WO2005077970A1

WO2005077970A1 - Novel heparin-binding peptide designed from heparin-binding site of snake venom-origin vascular endothelial growth factor (vegf)-like protein and use thereof

Info

Publication number: WO2005077970A1
Application number: PCT/JP2004/001494
Authority: WO
Inventors: Takashi Morita; Yasuo Yamazaki
Original assignee: Nec Soft, Ltd.
Priority date: 2004-02-12
Filing date: 2004-02-12
Publication date: 2005-08-25
Also published as: JPWO2005077971A1; WO2005077971A1

Abstract

As a novel heparin-binding peptide which inhibits the binding of VEGF-A165 to heparin or heparan sulfate proteoglycan and by which various physiological reactions caused by interactions between KDR and VEGF-A165 can be regulated, it is intended to provide a heparin-binding peptide which is designed based on the heparin-binding site of a snake venom-origin VEGF-like protein vammin, contains at least a first partial amino acid sequence: R-P-R-X-K-Q-G (wherein X represents one of R, W, H and K) originating in the heparin-binding site as described above and consists of from 7 to 20 amino acid residues.

Description

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 a specific binding property to vascular endothelial growth factor receptor type 2 (VEGF recptor2; KDR), while vascular endothelial growth factor receptor type 1 (V light

Does not show binding to EGFreceptor1; F1t-1), and is derived from the heparin binding site in vascular endothelial growth factor (VEGF) -like protein derived from snake venom

The present invention relates to heparin-binding peptides designed based on the amino acid sequence to come and their use in the medical field. More specifically, a VEGF-like protein isolated from the snake venom of Vipera ammo dytesa mm odytes; vammin, and a VEGF-like protein isolated from the snake venom of Daboiarussellirusse 11 /; A heparin-binding peptide designed based on an amino acid sequence derived from a heparin-binding site in VR-1 and a vascular endothelial growth factor (vascular endothelia 1 growth factor) utilizing 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, through suppression of heparin binding to VEGF-A).

Background technology

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 in the female sexual cycle I), while solid tumor growth, diabetic It is also known to be involved in various diseases such as retinopathy, retinopathy of prematurity, and psoriasis. VEGF-A is a glycoprotein in which a submit 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 growth factor (PI GF) (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); Yasushi Sato, Ed., Frontiers of Angiogenesis, Sheep See Shasha, 10—171 (1999)). VEGF binds with high affinity to VEGFR-l, fms-liketyrosinekinase-1: F1t-1) and VEGFR-2 (kinaseinserted oma in- containing receptor: KDR) of vascular endothelial cells. It has been suggested that the endothelial cell proliferation signal and blood pressure lowering effect of VEG F-A are mainly mediated by KDR (Li, B. et al., Hypertension, 39, 1095–1100 (2002)). See). In addition, NO produced by signal transmission via KDR has various physiological activities such as vasorelaxant, platelet aggregation inhibition, antithrombotic and anti-inflammatory, and is considered to work anti-atherosclerotically. (See Masashi Shibuya and Masahiko Kurabayashi, Experimental Medicine, 20 (special edition), 1070–1269 (2002)).

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, mature monomer, 121, 145, 165, 189, as well, of five with 206 amino acid residues iso- form; VEGF- A ₁₂ had VEGF one _{_{A 145, VEGF- A 165, VEGF-}} A 187, VEGF- A _2. ₆ is present, especially, VEGF-A ₁₆₅ lacks the partial amino acid sequence encoded by Ekison 6, also, VEGF-A ₁₂₁ is Ekison 6, lacking the partial amino acid sequence encoded by Ekison 7 Has been confirmed. Among them, VEGF- A ₁₆₅ multilingual It has been confirmed that it is expressed as free form in many tissues and functions as mature and active VEGF. VEGF-A ₁₆₅ also has binding properties to heparin or heparan sulfate proteoglycan, and in the presence of low concentration of heparin, increases the affinity of VEGF-A ₁₆₅ for KDR while increasing it. upon the presence of heparin concentration, it is also reported that VEGF- affinity enhancement of a ₁₆₅ is not permitted for KDR (see WO 01/7282 9 No. Panfuretsuto). In addition, it has been reported that neuropilin-1 also functions as a specific co-receptor for VEGF-A ₁₆₅ by binding to VEGF-A ₆₅ and enhancing affinity for KDR (Japanese Patent Laid-Open No. 2003 -1 No. 2541).

On the other hand, inhibition of interaction between KDR and VEGF-A _165, the growth or metastasis of solid tumors, diabetic retinopathy, angiogenesis is inhibited in retinopathy of prematurity, can inhibit the progression of these diseases . Thus, KDR and VEGF- interaction with A ₁₆₅ has the function of P且害or Antago two strike shows specific affinity for KDR, KDR with specific binding to VEGF- A ₁₆₅ The search for substances that inhibit the binding to is progressing.

Incidentally, other VEGF-E derived from the above-mentioned P Arapox viruses have Amino acid sequence similarity to VEGF one A i _65, as the protein showing the binding to KDR, bi toxin into the Ve p β raaspisaspis It was isolated as a VEGF-like molecule with homology to VEGF (hypotensive factor: HF) (Komori, Y. et al., ΤοΤ icon, 28, 359-369 (1990); K omori, Y. et al., Biochemistry, 38, 1 1 796-1 1803 (1990)). More recently, two VEGF-like molecules, snakevenom VEGF and increasing capillary perme ability protein (I CPP), have been isolated from other snake venoms, respectively, and VEGF from these two snake venoms has been isolated. -Like proteins are involved in vascular permeability and angiogenesis Has been shown to have activity (Jun qu eirade Az evedo

I .: L. et al., J. Biol. Chem., 276, 39836-3 9842 (2001); Gasmi, A. et al., Bioche m. Biophys. Res. Commun., 268. , 69-72 (2000); Gasmi, A. et al., J. Bio 1. Chem., 277, 299 92-29998 (2002)).

Disclosure of the invention

As described above, inhibition of interaction between KDR and VEGF-A _165, the growth or metastasis of solid tumors, diabetic retinopathy, neovascularization in retinopathy of prematurity is suppression, it can inhibit the progression of these diseases It is. Antagonists in the previous studies, as a means of inhibiting the interaction of KDR and VEGF-A _165, having a function of inhibiting the interaction of KDR and VEGF-A ₁₆ _5, showing the specific affinity to KDR or, VEGF-interest research to explore the binding inhibitor of the KD R with specific binding to a ₁₆₅ are concentrated.

