NZ752999B2 - Peptides derived from fibronectin with improved bioactivity and reduced susceptibility to neutrophil elastase degradation - Google Patents

Abstract

Polypeptides derived from fibronectin are presented that are neutrophil elastase-resistant and can bind to growth factors and/or enhance growth factor activity. These polypeptides are useful for enhancing wound healing in a patient.

Description

WO 71709 Peptides Derived from Fibronectin with Improved Bioactivity and Reduced Susceptibility to Neutrophil Elastase ation GO VERNMENT SUPPOR T This invention was made with government t d by US Army AFIRM 1 and JWMRP SI4 under grant numbers W81XWH2-0034 and W81XWHC-0043, tively. The government has certain rights in the invention.

TECHNICAL FIELD This invention is based on the discovery of polypeptides derived from fibronectin that are neutrophil elastase-resistant and can bind to growth factors and/or enhance growth factor activity. Previously known polypeptides d from fibronectin with the y to enhance growth factor activity are found to be susceptible to ation by human neutrophil elastase, reducing their effectiveness in in vivo uses. This invention s to novel growth factor enhancing polypeptides with increased resistance to degradation by neutrophil elastase. This invention also relates to the uses of such compounds in cosmetic treatments and the treatment of wounds and cancer.

BACKGROUND In the US civilian population, each year, approximately 500,000 patients with bums present to emergency departments. Of 40,000 annual hospital admissions, 25,000 bum victims are ed to specialized bum centers. Progressive extension of bums can have a devastating effect. Over the course of a few days to one week deep partial-thickness bums can become full-thickness bums, which in the short term, leads to increased tissue loss, longer healing time, excess morbidity and mortality. In the long term, increased scarring, wound contractures and poor quality of life become major issues. While the exact mechanism(s) leading to conversion of the zone of ischemia to full-blown necrosis is unclear, several processes, including oxidant and cytokine stress resulting from mation as well as ischemia/reperfusion, probably play a role. Therapies to improve blood flow, such as non-steroidal antiinflammatory agents (NSAIDS) and anti-coagulants (heparin) have not shown substantial benefit in preventing bum injury progression. Hence therapy to limit bum injury progression is an unmet need.

There is ce that fibronectin (FN) is involved in many biological processes including tissue repair, embryogenesis, blood clotting, cell ion, wound repair, and cell adhesion. There are two primary forms of fibronectin. The first is an insoluble glycoprotein dimer that serves as a linker in the extracellular matrix (ECM), and the second is a soluble disulfide-linked dimer found in plasma. The ECM form of fibronectin is sed by lasts, chondrocytes, endothelial cells, macrophages and certain lial cells. The plasma form of fibronectin is expressed by hepatocytes. Fibronectin can serve as a general cell adhesion molecule by anchoring cells to collagen or to proteoglycan substrates.

Fibronectin can also play a role in organizing cellular ctions by binding to ents of the ECM and to membrane-bound fibronectin receptors on cell surfaces. Forms of fibronectin are found in vertebrates, including mammals, birds, amphibians, fish, and reptiles.

FN, a 500 kDa rotein, circulates in the blood and is ed and deposited by tissue cells in the provisional extracellular matrix (ECM) during tissue formation. As a al component of the provisional ECM, FN plays a vital role in embryogenesis, morphogenesis and wound healing but is deficient in bum patients’ wounds and blood. FN is known to be degraded in bum wound fluids by the endopeptidase neutrophil elastase. See Grinnell, et al, Identification of Neutrophil Elastase as the Proteinase in Bum Wound Fluid Responsible for Degradation of Fibronectin. J Invest ology, 1994, 103(2):155-61.

Previously disclosed peptide "PI2" is 14-residue e that is cryptic within the immunoglobulin sandwich type of P-pleated sheet of fibronectin’s (FN) first type III repeat (FNHIi). Unlike FN, P12 in solution es mesenchymal cell growth, proliferation and migration intrinsically and synergistically with a variety of growth factors, especially platelet-derived growth factor-BB (PDGF-BB).

Furthermore, PI2 protects adult human dermal fibroblasts (AHDF) from cell death induced by oxidative and cytokine stress and/or nutrient withdrawal in the presence of PDGF-BB. P12 also limits bum injury progression in rat and porcine bum models and mitigates scarring in a al bum injury progression pig model. See, e.g., ; US Patent 8,759,300; Lin, et al, Fibronectin peptides that bind PDGF-BB enhance survival of cells and tissue under stress. J Invest Dermatol. 2014 Apr; 134(4): 1119-1127; and Asif et al.. Blood Vessel Occlusion in um Tissue is Secondary to Ervthrocvte Aggregation and Mitigated bv a Fibronectin-derived Peptide that Limits Bum Injury Progression.

Wbund Rep Reg (2016) 24 501-513. In particular, the fragment of fibronectin PSHISKYILRWRPK, or "P12" is disclosed in both Lin, et al. and Asif, et al. as being useful for the treatment of wounds, particularly the treatment of bums.

SUMMARY We present neutrophil elastate-resistant peptides derived from fragments of fibronectin for use in the ent of wounds. In particular, we have discovered that previously disclosed, biologically active, peptide fragments of fibronectin, such as PI 2, are readily degraded, in vitro, by neutrophil elastase, and do not promote healing when applied topically to wounds. Even cyclized forms of the previously disclosed, biologically active, peptide fragments of fibronectin, such as cyclic P12, remain sensitive to this endopeptidase. We present peptides that are fragments and/or tives of fragments of fibronectin that in their biological activity of binding growth factors, such as et-derived growth factor-BB (PDGF-BB) and enhancing fibroblast survival, and also have been modified to increase their resistance to ation by neutrophil elastase, in vitro and in vivo.

In some embodiments of the invention, the phil elastase resistant peptide is a linear or cyclic polypeptide ing to formula I: (I): H-X1-X2-K-Y-X3-X4-R-W-R-P-K-X5-X6-X7 wherein Xi is I or G or L, X2 is S or G, X3 is I or G or L, X4 is L or G, X5 is N or G, X6 is absent or S, and X? is absent or V; and n no two consecutive amino acids in the first 13 amino acids of the polypeptide differ from the sequence LRWRPKN. Further embodiments of the invention include HISKYIFRWRPKNSV (P46), FFGKYGFRWRPKNSV (cNP7), FRWRPKGSV (cNP8), HGSKYGFRWRPKNSV, fflGKYIGRWRPKNSV, HGSKYIGRWRPKNSV, HGSKYIGRWRPKGSV and cyclic forms thereof.

We also present methods of using these peptides for cosmetic treatments and the treatment of wounds and vascular disease. The wounds to be treated include surgical incision or extirpation, a tic injury, a thermal bum, a chemical bum, a lesion or ulceration of the patient's skin, mucosa, connective , fascia, ligament, tendon, cartilage, nerve or muscle and a wound to the patient's bone.

The treated wounds may be infected or uninfected. The cardiovascular incidents to be treated include blood vessels occluded with aggregates of red blood cell and/or fibrinogen and/or fibrin, such as can occur in bum wounds; myocardial infarction; multi-organ failure; diabetes; sickle cell anemia; polycythemia vera; and ibrinogenemia. In a particular embodiment, the neutrophil elastase resistant peptides of the invention are used to treat thermal and/or chemical bums.

The invention also features compositions (e.g., physiologically acceptable compositions) that include a phil elastase resistant peptide of the invention. The physiologically able composition may be a pharmaceutical composition that promotes a therapeutic response. As noted above, cosmetic compositions are also ed and can include the peptides described . The t compositions may also be armaceutical in the sense that they may e concentrated peptides and/or other ingredients that should be diluted or otherwise modified (e.g., mixed with other active or inactive ingredients) prior to use (e.g., in cell culture or as a cosmetic or therapeutic formulation).

BRIEF DESCRIPTION OF THE DRA WINGS is a diagram of the susceptibility of cP12 to digestion by human neutrophil elastase. : cP12 was incubated with purified human neutrophil elastase at 1:200 enzyme:substrate molar ratio, minimal intact peptide (MW=1789) was retained at 24h. FIG. IB: Elastase sensitive sites of cP12 cleaved by human neutrophil elastase and analyzed by MS. shows bioactivity for P12, P45 and P46. Adult human dermal fibroblasts CF31 cells at 1000 cells/well were seeded in a collagen-coated 96-well plate in DMEM overnight, then InM PDGF-BB with peptides at indicated doses was added and cells were incubated at 37°C for 6 days. Then cell metabolism was measured by XTT assay by reading OD at 450 nm. Each point represents the mean of 6 replicates with a l SD = 5-10% around the mean (not shown for graft clarity). shows P46 ion by neutrophil elastase at 4 hours and 24 hours. P46 was incubated with purified human neutrophil se at 1:100 enzyme:substrate molar ratio at 37°C for 4 h (A) or 24 h (B). Peptides stability was determined by MALDI-TOF analysis. Molecular weight of P46 is 1898. shows bioactivity of P12, P46, cNP7 and cNP8. Adult human dermal fibroblasts, CF31, at 1000 cells/well were incubated at 37 °C in a collagen-coated l plate with DMEM, InM PDGF-BB with peptides at indicated doses for 6 days and then cell metabolism was measured by XTT assay, reading OD at 450 nm. Data represents the mean of 6 replicates. shows that cNP8, derived from P46, is resistant to ion by neutrophil elastase. cNP8 was incubated with purified human neutrophil se at 1:100 enzyme:substrate molar ratio at 37°C for 4 h (A) or 24 h (B). Peptides' stability was determined by MALDI-TOF analysis. Molecular weight of cNP8 is 1736. Intact peptide 36) was retained at 24h. shows a comparison of effectiveness of cP12 and cNP8 peptide for promoting re-epithelialization of bum wounds with topical treatment in a porcine model. shows the increase of re-epithelialization on day 10 at the wound site with intravenous cNP8 treatment in a porcine bum model. shows the increase of re-epithelialization on day 14 at the wound site with intravenous cNP8 treatment in a porcine bum model.

DETAILED DESCRIPTION The details of one or more embodiments of the ion are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and gs, and from the claims.

Determination of peptides derived from fibronectin with improved ivity and resistance to neutrophil elastase digestion: usly disclosed e P12 is degraded by neutrophil elastase, which is present in wound fluids: To determine whether cyclic P12 ("cP12") is ive to endopeptidases, cP12 was digested with human neutrophil elastase. The reaction was stopped with formic acid and the reaction mixture was analyzed by mass spectroscopy. Analysis of results showed that cP12 has five se-cleavage sites (Figure IB). In addition, almost all intact cP12 substrate (1789mw) was eliminated (Figure 1A).

Determination of fragments created by digestion of fibronectin with human neutrophil elastase, and their bioactivity: To find an elastase-resistant cPl2-derivative with cP12-equivalent ivity, we digested fibronectin nii-C (FNnii-C), in which PI 2 e is found, with purified human neutrophil elastase at 1:100 enzyme:substrate molar ratio at 37°C for 4 h or24h. Digested samples were analyzed with MS. The results at both time points showed that elastase digestion of FN IIIi-C produced two peptides, P45 (SKYILRWRPKNSV) and P46 (fflSKYILRWRPKNSV) from the same region of fibronectin as PI 2.

Bioactivity assay demonstrated that P46 showed higher bioactivity than P12 as determined by fibroblast metabolism assay (Figure 2). On the other hand, P45 showed little bioactivity (Figure 2).

