WO2005077971A1

WO2005077971A1 - Novel heparin-binding peptide designed from heparin-binding site of snake poison-origin vascular endothelial growth factor (vegf)-like protein and use tehreof

Info

Publication number: WO2005077971A1
Application number: PCT/JP2004/008109
Authority: WO
Inventors: Takashi Morita; Yasuo Yamazaki
Original assignee: Nec Soft, Ltd.
Priority date: 2004-02-12
Filing date: 2004-06-10
Publication date: 2005-08-25
Also published as: WO2005077970A1; JPWO2005077971A1

Abstract

It is intended to provide a novel heparin-binding peptide being capable of inhibiting the binding of VEGF-A165 to heparin or heparan sulfate proteoglycan and thus suppressing various physiological reactions caused by interaction between KDR and VEGF-A165. Namely, a heparin-binding peptide which is designed based on the heparin-binding site of a snake poison-origin VEGF-like protein vammin, contains at least a first partial amino acid sequence R-P-R-X-K-Q-G (wherein X represents any of R, W, H and K) originating in the above-described heparin-binding site and has from 7 to 20 amino acid residues.

Description

Specification

Novel peptide with heparin binding ability designed from heparin binding site of vascular endothelial growth factor (VEGF) -like protein derived from snake venom and its use

Technical field

The present invention has specific binding properties to vascular endothelial growth factor receptor type 2 (VEGF receptor 2; KDR), while vascular endothelial growth factor receptor type 1 (VEGF receptor 1; Fit-1) A heparin-binding peptide designed based on the amino acid sequence derived from the heparin-binding site in a snake venom-derived vascular endothelial growth factor (VEGF) -like protein For use. More specifically, a VEGF-like protein isolated from the snake venom of Vipera ammodytes ammodytes; vammin, and a VEGF-like protein isolated from the snake venom of Daboia russelli russelli; heparin binding in VR-1 Heparin-binding peptide designed based on the amino acid sequence derived from the site, and suppression of heparin binding to vascular endothelial growth factor (VEGF-A) using the heparin-binding peptide The present invention relates to uses in the medical field, such as suppression of physiological effects caused by binding of VEGF to KDR via the steroid.

Background art

[0002] Vascular endothelial growth factor (VEGF-A) plays a central role in vasculogenesis and angiogenesis. Angiogenesis plays an important role in physiological phenomena such as wound healing, endometrium and luteal formation during the female sexual cycle, while growth of solid tumors, diabetic retinopathy, retinopathy of prematurity, and psoriasis. It is also known to be involved in various diseases. VEGF-A is a glycoprotein in which a subunit having a molecular weight of 23 kDa forms a homodimer through disulfide bonds. Growth factors similar to VEGF-A include VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental grow th factor (PIGF) (Masashi Shibuya, Masahiko Kurabayashi, Experimental Medicine, 20 (extra number), 1070 -1269 (2002); Mayumi Ono, Nobuhiko Kuwano, Angiogenesis and Biology of Cancer, Kyoritsu Shuppan 1-97 (2000); edited by Yasushi Sato, Frontiers of Angiogenesis, Yodosha, 10-171 (1999)). VEGF binds to VEGFR-l (fms-like tyrosine kinase_l: Flt_l) and VEGFR-2 (kmase insert domain-containing receptor: KDR) of vascular endothelial cells with high affinity. It has been suggested that the endothelial cell proliferation signal and blood pressure lowering effect of VEGF-A are mainly mediated by KDR (see Li, B. et al., Hypertension, 39, 1095-1100 (2002) '). See). In addition, signal transmission via KDR (NO produced by this has various physiological activities such as vasorelaxant action, platelet aggregation inhibitory action, antithrombotic action, anti-inflammatory action, etc. (See Masashi Shibuya and Masahiko Kurabayashi, Experimental Medicine, 20 (special edition), 1070-1269 (2002)).

[0003] The human gene encoding VEGF-A is composed of eight exons, and the existence of splicing variants derived from alternative splicing has been confirmed. That is, as the mature monomer, five iso_forms having 121, 145, 165, 189, and 206 amino acid residues; VEGF-A, VEGF-A, VEGF-A, VEGF-A, VEGF-A

121 145 165 187 207 206 present, in particular, VEGF-A lacks the partial amino acid sequence encoded by exon 6,

165

VEGF-A lacks the partial amino acid sequence encoded by exon 6 and exon 7.

121

Has been confirmed. In particular, VEGF-A is free in many tissues.

165

Has been confirmed to function as a mature and active form of VEGF. VEGF-A also binds to heparin or heparan sulfate proteodalican.

165

VEGF-A parental to KDR in the presence of heparin at low concentrations

165, it has been reported that when palin was present at a high concentration, no increase in the affinity of VEGF-A for KDR was observed (International Publication No. 01).

165

/ See No. 72829). In addition, neuropilin-1 also binds to VEGF-A,

165

By increasing the affinity for KDR, specific co-receive for VEGF-A

165

It has also been reported that it functions as a ptor (see JP-A-2003-12541).

[0004] On the other hand, if the interaction between KDR and VEGF-A is inhibited, solid tumor growth or

165

Angiogenesis in metastasis, diabetic retinopathy and retinopathy of prematurity is suppressed, and the progress of these diseases can be inhibited. Therefore, the function that inhibits the interaction between KDR and VEGF-A

165

An antagonist showing specific affinity for KDR, or VEGF- The search for a KDR-binding inhibitor that has specific binding properties to A

165

Yes.

In addition, in addition to VEGF-E derived from the above Parapox virus, similar to VEGF-A

A protein that has an amino acid sequence of 165 and that exhibits binding to KDR is a VEGF-like molecule that has a hypotensive factor (HF) power from Vepera as pis aspis snake toxin and has homology to VEGF. Isolated (Komori, Y. et al., Toxicon,

28, 359-369 (1990); Komori, Y. et al., Biochemistry, 38, 11796-11803 (1990)). More recently, two VEGF-like molecular forces, snake venom VEGF and increasing g capillary permeability protein (ICPP), have been isolated from other snake venoms, respectively. -Like proteins have been shown to have vascular permeability and angiogenic activities, tiunqueira de Azevedo, IL, et al., J. Biol. Chem., 276, 39836-39842 (2001); Gasmi,

A. et al., Biochem. Biophys. Res. Commun., 268, 69-72 (2000); Gasmi, A. et al., J. Biol. Chem., 277, 29992-29998 (2002)).

Disclosure of the invention

[0005] As described above, inhibition of the interaction between KDR and VEGF-A causes the growth of solid tumors.

165

Alternatively, angiogenesis in metastasis, diabetic retinopathy, and retinopathy of prematurity can be suppressed, and the progress of these diseases can be inhibited. In previous studies, KDR and VEGF-A

165 Function to inhibit the interaction between KDR and VEGF-A as a means to inhibit the interaction

165

Research interests in the search for antagonists with specific affinity for KDR or KDR with specific binding to VEGF-A

165

Is concentrated.

[0006] The present inventors have reported that, in addition to these conventional approaches, VEGF-A

165

Also has the ability to bind to heparan sulfate proteodarican, and enhances the affinity of VEGF-A for KDR in the presence of low levels and concentrations of free heparin.

165

Focusing on the binding between this heparin or heparan sulfate proteoglycan and VEGF-A,

165 Means to suppress the binding also make a significant contribution to the suppression of the interaction between KDR and VEGF-A. I came to that. However, at present, as a means to suppress the binding of heparin or heparan sulfate proteodarican present on the cell surface to VEGF-A,

165

It has also been found that a highly specific and effective means is an issue that has not been proposed and must be solved.

The present invention solves the above-mentioned problems, and an object of the present invention is to provide a detailed description of VEGF-A.

165 Inhibits binding to heparin or heparan sulfate proteodalican present on the cell surface, for example, due to the interaction between KDR and VEGF-A in the presence of heparin

165

It is to provide a new means capable of suppressing various physiological reactions. Specifically, when VEGF-A forms a bond with KDR on the cell membrane,

165

Binds and has a co_rece _P tor-like function on VEGF-A, present on the cell surface

165

To provide a novel substance that exhibits high binding to heparin or heparan sulfate proteodalican and competitively inhibits the complex formation between VEGF-A and heparin.

165

The

[0008] In the course of intensive research to solve the above-mentioned problems, the present inventors first involved in the binding of VEGF-A to heparin or heparan sulfate proteodalican.

165

It is noted that the site that is reported to be present at the C-terminal side (see Keyt, BA et al., J. Biol. Chem., Vol. 271 7788-7795 (1996), etc.) Specifically, in the recombinant production VEGF-a monomers, Ala ¹¹¹ - Department of Arg ¹⁶⁵

165

A C-terminally truncated protein monomer was prepared from which the position was removed by plasmin digestion, and the heparin binding property was verified. It was confirmed that the heparin binding property was lost. In addition, a potent homodimer of VEGF-derived C-terminally truncated proteins was produced in vitro.

165

Binding to KDR in VEGF-A

It was also confirmed that 16 homodimers were unsuitable. However, in vitro

Five

In the proliferation test of aortic endothelial cells, its growth-inducing effect was determined by the VEGF-derived C

165 The homodimer of the truncated variant protein is

165

It has also been found that it has dropped significantly. That is, in addition to binding VEGF to KDR,

165

, Which simultaneously binds to proteodalican-type heparin present on the surface of endothelial cells, or to heparan sulfate proteodalican. It is confirmed that it is indispensable for inducing a reactive reaction.

[0009] Furthermore, the present inventors have newly isolated and identified a VEGF-like protein derived from Vipera ammodytes ammodytes as a vascular endothelial growth factor VEGF-like protein derived from snake venom; vammin, a protein derived from Daboia russelli russelli For the VEGF-like protein; VR-1, the proteodalican-type peparin or heparan sulfate present on the cell surface is required to induce a physiological response in the cell following the binding to KDR. We found that it was necessary to bind to proteodalican at the same time. We searched for sites involved in binding to heparin or heparan sulfate proteodalican in these venom endothelial growth factor VEGF-like proteins derived from snake venom, and found that only the C-terminal part was bound to heparin. Found to be involved. Indeed, the C-terminal partial amino acid sequence of this vammin (A rg ⁹⁴ -Arg ^o) or VR-1 of the C-terminal partial amino acid sequence ^{^{(Arg 94 - Arg lc> 9}} ) was synthesized peptide fragment having the heparin binding to the When the ability (affinity) was evaluated, it was confirmed to show an amino acid sequence derived from the heparin binding site in vammin, VR-1. In addition, in addition to the amino acid sequence derived from the heparin binding site in Vammin and VR-1, a series of peptides having a modified amino acid sequence designed based on the amino acid sequence also have high heparin binding ability (affinity). It was also verified that the property was maintained. Furthermore, it was also confirmed that the amino acid sequence portion having a dominant function in the heparin binding ability (affinity) was a portion of RPR-X-KQ-G (X is R or W).

