Use of peptide derivatives

Abstract: The present invention is related to the use of a peptide derivative containing 1 to 20 units of peptide unit represented by the following general formula [I] or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for inhibiting tumor metastasis; [Z]-Arg-X-Asp-[Y] [I] wherein Arg represents L- or D-arginine residue, Asp represents L-aspartic acid residue, X represents L-phenylalanine or D-alanine residue, and [Z] and [Y] each represents an amino acid or a peptide residue, which may be present or absent, selected from glycine, L-serine, L-threonine, L- and D-aspartic acid, L-alanine, L- and D-glutamic acid, L-proline residues and a peptide residue constituted by the foregoing amino acid residues.

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to use of a peptide derivative comprising a tripeptide unit or a pharmaceutically acceptable salt thereof for manufacturing a pharmaceutical composition for inhibiting metastasis of tumor. 2. Prior Art Fibronectin is a protein involved in the cell-extracellular substrate cohesion and is likewise thought to be involved in coagulation of blood platelets and the metastasis of tumor. These interactions are mediated by a series of receptors present in the cell surface region. It is confirmed that these receptors can specifically recognize an amino acid sequence: arginine-glycine-aspartic acid (hereinafter abbreviated as "Arg-Gly-Asp") of the fibronectin although the fibronectin is a macromolecule having a molecular weight of about 250,000, and there has been reported that the sequence plays an important role in the interaction between the receptors and the fibronectin (Nature, 309, 1984, p30). The above tripeptide sequence, Arg-Gly-Asp, is present in other cohesive proteins such as bitronectin, and fibronectin associates with receptors of cells via the tripeptide sequence to transfer the information of fibronection to the adhered cells. Further, fibronectin is thought to have bonding ability to natural macromolecules such as heparin, collagen and fibrin and to be involved in adhesion of cells and interstitial connective tissue as well as differentiation and proliferation of cells. Since the proteins having the cell adhesion activity have various biological activities as described above, there have been conducted many studies on the applications of these proteins for pharmaceutical drugs and materials. For example, there has been reported a method for inhibiting the coagulation of blood platelets by the use of various linear and cyclic oligopeptides having an Arg-Gly-Asp sequence (Polymer Preprints, Japan, 38 , 1989, p. 3149; Japanese Unexamined Patent Publication (hereinafter referred to as "JP-A-2-174797); a method in which a peptide having an Arg-Gly-Asp sequence is used as a cell movement-inhibiting agent (JP-A-2-4716); and a method using, as a cell-adhesive membrane, a PMMA film on which Arg-Gly-Asp sequences are immobilized (Polymer Preprints, Japan, 37 , 1988, p. 705). In addition, JP-A-1-309682 and 1-305960 disclose a method which comprises covalently bonding peptides having Arg-Gly-Asp sequences as essential structural units to a polymer and in which the resulting product is used as a substrate for cultivating animal cells or for biological composite artificial organs and JP-A-64-6217 discloses a method in which a polypeptide having Arg-Gly-Asp-Ser sequences is used as a platelet protective agent for blood taken out of the body. Further, the cell-adhesive proteins have been paid attention as materials involved in metastasis of tumor. During the series of stages of the metastasis of tumor, tumor cells could contact with various host cells and biological macromolecules and, by the presence of the cell adhesive proteins such as fibronectin, the cells form cell coagulations to facilitate proliferation and survival of the tumor cells. In this regard, however, it is reported that if the adhesive core sequence of fibronectin, the tripeptide Arg-Gly-Asp, is present in such a circumstance, the tripeptide would associate with the receptors on the surface of the cells in a competitive manner with the cell adhesive proteins and thus may inhibit the metastasis of tumor (Science, 238 (1986) p467). Further, there has been known a method comprising inhibiting the metastasis of tumor by the use of an oligopeptide having Arg-Gly-Asp sequences or a polypeptide having the sequence as repeating units (Int. J. Biol. Macromol., 11, 1989, p23 ibid, 11, 1989, p. 226; and Jpn. J. Cancer Res., 60, 1939, p. 722). As described above, many studies on the applications of the activities of the cell adhesive proteins such as fibronectin or peptide fragments thereof for pharmaceutical drugs and related medical materials were actively conducted, and the applications of the adhesive core sequence, Arg-Gly-Asp-Ser, to tumor metastasis inhibiting agents are particularly intensively conducted. However, according to the studies so far, the core sequence was regarded as essential for the expression of the biological activity mediated by the receptor and the ligand and it was reported that any peptide analog having another amino acid residue instead of any of the constitiuent amino acid residues, particularly the Arg residue, could not exhibit the activity (Polymer Preprints, Japan, 38 (1989) p3149: J. Biol. Chem., 262 (1987) p17294). EP-A-368486 discloses peptides containing the tripeptide sequence Arg-Tyr-Asp as inhibitors of the binding of fibrinogen to platelets. Substitution of Tyr by D-Ala reduced the inhibitory capacity of the peptides according to EP-A-368486. Summary of invention It is an object of the present invention to provide a novel use for a peptide derivative or pharmaceutically acceptable salt thereof, which is stable in blood. To achieve the above object, we have systematically investigated various sequences of the adhesive core sequence of fibronectin, Arg-Gly-Asp-Ser, particularly analogues having other various amino acid residues instead of the Gly and Ser residue of the adhesive core sequence Arg-Gly-Asp-Ser and we have determined the biological activities thereof to find a novel use for peptide derivatives of the adhesive core sequence of fibronectin, which are stable in the bloodstream for a long period of time, which may be easily synthesized and which do not exhibit any serious side-effect, and finally achieved the present invention. According to the present invention, a peptide derivative containing 1 to 20 units of a peptide unit represented by the following general formula [I] or a salt thereof; [Z]-Arg-X-Asp-[Y] wherein Arg represents L- or D-arginine residue, Asp represents L-aspartic acid residue, X represents L-phenylalanine or D-alanine residue, and [Z] and [Y] each represent an amino acid or a peptide residue, which may be present or absent, selected from glycine, L-serine, L-threonine, L- and D-aspartic acid, L-alanine, L- and D-glutamic acid, L-proline residues and a peptide residue constituted by the foregoing amino acid residues, is used for the manufacture of a pharmaceutical composition for inhibiting tumor metastasis DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will hereinafter be explained in more detail. The peptide derivatives used according to the present invention are those containing 1 to 20 units of the peptide unit represented by the following general formula [I] or a salt thereof; [Z]-Arg-X-Asp-[Y] wherein Arg represents L- or D-arginine residue, Asp represents L-aspartic acid residue, X represents a L-phenylalanine or D-alanine residue, and [Z] and [Y] each represent an amino acid or a peptide residue, which may be present or absent, selected from glycine, L-serine, L-threonine, L- and D-aspartic acid, L-alanine, L- and D-glutamic acid, L-proline