JP2014001182A - Therapy or preventive of disease associated with bone, cartilage or skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a therapy or a preventive of a disease associated with bone, cartilage or skin, which contains a peptide derived from natural collagen and is improved than the prior art.SOLUTION: A therapy or a preventive of a disease associated with bone, cartilage or skin includes a therapy, a preventive or the like of osteoporosis, osteoarthritis or photoaging, the therapy or preventive containing Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof.

Description

  The present invention relates to a therapeutic or prophylactic agent for diseases related to bone, cartilage or skin, which contains peptides derived from natural collagen.

  It is known that collagen-derived peptide molecules have various pharmacological actions. For example, Patent Document 1 describes that a dipeptide such as Hyp-Pro has an osteoclast differentiation inhibitory action, an alkaline phosphatase inhibitory action, and the like. Non-Patent Document 1 describes that peptides such as Hyp-Gly-Pro have an antioxidant effect.

WO2010 / 038323

Abstracts of the 5th Annual Meeting of the Japanese Amino Acid Society (JSAAS2011), p74

  The problem to be solved by the present invention is to provide a therapeutic or prophylactic agent for diseases related to bone, cartilage or skin, which contains a peptide derived from natural collagen and is superior to the prior art.

  As a result of intensive studies, the present inventors have found that Hyp-Gly-Pro and Hyp-Gly-Pro-Hyp, which are peptides derived from natural collagen, have excellent hyaluronic acid production promoting action, pigmentation inhibiting action, alkaline phosphatase inhibiting action, and It has been found that it has an osteoclast differentiation inhibitory effect, and the present invention has been completed. That is, the present invention is as follows.

[1] A therapeutic or prophylactic agent for a disease related to bone, cartilage or skin, comprising Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof.
[2] The treatment or prevention agent according to [1], which is a hyaluronic acid production promoter, a pigmentation inhibitor, an alkaline phosphatase activity inhibitor, or a precursor osteoclast differentiation inhibitor.
[3] The treatment or prevention agent according to [1], which is a treatment or prevention agent for osteoporosis, osteoarthritis, or photoaging.
[4] A food, drink, cosmetic or feed containing Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof.

According to the present invention, it is possible to provide a therapeutic or prophylactic agent for diseases related to bone, cartilage or skin, which is superior to the prior art, containing a peptide derived from natural collagen.
Moreover, since the therapeutic or prophylactic agent of the present invention contains Hyp-Gly-Pro and Hyp-Gly-Pro-Hyp, which are peptides derived from natural collagen, it is extremely safe to be taken or applied on a daily basis. . Therefore, it is also useful as a food, drink, cosmetic or feed containing the peptide etc. by utilizing such effects of the peptide etc.

Hereinafter, the present invention will be described in detail.
1. Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof The peptides used in the present invention are Hyp-Gly-Pro and Hyp-Gly-Pro-Hyp. It may be a salt that is acceptable above. A preferred peptide is Hyp-Gly-Pro.
Examples of 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). In the case of the 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. First, 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. Subsequently, a peptide is synthesized on a solid phase by sequentially repeating the same reaction using an amino acid with an amino group protected. Finally, the peptide can be synthesized by digesting the solid phase with trifluoroacetic acid to separate the peptide from the solid phase.
This peptide can also be produced by hydrolyzing gelatin with a combination of two or more of endo-type protease and exo-type protease.

In the present invention, the peptide may be chemically modified. Chemical modification can be performed in amino acid units, and examples thereof include a hydroxyl group of hydroxyproline, an amino group of an N-terminal amino acid, and a carboxyl group of a C-terminal amino acid. By such chemical modification, it is possible to dissolve from weakly acidic to neutral, and to improve compatibility with other active ingredients described later.
Specifically, examples of the chemical modification of the hydroxyl group of hydroxyproline include O-acetylation. 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. Furthermore, when the present peptide is cationized, it can be subjected to ethylenediamine formation, spermination or the like.

