WO2024111590A1 - Composition containing ras inhibitor peptide - Google Patents

Composition containing ras inhibitor peptide Download PDF

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Publication number
WO2024111590A1
WO2024111590A1 PCT/JP2023/041845 JP2023041845W WO2024111590A1 WO 2024111590 A1 WO2024111590 A1 WO 2024111590A1 JP 2023041845 W JP2023041845 W JP 2023041845W WO 2024111590 A1 WO2024111590 A1 WO 2024111590A1
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acid
peptide
group
residue
ras
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PCT/JP2023/041845
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French (fr)
Japanese (ja)
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孝太郎 坂元
英次郎 都
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一丸ファルコス株式会社
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Publication of WO2024111590A1 publication Critical patent/WO2024111590A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/20—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing sulfur, e.g. dimethyl sulfoxide [DMSO], docusate, sodium lauryl sulfate or aminosulfonic acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to a composition containing a Ras inhibitor peptide.
  • the present invention relates to a composition that can suppress aggregation of a Ras inhibitor peptide by using a surfactant.
  • Ras protein (meaning the Ras subfamily, hereafter abbreviated as Ras) is a GTPase expressed in cells, and by binding to GDP or GTP, it functions as a molecular switch that controls the off (inactivation)/on (activation) of cell proliferation signaling.
  • Ras consists of a sequence of 188 to 189 amino acids, and any amino acid mutation greatly suppresses its own GTPase activity and the ability of GTP hydrolysis by GTPase activating protein (GAP), resulting in a bias toward the GTP-bound form. As a result, cell proliferation signals are extended.
  • GAP GTPase activating protein
  • K-Ras, N-Ras, and H-Ras are members that are attracting attention as targets for drug discovery.
  • amino acid mutations in K-Ras occur most frequently, occurring in approximately 20% of human tumors.
  • Patent Document 2 As an example of a method for suppressing protein aggregation, the addition of a surfactant to a solution containing an antibody has been proposed (see, for example, Patent Document 2).
  • the formation of aggregates and cloudiness are suppressed by adding a surfactant during ultrafiltration, and this method is effective for diagnostic and therapeutic agents containing antibodies, particularly for injectables. It is also important to suppress aggregation in aqueous solutions containing Ras inhibitors, but previously no composition capable of effectively preventing the formation of such aggregates was known.
  • the present invention has been made to solve the above 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 peptide with a specific surfactant. That is, the present invention includes the following embodiments.
  • composition according to (1) or (2) comprising at least 10% by mass of a surfactant.
  • surfactant is selected from the group consisting of polyoxyethylene castor oil and polyoxyethylene sorbitan fatty acid esters.
  • the composition according to any one of [1] to [4] comprising 25 to 45% by volume of 10-fold concentrated Dulbecco's PBS solution.
  • the surfactant is polyoxyethylene-35-ricinoleate or polysorbate 80.
  • the solubility of a Ras inhibitor peptide having a specific cyclic structure and amino acid sequence in an aqueous solution can be improved, thereby improving the efficacy of the peptide when administered to a living body.
  • FIG. 1 shows the chemical structures of representative Ras inhibitory peptides and representative surfactants of the present invention.
  • FIG. 2 shows the results of evaluating the solubility of a representative Ras inhibitory peptide of the present invention when it is mixed with a representative surfactant.
  • FIG. 3 shows the results of evaluating the anticancer activity of a representative Ras inhibitory peptide of the present invention mixed with a representative surfactant and administered into the tail vein of mice bearing subcutaneous CT26 tumors.
  • FIG. 4 shows the results of evaluating the anti-cancer activity of a representative Ras inhibitory peptide of the present invention mixed with a representative surfactant and administered into the tail vein of mice orthotopically transplanted with PANC-1.
  • FIG. 1 shows the chemical structures of representative Ras inhibitory peptides and representative surfactants of the present invention.
  • FIG. 2 shows the results of evaluating the solubility of a representative Ras inhibitory peptide of the present invention when it is mixed with a representative surfactant.
  • FIG. 3 shows
  • FIG. 5 shows the results of observing 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 a transmission electron microscope.
  • 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, by dynamic light scattering.
  • a peptide refers to two or more amino acids bound by amide bonds (peptide bonds), and can be, for example, 2 to 20 amino acids bound by amide bonds.
  • 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 called the C ⁇ carbon.
  • amino acid or a derivative thereof is used in its broadest sense and includes, in addition to natural amino acids, artificial amino acids having unnatural structures, chemically synthesized compounds having properties known in the art that are characteristic of amino acids, and also carboxylic acids having functional groups.
  • unnatural amino acids include D-amino acids, ⁇ / ⁇ -disubstituted amino acids whose main chain structure differs from that of natural amino acids (such as ⁇ -methylated amino acids such as 2-aminoisobutyric acid), N-alkyl-amino acids (such as N-methylated amino acids), N-substituted glycines (peptoids), amino acids whose main chains are extended (such as ⁇ homoamino acids and ⁇ homoamino acids), amino acids whose side chain structure differs from that of natural amino acids (such as cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, and 3-(1H-benzimidazol-2-yl)-alanine), amino acids whose side chains are partially substituted (such as norleucine, diaminopropanoic acid, and 3-(2-pyridyl)-alanine), amino acids having an extra functional group in the side chain; amino acids having an
  • 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 proteins.
  • 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 phosphorylation of extracellular signal-regulated kinase (Erk) in Ras-expressing cells, and (4) suppresses the proliferation of Ras-expressing cells, and a peptide that exhibits any one of these effects is called a "Ras inhibitory peptide.”
  • Erk extracellular signal-regulated kinase
  • Ras inhibitory peptides 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) related to Ras binding activity. Any of the peptides disclosed in these documents can be used to produce the composition of the present invention.
  • 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) (SEQ ID NO: 22).
  • X 1 represents a ⁇ -alanine or ⁇ -aminobutyric acid residue
  • X 3 represents a leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue
  • 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 a valine, phenylalanine, tryptophan, 1-naphthylalanine, or an N-methylated amino acid residue thereof
  • X 11 represents a D- or L-cysteine residue.
  • X1 and X11 form an amide bond between the amino group and carboxyl group of the main chain
  • X4 and X11 form a covalent bond between the -SH groups of the respective side chains via a linker of a methylene group, an ethylene group, a propylene group (trimethylene group) or a butylene group (tetramethylene group), so that the peptide of formula (1) has two cyclic structures in the molecule.
  • the "4fF” means 4-fluoro-L-phenylalanine.
  • XN means the amino acid at the N-position (Nth) in the target amino acid sequence.
  • a further preferred embodiment of the Ras inhibitory peptide has the following formula (2): c[ ⁇ Ala-Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-Trp- D Cys)] (2) (SEQ ID NO:23).
  • X3 represents a leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue
  • X5 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 side chain -SH groups via a propylene group (trimethylene group) linker, so that the peptide of formula (2) has two cyclic structures in the molecule.
  • the " D Cys” means a D-form cysteine residue.
  • m Val means a methylated valine residue.
  • Examples of the individual Ras inhibitory peptides included in the above formula (1) or (2) include the following. c[ ⁇ Ala-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val-Cys)] (SEQ ID NOs: 1 to 3) c[ ⁇ Ala-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NOs: 4 to 6) c[ ⁇ Aba-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val-Cys)] (SEQ ID NOs: 7 to 9) c[ ⁇ Aba-Pro-Nle-c(Cys-Ile-Ser-4fF-Asp-Pro-Val- D Cys)] (SEQ ID NOs: 10-12) c[ ⁇ Ala-Pro-Nle-c(Cys-Phg-Ser-4fF-
  • cysteine residues at positions 4 and 11 form a covalent bond between their respective side chain -SH groups via a propylene group (trimethylene group: -CH 2 -CH 2 -CH 2 -).
  • cysteine residues at positions 4 and 11 form a covalent bond between their respective side chain -SH groups via a butylene group (tetramethylene group: -CH 2 -CH 2 -CH 2 -CH 2 -).
  • cyclization means that two or more amino acids separated by one or more amino acids in one peptide are covalently bonded directly or indirectly via a linker to create one or more ring structures within the molecule.
  • this can be an amide bond between an amino group and a carboxy group, a disulfide bond between thiol groups, or a thioether bond between a linker having a halogen group and two thiol groups, but is not limited to these.
  • the direct covalent bond or the indirect covalent bond via a linker for cyclization may be between the main chain and the main chain, between the main chain and the side chain, between the side chain and the main chain, or between the side chain and the side chain.
  • the Ras inhibitory peptide according to this embodiment includes peptides that have the homology of the amino acid sequences shown in [1] and [2] above with one to several amino acids deleted, added, and/or substituted, but have binding activity to Ras.
  • 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 ends or in the middle of the peptide, and may occur at one or more locations.
  • the amino acid sequence in which one or more amino acids have been deleted, added, and/or substituted in the above amino acid sequence has an identity of at least 50%, preferably 70%, more preferably 80%, 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 or initial setting parameters).
  • BLAST Basic Local Alignment Search Tool at the National Center for Biological Information
  • the Ras inhibitory peptide according to the present embodiment also encompasses various derivatives and/or modifications thereof.
  • peptides in which some of the amino groups of the peptide have been acetylated, alkylated, or deaminated, and peptides in which some of the carboxy groups of the peptide have been amide or ester peptides in which S is a sulfoxide S( ⁇ O) or a sulfone S( ⁇ O).
  • peptides examples include, but are not limited to, peptides in which the peptide is fused with an alkyl chain, polyethylene glycol, an antibody, a lectin, a sugar chain, an enzyme, a membrane-permeable peptide, a low molecular weight compound, or a molecule that induces ubiquitination of a protein.
  • the Ras inhibitory peptide according to this embodiment also includes peptide salts.
  • a salt with a physiologically acceptable base or acid is used, and examples thereof include addition salts with inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts with organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts with inorganic bases (ammonium hydroxide, or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.
  • inorganic acids hydroochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.
  • organic acids p-toluenesulfonic
  • the Ras inhibitory peptide according to this embodiment may be a prodrug.
  • a prodrug is a compound that is converted into the peptide of the present invention by a reaction with an enzyme, gastric acid, or the like under physiological conditions in the body, i.e., a compound that is enzymatically oxidized, reduced, hydrolyzed, or the like to be converted into the peptide of the present invention, or a compound that is hydrolyzed by gastric acid or the like to be converted into the peptide of the present invention.
  • the Ras inhibitory peptide of this embodiment may be a crystal, and whether the crystalline form is a single one or a mixture of crystalline forms, the peptide of the present invention is included.
  • the crystals can be produced by crystallization using a crystallization method known per se.
  • Surfactants are generally substances that have hydrophilic and hydrophobic groups (lipophilic groups) in 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.
  • surfactants used in the compositions of the present invention include any surfactant or combination of surfactants that stabilize the Ras inhibitory peptides described herein and inhibit aggregation.
  • surfactants of the present invention include, but are not limited to, polyoxyethylene castor oil, polyoxyethylene sorbitan fatty acid esters, TritonTM X-100, nonoxynol-9, triethanolamine, triethanolamine polypeptide oleate, polyoxyethylene-660 hydroxystearate (PEG-15, Solutol H15), soy lecithin, poloxamer, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecyl ammonium bromide, methoxyhexadecylglycerol, pluronic polyols, polyamines (e.g., pyran, dextran sulfate, poly
  • Preferred surfactants are polyoxyethylene castor oils or polyoxyethylene sorbitan fatty acid esters.
  • Polyoxyethylene castor oils include: a) polyoxyethylene castor oil derivatives, including those commercially available under the trade name "CREMOPHOR” (BASFTWEEN), in particular CREMOPHOR EL or ELP (also referred to as 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 low water, potassium and free fatty acid content, which is a refined grade of CREMOPHOR EL), and CREMOPHOR RH 40 (also referred to as polyoxyl 40 hydrogenated castor oil, macrogolglycerol hydroxystearic acid, polyoxyethylene 40 hydrogenated castor oil, PEG-40 hydrogenated castor oil);
  • Polyoxyethylene sorbitan fatty acid esters including those commercially available 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 specifically polyoxyethylene-sorbitan-fatty acid mono-oleyl esters, (containing a mixture of fatty acids including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, mainly oleic acid), TWEEN 85 (85 [polyoxyethylene (20) sorbitan trioleate], also known as polysorbate 85); polyoxyethylene 20 sorbitan trioleate (polymerized with ethylene oxide), Partial fatty acid esters of sorbitol and its anhydrides), specifically polyoxyethylene
  • 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.
