HK1152717B - Angiogenic peptide - Google Patents

Description

Angiogenic peptides

Technical Field

The present invention relates to angiogenic peptides that cause intracellular calcium release in target cells and thereby induce proliferation, migration and capillary-like tube formation in primary cultured endothelial cells. In addition, the angiogenic peptides may be used for the prevention and/or treatment of angiogenesis-related disorders, in particular wound healing, treatment of foot and leg ulcers in a subject, and the like. In addition, the angiogenic peptides can be used in cosmetics as cosmetic components for skin aging, e.g., for anti-wrinkle and skin whitening.

Background

Angiogenesis, the outgrowth of new blood vessels from existing vasculature, is an essential process in a variety of physiological and pathological conditions including wound healing, embryonic development, chronic inflammation and tumor progression and metastasis (J.Folkman, Nat.Med.1(1995), pp.27-31; W.Risau, Nature 386(1997), pp.671-674). During angiogenesis, complex cellular processes occur, including extracellular matrix degradation, proliferation, migration and morphological differentiation of endothelial cells to form tubes. This entire process is coordinated by local factors that regulate neovascularization (f. bussolino, et al, Trends biochem. sci.22(1997), pp.251-256), and changes in angiogenic balance can mediate the "angiogenic switch". Interference with the angiogenesis switch can cause serious vascular problems.

Calcium plays a key role in the signaling events induced by a variety of extracellular stimuli and in the coordination of a large number of different cellular functions (m.j.berry, Nature 361(1993), pp.315-325; k.kiselyov, et al, Cell Signal 15(2003), pp.243-253). Intracellular Ca²⁺The increase is essential for the adhesion, collagenolytic activity, migration and proliferation of human endothelial cells, and for capillary growth in vivo (e.c.kohn, et al, proc.natl.acad.sci.usa92(1995), pp.1307-131). Indeed, Vascular Endothelial Growth Factor (VEGF), a Receptor Tyrosine Kinase (RTK) ligand, and sphingosine-1-phosphate (S1P), a G protein-coupled receptor (GPCR) ligand, are mediated by the modulation of intracellular Ca²⁺The levels induce neovascularization (M.Faehling, et al., FASEB J.16(2002), pp.1805-1807; M.Guidooni, et al., Cancer Res.65(2005), pp.587-595). Thus, Ca in endothelial cells²⁺Mobilization characteristics studies may provide important information for a comprehensive understanding of the physiological processes involved in angiogenesis.

Synthetic peptide combinatorial library position scanning strategy (PS-SPCL) has been used to isolate peptides with angiogenic potential (r.a. houghten, et al., Nature 354(1991), pp.84-86). Furthermore, the PS-SPCL method has been successfully applied to screen for useful peptides involved in various biological processes, thereby identifying several peptides, such as interleukin-8 specific antagonists (s.hayashi, et al, J immunol.154(1995), pp.814-824), inhibitors of nuclear factors of activated T cells (j.armaburu, et al, Science 285(1999), pp.2129-2133) and immunomodulatory peptides (y.s.bae, et al, Blood 97(2001), pp.2854-2862).

Disclosure of Invention

In screening synthetic peptide libraries for bioactive synthetic peptides that act on endothelial cells, the present inventors have identified novel peptides that strongly induce angiogenic activity under both in vitro (in vitro) and ex vivo (ex vivo) conditions. Furthermore, the present inventors provide evidence that SFKLRY-NH₂Is mediated by VEGF-A induction in endothelial cells.

One embodiment of the present invention provides an angiogenic peptide sequence of selected length from 6 to 15 amino acids, said angiogenic peptide sequence having the following activity: promoting cell migration, angiogenesis or collagen synthesis, or inhibiting melanin formation. The angiogenic peptide includes a basic hexapeptide and a linker peptide consisting of 1-9 amino acids. Optionally, the angiogenic peptide is modified by addition of-NH₂Substituted for the C-terminal carboxyl group. The angiogenic activity of the angiogenic peptide is mediated by upregulation of Vascular Endothelial Growth Factor (VEGF).

Another embodiment of the present invention provides a composition for healing wounds, promoting collagen synthesis, or inhibiting melanin synthesis comprising an angiogenic peptide. The composition is a pharmaceutical composition for wound healing comprising an angiogenic peptide as an active ingredient and a pharmaceutically acceptable carrier. The composition is a cosmetic composition for improving the condition of aged skin comprising an angiogenic peptide as an active ingredient and a cosmetically acceptable carrier. The cosmetic composition is an active composition having anti-wrinkle and skin whitening activities.

Yet another embodiment provides a method of promoting angiogenesis in a mammal, the method comprising administering to a subject in need thereof an effective amount of an angiogenic peptide.

Yet another embodiment provides a method of healing a wound in a subject, comprising the steps of: administering to the subject in need thereof a wound-healing effective amount of an angiogenic peptide or a peptidomimetic thereof.

In another aspect, the invention relates to a method of ameliorating wrinkles comprising administering to a subject in need thereof an effective amount of an angiogenic peptide or a peptidomimetic thereof.

In yet another aspect, the invention relates to a method of reducing skin pigmentation comprising administering to a subject in need thereof an effective amount of an angiogenic peptide or peptidomimetic thereof.

In another aspect, the invention relates to a method of identifying an anti-angiogenic molecule comprising the steps of: providing an endothelial cell; contacting said cell with a candidate antagonist compound; and identifying the candidate antagonist compound as an antagonist compound if the candidate antagonist compound inhibits the angiogenic activity of the polypeptide or a peptidomimetic thereof.

Drawings

These and other objects of the present invention will be more fully understood from the following description of the invention, the accompanying drawings thereof and the appended claims.

The present invention will become more fully understood from the detailed description given herein below and the accompanying drawings, which are given by way of illustration only, and thus are not limitative of the present invention, and wherein:

FIGS. 1A-1F show initial PS-SPCL screens for peptides that elevate intracellular calcium of MS-1 cells, where each figure shows the results from a peptide library (peptide pool) of known amino acids at each of the 6 positions of the hexapeptide. The 6 positions are defined by one of the 19L-amino acids (e.g.O 1, O2). The remaining 5 positions consist of the mixture (X) of the 19L-amino acids, excluding cysteine. The library consisted of 114 peptide libraries; PS-SPCL consists of a total of 47,045,881 different peptides. Determination of [ Ca ] by fluorescence analysis using Fura-2/AM as described in materials and methods²⁺]_iAnd (4) rising. The results are presented in one of 3 independent experiments.

FIGS. 2A-2C show SFKLRY-NH₂(SEQ ID NO: 2) induces HUVEC proliferation, migration and tube formation. Cells were treated with different concentrations of SFKLRY-NH₂(SEQ ID NO: 2) (0.1-10. mu.M). After 48 hours of incubation, the activity of DNA synthesis was counted in a liquid scintillation counter. Bars represent mean ± s.d. of 3 independent experiments.^*P is less than 0.05. (B) Different doses of SFKLRY-NH₂(SEQ ID NO: 2) induced HUVEC mobility. After wounding, HUVEC was dosed with indicated concentrations of SFKLRY-NH₂(SEQ ID NO: 2) (0.1-10. mu.M) for 16 hours, and then the number of migrating HUVECs that exceeded the reference line was counted. Values represent 3 independent experiments (mean ± s.d.) performed in 2 replicates.^*P＜0.05。(C)SFKLRY-NH₂(SEQ ID NO: 2) effects on HUVEC tube formation. HUVEC were seeded on growth factor-reduced Matrigel (Matrigel) and exposed to SFKLRY-NH₂(SEQ ID NO：2)(1μM)、FYSRLK-NH₂(10. mu.M) and S1P (100nM) for comparison. After 24 hours of incubation, the tube-like structure was photographed and the length of tube formation was measured. Values represent 3 independent experiments (mean ± s.d.) performed in 2 replicates.^*P＜0.05。

FIGS. 3A-3B illustrateBAPTA, U73122 and PTX inhibit SFKLRY-NH in MS-1 cells₂Induced [ Ca²⁺]_iAnd (4) rising. Measurement of SFKLRY-NH in the Presence and absence of the intracellular calcium chelator BAPTA-AM (A), PLC inhibitor U73122 and its inactive analogue U73433, and PTX (B)₂(SEQ ID NO: 2) induced intracellular Ca²⁺Mobilizing the activity. (A) The cells were preincubated with Fluo-4/AM in DMEM containing 10. mu.M BAPTA-AM for 30min at 37 ℃. Suspending the cells in Ca-free medium containing 0.2mM EGTA²⁺In Locke solution of (5) and SFKLRY-NH at the indicated concentration₂(SEQ ID NO: 2). The change in fluorescence intensity was determined by excitation at 488nm and emission at 520 nm. (B) Cells were preincubated with Fura-2 for 30min with or without U73122 (10. mu.M), U73433 (10. mu.M), and for 2 h with or without PTX (100ng/mL), respectively. Then, Fura-2-loaded cells were suspended in Ca-free medium²⁺In Locke solution of (5), then 1. mu.M SFKLRY-NH₂(SEQ ID NO: 2). Data represent mean ± s.d. of 3 independent experiments performed in 3 replicates.^*P＜0.05。

FIG. 4 shows SFKLRY-NH₂(SEQ ID NO: 2) induces in vitro vascular sprouting. Rat aortic explants were treated with SFKLRY-NH in matrigel₂(SEQ ID NO：2)(1μM)、FYSRLK-NH₂(1. mu.M), S1P (10nM), VEGF (10ng/mL), SFKLRY-NH containing U73122 (10. mu.M) or PTX (50ng/mL)₂(SEQ ID NO: 2) (1. mu.M) or 10% FBS in M-199, and photographs were taken after 7 days of incubation. Then 3 independent experiments were performed, each experiment being repeated twice.