Determination of sensitivity of P46 to human neutrophil elastase digestion: To determine the sensitivity of P46 to elastase digestion, P46 was synthesized and digested with elastase. The MS analysis results showed that P46 retained some sensitivity to se digestion (Figure 3). P46 was incubated with purified human neutrophil elastase at 1:100 enzyme:substrate molar ratio at 37°C for 4 h (Figure 3A) or 24 h (Figure 3B). The elastase-generated smaller peptides were ined by MALDI-TOF analysis. Molecular weight of P46 is 1898. cNP7 and cNP8 as biologically active, neutrophil elastase-resistant es: Based on the amino acid sequence of P46 and enzymatic cleavage properties of elastase, we designed and synthesized five engineered peptides, -HIGKYGLRWRPKNSV- (NP7) and GLRWRPKGSV- (NP8), fflSKYILGWRPKNSV (NP9), fflSKYILRGRPKNSV (NP10), HISKYILRWGPKNSV (NP11), and for resistance to elastase ion. Of these, NP8 demonstrated the best biological activity on screening experiment of adult human dermal fibroblast survival in medium without serum but with InM PDGF-BB after 6 days of incubation. NP9, NP10 and NP11 showed minimal ical activity in this screen.

PI 2, P46, cNP7 and cNP8 were compared for their ability to e the al of adult human dermal fibroblasts in the presence of PDGF-BB. Adult human dermal fibroblasts at 1000 cells/well were ted in a collagen-coated 96-well plate with DMEM, InM PDGF-BB with peptides at indicated doses for 6 days and then cell metabolism was measured by XTT assay. Data represents the mean of 6 replicates (Figure 4). This bioactivity assay demonstrated all four peptides promoted the al of adult human dermal fibroblasts and that cNP8 showed equivalent or better bioactivity, depending on the concentration, compared to P46 and cNP7.

Results from peptide incubation with elastase showed that cNP8 was resistant to elastase digestion. cNP8, derived from P46, was incubated with purified human neutrophil elastase at 1:100 enzyme: substrate molar ratio at 37°C for 4 h (Figure 5A) or 24 h (Figure 5B). Peptide's stability was determined by MALDI-TOF analysis. Molecular weight of cNP8 is 1736. A large majority of intact peptide was retained at 24h. Thus, cNP8 is a peptide with both bioactivity and ant to elastase digestion. p46 and cNP8 engineered fromP46 have the ability to bind growth factors: Previous studies showed that fibronectin-derived bioactive peptide PI2 interacts with platelet-derived growth factor-BB and enhances its activity to support fibroblasts al. We showed, above, that both fibronectin-derived peptide P46 and elastase-resistant engineered peptide cNP8 demonstrated much higher ivity than that of PI 2. To study the binding activities of these peptides, real time ctions of PI 2, P46, and cNP8 with growth factors were determined by plasmon surface resonance (Biacore T200). The results demonstrated that both P46 and cNP8 showed r binding activity as P12. They bind PDGF-BB and Transforming Growth Factor-Betal (TGF-pi), but did not bind to epidermal growth factor (EGF) and insulin-like growth factor-1 ).

Surface Plasmon Resonance: In the Biacore2000 system, binding nts from c data are determined by passing varying concentrations of FN peptides (analytes) over chip surfaces which are d with PDGF-BB (ligand), respectively. All kinetic experiments are carried out at 20° C. at a flow rate of 20 pl/min. For mass transport experiments, each analyte is injected at a fixed tration and run at flow rates ranging from 5 to 75 pl/min. All analytes are injected over PDGF-BB ligand surfaces as well as over a control surface for 120 s, followed by 300 s of dissociation in running buffer. Regeneration of the sensor chip for subsequent injections is accomplished by pulse of 0.1% SDS. Mass transport experiments have detected little difference in response at different flow rates thus validating data from c experiments. Sensorgrams are prepared and ly fitted using ear least-squares analysis and numerical integration of the differential rate equations with the Biacore Bioevaluation software. Each sensorgram generated using a control surface is subtracted from the corresponding experimental sensorgrams, and the resulting curves are transformed to concentration units using the molecular mass of the injected species, the equivalence of 1000 resonance units (RU) per 1 ng/mm2, and a matrix thickness of 100 nm. Each data set, which consists of a series of sensorgrams from injections of different concentration of e over the same surface, is analyzed using kinetic models from Bioevaluation software.

Novel peptides for use in wound healing: Linear or cyclic peptides according to Formula I: (I): H-Xj -X2-K-Y-X3-X4-R-W-R-P-K-X5-X6-X7 wherein Xi is I or G or L, X2 is S or G, X3 is I or G or L, X4 is L or G, X5 is N or G, X6 is absent or S, and X7 is absent or V; and wherein no two consecutive amino acids in the first 13 amino acids of the polypeptide differ from the sequence fflSKYILRWRPKN, are useful for the treatment of wounds. These peptides promote the survival, migration or growth of human adult dermal fibroblasts and human adult cardiomyocytes, and are resistant to neutrophil elastase, an endopeptidase found in wound .

Each linear or cyclic e selected from: fflSKYILRWRPKNSV, fflSKYILRWRPKNS, fflSKYILRWRPKN, LRWRPKGSV, HISKYILRWRPKGS, HISKYILRWRPKG, HGSKYILRWRPKNSV, HGSKYILRWRPKNS, HGSKYILRWRPKN, HGSKYILRWRPKGSV, HGSKYILRWRPKGS, HGSKYILRWRPKG, HLSKYILRWRPKNSV, HLSKYILRWRPKNS, HLSKYILRWRPKN, LRWRPKGSV, HLSKYILRWRPKGS, HLSKYILRWRPKG, fflGKYILRWRPKNSV, LRWRPKNS, HIGKYILRWRPKN, fflGKYILRWRPKGSV, fflGKYILRWRPKGS, fflGKYILRWRPKG, LRWRPKNSV, HISKYGLRWRPKNS, HISKYGLRWRPKN, HISKYGLRWRPKGSV, HISKYGLRWRPKGS, HISKYGLRWRPKG, HGSKYGLRWRPKNSV, HGSKYGLRWRPKNS, HGSKYGLRWRPKN, HGSKYGLRWRPKGSV, HGSKY GLRWRPKGS, HGSKY GLRWRPKG, HLSKYGLRWRPKNSV, HLSKY GLRWRPKNS, HLSKY GLRWRPKN, HLSKY GLRWRPKGSV, HLSKY GLRWRPKGS, HLSKY GLRWRPKG, HIGKYGLRWRPKNSV, HIGKYGLRWRPKNS, HIGKYGLRWRPKN, LRWRPKGSV, HIGKY GLRWRPKGS, HIGKY GLRWRPKG, HISKYLLRWRPKNSV, HISKYLLRWRPKNS, HISKYLLRWRPKN, fflSKYLLRWRPKGSV, fflSKYLLRWRPKGS, fflSKYLLRWRPKG, HGSKYLLRWRPKNSV, HGSKYLLRWRPKNS, HGSKYLLRWRPKN, HGSKYLLRWRPKGSV, HGSKYLLRWRPKGS, HGSKYLLRWRPKG, HLSKYLLRWRPKNSV, HLSKYLLRWRPKNS, LRWRPKN, HLSKYLLRWRPKGSV, HLSKYLLRWRPKGS, HLSKYLLRWRPKG, fflGKYLLRWRPKNSV, LLRWRPKNS, HIGKYLLRWRPKN, fflGKYLLRWRPKGSV, fflGKYLLRWRPKGS, fflGKYLLRWRPKG, fflSKYIGRWRPKNSV, IGRWRPKNS, HISKYIGRWRPKN, fflSKYIGRWRPKGSV, fflSKYIGRWRPKGS, fflSKYIGRWRPKG, HGSKYIGRWRPKNSV, HGSKYIGRWRPKNS, GRWRPKN, HGSKYIGRWRPKGSV, HGSKYIGRWRPKGS, HGSKYIGRWRPKG, HLSKYIGRWRPKNSV, HLSKYIGRWRPKNS, HLSKYIGRWRPKN, HLSKYIGRWRPKGSV, GRWRPKGS, HLSKYIGRWRPKG, HIGKYIGRWRPKNSV, HIGKYIGRWRPKNS, GRWRPKN, HIGKYIGRWRPKGSV, HIGKYIGRWRPKGS or HIGKYIGRWRPKG promotes the al, migration or growth of human adult dermal fibroblasts and/or human adult cardiomyocytes, is resistant to neutrophil elastase and is useful for the treatment of wounds and/or cardiovascular e.

In a particular embodiment, each linear or cyclic peptide: HISKYILRWRPKNSV (P46), HIGKYGLRWRPKNSV (NP7), HIGKYGLRWRPKGSV (NP8), HGSKYGLRWRPKNSV, HIGKYIGRWRPKNSV, HGSKYIGRWRPKNSV or HGSKYIGRWRPKGSV promotes the survival, migration or growth of human adult dermal fibroblasts and/or human adult cardiomyocytes, is resistant to neutrophil elastase and is useful for the treatment of wounds and/or cardiovascular disease.

In another particular embodiment, cyclic HIGKYGLRWRPKGSV (NP8) promotes the survival, migration or growth of human adult dermal fibroblasts and/or human adult cardiomyocytes, is resistant to neutrophil elastase and is useful for the treatment of wounds and/or cardiovascular disease.

All peptides described herein are presented in a linear format of rd, single-letter amino acid codes, reading from the N us on the left to the C terminus on the right. c" or "cyclized" peptides may be represented in linear form but have the N terminus amino acid bound to the C terminus amino acid by one or more standard methods, described below.

A biologically active fibronectin fragment or variant of a FN fragment described herein is one that functions as a PDGF-BB binding peptide, is resistant to human neutrophil elastase, and functions to a useful extent and in ntially the same manner as the corresponding FN nt. For example, where a FN fragment having a naturally occurring sequence binds a GF with a particular affinity and, upon administration to a patient, effectively enhances or alters the GF activity to at a site where the GF is needed, a biologically active variant of that FN fragment will be one that, although not identical to the FN fragment, will bind the same GF(s) with sufficiently useful affinity and rly enhances or alters the GF(s) at a site of need. For ease of reading, we do not repeat the term "or a biologically active variant thereof’ after every reference to a FN nt or other protein or peptide. It is to be understood that where FN fragments having a naturally occurring sequence are useful, so are biologically active variants of those fragments.

With respect to function, a fragment can bind a polypeptide growth factor with an affinity of at least or about 1 xlO-7M (e.g., at least 1 x 10-8M; 1x 10-9 M; or more). Alternatively, or in addition, a fragment may support FN-null cell survival and/or proliferation.

Alternatively, or in addition, the fragment can r include a substituent at the amino-terminus or carboxy-terminus. The substituent can be an acyl group or a substituted or un-substituted amine group (e.g., the substituent at the N-terminus can be an acyl group and the C-terminus can be ami dated with a tuted or un-substituted amine group (e.g., an amino group having one, two, or three substituents, which may be the same or different)). The amine group can be a lower alkyl (e.g., an alkyl having 1-4 carbons). The acyl group can be a lower acyl group (e.g., an acyl group having up to four carbon atoms), especially an acetyl group.

The fragments of fibronectin, including the modified nts described above, can be protease resistant and can include one or more types of protecting groups such as an acyl group, an amide group, a benzyl or benzoyl group, or a polyethylene glycol. More ically, a fragment of fibronectin, including the modified fragments described above, can be N-terminally acetylated and/or C-terminally ami dated.

The fragments of fibronectin can also be ed in order to improve absorption, including for example, an addition of sugar residues to enhance transport across the blood-brain barrier.

Any of the fragments can include at least one amino acid residue in the D-form.

