Peptide, peptide complex, pharmaceutical composition, composition for cell culture, and composition for medical use, diagnostic use, or research use

Abstract

The present invention relates to a peptide complex comprising a first peptide and a second peptide. The first peptide comprises an amino acid sequence represented by formula A1 or an amino acid sequence represented by formula A1 with one or more amino acid residues substituted, deleted, added or inserted. The second peptide comprises an amino acid sequence represented by formula B1 or an amino acid sequence represented by formula B1 with one or more amino acid residues substituted, deleted, added or inserted. Formula A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14. Formula B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14.

Description

Peptides, peptide complexes, pharmaceutical compositions, cell culture compositions, and compositions for medical, diagnostic, or research use

[0001] The present invention relates to peptides, peptide conjugates, pharmaceutical compositions, cell culture compositions, and compositions for medical, diagnostic, or research use. This application claims priority to Japanese Patent Application No. 2023-192587, filed November 10, 2023, the contents of which are incorporated herein by reference.

[0002] Wnt is a secreted glycoprotein with a molecular weight of approximately 40,000 that regulates early development and morphogenesis, as well as postnatal proliferation, differentiation, cell motility, and polarity. Nineteen species of Wnt have been identified in humans and mice. Wnt3a, a member of the Wnt family, binds to the seven-transmembrane Frizzled receptor and its single-transmembrane co-receptor, LRP5 or LRP6, to activate the Wnt signaling pathway. Chronic activation of this pathway results in uncontrolled cell proliferation and survival, leading to cell proliferative disorders, such as cancer, while significant inhibition of this pathway leads to bone loss and other bone disorders. Therefore, many drugs and antibodies targeting the Wnt signaling pathway have been reported for the treatment of the above-mentioned cancer and bone disorders (Patent Documents 1 to 9, Non-Patent Documents 1 to 2). On the other hand, activation of the Wnt signaling pathway plays an important role in early development and the maintenance, proliferation, and differentiation of normal tissue stem cells, and Wnt3a in particular has been used to create various organoids, such as those of the small intestine, large intestine, stomach, pancreas, and lung.

[0003] Japanese Patent Publication No. 2022-544308 Japanese Patent Publication No. 2022-544307 Japanese Patent Publication No. 2022-551380 Japanese Patent Publication No. 2022-137092 Japanese Patent Publication No. 2020-178701 Japanese Patent Publication No. 2020-063262 Japanese Patent Publication No. 2013-530929 Japanese Patent No. 6093692 Japanese Patent Publication No. 2009-142274

[0004] Shinji Matsumoto et al., "Development of novel anticancer drugs based on research into Wnt signaling," Interdisciplinary Review, December 27, 2018, Vol. 7, e009; A. Neiheisel et al., "Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials," International Journal of Cancer, March 2022, Vol. 150, No. 5, pp. 727-740

[0005] Wnt3a is a lipid-soluble protein, and therefore has low solubility in culture media and is unstable, but no chemically synthesizable Wnt3 alternative peptides have yet been provided.

[0006] An objective of the present invention is to provide a peptide complex that activates the Wnt signaling pathway.

[0007] As a result of extensive research aimed at solving the above problems, the present inventors discovered that a peptide complex containing a specific peptide activates the Wnt signaling pathway, and thus completed the present invention. That is, the present invention includes the following aspects.

[0008] [1] A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues in the amino acid sequence represented by formula A1 have been substituted, deleted, added, or inserted. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X6 is an amino acid residue having an optionally substituted aryl group in the side chain, X7 is an N-alkylamino acid residue having an optionally substituted aryl group or an optionally substituted cycloalkyl group in the side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, X12 is an amino acid residue having an aliphatic hydrocarbon group in the side chain, X13 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X14 is an L-cysteine ​​(C) residue, and in formula A1, the amino acid sequence is written from the N-terminus to the C-terminus. [2] In the formula A1, X1 is a D-phenylalanine (df) residue, a (R)-2-amino-3-(pyridin-4-yl)propanoic acid (d4py) residue, a (R)-2-amino-3-(pyridin-3-yl)propanoic acid (d3py) residue, a (R)-2-amino-3-(3-methoxyphenyl)propanoic acid (df3OMe) residue, a (R)-2-amino-3-(4-methoxyphenyl)propanoic acid (df4OMe) residue, a (R)-2-amino-5-ureidopentanoic acid (dicit) residue, a D-tyrosine (dy) residue, or a (R)-2-amino-3-(4-fluorophenyl)propanoic acid (df4F) residue; X2 is an L-serine (S) residue, an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-2-aminoadipic acid (Hgl) residue, an L-citrulline (Cit) residue, or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue;X3 is an L-aspartic acid (D) residue, an L-arginine (R) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, a (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, an L-asparagine (N) residue, or an L-serine (S) residue; X4 is an L-isoleucine (I) residue or an O-methyl-L-threonine (TMe) residue; X5 is an L-arginine (R) residue, an L-glutamic acid (E) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue, or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue; X6 is an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, an (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, an (S)-2-amino-3-(4-chlorophenyl)propanoic acid (F4C) residue, an (S)-2-amino-3-(pyridin-4-yl)propanoic acid (4Py) residue, or an (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue; X7 is a methyl-L-phenylalanine (MeF) residue, an (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue;X8 is an L-glutamine (Q) residue, an L-valine (V) residue, an L-2-aminoadipic acid (Hgl) residue, an 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an allothreonine (alT) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl) -L-lysine (KCOpipzaa) residue, (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp) residue, (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, (S)-2-amino-2-cyclohexylacetic acid (Chg) residue or O-methyl-threonine (TMe) residue, X10 is a methyl-L-phenylalanine (MeF) residue, an (S)-2-(methylamino)hexanoic acid (MeNle) residue, an (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue; The peptide according to [1], wherein X11 is an L-isoleucine (I) residue, an L-tyrosine (Y) residue, an (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (F4aao) residue, or an O-methyl-L-threonine (TMe) residue, X12 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue, and X13 is a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue.[3] A peptide comprising an amino acid sequence represented by formula A2, or an amino acid sequence in which at least one amino acid residue has been substituted, deleted, added, or inserted among 1 to 12 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 11th, 12th, and 13th amino acid residues in the amino acid sequence represented by formula A2. A2: df-S-D-I-R-Y-MeF-Q-T-MeF-I-V-G-C (SEQ ID NO: 164) where df is a D-phenylalanine residue, S is an L-serine residue, D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, G is a glycine residue, and C is an L-cysteine ​​residue. [4] The peptide according to any one of [1] to [3], further comprising a glycine residue at the C-terminus. [5] The peptide according to any one of [1] to [3], which is a cyclic peptide. [6] The peptide according to [4], which is a cyclic peptide. [7] The peptide according to any one of [1] to [6], which has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are bonded. [8] The peptide according to any one of [1] to [7], which further contains an additional amino acid residue. [9] The peptide according to any one of [1] to [8], which has binding ability to an Fzd receptor.

[10] The peptide according to [9], which has binding ability to at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7, and Fzd receptor 8.

[11] The peptide according to any one of [1] to [3], which consists of an amino acid sequence represented by formula A3. A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14wherein ClAc is a chloroacetyl group, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are amino acid residues shown in Table 1, in which: df is a D-phenylalanine residue, d4py is a (R)-2-amino-3-(pyridin-4-yl)propanoic acid residue, d3py is a (R)-2-amino-3-(pyridin-3-yl)propanoic acid residue, df3OMe is a (R)-2-amino-3-(3-methoxyphenyl)propanoic acid residue, df4OMe is a (R)-2-amino-3-(4-methoxyphenyl)propanoic acid residue, and dcit is a (R)-2-amino-5-ureidopentanoic acid residue, dy is a D-tyrosine residue, df4F is an (R)-2-amino-3-(4-fluorophenyl)propanoic acid residue, S is an L-serine residue, N is an L-asparagine residue, D is an L-aspartic acid residue, Hgl is an L-2-aminoadipic acid residue, Cit is an L-citrulline residue, Kmor is an (S)-2-amino-6-morpholinohexanoic acid residue, R is an L-arginine residue, KCOpipzaa is an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, Atp is a (2S)-2-amino-3-(oxan-4-yl)propanoic acid residue, I is an L-isoleucine residue, TMe is an O-methyl-L-threonine residue, E is an L-glutamic acid residue, Ahp is an (S)-2-aminoheptanoic acid residue, Hse is an L-homoserine residue, Hcit is an N6-carbamoyl-L-lysine residue, Ncit is an (S)-2-amino-4-ureidobutanoic acid residue, OrnAc is an (S)-5-acetamido-2-aminopentanoic acid residue, Y is an L-tyrosine residue, MeY is a methyl-L-tyrosine residue, F4F is an (S)-2-amino-3-(4-fluorophenyl)propanoic acid residue, F4C is an (S)-2-amino-3-(4-chlorophenyl)propanoic acid residue,4Py is a (S)-2-amino-3-(pyridin-4-yl)propanoic acid residue, F3COO is a (S)-3-(2-amino-2-carboxyethyl)benzoic acid residue, MeF is a methyl-L-phenylalanine residue, Me3Py is a (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid residue, Me4Py is a (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid residue, MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF4COO is a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeAtp is an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid residue, Q is an L-glutamine residue, V is an L-valine residue, W1aa is a 1-(carboxymethyl)-L-tryptophan residue, Hty is a homo-L-tyrosine residue, T is an L-threonine residue, alT is an allothreonine residue, Gthp is an (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid residue, G4pipaa is an (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid residue, Cbg is an (S)-2-amino-2-cyclobutylacetic acid residue, Chg is an (S)-2-amino-2-cyclohexylacetic acid residue, MeNle is an (S)-2-(methylamino)hexanoic acid residue, F4aao is an (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid residue, MeV is a methyl-L-valine residue, MeChg is an N-methyl-L-cyclohexylglycine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

[12] A peptide comprising an amino acid sequence represented by formula B1, or an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by formula B1. B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 with the proviso thatY1 is an optionally substituted L-phenylalanine (F) residue, Y2 is an optionally substituted L-arginine (R) residue, Y3 is an optionally substituted L-tryptophan (W) residue or an optionally substituted (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is an optionally substituted aromatic amino acid residue, aliphatic amino acid residue or L-proline (P) residue, Y6 is an optionally substituted aliphatic amino acid residue, Y7 is an N-alkylated aromatic amino acid residue, Y8 is an amino acid residue derived from an amino acid having a hydroxy group, a carboxy group or an amide group in the side chain, Y9 is any amino acid residue, Y10 is an optionally substituted L-tryptophan (W) residue, Y11 is an optionally substituted L-asparagine (N) residue or an L-glutamine (Q) residue,

[13] In Formula B1, Y1 is an L-phenylalanine (F) residue, (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, (S)-2-amino-3-(p-toluyl)propanoic acid (F4Me) residue, or (S)-2-amino-3-(m-toluyl)propanoic acid (F3Me) residue, Y2 is an L-arginine (R) residue, or an N6-carbamoyl-L-lysine (Hcit) residue,Y3 is an L-tryptophan (W) residue, a methyl-L-tryptophan (MeW) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid (W5N) residue, an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3 (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue, (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue, (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue or (S)-2-amino-3-(quinolin-4-yl)propanoic acid (Nal14N) residue; Y4 is an L-aspartic acid (D) residue, an L-glutamic acid (E) residue, an L-asparagine (N) residue, or an L-glutamine (Q) residue; Y5 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, an (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, an (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid (3Py5Ph) residue, an (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or an L-proline (P) residue; Y6 is an L-arginine (R) residue, a methyl-L-arginine (MeR) residue, an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue, or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue;Y7 is a methyl-L-phenylalanine (MeF) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeFCOO) residue, a methyl-L-tyrosine (MeY) residue, an (S)-2-(methylamino)-3-(p-toluyl)propanoic acid (MeFMe) residue, an (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (MeFC) residue, an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeFCOO) residue, an (S)-2-(methylamino)-3-(m-toluyl)propanoic acid (MeFMe) residue, or an (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (MeFC) residue; Y8 is an L-serine (S) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a homo-L-tyrosine (Hty) residue, an allothreonine (alT) residue, an L-threonine (T) residue, an L-asparagine (N) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue, or an L-glutamic acid (E) residue; Y9 is an L-asparagine (N) residue, a methyl-L-asparagine (MeN) residue, an L-2-aminoadipic acid (Hgl) residue, an (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, a homo-L-tyrosine (Hty) residue, an (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid (Hw7N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue, or an L-glutamic acid (E) residue; Y10 is an L-tryptophan (W) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (W6N) residue, an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue, or an (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue;Y11 is an L-asparagine (N) residue, an L-glutamine (Q) residue, or a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (Nmm) residue, Y12 is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, an (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, an (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue, or an (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue,

[14] A peptide comprising an amino acid sequence represented by formula B2, or an amino acid sequence obtained by substituting, adding, deleting or inserting at least one amino acid residue among 1 to 13 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th amino acid residues in the amino acid sequence represented by formula B2. B2: F-R-W-D-V-R-MeF-S-N-W-N-Tic-N-C (SEQ ID NO: 165) wherein F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue.

[15] The peptide according to any one of

[12] to

[14] , further comprising a glycine residue at the C-terminus.

[16] The peptide according to any one of

[12] to

[14] , which is a cyclic peptide.

[17] The peptide according to

[15] , which is a cyclic peptide.

[18] The peptide according to any one of

[12] to

[17] , having a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are bonded.

[19] The peptide according to any one of

[12] to

[18] , further comprising an additional amino acid residue.

[20] The peptide according to any one of

[12] to

[19] , having binding ability to LRP.

[21] The peptide according to

[20] , having binding ability to at least one selected from the group consisting of LRP5 and LRP6.

[22] The peptide according to any one of

[12] to

[19] , having DKK1-like activity.

[23] The peptide according to any one of

[12] to

[14] , consisting of an amino acid sequence represented by formula B3. B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 wherein ClAc is a chloroacetyl group, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are amino acid residues shown in Table 2, in which F is an L-phenylalanine residue, F4F is an (S)-2-amino-3-(4-fluorophenyl)propanoic acid residue, F4Me is an (S)-2-amino-3-(p-toluyl)propanoic acid residue, F3Me is an (S)-2-amino-3-(m-toluyl)propanoic acid residue, R is an L-arginine residue, Hcit is an N6-carbamoyl-L-lysine residue, W is an L-tryptophan residue, MeW is a methyl-L-tryptophan residue, W7N is an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, W5N is an (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid residue, W1Me7N is an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, W7N5Me is an (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, Nal1 is (S)-2-amino-3-(naphthalen-1-yl)propanoic acid residue,Na114N is an (S)-2-amino-3-(quinolin-4-yl)propanoic acid residue, D is an L-aspartic acid residue, E is an L-glutamic acid residue, N is an L-asparagine residue, Q is an L-glutamine residue, V is an L-valine residue, MeV is a methyl-L-valine residue, W1aa is a 1-(carboxymethyl)-L-tryptophan residue, Cha is an (S)-2-amino-3-cyclohexylpropanoic acid residue, 3Py5Ph is an (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid residue, Cbg is an (S)-2-amino-2-cyclobutylacetic acid residue, P is an L-proline residue, MeR is a methyl-L-arginine residue, Kmor is an (S)-2-amino-6-morpholinohexanoic acid residue, Cit is an L-citrulline residue, Ncit is an (S)-2-amino-4-ureidobutanoic acid residue, OrnAc is an (S)-5-acetamido-2-aminopentanoic acid residue, MeF is a methyl-L-phenylalanine residue, MeF4COO is an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeY is a methyl-L-tyrosine residue, MeF4Me is an (S)-2-(methylamino)-3-(p-toluyl)propanoic acid residue, and MeF4C is an (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid residue. MeF3COO is an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF3Me is an (S)-2-(methylamino)-3-(m-toluyl)propanoic acid residue, MeF3C is an (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid residue, S is an L-serine residue, Hty is a homo-L-tyrosine residue, alT is an allothreonine residue, T is an L-threonine residue, MeN is a methyl-L-asparagine residue, Hgl is an L-2-aminoadipic acid residue,Hw7N is an (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid residue, KCOpipzaa is an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, W6N is an (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid residue, Nmm is a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid residue, Tic is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic6C is an (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7H is an (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7C is an (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Ndm is an N4,N4-dimethyl-L-asparagine residue, C is an L-cysteine ​​residue, and G is a glycine residue.

[0009]

[24] A peptide complex comprising a first peptide and a second peptide, wherein the first peptide is a peptide according to any one of [1] to

[11] , and the second peptide is a peptide according to any one of

[12] to

[23] .

[25] The peptide complex according to

[24] , comprising the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

[26] The peptide complex according to

[25] , wherein the C-terminus of the first peptide and the C-terminus of the second peptide are bonded via the linker.

[27] The peptide complex according to

[25] , wherein the linker is selected from the linkers listed in Table 3, where -click- represents a bond formed by click chemistry.

[28] The peptide complex according to any one of

[24] to

[27] , wherein the first peptide is a peptide consisting of an amino acid sequence represented by Formula A1, and the second peptide is a peptide consisting of an amino acid sequence represented by Formula B1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 With the proviso that, in formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue or an (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, and X4 is an L-isoleucine (I) residue, X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue, X6 is an L-tyrosine (Y) residue, X7 is a methyl-L-phenylalanine (MeF) residue or an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue,X8 is an L-glutamine (Q) residue, an L-valine (V) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an L-citrulline (Cit) residue, or an O-methyl-L-threonine (TMe) residue, X10 is a methyl-L-phenylalanine (MeF) residue or an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue, X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue, X13 is a glycine (G) residue or a D-serine (ds) residue, and in formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, Y3 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, Y6 is an L-arginine (R) residue or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, Y7 is a methyl-L-phenylalanine (MeF) residue or an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue, Y9 is an L-asparagine (N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, or an L-glutamic acid (E) residue; Y10 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue; Y11 is an L-asparagine (N) residue; Y12 is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue; and Y13 is an L-asparagine (N) residue or an L-aspartic acid (D) residue.

