Peptides used in the treatment of sarcomas

Abstract

The present invention provides peptides for use in the treatment and / or prevention of sarcomas. [Solution] Peptides for use in the treatment and / or prevention of sarcomas, comprising the following group: (i) Peptides having the following amino acid sequence: Z1-LVRYTKKVPQVSTPTL-Z2(ALB-408) (where Z represents 0 to 10 amino acid residues) (ii) Peptides with biological activity of ALB408-423 obtained by multiplex synthesis and (iii) Biologically active fragments and / or variants and / or derivatives of (i) and (ii) above, in particular PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives It is a peptide selected from among them.

Description

[Technical Field]

[0001] The present invention relates to peptides used for the treatment and / or prevention of sarcoma, pharmaceutical compositions used for the treatment and / or prevention of sarcoma, and methods for treating sarcoma in patients requiring treatment. The peptide used in the present invention is a CXC chemokine receptor 4 (CXCR4) antagonist peptide (protein). The peptides used in the present invention include native human circulating antiviral albumin fragments (ALB408-423), peptides obtained by multiplex synthesis that have the biological activity of ALB408-423, and biologically active fragments and / or variants and / or derivatives thereof. [Background technology]

[0002] Sarcomas are malignant neoplasms of mesenchymal origin, that is, malignant neoplasms that arise from cells of mesenchymal supporting tissue. Along with neoplasms of epithelial tissue (carcinomas), neoplasms of blood (lymphomas and leukemias), neuroendocrine tumors, and dysplastic tumors, sarcomas form a subgroup of malignant tumors.

[0003] Sarcomas are rare diseases, accounting for only 1% of all adult cancer diagnoses and about 15% of childhood cancer diagnoses. In the United States, approximately 16,000 cases of sarcoma are diagnosed annually (approximately 4,000 osteosarcomas and 13,000 soft tissue sarcomas). In France, approximately 4,000 people are diagnosed with sarcoma each year. In Italy, the number is even higher, with an estimated 5,890 new sarcoma cases per year. Sarcomas can have adverse effects on both children and adults. Generally, soft tissue sarcomas often occur more frequently in adults. Diagnoses of osteosarcoma are more common in children, teenagers, and older adults (65 years and older). Sarcomas form when immature bone cells or soft tissue cells undergo DNA changes, developing into cancer cells that proliferate uncontrollably. Ultimately, they form lumps or tumors that can invade surrounding healthy tissue. If left untreated, the cancer can spread from its primary site to other organs via the bloodstream or lymphatic system (metastasis).

[0004] According to the current WHO classification of sarcomas, tumors are divided into more than 100 different types. This classification is based primarily on the morphology, origin, and molecular genetic variations of the tumor. Sarcomas are broadly classified into osteosarcoma, chondrosarcoma, and soft tissue sarcoma. Osteosarcoma is a primary malignant bone tumor. The three main types of osteosarcoma are osteosarcoma, Ewing's sarcoma, and chondrosarcoma. Osteosarcoma is the most commonly observed type of osteosarcoma. It is a malignant bone tumor consisting of cells that produce bone matrix, and is most commonly found in teenagers and young adults. Ewing's sarcoma is the third most common type of sarcoma, after childhood and adolescence, and chondrosarcoma is the most common type in adults.

[0005] For most sarcomas that have not metastasized to other parts of the body, surgery is the most common treatment. In cases of limb (arm or leg) sarcoma, limb-sparing surgery, as opposed to amputation, is used to preserve the limb in at least 90% of patients. Additional treatments, including chemotherapy, radiotherapy (also called "radiotherapy"), and proton therapy, may be administered before surgery (called "neoadjuvant chemotherapy" or "neoadjuvant radiotherapy") or after surgery (called "post-adjuvant chemotherapy" or "post-adjuvant radiotherapy"). Neoadjuvant or post-adjuvant chemotherapy, and neoadjuvant or post-adjuvant radiotherapy, significantly improve the prognosis for many sarcoma patients. Treatment can be a long and difficult process, lasting about a year for many patients. In pediatric sarcomas, the cytotoxic agent cyclophosphamide is widely used and exhibits excellent antitumor effects.

[0006] The first-line treatment for osteosarcoma is surgical resection, which completely and curatively removes the tumor en bloc. Approximately 90% of patients can undergo limb-sparing surgery, but complications, particularly infection, loosening or failure of prosthetic joints, or local tumor recurrence may necessitate further surgery or amputation. Standard treatments include combination therapy with limb-sparing orthopedic surgery (and sometimes amputation) whenever possible, or salvage therapy with high-dose methotrexate and intra-arterial administration of leucovorin or cisplatin, or adriamycin or ifosfamide with mesna, etoposide, or muramil tripeptide. Although chemotherapy for osteosarcoma has been successful, it is one of the diseases with the lowest survival rates among childhood cancers, for example.

[0007] Therefore, further development of compounds and pharmaceuticals for the treatment and / or prevention of sarcomas, particularly osteosarcoma, is necessary.

