JPH04217700A - Cm-chitin derivative and its use - Google Patents

Abstract

PURPOSE:To obtain a new carboxymethyl (CM)-chitin derivative, having tripeptide of arginine-glycine-aspartic acid as essential units and useful as an adhesion inhibitor for animal cells and a blood platelet agglutination.adhesion inhibitor, etc. CONSTITUTION:Carboxymethyl-chitin is dissolved in a phosphate buffer solution at pH7.4 and a peptide containing adhesive tripeptide units of arginine-glycine- aspartic acid is added in the presence of water-soluble carbodiimide while keeping the solution at 0 deg.C. Reaction is carried out overnight at 4 deg.C and dialysis is then performed against ion exchange water to remove low-molecular weight ingredients and purify the resultant product. The purified compound is subsequently freeze-dried to afford the objective compound containing an adhesive peptide, expressed by the formula [X and Y are amino acid residue or peptide residue such as Ser, Gly, Val, Asn or Pro; R<1> and R<2> are H, 1-9C alkyl, 6-9C aryl, etc.; Z is O, NH, etc.; (n) is 1-5] as an essential ingredient and linked through any of amide bond, ester bond, ether bond and urethane bond and to the side chain of the carboxymethyl-chitin.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] The present invention relates to Arg-Gly-As
The present invention relates to a CM-chitin derivative having a p tripeptide as an essential unit, a salt thereof, and an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing the same as an active ingredient.

0002

BACKGROUND OF THE INVENTION Fibronectin is a protein involved in cell-extracellular matrix adhesion, and is also thought to be involved in platelet aggregation and cancer metastasis. These interactions are mediated by a series of cell surface receptors, and although fibronectin is a large molecule with a molecular weight of approximately 250,000, these receptors
It was revealed that it specifically recognizes the Asp sequence,
It has been reported that it is important for interaction with receptors (Nature, No. 309).
Vol. 30, 1984). Since then, Arg-Gly-A
Research is underway using oligos or polypeptides having sp sequences.

For example, methods for inhibiting platelet aggregation using various linear and cyclic oligopeptides having the Arg-Gly-Asp sequence (Proceedings of the Society of Polymer Science and Technology (Pol.
ymer Preprints, Japan), No. 3
Volume 8, page 3149, 1989, JP-A-2-17479
No. 7), Method of using a peptide having an Arg-Gly-Asp sequence as a cell migration inhibitor (Unexamined Japanese Patent Publication No. 2-4
716), method of using PMMA membrane with immobilized Arg-Gly-Asp as a cell adhesion membrane (Polymer Preprints, Jap.
an), Vol. 37, p. 705, 1988). Furthermore, the polymer has Arg-Gly-Asp.
A method of covalently bonding peptides having as essential constituent units and using them as animal cell culture substrates and biocomposite artificial organ substrates (JP-A-1-309682, JP-A-1-305960)
JP-A No. 64-6217) discloses a method of using a polypeptide having the Arg-Gly-Asp-Ser sequence as a platelet protecting agent for extracorporeal blood. Furthermore, a method for suppressing cancer metastasis using an oligopeptide having an Arg-Gly-Asp sequence or a polypeptide having a repeating structure thereof is known ((In
t. J. Biol. Macromol. ), vol. 11, p. 23, 1989, same magazine, vol. 11, 226
Page, 1989, (Jpn. J. CancerRe
s. ) Vol. 60, p. 722, 1989).

Chitin is a polysaccharide in which N-acetyl-D-glucosamine is linked in a β-(1→4) manner, and is the main component of the exoskeleton of shellfish and insects. It is widely distributed among lower animals and invertebrates, and plays a supporting and protective role for living organisms, and corresponds to cellulose in the plant kingdom. Chitin is also called the last biomass, and research on its derivatives has been actively conducted in recent years, and in particular, many studies on chitin derivatives that are soluble in solvents have been reported. CM-chitin, in which a carboxylmethyl group is bonded to the hydroxyl group at the C-6 position, is water-soluble and is an important compound as a starting material for various chitin derivatives. Chitin and its derivatives are described in detail in ``Applications of Chitin and Chitosan'' (Gihodo), edited by the Chitin and Chitosan Study Group, and ``Chitin and Chitosan, the Last Biomass'' (Gihodo), edited by the Chitin and Chitosan Study Group.

[0005] Since deacetylation occurs in CM-chitin during carboxylation, amino groups are also present in addition to carboxyl groups. The amino groups of CM-chitin can be easily carboxylated with dibasic acids and their derivatives, preferably polybasic acid anhydrides. Furthermore, N,O-sulfation of CM-chitin can be easily carried out. However, as a derivative of CM-chitin, Arg-Gly-A
There are no known compounds into which an oligopeptide having sp as an essential unit or a polypeptide having a repeating structure thereof is introduced, and when introduced, it is expected that the ability to bind to a receptor will be enhanced and stabilization in blood will occur.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a novel CM-chitin derivative and an animal cell adhesion inhibitor and platelet aggregation/adhesion inhibitor containing the same as an active ingredient.

