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

Abstract

[Problem] To provide a peptide having TPO receptor binding ability, a peptide complex having intracellular signal activation ability, or a peptide complex having TPO / TPOR signal activation ability. [Solution] Provided are: a peptide comprising an amino acid sequence represented by a formula A1, or an amino acid sequence in which, in the amino acid sequence represented by a formula A1, a plurality of amino acid residues are substituted, deleted, added or inserted; a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof; a peptide complex having the peptide and having intracellular signal activation ability; or a peptide complex having TPO / TPOR signal activation ability. Formula A1: X1-X2-X3-MeG-X4-X5-X6-X7-df-X8-X9-X10-X11-X12-MeC.

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.

[0002] Thrombopoietin (TPO) is a cytokine primarily produced in the liver and kidney. Signal transduction by TPO via its TPO receptor (TPOR or c-MPL) plays an important role in regulating megakaryocytopoiesis and platelet production, as well as in the maintenance and self-renewal of hematopoietic stem cells. Therefore, research is being conducted to discover TPO substitutes or TPOR agonists that activate the TPO / TPOR signaling pathway, with the aim of treating hematologic disorders involving megakaryocytopoiesis and platelet production. Recombinant TPO has been proven effective in treating thrombocytopenia, but side effects such as thrombocytopenia and pancytopenia due to the production of neutralizing antibodies against TPO have been reported. In recent years, TPOR agonists, primarily consisting of low-molecular-weight compounds, have been reported to be effective in treating thrombocytopenia (Non-Patent Document 1, Non-Patent Document 2). Some of these drugs have been approved as therapeutic agents for immune thrombocytopenia, thrombocytopenia due to hepatitis C, bone marrow failure, etc.; however, side effects such as thrombocytosis, thrombosis, bone marrow reticular fibrosis, and recurrence of thrombocytopenia have also been reported (Non-Patent Document 3). In addition to therapeutic methods in which TPOR agonists are directly administered to patients, therapeutic methods have been investigated that efficiently induce differentiation of iPS cells ex vivo into megakaryocytes and platelets to stably supply platelets. In this case, the use of low-molecular-weight compounds that can substitute for TPO has been reported to be effective (Patent Document 1). It has been reported that upon binding to TPOR, TPO dimerizes TPOR, transduces signals into cells, and exerts physiological effects (Non-Patent Documents 4 and 5). Recently, TPO agonist antibodies that utilize these properties of TPOR have been reported (Non-Patent Document 6, Patent Document 2, Patent Document 3).

[0003] WO2013 / 051625 Pamphlet WO2002 / 033072 Pamphlet Japanese Patent No. 4708190

[0004] Blood 2006, 107, 4300. Experimental Hematology 2005, 33, 85. Drug 2021, 81, 1285. Science 2020, 367, 643. EMBO J. 2011, 30, 4398. PNAS 2021, 118, e2017849118.

[0005] An objective of the present invention is to provide a peptide capable of binding to a TPO receptor, a peptide complex capable of activating an intracellular signal, or a peptide complex capable of activating a TPO / TPOR signal.

[0006] As a result of intensive research aimed at solving the above problems, the present inventors have discovered that a specific peptide has the ability to bind to a TPO receptor, that a peptide complex containing a specific peptide has the ability to activate an intracellular signal, and that a peptide complex containing a specific peptide has the ability to activate a TPO / TPOR signal, and have thus completed the present invention. That is, aspects of the present invention are as follows:

[0007] [1] A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which a plurality of amino acid residues in the amino acid sequence represented by formula A1 have been substituted, deleted, added, or inserted, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. １ -X ２ -X ３ -MeG-X ４ -X ５ -X ６ -X ７ -df-X ８ -X ９ -X １０ -X １１ -X １２ -MeC where X １ is any amino acid residue, and X ２ is an amino acid residue having an optionally substituted aryl group in the side chain, ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain,５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ９ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or an optionally substituted 4- to 6-membered cyclic secondary amino acid residue, １０ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １１ is any amino acid residue, and X １２ is any amino acid residue, and in Formula A1, the amino acid sequence is written from the N-terminal side to the C-terminal side. [2] In Formula A1, X １ is F, A, Q, E, F4COO, F3Me, F3C, F4Me, F4C, F4OMe, 3Py, Cha, or F4aao; ２ is W, W7N, W1Me7N, or W1aa, and X ３ is V, R, Cit, T, alT, or S; X ４ is W, W1Me, or Na11, and X ５ is V, T, alT, or dMeS; X ６ is V, I, alI, Eva, Tbg, or Gcpe; ７ is MeG, MeA, Meda, MeKCOpipzaa, dp, EtG, or EtA; ８ is R, Q, or KCOpipzaa; X ９ is V, T, a1T, P, Hpr, Hyp, or Mor; X １０ is I, V, Gthp, or G4pipaa, and X １１ is S, E, I, F, W, Y, R, Cit, Atp, KCOpipzaa, or Hgl; X １２is D, H, R, S, Q, SMe, Cit, E, or Hgl. [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 in an amino acid sequence consisting of 1 to 12 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 11th, 12th, 13th, and 14th amino acid residues in the amino acid sequence represented by formula A2, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. A2: F-W-V-MeG-W-V-V-MeG-df-R-V-I-S-D-MeC (SEQ ID NO: 1) [4] In formula A1, X １ is an amino acid residue having an optionally substituted aryl group in the side chain, ２ is an amino acid residue having an optionally substituted aryl group in the side chain, ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ９ is an amino acid residue having an aliphatic hydrocarbon group in the side chain or a 4- to 6-membered cyclic secondary amino acid residue, １０ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, １１ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １２[5] The peptide according to [1], a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein, in formula A1, X is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain. １ is F or F4Me, and X ２ is W or W7N, X ３ are V, T, and a1T, and X ４ is W or Na11, X ５ is V, and X ６ is V, I, alI, or Gcpe, and X ７ is MeG, MeA, Meda, MeKCOpipzaa, or dp; X ８ is R, and X ９ is V or P, and X １０ is I or V, and X １１ is S, KCOpipzaa, or Hgl, and X １２ is D. The peptide according to [4], a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. [6] The peptide according to any one of [1] to [5], which is a cyclic peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. [7] The peptide according to any one of [1] to [5], which has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bonded to MeC, which is the 15th amino acid residue in Formula A1 or Formula A2 contained in the peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. [8] The peptide according to any one of [1] to [5], which further contains an additional amino acid residue, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. [9] The peptide according to [4] or [5], which has TPO receptor binding ability, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[10] A peptide comprising an amino acid sequence represented by formula B1, or an amino acid sequence in which a plurality of amino acid residues in the amino acid sequence represented by formula B1 have been substituted, deleted, added, or inserted, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. １ -Y ２ -Y３ -MeG-Y ４ -Y ５ -Y ６ -Y ７ -df-R-Y ８ -Y ９ -Y １０ -D-MeC where Y １ is any amino acid residue, and Y ２ is any amino acid residue, Y ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; Y ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or a 4- to 6-membered cyclic secondary amino acid residue, ９ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １０ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, and in Formula B1, the amino acid sequence is written from the N-terminal side to the C-terminal side.

[11] In Formula B1, Y １ is F, F4aao, F3COO, F3aao, 4Py, 3Py, or Cha, and Y ２ is Ahp, W1Me7N, W, or W7N, and Y ３ is V, I, alI, Q, T, TMe, or KCOpipzaa; ４ is W, W1Me, Na1, or Na12, and Y ５ is V, Tbg, TMe, alTMe, dMeS, Gthp, G4pipaa, or Cit, and Y ６ is V, Ahp, Nle, I, L, Tbg, IMe, Cbg, Gcpe, Chg, or alTMe; ７is MeG, EtG, MeA, Meda, dp, MeQ, or Medq; Y ８ is V, P, Hpr, Tic, TMe, or alTMe; Y ９ is I, alI, alTMe, dMeS, Gthp, or G4pipaa; Y １０is S or Hgl.

[12] The peptide according to

[10] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, 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, deleted, added, or inserted in an amino acid sequence consisting of 1 to 10 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 11th, 12th, and 13th amino acid residues in the amino acid sequence represented by formula B2, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. B2: F-Ahp-V-MeG-W-V-V-MeG-df-R-V-I-S-D-MeC (SEQ ID NO: 97)

[13] The peptide according to any one of

[10] to

[12] , which is a cyclic peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[14] The peptide according to any one of

[10] to

[12] , having a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid and MeC, which is the 15th amino acid residue in Formula B1 or B2 contained in the peptide, are bonded, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[15] The peptide according to any one of

[10] to

[12] , further comprising an additional amino acid residue, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[16] The peptide according to any one of

[10] to

[12] , having TPO receptor binding ability, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[17] A peptide complex comprising a first peptide and a second peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide may be the same or different and are the peptide according to [1] or

[10] .

[18] The peptide complex according to

[17] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the peptide complex comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide, and the second peptide may be the same as or different from the first peptide and has an amino acid sequence represented by Formula A1, or an amino acid sequence in which a plurality of amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by Formula A1.

[19] The peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the homology between the first peptide and the second peptide is 90% or more and 100% or less.

[20] The peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide are substantially the same peptide.

[21] The peptide conjugate according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide are each cyclic peptides.

[22] The peptide conjugate according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide each have a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bound to MeC contained in the first peptide or the second peptide.

[23] The peptide conjugate according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide further comprise an additional amino acid residue.

[24] The peptide conjugate according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the C-terminus of the first peptide and the C-terminus of the second peptide are linked via a linker.

[25] The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to

[18] , wherein the linker is a PEG linker or a linker consisting of PEG and an amino acid residue.

[26] The peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, which has the ability to activate an intracellular signal.

