WO2014092150A1 - 筋芽細胞分化促進剤 - Google Patents
筋芽細胞分化促進剤 Download PDFInfo
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- WO2014092150A1 WO2014092150A1 PCT/JP2013/083324 JP2013083324W WO2014092150A1 WO 2014092150 A1 WO2014092150 A1 WO 2014092150A1 JP 2013083324 W JP2013083324 W JP 2013083324W WO 2014092150 A1 WO2014092150 A1 WO 2014092150A1
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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
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
- A23L33/18—Peptides; Protein hydrolysates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
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- 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
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06026—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atom, i.e. Gly or Ala
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
- C07K5/06043—Leu-amino acid
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- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06104—Dipeptides with the first amino acid being acidic
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06139—Dipeptides with the first amino acid being heterocyclic
- C07K5/06165—Dipeptides with the first amino acid being heterocyclic and Pro-amino acid; Derivatives thereof
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0802—Tripeptides with the first amino acid being neutral
- C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
- C07K5/0808—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/08—Tripeptides
- C07K5/0821—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp
- C07K5/0823—Tripeptides with the first amino acid being heterocyclic, e.g. His, Pro, Trp and Pro-amino acid; Derivatives thereof
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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
Definitions
- the present invention relates to a myoblast differentiation promoter containing a peptide molecule or the like.
- Patent Document 1 describes that dipeptides such as Hyp-Gly have an osteoclast differentiation inhibitory action, an alkaline phosphatase inhibitory action, and the like.
- Non-Patent Document 2 describes that peptides such as Hyp-Gly-Pro have an antioxidant action.
- Patent Document 2 a peptide having up to 10 amino acids in the upstream region and / or the downstream region such as Pro-Gly and Hyp-Gly has an action of stimulating the growth, maintenance and repair of bone and the like.
- Patent Document 3 describes that prickly pear, soybean peptide, C12 peptide and the like have a myoblast activation action.
- Non-Patent Document 3 describes the rate of change in muscle weight when an American football player ingests an equal mixture of collagen peptide and whey protein for 3 months under exercise combined use. Ingestion of this equimolar mixture increases muscle weight (Fig. 2), but body weight also increases (Fig. 1), and when converted to the ratio of muscle weight per body weight, it decreases from the start. is doing. In contrast, in the present invention, as described in Test Example 2 described later, the muscle weight ratio per body weight is greatly increased.
- the present inventors have found that peptide molecules such as Ala-Hyp-Gly, Hyp-Gly-Pro, Leu-Hyp, Glu-Hyp, Gly-Pro-Hyp, Pro-Ala, Hyp-Gly and Pro. It was found that -Hyp has an excellent myoblast differentiation promoting action, and the present invention has been completed. That is, the present invention is as follows.
- a peptide selected from the group consisting of Ala-Hyp-Gly, Hyp-Gly-Pro, Leu-Hyp, Glu-Hyp, Gly-Pro-Hyp, Pro-Ala, Hyp-Gly and Pro-Hyp A myoblast differentiation promoter containing a pharmaceutically acceptable salt.
- the preparation is a preparation for oral administration, an injection for direct administration to muscle, a transdermal agent, a suppository, a nasal drop or an inhalant Or a myoblast differentiation promoter.
- a peptide selected from the group consisting of Ala-Hyp-Gly, Hyp-Gly-Pro, Leu-Hyp, Glu-Hyp, Gly-Pro-Hyp, Pro-Ala, Hyp-Gly and Pro-Hyp A food or drink or feed containing a pharmaceutically acceptable salt.
- Peptides used in the present invention are Ala-Hyp-Gly, Hyp-Gly-Pro, Leu-Hyp, Glu-Hyp, Gly-Pro-Hyp, Pro-Ala, Hyp-Gly and Pro-Hyp.
- the peptide can be a pharmaceutically acceptable salt.
- Preferred peptides include Hyp-Gly and Pro-Hyp.
- the “pharmaceutically acceptable salt” include inorganic acid salts such as hydrochloride, sulfate, and phosphate, and organic acid salts such as methanesulfonate, benzenesulfonate, succinate, and oxalate.
- Inorganic base salts such as sodium salt, potassium salt and calcium salt, and organic base salts such as triethylammonium salt.
