Pro-angiogenic peptides and uses thereof

Abstract

This present invention is directed to peptides, compositions, and methods for modulating endogenous cytokine expression in a subject. More specifically, the invention provides peptides useful in regulating the release of a specific pattern of cytokines that promote angiogenesis and/or can be used to modulate the immune system of a subject.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

VNSQH VSNQH (SEQ ID NO:9).

In a second aspect, the present invention provides a therapeutic angiogenic peptide comprising a construct and at least two arms. The construct has a central framework and each arm consists of a core sequence linked to the central framework via a linker. Each core sequence can be the same or different.

As used herein, “construct” is defined as the entire molecule and comprises the central framework linked with at least two arms. In a preferred embodiment, the construct comprises the central framework linked to 2 or more arms, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 arms, preferably 2 to 8 arms. In a further preferred embodiment, the construct comprises the central framework linked to 4 arms. Each arm within the construct may consist of the same or different core sequence and/or linker. In one preferred embodiment, the core sequence is the same between arms.

The “central framework” is defined as the structural core of the construct, providing a structure for attaching the arms to a central structure. The central framework is based on a core molecule which has at least two functional groups to which molecular branches having terminal functional groups are bonded, e.g., a tri-lysine to which the peptide arms. Such molecules may be developed or created to present a varying number of branches, depending on the number of monomers branched from the core molecule. Each non-terminal functional group on each branch provides a means of attachment to an arm. Non-limiting examples of preferred central framework include: ethylenediamine (1,2-ethanediamine), ethylene glycol (1,2-dihydroxyethane), polyols such as glycerol, 3,5-diaminobenzoic acid, 1,3,5-triaminobenzene, and monocarboxylic-diamino compounds of intermediate length. Preferably, the monocarboxylic-diamino compounds are within the range of 2 to 10 carbons in length. Non-limiting examples of such compounds are 2,3-diaminopropionic acid and 2,6-diaminocaproic acid. In a more preferred embodiment, the monocarboxylic-diamino compound is 6 carbons in length. Compounds that provide an aromatic central framework which absorbs light may be beneficial for determining peptide concentration as well. The carboxyl group of the monocarboxylic-diamino compounds allows the addition of C-terminal tags including biotin derivatives. In a preferred embodiment, the central framework comprises a tri-lysine core (a lysine residue as the core molecule bonded to two lysine residues, each through its carboxyl group, to one of the amino groups of the central lysine residue), providing a central frame work for up to four arms.

The “arm” is defined as the core sequence, defined below, plus the linker. The “linker” is defined as a peptide chain or other molecule that connects the central framework to the core sequence. In a preferred embodiment, the linker comprises, but is not limited to, certain linker peptide sequences, polyethylene glycol, 6-aminocaproic acid (6-aminohexanoic acid), 8-aminooctanoic acid, and dextran. In a most preferred embodiment, the linker is GGGS (SEQ ID NO:3), GGGSGGGS (SEQ ID NO:4), SSSS (SEQ ID NO:10), SSSSSSSS (SEQ ID NO:11), or variations thereof. The length of the linker can be adjusted, for example, the linker GGGS (SEQ ID NO:3) can be repeated to provide variable lengths, e.g., repeated twice (GGGSGGGS (SEQ ID NO:4)), or even three or more times; additional serine residues could be added to SSSS (SEQ ID NO:10) to also produce varying lengths of the linker.

The “core sequence” is defined as the functional portion of each arm that provides the therapeutic effect. The core sequence is preferably selected from the group of therapeutic peptides of 5 to 6 amino acids in length described above in the first aspect. In a most preferred embodiment, the core sequence is selected from the group consisting specifically of: WNSTL (SEQ ID NO:1), NQHTPR (SEQ ID NO:2), WNSTY (SEQ ID NO:5), YNSTL (SEQ ID NO:6), YQPSL (SEQ ID NO:7), VQATQS (SEQ ID NO:8), and VNSQH VSNQH (SEQ ID NO:9).

A specific illustration of a therapeutic peptide of the invention is set forth in FIG. 1. In FIG. 1, the therapeutic peptide is in the form of a multivalent immuno-regulatory peptide construct 10. The construct 10 can be synthesized with at least two arms 1, (e.g., two, three, four, eight or more arms 1). The same core peptide sequence 2 can be used for each arm or, alternatively, two or more different core peptide sequences 2 can be used for each arm 1 instead. The length of the linker 3 between the central framework 4 of the construct 10 and the core peptide sequence 2 determines the length of each of the arms 1. The arms 1 illustrated in FIG. 1, for example, are often about 3±0.5 nm in length depending on conformation, or approximately 7±0.5 nm across the molecule. Cell-surface domains of known receptor proteins are correspondingly about 3 to 4 nm in diameter. This distance can be adjusted by increasing or decreasing the length of the linker 3. Preferably, the length of each of the linkers 3 allows for and promotes cross-linking with receptors. The multidimensional nature of the structure illustrated in FIG. 1 was obtained using standard molecular modeling techniques.

