JP2003230382A - Age-rage antagonist - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a substance antagonizing interaction of an advanced glycation endproduct (AGE) associated with diabetic complications with its receptor (FRAGE) and expected as a therapeutic and a prophylactic agent related to diabetes. <P>SOLUTION: A peptide having an amino acid sequence corresponding to that selected from the group consisting of a chemically synthesizable oligopeptide, e.g. Lys-Gly-Ala-Pro-Lys-Lys-Pro-Pro-Gln-Arg-Leu-Glu-Trp-Lys-Leu- Asn and Trp-Lys-Leu-Asn-Thr-Gly-Arg-Thr-Glu-Ala-Trp-Lys-Val-Leu-Ser-Pro-Gln- Gly or its derivative or a salt thereof is active as an AGE-RAGE antagonist. The peptide or its derivative or the salt thereof is useful as a therapeutic agent for various kinds of diseases which are diabetic complications and the development thereof. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an AGE-RAGE antagonist.

[0002]

2. Description of the Related Art In recent years, the number of diabetic patients has been increasing,
According to the Ministry of Health and Welfare statistics in 1998, the estimated affected population in Japan was 6.9 million, including the reserve army, 14 million. The prognosis and quality of life (QOL) of diabetic patients are not directly influenced by the primary lack of insulin action but by the vascular disorders or vascular complications in various parts of the body that occur secondarily as a result of hyperglycemia. Therefore, clarifying the etiology of diabetic complications and elucidating how to overcome them is a national research task that requires an urgent solution. As an environmental factor involved in the onset and progression of diabetic complications, late glycation reaction products (adva
nced glycation end products: AGE), and cell surface receptors (receptor for AGE: RAGE) that specifically recognize AGE play an important role as cell-side factors that respond to this. .

AGE is a non-enzymatic reaction between a reducing sugar such as glucose and an amino group of a protein to produce a Shiff base,
Amadori is a general term for irreversible cross-linking substances formed through transfer compounds. Recent studies have shown that the highly reactive glyceraldehyde and glycolaldehyde
It is said that AGE is also formed from various intermediate metabolites such as (glycolaldehyde), methylglyoxal, glyoxal, and 3-deoxyglucosone. It is also said that AGE formation is caused by oxidative decomposition of Amadori transfer compound, autoxidation of glucose, and lipid peroxidation. In vivo, blood plasma proteins such as erythrocyte membrane protein, albumin, lipoprotein, antithrombin, thrombomodulin, collagen, elastin,
There are various proteins and nucleic acids such as extracellular matrix constituent proteins such as proteoglycan.
It is believed that GE causes functional disorders. As AGEs, carboxymethyllysine, carboxyethyllysine, pentosidine, pyraline, crosulin, imidazolium salt, imidazolone, dehydroimidazolone, fluorolink, etc. have been identified. The existence of various substances is also considered.

It has been pointed out that AGE is involved in various complications associated with diabetes and aging, and cells such as receptors expressed on the cell surface of monosites / macrophages, neurons, smooth muscle cells, endothelial cells, etc. It is also known to bind to surface receptors. AGE is believed to interact with these receptors and exert various physiological and biological effects on living organisms and cells. AGE is, for example,
For endothelial cells it proliferates and enhances permeability and thrombus formation. In addition, in monosite / macrophage, etc., it promotes the release of cytokines, and further promotes the release of various factors involved in cell growth, migration, and matrix synthesis. Furthermore, it is also suspected to be involved in the inflammatory response in the vascular wall. Recently, the present inventor Yamamoto has revealed that there is molecular diversity in RAGE proteins expressed in human vascular cells, which is due to alternative splicing of RAGE gene transcripts, and one of the major molecular species is soluble RAGE. It was discovered that the protein is secreted extracellularly because it lacks the membrane-bound region, but it has the same extracellular domain as the mature membrane-bound protein and can thus trap AGE. In fact, when recombinant human soluble RAGE protein was purified and binding experiments with AGE ligands were performed, it was proved that it binds to various AGE fractions with high affinity.

[0005]

AGE interacts with its receptor and exerts various physiological and biological effects on living organisms and cells, resulting in various diseases and illnesses. Providing evidence of deteriorating activity, it provides substances that influence the interaction between AGE and RAGE, enabling the prevention, treatment and diagnosis of various diseases and illnesses. Is required. In particular, if a substance that inhibits, suppresses, or antagonizes the interaction between the above RAGE protein and AGE can be provided, it is expected to contribute to the prevention, treatment, and diagnosis of various diseases and illnesses. .

[0006]

[Means for Solving the Problems] From the comparison between RAGE splice variants having a V region domain and those not having a V region domain, the present inventors have found that the AGE binding region exists in the V region domain. As a result of further earnest research, they succeeded in identifying a ligand binding site, and arrived at the present invention.

The present invention relates to [1] Lys-Gly-Ala-Pro-Lys-Lys-Pro-Pro-Gln-Arg-Le.
u-Glu-Trp-Lys-Leu-Asn (SEQ ID NO: 7 in the sequence listing) and
Trp-Lys-Leu-Asn-Thr-Gly-Arg-Thr-Glu-Ala-Trp-Lys-Va
[2] Lys-Gly A peptide having an amino acid sequence corresponding to the amino acid sequence selected from the group consisting of l-Leu-Ser-Pro-Gln-Gly (SEQ ID NO: 8 in the Sequence Listing), a derivative thereof or a salt thereof; -Ala-Pro-Lys-Lys-Pro-Pro-Gln-Arg-Le
u-Glu-Trp-Lys-Leu-Asn (SEQ ID NO: 7 in Sequence Listing) or a derivative thereof, the compound according to [1] above; [3] Trp-Lys-Leu-Asn-Thr-Gly -Arg-Thr-Glu-Ala-Tr
p-Lys-Val-Leu-Ser-Pro-Gln-Gly (SEQ ID NO: in Sequence Listing:
[1] which is a peptide or derivative thereof which is 8)
Described compound; [4] A pharmaceutical composition comprising the compound described in [1] above as an active ingredient; [5] containing the compound described in [1] above as an active ingredient, and AGE and RAGE An AGE-RAGE antagonist characterized by inhibiting the interaction between

[6] A method for inhibiting AGE-RAGE interaction, which comprises contacting a sample containing RAGE and / or cells expressing RAGE with the AGE-RAGE antagonist described in [5] above; [7] (i ) AGE-containing test sample and RAGE or RAGE in the presence or absence of the peptide of SEQ ID NO: 7 or 8
An AGE-RAGE interaction analysis method, which comprises contacting with an expressing cell and comparing the presence and absence of the peptide, or (ii) (a) the peptide or sequence of SEQ ID NO: 7 In the presence or absence of the peptide of No. 8 and (b) in the presence or absence of AGE derived from glyceraldehyde or AGE derived from glycolaldehyde,
Contacting the test sample with RAGE or RAGE-expressing cells, and comparing between the presence and absence of the peptide and the presence or absence of glyceraldehyde-derived AGE or glycolaldehyde-derived AGE. [8] The inhibition method according to [6] above, wherein the RAGE-expressing cells are endothelial cells, vascular smooth muscle cells,
Nerve cells, macrophages, lymphocytes, retinal vascular cells,
The method according to the above [6] or [7], which is selected from the group consisting of retinal nerve cells, gingival-related cells, skin-related cells, glomerular cells, tubular cells and connective tissue cells. ;

[9] Due to the fact that the compound according to [1] above is contained as an active ingredient, and selected from the group consisting of an interaction between AGE and its receptor, a change in the expression level of RAGE, and a change in the AGE scavenging activity. [10] Diabetes complications, various diseases associated with aging, Alzheimer's disease, arteriosclerosis, the onset of diseases or illnesses due to glycation of in vivo proteins, and And / or for a pathological condition or symptom selected from the group consisting of progression and invasion or spread of tumor

[9] The medicine described above; and [11] Diabetes, diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic angiopathy, diabetic microangiopathy, renal glomerulosclerosis, hyperlipid atherosclerosis. , Neurocytotoxicity, Down syndrome, dementia associated with head trauma, amyotrophic lateral sclerosis, multiple sclerosis, amyloidosis, autoimmune disease,
For the treatment and / or prevention of a substance selected from the group consisting of inflammation, tumor, cancer, erectile dysfunction in men, wound healing, periodontal disease, neuropathy and neurodegeneration.

A pharmaceutical according to [9] or [10] is provided.

Other objects, features, excellence and aspects of the present invention will be apparent to those skilled in the art from the following description. However, it is understood that the description of the present specification, including the description below and the description of specific examples and the like, shows the preferred embodiments of the present invention and is provided only for the purpose of explanation. I want to. Various changes and / or alterations (or modifications) within the spirit and scope of the invention disclosed herein can be made by those of ordinary skill in the art based on knowledge from the following description and other portions of the specification. Would be readily apparent. All patent and literature references cited in this specification are cited for the purpose of illustration and are to be construed as part of this specification, the contents of which are included herein. is there. In this specification,
The term “and / or” means that there are both (1) a merged connection relationship and (2) a selective connection relationship, eg in the case of “treatment and / or prevention” (1) It is used to mean both treatment and prevention and (2) treatment or prevention. In other cases, the term “and / or” is similarly used to include both (1) merged connection and (2) selective connection.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, "soluble RAGE"
Is a receptor for advanc closely related to diabetic complications
It is a peptide related to ed glycation end products (RAGE), which is a splicing variant of RAGE and does not have a transmembrane region. The soluble RAGE
Examples thereof include those disclosed in Japanese Patent Application No. 2001-78409, Japanese Patent Application No. 2001-243114 and 2001 Diabetes Society (April 16, 2001). The soluble RAGE is a peptide consisting of 347 amino acid residues and has a characteristic sequence Gl on its C-terminal side.
u-Gly-Phe-Asp-Lys-Val-Arg-Glu-Ala-Glu-Asp-Ser-Pro-
It has Gln-His-Met and is a transmembrane RAGE (membrane RAGE,
It is characterized in that it lacks the transmembrane domain that is present in membrane-bound RAGE or full-length RAGE). It has been observed that the soluble RAGE has an advanced glycation end products (AGE) binding activity, or has an inhibitory or inhibitory activity on the interaction between AGE and its receptor. Typically, the soluble RAGE is a naturally-occurring peptide (endogenous peptide or endogenous peptide) existing in the living body, and has 16 amino acid residues at the C-terminal portion.
It is different from RAGE protein. As the typical soluble RAGE, a polypeptide produced by being encoded by the DNA of SEQ ID NO: 1 in the sequence listing, for example, the amino acid sequence of SEQ ID NO: 2 in the sequence listing or an amino acid sequence substantially equivalent thereto And a polypeptide having Further, in the typical soluble RAGE, at least 1 to 16 of the amino acid sequences Glu ^{332 to} Met ³⁴⁷ of SEQ ID NO: 2 in the sequence listing are used.
Has consecutive amino acid residues on its C-terminal side and AG
E binding activity, having at least ^{1 to 117} consecutive amino acid residues of the amino acid sequence Met ^{1 to} Val ¹¹⁷ of SEQ ID NO: 2 in the sequence listing on the N-terminal side and having AGE binding activity Alternatively, those having these characteristics and having at least 60% homology with the amino acid sequence Tyr ^{118 to} Gly ³³¹ of SEQ ID NO: 2 in the sequence listing can be mentioned. Soluble RAGE includes human, chimpanzee, monkey, mouse, rat, cow, pig, goat, sheep, dog, cat,
Those derived from mammals such as rabbits may also be included.
As used herein, the term “RAGE” may include full-length RAGE, the soluble RAGE, and other splicing variants of RAGE having AGE binding activity.

