JP2016169171A - Osteoprotegerin derivatives and applications thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To find a novel osteogenesis promoting agent that is expected to have more safety and effectiveness, and to provide means for using this as an osteogenesis-promoting component or the like.SOLUTION: There is invented a peptide derivative having amidation at C-terminal of OP3-4 consisting of an amino acid sequence (Tyr-Cys-Glu-Ile-Glu-Phe-Cys-Tyr-Leu-Ile-Arg), and it is found that the above subject can be solved by providing an osteogenesis-promoting agent consisting of the peptide derivative, a medical composite carrier having the fixed osteogenesis-promoting agent to a carrier, and a pharmaceutical composition containing the osteogenesis promoting agent or an osteogenesis promoting agent.SELECTED DRAWING: None

Description

  The present invention relates to a peptide derivative having a bone formation promoting action and an application thereof.

  For example, in a modern society where an excessive consuming diet containing a large amount of ingredients such as sugar, such as sugar, and an aging society, bone loss per unit volume is deeply involved in various ways. There is a tendency that troubles related to bone and teeth such as periodontal disease accompanied by a decrease in alveolar bone, etc., due to bone fractures, etc. are increased.

  Of course, as countermeasures against these, it is indispensable to improve lifestyle habits such as consideration of dietary content and increased bone mass due to exercise, but the other approach recognizes the need for treatment and prevention with drugs.

  For example, osteoporosis is a collective term for pathological decrease in bone mass due to various causes, (1) senile and postmenopausal osteoporosis, (2) endocrine osteoporosis, (3) congenital osteoporosis, and (4) immobility. Sexual or traumatic osteoporosis and the like are included. For these, calcium preparations, vitamin D preparations, female hormone preparations, ipriflavones, vitamin K2 preparations and the like are provided.

  Recently, BMP (Bone morphogenetic protein) is attracting attention as an ingredient for promoting the formation of bones and teeth. BMP-2 is a glycoprotein having a molecular weight of about 30,000, which was discovered as a cytokine that induces ectopic ossification in muscle tissue, which belongs to the dpp subfamily of the BMP subfamily. It is known that it constitutes a subfamily in the superfamily (Wozney, JM et al., Science, 242, 2538 (1988): Non-Patent Document 1).

  Furthermore, an attempt has been made to use an osteogenic peptide having a specific sequence as a bone formation promoting component (Patent Document 1).

JP 2012-77093 A

Wozney, J.M. et al., Science, 242, 2538 (1988)

  Bone-related metabolic mechanisms are diverse, and there are still unclear points. Therefore, it is significant to take a more diverse approach for promoting bone formation. In addition, it has been pointed out that the above-mentioned BMP has difficulty in absorption into the body, instability, and risk of various side effects (Patent Document 1, etc.).

  Accordingly, it is an object of the present invention to find a novel bone formation promoting component that is expected to be further safe and effective, and to provide means for using this as a formation promoting agent.

  The present inventor has focused on osteoprotegerin (OPG) in order to solve the above problems. OPG is an in vivo protein that is deeply involved in bone remodeling. Remodeling is known to be performed through four stages of activation, absorption, reversal, and formation, and OPG is greatly related to its activation phase. That is, upon stimulation of bone resorption, RANKL (receptor activator of nuclear κB ligand) is produced in mesenchymal cells and osteoblasts or bone cells, and the reception of RANKL in preosteoclasts derived from hematopoietic stem cells. Differentiation into multinucleated osteoclasts is induced through interaction with the body RANK. Normally, this process is balanced by the production of OPG. OPG is a secreted protein in mesenchymal cells and osteoblasts that is similar in structure to RANK but does not have a transmembrane domain, and so to speak, has a function as a “decoy receptor” for RANK. That is, OPG and RANK, which are decoy receptors, bind to each other, thereby blocking the signal between RANKL and RANK and suppressing bone resorption.

  As a result of repeated studies on this OPG, the present inventor has found that a peptide derivative in which the C-terminal of the partial peptide consisting of the amino acid sequence of SEQ ID NO: 1 called OP3-4 of OPG is amidated has a very excellent promotion of bone formation. Ascertaining that the activity is recognized, the present invention has been completed.

  That is, in the present invention, the C-terminal of OP3-4 consisting of the amino acid sequence represented by SEQ ID NO: 1 (Tyr-Cys-Glu-Ile-Glu-Phe-Cys-Tyr-Leu-Ile-Arg) is amidated. The present invention provides a peptide derivative (hereinafter also referred to as a peptide derivative of the present invention).

  The present invention further provides (1) an osteogenesis promoter (hereinafter also referred to as the osteogenesis promoter of the present invention), characterized by comprising the peptide derivative of the present invention, and (2) the bone A medical composite carrier (hereinafter also referred to as a composite carrier of the present invention), wherein a formation promoter or a bone disease preventive or therapeutic agent (described later) is fixed to the carrier; and (3) the bone An invention that provides a pharmaceutical composition (hereinafter, also referred to as the pharmaceutical composition of the present invention) comprising the formation promoter, the bone formation promoter, or the bone disease preventive or therapeutic agent. is there.

