JP3059298B2 - Osteoporosis treatment - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a human interleukin.
(1) The present invention relates to a therapeutic agent for osteoporosis, comprising a peptide having binding ability or a salt thereof as an active ingredient. The therapeutic agent for osteoporosis provided by the present invention is obtained by binding a peptide as an active ingredient or a salt thereof to human interleukin-1 (hereinafter, abbreviated as human IL-1).
-1 inhibits the expression of activity, so that paraarticular osteoporosis associated with rheumatoid arthritis, alveolar bone absorption associated with periodontitis,
IL such as post-menopausal high-rotation osteoporosis and senile osteoporosis
-1 is useful for treating osteoporosis.

[0002]

2. Description of the Related Art Osteoporosis includes primary osteoporosis such as postmenopausal osteoporosis and senile osteoporosis, and secondary osteoporosis associated with rheumatoid arthritis and periodontal disease. In addition, osteoporosis is classified into high-rotation osteoporosis and low-rotation osteoporosis according to its causes. High-rotation osteoporosis is osteoporosis caused by the increase in bone resorption and bone formation, but the increase in bone resorption is greater. On the other hand, low-rotation osteoporosis is osteoporosis caused by a decrease in bone resorption and bone formation, but a greater decrease in bone resorption.
It is considered that secondary osteoporosis and postmenopausal osteoporosis associated with rheumatoid arthritis and periodontal disease are the former, and senile osteoporosis is the latter. IL-1 plays an important role in various inflammatory diseases and bone and joint diseases. For example, rheumatoid arthritis [P. Miossec et al., Arthritis Rheumatism
m.), Vol. 29, No. 4, pp. 461-470, (1986)
)], Mechanism of alveolar bone destruction in periodontitis [J. Hoeni
g et al., Journal of Periodont Res., 24, 362-367,
(1989)], high rotation osteoporosis after menopause [
R. Pacifici et al., Exp. Gerontol., 25, 309-316,
(1986)], and that IL-1 is involved (Interleukin 1, Protein Nucleic Acid Enzyme, Vol. 33, No. 10, 1728-1741).
1988). Osteoclasts and osteoblasts play major roles in bone metabolism, and various factors controlling them have been elucidated [DA Watrous et al., Seminars in Arsleitis. And Rheumatism (Semin. Arthr. Rheumat.), 19, 45-65.
, (1989)]. Of these factors, I
L-1 enhances collagenolytic enzyme production of osteoblasts,
Induces the formation and activation of osteoclasts and strongly promotes bone resorption. Furthermore, it is known that administration of a large amount of IL-1 to mice causes hypercalcemia. In addition, the presence of a large amount of IL-1 in the periodontitis tissue, the finding of high IL-1 activity in the synovial fluid of patients with rheumatoid arthritis, the peripheral blood monotherapy of postmenopausal patients with high rotational osteoporosis, That the spheres produce IL-1 without stimulation, significantly higher concentrations of IL- in the serum of postmenopausal patients with high-rotation osteoporosis
It has been reported that No. 1 was detected.
It is thought to exhibit bone resorption in vivo. IL
-1 was found in the culture supernatant of macrophages as a factor that enhances lectin-responsive proliferation of mouse thymocytes. IL-1 is a polypeptide having a molecular weight of 17.5 KD, and there are two kinds of IL-1α and IL-1β having different isoelectric points, both of which bind to the same receptor on a cell. The biological activity of IL-1 is extremely diverse and not only activates and induces differentiation of immunocompetent cells, but also forms joints such as nervous system, endocrine system, and synovial cells, bone / chondrocytes, and vascular endothelial cells. It also has various effects on cell groups. It is also known that the receptor for IL-1 is expressed on various cells.
E. Sims et al., Providing's of the Natural Academy of Sciences of the.
United States of America (Proc. Na
Acad. Sci. USA.), 86, 8946-8950.
P., (1989)] and on B cells [CJ McMaha
n et al., EMBO J., Vol. 10, No. 10, pp. 2821-2832 (1991)]
Of IL-1 receptors were successively cloned. It has been clarified that various signals are transmitted into cells by binding of IL-1 receptor on these cells to IL-1.