  • 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
  • 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 in the total amount of the composition of the present invention.
  • CREMOPHOR EL and/or TWEEN 80 in the total amount of the composition of the present invention.
  • composition contains the above-mentioned Ras inhibitory peptide and a surfactant, and is capable of suppressing the growth of malignant tumors and the like.
  • the administration form is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (transnasal, oral, ocular, pulmonary, vaginal, or rectal administration).
  • the peptide in the composition may be modified in various ways in consideration of its tendency to be metabolized and excreted.
  • the blood residence time can be increased and the antigenicity can be reduced.
  • biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. may be used as sustained-release bases, and the peptide may be encapsulated in them.
  • PLGA polylactic acid glycol
  • porous hydroxyapatite porous hydroxyapatite
  • liposomes liposomes
  • surface-modified liposomes emulsions prepared with unsaturated fatty acids
  • nanoparticles nanospheres, etc.
  • emulsions prepared with unsaturated fatty acids nanoparticles, nanospheres, etc.
  • a weak electric current can be passed through the skin surface to cause penetration through the stratum corneum (iontophoresis method).
  • compositions may be formulated by adding pharma- ceutically acceptable carriers, excipients, additives, etc. in addition to the surfactant.
  • 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 be immediate-release or sustained-release controlled-release formulations (e.g., sustained-release microcapsules).
  • Formulation may be performed by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption promoters, pH adjusters, preservatives, antioxidants, etc., as appropriate.
  • ingredients used in formulations include purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharma- ceutically 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 polymer, 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 myri
  • absorption enhancers that improve the absorption of poorly absorbed drugs may be used, such as 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.
  • 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 ca
  • 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, 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 in a suitable manner, for example in the form of a cake or powder by the addition of a cryoprotectant. Solid formulations can be dissolved, i.e., reconstituted in a suitable vehicle, to become a liquid suitable for administration.
  • Solvents suitable for reconstituting solid formulations include water, isotonic saline, buffered solutions such as phosphate buffered saline, Ringer's (lactated or dextrose) solution, essential mineral media, alcohol/aqueous solutions, dextrose solution, and the like.
  • the composition of the present invention contains 25-45% by volume of 10x concentrated Dulbecco's PBS solution (D-PBS).
  • D-PBS typically contains 0.02% potassium chloride, 0.02% sodium dihydrogen phosphate, 0.8% sodium chloride, and 0.115% disodium hydrogen phosphate.
  • 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.
  • the Ras inhibitor peptide may be first dissolved in DMSO and then diluted with an aqueous solution containing the above-mentioned surfactant.
  • concentration of DMSO in the composition of the present invention is preferably 50% or less, more preferably 20% or less.
  • the composition of the present invention can easily dissolve a Ras inhibitor peptide dissolved in an organic solvent such as DMSO or ethanol without causing turbidity or precipitation when diluted with an aqueous solution such as physiological saline, phosphate buffer or glucose solution, and improves the pharmaceutical effect when administered to a living body.
  • an organic solvent such as DMSO or ethanol
  • an aqueous solution such as physiological saline, phosphate buffer or glucose solution
  • the reason is not necessarily clear, and it is not bound by any theory, but it is considered that the Ras inhibitor, which is an active ingredient, does not become turbid or precipitate because the Ras inhibitor forms nanomicelles of about 5 to 100 nm in the aqueous solution.
  • the size of the nanomicelle is preferably a particle diameter of 5 to 50 nm, more preferably 5 to 25 nm.
  • nanomicelles can be measured from a photographic image of a transmission electron microscope or a scanning electron microscope.
  • nanomicelles of about 5 to 100 nm account for 50% or more of the total, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. It is believed that forming nanomicelles with such particle sizes will increase the stability in the bloodstream when administered to the body, without being filtered out by the kidneys and excreted. In addition, it is believed that having such particle sizes will allow the nanomicelles to easily enter target cells such as tumor cells.
  • the present invention will be described in more detail below with reference to examples, but the present invention is in no way limited to these examples.
  • the unit % of the numerical values showing the amount of each component added means mass %.
  • Synthetic Peptide KS-58 Aqueous Solution 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 Co., Ltd.
  • 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.) that did not contain a surfactant or contained 1-5% Cremophor EL.
  • the final concentration of KS-58 in this aqueous solution was 10 mg/mL, and the final concentration of DMSO was 10%.
  • Figure 2 (A) In D-PBS that did not contain a surfactant, a suspension with a clear cloudiness was obtained. In D-PBS containing 1% Cremophor EL, a thin cloudy liquid was obtained, and in D-PBS containing 3% or 5% Cremophor EL, a clear solution was obtained.
  • 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, Ltd.) with purified water to contain 70-10% 10x D-PBS.
  • the final concentration of KS-58 was 15 mg/mL, and the final concentration of DMSO was 20%.
  • Figure 2 (B) The solubility of KS-58 changed depending on the composition concentration of 10x D-PBS contained in the solution, and when 10x D-PBS was contained at 40-20%, a clear solution without precipitation or cloudiness was obtained.
  • 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 contain 45-25% 10x D-PBS.
  • the final concentration of KS-58 was 20 mg/mL, and the final concentration of DMSO was 20%.
  • Figure 2 (C) In both cases, a clear solution was obtained without any precipitation or cloudiness.
  • Example 2 In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 CT26 mouse colon cancer cell line was subcutaneously transplanted into BALB/cCrSlc mice (4 weeks old) to form tumors.
  • a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was prepared, diluted with saline, and administered in the same manner to a group as a comparison subject (control group).
  • the tumor size was measured every 3 days until the 17th day after the start of administration of the three types of solutions prepared above, and the results are shown in FIG. 3.
  • tumor growth was significantly suppressed in the groups administered KS-58 (10 mg/kg) and KS-58 (40 mg/kg) dissolved using Cremophor EL, but was particularly markedly suppressed in the group administered KS-58 (40 mg/kg).
  • the tumor volume was estimated to be 34%, assuming that the tumor volume of the control group on day 17 was 100%. It has been reported that when KS-58 was suspended in DMSO (10%)/physiological saline and administered subcutaneously at 40 mg/kg once every two days, the growth of a tumor derived from a subcutaneously implanted CT26 colon cancer cell line was 35%, assuming that the weight of the tumor on day 17 was 100% (Non-Patent Document 2). That is, it was shown that dissolution using moderate concentrations of 10xD-PBS and Cremophor EL improved the efficacy of KS-58 in the living body, and the number of administrations could be reduced while maintaining anticancer activity.
  • Example 3 In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 2.5 x 106 cells of PANC-1 cells expressing K-Ras (G12D) (catalog No. CRL-1469, manufactured by ATCC) were transplanted under the pancreatic tail capsule of BALB/cAJcl-nu/nu mice (male, 8 weeks old) (CLEA Japan, Inc.) and subjected to the test after 1 week.
  • a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was prepared, diluted with saline, and administered to a group for comparison (control group).
  • the sham group was a group in which transplantation was performed using a cell culture medium not containing cancer cells, and a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was administered.
  • the weights of the mouse organs (pancreas, liver, kidney) 6 weeks after the start of administration were measured, and the results are shown in Figure 4.
  • the weight of the pancreas in the control group was significantly increased due to the increase in the PANC-1 tumor ( ⁇ p ⁇ 0.001, Student's t-test).
  • Non-Patent Document 1 it was shown that dissolution using appropriate concentrations of 10xD-PBS and Cremophor EL improved the efficacy of KS-58 in vivo, maintained or improved anticancer activity, and further reduced the number of administrations.
  • Example 4 Measurement of particle size of a composition containing synthetic peptide KS-58
  • the particle size of particles formed in a solution in which KS-58 was dissolved at 20 mg/mL in a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution was observed using a transmission electron microscope FEI Tecnai F20 TEM. The results are shown in FIG. 5. The formation of uniform particles of about 10 nm was confirmed. The particle size was also evaluated by dynamic light scattering using a Zetasizer Nano ZSP (Malvern Instrument). The results are shown in FIG. 6. The formation of uniform particles of about 10 nm was confirmed.
  • composition of the present invention is useful for producing Ras inhibitor peptides as pharmaceuticals or diagnostic agents by improving the solubility of the Ras inhibitor peptides in aqueous solutions, or for conducting clinical research to develop Ras inhibitor peptides as pharmaceuticals.

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Abstract

The present invention improves the medicinal effect of a Ras inhibitor peptide when being administered to a living body, by improving the solubility of the Ras inhibitor peptide to an aqueous solution. This composition contains: a cyclic peptide having a specific amino acid sequence indicated in the description, or a derivative or a modification product of the peptide, or a pharmacologically acceptable salt thereof; and at least one surfactant.

Description

Ｒａｓ阻害ペプチドを含む組成物Compositions Comprising Ras Inhibitory Peptides
　本発明は、Ｒａｓ阻害ペプチドを含む組成物などに関する。特に、界面活性剤を用いることで、Ｒａｓ阻害ペプチドの凝集を抑制することができる組成物に関する。 The present invention relates to a composition containing a Ras inhibitor peptide. In particular, the present invention relates to a composition that can suppress aggregation of a Ras inhibitor peptide by using a surfactant.
　Ｒａｓタンパク質（Ｒａｓサブファミリーを意味し、以下Ｒａｓと略す）は、細胞内に発現するＧＴＰａｓｅであり、ＧＤＰまたはＧＴＰと結合することで、細胞増殖シグナル伝達のオフ（不活性化）／オン（活性化）を司る分子スイッチとして機能する。Ｒａｓは、１８８～１８９個のアミノ酸配列からなっており、任意のアミノ酸変異によって、自身のＧＴＰａｓｅ活性とＧＴＰａｓｅ活性化タンパク質（ＧＡＰ）によるＧＴＰの加水分解能が大きく抑制され、ＧＴＰ結合型に偏重する。この結果、細胞増殖シグナルが延長される。ヒト腫瘍の約３０％がアミノ酸変異を生じた変異型Ｒａｓを発現しており、腫瘍増殖のドライバーとしている。Ｒａｓの中で、創薬ターゲットとして注目されているメンバーにＫ－Ｒａｓ、Ｎ－Ｒａｓ、Ｈ－Ｒａｓが挙げられる。その中でもＫ－Ｒａｓのアミノ酸変異は、ヒト腫瘍の約２０％という最も高い頻度で発生することが知られている。 Ras protein (meaning the Ras subfamily, hereafter abbreviated as Ras) is a GTPase expressed in cells, and by binding to GDP or GTP, it functions as a molecular switch that controls the off (inactivation)/on (activation) of cell proliferation signaling. Ras consists of a sequence of 188 to 189 amino acids, and any amino acid mutation greatly suppresses its own GTPase activity and the ability of GTP hydrolysis by GTPase activating protein (GAP), resulting in a bias toward the GTP-bound form. As a result, cell proliferation signals are extended. Approximately 30% of human tumors express mutant Ras with amino acid mutations, which act as a driver of tumor growth. Among Ras, K-Ras, N-Ras, and H-Ras are members that are attracting attention as targets for drug discovery. Among them, it is known that amino acid mutations in K-Ras occur most frequently, occurring in approximately 20% of human tumors.