Any of the fragments can include at least one non-naturally ing or modified amino acid residue (e.g., 4-hydroxyproline, gamma-carboxyglutamic acid, o-phosphoserine, o-phosphotyrosine, or deltahydroxylysine ). Non-naturally ing amino acid residues are amino acid residues other than the 20 naturally occurring, genetically encoded amino acids. Other examples include ylalanine, which can be tuted for tryptophan to facilitate synthesis, L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, alpha-amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L-alpha-methylalanyl, betaamino acids, and isoquinolyl. Fragments having non-naturally occurring amino acid residues may be referred to as synthetic fragments and constitute one type of variant as described herein. Other variants include fragments of fibronectin in which a naturally occurring side chain of an amino acid residue is ed with a turally occurring side chain (in either the L- or D-form). In another aspect, the invention features polypeptides that include a sequence that is reversed with respect to the N- and C- termini of a sequence naturally found in a fibronectin polypeptide or a ically active variant thereof.

Any of the fragments in the present compositions can be one of a plurality of fragments present. These nts may be linked er by methods described herein. As noted, fragments of fibronectin, ing the variant forms described herein, can further include a heterologous ptide (i.e., a polypeptide having a sequence that does not appear in a fibronectin). The heterologous polypeptide can be a polypeptide that increases the circulating half-life, cell penetration or ermal penetration of the fragment to which it is attached.

The fragments can be contained within physiologically acceptable compositions, or they may be contained within compositions that are not suitable for administration to a living being (e.g., concentrated stocks or frozen or lyophilized itions).

The logically acceptable compositions can be pharmaceutical compositions, and methods of treating ts are described further below. The physiologically acceptable compositions can also be non-pharmaceutical compositions or pharmaceutical compositions that can be dispensed without a physician's iption. For example, they can be sold to a distributer or "over the counter" for cosmetic purposes (e.g., to reduce the risk of damage from the skin or to minimize or repair damage to the skin). For example, the fragments of fibronectin and compositions that include them or combinations of them (e.g., a FN-growth factor complex) can be incorporated in topical formulations sold as cosmetics, moisturizers and the like, sunscreens, shampoos or conditioners, soaps or other foaming cleansers, or lip balm.

The invention also encompasses nucleic acid molecules that encode the polypeptides bed herein. ic nucleic acid molecules, vectors (e.g., plasmid vectors or viral vectors), and host cells containing them are described further below, as are physiologically able compositions containing them.

Other compositions of the present invention are tissue engineered products that include a fragment of a fibronectin or a biologically active variant thereof. As in other compositions, the fragment or the t f can bind a polypeptide growth factor or e growth factor activity (as described above and further , which factor may subsequently retain biological activity and may be administered to a patient.

Other itions of the present invention comprise a solid support that is associated with (e.g., bound to or impregnated with) one or more of the fragments of fibronectin, or the biologically active variants thereof, described herein. The support can be, for example, a tissue culture vessel (e.g., a plate or flask) or device (e.g., a medical device such as one used in wound dressing (e.g., a bandage or gauze), wound repair (e.g., a suture or "steri-strip"), surgical repair (e.g., a surgical mesh), or a tissue implant (e.g. a stent). The fragment of fibronectin, or the biologically active variant thereof, can be bound to an active growth factor, including any of those described above.

The methods of the invention include s for promoting wound healing. These methods include a step of administering to a patient a therapeutically effective amount of a pharmaceutical composition comprising a fragment of fibronectin, or a biologically active variant f, as described herein. The fragment of fibronectin, or the biologically active variant thereof, can be present in a complex with one or more growth factors. The methods can optionally include a step of identifying a t in need of treatment. Such patients include patients who are suffering from a al extirpation or incision of the skin, mucosa, underlying connective tissue, fascia, nt, tendon, cartilage, bone, nerve or muscle; ts who are suffering from a traumatic tion or tissue loss of the skin, mucosa, underlying connective tissue, fascia, nerve or muscle; and patients who are suffering from a thermal bum, chemical bum, or ulceration of the skin, mucosa, underlying connective tissue, fascia, nerve or muscle.

As used herein, a "bum" is tissue damage due to exposure to heat or a caustic chemical. A "thermal bum" is tissue damage due to re to heat. A "chemical bum" is tissue damage due to re to a c chemical, often strong alkali or strong acid. Agents of chemical bums to be treated by the peptides defined by the invention include, but are not limited to, phenol, l, mustard gas, phosphoms, nitrogen mustard, arsenic compounds, ammonia, caustic potash, lime, sodium hydroxide, hloric acid, and sulphuric acid.

The methods of the invention include methods for treating cardiovascular disease, including decreasing blood vessel occlusion from aggregates of red blood cell and/or fibrinogen and/or fibrin, as can occur in bum wounds; myocardial infarction; multi-organ failure; diabetes; sickle cell anemia; polycythemia vera; and hyperfibrinogenemia. These methods include a step of administering to a patient a therapeutically effective amount of a pharmaceutical ition sing a fragment of fibronectin, or a biologically active variant thereof, as described herein. The fragment of fibronectin, or the biologically active variant thereof, can be present in a x with one or more growth factors. The methods can ally e a step of identifying a t in need of treatment.

Suitable formulations are described further below and, generally, take the form of a solution, lotion, ointment, gel, cream or salve. The fragments of fibronectin, whether or not complexed with a growth factor, can also be administered by way of their inclusion in an extracellular matrix (ECM; e.g., a natural or engineered ECM), a bandage, dressing, compress, or the like.

By other methods of the invention, one can localize an endogenous growth factor to a tissue of a patient.

These methods can be carried out by administering, to the t, a eutically effective amount of a composition that includes a fragment of fibronectin, or a biologically active variant thereof, as described herein. As in the more specific treatment methods described above, these compositions can be administered by way of l application of a ceutical composition, an engineered ECM, or a solid support. These methods can be described as methods of delivering one or more growth factors to a patient. The methods can optionally include a step of identifying a patient in need of treatment. Such patients e patients who are suffering from an injury to a tissue, a loss of a tissue or a disorder resulting in tissue disfigurement or dysfunction. More specifically, the t can be suffering from an injury or loss to the brain, spinal cord or nerves or a disorder resulting in brain, spinal cord or nerve dysfunction; an injury or loss to the heart or blood vessels or a disorder resulting in heart or blood vessel dysfunction; an injury or loss to the lung, nasopharyngeal tract, sinuses, trachea or airways or a disorder resulting in lung, nasopharyngeal tract, sinus, trachea or airway dysfunction; an injury or loss to the gastrointestinal tract, liver or pancreas or a disorder ing in gastrointestinal tract, liver or pancreas dysfunction; an injury or loss to a kidney, ureters, bladder or urethra or a disorder resulting in kidney, ureter, r or urethra dysfunction; an injury or loss to bone, cartilage, synovium, meniscus, ligament, tendon or nucleus pulposus or a disorder resulting in bone, age, um, meniscus, ligament, tendon or nucleus pulposus dysfunction; an injury or loss to lips, tongue or gums or a disorder resulting in lip, tongue or gum dysfunction; an injury or loss to the subcutaneous tissue or a er resulting in subcutaneous tissue dysfunction.

The ion can also be described in terms of "use," in which case it encompasses "use" of the compositions described herein, including FN fragments, peptide derivatives of FN fragments, complexes containing one or more of FN fragments and/or peptide derivatives of FN fragments, ing those with a bound GF, nucleic acids encoding the present FN fragments and/or e derivatives of FN nts, expression vectors, host cells, and tissue ered products, including those that contain biomaterials, for promoting tissue regeneration and/or tissue repair. For example, the present compositions can be used in promoting wound healing, or for the preparation of a ment for the promotion of tissue regeneration or wound healing. The tissue regeneration or repair may result in healing with little or no scarring, in contradistinction with usual adult wound healing.

As used herein, "growth factor binding peptide" (or "GFBP") and "growth factor enhancing e" (or "GFEP") are used synonymously.

As used herein, "cosmetic treatment" refers to the use of a physiologically acceptable composition to improve or maintain the appearance of an individual.

As detailed above, we have found, inter alia, that specific fragments of fibronectin and peptides derived from fibronectin can bind various growth factors (e.g., IGF-1, HGF, TGF-pi, TGF-P2, bFGF, FGF-7, PDGF-BB, VEGF-A, or NGF), and the bound growth factors can retain or show enhanced/decreased biological activity. The present invention features compositions that include such fragments and peptides, with or without bound growth factors in the represented families (i.e., in the IGF, TGF, FGF, PDGF, VEGF, and NGF families), in various formulations and configurations. The fragments and es may promote y with GFs to which the FN fragments or peptides do not bind. In one configuration, the FN fragments or peptides, or FN fragment or e/GF-containing complexes can be incorporated into engineered two- or three-dimensional extracellular matrices (which we may iate herein as engECM or refer to as synthetic matrices), and these can include any of; or any combination of, the peptides described herein (e.g., a peptide conforming to Formulas I) or biologically active variants thereof. The growth (s) incorporated can be, for example, IGF-1, TGF-pi, , bFGF, FGF-7, PDGF-BB, VEGF-A, or NGF; any combination or sub-combination thereof; or another specific growth factor in the same family as those listed. The growth factors can be exogenously added to the peptide-containing formulation (e.g., a FN fragment-containing matrix), or the formulation (e.g., the matrix) can be generated without growth factors. In the latter case, when placed in the vicinity of an endogenous supply of growth factors, the growth factors can be recruited by the matrix. The matrix can also recruit cells and induce them to entiate, produce tissue or proliferate (presumably by virtue of the ion or tment of growth factors, although the invention is not limited to compositions that on by any particular mechanism).

The matrix can include any type of biomaterial (e.g., a biopolymer). For example, the matrix can be or can include a hydrogel (e.g., an intramolecularly crosslinked hydrogel). The present es and GFs can be orated in or associated with many different types of materials (e.g., hyaluronan). The matrix can have, for example, a polycarbonate backbone, or include biodegradeable polyurethanes.

Further examples of suitable biopolymers are: proteins (e.g., collagen), protein-containing macromolecules (e.g., proteoglycans), silk (e.g., a derivatized silk), alginate, chitan and chitosan.

For preparation of pharmaceutical compositions containing one or more of the present peptides, for lactic and/or therapeutic treatments, the active ingredients (e.g., the peptide alone or the peptide bound to GF(s)) can be incorporated alone or in combination with other active agents into compositions suitable for administration to a patient. The formulations can be made using methods routine in the art and particular guidance may be provided by prior formulations of protein-based therapeutics. The compositions will be physiologically acceptable (i.e., substantially non-toxic) and may be formulated as iption medications or over-the-counter products. Pharmaceuticals or pharmaceutically acceptable compositions contain compounds (e.g., polypeptides), other materials (e.g., ts), and/or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and s without excessive toxicity, tion, allergic response, or other m or complication, commensurate with a reasonable benefit/risk ratio.

Nucleic acid molecules that encode the peptides described herein can also be formulated for use in cell culture or administration to a patient or subject. Such compositions commonly include a ceutically acceptable carrier, and carriers are contemplated in the present formulations. Any tional media or agent compatible with the active ingredients can be used in the present compositions. While formulations and s of use are described further below, we note here that application to human patients is intended, as is application to animals (e.g., domesticated, farm, or show animals). The ion extends to non-physiologically acceptable compositions in that it extends to preparatory compositions and compositions suitable for storage (e.g., concentrated stocks and frozen or lyophilized preparations).

The specific sequences described herein are d from human plasma fibronectin. In addition, one can use corresponding sequences (e.g., fragments having a corresponding sequence from any ectin isoform of any species).

