Peptides for the treatment of cancer and / or metastases

Abstract

The present invention is in the field of biomedical chemistry. The present invention provides modified peptides and dimers thereof derived from the amino acid sequence of the protein ADAM-12, their compositions, methods for their synthesis, and their use in medicine, in particular for inhibiting cancer growth and / or reducing or inhibiting the invasion and metastasis of cancer cells in various cancers.

Description

Detailed Description of the Invention

[0001] 〔Field of the Invention〕 The present invention is included in the field of biomedical chemistry. In particular, the present invention relates to the field of cancer and / or metastasis suppression.

[0002] 〔Background of the Invention〕 Metastasis is the cause of death in about 90% of cancer patients (see, for example, Reymond et al. Nature Reviews 2016, 13(12), 858 - 70).

[0003] With the seeding of cancer tumors due to metastasis, standard treatments are not at all effective, and research on new treatment targets and therapies is needed.

[0004] ADAM (A Disintegrin and Metalloproteinases) is a transmembrane protein family first reported in the 1990s. Multiple ADAM members, including ADAM - 9, ADAM - 10, ADAM - 12, ADAM - 15, and ADAM - 17, have been shown to play roles in either cancer formation or progression. Consistent with these findings, in several cancer types, it has been revealed that increased expression of specific ADAMs correlates with characteristics of invasive disease and poor prognosis (see, for example, Duffyet al. Clin Chim Acta 2009, 403(1 - 2), 31 - 6). Many ADAMs are involved in the regulation of growth factor activity and integrin function, which lead to the promotion of cell growth and invasion (see, for example, Mochizukiet al. Cancer Sci 2007, 98(5):621 - 8; Zadka et al. Neoplasma 2018, 65(6), 823 - 39).

[0005] ADAM-12 was identified in 1995 in a mouse myogenic cell line to investigate homologs of fertilin (see, for example, Yagami-Hiromasa et al. Nature 1995, 377(6550), 652-6). Human ADAM-12 was characterized in 1998 (see, for example, Gilpin et al. J Biol Chem 1998, 73(1), 157-66). Its structure consists of an N-terminal signal peptide, a pro-region, a metalloprotease (i.e., metalloprotease) region, a disintegrin region, a cysteine-rich region, an EGF region, a transmembrane region, and a C-terminal cytoplasmic tail. ADAM-12 includes a membrane-bound form (ADAM12-L) and a secreted form (ADAM12-S) that have all of the regions described above and lack the transmembrane and cytoplasmic regions (see, for example, Nyren-Erickson et al. Biochim Biophys Acta 2013, 1830(10), 4445-55).

[0006] ADAM-12 is highly expressed in many types of cancer and tumor cells. ADAM-12 can regulate cell-cell adhesion or cell-ECM interactions through binding to adhesion molecules such as integrins and syndecans (see, for example, Kveiborg et al. J Biochem Cell Biol 2008, 40(9), 1685-702). ADAM-12 can interact with syndecan-4 and promote cell seeding and the assembly of stress fibers (see, for example, Thodeti et al. J Biol Chem 2003, 278(11), 9576-84).

[0007] Overall, overexpression of ADAM-12 correlates with metastasis and poor survival rates for a wide variety of cancers.

[0008] In breast cancer, ADAM-12 is highly expressed and has been reported to act as a promoter of breast cancer metastasis (see, for example, Iba et al. Am J Pathol 1999, 154(5), 1489-501; Huang et al. Oncogene 2018, 37(49), 6316-26). Functional studies by Kveiborg et al. (see, for example, Kveiborg et al. Cancer Res 2005, 65(11), 4754-61) demonstrated that upregulation of ADAM-12 accelerates tumor progression in a mouse breast cancer model. In addition, ADAM-12 is upregulated in tumor tissues of breast cancer compared to adjacent normal tissues. Results from Kaplan-Meier survival curves showed that patients with high levels of ADAM-12 had shorter survival periods compared to patients with low levels of ADAM-12 (see, for example, Ma et al. Int J Clin Exp Pathol 2015, 8(10), 13279-83). It has also been noted that ADAM-12 in the urine of patients increases in proportion to the disease progression of breast cancer patients (see, for example, Roy et al. J Biol Chem 2004, 279(49), 51323-30).

[0009] High tumor levels of ADAM-12 indicate that ADAM-12 is the most prognostic factor associated with the worst prognosis in the invasive molecular subtype of high-grade serous ovarian cancer (see, for example, Cheon et al. Carcinogenesis 2015, 36(7), 739-47).

[0010] ADAM-12 is highly expressed in prostate cancer (see, e.g., Peduto et al. Oncogene 2006, 25(39), 5462-6), glioblastoma (see, e.g., Kodama et al. Am. J Pathol 2004, 165(5), 1743-53), and gastric cancer (see, e.g., Carl-McGrath et al. Int J Oncol 2005, 26(1), 17-24; Shimura et al. Cancer Prev Research 2015, 8(3), 240-8).

[0011] In patients diagnosed with pancreatic ductal adenocarcinoma, an increase in ADAM-12 levels was observed compared to healthy controls. A decrease in ADAM-12 levels during treatment was associated with an extended survival period (see, e.g., Veenstra et al. Oncogenesis 2018, 7(11), 87).

[0012] ADAM-12 has also been proposed as a biomarker for bladder cancer. Bladder cancer patients have a risk of progressing to muscle-invasive cancer of up to 60%. Its upregulation correlates with the grade and stage of bladder cancer in terms of mRNA levels and protein expression (see, e.g., Frolich et al. Clin Cancer Research 2006, 12(24), 7359-68).

[0013] ADAM-12 expression is hardly detected in normal liver. However, it increases in hepatocellular carcinoma and liver metastases (see, e.g., Mazzocca et al. Biochim Biophys Acta 2010, 1806(1), 74-81; Le Pabic, Hepatology 2003, 37(5), 1056-66).

[0014] Metastatic small cell lung cancer (SCLC) also showed an increase in ADAM-12 expression along with enhanced invasion and metastasis (see, e.g., Shao et al. Plos One 2014, 9(1), e85936).

[0015] ADAM-12 mRNA has been detected in 70% of giant cell tumors of bone. The significance of ADAM-12 in the cell fusion process of mononuclear stromal cells in giant cell tumors has also been described (see, for example, Tian et al. J Clin Pathol 2002, 55(6), 394-7). High expression of ADAM-12 is not only correlated with tumor growth, but is also associated with enhanced osteolysis and a significant decrease in the survival rate of animals, suggesting that ADAM-12 may be a new therapeutic target for osteosarcoma (see, for example, George et al. Eur J Cancer 2013, 49(9), 2253-63).

[0016] ADAM-12 is significantly overexpressed in advanced stage melanoma compared to early stage melanoma (see, for example, Cireap et al. Pathol Oncol Res 2013, 19(4), 755-62).

[0017] Iba et al. described the importance of the Cys-rich region of ADAM-12 when it supports cell adhesion in a panel of cancer cell lines (e.g., MDA-MB-231 breast cancer cells) (see, for example, Iba et al. Am J Pathol 1999, 154(5), 1489-501). According to Iba et al., in the case of human ADAM-12, the recombinant polypeptide of the cysteine-rich region supported cell adhesion in a panel of cancer cell lines, but the disintegrin-like region did not.

[0018] Subsequently, the involvement of the disintegrin domain of ADAM-12 (obtained from SEQ ID NO: 1, Gilpin et al. J Biol Chem 1998, 73(1), 157-66) was investigated. Dyczynska et al. showed that breast cancer-related mutations in the disintegrin domain of ADAM-12 interfere with the intracellular transport and processing of the protein (see, for example, Dyczynska et al. Int J Cancer 2008, 122(11), 2634-40). Li et al. also described the role of the disintegrin domain of ADAM-12 in the activation of epidermal growth factor receptor (EGFR) in triple-negative breast cancer and breast cancer-initiating cells (BTIC) (see, for example, Li et al. Breast Cancer Res Treat 2012, 135(3), 759-69; Li et al. Breast Cancer Res Treat 2013, 139(3), 691-703).

[0019] α9β1 integrin is expressed in a wide variety of cell types and interacts with many ligands such as fibronectin and tenascin-C, particularly ADAM-12. Aberrant expression of α9β1 can cause or exacerbate pathological conditions (such as cancer) (see, for example, Hoye et al. Adv Biol Regul 2012, 52(2), 326-39).

[0020] α9β1 integrin expression is associated with reduced survival in the basal-like breast cancer subtype. Therefore, it has been proposed as a novel marker for this tumor subtype (see, for example, Allen et al. J Pathol 2011, 223(5), 646-58). In contrast, downregulation in triple-negative breast cancer tumors correlates with decreased tumor angiogenesis, tumor growth, and metastasis (see, for example, Wang et al. Int J Cancer 2019, 145(10), 2767-80). Also, tumor-α9β1 integrin-mediated signaling plays a central role in creating a unique primary tumor tissue microenvironment that promotes breast cancer growth and lymphatic metastasis (see, for example, Majumder et al. Plos One 2012, 7(4), e35094; Ota et al. J Mol Med 2014, 92(12), 1271-81).

[0021] ADAM-12 has been described as a ligand for α9β1 integrin that supports α9β1 integrin-mediated cell adhesion and GPT-Rac-dependent migration in melanoma cells (see, for example, Lydolph et al. Exp Cell Research 2009, 315(19), 3312-24). However, it has also been disclosed that when this receptor is not expressed, it can bind to other members of the β1 integrin family.

[0022] The disintegrin domain of ADAM seems to play an important role in the interaction with integrin. Most ADAMs can interact with integrin via the RGD-motif or XCD-motif in their disintegrin domain. Indeed, the XCD sequence region is probably a major factor in the inhibition of platelet aggregation and integrin interaction (see, for example, Lu et al. Cardiovasc Hematol Agents Med Chem 2007, 5(1), 29-42).

[0023] Some ADAMs are hypothesized to play roles in the suppression of cell adhesion and metastasis. However, there is little literature on the clinical application of ADAM-12 protein or its peptides in the treatment of cancer and / or metastasis.

[0024] WO2015 / 028027A1 discloses monoclonal antibodies against the pro-region of ADAM-12 and their use for the treatment of cancer. The pro-region of ADAM-12 is a region distant from the cysteine-rich region and the disintegrin region.

[0025] WO2006 / 014903A2 discloses ADAM-12 polynucleotides highly expressed in cancer tissues or their encoded polypeptides, and their modulators (e.g., antibodies). The document proposes methods for the treatment and diagnosis of proliferative disorders including cancer and psoriasis. The document only discloses the expression of ADAM-12 in cancerous tissues and does not disclose examples of activity or treatment using any compounds.