[0010] Furthermore, the synthetic peptide fragment having heparin binding ability (affinity) designed based on the amino acid sequence derived from the heparin binding site in vammin and VR-1 is VEGFA or vammin. Inhibits vascular endothelial cell proliferation-inducing action in a concentration-dependent manner

165

In vivo, VEGF-A exhibits KDR-mediated

165

It was also verified that it exerts a concentration-dependent inhibitory effect on blood pressure lowering ability due to N〇 produced by signal transduction. Based on the above findings, the present inventors have completed the first embodiment of the present invention.

[0011] That is, the peptide having heparin binding ability according to the first aspect of the present invention is:

First partial amino acid sequence derived from the heparin binding site in vammin:

R_P_R_X_K_Q—including at least G (X is any of R, W, H, and K), It is a peptide having heparin binding ability characterized by consisting of 7 to 20 amino acid residues.

[0012] Accordingly, peptides having a heparin-binding ability that are effective in the first aspect of the present invention include:

R_P_R_X_K_Q—including at least G (X is any of R, W, H, and K), A -R-P-R-X-K-Q-G-A

N C

(A and A are each selected from a peptide chain having 0 to 13 amino acids;

N C N C

The total number of amino acids is 13 or less)

And a peptide having an amino acid sequence of 720 amino acid residues and having heparin binding ability.

[0013] Furthermore, in addition to the first partial amino acid system IJ: R—P_R_X—K_Q_G,

Second partial amino acid sequence from the heparin binding site in vammin:

Including K—E—K—P—R,

A peptide having heparin binding ability, wherein the second partial amino acid sequence is linked to the C-terminal of the first partial amino acid sequence from 416 amino acid residues via a linker sequence. You can also.

[0014] In particular, an amino acid sequence derived from the heparin binding site in vammin:

R-P-R-R-K-Q-G-E-P-D-G-P-K-E-K-P-R

A strong amino acid sequence | J, or a heparin-binding ability comprising at least one amino acid substitution in the sequence thereof and including a modified amino acid sequence retaining the first partial amino acid sequence. It can also be a peptide.

[0015] The present invention is directed to a method of using the peptide having heparin-binding ability according to the first aspect of the present invention described above, which inhibits the binding of VEGF-A to heparin.

165

We also provide inventions for use as harmful agents,

That is, for the binding between VEGF-A and heparin, which is effective in the first aspect of the present invention.

165

The inhibitor is

A competitive inhibitor of VEGF-A binding to heparin,

165

The competition-inhibiting active ingredient has a heparin-binding ability that is effective in the first aspect of the present invention described above. Inhibition of VEGF-A binding to heparin

165

Agent.

[0016] The inhibition of the binding between VEGF-A and heparin according to the first aspect of the present invention.

165

Agents, for example, when preparing in the form of injection solutions,

Dissolving an effective amount of a peptide having the ability to bind carboxyl to the above-mentioned first aspect of the present invention in a pharmaceutically acceptable liquid carrier suitable for intravenous administration to the subject. The composition can be

[0017] Alternatively, when preparing a dosage form of eye drops,

Effectiveness of the peptide having heparin binding ability according to the first aspect of the present invention described above in a pharmaceutically acceptable liquid carrier suitable for application to the eyeball or orbit of the subject to be administered. The composition can be obtained by dissolving the amount.

[0018] The novel peptide having a heparin-binding ability that is effective in the first embodiment of the present invention is a vascular endothelial growth factor VEGF-like protein derived from snake venom; vammin and a heparin-binding site in VR-1. A peptide compound consisting of 7-20 amino acid residues designed based on the amino acid sequence, induced by the binding of VEGF-A or vammin to KDR

165

Binds VEGF-A or vammin to heparin on the cell surface, which is necessary for exerting potent nitric oxide (NO) -dependent hypotensive activity and promoting angiogenesis.

165

Has competitive inhibitory action. As a result, VEGF-A, which causes various diseases such as growth and metastasis of solid tumors, diabetic retinopathy, retinopathy of prematurity, and psoriasis, has become

The novel peptide having heparin-binding ability according to the present invention can be used for therapeutic purposes for the purpose of suppressing the promotion of angiogenesis caused by 165. At this time, since it is a peptide of 720 amino acid residues containing many basic amino acids, it does not become an antigenic peptide presented by MHC and does not show immunogenicity. Therefore, there is no reduction in the therapeutic effect due to the above.

In short, the present inventors have further modified the amino acid sequence on the basis of a peptide having heparin-binding ability that is strong in the first embodiment of the present invention, and A synthetic peptide fragment having binding ability was created. Specifically, the heparin binding ability (affinity) of the peptide having heparin binding ability according to the first aspect of the present invention is controlled by R-PR—X—KQG (X is R or W) by modifying the above-mentioned first amino acid sequence portion having a common function, and RX-X-X-KQG (X is P or R , X is

01 02 03 01 02

R, K, and X have the same or better heparin-binding ability when converted to Κ, Η, or R).

03

I found that.

Further, the above R—X—X—X—K—Q—G (X is P or R, X is R or K,

01 02 03 01 02

X has a modified first amino acid sequence portion of Κ, Η or R), and has a heparin binding ability.

03

Similarly, a synthetic peptide fragment that retains (affinity) was obtained from vascular endothelium by VEGF-A.

165

It was also verified that the compound exhibited a concentration-dependent inhibitory effect on the growth induction of vesicles. Based on the above findings, the present inventors have completed the second embodiment of the present invention.

That is, the peptide having heparin-binding ability according to the second aspect of the present invention is:

First modified partial amino acid sequence derived from the heparin binding site in vammin: R-X-X-X-K-Q-G (X is P or R, X is R or K, X is Κ, Η

01 02 03 01 02 03

Or R)

It is a peptide having heparin binding ability characterized by consisting of 7 to 20 amino acid residues.

[0022] Accordingly, peptides having heparin-binding ability that are effective in the second aspect of the present invention include:

01 02 03 01 02 03

Or R)

A -R-X -X -X -K-Q-G-A

Nl 01 02 03 CI

(A and A are each selected from a peptide chain having 0 to 13 amino acids, and A and A

Nl CI The total number of amino acids in Nl CI is 13 or less.)

Further, the modified first partial amino acid sequence: R—X—X—X—added to K_Q_G

01 02 03

I mean,

Second partial amino acid sequence from the heparin binding site in vammin:

Including K—E—K—P—R, A peptide having heparin binding ability, which is linked to the c-terminus of the modified first partial amino acid sequence from the second partial amino acid sequence of 416 amino acid residues via a linker sequence, You can also.

In particular, E_P_D_G_P is selected as the linker sequence,

R-X -X -X -K-Q-G-E-P-D-G-P-K-E-K-P-R

01 02 03

(X is P or R, X is R or K, X is Κ, Η or R)

01 02 03

A peptide having heparin binding ability, comprising an acid sequence or a modified amino acid sequence having at least one amino acid substitution in the sequence and retaining the modified first partial amino acid sequence. You can also.

In summary, the present invention relates to a method of using the peptide having heparin-binding ability according to the second aspect of the present invention, which comprises inhibiting the binding of VEGF-A to heparin.

165

We also provide inventions for use as harmful agents,

That is, for the binding between VEGF-Α and heparin, which is exerted by the second aspect of the present invention.

165

The inhibitor is

A competitive inhibitor of VEGF-A binding to heparin,

165

The competition-inhibiting active ingredient is a peptide capable of binding to heparin, which is capable of binding heparin to the second embodiment of the present invention.

165

Agent.

[0026] Inhibition of VEGF-A binding to heparin according to the second aspect of the present invention

165

Agents, for example, when preparing in the form of injection solutions,

In a pharmaceutically acceptable liquid carrier suitable for intravenous administration to a subject, an effective amount of the peptide having the ability to bind carboxyl in the second form of the present invention is dissolved. The composition can be

Brief Description of Drawings

[FIG. 1] FIG. 1 shows the results of evaluating the heparin binding ability of six types of synthetic peptides, peptide 1 to peptide 6, in which 10 mL of 50 mM Tris-HCl ρΗ80 After application to a HiTrap Heparin HP column (column volume: 10 mL, Amersham Biosciences), the buffer was flowed through the column at a flow rate of 1 mL / min for 5 column volumes. The elution was performed with a linear gradient around 1.0 1 NaC, and the elution conditions (NaCl concentration) were measured.

Garden 2] FIG. 2 illustrates the basis of the design of a peptide having the ability to bind to heterologous protein according to the present invention. FIG. 2A shows the amino acid sequence alignment between vammin and the peptide chain of human VEGF-A.

165

The disulfide bond site within the vammin monomer chain and between the dimer chains, and the cysteine nut 'motif, which are predicted from the results, are also displayed for heparin binding in human VEGF-A.

165

The region to be assigned is indicated by a dotted line, and Fig. 2B compares the amino acid sequence of vammin C-terminal (indicated by an underline in A) with the amino acid sequence of peptide 13 designed based on it. Shown.

[Fig. 3] Fig. 3 shows the effect of VEGF-A of peptide 1 on the proliferation of vascular endothelial cells.

165

In addition, the results of evaluation of the inhibitory effect on the growth-promoting action of vascular endothelial cells by vammin were shown. Under low serum culture conditions, addition of VEGF-A or vammin (each final concentration InM) was performed.

165

Inhibition of co-supplementation of peptide 1 on the promotion of proliferation of E. coli aortic endothelial cells was evaluated by the number of cells after 6 days of culture. White: control, black: VEGF-A

165 or vammin alone (positive control), light gray, gray: 30 μM (final concentration) peptide 1 at 0, dark gray: 100 / i M (final concentration) peptide 1 results Are shown in contrast.

[Figure 4] Figure 4 shows the effect of peptide 1 on the blood pressure lowering effect of VEGF-A and vammin.

