WO2011148972A1 - Pharmaceutical composition containing biologically active peptide - Google Patents

Pharmaceutical composition containing biologically active peptide Download PDF

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Publication number
WO2011148972A1
WO2011148972A1 PCT/JP2011/061965 JP2011061965W WO2011148972A1 WO 2011148972 A1 WO2011148972 A1 WO 2011148972A1 JP 2011061965 W JP2011061965 W JP 2011061965W WO 2011148972 A1 WO2011148972 A1 WO 2011148972A1
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Prior art keywords
peptide
yvlsr
anxiolytic
receptor
acid
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PCT/JP2011/061965
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French (fr)
Japanese (ja)
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耕作 大日向
千尋 鈴木
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独立行政法人科学技術振興機構
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Publication of WO2011148972A1 publication Critical patent/WO2011148972A1/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
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/20—Hypnotics; Sedatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/22—Anxiolytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/24—Antidepressants

Definitions

  • the present invention relates to a pharmaceutical composition that acts on the nervous system.
  • the present invention also relates to a peptide or an analog thereof, and more specifically, a peptide that activates at least one of a ⁇ opioid receptor, a 5-HT 1A receptor, a D 1 receptor, and a GABA A receptor or an analog thereof.
  • the present invention relates to a food for improving anxiety or sleep.
  • anxiolytic compound can be produced inexpensively and is preferably effective by oral administration.
  • the purpose is to supply drugs and foods that have anti-anxiety action, antidepressant action, etc. even with relatively low doses and few side effects.
  • the present inventors have studied the emotion-regulating action of various food protein-derived peptides, and Tyr-Val-Leu-Ser-Arg derived from milk ⁇ -casein exhibits an anxiolytic action.
  • the present inventors have found that the ⁇ 1 receptor, 5-HT 1A receptor, D 1 receptor, or GABA A receptor is activated downstream of the ⁇ receptor, thereby completing the present invention.
  • Item 1 A pharmaceutical composition comprising a peptide represented by YVLSR (SEQ ID NO: 1) or an analog thereof as an active ingredient.
  • Item 2. The pharmaceutical composition according to Item 1, which is an anxiolytic agent, sleep-inducing agent, sleep-improving agent, schizophrenia therapeutic agent or antidepressant.
  • Item 3 An anti-anxiety or sleep improving food containing a peptide represented by YVLSR or an analog thereof.
  • Item 4 A peptide represented by YVLSR or an analog thereof.
  • Item 5 A peptide represented by YVLSR or an analog thereof for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant.
  • Item 6 A method for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant, comprising the step of administering the pharmaceutical composition according to item 1 or 2 to a patient.
  • the pharmaceutical composition and food of the present invention exhibit high anti-anxiety action, antidepressant action, etc., even at a relatively low dose, as peptides contained therein or their analogs. Moreover, the peptides contained in the pharmaceutical composition and food of the present invention are derived from food proteins with abundant food experience, and it can be expected that there are few side effects.
  • the present inventors are currently trying to identify a receptor to which this peptide directly binds, but if the target receptor of YVLSR, which is a very potent anxiolytic peptide, is clarified, a novel anxiolytic mechanism will be developed. Not only can it be elucidated, it can be a target for new drug discovery.
  • ⁇ Anorexia in the elderly is known to promote aging.
  • endogenous feeding stimulating peptides such as NPY and ghrelin were administered intraventricularly or intravenously, feeding stimulating effects were observed, but there were no reports of effective feeding stimulating peptides by oral administration.
  • the amount of food consumed in mice after oral administration 0.3 mg / kg
  • the delta opioid peptide rubiscolin-6 derived from Rubisco which is a major protein of green leaves and is known as the most abundant protein on earth.
  • ⁇ opioid has both a learning promoting action and an anxiolytic action. Therefore, the anti-anxiety peptide YVLSR that activates the ⁇ opioid receptor is expected to have a feeding-promoting action and a learning-promoting action, and may be used as a material for food for the elderly.
  • Rubisocolin-6 has recently been found to promote normal food intake while suppressing high-fat food intake as described above, and ⁇ opioid receptors have been shown to be involved in normalizing food preferences .
  • the YVLSR and its analogs of the present invention may also have a preference normalization ability.
  • the anxiolytic action is developed as an anxiety-related behavior evaluation method for screening an anxiolytic drug, and can be evaluated by an elevated plus maze test (FIG. 1). Specifically, an anxiolytic candidate substance is administered orally or intraperitoneally, and after 30 minutes, the mouse is placed in the elevated plus maze, and the change in the number of intrusions into the open arm and the staying time on the open arm is indicated. As described above, the strength of the anti-anxiety action can be evaluated.
  • YVLSR showed no affinity for the ⁇ receptor and also no ⁇ opioid activity using mouse vas deferens (MVD). From the above results, it is considered that YVLSR activates the central ⁇ receptor by promoting the release of endogenous ⁇ opioid ligand.
  • the anxiolytic action of the peptide which is the active ingredient of the medicament of the present invention is inhibited by antagonists to ⁇ 1 , serotonin 5-HT 1A , dopamine D 1 , and GABA A receptor. There was no affinity for the receptor. Therefore, the anxiolytic effect of YVLSR is through the release of neurotransmitters such as endogenous ⁇ 1 ligand, serotonin, dopamine, GABA and the activation of ⁇ 1 , 5-HT 1A , D 1 , and GABA A receptors. (Fig. 8).
  • a preferable route of administration of the peptide of the present invention or an analog thereof is oral administration.
  • the active ingredient of the anxiolytic agent of the present invention is a peptide represented by Y (Tyr) -V (Val) -L (Leu) -S (Ser) -R (Arg) or an analog thereof.
  • the amino acids constituting the peptide are L-form amino acids, D-form amino acids, or DL-form amino acids (if the D-form and L-form amino acids are mixed, the racemic form and any amino acid in which one of the enantiomers is excessive) Included).
  • Preferable is a peptide consisting only of L-form amino acids or only D-form amino acids, particularly a peptide consisting only of L-form amino acids.
  • the peptide used in the present invention when it contains two or more asymmetric carbons, it may be in the form of each enantiomer or diastereomer or a mixture of these in any ratio. Separation of enantiomers or diastereomers may be carried out using a normal column, or an optically active column is used, or an optically active group is introduced and optically resolved in the form of a derivative, and then the optically active group is removed. Alternatively, any known method such as optical resolution by forming a salt with an optically active acid or base may be used.
  • salts of peptides or analogs thereof include acid addition salts and base salts.
  • Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid And salts of organic acids such as benzenesulfonic acid, methanesulfonic acid and trifluoroacetic acid.
  • the base salt include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as calcium and magnesium.
  • Solvates include solvates such as water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. Things.
  • the active ingredient of the pharmaceutical composition and food according to the present invention not only the above peptides but also various peptide analogs can be used as long as a desired pharmacological effect is obtained.
  • the peptide active ingredient is a peptide analog
  • the peptide analog includes (1) N-terminal modification and (2) C-terminal modification analogs of the peptide of the active ingredient.
  • the N-terminal amino group of the peptide is linear or branched carbon such as methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, n-butylamino, di-n-butylamino, etc. It may be an amino group mono- or di-substituted with an alkyl group of 1 to 4.
  • the N-terminal amino group or the side chain amino group may be mono- or di-substituted with an aralkyl group such as a benzyl group or a phenethyl group, a formyl group, an acetyl group, or a propionyl group And may be modified with a linear or branched alkanoyl group having 1 to 6 carbon atoms, such as a butyryl group or an isobutyryl group, or an acyl group such as a benzoyl group.
  • an aralkyl group such as a benzyl group or a phenethyl group, a formyl group, an acetyl group, or a propionyl group
  • a linear or branched alkanoyl group having 1 to 6 carbon atoms such as a butyryl group or an isobutyryl group
  • an acyl group such as a benzoyl group.
  • the carboxyl group at the C-terminal of the peptide is an ester with a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, benzyl , Esters with aralkyl groups such as phenethyl, amino groups, methylamine, dimethylamine, ethylamine, diethylamine, propylamine, dipropylamine, n-butylamine, di-n-butylamine, etc.
  • An amine mono- or di-substituted with 4 alkyl groups or an amide with ammonia may be formed.
  • the peptide of the present invention can be obtained by hydrolysis of a natural protein or polypeptide, or can be obtained by chemical synthesis.
  • Examples of the protein or polypeptide to be hydrolyzed include casein derived from milk or human milk. These peptides derived from food materials can be used as they are, or as they are, as they are, by carrying out treatments such as concentration, desalting and purification as necessary.
  • Examples of protein hydrolysis include the use of hydrolytic enzymes derived from animals, plants or microorganisms such as trypsin, chymotrypsin, papain, pepsin, carboxypeptidase, thermolysin, and subtilisin. Accordingly, the peptide of the active ingredient of the present invention can be obtained by adjusting to an appropriate value and reacting at a temperature of about 20 to 40 ° C. for about 30 minutes to 48 hours.
  • YVLSR is partially generated by further digesting casoxin C generated by trypsin digestion of ⁇ -casein with pepsin.
  • the peptide of the present invention may be purified from the obtained reaction solution, and when the food material is enzymatically decomposed, it can be used as it is or added to another food material to form a food or a food composition.
  • Hydrolysis can be performed using strong acids (for example, hydrochloric acid, nitric acid, sulfuric acid, etc.) or strong bases (alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, alkali metal carbonates such as sodium carbonate, potassium carbonate, carbonates).