[0026] WO2011 / 100362A1 discloses a modified ADAM-derived polypeptide containing an ADAM polypeptide derived from the disintegrin-like region of ADAM for use in the treatment of cancer, and a fusion protein containing thioredoxin as the N-terminal segment. The sequence of the above polypeptide has a sequence length of about 80 amino acids close to the full-length sequence of the disintegrin region of ADAM.

[0027] Rhabdomyosarcoma (RMS) and neuroblastoma (NB) are examples of common pediatric cancers. Rhabdomyosarcoma (RMS), an early-onset malignant tumor, is the most common type among pediatric soft tissue sarcomas (see, for example, Masia et al. Br J Cancer 2012, 107(8), 1374-83). Neuroblastoma is the most common cancer in infants and the third most common cancer in children. Metastasis of the disease is recognized at the time of diagnosis in almost half of all patients presenting with neuroblastoma (see, for example, Maris et al. The Lancet 2007, 369 (9579), 2106-20).

[0028] Patients with metastatic disease have an extremely poor prognosis. Therefore, more potent treatment methods are needed. Furthermore, the major cause of death in these patients is the formation of distant metastases.

[0029] The involvement of α9β1 integrin in the processes of invasion and metastasis has also been reported in pediatric cancers such as rhabdomyosarcoma (see, for example, Masia et al. Br J Cancer 2012, 107(8), 1374-83). Rhabdomyosarcoma cell lines have been used as a model to establish the role of the ADAM-12 / α9β1 integrin interaction in myogenesis. However, the role of ADAM-12 in tumorigenesis of rhabdomyosarcoma has not been described. The role of ADAM-12 in neuroblastoma has not been disclosed either.

[0030] Therefore, there is an urgent need to identify new therapeutic targets and find new pharmacological treatments to improve the survival rate of patients with cancer and / or metastatic diseases.

[0031] 〔Detailed Description of the Invention〕 The present invention provides a solution to the need to reduce or suppress cancer growth, invasion, and / or metastasis formation in various cancers.

[0032] After extensive and thorough investigations, the inventors of the present invention surprisingly found a series of synthetic analogs of ADAM-12 that can reduce the metastatic potential of various cancers (e.g., rhabdomyosarcoma, neuroblastoma, and breast cancer).

[0033] In the prior art, short peptides derived from the disintegrin region of ADAM-12 substituted with non-natural amino acids for clinical use have not been disclosed or suggested in the suppression of cancer growth and / or the reduction of invasion and / or metastasis.

[0034] The present invention provides a modified peptide (and its dimer) derived from the amino acid sequence of the protein ADAM-12, specifically the disintegrin region of ADAM-12, more specifically the amino acid sequence -CRDSSNSCDLPEFC, i.e., Cys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-Cys (SEQ ID NO: 2) contained within the disintegrin region of ADAM-12. The modified peptide (and its dimer) is stable in plasma and can inhibit invasion in vitro. Thus, it can reduce invasion and metastasis in vivo. Specific modifications using synthetic amino acids (e.g., the introduction of mesityl amino acids (e.g., 3-mesitylalanine (2,4,6-trimethylphenylalanine, Msa)) at the phenylalanine position) optionally together with other modifications (e.g., substitution of cysteine residues and / or inclusion of non-natural amino acids (e.g., pyroglutamic acid in the amino terminus of the peptide)) provide novel compounds with improved pharmacological properties and activities for the suppression of cancer growth and / or the reduction or suppression of invasion and metastasis of cancer cells in various cancers. The cysteine residue can be substituted with methionine, cystine, or a basic amino acid (e.g., lysine).

[0035] In vitro studies using the ADAM-12 analog of the present invention showed a decrease in invasion in rhabdomyosarcoma cells overexpressing α9β1 integrin (RD) and rhabdomyosarcoma cells not overexpressing α9β1 integrin (RH30). From this fact, it became clear that integrin could not be a limited target of the ADAM-12 analog of the present invention.

[0036] 〔Definition〕 To facilitate the understanding of the present invention, the meanings of some terms and expressions used in connection with the present invention are given.

[0037] "Invasion" and "invasiveness" and their plurals relate to cell migration and define the ability of cells to become motile and pass through the extracellular matrix within a tissue or invade adjacent tissues. Cell invasion is the invasion of adjacent tissues and the destruction of adjacent tissues. Cancer cells that have become invasive may seed at secondary sites and form metastases.

[0038] "Metastasis" and its plural refer to foci of cancerous cells related to a pre-existing cancer called the primary, but which occur away from and are not continuous with the primary lesion. The seeding of these secondary lesions occurs via lymphatic or hematogenous pathways.

[0039] The term "treatment" and its plural, as used herein, mean the administration of a compound according to the present invention to prevent, ameliorate or eliminate the adverse clinical symptoms caused by a disease, or to reduce or eliminate the incidence or severity of said disease.

[0040] In the context of the present invention, the term "diagnosis" refers to a diagnostic method performed in the absence of a human body (i.e., an in vitro diagnostic method).

[0041] In the context of the present invention, the term "therapeutic dose" and its plural forms refer to the amount of the compounds disclosed in the present invention that must be administered to an individual in order to obtain a medical or biological positive response. This is for an individual cell, animal or human, and the compound is administered by a researcher, physician, veterinarian or the individual him / herself.

[0042] In the context of the present invention, the term "therapeutic agent" and its plural forms refer to any agent or compound that produces a desired pharmacological effect in an individual (cell, animal or human).

[0043] In the context of the present invention, the term "activity" or "pharmacological activity" and their plural forms refer to a biological or medical response as a result of treating an individual (cell, animal or human) with the compounds disclosed in the present invention. This is the compound administered by a researcher, physician, veterinarian or the individual him / herself.

[0044] The term "individual" and its plural forms refer to any organism to which the compounds described in the present invention can be administered. This is for administration for experimental, diagnostic, and / or therapeutic purposes. The individual can be a cell, animal or human.

[0045] In the context of the present invention, the term "peptide dimer" and its plural forms refer to a compound containing 2 monomer units of the peptides of the present invention linked by a disulfide bridge between cysteine residues. In a homodimeric peptide of 2 equal peptide subunits, the peptide dimer is represented as (peptide)2(disulfide bridge).

[0046] The term "peptide" or "peptide analogue" and their plural forms can be used as a general term for the compounds of the present invention that encompass both the peptides and peptide dimers of the present invention.

[0047] In this specification, the abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint Commission on Biochemical Nomenclature, as outlined in J. Biol. Chem. (1989) 264:633-673.

[0048] Thus, for example, Asn represents NH2-CH(CH2CONH2)-COOH. Therefore, a dash representing a peptide bond excludes the OH of the 1-carboxyl group of the amino acid (here represented in the non-ionized conventional form) when located to the right of the symbol, and excludes the H of the 2-amino group of the amino acid when located to the left of the symbol. The same symbol can be used for both modifications.

[0049] Amino acid structures, as well as their one-letter and three-letter naming codes, and / or the structures are shown in Table 1. Amino acids can have an L- or D-configuration.

[0050]

Table 1

[0051] The abbreviation "Ac-" is used in this specification to name the acetyl group (CH3-CO-). The octanoyl group refers to the n-octanoyl group or the capryloyl group.

[0052] In the context of the present invention, the terms "mesityl amino acid" and its plural forms include 3-mesitylalanine (2,4,6-trimethylphenylalanine, Msa) and 2-mesitylglycine (2,4,6-trimethylphenylglycine, Msg).

[0053] The term "acyclic aliphatic group" is used, for example, to include, but is not limited to, straight-chain or branched-chain alkyl groups, alkenyl groups, and alkynyl groups.

[0054] The term "alkyl group" relates to a saturated, straight-chain or branched group. It has from 1 to 24, preferably from 1 to 16, more preferably from 1 to 14, even more preferably from 1 to 12, and still even more preferably 1, 2, 3, 4, 5 or 6 carbon atoms. These are bonded to the rest of the molecule by a single bond (e.g., methyl, ethyl, isopropyl, isobutyl, tert-butyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, amyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, etc., but not limited thereto).

[0055] The term "alkenyl group" refers to a straight or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, and still even more preferably 2, 3, 4, 5 or 6 carbon atoms, and having one or more carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds, which are conjugated or non-conjugated. The alkenyl group is bonded to the rest of the molecule via a single bond (e.g., vinyl, oleyl, linoleyl and similar groups, but not limited thereto).

[0056] The term "alkynyl group" refers to a straight or branched group having from 2 to 24, preferably from 2 to 16, more preferably from 2 to 14, even more preferably from 2 to 12, and still even more preferably 2, 3, 4, 5 or 6 carbon atoms, and having one or more carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds, which are conjugated or non-conjugated. The alkynyl group is bonded to the rest of the molecule via a single bond (e.g., ethynyl group, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, pentynyl (e.g., 1-pentynyl) and similar groups, but not limited thereto).

[0057] The term "alicyclic group" is used in the present invention to include, for example, but not limited to, cycloalkyl groups, cycloalkenyl groups or cycloalkynyl groups.

[0058] The term "cycloalkyl" relates to a saturated monocyclic or polycyclic aliphatic group having 3 to 24, preferably 3 to 16, more preferably 3 to 14, even more preferably 3 to 12, and still even more preferably 3, 4, 5 or 6 carbon atoms. Cycloalkyl is bonded to the rest of the molecule via a single bond (e.g., cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrophenalene, etc., but not limited thereto).

[0059] The term "cycloalkenyl" relates to a non-aromatic monocyclic or polycyclic aliphatic group having 5 to 24, preferably 5 to 16, more preferably 5 to 14, even more preferably 5 to 12, and still even more preferably 5 or 6 carbon atoms and having one or more conjugated or non-conjugated carbon-carbon double bonds, preferably 1, 2 or 3 carbon-carbon double bonds. Cycloalkenyl is bonded to the rest of the molecule via a single bond (e.g., cyclopent-1-en-1-yl group and similar groups, but not limited thereto).

[0060] The term "cycloalkynyl" relates to a non-aromatic monocyclic or polycyclic aliphatic group having 8 to 24, preferably 8 to 16, more preferably 8 to 14, even more preferably 8 to 12, and still even more preferably 8 or 9 carbon atoms and having one or more conjugated or non-conjugated carbon-carbon triple bonds, preferably 1, 2 or 3 carbon-carbon triple bonds. Cycloalkynyl is bonded to the rest of the molecule via a single bond (e.g., cyclooct-2-yn-1-yl group, etc., but not limited thereto).

