KR100729210B1 - Peptide with a enhanced inhibitory activity against VEGF and a remedy thereof - Google Patents

Abstract

In the present invention, angiogenesis is induced by overexpression of an endothelial growth hormone (VEGF), which is an inducer of angiogenesis or neovascularization, and an overexpression of an endothelial growth hormone prepared therefrom. The present invention relates to a therapeutic agent for a developing disease, and peptides constituting the present invention (particularly, RRRRKRRRWR and MAP2-dRK6) may be used to treat various diseases related to angiogenesis such as cancer treatment and rheumatoid arthritis and diabetic retinopathy.

Angiogenesis, Endothelial Growth Hormone, Antagonism, Peptides, Cancer, Rheumatoid

Description

Peptide with a enhanced inhibitory activity against VEGF and a remedy about             

Figure 1 shows the sequence of peptide RRKRRR (hereinafter referred to as "RK6") when one amino acid is changed from L-type to D-type or when all amino acids are changed from L-type to D-type (rrkrrr). , Hereinafter referred to as "dRK6") is a result confirming that the degree of inhibiting the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor is similar to the existing RK6 peptide,

FIG. 2 shows the results of the first search to find a peptide that inhibits binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor by a peptide random sequence mixture of two sequences before and after RK6.

3 is a description of a process of selecting RRRRKRRRWR as a result of the secondary library search (B) synthesized based on the amino acid (A) selected by the primary search and the primary search result,

Figure 4 shows the degree of inhibition of binding between peptide (A), endothelial growth hormone (VEGF ₁₆₅ ) and its receptor, which cleave 1, 2, and 3 amino acids from the N-terminus or C-terminus of RK6, respectively. Results (B) and the degree of inhibiting the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor of one peptide selected here (C-terminal one residue cleavage peptide, hereinafter referred to as "CD1") ( C),

Figure 5 shows the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor of circular RK6 formed by attaching one cysteine to each of the N- and C-terminus of RK6, and then forming an SS disulfide bond. Is the result of checking the degree of suppression,

FIG. 6 shows four branched RK6 (hereinafter referred to as “MAP4-RK6”) and two branched RK6 (hereinafter referred to as “MAP2”), which are branched peptides (multiple antigenic peptides) using lysine. -RK6 ”is the result of confirming the degree of inhibition of the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor,

7 is a result showing that the peptide converting the amino acid of MAP2-RK6 into D-type (hereinafter referred to as “MAP2-dRK6”) inhibits the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor in proportion to concentration. ,

FIG. 8 shows the results of toxicity test of rat tail vein of dRK6 and selected RK6 modified derivatives (RRRRKRRRWR & MAP2-RK6).

9 shows that MAP2-dRK6 inhibits the growth of endothelial cells induced by endothelial growth hormone (VEGF ₁₆₅ ) in proportion to concentration (A), and the peptide itself does not directly affect human endothelial cells. (B),

10 is a result showing that MAP2-dRK6 has a much greater translocation ability into cells compared to dRK6,

11 is a result showing that peptide MAP2-dRK6 inhibits angiogenesis (A) in rabbit cornea induced by endothelial growth hormone (VEGF ₁₆₅ ),

FIG. 12 shows that the growth of human colon cancer cell lines (SW480R3) secreting endothelial growth hormone (SW480R3) by subcutaneously injected peptide MAP2-dRK6 was effectively inhibited in nude mice (A). Growth of human rectal cancer cells (HM7) is also shown to be effectively inhibited in nude mice (B),

FIG. 13 shows that subcutaneously injected peptide dRK6 inhibits the incidence (A) and severity (B) of collagen-induced arthritis in rats in proportion to concentration.

14 shows that subcutaneous injection of peptide dRK6 reduces the amount of antibody produced by type II collagen in mice (A) and lowers the stimulation index of T cells caused by type II collagen. Result (B),

FIG. 15 shows that dRK6 inhibits tumor necrosis factor- α , TNF- α production of rheumatoid arthritis synovial fluid mononuclear cells induced by endothelial growth hormone. Results (A) and results (B) of inhibiting the production of interleukin-6 (IL-6) of synovial fibroblasts induced by endothelial growth hormone,

16 shows that dRK6 (B) is more stable in rat serum than RK6 (A).

Fig. 17 shows the sequence of MAP2-RK6, a peptide made in two branches using lysine among derivatives of the peptide consisting of RRKRRR,

Fig. 18 shows the sequence of MAP4-RK6, a peptide made in four branches using lysine among derivatives of the peptide consisting of RKRRR.

