Compositions comprising ras-inhibitory peptides

Abstract

To improve the medicinal effect of a Ras-inhibiting peptide when administered to a living body by improving the solubility of the peptide in an aqueous solution.SOLUTION: A composition comprising a cyclic peptide consisting of a specific amino acid sequence described in the specification, a derivative or modified form thereof, or a pharmacologically acceptable salt thereof, and at least one surfactant.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a composition containing a Ras inhibitory peptide, and in particular to a composition that can suppress aggregation of a Ras inhibitory peptide by using a surfactant. [Background technology]

[0002] Ras proteins (the Ras subfamily, hereafter abbreviated as Ras) are intracellular GTPases that bind to GDP or GTP, acting as molecular switches that control the on / off (inactivation) and on / off (activation) of cell proliferation signaling. Ras consists of a sequence of 188–189 amino acids. Any amino acid mutation significantly inhibits its own GTPase activity and the ability of GTPase-activating proteins (GAPs) to hydrolyze GTP, resulting in a bias toward the GTP-bound form. This results in the prolongation of cell proliferation signals. Approximately 30% of human tumors express mutant Ras with amino acid mutations, which act as drivers of tumor growth. Among the Ras family, K-Ras, N-Ras, and H-Ras are attracting attention as potential drug targets. Among these, K-Ras amino acid mutations are known to occur most frequently in approximately 20% of human tumors.

[0003] The present inventors have discovered several cyclic peptides that exhibit Ras protein inhibitory activity and are stable in plasma, and have reported their inhibitory effect on the proliferation of Ras-expressing cells (see, for example, Patent Document 1 and Non-Patent Document 1). These documents demonstrate that KS-58, a bicyclic peptide with an unnatural amino acid structure, inhibits the in vitro proliferation of the human lung cancer cell line A427 and the human pancreatic cancer cell line PANC-1, which express K-Ras(G12D). Furthermore, KS-58 has been shown to have anticancer activity against mouse tumors derived from the colon cancer cell line CT26, which stably expresses K-Ras(G12D) (see Non-Patent Document 2). However, because KS-58 is a highly hydrophobic compound, it was dissolved in dimethyl sulfoxide (DMSO) and then diluted 10-fold with saline before use in the animal experiments. Generally, when highly hydrophobic peptides or antibody proteins are dissolved in aqueous solutions at high concentrations to confirm their therapeutic effects in vivo, aggregation and / or cloudiness occur, making their development into injectable formulations difficult.

[0004] As an example of a method for suppressing protein aggregation, adding a surfactant to a solution containing an antibody has been proposed (see, for example, Patent Document 2). In the method described in Patent Document 2, adding a surfactant during ultrafiltration suppresses the formation of aggregates and cloudiness, and is effective for antibody-containing diagnostic agents and therapeutic agents, particularly injectables. It is also important to suppress aggregation in aqueous solutions containing Ras inhibitors, but no composition capable of effectively preventing the formation of aggregates has been known to date. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kotaro Sakamoto, Teruaki Masutani and Takatsugu Hirokawa, Generation of KS-58 as the first K-Ras(G12D)-inhibitory peptide presenting anti-cancer activity in vivo. Scientific Reports volume 10, Article number: 21671 (2020) [Non-patent document 2] Kotaro Sakamoto,et al.,The K-Ras(G12D)-inhibitory peptide KS-58 suppresses growth of murine CT26 colorectal cancer cell-derived tumors.Scientific Reports volume12,Article number:8121(2022) [Patent documents]

[0006] [Patent Document 1] WO2020 / 230780 [Patent Document 2] WO2002 / 013859 Summary of the Invention [Problem to be solved by the invention]

[0007] The problem to be solved by the present invention is to improve the solubility of Ras inhibitory peptides in aqueous solutions, thereby improving the efficacy of the peptides when administered to a living body. [Means for solving the problem]

[0008] The present invention has been made to solve the above-mentioned problems, and relates to the unexpected and surprising result that the solubility of a Ras inhibitory peptide having a specific cyclic structure and amino acid sequence is improved by mixing the Ras inhibitory peptide with a specific surfactant. That is, the present invention includes the following embodiments.

