WO2005077970A1 - Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci - Google Patents

Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci Download PDF

Info

Publication number
WO2005077970A1
WO2005077970A1 PCT/JP2004/001494 JP2004001494W WO2005077970A1 WO 2005077970 A1 WO2005077970 A1 WO 2005077970A1 JP 2004001494 W JP2004001494 W JP 2004001494W WO 2005077970 A1 WO2005077970 A1 WO 2005077970A1
Authority
WO
WIPO (PCT)
Prior art keywords
heparin
amino acid
peptide
vegf
binding
Prior art date
Application number
PCT/JP2004/001494
Other languages
English (en)
Japanese (ja)
Inventor
Takashi Morita
Yasuo Yamazaki
Original Assignee
Nec Soft, Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nec Soft, Ltd. filed Critical Nec Soft, Ltd.
Priority to PCT/JP2004/001494 priority Critical patent/WO2005077970A1/fr
Priority to PCT/JP2004/008109 priority patent/WO2005077971A1/fr
Priority to JP2005517889A priority patent/JPWO2005077971A1/ja
Publication of WO2005077970A1 publication Critical patent/WO2005077970A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/06—Antipsoriatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • VEGF vascular endothelial growth factor
  • 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
  • VEGF vascular endothelial growth factor
  • 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).
  • VEGF-A Vascular endothelial growth factor 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.
  • VEGF-A 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)).
  • PI GF placental growth factor
  • 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.
  • KDR kinaseinserted oma in- containing receptor
  • VEGF-A 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.
  • 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).
  • 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).
  • KDR and VEGF-A 165 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 .
  • KDR and VEGF- interaction with A 165 has the function of P ⁇ 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.
  • 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 ⁇ raaspisaspis It was isolated as a VEGF-like molecule with homology to VEGF (hypotensive factor: HF) ( Komori, Y. et al., ⁇ icon, 28, 359-369 (1990); K omori, Y. et al., Biochemistry, 38, 1 1 796-1 1803 (1990)).
  • HF hypotensive factor
  • VEGF-like molecules snakevenom VEGF and increasing capillary perme ability protein (I CPP)
  • I CPP capillary perme ability protein
  • KDR and VEGF-A 165 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.
  • 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.
  • VEGF-A 165 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the peptides having heparin binding ability according to the present invention include:
  • 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.
  • 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.
  • 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
  • a peptide having a heparin-binding ability which contains a modified amino acid sequence retaining
  • 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.
  • the competitive inhibitory active product is the peptide having heparin binding ability according to the present invention described above.
  • 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.
  • 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.
  • VEGF-a 165 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.
  • NO nitric oxide
  • the novel peptide having the ability to bind heparin according to the present invention can be used.
  • 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.
  • 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
  • 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,
  • 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.
  • 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 Top: time course of rat carotid artery pressure after intravenous injection of V amm in (dose 0.1 g / g) (marked with ⁇ ), 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 ⁇ gZg),
  • 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,
  • VEGF 165 (dose 0.1 ⁇ g / g) alone (U mark) (positive control),
  • VEG After pre-administration of peptide2 (dose of 30 ⁇ g / g) (V mark), VEG
  • FA 165 dose 0.
  • the time course of the rat carotid artery pressure at the time of administration of 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.
  • 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.
  • heparin-binding site to the human VEGF-A 165 is given, is surrounded by a dotted line.
  • 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
  • Isolated VEGF-like protein Purified from snake venom of Vammin and boiarusse 1 1 irusse 11 1 ⁇
  • Sequence 2 Primary structure of peptide chain consisting of 109 amino acids of VR-1
  • 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
  • Sequence 3 Primary structure of peptide chain consisting of 110 amino acids of HF
  • Sequence 4 Primary structure of peptide chain consisting of 110 amino acids of I CPP
  • 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.
  • 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.
  • VEGF-realm of the C-terminus of the Ar g ⁇ Ar g 165 present in A 165 have a three-dimensional structure formed by a disulfide bond within the chain within such regions
  • 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.
  • 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.
  • 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,
  • 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)
  • 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.
  • VEGF 110 R-P-K-K-D-R
  • the protein exhibits at least heparin-binding property comparable to that of the synthetic peptide peptide2 at the same level.
  • 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.
  • peptides having heparin binding ability include:
  • 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.
  • peptides have a heparin binding ability to have the amino acid sequence of 7 to 20 amino acid residues are included, for example,.