With respect to on, the featured es can bind a polypeptide growth factor, for example PDGFBB , with an affinity of about or at least about 1x101x10-7 (e.g., about or at least about 5x10-7; 1x10-8; ; 1x10-9; or 5 x 10-9). Alternatively or in addition, the peptides support FN- null cell survival and/or proliferation secondary to intrinsic growth factor ty and/or growth factor enhancing activity.

Although ants do not wish to be bound by theory, the peptides described herein are useful in the treatment of skin-aging or photo-aging (e.g., for the treatment of wrinkles) and in other cosmetic treatments in that n fragments derived from fibronectin have been shown to promote fibroblast survival and proliferation. Furthermore, the peptides described herein may be used to deliver growth factors that promote fibroblast survival and proliferation to sites needing cosmetic treatment. For example, peptides may be incorporated into transdermal patches or any other device to facilitate their delivery with or without growth factors.

Although applicants do not wish to be bound by theory, the es bed herein are useful in the treatment of wounds insofar as they stimulate fibroblast al, proliferation and/or migration.

Additionally, the es described herein are useful, for example, as components of growth factor delivery devices such as engineered three-dimensional extracellular matrices.

Modifications of Peptides: The ed fragments and biologically active variants thereof can be modified in numerous ways. For example, agents, including additional amino acid es, other substituents, and protecting groups can be added to either the amino terminus, the carboxy terminus, or both. The modification can be made for the purpose of altering the fragments' form or altering the way the nts bind to or interact with one another, with non-identical fragments, or with other ptides. While the peptides of the present invention may be linear or cyclic, cyclic peptides generally have an advantage over linear peptides in that their cyclic structure is more rigid and hence their biological activity may be higher than that of the corresponding linear peptide (see, generally, Camarero and Muir, J. Am. Chem. Soc. 121:5597-5598, 1999).

Strategies for the ation of circular polypeptides from linear precursors have been described and can be employed with the present fragments. For example, a chemical cross-linking approach can be used to prepare a ne cyclized version of the peptide (Goldenburg and Creighton, J. Mol. Biol, 165:407-413, 1983). Other approaches include chemical intramolecular ligation methods (see, e.g., Camarero et al, Angew. Chem. Int. Ed., 37:347-349, 1998; Tam andLu, Pro! Sci., 7:1583-1592, 1998; Camarero and Muir, Chem. Commun., 1997:1369-1370, 1997; and Zhang and Tam, J. Am. Chem. Soc. 119:2363-2370, 1997) and enzymatic intramolecular ligation methods (Jackson et al, J. Am. Chem.

Soc., 117:819-820, 1995), which allow linear synthetic peptides to be ently cyclized under aqueous conditions. See also U.S. Pat. No. 7,105,341. atively, or in on, any of the t fragments can further include one or more substituents.

For example, the fragment can include a substitutent at the amino-terminus, carboxy-terminus, and/or on a reactive amino acid residue side-chain. The substituent can be an acyl group or a substituted or unsubstituted amine group (e.g., the substituent at the N-terminus can be an acyl group and the C- terminus can be ami dated with a substituted or unsubstituted amine group (e.g., an amino group having one, two, or three substituents, which may be the same or different)). The amine group can be a lower alkyl (e.g., an alkyl having 1-4 carbons), alkenyl, alkynyl, or haloalkyl group. The acyl group can be a lower acyl group (e.g., an acyl group having up to four carbon atoms), especially an acetyl group. The substituent can be a non-protein polymer, for example, a polyether, a polyethylene glycol (PEG), a polypropylene glycol, or a polyoxyalkylene, a polyalkylene glycol (for example, polypropylene glycol (PPG), a tylene glycol (PEG), or a PPG-PEG random polymer. The peptide can be modified by a non-protein polymer by methods known in the art and in the manner set forth in U.S. Pat.

No. 835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. The modification (e.g., PEGylation) can stabilize the peptide, reduce its antigenicity, decrease the required dosage, and/or augment its targeting y.

The non-protein r can vary in size and shape. For example, any of the non-protein polymers listed above (e.g., PEG) can be linear, branched, or comb-shaped. Regarding size, the molecular weight can vary. For example, the PEG can have a molecular weight of, for example, about 300 kDa, about 1,000 kDa, about 2,000 kDa, about 3,000 kDa, about 4,000 kDa, about 5,000 kDa, about 6,000 kDa, about 7,000 kDa, about 8,000 kDa, about 9,000 kDa, about 10,000 kDa, about 11,000 kDa, about 12,000 kDa about 13,000 kDa about 14,000 kDa about 15,000 kDa, about 20,000 kDa, about 30,000 kDa, about 40,000 kDa, or about 50,000 kDa. For example, the PEG can be of a molecular weight anywhere in between 300 kDA and 2000 kDA, 300 kDA and 3000 kDA, 1000 kDA and 2000 kDA and 1000 and 3000 kDA.

The otein polymer (e.g., PEG) can be linked to the fragment by any number of onal group chemistries (e.g., carboxylated-mPEGs, p-nitrophenyl-PEGs, aldehyde-PEGs, amino-PEGs, thiol-PEGs, maleimide-PEGs, aminoxy-PEGs, hydrazine-PEGs, tosyl-PEGs, iodoacetamide-PEGs, succinimidylsuccinate-PEGs, succinimidylglutarate-PEGS, succinimidylcarboxypentyl-PEGs, pnitrophenycarbonate-PEGs , or ethanethiol-PEGs). The non-protein polymer (e.g., PEG) can be linked to the fragment through any number of chemical groups including, but not limited to, amino-terminal amino acids, carboxy-terminal amino acids, free , and free sulfhydryl groups.

The non-protein polymer (e.g., PEG) may be a functionalized (for example, a monofunctional activated linear PEG, a homobifunctional activated linear PEG, a heterobifunctional activated linear PEG, a multiarmed activated PEG (e.g. 2-armed, 4-armed, 8-armed, etc.), a branched activated PEG and a comb-shaped activated PEG).

As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group, which is straightchained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, l), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 10, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.

As used herein, "alkenyl" refers to an alkyl group having one or more double carbon-carbon bonds.

Example alkenyl groups include l, propenyl, and the like. "Alkynyl" refers to an alkyl group having one or more triple carbon-carbon bonds. Example alkynyl groups include ethynyl, propynyl, and the like. "Haloalkyl" refers to an alkyl group having one or more halogen substituents. e haloalkyl groups include CF3, C2F5, CHF2, CC13, CHC12, C2C15, and the like.

As used herein, ther" refers to a polymer ning ether linkages. Examples include polyethylene glycol.

The fragments, including the modified fragments described above, can be protease resistant and can include one or more types of protecting groups such as an acyl group, an amide group, a benzyl or benzoyl group, or a polyethylene glycol. More specifically, a fragment, ing the modified fragments bed above, can be N-terminally acetylated and/or C-terminally amidated.

Where non-naturally occurring or modified amino acid residues are included they can be selected from the following or many others available in the art: 4-hydroxyproline, gamma-carboxyglutamic acid, ophosphoserine , o-phosphotyrosine, or delta-hydroxylysine. Other es include naphthylalanine, which can be substituted for tryptophan to facilitate synthesis, L-hydroxypropyl, L-3,4- dihydroxyphenylalanyl, amino acids such as L-alpha-hydroxylysyl and D-alpha-methylalanyl, L- alpha-methylalanyl, beta-amino acids, and isoquinolyl. Fragments having non-naturally occurring amino acid residues may be referred to as synthetic fragments and constitute one type of variant as described herein. Other variants include fragments in which a naturally occurring side chain of an amino acid residue (in either the L- or ) is replaced with a non-naturally occurring side chain.

In one embodiment, the fragments can have three extra amino acids (MetGlySer) at either terminus (or both) (e.g., at the N-terminus) and seven to eight extra amino acids (ThrSerHisHisHisHisHisHisCys) at either us (or both) (e.g., at the C-terminus).

For guidance on fragment modification by reduction/alkylation and/or acylation, one can consult Tarr, Methods of Protein Microcharacterization, J. E. Silver ed., Humana Press, Clifton NJ. 155-194, 1986; for guidance on chemical ng to an appropriate carrier, one can consult Mishell and Shiigi, eds, ed Methods in Cellular Immunology, WH Freeman, San Francisco, Calif (1980) and U.S. Pat.

No. 4,939,239; and for guidance on mild formalin treatment, one can consult Marsh, Int. Arch, of Allergy and Appl. Immunol, 41:199-215, 1971.

Any of the peptides in the featured compositions can be one of a plurality present in a multimeric form (e.g., a dimer). These multimers can be linear or branched. The eric form can also include one or more types of fragments and a backbone structure. Where two or more fragments are present, they may be identical or non-identical. A r structure, referred to as a , may also be present and may mediate attachment of the fragments to the backbone. Generally, the linker is smaller than the backbone.

The nature of the backbone structure is not critical, and many different types of molecules may be used.

One example of a linker structure is an oligolysine molecule having, for example, two or more lysine residues (e.g., 2, 3, 4, or more lysine residues). Two or more nts of the ion (e.g., two three or four polypeptides) may be attached to lysine residues by, for example, peptide bonds. These fragments, having a polylysine linker, can be linked to a ne structure. For example, the invention encompasses: Backbone-KKK HIGKY GLRWRPKGS V HIGKY GLRWRPKGS VKKK-B ackbone.

A backbone structure, for example, an oligolysine molecule, may be linear or ed. A multimeric peptide of the ion on a branched backbone molecule may be referred to herein as a "dendrimeric" peptide.

Any of the nts described herein, including the variant forms described herein, can further include a heterologous polypeptide (i.e., a polypeptide having a sequence that does not appear in a fibronectin).

The heterologous polypeptide can be a ptide that increases the circulating half-life of the fragment to which it is attached (e.g., fused, as in a fusion protein). The heterologous ptide can be an albumin (e.g., a human serum albumin or a portion thereof) or a portion of an immunoglobulin (e.g., the Fc region of an IgG). ptide growth factors that can be bound by the FN described herein can be within the insulin-like growth factor (IGF) family (e.g., IGF-1), within the transforming growth factor (TGF) family (e.g., TGF-pi or TGF-P2), within the fibroblast growth factor (FGF) family (e.g. bFGF or FGF-7), within the platelet-derived growth factor (PDGF) family (e.g., PDGF-BB), within the vascular endothelial growth factor (VEGF) subfamily (e.g., VEGF-A), or within the nerve growth factor (NGF) family. To determine whether fibronectin fragments bind growth factors that have retained a biological activity, standard biological assays can be carried out. For example, as outlined in the Examples below, migratory responses to bound growth factors that usually stimulate ion can be carried out. For example, one can compare the effect of a bound and unbound growth factor on fibroblast migration or granulation tissue formation. Specifically, if a growth factor is a PDGF (e.g., PDGF-BB), migration of AHDF cells can be analyzed.

Compounds mimicking the necessary mation of the fibronectin fragments described herein are contemplated as within the scope of this ion. A y of designs for such mimetics are possible.

U.S. Pat. No. 5,192,746; U.S. Pat. No. 5,169,862; U.S. Pat. No. 5,539,085; U.S. Pat. No. 5,576,423; U.S. Pat. No. 5,051,448; and U.S. Pat. No. 5,559,103, all hereby incorporated by nce, describe le methods for creating such compounds.

Physiologically Acceptable Compositions: A present pharmaceutical composition is formulated to be ible with its intended route of stration, for example, oral or parenteral (e.g., intravenous, intradermal, subcutaneous, intraperitoneal, intramuscular, by inhalation, transdermal (topical), and transmucosal administration).