[0008] Chemokine receptors are expressed on the surface of specific cells and interact with cytokines called chemokines. CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor that transmits the signal of its endogenous ligand, the chemokine CXCL12 (stromal cell-derived factor-1, SDF-1). Following the interaction between CXCR4 and CXCL12, intracellular calcium (Ca) 2+It induces an influx of ions. This triggers a chemotaxis-like cellular response in which cells can migrate within the body. CXCR4 is expressed in bone marrow cells, T lymphocytes, B lymphocytes, epithelial cells, endothelial cells, and dendritic cells. The chemokine CXCL12 is the only known agonist ligand for CXCR4. The interaction between CXCL12 and CXCR4 plays a crucial role in the migration of progenitor cells during developmental stages of the cardiovascular, hematopoietic, or central nervous system. This interaction is also known to be involved in several diseases, including HIV infection / AIDS, cancer cell metastasis, leukemia progression, pulmonary fibrosis, and rheumatoid arthritis. This interaction is thought to be a potentially important therapeutic target in all of these diseases. Substances that inhibit CXCR4 / CXCL12 signaling are thought to have potential as drugs in the treatment of HIV / AIDS, for example, or to inhibit cell migration processes involved in inflammatory diseases such as cancer metastasis, leukemia, pulmonary fibrosis, rheumatoid arthritis, and asthma (Non-Patent Literature 1). In contrast to receptor agonists that induce cellular responses, such as CXCL12, receptor antagonists, even when bound to the receptor, do not induce biological responses, namely cell migration or Ca2+. 2+ Receptor antagonists are ligands or drugs that do not induce signal transduction. Receptor antagonists are useful drugs already in clinical use that inhibit HIV-1 infection (CCR5 antagonists) or reduce agonist-mediated cellular responses (e.g., angiotensin antagonists, β-adrenergic antagonists, serotonin antagonists, CCR5 antagonists). The interaction between receptor antagonists and receptors inhibits the function of agonists. Most antagonists exert their efficacy by competing with endogenous ligands or substrates at structurally determined binding sites on the receptor.

[0009] It has already been shown both in vitro and in vivo that CXCR4 antagonists inhibit the migration of cancer cells and thus metastasis. CXCR4 is expressed on the surface of various cells (bone marrow cells, T lymphocytes, B lymphocytes, epithelial cells, endothelial cells, and dendritic cells), as well as on 23 different types of cancer cells. The interaction between CXCL12 and CXCR4 is involved in the metastasis of various types of cancer such as breast cancer, renal cancer, prostate cancer, lung cancer, pancreatic cancer, melanoma, neuroblastoma, non-Hodgkin lymphoma, multiple myeloma, ovarian cancer, and malignant brain tumors (Non-Patent Document 1). CXCR4 antagonists such as T140 analogs have been shown to inhibit CXCL12-induced pancreatic cell migration and invasion or the migration of breast cancer cells both in vitro and in vivo (Non-Patent Document 1). Also, it has been demonstrated that CXCR4 antagonists effectively inhibit the invasion and adhesion of small cell lung cancer (SCLC) in vitro (Non-Patent Document 1), supporting the involvement of the CXCL12-CXCR4 interaction in SCLC metastasis. The CXCR4 / CXCL12 interaction is also involved in the development of precursor B cell (pre-B cell) acute lymphoblastic leukemia (ALL) and chronic lymphocytic leukemia (CLL). CXCR4 antagonists also attenuate the migration of pre-B cell ALL cells (Non-Patent Document 1).

[0010] Patent Document 1 discloses that the chemokine receptor CXCR4 is a therapeutic target in the treatment of HIV / AIDS, cancer-related pathologies, and chronic inflammatory diseases such as asthma and pulmonary fibrosis. This Patent Document 1 discloses peptides having the amino acid sequence Z1-LVRYTKKVPQVSTPTL-Z2 (ALB-408), biologically active fragments and / or variants and / or derivatives of ALB408-423, and peptides obtained by multiple synthesis, and also discloses their use in the manufacture of pharmaceuticals for treating viral diseases, bacterial and fungal infections, inflammatory processes, inhibition of inflammatory reactions, neoplastic diseases, growth disorders, neurological diseases, blood coagulation and hematopoietic system diseases, vascular diseases, immune system diseases, and for treating wounds and for bone healing.

[0011] Patent Document 2 discloses a peptide, particularly a dimer, that is effective in inhibiting CXCR4-mediated HIV-1 NL4-3 (X4 tropic) infection with an IC 50 value of less than 50 μM.

[0012] Patent Document 3 discloses a peptide for use in the treatment of cancer, particularly cancer expressing the CRCX receptor, such as liver cancer, pancreatic cancer, prostate cancer, or breast cancer.

[0013] Patent Document 4 discloses a polypeptide for use in the treatment of cancer, particularly cancer expressing the CRCX receptor, such as liver cancer, pancreatic cancer, prostate cancer, or breast cancer.

Prior Art Documents

Non-Patent Documents

[0014]

Non-Patent Document 1

Patent Documents

[0015]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Summary of the Invention

Problems to be Solved by the Invention

[0016] An object of the present invention is to provide a peptide for use in the treatment and / or prevention of sarcoma, particularly osteosarcoma. In particular, the peptide to be used inhibits the growth and / or metastasis of sarcoma cancer cells.

[0017] A further object of the present invention is to provide a pharmaceutical composition for use in the treatment and / or prevention of sarcomas, particularly osteosarcomas. In particular, the pharmaceutical composition used inhibits the proliferation and / or metastasis of sarcoma cancer cells.