The present inventors, in addition to these conventional approaches, VEGF-A ₁₆₅ is to also have a binding to heparan sulfate Puroteodarikan to path phosphorus or Teire, the presence of heparin free concentration in, by focusing on the point making any affinity enhancement of VEGF-a ₁₆₅ for KDR, means for suppressing the binding of heparan sulfate proteoglycan and VEGF-a ₁₆₅ heparin or to to this also, KDR and VEGF-a It was conceived to have a significant contribution to the inhibition of the interaction of ₁₆ _5. However, in the current situation, as means for suppressing binding to VEGF-A ₁₆₅ heparin or heparan sulfate Puroteodarikan to present on the cell surface, has high specificity, effective means were or are proposed seven It was also found that this was a problem to be solved. The present invention is intended to solve the above problems, an object of the present invention, VEGF-to present on the cell surface of the A i ₆₅ heparin or to the heparan sulfate-flop port Teogurikan Against inhibit binding, for example, to the presence of heparin, which can suppress various physiological responses caused by the interaction of KDR and VEGF-A i _65, in subjecting Hisage new means. Specifically, when VEGF-A ₁₆₅ forms a bond with KDR present on the cell membrane, it binds at the same time and exists on the cell surface, which has a co-receptor-like function for VEGF-A ₁₆₅ to walk heparin is to show the heparan sulfate Puroteodarikan high binding to provide a novel substance that competitively inhibit the complex formation with VEGF-a ₁₆₅ Toepa phosphorus. The present inventors, in order to solve the above problems, in the course of intensive further research, previously not a, the sites involved in binding against the heparan sulfate Puroteodarikan VEGF-A ₁₆₅ to the heparin or to its C It has been reported that it exists on the terminal side (Keyt, BA eta 1., J. Biol. Chem., Vo 1.2 7 17 7 8 8-7 7 9 5 (1 9 9 focusing on 6) referring to a), specifically, in the recombinant production _VEGF-65 monomer, the site of a la ^ a rg ¹⁶ ⁵ was removed by trypsin digestion, regulating the C-terminal truncated version protein monomer papermaking When heparin binding was examined, it was confirmed that heparin binding was lost. In addition, homodimers of C-terminal truncated version proteins from force Cal VEGF ₁₆₅ is binding to KDR in in V Nitro, in the pair heparin absence, homodimerization of which a base VEGF-A ₁₆₅ It was also confirmed that it was comparable to the body. However, in the proliferation test of the © shea aortic endothelial cells in V Nitro, its growth inducing action, homodimers of C-terminal truncated version modified variant proteins from VEGF ₁₆₅ is a homodimer of VEGF ₁₆₅ has a basic It was also found that it had dropped significantly. That is, in addition to the binding of VEGF ₁₆₅ to KDR, simultaneous binding to proteoglycan-type paparin or heparan sulfate proteoglycan present on the surface of endothelial cells is a consequence of intracellular physiology following binding to KDR. It is confirmed that it is indispensable for inducing a reactive reaction.

Furthermore, the inventors have developed a vascular endothelial growth factor VEGF-like protein derived from snake venom. VEGF-like protein from peraa mmo dy tesa mm odytes identified; vamm in Daboiarusse 11 1 VEGF-like protein from irusse 11; VR-1 also has KDR and It has been found that the induction of a physiological response in the cell following the binding of is required to simultaneously bind to the proteoglycan-type peparin or heparan sulfate proteoglycan present on the cell surface. When we searched for sites involved in binding to heparin or heparan sulfate proteoglycan in these venom endothelial growth factor VEGF-like proteins derived from snake venom, only the C-terminal part was involved in binding to heparin. I found out. Indeed, by combining the peptide fragment having a C-terminal partial amino acid sequence of the ^{V mm in (Ar g 94 ~Ar} g 110) or VR- 1 of the C-terminal portion partial amino acid sequence ^{^{(Ar g 94 ~Ar g 109)}} , When the heparin-binding ability (affinity) was evaluated, it was confirmed that the protein exhibited an amino acid sequence derived from the heparin-binding site in vaccin, VR-1. In addition, in addition to the amino acid sequence derived from the heparin binding site in V ammin, VR-1, a series of peptides having a modified amino acid sequence designed based on the amino acid sequence are also high. Retention of heparin binding ability (affinity) was also verified. Furthermore, the amino acid sequence portion having a dominant function in its heparin binding ability (affinity) is a portion of R—P—R—X—K—Q—G (X is R or W) I also confirmed that. Furthermore, a synthetic peptide fragment having heparin binding ability (affinity) designed based on the amino acid sequence derived from the heparin binding site in the above-mentioned Vammin, VR-1 is VEGF- a _165, or V a mm in showing concentration dependent inhibits action on the growth-inducing effect of vascular endothelial cells by, in the vivo, indicated .VEGF- a _165, produced by signaling through KDR It also demonstrated that it exhibited a concentration-dependent inhibitory effect on blood pressure lowering ability due to NO. Based on the above knowledge, the present inventors have completed the present invention.

That is, the peptide having heparin binding ability according to the present invention is:

First partial amino acid sequence from heparin binding site in V ammin: At least RPRXKQG (X is R, W, H, K)

A peptide having heparin-binding ability, which is composed of 7 to 20 amino acid residues.

Therefore, the peptides having heparin binding ability according to the present invention include:

A first partial amino acid sequence derived from a heparin binding site in V amm in: at least R-P-R-X-K-Q-G (X is any one of R, W, H, and K);

A _N -RPRXKQGA _C

(A _N and A _c are each selected from a peptide chain having 0 to 13 amino acids, and the total number of amino acids of A _N and A _c is 13 or less.)

And a peptide having an amino acid sequence of 7 to 20 amino acid residues and capable of binding heparin.

Further, in addition to the first partial amino acid sequence: R—P—R—X—K—Q—G, a second partial amino acid sequence derived from the heparin binding site in V amm in:

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 4 to 6 amino acid residues via a linker sequence, may be used. it can.