[29] The peptide conjugate according to any one of

[24] to

[28] , which activates Wnt signaling.

[0010]

[30] A pharmaceutical composition comprising the peptide according to any one of [1] to

[23] .

[31] A pharmaceutical composition comprising the peptide conjugate according to any one of

[24] to

[29] .

[0011]

[32] A composition for cell culture, which comprises the peptide complex according to any one of

[24] to

[29] and is used for cell culture.

[0012]

[33] A composition for medical, diagnostic or research use, comprising the peptide conjugate according to any one of

[24] to

[29] .

[0013]

[34] A composition for medical, diagnostic or research use, comprising the peptide according to any one of [1] to

[11] and

[12] to

[23] .

[0014]

[35] The peptide according to any one of [1] to

[23] , for use in the treatment or prevention of cancer diseases such as breast cancer, colon cancer, lung cancer, or stomach cancer, or inflammatory bowel disease.

[0015]

[36] The peptide conjugate according to any one of

[24] to

[29] for use in the treatment or prevention of bone metabolism diseases such as osteoporosis.

[0016]

[37] Use of the peptide according to any one of [1] to

[23] for producing a pharmaceutical composition for treating or preventing cancer diseases such as breast cancer, colon cancer, lung cancer, or stomach cancer, or inflammatory bowel disease.

[0017]

[38] Use of the peptide conjugate according to any one of

[24] to

[29] for producing a pharmaceutical composition for treating or preventing a bone metabolic disease such as osteoporosis.

[0018]

[39] A method for treating or preventing cancer diseases such as breast cancer, colon cancer, lung cancer, or stomach cancer, or inflammatory bowel disease, comprising administering an effective amount of the peptide according to any one of [1] to

[23] to a subject in need of such treatment or prevention.

[0019]

[40] A method for treating or preventing a bone metabolic disease such as osteoporosis, comprising administering an effective amount of the peptide conjugate according to any one of

[24] to

[29] to a subject in need of such treatment or prevention.

[0020]

[41] Use of the peptide complex according to any one of

[24] to

[29] for producing organoids such as the small intestine, large intestine, stomach, pancreas, or lung.

[0021] The present invention provides peptide conjugates that activate the Wnt signaling pathway.

[0022] The following describes in detail embodiments of the peptides, peptide complexes, pharmaceutical compositions, cell culture compositions, and compositions for medical, diagnostic, or research use of the present invention. However, the present invention is not limited to the embodiments described below, and various modifications are possible without departing from the gist of the present invention.

[0023] [Abbreviation (general)] Å: Angstrom (unit, 10 －１０ m) AcOH: acetic acid BSA: bovine serum albumin Boc: tert-butoxycarbonyl group ClAc: chloroacetyl ClAcOSu: (2,5-dioxopyrrolidin-1-yl) 2-chloroacetate (CAS number: 27243-15-8) [Cu(CH ３ CN) ４ ]PF ６ : Tetrakis(acetonitrile)copper(I) hexafluorophosphate (CAS number: 64443-05-6) DCM: Dichloromethane or methylene chloride DIPCI or DIC: N,N'-diisopropylcarbodiimide DIPEA or DIEA: N,N-diisopropylethylamine DMA: N,N-dimethylacetamide Dmb: Dimethoxybenzyl group DMEM: Dulbecco's modified Eagle's medium DMF: N,N-dimethylformamide DMSO: Dimethyl sulfoxide DODT: 3,6-dioxa-1,8-octanedithiol EC50: 50% effective concentration FBS: Fetal bovine serum Fmoc: 9-fluorenylmethyloxycarbonyl Fmoc-Lys(Fmoc)-OH: N ２ , N ６-bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine g: gram (unit) HATU: O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate HPLC: high performance liquid chromatography LC-MS or LC / MS: liquid chromatography mass spectrometer M: molar (unit) MeCN: acetonitrile mg: milligram (unit) min: minute (unit) mL: milliliter (unit) mM: millimolar (unit) mm: millimeter (unit) Mpe group: 3-methyl-pent-3-yl group NHS: N-hydroxysuccinimide nm: nanometer (unit) μL: microliter (unit) OSu: oxysuccinimide Pbf: 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group PEG: polyethylene glycol rpm: revolutions per minute (units) Sub: dibenzosuberyl group TBTA: tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (CAS number: 510758-28-8) tBu: tert-butyl group TEAA: triethylamine acetate TFA: trifluoroacetic acid TIS: triisopropylsilane Trt or Tr: trityl group

[0024] [Abbreviations (unnatural amino acids)] 3Py5Ph: (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid (CAS number: 2973752-28-0) 3Py6NH2: (S)-2-amino-3-(6-aminopyridin-3-yl)propanoic acid (CAS number: 1269968-61-7) 4Py: (S)-2-amino-3-(pyridin-4-yl)propanoic acid (CAS number: 37535-49-2) Ahp: (S)-2-aminoheptanoic acid (CAS number: 44902-02-5) a1T: allo-threonine (CAS number: 28954-12-3) Atb: (S)-2-amino-4,4-dimethylpentanoic acid (CAS number: 57224-50-7) Atp: (2S)-2-amino-3-(oxan-4-yl)propanoic acid (CAS number: 1344910-91-3) AZ(L08)-P12-PS: 2,5-dioxopyrrolidin-1-yl 40-(33-azido-29-oxo-4,7,10,13,16,19,22,25-octaoxa-28-azatritriacontanamide)-40-(2,5,8,11,14,17,20,23,26,29,32,35,38-tridecaoxanonatriacontyl)-43-oxo-2,5,8,11,14,17,20,23,26,29,32,35,38,42-tetradecaoxa-44-azaheptatetracontane-47-oate (NOF Corporation) Cbg: (S)-2-amino-2-cyclobutylacetic acid (CAS number: 49607-08-1) Cha: (S)-2-amino-3-cyclohexylpropanoic acid (CAS number: 27527-05-5) Chg: (S)-2-amino-2-cyclohexylacetic acid (CAS number: 14328-51-9) Cit: L-citrulline (CAS number: 372-75-8) cPEG9c: 4,7,10,13,16,19,22,25,28-nonaoxahentriacontanedioic acid (CAS number: 1268488-70-5) cPEG13c: 4,7,10,13,16,19,22,25,28,31,34,37,40-tridecaoxatritetracontanedioic acid (CAS number: 2225903-66-0)cPEG17c: 4,7,10,13,16,19,22,25,28,31,34,37,40,43,46,49,52-heptadecaoxapentacontanedioic acid (CAS number: 2226897-74-9) d3py: (R)-2-amino-3-(pyridin-3-yl)propanoic acid (CAS number: 70702-47-5) d4py: (R)-2-amino-3-(pyridin-4-yl)propanoic acid (CAS number: 37535-50-5) da: D-alanine (CAS number: 338-69-2) dcit: (R)-2-amino-5-ureidopentanoic acid (CAS number: 13594-51-9) df: D-phenylalanine (CAS number: 673-06-3) df3OMe (R)-2-amino-3-(3-methoxyphenyl)propanoic acid (CAS number: 145306-65-6) df4F: (R)-2-amino-3-(4-fluorophenyl)propanoic acid (CAS number: 18125-46-7) df4OMe: (R)-2-amino-3-(4-methoxyphenyl)propanoic acid (CAS number: 39878-65-4) dkCOpipzaa: N6-(4-(carboxymethyl)piperazine-1-carbonyl)-D-lysine (CAS number: 2973757-42-3) ds: D-serine (CAS number: 312-84-5) dy: D-tyrosine (CAS number: 556-02-5) F3COO: (S)-3-(2-amino-2-carboxyethyl)benzoic acid (CAS number: 13861-02-4) F3Me: (S)-2-amino-3-(m-toluyl)propanoic acid (CAS number: 114926-37-3) F4aao: (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (CAS number: 24558-63-2) F4C: (S)-2-amino-3-(4-chlorophenyl)propanoic acid (CAS number: 14173-39-8) F4F: (S)-2-amino-3-(4-fluorophenyl)propanoic acid (CAS number: 1132-68-9) F4Me: (S)-2-amino-3-(p-toluyl)propanoic acid (CAS number: 1991-87-3) G4pipaa: (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (CAS number: 2973756-12-4)Gpra: (S)-2-aminopent-4-ynoic acid (CAS number: 23235-01-0) Gthp: (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (CAS number: 811842-25-8) Hcit: N6-carbamoyl-L-lysine (CAS number: 1190-49-4) Hgl: L-2-aminoadipic acid (CAS number: 1118-90-7) Hse: L-homoserine (CAS number: 672-15-1) Hty: Homo-L-tyrosine (CAS number: 221243-01-2) Hw7N: (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid (CAS number: 2349860-05-3) KCOpipzaa: N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (CAS number: 2913141-21-4) Kmor: (S)-2-amino-6-morpholinohexanoic acid (CAS number: 960135-14-2) KN3: N6-diazo-L-lysine (CAS number: 159610-92-1) Me3Py: (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (CAS number: 2651172-69-7) Me4Py: (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (CAS number: 2913141-25-8) MeAtp: (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (CAS number: 2973752-56-4) MeChg: (S)-2-cyclohexyl-2-(methylamino)acetic acid (CAS number: 145963-18-4) MeF: methyl-L-phenylalanine (CAS number: 2566-30-5) MeF3C: (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (CAS number: 2255324-91-3) MeF3COO: (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (CAS number: 2913141-26-9) MeF3Me: (S)-2-(methylamino)-3-(m-toluyl)propanoic acid MeF4C: (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (CAS number: 347851-70-1)MeF4COO: (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (CAS number: 2973752-03-1) MeF4Me: (S)-2-(methylamino)-3-(p-toluyl)propanoic acid (CAS number: 2307782-25-6) MeN: Methyl-L-asparagine (CAS number: 19026-58-5) MeNle: (S)-2-(methylamino)hexanoic acid (CAS number: 17343-27-0) MeR: Methyl-L-arginine (CAS number: 2480-28-6) MeV: Methyl-L-valine (CAS number: 2480-23-1) MeW: Methyl-L-tryptophan (CAS number: 526-31-8) MeY: Methyl-L-tyrosine (CAS number: 537-49-5) Na1: (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (CAS number: 55516-54-6) Na14N: (S)-2-amino-3-(quinolin-4-yl)propanoic acid (CAS number: 137855-78-8) Ncit: (S)-2-amino-4-ureidobutanoic acid (CAS number: 1190-47-2) Nmm: (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (CAS number: 7175-34-0) Ndm: (2S)-2-amino-4-(dimethylamino)-4-oxobutanoic acid (CAS number: 62937-43-3) OrnAc: (S)-5-acetamido-2-aminopentanoic acid (CAS number: 2185-16-2) PEG4c: 1-amino-3,6,9,12-tetraoxapentadecan-15-oic acid (CAS number: 663921-15-1) PEG8c: 1-amino-3,6,9,12,15,18,21,24-octaoxaheptacosane-27-oic acid (CAS number: 756526-04-2) PEG12c: 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontane-39-oic acid (CAS number: 1415408-69-3) SMe: O-methyl-L-serine (CAS number: 32620-11-4) Tbg: (S)-2-amino-3,3-dimethylbutanoic acid (CAS number: 20859-02-3) Tic: (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS number: 74163-81-8)Tic6C: (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS number: 1344407-69-7) Tic7C: (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS number: 1344492-44-9) Tic7H: (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (CAS number: 128502-56-7) TMe: O-methyl-L-threonine (CAS number: 4144-02-9) W1aa: 1-(carboxymethyl)-L-tryptophan (CAS number: 773823-50-0) W1Me7N: (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 1632971-18-6) W5N: (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid (CAS number: 149704-62-1) W6N: (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (CAS number: 149704-63-2) W7N: (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 49758-35-2) W7N5Me: (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 1270158-20-7)

[0025] [Wnt] Wnt is a secreted glycoprotein with a molecular weight of approximately 40,000 that controls early development and morphogenesis, as well as postnatal proliferation, differentiation, cell motility, and polarity. Nineteen types of Wnt (Wnt1-19) have been identified in humans and mice. Wnt binds to Frizzled (Fzd) receptor family members and co-receptors LRP5, LRP6, ROR, or RYK to activate the Wnt signaling pathway. The peptide complexes of the present invention bind to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRP (LRP5, LRP6) to activate the Wnt signaling pathway, and can therefore be used as a substitute for naturally occurring Wnt family members. Examples of Wnt family members that can be replaced by the peptide complexes of the present invention include Wnt3 and Wnt1, with Wnt3 being preferred.

[0026] [Wnt signal activation ability] Wnt signal activation ability refers to the ability to bring about an effect similar to that brought about by naturally occurring Wnt. It also refers to the ability to specifically activate the Wnt signaling pathway. Wnt signal activation ability may be evaluated, for example, by activation of the Wnt-β-catenin pathway. For example, but not limited to, evaluation can be performed by measuring the nuclear translocation of β-catenin in cells or by a TCF / LEF reporter assay. Examples of evaluation methods include, but are not limited to, evaluation using PathHunter eXpress β-Catenin Nuclear Translocation Assay (DiscoverX), TCF / LEF Reporter-HEK 293 cell line (BPS Bioscience), etc. In any evaluation system, when the ability to activate Wnt signaling can be detected using a peptide or peptide complex at an optimal concentration under optimal conditions according to standard procedures, it is said to have the ability to activate Wnt signaling.

[0027] [Wnt inhibitory activity] Wnt inhibitory activity refers to the activity of inhibiting the Wnt signaling pathway. For example, the peptide of the present invention binds to the Wnt co-receptors LRP5 and / or LRP6, inhibiting Wnt binding to these co-receptors, thereby inhibiting the Wnt signaling pathway. Wnt inhibitory activity may be evaluated, for example, by inhibiting activation of the Wnt-β-catenin pathway. For example, but not limited to, evaluation can be performed by measuring the nuclear translocation of β-catenin in cells or by a TCF / LEF reporter assay. Examples of evaluation methods include, but are not limited to, evaluation using PathHunter eXpress β-Catenin Nuclear Translocation Assay (DiscoverX), TCF / LEF Reporter-HEK 293 cell line (BPS Bioscience), etc. In any evaluation system, when Wnt inhibitory activity can be detected using a peptide or peptide complex at an optimal concentration under optimal conditions according to standard procedures, it is said to have Wnt inhibitory activity.

[0028] [Canonical Wnt signaling pathway and non-canonical Wnt signaling pathway] See Neiheisel, A., et al., "Wnt pathway modulators in cancer therapeutics: An update on completed and ongoing clinical trials," International Journal of Cancer, March 2022, Vol. 150, No. 5, pp. 727-740.

[0029] A signaling pathway generally refers to a biochemical chain of events that is initiated by a protein-protein interaction, e.g., the binding of a growth factor to a receptor, and results in the transmission of a signal from one part of a cell to another part of the cell. A Wnt signaling pathway refers to the biochemical chain of events that occurs when the growth factor is a Wnt.

[0030] The Wnt signaling pathway is classified into the canonical Wnt signaling pathway and the non-canonical Wnt signaling pathway.

[0031] <Classical Wnt Signaling Pathway> In the absence of Wnt ligands, a degradation complex consisting of APC, Axin, CK1, and GSK3β phosphorylates, ubiquitinates, and degrades β-catenin in the proteasome. As a result, β-catenin levels in the cytoplasm are maintained low, suppressing the transcription of Wnt-targeted genes. On the other hand, when Wnt ligands bind to the Frizzled receptor and its co-receptor, LRP5 or LRP6, Dvl proteins are attracted to the plasma membrane, destabilizing the β-catenin degradation complex. Binding of Wnt ligands to the above receptors leads to the accumulation of unphosphorylated β-catenin in the cytoplasm, which then translocates into the nucleus and forms a complex with TCF / LEF, resulting in the transcription of Wnt-targeted genes. It has also been reported that binding of R-spondin to LGR5 activates the above-mentioned classical Wnt signaling pathway.

[0032] The Wnt signaling pathway refers to the classical Wnt signaling pathway, in which members of the Wnt family of secreted protein ligands bind to a receptor complex of LRP and Frizzled (Fzd), which allows β-catenin to translocate into the nucleus, where it can interact with TCF / LEF transcription factors and activate the expression of target genes.

[0033] <Non-canonical Wnt signaling pathway> There are at least two types of non-canonical Wnt signaling pathways: the PCP pathway, which controls planar cell polarity (PCP), and the Ca2+ pathway, which promotes the mobilization of calcium ions within cells.

[0034] (PCP pathway) When a Wnt ligand binds to the Frizzled receptor and its co-receptor ROR or RYK, Dvl proteins are attracted, activating ROCK, which controls the cytoskeleton, or JNK, which activates Jun, a transcription factor that mediates cellular responses.

[0035] (Ca2+ pathway) When Wnt ligands interact with Frizzled receptors and their co-receptors, RORs, they activate the membrane-bound enzyme PLC, which converts the membrane-bound phospholipid PIP2 into IP3 and DAG. IP3 causes the release of calcium ions from the endoplasmic reticulum into the cytoplasm. Calcium ions then activate calmodulin and PKC. PKC activation results in cellular responses.

