Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Web – sheet or filament bases; compositions of bandages; or...
Reexamination Certificate
2000-11-15
2003-01-21
Page, Thurman K. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Web, sheet or filament bases; compositions of bandages; or...
C424S400000, C424S402000, C424S422000, C424S423000, C424S444000, C424S445000, C424S449000, C424S486000, C514S781000, C514S772400, C514S772600, C514S777000
Reexamination Certificate
active
06509031
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of crosslinking proteins, such as collagen, using peroxidase enzymes, such as horseradish peroxidase, in situ, to form biocompatible crosslinked semi-solid gels useful in a number of in vivo and in vitro applications, from wound healing, to drug delivery, to food processing.
BACKGROUND OF THE INVENTION
Various collagen powders, sponges or other artificial constructs including “artificial tissue” or organs for medical uses have been described in the literature and in patents. All of these materials have been prepared ex situ, crosslinked or not, for later application to wounds. The same may be said for the use of collagen for augmentation of processed foods (meats, poultry). The inventors are not aware of any currently available products using collagen, or collagen used in combination with other co-reactants or additives, that can be prepared as a solution and applied by injection or pouring of a liquid at the site of interest (wound tissue or chopped meat or poultry), where covalent polymerization and bonding to the site is accomplished in situ. The closest known such wound sealants are polymers of purified fibrinogen (Sierra, 1993), crosslinked by addition of thrombin, and cyanoacrylate cements. The former represent the natural plasma proteins, in purified form, involved in clot formation. For human use they must be prepared from human plasma due to immunological considerations, and are used primarily in Europe and Asia (e.g., Japan), but have not been approved by the FDA (as of 1996) for U.S. use. Cyanoacrylate cements are more commonly known, for household use, as ‘superglue’. Both the fibrin and cyanoacrylate wound sealants are used primarily as alternatives to sutures (i.e., to seal surgical wounds), and have the disadvantage of forming such a dense polymer as to be only slowly biodegradable, thus often retarding wound remodeling and repair (Lasa et al., 1993). Neither is suitable for filling of larger wound beds or for use as a delivery vehicle for other components. Minor soft tissue wounds normally are capable of good repair with minimal treatment. More serious injuries (e.g., trauma), recalcitrant wounds (e.g., decubitus ulcers) or tissue repair which is compromised by other factors (e.g., age, diabetes) require assistance to heal. Healing of such wounds often is further complicated by infection, and where repair is finally achieved it occurs via scarring and contracture, usually resulting in some loss of cosmetic appearance and/or function. Attempts to improve repair may be made by mechanical means, where wound strength and barrier function are aided by a bioinert material (e.g., bandage) or by a biologic approach (e.g., resorbable sponges, grafts, “artificial skin”) which encourages regenerative repair. The former materials eventually must be removed, while materials for the latter often are deficient in mechanical strength, elasticity, or availability, are immunogenic or cytotoxic, or are expensive.
A similar adhesive for food use is Fibrimex®, distributed in North America by F.N.A. Foods, Inc. It also is based on fibrinogen-thrombin reaction (materials obtained from bovine plasma) and will react at low temperature (4° C.), but must be mixed very quickly with the meat products being reformed since the reaction proceeds to completion in less than 30 minutes.
SUMMARY OF THE INVENTION
We have demonstrated the feasibility of a new collagen-based wound sealant which can be gently but covalently crosslinked to a wound bed via a catalyzed chemical reaction utilizing peroxidase enzyme (primarily, horseradish peroxidase) and hydrogen peroxide (H
2
O
2
). Unlike other collagen compositions that are prepared ex situ, this material is prepared as a thick liquid that is poured or injected into the site of interest immediately upon activation by the catalyst, whereupon it polymerizes in situ. Good mechanical strength and elasticity by the polymer is achieved, cellular compatibility and faster healing is demonstrated, and the sealant can be prepared at relatively low cost. In addition, the base material could be modified by inclusion of other matrix components, co-reactants, non-reactive materials, growth factors, antibiotics and microbeads (e.g., of potential use for delayed-release of additional components). This material is particularly useful for emergency wound repair (e.g., use ‘in the field’), for repair of recalcitrant wounds (e.g., decubitus or pressure ulcers), and long-term regenerative type repair of a wound. Additional uses for this material include a slow release depot for vaccines, adjuvants or drugs, bone repair, graft or prosthetic implant stabilization, and as a binding agent for restructured foods (e.g., sausages, ‘poultry rolls’, restructured meats). This material thus represents a new product and composition of matter as well as a potential improvement of existing products and compositions currently available.
This material has use throughout veterinary medical, human medical and dental practice, and the food industry.
Collagen may be crosslinked by a variety of chemical or physical means (for reviews, see Rault et al., 1996; Jain, 1992; Meade and Silver, 1990) and the products subsequently used (crosslinked or not) in various wound repair materials (Pachence, 1996; Choucair and Phillips, 1996; Jeter and Tintle, 1991). While these products may have acceptable mechanical strength, antigenicity and permeability to cellular ingrowth and remodeling, none of the crosslinking methods are compatible with living cells and therefore cannot be employed in situ. Crosslinking of collagen solutions into gels using lactoperoxidase (Tenovuo and Paunio, 1979) or horseradish peroxidase ( LaBella et al., 1968) has been demonstrated and the formation of small amounts of dityrosine reported, however, no use for these collagen gels was demonstrated or suggested.
We have explored the use of peroxide-peroxidase catalyzed crosslinking of collagen with the aim of developing one or more compositions or materials that may be used by the medical, pharmaceutical or food industries. The following are embodiments of the present invention.
One embodiment relates to a method of covalent crosslinking of acid-soluble Type I collagen from calf skin with horseradish peroxidase (HRP)-hydrogen peroxide (H
2
O
2
, O
2
, Px) (collagen polymer). Collagen solutions of 8-12 mg/ml can optimally be crosslinked into a semi-solid gel by addition of HRP and peroxide in molar ratios of 4-5:1:50 to 4-5:1:200 collagen:HRP:peroxide (molecular weight basis, e.g., moles of each component). Controls lacking either HRP or peroxide failed to form a gel. Greater ratios of collagen relative to HRP (e.g., 20:1:200) or substantially higher concentrations of peroxide (>400 parts) did not provide optimal polymerization.
That a crosslinked polymer was being formed was confirmed by decreased solubility of the matrices in hot (75° C.) sodium dodecyl sulfate (SDS) solution, and an increase in high molecular weight components observed by SDS agarose gel filtration and SDS-polyacrylamide gel electrophoresis with a concomitant decrease in lower molecular weight components (monomers).
The matrix was found to have significant mechanical strength and elasticity by Instron compression analysis and deformation testing, and was superior in these respects to glutaraldehyde crosslinked positive control preparations. Uncrosslinked materials had no demonstrable mechanical strength as measured by either method.
Other embodiments relate to the crosslinking of interstitial collagen types II, III and of type IV collagen (basement membrane type) in the presence of HRP: peroxide, therefore the method is useful for both interstitial and basement membrane collagens.
One embodiment relates to the solidification (crosslinking) of solutions of type I collagen using soybean peroxidase (Sigma®) and microbial peroxidase (Sigma®, from
Arthromyces ramosus
), although to a lesser apparent degree than with HRP. The method thus appears to be generally applicable to the use of any peroxidase.
Other
Keeton Jimmy T.
Miller Douglas R.
Prochaska Jerry F.
Tizard Ian R.
Board of Regents, Texas A&M University System
Evans Charesse
Fulbright & Jaworski L.L.P.
Page Thurman K.
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