[27] A pharmaceutical composition comprising the peptide according to any one of [1] to [5] and

[10] to

[12] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[28] A pharmaceutical composition comprising the peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[29] A composition for cell culture, which comprises the peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and is used for cell culture.

[30] A composition, which comprises the peptide complex according to

[18] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, and is used for medical, diagnostic, or research purposes.

[31] A composition for medical, diagnostic, or research use, comprising the peptide according to any one of [1] to [5] and

[10] to

[12] , a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

[0008] The present invention provides a peptide capable of binding to a TPO receptor, a peptide complex capable of activating an intracellular signal, or a peptide complex capable of activating a TPO / TPOR signal.

[0009] 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.

[0010] [General Abbreviations] Å as Angstrom (unit); AcOH as acetic acid; BSA as bovine serum albumin; Boc as tert-butoxycarbonyl group; ClAc as chloroacetyl; ClAcOSu as (2,5-dioxopyrrolidin-1-yl) 2-chloroacetate (CAS number: 27243-15-8); cPEG1c as 3,3'-oxydipropanoic acid (CAS number: 5961-83-1); CV as column volume; DCM as dichloromethane or methylene chloride; DIPCI or DIC as N,N'-diisopropylcarbodiimide; DIPEA or DIEA as N,N-diisopropylethylamine; DMA as N,N-dimethylacetamide; Dmb as dimethoxybenzyl group; DMEM as Dulbecco's modified Eagle's medium; DMF as N,N-dimethylformamide; DMSO as dimethyl sulfoxide; DODT as 3,6-dioxa-1,8-octanedithiol; EC50 as 50% effective concentration; FBS as fetal bovine serum; Fmoc as 9-fluorenylmethyloxycarbonyl; N ２ , N ６Fmoc-Lys(Fmoc)-OH as -bis(((9H-fluoren-9-yl)methoxy)carbonyl)-L-lysine; g as grams; HATU as O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; HPLC as high performance liquid chromatography; LC-MS or LC / MS as liquid chromatography mass spectrometry; M as molar; MeCN as acetonitrile; mg as milligrams; min as minutes; mL as milliliters; mM as millimolar; mm as millimeters; Mpe group as O-3-methyl-pent-3-yl group; NHS as N-hydroxysuccinimide; NHS-cPEG1c-NHS (CAS Number: 65869-64-9) as bis(2,5-dioxopyrrolidin-1-yl) 3,3'-oxydipropionate; nm as nanometers (units); μL as microliters (units); OSu as succinimide; Pbf as 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group; PEG as polyethylene glycol; rpm as revolutions per minute (units); Sub as dibenzosuberyl group; tBu as tert-butyl group; TEAA as triethylamine acetate; TFA as trifluoroacetic acid; TIS as triisopropylsilane; Trt or Tr as trityl group; [Abbreviation (unnatural amino acid)] 3Py (S)-2-Amino-3-(pyridin-3-yl)propanoic acid (CAS number: 64090-98-8) 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) alI L-Alloisoleucine (CAS number: 1509-34-8) alT L-Allothreonine (CAS number: 28954-12-3) alTMe O-Methyl-L-Allothreonine (CAS number: 104195-80-4) Atp (S)-2-Amino-3-(tetrahydro-2H-pyran-4-yl)propanoic acid (CAS number: 1344910-91-3)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 (S)-2-Amino-5-ureidopentanoic acid (CAS number: 372-75-8) de D-Glutamic acid (CAS number: 6893-26-1) df D-Phenylalanine (CAS number: 673-06-3) dMeS (S)-2-Amino-3-hydroxy-3-methylbutanoic acid (CAS number: 2280-27-5) dp D-Proline (CAS No.: 344-25-2) EtA Ethyl-L-alanine (CAS No.: 65278-03-7) EtG Ethylglycine (CAS No.: 627-01-0) Eva (S)-2-Amino-3-ethylpentanoic acid (CAS No.: 14328-49-5) F3aao (S)-2-Amino-3-(3-(carboxymethoxy)phenyl)propanoic acid (CAS No.: 2973751-62-9) F3C (S)-2-Amino-3-(3-chlorophenyl)propanoic acid (CAS No.: 80126-51-8) F3COO (S)-3-(2-Amino-2-carboxyethyl)benzoic acid (CAS No.: 13861-02-4) F3Me (S)-2-Amino-3-(m-tolyl)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) F4COO (S)-4-(2-Amino-2-carboxyethyl)benzoic acid (CAS number: 126109-42-0) F4Me (S)-2-Amino-3-(p-tolyl)propanoic acid (CAS number: 1991-87-3) F4OMe (S)-2-Amino-3-(4-methoxyphenyl)propanoic acid (CAS number: (6230-11-1) G4pipaa (S)-2-Amino-2-(1-(carboxymethyl)piperidin-4-yl)acetic acid (CAS number: N / A) Gcpe (S)-2-Amino-2-cyclopentylacetic acid (CAS number: 2521-84-8)Gthp (S)-2-Amino-2-(tetrahydro-2H-pyran-4-yl)acetic acid (CAS number: 811842-25-8) Hgl (S)-2-Aminoadipic acid (CAS number: 1118-90-7) Hpr (S)-Piperidine-2-carboxylic acid (CAS number: 3105-95-1) Hyp (2S,4R)-4-Hydroxypyrrolidine-2-carboxylic acid (CAS number: 51-35-4) IMe (S)-2-Amino-3,3-dimethylpentanoic acid (CAS number: 173831-84-0) KCOpipzaa N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (CAS number: N / A) MeA Methyl-L-alanine (CAS No.: 3913-67-5) MeC Methyl-L-cysteine ​​(CAS No.: 4026-48-6) Meda Methyl-D-alanine (CAS No.: 29475-64-7) Medq Methyl-D-glutamine (CAS No.: 862504-01-6) MeG Methylglycine (CAS No.: 107-97-1) MeKCOpipzaa N6-(4-(carboxymethyl)piperazine-1-carbonyl)-N2-methyl-L-lysine (CAS No.: N / A) MeQ Methyl-L-glutamine (CAS No.: 300560-56-9) Mor (S)-Morpholine-3-carboxylic acid (CAS number: 106825-79-0) Nal1 (S)-2-amino-3-(naphthalen-1-yl)propanoic acid (CAS number: 55516-54-6) Nal2 (S)-2-amino-3-(naphthalen-2-yl)propanoic acid (CAS number: 58438-03-2) Nle (S)-2-aminohexanoic acid (CAS number: 327-57-1) PEG10c 1-amino-3,6,9,12,15,18,21,24,27,30-decaoxatritriacontan-33-oic acid (CAS number: 2170987-85-4) PEG12c 1-amino-3,6,9,12,15,18,21,24,27,30,33,36-dodecaoxanonatriacontan-39-oic acid (CAS number: 1415408-69-3) 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) 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) TMe O-methyl-L-threonine (CAS number: 4144-2-9) W1aa 1-(carboxymethyl)-L-tryptophan (CAS number: 773823-50-0) W1Me 1-Methyl-L-tryptophan (CAS number: 21339-55-9) W1Me7N (S)-2-amino-3-(1-methyl-1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 1632971-18-6) W7N (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 49758-35-2)

[0011] [TPO] Thrombopoietin (TPO) is a ligand for c-MPL, constitutively produced in the liver and other organs, circulates in the bloodstream, and is transported to the bone marrow, where it stimulates early development, such as megakaryocytopoiesis. Blood TPO concentration is regulated by the amount of c-MPL on platelets and megakaryocytes. In addition to regulation by the number of TPO molecules, including regulation of TPO mRNA deficiency in the bone marrow, megakaryocytopoiesis and platelet production may also be regulated by modulating TPO activity through a proteolytic process that generates truncated forms of the molecule (Stem Cells 1998, 16, 322). In one embodiment, the peptide complex of the present invention preferably has the ability to activate intracellular signals, more preferably the ability to activate TPO / TPOR signals, and therefore can be used as a substitute for naturally occurring TPO.

[0012] [TPO Receptor] The TPO receptor, also known as c-MPL, is a 635-amino acid protein encoded by the c-MPL gene. The TPO receptor has three functional domains, including an extracellular cytokine-binding domain, a transmembrane domain, and a cytoplasmic domain that binds to signaling molecules, including Janus kinases (JAKs) and signal transducers and activators of transcription (STATs).

[0013] [TPO / TPOR signal activation ability] TPO / TPOR signal activation ability refers to the ability to produce an effect similar to that produced by naturally occurring TPO. It also refers to the ability to specifically activate the TPO / TPOR signaling pathway. TPO / TPOR signal activation ability can be evaluated by known methods, for example, by the ability to bind to the TPO receptor and the ability to activate intracellular signaling molecules that receive signals from the TPO receptor.

[0014] [TPO receptor binding ability] TPO receptor binding ability refers to the ability to specifically bind to the TPO receptor. The binding ability to the TPO receptor can be evaluated by known methods, including, but not limited to, SPR or ELISA using a recombinant TPO receptor protein. In either evaluation system, a substance is said to have TPO receptor binding ability (or TPOR binding activity) when binding to the TPO receptor can be detected using a peptide or peptide complex at an optimal concentration under optimal conditions according to standard procedures.

[0015] [Intracellular signal activation ability] The intracellular signal activation ability can be evaluated by phosphorylation of intracellular signaling molecules such as ERK, AKT, and STAT, and further expression of downstream reporter genes. Examples of such assays include, but are not limited to, AlphaLISA SureFire Ultra p-Erk1 / 2 (Thr202 / Tyr204) Assay kit (PerkinElmer), AlphaLISA SureFire Ultra phosho-STAT5 (Tyr694 / 699) kit (PerkinElmer), and HEK-Blue. ＴＭ Examples of evaluation methods include those using TPO Cells (InvivoGen). Regardless of the evaluation system, if activation of intracellular signals can be detected using the peptide or peptide complex at an optimal concentration under optimal conditions according to standard procedures, the peptide is said to have the ability to activate intracellular signals.