[0036] Fzd receptors are seven-transmembrane domain proteins of the G protein-coupled receptor (GPCR) superfamily that contain a large extracellular N-terminal ligand-binding domain with ten conserved cysteines known as the cysteine-rich domain (CRD) or Fri domain. Ten Fzd receptors (Fzd1-10) have been identified in humans and are involved in many important biological processes, such as development, cell proliferation, survival, migration, and stem cell maintenance. In adult animals, interaction of Wnt ligands with Fzd receptor family members activates the Wnt signaling pathway, regulating embryonic development, stem and progenitor cell renewal during tissue homeostasis, and cell differentiation.

[0037] [Fzd Binding Activity] Fzd (Frizzled) binding activity means the activity of specifically binding to an Fzd (Frizzled) receptor, preferably the activity of binding to an Fzd2, Fzd7, and / or Fzd8 receptor.

[0038] [LRP5 / 6] LRP is an abbreviation for low-density lipoprotein receptor-related protein, and includes LRP1-6, LRP1B, LRP8, and LRP10-12. LRP members LRP5 and LRP6 are single-pass transmembrane receptors present on the cell surface and form part of the LRP5 / LRP6 / Fzd coreceptor family involved in the canonical Wnt signaling pathway. LRP5 and LRP6 are highly homologous, with 73% and 64% identity in their extracellular and intracellular domains, respectively. They are widely co-expressed during embryogenesis and in adult tissues and are functionally redundant.

[0039] [LRP binding activity] LRP binding activity means the activity of specifically binding to LRP, preferably the activity of binding to LRP5 and / or LRP6.

[0040] [Dkk] Dkk (Dikkopff) is a nucleic acid and protein of a Dkk family member, including Dkk1-4, Soggy, and related Dkk proteins. In the present application, a preferred example is Dkk1, but is not limited thereto. Dkk1 binds to the Wnt co-receptors LRP5 and LRP6, and inhibits Wnt binding to these co-receptors, thereby inhibiting the Wnt signaling pathway. Dkk1 has been shown to inhibit both Wnt1- and Wnt3a-mediated signaling. As used herein, "DKK1-like activity" refers to the activity of inhibiting the Wnt signaling pathway by the above-mentioned mechanism.

[0041] [Conservative Amino Acid Substitutions] In the peptides and peptide complexes of the present invention, conservative amino acid substitutions are preferably made when one, two, or three amino acid residues are substituted, deleted, added, or inserted from a specific amino acid sequence.

[0042] A "conservative amino acid substitution" means a substitution with a functionally equivalent or similar amino acid.

[0043] Conservative amino acid substitutions in a peptide result in a silent change in the amino acid sequence of the peptide. For example, one or more amino acids of similar polarity act functionally equivalently and result in a silent change in the amino acid sequence of such a peptide. In general, substitutions within a group can be considered conservative in structure and function.

[0044] However, as will be apparent to those skilled in the art, the role played by a particular amino acid residue can be determined in terms of its significance in the three-dimensional structure of a molecule containing that amino acid.

[0045] Cysteine ​​residues can adopt an oxidized (disulfide) form which is less polar compared to the reduced (thiol) form.

[0046] The long aliphatic portion of the arginine side chain may constitute an important structural and functional feature.

[0047] Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic interactions or cation-pi interactions. In such cases, amino acids with these side chains can be substituted with amino acids belonging to acidic or nonpolar groups without structural and functional consequences.

[0048] Residues such as proline, glycine, and cysteine ​​(disulfide form) can have direct effects on the main-chain conformation and often cannot be substituted without structural distortion.

[0049] Conservative amino acid substitutions include specific substitutions based on side chain similarity (L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and typical substitutions, as shown below.

[0050] Furthermore, conservative amino acid substitution is preferably, for example, substitution with an amino acid that belongs to the same group as a certain amino acid, in which natural amino acids are divided into groups based on the properties of their common side chains, as shown below.

[0051] Hydrophobic (also called non-polar) amino acids: These are amino acids that exhibit hydrophobicity (non-polarity), and include, for example, L-alanine (A), glycine (G), L-valine (V), L-leucine (L), L-isoleucine (I), L-proline (P), L-phenylalanine (F), L-tryptophan (W), L-tyrosine (Y), and L-methionine (M). Hydrophobic amino acids can also be further divided into the following groups:

[0052] Aliphatic amino acid: An amino acid having an aliphatic hydrocarbon group or a hydrogen atom in the side chain, including, for example, L-alanine (A or Ala), glycine (G or Gly), L-valine (V or Val), L-isoleucine (I or Ile), and L-leucine (L or Leu).

[0053] Aliphatic / branched-chain amino acids: Amino acids having a branched aliphatic hydrocarbon group in the side chain, including, for example, L-valine (V), L-isoleucine (I), and L-leucine (L).

[0054] Aromatic Amino Acid: An amino acid that has an aromatic ring in the side chain, including, for example, L-tryptophan (W), L-tyrosine (Y), and L-phenylalanine (F).

[0055] Hydrophilic (also called polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including, for example, L-serine (S), L-threonine (T), L-cysteine ​​(C), L-asparagine (N), L-glutamine (Q), L-aspartic acid (D), L-glutamic acid (E), L-lysine (K), L-arginine (R), and L-histidine (H).

[0056] Hydrophilic amino acids can be further divided into the following groups:

[0057] Acidic Amino Acid: An amino acid whose side chain exhibits acidity, including, for example, L-aspartic acid (D) and L-glutamic acid (E).

[0058] Basic amino acids: Amino acids whose side chains exhibit basicity, including, for example, L-lysine (K), arginine (R), and L-histidine (H).

[0059] Neutral amino acid: An amino acid whose side chain is neutral, including, for example, L-serine (S), L-threonine (T), L-asparagine (N), L-glutamine (Q), and L-cysteine ​​(C).

[0060] Glycine (G) and L-proline (P) can also be classified as "amino acids that influence the orientation of the main chain."

[0061] L-cysteine ​​(C) and L-methionine (M), which contain a sulfur molecule in their side chains, can also be classified as "sulfur-containing amino acids."

[0062] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Unnatural amino acids include, for example, N-alkylamino acids in which the above-described natural amino acids are N-alkylated, and amino acids in which the nitrogen atom forming the peptide bond is modified with a branched or unbranched lower (e.g., C1-C5, preferably C1-C3, more preferably C1) alkyl group. N-alkylamino acids are preferably N-ethyl amino acids, N-butyl amino acids, or N-methyl amino acids, and more preferably N-methyl amino acids.

[0063] In addition, unnatural amino acids include D-amino acids (also referred to as D-amino acids), β-amino acids, γ-amino acids, amino acid mutants, chemically modified amino acids such as amino acid derivatives, and amino acids that do not become components of proteins in vivo, such as norleucine and ornithine.

[0064] Furthermore, the amino acids include naturally occurring amino acids having a functional group added to the side chain or substituted with another functional group (for example, amino acids having a substitution or addition in an arylene group, alkylene group, or other portion of the side chain, amino acids having an increased C number in the arylene group, alkylene group, or alkyl group of the side chain, amino acids having a substitution in an aromatic ring of the side chain, and heterocyclized or condensed cyclized amino acids).

[0065] Furthermore, by adding or substituting a structure such as a functional group to the side chain of a natural amino acid, properties different from those of the natural amino acid can be imparted. For example, (S)-2-amino-3-(pyridin-4-yl)propanoic acid (4Py) is an alanine with a pyridyl group (pyridine ring) added to the side chain, and the addition of the pyridyl group makes it basic, which is polar, unlike alanine, which belongs to the nonpolar amino acid group.

[0066] That is, unnatural amino acids with similar side chain properties can be included in the aforementioned groups, which are obtained by dividing natural amino acids based on the properties of their common side chains. For example, methyl-L-arginine (MeR), an N-methylated version of arginine, which belongs to the basic amino acids, is an unnatural amino acid, but it exhibits basicity and can therefore be classified as a basic amino acid. In this way, unnatural amino acids with similar side chain properties to a certain amino acid can also be included as targets for conservative amino acid substitution.

[0067] Non-naturally occurring amino acids include, but are not limited to, N-methyl amino acids, (S)-2-amino-3-(pyridin-4-yl)propanoic acid (4Py), allothreonine (alT), L-citrulline (Cit), O-methyl-L-serine (SMe), (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp), L-2-aminoadipic acid (Hgl), (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1), (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (W6N), and (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N). N-methylamino acids can also be classified as N-alkylamino acids, or can be classified according to the properties of the side chain of the original non-N-methylated amino acid.

[0068] Peptides and their pharmaceutically acceptable salts, esters, or solvates Examples of pharmaceutically acceptable salts include, but are not limited to, inorganic acid salts, organic acid salts, inorganic base salts, organic base salts, and acidic or basic amino acid salts.

[0069] Examples of inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates.

[0070] Examples of organic acid salts include, but are not limited to, acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.

[0071] Examples of the inorganic base salt include, but are not limited to, alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, aluminum salts, and ammonium salts.

[0072] Examples of said organic base salts include, but are not limited to, diethylamine salts, diethanolamine salts, meglumine salts, and N,N'-dibenzylethylenediamine salts.

[0073] Examples of the acidic amino acid salts include aspartate and glutamate, and examples of the basic amino acid salts include, but are not limited to, arginine salt, lysine salt, and ornithine salt.

[0074] Examples of pharmaceutically acceptable esters include, but are not limited to, acetate and sulfate esters.

[0075] An example of a pharmaceutically acceptable solvate includes, but is not limited to, a hydrate.

[0076] [Peptide] The peptide of the present invention may be a pharmaceutically acceptable salt, ester, or solvate. An example of a solvate is a hydrate. <First Embodiment> A first embodiment of the peptide of the present invention is a peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues in the amino acid sequence represented by formula A1 have been substituted, deleted, added, or inserted. In formula A1, the amino acid sequence is written from the N-terminus to the C-terminus.

[0077] A1:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14

[0078] wherein X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X6 is an amino acid residue having an optionally substituted aryl group in the side chain, X7 is an N-alkylamino acid residue having an optionally substituted aryl group or an optionally substituted cycloalkyl group in the side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, X12 is an amino acid residue having an aliphatic hydrocarbon group in the side chain, X13 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, and X14 is an L-cysteine ​​(C) residue.

[0079] In the peptide of the first embodiment, X1 is preferably a D-phenylalanine (df) residue, a (R)-2-amino-3-(pyridin-4-yl)propanoic acid (d4py) residue, a (R)-2-amino-3-(pyridin-3-yl)propanoic acid (d3py) residue, a (R)-2-amino-3-(3-methoxyphenyl)propanoic acid (df3OMe) residue, a (R)-2-amino-3-(4-methoxyphenyl)propanoic acid (df4OMe) residue, a (R)-2-amino-5-ureidopentanoic acid (dicit) residue, a D-tyrosine (dy) residue, or a (R)-2-amino-3-(4-fluorophenyl)propanoic acid (df4F) residue. The structures of d4py, d3py, df4F, df3OMe, df4OMe, dcit, df, and dy are shown below.

[0080]

[0081] In the peptide of the first embodiment, X2 is preferably an L-serine (S) residue, an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-2-aminoadipic acid (Hgl) residue, an L-citrulline (Cit) residue, or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue. The structures of Kmor, Hgl, and Cit are shown below.

[0082]

[0083] In the peptide of the first embodiment, X3 is preferably an L-aspartic acid (D) residue, an L-arginine (R) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, a (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, an L-asparagine (N) residue, or an L-serine (S) residue. The structures of Cit, Atp, and KCOpipzaa are shown below.

[0084]

[0085] In the peptide of the first embodiment, X4 is preferably an L-isoleucine (I) residue or an O-methyl-L-threonine (TMe) residue. The structure of TMe is shown below.

[0086]

[0087] In the peptide of the first embodiment, X5 is preferably an L-arginine (R) residue, an L-glutamic acid (E) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue, or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue. The structures of Ahp, Hse, Ncit, Hcit, Cit, and OrnAc are shown below.

[0088]

[0089] In the peptide of the first embodiment, X6 is preferably an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, an (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, an (S)-2-amino-3-(4-chlorophenyl)propanoic acid (F4C) residue, an (S)-2-amino-3-(pyridin-4-yl)propanoic acid (4Py) residue, or an (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue. The structures of F4F, F4C, F3COO, and 4Py are shown below.

[0090]

[0091] In the peptide of the first embodiment, X7 is preferably a methyl-L-phenylalanine (MeF) residue, an (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue. The structures of Me3Py, Me4Py, MeF3COO, MeF4COO, and MeAtp are shown below.

[0092]

[0093] In the peptide of the first embodiment, X8 is an L-glutamine (Q) residue, an L-valine (V) residue, an L-2-aminoadipic acid (Hgl) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an allothreonine (alT) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1- Preferably, the residue is a (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp) residue, a (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, a (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, a (S)-2-amino-2-cyclohexylacetic acid (Chg) residue, or an O-methyl-threonine (TMe) residue. The structures of Hgl, Ahp, Hty, Cit, W1aa, KCOpipzaa, TMe, alT, Cbg, Gthp, G4pipaa, and Chg are shown below.

[0094]

[0095]

[0096] In the peptide of the first embodiment, X10 is preferably a methyl-L-phenylalanine (MeF) residue, an (S)-2-(methylamino)hexanoic acid (MeNle) residue, an (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue. The structures of Me3Py, Me4Py, MeF4COO, MeAtp, and MeNle are shown below.

[0097]

[0098] In the peptide of the first embodiment, X11 is preferably an L-isoleucine (I) residue, an L-tyrosine (Y) residue, an (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (F4aao) residue, or an O-methyl-L-threonine (TMe) residue. The structures of F4aao and TMe are shown below.

[0099]

[0100] In the peptide of the first embodiment, X12 is preferably an L-valine (V) residue, a methyl-L-valine (MeV) residue, or an N-methyl-L-cyclohexylglycine (MeChg) residue. The structure of MeChg is shown below.

[0101]

[0102] In the peptide of the first embodiment, X13 is preferably a glycine (G) residue, a D-alanine (da) residue, or a D-serine (ds) residue.

[0103] The peptide of the first embodiment is preferably a peptide comprising an amino acid sequence represented by formula A2, or an amino acid sequence in which at least one amino acid residue has been substituted, deleted, added, or inserted among 1 to 12 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 11th, 12th, and 13th amino acid residues in the amino acid sequence represented by formula A2. In formula A2, the amino acid sequence is written from the N-terminus to the C-terminus.

[0104] A2: df-S-D-I-R-Y-MeF-Q-T-MeF-I-V-G-C (SEQ ID NO: 164)

[0105] where df is a D-phenylalanine residue, S is an L-serine residue, D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

[0106] The peptide of the first embodiment preferably further has a glycine residue at the C-terminus.

[0107] The peptide of the first embodiment is preferably a cyclic peptide.

[0108] When the peptide of the first embodiment is a cyclic peptide, it preferably has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are bound to each other.

[0109] A cyclic peptide refers to a peptide in which two amino acids are bonded together, forming a ring structure in whole or in part. In this specification, the term also includes peptides in which amino acids form a cross-linked structure, peptides in which a ring structure is formed by lactam ring formation or macrocyclization, and peptides having a lasso peptide-like structure. That is, in this specification, a cyclic peptide may be a peptide in which a portion thereof forms a ring structure, and may also have a linear portion.

[0110] In some cases, some amino acids may be modified for cyclization of the peptide of this embodiment. The peptide of this embodiment also encompasses peptides containing amino acids with such partial modifications. An example of a modification for cyclization is adding a chloroacetyl group to the amino acid located at the N-terminus, and binding it to a cysteine ​​residue in the peptide to cyclize it. Peptides containing various (natural / unnatural) amino acids to which a chloroacetyl group has been added are also encompassed by the peptides of this specification.

[0111] Peptides generally have poor metabolic stability in vivo and, due to their large size, have the drawback of being difficult to penetrate cell membranes. To address these issues, peptide cyclization has been used. It has been suggested that cyclization of peptides improves protease resistance, metabolic stability, and restricts conformational changes, thereby increasing rigidity and improving membrane permeability and affinity for target proteins.

[0112] Peptide cyclization can be carried out according to known methods. For example, but not limited to, by designing a peptide to contain two or more cysteine ​​residues, a cyclic structure can be formed by disulfide bonds after translation. Alternatively, cyclization can be achieved by synthesizing a peptide with a chloroacetyl group at the N-terminus and placing a cysteine ​​residue in the peptide using genetic code reprogramming technology (Goto, Y. et al., ACS Chem. Biol., 2008, Vol. 3, pp. 120-129). This allows spontaneous nucleophilic attack of the mercapto group on the chloroacetyl group after translation, resulting in cyclization of the peptide via a thioether bond. Using genetic code reprogramming technology, other combinations of amino acids that bond to form a cyclic structure can also be placed in the peptide for cyclization. Alternatively, a peptide with a cycloamide at the N-terminus can be synthesized, and an L-2-aminoadipic acid residue can be placed in the peptide, followed by bonding between them to form a cyclization. Thus, any known cyclization method can be used without particular limitations.

[0113] The peptide of the first embodiment preferably further contains additional amino acid residues. In this case, the additional amino acid residues are not particularly limited.

[0114] In the peptide of the first embodiment, the additional amino acid residues may be contained in the peptide forming a cyclic structure, or further amino acid residues may be added to the cyclic peptide in the form of a linker. The number of amide bonds (number of amino acids / length) in the peptide or peptide moiety is not particularly limited, but the total number of amino acid residues (referring to the number of amino acid residues contained in the peptide forming a cyclic structure; if further amino acid residues are added to the cyclic peptide in the form of a linker, these amino acids are not included) is preferably 20 or less. The preferred peptide length is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more amino acid residues, and preferably 19 or less or 18 or less amino acid residues. The more preferred peptide length is 13 to 16 amino acid residues, and most preferably 14 or 15 amino acids.