FIGS. 5A-5B illustrate SFKLRY-NH₂(SEQ ID NO: 2) resulting in upregulation of VEGF and VEGFR-1 mRNA. For the slave material with SFKLRY-NH₂(SEQ ID NO: 2) RT-PCR analysis of mRNA isolated from primary cultured HUVECs treated. The data shown are representative of 3 independent experiments. In HUVEC as SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) for 0, 1, 2, 3, 4, 8 or 12 hours (A) and at 0, 0.01, 0.1 or 10. mu.M for 2 hours (B), which was amplified using VEGF-A specific primers. GAPDH was used as a reference gene.

FIGS. 6A-6B show that anti-VEGF antibodies inhibit SFKLRY-NH2(SEQ ID NO: 2) -induced tube formation in HUVECs. Cells were cultured in the presence of anti-VEGF-A neutralizing antibody (0.1, 1 or 10. mu.g/mL) in the presence of SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) or VEGF-A (10ng/mL) for 24 hours. After 24 hours of incubation, the tube-like structure was photographed and the length of tube formation was measured. Data are presented as one out of 2 independent experiments, with values being the average of 2 independent experiments. Compared with carrier treatment^*P＜0.05。

Fig. 7A-7B show histology of healed wound tissue at day 14. (A) Sham-treated wounds exhibited severe edema and disordered microstructure. (B) By using SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) treatment almost complete restoration of the microstructure to normal was observed.

FIG. 8 shows the effect of SFKLRY-NH2(SEQ ID NO: 2) on collagen type I synthesis. Human fibroblasts were treated with SFKLRY-NH2(SEQ ID NO: 2) (0.1, 1, 10. mu.M).

FIGS. 9A-9B show the effect of SFKLRY-NH2(SEQ ID NO: 2) on melanin content in B16 melanoma cells.

Detailed Description

A series of induction [ Ca ] in endothelial cells was disclosed by the position scanning method (PS-SPCL) providing a combinatorial library of synthetic peptides²⁺]_iNovel peptides are raised. Among these peptides, the prototype peptide SFKLRY-NH₂(SEQ ID NO: 2) demonstrated that [ Ca ] is mediated by PTX-sensitive G protein/PLC in endothelial cells²⁺]_iElevated to induce DNA synthesis, migration and tube formation, which are key steps in angiogenesis. SFKLRY-NH as small peptides₂The identification of (SEQ ID NO: 2) is noteworthy in angiogenesis studies. SFKLRY-NH₂(SEQ ID NO: 2) not only showed angiogenic activity under in vitro and ex vivo conditions, but also significant wound healing activity in a rat model. Furthermore, it is possible to provide a liquid crystal display device,treatment with the peptide stimulates collagen synthesis and inhibits melanin synthesis in skin fibroblasts.

The present invention relates to a polypeptide having the sequence of SEQ ID NO: 2 and conservative variants or functional fragments thereof. The angiogenic peptide may be about 6 to 15 amino acids, 6 to 10 amino acids, 6 to 7 amino acids, or 6 amino acids in length, comprising the amino acid sequence X₁FX₂LRX₃As a basic moiety, and is linked to X₁FX₂LRX₃The C-terminal peptide of (1) to (9). The angiogenic peptide may be modified by reacting with-NH₂The modification is carried out in place of the terminal carboxyl group. Examples of angiogenic peptides include peptides consisting of SEQ ID NO: 1.2, 8, 12, 14 or 15-24.

Angiogenic peptides, as used herein, refer to oligopeptides that may be about 6 to about 15 amino acids in length and comprise hexapeptides having the amino acid sequence of formula I. In addition, the peptides are stimulating [ Ca ] in endothelial cells²⁺]_iCompounds that elevate and thereby induce the formation of capillary-like vessels.

X₁FX₂LRX₃

Wherein X1 is serine or threonine;

x2 is lysine, arginine, or isoleucine; and is

X3 is phenylalanine, tryptophan, tyrosine, arginine, or histidine.

The most active amino acids at each position are: first positions Ser (S) and Thr (T); second position Phe (F); third positions ile (i), lys (k) and arg (r); fourth position Leu (L); the fifth position Arg (R); the sixth positions Arg (R), Phe (F), Trp (W), Tyr (Y) and His (H).

The term "angiogenesis" as used herein refers to the growth of new blood vessels or "neovascularization," including the growth of new blood vessels of relatively small caliber composed of endothelial cells. Angiogenesis is an integral part of many important biological processes including cancer cell proliferation solid neoplasia, inflammation, wound healing, repair of damaged ischemic tissue, myocardial revascularization and remodeling, follicular maturation, menstrual cycle and fetal development. Neovascularization is required for the development of any new tissue, whether normal or pathological, and therefore it represents not only a therapeutically good opportunity to promote normal tissue growth and "normal" angiogenesis, but also a potential control point in the regulation of many disease states.

The complete process of angiogenesis is not yet fully understood, but it is known to affect endothelial cells of capillaries in the following ways:

(1) the attachment between endothelial cells and the surrounding extracellular matrix (ECM) is altered, presumably mediated by proteases and glycosidases, which disrupt the basal membrane around microvascular endothelial cells, thereby allowing the endothelial cells to extend beyond the cytoplasmic buds in the direction of chemokines;

(2) there is a "chemotactic process" of the endothelial cells to migrate to the tissue to be vascularized; and

(3) there is a "mitotic process" (e.g., the endothelial cells proliferate to provide additional cells for new blood vessels). Each of these angiogenic activities can be independently measured using in vitro endothelial cell cultures.

Wounds are internal or external bodily injuries or lesions caused by physical pathways, such as mechanical, chemical, bacterial or thermal pathways, that can disrupt the normal continuity of a structure. Such physical injuries include contusions-wounds in which the skin has not been breached; cut-a wound in which the skin is cut by a cutting instrument; and laceration, a wound in which the skin is damaged by an unsharpened or blunt instrument. The trauma may be caused by accident or by a surgical procedure.

Wound healing consists of a series of processes through which damaged tissue is repaired, specialized tissue is regenerated, and new tissue is recombined. Wound healing can generally be divided into 3 phases: inflammatory phase, proliferative phase and remodeling phase. Fibronectin has been reported to be involved in every stage of the wound healing process, particularly by generating scaffolds to which invading cells can attach. Initially, many regulatory factors, such as fibronectin and fibrinogen, are released to the wound site. Thereafter, angiogenesis and re-epithelialization occurred (U.S. patent 5,641,483). Repair of damaged tissue caused by ischemia is a form of wound healing that requires extensive remodeling and revascularization. Infarctions and ischemic necrosis of tissue regions are actually caused by the blockage of local blood circulation. The resulting necrotic lesions starve the damaged tissue of oxygen and nutrients. In the heart, obstruction of the coronary circulation in particular can form a myocardial infarction. With rapid hypoxia of ischemic myocardium, the hypoxic microenvironment of the affected myocardium leads to the synthesis of angiogenic factors in an attempt to carry out revascularization. For example, Vascular Endothelial Growth Factor (VEGF) is known to be produced in areas of the myocardium where infarction (Ref) occurs.

In one embodiment, the invention relates to the stimulation of [ Ca ] in endothelial cells²⁺]_iCompounds such as polypeptides, peptidomimetics or chemical compounds that elevate and thereby induce the formation of capillary-like vessels are screened. The compounds are expected to treat patients suffering from diseases induced by impaired blood supply, including foot and leg ulcers and retinopathy associated with diabetes or trauma.