The amino acid sequence and base sequence of human OPG are already known (Simonet, WS et al, Cell. 89 (2): 309-19 1997, Yasuda H et al, Endocrinology. 139 (3): 1329-37 1998. ), OPG is accompanied by three RANKL binding sites, OP1, OP2 and OP3 (Cheng et al, J Biol Chem. 279 (9): 8269-77 2004). OP3-4 is an essential site for forming a complex with RANKL in OP3. In the peptide derivative of the present invention, the C-terminus of OP3-4, that is, “—OH” of the carboxyl group “—C (═O) —OH” of “Arg” in the above sequence is replaced with “—NH ₂ ”. It is a secondary amide.

  By applying such an amidation modification at the C-terminal, the peptide derivative of the present invention, in which the osteoclast differentiation inhibitory action and the above-mentioned BMP action enhancement are observed, compared with the unmodified peptide of OP3-4, is produced. can do.

  Furthermore, by using the peptide derivative of the present invention in combination with BMP-2, it is possible to synergistically exert an osteogenesis promoting action. The present invention is based on the peptide derivative of the present invention and BMP-2. The present invention provides an osteogenesis promoter, and this embodiment is also the osteogenesis promoter of the present invention.

  `` Promotion of formation '' in the osteogenesis promoter of the present invention means literally promotion of bone formation and promotion of bone renewal. Specific effects include fracture treatment, fracture prevention, Examples include suppression of bone loss, and suppression of alveolar bone dissolution in periodontal diseases or recovery from the dissolution state, and new generation of alveolar bone. In particular, the efficacy of the osteogenesis promoter of the present invention is useful in the treatment for stabilizing dentures by restoring the bone mass of the alveolar bone and the implant treatment.

  Implant treatment refers to implanting an artificial object in the jawbone at the defect site caused by loss of teeth and attaching an alternative to the crown or gingiva using this as an abutment to impair the chewing function, aesthetics, and pronunciation The implant is also called an artificial tooth root. At present, the main treatment for implant treatment is an intraosseous implant method in which an implant body is embedded in an alveolar bone and integrated with an upper structure as an artificial tooth root. For the success of implant treatment, the connection between the implant body and the alveolar bone is greatly different from the natural tooth having periodontal ligament at the X-ray photograph and optical microscope level, and there is nothing at the interface, and both are in direct contact with each other. It is assumed that it is in a state (Osseointegration). In order to obtain this osseointegration state, the amount and quality of the alveolar bone are required to be at a certain level or higher in the implant body planned insertion portion. In other words, even if the amount of alveolar bone is small or the alveolar bone density is low, there is a strong possibility that it is inaccurate as the foundation of the implant body, and if it is suffering from osteoporosis, it may be excluded from the application of implant treatment There is sex. In addition, bone transplantation, bone regeneration induction method (GBR method), maxillary sinus floor elevation, socket lift, ridge expansion, callus extension method and the like are performed.

  The osteogenesis promoter of the present invention exhibits an effect of promoting the formation of alveolar bone, particularly when performing the above-described bone transplantation and GBR method, and the effect of successfully regenerating these alveolar bones is recognized. Alternatively, it is possible to increase bone mass without using the GBR method. The bone formation-promoting agent of the present invention can be used for promoting healing of fractures, treating cleft palate, and treating bone augmentation of the jawbone by promoting local bone formation.

  Thus, the osteogenesis promoter of the present invention can be used as a preventive or therapeutic agent for bone diseases (hereinafter also referred to as the bone disease therapeutic agent of the present invention). Specifically, the therapeutic agent for bone disease of the present invention includes osteoporosis [senile and postmenopausal osteoporosis, endocrine osteoporosis, congenital osteoporosis, and immobility or traumatic osteoporosis, etc.], osteomalacia, rheumatic bone disease, tooth It can be used as a prophylactic or therapeutic agent for alveolar bone dissolution associated with periodontal disease, bone disease associated with cancer, rickets, arthritis, osteoarthritis and the like.

  The composite carrier of the present invention obtained by immobilizing the bone formation promoter or bone disease therapeutic agent of the present invention on a carrier promotes bone formation at the site by implanting it in an affected area or a bone defect site. Thus, the target bone disease can be prevented or treated.

  The pharmaceutical composition of the present invention comprising the osteogenesis promoting agent or bone disease treating agent of the present invention is administered intravenously, subcutaneously, intraperitoneally, intraarticularly, transdermally, or a bone defect or affected part. It is possible to promote necessary bone formation and to treat or prevent bone diseases by filling the bones.

  According to the present invention, a peptide derivative having a very excellent osteogenesis promoting action is provided, and an osteogenesis promoter and a bone disease therapeutic agent comprising the peptide derivative are provided. And the medical composite carrier by which these agents were fix | immobilized by the support | carrier, and the pharmaceutical composition containing these agents are provided by this invention.