In another embodiment of the present invention, most of the full-length RAGE or soluble RAGE, which lacks AGE binding activity, is used.
A polypeptide having an amino acid sequence contained in is provided. Examples of the RAGE analog lacking AGE binding activity include N-terminal deletion type RAGE (lacking the amino acid residues 1-101 present in the full-length type and the C-terminal deletion type). RAGE-encoding nucleic acids used to produce such polypeptides include single-stranded DNA, double-stranded DNA, RNA, and DNA: RN.
It may be a nucleic acid such as A hybrid or synthetic DNA, and may be any of human genomic DNA, human genomic DNA library, cDNA complementary to mRNA derived from human tissue / cell, and synthetic DNA. The base sequence of the nucleic acid encoding RAGE may be modified (eg, added, removed, substituted, etc.), and such modified products may be included. Further, as described below, the nucleic acid may encode the peptide designed in the present invention or a part thereof, and preferred examples include DNA. In addition, the nucleic acid encodes a peptide having a biological activity substantially equivalent to that designed with attention paid to AGE binding activity, interaction between AGE and its receptor, or equivalent antigenicity. Any substance may be used as long as it contains a nucleotide sequence having the same effect. Here, the “equal effect base sequence” means, for example, 5 or more consecutive base sequences, preferably 10 or more base sequences of the base sequence under stringent conditions.
More preferably 15 or more nucleotide sequences, even more preferably
Examples include those that hybridize with 20 or more base sequences and encode an amino acid sequence substantially equivalent to the peptide.

In the present invention, it is possible to utilize gene recombination technology to isolate and sequence a predetermined nucleic acid, produce a recombinant, and obtain a predetermined peptide. Gene recombination techniques are described, for example, in J. Sambrook, EF Fritsch & T. M.
aniatis, "Molecular Cloning: A Laboratory Manual
(2nd edition) ", Cold Spring Harbor Laboratory Pres
s, Cold Spring Harbor, New York (1989); DM Glov
er et al. ed., "DNA Cloning", 2nd ed., Vol. 1 to
4, (The Practical Approach Series), IRL Press, Oxf
ord University Press (1995); “Biological Chemistry Experiment Course 1, Gene Research Method II”, edited by The Biochemical Society of Japan, Tokyo Kagaku Dojin (19
86); Japan Society for Biochemistry, "Shinsei Chemistry Laboratory 2, Nucleic Acid III"
(Recombinant DNA Technology) ", Tokyo Kagaku Dojin (1992); R. Wue
d., "Methods in Enzymology", Vol. 68 (Recombinant
DNA), Academic Press, New York (1980); R. Wu et a
l. ed., "Methods in Enzymology", Vol. 100 (Recombi
nantDNA, Part B) & 101 (Recombinant DNA, Part C),
Academic Press, New York (1983); R. Wu et al. Ed.,
"Methods in Enzymology", Vol. 153 (RecombinantDN
A, Part D), 154 (Recombinant DNA, Part E) & 155 (R
ecombinant DNA, PartF), Academic Press, New York
(1987); JH Miller ed., "Methods in Enzymology",
Vol. 204, Academic Press, New York (1991); R. Wu
et al. ed., "Methods in Enzymology", Vol. 218, Aca
The method described in demic Press, New York (1993) or the method described in the literature cited therein, or a method or a modification substantially similar thereto can be used (for the description in them, refer to it). Included in the disclosure of the present specification).

In the present specification, "polymerase chain.
Reaction (Polymerase Chain Reaction) "or" PC
“R 2” generally refers to methods such as those described in US Pat. No. 4,683,195 and the like, eg, for in vitro enzymatic amplification of a desired nucleotide sequence. In general, the PCR method involves repeating a cycle for primer extension synthesis using two oligonucleotide primers capable of preferentially hybridizing with a template nucleic acid.
Typically, the primer used in the PCR method can be a primer complementary to the nucleotide sequence to be amplified inside the template, and for example, the nucleotide sequence to be amplified and the complementary nucleotides at both ends thereof can be used. Those that are target or are adjacent to the nucleotide sequence to be amplified can be preferably used. The 5'-end primer contains at least a start codon,
Alternatively, it is preferable to select such that the amplification can be carried out including the start codon, and the primer at the 3′-end side is preferably selected so as to contain at least a stop codon or to be amplified so as to include the stop codon. The primer is preferably an oligonucleotide consisting of 5 or more bases, more preferably 10 or more bases, and still more preferably an oligonucleotide consisting of 18 to 25 bases. Preparation of a primer can be performed by a method known in the art, and is typically Angew. Chem. Int.
Ed. Engl., Vol. 28, p.716-734 (1989), known methods, for example, phosphophotoester method, phosphodiester method, phosphite method, phosphoramidite method, phosphite method. It can be chemically synthesized by a method such as a phonate method. It is generally known that the synthesis can be conveniently carried out on a modified solid support, for example, an automated synthesizer, such as model 381A DNA synthesizer (Applied Biosystem).
s) and the like. The oligonucleotide may contain one or more modified bases, for example, an unnatural base such as inosine or a tritylated base.

The PCR reaction can be carried out by a method known in the art or a method substantially similar thereto or a modified method, and for example, R. Saiki, et al., Science, 230: 13.
50, 1985; R. Saiki, et al., Science, 239: 487, 198.
8; HA Erliched., PCR Technology, Stockton Pres
s, 1989; DM Glover et al. ed., "DNA Cloning", 2
nd ed., Vol. 1, (The Practical Approach Series), I
RLPress, Oxford University Press (1995); MA In
nis et al. ed., "PCRProtocols: a guide to methods
and applications ", Academic Press, New York (199
0)); MJ McPherson, P. Quirke and GR Taylor
(Ed.), PCR: a practical approach, IRL Press, Oxfor
d (1991); MA Frohman et al., Proc. Natl. Acad.
Sci. USA, 85, 8998-9002 (1988) and the like, or modified or modified methods thereof. In addition, the PCR method can be performed using a commercially available kit suitable for the PCR method, or can be performed according to the protocol disclosed by the kit manufacturer or the kit vendor. The PCR product obtained is
It is usually subjected to 1-2% agarose gel electrophoresis and cut out from the gel as a unique band.
Extract the DNA using a commercially available extraction kit such as it (Bio 101). The extracted DNA is cleaved with an appropriate restriction enzyme, purified if necessary, and the 5 ′ end is further cleaved if necessary.
After phosphorylation by T4 polynucleotide kinase or the like, the plasmid is ligated with an appropriate plasmid vector such as pUC18 vector such as pUC18 to transform an appropriate competent cell. The nucleotide sequence of the cloned PCR product is analyzed. For cloning PCR products, for example, p-Direct (Clontech), pCR-Script ^TM SK
(+) (Stratagene), pGEM-T (Promega), pAmp ^TM (Gibco
-BRL) and other commercially available plasmid vectors can be used. For transformation of host cells, for example, a phage vector is used, calcium method, rubidium /
Calcium method, calcium / manganese method, TFB high efficiency method, FSB frozen competent cell method, rapid colony method, electroporation and the like known in the art or substantially similar methods can be used (D .
Hanahan, J. Mol. Biol., 166: 557, 1983). In order to isolate the desired DNA, reverse transcription PCR (polymer
ase chain reaction coupled reverse transcription;
RT-PCR), RACE (rapid amplificationof cDNA ends)
Can be applied. RACE is, for example, MA Inni
s et al.ed., "PCR Protocols" (MA Frohman, "a gu
ide to methods and applications "), pp.28-38, Acade
It can be performed according to the method described in mic Press, New York (1990) and the like.

A hybridization technique is used to identify a predetermined nucleic acid. In the hybridization, a predetermined DNA is transferred onto a membrane such as a nylon filter and, if necessary, a denaturation treatment, an immobilization treatment, After washing treatment and the like, the product transferred onto the membrane is reacted with a labeled probe DNA fragment modified as necessary in a hybridization buffer. The hybridization treatment is usually about 35 ° C to about 80 ° C.
C., more preferably about 50.degree. C. to about 65.degree. C., for about 15 minutes to about 36 hours, more preferably for about 1 hour to about 24 hours, but can be performed by selecting optimal conditions as appropriate. . For example, the hybridization treatment is performed at about 55 ° C. for about 18 hours. The hybridization buffer can be selected from those commonly used in the art and used, for example, Rapid hybridization buffer (Amersha
m) or the like can be used. Examples of the modification treatment of the transferred film include a method using an alkali denaturing solution,
After the treatment, it is preferable to treat with a neutralizing solution or a buffer solution. The immobilization treatment of the membrane is usually about 40 ° C to about 100 ° C, more preferably about 70 ° C to about 90 ° C for about 15 minutes to about 24 hours, more preferably about 1 hour to about 4 hours. It is carried out by baking, but it can be carried out by appropriately selecting preferable conditions. For example, the immobilization is performed by baking the filter at about 80 ° C for about 2 hours. As the washing treatment of the transferred film, a washing solution commonly used in this field, for example, 1M NaCl, 1 mM EDTA and 0.1% sodium dodecyl is used.
It can be performed by washing with 50 mM Tris-HC1 buffer containing sulfate (SDS), pH 8.0 and the like. Membranes such as nylon filters can be selected and used from those commonly used in the art, and examples thereof include nylon filters [Hybond-N, Amersham].

The alkali denaturing solution, the neutralizing solution, and the buffer solution can be selected from those commonly used in the art, and examples of the alkali denaturing solution include:
A solution containing 0.5M NaOH and 1.5M NaCl can be mentioned. Examples of the neutralizing solution include 1.5M NaCl.
0.5M Tris-HCl buffer solution, pH 8.0 and the like can be mentioned. Examples of the buffer solution include 2 × SSPE (0.36M NaCl,
20 mM NaH ₂ PO ₄ and 2 mM EDTA) and the like. In addition, prior to the hybridization treatment, it is preferable to pre-hybridize the transferred film in order to prevent non-specific hybridization reaction. This prehybridization treatment can be performed, for example, by prehybridization solution [50%
formamide, 5 x Denhardt's solution (0.2% bovine serum albumin, 0.2% polyvinyl pyrrolidone), 5 x SSPE,
Soaked in 0.1% SDS, 100 μg / ml heat-denatured salmon sperm DNA] etc., at about 35 ℃ to about 50 ℃, preferably about 42 ℃, about 4 to about
The reaction can be carried out for 24 hours, preferably for about 6 to about 8 hours, and those skilled in the art can appropriately determine preferable conditions by repeating experiments. Labeled probe used for hybridization
The denaturation of the DNA fragment is performed, for example, at about 70 ° C to about 100 ° C, preferably about 100 ° C, for about 1 minute to about 60 minutes, preferably about 5 ° C.
It can be performed by heating for minutes. The hybridization can be performed by a method known per se or a method analogous thereto, but the stringent conditions in the present specification include, for example, sodium concentration,
About 15 to about 50 mM, preferably about 19 to about 40 mM, more preferably about 19 to about 20 mM, and the temperature is about 35 to about 85 ° C, preferably about 50 to about 70 ° C, more preferably about 60 to about. The conditions of 65 ° C are shown. After the hybridization is completed, the filter is thoroughly washed to remove the labeled probe other than the labeled probe DNA fragment which has undergone the specific hybridization reaction. The filter cleaning process can be performed by selecting from those commonly used in the art,
For example, 0.5 x SSC containing 0.1% SDS (O.15M NaCl, 15 mM
It can be carried out by washing with a citric acid solution or the like. Labeled probe with specific hybridization reaction
The detection process can be performed after removing the labeled probe other than the DNA fragment.

