JP5395915B2 - Novel peptide having osteogenesis action and osteogenesis promoter obtained by immobilizing the peptide - Google Patents

Description

The present invention relates to a peptide having an osteogenesis action and an osteogenesis promoter comprising the peptide as an active ingredient.
Since the peptide provided by the present invention and the osteogenesis promoter containing this as an active ingredient have an osteogenic action, treatment of fractures, suppression of bone loss in osteoporosis and periodontal diseases, osteoporosis and rheumatic properties It is useful for prevention of fractures in arthritis.

  BMP (Bone morphogenetic protein) is a protein belonging to the TGF (transforming growth factor) β family (Wozney, J.M., et al., Science, 242, 1528 (1988)), and the active form exists as a homodimer having a molecular weight of about 18 kD. BMP acts on undifferentiated mesenchymal cells, induces differentiation into chondroblasts and osteoblasts, and causes cartilage and bone formation (Wang, EA et al., Proc. Natl. Acad. Sci. USA, 87, 2220 (1990)). Therefore, the effectiveness of BMP is expected in the treatment of fractures, the suppression of bone loss due to osteoporosis and periodontal diseases, and the prevention of fractures in osteoporosis and rheumatoid arthritis.

However, an effective amount of BMP is not absorbed into the body by oral administration or transdermal administration, and disappears from blood or tissue within a few minutes even when administered directly into blood vessels or tissues. On the other hand, when BMP is administered in a large amount, various side effects such as toxicity to the liver and kidney may occur. In addition, BMP is immunogenic due to its large molecular weight, and repeated administration may cause anaphylactic shock. Further, when BMP is used by impregnating a matrix of demineralized bone or collagen, an osteogenesis action is expressed, but problems of antigenicity and infectivity derived from these matrices themselves newly arise.
The present inventors have intensively studied for the purpose of providing a peptide having an osteogenesis action with reduced side effects, and a peptide variant derived from BMP has an osteogenesis action similar to that of BMP. This was recognized and the invention was completed.

According to the present invention, the above object is achieved by a peptide having at least the amino acid sequence represented by the sequence SEQ ID NO: 1 in the sequence listing.
The object of the present invention is also achieved by an osteogenesis promoter containing an effective amount of the above peptide.

ADVANTAGE OF THE INVENTION According to this invention, the osteogenesis promoter which consists of a peptide which has a bone formation effect, and containing this as an active ingredient is provided.
Since these peptides and osteogenesis promoters have low bone toxicity and excellent bone formation action, they are useful for the treatment of fractures, suppression of bone loss in osteoporosis and periodontal diseases, and prevention of fractures in osteoporosis and rheumatoid arthritis. It can be used effectively.

As long as the peptide of the present invention has an amino acid sequence represented by the sequence SEQ ID NO: 1 showing an osteogenesis action, any one or more amino acids may be added thereto.
That is, the sequence shown in SEQ ID NO: 1 is combined with SEQ ID NO: 2, combined with SEQ ID NO: 3, or combined with both SEQ ID NO: 2 and SEQ ID NO: 3. It may be.
Here, the N-terminus and C-terminus of each sequence of SEQ ID NO: 1 to SEQ ID NO: 3 are oriented in the usual manner, with the N-terminus being the left of the sequence and the C-terminus being the right of the sequence. The combination of ID NO: 1 and SEQ ID NO: 2 includes both N-terminal-KIPKA STLY-C-terminal and N-terminal-STLY KIPKA-C-terminal, so that SEQ ID NO: 1 to SEQ ID NO in the entire sequence The orientation of each sequence of: 3 is not limited, and all combinations that can be considered when the orientation of the whole sequence is performed by a conventional method are included in the scope of the present invention.

Further, in the above combination, between each sequence of SEQ ID NO: 1 to SEQ ID NO: 3 and / or from each sequence of SEQ ID NO: 1 to SEQ ID NO: 3 in the direction of each terminal in the entire sequence Amino acids may be present between 1 and 10 residues, and such sequences are also encompassed within the scope of the present invention. The amino acid may be any amino acid that does not substantially inhibit the bone forming action of SEQ ID NO: 1, specifically, Asn, Cys, Pro, Gly, Ala, Val, Leu, Ile, Ser, Thr, Glu , Asp, Lys, Tyr, the amino acids may be the same or different.
However, in any combination, SEQ ID NO: 1 is preferably located on the C-terminal side of the entire sequence, and specific examples of the combination are represented by SEQ ID NO: 4 to SEQ ID NO: 7. The amino acid sequence is preferably used.
The “bone formation action” in the present invention promotes the activation of alkaline phosphatase in osteoblasts (Akira Yamaguchi, Molecular Medicine, Vol. 30, No. 10, 1232 (1993)) and forms new bone. Or it can be regarded as an action that induces the growth of existing bone.

