WO2003014146A1

WO2003014146A1 - A novel peptide p1 inhibiting differentiation of osteoclasts

Info

Publication number: WO2003014146A1
Application number: PCT/KR2002/001508
Authority: WO
Inventors: Hong-Hee Kim; Zang-Hee Lee; Kyung-Soo Hahm
Original assignee: Komed Co., Ltd
Priority date: 2001-08-07
Filing date: 2002-08-07
Publication date: 2003-02-20
Also published as: KR20030013189A

Abstract

The present invention relates to a novel peptide P1 capable of inhibiting osteoclast differentiation or homologues thereof, to nucleic acid molecules encoding said peptides, and to a process for producing said peptides. The present invention also relates to an isolated and purified peptide P1 and functional homologues thereof, to a pharmaceutical composition for the treatment of metabolic bone disease comprising said peptide, and to a method for inhibiting osteoclast differentiation which comprises administering said composition to the patient suffering from said disease.

Description

A NOVEL PEPTIDE PI INHIBITING DIFFERENTIATION OF OSTEOCLASTS

TECHNICAL FIELD

The present invention relates to a novel peptide PI with osteoclast differentiation-inhibiting activity or homologues thereof, to nucleic acid molecules coding for said peptides, and to methods for producing peptides encoded by said nucleic acid molecules. It also relates to compositions for the treatment of metabolic bone disease comprising such peptide PI and/or functional homologues thereof as an active ingredient, and to methods for inhibiting osteoclast differentiation by administering such compositions.

BACKGROUND ART

Bones consists of bone cells including osteocytes, osteoclasts and osteoblasts, bone matrix including hydroxyapat,ite crystals, collagenous fibers and glycosaiainoglycans, and spaces including bone marrow cavity, vascular canals, canaliculi and lacunae (Stavros C. M., Endocrine Reveiws, 21(2), 115-137, 2000). Bones provide mechanical support and protection to the vital organs, create microsurrondings necessary for hemopoesis, and function to reserve various minerals including calcium.

Growth, development and maintenance of bones occur continuously throughout life. Old bones are destroyed and new bones are regenerated to replace them. Such turnover takes place mainly in basic ulticellular units (BMU) consisting of osteoclasts and osteoblasts and serves to repair micro damage of bone caused by growth and stresses and to maintain bone functions. Erosion or resorption of aged bones is the task of osteoclasts. Formation of new bones is accomplished by osteoblasts. Osteoclasts adhere to the bone surface and destruct the bone by secreting acids and lytic enzymes thereon and eroding bone matrix constituting bone, such as hydroxyapatite crystals and collagenous fibers. Osteoblasts form skeleton by synthesizing and secreting new bone matrix and controlling the concentration of calcium and phosphorous (Stavros C. M., Endocrine Reviews, 21(2), 115-137, 2000).

Metabolic bone disease is induced by an imbalance between osteoclast and osteoblast functions. Osteoporosis is a common metabolic bone disease that leads to the gradual loss of mineralized bone from the skeletal mass. This is in part because the actions of osteoclasts exceed those of osteoblasts. Once osteoporosis occurred, cortical bone becomes more porous and thinner as the width of cortical bone lessens, the cavity of the bone marrow expands, and the structural integrity of trabecular bone is impaired. As osteoporosis progresses, the physical strength of bone is decreased, whereby lumbago and arthralgia occurs and bone is more prone to breakage. In addition, the metabolic bone disease includes bone metastatic lesions in which tumors such as breast or prostate carcinomas have been transmitted to bone, tumors primarily generated in bone (for example, multiple myeloma) , rheumatoid or degenerative arthritis, periodontal disease having the alveolar bone destroyed by bacteria, inflammatory alveolar bone resorption disease caused by dental implants, inflammatory bone resorption disease caused by bony implants in orthopedic surgery, Paget's disease caused by various genetic factors and the like.

Myeloma is a disease accompanied with severe pain in which the actions of osteoclasts are increased by tumor cells, making it prone to fracture even after a minor injury. Breast or prostate carcinomas metastasize easily to bone and promote the actions of osteoclasts, causing the destruction of bone. In cases of rheumatoid arthritis or degenerative arthritis, a tumor necrosis factor, interleukin-1, interleukin-β and the like, which have been generated by immune response, increase the actions of osteoclasts existing in the articular cavity, thereby leading to local destruction of bone surrounding the joints. When inflammation occurs in response to periodontal disease-causing bacteria, inflammatory cytokines such as tumor necrosis factor, interleukin-1, and interleukin-6 are produced as a result of the immune response. These cytokines promote differentiation of osteoclasts, resulting in the destruction of alveolar bones anchoring teeth.

Recently, molecular biological studies of the treatment of metabolic bone diseases including osteoporosis have been actively conducted and bone-formation stimulating factors and bone-destruction inhibiting factors have been found. Examples of the bone-formation stimulating factors include fluorides, parathyroid hormone, TGF-β, bone- morphogenic protein, and insulin-like growth factor. Examples of the bone-destruction inhibiting factors include estrogen, calcitonin, vitamin D and analogues thereof, and bisphosphonate (Jardine et al., Annual Reports in Medicinal Chemistry, 31, 211, 1996) .

Until now, many anti-osteoporosis agents have been developed. Among them, estrogen is most commonly used. However, its efficacy has not yet been proven and, disadvantageously, its administration should be made during the entire lifetime of the patient. Moreover, its long- term administration induces adverse side effects including an increased incidence of breast cancer or uterine cancer. Alendronate also has yet to be proven of its efficacy, and has problems in that it is slowly absorbed in the digestive tract and may cause inflammation in the stomach, bowels and esophageal ucosa. It is known that calcium-containing agents have excellent anti-osteoporosis activities with few side effects but are used as prophylactics rather than therapeutics. Besides these, calcitonin and vitamin D may be candidates as anti-osteoporosis agents, but there is insufficient research into their efficacies and side effects.

