Peptides for the treatment of cancer and / or metastasis

Abstract

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

Description

Technical Field

[0001] This invention falls within the field of biomedical chemistry. In particular, this invention relates to the field of inhibiting cancer and / or metastasis. Background Technology

[0002] In approximately 90% of cases, metastasis is the cause of death in cancer patients [Reymond et al., Nature Reviews 2016, 13(12), 858-70].

[0003] As cancer tumors spread due to metastasis, conventional treatments become ineffective, necessitating the research of new therapeutic targets and methods.

[0004] ADAM (de-integrin metalloproteinases) is a family of transmembrane proteins first described in the 1990s. Several ADAM members, including ADAM-9, ADAM-10, ADAM-12, ADAM-15, and ADAM-17, have been shown to play a role in cancer formation or progression. Consistent with these findings, increased expression of specific ADAMs in several cancer types has been found to be associated with aggressive disease and poor prognosis [Duffy et al., Clin Chim Acta [Journal of Clinical Chemistry] 2009, 403(1-2), 31-6]. Many ADAMs are involved in the regulation of growth factor activity and integrin function, thereby promoting cell growth and invasion [Mochizuki et al., Cancer Sci [Cancer Science] 2007, 98(5): 621-8; Zadka et al., Neoplasia [Oncology] 2018, 65(6), 823-39].

[0005] ADAM-12 was identified in 1995 during the search for fertilization homologs in mouse myogenic cell lines [Yagami-Hiromasa et al., Nature, 1995, 377(6550), 652-6] and human ADAM-12 was characterized in 1998 [Gilpin et al., J Biol Chem, 1998, 73(1), 157-66]. Its structure consists of an N-terminal signal peptide, a prodomain, a metalloproteinase (or metalloprotease) domain, a detegrin domain, a cysteine-rich domain, an EGF domain, a transmembrane region, and a C-terminal cytoplasmic tail. ADAM-12 has a membrane-bound form with all of the above domains (ADAM12-L) and a secretory form lacking transmembrane and cytoplasmic domains (ADAM12-S) [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, where it can regulate cell-cell adhesion or cell-ECM interactions by binding to adhesion molecules such as integrins and polymers [Kveiborg et al. J Biochem Cell Biol 2008, 40(9), 1685-702]. ADAM-12 interacts with syndecan-4, and this interaction can promote cell diffusion and stress fiber assembly [Thodeti et al. J Biol Chem 2003, 278(11), 9576-84].

[0007] Overall, overexpression of ADAM-12 is associated with metastasis and poor survival in a variety of cancers.

[0008] In breast cancer, ADAM-12 has been published as a high-expression initiator of breast cancer metastasis [Iba et al., Am J Pathol, 1999, 154(5), 1489-501; Huang et al., Oncogene, 2018, 37(49), 6316-26]. A functional study by Kveiborg et al. [Kveiborg et al., Cancer Research, 2005, 65(11), 4754-61] demonstrated that upregulation of ADAM-12 accelerates tumor progression in a mouse model of breast cancer. It is also upregulated in breast cancer tumor tissue compared to adjacent normal tissue. Kaplan-Meier survival curve results showed that patients with higher levels of ADAM-12 had shorter survival times compared to patients with low levels of ADAM-12 [Ma et al., Int J Clin Exp Pathol, 2015, 8(10), 13279-83]. It also describes that the presence of ADAM-12 in the urine of breast cancer patients increases proportionally with the progression of the disease [Roy et al. J Biol Chem [Journal of Biochemistry] 2004, 279(49), 51323-30].

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

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

[0011] Elevated ADAM-12 levels were found in patients diagnosed with pancreatic ductal adenocarcinoma compared with healthy controls, and reduced ADAM-12 levels during treatment were associated with longer survival [Veenstra et al. Oncogenesis 2018, 7(11), 87].

[0012] ADAM-12 has also been proposed as a biomarker for bladder cancer. The risk of progression to muscle-invasive bladder cancer in bladder cancer patients is as high as 60%. Upregulation of ADAM-12 at both the mRNA and protein expression levels is associated with the grade and stage of bladder cancer. et al., Clin Cancer Research, 2006, 12(24), 7359-68.

[0013] ADAM-12 expression is almost undetectable in normal liver, but increased in hepatocellular carcinoma and liver metastases [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 increased ADAM-12 expression and enhanced invasion and metastasis [Shao et al. Plos One [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 [Tian et al. J Clin Pathol [Journal of Clinical Pathology] 2002, 55(6), 394-7]. Its high expression is associated not only with tumor growth but also with enhanced osteolysis, in which animal survival is significantly reduced, suggesting that ADAM-12 may be a novel therapeutic target for osteosarcoma [George et al. Eur J Cancer [European Journal of Cancer] 2013, 49(9), 2253-63].

[0016] Compared with early-stage melanoma, ADAM-12 is significantly overexpressed in late-stage melanoma [Cireap et al. Pathol Oncol Res [Pathological Oncology Research] 2013, 19(4), 755-62].

[0017] Iba et al. described the importance of the Cys-rich domain of ADAM-12 in supporting cell adhesion in a group of cancer cell lines, such as MDA-MB-231 breast cancer cells [Iba et al., Am J Pathol, 1999, 154(5), 1489-501]. According to Iba et al., in the case of human ADAM-12, recombinant peptides rich in cysteine ​​domains rather than integrin-like domains support cell adhesion in a group of cancer cell lines.

[0018] Subsequently, the involvement of the deintegrin domain of ADAM-12 (SEQ ID NO:1, obtained from [Gilpin et al. J Biol Chem, 1998, 73(1), 157-66]) was investigated. Dyczynska et al. showed that mutations in the deintegrin region of ADAM-12 associated with breast cancer interfere with intracellular transport and processing of the protein [Dyczynska et al. Int J Cancer, 2008, 122(11), 2634-40]. Li et al. also described the role of the deintegrin domain of ADAM-12 in activating epidermal growth factor receptor (EGFR) in triple-negative breast cancer and breast tumor initiation cells (BTIC) [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 multiple cell types and interacts with many ligands, such as fibronectin and tendinin-C, including ADAM-12. Abnormal α9β1 expression can lead to or worsen pathological conditions such as cancer. AdvBiol Regul et al. [Advances in Biological Regulation] 2012, 52(2), 326-39.

[0020] α9β1 integrin expression is associated with reduced survival in the basal-like breast cancer subtype and is therefore considered a novel biomarker for this tumor subtype [Allen et al. J Pathol [Journal of Pathology] 2011, 223(5), 646-58]. Conversely, its downregulation in triple-negative breast cancer tumors is associated with reduced tumor angiogenesis, tumor growth, and metastasis [Wang et al. Int J Cancer [International Journal of Cancer] 2019, 145(10), 2767-80]. Tumor-α9β1 integrin-mediated signaling also plays a crucial role in generating a unique primary tumor tissue microenvironment conducive to breast cancer growth and lymphatic metastasis [Majumder et al. Plos One [PLOS ONE] 2012, 7(4), e35094; Ota et al. J Mol Med [Journal of Molecular Biology] 2014, 92(12), 1271-81].

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

[0022] The de-integrin domain of ADAM appears to play a crucial role in its interaction with integrins. Most ADAMs can interact with integrins through the RGD- or XCD- motif in their de-integrin domain; in fact, the XCD sequence region may be primarily responsible for inhibiting platelet aggregation and integrin interaction [Lu et al. Cardiovasc Hematol Agents Med Chem, 2007, 5(1), 29-42].

[0023] Although some ADAMs have been assumed to play a role in inhibiting cell adhesion and metastasis, there is little literature on the clinical use of ADAM-12 protein or its peptides in the treatment of cancer and / or metastasis.

[0024] Patent application WO 2015 / 028027 A1 discloses a monoclonal antibody targeting the predomain of ADAM-12 and its use in the treatment of cancer. The predomain of ADAM-12 is a region distant from the cysteine-rich and detegrin-free domains.

[0025] WO 2006 / 014903 A2 discloses ADAM-12 polynucleotide or its encoded polypeptide, which is highly expressed in cancerous tissue, and its modulators, such as antibodies. This document proposes treatment and diagnostic methods for proliferative diseases, including cancer and psoriasis, although it only discloses the expression of ADAM-12 in cancerous tissue and does not show any examples of the activity of any compound or treatment.

[0026] WO 2011 / 100362 A1 discloses a modified ADAM-derived polypeptide for cancer treatment, comprising an ADAM-derived polypeptide with an integrin-like domain deintegrated from ADAM and a fusion protein containing thioredoxin as the N-terminal segment. The polypeptide sequence has a sequence length of approximately 80 amino acids, close to the full-length sequence of the integrin-deintegrated domain of ADAM.

[0027] Rhabdomyosarcoma (RMS) and neuroblastoma (NB) are common examples of pediatric cancers. Rhabdomyosarcoma (RMS) is an early-onset malignant tumor and the most common type of soft tissue sarcoma in children [Masià et al. Br J Cancer [International Journal of Cancer] 2012, 107(8), 1374-83]. Neuroblastoma is the most common cancer in infants and the third most common cancer in children. Nearly half of patients with neuroblastoma have disease dissemination at the time of diagnosis [Maris et al. The Lancet [The Lancet] 2007, 369(9579), 2106-20].

[0028] Patients with metastatic disease have a very poor prognosis and therefore require more intensive treatment. Furthermore, the leading cause of death in these patients is the formation of distant metastases.

[0029] The significance of α9β1 integrin in invasion and metastasis has also been described in pediatric cancers such as rhabdomyosarcoma [Masià et al. Br J Cancer [British Journal of Cancer] 2012, 107(8), 1374-83]. Rhabdomyosarcoma cell lines have been used as models to establish the role of ADAM-12 / α9β1 integrin interaction in myogenesis, but the role of ADAM-12 in rhabdomyosarcoma tumorigenesis has not been described. The role of ADAM-12 in neuroblastoma has not been disclosed.

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

[0031] This invention provides a solution for reducing or inhibiting cancer growth, invasiveness, and / or metastasis in various cancers.

[0032] After extensive and detailed research, the inventors of this invention unexpectedly discovered a series of synthetic ADAM-12 analogues that can reduce the metastatic potential of various cancers such as rhabdomyosarcoma, neuroblastoma, and breast cancer.

[0033] The prior art does not disclose or suggest the use of short peptides derived from non-naturally substituted amino acids of ADAM-12 with detegrin domains for clinical inhibition of cancer growth and / or reduction of invasiveness and / or metastasis.