　本発明者らは、Ｒａｓタンパク質の阻害活性を有し、血漿中で安定に存在するいくつかの環状ペプチドを見出し、それによるＲａｓ発現細胞の増殖抑制作用を報告している（例えば、特許文献１及び非特許文献１参照）。これらの文献では、非天然アミノ酸構造を持つ二環式ペプチドの一つであるＫＳ－５８が、Ｋ－Ｒａｓ（Ｇ１２Ｄ）を発現するヒト肺癌細胞株Ａ４２７及びヒト膵臓癌細胞株ＰＡＮＣ－１のインビトロでの増殖を抑制することが示されている。さらに、ＫＳ－５８は、Ｋ－Ｒａｓ（Ｇ１２Ｄ）を安定に発現する大腸癌細胞株ＣＴ２６由来のマウス腫瘍に対しても抗癌活性を有することが明らかになった。（非特許文献２参照）。しかしながら、ＫＳ－５８は疎水性の高い化合物であるため、ジメチルスルフォキシド（ＤＭＳＯ）に溶解した後、生理食塩水で１０倍希釈して上記の動物実験に用いている。一般に、疎水性の高いペプチドや抗体タンパク質などの生体内での治療効果を確かめるために水溶液中に高濃度で溶解すると、凝集及び／又は白濁が生じ、注射剤としての開発が難しいという問題がある。 The present inventors have found several cyclic peptides that have inhibitory activity against Ras proteins 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 show that KS-58, a bicyclic peptide with an unnatural amino acid structure, inhibits the in vitro proliferation of human lung cancer cell line A427 and human pancreatic cancer cell line PANC-1, which express K-Ras (G12D). Furthermore, it has been revealed that KS-58 also has 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, since KS-58 is a highly hydrophobic compound, it was dissolved in dimethyl sulfoxide (DMSO) and then diluted 10-fold with physiological saline before being used in the above animal experiments. In general, when highly hydrophobic peptides, antibody proteins, and other substances are dissolved in aqueous solutions at high concentrations to confirm their therapeutic effects in vivo, aggregation and/or cloudiness occur, making it difficult to develop them as injections.
　これまでにタンパク質の凝集を抑制する方法の例として、抗体を含む溶液に界面活性剤を添加することが提案されている（例えば、特許文献２参照）。特許文献２に記載の方法では限外濾過において界面活性剤を添加することで凝集物の生成や白濁化を抑制し、抗体を含む診断薬や治療剤、特に注射剤に効果を発揮している。Ｒａｓ阻害剤を含む水溶液においても、凝集を抑制することが重要であるが、従来この凝集物の生成を効果的に防止することのできる組成は知られていなかった。 As an example of a method for suppressing protein aggregation, the addition of 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, the formation of aggregates and cloudiness are suppressed by adding a surfactant during ultrafiltration, and this method is effective for diagnostic and therapeutic agents containing antibodies, particularly for injectables. It is also important to suppress aggregation in aqueous solutions containing Ras inhibitors, but previously no composition capable of effectively preventing the formation of such aggregates was known.
国際公開第２０２０／２３０７８０号公報International Publication No. 2020/230780 国際公開第２００２／０１３８５９号公報International Publication No. 2002/013859
　本発明が解決しようとする課題は、Ｒａｓ阻害ペプチドの水溶液に対する溶解性を改善することにより生体に投与した際の薬効を向上させることである。 The problem that the present invention aims to solve is to improve the solubility of Ras inhibitor peptides in aqueous solutions, thereby improving their efficacy when administered to a living body.
　本発明は、上記課題を解決するためになされたものであって、特定の環状構造とアミノ酸配列を有するＲａｓ阻害ペプチドと特定の界面活性剤とを混合することで、当該Ｒａｓ阻害ペプチドの溶解性が向上するという予想外で驚くべき結果に関する。すなわち、本発明は以下の実施形態を含む。 The present invention has been made to solve the above 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 peptide with a specific surfactant. That is, the present invention includes the following embodiments.
［１］下記式（１）：
ｃ［Ｘ１－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｘ１０－Ｘ１１）］　　　（１）（配列番号２２）
（式中、Ｘ１は、β－アラニン（βＡｌａ）又はγ－アミノ酪酸（γＡｂａ）残基を表し、Ｘ３は、ロイシン、ノルロイシン（Ｎｌｅ）、シクロヘキシルグリシン、フェニルグリシン（Ｐｈｇ）、２－アミノヘプタン酸（Ａｈｅ）、２－アミノオクタン酸（Ａｏｃ）、２－アミノノナン酸（Ａｎｏｎ）又は２－アミノデカン酸残基を表し、Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、Ｘ１０は、バリン、フェニルアラニン、トリプトファン、１－ナフチルアラニン（１ＮａｐｈＡ）又はそれらのＮ－メチル化アミノ酸残基を表し、Ｘ１１は、Ｄ体又はＬ体のシステイン残基を表し、Ｘ１（１位のアミノ酸残基）とＸ１１（１１位のアミノ酸残基）は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、Ｘ４（４位のシステイン残基）とＸ１１（１１位のシステイン残基）は、それぞれの側鎖－ＳＨ基の間で、メチレン基、エチレン基、プロピレン基（トリメチレン基）又はブチレン基（テトレメチレン基）のリンカーを介して共有結合を形成し、それによって式（１）のペプチドは分子内に２つの環状構造を有する。）で表されるアミノ酸配列からなる環状ペプチド、その誘導体若しくは修飾体又はその薬理学的に許容されるそれらの塩と、少なくとも１種類の界面活性剤と、を含む組成物。
［２］下記式（２）：
ｃ［βＡｌａ－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｔｒｐ－ＤＣｙｓ）］　　　（２）（配列番号２３）
（式中、Ｘ３は、ロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、１位と１１位の残基は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、４位と１１位の２つのシステイン残基は、それぞれの側鎖－ＳＨ基の間で、プロピレン基（トリメチレン基）のリンカーを介して共有結合を形成し、それによって式（２）のペプチドは分子内に２つの環状構造を有する。）で表されるアミノ酸配列からなる環状ペプチド、その誘導体若しくは修飾体又はその薬理学的に許容されるそれらの塩と、少なくとも１種類の界面活性剤と、を含む組成物。
［３］質量比で少なくとも１０％の界面活性剤が含まれる（１）又は（２）に記載の組成物。
［４］界面活性剤がポリオキシエチレンヒマシ油及びポリオキシエチレンソルビタン脂肪酸エステルからなる群より選択される［１］から［３］のいずれかに記載の組成物。
［５］１０倍濃縮ダルベッコＰＢＳ溶液を体積比で２５～４５％含む［１］から［４］のいずれかに記載の組成物。
［６］ジメチルスルホキシドを含む［１］から［５］のいずれかに記載の組成物。
［７］界面活性剤が、ポリオキシエチレン－３５－リシノール酸塩又はポリソルベート８０である［４］に記載の組成物。 [1] The following formula (1):
c[X 1 -Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-X 10 -X 11 )] (1) (SEQ ID NO: 22)
(wherein X 1 represents a β-alanine (βAla) or γ-aminobutyric acid (γAba) residue; X 3 represents a leucine, norleucine (Nle), cyclohexylglycine, phenylglycine (Phg), 2-aminoheptanoic acid (Ahe), 2-aminooctanoic acid (Aoc), 2-aminononanoic acid (Anon) or 2-aminodecanoic acid residue; 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 a valine, phenylalanine, tryptophan, 1-naphthylalanine (1NaphA) or an N-methylated amino acid residue thereof; X 11 represents a D- or L-cysteine residue; and X 1 (the amino acid residue at position 1) and X 11 (the amino acid residue at position 11) forms an amide bond between the amino group and carboxyl group of the main chain, and X 4 (the cysteine residue at position 4) and X 11 (the cysteine residue at position 11) form a covalent bond between the -SH groups in their side chains via a linker which is a methylene group, an ethylene group, a propylene group (trimethylene group) or a butylene group (tetramethylene group), thereby the peptide of formula (1) has two cyclic structures in the molecule.
[2] The following formula (2):
c[βAla-Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-Trp- D Cys)] (2) (SEQ ID NO: 23)
(wherein X3 represents a leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue; X5 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 the 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 chain via a propylene group (trimethylene group) linker, whereby the peptide of formula (2) has two cyclic structures in the molecule), or a derivative or modification thereof, or a pharmacologically acceptable salt thereof; and at least one surfactant.
[3] The composition according to (1) or (2), comprising 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 10-fold concentrated Dulbecco's PBS solution.
[6] The composition according to any one of [1] to [5], comprising dimethyl sulfoxide.
[7] The composition according to [4], wherein the surfactant is polyoxyethylene-35-ricinoleate or polysorbate 80.
　本発明によれば、特定の環状構造とアミノ酸配列を有するＲａｓ阻害ペプチドの水溶液に対する溶解性を改善することにより生体に投与した際の薬効を向上させることができる。 According to the present invention, the solubility of a Ras inhibitor peptide having a specific cyclic structure and amino acid sequence in an aqueous solution can be improved, thereby improving the efficacy of the peptide when administered to a living body.
図１は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤の化学構造である。FIG. 1 shows the chemical structures of representative Ras inhibitory peptides and representative surfactants of the present invention. 図２は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤を混合し、溶解性を評価した結果である。FIG. 2 shows the results of evaluating the solubility of a representative Ras inhibitory peptide of the present invention when it is mixed with a representative surfactant. 図３は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤を混合し、ＣＴ２６皮下担癌マウスに尾静脈内投与することによって、その抗癌活性を評価した結果である。FIG. 3 shows the results of evaluating the anticancer activity of a representative Ras inhibitory peptide of the present invention mixed with a representative surfactant and administered into the tail vein of mice bearing subcutaneous CT26 tumors. 図４は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤を混合し、ＰＡＮＣ－１同所移植マウスに尾静脈内投与することによって、その抗癌活性を評価した結果である。FIG. 4 shows the results of evaluating the anti-cancer activity of a representative Ras inhibitory peptide of the present invention mixed with a representative surfactant and administered into the tail vein of mice orthotopically transplanted with PANC-1. 図５は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤を混合し、溶液中に形成された粒子の粒子径を透過型電子顕微鏡で観察した結果である。FIG. 5 shows the results of observing 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 a transmission electron microscope. 図６は、本発明の代表的なＲａｓ阻害ペプチドと代表的な界面活性剤を混合し、溶液中に形成された粒子の粒子径を動的光散乱により測定した結果である。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, by dynamic light scattering.
　次に、本発明の各実施形態について、図面を参照して説明する。なお、以下に説明する各実施形態は、特許請求の範囲に係る発明を限定するものではなく、また、各実施形態の中で説明されている諸要素及びその組み合わせの全てが本発明の解決手段に必須であるとは限らない。 Next, each embodiment of the present invention will be described with reference to the drawings. Note that each embodiment described below does not limit the invention related to the claims, and not all of the elements and combinations thereof described in each embodiment are necessarily essential to the solution of the present invention.
（定義）
　本明細書においてペプチドは、２つ以上のアミノ酸がアミド結合（ペプチド結合）で結合しているものを指し、例えば２～２０アミノ酸がアミド結合したものとすることができる。また、ペプチド標記の慣例に従って左端がＮ末端（アミノ末端）、右端がＣ末端（カルボキシ末端）である。ペプチド結合を形成するカルボニル基に隣接する１番目の炭素原子をＣα炭素と称する。 (Definition)
In this specification, a peptide refers to two or more amino acids bound by amide bonds (peptide bonds), and can be, for example, 2 to 20 amino acids bound by amide bonds. In addition, according to the convention of 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 called the Cα carbon.