Various libraries, including phage display libraries or chemical libraries, can be used to screen for [ Ca ] in stimulated endothelial cells²⁺]_iAn elevated compound. Another method utilizes a two-hybrid system (e.g., a yeast or mammalian two-hybrid system) to identify compounds that induce the formation of blood vessels in the subject, including the treatment of diseases induced by impaired blood supply, including foot and leg ulcers and retinopathy associated with diabetes or trauma. Many of these methods are useful for high throughput analysis. In addition, methods are provided that allow for the identification of additional angiogenic compounds, and which also treat and/or prevent the foot and legUlcers, retinopathy and wounds. One approach involves applying a rational drug design technique. Thus, computer-based homology searches, protein modeling, and combinatorial chemistry are used to design and form molecules that are similar to the identified compounds and fragments or derivatives of these molecules. For example, a database containing nucleic acid or protein sequences corresponding to the X1FX2LRX3 peptide or fragments or derivatives of these molecules is accessed by a search program that aligns the sequences with other sequences in public or commercial databases to identify homologous ligands. By another rational approach, protein modeling techniques (e.g., X-ray crystallography, NMR, and computer modeling) are used to model the compounds. Rational drug design can be achieved from these models. Once the candidate compounds are designed and generated, they are preferably evaluated for their ability to stimulate [ Ca ] in endothelial cells²⁺]_iIncreased capacity. Methods for assessing the ability of a candidate and the resulting induction of angiogenesis can be accomplished using a variety of assays, such as matrigel assays.

Variant and mutant polypeptides

In order to improve or alter the characteristics of the polypeptide, amino acid engineering may be employed. Recombinant DNA techniques known to those skilled in the art can be used to generate novel mutant polypeptides or fusion proteins comprising single or multiple amino acid substitutions, deletions, insertions. Similar mutant polypeptides may also be produced by chemical synthesis, particularly short peptides. Such modified polypeptides may exhibit, for example, increased/decreased activity or increased/decreased stability. In addition, they can be purified in higher yields and exhibit better solubility than the corresponding native polypeptide, at least under certain purification and storage conditions.

Compounds similar to the X1FX2LRX3 peptide and fragments or derivatives of these molecules (e.g., X1FX2LRX3 peptide mimetics) include not only those molecules that contain the angiogenic peptide amino acid sequence in whole or in part as the primary amino acid sequence and the native fragments or derivatives of these molecules, but also altered sequences such as: wherein residues in the sequence are substituted with functionally equivalent amino acid residues, thereby producing a silent change. Thus, one or more amino acid residues in the sequence of the angiogenic peptide and fragments or derivatives of these molecules may be replaced by another amino acid of similar polarity that acts as a functional equivalent, thereby producing a silent change. Amino acid substitutions in the sequence may be selected from other members of the class to which the amino acid belongs. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Uncharged polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Aromatic amino acids include phenylalanine, tryptophan, and tyrosine. Aromatic amino acids include phenylalanine, tryptophan, and tyrosine. In another aspect of the invention, X1FX2LRX3 peptides are contemplated as well as fragments or derivatives of these molecules that are differentially modified during or after translation, for example by phosphorylation, glycosylation, cross-linking, acylation, proteolytic cleavage, attachment to antibody molecules, membrane molecules or other ligands. Thus, the protein sequence corresponding to the X1FX2LRX3 peptide or fragment or derivative of these molecules can be compared to known sequences. Candidate compounds greater than or equal to the X1FX2LRX3 peptide were identified and subsequently examined using a calcium mobilization assay.

Simulation object

The peptides used in some aspects of the invention may also be modified, for example the peptides may have substituents not normally present on the peptide, or the peptides may have substituents normally present on the peptide but introduced into regions of the abnormal peptide. For example, peptides used in some aspects of the invention may be acetylated, acylated, or aminated. For modification, substituents that may be included on the peptide include, but are not limited to, H, alkyl, aryl, alkenyl, alkynyl, aromatic, ether, ester, unsubstituted or substituted amine, amide, halogen, unsubstituted or substituted sulfonyl, or a 5-or 6-membered aliphatic or aromatic ring. A "SFKLRY peptide mimetic" is a compound similar to the SFKLRY peptide. The SFKLRY peptide mimetics can be peptidomimetics, peptides, modified peptides, and derivatized peptides.

Additional derivative compounds include peptidomimetics similar to the polypeptide of interest. The natural amino acids involved in the biological production of peptides all have the L configuration. Synthetic peptides can be prepared using conventional synthetic methods using various combinations of L amino acids, D amino acids, or two amino acids of different configurations. Such synthetic compounds are called "peptidomimetics" as follows: i.e., a particular peptide (e.g., an oligopeptide), once discovered, the compound mimics the conformation and desired characteristics of such a peptide, but avoids undesirable characteristics such as flexibility (loss of conformation) and bond cleavage.

In general, the design and synthesis of a peptidomimetic comprises starting with the sequence of the peptide and conformational data (e.g., geometric data such as bond length and bond angle) of the desired peptide (e.g., the most likely mimetic peptide) and using such data to determine the geometric parameters that should be designed into the peptidomimetic. There are a variety of methods and techniques known in the art for performing this step, any of which may be used.

Although mimetics are characterized as non-peptide ligands by the definition of peptidomimetic, there are many structures that lie between the authentic peptide and peptidomimetic that are composed of natural amino acids. The debate as to what a peptidomimetic is made of continues, however one skilled in the art can distinguish between a mimetic and a peptide. Peptide mimetics can generally be considered as modified peptide forms. Chemical modifications of peptides, such as reduction of peptide bonds, can improve their enzymatic stability. Incorporation of unnatural amino acids can also improve the activity and selectivity of the peptide. The more structurally and/or chemically the peptide changes, the more it conforms to a true peptidomimetic. Peptide mimetics include peptides, proteins, and derivatives thereof, for example: peptides comprising non-peptide organic moieties, synthetic peptides which may or may not comprise amino acids and/or peptide bonds but retain the structural and functional characteristics of the peptide ligand, and molecules comprising N-substituted glycines, i.e. peptoids and oligopeptides, such as Simonet et al, proc.natl.acad.sci.usa 89: 9367 (1992); and antibodies, including anti-idiotype antibodies.

In another aspect of the invention, the compounds of the invention can be prepared by synthetically incorporating a variety of alternative compounds such as scaffolds, turn-over mimetics, and alternatives to binding peptides. Amino acid synthesis using a variety of linear and heterocyclic scaffolds instead of a peptide backbone can be used. Chemical processes and methods include transient protection of charged peptides to form neutral prodrugs for improved penetration of the blood brain barrier, and replacement of peptide bonds with groups such as heterocycles, alkenes, fluoroalkenes, and ketomethylenes (ketomethylenes).

In one embodiment of the invention, the mimetic is highly specific for its target, low in toxicity, and refers to a peptidomimetic that penetrates the skin barrier to promote wound healing. Thus, the present invention encompasses compounds that are modified such that they are capable of penetrating the skin barrier.

Pharmaceutical composition

As used herein, "carrier" includes pharmaceutically acceptable carriers, excipients, or stabilizers which are non-toxic to the cells or mammals to which they are exposed at the dosages and concentrations employed. The pharmaceutically acceptable carrier is typically a pH buffered aqueous solution. Examples of pharmaceutically acceptable carriers include, without limitation, buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartyl, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugar alcohols, such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENPolyethylene glycol (PEG) and PLURONICS。

As used herein, "effective amount" refers to an amount sufficient to obtain a beneficial or desired clinical or biochemical result. An effective amount may be administered 1 or more times. For purposes of the present invention, an effective amount of an inhibitor compound is an amount sufficient to moderate, ameliorate, stabilize, reverse, slow or delay the progression of a disease state. In a preferred embodiment of the invention, an "effective amount" is defined as the amount of a compound that is capable of eliciting a response in a given set of experiments.

As used herein, a "subject" is a vertebrate, preferably a mammal, more preferably a human.

The term "treatment" as used herein is a method for obtaining a beneficial or desired clinical result. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or complete), whether detectable or undetectable. "treatment" may also refer to an increase in survival time compared to the expected survival time without treatment. "treatment" refers to both therapeutic treatment as well as prophylactic or defensive measures. Subjects in need of treatment include subjects already having the disorder, as well as subjects in whom the disorder is to be prevented. By "alleviating" a disease is meant that the extent and/or adverse clinical manifestations of the disease state are reduced and/or the time course of its development is slowed or prolonged as compared to an untreated situation.

The discovery of a number of neo-angiogenic peptides is provided in the present disclosure. These drugs may be delivered by any conventional route including, but not limited to, transdermal, topical, parenteral, bronchial and alveolar routes. Embodiments of the invention also include biotechnological tools, prophylactic methods, therapeutic methods, and methods of using the foregoing to study, treat and/or prevent foot and leg ulcers and retinopathy associated with diabetes, trauma, and skin aging.

Formulations of therapeutic compounds are well known in the art and reference is conveniently made to Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing co. For example, about 0.05 μ g to about 20mg per kilogram of body weight per day may be administered. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, multiple divided doses may be administered per day, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. The active compounds can be administered in a convenient manner, for example by oral, intravenous (where soluble in water), intramuscular, subcutaneous, intranasal, intradermal or embolic routes or by implantation (for example using slow release molecules by the intraperitoneal route, or by using cells such as monocytes or dendritic cells which are sensitized in vitro and adoptively transferred to the recipient). Depending on the route of administration, it may be desirable to coat the peptide in a substance to protect it from enzymes, acids and other natural conditions that might inactivate the component.