The DNA fragment obtained is then transformed into a suitable vector as described in detail below, for example plasmids pEX, pM.
AMneo, pKG5 and other vectors, suitable host cells as described in detail below, for example, E. coli, yeast,
It can be expressed in CHO cells, COS cells, etc. The DNA fragment may be used as it is or as a DNA fragment to which an appropriate control sequence is added, or it may be incorporated into an appropriate vector and then introduced into an animal to give a desired gene such as N-.
Transgenic animals expressing truncated RAGE can be generated. Examples of animals include mammals, such as mice, rats, rabbits, guinea pigs, and cows. Preferably, the DNA fragment can be introduced into a fertilized egg of an animal such as a mouse to prepare a transgenic animal. The foreign gene can be introduced into animal cells such as mammals by a method known in the art or a method substantially similar thereto, for example, calcium phosphate method (for example, FL Graham
et al., Virology, 52: 456, 1973), DEAE-dextran method (eg, D. Warden et al., J. Gen. Viro).
l., 3: 371, 1968, etc.), electroporation method (eg, E. Neumann et al., EMBO J, 1: 841, 1982).
Etc.), microinjection method, ribosome method,
Viral infection method, phage particle method and the like can be mentioned. The gene product produced by the animal cell thus transfected with the predetermined gene can be analyzed.

Predetermined gene, etc. (DNA obtained by the present invention
, Etc., as a plasmid that incorporates a genetically engineered host cell (eg, prokaryotic host such as Escherichia coli or Bacillus subtilis, eukaryotic host such as yeast, CHO cell, COS cell, Sf, etc.
Any plasmid can be used as long as it can express the DNA in an insect cell host such as 21). Within such a sequence, for example, codons suitably modified for expression in the selected host cell may be contained, restriction enzyme sites may be provided, and the desired gene Regulatory sequences and facilitating sequences for facilitating expression, such as linkers and adapters that help bind the target gene, antibiotic resistance, metabolism control, selection, etc. It may contain useful sequences (including those encoding hybrid proteins and fusion proteins) and the like. Preferably, a suitable promoter, for example, a plasmid using E. coli as a host, tryptophan promoter (trp), lactose promoter (lac), tryptophan-lactose promoter (tac), lipoprotein promoter
(lpp), λ phage P _L promoter, etc., in the plasmid using animal cells as a host, SV40 rate promoter,
The MMTV LTR promoter, RSV LTR promoter, CMV promoter, SRα promoter and the like can be used, and for plasmids using yeast as a host, GAL1, GAL10 promoter and the like can be used.

As a plasmid using E. coli as a host,
For example pBR322, pUC18, pUC19, pUC118, pUC119, pSP6
4, pSP65, pTZ-18R / -18U, pTZ-19R / -19U, pGEM-3, pG
EM-4, pGEM-3Z, pGEM-4Z, pGEM-5Zf (-), pBluescrip
t KS ^™ , (Stratagene) and the like. Suitable plasmid vectors for expression in E. coli include pAS and pKK2
23 (Pharmacia), pMC1403, pMC931, pKC30, pRSET-B
(Invitrogen) is also included. Examples of plasmids using animal cells as hosts include SV40 vector, polyoma virus vector, vaccinia virus vector, retrovirus vector, and the like, for example, pcD, pcD-SR.
α, CDM8, pCEV4, pME18S, pBC12BI, pSG5 (Stratage
ne) and so on. Examples of the plasmid using yeast as a host include YIp type vector, YEp type vector, YRp type vector, YCp type vector, and the like, for example, pGPD-2. When the host cell is E. coli, examples of the host cell include those derived from the Escherichia coli K12 strain, and examples thereof include NM533, XL1-Blue, C600, DH1, DH5, DH11.
S, DH12S, DH5α, DH10B, HB101, MC1061, JM109
Examples of the STBL2 and B834 strains include BL21 (DE3) pLysS. When the host cell is an animal cell, for example, African green monkey fibroblast-derived COS-7 cells, COS-1 cells, CV-1 cells, mouse fibroblast-derived COP cells, MOP
C derived from cells, WOP cells, Chinese hamster cells
HO cells, CHO DHFR ⁻ cells, human HeLa cells, mouse cell-derived C127 cells, mouse cell-derived NIH 3T3 cells and the like can be mentioned. Insect cells include silkworm nuclear polyhedrosis virus
(Bombyx mori nuclear polyhedrosis virus) or one derived therefrom can be used as a vector, and silkworm larvae or silkworm cultured cells such as BM-N cells can be used. It is also possible to use plant cells as host cells, together with suitable vectors, which are well known in the art.

The transformant transformed with the expression vector containing the nucleic acid encoding the protein of the present invention stabilizes its high expression ability by carrying out repeated cloning using an appropriate selection marker as necessary. Can be obtained. For example, in a transformant using an animal cell as a host cell, when the dhfr gene is used as a selection marker, the protein of the present invention is encoded by gradually increasing the MTX concentration and culturing and selecting a resistant strain. It is possible to amplify the DNA and obtain a cell line with higher expression. The transformant of the present invention can be cultured under conditions in which the nucleic acid encoding the protein of the present invention can be expressed to produce and accumulate the desired product. The transformant can be cultured in a medium commonly used in the art. For example, a liquid medium can be preferably used for a transformant using a prokaryotic host such as Escherichia coli or Bacillus subtilis or a yeast host. The medium contains a carbon source, a nitrogen source, an inorganic substance and the like necessary for the growth of the transformant. Examples of the carbon source include glucose, dextrin, soluble starch, and sucrose, and examples of the nitrogen source include ammonium salts, nitrates, corn steep liquor, peptone, casein,
Examples of inorganic or organic substances such as meat extract, malt extract, soybean meal, and potato extract, and inorganic substances include calcium chloride, sodium dihydrogen phosphate, magnesium chloride, calcium carbonate, and the like. In addition, yeast, vitamins, casamino acids, growth promoting factors and the like may be added. Further, if necessary, a drug such as 3β-indolylacrylic acid can be added in order to make the promoter work efficiently. The pH of the medium is preferably about 5-8.

The culture is usually about 15 to about 45 in E. coli.
It is carried out at a temperature of about 3 to about 75 hours, and if necessary, aeration and stirring can be added. When culturing a transformant whose host is an animal cell, as the medium, for example, MEM medium containing about 5 to about 20% fetal bovine serum, PRMI1640 medium, DMEM medium and the like are used. The pH is preferably about 6 to about 8. Culturing is usually performed at about 30 ° C to about 40 ° C for about 15 to about 72 hours, and aeration and agitation are added as necessary. When extracting from the cultured cells, after culturing, the cells or cells are collected by a known method, suspended in an appropriate buffer, and disrupted by ultrasonic waves, lysozyme and / or freeze-thawing. After that, a method of obtaining a crude extract by centrifugation or filtration can be appropriately used. The buffer contains protein denaturants such as urea and guanidine hydrochloride, and Triton X.
A surfactant such as -100 (trade name) or Tween-20 (trade name) may be added. When the desired product is secreted into the culture solution, after the completion of the culture, the cells or cells are separated from the supernatant by a method known per se, and the supernatant is collected.
The target product contained in the thus obtained culture supernatant or the extract can be purified by appropriately combining separation / purification methods known per se, for example, salts such as ammonium sulfate precipitation method. Gel filtration by Sephadex or Sephadex, for example, ion exchange chromatography using a carrier having a diethylaminoethyl group or carboxymethyl group, for example, a carrier having a hydrophobic group such as a butyl group, an octyl group or a phenyl group. It can be obtained by purification by the used hydrophobic chromatography method, dye gel chromatography method, electrophoresis method, dialysis, ultrafiltration method, affinity chromatography method, high performance liquid chromatography method and the like. Preferably, it can be purified and separated by polyacrylamide gel electrophoresis, affinity chromatography with immobilized ligand and the like. For example, gelatin-agarose affinity chromatography, heparin-agarose chromatography and the like can be mentioned.

It is also possible to express it as a fusion protein when it is produced by the gene recombination method, and purify the fusion protein thus obtained by affinity chromatography or the like using the fusion portion. Such fusion proteins include those fused to a histidine tag, β-galactosidase (β-gal), maltose binding protein (MBP), glutathione-S-transferase (GST), thioredoxin (TRX) or Cre Rec.
Examples include those fused to the amino acid sequence of ombinase. Similarly, the polypeptide can be tagged with a heterogeneous epitope so that it can be purified by immunoaffinity chromatography using an antibody that specifically binds to the epitope. In a more suitable embodiment, the epitope tag is, for example, AU5, c-Myc, CruzTag 09, CruzT.
ag 22, CruzTag 41, Glu-Glu, HA, Ha.11, KT3, FLAG
(registered trademark, Sigma-Aldrich), Omni-probe,
Examples include S-probe, T7, Lex A, V5, VP16, GAL4, VSV-G. (Field et al., Molecular and Cellular Bi
ology, 8: pp.2159-2165 (1988); Evan et al., Molecu
lar and Cellular Biology, 5: pp.3610-3616 (1985);
Paborsky et al., Protein Engineering, 3 (6): pp.547
-553 (1990); Hopp et al., BioTechnology, 6: pp.120.
4-1210 (1988); Martin et al., Science, 255: pp.192.
-194 (1992); Skinner et al., J. Biol. Chem., 266:
pp.15163-15166 (1991); Lutz-Freyermuth et al., Pro
c. Natl. Acad. Sci. USA, 87: pp.6393-6397 (1990))
Such. A two-hybrid method using yeast can also be used. Further, the fusion protein may be one with a marker attached so that it becomes a detectable protein. In a more preferred embodiment, the detectable marker is the biotin / streptavidin system BiotinAvi.
It may be a Tag, a substance that emits fluorescence, or the like. The substance that emits the fluorescence is Aequorea victorea.
Green fluorescent protein derived from luminescent jellyfish such as
orescent protein: GFP), a variant that modified it (GFP variant), for example, EGFP (Enhanced-humanized GFP),
rsGFP (red-shift GFP), yellow fluorescent protein (yellow
fluorescent protein: YFP), green fluorescent protein (gr
een fluorescent protein: GFP), indigo fluorescent protein
(cyan fluorescent protein: CFP), blue fluorescent protein (BFP), Renilla
Examples include GFP derived from (Renilla reniformis) (Miyawaki Atsushi, Ed., Experimental Medicine, Separate volume, Post-genomic era experimental course 3-GFP and bio-easing, Yodosha (2000)). Detection can also be performed using an antibody (including a monoclonal antibody and a fragment thereof) that specifically recognizes the above fusion tag.

The obtained protein or peptide can be solid-phased by binding it to a suitable carrier or solid phase by a known method such as enzyme immunoassay. The immobilized protein and immobilized peptide can be conveniently used for binding assay and screening of substances. In one embodiment of the present invention, the active compound and the active peptide (active ingredient of the present invention) are human full-length RAGE, human soluble RAGE, particularly human soluble type shown in SEQ ID NO: 2 in the sequence listing. 39-5 out of RAGE
It may include a peptide having an amino acid sequence corresponding to the 4th amino acid sequence, a derivative thereof, or a salt thereof, and a peptide having an amino acid sequence corresponding to the 51st to 68th amino acid sequence, a derivative thereof, or a salt thereof. The peptide can be a peptidomimetic, a synthetic peptide, or a peptide analog. The peptide is a non-natural peptide having optical activity that is not found in nature, that is,
D-amino acid or L-amino acid
It can also be d). The amino acids may be replaced by synthetic amino acids that have been modified to increase the half-life of the peptide, increase the potency of the peptide, or increase bioavailability. "peptide"
And the terms "polypeptide" are used interchangeably throughout this specification. Examples of the peptide include those having an amino acid region necessary for simulating the AGE binding site of sRAGE. The peptide may be a derivative of a natural peptide, a modified peptide, a labeled peptide, or a peptide including a non-natural peptide. The peptidomimetic is AG
Screens can be performed to determine compounds that can inhibit the interaction of E and RAGE and can be identified by screening large libraries containing various compounds that are peptidomimetics.