As results of years-long study of an agent for the treatment of metabolic bone disease, the present inventors have found a novel peptide PI with potent osteoclast differentiation-inhibiting activity.

DISCLOSURE OF INVENTION

Therefore, an object of the present invention is to provide a novel peptide which has more potent osteoclast differentiation-inhibiting activity but exhibits significantly reduced adverse side effects, as compared to known therapeutic agents for the treatment of metabolic bone disease.

It is another object of the present invention to provide a nucleic acid molecule encoding a novel peptide with ability to inhibit osteoclast differentiation.

It is a further object: of the present invention to provide a process for producing a novel peptide with ability to inhibit osteoclast differentiation. It is another further object of the present invention to provide a pharmaceutical composition for the treatment of metabolic bone disease containing an isolated and purified peptide with ability to inhibit osteoclast differentiation or functional homologues thereof. It is still another object of the present invention to provide a method for inhibiting osteoclast differentiation in a patient by administering an isolated and purified peptide with ability to inhibit osteoclast differentiation and/or functional homologues thereof to said patient.

Brief Description of the Drawings

Fig. 1 is a schematic view illustrating the differentiation and activation processes of osteoclasts.

Fig. 2 is a graph showing the effects of the peptide PI on osteoclast differentiation which progresses under the co-culture system of bone marrow cells and osteoblasts.

Fig. 3 is a graph showing the effects of the peptide PI on osteoclast differentiation which progresses under the culture of bone marrow cells.

Fig. 4 is a graph showing the effects of the peptide PI on differentiation of the osteoclast precursor cells.

Fig. 5 is a graph showing cytotoxicity of the peptide PI on osteoblasts.

Fig. 6 is a graph showing cytotoxicity of the peptide PI on bone marrow cells .

Fig. 7 is a graph showing cytotoxicity of the peptide PI on abdominal macrophages. Fig. 8 is a graph showing cytotoxicity of the peptide

PI on 293T cells, human embryonic kidney cell line.

Fig. 9 is a graph showing the effects of the peptide PI on the activity of JNK, MAP kinase.

Fig. 10 is a graph showing the effects of the peptide PI on nuclear transcription factor NF-κB.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides peptides, including peptide Pi, which inhibit osteoclast differentiation.

Osteoclast progenitor cells are hematopoietic as a member of monocyte/macrophage cell family originating from the bone marrow. These cells differentiate and develop into osteoclasts under the influence of growth factors and cytokines produced in the bone marrow (Roodman G. D., Endocr. Rev., 17, 308-332, 1996) (Fig. 1). The osteoclasts function to destroy or resorb bone.

Recently, osteoclast differentiation factor (ODF) , which is needed to differentiate osteoclasts, has been cloned. ODF is a member of the TNF ligand family bound to cell membranes. It has been shown that genetically engineered soluble ODF may induce formation of osteoclasts in the presence of M-CSF without osteoblasts or stromal cells. ODF also may be called TRANCE, OPGL or RANKL. As a member of TNF receptor, ODF interact with RANK existing in osteoclast progenitor cells and mature osteoclasts. In mouse, expression of the ODF is limited to bones, spleen thymus and lung. It has been reported that in cultured osteoblasts, stimulation of bone resorption increases expression of the ODF (Suda T. et al., Endocr. Rev., 20, 345-357, 1999; Yasuda et al., Proc. Natl. Acad. Sci. USA, 95(7) , 3597-3604, 1998) .

Osteoclastogenesis-inhibitory factor (OPG) , also called osteoprotogerin, is a protein which inhibits production of osteoclasts and action of mature osteoclasts. OPG acts as a decoy protein of the TNF receptor family and binds to membrane-bound ODF with high affinity. Bone remodeling is controlled by ODF and OPG (Fig. 1) .

Mature osteoclasts are multinucleated cells having a diameter of about 50 to 100 fi . They have a morphological property of ruffled surface and function to resorb calcified bone matrix (Boskey A. L., J. Cell. Biochem. Suppl., 30-31, 83-91, 1998). The mature osteoclasts adhere to the surface of the bone matrix and secret proteinase and acid into a sealing zone between their cell membrane and the bone matrix. Bones are destructed by the proteinase and acid.

The novel peptide PI of the present invention is capable of inhibiting osteoclast differentiation and comprises certain regions of amino acid sequences of cecropin A and magainin 2. Specifically, the peptide Pi comprises the amino acid sequence of NH₂- (amino acid residues 1 to 8 of cecropin A) - (Pro) - (amino acid residues 4 to 7 of magainin 2) - (Leu) - (amino acid residues 9 to 12 of magainin 2)-C00H. Preferably, the peptide PI of the present invention has the amino acid sequence of SEQ ID NO: 3 : NH₂-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Pro-Lys-Phe-Leu-His- Leu-Ala-Lys-Lys-Phe-COOH.

The following abbreviations are used herein: Ala: alanine; Arg: arginine; Cys: cysteine; lie: isoleucine; Gly: glycine; Leu: leucine; Lys: lysine; Orn: ornithine; Phe: phenylalanine; Pro: proline; Trp: tryptophan; Tyr: tyrosine; Val: valine; His: histidine; Gin: gluta ine; Thr: threonine; Met: methionine; Asp: aspartic acid. The cecropin which constitutes the N-terminal of the peptides according to the present invention is a family of antibacterial peptides isolated from Cecropia moth (Boman, H. G. and Steiner, H., Current Topics In Microbiology and Immunology, 94/95, 75-91, 1981) . Cecropin A, B and D have been isolated and purified from immune hemolymph and their amino acid sequences have been identified (Steiner, et al . , Nature, 292, 246-248 (1981); Hultmaker, D., European J. Biochem., 127, 207-217, 1982; U.S. Pat. No. 4,355,104). Cecropin A is a 37-residue linear polypeptide isolated from pupae of Hyalophora cecropia (Hultmark et al., Eur. J. Biochem., 106, 7-16, 1980). Cecropin A is used as an antibacterial agent and selectively acts on the cytoplasmic membrane of bacteria. Steiner reported that cecropin polypeptide binds to negatively charged membrane lipids of bacteria so that the cell membrane is rendered permeable, thereby killing bacteria (Steiner, et al., Nature, 292, 246-248, 1981) .

