GHR-binding peptides and compositions comprising same

Abstract

The present invention relates generally to peptides that bind to the growth hormone receptor (GhR), peptides that bind to the GhR and have antagonist activity, and compositions comprising such peptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 212,596, filed June 18, 2021, the contents of which are incorporated herein by reference in their entirety. [Technical field]

[0002] The present invention relates to peptides which bind to the growth hormone receptor (GhR), to peptides which bind to the GhR and have antagonist activity, and to compositions comprising such peptides. [Background technology]

[0003] Growth hormone (GH or Gh) is a hormone essential for growth and is secreted by somatotrophs in the anterior pituitary gland. Growth hormone is known to have effects on various tissues, including growth, e.g. bone elongation and muscle growth, and metabolism, e.g. glycogenolysis in the liver. Growth hormone is produced in the pituitary gland and secreted into the bloodstream, where it binds to growth hormone receptors (GhR) expressed on various cell surfaces, e.g. liver, muscle tissue, and bone tissue. GH binding to GhR induces the production of insulin-like growth factor-1 (IGF-1) in certain cells, especially liver cells. IGF-1 then stimulates whole body growth and exerts growth-promoting effects on somatic cells.

[0004] As an example of a compound that binds to GhR, a growth hormone variant compound that binds to human GhR is described in JP 2016-511275 A (PCT application translation), which is incorporated herein by reference, and further, a method for generating a subject's response to a drug that can bind to human GhR is described in JP 2006-525785 A (PCT application translation), which is incorporated herein by reference. Thus, various drugs that have avidity for GhR have been examined.

[0005] Using peptides that bind to GhR (GhR-binding peptides), the distribution and amount of GhR expression can be confirmed, for example, by measuring the binding of fluorescent or isotopically labeled peptides to GhR. Furthermore, the affinity of ligands to GhR or to GhR of different species can be determined by using GhR-binding peptides. In addition, GhR-binding peptides can be used to target and transport compounds with pharmacological activity to GhR, such as isotopes, low molecular weight compounds, peptides, proteins, antibodies, and nucleic acids.

[0006] Thus, both novel GhR-binding peptides and compositions containing GhR-binding peptides would be useful and desirable. Summary of the Invention

[0007] One embodiment of the present invention provides peptides that bind to GhR, in particular the human GhR, and compositions comprising such GhR-binding peptides.

[0008] In some embodiments, the peptide according to the present invention is an isolated peptide.

[0009] In some embodiments, the peptide according to the invention is a purified peptide.

[0010] In some embodiments, the peptide according to the invention has the following amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13 (SEQ ID NO:1), wherein: X1 is an amino acid having an aromatic ring or a substitute thereof; X2 is an N-alkylamino acid or a modified form thereof; X3 and X4 are each independently a branched chain amino acid; X5 is any amino acid; X6 is N(asparagine); X7 is W (tryptophan) or a substitution thereof; X8 is an amino acid or a substitute thereof having an aromatic ring in the side chain; X9 is K (lysine) or a substitute thereof or R (arginine) or a substitute thereof; X10 is an amino acid or a substitute thereof having an aromatic ring in the side chain; X11 is A (alanine) or a substitute thereof or K (lysine) or a substitute thereof; X12 is any amino acid; and X13 is C (cysteine), and The peptide or salt thereof may contain no substitutions, deletions, additions or insertions, or may contain one, two or three substitutions, deletions, additions or insertions, and the peptide or salt thereof has binding ability to GhR.

[0011] In some implementations of these embodiments, X2 is N-methyllysine or a modified version thereof; X3 and X4 are each V (valine); X5 is S (serine); X7 is a substituted W (tryptophan); X8 is F (phenylalanine) or a substitute thereof, or Y (tyrosine) or a substitute thereof; X9 is K (lysine) or a substitute thereof, or R (arginine) or a substitute thereof; X10 is a substituted W (tryptophan); X11 is A (alanine) or a substitute thereof, or K (lysine) or a substitute thereof; and X12 is V (valine).

[0012] In some implementations of these embodiments, X2 is N-methyllysine or a modification thereof; X7 is 1-(carboxymethyl)-L-tryptophan (W1aa), and the amino acid residues of X2 and X7 are linked.

[0013] In some implementations of these embodiments, X2 is an N-methyllysine having an albumin binder attached thereto. The albumin binder may be attached directly to the N-methyllysine or through another amino acid residue or linking group.

[0014] In some implementations of these embodiments, the albumin binder is any one of 4IphpCO, Biph4pCO, PhPeCO, PhpCO, cC14COO, and 4MePhpCO.

[0015] In some implementations of these embodiments, X1 is Y, 4Py, or F4COO; X8 is Y or F4COO; X9 is K, KCOpipzaa, Hgn, Ahp, or Har; X10 is W5H; and X11 is Aib or A4pipaa.

[0016] In some embodiments, the peptide or salt thereof according to the present invention has an amino acid sequence represented by F4COO-MeK-VVSN-W1aa-F4COO-K-W5H-Aib-VC (SEQ ID NO: 10), or an amino acid sequence in which one, two or three amino acid residues have been substituted, deleted, added or inserted from SEQ ID NO: 10, wherein the second and seventh amino acid residues in SEQ ID NO: 10 are linked, and the peptide or salt thereof has binding ability to hGhR.

[0017] In some embodiments, the peptide or salt thereof according to the present invention has an amino acid sequence represented by Y-MeK-VVSN-W5OMe-F4COO-K-W5H-A4pipaa-VC (SEQ ID NO: 30), or an amino acid sequence having one, two or three amino acid residues substituted, deleted, added or inserted from SEQ ID NO: 30, wherein the albumin binder is bound to the second amino acid residue, MeK, of SEQ ID NO: 30, and the peptide or salt thereof has binding ability to hGhR.

[0018] In some embodiments, the albumin binder is any one of 4IphpCO, Biph4pCO, PhPeCO, PhpCO, cC14COO, and 4MePhpCO. In some embodiments, the albumin binder is 4IphpCO. In some embodiments, the albumin binder is Biph4pCO. In some embodiments, the albumin binder is PhPeCO. In some embodiments, the albumin binder is PhpCO. In some embodiments, the albumin binder is cC14COO. In some embodiments, the albumin binder is Biph4pCO.

[0019] In some embodiments, the peptide or salt thereof according to the invention is a cyclic peptide or a salt of a cyclic peptide.

[0020] In some embodiments, the peptide or salt thereof according to the present invention has a cyclic structure with a chloroacetylated first amino acid residue and a cysteine ​​residue linked thereto.

[0021] In some embodiments, the peptide according to the present invention or a salt thereof has an amino acid sequence selected from SEQ ID NOs: 2-9, 11-29, and 31.

[0022] In some embodiments, the peptide or salt thereof according to the present invention has an amino acid sequence selected from SEQ ID NOs: 2 to 9, 11 to 29, and 31, and further comprises a linker at the C-terminus.

[0023] In some implementations of this embodiment, the linker has an amino acid sequence selected from SEQ ID NOs:32 and 35.

[0024] In some embodiments, the peptide or salt thereof according to the invention has hGhR antagonist activity.

[0025] In some embodiments, a peptide or salt thereof according to the present invention includes any combination described herein, and any peptide or salt thereof described herein.

[0026] In some aspects, the present invention relates to pharmaceutical compositions.

[0027] In some embodiments, a pharmaceutical composition according to the invention comprises any peptide described herein or a salt thereof, and a pharma- ceutically acceptable carrier, excipient, or additive.

[0028] In some embodiments, the pharmaceutical composition according to the present invention has hGhR antagonist activity.Therefore, the pharmaceutical composition is effective in treating diseases related to hypersecretion of human growth hormone (such as acromegaly or gigantism), and can be used as a pharmaceutical composition for treating these diseases.

[0029] In some aspects, the present invention relates to methods of treatment for diseases associated with hypersecretion of human growth hormone.

[0030] In some embodiments, the therapeutic method according to the present invention is for the treatment of a disease associated with hypersecretion of human growth hormone, the method comprising the step of administering the pharmaceutical composition described above (or the peptide or a salt thereof described above) to a patient having a disease associated with hypersecretion of human growth hormone.

[0031] In some embodiments, the disease associated with hypersecretion of human growth hormone is acromegaly or gigantism.

[0032] Due to the ability of the peptides according to the invention to bind to GhR, they are capable of targeting and delivering compounds having pharmacological activity against GhR, such as isotopes, low molecular weight compounds, peptides, proteins, antibodies and nucleic acids.

[0033] Other aspects and features of the present disclosure will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures. [Brief description of the drawings]

[0034] [Figure 1] FIG. 1 shows an overview of example peptide sequences according to one embodiment of the present invention.

[0035] [Diagram 2] FIG. 1 is a diagram showing an outline of an example of the sequence of a linker site in a peptide according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0036] All features of the embodiments described in this disclosure are not mutually exclusive and can be combined with one another. For example, elements of one embodiment can be utilized in other embodiments without further recitation. Detailed descriptions of specific embodiments are provided herein below with reference to the accompanying drawings.

[0037] It should be understood that both the general description and the detailed description below are merely exemplary and descriptive, and are not intended to limit the invention of the present application. In this specification, the use of the singular includes the plural, unless otherwise specified. In this specification, the use of "or" means "and / or" unless otherwise specified. Furthermore, terms such as "element" or "component" include both elements and components that include one unit, and elements and components that include two or more subunits, unless otherwise specified.

[0038] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described. All or portions of documents cited in this application, including but not limited to patents, patent applications, literature, books, and articles, are expressly incorporated by reference in whole or in part, from among the documents discussed herein.

[0039] The recitation herein of numerical ranges by endpoints are intended to include all numbers subsumed within that range (e.g., recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0040] The term "about" is used expressly or not in this specification, and all amounts given in this specification are intended to refer to the actual given amount, which also refers to approximations to such given values ​​that would be reasonably inferred based on ordinary skill in the art, including equivalents and approximations resulting from experimental and / or measurement conditions for such given values. For example, the term "about" in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, more preferably within 5% of the given value or range.

[0041] As used herein, the term "comprises" is used in its open-ended sense meaning that the items following the word are included but not excluding items not specifically listed.

[0042] Unless otherwise defined, the terminology used in analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein, as well as the procedures and techniques thereof, are well known and commonly used in the field according to the present invention. Standard techniques may be used for chemical synthesis and chemical analysis. Such techniques and procedures defined can be found, for example, in "KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013", which are incorporated by reference for all purposes. All patents, applications, published applications, and other publications, as well as other data, referenced throughout the disclosure are incorporated by reference, where permitted.