In the present invention, angiogenesis is induced by overexpression of an endothelial growth hormone (VEGF), which is an inducer of angiogenesis or neovascularization, and an overexpression of an endothelial growth hormone prepared therefrom. It relates to a therapeutic agent for the disease that occurs.

Angiogenesis, the process of forming new blood vessels from existing vessels, occurs in a number of normal or pathological processes. In normal conditions, such as embryo development and wound healing, angiogenesis is only briefly active and always inhibited by the balance between inducers and inhibitors (Loitta, LA et al. (1991) Cell 64 , 327). Many diseases, such as the growth and metastasis of solid cancer, rheumatism, diabetic retinopathy, and the development of various inflammatory diseases, are accelerated by faulty angiogenesis caused by abnormalities of these fine control mechanisms (Kohn, EC et al. (1995) Proc. Natl.Acad.Sci. USA 92, 1307; Folkman, J. et al. (1987) Science 235, 442; Risau, W. (1997) Nature 386, 671). Angiogenesis is particularly essential for cancer growth and metastasis, as it enables smooth nutrient supply through the blood and provides a pathway for metastatic cancer (Hanahan, D. et al. (1996) Cell 86, 353; Skobe, M., et al. (1997) Nature Med. 3, 1222). Thus, angiogenesis is an essential step in cancer growth as well as cancer metastasis, which is an indicator of malignancy.

Although many neovascular inducers have been identified to date, most of them do not act as growth hormones on endothelial cells (Bussolino, F., et al. (1997) Trends Biochem. Sci. 22, 251). In contrast, endothelial growth hormone acts as a potent endothelial specific growth factor in vitro (Gospodarowicz, D., et al. (1989) Proc. Natl. Acad. Sci. USA 86, 7311), and permeability of blood vessels (Leung, DW, et al. (1989) Science 246, 1306) and are known to induce angiogenesis associated with cancer progression in vivo (Plouet, J., et al. (1989) EMBO J. 8, 3801). Endothelial growth hormone binds to heparin and shows some amino acid sequence similarities with placental growth factor (PlGF) and platelet-derived growth factor (PDGF) (Conn, G., et al. (1990) Proc. Natl.Acad. Sci. USA 87, 2628; Keck, PJ, et al. (1989) Science 246, 1309; Maglione, D., et al. (1991) Proc. Natl. Acad. Sci. USA 88, 9267) . Endothelial growth hormone is expressed in the form of isoforms with 121, 165, 189, and 206 amino acids by alternative splicing (Tischer, E., et al. (1991) J. Biol. Chem. 266, 11947), of which VEGF ₁₂₁ does not bind heparin (Conn, G., et al. (1990) Proc. Natl. Acad. Sci. USA 87, 2628). Endothelial growth hormone binds to Flt-1 and KDR / Flk-1, which are receptors specifically expressed on the surface of endothelial cells, and are active (Millauer, B., et al. (1993) Cell 72, 835; De Vries, C., et al. (1992) Science 255, 989). In KDR / Flk-1 knock-out mice, mature endothelial cells are not found, whereas Flt-1 knock-out mice are found to have mature endothelial cells but do not function and form blood vessels (Fong, G-). H., et al. (1995) Nature 376, 66; Shalaby, F, et al. (1995) Nature 376, 62).

The importance of endothelial growth hormone (HGF) and its receptors in the development of angiogenesis (vasculogenesis) and angiogenesis has been investigated in the study of knock-out mice (Fong, G-.H., Et al. (1995) 376, 66; Shalaby, F, et al. (1995) Nature 376, 62; Carmeliet, P., et al. (1996) Nature 380, 435; Ferrara, N., et al. (1996) Nature 380, 439 ), Overexpression of endothelial growth hormone in cancer cells (Zhang, HT, et al. (1995) J. Natl. Cancer. Inst. 87, 213), neutralization of endothelial growth hormone Inhibition of growth of cancer cells by expression of neutralizing monoclonal antibody and soluble Flt-1 receptor (Kim, KJ, et al. (1993) Nature 362, 841; Goldman, CK, et al. (1998) Proc. Natl. Acad. Sci. USA 95, 8795).

Therefore, blocking the binding between endothelial growth hormone and its receptors is an attractive target for preventing cancer neovascularization, cancer growth and metastasis (Martiny-Baron, G. et al. (1995) Curr. Opin. Biotechnol. 6 , 675), substances that inhibit the binding between endothelial growth hormone and its receptors can not only inhibit angiogenesis induced by endothelial growth hormone, but also inhibit the growth and metastasis of cancer cells secreting endothelial growth hormone. Could be.