residues and a peptide residue constituted by the foregoing amino acid residues. The peptide derivatives used according to the present invention may be a peptide composed of one unit of the peptide sequence represented by the formula [I], but preferably they are either those composed of a oligopeptide or polypeptide comprising two or more of the above peptide units represented by the general formula [I] or those composed of a compound consisting of one or more of the peptide unit represented by the general formula [I] and one or more of pharmaceutically acceptable organic groups bonded to the units, which maintain the water-solubility of the compound and do not reduce the biological activity of the compound. When the peptide derivatives used according to the present invention consist of an oligopeptide or polypeptide, the carboxyl terminal of the peptides may be amidated. It has been known that stability of phermaceutical drugs in living bodies after the administration thereof, such as represented by the period of staying in the bloodstream and the period before the drugs are excreted from the living bodies, would have a great influence on whether the drugs may be maintained at a therapeutically effective concentration in the living bodies. That is, not only the biological activity but also the stability of the drugs in living bodies are very important for the drugs to be sufficiently effective. This may be also applied to the peptide sequence used according to the present invention, which is represented by the formula [I]. If the peptide sequences of the formula [I] are administered by themselves, the peptide sequences may be rapidly degraded and excreted and such rapid degradation and excretion are generally inherent to peptide compounds. Thus, it is not necessarily possible to expect sufficient biological activity of the peptide sequences of the formula [I] when the sequences are administered by themselves. This is the reason why we have investigated various peptide derivatives containing the peptide sequence of the formula [I], such as those composed of an oligopeptide containing the sequence as a repeating unit, those containing the sequences bonded to other organic groups and those containing D-amino acids instead of the corresponding L-amino acids, and we finally achieved the present invention. As explained above, the important purposes of the modification of the peptide sequence with the organic groups are to protect the effective sequence from the degradation by the enzymes in body fluid by modifying the neighborhood of the sequence, to obviate or aim at accumulation of the derivatives in a specific organ by modifying the sequence with acidic groups or saccharide groups, to endow the derivatives with the slow release property by making the sequence contained in high molecular weight compounds, and the like. Therefore, as far as the organic groups to be bonded to the sequence are pharmaceutically acceptable, able to maintain the water-solubility of the compound and do not deteriorate the biological activity of the sequence, various organic groups may be used depending on the purposes. The organic groups are preferably selected from substituted and unsubstituted acyl, alkyl, alkylamino groups, which groups preferably have 1 to 15 carbon atoms and may contain -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond, and groups derived from monosaccharides, oligosaccharides, preferably containing 2 to 7 monosaccharide units, polysaccharaide derivatives, polycarboxylic acids, preferably having 2 to 12 carboxyl groups, polyamines, preferably having 2 to 12 amino groups, polymers formed from monomers having an ethylenically unsaturated bond such as acrylic acid, methacrylic acid, ethacrylic acid and allylamine, polyethylene glycol, preferably having an average molecular weight of 2,000 to 10,000 and carboxylic acid derivative of polyethylene oxide (PEO Acid). When the organic groups are selected from those derived from the saccharides, the peptide derivatives of the invention preferably contain one to ten units of the peptide sequence of the formula [I]. When the organic groups are selected from those derived from the polycarboxylic acids or polyamines, the peptide derivatives of the invention preferably contain one to five units of the peptide sequence of the formula [I]. The constituent saccharides of the monosaccharides, oligosaccharides and polysaccharides are preferably selected from e.g. glucose, galactose, N-acetylglucosamine, glucosamine, N-acetylgalactosamine, galactosamine, uronic acid and sialic acid, and these saccharides may be used in any combination. Examples of the polysaccharide include chitin, chitosan, carboxymethylated chitin, sulfated carboxymethylated chitin, chondroitin sulfate, curdlan sulfate, heparin sulfate, keratin sulfate and hyaluronic acid. The polysaccharides preferably have a molecular weight of from 10,000 to 30,000. Those organic groups may have anionic groups such as - SO 3 (X), -OSO 3 (X), -OPO 3 (X) and -CO 2 (X), wherein X represents a hydrogen atom or pharmaceutically acceptable counter anion; preferably it represents an alkali metal anion. The peptide sequence and the organic groups are bonded e.g. with an ester bond, amide bond, urethane bond or urea bond. In particular, when the organic groups are selected form those derived from e.g. monosaccharides, oligosaccharides, polysaccharides or polymers, the peptide sequence of the formula [I] and the organic group may be bonded through an alkylene group or an arylene group. In such cases, the alkyl group and arlylene group are preferably linear and substituted, preferably have 1 to 15 carbon atoms and may contain an -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond. The organic groups are preferably bonded to the peptide sequence of the formula [I] at the carboryl terminal or the amino terminal of the peptide sequence. When the organic group is bonded to the peptide sequence at the amino terminal of the peptide sequence, the carboxyl terminal of the peptide sequence is preferably in the form of -OR a or -NR b R c , wherein each of R a , R b and R c represents a hydrogen atom or a linear or cyclic alkyl group and R b and R c may be bonded together to form a ring structure. Further, R a , R b and R c may contain -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond. R a , R b and R c are preferably selected from a methyl, ethyl, iso-propyl and cyclohexyl group. When the organic group is bonded to the peptide sequence at the carboxyl terminal of the peptide sequence, the amino terminal preferably has a hydrogen atom or it is a substituted or unsubstituted acyl group. The acyl group preferably has 1 to 15 carbon atoms. Preferred examples or the substituent of the acyl group are the foregoing anionic groups such as a carboxyl group and sulfonic group. The acyl group may contain an -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond. The molecular weight of the peptide derivatives of the present invention is not particularly limited as far as the biological activity of the derivatives is maintained and the whole derivative is maintained as water-soluble, but the average molecular weight thereof preferably ranges from 1,000 to 100,000. As the salts of the peptide derivatives of the present invention, there may preferably be mentioned, salts with inorganic acids, for instance, hydrochlorides, sulfates, nitrates, phosphates and borates, and salts with organic acids, for instance, acetates, trifluoroacetates, trifluoromethanesulfo nates, lactates and tartrates. Conversion of the derivatives into these salts may be carried out by a conventional manner. Preferred examples of the peptide derivative used according to the present invention including those