In particular, an amino acid sequence derived from a heparin binding site in Vammin: an amino acid sequence consisting of RPRRKQGEPDGPKEKPR, or at least one amino acid substitution in the sequence, and the first partial amino acid sequence And a peptide having a heparin-binding ability, which contains a modified amino acid sequence retaining

In addition, the present invention as an invention of a method for utilizing a peptide having heparin binding ability to according to the present invention described above, also provides a use invention as inhibitors for the binding of heparin VEGF-A i ₆₅ Metropolitan , Namely, inhibitors for the binding of heparin present invention to such VEGF-A i ₆₅ Metropolitan,

A competitive inhibitor of VEGF-A ₁₆₅ binding to heparin, wherein the competitive inhibitory active product is the peptide having heparin binding ability according to the present invention described above. - a INHIBITOR on the binding of heparin a ₁₆₅ Prefecture.

Inhibitors on the binding of heparin according to the present invention such VEGF-A i ₆₅ Prefecture, for example, in preparing the form of injection solutions,

A composition comprising an effective amount of the above-mentioned peptide having heparin-binding ability according to the present invention dissolved in a pharmaceutically acceptable liquid carrier suitable for intravenous administration of a subject to be administered. it can.

Alternatively, when preparing a dosage form for eye drops,

An effective amount of the above-mentioned peptide having heparin binding ability according to the present invention is dissolved in a pharmaceutically acceptable liquid carrier suitable for application to the eyeball or orbit of the subject to be administered. A composition comprising: The novel peptide having heparin binding ability 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 _165, or v is induced by binding to amm in the KDR, strong hypotensive activity of nitric oxide (NO) dependent Ya It has the action of competitively inhibiting the binding between VEGF-A ₁₆₅ or Vamin and heparin on the cell surface, which is necessary for exerting the angiogenesis promoting action. As a result, for example, solid tumors增殖and metastasis, diabetic retinopathy, retinopathy of prematurity, psoriasis, etc., causes a variety of diseases, for the purpose of suppression of angiogenesis promotion caused by VEGF-A ₁₆₅ treatment For the use, the novel peptide having the ability to bind heparin according to the present invention can be used. At this time, 7-20 amino acid residues containing a large number of basic amino acids Because it is a peptide, it does not become an antigenic peptide presented by the MHC and does not show immunogenicity, so that there is no reduction in the therapeutic effect due to antibody creation associated with repeated administration.

Brief Description of Drawings

Figure 1 shows the results of the evaluation of the heparin-binding ability of the six synthetic peptides of peptide :! to peptide 6, using 10 mL of 50 mM Tris—HC 1 pH8 After dissolving in 0, the buffer was applied to a Hi Trap He parin HP column (column volume: 10 mL, Amhersh Biosciences; The elution was performed with a linear gradient up to 1.0 M NaCl, and the elution conditions (NaCl concentration) were measured.

FIG. 2 illustrates the basis of the design of a heparin-binding peptide according to the present invention,

A in Fig. 2, V a mm in the predicted from peptide chain amino acid sequence Arain result of human VEGF- A _65, V a mm in monomer intrachain di Surufui de between the chains of the dimer The binding site and the cystein nut 'motif are displayed, and the region involved in heparin binding in human VEGF-A ₁₆₅ is indicated by a dotted line,

B in FIG. 2 shows the amino acid sequence of the C-terminal part of V amm in (underlined in A) and the amino acid sequences of peptid 1 to 3 designed based on it.

3, the growth promoting effects of vascular endothelial cells by VEGF-A ₁₆₅ of peptide 1, and shows the inhibitory capacity evaluation results of proliferation I AshiSusumu action of vascular endothelial cells by V a mm in, in low serum culture conditions, VEGF- for a ₁₆₅ or V amm in promoting growth of © shea aortic endothelial cells by hydrogenation mosquito卩of (each final concentration 1 nM), the inhibition by peptide 1 to codoped, commentary by 6 days the number of cells after culturing And valence, white: control, black: VEGF-A _165, or added only V a mm in (positive control), light gray: 3 0 addition of peptide 1 (final concentration), dark gray: 1 0 0 (final concentration) The results obtained with the addition of peptide 1 are shown in comparison.

Figure 4 is a hypotensive action by VEGF-A ₆₅ of peptide 1, shows the inhibitory capacity evaluation results of antihypertensive effect of V a mni in the list,

Figure 4 (A): Top: time course of rat carotid artery pressure after intravenous injection of V amm in (dose 0.1 g / g) (marked with 丄), lower: peptide 1 (dose 3 gZg) Time course of rat carotid artery pressure after administration (V mark), U mark after intravenous injection of V amm in (dose 0.1 μgZg),

FIG. 4 (B), upper: VEGF-A ₁₆₅ after intravenous injection of (a dose 0. 1 mu gg) Time course of rat carotid pulse pressure (1 mark), medium: peptide 1 (dose 3 mu g / g) after prior administration of (▼ mark), VEGF-a ₁₆₅ (dose 0. l GZG) of rat carotid pulse pressure after venous injection (丄印) of temporal transition, below: peptide 1 (dose 3 0 μ g / g ) mark after a prior administration) shows versus time transition of the rat carotid pulse pressure of VEGF-a ₁₆₅ (intravenously after U indicia dose 0. 1 μ g, g)) .

Figure 5 shows the results of comparative evaluation of their ability to inhibit bloody hypotensive action of VEGF-A ₆₅ of the three kinds peptide peptide. 1 to 3, in the drawing,

A: VEGF—A ₁₆₅ (dose 0.1 μg / g) alone (U mark) (positive control),

B: After pre-administration of peptide1 (dose of 30 g / g) (T mark), VEG

FA ₁₆₅ (dose 0. l ^ ug / g) was given U mark),

C: After pre-administration of peptide2 (dose of 30 μg / g) (V mark), VEG

F- upon administration A ₁₆₅ (dose 0. 1 μ g / g) (丄印)

D: After pre-administration of peptide3 (dose of 30 g / g) (T mark), VEG

FA ₁₆₅ (dose 0.