[0003]

In the treatment of high-rotation osteoporosis, drugs such as estrogen for the purpose of suppressing bone resorption are mainly used. On the other hand, drugs for improving calcium metabolism such as vitamin D derivatives are mainly used for treatment of low-rotation osteoporosis. However, vitamin D derivatives have side effects such as hypercalcemia, gastrointestinal dysfunction, and hepatic dysfunction, and particularly cause hypercalcemia and are not used for patients with renal dysfunction. On the other hand, estrogen is concerned about dangers such as side effects associated with female hormone action and carcinogenicity. In addition, fluorine (sodium fluoride), PTH
Attempts have been made to use drugs aimed at promoting bone formation, such as (parathyroid hormone) and vitamin K, but no sufficient effect has been observed. Therefore, at present, there is a demand for the development of a drug that is effective and highly safe and is effective for treating osteoporosis. Accordingly, an object of the present invention is to provide a therapeutic agent for osteoporosis containing, as an active ingredient, a peptide having an inhibitory activity on IL-1, which is a major causative substance of osteoporosis.

[0004]

According to the present invention, the above object is achieved by the following general formula: HX ¹ -Cys- A ¹ -B ¹ -A ² -B ² -A ³ -Ser-X ² −
Y (where A ¹ , A ² and A ³ each represent an amino acid residue selected from the group consisting of Val, Leu, Ile and Nle;
B ¹ and B ² are Arg, Lys, Gln, His and Ser, respectively.
X ¹ and X ² each represent a single bond or Gly, Ala, Val, Arg, As
n, Ser, Phe, Pro, Leu, Glu, Asp, Lys, Thr, His, Ty
a peptide fragment consisting of 1 to 10 amino acid residues selected from the group consisting of r, Nle and Ile, wherein Y represents a hydroxyl group or an amino group), and a peptide having human interleukin-1 binding ability Alternatively, the problem is solved by providing a therapeutic agent for osteoporosis containing a salt thereof.

[0005] Representative examples of the above peptides are shown below. (A): Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Il
e Lys Ile Ser Ala Lys (SEQ ID NO: 1), (b): Lys Asn Ser Ser Tyr Cys Leu Arg Ile Lys Il
e Ser Ala Lys Phe ValGlu (SEQ ID NO: 2), (c): Lys Tyr Cys Leu Arg Ile Lys Ile Ser Ala Ly
s Phe Val Glu Asn GluPro (SEQ ID NO: 3), (d): Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys
(SEQ ID NO: 4), (e): Tyr Cys Leu Arg Ile Lys Ile Ser (SEQ ID NO: 5), (f): Cys Leu Arg Ile Lys Ile Ser (SEQ ID NO:
6), (g): Lys Cys Leu Arg Ile Lys Ile Ser (SEQ ID NO: 7), (h): Cys Leu Gln Ile Lys Ile Ser (SEQ ID NO:
8), (i): Lys Cys Leu Gln Ile Gln Ile Ser (SEQ ID NO: 9), (j): Lys Ile Cys Ile Arg Ile Gln Ile Ser (SEQ ID NO: 10), (k): Lys Xaa Cys Xaa Arg Xaa Gln Xaa Ser (SEQ ID NO: 11) (where Xaa represents Nle), (l): Lys Ile Cys Ile His Ile Gln Ile Ser (SEQ ID NO: 12), (m): Lys Ile Cys Leu Arg Ile Gln Ile Ser (SEQ ID NO: 13), (n): Lys Cys Val Gln Val Gln Val Ser (SEQ ID NO: 14), (o): Lys Ile Cys Ile Arg Ile Gln Ile Xaa (SEQ ID NO: 15) ) (wherein, Xaa represents those with substitution of -COOH groups Ser at -CONH ₂ group) and (p): Lys Ile Cys Ile His Ile His Ile Ser ( SEQ ID NO: 16).

The salts of the above-mentioned peptides are physiologically acceptable salts. Examples of the salts include hydrochloric acid, sulfuric acid, phosphoric acid, lactic acid, tartaric acid, maleic acid, fumaric acid, oxalic acid, malic acid, and the like. Salts with acids such as citric acid, oleic acid, palmitic acid; salts with hydroxides or carbonates of alkali metals or alkaline earth metals such as sodium, potassium, calcium, aluminum; triethylamine, benzylamine, diethanolamine , T-butylamine, dicyclohexylamine, arginine and the like.