described in the Examples (Synthesis of the derivatives and investigation of the biological activities thereof) will be listed below, but the present invention is by no means limited to these specific examples. When the amino terminal of the peptide sequence has a hydrogen atom and when the carboxyl terminal of the peptide sequence has -OH, indications of the hydrogen atom and the -OH group are omitted in the formulae in accordance with the orthography in this field. Further, D-amino acids are shown with the indications of "D-", but the indications of "L-" of L-amino acids are omitted. Reference Compound 1 Arg-Leu-Asp-Ser Compound 6 Arg-Phe-Asp-Ser Compound 10 Arg-D-Ala-Asp-Thr-NH 2 Compound 27 Acetyl-Asp-Arg-Phe-Asp-Ser Compound 28 Acetyl-D-Asp-Arg-Phe-Asp-Ser Compound 45 (D-Arg-Phe-Asp) 20 Compound 51 NaO 3 SO(CH 2 ) 11 -CO-Gly-D-Arg-L-Phe-Asp-Ser-NH-CH 3 Compound 53 NaO 3 SO-CH 2 CH(OSO 3 Na)-CO-Arg-Phe-Asp-Ser Compounds 56 and 60 are those polymers formed from each of the following Monomers 56 and 60. Reference Monomer 56 CH 2 =CCH 3 -CONHCH 2 CH 2 -CO-Arg-Leu-Asp-Ser Monomer 60 CH 2 =CCH 3 -CONHCH 2 CH 2 -CO-Gly-Arg-Phe-Asp-Ser-NH-isoC 3 H 7 Compound 64 HOOC-C(CH 2 -O-COCH 2 CH 2 -CO-Gly-Arg-Phe-Asp-Ser) 3 Compound 66 NaO 3 SO-(CH 2 ) 11 -CO-Gly-Arg-Phe-Asp-Ser-NHCH 3 Compound 83 Reference Compound 87 Compound 89 Reference Compound 94 and Compound 96 Reference Compound 94 W=Asp-Arg-Leu-Asp-Ser n=4 Compound 96 W=Gly-Arg-Phe-Asp-Ser n=4 Reference Compound 98 and Compound 100 Reference Compound 98 W 1 =SO 3 H or CH 2 CO-Asp-Arg-Leu-Asp-Ser n= 1-10 Compound 100 W 1 =SO 3 H or CH 2 CO-Gly-Arg-Phe-Asp-Ser n=1-10 Reference Compound 102 Succinylated polyallylamine carrying -Arg-D-Leu-Asp-Ser Compound 103 Succinylated polyallylamine carrying -(Asp-D-Arg-Phe-Asp-Ser) 2 Reference Compound 105 CM-chitin carrying -Arg-Leu-Asp-Ser Reference Compound 113 Sulfated CM-chitin carrying -Gly-Arg-Nle-Asp-Ser-NH-CH 3 Compound 115 Sulfated Succinylated CM-chitin carrying -Arg-Phe-Asp-Ser-NH-iso-C 3 H 7 Reference Compound 117 Chondroitin sulfate carrying -Gly-D-Arg-Leu-Asp-Ser-NH-isoC 3 H 7 Compound 118 Succinylated chondroitin sulfate carrying -D-Arg-Phe-Asp-Ser Methods for synthesizing the peptide sequences are not particularly limited to any specific method and a suitable method may be selected from liquid phase methods, solid phase methods and methods utilizing an automatic synthesizer depending on the kind of the organic group to be bonded with the peptide sequences. In the solid phase methods, oligopeptides are synthesized using Boc groups, cleavage of the peptides from a resin carrier and elimination of the protection groups on the side chain are carried out using trifluoromethanesulfonic acid and the peptides or the derivatives thereof are purified by HPLC (high performance liquid chromatography) for fractionation and collected as fractions showing a single peak. For example, the peptide derivatives of the present invention, Compound 45 may be fractionated by using a YMC-Pack ODS column (length: 500 mm, inner diameter: 20 mm) and a suitable eluent such as 0.01N HCl/acetonitrile, 0.1% TFA/acetonitrile and 0.1% TFA/water. The eluent may have a suitable gradient. The peptides are then converted into e.g. hydrochloric acid salt or acetic acid salt by passing through an anion-exchange resin column such as Amberlite IRA-400; C1 form and IRA-93ZU; acetic acid salt form. Though thus obtained peptide may be directly reacted with the organic groups, they may be modified with the organic groups before the cleavage from the resin carrier. In the synthesis by the liquid phase methods, the peptides are generally synthesized in the properly protected form, optionally deprotected at a specific modification site or sites and modified, and then totally deprotected. Though only the representative examples of the synthesis of the protected peptides are described herein, the details of the synthesis methods of protected peptides are described in, for example, Lectures on Biochemical Experiments, "Chemistry of Proteins IV", p207 to 495, edited by the Biochemical Society of Japan, published by Tokyo Kagaku Dojin; Lectures on Biochemical Experiments, Second Series, "Chemistry of Proteins (the last volume)", edited by the Biochemical Society of Japan, published by Tokyo Kagaku Dojin and " Fundamental Knowledge and Experiments of Peptide Synthesis", edited by Izumiya et al., published by Maruzen. Alternatively, it is also possible to use commercially available synthetic peptides as intermediates. The peptides composed of repeating units may be easily synthesized by the continuous polymerization method by means of diphenylphosphoryl azide (DPPA) by N. Nishi et al., Int. J. Peptide Protein Res., 30 , p275 (1987). In addition, the peptides may be prepared by the methods of genetic engineering. Synthesis of the saccharide moiety may be carried out by using glycosylation reaction as a key reaction. Various methods of the glycosylation are known and the reaction conditions may be selected taking account of the kinds and availability of saccharides and the conditions of the deprotection. The synthesis of saccharide is carried out essentially by condensation reaction of a sugar donor and a sugar receptor of alcohol in the presence of a promoter. For example, when sugar chloride, sugar bromide and the like are used as the sugar donor, silver oxide, silver carbonate, silver perchlorate, silver trifluoromethanesulfonate, mercury (II) cyanide, mercury bromide and the like may be used as the promoter. When sugar fluoride and the like are used as the sugar donor, tin (II) chloride/silver perchlorate, zirconocene dichloride/silver perchlorate and the like may be used as the promoter. When alkylthio compounds of sugar are used as the sugar donor, dimethyl(methylthio)sulfonium triflate, methyl trifluoromethane sulfonate ester, phenylselenyltriflate and the like may be used as the promoter. When imidate compounds are used as the sugar donor, the reaction is carried out by using lewis acids such as boron trifluoride/diethyl ether complex. Dehydration desiccating agents such as molecular sieves and Drylite may be used in the glycosylation reactions explained above. When the sugar donor has a reducing terminal sugar of 2-acetoamido-2-deoxy sugar such as N-acetylglucosamine, the glycosylation reaction may be effectively carried out by the oxazoline method. Sugar residues of aryl glycoside may be synthesized by using a reaction of polyacetylated sugar and phenol which are heated in nonpolar solvent such as benzene and toluene as the key step. Further, as regards p-nitrophenyl glycoside compounds, many of such compounds are commercially available. S-glycosides may be synthesized by the Koenig-Knorr method wherein the sugar donors of sugar chloride, sugar bromide and the like are reacted with thiol compounds. Nucleophilic reactivity of thiol compounds is higher than that of alcohols, and therefore the reaction easily proceeds. When the peptide sequences and the pharmaceutically acceptable organic groups are bonded with amide bonds or ester bonds, the peptide derivatives of the present invention may be prepared e.g. by the active ester methods, the mixed acid anhydride method, the azide method, the acid chloride method, the symmetric acid anhydride method, the DCC method, the DCC-additive method or the carbonyldiimidazole method The peptide derivatives composed of polymers such as propeneamide derivatives may prepared e.g. by a conventional radical polymerization method or ion polymerization method. Resulted polymers may be fractionated depending on the molecular weight thereof e.g. by conventional methods such as gel filtration or dialysis. The mechanism of the potent in vivo activity for inhibiting tumor