The time course of the rat carotid artery pressure at the time of administration of U (marked with U) is shown in comparison. Figure 6 shows the amino acid sequence Arain results peptidyl de chain bicycloalkyl venom-derived VEGF-like protein and human VEGF-A ₁₆₅ to. In VEGF-like proteins derived from snake venom, matching amino acid residues are shaded, cysteine residues are shown in white nuclei, and intra- and inter-chain disulfide bonds are indicated. The portion of the loop within the human VEGF-A ₁₆₅ chain that forms is indicated by a horizontal line. Furthermore, heparin-binding site to the human VEGF-A ₁₆₅ is given, is surrounded by a dotted line. In human VEG F- A _165, a key residue for KGR receptor binding, in Hatashirushi, F lt - 1 residues important for binding, bi venom-derived VEGF to mouth mark, The amino acid substitution site contributing to the decrease in the binding to F1t-1 in the T-like protein is indicated by a T mark.

FV iperaaspisaspis 5fe hy potensive factor; I CPP: Vipera 1 ebetina snake venom derived increasingcapi 11 aryperme abi 1 ityprotein; VEGF 165: human vascular endothelial growth factor human VEG FA ₁₆₅ (Gen Bank registration number, AAM03108) Best mode for carrying out the invention

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

Prior to the present invention, the present inventors

\ 1 peraa mm oaytesa mm odytes hemitoxicity ・ Isolated VEGF-like protein: Purified from snake venom of Vammin and boiarusse 1 1 irusse 11 1 ・ Isolated VEGF-like protein: VR-1 is composed of a homodimer in which two peptide chains each consisting of 110 amino acids are linked by interchain disulfide bonds, and two peptide chains each consisting of 109 amino acids, In addition to being homodimers linked by disulfide bonds, in addition, these v ammin and VR-1 have the ability to bind to vascular endothelial growth factor receptor type 2 (VEGF receptor 2; KDR), Endothelial growth factor receptor type 1 (VEGF receptor 1; F1t-1), vascular endothelium We elucidated that it has characteristics that show no binding to growth factor receptor type 3 (VEGF receptor 3; Fit-4) and neuropilin-1 (neur 0 pi 1 in-1). Specifically, the primary structure (amino acid sequence) of a peptide chain consisting of 110 amino acids, which constitutes V ammin, is as follows: Sequence 1: Sequence 1: From 110 amino acids of V ammin Primary structure of the peptide chain

EVRP F LEVHE RS ACQARETL VP I LQEYPDE I SD I FRP S CV AVLRCSGCCT DE S LKCTP VG KHTVDLQ IMR VNPRTQS S KM E VMKFTEHTA CECRPRRKQG EPDGPKEKPR

In addition, the fire structure (amino acid sequence) of a peptide chain consisting of 109 amino acids that constitutes VR-l is represented by the following sequence 2:

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

EVRP F LDVYQ RS ACQTRETL VS I LQEHPDE I SD I FRP S CV AVLRCSGCCT DE SMKCTPVG KHTAD I Q I MR MNPRTHS S KM EVMKFMEHTA CECRPRWKQG E PEGPKE PR

It is.

In addition, the primary structure of a peptide chain consisting of VEGF-like protein isolated from the snake venom of the eye U i5 pe _raa mm odytesa mm odytes: V amm in 110 amino acids Primary structure of HF, isolated from eperaaspisaspis snake toxin, a homodimer in which two peptide chains consisting of amino acids are linked by interchain disulfide bonds.

3:

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

EVRPFLEVHE RSACQARETL V S_ I LQEYPDE

I SD I FRP S CV AVLRCSGCCT DE S LKCTP VG

KHTVDLQ IMR VNPRTQS S KM E VMKFTEHTA CECRPRRKQG EPDGPKEKPR

Alternatively, the primary structure of I CPP isolated from the snake venom of V i p e a la e e b e tina, sequence 4 below:

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

EVRP F PDVHE RS ACQARETL V S_ I LQE YPDE

I SD I FRPSCV AVLRCSGCCT DESLKCTPVG KHTVDMQ I MR VNPRTQ S S KM EVMKFTEHTA CECRPRRKQG EPDGPKEKPR

As shown in Fig. 6, they showed extremely high homology, and at the same time, they thoroughly examined the correlation between the physiological activities of these series of snake venom-derived VEGF-like proteins and their amino acid sequences. Among the amino acid sequences that match, amino acids that maintain high affinity with KDR, but do not have the ability to bind to F1t-1 or that contribute significantly or significantly contribute to the property One of the characteristics of the sequence is that between the presence of the five amino acids Alal3, Lys55, Arg74, Ser77, and Lys79 in sequence 1 and the high selectivity for KDR. Have already been found and reported (Yamazaki, Y. eta 1., J. Biol. Chem., Vol. 278 51985—51 988 ') (2003)).

On the other hand, is a homodimeric protein with a molecular weight 38. 2 kD a, vascular endothelial growth factor VEG.F- A ₁₆₅ of human also has a binding to heparin or to heparan sulfate Puroteodarikan to low in the presence of heparin concentration, by paying attention to that the affinity enhancement of against the KDR VEGF-a ₁₆₅ is Nasaru advances specific areas governing heparin binding to present in VEGF-a ₁₆₅ Was. In the process, it was found that a C-terminal excision product obtained by excision of the C-terminal Arg ^ Arg ¹⁶⁵ region by trypsin digestion lost heparin binding. Furthermore, it was confirmed that the C-terminal excision product (V EGF 110) has a markedly reduced vascular endothelial growth promoting activity, for example, as compared to the vascular endothelial growth promoting activity originally exhibited by VEGF ^ _A165. EGF- Banre binding to KD R of A _165, the various physiological activities expressed exerted, that binding of heparin also is an essential process to be present in a subject cell surface the KDR is expressed There was found.

Indeed, VEGF-realm of the C-terminus of the Ar g ^ Ar g ¹⁶⁵ present in A _165, as shown in FIG. 2, have a three-dimensional structure formed by a disulfide bond within the chain within such regions When the three-dimensional structure is constructed, a plurality of basic amino acid residues contained therein interact with the sulfate or amide sulfate structure of the acidic substituent in heparin, and as a result, the bond with heparin is reduced. It is inferred that it has been achieved.