[0061] The term "aryl group" relates to an aromatic group having 6 to 30, preferably 6 to 18, more preferably 6 to 10, and even more preferably 6 or 10 carbon atoms. The aryl group contains 1, 2, 3 or 4 aromatic rings and is bonded or fused by carbon-carbon bonds. The aryl group is bonded to the rest of the molecule via a single bond (for example, but not limited to, phenyl, naphthyl, diphenyl, indenyl, phenanthryl or anthryl).

[0062] The term "aralkyl group" relates to an alkyl group substituted by an aromatic group having 7 to 24 carbon atoms. The aralkyl group includes, for example, -(CH2) 1-6 -phenyl, -(CH2) 1-6 -(1-naphthyl), -(CH2) 1-6 -(2-naphthyl), -(CH2) 1-6 -CH(phenyl)2 and the like, but are not limited thereto.

[0063] The term "heterocyclic group" relates to a 3- to 10-membered heterocyclyl or hydrocarbon ring. In the heterocyclic group, one or more ring atoms, preferably one, two or more than three ring atoms, are elements different from carbon (for example, nitrogen, oxygen or sulfur), and may be saturated or unsaturated. For the purposes of the present invention, the heterocyclyl may be a cyclic, monocyclic, bicyclic or tricyclic system that may include a fused ring system; the nitrogen, carbon or sulfur atoms may optionally be oxidized in the heterocyclyl radical; the nitrogen atoms may optionally be quaternized; the heterocyclyl radical may be partially or completely saturated or may be aromatic. Preferably, the term heterocyclic ring relates to a 5- or 6-membered ring.

[0064] The term "heteroarylalkyl group" relates to an alkyl group substituted by a substituted or unsubstituted aromatic heterocyclyl group. The alkyl group has 1 to 6 carbon atoms, and the aromatic heterocyclyl group has 2 to 24 carbon atoms and 1 to 3 atoms other than carbon (for example, -(CH2) 1-6 -imidazolyl, -(CH2)1-6 -triazolyl, -(CH2) 1-6 -thienyl, -(CH2) 1-6 -furyl, -(CH2) 1-6- (such as pyrrolidinyl, etc., but not limited thereto).

[0065] When used in the relevant art, for the groups defined above, a certain degree of substitution may occur. Therefore, substitution may occur in any of the groups of the present invention. Regarding the groups of the present invention, a reference in this specification to a substituted group indicates that the specified radical may be substituted by one or more substituents at one or more available positions, preferably at 1, 2, or 3 positions, more preferably at 1 or 2 positions, and even more preferably at 1 position. These substituents include, for example, C1-C4 alkyl; hydroxyl; C1-C4 alkoxyl; amino; C1-C4 aminoalkyl; C1-C4 carbonyloxyl; C1-C4 oxycarbonyl; halogen (e.g., fluorine, chlorine, bromine, and iodine); cyano; nitro; azide; C1-C4 alkylsulfonyl; thiol; C1-C4 alkylthio; aryloxyl (e.g., phenoxyl); -NR b (C=NR b )NR b R C ; where R b and R C are independently selected from the group consisting of H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 alkynyl, C3-C 10 cycloalkyl, C6-C 18 aryl, C7-C 17 aralkyl, 3-10 membered heterocyclyl, or an amino protecting group.

[0066] The "percentage of identity" with respect to peptides, polypeptides, and proteins, as used herein, has the meaning generally ascribed in the art. Thus, it relates to the percentage of amino acids for which the two amino acid sequences being compared are identical after optimal alignment of these sequences. Here, the percentage is merely statistical, and the differences between the two amino acid sequences are randomly distributed throughout the entire sequence. "Optimal alignment" is understood as an alignment of amino acid sequences that results in a greater percentage of identity. The percentage of identity is calculated by measuring the number of positions at which the amino acids in the two sequences being compared are identical, dividing the number of identical positions by the number of positions compared, and multiplying the resulting value by 100 to obtain the percentage of identity between the two sequences. The comparison of the sequences between two amino acid sequences can be performed manually or by means of computer programs known in the art (e.g., the BLAST (Basic Local Alignment Search Tool) algorithm).

[0067] [Compound of the present invention] The first aspect of the present invention is a peptide of general formula (I): R1-AA1-AA2-Arg-Asp-Ser-Ser-Asn-Ser-AA3-Asp-Leu-Pro-Glu-AA4-AA5-R2 (I) its stereoisomers, mixtures thereof, and / or pharmaceutically acceptable salts thereof, characterized by the following; AA1 is Pyr, or a bond; AA2 is Lys, Cys, or a bond; AA3 is Cys, Met, or Cys(Cys) (Cystine); AA4 is 2,4,6-trimethylphenylalanine (Msa), or 2,4,6-trimethylphenylglycine (Msg); AA5 is Lys, Cys, or a bond; R1 is selected from the group consisting of H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic, a substituted or unsubstituted cycloaliphatic, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a polymer derived from polyethylene glycol, and R5-CO-, wherein R5 is selected from the group consisting of H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic, a substituted or unsubstituted aryl, a substituted or unsubstituted aralkyl, a substituted or unsubstituted heterocyclyl, and a substituted or unsubstituted heteroarylalkyl; R2 is independently selected from the group consisting of -NR3R4, -OR3, and -SR3, wherein R3 and R4 are selected from the group consisting of H, a substituted or unsubstituted acyclic aliphatic group, a substituted or unsubstituted alicyclic, a substituted or unsubstituted heterocyclyl, a substituted or unsubstituted heteroarylalkyl, a substituted or unsubstituted aryl, and a substituted or unsubstituted aralkyl, refers to a peptide, its stereoisomers, mixtures thereof, and / or its pharmaceutically acceptable salts.

[0068] In a preferred embodiment, AA4 in general formula (I) is 2,4,6-trimethylphenylalanine (Msa).

[0069] In another preferred embodiment, AA2 and AA5 in general formula (I) are independently selected from Lys or a bond.

[0070] The groups of R1 and R2 are respectively bonded to the amino terminus (N-terminus) and carboxy terminus (C-terminus) of the peptide sequence.

[0071] In a preferred embodiment of the present invention, R1 is selected from the group consisting of H or R5-CO-, wherein R5 is; substituted or unsubstituted C1-C 24 alkyl; substituted or unsubstituted C2-C 24 alkenyl; substituted or unsubstituted C2-C 24 alkynyl; substituted or unsubstituted C3-C24 Cycloalkyl; substituted or unsubstituted C5-C 24 Cycloalkenyl; substituted or unsubstituted C8-C 24 Cycloalkynyl; substituted or unsubstituted C6-C 30 Aryl; substituted or unsubstituted C7-C 24 Aralkyl; substituted or unsubstituted heterocyclyl having 3 to 10 ring members; and substituted or unsubstituted heteroarylalkyl having 2 to 24 carbon atoms, having 1 to 3 atoms other than carbon atoms, and having an alkyl chain having 1 to 6 carbon atoms; selected from the group consisting of. Preferably, R5 is substituted or unsubstituted C1-C 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl, and substituted or unsubstituted C3-C 24 Selected from the group consisting of cycloalkyl. More preferably, R1 is H, acetyl, tert-butanoyl, hexanoyl, 2-methylhexanoyl, cyclohexanecarboxyl, octanoyl, decanoyl, lauroyl, myristoyl, palmitoyl, stearoyl, oleoyl, and linoleoyl. Even more preferably, R1 is H, acetyl, octanoyl, lauroyl, myristoyl or palmitoyl. In an even more preferred embodiment, R1 is acetyl, octanoyl or palmitoyl.

[0072] According to another preferred embodiment, R2 is -NR3R4, -OR3 or -SR3, where R3 and R4 are independently H; substituted or unsubstituted C1-C 24 Alkyl; substituted or unsubstituted C2-C 24 Alkenyl; substituted or unsubstituted C2-C 24 Alkynyl; substituted or unsubstituted C3-C 24 Cycloalkyl; substituted or unsubstituted C5-C 24 Cycloalkenyl; substituted or unsubstituted C8-C 24 Cycloalkynyl; substituted or unsubstituted C6-C 30 Aryl; substituted or unsubstituted C7-C 24Aralkyl; a substituted or unsubstituted heterocyclyl having 3 to 10 ring members; and a substituted or unsubstituted heteroarylalkyl having 2 to 24 carbon atoms, having 1 to 3 atoms other than carbon, and having an alkyl chain with 1 to 6 carbon atoms; selected from the group consisting of. Preferably, R3 and R4 are H, substituted or unsubstituted C1-C 24 alkyl, substituted or unsubstituted C2-C 24 alkenyl, and substituted or unsubstituted C3-C 24 cycloalkyl, independently selected from the group consisting of. R3 and R4 can optionally be bonded via a saturated or unsaturated carbon-carbon bond to form a ring with the nitrogen atom. Preferably, R2 is -NR3R4 or -OR3. More preferably, R3 and R4 are selected from the group consisting of H, methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, decyl, dodecyl and hexadecyl. Even more preferably, R3 is H and R4 is selected from the group consisting of H, methyl, ethyl, hexyl, dodecyl and hexadecyl. In an even more preferred embodiment, R2 is selected from -OH and -NH2.

[0073] In certain embodiments, the invention also relates to a peptide dimer comprising two peptides of formula (I) of the invention, its stereoisomers, mixtures thereof, and / or pharmaceutically acceptable salts thereof. The peptide dimer is characterized in that it is a disulfide-bridged peptide dimer.

[0074] In a preferred embodiment, the peptide dimer is a homodimer peptide. Also preferably, the peptide dimer is formed by a disulfide bridge between two equal peptide monomers of formula (I) of the invention when AA3 is Cys and AA2 and AA5 are independently Lys or a bond.

[0075] Preferably, the peptide or peptide dimer of the invention is selected from the group consisting of: R1-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-R2 (R1-sequence number 3-R2) Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-R2 (sequence number 4-R2) R1-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2 (R1-sequence number 5-R2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2 (sequence number 6-R2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-R2 (sequence number 7-R2) (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2)2 (disulfide bridge)[(sequence number 6-R2)2 (disulfide bridge)] R1-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-R2 (R1-sequence number 8-R2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-R2 (sequence number 9-R2) Here, R1 and R2 are as defined above.