The peptide or polypeptide of the present invention may have changes in the sequences represented by SEQ ID NOs: 7 and 8. The peptides of the present invention include in their sequences alterations that do not negatively affect the function of the peptide but can increase the peptide function in the positive direction (for example, alterations that increase the potency of the peptide). be able to. Substantially equivalent substitutions (alterations) of amino acids in the amino acid sequence can be selected from other amino acids of the class to which the amino acid belongs.
For example, nonpolar (hydrophobic) amino acids include alanine, phenylalanine, leucine, isoleucine, valine, proline, tryptophan, methionine, and the like, and polar (neutral) glycine, serine, threonine, cysteine, tyrosine, Examples include asparagine and glutamine, and examples of positively charged amino acids (basic amino acids) include arginine, lysine, and histidine, and negatively charged amino acids (acidic amino acids).
Examples thereof include aspartic acid and glutamic acid. Also, replacing L-alanine with D-alanine,
It may be one in which L-lysine is replaced with D-lysine, L-lysine is replaced with L-arginine, or L-valine is replaced with D-valine. Furthermore, it will be appreciated that the peptides described above can be modified (modified) by chemical modification techniques well known to those skilled in the art. Many forms of modifications (modifications) to be added to the peptide are known, and they are described in detail in basic reference books and more detailed papers in this field as well as in many research literatures. , These are well known to those skilled in the art. Some particularly conventional alterations / modifications include, for example, glycosylation, lipid binding, sulfation, γ-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
For example TE Creighton, Proteins-Structure and Mole
cular Properties, Second Edition, WH Freeman an
d Company, New York, (1993); BCJohnson (Ed.), Po
sttranslational Covalent Modification of Proteins,
Academic Press, New York, (1983) (Wold, F., "Postt
ranslationalProtein Modifications: Perspective and
Prospects ", pp.1-12); Seifter et al.," Analysis fo
r Protein Modifications and nonprotein cofactors ",
Meth. Enzymol., 182: 626-646 (1990); Rattan et a
l., "Protein Synthesis: Posttranslational Modifica
tion and Aging ", Ann. NY Acad. Sci., 663: p.48-
62 (1992) etc. can be referred to.

The peptide having an amino acid sequence corresponding to SEQ ID NO: 7 or 8 of the present invention has, in addition to the naturally-occurring form, the same or stronger or more positive function as the peptide. Also included are other polypeptides such as peptide analogs. The analog may be more stable to hydrolysis (hence it may have a more pronounced or longer-lasting effect than the natural one) or one or more potential O-glycosylation and / or Those modified to remove or add N-glycosylation sites are included. The peptides of the invention may be peptidomimetic compounds that may be at least partially non-natural. The peptidomimetic compound may be a small molecule that mimics a portion of the amino acid sequence.
The compound has increased stability to mimic it,
It may have potency, bioavailability. In addition, the compound may have reduced toxicity. The peptidomimetic compound may have increased intestinal mucosal permeability. The compound may be prepared synthetically. The compounds of the present invention may include L-, D-, DL-, or unnatural amino acids, α, α-disubstituted amino acids, N-alkyl amino acids, lactic acid. The peptide backbone of the compound may have at least one bond replaced with PSI- [CH = CH]. Further, the compound contains trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, poly-L-propargylglycine, poly-D, L-allylglycine, or poly-L-allylglycine. You may

Further, the compound is a peptide mimicking compound having a biological activity of suppressing vascular complications which are problematic in diabetic complications such as diabetic nephropathy and diabetic retinopathy,
The compound has a bond, a peptide backbone, or an amino acid moiety that is replaced with a suitable mimetic. Non-natural amino acids that may be suitable amino acid mimetics include, for example, β-alanine, L-α-aminobutyric acid, L-γ-aminobutyric acid, L-α-aminoisobutyric acid, L-ε-aminocaproic acid, 7- Aminoheptanoic acid, L-aspartic acid, L-glutamic acid, N-ε-Boc
-N-α-CBZ-L-lysine, N-ε-Boc-N-α-Fmoc-L-lysine, L
-Methionine sulfone, L-norleucine, L-norvaline, N-α-Boc-N-δ-CBZ-L-ornithine, N-δ-Boc-N-α-
CBZ-L-ornithine, Boc-p-nitro-L-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline and the like can be mentioned. In the present specification, “substantially equivalent” means the activity of the peptide, for example, the inhibitory activity, physiological activity,
It means that the biological activities are substantially the same.
Furthermore, the meaning of the term may include the case of having substantially the same activity, and the substantially same activity includes the activity for the interaction between AGE and RAGE, For example, AGE-RAGE antagonistic activity against any one of AGEs, activity of suppressing or inhibiting the binding activity of transmembrane RAGE to any one of AGEs and the like can be mentioned. The "substantially the same activity" means that the activities are the same in nature, for example, physiologically, pharmacologically or biologically. For example, activities such as the activity of inhibiting the binding of AGE to transmembrane RAGE are equivalent (for example, about 0.001 to about 10
00 times, preferably about 0.01 to about 100 times, more preferably about
0.1 to about 20 times, more preferably about 0.5 to about 2 times), but the degree of these activities and quantitative factors may be different.

For the synthesis of the peptide of the present invention, methods known in the field of peptide synthesis can be used, for example, chemical synthesis methods such as liquid phase synthesis method and solid phase synthesis method. In such a method, for example, a resin for peptide synthesis is used, and an appropriately protected amino acid is sequentially bound to the desired amino acid sequence on the resin by various condensation methods known per se. For the condensation reaction, various activation reagents known per se are preferably used, and as such a reagent, for example, carbodiimides such as dicyclohexylcarbodiimide can be preferably used. When the product has a protecting group,
The desired product can be obtained by appropriately removing the protecting group. When the peptide of the present invention is obtained in a free form, it can be converted into a salt by a method known per se or a method analogous thereto, and when they are obtained as a salt, The compound can be converted into a free form or other salt by a method known per se or a method analogous thereto. The salt of the peptide of the present invention is preferably physiologically acceptable or pharmaceutically acceptable, but is not limited thereto. Examples of such salts include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
Salts with inorganic acids such as phosphoric acid, such as acetic acid, formic acid, maleic acid, fumaric acid, succinic acid, citric acid, tartaric acid, malic acid, benzoic acid, methanesulfonic acid, p-toluenesulfonic acid, benzenesulfonic acid, etc. Examples thereof include salts with organic acids. Further, as the salt, ammonium salts such as ethylamine, dimethylamine, trimethylamine,
Examples thereof include salts with organic bases such as hydroxyethylamine.

The peptide of SEQ ID NO: 7 and the peptide of SEQ ID NO: 8 have an activity of inhibiting the binding between AGE and RAGE. In particular, the peptide of SEQ ID NO: 7 has an activity of specifically inhibiting the binding between AGE derived from glyceraldehyde and RAGE. Further, the peptide of SEQ ID NO: 8 has an activity of specifically inhibiting the binding between glycolaldehyde-derived AGE and RAGE. Thus, the peptide or its derivative or its salt is useful for inhibiting the interaction between AGE and its receptor (RAGE), and is useful as an AGE-RAGE antagonist. The AGE-RAGE interaction can be inhibited by bringing the AGE-RAGE antagonist into contact with RAGE-expressing cells. The RAGE-expressing cell may be a eukaryotic cell,
Examples include those expressing RAGE or a variant thereof, for example, endothelial cells, smooth muscle cells (for example, vascular smooth muscle cells), nerve cells, glial cells, microglial cells, macrophages, lymphocyte cells, bone marrow cells, Examples thereof include retinal vascular cells, retinal nerve cells, gingival-related cells, skin-related cells, glomerular cells, tubular cells, connective tissue cells and the like. The peptide or its derivative or its salt is
It can be expected as a drug for diseases caused by an interaction between AGE and its receptor, a change in the expression level of soluble RAGE, and a change in the AGE scavenging activity, for example, diabetic complications, Prevention of pathological conditions or symptoms such as various diseases associated with aging, Alzheimer's disease, arteriosclerosis, the onset and / or progression of diseases or diseases caused by glycation of proteins in the body, and infiltration or spread of tumor And / or is useful in therapy. And diabetes, diabetic complications, diabetic nephropathy, diabetic retinopathy, diabetic angiopathy, diabetic microangiopathy, renal glomerulosclerosis, hyperlipid atherosclerosis, neurocytotoxicity, Down syndrome,
Dementia associated with head trauma, amyotrophic lateral sclerosis, multiple sclerosis, amyloidosis, autoimmune disease, inflammation, tumor,
It can be expected to be used for the prevention and / or treatment of cancer, erectile dysfunction in men, wound healing, periodontal disease, neuropathy and neurodegeneration. The peptide or its derivative or salt thereof, (1) the action of inhibiting or suppressing the breakdown of vascular endothelial cell growth control, (2) the action of suppressing the decrease in prostacyclin production ability, (3) vascular pericyte loss or Inhibition or inhibition of reduction, (4) inhibition or inhibition of angiogenesis, (5) inhibition or inhibition of thrombus formation,
(6) inhibitory or inhibitory action on the induction of autocrine vascular endothelial growth factor, (7) inhibitory or inhibitory action on the growth of microvascular endothelial cells, (8) inhibitory or inhibitory action on the lumen formation by microvascular endothelial cells, (9) Inhibition or inhibition of plasminogen activator inhibitor 1 enhancement, (10) Inhibition or inhibition of extracellular matrix protein production enhancement, (11) Inhibition or inhibition of vascular permeability enhancement, etc. Either action can be expected.

The active ingredient of the present invention [for example, a peptide having an amino acid sequence corresponding to the sequences represented by SEQ ID NOs: 7 and 8, a derivative thereof or a salt thereof, etc., (b) found using the present invention Active substance or the like] as a medicine, for example, an interaction inhibitor between AGE and RAGE, a salt thereof, or the like is ordinarily used alone or in admixture with various pharmaceutically acceptable formulation adjuvants to prepare a pharmaceutical composition. It can be administered as a product or a pharmaceutical preparation. It is preferably administered in the form of a pharmaceutical preparation suitable for oral administration, topical administration, parenteral administration or the like, and may be administered in any administration form (including inhalation method and rectal administration) depending on the purpose. Good. In addition, the active ingredient of the present invention is used for diabetic complications, arteriosclerosis agents, antihyperlipidemic agents, antitumor agents (anticancer agents), tumor transfer inhibitors, thrombus formation inhibitors, Alzheimer's treatment Agents, joint destruction therapeutic agents, anti-inflammatory agents and / or immunosuppressive agents can also be used in combination, and they can be used without limitation as long as they have an advantageous action, for example, those known in the art. You can choose from.

The parenteral administration form may include topical, transdermal, intravenous, intramuscular, subcutaneous, intradermal or intraperitoneal administration, but direct administration to the affected area is also possible.
It is also suitable in some cases. Preferably orally in mammals, including humans, or parenterally (eg, intracellular,
Tissue, intravenous, intramuscular, subcutaneous, intracutaneous, intraperitoneal, intrathoracic, spinal cavity, drip, enema, rectal, eardrop, eye drop or nose, tooth, skin or mucous membrane application, etc. ). Specific forms of preparation preparation include solution preparation,
Dispersed preparations, semi-solid preparations, powdered and granulated preparations, molded preparations, leaching preparations and the like can be mentioned, for example, tablets, coated tablets, sugar-coated agents, pills, troches, hard capsules, soft capsules, microcapsules. Capsules, implants, powders, powders, granules, fine granules, injections, solutions, elixirs, emulsions, irrigants, syrups, water solutions, emulsions, suspensions, liniments, lotions , Aerosol, spray,
Inhalants, sprays, ointments, plasters, patches, pasta, poultices, creams, oils, suppositories (for example, rectal suppositories), tinctures, water solutions for skin, eye drops, nasal drops, Powders for ear drops, coatings, infusions, injectables, etc.
Lyophilized preparations, gel preparations and the like can be mentioned. The pharmaceutical composition can be formulated according to a conventional method. For example, if necessary, a physiologically acceptable carrier,
Pharmaceutically acceptable carrier, adjuvant agent, excipient,
Excipients, diluents, flavors, fragrances, sweeteners, vehicles, preservatives, stabilizers, binders, pH adjusters, buffers, surfactants, bases, solvents, fillers, extenders, solubilizers Auxiliary agents, solubilizing agents, isotonic agents, emulsifying agents, suspending agents, dispersing agents, thickening agents, gelling agents, curing agents, absorbents, adhesives, elastic agents, plasticizers, disintegrating agents, propellants. , A preservative, an antioxidant, a light-shielding agent, a moisturizer, a emollient, an antistatic agent, a soothing agent, etc., alone or in combination, and generally admixed with the protein of the present invention. It can be manufactured in the unit dose form required for formulation implementation. Formulations suitable for parenteral use include sterile solutions of the active ingredient and water or other pharmaceutically acceptable medium, or suspensions, such as injections. Generally, water, saline, aqueous dextrose solution, other related sugar solutions, and glycols such as ethanol, propylene glycol, and polyethylene glycol are mentioned as preferred liquid carriers for injection. When preparing an injection, the solution is prepared by a method known in the art using a carrier such as distilled water, Ringer's solution, physiological saline, an appropriate dispersing agent or wetting agent and suspending agent. , Suspensions, emulsions, and injectable forms.