Given the ability of the t FN fragments, and GF-containing complexes g these fragments, to facilitate wound healing, topical ations are particularly oned.

Solutions or suspensions used for parenteral administration can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfate; ing agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or se. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The ition can be ted or packaged in ampules, disposable syringes, single or multiple dose vials made of glass or plastic, bottles, and the like, and such packaged forms, along with ctions for use, are within the scope of the present invention. Preferably, the compositions are sterile at a medically acceptable level in view of the intended route of administration.

Pharmaceutical compositions adapted for injection include, for example, sterile aqueous ons (where water e) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include, for example, physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, N. J.) and phosphate buffered saline (PBS). In all cases, the compositions prepared for administration should be e and should be fluid or convertible to a fluid at least sufficient for easy syringability. The composition and/or nucleic acid constructs should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi. Preservatives against microorganisms can include various antibacterial and ngal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.

The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures f. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required le size in the case of dispersions and by the use of surfactants.

In many cases, it will be desirable for the composition to be isotonic to blood. This can be accomplished using s isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the ition.

Delayed or extended absorption of the injectable itions can be desirable and can be achieved by including in the ition an agent which delays absorption, for example, aluminum monostearate and gelatin, or by coating micro- or nano-particles of active agent in the composition with materials that delayed or extended release of components.

Sterile injectable solutions can be prepared, for example, by solubilizing or suspending the active compound in the required amount in an appropriate solvent with one or a combination of additional ingredients. Typically creation of such on or suspension is followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the other desired ingredients. In the case of sterile powders for the ation of sterile injectable solutions, the preparation is dried, e.g., by vacuum drying and/or freeze- drying.

Pharmaceutical compositions adapted for topical administration may include, but are not limited to, compositions in the form of skin care, skin cleansing, or anti-wrinkle products, shampoos, p, ioners, s, aerosols, gels, mousses, dyes, or bleaches. These compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, ing, but not limited to, fillers, surfactants, thixotropic agents, antioxidants, preserving agents, dyes, pigments, fragrances, thickeners, ns, hormones, moisturizers, UV absorbing organic sunscreens, UV scattering inorganic sunscreens, wetting agents, cationic, anionic, nonionic or amphoteric polymers, and hair ng active substances. These adjuvants are well known in the field of cosmetics and are described in many publications, for example see Harry's Book of Cosmeticology, S.sup.th n, Martin Rieger, ed., Chemical Publishing, New York (2000). Exemplary compositions are described in, for example, in U.S. Pat. Application 2005008604, U.S. Pat. Application 20050025725 andU.S. Pat. Application 20040120918 which are herein incorporated by reference.

In certain embodiments, the ceutical compositions of this invention can include one or more chemical ation ers (as described, for example, in International Publication No.

W02005009510).

Exemplary chemical penetration ers include, but are not limited to, 1-dodecyl idone, benzyl dimethyl dodecyl ammonium chloride, dopropyl betaine, cocamidopropyl hydroxysultaine, oleyl betaine, cineole, cetyl trimethyl ammonium bromide, dodecyl amine, dodecyl pyridinium chloride, hexadecyl trimethyl ammoniopropane sulfonate, isopropyl myristate, lauric acid, limonene, linoleic acid, linolenic acid, menthol (terpene), methyl laurate, 1-methylpyrrolidone, N- lauryl sarcosine (CAS number 1376, also called sodium lauroyl sarcosinate), nicotine sulfate, oleic acid, octyl trimethyl ammonium e, poly ethyleneglycol dodecyl ether, 1-phenyl zine, sorbitan monolaurate, sodium lauryl ether e, sodium dodecyl sulfate, sodium oleate, sodium octyl e, tetracaine, and Tween-20™.

Chemical penetration enhancers increase skin permeability and are known in the art (see, for example, Shah et al. "Skin Penetration Enhancement: Clinical pharmacological and regulatory considerations." Pharmaceutical Skin Penetration Enhancement, ed. K. Walters. 1993, New York, Basel, Hong Kong: Marcel Dekker. 417-427).

The present peptides may be used in cosmetic compositions either as the es themselves or in the form of a premix in a suitable excipient and they may be used in the form of a solution, dispersion, emulsion, paste or . They may individually or with other active substances, including but not d to those specifically described herein, be carried by cosmetic vectors such as macro-, micro- or nanocapsules, liposomes or chylomicrons, macro-, micro- or nanoparticles or microsponges. They may also be adsorbed on powdered organic rs, talcs, bentonites and other inorganic carriers.

The peptides may be used in any form or in a form that is bound, incorporated, absorbed in or ed on macro-, micro- and nanoparticles, macro-, micro- and psules for the ent of textiles, synthetic or natural fibers, wools and all materials liable to be used in the manufacture of clothing or underwear for the day or night, intended for contact with the skin, such as pantyhose, underwear, handkerchiefs and wipes, in order to exert a cosmetic effect h the contact between the textile and skin and enable continuous topical ry.

The peptides can be used in topical compositions (e.g., therapeutic or ic compositions) at concentrations ranging from 0.00001% (w/w) ("w/w" is weight/weight) and 10% (w/w) (e.g., between about 0.0001% (w/w) and 1% (w/w)). r useful range is from about 0.001% and about 5% (w/w).

The peptides may also be used in the range of about 1 ppm to about 500 ppm (e.g., about 100 to about 400 ppm).

Compositions for oral administration typically include an inert or edible diluent or edible carrier. Such compositions can be ated in various ways, e.g., in liquid, capsule, or tablet form.

Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any one or more of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or es; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a ing agent such as peppermint, methyl salicylate, or orange flavoring.

For inhalation administration (e.g., for application to wounded tissues, such as mucosa, within the nasal es, nasopharynx, trachea or lungs or), the present compositions are delivered in the form of a wet or dry l spray, e.g., from a pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

Systemic administration can also be by transmucosal or transdermal routes. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are typically used in the formulation. A number of such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Administration may also be facilitated by horesis, microneedles and other devices designed to enhance transdermal ation.

Transmucosal administration can be accomplished through the use of nasal sprays or itories (e.g., using conventional suppository bases such as cocoa butter and other glycerides). For transdermal administration, the active compounds are ated into ointments, salves, gels, or creams as generally known in the art.

Such itions can also be formulated with carriers that will protect the compositions against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polycarbonates, and poly lactic acid. The als can also be obtained commercially, e.g., from Alza ation and Nova ceuticals, Inc. Liposomal suspensions (including liposomes targeted to particular cells (e.g., targeted to infected cells) with monoclonal antibodies) can also be used to prepare pharmaceutical compositions. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and mity of dosage. Dosage unit form as used herein refers to physically te units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound ated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active nd and the particular eutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the ent of individuals. In one embodiment of the fibronectin fragments and peptide derivatives of fibronectin fragments of the invention, the dosage unit form is about 0.1 to 5 mg of lyophilized peptide or peptide derivative. In r embodiment of the fibronectin fragments and peptide derivatives of fibronectin fragments of the ion, the dosage unit form is about 1 mg of lyophilized peptide or peptide derivative.

Toxicity and therapeutic efficacy of active compounds and ceutical compositions can be determined by standard ceutical procedures in cell cultures or experimental animals. For example, such procedures are routinely applied for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose eutically ive in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be sed as the ratio LD50/ED50. Compounds that exhibit large therapeutic indices are generally preferred. The data obtained from the cell culture assays and animal studies (including those described in the examples, below) can be used in ating a range of dosage for use in humans or other intended subjects. The dosage of such compounds is usually selected to produce a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays.

Thus, for example, a dose may be initially established in animal models to achieve a ating plasma concentration range that includes the EC50 (i.e., the concentration of the test compound which achieves a half-maximal response) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high mance liquid chromatography, or by other suitable analysis method adapted for the compound of interest.

As noted, peptides (e.g., tic or recombinantly produced peptides) with growth factor-binding and/or cing/inhibiting activity can be incorporated into a tissue engineered product. FN domains that promote fibroblast migration can also be included. Preferably, the products are robust (i.e., relatively resistant to rapid degradation). They can be used, for example, in treating wounds, including acute or non-healing wounds (e.g., chronic ). Patients amenable to treatment are described further below. Alternatively or in addition, growth factor-binding and/or enhancing peptides can be tethered to a biocompatible polymer for delivery of one or more growth factors to a cell, tissue or organ in need of treatment or for endogenous zation of growth factors. Alternatively or in addition, growth factorbinding and/or enhancing peptides can be incorporated in a polymer or ymer biomaterial for controlled e to an acute or non-healing wound.

We have ped an engineered ECM that is conductive and ive of new tissue formation in porcine cutaneous wounds utilizing molecular domains C, H, and HV or cell adhesion peptides of the blood protein fibronectin (FN) tethered to an olecularly crosslinked hyaluronan (HA) hydrogel.

Thus, in one implementation, the invention includes an engineered ECM that includes a fragment of a fibronectin (e.g., a plasma fibronectin) or a ically active variant thereof. The fragment can be tethered to (e.g., covalently or non-covalently bound to) a hydrogel (e.g., an HA hydrogel) and can be a fragment that binds and/or enhances a polypeptide growth . The fragment can be tethered according to attachment methods discussed in U.S. Pat. Application 20050282747, the contents of which are orated herein in their entirety.

The naturally-occurring ECM is comprised of diverse constituents such as glycoproteins, proteoglycans, complex ydrates, and other molecules. Major functions of the ECM include, but are not limited to, ing ural support, e strength or cushioning; providing substrates and pathways for cell adhesion and cell migration; and regulating cellular differentiation and metabolic function. ECM proteins include, for example, collagens, elastin, ectin, laminin, proteoglycans, vitronectin, thrombospondin, tenascin (cytoactin), entactin (nidogen), osteonectin (SPARC), anchorin CII, chondronectin, link protein, osteocalcin, bone sialoprotein, osteopontin, epinectin, hyaluronectin, amyloid P component, fibrillin, merosin, s-laminin, undulin, epilligrin, and kalinin.

The featured tissue engineered product (e.g., the engineered ECM) can e biological and/or synthetic components. It can include a biopolymer (e.g., hyaluronan (HA), a aminoglycan (GAG), fibrinogen, laminin, or collagen). The biocompatible polymer can be a synthetic biodegradable polymer, many of which are known in the art. For example, the biodegradable polymer can be a poly(lactide), a poly(glycolide), a poly(lactide-coglycolide), a poly(lactic acid), a poly(glycolic acid), a poly(lactic acidco-glycolic acid), a poly(caprolactone), a polycarbonate, a polyesteramide, a polyanhydride, a poly(amino acid), a poly(ortho ester), a poly cyanoacrylate, a polyamide, a polyacetal, a poly(ether ester), a copolymer of poly(ethylene glycol) and a poly(ortho ester), a ioxanone), a poly(alkylene alkylate)s, a biodegradable ethane, or any blend or mer thereof. Other useful polymers include an alginate polymer and a carboxy-vinyl polymer (e.g., a r including at least 90% acrylic acid monomers and about 0.1% to about 5.0% of a difunctional crosslinking .

A tissue engineered "smart" matrix that would be conductive and inductive of tissue cell lation of a wound site and the development of new tissue, respectively, can be ed of GFs, or active fragments thereof, in the context of an appropriate ECM that are required for optimal wound repair. In addition, FN GF-binding (s) may provide a useful tool for engineering many other GF localization (from endogenous or exogenous sources) and/or delivery systems for soft or hard tissue repair, augmentation and regeneration. Furthermore, growth factor FN/VN-binding peptides or WO 71709 molecularly engineered derivatives of the FN and VN GF-binding domains might become strongly inhibitory of GF activity and thus useful for proliferative or fibrotic disorders such as cancer, pulmonary fibrosis, GI or GU stenosis, bum contractures and autoimmune ted sclerosis.