[0018] A further object of the present invention is to provide a method for treating sarcomas, particularly osteosarcomas, in patients requiring treatment, in particular, a method that inhibits the proliferation and / or metastasis of sarcoma cancer cells. [Means for solving the problem]

[0019] Unexpectedly, (i) a CXCR4 antagonist polypeptide having the amino acid sequence Z1-LVRYTKKVPQVSTPTL-Z2(ALB-408) (wherein Z represents 0 to 10 amino acid residues), (ii) a peptide with biological activity of ALB408-423 obtained by multiplex synthesis, and (iii) these biologically active fragments and / or variants and / or derivatives, in particular PEGylated, HESlated, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives, were found to be effectively usable in methods for the treatment and / or prevention of sarcomas, especially osteosarcoma.

[0020] Therefore, the present invention relates to peptides for use in the treatment and / or prevention of sarcomas, wherein the peptides are comprised of the following: (i) The following amino acid sequence Z1-LVRYTKKVPQVSTPTL-Z2(ALB-408) (where Z represents 0 to 10 amino acid residues) Peptides having, (ii) A peptide with biological activity obtained by multiple synthesis, and (iii) Biologically active fragments and / or variants and / or derivatives of (i) and (ii), in particular PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives; It is selected from among them.

[0021] The peptides used in the present invention may be peptides having the same or identical biological or pharmacological activity as the peptides used in the present invention, in which one or more amino acid residues in the sequence are exchanged, deleted, or added, or in which a chemical modification is introduced to one amino acid of the peptide used in the present invention. In particular, these peptides can be easily obtained by converting the amino acids in the sequence using conventional methods, meaning that hydrophobic amino acids are exchanged with other hydrophobic amino acids, aromatic amino acids are exchanged with other aromatic amino acids, and basic amino acids are exchanged with other basic amino acids. Such methods are known to those skilled in the art.

[0022] Furthermore, the retroinversopeptides used in the present invention, as well as other derivatives that stabilize the peptide bond with respect to peptidases, are also included within the scope of the present invention.

[0023] The term "derivative" means peptides comprising fragments of any length including cleavage at the N-terminus and C-terminus, peptides comprising amino acid residue substitutions including D-amino acid residues and modified amino acid residues, and peptides comprising N-terminus and C-terminus disulfide bonds or extensions, and peptides in which at least one side chain of amino acids of said peptide is chemically modified. In particular, the term "derivative" means PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives of peptides (i) and (ii) used in the present invention.

[0024] The term "mutant" means a peptide having an amino acid sequence that has at least 70% sequence identity, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with either peptide (i) or (ii) used in the present invention. The terms "sequence identity" or "sequence identity rate" with respect to two or more peptides refer to two or more sequences or subsequences in which amino acids are either completely identical or identical at a certain ratio when compared and "aligned" using a sequence comparison algorithm to obtain maximum similarity. Optimal alignment for sequence comparison can be performed using the following algorithms, for example, local homology alignment by Smith & Waterman: Local Homology Alignment (Adv. Appl. Math. 2: 482 (1981)), homology alignment algorithm by Needleman & Wunsch: Homology Alignment Algorithm (J. Mol. Biol. 48: 443 (1970)), Proc. Nat’l. Acad. Sci. USA 85: 2444 (1988) by Pearson & Lipman, GAP, BESTFIT, FASTA, TFASTA of the Wisconsin Genetics Software Package by Madison, WI, Genetics Computer Group, 575 Science Dr., as well as visual observation (see Current Protocols in Molecular Biology (Ausubel, F. M. et al., eds.) John Wiley & Sons, Inc, New York (1987 - 1999, Supplement 46 (April 1999)) in molecular biology).

[0025] The peptide obtained by multiple synthesis and having the biological activity of ALB408 - 423 is a peptide selected from the group consisting of SEQ ID NOs: 1 - 31, and / or the following general formula in one - letter notation X 1 X 2 RX 4 X 5 X 6 KX 8 PX 10 X 11 S (where X 1 = L, or I, X 2 =V, M, or L, X 4 =Y or W, X 5 =T or S, X 6 =K, C, R, or Q, X 8 =V, M, L, F, or A, X 10 =Q or C, X 11 (=V, M, or F) It may also be a peptide having the amino acid sequence shown.

[0026] Preferably, the peptides obtained by multiplex synthesis that have the biological activity of ALB408-423 are peptides selected from the group consisting of sequence identification numbers 1 to 31, and / or a general formula represented by a single letter. X 1 VRX 4 X 5 X 6 KVPX 10 VS (However, X 1 =L or I, X 4 =Y or W, X 5 =T or S, X 6 =K or C, X 10 =Q or C) It is a peptide having the amino acid sequence shown.

[0027] In another preferred embodiment, the peptides having biological activity, obtained by multiplex synthesis, are expressed by a single-letter general formula: X 1 X 2 RX 4 X 5 X 6 KX 8 PX 10 X 11 S (However, X 1 =L, or I X2 =V, M, or L, X 4 =Y or W, X 5 =T or S, X 6 =K, C, R, or Q, X 8 =V, M, L, F, or A, X 10 =Q or C, X 11 =V, M, or F), More convenient general formula using single-character notation X 1 VRX 4 X 5 X 6 KVPX 10 VS (However, X 1 =L or I, X 4 =Y or W, X 5 =T or S, X 6 =K or C, X 10 It is a peptide having the amino acid sequence indicated by =Q or C).

[0028] In another preferred embodiment, the peptide used in the present invention has the following amino acid sequence IVRYTKKVPQVS (Sequence identification number 21) Peptides having, and It is selected from the group consisting of amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives thereof.