Examples of the aqueous solution for injection include physiological saline, isotonic solution containing glucose and other adjuvants (for example, D-sorbitol, D-mannitol, sodium chloride, etc.), etc. A suitable acceptable solubilizing agent, eg alcohol (eg ethanol),
You may use together with polyalcohol (for example, propylene glycol, polyethylene glycol, etc.), a nonionic surfactant (for example, Polysorbate 80 ^™ , HCO-50, etc.) and the like. Examples of the oily liquid include sesame oil and soybean oil, which may be used in combination with solubilizing agents such as benzyl benzoate and benzyl alcohol. In addition, buffers (eg, phosphate buffer, sodium acetate buffer, etc.) or reagents for adjusting osmotic pressure, soothing agents (eg, benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (eg, human) Serum albumin, polyethylene glycol, etc.), a preservative (eg, benzyl alcohol, phenol, etc.), an antioxidant such as ascorbic acid, an absorption promoter and the like may be added. The prepared injection solution is usually filled in a suitable ampoule.

For parenteral administration, in a sterile pharmaceutically acceptable liquid such as water, ethanol or oil, with or without the addition of surfactants and other pharmaceutically acceptable auxiliaries. It is formulated in the form of a solution or suspension. Examples of the oily vehicle or solvent used in the preparation include natural or synthetic or semi-synthetic mono-, di- or triglycerides, natural, semi-synthetic or synthetic fats and oils, such as peanut oil, corn oil, Examples include vegetable oils such as soybean oil and sesame oil. For example, this injection can be usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 10% by weight. Formulations suitable for topical, eg buccal, or rectal use include, eg, mouth washes, dentifrices, mouth sprays,
Inhalants, ointments, dental fillers, dental coating agents, dental paste agents, suppositories and the like can be mentioned. The mouthwash and other dental agents are prepared by a conventional method using a pharmacologically acceptable carrier. As an oral spray and an inhalant, the compound of the present invention itself or a pharmacologically acceptable inert carrier can be dissolved in a solution for an aerosol or a nebulizer, or can be administered as fine powder for inhalation to teeth and the like. The ointment is prepared by a conventional method by adding a commonly used base such as an ointment base (white petrolatum, paraffin, olive oil, Macrogol 400, Macrogol ointment, etc.).

Agents for topical application to the teeth, skin may be formulated in solutions or suspensions of suitably sterile water or non-aqueous vehicles. Additives include, for example, buffering agents such as sodium bisulfite or disodium edetate; preservatives such as acetic acid or phenylmercuric nitrate, benzalkonium chloride or chlorohexidine, and preservatives such as hypromelrose. Examples include thickeners. Suppositories are carriers well known in the art, preferably suitable non-irritating excipients such as polyethylene glycols, lanolin, cocoa butter, fatty acid triglycerides, etc., preferably solid at room temperature but at intestinal temperature. It is prepared by a conventional method using a liquid that melts in the rectum and releases the drug, and is usually prepared so as to contain the compound of the present invention in an amount of about 0.1 to 95% by weight. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Adjuvants such as local anesthetics, preservatives and buffers can be dissolved in the vehicle. Examples of formulations suitable for oral use include solid compositions such as tablets, pills, capsules, powders, granules, and troches, and liquid compositions such as solutions, syrups, and suspensions. Can be mentioned. When preparing the formulation,
A formulation auxiliary agent or the like known in the art is used. Tablets and pills can also be manufactured with enteric coating. When the unit dosage form is a capsule, a liquid carrier such as fat may be included in addition to materials of the above type.

The active ingredient of the present invention is bound to a detectable marker substance (for example, ^radiolabeled with ¹²⁵ I, or biotinylated), and the labeled substance is used as a receptor. It may be a reagent useful for detection and quantification in cells or tissues having a body and a liquid sample such as blood, cerebrospinal fluid, or urine. Substances such as peptides are often rapidly cleared from the systemic circulation when administered, and thus exhibit their pharmacological activity only in a relatively short period of time. As a result, relatively large doses and frequent administration of bioactive agents are required to maintain therapeutically effective conditions. Polyethylene glycol, copolymers of polyethylene glycol, and substances modified by covalently attaching a water-soluble polymer such as polypropylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, or polyproline to the corresponding unmodified It is known to exhibit a longer half-life in blood after intravenous administration, as compared with the substance described above. Such modifications may increase the solubility of the substance in aqueous solution, prevent aggregation, increase the physical and chemical stability of the substance, and also significantly reduce the immunogenicity and reactivity of the substance. As a result, the desired in vivo bioactivity can be achieved by administering such polymer-substance adducts less frequently or at lower doses than the unmodified substance.

The attachment of polyethylene glycol (PEG) is particularly useful because it is extremely toxic in mammals. In addition, the attachment of PEG may sometimes effectively reduce the immunogenicity and antigenicity of the heterologous compound. The compound may be provided in a microcapsule device. Polymers such as PEG include amino-terminal amino acid α-amino groups, lysine side-chain ε-amino groups, aspartic acid or glutamic acid side-chain carboxyl groups, and carboxy-terminal amino acid α-amino groups.
-Can be conveniently attached to an activated derivative of a glycosyl chain attached to a carboxyl group or certain asparagine, serine or threonine residues. Many activated forms of PEG are known which are suitable for direct reaction with proteins. PEG reagents useful for reacting with the amino groups of proteins include carboxylic acids, active esters of carbonate derivatives, especially where the leaving group is N-hydroxysuccinimide, p-nitrophenol, imidazole, or 1-hydroxy-2-. One that is nitrobenzene-4-sulfonate. Similarly, PEG reagents containing aminohydrazine or hydrazide groups are useful for reaction with aldehydes produced by periodate oxidation in proteins. The active ingredient of the present invention can be administered with a wide range of doses selected, and the dose, frequency of administration, etc. can be determined by the sex, age, weight, general health condition, diet, administration time and administration method of the treated patient. , Excretion rate, drug combination, and the degree of the medical condition being treated by the patient at that time, and these factors or other factors are taken into consideration.

In the manufacture of pharmaceutical products, the additives and the preparation method thereof are described in, for example, the Japanese Pharmacopoeia Commentary Book Editing Committee,
14th Revised Japanese Pharmacopoeia Manual, published June 27, 2001, Hirokawa Shoten Co., Ltd .; Takashi Ichibanse et al. Pharmaceutical Development Volume 12 (Drug formulation [I]), October 1990 Issued on the 15th,
Hirokawa Shoten Co., Ltd .; the same, Pharmaceutical Development Volume 12 (Formulation Material [II]) Issued on October 28, 1990, refer to the description of Hirokawa Shoten Co., Ltd., etc. and select from them as necessary. Can be applied. The active ingredient of the present invention is
There is no particular limitation as long as it has an AGE activity, particularly a biological activity such as suppressing or inhibiting the interaction between AGE and RAGE, but preferably one having an advantageous effect is mentioned. The active ingredient of the present invention is expected to be useful for suppressing or inhibiting changes in various tissues or cells due to the interaction between AGE and RAGE.
In addition, the active ingredient is useful for suppressing the expression of AGE activity, and is useful for preventing or treating disorders, abnormalities and / or diseases caused by the interaction between AGE and RAGE. Further, it is expected to be useful for controlling, for example, suppressing migration, invasion, migration and / or metastasis of tumor cells and the like, which involve the interaction between AGE and RAGE. The active ingredient is useful for preventing and / or suppressing the migration, invasion and / or metastasis of malignant tumor, that is, cancer, an angiogenesis / neogenesis inhibitor, an antitumor agent and / or a cancer metastasis inhibitor. Can be expected as It is also useful for preventing or treating AGE-related disorders, abnormalities, and / or diseases of blood cells, including diabetic complication treatment / prevention agent, arteriosclerosis treatment / prevention agent, thrombosis treatment / prevention agent, It can also be expected as an anti-inflammatory agent and / or an immunosuppressive agent. Furthermore, it can be expected as a therapeutic agent for Alzheimer's disease, a therapeutic agent for joint destruction and the like.

Further, in the present invention, a molecular design is performed based on the amino acid sequence represented by SEQ ID NO: 7 or 8 to obtain AG
It can be used to obtain a substance having an activity of suppressing or inhibiting the interaction between E and RAGE. The substances thus obtained are also within the scope of the idea of the present invention and can be treated as the active ingredient of the present invention. Whether a particular characteristic portion is selected from the sequence and (i) the pharmacophore among them is replaced with an isostere, or (ii) at least one of the constituent amino acid residues is replaced with a D-amino acid residue , (Iii) modify the side chain of the amino acid residue, or (i
v) Arrange and link amino acid residues that are different from the amino acid residues present in the sequence, or (v) analyze the three-dimensional structure and
This can be done by making full use of the technology adopted in the relevant field, such as designing a mic body (for example, Koichi Suto, Vol. 7: Pharmaceutical Development, June 25, 1990).
Issued daily, Hirokawa Shoten Co., Ltd. and references and articles cited therein). Some of these techniques include those described above.

In another embodiment of the present invention, it is shown that there are at least two types of regions as the AGE binding region of RAGE, and that a substance capable of distinguishing and antagonizing regions having different properties in terms of the binding property can be provided. ing. Therefore, a screening method or an assay method utilizing this feature, a reagent therefor, and a specific AGE-RAGE obtained by applying the method.
Antagonists are also provided herein, all of which are within the scope of the invention. Thus, the present invention provides an AGE-RAGE antagonist specific to a specific AGE, a screening method therefor, and a reagent therefor. For example, contacting an AGE-containing test sample with RAGE or RAGE-expressing cells in the presence or absence of the peptide of SEQ ID NO: 7 or 8 and performing comparison between the presence and absence of the peptide. An AGE-RAGE interaction analysis method characterized by the above can be provided. In another embodiment, in the presence or absence of the peptide of SEQ ID NO: 7 (or the peptide of SEQ ID NO: 8), and in the presence or absence of AGE derived from glyceraldehyde (or AGE derived from glycolaldehyde). The test sample was contacted with RAGE or RAGE-expressing cells, and comparison was made between the presence and absence of the peptide and the presence or absence of glyceraldehyde-derived AGE or glycolaldehyde-derived AGE. An AGE-RAGE interaction analysis method characterized by carrying out can be provided. In the method, for example, the biological activity (eg, the activity associated with the interaction between AGE and RAGE) is measured and compared. The substances in the system are
If necessary, a fluorescent substance such as fluorescein labeled with an enzyme or a radioactive substance can be used, or it can be used as it is. Examples of test samples include proteins, peptides, non-peptidic compounds, synthetic compounds, fermentation products, plant extracts, tissue extracts of animals and the like, cell extracts and the like. In particular, it may include synthetic compounds and the like. These compounds may be novel compounds or known compounds. Further, as the sample to be measured by the measuring method of the present invention, a solution or colloidal solution of any form, a non-fluid sample or the like can be used, but preferably a biological sample such as thymus, testis, intestine, kidney,
Brain, breast cancer, ovarian cancer, colorectal cancer, blood, serum, plasma,
Examples thereof include synovial fluid, cerebrospinal fluid, pancreatic fluid, bile fluid, saliva, amniotic fluid, urine and other body fluids, cell culture fluid, tissue culture fluid, tissue homogenate, biopsy sample, tissue and cells. This method can be carried out according to a conventional method for measuring binding activity, and can be carried out with reference to, for example, methods known in the art. In addition, it is possible to use various labels, buffer systems and other appropriate reagents, and to carry out according to the operations explained there. The measurement is carried out in a buffer solution or the like that does not adversely affect the reaction, for example, at a pH of about 4 to about 10 (preferably,
It can be carried out at a pH of about 6 to about 8). In screening each of these, the AGE-RAGE interaction analysis system of the present invention may be constructed by adding ordinary technical consideration to those skilled in the art to ordinary conditions and operating methods in each method. For details of these general technical means, reference can be made to reviews, textbooks, etc. [eg, Method
s in Enzymology, Academic Press (USA))).

