in an aqueous medium to give hydrogels and release the protein or peptide again from the hydrogel matrix. Moreover U.S. Pat. No. 5,000,955 describes polyurethane hydrogels for cosmetic, biological and medical applications.
Cubic phases made of glyceryl monooleate are able to immobilize enzymes by non-covalent linkages, as reported in WO 96/39125. In this case, the enzymatic activity is retained due to the immobilization and is even increased over a lengthy period by comparison with dissolved enzyme. It should furthermore be emphasized that the barrier function of the gel matrix suppresses the proteolytic degradation of the immobilized enzymes occurring in the wound fluid.
Hydrogels of particular interest are those in which a biomolecule is covalently incorporated, as described in U.S. Pat. No. 5,733,563 and in 1994 by Fortier (1994, Biotechnol.Techn., 8, 71). These hydrogels are formed by copolymerization of a polyethylene glycol which has been activated by 4-nitrophenyl chloroformate and of bovine serum albumin in borate buffer. In this case, the activated group may react with an amino, SH—, OH— or COOH group in the protein.
Disadvantages of the bioartificial hydrogels known in the state of the art are the long gel times of from 20 up to 270 minutes, the formation of cleavage products in the form of p-nitrophenols in the hydrogel matrix and the impossibility of crosslinking oligomeric proteins such as, for example dimeric SOD or tetrameric catalase stably with the polyethylene glycol which has been activated with 4-nitrophenyl chloroformate.
It is an object of the invention to make water-insoluble, water-swellable hydrogels which have short gel times, can be employed for medical purposes and are particularly suitable for promoting the healing of chronic wounds.
This object is achieved by a hydrogel which comprises at least one protein and/or enzyme and PEGs, the proteins and/or enzymes being connected to the PEGs via urea groups.
In the production of the hydrogel there is use of additional substances which interact with the oxygen species (ROS) present in wound fluids of chronic wounds, that is to say with factors which impede the wound healing process, these additional substances being covalently polymerized into the hydrogel. The invention describes the novel production of protein-containing hydrogels with which it is possible to render ROS in the wound fluid of chronic wounds harmless. It describes the production of collagenase-containing and trypsin-containing hydrogels for treating necrotic deposits covering wounds, and the production of lysozyme-containing hydrogels which can be employed for controlling bacteria which have infected wounds. The feasibility of incorporating biologically active substances such as, for example, antibiotics, growth factors and other active ingredients into the hydrogels is also described.
The present invention makes use of the reaction of, in particular, &agr;,&ohgr;-diisocyanato-polyethylene glycols with protein to form a protein-containing hydrogel in an aqueous medium. In this case, the crosslinking reaction of the isocyanate with the amino groups of the protein is a reaction which is preferred owing to the higher nucleophilicity by comparison with hydrolysis of the isocyanate to the amine with formation of carbon dioxide. The reaction represents one possibility for the three-dimensional crosslinking of polyethylene glycol and proteins without the occurrence of cleavage products resulting from the activated group. It is furthermore possible to form stable hydrogels with oligomeric proteins such as, for example, SOD and catalase.
Polyethylene glycols (PEGs) belong to the class of polyalkylene glycols which are polyethers of the general formula (Römpp Lexikon Chemie—Version 1.3, Stuttgart/New York: Georg Thieme Verlag 1997):
H&Brketopenst;O—CH
2
—CH
2
&Brketclosest;
n
OH
Polyethylene glycols (PEGS) are produced industrially by base-catalysed polyaddition of ethylene oxide (oxirane) in systems which usually contain small amounts of water with ethylene glycol as starter molecule. They have molecular weights in the range of about 200-5 000 000 g/mol, corresponding to degrees of polymerization n of about 5 to >100 000. In the wider sense, products with n=2−4 (di-, tri- and tetraethylene glycol) are also included in the PEGs; they can be produced mol. uniformly, whereas the PEGs with higher molecular weights are polymol, that is to say consist of populations of macromolecules with different molecular weights.
Liquid products with molecular weights <about 25 000 g/mol are referred to as proper polyethylene g
Ettner Norbert
Meier Wolfgang
Sauer Marc
Schink Michael
Schreiber Jörg
Beiersdorf AG
Monshipouri Maryam
Norris McLaughlin & Marcus PA
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