[0016] [ERK activation] ERK activation is a type of intracellular signal activation and refers to the ability to phosphorylate ERK (extracellular signal-regulated kinase), an intracellular signaling molecule. ERK is a factor involved in the MAPK signaling pathway, and is activated by phosphorylation via MEK in response to signals from cell surface receptors. For example, the peptide complex of the present invention phosphorylates ERK.

[0017] [STAT5-Dependent Transcriptional Activation] STAT5-dependent transcriptional activation is a type of intracellular signal activation, and refers to the ability of STAT5 (signal transducer and activator of transcription), an intracellular signal transduction factor and transcriptional activator, to activate the transcription of a target gene. STAT5 is a factor involved in the JAK-STAT signaling pathway, and after being activated via JAK by a signal from a receptor on the cell surface, it translocates into the nucleus and functions as a transcriptional activator. For example, the peptide complex of the present invention activates the transcription of a STAT5-dependent reporter gene.

[0018] [TPO / TPOR Signaling Pathway] A signaling pathway generally refers to a biochemical causal relationship that is initiated by a protein-protein interaction, such as 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 a cell. The TPO / TPOR signaling pathway refers to the biochemical causal relationship that occurs when the growth factor is TPO. The TPO receptor, also known as c-MPL, belongs to the type 1 cytokine receptor family. Like the erythropoietin (EPO) and granulocyte colony-stimulating factor (GCSF) receptors, TPO binding induces a conformational change in the homodimeric receptor. This is followed by phosphorylation of the intracellular domain of c-MPL and various secondary signaling molecules. Signaling molecules activated by TPO include Janus kinases (Jaks), signal transducers and activators of transcription (STATs), phosphatidylinositol-3-kinase (PI3K) / Akt, and Ras / mitogen-activated protein kinase (MAPK). Activation of these signaling pathways leads to the induction of megakaryocytopoiesis and thrombopoiesis from hematopoietic stem cells.

[0019] [Conservative Amino Acid Substitution] In the peptides and peptide complexes of the present invention, when one, two or three amino acid residues are substituted, deleted, added or inserted from a specific amino acid sequence, conservative amino acid substitutions are preferably made.

[0020] A "conservative amino acid substitution" means a substitution with a functionally equivalent or similar amino acid.

[0021] 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 terms of structure and function.

[0022] 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.

[0023] Cysteine ​​residues can adopt an oxidized (disulfide) form which is less polar compared to the reduced (thiol) form.

[0024] The long aliphatic portion of the arginine side chain may constitute an important structural and functional feature.

[0025] Additionally, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) can contribute to ion-aromatic 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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:

[0030] 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).

[0031] Aliphatic branched-chain amino acids: Amino acids having branched fatty acids in the side chain, including, for example, L-valine (V), L-isoleucine (I), and L-leucine (L).

[0032] 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).

[0033] 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).

[0034] Hydrophilic amino acids can be further divided into the following groups:

[0035] Acidic Amino Acid: An amino acid whose side chain exhibits acidity, including, for example, L-aspartic acid (D) and L-glutamic acid (E).

[0036] Basic amino acids: Amino acids whose side chains exhibit basicity, including, for example, L-lysine (K), arginine (R), and L-histidine (H).

[0037] 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).

[0038] Glycine (G) and L-proline (P) can also be classified as "amino acids that influence the orientation of the main chain."

[0039] 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."

[0040] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Examples of unnatural amino acids include N-alkylamino acids obtained by N-alkylating the above-described natural amino acids, lower amino acids in which the nitrogen atom forming the peptide bond is branched or unbranched (e.g., C １ -C ５ Preferably, C １ -C ３ , more preferably C １ The N-alkylamino acid is preferably an N-ethyl amino acid, an N-butyl amino acid, or an N-methyl amino acid, and more preferably an N-methyl amino acid.

[0041] 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.

[0042] 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).

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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 and basic amino acid salts.

[0047] Examples of inorganic acid salts include, but are not limited to, hydrochlorides, hydrobromides, sulfates, nitrates, and phosphates.

[0048] Examples of organic acid salts include, but are not limited to, acetate, succinate, fumarate, maleate, tartrate, citrate, lactate, stearate, benzoate, methanesulfonate, and p-toluenesulfonate.

[0049] Examples of said inorganic base salts 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.

[0050] Examples of said organic base salts include, but are not limited to, diethylamine salts, diethanolamine salts, meglumine salts, and N,N'-dibenzylethylenediamine salts.

[0051] 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.

[0052] Examples of pharmaceutically acceptable esters include, but are not limited to, acetate and sulfate esters.

[0053] An example of a pharmaceutically acceptable solvate includes, but is not limited to, a hydrate.

[0054] [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 a plurality of amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by Formula A1. In Formula A1, the amino acid sequence is written from the N-terminus to the C-terminus. Examples of a plurality of amino acid residues include one, two, or three. When amino acid residues are substituted, it is preferable that an amino acid residue other than MeG at position 4, df at position 9, and MeC at position 15 is substituted. It is preferable that this peptide, for example, a homodimer peptide complex, exhibits intracellular signal activation ability.

[0055] A1:X １ -X ２ -X ３ -MeG-X ４ -X ５ -X ６ -X ７ -df-X ８ -X ９ -X １０ -X １１ -X １２ -MeC

[0056] However, X １ is any amino acid residue, and X ２ is an amino acid residue having an optionally substituted aryl group in the side chain, ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ９is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or an optionally substituted 4- to 6-membered cyclic secondary amino acid residue, １０ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １１ is any amino acid residue, and X １２ is any amino acid residue.

[0057] X １ is preferably F, A, Q, E, F4COO, F3Me, F3C, F4Me, F4C, F4OMe, 3Py, Cha, or F4aao. ２ is preferably W, W7N, W1Me7N, or W1aa. ３ is preferably V, R, Cit, T, a1T, or S. ４ is preferably W, W1Me, or Na11. ５ is preferably V, T, alT, or dMeS. ６ is preferably V, I, alI, Eva, Tbg, or Gcpe. ７ is preferably MeG, MeA, Meda, MeKCOpipzaa, dp, EtG, or EtA. ８ is preferably R, Q, or KCOpipzaa. ９ is preferably V, T, a1T, P, Hpr, Hyp, or Mor. １０ is preferably I, V, Gthp, or G4pipaa. １１ is preferably S, E, I, F, W, Y, R, Cit, Atp, KCOpipzaa, or Hgl. １２ is preferably D, H, R, S, Q, SMe, Cit, E, or Hgl.

[0058] The peptide of the first embodiment is preferably a peptide comprising an amino acid sequence represented by Formula A2, or an amino acid sequence comprising 1 to 12 amino acid residues selected from the group consisting of the first, second, third, fifth, sixth, seventh, eighth, tenth, eleventh, twelfth, thirteenth, and fourteenth amino acid residues in the amino acid sequence represented by Formula A2, in which at least one amino acid residue has been substituted, deleted, added, or inserted. In Formula A2, the amino acid sequence is written from the N-terminus to the C-terminus. Examples of "at least one" include one, two, or three. When an amino acid residue is substituted, it is preferable that an amino acid residue other than MeG at position 4, df at position 9, and MeC at position 15 is substituted.

[0059] A2: FW-V-MeG-W-V-V-MeG-df-R-V-I-SD-MeC (SEQ ID NO: 1)

[0060] In a peptide containing the amino acid sequence represented by Formula A1 or an amino acid sequence in which multiple amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by Formula A1, preferred amino acid residues are as follows. This peptide preferably has, for example, TPO receptor binding activity. Furthermore, this peptide preferably has, for example, a homodimeric peptide complex that exhibits intracellular signal activation ability.

[0061] X １ is preferably an amino acid residue having an optionally substituted aryl group in the side chain. ２ is preferably an amino acid residue having an optionally substituted aryl group in the side chain. ３ is preferably an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain. ４ is preferably an amino acid residue having an optionally substituted aryl group in the side chain. ５ is preferably an amino acid residue having an aliphatic hydrocarbon group in the side chain. ６ is preferably an amino acid residue having an aliphatic hydrocarbon group in the side chain. ７is preferably any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue. ８ is preferably an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain. ９ is preferably an amino acid residue having an aliphatic hydrocarbon group in the side chain, or a 4- to 6-membered cyclic secondary amino acid residue. １０ is preferably an amino acid residue having an aliphatic hydrocarbon group in the side chain. １１ is preferably an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain. １２ is preferably an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain.

[0062] X １ is preferably F or F4Me. ２ is preferably W or WN. ３ is preferably V, T, or alT. ４ is preferably W or Na11. ５ is preferably V. ６ is preferably V, I, alI, or Gcpe. ７ is preferably MeG, MeA, Meda, MeKCOpipzaa, or dp. ８ is preferably R. ９ is preferably V or P. １０ is preferably I or V. １１ is preferably S, KCOpipzaa, or Hgl. １２ is preferably D.

[0063] Any of the above peptides is preferably a cyclic peptide.

[0064] 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 is bonded to MeC, which is the 15th amino acid residue in Formula A1 or Formula A2 contained in the peptide. The amino acid residue derived from a chloroacetylated amino acid is preferably an N-terminal amino acid residue. The peptide having a cyclic structure is preferably one in which the chloroacetyl group has been removed.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 acid residues.

[0071] The peptide of the first embodiment preferably has TPO receptor binding ability.