EngECM can be generated with or without growth factors, or active fragments thereof (e.g., growth factors and fragments described herein). In the former case, the dosage of growth factors in the engECM can vary, e.g., as described below, 100 ng/ml (15 ng total per wound) of PDGF-BB added to 2:1 ered ECM enhanced granulation formation at 4 days after injury and application of material. In the latter case, when placed in the vicinity of an endogenous supply of growth factors, the growth factors can be recruited by the matrix.

The invention further encompasses nucleic acid molecules, including DNA and RNA molecules, that encode the polypeptides described . The nucleic acid molecules can be formulated in physiologically acceptable compositions for administration. The invention also es vectors that include the t nucleic acid constructs. Of particular benefit are expression vectors, especially those for expression in eukaryotic cells. Such vectors can, for example, be viral, plasmid, cosmid, or artificial chromosome (e.g., yeast artificial chromosome) vectors. Typically, plasmids are circular, dsDNA elements that e one or more cloning sites for insertion of ed DNA sequences, e.g., coding sequences. Such plasmids may include a functional origin of replication and thus are replication competent, or may be replication defective.

In addition to plasmids, viral vectors (e.g., replication defective retroviruses, lentiviruses, adenoviruses and adeno-associated viruses) can also be advantageously used. A large number of such viral s have been developed having a broad variety of different properties. For example, such viral vectors may be replication defective retroviruses, adenoviruses and adeno-associated viruses. Techniques and procedures for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses are provided in Current ols in Molecular Biology, Ausubel, F. M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9.10-9.14 and other standard laboratory manuals. Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.

Examples of suitable packaging virus lines include psi.Crip, psi.Cre, psi.2 and psi.Am. The genome of irus can be manipulated such that it s and expresses a c acid construct, as described , but is inactivated in terms of its ability to ate in a normal lytic viral life cycle, (see, e.g., r et al., BioTechniques 6:616, 1988; Rosenfeld et al, Science 252:431-434, 1991; and Rosenfeld et al, Cell 68:143-155, 1992). Suitable adenoviral vectors d from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.

Alternatively, an adeno-associated virus vector such as that described in Tratschin et al. (Mol. Cell. Biol. :3251-3260, 1985) can be used to express a transactivator fusion protein. Other viral vector alternatives include lentiviral vectors. Such vectors and their preparation and use are described, for example, in U.S.

Pat. Nos. 6,924,123; 884; 6,830,892; 6,818,209; 6,808,923; 6,799,657, all of which are incorporated herein in their entireties.

The vectors of the invention can advantageously include a polypeptide fragment described . Other ts included in the design of a particular expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. The expression vectors of the invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein.

The s described herein can be introduced into cells or tissues by any one of a variety of known methods within the art. Such methods are described for example in Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York , which is hereby incorporated by nce. See, also, Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989); Hitt et al, ruction and Propagation of Human Adenovirus Vectors," in Cell Biology: ALaboratory Handbook, Ed. J. E. Cells., Academic Press. 2nd Edition, Volume 1, pp: 500-512, 1998; and Hitt et al, "Techniques for Human Adenovirus Vector Construction and Characterization," in Methods in Molecular Genetics, Ed. K. W. Adolph, Academic Press, Orlando, Fla., Volume 7B, pp: 12-30, 1995. The methods include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. The term "transfecting" or "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into host cells, including calcium phosphate co-precipitation, DEAF-dextran-mediated transfection, lipofection, electroporation and microinjection. Suitable methods for transfecting host cells can be found in Sambrook et al. ular Cloning: atory Manual, 2nd Edition, Cold Spring Harbor tory press (1989)), and other laboratory textbooks.

For plant cells, a Ti plasmid or viral vector is often used. For e, such plasmids and viral vectors can be used to transfect host plant cells via Agrobacterium ciens-mediated transfection (for plant cells susceptible to A. tumefaciens ion), or can be directly inserted in cells, e.g., using microinjection, particle bombardment, or electroporation. In other methods, protoplasts can be made from plant cells and then ected.

The number of host cells ormed with a c acid constructs of the invention will depend, at least in part, upon the type of recombinant expression vector and the type of transfection technique used. c acid can be introduced into a host cell ently, or for long-term expression. For long-term expression, the nucleic acid is stably integrated into the genome of the host cell or s as a stable al element.

For integration of c acid into host cell DNA, typically a gene is used that encodes a selectable marker (e.g., drug resistance) is introduced into the host cells along with the nucleic acid of interest. A variety of such selectable markers are commonly used, such as the drugs hygromycin and neomycin.

Selectable markers can be introduced on a separate plasmid or other vector from the nucleic acid of interest or, are introduced on the same vector. Host cells transfected with a nucleic acid construct of the invention (e.g., a inant expression vector) and a gene for a selectable marker can be identified by selecting for cells using the selectable marker.

The present nucleic acid constructs can be introduced into eukaryotic cells growing in culture in vitro by conventional transfection techniques (e.g., m phosphate precipitation, DEAE-dextran transfection, electroporation, and other methods). Cells can also be transfected in vivo, for example by application of a delivery mechanism suitable for introduction of nucleic acid into cells in vivo, such as viral vectors (see e.g., Ferry et al, Proc. Natl. Acad. Sci. USA 88:8377-8381, 1991, and Kay et al., Human Gene Therapy 3:641-647, 1992), adenoviral vectors (see e.g., Rosenfeld, Cell -155, 1992; andHerz and , Proc. Natl. Acad. Sci. USA 90:2812-2816, 1993), receptor-mediated DNA uptake (see e.g., Wu and Wu, J. Biol. Chem. 263:14621, 1988; Wilson et al, J. Biol. Chem. 267:963-967, 1992; and US. Pat.

No. 5,166,320), direct injection of DNA (see e.g., Acsadi et al, Nature 5-818, 1991; and Wolff et al, Science 247:1465-1468, 1990) or particle bombardment (see e.g., Cheng et al, Proc. Natl. Acad.

Sci. USA 90:4455-4459, 1993; and Zelenin et al, FEES Letters 315:29-32, 1993). Thus, in the present invention, cells can be transfected in vitro or ex vivo, and the sed peptide can be isolated there from by methods known in the art. The cells can also be administered to a subject or, alternatively, cells can be directly modified in vivo. In any of these ions, the nucleic acid construct used to express the peptide can include a signal ce to facilitate export from the cell.

Another aspect of the ion pertains to host cells into which a nucleic acid construct of the invention has been introduced, i.e., a "recombinant host cell." It is understood that the term "recombinant host cell" refers not only to the particular subject cell but to the progeny or potential progeny of such a cell.

Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell, although eukaryotic cells are preferred. Exemplary eukaryotic cells include ian cells (such as e hamster ovary cells (CHO) or COS cells).

Other suitable host cells are known in the art.

It is not intended that the present invention be limited by the particular nature of the eutic preparation, so long as the preparation comprises an appropriate fragment of fibronectin that binds a polypeptide growth factor or that has intrinsic survival or growth factor activity or an appropriate fragment of a growth factor that binds fibronectin. For e, such compositions can be provided together with physiologically tolerable liquid, gel or solid carriers, diluents, adjuvants and/or excipients.

These therapeutic preparations can be administered to mammals for nary use, such as with domestic animals, and clinical use in humans in a manner similar to other therapeutic agents. In l, the dosage ed for therapeutic efficacy will vary according to the type of use and mode of administration, as well as the particularized requirements of individual hosts.

Such compositions are typically prepared as liquid solutions or suspensions, or in solid forms.

Formulations can include such normally employed additives such as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers and excipients as, for e, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These itions take the form of solutions, suspensions, s, pills, capsules, sustained release formulations, or powders, and typically contain l%-95% of active ingredient, preferably 2%-70%.

The compositions are also prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for on in, or suspension in, liquid prior to injection may also be ed.

The fragments of the present ion are often mixed with ts or excipients which are physiologically tolerable and compatible. Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.

Additional formulations which are suitable for other modes of administration, such as topical administration, include salves, tinctures, , lotions, and, in some cases, suppositories. For salves and creams, traditional binders, rs and ents may include, for example, polyalkylene glycols or triglycerides.

Methods of Use: The fibronectin fragments and peptide derivatives of fibronectin fragments described herein are useful in promoting tissue regeneration, e.g., wound healing, and in ic and therapeutic formulations for the prevention and ent of poor skin appearance related to, for example, aging. Use in cell culture is also described. The polypeptides (or c acids or sion vectors encoding them or cells expressing them) can be incorporated into, for example, therapeutic ations for the indications described herein as well as into products and compositions for improving, for example, skin appearance and/or feel of skin exhibiting signs of skin aging.

For example, compositions of the present invention are useful for regulating the appearance of skin due to wrinkles and UVB photodamage by ing visual improvement in skin ance following application of the composition to the skin. Generally speaking, compositions of the present invention which further contain particulate materials will be most useful for providing the ate visual improvement.

The invention features cosmetic treatments including those for prophylactically regulating a skin ion and those for therapeutically regulating a skin condition. "Signs of skin aging," "poor skin appearance," and other phrases similarly referring to, for example, symptoms of aging and the like include, but are not limited to, all outward visibly and tactilely perceptible stations as well as any other macro or micro effects due to skin aging. Such signs may be induced or caused by intrinsic factors and/or extrinsic factors, e.g., logical aging and/or environmental damage (e.g., UVB photodamage, exposure to pollutants, and poor diet). These signs may result from processes which include, but are not limited to, the development of textural discontinuities such as wrinkles and coarse deep wrinkles, skin lines, crevices, bumps, large pores (e.g., associated with adnexal structures such as sweat gland ducts, sebaceous glands, or hair follicles), or unevenness or roughness, loss of skin elasticity (loss and/or inactivation of functional skin n), sagging (including puffiness in the eye area and , loss of skin firmness, loss of skin tightness, loss of skin recoil from deformation, discoloration (including undereye circles), blotching, sallowness, igmented skin regions such as age spots and freckles, keratoses, abnormal differentiation, hyperkeratinization, elastosis, collagen breakdown, and other histological changes in the stratum comeum, dermis, epidermis, the skin vascular system (e.g., telangiectasia or spider vessels), and underlying tissues, especially those proximate to the WO 71709 skin. Particularly preferred in ance with the present invention, the signs of skin aging are wrinkles and the compositions of the present invention are, in certain preferred ments, useful in fighting, treating or preventing wrinkles.

Wrinkles can result from numerous causes. For example, es can be caused from the natural aging process of the skin, from smoking, and from exposure to ultraviolet radiation (e.g., from chronic sun exposure). Wrinkles can be classified as described in Kligman et al. (Br. J. Derm. 113:37-42, 1985), herein incorporated by reference. Kligman classifies wrinkles into three classes: linear wrinkles, glyphic wrinkles, and crinkles, and any of these types of wrinkles, regardless of their cause, can be treated as described herein. Aside from wrinkles per se, the present compositions can be used to improve the skin's ance.

The methods disclosed herein are useful to prevent or treat or reduce wrinkles, including UY-induced wrinkles, and/or to improve skin quality and appearance in a subject. The methods can be carried out by administering to the subject a composition containing a fibronectin fragment or a ically active variant thereof. An ary treatment method can include ng a wrinkle or a potential site of wrinkling and applying a composition described herein.