The magainin, which makes up the C-terminal of the peptide according to the present invention, was isolated from the African clawed frog Xenopus laevis and has demonstrated broad activity against a variety of pathogens including gram negative bacteria, gram positive bacteria, protozoa, fungi and parasites (Soravia et al., Febs. Lett., 15, 228, 337-340, 1998) . The magainin exists in two active forms, i.e. magainin 1 and magainin 2. The magainin 2 is a linear peptide consisting of 23 amino acid residues. It is effective in a small amount due to its highly selective toxicity, but it exhibits a very low toxicity to. mammalian cells. In recent, it has been reported that magainin polypeptide has an anti-cancer effect and that it destroys cells by increasing permeability of the cell membrane, i.e., osmotic pressure.

The peptide of the present invention includes functional homologues of NH₂- (amino acid residues 1 to 8 of cecropin A) - (Pro) - (amino acid residues 4 to 7 of magainin 2) - (Leu) - (amino acid residues 9 to 12 of magainin 2)-C00H, specifically, functional homologues of peptide of SEQ ID NO: 1, and salts thereof. The "functional homologues" refer to peptides which comprise an amino acid sequence that has at least 70% sequence homology, preferably at least 80% sequence homology and most preferably at least 90% sequence homology to the PI peptide shown in SEQ ID NO: 3, through addition, substitution or deletion of amino acids and which show a physiological activity substantially equivalent to that of the peptide PI. The "functional homologues" of the present invention include derivatives of the peptide PI with the chemical structure being partially modified but the basic backbone and the osteoclast differentiation-inhibiting activity being conserved. Examples of structural modifications are those affecting stability, storability, volatility or solubility of the PI peptide.

The peptide of the present invention has an ability to inhibit osteoclast differentiation. An obstacle for studying osteoclast differentiation is that cell lines possessing the action of osteoclast cannot be prepared. Therefore, a co-culture system of bone marrow cells and osteoblasts is commonly used as a differentiation model system of osteoclasts. It has been found in this co- culture system that the differentiation amount of osteoclasts into ononucleated and multinucleated osteoclasts when osteoclasts were treated with the peptide PI is significantly lower than that when osteoclasts were not treated with the peptide PI (Fig. 2) . The differentiation was measured at 2nM, 4nM and 20nM of PI peptideare. The results reveal that the ability of the peptide PI to inhibit osteoclast differentiation is proportional to the concentration of the peptide PI . Since the peptide PI may inhibit osteoclast differentiation through a mechanism in which the peptide PI acts on osteoblasts to interfere with production of ODF, the present inventors isolated only the bone marrow cells from the co-culture system of Example 3, treated bone marrow cells with ODF, and then measured the ability of the peptide PI to inhibit osteoclast differentiation, Likewise, it was shown that the peptide PI inhibited osteoclast differentiation in dose-dependent manner (Fig. 3) .

The present inventors also isolated osteoclast precursor cells from bones, treated osteoclast precursor cells with ODF, and then measured the ability of the peptide PI to inhibit osteoclast differentiation. As a result, it was found that the peptide Pi inhibited both differentiation of osteoclasts derived from the bone marrow cells and differentiation of osteoclast precursor cells derived from the bone marrow cells.

Differentiation and proliferation of cells involve complex signal transduction mechanisms. In connection with this, MAP kinase has been studied extensively. The present inventors have examined the effects of the peptide Pi on

JNK, a mamber of MAP kinase family. After Osteoclasts were obtained from the co-culture system, they were treated with PI, ODF or TNF-α, alone, or a combination of PI and ODF. The results reveal that the treatment of osteoclasts with PI or TNF-α, alone, did not affect the activity of JNK. On the other hand, the treatment of osteoclasts with the combination of peptide Pi and ODF, an osteoclast differentiation-inducing factor, inhibits the activity of JNK (Fig. 9) . This suggests that the peptide PI potently prevent JNK from being activated by ODF. NF-κB is the most important nuclear transcription factor for osteoclast differentiation, and is activated by ODF. It has been reported that NF-κB knock-out mouse models completely lack osteoclast differentiation and, as a result, suffers from osteopetrosis. The peptide PI of the present invention also inhibits activation of NF- B by ODF (Fig. 10) .

The present invention provides nucleic acid molecules encoding the peptide PI. The nucleic acid molecules of the present invention include a group of nucleic acid sequences encoding the amino acid sequence of NH₂- (amino acid residues 1 to 8 of cecropin A) - (Pro) - (amino acid residues 4 to 7 of magainin 2) - (Leu) - (amino acid residues 9 to 12 of magainin 2)-COOH. Preferably, the nucleic acid molecule of the present invention has the nucleic acid sequence encoding the peptide having the amino acid sequence of SEQ ID NO: 3. The nucleic acid molecules of the present invention can be chemically synthesized by techniques well known to those of skill in the art.

The present invention provides an isolated peptide PI and a method for preparing the same. The peptide of the present invention may be chemically synthesized by techniques well known to those of skill in the art (Creighton, Proteins: Structures and Molecular Principles, W. H. Freeman and Co., N.Y., 1983) . The peptide may be prepared using conventional step-wise solution or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (Chemical Approaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Floria, 1997; A Practical Approach, Atherton & Sheppard, Eds., IRL Press, Oxford, England, 1989).