[0029] In another preferred embodiment, the peptide used in the present invention has the following amino acid sequence: IVRYTKKVPQVS (Sequence identification number 21) It possesses the following characteristics.

[0030] The sarcoma may be selected from the group consisting of osteosarcoma, chondrosarcoma, and soft tissue sarcoma. Preferably, the sarcoma is selected from the group consisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, and angiosarcoma. Osteosarcoma, Ewing's sarcoma, and chondrosarcoma are the three main types of osteosarcoma. Preferably, the sarcoma is osteosarcoma.

[0031] In preferred embodiments, the sarcoma is osteosarcoma, preferably osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, telangiectatic osteosarcoma, small cell osteosarcoma, low-grade central osteosarcoma, periosteal osteosarcoma, paraosteal osteosarcoma, secondary osteosarcoma, high-grade superficial osteosarcoma, or extraosseous osteosarcoma, and more preferably osteoblastic osteosarcoma, chondroblastic osteosarcoma, or fibroblastic osteosarcoma.

[0032] The peptide is administered, in preferred embodiments, via parenteral, intravenous, intramuscular, intrathecal, transpulmonary, intranasal, ophthalmic, auricular, topical, subcutaneous, or sublingual routes, preferably intranasal, ophthalmic, or auricular routes, and more preferably intranasal. The routes of administration and implementation of the pharmaceutical compounds and pharmaceutical compositions thereof are known to those skilled in the art.

[0033] The peptide used in this invention can be administered two or three times a day, preferably three times a day, for 1 to 3 months, preferably 4 to 6 weeks. The treatment period can be extended as needed.

[0034] In a preferred embodiment, the peptide is administered three times a day, preferably three times a day, via the intranasal route, for six weeks.

[0035] The peptide used in this invention is preferably administered intranasally using a nasal spray.

[0036] The peptide used in this invention can be administered at a concentration of 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL.

[0037] The peptide used in this invention is subjected to a sodium chloride solution, preferably physiological saline, or a citrate / bicarbonate buffer, preferably sodium citrate / sodium bicarbonate buffer. Suitable buffers according to the European Pharmacopoeia (Ph.Eur.) are known to those skilled in the art. The concentration of the peptide used in this invention may be 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL.

[0038] In a preferred embodiment, the present invention relates to a peptide having the amino acid sequence IVRYTKKVPQVS (sequence identification number 21) for use in the treatment and / or prevention of osteosarcoma.

[0039] Another object of the present invention is a pharmaceutical composition for use in the treatment and / or prevention of sarcoma. The pharmaceutical composition is (a) The following amino acid sequence Peptides having Z1-LVRYTKKVPQVSTPTL-Z2(ALB-408) (where Z is 0 to 10 amino acid residues), peptides obtained by multiplex synthesis that have the biological activity of ALB408-423, and biologically active fragments and / or variants and / or derivatives thereof, in particular PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives thereof, (b) A pharmaceutically acceptable carrier and It includes or consists of.

[0040] The pharmaceutical composition for use contains these peptides for use in the present invention. Therefore, it can be understood that all embodiments and features described herein with respect to the peptides for use in the present invention also apply to the pharmaceutical composition for use in the present invention.

[0041] In a preferred embodiment, this peptide has the following amino acid sequence IVRYTKKVPQVS (Sequence identification number: 21) It has.

[0042] The pharmaceutical composition for use is suitable for parenteral, intravenous, intramuscular, intrathecal, transpulmonary, intranasal, ophthalmic, auricular, topical, subcutaneous, or sublingual administration, preferably intranasal, ophthalmic, or auricular administration, and more preferably sublingual administration.

[0043] The pharmaceutical composition for use may be in liquid dosage form.

[0044] The peptide concentration may be 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL.

[0045] In preferred embodiments, the pharmaceutically acceptable carrier is a sodium chloride solution, preferably a physiological saline solution, or a citrate / bicarbonate buffer, preferably a sodium citrate / sodium bicarbonate buffer. Suitable buffers according to the European Pharmacopoeia (Ph.Eur.) are known to those skilled in the art. The pharmaceutically acceptable concentration of the peptide in the carrier is 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL.

[0046] A further object of the present invention is a method for treating sarcoma in patients requiring treatment, comprising means of administering an effective amount of peptide to the patient. The peptide is comprised of the following group: (i) The following amino acid sequence A peptide having Z1-LVRYTKKVPQVSTPTL-Z2(ALB-408) (where Z represents 0 to 10 amino acid residues), (ii) Peptides with biological activity of ALB408-423 obtained by multiple synthesis, and (iii) Biologically active fragments and / or variants and / or derivatives of (i) and (ii) above, in particular PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives Selected from.

[0047] In the method for treating sarcoma of the present invention, the peptide used in the present invention is a peptide administered in an effective amount. This peptide can also be administered in the dosage form of the pharmaceutical composition used in the present invention. Therefore, it will be understood that all embodiments and features described herein with respect to the peptide and pharmaceutical composition used in the present invention are also applicable to the method for treating sarcoma of the present invention.

[0048] The peptides used in this invention are supplied by a process comprising extraction from blood filtrate by cation exchange extraction, followed by elution of adsorbed substances, further cation exchange chromatography of the peptide-containing extract, and fractional reverse-phase chromatography.