In a further aspect of the invention, human full length RA
GE, human soluble RAGE, particularly, the human soluble RAGE shown in SEQ ID NO: 2 of the sequence listing, by identifying the region corresponding to the amino acid sequence at positions 39-54 or 51-68. , AGE-RAGE
Regarding antagonism. The present antagonism may be in any form as long as it substantially distinguishes the region, and substantially equivalent in terms of activity may be included without limitation. The AGE-
Substantially the same activity in RAGE antagonism means that the regions are distinct in that they are of the same nature, such as physiologically, pharmacologically, or biologically similar. Is shown. The treatment of the above-mentioned diseases or pathological symptoms using the present antagonistic method is also included in the present invention. It can be used to elucidate what kind of disease is induced depending on the difference in AGE, and to develop the therapeutic method and therapeutic agent. In the specification and drawings, the terms are IUPAC-IUB Comm.
Ission on Biochemical Nomenclature or based on the meaning of terms commonly used in the art. Among the abbreviations used herein, those commonly used for amino acids are: A = Ala = alanine; R = Arg = arginine; N = Asn = asparagine; D = Asp = Aspartic acid; C = Cys = Cysteine; Q = Gln =
Glutamine; E = Glu = Glutamic acid; G = Gly = Glycine; H = H
is = histidine; I = Ile = isoleucine; L = Leu = leucine;
K = Lys = lysine; M = Met = methionine; F = Phe = phenylalanine; P = Pro = proline; S = Ser = serine; T = Thr = threonine; W = Trp = tryptophan; Y = Tyr = tyrosine; V = Val = Valine.

[0040]

EXAMPLES The present invention will be specifically described with reference to examples below, but it is understood that the present invention is not limited to these examples and various embodiments based on the idea of the present specification are possible. It should be. All examples, unless otherwise indicated in detail, are carried out or can be carried out using standard techniques, which are well known and routine to those skilled in the art. .. In the following examples, unless otherwise specified, specific operations and treatment conditions are described in J. Sambrook, E.
F. Fritsch & T. Maniatis, "Molecular Cloning", 2nd
ed., Cold Spring Harbor Laboratory, Cold Spring Ha
rbor, NY (1989) and DM Glover et al. ed.,
"DNA Cloning", 2nd ed., Vol. 1 to 4, (The Practica
l Approach Series), IRLPress, Oxford University Pr
ess (1995); Especially in the PCR method, HA Erliched., PCR
Technology, Stockton Press, 1989; DM Glover et
al. ed., "DNA Cloning", 2nd ed., Vol. 1, (The Prac
tical Approach Series), IRLPress, Oxford Universit
y Press (1995) and MA Innis et al. ed., "PCR
Protocols ", Academic Press, New York (1990), and when using commercially available reagents or kits, attach the instructions (proto) attached to them.
(cols) and attached chemicals are used.

Example 1 [Cells] Primary cultured human skin microvascular endothelial cells were Cascad
e Biologics, Inc. (Portland, OR), poly
(A) ⁺ RNA was purified using cells at passage number 5-10. [Separation of polysome-derived poly (A) ⁺ RNA] Human skin microvascular endothelial cells cultured in a tissue culture flask were washed with ice-cold phosphate-buffered saline, and then scraped with a cell scraper. The cell suspension was centrifuged to collect the cells as a precipitate, which was then added to 0.25M KCl, 10mM MgCl ₂ , 1mM EDTA, 0.25M sucro.
se (RNase free), 0.1mM DTT, 2mM 4- (2-aminoethyl) -be
nzenesulfonyl fluoride, 1,000u / ml RNase inhibitor
(Ambion, Inc., Austin, TX) was suspended in a 10 mM Tris-HCl buffer solution (pH 7.6) containing (Ambion, Inc., Austin, TX) and then disrupted with a Dounce homogenizer. The cell lysate was centrifuged at 12,000 xg for 15 minutes to remove the cell nucleus and mitochondria, and the supernatant was collected. The resulting supernatant was centrifuged at 100,000 xg for 60 minutes, and the polysome fraction was collected as a precipitate. From the obtained polysome fraction, Quickprep micro mRNA isolation kit (Amersham Ph
poly (A) ⁺ RNA was purified using armacia Biotech).

[Separation of Soluble RAGE cDNA] Using human skin microvascular endothelial cell polysome-derived poly (A) ⁺ RNA as a template
cDNA using igo (dT) primer and AMV-derived reverse transcriptase
Was synthesized, and primers corresponding to exon 1 and exon 11 of the RAGE gene (SEQ ID NO: 3, 5'-GCCAGGACCC in the sequence listing
TGGAAGGAAGCA-3 '; Sequence Listing SEQ ID NO: 4, 5'-CTGATGGA
RAGE cDNA was amplified using TGGGATCTGTCTGTG-3 ') and TaKaRa La Taq polymerase. The amplified RAGE cDNA is
E. coli XL1-Blue was transformed by inserting into pCR2.1 (Invitrogen). Plasmid DNA was purified from 32 clones of the obtained recombinant E. coli colonies and the ABI377 sequencer (Aplie
d Biosystems Inc) was used to determine the nucleotide sequence to obtain a cDNA encoding soluble RAGE. The nucleotide sequence of the cDNA encoding the sequenced soluble RAGE and the amino acid sequence encoded by the open reading frame of the sequence are shown in SEQ ID NO: 1 of the sequence listing. The amino acid sequence of soluble RAGE is shown in SEQ ID NO: 2 in the sequence listing. Soluble RAGE (soluble RAGE) thus obtained is a natural type (nativ
e) Endogenous, full-length RAGE
And a so-called recombinant solubilized RAGE artificially produced from RAGE cDNA having a transmembrane domain (US 586401
It differs from 8) in 16 amino acids in the C-terminal part (sequence Glu ^{332 to} Met ³⁴⁷ of SEQ ID NO: 2).

[Preparation of Soluble Type RAGE Expression Vector] Soluble Type
5'-primer with EcoRI recognition sequence using cDNA encoding RAGE as a template (SEQ ID NO: 5, 5'-GAGA in the sequence listing
ATTCGCCAGGACCCTGGAAGGAAGCA-3 ') and 3'-primer having an XbaI recognition sequence (SEQ ID NO: 6, 5'-GATCTA in the sequence listing)
GAGATTGTTGACCATCCCCCCAG-3 ') was used for amplification. After the amplified DNA was purified, it was digested with EcoRI and XbaI, and EcoRI and Xb of the animal cell expression vector pCI-neo vector (Stratagen) were digested.
I inserted it in the aI site. The expression vector DNA is QIAGEN (Va
(lencia, CA), and the nucleotide sequence was confirmed using ABI377 sequencer (Aplied Biosystems Inc).

[Introduction of Soluble RAGE Expression Vector into COS 7 Cells and Separation of Stable Transformant] The soluble RAGE expression vector was prepared using Tfx-20 reagent (Promega Corp., Madiso
n, WI) was used to introduce into COS 7 cells. 48 hours after the introduction of the expression vector, G418 (Geneticin) was added to the cell culture medium, and two weeks later, a plurality of G418-resistant colonies were obtained. The obtained clones were separately cultured, and the cell extract and the culture medium were analyzed by Western blotting using an anti-human RAGE antibody to select a clone highly expressing soluble RAGE. [Transformant culture] Using a 150 mm dish for tissue culture, the soluble RAGE overexpressing COS7 cells were treated with 5% fetal bovine serum and
After culturing in Dulbecco's modified Eagle medium containing 500 μg / ml G418 until confluence under the conditions of 37 ° C. and 5% CO ₂ ,
The cell layer was washed twice with PBS (Ca ²⁺ / Mg ²⁺ -free) to completely remove the medium. Subsequently, the cells were cultured in serum-free Dulbecco's modified Eagle medium for 48 hours. Then, collect the culture medium,
The supernatant obtained by centrifugation at 10,000 rpm for 20 minutes was filtered through a 0.22 μm membrane filter, and soluble RAGE was purified from the resulting treated medium according to the following method.

[Purification of Soluble RAGE Protein]
HiTrap Heparin equilibrated with 20 mM Tris-HCl (pH 7.4)
(Column volume 3 x 5 ml, Amersham Pharmacia) Flow rate 3
20 mM containing 75 ml of 0.15 M NaCl, applied at ml / min
The column was washed with Tris-HCl (pH 7.4) at a flow rate of 2 ml / min. Then stepwise in 75 ml of 0.3 M, 0.5 M and 1 M Na.
Hepari bound to a column with 20 mM Tris-HCl pH 7.4 containing Cl
The n-binding substance was sequentially eluted at a flow rate of 2 ml / min and fractionated in 5 ml portions. The soluble RAGE fraction eluted was Western blotti.
It was identified by ng. Then 50 mM sodium acetate buffer
RESOURCE S 1 ml column (Amersh) equilibrated at pH 4.5
am Pharmacia) in 50 mM sodium acetate buffer (pH 4.5)
The soluble RAGE fraction diluted 5 times with 1 was applied at a flow rate of 1 ml / min, and the column was washed with 3 ml of 50 mM sodium acetate buffer containing 0.2 M NaCl at a flow rate of 1 ml / min. next
Using 5 ml of 50 mM sodium acetate buffer, flow rate 1 ml / m
A linear concentration gradient of 0.2 M to 1.0 M NaCl was applied to the column, and the anion substance bound to the column was sequentially eluted, and fractionated in 0.5 ml fractions. Soluble RAGE fraction is Western bl
Determined by otting. 2 ml of the soluble RAGE fraction obtained
Each was applied to a ^{PBS (Ca 2+ / Mg 2+ -free} ) equilibrated with HiTrap desalting column (column volume 2 × 5 ml, Amersham Pharmacia) , using ^{PBS (Ca 2 + / Mg 2+} -free) Flow rate 0.5 ml / min
Gel filtration was performed and the high molecular weight fraction was collected. Then, a soluble RAGE (sRAGE) concentrated solution was prepared by ultrafiltration using Centricon YM-3 (MILLIPORE) as needed.

The purity of the purified sRAGE protein was evaluated by silver staining of the gel after separating 100 ng protein corresponding to 10% SDS-PAGE. The above chromatograph

[Equation 1] The system (Amersham Pharmacia) was used and the eluent was 230
Absorbance at nm, 280 nm and 300 nm was measured and monitored.

[Binding Test between Purified Recombinant Soluble RAGE Protein and AGE] The AGE binding ability of soluble RAGE was confirmed by surface plasmon resonance method using BIACORE (Biacore, Sweden).
The purified soluble RAGE protein was immobilized on the Sensor chip CM5 (Biacore) using the amino coupling method. AGE-BSA prepared by aseptically incubating glucose and bovine serum albumin (BSA) for 12 weeks at 37 ° C was added as an analyte at a concentration of 500 µg / ml to the microchannel system of BIACORE. As a result, it did not react at all with the in-line reference (control chip) or the chip on which immunoglobulin was fixed, and showed specific binding only to the sensor chip on which soluble RAGE was fixed.