[0076] More preferably, the peptide or peptide dimer of the present invention is selected from the group consisting of: Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (Ac-sequence number 3-NH2) Octanoyl-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (Octanoyl-sequence number 3-NH2) Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (SEQ ID NO: 4-NH2) Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Ac-SEQ ID NO: 5-NH2) Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Octanoyl-SEQ ID NO: 5-NH2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (SEQ ID NO: 6-NH2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (SEQ ID NO: 7-NH2) (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2)2 (Disulfide bridge) [(SEQ ID NO: 6-NH2)2 (Disulfide bridge)] Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Octanoyl-SEQ ID NO: 8-NH2) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (SEQ ID NO: 9-NH2) The amino acids forming the peptide and the peptide dimer may have L- or D-configuration, or a combination thereof.

[0077] For example, when it is shown that AA5 can be Lys, it is understood that AA5 is selected from L-Lys or D-Lys. The preparation methods described herein serve to obtain each stereoisomer of the peptides or peptide dimers of the present invention by a person skilled in the art selecting amino acids having the appropriate configuration.

[0078] In a preferred embodiment, the amino acids of the peptide or peptide dimer of the present invention are L-amino acids. The preferred structure of the peptides and peptide dimers of the present invention is a pure isomer (i.e., an enantiomer or a diastereomer).

[0079] In the context of the present invention, the terms "uncodified amino acid" and its plural forms relate to amino acids that are not encoded by the natural or non-natural genetic code (e.g., pyroglutamic acid, or cystine, or the synthetic amino acids 2,4,6-trimethylphenylalanine (Msa), or 2,4,6-trimethylphenylglycine (Msg) (see Table 1)).

[0080] The amino acid sequences of the peptides and peptide dimers of the present invention may include modifications of a given sequence. Such modifications are known to those skilled in the art. For example, one or more L-amino acids of the amino acid sequences of the peptides and peptide dimers of the present invention may be replaced by D-amino acids in order to increase their stability. For example, N-acylation, and / or C-amidation or C-esterification of the amino acid sequences of the peptides and peptide dimers of the present invention may increase their resistance to proteolysis. For example, cyclization of one or more amino acid sequences of the peptides of the present invention may increase their stability and permeability. For example, one or more amino acids of the amino acid sequences of the peptides and peptide dimers of the present invention may be N-alkylated (generally N-methylated) in order to improve their stability. For example, one or more amino acid sequences of the peptides and peptide dimers of the present invention may be conjugated to one or more macromolecules (e.g., polyethylene glycol (PEG), albumin). As a result, the one or more amino acid sequences improve their stability and / or reduce renal clearance.

[0081] Pharmaceutically acceptable salts of the peptides and peptide dimers provided by the present invention are also included in the present invention. The term "pharmaceutically acceptable salts" means salts approved for their use in animals, particularly humans, and includes salts used for the formation of base addition salts. Pharmaceutically acceptable salts are inorganic (e.g., particularly, but not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum, etc.); or organic (e.g., particularly, but not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine, etc.) or base addition salts, and are organic (e.g., particularly, but not limited to, acetate, citrate, lactate, malonate, maleate, tartrate, fumarate, benzoate, aspartate, diaspartate, triaspartate, glutamate, succinate, etc.); or inorganic (e.g., particularly, but not limited to, chloride, sulfate, borate, or carbonate, etc.). The nature of the salt is not important as long as it is pharmaceutically acceptable. Pharmaceutically acceptable salts of the peptides and peptide dimers of the present invention can be obtained by conventional known methods in the prior art (see, for example, Berge et al. J Pharm Sci 1977, 66(1), 1-19).

[0082] Peptides or peptide dimers having conservative or non-conservative substitutions, more preferably conservative substitutions, that exhibit the activities described herein for the peptides of the present invention are also included in the present invention. With respect to any of the peptides or peptide dimers of the present invention, peptides or peptide dimers that match at a rate of 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 96%, 97%, 98%, or 99% are also included in the present invention. The said peptides exhibit the activities described herein, more preferably conservative substitutions, with respect to the peptides of the present invention.

[0083] [Preparation Method] The peptides and peptide dimers of the present invention, their stereoisomers, or their pharmaceutically acceptable salts can be synthesized according to conventional methods known in the art. In one embodiment of the present invention, the peptides and peptide dimers are synthesized by solution-phase or solid-phase peptide synthesis methods.

[0084] Solid-phase synthesis methods are described, for example, in Stewart J.M. and Young J.D., 1984, “Solid Phase Peptide Synthesis, 2nd edition” Pierce Chemical Company, Rockford, Illinois; Bodanszky M., and Bodanszky A., 1984 “The practice of Peptide Synthesis” Springer Verlag, Berlin; Lloyd-Williams P., Albericio F. and Giralt E. (1997) “Chemical Approaches to the Synthesis of Peptides and Proteins” CRC, Boca Raton, FL, USA. Combinations of solution-phase and solid-phase synthesis, as well as solution-phase or enzymatic synthesis, are described in Kullmann, J Biol Chem 1980, 255(17), 8234-8238.

[0085] For example, a method for obtaining the peptides and peptide dimers of the present invention includes the following steps: - Coupling an amino acid with a protected N-terminus and a free C-terminus to an amino acid with a free N-terminus and a protected or solid support-bound C-terminus; - Removing the N-terminal protecting group; - Repeating the sequence of coupling and removing the N-terminal protecting group until the desired peptide sequence is obtained; - Removing the C-terminal protecting group or cleaving from the solid support.

[0086] - Optionally, oxidizing the obtained peptide to obtain a disulfide-bridged peptide dimer.

[0087] In one embodiment of the present invention, the peptides and peptide dimers of the present invention, their stereoisomers, or their pharmaceutically acceptable salts are prepared by a method comprising the following steps: a) Performing solid-phase peptide synthesis in a polymer support; b) Cleaving the peptide from the polymer support (preferably by acid treatment); c) Optionally, oxidizing the peptide in solution to obtain a cyclic peptide or peptide dimer; d) Optionally, removing protecting groups (preferably using trifluoroacetic acid); Alternatively, i) Performing solid-phase peptide synthesis in a polymer support; ii) Optionally, performing solid-phase peptide cyclization or dimer formation in the polymer support; iii) Cleaving the peptide from the polymer support and, optionally, simultaneously removing protecting groups (preferably by treatment with trifluoroacetic acid); iv) Optionally, oxidizing the peptide in solution to obtain a peptide dimer.

[0088] In a preferred embodiment, the peptide dimer of the present invention is obtained by oxidizing the peptide of the present invention in the liquid phase. The free sulfhydryl groups of the cysteine residues of the peptide can be oxidized to form a disulfide-bridged peptide dimer. Preferably, oxidation is carried out using dimethyl sulfoxide (DMSO).

[0089] The C-terminus is attached to the polymer support, and the process preferably proceeds in the solid phase. Thus, the process comprises a coupling step of an amino acid having a protected N-terminus and a free C-terminus to an amino acid having a free N-terminus and a C-terminus attached to the polymer support; a step of removing the protecting group from the N-terminus; and repeating the said procedure the required number of times to obtain the target peptide sequence, and finally cleaving the synthesized peptide or peptide dimer from the original polymer support. Optionally, the peptide may be a cyclic peptide, and the cyclization step may be carried out before or after cleavage from the polymer support. The functional groups of the amino acid side chains are maintained appropriately using temporary or permanent protecting groups during synthesis. They can be deprotected simultaneously with or orthogonal to the process of cleaving the peptide from the polymer support.

[0090] Alternatively, solid-phase synthesis can be carried out by a convergent strategy by coupling peptide fragments on a polymer support or on a peptide fragment pre-attached to the polymer support. Convergent synthesis strategies are known to those skilled in the art and are described in Lloyd-Williams P. et al. Tetrahedron 1993, 49(48), 11065-11133.

[0091] The method may also include a step of deprotecting the N-terminus and C-terminus and / or an additional step of cleaving the peptide from the polymer support in an unspecified order using standard methods and conditions known in the art. Thereafter, the functional groups of the said termini may be modified. Any modification of the N-terminus and C-terminus can be carried out using the peptide of formula (I) of the present invention immobilized on the polymer support or after the peptide has been cleaved from the polymer support.

[0092] If necessary, R1 may be introduced by reacting the N-terminus of the peptide of the present invention with an R1-Z compound. Here, R1 has the meaning described above, and Z is a leaving group (for example, but not limited to, in particular, tosyl group, mesyl group, and halogen group); also, in the presence of an appropriate base and solvent, via a nucleophilic substitution reaction, the fragment does not participate in N-C bond formation and has a functional group that is conveniently protected by a temporary or permanent protecting group. R1 may also be introduced by reacting the N-terminus of the compound of the present invention with an R5COOH group or its ester, acid halide, or its anhydride. R5 is as already defined above.

[0093] If necessary and / or additionally, in the presence of an appropriate solvent, a base (for example, N,N-diisopropylethylamine (DIEA), or triethylamine), or an additive (for example, 1-hydroxybenzotriazole (HOBt), or 1-hydroxyazabenzotriazole (HOAt)), and a dehydrating agent (for example, in particular, carbodiimide, uronium salt, phosphonium salt, or amidinium salt), an R2 radical can be introduced by the reaction of an HR2 compound (where R2 is -OR3, -NR3R4, or -SR3) with a complementary fragment (where R2 is -OH) corresponding to the peptide of formula (I) of the present invention. Thereby, a peptide of the present invention of general formula (I) is obtained. Here, the said fragment does not participate in N-C, O-C, or S-C bond formation and has a functional group that is appropriately protected by a temporary or permanent protecting group. Alternatively, other R2 radicals can be incorporated simultaneously in the process of cleaving the peptide from the polymer support.

[0094] One skilled in the art can readily understand that the deprotection / cleavage steps at the C-terminus and N-terminus, and their subsequent derivatization, may be carried out in an unspecified order according to methods known in the prior art. (See, for example, Smith M. B. and March J., 1999 “March’s Advanced Organic Chemistry Reactions, Mechanisms and Structure”, 5th Edition, John Wiley & Sons, 2001).

[0095] The term “protecting group” and its plural forms refer to a group that blocks an organic functional group and can be removed under controlled conditions. Protecting groups, their relative reactivities, and the conditions under which they are maintained in an inert state are known to those skilled in the art.

[0096] Examples of representative protecting groups for amino groups include amides (e.g., amide acetate, amide benzoate, amide pivalate); carbamates (e.g., in particular, benzyloxycarbonyl (Cbz or Z), 2-chlorobenzyloxycarbonyl (ClZ), para-nitrobenzyloxycarbonyl (pNZ), tert-butyloxycarbonyl (Boc), 2,2,2-trichloroethoxycarbonyl (Troc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 9-fluorenylmethoxycarbonyl (Fmoc), or allyloxycarbonyl (Alloc)), trityl (Trt), methoxytrityl (Mtt), 2,4-dinitrophenyl (Dnp), N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-ylidene)ethyl] (Dde), 1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)-3-methyl-butyl (ivDde), 1-(1-adamantyl)-1-methylethoxy-carbonyl (Adpoc); preferably, Boc or Fmoc.