[0034] This invention provides modified peptides (and their dimers) derived from the amino acid sequence of the protein ADAM-12, particularly from the deintegrin domain of ADAM-12, and even more particularly from the amino acid sequence -CRDSSNSCDLPEFC- contained within the deintegrin domain of ADAM-12, namely Cys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-Cys (SEQ ID NO:2). The modified peptides (and their dimers) are stable in plasma and can inhibit invasiveness in vitro, thus reducing in vivo invasiveness and metastasis. Modifications to synthetic amino acids, such as the introduction of a trimethylphenyl amino acid, for example 3-trimethylphenylalanine (2,4,6-trimethylphenylalanine, Msa), at the phenylalanine position, optionally along with other modifications such as substitution of cysteine ​​residues and / or inclusion of a non-natural amino acid, such as pyroglutamic acid, at the amino terminus of the peptide, provide novel compounds with improved pharmacological properties and activities in inhibiting cancer growth and / or reducing or inhibiting cancer cell invasion and metastasis in various cancers. Cysteine ​​residues can be substituted with methionine, cystine, or basic amino acids such as lysine.

[0035] In vitro studies using the ADAM-12 analogue of the present invention showed reduced invasiveness in rhabdomyosarcoma cells that overexpressed (RD) and did not express (RH30) α9β1 integrin. This fact suggests that integrins cannot be the sole target of the ADAM-12 analogue of the present invention.

[0036] definition

[0037] For ease of understanding of the invention, the meanings of certain terms and expressions, as they are used in the context of the invention, are included.

[0038] "Invasion" and "invasiveness," along with their plural forms, are related to cell migration and define the ability of cells to become mobile and penetrate the extracellular matrix within tissues or infiltrate adjacent tissues. Cell invasion is the invasion and destruction of adjacent tissues. Cancer cells that become invasive may spread to secondary sites and form metastases.

[0039] "Metastasis" and its plural form are lesions of cancer cells that are associated with a pre-existing (called primary) cancer, but develop far from that primary lesion and are not continuous with it. The spread of these secondary lesions occurs via the lymphatic or hematogenous pathways.

[0040] As used herein, the term “treatment” and its plurals mean the application of compounds according to the invention to prevent, improve or eliminate adverse clinical symptoms caused by a disease, or to reduce or eliminate the incidence or severity of said disease.

[0041] In the context of this invention, the term "diagnosis" refers to a diagnostic method performed in the absence of a human body, i.e., an in vitro diagnostic method.

[0042] In the context of this invention, the term "therapeutic dose" and its plural form refer to the amount of a compound disclosed herein that must be administered to an individual in order to obtain a medical or biological positive response, the individual being a cell, animal, or human, and the compound being administered by a researcher, physician, veterinarian, or by the individual himself.

[0043] In the context of this invention, the term "therapeutic agent" and its plural form refer to any agent or compound that produces a desired pharmacological effect in an individual (cell, animal, or human).

[0044] In the context of this invention, the term "activity" or "pharmacological activity" and its plural form refer to a biological or medical response as a result of treating an individual (cell, animal, or human) with a compound disclosed herein, administered by a researcher, physician, veterinarian, or by the individual himself.

[0045] The term "individual" and its plural form refer to any organism to which the compounds described in this invention may be applied for experimental, diagnostic, and / or therapeutic purposes. The individual can be a cell, animal, or human.

[0046] In the context of this invention, the term "peptide dimer" and its plural form refer to a compound comprising two monomeric units of the peptide of this invention linked by a disulfide bond between cysteine ​​residues. In a homodimeric peptide with two equal peptide subunits, the peptide dimer is represented as: (peptide)2(disulfide bridge).

[0047] The terms “peptide” or “peptide analogue” and their plurals can be used as general terms for the compounds of the present invention, covering the peptides and peptide dimers of the present invention.

[0048] In this specification, the abbreviations used for amino acids follow the rules of the IUPAC-IUB Joint Committee on Biochemical Nomenclature as outlined in the Journal of Biochemistry (J. Biol. Chem.) (1989) 264:633-673.

[0049] Therefore, for example, Asn represents NH2-CH(CH2CONH2)-COOH. Thus, the hyphen representing the peptide bond eliminates the OH in the 1-carboxyl group of the amino acid (represented here in its non-ionic conventional form) when it is on the right side of the symbol, and eliminates the H in the 2-amino group of the amino acid when it is on the left side of the symbol; both modifications can be applied to the same symbol.

[0050] The amino acid structures and their one-letter and three-letter nomenclature codes and / or structures are shown in Table 1.

[0051] Amino acids can have L-configuration or D-configuration.

[0052]

[0053]

[0054] Table 1. Amino acid structures and their one-letter and three-letter nomenclature codes.

[0055] The abbreviation "Ac-" is used in this specification to name the acetyl group (CH3-CO-). The octyl group refers to an n-octyl or octyl group.

[0056] In the context of this invention, the term "trimethylphenyl amino acid" and its plural forms include 3-trimethylphenylalanine (2,4,6-trimethylphenylalanine, Msa) and 2-trimethylphenylglycine (2,4,6-trimethylphenylglycine, Msg).

[0057] The term "acyclic aliphatic group" is used in this invention to cover, for example, but not limited to, straight-chain or branched alkyl, alkenyl, and alkynyl groups.

[0058] The term "alkyl group" refers to a saturated, straight-chain or branched group having between 1 and 24, preferably between 1 and 16, more preferably between 1 and 14, even more preferably between 1 and 12, and still more preferably between 1, 2, 3, 4, 5, or 6 carbon atoms, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to, methyl, ethyl, isopropyl, isobutyl, tert-butyl, heptyl, octyl, decyl, dodecyl, lauryl, hexadecyl, octadecyl, pentyl, 2-ethylhexyl, 2-methylbutyl, 5-methylhexyl, and similar groups.

[0059] The term "alkenyl group" refers to a straight-chain or branched group having between 2 and 24, preferably between 2 and 16, more preferably between 2 and 14, even more preferably between 2 and 12, and still more preferably 2, 3, 4, 5, or 6 carbon atoms, wherein one or more carbon-carbon double bonds are present, preferably 1, 2, or 3 conjugated or non-conjugated carbon-carbon double bonds, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to, vinyl, oleyl, linoleyl groups, and similar groups.

[0060] The term "alkynyl group" refers to a straight-chain or branched group having between 2 and 24, preferably between 2 and 16, more preferably between 2 and 14, even more preferably between 2 and 12, even more preferably 2, 3, 4, 5 or 6 carbon atoms, wherein the group has one or more carbon-carbon triple bonds, preferably 1, 2 or 3 conjugated or non-conjugated carbon-carbon triple bonds, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to: ethynyl groups, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl, 3-butynyl, pentyynyl such as 1-pentynyl, and similar groups.

[0061] The term "alicyclic group" is used in this invention to cover, for example, but not limited to, cycloalkyl, cycloalkenyl, or cycloynyl groups.

[0062] The term "cycloalkyl" refers to a saturated monocyclic or polycyclic aliphatic group having between 3 and 24, preferably between 3 and 16, more preferably between 3 and 14, even more preferably between 3 and 12, and even more preferably still 3, 4, 5, or 6 carbon atoms, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, methylcyclohexyl, dimethylcyclohexyl, octahydroindene, decahydronaphthalene, dodecahydrofinaene, and similar groups.

[0063] The term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic aliphatic group having between 5 and 24, preferably between 5 and 16, more preferably between 5 and 14, even more preferably between 5 and 12, and even more preferably still 5 or 6 carbon atoms, wherein it has one or more carbon-carbon double bonds, preferably having 1, 2 or 3 conjugated or non-conjugated carbon-carbon double bonds, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to, cyclopent-1-en-1-yl groups and similar groups.

[0064] The term "cycloalkynyl" refers to a non-aromatic monocyclic or polycyclic aliphatic group having between 8 and 24, preferably between 8 and 16, more preferably between 8 and 14, even more preferably between 8 and 12, and even more preferably still 8 or 9 carbon atoms, wherein it has one or more carbon-carbon triple bonds, preferably having 1, 2 or 3 conjugated or non-conjugated carbon-carbon triple bonds, and the group is bonded to the rest of the molecule by single bonds. The group includes, for example and not limited to, cyclooctyl-2-yn-1-yl groups and similar groups.

[0065] The term "aryl group" refers to an aromatic group having between 6 and 30, preferably between 6 and 18, more preferably between 6 and 10, and even more preferably 6 or 10 carbon atoms, comprising 1, 2, 3 or 4 aromatic rings bonded or fused by carbon-carbon bonds, and the group being bonded to the rest of the molecule by single bonds, including, but not limited to, phenyl, naphthyl, biphenyl, indene, phenanthryl or anthracene, etc.

[0066] The term "aralkyl group" refers to an alkyl group substituted with an aromatic group, which has between 7 and 24 carbon atoms and includes, for example but not limited to, -(CH2). 1-6 -phenyl, -(CH2) 1-6 -(1-Naphthyl), -(CH2) 1-6 -(2-Naphthyl), -(CH2) 1-6 -CH(phenyl)2 and similar groups.

[0067] The term "heterocyclic group" refers to a heterocyclic ring or hydrocarbon ring having 3 to 10 members, wherein one or more atoms in the ring, preferably one, two, or three atoms in the ring, are elements other than carbon, such as nitrogen, oxygen, or sulfur, and the group can be saturated or unsaturated. For the purposes of this invention, the heterocyclic group can be a cyclic, monocyclic, bicyclic, or tricyclic system, which may include fused ring systems; and the nitrogen, carbon, or sulfur atoms may optionally be oxidized in the heterocyclic group; the nitrogen atom may optionally be quaternized; and the heterocyclic group may be partially or fully saturated or may be aromatic. With increasing preference, the term heterocyclic refers to a 5- or 6-membered ring.

[0068] The term "heteroarylalkyl group" refers to an alkyl group substituted with or unsubstituted aromatic heterocyclic groups having 1 to 6 carbon atoms, and the aromatic heterocyclic group having between 2 and 24 carbon atoms and 1 to 3 atoms other than carbon atoms, and the heteroarylalkyl group includes, for example, but is not limited to: -(CH2). 1-6 -imidazolium group, -(CH2) 1-6 -triazolyl, -(CH2) 1-6-Thienyl, -(CH2) 1-6 -furanyl, -(CH2) 1-6 -Pyrrolidinyl groups, and similar groups.