[0072] 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 a plurality of 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. Examples of a plurality of amino acid residues include one, two, or three. When amino acid residues are substituted, it is preferable that an amino acid residue other than MeG at position 4, df at position 9, and MeC at position 15 is substituted.

[0073] B1:Y １ -Y ２ -Y ３ -MeG-Y ４ -Y ５ -Y ６ -Y ７ -df-R-Y ８ -Y ９ -Y １０ -D-MeC

[0074] However, Y １ is any amino acid residue, Y ２ is any amino acid residue, Y ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; Y ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or a 4- to 6-membered cyclic secondary amino acid residue, ９ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １０is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain.

[0075] Y １ is preferably F, F4aao, F3COO, F3aao, 4Py, 3Py, or Cha. ２ is preferably Ahp, W1Me7N, W, or W7N. ３ is preferably V, I, alI, Q, T, TMe, or KCOpipzaa. ４ is preferably W, W1Me, Na1, or Na12. ５ is preferably V, Tbg, TMe, alTMe, dMeS, Gthp, G4pipaa, or Cit. ６ is preferably V, Ahp, Nle, I, L, Tbg, IMe, Cbg, Gcpe, Chg, or alTMe. ７ is preferably MeG, EtG, MeA, Meda, dp, MeQ, or Medq. ８ is preferably V, P, Hpr, Tic, TMe, or alTMe. ９ is preferably I, alI, alTMe, dMeS, Gthp, or G4pipaa. １０ is preferably S or Hgl.

[0076] The peptide of the second embodiment is preferably a peptide comprising an amino acid sequence represented by Formula B2, or an amino acid sequence comprising 1 to 10 amino acid residues selected from the group consisting of the first, second, third, fifth, sixth, seventh, eighth, eleventh, twelfth, and thirteenth amino acid residues in the amino acid sequence represented by Formula B2, in which at least one amino acid residue has been substituted, deleted, added, or inserted. In Formula B2, the amino acid sequence is written from the N-terminus to the C-terminus. Examples of "at least one" include one, two, or three. When an amino acid residue is substituted, it is preferable that an amino acid residue other than MeG at position 4, df at position 9, and MeC at position 15 is substituted.

[0077] B2: F-Ahp-V-MeG-W-V-V-MeG-df-R-V-I-SD-MeC (SEQ ID NO: 97)

[0078] 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 is bonded to MeC, which is the 15th amino acid residue in Formula B1 or Formula B2 contained in the peptide. The amino acid residue derived from a chloroacetylated amino acid is preferably an N-terminal amino acid residue. The peptide having a cyclic structure is preferably one in which the chloroacetyl group has been removed.

[0079] The peptide of the second embodiment preferably further contains additional amino acid residues, but the additional amino acid residues in this case are not particularly limited.

[0080] In the peptide of the second 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 acid residues.

[0081] The peptide of the second embodiment preferably has TPO receptor binding ability.

[0082] [Peptide Complex] The peptide complex of the present invention comprises a first peptide and has the ability to activate TPO / TPOR signaling. The peptide complex of the present invention may also have the ability to induce cell proliferation derived from the ability to activate TPO / TPOR signaling.

[0083] 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.

[0084] The peptide complex may further contain one or more partial peptides different from the first peptide. The peptide complex is preferably formed by linking the first peptide or partial peptides via a linker. The peptide complex is preferably a homomultimer containing only peptides having the same amino acid sequence. The peptide complex may also be a heteromultimer containing peptides having different amino acid sequences. The peptide complex is preferably a homodimer having a first peptide and a second peptide having the same amino acid sequence, 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 intracellular signal activation ability when they form a peptide complex structure via a linker.

[0085] 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.

[0086] 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 first embodiment or the peptide of the second embodiment, preferably the peptide of the first embodiment.

[0087] One embodiment of the peptide complex of the present invention is a peptide complex comprising a first peptide and a second peptide, and it is preferable that the homology between the first peptide and the second peptide is 90% or more and 100% or less.

[0088] One embodiment of the peptide complex of the present invention is a peptide complex comprising a first peptide and a second peptide, and it is preferable that the first peptide and the second peptide are substantially the same peptide.

[0089] One embodiment of the peptide complex of the present invention is a peptide complex comprising a first peptide and a second peptide, and it is preferable that the first peptide and the second peptide are each a cyclic peptide.

[0090] 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 and the second peptide each preferably have a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bound to MeC contained in the first peptide or the second peptide. The amino acid residue derived from the chloroacetylated amino acid is preferably an N-terminal amino acid residue. The peptide having a cyclic structure is preferably one in which the chloroacetyl group has been removed.

[0091] One embodiment of the peptide conjugate of the present invention is a peptide conjugate comprising a first peptide and a second peptide, wherein the first peptide and the second peptide may further comprise additional amino acid residues, in which case the additional amino acid residues are preferably contained in the peptides forming a cyclic structure.

[0092] The peptide complex preferably comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide.

[0093] 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.

[0094] An example of a chemical linker is a PEG (Polyethyleneglycol) linker, which may be a linker consisting of 1 to 36 ethylene glycol units.

[0095] 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.

[0096] Without limitation, the addition of a linker may change the physical properties (e.g., solubility) of the peptide. Furthermore, the linker may be a combination of the above linkers. For example, a glycine (G)-L-lysine (K) may be bound as an amino acid linker, and a PEG linker may be further bound to the side chain terminal of the Lys. Furthermore, the linker may have a structure in which amino acids and PEGs are bound alternately, such as PEG-amino acid-PEG. Here, PEG refers to a PEG linker.

[0097] 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.

[0098] The linker may be added to any position in the peptide. For example, it may be attached to L-cysteine ​​(C) or methyl-L-cysteine ​​(MeC) at the C-terminus of the peptide, which is bonded 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 preferred that the linker be attached to L-cysteine ​​(C) or methyl-L-cysteine ​​(MeC) at the C-terminus, or to the side chain of an amino acid contained in the cyclic peptide.

[0099] For example, in the case where two cyclic peptide structures in which the first amino acid in the amino acid sequences shown in SEQ ID NOs: 1 to 145 is bonded to the 15th amino acid, methyl-L-cysteine ​​(MeC), form a dimer structure via methyl-L-cysteine ​​(MeC) as shown in Table 3, the linker structure can be said to be the structure shown in Table 3. Preferably, the dimer is one in which the C-terminus of the first peptide and the C-terminus of the second peptide are bonded via a linker.

[0100] In the peptide complex of this embodiment, the first peptide and the second peptide are peptides consisting of the amino acid sequence shown in formula A1 or the amino acid sequence shown in formula B1, and are preferably peptides consisting of the amino acid sequence shown in formula A1.

[0101] A1:X １ -X ２ -X ３ -MeG-X ４ -X ５ -X ６ -X ７ -df-X ８ -X ９ -X １０ -X １１ -X １２ -MeC

[0102] However, in Formula A1, X １ is F, A, Q, E, F4COO, F3Me, F3C, F4Me, F4C, F4OMe, 3Py, Cha, or F4aao; ２is W, W7N, W1Me7N, or W1aa, and X ３ is V, R, Cit, T, alT, or S; X ４ is W, W1Me, or Na11, and X ５ is V, T, alT, or dMeS; X ６ is V, I, alI, Eva, Tbg, or Gcpe; ７ is MeG, MeA, Meda, MeKCOpipzaa, dp, EtG, or EtA; ８ is R, Q, or KCOpipzaa; X ９ is V, T, a1T, P, Hpr, Hyp, or Mor; X １０ is I, V, Gthp, or G4pipaa, and X １１ is S, E, I, F, W, Y, R, Cit, Atp, KCOpipzaa, or Hgl; X １２ is D, H, R, S, Q, SMe, Cit, E, or Hgl.

[0103] B1:Y １ -Y ２ -Y ３ -MeG-Y ４ -Y ５ -Y ６ -Y ７ -df-R-Y ８ -Y ９ -Y １０ -D-MeC

[0104] However, in formula B1, Y １ is F, F4aao, F3COO, F3aao, 4Py, 3Py, or Cha, and Y ２ is Ahp, W1Me7N, W, or W7N, and Y ３ is V, I, alI, Q, T, TMe, or KCOpipzaa; ４ is W, W1Me, Na1, or Na12, and Y ５ is V, Tbg, TMe, alTMe, dMeS, Gthp, G4pipaa, or Cit, and Y ６is V, Ahp, Nle, I, L, Tbg, IMe, Cbg, Gcpe, Chg, or alTMe; ７ is MeG, EtG, MeA, Meda, dp, MeQ, or Medq; Y ８ is V, P, Hpr, Tic, TMe, or alTMe; Y ９ is I, alI, alTMe, dMeS, Gthp, or G4pipaa; Y １０ is S or Hgl.

[0105] The peptide complex of this embodiment has the ability to activate an intracellular signal, and preferably has the ability to activate a TPO / TPOR signal.

[0106] [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.

[0107] 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 disease targeted by the pharmaceutical composition refers to any disease caused by, exacerbated by, or otherwise associated with increased or decreased expression or activity of the TPO receptor, or any disease caused by, exacerbated by, or otherwise associated with increased or decreased TPO / TPOR signaling or any other intracellular signaling cascade activated via the TPO receptor. Examples include, but are not limited to, thrombocytopenic syndrome, aplastic anemia, etc.

[0108] 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) administration.

[0109] 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).

[0110] 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] 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.

[0115] The pharmaceutical compositions of the present invention may be administered in combination with other medications or therapies useful for treating the above diseases.

[0116] 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.

[0117] [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.

[0118] In one embodiment, the peptide complex has the ability to activate intracellular signals, preferably the ability to activate TPO / TPOR signals, and therefore can be used as a medium reagent or additive for cell culture, preferably a medium reagent or additive for culture of mammalian cells, more preferably human cells.