[0049] Alternatively, the peptide used in the present invention can be produced by solid-phase synthesis using Merrifield synthesis, i.e., liquid-phase synthesis using protected amino acids by a method known to those skilled in the art, and purification thereof.

[0050] A further method for producing peptides for use in accordance with the present invention is to use a mutagenesis method using a common bioengineered vector, which is known to those skilled in the art.

[0051] The present invention will be described in more detail using the peptide IVRYTKKVPQVS (sequence identification number 21) as a specific example. Similarly, it will be readily apparent that the peptides used in the present invention can be used in place of IVRYTKKVPQVS (sequence identification number 21) in the following description. [Modes for carrying out the invention]

[0052] ALB408-423 could be isolated from human blood filtrate by chromatography and biological assays. The peptide's biochemical characterization was performed by mass spectrometry along with complete amino acid sequence analysis.

[0053] The peptide has the following amino acid sequence (sequence identification number 8) LVRYTKKVPQVSTPTL It possesses the following characteristics.

[0054] The molecular weight of peptide ALB408-423 is 1830.2 Da. The isoelectric point (pI) of peptide ALB408-423 is 10.3.

[0055] Peptide ALB408-423 is a 16-amino acid fragment of the known human plasma protein serum albumin (accession number NP000468), which in its processed form consists of 585 amino acids. Human albumin is a soluble monomeric serum protein with a molecular weight of approximately 65,000, accounting for more than half of the total plasma protein (concentration: 3.5-5 g / dL). The main function of human albumin is described as that of a carrier molecule for all kinds of hydrophobic and hydrophilic substances, such as steroids, peptide hormones, fatty acids, vitamins, pharmaceuticals, and cations. Due to its very high serum concentration, human albumin substantially contributes to the stabilization of blood pH, extracellular fluid volume, and colloidal osmotic pressure. Albumin has a spherical structure stabilized by numerous disulfide crosslinks and is normally not glucosylated, but partial modifications by acetylation, enzymatic glucosylation, and non-enzymatic glucosylation frequently occur with molecular aging and pathophysiological changes. Albumin is synthesized in the liver as pre-proalbumin, consisting of 609 amino acids. The N-terminal signal peptide, consisting of 18 amino acids, is cleaved intracellularly upon entering the endoplasmic reticulum. After further removal of 6 amino acids in the Golgi apparatus, human serum albumin, consisting of 585 amino acids, is secreted from hepatocytes. Albumin clearance occurs via tissue cells in the kidneys, gastrointestinal tract, and liver.

[0056] The peptide sequence of ALB408-423 used in this invention begins with amino acid 408 and therefore contains amino acids 408-423 in circulating albumin. This sequence is clearly generated by the spontaneous process of albumin precursors mediated by the corresponding protease.

[0057] The peptide ALB408-423 used in this invention is obtained from human blood filtrate (HF) by chromatographic purification. HF is obtained in large quantities during ultrafiltration of blood from renal patients (Example 1). HF contains all peptides and proteins with a molecular weight of less than 30 kDa that circulate in human blood. Peptides and proteins in HF were extracted using cation exchange chromatography. The peptides and proteins bound to the column were eluted with buffer systems of various pH values, and the eluates were subjected to reverse-phase chromatography (Example 1). After four purifications, mass spectrometry of the active fraction 31 revealed it to be a single peptide with a molecular weight of 1830 Da (Example 1). Sequence analysis identified LVRYTKKVPQVSTPTL, and sequence comparison showed 100% homology to the serum protein "human serum albumin (ALB)," which is very rich in amino acid residues 408-423 (ALB408-423).

[0058] The preferred peptide used in this invention, obtained by multiple synthesis and possessing the biological activity of ALB408-423, is a 12-amino acid derivative of the 16-amino acid peptide ALB408-423. Starting with the C-terminal cleavage of ALB408-423 (LVRYTKKVPQVS; sequence identification number 12), peptides were subsequently generated by amino acid substitution. First, leucine was substituted with isoleucine to obtain IVRYTKKVPQVS (sequence identification number 21). Next, tyrosine in sequence identification number 21 was substituted with tryptophan to obtain IVRVTKKVPQVS (sequence identification number 32). Lysine in sequence identification number 32 was substituted with cysteine ​​to obtain IVRVTCKVPQVS (sequence identification number 33). By substituting threonine, cysteine, and glutamine in sequence identification number 33 with serine, lysine, and cysteine, respectively, IVRWSKKVPCVS (sequence identification number 34) was obtained. The 12-amino acid length derivatives from ALB408-423 derived from the above derivatives are shown in Table 1 (Example 4; sequence identification numbers 35-45).

[0059] Further peptides used in the present invention, obtained by multiple synthesis and possessing the biological activity of ALB408-423, are the peptides with sequence identification numbers 1-7, 9-11, 13-20, 22-24, and 25-31 shown in Table 1 (Example 4).

[0060] The peptide of the present invention is an antagonist of CXC chemokine receptor 4 (CXCR4).

[0061] Unexpectedly, the peptide IVRYTKKVPQVS (sequence identification number 21) was found to be effective in treating sarcoma. Patients with osteosarcoma were treated with informed consent. Unexpectedly, the patients' motor function recovered during treatment, and pain disappeared after 6 weeks. Furthermore, tumor regression was observed. Fortunately, no side effects were observed (Example 5). Unless otherwise noted, all measurements were taken at 23°C. [Examples]

[0062] The present invention will be further explained by the following examples.