[0069] As used in this technical field, a certain degree of substitution may be present in the groups defined above. Therefore, substitution may be present in any group in this invention. The substituted groups mentioned in this document in the groups of this invention indicate that the specified group may be substituted 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 by one or more substituents. These substituents include, for example but not limited to, C1-C4 alkyl; hydroxyl; C1-C4 alkoxy; amino; C1-C4 aminoalkyl; C1-C4 carbonyloxy; C1-C4 oxycarbonyl; halogens such as fluorine, chlorine, bromine, and iodine; cyano; nitro; azide; C1-C4 alkylsulfonyl; thiol; C1-C4 alkylthio, aryloxy such as phenoxy; -NR b (C=NR) b )NR b R c ;where R b and R c Independently selected from the group consisting of: H, C1-C4 alkyl, C2-C4 alkenyl, C2-C4 ynyl, C3-C 10 cycloalkyl, C6-C 18 Aryl, C7-C 17 Aryl groups, 3-10 membered heterocyclic groups, or amino protecting groups.

[0070] As used herein, the "percentage of identity" for peptides, polypeptides, and proteins has the meaning commonly assigned in the art and thus refers to the percentage of identical amino acids between two amino acid sequences compared after optimal alignment of these sequences, where the percentage is statistically significant only and the differences between the two amino acid sequences are randomly distributed throughout the sequence. "Optimal alignment" is understood as an amino acid sequence alignment that yields a greater percentage of identity. The percentage of identity is obtained by determining the number of identical positions in the two compared sequences where amino acids are identical, dividing that number by the number of positions compared, and multiplying the result by 100. Sequence comparisons between two amino acid sequences can be performed manually or by computer programs known in the art, such as the BLAST (Basic Local Alignment Search Tool) algorithm.

[0071] The compounds of the present invention

[0072] The first aspect of the present invention relates to compounds of general formula (I):

[0073] R1-AA1-AA2-Arg-Asp-Ser-Ser-Asn-Ser-AA3-Asp-Leu-Pro-Glu-AA4-AA5-R2 (I)

[0074] The stereoisomers of the compound, mixtures thereof, and / or pharmaceutically acceptable salts of the compound, characterized in that:

[0075] AA1 is a Pyr OR key;

[0076] AA2 is Lys, Cys, or a bond;

[0077] AA3 is Cys, Met, or Cys(Cys)(cystine);

[0078] AA4 is 2,4,6-trimethylphenylalanine (Msa) or 2,4,6-trimethylphenylglycine (Msg);

[0079] AA5 is Lys, Cys, or a bond;

[0080] R1 is selected from the group consisting of: H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroarylalkyl groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, polymers derived from polyethylene glycol, and R5-CO-, wherein R5 is selected from the group consisting of: H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted aryl groups, substituted or unsubstituted aralkyl groups, substituted or unsubstituted heterocyclic groups, and substituted or unsubstituted heteroarylalkyl groups;

[0081] R2 is selected from the group consisting of -NR3R4, -OR3 and -SR3, wherein R3 and R4 are independently selected from the group consisting of H, substituted or unsubstituted acyclic aliphatic groups, substituted or unsubstituted alicyclic groups, substituted or unsubstituted heterocyclic groups, substituted or unsubstituted heteroarylalkyl groups, substituted or unsubstituted aryl groups and substituted or unsubstituted aralkyl groups.

[0082] In a preferred embodiment, AA4 in general formula (I) is 2,4,6-trimethylphenylalanine (Msa).

[0083] In another preferred embodiment, AA2 and AA5 in general formula (I) are independently selected from Lys or bonds.

[0084] The R1 and R2 groups are attached to the amino terminus (N-terminus) and carboxyl terminus (C-terminus) of the peptide sequence, respectively.

[0085] According to a preferred embodiment of the invention, R1 is selected from the group consisting of H or R5-CO-, wherein R5 is selected from the group consisting of substituted or unsubstituted C1-C. 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkyne group, substituted or unsubstituted C 3- C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloynyl, substituted or unsubstituted C6-C 30 Aryl, substituted or unsubstituted C7-C 24 Aryl groups, substituted or unsubstituted heterocyclic groups having 3-10 ring members, and substituted or unsubstituted heteroarylalkyl groups having 2 to 24 carbon atoms and 1 to 3 atoms other than carbon atoms and 1 to 6 carbon atoms in an alkyl chain. Preferably, R5 is selected from the group consisting of: substituted or unsubstituted C1-C 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl and substituted or unsubstituted C3-C 24 Cycloalkyl. More preferably, R1 is selected from H, acetyl, tert-butyryl, 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.

[0086] According to another preferred embodiment, R2 is -NR3R4, -OR3, or -SR3, wherein R3 and R4 are independently selected from the group consisting of: H, substituted or unsubstituted C1-C 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl, substituted or unsubstituted C2-C 24 Alkyne group, substituted or unsubstituted C3-C 24 Cycloalkyl, substituted or unsubstituted C5-C 24 Cycloalkenyl, substituted or unsubstituted C8-C 24 Cycloynyl, substituted or unsubstituted C6-C 30 aryl, substituted or unsubstituted C7-C 24Aryl groups, substituted or unsubstituted heterocyclic groups having 3-10 ring members, and substituted or unsubstituted heteroarylalkyl groups having 2 to 24 carbon atoms and 1 to 3 atoms other than carbon atoms and 1 to 6 carbon atoms in an alkyl chain. Preferably, R3 and R4 are independently selected from the group consisting of: substituted or unsubstituted C1-C 24 Alkyl, substituted or unsubstituted C2-C 24 Alkenyl and substituted or unsubstituted C3-C 24 Cycloalkyl. Optionally, R3 and R4 may be linked by saturated or unsaturated carbon-carbon bonds to form a ring with a 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. According to one or even more preferred embodiment, R2 is selected from -OH and -NH2.

[0087] In one particular aspect, the invention also relates to peptide dimers comprising two peptides of formula (I) of the invention, stereoisomers of the peptide dimer, mixtures thereof, and / or pharmaceutically acceptable salts of the peptide dimer, characterized in that the peptide dimer is a disulfide-bridged peptide dimer.

[0088] In a preferred embodiment, the peptide dimer is a homodimeric peptide. More preferably, when AA3 is Cys and AA2 and AA5 are independently Lys or bonds, the peptide dimer is formed by a disulfide bond bridge between two equal peptide monomers of formula (I) of the present invention.

[0089] Preferably, the peptide or peptide dimer of the present invention is selected from the group consisting of the following items:

[0090] R1-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-R2(R1-SEQ ID NO:3-R2)

[0091] Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-R2 (SEQ ID NO: 4-R2)

[0092] R1-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2(R1-SEQID NO:5-R2)

[0093] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2 (SEQ IDNO: 6-R2)

[0094] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-R2 (SEQ IDNO:7-R2)

[0095] (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-R2)2(disulfide bridge)[(SEQ ID NO:6-R2)2(disulfide bridge)]

[0096] R1-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-R2(R1-SEQID NO:8-R2)

[0097] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-R2 (SEQ ID NO:9-R2)

[0098] Where n, R1, and R2 are defined as above.

[0099] More preferably, the peptide or peptide dimer of the present invention is selected from the group consisting of the following items:

[0100] Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (Ac-SEQ ID NO: 3-NH2)

[0101] Octanoyl-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (Ocanoyl-SEQID NO:3-NH2)

[0102] Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2 (SEQ ID NO: 4-NH2)

[0103] Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2(Ac-SEQID NO:5-NH2)

[0104] Octyl

[0105] -Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2(Octayl-SEQ ID NO:5-NH2)

[0106] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2(SEQID NO:6-NH2)

[0107] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2(SEQID NO:7-NH2)

[0108] (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)]

[0109] Octyl

[0110] -Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2(Octayl-SEQ ID NO:8-NH2)

[0111] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (SEQ ID NO:9-NH2)

[0112] The amino acids that form peptides and peptide dimers can have L- or D-configurations, or combinations thereof.

[0113] For example, when it is stated that AA5 can be Lys, it should be understood that AA5 is selected from L-Lys or D-Lys. The preparation methods described in this document allow those skilled in the art to obtain each stereoisomer of the peptide or peptide dimer of the present invention by selecting amino acids having suitable configurations.

[0114] In a preferred embodiment, the amino acids of these peptides or peptide dimers of the present invention are L-amino acids. The preferred structures of these peptides and peptide dimers of the present invention are pure isomers, i.e., enantiomers or diastereomers.

[0115] In the context of this invention, the term “unencoded amino acid” and its plural form refer to natural or non-natural amino acids that are not encoded by the genetic code, such as pyroglutamic acid or cysteine, or synthetic amino acids 2,4,6-trimethylphenylalanine (Msa) or 2,4,6-trimethylphenylglycine (Msg) (see Table 1).

[0116] The amino acid sequences of the peptides and peptide dimers of the present invention may include modifications to a given sequence. Such modifications are well known to those skilled in the art. For example, one or more L-amino acids in the amino acid sequences of the peptides and peptide dimers of the present invention may be substituted with D-amino acids 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 in the amino acid sequences of the peptides and peptide dimers of the present invention may be N-alkylated (typically N-methylated) 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) to improve their stability and / or reduce renal clearance.

[0117] Pharmaceutically acceptable salts of these peptides and peptide dimers provided by this invention are also within the scope of this invention. The term "pharmaceutically acceptable salt" refers to salts generally accepted for use in animals, and more specifically in humans, and includes salts used to form: base addition salts, whether they are inorganic salts, such as, but not limited to, lithium, sodium, potassium, calcium, magnesium, manganese, copper, zinc, or aluminum; or organic salts, such as, but not limited to, ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, arginine, lysine, histidine, or piperazine; or acid addition salts, whether organic salts, such as, but not limited to, acetates, citrates, lactates, malonates, maleates, tartrates, fumarates, benzoates, aspartates, tripartites, glutamates, succinates, oleates, trifluoroacetates, oxalates, dihydroxynaphthyl salts, or gluconates; or inorganic salts, such as, but not limited to, chlorides, sulfates, borates, or carbonates. The properties of the salt are not critical, as long as it is pharmaceutically acceptable. The pharmaceutically acceptable salts of these peptides and peptide dimers of the present invention can be obtained by conventional methods well known in the art [Berge et al., J. Pharm. Sci. 1977, 66(1), 1-19].

[0118] The present invention also includes peptides or peptide dimers having conserved or non-conserved substitutions that still exhibit the activity of the peptides of the present invention described herein, more preferably with conserved substitutions. The present invention also includes peptides or peptide dimers having 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 96%, 97%, 98%, or 99% percentage identity with any peptide or peptide dimer of the present invention, and these peptides or peptide dimers still exhibit the activity of the peptides of the present invention described herein, more preferably with conserved substitutions.

[0119] Preparation method

[0120] The peptides or peptide dimers, their stereoisomers, or their pharmaceutically acceptable salts of the present invention can be synthesized according to conventional methods known in the art. In embodiments of the present invention, these peptides and peptide dimers are synthesized by solution or solid-phase peptide synthesis methods.