In a third aspect, the present invention provides a therapeutic composition, preferably pro-angiogenic. The composition preferably comprises one or more of the therapeutic peptides disclosed herein, and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. There term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which an active ingredient is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Preferably, the pharmaceutically acceptable carrier comprises, but is not limited to, a saline solution, a polyether, and/or water. Examples of suitable carriers, include water, phosphate-buffered saline, sodium chloride solutions, polyethylenelglycol solutions, etc. The type and amount of carrier is typically influenced by the route of administration. For example, when the peptides are administered via injection, preferable carriers comprise, but are not limited to, a phosphate-buffered saline solution having a pH between 6.5 and 7.5 (e.g., about 7.2) or a sodium chloride solution (e.g., 100-150 mM); whereas when administered via a patch, the carrier preferably selected from the group consisting of: polyethyleneglycol solution (e.g., 250 mg/mL of PEG8000), carbopol gel base, propylene glycol, methyl paraben, ethyl paraben, HPMC gel base (hydroxypropylmethyl cellulose), PEG 4000, PEG 300, DMSO, isopropyl myristate, mineral oil, white petrolatum, bees wax, glycerine and water.

In a fourth aspect, the present invention provides a method for modulating the cytokine expression in a subject. Being able to modulate the expression of endogenous cytokines provides a means of regulating and treating a significant number of diseases. The method comprises the steps of administering to the subject one or more of the therapeutic peptides described herein, wherein the peptide is administered in an amount sufficient to increase or decrease the expression of at least one endogenous cytokine in the subject. Preferably, the subject being treated by the method is an animal, more preferably a mammal, e.g., monkey, dog, cat, horse, cow, sheep, pig, and most preferably the subject is human.

In a preferred embodiment, the peptide modulates the expression of at least one cytokine selected from the group consisting of: Eotaxin, Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10, IL-11, IL-15, IL-16, IL-17, IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40, IL-12p70, M-CSF, MCP-2, MIG, PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2. More specifically, preferably the peptides increase the endogenous expression of at least one cytokine selected from the group consisting of: IL-11, IL-12p40, IL-12p70, RANTES, sTNF RI, PDGF-BB, Eotaxin, Eotaxin-2, IL-15, IL-16, IL-17, MCP-2, M-CSF, MIG, TNF-β, sTNF RII, TIMP-2. Most preferably, the peptide increases at least two, three, four, or more of these cytokines. Moreover, expression of these cytokines is preferably increased by at least 20%, more preferably 50%, and most preferably 80%. Sometimes the beneficial endogenous cytokine is increased by even more than 100%.

In addition, preferably the peptide decreases the endogenous expression of at least one cytokine selected from the group consisting of: IL-7, IL-8, Ecotaxin-2, GCSF, ICAM-1, INF-γ, IL-6SR, TIMP-2, MCP-1, and MIP-1b. Likewise, expression of these cytokines is preferably decreased by at least 20%, more preferably 50%, and most preferably 80%. Sometimes the beneficial endogenous cytokine is decreased by even more than 100%.

In a further preferred embodiment, the peptide stimulates the release of those cytokines that induce angiogenesis. In another preferred embodiment, the peptide does not stimulate the release of those cytokines that cause or exacerbate inflammation. In a specific embodiment, the peptide decreases at least one cytokine selected from the group consisting of: IL-1a, IL-8, IL-13, IL-11, IL-12p40, and IL-12p70. It is most preferable that the peptide does not stimulate the release of IL-8. In this context, “does not stimulate” means levels of IL-10 are not statistically greater (preferably p>0.20; more preferably p>0.10; and most preferably p>0.05) between treatments and control samples when examined in experiments similar to those described in Example 3.

Preferably the “amount sufficient” as used herein, is the amount necessary to modulate cytokine expression in a subject. In a more specific embodiment, the amount sufficient is an amount within the range of 1 pmole to 1 nmole per g of body weight and/or within the range of 0.1 to 300 mg per dose. For a typical adult human, the amount sufficient is usually within the range of 1 to 100 mg, more preferably, 1 to 70 mg, and most preferably 1 to 50 mg per dose. Based on the subject's body weight, preferably the amount is 0.01 to 1.4 mg/kg; more preferably 0.01 to 1 mg/kg; and most preferably between 0.01 to 0.7 mg/kg of the subject's body weight per dose. As a nonlimiting example, an amount sufficient to treat the disease in a typical 70 kg adult human would be 0.1 mg/kg of the subject's body weight, 2 μmole, or 7 mg per dose. As would be known to one skilled in the art, the lifetime of activated macrophages suggests that a dose should be administered once about every 2 to 6 days, more preferably 1 or 2 times a week, until the disease is treated, resulting in an improvement of at least one symptom, and preferably by eradication from the body of the subject.

In a fifth aspect, the present invention provides a method for stimulating wound healing via induction of angiogenesis in a subject. The method comprises the steps of administering to the subject a composition comprising a peptide of the invention, wherein the peptide is in an amount sufficient to stimulate angiogenesis in an area substantially near a subject's wound. An area substantially near a subject's wound” refers to the area of the body, preferably within 6 inches, more preferably within 4 inches, and most preferably within 2 inches from the outer edge of the wound being treated. Preferably, the subject being treated by the method is an animal, more preferably a mammal, and most preferably a human.

Administration of the composition to the subject comprises transferring the composition into the body of the subject in an amount sufficient to stimulate wound healing via induction of angiogenesis. The composition of the invention can be administered via any suitable route that achieves the intended purpose. For example, administration can be by subcutaneous, intravenous, intramuscular, intraperitoneal, buccal, or ocular routes, rectally, parenterally, intrasystemically, topically (as by powders, ointments, drops or transdermal patch), or as an oral or nasal spray. Alternatively, or concurrently, administration can be by the oral route. The dosage administered will be dependent upon the age, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired.