Meanwhile, as a prior art regarding peptides that inhibit vascular wall cell growth hormone activity, the RK6 is a peptide described in the registered publication of the patent (the Republic of Korea Patent Publication No. 10-0319828, published March 13, 2002) (RRKRRR)

However, there is a lot of possibility to further increase the stability in vivo to the activity that inhibits the cell growth hormone activity of the peptide.

Accordingly, an object of the present invention is a derivative of enhanced activity or stability of RK6 (RRKRRR) as a therapeutic agent for the treatment of diseases caused by angiogenesis induced by the overexpression of new peptides and endothelial growth hormone prepared therefrom. To provide.

In order to achieve the above object, the present invention provides a antagonistic peptide for endothelial growth hormone, characterized in that the peptide described in SEQ ID NO: 1 comprising a peptide whose amino acids are D-type derivatives,

An antagonistic peptide for endothelial growth hormone, comprising the peptide set forth in SEQ ID NO: 2,

An antagonistic peptide for endothelial growth hormone, characterized in that the amino acid at the N-terminal end of the peptide described in SEQ ID NO: 1 comprises a peptide that has been cut by any one selected from one, two, and three,

An antagonistic peptide for endothelial growth hormone, characterized in that the amino acid at the C-terminal end of the peptide described in SEQ ID NO: 1 comprises a peptide cleaved by any one selected from one, two, and three,

Antagonistic peptides against endothelial growth hormone, characterized in that it comprises a peptide made by connecting Cysteine to the N- and C-terminal peptides of SEQ ID NO: 1,

Antagonistic peptides against endothelial growth hormone, comprising the peptide described in FIG.

Provided is an antagonistic peptide for endothelial growth hormone, comprising the peptide described in FIG.

The present invention also provides a therapeutic agent for a disease caused by angiogenesis induced by overexpression of endothelial growth hormone, characterized in that it contains an antagonistic peptide to the endothelial growth hormone as an active ingredient.

Hereinafter, the present invention will be described in more detail.

In the present invention, dRK6, a D-type peptide of RK6 (RRKRRR), which is one of the AR peptides mentioned in a previously filed invention (Korean Patent Publication No. 10-0319828, published March 13, 2002), is endothelial cell growth. It has been further found in contrast to the previous patents that the hormones reduce the incidence and symptom of arthritis, inhibit the autoimmune response, and inhibit the production of immune substances (tumor necrosis factor-alpha and interleukin-6). .

And, when the amino acid of RK6 was changed to D-form (dRK6), its activity in vitro was similar, and peptide library (peptide random sequence mixture, Positional) which was added by adding two amino acids on both sides based on RK6 Peptide RRRRKRRRWR, found in the Scanning-Synthetic Peptide Combinatorial Library, was toxic during intravenous rat injection, but showed a 10-fold improvement in activity. However, it was found that a certain level of activity was observed, and that the peptides formed by attaching cysteine to both sides of the RK6 peptide showed no difference in activity, but showed a level of activity.

In addition, the present inventors examined the activity of two kinds of RK6 derivatives (two and four: MAP4-RK6 and MAP2-RK6, respectively) made into branches using lysine.

Both activities improved, but the bimodal peptide MAP2-RK6 and its amino acid-modified peptide, DAP-type MAP2-dRK6, combined with endothelial growth hormone and endothelial growth hormone and endothelial cells derived from human umbilical cord (Human Umbilical Vein Endothelial Cells, HUVECs) showed a 20-fold improvement in activity compared to dRK6 in inhibiting binding between the receptors on the surface of the cell membrane. Intracellular translocation activity was higher than that of dRK6, inhibited endothelial cell proliferation by endothelial growth hormone, inhibited angiogenesis in rabbit cornea induced by endothelial growth hormone. Inhibition of angiogenesis inhibited the growth and metastasis of the hormone-producing human rectal cancer cells (SW480R3 and HM7).

The pharmaceutical composition of the present invention may be administered parenterally during actual clinical administration, and parenteral administration may be performed by subcutaneous injection, intraperitoneal injection, subcutaneous injection or ointment. The dosage of the active ingredient according to the present invention is appropriately selected depending on the stability of the active ingredient in the body, the rate of excretion, the age, sex and condition of the patient, and the severity of the disease to be treated. It is expected that both dRK6 and MAP2-dRK6 are 2.5-10 mg / kg for subcutaneous and intraperitoneal injection and 2.5-5 mg / kg for intravenous injection. In the case of the ointment method, it is necessary to determine the dose later. The exact amount, route of administration, and frequency of the agent can be readily determined according to the nature of the agent, the weight and condition of the subject to be administered, and the nature of the particular derivative to be used.

