WO2013015520A1 - Novel peptide for increasing vegf expression and pharmaceutical composition including same - Google Patents

Abstract

Provided is a novel peptide for increasing the transcriptional activity of vascular endothelial growth factor (VEGF), which is important for wound treatment by means of activation of the HIF-1α gene, and a pharmaceutical composition including the peptide. The pharmaceutical composition of the present invention has the effects of wound treatment, skin tissue neogenesis, hair growth promotion, and the like.

Description

Novel peptides for increasing VEGF expression and pharmaceutical compositions containing them

The present invention relates to a novel peptide that increases VEGF expression, and more particularly, to a pharmaceutical composition for wound treatment or skin tissue regeneration including the peptide.

Every cell of human tissue needs a supply of oxygen and nutrients in the blood vessels. Angiogenesis is a method for maintaining intracellular oxygen partial pressure due to hypoxia during this phenomenon (Chandan et al., 2009). In experimental animals lacking HIF-1α, expression of vascular endothelial growth factor (VEGF) decreases, and cell death occurs in fetal conditions due to vascular and cardiac abnormalities (Iyer et al., 1998). Wounds also include vascular damage, which is mainly caused by the inhibition of microcirculation, which impedes the oxygen supply to the site of injury, so hypoxia plays an important role in the mechanism of wound healing. In hypoxic conditions, it has been reported that TGF-β1 (transforming growth factor-β1) production is increased in dermal fibroblasts and that kratinocyte motility is increased in connective tissues, which is involved in collagen synthesis in fibroblasts. In addition, it promotes angiogenesis and increases the transcriptional activity of VEGF, an angiogenic growth factor important for wound healing, thereby facilitating oxygen supply to oxygen-dependent therapeutic tissues (Trentin et al., 2006; Sheid et al., 2000 Hirota et al., 2009).

Hypoxia-inducible factor-1 (hypoxia-inducible factor-1) is induced in hypoxia and functions to express genes related to angiogenesis, erythropoiesis, glycolytic metabolism, etc. in order to maintain homeostasis in cells. Since HIF-1 activity is closely related to the pathological mechanisms of various chronic diseases such as ischemic stroke, arteriosclerosis, and rheumatoid arthritis, it has been used as a major drug target. Recently, RORα expression is increased in the atherosclerotic site where hypoxic partial pressure is formed, and it has been reported to be a nuclear receptor capable of regulating HIF-1 (Kim et al., 2008).

RORα (retinoic acid related orphan nuclear receptor α NR1F1) is an orphan nuclear receptor belonging to the NR1 family and is expressed in many organs such as liver, adipose tissue, blood vessels, heart and muscle (Jetten, 2009). RORα has been shown to significantly increase expression in hypoxic endothelial cells (Besnard et al., 2002), and the presence of a hypoxia-responsive element (HRE) in the RORα promoter increases transcriptional activity in the hypoxic state (Miki et. al., 2004). It has been reported that RORα and its ligands can be used to treat hypoxia-related vascular diseases by promoting hypoxic signal transduction and VEGF production. In this regard, it was also confirmed that the DNA-binding domain (DBD) portion of RORα binds to the inhibitory domain (ID) portion of HIF-1 to stabilize HIF-1α and increase VEGF expression (Kim et al., 2008).

The present inventors have a wide range of triggers, and as a result of diligent efforts to develop a therapeutic agent that is simple to use and has various applications, which can inhibit a wound, a disease that can be exposed to various environments during the treatment process, HIF-1α A peptide encoding a nucleotide sequence or a mutated nucleotide sequence of RORα DBD that increases the activity of VEGF, fibronectin and collagen through increased expression was produced. Accordingly, it is an object of the present invention to provide a novel peptide for wound healing or skin tissue regeneration by preparing an effective peptide via VEGF expression.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problem, another task that is not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention provides a novel peptide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

 In one embodiment of the invention, the peptide is characterized by increasing the expression of hypoxic inducer (HIF) -1α, vascular endothelial growth factor (VEGF), fibronectin, and / or collagen (collagen) It is done.

In another embodiment of the present invention, the peptide is characterized in that proliferation of vascular endothelial cells.

In another embodiment of the invention, the peptides are characterized by increasing cell migration.