In a preferred embodiment, the composition is administered orally. In this embodiment, the composition is in an edible form, including for example, powders, granules, capsules, pills, tablets, elixirs, suspensions, emulsions, syrups and the like. These preparations may be subjected to modification such as sustained-release, stabilization, easy disintegration, poor disintegration, enteric coating, easy absorption and the like. Preferably in this embodiment, the composition is in a form that allows for passage through the stomach and release in the intestine for absorption in intestinal lumen, e.g., enteric coated formations based on pH or timed release. Additionally, the dosage may be in the form of chewable preparations, sublingual preparations, buccal preparations, troches, ointments, patches, solutions and the like. These preparations may be also subjected to modification such as sustained-release, stabilization, easy disintegration, poor disintegration, enteric coating, easy absorption and the like.

In another embodiment, the composition is administered via injection, e.g., subcutaneous, intramuscular, intravenous, and intraperitoneal injection, preferably subcutaneously. When the composition is formulated for transdermal administration, the composition preferably comprises PEG8000, but may be comprised of other suitable carriers such as carbopol gel base, propylene glycol, methyl paraben, ethyl paraben, HPMC gel base, PEG 4000, PEG 300, DMSO, isopropyl myristate, mineral oil, white petrolatum, bees wax, glycerine and water in a medical patch. The patch preferably comprises 1 to 8 mg, more preferably 2 to 6 mg, and most preferably about 4 mg of therapeutic peptides per mL of solution in the patch. A patch typically comprises 1 to 75 mL, and more preferably 1 to 18 mL of solution within the patch. When administering to the subject, the patch should be in contact with the subject's skin for a period of at least 2 to 72 hours. A typical patch would be in contact with the subject's skin for approximately 24 to 48 hours.

As would be known by one skilled in the art, the PDGF class of cytokines is instrumental in the process of angiogenesis. Thus, in a preferred embodiment, the stimulation of wound healing via the induction of angiogenesis increases the production of cytokines comprising but not limited to the PDGF class. In a further preferred embodiment, the administration of the composition stimulates the production of the cytokine PDGF-BB. “Stimulates the production” in this instance means PDGF-BB production is preferably increased by at least 20%, more preferably 50%, and most preferably 80%. Data from TABLE 3 suggests PDGF-BB production may increase by at least 90%.

Preferably the administration of the compositions of the invention does not cause inflammation in the subject. As would be known by one skilled in the art, inflammation in response to a wound is often caused by the release of the cytokines IL-8 and ICAM-1 in the body. Thus, in a further preferred embodiment, the administration of the composition inhibits the production of cytokines that cause inflammation or at least does not increase the production of these cytokines. More preferably, the administration of the composition inhibits the production of IL-8. In certain embodiment, the composition actually promotes the release and/or production of anti-inflammatory cytokines.

As would be known by one skilled in the art, selective cross-linking of cell-surface receptors by a multivalent structure incorporating at least one peptide embodiment of the present invention may attenuate bacterial infections by stimulating activity of phagocytic cells that are recruited to the injured or infected tissue. Phagocytic cells respond to the presence of bacterial cells containing lipopolysaccharide (LPS) on their surface by engulfing the cells into phagocytic vacuoles and digesting the bacterial cells. In addition, the phagocytic cells respond to the presence of antibodies directed toward—and bound to—a pathogen such as a bacterial cell, fungal cell or virus. Thus, in another preferred embodiment, the administration of the composition enhances the ability of the subject's immune system to ward off or attenuate infection. In a most preferred embodiment, the infection is attenuated or prevented at or substantially near the target wound area. An area substantially near a subject's wound” refers to the area of the body, preferably within 6 inches, more preferably within 4 inches, and most preferably within 2 inches from the outer edge of the wound being treated.

EXAMPLES

Elements and acts in the examples are intended to illustrate the invention for the sake of simplicity and have not necessarily been rendered according to any particular sequence or embodiment. The examples are further intended to establish possession of the invention by the Inventors.

Example 1

Peptide Design and Synthesis

A screen of peptide sequences identified one set of sequences of interest. The corresponding peptides were synthesized by solid-phase methods using standard Fmoc side chain protection. Branched peptides were constructed on a central tri-lysine framework, which allows four identical sequences within the same structure. A (Gly)₃-Ser (GGGS, SEQ ID NO:3) linker sequence was included to distance the active sequence from the central framework. Distances between the active sequences can be adjusted by decreasing or increasing the length of the linker, including without limitation the use of two linkers in tandem (GGGSGGGS, SEQ ID NO:4) or by inserting any inert linker such as polyethylene glycol (PEG) of a variable length. The branched structure was designed to have enhanced activity by causing receptor clustering (cross-linking) on the surface of responsive cells.

The peptides were synthesized on PAL-PEG-polystyrene resin (Applied Biosystems, Foster City, Calif.) utilizing Fmoc (9-fluorenylmethoxycarbonyl)-protected amino acids and a Milligen Biosearch 9050+ continuous flow peptide synthesizer (Millipore Corporation, Billerica, Mass.).

The C-terminus of the central framework is typically a lysine residue containing an amide derivative of the carboxyl group. However, the C-terminus can be modified to include additional C-terminal amino acids such as a cysteine residue, to which tags such as fluorescent groups can be added, or an ϵ-biotinyl-N-lysine (biotinyl-K) residue useful for subsequent purification processes. In addition, an amino acid such as β-alanine (βA) or tryptophan can be inserted between the added C-terminal amino acid and the C-terminal lysine residue of the central framework in order to provide a spacer or a means to determine concentration by absorbance. Non-limiting examples of such modified C-terminal lysine residues on the central framework include K-βA-C and K-W-biotinyl-K, respectively. Furthermore, additional lysine residues can be added to either one or both of the α- and ϵ-amino groups of a modified C-terminal lysine on the central framework to yield, for example, (K)₂K, (K)₂K-βA-C or (K)₂K-W-biotinyl-K, thereby forming branched structures in which the α- and ϵ-amino groups are available for extension.