[0007] The peptide in the present invention is a novel peptide. These peptides are prepared by a method usually used in peptide synthesis, for example, a solid phase synthesis method or a liquid phase synthesis method, and the solid phase synthesis method is easy to operate. Chemistry Experiment Course 2 Chemistry of Protein (2) "(May 20, 1987, published by Tokyo Chemical Co., Ltd.), pp. 641-694).
In addition, the salt of the peptide is prepared by utilizing a usual salt formation reaction.

[0008] The peptides and salts thereof (hereinafter, these are abbreviated as peptides) in the present invention are human IL-1
Have the ability to bind to IL-1 and IL-1
It can inhibit receptor binding, thereby suppressing various immunological and physiological activities caused by IL-1.

[0009] The peptides of the present invention also have a bone formation promoting action.

The results of the test of the peptide of the present invention for inhibiting bone resorption and for promoting bone formation are shown below.

Test Example 1 Bone Resorption Inhibition Test Associated with Adjuvant Arthritis Bone resorption inhibition test associated with adjuvant arthritis of the following peptide was carried out using 6 rats each of 6-week-old female Lewis rats. Peptide-1: Lys Ile Cys Ile Arg Ile Gln Ile Ser
(SEQ ID NO: 10)

Mycobacterium butyricum (M. buty)
ricum) containing 0.05 mg of dead bacteria.
05 ml (adjuvant) was injected into the right footpad skin of each rat. Three weeks later, the right radial bone was collected, and the bone mineral density at the proximal end of the ankle joint was measured using DEXA (Dualenergy X-ray absorptio).
measurement). During the test period, 5
% Glucose solution at a rate of 3.6 mg / kg 3 per week
The dose was subcutaneously administered to the right footpad of each rat. The same measurement of bone density was performed for control rats to which the same amount of 5% glucose solution alone was administered and healthy rats to which no adjuvant was injected, for comparison.

The bone density of healthy rats was 57.8 mg / cm.
^In rats to which the same amount of 5% glucose solution alone was administered, the dose was 48.4 mg / cm ² , and a reduction of 16% was observed. In rats to which peptide-1 was administered, 53.9 m / cm ² was observed.
g / cm ² , a decrease of only 7%, and bone resorption was suppressed.

Furthermore, in the rats to which the peptide was administered, the body weight was significantly increased as compared with the control group, and in addition, improvement in systemic bone destruction such as suppression of deformity of the coccyx joint and vertebra was observed.

Test Example 2 Bone formation promoting action in ovariectomized rats 6 to 4 weeks old Wistar female rats 3 to 4 each
A test was performed on the osteogenesis-promoting effect of the following peptide in ovariectomized rats using the mice. Peptide-1: Lys Ile Cys Ile Arg Ile Gln Ile Ser
(SEQ ID NO: 10)

The ovaries of 6-week-old Wistar female rats were excised to prepare experimental osteoporotic rats. After exposing the ovaries, a group in which only the ovariectomy operation was not performed was set as a control (sham control). Peptide-1 was administered for one month from 3 months after ovariectomy. Peptide-1
After completion of the administration, bone marrow cells were collected from the femur and the tibia, and were placed in an αMEM medium containing 10% fetal calf serum at 37 ° C.
The cells were cultured in the presence of% CO ₂ for 7 days. The total amount of the medium was changed every three days from 24 hours after the start of the culture. On the 7th day, the bone marrow cells were stained with an alkaline phosphatase staining kit manufactured by Sigma, and the number of alkaline phosphatase-positive fibroblast-like colonies was determined as the number of osteoblast colonies. During the test period, peptide-1 was dissolved in 5% glucose solution and
At a dose of 10 mg / kg intraperitoneally or 1
The drug was subcutaneously administered at a rate of mg / kg to the right footpad of the rat. 5%
The number of osteoblast colonies was calculated in the same manner for control rats and sham controls to which only the same amount of glucose solution was intraperitoneally administered for comparison.

The number of colonies of osteoblasts was significantly reduced to 25 in control rats to which 5% glucose solution was administered, compared to 60 in sham control.
In the group administered intraperitoneally at a rate of mg / kg, 130 mice, and in the group administered the same peptide subcutaneously in the right footpad at a rate of 1 mg / kg, 115 mice showed significant bone loss compared to control rats and sham controls. It was shown that the number of blast colonies increased and bone formation was promoted.

Further, it has been confirmed that the peptides of the present invention have low toxicity in toxicity tests.