Hereinafter, the configuration of the present invention will be described in more detail with reference to the following examples, but the scope of the present invention is not limited only to the following examples.

Example 1: A peptide wherein each amino acid in RK6 and RK6 is substituted with a D-type amino acid ¹²⁵ Endothelial growth hormone labeled with I ( ¹²⁵ I-VEGF ₁₆₅ ) And inhibition of binding between receptors on endothelial cells

In order to determine the extent to which peptides substituted with D-type amino acids in RK6 and RK6 inhibit the binding capacity between endothelial growth hormone and its receptor, the following experiment was performed. Endothelial cells (2 × 10 ⁴ cells / well) were placed in a gelatin-fixed culture vessel (48-well, Nunc) and cultured overnight, followed by binding assay medium (medium 199/25 mM HEPES / 0.1% bovine serum albumin, pH 7.4). ) Was added and incubated at 37 ° C. for 1 hour. Meanwhile, each peptide was pretreated with ¹²⁵ I-labeled endothelial growth hormone for 1 hour at 4 ° C. Thereafter, the medium in the culture vessel was changed to endothelial growth hormone (20 nCi / well) labeled with each peptide / ¹²⁵ I and incubated at 4 ° C. for 3 hours. To remove unbound labeled endothelial growth hormone, the cells were washed twice and once with phosphate buffer containing 0.1% bovine serum albumin and binding medium.

To determine the amount of ¹²⁵ I labeled endothelial growth hormone bound to receptors present in endothelial cells, 150 μl of lysis solution (20 mM Tris-HCl / 1% Triton X-100, pH 7.4) was added at room temperature for 20 minutes. After treatment, the amount of radioactivity in the lysed cell mixture was measured using γ- counter, and the binding inhibitory activity between endothelial growth hormone ( ¹²⁵ I-VEGF ₁₆₅ ) and receptors on endothelial cells was examined.

Figure 1 shows the sequence of peptide RRKRRR (hereinafter referred to as "RK6") when one amino acid is changed from L-type to D-type or when all amino acids are changed from L-type to D-type (rrkrrr). , Hereinafter referred to as “dRK6”), the degree of inhibition of binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor is compared with the conventional RK6 peptide. Where R is L-arginine, K is L-lysine, r is D-arginine, k is D-lysine, 1r is substituted with D-arginine in the first position, and 2r is substituted with D-arginine in the second position. RK6, 3r is RK6 with 3rd position substituted with D-arginine, 4r is RK6 with 4th position substituted with D-arginine, 5r is RK6 with 5th position substituted with D-arginine, 6r is 6th position D- RK6 substituted with arginine, dRK6 are peptides in which all of RK6 is substituted with D-amino acid, 6R is hexaarginine (RRRRRR), and 6K is hexalysine (KKKKKKK).

As shown in FIG. 1, when the amino acid of RK6 was changed to D-type, the activity was found to be similar. From these results, the activity of peptides consisting of D-type and L-type amino acids is similar, so considering the stability in vivo, it is advantageous to use RK6 (dRK6) consisting of D-type amino acids in animal experiments. I think so.

Example 2: By peptide extended two sequences before and after RK6 ¹²⁵ Endothelial growth hormone labeled with I ( ¹²⁵ I-VEGF ₁₆₅ ) And inhibition of binding between receptors on endothelial cells

RK6 performed by the following test to determine the extent to which inhibits the binding between a receptor on the sides two by two amino acids is a spun peptide sequence labeled with ¹²⁵ I endothelial growth hormone ⁽¹²⁵ I-VEGF ₁₆₅₎ and endothelial cells Binding inhibition was tested in the same manner as in Example 1.

Figure 2 is a result of measuring the binding inhibitory ability of the VEGF and the receptor of the positional scanning-synthetic peptide combinatorial library (PS-SPCL) extending two amino acid sequences on both sides of RK6.

As shown in Figure 2 A, the first amino acid was O ₂ series {arginine (R), lysine (K), histidine (H)} was excellent in inhibition,

As shown in FIG. 2B, the second amino acid also had excellent inhibition when O ₂ series {arginine (R), lysine (K), histidine (H)},

As shown in Figure 3 C, the seventh amino acid was excellent in inhibition when O ₃ series {phenylalanine (F), tryptophan (W), tyrosine (Y)},

As shown in Figure 2 D, the eighth amino acid was excellent in inhibition when O ₂ series (arginine (R), lysine (K), histidine (H)).