[0007]

[Means for Solving the Problems] The present invention provides adhesive peptides represented by the following general formula [I] that are bonded to the side chains of CM-chitin through any one of amide bonds, ester bonds, ether bonds, and urethane bonds. The object of the present invention is to provide a chitin derivative having as an essential unit and a salt thereof. General formula [I] -[R1]-[CO]-([X]-Arg-Gly-A
sp-[Y])n-[Z]-[R2]- [ ] may or may not exist, and if present, X, Y are Ser, Gly, Val, As
represents an amino acid residue or a peptide residue selected from n, Pro, and Z represents -O- or -NH-. R1
, R2 is hydrogen, a linear or branched alkyl group having 1-9 carbon atoms, or an aryl group having 6-9 carbon atoms, and may have a substituent. The other is hydrogen, a linear or branched alkylene group having 1 to 9 carbon atoms, or an arylene group having 6 to 9 carbon atoms, and may have a substituent. n represents an integer of 1-5.

Substituents for R1 and R2 include carbonyl groups, carboxyl groups, amino groups, hydroxyl groups, sulfo groups, halogen atoms, aryl groups, nitro groups, cyano groups, double bonds and triple bonds of unsaturated groups. etc. are mentioned,
The same chain may have two or more. The present invention further provides an animal cell adhesion inhibitor and a platelet aggregation/adhesion inhibitor containing the above-mentioned CM-chitin derivative or its salt as an active ingredient.

The present invention provides Arg-G to sulfated CM-chitin, carboxylated CM-chitin or CM-chitin.
This is a CM-chitin derivative formed by covalently bonding an adhesive peptide having ly-Asp as an essential unit. CM-
The molecular weight of the chitin derivative is preferably 200,000 or less, particularly 3
000-100,000 and is preferably water-soluble at room temperature. Examples of the carboxylating agent include succinic anhydride, maleic anhydride, phthalic anhydride, itaconic anhydride, citraconic anhydride, pyromellitic anhydride, trimellitic anhydride, and the like. The amino acid used in the adhesive peptide according to the present invention may be either L-form or D-form, but preferably L-form.

Examples of the salts of the CM-chitin derivative of the present invention include salts with inorganic acids such as hydrochloride, sulfate, nitrate, phosphate, and borate, acetate, trifluoroacetate,
Examples include salts with organic acids such as trifluoromethanesulfonate, lactate, and tartrate. The peptide synthesis method is not particularly limited, but includes a liquid phase method, a solid phase method, and a synthesis method using an automatic synthesizer. For details on these synthesis methods, please refer to the Biochemistry Experimental Course “Protein Chemistry IV” p207-495 (edited by the Japanese Biochemical Society, Tokyo Kagaku Dojin), the “Continued Biochemistry Experimental Course Protein Chemistry (Part 2)” (
It is described in "Basics and Experiments of Peptide Synthesis" (edited by the Japanese Biochemical Society, Tokyo Kagaku Doujin) and Izumiya et al. (Maruzen). Also,
It is also possible to utilize commercially available synthetic peptides.

CM-chitin and carboxylated CM-
Examples of bonding between chitin and adhesive peptide include amide bond synthesis methods using cyanogen bromide, acid azide, water-soluble carbodiimide, and the like. The CM-chitin derivative of the present invention is
Core sequence of cell adhesion protein Arg-Gly-Asp
It adheres to cells via the core sequence using a mechanism similar to that of cell adhesion proteins. Therefore, it exhibits various biological activities as an agonist or antagonist of cell adhesion proteins, and has a wide range of biological activities such as immunomodulation, wound healing, inhibition of platelet aggregation by cancer cells that occur in capillaries, and healing of neurological diseases. It recognized.

[0012] Therefore, the CM-chitin derivative of the present invention is
At least one of them can be administered to a patient as a cancer metastasis inhibitor, wound healing agent, immunomodulator, or platelet aggregation/adhesion inhibitor, optionally together with a conventional carrier or pharmaceutical auxiliary. In particular, use as an animal cell adhesion inhibitor or platelet aggregation/adhesion inhibitor is preferred. The dosage ranges from 0.2 μg/kg to 400 mg/kg,
Determined based on symptoms, age, weight, etc.

The CM-chitin derivative of the present invention is preferably administered by a method commonly used for peptide drugs, that is, by parenteral administration, such as intravenous administration, intramuscular administration, subcutaneous administration, etc. When producing such an injectable preparation, the chitin derivative of the present invention is dissolved in PBS or physiological saline, for example, as shown in the Examples below.
It may be prepared as an injection preparation, or it may be prepared as a lyophilized preparation after being dissolved in approximately 0.1N acetic acid water or the like. Conventional stabilizers such as glycine and albumin may be added to such formulations. Furthermore, the chitin derivative of the present invention can be administered orally, for example, in the form of microcapsules encapsulated in liposomes, and can be administered orally in the form of suppositories, sublingual tablets, nasal sprays, etc. It is also possible to absorb it through mucous membranes other than the ducts.