165

Fig. 4 (A) shows the time course of rat carotid artery pressure after intravenous injection of vammin (dose 0. lig / g) (! Mark), lower: Time course of rat carotid artery pressure after pre-administration of peptide 1 (dose 3 / g / g) (marked with ▼) and after intravenous injection of vammin (dose 0.1 / g / g) (marked with i), Figure 4 ( B) Top: VEGF-A (dose 0.1

165

μg / g) after intravenous injection (marked with 丄), time course of rat carotid artery pressure, middle: after pre-administration of peptide 1 (dose 3 μg / g) (marked with T), VEGF-A (dose 0. 1 μg / g) after intravenous injection (

165

Time course of rat carotid artery pressure in 丄 mark, lower: after pre-administration of peptide 1 (dose 30 z gZg) (T mark), after intravenous injection of VEGF-A (dose 0.1 μg / g) (丄 mark) Mark) rat carotid artery pressure

165

Are shown in comparison.

[Figure 5] Figure 5 shows the effect of VEGF-A on the blood pressure lowering of three peptides, peptide 1-3. The results of comparative evaluation of inhibitory activity against A are shown in the figure, where A: VEGF-A (dose 0.1 μg /

165

g) only) (positive control), B: mark after administration of peptide 1 (dose 30 ig / g) beforehand, and VEGF-A (dose 0.1 / g / g) ), C: peptide

165

2 After administering (dose 30 x gZg) (T mark), VEGF-A (dose 0.1 z gZg) was administered.

165

), D: peptide 3 (dose 30 μg / g) before administration (T), and VEGF-A (dose 0. lzg / g) before administration (!) Time course of arterial pressure

165

Are shown in comparison.

[Fig. 6] Fig. 6 shows the relationship between the VEGF-like protein derived from snake venom and the peptide chain of human VEGF-A.

165

7 shows the results of amino acid sequence alignment. In VEGF-like proteins derived from snake venom, matching amino acid residues are shaded, cysteine residues are shown in white nuclei, indicating intra- and inter-chain disulfide bonds, and cysteine nut 'motifs. Form human VEGF—in the A chain

The loop portion of 165 is indicated by a horizontal line. The heparin binding site of human VEGF-A is

165

, Surrounded by a dotted line. Important residue for KGR receptor binding in human VEGF-A

165

The group is indicated by Hata, the residue important for Fit-1 binding is indicated by the mouth, and the amino acid substitution site contributing to reduced binding to Flt-1 in the snake venom-derived VEGF-like protein. Is indicated by a ▼ mark. HF: Hypotensive factor derived from Vipera aspis aspis snake venom; ICP P: Increasing capillary permeability protein from Vipera lebetma heme venom; VEGF165: Human vascular endothelial growth factor human VEGF-A (GenBank registration number

165

, AAM03108)

[FIG. 7] FIG. 7 shows the relationship between heparin co-added to the medium, VEGF-A and vammin.

165

VEGF-A or vammin-stimulated vascular endothelial cell proliferation due to binding

165

Shows the suppression effect. In serum-containing culture conditions, various concentrations of unfractionated heparin and V

Number of surviving cells after 3 days of culture with EGF-A or vammin (final concentration InM)

165

Was evaluated. Kuromaru Okina: vammin, Shiramaru I: Increase of endothelial cells by VEGF-A stimulation

165

Fig. 3 shows the absorption coefficient A (number of viable cells) of color development in the WST-8 method when heparin was co-added to the medium at various concentrations during growth promotion.

405-630

FIG. 8 shows the results of evaluation of the inhibitory ability of VEGF-A on the growth-promoting action of vascular endothelial cells, showing the strength of each of peptide 1, peptide 2, and peptide 3. Serum addition

165 In culture conditions, human umbilical vein endothelial cells were added by adding VEGF-A (final concentration InM).

165

Suppression of cell growth promotion by the co-added peptides was evaluated by the number of cells after 3 days of culture. Closed circle: peptide 1, open circle: peptide 2, closed triangle ▲: peptide

3 shows the growth promotion rate when each concentration was added.

[Fig. 9] Fig. 9 shows the results of evaluation of the inhibitory ability of VEGF-A against the vascular endothelial cell growth-promoting effect, which is shown by peptide 1 and peptide 9 (TFPI ^{254 265} ). Serum addition

165

Human umbilical vein endothelium with VEGF-A (final concentration InM)

165

The suppression of cell growth promotion by each co-added peptide was evaluated by the number of cells after 3 days of culture. Black: peptide 1, gray: peptide 9 (TFPI ^{254 265} ) shows the growth promotion rate when each concentration was added.

[Fig. 10] Fig. 10 shows the results of evaluation of the inhibitory ability of VEGF-A on the promotion of vascular endothelial cell growth, showing the peptide strength of peptide 1, peptide 7, and peptide 8. serum

165

In addition culture conditions, human umbilical vein by addition of VEGF-A (final concentration InM)

165

Suppression of the proliferation of skin cells by the co-added peptides was evaluated by the number of cells after 3 days of culture. Black circles: peptide 1, white circles: peptide 7, black triangles A: peptide 8 show the growth promotion rate when each concentration was added.

[Fig. 11] Fig. 11 shows that VEGF-A or base caused by peptide 1 co-added in the medium.

165

4 shows the inhibitory effect of the vascular endothelial cell growth effect by stimulating inflammatory fibroblast growth factor (bFGF). In serum-containing culture conditions, various concentrations of peptide 1 and VEGF-A or

165 added bFGF (final concentration InM) and evaluated the number of surviving cells after 3 days of culture. Black circle Hata: VEGF-A, white circle 〇: In bFGF-stimulated vascular endothelial cell growth, in medium

165

Shows the growth promotion rate when peptide 1 was co-added at each concentration.

BEST MODE FOR CARRYING OUT THE INVENTION

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

[0029] Prior to the present invention, the present inventors

Purified from snake venom of Vipera ammodytes ammodytes · VEGF-like protein isolated: Purified from snake venom of vammin and Daboia russelli russelli 'Isolated VEGF-like protein: VR-1 is 110 amino acids each. Peptide chain consisting of two chains Homodimer linked by a disulfide bond between them, and that two peptide chains consisting of 109 amino acids are homodimers linked by an interchain disulfide bond. VR-1 has binding properties to vascular endothelial growth factor receptor type 2 (VEGF receptor 2; KDR), vascular endothelial growth factor receptor type 1 (VEGF receptor l; Flt_l), vascular endothelial growth factor receptor 3 It has been revealed that it has characteristics that show no binding to the type (VEGF receptor 3; Flt_4) and neuropilin-1 (neuropilin-1). Specifically, the primary structure (amino acid sequence) of a 110-amino acid peptide chain constituting vammin has the following sequence 1:

Sequence 1: Primary structure of vammin 110 amino acid peptide chain

EVRPFLEVHE RSACQARETL VPILQEYPDE ISDIFRPSCV AVLRCSGCCT DESLKCTPVG KHTVDLQIMR VNPRTQSSKM EVMKFTEHTA CECRPRRKQG EPDGPKEKPR

In addition, the primary structure of the peptide chain consisting of 109 amino acids (amino acid sequence IJ) constituting VR-1 is represented by the following sequence 2:

Sequence 2: Primary structure of peptide chain consisting of 109 amino acids of VR-1

EVRPFLDVYQ RSACQTRETL VSILQEHPDE ISDIFRPSCV AVLRCSGCCT DESMKCTPVG KHTADIQIMR MNPRTHSSKM EVMKFMEHTA CECRPRWKQG EPEGPKEPR

It is.

The primary structure of the VEGF-like protein isolated from the snake venom of the Vipera ammodytes ammodytes: a peptide chain consisting of vamminl lO amino acids is also a disulfide between two peptide chains consisting of 110 amino acids. Primary structure of HF, isolated from the snake toxin of Vepera aspis aspis, which is a homodimer linked by a bond.

Sequence 3: Primary structure of peptide chain consisting of 110 amino acids of HF

EVRPFLEVHE RSACQARETL VSILQEYPDE ISDIFRPSCV AVLRCSGCCT DESLKCTPVG

KHTVDLQIMR VNPRTQSSKM EVMKFTEHTA CECRPRRKQG EPDGPKEKPR

Alternatively, the primary structure of ICPP isolated from Vipera lebetina snake venom has the following structure:

Sequence 4: Primary structure of peptide chain consisting of 110 amino acids of ICPP

EVRPFPDVHE RSACQARETL VSILQEYPDE ISDIFRPSCV AVLRCSGCCT DESLKCTPVG KHTVDMQIMR VNPRTQSSKM EVMKFTEHTA CECRPRRKQG EPDGPKEKPR

As shown in Fig. 6, it showed extremely high homology, and at the same time, the correlation between the physiological activities of these series of snake venom-derived VEGF-like proteins and their amino acid sequences was thoroughly examined. Amino acid sequences that maintain high affinity for KDR and have no affinity for Flt-1 or that contribute significantly to the amino acid sequence, even among the consensus sequences. As one of them, we have already elucidated that a high correlation was found between the presence of the five amino acids Alal 3, Lys55, Arg 74, Ser77 and Lys79 in sequence 1 and high selectivity for KDR. (See Yamazaki, Y. et al., J. Biol. Chem., Vol. 278 51985-51988 (2003)). On the other hand, human vascular endothelial growth factor VEGF-A, a homodimeric protein with a molecular weight of 38.2 kDa, has an ability to bind to heparin or heparan sulfate proteodalican.

165

VEGF-A affinity for KDR in the presence of heparin at low concentrations

165 Controls heparin binding in VEGF-A

165

The area to be used was advanced. In the process, by plasmin digestion Arg ¹¹¹ at the C-terminus - was resected Arg ¹ ⁶⁵ region of, C-terminal ablation member has been found that loss of heparin binding to. Furthermore, the C-terminal excision body (VEGF110) is, for example, a vascular endothelium originally shown by VEGF-A.

165

It has been confirmed that the vascular endothelial growth promoting activity is significantly reduced as compared with the growth promoting activity, and that various physiological activities exerted by the binding of VEGF-A to KDR can be suppressed.

165

In addition, binding to heparin present on the surface of the target cell in which the KDR is expressed is indispensable. It turned out to be about.

[0032] Indeed, the C-terminal present in the VEGF-A Arg ¹¹¹ - area of the Arg ¹⁶⁵ is shown in Figure 2

165

Thus, it has a three-dimensional structure formed by intra-chain disulfide bonds inside the force region, and when the three-dimensional structure is constructed, a plurality of basic amino acid residues contained in heparin It is conjectured that, as a result of interaction with the sulfuric acid ester and sulfuric acid amide structures of the acidic substituent, binding to heparin is achieved.