  The peptide of the present invention is prepared by a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method, but the solid phase synthesis method is simple in operation [for example, “Continued in Japan Biochemical Society” Biochemistry Experiment Course 2 Protein Chemistry (below) "(May 20, 1987, published by Tokyo Chemical Co., Ltd.), pages 641-694, and Atherton, E. et al.," Solid Phase Peptide Synthesis-Practical Method "(1989, IRL Press, Oxford), pages 152-154]. Solid phase synthesis is usually carried out with the amino group protected with a suitable protecting group such as Boc (tert-butoxycarbonyl) or Fmoc (9-fluorenylmethyloxycarbonyl), or a combination thereof. it can.

Preparation of the peptide of the present invention by a solid phase synthesis method, for example, using a polymer insoluble in the reaction medium, 1) α-COOH group having an amino acid corresponding to the C-terminus of the target peptide in the polymer 2) Next, α-amino group other than α-COOH group that the amino acid or peptide fragment has corresponding amino acid or peptide fragment toward the amino acid toward the N-terminus of the target peptide, etc. 3) by sequentially repeating the operation of removing the protecting group of the amino group that forms a peptide bond such as α-amino group in the bound amino acid or peptide fragment. The peptide chain is elongated to form a peptide chain corresponding to the target peptide.
The peptide chain thus formed can be obtained by appropriately purifying the peptide by removing it from the polymer and removing the protecting group from the protected functional group.

Examples of polymers include styrene-divinylbenzene copolymer, Maryfield resin, chloromethyl resin, won resin, siever resin, link amide resin, link acid resin, 2-chlorotrityl chloride resin, HMBA-MBHA resin, and MBHA resin. An oxime resin or the like can be used. Of these, a styrene-divinylbenzene copolymer is preferred.
In addition, it is preferable from the viewpoint of suppressing side reactions that the peptide chain is removed from the polymer and the protecting group is removed simultaneously using trifluoroacetic acid or hydrogen fluoride.
As the solvent and condensing agent in peptide synthesis, those generally known in the art can be used as appropriate. Examples of the solvent include DMF (dimethylformamide), trichloroethanol, N-methylpyrrolidone, and the like. Examples of the condensing agent include DCC, HATU (O- (7-azabenzotriazol-1-yl) -1,1,3 , 3-tetramethyluronium hexafluorophosphate), HOBt (1-hydroxybenzotriazole), HBTU (O-benzotriazol-1-yl-N, N, N ', N'-tetramethyluronium hexafluorophosphate) PyBOP (benzotriazol-1-yl-oxy-tris-pyrrolidinophosphonium hexafluorophosphate), CF ₃ —NO ₂ —PyBOP, and the like.
It is effective to purify the obtained peptide by reverse phase liquid chromatography.

The N-terminus or C-terminus of the peptide of the present invention prepared as described above, or both of them may be appropriately modified chemically. Specifically, for example, the N-terminus is acetylated and the C-terminus is It may be amidated or esterified.
Moreover, the peptide of the present invention may form a physiologically acceptable salt by utilizing a normal salt formation reaction. Examples of such salts include salts with acids such as hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, citric acid, oleic acid, and palmitic acid; sodium, potassium, And salts with hydroxides or carbonates of alkali metals or alkaline earth metals such as calcium and aluminum; salts with amines such as triethylamine, benzylamine, diethanolamine, t-butylamine, dicyclohexylamine and arginine.

The peptide of the present invention has an osteogenic action and has low toxicity in toxicity tests.
Therefore, the peptide of the present invention may contain any additive such as a stabilizer, a preservative, a thickener, a solubilizer or the like which is acceptable by itself or pharmacologically for the purpose of bone formation. It can be used as an osteogenesis promoter obtained by fixing to an appropriate carrier, or by dissolving or suspending in an aqueous solvent.
The osteogenesis promoter according to the present invention is particularly preferably one obtained by immobilizing a peptide on a carrier.