In another aspect, the present invention provides a pharmaceutical composition for wound treatment or skin tissue regeneration comprising a peptide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

The present invention also provides a method of treating wounds or regenerating skin tissue by administering the pharmaceutical composition to a subject.

The novel peptides according to the invention affect cell proliferation and migration by activating the VEGF gene through the activity of HIF-1α. Therefore, it shows effects such as wound healing, skin tissue regeneration, and hair growth promotion, and ultimately, it is expected to be used for the development of various therapeutic agents while being simple to use. In addition, considering that the novel peptides according to the present invention significantly increase epithelial regeneration and greatly reduce the number of inflammatory cells based on experiments in vivo, existing ointments have an effective effect in the inflammatory phase in the early development of wounds. It is expected to have a faster wound healing effect.

Figure 1 shows the effect of NR1, NR2 and NR3 on the transcriptional activity of HIF-1α.

Figure 2 shows the effect of NR1, NR2 and NR3 on the protein expression of HIF-1α and VEGF.

3 shows the effects of NR1, NR2 and NR3 on tube formation, which is an important morphological change in neovascular formation in vascular endothelial cells.

Figure 4 shows the effect of NR1, NR2 and NR3 on the protein expression of collagen 1A2, fibronectin and IF-1α in human dermal fibroblast (HDF), and the transcriptional activity of collagen 1A2, fibronectin and VEGF.

5 shows the effects of NR2 and NR3 on cell migration in HDF over time.

Figure 6 shows the effect of NR3 on wound healing in animal experiments as a wound size reduction rate compared to the base and commercial ointments (fucidin, anabolic drugs).

Figure 7 shows the results of histopathological observation of the effect of NR3 on wound healing in animal experiments, the epithelial portion of the epithelium, the neo-capillary and infiltrating inflammatory cells in granulation tissue, and collagen expression rate.

The present invention seeks to provide novel peptides that affect cell proliferation and migration by activating the VEGF gene through the activity of HIF-1α. Specifically, by increasing the expression of HIF-1α, VEGF, fibronectin and collagen through the novel peptide, induce vascular endothelial cell proliferation in human umbilical vascular endothelial cells (HUVECs), by increasing cell migration in fibroblast To provide a therapeutic agent for the treatment of diseases related to wound healing and skin tissue regeneration.

It is known that in hypoxic state, TGF-β1 (transforming growth factor-β1) production is increased and keratinocyte motility is increased in connective tissue, which is involved in collagen synthesis in fibroblast. In addition, it promotes angiogenesis and increases the transcriptional activity of VEGF, an angiogenic growth factor important for wound healing, thereby facilitating oxygen supply to oxygen-dependent therapeutic tissues (Trentin et al., 2006; Sheid et al., 2000; Hirota et al., 2009).

The present inventors have made a thorough research to develop a substance that effectively treats wounds. As a result, the present inventors have found that the base sequence of the RORα DBD (DNA-binding domain), which increases the activity of VEGF, fibronectin and collagen, is increased by increasing HIF-1α expression. A peptide encoding a partial or mutated sequence was produced, and the peptide was found to be effective in wound healing. That is, the present inventors observed that HF-1α transcriptional activity, HIF-1α and VEGF protein expression increased and proliferation of vascular endothelial cells increased when the effective peptide was treated. In addition, the present inventors observed that protein expression and transcriptional activity of collagen 1A2, fibronectin and VEGF increased in fibroblasts during the effective peptide treatment, and were involved in promoting cell migration, and in vivo (in vivo) controls. Compared with the increase of epithelial regeneration rate, reduction of neocapillary and infiltrating inflammatory cells in granulation tissue, it showed excellent wound healing promoting effect.

Accordingly, the present invention provides the novel effective peptide having the nucleotide sequence selected from the group consisting of the nucleotide sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 3 and a pharmaceutical composition comprising the peptide.

The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may include physiological saline, polyethylene glycol, ethanol, vegetable oil, isopropyl myristate, and the like, but is not limited thereto.