It is the data containing the chart of the MS spectrum of the peptide derivative of the present invention. It is the data containing the chart of HPLC of the peptide derivative of this invention. It is the data containing the chart of the MS spectrum of non-amidated peptide OP3-4. It is the data containing the chart of HPLC of non-amidated peptide OP3-4. Data including a chart of the MS spectrum of W9. It is the data containing the chart of HPLC of W9. It is an image which shows the dyeing | staining result which shows the influence on the inducibility to an osteoclast from mouse | mouth bone marrow cells regarding use of the peptide derivative of this invention. FIG. 8 is an enlarged view of a representative example of the staining result of FIG. 7. It is drawing which shows the fixed_quantity | quantitative_assay result when the number of the cells which carry out the TRAP dyeing | staining shown in FIG. It is an image which shows the result of having examined the influence with respect to the differentiation of the osteoblast by use of the peptide derivative of this invention by alkaline phosphatase dyeing | staining. It is drawing which shows the result of having quantified the dyeing density in the image shown in FIG. It is the image which showed the effectiveness with respect to the arthritis of the peptide derivative of this invention by the arthritis model using the tibia. It is drawing which shows the result of having examined the effectiveness with respect to the arthritis of the peptide derivative of this invention by the bone density determination using the quantitative CT for peripheral bone in the tibia of the model shown in FIG. It is drawing which shows the result examined about suppression of the bone resorption activity of the peptide derivative of this invention. It is drawing which shows the result examined about promotion of the deposition to the bone formation site of calcein by administration of the peptide derivative of this invention. It is drawing which shows the result of having examined the influence on the width | variety of the double label of calcein by administration of the peptide derivative of this invention. It is drawing which shows the result of having examined about the bone formation rate based on FIG. It is a micro CT image which shows the result of having examined the local bone formation in the mouse skull of the peptide derivative of this invention. It is drawing which shows the result of having measured the bone density of the new bone of FIG. 18 by DXA (dual energy X-ray measuring method). It is a micro CT image which shows the result of having examined the effect with respect to bone formation when the carrier containing the peptide derivative of this invention and BMP-2 was inject | poured locally with respect to the maxilla of a mouse | mouth. In the test system of FIG. 20, it is drawing which shows the result of having analyzed about the bone density (1) and bone mass (2) of a new bone using pQCT (quantitative CT for peripheral bone). In the test system of FIG. 20, it is a fluorescence microscope image which shows the result of having verified the bone formation using the fluorescence microscope image of a mouse | mouth maxillary part non-demineralized section. FIG. 22 shows a comparison of the BMP-2 administration group and the combined use group of the peptide derivative of the present invention and BMP-2 with respect to the area of the demeclocycline stained portion 2 days before sacrifice in the fluorescence microscopic image of FIG. is there. It is a micro CT image which shows the result of having examined the ectopic bone formation activity at the time of using the peptide of this invention, and BMP-2 in combination. It is drawing which shows the result of having measured the quantity of the bone formed under the fascia of FIG. 24 as the amount of bone mineral by DXA.

(1) Peptide derivative of the present invention The peptide derivative of the present invention is a peptide derivative in which the C-terminal in the amino acid sequence of SEQ ID NO: 1 is amidated as described above.

  The method for producing the peptide derivative of the present invention is not particularly limited, and can be mainly produced by making full use of a chemical synthesis method.

  Since the number of amino acids in the peptide derivative of the present invention is 11 and a relatively short chain, it is advantageous to directly produce the peptide derivative of the present invention by carrying out a chemical peptide synthesis method.

  The peptide derivative of the present invention can be produced according to known peptide chemical synthesis methods and C-terminal amidation modification methods. Regarding peptide synthesis, it can be produced using a liquid phase peptide synthesis method or a solid phase peptide synthesis method established as a conventional method. A solid phase peptide synthesis method generally recognized as a suitable chemical synthesis method can also use the Boc solid phase method or the Fmoc solid phase method, and a ligation method can be used as necessary. It is. Moreover, each amino acid which comprises an oligopeptide etc. can be manufactured by a well-known method, and it is also possible to use a commercial item.

  As described above, the peptide derivative of the present invention requires C-terminal amidation. As a method suitable for this amidation, synthesis using a synthetic resin suitable for amide formation can be mentioned. Examples of the resin for synthesis include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin, 4- ( 2 ', 4'-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin and the like. Among these, 4- (2 ', 4'-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy resin and 4- (2', 4'-dimethoxyphenyl-Fmoc-aminoethyl) phenoxy resin are preferable. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin in accordance with the sequence of the target peptide according to a known condensation method. At the end of the reaction, the peptide is excised from the resin, and at the same time, various protecting groups are removed to obtain the peptide derivative of the present invention in which the target C-terminal is amidated.

  The peptide derivative of the present invention can also be obtained by amidating the α-carboxyl group of arginine, which is the carboxyl terminal amino acid, and then extending the peptide to the amino group side according to the amino acid sequence of the peptide derivative of the present invention. be able to. The amidation can be performed by a conventional method, for example, contact with ammonia in an aqueous solution.

  In the above-described condensation of protected amino acids, various known activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide and the like. For activation by these, a protected amino acid together with a racemization inhibitor (eg, HOBt, HOBt, etc.) is added directly to the resin, or a preprotected amino acid as a symmetric anhydride or HOBt ester or HOBt ester. Can be added to the resin after activation. Examples of solvents used for the activation of protected amino acids and condensation with resins include acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide and N-methylpyrrolidone; methylene chloride, chloroform and the like Halogenated hydrocarbons; alcohols such as trifluoroethanol; sulfoxides such as dimethyl sulfoxide; tertiary amines such as pyridine; ethers such as dioxane and tetrahydrofuran; nitriles such as acetonitrile and propionitrile; methyl acetate , Esters such as ethyl acetate; or a mixture thereof. The reaction temperature is about −20 ° C. to 50 ° C. employed in the peptide bond forming reaction.