For example, the low lipophilicity of the peptides makes them destructible by enzymes capable of cleaving peptide bonds in the gastrointestinal tract and by acid hydrolysis in the stomach. For administration of the peptide by a route other than parenteral administration, the peptide will be coated with or administered with a substance to prevent its inactivation. For example, the peptide may be administered in a vehicle, co-administered with an enzyme inhibitor, or administered in liposomes. Adjuvants contemplated herein include resorcinol, non-ionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether. Enzyme inhibitors include trypsin inhibitor, diisopropyl fluorophosphate (DEP) and tesla (trasylol). Liposomes include conventional liposomes and water-in-oil-in-water CGF emulsions.

The active compounds can also be administered parenterally or intraperitoneally. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof, as well as in oils. Under normal conditions of storage and use, these preparations contain preservatives to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water is soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that the syringe is easily expelled. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium including, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof, and vegetable oils. Suitable fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size for the dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, chlorobutanol, phenol, sorbic acid, thimerosal (theomersal), and the like. In many cases it will preferably contain isotonic agents, for example sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

If the peptide is suitably protected, the active compound may be administered orally, for example with an inert diluent or with an absorbable edible carrier; or it may be enclosed in hard or soft shell gelatin capsules; or it may be compressed into tablets; or it may be incorporated directly into the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and articles should contain at least 1 part by weight of the active compound. The percentages of the compositions and articles may of course vary. The amount of active compound in such therapeutically useful compositions is such that a suitable dosage can be obtained. Preferred compositions or articles of manufacture of the invention are prepared such that an oral dosage unit form contains from about 0.1 μ g to 2000mg of the active compound.

The tablets, pills, capsules, etc. may also comprise the following: binders, such as tragacanth, acacia, corn starch or gelatin; excipients, such as dicalcium phosphate; disintegrating agents such as corn starch, potato starch, alginic acid, and the like; lubricants, such as magnesium stearate; and sweetening agents such as sucrose, lactose or saccharin may be added, or flavoring agents such as peppermint, oil of wintergreen or cherry flavoring. If the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to modify the physical form of the dosage unit. For example, tablets, pills or capsules may be coated with shellac and/or sugar. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compounds may be incorporated into sustained release preparations and formulations.

As used herein, "pharmaceutically acceptable carrier and/or diluent" includes any and all solvents, dispersion media, coated antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients may also be incorporated into the composition.

Various delivery systems are known and may be used to administer the compounds of the invention, such as liposomes, microparticles, microencapsulation, receptor mediated endocytosis. Methods of introduction include, but are not limited to, intradermal routes, intramuscular routes, intraperitoneal routes, intravenous routes, subcutaneous routes, intranasal routes, epidural routes, and oral routes. The compounds or compositions may be administered by any conventional route, for example by infusion or bolus injection, by absorption through epithelial or mucosal linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Can be administered systemically or locally. In addition, it may be desirable to introduce a pharmaceutical compound or composition of the invention into the central nervous system by any suitable route, including intraventricular injection and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, connected to a container such as an omaya reservoir (omaya reservoir). Pulmonary administration may also be used, for example by use of an inhaler or nebulizer, as well as formulations containing a nebulizing agent.

Cosmetic composition

As used herein, "cosmetically acceptable" means that the ingredients are suitable for use in contact with tissue (e.g., skin or hair) without excessive toxicity, incompatibility, instability, irritation, allergic response, and the like.

As used herein, "effective amount" refers to an amount sufficient to improve aged skin such as wrinkles and darkening in humans, but low enough to avoid serious side effects. The safe and effective amount of the composition may vary depending on the following factors: the area of treatment, the age and skin type of the end user, the duration and nature of the treatment, the particular ingredients or compositions applied, the particular cosmetically acceptable carrier used, and the like.

In one embodiment, the topical composition comprises another cosmetic active in addition to the polymer. "cosmetic active agent" refers to a compound (e.g., a synthetic compound or a compound isolated from a natural source or natural extract) that has a cosmetic or therapeutic effect on the skin or hair, including, but not limited to, anti-acne agents, oil control agents, antimicrobial agents, anti-inflammatory agents, sunscreens, photoprotective agents, antioxidants, keratolytic agents, detergents/surfactants, moisturizers, nutrients, vitamins, energy enhancers, antiperspirants, astringents, deodorants, firming agents, anti-keratolytic agents, and agents for regulating the condition of the hair and/or skin.

The present invention is further described in more detail with reference to the following examples. These examples, however, should not be construed as limiting the scope of the invention in any way.

Example 1

Materials and methods for characterizing FPRL1 antagonist peptides

1.1. Material

Fura-2 pentaacetoxymethyl ester (pentaacetoxymethyl ester) (Fura-2-AM) and 1, 2-bis (2-aminophenoxy) ethane-N, N, N ', N' -tetraacetoxymethyl ester (BAPTA-AM) were purchased from Molecular Probes (Eugene, OR). Pertussis Toxin (PTX), U73122 and U73433 were from Calbiochem (San Diego, CA). Matrigel (Matrigel) was from Becton Dickinson (Bedford, MA). Recombinant human VEGF and anti-VEGF neutralizing antibodies were from R & D systems (Minneapolis, MN). Peptides were synthesized by Peptron Inc (korea). PS-SPCL was prepared in Peptide Library Support Facility (Korea) of the university of Otsu science as described previously (S.H.Baek, et al, J Biol Chem 271(1996), pp.8170-8175; R.A.Houghten, et al, Nature 354(1991), pp.84-86).

1.2. Cell culture

At 37 deg.C, 95% air and 5% CO were supplied₂MS-1 cells and B16F1 murine melanoma cells were cultured in DMEM containing 10% FBS. HUVECs were prepared from fresh human umbilical cord by collagenase digestion as previously described (E.A. Jaffe, et al, J Clin Invest 52(1973), pp.2745-2756) and maintained in M-199 medium containing 20% FBS. All HUVECs used in this study did not exceed passage 5. Human normal fibroblasts were purchased from the American Type Culture Center (ATCC). At 37 ℃ in 5% CO₂In a humidified atmosphere, the cells were cultured in DMEM containing 10% FBS and 1% antibiotics. Then, after replacing the fresh medium every 2 or 3 days, the medium is addedCells were subcultured with 0.05% trypsin-0.53 mM EDTA.

1.3. Using [ Ca ]²⁺]_iMobilization assay PS-SPCL screen was repeated in MS-1 cells

Cells were incubated with 4. mu.M Fura-2 AM and 250. mu.M sulpirtone (sulfofinpyrazone) in serum-free DMEM medium for 30min at 37 ℃ with continuous stirring. The cells were then washed with a Locke solution (M.Faehling, et al, FASEB J16 (2002), pp.1805-1807) and diluted to 2X 10⁶cells/mL. Aliquots of 50 μ L of cell suspension were added to each well of a 96-well plate and the change in fluorescence ratio at both excitation wavelengths of 340 and 380nm and at the emission wavelength of 500nm was determined after addition of the peptide. Plates were read immediately after peptide library addition with a time delay of approximately 5 seconds between peptide addition and FLEXstation (molecular devices) detection. The negative and positive controls were run simultaneously with the test samples to ensure that the conditions were the same for all samples. According to Grynkiewicz et al J BiolChem 260(1985), pp.3440-3450 [ Ca [²⁺]_iThe fluorescence ratio was corrected.

1.4.[³H]Thymidine incorporation assay

HUVEC at 2X 10⁴The density of individual cells/well was plated in 24-well dishes and allowed to attach overnight. After 12 hours of serum starvation, the cells were treated with or without indicated concentrations of SFKLRY-NH2(SEQ ID NO: 2) for 48 hours. Prior to performing the assay, the cell is contacted with³H]Thymidine (25 mCi/mmol; Amersham, Aelesbury, United kingdom) was labelled for 4 hours (M.Guidooni, et al., Cancer Res 65(2005), pp.587-595). The non-incorporated [ 2 ], [ solution ] was removed by washing with 10% trichloroacetic acid³H]Thymidine, followed by extraction of the incorporated [ 2 ], [ solution ] in 0.2M NaOH and 0.1% SDS at 37 ℃, [ solution ]³H]Thymidine for 1 hour. The radioactivity of the cells was counted in a liquid scintillation counter (Beckman Instruments, Fullerton, CA).

1.5. Tube formation assay

E.g., M.S.Lee, et al, supraHUVEC and SFKLRY-NH2(SEQ ID NO: 2), their scrambled sequence FYSRLK-NH 2, described in J Immunol 177(2006), pp.5585-5594, in the presence or absence of VEGF neutralizing antibodies₂S1P or VEGF together on an already polymerized matrigel layer. After 24 hours of incubation, the change in cell morphology was observed by phase contrast microscopy and photographed. To measure the formation of the capillary network, the total length of the tubes in each field of view was measured with a ruler at 40 x magnification. Each well analyzed 3 different fields.