The carrier is preferably one that can be decomposed and absorbed in vivo, such as various ceramics, alginic acid covalently crosslinked gel (Suzuki, Y. et al., J. Biomed. Mater. Res., 39, 317 (1998)), etc. A gel made of a polysaccharide and a gel made of a protein such as collagen can be used alone or in combination of two or more.
Of these carriers, polysaccharide gels are preferred from the viewpoint of non-inflammatory and non-immunogenic, covalent bonds formed by immersion, spraying, coating, dripping, etc. using peptide-containing solutions such as dimethylformamide, The peptide can be immobilized on the carrier using ionic bond, hydrophobic bond, hydrogen bond, SS bond and the like.
Immobilization is preferably performed by a covalent bond from the viewpoint of stability and long-lasting action, and can usually be performed by a method used for immobilizing a physiologically active protein such as an enzyme (for example, Scouten, WH, Methods in Enzymol., 135, Mosbach, K. Ed., 1987, Academic Press, NY, pp 30-65).
The solid form osteogenesis promoter thus obtained can be used for implantation in a bone defect site or the like.

On the other hand, the aqueous solvent used for the preparation of the osteogenesis promoter in liquid form includes physiologically acceptable aqueous solutions such as physiological saline, mannitol, sucrose, lactose, maltose, glucose and fructose, preferably 5% glucose. Examples include liquid and physiological saline.
Depending on the type of formulation, osteogenesis promoters obtained by dissolving or suspending peptides in these aqueous solvents can be administered intravenously, subcutaneously, intraperitoneally, intraarticularly, transdermally, or to bone defect sites. It can be used for filling and the like, and can be made into an oral dosage form by encapsulating or liposome-forming by a conventional method.

The peptide and osteogenesis promoter of the present invention can be administered to patients such as exogenous fractures, rheumatoid arthritis and osteoporotic fractures, or can be promoted to treat fractures by being implanted or filled in fracture sites. it can.
In addition, by administering these to patients with osteoporosis, periodontal disease and rheumatoid arthritis, bone loss due to osteoporosis and periodontal disease can be suppressed, and fractures caused by osteoporosis and rheumatoid arthritis can be prevented. it can.

The peptide of the present invention is appropriately increased or decreased depending on the bone weight desired to be formed, the site of bone damage, the condition of the bone, the age, sex, weight, etc. of the patient, but is usually 0.01 μg / kg to 2 g / kg (adult) Preferably, osteogenic activity is expressed by implanting or administering an amount of 0.01 μg / kg to 200 mg / kg (adult) as an active ingredient.
Hereinafter, the present invention will be described specifically by way of examples. In addition, this invention is not limited by these Examples.

Example 1
A peptide having the sequence shown in SEQ ID NO: 4 of the sequence listing (amino group at the N terminus and carboxyl group at the C terminus) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer.
That is, a styrene-divinylbenzene copolymer having a 4- (Nα-9- (fluorenylmethoxycarbonyl) -L-leucyl) -oxymethyl-phenoxy-methyl group at a rate of 0.74 mmol / g (resin) [styrene Amino acid corresponding sequentially from the carboxyl terminus to the amino terminus of the target peptide using 0.1 millimole of granular resin (HMP leucine, manufactured by Applied Biosystems, USA) Were combined. In the binding reaction, Nα-9- (fluorenylmethoxycarbonyl) -Nβ-trityl-L-asparagine (Fmoc asparagine), Nα-9- (fluorenylmethoxycarbonyl) manufactured by Applied Biosystems, Inc. -Ot-butyl-L-serine [Fmoc serine], Nα-9- (fluorenylmethoxycarbonyl) -valine [Fmoc valine], Nα-9- (fluorenylmethoxycarbonyl) -Nε-t-butyloxycarbonyl -L-lysine (Fmoc lysine), Nα- (fluorenylmethoxycarbonyl) -L-isoleucine (Fmoc isoleucine), Nα- (fluorenylmethoxycarbonyl) -L-proline (Fmoc proline), Nα- (fluorene Nylmethoxycarbonyl) -L-alanine [Fmoc alanine], Nα-9- (fluorenylmethoxycarbonyl) -S-trityl-L-cysteine [Fmoc cysteine], Nα-9- (fluorenylmethoxycarbonyl) -Ot - Tyl-L-threonine (Fmoc threonine), Nα-9- (fluorenylmethoxycarbonyl) -γ-butyl-L-glutamic acid (Fmoc glutamic acid), Nα- (fluorenylmethoxycarbonyl) -L-leucine (Fmoc leucine Nα- (fluorenylmethoxycarbonyl) -Ot-butyl-L-tyrosine [Fmoc tyrosine] was used in an amount of 1 mmol for each binding step.
HOBt and HBTU were used to generate each amino acid bond.