  • the peptide of the active ingredient of the present invention can be obtained by reacting in water at a temperature of 1 to 100 ° C. for 30 minutes to 48 hours in the presence of an alkali metal hydrogen carbonate such as sodium hydrogen or potassium hydrogen carbonate). It can.
  • the reaction product of the hydrolysis may be used as it is after the pH is adjusted, or the peptide of the active ingredient may be separated and used by purification.
  • the peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a commonly used method for peptide synthesis, a raw material having a reactive carboxyl group and a raw material having a reactive amino group are combined with a method using an active ester such as HBTU, or carbodiimide In a peptide synthesis such as a method using a coupling agent such as When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
  • the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, p- It is bound to a carrier such as an alkoxybenzyl alcohol resin.
  • the condensation reaction is performed in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.
  • the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved.
  • the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like.
  • the synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by Edman degradation, LC-MS, GC-MS, or the like.
  • the peptide of the present invention can also be synthesized by an enzymatic method (see WO2003 / 010307).
  • the administration route of the peptide of the present invention is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, and it can be administered orally or parenterally.
  • the dosage of this peptide varies depending on the type of compound, the administration method, the condition and age of the administered person, etc., but is usually 0.001 to 500 mg / kg, preferably 0.005 to 100 mg / kg per day for an adult. More preferably, it is 0.01 to 30 mg / kg.
  • the peptide (active ingredient) of the present invention is usually administered in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier.
  • a pharmaceutical carrier a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
  • the peptide of the present invention can be used as a food or a medicine per se, or alone or together with a suitable non-toxic carrier for ingestion, diluent or excipient (tablet, uncoated tablet, dragee, effervescent tablet, Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) Or a preparation for food or medicine such as sustained release preparations such as enteric tablets, capsules and granules.
  • the content of the peptide in the food can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.
  • Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method.
  • the liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method.
  • injection it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.
  • These preparations may contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.
  • an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules.
  • these powders and granules may be tableted as they are or with the addition of lubricants such as magnesium stearate and talc.
  • granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc., and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also.
  • an enteric solvent base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc.
  • enteric solvent preparation or with ethylcellulose, carnauba wax, hardened oil, etc.
  • an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension.
  • a transparent syrup such as sucrose, sorbitol, and glycerin
  • active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, pH adjusters, sodium chloride, glucose etc.
  • active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, pH adjusters, sodium chloride, glucose etc.
  • Dissolve in distilled water for injection with an isotonic agent filter aseptically and fill into ampoules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. Good.
  • lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. may be added to the active ingredient and emulsified in water to give an emulsion for injection.
  • the active ingredient is moistened with a suppository base such as cacao butter, fatty acid tri, di and monoglycerides, polyethylene glycol, etc., poured into a mold and cooled,
  • a suppository base such as cacao butter, fatty acid tri, di and monoglycerides, polyethylene glycol, etc.
  • the active ingredient may be dissolved in polyethylene glycol, soybean oil, etc. and then covered with a gelatin film.
  • the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to make an ointment, or rosin, alkyl acrylate polymer After being kneaded with an adhesive such as polyalkyl, it is spread on a non-woven fabric such as polyalkyl to obtain a tape.
  • beverages coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, tea, oolong tea, and milk drinks.
  • Lactic acid bacteria beverage yogurt beverage, carbonated beverage
  • gum gummi
  • jelly candy
  • cookies crackers
  • biscuits ice confectionery (ice cream, ice candy, sorbet, shaved ice, etc.)
  • retort food jelly-like food (je
  • Foods that can be prepared by adding and blending the peptides of the present invention include so-called health foods, functional foods, dietary supplements, supplements, foods for specified health use, combined foods for the sick and the sick (Ministry of Health, Labor and Welfare) , Special-purpose foods) or elderly foods (Ministry of Health, Labor and Welfare, special-purpose foods), uncoated tablets, film-coated tablets, dragees, granules, powders, tablets, capsules (both hard capsules and soft capsules) Including chewable type, syrup type, and drink type.
  • the preparation of food containing the peptide according to the present invention can be carried out by a method known per se.
  • Peptide YVLSR was synthesized by solid phase by Fmoc method, purified using reverse phase HPLC, and further freeze-dried. Male ddY mice were used as experimental animals. The anxiolytic activity was measured by the elevated plus maze test and the open field test described below. The result of the mass spectrum of peptide YVLSR is shown below. Ion Measured value Theoretical value [YVLSR + H] 637.39 637.37 [YVLSR + 2H] 319.20 319.19
  • the elevated plus maze (EPM) consists of two open arms (25cm x 5cm) and two closed arms (25cm x 5cm x 15cm), which are off the floor Combined with the central platform raised 50 cm (see Figure 1).
  • EPM elevated plus maze
  • the periphery of the open arm is open and there are no walls, the mouse walking on the open arm feels uneasy that the mouse falls from a high position. For this reason, the longer the mouse stays in the open arm or the greater the number of times of entry, the less the anxiety of the mouse, which becomes an index of anxiolytic activity.
  • the test was started by placing the mouse on the central platform facing one of the open arms. Cumulative time spent in open arms (time in open arms), number of visits to open arms (visit to open arms), total number of visits to any arm (total visits) was recorded. The percentage of time spent in the open arm and the percentage of visits to the open arm were calculated as an indicator of anxiety.
  • the peptide when administered into the ventricle, it was administered 20 minutes before the test, and when administered intraperitoneally and orally, 30 minutes before the test.
  • the open field used in this experiment is a cylindrical gray device with a diameter of 60 cm and a height of 50 cm, divided into 25 sections by black lines.
  • the mouse performs a search action when placed in a new environment, but normally, there are few searches to the center of the device. However, treatment with anxiolytics increases the time spent in the center circle and the number of entries. Thirty minutes after the administration of the peptide, the mouse was placed in the center of the apparatus and the behavior was observed for 5 minutes.
  • YVLSR Central ⁇ receptor activation by YVLSR
  • the anxiolytic effect of YVLSR by oral administration was inhibited by intracerebroventricular administration (10 nmol / mouse) of the ⁇ opioid receptor antagonist naltrindole (FIG. 5).
  • the ⁇ antagonist naloxone was not inhibited (data not shown).
  • YVLSR showed no affinity for the ⁇ receptor and also no ⁇ opioid activity using mouse vas deferens (MVD). From the above results, it is considered that YVLSR activates the central ⁇ receptor by promoting the release of endogenous ⁇ opioid ligand.
  • the anxiolytic effect of YVLSR is through the release of neurotransmitters such as endogenous ⁇ 1 ligand, serotonin, dopamine, GABA and the activation of ⁇ 1 , 5-HT 1A , D 1 , and GABA A receptors. It became clear that. As a preliminary study, the anxiolytic effect of the specific ⁇ agonist DPDPE is inhibited by antagonists to ⁇ 1 , 5-HT 1A , D 1 , and GABA A receptors, and is consistent with the anxiolytic pathway of YVLSR.
  • YVLSR exhibits an anxiolytic effect upon intraventricular and intraperitoneal administration. Furthermore, this peptide was also effective when administered orally.
  • YVLSR activates central ⁇ receptors, and further activates ⁇ 1 , 5-HT 1A , D 1 , and GABA A receptors, and is considered to exhibit an anxiolytic action.
  • Benzodiazepines such as diazepam, known as common anxiolytic drugs, bind to the benzodiazepine binding site of the GABA A receptor and show anxiolytic activity, whereas YVLSR does not show affinity for this binding site Therefore, it is considered that GABA release is promoted and an anxiolytic action is exhibited. Therefore, it was found that the pathway is different from that of conventional anxiolytic drugs.
  • the anti-anxiety drug of the present invention may have a different mechanism of action from conventional anxiolytic drugs, and can provide a new type of drug.
  • drugs that have a mental stress relieving action often have a sleep-inducing action, and a typical anxiolytic drug diazepam is prescribed as a sleep inducer.
  • YVLSR has also been shown to activate GABA A receptors that mediate sleep-induced effects. Therefore, the pharmaceutical composition of the present invention can also be used for sleep induction and sleep improvement.
  • the present inventors have found a plurality of anxiolytic peptides derived from milk protein.
  • SEQ ID NO: 1 is a designed peptide.

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Abstract

Disclosed is a pharmaceutical composition which contains a peptide that is represented by YVLSR or an analogue thereof as an active ingredient.

Description

生理活性ペプチドを含む医薬組成物Pharmaceutical composition comprising bioactive peptide
　本発明は神経系に作用する医薬組成物に関する。また、本発明は、ペプチドまたはその類縁体に関し、詳しくは、δオピオイド受容体、５－ＨＴ１Ａ受容体、Ｄ１受容体およびＧＡＢＡＡ受容体の少なくとも1種を活性化するペプチドまたはその類縁体に関する。さらに本発明は、抗不安または睡眠改善用食品に関する。 The present invention relates to a pharmaceutical composition that acts on the nervous system. The present invention also relates to a peptide or an analog thereof, and more specifically, a peptide that activates at least one of a δ opioid receptor, a 5-HT 1A receptor, a D 1 receptor, and a GABA A receptor or an analog thereof. About. Furthermore, the present invention relates to a food for improving anxiety or sleep.
　現代のストレス社会を反映し、不安障害、統合失調症、うつ病などの精神疾患の増加が問題となっている。不安感は、生体において危険を回避するための警告として本来必要なものであるが、過剰な不安感は上記精神疾患の発症や症状の進行に関与するとともに、生活習慣病の発症リスクを上昇させることが知られており、精神的ストレスを緩和する食品や医薬品の開発が期待されている。このような抗不安作用を有する化合物は、安価に製造が可能で、経口投与で有効なものが望ましい。 Reflecting the modern stress society, an increase in mental illnesses such as anxiety disorder, schizophrenia and depression has become a problem. Anxiety is essential as a warning to avoid danger in the living body, but excessive anxiety is involved in the onset of the above mental illnesses and progression of symptoms, and increases the risk of developing lifestyle-related diseases. It is known that development of foods and medicines that relieve mental stress is expected. Such an anxiolytic compound can be produced inexpensively and is preferably effective by oral administration.