The lysine residues used at the branch points are incorporated with Fmoc protection on both the α- and ϵ-amino groups, so that both become available for amide bond formation after the standard deprotection reaction with piperidine. A dansyl group (or other fluorescent tag) may be incorporated by reaction with the thiol group on the C-terminal cysteine residue using 5-((((2 iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS) following a standard protocol for thiol-reactive probes (Invitrogen Corp., Carlsbad, Calif.). Biotin is attached to lysine through an amide linkage to the side chain amino group which, because of its high affinity with streptavidin, provides a means to retrieve the peptide with associated proteins from reaction mixtures in order to study the interaction of the peptide with cellular components.

The peptides were built attached to a solid-phase resin, which was chosen such that when the peptide is cleaved from the resin, the carboxyl group at the C-terminus of the peptide is released as the amide. Each of the four amino groups of the tri-lysine central framework was extended by addition of the linker, GGGS (SEQ ID NO:3), followed by the core sequence.

After cleavage from the resin bed, the product can be substantially or completely purified by HPLC on a preparative Jupiter Proteo C12 column (21.2 mm×250 mm) (Phenomenex, Inc., Torrance, Calif.) using a gradient from 8% to 18% acetonitrile in water containing 10 mM trifluoroacetic acid (TFA). The purity of the final peptide product was confirmed by mass spectroscopy performed using a Voyager DE STR mass spectrometer (Applied Biosystems, Foster City, Calif.). HPLC-purified peptide was dried under vacuum, dissolved in sterile phosphate buffered saline, pH 7.2 (PBS) and passed through a gel filtration column of Sephadex G 15 or G 25 (1×48 cm for small samples) to separate TFA from the peptide. The column is then eluted with sterile PBS. Endotoxin is removed by passage of the peptide through a DEAE-Sephadex A-25 column.

Alternatively, the product is purified by use of a C18 reverse-phase cartridge, ion exchange chromatography, and gel filtration chromatography to remove side products of synthesis. Concentration can be determined by absorbance of the fluorophore (e.g., dansyl group, extinction coefficient, ϵ_mM=5.7 cm⁻¹ at 336 nm), absorbance of the peptide bond at 210 nm (ϵ_mg/mL≈31 cm⁻¹), absorbance of aromatic amino acids (e.g., tryptophan, ϵ_mM=5.6 cm⁻¹ at 280 nm) in the peptide (when present) and/or absorbance of the bicinchoninic acid reagent (Pierce Biotechnology, Rockford, Ill.). The peptide solutions can be adjusted to the desired concentration and filter-sterilized prior to use.

Example 2

Core Peptide Sequences: WNSTL (SEQ ID NO:1) and NQHTPR (SEQ ID NO:2)

The peptides (WNSTL, SEQ ID NO:1) and (NQHTPR, SEQ ID NO:2) were identified through a screen of peptide sequences as potentially of interest. The sequences were synthesized on a tri-lysine core according to Posnett et al., J. Biol. Chem., 263: 1719-25, 1988, with a linker (GGGS, SEQ ID NO:3) included with the sequence to extend the active peptide away from the core.

FIGS. 2A-B illustrate the chemical structure of these two embodiments of the invention. In these embodiments, R═H or can be an adduct containing a fluorescent tag such as the dansyl group shown in FIG. 3. The peptide constructs illustrated contain four identical sequences, each of which is connected to a branched central tri-lysine framework via a (Gly)₃Ser (GGGS, SEQ ID NO:3) linker. FIG. 2A is a branched peptide construct according to one embodiment of the invention, a peptide construct which contains four copies of the core sequence WNSTL (SEQ ID NO:1). The peptide embodiment containing R═H has a molecular mass of 3,841.16 Daltons.

FIG. 2B illustrates the structure of the construct containing four copies of the N-terminal core sequence NQHTPR (SEQ ID NO:2). This peptide embodiment containing R═H has a molecular mass of 4,543.89. In another embodiment, a C-terminal extension containing β-alanine, cysteine and a dansyl tag, as shown in FIG. 3, is covalently added to the construct illustrated in FIG. 2B, resulting in a peptide having a molecular mass of 4,850.23 Daltons.

Example 3

Regulation of Cytokine Release

To determine whether the peptides regulate induction or inhibition of release of cytokines, cultured peripheral blood mononuclear cells (PBMCs) were treated with one peptide embodiment of the invention and, after 4 hour incubation, the medium was collected and assayed for changes in the amounts of 40 different cytokines. The peptide construct containing four copies of WNSTL (SEQ ID NO:1), illustrated in FIG. 2A, was added at a concentration of 100 nM in each of the assays. The PBMC cultures were established with cells from Cellular Technology, Ltd. (Shaker Heights, Ohio). Approximately 3 million cells of frozen human PBMCs were thawed at 37° C. and transferred to a 50 mL conical tube where 8 mL of wash medium were added slowly. Then an additional 8 mL were added and swirled to mix. The cells were then centrifuged at 330 g for 10 min, the supernatant was removed and the pellet was resuspended in 10 mL wash medium and centrifuged as above. The washed cells were then resuspended in RPMI 1640 medium containing 10% FBS to about 6 million cells per mL and 100 μL of the suspension were added into each well of a 96-well microtiter plate and incubated overnight at 37° C. in humidified 5% CO₂. After 24 hr, the medium was replaced with 200 μL fresh RPMI 1640 medium containing 10% FBS and incubated at 37° C. in humidified 5% CO₂ for 2 days. For the data shown in TABLE 1, an aliquot of the peptide construct was added to samples in duplicate at a final concentration of 5 nM or 100 nM and incubated at 37° C. in humidified 5% CO₂ for 4 hr. For other experiments (data not included), the incubation was continued for 24 hr. The medium was then removed and stored at 80° C. The samples were analyzed for production of cytokines. One set of control cells was not treated with an experimental agent. A second set of control cells was treated with LPS, the agent commonly used to induce production of a variety of inflammatory cytokines. The positive control for inflammation provided by this second set of control cells was essential to ensure that the peptides do not produce an unregulated inflammatory response.