  As specific means and processing conditions for chemical modification, ordinary peptide chemical modification techniques are applied. For example, O-acetylation of the hydroxyl group of hydroxyproline can be performed by reacting acetic anhydride in an aqueous solvent or a non-aqueous solvent. For example, esterification of the carboxyl group of the C-terminal amino acid can be performed by passing dry hydrogen chloride gas after suspension in methanol, and amidation can be performed by acting carbodiimide or the like. Furthermore, as other specific examples of chemical modification, the chemical modification techniques described in Japanese Patent Publication No. Sho 62-44522 and Japanese Patent Publication No. 5-79046 can be applied.

2. Treatment or preventive agent for diseases related to bone, cartilage or skin As described in the evaluation test described later, this peptide and the like are used to promote hyaluronic acid production of fibroblasts, alkaline phosphatase inhibitory action of precursor chondrocytes, and osteoclast Has an effect of inhibiting cell differentiation. Therefore, this peptide etc. can treat or prevent diseases related to bone, cartilage or skin.
“Diseases related to bone, cartilage or skin” include, for example, osteoporosis, bone Paget's disease, treatment of bone calcium metabolic diseases such as hypercalcemia associated with malignant tumors; arthritis, chronic progressive arthritis, osteoarthritis, Collagen-induced arthritis, juvenile chronic arthritis, spondyloarthritis, rheumatoid arthritis, juvenile chronic rheumatoid arthritis and other rheumatic diseases; photoaging, cuts, stab wounds, surgical wounds, pressure ulcers, burns , Electrical and radiation, etc.), abrasion or assault of the skin, osteomyelitis, orthopedic wounds, rough skin, etc.

The therapeutic or prophylactic agent 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. when administered orally, and when administered parenterally, for example, injection Agents, transdermal agents, suppositories, nasal drops, inhalants and the like. Preferable examples include tablets, granules, capsules, liquids directly administered to the affected area, and the like. In addition, since this peptide hardly decomposes into amino acids in the gastrointestinal tract and is rapidly absorbed in the intestinal tract, it is preferably taken by oral administration.
The dose of this peptide varies depending on the patient's condition and body weight, the type of compound, the route of administration, etc., but in the case of oral administration per day for adults, for example, about 0.1 to 1000 mg, preferably about 1 to 500 mg, More preferably, about 10-200 mg is mentioned, When administering directly to an affected part, about 0.01-200 mg, Preferably it is about 0.1-100 mg, More preferably, about 1-50 mg is mentioned, for example. Formulations in other forms can be appropriately determined with reference to these dosages. These preparations can be administered divided into 1 to several times a day, or once to several days.

The therapeutic or prophylactic agent of the present invention may contain other active ingredients and ingredients for preparation as appropriate as long as the effects of the present invention are not impaired.
Examples of other active ingredients include glucosamine or a pharmaceutically acceptable salt thereof, chondroitin sulfate, and the like. In particular, glucosamine or a pharmaceutically acceptable salt thereof is preferable because it has a function of improving the effect of the peptide. As other active ingredients, calcium, sugar-transferred hesperidin and the like can be used for promoting the deposition of bone salt, and vitamin C and the like can be used for promoting the synthesis and deposition of collagen. Furthermore, it may contain a peptide or amino acid other than the present peptide as another active ingredient. In that case, collagen or gelatin can be hydrolyzed to obtain a collagen peptide containing the present peptide, which can be directly used as a mixture without purification.

  The amount of other active ingredients can be appropriately changed according to each action, for example, 0.001 to 20 parts by weight with respect to the therapeutic or prophylactic agent of the present invention, Preferably the ratio of 0.01-20 weight part is mentioned. In particular, in the case of glucosamine or a pharmaceutically acceptable salt thereof, the proportion is preferably 5 to 15 parts by weight. If the amount is less than 5 parts by weight, the effect of improving the effect of the peptide molecule having a specific structure may not be sufficiently exhibited. If the amount exceeds 15 parts by weight, the peptide molecule may be discharged into urine or feces, resulting in excessive intake.