1.6. Wound migration assay

To determine the effect of SFKLRY-NH2(SEQ ID NO: 2) on HUVEC migration, an in vitro wound healing repair assay was performed as described previously in M.S. Lee, et al, J Immunol 177(2006), pp.5585-5594. Briefly, HUVECs plated on 35-mm petri dishes at 90% confluence were wounded with a 2-mm wide razor blade and the line of injury was marked. After wounding, the cells were washed in serum-free medium and further incubated in M199 containing 1% serum and/or the indicated amount of SFKLRY-NH2(SEQ ID NO: 2). HUVECs were allowed to migrate for 16 hours, washed with serum-free medium, fixed with anhydrous methanol and stained with Giemsa dye (Giemsa). Migration was quantified by counting the number of cells that moved beyond the lesion line.

1.7. Aortic ring measurement

The method developed In In Vitro Cell Dev Biol 26(1990), pp.119-128, by Nicosia and Ottinetiti was used, with minor modifications. Aortas were collected from 6-week-old Sprague-Dawley rats. After removal of surrounding fibroadipose tissue, the aortic annulus was immersed in matrigel in the well of the petri dish. Culturing the aortic annulus in the presence or absence of PTX or U73122 in a culture medium comprising SFKLRY-NH2(SEQ ID NO: 2), S1P, FYSRLK-NH₂Or VEGF in serum free M199 (5% CO)₂At 37 ℃ C. On day 7, sprouting of aortic explants was measured.

RT-PCR analysis

Use of easy-BLUE^TMTotal RNA extraction kit (Intron Biotechnology, Inc.) total RNA was isolated from HUVEC according to the manufacturer's instructions. 3 μ g of DNA-free total RNA and oligo (dT) by Moloney murine leukemia virus reverse transcriptase (MMLV-RT)₁₅Primers (Promega) were used to synthesize single-stranded cDNA. The sequences of the primers used were: human VEGF (150-bp product): forward, 5'-GAGGAGGGCAGAATCATCACG-3' (SEQ ID NO: 25); reverse, 5'-ATCGCATGAGGGGCACACAGG-3' (SEQ ID NO: 26). The PCR products were electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining.

1.9. Conditioned Medium and ELISA

Conditioned media were produced as follows: to an 80% confluent HUVEC in 6 well dishes was added 2mL of serum-free M199 per dish and incubated for the indicated time. The collected medium was centrifuged to remove any residual cells and frozen at-80 ℃. Enzyme-linked immunosorbent assay (ELISA) analysis of VEGF (R & D system) was performed according to the manufacturer's instructions.

1.10. Statistical analysis

Data are presented as mean ± s.d. Statistical comparisons were made between groups using Sigma Plot followed by Student's st-test.

Example 2

2.1. Identification of [ Ca ] in induced MS-1 cells²⁺]_iElevated peptides

To identify peptides that stimulate intracellular calcium mobilization in murine endothelial cells (MS-1 cells), the inventors screened a library of 114C-terminally amidated synthetic hexapeptides. The results of the initial screening of the peptide library in MS-1 cells are shown in FIGS. 1A-1F.

FIGS. 1A-1F show initial PS-SPCL screens for peptides that elevate intracellular calcium of MS-1 cells. Each figure shows the results obtained for a library of peptides with known amino acids at each of the 6 positions of the hexapeptide. The 6 positions are defined by one of the 19L-amino acids (e.g.O 1, O2). The remaining 5 positions are composed ofThe mixture (X) of 19L-amino acids, excluding cysteine. The library consisted of 114 peptide libraries; PS-SPCL consists of a total of 47,045,881 different peptides. As described in example 1, [ Ca ] was determined by fluorescence analysis using Fura-2/AM²⁺]_iAnd (4) rising. The results are presented in one of 3 independent experiments.

The most active amino acids at each position are: first positions Ser (S) and Thr (T); second position Phe (F); the third positions are Ile (I), Lys (K) and Arg (R); fourth position Leu (L); the fifth position Arg (R); the sixth positions Arg (R), Phe (F), Trp (W), Tyr (Y) and His (H).

Based on the results from the initial screening of the peptide library, SFKLRY-NH2(SEQ ID NO: 2) was selected and synthesized as a prototype peptide for further analysis. The C-terminally amidated form of SFKLRY-NH2(SEQ ID NO: 2) showed stronger activity than the carboxylated peptide (SFKLRY-COOH) and modification of the first amino terminal residue (either deleted or substituted with a D-form amino acid) resulted in a complete loss of intracellular calcium mobilization activity, indicating that intracellular calcium mobilization activity is sequence specific and that the amino terminal residue is more important than the C-terminal residue for activity. Moreover, the results for the other hexapeptides were also correlated with the initial screening results (table 1). Many peptide ligands exist in C-terminally amidated form, this modification being essential for the expression of activity in some cases (y.in, m.fujii, et al, Acta Crystallogr B57 (2001), pp.72-81); thus, the peptides of the invention have the characteristic of acting as ligands for certain receptor(s) in the cell.

TABLE 1

EC of peptides tested on MS-1 cells₅₀This is from intracellular Ca²⁺Is determined by the dose-dependent variation of (a). Peptide sequence and EC from 3 experiments₅₀And s.e.m together.

SEQ ID NO	Sequence of	Average EC50(μM)	S.E.M.
				1	SFKLRY-COOH	＞10	N/A
2	SFKLRY-NH2	1.21	0.01
				3	sFKLRY-NH2(type d)	N/D	N/A
4	SfKLRY-NH2(type d)	N/D	N/A
				5	SFkLRY-NH2(type d)	N/D	N/A
6	SFKlRY-NH2(type d)	＞100	N/A
				7	SFKLrY-NH2(type d)	＞100	N/A
8	SFKLRy-NH2(type d)	1.14	0.09
				9	FKLRY-NH2	N/D	N/A
10	KLRY-NH2	N/D	N/A
				11	LRY-NH2	N/D	N/A
12	SFKLR-NH2	6.19	0.84
				13	SFKL-NH2	＞50	N/A
14	SFK-NH2	N/D	N/A
				15	SFRLRY-NH2	0.97	0.14
16	SFKLRR-NH2	2.66	0.36
				17	SFILRY-NH2	0.99	0.2
18	SFILRR-NH2	0.97	0.09
				19	SFKLRW-NH2	2.37	0.4
20	SFILRW-NH2	1.98	0.03
				21	SFKLRF-NH2	3.16	0.91
22	SFILRF-NH2	2.01	0.18
				23	SFKLRH-NH2	3.6	0.11
24	TFKLRY-NH2	3.87	1.15

Note: in SEQ ID NO: in 3 to 8, lower case letters indicate D-form amino acids.

HUVEC-inducing [ Ca ] by SFKLRY-NH2(SEQ ID NO: 2)²⁺]_iElevation, proliferation, migration and tubular structure formation

To confirm that SFKLRY-NH2(SEQ ID NO: 2) can induce [ Ca ] in human endothelial cells²⁺]_iThe intracellular Ca of primary cultured Human Umbilical Vein Endothelial Cells (HUVEC) was measured²⁺And (5) mobilizing. The peptide was substituted with SFKLRY-NH2(SEQ ID NO:2) the treatment performed induces [ Ca ] in HUVEC²⁺]_iElevated, half maximal effect at 1.4. + -. 0.15. mu.M (data not shown), but scrambled sequence FYSRLK-NH₂(10. mu.M) not triggering Ca in HUVEC²⁺And (5) mobilizing. Ca triggered by said peptide²⁺The released dose-response curve was very similar to that observed in the mouse endothelial cell line (fig. 2A-2C).

FIGS. 2A-2C show SFKLRY-NH₂(SEQ ID NO: 2) induces HUVEC proliferation, migration and tube formation. (A) SFKLRY-NH₂Effect on HUVEC proliferation. Cells were treated with different concentrations of SFKLRY-NH₂(SEQ ID NO: 2) (0.1-10. mu.M). After 48 hours of incubation, the activity of DNA synthesis was counted in a liquid scintillation counter. Bars represent mean ± s.d. of 3 independent experiments.^*P is less than 0.05. (B) Different doses of SFKLRY-NH₂(SEQ ID NO: 2) induced HUVEC mobility. After wounding, HUVEC was dosed with indicated concentrations of SFKLRY-NH₂(SEQ ID NO: 2) (0.1-10. mu.M) for 16 hours, and then the number of migrating HUVECs that exceeded the reference line was counted. Values represent 3 independent experiments (mean ± s.d.) performed in 2 replicates.^*P＜0.05。(C)SFKLRY-NH₂(SEQ ID NO: 2) Effect on HUVEC tube formation. HUVEC were seeded on growth factor-reduced matrigel and SFKLRY-NH₂(SEQ ID NO：2)(1μM)、FYSRLK-NH₂(10. mu.M) and S1P (100nM) for comparison. After 24 hours of incubation, the tube-like structure was photographed and the length of tube formation was measured. Values represent 3 independent experiments (mean ± s.d.) performed in 2 replicates.^*P＜0.05。

The process of angiogenesis is complex and involves a number of different steps, including extracellular matrix degradation, proliferation, migration and morphological differentiation of endothelial cells to form tubes (f. bussolino, et al, Trends Biochem Sci 22(1997), pp.251-256). To determine whether SFKLRY-NH2(SEQ ID NO: 2) induces angiogenesis, the ability of SFKLRY-NH2(SEQ ID NO: 2) as a stimulator of angiogenesis was evaluated in an in vitro model of angiogenesis. The inventors have tested SFKLRY-NH2(SEQ ID NO: 2) for the first time on the angiogenic cascade: the effect of this aspect of HUVEC proliferation.

