AU7737101A - Peptides promoting the activation of latent TGF-b and method for screening TGF-b activity regulators - Google Patents

Description

S&FRef: 448955D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

ORIGINAL

Name and Address of Applicant:

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Actual Inventor(s): Kyowa Hakko Kogyo Co., Ltd.

6-1, Ohtemachi 1-chome Chiyoda-ku Tokyo 100-8185 Japan Motoo Yamasaki, Kenji Shibata, Yasufumi Sato Spruson Ferguson St Martins Tower,Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Peptides Promoting the Activation of Latent TGF-B and Method for Screening TGF-B Activity Regulators Address for Service: Invention Title: The following statement is a full description of this invention, including the best method of performing it known to me/us:o o 5845c

SPECIFICATION

PEPTIDES PROMOTING THE ACTIVATION OF LATENT TGF B AND METHOD FOR SCREENING TGF B ACTIVITY REGULATORS Technical Field The present invention relates to novel peptides which promote conversion of latent TGF-0 (TGF-0 type ordinarily secreted) into active transforming growth factor- 1 (hereinafter occasionally abbreviated as active TGF- 0 or merely as TGF- having a variety of physiological activities such as inhibition of cell growth, promotion of cell differentiation, immunosuppression,' and stimulation of chemotaxis of fibroblasts and which are useful as therapeutic agents for diseases.pointed out to be related to the lack of TGF- 8 activity and diseases against which administration of exogenous TGF-1 is considered to be effective, such as cancer, bone fracture, myocardial infarction, myocardial disorder after ischemia reperfusion, cerebral infarction and retinal detachment.

The present invention also relates to methods of screening 20 compounds which regulate the binding of latent transforming growth factor- B (hereinafter occasionally abbreviated as LTGF- 8 to cells or compounds which regulate the release of active TGF-3 from latent TGF-3, and to compounds obtainable by the above methods which are useful for the treatment or prevention of TGF-1-related diseases.

Background Art In mammals including humans exist some types of TGF- 3 such as TGF- 1 1, 6 2 and 3, and all of them are secreted as inactive 30 LTGF-0 [Robert, A.B. Sporn, Peptide Growth Factors and Their Roceptors, Handbook of Experimental Pharmacology, Part 1, SPRINGER-VERLAG, Berlin, p. 419-472- (1990)] and need to be activated after the secretion to exhibit their activities. LTGF- 3 is divided into two types: small molecular weight latent TGF- 3 (hereinafter abbreviated as SLTGF- wherein a latency associated peptide (hereinafter occasionally abbreviated as

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LAP) is non-covalently bound to TGF-3 and large molecular weight latent TGF-3 (hereinafter occasionally abbreviated as LLTGF- 3) wherein latent TGF-13 binding protein (hereinafter occasionally abbreviated as LTBP) is bound to SLTGF-3 by SS bond with LAP. LTGF-3 is secreted mostly in the form of LLTGF-3 [EMBO Journal, 10, 1091 (1991)]. TGF-3 and LAP are biosynthesized as the same protein molecule (TGF-3 precursor) having a signal peptide and the amino acid sequence thereof is known [Nature, 316, 701 (1985)].

Some protease enzymes have been pointed out to participate in the activation of latent TGF-j3, and plasmin has been analyzed most closely among these enzymes. That is, non-covalently bound TGF- is released by the limited degradation of LAP by plasmin [Journal of Cell Biology, 110, 1361 (1990)]. The analysis of the activation of latent TGF-j by plasmin at the cell level has revealed the following: the activation by plasmin is carried out on the surface of the cell membrane [Journal of Cell Biology, 109, 309 (1989)], binding of latent TGF-3 to the cell membrane is necessary for the activation [Journal of Cell Biology, 121, 20 439 (1993), ibid., 120, 995 (1993), ibid., 123, 1249 (1993)], and latent TGF- 3 is bound to the cell membrane via LAP [Journal of Cell Biology, 123, 1249 (1993), Tohoku Journal of Experimental Medicine, 179, 23 (1996)]. However, it is not clear how the regulation of the binding of latent TGF-3 to a cell membrane 25 is associated with the regulation of TGF-3 activity.

It is recognized that latent TGF-3 is bound to vascular smooth muscle cells, but not to vascular endothelial cells [Journal of Cell Biology, 123, 1249 (1993)].

TGF-0 has a variety of physiological activities such as 30 inhibition of cell growth, promotion of cell differentiation, immunosuppression, and stimulation of chemotaxis of fibroblasts. TGF-3 is considered to be associated with various diseases. For example, it has been reported that lack of TGF- 3 activity is related to diabetic retinopathy [Journal of Cell Biology, 19,. 309 (1989), Archives of Ophthalmology, 66, 366 (1961)] and initial lesion of atherosclerosis [Nature Medicine, 0

S

S

S

S 0

S

3 1, 1067 (1995)]. TGF-/ itself is expected to have a therapeutic effect on bone fracture, myocardial infarction, myocardial disorder after ischemia reperfusion, cerebral infarction and retinal detachment [Journal of Cell Biology, 11, 1017 (1992)] Further, TGF-3 is known to inhibit the growth of various cancer cells [Endocrinology, 128, 1981 (1991), Journal of Clinical Investigation, 82, 277 (1991), Cell Growth Differentiation, 1, 549 (1990)] and is expected as an anti-tumor agent [Proceedings of the National Academy of Science 92, 4254 (1995)].

Only a part of latent TGF-/ produced and secreted in vivo is activated and exhibits its 4ctivity, and accordingly, it is f considered that the activity of TGF-3 can be enhanced by increasing the activation efficiency of latent TGF-/ in vivo.

Therefore, a compound which promotes the activation of latent TGF-8 is expected to be effective as a therapeutic agent for diseases pointed out to be related to the lack of TGF- activity and diseases against which administration of exogenous

TGF-

is considered to be effective, for example, cancer, diabetic 20 retinopathy, atherosclerosis, bone fracture, myocardial infarction, myocardial disorder after ischemia reperfusion, cerebral infarction and retinal detachment.

On the other hand, there have been known various diseases basically accompanied by development of extracellular matrix which are caused by advance of TGF- activation. A substance which inhibits the TGF- activation is expected to be effective as a therapeutic agent for diseases such as glomerulonephritis diabetic nephropathy, renal graft rejection, HIV nephropathy, sudden pulmonary fibrosis, autoimmune pulmonary fibrosis, 30 hepatic cirrhosis, venous constrictive hepatopathy (often occurring after treatments of cancer), systemic sclerosis, S keloid, eosinophilia-muscle ache syndrome, re-stricture after angioplasty, intraocular fibrosis, rheumatic arthritis and fibrosis such as nasal polyp [Border W.A. Noble

N.A.,

Transforming growth.factor-O in tissue fibrosis, New Engl.

J. Med., 331, 1286 (1994) and Border W.A. Rouslahti

E.,

1. 1.

4 Transforming growth factor-p in disease: The dark side of tissue repair, J. Clin, Invest., 1, (1992)].

Disclosure of the Invention According to this invention there is provided a method of screening a compound to be used for treatment or prevention of TGF-P-related diseases, which comprises: measuring the amount of latent TGF-p bound to animal cells after addition of latent TGFp to said cells; measuring the amount of latent TGF-P bound to animal cells after addition of latent TGF-p and a compound to be evaluated to said cells; and evaluating the inhibiting activity or promoting activity of said compound on the binding of latent TGF-P 0o to animal cells from the change in the amount of latent TGF-p bound to animal cells caused by the addition of said compound.

In another embodiment, the present invention provides a peptide having an activity to promote the release of active TGF-P from latent TGF-P or an activity to promote the binding of latent TGF-P or an activity to promote the binding of latent TGF-P to a cell S 15 membrane which is represented by general 0 0 00 *oo o [R:\LIBUU]02161.doc:MCN formula wherein A is an amino acid sequence selected from partial sequences of an amino acid sequence selected from the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF- 13 1 precursor sequence and the sequences of TGF- 3 precursors other than human TGF-01 corresponding to the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF-31 precursor sequence when aligned with the human TGF- 1 sequence, and 1 to 5 amino acid residues in said partial sequence may be deleted, substituted or added, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a peptide having an activity to promote the release of active

TGF-

3 from latent TGF-1 or an activity to promote the binding of latent TGF-3 to a cell membrane which is represented by general formula wherein A is any one of the amino acid sequences of SEQ ID NOS: 1-16 in which 1 to 5 amino acid residues may be deleted, substituted or added, or a pharmaceutically acceptable salt thereof.

In another embodiment, the present invention provides a 20 method of screening a compound to be used for the treatment or prevention of TGF- 3 -related diseases which comprises measuring the amount of latent TGF-13 bound to animal cells after addition of latent TGF-1 to said cells, measuring the amount of latent TGF-3 bound to animal cells after addition of latent TGF-3 and a compound to be evaluated to said cells, and evaluating the 4040: inhibiting activity or promoting activity of said compound on the binding of latent TGF-3 to animal cells from the change in the amount of latent TGF-1 bound to animal cells caused by the addition of said compound.

30 In another embodiment, the present invention provides a method of screening a compound to be used for the treatment or prevention of TGF-13 -related diseases which comprises measuring the amount of TGF-1 after addition of a peptide represented by general formula or a pharmaceutically acceptable salt thereof to animal cells, measuring the amount of TGF-0 after addition of a compound to be evaluated and a peptide represented by general formula or a pharmaceutically acceptable salt thereof to animal cells, and evaluating the inhibiting activity or promoting activity of said compound on the conversion of latent TGF-P into TGF-P from the change in the amount of TGF-P caused by the addition of said compound.

In another embodiment of the present invention, a compound having inhibiting activity or promoting activity on the binding of latent TGF-P to cells or on the conversion of latent TGF-P into TGF-P is obtainable according to either of the above two methods, and a compound to be used for the treatment or prevention of TGF-p-related diseases or a pharmaceutically acceptable salt thereof is provided.

The peptides represented by general formula are hereinafter referred to as Compounds In the definitions of the groups in general formula the alkanoyl includes alkanoyl groups having 1 to 20 carbon atoms, such as formyl, acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl, pivaloyl, hexanoyl, heptanoyl, lauroyl and icosanoyl.

Examples of the aryl moiety of the aroyl and the aryloxycarbonyl are phenyl and naphthyl.

Examples of the heteroaryl moiety of the heteroarylcarbonyl and the heteroaryloxycarbonyl are furyl, thienyl, pyridyl, pyrrolyl, pyrazolyl, pyrimidinyl, pyrazinyl, indolyl, quinolyl, isoquinolyl and quinazolinyl.

The alkyl moiety of the alkoxycarbonyl and the alkoxy includes alkyl groups having S 1 to 20 carbon atoms, such as methyl, ethyl, priopyl, isopropyl, butyl, pentyl, hexyl, heptyl, decyl, dodecyl and icosyl.

The substituted alkanoyl, the substituted alkoxycarbonyl and the substituted alkoxy each has 1 to 3 substituents which are the same or different. Examples of the substituents are hydroxy, carboxyl, alicyclic alkyl groups having 3 to 8 carbon atoms (e.g.

S* cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl), substituted or unsubstituted phenyl, and fluorenyl. The substituted phenyl has 1 to 3 IN:\LIBCI04308:SAK substituents which are the same or different. Examples of the substituents are alkyl, alkoxy, hydroxy, nitro, sulfo, cyano and halogen. The halogen includes fluorine, chlorine, bromine and iodine. The alkyl moiety of the alkyl and the alkoxy as the substituents of the substituted phenyl has the same significance as the above-mentioned alkyl moiety of the alkoxycarbonyl and the alkoxy.

The substituted aroyl, the substituted aryloxycarbonyl, the substituted heteroarylcarbonyl and the substituted heteroaryloxycarbonyl each has 1 to 3 substituents which are the same or different. The substituents are the same as the substituents of the above substituted phenyl.

I The substituted amino has 1 to 2 substituents which are the same or different, and examples of the substituents are substituted or unsubstituted alkyl and substituted or unsubstituted aryl. The alkyl has the same significance as the above-mentioned alkyl moiety of the alkoxy, etc., including the substituents thereof. The aryl group has the same significance as the above-mentioned aryl moiety of the aroyl and the S* 20 aryloxycarbonyl, including the substituents thereof.

As the TGF-/ precursor sequence, any kind of TGF-3 sequence derived from any animal may be employed. Suitable examples are human TGF-1 (J05114) [Nature, 316, 701 (1985)] (SEQ IDNO: 17), humanTGF-f 2 (Y00083) [EMBO. 6, 3673 (1987) (SEQ IDNO: 18), human TGF- J3 (J03241) [Proc. Natl. Acad. Sci.

USA, 85, 4715 (1988)] (SEQ ID NO: 19), murine TGF-31 (M13177) Biol. Chem., 261, 4377 (1986)] (SEQ ID NO: 20), murine

TGF-

32 (X57413) [Mol. Endocrinol., 3, 1108 (1989)] (SEQ ID NO: 21), murine TGF-33 (M32745) [Mol. Endocrinol, 3, 1926 (1989)]

(SEQ

30 ID NO: 22), rat TGF-81 (X52498) [Nucleic Acids Res., 18, 3059 (1990)] (SEQ ID NO: 23), rat TGF-33 (U03491) Biol. Chem., 210, 2722 (1995)] (SEQ ID NO: 24), bovine TGF- 1 (M36271) [Mol.

Endocrinol., 1, 693 (1987)] (SEQ ID NO: 25), porcine TGF-01 (Y00111) [Nucleic Acids Res., 15, 3187 (1987)] (SEQ ID NO: 26), porcine TGF-83 (X14150) [EMBO 7, 3737 (1988)] (SEQ ID NO: 2 7 canine TGF-31 (L34956) [Gene, 155, 307 (1995)] (SEQ IDNO: 28), ovine TGF-31 (X76916) [Gene, 150, 371 (1994)] (SEQ ID NO:29), chicken TGF-p2 (X58071) [Mol. Endocrinol., 7, 175 (1991)] (SEQ ID NO:30), chicken TGF-p3 (M31154) [Mol. Endocrinol., 2, 747 (1988)] (SEQ ID NO:31), chicken TGF-p4 (M31160) [Mol. Endocrinol., 6, 989 (1992)] (SEQ ID NO: 32), Simian (African green monkey) TGF-P (M16658) [DNA, 6, 239 (1987)] (SEQ ID NO:33) and frog (Xenopus laevis) TGF-35 (J05180) Biol. Chem., 265, 1089 (1990)] (SEQ ID NO:34). The numbers in parentheses following the names of TGF-P precursor sequences indicate the accession numbers of GenBank.

There is no restriction in employing A as long as A is an amino acid sequence which is selected from partial sequences of a TGF-P precursor sequence in which 1 to amino acid residues may be deleted, substituted or added. It is preferable that A is an amino acid sequence which is a partial sequence of a sequence selected from the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF-P1 precursor sequence and the sequences of TGF-P precursors other than human TGF-pl 1i corresponding to the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF-11 precursor sequence when aligned with the human TGF-pl sequence, and in which 1 to 5 amino acid residues may be deleted, substituted or added.

It is particularly preferably that A is a partial sequence of a sequence selected from the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF-p31 precursor sequence and the sequences of TGF-p precursors other than human TGF-l1 eo corresponding to the sequences of amino acids 30 to 60, 142 to 186, and 269 to 297 in the human TGF-1 precursor sequence when aligned with the human TGF-p1 sequence.

The above animal-derived TGF-p precursors which were aligned with the human TGF-1 precursor sequence are shown in Tables 1-1 to 1-8. The figures before and after each sequence indicate the position numbers of amino acids, and in the amino acid sequences indicates gap positions.