Also, as schematically shown below, the polysaccharide chain of heparin has structural units of L-iduronic acid, D-glucuronic acid, and D-dalcosamine, and sulfation is performed by almost all the amino groups of D-glucosamine. In addition, it is also present in some 6-hydroxy groups and some 2-peronic acid hydroxy groups. In cells, most are present in the form of proteoglycans attached on 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 in the sequence of constituent sugar units, and are involved in various physiological activities.

a-D-glucuronic acid] 3-L-iduronic acid-2-sulfate α-D-darcosamine

-V, 0- disulfate other hand, V a mm in the, from the comparison shown in FIG. 2, the C-terminal that exists in VEGF-A ₁₆₅ Ar

Heparin with a three-dimensional structure, such as the ^g165 region Despite the absence of a binding site, it also exhibits binding to heparin,

Elucidation that binding of heparin present on the surface of target cells expressing KDR is also an essential process in the expression of various physiological activities exerted by the binding of Vammin to KDR did. Furthermore, VR-1 also exhibits binding to heparin, and the KDR is also expressed in the expression of various physiological activities exerted by the binding of VR-1 to KDR. It has been clarified that binding to heparin present on the surface of target cells is also an essential process. These new facts, coupled with the heparin is found in V amm in or VR- 1 is that their C-terminal portion, i.e., C-terminal partial amino acid sequence of the ^{v amm in (A rg 94 ~A} rg 110) or in the inspired as being governed by the C-terminal partial amino acid sequence of ^{VR-1 (Ar g 94 ~Ar} g 109), actually, as shown in the examples below, or mow the C-terminal partial amino acid sequence The corresponding synthetic peptide peptide 1 was prepared, and it was verified that all of them were peptides showing binding ability to heparin. Therefore, V amm in or VR- 1 well, VEGF-like proteins HF from bi poison to others, even in the I CPP, in common, such C-terminal partial amino acid sequence (Ar g ⁹⁴ ~Ar g ¹¹⁰ ) Was also able to bind to heparin.

Subsequently, the heparin has been dominated by the C-terminal partial amino acid sequence of the ^{V amm in (A rg 9 '} 4 ~A rg 110) or VR-1 of the C-terminal partial amino acid sequence (Ar g ⁹⁴ ~Ar g ¹⁰⁹⁾ In order to identify the major partial region in the binding of, two types of partial fragment peptides of peptide 2 and peptide 3 shown in Fig. 2B were prepared and their heparin binding was evaluated. Peptide peptide 2 showed heparin binding property comparable to that of synthetic peptide peptide 1, but it was found that synthetic peptide peptide 3 lost heparin binding property.

Furthermore, included in the synthetic peptide peptide 2, parts partial amino acid sequence derived from va mm in: R- P- R- R- K- Q- G- E and, from VEGF-A ₁₆₅ The partial amino acid sequence remaining at the C-terminus in the C-terminal truncated product (VEGF110): When compared with R-P-K-K-D-R, both are amino acid sequences rich in basic amino acid residues. The partial amino acid sequence: R—P—K—K—D—R has a decisive difference that heparin binding is impaired.

That is, a partial amino acid sequence derived from V ammin: R—P—R—R—K—Q—G—E, the corresponding region of VR-1, and VEGF 110 that does not show heparin binding When the partial amino acid sequences remaining at the C-terminus are compared with each other,

V a mm inn: R— P— R— R— K— Q— G— E

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

6

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

Assumed partial array: One — — X— J £ — Q— G (X is any of R, W, H, K)

When the above-mentioned assumed partial sequence (first partial amino acid sequence) is retained, it is determined that the protein exhibits at least heparin-binding property comparable to that of the synthetic peptide peptide2 at the same level. In particular, the four underlined amino acids are basic amino acids 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. It is determined that it plays a role in arranging residues. Therefore, in the peptide having heparin binding ability according to the present invention,

The first partial amino acid sequence: R—P—R—X—K—Q—G (where X is any force of R, W, H, and K), and the total number of amino acid residues is: A peptide consisting of 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, such as 8 amino acid residues of angiotensin II or 21 amino acid residues of insulin A chain. It corresponds to an appropriate peptide chain length for maintaining water solubility while conducting.

As described above, peptides having heparin binding ability according to the present invention include:

First partial amino acid sequence from the heparin binding site in V ammin: RPRXKQG (X is any of R, W, H, K)

A _N -RPRXKQGA _C

Consisting, although peptides have a heparin binding ability to have the amino acid sequence of 7 to 20 amino acid residues are included, for example,., As illustrated in the examples below to synthetic peptide peptide 4, 5, A _N Can be a form in which no amino acid is present. Generally, as an amino acid for protection at the N-terminal, for example, Gly

1,

7

It is desirable to add a small-volume amino acid such as A1a to form a form having a minimum number of amino acids of 8 or more.

In addition, the synthetic peptide peptide 1 also has a partial sequence of K-Ε-Κ-Ρ-R at the C-terminus, and has a slightly better parin binding property than the synthetic peptide peptide 2. It is desirable to also have a second partial amino acid sequence: Ji-E-K-P-R. 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, and as a linker sequence, about 5 amino acid residues, and thus 4 to 6 amino acid residues To provide a linker sequence, more preferably a linker sequence from 5 amino acid residues,

R- P- R- X- K one Q-G-X _{_{_{"X 2 - X 3 - X 4}}} - X 5 - K- E- K- P one R

(X is one of R, W, H, and K, and one — X ₂ — X ₃ — X ₄ — X ₅ — is a linker sequence)

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

An amino acid sequence consisting of RPRRKQGEPDGPKEKPR, or a modified amino acid sequence having at least one amino acid substitution in the sequence and retaining the first partial amino acid sequence. Alternatively, a peptide having heparin binding ability can be used. As a preferred example of this type of modified amino acid sequence,

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 at the C terminal,

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

R

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

And the like.

8

In addition, in place of the second partial amino acid sequence: Κ-Ε-Κ-Ρ-R, the first partial amino acid sequence: R—P—R—X—K—Q—G (X is R, W, Η, Κ) are linked by a linker sequence

R— Ρ— R— X— Κ— Q— G— Χ “Χ ₂ — Χ ₃ — Χ ₄ — Χ ₅ — R— Ρ— R— X— KQG

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. Alternatively, a portion of A _c, 5 amino acid residues at the C-terminus present in _{^{VEGF- A 165 (As p 161 ~A}} rg 165): D- K- P- R- in correspondence with R, wherein K once K-P-R-R was converted to K once K-P-R-R,

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

R

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

May have an amino acid sequence such as

Further, as the first partial amino acid sequence portion, it is more preferable to use R-P-R-R-K-Q-G, wherein R is selected as X.