Examples of the protective group for the amino group of the starting amino acid include benzyloxycarbonyl group, t-butoxycarbonyl group, t-pentyloxycarbonyl group, isobornyloxycarbonyl group, 4-methoxybenzyloxycarbonyl group, 2-Cl- Benzyloxycarbonyl group, 2-Br-benzyloxycarbonyl group, adamantyloxycarbonyl group, trifluoroacetyl group, phthaloyl group, formyl group, 2-nitrophenylsulfenyl group, diphenylphosphinothioyl group, N-9-fur Examples include oleenylmethoxycarbonyl group. Examples of the protecting group for the carboxyl group include a C _1-6 alkyl group, a C _3-8 cycloalkyl group, a C _7-14 aralkyl group, a 2-adamantyl group, a 4-nitrobenzyl group, a 4-methoxybenzyl group, 4- Examples include a chlorobenzyl group, a phenacyl group, a benzyloxycarbonyl hydrazide group, a t-butoxycarbonyl hydrazide group, and a trityl hydrazide group.

  Examples of the protecting group for the phenolic hydroxyl group of tyrosine include benzyl group, Cl-benzyl group, 2-nitrobenzyl group, Br-benzyloxycarbonyl group, t-butyl group and the like.

  Examples of methods for removing (eliminating) protecting groups include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, and anhydrous hydrogen fluoride, methanesulfonic acid, trifluoromethane. Examples include acid treatment with sulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like. The elimination reaction by the acid treatment is generally performed at a temperature of −20 ° C. to 40 ° C. In the acid treatment, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1,4-butanedithiol, 1 It is effective to add a cation scavenger such as 1,2-ethanedithiol. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide, dilute ammonia or the like.

  The protection of the functional group that should not be involved in the reaction of the raw material and the protection group, the removal of the protective group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

  The peptide derivative of the present invention synthesized as described above is obtained as a purified product of the peptide derivative of the present invention by a conventional method such as reverse phase high performance liquid chromatography (reverse phase HPLC) after the steps such as deprotection according to a conventional method. It is possible. Then, identification can be performed by mass spectrometry (matrix-assisted laser desorption ionization time-of-flight: MALDI-TOF or liquid chromatography-electrospray ionization: LC-ESI). Finally, the peptide derivative can be hydrolyzed to confirm the amino acid composition and content.

  The peptide derivative of the present invention may be a physiologically acceptable salt. Such salts can be obtained through normal salt formation reactions, such as hydrochloride, sulfate, phosphate, lactate, tartrate, maleate, fumarate, oxalate. , Salts with acids such as malate, citrate, oleate, palmitate; salts with hydroxides or carbonates of alkali metals or alkaline earth metals such as sodium, potassium, calcium, aluminum; Examples thereof include salts with amines such as triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine and arginine. Such salts are also within the category of the peptide derivatives of the present invention.

  As described above, the peptide derivative of the present invention can be produced. In addition, today, the peptide derivative of the present invention can be obtained by outsourcing to a peptide synthesis contract service. For example, Wako Pure Chemical Industries, Ltd. (http://wako-chem.co.jp/siyaku/jutaku/peptide/index.htm), Toray Research Center, Inc. (http://www.toray-research.co.jp) /bio/taishouta/tai_002.html) and the like, it is described that a peptide synthesis contract service is provided and that it is possible to accept the manufacture of a peptide derivative having an amidated C-terminus.

(2) Bone formation promoter or bone disease therapeutic agent of the present invention The peptide derivative of the present invention described above is referred to as “bone formation promoting use” and “bone disease treating use”, that is, “bone formation promoting agent”. And as a “bone disease therapeutic agent”.

  The contents of “use for promoting bone formation” and “use for treating bone disease” are as disclosed in the above “Means for Solving Problems”. The peptide derivative of the present invention can be used with these uses. In addition, the peptide derivative of the present invention can be used as an osteogenesis promoter or a bone disease treatment agent in combination with an existing bone or tooth formation promoting component such as BMP-2 described above. In particular, by using a combination of the peptide derivative of the present invention and BMP-2, it is possible to further promote local bone formation.

(3) Embodiment of osteogenesis promoter or bone disease therapeutic agent of the present invention [Composite carrier]
As described above, the composite carrier of the present invention is “a medical composite carrier in which the osteogenesis promoter or bone disease therapeutic agent of the present invention is fixed to the carrier”. In particular, this aspect is suitable for the bone formation application that promotes the formation of bone in a specific part of a damaged part or a lesion part. Specifically, a medical composite carrier formed by binding the peptide derivative of the present invention to a carrier of a material that is compatible with a living body, particularly preferably a carrier of a biodegradable material that can be easily processed and molded. It is. Examples of the carrier material include various ceramics, artificial bone, alginate covalently crosslinked gel (Suzuki, Y. et al., J. Biomed. Mater. Res., 39, 317 (1998)), hyaluronic acid gel, etc. Examples include polysaccharide gel, protein gel such as collagen, calcium sulfate, polylactic acid, polylactic acid / gelatin complex, polyglycolic acid, hydroxyapatite, tricalcium phosphate, etc. Is not to be done. As long as the effects of the present invention are not impaired, the presently provided carrier can of course be used in the future. The method for immobilizing the peptide derivative of the present invention on these carriers can be appropriately selected depending on the specific material, and forms a covalent bond, an ionic bond, a hydrophobic bond, a hydrogen bond, an SS bond, etc. Immobilization methods such as immersion, impregnation, spraying, coating, and dropping using a peptide derivative-containing solution can be used. Among these immobilization methods, the covalent immobilization method is particularly preferred from the viewpoint of stability and long-lasting action, and is usually desired depending on the method used for immobilizing physiologically active proteins such as enzymes. Covalent immobilization can be performed (Scouten, WH, Method in Enzymol., 135, Mosbach, K. Ed., 1987, Academic Press, NY, pp30-65, etc.). In the composite carrier of the present invention, the peptide derivative of the present invention is preferably about 0.01 to 50 parts by mass with respect to 100 parts by mass of the dry carrier. Furthermore, when BMP-2 is used in combination, it is preferable to use about 10 ⁻⁵ to 1 part by mass of BMP-2 together with the use of the peptide derivative.