metastasis of the peptide derivatives or the salts thereof according to the present invention is not completely elucidated. However, it was clearly shown that the cell-migration (infiltration) inhibitory activity in an in vitro system of the present peptide derivatives is superior to that of the peptide Arg-Gly-Asp-Ser, which is the minimum unit of the cell-adhesive proteins for exhibiting the adhesive function, though the cell-adhesion inhibitory activity and the platelet-coagulation inhibitory activity in an in vitro system of the present peptide derivatives are not necessarily superior to those of Arg-Gly-Asp-Ser. Therefore, the peptide derivatives or the salts thereof according to the present invention can be administered to patients optionally together with conventional carriers or pharmaceutical auxiliary agents, i.e., as a composition containing at least one of the peptide derivatives of the present invention, as tumor or cancer metastasis inhibiting agents. The administration dose thereof may vary depending on conditions as the disease to be treated, the age and body weight of patients but generally ranges from 0.2 µ g/kg to 400 mg/kg a day. The peptide derivatives used according to the present invention may be administered through various routes which are generally used for the administration of peptide-containing medicines. For example, it is preferably administered parenterally, e.g., intravenously, intramuscularly and subcutaneously. In the preparation of injectable pharmaceutical preparations containing the peptide derivatives used according to the present invention the derivatives are dissolved in, for example, PBS and physiological saline solution as described hereinbelow, or dissolved in about 0.1N aqueous solution of acetic acid and then lyophilized to give a lyophilized preparation. These pharmaceutical preparations may comprise a conventional stabilizer such as glycine and albumin. In addition, the peptide derivative used according to the present invention may be orally administered in the form of a microcapsule by encapsulating the derivatives in, for example, liposomes and microspheres or in the form of a hydrogel. Further, if the derivatives are formulated in the form of , for example, a suppository, sublingual tablets and nasal sprays, they can be absorbed through mucous other than in the digestive tracts. The present invention will hereinafter be explained in more detail with reference to the following non-limitative examples, but the present invention is by no means limited to these specific Examples. The abbreviations used herein are listed below. In the descriptions hereinbelow, D-amino acid residues are described with the indications of "D-", but, as regards L-amino acid residues, the indications of "L-" are omitted. Boc : t-butoxycarbonyl Bn : benzyl Z : benzyloxycarbonyl Ac : acetyl Su : succinyl Me : methyl Et : ethyl iPr : isopropyl OSu : N-hydroxysuccinimide ONb : nitrobenzyl ester Mts : mesitylenesulfonyl HOBt : 1-hydroxybenzotriazole DCC : dicyclohexylcarbodiimide iPr2NEt : diisopropylethylamine AcOH : acetic acid AcOEt : ethyl acetate DMF : dimethylformamide CDI : carbonyldiimidazole TFA : trifluoroacetic acid DC-urea : dicyclohexylurea Thr : threonine Arg : arginine Gly : glycine Asp : aspartic acid Ser : serine Pro : proline Val : valine Leu : leucine Phe : phenylalanine Phg : phenylglycine Ile : isoleucine Nle : norleucine Glu : glutamic acid Ala : alanine Example 1: Synthesis of Reference Compound 1 Reference Compound 1 was synthesized by following Route 1 below. Synthesis of Reference Compound (1a) To a DMF solution (150 ml) containing t-Boc-Ser(Bn) (14.8 g, 50 mmol),and NaHCO 3 (8.4 g, 0.1 mol), benzyl bromide (8.55 g, 50 mmol) was added dropwise, and the solution was stirred for 24 hours at room temperature. The reaction solution was extracted by adding 4% aqueous solution of Na 2 CO 3 and ethyl acetate and the organic and aqueous layers were separated. The ethyl acetate layer was washed with water and saline solution successively and dried over sodium sulfate. The organic solvent was evaporated under reduced pressure to give 17.0 g (4.4 mol) Reference Compound (1a) as an oily product. Yield: 88%. Synthesis of Reference Compound (1b) Reference Compound (1a) (17.0 g, 44 mmol) was dissolved in methylene chloride (30 ml), added with TFA (30 ml) and dried over 3 hours at room temperature. After removing methylene chloride and TFA by means of a rotatory evaporator, the residue was extracted by adding ethyl acetate and 4% aqueous solution of Na 2 CO 3 and the organic layer was separated. The organic layer was washed with water and saline solution successively and dried over sodium sulfate. After evaporating the solvent under reduced pressure, the residue was dissolved in a mixed solvent of methylene chloride (60 ml) and DMF (40 ml), added with t-Boc-Asp(OBn) (15 g, 46 mmol) and HOBt monohydrate (HOBt · H 2 O, 6.7 g, 44 mmol) and stirred with ice cooling. The reaction mixture was added with DCC (10.3 g) and stirred for 2 hours with ice cooling and overnight at room temperature. Then, the precipitated urea derivative was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was extracted by adding ethyl acetate and 4% aqueous solution of Na 2 CO 3 and the organic layer was separated. The ethyl acetate layer was washed with water and saline solution successively and dried over sodium sulfate. After removing newly precipitated urea derivative by filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate = 2/1) to give Reference Compound (1b) (15.8 g, 26.7 mmol) as a white solid. Yield: 61% Synthesis of Reference Compound (1c) Reference Compound (1c) (15.5 g, 26.2 mmol) was dissolved in methylene chloride (30 ml), added with TFA (30 ml) and stirred for 30 minutes at room temperature. After removing ethylene chloride and TFA by means of a rotary evaporator, the residue was added with a mixed solvent of n-hexane and ether (5:1) and precipitated white solid of Reference Compound (1c)(15.0 g, 24.8 mmol) was taken by filtration. Yield: 95%. Synthesis of Reference Compound (1d) A reaction mixture containing Reference Compound (1c)(1.5 g, 2.48) mmol), t-Boc-Leu · H 2 O (690 mg, 2.96 mmol), HOBt · H 2 O (380 mg, 2. 5 mmol) and diisopropyl ether (0.470 µl, 2.7 mmol) in methylene chloride (15 ml) and DMF (7 ml) was stirred with ice cooling. This reaction mixture was added with DCC (570 mg) and stirred for 2 hours with ice cooling and overnight at room temperature. After removing precipitated urea derivative by filtration, the filtrate was concentrated under reduced pressure. The residue was added with ethyl acetate and washed with 10% aqueous solution of citric acid, 4% aqueous solution of Na 2 CO 3 , water and saline solution successively. After removing newly precipitated urea derivative by filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (n-hexane/ethyl acetate = 10/1) to give Reference Compound (1d)(1.39 g, 2 mmol) as a white solid. Yield: 80%. Synthesis of Reference Compound (1e) Reference Compound (1d) (1.15 g, 1.6 mmol) was dissolved in methylene chloride (6 ml), added with TFA (6 ml) and stirred for 1 hour at room temperature. After evaporating methylene chloride and TFA by means of a rotatory evaporator, the residue was extracted by adding ethyl acetate and 4% aqueous solution of Na 2 CO 3 and the organic layer was separated. The ethyl acetate layer was washed with water and saline solution successively and dried over sodium sulfate. After evaporating the ethyl acetate under reduced pressure, the residue was dissolved in a mixed solvent of methylene chloride (15 ml) and DMF (8 ml), added with t-Boc-Arg(Z 2 ) (970 mg, 1.8 mmol) and HOBt · H 2 O (220 mg, 1.4 mmol) and stirred with ice cooling. The reaction mixture