- This peptide can be synthesized by, for example, “solid phase synthesis method” and “liquid phase synthesis method” (for example, JP-A No. 2003-183298).
- solid phase synthesis method methods of the Fmoc method and the Boc method are further known, and this peptide may be synthesized by any method.
- An example of the solid phase synthesis method will be specifically described below.
- a polystyrene polymer gel bead having a diameter of about 0.1 mm whose surface is modified with an amino group is used as a solid phase, and diisopropylcarbodiimide is used as a condensing agent.
- the amino group of the C-terminal amino acid is protected with an Fmoc group or a Boc group to form a peptide bond with the amino group of the polystyrene polymer gel.
- the solid phase is thoroughly washed with a solvent, and the remaining reagent and amino acid are washed and removed, and then the amino group protecting group of the amino acid bonded to the solid phase is removed.
- a peptide is synthesized on a solid phase by sequentially repeating the same reaction using an amino acid with an amino group protected.
- the peptide can be separated from the solid phase and the peptide can be synthesized.
- This peptide can also be produced by hydrolyzing gelatin with a combination of two or more of endo-type protease and exo-type protease.
- the hydrolyzed peptide mixture itself or a partially purified mixture thereof can also be used as a myoblast differentiation promoter.
- Examples of chemical modification of the amino group of the N-terminal amino acid include polypeptidylation, succinylation, maleylation, acetylation, deamination, benzoylation, alkylsulfonylation, allylsulfonylation, dinitrophenylation, trinitrophenylation, Examples thereof include carbamylation, phenylcarbamylation, and thiolation.
- Examples of the chemical modification of the carboxyl group of the C-terminal amino acid include esterification and amidation.
- the present peptide is cationized, it can be subjected to ethylenediamine formation, spermination or the like.
- Myoblast differentiation promoting agent The peptide and the like have a myoblast differentiation promoting action as described in the following test examples. Therefore, this peptide etc. can be used for the treatment or prevention of various diseases which require muscle strengthening.
- the myoblast differentiation promoter of the present invention is, for example, treatment of locomotive syndrome, treatment of sarcopenia, improvement of training effect of athletes, students, etc., enhancement of physical strength of elderly and long-term hospitalized patients, improvement of meat quality of livestock, etc. Can be used.
- the myoblast differentiation promoter of the present invention can be administered orally or parenterally in various forms of pharmaceutical preparations.
- the form includes, for example, tablets, granules, capsules, powders, liquids, suspension preparations, emulsion preparations, etc.
- the myoblast differentiation promoter of the present invention may contain other active ingredients and ingredients for preparations as appropriate as long as the effects of the present invention are not impaired.
- active ingredients include muscle enhancing agents such as male hormones, and muscle enhancing supplements such as amino acid mixtures.
- muscle enhancing agents such as male hormones
- muscle enhancing supplements such as amino acid mixtures.
- the blending amount of other active ingredients can be appropriately changed according to each action.
- the food and drink or the feed peptide is a peptide derived from gelatin, it is extremely safe even if it is taken or applied on a daily basis. Then, it is useful also as food-drinks or feed containing this peptide etc. which utilized the effect of the outstanding myoblast differentiation promotion effect
- the present invention can be used to increase the physical strength of elderly people and long-term hospitalized patients, to increase the effect of training of athletes and students, and to improve the quality of livestock.
- the peptide or the like in the food or drink or feed of the present invention can be used by changing the content as appropriate according to the effect to be used.
- Examples 1 to 22 and Comparative Examples 1 to 4 The peptides of Examples 1 to 8 and Comparative Example 1 below were synthesized using the peptide solid phase synthesis method described above. Using these peptides, equimolar ratio mixtures of the following two peptides of Examples 9 to 21 were prepared, and equimolar ratio mixtures of the following two amino acids of Comparative Examples 2 and 3 were prepared. In addition, commercially available collagen peptides of Example 22 and Comparative Example 4 below were used.