[0097] Examples of representative protecting groups for carboxyl groups are esters (e.g., in particular, tert-butyl (tBu) ester, allyl (All) ester, triphenylmethyl ester (trityl ester, Trt ester), cyclohexyl (cHx) ester, benzyl (Bzl) ester, ortho-nitrobenzyl ester, para-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, fluorenylmethyl (Fm) ester, 4-(N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl)benzyl (Dmab) ester; preferred protecting groups of the present invention are All, tBu, cHex, Bzl, and Trt esters.

[0098] The trifunctional amino acids can be protected using temporary or permanent protecting groups that are orthogonal to the protecting groups at the N-terminus and C-terminus during the synthesis process. The protecting groups for the amino groups described above are used to protect the amino groups in the lysine side chains; the guanidine groups in the arginine side chains can be protected with nitro groups, allyloxycarbonyl (Alloc), para-toluenesulfonyl (tosyl, Tos), 2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), or 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr), the side chains of serine and threonine can be protected with tert-butyl (tBu) esters; the cysteine side chains can be protected with a protecting group selected from the group consisting of trityl and acetamidomethyl; and the asparagine side chains can be protected with a protecting group selected from the group consisting of methoxytrityl, trityl, and xanthyl, or the asparagine side chains may not be protected. To protect the carboxyl groups in the side chains of aspartic acid and glutamic acid, esters (e.g., in particular, tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (trityl ester, Trt), cyclohexyl ester (cHx), benzyl ester (Bzl), ortho-nitrobenzyl ester, para-nitrobenzyl ester, para-methoxybenzyl ester, trimethylsilylethyl ester, 2-phenylisopropyl ester, fluorenylmethyl ester (Fm), 4-(N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl)benzyl ester (Dmab)) can be used. The methionine side chains are either protected with sulfoxide or used without protection.

[0099] Preferred trifunctional amino acid protecting groups of the present invention are tBu esters in serine and threonine, and in the side chains of aspartic acid and glutamic acid; Boc in the lysine side chain, Trt in the cysteine side chain, and Fmoc or Boc as a temporary protecting group at the N-terminus.

[0100] Examples of these and additional protecting groups, their introduction and removal, can be found in the literature Greene T.W. and Wuts P.G.M., (1999) “Protective groups in organic synthesis” John Wiley & Sons, New York; Atherton B. and Sheppard R.C. (1989) “Solid Phase Peptide Synthesis: A practical approach” IRL Oxford University Press. The term “protecting group” and its plural forms also include the polymeric supports used in solid phase synthesis.

[0101] When the synthesis is carried out completely or partially in the solid phase, solid supports that can be used in the procedures of the present invention include, but are not limited to, polystyrene supports, polyethylene glycol grafted onto polystyrene, etc. (for example, see p-methylbenzhydrylamine resin (MBHA) (Matsueda et al. Peptides 1981, 2(1), 45-50), 2-chlorotrityl resin (see Barlos et al. Tetrahedron Lett 1989, 30, 3943-3946; Barlos K. et al. Tetrahedron Lett. 1989, 30, 3947-3951), TentaGel resin (Rapp Polymere GmbH), ChemMatrix resin (Matrix Innovation, Inc), etc.). These may or may not contain labile linkers (for example, see 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valeric acid (PAL) (Albericio et al. J Org Chem 1990, 55(3), 3730-3743), 2-[4-aminomethyl-(2,4-dimethoxyphenyl)]phenoxylacetic acid (AM) (see Rink 1987, Tetrahedron Lett 28(33), 3787-90), Wang (see Wang, J Am Chem Soc 1973, 95(4), 1328-33), etc.). These are useful for the cleavage and cyclization of peptides that are half-protected in solution using a deprotection step, even in solution or in solid-phase cyclization and subsequent simultaneous deprotection and cleavage of the peptide.

[0102] [Pharmaceutical Composition] The peptides and peptide dimers of the present invention can be administered in the form of a composition containing them by any means that causes contact between the peptides and peptide dimers and their sites of action in the body of a mammal, preferably the human body.

[0103] In this regard, another aspect of the present invention is a pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide and / or at least one peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts. The pharmaceutical composition of the present invention can contain the peptide or peptide dimer of the present invention obtained by freeze-drying or spray-drying and can be reconstituted in a solvent suitable for its administration.

[0104] The peptides and peptide dimers of the present invention have variable water solubility according to the nature of their sequences or any possible modifications at the N-terminus and / or C-terminus. Therefore, the peptides and peptide dimers of the present invention can be incorporated into the composition by a method using an aqueous solution. Those that are not soluble in water can be solubilized in conventional pharmaceutically acceptable solvents (such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol, or polyethylene glycol, or any combination thereof).

[0105] The pharmaceutically effective amount of the peptides and peptide dimers of the present invention that must be administered, and their dosages, depend on a number of factors, including age, the condition of the patient, the nature or severity of the disorder or disease to be treated or prevented, the route and frequency of administration, and the specific nature of the peptide to be used.

[0106] "Pharmaceutically effective amount" is understood to mean a non-toxic but sufficient amount of the peptide or peptide dimer of the present invention to provide the desired pharmaceutical effect (i.e., prophylaxis and / or improvement of the pathology to be treated). The peptides and peptide dimers of the present invention are used in the pharmaceutical compositions of the present invention at a pharmaceutically effective concentration to achieve the desired pharmaceutical effect; in their preferred forms, the effective daily dose in humans is 0.001 mg / kg to 250 mg / kg, more preferably 0.005 mg / kg to 100 mg / kg, more preferably 0.01 mg / kg to 50 mg / kg, and even more preferably 0.01 mg / kg to 10 mg / kg.

[0107] The frequency of administration of the pharmaceutical composition can be, for example, once a month, once every two weeks, once a week, twice a week, three times a week, or once a day, but is not limited thereto.

[0108] The peptides and peptide dimers of the present invention, their stereoisomers, mixtures thereof, and / or their pharmaceutically acceptable salts may also be incorporated into delivery systems and / or pharmaceutical sustained release systems.

[0109] The pharmaceutical compositions of the present invention may contain at least one pharmaceutically acceptable excipient and / or adjuvant. The number and nature of the pharmaceutically acceptable excipients depend on the desired mode of administration. Pharmaceutically acceptable excipients are known to those skilled in the art (see Rowe R.C., Sheskey P.J., Quinn, M.E. (2009) “Handbook of Pharmaceutical Excipients, 6th Edition”, Pharmaceutical Press and American Pharmacists Association). The compositions can be prepared using conventional methods known in the art.

[0110] The terms "delivery system" and its plural forms relate to diluents, auxiliary excipients or carriers for administering the peptides or peptide dimers of the present invention. These pharmaceutical carriers may be liquids (e.g., water, oils, or surfactants (derived from petroleum, animal, plant, or synthetic sources (e.g., peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitan ester, ether sulfate, sulfate, betaine, glycoside, maltoside, fatty alcohol, nonoxynol, poloxamer, polyoxyethylene, polyethylene glycol, dextrose, glycerol, digitonin, etc., but not limited thereto))). Those skilled in the art know the diluents, adjuvants, or excipients that can be used in various delivery systems for administering the peptides and peptide dimers of the present invention.

[0111] The term "sustained release" is used in its conventional meaning to refer to a delivery system of a compound that provides a slow release of the compound over a period of time, preferably having a relatively constant compound release level over a period of time, although not necessarily so.

[0112] Examples of delivery systems or sustained release systems include, but are not limited to, liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, milliparticles, microparticles, nanoparticles, and solid lipid nanoparticles, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed micelles of surfactants, surfactant-lipid mixed micelles, myristospheres, microspheres, and nanospheres, lipospheres, millicapsules, microcapsules, and nanocapsules, as well as microemulsions and nanoemulsions. These may be added to achieve greater biological utility of the active ingredient and / or to improve its pharmacokinetic and pharmacodynamic properties.

[0113] The pharmaceutical compositions of the peptides and peptide dimers of the present invention, their stereoisomers, mixtures thereof, and / or their pharmaceutically acceptable salts include pharmaceutically acceptable excipients necessary for formulating the desired dosage form and can be administered by any suitable route, including local or systemic use (e.g., but not limited to local, enteral, or parenteral routes). In the context of the present invention, the term "local" route includes transdermal and ocular routes. The term "enteral" route includes administration to the digestive system (e.g., oral, buccal, gastric, sublingual, and rectal routes). The term "parenteral" refers to nasal, auricular, ocular, rectal, urethral, vaginal, subcutaneous, intradermal, intravascular injection (e.g., intravenous, intramuscular, intraocular, intraspinal, intracranial, intranasal, intracerebral, intrathecal, intraarticular, intrahepatic, intrathoracic, intratracheal, intramedullary, and intraperitoneal, and any other similar injection or infusion techniques). Treatments in vitro are also contemplated, e.g., treatments in damaged cell cultures and / or stem cells, and ex vivo treatments.

[0114] More specifically, treatment with the peptides, peptide dimers, and compositions of the present invention is preferably carried out in vivo because the route of administration is preferably subcutaneous.

[0115] The pharmaceutical compositions of the peptides and peptide dimers of the present invention, their stereoisomers, mixtures thereof, and / or their pharmaceutically acceptable salts can be administered in combination therapy with other therapeutic agents and / or treatment procedures. Preferably, the above therapeutic agents and / or treatment procedures are anti-cancer agents and / or treatments.

[0116] 〔Use〕 In another aspect, the present invention relates to the peptides or peptide dimers of the present invention, their stereoisomers, mixtures thereof, and / or their pharmaceutically acceptable salts, or the pharmaceutical compositions of the present invention for use as a medicament.

[0117] Another aspect of the present invention relates to the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention for use in the treatment and / or diagnosis of cancer and / or metastasis. Specifically, the peptides and peptide dimers of the present invention can suppress cancer growth in various cancers and / or reduce or suppress the invasion and metastasis of cancer cells.

[0118] The present invention relates to a method for the treatment and / or diagnosis of cancer and / or metastasis. The method includes the pharmaceutical administration of an effective amount of the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention, to an individual (preferably a human) in need thereof.

[0119] The present invention relates to the use of the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention for the manufacture of a medicament.