[0063] (Example 1: Isolation of ALB408-423 from human blood filtrate) Human blood filtrate was diluted with water as needed and acidified. The pH value was preferably 1.5 to 3.5, particularly 2.5 to 3.0. The blood filtrate was then passed through a cation exchanger, for example, a carrier modified with sulfonic acid groups (Fraktogel SP-650(M), Merck, Darmstadt, Germany). Peptides bound to the cation exchanger were eluted with a relatively high concentration salt solution. The ionic strength of the eluate was approximately that of a 0.5 to 1 M ammonium acetate solution. The collected eluate was subjected to cation exchange chromatography again. This chromatography preferably involved fractional elution using a buffer solution with an increased pH value. The fraction containing the peptide according to the present invention was further purified by preparative reverse-phase chromatography, followed by semi-preparative reverse-phase chromatography using, for example, a C18-modified support. It is preferable to monitor the degree of purification using, for example, analytical reverse-phase chromatography using a C18-modified support.

[0064] [Step 1: Batch extraction of blood filtration solution] 800-1,000 liters of blood filtration solution were adjusted to a pH of 2.7 with HCl, diluted with water to achieve a conductivity of 5.5 mS / cm, and then charged onto a strong cation exchanger at a flow rate of 3 liters / min. • Schematic conditions: Column: Vantage VA 250 (manufactured by Amicon, Witten, Germany) Column material: Fractogel TSK SP 650(M), 25cm x 20cm Flow rate: 3L / min Detection: 280nm, pH, conductivity Buffer A: Hemofiltration solution has a pH of 2.7 and a conductivity of 5.5 mS / cm². Buffer solution B: 0.5M ammonium acetate • Equipment: Autopilot chromatography system (Perseptive (Manufactured by Biosystems, Wiesbaden, Germany) After charging a total of 1,000 L of solution overnight, the column was rinsed with 5 mM HCl at a concentration several times the column volume. Elution of the bound peptide was performed as batch elution using 0.5 M ammonium acetate. Complete elution of the peptide was achieved by gradually changing the pH (from 6.8 to 7.2) and conductivity (to 56 mS / cm) of approximately 5 liters of eluate.

[0065] [Step 2: First preparative separation (Batch 01 / 2003)] The ammonium acetate eluate from the batch extract was mixed with 10,000 liters of hemofilate peptide. After adjusting the pH to 2.7, the peptide extract was charged onto a preparative cation exchanger, and completely desalted water was added to achieve a conductivity of 5.5 mS / cm. • Schematic conditions: Column: Vantage 250 VA Column material: Fractogel TSK SP 650M, 25cm x 20cm Flow rate: Maximum 3 L / min during filling, 0.5-1 L / min during dissolution. Detection: 280nm, pH, electrical conductivity Sample: Blood filtrate has a pH of 2.7 and an electrical conductivity of 5.5 mS / cm². • Equipment: Autopilot chromatography system (Perseptive (Manufactured by Biosystems, Wiesbaden, Germany) The stock extract was charged into the column over 240 minutes, and then the column was rinsed with 0.01 M HCl until the conductivity was less than 1 mS / cm. Elution was performed in several stages using the buffer solutions listed below.

[0066] [Table I]

[0067] Elutions 1-7 were collected in pH-separated reservoirs designated I-VII. These were collected separately and finally washed with completely demineralized water. Elution was continued until a new baseline was reached, resulting in elution volumes of 10-25 liters for each pH-separated reservoir I-VII.

[0068] [3rd step: 2nd preparative separation] Each pH-separated reservoir was separated by reverse-phase chromatography, and desalting was performed simultaneously with fractionation. • Schematic conditions: Column: FineLine 100 (manufactured by Pharmacia) Freiburg, Germany) Column material: Source RPC, 15 μm, 10 × 12.5 cm (Fine Line 100) Flow rate: 150ml / min (FineLine100) Detection: 280nm, pH, conductivity Buffer A: 10mM HCl Buffer B: 80% acetonitrile in 10 mM HCl Gradient: 0-60% of buffer B (5 times the column volume) After charging the columns with the collected material from each pH-specific reservoir, the columns were washed with buffer A. 200 ml of fraction was collected during elution. Each fraction was freeze-dried and stored at -20°C. Peptides ALB408-423 were found in fractions 6-8 obtained from pH-specific reservoir II.

[0069] [Step 4: Semi-preparative reverse-phase C18 chromatography] A total of 200 mg (equivalent to 1087 liters of blood filtrate) of fractions 6-8 obtained from pH pool II was separated using a semi-preparative reverse-phase column. Fractions 33 and 34 contained ALB408-423. • Schematic conditions: Column: 4.7cm x 30cm steel column Filler: Bakerbond RP-C18, 15-30 μm, 300 Å Buffer A: 100% water, 10mM HCl Buffer B: 80% acetonitrile, 20% water, 10 mM HCl Gradient: 0-30% of B in 2000ml Flow rate: 40 ml / min (Pressure: 40 bar) Detection: 214nm and 280nm • Schema Equipment: BioCad 250, Perseptive (manufactured by Biosystems) Fractions: 50 ml each, starting from the gradient start (minimum 10.75).