Assays of cytokine levels in samples of culture media were performed using methods developed by RayBiotech, Inc. (Norcross, Ga.). In this technology, membrane arrays of antibodies against cytokines were incubated with samples of media. After washing, the array was incubated with a cocktail of all antibodies tagged with biotin. The membrane was then washed free of unbound antibodies and incubated with streptavidin labeled with a fluorescent dye. After a final wash, the membrane arrays were read in a fluorescence detector and the intensities of the spots quantitated to obtain relative values.

TABLES 1-3 list a number of cytokines whose concentrations in a medium of PBMC cultures can be altered as the result of treatment of the cells with peptide embodiments of the present invention. The cytokines thus affected include without limitation:

Eotaxin (chemoattractant, induces substantial accumulation of eosinophils);

Eotaxin-2 (induces chemotaxis of eosinophils and basophils, release of histamine);

GCSF (granulocyte colony stimulating factor, growth factor);

ICAM-1 (intercellular adhesion molecule-1, binds to integrins, human rhinovirus receptor);

IL-1β (Interleukin 1β, a mediator of inflammatory reactions);

IL-4 (promotes proliferation and differentiation of B-cells and inhibits production of inflammatory cytokines such as IL-1, IL-6 and TNF-α);

IL-6SR (soluble receptor for IL-6);

IL-7 (stimulates proliferation of precursor B and T cells);

IL-8 (chemoattractant and activator of neutrophils);

IL-10 (inhibits synthesis of inflammatory cytokines such as INF-γ, IL-2 and TNF-β);

IL-11 (induces inflammatory responses, promotes immune responses);

IL-12 (contains subunits of 40 and 70 kDa, activates NK-cells and stimulates proliferation of lymphoblasts);

IL-15 (has many of the same properties as IL-2, may contribute to T-cell mediated immune responses);

IL-16 (chemoattractant and activator for cells that express CD4);

IL-17 (functions as a mediator of angiogenesis that stimulates migration of vascular endothelial cells and cord formation and regulates production of a variety of growth factors promoting angiogenesis);

INF-γ (Interferon-gamma, has antiviral, immunoregulatory and anti-tumor properties);

MCP-1,2 (monocyte chemotactic proteins);

M-CSF (induces proliferation and stimulates monocytes and macrophages); MIG (chemoattractant for stimulated T cells but not active on neutrophils or monocytes);

MIP-1b (macrophage inflammatory protein, involved in cell activation of granulocytes and killer cells);

PDGF-BB (platelet-derived growth factor, BB homodimer);

RANTES (regulated on activation, normal T cell expressed, and secreted; chemotactic for T cells);

sTNF RI, Rh (soluble forms of receptor RI or RII for tumor necrosis factor (TNF));

TIMP-2 (tissue inhibitor of metalloproteinases of the extracellular matrix); and

TNF-β (promotes the proliferation of fibroblasts and is involved in wound healing).

TABLE 1 contains data showing cytokines that are released at a significantly higher or lower rate (compared to untreated controls) during a 4-hour incubation of PBMCs with the branched peptide construct containing four copies of WNSTL (SEQ ID NO:1) in the presence of serum. One set of control samples was not treated with peptide and a second set of control samples was treated with lipopolysaccharide (LPS) in the absence of the peptide construct. The structure of the construct is illustrated in FIG. 2A. Among the cytokines that are induced to more than two-fold higher concentrations as a result of incubation with the construct containing four copies of WNSTL (SEQ ID NO:1) are PDGF-BB, IL-1β, IL-4, IL-1, IL-12 and RANTES. In contrast, several cytokines show more than a two-fold decrease in concentration as compared to untreated control samples with this peptide. The decrease in IL-8 concentration in the peptide-treated sample, as compared with the amount of IL-8 in the sample treated with the proto-typical inflammatory agent LPS, is particularly notable. Furthermore, when compared to untreated control samples, the peptides did not induce a change in the amount of the inflammatory cytokine IL-6. However, IL-6 levels were significantly elevated in samples treated with LPS in the absence of peptide. In one experiment, for example, the peptide treated sample had a relative IL-6 concentration of 93, the untreated control sample, 98, and the LPS-treated sample (in the absence of peptide), 5,879. Because the concentration of IL-6 in the peptide treated sample was not significantly different from the untreated control sample, the data for IL-6 are not included in TABLES 1 or 2. The increase in the proangiogenic PDGF-BB and the decrease in the inflammatory IL-8, without concomitant stimulation of inflammation, are of particular importance with respect to wound healing.

