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.
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1. A therapeutic peptide consisting of between 5 to 6 amino acids and having 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.
3. The therapeutic peptide of
4. A therapeutic composition comprising the therapeutic peptide of
5. A method of modulating a cytokine expression in a subject, the method comprising administering to a subject one or more therapeutic peptides of
6. The method of
7. The method of
8. A method of stimulating wound healing in a subject via induction of angiogenesis, the method comprising: administering the composition of
9. The method of
10. The method of
11. The method of
12. The method of
13. The method of
14. The method of
15. The method of
16. The method of
17. The method of
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The present application 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
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.
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.
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, εmM=5.7 cm−1 at 336 nm), absorbance of the peptide bond at 210 nm (εmg/mL≈31 cm−1), absorbance of aromatic amino acids (e.g., tryptophan, εmM=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.
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.
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
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
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
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
Un-
Cytokine
VQATQS
VSNQH
NQHTPR
WNSTL
treated
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).
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.
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.
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.
Eggink, Laura L., Hoober, J. Kenneth
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