[0070] [Step 5: Semi-preparative reverse-phase C18 chromatography] Fractions 33 and 34 obtained in the aforementioned chromatography process were separated using different mobile phases with a similar semi-preparative reverse-phase column. Fractions 5 and 6 contained ALB408-423. • Schematic conditions: Column: 4.7cm x 30cm Steel Column Filler: Bakerbond RP-C18, 15-30 μm, 300 Å Buffer A: 30% methanol, 70% water, 10 mM HCl Buffer B: 100% methanol, 10 mM HCl Gradient: 0-15% B (40 mL), 15-60% B (1900 mL) Flow rate: 40 mL / min (Pressure: 30 bar) Detection: 214nm and 280nm • Schema Equipment: BioCad 250, Perseptive (manufactured by Biosystems) Fractions: 50 mL each, starting from the gradient start (minimum 9.75).

[0071] [Step 6: Analytical Reverse-Phase C4 Chromatography] When fractions 5 and 6 obtained by the above chromatography were separated using an analytical reverse-phase column, fractions 51-57 contained ALB408-423. • Schematic conditions: Column: 2cm x 25cm Steel Column Packing material: RP-C4, 5 μm, 100 Å, manufactured by Biotek Silica. (Ostringen, Austria) Buffer A: Water, 0.1% TFA Buffer B: 80% acetonitrile, 20% water, 0.1% TFA Gradient: 0-5% B in 2 minutes, 5-35% B in 60 minutes. 35-100% B in 3 minutes Flow rate: 7ml / min Detection: 214nm and 280nm • Schema Device: Kontron Fractions: Every minute from minute 1

[0072] [Step 7: Analytical reversed-phase C18 chromatography] Fractions 51–57 obtained from the previous chromatography were separated using an analytical reverse-phase column. Fraction 31 contained ALBs 408–423. • Schematic conditions: Column: 1cm x 25cm Steel Column Filling material: RP-C18, 5μm, 300Å, manufactured by Vydac (Hesperior, USA) Buffer A: water, 0.1% TFA Buffer B: 80% acetonitrile, 20% water, 0.1% TFA Gradient: 0-15% B in 5 minutes, 15-45% B in 60 minutes, 45-100% B in 1 minute Flow rate: 2ml / min Detection: 214nm and 280nm • Schema Device: Kontron Fraction: Every minute from minute 1 ALB408-423 was included in fraction 31.

[0073] (Example 2: Chemical synthesis of ALB408-423) The chemical synthesis of ALB408-423 was carried out using a conventional solid-phase synthesis method with a peptide synthesizer 9050 (Applied Biosystems) employing the known Fmoc method. The obtained peptide was purified by reverse-phase chromatography, and its identification and purity were confirmed by analytical RP-HPLC and MALDI-MS mass spectrometry (Example 3).

[0074] (Example 3) [Mass measurement] The peptides obtained from fraction 31 of step 7 of Example 1, isolated from blood filtrate, and chemically synthesized peptides (Examples 2 and 4) were measured by mass analysis using a MALDI mass spectrometer (Voyager DE-Pro). The molecular weight of the peptides was determined by corresponding them to the following mass numbers (MW). ALB408-423 (isolated from human blood filtrate): 1830.9Da ALB408-423 (chemically synthesized peptide): 1830.6 Da [Arrangement determination] The purified natural peptides were analyzed using MS-MS coupling analysis (ESI-TRAP) provided by PROTEOMEFACTORY AG (Dorotheenstr. 94, 10117, Berlin, Germany). Using the Mascot search engine, the results compared existing ESI MS-MS masses with a database, and the following sequence was identified with the highest probability. LVRYTKKVPQVSTPTL We made that decision. [Database Comparison] Further database comparison with the SwissProt database revealed that this peptide sequence exhibits 100% homology to amino acids 408-423 of human serum albumin (accession number NP000468), and that this sequence contains the amino acids LVRYTKKVPQVSTPTL.

[0075] (Example 4: Synthesis of a bioactive peptide of ALB408-423) Various ALB408-423 derivatives, including deletions of the N-terminus or C-terminus, or amino acid substitutions, were chemically synthesized (Tables 1-1 and 1-2). These peptides were obtained by multiplex synthesis and possess the biological activity of ALB408-423.

[0076] [Table 1-1]

[0077] [Table 1-2]

[0078] (Example 5: Use of IVRYTKKVPQVS (sequence identification number 21) in the treatment of sarcoma) A 21-year-old male patient with osteosarcoma was treated under the conditions of a clinical trial conducted on humanitarian grounds. Peptide IVRYTKKVPQVS (sequence identification number 21) was dissolved in citrate / bicarbonate buffer (European Pharmacopoeia, Ph.Eur.) and supplied at a concentration of 1 mg / mL (1 mg of peptide per 1 mL of buffer). This composition was administered intranasally as a nasal spray. The nasal spray was administered three times a day (morning, noon, and evening) with two strokes into each nostril for 6 weeks. The patient's medical condition and clinical observations were monitored weekly using an immunotherapy panel. Blood cell counts, including red blood cell count and platelet count, were measured. Four weeks after the start of treatment, blood tests showed normalization. Before treatment, the patient suffered from severe pain and was unable to move, but during the course of treatment, the patient recovered to the point where they could move, and the pain disappeared six weeks after the start of treatment. MRT measurements showed tumor regression, particularly in the spine and intervertebral foramen regions. No side effects were observed.