[0119] 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.

[0120] 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.

[0121] 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.

[0122] 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.

[0123] [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").

[0124] <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.

[0125] 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 targeted by the medical composition refers to any disease caused by, exacerbated by, or otherwise related to an increase or decrease in the expression or activity of the TPO receptor, or any disease caused by, exacerbated by, or otherwise related to an increase or decrease in TPO / TPOR signaling or any other intracellular signaling cascade activated via the TPO receptor. Examples include, but are not limited to, thrombocytopenic syndrome, aplastic anemia, etc.

[0126] 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) administration.

[0127] 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).

[0128] 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.

[0129] 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.

[0130] 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.

[0131] 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.

[0132] 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.

[0133] The medical composition of the present invention may be administered in combination with other medicines or treatments useful for the above diseases.

[0134] 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.

[0135] <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.

[0136] The peptide binds to the TPO receptor. Therefore, it can also be used as a diagnostic agent for detecting the TPO receptor. The diagnostic agent may be a detection agent for detecting the expression level of the TPO receptor, and when used as a detection agent, the peptide of the present invention may be detectably labeled. In this way, the peptide or a composition containing it can be used as a diagnostic agent for detecting the TPO receptor.

[0137] <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.

[0138] In one embodiment, the peptide and / or peptide complex binds to the TPO receptor and can therefore be preferably used in research involving the TPO receptor.

[0139] The research composition of this embodiment is preferably used in a test method for testing at least one of the following for the peptide, the peptide conjugate, or a conjugate containing a substance further bound to the peptide or the peptide conjugate via a linker: a) solubility in a solvent, b) binding ability to a TPO receptor, c) toxicity to cells and / or tissues, and d) toxicity to experimental animals.

[0140] Regarding the test method, the test for the solubility in a solvent of the peptide, the peptide complex, or a complex containing a substance further bound to the peptide or the peptide complex via a linker may be a measurement of solubility. When measuring solubility, the solvent is not limited and may be freely selected depending on the purpose. Furthermore, regarding the method for measuring solubility, a known method may be appropriately selected depending on the type of solvent.

[0141] The test for the binding ability to the TPO receptor may be a measurement of the binding ability to the TPO receptor, and known methods can be preferably used, including, 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.

[0142] 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.

[0143] The method for testing toxicity in laboratory animals may be a known toxicity evaluation test using laboratory animals. Laboratory 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.

[0144] [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.

[0145] Peptide Drug Conjugate (PDC) Another embodiment of the present invention is a conjugate (peptide drug conjugate, PDC) comprising the above-described peptide or peptide conjugate, a substance to be delivered to the TPO receptor, and a linker for connecting the peptide or peptide conjugate to the substance. The peptide or peptide conjugate binds to the TPO receptor. Thus, the peptide or peptide conjugate is capable of delivering the substance to the TPO receptor. The substance may be any substance desired by those skilled in the art as long as it is a substance desired to be delivered to the TPO receptor. The substance is not particularly limited, but examples thereof include compounds, peptides, RI (radioisotopes), proteins, nucleic acids, and DDS (drug delivery systems). The compound is not particularly limited, but is 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 is preferably a peptide that binds to a target in the body and exerts 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 an enzyme. For example, an enzyme used in enzyme replacement therapy is included. Nucleic acid: may be DNA, RNA, or a chimera thereof, but is not particularly limited. For example, a nucleic acid drug is included. The molecule used in the DDS is not particularly limited, but is preferably a known molecule used in DDS such as a liposome or micelle. The DDS molecule may further contain a compound such as a pharmaceutical drug. Furthermore, the substance desired to be delivered to the TPO receptor may be a complex of the above-listed substances. [Example]

[0146] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. 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.

[0147] 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 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 is. 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. 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 resin used was NovaPEG Rink Amide resin or Seiber Amide resin, and the amount used ranged from 5 mg to 2 g depending on the peptide. The reagent cocktail used for side chain deprotection and cleavage 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 below with side chain protecting groups indicated in brackets.

[0148]

[0149] Unless otherwise specified, the obtained crude peptides were purified using one of the following reversed-phase separation and 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.

[0150] Unless otherwise specified, the columns used were any of the following a) to k). a) Kinetex EVO C18 30x150mm b) XBridge C18 5μm 19x150mm c) XBridge C18 5μm 30x150mm d) XBridge C18 5μm 50x150mm e) XBridge C18 5μm 50x250mm f) XSelect C18 5μm 19x150mm g) XSelect C18 5μm 30x150mm h) XSelect CSH PrepC18 5μm OBD 50x250mm i) XSelect Fluoro-Phenyl 5μm 10x150mm j) YMC-Actus Triart Prep C18-S 10μm 20x250mm k) YMC-Triart Prep C18-S 10μm 10x250mm

[0151] 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 1,000 were frequently detected as multiply charged ions.

[0152] 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 either x / y / z conditions.

[0153] Apparatus: Waters AutoPurification System-SQD2 single quadruple mass spectrometer 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 (%): x: 5-95% / 2.10 min, 95-95% / 0.75 min;

[0154] Equipment: Shimadzu LC / MS system (LC-20ADXR, CTO-20AC, SPD-M20A, SIL-20AXR, CBM-20A and LCMS-2020) Column: Kinetex EVO C18 2.6 μm 2.1x150mm, 100Å Column temperature: 60°C Mobile phase A: 0.025% TFA in H ２ O Mobile phase B: 0.025% TFA in MeCN Flow rate: 0.5 mL / min Wavelength: 225 nm PDA Gradient B (%): y: 20-60% / 7.15 min, 60-95% / 0.3 min, 95-95% / 1.55 ​​min; z: 40-80% / 7.15min, 80-95% / 0.3min, 95-95% / 1.55min;

[0155] Synthesis of Monomer Structure No. 58-2 (See the table below for the structure. The same applies below.)

[0156] 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 / 8 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 third, sixth, seventh, eleventh, and twelfth residues were reacted twice for 30 minutes at 75°C. The tenth and fifteenth residues were reacted twice for 30 minutes at 50°C. The 17th residue, Fmoc-PEG10c, was introduced once for 60 minutes at 25°C. When Fmoc-W7N-OH and Fmoc-Hgl(tBu)-OH were introduced, the amino acids were dissolved in NMP. The Fmoc group was removed by reacting the residue with a 20% piperidine solution in DMF at room temperature for 5 minutes, then removing the solution and reacting it again with a 20% piperidine solution in DMF at room temperature for 5 minutes. The chloroacetyl group was introduced 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 using the method described above, then adding a DMF solution of ClAcOSu (20 equivalents) to the solid-phase resin and shaking for 60 minutes at room temperature. The side chain deprotection and cleavage from the solid-phase resin were carried out by adding reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of DIET / TIS / DODT was added and shaken well, followed by shaking at room temperature for 60 minutes. The reaction solution was filtered 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 diethyl ether / hexane (1 / 1) mixed solvent and then dried for 60 minutes. The resulting solid was used in the subsequent cyclization reaction. The peptide cyclization reaction was carried out in DMSO / H2SO4 at a final peptide concentration of 1.25 mM based on the molar number of the solid phase resin. ２After dissolving the peptide in HCl (9 / 1), triethylamine (21 equivalents) was added and the mixture was left to stand at room temperature for 3 hours. The resulting reaction solution was concentrated under reduced pressure using a Genevac EZ-2 Elite, and DMSO was added to adjust the peptide concentration to 12.5 mM. The resulting crude product was subjected to solid-phase extraction using a Gilson column (column: Gilson ASPEC C18 50 mg 1 mL): (1) The column was loaded 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 The column was equilibrated with 0.02 mL of the above solution (0.3 mL). (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. The purity of one of the main peaks of the target compound 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 65%. Analytical conditions: retention time = 1.47 min; column: Kinetex EVO C18 1.7 μm 2.1 × 50 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ 0, 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 m / z = 1202 (M+3H)3+.

[0157] Synthesis of dimer structure number 18

[0158] The target peptide was synthesized using NovaPEG Rink 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 (12.6 equivalents / 11.22 equivalents / 25.2 equivalents) was used per equivalent of resin, and the reaction was carried out twice for 20 minutes at 75°C in DMF. However, the 3rd, 8th, 10th, 12th, 13th, 14th, and 15th residues were reacted twice for 30 minutes at 50°C. The 17th residue, Fmoc-PEG10c, was introduced once for 60 minutes at 25°C. When Fmoc-SMe-OH was introduced, the amino acid was dissolved in NMP. The Fmoc group was removed by reacting the solid-phase resin with a 20% DMF solution at room temperature for 5 minutes, then removing the solution and reacting again with a 20% DMF solution at room temperature for 15 minutes. The chloroacetyl group was introduced 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 using the method described above, followed by adding chloroacetic acid (0.3 M DMF solution, 12.6 equivalents), HCTU (0.28 M DMF solution, 12 equivalents), and DIEA (1.05 M DMF solution, 25 equivalents) to the solid-phase resin and shaking for 30 minutes at room temperature twice. The side chain was deprotected and cleaved from the solid-phase resin by adding reagent cocktail A (TFA / H ２A mixture of 92.5:2.5:2.5:2.5 (volume ratio of 0.01:0.01:0.01:0.01) of 1,000 sucrose, 1,000 sucrose, and 1,000 sucrose was added to the reaction mixture. The mixture was then filtered through a frit. The filtrate was added to an excess of chilled diisopropyl ether, resulting in a cloudy white precipitate. 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. For the peptide cyclization reaction, the peptide was dissolved in DMSO to a final concentration of 2.5 mM based on the molar number of the solid-phase resin. Triethylamine (10 equivalents) was added, the mixture was stirred, and the mixture was left to stand overnight at room temperature. The resulting reaction solution was concentrated under reduced pressure to 12.5 mM using a Genevac EZ-2 Elite. The obtained crude product was subjected to solid-phase extraction using a Gilson column (column: Gilson ASPEC C18 500 mg 3 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, 3 mL) was used. ２ The column was equilibrated with 1 mL of HCl (3 mL of HCl). (3) 0.2 mL of the above solution was loaded onto the column. (4) The column was washed with Extraction Solution B (4 mL). (5) Extraction was performed with Extraction Solution A (4 mL). The resulting extract was concentrated under reduced pressure using an EZ-2 Elite. The purity of one of the main peaks of the target compound 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 60%. Analytical conditions: retention time = 1.77 min; column: Kinetex EVO C18 1.7 μm 2.1 × 50 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ 0, 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 m / z = 1276 (M+4H)4+.