The above linker sequence, for example, a linker sequence consisting of 5 amino acid residues: 1 X -x ₂ -x ₃ -x _4- x ₅ —The role of the moiety is to

And the second partial amino acid sequence portion is linked, and the contribution of the weak interaction between the second partial amino acid sequence portion and heparin is added to the heparin-binding property where the first partial amino acid sequence portion is the main component. It is intended to do so. Thus, present in the body of the administration subject, proteases, by the action of peptidases, such linker one sequence: A _{_{X i- x 2 - x 3 -}} x 4 - x 5 - utilize what portion of the cutting is not performed can do. In addition, by using a linker sequence consisting of D-form amino acids instead of a linker sequence consisting of L-form amino acids, it is also possible to suppress digestion by proteases in the body. In addition, by using a peptide bond mimic structure used in various peptide reagents for such a linker sequence, it is possible to suppress digestion by in vivo proteases. However, even if the first partial amino acid sequence portion itself is not cleaved even if the first partial amino acid sequence portion itself is cleaved in the linker sequence, the first partial amino acid sequence partial fragment retaining heparin-binding property is removed. There is no particular problem because it produces by-products.

In general, in order to prevent peptide compounds from decomposing in vivo, it is effective to add or modify extra amino acids at the N-terminal and C-terminal. For example, the N-terminal amino group can be modified with various acyl groups, or the C-terminal carboxyl group can be amidated. Since the peptide having heparin binding ability according to the present invention can be produced by using a chemical synthesis method, for example, the solid-phase synthesis method can be used to convert a peptide chain from the C-terminal side. When the extension is performed, it is preferable from the viewpoint of synthesis that C-terminal Amidig fixed on the resin is used. Further, the linker one _{_{sequence: - - x 2 - x 3}} - x 4 - x 5 - moiety, etc., in place of the naturally occurring amino acids can be made comprising amino acids of human E. By using artificial amino acids, it is also possible to design the linker sequence to one that suppresses enzymatic cleavage of the peptide chain.

In addition, in the amino acid sequence derived from the heparin binding site in V ammin, an amino acid sequence corresponding to the linker sequence: 1 X—X ₂ —X ₃ —X ₄ —X ₅ — Is one E—P—D—G—P—, which is included in one D—G— (A sp -G 1 y) sequence, in some cases, on the side chain of Asp. Between the carboxy group of-(COOH) and the imino nitrogen at the N-terminus of G1y (-N-), the formation of a succinimide structure or rearrangement to the / 3 position is known. I have. Formation of succinimide structure

To:

In order to suppress the structural change accompanying the reaction in this kind of peptide molecule, for example, the sequence of one DG- (Asp-G1y) is replaced with one D—A— (Asp—A1a) or one Although structurally similar, such as NG- (AsnGly), amino acid substitutions that suppress unwanted intramolecular reactions can also be performed.

The peptide having heparin binding ability according to the present invention is a peptide having a total number of amino acid residues of 7 to 20 amino acid residues, and contains a large number of hydrophilic amino acids, particularly, basic amino acids. Therefore, it is possible to use an aqueous solution in a wide concentration range. When applied to pharmaceutical applications, aqueous media such as various peptides It is possible to prepare a composition using an aqueous medium used for preparing an injection solution containing lumon, or an aqueous medium used for preparing a liquid preparation containing an orally administrable peptide bioactive substance as a carrier. preferable. 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 produced. In general, the water solubility tends to decrease as the number of amino acid residues constituting the peptide increases, but the peptide having heparin binding ability according to the present invention has the first partial amino acid sequence portion, Furthermore, since the second partial amino acid sequence portion also has a high ratio of amino acids with high hydrophilicity, its solubility in water is at least more than 2 Omg / mL. Peptide having heparin binding activity according to the novel to the present invention, VEGF-A i required for exhibiting binding by angiogenesis promoting action induced to KDR of _65, VEGF - A ₁₆₅ and the vascular endothelial cell surface the bond between the heparin to have a work to competitively inhibit, available as an anti-VEGF-a ₁₆₅ agents, for example, solid month heavy瘍of增-growth and metastasis, diabetic retinopathy, retinopathy of prematurity, such as psoriasis, which causes various diseases, VEGF-therapeutic agents aimed at suppression of pro-angiogenic caused by a _165, on the application of the prophylactic agent is applicable. In therapeutic applications aimed at suppressing the promotion of angiogenesis caused by these endogenous vascular endothelial growth factors V EGF-A ₁₆₅ , the peptide having heparin binding ability according to the present invention is directly administered into the bloodstream. However, it is suitable for administration in the form of being supplied to the surface of vascular endothelial cells at the site of action, and it is usually preferable to prepare a pharmaceutical composition in a dosage form suitable for intravenous administration. Specifically, dosage forms used for intravenous administration of peptide preparations having various physiological activities are, for example, dosage forms such as intravenous injections and infusions. It is determined appropriately according to In addition, the desired physiological activity is exhibited in the estimated total blood volume in consideration of the situation of the administration target (patient), the severity of the symptoms, sex, age, weight, and other health conditions. It will be blood concentration As described above, it is preferable to set an administration dose. When the peptide having heparin-binding ability according to the present invention is used in the form of an intravenous injection, it is generally possible to administer the peptide in multiple doses, but the total dose is It is desirable to set it in the range of 1 to 10 OmgZkg body weight, preferably in the range of 3 to 5 OmgZkg body weight. For example, assuming that the dose immediately after administration is evenly dispersed in consideration of the total blood volume, the average blood concentration is in the range of 0.1; uM to l0, preferably 1 μΜ '. It is desirable to set the total dose within the range of ~ 3 μΜ.