[0014]

[Examples] The present invention will be further explained below with reference to Examples, but the present invention is not limited thereto. Production Example 1 Synthesis of adhesive peptide by solid phase method Mer
Synthesis was performed using a peptide synthesizer using the Rifield method. A Boc group was used to protect the α-amino group, and after cutting it out from the resin, it was purified by preparative HPLC (high performance liquid chromatography) to obtain an adhesive synthetic peptide showing a single peak. Adhesive synthetic peptide
Name Structural formula
Abbreviation Sequence number Yield
Peptide-1 H-Arg-Gly-As
p-OH RGD 1
37% Peptide-2H-(A
rg-Gly-Asp)2-OH (RGD)2
2 28% Peptide-3H-(Arg-Gly-Asp)3-OH
(RGD) 3 3 19%
Peptide-4H-(Arg-Gly-As
p)5-OH (RGD)5 4
11%

Production Example 2 Peptide-5 H-(Arg-Gly-Asp-Ser
-Gly)-NH2 (SEQ ID NO: 5) Peptide-5 was synthesized by a liquid phase method using a sequential extension method. (1) Synthesis of BocSer(Bzl)GlyNH2 59g (0.2mol) of BocSer(Bzl) was
Dissolve in 2400 ml of H2Cl and dissolve 41.2 g of DCC (0
．． 2 mol) was added under ice cooling. Add GlyN to this solution.
After 22.1 g of H2.HCl was dissolved in 400 ml of CH2Cl2, a solution neutralized with 20.2 g of N-methylmorpholine under ice cooling was added. The mixture was stirred for 3 hours under ice cooling, and then further stirred at room temperature overnight. The precipitate was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. NaHCO3 aqueous solution,
Wash with 1M citric acid aqueous solution and NaCl aqueous solution in order, and
Dry with a2SO4 and dry under reduced pressure to obtain a white powder 58.
3 g (yield 83%) was obtained.

(2) BocAsp(OBzl)Ser(
Synthesis of Boc Ser(Bzl)GlyNH2
)GlyNH2 56.2g (0.16mol) with TF
Add 400ml of A/CH2Cl2=1/1 and mix at room temperature.
After stirring for an hour, TFA and CH2Cl2 were concentrated under reduced pressure. This was dissolved in ethyl acetate, neutralized with an aqueous NaHCO3 solution, and then washed with an aqueous NaCl solution. After drying with Na2SO4, ethyl acetate was distilled off under reduced pressure.

[0017] This compound and BocAsp(OBzl)
51.7g (0.16mol) of CH2Cl2 80
33 g (0.16 mol) of DCC was added under ice cooling, stirred for 3 hours, and further stirred overnight at room temperature. After CH2Cl2 was distilled off under reduced pressure, the residue was dissolved in ethyl acetate. NaHCO3 aqueous solution, 1M citric acid aqueous solution, N
The mixture was washed with an aCl aqueous solution, dried over Na2SO4, and then evaporated to dryness to obtain 71.2 g of white powder (yield: 80%).

(3) BocGlyAsp(OBzl)S
Synthesis of er(Bzl)GlyNH2 BocAsp(OBzl)Ser(Bzl)GlyN
TFA:CH2 to H2 66.7g (0.12mol)
After adding 400 ml of Cl2=1:1 and stirring at room temperature for 1 hour, TFA was concentrated under reduced pressure. This was dissolved in ethyl acetate, neutralized with an aqueous NaHCO3 solution, and then washed with an aqueous NaCl solution. After drying with Na2SO4, ethyl acetate was distilled off under reduced pressure.

[0019] This compound and BocGly51.7g (0
．． 12 mol) was dissolved in 700 ml of CH2Cl2,
Add 24.7 g (0.12 mol) of DCC under ice cooling 3
After stirring for an hour, the mixture was further stirred at room temperature overnight. DCu
The rea was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. NaHCO3 aqueous solution, 1M citric acid aqueous solution, NaCl
The mixture was washed with an aqueous solution, dried over Na2SO4, and then evaporated to dryness to obtain 61.8 g of white powder (yield: 84%).

(4) BocArg(Mts)GlyAs
Synthesis of p(OBzl)Ser(Bzl)GlyNH2BocGlyAsp(OBzl)Ser(Bzl)Gl
y NH2 61.3g (0.1mol) TFA:C
After adding 400 ml of H2Cl2=1:1 and stirring at room temperature for 1 hour, TFA and CH2Cl2 were concentrated under reduced pressure. This was dissolved in ethyl acetate, neutralized with an aqueous NaHCO3 solution, and then washed with an aqueous NaCl solution. After drying with Na2SO4, ethyl acetate was distilled off under reduced pressure.