[0159] Synthesis of Monomer Structure No. 53-1

[0160] 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 at 90°C in DMF. The third and seventh residues were reacted twice for 30 minutes at 75°C. The tenth residue was reacted twice for 15 minutes at 50°C. The fourteenth residue was reacted twice for 10 minutes at 90°C. The fifteenth residue was reacted once for 15 minutes at 50°C. When Fmoc-W7N-OH was introduced, the amino acid was dissolved in NMP. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute, or by reacting twice consecutively at room temperature for 1 minute. The introduction of a chloroacetyl group was performed by adding a DMF solution of ClAcOSu (10 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes. The side chain was deprotected and cleaved from the solid-phase resin by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride and diethyl ether, drying under reduced pressure, and then adding reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of 2.5-diisopropyl ether / TIS / DODT was added and the mixture was shaken at room temperature for 90 minutes. The reaction solution was filtered through a frit. When the filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether / hexane (1 / 1), a cloudy white precipitate was formed. 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. The peptide cyclization reaction was carried out using MeCN / H2SO4 so that the final peptide concentration was 5 mM based on the molar number of the solid phase resin. ２After dissolving the product in 1:1 ethanol, triethylamine (10 equivalents) was added, and the mixture was shaken at room temperature for 3 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using an EZ-2 Elite. The resulting crude product was purified using the following conditions: Column: YMC-Triart Prep C18-S 20 μm 20 × 250 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 50°C; gradient (% B conc): 33-38% over 6 CV, then 38% over 1 CV; flow rate: 17 mL / min. 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 93.51%. Analytical conditions: retention time = 4.02 min; column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ O, B = 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 = 1068 (M+2H)2+.

[0161] Synthesis of Monomer Structure No. 88-7

[0162] The target peptides were 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 / 4 equivalents / 4 equivalents) was used per equivalent of resin, and the reaction was carried out once for 20 minutes in DMF at 50°C. The third and seventh residues were reacted twice for 30 minutes at 75°C. The tenth, eleventh, and fourteenth residues were reacted twice for 20 minutes at 50°C. The 19th and 20th residues were reacted once for 10 minutes at 75°C. When Fmoc-W7N-OH was introduced, the amino acid was dissolved in NMP. Fmoc removal was performed by reacting the solid-phase resin with a 10% DMF solution at 25°C for 1 minute, twice in succession. The introduction of a chloroacetyl group was performed by adding a DMF solution of ClAcOSu (5 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes. The side chain was deprotected and the solid-phase resin was cleaved by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride, and then diethyl ether, drying under reduced pressure, and then adding reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of DISO / TIS / DODT was added and the mixture was shaken at room temperature for 70 minutes. The reaction solution was filtered through a frit. When the filtrate was added to a cooled excess of a mixed solvent of diisopropyl ether / hexane (1 / 1), a cloudy white precipitate was formed. 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. The peptide cyclization reaction was carried out in DMSO / H2SO4 at a final peptide concentration of 3.8 mM based on the molar number of the solid phase resin. ２After dissolving the residue in HCl (9 / 1), triethylamine (20 equivalents) was added, and the mixture was shaken at room temperature for 3 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using a Genevac HT-12. The resulting crude product was purified using the following conditions: Column: Waters XBridge C18 5 μm 50 × 150 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B conc): 8% over 2 minutes, 8-33% over 1 minute, then 33-38% over 8 minutes, then 38-60% 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. 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 94.21%. Analytical conditions: retention time = 4.28 min; column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ O, B = 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 = 1131 (M+3H)3+.

[0163] Synthesis of peptide conjugate (dimer structure no. 108)

[0164] The peptide conjugate was synthesized by dissolving 49 mg of monomer structure number 88-7 in DMSO, adding 0.4 equivalents of NHS-cPEG1c-NHS and 10 equivalents of DIEA, and stirring at room temperature for 1 hour. After this, acetic acid was added. The resulting crude product was purified using the following conditions: Column: Waters XSelect CSH Prep C18 5 μm OBD 50 x 250 mm; Mobile phase: A = 1% AcOH in H ２ O, B=1% AcOH in MeCN, C=0.2M TEAA in H ２O, D = MeCN; Temperature: 50°C; Main pump gradient (% A conc): 0.1% over 5.0 min, then 0.1-100% over 0.1 min, (100% - %B) from 5.1 min onwards; (% B conc): 0% over 5.1 min, then 0-4.2% over 1.9 min, then 4.2-60% over 20 min, then 60-90% over 4 min; (% C conc): 99.9% over 5.0 min, then 99.9-0% over 0.1 min, 0% from 5.1 min onwards; (% D conc): 0%. Flow rate: 18 mL / min over 8 min, then (18 mL / min - 118 mL / min) over 2 min, then 118 mL / min. At-column-dilution pump 1% AcOH in H ２ O / MeCN (1 / 1). Flow rate: 2 mL / min. The purity of the target substance 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 96.29%. Analytical conditions: retention time = 5.21 min; column: Kinetex EVO C18 2.6 μm 2.1 × 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２ O, B = 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 = 1383 (M+5H)5+.

[0165] Synthesis of Peptide Complex (Dimer Structure No. 103) <6-1> Synthesis of Monomer Structure No. 71-8

[0166] 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 at 90°C in DMF. However, the third and seventh residues were reacted twice for 30 minutes at 75°C. The fifth and fourteenth residues were reacted twice for 10 minutes at 90°C. The tenth residue was reacted twice for 15 minutes at 50°C. The fifteenth residue was reacted once for 15 minutes at 50°C. When Fmoc-W7N-OH was introduced, the amino acid was dissolved in NMP. Fmoc removal was performed by reacting with a 10% pyrrolidine solution in DMF at 90°C for 1 minute, or by reacting twice consecutively at room temperature for 1 minute. The introduction of a chloroacetyl group was performed by adding a DMF solution of ClAcOSu (10 equivalents) to the solid-phase resin and shaking at room temperature for 60 minutes. The side chain was deprotected and cleaved from the solid-phase resin by first washing the resin obtained after the chloroacetyl group introduction step with DMF, followed by methylene chloride and diethyl ether, drying under reduced pressure, and then adding reagent cocktail A (TFA / H ２ A mixture of 92.5:2.5:2.5:2.5 by volume of 2.5-3.0% DISO / TIS / DODT was added and the mixture was shaken at room temperature for 60 minutes. The reaction mixture was filtered through a frit. When the filtrate was added to a cooled excess of a mixed solvent of diethyl ether and hexane (1 / 1), a cloudy white precipitate was formed. 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. The peptide cyclization reaction was carried out in DMSO / 2-propanol / H2SO4 such that the final peptide concentration was 2.5 mM based on the molar number of the solid phase resin. ２After dissolving the residue in HCl (90 / 5 / 5), triethylamine (10 equivalents) was added and the mixture was shaken at room temperature for 3 hours, followed by the addition of acetic acid. The resulting reaction solution was concentrated using a Genevac HT-12. The resulting crude product was purified using the following conditions: Column: Waters XBridge C18 5 μm 50 x 150 mm; Mobile phase: A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; Temperature: 40°C; Gradient (% B conc): 6% over 2 min, 6-31% over 1 min, then 31-36% over 8 min, then 36-60% over 1 min; Flow rate: 20 mL / min over 1 min, then (20 mL / min-120 mL / min) over 1 min, then 120 mL / min. After lyophilization, monomer structure number 71-8 (15 mg) was obtained.

[0167] <6-2> Synthesis of peptide complex (dimer structure number 103)

[0168] The peptide conjugate was synthesized by dissolving 15 mg of monomer structure number 71-8 in DMSO, adding NHS-cPEG1c-NHS (0.3 equivalents) and DIEA (5 equivalents), stirring at room temperature for 30 minutes, and then adding acetic acid. The resulting crude product was purified using the following conditions: Column: Waters XSelect C18 5 μm 30 × 150 mm; A = 0.1% TFA in H ２ O, B = 0.1% TFA in MeCN; temperature: 40°C; gradient (% B conc): 9-34% over 3 minutes, then 34-39% over 8 minutes, then 39-60% over 1 minute; flow rate: 45 mL / min. 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 99.21%. Analytical conditions: retention time = 4.81 min; column: Kinetex EVO C18 2.6 μm 2.1 x 150 mm, 100 Å; mobile phase: A = 0.025% TFA in H ２O, B = 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 = 1367 (M+5H)5+.