Further, the organ site to be applied is specified, diabetic retinopathy, against the retinopathy of prematurity, therapeutic agents aimed at suppression of pro-angiogenic due to VEGF_A _165, as a pre Boyaku, eye drops Can be prepared. For the subject to be administered, an ophthalmic solution prepared by dissolving an effective amount of a peptide capable of binding heparin 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 liquid in the range of 0.1 μΜ to: L 0 μΜ, preferably in the range of 1 ^ Μ to 3 μΜ. Example

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

(Peptide synthesis and purification)

Vascular endothelial growth factor VEGF-like protein from snake venom; C-terminal portion of Vammin, which is assumed to be involved in heparin binding by referring to the amino acid sequence of Vamin and VR-1 based on the amino acid sequence (Ar g ⁹⁴ ~Ar g ¹¹⁰⁾ or VR- 1 of the C-terminal partial amino acid sequence ^{^{(Ar g 94 ~Ar g 109)}} , designed six peptides peptide. 1 to peptide 6 shown in table 1 did. Based on the designed amino acid sequence, each peptide was prepared by a solid phase synthesis method by the F-moc method using Peptide Synthesizer (Applied Biosystems, Modenole 431 A). According to a conventional method, the synthesized peptide was deprotected and separated and eluted from the base resin by the F-moc cleavage method, and the recovered crude peptide was lyophilized.

Purification of the crude peptide was carried out using AKTAxplorer10S (AmershamBiosciencs) and an HP LC system (JASCO). The peptide was detected in the column elution fraction by measuring absorbance at 240 nm.

4 Omg of the obtained crude peptide was dissolved in 1 OmL of 5 OmM Tris—HC 1 pH 8.0, and then a Hi Trap He parin HP column (column capacity: 10 mL _s Am ersh am Biosciences) did. Elution was carried out from the henolin affinity column at a flow rate of 1 mLZmin with a linear gradient up to 1.0 MNaCl at 5 column volumes.

The peptide fraction showing the binding property to heparin was pooled and collected, and then applied to Cosmo si 15 C18AR-300 (2 οπι Χ 25 οιη (L)), and acetonitrile 0% to 30% linearity was applied. Elution was performed with a gradient, and a 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)

Confirmation of the amino acid sequence of the purified peptide was performed using Protein 'sequencer (Applied Biosternsmodeles 473A, 477 Shimadzu model PPSQ-21A).

(Evaluation of heparin binding) The purified peptide (100 g) was dissolved in 1 OmL of 50 mM Tris-HC1 pH 8.0, and then applied to a Hi Trap He parin HP column (column volume 1 OmL, Amhersh Biosciences). . Thereafter, the buffer solution was flowed through each column at a flow rate of ImL / min, followed by elution with a linear gradient up to 1.0 M NaCl and 1.0 M NaCl, and elution conditions (NaCl concentration) were determined. It was measured.

Figure 1 shows the elution peak position (thick line) of the peptide fraction showing binding properties to heparin for each peptide. Table 1 summarizes the concentration of NaCl dissolved at the peak maximum.

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 0.41

Peptide 2 G R P R R K Q G E 0.38

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 0.40

18 residues

Peptide 5 R P R W K Q G E P D G P K E-P R G 0.26

17 residues ―

Peptide 6 R P R R K Q G E P D G P K E K P R R 0.45

Similarly, for the vascular endothelial growth factor VEGF-like protein derived from snake venom; v ammin and VR-1, similarly, the peak maximum of the protein fraction eluted from the heparin 'abundity column. Table 2 shows the results of the measurement of the eluted NaC1 concentration in. Table 2 Heparin binding properties of va 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 0.30

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

(Evaluation of the inhibitory ability of VEGF-A ₁₆₅ for promoting the growth of vascular endothelial cells in vitro)

Peptides with heparin binding ability to the above, to suppress the growth promoting effects of vascular endothelial cells by VEGF-A _165, was verified have your evaluation system of invitro to below. At the same time, it was also verified that the inhibitory effect of vammin on the proliferation of vascular endothelial cells was suppressed.

Cell suspensions of Escherichia coli aortic endothelial cells (BAECs) were seeded at a density of 5,000 cells / well in 96-well cell culture plates. After adhesion of cells in the well, 0.

The medium was replaced with a medium supplemented with 1% fetal serum and cultured for a total of 18 hours. At that time, the medium, the final concentration of 1 nM for VEGF-A ₁₆₅ or V amm in vascular endothelial growth factor protein, as well as peptide 1, respectively, were added at a predetermined final concentration. Then, after culturing was continued for 6 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).

The absorption coefficient A405-630 of color development in the WS WS-8 method was used as an indicator of the number of viable cells. Figure 3 shows the parallel evaluation on the same plate,

Left: vascular endothelial growth factor protein and peptidel-free supplemented gel (negative control: white column)

Center: In addition to V ammin, peptide 1 was added to a final concentration of 100 (dark gray column: right of center), 30 μΜ (light gray column: middle), 0 μΜ (uncoated pod: positive control; black column) : Center left)

Right: VEGF-in addition to Alpha _165, a peptide 1 final concentration of 100 (conc. Gray column: middle right), 30 / M (light gray column: middle), 0 μΜ (no addition: positive control; black column: center left)

3 shows the measurement results.

Comparing the results shown in FIG. 3, but is added by VEGF-Alpha ₁₆₅ and V a mm in which the growth promoting effects of vascular endothelial cells, in the presence of peptide 1, addition concentration-dependent manner that, The cell growth-promoting effect is suppressed. Accordingly, p, eptide 1 having a heparin binding ability to, by binding to heparin present in the B AEC cell surface, VEGF-A ₁₆₅ and V am min exerts growth-promoting effect of vascular endothelial cells required above, among the binding of heparin and bind to KDR, the results to competitively inhibit binding of the heparin, inhibit the growth promoting effects of vascular endothelial cells indicated by VEGF one a ₁₆₅ and V amm in Is determined to be

(inhibitory capacity evaluation of hypotensive action by VEGF-A ₁₆₅ in the in V ivo)

Peptide having heparin binding ability to the above, to suppress the hypotensive action by VEGF-A i _65, was examined in the evaluation system of the in V ivo to below. At the same time, the suppression of the blood pressure lowering effect of Vammin was also verified.

The measurement of blood pressure lowering ability by VEGF-A ₁₆₅ or Vammin was performed using Vos Wisratrat (n = 3 or 5,150 to 220 g in each group). After anesthesia with intraperitoneal injections for each individual ,, force Rubamin acid Echiruesuteru (1 _{g (kg),} and揷入a polyethylene tube filled with 25% M g SO ₄ the carotid artery, a pressure transducer (Model P 1 OEZ, (Becton Dickinson), and the carotid artery pressure was monitored.The carotid artery pressure measured by a pressure transducer was recorded by a recorder connected to an amplifier ( _model AP-621 Nippon Koden).