Particularly, a preferred method for producing the peptide PI employs a solid phase synthesis. The peptide PI may be chemically synthesized by a condensation reaction between protected amino acids according to a conventional solid phase method. The condensation reaction is conducted by attaching the C-terminal amino acid of the sequence shown in SEQ ID NO: 3 to an insoluble support followed by sequential addition of the remaining amino acids in the sequence. After completion of the condensation reaction, both the protective groups and the carrier to which the C- terminal amino acid residue has been linked can be removed by known methods such as acid decomposition and aminolysis. The aforementioned peptide synthesis method is described in detail in various pertinent literatures (e.g., Gross and Meienhofer's, The Peptides, Vol 2., Academic Press, 1980). The solid phase carriers for the peptide synthesis may be selected from those commonly used in the art. Specific examples of such carriers are polystyrene resins of substituted benzyl type, polystyrene resins of hydroxymethylphenylacetic amide type, substituted benzhydrylpolystyrene resins, and polyacrylamide resins having functional groups capable of binding to the peptide. Also, amino acid condensation may be carried out by conventional methods, for example, dicyclohexylcarbodiimide (DDC) , acid anhydride, and activated ester methods.

The protective groups of the starting protected amino acids are those which are commonly used in conventional peptide synthesis methods and can be readily removed by conventional means such as acid decomposition, reduction or aminolysis. Specific examples of the amino protective group are formyl; trifluoroacetyl; benzyloxycarbonyl; substituted benzyloxycarbonyls such as (ortho- or para-) chlorobenzyloxycarbonyl and (ortho- or para-) bromobenzyloxycarbonyl; and aliphatic oxycarbonyl such as t-butoxycarbonyl and t-amyloxycarbonyl . A carboxyl group of an amino acid can be protected by esterification reaction. As the ester group, there may be mentioned benzyl ester; substituted benzyl ester such as methoxybenzyl ester; alkyl esters such as cyclohexyl ester, cycloheptyl ester or t-butyl ester; and the like. The guanidino group does not require a protective group, but may be protected by nitro; or arylsulfonyl such as tosyl, methoxybenzenesulfonyl or methylenesulfonyl. The protective group .for imidazole includes tosyl, benzyl, and dinitrophenyl . The indole group of tryptophan may be non- protected or protected by formyl and the like.

The deprotection of the resulting peptide and the detachment of the resulting peptide from the carrier can be conducted using anhydrous hydrogen fluoride in the presence of various scavengers. Examples of the scavengers include anisole, ortho-, metha- or para-cresol, dimethylsulfide, thiocresol, ethanediol, and mercaptopyridine, which are all conventionally used in. peptide synthesis methods .

The peptides of the present invention can be isolated and purified by methods well known to those of skill in the art, for example, extraction, recrystallization, various chromatographies including gel-filtration, ion-exchange, precipitation, adsorption, and reverse phase, electrophoresis, counter current distribution and the like. The reverse-phase high performance liquid chromatography is most efficious. The solid phase method is advantageous in that the intermediate products obtained from respective steps need not to be isolated and purified, because they are attached to beads. By using the foregoing methods, it is possible to synthesize the 18-mer peptide of the present invention with a high purity at a high yield.

Alternatively, the peptides of the present invention can be prepared by a genetic engineering method using the nucleic acid sequences coding for the peptide PI. The method involves constructing an expression vector comprising a nucleic acid molecule encoding the peptide PI, expressing the expression vector in a suitable host cell and isolating the peptide from the host cell culture. Such genetic engineering methods for producing a polypeptide are well-known to those skilled in the art (Maniatis et al . ,

Molecular cloning: A laboratory Manual, Cold Spring Harbor Laboratory, 1982; Sambrook et al . , supra; Gene Expression Technology, Methods in Enzymology, Genetics and Molecular Biology, Methods in Enzymology, Guthrie & Fink (eds.), Academic Press, San Diego, Calif., 1991; Hitzeman et al., J. Biol. Chem., 255, 12073-12080, 1980).

The present invention provides a pharmaceutical composition for the treatment of metabolic bone disease, toothpaste or mouthwash comprising the isolated and purified PI peptide, and salt and functional homologues thereof, and the use thereof. Examples of metabolic bone disease include osteoporosis, bone metastatic lesions in which tumors such as breast or prostate carcinomas have been transmitted to bone, tumors primarily .generated in bone (for example, multiple myeloma) , rheumatoid or degenerative arthritis, periodontal disease having the alveolar bone destroyed by bacteria, inflammatory alveolar bone resorption disease caused by dental implants, inflammatory bone resorption disease caused by bony implants in orthopedic surgery, Paget's disease caused by various genetic factors and the like.

The pharmaceutical composition for treatment of the present invention can be prepared into various formulations and administered ^'through many routes. For example, it can be prepared by mixing the peptide of the present invention with carriers commonly used in pharmaceutical field. For oral administration, the composition can be formulated in a variety of forms such as tablet, troche, capsule, elixir, suspension, syrup, and wafer using binder, lubricant, disintegrant, excipient, stabilizer, suspending agent, colorant and/or flavor. For injection, the composition can be used in combination with preservative, analgesics, solubilizer and/or stabilizer. For topical application, it can be prepared by mixing with base, excipient, lubricant and preservative. As a therapeutic agent for periodontal disease, forms of slow release formulation or dental implant formulation coated with delayed delivery substance can be used.

The pharmaceutical composition prepared as described above can be administered orally, intravenously, subcutaneously, intranasaly or intraperitoneally. Other dosage forms can be prepared by techniques commonly used in the art. The dose can be suitably selected depending on absorption degree of the active ingredient in the body, inactivation rate and elimination rate, the age, sex and conditions of the patient, and severity of disease to be treated. The pharmaceutically effective amount of the peptide of the present invention is generally from about 25 g to about 5 mg per kg _Qf weight. A suitable dose size is typically from about 0.1 m£ to about 5 m0, although it varies with patients.