[0009] [1] Formula (1): c[X 1 -Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-X 10 -X 11 )] (1) (In the formula, X 1 represents a β-alanine or γ-aminobutyric acid residue, and X 3 represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid; X 5 represents an isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue; X 10 represents valine, phenylalanine, tryptophan, 1-naphthylalanine or an N-methylated amino acid residue thereof, and X 11 represents a D- or L-cysteine ​​residue, and X 1 and X 11 forms an amide bond between the amino group and the carboxyl group of the main chain, and X 4 and X 11 form a covalent bond between the -SH groups in their side chains via a linker of a methylene group, an ethylene group, a propylene group, or a butylene group, thereby causing the peptide of formula (1) to have two cyclic structures in the molecule. A composition comprising a cyclic peptide consisting of an amino acid sequence represented by the following formula: [2] Formula (2): c[β-Ala-Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-Trp- D Cys)] (2) (In the formula, X 3represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid; X 5 represents isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue, and the residues at positions 1 and 11 form an amide bond between the amino group and carboxyl group of the main chain, and the two cysteine ​​residues at positions 4 and 11 form a covalent bond between their respective -SH groups in the side chain via a propylene group linker, thereby causing the peptide of formula (2) to have two cyclic structures within the molecule. A composition comprising a cyclic peptide consisting of an amino acid sequence represented by the following formula (1), a derivative or modified form thereof, or a pharmacologically acceptable salt thereof, and at least one surfactant. [3] The composition according to (1) or (2), which contains at least 10% by mass of a surfactant. [4] The composition according to any one of [1] to [3], wherein the surfactant is selected from the group consisting of polyoxyethylene castor oil and polyoxyethylene sorbitan fatty acid esters. [5] The composition according to any one of [1] to [4], comprising 25 to 45% by volume of 10x concentrated Dulbecco's PBS solution. [6] The composition according to any one of [1] to [5], which contains dimethyl sulfoxide. [7] The composition described in [4], wherein the surfactant is polyoxyethylene-35-ricinoleate or polysorbate 80. [Effects of the Invention]

[0010] According to the present invention, by improving the solubility in aqueous solution of a Ras inhibitory peptide having a specific cyclic structure and amino acid sequence, it is possible to improve the efficacy when administered to a living body. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the chemical structures of representative Ras inhibitory peptides and representative surfactants of the present invention. [Figure 2] FIG. 2 shows the results of evaluating the solubility of a representative Ras inhibitory peptide of the present invention mixed with a representative surfactant. [Figure 3] FIG. 3 shows the results of evaluating the anticancer activity of a mixture of a representative Ras inhibitory peptide of the present invention and a representative surfactant, which was administered intravenously to the tail vein of CT26 subcutaneous tumor-bearing mice. [Figure 4] FIG. 4 shows the results of evaluating the anti-cancer activity of a mixture of a representative Ras inhibitory peptide of the present invention and a representative surfactant, which was administered intravenously into the tail vein of PANC-1 orthotopically transplanted mice. [Figure 5] FIG. 5 shows the results of observation with a transmission electron microscope of the particle size of particles formed in a solution obtained by mixing a representative Ras inhibitory peptide of the present invention with a representative surfactant. [Figure 6] FIG. 6 shows the results of measuring the particle size of particles formed in a solution obtained by mixing a representative Ras inhibitory peptide of the present invention with a representative surfactant, using dynamic light scattering. DETAILED DESCRIPTION OF THE INVENTION

[0012] Next, various embodiments of the present invention will be described with reference to the drawings. Note that the various embodiments described below do not limit the scope of the invention as claimed, and not all of the elements and combinations thereof described in the various embodiments are necessarily essential to the solution of the present invention.

[0013] (definition) As used herein, a peptide refers to two or more amino acids bonded together via amide bonds (peptide bonds), and can be, for example, 2 to 20 amino acids bonded together via amide bonds. In addition, according to the convention for peptide notation, the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxy terminus). The first carbon atom adjacent to the carbonyl group that forms the peptide bond is referred to as the Cα carbon.

[0014] As used herein, the term "amino acid or a derivative thereof" is used in its broadest sense and includes not only naturally occurring amino acids, but also artificial amino acids with unnatural structures, chemically synthesized compounds having properties known in the art that are characteristic of amino acids, and carboxylic acids having functional groups. Examples of unnatural amino acids include D-amino acids, α / α-disubstituted amino acids with a backbone structure that differs from that of natural amino acids (such as α-methylated amino acids such as 2-aminoisobutyric acid), N-alkyl-amino acids (such as N-methylated amino acids), N-substituted glycines (peptoids), amino acids with extended backbones (such as homo-β and homo-γ amino acids), amino acids with a side chain structure that differs from that of natural amino acids (such as cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, and 3-(1H-benzimidazol-2-yl)-alanine), amino acids with partially substituted side chains (such as norleucine, diaminopropanoic acid, and 3-(2-pyridyl)-alanine), amino acids with extra functional groups in the side chain; amino acids with extra C, alkyl groups, or methyl groups in the side chain (such as homonorleucine and γ-methylleucine), amino acids with halogen atoms (F, Cl, Br, I) in the side chain (such as 3-chloro-alanine), and amino acids with halogen atoms (F, Examples of amino acids include, but are not limited to, carboxylic acids having an extra N or amino group in the side chain (such as 3-azidoalanine and ornithine), amino acids having an extra O or methoxy group in the side chain (such as O-methyl-serine and O-methyl-threonine), amino acids having an extra hydroxy group in the side chain (such as 3-hydroxy-phenylalanine), amino acids having an extra carboxy group (-COOH) in the side chain (such as 3-carboxy-phenylalanine), amino acids having an extra S in the side chain (such as ethionine), amino acids in which the carboxylic acid functional group in the side chain is protected with an ester (such as aspartic acid-4-methyl ester), and amino acids in which the thio group (-S-) in the side chain has been oxidized to a sulfinyl group (-S(=O)-) or a sulfonyl group (-S(=O)2-) (methionine sulfoxide).