The therapeutic agent for osteoporosis according to the present invention is characterized in that the peptide or its salt as an active ingredient binds to IL-1 in a body fluid and can neutralize it, and further has an osteogenesis promoting action. By administering to a patient suffering from osteoporosis involving IL-1, such as paraarticular osteoporosis associated with rheumatoid arthritis, alveolar bone resorption associated with periodontitis, high-rotation osteoporosis after menopause, and senile osteoporosis. Can suppress bone resorption and promote bone formation.

The dose of the peptide, which is an active ingredient of the therapeutic agent for osteoporosis of the present invention, for expressing effective activity is usually 2 g.
/ Kg to 0.1 μg / kg (adult), preferably 200 mg / kg to 1 μg / kg (adult).

The method for administering the therapeutic agent for osteoporosis of the present invention includes intravenous administration, subcutaneous administration, intramuscular administration, joint administration, oral administration, nasal administration, and enteral administration.

The preparation of the therapeutic agent for osteoporosis of the present invention can be carried out by appropriately selecting a method commonly used for preparing various preparations.

Examples of the oral preparation include tablets, granules, powders, hard capsules, soft capsules, and liquid preparations for oral use. Oral preparations include syrup, gum arabic, gelatin, sorbit, tragacanth, polyvinylpyrrolidone, etc. as binders; lactose, sugar, starch, calcium phosphate, sorbit, glycine, etc. as excipients; magnesium stearate, talc, Polyethylene glycol, silica, etc .; may contain starch, calcium carboxymethylcellulose, etc. as disintegrants.

Examples of the injection include an aqueous solution in an ampoule or a vial, a saline solution, a physiologically acceptable solution such as a 5% dextrose solution, or a lyophilized powder thereof. These may contain pharmacologically acceptable additives such as preservatives, solubilizers, dispersants and the like. In order to further provide sustained release properties, it may be included in a biodegradable compound such as polylactic acid or collagen.

Examples of the nasal preparation include aqueous preparations capable of dropping or spraying a predetermined amount into the nasal cavity. The aqueous preparation may contain pharmacologically acceptable stabilizers, thickeners, solubilizing agents, surfactants, preservatives, bulking agents, tonicity agents, and the like, if necessary.

Aqueous solutions or suspensions are used as eye drops, and may contain stabilizers, solubilizers, suspending agents, emulsifiers, buffers, thickeners, preservatives, and the like as necessary. Good.

The suppository may contain a surfactant, a preservative, a fragrance, a stabilizer and the like based on cocoa butter, macrogol and the like.

[0028]

The present invention will be described below in detail with reference to examples. Note that the present invention is not limited by these examples.

Reference Example 1 A peptide represented by the formula (1): Lys Ile Cys Ile Arg Ile Gln Ile Ser (SEQ ID NO: 10) was synthesized by a solid phase synthesis method using an automatic peptide synthesizer. That is, 4
-[N- (t-butoxycarbonyl) -O-benzyl-
Particulate resin comprising a styrene-divinylbenzene copolymer having a ratio of L-seryloxymethyl] -phenylacetamidomethyl group of 0.72 mmol / g (resin) [constituent molar ratio of styrene to divinylbenzene: 99: 1] Amino acids were sequentially bound toward the N-terminus of the peptide using 0.1 mmol of [PAM serine, t-Boc-L-Ser (Bzl) manufactured by Applied Biosystems, USA]. In the binding reaction, N ^α- (t-butoxycarbonyl)-manufactured by Applied Biosystems, USA
N ^ε- (p-chlorobenzyloxy) -L-lysine [t
-Boc lysine], N ^α- (t-butoxycarbonyl)
- N ^G - (mesitylene-2-sulfonyl) -L- arginine [t-Boc-arginine], N- (t- butoxycarbonyl) -L- isoleucine [t-Boc isoleucine], N- (t- butoxycarbonyl) - S- (p-methoxybenzyl) -L-cysteine [t-Boc cysteine] and N- (t-butoxycarbonyl) -L-glutamine [t-Boc glutamine] were each used in an amount of 1 mmol.

The obtained peptide was subjected to deprotection and elimination from the solid phase using an HF reaction apparatus type I manufactured by Peptide Research Institute. The crude product was subjected to high performance liquid chromatography for separation by Millipore Waters [column: Deltapack C18 47 × 300 mm Preppack 100
0 with pressure module]. The obtained purified peptide was subjected to high performance liquid chromatography for LC6A analysis manufactured by Shimadzu Corporation [Column: TSKgel manufactured by Tosoh Corporation]
ODS-80TM CTR, mobile phase: mixed solvent of acetonitrile and water containing 0.05% by volume of trifluoroacetic acid (acetonitrile concentration from 5% by volume to 50% in 30 minutes)
Volume was changed to 1%).
n showed a single peak. The molecular weight of the purified peptide determined by the FAB mass spectrum was 1073 (theoretical value: 1073.35).