　かかる抗不安薬の候補として、いくつかのオリゴペプチドが試験されている。そして、このような生理活性を示すオリゴペプチドとしては鎮痛作用を有するオピオイドペプチドが知られている。さらに、本発明者らは、ダイズの主要タンパク質であるβ-コングリシニンに由来するsoymorphinが抗不安作用を有することを明らかにしている(特許文献１)。しかし、より低い用量で効果を示す化合物が所望されていた。 Several oligopeptides have been tested as candidates for such anxiolytic drugs. And as an oligopeptide which shows such physiological activity, the opioid peptide which has an analgesic effect is known. Furthermore, the present inventors have revealed that soymorphin derived from β-conglycinin, which is a major protein of soybean, has an anxiolytic action (Patent Document 1). However, compounds that would be effective at lower doses were desired.
特開2007-91656JP2007-91656
　副作用が少なく、かつ比較的低用量であっても、抗不安作用、抗うつ作用等の作用を有する薬剤及び食品を供給することを目的とする。 The purpose is to supply drugs and foods that have anti-anxiety action, antidepressant action, etc. even with relatively low doses and few side effects.
　本発明者らは、上記課題を解決すべく、種々の食品タンパク質由来ペプチドの情動調節作用を検討したところ、牛乳κ-カゼイン由来のTyr-Val-Leu-Ser-Argが抗不安作用を示すとともに、δ受容体の下流で、σ１受容体、5-HT1A受容体、D1受容体、又はGABAA受容体を活性化することを見出し、本発明を完成させた。 In order to solve the above-mentioned problems, the present inventors have studied the emotion-regulating action of various food protein-derived peptides, and Tyr-Val-Leu-Ser-Arg derived from milk κ-casein exhibits an anxiolytic action. The present inventors have found that the σ 1 receptor, 5-HT 1A receptor, D 1 receptor, or GABA A receptor is activated downstream of the δ receptor, thereby completing the present invention.
　従って、本発明は、以下の項１～６を提供する：
　項１．ＹＶＬＳＲ（配列番号１）で表されるペプチド又はその類縁体を有効成分とする、医薬組成物。 Accordingly, the present invention provides the following items 1 to 6:
Item 1. A pharmaceutical composition comprising a peptide represented by YVLSR (SEQ ID NO: 1) or an analog thereof as an active ingredient.
　項２．抗不安剤、睡眠導入剤、睡眠改善剤、統合失調症治療薬または抗うつ薬である、項１に記載の医薬組成物。 Item 2. Item 2. The pharmaceutical composition according to Item 1, which is an anxiolytic agent, sleep-inducing agent, sleep-improving agent, schizophrenia therapeutic agent or antidepressant.
　項３．ＹＶＬＳＲで表されるペプチド又はその類縁体が配合された、抗不安または睡眠改善用食品。 Item 3. An anti-anxiety or sleep improving food containing a peptide represented by YVLSR or an analog thereof.
　項４．ＹＶＬＳＲで表されるペプチド又はその類縁体。 Item 4. A peptide represented by YVLSR or an analog thereof.
　項５．抗不安、睡眠導入、睡眠改善、統合失調症治療または抗うつのための、ＹＶＬＳＲで表されるペプチド又はその類縁体。 Item 5. A peptide represented by YVLSR or an analog thereof for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant.
　項６．患者に、項１又は２に記載の医薬組成物を投与する工程を含む、抗不安、睡眠導入、睡眠改善、統合失調症治療または抗うつのための方法。 Item 6. A method for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant, comprising the step of administering the pharmaceutical composition according to item 1 or 2 to a patient.
　本発明の医薬組成物及び食品は、これらに含まれるペプチド又はその類縁体として、比較的低用量であっても、高い、抗不安作用、抗うつ作用等の作用を示す。また、本発明の医薬組成物及び食品に含まれるペプチドは、食経験の豊富な食品タンパク質に由来するものであり、副作用が少ないことが期待できる。 The pharmaceutical composition and food of the present invention exhibit high anti-anxiety action, antidepressant action, etc., even at a relatively low dose, as peptides contained therein or their analogs. Moreover, the peptides contained in the pharmaceutical composition and food of the present invention are derived from food proteins with abundant food experience, and it can be expected that there are few side effects.
　本発明者らは、現在、本ペプチドの直接結合する受容体の同定を試みているが、非常に強力な抗不安ペプチドであるYVLSRの標的受容体が明らかになれば、新規の抗不安機構を解明することができるだけでなく、新しい創薬のターゲットになりうる。 The present inventors are currently trying to identify a receptor to which this peptide directly binds, but if the target receptor of YVLSR, which is a very potent anxiolytic peptide, is clarified, a novel anxiolytic mechanism will be developed. Not only can it be elucidated, it can be a target for new drug discovery.
　高齢者における食欲不振が、老化を促進することが知られている。NPYやghrelinを代表とする内因性の摂食促進ペプチドを脳室内あるいは静脈内投与した場合に、摂食促進作用が認められるが、経口投与で有効な摂食促進ペプチドの報告はなかった。しかしながら最近我々は、緑葉の主要なタンパク質であり、地球上で最も多く存在するタンパク質として知られるRubiscoから派生するδオピオイドペプチドrubiscolin-６を経口投与（0.3 mg/kg）した場合に、マウス摂食量が顕著に増加することを見出した。さらに、δオピオイドは学習促進作用及び抗不安作用を併せ持つことが知られている。したがって、δオピオイド受容体を活性化する抗不安ペプチドYVLSRには、摂食促進作用及び学習促進作用が期待され、高齢者対応食品の素材として利用できる可能性が考えられる。 不 Anorexia in the elderly is known to promote aging. When endogenous feeding stimulating peptides such as NPY and ghrelin were administered intraventricularly or intravenously, feeding stimulating effects were observed, but there were no reports of effective feeding stimulating peptides by oral administration. Recently, however, we found that the amount of food consumed in mice after oral administration (0.3 mg / kg) of the delta opioid peptide rubiscolin-6 derived from Rubisco, which is a major protein of green leaves and is known as the most abundant protein on earth. Has been found to increase significantly. Furthermore, it is known that δ opioid has both a learning promoting action and an anxiolytic action. Therefore, the anti-anxiety peptide YVLSR that activates the δ opioid receptor is expected to have a feeding-promoting action and a learning-promoting action, and may be used as a material for food for the elderly.
　rubisocolin-6は上記のとおり普通食摂取を促進する一方、高脂肪食摂取を抑制することを最近見出しており、δオピオイド受容体は食の嗜好性の正常化に関与することが判明している。本発明のＹＶＬＳＲおよびその類縁体も嗜好性正常化能を有する可能性がある。 Rubisocolin-6 has recently been found to promote normal food intake while suppressing high-fat food intake as described above, and δ opioid receptors have been shown to be involved in normalizing food preferences . The YVLSR and its analogs of the present invention may also have a preference normalization ability.
高架式十字迷路Elevated cross maze ＹＶＬＳＲの脳室内投与による抗不安作用Anti-anxiety effect by intraventricular administration of YVLSR ＹＶＬＳＲの腹腔内投与による抗不安作用Anti-anxiety effect by intraperitoneal administration of YVLSR ＹＶＬＳＲの経口投与による抗不安作用Anti-anxiety effect by oral administration of YVLSR ＹＶＬＳＲの抗不安作用に影響を及ぼすδオピオイド受容体アンタゴニストのnaltrindoleの影響Effect of naltrindole, a δ opioid receptor antagonist, affecting the anxiolytic effect of YVLSR ＹＶＬＳＲの抗不安作用に影響を及ぼすσ１、セロトニン5-HT1A、ドーパミンD1、及びGABAA受容体に対するアンタゴニストの影響Effects of antagonists on σ 1 , serotonin 5-HT 1A , dopamine D 1 , and GABA A receptors affecting the anxiolytic effects of YVLSR ＹＶＬＳＲの抗不安作用に影響を及ぼすσ１、セロトニン5-HT1A、ドーパミンD1、及びGABAA受容体に対するアンタゴニストの影響Effects of antagonists on σ 1 , serotonin 5-HT 1A , dopamine D 1 , and GABA A receptors affecting the anxiolytic effects of YVLSR ＹＶＬＳＲの抗不安機構Anti-anxiety mechanism of YVLSR
　薬理作用
　本発明において、抗不安作用は、抗不安薬をスクリーニングするための不安関連行動評価法として開発され、広く用いられている高架式十字迷路試験により評価することができる（図１）。具体的には抗不安薬の候補物質を経口投与または腹腔内投与し、３０分後に高架式十字迷路にマウスを置いて、オープンアームに侵入した回数とオープンアーム上での滞在時間の変化を指標として、抗不安作用の強さを評価することができる。 Pharmacological Action In the present invention, the anxiolytic action is developed as an anxiety-related behavior evaluation method for screening an anxiolytic drug, and can be evaluated by an elevated plus maze test (FIG. 1). Specifically, an anxiolytic candidate substance is administered orally or intraperitoneally, and after 30 minutes, the mouse is placed in the elevated plus maze, and the change in the number of intrusions into the open arm and the staying time on the open arm is indicated. As described above, the strength of the anti-anxiety action can be evaluated.