0 0 [N:\LIBC104308:SAK Table 1-1I Hurane 1: IPPSGLRLLPLLLPLLILLTLTPGPAG SKTIDJ[LVKRKIEAIRGQILKLIA TGF-,8 1 Hurine 1: IIYCY STFILLVYVAI S CSTLDIO M IEAIRGQILSKLKI., 51 TGF-,8 2 Humane -KHQAVLLNA~LLTTTDGIKREIGISLL 52

TGF-

1 8 3 MRte 1:MPS LPLPPLVIGPALSCTDEVRREIGISLL TGF-fi 1 TGF- fl3 TGF- fl 1 ~6 PRcin 1: -MLQRALVVLALLNFAT VSLSLIMSTTLFGIKRVEAIRGQILSKLRLT 52 TGF-fI 3 Canine 1: IPPSGLRLLPLLLPLLRLL VLTPGRPAAG STKTIDMLVKRKRIEAIRGQILK1,L TGF- R 1 Ovine 1: MPPSGLRLLPLLLPLL WLLMLT GRP VAG STKTDIELYVRGEAIRGQILKJJJ TGF- fl 1 Chicken 1:--------ICYLLSVLTLDLAA VALSLSTCSTL DQIARIEAIRGQILSKJIJ.T 51 *see TF-2 0*0TGF-fl 2 Chicken 1: ALSTCORDLEAAKKKR JEAYRGQILSKLR1,T 32 TGF-fl 4 S Simian 1: MPPSGLRLLPLLLPLL WLL VLTPSRPAAGLSTCKTIDMEL1/VKR rIRGQILSK1LRLA TGF- fl Frog 1 :-------NVWLLVHSLNLTKADE~KREIGISLL 52 TGF- fl Table 1-2 Origin Sequence Human 61: SPPSQGEYPPGpLpEAVAyNS RDVGEAPPEEAY KVR 115

GF-

6 8 -1 Human 52: SPP-EDYPEPEE VPPE VISTY SRDLLQEKASRRAICERERSDEEyyKEYKDIP 110 GF-fi 2 Human 112sRLLEUERECQETS Murine 61:SPQEPGpEVAYT DVGEAPPEEDYKVRLV 115

GF-

1 8 1 Murine 5 2 :SPP-EDYPEPDEVPPEVISIYTRDLLQEKASRRAcERERSEQ~yAKEVYKIDMP 110 GF-f 2 Murine, 53:SP-ES MYVAY~TELEHEEGTQTEEYKIKDI 110 GF-fi 3 Rat 61: SPPSQGE VPPGPLPEAY AYNST RDR VAG-Ap~pEPEDYYAKEVTRVLV 115 TGF-8 1 **Rat 55: YYKEWDIIQ 112 TGF-fl 3 1 Bvine 1: AILAJYNST RDR VAG-S P-P A LMyjj~4 TGF-fi 14 *Porcine 61:SPQDPGLEVAyS RRA-SEE-EEDYKVRL 115 TGF-,8 1 Porcine 53: S 110 TGF- ft 3 Canine 61: SPPSQGE VPPYPLPEAYLALYNS RRVAGESAEPEPEPEyyKEVRVL 115 TGF-fi 1 SOvin*'~F 61: SPPSQGD VPPGPLPEAIILYNS RVAGESAETEPEPEA~yyAVTVJ 115j 0*0 Chicken TEESEYAEYIMP 1 TGF-,8 2 Cicken 55:SP-EVPMyIAySR ETSYAEHF I 112 3 Chiken 33APPPASETPPRPLPDD RALY LLKQLPPDGPDEWAKLRRpE 88 *TGF- R84 **Simian 61SPQEPGLEVAyS 115 TGF-8 R,9IIAEPPEEDYKETVI 1 Frog 53: KTP-DVDSEKIITVPSEAIFLYNsMIEARE~f STIEAR~ GU1QNQDYYAKQ--- 104 TGF- fl 5 Table 1-3 OriginSeun Human 116: THEIYDKFKQSTHSIYIIFFNTSELREAVPEP VI A-ELRLL-RR -LKLKVEQW 169 TGF- 0 1 Human I11: FFPSENAIPPTFYRPYFRI VRFD VSAMEKNASNLYUAFRVFRLQNPKARVPEQRIELYQ 170 TGF-fi 2 Human 113: GLAEHNELAVCPKGITSKYFRFNVSSVEKNTNFRAEFRVLRV1PSSKRNEQRIELFQ 172 TGF- ,8 3 Murine 116: RNNAIYEKTKDISIIsIyMFNTSDIREA VPEppLLJ -ELR L'QR -LKSSVEQIV 168 TOF-f1 Mturine 111:H SNIPF~yRVFVTEKANV FVRW VEREY 17 0 TGF- R 2 Murine .113: GLAEHNELA VCPKGITSK VFRFN-VSSVEKGNFRAEFRVLRVPNPSSKI1'EQRIEL.,Q 172 TGF-,8 3 Rat 111: GLAEHNELAVCPKGITSKVFRFNVSSVEKNTNFRAEFRVLRVpNSRTQRIELpQ17 TGF-,8 1 7 Rat 116: RNNAIYDKTKDITHSIYIMFFNTSDI1?EAVPEPPLLI A-ELR-L-QR -FKSTVQ 168

TGF-

1 8 3 S Bovine 4 :YNIDN&SSYFNSLE~ppLSA-DRL----KKEH 93 TGF-fil1 Porcine 116:SGQYKKIHLY[FTERAPPLSA-LL-R--LLW 168.

TGF-fi 1 Porcine 111: GLEE11 DLAVCPKGITSKIFRFNYSSVEKENFRAEFRIRPNPSSKRSEQRIELFQ 170 TGF- fl 3 Canine 11:TKYKKSIS~[FTkRAVEVLR-ERL---KKEH 168 TGF- fl 1 roe Ovine 116: YGNKIYDKKSSSHSIYi FFNTSELkEAVPEPV HSA--DVRLLR -LKLKEQW 168 TGF- fl 1 seike e -FPENIPYSLFIR SAMEKNASNLVKAEFRVFRLOJSKRVSEQRIELYQ 16 9 Chicke 113,2 GLPEHNELGICPKGVTSNVFRFNSSAKNSNLFRAEFRVLRVPNPSSKRSEQRIELFQ 172 TGF-f8 3 Chicken 89: TWDGAME111!QPs1IFFVFNYSR GRPTLLIRAELMRQKAJMAGJEQL 145 TGF- fl 4 Simian 116:TNYDFsnjy NTEREppVLR-LRR KKEH 168 TGF-fi Frog 105: -YFSTLDEKKNSM VMSLW RY-KIKMQiEF 161 TGF- ,8 0 ~00 0

B

a..

0 00 Table 1-4 Origin Sequence Human 170: ELYQXYSNNSW-RYLSNRLLAPSDSPEWLSFDVTGVVRQW'RGGEIEGFRLSACSC-- 226 TGF- 1 Human 171: ILKSKDLTSPTQRYIDSKVKTRAEGEWLSFDVTDAVIIEWLHHKRNLGFKISLHCPCCT 230 TGF- 06 2 H uman 173: ILRPDE-IIAKQRYIGGKNLPTRGTAEWLSFDVTDTYREILLRRESNLGLEISIHCPCHT 231

TGF-

4 6 3 Murine 169: ELYQKYSNNSI!-RYLGNRLLTPTDTPELSFDVTGYVRQWLNQGDGIQGFRFSAUCSC-- 225 TGF- 1 Murine 171 :ILKSKDLTSPTQRYI DSKVYKTRAEGEWLSFDVTDAVQEWLHHKRNLGFKISLllCPCCT 230 TGF- R8 2 Murine 171: ILRPDE-IIAKQRYIGGKNLPTRGTAErLSFDVT.DTVREWLLRRESNLGLEISIHCPCHT 229 TGF- ft 3 Rat 169: ELYQKYSNNS!-RYLGNRLLTPTDTPEI.LSFDVTGVVRQWLNQGDGIQGFRFSAHCSC-- 225 TGF-46 1 Rat 173: ILRPDE-HIAKQRYIGGKNLPTRGTAEILSFDVT.DTVRE1'LLRRESNLGLEISIHCpCHIT 231 TGF- 46O 3 Bovine 94: ELYQKYSNNS-RYLSNRLLAPSDSPE!ISFDVTGVVRQULTRREEIEGFRLSAlCSC-- 150 TGF- #6 1 Porcine 169: ELYQKYSNDSW-RYLSNRLLAPSDSPEWLSFDVTGVRQLTRREAIEGFRLSAICSC-- 225 TGF-46 1 Porcine 171: JLQPDE-HIAKQRYIDGKNLPTRGAAEU'LSFDVTDTVRE!LLRRESNLGLEISIHCPCHT 229 TGF-46 3 Canine 169: ELYQKYSNDSV-RYLSNRLLAPSDTPEWLSFDVTGVYRQW'LHGGEVEGFRLSACSC-- 225 TGF-46 1 Ovine 169: ELYQKYSNNSJ-RYLSNRLLAPSDSPE!LSFDVTGYYRQWLTHREEIEGFRLSAHCSC-- 225 TGF-461I Chicken 170: YLKSKELSSPGQRYIDSKYVKTRAEGEWL-SFDVTEAVTIEWLIIRRNLGFKISLHCPCCT 229 TGF- R 2 Chicken 173: ILRPDE-HIAKQRYLSGRNVQTRGSPEWLSFDVTIyfYREWLLHRESNLGLEISIHCPCHT 231 TGF-46 3 Chicken 146: ELYQGYGNASW-RYLHGRSVRATADDEWLSFDVTDAHQLSGSELLGyFKLSyL1CPC-- 202 TGF- PA' Simian. 169:ELYQKYSNNSJ-RYLSNRLLAPSNSPEWLSFDVTGVVRQI!LSRGGEIEGFRLSAIICSC-- 2 TGF- 46 Frog 162: K-YQENGITHS-RYbESKYITPVTDEWMSFDVTKTVNEWLKRAENEQFGLQPACKCPT 219 TGF-46 00 a 0 *0O *0 0 0 0 0@*0 a 0 0 Table Origin Sequence Human SRDNTLQYDI-N--GFTTGRRGDLATIHGN R-PFLLLIATPLER 268 TGF-8 1t Human. 231: :FPSNNYHPNKSEELEARFAGDGTSTYTSGDQKTIKSTRKKNSGKIPHLLLILLPSYR 290 TGF-,8 2 Human 232: FQPNGD-ILENIIEVMEIKFKGYDNEDDIIGRGD--LGR-LKKQKDIIHNPHLILMIIPPHR 287 TGF-m8 3 MKurine 226: SKDNKLHYEI-N--GISPKRRGDLGTIHDMN--R-PFLLLhIATPLER 267 TGF- ft Murine 231 :FVPSNNYIIPNKSEELEARFAGIDGTSTYASGDQKTIKSTRKKTSGKTPHLLLMLLPSYR 290 TGF- 9 2 Murine 230: FQPNGD-ILENYHEVMEIKFKGYDNEDDHGRGD--LGR-LKKQKDHNPHLILUMIPPIR 285 TGF-A8 3 Rat SKDNVLIVEI-N--GISPKRRGDLGTIHD N R-PFLLLMATPLER 267 TGF-,8 1 Rat 232: FQPNGD-ILENVHEYMEIKFKGVDNEDDHGRGD--LGR-LKKQKDHRNpHLILMIflPPHR 287 TGF-,8 3 Bovine 151: SKDNTQYDI-N--GFSSGRRGDLATIGMN--R-PFLLLMATPLER 192 TGF-fi 1 Porcine SKDNTLHWEI-N-GFNSGRRGDLATIHGMN--R-PFLLLI[ATPLER 267 TGF- 1 Porcine 230: FQPNGD-ILENIQEYIIEIKFKGYDSEDDPGRGD--LGR-LKKKKE-IISPHLILMMIPPDR 284

TGF-

1 8 3 Canine 226:---D SKDNTLQVDI-N--GFSSSRRGDLATIHGMN--R-PFLLITPLER 267 TGF-fi 1 Ovine 226: SKDNTLQVDI-N--GFSSRRGDLATIHGMN--R-PFLLLIATPLER 267 TGF-fil1 Chicken 230: FVPSNNYIIPNKSEEPEARFAGIDD-YTYSSGDVKAKSNRKKYSGK1pIJLLLMLLPSYR 288

TGF-

1 8 2 Chicken 232: FQPNGD-ILENtLHEYLEIKKGIDSEDDYGRGD--LGR-LKKQKDLENPHLILAILPPllR 287 TGF- fl.3 Chicken 203: EM--GPGHAEEMRI-SI-EGFEQQRGDMQSIAKKIIR----RYLJJALPAER 248 TGF-fi 4 Simian skDNTLQvDi-N--GFTGRRGDLATIHGMN RPFLLLMATPLER 267 TGF- fi Frog 220: QAK.DIDIE-GFPALRGD-LASLSSKENTKPYL-MITSM--PAER 259 TGF- P 00000 *0 000 0 00 @0: *0 S 0 000 Table 1-6 Origin Sequence Human 269: AQIQSRHRRALDTNYCF-SST-EKNCCVRQLYIDFRKDLGWKWIHEPKGYHANCL 324

TGF-

1 8 1 Human 291 :1-ES QNRKRLAAYCF--RNYDNCCLRPLYIDKRDLGKWIlEPKGYNANFCA 347 TGF- R8 2 Human 288 :LDNPGQGGQRKKRALTNYCF--RNLEENCCVRLYIDFRQDLGWKWyMjPKGYYAFCS 345 TGF- A8 3 Murine 268: AQHLHSSRHRRAL.DTNYCF-SST-EKNCCVRQLYIDFRKDLWKWIIIEPKGYIIAWCL 323 TGF-fi 1 lurine 291 :L-ESQQSSRRKKRAIJDAAYCF--RNVQDNCCLRPLYIDFKRDLGvKWIHEPKGYNANFCA 347 TGF-fi 2 AIurine .286: LDSPGQGSQRKKRALD1NYCF--RNLEENcCVRPLYIDFRQDLGKWVHPKGYYAJNFCS 343

TGF-

1 8 3 Rat 26: AQIHSSRHRAL'1NYCF-SST-EKNCCVRQLYIDFRKDLGKWIHEPKGYJIJNFCL 323

TGF-

4 8 1.28 Rat .288 LDSPGQGGQRKKRALDTNYCF--RNLEENCCVRPLYIDFRQDLGWKWVHPKGYYANFCS 345

TGF-

4 8 3 Bovine .193: AQHLHSSRIIRRALDTNYCF-SST-EKNCCVRQLYIDFRKDLGKWIEPKGYIIMNFCL 248 TGF-;8 1 Porcine 268 AQflLHSSRIIRRALDTNYCF-SST-EKNCCVRQLYIDFRKDLG JWIHEPKGYIIANFCL 323 TGF-fi 1.

Porcine 285 :LDNPGLGAQRKRAL DTNYCF--RNLEENCCVRPLYIDFRQDLGWKWVHEPKGYYAJN'CS 342

TGF-

1 8 3 Canine 268 AQHIISSRQRRADTNYCF-SST-EKNiCCyRQLYDFRKDLG KIHEPKGYIIAFCL 323 TGF-fi 1 Ovine 268: AQfLHSSRHRRALDTNYCF-SST-EKNCCVROJYIDFRKDGWKWIIIEPKGYHNJJFCL 323 TGF- R 1 Chicken 289: L-ESQQPSRRKKRALDAAYCF--RNVQDNCCLRPLYDFKRDLGWKWIHEPKGYHANFCA 345 TGF-fi 2 Chicken 288: LESPTLGGQRKRALDTNYCF--RNLEENCCRPLYIDFRQDWKWylEpKGYFANFC~S 345 TGF-fi 3 Chicken 249: ANELHSARRRRLDTDYCFGPGIDEKNCCVRPLYIDFRKDIQWKWIEPKG-YiMWCM 306 TGF-fi 4 Simian 268: AQffl~SSRRRALDTNYCF-SS'f-EKNCCVRLYIDFRKDG!KIHEPKGYHANFCL 323 TGF- 0 Frog 260: I--DTVTSSRKRGVG EYCFG--NNGPNcCKPLYINFRKDIG IIHEPKGYEANYCL 315 TGF-fi 0 0

S.