[0169] Synthesis of Various Peptides In this example, various peptides and peptide conjugates were chemically synthesized in the same manner as in Examples 1-6. The sequences of the synthesized cyclic peptides are shown in Tables 2-1 and 2-2, the linker structures are shown in Table 3, and peptide conjugates in which cyclic peptides are dimerized via a linker are shown in Table 4. The synthesized peptides and peptide conjugates were analyzed using the basic analytical equipment and under the analytical conditions described in the basic conditions above, and their structures were confirmed by ESI-MS(+) 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 2-1, 2-2, and 4. In Tables 2-1, 2-2, and 4, when the "monomer structure number" is listed as "A-B," A represents the peptide sequence number and B represents the linker structure number. Furthermore, dimer structure numbers 68 to 117 in Table 4 indicate that the compound represented by the "monomer structure number" forms a homodimer via the linker represented by the "linker structure number." In the table, ClAc at the N-terminus means a chloroacetyl group. However, a peptide identified by a certain sequence number may have no ClAc substitution at the N-terminus (and may also be a pharmaceutically acceptable salt or solvate of the peptide). In particular, when the peptide means a cyclic peptide, ClAc at the N-terminus may be removed when binding to any group in the sequence to form the cyclic peptide. In particular, when the peptide is a cyclic peptide, it is preferable that the N-terminus is not substituted with ClAc. In addition, if there is a discrepancy between the sequence listing and the following table, the table is interpreted as correct. The sequence numbers in the table mean the peptide sequence numbers.

[0170] Table 2-1 Sequences of synthesized cyclic peptides

[0171] Table 2-2 Sequences of synthesized cyclic peptides

[0172] Table 3. Linker structures

[0173] Table 4. Peptide conjugates

[0174] Evaluation of binding activity to TPOR by ELISA To evaluate the binding activity of the peptides of the present invention to TPOR, binding activity was evaluated using ELISA. The specific test method is as follows. His-tag fused TPOR (R&D systems; 4444-TR) was added at 3 pmol per well to an anti-his-tag antibody-coated 96-well plate (GenScript; L00440C) and allowed to stand for 30 minutes to immobilize TPOR on the plate. After washing three times with PBS-T, HA-tagged peptide diluted to 5 nM was added and allowed to stand for 1 hour. After washing three times with PBS-T, Anti-HA-tag mAb-HRP-Direct T (MBL; M180-7) diluted 5000-fold with PBS-T containing 1% BSA was added and allowed to stand for 30 minutes. After washing three times with PBS-T, the detection reagent SureBlue (registered trademark) TMB 1-Component Microwell Peroxidase Substrate (SeraCare; 5120-0077) was added and allowed to stand for 10 minutes. The reaction was stopped by adding an equal volume of TMB Stop Solution (SeraCare; 5150-0021). The absorbance at 450 nm was measured using EnSpire (PerkinElmer). The binding signal for each peptide was calculated by subtracting the absorbance in wells to which no peptide was added from the absorbance in wells to which the peptide was added. The calculated binding signals for monomer structure numbers 1-2, 2-2, 25-2 to 29-2, and 52-2 to 58-2 are shown in Table 5-1. The calculated binding signals for monomer structure numbers 97-2 to 145-2 are shown in Table 5-2. In Tables 5-1 and 5-2, when "monomer structure number" is listed as "A-B," A represents the peptide sequence number and B represents the linker structure number. As shown in Tables 5-1 and 5-2, these peptides were shown to bind to TPOR.

[0175] Table 5-1 Evaluation of binding activity to TPOR

[0176] Table 5-2 Evaluation of binding activity to TPOR

[0177] Evaluation of intermolecular interactions between TPOR and peptides by surface plasmon resonance (SPR) To evaluate the binding activity of the peptides of the present invention to TPOR, intermolecular interactions were evaluated by surface plasmon resonance (SPR). The specific test method is described below. [SPR Measurement] An NTA sensor chip (Cytiva) was inserted into a Biacore T200 (Cytiva), primed three times with running buffer: HBS-P+, pH 7.4 (Cytiva), and equilibrated at a flow rate of 30 μL / min. Ligand immobilization was performed at a flow rate of 10 μL / min. The NTA sensor chip was washed by pumping 350 mM EDTA solution (Nacalai Tesque) for 30 seconds. Then, 500 μM nickel chloride solution (Hampton Research) was pumped for 30 seconds to coordinate nickel ions onto the carbomethyldextran chains. The column was washed with 50 mM EDTA solution (Nacalai Tesque). 50 μL each of 60 mM EDC solution (Cytiva) and 650 mM NHS solution (Cytiva) was mixed and reacted for 420 seconds at a flow rate of 10 μL / min. A 100 nM TPOR solution was reacted for 420 seconds at a flow rate of 10 μL / min to achieve approximately 2000 RU. Amine coupling was performed by crosslinking NTA with nickel ions. A 1.0 M aqueous ethanolamine solution (Cytiva) was reacted for 420 seconds at a flow rate of 10 μL / min for capping. A peptide solution prepared at 10 mM in DMSO was diluted with running buffer to a final peptide concentration of 10 μM, and peptide solutions of 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM were prepared. The peptide sample was reacted at a flow rate of 30 μL / min for 120 seconds, and dissociation was performed for 600 seconds. The kinetics of the peptide relative to TPOR were obtained by SPR measurement. The kinetics evaluation model was Single Cycle Kinetics, and curve fitting was performed using Biacore T200 Evaluation Software Version 3.0 (Cytiva). The resulting sensorgram was subjected to least squares curve fitting, and the binding of the peptide to TPOR was evaluated by calculating the KD value.The calculated KD values ​​for monomer structure numbers 1-1, 53-1, and 88-7 are shown in Table 5-1. As shown in Table 5-1, it was demonstrated that these peptides bind to TPOR.

[0178] ERK Activation Assessment by AlphaLISA To evaluate the ERK activation ability of the peptide complex of the present invention, ERK phosphorylation was assessed using AlphaLISA. The specific test method is as follows: TPO Reporter HEK293 Cells (InvivoGen; hkb-tpo) were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS, 100 μg / mL Nomocin (InvivoGen), 50 μg / mL Gentamicin (Nakarai Tesque), and 1×HEK-Blue selection (Invivogen). After detaching the cells using PBS, they were seeded onto a Poly-D lysine-coated 96-well plate for adherent cells at 30,000 cells per well and cultured for 24 hours. For starvation, the medium was replaced with DMEM containing 0.1% BSA (Sigma) and 50 μg / mL Gentamicin (Nakarai Tesque), and the cells were cultured for 20 hours. After that, recombinant human thrombopoietin (R&D systems) or peptide was added, and the cells were incubated for 20 hours in CO. ２ 37 in the incubator ｏ C, stimulation for 20 minutes. Cells were then lysed using the lysis buffer included with the AlphaLISA SureFire Ultra p-Erk1 / 2 (Thr202 / Tyr204) Assay Kit (PerkinElmer). The assay was performed according to the kit's protocol, and a SpectraMax Paradigm multimode microplate reader (Molecular Devices) was used for signal detection. The resulting signals were analyzed using GraphPad Prism, and the % activation was calculated by setting the maximum signal induced by TPO as 100% and the unstimulated signal as 0%.

[0179] The test was performed with TPO added at concentrations ranging from 0.002 nM to 10 nM, and the maximum induced signal (Emax) was calculated. For dimer structure numbers 1 to 34, the test was performed with peptide added at concentrations of 10, 100, and 1000 nM. Table 6 shows the results: A-1 indicates a % activation of 50% or more at 10 nM, A-2 indicates a % activation of 50% or more at 100 nM, A-3 indicates a % activation of 50% or more at 1000 nM, and A-4 indicates a % activation of 5% or more but less than 50% at 1000 nM. For dimer structure numbers 35 to 67, tests were performed at peptide concentrations of 1, 10, and 100 nM, and the results are shown in Table 6: those with a % activation of 50% or more at 1 nM addition were designated B-1, those with a % activation of 50% or more at 10 nM addition were designated B-2, those with a % activation of 50% or more at 100 nM addition were designated B-3, and those with a % activation of 5% or more but less than 50% at 100 nM addition were designated B-4. There were no samples that fell into the B-1 category. For dimer structure numbers 68 to 107 and 110 to 117, tests were performed at peptide concentrations of 1, 10, and 100 nM. Table 6 shows the results as follows: C-1 indicates a % activation of 50% or more when 1 nM is added; C-2 indicates a % activation of 50% or more when 10 nM is added; C-3 indicates a % activation of 50% or more when 100 nM is added; and C-4 indicates a % activation of 5% or more but less than 50% when 100 nM is added.

[0180] For dimer structure numbers 108 and 109, tests were performed at peptide concentrations of 0.039, 0.156, 0.625, and 2.5 nM, and the samples that showed a % activation of 50% or more at 0.039 nM were designated D-1, those that showed a % activation of 50% or more at 0.156 nM were designated D-2, those that showed a % activation of 50% or more at 0.625 nM were designated D-3, those that showed a % activation of 50% or more at 2.5 nM were designated D-4, and those that showed a % activation of 5% or more but less than 50% at 2.5 M were designated D-5, as shown in Table 6. There were no samples that met the criteria for D-1, D-3, D-4, or D-5. As shown in Table 6, these peptides were shown to have the ability to activate ERK signaling in TPO Reporter HEK293 cells.

[0181] Table 6. ERK activation assessment and JAK-STAT pathway activation assessment

[0182] Evaluation of STAT5-dependent transcription activation by STAT5-inducible secreted embryonic alkaline phosphatase (SEAP) reporter assay To evaluate the STAT5-dependent transcription activation ability of the peptide complex of the present invention, transcription activity was evaluated using a STAT5-inducible SEAP reporter. The specific test method is shown below. TPO Reporter HEK293 cells used in AlphaLISA were cultured in DMEM (Thermo Fisher Scientific) containing 10% FBS, 100 μg / mL Nomocin (InvivoGen), 50 μg / mL Gentamicin (Nakarai Tesque), and 1X HEK-Blue selection (Invivogen). After detachment with PBS, the cells were suspended in DMEM containing 0.1% BSA (Sigma) and 50 μg / mL Gentamicin (Nakarai Tesque) and seeded into a 96-well plate for adherent cells at 54,000 cells per well. Immediately after seeding, TPO or peptide was added and CO ２ 37 in the incubator ｏ C, and cultured for 24 hours. 20 μL of the culture supernatant was transferred to a new 96-well plate for adherent culture (ThermoFisher Scientific), and 180 μL of QUANTI-BLUE (InvivoGen) was added. ２ 37 in the incubator ｏ C: After 2 hours of incubation, the absorbance at 620 nm was measured. The obtained values ​​were analyzed using GraphPad Prism, and the % activation was calculated by setting the maximum signal induced by TPO as 100% and the unstimulated signal as 0%.