When in use³H]Thymidine incorporation assay SFKLRY-NH2(SEQ ID NO: 2) when evaluated for its effect on DNA synthesis of HUVEC, SFKLRY-NH2(SEQ ID NO: 2) promoted the proliferative activity of HUVEC in a dose-dependent manner, increasing HUVEC DNA synthesis by about 2.5-fold at 1. mu.M. The proliferative activity of 10 μ M of the peptide was comparable to that of 10% FBS (fig. 2A).

Since angiogenesis is highly dependent on endothelial cell motility, the inventors next examined the effect of SFKLRY-NH2(SEQ ID NO: 2) on HUVEC migration in an in vitro wound migration assay. As shown in FIG. 2B, the migratory activity of HUVEC was enhanced in a dose-dependent manner by the addition of SFKLRY-NH2(SEQ ID NO: 2), approaching maximal activity at 10. mu.M. The migration activity of 10. mu.M SFKLRY-NH2(SEQ ID NO: 2) was 1.6 times that of the control (vehicle or untreated), and the effect of SFKLRY-NH2(SEQ ID NO: 2) was comparable to that of 10% FBS, which is known to be a stimulator of HUVEC migration.

To provide further evidence for the functional role of SFKLRY-NH2(SEQ ID NO: 2) in endothelial cells, the effect of SFKLRY-NH2(SEQ ID NO: 2) on HUVEC morphological differentiation was examined in an in vitro tube formation assay (FIG. 2C). Although control cells aggregated and formed clusters, HUVECs treated with SFKLRY-NH2(SEQ ID NO: 2) (10. mu.M) showed morphological changes such as elongation and threadiness, which resulted in network formation. Tube formation activity (20.25. + -. 1.31) of SFKLRY-NH2(SEQ ID NO: 2) was 2 times more than that of control cells (9.4. + -. 0.67), activity of 10. mu.M SFKLRY-NH2(SEQ ID NO: 2) was similar to that of 100nM S1P (19.69. + -. 0.32), but 10. mu.M of the scrambling sequence FYSRLK-NH₂(8.96. + -. 1.14) had no effect on the morphological differentiation of HUVEC (FIG. 2C). These results support the sequence-specific property of SFKLRY-NH2(SEQ ID NO: 2) for neovascularization in an in vitro human endothelial cell culture system.

[ Ca ] induced by SFKLRY-NH2(SEQ ID NO: 2)²⁺]_iElevation is mediated by PTX-sensitive G protein-PLC signaling pathway

To elucidate in endothelial cellsSFKLRY-NH2(SEQ ID NO: 2) -mediated signaling pathway, and the present inventors investigated interaction with intracellular Ca²⁺Raising the associated upstream signaling mechanisms. PLC is known to produce IP3 and diacylglycerol, which activate intracellular Ca²⁺Mobilisation (M.J.Berridge et al, Nature 312(1984), pp.315-321; P.W.Majerus, et al, Biochem Biophys Res Commun 268(2000), pp.47-53; and Y.Nishizuka, Science 258(1992), pp.607-614). Ca induced in SFKLRY-NH2(SEQ ID NO: 2) for the PLC-mediated signaling pathway²⁺The possible role in signaling was studied, investigating the effect of the PLC inhibitor U73122 and its inactive analogue U73433 on the intracellular calcium mobilisation induced by the peptide.

FIGS. 3A-3B show that BAPTA, U73122 and PTX inhibit SFKLRY-NH in MS-1 cells₂Induced [ Ca²⁺]_iAnd (4) rising. Measurement of SFKLRY-NH in the Presence and absence of the intracellular calcium chelator BAPTA-AM (A), PLC inhibitor U73122 and its inactive analogue U73433, and PTX (B)₂(SEQ ID NO: 2) induced intracellular Ca²⁺Mobilizing the activity. (A) The cells were preincubated with Fluo-4/AM in DMEM containing 10. mu.M APTA-AM for 30min at 37 ℃. Cells were suspended in Ca-free medium containing 0.2mM EGTA²⁺In Locke solution of (5) and SFKLRY-NH at the indicated concentration₂(SEQ ID NO: 2). The change in fluorescence intensity was determined by excitation at 488nm and emission at 520 nm. (B) Cells were preincubated with Fura-2 for 30min with or without U73122 (10. mu.M), U73433 (10. mu.M), and for 2 h with or without PTX (100ng/mL), respectively. Then, Fura-2-loaded cells were suspended in Ca-free medium²⁺In Locke solution of (5), then 1. mu.M SFKLRY-NH₂(SEQ ID NO: 2). Data represent mean ± s.d. of 3 independent experiments with 2 replicates.^*P＜0.05。

U73122(10 μ M) completely abolished the peptide-induced intracellular calcium mobilization, but there was little inhibition by U73433(10 μ M) (fig. 3B). Due to the passage of G_iCoupled receptor-PLC-intracellular Ca²⁺Induction of angiogenic activity of HUVEC of signaling pathwayDetailed descriptions have been given (D.English, et al., Biochim Biophys Acta 1582(2002), pp.228-239; O.H.Lee, et al., Biochem Biophys Res Commun 268(2000), pp.47-53; F.Wang, et al., J Biol Chem 274(1999), pp.35343-35350), and thus it was examined that G protein participates in SFKLRY-NH2(SEQ ID NO: 2) induced intracellular Ca²⁺The case of mobilization. When MS-1 cells were treated with Pertussis Toxin (PTX) (100ng/mL) for 2 hours followed by SFKLRY-NH2(SEQ ID NO: 2), the peptide-dependent intracellular Ca was shown in FIG. 3B²⁺Mobilization was significantly attenuated by PTX pretreatment, indicating that the reaction was largely driven by PTX-sensitive G proteins. In general, [ Ca ]²⁺]_iThe increase is achieved by releasing Ca from the endosome²⁺Or by influx from the extracellular environment.

To determine the Ca²⁺Source of pool in Ca-free containing 0.2mM EGTA²⁺The [ Ca ] induced by the peptide in MS-1 cells was measured in Locke solution of²⁺]_iAnd (4) rising. Although intracellular Ca in the absence of BAPTA-AM²⁺The concentration was shown to rise in a dose-dependent manner (FIG. 3A), but by the intracellular Ca²⁺Chelator BAPTA-AM (10. mu.M) Pre-Loading of MS-1 cells to deplete intracellular Ca²⁺Completely eliminate [ Ca²⁺]_iIncreased, even in cells treated with the maximum effective peptide concentration (fig. 3A). These results indicate that SFKLRY-NH2(SEQ ID NO: 2) can trigger a signal from intracellular Ca²⁺Of [ Ca ] library²⁺]_iElevated, and PTX-sensitive G proteins may be involved in PLC-mediated intracellular calcium mobilisation within MS-1 cells.

If it is assumed that SFKLRY-NH2(SEQ ID NO: 2) binds to one or more membrane receptors and induces intracellular Ca²⁺It is necessary to understand the signal transduction mechanisms involved. Inhibition of PTX of G proteins by ADP ribosylation of the alpha subunit strongly attenuates intracellular Ca²⁺Elevated (FIG. 3B) and significantly abolished the activity of SFKLRY-NH2(SEQ ID NO: 2) in rat aortic annuli leading to the outgrowth of endothelial cells from vascular explants (FIG. 4). Thus, it is possible to provideThe data of the present inventors indicate that the mediation of PTX sensitive G-protein is an important signal in the angiogenesis induced by SFKLRY-NH2(SEQ ID NO: 2). Signalling following G proteins can lead to a variety of cellular responses, including activation of phospholipase C, which hydrolyses PIP₂And subsequently form intracellular Ca mediating intracellular calcium release from the cell²⁺IP released in repository₃And a DAG. Hormones, growth factors and neurotransmitters are involved in this signaling pathway (M.J.Berridge., Nature 312(1984), pp.315-321; P.W.Majerus, et al., Cell 63(1990), pp.459-465; Y.Nishizuka, Science 258(1992), pp.607-614). Phospholipase C inhibitor U73122 abolished SFKLRY-NH2(SEQ ID NO: 2) -initiated intracellular calcium mobilization (FIG. 3B), thus indicating [ Ca²⁺]_iThe increase is dependent on the activation of the PLC. Based on these results, SFKLRY-NH2(SEQ ID NO: 2) might trigger its reaction by: PTX-sensitive G protein-coupled cell surface receptors were activated, followed by PLC-mediated intracellular calcium mobilization from internal storage (fig. 3A).