This compound and BocArg(Mts)(M
ts represents a mesitylene-2-sulfonyl group)45.
Dissolve 6g (0.1mol) in DMF 800ml, DCC22.5g (0.1mol), HOBt14
g (0.1 mol) was added under ice cooling, stirred for 3 hours, and then
The mixture was further stirred at room temperature overnight. DCurea was filtered off, concentrated under reduced pressure, and dissolved in ethyl acetate. Washed with NaHCO3 aqueous solution, 1M citric acid aqueous solution, and NaCl aqueous solution in this order, dried with Na2SO4, and dried under reduced pressure to obtain white powder 4.
2.8 g (yield 45%) was obtained.

(5) Deprotection of Peptide-5 Protected Form BocArg(Mts)GlyAsp(OBzl)Se
r(Bzl)GlyNH2 5g (5.3mmol)
A TFA solution of 1M trifluoromethanesulfonic acid-thioanisole-m-cresol was added to the TFA solution under ice cooling and allowed to react for 1 hour to deprotect the peptide side chain and terminal protecting group. The reaction solution was poured into ether, and the oily precipitate was dissolved in distilled water and washed with ethyl acetate.
A-400Cl type) to form a hydrochloride and freeze-dry it. 2.17 g (86% yield) of white solid was obtained. Amino acid analysis (nmol/50 μl) Arg
4.9877, Gly 10.3916, Asp 5.0199, Ser 4.8891, Mass spectrum M+ 404

Production Example 3 Peptide-6 H-(Gly-Arg-Gly-As
p-Ser-Pro)-OH (SEQ ID NO: 6) Merri
Synthesis was performed using a field system peptide synthesizer. A Boc group was used to protect the α-amino group, and after being excised from the resin, it was purified by preparative HPLC (high performance liquid chromatography) to obtain an adhesive synthetic peptide showing a single peak. Yield 25%

Synthesis Example 1 Synthesis of CM-chitin-RGDS (SEQ ID NO: 7) Viscosity: 9 cps (1% solution, 20°C), degree of etherification: 0.
78, 0.30 g of CM-chitin (manufactured by Yaizu Suisan Kagaku Kogyo) with a degree of deacetylation of 0.5 was dissolved in a phosphate buffer of pH 7.4, and water-soluble DCC [1-ethyl-3, A solution of 128 mg of 3-(dimethylaminopropyl)-carbodiimide in 2.6 ml of phosphate buffer was added, and the mixture was reacted for 1.5 hours. Then, the adhesive peptide Arg-G dissolved in 8 ml of phosphate buffer
400 mg of ly-Asp-Ser (RGDS) (manufactured by Kokusan Kagaku Kogyo) was added and reacted overnight at 4°C. Put the reaction solution into a Visking tube, add ion exchange water,
Then, it is purified by dialysis against pure water to remove low molecular weight components.
Lyophilized. Yield: 0.24g Structure confirmed by IR
and amino acid analysis. CM-Chitin RGDS Amino acid analysis (nmol/50μl) Glucosamine 20.5558Arg
2.0556Gly
2.1352Asp
1.9854Ser
The introduction rate of the RGDS fragment was calculated from the ratio of the arginine residue concentration to the glucosamine concentration according to the calculation formula below 1.8792, and a value of about 10% was obtained.

[0025] Introduction rate = [Arg] / [glucosamine] ×10
0 IR: Stretching vibration of amide carbonyl (C=O)
1652cm-1 Synthesis Example 2 Succinylated CM-chitin-RGDS
20.0 g of CM-chitin from Synthesis Example 1 was dissolved in 100 ml of 1% triethylamine solution, and 34.0 g of CM-chitin from Synthesis Example 1 was dissolved in 100 ml of 1% triethylamine solution.
0g, 2.00g of 4-dimethylaminopyridine was added, and the mixture was stirred at room temperature all day and night. After the reaction was completed, the solution was poured into a large excess of acetone to reprecipitate succinylated CM-chitin. The precipitate was collected, washed with a large amount of methanol, then washed with ether, and dried under vacuum. Yield 22.40g Succinylated CM-chitin 0.30g was dissolved in pH 7.4 phosphate buffer, and water-soluble DCC was added while keeping it at 0°C.
(1-ethyl-3,3-(dimethylaminopropyl)-
A solution of 128 mg (carbodiimide) in 2.6 ml phosphate buffer was added and reacted for 1.5 hours. Then, 8
400 mg RGDS dissolved in ml phosphate buffer
was added and reacted overnight at 4°C. The reaction solution was placed in a Visking tube and dialyzed against ion-exchanged water and then against pure water, purified to remove low molecular weight components, and freeze-dried. Yield: 0.26g Structure confirmation:
This was done by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 10%.

The structural formula of succinylated CM-chitin-RGDS is shown below.