　本発明のペプチド又はその類縁体の抗不安作用は、δオピオイド受容体アンタゴニストのnaltrindoleの脳室内投与により、阻害される。なお、μアンタゴニストのnaloxoneでは阻害されなかった（データ示さず）。YVLSRは、δ受容体に対する親和性を示さず、かつ、マウス輸精管（MVD）を用いたδオピオイド活性も示さなかった。以上の結果より、YVLSRは、内因性δオピオイドリガンドの遊離促進により、中枢のδ受容体を活性化しているものと考えられる。 The anxiolytic action of the peptide of the present invention or an analog thereof is inhibited by intracerebroventricular administration of the δ opioid receptor antagonist naltrindole. The μ antagonist naloxone was not inhibited (data not shown). YVLSR showed no affinity for the δ receptor and also no δ opioid activity using mouse vas deferens (MVD). From the above results, it is considered that YVLSR activates the central δ receptor by promoting the release of endogenous δ opioid ligand.
　また、本発明において、本発明の医薬の有効成分であるペプチドの抗不安作用は、σ１、セロトニン5-HT1A、ドーパミンD1、及びGABAA受容体に対するアンタゴニストにより阻害されるが、これらの受容体には親和性を示さなかった。したがって、YVLSRの抗不安作用は、内因性σ1リガンド、セロトニン、ドーパミン、GABA等の神経伝達物質の放出ならびに、σ１、5-HT1A、D1、及びGABAA受容体の活性化を介していることが明らかとなった(図８)。なお、予備的検討結果であるが、特異的δアゴニストDPDPEによる抗不安作用は、σ１、5-HT1A、D1、及びGABAA受容体に対するアンタゴニストで阻害され、YVLSRの抗不安経路と一致することがわかった。また、これらの受容体に対するアゴニスト及びアンタゴニストを用いた薬理学的検討及び既知の報告より、δ受容体→σ１受容体→5-HT1A受容体→D1受容体→GABAA受容体の順番で活性化されるものと考えられる。 In the present invention, the anxiolytic action of the peptide which is the active ingredient of the medicament of the present invention is inhibited by antagonists to σ 1 , serotonin 5-HT 1A , dopamine D 1 , and GABA A receptor. There was no affinity for the receptor. Therefore, the anxiolytic effect of YVLSR is through the release of neurotransmitters such as endogenous σ 1 ligand, serotonin, dopamine, GABA and the activation of σ 1 , 5-HT 1A , D 1 , and GABA A receptors. (Fig. 8). As a preliminary study, the anxiolytic effect of the specific δ agonist DPDPE is inhibited by antagonists to σ 1 , 5-HT 1A , D 1 , and GABA A receptors, and is consistent with the anxiolytic pathway of YVLSR. I found out that In addition, pharmacological studies using agonists and antagonists for these receptors and known reports show that the order of δ receptor → σ 1 receptor → 5-HT 1A receptor → D 1 receptor → GABA A receptor It is thought that it is activated by.
　一方、YVLSRの抗不安作用は、プロスタグランジン（PG）の生合成に関与するシクロオキシゲナーゼの阻害剤indomethacinではブロックされない。従って、本ペプチドの抗不安作用にはPG類は関与しないものと考えられる。 On the other hand, the anxiolytic effect of YVLSR is not blocked by the cyclooxygenase inhibitor indomethacin involved in prostaglandin (PG) biosynthesis. Therefore, it is considered that PGs are not involved in the anxiolytic action of this peptide.
　また、本発明のペプチド又はその類縁体の好ましい投与経路としては、経口投与が挙げられる。 In addition, a preferable route of administration of the peptide of the present invention or an analog thereof is oral administration.
　有効成分
　本発明の抗不安剤の有効成分は、Ｙ(Tyr)－Ｖ(Val)－Ｌ(Leu)－Ｓ(Ser)－Ｒ(Arg)で表わされるペプチドないしその類縁体である。 Active ingredient The active ingredient of the anxiolytic agent of the present invention is a peptide represented by Y (Tyr) -V (Val) -L (Leu) -S (Ser) -R (Arg) or an analog thereof.
　ペプチドを構成するアミノ酸は、Ｌ体のアミノ酸、Ｄ体のアミノ酸又はＤＬ体のアミノ酸（Ｄ体とＬ体が混合されたアミノ酸であればラセミ体といずれか一方のエナンチオマーが過剰なアミノ酸のいずれも含まれる）のいずれであってもよい。好ましくはＬ体のアミノ酸のみ、又はＤ体のアミノ酸のみからなるペプチド、特にＬ体のアミノ酸のみからなるペプチドがよい。 The amino acids constituting the peptide are L-form amino acids, D-form amino acids, or DL-form amino acids (if the D-form and L-form amino acids are mixed, the racemic form and any amino acid in which one of the enantiomers is excessive) Included). Preferable is a peptide consisting only of L-form amino acids or only D-form amino acids, particularly a peptide consisting only of L-form amino acids.
　また、本発明で使用するペプチドが２以上の不斉炭素を含む場合、各エナンチオマーないしジアステレオマー又はこれらの任意の比率の混合物のいずれの形態であってもよい。エナンチオマー又はジアステレオマーの分離は、通常のカラムで行ってもよく、光学活性カラムを使用したり、光学活性基を導入して誘導体の形態で光学分割した後、その光学活性基を除去する方法や、光学活性の酸又は塩基との塩を形成して光学分割するなどの公知のいずれの方法を用いてもよい。 In addition, when the peptide used in the present invention contains two or more asymmetric carbons, it may be in the form of each enantiomer or diastereomer or a mixture of these in any ratio. Separation of enantiomers or diastereomers may be carried out using a normal column, or an optically active column is used, or an optically active group is introduced and optically resolved in the form of a derivative, and then the optically active group is removed. Alternatively, any known method such as optical resolution by forming a salt with an optically active acid or base may be used.
　ペプチド又はその類縁体の塩としては、酸付加塩と塩基塩が挙げられる。酸付加塩としては、塩酸、硫酸、硝酸、リン酸、臭化水素酸、過塩素酸などの無機塩、クエン酸、コハク酸、マレイン酸、フマル酸、リンゴ酸、酒石酸、ｐ－トルエンスルホン酸、ベンゼンスルホン酸、メタンスルホン酸、トリフルオロ酢酸などの有機酸の塩が挙げられる。塩基塩としては、ナトリウム、カリウム、リチウムなどのアルカリ金属塩、カルシウム、マグネシウムなどのアルカリ土類金属塩などが挙げられる。 Examples of salts of peptides or analogs thereof include acid addition salts and base salts. Acid addition salts include inorganic salts such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, perchloric acid, citric acid, succinic acid, maleic acid, fumaric acid, malic acid, tartaric acid, p-toluenesulfonic acid And salts of organic acids such as benzenesulfonic acid, methanesulfonic acid and trifluoroacetic acid. Examples of the base salt include alkali metal salts such as sodium, potassium and lithium, and alkaline earth metal salts such as calcium and magnesium.
　溶媒和物としては、水(水和物の場合)、メタノール、エタノール、イソプロパノール、酢酸、テトラヒドロフラン、アセトン、ジメチルホルムアミド、ジメチルスルホキシド、ジメチルアセトアミド、アセトアミド、エチレングリコール、プロピレングリコール、ジメトキシエタンなどの溶媒和物が挙げられる。 Solvates include solvates such as water (in the case of hydrates), methanol, ethanol, isopropanol, acetic acid, tetrahydrofuran, acetone, dimethylformamide, dimethyl sulfoxide, dimethylacetamide, acetamide, ethylene glycol, propylene glycol, and dimethoxyethane. Things.
　本発明に係る医薬組成物及び食品の有効成分としては、上記ペプチドだけでなく、所望の薬理効果が得られる限り、種々のペプチド類縁体を用いることもできる。ペプチド有効成分がペプチド類縁体の場合、当該ペプチド類縁体には、有効成分の上記ペプチドの(1)Ｎ末端の修飾、及び(2)Ｃ末端の修飾の類縁体が含まれる。 As the active ingredient of the pharmaceutical composition and food according to the present invention, not only the above peptides but also various peptide analogs can be used as long as a desired pharmacological effect is obtained. When the peptide active ingredient is a peptide analog, the peptide analog includes (1) N-terminal modification and (2) C-terminal modification analogs of the peptide of the active ingredient.
(1)ペプチドのＮ末端のアミノ基は、メチルアミノ、ジメチルアミノ、エチルアミノ、ジエチルアミノ、プロピルアミノ、ジプロピルアミノ、ｎ－ブチルアミノ、ジｎ－ブチルアミノ、などの直鎖又は分岐を有する炭素数１～４のアルキル基でモノ置換又はジ置換されたアミノ基でもよい。又は、Ｎ末端のアミノ基又は側鎖のアミノ基（Ｌｙｓを含む場合）は、ベンジル基、フェネチル基などのアラルキル基でモノ置換又はジ置換されていてもよく、ホルミル基、アセチル基、プロピオニル基、ブチリル基、イソブチリル基などの炭素数１～６の直鎖又は分岐を有するアルカノイル基、ベンゾイル基などのアシル基で修飾されていてもよい。 (1) The N-terminal amino group of the peptide is linear or branched carbon such as methylamino, dimethylamino, ethylamino, diethylamino, propylamino, dipropylamino, n-butylamino, di-n-butylamino, etc. It may be an amino group mono- or di-substituted with an alkyl group of 1 to 4. Alternatively, the N-terminal amino group or the side chain amino group (when Lys is included) may be mono- or di-substituted with an aralkyl group such as a benzyl group or a phenethyl group, a formyl group, an acetyl group, or a propionyl group And may be modified with a linear or branched alkanoyl group having 1 to 6 carbon atoms, such as a butyryl group or an isobutyryl group, or an acyl group such as a benzoyl group.