Reference Example 2 A peptide represented by the formula (2): Lys Ile Cys Ile His Ile His Ile Ser (SEQ ID NO: 16) was synthesized in the same manner as in Reference Example 1. Amino acids were N- (t) manufactured by Applied Biosystems, USA in addition to those used in Reference Example 1.
-Butoxycarbonyl) -N ^Im -dinitrophenyl-L
-Histidine [t-Boc histidine] was used.

The obtained peptide was subjected to deprotection and elimination from the solid phase in the same manner as in Reference Example 1, and the crude product was purified. For the purified peptide, the elution time in the analytical high performance liquid chromatograph was 17.7 min, and the molecular weight measurement result by FAB mass spectrum was 1
063 (theoretical value: 1063.32).

Reference Example 3 Test for Binding to Human IL-1 In 5 ml of dioxane obtained by distillation in the presence of sodium metal, 1 g of cellulose particles (CM-Cellulofine CH, sold by Seikagaku Corporation) was added. After suspending, the resulting suspension was treated with 0.05 g of N-hydroxysuccinimide and 0.1 g of dicyclohexylcarbodiimide.
1 g was added, and the mixture was shaken and stirred at room temperature overnight. The resulting mixture was treated with 0.02 mol / l phosphate buffer (p
H: 7.4), followed by suction filtration. The particles obtained were 1 m of the peptides obtained in Reference Examples 1 and 2.
g of an aqueous solution containing 1 g.
And stirred overnight. The resulting mixture was suction filtered. The filtrate was subjected to analytical reversed-phase high-performance liquid chromatography, and no unreacted peptide remained. (Immobilization ratio of peptide on carrier: about 100
%). Thus, 1 g of the adsorbent to which 1 mg of each of the peptides obtained in Reference Examples 1 and 2 was immobilized was obtained.

100 mg of the adsorbent was weighed and added to PBS (0.15 M NaCl) containing 5% bovine serum albumin.
10 mM phosphate buffer, pH 7.4) 100
Add 1 μl and incubate at 4 ° C. overnight, then add ¹²⁵ I
About 300 Bq (16,300 cpm) of labeled human IL-1β (purchased from Amersham Japan) was added and incubated at room temperature for 3 hours. 0.2% of the resulting mixture
Triton buffer containing Triton X-100 (pH: 7.
After washing 5 times in 4), the radioactivity remaining on the adsorbent was measured with a γ counter. As a result, the adsorbent on which the peptide obtained in Reference Example 1 was immobilized was 1,850 cp.
m, a count of 2,400 cpm was obtained from the adsorbent on which the peptide obtained in Reference Example 2 was immobilized, and 45 cp of the adsorbent on which glycine (H-Gly-OH) (control) was immobilized.
m, a significantly higher radioactivity was observed as compared to m.
And it was confirmed that the peptide obtained in Reference Example 2 had human IL-1 binding ability.

Example 1 Injection A 5% glucose solution obtained by dissolving the peptide obtained in Reference Example 1 at a ratio of 4 mg / 1 ml was sterilized by filtration through a filter having a pore size of 0.22 μm, and then 5 ml was added to a heat-sterilized vial. Each was aseptically dispensed to give an injection.

Example 2 Sustained Release Injection Distilled water in which 10 mg of the peptide obtained in Reference Example 2 was dissolved was sterilized by filtration through a filter having a pore size of 0.22 μm, and aseptically prepared from poly (D, L) lactic acid and glycolic acid. Copolymer (composition ratio, 75:25, average molecular weight about 10,000)
Was mixed with a methylene chloride solution to prepare a W / O emulsion. Further, after the above emulsion was suspended in hexane, the solvent was removed under reduced pressure to obtain polylactic acid microcapsules containing the peptide.

Example 3 Nasal Agent 10 mg of the peptide prepared in Reference Example 2, 1.36 mg of methyl parahydroxybenzoate, 0.16 mg of propyl paraoxybenzoate and 50 mg of glucose were dissolved in 1 ml of distilled water, and the solution having a pore size of 0.22 μm was dissolved. It was sterilized by filtration through a filter to obtain a liquid for nasal administration.