  Pharmaceutically acceptable carriers used in formulating pharmaceutical preparations include diluents, binders (syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone), excipients (lactose, sucrose, corn starch, Examples include potassium phosphate, sorbit, glycine), lubricants (magnesium stearate, talc, polyethylene glycol, silica), disintegrants (potato starch), wetting agents (sodium lauryl sulfate), and the like. The pharmaceutical preparation can be produced by mixing the peptide, other active ingredients, a pharmaceutically acceptable carrier and the like according to a conventionally known method.

3. Food / beverage products, cosmetics, or feed This peptide is a peptide derived from natural collagen, so it is extremely safe to be ingested or applied on a daily basis. Therefore, it is also useful as a food / drink, cosmetics or feed containing the peptide etc. utilizing the effects such as the superior hyaluronic acid production promoting action, alkaline phosphatase inhibiting action and osteoclast differentiation inhibiting action such as the peptide. The peptide or the like in the food / beverage product, cosmetic product or feed of the present invention can be used by changing the content as appropriate according to the effect to be used.

  EXAMPLES Hereinafter, although an Example, a comparative example, and an evaluation test demonstrate this invention further in detail, this invention is not limited to these at all.

Examples 1 and 2 and Comparative Examples 1 and 2
The following peptides were synthesized using the peptide solid phase synthesis method described above.
(Example 1) Hyp-Gly-Pro
(Example 2) Hyp-Gly-Pro-Hyp
(Comparative Example 1) Pro-Hyp-Gly
(Comparative Example 2) Gly-Pro-Hyp
These peptides are all known peptides.

Evaluation test 1
[Promoting hyaluronic acid production]
Evaluation was performed according to the method of hyaluronic acid measurement by JR Martins et al. (Anal Biochem., 319 (1): 65-72 (2003)).
Hyaluronic acid is an anionic polysaccharide having a molecular weight of 1000 to 5000 kDa, and is composed of repeating glucuronic acid-acetylglucosamine disaccharides. The amount of hyaluronic acid production per cell in normal human skin fibroblast NHDF (manufactured by Kurabo Industries) was measured to evaluate the hyaluronic acid production promoting effect on the control.
In this test, R & D DuoSet (product code DY3614) was used as the measurement kit. NHDF cells were seeded in a 96-well plate at 8 × 10 ³ cells / well, and samples were added after adhesion was stabilized. The culture supernatant 24 hours after the addition was used as a test sample. Using the property that aggrecan and hyaluronic acid form aggregates, the amount of hyaluronic acid in the sample was specifically detected and quantified using the sandwich method. The test results are described below.

Evaluation test 2
[Inhibition of pigmentation]
Recently, TRP-1 and TRP-2, tyrosinase-related proteins, have been shown to be involved in black eumelanin production at the gene and protein levels. In particular, TRP-1 is a product of the brown locus, having 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidation activity, TRP-2 is a product of the slaty locus, It was shown to be identical to the enzyme that catalyzes the isomerization to. Black melanin is produced by the coordinated expression of these three enzymes. In this test, mouse B16 melanoma cells were used to confirm the effect of suppressing the expression of the enzymes TRP-1 and TRP-2.
Melanoma cells were seeded in a 100 mm petri dish at a concentration of 2 × 10 ⁴ cells / ml × 10 ml, pre-cultured for 2 days, and then the medium was changed to a test serum-free DMEM / F12 (1: 1) medium. Was added and cultured for 3 days. After culturing, 1 ml of Trizol solution was added to the collected cells, and total RNA was prepared according to the protocol. And it reverse-transcribed with reverse transcriptase and measured each gene expression level by real-time PCR. Each value was corrected with GAPDH, control was set to 1.0, and each gene expression level was calculated.