(2)ペプチドのＣ末端のカルボキシル基は、メチル、エチル、ｎ－プロピル、イソプロピル、ｎ－ブチル、イソブチル、ｔ－ブチル、ペンチル、ヘキシルなどの炭素数１～６のアルキル基とのエステル、ベンジル、フェネチルなどのアラルキル基とのエステル、アミノ基、メチルアミン、ジメチルアミン、エチルアミン、ジエチルアミン、プロピルアミン、ジプロピルアミン、ｎ－ブチルアミン、ジｎ－ブチルアミンなどの直鎖又は分岐を有する炭素数１～４のアルキル基でモノ置換又はジ置換されたアミン、又はアンモニアとのアミドを形成してもよい。 (2) The carboxyl group at the C-terminal of the peptide is an ester with a C 1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, benzyl , Esters with aralkyl groups such as phenethyl, amino groups, methylamine, dimethylamine, ethylamine, diethylamine, propylamine, dipropylamine, n-butylamine, di-n-butylamine, etc. An amine mono- or di-substituted with 4 alkyl groups or an amide with ammonia may be formed.
　本発明のペプチドは、天然のタンパク質ないしポリペプチドの加水分解により得ることもでき、化学合成により得ることもできる。 The peptide of the present invention can be obtained by hydrolysis of a natural protein or polypeptide, or can be obtained by chemical synthesis.
　加水分解されるタンパク質ないしポリペプチドとしては、牛乳又は人乳由来のカゼインなどが挙げられる。これらの食品素材由来のペプチドは、そのまま又は必要に応じて濃縮、脱塩、精製等の処理を行うことにより、そのまま食品とすることができる。タンパク質の加水分解には、トリプシン、キモトリプシン、パパイン、ぺプシン、カルボキシペプチダーゼ、サーモリシン、サチライシンなどの動物、植物ないし微生物由来の加水分解酵素の使用が例示され、これらの酵素を用い、ｐＨを酵素に応じて適切な値に調製し、２０～４０℃程度の温度下に３０分から４８時間程度反応させることにより、本発明の有効成分のペプチドを得ることができる。例えば、YVLSRは、κ-カゼインのトリプシン消化により生成するcasoxin Cを、さらにペプシンで消化することにより部分的に生成する。得られた反応液から本発明のペプチドを精製して用いてもよく、食品素材を酵素分解した場合には、そのまま又は他の食品素材に添加して食品ないし食品組成物とすることもできる。加水分解は、強酸(例えば塩酸、硝酸、硫酸など)又は強塩基(水酸化ナトリウム、水酸化カリウム、水酸化リチウムなどのアルカリ金属水酸化物、炭酸ナトリウム、炭酸カリウムなどのアルカリ金属炭酸塩、炭酸水素ナトリウム、炭酸水素カリウムなどのアルカリ金属炭酸水素塩)などの存在下に水中で、１～１００℃の温度で、３０分から４８時間反応させることにより、本発明の有効成分のペプチドを得ることができる。加水分解の反応生成物は、ｐＨを調製した後、そのまま使用してもよく、精製により有効成分のペプチドを分離して使用してもよい。 Examples of the protein or polypeptide to be hydrolyzed include casein derived from milk or human milk. These peptides derived from food materials can be used as they are, or as they are, as they are, by carrying out treatments such as concentration, desalting and purification as necessary. Examples of protein hydrolysis include the use of hydrolytic enzymes derived from animals, plants or microorganisms such as trypsin, chymotrypsin, papain, pepsin, carboxypeptidase, thermolysin, and subtilisin. Accordingly, the peptide of the active ingredient of the present invention can be obtained by adjusting to an appropriate value and reacting at a temperature of about 20 to 40 ° C. for about 30 minutes to 48 hours. For example, YVLSR is partially generated by further digesting casoxin C generated by trypsin digestion of κ-casein with pepsin. The peptide of the present invention may be purified from the obtained reaction solution, and when the food material is enzymatically decomposed, it can be used as it is or added to another food material to form a food or a food composition. Hydrolysis can be performed using strong acids (for example, hydrochloric acid, nitric acid, sulfuric acid, etc.) or strong bases (alkali metal hydroxides such as sodium hydroxide, potassium hydroxide, lithium hydroxide, alkali metal carbonates such as sodium carbonate, potassium carbonate, carbonates). The peptide of the active ingredient of the present invention can be obtained by reacting in water at a temperature of 1 to 100 ° C. for 30 minutes to 48 hours in the presence of an alkali metal hydrogen carbonate such as sodium hydrogen or potassium hydrogen carbonate). it can. The reaction product of the hydrolysis may be used as it is after the pH is adjusted, or the peptide of the active ingredient may be separated and used by purification.
　また本発明のペプチドは、ペプチド合成法で取得することもできる。即ち、ペプチド合成に通常用いられる方法である液相法又は固相法で、反応性カルボキシル基を有する原料と、反応性アミノ基を有する原料とをＨＢＴＵ等の活性エステルを用いた方法や、カルボジイミドなどのカップリング剤を用いた方法等のペプチド合成において通常の方法により縮合させることができる。生成する縮合物が保護基を有する場合、その保護基を除去することによっても製造し得る。 The peptide of the present invention can also be obtained by peptide synthesis. That is, in a liquid phase method or a solid phase method, which is a commonly used method for peptide synthesis, a raw material having a reactive carboxyl group and a raw material having a reactive amino group are combined with a method using an active ester such as HBTU, or carbodiimide In a peptide synthesis such as a method using a coupling agent such as When the resulting condensate has a protecting group, it can also be produced by removing the protecting group.
　この反応工程において反応に関与すべきでない官能基は、保護基により保護される。アミノ基の保護基としては、例えばベンジルオキシカルボニル（ＣＢＺ）、ｔ－ブチルオキシカルボニル（Ｂｏｃ），９－フルオレニルメチルオキシカルボニル（Ｆｍｏｃ）等が挙げられる。カルボキシル基の保護剤としては例えばアルキルエステル、ベンジルエステル等を形成し得る基が挙げられるが、固相法の場合は、Ｃ末端のカルボキシル基はクロロトリチル樹脂、クロルメチル樹脂、オキシメチル樹脂、ｐ－アルコキシベンジルアルコール樹脂等の担体に結合している。縮合反応は、カルボジイミド等の縮合剤の存在下にあるいはＮ－保護アミノ酸活性エステル又はペプチド活性エステルを用いて実施する。 In this reaction step, functional groups that should not participate in the reaction are protected by protecting groups. Examples of amino-protecting groups include benzyloxycarbonyl (CBZ), t-butyloxycarbonyl (Boc), 9-fluorenylmethyloxycarbonyl (Fmoc) and the like. Examples of the carboxyl group-protecting agent include groups capable of forming alkyl esters, benzyl esters, and the like. In the solid phase method, the C-terminal carboxyl group is a chlorotrityl resin, chloromethyl resin, oxymethyl resin, p- It is bound to a carrier such as an alkoxybenzyl alcohol resin. The condensation reaction is performed in the presence of a condensing agent such as carbodiimide or using an N-protected amino acid active ester or peptide active ester.
　縮合反応終了後、保護基は除去されるが、固相法の場合はさらにペプチドのＣ末端と樹脂との結合を切断する。更に、本発明のペプチドは通常の方法に従い精製される。例えばイオン交換クロマトグラフィー、逆相液体クロマトグラフィー、アフィニティークロマトグラフィー等が挙げられる。合成したペプチドの合成はエドマン分解法でＣ－末端からアミノ酸配列を読み取るプロテインシークエンサー、ＬＣ－ＭＳ、ＧＣ－ＭＳ等で分析される。 After completion of the condensation reaction, the protecting group is removed, but in the case of the solid phase method, the bond between the C-terminus of the peptide and the resin is further cleaved. Furthermore, the peptides of the present invention are purified according to conventional methods. Examples thereof include ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like. The synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by Edman degradation, LC-MS, GC-MS, or the like.
　本発明のペプチドは、酵素法によっても合成することが可能である（WO2003/010307参照）。 The peptide of the present invention can also be synthesized by an enzymatic method (see WO2003 / 010307).
　本発明のペプチドの投与経路は特に限定されるものではなく、経口投与、非経口投与、直腸内投与のいずれを採用することも可能であり、経口的あるいは非経口的に投与することができる。本ペプチドの投与量は化合物の種類、投与方法、投与される者の状態や年齢等により異なるが、成人１日あたり通常は０．００１～５００ｍｇ／ｋｇ、好ましくは０．００５～１００ｍｇ／ｋｇ、より好ましくは０．０１～３０ｍｇ／ｋｇである。本発明のペプチド(有効成分)は通常、製剤用担体と混合して調製した医薬組成物の形で投与される。製剤用担体としては、製剤分野において常用され、かつ本発明のペプチドと反応しない物質が用いられる。 The administration route of the peptide of the present invention is not particularly limited, and any of oral administration, parenteral administration, and rectal administration can be adopted, and it can be administered orally or parenterally. The dosage of this peptide varies depending on the type of compound, the administration method, the condition and age of the administered person, etc., but is usually 0.001 to 500 mg / kg, preferably 0.005 to 100 mg / kg per day for an adult. More preferably, it is 0.01 to 30 mg / kg. The peptide (active ingredient) of the present invention is usually administered in the form of a pharmaceutical composition prepared by mixing with a pharmaceutical carrier. As a pharmaceutical carrier, a substance that is commonly used in the pharmaceutical field and does not react with the peptide of the present invention is used.