TABLE 1
Relative Cytokine Concentration after Incubation of PBMCs in
Serum with a Peptide Construct Containing Four Copies of the
Core Sequence WNSTL (SEQ ID NO: 1).
	WNSTL
Cytokine	(SEQ ID NO: 1)	Untreated (control)	LPS (control)
	Increased:
PDGF-BB	159	43	56
IL-1β	120	44	47
IL-4	97	30	49
IL-11	49	17	26
IL-12p40	228	108	46
IL-12p70	131	90	89
RANTES	145	80	114
STNF RI	78	42	58
	Decreased:
IL-7	93	154	178
IL-8	144	417	840
IL-10	43	101	218
Eotaxin-2	109	1934	469
GCSF	50	108	120
ICAM-1	44	57	53
INF-γ	103	134	158
IL-6sR	32	52	52
TIMP-2	25	58	92
MCP-1	968	1464	1844

Relative cytokine concentration data for the peptide construct containing four copies of the core sequence NQHTPR (SEQ ID NO:2) is similarly outlined in TABLE 2. The structure of the construct is illustrated in FIG. 2B. Cytokines were again observed at significantly higher or lower concentrations (relative to controls) after a 4-hour incubation with the branched construct containing four copies of the core sequence NQHTPR (SEQ ID NO:2). The data in TABLE 2 show that, although the overall pattern of cytokine response to this peptide is somewhat different from that of the quadravalent peptide construct containing the core sequence WNSTL (SEQ ID NO:1) shown in TABLE 1, it similarly induces a higher amount of PDGF and a lower amount of IL-8.

TABLE 2
Relative Cytokine Concentration after Incubation of PBMCs in
Serum with the Peptide Construct Containing Four Copies of the
Core Sequence NQHTPR (SEQ ID NO: 2).
	Cytokine	NQHTPR (SEQ ID NO: 2)	Untreated (control)
	Increased:
	PDGF-BB	84	43
	Eotaxin	57	32
	Eotaxin-2	310	193
	IL-15	146	97
	IL-16	8	1
	IL-17	14	5
	MCP-2	90	56
	M-CSF	116	44
	MIG	100	54
	TNF-β	84	38
	sTNF RII	26	8
	TIMP-2	110	58
	Decreased:
	IL-8	261	417
	MIP-1b	777	1172

TABLE 3 is based on the same data as TABLES 1-2, and shows the effects of constructs containing four copies of VQATQS (SEQ ID NO:8), VSNQH (SEQ ID NO:9), NQHTPR (SEQ ID:2) and WNSTL (SEQ ID NO:1) on the relative concentrations of cytokines in PBMCs treated for four hours with each peptide construct as compared with untreated control cultures and LPS-treated cells.

TABLE 3
Relative Concentrations of Cytokines in PBMCs Treated for
4 hr with each Peptide Construct as Compared with Untreated
Control Cultures and LPS-treated Cells.*
	Core Sequence of Peptide
Cytokine	VQATQS	VSNQH	NQHTPR	WNSTL	Untreated	LPS
Eotaxin		47	57		32	31
Eotaxin-2	401	470	310	109	193	469
GCSF				50	108	120
GM-CSF		96			57	106
ICAM-1		70		44	57	53
IFN-γ		170		103	134	158
I-309	101				26	39
IL-1α		322			225	246
IL-1β				120	44	47
IL-2					86	90
IL-3					130	132
IL-4	64			97	30	49
IL-6	167				98	4,375
IL-6sR				32	52	52
IL-7				93	154	178
IL-8			261	144	417	840
IL-10	67			43	101	218
IL-11				49	17	26
IL-12p40	74	52	45	228	108	46
IL-12p70	112	117	65	131	90	89
IL-13					138	125
IL-15	134	137	146		97	104
IL-16	10		8		1	2
IL-17	21	21	14		5	10
IL-21	90	130	200	—	50	(IFN-γ: 100)
IP-10	334	377			230	268
MCP-1	1,966			968	1,464	1,844
MCP-2		97	90		56	177
M-CSF	90		116		44	59
MIG		94	100		54	66
MIP-1a					81	237
MIP-1b			777		1,172	1,828
MIP-1d					40	38
RANTES				145	80	114
TGF-β1	83				62	62
TNF-α	98	138			73	93
TNF-β	91	93	84		38	95
sTNF RI	75			78	42	58
sTNF RII	20		26		8	26
PDGF-BB	83	82	84	159	43	56
TIMP-2	175	285	110	25	58	92
*The absence of a number indicates no significant change from untreated control cultures.

Toxicity of the peptide in vivo can be tested by injection of a peptide into animal subjects, including without limitation mice. The peptides can be administered in a number of ways, including without limitation by injection (intravenously, subcutaneously, intramuscularly or intraperitoneally), topically (transmucosally, transbuccally, or transdermally) and/or orally (liquid, tablet or capsule). In preliminary studies on mice, no adverse effects of the peptide on the growth rate of the animals have been observed after injection of an effective dose on alternate days for 1 month (data not shown).

Example 4

Pro-Angiogenic Activity of Peptides

Tumors require vascularization to obtain nutrients to support growth. Therefore, stimulation of growth of a tumor in response to administration of a construct of this invention is an indication of angiogenesis. For this example, a xenograph model system with the nude mouse (nu/nu) was used to determine the effect of peptide on growth of 786-0 human renal cell adenocarcinoma cell line injected into the flank of a mouse so as to induce a tumor. After the tumor was established, peptide was injected subcutaneously on alternate days. The weight of the tumor was estimated by calculation of the volume.