[0043] Abbreviation: Unless otherwise stated herein, the following abbreviations are used in accordance with the following meanings: BiPh4pCO: 4-([1,1'-biphenyl]-4-yl)butanoic acid (CAS number: 6057-60-9); Boc: Tert-butoxycarbonyl; cC12COO: tetradecanedioic acid (CAS number: 821-38-5); cC13COO: pentadecanedioic acid (CAS number: 1460-18-0); cC14COO: hexadecanedioic acid (CAS number: 505-54-4); cC15COO: heptadecanedioic acid (CAS number: 2424-90-0); ClAc: chloroacetyl; DCM: dichloromethane; DIC: N,N'-diisopropylcarbodiimide; DMSO: dimethyl sulfoxide; DMF: dimethylformamide; DIPEA or DIEA: N,N-diisopropylethylamine; DODT: 6-dioxa-1,8-octanedithiol; E_cC14COO: N-(15-carboxy-1-oxopentadecyl)-L-glutamic acid (CAS number: 1472005-57-4); Fmoc: 9-fluorenylmethyloxycarbonyl; g: grams (unit); HOSu: N-hydroxysuccinimide; HPLC: high performance liquid chromatography; ivDde: 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)-3-methylbutyl; LC-MS or LC / MS: Liquid Chromatography Mass Spectrometer; MeCN: acetonitrile; mL: milliliters (unit); M: Molar concentration (units); μL: microliter (unit); mM: millimolar concentration (unit); μΜ: micromolar concentration (unit); mg: milligrams (unit); mm: millimeters (unit); nm: nanometers (units); nM: nanomolar concentration (units); Oxyma pure: Ethyl cyano(hydroxyimino)acetate; PhpCO: 4-phenylbutanoic acid (CAS number: 1821-12-1); PhPeCO: 6-phenylhexanoic acid (CAS number: 5581-75-9); qPCR: quantitative PCR; rpm: Revolutions per minute (unit); tBu: tert-butyl; TFA: trifluoroacetic acid; TIS: triisopropylsilane; Trt or Tr: trityl group; 4IPhpCO: 4-(p-iodophenyl)butyric acid (CAS number: 27913-58-2, Merck); 4MePhpCO: 4-(p-tolyl)butanoic acid (CAS number: 4521-22-6). Abbreviations (unnatural amino acids): W5OMe: 5-Methoxy-L-tryptophan (CAS number: 25197-96-0); F4COO: 4-carboxy-L-phenylalanine (CAS number: 126109-42-0); W5H: 5-hydroxy-L-tryptophan (CAS number: 2382808-45-7); Aib: alpha-methylalanine (CAS number: 62-57-7); ds: D-serine (CAS number: 312-84-5); W1aa: 1-(carboxymethyl)-L-tryptophan (CAS number: 773823-50-0); A4pipaa: 4-amino-1-(carboxymethyl)piperidine-4-carboxylic acid (Kishida Chemical Inc.); Hgn: (S)-2,6-diamino-6-oxohexanoic acid (CAS number: 1263046-43-0); Har: N6-carbamimidoyl-L-lysine (CAS number: 214852-52-5); KCOpipzaa: N6-(4-(carboxymethyl)piperazine-1-carbonyl)-L-lysine (Kishida Chemical Inc.); PEG2Ac: 2-(2-(2-aminoethoxy)ethoxy)acetic acid (CAS number: 134978-97-5); 4Py: 4-pyridyl-L-alanine (CAS number: 169555-95-7); W7N: (S)-2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic acid (CAS number: 737007-45-3); Ahp: (s)-2-aminoheptanoic acid (CAS number: 44902-02-5); Me: N-methyl; MeK: N-methyl-L-lysine (CAS number: 7431-89-2);

[0044] peptide: In one embodiment, the peptide according to the invention has the amino acid sequence: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13, as shown in SEQ ID NO:1, where: X1 is an amino acid or a substitute thereof having an aromatic ring in the side chain; X2 is an N-alkylamino acid or a modified form thereof; X3 and X4 are each a branched chain amino acid; X5 is any amino acid; X6 is N(asparagine); X7 is W (tryptophan) or a substitution thereof; X8 is an amino acid or a substitute thereof having an aromatic ring in the side chain; X9 is K (lysine) or a substitute thereof or R (arginine) or a substitute thereof; X10 is an amino acid or a substitute thereof having an aromatic ring in the side chain; X11 is A (alanine) or a substitute thereof or K (lysine) or a substitute thereof; X12 is any amino acid; X13 is C (cysteine) having The peptides may contain no amino acid substitutions, deletions, additions or insertions, or may contain one, two or three amino acid substitutions, deletions, additions or insertions. In the practice of this embodiment, the peptides according to the invention have binding to GhR.

[0045] In some embodiments, the peptide according to the present invention having the amino acid sequence depicted in SEQ ID NO:1 comprises substitutions, additions, deletions, or insertions. The number of amino acid substitutions, deletions, additions, and / or insertions can be 1 or more and 3 or less, with the lower limit being 1. The upper limit being 2 and the minimum being 1. In some embodiments, the amino acid substitutions are conservative amino acid substitutions. In some further implementations, 1 to 3 such amino acid substitutions are present at positions selected from X1, X2, X7, X8, X9, X10, and X11 in SEQ ID NO:1.

[0046] As used herein, the phrase "conservative amino acid substitution" refers to the substitution of a functionally equivalent or similar amino acid. Conservative amino acid substitution in a peptide results in a static change in the amino acid sequence of the peptide. For example, one or more amino acids with similar polarity act functionally equivalent to each other, resulting in a static change in the amino acid sequence of the peptide. In general, substitutions within a certain group may be considered conservative in terms of structure and function. However, as will be apparent to those skilled in the art, the role played by a defined amino acid residue may be determined by its effect on the three-dimensional structure of the molecule containing the amino acid. For example, a cysteine ​​residue may be in an oxidized (disulfide) form that has a lower polarity than that of the reduced (thiol) form. The long aliphatic portion of the arginine side chain may constitute a structurally and functionally important feature. Furthermore, side chains containing aromatic rings (tryptophan, tyrosine, phenylalanine) may contribute to non-aromatic interactions or cation-pi interactions. In such cases, amino acids with these side chains may be structurally and functionally preserved even if they are substituted with amino acids belonging to acidic or non-polar groups. Residues such as proline, glycine, and cysteine ​​(in disulfide form) have direct effects on the three-dimensional structure of the main chain and often cannot be substituted without distortion of the structure.

[0047] As set forth below, conservative amino acid substitutions include specific substitutions based on side chain similarity (e.g., those substitutions described in Lehninger, Biochemistry, 2nd revised edition, 1975, pp. 73-75: L. Lehninger, Biochemistry, 2nd edition, pp. 73-75, Worth Publisher, New York (1975)) and exemplary substitutions, which are incorporated by reference into this disclosure.

[0048] In addition to conservative amino acid substitutions, substitutions of amino acids that belong to the same group as a particular amino acid, e.g., in groups obtained by classifying natural amino acids based on the properties of their common side chains as follows, are preferred.

[0049] Hydrophobic (also referred to as non-polar) amino acids: Amino acids that exhibit hydrophobicity (non-polarity) include alanine (also referred to as "Ala" or simply "A"), glycine (also referred to as "Gly" or simply "G"), valine (also referred to as "Val" or simply "V"), leucine (also referred to as "Leu" or simply "L"), isoleucine (also referred to as "Ile" or simply "I"), proline (also referred to as "Pro" or simply "P"), phenylalanine (also referred to as "Phe" or simply "F"), tryptophan (also referred to as "Trp" or simply "W"), tyrosine (also referred to as "Tyr" or simply "Y"), and methionine (also referred to as "Met" or simply "M").

[0050] The hydrophobic amino acids can be further divided into the following groups: - Aliphatic Amino Acids: Amino acids that have a fatty acid or hydrogen in the side chain, including Ala, Gly, Val, Ile, and Leu. - Aliphatic / Branched Chain Amino Acids: Amino acids that have branched fatty acids in their side chains, including Val, Ile, and Leu. - Aromatic Amino Acids: Amino acids that have an aromatic ring in the side chain, including Trp, Tyr, and Phe.

[0051] Hydrophilic (also referred to as polar) amino acids: Amino acids that exhibit hydrophilicity (polarity), including serine (also referred to as "Ser" or simply "S"), threonine (also referred to as "Thr" or simply "T"), cysteine ​​(also referred to as "Cys" or simply "C"), asparagine (also referred to as "Asn" or simply "N"), glutamine (also referred to as "Gln" or simply "Q"), aspartic acid (also referred to as "Asp" or simply "D"), glutamic acid (also referred to as "Glu" or simply "E"), lysine (also referred to as "Lys" or simply "K"), arginine (also referred to as "Arg" or simply "R"), and histidine (also referred to as "His" or simply "H").

[0052] The hydrophilic amino acids can be further divided into the following groups: - Acidic amino acids: amino acids whose side chains are acidic, including Asp and Glu. - Basic Amino Acids: Amino acids whose side chains are basic, including Lys, Arg, and His. -Neutral Amino Acids: amino acids whose side chains are loyal, including Ser, Thr, Asn, Gln, and Cys.

[0053] Furthermore, Gly and Pro are sometimes classified as "amino acids that influence the direction of the main chain", and the amino acids Cys and Met, which contain a sulfur atom in their side chains, are sometimes classified as "sulfur-containing amino acids".

[0054] As used herein, the term "amino acid" includes not only natural amino acids but also unnatural amino acids. Unnatural amino acids include, for example, N-alkylamino acids in which the natural amino acids described above are N-alkylated; and those in which the nitrogen forming the peptide bond is modified with a branched or unbranched lower alkyl group (e.g., C1-C5, preferably C1-C3, more preferably C1). Among N-alkylamino acids, N-ethyl amino acids, N-butyl amino acids, or N-methyl amino acids are preferred, and N-methyl amino acids are more preferred. Furthermore, unnatural amino acids also include D-amino acids (also called D-amino acids), chemically modified amino acids, such as β-amino acids, γ-amino acids, amino acid variants, amino acid derivatives, and the like; amino acids that are not building blocks for proteins in vivo, such as norleucine, ornithine, and the like; and the like. Also included are amino acids in which functional groups have been added to the side chain of a natural amino acid or have been substituted with another functional group (e.g., amino acids having substitutions or additions to moieties such as arylene groups, alkylene groups, etc. in the side chain; amino acids in which the arylene or alkyl groups in the side chain have increased C numbers; amino acids having substitutions in the aromatic ring in the side chain; heterocyclic or fused ring amino acids; etc.).

[0055] 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 a natural amino acid can be imparted. For example, A4p is an amino acid having a piperidyl group in the side chain of alanine, but by adding a piperidyl group, it exhibits the properties of a polar amino acid having basicity, unlike alanine belonging to the nonpolar amino acid group. That is, the above-described groups obtained by classifying natural amino acids based on the properties of their common side chains may include unnatural amino acids having the same properties of the side chain. For example, N-methyllysine (MeK), an amino acid in which the nitrogen atom of the main chain of lysine, which belongs to basic amino acids, is methylated, is an unnatural amino acid, but it exhibits basicity and can be classified as a basic amino acid. Therefore, unnatural amino acids that exhibit the same properties of the side chain as those of a certain amino acid may also be included as targets of conservative amino acid substitution.

[0056] In a non-limiting manner, unnatural amino acids include, but are not limited to, N-methyl amino acids, W5OMe, F4COO, W5H, Aib, ds, W1aa, A4pipaa, Hgn, Har, KCOpipzaa, 4Py, W7N, Ahp, etc. For example, W5OMe, F4COO, W5H, Aib, W1aa, W7N, and Ahp can be classified as hydrophobic amino acids; 4Py, A4pipaa, ds, Har, and KCOpipzaa can be classified as hydrophilic amino acids; furthermore, Ahp and Aib can be classified as aliphatic amino acids; KCOpipzaa can be classified as acidic amino acids; Har, 4Py, and A4pipaa can be classified as basic amino acids; Hgn and ds can be classified as neutral amino acids; W5OMe, W5H, W1aa, F4COO, and W7N can be classified as aromatic amino acids. It should be noted that D-amino acids such as ds may be classified as D-amino acids, but they may also be classified according to the nature of their side chains, and N-methyl amino acids may be classified as N-alkyl amino acids, also according to the nature of the side chain of the original amino acid that has not undergone N-methylation.