[0115] The peptide of the first embodiment preferably has binding ability to an Fzd receptor. The Fzd receptor is preferably at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7, and Fzd receptor 8.

[0116] The peptide of the first embodiment is preferably a peptide consisting of an amino acid sequence represented by formula A3: A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, where ClAc is a chloroacetyl group, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are amino acid residues shown in Table 1. The abbreviations for the amino acid residues in Table 1 are as described above.

[0117]

[0118]

[0119]

[0120]

[0121] Second Embodiment A second embodiment of the peptide of the present invention is a peptide comprising an amino acid sequence represented by formula B1, or an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by formula B1. In formula B1, the amino acid sequence is written from the N-terminus to the C-terminus.

[0122] B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14

[0123] wherein Y1 is an optionally substituted L-phenylalanine (F) residue, Y2 is an optionally substituted L-arginine (R) residue, Y3 is an optionally substituted L-tryptophan (W) residue or an optionally substituted (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is an optionally substituted aromatic amino acid residue, aliphatic amino acid residue or L-proline (P) residue, Y6 is an optionally substituted aliphatic amino acid residue, Y7 is an N-alkylated aromatic amino acid residue, Y8 is an amino acid residue derived from an amino acid having a hydroxy group, a carboxy group or an amide group in the side chain, Y9 is any amino acid residue, Y10 is an optionally substituted L-tryptophan (W) residue, and Y11 is an optionally substituted L-asparagine (N) residue or glutamine (Q) residue, Y12 is an optionally substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue; Y13 is an optionally substituted neutral amino acid residue or an acidic amino acid residue; and Y14 is an L-cysteine ​​(C) residue.

[0124] In the peptide of the second embodiment, Y1 is preferably an L-phenylalanine (F) residue, an (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, an (S)-2-amino-3-(p-toluoyl)propanoic acid (F4Me) residue, or an (S)-2-amino-3-(m-toluoyl)propanoic acid (F3Me) residue. The structures of F3Me, F4F, and F4Me are shown below.

[0125]

[0126] In the peptide of the second embodiment, Y2 is preferably an L-arginine (R) residue or an N6-carbamoyl-L-lysine (Hcit) residue. The structure of Hcit is shown below.

[0127]

[0128] In the peptide of the second embodiment, Y3 is an L-tryptophan (W) residue, a methyl-L-tryptophan (MeW) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid (W5N) residue, an (S)-2-amino-3-(1-methyl-1H-pyro Preferably, the residue is a (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue, a (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue, a (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, or a (S)-2-amino-3-(quinolin-4-yl)propanoic acid (Nal14N) residue. The structures of W7N, W5N, W1Me7N, W7N5Me, Nal1, and Nal14N are shown below.

[0129]

[0130]

[0131] In the peptide of the second embodiment, Y4 is preferably an L-aspartic acid (D) residue, an L-glutamic acid (E) residue, an L-asparagine (N) residue, or an L-glutamine (Q) residue.

[0132] In the peptide of the second embodiment, Y5 is preferably an L-valine (V) residue, a methyl-L-valine (MeV) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, an (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, an (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid (3Py5Ph) residue, an (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, or an L-proline (P) residue. The structures of Na1, 3Py5Ph, W1aa, Cha, and Cbg are shown below.

[0133]

[0134] In the peptide of the second embodiment, Y6 is preferably an L-arginine (R) residue, a methyl-L-arginine (MeR) residue, an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue, or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue. The structures of Kmor, Cit, Hcit, Ncit, and OrnAc are shown below.

[0135]

[0136] In the peptide of the second embodiment, Y7 is preferably a methyl-L-phenylalanine (MeF) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeFCOO) residue, a methyl-L-tyrosine (MeY) residue, an (S)-2-(methylamino)-3-(p-toluyl)propanoic acid (MeFMe) residue, an (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (MeFC) residue, an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeFCOO) residue, an (S)-2-(methylamino)-3-(m-toluyl)propanoic acid (MeFMe) residue, or an (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (MeFC) residue. The structures of MeF4COO, MeF4C, MeF4Me, MeF3Me, MeF3COO, and MeF3C are shown below.

[0137]

[0138] In the peptide of the second embodiment, Y8 is preferably an L-serine (S) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a homo-L-tyrosine (Hty) residue, an allothreonine (alT) residue, an L-threonine (T) residue, an L-asparagine (N) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue, or an L-glutamic acid (E) residue. The structures of alT, Hty, and W1aa are shown below.

[0139]

[0140] In the peptide of the second embodiment, Y9 is preferably an L-asparagine (N) residue, methyl-L-asparagine (MeN) residue, L-2-aminoadipic acid (Hgl) residue, (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, homo-L-tyrosine (Hty) residue, (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid (Hw7N) residue, N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, L-glutamine (Q) residue, L-aspartic acid (D) residue, or L-glutamic acid (E) residue. The structures of Cha, Hty, Hgl, Hw7N, and KCOpipzaa are shown below.

[0141]

[0142] In the peptide of the second embodiment, Y10 is preferably an L-tryptophan (W) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (W6N) residue, an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue, or an (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue. The structures of W1Me7N, W7N, W7N5Me, and W6N are shown below.

[0143]

[0144] In the peptide of the second embodiment, Y11 is preferably an L-asparagine (N) residue, an L-glutamine (Q) residue, or a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (Nmm) residue. The structure of Nmm is shown below.

[0145]

[0146] In the peptide of the second embodiment, Y12 is preferably an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, an (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, an (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue, or an (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue. The structures of Tic, Tic6C, Tic7C, and Tic7H are shown below.

[0147]

[0148] In the peptide of the second embodiment, Y13 is preferably an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-glutamine (Q) residue, a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (Nmm) residue, or an N4,N4-dimethyl-L-asparagine (Ndm) residue. The structures of Nmm and Ndm are shown below.

[0149]

[0150] The peptide of the second embodiment is preferably a peptide comprising an amino acid sequence represented by formula B2, or an amino acid sequence in which at least one amino acid residue has been substituted, added, deleted, or inserted among 1 to 13 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, and 13th amino acid residues in the amino acid sequence represented by formula B2. In formula B2, the amino acid sequence is written from the N-terminus to the C-terminus.

[0151] B2: F-R-W-D-V-R-MeF-S-N-W-N-Tic-N-C (SEQ ID NO: 165)

[0152] wherein F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue.

[0153] The peptide of the second embodiment preferably further comprises a glycine residue at the C-terminus. The number of glycine residues is not particularly limited as long as it is one or more.

[0154] The peptide of the second embodiment is preferably a cyclic peptide. When the peptide of the second embodiment is a cyclic peptide, it preferably has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are bonded.

[0155] The peptide of the second embodiment preferably further comprises additional amino acid residues. The additional amino acid residues are not particularly limited.

[0156] The peptide of the second embodiment preferably has binding ability to LRP. The LPR is preferably at least one selected from the group consisting of LRP5 and LRP6.

[0157] The peptide of the second embodiment preferably has DKK1-like activity.

[0158] The peptide of the second embodiment preferably consists of the amino acid sequence shown in formula B3.

[0159] B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14

[0160] wherein ClAc is a chloroacetyl group, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are amino acid residues shown in Table 2. The abbreviations for the amino acid residues in Table 2 are as described above.

[0161]

[0162]

[0163]

[0164]

[0165]

[0166] [Peptide Complex] The peptide complex of the present invention comprises a first peptide and has the ability to activate Wnt signaling. The peptide complex of the present invention may also have the ability to induce cell proliferation derived from the ability to activate Wnt signaling.

[0167] The peptide complex is a peptide, a peptide-containing compound, or a pharmaceutically acceptable salt thereof, which contains a first peptide and another peptide or compound. The peptide complex may contain one or more (three or more, or four or more) first peptides.

[0168] The peptide complex may further contain one or more partial peptides different from the first peptide. The peptide complex preferably has the first peptide or partial peptides linked via a linker. The peptide complex may be a homomultimer containing only peptides having the same amino acid sequence. The peptide complex may be a heteromultimer containing peptides having different amino acid sequences. The peptide complex is preferably a heterodimer having a first peptide and a second peptide having different amino acid sequences, the first peptide and the second peptide being linked via a linker. As shown in the examples, the first peptide and the second peptide exhibit Wnt signal activation ability by forming a peptide complex structure via a linker.

[0169] The peptide conjugate of the present invention may comprise a first peptide, a second peptide, and a linker connecting the first and second peptides. In this case, the second peptide may be the same as or different from the first peptide.

[0170] One embodiment of the peptide complex of the present invention is a peptide complex comprising a first peptide and a second peptide, wherein the first peptide is the peptide of the first embodiment and the second peptide is the peptide of the second embodiment.

[0171] The peptide complex preferably comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

[0172] The linker may be a structure that connects multiple peptides to each other in the peptide complex. Examples of the linker include an amino acid linker (peptide linker), a chemical linker, a fatty acid linker, a nucleic acid linker, and a sugar chain linker, and may also be a complex of, for example, a chemical linker and a peptide linker.

[0173] An example of a chemical linker is a PEG (Polyethyleneglycol) linker, which may be a linker consisting of 1 to 36 ethylene glycol units.

[0174] The linker may also be a fatty acid linker, which comprises a divalent chemical moiety derived from a fatty acid. An amino acid (peptide) linker is a linker comprising at least one amino acid, for example, the sequence [GGGGGS], as described in U.S. Pat. No. 7,271,149. ｎ Glycine-rich peptides such as peptides having the formula: where n is 1, 2, 3, 4, 5, or 6, or serine-rich peptide linkers as described in US Pat. No. 5,525,491 can be used.

[0175] Addition of a linker may, without limitation, change the physical properties (e.g., solubility) of a peptide. Furthermore, the linker may be a combination of the above linkers. For example, a linker may have a structure in which glycine (G)-L-lysine (K) is bound as an amino acid linker, and a PEG linker is further bound to the side chain terminal of the Lys. Furthermore, the linker may have a structure in which amino acids and PEG are bound alternately, such as PEG-amino acid-PEG. Here, PEG refers to a PEG linker.

[0176] Another example of a linker is a linker in which 1 to 6 amino acids are added to a PEG linker moiety. This linker may have a structure in which an amino acid is added to one end of the PEG linker moiety, or may have a structure in which amino acids are added to both ends of the PEG linker moiety. A preferred example of the amino acid added to the PEG moiety is L-lysine (K), but is not limited to L-lysine (K) and may be another amino acid.

[0177] The linker may be added to any position in the peptide. For example, it may be attached to L-cysteine ​​(C) at the C-terminus of the peptide, which is attached to the first amino acid to form a cyclic structure, or to an amino acid contained in the cyclic peptide. Although not limited thereto, it is preferable that the linker be attached to L-cysteine ​​(C) at the C-terminus or to the side chain of an amino acid contained in the cyclic peptide.

[0178] For example, in the amino acid sequences shown in SEQ ID NOs: 1 to 163, the glycine (G) at the 15th position can also be considered to be included in the linker. Preferably, the glycine (G) at the 15th position is included in the linker. In the case of a cyclic peptide in which the first amino acid in the amino acid sequences shown in SEQ ID NOs: 1 to 163 is bound to the 14th amino acid, L-cysteine ​​(C), and which forms a dimer structure via the structure shown in the following formula (the following formula includes the 15th glycine added to the 14th amino acid, L-cysteine ​​(C)), the linker structure is the structure shown in the following formula. Preferably, the dimer is one in which the C-terminus of the first peptide and the C-terminus of the second peptide are bound via a linker.

[0179]

[0180]

[0181]

[0182] The linker is also preferably selected from the linkers listed in Table 3, where -click- represents a bond formed by click chemistry.

[0183]

[0184] An example of click chemistry is the Fusgen reaction (see formula below), in which an alkyne and an azide compound undergo a cycloaddition reaction to form a 1,2,3-triazole ring.

[0185]

[0186] In the peptide complex of this embodiment, it is preferable that the first peptide is a peptide consisting of the amino acid sequence shown in formula A1, and the second peptide is a peptide consisting of the amino acid sequence shown in formula B1.

[0187] A1:X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14

[0188] provided that in formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue or a (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, X4 is an L-isoleucine (I) residue, X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue, X6 is an L-tyrosine (Y) residue, X7 is a methyl-L-phenylalanine (MeF) residue or a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeFCOO) residue, X8 is an L-glutamine (Q) residue, an L-valine (V) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an L-citrulline (Cit) residue, or an O-methyl-L-threonine (TMe) residue; X9 is an L-threonine (T) residue; X10 is a methyl-L-phenylalanine (MeF) residue or an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue; X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue; X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue; X13 is a glycine (G) residue or a D-serine (ds) residue; and X14 is an L-cysteine ​​(C) residue.

[0189] B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14

[0190] wherein in formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, Y3 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, Y6 is an L-arginine (R) residue or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, Y7 is a methyl-L-phenylalanine (MeF) residue or an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue, Y9 is an L-asparagine (N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue or an L-glutamic acid (E) residue, Y10 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, Y11 is an L-asparagine (N) residue, Y12 is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, Y13 is an L-asparagine (N) residue or an L-aspartic acid (D) residue, and Y14 is an L-cysteine ​​(C) residue.

[0191] The peptide complex of this embodiment preferably activates Wnt signaling.

[0192] [Pharmaceutical Composition] Another embodiment of the present invention is a pharmaceutical composition. One aspect of the pharmaceutical composition of this embodiment is a pharmaceutical composition comprising at least one selected from the group consisting of the above-mentioned peptides and pharmaceutically acceptable salts, esters, or solvates thereof (hereinafter, for simplicity, also referred to simply as "peptides of the present invention"). Another aspect is a pharmaceutical composition comprising at least one selected from the group consisting of the above-mentioned peptide conjugates and pharmaceutically acceptable salts, esters, or solvates thereof (hereinafter, for simplicity, also referred to simply as "peptide conjugates of the present invention"). Yet another aspect is a pharmaceutical composition comprising at least one selected from the group consisting of the above-mentioned peptides and peptide conjugates and pharmaceutically acceptable salts, esters, or solvates thereof.

[0193] The pharmaceutical composition preferably contains an effective amount of the peptide of the present invention or the peptide conjugate of the present invention as an active ingredient. The target disease of the pharmaceutical composition refers to any disease caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), or any disease caused by, exacerbated by, or otherwise associated with increased or decreased Wnt signaling or any other intracellular signaling cascade activated via the Frizzled receptors and / or LRPs. For example, bone metabolic diseases such as osteoporosis (see Manolagas, S.C., "Wnt signaling and osteoporosis," Maturitas, 2014, Vol. 78, No. 3, pp. 233-237), inflammatory bowel disease (see Moparthi, L. et al., "Wnt signaling in intestinal inflammation," Differentiation, 2019, Vol. 108, pp. 24-32), cancer diseases such as breast cancer, colon cancer, lung cancer, and stomach cancer (see Park, W.-J. et al., "A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities”, Cells, 2023, Vol. 12, 1110), but is not limited thereto.

[0194] The administration route of the pharmaceutical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0195] The peptides in the pharmaceutical composition can be modified in various ways, taking into account their susceptibility to metabolism and excretion. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood residence time and reduce its antigenicity. Alternatively, the polypeptide can be encapsulated in biodegradable polymers such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc., which can be used as sustained-release bases. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0196] The pharmaceutical composition may contain an active ingredient as it is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

[0197] The formulation can be carried out by a conventional method using, for example, an excipient, a binder, a disintegrant, a lubricant, a solubilizer, a solubilizing agent, a colorant, a flavoring agent, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, a preservative, an antioxidant, and the like, as appropriate.

[0198] Examples of ingredients used in the formulation include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, trehalose, polysorbate and the like.

[0199] The absorption enhancer can be used to improve the absorption of poorly absorbed drugs. Examples of the absorption enhancer that can be used include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0200] The pills or tablets may be coated with sugar, gastric, or enteric coating materials. The injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. may be added.

[0201] The pharmaceutical compositions of the present invention may be administered in combination with other medications or therapies useful for treating the above diseases.

[0202] The dosage when the pharmaceutical composition of the present invention is administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), particularly humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but can be, for example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg administered once or in divided doses. In the case of injection, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg may be administered once or in divided doses, depending on the patient's weight.

[0203] [Cell Culture Composition] Another embodiment of the present invention is a cell culture composition used for cell culture, which contains the peptide complex described above.

[0204] Because the peptide complex has the ability to activate Wnt signaling, it can also be used as a medium reagent or additive for cell culture, preferably for culturing mammalian cells, more preferably for culturing human cells. The cell culture medium reagent or additive may be a medium reagent or additive for culturing cells for the production of cell-cultured meat.

[0205] The peptide complex can also be used as a reagent or additive for producing gastrointestinal organoids, lung organoids, and the like. The organoids are produced, but not limited to, by inducing differentiation from pluripotent stem cells into endoderm and then into cells of various tissues. Since Wnt signaling plays an important role in this differentiation induction, the peptide complex can be used in the differentiation induction step of pluripotent stem cells into endoderm and into various cells, but is not limited thereto (see Yiangou, L., et al., "Human Pluripotent Stem Cell-Derived Endoderm for Modeling Development and Clinical Applications," Cell Stem Cell, 2018, Vol. 22, No. 4, pp. 485-499).

[0206] The medium is not particularly limited as long as it is a medium for culturing cells or tissues. The medium may be a serum medium, and is preferably a serum-free medium or a low-serum medium.

[0207] The culture medium additive may be in the form of a solution or a dried solid (e.g., solid, powder, etc.). When in the form of a solution, it may be used as a culture medium as is, or it may be diluted with a solvent and, if necessary, the above-mentioned additives may be added thereto, and then used as a culture medium. Examples of solvents used for dilution include water, buffer solutions, physiological saline, and media used for various cell and tissue cultures, and these may be used alone or in combination of two or more.