Secondary peptide libraries (sub 1 to sub 11) synthesized based on the amino acid selected by the first search of FIG. 2 were prepared (FIG. 3 A), and the inhibitory ability of VEGF and the receptor was again measured (FIG. 3 B). As a result, it was confirmed that the peptide sequence RRRRKRRRWR was the best from the excellent binding inhibitory activity of sub 1, sub 4, sub 8, sub 10, and the activity was improved by about 10 times compared to RK6.

Example 3: with a peptide in truncated form of RK6 ¹²⁵ Endothelial growth hormone labeled with I ( ¹²⁵ I-VEGF ₁₆₅ ) And inhibition of binding between receptors on endothelial cells

As a result of investigating the binding inhibition between VEGF and the receptor by a peptide such as FIG. 4A in the same manner as in Example 1 (FIG. 4B), CD1 (peptide sequence RRKRR) showed the highest activity and similar activity to RK6. (FIG. 4C, FIG. 4D).

Example 4: by circular RK6 peptide ¹²⁵ Endothelial growth hormone labeled with I ( ¹²⁵ I-VEGF ₁₆₅ ) And inhibition of binding between receptors on endothelial cells

Figure 5 shows the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor of circular RK6 formed by attaching one cysteine to each of the N- and C-terminus of RK6, and then forming an SS disulfide bond. This is the result of checking the degree to suppress.

As shown in Figure 5, the activity of the circular RK6 peptide showed similar results to the activity of RK6.

Example 5: by RK6 (MAP2-dRK6) and RK6 (MAP4-dRK6) Peptides ¹²⁵ Endothelial growth hormone labeled with I ( ¹²⁵ I-VEGF ₁₆₅ ) And inhibition of binding between receptors on endothelial cells

FIG. 6 shows four branched RK6 (hereinafter referred to as “MAP4-RK6”) and two branched RK6 (hereinafter referred to as “MAP2”), which are branched peptides (multiple antigenic peptides) using lysine. -RK6 ”) confirmed the degree of inhibition of the binding between endothelial growth hormone (VEGF ₁₆₅ ) and its receptor.

As shown in FIG. 6, the activity of two kinds of RK6 derivatives made of a branch is improved, and in particular, the bifurcated RK6 (MAP2-dRK6) made of D-type amino acid has an increase in activity in proportion to its concentration. It showed a 20-fold increase in activity compared to dRK6 (Fig. 7, IC ₅₀ approximately 0.35 μM).

Example 6: Toxicity Tests Through Intravenous Rat Injection

A certain amount (200 μg) of RK6 peptide was subcutaneous or intraperitoneal to mice, but not intravenously. To determine whether the RK6 peptides had decreased toxicity during intravenous injection of rats, the toxicity of various RK6 derivative peptides was examined by intravenous injection. Four hundred-week-old BALB / c female rats were injected with 200 μg of dRR6 and RRRRKRRRWR and MAP2-dRK6 peptides with increased activity in 100 μl of saline and examined for mortality.

As a result (Fig. 8), mice injected with dRK6 and RRRRKRRRWR died, but mice injected with MAP2-dRK6 did not die, indicating that the toxicity of MAP2-dRK6 was reduced than that of dRK6.

Example 7 Inhibition of Endothelial Cell Proliferation by Endothelial Growth Hormone of MAP2-dRK6 Assay

To determine whether MAP2-dRK6 inhibits DNA synthesis of endothelial cells induced by endothelial growth hormone (HUVEC), the following experiment was performed. Endothelial Growth Hormone (final concentration 5 ng / ml, R & D system) and MAP2-dRK6 peptide in medium cultured gel (96-well plate, Nunc) culture medium (medium 199/10% fetal bovine serum / 10 mM HEPES, pH 7.4) was mixed and pretreated at 37 ° C. for 1 hour, and then endothelial cells (5 × 10 ³ cells / well) were added and incubated at 37 ° C. for 2 days. Then, incubated for 3 hours with ³ H-labeled thymidine (3H-Thymidine, 0.5 μCi / well), washed three times with a phosphate buffer solution containing 0.1% of BSA, and washed at room temperature with 0.4 N NaOH. Cells were lysed for a minute, neutralized with 2N HCl solution, 1.25 ml of scintillation cocktail solution was added, and the amount of radioactivity used for DNA synthesis was measured by a liquid scintillation counter.

MAP2-dRK6 inhibited DNA synthesis of endothelial cells induced by endothelial growth hormone in a concentration-dependent manner, and the IC ₅₀ value was about 9 μM (FIG. 9A).