　本発明のペプチドはそれ自体食品又は医薬として利用することができ、又は単独で、もしくは適当な無毒性の経口摂取用担体、希釈剤又は賦形剤とともに、タブレット（素錠、糖衣錠、発泡錠、フィルムコート錠、チュアブル錠など）、カプセル、トローチ、粉末、細粒剤、顆粒剤、液剤、懸濁液、乳濁液、ペースト、クリーム、注射剤（アミノ酸輸液、電解質輸液等の輸液に配合する場合を含む）、又は腸溶性の錠剤、カプセル剤、顆粒剤などの徐放性製剤などの食品用もしくは医薬用の製剤にすることが可能である。食品中のペプチドの含有量は適宜選択が可能であるが一般に、０．０１～１００重量％の範囲である。 The peptide of the present invention can be used as a food or a medicine per se, or alone or together with a suitable non-toxic carrier for ingestion, diluent or excipient (tablet, uncoated tablet, dragee, effervescent tablet, Film-coated tablets, chewable tablets, etc.), capsules, troches, powders, fine granules, granules, solutions, suspensions, emulsions, pastes, creams, injections (amino acid infusions, electrolyte infusions, etc.) Or a preparation for food or medicine such as sustained release preparations such as enteric tablets, capsules and granules. The content of the peptide in the food can be appropriately selected, but is generally in the range of 0.01 to 100% by weight.
　具体的には、医薬又は食品に加えることができる製剤用担体ないし経口摂取用担体、希釈剤又は賦形剤のような物質の例として乳糖、ブドウ糖、マンニット、デキストリン、シクロデキストリン、デンプン、蔗糖、メタケイ酸アルミン酸マグネシウム、合成ケイ酸アルミニウム、カルボキシメチルセルロースナトリウム、ヒドロキシプロピルデンプン、カルボキシメチルセルロースカルシウム、イオン交換樹脂、メチルセルロース、ゼラチン、アラビアゴム、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース、ポリビニルピロリドン、ポリビニルアルコール、軽質無水ケイ酸、ステアリン酸マグネシウム、タルク、トラガント、ベントナイト、ビーガム、酸化チタン、ソルビタン脂肪酸エステル、ラウリル硫酸ナトリウム、グリセリン、脂肪酸グリセリンエステル、精製ラノリン、グリセロゼラチン、ポリソルベート、マクロゴール、植物油、ロウ、流動パラフィン、白色ワセリン、フルオロカーボン、非イオン性界面活性剤、プロピレングルコール、水等が挙げられる。 Specifically, examples of substances such as pharmaceutical carriers or oral intake carriers, diluents or excipients that can be added to medicines or foods include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, and sucrose. , Magnesium aluminate metasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, carboxymethylcellulose calcium, ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light Silica, magnesium stearate, talc, tragacanth, bentonite, bee gum, titanium oxide, sorbitan fatty acid ester, sodium lauryl sulfate Arm, glycerin, fatty acid glycerin ester, purified lanolin, glycerogelatin, polysorbate, macrogol, vegetable oils, waxes, liquid paraffin, white petrolatum, fluorocarbons, nonionic surfactants, propylene glycol, water and the like.
　剤型としては、錠剤、カプセル剤、顆粒剤、散剤、シロップ剤、懸濁剤、坐剤、軟膏、クリーム剤、ゲル剤、貼付剤、吸入剤、注射剤等が挙げられる。これらの製剤は常法に従って調製される。尚、液体製剤にあっては、用時、水又は他の適当な溶媒に溶解又は懸濁する形であってもよい。また錠剤、顆粒剤は周知の方法でコーティングしてもよい。注射剤の場合には、本発明のペプチドを水に溶解させて調製されるが、必要に応じて生理食塩水あるいはブドウ糖溶液に溶解させてもよく、また緩衝剤や保存剤を添加してもよい。 Examples of the dosage form include tablets, capsules, granules, powders, syrups, suspensions, suppositories, ointments, creams, gels, patches, inhalants, injections, and the like. These preparations are prepared according to a conventional method. The liquid preparation may be dissolved or suspended in water or other appropriate solvent at the time of use. Tablets and granules may be coated by a known method. In the case of injection, it is prepared by dissolving the peptide of the present invention in water, but it may be dissolved in physiological saline or glucose solution as necessary, and a buffer or preservative may be added. Good.
　これらの製剤は、本発明のペプチドを０．０１％～１００重量％、好ましくは１～９０重量％の割合で含有することができる。これらの製剤はまた、治療上価値のある他の成分を含有していてもよい。 These preparations may contain the peptide of the present invention in a proportion of 0.01% to 100% by weight, preferably 1 to 90% by weight. These formulations may also contain other therapeutically valuable ingredients.
　経口投与用の固形製剤を製造するには、有効成分と賦形剤成分例えば乳糖、澱粉、結晶セルロース、乳酸カルシウム、無水ケイ酸などと混合して散剤とするか、さらに必要に応じて白糖、ヒドロキシプロピルセルロース、ポリビニルピロリドンなどの結合剤、カルボキシメチルセルロース、カルボキシメチルセルロースカルシウムなどの崩壊剤などを加えて湿式又は乾式造粒して顆粒剤とする。錠剤を製造するには、これらの散剤及び顆粒剤をそのまま又はステアリン酸マグネシウム、タルクなどの滑沢剤を加えて打錠すればよい。これらの顆粒又は錠剤はヒドロキシプロピルメチルセルロースフタレート、メタクリル酸－メタクリル酸メチルポリマーなどの腸溶剤基剤で被覆して腸溶剤製剤、あるいはエチルセルロース、カルナウバロウ、硬化油などで被覆して持続性製剤とすることもできる。また、カプセル剤を製造するには、散剤又は顆粒剤を硬カプセルに充填するか、有効成分をそのまま又はグリセリン、ポリエチレングリコール、ゴマ油、オリーブ油などに溶解した後ゼラチン膜で被覆し軟カプセルとすることができる。 In order to produce a solid preparation for oral administration, an active ingredient and excipient components such as lactose, starch, crystalline cellulose, calcium lactate, anhydrous silicic acid and the like are mixed to form a powder, or if necessary, sucrose, Add a binder such as hydroxypropylcellulose and polyvinylpyrrolidone, a disintegrant such as carboxymethylcellulose and carboxymethylcellulose calcium, and wet or dry granulate to form granules. In order to produce tablets, these powders and granules may be tableted as they are or with the addition of lubricants such as magnesium stearate and talc. These granules or tablets should be coated with an enteric solvent base such as hydroxypropylmethylcellulose phthalate, methacrylic acid-methyl methacrylate polymer, etc., and coated with an enteric solvent preparation, or with ethylcellulose, carnauba wax, hardened oil, etc. You can also. In order to produce capsules, powders or granules are filled into hard capsules, or active ingredients are dissolved as they are or dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc., and then coated with a gelatin film to form soft capsules. Can do.
　経口投与用の液状製剤を製造するには、有効成分と白糖、ソルビトール、グリセリンなどの甘味剤とを水に溶解して透明なシロップ剤、更に精油、エタノールなどを加えてエリキシル剤とするか、アラビアゴム、トラガント、ポリソルベート８０、カルボキシメチルセルロースナトリウムなどを加えて乳剤又は懸濁剤としてもよい。これらの液状製剤には所望により矯味剤、着色剤、保存剤などを加えてもよい。 In order to produce a liquid preparation for oral administration, an active ingredient and a sweetener such as sucrose, sorbitol, and glycerin are dissolved in water to add a transparent syrup, further essential oil, ethanol, etc. to make an elixir, Gum arabic, tragacanth, polysorbate 80, sodium carboxymethyl cellulose and the like may be added to form an emulsion or suspension. These liquid preparations may contain a flavoring agent, a coloring agent, a preservative and the like as desired.
　注射剤を製造するには、有効成分を必要に応じて塩酸、水酸化ナトリウム、乳糖、乳酸、ナトリウム、リン酸一水素ナトリウム、リン酸二水素ナトリウムなどのｐＨ調整剤、塩化ナトリウム、ぶどう糖などの等張化剤と共に注射用蒸留水に溶解し、無菌濾過してアンプルに充填するか、更にマンニトール、デキストリン、シクロデキストリン、ゼラチンなどを加えて真空凍結乾燥し、用時溶解型の注射剤としてもよい。また、有効成分にレシチン、ポリソルベート８０、ポリオキシエチレン硬化ヒマシ油などを加えて水中で乳化せしめ注射剤用乳剤とすることもできる。 In order to produce injections, active ingredients such as hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, sodium monohydrogen phosphate, sodium dihydrogen phosphate, pH adjusters, sodium chloride, glucose etc. Dissolve in distilled water for injection with an isotonic agent, filter aseptically and fill into ampoules, or add mannitol, dextrin, cyclodextrin, gelatin, etc. Good. In addition, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, etc. may be added to the active ingredient and emulsified in water to give an emulsion for injection.
　直腸投与剤又は膣投与剤を製造するには、有効成分をカカオ脂、脂肪酸のトリ、ジ及びモノグリセリド、ポリエチレングリコールなどの坐剤用基材と共に加湿して溶解し型に流し込んで冷却するか、有効成分をポリエチレングリコール、大豆油などに溶解した後、ゼラチン膜で被覆すればよい。 To produce a rectal or vaginal dosage, the active ingredient is moistened with a suppository base such as cacao butter, fatty acid tri, di and monoglycerides, polyethylene glycol, etc., poured into a mold and cooled, The active ingredient may be dissolved in polyethylene glycol, soybean oil, etc. and then covered with a gelatin film.