was added with DCC (420 mg) and stirred for 2 hours with ice cooling and overnight at room temperature. Then, the precipitated urea derivative was removed by filtration and the filtrate was concentrated under reduced pressure. The residue was extracted by adding ethyl acetate and 4% aqueous solution of Na 2 CO 3 and the organic layer was separated. The ethyl acetate layer was washed with water and saline solution successively and dried over sodium sulfate. After removing newly precipitated urea derivative by filtration, the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform/ethyl acetate = 10/1) and recrystallized from a mixed solvent of n-hexane and ethyl acetate (2/1) to give Compound (1e) (690 mg, 0.61 mmol) as a white solid. Yield: 38%, M.P.: 100-104°C. Synthesis of Reference Compound 1 Reference Compound (1e) (590 mg, 0.53 mmol) was dissolved in methylene chloride (6 ml), added with TFA (6 ml) and stirred for 30 minutes at room temperature. After removing ethylene chloride and ethyl acetate by means of a rotatory evaporator, the residue was added with acetic acid (10 ml) and 10% palladium/carbon (Pd/C, 60 mg) and hydrogenolysis was carried out overnight at room temperature. After removing the catalyst by filtration and evaporating acetic acid under reduced pressure, the residue was added with ether and precipitate was taken by filtration. Thus obtained crude peptide was dissolved in a small amount of water (pH 1 - 2) and developed in a column of Amberlite IRA93ZU (acetic acid form) with pure water. Positive fractions in the ninhydrin reaction (pH 4 - 5) were combined, concentrated and finally lyophilized to give Compound 1 (240 mg) as a white amorphous product. In the proton NMR, a peak of methyl group of acetic acid was observed at 2.02 ppm, of which integral intensity showed that Reference Compound 1 was obtained as a monosalt of acetic acid. Yield: 82%. FAB Mass: 490 (M+H) + Example 2: Synthesis of Compound 6 Compound (6d) was prepared in the same manner as used in the synthesis of the intermediate compound for the synthesis of Reference Compound 1, Reference Compound (1d), by using t-Boc-Phe instead of t-Boc-Leu respectively. Then, Compound (6e) was prepared in the same manner as used in the synthesis of Reference Compound (1e) by using Compound (6d) respectively instead of Reference Compound (1d). Finally, deprotection and purification of Compounds (6e) was carried out in the same manner as used in Example 1 to obtain Compound 6 in the form of monosalt of acetic acid. Yields of the reactions, melting points and mass spectrum data of the above-obtained compound according to the present invention are listed below. Yield (%) Compound (6d) 91 Compound (6e) 65 Compound 6 82 Melting point (°C) Compound (6e) 124 - 127 FAB Mass Compound 6 524 (M+H) + , 546 (M+Na) + Example 3 : Synthesis of Reference Compound 56 (a) An aqueous solution of sodium hydroxide containing β - alanine (17.8g, 0.2 mol) was added with methacryloyl chloride (20.9 g, 0.2 mol) with ice cooling, stirred for 4 hours and neutralized with hydrochloric acid. After concentrating the reaction mixture under reduced pressure, precipitated sodium chloride was removed by filtration. The concentrate was extracted with chloroform and dried. After evaporating the chloroform, the concentrate was washed with ether to obtain carboxymethylmethacrylamide as a white solid product (17.6 g). Yield: 56%. CH 2 =CCH 3 -CONHCH 2 CH 2 -COOH In the same manner as described above, various propenoic acid derivatives may be prepared, for example, from combinations of methacryloyl chloride and 4-aminobutyric acid, ethacryloyl chloride and 5-aminovaleric acid, methacrylic acid and 6-aminocaproic acid, acrylic acid and 12-aminolauric acid, acrylic acid and leucine, methacrylic acid and glutamine, acrylic acid and 2-(2-aminoethoxy)propyonic acid, methacrylic acid and 2-(2-aminoethoxy)acetic acid, methacrylic acid and glycylglycine and the like. (b) Synthesis of Boc-Arg(Mts)-Leu-Asp(OBn)-Ser(Bn)-OBn Boc-Lue-Asp(OBn)-Ser(Bn)-OBn(Reference Compound (1d)) (28 g, 39 mmol) was added with a mixed solvent (200 ml) of TFA and CH 2 Cl 2 (1:1) and stirred for 1 hour at room temperature, and then the TFA and CH 2 Cl 2 were evaporated under reduced pressure. The residue was dissolved in ethyl acetate, neutralized with NaHCO 3 aqueous solution and washed with NaCl aqueous solution. The mixture was dried over sodium sulfate, and the ethyl acetate was evaporated under reduced pressure. Thus obtained product and Boc-Arg(Mts) (17.8 g, 39 mmol) were dissolved in DMF (400 ml), added with DCC (8.0 g, 39 mmol) and HOBt (6.8 g, 45 mmol) with ice cooling and stirred for 3 hours with ice cooling and overnight at room temperature. After removing DC-urea by filtration, the reaction mixture was concentrated under reduced pressure and the residue was dissolved in ethyl acetate. This solution was washed with NaHCO 3 aqueous solution, 1M citric acid aqueous solution and NaCl aqueous solution successively, dried over sodium sulfate and evaporated to dryness under reduced pressure to obtain the desired compound as white powder (19.5 g). Yield: 48%. (c) Synthesis or Reference Monomer 56 Boc-Arg(Mts)-Leu-Asp(OBn)-Ser(Bn)-OBn (15.6 g, 15 mmol) was added with a mixed solvent (100 ml) of TFA and CH 2 Cl 2 (1:1) and stirred for 1 hour at room temperature, and then the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate, neutralized with NaHCO 3 aqueous solution and washed with NaCl aqueous solution. The mixture was dried over sodium sulfate, and the ethyl acetate was evaporated under reduced pressure. Thus obtained product and carboxyethylmethacrylamide (2.4 g, 15 mmol) were dissolved In CH 2 Cl 2 (200 ml), added with DCC (3.1 g, 15 mmol) and stirred for 3 hours with ice cooling and further stirred overnight at room temperature. The reaction mixture was concentrated under reduced pressure and added with acetone, and formed DC-urea was removed by filtration. The mixture was concentrated under reduced pressure, washed with ether and dried under reduced pressure to obtain white powder (10.0 g). Yield: 62%. The above obtained product (10.0 g, 9.3 mmol) was dissolved in TFA, added with a solution of 1M-trifluoromethanesulfonic acid/thioanisole/m-cresol in TFA with ice cooling and allowed to react for 1 hour to eliminate the protection groups present on the side chain and the terminal of the peptide. The reaction solution was poured into ether, resulted oily precipitate was dissolved in distilled water, then washed with ethyl acetate, passed through an anion-exchange resin column (Amberlite IRA-400;C1 form) to convert into a hydrochloride and lyophilized to give the desired monomer as a white solid product (4.4 g). Yield: 76%. Amino Acid Analysis (nmol/50 µl) Arg: 0.9877 Leu: 0.9916 Asp: 0.9899 Ser: 0.8891 β-Ala: 1.0115 Mass Spectra (MS): M + 573 (d) Synthesis of Reference Compound 56 (Polymerization of Reference Monomer 56) Monomer 56 (500 mg) was dissolved in water (5 ml), the pH value adjusted at 7.4 with 1N NaOH and added with potassium persulfate (2.5 mg) and sodium hydrogensulfite (1.0 mg) as initiators and allowed to cause polymerization reaction under nitrogen flow at 20 °C for 20 hours. After the reaction, the reaction mixture was dialyzed against pure water using Spectrapore 7 (MWCO: 3000) to eliminate low molecular weight compounds and lyophilized to obtain the desired Reference Compound 56 (240 mg). The obtained Reference Compound 56 was fractionated according to the molecular weight thereof by gel permeation chromatography which was carried out by using TSK Gel G3000SW column available from Toso Co.,Ltd., and 0.2 M phosphate buffer (pH 7.4) as a mobile phase at a flow rate of 1.0 ml/min. The determined molecular weight of the fractions of Reference Compound 56 are as follows (Standard: PEG). Fraction 1 Molecular weight: about 48,000 (Reference Compound 56-1) Fraction 2 Molecular weight: about 21,000 (Reference Compound 56-2) Fraction 3 Molecular weight: about 12,000 (Reference Compound 56-3) Example 4 : Synthesis of Reference Compound 56-4 (synthesis of low molecular weight polymer by altering the amount of initiator) Reference Compound 56-4 was prepared in the same manner as used in Example 10 by using the following initiators. Initiator: Potassium persulfate 10 mg Sodium hydrogensulfite 4 mg Yield: 180 mg Molecular weight: about 5,000 Example 5 : Synthesis of Reference Polymer A The above-synthesized carboxyethylmethacrylamide was polymerized through radical polymerization reaction as follows. Carboxyethylmethacrylamide (2 g) was dissolved in DMF (20 ml), added with a radical initiator V65 (2,2-azobis(2,4-dimethylvaleronitrile, Wako Junyaku Co.,Ltd., 10 mg) and allowed to cause polymerization under nitrogen flow at 65 °C for 4 hours. The resulted polymer was precipitated with ethyl acetate, dialyzed against pure water using Spectrapore (MWCO: 3000) to eliminate low molecular weight fraction and lyophilized to obtain Reference Polymer A (1.24 g). Reference Polymer A H-(CH 2 -CHCH 3 (CONHCH 2 CH 2 COOH)) n Molecular weight (Standard: PEG): about 30,000 Example 6 : Synthesis of Compound 60 (Polymerization of Monomer 60 Polymerization of Monomer 60 was carried out in the same manner as used in Example 3 (d). Compound No. Yield (mg) Molecular weight 60 190 11,000 Example 7 : Synthesis of succinylated polyallylamine Polyallylamine (5.2 g, Nitto Boseki Co.,Ltd.) and triethylamine (15.2 g) were dissolved in water (100 ml), added with 4-dimethylaminopyridine (1.7 g) and succinic anhydride (15.0 g) and stirred overnight at room temperature. The desired compound, succinylated polyallylamine, was precipitated with acetone, washed with ether and dried. Yield: 1.26 g, Molecular weight 11,000. Example 8 : Synthesis of Reference Compound 102 Succinylated polyallylamine carrying -Arg-D-Leu-Asp-Ser The succinylated polyallylamine (0.39 g) obtained in Example 7 was dissolved in phosphate buffer (25 ml, pH 7.4), added with a solution of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (0.26 g) in phosphate buffer (5.0 ml) at 0°C and allowed to react for 1.5 hours. Then, the reaction solution was added with a solution of Arg-D-Leu-Asp-Ser (0.71 g) dissolved in phosphate buffer (10 ml) and allowed to react overnight at 4 °C. The reaction solution was dialyzed against ion-exchanged water and then against pure water using Extrapore 7 (MWCO: 8000) to eliminate low molecular weight compounds, purified and lyophilized. Yield: 0.71 g. The structure of the product was confirmed by IR and elemental analysis. As a result of the elemental analysis, the introduction rate of the peptide fragment was found to be about 15 %. The introduction rate was calculated as described in Example 44. Elemental Analysis: N; 19.80% IR: Stretching vibration of amidocarbonyl (C=O) 1648 cm -1 Example 9 : Synthesis of Compound 103 Succinylated polyallylamine carrying -(Asp-D-Arg-Phe-Asp-Ser) 2 By using succinylated polyallylamine (0.66 g) obtained in Example 7 , (Asp-D-Arg-Phe-Asp-Ser) 2 (0.85 g) as the peptide fragment and water-soluble DCC (0.26 g), Compound 103 was prepared in the same manner as used in Example 8 . Yield: 0.71 g. The structure of the product was confirmed by IR and the elemental analysis. As a result of the elemental analysis, the introduction rate of the peptide fragment, (Asp-D-Arg-Phe-Asp) 2 , was found to be about 9%. Elemental Analysis: N; 19.44% IR: Stretching vibration of amidocarbonyl (C=O) 1648 cm -1 Example 10: Synthesis of Compound 89 Compound 89 was prepared by using, as starting materials, 4-nitrophenyl-N-acetyl-β-glucosaminide, 4-nitrophenyl-di-N-acetyl-β-chitobiose, 4-nitrophenyl-di-N-acetyl-β-chitotriose, 4-nitrophenyl-α-D-glucopyranoside and 4-nitrophenyl-α-D-maltotrioside, which are commercially available from Seikagaku Kogyo Co.,Ltd. and Boeringer Mannheim & Yamanouchi Co.,Ltd. As the representative examples of the synthesis of Compound 89, synthesis of Reference Compound 87 is described below. Synthesis of Reference Compound 87 Reference Compound 87 was synthesized by following Route 4 below. 4-nitrophenyl-N-acetyl-β-glucosaminide (3.4 g, 0.01 mol) was added to acetic anhydride (50 ml) and pyridine (100 ml), stirred for 16 hours at room temperature and treated in a conventional manner to give a white powder. This was repeatedly precipitated in ether/hexane to give 3.8 g of Intermediate Compound 1. Intermediate Compound 1 (3.0 g, 0.0064mol) was dissolved in ethyl acetate (100 ml) and added with 10% palladium/carbon (0.3 g) and hydrogenolysis was carried out for 6 hours under hydrogen atmosphere. After removing the catalyst by filtration, the filtrate was concentrated to give 2.7 g of the objective Intermediate Compound 2 as an amorphous product. Because of unstability of this product, it was used in the next step without purification. Intermediate Compound 2 (2.5 g, 0.0057mol) was dissolved in pyridine (50 ml), added with glutaric anhydride (684 mg, 0.006mol) and catalytic amount of dimethylaminopyridine and warmed for 14 hours in a water bath at 50 °C . After a conventional post-treatment, the resulted residue was purified by silica gel column chromatography (eluent: chloroform/methanol = 30/1) to give 2.1 g of the objective Intermediate Compound 3 as powder. Intermediate Compound 3 (2.0 g, 0.0036 mol) and carbonyldiimidazole (640 mg, 0.004 mol) were dissolved in dry tetrahydrofuran (40 ml) and cooled at 0°C. Boc-eliminated Compound 2 described in Example 2 (4.1 g, 0.004 mol), which has a free amino group, was dissolved in dry DMF (10 ml), cooled and added to the solution of Intermediate Compound 3 dropwise. The reaction mixture was stirred for 16 hours at room temperature and treated in a conventional manner. The resulted residue was purified by silica gel column chromatography (eluent: chloroform/methanol = 20/1) to give 4.8 g of the objective Intermediate Reference Compound 4. Intermediate Reference Compound 4 (2.0 g, 0.0013mol) was dissolved in ethanol (20 ml), added with 1N NaOH aqueous solution (10 ml) and stirred for 8 hours at room temperature. After neutralization with 1N HCl, the solvent was evaporated and the residue was dried intensively. The residue was dissolved in dry DMF (15 ml), added with sulfur trioxide/trimethylamine complex (500 mg, 0.0036 mol) and stirred for 20 hours at room temperature. After confirming the disappearance of the starting materials by TLC, the reaction mixture was added with a proper amount of saturated NaHCO 3 aqueous solution to stop the reaction and neutralized. After evaporating a major part of the solvent, the residue was crystallized with ether, washed with water and dried to give a sulfate ester compound. This product was dissolved in acetic acid (20 ml), added with 10% Pd/C (100 mg) and stirred for 40 hours at room temperature under hydrogen atmosphere. The catalyst was removed by filtration through a Celite layer, the Celite layer was washed with water. The filtrate and the washing water were combined and concentrated under reduced pressure. The residue was dissolved in a small amount of water, purified by gel permeation chromatography using Sephadex G-10 and lyophilized to give 710 mg of Compound 87 as colorless powder. FAB-MS: (M-H) + 998 Compound 89 was prepared in the same manner as described above. FAB-MS data of those compounds are shown below. Compound 89 (M-H) - 700 Example 11: Synthesis of Reference Compound 94 6-O-carboxymethyl-N-acetylchitohexaose carrying -Asp-Arg-Leu-Asp-Ser 6-O-carboxymethyl-N-acetylchitohexaose (0.5 g) was