[0120] The present invention relates to the use of the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention for the manufacture of a medicament for the purpose of diagnosing and / or treating cancer and / or metastasis.

[0121] In one embodiment, the present invention relates to the use of the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention in the diagnosis of cancer and / or metastasis. The diagnosis is performed without using the human body. This is called an in vitro diagnosis.

[0122] Accordingly, in a preferred embodiment, the present invention refers to an in vitro diagnostic method for cancer and / or metastasis, which comprises the use of the peptide or peptide dimer of the present invention, its stereoisomers, its mixtures, and / or its pharmaceutically acceptable salts, or the pharmaceutical composition of the present invention.

[0123] Cancer and / or metastasis include, but are not limited to, neuroblastoma, sarcoma, soft tissue sarcoma (e.g., rhabdomyosarcoma (including fetal, alveolar, pleomorphic, and spindle cell / sclerosing)), endometrial sarcoma, undifferentiated spindle cell sarcoma, undifferentiated pleomorphic sarcoma, undifferentiated round cell sarcoma, undifferentiated epithelioid sarcoma, liposarcoma, atypical lipomatous tumor, malignant solitary fibrous tumor, inflammatory myofibroblastic tumor, low-grade myofibroblastic sarcoma, fibrosarcoma (including adult and sclerosing epithelioid variants), myxofibrosarcoma, low-grade fibromyxoid sarcoma, giant cell tumor of soft tissue, leiomyosarcoma, malignant glomus tumor, angiosarcoma (including reticular, pseudomyogenic, and epithelioid), angiosarcoma of soft tissue, extraskeletal osteosarcoma, malignant gastrointestinal stromal tumor (GIST), malignant peripheral nerve sheath tumor (including epithelioid variants), malignant triton tumor, malignant granular cell tumor, malignant ossifying mucinous tumor, stromal sarcoma, myoepithelial tumor, malignant phosphaturic mesenchymal tumor, synovial sarcoma (including spindle cell and biphasic synovial sarcoma), epithelioid sarcoma, alveolar soft part sarcoma, clear cell sarcoma of soft tissue, extraskeletal myxoid chondrosarcoma, extraskeletal Ewing sarcoma, desmoplastic small round cell tumor, extrarenal rhabdoid tumor, perivascular epithelioid cell tumor, bone sarcomas (e.g., osteosarcoma), chondrosarcoma, and Ewing sarcoma, angiosarcoma, angiosarcoma, fibrosarcoma, and myofibrosarcoma, chordoma, ameloblastoma, breast cancer (e.g., ductal carcinoma, lobular carcinoma, and papillary carcinoma), colon cancer, rectal cancer, anal cancer, colorectal cancer, brain tumor (e.g., glioblastoma, astrocytoma, or medulloblastoma), malignant glioma, prostate cancer, melanoma and other skin cancers, cervical cancer, uterine cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), endometrial cancer, lymphoma, head and neck cancer, oral cancer, salivary gland cancer, retinoblastoma, gastrointestinal cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer (e.g., hepatocellular carcinoma (HCC)), kidney cancer or renal cancer (e.g., Wilms tumor or nephroblastoma), bladder cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer), non-Hodgkin lymphoma, multiple myeloma, pituitary adenoma, squamous cell carcinoma, testicular cancer, leukemia (e.g., acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, plasmacytoma or multiple myeloma).

[0124] To better understand the present invention, reference is made to the accompanying drawings, in which the invention will be described in more detail below. The drawings are shown by way of example and with reference to exemplary and non-limiting examples.

[0125] [Brief Description of the Drawings] Figure 1. Western blot image showing the expression of ITGA9 in 6 rhabdomyosarcoma cell lines. Actin was used as a control.

[0126] Figure 2. Western blot image showing the expression of ITGA9 in 3 neuroblastoma cell lines. Actin was used as a control.

[0127] Figure 3: Western blot image showing the expression of ITGA9 in 6 different breast cancer cell lines. Actin was used as a control.

[0128] Figure 4. Kaplan-Meier curve of event-free survival along a 27-week study of rhabdomyosarcoma metastatic mice treated with vehicle (PBS, solid line, -), 0.5 mg / kg of SEQ ID NO: 7-NH2 (dotted line, ···), or 2 mg / kg of SEQ ID NO: 7-NH2 (dashed line, ---). The y-axis indicates the event-free survival rate (%). The x-axis indicates weeks (number of weeks).

[0129] Figure 5. Average number of metastases per mouse along a 27-week study of mice injected with RD cells into the tail vein and treated with vehicle (PBS), 0.5 mg / kg of SEQ ID NO: 6-NH2, and 2 mg / kg of SEQ ID NO: 6-NH2, or 0.5 mg / kg of SEQ ID NO: 7-NH2, and 2 mg / kg of SEQ ID NO: 7-NH2. The horizontal line indicates the mean ± SEM. Statistical significance: * p-value < 0.05; ** p-value < 0.01. The y-axis indicates the number of metastases per mouse. The x-axis indicates the treatment groups, and the various groups are, from left to right, vehicle (PBS), 0.5 mg / kg of SEQ ID NO: 6-NH2, 2 mg / kg of SEQ ID NO: 6-NH2, 0.5 mg / kg of SEQ ID NO: 7-NH2, 2 mg / kg of SEQ ID NO: 7-NH2.

[0130] Figure 6. Kaplan–Meier curves of event-free survival along a 13-week study of neuroblastoma metastatic mice treated with excipient (PBS, solid line, -), 1 mg / kg of SEQ ID NO:7-NH2 (dotted line, ···), or 2 mg / kg of SEQ ID NO:7-NH2 (dashed line, ---). The y-axis indicates the event-free survival rate (%). The x-axis indicates weeks (number of weeks).

[0131] 〔Example〕 The following specific examples provided herein serve to illustrate the nature of the present invention. These examples are given for illustrative purposes only and should not be construed as limiting the invention claimed herein.

[0132] 〔Abbreviations〕 The abbreviations used herein have the following meanings: Ac2O, acetic anhydride; ACN, acetonitrile; AcOH, acetic acid; Boc, tert-butyloxycarbonyl; DCM, dichloromethane; DMEM, Dulbecco’s Modified Eagle Medium; DIEA, N,N’-diisopropylethylamine; DIPCDI, diisopropylcarbodiimide; DMF, N,N-dimethylformamide; Et2O, diethyl ether; eq, equivalents; ESI-MS, electrospray ionization mass spectrometry; Et2O, diethyl ether; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBT, N-hydroxybenzotriazole; HPLC, high performance liquid chromatography; ITGA9, integrin α9β1; i.v., intravenous; p-MBHA resin, 4-methylbenzhydrylamine resin; MeOH, methanol; MW, molecular weight; μL, microliter; PBS, phosphate buffered saline; RP-HPLC, reverse phase HPLC; rpm, revolutions per minute; s.c, subcutaneous; tBu, tert-butyl; TFA, trifluoroacetic acid; TIS, triisopropylsilane; TIO, thioanisole; tr, retention time; Trt, trityl.

[0133] Example 1. Synthesis of peptides and peptide dimers.

[0134] Ac-sequence number 10-NH2: Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-NH2 Ac-sequence number 3-NH2: Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 Octanoyl-sequence number 3-NH2: Octanoyl-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 Sequence number 4-NH2: Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 Ac-sequence number 5-NH2: Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 Octanoyl-sequence number 5-NH2: Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 Sequence number 6-NH2: Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 Sequence number 7-NH2: Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Sequence number 6-NH2)2 (disulfide bridge): (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2)2 (disulfide bridge) Octanoyl-sequence number 8-NH2: Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2 Sequence number 9-NH2: Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-NH2.

[0135] Solid-phase peptide synthesis was used for the synthesis of peptides. The peptide Ac-sequence number 10-NH2 (a 12-amino acid sequence contained within the native disintegrin region of ADAM-12 and, in particular, within sequence number 2) was also synthesized as a control compound.

[0136] All peptides were synthesized manually or using an automated peptide synthesizer (Liberty, CEM) by solid-phase peptide synthesis according to the standard Fmoc / tBu strategy using 4-methylbenzhydrylamine resin (p-MBHA) on various scales (0.3 mmol to 30 mmol). For each sequence, the resin was placed in an appropriate reaction vessel equipped with a filter plate. It was dissolved in DMF:DCM (1:1) stirred until the Fmoc-AM-OH linker (2 eq) and HOBT (2 eq) were dissolved, and then DIPCDI (2 eq) was added. The solution was transferred to the reaction vessel and reacted for 40 to 60 minutes. Incorporation of the linker was controlled by the ninhydrin test. If the ninhydrin test was positive, a reactivation or recoupling step was carried out until the coupling was complete.

[0137] Once coupling was complete, synthesis proceeded with removal of the Fmoc group by treatment with a 20% piperidine solution in DMF (twice for 5 minutes and twice for 10 minutes). The peptide resin was washed five times with DMF, filtered, the washings discarded, and the next amino acid coupled. Coupling was carried out using Fmoc-AA-OH:DIPCDI:HOBt (3 eq: 3 eq: 3 eq) in DMF (coupling Fmoc-Msa-OH at 1.5 eq). All of these coupling reactions were allowed to react for 40 to 60 minutes. Completion of each coupling reaction was monitored by the ninhydrin test (or chloranil if the coupling is on a secondary amine of proline). If necessary, a reactivation step or a recoupling step was carried out. Synthesis performed on an automated synthesizer was not monitored by the ninhydrin test (nor by chloranil). If necessary, at the end of the synthesis, acetylation was carried out by using Ac2O (2.5 eq) and DIEA (5 eq) in DMF. For these sequences with an N-terminal octanoyl, 5 eq of octanoic acid, HOBT and DIPCDI were used for acylation. After completion of the peptide sequence, the peptidyl resin was washed with DMF, MeOH, and Et2O and dried.

[0138] After synthesis, cleavage of the peptide was carried out according to the following procedure. The peptidyl resin was reacted for 2 to 4 hours in a suitable TFA mixture (see Table 2 below) at room temperature. The resin was washed with TFA and Et2O. All of the filtrate was poured into cold ether 81:7 (v:v) and allowed to stand for 15 to 30 minutes. The resulting suspension was filtered through a filter plate and the filter discarded. The residue was washed with ether and the filter used for each washing discarded. The solid was lyophilized to obtain the crude peptide product. The following table (Table 2) shows the TFA cocktails used for various peptides and peptide dimers.