　本明細書において「アミノ酸又はその誘導体」は、その最も広い意味で用いられ、天然アミノ酸に加え、非天然構造を有する人工のアミノ酸、アミノ酸の特徴である当業界で公知の特性を有する化学的に合成された化合物、さらに官能基を有するカルボン酸も含む。非天然アミノ酸の例として、Ｄ体アミノ酸、主鎖の構造が天然型と異なるα／α－二置換アミノ酸（２－アミノイソ酪酸といったα－メチル化アミノ酸など）、Ｎ－アルキル－アミノ酸（Ｎ－メチル化アミノ酸など）、Ｎ－置換グリシン（ペプトイド）、主鎖が伸長しているアミノ酸（βホモアミノ酸やγホモアミノ酸）、側鎖の構造が天然型と異なるアミノ酸（シクロヘキシルアラニンやアリルグリシンや２－（２－ピリジル）－グリシンや３－（１Ｈ－ベンゾイミダゾール－２－イル）－アラニンなど）、側鎖の一部が置換されているアミノ酸（ノルロイシンやジアミノプロパン酸や３－（２－ピリジル）－アラニンなど）、側鎖に余分の官能基を有するアミノ酸；側鎖に余分のＣやアルキル基やメチル基を有するアミノ酸（ホモノルロイシンやγ－メチルロイシンなど）、側鎖にハロゲン原子（Ｆ、Ｃｌ、Ｂｒ、Ｉ）を有するアミノ酸（３－クロロ－アラニンなど）、側鎖にハロゲン原子（Ｆ、Ｃｌ、Ｂｒ、Ｉ）を有するカルボン酸（３－クロロプロパン酸など）、側鎖に官能基を有するカルボン酸（３－ブテン酸など）、側鎖に余分のＮやアミノ基を有するアミノ酸（β－アジドアラニンやオルニチンなど）、側鎖に余分のＯやメトキシ基を有するアミノ酸（Ｏ－メチル－セリンやＯ－メチル－スレオニンなど）、側鎖に余分のヒドロキシ基を有するアミノ酸（３－ヒドロキシ－フェニルアラニンなど）、側鎖に余分のカルボキシ基（－ＣＯＯＨ）を有するアミノ酸（３－カルボキシ－フェニルアラニンなど）、側鎖に余分のＳを有するアミノ酸（エチオニンなど）、側鎖中のカルボン酸官能基がエステルで保護されているアミノ酸（アスパラギン酸－４－メチルエステルなど）、側鎖のチオ基（－Ｓ－）が酸化されてスルフィニル基（－Ｓ（＝Ｏ）－）やスルホニル基（－Ｓ（＝Ｏ）２－）に変換されているアミノ酸（メチオニンスルホキシド）などが挙げられるが、これらに限定されない。 As used herein, the term "amino acid or a derivative thereof" is used in its broadest sense and includes, in addition to natural amino acids, artificial amino acids having unnatural structures, chemically synthesized compounds having properties known in the art that are characteristic of amino acids, and also carboxylic acids having functional groups. Examples of unnatural amino acids include D-amino acids, α/α-disubstituted amino acids whose main chain structure 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 whose main chains are extended (such as β homoamino acids and γ homoamino acids), amino acids whose side chain structure differs from that of natural amino acids (such as cyclohexylalanine, allylglycine, 2-(2-pyridyl)-glycine, and 3-(1H-benzimidazol-2-yl)-alanine), amino acids whose side chains are partially substituted (such as norleucine, diaminopropanoic acid, and 3-(2-pyridyl)-alanine), amino acids having an extra functional group in the side chain; amino acids having an extra C, alkyl group, or methyl group in the side chain (such as homonorleucine and γ-methylleucine), amino acids having a halogen atom (F, Cl, Br, I) in the side chain (such as 3- chloro-alanine, etc.), carboxylic acids having halogen atoms (F, Cl, Br, I) in the side chain (3-chloropropanoic acid, etc.), carboxylic acids having functional groups in the side chain (3-butenoic acid, etc.), amino acids having extra N or amino groups in the side chain (β-azidoalanine, ornithine, etc.), amino acids having extra O or methoxy groups in the side chain (O-methyl-serine, O-methyl-threonine, etc.), amino acids having extra hydroxy groups in the side chain (3-hydroxy-phenylalanine, etc.), amino acids having extra carboxy groups (—COOH) in the side chain (3-carboxy-phenylalanine, etc.), amino acids having extra S in the side chain (ethionine, etc.), amino acids in which the carboxylic acid functional group in the side chain is protected with an ester (aspartic acid-4-methyl ester, etc.), 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), but are not limited to these.
　本明細書において、「Ｒａｓ」はマウス、ラット、イヌ、サル、ヒトなどの哺乳類のＫ－Ｒａｓ、Ｎ－Ｒａｓ、Ｈ－ＲａｓといったＲａｓサブファミリーの野生型タンパク質及びアミノ酸変異型タンパク質全般を意味し、ＧＤＰ結合型とＧＴＰ結合型のいずれも包含する。 In this specification, "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 proteins.
　本明細書において、「Ｒａｓ阻害ペプチド」とは、インビトロ試験において、当該ペプチドが、（１）既存のＲａｓ阻害ペプチドのＲａｓタンパク質に対する結合を阻害すること、（２）Ｒａｓタンパク質に対して濃度依存的に結合すること、（３）Ｒａｓ発現細胞内の細胞外シグナル調節キナーゼ（extracellular signal-regulated kinase：Ｅｒｋ）のリン酸化を阻害すること、（４）Ｒａｓ発現細胞の増殖を抑制すること、などを意味し、これらの効果のうちいずれか一つでも示すペプチドを、「Ｒａｓ阻害ペプチド」という。Ｒａｓ阻害活性の有無は、当業者が公知の方法に従って確認することができる。 In this specification, 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 phosphorylation of extracellular signal-regulated kinase (Erk) in Ras-expressing cells, and (4) suppresses the proliferation of Ras-expressing cells, and 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 a person skilled in the art according to a known method.
（Ｒａｓ阻害ペプチド）
　Ｒａｓを阻害するための有効成分である環状ペプチドは特許文献１や非特許文献１及び２に開示されており、その内容は全て参照により本願に組み込まれるものとする。特許文献１に開示されたペプチドは、Ｒａｓ結合活性にかかわる特徴（ファーマコフォア）を維持又は増強しつつ、二環状化により安定化されたものである。これらの文献に開示されたペプチドはいずれも本発明の組成物を製造するために用いることが可能である。 (Ras inhibitory peptides)
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) related to Ras binding activity. Any of the peptides disclosed in these documents can be used to produce the composition of the present invention.
［１］１つの実施形態におけるＲａｓ阻害ペプチドは、下記式（１）：
ｃ［Ｘ１－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｘ１０－Ｘ１１）］　　　（１）で表されるアミノ酸配列（配列番号２２）からなる。
式（１）において、Ｘ１は、β－アラニン又はγ－アミノ酪酸残基を表し、Ｘ３は、ロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、Ｘ１０は、バリン、フェニルアラニン、トリプトファン、１－ナフチルアラニン又はそれらのＮ－メチル化アミノ酸残基を表し、Ｘ１１は、Ｄ体又はＬ体のシステイン残基を表わす。また、Ｘ１とＸ１１は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、Ｘ４とＸ１１は、それぞれの側鎖－ＳＨ基の間で、メチレン基、エチレン基、プロピレン基（トリメチレン基）又はブチレン基（テトレメチレン基）のリンカーを介して共有結合を形成し、それによって式（１）のペプチドは分子内に２つの環状構造を有する。なお、本明細書において、前記「４ｆＦ」は、４－フルオロ－Ｌ－フェニルアラニンを意味する。また、「ＸＮ」は、対象のアミノ酸配列におけるＮ位（Ｎ番目）のアミノ酸を意味する。 [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) (SEQ ID NO: 22).
In formula (1), X 1 represents a β-alanine or γ-aminobutyric acid residue, X 3 represents a leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue, 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 a valine, phenylalanine, tryptophan, 1-naphthylalanine, or an N-methylated amino acid residue thereof, and X 11 represents a D- or L-cysteine residue. Furthermore, X1 and X11 form an amide bond between the amino group and carboxyl group of the main chain, and X4 and X11 form a covalent bond between the -SH groups of the respective side chains via a linker of a methylene group, an ethylene group, a propylene group (trimethylene group) or a butylene group (tetramethylene group), so that the peptide of formula (1) has two cyclic structures in the molecule. In this specification, the "4fF" means 4-fluoro-L-phenylalanine. Furthermore, " XN " means the amino acid at the N-position (Nth) in the target amino acid sequence.
［２］さらに好ましい実施形態のＲａｓ阻害ペプチドは、下記式（２）：
ｃ［βＡｌａ－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｔｒｐ－ＤＣｙｓ）］　　　（２）で表されるアミノ酸配列（配列番号２３）からなる。
式（２）において、Ｘ３は、ロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表わす。また、１位と１１位の残基は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、４位と１１位の２つのシステイン残基は、それぞれの側鎖－ＳＨ基の間で、プロピレン基（トリメチレン基）のリンカーを介して共有結合を形成し、それによって式（２）のペプチドは分子内に２つの環状構造を有する。なお、本明細書において、前記「ＤＣｙｓ」は、Ｄ体のシステイン残基を意味する。また、「ｍＶａｌ」は、メチル化バリン残基を意味する。 [2] A further 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) (SEQ ID NO:23).
In formula (2), X3 represents a leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid, or 2-aminodecanoic acid residue, and X5 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 side chain -SH groups via a propylene group (trimethylene group) linker, so that the peptide of formula (2) has two cyclic structures in the molecule. In this specification, the " D Cys" means a D-form cysteine residue. In addition, " m Val" means a methylated valine residue.
　上記式（１）又は式（２）に含まれる個々のＲａｓ阻害ペプチドとしては、例えば、以下のような例を挙げることができる。
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ｉｌｅ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－Ｃｙｓ）］（配列番号１～３）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ｉｌｅ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－ＤＣｙｓ）］（配列番号４～６）
ｃ［γＡｂａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ｉｌｅ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－Ｃｙｓ）］（配列番号７～９）
ｃ［γＡｂａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ｉｌｅ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－ＤＣｙｓ）］（配列番号１０～１２）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ｐｈｇ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－ＤＣｙｓ）］（配列番号１３）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｏｃ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－ＤＣｙｓ）］（配列番号１４）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｎｏｎ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｖａｌ－ＤＣｙｓ）］（配列番号１５）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｎｏｎ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｐｈｅ－ＤＣｙｓ）］（配列番号１６）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｎｏｎ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｔｒｐ－ＤＣｙｓ）］（配列番号１７）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｎｏｎ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－１ＮａｐｈＡ－ＤＣｙｓ）］（配列番号１８）
ｃ［βＡｌａ－Ｐｒｏ－Ａｎｏｎ－ｃ（Ｃｙｓ－Ａｎｏｎ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｔｒｐ－ＤＣｙｓ）］（配列番号１９）
ｃ［βＡｌａ－Ｐｒｏ－Ｎｌｅ－ｃ（Ｃｙｓ－Ａｏｃ－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－ｍＶａｌ－ＤＣｙｓ）］（配列番号２０～２１）
　上記配列番号１～２１のアミノ酸において、配列番号１、４、７、１０、及び２０は、４位及び１１位のシステイン残基が、それぞれの側鎖－ＳＨ基の間で、エチレン基（-CH2-CH2-）を介して共有結合を形成している。
　上記配列番号１～２１のアミノ酸において、配列番号２、５、８、１１、及び１３－１９、は、４位及び１１位のシステイン残基が、それぞれの側鎖－ＳＨ基の間で、プロピレン基（トリメチレン基：-CH2-CH2-CH2-）を介して共有結合を形成している。
　上記配列番号１～２１のアミノ酸において、配列番号３、６、９、１２、及び２１は、４位及び１１位のシステイン残基が、それぞれの側鎖－ＳＨ基の間で、ブチレン基（テトレメチレン基：-CH2-CH2-CH2-CH2-）を介して共有結合を形成している。 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: 10-12)
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- mVal -D Cys)] (SEQ ID NOs: 20-21)
Among the amino acids of SEQ ID NOs: 1 to 21, in SEQ ID NOs: 1, 4, 7, 10, and 20, the cysteine residues at positions 4 and 11 form a covalent bond between the respective side chain --SH groups via an ethylene group (-CH 2 -CH 2 -).
Among the amino acids of SEQ ID NOs: 1 to 21, in SEQ ID NOs: 2, 5, 8, 11, and 13-19, the cysteine residues at positions 4 and 11 form a covalent bond between their respective side chain -SH groups via a propylene group (trimethylene group: -CH 2 -CH 2 -CH 2 -).
Among the amino acids of SEQ ID NOs: 1 to 21, in SEQ ID NOs: 3, 6, 9, 12, and 21, the cysteine residues at positions 4 and 11 form a covalent bond between their respective side chain -SH groups via a butylene group (tetramethylene group: -CH 2 -CH 2 -CH 2 -CH 2 -).