[0027]

[Chemical formula 1]

Succinylated CM-chitin RGDS Amino acid analysis (nmol/50 μl) Glucosamine 23.6218Arg
2.3622Gly
2.1253Asp
2.2391Ser
2.0031IR: Amidocarbonyl (C=O)
Stretching vibration of 1652cm-1

Synthesis Example 3 Maleylated CM-Chitin RGDS Maleylated C
21.60 g of M-chitin was obtained. 0.30 g of maleylated CM-chitin was dissolved in a phosphate buffer of pH 7.4, and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 2. Yield: 0.33g The structure was confirmed by IR and amino acid analysis. Amino acid analysis results R
The introduction rate of GDS fragment was about 11%. Maleylated CM-chitin RGDS Amino acid analysis (nmol/50 μl) Glucosamine 28.4956Arg
3.1345Gly
2.7751Asp
2.7213Ser
2.5694IR: Amidocarbonyl (C=O)
The stretching vibration of 1648 cm-1

Synthesis Example 4 Phthaloylated CM-Chitin-RGDS 20.00 g of CM-chitin from Synthesis Example 1 and 50.0 g of phthalic anhydride were reacted in the same manner as in Synthesis Example 2 to obtain 22.31 g of phthaloylated CM-chitin. Obtained.

[0031] 0.30 g of phthaloylated CM-chitin was added to p
It was dissolved in H7.4 phosphate buffer, and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 2. Yield 0
．． The structure of 44g was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 12%. Phthaloylated CM-Chitin RGDS Amino acid analysis (nmol/50μl) Glucosamine 19.1856Arg
2.3023Gly
2.2231Asp
1.8937Ser
1.7632IR: Amidocarbonyl (C=O)
The stretching vibration of 1652 cm-1

Synthesis Example 5 Itaconylated CM-Chitin-RGDS 20.00 g of CM-chitin from Synthesis Example 1 and 38.0 g of itaconic anhydride were reacted in the same manner as in Synthesis Example 2 to obtain 21.45 g of itaconylated CM-chitin. Ta. Itaconylated CM-
0.30 g of chitin was dissolved in a phosphate buffer of pH 7.4, and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 2. Yield: 0.36g Structure confirmed by IR
and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 9%. Itaconylated CM-chitin RGDS Amino acid analysis (nmol/50μl) Glucosamine 35.2316Arg
3.1708Gly
3.2511Asp
3.1005Ser
2.8862IR: Amidocarbonyl (C=O)
The stretching vibration of 1650cm-1

Synthesis Example 6 Trimethylated CM-chitin-RGDS 20.00 g of CM-chitin from Synthesis Example 1 and 64.9 g of trimellitic anhydride were reacted in the same manner as in Synthesis Example 2 to obtain 23.75 g of trimerytylated CM-chitin. Obtained. 0.30 g of trimerytylated CM-chitin was dissolved in a phosphate buffer of pH 7.4, and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 2. Yield: 0.37g Structure confirmation:
This was done by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 14%. Trimethylated CM-chitin RGDS Amino acid analysis (nmol/50μl) Glucosamine 18.7612Arg
2.6266Gly
2.7899Asp
2.5532Ser
2.2689IR: Amidocarbonyl (C=O)
The stretching vibration of 1656 cm-1

Synthesis Example 7 CM-chitin-GRGD
S (SEQ ID NO: 8) Gly-Arg-G as adhesive peptide fragment
CM-chitin-GRGDS was synthesized in the same manner as in Synthesis Example 1 using 460 mg of ly-Asp-Ser (GRGDS) (manufactured by Kokusan Kagaku Kogyo). Yield: 0.36g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of GRGDS fragment is approximately 1
It was 0%. CM-Chitin-GRGDS Amino acid analysis (nmol/50μl) Glucosamine 15.3319Arg
1.5332Gly
3.2132Asp
1.3468Ser
1.1132IR: Amidocarbonyl (C=O)
The stretching vibration of 1654 cm-1

Synthesis Example 8 Succinylated CM-Chitin-GRGDS GRGDS460m as an adhesive peptide fragment
Succinylated CM-chitin-GRGDS was synthesized in the same manner as in Synthesis Example 2 using g. Yield: 0.39g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the GRGDS fragment was approximately 12%. Succinylated CM-chitin-GRGDS amino acid analysis
(nmol/50μl) Glucosamine
30.3268Arg
3.6392Gly
7.0624Asp 3.
5691Ser 3.30
06IR: Stretching vibration of amide carbonyl (C=O) 16
48cm-1

Synthesis Example 9 Succinylated CM-Chitin-RGD (SEQ ID NO: 9) 460 mg of RGD as an adhesive peptide fragment
Succinylated CM-chitin-RGD was synthesized in the same manner as in Synthesis Example 2 using. Yield: 0.34g Confirmation of structure:
This was done by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of RGD fragment was approximately 16%. Succinylated CM-chitin-RGD Amino acid analysis (nmol/50μl) Glucosamine 27.8867Arg
4.4619Gly
4.5518Asp
4.4911IR: Amidocarbonyl (C=
O) stretching vibration 1650cm-1