As used herein, prophylactically regulating a skin condition includes delaying, minimizing and/or preventing visible and/or tactile discontinuities in skin (e.g., texture irregularities in the skin which may be detected visually or by feel), including signs of skin aging.

As used , therapeutically regulating skin condition includes ameliorating, e.g., diminishing, zing and/or ng, tinuities in skin, including signs of skin aging. Some of the products produced using the compositions of the present invention and indeed the compositions themselves may be used for prophylactically or eutically regulating a skin condition.

In certain preferred aspects, the present invention is useful for improving the physiological state and/or the physical appearance of human skin, in particular to reduce the signs of skin aging that are generated by sun exposure (e.g., UVB photodamage), physical and hormonal stress, abrasion, nutritional effects and other similar causes. The itions may often be used to prevent the signs of aging and/or to treat them in order to afford the consumer who uses them, a more youthful appearance.

All terms such as "skin aging," "signs of skin aging," "poor skin appearance," "topical application," and the like are used in the sense in which they are generally and widely used in the art of developing, testing and marketing cosmetic and personal care products. "Wrinkles" means furrows in the otherwise smooth e of the facial skin, visible to the naked eye, in the average depth of 50 to more than 200 pm and essentially appearing with progressive age. The term "cosmetic composition" in accordance with the present invention relates to a formulation that can be used for cosmetic purposes, purposes of hygiene or as a basis for delivery of one or more ceutical ingredients. This includes cosmetics, personal care ts and pharmaceutical preparations. It is also possible that these formulations are used for two or more of these same purposes at one time. A medicated dandruff shampoo, for example, has pharmacological properties and is used as a personal care product to provide clean hair. These compositions may also include additional ingredients such as a dermatologically acceptable r.

"Cosmetics," as used herein, include without limitation, lipstick, mascara, rouge, foundation, blush, eyeliner, er, lip gloss, facial or body powder, sunscreens and blocks, nail polish, mousse, sprays, styling gels, nail conditioner, r in the form of , lotions, gels, ointments, emulsions, colloids, solutions, suspensions, compacts, , pencils, spray-on formulations, brush-on formulations and the like. "Personal care products" include, without limitation, bath and shower gels, shampoos, conditioners, cream rinses, hair dyes and coloring products, leave-on conditioners, sunscreens and sunblocks, lip balms, skin ioners, cold creams, moisturizers, hair sprays, soaps, body scrubs, exfoliants, astringents, depilatories and permanent waving solutions, anti dandruff formulations, antisweat and antiperspirant compositions, shaving, preshaving and after shaving products, moisturizers, deodorants, cold creams, cleansers, skin gels, rinses, whether in solid, powder, liquid, cream, gel, ointment, lotion, emulsions, colloids, solutions, suspensions, or other form. "Pharmaceutical preparations" in accordance with the t invention include, t limitation, carriers for dermatological purposes, including topical and transdermal ation of pharmaceutically active ingredients. These can be in the form of gels, s, , nose sprays, ointments, s, emulsions, colloids, solutions, sions, powders and the like. Compositions in accordance with the invention include cosmetics, personal care products and pharmaceutical preparations.

The invention features methods for ing tissue regeneration, including, for example, wound healing. As used herein, tissue regeneration is used to refer to the replacement of damaged tissue by the proliferation and differentiation of cells into a tissue. Tissue damage can occur by any means, including physical injury, disease, and infection. As described herein, "wound-healing" is used as anon-limiting example of tissue regeneration.

The primary goal in the treatment of wounds is to achieve wound closure. Open cutaneous wounds represent one major category of wounds and include thermal and/or chemical bum wounds, neuropathic ulcers, re sores, venous stasis ulcers, and diabetic ulcers. Open cutaneous wounds ely heal by a process which ses six major components: i) inflammation, ii) fibroblast proliferation, iii) blood vessel proliferation, iv) connective tissue sis v) lialization, and vi) wound ction. Wound healing is impaired when these components, either individually or as a whole, do not function properly. Numerous factors can affect wound healing, including malnutrition, infection, pharmacological agents (e.g., actinomycin and steroids), diabetes, and advanced age (see Hunt and Goodson in Current Surgical Diagnosis & Treatment (Way; Appleton & Lange), pp. 86-98, 1988).

The term "wound" refers broadly to injuries to the skin and subcutaneous tissue initiated in different ways (e.g., pressure sores from extended bed rest and wounds d by trauma) and with varying teristics as well as to injuries of other s and bone, including tissues and bone in or around the vicinity of a primary wound site. Of course, wounds can also be made surgically or by disease (e.g.

). Wounds may be classified into one of four grades ing on the depth of the wound: i) Grade I: wounds d to the epithelium; ii) Grade II: wounds extending into the dermis; iii) Grade III: wounds extending into the subcutaneous tissue; and iv) Grade IV (or full-thickness wounds): wounds wherein bones are exposed (e.g., a bony pressure point such as the greater trochanter or the sacrum).

The term "partial thickness wound" refers to wounds that encompass Grades I-III; examples of partial thickness wounds include thermal or chemical bum wounds, pressure sores, venous stasis , and diabetic ulcers. The term "deep wound" is meant to include both Grade III and Grade IV wounds. The present invention contemplates treating all wound types, including deep wounds and chronic wounds.

The phrases "promote wound healing," "enhance/improve wound healing," and the like refer to either the induction of the formation of granulation tissue and/or the induction of lialization (i.e., the generation of new cells in the epithelium), and/or reduction of scarring. Wound healing is conveniently measured by decreasing wound area. It is not intended that phrases such as "promote wound healing" or "enhance/improve wound healing" require a quantitative ison with controls. In the case of ent of a chronic wound, it is sufficient that evidence of wound healing begins after ent.

Many traumatic wounds and cancer extirpations must be left open to heal by secondary intention, and patients having such wounds and extirpations can be treated with the compositions described herein that promote wound healing.

The phrase "therapeutically effective amount" of the fibronectin fragments or peptide derivatives of fibronectin fragments of the invention, when referring to wound healing, promoting wound healing or enhancing wound healing, is that amount that promotes induction of the formation of granulation tissue and/or the induction of epithelialization and/or reduction of scarring. For example, fibronectin fragments WO 71709 or peptide derivatives of fibronectin fragments of the invention can be used to promote would healing in i.v. formulations in an amount of from about 0.1 pg/kg to about 1 mg/kg of patient body weight; in some embodiments, from about 1 pg/kg to about 1 mg/kg of patient body ; in some embodiments, from about 1 pg/kg to about 0.1 mg/kg of patient body weight; in some embodiments, from about 0.01 mg/kg to about 1 mg/kg of patient body ; and in some embodiments, from about 0.01 mg/kg to about 0.1 mg/kg of patient body weight.

The incidence of chronic wounds, mes referred to as non-healing wounds, is rising due to events such as aging populations; an increase in age-related diseases in those populations; an increase in the incidence of AIDS; an increase in the incidence of diabetes, and an increase in radiation wounds secondary to cancer intervention. Patients who have chronic wounds, ing those associated with the events just described, can be treated with the compositions described herein that promote wound healing.

The present itions can be used either instead of or to supplement ng wound-care procedures such as skin grafting and tissue flaps, debridement, and the administration of anti-inflammatory, antibacterial and/or anti-pain medications. Patients amenable to treatment include those who have chronic dermal ulcerations, as can occur in association with diabetes. Diabetic ulcers, however, are just one part of the chronic wound picture. It is estimated that 5.5 million people in the United States have chronic, nonhealing wounds.

The s of the invention include a step of administering to a patient a therapeutically effective amount of a pharmaceutical composition comprising a peptide fragment of fibronectin, or a biologically active variant f, as described herein. The peptide fragment of fibronectin, or the biologically active variant thereof, can be present in a complex with one or more growth factors. The methods can optionally include a step of identifying a patient in need of treatment. Such patients include patients who are ing from a surgical extirpation or incision of the skin, mucosa, underlying connective , fascia, nerve or muscle; ts who are suffering from a traumatic laceration or tissue loss of the skin, , underlying connective tissue, fascia, nerve, muscle or bone; and patients who are suffering from a thermal or chemical bum or ulceration of the skin, mucosa, underlying connective , fascia, nerve or muscle.

Suitable formulations are described herein and, generally, take the form of a solution, ointment or salve.

The nts of fibronectin, whether or not complexed with a growth factor, can also be administered by way of their inclusion in a biomaterial, such as a synthetic polymer, an engineered ECM, a bandage, dressing, compress, or the like.

By other methods of the invention, one can localize an endogenous growth factor to a tissue of a patient.

These methods can be carried out by administering, to the patient, a eutically effective amount of a composition that includes a fragment of fibronectin or a biologically active t thereof, as described herein. As in the more specific treatment methods described herein, these compositions can be administered by way of topical application of a pharmaceutical composition, a biomaterial, or a solid support, or by other local and systemic routes (e.g., orally, intravenously, intramuscularly, subcutaneously, intradermally, pericutaneously, or transmucosally). These methods can be described as methods of delivering one or more growth s to a patient. The methods can optionally include a step of identifying a patient in need of treatment. Such patients include patients who are suffering from an injury to a tissue, a loss of a tissue or a disorder ing in tissue disfigurement or dysfunction.

More specifically, the t can be suffering from an injury or loss to the brain, spinal cord or nerves or a disorder resulting in brain, spinal cord or nerve dysfunction; an injury or loss to the heart or blood s or a disorder resulting in heart or blood vessel dysfunction; an injury or loss to the lung, nasopharyngeal tract, sinuses, trachea or airways or a disorder resulting in lung, nasopharyngeal tract, sinus, trachea or airway dysfunction; an injury or loss to the gastrointestinal tract, liver or as or a disorder resulting in gastrointestinal tract, liver or pancreas dysfunction; an injury or loss to a kidney, ureters, r or urethra or a disorder resulting in kidney, ureters, bladder or urethra dysfunction; an injury or loss to cartilage, um, menicus, ligament, tendon or nucleus pulposis or a disorder resulting in cartilage, um, meniscus, ligament, tendon or nucleus pulposis dysfunction; an injury or loss to bone; an injury or loss to lips, tongue or gums or a disorder resulting in lip, tongue and gum dysfunction; an injury or loss to the subcutaneous tissue or a disorder resulting in subcutaneous tissue dysfunction.

In Vitro and In Vivo Model s: Test compounds may be further characterized in in vitro and in vivo model systems. For example, test compounds can be tested for s on cell migration using Adult Human Dermal Fibroblasts , human microvascular endothelial cells (HEDMC), or other cell types. For example, test compounds can be tested for effects on wound healing using the porcine re-injury model, excisional wound model in pigs or mice, hot comb bum wound model in pigs or rats, vertical injury progression bum models in pigs, chemical bums in pigs.

EXAMPLES The following examples further describe and demonstrate embodiments within the scope of the present invention. The examples are given solely for the purpose of ration and are not to be construed as limitations of the present invention, as many variations thereof are le t departing from the spirit and scope of the invention.

Studies of Topical Treatment of Porcine Bums with PDGF-BB and Formula I Peptide Formulations: Four female, 20-30 kg, domestic pigs were used for cutaneous wound procedures.

Study Protocol: The animals were sedated with Talazine amine and pam, Fort Dodge Lab, Fort Dodge, Iowa) 5 mg/kg IM. The pigs were then intubated endotracheally and maintained under a surgical plane of anesthesia with isoflurane 0.5-2.5% in room air. The flank and back hair were clipped with electric hair clippers and the skin was scrubbed with a povidone iodine solution.