[0121] Solid-phase synthesis methods are described, for example, in [Stewart JM and Young JD, 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]. In-solution synthesis methods, as well as combinations of solid-phase synthesis methods and in-solution methods, or enzyme synthesis, are described in [Kullmann, J Biol Chem, 1980, 255(17), 8234-8238].

[0122] For example, a method for obtaining these peptides and peptide dimers of the present invention includes the following steps:

[0123] - Couple amino acids with a protected N-terminus and a free C-terminus to amino acids with a free N-terminus and a protected or solid-supported C-terminus.

[0124] -Remove the protecting group at the N-terminus;

[0125] - Repeat the coupling sequence and remove the N-terminal protecting group until the desired peptide sequence is obtained;

[0126] -Remove the C-terminal protecting group or cut from the solid support.

[0127] - Optionally, the obtained peptide is oxidized to produce a disulfide-bridged peptide dimer.

[0128] In embodiments of the present invention, the peptides and peptide dimers, their stereoisomers, or their pharmaceutically acceptable salts are prepared by a method comprising the following steps:

[0129] a) Solid-phase peptide synthesis in a polymer support;

[0130] b) The peptide is cleaved from the polymer support, preferably by acid treatment;

[0131] c) Optionally, the peptide in solution is oxidized to obtain a cyclic peptide or a peptide dimer;

[0132] d) If necessary, remove the protecting group, preferably using trifluoroacetic acid;

[0133] Alternatively,

[0134] i) Solid-phase peptide synthesis in a polymer support;

[0135] ii) Optionally, solid-phase peptide cyclization or dimer formation is carried out in a polymer support;

[0136] iii) The peptide is cleaved from the polymer support, and if necessary, the protecting groups are removed, preferably by treatment with trifluoroacetic acid;

[0137] iv) Optionally, the peptide in solution is oxidized to obtain a peptide dimer;

[0138] In a preferred embodiment, the peptide dimer of the present invention is obtained by oxidizing the peptide of the present invention in a solution phase. The free thiol groups of the cysteine ​​residues of the peptide can be oxidized to form a disulfide-bridged peptide dimer. Preferably, the oxidation is performed using dimethyl sulfoxide (DMSO).

[0139] Preferably, the C-terminus is bound to a polymer support, and the process is carried out in a solid phase and therefore includes the coupling 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 bound to the polymer support; removal of the N-terminal protecting group; and repeating this sequence multiple times as needed to obtain the target peptide sequence, 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 performed before or after cleavage from the polymer support. Throughout the synthesis, the functional groups of these amino acid side chains are appropriately protected with temporary or permanent protecting groups, and they may be deprotected simultaneously or orthogonally during the cleavage of the peptide from the polymer support.

[0140] Alternatively, solid-phase synthesis can be performed via a convergent strategy, either by coupling a peptide fragment to the polymer support or by coupling a peptide fragment pre-bound to the polymer support. Convergent synthesis strategies are well known to those skilled in the art and are described in Lloyd-Williams P. et al., Tetrahedron 1993, 49(48), 11065-11133.

[0141] The method may include additional steps of deprotecting the N-terminus and C-terminus in an indeterminate order and / or cleaving the peptide from the polymer support using standard methods and conditions known in the art, after which the functional groups of the terminals may be modified. Optional N-terminal and C-terminal modifications may be performed on the peptide of the present invention having formula (I) anchored to the polymer support, or optional N-terminal and C-terminal modifications may be performed once the peptide has been cleaved from the polymer support.

[0142] Optionally, R1 can be introduced by reacting the N-terminus of the peptide of the present invention with compounds R1-Z, wherein R1 has the meaning described above and Z is a leaving group, such as, but not limited to, toluenesulfonyl, methanesulfonyl, and halogen groups; this is by nucleophilic substitution reaction in the presence of a suitable base and solvent, wherein the fragment has a functional group that does not participate in the formation of NC bonds and is conveniently protected with temporary or permanent protecting groups. R1 can also be introduced by reacting the N-terminus of the compound of the present invention with an R5COOH group or its ester, acyl halide, or anhydride, as defined above.

[0143] Optionally and / or additionally, these R2 groups can be introduced by reacting an HR2 compound (where R2 is -OR3, -NR3R4, or -SR3) with a complementary fragment of a peptide corresponding to formula (I) of the present invention (where R2 is -OH) in the presence of a suitable solvent and a base (such as N,N-diisopropylethylamine (DIEA) or triethylamine) or an additive (such as 1-hydroxybenzotriazole (HOBt) or 1-hydroxyazabenzotriazole (HOAt)) and a dehydrating agent (such as carbodiimide, urea cationic salt, phosphonium salt, or amidine salt, etc.) to obtain a peptide of general formula (I) according to the present invention, wherein the fragment has functional groups not involved in the formation of NC, OC, or SC bonds and suitably protected with temporary or permanent protecting groups. Alternatively, other R2 groups can be incorporated simultaneously with the peptide cleavage process from the polymer support.

[0144] Those skilled in the art will readily understand that the deprotection / cleavage of C-terminus and N-terminus, and their subsequent derivatization, can be performed in an indeterminate order according to methods known in the prior art. [Smith MB and March J., 1999 March's Advanced Organic Chemistry Reactions, Mechanisms and Structure, 5th ed., John Wiley & Sons, 2001].

[0145] The term "protecting group" and its plurals refer to groups that close off organic functional groups and can be removed under controlled conditions. These protecting groups, their relative reactivity, and the conditions under which they remain inert are known to those skilled in the art.

[0146] Representative examples of protecting groups for the amino group are amides, such as acetamide, benzoamide, and neopentanoic acid amide; and carbamates such as benzyloxycarbonyl (Cbz or Z), 2-chlorobenzyloxycarbonyl (CIZ), p-nitrobenzyloxycarbonyl (pNZ), tert-butoxycarbonyl (Boc), 2,2,2-trichloroethoxycarbonyl (Troc), 2-(trimethylsilyl)ethoxycarbonyl (Teoc), 9-fluorenylmethoxycarbonyl (Fmoc), or allyloxycarbonyl. (Alloc), triphenylmethyl (Trt), methoxytriphenylmethyl (Mtt), 2,4-dinitrophenyl (Dnp), N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl-1-ethylene)ethyl] (Dde), 1-(4,4-dimethyl-2,6-dioxo-cyclohexylene)-3-methylbutyl (ivDde), 1-(1-adamantyl)-1-methylethoxycarbonyl (Adpoc), etc.; preferably Boc or Fmoc.

[0147] Examples of representative protecting groups for the carboxyl group are esters, such as tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (trityl ester, Trt), cyclohexyl ester (cHx), benzyl ester (Bzl), o-nitrobenzyl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, trimethylsilyl ethyl ester, 2-phenylisopropyl ester, fluorenyl methyl ester (Fm), 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methylbutyl]amino)benzyl ester (Dmab), etc.; the preferred protecting groups of the present invention are All, tBu, cHex, Bzl, and Trt esters.

[0148] Trifunctional amino acids can be protected during synthesis using temporary or permanent protecting groups orthogonal to the N-terminal and C-terminal protecting groups. The aforementioned amino protecting groups are used to protect the amino group of the lysine side chain; the guanidino group of the arginine side chain can be protected by a nitro group, allyloxycarbonyl (Alloc), tosyl (Tos), 2,2,5,7,8-pentamethylbenzodihydropyran-6-sulfonyl (Pmc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) or 4-methoxy-2,3,6-trimethylbenzenesulfonyl (Mtr); the serine and threonine side chains are protected by tert-butyl (tBu) esters; the cysteine ​​side chain can be protected by a protecting group selected from the group consisting of triphenylmethyl and acetamoxymethyl; the asparagine side chain can be protected by a protecting group selected from the group consisting of methoxytriphenylmethyl, triphenylmethyl or xanthyl; or it can be unprotected. To protect the carboxyl groups of the aspartic acid and glutamic acid side chains, esters such as tert-butyl ester (tBu), allyl ester (All), triphenylmethyl ester (trityl ester, Trt), cyclohexyl ester (cHx), benzyl ester (Bzl), o-nitrobenzyl ester, p-nitrobenzyl ester, p-methoxybenzyl ester, trimethylsilyl ethyl ester, 2-phenylisopropyl ester, fluorenyl methyl ester (Fm), 4-(N-[1-(4,4-dimethyl-2,6-dioxocyclohexyl)-3-methylbutyl]amino)benzyl ester (Dmab) can be used. The methionine side chain can be used with or without protection using sulfoxide.

[0149] The preferred trifunctional amino acid protecting groups of this invention are tBu esters in the side chains of serine and threonine, as well as aspartic acid and glutamic acid; Boc in the side chain of lysine, Trt and Fmoc or Boc in the side chain of cysteine ​​are used as temporary protecting groups at the N-terminus.

[0150] Examples of these and other protecting groups, their introduction and removal, can be found in the following literature: [Greene T.W. and Wuts PGM, (1999) Protective groups in organic synthesis, John Wiley & Sons, New York; Atherton B. and Sheppard RC, (1989) Solid Phase Peptide Synthesis: A practical approach, IRL Oxford University Press]. The term “protecting group” and its plural form also include the polymer support used in solid-phase synthesis.

[0151] When the synthesis is carried out entirely or partially in the solid phase, possible solid supports used in the procedure of this invention may include polystyrene supports, polyethylene glycol grafted onto polystyrene, etc., such as, but not limited to, p-methyldiphenylmethylamine (MBHA) resin [Matsueda et al. Peptides 1981, 2(1), 45-50], 2-chlorotriphenylmethyl resin [Barlos et al. Tetrahedron Lett 1989, 30, 3943-3946; Barlos K. et al. Tetrahedron Lett 1989, 30, 3947-3951], Resin (Rapp Polymere GmbH), Resins (Matrix Innovation, Inc.) and the like, which may or may not include unstable linking agents, such as 5-(4-aminomethyl-3,5-dimethoxyphenoxy)valerate (PAL) [Albericio et al. J Org Chem 1990, 55(3), 3730-3743], 2-[4-aminomethyl-(2,4-dimethoxyphenyl)]phenoxyacetic acid (AM) [Rink 1987, Tetrahedron Lett 28(33), 3787-90], Wang [Wang, J Am Chem Soc 1973, 95(4), 1328-33], etc., which enable the cleavage of hemiprotected peptides and the formation of rings in solution, wherein there is a deprotection step in solution or even solid-phase cyclization and subsequent simultaneous deprotection and cleavage of the peptide.