[0033] Further, as schematically shown below, the polysaccharide chain of heparin has L-iduronic acid, D-darconic acid, and D-darcosamine as structural units, and sulfation is almost all of D-darcosamine. It is also present in amino groups, other hydroxyl groups in part 6 and hydroxyl groups in part 2 of peronic acid. In cells, most are present in the form of proteanodolecan attached on the amino acid side chains of proteins. In addition, depending on the cell type, there are differences in the sulfated site present on the polysaccharide chain of heparin and the sequence of the constituent saccharide units, which are involved in various physiological activities.

[0034] [Formula 1]---

α-D-glucuronic acid) 3-L-iduronic acid-2-sulfuric acid a-D-Darcosamine-Ν, Ο-disulfuric acid

On the other hand, in the case of vammin, the C-terminal Ar present in VEGF-A

165

g ¹¹¹ - Arg ^165, such as area, heparin binding site to have a three-dimensional structure despite the absence, it shows the binding of the heparin to the same ingredients, also accompanied Rere exerted binding to KDR vammin It has been elucidated that the binding to heparin present on the surface of the target cell in which the KDR is expressed is also an essential process in the expression of various physiological activities. In addition, VR_1 also exhibits binding properties to heparin, and the subject expressing the KDR is also involved in the expression of various physiological activities exerted upon the binding of VR-1 to the KDR. It has been clarified that binding to heparin present on the cell surface is also an essential process. these According to the new fact, the binding to heparin found in vammin and VR-1 is due to their C-terminal part, that is, the amino acid arrangement of the C-terminal part of vammin (Arg ⁹⁴ — Arg ^11Q ). Based on the idea that it is governed by the C-terminal partial amino acid sequence 1 (Arg ⁹⁴ — Arg ¹⁰⁹ ), it actually corresponds to the C-terminal partial amino acid sequence as shown in the Examples below. Synthetic peptide peptide 1 was prepared, and it was verified that all of them were peptides showing binding ability to heparin.

[0036] Thus, not vammin and VR-1 alone, VEGF-like proteins HF from bi poison to others, even in Icpp, commonly, force mow C-terminal partial amino acid Hai歹lJ (Arg ⁹⁴ Arg ^11Q) is It was also concluded that it allowed binding to heparin.

[0037] § I-out continues, C-terminal, partial amino acid sequence of vammin (Arg ⁹⁴ Arg ^u °) or VR_1 C-terminal partial amino acid Hai歹lJ of - binding to heparin is dominated by (Arg ⁹⁴ Arg ¹⁰⁹⁾ In order to identify the main partial region, two partial fragment peptides of peptide 2 and peptide 3 shown in FIG. 2B were prepared and their heparin binding properties were evaluated. Shows heparin-binding property comparable to that of the synthetic peptide peptide 1, but it was found that the synthetic peptide peptide 3 lost the heparin-binding property.

[0038] Furthermore, the partial amino acid sequence derived from vammin, R-P-R-R-K-Q-G-E, included in the synthetic peptide peptide 2, and a C-terminal truncated product (VEGF110) derived from VEGF-A are also included.

165

When compared with the partial amino acid sequence remaining at the C-terminus, the amino acid sequences are both rich in basic amino acid residues when compared to the partial amino acid sequence: R—P—K—K—D—R. — There is a crucial difference between P-K-K-D-R that heparin binding is impaired.

[0039] That is, when the partial amino acid sequence derived from vammin: R_P_R_R_K_Q_G_E, the corresponding region of VR-1, and the partial amino acid sequence remaining at the C-terminus of VEGF110 that does not show heparin binding are compared with each other,

vammin: R—P—R—R—K—Q_G—E

VR-1: R-P-R-W-K-Q-G-E

VEGF 110: R-P-K-K-D-R

Assumed partial array: R_P_R_X_K_Q_G (X is R, W, H, or K) When the above-mentioned assumed partial sequence (first partial amino acid sequence) is retained, it is determined that the synthetic peptide peptide 2 exhibits at least heparin-binding property comparable to that of the synthetic peptide peptide 2. In particular, the basic amino acid residues are placed in a relative configuration suitable for interaction with the sulfate ester structure and the sulfate amide structure in the sulfated sugar chain in heparin, as exemplified above in the underlined 4 amino acid power. It is determined that it plays the role of placing. Therefore, in the peptide having the ability to bind to the first form of the present invention,

The first partial amino acid system IJ: R_P_R_X_K_Q_G (X is any one of R, W, H, and K, and the total number of amino acid residues is 7 to 20 amino acid residues. The range of the total number of amino acid residues is determined by the length of peptide chains constituting various peptide 'hormones, for example, 8 amino acid residues of angiotensin II or 21 amino acid residues of insulin A chain. This is equivalent to an appropriate peptide chain length for maintaining water solubility while exerting the above properties.

[0040] As described above, peptides having heparin-binding ability that are effective in the first aspect of the present invention include:

R-P-R-X-K-Q_G (X includes at least any of R, W, H, and K, and A -R-P-R-X-K-Q-G-A

N C

(A and A are each selected from a peptide chain having 0 to 13 amino acids, and the A and A

N C N C

The total number of amino acids is 13 or less)

The ability to include a peptide having an amino acid sequence of 7-20 amino acid residues and having heparin binding ability. For example, as shown in the following examples of synthetic peptides peptides 4 and 5, the portion of A Can be in a form in which no amino acid is present. Generally, N end

N

It is preferable to add a small amino acid such as Gly or Ala as a protective amino acid to form a form having at least the minimum number of 8 amino acids.

[0041] In addition, the synthetic peptide peptide 1 also has a partial sequence of K_E_K_P_E at the C-terminus, has slightly better heparin binding properties than the synthetic peptide peptide 2, and has the second partial amino acid sequence IJ : K_E_K_P_E is also desirable. In that case, it is preferable to arrange at an appropriate interval between the first partial amino acid sequence and the second partial amino acid sequence. As a linker sequence, about 5 amino acid residues, therefore, a linker sequence from 416 amino acid residues, more preferably, a linker sequence from 5 amino acid residues,

R-P-R-X-K-Q-G-X -X -X—X—X -K—E—K—P-R

1 2 3 4 5

(X is any of R, W, H, K, and -X -X -X -X -X— is a linker sequence)

1 2 3 4 5

Preferably, the amino acid sequence includes In particular, the amino acid sequence derived from the heparin binding site in vammin:

R-P-R-R-K-Q-G-E-P-D-G-P-K-E-K-P-R

A heparin-binding ability, comprising a modified amino acid sequence having at least one amino acid substitution in its sequence and retaining the first partial amino acid sequence. Can also be used as the peptide. This type of modified amino acid sequence is preferred, for example,

R-P-R-X-K-Q-G-E-P-D-G-P-K-E-K-P-R

(X is one of W, H, K)

In addition, R is added to the C-terminal,

R-P-R-X-K-Q— G_E_P_D_G_P_K_E— K-P-R-R

(X is one of R, W, H, K)

And the like.

Calorie, the second partial amino acid sequence ΐ ": Κ_Ε_Ι £ _Ρ_β instead of the first partial amino acid sequence IJ: RPRXKQG (X is any one of R, W, H, K Are linked by

R-P-R-X-K-Q-G-X -X -X -X -X -R-P-R-X—K-Q—G

1 2 3 4 5

Can be selected. That is, the first partial amino acid sequence is tandemly linked via the linker sequence, and the number of sites that can participate in binding to heparin is increased. Or, as part of A, VEGF— in A

C-terminal 5 amino acid residues present in C 165 (Asp ¹⁶¹ — Arg ¹⁶⁵ ): corresponding to D_K_P_R_R, the K-I E_K_P_R_R was converted to Κ_β_Κ—P_R_R,

R-P-R-X-K-Q-G-E-P-D-G-P-K-D-K-P-R-R

(X is any of R, W, H, K) May have an amino acid sequence such as

[0043] Furthermore, as the first partial amino acid sequence portion, R is selected as X, and R-PR-R-I K_Q_G is more preferably used.

[0044] The above linker sequence, for example, from 5 amino acid residues to a linker system IJ: 1 X -X -X -X

one two Three

The role of the -X-part is to compare the second partial amino acid sequence to the first partial amino acid sequence portion.

4 5

The purpose of the present invention is to link the column portions and to add the contribution of a weak interaction between the second partial amino acid sequence portion and heparin to the heparin binding property mainly composed of the first partial amino acid sequence portion. Things. Accordingly, cleavage of such a linker sequence: one X--X--X--X--X portion is prevented by the action of proteases and peptidases present in the body of the administration subject.

1 2 3 4 5

You can use what is not done. By using a linker sequence consisting of D-amino acids instead of a linker sequence consisting of L-amino acids, it is possible to suppress digestion by proteases in the body. In addition, by using the mimic structure of peptide bond used in various peptide reagents for a powerful linker sequence portion, it is possible to suppress digestion by proteases in the body. However, even when cleavage is performed within this linker sequence, the first partial amino acid sequence partial fragment that retains heparin binding properties is not modified unless the first partial amino acid sequence portion itself is cleaved. This is not a problem, especially because it is produced.

In general, in order to prevent in vivo degradation of a peptide compound, a method of adding or modifying an extra amino acid at the N-terminus and C-terminus is effective. For example, various N-terminal amino groups can be modified with various acyl groups, or the C-terminal carboxy group can be amidated. Since the peptide having heparin-binding ability according to the first aspect of the present invention can be produced by using a chemical synthesis technique, for example, using a solid phase synthesis method, When extending the peptide chain, amidation of the C-terminal immobilized on the resin is also suitable for synthesis. In addition, the linker is arranged as follows:

one two Three

-X—X—parts should contain artificial amino acids instead of natural amino acids

4 5

Can do. By using artificial amino acids, it is also possible to design the linker sequence to one that suppresses enzymatic cleavage of the peptide chain.

[0046] In the amino acid sequence derived from the heparin binding site in vammin, the linker One sequence: One X-X-X-X-X part of the amino acid sequence is _E_P_D_G_P—

1 2 3 4 5

In the sequence of —D—G— (Asp—Gly) contained in it, in some cases, the carboxy group (one COOH) on the side chain of Asp and the N-terminal imino nitrogen (one N It is known that the reaction between-) causes the formation of a succinimide structure or the rearrangement to the j3 position.

[Formula 2] Formation of succinimide structure:

[0048]

/ 3rd transposition

[0049] In order to suppress the structural change accompanying the reaction in this kind of peptide molecule, for example, the sequence of -DG-(Asp-Gly) is replaced with -DA- (Asp-Ala) or -N_G_ (Asn_Gly). Although structurally similar, amino acid substitutions that suppress unwanted intramolecular reactions can also be made.