[0208] When the culture medium additive is in the form of a dry solid, it may be dissolved in a solvent such as water, a buffer solution, physiological saline, or a medium used for various cell or tissue cultures, and the above-mentioned additives may be added as needed to be used as a culture medium.

[0209] The content of the peptide conjugate of the present invention in a medium for culturing the cells or tissues, or in a medium for cells obtained therefrom, can be, for example, about 0.01 to about 10,000 nmol / L, preferably about 0.1 to about 1,000 nmol / L, more preferably about 0.5 to about 1,000 nmol / L, and even more preferably about 1 to about 100 nmol / L, as a final concentration relative to the total volume of the composition or the total volume of the medium.

[0210] [Composition for medical, diagnostic, or research use] Another embodiment of the present invention is a composition for medical, diagnostic, or research use. The composition for medical, diagnostic, or research use of this embodiment is a pharmaceutical composition comprising at least one selected from the group consisting of the above-mentioned peptides and pharmaceutically acceptable salts, esters, or solvates thereof (hereinafter, for simplicity, also referred to simply as "the peptide of the present invention"). Another aspect is a composition comprising at least one selected from the group consisting of the above-mentioned peptide conjugates and pharmaceutically acceptable salts, esters, or solvates thereof (hereinafter, for simplicity, also referred to simply as "the peptide conjugate of the present invention").

[0211] <Composition for medical use> The composition for medical use of this embodiment (hereinafter also simply referred to as "medical composition") contains at least one selected from the group consisting of the peptide and peptide complex of the present invention.

[0212] The medical composition preferably contains an effective amount of the peptide of the present invention or the peptide conjugate of the present invention as an active ingredient. The disease that the medical composition is intended to treat refers to any disease caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), or any disease caused by, exacerbated by, or otherwise associated with increased or decreased Wnt signaling or any other intracellular signaling cascade activated via the Frizzled receptors and / or LRPs. For example, bone metabolic diseases such as osteoporosis (see Manolagas, S.C., "Wnt signaling and osteoporosis," Maturitas, 2014, Vol. 78, No. 3, pp. 233-237), inflammatory bowel disease (see Moparthi, L. et al., "Wnt signaling in intestinal inflammation," Differentiation, 2019, Vol. 108, pp. 24-32), cancer diseases such as breast cancer, colon cancer, lung cancer, and stomach cancer (see Park, W.-J. et al., "A New Wave of Targeting 'Undruggable' Wnt Signaling for Cancer Therapy: Challenges and Opportunities”, Cells, 2023, Vol. 12, 1110), but is not limited thereto.

[0213] The administration route of the medical composition is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal).

[0214] The peptides in the medical composition can be modified in various ways, taking into account their susceptibility to metabolism and excretion. For example, polyethylene glycol (PEG) or sugar chains can be added to the polypeptide to increase its blood residence time and reduce its antigenicity. Furthermore, biodegradable polymers such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. can be used as sustained-release bases, and the polypeptides can be encapsulated in these. For transdermal administration, a weak electric current can be applied to the skin surface to penetrate the stratum corneum (iontophoresis).

[0215] The medical composition may use the active ingredient as it is, or may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. Examples of dosage forms include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices.

[0216] The formulation can be carried out by a conventional method using, for example, an excipient, a binder, a disintegrant, a lubricant, a solubilizer, a solubilizing agent, a colorant, a flavoring agent, a stabilizer, an emulsifier, an absorption enhancer, a surfactant, a pH adjuster, a preservative, an antioxidant, and the like, as appropriate.

[0217] Examples of ingredients used in the formulation include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, silicic anhydride, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, human serum albumin, trehalose, polysorbate and the like.

[0218] The absorption enhancer can be used to improve the absorption of poorly absorbed drugs. Examples of the absorption enhancer that can be used include surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0219] The pills or tablets may be coated with sugar, gastric, or enteric coating materials. The injections may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohols, etc. Furthermore, wetting agents, emulsifiers, dispersants, stabilizers, solubilizers, solubilizers, preservatives, etc. may be added.

[0220] The medical composition of the present invention may be administered in combination with other medicines or treatments useful for the above diseases.

[0221] The dosage when the medical composition of the present invention is administered to mammals (e.g., humans, mice, rats, guinea pigs, rabbits, dogs, horses, monkeys, pigs, sheep, etc.), particularly humans, varies depending on the symptoms, the patient's age, sex, weight, sensitivity, administration method, administration interval, type of active ingredient, and type of formulation, and is not particularly limited, but can be, for example, 30 μg to 1000 mg, 100 μg to 500 mg, or 100 μg to 100 mg administered once or in divided doses. When administered by injection, 1 μg / kg to 3000 μg / kg or 3 μg / kg to 1000 μg / kg may be administered once or in divided doses, depending on the patient's weight.

[0222] <Composition used for diagnosis> The composition used for diagnosis in this embodiment (hereinafter also simply referred to as "diagnostic composition") contains at least one selected from the group consisting of the peptide and peptide complex of the present invention.

[0223] The peptides and peptide complexes bind to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6). Therefore, they can also be used as diagnostic agents for detecting the Frizzled receptor and / or LRP. The diagnostic agent may be a detection agent for detecting the expression level of the Frizzled receptor and / or LRP. When used as a detection agent, the peptide or peptide complex of the present invention may be detectably labeled. In this way, the peptide or peptide complex, or a composition containing them, can be used as a diagnostic agent for detecting the Frizzled receptor and / or LRP.

[0224] <Composition used for research> The composition used for research in this embodiment (hereinafter also simply referred to as "research composition") contains at least one selected from the group consisting of the peptides and peptide complexes of the present invention.

[0225] The peptides and peptide complexes bind to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), and can therefore be used favorably in research involving the Frizzled receptors and / or LRPs.

[0226] The research composition of this embodiment is preferably used in a test method for testing at least one of the following for the peptide or peptide conjugate, and / or a conjugate containing a substance further bound to the peptide or peptide conjugate via a linker: a) solubility in a solvent, b) binding ability to Frizzled receptors (Fzd2, Fzd7, Fzd8) and / or LRPs (LRP5, LRP6), c) toxicity to cells and / or tissues, and d) toxicity to experimental animals.

[0227] Regarding the test method, the test of the solubility in a solvent of the peptide or the peptide complex, and / or a complex containing a substance further bound to the peptide or the peptide complex via a linker, may be a measurement of solubility. The solvent used in the solubility measurement is not limited and may be freely selected depending on the purpose. Furthermore, a known method for measuring solubility may be appropriately selected depending on the type of solvent.

[0228] The test for binding ability to the Frizzled receptor and / or the LRP may be a measurement of binding ability to the Frizzled receptor and / or the LRP, and known methods such as, but not limited to, surface plasmon resonance (SPR) assay, Scatchard analysis, and / or competitive binding assays such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay can be preferably used.

[0229] The test for toxicity to cells and / or tissues may be a known toxicity evaluation test using cells and / or tissues, for example, an in vitro method. The cells and tissues may be, but are not limited to, cells and / or tissues typically used in toxicity evaluation tests for pharmaceuticals.

[0230] The toxicity test method for experimental animals may be a known toxicity evaluation test using experimental animals. The experimental animals are not particularly limited as long as they are commonly used, and examples include mice, rats, guinea pigs, gerbils, hamsters, ferrets, rabbits, dogs, cats, pigs, goats, horses, cows, birds (e.g., chickens, quails, etc.), monkeys, and non-human primates (e.g., cynomolgus monkeys, marmosets, rhesus monkeys, etc.). The toxicity evaluation test may be, but is not limited to, a safety test typically performed in non-clinical studies of pharmaceuticals, and examples include general toxicity tests (single-dose toxicity tests / repeated-dose toxicity tests), genotoxicity tests (Ames tests / chromosomal aberration tests / in vitro micronucleus tests), carcinogenicity tests, reproductive and developmental toxicity tests (ICH-I, II, III), local irritation tests (eye irritation tests, skin irritation tests, etc.), other toxicity tests (skin sensitization tests, phototoxicity tests, antigenicity tests), chemical analysis / biological analysis (TK / PK), etc.

[0231] [Nucleic Acid] Yet another embodiment of the present invention is a nucleic acid encoding the above-described peptide or peptide. The nucleic acid of this embodiment may be natural or non-natural. Nucleic acids include, but are not limited to, DNA, RNA, and chimeras thereof. The nucleic acid of this embodiment can be designed and produced by known methods based on the amino acid sequence of the above-described peptide or peptide complex.

[0232] Peptide Drug Conjugate (PDC) Yet another embodiment of the present invention is a conjugate (peptide drug conjugate, PDC) comprising the above-described peptide or peptide conjugate, a desired substance to be delivered to a Frizzled receptor (Fzd2, Fzd7, Fzd8) and / or LRP (LRP5, LRP6), and a linker for linking the peptide or peptide conjugate to the substance. The peptide binds to the Frizzled receptor or the LRP. The peptide conjugate binds to the Frizzled receptor and the LRP. Thus, the peptide or peptide conjugate is capable of delivering a substance to the Frizzled receptor and / or the LRP. The substance may be any substance desired by those skilled in the art, as long as it is a substance that is desired to be delivered to the Frizzled receptor and / or the LRP. The substance is not particularly limited, but examples thereof include compounds, peptides, RIs (radioisotopes), proteins, nucleic acids, and molecules used in DDSs (drug delivery systems). The compound is not particularly limited, but preferably a low-molecular-weight or medium-molecular-weight compound, such as a known low-molecular-weight drug. The peptide is not particularly limited, but preferably a peptide that binds to a target in the body and exhibits some effect, such as a cyclic peptide. The RI is not particularly limited, but may be a radioisotope-labeled compound such as a low-molecular-weight compound, a medium-molecular-weight compound, or an antibody, for example, a compound used in PET (positron emission tomography) examination. The protein is not particularly limited, but may be any protein that exhibits a useful function in the body, such as an antibody or enzyme. Examples include enzymes used in enzyme replacement therapy. The nucleic acid may be DNA, RNA, or a chimera thereof, but is not particularly limited. Examples include nucleic acid pharmaceuticals. The molecule used in the DDS is not particularly limited, but preferably a known molecule used in DDSs, such as a liposome or micelle. The molecule used in the DDS may further contain a compound such as a drug. Furthermore, the substance desired to be delivered to the Frizzled receptor and / or the LRP may be a complex of the above-listed substances.

[0233] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below. Those skilled in the art can easily modify and alter the present invention based on the description in this specification, and such modifications and alterations are within the technical scope of the present invention.

[0234] [Chemical Synthesis] All raw materials, building blocks, reagents, acids, bases, solid-phase resins, and solvents used in the chemical synthesis in the following examples were either commercially available products or could be synthesized by those skilled in the art using organic chemistry techniques. Unless otherwise specified, commercially available amino acids containing protecting groups were used as they were.

[0235] Peptide chain elongation on solid-phase resins was carried out using the resins described in each Example as starting materials under commonly used peptide coupling reaction conditions and Fmoc removal reaction conditions. The reactions were carried out using an automated peptide synthesizer, such as a Biotage Syro I, a Biotage Syro II, a CEM Liberty Blue, a CEM Liberty Blue HT12, or a CEM Liberty Prime, according to the manufacturer's instructions. The resins used were NovaPEG Rink Amide resin, HMPB-MBHA resin, or Seiber Amide resin, and the amounts used ranged from 5 mg to 2 g depending on the peptide.

[0236] The reagent cocktail used for deprotecting the side chains and cleaving from the solid phase resin was 4 mL to 50 mL depending on the peptide, and a solution with the following composition was used: A: TFA / H ２ O / TIS / DODT (92.5 / 2.5 / 2.5 / 2.5) B:TFA / H ２ O / TIS / DODT (90 / 2.5 / 2.5 / 5) Common Fmoc amino acids used are listed in Table 4, with side chain protecting groups indicated in brackets.

[0237]

[0238]

[0239]

[0240]

[0241] Unless otherwise specified, the resulting crude peptides were purified using one of the following reversed-phase preparative purification systems: A) Shimadzu prep-HPLC system (LC-20AP, SPD-M20A, CTO-20AC, and CBM-20A). B) Waters AutoPurification System. C) Waters AutoPurification System with SQD. D) Waters Preparative HPLC System. E) YMC Contichrom CUBE.

[0242] Unless otherwise specified, the columns used were one of the following a) to k): a) Jeanious One-Column 20 mm ID x 150 mm L; b) Kinetex EVO C18 5 μm 21.2 x 150 mm; c) Waters XBridge C18 5 μm 19 x 150 mm; d) Waters XBridge C18 5 μm 30 x 150 mm; e) Waters XBridge C18 5 μm 50 x 150 mm; f) Waters XBridge C18 5 μm 50 x 250 mm; g) Waters XSelect C18 30 x 150 mm. h) Waters XSelect C18 5μm 19x150mm. i) Waters XSelect C18 5μm 30x150mm. j) Waters XSelect CSH Prep C18 5μm OBD 50x250mm. k) YMC-Triart Prep C18-S 10μm 10x250mm.

[0243] The structure of the chemically synthesized peptides was determined by ESI-MS(+) mass spectrometry, where the molecular weight was calculated based on the amino acids used in the target sequence and the building blocks used as needed. "ESI-MS(+)" refers to electrospray ionization mass spectrometry performed in positive ion mode. Detected masses were reported in "m / z" units. Compounds with molecular weights greater than approximately 1000 were frequently detected as multiply charged ions.

[0244] Basic analytical equipment and basic conditions For mass spectral analysis of the peptides synthesized in the following examples, the following basic analytical equipment and basic conditions were used, unless otherwise specified: Analysis was performed using gradient B (%) under one of the following conditions: w / x / y / z.

[0245] (Basic analytical equipment) Waters AutoPurification System-SQD2 single quadruple mass spectrometer (Basic conditions) Column: Kinetex EVO C18 (1.7 μm, 2.1 × 50 mm, 100 Å) Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２ O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.6 mL / min Wavelength: 220 nm Gradient B (%): w) 5-95% / 2.10 min, 95-95% / 0.75 min

[0246] (Basic analysis equipment) Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) (Basic conditions) Column: Kinetex EVO C18 (2.6 μm, 2.1 x 150 mm, 100 Å) Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA Gradient B (%): x) 5-45% / 7.15 min, 45-95% / 0.3 min, 95-95% / 1.55 ​​min y) 20-60% / 7.15min, 60-95% / 0.3min, 95-95% / 1.55min z) 20-60% / 54.15min, 60-95% / 0.3min, 95-95% / 1.55min

[0247] Example 1 Synthesis of Peptide Conjugate (Peptide Conjugate with Linker Structure No. 9 Added to Peptide SEQ ID NO: 66)

[0248]

[0249] The target peptide was synthesized using Sieber amide resin, starting with the removal of the Fmoc group using the general method described above. A Biotage Syro II solid-phase synthesizer was used, and synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / HATU / DIEA (8.4 equivalents / 7.84 equivalents / 16.8 equivalents) was used per equivalent of resin, and the reaction was carried out twice for 20 minutes at 75°C in DMF. The fifth and fourteenth residues were reacted twice for 30 minutes at 50°C. The sixth, seventh, eighth, ninth, tenth, eleventh, and twelfth residues were reacted three times for 20 minutes at 75°C. The 16th residue, Fmoc-PEG10c, was introduced once for 60 minutes at 25°C.

[0250] The Fmoc group was removed by reacting with a 10% pyrrolidine solution in DMF at 25° C. for 3 minutes.

[0251] The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step by the method described above, followed by adding a DMF solution of ClAcOSu (5 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes.

[0252] For side chain deprotection and cleavage from the solid-phase resin, Reagent Cocktail A (a mixture of TFA / HO / TIS / DODT in a volume ratio of 92.5:2.5:2.5:2.5) was added to the reaction vessel containing the solid-phase resin, and the mixture was shaken vigorously and then shaken at room temperature for 60 minutes. The reaction solution was collected by filtration through a frit. When this filtrate was added to an excess of chilled diisopropyl ether, a cloudy white precipitate formed. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with a chilled diisopropyl ether / hexane (1 / 1) mixed solvent and then dried for 60 minutes. The resulting solid was used in the subsequent cyclization reaction.

[0253] The peptide cyclization reaction was carried out in DMSO / H 2 SO 4 at a final peptide concentration of 1.25 mM based on the molar number of the solid phase resin. ２ After dissolving the peptide in 0 (7 / 3), triethylamine (10 equivalents) was added and the mixture was left to stand at room temperature overnight. The resulting reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite, and then DMSO was added to adjust the peptide concentration to 12.5 mM.

[0254] The obtained crude product was subjected to solid phase extraction using a Gilson column (column: Gilson ASPEC C18, 50 mg, 1 mL). (1) The column was extracted with extraction solution A (0.1% TFA in 95% MeCN / H ２ (2) Extraction solution B (0.1% TFA in 5% MeCN / H2O, 0.3 mL) was used. ２ (3) 0.02 mL of the above solution was loaded onto the column. (4) The column was washed with Extraction Solution B (0.4 mL). (5) Extraction was performed with Extraction Solution A (0.4 mL). The obtained extract was concentrated under reduced pressure using an EZ-2 Elite.

[0255] The purity of one of the main peaks of the target product was calculated from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions, and was found to be 55%.