[0152] Pharmaceutical Composition

[0153] The peptides and peptide dimers of the present invention can be applied by any means of bringing these peptides and peptide dimers into contact with their sites of action in mammals (preferably human sites of action) and in the form of compositions containing them.

[0154] Therefore, 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, a stereoisomer of the peptide or peptide dimer, a mixture thereof, and / or a pharmaceutically acceptable salt of the peptide or peptide dimer. The pharmaceutical compositions of the present invention may comprise the peptides and peptide dimers of the present invention obtained by freeze-drying or spray-drying, and may be reconstituted in a solvent suitable for their application.

[0155] The peptides and peptide dimers of the present invention have variable solubility in water depending on the nature of their sequence or any possible modifications to the N-terminus and / or C-terminus. Therefore, the peptides and peptide dimers of the present invention can be incorporated into these compositions via aqueous solutions, and those insoluble in water can be dissolved in pharmaceutically acceptable conventional solvents such as, but not limited to, ethanol, propanol, isopropanol, propylene glycol, glycerol, butylene glycol, or polyethylene glycol, or any combination thereof.

[0156] The pharmaceutically effective amount of these peptides and peptide dimers of the present invention that must be administered, and their dosage, will depend on many factors, including age, patient condition, nature or severity of the condition or disease to be treated or prevented, route and frequency of administration, and the specific nature of the peptides to be used.

[0157] "Pharmaceutically effective amount" is understood to mean that the peptides or peptide dimers of the present invention are non-toxic but sufficient to provide the desired pharmaceutical effect (i.e., preventive treatment 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 pharmaceutically effective concentrations to achieve the desired efficacy; in their preferred forms, the effective daily dose for humans is 0.001 to 250 mg / kg, more preferably 0.005 to 100 mg / kg, more preferably 0.01 to 50 mg / kg, and even more preferably 0.01 to 10 mg / kg.

[0158] The frequency of administration of this pharmaceutical composition may be, for example, but not limited to, once a month, once every two weeks, twice a week, three times a week, or daily.

[0159] The peptides and peptide dimers of the present invention, stereoisomers of the peptide or peptide dimer, mixtures thereof, and / or pharmaceutically acceptable salts of the peptide or peptide dimer may also be incorporated into delivery systems and / or pharmaceutically sustained-release systems.

[0160] The pharmaceutical compositions of the present invention may comprise at least one pharmaceutically acceptable excipient and / or adjuvant. The quantity and properties of these pharmaceutically acceptable excipients depend on the desired method of administration. Pharmaceutically acceptable excipients are well known to those skilled in the art [Rowe RC, Sheskey PJ, Quinn, ME (2009) "Handbook of Pharmaceutical Excipients, 6th Edition", Pharmaceutical Press and American Pharmacists Association]. The compositions may be prepared using conventional methods known in the prior art.

[0161] The term "delivery system" and its plural form refer to diluents, adjuvants, excipients, or carriers applied in conjunction with the peptides and peptide dimers of the present invention. These pharmaceutical carriers can be liquids, such as water, oils, or surfactants, including those of petroleum, animal, plant, or synthetic origin, such as, but not limited to, peanut oil, soybean oil, mineral oil, sesame oil, castor oil, polysorbate, sorbitol esters, ether sulfates, sulfates, betaine, glucosides, maltodextrins, fatty alcohols, nonyl alcohol ethers, poloxamer, polyoxyethylene, polyethylene glycol, dextran, glycerol, digitalis saponins, and the like. Those skilled in the art are familiar with the various diluents, adjuvants, or excipients that can be used in different delivery systems in which the peptides and peptide dimers of the present invention can be applied.

[0162] The term "slow release" is used in its conventional sense with respect to a delivery system for a compound that provides the gradual release of the compound over a period of time, and preferably, but not necessarily, has a relatively constant level of compound release over that period of time.

[0163] Examples of delivery or sustained-release systems include, but are not limited to, liposomes, mixed liposomes, oleosomes, niosomes, liposomes, millimeter particles, microparticles, nanoparticles and solid lipid nanoparticles, nanostructured lipid carriers, sponges, cyclodextrins, vesicles, micelles, mixed micelles of surfactants, mixed micelles of surfactants and phospholipids, millimeter spheres, microspheres and nanospheres, lipospheres, millimeter capsules, microcapsules and nanocapsules, as well as microemulsions and nanoemulsions. These delivery or sustained-release systems can be added to achieve greater bioavailability of the active ingredient and / or improve its pharmacokinetic and pharmacodynamic properties.

[0164] Pharmaceutical compositions of peptides and peptide dimers, stereoisomers of the peptide or peptide dimer, mixtures thereof, and / or pharmaceutically acceptable salts of the peptide or peptide dimer of the present invention may be administered via any suitable route, for which will include pharmaceutically acceptable excipients necessary for formulation of the desired form of administration; by local or systemic administration, such as, but not limited to, local, enteric, or parenteral routes. In the context of the present invention, the term “local” includes skin and ocular routes; the term “enteric” includes administration to the digestive system, such as oral, buccal, gastric, sublingual, and rectal routes; and the term “parenteral” refers to nasal, ear, eye, rectum, urethra, vagina, subcutaneous, intradermal, intravascular injection such as intravenous, intramuscular, intraocular, intraspinal, intracranial, intracervical, intracerebral, intrameningeal, intra-articular, intrahepatic, intrathoracic, intratracheal, intrathecal, and intraperitoneal routes, as well as any other similar injection or infusion techniques. In vitro therapy, such as in damaged cell cultures and / or stem cells, and ex vivo therapy are also contemplated.

[0165] More precisely, treatment using the peptides, peptide dimers, and compositions of the present invention is performed in vivo, as the preferred route of administration is subcutaneous.

[0166] Pharmaceutical compositions of peptides and peptide dimers, stereoisomers of the peptide or peptide dimer, mixtures thereof, and / or pharmaceutically acceptable salts of the peptide or peptide dimer of the present invention can be administered in combination with other therapeutic agents and / or therapeutic treatments. Preferably, the therapeutic agent and / or therapeutic treatment is an anticancer agent and / or treatment.

[0167] use

[0168] In another respect, the present invention relates to peptides or peptide dimers of the present invention used as pharmaceuticals, stereoisomers of the peptides or peptide dimers, mixtures thereof and / or pharmaceutically acceptable salts of the peptides or peptide dimers, or pharmaceutical compositions of the present invention.

[0169] Another aspect of the invention relates to peptides or peptide dimers of the invention, stereoisomers of the peptides or peptide dimers, mixtures thereof, and / or pharmaceutically acceptable salts of the peptides or peptide dimers, or pharmaceutical compositions of the invention, for the treatment and / or diagnosis of cancer and / or metastasis. In particular, the peptides and peptide dimers of the invention are capable of inhibiting cancer growth and / or reducing or inhibiting the invasion and metastasis of cancer cells in various cancers.

[0170] This invention relates to a method for treating and / or diagnosing cancer and / or metastasis, comprising administering a pharmaceutically effective amount of the peptide or peptide dimer of the invention, a stereoisomer of the peptide or peptide dimer, a mixture thereof, and / or a pharmaceutically acceptable salt of the peptide or peptide dimer, or a pharmaceutical composition of the invention to an individual in need (preferably a person).

[0171] This invention relates to the use of the peptide or peptide dimer of the present invention, stereoisomers of the peptide or peptide dimer, mixtures thereof and / or pharmaceutically acceptable salts of the peptide or peptide dimer, or the pharmaceutical compositions of the present invention in the preparation of pharmaceuticals.

[0172] This invention relates to the use of the peptides or peptide dimers of the present invention, stereoisomers of the peptides or peptide dimers, mixtures thereof, and / or pharmaceutically acceptable salts of the peptides or peptide dimers, or pharmaceutical compositions of the present invention in the preparation of medicaments for the diagnosis and / or treatment of cancer and / or metastasis.

[0173] In the embodiments, the present invention relates to the use of the peptides or peptide dimers of the present invention, stereoisomers of the peptides or peptide dimers, mixtures thereof, and / or pharmaceutically acceptable salts of the peptides or peptide dimers, or pharmaceutical compositions of the present invention in the diagnosis of cancer and / or metastasis, said diagnosis being performed without a human subject. That is, the diagnosis is performed in vitro.

[0174] Therefore, in a preferred aspect, the present invention relates to an in vitro diagnostic method for cancer and / or metastasis, the method comprising using a peptide or peptide dimer of the present invention, a stereoisomer of the peptide or peptide dimer, a mixture thereof and / or a pharmaceutically acceptable salt of the peptide or peptide dimer, or a pharmaceutical composition of the present invention.

[0175] Cancer and / or metastasis include, but are not limited to, neuroblastoma, sarcoma, soft tissue sarcoma such as rhabdomyosarcoma (including embryonal, alveolar, pleomorphic, and spindle cell / sclerosing rhabdomyosarcoma), endometrial sarcoma, undifferentiated spindle cell sarcoma, undifferentiated pleomorphic sarcoma, undifferentiated round cell sarcoma, undifferentiated epithelioid sarcoma, liposarcoma, atypical lipoma, malignant solitary fibroma, inflammatory myofibroblastic tumor, low-grade malignant myofibroblastic sarcoma, and fibrosarcoma (including adult and sclerosing epithelioid variants). Myxofibrosarcoma, low-grade malignant fibromyxoid sarcoma, soft tissue giant cell tumor, leiomyosarcoma, malignant glomus tumor, hemangioendothelioma (including reticular, pseudomyogenic, and epithelioid types), soft tissue angiosarcoma, extraosseous osteosarcoma, malignant gastrointestinal stromal tumor (GIST), malignant peripheral nerve sheath tumor (including epithelioid variants), malignant salamander tumor, malignant granular cell tumor, malignant ossifying fibromyxoid tumor, stromal sarcoma, myoepithelial carcinoma, malignant hyperphosphatemic mesenchymal tumor, synovial sarcoma (including... Spindle cell and biphasic), epithelioid sarcoma, alveolar soft tissue sarcoma, clear cell sarcoma of soft tissue, extraosseous myxoid chondrosarcoma, extraosseous Ewing sarcoma, fibroproliferative small round cell tumor, extrarenal rhabdoid tumor, perivascular epithelioid cell tumor, skeletal sarcoma such as osteosarcoma, chondrosarcoma and Ewing sarcoma, hemangioendothelioma, angiosarcoma, fibrosarcoma and myofibroblastic sarcoma, chordoma, ameloblastoma, breast cancer (such as ductal, lobular and papillary carcinoma), colon cancer, rectal cancer, anal cancer, colorectal cancer Brain cancers such as glioblastoma, astrocytoma, or medulloblastoma; malignant glioma; prostate cancer; melanoma and other skin cancers; cervical cancer; uterine cancer; ovarian cancer (such as high-grade serous ovarian cancer); endometrial cancer; lymphoma; head and neck cancer; oral cancer; salivary gland cancer; retinoblastoma; gastrointestinal cancer; esophageal cancer; stomach cancer; pancreatic cancer; gallbladder cancer; liver cancer (such as hepatocellular carcinoma, HCC); kidney or renal cancer such as Wilms' tumor or nephroblastoma; bladder cancer; lung cancer such as small cell lung cancer and non-small cell lung cancer; non-Hodgkin's lymphoma; multiple myeloma; pituitary adenoma; squamous cell carcinoma; testicular cancer; leukemia (such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, and chronic myeloid leukemia); plasmacytoma or multiple myeloma. Preferably, the cancer and / or metastatic tumor is selected from neuroblastoma, rhabdomyosarcoma, or breast cancer.