[0015] As used herein, "Ras" refers to wild-type proteins and amino acid mutant proteins of the Ras subfamily, such as K-Ras, N-Ras, and H-Ras, in mammals such as mice, rats, dogs, monkeys, and humans, and includes both GDP-bound and GTP-bound forms.

[0016] As used herein, the term "Ras inhibitory peptide" refers to a peptide that, in an in vitro test, (1) inhibits the binding of existing Ras inhibitory peptides to Ras protein, (2) binds to Ras protein in a concentration-dependent manner, (3) inhibits Erk phosphorylation in Ras-expressing cells, or (4) suppresses the proliferation of Ras-expressing cells. A peptide that exhibits any one of these effects is called a "Ras inhibitory peptide." The presence or absence of Ras inhibitory activity can be confirmed by those skilled in the art using known methods.

[0017] (Ras inhibitor peptide) Cyclic peptides that are active ingredients for inhibiting Ras are disclosed in Patent Document 1 and Non-Patent Documents 1 and 2, the contents of which are incorporated herein by reference in their entirety. The peptides disclosed in Patent Document 1 are stabilized by bicyclization while maintaining or enhancing the characteristics (pharmacophore) associated with Ras binding activity. Any of the peptides disclosed in these documents can be used to produce the composition of the present invention.

[0018] [1] In one embodiment, the Ras inhibitory peptide has the following formula (1): c[X 1 -Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-X 10 -X 11 )] (1). In formula (1), X 1 represents a β-alanine or γ-aminobutyric acid residue, and X 3represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid; X 5 represents an isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue; X 10 represents valine, phenylalanine, tryptophan, 1-naphthylalanine or an N-methylated amino acid residue thereof, and X 11 represents a D- or L-cysteine ​​residue. 1 and X 11 forms an amide bond between the amino group and the carboxyl group of the main chain, and X 4 and X 11 form a covalent bond between the -SH groups of each side chain via a linker of a methylene group, an ethylene group, a propylene group, or a butylene group, so that the peptide of formula (1) has two cyclic structures within the molecule.

[0019] [2] A more preferred embodiment of the Ras inhibitory peptide has the following formula (2): c[βAla-Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-Trp- D Cys)] (2). In formula (2), X 3 represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid; X 5represents an isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue. The residues at positions 1 and 11 form an amide bond between the amino group and carboxyl group of the main chain, and the two cysteine ​​residues at positions 4 and 11 form a covalent bond between their respective -SH groups in the side chain via a propylene group linker, so that the peptide of formula (2) has two cyclic structures within the molecule.

[0020] Examples of the individual Ras inhibitory peptides included in the above formula (1) or (2) include the following. c[βAla-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val-Cys)] (SEQ ID NOs: 1 to 3) c[βAla-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NOs: 4 to 6) c[γAba-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val-Cys)] (SEQ ID NOs: 7 to 9) c[γAba-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NOs: 10 to 12) c[βAla-Pro-Nle-c(Cys-Phg-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NO: 13) c[βAla-Pro-Nle-c(Cys-Aoc-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NO: 14) c[βAla-Pro-Nle-c(Cys-Anon-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NO: 15) c[βAla-Pro-Nle-c(Cys-Anon-Ser-4fF-Asp-Pro-Phe- D Cys)] (SEQ ID NO: 16) c[βAla-Pro-Nle-c(Cys-Anon-Ser-4fF-Asp-Pro-Trp- D Cys)] (SEQ ID NO: 17) c[βAla-Pro-Nle-c(Cys-Anon-Ser-4fF-Asp-Pro-1NaphA- D Cys)] (SEQ ID NO: 18) c[βAla-Pro-Anon-c(Cys-Anon-Ser-4fF-Asp-Pro-Trp- D Cys)] (SEQ ID NO: 19) c[βAla-Pro-Nle-c(Cys-Aoc-Ser-4fF-Asp-Pro- m Val- D Cys)] (SEQ ID NOs: 20-21)

[0021] The Ras inhibitory peptide of this embodiment is cyclized in one aspect. As used herein, cyclization refers to the covalent bonding of two or more amino acids separated by one or more amino acids within a peptide molecule, either directly or indirectly via a linker, to form one or more ring structures within the molecule. Examples of cyclization include, but are not limited to, an amide bond between an amino group and a carboxyl group, a disulfide bond between thiol groups, or a thioether bond between a linker containing a halogen group and two thiol groups. The direct or indirect covalent bond via a linker for cyclization may be between main chains, between main chains and side chains, between side chains and main chains, or between side chains and side chains.