Synthesis Example 10 Succinylated CM-chitin-(RGD) 2 (SEQ ID NO: 10) (RGD) 2460 as an adhesive peptide fragment
Succinylated CM-
Chitin-(RGD)2 was synthesized. Yield 0.31g
The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis (RGD), the introduction rate of the 2 fragment was approximately 12%. Succinylated CM-chitin (RGD)2 Amino acid analysis (nmol/50 μl) Glucosamine
25.1913Arg
6.0459Gly
5.9883Asp
5.8996IR: Stretching vibration of amide carbonyl (C=O) 1654 cm-1

Synthesis Example 11 Succinylated CM-chitin-(RGD) 3 (SEQ ID NO: 11) (RGD) 3460 as an adhesive peptide fragment
Succinylated CM-
Chitin-(RGD)3 was synthesized. Yield 0.33g
The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis (RGD), the introduction rate of 3 fragments was approximately 10%. Succinylated CM-chitin (RGD)3 Amino acid analysis (nmol/50 μl) Glucosamine
22.3161Arg
6.6949Gly
6.5132Asp
6.2323IR: Stretching vibration of amide carbonyl (C=O) 1648 cm-1

Synthesis Example 12 Succinylated CM-chitin-(RGD) 5 (SEQ ID NO: 12) as an adhesive peptide fragment (RGD) 5460
Succinylated CM-
Chitin-(RGD)5 was synthesized. Yield 0.28g
The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis (RGD), the introduction rate of the 5 fragment was approximately 15%. Succinylated CM-chitin-(RGD)5 Amino acid analysis (nmol/50μl) Glucosamine 23.6811Arg
23.0108Gly
21.0993Asp 20.
3332IR: Stretching vibration of amide carbonyl (C=O) 1656 cm-1

Synthesis Example 13 Sulfated CM-Chitin-RG
DS (SEQ ID NO: 7) CM with degree of etherification 0.50 and degree of deacetylation 0.05
- Chitin was prepared using the method of Togura et al. (Jpn. J. Cance
rRes. , 80, 866-872 (1989)
, Cancer Res. , 50, 3631-36
37 (1990)), and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 1. Yield 0
．． The structure of 36g was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 12%.

Sulfated CM-chitin-RGDS Amino acid analysis (nmol/50 μl) Glucosamine 33.1569Arg
3.9780Gly
3.9251Asp
3.6053Ser
3.4921IR: Amidocarbonyl (C=O)
The stretching vibration of 1650cm-1

Synthesis Example 14 Sulfated CM-Chitin-GR
GDS (SEQ ID NO: 8) 460 mg of adhesive peptide fragment GRGDS and sulfated CM-chitin of Synthesis Example 13 were covalently bonded in the same manner as in Synthesis Example 1 to synthesize sulfated CM-chitin-GRGDS. Yield: 0.35g The structure was confirmed by IR and amino acid analysis. Amino acid analysis result GR
The introduction rate of GDS fragment was about 10%. Sulfated CM-chitin-GRGDS Amino acid analysis (nmol/50μl) Glucosamine 25.1515Arg
2.51 Gly
5.2134Asp
2.4251Ser
2.1111IR: Stretching vibration of amide carbonyl (C=O) 1655 cm-1

Synthesis Example 15 Sulfated succinylated CM-
Chitin-RGDS (SEQ ID NO: 7) The succinylated CM-chitin of Synthesis Example 2 was sulfated in the same manner as in Synthesis Example 13, and the RGDS fragment was covalently bonded in the same manner as in Synthesis Example 2. Yield: 0.37g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDS fragment was approximately 14%. Sulfated succinylated CM-chitin-RGDS amino acid analysis (nmol/50 μl) glucosamine
18.6932Arg
2.6170Gly
2.7739Asp
2.5931Ser 2.
2168IR: Stretching vibration of amide carbonyl (C=O) 1654 cm-1

Synthesis Example 16 Sulfated succinylated CM-
Chitin-GRGDS (SEQ ID NO: 8) The succinylated CM-chitin of Synthesis Example 2 was sulfated in the same manner as in Synthesis Example 13, and the GRGDS fragment was covalently bonded in the same manner as in Synthesis Example 7. Yield: 0.31 g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of RGDS fragment was approximately 17%.
Met. Sulfated succinylated CM-chitin-GRGDS amino acid analysis (nmol/50μl) Glucosamine 22.5661Arg
3.8362Gly
7.4963Asp
3.6811Ser 3
．． 2593IR: Stretching vibration of amide carbonyl (C=O) 1652 cm-1

Synthesis Example 17 CM-Chitin-RGDSG-NH2 (SEQ ID NO: 13)
Peptide-5 (
CM-chitin-RGDSG-NH2 was synthesized in the same manner as in Synthesis Example 1 using 460 mg of RGDSG-NH2). Yield: 0.36g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the RGDSG-NH2 fragment was approximately 10%. CM-Chitin RGDSG-NH2 Amino acid analysis (nmol/50μl) Glucosamine 17.6368Arg
1.8166Gly
3.8243Asp
1.8468Ser
1.6112IR: Amidocarbonyl (C=O)
The stretching vibration of 1658 cm-1

Synthesis Example 18 CM-Chitin-RGDS (SEQ ID NO: 7) Using 1.5 g of RGDS as the adhesive peptide fragment, Synthesis Example 1
CM-chitin-RGDS was synthesized in the same manner. Yield: 0.32g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of RGDS was approximately 20%.