dissolved in pure water, added with WSC (0.37 g) and stirred for 1 hour. Then, the reaction mixture was added with Asp-Arg-Leu-Asp-Ser (1.0 g) and stirred for a day. After concentrating the reaction mixture under reduced pressure, the residue was washed with ether and purified with an ion-exchange resin, Amberlite IRA-93 and Sephadex G-50 to give 0.6 g of the objective compound. Amino Acid Analysis (nmol/50 µl) Asp : 2.00 Arg : 0.98 Leu : 0.95 Ser : 0.78 Example 12 : Synthesis of Compound 96 6-O-carboxymethyl-N-acetylchitohexaose carrying -Gly-Arg-Phe-Asp-Ser Compound 96 was prepared in the same manner as in Example 11 by using 6-O-carboxymethyl-N-acetylchitohexaose and Gly-Arg-Phe-Asp-Ser. Amino Acid Analysis (nmol/50 µl) Gly : 0.91 Arg : 0.92 Phe : 0.94 Ser : 0.82 Asp : 1.00 Example 13: Synthesis of Reference Compound 98 6-O-sulfated-carboxymethyl-N-acetylchitooligosaccharide carrying -Asp-Arg-Leu-Asp-Ser 6-O-sulfated-carboxymethyl-N-acetylchitooligosaccharide (0.5 g) was dissolved in pure water, added with WSC (0.2 g) and stirred for 1 hour. Then, the reaction mixture was added with Asp-Arg-Leu-Asp-Ser (1.0 g) and stirred for a day. After concentrating the reaction mixture under reduced pressure, the residue was washed with ether and purified with an ion-exchange resin, Amberlite IRA-93 and Sephadex G-50 to give 0.3 g of the objective Reference compound. Amino Acid Analysis (nmol/50 µl) Asp : 2.00 Arg : 0.96 Leu : 0.96 Ser : 0.81 Example 14 : Synthesis of Compound 100 6-O-sulfated carboxymethyl-N-acetylchitooligosaccharide carrying -Gly-Arg-Phe-Asp-Ser Compound 100 was prepared in the same manner as in Example 13 by using Gly-Arg-Phe-Asp-Ser. Amino Acid Analysis (nmol/50 µl) Gly : 1.04 Arg : 1.12 Phe : 0.94 Asp : 1.00 Ser : 0.84 Example 15 : Synthesis of Reference Compound 105 CM-chitin carrying -Arg-Leu-Asp-Ser CM-chitin (Viscosity: 9 cps (1% solution, 20 °C ), etherification degree: 0.78, deacetylation degree: 0.5, available from Yaizu Suisan Kagaku Kogyo Co.,Ltd., 0.30 g) was dissolved in phosphate buffer (pH 7.4), added with a solution of water-soluble DCC (1-ethyl-3-(dimethylaminopropyl)-carbodiimide, 128 mg) in phosphate buffer (2.6 ml) while maintained at 0°C and allowed to react for 1.5 hours. Then, the reaction mixture was added with the peptide, Arg-Leu-Asp-Ser (400 mg), dissolved in phosphate buffer (8 ml) and allowed to react overnight at 4 °C. The reaction solution was packed in a Visking tube, purified by dialysis against deionized water and then pure water to eliminate low molecular weight compounds and lyophilized. Yield: 0.24 g. The structure of the product was confirmed by IR and amino acid analysis. Amino Acid Analysis (nmol/50 µl) Glucosamine : 20.5558 Arg : 2.0556 Leu : 2.1352 Asp : 1.9854 Ser : 1.8792 Introduction rate of peptide fragment was determined from the ratio of the concentration of arginine residue to that of glucosamine according to the following relation and found to be about 10%. Introduction rate = [Arg] / [Glucosamine] × 100 IR: Stretching vibration of amidocarbonyl (C=O) 1652 cm -1 Example 16 : Synthesis of Reference Compound 113 Sulfated CM-chitin carrying -Gly-Arg-Nle-Asp-Ser-NHCH 3 CM-chitin (etherification degree: 0.50, deacetylation degree: 0.05) was sulfated according to the method of Tokura et al., Jpn. J. Cancer Res., 80, p866-872 (1989) and Cancer Res., 50 , p3631-3637 (1990) and covalently bonded to Gly-Arg-Nle-Asp-Ser-NHCH 3 fragment in the same manner as used in Example 15 Yield: 0.36 g. The structure of the product was confirmed by IR and amino acid analysis. By the amino acid analysis, the introduction rate of the peptide fragment was found to be about 12%. Amino Acid Analysis (nmol/50 µl) Glucosamine : 33.1569 Arg : 3.9780 Gly : 3.9251 Asp : 3.6053 Ser : 3.4921 Nle : 3.5548 IR: Stretching vibration of amidocarbonyl (C=O) 1650 cm -1 Example 17 : Synthesis of Compound 115 Sulfated succinylated CM-chitin carrying -Arg-Phe-Asp-Ser-NH-iso-C 3 H 7 Succinylated CM-chitin was sulfated in the same manner as used in Example 16 and covalently bonded with the peptide fragment Arg-Phe-Asp-Ser-NH-iso-C 3 H 7 to give Compound 115. Yield: 0.37 g. The structure of the product was confirmed by IR and amino acid analysis. By the amino acid analysis, the introduction rate of the peptide fragment was found to be about 14%. Amino Acid Analysis (nmol/50 µl) Glucosamine : 18.6932 Arg : 2.6170 Phe : 2.7739 Asp : 2.5931 Ser : 2.2168 IR: Stretching vibration of amidocarbonyl (C=O) 1654 cm -1 Example 18: Synthesis of Reference Compound 117 Chondroitin sulfate carrying -Gly-D-Arg-Leu-Asp-Ser-NH-isoC 3 H 7 Chondroitin sulfate (viscosity: 9 cps (1% solution, 20°C ), etherification degree: 0.78, available from Yaizu Suisan Kagaku Kogyo Co.,Ltd., 0.30 g) was dissolved in phosphate buffer (pH 7. 4), added with a solution of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (128 mg) in phosphate buffer (2.6 ml) and allowed to react 1.5 hours. Then, the reaction mixture was added with the peptide Gly-D-Arg-Leu-Asp-Ser-NHisoC 3 H 7 (400 mg) dissolved in phosphate buffer (8 ml) and allowed to react overnight at 4 °C . The reaction solution was packed in a Visking tube, purified by dialysis against deionized water and then pure water to eliminate low molecular weight compounds and lyophilized. Yield: 0.24 g. The structure of the product was confirmed by IR and amino acid analysis. By the amino acid analysis, the introduction rate of the peptide fragment was found to be about 10%. Amino Acid Analysis (nmol/50 µl) Glucosamine : 20.5558 D-Arg : 2.0556 Gly : 2.1352 Asp : 1.9854 Ser : 1.8792 Leu : 1.9790 IR: Stretching vibration of amidocarbonyl (C=O) 1652 cm -1 Example 19 : Synthesis of Compound 118 Succinylated chondroitin sulfate carrying -D-Arg-Phe-Asp-Ser The chondroitin sulfate (20.0 g) the same as used in Example 18 was dissolved in 1% triethylamine solution (10 ml), added with succinic anhydride (34.0 g) and 4-dimethylaminopyrid ine (2.00 g) and stirred at room temperature for 24 hours. After the completion of the reaction, the reaction solution was poured into a large excess of acetone to precipitate the succinylated chondroitin sulfate. The recovered precipitates were washed with large amount of methanol and then with ether and dried in vacuo. Yield: 22.40 g. The succinylated chondroitin sulfate (0.30 g) was dissolved in phosphate buffer (pH 7.4), added with a solution of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (128 mg) in phosphate buffer (2.6 ml) while maintained at 0°C and allowed to react 1.5 hours. Then, the reaction mixture was added with the peptide D-Arg-Phe-Asp-Ser (400 mg) dissolved in phosphate buffer (8 ml) and allowed to react overnight at 4 °C . The reaction solution was packed in a Visking tube, purified by dialysis against deionized water and then pure water to eliminate low molecular weight compounds and lyophilized. Yield: 0.26 g. The structure of the product was confirmed by IR and amino acid analysis. By the amino acid analysis, the introduction rate of the peptide fragment was found to be about 10%. Amino Acid Analysis (nmol/50 µl) Glucosamine : 23.6218 D-Arg : 2.3622 Phe : 2.1253 Asp : 2.2391 Ser : 2.0031 IR: Stretching vibration of amidocarbonyl (C=O) 1652 cm -1 Pharmacological Test: Experimental tumor metastasis in lungs, liver and spleen The tumor metastasis inhibitory activities of the peptide derivatives of the present invention were examined as follows. Each of the following peptide derivatives according to the present invention, Compounds 6, 10, 89, 96, 103 and 118 was mixed with B16-BL6 melanoma cells having a potent metastatic tendency in PBS so that 0.2 ml of the mixture contained 3000, 1000 or 500 µg of the peptide derivative and 5 × 10 4 of B16-BL6 melanoma cells. Then, 0.2 ml of