The obtained peptide resin was treated with 10 ml of trifluoroacetic acid containing 2.5% water and 2.5% ethanedithiol for 3 hours. The resulting solution was added to diethyl ether, and the resulting precipitate was further washed several times with diethyl ether to deprotect the peptide and desorb from the resin. The crude product was purified by high-performance liquid chromatograph for reverse phase preparative separation (column: Novapack HR C18 25 × 100 mm, with RCM25 × 10 pressure module, manufactured by Japan Waters Co., Ltd.).
AKTA explorer 10XT manufactured by Pharmacia Biotech Co., Ltd. [Column: Novapack C18 manufactured by Japan Waters Co., Ltd. C18 3.9 × 150 mm, mobile phase: a mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (acetonitrile concentration (Changed from 5% to 50% by volume in 30 minutes) and a flow rate of 1 ml / min] showed a single peak at 21.6 minutes. The molecular weight of the purified peptide determined by FAB mass spectrum was 2637 (theoretical value: 2636.06).

Examples 2-5
4- (2 ', 4'-Dimethoxyphenyl-fluorenylmethoxycarbonyl-aminoethyl) phenoxyacetamido-ethyl copolymer having a ratio of 0.62 mmol / g (resin) [styrene and divinylbenzene In the same manner as in Example 1 except that 0.1 mmol of a granular resin (Fmoc amide resin, manufactured by Applied Biosystems, USA) is used. A peptide having ID NO: 5 and SEQ ID NO: 6, N-terminal amino group and C-terminal amide group (Examples 2 to 4) and SEQ ID NO: 7 having N-terminal amino group A peptide having an amide group at the C-terminus (Example 5) was synthesized.
Each of the obtained peptide resins was deprotected and desorbed from the solid phase in the same manner as in Example 1 to purify the crude product.
For each purified peptide, the elution time in the analytical high performance liquid chromatograph and the molecular weight measurement results by FAB method mass spectrum are shown together in Table 1.

Test Example 1 : Alkaline phosphatase activity induction test for C3H10T1 / 2 cell line An undifferentiated mouse mesenchymal cell line C3H10T1 / 2 (purchased from Dainippon Pharmaceutical Co., Ltd.) Eagle's MEM containing 10% fetal bovine serum A cell culture solution was prepared by dispersing in a medium so that the cell concentration was 3.75 × 10 ⁴ cells / ml. 100 μL was dispensed into each well and cultured at 37 ° C. in the presence of 5% CO ₂ .
As a control, the same number of cells were dispensed into wells where the peptide was not immobilized, and then 50 ng of human recombinant BMP-2 (R & D Systems, Inc.) was added. Wells in which no peptide was immobilized and human recombinant BMP-2 was not added were used as blanks.
Three days after the start of the culture, the culture supernatant was removed and washed once with a phosphate buffer (PBS: 10 mM, containing 0.15 M sodium chloride, pH 7.4) solution. Tris buffer solution (20 mM, pH 8.5) containing 100 μL of 1% Triton X-100 was added to each well of the 96-well plate, and the cells were lysed by allowing to stand for 30 minutes. 100 μL of cell lysate 100 μL of Tris buffer solution (1.5 M, 1 mM ZnCl ₂ and 1 mM MgCl ₂ , pH 8.5) containing 7.5 mM paranitrophenyl phosphate (Wako Pure Chemical Industries, Ltd.) And the increase in absorbance at 405 nm was measured to determine the alkaline phosphatase activity in the cell lysate. The protein concentration of the cell lysate was determined using a BCA assay kit (Pierce).
The alkaline phosphatase activity of the blank well was 0.24 ± 0.11 nmol / min · mg protein, whereas the alkaline phosphatase activity of the wells on which the peptides of Examples 1 to 5 were immobilized was 1.3 ± 0.9 nmol / min · mg protein, 1.5 ± 0.6 nmol / min · mg protein, 1.5 ± 0.8 nmol / min · mg protein, 1.7 ± 0.1 nmol / min · mg protein, 0.61 ± 0.07 nmol / min · mg protein.
On the other hand, in the wells to which human recombinant BMP-2 was added, the protein was 1.7 ± 0.4 nmol / min · mg, and the peptides shown in Examples 1 to 4 were as remarkable as human recombinant BMP-2. Caused an increase in alkaline phosphatase activity.
Moreover, by comparing with known sequences of the BMP family, it was inferred from the results of Example 5 that the peptide STLY had a good influence on the activity of alkaline phosphatase.