[0022] The Ras inhibitory peptides of this embodiment include peptides that have homology to the amino acid sequences represented by [1] and [2] above, in which one to several amino acids have been deleted, added, and / or substituted, but still have Ras-binding activity. As used herein, when referring to a "peptide in which one to several amino acids have been deleted, added, and / or substituted," the number of amino acids is not particularly limited as long as the peptide has Ras-binding activity, but is preferably one to five, and more preferably one or two. The deletions, additions, and / or substitutions may be at the terminus or in the middle of the peptide, and may occur at one or more positions.

[0023] Such amino acid sequences in which one to several amino acids have been deleted, added, and / or substituted in the above amino acid sequences include those that have an identity of at least 50% or more, preferably 70% or more, more preferably 80% or more, and particularly preferably 90% or more with the above amino acid sequence when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, using default, i.e., initial setting parameters).

[0024] The Ras inhibitory peptide according to this embodiment also encompasses various derivatives and / or modifications thereof. Such derivatives include those in which the saturated fatty chain of the peptide is replaced with an unsaturated fatty chain, those in which some of the atoms of the peptide are replaced with other atoms including radioactive or non-radioactive isotope atoms, those in which the amide bond of the peptide is replaced with a thioamide bond (-NH-C(=S)-), those in which the amide bond of the peptide is replaced with an alkene (-C=C-), those in which the amide bond of the peptide is replaced with an alkyl (-CC-), those in which the amide bond of the peptide is replaced with a hydroxyethylene (-C(-OH)-C-), those in which the amide bond of the peptide is replaced with an ester (-OC(=O)-), those in which the amide bond of the peptide is replaced with an alkene (-C=C-), those in which the amide bond of the peptide is replaced with (-C-NH-), or those in which the amide bond of the peptide is replaced with (-C(=O)-C-).Such modified forms include those in which the α-carbon of the peptide is disubstituted, and those in which the amide bond of the peptide is replaced with an N-alkene. Examples of such peptides include, but are not limited to, peptides in which some of the peptide's functional groups have been modified by halogenation, cyanation, nitration, oxo-, hydroxylation, amination, deamination, dehydrogenation, amidation, acetylation, methoxylation, prenylation, alkylation, or the like (for example, peptides in which some of the amino groups of the peptide have been acetylated, alkylated, or deaminated, or peptides in which some of the carboxyl groups of the peptide have been amide or esterified), peptides in which S in the peptide has been changed to sulfoxide S(=O) or sulfone S(=O)2, peptides in which the peptide has been polymerized via a chemical linker, peptides in which the peptide has been labeled with biotin, peptides in which the peptide has been fluorescently labeled, peptides in which the peptide has been luminescently labeled, and peptides in which the peptide has been fused with alkyl chains, polyethylene glycol, antibodies, lectins, sugar chains, enzymes, membrane-permeable peptides, low-molecular-weight compounds, or molecules that induce protein ubiquitination.

[0025] The Ras inhibitory peptide of this embodiment also encompasses peptide salts, including salts with physiologically acceptable bases or acids, such as addition salts with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid), addition salts with organic acids (e.g., p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acids, succinic acid, citric acid, benzoic acid, acetic acid), addition salts with inorganic bases (e.g., ammonium hydroxide, alkali or alkaline earth metal hydroxides, carbonates, bicarbonates), and addition salts with amino acids.

[0026] The Ras inhibitory peptide of this embodiment may be a prodrug. A prodrug is a compound that is converted into a peptide of the present invention by a reaction with an enzyme, gastric acid, or the like under physiological conditions in a living body, i.e., a compound that is converted into a peptide of the present invention by enzymatic oxidation, reduction, hydrolysis, or the like, or a compound that is converted into a peptide of the present invention by hydrolysis with gastric acid or the like.

[0027] The Ras inhibitory peptide of this embodiment may be in the form of a crystal, and the peptide of the present invention includes both a single crystal form and a mixture of crystal forms. The crystal can be produced by crystallization using a known crystallization method.

[0028] (surfactant) Surfactants are generally substances that have hydrophilic and hydrophobic groups (lipophilic groups) within their molecules, and are not particularly limited as long as they form micelles or vesicles at a certain concentration or above, have the effect of weakening surface tension, and have the effect of suppressing aggregation of the Ras inhibitory peptide of the present invention.