　皮膚用外用剤を製造するには、有効成分を白色ワセリン、ミツロウ、流動パラフィン、ポリエチレングリコールなどに加えて必要ならば加湿して練合し軟膏剤とするか、ロジン、アクリル酸アルキルエステル重合体などの粘着剤と練合した後ポリアルキルなどの不織布に展延してテープ剤とする。 In order to produce an external preparation for skin, the active ingredient is added to white petrolatum, beeswax, liquid paraffin, polyethylene glycol, etc., and if necessary, moistened and kneaded to make an ointment, or rosin, alkyl acrylate polymer After being kneaded with an adhesive such as polyalkyl, it is spread on a non-woven fabric such as polyalkyl to obtain a tape.
　本発明に係るペプチドを添加・配合して調製しうる食品の具体的形態としては、例えば、飲料類（コーヒー、ココア、ジュース、清涼飲料、ミネラル飲料、茶飲料、緑茶、紅茶、烏龍茶、乳飲料、乳酸菌飲料、ヨーグルト飲料、炭酸飲料）、ガム、グミ、ゼリー、キャンデー、クッキー、クラッカー、ビスケット、氷菓（アイスクリーム、アイスキャンディ、シャーベット、かき氷等）、レトルト食品、ゼリー状食品（ゼリー、寒天、ゼリー状飲料等）、等を挙げることができる。本発明のペプチドを添加・配合して調製しうる食品としては、いわゆる健康食品、機能性食品、栄養補助食品、サプリメント、特定保健用食品、病者用食品・病者用組合わせ食品（厚生労働省、特別用途食品の一種）又は高齢者用食品（厚生労働省、特別用途食品の一種）としてもよく、素錠、フィルムコート錠、糖衣錠、顆粒、粉末、タブレット、カプセル（ハードカプセルとソフトカプセルとのいずれも含む。）、チュアブルタイプ、シロップタイプ、ドリンクタイプ等とすることもできる。本発明に係るペプチドを添加・配合した食品の調製は、それ自体公知の方法で行うことができる。 Specific examples of foods that can be prepared by adding and blending the peptide according to the present invention include, for example, beverages (coffee, cocoa, juice, soft drinks, mineral drinks, tea drinks, green tea, tea, oolong tea, and milk drinks. , Lactic acid bacteria beverage, yogurt beverage, carbonated beverage), gum, gummi, jelly, candy, cookies, crackers, biscuits, ice confectionery (ice cream, ice candy, sorbet, shaved ice, etc.), retort food, jelly-like food (jelly, agar, Jelly-like beverages). Foods that can be prepared by adding and blending the peptides of the present invention include so-called health foods, functional foods, dietary supplements, supplements, foods for specified health use, combined foods for the sick and the sick (Ministry of Health, Labor and Welfare) , Special-purpose foods) or elderly foods (Ministry of Health, Labor and Welfare, special-purpose foods), uncoated tablets, film-coated tablets, dragees, granules, powders, tablets, capsules (both hard capsules and soft capsules) Including chewable type, syrup type, and drink type. The preparation of food containing the peptide according to the present invention can be carried out by a method known per se.
　次に実施例により本発明を更に具体的に説明する。しかし下記の実施例は本発明の範囲を限定するものではない： Next, the present invention will be described more specifically with reference to examples. However, the following examples do not limit the scope of the invention:
（ペプチド及び実験動物）
　ペプチドＹＶＬＳＲは、Fmoc法により固相合成し、逆相HPLCを用いて精製し、さらに凍結乾燥したものを使用した。実験動物として雄性ddＹマウスを用いた。以下に示す高架式十字迷路試験及びオープンフィールド試験により抗不安活性を測定した。
ペプチドＹＶＬＳＲのマススペクトルの結果を以下に示す。
イオン　　　　 測定値　　理論値
[YVLSR+H]　　　637.39　　637.37
[YVLSR+2H] 　　319.20　　319.19 (Peptides and experimental animals)
Peptide YVLSR was synthesized by solid phase by Fmoc method, purified using reverse phase HPLC, and further freeze-dried. Male ddY mice were used as experimental animals. The anxiolytic activity was measured by the elevated plus maze test and the open field test described below.
The result of the mass spectrum of peptide YVLSR is shown below.
Ion Measured value Theoretical value
[YVLSR + H] 637.39 637.37
[YVLSR + 2H] 319.20 319.19
（高架式十字迷路実験）
　高架式十字迷路（Eleveted plus maze：EPM）は、２つのオープンアーム（open arm; 25cm×5cm）と２つのクローズドアーム（closed arm; 25cm×5cm×15 cm）からなり、それらのアームは床から50cm高くなった中央プラットフォームと結合している（図１参照）。高い位置にあるにも関わらず、クローズドアームの周りには壁があるために、マウスは安全に歩行する事ができる。一方、オープンアームの周囲は開放されていて壁がないために、オープンアームを歩行するマウスは高い位置から転落するという不安感を感じる。そのために、マウスがオープンアームにいる時間が長いほど、あるいは進入回数が多いほど、マウスの不安感は緩和されており、抗不安活性の指標となる。 (Elevated cross maze experiment)
The elevated plus maze (EPM) consists of two open arms (25cm x 5cm) and two closed arms (25cm x 5cm x 15cm), which are off the floor Combined with the central platform raised 50 cm (see Figure 1). Despite being in a high position, there is a wall around the closed arm, so the mouse can walk safely. On the other hand, since the periphery of the open arm is open and there are no walls, the mouse walking on the open arm feels uneasy that the mouse falls from a high position. For this reason, the longer the mouse stays in the open arm or the greater the number of times of entry, the less the anxiety of the mouse, which becomes an index of anxiolytic activity.
　オープンアームの一つに面している中央プラットフォーム上にマウスを置いて試験を開始した。５分の試験時間の間、オープンアーム内で過ごした累積時間(time in open arms)、オープンアームを訪れた回数（visit to open arms）、いずれかのアームを訪れた回数の総数(total visits)を記録した。オープンアーム内で過ごした時間のパーセンテージ、オープンアームを訪れた回数のパーセンテージを不安の指標として計算した。 The test was started by placing the mouse on the central platform facing one of the open arms. Cumulative time spent in open arms (time in open arms), number of visits to open arms (visit to open arms), total number of visits to any arm (total visits) Was recorded. The percentage of time spent in the open arm and the percentage of visits to the open arm were calculated as an indicator of anxiety.
　なお、ペプチドを脳室内投与する場合には、試験20分前に、腹腔内及び経口投与する場合は試験30分前に投与した。 In addition, when the peptide was administered into the ventricle, it was administered 20 minutes before the test, and when administered intraperitoneally and orally, 30 minutes before the test.
（オープンフィールド試験）
　本実験に用いたオープンフィールドは、直径60 cm、高さ50 cm円筒状の灰色の装置で、黒い線で25区画に分割されている。マウスは新規環境におかれた場合に探索行動を行なうが、通常であれば装置の中央への探索は少ない。しかし抗不安薬を処置すると、中央の円への滞在時間及び進入回数が増加する。ペプチドを投与して30分後にマウスを装置の中央に置き、5分間行動を観測した。 (Open field test)
The open field used in this experiment is a cylindrical gray device with a diameter of 60 cm and a height of 50 cm, divided into 25 sections by black lines. The mouse performs a search action when placed in a new environment, but normally, there are few searches to the center of the device. However, treatment with anxiolytics increases the time spent in the center circle and the number of entries. Thirty minutes after the administration of the peptide, the mouse was placed in the center of the apparatus and the behavior was observed for 5 minutes.
（統計解析）
　高架式十字迷路試験で得たデータを、平均とSEMで表した。データを１方向ANOVAにより解析し、引き続いて多重比較のためのFisher試験を行った。 (Statistical analysis)
Data obtained from the elevated plus maze test were expressed as mean and SEM. Data were analyzed by one-way ANOVA, followed by a Fisher test for multiple comparisons.
　実施例
（実験及び結果）
　1. YVLSRの抗不安作用試験
　マウスを高架式十字迷路上に置く前に、ペプチドＹＶＬＳＲを各々図２及び３に示される量で脳室内投与（図２）（n =5）及び腹腔内投与（図３）した（n =4-10）。図２及び３に示されるように、YVLSRの脳室内投与及び腹腔内投与により、オープンアームへの滞在時間の割合及び進入回数の割合が増加した。行動量を示す両アームへの進入回数の合計には差は認められなかった。したがって、高架式十字迷路試験においてYVLSRが抗不安作用を示すことがわかった。さらに、オープンフィールド試験を行ったところ、YVLSR投与により、中心部分の滞在時間が延長し、本試験においても抗不安作用を示すことが判明した（表１）。したがって、二つの評価系において、YVLSRは抗不安作用を示すことが明らかとなった。 Examples (experiment and results)
1. Anti-anxiety test of YVLSR Before placing the mouse on the elevated plus maze, peptide YVLSR was administered intracerebroventricularly (Figure 2) (n = 5) and intraperitoneally in the amounts shown in Figures 2 and 3, respectively ( Fig. 3) (n = 4-10). As shown in FIGS. 2 and 3, the rate of staying in the open arm and the rate of entry were increased by intraventricular and intraperitoneal administration of YVLSR. There was no difference in the total number of times of entry into both arms indicating the amount of activity. Therefore, it was found that YVLSR exhibits an anxiolytic effect in the elevated plus maze test. Furthermore, when an open field test was conducted, it was found that the residence time of the central part was prolonged by administration of YVLSR, and an anxiolytic action was also exhibited in this test (Table 1). Therefore, it became clear that YVLSR shows an anxiolytic action in two evaluation systems.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
次に、YVLSRの経口投与による影響を高架式十字迷路試験で評価した（図４）（n =4-8）。図４に示されるように、オープンアームでの滞在時間及び進入回数の割合が増加し、本ペプチドは経口投与でも作用することがわかった。以後、高架式十字迷路試験により作用機構を検討した。 Next, the influence of oral administration of YVLSR was evaluated by an elevated plus maze test (FIG. 4) (n = 4-8). As shown in FIG. 4, the ratio of staying time in the open arm and the number of times of entry increased, and it was found that this peptide also acts by oral administration. Thereafter, the mechanism of action was examined by an elevated plus maze test.