The present invention provides a method for treating metabolic bone disease by administering the pharmaceutical composition of the present invention to the patient suffering from said disease. The pharmaceutical composition of the present invention is effective in the treatment of various diseases associated with over-action of osteoclasts. The diseases include osteoporosis bone metastatic lesions in which tumors such as breast or prostate carcinomas have been transmitted to bone, tumors primarily generated in bone (for example, multiple myeloma), rheumatoid or degenerative arthritis, periodontal disease having the alveolar bone destroyed by bacteria, inflammatory alveolar bone resorption disease caused by dental implants, inflammatory bone resorption disease caused by bony implants in orthopedic surgery, Paget's disease caused by various genetic factors and the like. The peptides of the present invention did not exhibit cytotoxicity in toxicity tests using osteoblasts, bone marrow cells, abdominal acrophage cells, and 293T cells, the human embryonic kidney cell line.

Now, the present invention will be explained in detail by the following examples. However, the examples are given for purposes of illustration and are not intended to be limiting of the present invention.

Example 1

Preparation of peptide PI The chemically synthesized peptide PI of the present invention consists of 18 amino acids as follows:

N-terminus- (amino acid residues 1 to 8 of cecropin

A) - (Pro) - (amino acid residues 4 to 7 of magainin 2 ) - (Leu) -(amino acid residues 9 to 12 of magainin 2 ) -

COOH.

Cecropin A has the following amino acid sequence (SEQ ID NO: 1) :

NH₂-Lys-Trp-lys-Leu-Phe-Lys-Lys-Ile-Glu-Lys-Val-Gly- Gln-Asn-Ile-Arg-Asp-Gly-Ile-Ile-Gly-Ala-Gly-Pro-Ala- Val-Ala-Val-Val-Gly-Gln-Ala-Thr-Gln-Ile-Ala-Lys-COOH.

Magainin 2 has the following amino acid sequence (SEQ ID NO : 2 ) :

NH₂-Gly-Ile-Gly-Lys-Phe-Leu-His-Ser-Ala-Lys-Lys-Phe- Gly-Lys-Ala-Phe-Val-Gly-Glu-Ile-Met-Asn-Ser-COOH .

Peptide PI of the present invention has the following amino acid sequence (SEQ ID NO: 3) :

NH₂-Lys-Trp-Lys-Leu-Phe-Lys-Lys-Ile-Pro-Lys-Phe-Leu- His-Leu-Ala-Lys-Lys-Phe-COOH.

The solid phase synthesis method was carried out as described above, and then the products obtained thereby were purified by reverse-phase high performance liquid chromatography to afford the peptide PI of the present invention having the amino acid sequence of SEQ ID NO: 3. The resulting synthetic peptide was confirmed by mass spectrometry.

Example 2

Cell culture

Primary osteoblasts, bone marrow cells, and osteoclast precursor cells were separately cultured in α- MEM (α-minimum essential medium, Gibo BRL) supplemented with 10% (v/v) fetal bovine serum (Gibco BRL) -and antibiotics (Gibco, BRL) containing IX penicillin and streptomycin.

Example 3 Inhibitory effect of peptide Pi on osteoclast differentiation: Co-culture of bone marrow cells and osteoblasts

Because there is no cell line in which osteoclast functions have been conserved, it. is very difficult to study the osteoclast differentiation. The osteoclasts are derived from hematopoietic stem cells which have their origin in the bone marrow and are differentiated with the aid of osteoblasts/basal cells. As such, a co-culture system of bone marrow cells and osteoblasts is commonly used as a differentiation model system of osteoclasts. In this example, bone marrow cells and osteoblasts were co- cultured to construct a system in which the bone marrow cells would be differentiated into osteoclasts. The system was used to examine whether the peptide Pi of the present invention could inhibit osteoclast differentiation.

1) Isolation of osteoblasts

A one-day old ICR mouse was sacrificed and washed in 70% ethanol for disinfection. The cranial bone was separated and cut into pieces using scissors and tweezers and collected in a 6 cm culture dish containing 3X HBSS . 0.1% collagenase (Gibco BRL) and 0.2% dispase (Boehringer Mannheim) were added to the dish. Then, the dish was treated 5 times in an incubator at 37^°C, each time for 15 minutes. After the second treatment, cells were collected and centrifuged (1,600 rpm, 5 minutes) to obtain osteoblasts. The cells were adjusted to the number of about 1 to 2 X 10⁵ and transferred to a 10 cm culture dish containing 15 ml of α-MEM with 10% FBS, followed by cultivation for 3 days . After cultivation, the culture was dispensed in a freezing vial and stored frozen in a nitrogen tank until use in the co-culture experiment.

2) Isolation of bone marrow cells A 6 to 7-week old ICR female mouse was sacrificed by neck torsion. A rear leg part was disinfected with 70% ethanol and the tibia was aseptically separated. The tibia was put in 3X HBSS (Gibco BRL) to neatly remove soft tissues. The tibia was cut at both ends and IX α-MEM was injected into the bone marrow using lcc syringe to obtain bone marrow cells, which were pipetted several times to ensure detachment of cells. Then, the suspension was centrifuged (1600 rpm, 5 minutes) and the supernatant was removed to obtain cell elements (bone marrow cells and hematocytes) which were precipitated at the bottom. The cells were treated with about 15 to 20 ml of ACK buffer solution (155 mM NH₄C1, 11 mM KHC0₃, 0.01 mM EDTA) for 2 minutes and phosphate buffer solution was added to dissolve hematocytes while minimizing damage to bone marrow cells. Then, the solution was centrifuged (1,600 rpm, 5 minutes) and the cells were resuspended in 10% α-MEM.