[0256] (Analysis conditions) Retention time: 1.52 minutes Column: Kinetex EVO C18 (1.7 μm 2.1×50 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２O, B) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 5-95% over 2.10 min, then 95-95% over 0.75 min Flow rate: 0.6 mL / min ESI-MS (+) Observed value m / z = 1144 (M+3H)

[0257] Example 2 Synthesis of Peptide Conjugate (Peptide Conjugate with Linker Structure No. 9 Added to Peptide SEQ ID NO: 99)

[0258]

[0259] The target peptide was synthesized using Sieber amide resin, starting with the removal of the Fmoc group using the general method described above. A Biotage Syro II solid-phase synthesizer was used, and synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / HATU / DIEA (4.2 equivalents / 3.92 equivalents / 8.4 equivalents) was used per equivalent of resin, and the reaction was carried out twice at 75°C for 20 minutes in DMF. However, the second and fourteenth residues were reacted twice at 50°C for 30 minutes. The sixth residue was reacted twice at 50°C for 60 minutes. The seventh residue was reacted twice at 75°C for 30 minutes. The 16th residue, Fmoc-PEG10c, was introduced once at room temperature for 60 minutes.

[0260] The Fmoc group was removed by reacting with a 20% piperidine solution in DMF at room temperature for 5 minutes, removing the solution, and then reacting again with a 20% piperidine solution in DMF at room temperature for 15 minutes.

[0261] The introduction of a chloroacetyl group was carried out by removing the Fmoc group from the α-amino group of the solid-phase resin carrying the Fmoc-protected peptide obtained in the previous step by the method described above, followed by adding a DMF solution of ClAcOSu (5 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes.

[0262] The deprotection of the side chain and the cleavage from the solid-phase resin were carried out by adding a reagent cocktail A (TFA / H ２A mixture of 2.5:2.5:2.5:2.5 (volume ratio of 2.5:2.5:2.5) of 2.5-dihydroxybenzoic acid (DPO) / TIS / DODT was added and shaken vigorously, followed by shaking at room temperature for 90 minutes. The reaction solution was filtered through a frit. When the filtrate was added to an excess of chilled diisopropyl ether, a cloudy white precipitate formed. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with a chilled diisopropyl ether / hexane (1 / 1) mixed solvent and then dried for 60 minutes. The resulting solid was used in the subsequent cyclization reaction.

[0263] The peptide cyclization reaction was carried out in DMSO / H2SO4 solution so that the final peptide concentration was 2.5 mM based on the molar number of the solid phase resin. ２ After dissolving the mixture in 200 ml of ethanol (7 / 3), triethylamine (20 equivalents) was added and the mixture was stirred, then allowed to stand at room temperature overnight. The resulting reaction solution was concentrated under reduced pressure using Genevac EZ-2 Elite to a concentration of 12.5 mM.

[0264] The obtained crude product was subjected to solid phase extraction using a Gilson column (column: Gilson ASPEC C18, 50 mg, 1 mL). (1) The column was extracted with extraction solution A (0.1% TFA in 95% MeCN / H ２ (2) Extraction solution B (0.1% TFA in 5% MeCN / H2O, 0.3 mL) was used to wash the extract. ２ (3) 0.02 mL of the above solution was loaded onto the column. (4) The column was washed with Extraction Solution B (0.4 mL). (5) Extraction was performed with Extraction Solution A (0.4 mL). The obtained extract was concentrated under reduced pressure using an EZ-2 Elite.

[0265] The purity of one of the main peaks of the target product was calculated from the area ratio of the LC / MS (UV wavelength 220 nm) chromatogram under the following analytical conditions, and was found to be 50%.

[0266] (Analysis conditions) Retention time: 1.24 minutes Column: Kinetex EVO C18 (1.7 μm, 2.1 × 50 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２0 B) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 5-95% over 2.10 minutes, then 95-95% over 0.75 minutes Flow rate: 0.6 mL / min ESI-MS (+) Observed value m / z = 1204 (M+3H)

[0267] Example 3: Synthesis of peptide conjugate (dimer of dimer structure number 62)

[0268] I. Synthesis of peptide conjugate (peptide conjugate in which a part of linker structure number 2 is added to peptide SEQ ID NO: 74)

[0269]

[0270] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.6 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue HT solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes in DMF at 90°C. The fifth residue, however, was reacted twice for 15 minutes at 50°C. The sixth and ninth residues were reacted twice for 10 minutes at 90°C. The eleventh residue was reacted twice for 30 minutes at 75°C. The 12th, 13th, 15th, and 16th residues, Fmoc-PEG4c, and the 17th residue, Fmoc-Gpra-OH, were reacted once at 75°C for 10 minutes. The 14th residue was reacted once at 50°C for 15 minutes. The 10th residue was reacted once at 40°C for 30 minutes, using condensation conditions of Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents).

[0271] Fmoc removal was carried out by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute, or at 50°C for 90 seconds, or at 75°C for 3 minutes, or at room temperature for 1 minute.

[0272] The introduction of a chloroacetyl group was carried out by adding a DMF solution of ClAcOSu (5 equivalents) to the solid phase resin and shaking at room temperature for 60 minutes.

[0273] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride and then diethyl ether, and then dried under reduced pressure. Reagent cocktail A (TFA / H ２ A mixture of 2.5:2.5:2.5:2.5 (volume ratio of 2.5:2.5:2.5) of 2.5-diisopropyl ether (DIS) and 2.5-diisopropyl ether (DODT) was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether and hexane (1 / 1), producing a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.

[0274] The peptide cyclization reaction was carried out in MeCN / H2SO4 at a final peptide concentration of 3.1 mM based on the molar number of the solid-phase resin. ２ After dissolving the residue in 1:1 ethanol, triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, followed by addition of acetic acid. The resulting reaction solution was concentrated using Genevac HT-12.

[0275] The obtained crude product was purified under the following conditions: (Purification conditions) Column: Waters XBridge C18 (5 μm, 50 × 150 mm) Mobile phase: A) 0.1% TFA in H ２ OB) 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc.): 5% over 2 minutes, 5-28% over 1 minute, then 28-33% over 8 minutes, then 33-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then 20 mL / min-120 mL / min over 1 minute, then 120 mL / min

[0276] After freeze-drying, a peptide conjugate (190 mg) in which a part of linker structure number 2 was added to peptide sequence number 74 was obtained.

[0277] II. Synthesis of peptide conjugate (peptide conjugate in which a part of linker structure number 2 is added to peptide SEQ ID NO: 162)

[0278]

[0279] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty Blue solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 3 minutes in DMF at 90°C. However, the second residue was reacted twice for 15 minutes at 50°C. The 11th and 12th residues were reacted twice for 10 minutes at 90°C. The 14th residue was reacted twice for 15 minutes at 50°C. The sixth residue was reacted twice for 60 minutes at room temperature, using the condensation conditions of Fmoc-AA / HATU / DIEA (4.2 equivalents / 4 equivalents / 8 equivalents). Furthermore, when Fmoc-W7N-OH, Fmoc-Kmor-OH, and Fmoc-KN3-OH were introduced, the amino acids were dissolved in NMP. Fmoc removal was performed by reacting with a 10% DMF solution at 90°C for 1 minute, at 50°C for 90 seconds, or at room temperature for 1 minute, followed by two consecutive cycles.

[0280] The introduction of a chloroacetyl group was carried out by adding a solution of ClAcOSu (5 equivalents) in DMF / DCM (1 / 1) to the solid phase resin and shaking at room temperature for 30 minutes.

[0281] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride and then diethyl ether, and then dried under reduced pressure. Reagent cocktail A (TFA / H ２A mixture of 2.5:2.5:2.5:2.5 (volume ratio of 2.5:2.5:2.5) of 2.5-diisopropyl ether (DIS) and 2.5-diisopropyl ether (DODT) was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether and hexane (1 / 1), producing a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.

[0282] The peptide cyclization reaction was carried out in MeCN / H2SO4 at a final peptide concentration of 5 mM based on the molar number of the solid phase resin. ２ After dissolving the residue in 1:1 ethanol, triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 1 hour, followed by addition of acetic acid. The resulting reaction solution was concentrated using Genevac HT-12.

[0283] The obtained crude product was purified under the following conditions: (Purification conditions) Column: Waters XBridge C18 (5 μm, 50 × 150 mm) Mobile phase: A) 0.1% TFA in H ２ OB) 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc): 5% over 2 minutes, 5-21% over 1 minute, then 21-26% over 8 minutes, then 26-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then 20 mL / min-120 mL / min over 1 minute, then 120 mL / min

[0284] After freeze-drying, a peptide conjugate (161 mg) in which a part of linker structure number 2 was added to peptide sequence number 162 was obtained.

[0285] III. Synthesis of Peptide Conjugate (Dimer of Dimer Structure No. 62)

[0286]

[0287] The peptide conjugate was synthesized by dissolving 30 mg of the peptide conjugate (peptide conjugate in which a part of linker structure number 2 was added to peptide SEQ ID NO: 162) in DMF / H ２After dissolving in 100 ml of HCl (3 / 1), a peptide complex (a peptide complex in which a part of linker structure number 2 is added to the peptide of SEQ ID NO: 74) (27 mg, 1.1 equivalents), [Cu(CH ３ CN) ４ ]PF ６ (4 equivalents) was added and the mixture was stirred at room temperature for 30 minutes.

[0288] The obtained crude product was purified under the following conditions: (Purification Conditions) Column: Waters XSelect CSH Prep C18 (5 μm, OBD, 50 × 250 mm) Mobile phase: A) 1% AcOH in H ２ OB) 1% AcOH in MeCN C) 0.2M TEAA in H ２ OD) MeCN Temperature: 50°C; Main pump gradient (%A conc.): 0.1% over 5.0 minutes, then 0.1 to 100% over 0.1 minutes, 100%-%B from 5.1 minutes onwards (%B conc.): 0% over 5.1 minutes, then 0 to 4.2% over 1.9 minutes, then 4.2 to 50% over 20 minutes, then 50 to 90% over 4 minutes (%C conc.): 99.9% over 5.0 minutes, then 99.9 to 0% over 0.1 minutes, 0% from 5.1 minutes onwards (%D conc.): 0%. Flow rate: 18 mL / min over 5.1 min, then 18 mL / min to 118 mL / min over 1.9 min, then 118 mL / min. At-column-dilution pump 1% AcOH in H ２ O / MeCN (1 / 1) flow rate: 2 mL / min

[0289] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 95.06%.

[0290] (Analysis conditions) Retention time: 5.80 min Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc.): 5-45% over 7.15 min, then 45-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed m / z = 1186 (M+4H)

[0291] Example 4: Synthesis of peptide conjugate (dimer of dimer structure number 46)

[0292] I. Synthesis of peptide conjugate (peptide conjugate in which a part of linker structure number 8 is added to peptide SEQ ID NO: 35)

[0293]

[0294] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty PRIME solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 2 minutes in DMF at 105°C. The fifth residue, however, was reacted twice for 15 minutes at 50°C. The sixth, ninth, and eleventh residues were reacted twice for 30 minutes at 75°C. The fourteenth residue was reacted once for 15 minutes at 50°C.

[0295] Fmoc removal was carried out under the basic condition of reacting the product with a 4% pyrrolidine solution of 83 mM Oxyma pure in DMF at 110°C for 90 seconds, and also under the condition of reacting the product with a 10% pyrrolidine solution in DMF at room temperature for 1 minute twice in succession.

[0296] The introduction of a chloroacetyl group was carried out by adding a DMF solution of ClAcOSu (5 equivalents) to the solid phase resin and shaking at room temperature for 60 minutes.

[0297] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride and then diethyl ether, and then dried under reduced pressure. Reagent cocktail A (TFA / H ２ A mixture of 2.5:2.5:2.5:2.5 (volume ratio of 2.5:2.5:2.5) of 2.5-diisopropyl ether (DIS) and 2.5-diisopropyl ether (DODT) was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether and hexane (1 / 1), producing a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.

[0298] The peptide cyclization reaction was carried out in MeCN / H2SO4 at a final peptide concentration of 2.5 mM based on the molar number of the solid phase resin. ２ After dissolving the residue in 1:1 ethanol, triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 4 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using a Genevac EZ-2 Elite. The resulting crude product was purified under the following conditions to obtain a peptide conjugate (a peptide conjugate in which a portion of linker structure number 8 was added to the peptide of SEQ ID NO: 35).

[0299] (Purification conditions) Column: Waters XBridge C18 (5 μm, 50 × 150 mm) Mobile phase: A) 0.1% TFA in H ２ OB) 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc.): 9% over 2 minutes, 9-34% over 1 minute, then 34-39% over 8 minutes, then 39-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then 20 mL / min-120 mL / min over 1 minute, then 120 mL / min

[0300] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 97.80%.

[0301] (Analysis conditions) Retention time: 4.25 minutes Column: Kinetex EVO C18 (2.6 μm, 2.1×150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２ OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 988 (M+2H)

[0302] II. Synthesis of peptide conjugate (peptide conjugate in which a part of linker structure number 8 is added to peptide SEQ ID NO: 79)

[0303]

[0304] The target peptide was synthesized using Sieber amide resin (Watanabe Chemical, 0.54 mmol / g) by the general method described above, starting with the removal of the Fmoc group. A CEM Liberty PRIME solid-phase synthesizer was used, and the synthesis was performed according to the manufacturer's instructions. To introduce each residue, Fmoc-AA / DIPCI / Oxyma pure (4.2 equivalents / 8 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 2 minutes in DMF at 105°C. However, the second and sixth residues were reacted twice for 15 minutes at 50°C. The eleventh and twelfth residues were reacted twice for 3 minutes at 105°C. The fourteenth residue was reacted once for 15 minutes at 50°C.

[0305] Fmoc removal was carried out under the basic conditions of reacting with a 4% pyrrolidine solution of 83 mM Oxyma pure in DMF at 110°C for 90 seconds, but also under the conditions of reacting with a 10% pyrrolidine solution in DMF at room temperature for 1 minute twice in succession, or reacting with a 10% pyrrolidine solution in DMF at 50°C for 90 seconds.

[0306] The introduction of a chloroacetyl group was carried out by adding a DMF solution of ClAcOSu (5 equivalents) to the solid phase resin and shaking at room temperature for 60 minutes.

[0307] For the deprotection of the side chain and the cleavage from the solid-phase resin, the resin obtained after the chloroacetyl group introduction step was first washed with DMF, followed by methylene chloride and then diethyl ether, and then dried under reduced pressure. Reagent cocktail A (TFA / H ２ A mixture of 2.5:2.5:2.5:2.5 (volume ratio of 2.5:2.5:2.5) of 2.5-diisopropyl ether (DIS) and 2.5-diisopropyl ether (DODT) was added and the mixture was shaken at room temperature for 60 minutes. The reaction solution was filtered through a frit. The filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether and hexane (1 / 1), producing a cloudy white precipitate. This mixture was centrifuged, and the solution was decanted. The resulting solid was washed again with cooled diethyl ether and then dried under reduced pressure. The resulting solid was used in the subsequent cyclization reaction.

[0308] The peptide cyclization reaction was carried out in MeCN / H2SO4 at a final peptide concentration of 2.5 mM based on the molar number of the solid phase resin. ２ After dissolving the product in 1:1 ethanol, triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 4 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using a Genevac EZ-2 Elite. The resulting crude product was purified under the following conditions to obtain a peptide conjugate (a peptide conjugate in which a portion of linker structure number 8 was added to the peptide of SEQ ID NO: 79).

[0309] (Purification conditions) Column: Waters XBridge C18 (5 μm, 50 × 150 mm) Mobile phase: A) 0.1% TFA in H ２ OB) 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc.): 5% over 2 minutes, 5-27% over 1 minute, then 27-32% over 8 minutes, then 32-60% over 1 minute Flow rate: 20 mL / min over 1 minute, then 20 mL / min-120 mL / min over 1 minute, then 120 mL / min

[0310] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 95.95%.

[0311] (Analysis conditions) Retention time: 5.85 minutes Column: Kinetex EVO C18 (2.6 μm, 2.1×150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２ OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 5-45% over 7.15 min, then 45-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1055 (M+2H)

[0312] III. Synthesis of Peptide Conjugate (Dimer of Dimer Structure No. 46)

[0313]

[0314] The peptide conjugate was synthesized by adding 0.0175 M AZ(L08)-P12-PS in DMA (0.36 mL) and DIPEA (0.012 mL) to 20 mg of the peptide conjugate (peptide conjugate in which a portion of linker structure number 8 was added to peptide SEQ ID NO: 35) at 40 °C, stirring for 2 hours at the same temperature, followed by the addition of 0.0175 M AZ(L08)-P12-PS in DMA (0.13 mL) and stirring for 1 hour at the same temperature. Water (0.36 mL), a DMA solution (0.36 mL) of the peptide conjugate (peptide conjugate in which a portion of linker structure number 8 was added to the peptide of peptide SEQ ID NO: 79) (20.05 mg), TBTA (7.2 mg), and [Cu(CH3CN)4]PF6 (3.4 mg) were added to the reaction mixture and stirred at room temperature for 1 hour, followed by the addition of acetic acid. The obtained crude product was purified under the following conditions to obtain the target product (17 mg).

[0315] (Purification conditions) Column: Waters XBridge C18 (5 μm, 19 × 150 mm) Mobile phase: A) 0.1% TFA in H ２ OB) 0.1% TFA in MeCN Temperature: 40°C Gradient (%B conc.): 14-39% over 3 minutes, then 39-44% over 8 minutes, then 44-60% over 1 minute Flow rate: 17 mL / min

[0316] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 94.64%.

[0317] (Analysis conditions) Retention time = 5.40 minutes Column: Kinetex EVO C18 (2.6 μm, 2.1 × 150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２ OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed m / z = 1484 (M+4H)

[0318] Example 5: Synthesis of peptide conjugate (dimer of dimer structure number 63)

[0319]

[0320] The target compound was obtained by synthesis in the same manner as in Example 3.

[0321] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 97.49%.