[0176] For a better understanding, the invention will now be described in more detail with reference to the accompanying drawings, which are presented by way of example and with reference to illustrative and non-limiting examples. Attached Figure Description

[0177] Figure 1 Western blot images of ITGA9 expression in six rhabdomyosarcoma cell lines are shown. Actin was used as a control.

[0178] Figure 2 Western blot images of ITGA9 expression in three neuroblastoma cell lines are shown. Actin was used as a control.

[0179] Figure 3 This image shows Western blot images of ITGA9 expression in six different breast cancer cell lines. Actin was used as a control.

[0180] Figure 4 In a 27-week study, Kaplan-Meier curves were used to measure event-free survival in rhabdomyosarcoma metastatic mice treated with PBS (solid line, —), 0.5 mg / kg SEQ ID NO:7-NH2 (dotted line, ...), or 2 mg / kg SEQ ID NO:7-NH2 (long dashed line, ---). The y-axis represents event-free survival as a percentage. The x-axis represents the number of weeks.

[0181] Figure 5 In a 27-week study, the mean number of metastases formed per mouse after tail vein injection of RD cells and treatment with either SEQ ID NO:6-NH2 (0.5 mg / kg and 2 mg / kg) or SEQ ID NO:7-NH2 (0.5 mg / kg and 2 mg / kg). Horizontal lines represent mean ± SEM. Statistical significance: *p-value < 0.05; **p-value < 0.01. The y-axis represents the number of metastases per mouse. The x-axis represents the treatment groups, from left to right, with the groups being either SEQ ID NO:6-NH2 (0.5 mg / kg and 2 mg / kg) or SEQ ID NO:7-NH2 (0.5 mg / kg and 2 mg / kg).

[0182] Figure 6 In a 13-week study, Kaplan-Meier curves were used to measure event-free survival in neuroblastoma metastatic mice treated with PBS (solid line, —), 1 mg / kg SEQ ID NO:7-NH2 (dashed line, ...), or 2 mg / kg SEQ ID NO:7-NH2 (long dashed line, ---). The y-axis represents event-free survival as a percentage. The x-axis represents the number of weeks.

[0183] Example

[0184] The following specific examples provided in this patent document are used to illustrate the nature of the invention. These examples are included for illustrative purposes only and should not be construed as limiting the invention claimed herein.

[0185] abbreviation

[0186] The abbreviations used in this description have the following meanings:

[0187] Ac₂O, acetic anhydride; ACN, acetonitrile; AcOH, acetic acid; Boc, tert-butoxycarbonyl; DCM, dichloromethane; DMEM, Duchenne-modified Eagle's medium; DIEA, N,N'-diisopropylethylamine; DIPCDI, diisopropylcarbodiimide; DMF, N,N-dimethylformamide; Et₂O, diethyl ether; eq, equivalent; ESI-MS, electrospray ionization mass spectrometry; Et₂O, diethyl ether; Fmoc, 9-fluorenylmethyloxycarbonyl; HOBT, N-hydroxybenzotriazole HPLC, High Performance Liquid Chromatography; ITGA9, Integrin α9β1; IV, Intravenous Injection; p-MBHA Resin, 4-Methyldiphenylmethylamine Resin; MeOH, Methanol; MW, Molecular Weight; μL, Microliter; PBS, Phosphate-Buffered Saline; RP-HPLC, Reversed-Phase HPLC; rpm, Revolutions per Minute; sc, Subcutaneous; tBu, Tert-Butyl; TFA, Trifluoroacetic Acid; TIS, Triisopropylsilane; TIO, Phenylenol; tr, Retention Time; Trt, Triphenylmethyl.

[0188] Example 1. Synthesis of peptides and peptide dimers.

[0189] Ac-SEQ ID NO:10-NH2:

[0190] Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Phe-NH2

[0191] Ac-SEQ ID NO:3-NH2:

[0192] Ac-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2

[0193] Octyl-SEQ ID NO:3-NH2: Octyl

[0194] -Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2

[0195] SEQ ID NO:4-NH2:

[0196] Pyr-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-NH2

[0197] Ac-SEQ ID NO:5-NH2:

[0198] Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0199] Octyl-SEQ ID NO:5-NH2: Octyl

[0200] -Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0201] SEQ ID NO:6-NH2:

[0202] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0203] SEQ ID NO:7-NH2:

[0204] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0205] (SEQ ID NO:6-NH2)2 (disulfide bridge):

[0206] (Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2)2 (disulfide bridge)

[0207] Octyl-SEQ ID NO:8-NH2: Octyl

[0208] -Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0209] SEQ ID NO:9-NH2:

[0210] Pyr-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys(Cys)-Asp-Leu-Pro-Glu-Msa-Lys-NH2

[0211] Solid-phase peptide synthesis was used for peptide synthesis. Peptide Ac-SEQ ID NO:10-NH2, a 12-amino acid sequence contained in the natural deintegrin domain of ADAM-12 and specifically contained in SEQ ID NO:2, was also synthesized as a control compound.

[0212] All peptides were synthesized manually or using an automated peptide synthesizer (Liberty, CEM): solid-phase peptide synthesis was performed using 4-methyldiphenylmethylamine resin (p-MBHA) at varying scales (0.3 to 30 mmol) following the standard Fmoc / tBu strategy. For each sequence, the resin was placed in an appropriate reaction vessel equipped with a filter plate. Fmoc-AM-OH linker (2 equivalents) and HOBT (2 equivalents) were dissolved in DMF: DCM (1:1) was stirred until dissolved, then DIPCDI (2 equivalents) was added. The solution was transferred to a reactor and allowed to react for 40–60 minutes. Linker incorporation was controlled by a ninhydrin test. If the ninhydrin test was positive, a reactivation or recoupling step was performed until coupling was complete.

[0213] Once coupling is complete, the synthesis continues by removing the Fmoc group through treatment with a 20% piperidine solution in DMF (twice for 5 minutes and twice for 10 minutes). The peptide-resin is washed five times with DMF, filtered, the washes are discarded, and the following amino acids are coupled. Coupling is performed in DMF using Fmoc-AA-OH:DIPCDI:HOBt (3 equivalents: 3 equivalents: 3 equivalents) (using 1.5 equivalents to couple Fmoc-Msa-OH). All these coupling reactions are carried out for 40–60 minutes. The completion of each coupling reaction is controlled by a ninhydrin test (or by chlorobenzene when the coupling is on a secondary amine of proline). Reactivation or recoupling steps are performed as needed. Syntheses performed using an automated synthesizer are not controlled by the ninhydrin test or chlorobenzene. At the end of the synthesis, acetylation is performed in DMF using Ac2O (2.5 equivalents) and DIEA (5 equivalents) as needed. For sequences with an N-terminal octanoyl group, acylation was performed using 5 equivalents of octanoic acid, HOBT, and DIPCDI. After peptide sequencing, the peptide resin was washed with DMF, MeOH, and Et2O and dried.

[0214] After synthesis, peptide cleavage was performed according to the following procedure. The peptide-based resin was reacted at room temperature for 2 to 4 hours in a suitable TFA mixture (see Table 2 below). The resin was washed with TFA and Et₂O. All filtrates were immersed in cold diethyl ether 81:7 (v:v) and allowed to stand for 15–30 minutes. The resulting suspension was filtered through a filter plate and the filtrate was discarded. The residue was washed with ether and the filtrate from each wash was discarded. The solid was freeze-dried to obtain the crude peptide product. Table 2 below shows the TFA mixtures used for different peptides and peptide dimers.

[0215]

[0216]

[0217] Table 2. TFA mixtures of different peptides and peptide dimers used in the synthesis of Example 1.

[0218] The peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge) was obtained by oxidizing 20 mg of peptide SEQ ID NO: 6-NH2 at a concentration of 10 mg / mL in 15% DMSO in 10% AcOH aqueous solution for 48-72 hours, followed by recovery using a Seppack C18 filter cartridge (Waters). The peptide dimer was held in a C18 column and then recovered with a mixture of water:acetonitrile (80:20) and lyophilized to give 17 mg of peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge) (81% yield).

[0219] The crude peptide and peptide dimer were purified in a semi-preparative system equipped with an NW50 column packed with 10 μm Kromasil silica, and the pure fractions analyzed by analytical RP-HPLC were lyophilized.

[0220] The table below (Table 3) shows the synthetic scale, crude peptide and pure peptide yields, and MW for each compound.

[0221]

[0222] * Use the Liberty (CEM) autosynthesizer for synthesis

[0223] Table 3. Synthetic scale, yield and MW of each peptide and peptide dimer synthesized in Example 1.

[0224] The ion exchange procedure is performed as follows: The lyophilized pure compound is dissolved in a mixture of AcOH. The acetic acid solution containing the compound is then treated with DOWEX resin to obtain peptides or peptide dimers with acetate balance ions. The final acetate compound is recovered by filtration and then lyophilized.

[0225] Peptides and peptide dimers were characterized by mass spectrometry in an ESI-MS instrument, as shown in Table 4 below.

[0226]

[0227] Table 4. Mass spectrometry results of the peptides and peptide dimers synthesized in Example 1.

[0228] Example 2. In vitro stability determination of the half-life (t1 / 2) of peptides and peptide dimers in human plasma.

[0229] The peptides and peptide dimers synthesized and prepared according to Example 1 were dissolved in water at a concentration of 6 mg / mL and heated to 37°C.

[0230] Human plasma (K3 EDTA plasma, BBI solution, code S112-1) was obtained as a lyophilized solid, reconstituted with sterile 0.9% sodium chloride solution, and stored at -20°C. Before use, the human plasma was thawed and incubated at 37°C.