Another aspect of the present invention provides a method of treating wounds or regenerating skin tissue by administering to a subject a pharmaceutically effective amount of a pharmaceutical composition comprising the novel peptide as an active ingredient. As used herein, "individual" means a subject in need of treatment for a disease, and more specifically, a human or non-human primate, mouse, rat, dog, cat, horse, and cattle Means such mammals. In addition, in the present invention, "pharmaceutical effective amount" may be adjusted in various ways depending on the weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of the disease of the patient. It is obvious to

The preferred dosage of the pharmaceutical composition of the present invention depends on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration, and the duration, and may be appropriately selected by those skilled in the art. However, preferably, it is administered at 0.001 to 100 mg / kg body weight per day, more preferably 0.01 to 30 mg / kg body weight. Administration may be administered once a day or may be divided several times.

The pharmaceutical composition of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. The method of administration is not limited, and may be administered by oral, rectal, or intravenous, intramuscular, subcutaneous, intrauterine dural, or intra cerbroventricular injection.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

New NR Peptide Construction

The novel NR peptide of the present invention was chemically synthesized according to the solid peptide synthesis method using Fmoc-chemistry (synthesis of peptide after blocking the amine portion of amino acid with 9-Fluorenylmethyloxy-carbonyl group).

TABLE 1

EXAMPLE

The following experiment was conducted using the novel NR peptides, NR1, NR2 and NR3.

Example 1 Effects of NR1, NR2 and NR3 on Transcription Activity of HIF-1α

HepG2 cells were purchased from the American Type Culture Collection (ATCC). HepG2 cells (1 × 10 ⁵ cells / well) were seeded in 12-well culture plates and incubated overnight in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% FBS (fetal bovine serum). HepG2 cells were maintained at 37 ° C. in a humidified thermostat with 5% CO ₂ and 95% air.

After incubation, the cells were treated with Welfect-EX ^™ Plus (WelGENE Inc., Korea) using HRE-Luc. (100 ng) with reporter plasmid. 24 hours after transformation, NR1 (0.2 μM), NR2 and NR3 (0.2 or 2 μM), solvent (control: DMSO, Dimethyl Sulfoxide) were treated. After 24 hours of treatment, luciferase activity was measured using an Analytical Luminescence luminometer. To confirm transformation efficiency, luciferase activity was normalized using the activity of 200 ng of β-galactosidase (β-gal) expression vector. The results are shown in FIG.

As shown in Figure 1, NR1, NR2 and NR3 increased the transcriptional activity of HIF-1α. In particular, it was observed to be 20 times more active than the solvent-treated control group.

Example 2 Effects of NR1, NR2 and NR3 on Protein Expression of HIF-1α and VEGF

HepG2 cells (1 × 10 ⁶ cells / well) were seeded in 60-cm ² dishes and incubated overnight in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS (fetal bovine serum). After incubation, HepG2 cells were treated with control and 2 μM of each of NR1, NR2 and NR3 for 24 hours. Protein expression was analyzed by Western blotting assay after treatment.

Specifically, after treatment, HepG2 cells were destroyed for 30 minutes on ice in lysis buffer containing 50 mM NaCl, 50 mM Tris pH 7.4, 5 mM EDTA, 1% NP-40 and protease inhibitors, and centrifuged to dissociate the whole cells. A solution was obtained. 20-30 μg protein from total cell lysate was transferred to 9% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane (Millipore, Bedford, Mass., USA). Blot with 5% or 10% (w / v) non-fat dry milk in PBS containing 0.1% Tween-20, for HIF-1α, VEGF (Santa Cruz Biotechnology), α-tubulin (Calbiochem) React with specific antibodies. Immunoreactive proteins were detected with Amersham ECL Western Blotting Detection Reagents using horseradish peroxidase (HRP) -conjugated secondary antibody (Zymed Lab). Protein concentration was quantified by BCA (bicinchoninic acid) (Pierce) assay and expression of α-tubulin was monitored as a control. The results are shown in FIG.

As shown in Figure 2, it was observed that the protein expression of HIF-1α and VEGF increased by NR1, NR2 and NR3.

Example 3 Effects of NR1, NR2 and NR3 on Tube Formation in Vascular Endothelial Cells

HUVEC (human umbilical vein endothelial cells (Lonza) cells contain 10% FBS (fetal bovine serum), insulin (5 μg / ml), rhFGF (recombinant human basic fibroblast growth factor (1 μg / ml), rhEGF (recombinant human epithermal growth) factor; 0.1 μg / ml), gentamycin (50 μg / ml), and amphotericin B (50 μg / ml) were added at 37 ° C. in a humidified thermostat with 5% CO ₂ and 95% air in the endothelial cell growth medium. . Matrigel (10 mg / ml) was added to the 96-well culture plate and polymerized in the incubator at 37 ° C. for 30 minutes, inoculated with HUVECs (1 × 10 ⁵ cells / well). After 24 hours of treatment was observed tube formation through a microscope and the results are shown in FIG.