Evaluation test 3
[Alkaline phosphatase inhibitory action]
Evaluated according to the chondrocyte differentiation culture method (J Pharmacol Sci 109, 413-423 (2009)) of Y. Nakamura et al.
Pre-chondrocyte ATDC5 is a cell that has the property of causing calcification by culturing for several days (about 5 to 7 days), and further cultivating, calcification, and finally differentiation to apoptosis. is there. Inhibiting calcification of pre-chondrocytes is assumed to be able to alleviate or delay the cause of pain (calcification) caused by arthritis such as osteoarthritis when assuming articular cartilage. It is widely used as a system that can evaluate regeneration in virto. It is known that alkaline phosphatase (ALP: phosphate monoester hydrolase) is discharged onto the cell membrane as a precursor to ATDC5 causing calcification. Naphthol or a derivative of naphthol or a derivative thereof serving as a substrate of ALP is decomposed into ALP to cause a diazo coupling reaction to produce an insoluble azo dye. It is a screening system that verifies whether or not it is possible to suppress the process before the ATDC5 shifts to the calcification step using these reactions. ATDC5 cells were seeded at 1 × 10 ⁴ cells / well and cultured for 7 days.
During the test, the medium was discarded, washed with PBS twice, and 0.1 μM Tris-HCl buffer (pH 7.5) 100 μl / well containing 0.1% TritonX-100 was added to prepare a cell suspension A. Next, staining solution B of 0.05 M 2-amino-2-methylpropanol, 2 mM MgCl ₂ and 10 mM p-nitrophenylphosphonic acid was prepared. 10 μl of cell suspension A was added to 200 μl of this staining solution B. The reaction was carried out at 37 ° C. for 30 minutes, and immediately after the reaction, the absorbance of p-nitrophenol at 405 nm was measured. The value of ALP activity of each peptide with the control ALP activity as 100% is shown below.

Evaluation test 4
[Osteoclast differentiation inhibitory action]
Evaluation was performed according to the osteoclast differentiation culture method (J. Bone Miner. Metab., 22: 318-328 (2004)) of Y. Kobayashi et al.
Experiments were performed in an osteoclast-forming system using hematopoietic cells. The femur and tibia were removed from 8-week-old male ddy mice (Tokyo experimental animals), and the muscles and cartilage were removed cleanly. Bone marrow cells were collected from the bone marrow and α-MEM (Minimum Essential) supplemented with 10% FBS (GIBCO), leucoprole (Kyowa Hakko) 2 uint / ml, penicillin G potassium (Meiji Seika) 100 units / ml on a petri dish. Medium Alpha Medium (GIBCO) was used and cultured under conditions of 37 ° C. and 5% CO ₂ . Three days later, macrophage-like hematopoietic cells were collected by T-EDTA (SIGMA). After washing, centrifugation was performed at 1400 G for 4 minutes, the medium used for the experiment was changed, and culture was started. The medium was changed after 2 days.
The separated macrophage-like hematopoietic cells were seeded in 1 × 10 4 ^/ 100μL ^/ well increments 96well cell culture plates. As a control, 20 ng / mL M-CSF (R & D) and 10 ng / mL RANKL (WAKO) were added, and each peptide (total) was added at 1 mM. Those seeded on a 96-well cell culture plate were fixed with 100% methanol (WAKO) after about 64 hours of culture. After washing with water 3 or 4 times, the mixture was reacted with tartrate-resistant acid phosphatase (TRAP) staining reagent SIGMA-ALDRICH 387A-IKT (SIGMA) at 37 ° C. for 5 minutes and observed under an optical microscope. After measuring the number of TRAP-positive multinucleated cells and the number of cells, capture the cell image using a microscope digital camera system Penguin 600CL (Pixela Corporation), and perform image analysis using In Studio (Pixera Corporation), a software dedicated to the above system. It was.

  According to the present invention, it is possible to provide a therapeutic or preventive agent for diseases related to bone, cartilage or skin, which is superior to that of the prior art, and it is possible to provide a food, drink, cosmetic or feed utilizing the effect.

Claims (4)

  A therapeutic or prophylactic agent for diseases related to bone, cartilage or skin, comprising Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof.   The therapeutic or prophylactic agent according to claim 1, which is a hyaluronic acid production promoter, a pigmentation inhibitor, an alkaline phosphatase activity inhibitor or a precursor osteoclast differentiation inhibitor.   The therapeutic or prophylactic agent according to claim 1, which is a therapeutic or prophylactic agent for osteoporosis, osteoarthritis or photoaging.   A food, drink, cosmetic or feed containing Hyp-Gly-Pro, Hyp-Gly-Pro-Hyp or a pharmaceutically acceptable salt thereof.