[0231] Peptides and peptide dimers were incubated in 90% human plasma at 37°C for different times, followed by precipitation with two volume equivalents of methanol. Samples were cooled in an acetone-carbon dioxide bath for a few seconds, then centrifuged at approximately 10,000 rpm for 12 minutes at 4°C. The supernatant was filtered through a 0.45 μm PVDF filter and analyzed in triplicate by RP-HPLC using isocratic method (elution A = 0.1% TFA aqueous solution; eluent B = 0.07% TFA acetonitrile solution, column = Kromasil C8). 5 μm, 250 x 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 initial time area to calculate its half-life.

[0232] The table below (Table 5) shows the t1 / 2 data for peptides and peptide dimers.

[0233]

[0234] Table 5. t1 / 2 data obtained for the peptides and peptide dimers studied in Example 2.

[0235] Example 3. Integrin α9β1 (ITGA9) protein levels. ITGA9 protein expression was assessed by Western blotting.

[0236] Rhabdomyosarcoma cell lines RH30, CW9019, RH4, RH18, RD, and HTB82 were cultured in a highly essential medium containing El salts (BioWest) 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 reagents were from BioWest). Neuroblastoma cell lines CHLA-90, BE(2)-C, and SK-N-BE(2)-C were grown in Iscove modified DuPont medium (Thermo Fisher Scientific) supplemented with 10% FBS (Sigma-Aldrich), 1% insulin-transferrin-selenoside G supplement (Thermo Fisher Scientific), 100 U / mL penicillin, and 0.1 mg / mL streptomycin (BioWest). Breast cancer cell lines MDA-MB-231, MDA-MB-468MCF7, T47D, BT474, and MCF10A were cultured in DuPont modified Eagle's 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 reagents were from BioWest). All cell lines were maintained at 37°C in a controlled environment with 5% CO2.

[0237] At 80% confluence, cells were washed with PBS and scraped in RIPA protein cleavage buffer (Thermo Fisher Scientific) supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Sigma). Cell lysates were incubated at 95°C for 5 minutes. After centrifugation at 13,000 rpm for 15 minutes at 4°C, cell debris was discarded. Protein concentration in the supernatant was quantified using a DC protein assay (Bio-Rad Laboratories) according to the manufacturer's instructions.

[0238] Proteins were separated by 8% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) and transferred to 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 a primary antibody dilution. The antibodies used were: 1:1000 dilution of anti-α9-integrin monoclonal antibody clone 3E4 (NovusBiologicals), 1:1000 dilution of anti-FAK (Cell Signaling), 1:1000 dilution of Tyr397 anti-Phospho-FAK (Cell Signaling), and 1:10000 dilution of anti-actin (Santa Cruz Biotechnology). After washing with TBS-T, the membrane was incubated with the corresponding peroxidase-conjugated secondary antibody at room temperature for 1 hour. The immunoreaction bands were observed using ECL Prime chemiluminescence assay kit (GE Healthcare).

[0239] like Figure 1 , 2 As shown in the Western blot images of RD and 3, ITGA9 expression 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 higher in MDA-MB-468 cells among other breast cancer cell lines evaluated.

[0240] Example 4. In vitro invasiveness assay of rhabdomyosarcoma cell lines (RH30 and RD).

[0241] In 0.1 mL of FBS-free culture medium, 10 5 RH30 or RD cells were seeded in the upper chamber of a Matrigel-coated Transwell (Corning) cell culture medium. Cells were pre-incubated for 48 hours with different concentrations (0 nM, 100 nM, 200 nM, 500 nM, and 1000 nM) of peptides and peptide dimers, which were also present in the medium during cell invasion assays. In the lower chamber, 0.6 mL of FBS-containing medium was added. Cells were cultured at 37°C for 24 hours.

[0242] Cells were then fixed with 4% paraformaldehyde (Sigma) for 10 minutes and washed with PBS. After removing Matrigel and remaining cells from the upper surface of the membrane with cotton swabs, cells present on the lower surface were stained with Hoechst dilution (Sigma) and counted using a Nikon inverted fluorescence microscope with a 10x objective (5 fields of view per well). All experiments were performed in triplicate. In Table 6, invasiveness% represents the average percentage of invasive cells relative to its control.

[0243]

[0244] nd: Undetermined

[0245] * p-value < 0.05 compared to untreated cells.

[0246] Table 6. Percentage of invasiveness of rhabdomyosarcoma cell lines (RH30 and RD) at different concentrations from 100 to 1000 nM in the absence and presence of peptides and peptide dimers, as assessed by transwell assay.

[0247] Compared to untreated cells, all peptide analogs significantly reduced the invasive percentage (invasiveness %) of RH30 and / or RD rhabdomyosarcoma cells, achieving significance at different concentrations. Ac-SEQ ID NO:5-NH2 and (SEQ ID NO:6-NH2)2 showed a significant reduction in invasiveness % of RH30 cells at a peptide concentration of 100 nM, and octanoyl-SEQ ID NO:5-NH2 and SEQ ID NO:6-NH2 showed a significant reduction at 500 nM. Treatment with octanoyl-SEQ ID NO:5-NH2 at 500 nM and with (SEQ ID NO:6-NH2)2 and octanoyl-SEQ ID NO:8-NH2 at 1 μM resulted in a significant reduction in invasiveness % of RD cells. SEQ ID NO:7-NH2 showed a significant effect on RD cell invasiveness, with invasiveness below 50% even at the lowest dose (100 nM). Its effect was milder in RH30 cell lines with low ITGA9 expression, reducing invasiveness by % but not to a significant degree.

[0248] Example 5. In vitro invasiveness assay of neuroblastoma cell lines (CHLA-90 and BE(2)-C).

[0249] Transwell assays using CHLA-90 and BE(2)-C cell lines were performed under the same conditions as those used to determine the percentage of invasiveness in rhabdomyosarcoma cells.

[0250] As observed in Table 7, neuroblastoma cell lines also showed sensitivity to the peptide SEQ ID NO:6-NH2, with significantly reduced invasiveness% in both CHLA-90 and BE(2)-C cell lines at concentrations of 200 nM and 500 nM.

[0251]

[0252] * p-value < 0.05 compared to untreated cells.

[0253] Table 7. Percentage of invasion of neuroblastoma cell lines (CHLA-90 and BE(2)-C) in the absence or presence of different concentrations (from 100 to 1000 nM) of peptide SEQ ID NO:6-NH2, as assessed by transwell assay.

[0254] Example 6. Results of in vitro invasiveness of peptide SEQ ID NO:7-NH2 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).

[0255] Transwell assays using 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) were performed under the same conditions as those used to determine the % invasiveness in rhabdomyosarcoma cells.

[0256] 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, with significantly reduced cell invasiveness. Further invasiveness studies using this peptide in a third neuroblastoma cell line showed that at a concentration of 500 nM, this treatment also effectively inhibited invasiveness in the SK-N-BE(2)-C cell line, which does not overexpress ITGA9. In breast cancer cell lines, the MDA-MB-468 cell line showed a significant reduction in invasiveness upon treatment, while the MDA-MB-231 cell line (with low ITGA9 expression) showed a modest reduction (not statistically significant) at the highest treatment dose.

[0257]

[0258] Table 8. Percentage of invasiveness of neuroblastoma cell lines (CHLA-90, BE(2)-C and SK-N-BE(2)-C) and breast cancer cell lines (MDA-MB-231 and MBA-MB-468) in the absence or presence of different concentrations of peptide SEQ ID NO:7-NH2, as assessed by transwell assay.

[0259] In summary, these results do not indicate a unique correlation between higher ITGA9 expression levels and the better efficacy of peptide SEQ ID NO:7-NH2 in reducing invasiveness. Therefore, interaction with ITGA9 must not be the only molecular mechanism by which peptide analogs exhibit efficacy, as, for example, SEQ ID NO:7-NH2 at a concentration of 500 nM induced a significant reduction in invasiveness in SK-N-BE(2)-C (a cell line with low ITGA9 expression levels).

[0260] Example 7. In vivo invasiveness in a mouse model of rhabdomyosarcoma metastasis.

[0261] Five-week-old female SCID / Beige mice were administered the first dose of treatment subcutaneously (sc) on day -1, either a mediator (PBS, phosphate-buffered saline) or a peptide. On day 0, 2 x 10^6 mg / L mice were administered intravenously (iv) via the tail vein. 6 RD rhabdomyosarcoma cells. Mice were then subcutaneously treated three times a week (every 2–3 days) for 27 weeks with the following: 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).

[0262] Weight was assessed twice weekly for 27 weeks. Overall survival and event-free survival were determined. At necropsy, the total number of metastases was quantified, and the number and location of metastases per mouse were reported.

[0263] In the study, mice treated with the methylated peptide showed statistically significant lower body weight compared to mice treated with 0.5 mg / kg SEQ ID NO:6-NH2 (p<0.0001), 2 mg / kg SEQ ID NO:6-NH2 (p<0.0001), 0.5 mg / kg SEQ ID NO:7-NH2 (p=0.0006), and 2 mg / kg SEQ ID NO:7-NH2 (p=0.0012). These differences were primarily due to the lower percentage of mice undergoing metastasis in the groups treated with SEQ ID NO:6-NH2 and SEQ ID NO:7-NH2 peptides.

[0264] As shown in Table 9, metastasis occurred in all PBS-treated mice. Treatment with two test doses of SEQ ID NO:6-NH2 and a low dose of SEQ ID NO:7-NH2 resulted in a 15% reduction in metastasis formation. Meanwhile, in the group treated with SEQ ID NO:7-NH2 2 mg / kg, only 2 out of 7 mice developed metastasis, which was statistically significant compared to the PBS-treated group (p = 0.0048).

[0265]

[0266]

[0267] ns: not significant

[0268] Table 9. Number and percentage of mice that developed metastases in each treatment group during a 27-week study following a single tail vein injection of RD cells.

[0269] The expression of event-free survival in each treatment group compared to the control group treated with the mordant also showed a significant difference in the groups treated with SEQ ID NO:7-NH2 at a dose of 2 mg / kg (log-rank p-value = 0.0006). No statistically significant differences were found between the lower dose of SEQ ID NO:7-NH2 and the two detection doses of SEQ ID NO:6-NH2 compared to the mordant-treated groups. Figure 4 Results of animals treated with PBS and SEQ ID NO:7-NH2 are shown.

[0270] Furthermore, assessment of the number of metastases per mouse following a single intravenous administration of RD cells and subcutaneous treatment three times weekly with methylated or different treatments showed that, compared with control mice treated with methylated (PBS), SEQ ID NO:6-NH2 and SEQ ID NO:7-NH2 at 2 mg / kg statistically reduced metastasis formation per mouse (SEQ ID NO:6-NH2 2 mg / kg, p = 0.0058; SEQ ID NO:7-NH2 2 mg / kg, p = 0.0184). Figure 5 ).