As shown in FIG. 3, it was observed that tube formation, an important morphological change of increased neovascularization due to the expression of VEGF in vascular endothelial cells, was increased by NR1, NR2 and NR3.

Example 4 Effect of NR1, NR2 and NR3 on Expression of Collagen 1A2, Fibronectin, HIF-1α Proteins, and Collagen 1A2, Fibronectin, VEGF Transcriptional Activities in Human Dermal Fibroblasts (HDF)

Human dermal fibroblast (HDF) cells (1 × 10 ⁶ cells / well) were seeded in 60-cm ² petri dishes and incubated overnight in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% FBS. HDF cells were maintained at 37 ° C. in a humidified thermostat with 5% CO ₂ and 95% air. After incubation, HDF cells were treated with 2 μM of each of NR1, NR2 and NR3 for 24 hours. After treatment, expression of collagen 1A2, fibronectin and HIF-1α (Santa Cruz Biotechnology) proteins were analyzed by Western blotting analysis, and expression of α-tubulin was monitored as a control. The results are shown in Figure 4a.

As shown in FIG. 4A, protein expression of collagen 1A2, fibronectin and HIF-1α was increased by NR1, NR2 and NR3.

In addition, 2 hours of NR1, NR2 and NR3 in HDF cells were treated for 24 hours under the same conditions, and then the effects on the transcriptional activity were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) assay. PCR reactions were performed using primers specific for collagen 1A2, fibronectin and VEGF, respectively; collagen 1A2 (forward: 5'-TCCAAGGACAAGAAACAC-3 ', reverse: 5'-GCAGCCATCTACAAGAAC-3'), fibronectin (forward: 5'-TGAAGAGGGGCACATGCTGA-3 ', reverse: 5'-GTGGGAGTTGGGCTGACTCG-3'), VEGF (forward : 5'-CTGCTGTCTTGGGTGCATTGG-3 ', reverse: 5'-CACCGCCTCGGCTTGTCACAT-3') and β-actin (5'-CGTGGGCCGCCCTAGGCACCA-3 ', reverse: 5'-TTGGCTTAGGGTTCAGGGGGG-3').

As shown in Figure 4b, it was observed that the transcriptional activity of collagen 1A2, fibronectin and VEGF increased in HDF cells by NR1, NR2 and NR3. The results indicate that NR1, NR2 and NR3 may promote the production of VEGF, an important growth factor for wound healing, and collagen and fibronectin, which cause rapid growth of granulation tissue.

Example 5 Effects of NR2 and NR3 on Cell Migration in HDF (human dermal fibroblast)

HDF cells (3 × 10 ⁵ cells / well) were seeded in Culture-insert culture dishes (iBidi) and incubated overnight in Dulbecco's modified Eagle's medium (DMEM) medium containing 10% FBS (fetal bovine serum). HDF cells were maintained at 37 ° C. in a humidified thermostat with 5% CO ₂ and 95% air. After incubation, the culture-insert membrane in the culture-insert culture dish was removed and then treated with 2 μM of NR2 and NR3, respectively. After peptide treatment, after 6 hours and 24 hours, the degree of cell migration was observed through a microscope, and the results are shown in FIG. 5.

As shown in FIG. 5, it was observed that cell migration occurred more actively compared to the control group treated with NR2 and NR3 for 24 hours.

Example 6. Effect of NR3 on wound healing in vivo

The dorsal hair of 250-280 g male Sprague Dawley rats was removed and anesthetized by IP injection with Zoletil50 ^® (Tiletamine / Zolazepam) / Rompun ^® (Xylazine) mixture. Two skin wounds (1.5 cm X 1.5 cm) were made in the rat dorsum and gauze, base (polyethylene glycol 400: polyethylene glycol 3350 = 4: 1), 500 µg / g NR3, After treatment with a commercial ointment (fucidin, sodium fusidate; anabolic drug), respectively, the wound size reduction after 3, 6, 9, 12 and 15 days was measured using the following formula and the results are shown in FIG. 7.