[0183] Tests were performed with TPO concentrations ranging from 0.002 nM to 10 nM, and the maximum induced signal (Emax) was calculated. For dimer structure numbers 68 and 69, tests were performed with peptide concentrations of 1, 10, and 100 nM. The samples with % activation of 50% or greater at 1 nM were designated E-1, those with % activation of 50% or greater at 10 nM were designated E-2, those with % activation of 50% or greater at 100 nM were designated E-3, and those with % activation of 5% to less than 50% at 100 nM were designated E-4, as shown in Table 6. There were no samples that met the criteria for E-2, E-3, or E-4. For dimer structure numbers 70, 88, 92, 93, 108, and 109, tests were performed at peptide concentrations of 0.0156, 0.0625, 0.25, and 1 nM. Table 6 shows the results for which the % activation was 50% or greater at 0.0156 nM, F-1; F-2; F-3; F-4; and F-5; respectively. There was no sample that met the F-5 criteria. NT indicates that the test was not performed. As shown in Table 6, these peptides were shown to have the ability to activate STAT5-dependent gene transcription in TPO Reporter HEK293 cells.

[0184] The present invention can be used in fields such as the pharmaceutical industry.

Claims

1. A peptide comprising an amino acid sequence represented by formula A1, or an amino acid sequence in which multiple amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by formula A1, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. A1:X １ -X ２ -X ３ -MeG-X ４ -X ５ -X ６ -X ７ -df-X ８ -X ９ -X １０ -X １１ -X １２ -MeC where X １ is any amino acid residue, and X ２ is an amino acid residue having an optionally substituted aryl group in the side chain, ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ９ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or an optionally substituted 4- to 6-membered cyclic secondary amino acid residue, １０ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １１ is any amino acid residue, and X １２ is any amino acid residue, and in Formula A1, the amino acid sequence is written from the N-terminal side to the C-terminal side.

2. In formula A1, X １ is F, A, Q, E, F4COO, F3Me, F3C, F4Me, F4C, F4OMe, 3Py, Cha, or F4aao; ２ is W, W7N, W1Me7N, or W1aa, and X ３ is V, R, Cit, T, alT, or S; X ４ is W, W1Me, or Na11, and X ５ is V, T, alT, or dMeS; X ６ is V, I, alI, Eva, Tbg, or Gcpe; ７ is MeG, MeA, Meda, MeKCOpipzaa, dp, EtG, or EtA; ８ is R, Q, or KCOpipzaa; X ９ is V, T, a1T, P, Hpr, Hyp, or Mor; X １０ is I, V, Gthp, or G4pipaa, and X １１ is S, E, I, F, W, Y, R, Cit, Atp, KCOpipzaa, or Hgl; X １２ is D, H, R, S, Q, SMe, Cit, E, or Hgl. The peptide of claim 1, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

3. A peptide comprising the 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 in the amino acid sequence represented by formula A2, consisting of 1 to 12 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 10th, 11th, 12th, 13th, and 14th amino acid residues, or a pharmaceutically acceptable salt or solvate thereof. A2: F-W-V-MeG-W-V-V-MeG-df-R-V-I-S-D-MeC (SEQ ID NO: 1) 4. In formula A1, X １ is an amino acid residue having an optionally substituted aryl group in the side chain, ２ is an amino acid residue having an optionally substituted aryl group in the side chain, ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ９ is an amino acid residue having an aliphatic hydrocarbon group in the side chain or a 4- to 6-membered cyclic secondary amino acid residue, １０ is an amino acid residue having an aliphatic hydrocarbon group in the side chain, １１ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １２ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, a peptide according to claim 1, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

5. In formula A1, X １ is F or F4Me, and X ２ is W or W7N, X ３ are V, T, and a1T, and X ４ is W or Na11, X ５ is V, and X ６ is V, I, alI, or Gcpe, and X ７ is MeG, MeA, Meda, MeKCOpipzaa, or dp; X ８ is R, and X ９ is V or P, and X １０ is I or V, and X １１ is S, KCOpipzaa, or Hgl, and X １２ is D. The peptide according to claim 4, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

6. The peptide according to any one of claims 1 to 5, which is a cyclic peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

7. A peptide according to any one of claims 1 to 5, having a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bonded to MeC, which is the 15th amino acid residue in Formula A1 or Formula A2 contained in the peptide, or a pharmaceutically acceptable salt or solvate thereof.

8. A peptide according to any one of claims 1 to 5, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, further comprising additional amino acid residues.

9. A peptide according to claim 4 or 5, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, which has TPO receptor binding ability.

10. A peptide comprising an amino acid sequence represented by formula B1, or an amino acid sequence in which multiple amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by formula B1, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. B1:Y １ -Y ２ -Y ３ -MeG-Y ４ -Y ５ -Y ６ -Y ７ -df-R-Y ８ -Y ９ -Y １０ -D-MeC where Y １ is any amino acid residue, Y ２ is any amino acid residue, Y ３ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ４ is an amino acid residue having an optionally substituted aryl group in the side chain, ５ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ６ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, ７ is any N-alkylated amino acid residue or a 4- to 6-membered cyclic secondary amino acid residue; Y ８ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, or a 4- to 6-membered cyclic secondary amino acid residue, ９ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, １０ is an amino acid residue having an optionally substituted aliphatic hydrocarbon group in the side chain, and in formula B1, the amino acid sequence is written from the N-terminal side to the C-terminal side.

11. In formula B1, Y １ is F, F4aao, F3COO, F3aao, 4Py, 3Py, or Cha, and Y ２ is Ahp, W1Me7N, W, or W7N, and Y ３ is V, I, alI, Q, T, TMe, or KCOpipzaa; ４ is W, W1Me, Na1, or Na12, and Y ５ is V, Tbg, TMe, alTMe, dMeS, Gthp, G4pipaa, or Cit, and Y ６ is V, Ahp, Nle, I, L, Tbg, IMe, Cbg, Gcpe, Chg, or alTMe; ７ is MeG, EtG, MeA, Meda, dp, MeQ, or Medq; Y ８ is V, P, Hpr, Tic, TMe, or alTMe; Y ９ is I, alI, alTMe, dMeS, Gthp, or G4pipaa; Y １０ The peptide according to claim 10, or a pharmaceutically acceptable salt or solvate thereof, wherein: is S or Hgl.

12. The peptide according to claim 10, 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, deleted, added, or inserted in an amino acid sequence consisting of 1 to 10 amino acid residues selected from the group consisting of the 1st, 2nd, 3rd, 5th, 6th, 7th, 8th, 11th, 12th, and 13th amino acid residues in the amino acid sequence represented by Formula B2, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof. B2: F-Ahp-V-MeG-W-V-V-MeG-df-R-V-I-S-D-MeC (SEQ ID NO: 97) 13. The peptide according to any one of claims 10 to 12, which is a cyclic peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

14. The peptide according to any one of claims 10 to 12, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, which has a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bonded to MeC, which is the 15th amino acid residue in Formula B1 or Formula B2 contained in the peptide.

15. A peptide according to any one of claims 10 to 12, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, further comprising additional amino acid residues.

16. A peptide according to any one of claims 10 to 12, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, which has TPO receptor binding ability.

17. A peptide complex comprising a first peptide and a second peptide, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, wherein the first peptide and the second peptide may be the same or different and are the peptides described in claim 1 or claim 10. A peptide complex, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

18. The peptide conjugate according to claim 17, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate, wherein the peptide conjugate comprises the first peptide, the second peptide, and a linker connecting the first peptide and the second peptide, and the second peptide may be the same as or different from the first peptide, and has an amino acid sequence represented by formula A1, or an amino acid sequence in which a plurality of amino acid residues have been substituted, deleted, added, or inserted in the amino acid sequence represented by formula A1.

19. The peptide complex, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the homology between the first peptide and the second peptide is 90% or more and 100% or less.

20. The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the first peptide and the second peptide are substantially the same peptide.

21. The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the first peptide and the second peptide are each a cyclic peptide.

22. The peptide complex, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the first peptide and the second peptide each have a cyclic structure in which an amino acid residue derived from a chloroacetylated amino acid is bound to MeC contained in the first peptide or the second peptide.

23. The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate of claim 18, wherein the first peptide and the second peptide further comprise additional amino acid residues.

24. The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the C-terminus of the first peptide and the C-terminus of the second peptide are linked via a linker.

25. The peptide conjugate, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate according to claim 18, wherein the linker is a PEG linker or a linker consisting of PEG and an amino acid residue.

26. The peptide complex of claim 18, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof, which has the ability to activate an intracellular signal.

27. A pharmaceutical composition comprising a peptide according to any one of claims 1 to 5 and 10 to 12, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

28. A pharmaceutical composition comprising the peptide conjugate of claim 18, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

29. A composition for cell culture used in cell culture, comprising the peptide complex according to claim 18, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

30. A composition for medical, diagnostic or research use, comprising the peptide conjugate of claim 18, a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable solvate thereof.

31. A composition for medical, diagnostic or research use, comprising the peptide according to any one of claims 1 to 5 and 10 to 12, its pharmaceutically acceptable salt, or its pharmaceutically acceptable solvate.