FIG. 4 illustrates data resulting from assays for pro-angiogenic activity for one embodiment of the invention. The bar graph in FIG. 4 shows the average weight of the tumor in mice treated with the peptide construct containing four copies of the core sequence WNSTL (SEQ ID NO:1) at a dose of 0.05 nmole/gm as compared with a control group. The peptide was also assayed in combination with Sorafenib, an anti-angiogenic drug. The results shown in FIG. 4 indicate that growth of the tumor was significantly enhanced, by a factor of 1.7, over the control in mice to which the construct with four copies of the core sequence WNSTL (SEQ ID NO:1) was administered. The drug Sorafenib, an inhibitor of angiogenesis, strongly (but not completely) inhibited the effect of the peptide.

Example 5

Stimulation of Phagocytosis by Peptides

The activity of the peptides to stimulate phagocytosis was assessed by the ability of macrophages to engulf microspheres opsonized with anti-HIV antibodies. A biotin-tagged peptide epitope of a surface protein of HIV-1 was bound to streptavidin on the surface of the beads. An antibody preparation that was raised against this epitope was then bound to the HIV peptide. The beads were then washed and presented to PBMC cultures pretreated with peptides. Macrophages in cultures not treated with peptides had little, if any, phagocytic activity. In multiple control cultures, the number of beads within a macrophage-like cell ranged from 0 to 3. In the cultures treated with peptide constructs containing four copies of the peptide sequences WNSTL (SEQ ID NO:1), NQHTPR (SEQ ID NO:2), or VQATQS (SEQ ID NO:8), an average of 10 beads were counted in each phagocytic cell, with no substantial difference between peptide constructs. These findings suggest a stimulation of phagocytic activity by the peptide constructs compared to untreated cultures. Cultures treated with the cytokine INF-γ exhibited an average of roughly 15 beads per phagocytic cell.

Example 6

Devices or Materials Containing the Peptides

The peptides of the present invention may also be deposited to provide an appropriate coating to a surface, including without limitation bioactive surfaces or inert, non-biological surfaces of a device or materials designed for implantation. The peptides can thus promote healing around the implanted materials in order to achieve vascularization without scarring. They could similarly be used in other in vitro or in vivo applications, including without limitation, with embedded sensors.

The embodiments and examples set forth herein were presented in order to best explain the present invention and its practical application and to thereby enable those of ordinary skill in the art to make and use the invention. Although the examples herein disclose the therapeutic efficacy of the peptides of the present invention with respect to wound healing, for example, the peptides may also be useful for restoration of circulation generally, including circulation compromised by chronic conditions such as diabetes, circulation to damaged tissues, and other similar disorders. Furthermore, the use of larger peptides containing active core sequences could potentially enhance the therapeutic benefits disclosed herein.

Claims

1. A therapeutic peptide consisting of between 5 to 6 amino acids and a peptide sequence selected from the group consisting of:

X1-Q-X2-X3-X4-X5; and

X1-N-S-X3-X4-X5

wherein X1 is selected from the group consisting of N, V, W, and Y;

X2 is selected from the group consisting of H, A, and P;

X3 is selected from the group consisting of T, Q, and S;

X4 is selected from the group consisting of P, Q, H, L, and Y; and

X5 is selected from the group consisting of R and S, or is absent.

2. The therapeutic peptide of claim 1, in a substantially pure form of at least 80% by weight.

3. The therapeutic peptide of claim 1, wherein the N-terminus is acetylated and the peptide is pro-angiogenic.

4. A therapeutic composition comprising the therapeutic peptide of claim 1 and a pharmaceutically acceptable carrier.

5. A method of modulating a cytokine expression in a subject, the method comprising administering to a subject one or more therapeutic peptides of claim 1, wherein the therapeutic peptide is administered in an amount sufficient to modulate the expression of at least one endogenous cytokine.

6. The method of claim 5, wherein the at least one cytokine is selected from the group consisting of: Eotaxin, Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10, IL-11, IL-15, IL-16, IL-17, IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40, IL-12p70, M-CSF, MCP-2, MIG, PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2.

7. The method of claim 5, wherein the therapeutic peptide increases the endogenous expression of at least one cytokine selected from the group consisting of: IL-11, IL-12p40, IL-12p70, RANTES, sTNF RI, PDGF-BB, Eotaxin, Eotaxin-2, IL-15, IL-16, IL-17, MCP-2, M-CSF, MIG, TNF-β, sTNF RII, and TIMP-2; and/or decreases the endogenous expression of at least one cytokine selected from the group consisting of: IL-7, IL-8, IL-10, Eotaxin-2, GCSF, ICAM-1, INF-γ, IL-6sR, TIMP-2, MCP-1, and MIP-1b.

8. A method of stimulating wound healing in a subject via induction of angiogenesis, the method comprising: administering the composition of claim 4 to the subject in an amount sufficient to stimulate angiogenesis of a wound area.

9. The method of claim 8, wherein the method comprises modulating the release of a profile of cytokines in the subject, comprising increasing the release in the subject of at least one therapeutically beneficial cytokine and/or inhibiting in the subject the production or release of at least one therapeutically deleterious cytokine.

10. The method of claim 9, further comprising stimulating the activity of phagocytic cells and modulating the immune system of that subject to further promote wound healing and decrease inflammation of a wound area.

11. The method of claim 9, wherein the cytokine profile released is indicative of a reduction of inflammation.

12. The method of claim 9, wherein the at least one beneficial cytokine is PDGF-BB and the at least one deleterious cytokine is IL-8.

13. The method of claim 9, wherein the administration of the composition enhances the ability of the immune system to ward off or attenuate infection at the wound.

14. The method of claim 9, wherein the amount is within the range of 1 pmole to 1 nmole per g of body weight of the subject.