3) Co-culture

The bone marrow cells and osteoblasts isolated and cultured as above were co-cultured. The respective cells were placed in a 48 well plate containing α-MEM with 10% FBS at densities of 2 X 10⁵ and 2 X 10⁴ per well, respectively, and co-cultured. The culture of each well was treated with vitamin D3 (10^~8) , PGE 2 (10^~6) and then peptide PI at concentrations of 20nM, 4nM and 2nM. Control was not treated with peptide PI . After 3 days of cultivation, when the medium was exchanged with fresh medium, vitamin D3 (10^~8) , PGE 2 (10^~6) and peptide PI were also added in the same manner as above. After 6 days of cultivation, the culture medium was removed from the wells containing osteoclasts which were completely differentiated and the remaining cells were treated with 10% formalin for 5 minutes to fix the cells. Formalin was removed and 0.1% Triton X-100 was added for 10 minutes. The Triton X-100 solution was then drained and the cells were stained for 5 minutes by the TRAP (tartrate- resistant acid phosphatase) staining method. The TRAP staining method was performed using Leukocyte Acid Phosphatase Kit (Sigma, Cat. No. 387-A) . The TRAP staining solution was removed and the cells were washed twice with distilled water and dried. The number of TRAP-positive osteoclasts counted under an optical microscope (X100) . Here, the osteoclasts were classified into multinucleated osteoclasts having 10 or more -nuclei and mononucleated osteoclasts having less than 10 nuclei and the number of each kind was counted.

As -a result, it was shown that the number of the TRAP positive cells in the control group was 314166.9 mononucleated osteoclasts and 157.6+52.3 multinucleated osteoclasts . On the other hand, in the group treated with 20nM the peptide PI of the present invention, no osteoclasts were detected, in the group treated with 4nM the peptide PI of the present invention, the numbers of the mononucleated osteoclasts and the multinucleated osteoclasts were 168.3154.15 and 80.6138.7, respectively, and in the group treated with 2nM the peptide PI of the present invention, the numbers of the mononucleated osteoclasts and the multinucleated osteoclasts were 175.2517.1 and 83.2519.18, respectively (Fig. 2). Therefore, it was noted that the peptide PI dose- dependently inhibited differentiation of both mononucleated osteoclasts and multinucleated osteoclasts upon co- culturing of bone marrow cells with osteoblasts.

Example 4

Inhibitory effect of peptide PI on osteoclast differentiation: Culture of bone marrow cells

In this example, only bone marrow cells were isolated, treated with recombinant protein ODF, and then measured for the ability of the peptide PI to inhibit differentiation, since the peptide PI might have affected osteoblasts, thereby indirectly inhibiting differentiation of osteoclasts in the co-culture experiment of bone marrow cells and osteoblasts performed in Example 3. Bone marrow cells isolated by the same method as in Example 3 were placed in a 48 well plate containing α-MEM with 10% FBS at densities of 4 X 10⁵ per well and cultured. The culture were treated with ODF (50 ng/m^β) , M-CSF (30 ng/m^β) and then, peptide PI at concentrations of 20nM, 4nM and 2nM. After 3 days of cultivation, when the culture medium was exchanged with fresh medium, ODF, M-CSF and the peptide Pi were also added in the same manner as above. After 6 days of cultivation, the culture medium was exchanged in the same manner as above and the culture was further performed for 2 days . The osteoclasts which were completely differentiated were confirmed by the TRAP (tartrate-resistant acid phosphatase) staining method. The TRAP staining method was performed using Leukocyte Acid Phosphatase Kit (Sigma, Cat. No. 387-A) . The culture medium was removed from the wells containing osteoclasts which were completely differentiated and treated with 10% formalin for 5 minutes to fix the cells. Formalin was removed and 0.1% Triton X-100 was added for 10 minutes. The Triton X-100 solution was then drained and the cells were treated with the TRAP (tartrate- resistant acid phosphatase) staining solution contained in the kit for 5 minutes. The TRAP staining solution was removed and the cells were washed twice with distilled water and dried. The number of TRAP-positive osteoclasts was counted under an optical microscope (X100) .

As a result, mononucleated osteoclasts were observed, unlike the co-culture in Example 3 and the culture of osteoclast precursor cells in Example 5. For the control group, the number of the TRAP positive cells was found to be 453167.5. On the other hand, in the group treated with 20nM the peptide Pi of the present invention, the number of the osteoclasts was 255117.52 and in the group treated with 4nM and 2nM the peptide PI of the present invention, the numbers of the mononucleated osteoclasts were 274.3135.79 and 383145.9, respectively (Fig. 3). Therefore, it was noted that the peptide PI dose-dependently inhibited differentiation of both mononucleated osteoclasts and multinucleated osteoclasts during the differentiation process of osteoclasts by the treatment of ODF in bone marrow cells.

Example 5

Inhibitory effect of peptide PI on osteoclast differentiation: Inhibitory effect of peptide PI on differentiation of osteoclast precursor cells

The osteoclast precursors derived from bone marrow cells ultimately move to bones, in which they become osteoclast precursor cells and then further differentiate to be active osteoclasts with a function of resorption. Thus, the osteoclast precursor cells existing in the bone are different from osteoclasts in their differentiation step. Recently, ODF, also called OPGL or RANKL, a very important cytokine in differentiation of osteoclasts, has been identified and a method for producing its recombinant protein has become available. Therefore, it is easy to achieve the differentiation of osteoclasts in primary culture.

Osteoclast precursor cells isolated from bone were treated with ODF recombinant protein and used to investigate the inhibitory effect of the synthetic peptide PI on differentiation of the osteoclast precursor cells at this state. The osteoclast precursor cells were isolated from a long bone .