Standardized deep partial-thickness bums were created on the animals' backs and flanks by applying a 2.5-cm by , 150-gram aluminum bar ted in hot water to 80° C. The bums were created on either side of the vertebral column between the forelegs and hind legs. The heated bar was wiped dry just prior to application to prevent water droplets from creating a steam bum on the skin. The bar was then placed at a vertical on perpendicular to the skin's surface and applied for a period of 20 seconds with all pressure supplied by gravity. This bum model results in damage to the upper 30-50% of the dermis and has been shown to be highly reproducible (Singer et al, Acad. Emerg. Med. 7:1-6, 2000). 24 bums were evenly distributed on both sides of the back of four pigs. Since pigs do not form blisters after thermal injury, debridement of the ic epidermis was performed immediately after injury in order to simulate bums in humans where rs may form and subsequently e (Singer et al, Acad. Emerg. Med., 7:114-119, 2000). Debridement was performed by gently rubbing dry gauze against the surface of the bum until the necrotic epidermis was peeled away from the entire bum e. Interventions: On the back skin of each pig, equal sets of 4 bums were be randomly treated with one of the 6 study treatments. Each treatment of pluronic lecithin organogel ("PLO") gels, PLO gels containing PDGF-BB, PLO gels containing cP12 or cNP8 and PLO gels containing either cP12 or cNP8 and PDGF-BB, was applied to 4 bum wounds per pig after either 24 hour post-bum or 48 hour post-bum. Peptides selected for testing were sized in a GMP y (American Peptide, Vista, Calif) and diluted in sterile, endotoxin-free PBS with sterile, endotoxin-free 2% porcine serum (HyClone, Logan, Utah) to avoid peptide loss via nonspecific surface adsorption. Sterile, endotoxin-free recombinant PDGF-BB (R&D Systems) was also diluted in PBS with 2% porcine serum. Final concentrations of es with and without PDGF-BB were compounded in a 30% pluronic lecithin gel using a sterile, endotoxin-free PLO kit (Transderma, Coquitlam, BC, Canada). PBS with 2% porcine serum in a 30% pluronic gel was used as a treatment l. Wounds received 150 pi of treatment gels d topically on a daily basis for the first week and twice weekly thereafter. Then bums were covered with dry non-adherent gauze (Telfa, Kendall Company, Mansfield, Mass.) and the burned areas covered with a gauze bandage roll (Conform, Kendall Healthcare Products Company, eld, Mass.) and an adhesive elastic bandage (Elastoplast, Beiersdorf-Jobst, Inc., ford College, N.C.). In order to prevent dressing removal, staples were applied to the periphery of the dressings. Dressing changes were d as above after each treatment application. All of the animals were treated with a Fentanyl transdermal patch post operatively for analgesia management.

Survival y of pigs and wound site harvest was done under general anesthesia. Pigs were fasted for 24 hours before the surgical procedures. Atropine was given pre-op at a dose of 0.05 mg/kg. For induction of general anesthesia 4.4 mg/kg Telazol and, 2.2 mg/kg Xylazine and 0.22 mg/kg Butorphanol were administered EVL The animal was then intubated and held at the stage of surgical anesthesia with Isoflurane (1-3%) and oxygen. Since covered cutaneous wounds cause minor pain to humans that require at most acetominophin, animals were treated likewise receiving 10-20 mg/kg acetominophen twice daily after al surgery.

Euthanasia is accomplished with intravenous 100 mg/kg pentobarbital and 2 mg/kg ne.

As shown in Figure 6, re-epithelialization of 48-hour debrided wounds, 10 days after ng was markedly increased for cNP8-treated wounds, as compared to control or cP12-treated wounds.

Studies of LV. Treatment of e Bums with PDGF-BB and Formila I Peptide Formulations: The vertical progression bum model (as shown in above topical experiment) was used to create bums on the backs of each of 4 pigs. Twenty bums 20 seconds) were made on the backs of each pig - one set of bums was made 8 hours prior to infusion, the second set was made 4 hours prior to on.

Three pigs were treated with infusions of 0.001, 0.01 or 0.1 mg/kg of cNP8 and one pig was treated with an infusion of buffer, as control. Lyophilized cNP8 was reconstituted in PBS (11.6mg/ml corrected- based on 75% pure) to get a 5 mM stock solution in the laboratory and filtered with a syringe filter with 0.22 um membrane. The concentration of ed cNP8 solution was determined by reading OD280 and the concentration was calculated based on OD280=6.76 per mM cNP8. The cNP8 was diluted to 5 mM with PBS as necessary. The fdtered cNP8 solution was ted and stored at -80°C. Just before on, cNP8 was further diluted: a) 1:270 with PBS to get a 0.019 mM cNP8 solution. The injection amount was 3 ml/kg body weight of 0.019 mM cNP8 which is equal to 0.1 mg/kg body weight, b) 1:2700 with PBS to get a 0.0019 mM cNP8 solution. The injection was 3 ml/kg body weight of 0.0019 mM cNP8 which is equal to 0.01 mg/kg body weight, c) 1:27000 with PBS to get a 0.00019 mM cNP8 solution. The injection amount was 3 ml/kg body weight of 0.00019 mM cNP8 which is equal to 0.001 mg/kg body weight. The uffer solution was intravenously administrated to each pig - 4 hours or 8 hours after the bums are created on each pig. The room temperature infusion was stered via ear vein over a period of 30 s to the pig. General anesthesia was used during all procedures. Post injury biopsies were collected at various time points for histological analysis to ine percent reepithelialization.

As shown in Figures 7 and 8, re-epithelialization was markedly increased at 10 and 14 days post-injury with cNP8.

Pharmaceutical and Cosmetic Compositions: As an illustration of the invention, several cosmetic formulae will be cited. The formulae are entative of, but do not restrict, the ion: 1 g/100 g White soft paraffin 1.5 Cyclomethicone 6.0 Crodacol C90 0.5 Lubrajel MS 10 Triethanolamine 0.3 Palmitoyl-HISKYILRWRPKNSY-OH 0.0005 Water, preservatives, fragrance q.s. 100 g The gel can be made by dissolving the peptide in the water at 80° C., mixing the first three components (paraffin, silicone and Crodacol) at 80° C., then blending the two phases, cool to 30° C., add the lubrajel, the preservatives and the fragrance. This gel may be used for daily application to the skin of the face, in particular around the eyes to reduce edematous infiltrations.

Cream: 2 g/100 g Volpo S2 2.4 Volpo S20 2.6 Prostearyl 15 8.0 Beeswax 0.5 Stearoxydimethicone 3.0 Propylene glycol 3.0 Carbomer 0.25 Triethanolamine 0.25 Ceramide H03 (SEDERMA) 0.5 Acetyl- HIGKYGLRWRPKNSV-OH 0.001 Water, preservatives, fragrance q.s. 100 g This emulsion can be used to moisturize, restructure and soothe the facial skin, in particular on areas of fragile skin and to treat wrinkles. To produce the emulsion, one can dissolve ceramide H03 in volpo 52, WO 71709 S20 and prostearyl 15 at 85° C., add beeswax and stearoxydimethicone; mix in the other ingredients in the water phase at 75-80° C., then blend the two phases, cool, and add nce. Ceramide H03 is Tirhydroxypalmitamido myristyl ether.

Moisturizing and Anti-Wrinkle Foundation: Compound % (w/w) Demineralized water 53.36 10% KOH 1.30 Polysorbate 80 0.10 Titanium dioxide 6.00 Talc 3.05 Yellow iron oxide 1.80 Red iron oxide 1.00 Black iron oxide 0.15 Propylene glycol 6.00 Magnesium aluminum silicate 1.00 Sodium carboxymethylcellulose 0.12 DiPPG3 myristyl ether e 12.00 Isostearyl neopentanoate 4.00 Crodafos CS 20 4.00 Steareth-10 2.00 Cetyl alcohol 0.50 Steareth-2 0.50 Ceramide 2 (N-stearoyl-0.10 sphinganine) LRWRPKGSV-OH 0.0004 Preservatives q.s.

Subjects can be enrolled in a study on the use of a foundation cream as per above. The wrinkles around the eyes can be evaluated by self-evaluation/questionnaire and by the sion method. The product is applied to the target areas once daily for 56 days. The determinations are conducted on day 0 and day 56. As a control, the sites are treated with the same foundation cream devoid of e and are evaluated for improvement in the symptoms of cutaneous aging.

A number of embodiments of the invention have been described. Nevertheless, it will be understood that s modifications may be made without departing from the spirit and scope of the invention.

Accordingly, other embodiments are within the scope of the following claims.

Claims (15)

1. A polypeptide ing to Formula I: (I): H – X1 - X2 – K – Y – X3 – X4 – R – W – R – P – K – X5 – X6 – X7 wherein X1 is I or G or L, X2 is S or G, X3 is I or G or L, X4 is L or G, X5 is N or G, X6 is absent or S, and X7 is absent or V; and wherein no two consecutive amino acids in the first 13 amino acids of the polypeptide differ from the sequence HISKYILRWRPKN, wherein the polypeptide is optionally substituted with at least one of a lower acyl group at the N-terminus, a lower alkyl-, alkenyl-, l- or haloalkyl- amine group at the C-terminus and polyethylene glycol, wherein the polypeptide is further optionally a linear or branched multimer of Formula I with an al linker between each independently determined sequence of Formula I; or a cyclized form thereof.

2. The polypeptide of claim 1, which is not a linear or ed multimer of Formula I.

3. The polypeptide of claim 2, selected from the group ting of HISKYILRWRPKNSV (P46), HIGKYGLRWRPKNSV (NP7), HIGKYGLRWRPKGSV (NP8), LRWRPKNSV, HIGKYIGRWRPKNSV, HGSKYIGRWRPKNSV, HGSKYIGRWRPKGSV and cyclized forms thereof.

4. The polypeptide of claim 2 or claim 3, selected from the group consisting of HIGKYGLRWRPKGSV (NP8) and cyclized forms thereof.

5. The polypeptide of claim 1, wherein the polypeptide is a linear or branched multimer of Formula I.

6. The polypeptide of claim 5, wherein the linear or ed multimer of Formula I comprises a linker between each independently determined ce of Formula I.

7. The polypeptide of any one of claims 1 to 6, wherein the polypeptide is substituted with at least one of a lower acyl group at the N-terminus, a lower alkyl-, alkenyl-, alkynyl- or haloalkylamine group at the C-terminus, and polyethylene glycol.

8. A ition comprising the polypeptide of any one of claims 1 to 7, and a pharmaceutically acceptable excipient, carrier or diluent.

9. The composition of claim 8, wherein the composition is suitable for a route of administration selected from injection, intravenous administration and topical administration to a patient.

10. Use of the polypeptide of any one of claims 1 to 7, or the composition of claim 8 or claim 9, in the manufacture of a medicament for treating a t with a wound selected from the group consisting of a surgical incision or extirpation, a traumatic injury, a thermal burn, a chemical burn, a lesion or ulceration of the patient's skin, mucosa, connective tissue, fascia, ligament, tendon, cartilage, nerve or muscle and a wound to the t's bone.

11. The use of claim 10, wherein the wound is a thermal burn or a chemical burn.

12. The use of claim 10 or claim 11, wherein the wound is a l burn.

13. The ptide of any one of claims 1 to 7 substantially as hereinbefore described with reference to the es and excluding, if any, comparative Examples.

14. The composition of claim 8 or claim 9 substantially as hereinbefore described with reference to the Examples and excluding, if any, comparative es.

15. The use of any one of claims 10 to 12 substantially as hereinbefore described with reference to the Examples and ing, if any, comparative examples.