0 000

S.

S S 0

S.

S

S..

S

*5* 0 0050

*SSS

S

@055O5 0 *0 SO S 0 0 Table 1-7 Origin Sequence Human 325:GPCPYIWSLDTYSKVLALYNQIINPGASAAPCCVPQALEPLPIYYYGRKPKYEQLNMI 384 TGF-fi 1 Human 348: GACPYLWSSDTQHSRVLSLYNTINPEASASPCCVSQDLEPLTILYYIGKPKIEQLSNII 407 TGF- 2 Human 346 GPCPYLRSADTTHSTYLGLYNTLNPEASASPCCVPQJ)LEPLTILYYVGRTPKVEOJNIV 405 TGF- 18 3 Murine 324: GPCPYIWSLDTQYSKVLALYNQIINPGASASPCYPQAMEPLPIVYYVGRKPKVEOJSNII 383

TGF-

1 8 1 Murine .348: GACPYLISSDTQHTKYLSLYNTINPEASASPCCVSQDLEPLTILYYIGNTPKLEQLSNIII 407

TGF-

1 8 2 Murine .344: -GPCPYLRSADTTHSTYLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKYEQLSNIV 403 TGF- R8 3 Rat 324: GPCPIYIWSLDTQYSKVLALYNQLINPGASASPCCVPQALEPLPIVyYGRKPKVEQLSNMI 383

TGF-

1 8 1 Rat 346: GPCPYLRSSDTITHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRTPKVEQLNIV 405 TGF- R8 3 Bovine 249: GPCPYISDTQYSKVLALYNQIINPGASAAPCCVPQALEPLPIYYYVGRKPKVEQLSNII 308 TGF-18 1 Porcine 324: GPCPYIWSLDQYSKVLALYNQHNPGASAAPCPQALEPLPIVYYGRKPKVEQLSNAI 383

TGF-

1 8 1 Porcine 343: GPCPYLRSADTTHSSVLGLYNTLNPEASASPCCyPOJDLEPLTILYYVGRTAKVE MS NMV 402 TGF- #8 3 Canine 324: GPCPYISLDTQYSKVLALYNQHNPGASAAPCCVPQALEPLPIYYVGRKPKVEQLSNLI 383

TGF-

1 6 1 Ovine .324: GPCPYISLDTQYSKYLALYNO~iPGASAA PpQALEPLPIVYYVGRKPKVEQLNI 383 TGF-18 1 Chicken 346: GACPYLWSSDTQilSRVLSLYNTINPEASASPCCVSQDLEPLTnhYYIG[TpKIEQLSNAI 405 TGF-18 2 Chicken 346: GPCPYLRSADTTHSTVLGLYNTLNPEASASPCCVPQDLEPLTILYYVGRIpKYEQLSNV 405 TGF-18 3 Chicken 307: GPCPYIWSADTQYTKVLALYNQllNPGASAAPC~CVPQTLDPLPIIYYV*GRNVRVEQL.NIV 366 TGF-18 4 Simian 324: GPCPYIWSLDQYSKVLALYNQHNPGASAAPCCVPQAIPLPIYYVGRKPKVEQLSNII 383 TGF- 18 Frog 316: G1CPYIWSWTQYSKVLSLYNQNNPGASISPCPDVLEPLPIIYYGRTAKVEQLSNMV 375 TGF-18 0 00000* 0 0* 000 S. 06 0 0 0 *0* 0@ 000

S

*05000

S

0 @0 0~e @005 0.

S

@00060

S

Se @5 0

S

S..

S

Table 1-8 Origin Sequence Human 385:YRSCKCS -391

TGF-

4 8:1 Human 408:YKSCKCS 414

TGF-

4 8 2 Human 406:YKSCKCS 412 TGF- lB 3 Murine 384:VRSCKC2S 390 TGF- R 1 Murine 408:YKSCKC2S 414 TGF-fi 2 Murine 404:VKSCKCS 410 TGF-fi 3 Rat 384:VRSCKCJS 390 TGF-,8 1 Rat 406:VKSCKCS 412

TGF-

1 6 3 Bovine 309:VRSCKCS 315 TGF-fi 1 Porcine 384:VRSCKCS 390

TGF-

1 6 1 Porcine 403:VKSCKCS 409

TGF-

1 8 3 Canine 384:VRSCKCS 390

TGF-

1 8 1 Ovine. 384:VRSCKCS 390 TGF- ,6 1 Chicken 406:VKSCKCS 412 TGF-,8 2 Chicken 4O6:VKSCKCS 412

TGF-

1 8 3 Chicken 367:VRACKC2S 373 TGF- R 4 Simian 384:VRSCKCJS 390 TGF- R6 Frog 376:VRSCNCS 382 TGF-lB The parts corresponding to the sequences of amino acids to 60, 142 to 186, and 269 to 297 in the human TGF- 1 precursor sequence (the underlined amino acid sequences in the human TGF- S1 precursor sequence in Table 1) are, for example, the sequences of amino acids 21 to 51, 137 to 188, and 291 to 320, respectively, in the human TGF-3 2 precursor sequence, and the sequences of amino acids 24 to 54, 139 to 190, and 288 to 318, respectively, in the human TGF- 33 precursor sequence. This kind of alignment can be carried out by the method of Barton Sternberg [Journal of Molecular Biology, 198, 327 (1987)].

Preferred.Compounds are peptides wherein A is an amino acid sequence selected from the sequences of SEQ ID NOS: 1 to 16 in which 1to 5 amino acid residues may be deleted, substituted or added, and pharmaceutically acceptable salts thereof.

Particularly preferred are peptides wherein A is an amino acid sequence selected from the sequences of SEQ ID NOS: 1 to 16.

The expression "1 to 5 amino acid residues may be deleted, substituted or added in the sequence" herein means that the sequence may contain deletion, substitution or addition of a 20 single or plural amino acid residues at a single or plural arbitrarily selected positions therein, and the total number of such residues deleted, substituted or added is 1 to 5, which deletion, substitution and addition may be simultaneously contained in the sequence. It does not matter whether or not the substituted or added amino acid is a natural one.

Examples of the addition are addition of amino acids having a thiol group cysteine and homocysteine) or organic groups at both ends of the sequence. Examples of the substitution are substitution of a cysteine residue existing in the sequence to 30 a serine or alanine residue, and substitution of a serine or alanine residue to a cysteine residue. A disulfide bond may be formed between two thiol groups contained in the sequence for S cyclization. An amide bond represented by CO-NH or a reversed amide bond represented by NH-CO may be formed between the N-terminal amino group or a side-chain amino group and the C-terminal carboxyl group or a side-chain carboxyl group for cyclization.

Examples of the natural amino acids areglycine, L-alanine, L-threonine, L-aspartic acid, L-asparagine, L-glutamic acid, L-glutamine, L-valine, L-leucine, L-serine, L-methionine, Lisoleucine, L-phenylalanine, L-tyrosine, L-lysine, L-arginine, L-histidine, L-proline, L-cysteine and L-tryptophan.

The term latent TGF-3 includes small molecular weight latent. TGF- 0 (SLTGF- comprising TGF- 3 and LAP which inhibits the activity thereof, and large molecular weight latent TGF- 3 (LLTGF- comprising TGF- LAP which inhibits the activity thereof, and LTBP.

The pharmaceutically acceptable salts of the compounds obtainable by the method of the present invention and Compounds include acid addition salts, metal salts, organic base addition salts, etc. Examples of the pharmaceutically acceptable acid addition salts are inorganic acid addition salt such as hydrochloride, sulfate and phosphate, and organic acid addition salts such as acetate, maleate, fumarate, tartrate and citrate. Examples of the pharmaceutically acceptable metal salts are alkali metal salts such as sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of the Soo pharmaceutically acceptable organic base addition salts are salts with primary amines, e.g. methylamine, ethylamine and aniline, secondary amines, e.g. dimethylamine, diethylamine, pyrrolidine, piperidine, morpholine and piperazine, and tertiary amines, e.g. trimethylamine, triethylamine, N,Ndimethylaniline and pyridine, and ammonium salt.

30 The abbreviations for amino acids and their protecting groups used herein are described below.

The abbreviations for amino acids and their protecting groups follow the recommendations by IUPAC-IUB Joint Commission on Biochemical Nomenclature [European Journal of Biochemistry, 138, 9 (1984)].

19 The abbreviations for amino acids and their protecting groups are as follows, unless otherwise specified.

Gly or G; Glycine Ala or A; L-Alanine Thr or. T; L-Threonine Asp or D; L-Aspartic acid Asn or N; L-Asparagine Asx; L-Aspartic acid or L-asparagine Glu'or E; L-Glutamic acid Gln or Q; L-Glutamine Gix; L-Glutarnic acid or L-glutamine Val or V; L-Valine Leu or L; .L-Leucine Ser or S; L-Serine Met or L-Methionine Ile or I; L-Isoleucine Phe or F; L-Phenylalanine Tyr or Y; L-Tyrosine or K; L-Lysine Arg or R; L-Arginine 0*His or H; L-Histidine :*Pro or P; *L-Proline Cys or C; L-Cysteine* Trp or W; L-Tryptophan Fmoc; 9-Fluorenylmethyloxycarbonyl t-Bu; t-Butyl Trt; Trityl C...Pmc; 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Boc; t-Butyloxycarbonyl *see*:The abbreviations for side-chain-protected amino acids are as follows.

.Fmoc-Asp(Ot-Bu)-OH; NO -9-Fluorenylmethyloxycarbonyl-Laspartic acid 13-t-butyl ester Fmoc-Glu (Ot-Bu) -OH; N( -9-Fluorenylrnethyloxycarbonyl-Lglutamic acid -T-t-butyl ester Fmoc-Thr (t-Bu) -OH; Emoc-Ser (t-Bu) -OH-; Fmoc-Tyr (t-Bu) -OH; Fmoc-Lys (Boo) -OH; Fmoc-Asn (Trt) -OH; Fmoc-Gln (Trt) -OH; Fmoc-Arg (Pmc) -OH; NOa -9-Fluorenylmethyloxycarbonyl-Ot -butyl -L-threonine Na' -9-Fluorenylmethyloxycarbonyl-Ot-butyl-L-serine N a 1-9-Fluorenylmethyloxycarbonyl-Ot -butyl-L-tyrosime Na-9-Fluorenylmethyloxycarbonyl-N 6 t-butyloxycarbonyl-L-lysine N a -9-Fluorenylmethyloxycarbonyl-N-T trityl -L--asparagine Na -9-Fluorenylmethyloxycarbonyl-N trityl-L-glutamine Na -9-Fluorenylmethyloxycarbonyl-Ng- 2,2,5,7, B-pent-amethylchroman-6sulfopyl-L-arginine N (V -9-Fluorenylmethyloxycarbonyl-Nimtrityl-L-glutamine NOa -9-Fluorenylmethyloxycarbonyl-Strityl-L-cysteine *NO' 9-Fluorenylmethyloxycarbonyl -Nindt-butyloxycarbonyl-L-t ryptophan 0 o s**0 *00 *04 0 0* 0 Fmoc-His (Trt) -01-; Fmoc-Cys (Trt) -OH; Fmoc-Ttp (Boc) -OH; The abbreviations for reaction solvents, reaction reagents, etc. are as follows.

PyBOP; Benzotriazol-1-yloxytrispyrroliclinophosphonium hexafluorophosphate HOBt; N-Hydroxybenzotriazole Dcc; Dicyclohexylcarbodiimide NMM; N-Methylmorpholine DMF: N, N-Dimethylformanide NMP; N-Methylpyrrolidone TFA; Trifluoroacetic acid DTT; Dithiothreitol HBTU; 2- (lH-Benzotriazoi-1-yl) -1,1,3,3tetramethyluronium hexafluorophosphate DIPC; N, N' -Diisopropylcarbodiimide DIEA; N,N-Diisopropylethylamine DCM; Dichloromethane The processes for producing Compounds are described below.

Compounds can be synthesized by general liquid phase or solid phase peptide synthetic methods [Fundamentals and Experiments of Peptide Synthesis, Nobuo Izumiya, et al., Maruzen (1985)], or appropriate combinations thereof. Compounds can also be synthesized by using an automatic peptide synthesizer.

That is, the peptide synthesis can be carried out by the use of commercially available peptide synthesizers from Shimadzu Corporation, Applied Biosystems Inc., U.S.A. (ABI), Advanced ChemTech Inc., U.S.A. (ACT), etc. using an appropriately side-chain-protected N

O

-9-fluorenylmethyloxycarbonyl amino acid or NO-t-butyloxycarbonyl amino acid according to respective synthesis programs.

.The protected amino acids which are starting materials for the synthesis of Compounds and carrier resins are available 20 from ABI, Shimadzu Corporation, Kokusan Chemical Works Co., Ltd., NovaBiochem Co., Watanabe Chemical Industries, Ltd., ACT, and Peptide Institute Inc.

Cyclization may be carried out after all the constituent amino acid residues and organic groups are bonded by a liquid phase method, a solid phase method or a combination thereof, or in the course of elongation of the peptide chain. In the latter case, the obtained cyclization product is subjected to further condensation with amino acid residues or organic groups to prepare Compound The cyclic structure may be formed by 30 forming, at the final step of the process, a disulfide bond, an amide bond or a reversed amide bond which forms a cyclic structure in general formula or by forming an amide bond in an ordinary sequence, after the above bonds are formed, between an amino acid *residue and the adjacent amino acid residue which are to be constituents of the cyclic structure. The cyclization process is described in detail below.

1. Cyclization by disulfide bond formation First a peptide which has, at two positions in the sequence, amino acid residues having appropriately protected thiol groups is prepared by a solid phase method, a liquid phase method or a combination thereof. Then, protecting groups other than the thidl-protecting groups are removed, followed by removal of the thiol-protecting groups. The thus obtained precursor peptide is subjected to oxidation reaction and the product is purified by general purification steps in organic chemical reactions to give the desired peptide having a cyclic structure with a disulfide bond.

1-1 Cyclization by disulfide bond formation according to a liquid phase method The peptide having a cyclic structure with a disulfide bond can be prepared by subjecting the above precursor peptide to air oxidation or reaction with an oxidizing agent in an inert S solvent. The reaction is carried out at a peptide concentration 0 S* of 0.5-5000 /Amol/l, preferably 50-500 Imol/1. As the solvent, 20 buffers such as 50 mM 1 M tris(hydroxymethyl)aminomethanehydrochloric acid (Tris-HC1) buffer adjusted to pH 4-9, preferably pH 6-8, 5-50% aqueous acid, water, and organic solvents such as DMF, DMSO, acetonitrile, tetrahydrofuran, methanol and ethanol can be used alone or in combination.

Examples of the oxidizing agents are potassium ferricyanide and iodine, which are used respectively in the amounts of 0.1-1 time and 0.5-5 times (preferably one time) that of the precursor peptide (weight:weight) DMSO can also be used as the oxidizing agent at a concentration of 10-50%. The reaction is usually 30 carried out at 0-40'C for one hour to one week. In some cases, the yield of oxidation product can be increased by addition of glutathione, and the reaction may be carried out in the presence of oxidized glutathione in an amount of 0.5-5 times that of the precursor peptide (weight :weight) and reduced glutathione in an amount of one-half the weight of oxidized glutathione [Journal of American Chemical Society, 103, 5867 (1981); Development of 4- 23 Medicines, second series, vol. 14, Peptide Synthesis, p. 239, compiled under the supervision of Haruaki Yajima, Hirokawa Shoten (1991) When iodine is used as the oxidizing agent, zinc powder is added after the completion of reaction until the color of iodine disappears from the reaction mixture, and the mixture is purified as such, or after concentration under reduced pressure, by means of various kinds of chromatography. When potassium ferricyanide is used as the oxidizing agent, the reaction mixture is made weakly acidic by addition of acetic acid and then is purified as such, or after concentration under .reduced pressure 1 by means of various kinds of chromatography.