[0271] The results above indicate that lower doses of SEQ ID NO:6-NH2 or SEQ ID NO:7-NH2 did not show significant effects in assessing the delay in the onset of metastases for 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 showed effectiveness in reducing the number of metastases observed at autopsy.

[0272] In terms of location, metastases developed in different sites: intestines, uterus, paws, back of spleen, ovaries, and adrenal glands. Metastases to the ovaries, adrenal glands, back, and spleen were more common across all treatment groups.

[0273] Example 8. Effect of peptide SEQ ID NO:7-NH2 on a mouse model of neuroblastoma metastasis.

[0274] Five-week-old female SCID / Beige mice were treated subcutaneously on day -1 with either a mediator (PBS) or 1 mg / kg and 2 mg / kg doses of the peptide SEQ ID NO:7-NH2. On day 0, 2 x 10⁻⁶ mg / kg of the peptide was administered intravenously via the tail vein. 5 BE(2)-C neuroblastoma cells. Mice were then subcutaneously treated for 13 weeks with SEQ ID NO:7-NH2 three times a week (every 2-3 days) with PBS (n=8) and SEQ ID NO:7-NH2 at 1 mg / kg (n=9) and 2 mg / kg (n=9) mediators.

[0275] As shown in Table 10, although 7 out of 8 mice (88%) in the PBS-treated group developed metastasis, the number of mice with metastasis was often lower in the group treated with SEQ ID NO:7-NH2 2 mg / kg (4 out of 9 mice (44%), p = 0.1312).

[0276]

[0277] Table 10. Number and percentage of mice that developed metastases in each treatment group during a 13-week study following a single tail vein injection of BE(2)-C cells.

[0278] Furthermore, event-free survival indicated significant differences among the mediator-treated group (survival: 1 of 8 mice, 13%), the group treated with 1 mg / kg peptide SEQ ID NO:7-NH2 (survival: 2 of 9 mice (22%), log-rank p = 0.6294), and the group treated with 2 mg / kg peptide (survival: 5 of 9 mice (56%), log-rank p = 0.0658), suggesting the efficacy of peptide SEQ ID NO:7-NH2 at the highest dose. Figure 6 ). sequence list <110> BCN Peptides, SA <120> Peptides for treating cancer and / or metastasis <130> RA <150> EP 19383084.1 <151> 2019-12-05 <160> 10 <170> PatentIn version 3.5 <210> 1 <211> 96 <212> PRT <213> Human ADAM-12, deintegration domain <400> 1 Glu Val Arg Glu Ser Phe Gly Gly Gln Lys Cys Gly Asn Arg Phe Val 1 5 10 15 Glu Glu Gly Glu Glu Cys Asp Cys Gly Glu Pro Glu Glu Cys Met Asn 20 25 30 Arg Cys Cys Asn Ala Thr Thr Cys Thr Leu Lys Pro Asp Ala Val Cys 35 40 45 Ala His Gly Leu Cys Cys Glu Asp Cys Gln Leu Lys Pro Ala Gly Thr 50 55 60 Ala Cys Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Phe Cys Thr 65 70 75 80 Gly Ala Ser Pro His Cys Pro Ala Asn Val Tyr Leu His Asp Gly His 85 90 95 <210> 2 <211> 14 <212> PRT <213> Human ADAM-12, a sequence derived from the deintetrodotium domain. <400> 2 Cys Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Phe Cys 1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (12)..(12) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 3 Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Xaa 1 5 10 <210> 4 <211> 13 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (1)..(1) <223> Xaa is pyroglutamic acid (Pyr). <220> <221> variants <222> (13)..(13) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 4 Xaa Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Xaa 1 5 10 <210> 5 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (13)..(13) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 5 Lys Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Xaa Lys 1 5 10 <210> 6 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (1)..(1) <223> Xaa is pyroglutamic acid (Pyr). <220> <221> variants <222> (14)..(14) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 6 Xaa Lys Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Xaa Lys 1 5 10 15 <210> 7 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (1)..(1) <223> Xaa is pyroglutamic acid (Pyr). <220> <221> variants <222> (14)..(14) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 7 Xaa Lys Arg Asp Ser Ser Asn Ser Met Asp Leu Pro Glu Xaa Lys 1 5 10 15 <210> 8 <211> 14 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (13)..(13) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 8 Lys Arg Asp Ser Ser Asn Ser Met Asp Leu Pro Glu Xaa Lys 1 5 10 <210> 9 <211> 15 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <220> <221> variants <222> (1)..(1) <223> Xaa is pyroglutamic acid (Pyr). <220> <221> variants <222> (9)..(9) <223> Xaa is cystine, Cys (Cys) <220> <221> variants <222> (14)..(14) <223> Xaa is 2,4,6-trimethylphenylalanine (Msa). <400> 9 Xaa Lys Arg Asp Ser Ser Asn Ser Xaa Asp Leu Pro Glu Xaa Lys 1 5 10 15 <210> 10 <211> 12 <212> PRT <213> Artificial sequence <220> <223> synthetic sequence <400> 10 Arg Asp Ser Ser Asn Ser Cys Asp Leu Pro Glu Phe 1 5 10

Claims

1. A peptide and / or a pharmaceutically acceptable salt thereof, wherein said peptide is selected from the group consisting of: Ac-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Ac-SEQ IDNO: 5-NH2); Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Cys-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Ocanoyl-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)]; and Octanoyl-Lys-Arg-Asp-Ser-Ser-Asn-Ser-Met-Asp-Leu-Pro-Glu-Msa-Lys-NH2 (Ocanoyl-SEQ ID NO: 8-NH2).

2. A method for preparing the peptide according to claim 1 and / or a pharmaceutically acceptable salt thereof, the method being carried out using solid-phase peptide synthesis or in-solution peptide synthesis.

3. The method according to claim 2, wherein the peptide is Ac-SEQ ID NO: 5-NH2, octanoyl-SEQ ID NO: 5-NH2, SEQ ID NO: 6-NH2, SEQ ID NO: 7-NH2 or octanoyl-SEQ ID NO: 8-NH2, and the method comprises the following steps: a) Solid-phase peptide synthesis in a polymer support; b) Cut the peptide from the polymer support; c) Eliminate protecting groups.

4. The method according to claim 2, wherein the peptide is a peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge), and the method further comprises the following steps: a) Solid-phase peptide synthesis in a polymer support; b) Cut the peptide from the polymer support; c) Oxidize the peptide in solution to obtain the peptide dimer; d) Eliminate protecting groups.

5. The method according to claim 2, wherein the peptide is Ac-SEQ ID NO: 5-NH2, octanoyl-SEQ ID NO: 5-NH2, SEQ ID NO: 6-NH2, SEQ ID NO: 7-NH2 or octanoyl-SEQ ID NO: 8-NH2, and the method comprises the following steps: i) Solid-phase peptide synthesis in a polymer support; ii) The peptide is cleaved from the polymer support, and the protecting group is removed at the same time.

6. The method according to claim 2, wherein the peptide is a peptide dimer (SEQ ID NO: 6-NH2)2 (disulfide bridge), and the method comprises the following steps: i) Solid-phase peptide synthesis in a polymer support; ii) Solid-phase peptide dimer formation is carried out in a polymer support; iii) The peptide dimer is cleaved from the polymer support, and the protecting group is removed at the same time.

7. A pharmaceutical composition comprising a pharmaceutically effective amount of at least one peptide according to claim 1 and / or a pharmaceutically acceptable salt of the peptide.

8. The pharmaceutical composition of claim 7, wherein the peptide and / or a pharmaceutically acceptable salt of the peptide is incorporated into a delivery system and / or a sustained-release system selected from the group consisting of: Vesicles, millimeter particles, microparticles, nanoparticle sponges, cyclodextrins, micelles, microemulsions, and nanoemulsions.

9. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is a vesicle selected from the group consisting of: Liposomes, liposomes, lipid vesicles, and alcohol bodies.

10. The pharmaceutical composition according to claim 8, wherein the delivery system and / or sustained-release system is a hybrid liposome.

11. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system are millimeter particles selected from the group consisting of: Millimeter balls and millimeter capsules.

12. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is microparticles selected from the group consisting of: Microspheres and microcapsules.

13. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is a lipid sphere.

14. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is nanoparticles selected from the group consisting of: Nanospheres and nanocapsules.

15. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is a solid lipid nanoparticle.

16. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is a nanostructured lipid carrier.

17. The pharmaceutical composition of claim 8, wherein the delivery system and / or sustained-release system is micelles selected from the group consisting of: Mixed micelles of surfactants and mixed micelles of surfactant-phospholipids.

18. The peptide of claim 1 and / or a pharmaceutically acceptable salt thereof, used as a medicament, wherein the peptide is Ac-SEQ ID NO:5-NH2, octanoyl-SEQ ID NO:5-NH2, SEQ ID NO:6-NH2, SEQ ID NO:7-NH2, (SEQ ID NO:6-NH2)2 (disulfide bridge), octanoyl-SEQ ID NO:8-NH2, and the medicament is used to inhibit rhabdomyosarcoma invasion.

19. The peptide of claim 1 and / or a pharmaceutically acceptable salt thereof, for inhibiting neuroblastoma invasion and rhabdomyosarcoma metastasis, wherein the peptide is SEQ ID NO:6-NH2.

20. The peptide of claim 1 and / or a pharmaceutically acceptable salt thereof, for inhibiting neuroblastoma and breast cancer invasion, rhabdomyosarcoma metastasis and neuroblastoma metastasis, wherein the peptide is SEQ ID NO:7-NH2.

21. Use of the peptide of claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the peptide is Ac-SEQ ID NO:5-NH2, octanoyl-SEQ ID NO:5-NH2, SEQ ID NO:6-NH2, SEQ ID NO:7-NH2, (SEQ ID NO:6-NH2)2 (disulfide bridge), or octanoyl-SEQ ID NO:8-NH2, and the medicament is used to inhibit the invasion of rhabdomyosarcoma.

22. Use of the peptide of claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the peptide is SEQ ID NO:6-NH2, and the medicament is used to inhibit neuroblastoma invasion and rhabdomyosarcoma metastasis.

23. Use of the peptide of claim 1 and / or a pharmaceutically acceptable salt thereof in the preparation of a medicament, wherein the peptide is SEQ ID NO:7-NH2, and the medicament is used to inhibit the invasion of neuroblastoma and breast cancer, rhabdomyosarcoma, and metastasis of neuroblastoma.

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