　2. YVLSRによる中枢δ受容体活性化
　YVLSRの経口投与による抗不安作用は、δオピオイド受容体アンタゴニストのnaltrindoleの脳室内投与（10 nmol/mouse）により、阻害された（図５）。なお、μアンタゴニストのnaloxoneでは阻害されなかった（データ示さず）。YVLSRは、δ受容体に対する親和性を示さず、かつ、マウス輸精管（MVD）を用いたδオピオイド活性も示さなかった。以上の結果より、YVLSRは、内因性δオピオイドリガンドの遊離促進により、中枢のδ受容体を活性化しているものと考えられる。 2. Central δ receptor activation by YVLSR The anxiolytic effect of YVLSR by oral administration was inhibited by intracerebroventricular administration (10 nmol / mouse) of the δ opioid receptor antagonist naltrindole (FIG. 5). The μ antagonist naloxone was not inhibited (data not shown). YVLSR showed no affinity for the δ receptor and also no δ opioid activity using mouse vas deferens (MVD). From the above results, it is considered that YVLSR activates the central δ receptor by promoting the release of endogenous δ opioid ligand.
　3． YVLSRによる、σ１、セロトニン5-HT1A、ドーパミンD1、及びGABAA受容体の活性化
　情動調節に関与する種々の受容体に対するアンタゴニストを用いて、YVLSRの抗不安機構を検討した。その結果、YVLSRの抗不安作用は、σ１、セロトニン5-HT1A、ドーパミンD1、及びGABAA受容体に対するアンタゴニスト（それぞれ、0.5、10、0.03及び5 ｍｇ／ｋｇ投与）により阻害されたが（図６,７）、これらの受容体には親和性を示さなかった。したがって、YVLSRの抗不安作用は、内因性σ1リガンド、セロトニン、ドーパミン、GABA等の神経伝達物質の放出ならびに、σ１、5-HT1A、D1、及びGABAA受容体の活性化を介していることが明らかとなった。なお、予備的検討結果であるが、特異的δアゴニストDPDPEによる抗不安作用は、σ１、5-HT1A、D1、及びGABAA受容体に対するアンタゴニストで阻害され、YVLSRの抗不安経路と一致することがわかった。また、これらの受容体に対するアゴニスト及びアンタゴニストを用いた薬理学的検討及び既知の報告より、δ受容体 → σ１受容体 → 5-HT1A受容体 → D1受容体 → GABAA受容体の順番で活性化されるものと考えられる。 3． Activation of σ 1 , Serotonin 5-HT 1A , Dopamine D 1 , and GABA A Receptor by YVLSR The anti-anxiety mechanism of YVLSR was examined using antagonists for various receptors involved in emotion regulation. As a result, the anxiolytic effect of YVLSR was inhibited by antagonists to σ 1 , serotonin 5-HT 1A , dopamine D 1 , and GABA A receptors (0.5, 10, 0.03, and 5 mg / kg, respectively). (FIGS. 6 and 7), no affinity was shown for these receptors. Therefore, the anxiolytic effect of YVLSR is through the release of neurotransmitters such as endogenous σ 1 ligand, serotonin, dopamine, GABA and the activation of σ 1 , 5-HT 1A , D 1 , and GABA A receptors. It became clear that. As a preliminary study, the anxiolytic effect of the specific δ agonist DPDPE is inhibited by antagonists to σ 1 , 5-HT 1A , D 1 , and GABA A receptors, and is consistent with the anxiolytic pathway of YVLSR. I found out that In addition, pharmacological studies using agonists and antagonists for these receptors and known reports indicate that δ receptor → σ 1 receptor → 5-HT 1A receptor → D 1 receptor → GABA A receptor. It is thought that it is activated by.
　一方、YVLSRの抗不安作用は、プロスタグランジン（PG）の生合成に関与するシクロオキシゲナーゼの阻害剤indomethacinではブロックされなかったことから、本ペプチドの抗不安作用にはPG類は関与しないことが判明した。 On the other hand, the anxiolytic effect of YVLSR was not blocked by indomethacin, a cyclooxygenase inhibitor involved in the biosynthesis of prostaglandins (PG). did.
　前述のように、YVLSRは、脳室内投与及び腹腔内投与により抗不安作用を示す。さらに本ペプチドは経口投与でも有効であった。 As described above, YVLSR exhibits an anxiolytic effect upon intraventricular and intraperitoneal administration. Furthermore, this peptide was also effective when administered orally.
　YVLSRは中枢δ受容体を活性化し、さらに、σ１、5-HT1A、D1、及びGABAA受容体を活性化し、抗不安作用を示すものと考えられる。一般的な抗不安薬として知られるジアゼパムなどのベンゾジアゼピン系の薬剤はGABAA受容体のベンゾジアゼピン結合部位に結合し、抗不安作用を示すのに対し、YVLSRは、この結合部位に親和性を示さないことから、GABA遊離を促進し、抗不安作用を示すものと考えられる。したがって、従来の抗不安薬とは異なる作用経路を介することがわかった。 YVLSR activates central δ receptors, and further activates σ 1 , 5-HT 1A , D 1 , and GABA A receptors, and is considered to exhibit an anxiolytic action. Benzodiazepines such as diazepam, known as common anxiolytic drugs, bind to the benzodiazepine binding site of the GABA A receptor and show anxiolytic activity, whereas YVLSR does not show affinity for this binding site Therefore, it is considered that GABA release is promoted and an anxiolytic action is exhibited. Therefore, it was found that the pathway is different from that of conventional anxiolytic drugs.
　本発明の抗不安薬は、従来の抗不安薬とは異なる作用メカニズムを持っている可能性があり、新しいタイプの薬剤を提供できる。一般に精神的ストレス緩和作用を示す薬品は睡眠誘発作用を併せ持つことが多く、代表的な抗不安薬ジアゼパムは睡眠導入剤として処方されている。また、YVLSRは睡眠誘発作用を仲介するGABAA受容体を活性化することを明らかにしている。従って、本発明の医薬組成物は、睡眠導入及び睡眠改善のために用いることもできる。本発明者らは、YVLSR以外にも、乳タンパク質由来の抗不安ペプチドを複数見出している。これらの抗不安ペプチドの作用経路は異なり、複数のペプチド投与による相加作用及び相乗作用が期待できる。したがって、乳タンパク質を摂取した際には、複数の低分子の抗不安ペプチドが派生し、相加効果及び相乗作用を示しているものかもしれない。 The anti-anxiety drug of the present invention may have a different mechanism of action from conventional anxiolytic drugs, and can provide a new type of drug. In general, drugs that have a mental stress relieving action often have a sleep-inducing action, and a typical anxiolytic drug diazepam is prescribed as a sleep inducer. YVLSR has also been shown to activate GABA A receptors that mediate sleep-induced effects. Therefore, the pharmaceutical composition of the present invention can also be used for sleep induction and sleep improvement. In addition to YVLSR, the present inventors have found a plurality of anxiolytic peptides derived from milk protein. The action pathways of these anxiolytic peptides are different, and an additive action and synergistic action by administration of a plurality of peptides can be expected. Therefore, when milk protein is ingested, a plurality of low-molecular anxiolytic peptides may be derived and exhibit additive effects and synergistic effects.
配列番号１は、設計ペプチド（designed peptide）である。 SEQ ID NO: 1 is a designed peptide.

Claims (6)

  1. ＹＶＬＳＲ（配列番号１）で表されるペプチド又はその類縁体を有効成分とする、医薬組成物。 A pharmaceutical composition comprising a peptide represented by YVLSR (SEQ ID NO: 1) or an analog thereof as an active ingredient.
  2. 抗不安剤、睡眠導入剤、睡眠改善剤、統合失調症治療薬または抗うつ薬である、項１に記載の医薬組成物。 Item 2. The pharmaceutical composition according to Item 1, which is an anxiolytic agent, sleep-inducing agent, sleep-improving agent, schizophrenia therapeutic agent or antidepressant.
  3. ＹＶＬＳＲで表されるペプチド又はその類縁体が配合された、抗不安または睡眠改善用食品。 An anti-anxiety or sleep improving food containing a peptide represented by YVLSR or an analog thereof.
  4. ＹＶＬＳＲで表されるペプチド又はその類縁体。 A peptide represented by YVLSR or an analog thereof.
  5. 抗不安、睡眠導入、睡眠改善、統合失調症治療または抗うつのための、ＹＶＬＳＲで表されるペプチド又はその類縁体。 A peptide represented by YVLSR or an analog thereof for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant.
  6. 患者に、項１又は２に記載の医薬組成物を投与する工程を含む、抗不安、睡眠導入、睡眠改善、統合失調症治療または抗うつのための方法。 A method for anxiety, sleep induction, sleep improvement, schizophrenia treatment or antidepressant, comprising the step of administering the pharmaceutical composition according to item 1 or 2 to a patient.
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