  • a N Can be a form in which no amino acid is present.
  • amino acid for protection at the N-terminal for example, Gly
  • the synthetic peptide peptide 1 also has a partial sequence of K- ⁇ - ⁇ - ⁇ -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,
  • 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.
  • a peptide having heparin binding ability can be used.
  • this type of modified amino acid sequence is a preferred example of this type of modified amino acid sequence.
  • R is added at the C terminal
  • the first partial amino acid sequence: R—P—R—X—K—Q—G (X is R, W, ⁇ , ⁇ ) are linked by a linker sequence
  • 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.
  • 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-R-K-Q-G wherein R is selected as X.
  • 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
  • the N-terminal amino group can be modified with various acyl groups, or the C-terminal carboxyl group can be amidated.
  • 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.
  • the extension it is preferable from the viewpoint of synthesis that C-terminal Amidig fixed on the resin is used.
  • 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.
  • linker sequence it is also possible to design the linker sequence to one that suppresses enzymatic cleavage of the peptide chain.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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 ⁇ of ⁇ -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.
  • an anti-VEGF-a 165 agents for example, solid month heavy ⁇ of ⁇ -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 prophy
  • the peptide having heparin binding ability according to the present invention is directly administered into the bloodstream.
  • 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.
  • dosage forms used for intravenous administration of peptide preparations having various physiological activities are, for example, dosage forms such as intravenous injections and infusions.
  • 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.
  • 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.
  • the average blood concentration is in the range of 0.1; uM to l0, preferably 1 ⁇ '. It is desirable to set the total dose within the range of ⁇ 3 ⁇ .
  • 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.
  • 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.
  • it is desirable to set the concentration in the liquid in the range of 0.1 ⁇ to: L 0 ⁇ , preferably in the range of 1 ⁇ ⁇ to 3 ⁇ .
  • 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.
  • the peptide fraction showing the binding property to heparin was pooled and collected, and then applied to Cosmo si 15 C18AR-300 (2 ⁇ ⁇ 25 ⁇ (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.
  • 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.
  • BAECs Escherichia coli aortic endothelial cells
  • 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).
  • 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
  • FIG. 4A upper: when only V amm in (dose 0.1 ⁇ g / g) was administered, lower: after pre-administration of peptide 1 (dose 3 / ig / g), va mm in (dose) 0. 1 ⁇ 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 ⁇ gg) only when administered, medium: peptide after previously administered (dose 3 ⁇ gZg), VEGF-a 165
  • 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.
  • 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.
  • NO nitric oxide
  • VEGF- 65 and V amm in only by the R—P—R—XKQG (X is any one of R, W, H, and K).
  • X is any one of R, W, H, and K.
  • FIG. 5 A: VEGF- A 165 (dose 0. 1
  • D peptide 3 (dose 30 ⁇ g / g) after previously administered, definitive when administered VEGF-A 65 (dose 0. 1 ⁇ g / g), and the amount of decrease in carotid pulse pressure shows, respectively it .
  • 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.
  • RPRXKQG is one of R, W, H, and K
  • 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.
  • the novel peptide having heparin binding ability 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Diabetes (AREA)
  • Emergency Medicine (AREA)
  • Toxicology (AREA)
  • Vascular Medicine (AREA)
  • Oncology (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Endocrinology (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Ophthalmology & Optometry (AREA)
  • Dermatology (AREA)
  • Urology & Nephrology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Comme nouveau peptide se liant à l'héparine qui inhibe la liaison du VGEF-A165 à l'héparine ou au protéoglycane d'héparane-sulfate et par lequel on peut réguler différentes réactions physiologiques provoquées par des interactions entre le récepteur KDR et le VGEF-A165, on a l'intention de fournir un peptide se liant à l'héparine qui est conçu sur la base du site de liaison à l'héparine d'une protéine vammine ressemblant au VGEF provenant du venin d'un serpent, qui contient au moins une première séquence partielle d'acides aminés: R-P-R-X-K-Q-G (dans laquelle X représente l'un de R, W, H et K) trouvant son origine dans le site de liaison à l'héparine tel que décrit ci-dessus et qui est constitué de 7 à 20 résidus d'acides aminés.
PCT/JP2004/001494 2004-02-12 2004-02-12 Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci WO2005077970A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
PCT/JP2004/001494 WO2005077970A1 (fr) 2004-02-12 2004-02-12 Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci
PCT/JP2004/008109 WO2005077971A1 (fr) 2004-02-12 2004-06-10 Peptide a croissance de fibroblastes inedit concu a partir d'un site a croissance de fibroblastes de proteine a facteur de croissance endothelial vasculaire (vegf) de venin de serpent et son utilisation
JP2005517889A JPWO2005077971A1 (ja) 2004-02-12 2004-06-10 ヘビ毒由来の血管内皮増殖因子（ｖｅｇｆ）様タンパク質のヘパリン結合部位より設計された新規なヘパリン結合能を有するペプチドとその用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2004/001494 WO2005077970A1 (fr) 2004-02-12 2004-02-12 Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci

Publications (1)

Publication Number Publication Date
WO2005077970A1 true WO2005077970A1 (fr) 2005-08-25

Family

ID=34857515

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/JP2004/001494 WO2005077970A1 (fr) 2004-02-12 2004-02-12 Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci
PCT/JP2004/008109 WO2005077971A1 (fr) 2004-02-12 2004-06-10 Peptide a croissance de fibroblastes inedit concu a partir d'un site a croissance de fibroblastes de proteine a facteur de croissance endothelial vasculaire (vegf) de venin de serpent et son utilisation

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/JP2004/008109 WO2005077971A1 (fr) 2004-02-12 2004-06-10 Peptide a croissance de fibroblastes inedit concu a partir d'un site a croissance de fibroblastes de proteine a facteur de croissance endothelial vasculaire (vegf) de venin de serpent et son utilisation

Country Status (2)

Country Link
JP (1) JPWO2005077971A1 (fr)
WO (2) WO2005077970A1 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072829A2 (fr) * 2000-03-31 2001-10-04 Institut Pasteur Peptides inhibant l'angiogenese induite par le vegf (facteur de croissance endotheliale), polynucleotides codant pour ces peptides et leurs procedes d'utilisation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001072829A2 (fr) * 2000-03-31 2001-10-04 Institut Pasteur Peptides inhibant l'angiogenese induite par le vegf (facteur de croissance endotheliale), polynucleotides codant pour ces peptides et leurs procedes d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GASMI A. ET AL: "Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom", J. OF BIOLOGICAL CHEMISTRY, vol. 277, no. 33, 16 August 2002 (2002-08-16), pages 29992 - 29998, XP002979509 *
KOMORI Y. ET AL: "Vascular endothelial growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis aspis (Aspic viper)", BIOCHEMISTRY, vol. 38, no. 36, 1999, pages 11796 - 11803, XP002196145 *
YAMAZAKI Y. ET AL: "Snake venom vascular endothelial growth factors (VEGFs) exhibit potent activity through their specific recognition of KDR (VEGF receptor 2)", J. OF BIOLOGICAL CHEMISTRY, vol. 278, no. 52, 2003, pages 51985 - 51988, XP002979508 *

Also Published As

Publication number Publication date
WO2005077971A1 (fr) 2005-08-25
JPWO2005077971A1 (ja) 2008-01-10

Similar Documents

Publication Publication Date Title
JP3281370B2 (ja) 血小板凝集阻害剤
AU666853B2 (en) Platelet aggregation inhibitors having high specificity for GP IIbIIIa
US7696309B2 (en) Protease resistant mutants of stromal cell derived factor-1 in the repair of tissue damage
AU2004216680B2 (en) Venom-derived vascular endothelial growth factor-like protein having binding activity specific to vascular endothelial growth factor receptor type 2 and use thereof
KR20150121715A (ko) Ｃｓｆ１ 치료제
CN105339383A (zh) 新型α4β7肽二聚体拮抗剂
EP2412727A2 (fr) Fragments de peptides dérivés de la métallothionéine
KR20180012856A (ko) 면역반응의 조절을 위한 방법 및 폴리펩타이드
JP2004203890A (ja) マクロファージ炎症蛋白変種
CN102690352A (zh) 含有glp-1的融合蛋白、其药物组合物及用途
US8501680B2 (en) Antagonists against interaction of PF4 and RANTES
US10336813B2 (en) Fusion protein SLIT2D2-HSA and its use in treatment of sepsis
US20050181995A1 (en) Venom-derived vascular endothelial growth factor-like protein having binding activity specific to vascular endothelial growth factor receptor type 2 and use thereof
US5338725A (en) Anti-aggregatory agents for platelets
KR101909052B1 (ko) 인슐린의 a 사슬로부터 유래된 펩타이드 단편 및 이를 포함하는 당뇨병 또는 당뇨병성 창상의 예방 또는 치료용 약학 조성물
US5587457A (en) Neutrophil stimulating peptides
WO2005077970A1 (fr) Nouveau peptide se liant à l'héparine conçu à partir d'un site de liaison à l'héparine d'une protéine ressemblant à un facteur de croissance vasculaire endothélial (vegf) provenant du venin d'un serpent et utilisation de celui-ci
EP0333517A2 (fr) Méthode et agent pour inhiber la liaison des leucocytes humains polymorphonucléaires à l'endothélium et les compositions les contenant
US20120295851A1 (en) Peptides, compositions and uses thereof
CN113501862A (zh) 一种多肽及其在制备免疫调节药物中的应用
CN114106194B (zh) 一种用于治疗糖尿病和/或肥胖症的融合蛋白
JP2010120937A (ja) ヒトアポリポタンパク質ａ−ｉｉのアミノ酸断片又はその改変体を含有する炎症性疾患予防・治療剤
EP3333177A1 (fr) Composé multicible présentant une activité d'anticoagulation et antiplaquettaire, son procédé de préparation et son utilisation
JPH06172203A (ja) フィブロネクチンレセプター産生異常細胞抑制剤
AU676842B2 (en) Neutrophil stimulating peptides

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): BW GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
NENP Non-entry into the national phase

Ref country code: DE

WWW Wipo information: withdrawn in national office

Country of ref document: DE

122 Ep: pct application non-entry in european phase