Example 8: Determination of MAP2-dRK6 Toxicity to Endothelial Cells

Inhibition of endothelial proliferation by MAP2-dRK6 inhibited the action of endothelial growth hormone, and MTT (3- [4,5-dimethylthiazol-2-yl ] -2,5-diphenyltetrazolium bromide) assay was performed. Endothelial cells (5 × 10 ³ cells / well) were incubated overnight in a gelatin-fixed culture vessel (96-well plate, Nunc). The medium was changed to the growth test medium containing the concentration-specific MAP2-dRK6, and then cultured at 37 ° C. for 2 days, and 5 mg / ml MTT solution was added 10% of the total volume. After incubating at 37 ° C. for 4 hours, the solution in the container was removed, and 100 μl of lysis solution (10% sodium dodecyl sulfate / 25% dimethylformamide) was added and then dissolved overnight. The next day, the absorbance at 570 nm was measured to assess the viability of the cells.

As shown in FIG. 9B, it was found that there was no direct toxicity to the cells until the peptide concentration was 25 μM. Based on the above experimental results, we could confirm that MAP2-dRK6 has specific activity of inhibiting endothelial cell growth by endothelial growth hormone by inhibiting binding between endothelial growth hormone and its receptor.

Example 9 Intracellular Translocation Activity Comparison of dRK6 and MAP2-dRK6

The translocation activity of the two peptides into cells was tested in anticipation that the difference in virulence of dRK6 and MAP2-dRK6 upon intravenous injection of mice was due to the difference in intracellular dislocation activity. Fluorescein isothiocyanate (FITC) labeled dRK6 and MAP2-dRK6 were mixed in medium (DMEM / 10% FBS) at a concentration of 1 μM, treated with human rectal cancer cells (SW480R3) for 3 hours, followed by FACS (fluorescence-activated cell sorter) 10,000 cells were selected using to measure the degree of translocation into the cells.

As shown in FIG. 10, MAP2-dRK6 was found to have a much higher translocation activity into cells than dRK6. In this case, it is thought that the dRK6 peptide does not cause toxicity during intravenous injection due to the translocation activity into cells, and it can be used as a deliverer if the high translocation activity of MAP2-dRK6 is used in the cells.

Example 10: Angiogenesis Inhibition Assay by Endothelial Growth Hormone of MAP2-dRK6 Peptide through a Bio-Model Experiment

After confirming endothelial growth inhibitory activity induced by endothelial growth hormone, angiogenesis biomodel experiments using rabbit corneas were performed to test the antiangiogenic activity of MAP2-dRK6 peptide. New Zealand male white rabbits (3 kg, SLC, Japan) were anesthetized (intramuscular ketamine anesthesia (44 mg / kg)), and the corneal dome was cut 3 mm using a Bard-Parker # 11 surgical knife. Endothelial growth hormone (10 ng, R & D system) alone, or a solution containing MAP2-dRK6 (1 μg) and endothelial growth hormone (10 ng) was added dropwise and the dried Thermanox coverslip (Nunc) was added to this incision site. Natural healing. Sixteen days after angiogenesis was clearly observed, pictures of newly formed blood vessels were taken on the cornea.

In the experimental group treated only with endothelial growth hormone (Fig. 11 A), distinct angiogenesis was observed, but the angiogenesis was completely inhibited in the experimental group treated with MAP2-dRK6 peptide and endothelial growth hormone simultaneously (Fig. 11 B). According to the results of the present example and FIG. 9, the MAP2-dRK6 peptide does not directly affect endothelial cell growth and prevents endothelial cell growth hormone-induced endothelial cell growth hormone from binding to its receptor. It can be seen that angiogenesis can be inhibited in vivo .

Example 11: Effect of MAP2-dRK6 Peptide on Growth of Human Rectal Cancer Cells (SW480R3 & HM7)

Acquiring angiogenic capacity is a critical step in cancer progression and has been reported to be essential for continued growth of cancerous tissues (Hanahan, D. et al. (1996) Cell 86, 353; Skobe, M., et al. (1997) Nature Med. 3, 1222), Example 6 shows that the MAP2-dRK6 peptide can effectively inhibit angiogenesis in vivo by VEGF. Investigated if it could inhibit the growth of cancer. Human rectal cancer cells (SW480R3, 5 × 10 ⁶ cells or HM7, 5 × 10 ⁵ cells) were mixed in serum-free DMEM, followed by 4 week old female mice (BALB / c / nu / nu, Charles River, Japan). Each peptide (100 μg / 100 μl / day) dissolved in phosphate buffer solution was injected subcutaneously for 14 days from the next day. Tumor size was measured regularly and tumor volume was calculated using the following formula (volume = 0.5 x (short) ² x long axis). When examining the effect on human rectal cancer cells using MTT assay, it was confirmed that MAP2-dRK6 itself does not directly affect the growth of cancer cells, so MAP2-dRK6 may be inhibited by the toxicity of cancer cells. Could be excluded.

After 15 days of subcutaneous injection, the MAP2-dRK6 peptide showed tumor suppression of tumor growth compared to the control group injected with only phosphate buffer for tumors caused by SW480R3 and HM7 (FIG. 12). From the above results, it can be seen that the MAP2-dRK6 peptide may inhibit the growth of malignant tumors by blocking the signaling of endothelial growth hormone.

Example 12 Effect Assay of dRK6 on Arthritis Induced by Collagen

Angiogenesis by endothelial growth hormone in arthritis is known to play an important role in maintaining and promoting arthritis (Palelog, E.M., (2002) Arthritis Res 4 (suppl 3), S81). Previously, RK6 inhibited angiogenesis caused by endothelial growth hormone, and thus, it was expected that a good effect would be obtained when treated with arthritis. 0.1 ml of emulsion (1: 1 ratio of CII / 0.05 N) containing 100 μg of type II collagen (CII) in male DBA / 1 rats (Jackson Laboratories, Bar Harbor, ME), aged 8-12 weeks. Arthritis was induced by injection of acetic acid and complete Freund's adjuvant), and two weeks later, 50 μg of CII. Three weeks after the first injection, 25 or 50 μg of dRK6 peptide was injected subcutaneously for three weeks every two days and the incidence and severity of arthritis was observed.

As shown in Fig. 13, it can be seen that the incidence of collagen-induced arthritis (Fig. 13A) and the symptom (Fig. 13B) are reduced when dRK6 peptide is injected, which is effective in the treatment and prevention of arthritis. Could.

Example 13: Effect of dRK6 on Antibody Formation by Type II Collagen

One symptom of arthritis from injection of type II collagen is the production of antibodies against it. The effects of dRK6 on the incidence and symptom of arthritis on the production of antibodies to type II collagen were investigated through the following experiments. Serum was obtained from each group of rats 42 days after CII injection. The level of anti-CII immunoglobulin G (IgG anti-CII) produced in the obtained serum was measured using Eliza kit (Chondrex, Redmond, WA).

As shown in FIG. 14A, the production of antibody against CII was reduced by dRK6 peptide injection, which may be one explanation for the reduction in the incidence and symptom of collagen-induced arthritis in rat experiments. have.

Example 14 Effect of dRK6 on Proliferation of T Cells by Type II Collagen

Another one of the symptoms of arthritis caused by the injection of type II collagen is that T cells proliferate accordingly. The following experiments were performed to determine whether the activity of pRK and the symptom relief of arthritis of dRK6 is related to the inhibition of T cell proliferation by type II collagen. 42 days after injection of CII, lymph nodes of inguinal and slaw were removed from each group of rats and washed with RPMI 1640 medium. Tissues were obtained from 4-5 rats, separated into individual cells, and placed in or without CII (40 μg / well) in culture vessels (96-well, Nunc) (5 × 10 ^5). cells / 0.3 ml of Click's medium / 0.5% mouse serum / well). After incubation at 37 ° C. for 4 days, thymidine (1 μCi / well) labeled with H ³ was added thereto for 18 hours. Radioactivity was measured by Matrix 96 direct ionization β-counter (Packard Instrument Co., Meridan, CT), and the values of the experimental group including CII were divided by the values of the experimental group without CII and expressed as stimulation indexes.

As shown in FIG. 14B, the degree of proliferation of T cells obtained from mice treated with dRK6 peptide was lower than that of T cells obtained from mice not treated with peptide, and thus the incidence and symptoms of arthritis induced by CII. It can be said that the degree is lowered.

The two examples above show that RK6 peptides that inhibit endothelial growth hormone effectively inhibit the body's specific immune response to CII in collagen-induced arthritis animals.

Example 15 Tumor Necrosis Factor-alpha Induced by Endothelial Growth Hormone (TNF-) α ) And the effect of RK6 on the production of interleukin-6 (IL-6)

In order to examine the effect of RK6 peptide on human rheumatoid arthritis, specifically, it is possible to control the activity of monocytes stimulated by endothelial growth hormone, It was. Synovial fluid (SF) was taken from 11 patients with arthritis and mononuclear cells were isolated. These cells were placed in a culture vessel (96-well, Nunc) (1 × 10 ⁶ cells / well), and endothelial growth hormone (10 ng / ml) pretreated with RK6 peptide (0-100 μM) for 1 hour at room temperature. VEGF ₁₆₅ , R & D system). After culturing at 37 ° C. for 24 hours, the amount of TNF- α and IL-6 produced by taking the medium was confirmed by a pathological diagnosis test (ELISA).

As shown in FIG. 15, the RK6 peptide inhibited the production of TNF- α and IL-6 from synovial mononuclear cells of rheumatoid arthritis patients by endothelial growth hormone. Based on these results, it can be seen that the RK6 peptide has an activity of inhibiting the production of cytokines causing inflammation induced by endothelial growth hormone by preventing binding between endothelial growth hormone and its receptor.

Example 16: Stability Comparison Assay of RK6 and D-type Derivative dRK6 in Rat Serum

In FIG. 1, a D-type amino acid derivative dRK6 showing the same activity as RK6 was obtained to improve stability in vivo. The following experiment was performed to compare the stability of RK6 and dRK6 in serum using serum obtained from experimental rats (SD rat). Four male 16-week-old rats were anesthetized with diethyl ether, and 30 ml of blood was obtained through the portal vein after opening. After incubation at 4 ° C. for 30 minutes to induce blood coagulation, centrifugation (4 ° C., 3000 rpm, 10 minutes) to obtain a supernatant serum, and the aliquot was stored at -70 ° C. until just before use. For comparison of stability in serum, each peptide (100 μg in PBS) was co-cultured with 50 μl serum at 37 ° C. for various time periods, and the amount of peptide remaining undissolved was determined using a C18 reversed phase column (Vydac Protein). & Peptide C18, Equilibration: 0.1% Trifluoroacetic acid in water; Elution: 0.1% Trifluoroacetic acid in acetonitrile, Detection at 215 nm, 1 ml / min of flow rate.

When 100 μg of peptide was injected in a C18 reversed phase column, both RK6 and dRK6 were detected at the retention time of the same 4 minutes (16% acetonitrile) (second graph of FIGS. 16A and B). When only serum was injected into this column (top graphs in Figures 16A and B), a peak with a retention time of 4 minutes and a peak with a retention time of 5.5 minutes were found at similar times as the peptide peak. . In the case of the 4 minute peak, there was no problem in the detection of RK6 or dRK6 because it was very small compared to the peptide used in this experiment. Since a constant amount was always maintained at each serum injection, the 5.5 minute peak was used as a reference peak for the amount of serum injected in this experiment. After culturing each peptide and serum for various times, the amount of each peptide was detected.The half-life in serum was about 3 minutes for RK6, whereas dRK6 degraded more than 50% after 12 hours. Remained unsuccessful. When the serum or only peptide was incubated at 37 ° C under these experimental conditions, there was no change in the peaks at 4 minutes and 5.5 minutes. Therefore, the decrease of the RK6 peak with increasing incubation time resulted in various proteins in the serum. The low stability of RK6 showed a marked improvement in dRK6, a D-type amino acid derivative.

As described above, by using the peptides constituting the present invention (particularly, RRRRKRRRWR and MAP2-dRK6) to improve the binding strength and in vivo stability, cancer treatment, angiogenesis and rheumatoid arthritis, diabetic retinopathy, It can be used as a treatment for many related diseases. In particular, MAP2-dRK6 is likely to have as a deliverer due to its reduced toxicity and its high potential activity.

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Claims (12)

An antagonistic peptide against endothelial cell growth hormone consisting of a peptide of SEQ ID NO: 1 consisting of one or more D-type peptides. An antagonistic peptide for endothelial growth hormone consisting of the peptide set forth in SEQ ID NO: 2. An antagonistic peptide against endothelial growth hormone consisting of peptides in which the amino acid at the N-terminus of the peptide of SEQ ID NO: 1 is cleaved by any one selected from one, two and three. An antagonistic peptide against endothelial growth hormone consisting of peptides whose amino acids at the C terminus of the peptide of SEQ ID NO: 1 are cut by any one selected from one, two and three. An antagonistic peptide against endothelial growth hormone consisting of a peptide made by connecting Cysteine to the N- and C-terminals of the peptide of SEQ ID NO: 1. An antagonistic peptide derivative for endothelial growth hormone, consisting of the following formula (1). [Formula 1]

The method of claim 6, An amino acid constituting the peptide is an antagonistic peptide derivative for endothelial growth hormone, characterized in that at least one D-type. An antagonistic peptide derivative for endothelial growth hormone, consisting of the following formula (2). [Formula 2]

The method of claim 8, An amino acid constituting the peptide is an antagonistic peptide derivative for endothelial growth hormone, characterized in that at least one D-type. Angiogenesis is induced by overexpression of endothelial growth hormone, characterized by containing an antagonistic peptide against endothelial growth hormone as described in any one of claims 1 to 9 as an active ingredient. Drugs for developing cancer or arthritis. delete delete

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