[0057] Among the amino acids having an aromatic ring or a substitution thereof in the side chain, the amino acid having an unsubstituted aromatic ring in the side chain is an amino acid having an aromatic ring in the side chain, and includes natural amino acids belonging to the aromatic amino acid group; non-natural amino acids, such as N-acetylated aromatic amino acids; or amino acids having an aromatic ring added or substituted in the side chain of a natural amino acid. Furthermore, the amino acid having an aromatic ring substituted in the side chain is an amino acid having an aromatic ring in the side chain, and includes an amino acid having a ring in which a part of the aromatic ring of a natural amino acid or a non-natural amino acid, such as N-acetylated aromatic amino acid, is substituted with another molecule, a functional group, or a heterocyclic ring, or an amino acid having a fused ring. For example, the amino acid also includes an amino acid having a substitution in the side chain hydroxy group of Tyr; an amino acid having a substitution in the benzene ring of Phe; or an amino acid having a ring containing a heteroatom in the side chain indole ring of Trp; an amino acid having a substitution; and an amino acid having a functional group added thereto.

[0058] Furthermore, among W or its substituted derivatives, unsubstituted W is the natural amino acid tryptophan, and substituted W includes an amino acid derivative of W having a heteroatom in the indole ring of W in the side chain; an amino acid derivative of W in which the hydrogen contained in the NH of the indole ring is substituted; an amino acid derivative of W having a substituent on the benzene ring; and the like.

[0059] Among K or its substituted derivatives, unsubstituted K is lysine, a natural amino acid, and substituted K includes an amino acid derivative of K having a substituent on the amino group of K in the side chain (including a derivative in which the amino group is substituted with hydrogen); an amino acid derivative of K in which the aminobutyl group of K in the side chain is substituted with an aminoalkyl group having a branched or linear structure; and an amino acid derivative of K in which the alkyl group has a substituent.

[0060] Among R or its substituted derivatives, unsubstituted R is arginine, a natural amino acid, and substituted R includes an amino acid derivative of R having a substituent on the guanidino group of R in the side chain (including a derivative in which the guanidino group is substituted with hydrogen); an amino acid derivative of R in which the pentyl group of R in the side chain is substituted with an aminoalkyl group having a branched or linear structure; and an amino acid derivative of R in which the alkyl group has a substituent.

[0061] Among A or its substituted derivatives, unsubstituted A is the natural amino acid alanine, and substituted A includes amino acid derivatives of A in which the α-hydrogen of A is substituted; and the like.

[0062] Among Y or its substituted derivatives, unsubstituted Y is the natural amino acid tyrosine, and substituted Y includes an amino acid derivative of Y in which the phenolic hydroxyl group of Y is substituted in the side chain; an amino acid derivative of Y having a heterocyclic ring, or an amino acid derivative of Y having a fused polycyclic structure; and the like.

[0063] Among F or its substitutes, unsubstituted F is the natural amino acid phenylalanine, and substituted F includes amino acids having a substituent on the benzene ring of phenylalanine in the side chain; amino acid derivatives of F having a heterocyclic ring, or amino acid derivatives of F having a condensed polycyclic structure; and the like.

[0064] Furthermore, the N-alkylamino acid or its modification is an amino acid in which a functional group or an amino acid, a compound, etc. may be added to the side chain of the N-alkylamino acid. With regard to the example of the N-alkylamino acid or its modification, the N-alkylamino acid is N-alkyllysine or N-methyllysine. Another example of the modified N-alkylamino acid is N-methyllysine with an albumin binder attached, or N-methyllysine with one or more of any amino acids attached. Another example of the modified N-alkylamino acid is N-methyllysine with an albumin binder attached to the amino group in the side chain of the N-methyllysine, or N-methyllysine with one or two of glycine, N-methylglycine, or proline attached.

[0065] The above options for X1 to X11 in SEQ ID NO: 1 may be selected in any combination.

[0066] In one embodiment, X1 of SEQ ID NO:1 is Y, 4Py, or F4COO.

[0067] In one embodiment, X2 of SEQ ID NO: 1 is N-methyllysine or a modified version thereof. In a further embodiment, X2 of SEQ ID NO: 1 is N-methyllysine or a modified N-methyllysine.

[0068] In one embodiment, X3 and X4 of SEQ ID NO:1 are each V.

[0069] In one embodiment, X5 of SEQ ID NO:1 is S.

[0070] In one embodiment, X6 of SEQ ID NO:1 is N.

[0071] In one embodiment, X7 of SEQ ID NO:1 is a substituted W. In a further embodiment, X7 of SEQ ID NO:1 is W5OMe, W7N, or W1aa.

[0072] In one embodiment, X8 of SEQ ID NO:1 is substituted F or substituted Y. In a further embodiment, X8 of SEQ ID NO:1 is Y or F4COO.

[0073] In one embodiment, X9 of SEQ ID NO:1 is substituted K or R. In a further embodiment, X9 of SEQ ID NO:1 is K, KCOpipzaa, Hgn, Ahp, or Har.

[0074] In one embodiment, X10 of SEQ ID NO:1 is a substituted W. In a further embodiment, X10 of SEQ ID NO:1 is W5H.

[0075] In one embodiment, X11 of SEQ ID NO: 1 is a substituted A or K. In a further embodiment, X11 of SEQ ID NO: 1 is Aib or A4pipaa.

[0076] In one embodiment, X12 of SEQ ID NO:1 is V.

[0077] In one embodiment, X13 of SEQ ID NO:1 is C.

[0078] The options of one embodiment for X1 to X13 in SEQ ID NO: 1 may be selected in any combination. In this specification, the expressions "in a non-limiting manner" and "in one embodiment" may be used interchangeably.

[0079] In one embodiment, the peptide according to the present invention is a cyclic peptide.As used herein, the expression "cyclic peptide" refers to a peptide that is cyclic in its entirety or in part by binding two amino acids.This peptide also includes the amino acids in peptides that form bridged structures; form cyclic structures by lactam ring formation or macrocyclization; have lassopeptide-like structures, etc.That is, a part of cyclic peptides can form cyclic structures or have linear parts.

[0080] In some cases, some peptides show poor metabolic stability in vivo, and some peptides are large in size, making them difficult to penetrate cell membrane.The method for cyclizing peptide is adapted to take such problems into account.It is suggested that when peptide is cyclized, protease resistance is improved, metabolic stability is improved, and the restriction is added to conformational change, resulting in increased rigidity, and improved membrane permeability and affinity to target protein.

[0081] In one embodiment, the peptide according to the present invention has a cyclic structure in which the chloroacetylated amino acid and the cysteine ​​residue present in the peptide are bonded. In one embodiment, the peptide has a cyclic structure in which the N-terminal amino acid (the first amino acid residue) and the cysteine ​​residue present in the peptide are bonded. In one embodiment, the peptide has a cyclic structure in which the N-terminal amino acid (the first amino acid residue) and the 13th cysteine ​​residue present in the peptide are bonded. In one embodiment, the peptide has a cyclic structure in which the chloroacetylated N-terminal amino acid (the first amino acid residue) and the 13th cysteine ​​residue present in the peptide are bonded. "Chloroacetylation" may be "halogen acetylation" using another halogen. Furthermore, "acetylation" may be "acylation" using an acyl group other than an acetyl group.

[0082] In some embodiments, the peptide according to the invention has an amino acid sequence according to any one of SEQ ID NOs: 2-31.

[0083] In some embodiments, the peptide according to the present invention consists of an amino acid sequence according to any one of SEQ ID NOs: 2-31.

[0084] In some embodiments, the peptide according to the invention is a cyclic peptide having an amino acid sequence according to any one of SEQ ID NOs: 2-31.

[0085] In some embodiments, the peptide according to the present invention is a cyclic peptide consisting of an amino acid sequence according to any one of SEQ ID NOs: 2-31.

[0086] The number of amide bonds (number and length of amino acids) and peptide sites of the peptides contained in the peptides according to the present invention are not particularly limited. The total number of amino acid residues (referring to the number of amino acid residues contained in the peptides forming a cyclic structure, and not including the case where the amino acid residues are further added in the linker to form a cyclic peptide) is preferably 20 residues or less. In some implementations, the number of amino acids is 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or 11 or more. In some other implementations, the number of amino acids is 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, or 12 or less.

[0087] In some embodiments, the peptides according to the invention comprise between 6 and 19 amino acids. In some embodiments, the peptides according to the invention comprise between about 6 and 17 amino acids. In some embodiments, the peptides according to the invention comprise between about 6 and 15 amino acids. In some embodiments, the peptides according to the invention comprise between about 6 and 14 amino acids. In some embodiments, the peptides according to the invention comprise between about 6 and 13 amino acids. In some embodiments, the peptides according to the invention comprise between about 6 and 12 amino acids.

[0088] In some embodiments, the peptides according to the invention comprise between 8 and 19 amino acids. In some embodiments, the peptides according to the invention comprise between about 8 and 17 amino acids. In some embodiments, the peptides according to the invention comprise between about 8 and 15 amino acids. In some embodiments, the peptides according to the invention comprise between about 8 and 14 amino acids. In some embodiments, the peptides according to the invention comprise between about 8 and 13 amino acids. In some embodiments, the peptides according to the invention comprise between about 8 and 12 amino acids.

[0089] In some embodiments, the peptides according to the invention comprise between 10 and 19 amino acids. In some embodiments, the peptides according to the invention comprise between about 10 and 17 amino acids. In some embodiments, the peptides according to the invention comprise between about 10 and 15 amino acids. In some embodiments, the peptides according to the invention comprise between about 10 and 14 amino acids. In some embodiments, the peptides according to the invention comprise between about 10 and 13 amino acids. In some embodiments, the peptides according to the invention comprise between about 10 and 12 amino acids.

[0090] The linker may also be added from a cyclic peptide. Examples of the linker include the above-mentioned amino acid linker (peptide linker), chemical linker, fatty acid linker, nucleic acid linker, sugar chain linker, etc., or may be a complex, such as a chemical linker, peptide linker, etc. An example of a chemical linker is a PEG (polyethylene glycol) linker. For example, the PEG linker may contain 1 to 24 ethylene glycol units. Furthermore, the linker may be a fatty acid linker containing a divalent chemical moiety derived from a fatty acid. The linker may contain at least one amino acid, such as a glycine-rich peptide, such as a peptide having the sequence [Gly-Gly-Gly-Gly-Ser]n (wherein n is 1, 2, 3, 4, 5, or 6) as described in U.S. Pat. No. 7,271,149, which is incorporated herein by reference, or a serine-rich peptide linker as described in U.S. Pat. No. 5,525,491, which is incorporated herein by reference. In a non-limiting manner, there are several cases in which the physical properties (e.g., solubility) of a peptide can be altered by the addition of a linker. In one embodiment, the amino acid linker comprises an amino acid sequence according to any one of SEQ ID NOs: 32-35.

[0091] Linker can be added at any position.For example, linker can be attached to Cys located at N-terminus side, or can be attached to amino acid contained in cyclic peptide.In some examples, linker is attached to Cys located at N-terminus side.

[0092] GhR-binding peptides and peptides with GhR antagonist activity In some embodiments, the peptides according to the invention bind to GhR. In some implementations of these embodiments, the peptides have GhR antagonist activity. In some examples, the peptides bind to human GhR (hGhR) and have hGhR antagonist activity.

[0093] As used herein, the term "GhR" refers to any form of GhR and its variants to retain at least a portion of the activity of GhR.Unless specifically stated otherwise as human GhR (hGhR), GhR includes all of the native sequences of GhR in mammals, such as human, dog, cat, horse and cow.One example of GhR is hGhR (gene ID: 2690), which is human GhR, a protein with two disulfide bridges, a molecular weight of 22 kDa, and a length of 191 amino acids.