　本実施形態のＲａｓ阻害ペプチドは、一態様として環状化されている。本明細書において環状化とは、１つのペプチド内において、１アミノ酸以上離れた２つ以上のアミノ酸が直接的に、又はリンカーを介して間接的に共有結合し、分子内に１つ以上の環状構造を作ることを意味する。例えば、アミノ基とカルボキシ基間のアミド結合、チオール基とチオール基間のジスルフィド結合、ハロゲン基を有するリンカーと二つのチオール基間のチオエーテル結合などによることができるが、これらに限定されない。環状化のための直接的な又はリンカーを介した間接的な共有結合は、主鎖－主鎖間、主鎖－側鎖間、側鎖－主鎖間、側鎖－側鎖間のいずれであってもよい。 The Ras inhibitory peptide of this embodiment is cyclized as one aspect. In this specification, cyclization means that two or more amino acids separated by one or more amino acids in one peptide are covalently bonded directly or indirectly via a linker to create one or more ring structures within the molecule. For example, this can be an amide bond between an amino group and a carboxy group, a disulfide bond between thiol groups, or a thioether bond between a linker having a halogen group and two thiol groups, but is not limited to these. The direct covalent bond or the indirect covalent bond via a linker for cyclization may be between the main chain and the main chain, between the main chain and the side chain, between the side chain and the main chain, or between the side chain and the side chain.
　本実施形態に係るＲａｓ阻害ペプチドは、上記［１］及び［２］で表されるアミノ酸配列において、１～数個のアミノ酸が欠失、付加、及び／又は置換された相同性を有するペプチドであっても、Ｒａｓに対する結合活性を有するものは包含する。本明細書において、「１～数個のアミノ酸が欠失、付加、及び／又は置換されているペプチド」という場合、それらのアミノ酸の個数は、そのペプチドがＲａｓ結合活性を有する限りは特に限定されないが、好ましくは１～５個、さらに好ましくは１個若しくは２個である。欠失、付加、及び／又は置換されている場所は、ペプチドの末端であっても、中間であってもよく、１ヶ所であっても２ヶ所以上であってもよい。 The Ras inhibitory peptide according to this embodiment includes peptides that have the homology of the amino acid sequences shown in [1] and [2] above with one to several amino acids deleted, added, and/or substituted, but have binding activity to Ras. In this specification, when referring to a "peptide with one to several amino acids 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 ends or in the middle of the peptide, and may occur at one or more locations.
　このような上記アミノ酸配列において１～数個のアミノ酸が欠失、付加、及び／又は置換されたアミノ酸配列として、前記アミノ酸配列と、ＢＬＡＳＴ（Ｂａｓｉｃ　Ｌｏｃａｌ　Ａｌｉｇｎｍｅｎｔ　Ｓｅａｒｃｈ　Ｔｏｏｌ　ａｔ　ｔｈｅ　Ｎａｔｉｏｎａｌ　Ｃｅｎｔｅｒ　ｆｏｒ　Ｂｉｏｌｏｇｉｃａｌ　Ｉｎｆｏｒｍａｔｉｏｎ（米国国立生物学情報センターの基本ローカルアラインメント検索ツール））等（例えば、デフォルトすなわち初期設定のパラメータを用いて）を用いて計算したときに、少なくとも５０％以上、好ましくは７０％以上、さらに好ましくは８０％以上、特に好ましくは９０％以上の同一性を有しているものが挙げられる。 The amino acid sequence in which one or more amino acids have been deleted, added, and/or substituted in the above amino acid sequence has an identity of at least 50%, preferably 70%, more preferably 80%, 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 or initial setting parameters).
　本実施形態に係るＲａｓ阻害ペプチドは、その種々の誘導体、及び／又は修飾体も包含する。係る誘導体としては、ペプチドの飽和脂肪鎖が不飽和脂肪鎖に置換されているもの、ペプチドの原子の一部が放射性または非放射性の同位体原子を含む他の原子に置換されているもの、ペプチドのアミド結合がチオアミド結合（－ＮＨ－Ｃ（＝Ｓ）－）に置換されているもの、ペプチドのアミド結合がアルケン（－Ｃ＝Ｃ－）に置換されているもの、ペプチドのアミド結合がアルキル（－Ｃ－Ｃ－）に置換されているもの、ペプチドのアミド結合がヒドロキシエチレン（－Ｃ（－ＯＨ）－Ｃ－）に置換されているもの、ペプチドのアミド結合がエステル（－Ｏ－Ｃ（＝Ｏ）－）に置換されているもの、ペプチドのアミド結合がアルケン（－Ｃ＝Ｃ－）に置換されているもの、ペプチドのアミド結合が（－Ｃ－ＮＨ－）に置換されているもの、又はペプチドのアミド結合が（－Ｃ（＝Ｏ）－Ｃ－）に置換されているものなどが挙げられ、係る修飾体としては、ペプチドのα位炭素が二置換されているもの、ペプチドのアミド結合がＮ－アルキル化されているもの、ペプチドの官能基の一部がハロゲン化、シアノ化、ニトロ化、オキソ化、ヒドロキシ化、アミノ化、デアミノ化、デヒドロ化、アミド化、アセチル化、メトキシ化、プレニル化、アルキル化などの修飾を受けているもの（例えば、ペプチドのアミノ基の一部がアセチル化やアルキル化やデアミノ化されているもの、ペプチドのカルボキシ基の一部がアミドやエステルになっているものなど）、ペプチドのＳがスルホキシドＳ（＝Ｏ）又はスルホンＳ（＝Ｏ）２になっているもの、ペプチドがケミカルリンカーを介して多量体化しているもの、ペプチドがビオチン標識化されているもの、ペプチドが蛍光標識化されているもの、ペプチドが発光標識化されているもの、さらにはアルキル鎖、ポリエチレングリコール、抗体、レクチン類、糖鎖、酵素、膜透過性ペプチド、低分子化合物、又はタンパク質のユビキチン化を誘導する分子などとペプチドを融合させたもの等が挙げられるが、これらに限定されない。 The Ras inhibitory peptide according to the present 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 (-C-C-), 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 (-O-C(=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 an (-C- Examples of such modifications include peptides in which the α-carbon of the peptide is disubstituted, peptides in which the amide bond of the peptide is N-alkylated, peptides in which some of the functional groups of the peptide have been modified by halogenation, cyanation, nitration, oxo-ation, hydroxylation, ammination, deamination, dehydrogenation, amidation, acetylation, methoxylation, prenylation, alkylation, etc. (for example, peptides in which some of the amino groups of the peptide have been acetylated, alkylated, or deaminated, and peptides in which some of the carboxy groups of the peptide have been amide or ester), peptides in which S is a sulfoxide S(═O) or a sulfone S(═O). Examples of the peptide include, but are not limited to, peptides in which the peptide is fused with an alkyl chain, polyethylene glycol, an antibody, a lectin, a sugar chain, an enzyme, a membrane-permeable peptide, a low molecular weight compound, or a molecule that induces ubiquitination of a protein.
　本実施形態に係るＲａｓ阻害ペプチドは、ペプチドの塩も包含する。ペプチドの塩としては、生理学的に許容される塩基や酸との塩が用いられ、例えば、無機酸（塩酸、臭化水素酸、ヨウ化水素酸、硫酸、リン酸等）の付加塩、有機酸（ｐ－トルエンスルホン酸、メタンスルホン酸、シュウ酸、ｐ－ブロモフェニルスルホン酸、カルボン酸、コハク酸、クエン酸、安息香酸、酢酸等）の付加塩、無機塩基（水酸化アンモニウム、又はアルカリ、若しくはアルカリ土類金属水酸化物、炭酸塩、重炭酸塩等）、アミノ酸の付加塩等が挙げられる。 The Ras inhibitory peptide according to this embodiment also includes peptide salts. As the peptide salt, a salt with a physiologically acceptable base or acid is used, and examples thereof include addition salts with inorganic acids (hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid, etc.), addition salts with organic acids (p-toluenesulfonic acid, methanesulfonic acid, oxalic acid, p-bromophenylsulfonic acid, carboxylic acid, succinic acid, citric acid, benzoic acid, acetic acid, etc.), addition salts with inorganic bases (ammonium hydroxide, or alkali or alkaline earth metal hydroxides, carbonates, bicarbonates, etc.), and addition salts of amino acids.
　本実施形態に係るＲａｓ阻害ペプチドは、プロドラッグであってもよい。プロドラッグは、生体内における生理条件下で酵素や胃酸などによる反応によって本発明のペプチドに変換される化合物、すなわち酵素的に酸化、還元、加水分解などを起こして本発明のペプチドに変化する化合物、胃酸などにより加水分解などを起こして本発明のペプチドに変化する化合物をいう。 The Ras inhibitory peptide according to this embodiment may be a prodrug. A prodrug is a compound that is converted into the peptide of the present invention by a reaction with an enzyme, gastric acid, or the like under physiological conditions in the body, i.e., a compound that is enzymatically oxidized, reduced, hydrolyzed, or the like to be converted into the peptide of the present invention, or a compound that is hydrolyzed by gastric acid or the like to be converted into the peptide of the present invention.
　本実施形態に係るＲａｓ阻害ペプチドは、結晶であってもよく、結晶形が単一であっても結晶形混合物であっても本発明のペプチドに包含される。結晶は、自体公知の結晶化法を適用して、結晶化することによって製造することができる。 The Ras inhibitory peptide of this embodiment may be a crystal, and whether the crystalline form is a single one or a mixture of crystalline forms, the peptide of the present invention is included. The crystals can be produced by crystallization using a crystallization method known per se.
（界面活性剤）
　界面活性剤は、一般に分子内に親水基及び疎水基（親油基）を持つ物質で、一定濃度以上でミセルやベシクルを形成し、表面張力を弱める作用を持ち、本発明のＲａｓ阻害ペプチドの凝集を抑制する作用を有する界面活性剤であれば特に限定はない。 (Surfactant)
Surfactants are generally substances that have hydrophilic and hydrophobic groups (lipophilic groups) in 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.
　本発明の組成物において使用される界面活性剤は、本明細書において記載されるＲａｓ阻害ペプチドを安定化させ、凝集を阻害する任意の界面活性剤又は界面活性剤の組み合わせを含む。従って、本発明の界面活性剤としては、これに限定されないが、ポリオキシエチレンヒマシ油、ポリオキシエチレンソルビタン脂肪酸エステル、Ｔｒｉｔｏｎ（商標）Ｘ－１００、ノノキシノール－９、トリエタノールアミン、トリエタノールアミンポリペプチドオレエート、ポリオキシエチレン－６６０ヒドロキシステアレート（ＰＥＧ－１５、ＳｏｌｕｔｏｌＨ１５）、大豆レシチン、ポロキサマー、ヘキサデシルアミン、オクタデシルアミン、オクタデシルアミノ酸エステル、リソレシチン、ジメチル－ジオクタデシルアンモニウムブロミド、メトキシヘキサデシルグリセロール、プルロニック（登録商標）（ｐｌｕｒｏｎｉｃ）ポリオール、ポリアミン（例えば、ピラン、硫酸デキストラン、ポリＩＣ、カーボポール）、油エマルジョン、及びミネラルゲル（例えば、リン酸アルミニウム）が挙げられる。 Surfactants used in the compositions of the present invention include any surfactant or combination of surfactants that stabilize the Ras inhibitory peptides described herein and inhibit aggregation. Thus, 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), soy lecithin, poloxamer, hexadecylamine, octadecylamine, octadecyl amino acid esters, lysolecithin, dimethyl-dioctadecyl ammonium bromide, methoxyhexadecylglycerol, pluronic polyols, polyamines (e.g., pyran, dextran sulfate, poly IC, carbopol), oil emulsions, and mineral gels (e.g., aluminum phosphate).