A 6 to 7-week old ICR female mouse was sacrificed by neck torsion. A rear leg part was disinfected with 70% ethanol and the tibia and femur were aseptically separated. Soft tissues were removed from the separated tibia and femur by the same method as in Example 2. 3X HBSS (Gibco BRL) was injected into the bone marrow several times to remove bone marrow cells. The bones were cut into small pieces using operating scissors and collected in a culture dish containing an enzyme solution containing collagenase (1 mg/m£ collagenase type II, 0.05% trypsin, 4 mM EDTA, Gibco BRL) , followed by treatment 5 times in an incubator at 37^°C , each time for 15 minutes. After the third treatment, the bone pieces were finely cut once more. After completion of the enzyme treatment, the culture was treated 5 times with 3X HBSS, centrifuged to completely remove the enzyme solution, suspended in cold α-MEM and left on ice for 15 minutes. The suspension was vortexed for 1 minute, mixed with an equal volume of cold α-MEM and left on ice for 15 minutes. The resulting suspension was passed through a sterilized sieve to obtain only cell elements, which were cultured in α-MEM containing 10% FBS. The isolated and cultured osteoclast precursor cells were placed in a 48 well plate containing α-MEM with 10% FBS at densities of 0.5xlO^δ per well and cultured. At this time, the culture of each well was treated with ODF (100 ng/m^β), M-CSF (30 ng/m^β) and then peptide Pi at concentrations of 20nM, 4nM and 2hM. Control was not treated with peptide PI. After 3 days of cultivation, when the medium was exchanged with fresh medium, ODF, M-CSF and peptide PI were also added in the same manner as above. After 6 days of cultivation, the culture medium was changed in the same manner as above and at the 9th day of cultivation the TRAP staining was performed to count the number of TRAP-positive osteoclasts under an optical microscope (X100) . Here, the osteoclasts were classified into multinucleated osteoclasts having 10 or more nuclei and mononucleated osteoclast having less than 10 nuclei and the number of each kind was counted.

As a result, it was shown that the number of the TRAP positive cells in the control group was 224148.5 mononucleated osteoclasts and 97.6617.57 multinucleated osteoclasts . On the other hand, in the group treated with 20nM the peptide PI of the present invention, no osteoclasts were detected, in the group treated with 4nM the peptide PI of the present invention, the numbers of the mononucleated osteoclasts and the multinucleated osteoclasts were 126.3122.66 and 40.45111.59, respectively, and in the group treated with 2nM the peptide PI of the present invention, the numbers of the mononucleated osteoclasts and the multinucleated osteoclasts were 121.33126.66 and 53.15110.12, respectively (Fig. 4). Therefore, it was noted that the peptide PI dose- dependently inhibited differentiation of both mononucleated osteoclasts and multinucleated osteoclasts in the differentiation of osteoclast precursor cells isolated from bone.

Example 6

Cytotoxicity test of peptide PI

In order to confirm whether the peptide PI is toxic to cells, the MTT (3- [4, 5-dimethylthiazol-2-yl] -2, 5- diphenyltetrazolium bromide) assay was performed for various cells.

In the MTT assay, osteoblasts isolated in Example 3, mouse bone marrow cells, abdominal macrophages and 293T cells, the human embryonic kidney cell line were used. Respective cells were seeded in a 96 well plate at densities of 1.5 X 10⁴, 1 X 10⁵, 1 X 10⁵ and 1.5 X 10⁴ per well, respectively. The cells were treated with the peptide PI at concentrations of 20nM, 4nM and 2nM, and cultured. After 24 hours , 50 f& of the MTT assay solution (a mixture of 1 m£ of an MTT labeling reagent and 0.2 m& of an electron coupling reagent) was added to each well. After 4 hour-culture at room temperature, absorbance was measured at a wavelength of 450 and 630 n using an ELISA reader (Bio-Tek Instrument, Winooski, VT) . The above- described procedure was repeated 3 times.

As a result, the peptide Pi did not show toxicity in any of osteoblasts, bone marrow cells, abdominal macrophages or 293T cells (Fig. 5 to Fig. 8) .

Example 7

Effect of peptide PI on activity of JNK According to the report that MAP kinases are involved in differentiation and proliferation of cells, the JNK kinase assay was performed to investigate the effect of the peptide PI on activity of c-Jun NH₂-terminal kinase (JNK), a kind of MAP kinase.

The osteoblasts and bone marrow cells of Example 3 were plated at a density of 1 X 10⁶ and 1 X 10⁷, respectively, in a collagen gel-coated culture dish and co- cultured in the presence of α-MEM containing 10% FBS. At this time, vitamin D3 (10^~8) and PGE 2 (10^"6) were added to the culture of each well. After 3 days, the medium was changed and then cultured for another 3 days. All the cells were harvested by treatment with 0.2% collagenase, re-plated on a 6-well culture plate and cultured again. On the next day, osteoblasts were then removed by treatment with 0.1% collagenase to obtain pure osteoclasts.

The pure osteoclasts were treated with the peptide PI (20nM) , ODF (500 ng/m£) and TNF (100 ng/m^β) . Here, about one hour later, after the osteoblasts were removed, the osteoclasts were pre-treated with PI for 30 minutes, treated with ODF or TNF for 30 minutes, rinsed in PBS and collected. The collected cells were lysed in lysis buffer and total proteins were quantified. According to the kinase assay, anti-JNK antibody was added to protein A agarose beads (Pierce) and rocked for 1 hour at 4^°C. The beads were washed twice with the buffer solution, centrifuged (3000 rpm, 4 minutes) , added to the solution of lysed osteoclasts and rocked overnight. After rocking, the beads were washed twice with the lysis buffer solution and centrifuged (3000 rpm, 4 minutes) . Then, the beads were washed twice with the kinase assay buffer solution, centrifuged (3000 rpm, 4 minutes) , treated with a kinase reactive mixture (γ³²P-ATP-l/t#, 1 mM ATP-1.5^, c-Jun-1 tg mixed with IX kinase buffer to a final volume of 10/t^β ) and reacted at 30°C for 30 minutes. 15 βi of 3X sample buffer solution was added thereto, boiled for 5 minutes, and subjected to electrophoresis and the gel was dried. For quantitative analysis of respective bands formed on the gel, an amount of a binding radioactive isotope (cpm) was measured using the Phosphoimager (BAS 1500, Fuji film) . As a result, it was noted that the treatment with peptide PI alone or TNF-α alone did not affect the activity of JNK in the differentiation of osteoclasts. On the other hand, it was confirmed that the treatment of the peptide PI in combination with ODF, a factor inducing differentiation of osteoclasts, impeded the activity of JNK. This suggests that the peptide PI strongly inhibits the activation of JNK by ODF. Therefore, it is appreciated that the peptide PI is involved in the signal transduction pathway of JNK (Fig. 9) .