- Example 1 Ala-Hyp-Gly (AOG) (Example 2) Hyp-Gly-Pro (OGP) (Example 3) Leu-Hyp (LO) (Example 4) Glu-Hyp (EO)
- Example 5 Gly-Pro-Hyp (GPO)
- Example 6 Pro-Ala (PA) (Example 7) Hyp-Gly (OG) (Example 8) Pro-Hyp (PO)
- Example 9 Mixture of OG and PO (OG + PO) (Example 10) Mixture of OG and AOG (OG + AOG) (Example 11) Mixture of OG and OGP (OG + OGP) (Example 12) Mixture of OG and LO (OG + LO) (Example 13) Mixture of OG and EO (OG + EO) (Example 14) Mixture of OG and GPO (OG + GPO) (Example 15) Mixture of OG and PA (OG + PA) (Example 16) Mixture of PA and AOG (PA + AOG) (Example 17: Mixture of PA and OGP (PA + OGP) Example 18: Mixture of PA and LO (PA + LO) Example 19: Mixture of PA and EO (PA + EO) (Example 20) Mixture of PA and GPO (PA + GPO) (Example 22) Collagen peptide "Type-M (manufact
- Test example 1 Myoblast differentiation promotion test in myoblast culture
- Mouse-derived C2C12 myoblasts were cultured in D-MEM medium containing 10% FBS, 100 units / ml penicillin G sodium, 100 ⁇ g / ml streptomycin, 1.0 g / l NaHCO 3.
- D-MEM medium (10% FBS, + P / S)
- the culture was performed in an incubator at 95% air, 5% CO 2 and 37 ° C.
- D-MEM medium containing 2% horse serum (HS), 100 units / ml penicillin G sodium, 100 ⁇ g / ml streptomycin, 1.0 g / l NaHCO 3 with 90% confluent C2C12 myoblasts
- the medium was changed to medium (2% HS + P / S), and thereafter, D-MEM medium (2% HS + P / S) was changed every two days to differentiate into myotube cells.
- Examples 1-21 and Comparative Examples 1-3 were added to the medium at every final medium exchange at a final concentration of 100 ⁇ M, and the cells were kept exposed for 8 days.
- the Western blotting method was performed according to the following procedure.
- the cells used for protein measurement were washed twice with PBS at the time of recovery, peeled off with a cell scraper, transferred to a tube, centrifuged at 4 ° C., 250 ⁇ g for 3 minutes, and then PBS was completely removed, and a buffer ( 20 mM HEPES-NaOH (pH 7.5), 0.5% NP-40, 1 mM EDTA, 100 ⁇ M AEBSF, 1 ⁇ g / ml aprotinin, 10 ⁇ g / ml leupeptin, 1 mM DTT, 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 ⁇ M molybdenum
- the suspension was suspended in 100 ⁇ l of anthracic acid, 10 mM sodium pyrophosphate), and pulverized 3 times for 5 seconds for each sample using an ultrasonic disperser while cooling with ice, and then the amount of protein was quant
- Protein quantification was performed using bovine serum albumin as a standard protein according to the method of Bradford. After SDS-PAGE finished, equilibrated with methanol transfer buffer (48 mM Tris, 39 mM glycine) to PVDF membrane was carried out prewetting infiltrated and using blotting apparatus, 1 hour electro at 1.5mA per membrane 1 cm 2 A transfer was performed. After the transfer, the PVDF membrane was blocked with PBS (-) containing 6% skim milk for 1 hour. The primary antibody was diluted as follows, and the antigen-antibody reaction was performed overnight at 4 ° C.
- Peroxidase-conjugated secondary antibodies for each primary antibody were mouse anti-myosin heavy chain monoclonal antibody (MF20) culture supernatant 1/2000, mouse anti-tropomyosin monoclonal antibody (CH1) culture supernatant 1/2000, rabbit anti-GAPDH polyclonal antibody 1 / 3000, peroxidase-conjugated anti-mouse IgG 1/5000, peroxidase-conjugated anti-rabbit IgG 1/2000 at a ratio of 6% skim milk diluted with PBS (-), reacted for 30 minutes, PBS (-) for 10 minutes, 3 times Washed.
- MF20 mouse anti-myosin heavy chain monoclonal antibody
- CH1 mouse anti-tropomyosin monoclonal antibody
- rabbit anti-GAPDH polyclonal antibody 1 / 3000 rabbit anti-GAPDH polyclonal antibody 1 / 3000
- tropomyosin and myosin heavy chain are used as differentiation markers for myoblasts. From the above test results, it was confirmed that each of the peptides of Examples 1 to 8 produced more myoblast differentiation markers, tropomyosin and myosin heavy chain, than the control. Therefore, it is understood that these peptides have a myoblast differentiation promoting action. Synergistic for two peptide mixtures selected from Ala-Hyp-Gly, Hyp-Gly-Pro, Leu-Hyp, Glu-Hyp, Gly-Pro-Hyp, Pro-Ala, Hyp-Gly and Pro-Hyp It was shown to have an effect of promoting myoblast differentiation.