each mixture was intravenously administered to each individual of a group of C57BL/6 female mice consisting of 5 mice. Fourteen days after the administration, the number of melanoma cell colony in lungs was determined and compared with that of the control mice, which were administered with PBS. The results are shown in Table 4 below. For comparison, the peptide fragments known to have tumor metastasis inhibition activity, Arg-Gly-Asp-Ser (Comparison A), Gly-Arg-Gly-Asp-Ser (Comparison B) and (Arg-Gly-Asp)n (n=5) as well as Reference Polymer A were also administered to the mice in the same manner as above. As seen from the results shown in Table 4 , the peptide derivatives used according to the invention more effectively inhibited the metastases of the tumor cells to the lungs as compared with the conventional adhesive peptide sequences. This clearly shows that the peptide derivatives used according to the present invention have higher tumor metastasis inhibitory activity than the conventional peptide compounds. In particular, the peptide derivatives used according to the present invention comprising the peptide sequences modified with the organic groups show high inhibitory activity with a small administration dose, and it means that the enhancement of the activity by the modification with organic groups, which is one of the important object of the present invention, has been successfully achieved. Further, it was also found that the peptide derivatives used according to the present invention can exhibit high tumor metastasis inhibitory activity even though the derivatives were administered 5 minutes after the administration of B16-BL6 cells without mixing the derivatives and tumor cells This means that the peptide derivatives used according to the present invention can exhibit the tumor metastasis inhibitory effect in patients having cancer, when the derivatives are administered to the patients by a proper way such as intravenous injection. Inhibitory Effect on Experimental Melanoma Metastasis in Lungs Caused by Injection of B16-BL6 Melanoma Cell Compound Dose (µg) Number of Colony Average ± SD (Range) Experiment 1 Control(PBS) --- 116 ± 27 (84 - 160) 6 3000 51 ± 10 (10 - 29)** 10 3000 58 ± 15 (41 - 72) Comparison A 3000 70 ± 8 (59 - 78) Comparison B 3000 50 ± 10 (35 - 63) Comparison C 1000 40 ± 15 (26 - 59) Experiment 2 Control (PBS) --- 128 ± 24 (100 - 156) 89 500 29 ± 11 (16 - 39) Comparison A 3000 88 ± 20 (65 - 112) Comparison B 1000 76 ± 6 (65 - 87) Comparison C 500 57 ± 12 (44 - 71) Experiment 3 Control (PBS) --- 85 ± 20 (64 - 107) 96 500 9 ± 4 (3 - 11) 103 500 24 ± 8 (14 - 33) 118 500 43 ± 8 (33 - 49) Comparison A 3000 51 ± 6 (45 - 59) Comparison B 1000 50 ± 8 (40 - 56) Comparison C 500 44 ± 16 (28 - 63) Toxicity In the above in vivo tests, it was confirmed that the peptide derivatives used according to the present invention have no cytotoxicity against the erythrocyte cells, the spleen cells and the thymus cells of the host mice and no undesirable serum protein coagulation activity. As described above, the peptide derivatives used according to the present invention have higher tumor metastasis inhibitory activities as compared with the core sequences of the cell adhesive proteins and suffer almost no problem of toxicity. The structures of the compounds are rather simple and, therefore, the peptide derivatives of the present invention are extremely valuable as pharmaceutical drugs.

Claims: Use of a peptide derivative containing 1 to 20 units of peptide unit represented by the following general formula (I) or a pharmaceutically acceptable salt thereof for the manufacture of a pharmaceutical composition for inhibiting tumor metastasis; [Z]-Arg-X-Asp-[Y] wherein Arg represents L- or D-arginine residue, Asp represents L-aspartic acid residue, X represents L-phenylalanine or D-alanine residue, and [Z] and [Y] each represents an amino acid or a peptide residue, which may be present or absent, selected from glycine, L-serine, L-threonine, L- and D-aspartic acid, L-alanine, L- and D-glutamic acid, L-proline residues and a peptide residue constituted by the foregoing amino acid residues. The use of claim 1 wherein the peptide derivative is an oligopeptide or polypeptide composed of one or more of the peptide sequence of the formula [I] or pharmaceutically acceptable salts thereof, of which carboxyl terminal may be amidated. The use of claim 2 wherein the peptide derivative comprises 2 to 20 units of the peptide sequence of the formula [I] or pharmaceutically acceptable salts thereof. The use of claim 1 wherein the peptide derivative is a compound consisting of one or more of the peptide units represented by the general formula [I] and one or more of pharmaceutically acceptable organic groups bonded to the units, which organic groups maintain the water-solubility of the compound and do not reduce the biological activity of the compound, wherein the organic group is selected from; (i) acyl, alkyl and alkylamino groups, which groups may contain -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond, and (ii) groups derived from monosaccharides, oligosaccharides, polysaccharide derivatives, polycarboxylic acids, polyamines, polymers formed from monomers having an ethylenically unsaturated bond, polyethylene glycol and carboxylic acid derivative of polyethylene oxide, which groups may be bonded to the peptide sequences through an alkylene group or an arylene group, which alkylene and arlylene groups may contain -O-, -NH-, -S-, ester bond, amide bond, urethane bond or urea bond. The use of claim 4 wherein the organic group is bonded to the peptide sequences at the amino terminals of the peptide sequences and the carboxyl terminals of the peptide sequences have a group of -OR a or -NR b , R c , where each of R a , R b and R c , represents a hydrogen atom or a linear or cyclic alkyl group and R b and R c may be bonded together to form a ring structure. The use of claim 4 wherein the organic group is bonded to the peptide sequences at the carboxyl terminals of the peptide sequences and the amino terminals have a hydrogen atom or substituted or unsubstituted acyl group. The use of claim 4 wherein the peptide derivative is a polypeptide comprising 2 to 20 units of the peptide sequence of the formula [I], the amino terminal of the polypeptide having a substituted or unsubstituted acyl group and the carboxyl terminal of the polypeptide having a substituted or unsubstituted linear or cyclic alkylamino group. The use of claim 4 wherein the peptide derivative comprises 1 to 5 units of the peptide sequence of the formula [I] or pharmaceutically acceptable salt thereof and the organic group is selected from those derived form polycarboxylic acids or polyamines. The use of claim 4 wherein the peptide derivative comprises 1 to 10 units of the peptide sequence of the formula [I] or pharmaceutically acceptable salt thereof and the organic group is selected from those derived form polyethylene glycol. The use of claim 4 wherein the peptide derivative comprises 1 to 10 units of the peptide sequence of the formula [I] or pharmaceutically acceptable salt thereof and the organic group is selected from those derived form monosaccharides, oligosaccharides and polysaccharide derivatives.

Inventors: AZUMA ICHIRO, KITAGUCHI HIROSHI, KOJIMA MASAYOSHI, KOMAZAWA HIROYUKI, MORI HIDETO, NISHIKAWA NAOYUKI, ONO MITSUNORI, ORIKASA ATSUSHI, SAIKI IKUO, SATOH HIDEAKI, AZUMA, ICHIRO, KITAGUCHI, HIROSHI, KOJIMA, MASAYOSHI, KOMAZAWA, HIROYUKI, MORI, HIDETO, NISHIKAWA, NAOYUKI, ONO, MITSUNORI, ORIKASA, ATSUSHI, SAIKI, IKUO, SATOH, HIDEAKI
Applicants: FUJI PHOTO FILM CO LTD, FUJI PHOTO FILM CO., LTD.
Application year: 1992
Main classification: A61K38
IPC codes: A61K38, C07K5, C07K14, C07K9