Alternatively, an anion exchange resin such as Dowex IX2'(AcO-) i (Dow Chemical Co.) may be added to the reaction mixture to remove excess potassium ferricyanide (ferricyan ion and ferrocyan ion) by adsorption, and then the mixture is purified as such, or after concentration under reduced pressure, by means of various kinds of chromatography.

It is also possible to pyridylsulfenylate or 2nitr.opyridylsulfenylate one of the thiol groups and then force 20 the cyclization reaction to proceed to completion simultaneously Swith the selective removal of the other thiol protecting group [International Journal of Peptide and Protein Research, 29, 162 (1987)]. The solvent, reaction temperature, reaction time, etc.

for the cyclization reaction are substantially the same as described above. Further, after the protection groups of the two thiol groups are removed, an equivalent amount of a reagent for pyridylsulfenylation or 2-nitropyridylsulfenylation may be introduced. Pyridylsulfenylation can be carried out by adding.

1-3 equivalents of a reagent such as 2,2'-dithiodipyridine to 30 a solvent containing the peptide, followed by stirring., 2- Nitropyridylsulfenylation can be carried out in a similar manner. The solvent, reaction temperature, reaction time, etc.

Sfor the reaction are substantially the same as described above [Peptide Chemistry, 1991, 125 (1992)] 1-2 Cyclization by disulfide bond formation according to a solid phase method Apeptide which has, at two positions in the sequence, amino acid residues having appropriately protected thiol groups is elongated by a solid phase method. Before cleavage of the peptide from the resin, the thiol-protecting groups are selectively removed and the peptide is subjected to oxidation reaction to prepare a peptide moiety having a cyclic structure. Then, the peptide is cleaved from the resin and the remaining protecting groups are removed, whereby the desired peptide having a cyclic structure is obtained.

Examples of the thiol-protecting groups include acetamidomethyl (Acm) group and trityl (Trt) group. By reaction of the protected peptide on the resin with iodine in an appropriate solvent such as DMF or DCM, Acm group and Trt group are removed and an intramolecular disulfide bond is formed. The reaction is carried out using 0.5-2 ml of a solvent for 50 mg of the resin, and iodine in an amount of 0.5-5 times, preferably one time the calculated weight of the peptide on the resin. The 20 reaction is usually carried out at 0-400C for one hour to one week. After the completion of reaction, the resin is subjected S*o to a usual treatment in a solid phase method, that is, washing with a small amount of a solvent such as DMF or DCM, and then subjected to the subsequent reaction.

The pyridylsulfenylation or 2-nitropyridylsulfenylation of one of the thiol groups as described in 1-1 above can be applied to a solid phase method. The solvent, reaction temperature, reaction time, etc. for the cyclization reaction are substantially the same as described above. Further, similarly 30 to the above-described liquid phase method, an equivalent aiount of a reagent for pyridylsulfenylation or 2nitropyridylsulfenylation may be introduced after the t protecting groups of the two thiol groups. are removed.

Pyridylsulfenylation can be carried out by adding 1-3 equivalents of a reagent such as 2,2'-dithiodipyridine to the resin swollen with a solvent, followed by stirring. 2- Nitropyridylsulfenylation can be carried out in a similar manner. The solvent, reaction temperature, reaction time, etc.

for the reaction are substantially the same as in the above cyclization reaction in the solid phase method.

2. Cyclization by amide bond or reversed amide bond formation By a solid,phase method, a liquid phase method, or a combination thereof, a peptide is prepared which has, at two positions in the sequence, :an amino acid residue having an appropriately protected amino group and an amino acid residue having an appropriately protected carboxyl group and in.which the side chains, N-terminus and C-terminus are protected.. After the amino- and carboxyl-protecting groups are selectively removed, the peptide is subjected to itramolecular condensation, followed by general purification steps in organic chemical reactions to give a peptide which has a cyclic structure and in which the side chains, N-terminus and C-terminus are protected. Then, the remaining protecting groups are rdmoved, whereby the desired peptide is obtained. The desired peptide 20.. can also be prepared by first preparing a peptide moiety having a cyclic structure and then elongating it.

O

2-1 Cyclization by amide bond or reversed amide bond formation according to a liquid phase method By a solid phase method, a peptide is prepared which has, at two positions in the sequence, an amino acid residue having an appropriately protected amino group and an amino acid residue having an appropriately protected carboxyl group. Before cleavage of the peptide from the resin, the amino- and 30 carboxyl-protecting groups are selectively removed, and the obtained peptide having free amino group and free carboxyl group is subjected to condensation reaction to give a peptide moiety having a cyclic structure. Then, the peptide is cleaved from the.resin and the remaining side-chain-protecting groups are removed, whereby the desired peptide having a cyclic structure is obtained.

26 When 4-methyltrityi group is used as the amino-protecting group, it can be removed by reaction using acetic acid/trifluoroethanol/DCM The reaction is.usually carried out at 0-40C for 0.5-6 hours. After the completion of reaction, the peptide is precipitated by addition of diethyl ether, etc., followed by removal of the solvent, if necessary under reduced pressure. General 'purification steps in organic chemical reactions including such step are applicable as may be required.

When allyloxycarbonyl group is used as the aminoprotecting group and allyl ester group is used as the carboxyl-protecting group, these protecting groups can be simultaneously removed by reaction with a reducing agent in the presence of a palladium catalyst. It is also possible to use only allyl ester group as the carboxyl-protecting group. Any zerovalent palladium catalysts for: homogeneous system can be used in the reaction. Suitable catalysts include tetrakis(triphenylphosphine).palladium(0) and palladium (II) acetate-triphenylphosphine. The catalyst is used in an amount 20 of 0.01-1 equivalent, preferably 0.1-0.5 equivalent, based on o the above protecting groups. Further, additives such as formic acid, formic acid-triethylammonium, tributyltin. hydride, triphenyltin hydride, trimethylhydrosilane, sodium borohydride, acetic acid, and acetic acid-NMM are added in an amount of one equivalent to excess based on the above protecting groups. As a solvent, ether, tetrahydrofuran, acetonitrile, DMF, chloroform, etc. are used alone or in combination. For 1 mM of allyloxycarbonyl group and allyl ester group are added the S above-reagents and 3-10 ml of the solvent. The reaction is 30 carried-out at -20 to 80C, preferably 0 to.30cC for 10 minutes to 6 hours. After the completion of reaction, genera'l purification steps in organic chemical reactions can be applied.

The obtained peptide is then subjected. to reaction. for S* forming an intermolecular amide bond between the free amino group and the free carboxyl group. Typical amide bond.formation reactions for cyclization are described below. Common reaction conditions are as follows. As a solvent, DMF, NMP, methylene chloride, chloroform, acetonitrile, tetrahydrofuran, etc. are used alone or in combination. The peptide is used at a concentration of 0.5-5000 Imol/1, preferably 50-500 Atmol/l.

The reaction is carried out usually at 0-40cC, preferably 4with stirring for 3 hours to one week. After the completion of reaction, general purification steps in organic chemical reactions can be applied.

Amide bond formation reaction can be carried out by using carbodiimide such as dicyclohexylcarbodiimide (DCC) or water-soluble carbodiimide (WSC) in an amount of 1-10 equivalents based on the carboxyl group.. NMM, DIEA or sodium hydrogencarbonate is added in an amount of 1.5-2 equivalents based on carbodiimide. If necessary, HOBt or HONSu may be added in an equimolar amount based on carbodiimide.

Amide bond formation reaction can also be carried out by using diphenylphosphoryl azide (DPPA) or diethyl phosphorocyanidate (DEPC) in an amount of 1-10 equivalents based on the carboxyl group. NMM, DIEA or sodium hydrogencarbonate is 20 added in an amount of 1.5-2 equivalents based on carbodiimide.

Further, amide bond formation reaction can be carried out by using PyBOP or HBTU in. an amount of 1-10 equivalents, preferably 2-5 equivalents based on the carboxyl group, and HOBt in an equimolar amount based on PyBOP or HBTU. NMM, DIEA or sodium hydrogencarbonate is added in an amount of 1.5-2 equivalents based on PyBOP or HBTU.

SIt is also possible to convert the carboxyl group into an active ester, selectively remove the amino-protecting group, and then form an amide bond. Examples of the active esters are 30 p-nitrophenyl ester, pentafluorophenyl ester, and Noxysuccinimide ester. The active esters can be formed by various ,0 methods. For example, DCC is added in an amount of 1-10 equivalents based on the carboxyl group, together with an equimolar amount of p-nitrophenol, pentafluorophenol or HONSu, and the mixture is stirred at 0-5C for one hour to one day, followed by removal of the formed dicyclohexylurea (DCUrea) by

S

@005 0

S.

0 0@O 00 0 *0S

OS

S

s.

0 filtration. Then, purification can be carried out by general purification steps in organic chemical reactions. The solvent, reaction temperature, reaction time, etc. for the formation of active esters are substantially the same as in the above amide bond formation reactions.

2-2 Cyclization by amide bond or reversed amide bond formation according to a solid phase method By -a solid phase method, a peptide is prepared which has, at two positions in the sequence, an amino acid residue or an organic group having an appropriately protected amino group and an amino acid residue or an organic group having anappropriately protected carboxyl group. Before cleavage of the peptide from the resin, the amino- and carboxyl-protecting groups are selectively removed, and the obtained peptide having free amino group and free carboxyl group is subjected to condensation reaction to give a peptide moiety having a cyclic structure.

Then, the peptide is cleaved from the resin and the remaining side-chain-protecting groups are removed, whereby the desired 20 peptide having a cyclic structure is obtained.

When 4-methyltrityl group is used as the amino-protecting group, it can be removed by reaction using 0.5-2 ml of acetic acid/trifluoroethanol/DCM for 50 mg of the resin. The reaction is usually carried out at 0-40'C for 0.5-6 hours. After 25 the completion of reaction, the resin is subjected to a usual treatment in a solid phase method, that is, washing with a small amount of a solvent such as DMF, and then subjected to the subsequent reaction.

When allyloxycarbonyl group is used as the amino- 30 protecting group and allyl ester group is used as the carboxyl-protecting group, these protecting groups can be removed by reaction using, for example, a chloroform solution containing 0.1-0.2 M tetrakis (triphenylphosphine)palladium(0), 5% acetic acid and 2.5% NMM.

For 1 mM of allyloxycarbonyl group and allyl ester group is added 3-10 ml of the above chloroform solution. The reaction is usually carried out at 0-40'C for 0.5-6 hours. After the completion of reaction, the resin is subjected to a usual treatment in a solid phase method, that is, washing with a small amount of a solvent such as DMF, and then subjected to the subsequent reaction.

The subsequent reaction is carried out by using, for mg of the resin, 1 ml of an organic solvent such as DMF, DCM.or NMP containing PyBOP or HBTU in an amount of 1-10 equivalents, preferably 2-5 equivalents based on the calculated quantity of the carboxyl group on the resin, HOBt in an equimolar amount based on PyBOP or HBTU, and NMM or DIEA in an amount of 1.5-2 equivalents based on PyBOP or HBTU. The reaction is carried out usually at 0-40C, preferably 4-250C, with stirring for 3 hours to one week.

S After the completion of reaction, the resin is subjected to a usual treatment in a solid phase method, that is, washing with a small amount of a solvent such as DMF, and then subjected to the subsequent reaction.

.Compounds obtained by the- above processes can be purified by high performance liquid chromatography (hereinafter 20 referred to as HPLC) using reversed-phase silica gel columns, such as C-4, C-8 and C-18, or column chromatography or thin layer chromatography such as gel filtration\using partition resins, adsorption resins, ion-exchange resins, silica gel, chemically-modified silica gel, reversed-phase silica gel, alumina, diatomaceous earth or magnesium silicate.

The pharmaceutically acceptable salts of Compound (I).are obtained according to an ordinary method. That is, the acid addition salts and organic base addition salts of Compound (I) are obtained by dissolving Compound in an aqueous solution 30 of the corresponding acid or organic base, followed by freeze-drying. The metal salts of Compound are obtained by dissolving Compound in an aqueous solution containing the corresponding metal ion, followed by purification by gel filtration or HPLC.

Specific examples of Compounds are shown in Table 2.

S

0

S.

00 0 0 0@*

S

0eS 0 050e 0

*OS@

S

S

S* Se S S 0

OSS

Table 2 Compound Sequence 1 H-Leu-Gn-Ser--Ser-Arg-His-Arg-Arg-Ala-Leu-Asp-Thr-Asn-Tyr- ________Ser-Phe-Ser-Ser-Thr-Glu-Lys-Asn-Cys-OI 2 H-Pro-Val -Leu-Leu-Ser-rg-Ala-Glu-Leu-Arg-Leu-Leu-Arg-Arg- ________Leu-Lys--Leu-Lys-Val-Glu-Gln-His-Val-Cys-OH 3 H-Leu-Ser-Thr-Ser-Lys-Thr-J le-Asp-Met-Glu-Leu-Val-Lys-Argle-Glu-Ala-Il]e-Arg-Gly-Cys-OH 4 H-Leu-Ser-Thr-Ser-Lys-Thr-Ile-Asp-Met-Glu-Leu-Val-Lys-Arg- Lys-Arg-I le-Glu-Ala-I le-Arg-Gly-OH H-Thr-I le-Asp-Met-Glu-Leu-Yal-Lys-Arg-Lys-Arg-I le-Glu-Alale-Arg-Gly-OH 6 l-Leu-Val-Lys-Arg--Lys-Arg-Ile-Gli-Ala-I le-Arg-GyO 7 H-Leu-Ser-Thr-Ser-Lys-Thr-I le-Asp-Met-Glu-Leu-Val-Lys-Argle-Ofi 8 H-Leu-Ser-Thr-Ser-Lys-Thr-I le-Asp-Met-Glu-Leu-Yal-Lys-Arg-OHf 9 H-Leu-Ser-Thr-Ser-Lys-Thr-I le-Asp-Met-Glu-Leu-Val-OH 1 0 1-cyclo (Cys-Leu-Ser-Thr-Ser-Lys-Thr-J le-Asp-Met-Glu-Leu-Yal- Lys-Arg-Lys-Arg-I le-Glu-Ala-I le-Arg-Gly-Cys) -Oil 1 1 H-Leu-Ser-Thr-cyclo (Cys-Lys-Thr -I le-Asp-Met-Glu-Leu-Val-Lys- Arg-Lys-Arg-I le-Glu-Ala-I le-Arg- Gly-Cys) -OH 1 2 il-cyclo (Cys-Yal-Leu-Leu-Ser-Arg-Ala-Glu-Leu-Arg-Leu-Leu-Arg- ________Arg-Leu-Lys-Leu-Lys--Cys) -OH 1 3 Biotinyl-cyclo (Cys-Val-Leu-Leu-Ser-Arg-Ala-Glu-Leu-Arg-Leu- Leu-Arg-Arg-Leu-Lys-Leii-Lys-Cys) -OHl 1 4 H-Leu-Ser-Thr--Cys-Lys-Thr-I le-Asp-Met-Glu-Leu-Val-Lys-Arg- Lys-Arg-OHf 1 5 H-Leu-Lys-Leu-Lys-Val-Glu-Gln-His-Val-Glu-Leu-Tyr-Gln-Lys- Tyr-Ser-Asn-Asn-Ser-Trp-Arg-Oll 1 6 Biotinyl-Gly-Arg-Arg-Leu-Lys-Leu-Lys-Val-Glu-Gln-His-ValGlu- ________Leu-Tyr-Gln-Lys-Tyr-Ser-Asn-Asn-Ser-Trp-Arg-Ol 31 The method of screening a compound to be used for the treatment or prevention of TGF- 3 -related diseases is described below which comprises measuring the amount of latent TGF- 3 bound to animal cells after addition of latent TGF-0 to said cells, measuring the amount of latent TGF- 3 bound to animal cells after addition of latent TGF- 3 and a compound to be evaluated to said cells, and evaluating the inhibiting activity or promoting activity of said compound on the binding of latent TGF-1 to animal cells from the change in the amount of latent TGF- 3 bound to animal cells caused by the addition of said compound.