　好ましい界面活性剤は、ポリオキシエチレンヒマシ油又はポリオキシエチレンソルビタン脂肪酸エステルである。ポリオキシエチレンヒマシ油としては：
ａ）商標名「ＣＲＥＭＯＰＨＯＲ」（ＢＡＳＦＴＷＥＥＮ社）で市販のものを含む、ポリオキシエチレンヒマシ油誘導体、特に、ＣＲＥＭＯＰＨＯＲ　ＥＬ又はＥＬＰ（ＰＥＧ　３５ヒマシ油、ポリエトキシル化ヒマシ油、マクロゴグリセロリ・リシノレアス、マクロゴグリセロリ・ヒドロキシステアラス、ＰＯＥ－３５ヒマシ油、ＰＥＧリシノレアートとも称される）；ＣＲＥＭＯＰＨＯＲ　ＥＬＰ（水、カリウム及び遊離脂肪酸が低含量であり、ＣＲＥＭＯＰＨＯＲ　ＥＬの精製グレードである、ポリオキシエチレングリコール化ヒマシ油）、及びＣＲＥＭＯＰＨＯＲ　ＲＨ　４０（ポリオキシル４０水素添加ヒマシ油、マクロゴールグリセロールヒドロキシステアリン酸、ポリオキシエチレン４０水素添加ヒマシ油、ＰＥＧ－４０水素添加ヒマシ油とも称される）、並びに Preferred surfactants are polyoxyethylene castor oils or polyoxyethylene sorbitan fatty acid esters. Polyoxyethylene castor oils include:
a) polyoxyethylene castor oil derivatives, including those commercially available under the trade name "CREMOPHOR" (BASFTWEEN), in particular CREMOPHOR EL or ELP (also referred to as 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 low water, potassium and free fatty acid content, which is a refined grade of CREMOPHOR EL), and CREMOPHOR RH 40 (also referred to as polyoxyl 40 hydrogenated castor oil, macrogolglycerol hydroxystearic acid, polyoxyethylene 40 hydrogenated castor oil, PEG-40 hydrogenated castor oil);
ｂ）商標名「ＴＷＥＥＮ（登録商標）」（ＩＣＩ　Ａｍｅｒｉｃａｓ社）で市販のものを含む、ポリオキシエチレンソルビタン脂肪酸エステル、特に、ＴＷＥＥＮ８０（８０［ポリオキシエチレン（２０）モノオレイン酸ソルビタン］、ポリソルベート８０としても公知）；ポリオキシエチレン２０モノオレイン酸ソルビタン（エチレンオキシドと重合させたソルビトール及びその無水物の部分的な脂肪酸エステル）、より具体的にはポリオキシエチレン－ソルビタン－脂肪酸モノ－オレイルエステル、（ミリスチン酸、パルミチン酸、パルミトレイン酸、ステアリン酸、オレイン酸及びリノレン酸、主にオレイン酸を含む脂肪酸の混合物を含む）、ＴＷＥＥＮ８５（８５［ポリオキシエチレン（２０）トリオレイン酸ソルビタン］、ポリソルベート８５としても公知）；ポリオキシエチレン２０トリオレイン酸ソルビタン（エチレンオキシドと重合させたソルビトールおよびその無水物の部分的な脂肪酸エステル）、具体的には、ポリオキシエチレン－ソルビタン－脂肪酸トリ－オレイルエステル（ミリスチン酸、パルミチン酸、パルミトレイン酸、ステアリン酸、オレイン酸およびリノレン酸、主にオレイン酸を含む脂肪酸の混合物を含む）、ＴＷＥＥＮ２０（ポリソルベート２０、ポリ（オキシ－１，２－エタンジイル）誘導体としても公知）；ポリオキシエチレン２０ラウレート；ポリオキシエチレン２０モノラウリン酸ソルビタン、ソルビタンモノドデカノアート；ＴＷＥＥＮ６０（ポリソルベート６０、ポリオキシエチレン２０ステアレート（ソルビタンモノオクタデカノアートポリ（オキシ－１，２－エタンジイル）誘導体としても公知）、並びにＴＷＥＥＮ４０（ポリソルベート４０、ポリオキシエチレン２０ソルビタンモノパルミテート、ソルビタンモノヘキサデカノアートとしても公知）。 b) Polyoxyethylene sorbitan fatty acid esters, including those commercially available 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 specifically polyoxyethylene-sorbitan-fatty acid mono-oleyl esters, (containing a mixture of fatty acids including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, mainly oleic acid), TWEEN 85 (85 [polyoxyethylene (20) sorbitan trioleate], also known as polysorbate 85); polyoxyethylene 20 sorbitan trioleate (polymerized with ethylene oxide), Partial fatty acid esters of sorbitol and its anhydrides), specifically polyoxyethylene-sorbitan-fatty acid tri-oleyl esters (containing a mixture of fatty acids including myristic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid and linoleic acid, mainly oleic acid), TWEEN 20 (also known as polysorbate 20, poly(oxy-1,2-ethanediyl) derivative); 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) derivative), and TWEEN 40 (polysorbate 40, polyoxyethylene 20 sorbitan monopalmitate, sorbitan monohexadecanoate).
　いくつかの実施形態において、界面活性剤は、ポリオキシエチレンヒマシ油誘導体（特にＣＲＥＭＯＰＨＯＲ　ＥＬ（ポリオキシエチレン－３５－リシノール酸塩、別名は、ＰＥＧ－３５ヒマシ油、ポリオキシル－３５ヒマシ油、ポリオキシル－３５硬化ヒマシ油、マクロゴルグリセロールリシノール酸と称する場合もある。）、ポリオキシエチレンソルビタン脂肪酸エステル（特にＴＷＥＥＮ８０）、及びそれらの混合物から選択される。 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.
　好ましい実施形態において、界面活性剤は、本発明の組成物の総量において、少なくとも約１０質量％、好ましくは、約１０～２０（例えば約１５）質量％のＣＲＥＭＯＰＨＯＲ　ＥＬ、及び／又はＴＷＥＥＮ８０を含む。なお、図１に、この実施形態における組成物を構成するＲａｓ阻害ペプチド及び２種類の界面活性剤の構造式を示した。 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 in the total amount of the composition of the present invention. Note that the structural formulae of the Ras inhibitor peptide and two types of surfactants constituting the composition in this embodiment are shown in Figure 1.
（組成物）
　本発明に係る組成物は、上述したＲａｓ阻害ペプチドと、界面活性剤とを含み、悪性腫瘍などの増殖を抑制することが可能である。その投与形態は特に限定されず、経口的投与でも非経口的投与でもよい。非経口投与としては、例えば、筋肉内注射、静脈内注射、皮下注射等の注射投与、経皮投与、経粘膜投与（経鼻、経口腔、経眼、経肺、経膣、又は経直腸投与）等が挙げられる。組成物中のペプチドは、代謝及び排泄されやすい性質に鑑みて、各種の修飾を行うことができる。例えば、ペプチドにアルキル鎖、ポリエチレングリコール、又は糖鎖などを付加することで、血中滞留時間を長くする、抗原性を低下させることができる。また、ポリ乳酸・グリコール（ＰＬＧＡ）などの生体内分解性の高分子化合物、多孔性ヒドロキシアパタイト、リポソーム、表面修飾リポソーム、不飽和脂肪酸で調製したエマルジョン、ナノパーティクル、ナノスフェア等を徐放化基剤として用い、これにペプチドを内包させてもよい。経皮投与する場合、弱い電流を皮膚表面に流して角質層を透過させることもできる（イオントフォレシス法）。 (Composition)
The composition according to the present invention contains the above-mentioned Ras inhibitory peptide and a surfactant, and is capable of suppressing the growth of malignant tumors and the like. The administration form is not particularly limited, and may be oral or parenteral. Examples of parenteral administration include injection administration such as intramuscular injection, intravenous injection, and subcutaneous injection, transdermal administration, and transmucosal administration (transnasal, oral, ocular, pulmonary, vaginal, or rectal administration). The peptide in the composition may be modified in various ways in consideration of its tendency to be metabolized and excreted. For example, by adding an alkyl chain, polyethylene glycol, or sugar chain to the peptide, the blood residence time can be increased and the antigenicity can be reduced. In addition, biodegradable polymer compounds such as polylactic acid glycol (PLGA), porous hydroxyapatite, liposomes, surface-modified liposomes, emulsions prepared with unsaturated fatty acids, nanoparticles, nanospheres, etc. may be used as sustained-release bases, and the peptide may be encapsulated in them. In the case of transdermal administration, a weak electric current can be passed through the skin surface to cause penetration through the stratum corneum (iontophoresis method).
　上記の組成物は、界面活性剤に加えて、薬学的に許容できる担体、賦形剤、添加剤等を加えて製剤化してもよい。剤形としては、例えば、液剤（注射剤など）、分散剤、懸濁剤、錠剤、丸剤、粉末剤、坐剤、散剤、細粒剤、顆粒剤、カプセル剤、シロップ剤、トローチ剤、吸入剤、軟膏剤、点眼剤、点鼻剤、点耳剤、パップ剤等が挙げられる。これらの製剤は、速放性製剤又は徐放性製剤等の放出制御製剤（徐放性マイクロカプセルなど）であってもよい。製剤化は、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、溶解剤、溶解補助剤、着色剤、矯味矯臭剤、安定化剤、乳化剤、吸収促進剤、ｐＨ調整剤、防腐剤、抗酸化剤などを適宜使用し、常法により行うことができる。製剤化に用いられる成分の例としては、精製水、食塩水、リン酸緩衝液、デキストロース、グリセロール、エタノール等の薬学的に許容される有機溶剤、動植物油、乳糖、マンニトール、ブドウ糖、ソルビトール、結晶セルロース、ヒドロキシプロピルセルロース、デンプン、コーンスターチ、無水ケイ酸、ケイ酸アルミニウムマグネシウム、コラーゲン、ポリビニルアルコール、ポリビニルピロリドン、カルボキシビニルポリマー、カルボキシメチルセルロースナトリウム、ポリアクリル酸ナトリウム、アルギン酸ナトリウム、水溶性デキストラン、カルボキシメチルスターチナトリウム、ぺクチン、メチルセルロース、エチルセルロース、キサンタンガム、アラビアゴム、トラガント、カゼイン、寒天、ポリエチレングリコール、ジグリセリン、グリセリン、プロピレングリコール、ワセリン、パラフィン、ミリスチン酸オクチルドデシル、ミリスチン酸イソプロピル、高級アルコール、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン等が挙げられるが、これらに限定されない。ペプチドが経粘膜吸収されにくい場合は、難吸収性薬物の吸収を改善する吸収促進剤として、ポリオキシエチレンラウリルエーテル類、ラウリル硫酸ナトリウム、サポニン等の界面活性剤；グリココール酸、デオキシコール酸、タウロコール酸等の胆汁酸塩；ＥＤＴＡ、サリチル酸類等のキレート剤；カプロン酸、カプリン酸、ラウリン酸、オレイン酸、リノール酸、混合ミセル等の脂肪酸類；エナミン誘導体、Ｎ－アシルコラーゲンペプチド、Ｎ－アシルアミノ酸、シクロデキストリン類、キトサン類、一酸化窒素供与体等を用いてもよい。 The above compositions may be formulated by adding pharma- ceutically 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 be immediate-release or sustained-release controlled-release formulations (e.g., sustained-release microcapsules). Formulation may be performed by conventional methods using, for example, excipients, binders, disintegrants, lubricants, solubilizers, solubilizers, colorants, flavorings, stabilizers, emulsifiers, absorption promoters, pH adjusters, preservatives, antioxidants, etc., as appropriate. Examples of ingredients used in formulations include purified water, saline, phosphate buffer, dextrose, glycerol, ethanol and other pharma- ceutically 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 polymer, 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, human serum albumin and the like, but are not limited thereto. In cases where a peptide is poorly absorbed through the mucosa, absorption enhancers that improve the absorption of poorly absorbed drugs may be used, such as 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.
　本発明の組成物は、液体又は固体であることができる。液体製剤は、水又は、水・アルコール混合物のような水性／有機性混合物のような好適な水性溶媒にて調製される水溶液又は水性懸濁液であることができる。液体製剤は、約５．５～約７．５の間、約６．０～約７．０の間、約６．０～約６．５の間、たとえば、約６．０、６．１、６．２、６．３、６．４又は６．５のｐＨを有することができる。液体製剤は室温、冷蔵（たとえば、２～８℃）又は冷凍（たとえば、－２０℃又は－８０℃）で保存することができる。固体製剤は好適な方法で調製することができ、たとえば、凍結保護剤の添加によってケーキ又は粉末の形態であることができる。固体製剤は溶解することができ、すなわち、好適な媒体で再構成し、投与に好適な液体になることができる。固体製剤を再構成するのに好適な溶媒には、水、等張の生理食塩水、緩衝液、たとえば、リン酸緩衝化生理食塩水、リンガー（乳酸加又はデキストロース）溶液、必須無機質媒体、アルコール／水溶液、デキストロース溶液等が挙げられる。 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, 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 in a suitable manner, for example in the form of a cake or powder by the addition of a cryoprotectant. Solid formulations can be dissolved, i.e., reconstituted in a suitable vehicle, to become a liquid suitable for administration. Solvents suitable for reconstituting solid formulations include water, isotonic saline, buffered solutions such as phosphate buffered saline, Ringer's (lactated or dextrose) solution, essential mineral media, alcohol/aqueous solutions, dextrose solution, and the like.