[0139]

Table 2

[0140] The peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge) was obtained by oxidizing 20 mg of the peptide (SEQ ID NO: 6-NH2) at a concentration of 10 mg / mL in water, 10% AcOH, and 15% DMSO for 48 to 72 hours. Subsequently, it was recovered using a Sep pack C18 cartridge (Waters). The peptide dimer was retained on a C18 column. Then, it was recovered with a mixture of water:acetonitrile (80:20) and lyophilized to obtain 17 mg of the peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge) (yield 81%).

[0141] The crude peptide and peptide dimer were purified using a semi-preparative system equipped with an NW50 column packed with 10 micrometer Chromasil silica, and the pure fractions analyzed by analytical RP-HPLC were lyophilized.

[0142] The following table (Table 3) shows the yields of the synthetic weight, crude peptide, and pure peptide, as well as the MW of each compound.

[0143]

Table 3

[0144] The ion exchange step was performed as follows. The lyophilized pure compounds were dissolved in a mixture of AcOH. Subsequently, the acetic acid solutions containing them were treated with DOWEX resin to obtain peptides or peptide dimers with acetate counterions. Finally, the acetate compounds were filtered, recovered, and lyophilized.

[0145] As shown in the following table (Table 4), the peptides and peptide dimers were characterized by mass spectrometry in an ESI-MS instrument.

[0146]

Table 4

[0147] Example 2. In vitro stability. Measurement of the half-life (t1 / 2) of the peptide and peptide dimer in human plasma.

[0148] The peptides and peptide dimers synthesized and prepared according to Example 1 were dissolved in water at a concentration of 6 mg / mL and warmed to 37°C.

[0149] Human plasma was obtained as a lyophilized solid (K3 EDTA plasma, BBI solution, code S112-1). It was reconstituted with sterile 0.9% sodium chloride solution and stored at -20°C. The human plasma was thawed and incubated at 37°C before use.

[0150] The peptides and peptide dimers were incubated in 90% human plasma at 37°C for various periods and then precipitated with 2 volume equivalents of methanol. The samples were cooled in an acetone-carbon dioxide bath for several seconds and centrifuged at approximately 10,000 rpm at 4°C for 12 minutes. The supernatant was filtered through a 0.45 μm PVDF filter and analyzed in triplicate by RP-HPLC using an isocratic method (eluent A = 0.1% TFA in water; eluent B = 0.07% TFA in ACN, column = Kromasil C8, 100 Å, 5 μm, 250 × 4.6 mm, flow rate = 1 mL / min, wavelength: 220 nm, injection volume = 20 μL, temperature = 60°C). The disappearance of the compound was determined in relation to the area of the initial period for calculating its half-life.

[0151] The following table (Table 5) shows the t1 / 2 data for the peptides and peptide dimers.

[0152]

Table 5

[0153] Example 3. Integrin α9β1 (ITGA9) protein level. Evaluation of ITGA9 protein expression by Western blot.

[0154] The rhabdomyosarcoma cell lines RH30, CW9019, RH4, RH18, RD, and HTB82 were cultured in minimum essential medium supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich), 2 mM L-glutamine, 1 mM sodium pyruvate, 1x non-essential amino acids, 100 U / mL penicillin, and 0.1 mg / mL streptomycin (all Biowest reagents) and Earle's salts (Biowest). The neuroblastoma cell lines CHLA-90, BE(2)-C, and SK-N-BE(2)-C were grown in Iscove's Modified Dulbecco's Medium (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich), 1% insulin-transferrin-selenium G supplement (Thermo Fisher Scientific), 100 U / mL penicillin, and 0.1 mg / mL streptomycin (Biowest). The breast cancer cell lines MDA-MB-231, MDA-MB-468, MCF7, T47D, BT474, and MCF10A were cultured in Dulbecco's Modified Eagle Medium (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma), 1x non-essential amino acids, 100 U / ml penicillin, and 0.1 mg / ml streptomycin (all Biowest reagents). All cell lines were maintained at 37°C in an atmosphere controlled with 5% CO2.

[0155] At 80% confluence, the cells were washed with PBS and scraped in RIPA protein lysis buffer (Thermo Fisher Scientific) supplemented with protease inhibitor (Roche) and phosphatase inhibitor (Sigma). The cell lysates were incubated at 95°C for 5 minutes. After centrifugation at 13,000 rpm for 15 minutes at 4°C, the cell debris was discarded. The supernatant protein concentration was quantified by the DC protein assay (Bio-Rad Laboratories) according to the manufacturer's instructions.

[0156] The proteins were separated by 8% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred onto a PVDF (polyvinylidene fluoride) membrane (GE Healthcare). After blocking with 5% BSA (bovine serum albumin) in TBS-T (Tris-buffered saline-Tween) for 1 hour, the membrane was incubated overnight at 4 °C with the primary antibodies diluted as follows: anti-α9-integrin monoclonal antibody clone 3E4 (Novus Biologicals) diluted 1:1000, anti-FAK (Cell Signaling) diluted 1:1000, anti-phospho-FAK of Tyr397 (Cell Signaling) diluted 1:1000, and anti-actin (Santa Cruz Biotechnology) diluted 1:10,000. After washing with TBS-T, the membrane was incubated with the corresponding peroxidase-conjugated secondary antibody for 1 hour at room temperature. Immunoreactive bands were visualized with ECL prime chemiluminescent detection reagent (GE Healthcare).

[0157] As shown in the Western blot images of FIGS. 1, 2, and 3, the expression of ITGA9 was higher in RD than in RH30 cells, higher in BE(2)-C and CHLA-90 cells than in SK-N-BE(2)-C cells, and also high in MDA-MB-468 cells among the remaining breast cancer cell lines evaluated.

[0158] Example 4. Measurement of in vitro invasiveness in rhabdomyosarcoma cell lines (RH30 and RD).

[0159] 10 5 Ten individual RH30 or RD cells were plated in the upper chamber of a Matrigel-coated transwell (Corning) in 0.1 mL of serum-free medium. The cells were pre-incubated for 48 hours with peptides and peptide dimers at various concentrations (0 nM, 100 nM, 200 nM, 500 nM, and 1000 nM) and were also present in the medium during the cell invasion assay. 0.6 mL of medium containing FBS was added to the lower chamber. The cells were incubated at 37 °C for 24 hours.

[0160] Thereafter, the cells were fixed with 4% paraformaldehyde (Sigma) for 10 minutes. Then, they were washed with PBS. Matrigel and the remaining cells were removed from the upper surface of the membrane with a cotton swab. Thereafter, the cells present on the lower surface were stained with Hoechst dilution solution (Sigma) and counted using an inverted fluorescence microscope Nikon equipped with a 10x objective lens (5 fields for each well). All experiments were performed in triplicate. In Table 6, % invasiveness indicates the average ratio of invasive cells to their control.

[0161]

Table 6

[0162] All peptide analogs caused a significant decrease in the ratio of invasiveness (% invasiveness) of RH30 and / or RD rhabdomyosarcoma cells compared to untreated cells. This was significantly achieved with various concentrations of the peptide analogs. Ac-sequence number 5-NH2 and (sequence number 6-NH2)2 showed a significant decrease in the % invasiveness of RH30 cells at a peptide concentration of 100 nM. Octanoyl-sequence number 5-NH2 and sequence number 6-NH2 showed a significant decrease at 500 nM. A significant decrease in the % invasiveness of RD cells was observed with treatment with Octanoyl-sequence number 5-NH2 at a concentration of 500 nM, and with (sequence number 6-NH2)2 and Octanoyl-sequence number 8-NH2 at a concentration of 1 μM. Sequence number 7-NH2 showed a significant effect on RD cell invasiveness with less than 50% invasiveness even at the lowest dose (100 nM). In the RH30 cell line showing low ITGA9 expression, the effect was milder and decreased the % invasiveness without reaching significance.

[0163] Example 5. Measurement of in vitro invasiveness in neuroblastoma cell lines (CHLA-90 and BE(2)-C).

[0164] Transwell assays were performed under the same detailed conditions using the CHLA-90 and BE(2)-C cell lines, and the % invasiveness in rhabdomyosarcoma cells was measured.

[0165] As described in Table 7, the neuroblastoma cell lines also showed sensitivity to the peptide (SEQ ID NO: 6-NH2), reaching a significant decrease in % invasiveness for both cell lines CHLA-90 and BE(2)-C at concentrations of 200 nM and 500 nM, respectively.

[0166]

Table 7

[0167] Example 6. Results of the peptide (SEQ ID NO: 7-NH2) on in vitro invasiveness in neuroblastoma cells (CHLA-90, BE(2)-C and SK-N-BE(2)-C) and breast cancer cell lines (MDA-MB-231 and MDA-MB-468).

[0168] Transwell assays were performed under the same detailed conditions using the neuroblastoma cell lines (CHLA-90, BE(2)-C and SK-N-BE(2)-C) and breast cancer cell lines (MDA-MB-231 and MDA-MB-468), and the % invasiveness in rhabdomyosarcoma cells was measured.

[0169] As shown in Table 8, the neuroblastoma cell lines CHLA-90 and BE(2)-C also showed sensitivity to the peptide (SEQ ID NO: 7-NH2), and cell invasiveness decreased significantly. In a further study of invasiveness using the peptide in the third neuroblastoma cell line, it was shown that this treatment was also effective in suppressing invasion at a concentration of 500 nM in the SK-N-BE(2)-C cell line that does not overexpress ITGA9. In the breast cancer cell lines, the cell line MDA-MB-468 showed a clear decrease in invasion under treatment. On the other hand, the cell line MDA-MB-231 (with low ITGA9 expression) showed a mild decrease at the maximum treatment dose (not statistically significant).

[0170]

Table 8

[0171] Collectively, these results do not show a specific association between higher ITGA9 expression levels and better efficacy of the peptide that reduces invasion (SEQ ID NO: 7-NH2). Therefore, the interaction with ITGA9 should not be the only molecular mechanism by which the peptide analog exerts its efficacy. This is because, for example, SEQ ID NO: 7-NH2 at a concentration of 500 nm caused a significant decrease in the invasiveness of SK-N-BE(2)-C, a cell line with low ITGA9 expression levels.

[0172] Example 7. In vivo invasiveness in a mouse model of rhabdomyosarcoma metastasis.

[0173] On day -1, 5-week-old SCID / Beige female mice were subcutaneously (s.c.) administered with the vehicle (PBS, phosphate-buffered saline) or the peptide at the first treatment dose. On day 0, 2×10 6 RD rhabdomyosarcoma cells were intravenously (i.v.) injected into the tail vein of the mice. Thereafter, the mice were treated subcutaneously three times a week (every 2 - 3 days) for 27 weeks with the vehicle PBS (n = 9); SEQ ID NO: 6-NH2 0.5 mg / kg (n = 6); SEQ ID NO: 6-NH2 2 mg / kg (n = 7); SEQ ID NO: 7-NH2 0.5 mg / kg (n = 6) and SEQ ID NO: 7-NH2 2 mg / kg (n = 7).