[0094] As used herein, the expression "bind to GhR" refers to having the activity of binding to GhR. Binding to GhR can be measured by any known method for measuring intermolecular binding. In a non-limiting manner, for example, it can be determined by competitive binding assay, such as surface plasmon resonance (SPR) assay, scattering analysis, and / or radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich and competitive assay, in any suitable manner, including different variants of given examples known in the art.

[0095] As used herein, the expression "GhR antagonist activity" refers to the activity of inhibiting the biological activity of GhR and / or downstream pathway(s) mediated by GhR signal transduction. Peptides with GhR antagonist activity include peptides that can block, antagonize, and suppress or reduce (to any extent, including to a significant extent) the biological activity of GhR, including downstream pathways mediated by GhR signal transduction, and for example, GH interaction and / or induction of cellular response to GH. For the purposes of the present invention, it will be clearly understood that the phrase "peptide with GHR antagonist activity" encompasses all of the previously defined terms, titles, and functional states and properties, whereby GHR itself, GHR biological activity (including but not limited to its ability to mediate any aspect of insulin-like growth factor-1 (IGF-1) expression), or the result of biological activity is substantially abolished, reduced, or neutralized to any degree of meaning. In one embodiment, the peptide with GhR antagonist activity binds to GhR and affects and / or prevents the interaction between growth hormone and GhR. In some embodiments, the peptide with GhR antagonist activity binds to GhR and affects and / or prevents GhR dimerization.

[0096] In some embodiments, the peptide according to the present invention has a bicyclic structure. As used herein, the expression "bicyclic structure" refers to a peptide having two or more cyclic structures. As an example, a peptide having a bicyclic structure is a cyclic peptide having a cyclic structure in which the chloroacetylated amino acid and cysteine ​​residue present in the peptide are bonded, and the cyclic peptide also has a bridge structure in the ring. In one embodiment, a peptide having a bicyclic structure is a peptide in which the amino acid residue of X2 in SEQ ID NO: 1 is N-methyllysine and the amino acid residue of X7 is W1aa, where the peptide has a cyclic structure in which the chloroacetylated X1 amino acid residue and the cysteine ​​residue of X13 are bonded, and also has a structure in which the amino acid residues of X2 and X7 are bonded. Furthermore, in one embodiment, one or more amino acids may be present between the amino acid residues of X2 and X7. For example, the amino group in the side chain of the X2 amino acid residue, N-methyllysine, may be linked to the carboxylic acid in the side chain of the X6 amino acid residue, W1aa, to form a bicyclic structure, or the amino group in the side chain of the N-methyllysine and the carboxylic acid in the side chain of the X6 amino acid residue, W1aa, may be linked via glycine, N-methylglycine, or proline.

[0097] In some embodiments, the peptide according to the present invention comprises an albumin binder. As used herein, the term "albumin binder" refers to a compound that does not bind covalently to human serum albumin. Binding to human serum albumin can be measured by known methods for measuring binding, such as surface plasmon resonance (SPR) as described above. Exemplary albumin binders suitable for use in the present invention include fatty acids such as myristic acid or palmitic acid, derivatives thereof, or diphenylcyclohexane derivatives. In one embodiment, the albumin binder is a compound that includes linear and branched chain lipophilic groups having 12 to 40 carbon atoms and a distal acidic group. In one embodiment, the albumin binder is any of 4IphpCO, cC14COO, BiPh4pCO, PhpCO, PhPeCO, and 4MePhpCO. Furthermore, as used herein, the albumin binder may have a structure that includes one or more glutamic acids. In some examples, the albumin binder has a structure in which the albumin binder is bound to the N-terminus of a peptide having one glutamic acid or 2 to 5 glutamic acids bound thereto.

[0098] In one embodiment, the albumin binder may be bound to the amino acid present in the cyclic structure of the peptide, or may be further bound to a linker bound to the peptide.As an example, in the case of binding to the amino acid residue of X2 in SEQ ID NO: 1, the N-methyllysine, which is the amino acid residue of X2, and the compound that is not covalently bound to human serum albumin are bound through a structure having one or more glutamic acids.A preferred embodiment is that one or two glutamic acids are bound to the side chain of N-methyllysine, and the albumin binder is bound to its N-terminus.

[0099] Furthermore, as an example, an amino acid linker having human serum albumin containing a structure having one or more glutamic acids is bound to the peptide. In a preferred embodiment, an amino acid linker having K (SEQ ID NO: 32) or GP-(ds)3-K (SEQ ID NO: 35) is bound to Cys present at the C-terminus of the peptide, one or two glutamic acids are bound to the side chain of lysine of the amino acid linker, and an albumin binder is bound to its N-terminus.

[0100] Aspects of payload-bound PDC In one embodiment, the present invention relates to a complex comprising any of the peptides described herein, a linker attached to the peptide, and a substance attached to the linker, wherein the peptide is capable of binding to the GhR, such that the complex is capable of delivering the substance to the GhR.

[0101] The substance can be any substance desired by one of skill in the art, so long as it is a substance that one of skill in the art desires to deliver to the GhR. Examples of substances include, but are not limited to, the following: - Compounds: include low molecular weight compounds, medium molecular weight compounds, examples of which include known low molecular weight drugs. - Peptides: They may be peptides, for example cyclic peptides, that bind to targets in the body and exert some type of effect. - RI: Any compound that can be labeled with a radioisotope, such as a low or medium molecular weight compound or an antibody labeled with a radioisotope. Examples include compounds for PET scans. - Protein: Any protein that exhibits a useful function in the body, such as an antibody or an enzyme. Examples include enzymes used in enzyme replacement therapy. - Nucleic acid: any substance having a base sequence, such as DNA and RNA. Examples include nucleic acid medicines. - Molecules used in drug delivery systems (DDS): may be known molecules used in DDS, such as liposomes or micelles. The DDS molecule may further contain compounds therein, such as pharmaceutical agents.

[0102] The DDS molecule may also be a complex combining several of the examples given above.

[0103] Peptide production The peptides according to the invention can be produced by any known method for producing peptides, such as, for example: - Chemical synthesis methods such as liquid phase, solid phase, and hybrid methods combining liquid and solid phase methods; - Genetic engineering methods; etc. In some instances where the peptides according to the invention are produced by chemical synthesis methods, the peptides according to the invention can be said to be synthetic peptides.

[0104] In the solid phase method, for example, the hydroxy group of the resin having the hydroxy group and the carboxy group of the first amino acid (usually the C-terminal amino acid of the target peptide) whose α-amino group is protected by a protecting group are subjected to an esterification reaction. For the esterification catalyst, known dehydrating agents and condensing agents, such as 1-mesitylenesulfonyl-3-nitro-1,2,4-triazole (MSNT), dicyclohexylcarbodiimide (DCC), and diisopropylcarbodiimide (DIC), can be used.

[0105] Next, the protecting group of the α-amino group of the first amino acid is removed, a second amino acid in which all functional groups except the carboxy group of the main chain are protected is added, the carboxy group is activated, and the first and second amino acids are linked. Furthermore, the α-amino group of the second amino acid is deprotected, a third amino acid in which all functional groups except the carboxy group of the main chain are protected is added, the carboxy group is activated, and the second and third amino acids are linked. This is repeated until a peptide of the target length is synthesized, after which all functional groups are deprotected.

[0106] Examples of resins for solid phase synthesis include Merrifield resin, MBHA resin, Cl-Trt resin, SASRIN resin, Wang resin, Rink amide resin, HMFS resin, amino-PEGA resin (Merck KGaA), HMPA-PEGA resin (Merck KGaA), etc. These resins may be used after being washed with a solvent (dimethylformamide (DMF), 2-propanol, methylene chloride, etc.).

[0107] Examples of protecting groups for α-amino groups include benzyloxycarbonyl (Cbz or Z) groups, tert-butoxycarbonyl (Boc) groups, fluorenylmethoxycarbonyl (Fmoc) groups, benzyl groups, allyl groups, allyloxycarbonyl (Alloc) groups, etc. Cbz groups can be deprotected by treatment with hydrofluoric acid, hydrogenation, etc., Boc groups can be protected by treatment with trifluoroacetic acid (TFA), and Fmoc groups can be deprotected by treatment with piperidine or pyrrolidine.

[0108] The α-carboxy group may be protected using such examples as methyl esters, ethyl esters, allyl esters, benzyl esters, tert-butyl esters, cyclohexyl esters, and the like.

[0109] The activation of carboxyl group can be carried out using a condensation agent.Examples of the condensation agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU) and the like.

[0110] Cleavage of the peptide chain from the resin can be carried out by treating the peptide chain with an acid, such as TFA, hydrogen fluoride (HF), or the like.

[0111] The production of peptides by recombinant gene methods (translation / synthesis systems) can be carried out using nucleic acids that code for the peptides. The nucleic acids that code for the peptides can be DNA or RNA.

[0112] Nucleic acids encoding peptides can be prepared by known methods or equivalent methods. For example, peptides can be synthesized by an automatic synthesizer. Restriction enzyme recognition sites can be added and the resulting DNA can be inserted into a vector. Alternatively, a base sequence encoding an amino acid sequence for splicing the formed peptide chain can be incorporated using an enzyme or the like.

[0113] As described above, when the peptide is fused to a cell penetrating peptide or the like, the nucleic acid also includes a nucleic acid encoding the cell penetrating peptide.

[0114] Degradation by host-derived proteases can also be suppressed using chimeric protein expression methods to express a target peptide as a chimeric peptide of another peptide. In this case, a nucleic acid encoding the target peptide and a peptide bound to it can be used as the nucleic acid.

[0115] Then, an expression vector is prepared using the nucleic acid encoding the peptide. The nucleic acid may be directly or by digestion with a restriction enzyme, or the nucleic acid may be inserted downstream of the promoter of the expression vector by adding a linker or the like. Examples of vectors include E. coli-derived plasmids (pBR322, pBR325, pUC12, pUC13, pUC18, pUC19, pUC118, pBluescript II, etc.), Bacillus subtilis-derived plasmids (pUB110, pTP5, pC1912, pTP4, pE194, pC194, etc.), yeast-derived plasmids (pSH19, pSH15, YEp, YRp, YIp, YAC, etc.), bacteriophages (e phage, M13 phage, etc.), viruses (retroviruses, vaccinia viruses, adenoviruses, adeno-associated viruses (AAV), cauliflower mosaic viruses, tobacco mosaic viruses, baculoviruses, etc.), cosmids, etc.

[0116] The promoter can be appropriately selected according to the type of the host. When the host is an animal cell, for example, a promoter derived from SV40 (simian virus 40) or a promoter derived from CMV (cytomegalovirus) can be used. When the host is E. coli, a trp promoter, a T7 promoter, a lac promoter, etc. can be used.

[0117] The expression vector may incorporate, for example, a DNA replication origin (ori), a selection marker (antibiotic resistance, auxotrophy, etc.), an enhancer, a splicing signal, a polyA addition signal, a nucleic acid encoding a tag (FLAG, HA, GST, GFP, etc.), and the like.

[0118] Next, a suitable host cell is transformed by the expression vector. The host can be appropriately selected in relation to the vector. Examples such as E. coli, Bacillus subtilis, yeast, insect or insect cells, animal cells, etc. can be used as hosts. As animal cells, for example, HEK293T cells, CHO cells, COS cells, myeloma cells, HeLa cells, and Vero cells can be used. Depending on the type of host, transformation can be performed according to known methods, such as lipofection, calcium phosphate, electroporation, microinjection, gene gun, etc. The target peptide is expressed by culturing the transformant according to conventional methods.

[0119] For purification of peptides from transformant cultures, the cultured cells are harvested and then suspended in an appropriate buffer solution, followed by cell disruption by methods such as sonication, freeze-thawing, etc., and then the crude extract is obtained by centrifugation or filtration. If the peptide is secreted into the culture solution, the supernatant is collected.