[0050] The peptide having heparin-binding ability that is effective in the first aspect of the present invention is a peptide having a total number of amino acid residues of 7 to 20 amino acid residues, and a hydrophilic amino acid, particularly Since it contains a large amount of basic amino acids, it can be used as an aqueous solution in a wide concentration range. When applied to pharmaceutical uses, aqueous media such as aqueous media used for preparing injection solutions containing various peptide 'hormones, or orally administrable peptide It is preferable to prepare a composition using an aqueous medium used for preparing a liquid preparation containing a physiologically active substance as a carrier. Further, by adding a predetermined amount of water, it is also possible to prepare a lyophilized mixture in which a composition using the aqueous medium can be prepared. In general, the water solubility tends to decrease as the number of amino acid residues constituting the peptide increases. Since the amino acid sequence portion and the second partial amino acid sequence portion also have a high ratio of amino acids with high hydrophilicity, the solubility in water is at least more than 20 mgZmL.

On the other hand, the peptide having heparin binding ability according to the second aspect of the present invention is

The first partial amino acid system U: R—P_R_X—K_Q_G (X is R, W, H, A modified first partial amino acid sequence in place of any force of K:

R-X -X -X -K-Q-G (X is P or R, X is R or K, X is Κ, Η

01 02 03 01 02 03

Or R), and the total number of amino acid residues is 7 to 20 amino acid residues. As described above, the range of the total number of amino acid residues is determined by the length of peptide chains constituting various peptide 'hormones, for example, 8 amino acid residues of angiotensin II or 21 amino acid residues of insulin chain. Group, and so on, while maintaining their water solubility while exhibiting their functions.

[0052] As described above, peptides having heparin-binding ability that are effective in the second aspect of the present invention include:

01 02 03 01 02 03

Or R)

A -R-X -X -X -K-Q-G-A

Nl 01 02 03 CI

Nl CI The total number of amino acids in Nl CI is 13 or less.)

Of a peptide having an amino acid sequence of 720 amino acid residues and having a heparin binding ability.For example, the following synthetic peptide peptides 7 and 8 are exemplified. As described above, the portion A may be in a form in which no amino acid is present. Generally, N end

N

When a protective amino acid is added to the form, it is desirable to add a bulky amino acid such as Gly or Ala to form a form having a minimum number of amino acids of 8 or more.

[0053] In addition, the synthetic peptides peptide 7 and 8 also have a partial sequence of K_E_K_P_E at the C-terminal, similar to the synthetic peptide peptide 1, and have a slightly higher heparin binding property than the synthetic peptide peptide 2. Therefore, it is desirable to have the second partial amino acid sequence IJ: K_E_K_P_E as well. At this time, it is preferable that the modified first partial amino acid sequence and the second partial amino acid sequence are arranged at an appropriate interval, and the linker sequence is about 5 amino acid residues, A linker sequence is provided from sixteen amino acid residues, more preferably a linker sequence is provided from five amino acid residues.

R-X —X —X — K— Q— G— X— X— X— X— X— K— E— K— P— R

01 02 03 1 2 3 4 5

(X is P or R, X is R or K, X is Κ, Η or R, -X -X -X _

01 02 03 1 2 3

X-X is the linker

4 5 arrangement)

Preferably, it contains an amino acid sequence represented by:

[0054] In particular, the modified first partial amino acid sequence and the second partial amino acid sequence are linked, and EP-D-GG- is selected as a linker sequence.

R-X -X -X -K-Q-G-E-P-D-G-P-K-E-K-P-R

01 02 03

(X is Ρ or R, X is R or K, and X is Κ, Η or R)

01 02 03

A peptide having heparin binding ability, comprising an acid sequence or a modified amino acid sequence having at least one amino acid substitution in the sequence and retaining the modified first partial amino acid sequence. You can also. This type of modified amino acid sequence is preferred, for example,

R-X -X -X -K-Q-G-E-P-D-G-P-K-E-K-P-R

01 02 03

(X is Ρ or R, X is R or K, Κ

02, X is

03, Η or R)

01

For, R is added to the C-terminal,

R-X -X -X -K-Q-G-E-P-D-G-P-K-E-K-P-R-R

01 02 03

(X is Ρ or R, X is R or K, X is Κ, Η or R)

01 02 03

Can be mentioned. Alternatively, the second partial amino acid sequence contained in the portion of Α Instead of 5 amino acid residues at the C-terminus present in VEGF-A (Asp ¹⁶¹ — Arg ¹⁶⁵ ): D

165

_K-P_R_R was converted from K-E-K_P_R_R to K-D-K-P-R-R in correspondence with _K-P_R_R,

R-X -X -X -K-Q-G-E-P-D-G-P-K-D-K-P-R-R

01 02 03

(X is P or R, X is R or K, X is Κ, Η or R)

01 02 03

May have an amino acid sequence such as

[0055] Furthermore, as the first partial amino acid sequence portion, R is selected as X, and R-PR-R-I K_Q_G is more preferably used.

[0056] The above linker sequence, for example, from 5 amino acid residues to a linker system IJ: 1 X -X -X -X

one two Three

4 5

The purpose of the present invention is to link the column portions and to add the contribution of a weak interaction between the second partial amino acid sequence portion and heparin to the heparin binding property mainly composed of the first partial amino acid sequence portion. Things. Therefore, due to the action of proteases and peptidases existing in the body of the administration subject, cleavage of such a linker sequence: one X-X-X-X-X is not possible.

1 2 3 4 5

In general, in order to prevent in vivo degradation of a peptide compound, a method of adding or modifying an extra amino acid at the N-terminus and C-terminus is effective. For example, various N-terminal amino groups can be modified with various acyl groups, or the C-terminal carboxy group can be amidated. The peptide having heparin-binding ability according to the second embodiment of the present invention can also be produced by using a chemical synthesis technique. When extending the peptide chain, C-terminal immobilized on the resin The terminal amidation is also synthetically suitable. Further, the linker system ij: X-X-X-X

1 2 3 X-parts may contain artificial amino acids instead of natural amino acids

4 5

it can. By using artificial amino acids, it is also possible to design the linker system [J] for those that suppress enzymatic cleavage of the peptide chain.

[0058] That is, in the peptide having heparin binding ability that is strong in the second embodiment of the present invention, the peptide chain portion other than the modified first partial amino acid sequence is the same as the above-described first embodiment of the present invention. It is also possible to adopt an embodiment in which various mutations, modifications, and alterations described in the description of the peptide having the ability to bind to heparin and the like can be used in the same manner.

[0059] A novel peptide having heparin binding ability that is useful in the present invention is VEGF-A KDR.

VEGF-A and vasculature are required for exerting the pro-angiogenic effect induced by binding to 165

165 It has the effect of competitively inhibiting the binding to heparin on the surface of endothelial cells and can be used as an anti-VEGF-A agent.For example, the growth and metastasis of solid tumors, diabetic retinopathy

165

Blood caused by VEGF-A, which causes various diseases such as retinopathy of prematurity, retinopathy of prematurity, and psoriasis

165

The present invention is applicable to the use of a therapeutic drug or a prophylactic drug for suppressing the promotion of angiogenesis.

[0060] Suppression of promotion of angiogenesis caused by these endogenous vascular endothelial growth factors VEGF-A

165

For therapeutic purposes, the peptide having heparin binding ability according to the present invention is suitably administered directly into the bloodstream and administered to the surface of vascular endothelial cells at the site of action. It is preferable to prepare a pharmaceutical composition in a dosage form suitable for intravenous administration. Specifically, the dosage form is a dosage form used for intravenous administration of peptide preparations having various physiological activities, for example, dosage forms such as intravenous injections and infusions. It is determined appropriately according to the application. In addition, taking into account the condition of the subject to be administered (patient), the severity of symptoms, gender, age, weight, and other health conditions, etc., the desired physiological activity is exhibited in the estimated total blood volume. It is preferable to set the administered dose so that the blood concentration is as follows. When the peptide having heparin-binding ability according to the present invention is used in the form of an intravenous injection, it is usually possible to administer the peptide in a plurality of divided doses. It is desirable to set the dose in the range of 100 mg / kg body weight, preferably in the range of 3-50 mg / kg body weight. For example, taking into account the total blood volume and assuming that the dose immediately after administration is evenly distributed, the average blood concentration is in the range of 0.1 μM ΙΟ μ Μ. Preferably, the total dose is set in the range of 1 / iM-3 / iM.

[0061] Further, a therapeutic or prophylactic agent for the purpose of suppressing the promotion of angiogenesis caused by VEGF-A for diabetic retinopathy and retinopathy of prematurity in which the organ site to be applied is specified.

165

Alternatively, it can be prepared in the form of eye drops. For this administration subject, an ophthalmic solution prepared by dissolving an effective amount of a peptide having heparin binding ability in a pharmaceutically acceptable liquid carrier suitable for application to the eyeball or orbit is available. Usually, it is desirable to set the concentration in the solution in the range of 0.1 μM to 10 μM, preferably in the range of 1 μm 3 μm.

Example

Hereinafter, the present invention will be described more specifically with reference to examples. Although the specific examples shown here are one example of the best embodiment working on the present invention, the present invention is not limited to these specific examples.

Example 1

(Peptide synthesis and purification)

A vascular endothelial growth factor VEGF-like protein derived from snake venom; the C-terminal partial amino acid sequence of vammin (Arg ⁹⁴ — Arg), which is assumed to be involved in heparin binding by referring to the amino acid sequences of vammin and VR-1 ^{11 ())} or VR - 1 of the C-terminal partial amino acid sequence (Arg ^9, according to one Arg ^1Q9), it was designed six peptidase de of peptide 1-peptide 6 shown in table 1 below.

[0064] Based on the designed amino acid sequence, each peptide was prepared by a solid phase synthesis method using the F_moc method, using a peptide synthesizer (Applied Biosystems, model 431A). According to a conventional method, the synthetic peptide was deprotected and separated and eluted from the base resin by the F-moc tarry vedge method, and the recovered crude peptide was lyophilized.

[0065] The crude peptide was purified using AKTAexplorer 10S (Amersham Biosciences) and an HPLC system (JASCO). The detection of the peptide in the column elution fraction was performed by measuring the absorbance at 240 nm.

[0066] After dissolving 40 mg of the obtained crude peptide in 10 mL of 50 mM Tris-HCl pH 8.0, Hi Trap Heparin HP Kahum Kahum valley lOmL, Amersham Biosciences Nokoano. I lied. Elution was performed from the heparin affinity column at a flow rate of 1 mL / min with a linear gradient up to 1.0 M NaCl in 5 column volumes.