Thus, the data of the present inventors indicate that the mediation of pertussis toxin-sensitive G-protein is an important signal in SFKLRY-NH2(SEQ ID NO: 2) -induced angiogenesis, which involves receptors belonging to the GPCR class, rather than direct G-protein receptor activators such as melittin (R.Weingarten, J Biol Chem 265(1990), pp.11044-11049). The melittin is an amphiphilic peptide and has intracellular Ca in various cells including immune and neural cell lines²⁺Mobilisation activity (J.F. Klinker, et al., Biochem J304 (1994), pp.377-383; T.Murayama, et al., J Cell Physiol 169(1996), pp.448-454). However, SFKLRY-NH2(SEQ ID NO: 2) is a hydrophilic peptide that is difficult to penetrate the cell membrane, and the intracellular calcium mobilisation activity occurs in cells such as MC3T3-E1, C6bu1, NIH/3T3 and C2C12 cells, but not in neurons or immune cells such as PC12 and U937 (data not shown). However, in order to achieve the same level of calcium response in other cells, 10-fold or higher doses of SFKLRY-NH2(SEQ ID NO: 2) were required. Results of the present inventorsIt was shown that the putative receptor for SFKLRY-NH2(SEQ ID NO: 2) is widely expressed in mesenchymal cells, and that the role of SFKLRY-NH2(SEQ ID NO: 2) in endothelial cells is more specific.

Recently, several studies have described the switching on of G protein-coupled receptors (GPCR) after activation of the PLC-Ca during the regulation of the angiogenic process²⁺The results of the signaling pathway (D.English, et al., Biochim Biophys Acta 1582(2002), pp.228-239; D.S.Gelinas, et al., Br J Pharmacol 137(2002), pp.1021-1030; Y.M.Kim, et al., J Biol Chem 277(2002), pp.6799-6805; F.Wang, et al., J Biol Chem 274(1999), pp.35343-35350). Already known as G_iCoupled receptor mediated PLC-Ca²⁺The signaling pathway is important in S1P-stimulated focal adhesion formation and endothelial cell migration (O.H.Lee, et al, Biochem Biophys Res Commun 268(2000), pp.47-53; F.Wang, et al, J Biol Chem 274(1999), pp.35343-35350). In the initial stage of neovascularization, the sprouting of endothelial cells is a critical step requiring cell proliferation, cell migration and tube formation (w. rosau, et al., Nature 386(1997), pp.671-674). The results of the present inventors show that cell proliferation, cell migration and tube formation of HUVECs are enhanced in a dose-dependent manner by SFKLRY-NH2(SEQ ID NO: 2) treatment (FIG. 2). In addition, the results of the outgrowth of endothelial cells from the aortic annulus of isolated rats stimulated by SFKLRY-NH2(SEQ ID NO: 2) were similar to those obtained by VEGF treatment (FIG. 4). Sprouting of endothelial cells from vascular explants by SFKLRY-NH2(SEQ ID NO: 2) was significantly reduced under PTX or U73122 treatment, which is in contrast to the peptide-induced [ Ca ] by these inhibitors²⁺]_iThe effect of mobilization is consistent. These results suggest that PTX sensitive GPCR-PLC-Ca²⁺The signaling pathway is essential for SFKLRY-NH2(SEQ ID NO: 2) induced endothelial cell sprouting.

Induction of sprouting in isolated blood vessels by SFKLRY-NH2

To elucidate whether SFKLRY-NH2(SEQ ID NO: 2) plays a role in the sprouting of vascular endothelial cells ex vivo, the presence includedAortic rings were analyzed under multiple stimuli with 1. mu.M SFKLRY-NH2(SEQ ID NO: 2), 10ng/mL VEGF, 10nM S1P, and 10% FBS. SFKLRY-NH2(SEQ ID NO: 2) (1. mu.M) caused significant outgrowth of endothelial cells from vascular explants with vascular sprouting activity greater than that of 10nM S1P and 10ng/mLVEGF treatment. FIG. 4 shows SFKLRY-NH₂(SEQ ID NO: 2) induces in vitro vascular sprouting. Rat aortic explants were treated with SFKLRY-NH in matrigel₂(SEQ ID NO：2)(1μM)、FYSRLK-NH₂(1. mu.M), S1P (10nM), VEGF (10ng/mL), SFKLRY-NH containing U73122 (10. mu.M) or PTX (50ng/mL)₂(SEQ ID NO: 2) (1. mu.M) or 10% FBS in M-199, and photographs were taken after 7 days of incubation. Then 3 independent experiments were performed, each experiment being repeated twice.

In addition, the scrambled sequence FYSRLK-NH₂The activity (1. mu.M) was negligible. With PTX and U73122 to [ Ca ]²⁺]_iConsistent with the mobilization effect, the vascular sprouting activity promoted by SFKLRY-NH2(SEQ ID NO: 2) was significantly attenuated by its co-treatment with PTX (50ng/mL) or U73122 (10. mu.M) (FIG. 4). Thus, these results show that PLC- [ Ca²⁺]_iSignaling pathway-PTX sensitive G proteins are involved in the enhancement of vascular sprouting by the peptides.

Upregulation of VEGF mRNA by SFKLRY-NH2(SEQ ID NO: 2)

To clarify the possibility that the VEGF signaling pathway is involved in the process of angiogenesis induced by SFKLRY-NH2(SEQ ID NO: 2), the expression of angiogenic factors was measured by RT-PCR in HUVECs treated with SFKLRY-NH2(SEQ ID NO: 2). FIGS. 5A-5B illustrate SFKLRY-NH₂(SEQ ID NO: 2) caused upregulation of VEGF and VEGFR-1 mRNA. For the slave material with SFKLRY-NH₂(SEQ ID NO: 2) RT-PCR analysis of mRNA isolated from primary cultured HUVEC treated. The data shown are representative of 3 independent experiments. In HUVEC as SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) for 0, 1, 2, 3, 4, 8 or 12 hours (A) and at 0, 0.01, 0.1 or 10. mu.M for 2 hours, VEGF-A was amplified using primers specific for VEGF-A. GAPDH was used as a reference gene.

As shown in fig. 5A, the expression level of the strong angiogenic factor VEGF-a increased 3.77-fold and exhibited induction in a time-dependent manner, reaching maximum expression within 2 hours.

The induction of VEGF-A lasted 8 hours and more than 4 hours. On the other hand, SFKLRY-NH2(SEQ ID NO: 2) stimulation did not affect the expression levels of bFGF and FGFR-2 (data not shown). The peptide elicited induction of VEGF-a in a dose-dependent manner (fig. 5B). These data indicate that enhanced VEGF expression may be involved in the angiogenic effects of SFKLRY-NH2(SEQ ID NO: 2) in HUVEC.

Induction of angiogenesis by VEGF of SFKLRY-NH2(SEQ ID NO: 2)

By assessing the increase in the VEGF message using RT-PCR analysis, the inventors showed that the up-regulation of these proteins might be involved in SFKLRY-NH2(SEQ ID NO: 2) -induced angiogenesis (FIG. 5A)&5B) In that respect To further support the induction of VEGF-A in SFKLRY-NH₂(SEQ ID NO: 2) possible role in induced angiogenesis, the effect of VEGF neutralizing antibodies on the peptide-induced angiogenesis was examined in a tube formation assay.

FIGS. 6A-6B show that anti-VEGF antibodies inhibit SFKLRY-NH2(SEQ ID NO: 2) -induced tube formation in HUVECs. In the presence of SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) or VEGF-A (10ng/mL) for 24 hours with anti-VEGF-A neutralizing antibody (0.1, 1 or 10. mu.g/mL). After 24 hours of incubation, the tube-like structure was photographed and the length of tube formation was measured. Data are presented as one out of 2 independent experiments and values are the average of 2 independent experiments. Compared with carrier treatment^*P＜0.05。

HUVEC tube formation induced by SFKLRY-NH2(SEQ ID NO: 2) (18.2. + -. 0.77) was shown to be statistically significantly attenuated by co-treatment with VEGF neutralizing antibody, inhibiting approximately half, at a level similar to control (VEGF + VEGF neutralizing antibody, 12.1. + -. 0.63) (FIG. 6A). In addition, the increase in VEGF-A mRNA expression to a maximum of 3.77-fold following SFKLRY-NH2(SEQ ID NO: 2) stimulation was accompanied by a significant increase in VEGF-A production by HUVEC, as determined by ELISA (FIG. 6B). These results indicate that VEGF induction is involved in SFKLRY-NH2(SEQ ID NO: 2) induced angiogenesis.