  In addition, the composite carrier of the present invention can exhibit an extremely excellent bone formation promoting effect and osteogenesis effect regardless of whether it is locally implanted or injected by injection.

  That is, the composite carrier of the present invention can be implanted or injected into a bone defect site, an alveolar bone complementation site, or the like and used for the treatment of fractures or the regeneration of alveolar bone.

[Pharmaceutical composition]
As described above, the pharmaceutical composition of the present invention is a “pharmaceutical composition containing the osteogenesis promoter or bone disease therapeutic agent of the present invention”.

  As described above, the peptide derivative of the present invention which is the osteogenesis promoter or bone disease therapeutic agent of the present invention is administered to the human body as an active ingredient of the “pharmaceutical composition”. In the case of direct administration of the peptide derivative, since an injection is mixed at the time of use, it is also included in the pharmaceutical composition.

  The pharmaceutical composition of the present invention is prepared in the form of a pharmaceutical composition by blending an appropriate pharmaceutical preparation carrier with the peptide derivative of the present invention which is an active ingredient. The pharmaceutical carrier can be selected depending on the form of use, and excipients or diluents such as fillers, extenders, binders, moisturizers, disintegrants, surfactants, etc. should be used. Can do. The form of the composition is not particularly limited as long as it can effectively contain the peptide derivative of the present invention, and is a solid or ointment such as a tablet, powder, granule, or pill. However, in general, it is preferably in the form of injections such as liquids, suspensions, and emulsions. In addition, the peptide derivative of the present invention can be made into a dry product that can be made liquid at the time of use by adding an appropriate carrier. Furthermore, in the pharmaceutical composition of the present invention, drug delivery systems such as nanoparticles composed of cyclodextrin-containing polymers, polymer micelles, stable nucleic acid lipid particles (SNALP), and multifunctional envelope nanostructures (MEND) are utilized. Thus, the effect as an active ingredient of the peptide derivative of the present invention can be further improved.

  The obtained pharmaceutical composition is administered in an appropriate administration route according to its form, for example, an injectable pharmaceutical composition is administered in a solid form by intravenous, intramuscular, subcutaneous, intradermal, intraperitoneal administration, etc. The pharmaceutical composition is administered orally or enterally. The amount of the peptide derivative of the present invention in the pharmaceutical composition is appropriately selected according to the administration method, dosage form, purpose of use, patient symptom and the like of the composition. , Prepared in the form of a composition containing about 0.1 to 95% by mass, and an appropriate dose (about 0.01 μg to 10 mg per adult per day, once to 2 to 5 times a day, several more It is also possible to carry out administration every other day). The administration can be performed once to 2 to 5 times a day, and every several days.

  Examples of the present invention will be disclosed below.

[Example 1] Production and acquisition of peptide derivative (1) Peptide derivative of the present invention, “non-amidated peptide OP3-4” used as a comparative example, and “W9: Tyr-Cys-Trp-Ser-Gln-” As for “Tyr-Leu-Cys-Tyr (SEQ ID NO: 2)”, those manufactured by consigning to American Peptide Company (CA USA) were used. The proof that each contract manufacturing substance is indeed intended is made by the following physical property values, HPLC data, and MS spectrum data.

(A) Peptide derivative of the present invention Molecular weight: 1448.7
MS spectrum: FIG.
HPLC data: FIG. 2 (peptide purity 97.8%)
Solubility: 1.0 mg / ml (30% acetone aqueous solution)
Appearance: White freeze-dried product

  In the following drawings and the like, the peptide derivative of the present invention may be abbreviated as OP3-4 (NH2), OP3-4 NH2, or simply NH2.

(B) Non-amidated peptide OP3-4
Molecular weight: 1449.7
MS spectrum: FIG.
HPLC data: FIG. 4 (peptide purity is 96.4%)
Solubility: 1.0 mg / ml (30% acetone aqueous solution)
Appearance: White freeze-dried product

  In the following drawings and the like, the non-amidated peptide OP3-4 may be abbreviated as OP3-4, OP3-4 (COOH), OP3-4 COOH, or simply COOH.

(C) W9
Molecular weight: 1226.4
MS spectrum: FIG.
HPLC data: FIG. 6 (peptide purity 97.7%)
Solubility: 1 mg / ml (20% acetone aqueous solution)
Appearance: White freeze-dried product

(2) Recombinant human BMP-2
It was obtained as a 1 mg / ml solution of recombinant human BMP-2 having a molecular weight of 26 KD (originally obtained from Yamanouchi Pharmaceutical, but is currently available from Bioventus (USA)). In this solution, recombinant human BMP-2 was dissolved in LF6 buffer (5 mM glutamic acid, 5 mM NaCl, 2.5% glycine, 0.5% sucrose, 0.01% Tween 80: pH 4.5). It is.
The amino acid sequence of the human BMP-2 is shown in SEQ ID NO: 3.