[0120] The crude extract or culture supernatant can also be purified by known methods or equivalent methods (e.g., salting out, dialysis, ultrafiltration, gel filtration, SDS-PAGE, ion exchange chromatography, affinity chromatography, reverse-phase high performance liquid chromatography, etc.).

[0121] The resulting peptide may be converted from the free form to a salt, or from a salt to the free form, by known methods or methods equivalent thereto.

[0122] In one embodiment, the translation / synthesis system can be a cell-free translation system. Cell-free translation systems can obtain highly pure forms of expression products, generally without purification. Cell-free translation systems include, for example, ribosomal proteins, aminoacyl-tRNA synthase (ARS), ribosomal RNA, amino acids, rRNA, GTP, ATP, translation inhibitors (IF), elongation factors (EF), release factors (RF), and ribosome recycling factors (RRF), or other factors required for translation. E. coli extract or wheat germ extract may be added to increase expression efficiency. In addition, rabbit erythrocyte extract or insect cell extract may be added.

[0123] By continuously supplying energy to the system containing these using dialysis, hundreds of μg to several mg / mL of protein can be produced in a non-limiting manner. The system may also contain RNA polymerase to transcribe genomic DNA. Examples of commercially available cell-free translation systems that can be used include RTS-100 (registered trademark) from Roche Diagnostics KK, PURE System from GeneFrontier Corporation, PURExpress in vitro protein synthesis kit from New England Biolabs Inc., and the like for systems derived from E. coli, and ZOIGENE, a system from CellFree Sciences Co., Ltd., for systems using wheat germ extract.

[0124] In a cellular translation system, an artificial aminoacyl-tRNA may be used, in which a desired amino acid or hydroxy acid may be linked (acylated) to a tRNA instead of the aminoacyl-tRNA synthesized by the natural aminoacyl-tRNA synthase. The aminoacyl-tRNA may be synthesized using an artificial ribozyme.

[0125] Examples of ribozymes include flexizyme (H. Murakami, H. Saito, and H. Suga, (2003), Chemistry & Biology, Vol. 10, 655-662; and WO2007 / 066627, etc.), all of which are incorporated herein by reference. Flexizyme is also known by the names prototype flexizyme (Fx), newly modified dinitrobenzyl flexizyme (dFx), enhanced flexizyme (eFx), aminoflexizyme (aFx), etc.

[0126] The desired codon can be translated in association with the desired amino acid or hydroxy acid by using the tRNA generated by flexizyme, to which the desired amino acid or hydroxy acid is linked. A special amino acid can also be used as the desired amino acid. For example, the unnatural amino acid required for cyclization described above can also be introduced into the linked peptide by this method.

[0127] For example, various methods commonly used in the art can be used for chemical synthesis of peptides, including stepwise solid-phase synthesis, semi-synthesis of peptide fragments undergoing conformationally supported religation, and chemical ligation. Synthesis of peptides is chemical synthesis using various solid-phase techniques, such as those described in KJ Jensen, PT Shelton, SL Pedersen, Peptide Synthesis and Applications, 2nd Edition, Springer, 2013, etc. A preferred strategy is based on a combination of protecting groups, such as the Fmoc group, which can temporarily protect the α-amino group and can be selectively removed using base, and the Fmoc group, which temporarily protects the side chain functional groups and is stable under Fmoc deprotection conditions. This type of general peptide side chain selection is described in the aforementioned Peptide Synthesis and Applications, 2nd Edition; GB Fields, RL Noble, Solid Phase Peptide Synthesis Utilizing 9-Fluorenylmethoxycarbonyl Amino Acids, Int.J.Peptide Protein. Res. 35, 1990, 161-214; however, preferred peptide side chain protecting groups include, for example, benzyl, tert-butyl and trityl (Trt) groups for the hydroxyl groups of serine or threonine; 2-bromobenzyloxycarbonyl or tert-butyl groups for the hydroxyl groups of tyrosine; Boc, methyltetrazolethiol (Mtt), Alloc and ivDde groups for the amino groups of lysine side chains; Trt or Boc groups for midazole groups; 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) group for the guanidyl group of arginine; tert-butyl, allyl, and 3-methylpentane (Mpe) groups for carboxyl groups such as glutamic acid and aspartic acid; Trt group for the carboxamide group of glutamine or asparagine; or Trt and monomethoxytrityl (Mmt) groups for the thiol group of cysteine.

[0128] Peptides may be synthesized by a stepwise method on the solid-phase resin described above. The C-terminal amino acid used and all of the amino acids or peptides used for synthesis must have their α-amino protecting groups selectively removed during the synthesis process. Preferably, the solid-phase resin described above is used, and the C-terminal carboxyl group of a peptide with its N-terminus appropriately protected by Fmoc or the C-terminal carboxyl group of an amino acid with its N-terminus protected by Fmoc is made into an activated ester by a suitable reagent, which is then added to the amino group on the solid-phase resin to initiate. Subsequent elongation of the peptide chain can be achieved by removing the N-terminal protecting group (Fmoc group), followed by successive repetitions of condensation of the protected amino acid derivative with the amino acid sequence of the target peptide. It should be noted that these may release the target peptide in the final step. Examples of releasing conditions are given in Teixeira, WEBenckhuijsen, PEde Koning, ARPMValentijn, JW Drijfhout, Protein Pept.Lett., 2002, 9, 379-385, etc., and the peptide can be released into a TFA solution containing water / silyl hydride / thiol as a scavenger in TFA. A typical example is TFA / water / TIS / DODT (volume ratio 92.5:2.5:2.5:2.5).

[0129] Synthesis of the peptides described herein may be carried out using a single or multi-channel peptide synthesizer, such as a Liberty Blue synthesizer manufactured by CEM Corporation, a Syro I synthesizer manufactured by Biotage Japan, Ltd., or a successor thereof.

[0130] The activation of carboxyl group can be carried out using a condensation agent.Examples of the condensation agent include dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIPCDI), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC or WSC), (1H-benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP), 1-[bis(dimethylamino)methyl]-1H-benzotriazolium-3-oxide hexafluorophosphate (HBTU) and the like.

[0131] Cyclization of peptides can be performed according to known methods. In a non-limiting manner, by designing a peptide to contain two or more cysteine ​​residues, for example, a ring structure can be formed post-translationally by disulfide bonds. Furthermore, according to the method of Goto et al. (Y.Goto et al. ACS Chem.Biol.3 120-129(2008)), a peptide with a chloroacetyl group at its N-terminus can be synthesized by genetic code reprogramming technology and cyclized by placing a cysteine ​​residue containing a sulfur molecule in the peptide. Therefore, the mercapto group spontaneously performs a nucleophilic attack on the chloroacetyl group post-translationally, and the peptide is cyclized by a thioether bond. A combination of other amino acids that bind to form a ring can be placed in the peptide and cyclized by genetic code reprogramming technology. Alternatively, cyclization can be performed by placing an L-2-aminoadipic acid residue in the peptide and binding it to the main chain amino acid at the N-terminus. In this manner, known cyclization methods can be used without any particular restrictions.

[0132] Pharmaceutical Compositions The present invention also relates to a pharmaceutical composition comprising the peptide according to the present invention. The disease targeted by the pharmaceutical composition according to the present invention is related to the hypersecretion of human growth hormone, which shows various symptoms due to the hypersecretion of growth hormone, preferably hypersecretion of pituitary somatotropin, which is caused by tumors and inflammation of the pituitary gland, etc. Furthermore, the target includes diseases caused by the hypersecretion of growth hormone, such as acromegaly or gigantism. The peptide according to the present invention is useful as an active ingredient of a pharmaceutical composition for treating diseases related to the hypersecretion of human growth hormone, as well as acromegaly and gigantism.

[0133] In some embodiments, the pharmaceutical composition has GhR antagonist activity.In some embodiments, the pharmaceutical composition has hGhR antagonist activity.

[0134] When growth hormone is produced in the pituitary gland, it is secreted into the bloodstream and binds to growth hormone receptor (GhR) expressed on various cell surfaces, such as liver, muscle tissue, and bone tissue.The binding of GH and GhR induces the production of insulin-like growth factor-1 (IGF-1) in cells, especially liver cells.It is known that in acromegaly and gigantism described below, the increase in growth hormone in plasma and / or tissue is associated with the increase in IGF-I level in plasma and / or tissue.

[0135] Excessive or insufficient secretion of GH is known to cause disease. Excessive secretion of GH leads to a condition called acromegaly (sometimes called gigantism when occurring in children), which presents symptoms such as excessive bone elongation, soft tissue hypertrophy, cardiovascular and gastrointestinal symptoms, and insulin resistance. In many cases, this condition is due to growth hormone-secreting cells becoming tumorigenic (pituitary tumors) while maintaining their secretory function, and is rare with a frequency of occurrence of 4-24 per 100,000 people. If not treated properly, there is a high possibility of complications such as metabolic diseases, e.g. diabetes and hypertension, angina, myocardial infarction, cerebrovascular conditions, colon and thyroid cancer, etc., which therefore require early diagnosis and treatment.

[0136] The most common treatment for acromegaly is surgical removal of the pituitary tumor, however, the tumor may be large and therefore difficult to remove, or GH secretion may remain excessive even after removal, in which case drug therapy is used.

[0137] Compounds having activity of inhibiting GhR and compositions containing such compounds for the treatment of acromegaly and gigantism are desired.Therefore, the present specification also discloses the aforementioned peptides having GhR antagonist activity and pharmaceutical compositions for the treatment of diseases associated with hypersecretion of human growth hormone, preferably acromegaly and gigantism.In some embodiments, the pharmaceutical composition according to the present invention is a pharmaceutical composition for the treatment of diseases associated with hypersecretion of human growth hormone.In some embodiments, the pharmaceutical composition according to the present invention is for the treatment of acromegaly or gigantism.

[0138] As used herein, the expression "diseases associated with hypersecretion of human growth hormone" refers to diseases that exhibit various symptoms mainly caused by excess peripheral hormones resulting from the hypersecretion of one or more hormones from the pituitary gland.The causes of the disease include disorders of the pituitary gland itself, disorders of the hypothalamus that controls the secretion of pituitary hormones, and disorders of the pituitary stalk that connects the pituitary gland and the hypothalamus, as well as combinations thereof.Since the peptide according to the present invention induces therapeutic effects by binding to growth hormone receptor, the causes of the disease are not particularly limited.

[0139] "Acromegaly" is a condition caused by excessive secretion of growth hormone, and may be referred to as gigantism when it occurs during childhood. In children, when acromegaly occurs before puberty, the long bones do not stop growing, and the height and limbs grow to abnormal lengths. However, in acromegaly occurring in adults, the bones are deformed or enlarged rather than elongated. Enlargement of non-bone tissues may also occur, for example, acromegaly may cause heart failure due to enlargement of the heart, vision problems due to enlargement of tissue compressing nerves, weakening of the limbs, etc. In addition, it is known that diabetes, high blood pressure, sleep apnea syndrome, tumors that may become cancer, etc. are more likely to occur. Therefore, treatment using a pharmaceutical composition containing the peptide according to the present invention for diseases associated with excessive secretion of human growth hormone (such as, but not limited to, diseases associated with increased expression, production, and / or secretion of human growth hormone) may result in the prevention of heart failure, diabetes, high blood pressure, sleep apnea syndrome, and certain (especially colon) tumors caused by acromegaly and gigantism.

[0140] In some embodiments, the pharmaceutical composition according to the present invention may contain the peptide itself or a pharma- ceutically acceptable salt of the peptide. The term "peptide" as used herein may include a pharma- ceutically acceptable salt of the peptide, unless otherwise specified. The pharmaceutical composition preferably contains an effective amount of the peptide as an active ingredient.