Example 2 [AGE binding test of RAGE splice variant] HiTrap N
HS-activated column (Amersham Pharmacia Biotech)
Then, according to the instructions attached to the column, AGE2
-BSA was bound at a density of about 20 mg / ml gel to prepare an AGE affinity column. The RAGE splice variant protein used in the binding test was prepared as follows. For full-length type and N-terminal deletion type RAGE (lacking amino acid residues 1-101 present in the full-length type and C-terminal deletion type),
Over-expressing COS-7 cells with introduced cDNA were disrupted with lysis buffer (0.25
M sucrose, 50 mM Tris-HCl (pH7.4), 10 mM KCl, 5 m
(M MgCl ₂ , 1 mM phenylmethylsulfonyl fluoride), and the resulting disrupted suspension was centrifuged at 4 ° C, 600 xg for 5 minutes to remove nuclei, and then at 4 ° C, 100,000 xg, 30
The membrane fraction was recovered by centrifugation for a minute. Extraction buffer of the obtained full-length and N-terminal deleted RAGE-expressing cell-derived membrane fractions
(20 mM Tris-HCl (pH 7.4), 0.1 M NaCl, 1% 1-On-oct
yl-bD-glucopyranoside, 1 mM phenylmethylsulfonyl
fluoride), extract at 4 ℃ for 60 minutes, then at 4 ℃, 100,000
Insoluble matter was removed by centrifugation at xg for 30 minutes to obtain crude purified full-length type and N-terminal deleted type RAGE fractions, respectively.

For soluble RAGE, the soluble RAGE cDNA
The culture supernatant of the overexpressed COS-7 cells introduced with
Insoluble matter was removed by centrifugation at 20,000 xg for 15 minutes at ℃, and it was used as a soluble RAGE fraction. Subsequently, the amount of RAGE protein contained in each fraction was assayed by Western blot analysis using an anti-RAGE antibody that commonly recognizes three types of splice variants, and a sample containing almost the same amount of RAGE protein was equilibrated and buffered. Liquid (2
0 mM Tris-HCl (pH 7.4) containing 0.1 M NaCl and 0.
AGE equilibrated with 5% 1-On-octyl-bD-glucopyranoside)
Added to the affinity column. After washing the column with 10 volumes of equilibration buffer, the bound protein is washed with elution buffer (2
0 mM Tris-HCl (pH 7.4) containing 2 MNaCl and 0.5%
It was eluted with 1-On-octyl-bD-glucopyranoside). 3
Western blot analysis using an anti-RAGE antibody that commonly recognizes splice variants of each species analyzes the sample before addition to the column, the flow-through fraction of the column, and the elution fraction to test the AGE-binding ability of each variant. did. As a result, the full-length (Full) and soluble (Soluble) RAGE proteins were found in the elution fraction (Bound) at the N-truncated RAGE protein.
The protein was recovered in the pass through (Fig. 1). This indicates that the N-terminal immunoglobulin V region-like domain in the former two and not in the latter has AGE binding ability.

Example 3 [Synthesis of oligopeptides and competitive test] On a RAGE protein by a peptide antagonistic assay by the surface plasmon resonance method.
The AGE binding site was determined. That is, V-N1, V-N2, VC covering the human RAGE N-terminal immunoglobulin-like V region
Four oligopeptides of 1, V-C2: V-N1, KGAPKKPPQRLEWKLN 39-54 (SEQ ID NO: 7 in the sequence listing) V-N2, WKLNTGRTEAWKVLSPQG 51-68 (SEQ ID NO: 8 in the sequence listing) V-C1, SPQGGGPWDSVARVLPNGSL 65-84 (SEQ ID NO: 9 in the sequence listing) V-C2, PNGSLFLPAVGIQDEGIFR 80-98 (SEQ ID NO: 10 in the sequence listing) (In the above formula, the number shown next to the one-letter amino acid sequence is the sequence (Showing the position of the amino acid residue on the amino acid sequence of SEQ ID NO: 2) was synthesized, and RA was prepared by preincubating AGE with each peptide for 2 hours before the assay.
A specific peptide showing a binding inhibitory activity with GE was identified.

Specifically, a sensor chip CM5 on which purified soluble RAGE protein was immobilized by the amino coupling method was used in an amount of 10 mM.
After equilibration with HEPES (pH 7.4) and 0.15 M NaCl, flow rate at 25 ℃ 20
50 mg / ml AGE was injected under the condition of ml / min, and the mass change was investigated using the BIAcore 2000 system (Biacore, Tokyo). 10 mMN for regeneration of sensor chip
AOH, 0.5% SDS solution was used. The results are shown in FIGS.
As a result, the binding between AGE-2 derived from glyceraldehyde and RAGE was inhibited by oligopeptide V-N1 in a concentration-dependent manner (Fig. 2), but it was affected by V-N2, V-C1, and V-C2. (Figs. 3 and 4). On the other hand, the binding between glycol AGE-derived AGE-3 and RAGE was inhibited by V-N2 (Fig. 6), but not affected by V-N1, V-C1, V-C2 (Figs. 5 and 7). . Therefore, the AGE binding region on the RAGE protein exists in the N-terminal half of the immunoglobulin V region-like domain, and furthermore, AGE-2 has V-N1 and AGE-3 has V-N2 and a different binding site depending on the ligand. It was estimated to have. In the above, AGE-2 (AGE2) is derived from glyceraldehyde
AGE (Watkins NG, Thorpe SR, Baynes JW: J. Biol. Ch
em. 260, 10629-10636,1985; Acharya AS, Cho YJ, Manj
ula BN: Biochemistry 27, 4522-4529, 1988; Bai Y, U
eno H, Manning JM: J. Protein Chem. 8, 299-315, 19
89; Syrovy I, J. Biochem. Biophys. Methods 28, 115
-121, 1994), and AGE-3 is AGE derived from glycol aldehyde (Namiki M, Hayashi T: J. Argric. Food Ch
em. 23, 487-491, 1975; Acharya AS, Manning JM: Pro
c. Natl. Acad. Sci. USA 80, 3590-3594, 1983; Gl
omb MA, Monnier VM: J. Biol. Chem. 270, 10017-1002
6, 1995). In addition, glucose-derived AGE (Brownlee
M, Cerami A, Vlassara H: N. Eng. J. Med. 318, 131
5-1321,1988) can also be used for measurement.

[0052]

INDUSTRIAL APPLICABILITY According to the present invention, a substance useful as an AGE-RAGE antagonist is provided, thereby preventing diabetic complications.
A drug for preventing progress can be provided. Obviously, the present invention may be carried out other than as specifically described in the above description and examples. Many modifications and variations of the present invention are possible in light of the above teachings, and are therefore within the scope of the claims appended hereto.

[0053]

[Sequence list]                                SEQUENCE LISTING        <110> President of Kanazawa University <120> AGE-RAGE Antagonists <130> P-02NF371 <140> <141> <160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 1223 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (25) .. (1068) <400> 1 gccaggaccc tggaaggaag cagg atg gca gcc gga aca gca gtt gga gcc 51                            Met Ala Ala Gly Thr Ala Val Gly Ala                              1 5 tgg gtg ctg gtc ctc agt ctg tgg ggg gca gta gta ggt gct caa aac 99 Trp Val Leu Val Leu Ser Leu Trp Gly Ala Val Val Gly Ala Gln Asn  10 15 20 25 atc aca gcc cgg att ggc gag cca ctg gtg ctg aag tgt aag ggg gcc 147 Ile Thr Ala Arg Ile Gly Glu Pro Leu Val Leu Lys Cys Lys Gly Ala                  30 35 40 ccc aag aaa cca ccc cag cgg ctg gaa tgg aaa ctg aac aca ggc cgg 195 Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu Asn Thr Gly Arg              45 50 55 aca gaa gct tgg aag gtc ctg tct ccc cag gga gga ggc ccc tgg gac 243 Thr Glu Ala Trp Lys Val Leu Ser Pro Gln Gly Gly Gly Pro Trp Asp          60 65 70 agt gtg gct cgt gtc ctt ccc aac ggc tcc ctc ttc ctt ccg gct gtc 291 Ser Val Ala Arg Val Leu Pro Asn Gly Ser Leu Phe Leu Pro Ala Val      75 80 85 ggg atc cag gat gag ggg att ttc cgg tgc cag gca atg aac agg aat 339 Gly Ile Gln Asp Glu Gly Ile Phe Arg Cys Gln Ala Met Asn Arg Asn  90 95 100 105 gga aag gag acc aag tcc aac tac cga gtc cgt gtc tac cag att cct 387 Gly Lys Glu Thr Lys Ser Asn Tyr Arg Val Arg Val Tyr Gln Ile Pro                 110 115 120 ggg aag cca gaa att gta gat tct gcc tct gaa ctc acg gct ggt gtt 435 Gly Lys Pro Glu Ile Val Asp Ser Ala Ser Glu Leu Thr Ala Gly Val             125 130 135 ccc aat aag gtg ggg aca tgt gtg tca gag gga agc tac cct gca ggg 483 Pro Asn Lys Val Gly Thr Cys Val Ser Glu Gly Ser Tyr Pro Ala Gly         140 145 150 act ctt agc tgg cac ttg gat ggg aag ccc ctg gtg cct aat gag aag 531 Thr Leu Ser Trp His Leu Asp Gly Lys Pro Leu Val Pro Asn Glu Lys     155 160 165 gga gta tct gtg aag gaa cag acc agg aga cac cct gag aca ggg ctc 579 Gly Val Ser Val Lys Glu Gln Thr Arg Arg His Pro Glu Thr Gly Leu 170 175 180 185 ttc aca ctg cag tcg gag cta atg gtg acc cca gcc cgg gga gga gat 627 Phe Thr Leu Gln Ser Glu Leu Met Val Thr Pro Ala Arg Gly Gly Asp                 190 195 200 ccc cgt ccc acc ttc tcc tgt agc ttc agc cca ggc ctt ccc cga cac 675 Pro Arg Pro Thr Phe Ser Cys Ser Phe Ser Pro Gly Leu Pro Arg His             205 210 215 cgg gcc ttg cgc aca gcc ccc atc cag ccc cgt gtc tgg gag cct gtg 723 Arg Ala Leu Arg Thr Ala Pro Ile Gln Pro Arg Val Trp Glu Pro Val         220 225 230 cct ctg gag gag gtc caa ttg gtg gtg gag cca gaa ggt gga gca gta 771 Pro Leu Glu Glu Val Gln Leu Val Val Glu Pro Glu Gly Gly Ala Val     235 240 245 gct cct ggt gga acc gta acc ctg acc tgt gaa gtc cct gcc cag ccc 819 Ala Pro Gly Gly Thr Val Thr Leu Thr Cys Glu Val Pro Ala Gln Pro 250 255 260 265 tct cct caa atc cac tgg atg aag gat ggt gtg ccc ttg ccc ctt ccc 867 Ser Pro Gln Ile His Trp Met Lys Asp Gly Val Pro Leu Pro Leu Pro                 270 275 280 ccc agc cct gtg ctg atc ctc cct gag ata ggg cct cag gac cag gga 915 Pro Ser Pro Val Leu Ile Leu Pro Glu Ile Gly Pro Gln Asp Gln Gly             285 290 295 acc tac agc tgt gtg gcc acc cat tcc agc cac ggg ccc cag gaa agc 963 Thr Tyr Ser Cys Val Ala Thr His Ser Ser His Gly Pro Gln Glu Ser         300 305 310 cgt gct gtc agc atc agc atc atc gaa cca ggc gag gag ggg cca act 1011 Arg Ala Val Ser Ile Ser Ile Ile Glu Pro Gly Glu Glu Gly Pro Thr     315 320 325 gca ggt gag ggg ttt gat aaa gtc agg gaa gca gaa gat agc ccc caa 1059 Ala Gly Glu Gly Phe Asp Lys Val Arg Glu Ala Glu Asp Ser Pro Gln 330 335 340 345 cac atg tga ctggggggat ggtcaacaag aaaggaatgg aaggccccag 1108 His Met aaaaccagga ggaagaggag gagcgtgcag aactgaatca gtcggaggaa cctgaggcag 1168 gcgagagtag tactggaggg ccttgagggg cccacagaca gatcccatcc atcag 1223 <210> 2 <211> 347 <212> PRT <213> Homo sapiens <400> 2 Met Ala Ala Gly Thr Ala Val Gly Ala Trp Val Leu Val Leu Ser Leu   1 5 10 15 Trp Gly Ala Val Val Gly Ala Gln Asn Ile Thr Ala Arg Ile Gly Glu              20 25 30 Pro Leu Val Leu Lys Cys Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg          35 40 45 Leu Glu Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu      50 55 60 Ser Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro  65 70 75 80 Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly Ile                  85 90 95 Phe Arg Cys Gln Ala Met Asn Arg Asn Gly Lys Glu Thr Lys Ser Asn             100 105 110 Tyr Arg Val Arg Val Tyr Gln Ile Pro Gly Lys Pro Glu Ile Val Asp         115 120 125 Ser Ala Ser Glu Leu Thr Ala Gly Val Pro Asn Lys Val Gly Thr Cys     130 135 140 Val Ser Glu Gly Ser Tyr Pro Ala Gly Thr Leu Ser Trp His Leu Asp 145 150 155 160 Gly Lys Pro Leu Val Pro Asn Glu Lys Gly Val Ser Val Lys Glu Gln                 165 170 175 Thr Arg Arg His Pro Glu Thr Gly Leu Phe Thr Leu Gln Ser Glu Leu             180 185 190 Met Val Thr Pro Ala Arg Gly Gly Asp Pro Arg Pro Thr Phe Ser Cys         195 200 205 Ser Phe Ser Pro Gly Leu Pro Arg His Arg Ala Leu Arg Thr Ala Pro     210 215 220 Ile Gln Pro Arg Val Trp Glu Pro Val Pro Leu Glu Glu Val Gln Leu 225 230 235 240 Val Val Glu Pro Glu Gly Gly Ala Val Ala Pro Gly Gly Thr Val Thr                 245 250 255 Leu Thr Cys Glu Val Pro Ala Gln Pro Ser Pro Gln Ile His Trp Met             260 265 270 Lys Asp Gly Val Pro Leu Pro Leu Pro Pro Ser Pro Val Leu Ile Leu         275 280 285 Pro Glu Ile Gly Pro Gln Asp Gln Gly Thr Tyr Ser Cys Val Ala Thr     290 295 300 His Ser Ser His Gly Pro Gln Glu Ser Arg Ala Val Ser Ile Ser Ile 305 310 315 320 Ile Glu Pro Gly Glu Glu Gly Pro Thr Ala Gly Glu Gly Phe Asp Lys                 325 330 335 Val Arg Glu Ala Glu Asp Ser Pro Gln His Met             340 345 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 3 gccaggaccc tggaaggaag ca 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 4 ctgatggatg ggatctgtct gtg 23 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 5 gagaattcgc caggaccctg gaaggaagca 30 <210> 6 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence:       Oligonucleotide to act as a primer for PCR <400> 6 gatctagaga ttgttgacca tccccccag 29 <210> 7 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       peptide to act as an AGE-RAGE antagonist <400> 7 Lys Gly Ala Pro Lys Lys Pro Pro Gln Arg Leu Glu Trp Lys Leu Asn   1 5 10 15 <210> 8 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       peptide to act as an AGE-RAGE antagonist <400> 8 Trp Lys Leu Asn Thr Gly Arg Thr Glu Ala Trp Lys Val Leu Ser Pro   1 5 10 15 Gln Gly          <210> 9 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       peptide to act as an AGE-RAGE antagonist <400> 9 Ser Pro Gln Gly Gly Gly Pro Trp Asp Ser Val Ala Arg Val Leu Pro   1 5 10 15 Asn Gly Ser Leu              20 <210> 10 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Designed       peptide to act as an AGE-RAGE antagonist <400> 10 Pro Asn Gly Ser Leu Phe Leu Pro Ala Val Gly Ile Gln Asp Glu Gly   1 5 10 15 Ile Phe Arg

[Brief description of drawings]

Figure 1: Pass through to show the binding properties of RAGE splice variants to AGE affinity columns.
・ Polyacrylamide gel electrophoresis of the elution (Bound) fraction
The results of Western analysis using RAGE antibody after (PAGE) are shown.

FIG. 2 shows the results of a peptide competitive inhibition assay. Synthetic peptide V-N1 and glyceraldehyde-derived AGE (50 μg / ml)
The surface plasmon resonance was measured after preincubation for 2 hours at the molar ratio shown on the horizontal axis.

FIG. 3 shows the results of a peptide competitive inhibition assay. Synthetic peptide V-N2 and AGE derived from glyceraldehyde (50 μg / ml)
The surface plasmon resonance was measured after preincubation for 2 hours at the molar ratio shown on the horizontal axis.

FIG. 4 shows the results of a peptide competitive inhibition assay. Synthetic peptide V-C1 or V-C2 and glyceraldehyde-derived AG
After preincubating E (50 μg / ml) for 2 hours at the molar ratio shown on the horizontal axis, surface plasmon resonance was measured.

FIG. 5 shows the results of a peptide competitive inhibition assay. Synthetic peptide V-N1 and AGE derived from glycol aldehyde (50 μg / m
The surface plasmon resonance was measured after pre-incubation for 2 hours at the molar ratio (l) shown on the horizontal axis.

FIG. 6 shows the results of a peptide competitive inhibition assay. Synthetic peptide V-N2 and AGE derived from glycolaldehyde (50 μg / m
The surface plasmon resonance was measured after pre-incubation for 2 hours at the molar ratio (l) shown on the horizontal axis.

FIG. 7 shows the results of a peptide competitive inhibition assay. Derived from synthetic peptide V-C1 or V-C2 and glycolaldehyde
Surface plasmon resonance was measured after preincubation of AGE (50 μg / ml) for 2 hours at the molar ratio shown on the horizontal axis.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) A61P 9/10 101 A61P 15/10 13/12 17/02 15/10 21/04 17/02 25/00 21/04 25/02 25/00 25/14 25/02 25/28 25/14 27/02 25/28 29/00 27/02 35/00 29/00 35/04 35/00 37/02 35 / 04 43/00 111 37/02 C07K 7/08 ZNA 43/00 111 C12N 15/00 A C07K 7/08 ZNA A61K 37/02 (72) Inventor Takuo Watanabe 2-11-5 Wakunami, Kanazawa, Ishikawa Prefecture Wakunami lodging (1) 42 (72) Inventor Yasuhiko Yamamoto 2710 Tateno, Takaoka City, Toyama Prefecture (72) Inventor Shigeru Sakurai 3-6-19 Nagasaka, Kanazawa City, Ishikawa Sunny Hill III 102 F term (reference) 4B024 AA01 BA63 CA04 CA05 DA02 DA03 EA04 GA11 GA18 HA03 HA14 4C084 AA02 AA03 AA07 BA18 BA23 CA18 CA53 CA59 MA13 MA17 MA22 MA23 MA28 MA31 MA3 2 MA35 MA36 MA37 MA38 MA41 MA43 MA44 MA52 MA56 MA58 MA59 MA60 MA63 MA66 MA67 NA14 ZA022 ZA152 ZA162 ZA202 ZA222 ZA332 ZA362 ZA452 ZA672 ZA812 ZA892 ZA942 ZB072 ZB112 ZB262 ZC352 ZC422 4H045 AA40 FA50A17 BA17 BA17 BA50

Claims (11)

[Claims] 1. Lys-Gly-Ala-Pro-Lys-Lys-Pro-Pro-Gln-Ar
g-Leu-Glu-Trp-Lys-Leu-Asn (SEQ ID NO: 7 in Sequence Listing) and Trp-Lys-Leu-Asn-Thr-Gly-Arg-Thr-Glu-Ala-Trp-Lys-
A peptide having an amino acid sequence corresponding to the amino acid sequence selected from the group consisting of Val-Leu-Ser-Pro-Gln-Gly (SEQ ID NO: 8 in the sequence listing), a derivative thereof or a salt thereof. 2. Lys-Gly-Ala-Pro-Lys-Lys-Pro-Pro-Gln-
Arg-Leu-Glu-Trp-Lys-Leu-Asn (SEQ ID NO: 7 in Sequence Listing)
The compound according to claim 1, which is a peptide or a derivative thereof. 3. Trp-Lys-Leu-Asn-Thr-Gly-Arg-Thr-Glu-
The compound according to claim 1, which is a peptide which is Ala-Trp-Lys-Val-Leu-Ser-Pro-Gln-Gly (SEQ ID NO: 8 in the sequence listing) or a derivative thereof. 4. A pharmaceutical composition comprising the compound according to claim 1 as an active ingredient. 5. A late saccharification reaction product (AGE) and its receptor (R) containing the compound according to claim 1 as an active ingredient.
AGE- characterized by inhibiting the interaction with AGE-
RAGE antagonist. 6. A method for inhibiting AGE-RAGE interaction, which comprises contacting a sample containing RAGE and / or cells expressing RAGE with the AGE-RAGE antagonist according to claim 5. 7. (i) Contacting an AGE-containing test sample with RAGE or RAGE-expressing cells in the presence or absence of the peptide of SEQ ID NO: 7 or 8 to obtain the presence or absence of the peptide. AGE-RAGE interaction analysis method characterized by performing a comparison between, or (ii) (a) in the presence or absence of the peptide of SEQ ID NO: 7 or the peptide of SEQ ID NO: 8, further, ( b) Contacting the test sample with RAGE or RAGE-expressing cells in the presence or absence of glyceraldehyde-derived AGE or glycolaldehyde-derived AGE, and between the presence and absence of the peptide and glyceraldehyde-derived From AGE or glycol aldehyde
The inhibition method according to claim 6, which is an AGE-RAGE interaction analysis method, characterized in that comparison is performed in the presence or absence of AGE. 8. RAGE-expressing cells are endothelial cells, vascular smooth muscle cells, nerve cells, macrophages, lymphocytes, retinal vascular cells, retinal nerve cells, gingival-related cells, skin-related cells, glomerular cells, renal tubular cells, and The method according to claim 6 or 7, wherein the method is selected from the group consisting of connective tissue cells. 9. A compound containing the compound according to claim 1 as an active ingredient, wherein the interaction between AGE and its receptor, RAGE
A medicament for treating a disease caused by a substance selected from the group consisting of a change in the expression level and a change in the AGE scavenging activity. 10. From the onset and / or progress of diabetic complications, various diseases associated with aging, Alzheimer's disease, arteriosclerosis, diseases or diseases caused by glycation of in vivo proteins, and invasion or diffusion of tumors. The medicament according to claim 9, which is for a pathological condition or symptom selected from the group consisting of: 11. Diabetes, diabetic complications, diabetic nephropathy,
Diabetic retinopathy, diabetic angiopathy, diabetic microangiopathy, renal glomerulosclerosis, hyperlipid atherosclerosis, neuronal toxicity, Down syndrome, dementia associated with head trauma, amyotrophic lateral sclerosis , Multiple sclerosis, amyloidosis, autoimmune disease, inflammation, tumor, cancer, erectile dysfunction in men, wound healing,
The medicament according to claim 9 or 10, which is for preventing and / or treating a substance selected from the group consisting of periodontal disease, neuropathy and neurodegeneration.