CM-Chitin RGDS Amino acid analysis (nmol/50μl) Glucosamine 20.4598Arg
4.1052Gly
4.2688Asp
3.9808Ser
3.7784IR: Amidocarbonyl (C=O)
The stretching vibration of 1655cm-1

Synthesis Example 19 CM-Chitin-GRGDSP (SEQ ID NO: 14) Adhesive Peptide Fragment-6 (Production Example 3), Gly-Arg
-Gly-Asp-Ser-Pro (GRGDSP)4
CM-chitin-
GRGDSP was synthesized. Yield: 0.35g The structure was confirmed by IR and amino acid analysis. As a result of amino acid analysis, the introduction rate of the GRGDSP fragment was approximately 10%.

CM-Chitin GRGDSP Amino acid analysis (nmol/50μl) Glucosamine 15.3319Arg
1.5332Gly
3.2132Asp
1.3468Ser
1.1132Pro
1.3326IR: Stretching vibration of amide carbonyl (C=O) 1657 cm-1

Test Example 1 ``Measurement of cell adhesion inhibitory activity'' A method for measuring the activity of the CM-chitin derivative of the present invention to inhibit cell adhesion to fibronectin and vitronectin is shown below. The competition method used here is basically one that is widely used in the field of biochemistry; for example, ``M
methods in Enzymology” 82
pp. 803-831 (1981), JP-A-1-3096
82, 2-174797.

Experimental method 1. Preparation of adsorption plate Commercially available fibronectin (human origin: Seikagaku Corporation)
) or vitronectin (human origin: purchased from Funakoshi Co., Ltd.) in PBS (NaH2PO4 0.0
05 M + NaCl 0.07 M), 1.0 μl each
/ml, diluted to 2.0μl/ml and diluted the diluted solution to 0.0μl/ml.
Pour 5ml into a 24-well plastic plate and add 3
It was coated by keeping it warm at 7°C overnight. Next, in order to prevent non-specific adsorption, bovine serum albumin (BSA 1%) was added and kept at 37°C for 1 hour, followed by normal washing operations (PBS
) and drained thoroughly to prepare an adsorption plate.

2. Adhesion inhibition experiment The CM chitin derivative obtained by freeze-drying was
o ,s Modified Eagles Medi
um (hereinafter abbreviated as DMEM) and diluted with 0,0.
CM-chitin derivative solutions were prepared at concentrations of 25, 0.5, 1.0 and 1.5 mg/ml. 0.25ml of this solution
were placed in the plate prepared by the above method, and a suspension of vascular endothelial cells (4 x 106 cells/ml) was added thereto at 0.0%.
25 ml was added and kept at 37°C for 1 hour to allow cells to adhere. After washing three times with DMEM medium to remove unattached cells, the adhered cells were detached with a 0.025% EDTA trypsin solution and stained with 2% trypan blue to count the number of cells. The results are shown in Tables 1 and 2 below.

Table-1 Inhibition of cell adhesion to fibronectin (cells/well)
━━━━━━━━━━━━━ Concentration Addition (mg/ml) 0
0.25 0.5 1.0
1.5 Compounds
━━━━━━━ CM-Chitin
160 171 157
165 152 CM-chitin derivative-1
160 131 106
79 69 〃
2 160 135
99 82 75
〃 3 160
123 100 77
55 〃 4
160 141 121
80 67 〃
5 160 136
105 86 72
〃 6 160
127 107 93
66 〃 7
160 119 98
84 71 〃
8 160 130 1
13 80 59
9 160 1
21 97 85 6
5 〃 10
160 122 110
87 73 〃
11 160 143
125 78 70
〃 12 160
125 101 89
77 Sulfated CM-chitin 160
150 155 147
141 CM-chitin derivative-13 1
60 97 77 5
7 33 〃
14 160 111
93 68 41
〃 15 160
105 89 65
37 〃 16
160 103 81
75 50 〃
17 160 99
84 61 35
〃 18 160
133 101 75
52 〃 19
160 127 106
79 56 RGD
160 157
162 141 83
GRGDS 160 15
4 140 95 80
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━

Table 2 Cell adhesion inhibition against vitronectin (
cells/well)━━━━━━━━━━━━━━━━━━
━━━━━━━━━━━━━━ Concentration Addition (mg/ml) 0
10 50 100
300 Compounds
━━━━━━━ CM-Chitin
249 260 243
240 255 CM-chitin derivative-1
249 148 89
72 41 〃
2 249 137
96 78 59
〃 3 249
141 87 69
49 〃 4
249 145 90
67 45 〃
5 249 129
91 80 48
〃 6 249
133 98 71
56 〃 7
249 143 85
79 55 〃
8 249 130
95 69 47
9 249 1
39 88 77 5
8 〃 10
249 136 86
71 43 〃
11 249 147
100 73 45
〃 12 249
150 101 80
59 Sulfated CM-chitin 249
231 235 222
200 CM-chitin derivative -13 2
49 103 70 4
7 31 〃
14 249 107
78 43 34
〃 15 249
115 69 54
38 〃 16
249 122 81
63 40 〃
17 249 114
76 48 33
〃 18 249
125 83 56
44 〃 19
249 130 92
63 39 RGD
249 171
132 104 73
GRGDS 249 15
7 116 87 61
━━━━━━━━━━━━━━━━━━━━━━━━━
━━━━━━━

Test Example 2 ``Platelet aggregation inhibitory activity test'' The platelet aggregation inhibitory effect of the CM-chitin derivative of the present invention in the IN VITRO system was tested using human platelet-rich plasma. The experimental method is shown below.

Experimental Method Fresh human blood was added with 1/9 volume of 3.8% sodium citrate and centrifuged (1000 rpm, 10 minutes), and the upper layer was collected as platelet-rich plasma. CM obtained by freeze-drying
- The chitin derivative was dissolved in physiological saline at various concentrations from 0 to 1.5 mg/ml. Add 25 μl of CM-chitin derivative solution to 200 μl of plasma, incubate at 37°C for 3 minutes, then add 25 μl of 50 μM ADP (adenosine diphosphate) solution or 200 μg/ml collagen solution and measure the degree of aggregation using an aggregometer. The test was carried out by measuring the permeability. The results are shown in Table-3. Aggregation inhibition rate (1-T/T0) x 100%T0 = C
Transmittance T of M-chitin derivative when no salt is added = CM-
Permeability of chitin derivatives when salt is added

[0057]

[0058]

[Sequence list]

[0059] Sequence number: 1 Array length: 3 Sequence type: amino acid Topology: linear Sequence type: Peptide array

[0060] Sequence number: 2 Array length: 6 Sequence type: amino acid Topology: linear Sequence type: Peptide array

[0061] Sequence number: 3 Array length: 9 Sequence type: amino acid Topology: linear Sequence type: Peptide array

SEQ ID NO: 4 Sequence length: 15 Sequence type: Amino acid Topology: Linear Sequence type: Peptide sequence Arg Gly Asp Arg Gly Asp A
rg Gly Asp Arg Gly Asp Ar
g Gly Asp 1
5 10
15

[0063] Sequence number: 5 Array length: 5 Sequence type: amino acid Topology: linear Sequence type: Peptide array

[0064] Sequence number: 6 Array length: 6 Sequence type: amino acid Topology: linear Sequence type: Peptide array

[0065] Sequence number: 7 Array length: 4 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0066] Sequence number: 8 Array length: 5 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0067] Sequence number: 9 Array length: 3 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0068] Sequence number: 10 Array length: 6 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0069] Sequence number: 11 Array length: 9 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0070] Sequence number: 12 Array length: 15 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0071] Sequence number: 13 Array length: 5 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

[0072] Sequence number: 14 Array length: 6 Sequence type: amino acid Topology: linear Sequence type: Peptide Fragment type: C-terminal fragment array

Claims (4)

[Claims] Claim 1: A chitin derivative or a chitin derivative having an adhesive peptide represented by the following general formula [I] as an essential unit in the side chain of CM-chitin via any one of an amide bond, an ester bond, an ether bond, and a urethane bond. That salt. General formula [I] -[R1]-[CO]-([X]-Arg-Gly-A
sp-[Y])n-[Z]-[R2]- [ ] may or may not exist, and if it exists, then X, Y is Ser, Gly, Val, A
It represents an amino acid residue or a peptide residue selected from sn, Pro, and Z represents -O- or -NH-. R1
, R2 is hydrogen or has 1-9 carbon atoms.
straight chain or branched alkyl group, or having 6-9 carbon atoms
is an aryl group which may have a substituent. The other is hydrogen, a linear or branched alkylene group having 1-9 carbon atoms, or an arylene group having 6-9 carbon atoms,
It may have a substituent. n represents an integer of 1-5. Claim 2: Molecular weight of about 3,000-100,00
2. The CM-chitin derivative or salt thereof according to claim 1, wherein the CM-chitin derivative is in the range of 0. 3. An animal cell adhesion inhibitor comprising the CM-chitin derivative or its salt according to claim 1 or 2 as an active ingredient. 4. A platelet aggregation/adhesion inhibitor comprising the CM-chitin derivative or its salt according to claim 1 or 2 as an active ingredient.