[0141] The salt of the peptide (the pharma- ceutically acceptable salt) is preferably an acid addition salt. For example, salts of inorganic acids (such as hydrochloric acid, phosphoric acid, hydrobromic acid, and sulfuric acid) and salts of organic acids (such as acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, and benzenesulfonic acid) are used as such salts. The peptide or its salt also includes solvates such as hydrates.

[0142] In this specification, the administration form of pharmaceutical composition is not particularly limited, and can be oral or parenteral.Examples of parenteral administration include injection, such as intramuscular injection, intravenous injection, or subcutaneous injection; transdermal administration; transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, or rectal); etc.

[0143] Pharmaceutical compositions may be modified in various ways, taking into account the properties of polypeptides that are easily metabolized and excreted. For example, polyethylene glycol (PEG) or sugar chains may be added to the polypeptide to extend its retention time in blood and reduce antigenicity. Furthermore, biodegradable polymeric compounds, such as polylactic acid / glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, and emulsions, nanoparticles, nanospheres, etc. prepared in unsaturated fatty acids may be used as release control substrates, and the polypeptide may be present in the substrate. In the case of transdermal administration, a weak electric current passes through the skin surface and allows it to penetrate the stratum corneum (iontophoresis).

[0144] Regarding the pharmaceutical composition, the active ingredient may be used as it is, or the pharmaceutical composition may contain pharma- ceutically acceptable carriers, excipients, additives, etc., or may be formulated. Examples of dosage forms include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powdered drugs, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, patches, etc. Formulation may be carried out by a general method, for example, using excipients, binders, disintegrants, lubricants, dissolving agents, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption promoters, surfactants, pH adjusters, preservatives, antioxidants, etc., as necessary.

[0145] Examples of ingredients used for formulation include, but are not limited to, purified water, physiological saline, phosphate buffer solution, dextrose, glycerol, pharma- ceutically acceptable organic solvents, such as ethanol, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, 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, and human serum albumin.

[0146] Considering the fact that peptides are generally difficult to absorb through mucous membranes, the pharmaceutical composition may contain an absorption enhancer to improve the absorption of poorly absorbed drugs.The following may be used as absorption enhancers: surfactants, such as polyoxyethylene lauryl ether, sodium lauryl sulfate, and saponin; bile salts, such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents, such as EDTA and salicylic acid; fatty acids, such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosan, nitric oxide donors, etc.

[0147] When the pharmaceutical composition is a pill or tablet, it may be coated using a sugar coating, or a gastric or enteric coating material.

[0148] When the pharmaceutical composition is an injection, it may contain distilled water for injection, physiological saline, propylene glycol, polyethylene glycol, vegetable oil, alcohol, etc. In addition, a water retaining agent, an emulsifier, a dispersing agent, a stabilizer, a dissolving agent, a solubilizing agent, a preservative, etc. may be added.

[0149] Furthermore, the pharmaceutical composition may target not only humans, but also non-human mammals or birds, including non-human primates (monkeys, chimpanzees, gorillas, etc.), livestock animals (pigs, cows, horses, sheep, etc.), dogs, cats, rats, mice, guinea pigs, rabbits, etc.

[0150] In particular, the dosage when administered to humans varies depending on symptoms, the patient's age, sex and weight, sensitivity differences, administration method, administration interval, type of active ingredient, and type of formulation, and it may be administered in a non-limiting manner, for example, by administering once or divided into several doses, between about 30 μg to about 100 g, between about 1 μg to about 10 g, between about 1 μg to about 1 g, between about 10 μg to about 1 g, between about 10 μg to about 1 g, between about 10 μg to about 500 mg, between about 100 μg to about 10 g, between about 100 μg to about 1 g, between about 10 μg to about 500 mg, between about 100 μg to about 500 mg, or between about 100 μg to about 100 mg. In the case of injections, about 1 μg / kg to about 3,000 μg / kg or about 3 μg / kg to about 1,000 μg / kg may be administered at once or divided into several doses depending on the patient's body weight.

[0151] The present invention also relates to a method for treating a disease associated with hypersecretion of human growth hormone by administering to a subject a peptide according to the invention.

[0152] The present invention also relates to the use of a peptide according to the invention for the treatment of diseases associated with hypersecretion of human growth hormone.

[0153] The invention also relates to the use of the peptides for preparing a pharmaceutical composition for the treatment of diseases associated with the hypersecretion of human growth hormone.

[0154] The present invention also relates to a peptide according to the invention for use in a method for treating a disease associated with hypersecretion of human growth hormone. EXAMPLES

[0155] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples. Those skilled in the art can easily make modifications and changes to the present invention based on the description in this specification, and these are included in the technical scope of the present invention.

[0156] Example 1 - Chemical Synthesis All raw materials, building blocks, reagents, acids, bases, solid phase resins, and solvents used in the chemical synthesis in the following examples can be obtained commercially or the synthesis can be completed by one skilled in the art using organic chemistry techniques. It is noted that commercially available products were used for amino acids containing protecting groups unless otherwise specified.

[0157] The extension of the peptide chain on the solid phase resin is carried out by using the resin described in each example as the starting material and by using standard peptide coupling and Fmoc removal reaction conditions. The reactions were carried out using an automated peptide synthesizer Liberty Blue manufactured by CEM according to the manufacturer's manual. As examples, some of the common amino acids used are listed below, with the side chain protecting groups indicated in brackets: - Fmoc-N-Me-Lys(alloc)-OH; - Fmoc-Tyr(tBu)-OH; - Fmoc-F4COO(tBu)-OH; - Fmoc-Val-OH; - Fmoc-Ser(tBu)-OH; - Fmoc-Asn(Trt)-OH; - Fmoc-W1aa(allyl)-OH; - Boc-Lys(Fmoc)-OH; - Fmoc-Lys(ivDde)-OH; - Fmoc-Lys(Boc)-OH; - Fmoc-Lys(alloc)-OH; - Fmoc-W5H-OH; - Fmoc-Glu-OtBu; - Fmoc-Aib-OH; - Fmoc-A4pipaa(tBu)-OH; - Fmoc-W7N-OH; - Fmoc-W5OMe-OH; - Fmoc-Cys(Trt)-OH; - Fmoc-Gly-OH; - Fmoc-Pro-OH; and - Fmoc-ds(tBu)-OH.

[0158] Reverse phase separation HPLC was performed as a method for purifying the obtained crude peptide using an AutoPurification system-SQD2 single quadrupole mass spectrometer manufactured by Waters, and elution was performed while monitoring the m / z ions derived from the target product. It was confirmed that the mass spectrum obtained in ESI positive scan mode and the mass spectrum containing multiply charged ions calculated by the molecular formula of the target product were consistent within the error range of the mass spectrometer used. It should be noted that the purification conditions including the column used are shown in each example.

[0159] For the structural determination of chemically synthesized peptides, the molecular weights calculated taking into account the amino acids used according to the target sequence and, if necessary, the building blocks used, were confirmed by ESI-MS(+) in mass spectrometry. Note that "ESI-MS(+)" indicates electrospray ionization mass spectrometry performed in positive ion mode. The detected masses are reported in "m / z" units. It should be noted that compounds with molecular weights greater than approximately 1,000 are frequently detected as doubly or triply charged ions.

[0160] Example 2 - Identification of hGhR-binding peptides Growth hormone receptor (GhR) binding peptides were screened and identified by a screening method similar to that described in patent documents WO2014 / 119600, WO2012 / 033154, and WO2007 / 066627, all of which are incorporated herein by reference. In this screening, recombinant human GhR protein (R&D systems) fused to the Fc portion of human IgG was used as a bait protein to enrich GhR binding peptides. The binding of peptides obtained from the screening was first tested using in vitro translated peptides fused to DNA / RNA tags expressed from singleton DNA templates. The in vitro translated peptides were incubated with human GhR-Fc immobilized on protein G magnetic beads, and the amount of peptides coprecipitated with the beads was quantified by qPCR. The peptides were then chemically synthesized to quantitatively confirm whether they have binding activity to GhR.

[0161] Example 3 - Synthesis of GhR-binding peptides All raw materials, building blocks, reagents, acids, bases, solid phase resins, and solvents used in the chemical syntheses in the following examples were either commercially available products used as received or organic chemistry methods used by those skilled in the art. Unless otherwise stated, commercially available amino acids containing protecting groups were used as received.

[0162] For structural determination of chemically synthesized peptides, the molecular weight calculated considering the amino acids used according to the target sequence and, if necessary, the building blocks used, was confirmed by ESI-MS(+) in mass spectrometry. Please note that "ESI-MS(+)" indicates electrospray ionization mass spectrometry performed in positive ion mode. The detected mass was reported in "m / z" units. Compounds with molecular weights greater than about 1000 were frequently detected as doubly or triply charged ions. Column: CORTECS® UPLC® C18 column (Japan Waters), 9 Å, 1.6 μm, 2.1×100 mm; Mobile phase: 0.025% TFA in MeCN / H2O; Temperature: 40°C; Gradient: 0.025% TFA in MeCN / H2O from 5 to 95% in 5.56 min; Linear gradient, Flow rate: 0.4 mL / min, Detection: UV 220 nm. Peptide synthesis was carried out according to standard solid phase synthesis methods using Sieber Amide resin (product of Novabiochem); 9-fluorenylmethoxycarbonyl group (Fmoc) as the protecting group of the α-amino group; and automated Liberty Blue (CEM Inc.).

[0163] GhR-binding peptides were synthesized: peptide synthesis was performed according to general solid-phase synthesis methods using Sieber Amide resin (a product of Novabiochem); 9-fluorenylmethoxycarbonyl group (Fmoc) as the protecting group for the α-amino group; 20% piperidine in DMF for Fmoc deprotection; 4.2 equivalents of Fmoc-amino acid, 4 equivalents of Oxyma Pure, and 8 equivalents of N,N''-diisopropylcarbodiimide (DIC) as coupling reagents for peptide elongation; and automated Liberty Blue (CEM Inc.).

[0164] The cyclic peptide was purified by reversed-phase high performance liquid chromatography (HPLC) using an AutoPurification system-SQD2 single quadrupole mass spectrometer (Waters product) to obtain the intended product. The peptide thus obtained was identified by the mass spectrum obtained in ESI-positive scan mode, and the mass spectrum containing the multiply charged ions calculated from the molecular formula of the target object matched within the error range of the mass spectrometer used. Column: Kinetex EVO C18 2.6um, 2.1ID×150mm, 100 angstroms (with guard cartridge 2.1mmID), mobile phase A: 0.025% TFA in H2O, mobile phase B: 0.025% TFA in MeCN. Temperature: 60°C, Gradient: 20-60% B over 7.15 min, 60-95% B over 0.3 min, 95% B over 1.55 min, 95-20% B over 0.01 min, then 20% B over 3.49 min, Flow rate: 0.5 mL / min, Detection: UV 225 nm, 20-60 / 7.15 min, 60-95 / 0.3 min, 95-95 / 1.55 ​​min, 95-20 / 0.01 min, 20-20 / 3.49 min.

[0165] The synthesis of bicyclic peptides (compound numbers 1 to 32 in Table 1-1) is carried out as described below. Synthesis of a compound comprising PD-217 (Compound No. 9 in Table 1-1); a bicyclic peptide (peptide SEQ ID NO: 10) and an albumin binder (E_E_4IPhpCO) conjugated linker (SEQ ID NO: 32) [ka]

[0166] The Fmoc amino acids used in the synthesis included Boc-Lys(Fmoc)-OH; Fmoc-Cys(Trt)-OH; Fmoc-Val-OH; Fmoc-Aib-OH; Fmoc-W5H-OH; Fmoc-Lys(ivDde)-OH; Fmoc-F4COO(tBu)-OH; Fmoc-W1aa(allyl)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Ser(tBu)-OH; Fmoc-MeK(alloc)-OH.