For each test subject, injection of physiological saline (600 μL) stabilized blood pressure. After confirming that they were present, a peptide having heparin binding ability (600 ^ L) and VEGF-A ₁₆₅ or Vammin (600 µL) were administered from the left femoral vein.

In FIG. 4A, upper: when only V amm in (dose 0.1 μg / g) was administered, lower: after pre-administration of peptide 1 (dose 3 / ig / g), va mm in (dose) 0. 1 μ gZg) definitive upon administration to show the amount of decrease in carotid pulse pressure, B in FIG. 4, upper: VEGF-a ₁₆₅ (dose 0. 1 μ gg) only when administered, medium: peptide after previously administered (dose 3 μ gZg), VEGF-a 165

(Dose 0.1 μg / g)

Lower: peptide 1 (dose 30 g / g) after previously administered, definitive when administered VEGF-A ₆₅ (dose 0. 1 μ g / g), shows a decrease in the carotid pulse pressure.

Comparing the results shown in FIG. 4, VEGF-A ₁₆₅ and V amm in to be administered although induced hypotensive, in the presence of peptide 1, the addition of its concentration-dependent manner, the antihypertensive Induced action is suppressed. Here, evaluated the VEGF-A ₁₆₅ and V a mm in the hypotensive activity shown is induced by binding to KDR V EG F protein, strong hypotensive of nitric oxide (NO) dependent are based on the active, therefore, peptide 1 having a heparin binding ability to, by binding to pair the heparin present on the cell surface expressing KDR, VEGF-a ₁₆₅ and V a mm in There needed to exert hypotensive action, among the binding of heparin and bind to KDR, the binding of heparin competitively inhibit result to, VEGF-a ₁₆₅ and under the blood pressure drop by V amm in It is determined that the action is suppressed. In the peptide having heparin-binding ability according to the present invention, in particular, VEGF- ₆₅ and V amm in only by the R—P—R—XKQG (X is any one of R, W, H, and K). Is necessary to exert blood pressure lowering effect, K Of binding of heparin and bind to DR, the results to competitively inhibit binding of heparin, VEGF-A ₁₆₅ and verification that the suppressing effect of a blood pressure lowering effect is achieved by the V amm in I also went.

Specifically, in addition to peptide 1, the peptide 2, and peptide 3 corresponding to the C-terminal region corresponding to the N-terminal region of the peptide 1, for the presence or absence of the effect of suppressing the antihypertensive effect of VEGF-A i ₆₅ The evaluation was performed in parallel with the above-mentioned in vivo evaluation system.

In FIG. 5, A: VEGF- A ₁₆₅ (dose 0. 1 | g Z g) only upon administration of (positive resistance control)

B: After administering peptid e1 (dose 30 g / g) in advance,

65 (dose of 0.1 g / g)

C: After pre-administration of peptide 2 (dose 30 _μg / g),

_{When 65} (dose 0.1 / ig / g) was administered,

D: peptide 3 (dose 30 μ g / g) after previously administered, definitive when administered VEGF-A ₆₅ (dose 0. 1 μ g / g), and the amount of decrease in carotid pulse pressure shows, respectively it .

Comparing the results shown in FIG. 5, with respect to lower blood pressure reducing action of VEGF-A ₁₆₅ to be administered are induced, peptide 1 and peptide 2 are administered in advance, the effect of suppressing the antihypertensive effect As shown, no inhibitory effect was observed for peptide 3. Therefore, peptide 1 and peptide 2 having heparin binding ability are required for VEGF_A ₁₆₅ to exert a blood pressure lowering effect by binding to heparin present on the cell surface expressing KDR. Do, of the binding between heparin and binding to KDR, a result that competitively inhibit the binding of heparin is determined to be suppressed hypotensive action by VEGF-a _165. On the other hand, peptide 3 does not show heparin binding ability, and thus it is judged that no inhibitory effect was observed.

In conclusion, in the peptide having heparin binding ability according to the present invention, in particular, RPRXKQG (is one of R, W, H, and K) is the site that controls heparin-binding ability. In addition, by using this part, KDR is expressed in vivo and which by binding with respect to heparin present on the cell surface and, VEGF-a ₁₆₅ have various physiological activities, necessary for exerting the effect, among binding of heparin and bind to KDR , the result of competitively inhibit the binding of heparin, VEGF-physiological activity by a _165, is judged when is suppressed activity. Industrial potential

The novel peptide having heparin binding ability 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. is, 7-20 is a peptide compound consisting of amino acid residues, associated with binding to KDR _VEGF-65, VEG F- binding between heparin a i ₆₅ and cell surface Ueno competitively have a function of inhibiting, as a result, VEGF-requires both binding of heparin binding to KDR a ₁₆₅ and the cell surface Ueno, suppresses exhibits angiogenesis promoting action peptide of Pharmaceuticals Available as

Claims

The scope of the claims

1. a first partial amino acid sequence derived from a heparin binding site in v amm ίn: at least R-P-R-X-K-Q-G (X is any of R, W, H, K);

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 sequence: R—P—R—X—K—Q—G,

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

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 via a linker sequence from 4 to 6 amino acid residues.

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

3. Amino acid sequence derived from the heparin binding site in V amm in:

An amino acid sequence consisting of R-P-R-R-K-Q-G-E-P-D-G-P-K-E-K-P-R, or a peptide having at least one amino acid substitution in the sequence and having 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. A first partial amino acid sequence derived from a heparin binding site in V amm.in: R-P-R-X-K-Q-G (X is any one of R, W, H, and K);

A _N -RPRXKQGA _C

(A _N, A _c is selected from each of the peptide chain from amino acid number 0 to 13, A _N, the total number amino acids of A _c is 13 or less)

Having an amino acid sequence of 7 to 20 amino acid residues

5. A competitive inhibitor of the binding of \ £ &?- ₁₆₅ to heparin, wherein the competitive inhibiting active ingredient is the heparin according to any one of claims 1 to 4. It is a peptide with binding ability

Inhibitors on the binding of heparin characterized that VEGF-A ₁₆₅ Metropolitan that.

6. A pharmaceutical composition that have a use as an inhibitor for the binding of heparin VEGF-A ₁₆₅ Prefecture,

The inhibitory active ingredient is a peptide having 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: