Title:  Wound healing compositions and methods using tropoelastin and lysyl oxidase

United States Patent:  6,808,707

Issued: October 26, 2004

Inventors:  Ensley; Burt D. (Newtown, PA)

Assignee:  Matrix Design (New York, NY)

Appl. No.:  498305

Filed:  February 4, 2000

Abstract

The present invention provides compositions and methods for promoting wound healing. The composition comprises virgin monomers of tropoelastin and lysyl oxidase. When the lysyl oxidase comes in contact with the tropoelastin, cross-linking of the tropoelastin monomers will occur to form elastin. Contacting the tropoelastin and lysyl oxidase together and applying the mixture to a wound before substantial cross-linking has occurred promotes wound healing by holding the damaged tissue together, increasing the rate of healing, and decreasing the amount of scarring.

SUMMARY OF THE INVENTION

The present invention provides compositions and methods useful in the promotion of wound healing. These compositions comprise virgin monomers of tropoelastin and the cross-linking enzyme, lysyl oxidase. The method comprises mixing these two components of the composition together and applying them to a wound before substantial cross-linking has occurred. The tropoelastin monomers and lysyl oxidase only come in contact with each other immediately before application to the wound or during application to the wound.

Without wishing to be bound by any particular theory, we propose that the lysyl oxidase catalyzes the oxidative deamination of the lysine residues of the tropoelastin monomers at the site of the wound. Then in a non-enzymatic step, cross-links form between the tropoelastin monomers as well as between tropoelastin monomers and other proteins of the extracellular matrix such as collagen. The cross-linked elastin at the site of injury helps to hold the injured tissue together and thereby promotes healing. The elastin is also chemotactic for fibroblasts, endothelial cells, and inflammatory cells, thereby promoting healing in another manner. Elastin at the site of injury also helps to lessen scarring since scar tissue is devoid of elastin, and elastin is an important component of uninjured skin. The cross-linked elastin also generally provides a favorable environment for the cells that participate in the healing process.

In preferred embodiments of the invention, one or both of the tropoelastin monomers and lysyl oxidase are made recombinantly and purified to homogeneity using standard techniques. The purified tropoelastin and lysyl oxidase may then be suspended in a liquid, such as an aqueous solution (e.g., water or saline) or an organic solvent, or provided in a dry powder form, or in a lyophilized form. These two components, tropoelastin and lysyl oxidase, are kept separate from each other until right before use. In another embodiment, the lysyl oxidase is kept in an inactive from in the presence of the tropoelastin, and the lysyl oxidase not activated until right before use. In yet another embodiment, the two components are applied to the wound separately.

In the method of the present invention, the composition may be applied only once at the time of the injury or more than once over the course of wound healing. The composition of tropoelastin and lysyl oxidase may also be used in conjunction with sutures, staples, or adhesive strips in closing the wound.

The composition of the present invention may also be used in promoting the healing of wounds involving structures with elastic fibers such as arteries, lung tissue, or skin. In particular, the composition may be used in surgeries involving arteries, lungs, or the skin.

In another embodiment of the present invention, the tropoelastin comprises only portions of the tropoelastin protein, preferably containing at least one cross-linking domain. In another embodiment, the lysyl oxidase comprises only an active portion of the enzyme.

The preferred compositions of the present invention are biocompatible, non-toxic, and non-immunogenic, and potentially inflammatory or immunogenic reaction products at the site of administration are avoided.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

As described above, the present invention provides compositions and methods for promoting wound healing. These compositions comprise tropoelastin and lysyl oxidase. The method of the present invention involves contacting these two components together and applying the mixture to a wound before substantial cross-linking of the tropoelastin has taken place. In another embodiment, the tropoelastin and lysyl oxidase are applied separately to the wound. A key aspect of the invention is that the tropoelastin must be applied to the wound before substantial cross-linking of the tropoelastin has occurred. To those skilled in the art, a variety of ways of applying the two substances wherein the tropoelastin is not substantially cross-linked will be clear. With the cross-linking reaction taking place at the wound site, cross-links will form that will hold the tissue together. Also, the new formation of elastin will attract fibroblasts, inflammatory cells, and endothelial cell by chemotaxis, will result in less scarring, and will provide a suitable environment for the cells involved in the healing process.

Elastin

Elastic fibers in the extracellular space of the lungs, dermis, and large blood vessels contribute to the elasticity and resilience of these tissues. These elastic fibers may comprise only a small (2-4%) portion of the dry weight of skin but may comprise greater than 50% of the dry weight of large arteries (Rosenbloom et al., FASEB J. 7:1208, 1993). These fibers are composed of amorphous elastin and microfibrils. Microfibrils are a complex of glycoproteins organized as small, 10- to 12-nm-diameter fibrils and serve as the scaffold onto which elastin is assembled. Elastin is the major component (>90%) of the mature elastic fibers and has been well characterized. The elastin protein is initially synthesized as tropoelastin monomers, soluble polypeptides of ca. 72 kDa (Parks et al., Am. J. Respir. Cell Mol. Biol. 2:399, 1990).

The tropoelastin gene exists as a single copy in the genome. The gene has been isolated from several species, including human, bovine, chick, and rat (Bressan et al. "Repeating structure of chick tropoelastin revealed by complementary DNA cloning" Biochemistry 26:1497-1503, 1987; Raju et al. "Primary structures of bovine elastin a, b, and c deduced from the sequences of cDNA clones" J. Biol. Chem. 262:5755-5762, 1987; Indik et al. "Alternative splicing of human elastin mRNA indicated by sequence analysis of cloned genomic and complementary DNA" Proc. Natl. Acad. Sci. USA 84:5680-5684, 1987; Yeh et al. "Structure of the bovine elastin gene and S1 nuclease analysis of alternative splicing of elastin mRNA in the bovine nuchal ligament" Biochemistry 28:2365-2370, 1989; Pierce et al. "Heterogeneity of rat tropoelastin mRNA revealed by cDNA cloning" Biochemistry 29:9677-9683, 1990; each of which is incorporated herein by reference); the entire bovine and human elastin genes have been isolated and sequenced (Indik et al., Proc. Natl. Acad. Sci. USA 84:5680, 1987; Yeh et al., Biochemistry 28:2365, 1989; Bashir et al., J. Biol. Chem. 264:8887, 1989; each of which is incorporated herein by reference). Elucidation of the amino acid sequence indicates alternating segments of cross-link domains and hydrophobic domains. The cross-link domains are characterized by the presence of lysine residues separated by two to three alanine residues, and the hydrophobic domains are found to be rich in hydrophobic residues such as glycine, alanine, valine, and proline. This composition results in hydrophobic interactions which are thought to be responsible for the elasticity of the fibers. The cross-link and hydrophobic domains are encoded by separate exons of the gene.

Extensive alternative splicing of the primary tropoelastin transcript yields the diversity of tropoelastin isoforms seen at the protein level. The human tropoelastin gene consists of 34 separate exons spanning a total of ca. 45 kB of genomic DNA. Six of these exons have been reported to be subject to alternative splicing (Uitto et al., Biochem. Soc. Transact. 19:824, 1991).

Newly synthesized pre-tropoelastin (containing the signal peptide) undergoes intracellular post-translational modifications including hydroxylation of certain proline residues to form 4-hydroxyproline and removal of the signal peptide. The tropoelastin monomers are then secreted into the extracellular milieu where they assemble into functional fibers (fibrillogenesis) and are cross-linked to form insoluble elastin (Uitto et al., Biochem. Soc. Transact. 19:824-829, 1991).

The assembly of elastic fibers (fibrillogenesis) takes place at unique sites close to the cell membrane, generally in infoldings of the cell surface. Microfibrils are the first component to appear and are found to be grouped in small bundles near the plasma membrane. Elastin then appears as amorphous material in discrete loci within each microfibrillar bundle. The microfibrils are thought to align the tropoelastin molecules so that the cross-linking regions are juxtaposed. Lysyl oxidase then oxidizes the terminal amino groups on the side chains of the lysine residues of the cross-linking regions. The oxidized side chains then undergo non-enzymatic condensation reactions to form the cross-links. The notion that tropoelastin monomers are secreted from the cell and diffuse onto the surface of growing fibers seems to be inadequate to explain the efficiency of the whole process and the variable forms of elastic fibers in different tissues. Rather, increasing evidence has supported the idea that helper proteins are needed in the secretion and fiber assembly steps. Given that tropoelastin monomers produced in organisms are rapidly incorporated into insoluble elastin fibers, purification of tropoelastin from anything other than recombinant sources is not practical. Thus, in accordance with the present invention, virgin tropoelastin monomers may be provided by any available method, including, for example, chemical synthesis and standard recombinant techniques known in the art.

In one preferred embodiment, the tropoelastin monomers are synthesized using available chemical synthetic methods. For example, tropoelastin monomers can be synthesized using an appropriate solid state synthetic procedure (Steward et al., Solid Phase Synthesis, Freemantle, San Francisco, Calif., 1968). A preferred method is the Merrifield process (Merrifield, Recent Prog. Hormone Res. 23:451, 1967).

Alternatively, tropoelastin monomers may be prepared by recombinant techniques. Techniques for the overexpression and purification of recombinant proteins in a wide variety of cell types are well known in the art ("Gene Expression Technology," Methods in Enzymology, vol. 185 (D. V. Goeddel, ed.), Academic Press Inc., 1990; incorporated herein by reference). Any such techniques can be employed in accordance with the present invention. Since tropoelastin monomers are normally secreted from the cell, a preferred way to produce the monomers for the present invention would be to introduce the gene with the signal sequence intact and under the control of a strong promoter into a cell and grow the recombinant cells in culture. Preferably, the recombinant cells are eukaryotic (i.e., Saccharomyces cerevisiae, Pichia pastoris), more preferably the cells are of mammalian origin (i.e., COS cell line, CHO cell line), and most preferably the cells are of a human origin. In certain preferred embodiments of the invention, the recombinant cells are derived from human fibroblasts. In another preferred embodiment, the recombinant cells are recombinant bacteria (e.g., E. coli). The desired tropoelastin monomers would be secreted into the media, and the protein could be purified to homogeneity from the media. To improve the ease at which the protein could be purified, the cells expressing the tropoelastin would be grown in serum-free media, thereby minimizing the amount of protein in the media to start with. Production of the tropoelastin monomers in mammalian cells such as fibroblasts would insure correct folding and post-translational modification of the protein. This could be important since some of the proline residues in tropoelastin are hydroxylated posttranslationally.

Once the recombinant tropoelastin of the present invention is expressed, the protein may be isolated and purified by standard methods including chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), centrifugation, or differential solubility, or by any other available technique for the purification of proteins (Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; incorporated herein by reference). For immunoaffinity chromatography in particular, the protein may be isolated by binding it to an affinity column comprising antibodies that were raised against that protein and were affixed to a stationary support. Alternatively, affinity tags such as an influenza coat sequence, poly-histidine, or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow for easy purification by passage over the appropriate affinity column.

Any method of producing tropoelastin known in the art can be used as long as the tropoelastin is able to be cross-linked to form elastin and does not initiate negative (e.g., immune reactions) reactions once introduced into or applied onto the recipient's body.

If particular isoforms of tropoelastin are needed, the appropriate cDNA(s) can be introduced into the cells for overexpression. Different isoforms of tropoelastin are found in differenct individuals within a species, in different locations within one individual, and in different tissues within an individual. In preferred embodiments of the present invention, a certain isoform of tropoelastin or mixture of isoforms is used for a particular individual being treated, a particular type of wound, or a particular type of tissue. For example, it may be desirable to provide a composition comprising multiple different tropoelastin isoforms, whose identity and/or relative quantity are selected to match as closely as possible the profile of isoforms naturally produced by the individual to whom the composition is being applied, at the site of application. If the natural isoform distribution in that individual (or a representative thereof, e.g., another member, or an averaged collection of members, of the same species) and/or at that site, is not known in advance it may readily be determined by one of ordinary skill in the art using standard techniques such as, for example, gene sequencing, restriction fragment length polymorphisms, Western blotting, immunoassay, Northern blotting, Southern Blotting, isoelectric focusing, SDS-polyacrylamide gel electrophoresis, etc. In a preferred embodiment, the individual isoforms of tropoelastin might be prepared separately and mixed at specific ratios later to simulate the ratio of isoforms in a specific tissue of an individual. The use of tropoelastin isoforms native to the tissue would minimize any immune reactions.

In another preferred embodiment of the invention, a modified version of tropoelastin is provided which is not susceptible to cross-linking. The tropoelastin may not be susceptible to cross-linking for a number of reasons including, for example, its binding another protein or peptide, or its not being folded correctly. In order for the tropoelastin to be cross-linked, the binding protein or peptide would need to be removed, or the tropoelastin would need to be refolded. The binding peptide or protein may be removed by competing off the peptide or protein; cleavage of the binding peptide or protein; conformational change of one of the proteins; etc. If the tropoelastin is not folded correctly, a protein from the family of chaperonins (e.g., GroEL, GroES) or heat shock proteins (e.g., Hsp60, Hsp40, Hsp70) may be added to re-fold the tropoelastin so that it may be cross-linked. In this embodiment, the virgin tropoelastin monomers and modified lysyl oxidase can be stored together without the risk of cross-linking the tropoelastin monomers.

Lysyl Oxidase

The high degree of cross-linking found in the elastic fibers contributes to their proper function. Lysyl oxidase is the enzyme that catalyzes the oxidative deamination of lysine residues leading to the non-enzymatic condensation of the modified lysine side chains. This same enzyme is involved in collagen cross-link formation as well. All but about 5 of the 34 lysine residues of tropoelastin participate in some form of cross-link resulting in a highly insoluble polymer (Rosenbloom et al., FASEB J. 7:1208, 1993).

Lysyl oxidase is an extracellular, copper-requiring enzyme which has been purified to homogeneity from several animal sources and found to have a molecular weight of ca. 30 kDa (Stassen, Biochim. Biophys. Acta 438:49, 1976; Siegel, Int. Rev. Connect. Tissue Res. 8:73, 1979; Sullivan et al., J. Biol. Chem. 257:13520, 1982; Kagan, Biology of Extracellular Matrix 1:321, 1986; Kuivaniemi et al., J. Biol. Chem. 259:6996, 1984). The genes encoding lysyl oxidase have been cloned from human and rat cDNA libraries (Hamalainen et al., Genomics 11:508 (1991); Trackman et al., Biochemistry 29:4863 (1990)).

Oxidation of lysine residues in tropoelastin leads to the formation of .alpha.-aminoadipic-.delta.-semialdehyde (AAS). Reaction of a lysine residue with AAS leads to the formation of a dehydrolysinonorleucine cross-link. Reaction of two AAS residues leads to an Aldol condensation product. Tetrafunctional cross-linkages can be formed in elastin from three peptidyl aldehydes (AAS) and one unmodified lysine residue (FIG. 1) (Kagan, Path. Res. Pract. 190:910, 1994; incorporated herein by reference).

For the purposes of the present invention, lysyl oxidase can be produced by any available method, including, for example, chemical synthesis, recombinant methods, and purification from a natural source. In a preferred embodiment, lysyl oxidase is produced recombinantly in much the same manner as the tropoelastin (discussed supra). Again, lysyl oxidase is normally secreted so a preferred method of producing and purifying the enzyme would be to secrete it into the media and then purify the protein from the media. If a modified version (i.e., change in amino acid sequence) was needed, the appropriate cDNA could be introduced into the cell to produce the desired mutant protein. Standard techniques for these procedures are known in the art.

In a preferred embodiment of the invention, a modified version of the lysyl oxidase is produced which is inactive initially and can subsequently be converted into an active form. This particular embodiment of the invention allows the tropoelastin and inactive lysyl oxidase to be stored together. Then, prior to application to a wound, the inactive lysyl oxidase is converted to the active form by cleavage of the protein or by a change in pH, temperature, salt concentration, metal ion concentration, etc. The conversion from the inactive form to the active form might even take place at the site of the wound and be caused by a protease or change in pH at the wound site per se.

In another preferred embodiment of the invention, a modified version of lysyl oxidase would be provided which binds to another protein or peptide (binding peptide or protein). The binding of this binding protein or peptide to the modified lysyl oxidase would lead to inactivation of the lysyl oxidase enzyme. In order for the lysyl oxidase to regain its enzymatic activity, the binding protein or peptide would need to be removed. Examples of this approach include competing off the modified lysyl oxidase using another peptide or protein, a small organic molecule, a metal, nucleic acid, polysaccharide, etc.; cleavage of the binding peptide or protein; conformational change of modified lysyl oxidase; and conformational change of the binding protein or peptide.

In a particulary preferred embodiment, a protein from the family of chaperonins (e.g., GroEL, GroES) or heat shock proteins (e.g., Hsp60, Hsp40, Hsp70) may be used as the binding protein, and the lysyl oxidase may be modified by techniques well known in the art to include a binding domain specific for a chaperonin or heat shock protein (Ranson et al, "Chaperonins" Biochem. J. 333 (Pt. 2):233-242, Jul. 15, 1998; Fink et al., "Chaperone-mediated protein folding" Physiol. Rev. 79(2):425-449, April 1999; each of which is incorporated herein by reference). In this way, the virgin tropoelastin monomers and modified lysyl oxidase bound to a chaperonin or heat shock protein can be stored together without the risk of cross-linking the tropoelastin monomers. After the tropoelastin and lysyl oxidase are applied to a wound or immediately before they are applied to a wound, an agent such as a small molecule (e.g., estrogen) is added to restore lysyl oxidase activity and begin the cross-linking of tropoelastin (Bohen et al., "Hold 'em and fold 'em: chaperones and signal transduction" Science 268(5125):1303-1304, Jun. 2, 1995; incorporated herein by reference).