Either a synthetic compound or a natural substance can be subjected to screening according to this method without any specific restriction. For example, a natural or synthetic peptide, a peptide obtained by hydrolyzing a natural protein with an enzyme, etc. can be evaluated.

The latent TGF- f may be those extracted and purified from animal cells, for example, bythe methodof Okada, et al. [Journal of Biochemistry, 106, 304 (1989)] or those produced by recombinant DNA techniques [Journal of Biological Chemistry, 20 271, 29891 (1996) Animal cells suitable for use in this method are. those to which latent TGF- 1 can be bound. Examples of such cells are platelets, vascular smooth muscle cells, capillary endothelial cells, aortic endothelial cells, fibroblasts, epithelial cells and macrophages. The cells may be those isolated and purified from animals such as a human, a cow, a pig and a rat, or cultured cells derived from such cells. The cells can be isolated and purified by the method of Okada, et al.

[Journal of Biochemistry, 106, 304 (1989)], or the like. The binding of latent TGF- 3 to cells can be carried out, for example, 30 by first culturing cells in a medium and adding latent TGF- 13 thereto, followed by incubation, and then washing said cells and measuring the amount of latent TGF-0 bound to the cells.

It is preferred to use latent TGF-0 which has been 1 25 I-labeled according to the chloramine T method [Molecular Cellular Biology, 2, 599 (1982)], or the-like. The amount of latent TGF-0 bound to cells can be determined by measuring the radioactivity.

The amount of latent TGF-3 bound to cells can also be determined by measuring the amount of active TGF-3 because latent TGF-3 is activated by being bound to the cells. The determination of active TGF-/3 can be carried out by any method.

For example, the determination can be carried out by methods such as enzyme immunoassay directly using an anti-TGF-0 antibody [Methods inEnzymology, 198, 303 (1991) and the luciferase assay system of Abe, etal. [Analytical Biochemistry, 216, 276 (1994)], or by measuring the degree of migration of vascular endothelial cells [Journal of Cell Biology, 123, 1249 (1993)], growth of vascular smooth muscle cells [Tohoku Journal of Experimental Medicine, 179, 23 (1996)], growth inhibition of various cancer cells [Journal of Clinical Investigation, 87, 277 (1991), Endocrinology, 128, 1981 (1991)] and growth inhibition of mink lung epithelial cells [Methods in Enzymology, 198, 317 (1991)] The judgment as to whether or not a compound to be evaluated is useful for the treatment or prevention of TGF-3-related 20 diseases is made from the difference between the amount of latent.

TGF- 0 bound to the cells or active TGF-j3 in the absence of said compound and that in the presence of said compound. The desired S" compound can be preferably obtainable by screening a compound, for example, which increases or decreases the amount of active S 25 TGF-03 by 10% or more when added at a concentration of 1 mM compared with that measured without addition of the compound.

The method of screening a compound to be used for the treatment or prevention of TGF-3 -related diseases is described below which comprises measuring the amount of TGF-3 after 30 addition of a peptide shown by Compound or a pharmaceutically S* acceptable salt thereof to animal cells, measuring the amount of TGF-3 after addition of a compound to be evaluated and a S peptide shown by Compound or a pharmaceutically acceptable salt thereof to animal cells, and evaluating the inhibiting activity or. promoting activity of said compound on the conversion of latent TGF-0 into TGF-03 from the change in the amount of TGF-3 caused by the addition of said compound.

Either a synthetic compound or a natural substance can be subjected to screening according to this method without any specific restriction. For example, a natural or synthetic peptide, a peptide obtained by hydrolyzing a natural protein with an enzyme, etc. can be evaluated.

Cells suitable for use in this mthod are those which secrete latent TGF- 3 themselves. Such cell lines are preferred because the desired compound can be selected without addition of latent TGF-3 to the test system. Examples of the cells which secrete latent TGF-3 are vascular endothelial cells, vascular smooth muscle cells and macrophages, and cultured cells derived therefrom.

The amount of active TGF- 3 can be determined by any method, for example, the methods mentioned above.

The compounds selected according to the present invention include not only the above-defined peptides but also all compounds which have an activity to promote the release of active 20 TGF-3 from latent TGF-3 or an activity to promote the binding of latent TGF-3 to a cell membrane.

The compounds obtainable according to the screening methods of the present invention, Compounds and pharmaceutically acceptable salts thereof can be used for treating or preventing diseases such as cancer, diabetic S retinopathy, atherosclerosis, bone fracture, myocardial o infarction, myocardial disorder after ischemia reperfusion, cerebral infarction, retinal detachment, glomerulonephritis, *diabetic nephropathy, renal graft rejection, HIV nephropathy, 30 sudden pulmonary fibrosis,. autoimmune pulmonary fibrosis, hepatic cirrhosis, venous constrictive hepatopathy (often occurring after treatments of cancer), systemic sclerosis, keloid, eosinophilia-muscle ache syndrome, re-stricture after angioplasty, intraocular fibrosis, rheumatic arthritis-and nasal polyp. Specifically, they can be preferably used as anti-fibrosis agents, anti-tumor agents and anti- 34 employed as such or in various administration forms. For example, a pharmaceutical composition which is appropriate as an injection can be prepared by dissolving a compound obtained according to the screening method of the present invention, Compound or a pharmaceutically acceptable salt thereof in physiological saline, or an aqueous solution of glucose, lactose or mannitol. A powdery composition for injection can be prepared by freeze-drying a compound obtained according to the screening method of the present invention, Compound or a pharmaceutically acceptable salt thereof and adding sodium chloride thereto. These pharmaceutical compositions may contain as may be appropriate an additive known in the pharmaceutical

I-.

field, for example, a pharmaceutically acceptable salt.

A pharmaceutical composition for oral administration such as a tablet, granule, powder or syrup can be prepared by mixing a compound obtained according to the screening method of the present invention, Compound or a pharmaceutically acceptable salt thereof with an appropriate excipient, disintegrating.

agent, binder, lubricant, or the like. Further, a suppository 20 for rectal administration can be prepared by mixing a compound .obtained according to the screening method of the present invention, Compound or a pharmaceutically acceptable salt thereof with a conventional carrier.

The effective dose will vary depending upon the mode of administration,. the kind of a compound obtained according to the screening method of the present invention, Compound or a O0 •O pharmaceutically acceptable salt thereof, the age and symptoms of apatient, etc. The mode of administration can also be changed r .o according to the symptoms and the dose. For example, a compound 30 obtained according to the screening method of the present invention, Compound or a pharmaceutically acceptable salt thereof can be administered in a daily dose of 0.00001-100mg/kg, 5 preferably 0.01-10 mg/kg.

0 Brief Description of the Drawings Fig. .1 shows the degree of binding of active TGF-3 to porcine vascular smooth muscle cells (PSMC) as determined by measuring the radioactivity. "Vehicle" lane shows the radioactivity when a solution without a test compound was added, and the other lanes show the radioactivity when the respective test compound was added at a concentration of 100 ILg/ml.

Fig. 2 shows the degree of binding of active TGF- 13 to bovine vascular smooth muscle cells (BSMC) as determined by measuring the radioactivity. "Vehicle" lane shows the radioactivity when a solution without a test compound was added, "cell free" lane shows the radioactivity when 1 2 5 I-LLTGF- 3 alone was added to a S plate without a cell, and the other lanes show the radioactivity when the respective test compound was added at.a concentration of 100 .g/ml.

Fig. 3 shows the migration inhibitory activity of active TGF-3 on bovine capillary endothelial cells (BCEC) as determined by counting the number of the cells which migrated into a field of a microscope. "Vehicle" lane shows the number 20 of the cells when a solution without a test compound was added, ,and the other lanes show the number of the cells when the respective test compound. was added at a concentration of 50 it g/ml.

Fig. 4 shows the migration inhibitory activity of active TGF- 0 on bovine capillary endothelial cells (BCEC) as determined by counting the number of the cells which migrated into a field of a microscope. "Vehicle" lane shows the number *ro r.o of the cells when a solution without a test compound was added, and the other lanes show the number of the cells when the So" 30 respective test compound was added at a concentration of 100 it ,g/ml.

Fig. 5 shows the migration inhibitory activity of active TGF-1 on bovine capillary endothelial cells (BCEC) as determined by counting the number of the cells which migrated into a field of a microscope. "Vehicle" lane shows the number of the cells when a.solution without a test compound was added, 36 and the other lanes show the number of the cells when the respective test compound was added at the concentration indicated.

Fig. 6 shows the luminescence intensity as measured by the luciferase assay system for the determination of the amount of active TGF-3 "STD" lane shows the luminescence intensity at stationary state, "vehicle" lane shows the luminescence intensity when a solution without a test compound was added, and the other lanes show the luminescence intensity when the respective compound was added at the concentration indicated.

Fig. 7 shows the amount of active TGF-3 converted from the luminescence intensity shown in Fig. 6. Each lane has the same significance as that in Fig. 6.

Best Modes for Carrying Out the Invention The physicochemical properties of the compounds in S- Examples below were determined according to the following methods. Mass spectrometric analysis was carried out according to the FAB method using JEOL JMS-SX102A. Amino acid analysis 20 was carried out according to the method of Bidlingmeyer, B.A., Set al. [Journal of Chromatography, 336, 93 (1984)]. Hydrolysis was carried out in hydrochloric acid vapor at 110C for 22 hours.

The amino acid compositions of the resulting hydrolyzates were analyzed with Pico Tag amino acid analyzer (Waters Associates) Example 1 Synthesis of Compound 1 (SEQ ID NO: 1) (H-Leu- Gln-Ser-Ser-Arg-His-Arg-Arg-Ala-Leu-Asp-Thr-Asn- Tyr-Ser-Phe-Ser-Ser-Thr-Glu-Lys-Asn-Cys-OH) *o A carrier resin (Wang resin, 123 mg) combined with 62.5 30 tmol of Fmoc-Cys(Trt) was put in a reactor of an automatic synthesizer (ABI, model 430A) and the following treatments were carried out by the Fmoc method according to the synthesis program developed by ABI.

To the carrier resin was added a 20% piperidine-NMP solution, and the mixture was stirred for 20 minutes, followed by discharge of said solution.

The carrier resin was washed with NMP for 5 minutes, and the rinsings were discharged. The carrier resin combined with Cys(Trt) without Fmoc group was thus obtained.

A solution previously prepared by stirring 250 jmol (4 equivalents based on the amino acid on the resin) of Fmoc-Asn(Trt)-OH, DCC and HOBt in NMP for 50 minutes was added to the resin, and the resulting mixture was stirred for 60 minutes, followed by discharge of said solution.

The carrier resin was washed with NMP for 3 minutes.

Fmoc-Asn(Trt)-Cys (Trt) was thus synthesized on the carrier resin.

Subsequently, deprotection and washing steps and (b) were carried out, and condensation reaction was carried out using Fmoc-Lys (Boc)-OH in step followed by washing step to synthesize Fmoc-Lys(Boc)-Asn(Trt)-Cys(Trt) on the carrier resin. Then, steps were repeated to obtain the carrier S resin .to which a protected peptide was bound. In step in the repeated procedures, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Thr(t-Bu)- OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Phe-OH, Fmoc- 20 Ser(t-Bu)-OH, Fmoc-Tyr(t-Bu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Thr(t-Bu)-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc-His(Trt)-OH, Fmoc- Arg(Pmc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc- Gln(Trt)-OH and Fmoc-Leu-OH were used in turn. After 25 deprotection and washing steps and were carried out, the 00." carrier resin was washed successively with methanol and butyl ether, followed by drying under reduced pressure for 12 hours to obtain 310.3 mg of the carrier resin to which a sidechain-protected peptide was bound. To the obtained carrier resin 30 was added 10 ml of a mixture of TFA thioanisole water ethyl methyl sulfide 1,2-ethanedithiol and thiophenol and the resulting mixture was allowed to stand at room temperature for 8 hours to remove the sidechain-protecting groups and to cleave the peptide from the resin.

After the resin was separated by filtration, the filtrate was concentrated to about 2 ml under reduced pressure, and about ml of ether was added thereto. The deposited precipitate was collected by centrifugation and decantation to obtain 145 mg of the crude peptide. A part of the obtained crude peptide (70 mg) was dissolved in 2 M acetic acid and then purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 mm I.D. x 25 mm). Elution was carried out with a linear concentration gradient by adding 90% aqueous acetonitrile containing 0.1% TFA to 0.1% aqueous TFA, followed by detection at 220 nm to give a fraction containing Compound 1. The obtained fraction was lyophilized to give 25.5 mg of Compound 1.

Mass spectrum [FABMS]: m/z=2701.1 Amino acid analysis: Asx 3.1 Glx 2.1 Ser 5.0 His 1.0 Arg 2.9 Thr 2.0 Ala 1.0 Tyr Leu 1.9 Phe 1.0 Lys 1.1 Cys 1.1 (1) Example 2 Synthesis of Compound 2 (SEQ ID NO: 2) (H-Pro- Val-Leu-Leu-Ser-Arg-Ala-Glu-Leu-Arg-Leu-Leu-Arg- Arg-Leu-Lys-Leu-Lys-Val-Glu-Gln-His-Val-Cys-OH) 20 Condensation was carried out in the same manner as in Example 1 using 181.8 mg of a carrier resin (Wang resin) combined with 100 /mdl of Fmoc-Cys (Trt) as a starting material, and using Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Glu(Ot-Bu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, 25 Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc)-OH, Fmoc- Arg(Pmc)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pmc)- OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Val-OH and Fmoc-Pro-OH in turn. The condensation product was washed and dried to obtain 568.4 mg of the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 320 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I. D. x 250 mm) to give 26.4 mg of Compound 2.

Mass spectrum [FABMS]: m/z=2870.4 Amino acid analysis: Glx 3.0 Ser 1.1 His 1.0 Arg 3.8 Ala 1.1 Pro 0.9 Val 2.6 Leu 7.3 Lys 2.1 Cys 1.2 (1) Example 3 Synthesis of Compound 3 (SEQ ID NO: 3) (H-Leu- Ser-Thr-Ser-Lys-Thr-Ile-Asp-Met-Glu-Leu-Val-Lys- Arg-Lys-Arg-Ile-Glu-Ala-Ile-Arg-Gly-Cys-OH) A carrier resin (Wang resin, 60 mg) combined with 33 jmol of Fmoc-Cys(Trt) was put in a reactor of an automatic synthesizer (Shimadzu Corporation, PSSM-8), and the following treatments were carried out according to the synthesis program.

The carrier resin was washed with 500 ILl of DMF for 3 minutes, and the rinsings were discharged.

o To the carrier resin was added 500 I 1 of a 30% piperidine-DMF solution, and the mixture was stirred for 4 minutes, followed by discharge of said solution. The same treatment 20 was repeated.

The carrier resin was washed with 500 al of DMF for one minute, and the rinsings were discharged. The same treatment was repeated 5 times. The carrier resin combined with Cys(Trt) without Fmoc was thus obtained.

25 DMF (1155 al) containing 330 mol of Fmoc-Gly-OH, 330 a mol of PyBOP, 330 Umol of HOBt monohydrate and 495 /mol ""of NMM was stirred for 3 minutes. The resulting solution was added to the carrier resin and the mixture was stirred for 30 minutes, followed by discharge of the solution.

30 The carrier resin was washed with 500 IL1 of DMF for one minute. The same treatment was repeated 5 times.

Fmoc-Gly-Cys(Trt) was thus synthesized on the carrier resin.