  In the following drawings and the like, recombinant human BMP-2 may be abbreviated as BMP.

  Up to Test Example 3 below, peptides including the peptide derivative of the present invention used as the test object were used without dissolving the carrier in amounts corresponding to the respective concentrations in the medium. In Test Examples 4 and 5, tests using a carrier were performed.

  In addition, statistical processing of quantitative data in each test was performed by performing an analysis of variance by ANOVA, and then a significant difference between each group was tested using Fisher's PLSD test as a posthoc test. The analysis data was expressed as an average value ± standard deviation (SD), and a risk rate of 5% or less was determined to be significant.

[Test Example 1] In vitro test using osteoclast differentiation induction system FIG. 7 shows the results of culture for 4 days according to a conventional method for inducing osteoclasts from mouse bone marrow cells.

  First, osteoclasts were induced by adding 50 ng / ml RANKL and 25 ng / ml M-CSF to a medium using 10 FBS for αMEM. A group in which DMSO (dimethyl sulfoxide), which is a peptide solvent, was added to a concentration of 0.05% from the first day of the culture was defined as a vehicle control (Veh Ctr) group, and a group in which 5 μM of a W9 peptide, which is a RANKL-binding peptide, was added. Was used as a positive control. Further, OP3-4 amidated at the C end was added at a concentration of 10 μM and 30 μM, and OP3-4 not amidated at the C end was added at a concentration of 10 μM and 30 μM, and the medium was changed on the third day. The results of fixing with formalin the next day and staining with tartrate-resistant acid phosphatase (TRAP) are shown in FIG. All the peptides used use DMSO as a solvent.

  FIG. 8 is a representative enlarged view of each post-staining result shown in FIG. Cells that are darkly stained are TRAP staining positive cells. FIG. 9 is a quantification result when the number of cells (TRAP Positive MNCs) positive for TRAP staining and possessing 3 or more nuclei shown in FIG. 8 is counted as the number of osteoclasts. Apparently, the number of osteoclasts induced in the medium supplemented with the peptide derivative of the present invention in which the C-terminal is amidated is almost zero in the formation of the multinucleated cells. On the other hand, in the group to which OP3-4 which was not amidated was added, the degree of inhibition of the formation of the multinucleated cells was clearly lower than that of the amidated peptide derivative of the present invention.

[Test Example 2] Test using in vitro osteoblast differentiation system Examining the effect of OP3-4 on osteoblast differentiation using osteoblast-like cells isolated from the skull of C57BL / 6J mice a day after birth did. Alkaline which is a marker for osteoblast differentiation after being cultured for 7 days using osteoblast differentiation medium (αMEM medium, 10% FBS, 10 nM dexamethasone, 50 ng / ml ascorbic acid, 10 μM β glycerphosphate). FIG. 10 shows the result of phosphatase staining, and FIG. 11 shows the result of quantifying the staining concentration. The symbols at the top of the bar graph in FIG. 11 represent the following statistical values.

*, P <0.05 vs vehicle
**, p <0.01 vs vehicle
$, P <0.05 vs 200 μM OP3-4 NH2
$$, p <0.01 vs 200 μM OP3-4 NH2

  Although the peptide derivative of the present invention promoted osteoblast differentiation in a concentration-dependent manner, the action of OP3-4 that had not been amidated to promote osteoblast differentiation was significantly lower than the amidated peptide derivative of the present invention. It was. Under these conditions, 200 μM of the peptide derivative of the present invention showed an osteoblast differentiation promoting effect equal to or better than 25 ng / ml BMP-2.

[Test Example 3] Examination using an arthritis model The first sensitization was performed by intradermal injection of an emulsion of bovine type 2 collagen with adjuvant into a DBA / 1J mouse ridge, and a second time after 3 weeks. In the CIA model that develops arthritis by sensitizing, apparent bone loss is caused 3 weeks after the second sensitization. On the 7th day after the second sensitization, an osmotic pump (Alzet pump) filled with a drug such as the peptide derivative of the present invention was implanted in the mouse subcutaneously, and the drug was gradually released for 3 weeks, and 28 days after the sensitization. Slaughtered in the eyes. In the CIA model, arthritis develops around the 5th day after the second sensitization, and therefore arthritis has already developed when the drug administration is started.

(1) Micro CT image FIG. 12 is a micro CT image of the mesial secondary cancellous bone portion of the tibia taken out after sacrifice. The bone (b) of the CIA model mouse clearly had less bone than the bone (a) of the mouse not sensitized with collagen. In the peptide derivative administration groups (c) and (d) of the present invention, a clear increase in bone mass was observed compared to the CIA group (b). Among these, there was no difference in bone mass between the low dose (9 mg / kg / day) group (c) and the high dose group (18 mg / kg / day) (d) of the peptide derivative of the present invention. On the other hand, in the group (e) to which OP3-4 in which the C-terminal was not amidated was administered, fewer bones were observed than in (c) and (d). In addition, an osmotic pump filled with 20% DMSO as a solvent of OP3-4 was implanted subcutaneously in a mouse not sensitized with collagen and a CIA mouse not administered with a drug.