　一実施形態では、本発明の組成物は１０倍濃縮ダルベッコＰＢＳ溶液（Ｄ－ＰＢＳ）を体積比で２５～４５％含む。Ｄ－ＰＢＳは、通常、塩化カリウム０．０２％、リン酸二水素ナトリウム０．０２％、塩化ナトリウム０．８％、リン酸水素二ナトリウム０．１１５％を含む。 In one embodiment, the composition of the present invention contains 25-45% by volume of 10x concentrated Dulbecco's PBS solution (D-PBS). D-PBS typically contains 0.02% potassium chloride, 0.02% sodium dihydrogen phosphate, 0.8% sodium chloride, and 0.115% disodium hydrogen phosphate.
　一実施形態では、本発明の組成物はジメチルスルホキシド（ＤＭＳＯ）を含む。ＤＭＳＯは、難溶性物質を溶解するための比較的毒性の少ない有機溶媒として知られている。本発明の組成物の製造工程において、最初にＲａｓ阻害ペプチドをＤＭＳＯに溶解し、その後、上記界面活性剤を含む水溶液で希釈してもよい。本発明の組成物中におけるＤＭＳＯの濃度は、好ましくは、５０％以下であり、より好ましくは２０％以下である。 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 manufacturing process of the composition of the present invention, the Ras inhibitor peptide may be first dissolved in DMSO and then diluted with an aqueous solution containing the above-mentioned surfactant. The concentration of DMSO in the composition of the present invention is preferably 50% or less, more preferably 20% or less.
（作用効果）
　本発明の組成物は、ＤＭＳＯやエタノールなどの有機溶媒に溶かしたＲａｓ阻害ペプチドを、生理食塩水、リン酸緩衝液又はグルコース溶液などの水溶液で希釈したときに、白濁や沈殿を生じることなしに容易に溶解することができ、生体に投与した際の薬効を向上させるものである。その理由は必ずしも明らかではなく、また如何なる理論にも拘束されるものではないが、本発明の組成物は、Ｒａｓ阻害剤が水溶液中で約５～１００ｎｍのナノミセルを形成するために、有効成分であるＲａｓ阻害剤が白濁、沈殿しないものと考えられる。ナノミセルの大きさは好ましくは５～５０ｎｍの粒子径であり、より好ましくは５～２５ｎｍである。このようなナノミセルの粒子径は、透過型電子顕微鏡または走査型電子顕微鏡の写真像から測定することができる。また、レーザー光回折法を用いた粒子径測定により得られる個数換算の粒子径分布において約５～１００ｎｍのナノミセルが全体の５０％以上、好ましくは７０％以上、より好ましくは８０％以上、さらに好ましくは９０％以上である。なお、このような粒子径のナノミセルを形成することにより、生体内に投与したときに腎臓で濾過されて排出されることなく血流中での安定性を高めることができると考えられる。また、この程度の粒子径を有することにより腫瘍細胞などの標的細胞内へも容易に入り込めると考えられる。 (Action and Effect)
The composition of the present invention can easily dissolve a Ras inhibitor peptide dissolved in an organic solvent such as DMSO or ethanol without causing turbidity or precipitation when diluted with an aqueous solution such as physiological saline, phosphate buffer or glucose solution, and improves the pharmaceutical effect when administered to a living body. The reason is not necessarily clear, and it is not bound by any theory, but it is considered that the Ras inhibitor, which is an active ingredient, does not become turbid or precipitate because the Ras inhibitor forms nanomicelles of about 5 to 100 nm in the aqueous solution. The size of the nanomicelle is preferably a particle diameter of 5 to 50 nm, more preferably 5 to 25 nm. The particle diameter of such nanomicelles can be measured from a photographic image of a transmission electron microscope or a scanning electron microscope. In addition, in the particle diameter distribution calculated by number obtained by particle diameter measurement using a laser light diffraction method, nanomicelles of about 5 to 100 nm account for 50% or more of the total, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. It is believed that forming nanomicelles with such particle sizes will increase the stability in the bloodstream when administered to the body, without being filtered out by the kidneys and excreted. In addition, it is believed that having such particle sizes will allow the nanomicelles to easily enter target cells such as tumor cells.
　次に実施例を挙げ、本発明を更に詳しく説明するが、本発明はこれら実施例に何ら制約されるものではない。なお、以下の実施例において、各種成分の添加量を示す数値の単位％は、質量％を意味する。 The present invention will be described in more detail below with reference to examples, but the present invention is in no way limited to these examples. In the following examples, the unit % of the numerical values showing the amount of each component added means mass %.
（実施例１）合成ペプチドＫＳ－５８水溶液の調製
　合成ペプチドＫＳ－５８（配列番号１７の環状ペプチド）は、株式会社スクラムでＦｍｏｃベースの固相ペプチド合成を行い、逆相高速液体クロマトグラフィー（ＲＰ－ＨＰＬＣ）により精製した。ペプチドの純度は分析用ＲＰ－ＨＰＬＣで確認し、構造の帰属はＭＡＬＤＩ－ＴＯＦ質量分析法で行った。ハロゲン化化学リンカーによるジスルフィド結合形成及びチオエーテル結合形成は、それぞれ既報の方法に準じて行った（特許文献１）。Ｔｗｅｅｎ８０（カタログ番号９００５－６５－６）は、東京化成より購入し、Ｃｒｅｍｏｐｈｏｒ　ＥＬ（カタログ番号０９７２７－１４）はナカライ社より購入した。 Example 1 Preparation of Synthetic Peptide KS-58 Aqueous Solution 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 Co., Ltd.
　ＫＳ－５８を１００ｍｇ／ｍＬとなるようにＤＭＳＯに溶解させた後、界面活性剤を含まない、又はＣｒｅｍｏｐｈｏｒ　ＥＬを１～５％含むＤ－ＰＢＳ（カタログ番号０４５－２９７９５、富士フィルム和光純薬）で１０倍希釈した。この水溶液中におけるＫＳ－５８の終濃度は１０ｍｇ／ｍＬ、ＤＭＳＯの終濃度は１０％である。その結果を図２（Ａ）に示す。界面活性剤を含まないＤ－ＰＢＳでは、明確な白濁を呈する懸濁液となった。Ｃｒｅｍｏｐｈｏｒ　ＥＬを１％含むＤ－ＰＢＳでは、薄い白濁液となり、Ｃｒｅｍｏｐｈｏｒ　ＥＬを３％又は５％含むＤ－ＰＢＳでは、澄明な溶解液となった。 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.) that did not contain a surfactant or contained 1-5% Cremophor EL. The final concentration of KS-58 in this aqueous solution was 10 mg/mL, and the final concentration of DMSO was 10%. The results are shown in Figure 2 (A). In D-PBS that did not contain a surfactant, a suspension with a clear cloudiness was obtained. In D-PBS containing 1% Cremophor EL, a thin cloudy liquid was obtained, and in D-PBS containing 3% or 5% Cremophor EL, a clear solution was obtained.
　ＫＳ－５８を７５ｍｇ／ｍＬとなるようにＤＭＳＯに溶解させた後、Ｃｒｅｍｏｐｈｏｒ　ＥＬを１０％含むＤ－ＰＢＳ溶液で５倍希釈した。この際、Ｄ－ＰＢＳ溶液は、１０×Ｄ－ＰＢＳ（カタログ番号０４８－２９８０５、富士フィルム和光純薬）を精製水で希釈し、１０×Ｄ－ＰＢＳを７０～１０％含むように調製した。ＫＳ－５８の終濃度は１５ｍｇ／ｍＬ、ＤＭＳＯの終濃度は２０％である。その結果を図２（Ｂ）に示す。溶液中に含まれる１０×Ｄ－ＰＢＳ組成濃度に応じて、ＫＳ－５８の溶解性が変化し、１０×Ｄ－ＰＢＳが４０～２０％含まれる場合では、析出や白濁がみられない澄明な溶解液となった。 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, Ltd.) with purified water to contain 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 changed depending on the composition concentration of 10x D-PBS contained in the solution, and when 10x D-PBS was contained at 40-20%, a clear solution without precipitation or cloudiness was obtained.
　ＫＳ－５８を２００ｍｇ／ｍＬとなるようにＤＭＳＯに溶解させた後、Ｃｒｅｍｏｐｈｏｒ　ＥＬを１０％含むＤ－ＰＢＳ溶液で５倍希釈した。この際、Ｄ－ＰＢＳ溶液は、１０×Ｄ－ＰＢＳを精製水で希釈し、１０×Ｄ－ＰＢＳを４５～２５％含むように調製した。ＫＳ－５８の終濃度は２０ｍｇ／ｍＬ、ＤＭＳＯの終濃度は２０％である。その結果を図２（Ｃ）に示す。いずれも、析出や白濁がみられない澄明な溶解液となった。以上の結果から、適度な濃度のＤ－ＰＢＳとＣｒｅｍｏｐｈｏｒ　ＥＬといった界面活性剤と混合することで、ＫＳ－５８の澄明な水溶液を調製できることが示された。 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 contain 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 show 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.
（実施例２）合成ペプチドＫＳ－５８を含む組成物のインビボ薬効評価
　ＢＡＬＢ／ｃＣｒＳｌｃマウス（４週齢）の皮下にＣＴ２６マウス大腸癌細胞株を移植し、腫瘍を形成させた。ＫＳ－５８をＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液に２０ｍｇ／ｍＬで溶解させた溶液を、生理食塩水を用いて希釈し、皮下担癌マウスに対して１０又は４０ｍｇ／ｋｇとなるように週に一回（１、７、１４日目）尾静脈内投与した（ｎ＝４）。この際、ＫＳ－５８を含まないＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液を調製し、生理食塩水を用いて希釈し、同様に投与する群を設けて、比較対象（コントロール群）とした。上記で調製した３種類の溶液の投与開始後１７日目までの間３日ごとに腫瘍の大きさを測定した結果を図３に示す。コントロール群と比較して、Ｃｒｅｍｏｐｈｏｒ　ＥＬを用いて溶解されたＫＳ－５８（１０ｍｇ／ｋｇ）及びＫＳ－５８（４０ｍｇ／ｋｇ）を投与した群では、腫瘍の成長が有意に抑制されたが、特にＫＳ－５８（４０ｍｇ／ｋｇ）を投与した群で著しく抑制された。１７日目のコントロール群の腫瘍の体積を１００％とすると、その腫瘍の体積は３４％と見積もられた。ＫＳ－５８をＤＭＳＯ（１０％）／生理食塩水に懸濁し、４０ｍｇ／ｋｇで二日に一回皮下投与した場合、皮下に移植されたＣＴ２６大腸癌細胞株由来の腫瘍の成長は、１７日目の腫瘍の重量を１００％とすると、３５％であると報告されている（非特許文献２）。すなわち、適度な濃度の１０×Ｄ－ＰＢＳとＣｒｅｍｏｐｈｏｒ　ＥＬを用いた溶解によって、ＫＳ－５８の生体における薬効が向上し、抗癌活性を維持したまま投与回数を低減できることが示された。 Example 2: In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 CT26 mouse colon cancer cell line was subcutaneously transplanted into BALB/cCrSlc mice (4 weeks old) to form tumors. KS-58 was dissolved in a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution at 20 mg/mL, and the solution was diluted with saline and administered to the tail vein of subcutaneous tumor-bearing mice once a week (1st, 7th, and 14th days) at 10 or 40 mg/kg (n=4). At this time, a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was prepared, diluted with saline, and administered in the same manner to a group as a comparison subject (control group). The tumor size was measured every 3 days until the 17th day after the start of administration of the three types of solutions prepared above, and the results are shown in FIG. 3. Compared to the control group, tumor growth was significantly suppressed in the groups administered KS-58 (10 mg/kg) and KS-58 (40 mg/kg) dissolved using Cremophor EL, but was particularly markedly suppressed in the group administered KS-58 (40 mg/kg). The tumor volume was estimated to be 34%, assuming that the tumor volume of the control group on day 17 was 100%. It has been reported that when KS-58 was suspended in DMSO (10%)/physiological saline and administered subcutaneously at 40 mg/kg once every two days, the growth of a tumor derived from a subcutaneously implanted CT26 colon cancer cell line was 35%, assuming that the weight of the tumor on day 17 was 100% (Non-Patent Document 2). That is, it was shown that dissolution using moderate concentrations of 10xD-PBS and Cremophor EL improved the efficacy of KS-58 in the living body, and the number of administrations could be reduced while maintaining anticancer activity.