- Test example 2 Clinical test A clinical test was conducted using Example 22 and Comparative Example 4 as collagen peptides.
- the subjects were 34 healthy women who were not athletes and were 24 to 61 years old. They were randomly assigned to the collagen peptide intake group of Example 22 (17 persons) and the collagen peptide intake group of Comparative Example 4 (17 persons), and a double-blind test was conducted. 5 g of each collagen peptide was ingested continuously for 10 weeks per day.
- the evaluation method was as follows. Muscle weight (kg: water content included) in each subject was measured using a TANITA body composition meter inner scan 50V BC-622-BK according to left and right parts.
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Abstract
Description
筋肉細胞分化の初期過程で、未分化な細胞が筋線維由来の細胞である筋芽細胞に分化する。筋芽細胞はさらに分化をして筋肉細胞特異的なタンパク質が発現される。筋肉細胞の分化で特徴的な現象は細胞融合であり、単核細胞の筋芽細胞が融合して、多核細胞である筋管細胞に分化する。さらに成熟した筋管細胞から、収縮能を持つ筋線維を形成する段階を経て、筋肉が完成する。上記の筋芽細胞の分化の際に、トロポミオシン、ミオシン重鎖等の特徴的なタンパク質が生成され、これらは分化マーカーとして用いられる(非特許文献1)。
特許文献3には、刺梨、大豆ペプチド、C12ペプチド等が筋芽細胞活性化作用を有することが記載されている。しかし、これらペプチド分子に筋芽細胞の分化促進作用があることは知られていない。
非特許文献3には、アメリカンフットボール選手がコラーゲンペプチドとホエイプロテインの等量混合物を3ヶ月間、運動併用下で摂取した場合の筋肉重量の変化率が記載されている。この等量混合物の摂取によって筋肉重量が増加(図2)しているが、体重もまた同様に増加(図1)しており、体重当たりの筋肉重量比率に換算すれば開始時よりも却って減少している。これに対して、本願発明では、後述の試験例2に記載の通り、体重当たりの筋肉重量比率が大きく増加している。
[1] Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、筋芽細胞分化促進剤。
[2] Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、[1]記載の筋芽細胞分化促進剤。
[3] Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択される2以上のペプチドまたはそれらの薬学上許容される塩を含有する、[1]記載の筋芽細胞分化促進剤。
[4] 製剤が、経口的に投与するための製剤、筋肉に直接投与するための注射剤、経皮剤、坐剤、点鼻剤または吸入剤である、[1]~[3]のいずれか記載の筋芽細胞分化促進剤。
[5] Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、飲食品または飼料。
1.ペプチド
本発明に用いられるペプチドは、Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypであり、本ペプチドは薬学上許容される塩とすることができる。好ましいペプチドとして、Hyp-GlyおよびPro-Hypが挙げられる。
「薬学上許容される塩」としては、例えば、塩酸塩、硫酸塩、リン酸塩等の無機酸塩、メタンスルホン酸塩、ベンゼンスルホン酸塩、コハク酸塩、シュウ酸塩等の有機酸塩、ナトリウム塩、カリウム塩、カルシウム塩等の無機塩基塩、トリエチルアンモニウム塩等の有機塩基塩等が挙げられる。
本ペプチドは、ゼラチンにエンド型プロテアーゼおよびエキソ型プロテアーゼの2種以上を組み合わせて加水分解することによっても製造することができる。また、上記加水分解をしたペプチド混合物自体またはこれを部分精製した混合物を筋芽細胞分化促進剤として用いることもできる。