[0029] The surfactants used in the compositions of the present invention include any surfactant or surfactant combination that stabilizes the Ras inhibitory peptides described herein and inhibits aggregation.Accordingly, surfactants of the present invention include, but are not limited to, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid esters, Triton® X-100, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), soybean lecithin, poloxamer, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecylammonium bromide, methoxyhexadecylglycerol, Pluronic® polyols, polyamines (e.g., pyran, dextran sulfate, poly IC, carbopol), oil emulsions, and mineral gels (e.g., aluminum phosphate).

[0030] Preferred surfactants are polyoxyethylene castor oil or polyoxyethylene sorbitan fatty acid esters. Polyoxyethylene castor oils include: a) Polyoxyethylene castor oil derivatives, including those sold under the trade name "CREMOPHOR" (BASFTWEEN), in particular CREMOPHOR EL or ELP (also called PEG 35 castor oil, polyethoxylated castor oil, macrogoglycerol ricinoleate, macrogoglycerol hydroxystearic acid, POE-35 castor oil, PEG ricinoleate); CREMOPHOR ELP (a polyoxyethylene glycolated castor oil with a low content of water, potassium and free fatty acids, which is a refined grade of CREMOPHOR EL), and CREMOPHOR RH 40 (also called polyoxyl 40 hydrogenated castor oil, macrogolglycerol hydroxystearic acid, polyoxyethylene 40 hydrogenated castor oil, PEG-40 hydrogenated castor oil), and

[0031] b) Polyoxyethylene sorbitan fatty acid esters, including those sold under the trade name "TWEEN®" (ICI Americas), in particular TWEEN 80 (80 [polyoxyethylene (20) sorbitan monooleate], also known as polysorbate 80); polyoxyethylene 20 sorbitan monooleate (partial fatty acid esters of sorbitol and its anhydrides polymerized with ethylene oxide), more particularly polyoxyethylene-sorbitan-fatty acid mono-oleyl esters (comprising a mixture of fatty acids, including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linolenic acid, primarily oleic acid), TWEEN 85 (85 [polyoxyethylene (20) sorbitan trioleate], also known as polysorbate 85); polyoxyethylene 20 sorbitan trioleate (partial fatty acid esters of sorbitol and its anhydrides polymerized with ethylene oxide), more particularly polyoxyethylene-sorbitan-fatty acid mono-oleyl esters (comprising a mixture of fatty acids, including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linoleic acid, primarily oleic acid); partial fatty acid esters of anhydrides), specifically polyoxyethylene-sorbitan-fatty acid tri-oleyl esters (comprising a mixture of fatty acids including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, and linolenic acid, primarily oleic acid), TWEEN 20 (also known as polysorbate 20, poly(oxy-1,2-ethanediyl) derivatives); polyoxyethylene 20 laurate; polyoxyethylene 20 sorbitan monolaurate, sorbitan monododecanoate; TWEEN 60 (polysorbate 60, polyoxyethylene 20 stearate (also known as sorbitan monooctadecanoate poly(oxy-1,2-ethanediyl) derivatives), and TWEEN 40 (polysorbate 40, polyoxyethylene 20 sorbitan monopalmitate, sorbitan monohexadecanoate).

[0032] In some embodiments, the surfactant is selected from polyoxyethylene castor oil derivatives (particularly CREMOPHOR EL (polyoxyethylene-35-ricinoleate, also known as PEG-35 castor oil, polyoxyl-35 castor oil, polyoxyl-35 hydrogenated castor oil, macrogolglycerol ricinoleate)), polyoxyethylene sorbitan fatty acid esters (particularly TWEEN 80), and mixtures thereof.

[0033] In a preferred embodiment, the surfactant comprises at least about 10% by mass, preferably about 10 to 20% (e.g., about 15%) by mass of CREMOPHOR EL and / or TWEEN 80, based on the total mass of the composition of the present invention. The structural formulae of the Ras inhibitory peptide and two surfactants that make up the composition in this embodiment are shown in Figure 1.

[0034] (composition) The composition of the present invention contains the above-described Ras inhibitory peptide and a surfactant and is capable of suppressing the growth of malignant tumors and other diseases. The administration route is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injections such as intramuscular injection, intravenous injection, and subcutaneous injection, as well as transdermal and transmucosal administration (nasal, oral, ocular, pulmonary, vaginal, and rectal administration). The peptides in the composition can be modified in various ways to accommodate their susceptibility to metabolism and excretion. For example, adding alkyl chains, polyethylene glycol, or sugar chains to the peptides can increase their blood residence time and reduce antigenicity. Furthermore, biodegradable polymers such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, and the like can be used as sustained-release vehicles, and the peptides can be encapsulated in these. When administered transdermally, a weak electric current can be passed through the skin surface to penetrate the stratum corneum (iontophoresis method).