Example 8

Effect of peptide PI on nuclear transcription factor NF-KB

In this example, effects of the peptide PI on the activity of NF-κB, a nuclear transcription factor involved in differentiation of osteoclasts, were investigated. The inhibitory effect of the peptide PI on the nuclear transcription factor NF-κB was measured by performing the EMSA (Electrophoresis Mobility Shift Assay) . Osteoclasts prepared as in Example 6 were cultured in a hypotonic lysis buffer solution (10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 10 mM KC1, 0.5 mM DTT, 0.5 mM PMSF) on ice for 10 minutes. The culture was transferred to a microcentrifuge tube and NP-40 was added to 0.1%. The tube was incubated for 15 minutes with intermittent shaking. The culture medium was centrifuged (4000 rpm, 15 minutes) and the cell mass was mixed with 15 β& of a high salt buffer solution (20 mM HEPES, pH 7.9, 420 mM NaCl, 25% glycerol, 1.5 mM

MgCl₂, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) and incubated on ice for 20 minutes. 75 ≠ of a storage buffer solution (20 mM HEPES, pH 7.9, 100 mM NaCl, 20% glycerol, 0.2 mM EDTA, 0.5 mM PMSF, 0.5 mM DTT) was added to the culture, stirred for 10 seconds and centrifuged (14,000 rpm, 20 minutes) . The supernatant was removed and quantified for protein. The quantification of protein was performed using the DC Protein Assay Kit (Bio-Rad) .

NF-KB site binding oligomer (5^f- AGTTGAGGGGACTTTCCCAGGC-3' , Santa Cruz) was labeled with γ- P32 ATP and Klenow enzyme to prepare a probe. 10 MS of the protein labeled probe was mixed with 20 β^Q> of a reaction buffer solution (10 mM Tris-HCl, 50 mM KC1, 1 mM EDTA, 5% glycerol, 2 mM DTT) containing l^g of poly(dldC) to about 20,000 cpm and reacted at room temperature for 30 minutes.

Protein binding to DNA was subjected to electrophoresis on

4 to 5% polyacrylamide gel and the gel was dried and analyzed by the magnetic radiation method.

As a result, it was noted that the peptide PI inhibited the activation of the nuclear transcription factor NF-KB by the osteoclast differentiation-inducing factor ODF (Fig. 10) .

As described above, it will be readily apparent to those skilled in the art to which the present invention belongs that the present invention may be realized into other embodiments without changing the technical spirit or indispensable features of the invention. In connection with this, it should be understood that the foregoing examples and experimental examples are offered to illustrate this invention and are not to be construed in any way as limiting the scope of this invention. Thus, it is intended that the -present invention covers all the modifications and variations of the present invention derived from the meanings and scope of the appended claims and their homologues rather than the foregoing detailed description.

The peptides of the present invention have the ability to inhibit osteoclast differentiation. Therefore, the peptides of the present invention are effective in the treatment of metabolic bone diseases. Additionally, the peptides of the present invention may be useful for research and study of molecular biological mechanisms in which osteoclasts are differentiated and osteoclast differentiation is inhibited.

Claims

What is claimed is:

1. A peptide capable of inhibiting osteoclast differentiation wherein said peptide comprises an amino acid sequence that has at least 70% sequence homology to the peptide PI having the amino acid sequence of SEQ ID NO: 3.

2. A peptide capable of inhibiting osteoclast differentiation wherein said peptide comprises an amino acid sequence having contiguous residues 1 to 8 of cecropin A, an amino acid sequence having contiguous residues 4 to 7 of magainin 2, and an amino acid sequence having contiguous residues 9 to 12 of magainin 2, and wherein said peptide has at least 70% amino acid sequence homology to the peptide PI having the amino acid sequence of SEQ ID NO: 3.

3. A peptide PI having the amino acid sequence of SEQ ID NO: 3 and salts thereof.

4. A nucleic acid molecule encoding the peptide PI having the amino acid sequence of SEQ ID NO: 3.

5. A method for producing the peptide according to any one of claims 1 to 3.

6. The method of claim 5, wherein the method is performed by a solid phase synthesis.

7. A pharmaceutical composition for the treatment or prevention of metabolic bone disease comprising the peptide defined in any one of claims 1 to 3 as an active ingredient.

8. The pharmaceutical composition of claim 7, wherein the metabolic bone disease is selected from bone metastatic lesions in which tumors such as breast or prostate carcinomas have been transmitted to bone, tumors primarily generated in bone (for example, multiple myeloma) , rheumatoid or degenerative arthritis, periodontal disease having the alveolar bone destroyed by bacteria, inflammatory alveolar bone resorption disease caused by dental implants, inflammatory bone resorption disease caused by bony implants in orthopedic surgery, and Paget's disease caused by various genetic factors.

9. The pharmaceutical composition of claim 7, which is a therapeutic preparation for periodontal disease in a form of slow release formulation or dental implant formulation coated with delayed delivery substance.

10. A toothpaste or mouthwash comprising the peptide of any one of claims 1 to 3.