The peptide fraction showing the binding property to heparin was pooled and collected, and then Cosmosil 5ΑΚ18ΑΚ_300 (2αηφX 25 cm (L)) was applied, and a linear gradient of acetonitrile 0 ^ο / ο 30% was obtained. And the peptide fraction having the desired number of amino acids was isolated. The purified peptide was freeze-dried, the amino acid sequence was confirmed by an amino acid sequencer, and the peptide concentration was quantified by amino acid analysis.

(Amino acid sequence analysis)

The amino acid sequence of the purified peptide was confirmed using Protein 'Sequencer (Applied Biosystems models 473A, 477, Shimadzu model PPSQ-21A).

(Evaluation of Heparin Binding Property)

The purified peptide (100 / ig) was dissolved in 10 mL of 50 mM Tris-HC1 ρΗ8.0 and then applied to Hi Trap Heparin HP Kahum (10 mL of sword Hum, Amersham Biosciences). Thereafter, the buffer was flowed through 5 column volumes at a flow rate of 1 mL / min, followed by elution with a linear gradient of about 1. OM NaC in 10 column volumes, and the elution conditions (NaCl concentration) were measured.

[0070] Fig. 1 shows the elution peak position (thick line part) of the peptide fraction showing the binding property to heparin for each peptide. Table 1 summarizes the concentration of eluted NaCl at the peak maximum.

[Table 1]

table 1

Heparin binding properties of various peptides.

Peptide Amino acid sequence Elution conditions with NaCl (M)

Peptide 1 G R P R R K Q G E P D G P K E K P R G 0.36

Ί 9 residues

Peptide 2 G R P R R K Q G E 0.34

9 residues

Peptide 3 G P D G P K E K P R G <0.1

1 1 residue

Peptide 4 R P R R K Q G E P D G P K E K P R G 0.37

18 residues

Peptide 5 R P R K Q G E P D G P K E-P R G 0.25

1 7 residues

Peptide 6 R P R R K Q G E P D G P K E K P R R 0.40

18 residues

[0072] Similarly, for the venous endothelial growth factor VEGF-like protein derived from snake venom; vammin and VR_1, the elution NaCl concentration at the peak maximum point of the protein fraction eluted from the heparin 'affinity column was similarly determined. Table 2 shows the measurement results.

[Table 2]

Table 2

Heparin binding properties of V a mm in and VR-1

Amino acid sequence of protein C-terminal region Elution conditions with NaCl (M)

V a mm inn R P R R K Q G E P D G P K E K P R 0.33

VR- 1 R P R * K Q G E P D G P K E-P R 0.16

[0074] (Inhibition of the in vitro effect of VEGF-A on promoting the growth of vascular endothelial cells.

165

Injury evaluation)

The peptide with heparin-binding ability described above is used to increase vascular endothelial cells by VEGF-A.

165

The suppression of the growth promoting effect was verified in the following in vitro evaluation system. At the same time, it also verified that vammin suppresses the growth promoting effect of vascular endothelial cells.

[0075] A cell suspension of P. aortic endothelial cells (BAEC) was seeded at a density of 5,000 cells / P in a 96 P cell culture plate. 0.1 0.1 after cell attachment in the well. The medium was replaced with a medium supplemented with / ₀ゥ fetal serum and cultured for a total of 18 hours. At that time, the medium was supplemented with a final concentration of 1 nM of the vascular endothelial growth factor protein VEGF-A or vammin, and

165

The peptide 1 was added thereto at a predetermined final concentration. After continuing the culture for 6 days, the number of cells in each well was determined using Tetra Color One (Seikagaku Corporation). The viable cell number density was evaluated by the WST-8 method.

[0076] The absorption coefficient A of color development in the WST-8 method was used as an index of the number of viable cells. Fig 3

405-630

Was evaluated in parallel on the same plate,

Left: Pell without vascular endothelial growth factor protein and peptide 1 (negative control: white column),

Center: In addition to vammin, peptide 1 was added at a final concentration of 100 μM (dark gray column: right of center), 30 μ μ (light gray, gray column: middle), 0 μΜ (no addition: positive control; Black column: center left) added gel,

Right: VEGF-A plus peptide 1 at a final concentration of 100 μM (dark gray column: medium

165

Center well (right), 30 μΜ (light, gray column: middle), 0 μΜ (no addition: positive control; black column: left of center)

3 shows the measurement results.

[0077] Comparing the results shown in Fig. 3, the VEGF-— and vammin added were intravascular.

165

It promotes the growth of skin cells, but in the presence of peptide 1, the growth-promoting effect of the cells is suppressed in a concentration dependent manner. Therefore, peptide 1 having heparin-binding ability binds to heparin present on the surface of BAEC cells, and VEGF-A and vammin exert the effect of promoting the growth of vascular endothelial cells.

165

VEGF-A and vammin show the growth-promoting action of vascular endothelial cells as a result of competitively inhibiting the binding to heparin out of the binding to KDR and the binding to heparin required in

165

Is determined to be suppressed.

[0078] (Evaluation of in vivo inhibitory activity against blood pressure lowering effect of VEGF-A)

165

The peptide with heparin binding ability described above suppresses the blood pressure lowering effect of VEGF-A.

165

Was controlled in the following in vivo evaluation system. At the same time, suppression of the blood pressure lowering effect of vammin was also verified.

[0079] Measurement of blood pressure lowering ability by VEGF-A or vammin was performed using Wis

165

This was performed using n = 3 or 5, 150-220 g) in each group. After anesthesia by intraperitoneal injection of ethyl carbamic acid ester (1 g / kg), 25% MgSO

A polyethylene tube filled with 4 was introduced into the carotid artery and a pressure transducer (model P10EZ, Becton Dickinson) to monitor carotid artery pressure. The carotid pressure measured by a pressure transducer was recorded by a recorder connected to an amplifier (model AP-621 Nihon Kohden).

After confirming that the blood pressure of each test individual was stable by injection of physiological saline (600 zL), a peptide having heparin binding ability (600 μL) and VEGF-A and

165 or vammin (600 μL) was administered through the left femoral vein.

[0081] A in FIG. 4 shows the top: when only vammin (dose 0.1 μg / g) was administered,

Bottom: Decrease in carotid artery pressure when vammin (dose 0.1 l x g / g) was administered after peptide 1 (dose 3 x g / g) was administered in advance.

B in Fig. 4 shows the top: When only VEGF-A (dose 0.1 x gZg) was administered,

165

Middle: VEGF-A (dose 0.1 μgZg) after pre-administration of peptide 1 (dose 3 x g / g)

165

When administered

Bottom: VEGF-A (dose 0.1 / ig / g) after pre-administration of peptide 1 (dose 30 ig / g)

165

) Shows the amount of decrease in carotid artery pressure after administration.

[0082] Comparing the results shown in Fig. 4, the administered VEGF-A and vammin showed lower blood pressure.

165

In the co-presence of the peptide peptide 1 which induces the lowering, its effect of lowering blood pressure is suppressed in a concentration dependent manner. The VEGF-A evaluated here

165 In addition, the blood pressure lowering effect of vammin is based on the potent nitric oxide (NO) -dependent blood pressure lowering activity induced by the binding of VEGF protein to KDR. VEGF-A and vammin lower blood pressure by binding to peptide 1 having heparin present on the surface of KDR-expressing cells.

165

Competitively inhibits the binding to heparin out of the binding to KDR and the binding to heparin, which are necessary for its action. As a result, VEGF-A and vammin lower blood pressure.

165

Is determined to be suppressed.

[0083] Amino acid sequence of R_P_R_X_K_Q_G_X -X—X—X—X _K_E_K_P_R

1 2 3 4 5

In particular, in the peptide having a heterogeneous binding ability according to the present invention, VEGF-A is formed only by a portion of R-P-R_X_K_Q_G (X is any one of R, W, H and K).

165 and binding to KDR and heparin, necessary for vammin to exert blood pressure lowering effect Competitively inhibits heparin binding, resulting in VEGF-A and va

165

It was also verified that the effect of suppressing the blood pressure lowering effect of mmin was achieved.

[0084] Specifically, VEGF-A caused a decrease in blood pressure of peptide 2 corresponding to the N-terminal region of peptide 1 and peptide 3 corresponding to the C-terminal region.

165

The presence or absence of the effect of suppressing the action was evaluated in parallel with the in vivo evaluation system described above.

[0085] In Fig. 5, when A: VEGF-A (dose 0.1 μgZg) alone was administered (positive control),

165

B: VEGF-A (dose 0.1 ^ gZg) after pre-administration of peptide 1 (dose 30 g / g)

165

When administered

C: After pre-administration of peptide 2 (dose 30 ^ g / g), VEGF-A (dose 0.1 gZg)

165

When administered

D: VEGF-A (dose 0.1 μgZg) after pre-administration of peptide 3 (dose 30 μg / g)

165

Shows the amount of decrease in the carotid artery pressure upon administration of.

[0086] Comparison of the results shown in Fig. 5 shows that the administered VEGF-A induced hypotensive effects.

165

In contrast, peptide 1 and peptide 2, which are administered beforehand, have the effect of suppressing the blood pressure lowering effect. Therefore, peptide 1 and peptide 2, which have heparin binding ability, bind to heparin present on the surface of cells expressing KDR, so that VEGF-A

165 Of the binding to KDR and the binding to heparin, which are necessary for exerting the antihypertensive action, competitively inhibits the binding to heparin, thereby suppressing the hypotensive action of VEGF-A.

165

Is determined to be. On the other hand, peptide 3 does not show heparin-binding ability, and therefore, the inhibitory effect was observed, and it was judged to be poor.

[0087] In conclusion, the above R_P_R_X_K_Q_G_X -X -X -X -X _K_E_K_P_R

1 2 3 4 5

In the peptide having an amino acid sequence and having heparin-binding ability according to the present invention, in particular, R_P_R_X_K_Q_G (X is a site where any part of R, W, H, or K governs heparin-binding ability. In addition, the RPRXKQ-G (X is present on the surface of a KDR-expressing cell even in vivo by using any part of R, W, H, and K. By binding to heparin, VEGF-A

165 Of the binding to KDR and the binding to heparin, which are necessary for As a result of competitive inhibition of binding to heparin, physiological activity and action by VEGF-A

165

Is determined to be suppressed.

[0088] Reference Example 1

In this example, VEGF-A and vammin exert the effect of promoting vascular endothelial cell proliferation.

165

Inhibiting the binding of heparin on the vascular endothelial cell surface to the binding of KDR and heparin, which are necessary for

165

It was verified that the cell growth promoting effect was suppressed.