[0035] The above-mentioned compositions may be formulated by adding pharmaceutically acceptable carriers, excipients, additives, etc. in addition to the surfactant. Examples of dosage forms include liquids (e.g., injections), dispersions, suspensions, tablets, pills, powders, suppositories, powders, fine granules, granules, capsules, syrups, lozenges, inhalants, ointments, eye drops, nasal drops, ear drops, and poultices. These formulations may also be controlled-release formulations such as immediate-release formulations or sustained-release formulations (e.g., sustained-release microcapsules). Formulation can be carried out by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption enhancers, pH adjusters, preservatives, antioxidants, etc., as appropriate. Examples of ingredients used in formulations include, but are not limited to, purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharmaceutically acceptable organic solvents, animal and vegetable oils, lactose, mannitol, glucose, sorbitol, crystalline cellulose, hydroxypropyl cellulose, starch, corn starch, anhydrous silicic acid, magnesium aluminum silicate, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium carboxymethylcellulose, sodium polyacrylate, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, methylcellulose, ethylcellulose, xanthan gum, gum arabic, tragacanth, casein, agar, polyethylene glycol, diglycerin, glycerin, propylene glycol, petrolatum, paraffin, octyldodecyl myristate, isopropyl myristate, higher alcohols, stearyl alcohol, stearic acid, and human serum albumin. When a peptide is poorly absorbed transmucosally, absorption enhancers that improve the absorption of poorly absorbed drugs may be used, including surfactants such as polyoxyethylene lauryl ethers, sodium lauryl sulfate, and saponin; bile salts such as glycocholic acid, deoxycholic acid, and taurocholic acid; chelating agents such as EDTA and salicylic acids; fatty acids such as caproic acid, capric acid, lauric acid, oleic acid, linoleic acid, and mixed micelles; enamine derivatives, N-acyl collagen peptides, N-acyl amino acids, cyclodextrins, chitosans, and nitric oxide donors.

[0036] The compositions of the present invention can be liquid or solid. Liquid formulations can be aqueous solutions or suspensions prepared in a suitable aqueous solvent, such as water or an aqueous / organic mixture, such as a water-alcohol mixture. Liquid formulations can have a pH between about 5.5 and about 7.5, between about 6.0 and about 7.0, or between about 6.0 and about 6.5, for example, about 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. Liquid formulations can be stored at room temperature, refrigerated (e.g., 2-8°C), or frozen (e.g., -20°C or -80°C). Solid formulations can be prepared by any suitable method, for example, in the form of a cake or powder, by the addition of a cryoprotectant. Solid formulations can be dissolved, i.e., reconstituted with a suitable vehicle, to form a liquid suitable for administration. Suitable solvents for reconstituting solid formulations include water, isotonic saline, buffered solutions such as phosphate-buffered saline, Ringer's (lactated or dextrose) solution, essential mineral media, alcoholic / aqueous solutions, dextrose solution, and the like.

[0037] In one embodiment, the composition contains 25-45% by volume of 10x concentrated Dulbecco's PBS solution (D-PBS), which typically contains 0.02% potassium chloride, 0.02% sodium dihydrogen phosphate, 0.8% sodium chloride, and 0.115% disodium hydrogen phosphate.

[0038] In one embodiment, the composition of the present invention contains dimethyl sulfoxide (DMSO). DMSO is known as a relatively non-toxic organic solvent for dissolving poorly soluble substances. In the preparation process of the composition of the present invention, the Ras inhibitory peptide may be first dissolved in DMSO and then diluted with an aqueous solution containing the surfactant. The concentration of DMSO in the composition of the present invention is preferably 50% or less, more preferably 20% or less.

[0039] (Action and effect) The compositions of the present invention allow Ras inhibitory peptides dissolved in organic solvents such as DMSO or ethanol to be easily dissolved without forming cloudiness or precipitation when diluted with aqueous solutions such as physiological saline, phosphate buffer, or glucose solution, thereby improving the efficacy of the compositions when administered to living organisms. The reason for this is not entirely clear, and without being bound by any theory, it is believed that the Ras inhibitor forms nanomicelles of approximately 5 to 100 nm in aqueous solution, preventing the active ingredient, Ras inhibitor, from becoming cloudy or precipitating. The nanomicelles preferably have a particle diameter of 5 to 50 nm, more preferably 5 to 25 nm. The particle diameter of such nanomicelles can be measured from photographs taken with a transmission electron microscope or a scanning electron microscope. Furthermore, in the particle size distribution calculated by number obtained by particle size measurement using laser diffraction, nanomicelles of approximately 5 to 100 nm account for at least 50%, preferably at least 70%, more preferably at least 80%, and even more preferably at least 90% of the total. Furthermore, by forming nanomicelles with such particle sizes, it is believed that when administered to the body, they will be more stable in the bloodstream and will not be filtered out by the kidneys.Furthermore, it is believed that nanomicelles with such particle sizes can easily enter target cells such as tumor cells.