[0322] (Analysis conditions) Retention time: 4.10 minutes Column: Kinetex EVO C18 (2.6 μm, 2.1×150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２ OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (%B conc.): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed m / z = 1242 (M+5H)

[0323] Example 6: Synthesis of peptide conjugate (dimer of dimer structure number 66)

[0324]

[0325] A peptide conjugate (a peptide conjugate in which a portion of linker structure number 3 is added to the peptide of peptide SEQ ID NO: 163) synthesized in the same manner as in Example 3 using HMPB-MBHA resin (Novabiochem, 0.69 mmol / g) and Fmoc-Gpra-HMPB-MBHA resin synthesized by reacting 1 equivalent of resin with Fmoc-Gpra-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.5 equivalents) in DMF / DCM (1 / 2) at room temperature for 1 hour once, and HMPB-MBHA The target product was obtained by synthesis in the same manner as in Example 3 using a peptide conjugate (a peptide conjugate in which a part of linker structure number 3 is added to the peptide of peptide SEQ ID NO: 76) synthesized in the same manner as in Example 3 using Fmoc-KN3-HMPB-MBHA resin (Novabiochem, 0.69 mmol / g) and Fmoc-KN3-OH / DIC / DMAP (4 equivalents / 4 equivalents / 0.5 equivalents) synthesized in a reaction once for 1 hour in DMF / DCM (1 / 2) with 1 equivalent of the resin.

[0326] The purity of the target product was calculated from the area ratio of the LC / MS (UV wavelength 225 nm) chromatogram under the following analytical conditions and was found to be 95.04%.

[0327] (Analysis conditions) Retention time: 2.80 minutes Column: Kinetex EVO C18 (2.6 μm, 2.1×150 mm, 100 Å) Mobile phase: A) 0.025% TFA in H ２ OB) 0.025% TFA in MeCN Temperature: 60°C Gradient (% B conc.): 20-60% over 7.15 min, then 60-95% over 0.30 min, then 95-95% over 1.55 min Flow rate: 0.5 mL / min ESI-MS (+) observed value m / z = 1176 (M+4H)

[0328] Example 7 Synthesis of Various Peptides In this example, various peptide conjugates were chemically synthesized in the same manner as in Examples 1 to 6. The amino acid sequences of the synthesized cyclic peptides are shown in Tables 5 and 6, the linker structures are shown in the formula below, and peptide conjugates in which the cyclic peptides are dimerized via the linker are shown in Table 7. The synthesized peptide conjugates were analyzed under any of the analytical conditions described in Examples 1 to 6, and their structures were confirmed by ESI-MS(+) in mass spectrometry. The obtained ESI-MS(+) observed values, retention times, charge numbers, and the concentration gradient (%) of mobile phase B used in the analysis are shown in Tables 5, 6, and 7.

[0329] The analytical results shown in Tables 5 and 6 are values ​​obtained as a result of analyzing a peptide complex in which a linker represented by linker structure number 9 is added to a peptide represented by a peptide sequence number.

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345] Example 8 Evaluation of binding activity to Frizzled by ELISA To evaluate the binding activity of the synthesized peptide conjugates (peptide conjugates in which linker structure number 9 is added to peptides SEQ ID NOS: 1 to 78) to Frizzled, binding activity was evaluated using ELISA in the following steps.

[0346] Biotin-labeled polyclonal anti-human IgG (eBioscience; 13-4998) was added to a streptavidin-coated 96-well plate (Thermo; 436014) at 200 μg per well and allowed to stand for 30 minutes.

[0347] After washing three times with PBS-T, 3 pmol of Fc-tagged Frizzled-2 (R&D systems; 1307-FZ), Frizzled-7 (R&D systems; 6178-FZ), or Frizzled-8 (R&D systems; 6129-FZ) was added per well and allowed to stand for 30 minutes to immobilize each Frizzled on the plate.

[0348] After washing three times with PBS-T, the peptide complex diluted to 10 nM was added to Frizzled-2 and Frizzled-7, and to 500 nM to Frizzled-8, and the mixture was allowed to stand for 1 hour.

[0349] After washing three times with PBS-T, Anti-HA-tag mAb-HRP-Direct T (MBL; M180-7) diluted 5000-fold with PBS-T was added and allowed to stand for 30 minutes.

[0350] After washing three times with PBS-T, SureBlue TMB 1-Component Microwell Peroxidase Substrate (SeraCare; 5120-0077) was added and allowed to stand for 10 minutes, after which the reaction was stopped by adding an equal volume of TMB Stop Solution (SeraCare; 5150-0021).

[0351] The absorbance at 450 nm was measured using Infinite M Nano* (TECAN). The binding signal for each peptide conjugate was calculated by subtracting the absorbance in wells without peptide conjugate addition from the absorbance in wells with peptide addition.

[0352] The results are shown in Table 8. The synthesized peptide conjugates were shown to have binding activity to Frizzled-2 (FZD2), Frizzled-7 (FZD7), and Frizzled-8 (FZD8).

[0353]

[0354]

[0355]

[0356] Example 9 Evaluation of binding activity to LRP by ELISA To evaluate the binding activity of the synthesized peptide complexes (peptide complexes in which linker structure number 9 is added to peptide SEQ ID NOs: 79 to 163) to LRP, binding activity was evaluated using the ELISA method by the following steps.

[0357] I. Evaluation of binding activity of peptide complex to LRP5 Biotin-labeled LRP5 (ACROBiosystems; LR5-H82E6) was added to a streptavidin-coated 96-well plate (Thermo; 436014) at 3 pmol per well and allowed to stand for 30 minutes to immobilize LRP5 on the plate.

[0358] After washing three times with PBS-T, the peptide complex diluted to 10 nM was added and allowed to stand for 1 hour.

[0359] After washing three times with PBS-T, anti-HA-tag mAb-HRP-Direct T (MBL; M180-7) diluted 5000-fold with PBS-T was added and the plate was left to stand for 30 minutes.

[0360] After washing three times with PBS-T, the detection reagent SureBlue TMB 1-Component Microwell Peroxidase Substrate (SeraCare; 5120-0077) was added and allowed to stand for 10 minutes, after which the reaction was stopped by adding an equal volume of TMB Stop Solution (SeraCare; 5150-0021).

[0361] The absorbance at 450 nm was measured using Infinite M Nano* (TECAN). The binding signal of each peptide conjugate was calculated by subtracting the absorbance of the well without peptide conjugate from the absorbance of the well with peptide conjugate.

[0362] II. Evaluation of binding activity of peptide complex to LRP6 Biotin-labeled Polyclonal Anti-Human IgG (eBioscience; 13-4998) was added to a streptavidin-coated 96-well plate (Thermo; 436014) at 200 μg per well and allowed to stand for 30 minutes.

[0363] After washing three times with PBS-T, 3 pmol of Fc tag-fused LRP6 (R&D systems; 1505-LR) was added per well to immobilize LRP6 on the plate.

[0364] After washing three times with PBS-T, the peptide complex diluted to 100 nM was added and allowed to stand for 1 hour. Thereafter, antibody reaction and absorbance detection were carried out in the same manner as in "I. Evaluation of the binding activity of peptide complexes to LRP5".

[0365] The results are shown in Table 9. The synthesized peptide conjugates were shown to have binding activity to LRP5 and LRP6.

[0366]

[0367]

[0368]

[0369] [Example 10] Evaluation of Wnt3a inhibitory activity

[0370] To evaluate the Wnt3a inhibitory activity of the synthesized peptides (peptide SEQ ID NOS: 79, 81, 83, 85-96, 98-100), inhibition of transcriptional activity using a TCF / LEF reporter was assessed.

[0371] TCF / LEF Reporter-HEK 293 cells (BPS Bioscience) were cultured in EMEM (Fuji FILM) containing 10% FBS (Thermo Fisher Scientific), 50 μg / mL Gentamicin (Nacalai tesque), and 400 μg / mL Geneticin (Thermo Fisher Scientific).

[0372] The cells were detached using TrypLE (Thermo Fisher Scientific) and then suspended in EMEM containing 10% FBS, 50 μg / mL Gentamicin, and 10 mM LiCl (Sigma).

[0373] The cells were seeded into a white 96-well plate for cell adhesion and luminescence detection (Thermo Fisher Scientific) at 35,000 cells per well and cultured overnight.

[0374] After the addition of the peptide, 1 nM Recombinant Human R-spondin 1 (R&D systems) and 10 nM Wnt3a were added. 37 ｏ C's CO ２ The cells were stimulated in an incubator for 5 hours and 30 minutes.

[0375] ONE-Glo Luciferase Assay System (Peomega) was added and the mixture was shaken for 4 minutes. Luminescence signals were detected using a SpectraMax Paradigm multimode microplate reader (Molecular Devices). The resulting signals were analyzed using GraphPad Prism, and the % inhibition of each peptide was calculated by defining the signal induced by Wnt3a and R-spondin as 0% inhibition and the signal without Wnt3a or R-spondin as 100% inhibition.

[0376] The test was carried out with the peptide added at concentrations of 1 nM, 10 nM, and 100 nM.

[0377] Regarding % inhibition, peptides that exhibited 50% or more inhibitory activity when 1 nM of peptide was added were designated 1-a, peptides that exhibited 50% or more inhibitory activity when 10 nM of peptide was added were designated 1-b, peptides that exhibited 50% or more inhibitory activity when 100 nM of peptide was added were designated 1-c, and peptides that exhibited 1% or more but less than 50% inhibitory activity when 100 nM of peptide was added were designated 1-d.

[0378] The results are shown in Table 10. The synthesized peptides were shown to have Wnt3a inhibitory activity.

[0379]

[0380] Example 11 Evaluation of Wnt signal activation ability

[0381] To evaluate the Wnt signal activation ability of the synthesized peptide complexes (dimer structure numbers 1 to 67), transcriptional activity was assessed using a TCF / LEF reporter.

[0382] TCF / LEF Reporter-HEK 293 cells (BPS Bioscience) were cultured in EMEM (Fuji FILM) containing 10% FBS (Thermo Fisher Scientific), 50 μg / mL Gentamicin (Nacalai tesque), and 400 μg / mL Geneticin (Thermo Fisher Scientific).

[0383] The cells were detached using TrypLE (Thermo Fisher Scientific) and then suspended in EMEM containing 10% FBS, 50 μg / mL Gentamicin, and 10 mM LiCl (Sigma).

[0384] The cells were seeded into a white 96-well plate for cell adhesion and luminescence detection (Thermo Fisher Scientific) at 35,000 cells per well and cultured overnight.

[0385] Then, Recombinant Human Wnt-3a Protein (R&D systems) or peptide complex was added in the presence of 1 nM Recombinant Human R-spondin 1 (R&D systems), and the cells were incubated for 37 ｏ C's CO ２ The cells were stimulated in the incubator for 5 hours and 30 minutes.

[0386] ONE-Glo Luciferase Assay System (Promega) was added and the mixture was shaken for 4 minutes, and the luminescent signal was detected using a SpectraMax Paradigm multimode microplate reader (Molecular Devices).

[0387] The resulting signals were analyzed using GraphPad Prism, and the % activity was calculated by setting the maximum signal induced by Wnt-3a as 100% and the unstimulated signal as 0%. Wnt concentrations were evaluated at 0.03 to 30 nM, and the 100% value was determined.

[0388] For each dimer structure number, tests were performed at the following concentrations of peptide complex added: a) For dimer structure number 1, 1 nM, 10 nM, 100 nM, 1000 nM b) For dimer structure numbers 2 to 4, 1 nM, 10 nM, 100 nM c) For dimer structure numbers 5 to 14, 0.1 nM, 1 nM, 10 nM, 100 nM d) For dimer structure numbers 15 to 43, 0.1 nM, 1 nM, 10 nM e) For dimer structure numbers 44 to 56, 6 concentrations (0.03 nM, 0.12 nM, 0.47 nM, 1.9 nM, 7.5 nM, 30 nM) were added at a common ratio of 1 / 4 from 30 nM for dimer structure numbers 44 to 56. For peptides SEQ ID NOs: 57 to 67, six points were chosen from 30 nM at a common ratio of 1 / 5 (0.0096, 0.048, 0.24, 1.2, 6.0, 30 nM).

[0389] Regarding % activity, those that showed 50% or more activity when 0.1 nM of the peptide complex was added were designated 2-A, those that showed 50% or more activity when 1 nM of the peptide complex was added were designated 2-B, those that showed 50% or more activity when 10 nM of the peptide complex was added were designated 2-C, those that showed 50% or more activity when 100 nM of the peptide complex was added were designated 2-D, and those that showed 1% or more but less than 50% activity when 100 nM of the peptide complex was added were designated 2-E.

[0390] The peptide complex that exhibited 50% or more activity when 0.03 nM of the peptide complex was added was designated 3-A, the peptide complex that exhibited 50% or more activity when 0.12 nM of the peptide complex was added was designated 3-B, the peptide complex that exhibited 50% or more activity when 0.47 nM of the peptide complex was added was designated 3-C, the peptide complex that exhibited 50% or more activity when 1.9 nM of the peptide complex was added was designated 3-D, the peptide complex that exhibited 50% or more activity when 7.5 nM of the peptide complex was added was designated 3-E, the peptide complex that exhibited 50% or more activity when 30 nM of the peptide complex was added was designated 3-F, and the peptide complex that exhibited 1% or more but less than 50% activity when 30 nM of the peptide complex was added was designated 3-G.

[0391] The peptide complex that exhibited 50% or more activity when 0.0096 nM of the peptide complex was added was designated 4-A, the peptide complex that exhibited 50% or more activity when 0.048 nM of the peptide complex was added was designated 4-B, the peptide complex that exhibited 50% or more activity when 0.24 nM of the peptide complex was added was designated 4-C, the peptide complex that exhibited 50% or more activity when 1.2 nM of the peptide complex was added was designated 4-D, the peptide complex that exhibited 50% or more activity when 6 nM of the peptide complex was added was designated 4-E, the peptide complex that exhibited 50% or more activity when 30 nM of the peptide complex was added was designated 4-F, and the peptide complex that exhibited 1% or more but less than 50% activity when 30 nM of the peptide complex was added was designated 4-G.

[0392] The results are shown in Table 11. The synthesized peptide complex was shown to have the ability to activate Wnt signaling, similar to the recombinant protein Wnt3a.

[0393]

[0394]

[0395]

[0396] The peptide complex of the present invention binds to the Frizzled receptor and co-receptor LRP5 and / or LRP6 to activate the Wnt signaling pathway. Therefore, the peptide complex of the present invention is useful for producing various organoids such as the small intestine, large intestine, stomach, pancreas, and lung. In addition, the peptide of the present invention binds to LRP5 and / or LRP6 and has Wnt inhibitory activity. Therefore, the peptide of the present invention can be used as a drug that inhibits the Wnt signaling pathway.

Claims

1. A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added or inserted in the amino acid sequence represented by formula A1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein X1 is any D-amino acid residue, X2 and X3 are each independently any amino acid residue, X4 and X5 are each independently an amino acid residue having an optionally substituted aliphatic hydrocarbon group at the side chain, X6 is an amino acid residue having an optionally substituted aryl group at the side chain, X7 is an N-alkylamino acid residue having an optionally substituted aryl group or an optionally substituted cycloalkyl group at the side chain, X8 is any amino acid residue, X9 is an L-threonine (T) residue, X10 is any N-alkylated amino acid residue, X11 is any amino acid residue, and X12 is an amino acid residue having an aliphatic hydrocarbon group at the side chain, X13 is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, X14 is an L-cysteine ​​(C) residue, and in formula A1, the amino acid sequence is written from the N-terminus to the C-terminus.

2. In the formula A1, X1 is a D-phenylalanine (df) residue, a (R)-2-amino-3-(pyridin-4-yl)propanoic acid (d4py) residue, a (R)-2-amino-3-(pyridin-3-yl)propanoic acid (d3py) residue, a (R)-2-amino-3-(3-methoxyphenyl)propanoic acid (df3OMe) residue, a (R)-2-amino-3-(4-methoxyphenyl)propanoic acid (df4OMe) residue, a (R)-2-amino-5-ureidopentanoic acid (dicit) residue, a D-tyrosine (dy) residue, or a (R)-2-amino-3-(4-fluorophenyl)propanoic acid (df4F) residue; X2 is an L-serine (S) residue, an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-2-aminoadipic acid (Hgl) residue, an L-citrulline (Cit) residue or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, X3 is an L-aspartic acid (D) residue, an L-arginine (R) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, a (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, an L-asparagine (N) residue or an L-serine (S) residue, X4 is an L-isoleucine (I) residue or an O-methyl-L-threonine (TMe) residue, X5 is an L-arginine (R) residue, an L-glutamic acid (E) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, an L-serine (S) residue, an L-homoserine (Hse) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue, or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue; X6 is an L-tyrosine (Y) residue, a methyl-L-tyrosine (MeY) residue, an (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, an (S)-2-amino-3-(4-chlorophenyl)propanoic acid (F4C) residue, an (S)-2-amino-3-(pyridin-4-yl)propanoic acid (4Py) residue or an (S)-3-(2-amino-2-carboxyethyl)benzoic acid (F3COO) residue;X7 is a methyl-L-phenylalanine (MeF) residue, an (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, or an (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue; X8 is an L-glutamine (Q) residue, an L-valine (V) residue, an L-2-aminoadipic acid (Hgl) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-aminoheptanoic acid (Ahp) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an allothreonine (alT) residue, an L-citrulline (Cit) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl) -L-lysine (KCOpipzaa) residue, (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (Gthp) residue, (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (G4pipaa) residue, (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue, (S)-2-amino-2-cyclohexylacetic acid (Chg) residue or O-methyl-threonine (TMe) residue, X10 is a methyl-L-phenylalanine (MeF) residue, a (S)-2-(methylamino)hexanoic acid (MeNle) residue, a (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid (Me3Py) residue, a (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue or a (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (MeAtp) residue; X11 is an L-isoleucine (I) residue, an L-tyrosine (Y) residue, an (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid (F4aao) residue, or an O-methyl-L-threonine (TMe) residue;2. The peptide according to claim 1, wherein X12 is an L-valine (V) residue, a methyl-L-valine (MeV) residue or an N-methyl-L-cyclohexylglycine (MeChg) residue, and X13 is a glycine (G) residue, a D-alanine (da) residue or a D-serine (ds) residue.