[0089] (In vitro promotion of proliferation of vascular endothelial cells by VEGF-A or vammin

165

Evaluation of the inhibitory ability of unfractionated heparin on the action)

As mentioned above, VEGF-A and vammin show binding ability to free heparin.

165

Therefore, beforehand, bind the free form of parin to VEGF-A and vammin.

165

Then, as a result, it becomes possible to inhibit the binding to heparin on the vascular endothelial cell surface.

[0090] VEGF-A and unfractionated heparin exhibiting binding ability to vammin were obtained from VEGF-A

165 The effect of suppressing the growth promoting effect of vascular endothelial cells by 165 or vammin was demonstrated in the following in vitro evaluation system.

[0091] A cell suspension of human umbilical vein endothelial cells (HUVEC) was seeded at a density of 5,000 cells / well in a collagen-coated 96-well cell culture plate. After seeding, the cells were cultured for 6 hours. After the cells in the wells were adhered, the medium was replaced with a medium supplemented with 1% human serum and cultured overnight (total 18 hours). At that point, the medium was supplemented with a final concentration of 1 nM of vascular endothelial growth factor protein, VEG F-A or vammin, and untreated fractions of parin.

165

It was added at a concentration. Thereafter, after culturing was continued for 3 days, the number of viable cells was evaluated for the number of cells in each well by the WST-8 method using Tetra Color One (Seikagaku Corporation).

[0092] The absorption coefficient A of color development in the WST-8 method was used as an index of the number of viable cells. VEG

405-630

F-A or vammin, and the 1%

165

The number of surviving cells when cultured in a clarified medium was measured using a negative control (unstimulated), without VEGF-A or vammin, without adding palin to unfractionated cells.

165 The number of surviving cells when cultured in the 1% serum-supplemented medium is used as a positive control (stimulate d), and the inhibitory effect on growth promotion is estimated based on the estimated number of surviving cells.

[0093] FIG. 7 shows that unfractionated heparin co-supplemented in the medium, VEGF-A and vammin

165

VEGF-A or vammin-stimulated vascular endothelial cell proliferation caused by binding of

165

Shows the suppression effect. Under serum-containing culture conditions, add various concentrations of unfractionated heparin and VEGF-A or vammin (final concentration InM), and cultivate for 3 days.

165

The number was evaluated. Closed circles: vammin, open circles: VEGF-A stimulation of vascular endothelial cells

165

Fig. 3 shows the absorption coefficient A (the number of viable cells) of color development in the WST-8 method when palin was co-added to the unfractionated medium at each concentration during growth promotion.

405-630

[0094] Depending on the concentration of unfractionated heparin added to the medium, VEGF-A or vamm

165

The vascular endothelial cell proliferation action by in stimulation is suppressed. That is, VEGF— A

If 165 or vammin is preliminarily bound to unfractionated pallin, the binding to KDR and the binding to heparin, which are necessary for promoting the proliferation of vascular endothelial cells, The binding to heparin is inhibited, resulting in the binding to KDR, but the effect of VEGF-A or vammin to proliferate vascular endothelial cells is exhibited.

165

Absent.

[0095] The above results indicate that VEGF-A or vammin stimulation

165

Demonstrates binding between VEGF-A or vammin and heparin on the surface of vascular endothelial cells.

165

It is a visa that inhibition can be suppressed.

[0096] Example 2

Further, based on the aforementioned peptide 6: R—P—R—R—K—Q—G_E_P_D_G_P_K—E—K—P—R—R, based on the R—P_R_R_K_Q_G portion corresponding to the first partial amino acid sequence portion After modification, two peptides, peptide 7 and peptide 8, shown in Table 3 below were designed. Additionally, Tissue factor pathway inhibitor (TFPI) partial amino acid sequence in a protein having a heparin binding to - a peptide 9 corresponding to (Lys ²⁵⁴ Lys ^265), was designed as a path phosphorus binding peptides to control.

[0097] (Evaluation of heparin binding property)

According to the procedure of (Peptide Synthesis and Purification) and (Amino Acid Sequence Analysis) described in Example 1, Purified samples of three peptides, peptide 7, peptide 8, and peptide 9, were prepared.

[0098] Next, according to the method described in Example 1 (evaluation of heparin binding property), elution conditions (NaCl concentration) for the purified peptide bound on the HiTrap Heparin HP column were measured.

[0099] Table 3 summarizes the measurement results of the eluted NaCl concentration at the peak maximum of the elution peak for the three peptides.

[0100] [Table 3]

Table 3

—Heparin binding properties of various peptides—

Elution conditions with peptide amino acid sequence NaCl (M)

Peptide 7 ~ R P K K K Q G E P D G P K E K P R R 0.36

18 residues

Peptide 8 R P H K Q G E P D G P K E K P R R 0.52

18 residues

Peptide 9 K T R K R K K Q R V K 0.73

1 2 residues

[0101] (Inhibition of the in vitro effect of VEGF-A on promoting the growth of vascular endothelial cells.

165

Injury evaluation)

The peptide having heparin-binding ability described above is used to increase vascular endothelial cells by VEGF-A.

165

The suppression of the growth promoting effect was verified in the following in vitro evaluation system.

[0102] A cell suspension of human umbilical vein endothelial cells (HUVEC) was seeded at a density of 5,000 cells / well in a collagen-coated 96-well cell culture plate. After seeding, the cells were cultured for 6 hours. After the cells in the wells were adhered, the medium was replaced with a medium supplemented with 1% human serum, and the cells were cultured overnight (total 18 hours). At that time, the medium was supplemented with the vascular endothelial growth factor protein, VEG F-A, at a final concentration of 1 nM and each peptide at the predetermined final concentration.

165

Added. Then, after culturing was continued for 3 days, the number of cells in each well was evaluated for the number of viable cells by the WST-8 method using Tetra Color One (Seikagaku Corporation).

[0103] The absorption coefficient A of color development in the WST-8 method was used as an index of the number of viable cells. VEG

405-630

F-A and the test peptide were not added together, but in the 1% serum-supplemented medium.

165

Of the number of surviving cells when cultured in a negative control: 0% growth promotion, no VEGF-A, without VEGF-A, The number of surviving cells in the culture was defined as a positive control: proliferation promotion 100%, and the proliferation promotion rate was calculated based on the estimated number of surviving cells.

[0104] FIG. 8 shows the peptide power S of peptide 1, peptide 2, and peptide 3, VEGF-A

16 shows the results of evaluation of the inhibitory ability of 165 on the growth promoting effect of vascular endothelial cells. Human umbilical vein endothelial cells with VEGF-A (final concentration InM)

165

The inhibitory effect of each of the co-added peptides on the promotion of cell proliferation was evaluated by the number of surviving cells after 3 days of culture.

[0105] The serum added to the medium originally contained a small amount of VEGF, and under the conditions that completely inhibited the growth promoting effect of vascular endothelial cells by VEGF-A,

165

The growth-promoting effect due to this serum-derived VEGF is similarly inhibited. Therefore, if VEGF-A completely inhibits the growth-promoting effect of vascular endothelial cells,

165

In addition, the growth promotion rate shows a negative value. In general, when "the growth promotion rate shows a negative value", it is inferred that the test compound (peptide) shows "cytotoxicity" to the target cell. However, in an evaluation system using these serum-added culture conditions, the "negative growth promotion rate" shown in this figure does not mean "cytotoxicity". It is confirmed by measuring the number of surviving cells when cultured in a medium containing a low concentration of serum containing 0.1% or less.

[0106] In Fig. 8, the concentration dependence of the addition of peptide 1, indicated by black circles, was due to VEGF-A-induced blood.

165 shows a concentration-dependent inhibitory activity on the growth promoting effect of endothelial cells. On the other hand, compared to Peptide 1, heparin binding ability is inferior. When white circles: peptide 2 and black triangle A: peptide 3 are added, the difference in heparin binding ability is reflected. The addition concentration (ED) at which% inhibition can be achieved is high. Therefore, peptide 1, peptide 2, p

50

All peptides of eptide 3 competitively inhibit the binding of heparin to KDR and the binding to heparin on the surface of vascular endothelial cells.

It is judged that the growth promoting effect of vascular endothelial cells is suppressed. Also 50. The difference in the concentration (ED) of the inhibitory addition of / o is indicated by the peptide 1, peptide 2, and peptide 3.

50

It is also confirmed that the difference is due to a difference in binding ability to heparin on the surface of vascular endothelial cells.

[0107] For free heparin used in a commercially available heparin affinity column, peptide 9 (TFPI ²⁵⁴ - ^265), although capable of binding equivalent to peptide 1, peptide 1 is capable of binding specific for Roh ^ emissions to the vascular endothelial cell surface Ueno, whereas, peptide 9 ( TFPI ²⁵⁴ - ^265), to the heparin surface of vascular endothelial cells Ueno, do not show specific binding ability, it was verified by the following evaluation method.

In contrast to the above-mentioned in vitro effect of VEGF-A on promoting the growth of vascular endothelial cells

165

The inhibitory activity of peptide 9 (TFPI ^254-265 ) was compared to the inhibitory activity of peptide 1 in the evaluation system for inhibitory activity.

[0108] Fig. 9 shows VEGF-A, which is shown by each of peptide 1 and peptide 9 (TFPI ^{254 265} ).

165 shows the results of evaluating the inhibitory ability of human umbilical vein endothelial cells on the growth promoting effect of stimulus

. Depending on the culture conditions with serum, human umbilical cord by adding VEGF-A (final concentration InM)

165

Suppression of vein endothelial cell growth promotion by each co-added peptide was evaluated by cell number after culturing for 3 days. Black: peptide 1, Gray: peptide 9 - a (TFPI ²⁵⁴ ^265), definitive when added each concentration, showing the growth-promoting factor.

Peptide 1 increases human umbilical vein endothelial cells upon VEGF-A stimulation in a concentration-dependent manner.

165

Although the growth was suppressed, peptide 9 (TFPI ²⁵⁴ " ²⁶⁵ ) showed only a slight inhibitory effect.

[0109] That is, since peptide 1 has specific binding ability to heparin on the surface of vascular endothelial cells, peptide 1 binds to KDR on the surface of vascular endothelial cells and As a result of competitive inhibition of heparin binding, VEGF-A shows that vascular endothelial cells

165

It effectively suppresses the growth promoting effect. On the other hand, peptide 9 (TFPI ^254-265 ) does not have a specific binding ability to heparin on the surface of vascular endothelial cells, so that heparin on the surface of vascular endothelial cells at the same concentration added. Only a part of. In that case, VEGF-A, when bound to KDR on the surface of vascular endothelial cells,

165

Is capable of binding to heparin on the vesicle surface and consequently VEGF-A

165

Most of the skin cell proliferation promoting action is maintained.