Subsequently, washing and deprotection steps were carried out, and condensation reaction was carried out using Fmoc-Arg(Pmc)-OH in step followed by washing step to synthesize Fmoc-Arg(Pmc)-Gly-Cys(Trt) on the carrier resin.

Then, steps were repeated to obtain the carrier resin to which a protected peptide was bound. In step in the repeated procedures, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Glu(Ot- Bu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH,-Fmoc-Lys(Boc)-OH, Fmoc-Arg (Pmc) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc- Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)- OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH and Fmoc-Leu-OH were used in turn. Subsequently, washing and deprotection steps were carried out, and the carrier resin was washed with 500 Itl of DMF for one minute. The same treatment was repeated times, and the carrier resin was washed successively with methanol and butyl ether, followed by drying under reduced pressure for 12 hours to obtain the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 103.2 mg of the crude peptide. The obtained crude S 20 peptide was purified by HPLC using a reversed-phase column (YMC, YMC-Pack ODS-AM 30 mm I.D. x 250 mm) to give 25.4 mg of Compound 3.

Mass spectrum [FABMS]: m/z=2648.1 Amino acid analysis: Asx 1.1 Glx 2.1 Ser 1.9 0 25 Gly 1.1 Arg 2.5 Thr 1.8 Ala 1.1 Val 0.9 Met 1.0 Ile 3.0 Leu 2.1 Lys 3.1 e Cys 1.2 (1) ease Example 4 Synthesis of Compound 4 (SEQ ID NO: 4) (H-Leu- 506060 30 Ser-Thr-Ser-Lys-Thr-Ile-Asp-Met-Glu-Leu-Val-Lys- Arg-Lys-Arg-Ile-Glu-Ala-Ile-Arg-Gly-OH) Condensation was carried out in the same manner as in Example 1 using 60 mg of a carrier resin (Wang resin) combined with 33 Imol of Fmoc-Gly as a starting material, and using Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Glu(Ot-Bu)- OH, -Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc- Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc- Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)- OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 115.3 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (YMC, YMC-Pack ODS-AM 30 mm I.D. x 250 mm) to give 63.3 mg of Compound 4.

Mass spectrum [FABMS]: m/z=2545.1 (M+H) Amino acid analysis: Asx 1.1 Glx 2.1 Ser 1.9 Gly 1.1 Arg 2..6 Thr 1.9 Ala 1.1 Val 0.9 Met 0.9 Ile 3.0 Leu 2.1 Lys (3)

B

Example 5 Synthesis of Compound 5 (SEQ ID NO: 5) (H-Thr- 20 Ile-Asp-Met-Glu-Leu-Val-Lys-Arg-Lys-Arg-Ile-Glu- Ala-Ile-Arg-Gly-OH) The following treatments were carried out using 51.0 mg of a carrier resin (Wang resin) combined with 25 fmol of Fmoc-Gly as a starting material by the use of a peptide synthesizer (ACT, ACT357) The carrier resin was washed with 1 ml of DMF for 30 seconds, and the rinsings were discharged.

To the carrier resin was added 1 ml of a 25% piperidine-DMF solution and the mixture was stirred for 2 minutes, followed S* 30 by discharge of said solution. To the carrier resin was added again 1 ml of a 25% piperidine-DMF solution and the mixture was stirred for 10 minutes, followed by discharge of said solution.

The carrier resin was washed with DMF for 12 seconds, followed by discharge of the rinsings. The same treatment 42 was repeated 7 times. The carrier resin combined with Gly without Fmoc was thus obtained.

To the carrier resin were added 125 Ll of DMF, 500 ILi of NMP solution containing 0.25 M Fmoc-Arg(Pmc)-OH and 0.25 M HOBt monohydrate, 500 1 of DMF solution containing 0.25 M PyBOP and 125 I11 of DMF solution containing 2.0 M NMM.

After stirring for 60 minutes, the solutions were discharged.

The reactor was washed with 500 1 of DMF, and .then with 1 ml of DMF for 30 seconds with stirring, followed by further washing with 500 IL1 of DMF. Fmoc-Arg(Pmc)-Gly was thus synthesized on the carrier resin.

Subsequently, washing and deprotection steps were carried out, and condensation reaction was carried out using a solution containing Fmoc-Ile-OH in place of Fmoc-Arg (Pmc) -OH in step followed by washing step to synthesize Fmoc- Ile-Arg(Pmc)-Gly on the carrier resin. Then, condensation was carried out using, instep Fmoc-Ala-OH, Fmoc-Glu (Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc- 20 Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, o** Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc- Ile-OH and Fmoc-Thr (t-Bu) -OH in turn. The condensation product was washed and dried to obtain 132.5 mg of the carrier resin (Wang resin) to which a side-chain-protected peptide was bound.

25 Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 58.3 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (YMC, YMC-Pack ODS-AM 30 mm I.D. x 250 mm) in the same manner 30 as in Example 1 to give 22.6 mg of Compound Mass spectrum [FABMS]: m/z=2029.2 Amino acid analysis: Asx 1.0 Glx 2.0 Gly 1.0 Arg 3.0 Thr 1.0 Ala 1.1 Val 0.9 Met 1.1 Ile 2.9 Leu 1.1 Lys 2.0 (2) Example 6 Synthesis of Compound 6 (SEQ ID NO: 6) (H-Leu- Val-Lys-Arg-Lys-Arg-Ile-Glu-Ala-Ile-Arg-Gly-OH) Condensation was carried out in the same manner as in Example 5 using 51.0 mg of a carrier resin (Wang resin) combined with 25.0 Atmol of Fmoc-Gly as a starting material, and using Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Glu(Ot-Bu)- OH, Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc- Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain 112.2 mg of the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 48.7 mg of the crude peptide. The obtained crude peptide was dissolved in 1 ml of TFA, and the resulting solution was added dropwise into 50 ml of ether. The deposited precipitate was separated by filtration and then dried to give 35.5 mg of Compound 6.

Mass spectrum [FABMS]: m/z=1439.0 Amino acid analysis: Glx 1-.1 Gly 1.0 Arg 3.0 S 20 Ala 1.1 Val 0.8 Ile 1.9 Leu 1.0 Lys Goo (2) Example 7 Synthesis of Compound 7 (SEQ ID NO: 7) (H-Leu- Ser-Thr-Ser-Lys-Thr-Ile-Asp-Met-Glu-Leu-Val-Lys- 25 Arg-Lys-Arg-Ile-OH) Condensation was carried out in the same manner as in Example 5 using 56.8 mg of a carrier resin (Wang resin) combined with 25.0 Umol of Fmoc-Ile as a starting material, and using Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pmc)-OH, Fmoc- 30 Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Thr(t- SBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)- OH, Fmoc-Ser (t-Bu)-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain 280.9 mg of the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 123.1mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversedphase column (YMC, YMC-Pack ODS-AM 30mm I.D. x 250mm) in the same manner as in Example 1 to give 26.7mg of Compound 7.

Mass spectrum [FABMS]: m/z=2019.2 (M+H Amino acid analysis: Asx 1.1 Glx 1.1 Ser 1.7 Arg 2.2 Thr 1.7 Val 1.0 Met 1.0 Ile 2.1 Leu 2.1 Lys 3.1 (3) Example 8 Synthesis of Compound 8 (SEQ ID NO:8) (H-Leu-Ser-Thr-Ser-Lys-Thr- Ile-Asp-Met-Glu-Leu-Val-Lys-Arg-OH) 1o Condensation was carried out in the same manner as in Example 5 using 48.1mg of a carrier resin (Wang resin) combined with 25.0tmol of Fmoc-Arg (Pmc) as a starting material, and using Fmoc-Lys (Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu (Ot- Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain 79.3mg of the carrier resin to which a side-chain-protected peptide was bound. Removal of the S side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 18.3mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (YMC, YMC-Pack 20 ODS-AM 30mm I.D. x 250mm) in the same manner as in Example 1 to give 4.7mg of Compound 8.

Mass spectrum [FABMS]: m/z= 1621.0 Amino acid analysis: Asx 1.0 Glx 1.1 Ser 1.8 Arg 1.1 Thr 1.9 Val 0.9 Met 1.0 Ile 1.0 Leu 2.1 Lys 2.0 (2) i: 25 Example 9 Synthesis of Compound 9 (SEQ ID NO:9) (H-Leu-Ser-Thr-Ser-Lys-Thr- Ile-Asp-Met-Glu-Leu-Val-OH) 00 *0 0 INALIBC104308:SAK Condensation was carried out in the same manner as in Example 5 using 48.1 mg of a carrier resin (Wang resin) combined with 25.0 /mol of Fmoc-Val as a starting material, and using Fmoc-Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot- Bu)-OH, Fmoc-Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain 120.3 mg of the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide .from the resin were carried out in the same manner as in Example 1, except that a mixture of 90% TFA, 5% 1,2-ethanedithiol and thioanisole was used and the standing time was 2 hours, to obtain 49.0 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (YMC, YMC-Pack ODS-AM 30 mm I.D. x 250 mm) in the same manner as in Example 1 **Sao: to give 20.2 mg of Compound 9.

0& Mass spectrum [FABMS]: m/z=1336.7 a" .a Amino acid analysis: Asx 1.1 Glx 1.1 Ser 1.8 Thr 1.8 Val 1.0 Met 1.0 Ile 1.0 Leu 2.1 Lys 1.0 (1) Example 10 Synthesis of Compound 10 (SEQ.ID NO: 10] [Hcyclo(Cys-Leu-Ser-Thr-Ser-Lys-Thr-Ile-Asp-Met- Glu-Leu-Val-Ly-Arg-Lys-Arg-Ile-Glu-Ala-I le-Arg- Gly-Cys)-OH] Condensation was carried out in the same manner as in Example 3 using 30 mg of a carrier resin (Wang resin) combined with 16.5 /mol of Fmoc-Cys (Trt) as a starting material, and using Fmoc-Gly, Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc- .Glu(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)- OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Glu(Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc- Ser(t-Bu)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH, Fmoc- Leu-OH and Fmoc-Cys(Trt)-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. The reaction time for condensation with each amino acid was 60 minutes. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 54.5 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 14.0 mg of an uncyclized form of Compound Mass spectrum [FABMS]: m/z=2751.7 The obtained compound was dissolved in 10 ml of DMSO, and aqueous ammonia was added thereto to adjust the pH of the solution to 7.1, followed by stirring at room temperature for 29 hours.

The resulting solution was lyophilized and then dissolved in a 2 M aqueous solution of acetic acid, followed by freeze-drying to give 13.5 mg of Compound Mass spectrum [FABMS]: m/z=2749.5 (M+H Amino acid analysis: Asx 1.2 Glx 2.2 Ser 2.0 Gly 1.1 Arg 3.0 Thr 2.0 Ala 1.1 Val 1.0 Met 1.1 Ile 3.1 Leu 2.2 Lys 3.1 Cys 1.8 (2) Example 11 Synthesis of Compound 11 (SEQ ID NO: 11) [H-Leu- Ser-Thr-cyclo (Cys-Lys-Thr-Ile-Asp-Met-Glu-Leu- 06O Val-Lys-Arg-Lys-Arg-Ile-Glu-Ala-Ile-Arg-Gly- Cys)-OH] *0 Condensation was carried out in the same manner as in 5000: 30 Example 3 using 30 mg of a carrier resin (Wang resin) combined with 16.5 /mol ofFmoc-Cys(Trt) as a starting material, and using 0 Fmoc-Gly, Fmoc-Arg(Pmc)-OH, Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc- Glu(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)- OH, Fmoc-Arg(Pmc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc- Leu-OH, Fmoc-Glu (Ot-Bu)-OH, Fmoc-Met-OH, Fmoc-Asp(Ot-Bu)-OH, Fmoc-Ile-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Lys(Boc)-OH, Fmoc- 47 Cys(Trt)-OH, Fmoc-Thr(t-Bu)-OH, Fmoc-Ser(t-Bu)-OH and Fmoc- Leu-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. The reaction time for condensation with each amino acid was 60 minutes. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 55.2 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 9.7 mg of an uncyclized form of Compound 11.

Mass spectrum [FABMS]: m/z=2664.5 (M+H The obtained compound was dissolved in 10 ml of DMSO, and aqueous ammonia was added thereto to adjust the pH of the solution to 7.3, followed by stirring at room temperature for 20 hours.

The resulting solution was lyophilized and then dissolved in a 2 M aqueous solution of acetic acid, followed by freeze-drying to give 9.9 mg of Compound 11.

20 Mass spectrum [FABMS]: m/z=2662.5 (M+H Amino acid analysis: Asx 0.9 Glx 2.0 Ser 0.9 Gly 1.1 Arg 3.0 Thr 1.9 Ala 1.1 Val 0.9 Met 1.0 Ile 3.2 Leu 2.1 Lys 2.9 Cys 1.5 (2) 0 0 *0 .900 *see di I 0000 N 0 :0 eL 0n 00 -0 0 Example 12 Synthesis of Compound 12 (SEQ ID NO: 12) [Hcyclo(Cys-Val-Leu-Leu-Ser-Arg-Ala-Glu-Leu-Arg- Leu-Leu-Arg-Arg-Leu-Lys-Leu-Lys-Cys)-OH] The following treatments were carried out using 250 mg of a carrier resin (Cl-Trt resin) combined with 140 /Amol of H- Cys(Trt) as a starting material by the use of a peptide synthesizer (ACT, ACT357).

The carrier resin was washed with 2.5 ml of DMF for 3 minutes, and the rinsings were discharged. The same treatment was repeated.

48 To the carrier resin were added 250 1/L of DMF, 1.4 ml of NMP solution containing 0.5 M Fmoc-Lys (Boc)OH and 0.5 M HOBt monohydrate and 1.4 ml of NMP solution containing 0.5 M DIPC.

After stirring for 40 minutes, the solutions were discharged.

The carrier resin was washed with 2.5 ml of DMF for one minute, and the rinsings were discharged. The same treatment was repeated twice.

To the carrier resin were added 250 a1 of DMF, 1.4 ml of NMP solution containing 0.5 M Fmoc-Lys(Boc)-OH and 0.5 M HOBt monohydrate, 1,.4 ml of DMF solution containing 0.5 M HBTU, and0.7 ml of NMP solution containing 2.0 M DIEA. After stirring for 20 minutes, the solutions were discharged.

The same treatment as in was carried out. Fmoc- Lys(Boc)-Cys(Trt) was thus synthesized on the carrier resin.

To the carrier resin was added 2.5 ml of DMF containing piperidine, and the resulting mixture was stirred for two minutes, followed by discharge of said solution. To the S 20 carrier resin was added again 2.5 ml of the same solution, I. and the resulting mixture was stirred for 10 minutes, followed by discharge of said solution.

The carrier resin was washed with 2.5 ml of DMF for one minute, and the rinsings were discharged. The same treatment was repeated 7 times. Thus, the carrier resin combined with Lys (Boc) -Cys (Trt) without Fmoc was obtained.

Subsequently, condensation reaction was carried out using a solution containing Fmoc-Leu-OH in place of Fmoc-Lys(Boc)- OH in steps followed by deprotection and washing steps 30 and to synthesize Leu-Lys(Boc)-Cys(Trt) on the carrier 06. 0 resin. Then, steps were repeated using Fmoc- Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc) -OH, Fmoc-Arg(Pmc) -OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Arg(Pmc)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Ala-OH, Fmoc-Arg (Pmc)-OH, Fmoc- Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Val-OH and Fmoc- Cys(Trt)-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 using 4/5 the amount of the obtained resin to give 137.2 mg of the crude peptide. The obtained crude peptide and 100 mg of DTT were dissolved in 2 ml of DMF, and the solution was allowed to stand at 50C for one hour. The resulting solution was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 mm I.D. X 250 mm) in the same manner as in Example 1 to give 12.2 mg of a peptide having two free SH groups.