[0167] To the reaction vessel containing the resin, 20% piperidine in DMF was added and the mixture was stirred. Synthesis was initiated from Boc-Lys(Fmoc)-OH using general solid phase synthesis. Double couplings were performed at positions 1, 3, 10, and 11 from the N-terminal amino acid.

[0168] After the N-terminal Fmoc-F4COO(tBu)-OH was linked, 0.25 equivalents of (Pd(PPh3)4), 15 equivalents of PhSiH3, and DCM were added and the resulting mixture was vortexed to remove the alloc and allyl groups on the side chains of N-methyl-L-lysine and 1-(carboxymethyl)-L-tryptophan, respectively. The peptide resin thus obtained was treated with 16 equivalents of DIC and 8 equivalents of Oxyma pure in DMF under microwave irradiation.

[0169] After the Fmoc group was deprotected with 20% piperidine in DMF, the resin was washed with DMF. 5 equivalents of 2-chloroacetic acid, 5 equivalents of DIC, and 5 equivalents of HOSu in DCM / NMP (1:1 v / v) were then added to the resin. The resin was washed successively with DMF and DCM, and then dried.

[0170] A mixture of TFA-water-TIS-DODT (92.5:2.5:2.5:2.5 v / v / v / v) was added and the resulting mixture was stirred at room temperature for 1.25 h.

[0171] The crude peptide was cleaved from the resin and collected by ether precipitation. After washing three times with diisopropyl ether and drying, DMSO-water-MeCN (1:1:1 v / v / v) containing 15 equivalents of triethylamine was added to give a final concentration of 5 mM, followed by stirring at room temperature for 2 h.

[0172] After removal of the solvent, a solution of DMSO-water (9:1 v / v), 1.02 equivalents of 1-(tert-butyl)5-(2,5-dioxopyrrolidin-1-yl)((S)-5-(tert-butoxy)-4-(4-(4-iodophenyl)butanamido)-5-oxopentanoyl)-L-glutamate, and 3 equivalents of N-ethyl-N-isopropylpropan-2-amine was added and the mixture was stirred for 1 h. The solution was concentrated and the product was reprecipitated from ether and dried.

[0173] A mixture of TFA-water-TIS-DODT (92.5:2.5:2.5:2.5 v / v / v / v) was added and the mixture was stirred at room temperature for 30 min. The peptide was collected by ether precipitation and dried under reduced pressure.

[0174] The ivDde group on the lysine at position 9 from the N-terminal amino acid was cleaved using 25 equivalents of hydrazine monohydrate in DMSO. After stirring the mixture for 2.5 hours, the reaction was quenched with 40 equivalents of acetic acid. The crude peptide was confirmed by analytical conditions by LCMS: retention time = 1.48 min.

[0175] The crude peptide was purified by reverse phase HPLC and the product was confirmed by HPLC under the conditions as shown below: Analytical HPLC conditions: retention time = 4.220 min. MS(ESI+);[M+2H] 2+ The calculated value was 1242.8, and the actual value was 1242.6. Synthesis of PD-233 (Compound No. 11 in Table 1-1); a compound containing a bicyclic peptide (peptide SEQ ID NO: 10) and an albumin binder (EE-cC14COO) conjugated linker (SEQ ID NO: 32) [ka]

[0176] The Fmoc amino acids used in the synthesis included Fmoc-Lys(alloc)-OH; Fmoc-Glu-OtBu; Fmoc-Cys(Trt)-OH; Fmoc-Val-OH; Fmoc-Aib-OH; Fmoc-W5H-OH; Fmoc-Lys(Boc)-OH; Fmoc-F4COO(tBu)-OH; Fmoc-W1aa(allyl)-OH; Fmoc-Asn(Trt)-OH; Fmoc-Ser(tBu)-OH; Fmoc-MeK(alloc)-OH.

[0177] To the reaction vessel containing the resin, 20% piperidine in DMF was added and the mixture was stirred. Synthesis was initiated from Fmoc-Lys(alloc)-OH using general solid phase synthesis methods. Double couplings were performed at positions 1, 3, and 11 from the N-terminal amino acid.

[0178] After two Fmoc-Glu-OtBu monomers (CAS: 84793-07-7) and 16-(tert-butoxy)-16-oxohexadecanoic acid (CAS: 843666-27-3) were linked to Fmoc-Lys(alloc)-OH, 0.25 equivalents of Pd(PPh3)4, 15 equivalents of PhSiH3, and DCM were added, and the resulting mixture was stirred to remove the alloc group on the side chain of lysine. The peptide resin thus obtained was used in further peptide synthesis.

[0179] After the N-terminal Fmoc-F4COO(tBu)-OH was linked, 0.25 equivalents of Pd(PPh3)4, 15 equivalents of PhSiH3, and DCM were added and the resulting mixture was vortexed to remove the alloc and allyl groups on the side chains of MeK and W1aa, respectively. The peptide resin thus obtained was treated with 16 equivalents of DIC and 8 equivalents of Oxyma pure in DMF under microwave irradiation.

[0180] After the Fmoc group was deprotected with 20% piperidine in DMF, the resin was washed with DMF. 5 equivalents of 2-chloroacetic acid, 5 equivalents of DIC, and 5 equivalents of HOSu in DCM / NMP (1:1 v / v) were then added to the resin. The resin was washed successively with DMF and DCM, and then dried.

[0181] A mixture of TFA-water-TIS-DODT (90:2.5:2.5:5 v / v / v / v) was added and the resulting mixture was stirred at room temperature for 1.25 h.

[0182] The crude peptide was cleaved from the resin and collected by ether precipitation. After washing with diisopropyl ether three times and drying, DMSO-water-MeCN (2:1:1 v / v / v) containing 15 equivalents of triethylamine was added to obtain a final concentration of 5 mM, followed by stirring at room temperature for 4 hours. The crude peptide was confirmed by LCMS analysis conditions: retention time = 2.36 min.

[0183] The crude peptide was purified by reverse phase HPLC and the product was confirmed by HPLC under the following conditions as shown below: Analytical HPLC conditions: retention time = 4.892 min. MS(ESI+);[M+2H] 2+ The calculated value was 1240.1, and the actual value was 1240.8.

[0184] The synthesis of monocyclic peptides (compound numbers 34 to 58 in Table 1-2) is carried out as described below. PD-209 (Compound No. 54 in Table 1-2); Synthesis of a compound containing an albumin binder (E-4IPhpCO) conjugated monocyclic peptide (peptide SEQ ID NO: 31) and a linker (SEQ ID NO: 34) [ka]

[0185] The Fmoc amino acids used in the synthesis included Fmoc-ds(tBu)-OH; Fmoc-Pro-OH; Fmoc-Gly-OH; Fmoc-Cys(Trt)-OH; Fmoc-Val-OH; Fmoc-Aib-OH; Fmoc-W5H-OH; Fmoc-Lys(Boc)-OH; Fmoc-F4COO(tBu)-OH; Fmoc-W5OMe-OH; Fmoc-Asn(Trt)-OH; Fmoc-Ser(tBu)-OH; Fmoc-MeK(alloc)-OH; Fmoc-Tyr(tBu)-OH.

[0186] To the reaction vessel containing the resin, 20% piperidine in DMF was added and the mixture was vortexed. Synthesis was initiated from Fmoc-ds(tBu)-OH using general solid phase synthesis methods. Double couplings were performed at positions 1, 3, 5, 10, 11, 14, and 16 from the N-terminal amino acid.

[0187] After the N-terminal Fmoc-Tyr(tBu)-OH was coupled, 20% piperidine in DMF was added. The resin was washed with DMF, and then 5 equivalents of 2-chloroacetic acid, 5 equivalents of DIC, and 5 equivalents of N-hydroxysuccinimide (HOSu) in methylene chloride (DCM) / 1-methyl-2-pyrrolidine (NMP) (1:1 v / v) were added. The resin was washed successively with DMF and DCM.

[0188] To the reaction vessel containing the resin, 0.25 equivalents of tetrakis(triphenylphosphine)palladium(0) (Pd(PPh3)4), 15 equivalents of phenylsilane, and DCM were added, and the resulting mixture was vortexed to remove the allyloxycarbonyl (alloc) group on the side chain of MeK. The peptide resin thus obtained was treated with 4 equivalents of (4-(4-iodophenyl)butanoyl)-L-glutamic acid, 8 equivalents of DIC, and 4 equivalents of Oxyma pure in DMF. The resin was washed three times with DMF and DCM, then dried.

[0189] A trifluoroacetic acid (TFA)-water-triisopropylsilane (TIS)-3,6-dioxa-1,8-octanedithiol (DODT) mixture (92.5:2.5:2.5:2.5 v / v / v / v) was added and the resulting mixture was stirred at room temperature for 1.5 hours.

[0190] The crude peptide was cleaved from the resin and collected by ether precipitation. After washing with diisopropyl ether three times and drying, DMSO-water-MeCN (1:1:1 v / v / v) containing 15 equivalents of triethylamine was added to obtain a final concentration of 5 mM, followed by stirring at room temperature for 2 hours. The crude peptide was confirmed by LCMS analysis conditions: retention time = 1.55 min.

[0191] The crude peptide was purified by reverse phase HPLC and the product was confirmed by HPLC under the conditions as shown below. Analytical HPLC conditions: retention time = 4.236 min. MS (ESI+); [M+2H] 2+ The calculated value was 1302.0 and the actual value was 1302.6. Synthesis of compounds containing PD-224 (Compound No. 36 in Table 1-2); Albumin binder (E-4IPhpCO) conjugated monocyclic peptide (Peptide SEQ ID NO: 23) [ka]

[0192] PD-224 was prepared by the same procedure as described for PD-209. Double coupling was performed at positions 1 and 3 from the N-terminal amino acid.

[0193] The Fmoc amino acids used in the synthesis included Fmoc-Cys(Trt)-OH; Fmoc-Val-OH; Fmoc-A4pipaa(tBu)-OH; Fmoc-W5H-OH; Fmoc-Lys(Boc)-OH; Fmoc-F4COO(tBu)-OH; Fmoc-W5OMe-OH; Fmoc-Asn(Trt)-OH; Fmoc-Ser(tBu)-OH; Fmoc-N-Me-Lys(alloc)-OH.

[0194] The final product was confirmed by HPLC under the following conditions as shown below.

[0195] Analytical HPLC conditions: retention time = 3.812 minutes. MS(ESI+);[M+2H] 2+ The calculated value was 1158.0 and the actual value was 1158.5.

[0196] Example 4 - Binding activity of hGhR binding peptides using surface plasmon resonance (SPR) SPR assays were performed using Biacore T200 (cytiva: formerly GE Healthcare). After equilibration of a series S sensor chip CM5 (cytiva: formerly manufactured by GE Healthcare) with running buffer (HBS-EP with 1% (v / v) DMSO), the EDC / NHS mixture was injected at a flow rate of 10 μL / min for 7 min, thereby activating the functional groups on the sensor chip. Recombinant human GhR-Fc protein (R&D Systems) in 10 mM acetate (pH 4.0) was injected and immobilized at a flow rate of 5 μL / min. It took 7 min to immobilize GhR on the substrate surface of the sensor chip. Ethanolamine was then injected at a flow rate of 10 μL / min for 7 min. 1 M ethanolamine (aqueous) was injected at a flow rate of 10 μL / min for 420 s for capping. 10 mM peptide in DMSO was diluted with running buffer to obtain 10 uM, and 100 nM, 50 nM, 25 nM, 10 nM, and 5 nM peptide solutions (peptide samples) were prepared.

[0197] The peptide samples were used to measure the kinetics of peptides to human GhR-Fc protein. The method adapted for the sample measurement was the single cycle kinetic method. The analysis was carried out using the evaluation software provided with the Biacore T200. A DMSO correction curve obtained by solvent correction measurement was applied for the analysis. Kinetic fitting was performed on the differential data obtained by subtracting the baseline data from the sample measurement data. The KD value was calculated based on the association rate constant (ka) and the dissociation rate constant (kd). The results obtained are shown in Tables 1, 2 and 3.

[0198] "Peptide SEQ ID NO" in Tables 1, 2 and 3 indicates the respective SEQ ID NO described in the sequence table, which indicates that the cyclic peptide of the compound has the amino acid sequence of the available SEQ ID NO. Furthermore, "Linker" indicates the SEQ ID NO described in the sequence table, which indicates that the linker has the amino acid sequence of the available SEQ ID NO to which it is attached. "Albumin binder" indicates a structure containing an albumin binder attached to the cyclic peptide. Please note that when "NO" is written, this indicates that the compound does not contain this structure. Although the synthesis is shown in separate examples, all of the peptides in Tables 1, 2 and 3 are cyclic peptides with the first amino acid residue and the 13th cysteine ​​residue (C) attached, and they have a structure in which a linker having the amino acid of the SEQ ID NO shown in "Linker" is further attached to the cysteine ​​residue.

[0199] In the table, "KD" is the value of the binding test result between the compound and hGhR using SPR, expressed in KD, in nM. Furthermore, "Observed m / z [M+2H]2+" indicates the ESI-MS (+) observed value. Unless otherwise specified, this is a divalent ([M+2H] 2+ ).

[0200] In Table 1, "albumin binder" indicates the type of albumin binder bound to the amino group of the lysine side chain located at the N-terminus of the linker. For example, when (E_E_4IPhoCO) is written, this indicates that an albumin binder called 4IphpCO is bound to the amino group of the lysine side chain located at the N-terminus of the linker through two glutamic acids. Please note that when NO is written for compound numbers 1 to 4 in "albumin binder", this indicates that these compounds do not contain an albumin binder.

[0201] Furthermore, all of Compound Nos. 1 to 32 and 58 to 65 shown in Table 1 are bicyclic peptides in which the amino group of the side chain of N-methyllysine, which is the second amino acid residue (X2), is bound to W1aa, which is the seventh amino acid residue (X7) (Compound Nos. 2, 5 to 32, and 58 to 65), or G / MeG / P is bound to the amino group of the side chain of the same N-methyllysine, and an amino acid is further bound to W1aa, which is the seventh amino acid residue (X7) (Compound Nos. 1 to 4).

[0202] In Table 2, "albumin binder" indicates the type of albumin binder bound to the amino group of the side chain of N-methyllysine, the second amino acid residue (X2). For example, when "albumin binder" is described as (E_ 4IPhpCO), this indicates that an albumin binder called 4IphpCO is bound to the amino group of the side chain of lysine, the second amino acid residue, through one glutamic acid. Furthermore, when "linker" is described as NO for compound numbers 33 to 40, 43 to 45, 47, 50, and 53, this means that they do not contain a linker. Please note that compound numbers 33 to 57 shown in Table 2 are all monocyclic peptides.

[0203] As shown in Tables 1 and 2, all compounds can effectively bind to human GhR-Fc protein, indicating that the addition of albumin binders to the peptides does not affect their binding activity. [Table 1] TIFF2024523280000006.tif69170 [Table 2] 1-(tert-butyl)5-(2,5-dioxopyrrolidin-1-yl)((S)-5-(tert-butoxy)-4-(4-(4-iodophenyl)butanamido)-5-oxopentanoyl)-L-glutamate [ka]

[0204] 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (4.22 g, 22.00 mmol) was added to a solution of 4-(4-iodophenyl)butanoic acid (5.80 g, 20 mmol) and 1-hydroxypyrrolidine-2,5-dione (2.76 g, 24.00 mmol) in DCM (40.0 ml) and the mixture was stirred at 0° C. for 1 h. The solution was quenched with 1N aqueous HCl and extracted twice with DCM. The combined organic layers were washed with brine, dried over Na2SO4 and the filtrate was evaporated under reduced pressure.

[0205] A solution of the resulting residue and DIPEA (6.99 ml, 40.0 mmol) in DMF (40.0 ml) was stirred at room temperature overnight. The solution was diluted with EtOAc, extracted with 1N aqueous HCl, and washed twice with EtOAc. The combined organic layers were washed with water and brine, dried over Na2SO4, and the filtrate was evaporated under reduced pressure.

[0206] The crude residue was purified by flash column chromatography (0% to 10% MeOH in DCM) to give a pale yellow oil.

[0207] EDC (4.22 g, 22.00 mmol) was added to a solution of the pale yellow oil obtained above and 1-hydroxypyrrolidine-2,5-dione (2.53 g, 22.00 mmol) in DCM (66.7 ml) and the mixture was stirred at 0° C. for 1 h. The solution was quenched with 1N aqueous HCl and extracted twice with DCM. The combined organic layers were washed with saturated aqueous NaHCO3, water and brine, dried over Na2SO4, and the filtrate was evaporated under reduced pressure.

[0208] A solution of the crude product, 1-(tert-butyl) 5-(2,5-dioxopyrrolidin-1-yl)(4-(4-iodophenyl)butanoyl)-L-glutamate and N-ethyl-N-isopropylpropan-2-amine (5.17 g, 40.0 mmol) in DMF (67 ml) was stirred at room temperature overnight. The solution was diluted with EtOAc, extracted with 1N aqueous HCl, and washed twice with EtOAc. The combined organic layers were washed with water and brine, dried over Na2SO4, and the filtrate was dried under reduced pressure to give a pale yellow oil.

[0209] To a solution of the pale yellow oil and 1-hydroxypyrrolidine-2,5-dione (2.014 g, 17.50 mmol) in DCM (28.0 ml) was added 3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine hydrochloride (3.35 g, 17.50 mmol) and the mixture was stirred at 0° C. for 1 h. The solution was quenched with a 1:1 mixture of 1N aqueous HCl and brine and extracted twice with DCM. The combined organic layers were dried over Na2SO4 and the filtrate was dried under reduced pressure to give the title compound as a white solid. Analysis by LCMS: MS (ESI+); [M+H] + The calculated value was 758.2, and the measured value was 758.4.

[0210] The present invention can be used in the bio-industry and pharmaceutical industry.

[0211] All references cited herein, and the references therein, where appropriate for teaching additional or alternative details, features, and / or technical background, are hereby incorporated by reference in their entirety into the disclosure of this specification.

[0212] While the present disclosure has been particularly shown and described with reference to certain embodiments, it will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications, and various presently unforeseen or unexpected substitutions, modifications, variations or improvements therein may subsequently be made by those skilled in the art, which are also intended to be encompassed by the following claims.

Claims

1. A peptide of the following amino acid sequence or a salt thereof: X1-X2-X3-X4-X5-X6-X7-X8-X9-X10-X11-X12-X13 (SEQ ID NO: 1) (wherein: X1 is an amino acid having an aromatic ring or a substituent thereof; X2 is N-methyllysine (MeK) or a modified form thereof; X3 and X4 are each V; X5 is S; X6 is N; X7 is W or substituted W; X8 is F or a substituent thereof, or Y or a substituent thereof; X9 is K or a substituent thereof, or R or a substituent thereof; X10 is substituted W; X11 is A or a substituent thereof, or K or a substituent thereof; X12 is V; and X13 is C) provided that the peptide or a salt thereof does not contain conservative amino acid substitutions, or contains one, two, or three conservative amino acid substitutions, and the peptide or a salt thereof has binding affinity for GhR, a peptide or a salt thereof.

2. The peptide or a salt thereof according to claim 1, wherein X2 is N-methyllysine or a modified form thereof; X7 is 1-(carboxymethyl)-L-tryptophan (W1aa); and the amino acid residues of X2 and X7 are linked.

3. The peptide or a salt thereof according to claim 1, wherein X2 is N-methyllysine to which an albumin binder is bound.

4. The peptide or a salt thereof according to claim 3, wherein the albumin binder is 4IphpCO, Biph4pCO, PhPeCO, PhpCO, cC14COO, or 4MePhpCO.

5. X1 is Y, 4Py, or F4COO; X8 is Y or F4COO; X9 is K, KCopipzaa, Hgn, Ahp, or Har; X10 is W5H; X11 is Aib or A4pipaa, the peptide or a salt thereof according to claim 3.

6. The peptide or a salt thereof has a sequence represented by F4COO-MeK-V-V-S-N-W1aa-F4COO-K-W5H-Aib-V-C (SEQ ID NO: 10); the peptide or a salt thereof contains one, two, or three substitutions, deletions, additions, or insertions; the second and seventh amino acid residues in SEQ ID NO: 10 are linked; The peptide or a salt thereof according to claim 1, wherein the peptide or a salt thereof has binding affinity for a human growth factor receptor (hGHR). **Claim 7** The peptide or a salt thereof has a sequence represented by Y-MeK-V-V-S-N-W5OMe-F4COO-K-W5H-A4pipaa-V-C (SEQ ID NO: 30); The peptide or a salt thereof may contain one, two, or three substitutions, deletions, additions, or insertions; The albumin binder is bound to MeK; The peptide or a salt thereof according to claim 1, wherein the peptide or a salt thereof has binding affinity for hGHR. **Claim 8** The peptide or a salt thereof according to claim 7, wherein the albumin binder is 4IphpCO, Biph4pCO, PhPeCO, PhpCO, cC14COO, or 4MePhpCO. **Claim 9** The peptide or a salt thereof according to claim 1, wherein the peptide or a salt thereof is a cyclic peptide. **Claim 10** The peptide or a salt thereof according to claim 6, wherein the peptide or a salt thereof is a cyclic peptide. **Claim 11** The peptide or a salt thereof according to claim 7, wherein the peptide or a salt thereof is a cyclic peptide. **Claim 12** The peptide or a salt thereof according to claim 8, wherein the peptide or a salt thereof is a cyclic peptide. **Claim 13** The peptide or a salt thereof according to claim 9, having a cyclic structure with a chloroacetylated amino acid at X1 and a cysteine residue; the chloroacetylated amino acid at X1 and the cysteine residue are bonded. **Claim 14** The peptide or a salt thereof according to claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 2-9, 11-29, and 31. **Claim 15** The peptide or a salt thereof according to claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 2-9, 11-29, and 31, with a linker added to the C-terminus. **Claim 16** The peptide or a salt thereof according to claim 15, wherein the linker contains an amino acid sequence selected from SEQ ID NOs: 32 and 35. **Claim 17** The peptide or a salt thereof according to claim 1, selected from the following: 【Chemical 1】 [Chemical 2] [Chemical Formula 3] 【Chemical Formula 4】 **Claim 18** A pharmaceutical composition comprising the peptide according to any one of claims 1 to 17 or a salt thereof, and a pharmaceutically acceptable carrier, excipient, or additive.

19. The peptide according to any one of claims 1 to 17 or a salt thereof for use in therapy.

20. The peptide according to any one of claims 1 to 17 or a salt thereof for the treatment of a disease associated with excessive secretion of human growth hormone in a patient.

21. The peptide according to claim 20 or a salt thereof, wherein the disease associated with excessive secretion of the human growth hormone is acromegaly or gigantism.

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