[0110] In other words, peptide 1, said peptide 9 - are Mashimashi inner amino acid sequence of (TFPI ²⁵⁴ ^265), to form a Tissue factor pathway inhibitor (TFPI) protein and specifically complex, blood Specificity for free heparin molecule that exerts anticoagulant effect Is not indicated. Due to differences in the structure and sulfated sites of heparin chains depending on their origin, peptide 1 has specific binding ability to proteodalican-type heparin chains present on the surface of vascular endothelial cells. However, it is judged that heparin does not have specific binding ability to heparin of other origin.

[0111] Similarly to peptide 1 and peptide 8 produced in Example 2, it was confirmed that the inhibitory effect of VEGF_A on the promotion of vascular endothelial cell growth was effectively suppressed by the above-mentioned in vit.

165

It was verified using the evaluation system of ro.

[0112] FIG. 10 shows the strength of each peptide of peptide 1, peptide 7, and peptide 8. VEGF-A

16 shows the results of evaluation of the inhibitory ability of No. 16 against the growth promoting effect of vascular endothelial cells. Serum added calo

Five

In culture conditions, human umbilical vein endothelial cells were treated with VEGF-A (final concentration InM).

165

Suppression of cell growth promotion by the co-added peptides was evaluated by the number of cells after 3 days of culture. Solid circles: peptide 1, open circles: peptide 7, solid triangles: peptide 8 show the growth promotion rate when each concentration was added. As with peptide 1, peptide 7 and peptide 8 were exposed to VEGF-A-stimulated human umbilical vein endothelium in a concentration-dependent manner.

165

Suppresses the growth of vesicles. In addition, it is also confirmed that the inhibitory activities exhibited by these three peptides have substantial differences.

[0113] The results shown in Fig. 10 show that at a high concentration of the test peptide,

"The growth promotion rate shows a negative value," but the serum added to the medium originally contained a small amount of VEGF, and the "negative growth promotion Rate "

The point that does not mean “cytotoxicity” is as described above.

[0114] Example 3

The peptide having heparin-binding ability described above is used for the activation of vascular endothelial cells by VEGF-A.

165

It was verified in an in vitro evaluation system described below that it inhibits the growth promoting effect but does not show the inhibitory effect of basic fibroblast growth factor (bFGF) on the growth promoting effect of vascular endothelial cells.

[0115] A cell suspension of human umbilical vein endothelial cells (HUVEC) was seeded at a density of 5,000 cells / well on a collagen-coated 96-well cell culture plate. After seeding, incubate for 6 hours, allow cells in the wells to adhere, replace the medium with 1% human serum, and overnight ( (18 hours in total). At that time, VEGF-A or bFGF was added to the medium at the final concentration.

165

The test peptide was added at a final concentration of 1 nM, as well as the test peptide. After continuing the culture for 3 days, the number of cells in each well was evaluated for the viable cell number density by the WST-8 method using Tetra Color One (Seikagaku Corporation).

[0116] In the WST-8 method, the color absorption coefficient A was used as an index of the number of viable cells. VEG

405-630

Without adding F-A or bFGF and the test peptide, add 1% serum as described above.

165

The number of surviving cells when cultured in a supplemented medium was determined using the negative control: 0% growth promotion, 1% blood growth without VEGF-A or bFGF, without the addition of the test peptide.

165

The number of surviving cells when cultured in a fresh medium was defined as a positive control: proliferation promotion 100%, and the proliferation promotion rate was calculated based on the evaluated number of surviving cells.

[0117] FIG. 11 shows that VEGF-A or bFGF ligated peptide 1 was added to the medium.

165

It shows an inhibitory effect on vascular endothelial cell proliferation by vigorous activity. Under serum-containing culture conditions, various concentrations of peptide 1 and VEGF-A or bFGF (final concentration InM) were added.

165

After 3 days of culture, the number of surviving cells was evaluated. Black circle ·: VEGF-A, white circle 〇: bFGF stimulation

165

The figure shows the growth promotion rate when the peptide 1 was co-added to the medium at each concentration when the growth of vascular endothelial cells was promoted by the method.

[0118] Peptide 1 was added at a concentration that inhibited the growth of vascular endothelial cells by VEGF-A stimulation.

165

Although the inhibitory effect was shown in a dependent manner, the inhibitory effect on bFGF-stimulated vascular endothelial cell proliferation was only slightly apparent. The serum added to the medium originally contains a small amount of VEGF, and when peptide 1 is added, the proliferation of vascular endothelial cells caused by VEGF derived from this serum is suppressed. For this reason, the vascular endothelial cell proliferation effect due to bFGF stimulation is not suppressed, but the overall growth promotion rate is slightly apparently suppressed. Also, as explained above, VEGF-A stimulation

In the system in which vascular endothelial cells are grown by 165, when peptide 1 is added at a high concentration, the evaluation result is apparently that the growth promotion rate shows a negative value. In other words, in a system in which vascular endothelial cells are proliferated by bFGF stimulation, when peptide 1 is added at a high concentration, the apparent “slightly reduced growth promotion rate” as shown in FIG. Peptide 1 shows no inhibitory effect on skin cell proliferation even at high concentration It is determined that they have not.

[0119] Therefore, from the above results, peptide 1 has specific binding ability to proteoglycan-type heparin chains present on the surface of vascular endothelial cells, and in particular, VEGF-A

165 competitively inhibits the binding of heparin on the vascular endothelial cell surface to the binding of KDR on the vascular endothelial cell surface and the binding to heparin, which are necessary for 165 to exert the vascular endothelial cell proliferation action On the other hand, the presence of peptide 1 indicates that VEGF-A can specifically bind

165

It can be seen that heparin on the surface of vascular endothelial cells is not directly involved in the mechanism of bFGF-stimulated vascular endothelial cell proliferation.

Industrial applicability

[0120] The novel heparin-binding peptide according to the present invention is designed based on the amino acid sequence of the heparin-binding site in snake venom-derived vascular endothelial growth factor VEGF-like protein; vammin and VR-1. It is a peptide compound consisting of 7-20 amino acid residues. VEGF-A and heparin on the cell surface are associated with the binding of VEGF-A to KDR.

165 165

Has the effect of competitively inhibiting the binding between VEGF-A and KDR.

165

It can be used as a peptidic drug that requires both binding and binding to heparin on the cell surface and suppresses the effect of promoting angiogenesis.

Claims

The scope of the claims

[1] First partial amino acid sequence derived from the heparin binding site in vammin:

R_P_R_X_K_Q—contains at least G (X is any of R, W, H, K) and consists of 7-20 amino acid residues

A peptide having heparin binding ability, which is characterized in that:

[2] Further, in addition to the first partial amino acid system IJ: R—P_R_X—K_Q_G,

Second partial amino acid sequence from the heparin binding site in vammin:

Including K—E—K—P—R,

The second partial amino acid sequence is linked to the C-terminal of the first partial amino acid sequence from 416 amino acid residues via a linker sequence.

The peptide having heparin-binding ability according to claim 1, characterized in that:

[3] Amino acid sequence derived from the heparin binding site in vammin:

R-P-R-R-K-Q-G-E-P-D-G-P-K-E-K-P-R

A strong amino acid sequence | J or a peptide comprising at least one amino acid substitution in the sequence thereof and containing a modified amino acid sequence retaining the first partial amino acid sequence.

3. The peptide having heparin-binding ability according to claim 1 or 2, wherein the peptide has heparin-binding ability.

[4] First partial amino acid sequence derived from the heparin binding site in vammin:

N C

(A and A are each selected from a peptide chain having 0 to 13 amino acids;

N C N C

The total number of amino acids is 13 or less)

Powerful, with an amino acid sequence of 720 amino acid residues

[5] a competitive inhibitor of VEGF-A binding to heparin,

165

The competition-inhibiting active ingredient is the peptide having heparin-binding ability according to any one of claims 1 to 4. An inhibitor of VEGF-A binding to heparin.

165

[6] A pharmaceutical composition having use as an inhibitor for the binding between VEGF-A and heparin

165

And

The inhibitory active ingredient is a peptide having a heparin-binding ability according to any one of claims 1 to 4,

An effective amount of the peptide having heparin-binding ability is dissolved in a pharmaceutically acceptable liquid carrier suitable for intravenous administration to a subject.

A composition comprising:

[7] A modified first partial amino acid sequence derived from a heparin binding site in vammin:

R-X -X -X -K-Q-G (X is P or R, X is R or K, X is Κ, Η

01 02 03 01 02 03 or R)

Consists of 7-20 amino acid residues

A peptide having heparin binding ability, which is characterized in that:

[8] Further, the modified first partial amino acid sequence: R—X-X-X-K-Q-G (X

01 02 03 01 is P or R, X is R or K, X is Κ

02 03, Η or R)

Second partial amino acid sequence from the heparin binding site in vammin:

Including K—E—K—P—R,

The second partial amino acid sequence is linked to the C-terminal of the first partial amino acid sequence from 4-6 amino acid residues via a linker sequence.

8. The peptide having heparin binding ability according to claim 7, wherein the peptide has heparin binding ability.

[9] E_P_D_G_P is selected as the linker sequence,

R-X -X -X -K-Q-G-E-P-D-G-P-K-E-K-P-R

01 02 03

(X is P or R, X is R or K, X is Κ, Η or R)

01 02 03

A peptide comprising an acid sequence or a modified amino acid sequence having at least one amino acid substitution in the sequence and retaining the modified first partial amino acid sequence.

9. The peptide having heparin-binding ability according to claim 8, wherein the peptide has heparin-binding ability.

[10] A modified first partial amino acid sequence derived from a heparin binding site in vammin: RX -X -X -KQG (X is P or RK

01 02 03 01, X is R or

02, X is K

03, H or R)

A -R-X -X -X -K-Q-G-A

Nl 01 02 03 CI

(A and A are each selected from a peptide chain having 0 to 13 amino acids;

CI Nl, A

The total number of amino acids in Nl CI is 13 or less.)

Powerful, with an amino acid sequence of 720 amino acid residues

[11] A competitive inhibitor of VEGF-A binding to heparin,

165

The competition-inhibiting active ingredient is the peptide having heparin-binding ability according to any one of claims 7 to 10.

An inhibitor of VEGF-A binding to heparin.

165

[12] A pharmaceutical composition having use as an inhibitor for the binding between VEGF-A and heparin

165

And

The inhibitory active ingredient is a peptide having heparin binding ability according to any one of claims 7 to 10,

A composition comprising: