TECHNICAL FIELD

This application claims priority to and the benefit of Korean Patent Application No. 10-2013-0086939 filed in the Korean Intellectual Property Office on 23 Jul. 2013, the entire contents of which are incorporated herein by reference.

The present invention relates to a peptide for inhibiting osteoclast differentiation and a use thereof.

BACKGROUND ART

Bone homeostasis and the skeletal structure are maintained by organized activity between osteoclasts for bone resorption and osteoblasts for bone formation. Osteoclasts, which are multinucleated cells, are differentiated from hematopoietic stem cells [T. Miyamoto, O. Ohneda, F. Arai, et al., Blood 98 (2001) 2544-2554]. The differentiation into osteoclasts is regulated by RANK ligand that is secreted from osteoblasts and activated T lymphocytes [Y. Y. Kong, U. Feige, I. Sarosi, et al., Nature 402 (1999) 304-309].

The receptor activator of nuclear factor kappa-B ligand (RANKL) binds to RANK, which is a receptor that is present in the osteoclast precursor, and osteoclast differentiation is induced in the presence of macrophage-colony stimulating factor (M-CSF). RANKL activates signaling pathways that regulate the formation of osteoclasts and the absorption of bones [J. Li, I. Sarosi, X. Q. Yan, et al., Proc. Natl. Acad. Sci. USA 97 (2000) 1566-1571]. RANK has no tyrosine kinase activity, and induces signaling through adaptor proteins, known as TNF receptor-associated factors (TRAFs) [L. Galibert, M. E. Tometsko, D. M. Anderson, et al., J. Biol. Chem. 273 (1998) 34120-34127]. TRAF6, which is a part of the intercellular molecule, plays a key role in the generation of osteoclasts and activates various downstream signals [M. A. Lomaga, W. C. Yeh, I. Sarosi, et al., Genes Dev. 13 (1999) 1015-1024]. TRAF6 binds to cytoplasmic domains of RANK, and then activates NF-κB and activator protein-1 (AP-1) [S. L. Teitelbaum, J. Clin. Invest. 114 (2004) 463-465].

Tumor necrosis factor-α (TNF-α), which is TNF ligand-based protein, is secreted in several types of cells including monocytes/macrophages or osteoclasts, and induces a number of biological responses through two cell-surface receptors termed TNFR1 and TNFR2 (also called TNFR p55 and TNFR p75, respectively). Both TNFR1 and TNFR2 induce intracellular signals that can stimulate the proteolytic breakdown of kappa B (IκB), a cytoplasmic inhibitor of NF-κB [Verma, I. M., Stevenson, J. K., Schwarz, E. M., Van Antwerp, D., and Miyamoto, S. (1995) Genes Dev. 9, 2723-2735].

TNF-α modulates a varied range of responses, such as inflammation, immune modulation, cell proliferation and differentiation, and apoptosis [Ledgerwood, E. C., Pober, J. S., and Bradley, J. R. (1999) Lab. Invest. 79, 1041-1050]. TNF-α also promotes bone resorption in vitro and in vivo [Bertolini, D. R., Nedwin, G. E., Bringman, T. S., Smith, D. D., and Mundy, G. R. (1986) Nature 319, 516-518], and can induce the secretion of RANKL in osteoblasts [Hofbauer, L. C., Lacey, D. L., Dunstan, C. R., Spelsberg, T. C., Riggs, B. L., and Khosla, S. (1999) Bone 25, 255-259]. In addition, TNF-α is crucial to the pathogenesis of the bone and joint destructions that occur in rheumatoid arthritis, and has been implicated in the bone loss inperiodontitis, orthopedic implant loosening, and other forms of chronic inflammatory osteolysis. TNF-α is mediated by lipopolysaccharide-stimulated osteoclasts [Abu-Amer, Y., Ross, F. P., Edwards, J., and Teitelbaum, S. L. (1997) J. Clin. Invest. 100, 1557-1565]. TNF-α plays an important role in estrogen deficiency-induced bone loss in postmenopausal osteoporosis [Cenci, S., Weitzmann, M. N., Roggia, C., Mamba, N., Novack, D., Woodring, J., and Pacifici, R. (2000) J. Clin. Invest. 106, 1229-1237].

Interleukin-3 (IL-3), which is the cytokine secreted mainly by activated T lymphocytes, may be used as a connection link between the immune system and the hematopoietic stem cell system [J. W. Schrader, Interleukin-3, in: A. W. Thomson, M. T. Lotze (Eds.), Academic Press, London, UK, 2003, pp. 201-225]. IL-3 directly acts on mouse osteoclast precursors, and promotes cell differentiation into macrophages, thereby inhibiting the RANKL-induced osteoclast differentiation [S. M. Knapli, L. S. Mangashetti, S. D. Yogesha, M. R. Wani, J. Immunol. 171 (2003) 142-151]. In the osteoclast precursor, IL-3 inhibits the phosphorylation and degradation of IκB, thereby preventing the nuclear translocation of NF-κB, which is induced by RANKL. In addition, IL-3 inhibits RANKL-induced c-Jun N-terminal kinase (JNK) activity, and down-regulates the expression of transcriptional factors, c-Fos and NFATc1. IL-3 inhibits the expression of RANK at the post-transcriptional stage, and this procedure was confirmed to be irreversible, through the in vivo experiment using mice.

Throughout the entire specification, many papers and patent documents are referenced and their citations are represented. The disclosure of the cited papers and patent documents are entirely incorporated by reference into the present specification, and the level of the technical field within which the present invention falls, and the details of the present invention are explained more clearly.

DETAILED DESCRIPTION OF THE INVENTION

Technical Problem

The present inventors have endeavored to develop a material that has excellent activity and stability compared with naturally occurring interleukin-3 (IL-3) protein while retaining the same or similar functions to IL-3. As a result, the present inventors have selected an IL-3-derived peptide having excellent physiological activity (e.g., osteoclast differentiation inhibitory ability, osteogenic differentiation promoting ability, etc.) among a lot of peptide candidates, and have then completed the present invention.

Therefore, an aspect of the present invention is to provide a peptide including an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

Another aspect of the present invention is to provide a pharmaceutical composition for alleviating or treating bone diseases.

Still another aspect of the present invention is to provide a composition for promoting osteogenic differentiation.

Still another aspect of the present invention is to provide a method for alleviating or treating bone diseases.

Still another aspect of the present invention is to provide a method for promoting osteogenic differentiation.

Other purposes and advantages of the present disclosure will become more obvious with the following detailed description of the invention, claims, and drawings.

Technical Solution

In accordance with an aspect of the present invention, there is provided a peptide including an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The present inventors have endeavored to develop a material that has excellent activity and stability compared with naturally occurring interleukin-3 (IL-3) protein while retaining the same or similar functions to IL-3. As a result, the present inventors have selected an IL-3-derived peptide having excellent physiological activity (e.g., osteoclast differentiation inhibitory ability, osteogenic differentiation promoting ability, etc.) among a lot of peptide candidates.

The present inventors synthesized a plurality of IL-3-derived peptides that exhibit the above-described characteristics, based on moieties involved in functions of human IL-3. More specifically, the peptide of the present invention was selectively prepared by arbitrarily partial-synthesizing several moieties of IL-3 protein to first search for moieties which is bindable to receptor proteins and then optimizing amino acid sequences of the predicted moieties, and then, of these candidate peptides, the peptides having the most excellent activity were screened, and thus the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention were prepared.

According to some embodiments, the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 of the present invention were derived from human IL-3 (GenBank Accession Number, AAH66275.1; SEQ ID NO: 3), which are shown in table 1. The peptides of SEQ ID NO: 1 and SEQ ID NO 2 of the present invention have activity, such as growth factors, by performing similar functions to naturally occurring IL-3 to bind to receptors thereof.

The peptides of the present invention did not only have strong osteoclast differentiation inhibitory ability in a dose-dependent manner, but also significantly promoted osteoblast differentiation (seeFIGS. 3 to 8).

According to a certain embodiment of the present invention, the peptides of the present invention inhibit the receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK signaling pathway.

The peptides of the present invention remarkably inhibited RANKL-mediated NF-κB activation and nuclear translocation (seeFIGS. 7aand 7b).

According to a certain embodiment of the present invention, the peptides of the present invention inhibit the RANKL- or inflammatory cytokine-induced osteoclast differentiation, and the RANKL- or inflammatory cytokine-induced expressions of tartrate-resistant acid phosphatase (TRAP), cathepsin K, or type 1 or type 2 TNF receptor.

According to a certain embodiment of the present invention, the foregoing inflammatory cytokines include tumor necrosis factor-α(TNF-α), macrophage colony-stimulating factor (M-CSF), interleukin-1β (IL-1β), IL-6, and IL-7; more specifically, TNF-α, IL-1β, and IL-6; still more specifically, TNF-α, and IL-1β; and most specifically, TNF-α.

The peptides of the present invention can promote osteoblast differentiation.

According to a certain embodiment of the present invention, the peptides of the present invention promote the expressions of osteoblast differentiation markers, such as osteocalcin (OCN), osteoprotegerin (OPG), bone sialoprotein (BSP), and osteopontin (OPN).

As used herein, the term “peptide” refers to a molecule in which amino acid residues bind to each other via a peptide linkage. The peptides of the present invention may be prepared by, for example, the solid-phase synthetic method (solid-phase synthesis techniques; Merrifield,J. Amer. Chem. Soc.85:2149-54(1963); Stewart, et al.,Solid Phase Peptide Synthesis,2nd. ed., Pierce Chem. Co.: Rockford, 111(1984)) or the liquid-phase synthetic method (U.S. Pat. No. 5,516,891).

The peptides of the present invention per se have excellent stability compared with naturally occurring IL-3 protein, but the stability thereof can be further improved through amino acid modification (seeFIGS. 2aand 2b).

According to the present invention, the peptides of the present invention have very excellent thermal stability compared with naturally occurring IL-3 protein. The naturally occurring IL-3 protein has low stability in long-term storage and temperature, is difficult to prepare, and has a high production cost. However, the peptides of the present invention can be mass-produced very cheaply, can prevent the deterioration in bioactivity as much as possible due to physiochemical stability thereof at high temperatures, and has further improved therapeutic effects by increasing the remaining period in vivo and in vitro. Therefore, the peptides of the present invention can be favorably applied to products that require long-term storage, such as medicines, quasi-medicines, and cosmetics.

According to a certain embodiment of the present invention, a protective group, which is selected from the group consisting of an acetyl group, a fluorenyl methoxy carbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, and polyethylene glycol (PEG), may be linked to the N- or C-terminal of the peptides.

The foregoing amino acid modification significantly improves the stability of the peptides of the present invention. As used herein, the terra “stability” refers to storage stability (e.g., room-temperature stability) as well as “in vivo” stability. The foregoing protective group protects the peptides of the present invention from the attack of in vivo protein cleavage enzymes.

In accordance with another aspect of the present invention, there is provided a pharmaceutical composition for alleviating or treating bone diseases, the composition containing the foregoing peptide as an active ingredient.

In accordance with still another aspect of the present invention, there is provided a composition for promoting osteogenic differentiation, the composition containing the peptide as an active ingredient.

Since the composition of the present invention contains the foregoing IL-3-related peptides of the present invention as an active ingredient, descriptions of overlapping contents between the two are omitted to avoid excessive complication of the specification due to repetitive descriptions thereof.

As used herein, the terra “bone diseases” refer to diseases, disorders, or conditions associated with RANKL-mediated signaling, and include diseases, disorders, conditions related to the regulation of bone formation and resorption, as well as bone mass reductive disorders, including the reduction of bone mass, osteoporosis, and osteolysis.

According to a certain embodiment of the present invention, the bone diseases, which can be alleviated or treated by the pharmaceutical composition of the present invention, include osteoporosis, childhood osteoporosis, osteogenesis imperfecta, osteomalacia, bone necrosis, rickets, osteomyelitis, alveolar bone loss, Paget's disease, hypercalcemia, primary hyperparathyroidism, metastatic bone diseases, myeloma, bone loss in rheumatoid arthritis, bone loss resulting from cancers, fibrous dysplasia, aplastic bone disease, metabolic bone diseases, or bone loss with age, but are not limited thereto.

The composition of the present invention may be used as a pharmaceutical composition containing: (a) a pharmaceutically effective amount of the foregoing peptide exhibiting IL-3 protein activity, of the present invention; and (b) a pharmaceutically acceptable salt.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to attain efficacy or activity of the foregoing IL-3-related peptide.

The pharmaceutically acceptable carrier contained in the pharmaceutical composition of the present invention is conventionally used for the formulation, and examples thereof may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like, in addition to the above ingredients. Suitable pharmaceutically acceptable carriers and preparations are described in detail inRemington's Pharmaceutical Sciences(19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and examples of the parenteral administration may include intravenous, subcutaneous, intramuscular, intraperitoneal, local, and transdermal injections.