(2) Quantification of bone density using pQCT Next, FIG. 13 shows the bone density quantification results of the same site using pQCT (quantitative CT for peripheral bone). This is a measurement result of bone density in the tibia mesial secondary cancellous bone.

  The CIA model mice showed a significant decrease compared to the bone density of unsensitized mice, whereas the peptide derivatives of the present invention restored the decreased bone density in a dose-dependent manner. On the other hand, in the OP3-4 peptide in which the C-terminal is not amidated, recovery of bone density as in the peptide derivative of the present invention was not observed. The symbols at the top of the bar graph in FIG. 13 represent the following statistical values, respectively.

$$, p <0.01 vs Ctr + vehicle
$, P <0.05 vs Ctr + vehicle
#, P <0.05 vs CIA + vehicle
&, P <0.05 vs CIA + NH2 18 mg / kg / day (amidated OP3-4)

  In addition, all of the following tests including this test are conducted using the same statistical analysis technique using 6 mice per group.

(3) Quantification of bone resorption activity using CTX value FIG. 14 is a graph showing bone resorption activity at the time of sacrifice of CIA model mice or the like.

  The CTX value (degradation product of type 1 collagen), a bone resorption marker in serum, was significantly higher than that of mice not sensitized in the CIA model. In the peptide derivative of the present invention, the bone resorption marker decreased in a dose-dependent manner as compared with the CIA mice administered with the solvent. On the other hand, even with OP3-4 in which the C-terminal is not amidated, suppression of bone resorption activity was observed at a high dose of 18 mg / kg / day. However, the degree of inhibition of bone resorption appeared more strongly with the peptide derivative of the present invention in which the C-terminal was amidated.

(4) Evaluation using dynamic parameters FIG. 15 shows a fluorescence micrograph image of the tibia mesial secondary cancellous bone.

  We observed the deposition of calcein, a fluorescent substance, on the osteogenic active site, which was injected subcutaneously 7 and 2 days before the sacrifice of mice.

  As shown by the white arrow, between the two calcein labels, the length of bone mineralized in 5 days is shown, but in CIA mice, it becomes shorter compared to the control group, and administration of the peptide derivative of the present invention causes A shortened width recovery was observed. The administration of OP3-4 with no C-terminal amidation was observed to be hardly recovered. FIG. 16 and FIG. 17 show the results of measuring and quantifying the double label.

  FIG. 16 shows the result of measuring the width of the double label of calcein. In other words, this is the result of measuring the width between calcein labeled on the cancellous bone of the tibia secondary cancellous bone, showing the width of the bone formed in 5 days, and the bone forming ability of osteoblasts Show.

  In the CIA model mouse, the width of the double label decreased, but the peptide derivative of the present invention recovered in a dose-dependent manner, and at a high dose, it completely recovered to the level of the control group (control). On the other hand, OP3-4 in which the C-terminal was not amidated could not recover the decrease in osteogenic activity shown in CIA model mice.

  FIG. 17 shows the result of the bone formation ability of each of the osteoblasts shown in FIG. 16 and the ratio of the length of osteoblasts forming bones side by side on the bone surface to the bone surface length. The bone formation rate (Bone Formation Rate) calculated by multiplication with an index (referred to as a calcified surface but not shown here) is shown.

  The Bone Formation Rate is an index indicating the osteogenic activity of the entire measurement site, and reflects the amount of bone formed per day. The CIA model mice showed a significant decrease in bone formation rate compared to the control group, but the decrease was significantly recovered by administration of the peptide derivative of the present invention. On the other hand, administration of OP3-4 in which the C-terminal was not amidated did not recover the decrease in the bone formation rate decreased in the CIA model mice.

[Test Example 4] Local bone regeneration test A bone defect was created on the skull of a 5-week-old male C57BL / 6J mouse with a 3.5 mm diameter skin biopsy cutter (Biopsy Punch, Kai medical). Gelatin hydrogel (Medgel PI9, provided by Prof. Yasuhiko Tabata, Kyoto University, now available as Medgel (Medgel)) as a carrier, BMP-2, W9 peptide (0.56 mg), peptide derivative of the present invention ( 0.28 mg, 0.56 mg) and placed on the defect, and after closing the skin, the mice were sacrificed 4 weeks later and micro-CT was taken (FIG. 18). DMSO (5 μL) was used as a peptide solvent. Compared to the W9 peptide, the amount of bone formed by the peptide derivative of the present invention in which the C-terminus was amidated was observed more than the others. Although the molecular weight of the peptide derivative of the present invention is larger, double bone formation was observed even at the same 0.56 mg.

  FIG. 19 shows the result of measuring the bone density of the new bone by DXA (dual energy X-ray measurement method). The number of tests is 4 in each group.

The significant difference symbols in FIG. 19 are as follows.
#: P <0.05 vs BMP-2
$: P <0.05
$: P <0.01 vs W9 (0.56 mg)

  An excellent bone regeneration effect was observed when the peptide derivative of the present invention was locally administered via a carrier.

[Test Example 5] Local bone formation test by injection of carrier by injection (1) Verification using micro-CT The carrier containing peptide derivative of the present invention and BMP-2 was injected into the maxilla of the mouse. The effect on bone formation when injected locally was examined.

  (A) Solvent (5 μL DMSO + 1 μL LF6 buffer), (b) BMP-2 (1 μg), (c) BMP-2 (1 μg) and the peptide derivative (0.56 mg) of the present invention in which the C-terminal is amidated Each mixture is diluted to 50 μL with a phosphate buffer solution and then added to a gelatin hydrogel (pI9 particle type, provided by Yasuhiko Tabata, Kyoto University, now available from Medgel), 5 weeks old male C57BL / 6J mouse The injection was performed under the periosteum of the maxilla. FIG. 20 shows a result of sacrifice of the mouse 4 weeks after the injection and analysis by micro CT at that time.

  (A) No osteogenesis was observed in the group injected with the carrier containing the solvent (carrier + solvent), and (b) the group injected with the carrier containing BMP-2 (carrier + BMP-2). However, only slight bone formation was observed. On the other hand, in the group in which (c) the carrier containing BMP-2 and the peptide derivative of the present invention (carrier + BMP-2 + OP3-4 NH2) was injected, clear bone formation was observed.

(2) Verification using quantitative CT (pQCT) for peripheral bone In the test system of carrier injection using the maxillary bone of the above mouse, the amount of hydroxyapatite 395 mg / cm ³ using pQCT as a threshold for new bone, The bone density (FIG. 21 (1)) and bone mass (FIG. 21 (2)) of the new bone were analyzed. As a result, the bone density was significantly higher in the combination group of the peptide derivative of the present invention and BMP-2 than in the BMP-2 single group. Also in bone mass, the combination group of the peptide derivative of the present invention and BMP-2 showed a significantly higher value than the BMP-2 single group.

The significant difference symbols in FIG. 21 are as follows.
#: P <0.05 vs carrier + solvent *: p <0.05 vs carrier + BMP-2

(3) Verification using a fluorescence microscope In the above-described test system for carrier injection using the maxilla of the mouse, bone neogenesis was verified using a fluorescence microscopic image of a non-decalcified slice of the mouse maxilla (Fig. 22). . In FIG. 22, the left column is an overall image of the fluorescence microscope, and the right column is an enlarged image of a lumped bone nascent region indicated by a square box.

  In this test system, calcein showing a green fluorescent color 16 days before sacrifice of mice and demeclocycline showing a yellow fluorescent color 2 days before were subcutaneously administered to the mice and deposited at the bone formation site. FIG. 22 is a black-and-white image, where the dark portion is a green calcein stained portion and the white portion is a yellow demeclocycline stained portion.

  In the solvent (+ carrier) group used as a control, no fluorescent staining portion was observed. In both the BMP-2 single group and the combination group of the peptide derivative of the present invention and BMP-2, an osteogenic region was observed, but the combination group had a larger area than the single group, and demecro The cyclin-stained portion is conspicuous, and it is shown that bone formation was vigorous even after 2 days before sacrifice.

  FIG. 23 shows the result of measuring the area of the portion showing the fluorescence color of this demeclocycline. As visually shown in FIG. 22, the combination group of the peptide derivative of the present invention and BMP-2 has a significantly larger area of the fluorescence color of demeclocycline than the BMP-2 alone group, and it is very prosperous. It is shown that bone formation was performed. In FIG. 23, # represents p <0.05 vs carrier + BMP-2.

[Test Example 6] Examination of ectopic bone formation activity BMP-2, BMP-2 and collagen (CollaPlug (registered trademark), Zimmer) were used as carriers under the back fascia of 5-week-old male C57BL / 6J mice. A combination of W9 peptide (0.56 mg), BMP-2 and a peptide derivative of the present invention (0.28 mg, 0.56 mg) was added, and the mice were sacrificed 12 days later. Thereafter, the micro-CT image taken out and photographed is shown in FIG. The amount of bone formed under the fascia was formed more when W9 peptide was added than BMP-2. However, when BMP-2 and the peptide derivative of the present invention (OP3-4 NH2) were added, W9 peptide was formed. Larger bones were formed compared to the case of adding. When only the carrier was buried, no bone formation was observed (not shown).

  The result of measuring the amount of bone formed under the fascia as the amount of bone mineral by DXA is shown in FIG. The number of tests is 4 in each group.

Significant difference symbols in FIG. 25 are as follows.
#: P <0.05 vs BMP-2
$: P <0.05
$: P <0.01 vs W9 (0.56 mg)

  When the peptide derivative of the present invention and BMP-2 were administered locally via a carrier, an excellent ectopic bone forming action was observed. This excellent action is very useful, for example, in the formation of alveolar bone necessary for implant treatment.

Claims (7)

  A peptide derivative, wherein the C-terminal of OP3-4 consisting of the amino acid sequence represented by SEQ ID NO: 1 is amidated.   An osteogenesis promoter comprising the peptide derivative according to claim 1.   An osteogenesis promoter comprising the peptide derivative according to claim 1 and human BMP-2.   A preventive or therapeutic agent for bone diseases, comprising the peptide derivative according to claim 1.   A preventive or therapeutic agent for bone diseases, comprising the peptide derivative according to claim 1 and human BMP-2.   A composite carrier for medical use, wherein the osteogenesis promoter according to claim 2 or 3 or the preventive or therapeutic agent for bone disease according to claim 4 or 5 is fixed to a carrier.   A pharmaceutical composition comprising the osteogenesis promoter according to claim 2 or 3, or the preventive or therapeutic agent for bone disease according to claim 4 or 5.