To investigate whether SFKLRY-NH2(SEQ ID NO: 2) might influence angiogenesis by inducing other angiogenic factors, RT-PCR analysis was performed on well known signaling molecules involved in angiogenesis. The inventors observed an increase in VEGF-A expression in HUVEC as early as 2 hours after SFKLRY-NH2(SEQ ID NO: 2) treatment (FIG. 5), while the expression levels of bFGF and FGFR-2 were unchanged (data not shown). In addition, the VEGF neutralizing antibody highly inhibited HUVEC tube formation induced by SFKLRY-NH2(SEQ ID NO: 2) (FIG. 6A). ELISA analysis to monitor VEGF-A protein secreted into the medium also showed that stimulation by SFKLRY-NH2(SEQ ID NO: 2) in HUVEC resulted in a 3-fold increase in VEGF accumulation (FIG. 6B). These results led the inventors to believe that the mechanism of SFKLRY-NH2(SEQ ID NO: 2) -induced angiogenic activity might be mediated by the induction of VEGF, and that the semi-inhibitory results of VEGF neutralizing antibodies (FIG. 6A) also indicate the presence of uncharacterized mediators in SFKLRY-NH2(SEQ ID NO: 2) -stimulated angiogenic activity.

Recent studies have shown the importance of VEGF induction in the following clinical setting: in animal models, transfer of plasmid or adenoviral DNA encoding VEGF has a beneficial effect in myocardial infarction and duodenal ulcer healing (X.Deng, et al, J Pharmacol Exp THER311(2004), pp.982-988; J.Rutanen, et al, Circulation 109(2004), pp.1029-1035; Y.S.Yoon, et al, Mol Cell Biochem 264(2004), pp.63-74). Although it should be tested whether SFKLRY-NH2(SEQ ID NO: 2) can induce VEGF expression in vivo, peptide treatment would be more advantageous than gene delivery, since the delivery of therapeutic plasmid genes to target organs is difficult and often transient. Furthermore, of the known VEGF inducers such as TNF- α, transforming growth factor β, interleukin 1 β and endothelin (F. Bussolino, et al, J Pharmacol Exp Ther311(2004), pp.982-988), SFKLRY-NH2(SEQ ID NO: 2) is the smallest peptide that has several advantages over other proteins in the following respects: easier synthesis, lower cost than protein expression and not necessarily an expression system; the present inventors simply obtained a peptide of high purity. Such potential benefits of SFKLRY-NH2(SEQ ID NO: 2) for vascular remodeling could indicate the potential use of SFKLRY-NH2(SEQ ID NO: 2) in human diseases induced by impaired blood supply, including foot and leg ulcers associated with diabetes or trauma.

Example 3: promotion of wound healing in animal models

Wound healing experiments were performed with Sprague-Dawley (6 weeks old, male, body weight 140-160 grams) and the animals were randomized into 3 groups. Under gerolan anesthesia, the back was depilated and the skin was disinfected with 70% ethanol. Full thickness wounds were created on the dorsal skin using an 8-mm skin biopsy punch. The wound site was covered with Tegaderm. The inventors administered the control (HBSS), SFKLRY 10uM and fysrlk 10uM topically to each group once a day for 14 days. After 14 days, the rats were sacrificed and then the wound tissue was removed. These samples were then individually fixed in 4% formaldehyde, dehydrated through a gradient ethanol series, cleared in xylene and embedded in paraffin. 4 μm serial sections were cut and stained with hematoxylin and eosin (H & E).

The effect of SFKLRY-NH2(SEQ ID NO: 2) on wound was examined in a rat model based on the positive effect of SFKLRY-NH2(SEQ ID NO: 2) on HUVEC migration in an in vitro wound migration assay. Histology of biopsied wound tissue at day 14 is shown in FIG. 7A&7B. Fig. 7A-7B show histology of healed wound tissue at day 14. In fig. 7A, the sham-treated wounds exhibited severe edema and disorganized microstructure. In FIG. 7B, the reaction is performed by using SFKLRY-NH₂(SEQ ID NO: 2) (10. mu.M) treatment almost complete restoration of the microstructure to normal was observed.

Wounds treated with SFKLRY-NH2(SEQ ID NO: 2) were almost completely restored compared to controls (PBS treatment).

Example 4: induction of collagen synthesis

Considering the possible link between angiogenesis and skin ageing, the possible use of SFKLRY-NH2(SEQ ID NO: 2) as an anti-wrinkle cosmetic component was investigated. Type I collagen expression was determined by western blotting. Fibroblasts from each group were pelleted and extracted with ice-cold Cell lysis buffer (Cell Signaling Technologies). The cell lysate was centrifuged at 15000g for 15 min at 4 ℃ and the supernatant from each group was separated by 8% SDS-PAGE before being transferred to nitrocellulose membrane. After incubation in blocking solution (5% skim milk), the membrane was incubated overnight with primary antibody (Sigma-Aldrich) at 4 ℃. Membranes were washed with 1 XTSST solution before incubation with secondary antibody (1: 5000 dilution, Amersham Life Sciences) for 2 hours. The membranes were tested on an ECL system (Amersham Life Sciences) and the relative intensities of the protein bands were analyzed by Scan-gel-it software.

As shown in FIG. 8, SFKLRY-NH2(SEQ ID NO: 2) significantly enhanced type I collagen expression with the greatest response occurring at a concentration of 10 μ M. FIG. 8 shows the effect of SFKLRY-NH2(SEQ ID NO: 2) on collagen type I synthesis. Human fibroblasts were treated with SFKLRY-NH2(SEQ ID NO: 2) (0.1, 1, 10. mu.M). After 24 hours, the expression of type I collagen was determined by western blotting. The experiment was repeated 3 times and the results were reproducible.

Example 5: inhibition of melanin formation in melanoma cells

To investigate the possible effect of SFKLRY-NH2(SEQ ID NO: 2) on melanin production, B16 melanoma cells stimulated with α -MSH were cultured for 5 days in the presence of SFKLRY-NH2(SEQ ID NO: 2) at concentrations of 1, 10 or 50 μ M. B16 melanoma cells were treated with SFKLRY-NH2(SEQ ID NO: 2) at the given concentration followed by α -MSH (10nM) for 5 days. After treatment, they were rendered non-adherent by a brief incubation with trypsin/EDTA. After precipitation, the cell pellet was photographed and dissolved in boiling 2M NaOH for 20 minutes. Spectrophotometric analysis of melanin content was performed at 405 nm.

FIGS. 9A-9B show the effect of SFKLRY-NH2(SEQ ID NO: 2) on melanin content in B16 melanoma cells. In FIG. 9A, B16 melanoma cells were treated with SFKLRY-NH2(SEQ ID NO: 2) at a given concentration followed by α -MSH (10nM) for 5 days. After treatment, they were rendered non-adherent by a brief incubation with trypsin/EDTA. After precipitation, the cell pellet was photographed (fig. 9A) and dissolved in boiling 2M NaOH for 20 minutes. Spectrophotometric analysis of melanin content was performed at 405nm (fig. 9B). The results are expressed as a percentage of the control (. alpha. -MSH treatment).

As shown in FIGS. 9A and 9B, the color of the cell pellet was discolored, and treatment with SFKLRY-NH2(SEQ ID NO: 2) produced a significant inhibition of melanin formation (inhibition of approximately 60%) and exhibited a saturation response at 1 μ M.

All references cited herein are incorporated by reference in their entirety. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be encompassed by the scope of the claims.

Claims (9)

1. An angiogenic peptide selected from the amino acid sequence of SEQ ID NO: 2. 8, 12, 14 or 15-24, and

wherein the angiogenic peptide is attached to the peptide via a linker selected from the group consisting of-NH₂Substituted for the C-terminal carboxyl group.

2. A composition for healing wounds, promoting collagen synthesis or inhibiting melanin synthesis comprising the angiogenic peptide of claim 1.

3. The composition of claim 2, wherein the composition is a pharmaceutical composition for wound healing comprising the angiogenic peptide of claim 1 as an active ingredient and a pharmaceutically acceptable carrier.

4. The composition of claim 2, wherein the composition is a cosmetic composition for improving the condition of aged skin comprising the angiogenic peptide of claim 1 as an active ingredient and a cosmetically acceptable carrier.

5. Use of a cosmetic composition comprising the angiogenic peptide according to claim 1 as an active ingredient and a cosmetically acceptable carrier for anti-wrinkle and skin whitening.

6. Use of the angiogenic peptide of claim 1 in the manufacture of a medicament for promoting angiogenesis in a mammalian subject.

7. The use of claim 6, wherein the subject is in need of angiogenesis for epithelial wound healing.

8. The use of claim 6, wherein the subject is in need of angiogenesis for inducing collagen synthesis.

9. The use of claim 6, wherein the medicament is administered by a topical, systemic, oral or parenteral route.