Claims

1. (i) The following amino acid sequence Z １ -LVRYTKKVPQVSPTL-Z ２ (ALB-408) Peptides having (where Z represents 0 to 10 amino acid residues), (ii) A peptide with biological activity obtained by multiplex synthesis, ALB408-423 and (iii) Biologically active fragments and / or variants and / or derivatives of (i) and (ii) above, in particular PEGylated, HES-lated, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives A peptide for use in the treatment and / or prevention of sarcoma, characterized by being a peptide selected from the group consisting of the following.

2. The peptide ALB408-423, obtained by the above multiplex synthesis, has biological activity. A peptide selected from the group consisting of sequence identification numbers 1 to 31, and / or The following general formula X １ X ２ RX ４ X ５ X ６ KX ８ PX １０ X １１ S (However, X １ = L, or I, X ２ = V, M, or L, X ４ = Y, or W, X ５ = T, or S, X ６ = K, C, R, or Q, X ８ = V, M, L, F, or A, X １０ = Q, or C, X １１ Peptides having an amino acid sequence represented by a single letter (V, M, or F) The peptide for use according to claim 1, characterized in that it is the same as described in claim 1.

3. The peptide having biological activity obtained by the above multiplex synthesis is the peptide selected from the group consisting of sequence identification numbers 1 to 31, and / or the following general formula X １ VRX ４ X ５ X ６ KVPX １０ VS (However, X １ = L, or I, X ４ = Y, or W, X ５ = T, or S, X ６ = K, or C, X １０ = Q, or C) The peptide having an amino acid sequence represented by a single letter is a peptide A peptide for use according to claim 1 or 2, characterized in that it is the same as the peptide described in claim 1 or 2.

4. The aforementioned peptide has the following amino acid sequence IVRYTKKVPQVS (Sequence Identification Number 21) The peptide for use according to any one of claims 1 to 3, characterized in that it is a peptide selected from a peptide having and derivatives thereof that are amidated, acetylated, sulfated, phosphorylated, and / or glycosylated.

5. The aforementioned peptide has the following amino acid sequence IVRYTKKVPQVS (Sequence Identification Number 21) A peptide for use according to any one of claims 1 to 4, characterized in that it is a peptide having [a certain characteristic].

6. The peptide for use according to any one of claims 1 to 5, characterized in that the sarcoma is selected from the group consisting of osteosarcoma, Ewing's sarcoma, chondrosarcoma, fibrosarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, and angiosarcoma.

7. The peptide for use according to any one of claims 1 to 6, characterized in that the sarcoma is osteosarcoma, preferably osteoblastic osteosarcoma, chondroblastic osteosarcoma, fibroblastic osteosarcoma, telangiectatic osteosarcoma, small cell osteosarcoma, low-grade central osteosarcoma, periosteal osteosarcoma, paraosteal osteosarcoma, secondary osteosarcoma, high-grade superficial osteosarcoma, or extraosseous osteosarcoma, and more preferably osteoblastic osteosarcoma, chondroblastic osteosarcoma, or fibroblastic osteosarcoma.

8. The peptide for use according to any one of claims 1 to 7, characterized in that the peptide is administered via parenteral, intravenous, intramuscular, intrathecal, transpulmonary, intranasal, ophthalmic, auricle, topical, subcutaneous, or sublingual route, preferably via intranasal, ophthalmic, or auricle route, and more preferably via intranasal route.

9. The peptide for use according to any one of claims 1 to 8, characterized in that the peptide is administered three times a day via an intranasal route.

10. (a) The following amino acid sequence Z １ -LVRYTKKVPQVSPTL-Z ２ (ALB-408) (However, Z represents 0 to 10 amino acid residues.) A peptide having, A peptide having the biological activity of ALB408-423 obtained by multiple synthesis, or a biologically active fragment and / or variant and / or derivative thereof, particularly PEGylated, HES-modified, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives, comprises or consists of: (b) containing a pharmaceutically acceptable carrier A pharmaceutical composition for use in the treatment and / or prevention of sarcoma.

11. The aforementioned peptide has the following amino acid sequence IVRYTKKVPQVS (Sequence Identification Number 21) A pharmaceutical composition for use according to claim 10, characterized in that it is a peptide having

12. The pharmaceutical composition according to claim 10 or 11, characterized in that the concentration of the peptide is 0.5 mg / mL to 2.0 mg / mL, preferably 1.0 mg / mL.

13. A pharmaceutical composition according to any one of claims 10 to 12, characterized in that it is suitable for parenteral, intravenous, intramuscular, intrathecal, transpulmonary, intranasal, ophthalmic, auricular, topical, subcutaneous, or sublingual administration, preferably suitable for intranasal, ophthalmic, or auricular administration, and more preferably suitable for intranasal administration.

14. The pharmaceutical composition according to any one of claims 10 to 13, characterized in that the pharmaceutically acceptable carrier is a sodium chloride solution or a citrate / bicarbonate buffer.

15. A method of treating sarcoma in patients who require treatment, The process includes administering an effective amount of peptide to the patient. The peptides are grouped as follows: (i) The following amino acid sequence Z １ -LVRYTKKVPQVSPTL-Z ２ (ALB-408) (However, Z represents 0 to 10 amino acid residues.) Peptides having, (ii) Peptides with biological activity obtained by multiplex synthesis, ALB408-423 and (iii) Biologically active fragments and / or variants and / or derivatives of (i) and (ii), in particular PEGylated, HES-lated, amidated, acetylated, sulfated, phosphorylated, and / or glycosylated derivatives A method for treating sarcoma, characterized by being selected from among the following.

Images