Mass spectrum [FABMS]: m/z=2284.3 The obtained peptide was dissolved in 5 ml of a 2 M aqueous S 15 solution of acetic acid. After the resulting solution was diluted with water to 50 ml, dilute aqueous ammonia was added S. thereto to adjust the pHof the solution to 5.7. To the resulting *o mixture was added 0.5 ml of a 0.1 M aqueous solution of K 3 Fe (CN), 6 followed by stirring at room temperature for 2.5 hours. After addition of 1 ml of acetic acid, the reaction mixture was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 7.3 mg of Compound 12.

Mass spectrum [FABMS]: m/z=2282.5 Amino acid analysis: Glx 1.0 Ser 1.0 Arg 3.9 Ala 1.1 Val 0.7 Leu 7.2 Lys 2.1 Cys 2.7 (2) Example 13 Synthesis of Compound 13 (SEQ ID NO: 13) [Biotinyl-cyclo(Cys-Val-Leu-Leu-Ser-Arg-Ala-Glu- Leu-Arg-Leu-Leu-Arg-Arg-Leu-Lys-Leu-Lys-Cys)-OH] After 1/5 the amount of the carrier resin with the side-chain-protected peptide obtained in Example 12 was washed with 1 ml of DMF, 440 iL1 of DMF suspension containing 68.4 mg of D-biotin (Nakalai Tesque, Inc.) and NMP solution containing M DIPC were added thereto, followed by stirring at room temperature for 2 days. After the solutions were discharged, the carrier resin was washed and dried to obtain the resin combined with a side-chain-protected peptide having the biotinylated N-terminus. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 47.3 mg of the crude peptide. The obtained crude peptide and 50 mg of DTT were .dissolved in 1 ml of DMF, and the solution was allowed to stand at 50C for one hour. The resulting solution was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 11.8 mg of a peptide having two free SH groups.

i Mass spectrum [FAB-MS]: m/z=2510.8 15 The obtained peptide was dissolved in 5 ml of a 2 Maqueous o solution of acetic acid. After the resulting solution was S* diluted with water to 50 ml, dilute aqueous ammonia was added thereto to adjust the pHof the solution to 5.5. To the resulting mixture was added 0.5 ml of a 0.1 M aqueous solution of K 3 Fe (CN)6, followed by stirring at room temperature for 3 hours. After addition of 1 ml of acetic acid, the reaction mixture was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 3.8 mg of Compound 13.

Mass spectrum [FABMS]: m/z=2508.8 so Amino acid analysis: Glx 1.1 Ser 1.0 Arg 3.8 Ala 1.1 Val 0.8 Leu 6.8 Lys 2.0 Cys 2.1 (2) 30 Example 14 Synthesis of Compound 14 (SEQ ID NO: 14) (H-Leu- Ser-Thr-Cys-Lys-Thr-Ile-Asp-Met-Glu-Leu-Val-Lys- Arg-Lys-Arg-OH) Condensation was carried out in the same manner as in Example 3 using 50 mg of a carrier resin (Wang resin) combined with 23 mol of Fmoc-Arg(Pmc) as a starting material, and using Fmoc-Lys (Boc) -OH, Fmoc-Arg(Pmc) -OH, Fmoc-Lys (Boc)-OH, Fmoc- Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc- Asp(OtBu)-OH, Fmoc-Ile-OH, Fmoc-Thr(tBu)-OH, Fmoc-Lys(Boc)- OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. The reaction time for condensation with each amino acid was 60 minutes. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1 to obtain 59.8 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 7.7 mg of Compound 14.

Mass spectrum [FABMS]: m/z=1921.7 15 Amino acid analysis: Asx 1.0 Glx 1.1 Ser 0.9 Arg 2.2 Thr 1.6 Val 0.9 Met 1.1 Ile Leu 2.1 Lys 3.1 Cys 1.3 (1) Example 15 Synthesis of Compound 15 (SEQ ID NO: 15) (H-Leu- Lys-Leu-Lys-Val-Glu-Gln-His-Val-Glu-Leu-Tyr-Gln- Lys-Tyr-Ser-Asn-Asn-Ser-Trp-Arg-OH) Condensation was carried out in the same manner as in Example 3 using 50 mg of a carrier resin (Wang resin) combined with 23 /Lmol of Fmoc-Arg (Pmc) as a starting material, and using 25 Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr (tBu)-OH, Fmoc- Lys (Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr (tBu)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys(Boc)- OH, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH and Fmoc-Leu-OH in turn. The condensation product was washed and dried to obtain the carrier resin to which a side-chain-protected peptide was bound. The reaction time for condensation with each amino acid was minutes. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1, except that a mixture of the same composition as in Example 1 additionally containing 5 mg/ml 2-methylindole was used and the standing time was 6 hours, to obtain 20.9 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. x 250 mm) in the same manner as in Example 1 to give 7.8 mg of Compound Mass spectrum [FABMS]: m/z=2663.5 Amino acidanalysis: Asx 2.0 Glx3.9 Ser 2.0 His 1.1 Arg 1.1 Tyr 2.2 Val 1.8 Leu 3.0 Lys 3.0 (3) Example 16 Synthesis of Compound 16 (SEQ ID NO: 16) (Biotinyl-Gly-Arg-Arg-Leu-Lys-Leu-Lys-Val -Glu- Gln-His-Val-Glu-Leu-Tyr-Gln-Lys-Tyr-Ser-Asn-Asn- 15 Ser-Trp-Arg-OH) Condensation was carried out in the same manner as in Example 3 using 30 mg of a carrier resin (Wang resin) combined with 13.8 ILmol of Fmoc-Arg (Pmc) as a starting material, and using Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc- Asn(Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Tyr (tBu)-OH, Fmoc- Lys(Boc)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, S Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc- Gln(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Fmoc-Lys (Boc)- OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Leu-OH, Fmoc- 25 Arg(Pmc)-OH, Fmoc-Arg(Pmc)-OH and Fmoc-Gly-OH in turn.

00 ~To the condensation product was added a solution which had been Soo*oo O previously prepared by adding 562 tl of DMF containing 0.5 M HBTU and 0.5 M HOBt monohydrate and 562 iLl of DMF containing 1 M DIEA to 61.9 mg of D-biotin (Nakalai Tesque, Inc.), and by stirring the mixture at room temperature for 5 minutes, and the resulting mixture was stirred for 4 hours. After the solution was discharged, the product was washed and dried to obtain the carrier resin to which a side-chain protected peptide having the biotinylated N-terminus was bound. In the condensation of an amino acid in step HBTU was used in place of PyBOP and DIEA was used in place of NMM. Removal of the side-chain-protecting groups and cleavage of the peptide from the resin were carried out in the same manner as in Example 1, except that a mixture of the same composition as in Example 1 additionally containing mg/ml 2-methylindole was used andthe standing time was 6 hours, to obtain 44.2 mg of the crude peptide. The obtained crude peptide was purified by HPLC using a reversed-phase column (Shiseido Co., Ltd., CAPCELL PAK C18 30 mm I.D. 250 mm) in the same manner as in Example 1 to give 10.3 mg of Compound 16.

Mass spectrum [FABMS]: m/z=3260.0 (M+H Amino acid analysis: Asx 2.1 Glx 4.1 Ser 2.0. Gly 1.0 His 1.0 Arg 2.8 Tyr 2.1 Val 1.9 Leu 3.0 Lys 2.9 (3) eee S Example 17 Conversion of Compound 10 into acetate 15 Compound 10 obtained according to the. process of Example 10 (22.1 mg) was dissolved in 5 ml of a 1% aqueous solution of acetic acid, and the solution was passed through an anion **o exchange column (Asahi Chemical Industry Co., Ltd., Asahi Pack ES-502N 21.5 mm I.D. x 100 mm) to remove TFA and convert the compound into acetate. Elution of the peptide was carried out with a 1% aqueous solution of acetic acid, followed.by detection at 220 nm. The eluate was lyophilizedto give 20.0 mg of acetate of Compound 25 Example 18 Conversion of Compound 12 into acetate Compound 12 obtained according to the process of Example 12 (17.5 mg) was dissolved in 5 ml of a 1% aqueous solution of acetic acid, and the solution was passed through an anion exchange column (Asahi Chemical Industry Co., Ltd., Asahi Pack 30 ES-502N 21.5 mm I.D. x 100 mm) to remove TFA and convert the compound into acetate. Elution of the peptide was carried out with a 1% aqueous solution of acetic acid, followed by detection at 220 nm. The eluate was lyophilizedto give 13.0 mg of acetate of Compound 12.

000000 6 0 0 0 0 0 '0 0 0 0 The biological activities of Compounds and the screening methods are described by the following Examples.

Example 19 Activity to release active TGF-3 from latent TGF- 3 examined by measuring the degree of binding of TGF-3 to bovine vascular smooth muscle cells, porcine vascular smooth muscle cells, bovine capillary endothelial cells or bovine aortic endothelial cells 1 25 I-Labeling of LLTGF-0 LLTGF-13 isolated and purified from human platelets by the method of Okada, et al. [Journal of Biochemistry, 106, 304 (1989) was 1 25 I-labeled according to the chloramine T method [Molecular Cellular Biology, 2, 599 (1982)] in the following manner.

To 2 ag of LLTGF-0 were added 25 a1 of 1 M K-phosphate buffer (pH 37 MBq of Na-1 25 I (DuPont NEN) and 10 1l of chloramine-T (0.5 mg/100 1i in 0.05 MK-phos. buffer, Wako Pure Chemical Industries, Ltd.), followed by stirring at room temperature for 40 seconds. After 15 L1 of Na 2

S

2 05 (1 mg/100 zl in 0.05 M K-phos. buffer) was added, the mixture was stirred at room temperature for 5 seconds, followed by addition of 100 I1 of an aqueous solution of tyrosine (I mg/ml) The reaction mixture was applied to a Sephadex G25 column and the eluate was taken in 500 a1 fractions. Each fraction was subjected to measurement of radioactivity using a Y-counter (ARC-2000, Aloka Co., Ltd.). The fractions containing labeled protein were selected for use in the experiment.

Preparation of cells The cells to be used in the experiment were obtained according to the method of Sato, et al. [Journal of Cell Biology, 123, 1249 (1993)]. Bovine vascular smooth muscle cells (BSMC) and porcine vascular smooth muscle cells (PSMC) were isolated from bovine aorta and porcine aorta, respectively, and cultured by the explant method [Journal of Cell Biology, 50, 172 (1971)].

Bovine capillary endothelial cells (BCEC) were isolated from bovine adrenal capillary and cultured. Bovine aortic endothelial cells (BAEC) were isolated from bovine aortic and cultured.

Measurement of the degree of TGF-3 binding The following procedure was carried out according to the method of Sato, et al. [Journal of Cell Biology, 123, 1249 (1993)].

BSMC, PSMC and BCEC, isolated and cultured in the above-described manner were respectively put into 35 mm dishes in an amount of 4 x 104 cells/dish. On the next day, the medium was replaced by Dulbecco's Modified Eagle's Medium (DMEM, Nissui S Pharmaceutical Co.) containing 0.1% bovine serum albumin (BSA), Sfollowed by incubation for 5 hours. After the cells were washed 15 with 2 ml of phosphate-buffered saline containing 0.1 g/l each of MgC 2 6H 2 0 and CaC12 [hereinafter sometimes referred to as phosphate-buffered saline containing neither MgC1 2 6H 2 0 nor CaCI 2 is referred to as PBS(-) the medium was replaced by 1 ml of ice-cold DMEM containing 0.1% BSA, 2 ng/ml 1 25

I-LLTGF-

3 and a test compound, followed by incubation at 4C for 3hours.

The test compound was added as a solution in dimethyl sulfoxide (DMSO) to give a final DMSO concentration in the system of 0.1%.

After being washed three times with 2 ml of ice-cold PBS(+) the se*o cells were lysed with 900 a1l of 0.5% Triton X-100 (room 25 temperature, 0.5-1 hour), and 800 Il of the lysate was subjected S to measurement of radioactivity using a T-counter.

The results are shown in Figs. 1 and 2 and Tables 3 to 6.

Table 3 Table 3 Test group Radioactivity (cpm), n=4 Vehicle 163.1 28.3 Cell free 70.6 10.2 Compound 12 2739.9 182.8 Table 3 shows the degree of binding of active TGF-3 to bovine capillary endothelial cells (BCEC) as determined by measuring

I

56 the radioactivity. "Vehicle" lane shows the radioactivity when a solution without a test compound was added, "cell free" lane shows the radioactivity when 125 I-LLTGF-0 alone was added to a plate without a cell, and the other lane shows the radioactivity when the test compound was added at a concentration of Table 4 Test group Radioactivity (cpm), n=4 Vehicle 416.8 89.6 Cell free 70.6 10.2 Compound 14 3369.9 166.8 Compound 15 5621.1 889.2 Table 4 shows the degree of binding of active TGF-P to bovine capillary endothelial cells (BCEC) as determined by measuring the radioactivity. "Vehicle" lane shows the S. radioactivity when a solution without a test compound was added, "cell free" lane shows the radioactivity when 125 I-LLTGF-P alone was added to a plate without a cell, and the 1o other lanes show the radioactivity when the respective test compound was added at a concentration of 100[tg/ml.

Table

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15 0 S. 9 Test group Radioactivity (cpm), n=4 Vehicle 1076.5 12.0 Compound 13 4704.0 939.9 Table 5 shows the degree of binding of active TGF-P to bovine aortic endothelial cells (BAEC) as determined by measuring the radioactivity. "Vehicle" lane shows the radioactivity when a solution without a test compound was added, and the other lane shows IN:\IBC1O4308:SAK the radioactivity when the test compound was added at a concentration of 100jpg/ml.

Table 6 Test group Radioactivity (cpm), n=4) Vehicle 792.0 107.0 Compound 16 10495.0 803.0 Table 6 shows the degree of binding of active TGF-P to bovine aortic endothelial cells (BAEC) as determined by measuring the radioactivity. "Vehicle" lane shows the radioactivity when a solution without a test compound was added, and the other lane shows the radioactivity when the test compound was added at a concentration of 100 g/ml.

As shown in Figs. 1 and 2 and Tables 3 to 6, the degree of binding of TGF-p to 10 cells was increased and the release of active TGF-p from latent TGF-P was promoted by SCompounds 1 to 3, 5 to 9 and 13 to 16 at a concentration of 100ug/ml and Compound 12 at a concentration of Compounds promoting the release of active TGF-P from latent TGF-p and increasing the degree of binding of TGF-P to cells can be obtainable by the method of the present invention.

Example 20 Activity to release active TGF-P from latent TGF-P examined by measuring the degree of migration of bovine vascular endothelial cells.

The following procedure was carried out according to the method of Sato et al.

[Journal of Cell Biology, 1249 (1993)].

**00 0 [I:\DAYLIB\LIBUU]02149.doc:MCN BCEC were cultured in a 35mm dish to make a confluent layer and then the medium was replaced by DMEM containing 0.1% BSA, followed by incubation for 5 hours. After a part of the cells were scraped with a razor, the remaining cells were washed with PBS(-), followed by incubation in DMEM containing 0.1% BSA and a test compound. After 24 hours, the number of the cells which migrated in four fields of a microscope (1X70, Olympus) was counted for each dish. The test compound was added in the same manner as in Example 19. The results are shown in Figs. 3 to 5. The migration of cells was inhibited and the release of active TGF-P from latent TGF-P was promoted by Compounds 3 and 5 to 9 at a concentration of 50pg/ml. The migration of cells was also 1 o inhibited by Compound 4 at a concentration of 100p.g/ml, and Compound 10 showed the migration inhibitory effect even at Compounds promoting the release of active TGF-P from latent TGF-p and increasing the degree of binding of TGF-P to cells can be screened by the method of the present invention.

15 Example 21 Determination of active TGF-P by luciferase assay The amount of active TGF-P was determined by measuring the luminescence of Smink lung epithelial cells (MLEC) carrying the luciferase gene introduced downstream of the PAI-I promoter by luciferase assay system (Promega) according to the method of Abe, et al. [Analytical Biochemistry, 216, 276 (1994)] as described below.