具体的には、ヒドロキシプロリンの水酸基の化学修飾としては、例えばO-アセチル化等が挙げられる。N末アミノ酸のアミノ基の化学修飾としては、例えばポリペプチジル化、スクシニル化、マレイル化、アセチル化、脱アミノ化、ベンゾイル化、アルキルスルホニル化、アリルスルホニル化、ジニトロフェニル化、トリニトロフェニル化、カルバミル化、フェニルカルバミル化、チオール化等が挙げられる。C末アミノ酸のカルボキシル基の化学修飾としては、例えばエステル化、アミド化等が挙げられる。さらに、本ペプチドをカチオン化する場合は、エチレンジアミン化、スペルミン化などを行うことができる。
本ペプチド等は、後述の試験例に記載の通り、筋芽細胞の分化促進作用を有する。従って、本ペプチド等は、筋肉増強が必要な各種疾患の治療または予防に用いることができる。本発明の筋芽細胞分化促進剤は、例えば、ロコモティブシンドロームの治療、サルコペニアの治療、運動選手、学生等のトレーニングの効果の向上、老人および長期入院患者等の体力増強、家畜の肉質の向上等に用いることができる。
本発明の筋芽細胞分化促進剤は、経口的にまたは非経口的に種々の形態の医薬製剤で投与することができる。その形態としては、経口的に投与する場合は、例えば、錠剤、顆粒剤、カプセル剤、粉剤、液剤、懸濁製剤、乳化製剤等が挙げられ、非経口的に投与する場合は、例えば、注射剤、経皮剤、坐剤、点鼻剤および吸入剤等が挙げられる。好ましくは、錠剤、顆粒剤、カプセル剤、筋肉等の患部に直接投与する液剤等が挙げられる。なお、本ペプチドは、消化管でアミノ酸への分解もほとんど起こらず、腸管で迅速に吸収されるため、経口投与による摂取が好適である。また、本ペプチドは食事または飲料に混ぜて摂取させても良い。
本発明の筋芽細胞分化促進剤は、本発明の効果を害しない範囲で、適宜他の有効成分や製剤用の成分を含有させても良い。他の有効成分として、例えば男性ホルモン等の筋肉増強剤、アミノ酸混合物等の筋肉増強サプリメント等が挙げられる。他の有効成分の配合量としては、各々の作用に応じて適宜、変更することができる。
本ペプチド等は、ゼラチンに由来するペプチドであるため、日常的に摂取または塗布しても極めて安全である。そこで、本ペプチド等の優れた筋芽細胞分化促進作用の効果を利用した、本ペプチド等を含有する飲食品または飼料としても有用である。例えば、老人および長期入院患者等の体力増強のために、運動選手、学生等のトレーニングの効果を高めるために、また家畜の肉質の向上のためなどに、本発明を用いることができる。本発明の飲食品または飼料における本ペプチド等は、利用する効果に応じて適宜、含有量を変更して用いることができる。
実施例1~22および比較例1~4
前記のペプチド固相合成法を用いて、以下の実施例1~8および比較例1のペプチドを合成した。これらペプチドを用いて、以下の実施例9~21の2種のペプチドの等モル比混合物を調製し、また以下の比較例2および3の2種のアミノ酸の等モル比混合物を調製した。また、以下の実施例22および比較例4の市販のコラーゲンペプチドを用いた。
(実施例1)Ala-Hyp-Gly(AOG)
(実施例2)Hyp-Gly-Pro(OGP)
(実施例3)Leu-Hyp(LO)
(実施例4)Glu-Hyp(EO)
(実施例5)Gly-Pro-Hyp(GPO)
(実施例6)Pro-Ala(PA)
(実施例7)Hyp-Gly(OG)
(実施例8)Pro-Hyp(PO)
(実施例10)OGとAOGの混合物(OG+AOG)
(実施例11)OGとOGPの混合物(OG+OGP)
(実施例12)OGとLOの混合物(OG+LO)
(実施例13)OGとEOの混合物(OG+EO)
(実施例14)OGとGPOの混合物(OG+GPO)
(実施例15)OGとPAの混合物(OG+PA)
(実施例16)PAとAOGの混合物(PA+AOG)
(実施例17)PAとOGPの混合物(PA+OGP)
(実施例18)PAとLOの混合物(PA+LO)
(実施例19)PAとEOの混合物(PA+EO)
(実施例20)PAとGPOの混合物(PA+GPO)
(実施例21)PAとPOの混合物(PA+PO)
(実施例22)コラーゲンペプチド「Type-M(新田ゼラチン製)」
LC-MS/MSで分析したところ、本コラーゲンペプチドには以下のペプチドがそれぞれ含まれていた。
Hyp-Gly:7573ppm,Pro-Ala:2541ppm,Ala-Hyp-Gly:331ppm,Pro-Hyp:184ppm,Gly-Pro-Hyp:85ppm,Glu-Hyp:72ppm,Hyp-Gly-Pro:7ppm
(比較例2)HypとGlyの混合物(O+G)
(比較例3)ProとHypの混合物(P+O)
(比較例4)コラーゲンペプチド「HDL-50SP(新田ゼラチン製)」