[0174] Body weight was evaluated twice a week for 27 weeks. All survival rates and event-free survival rates were measured. At autopsy, the total number of metastases was quantified, and the number of metastases per mouse and their localization were also reported.

[0175] In accordance with the study, the excipient-treated mice showed significantly lower body weights compared to mice treated with SEQ ID NO: 6-NH2 (p<0.0001) at 0.5 mg / kg, SEQ ID NO: 6-NH2 (p<0.0001) at 2 mg / kg, SEQ ID NO: 7-NH2 (p = 0.0006) at 0.5 mg / kg, and SEQ ID NO: 7-NH2 (p = 0.0012) at 2 mg / kg. These differences were mainly due to the lower proportion of mice with metastases in the groups treated with SEQ ID NO: 6-NH2 and SEQ ID NO: 7-NH2 peptides.

[0176] As shown in Table 9, all PBS-treated mice developed metastases. Treatment with the two test doses of SEQ ID NO: 6-NH2 and the low dose of SEQ ID NO: 7-NH2 caused an approximately 15% reduction in metastasis formation. On the other hand, in the group treated with 2 mg / kg of SEQ ID NO: 7-NH2, only 2 out of 7 mice developed metastases, showing a statistically significant decrease compared to the PBS-treated group (p = 0.0048).

[0177]

Table 9

[0178] The manifestation of event-free survival per treatment group also showed a significant difference in the group treated with 2 mg / kg of SEQ ID NO: 7-NH2 when compared to the control group treated with the excipient (log-rank p-value = 0.0006). The lower doses of SEQ ID NO: 7-NH2 and both doses of SEQ ID NO: 6-NH2 tested did not show statistically significant differences compared to the group treated with the excipient (Figure 4 shows the results for animals treated with PBS and SEQ ID NO: 7-NH2).

[0179] Furthermore, after a single intravenous administration of RD cells and after subcutaneous administration of the excipient or various treatments three times a week for one week, when the number of metastases formed per mouse was evaluated, 2 mg / kg of SEQ ID NO: 6-NH2 and SEQ ID NO: 7-NH2 showed a statistically significant decrease in metastasis formation per mouse compared to control mice treated with the excipient (PBS) (SEQ ID NO: 6-NH2 2 mg / kg, p = 0.0058; SEQ ID NO: 7-NH2 2 mg / kg, p = 0.0184) (Figure 5).

[0180] The above results indicate that lower doses of SEQ ID NO: 6-NH2 or SEQ ID NO: 7-NH2 did not show a significant effect on either the delay in metastasis expression evaluated as event-free survival or the number of mice without metastases. However, treatment with higher doses of SEQ ID NO: 6-NH2 and SEQ ID NO: 7-NH2 was shown to be useful in reducing the number of metastases observed at autopsy.

[0181] In terms of the relationship with the localization site, metastases were expressed at various localization sites in the intestine, uterus, foot, dorsal spleen, ovary, and adrenal gland. In all treatment groups, metastases to the ovary, adrenal gland, back, and spleen were more frequent.

[0182] Example 8. Effect of peptide (SEQ ID NO: 7-NH2) on a neuroblastoma metastatic mouse model.

[0183] - On day 1, 5-week-old SCID / Beige female mice were subcutaneously (s.c.) administered the excipient (PBS) or peptide (SEQ ID NO: 7-NH2) at doses of 1 mg / kg and 2 mg / kg as the first treatment dose. On day 0, 2 × 10 5 BE(2)-C neuroblastoma cells were intravenously (i.v.) injected into the tail vein of the mice. Thereafter, the mice were treated subcutaneously three times a week (every 2 - 3 days) for 13 weeks with the excipient PBS (n = 8) and SEQ ID NO: 7-NH2 at doses of 1 mg / kg (n = 9) and 2 mg / kg (n = 9).

[0184] As shown in Table 10, 7 out of 8 (88%) mice in the PBS treatment group developed metastases, while in the group treated with SEQ ID NO: 7-NH2 at 2 mg / kg, the number of mice with metastases tended to be lower (4 out of 9 (44%), p = 0.1312).

[0185]

Table 10

[0186] Furthermore, the manifestation of event-free survival was significantly different among the group treated with the excipient (survival rate: 1 out of 8, 13%), the group treated with peptide 1 mg / kg (SEQ ID NO: 7-NH2) (survival rate: 2 out of 9 (22%), log-rank p-value = 0.6294), and the group treated with peptide 2 mg / kg (survival rate: 5 out of 9 (56%), log-rank p-value = 0.0658), indicating the usefulness of the peptide (SEQ ID NO: 7-NH2) at the maximum dose (Figure 6).

Brief Description of the Drawings

[0187]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Claims

1. A peptide or peptide dimer, a mixture thereof, and / or a pharmaceutically acceptable salt thereof, selected from the group consisting of: R １ -Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R ２ (R １ -SEQ ID NO: 5-R ２ ) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R ２ (SEQ ID NO: 6-R ２ ) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-R ２ (SEQ ID NO: 7-R ２ ) (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R ２ ) ２ (Disulfide bridge)[(SEQ ID NO: 6-R ２ ) ２ (Disulfide bridge)] Here, R １ is selected from the group consisting of H, acetyl, and octanoyl, and, R ２ is NH ２ .

2. A peptide or peptide dimer, a mixture thereof, and / or a pharmaceutically acceptable salt thereof, according to claim 1, selected from the group consisting of: Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH ２ (Ac-SEQ ID NO: 5-NH ２ ) Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH ２ (Octanoyl-sequence number 5-NH ２ ) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH ２ (sequence number 6-NH ２ ) Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH ２ (sequence number 7-NH ２ ) (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH ２ ) ２ (disulfide bridge)[(sequence number 6-NH ２ ) ２ (disulfide bridge)].

3. A method for preparing the peptide or peptide dimer according to claim 1, a mixture thereof, and / or a pharmaceutically acceptable salt thereof, comprising: A method performed using solid-phase peptide synthesis or peptide synthesis in solution.

4. The method according to claim 3, comprising the following steps: a) Performing solid-phase peptide synthesis in a polymer support; b) Cleaving the peptide from the polymer support; c) Oxidizing the peptide in solution to obtain a cyclic peptide or peptide dimer; d) Removing protecting groups; Or, i) Performing solid-phase peptide synthesis in a polymer support; ii) Performing solid-phase peptide cycling or dimer formation in the polymer support; iii) cleaving the peptide from the polymer support and simultaneously removing the protecting group; iv) oxidizing the peptide in solution to obtain a peptide dimer.

5. A pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide or peptide dimer according to claim 1, a mixture thereof, and / or a pharmaceutically acceptable salt thereof.

6. The peptide, or the peptide dimer, is incorporated into a delivery system and / or sustained release system selected from the group consisting of liposomes, mixed liposomes, oleosomes, niosomes, ethosomes, mili particles, micro particles, nano particles, solid lipid nanoparticles, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed micelles of surfactants, surfactant - phospholipid mixed micelles, milispheres, microspheres, nanospheres, lipospheres, mili capsules, micro capsules, nano capsules, micro emulsions, and nano emulsions, the pharmaceutical composition according to claim 1.

7. The peptide or peptide dimer according to claim 1, a mixture thereof, and / or a pharmaceutically acceptable salt thereof for use as a medicament.

8. The peptide or peptide dimer according to claim 1, a mixture thereof, and / or a pharmaceutically acceptable salt thereof for use in the treatment and / or diagnosis of cancer and / or metastasis.

9. The cancer and / or metastasis as described above is selected from the group consisting of neuroblastoma, sarcoma, soft tissue sarcoma, rhabdomyosarcoma (including fetal, alveolar, pleomorphic, and spindle cell / sclerosing rhabdomyosarcoma), endometrial sarcoma, undifferentiated spindle cell sarcoma, undifferentiated pleomorphic sarcoma, undifferentiated round cell sarcoma, undifferentiated epithelioid cell sarcoma, liposarcoma, atypical lipomatous tumor, malignant solitary fibrous tumor, inflammatory myofibroblastic tumor, low-grade myofibrosarcoma, fibrosarcoma (including adult and sclerosing epithelioid fibrosarcoma variants), myxofibrosarcoma, low-grade fibromyxoid sarcoma, giant cell tumor of soft tissue, leiomyosarcoma, malignant glomus tumor, angiosarcoma (including reticular, pseudomyogenic, and epithelioid angiosarcoma), angiosarcoma of soft tissue, extraskeletal osteosarcoma, gastrointestinal stromal tumor (GIST), malignant peripheral nerve sheath tumor, malignant triton tumor, malignant granular cell tumor, malignant ossifying mucoid tumor, stromal sarcoma, myoepithelial tumor, malignant phosphaturic mesenchymal tumor, synovial sarcoma (including spindle cell and biphasic synovial sarcoma), epithelioid sarcoma, alveolar soft part sarcoma, clear cell sarcoma of soft tissue, extraskeletal myxoid chondrosarcoma, extraskeletal Ewing sarcoma, desmoplastic small round cell tumor, extrarenal rhabdoid tumor, perivascular epithelioid cell tumor, bone sarcomas, osteosarcoma, chondrosarcoma, Ewing sarcoma, angiosarcoma, angiosarcoma, fibrosarcoma, myofibrosarcoma, chordoma, ameloblastoma, breast cancer (including ductal carcinoma, lobular carcinoma, and papillary carcinoma), colon cancer, rectal cancer, anal cancer, colorectal cancer, brain tumor, glioblastoma, astrocytoma, medulloblastoma, malignant glioma, prostate cancer, melanoma, skin cancer, cervical cancer, uterine cancer, ovarian cancer (including high-grade serous ovarian cancer), endometrial cancer, lymphoma, head and neck cancer, oral cancer, salivary gland cancer, retinoblastoma, digestive tract cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, liver cancer (including hepatocellular carcinoma (HCC)), kidney cancer or renal cancer, Wilms tumor, nephroblastoma, bladder cancer, lung cancer, small cell lung cancer, non-small cell lung cancer, non-Hodgkin lymphoma, multiple myeloma, pituitary adenoma, squamous cell carcinoma, testicular cancer, leukemia (including acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, plasmacytoma, and multiple myeloma), the peptide or peptide dimer according to claim 8.

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