Packaging

In one preferred embodiment of the invention, the tropoelastin monomers are be provided in one vial, and the lysyl oxidase are provided in another vial. These proteins might be provided in a dry powder form, in solid form (i.e., lyophilized), in solution, or in suspension. To the proteins may have been added emulsifiers, salts, preservatives, other proteins, nucleic acids, protease inhibitors, antibiotics, perfumes, polysaccharides, adhesive agents, polymers, microfibrils, oils, etc.

In another preferred embodiment, the tropoelastin or the lysyl oxidase, or both, is encapsulated in a biodegradable polymer so that when the composition is applied to a wound, the polymer degrades and the tropoelastin and/or lysyl oxidase is released; the released tropoelastin can then be crosslinked.

Biodegradable polymers are usually based on functional groups such as esters, anhydrides, orthoesters, and amides. Rapidly biodegradable polymers include poly[lactide-co-glycolide], polyanhydrides, and polyorthoesters. Preferred bioerodible polymers include polylactides, polyglycolides, and copolymers thereof, poly(ethylene terephthalate), poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), poly(lactide-co-glycolide), polyanhydrides, polyphosphazenes, poly(.epsilon.-caprolactone), poly(dioxanone), poly(hydroxybutyrate), poly(hydroxyvalerate), polyorthoesters, blends, and copolymers thereof. Examples of biodegradable and biocompatible polymers of acrylic and methacrylic acids or esters include poly(methyl methacrylate), poly(ethyl methacrylate), poly(butyl methacrylate), poly(isobutyl methacrylate), poly(hexyl methacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), etc. Other polymers which can be used in the present invention include polyalkylenes such as polyethylene and polypropylene; polyarylalkylenes such as polystyrene; poly(alkylene glycols) such as poly(ethylene glycol); poly(alkylene oxides) such as poly(ethylene oxide); and poly(alkylene terephthalates) such as poly(ethylene terephthalate). Additionally, polyvinyl polymers can be used which include polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, and polyvinyl halides. Exemplary polyvinyl polymers include poly(vinyl acetate), polyvinyl phenol, and polyvinylpyrrolidone. Mixtures of two or more of the above polymers could also be used in the present invention.

Some polymeric materials are known to release entrapped compounds upon exposure to a stimulus such as a change in pH or temperature. An examples of microparticles that release as a function of a change in pH include the diketopiperazine particles describes in U.S. Pat. No. 5,352,461 issued on Oct. 4, 1994, to Steiner et al., and the proteinoid formulations described in U.S. Patent Reissue no. 35,862, issued on Jul. 28, 1998.

Application

Methods for using the composition include, for example, mixing the two separate components together immediately before application to the wound (i.e., before substantial cross-linking has occurred). Other preferred embodiments include applying either the tropoelastin or lysyl oxidase and then the other one to the wound site. One particularly preferred embodiment involves application of a composition comprising lysyl oxidase and tropoelastin to a wound and then use of sutures, staples, adhesive strips, or tissue adhesive to hold the tissue together during the healing process.

Another particularly preferred embodiment involves application of the composition more than once during the healing process. Preferred regiments for applying the lysyl oxidase and tropoelastin include several times a day, once a day, once a week, twice a week, once a month, and twice a month. In following each of these regiments, the agents would be mixed immediately before application, or they would be applied separately to the wound.

The lysyl oxidase and tropoelastin are delivered to the wound site using any means to apply a liquid, paste, gel, or solid (e.g., powder). These means include, for example, a brush, a syringe, a spatula, and a container specifically designed to delivery the agents such as a tube with a narrow tip.

In another preferred embodiment of the invention, the composition is applied to a wound resulting from deep trauma or surgery involving the lungs, large arteries, or other tissues with significant amounts of elastic fibers.

Other Embodiments

The foregoing has been a description of certain non-limiting preferred embodiments of the invention. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

Claim 1 of 10 Claims

What is claimed is:

1. A method for promoting healing of a skin wound comprising steps of:

providing isolated tropoelastin wherein the tropoelastin has not previously been crosslinked and is therefore available for crosslinking, and isolated lysyl oxidase, which components are kept separated from each other; and

applying both said tropoelastin and said lysyl oxidase to a skin wound simultaneously or sequentially, so that said tropoelastin is not crosslinked to itself prior to application to the wound.

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