LC-MS/MSで分析したところ、本コラーゲンペプチドには以下のペプチドがそれぞれ含まれていた。
Hyp-Gly:11ppm,Pro-Hyp:8ppm
筋芽細胞培養における筋芽細胞分化促進試験
マウス由来C2C12筋芽細胞の培養は、10%FBS、100units/ml ペニシリンGナトリウム、100μg/ml ストレプトマイシン、1.0g/l NaHCO3を含むD-MEM培地(D-MEM培地(10%FBS,+P/S))を用いた。培養は95%空気、5%CO2、37℃のインキュベーター内で行った。C2C12筋芽細胞が90%コンフルエントになった状態で2%ウマ血清(HS)、100units/ml ペニシリンGナトリウム、100μg/mlストレプトマイシン、1.0g/l NaHCO3を含むD-MEM培地(D-MEM培地(2%HS+P/S))に交換し、以降2日ごとにD-MEM培地(2%HS+P/S)交換をつづけ、筋管細胞へ分化させた。実施例1~21および比較例1~3を終濃度100μMで培地交換毎に培地に添加し、細胞に8日間暴露し続けた。
Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypから選択される2種のペプチド混合物には相乗的な筋芽細胞分化促進作用があることが示された。特に、実施例9のHyp-GlyとPro-Hypの混合物は筋芽細胞分化促進作用が顕著であった。比較例2および3のアミノ酸のみの混合物では同様の作用はなく、ペプチド分子としてのPro-HypとHyp-Glyの混合物で初めて、筋芽細胞分化促進作用が生じることが示された。
臨床試験
試験コラーゲンペプチドとして実施例22と比較例4を用いて、臨床試験を行った。被験者は、運動選手ではない健康な女性34人であり、24歳~61歳であった。実施例22のコラーゲンペプチド摂取群(17人)と比較例4のコラーゲンペプチド摂取群(17人)に無作為に割付し、二重盲検試験を行った。1日当たり5gの各コラーゲンペプチドを10週間連続摂取した。評価方法は、TANITA体組成計左右部位別インナースキャン50V BC-622-BKを用いて各被験者での筋肉重量(kg:水分量含有)を測定した。また同時に測定した水分量(kg)を差し引き、体重当たりの筋肉重量比率(%)を計算し、評価した。結果は平均値±標準偏差で表2に示す。表中の**はTwo-way-ANOVAにおいて、比較例4に対してP<0.001で有意であることを示す。
Claims (5)
- Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、筋芽細胞分化促進剤。
- Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、請求項1記載の筋芽細胞分化促進剤。
- Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択される2以上のペプチドまたはそれらの薬学上許容される塩を含有する、請求項1記載の筋芽細胞分化促進剤。
- 製剤が、経口的に投与するための製剤、筋肉に直接投与するための注射剤、経皮剤、坐剤、点鼻剤または吸入剤である、請求項1~3のいずれか記載の筋芽細胞分化促進剤。
- Ala-Hyp-Gly、Hyp-Gly-Pro、Leu-Hyp、Glu-Hyp、Gly-Pro-Hyp、Pro-Ala、Hyp-GlyおよびPro-Hypからなる群から選択されるペプチドまたはその薬学上許容される塩を含有する、飲食品または飼料。
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CN201380022730.5A CN104271144B (zh) | 2012-12-13 | 2013-12-12 | 成肌细胞分化促进剂 |
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WO2023022174A1 (ja) * | 2021-08-18 | 2023-02-23 | 株式会社 ニッピ | 筋肉疲労および/または即発性筋肉痛を抑制する食品組成物 |
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CN107759660A (zh) * | 2017-12-05 | 2018-03-06 | 陕西慧康生物科技有限责任公司 | 一种三肽‑29的液‑固相合成方法 |
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CN113929769B (zh) * | 2021-09-07 | 2023-11-28 | 中国科学院理化技术研究所 | 一种促进成肌细胞增殖的活性肽及其应用 |
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