[0040] The present invention will now be described in more detail with reference to the following examples, but the present invention is not limited to these examples. In the following examples, the unit % used to indicate the amount of each component added means % by mass. [Example]

[0041] Example 1: Preparation of an aqueous solution of synthetic peptide KS-58 The synthetic peptide KS-58 (cyclic peptide of SEQ ID NO: 17) was synthesized by Fmoc-based solid-phase peptide synthesis at Scrum Co., Ltd. and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The purity of the peptide was confirmed by analytical RP-HPLC, and the structure was assigned by MALDI-TOF mass spectrometry. Disulfide bond formation and thioether bond formation using halogenated chemical linkers were performed according to previously reported methods (Patent Document 1). Tween 80 (catalog number 9005-65-6) was purchased from Tokyo Kasei, and Cremophor EL (catalog number 09727-14) was purchased from Nakarai.

[0042] KS-58 was dissolved in DMSO to a concentration of 100 mg / mL and then diluted 10-fold with D-PBS (catalog number 045-29795, Fujifilm Wako Pure Chemical Industries, Ltd.) containing either no surfactant or 1-5% Cremophor EL. The final concentration of KS-58 in this solution was 10 mg / mL, and the final concentration of DMSO was 10%. The results are shown in Figure 2(A). A clear cloudy suspension was obtained with D-PBS containing no surfactant. A slightly cloudy solution was obtained with D-DBS containing 1% Cremophor EL, while a clear solution was obtained with D-DBS containing 3% or 5% Cremophor EL.

[0043] KS-58 was dissolved in DMSO to a concentration of 75 mg / mL and then diluted 5-fold with a D-PBS solution containing 10% Cremophor EL. The D-PBS solution was prepared by diluting 10x D-PBS (catalog number 048-29805, Fujifilm Wako Pure Chemical Industries) with purified water to a concentration of 70-10% 10x D-PBS. The final concentration of KS-58 was 15 mg / mL, and the final concentration of DMSO was 20%. The results are shown in Figure 2(B). The solubility of KS-58 varied depending on the concentration of 10x D-PBS in the solution. When the 10x D-PBS concentration was 40-20%, a clear solution was obtained without precipitation or cloudiness.

[0044] KS-58 was dissolved in DMSO to a concentration of 200 mg / mL and then diluted 5-fold with a D-PBS solution containing 10% Cremophor EL. The D-PBS solution was prepared by diluting 10x D-PBS with purified water to a concentration of 45-25% 10x D-PBS. The final concentration of KS-58 was 20 mg / mL, and the final concentration of DMSO was 20%. The results are shown in Figure 2(C). In both cases, a clear solution was obtained without any precipitation or cloudiness. These results demonstrate that a clear aqueous solution of KS-58 can be prepared by mixing an appropriate concentration of D-PBS with a surfactant such as Cremophor EL.

[0045] (Example 2) In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 CT26 mouse colon cancer cell line was implanted subcutaneously in BALB / cCrSlc mice (4 weeks old) to allow tumor formation. KS-58 was dissolved at 20 mg / mL in 20% DMSO / 10% Cremophor EL / 35% 10x D-PBS at 20 mg / mL, diluted with saline, and administered intravenously to tumor-bearing mice at 10 or 40 mg / kg once a week (days 1, 7, and 14) via the tail vein (n = 4). A control group was also administered a 20% DMSO / 10% Cremophor EL / 35% 10x D-PBS solution containing no KS-58, diluted with saline, and administered in the same manner. Tumor size was measured every 3 days for up to 17 days after administration of the three solutions. The results are shown in Figure 3. Compared with the control group, tumor growth was significantly suppressed in the groups administered KS-58 (10 mg / kg) and KS-58 (40 mg / kg) dissolved with Cremophor EL, with the suppression being particularly pronounced in the KS-58 (40 mg / kg) group. The tumor volume in the control group on day 17 was estimated to be 34% of the control group's tumor volume. It has been reported that when KS-58 was suspended in DMSO (10%) / physiological saline and administered subcutaneously at 40 mg / kg every other day, tumor growth from subcutaneously implanted CT26 colon cancer cell lines was 35% of the tumor weight on day 17 (Non-Patent Document 2). This suggests that dissolution using appropriate concentrations of 10x D-PBS and Cremophor EL improves the in vivo efficacy of KS-58, allowing for a reduction in the frequency of administration while maintaining anticancer activity.