15. The method of claim 9, wherein the amount administered is within the range of 0.1 to 300 mg per dose, and the dose is administered one or two times per week.

16. The method of claim 9, wherein the composition is in a formulation suitable for oral administration to the subject, the dosage designed to pass through the subject's stomach before releasing the peptide in the subject's intestine for absorption through the intestinal epithelium.

17. The method of claim 9, wherein the composition is administered via a patch comprising: polyethyleneglycol, propylene glycol, methyl paraben, ethyl paraben, hydroxypropylmethyl cellulose, DMSO, isopropyl myristate, mineral oil, white petrolatum, bees wax, or glycerine.

18. A therapeutic composition comprising:

a pharmaceutically acceptable carrier; and

a therapeutic peptide having the peptide sequence of VSNQH (SEQ ID NO:9) or a polypeptide comprising a construct having a central framework and at least two arms consisting of a core sequence having the peptide sequence of VSNQH (SEQ ID NO:9), wherein the core sequence is linked to the central framework via a linker.

19. The therapeutic composition of claim 18, wherein the central framework consists of a tri-lysine core and the linker is selected from the group consisting of GGGS (SEQ ID NO:3), GGGSGGGS (SEQ ID NO:4), SSSS (SEQ ID NO:10), and SSSSSSSS (SEQ ID NO:11).

20. The therapeutic composition of claim 18, wherein the polypeptide comprises four arms.

21. The therapeutic composition of claim 18, wherein the therapeutic peptide has at least two arms consisting of a core sequence having the peptide sequence of VSNQH (SEQ ID NO:9) and wherein the core sequence is linked to the central framework via a linker.

22. The therapeutic polypeptide of claim 21, wherein the central framework consists of a tri-lysine core and the linker is selected from the group consisting of GGGS (SEQ ID NO:3), GGGSGGGS (SEQ ID NO:4), SSSS (SEQ ID NO:10), and SSSSSSSS (SEQ ID NO:11).

23. The therapeutic composition of claim 18, wherein the polypeptide comprises eight arms.

24. A therapeutic peptide having a peptide sequence of VSNQH (SEQ ID NO:9) or a polypeptide comprising a construct having a central framework and at least two arms consisting of a core sequence having the peptide sequence of VSNQH (SEQ ID NO:9), wherein the core sequence is linked to the central framework via a linker.

25. The therapeutic peptide of claim 24, in a substantially pure form of at least 80% by weight.

26. The therapeutic peptide of claim 24, wherein the N-terminus is acetylated and the peptide is pro-angiogenic.

27. A method of modulating a cytokine expression in a subject, the method comprising administering to a subject one or more therapeutic peptides of claim 24, wherein the therapeutic peptide is administered in an amount sufficient to modulate the expression of at least one endogenous cytokine.

28. The method of claim 27, wherein the at least one cytokine is selected from the group consisting of: Eotaxin, Eotaxin-2, ICAM-1, 1-309, IL-4, IL-8, IL-10, IL-11, IL-15, IL-16, IL-17, IL-21, RANTES, sTNF RI, sTNF RII, IL-12p40, IL-12p70, M-CSF, MCP-2, MIG, PDGF-BB, TNF-β, MIP-1b, GCSF, and TIMP-2.

29. The method of claim 27, wherein the therapeutic peptide increases the endogenous expression of at least one cytokine selected from the group consisting of: IL-11, IL-12p40, IL-12p70, RANTES, sTNF RI, PDGF-BB, Eotaxin, Eotaxin-2, IL-15, IL-16, IL-17, MCP-2, M-CSF, MIG, TNF-β, sTNF RII, and TIMP-2; and/or decreases the endogenous expression of at least one cytokine selected from the group consisting of: IL-7, IL-8, IL-10, Eotaxin-2, GCSF, ICAM-1, INF-γ, IL-6sR, TIMP-2, MCP-1, and MIP-1b.

30. A method of stimulating wound healing in a subject via induction of angiogenesis, the method comprising: administering the composition of claim 18 to the subject in an amount sufficient to stimulate angiogenesis of a wound area.

31. The method of claim 30, wherein the method comprises modulating the release of a profile of cytokines in the subject, comprising increasing the release in the subject of at least one therapeutically beneficial cytokine and/or inhibiting in the subject the production or release of at least one therapeutically deleterious cytokine.

32. The method of claim 31, further comprising stimulating the activity of phagocytic cells and modulating the immune system of that subject to further promote wound healing and decrease inflammation of a wound area and/or the cytokine profile released is indicative of a reduction of inflammation and the at least one beneficial cytokine is PDGF-BB and the at least one deleterious cytokine is IL-8.

33. The method of claim 31, wherein the administration of the composition enhances the ability of the immune system to ward off or attenuate infection at the wound.

34. The method of claim 31, wherein the amount is within the range of 1 pmole to 1 nmole per g of body weight of the subject.

35. The method of claim 31, wherein the amount administered is within the range of 0.1 to 300 mg per dose, and the dose is administered one or two times per week.

36. The method of claim 31, wherein the composition is in a formulation suitable for oral administration to the subject, the dosage designed to pass through the subject's stomach before releasing the peptide in the subject's intestine for absorption through the intestinal epithelium.

37. The method of claim 31, wherein the composition is administered via a patch comprising: polyethyleneglycol, propylene glycol, methyl paraben, ethyl paraben, hydroxypropylmethyl cellulose, DMSO, isopropyl myristate, mineral oil, white petrolatum, bees wax, or glycerine.