Example 6
0.6 g (10 mmol) of ethylenediamine (EDA, Wako Pure Chemical Industries, Ltd.) dissolved in 10 ml of methanol was dissolved in 2.3 g (20 mmol) of N-hydroxysuccinimide (HOSu, Peptide Institute, Inc.). The mixture was added dropwise to 150 ml of methanol with stirring at room temperature. Stirring was continued for another hour after the completion of dropping. The precipitated crystals were collected by filtration and dried under reduced pressure to obtain 2.6 g (yield: about 90%) of ethylenediamine 2N-hydroxysuccinimide salt (EDA · 2HOSu).
In 30 ml of 1% by weight aqueous solution of sodium alginate (Funakoshi Co., Ltd., viscosity 550 cp, M / G ratio 1.0), 66 mg of EDA · 2HOSu and 0.48 g of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide hydrochloride (WSCD · HCl, Peptide Laboratory Co., Ltd.) Dissolve, cast on a 10cm x 10cm Teflon (registered trademark) coated aluminum tray and let stand at 25 ° C for 48 hours to form a covalently crosslinked alginic acid gel Got.
This was thoroughly washed with water for injection (Otsuka Pharmaceutical) in which 2.5 mM CaCl ₂ and 143 mM NaCl were dissolved, and then washed only with water for injection. The washed alginate gel was lyophilized to obtain a white sponge gel.
0.2 g of the resulting sponge gel was immersed in 4 ml of dimethylformamide, 6 mg of N-hydroxysuccinimide and 10 mg of WSCD · HCl were added, and the mixture was shaken overnight at room temperature. The sponge gel was thoroughly washed with methanol and dimethylformamide, 1 ml of a dimethylformamide solution containing 13 mg of the peptide shown in Example 2 and 0.86 μL of diisopropylethylamine were added, and the mixture was shaken overnight at room temperature. After thoroughly washing with methanol and ethanol, an osteogenesis promoter having the peptide shown in Example 2 immobilized thereon was obtained.

Test Example-2 : Rat Intramuscular Implantation Test 0.01 g of the osteogenesis promoter obtained in Example 6 was implanted in the lower leg muscle of a 6-week-old male Wistar rat (Charles River Japan). Four weeks later, the surrounding tissue including the implanted part was taken out and tissue staining was performed.
As a result, apparent Ca deposition was observed at the implantation site in von Kossa staining. For comparison, no Ca deposition was observed at the site where a sponge-like gel that did not immobilize the peptide was implanted on the opposite side of the rat.
Test Example-3 : Rat Tibial Defect Site Implantation Test 0.01 g of the osteogenesis promoter obtained in Example 6 was artificially prepared on the tibia of a 6-week-old male Wistar rat (Charles River Japan). Implanted in a 3 mm circular bone defect site.
At 4 weeks after implantation, the tissue including the implantation site was taken out and stained, and as a result, clear new bone formation was observed. As a comparison, the formation of new bone at the site where a sponge-like gel not immobilizing the peptide was implanted on the opposite side of the rat was weak.

Claims (4)

SEQ ID NO: 2, Cys, SEQ ID NO: 3, and SEQ ID NO: 7 have at least an amino acid sequence linked in this order from the N-terminal side to the C-terminal side , and have an osteogenic effect. Having peptide. The peptide according to claim 1, wherein the amino acid sequence represented by SEQ ID NO: 7 is located at the C-terminus. An osteogenesis promoter containing the peptide according to claim 1 or 2 as an active ingredient. The osteogenesis promoter according to claim 3 , wherein the peptide is immobilized on a carrier.