[0046] (Example 3) In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 PANC-1 cells expressing K-Ras (G12D) (Catalog No. CRL-1469, ATCC) were cultured at 2.5 × 10 6KS-58 cells were transplanted into the subcapsular space of the pancreatic tail of BALB / cAJcl-nu / nu mice (male, 8 weeks old) (CLEA Japan, Inc.) and subjected to the study 1 week later. KS-58 was dissolved at 20 mg / mL in 20% DMSO / 10% Cremophor EL / 35% 10x D-PBS solution, diluted with saline, and administered intravenously to tumor-bearing mice at 10 or 40 mg / kg once a week (days 1, 7, 14, 21, 28, and 35) (n=8). A control group was also administered a 20% DMSO / 10% Cremophor EL / 35% 10x D-PBS solution containing no KS-58, diluted with saline, and administered in the same manner. The Sham group was a group in which the transplantation procedure was performed using cell culture medium containing no cancer cells, and a DMSO (20%) / Cremophor EL (10%) / 10xD-PBS (35%) solution containing no KS-58 was administered. Six weeks after the start of administration, the weights of the mouse organs (pancreas, liver, and kidneys) were measured, and the results are shown in Figure 4. Compared to the Sham group, the weight of the pancreas in the control group was significantly increased due to the growth of the PANC-1 tumor ( †††p<0.001, Student's t-test). Furthermore, tumor growth was significantly suppressed in the groups treated with KS-58 (20 mg / kg) and KS-58 (40 mg / kg) dissolved in Cremophor EL compared with the control group (*p<0.05, ***p<0.001, Dunnett's). There were no significant differences in liver and kidney weights between groups. The pancreas weights in the sham group and control group were estimated to be 68% and 50%, respectively, of the sham group and 100% of the control group. It has been reported that when KS-58 was suspended in DMSO (10%) / physiological saline and administered subcutaneously at 40 mg / kg every other day, the weight of the pancreas in mice orthotopically transplanted with PANC-1 was 66%, with the weight of the pancreas in the sham group being 0% and the weight of the pancreas in the control group being 100% (Non-Patent Document 1). In other words, dissolution using appropriate concentrations of 10x D-PBS and Cremophor EL improved the in vivo efficacy of KS-58, maintained or improved its anticancer activity, and further reduced the frequency of administration.

[0047] Example 4: Measurement of particle size of a composition containing synthetic peptide KS-58 The particle size of particles formed in a solution of KS-58 dissolved at 20 mg / mL in DMSO (20%) / Cremophor EL (10%) / 10xD-PBS (35%) was observed using a transmission electron microscope (FEI Tecnai F20 TEM). The results are shown in Figure 5. The formation of uniform particles of approximately 10 nm was confirmed. The particle size was also evaluated by dynamic light scattering using a Zetasizaer Nano ZSP (Malvern Instrument). The results are shown in Figure 6. The formation of uniform particles of approximately 10 nm was confirmed. [Industrial Applicability]

[0048] The compositions of the present invention are useful for producing Ras inhibitory peptides as pharmaceuticals or diagnostic agents by improving the solubility of Ras inhibitory peptides in aqueous solutions, or for conducting clinical research to develop Ras inhibitory peptides as pharmaceuticals.

Claims

1. The following formula (1): c[X １ -Pro-X ３ -c(Cys-X ５ -Ser-4fF-Asp-Pro-X １０ -X １１ )] (1) (In the formula, X １ represents a β-alanine or γ-aminobutyric acid residue, X ３ represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid, X ５ represents an isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid residue, X １０ represents valine, phenylalanine, tryptophan, 1-naphthylalanine, or an N-methylated amino acid residue thereof, X １１ represents a D- or L-cysteine ​​residue, X １ and X １１ forms an amide bond between the amino group and the carboxyl group of the main chain, and X ４ and X １１ form a covalent bond between the -SH groups in their side chains via a linker of a methylene group, an ethylene group, a propylene group, or a butylene group, thereby causing the peptide of formula (1) to have two cyclic structures in the molecule.

2. The following formula (2): c[β-Ala-Pro-X ３ -c(Cys-X ５ -Ser-4fF-Asp-Pro-Trp- Ｄ Cys)] (2) (In the formula, X ３ represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid, X ５ represents an isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid residue, The residues at positions 1 and 11 form an amide bond between the amino group and carboxyl group of the main chain, and the two cysteine ​​residues at positions 4 and 11 form a covalent bond between the respective -SH groups in the side chains via a propylene group linker, thereby causing the peptide of formula (2) to have two cyclic structures within the molecule.

3. 3. The composition according to claim 1, further comprising at least 10% by weight of a surfactant.

4. 3. The composition according to claim 1, wherein the surfactant is selected from the group consisting of polyoxyethylene castor oil and polyoxyethylene sorbitan fatty acid esters.

5. 3. The composition according to claim 1, comprising 25 to 45% by volume of 10-fold concentrated Dulbecco's PBS solution.

6. 3. The composition of claim 1, further comprising dimethyl sulfoxide.

7. 5. The composition according to claim 4, wherein the surfactant is polyoxyethylene-35-ricinoleate or polysorbate 80.

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