3. A peptide comprising an amino acid sequence represented by formula A2, or an amino acid sequence in which at least one amino acid residue has been substituted, deleted, added or inserted among 1 to 12 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 10th, 11th, 12th and 13th amino acid residues in the amino acid sequence represented by formula A2. A2: df-S-D-I-R-Y-MeF-Q-T-MeF-I-V-G-C (SEQ ID NO: 164) where, df is a D-phenylalanine residue, S is an L-serine residue, D is an L-aspartic acid residue, I is an L-isoleucine residue, R is an L-arginine residue, Y is an L-tyrosine residue, MeF is a methyl-L-phenylalanine residue, Q is an L-glutamine residue, T is an L-threonine residue, V is an L-valine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

4. A peptide according to any one of claims 1 to 3, further comprising a glycine residue at the C-terminus.

5. A peptide according to any one of claims 1 to 3, which is a cyclic peptide.

6. The peptide of claim 4, which is a cyclic peptide.

7. The peptide according to any one of claims 1 to 3, which has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are linked together.

8. A peptide according to any one of claims 1 to 3, further comprising additional amino acid residues.

9. A peptide according to any one of claims 1 to 3, which has binding activity to an Fzd receptor.

10. The peptide according to claim 9, which has the ability to bind to at least one selected from the group consisting of Fzd receptor 2, Fzd receptor 7 and Fzd receptor 8.

11. A peptide according to any one of claims 1 to 3, consisting of the amino acid sequence represented by formula A3. A3: ClAc-X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14, wherein ClAc is a chloroacetyl group, and X1, X2, X3, X4, X5, X6, X7, X8, X9, X10, X11, X12, X13, and X14 are amino acid residues shown in Table 1, in which df is a D-phenylalanine residue, d4py is a (R)-2-amino-3-(pyridin-4-yl)propanoic acid residue, d3py is a (R)-2-amino-3-(pyridin-3-yl)propanoic acid residue, and df3OMe is a (R)-2-amino-3-(3-methoxyphenyl)propanoic acid residue, df4OMe is the (R)-2-amino-3-(4-methoxyphenyl)propanoic acid residue, dcit is the (R)-2-amino-5-ureidopentanoic acid residue, dy is the D-tyrosine residue, df4F is the (R)-2-amino-3-(4-fluorophenyl)propanoic acid residue, S is the L-serine residue, N is the L-asparagine residue, D is the L-aspartic acid residue, Hgl is the L-2-aminoadipic acid residue, Cit is the L-citrulline residue, Kmor is the (S)-2-amino-6-morpholinohexanoic acid residue, R is the L-arginine residue, KCOpipzaa is the N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, Atp is a (2S)-2-amino-3-(oxan-4-yl)propanoic acid residue, I is an L-isoleucine residue, TMe is an O-methyl-L-threonine residue, E is an L-glutamic acid residue, Ahp is an (S)-2-aminoheptanoic acid residue, Hse is an L-homoserine residue, Hcit is an N6-carbamoyl-L-lysine residue, Ncit is an (S)-2-amino-4-ureidobutanoic acid residue, OrnAc is an (S)-5-acetamido-2-aminopentanoic acid residue, Y is an L-tyrosine residue, MeY is a methyl-L-tyrosine residue,F4F is a (S)-2-amino-3-(4-fluorophenyl)propanoic acid residue, F4C is a (S)-2-amino-3-(4-chlorophenyl)propanoic acid residue, 4Py is a (S)-2-amino-3-(pyridin-4-yl)propanoic acid residue, F3COO is a (S)-3-(2-amino-2-carboxyethyl)benzoic acid residue, MeF is a methyl-L-phenylalanine residue, Me3Py is a (S)-2-(methylamino)-3-(pyridin-3-yl)propanoic acid residue, Me4Py is a (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid residue, MeF3COO is a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF4COO is (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeAtp is (S)-2-(methylamino)-3-(tetrahydro-2H-pyran-4-yl)propanoic acid residue, Q is L-glutamine residue, V is L-valine residue, W1aa is 1-(carboxymethyl)-L-tryptophan residue, Hty is homo-L-tyrosine residue, T is L-threonine residue, alT is allothreonine residue, Gthp is (S)-2-amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid residue, G4pipaa is (S)-2-amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid residue, Cbg is the (S)-2-amino-2-cyclobutylacetic acid residue, Chg is the (S)-2-amino-2-cyclohexylacetic acid residue, MeNle is the (S)-2-(methylamino)hexanoic acid residue, F4aao is the (S)-2-amino-3-(4-(carboxymethoxy)phenyl)propanoic acid residue, MeV is the methyl-L-valine residue, MeChg is the N-methyl-L-cyclohexylglycine residue, G is a glycine residue, and C is an L-cysteine ​​residue.

12. A peptide comprising an amino acid sequence represented by formula B1, or an amino acid sequence in which one or more amino acid residues have been substituted, deleted, added or inserted in the amino acid sequence represented by formula B1. B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14, wherein Y1 is an optionally substituted L-phenylalanine (F) residue, Y2 is an optionally substituted L-arginine (R) residue, Y3 is an optionally substituted L-tryptophan (W) residue or an optionally substituted (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, Y4 is an acidic amino acid residue or a neutral amino acid residue, Y5 is an optionally substituted aromatic amino acid residue, aliphatic amino acid residue or L-proline (P) residue, Y6 is an optionally substituted aliphatic amino acid residue, Y7 is an N-alkylated aromatic amino acid residue, and Y8 is an amino acid residue derived from an amino acid having a hydroxy group, a carboxy group or an amide group in the side chain, Y9 is any amino acid residue, Y10 is an optionally substituted L-tryptophan (W) residue, Y11 is an optionally substituted L-asparagine (N) residue or glutamine (Q) residue, Y12 is an optionally substituted (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, Y13 is an optionally substituted neutral amino acid residue or acidic amino acid residue, and Y14 is an L-cysteine ​​(C) residue. In formula B1, the amino acid sequence is written from the N-terminus to the C-terminus.

13. In the formula B1, Y1 is an L-phenylalanine (F) residue, an (S)-2-amino-3-(4-fluorophenyl)propanoic acid (F4F) residue, an (S)-2-amino-3-(p-toluyl)propanoic acid (F4Me) residue or an (S)-2-amino-3-(m-toluyl)propanoic acid (F3Me) residue, Y2 is an L-arginine (R) residue or an N6-carbamoyl-L-lysine (Hcit) residue, Y3 is an L-tryptophan (W) residue, a methyl-L-tryptophan (MeW) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid (W5N) residue, an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3 (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue, (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue, (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue or (S)-2-amino-3-(quinolin-4-yl)propanoic acid (Nal14N) residue; Y4 is an L-aspartic acid (D) residue, an L-glutamic acid (E) residue, an L-asparagine (N) residue or an L-glutamine (Q) residue; Y5 is an L-valine (V) residue, a methyl-L-valine (MeV) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, an (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, an (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (Nal1) residue, an (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid (3Py5Ph) residue, an (S)-2-amino-2-cyclobutylacetic acid (Cbg) residue or an L-proline (P) residue; Y6 is an L-arginine (R) residue, a methyl-L-arginine (MeR) residue, an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, an L-citrulline (Cit) residue, an N6-carbamoyl-L-lysine (Hcit) residue, an (S)-2-amino-4-ureidobutanoic acid (Ncit) residue or an (S)-5-acetamido-2-aminopentanoic acid (OrnAc) residue;Y7 is a methyl-L-phenylalanine (MeF) residue, a (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF4COO) residue, a methyl-L-tyrosine (MeY) residue, a (S)-2-(methylamino)-3-(p-toluyl)propanoic acid (MeF4Me) residue, a (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid (MeF4C) residue, a (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, a (S)-2-(methylamino)-3-(m-toluyl)propanoic acid (MeF3Me) residue, or a (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid (MeF3C) residue; Y8 is an L-serine (S) residue, a 1-(carboxymethyl)-L-tryptophan (W1aa) residue, a homo-L-tyrosine (Hty) residue, an allothreonine (alT) residue, an L-threonine (T) residue, an L-asparagine (N) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue or an L-glutamic acid (E) residue; Y9 is an L-asparagine (N) residue, a methyl-L-asparagine (MeN) residue, an L-2-aminoadipic acid (Hgl) residue, an (S)-2-amino-3-cyclohexylpropanoic acid (Cha) residue, a homo-L-tyrosine (Hty) residue, an (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid (Hw7N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue, an L-glutamine (Q) residue, an L-aspartic acid (D) residue or an L-glutamic acid (E) residue; Y10 is an L-tryptophan (W) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, an (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid (W6N) residue, an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W1Me7N) residue or an (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N5Me) residue;Y11 is an L-asparagine (N) residue, an L-glutamine (Q) residue, or a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (Nmm) residue; Y12 is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, an (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic6C) residue, an (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7H) residue, or an (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic7C) residue; 13. The peptide according to claim 12, wherein Y13 is an L-asparagine (N) residue, an L-aspartic acid (D) residue, an L-glutamine (Q) residue, a (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid (Nmm) residue, or an N4,N4-dimethyl-L-asparagine (Ndm) residue.

14. A peptide comprising an amino acid sequence represented by formula B2, or an amino acid sequence in which at least one amino acid residue has been substituted, added, deleted or inserted among 1 to 13 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th and 13th amino acid residues in the amino acid sequence represented by formula B2. B2: F-R-W-D-V-R-MeF-S-N-W-N-Tic-N-C (SEQ ID NO: 165) where F is an L-phenylalanine residue, R is an L-arginine residue, W is an L-tryptophan residue, D is an L-aspartic acid residue, V is an L-valine residue, MeF is a methyl-L-phenylalanine residue, S is an L-serine residue, Tic is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, N is an L-asparagine residue, and C is an L-cysteine ​​residue.

15. A peptide according to any one of claims 12 to 14, further comprising a glycine residue at the C-terminus.

16. The peptide according to any one of claims 12 to 14, which is a cyclic peptide.

17. The peptide of claim 15, which is a cyclic peptide.

18. The peptide according to any one of claims 12 to 14, which has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and an L-cysteine ​​residue contained in the peptide are linked together.

19. The peptide according to any one of claims 12 to 14, further comprising additional amino acid residues.

20. A peptide according to any one of claims 12 to 14, having binding activity to LRP.

21. The peptide according to claim 20, having the ability to bind to at least one selected from the group consisting of LRP5 and LRP6.

22. A peptide according to any one of claims 12 to 14, having DKK1-like activity.

23. A peptide according to any one of claims 12 to 14, consisting of the amino acid sequence represented by formula B3. B3: ClAc-Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14, wherein ClAc is a chloroacetyl group, and Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, and Y14 are amino acid residues shown in Table 2, in which F is an L-phenylalanine residue, F4F is an (S)-2-amino-3-(4-fluorophenyl)propanoic acid residue, F4Me is an (S)-2-amino-3-(p-toluoyl)propanoic acid residue, F3Me is an (S)-2-amino-3-(m-toluoyl)propanoic acid residue, R is an L-arginine residue, Hcit is an N6-carbamoyl-L-lysine residue, W is an L-tryptophan residue, MeW is a methyl-L-tryptophan residue, W7N is an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, W5N is an (S)-2-amino-3-(1H-pyrrolo[3,2-c]pyridin-3-yl)propanoic acid residue, W1Me7N is an (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, W7N5Me is an (S)-2-amino-3-(5-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid residue, Nal1 is (S)-2-amino-3-(naphthalen-1-yl)propanoic acid residue, Nal14N is (S)-2-amino-3-(quinolin-4-yl)propanoic acid residue, D is L-aspartic acid residue, E is L-glutamic acid residue, N is L-asparagine residue, Q is L-glutamine residue, V is L-valine residue, MeV is methyl-L-valine residue, W1aa is 1-(carboxymethyl)-L-tryptophan residue, Cha is (S)-2-amino-3-cyclohexylpropanoic acid residue, 3Py5Ph is (S)-2-amino-3-(5-phenylpyridin-3-yl)propanoic acid residue,Cbg is an (S)-2-amino-2-cyclobutylacetic acid residue, P is an L-proline residue, MeR is a methyl-L-arginine residue, Kmor is an (S)-2-amino-6-morpholinohexanoic acid residue, Cit is an L-citrulline residue, Ncit is an (S)-2-amino-4-ureidobutanoic acid residue, OrnAc is an (S)-5-acetamido-2-aminopentanoic acid residue, MeF is a methyl-L-phenylalanine residue, MeF4COO is an (S)-4-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeY is a methyl-L-tyrosine residue, and MeF4Me is an (S)-2-(methylamino)-3-(p-toluyl)propanoic acid residue. MeF4C is (S)-3-(4-chlorophenyl)-2-(methylamino)propanoic acid residue, MeF3COO is (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid residue, MeF3Me is (S)-2-(methylamino)-3-(m-toluyl)propanoic acid residue, MeF3C is (S)-3-(3-chlorophenyl)-2-(methylamino)propanoic acid residue, S is L-serine residue, Hty is homo-L-tyrosine residue, alT is allosethreonine residue, T is L-threonine residue, MeN is methyl-L-asparagine residue, Hgl is L-2-aminoadipic acid residue, Hw7N is the (S)-2-amino-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)butanoic acid residue, KCOpipzaa is the N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine residue, W6N is the (S)-2-amino-3-(1H-pyrrolo[2,3-c]pyridin-3-yl)propanoic acid residue, Nmm is the (2S)-2-amino-4-(methylamino)-4-oxobutanoic acid residue, Tic is the (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic6C is the (S)-6-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue,Tic7H is the (S)-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Tic7C is the (S)-7-chloro-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid residue, Ndm is the N4,N4-dimethyl-L-asparagine residue, C is the L-cysteine ​​residue, and G is a glycine residue.

24. A peptide complex comprising a first peptide and a second peptide, wherein the first peptide is a peptide according to claim 1, and the second peptide is a peptide according to claim 12.

25. The peptide conjugate of claim 24, wherein the peptide conjugate consists of the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

26. The peptide conjugate of claim 25, wherein the C-terminus of the first peptide and the C-terminus of the second peptide are linked via the linker.

27. The peptide conjugate of claim 25, wherein the linker is selected from those listed in Table 3, where -click- represents a bond formed by click chemistry.

28. The peptide complex according to claim 25, wherein the first peptide is a peptide consisting of an amino acid sequence represented by formula A1, and the second peptide is a peptide consisting of an amino acid sequence represented by formula B1. A1: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13-X14 B1: Y1-Y2-Y3-Y4-Y5-Y6-Y7-Y8-Y9-Y10-Y11-Y12-Y13-Y14 With the proviso that, in formula A1, X1 is a D-phenylalanine (df) residue, X2 is an L-serine (S) residue or an L-2-aminoadipic acid (Hgl) residue, X3 is an L-aspartic acid (D) residue, an L-citrulline (Cit) residue or an (2S)-2-amino-3-(oxan-4-yl)propanoic acid (Atp) residue, and X4 is an L-isoleucine (I) residue, X5 is an L-arginine (R) residue or an L-citrulline (Cit) residue, X6 is an L-tyrosine (Y) residue, X7 is a methyl-L-phenylalanine (MeF) residue or an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, X8 is an L-glutamine (Q) residue, an L-valine (V) residue, a homo-L-tyrosine (Hty) residue, an L-threonine (T) residue, an L-citrulline (Cit) residue or an O-methyl-L-threonine (TMe) residue, X10 is a methyl-L-phenylalanine (MeF) residue or an (S)-2-(methylamino)-3-(pyridin-4-yl)propanoic acid (Me4Py) residue, X11 is an L-isoleucine (I) residue or an L-tyrosine (Y) residue, X12 is an L-valine (V) residue or a methyl-L-valine (MeV) residue, X13 is a glycine (G) residue or a D-serine (ds) residue, and in formula B1, Y1 is an L-phenylalanine (F) residue, Y2 is an L-arginine (R) residue, and Y3 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,Y4 is an L-aspartic acid (D) residue, Y5 is an L-valine (V) residue, Y6 is an L-arginine (R) residue or an (S)-2-amino-6-morpholinohexanoic acid (Kmor) residue, Y7 is a methyl-L-phenylalanine (MeF) residue or an (S)-3-(2-carboxy-2-(methylamino)ethyl)benzoic acid (MeF3COO) residue, Y8 is an L-serine (S) residue or an L-glutamic acid (E) residue, Y9 is an L-asparagine (N) residue, an N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (KCOpipzaa) residue or an L-glutamic acid (E) residue, Y10 is an L-tryptophan (W) residue or an (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (W7N) residue, Y11 is an L-asparagine (N) residue, Y12 is an (S)-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid (Tic) residue, and Y13 is an L-asparagine (N) residue or an L-aspartic acid (D) residue.

29. The peptide complex of claim 25, which activates Wnt signaling.

30. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 3 and 12 to 14.

31. A pharmaceutical composition comprising the peptide conjugate of claim 25.

32. A composition for cell culture, comprising the peptide complex according to claim 25, for use in cell culture.

33. A composition comprising the peptide conjugate of claim 25 for medical, diagnostic or research use.

34. A composition comprising a peptide according to any one of claims 1 to 3 and 12 to 14 for medical, diagnostic or research use.