（実施例３）合成ペプチドＫＳ－５８を含む組成物のインビボ薬効評価
　Ｋ－Ｒａｓ（Ｇ１２Ｄ）を発現するＰＡＮＣ－１細胞（カタログＮｏ．ＣＲＬ－１４６９、ＡＴＣＣ社製）を２．５×１０６細胞でＢＡＬＢ／ｃＡＪｃｌ－ｎｕ／ｎｕマウス（雄、８週齢）（日本クレア株式会社）の膵臓尾部被膜下に移植し、１週間後、試験に供した。ＫＳ－５８をＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液に２０ｍｇ／ｍＬで溶解させた溶液を、生理食塩水を用いて希釈し、皮下担癌マウスに対して１０又は４０ｍｇ／ｋｇとなるように週に一回（１、７、１４、２１、２８、３５日目）尾静脈内投与した（ｎ＝８）。この際、ＫＳ－５８を含まないＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液を調製し、生理食塩水を用いて希釈し、同様に投与する群を設けて、比較対象（コントロール群）とした。Ｓｈａｍ群とは、癌細胞を含まない細胞培地で移植手技を実施しＫＳ－５８を含まないＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液を投与した群である。投与開始から６週間後のマウスの臓器（膵臓、肝臓、腎臓）の重量を測定した結果を図４に示す。Ｓｈａｍ群と比較して、コントロール群の膵臓の重量は、ＰＡＮＣ－１腫瘍の増大に由来する有意な重量の増加が確認された（†††ｐ＜０．００１、Ｓｕｔｕｄｅｎｔ’ｓ　ｔ－ｔｅｓｔ）。そして、コントロール群と比較して、Ｃｒｅｍｏｐｈｏｒ　ＥＬを用いて溶解されたＫＳ－５８（２０ｍｇ／ｋｇ）及びＫＳ－５８（４０ｍｇ／ｋｇ）を投与した群では、腫瘍の成長が有意に抑制された（＊ｐ＜０．０５、＊＊＊ｐ＜０．００１、Ｄｕｎｎｅｔｔ’ｓ）。肝臓と腎臓の重量には、各郡間で有意な差はみられなかった。Ｓｈａｍ群の膵臓の重量を０％、コントロール群の膵臓の重量を１００％とすると、ＫＳ－５８（２０ｍｇ／ｋｇ）及びＫＳ－５８（４０ｍｇ／ｋｇ）を投与した群では、膵臓の重量は６８％と５０％と見積もられた。ＫＳ－５８をＤＭＳＯ（１０％）／生理食塩水に懸濁し、４０ｍｇ／ｋｇで二日に一回皮下投与した場合、ＰＡＮＣ－１同所移植マウスの膵臓の重量は、Ｓｈａｍ群の膵臓の重量を０％、コントロール群の膵臓の重量を１００％とすると、６６％であると報告されている（非特許文献１）。すなわち、適度な濃度の１０×Ｄ－ＰＢＳとＣｒｅｍｏｐｈｏｒ　ＥＬを用いた溶解によって、ＫＳ－５８の生体における薬効が向上し、抗癌活性を維持または向上し、さらに投与回数を低減できることが示された。 Example 3: In vivo efficacy evaluation of a composition containing synthetic peptide KS-58 2.5 x 106 cells of PANC-1 cells expressing K-Ras (G12D) (catalog No. CRL-1469, manufactured by ATCC) were transplanted under the pancreatic tail capsule of BALB/cAJcl-nu/nu mice (male, 8 weeks old) (CLEA Japan, Inc.) and subjected to the test after 1 week. KS-58 was dissolved at 20 mg/mL in a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution, and the solution was diluted with saline and administered to subcutaneous tumor-bearing mice at 10 or 40 mg/kg into the tail vein once a week (1st, 7th, 14th, 21st, 28th, 35th days) (n=8). In this case, a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was prepared, diluted with saline, and administered to a group for comparison (control group). The sham group was a group in which transplantation was performed using a cell culture medium not containing cancer cells, and a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution not containing KS-58 was administered. The weights of the mouse organs (pancreas, liver, kidney) 6 weeks after the start of administration 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 increase in the PANC-1 tumor ( ††† p<0.001, Student's t-test). Compared to the control group, tumor growth was significantly suppressed in the groups administered KS-58 (20 mg/kg) and KS-58 (40 mg/kg) dissolved with Cremophor EL (*p<0.05, ***p<0.001, Dunnett's). No significant difference was observed in liver and kidney weights between the groups. The pancreas weights in the sham group and control group were estimated to be 68% and 50%, respectively, of the sham group and control group, respectively. It has been reported that when KS-58 was suspended in DMSO (10%)/physiological saline and subcutaneously administered at 40 mg/kg once every two days, the weight of the pancreas in mice orthotopically transplanted with PANC-1 was 66%, assuming that the weight of the pancreas in the Sham group was 0% and that in the control group was 100% (Non-Patent Document 1). In other words, it was shown that dissolution using appropriate concentrations of 10xD-PBS and Cremophor EL improved the efficacy of KS-58 in vivo, maintained or improved anticancer activity, and further reduced the number of administrations.
（実施例４）合成ペプチドＫＳ－５８を含む組成物の粒子径の測定
　ＫＳ－５８をＤＭＳＯ（２０％）／Ｃｒｅｍｏｐｈｏｒ　ＥＬ（１０％）／１０×Ｄ－ＰＢＳ（３５％）溶液に２０ｍｇ／ｍＬで溶解させた溶液中に形成される粒子の粒子径を透過型電子顕微鏡ＦＥＩ　Ｔｅｃｎａｉ　Ｆ２０　ＴＥＭで観察した。この結果を図５に示す。約１０ｎｍの均一な粒子の形成が確認された。また、粒子径をＺｅｔａｓｉｚａｅｒ　Ｎａｎｏ　ＺＳＰ（Ｍａｌｖｅｒｎ　Ｉｎｓｔｒｕｍｅｎｔ）を用いた動的光散乱でも評価した。この結果を図６に示す。約１０ｎｍの均一な粒子の形成が確認された。 Example 4 Measurement of particle size of a composition containing synthetic peptide KS-58 The particle size of particles formed in a solution in which KS-58 was dissolved at 20 mg/mL in a DMSO (20%)/Cremophor EL (10%)/10xD-PBS (35%) solution was observed using a transmission electron microscope FEI Tecnai F20 TEM. The results are shown in FIG. 5. The formation of uniform particles of about 10 nm was confirmed. The particle size was also evaluated by dynamic light scattering using a Zetasizer Nano ZSP (Malvern Instrument). The results are shown in FIG. 6. The formation of uniform particles of about 10 nm was confirmed.
　以上、実施形態および実施例を参照して本発明を説明したが、本開示は、上記実施形態および実施例に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解し得る様々な変更をすることができる。 The present invention has been described above with reference to embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.
　この出願は、２０２２年１１月２２日に出願された日本出願特願２０２２－１８６７８１を基礎とする優先権を主張し、その開示の全てをここに取り込む。 This application claims priority based on Japanese Patent Application No. 2022-186781, filed on November 22, 2022, the entire disclosure of which is incorporated herein by reference.
　本発明の組成物は、Ｒａｓ阻害ペプチドの水溶液に対する溶解性を改善することによりＲａｓ阻害ペプチドを医薬品又は診断薬として製造するために、又はＲａｓ阻害ペプチドを医薬品として開発するための臨床研究を行うために有用である。 The composition of the present invention is useful for producing Ras inhibitor peptides as pharmaceuticals or diagnostic agents by improving the solubility of the Ras inhibitor peptides in aqueous solutions, or for conducting clinical research to develop Ras inhibitor peptides as pharmaceuticals.

Claims (7)

  1. 　下記式（１）：
    ｃ［Ｘ１－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｘ１０－Ｘ１１）］　　　（１）
    （式中、Ｘ１は、β－アラニン又はγ－アミノ酪酸残基を表し、
    　Ｘ３は、ロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、
    　Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、
    　Ｘ１０は、バリン、フェニルアラニン、トリプトファン、１－ナフチルアラニン又はそれらのＮ－メチル化アミノ酸残基を表し、
    　Ｘ１１は、Ｄ体又はＬ体のシステイン残基を表し、
    　Ｘ１とＸ１１は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、Ｘ４とＸ１１は、それぞれの側鎖－ＳＨ基の間で、メチレン基、エチレン基、プロピレン基又はブチレン基のリンカーを介して共有結合を形成し、それによって式（１）のペプチドは分子内に２つの環状構造を有する。）で表されるアミノ酸配列からなる環状ペプチド、その誘導体若しくは修飾体又はその薬理学的に許容されるそれらの塩と、少なくとも１種類の界面活性剤と、を含む組成物。     The following formula (1):
    c[X 1 -Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-X 10 -X 11 )] (1)
    (wherein X1 represents a β-alanine or γ-aminobutyric acid residue;
    X3 represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid;
    X5 represents a residue of isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid;
    X10 represents valine, phenylalanine, tryptophan, 1-naphthylalanine or an N-methylated amino acid residue thereof;
    X11 represents a D- or L-cysteine residue;
    X1 and X11 form an amide bond between the amino group and carboxyl group of the main chain, and X4 and X11 form a covalent bond between the -SH groups of the respective side chains via a linker of a methylene group, an ethylene group, a propylene group or a butylene group, whereby the peptide of formula (1) has two cyclic structures in the molecule.
  2. 　下記式（２）：
    ｃ［βＡｌａ－Ｐｒｏ－Ｘ３－ｃ（Ｃｙｓ－Ｘ５－Ｓｅｒ－４ｆＦ－Ａｓｐ－Ｐｒｏ－Ｔｒｐ－ＤＣｙｓ）］　　　（２）
    （式中、Ｘ３は、ロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、
    　Ｘ５は、イソロイシン、ノルロイシン、シクロヘキシルグリシン、フェニルグリシン、２－アミノヘプタン酸、２－アミノオクタン酸、２－アミノノナン酸又は２－アミノデカン酸残基を表し、
    　１位と１１位の残基は、主鎖のアミノ基とカルボキシル基との間でアミド結合を形成し、４位と１１位の２つのシステイン残基は、それぞれの側鎖－ＳＨ基の間で、プロピレン基のリンカーを介して共有結合を形成し、それによって式（２）のペプチドは分子内に２つの環状構造を有する。）で表されるアミノ酸配列からなる環状ペプチド、その誘導体若しくは修飾体又はその薬理学的に許容されるそれらの塩と、少なくとも１種類の界面活性剤と、を含む組成物。     The following formula (2):
    c[βAla-Pro-X 3 -c(Cys-X 5 -Ser-4fF-Asp-Pro-Trp- D Cys)] (2)
    (wherein X3 represents a residue of leucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid;
    X5 represents a residue of isoleucine, norleucine, cyclohexylglycine, phenylglycine, 2-aminoheptanoic acid, 2-aminooctanoic acid, 2-aminononanoic acid or 2-aminodecanoic acid;
    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 -SH groups of the respective side chains via a propylene group linker, so that the peptide of formula (2) has two cyclic structures within the molecule.
  3. 　質量比で少なくとも１０％の界面活性剤が含まれる請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, which contains at least 10% by mass of a surfactant.
  4. 　前記界面活性剤がポリオキシエチレンヒマシ油及びポリオキシエチレンソルビタン脂肪酸エステルからなる群より選択される請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, wherein the surfactant is selected from the group consisting of polyoxyethylene castor oil and polyoxyethylene sorbitan fatty acid esters.
  5. 　１０倍濃縮ダルベッコＰＢＳ溶液を体積比で２５～４５％含む請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, which contains 25 to 45% by volume of 10x concentrated Dulbecco's PBS solution.
  6. 　ジメチルスルホキシドを含む請求項１又は２に記載の組成物。 The composition according to claim 1 or 2, which contains dimethyl sulfoxide.
  7. 　前記界面活性剤が、ポリオキシエチレン－３５－リシノール酸塩又はポリソルベート８０である請求項４に記載の組成物。

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