The appropriate dose of the pharmaceutical composition of the present invention varies depending on factors, such as the formulating method, manner of administration, patient's age, body weight, gender, morbidity, and food, time of administration, route of administration, excretion rate, and response sensitivity. An ordinarily skilled practitioner can easily determine and prescribe the dose that is effective for the desired treatment or prevention. According to a preferable embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0005-1,000 mg/kg.

In addition, the pharmaceutical composition of the present invention is formulated in a unit dosage form or into a multidose container, using a pharmaceutically acceptable carrier and/or excipient according to the method that is easily conducted by a person having an ordinary skill in the art to which the present invention pertains. Here, the dosage form may be a solution in an oily or aqueous medium, a suspension, an emulsion, an extract, a powder, granules, a tablet, a capsule, or a gel (e.g., a hydrogel), and may further include a dispersant or a stabilizer.

The composition of the present invention may be used as a cosmetic composition containing: (a) a cosmetically effective amount of the foregoing IL-3-derived peptide; and (b) a cosmetically acceptable carrier. As used herein, the term “cosmetically effective amount” refers to an amount that is sufficient to attain the efficacy of the composition of the present invention described above.

The cosmetic composition of the present invention may be formulated into any dosage form that is conventionally prepared, and examples thereof may include a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing, an oil, a powder foundation, an emulsion foundation, a wax foundation, and a spray, but are not limited thereto. More specifically, the cosmetic composition of the present invention may be prepared in a dosage form of an emollient lotion, nutritional emulsion, nutritional cream, message cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, facial pack, spray or powder.

In cases where the dosage form of the present invention is a paste, a cream, or a gel, the carrier component thereof may include animal fibers, vegetable fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide.

In cases where the dosage of the present invention is a powder or a spray, the carrier component thereof may include lactose, talc, silica, aluminum hydroxide, calcium silicate, or a polyamide powder. In cases where the dosage form of the present invention is especially a spray, the dosage form may additionally include a propellant, such as, chlorofluorohydrocarbon, propane/butane, or dimethyl ether.

In cases where the dosage form of the present invention is a solution or an emulsion, the carrier component thereof may include a solvent, a solubilizer, or an emulsifier, for example water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty esters, polyethylene glycol, or fatty acid esters of sorbitan.

In cases where the dosage form of the present invention is a suspension, the carrier component thereof may include liquid diluents, such as water, ethanol, and propylene glycol; suspending agents, such as, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester; microcrystalline cellulose; aluminum metal hydroxide; bentonite; agar; or tragacanth.

In cases where the dosage form of the present invention is a surfactant-containing cleansing, the carrier component thereof may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isothinate, imidazolium derivatives, methyl taurate, sarcosinate, fatty acid amide ether sulfate, alkyl amido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanoline derivatives, or ethoxylated glycerol fatty acid ester.

The components contained in the cosmetic composition of the present invention include compositions that are commonly used in the cosmetic composition, in addition to the peptides, as active ingredients, and the carrier component thereof, and for example, may include common aids, such as an antioxidant, a stabilizer, a solubilizer, vitamins, a pigment, and a flavor.

In accordance with another aspect of the present invention, there is provided a method for alleviating or treating bone diseases, the method including administering to a subject a composition containing the foregoing peptide as an active ingredient.

In accordance with still another aspect of the present invention, there is provided a method for promoting osteogenic differentiation, the method including contacting cells with a composition containing the foregoing peptide as an active ingredient.

Since the method of the present invention uses the above-described composition, descriptions of overlapping contents between the two are omitted to avoid excessive complication of the specification due to repetitive descriptions thereof.

Advantageous Effects

Features and advantages of the present invention are summarized as follows:

(i) The peptides of the present invention can exert the same or similar functions to naturally occurring interleukin-3 (IL-3), and have very excellent skin penetration due to a small size thereof.

(ii) The peptides of the present invention inhibit the activation and nuclear translocation of NF-κB through the inhibition of the receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK signaling pathway, and inhibit RANKL- or inflammatory cytokine-induced expression of tartrate-resistant acid phosphatase (TRAP), cathepsin K, or type 1 or type 2 TNF receptor, thereby inhibiting osteoclast differentiation, in a concentration-dependent manner.

(iii) Further, the peptides of the present invention can contribute to osteoblast differentiation by promoting the expressions of osteoblast differentiation markers, such as osteocalcin (OCN), osteoprotegerin (OPG), bone sialoprotein (BSP), and osteopontin (OPN).

(iv) Therefore, excellent activity and stability of the peptide of the present invention can be very favorably applied to medicines, quasi-medicines, and cosmetics.