Wound size reduction (%) = [(A _O -At) / A _O ] x 100

(A _O : initial wound size, At: wound size on observation day)

As shown in Figure 6, compared to the gauze control, a significant decrease in epithelial defect sites was recognized in NR3 and commercial ointments. In addition, the dermis and subcutaneous tissue of the rat wounds were taken, fixed in a 10% carbonic acid buffer-formalin solution, then embedded in paraffin using an embedding center to make thin slices, and then the slices were hematoxylin- Staining with eosin (haematoxylin-eosin) and histological examination under a microscope were performed.

To determine more detailed histological in vivo wound healing, the size of desquamated epithelium regions, epithelial regeneration rate, number of neocapillaries in granulation tissue, and infiltrating inflammatory cells using a digital image analyzer (DMI-300, DMI) The number of, collagen expression area and granulation tissue area were measured and the results are shown in Table 2.

TABLE 2

In addition, histopathological observation through a microscope is shown in FIG. 9. Each represents a gauze treated control group (A-C), a base treated group (D-F), a commercial ointment (G-I) and NR3 (J-L). Asterisks of A, D, G, and J indicate desquamated epithelium regions, and arrowed portions of B, E, H, and K represent neocapillaries and infiltrating inflammatory cells in granulation tissue. The green part of C, F, I, L represents the collagen expression area. The regenerated epithelium of the wound was calculated by the following equation.

Re-epithelization (%) = [(A-B) / C] x 100

(A, B and C represent 'total length total wound (10 mm)', 'Desquamated epithelium regions (mm)' and 'total wound (10 mm)').

As shown in Table 2 and Figure 7, gauze treatment control (A ~ C) Compared with NR3 ointment (J ~ L), a significant (p <0.01) reduction in epithelial defect site And commercial ointment treatment groups (G ~ I) And as a result, a significant (p <0.01) increase in epithelial regeneration rate was recognized, respectively. In addition, the NR3 ointment and the commercial ointment applied group showed a significant decrease in neonatal capillary and infiltrating inflammatory cells (p <0.01 or p <0.05) compared with the gauze treatment control in the granulation tissue, and the area of the granulation tissue itself was also significantly ( p <0.01). The NR3 ointment was found to have a significant decrease in neocapillary and infiltrating inflammatory cells in granulation tissue compared to the base treatment group, and the area of the granulation tissue itself was also significantly decreased (p <0.01). On the other hand, in the NR3 group (D ~ F) treatment group, significant wound healing effect was not recognized compared to the gauze control group.

The foregoing description of the present invention is intended for illustration, and it will be understood by those skilled in the art that the present invention may be easily modified in other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, the embodiments described above are to be understood in all respects as illustrative and not restrictive.

The novel peptides according to the invention affect cell proliferation and migration by activating the VEGF gene through the activity of HIF-1α. Therefore, it shows effects such as wound healing, skin tissue regeneration, and hair growth promotion, and ultimately, it is expected to be used for the development of various therapeutic agents while being simple to use.

SEQ. ID. NO. 1 (NR peptide 1): Lys Asn Cys Leu Ile Asp Arg Thr Ser Arg Asn Arg

SEQ. ID. NO. 2 (NR peptide 2): Lys Asn Cys Leu Ala Asp Arg Thr Ser Arg Ala Arg

SEQ. ID. NO. 3 (NR peptide 3): Lys Asn Cys Leu Ile Asp Ala Thr Ser Arg Ala Arg

Claims (6)

1. Pharmaceutical composition for wound treatment or skin tissue regeneration comprising a peptide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.
2. The method of claim 1,

   The peptide is characterized in that to increase the expression of hypoxic factor (HIF) -1α, vascular endothelial growth factor (VEGF), fibronectin, or collagen (collagen).
3. The method of claim 1,

   The peptide is characterized in that proliferation of vascular endothelial cells, pharmaceutical compositions.
4. The method of claim 1,

   The peptide is characterized in that to increase cell migration (migration), pharmaceutical composition.
5. Peptide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.
6. A method of treating wounds or regenerating skin tissue by administering to a subject a pharmaceutical composition comprising a peptide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3.

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