BCEC were cultured in a 24-well plate to make a confluent layer and then the medium was replaced by DMEM containing 0.1% BSA. After 6 hours, the cells were scraped reticulately with a comb and the remaining cells were washed with PBS(-), followed by incubation in DMEM containing 0.1% BSA and a test compound for 24 hours. The resulting culture supernatant was taken as a sample. Six hours before the sampling of this culture supernatant, the abovementioned MLEC were put into wells of a 96-well plate in an amount of 2.8 x 104 cells/well. After the cells were cultured in DMEM containing 10% fetal calf serum (FCS) for 6 hours, the medium was replaced by the above culture supernatant sample, followed by incubation for 16 hours. The cells S* were washed twice with PBS(-) and then subjected to N:\LIBC104308:SAK measurement of active TGF-0 concentration by lucuferase assay system. The results are shown in Fig. 6. Active TGF- was released by the addition of Compounds 3 and 10. Particularly, Compound 10 showed a remarkable effect even at a concentration of 10 ag/ml.

Compounds promoting the release of active TGF-0 from latent TGF- 0 and increasing the degree of binding of TGF-13 to cells can be screened by the method of the present invention.

Industrial Applicability The present invention provides novel peptides having an activity to promote the activation of latent TGF- by enhancing S the binding of latent TGF-3 to a cell membrane, and 0 Spharmaceutically acceptable salts thereof.

15 According to the methods of screening compounds of the S* present invention, compounds having an activity to promote the Se* release of active TGF-0 from latent TGF-/ or an activity to promote the binding of latent TGF-13 to a cell membrane can be See selected.

The compounds obtained by the screening methods of the present invention, Compounds and pharmaceutically acceptable salts thereof have inhibiting activity or promoting 0 activity on the binding of latent TGF-P to cells or on the *a conversion of latent TGF-0 into TGF-13 Thus, they are useful 25 for the treatment or prevention of diseases such as cancer, diabetic retinopathy, atherosclerosis, bone fracture, myocardial infarction, myocardial disorder after ischemia reperfusion, cerebral infarction, retinal detachment, glomerulonephritis, diabetic nephropathy, renal graft rejection, HIV nephropathy, sudden pulmonary fibrosis, autoimmune pulmonary fibrosis, hepatic cirrhosis, venous constrictive hepatopathy (often occurring after treatments of cancer), systemic sclerosis, keloid, eosinophilia-muscle ache syndrome, re-stricture after angioplasty, intraocular fibrosis, rheumatic arthritis and nasal polyp.

Page(s) q 6 are claims pages they appear after the sequence listing (1)GENERAL INFORMATION

APPLICANT

KYOVA HAIKO KOGYO CO. LTD.

TITLE OF INVENTION.

PEPTIDES WHICH PROMOTE ACTIVATION OF LATENT TGF- O AND METHOD OF SCRE ENING TGF- j6 ACTIVITY-REGULATING COMPOUNDS.

FILE REFERENCE 1060 CURRENT FILING DATE 1998. 5.12 PRIOR APPLICATION NUMBER JP 120683/97 PRIOR APPLICATION FILING DATE 1997. 5.12 NUMBER OF SEQUENCES 0 0

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(2INFORIATION FOR SEQUENCES INFORMATION FOR SEQ ID NO:1: LENGTH: 23 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Leu Gin Ser Ser Arg His Arg Arg Ala Leu Asp Thr Asn Tyr Ser Phe 1. 5 10 Ser Ser Thr Glu Lys Asn Cys 61 INFORMATION FOR SEQ ID NO:2: LENGTH: 24 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Pro Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Arg Leu Lys 10 Leu Lys Val Glu Gln His Val Cys INFORMATION FOR SEQ ID NO:3: LENGTH: 23 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Leu Ser Thr Ser Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg 1 5 10 Ile Glu Ala Ile Arg Gly Cys INFORMATION FOR SEQ ID NO:4: LENGTH: 23 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Leu Ser Thr Ser 1 Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg 5 10 Ile Gin Ala Ile Arg Gly INFORMATION FOR LENGTH: 17.

SEQ ID TYPE: amino acid STR AND EDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Thr Ile Asp Met Glu Leu

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SEQUENCE DESCRIPTION: Leu Ser Thr Ser Lys Thr Ile Asp Met Glu Leu Val Lys Arg Lys Arg 1 5 10 le INFORMATION FOR SEQ ID NO:8: LENGTH: 14 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: *Leu Ser Thr Ser Lys Thr Ile Asp Met Glu Leu Val Lys Arg *1 5 10 *INFORMATION FOR SEQ ID NO:9: LENGTH: 12 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide, *see SEQUENCE DESCRIPTION: Leu Ser Thr Ser Lys Thr Ile Asp Met Glu Leu Val .1 5 INFORMATION FOR SEQ ID NO:i0: LENGTH: 24 TYPE: amino acid STRAINDEDNES: single TOPOLOGY: linear 64 MOLECULE TYPE:.peptide

FEATURE:

NAME/KEY: disuif ide-bonds LOCATION: 1 and 24 SEQUENCE DESCRIPTION: Cys Leu Ser Thr Ser Lys Thr le Asp Met Glu Leu Val Lys Arg Lys 1 5 10 Arg Ile Glu Ala Ile Arg Gly Cys INFORMATION FOR SEQ ID NO:11: 0 06 00 0S. 0 0 .0 0 S

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NAME/KEY: disulf ide-bonds LOCATION: 4 and 23 SEQUENCE DESCRIPTION: Leu Ser Thr Cys Lys Thr le Asp Met Glu Leu Yal Lys Arg Lys Arg 1 5 10 Ile Glu Ala Ile Arg Gly Cys INFORMATION FOR SEQ ID NO::12 LENGTH: 19 TYE: amino acid SIR AND EDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide

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NAME/KEY: disulf ide-bond LOCATION: 1 and 19 SEQUENCE DESCRIPTION: Cys Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Arg Leu Lys 1 5 10 Leu Lys Cys INFORMATION FOR SEQ ID NO:13: LENGTH: 19 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide

FEATURE:

NAME/KEY: disulfide-bond LOCATION: 1 and 19 NAME/KEY: modified site LOCATION: 1 OTHER INFORMATION: Xaa represents N a-biotinyl-L-systeine g SEQUENCE DESCRIPTION: Xaa Val Leu Leu Ser Arg Ala Glu Leu Arg Leu Leu Arg Arg Leu Lys 1 5 10 Leu Lys Cys INFORMATION FOR SEQ ID NO:14: LENGTH: 16 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide

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FEATURE:

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Val Pro Gh Ile Giy Lys Val Thr 205 Giy Leu 220 Gly Asp Gly Val Lys Lys Len Leu Asn Leu Asp Phe Gin Ile Leu Met Thr His Gin Leu Leu Glu Asn Thr Met Ile Gin 110 Gly le Thr Asn Arg Thr Asn Pro Ser 160 Leu Arg Pro 175 Asn Leu Pro 190 Asp Thr Val Giu Ile Ser Ile Leu Gin 240 Asp Asn Gin 255 Gin Lys Asp 260 265 270 His His Asn Pro His Len Ile Len Met Met Ile Pro Pro His Arg Leu o* 5 0 275 Asp Asn Pro Gly Gln Gly Gly 290 295 Asn Tyr Cys Phe Arg Asn Leu 305 310 Tyr Ile Asp Phe Arg Gin Asp 325 Lys Gly Tyr Tyr Ala Asn Phe 340 Ser Ala Asp Thr Thr His Ser 355 Asn Pro Glu Ala Ser Ala Ser 370 375 Pro Leu Thr Ile Leu Tyr Tyr 385 390 Leu Ser Asn Met Val Val Lys 405 INFORMATION FOR SEQ ID NO:2( LENGTH: 390 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Met Pro Pro Ser Gly Leu Arg '1 5 Trp Leu Leu Val Leu Thr Pro 20 Cys Lys Thr Ile Asp Met Glu 35 Ile Arg Gly Gln Ile Leu Ser 55 Gin Gly Glu Val Pro Pro Gly 70 Tyr Asn Ser Thr Arg Asp Arg

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INFORMATION

Gin Asp 325 Asn -Phe 340 His Ser Ala Ser Tyr Tyr Val Lys 405 FOR SEQ LeuGly Trp, Lys Cys Ser Gly Pro 345 Thr Val Leu Gly 360 Pro Cys Cys Val 375 Val Gly Arg Thr 390 Ser Cys Lys Cys ID NO:23: Trp Val 330 Cys Pro Leu Tyr Pro Gin Pro Lys 395 Ser 410 His Glu Tyr Leu Asn Thr 365 Asp Leu 380 Val Giu 41 0 0 0*00 0 0 S0. 0 e LENGTH: 390 TYPE: amino acid STRANDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: Met Pro Pro Ser Gil Trp Leu Cys Lys Ile Arg Gin Gly Tyr Asn Pro Glu Met Val His Ser Leu Val 20 Thr Ile 35 Gly Gin Giu Val Ser Thr Pro Glu 100 Asp Arg 115 Ile Tyr Ii P1 Al As Me .y Leu Arg Leu Leu Pro Leu ,u Thr Pro Gly Arg Pro Ala ,p Met Giu Leu Val Lys Arg 40 .e Leu Ser Lys Leu Arg Leu 55 -o Pro Gly Pro Leu Pro Giu 70 gAsp Arg Val Ala Gly Giu a Asp Tyr Tyr Ala Lys Giu 105 .n Asn Ala Ile Tyr Asp Lys 120 t Phe Phe Asn Thr Ser Asp Leu Leu Ala Gly Lys Arg Ala Ser Ala Val Ser Ala Val Thr Thr Lys 125 le Arg 130 Pro Glu 135 Ser Arg 140, Arg Leu Pro Pro Leu Len Ala Gin Len Gin Arg Phe S. S See

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INFORMATION

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Arg Giu Giu Giy Cys Thr Gin 90 Lys Giu Ile His Lys Phe Asp 105 Giu Leu Ala Val Cys Pro Lys 120 125 Asn Val Ser Ser Val Giu Lys 135 140 Phe Arg Val Leu Arg Val Pro 155 Arg Ile Giu Leu Phe Gin Ilie 170 Gin Arg Tyr Ile Gly Gly Lys 185 Trp Leu Ser Phe Asp Val Thr 200 205 Arg. Giu Ser Asn Leu Gly Leu 215 220 Thr Phe Gin Pro Asn Gly Asp 235 Giu Ile Lys Phe Lys Gly Val 250 Gin Met Giu Giu Met 110 Gly Asn Asn Leu Asn 190 Asp Giu Ile Asp Ile. Leu Thr His Leu Leu Thr Ser Ile Gin Ile Thr Gly Thr Pro Ser 160 Arg Pro 175 Leu Pro Thr Val Ile Ser Leu Giu 240 Asn Giu 255 ,eu Leu Asn ,eu Asp Phe Asp Asp His Gly Arg Gly Asp Leu 260 His His Asp Ser 290 Asn Tyr 305 Tyr le Lys Gly Ser Ser Asn Pro 370 Asn Pro His 275 Pro Gly Gin Cys Phe Arg Asp Phe Arg 325 Tyr Tyr Ala 340 Asp Thr Thr 355 Giu Ala Ser Leu Ile Leu 280 Gly Gly Gin 295 Asn Leu Glu 310 Gin Asp Leu Asn Phe Cys His Ser Thr 360 Ala Ser Pro 375 Gly Arg Leu Lys Lys 265 Met Met Ile Pro Pro 285 Arg Lys Lys Arg Ala 300 Giu Asn Cys Cys Val 315 Gly Trp Lys Trp Val 330 Ser Gly Pro Cys Pro 345 Val Leu Gly Leu Tyr 365 Cys Cys Val Pro Gin 380 Gly Arg Thr Pro Lys 395 Cys Lys Cys Ser 410 9 9* 9

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Glu Leu 170 Arg Leu 185 Thr Gly Phe Arg Gin Val Thr Ile 250 Leu Glu 265 Asp Thr Gin Leu Glu Pro Ile Trp Leu Ala Giu Ala 75 Glu Ser Glu Val Lys Met Glu Leu 140 Val Arg 155 Tyr Gin Leu Ala Val Vai Leu Ser 220 Asp Ile 235 His Gly Arg Ala Asn Tyr Tyr Ile 300 Lys Gly 315 Ser Leu Ser Pro Pro Ser Ile Leu Ala Leu Ala Giu Thr Glu Thr Arg Val Leu 110 Lys Ser Ser Ser 125 Arg Glu Ala Val Leu Leu Arg Leu 160 Lys Tyr Ser Asn 175 Pro Ser Asp Ser 190 Arg Gin Trp Leu, 205 Ala His Cys Ser Asn Gly Phe Ser 240 Met Asn Arg Pro 255 Gin His Leu His 270 Cys Phe Ser Ser 285 Asp Phe Arg Lys Tyr.His Ala Asn 320 Asp Thr Gin Tyr 325 Ser Lys Val Leu Ala Leu Tyr A~ 340 Ala Pro Cys Cys Val Pro Gin Al 355 31 Tyr Val Gly Arg Lys Pro Lys Y; 370 375 Arg Ser Cys Lys Cys Ser 385 390 INFORMATION FOR:SEQ ID LENGTH: 412 TYPE: amino acid STRAiNDEDNES: single TOPOLOGY: linear MOLECULE TYPE: peptide SEQUENCE DESCRIPTION: 330 335 sn Gin His Asn Pro Gly Ala Ser Ala 345 350 la Leu Giu Pro Leu Pro Ile Val Tyr 60 365 1l Giu Gin Leu Ser Asn Met Ile Yal 380 0 0 9.

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Ala Pro Pro Pro Ala Ser Giu Thr Pro 40 Val Arg Ala Leu Tyr Asn Ser Thr Gin 55 Arg Leu Arg Pro Pro Pro Asp Gly Pro 70 270 Met Leu Pro Pro His 28.5 Lys Lys Arg Ala L eu 300 Asn Cys Cys Val Arg 315 Trp Lys Trp Val His 330 Gly Pro Cys Pro Tyr 350 Len Gly Leu Tyr Asn 365 Cys Val Pro Gin Asp 380 Arg Thr Pro Lys Val 395 Lys Cys Ser 410 Arg Leu Asp Thr Pro Leu .320 Gin Pro 335 Leu Arg Thr Len Leu Gin Gin Gin 400 Lys Lys Len Thr Asp Asp Arg Ala Lys Gin Len Glu Ala 10 Leu Ser Lys Pro Arg Pro Gin Len Len Asp Glu Tyr 75 Ala Lys Len Arg Len Pro Lys Gin Trp Ala Leu Arg Argle Pro Met Giu Thr Thr Trp Asp Gly Ala Met Giu His 0 0 0.

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Claims (5)

1. A method of screening a compound to be used for treatment or prevention of TGF-p-related diseases, which comprises: measuring the amount of latent TGF-P bound to animal cells after addition of latent TGF-P to said cells; measuring the amount of latent TGF-P bound to animal cells after addition of latent TGF-P and a compound to be evaluated to said cells; and evaluating the inhibiting activity or promoting activity of said compound on the binding of latent TGF-P to animal cells from the change in the amount of latent TGF-P o0 bound to animal cells caused by the addition of said compound.

2. The method according to claim 1, wherein said animal cells are vascular endothelial cells.

3. The method according to claim 1 or claim 2, wherein the promoting activity of said compound on the binding of latent TGF-P to animal cells is evaluated. S is

4. The method of screening a compound to be used for the treatment or prevention of TGF-p-related diseases, said method substantially as hereinbefore described with reference to Example 20 or Example 21.

5. A compound obtained by the method according to any one of claims 1 to 4. ~Dated 27 September, 2001 Kyowa Hakko Kogyo Co., Ltd Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON Orp. o• [R:\LIBUU]02161.doc:MCN