Example 4 Enteric Capsules An enteric capsule prepared from an aqueous solution of 65 parts of polyvinyl phthalate and 35 parts of gelatin is filled with a mixture of 50 mg of the peptide obtained in Reference Example 2, 250 mg of glucose and 1 g of crystalline cellulose. Thus, an enteric capsule was obtained.

[0039]

According to the present invention, IL-1 such as paraarticular osteoporosis associated with rheumatoid arthritis, alveolar bone absorption associated with periodontitis, high rotation osteoporosis after menopause, senile osteoporosis, etc.
Provided is a therapeutic agent for osteoporosis which is effective for the treatment of osteoporosis associated with osteoporosis. Osteoporosis therapeutic agent provided by the present invention,
As is clear from the above test examples, the peptide or the salt thereof as an active ingredient has the ability to specifically bind to human IL-1, and IL-1 and IL-1 receptor are in vivo in vivo. Can inhibit the binding of, and can be administered to a patient suffering from the above-mentioned disease to neutralize IL-1 in body fluids and reduce the symptoms of the disease.

[0040]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 16 Sequence type: Amino acid Topology-: Linear Sequence type: Peptide sequence Val Val Arg Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys 1 5 10 15

SEQ ID NO: 2 Sequence length: 17 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Lys Asn Ser Ser Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val 1 5 10 15 Glu

SEQ ID NO: 3 Sequence length: 17 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Lys Tyr Cys Leu Arg Ile Lys Ile Ser Ala Lys Phe Val Glu Asn Glu 1 5 10 15 Pro

SEQ ID NO: 4 Sequence length: 10 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 5 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 6 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 7 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 8 Sequence length: 7 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 9 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 10 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 11 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide Sequence characteristics Characteristic symbol: modified site Location: 2, 4, 6, 8 Characteristics Method for determining E: E Other information: Xaa represents Nle.

SEQ ID NO: 12 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 13 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 14 Sequence length: 8 Sequence type: amino acid Topology: linear Sequence type: peptide

SEQ ID NO: 15 Sequence Length: 9 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Characteristic Characteristic Symbol: modified site Location: 9 Characteristic Determination Method: E other information: Xaa represents those with substitution of -COOH groups Ser at -CONH ₂ group. Sequence Lys Ile Cys Ile Arg Ile Gln Ile Xaa 1 5

SEQ ID NO: 16 Sequence length: 9 Sequence type: amino acid Topology: linear Sequence type: peptide

Continuing from the front page (72) Inventor Etsuko Abe 1-5-8 Flag Nodai, Shinagawa-ku, Tokyo Showa University Campus (72) Inventor Yutaka Mizushima 2-16-1 Sugo, Miyamae-ku, Kawasaki-shi, Kanagawa Research for the treatment of intractable disease at St. Marianna University School of Medicine Center (72) Inventor Yasuo Suzuki 2-16-1 Sugo, Miyamae-ku, Kawasaki City, Kanagawa Prefecture Research Center for Intractable Diseases, St. Marianna University School of Medicine (72) Inventor Chiyuki Abe 2-16 Sugo, Miyamae-ku, Kawasaki City, Kanagawa Prefecture -1 St. Marianna University School of Medicine Research Center for Intractable Diseases Investigator Ikuharu Oya (58) Field surveyed (Int. Cl. ⁷ , DB name) A61K 38/00-38/58 GenBank / EMBL / DDBJ (G ENETYX)

Claims (1)

(57) [Claims] [Claim 1] The general formula H-X ¹ -Cys- A ¹ -B ¹ -A ² -B ² -A ³ -Ser-X ^2-
Y (where A ¹ , A ² and A ³ each represent an amino acid residue selected from the group consisting of Val, Leu, Ile and Nle;
B ¹ and B ² are Arg, Lys, Gln, His and Ser, respectively.
X ¹ and X ² each represent a single bond or Gly, Ala, Val, Arg, As
n, Ser, Phe, Pro, Leu, Glu, Asp, Lys, Thr, His, Ty
a peptide fragment consisting of 1 to 10 amino acid residues selected from the group consisting of r, Nle and Ile, wherein Y represents a hydroxyl group or an amino group), and a peptide having human interleukin-1 binding ability Or a therapeutic agent for osteoporosis comprising a salt thereof as an active ingredient.