Peptide for suppressing osteoclast differentiation and use thereof

Abstract

The peptide of the present invention performs a function, which is the same as or similar to that of natural interleukin (IL)-3, and has superior skin permeability due to the small size thereof. In addition, the peptide of the present invention suppresses the activation of NF-κB and nuclear transition by inhibiting the receptor activator of nuclear factor kappa-B ligand (RANKL)-RANK signaling pathway, and suppresses the expression of a RANKL or an inflammatory cytokine-induced tartrate-resistant acid phosphatase (TRAP), cathepsin K, or TNF receptor type 1 or type 2, thereby inhibiting osteoclast differentiation depending on the treatment concentration. Moreover, the peptide of the present invention can contribute to osteoblast differentiation by promoting the expression of osteoblast differentiation markers such as osteocalcin (OCN), osteoprotegerin (OPG), bone sialoprotein (BSP), or osteopontin (OPN). Therefore, the superior activity and stability of the peptide of the present invention are useful for medicines, sanitary aids, or cosmetics.

Claims

1. A method for alleviating or treating a bone disease in a subject suffering from a bone disease characterized by excessive bone resorption or insufficient bone formation, the method comprising administering to the subject a composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 2, wherein the bone disease is characterized by excessive bone loss or insufficient bone formation.

2. The method of claim 1, wherein the bone disease is selected from the group consisting of 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 diseases, metabolic bone diseases, and bone loss with age.

3. A method for promoting osteogenic differentiation, the method comprising contacting cells with a composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 2.

4. The method of claim 3, wherein the composition promotes the expression of osteocalcin (OCN), osteoprotegerin (OPG), bone sialoprotein (BSP) or osteopontin (OPN).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 illustrates graphs showing high-performance liquid-phase chromatography assay results of peptides of SEQ ID NO: 1 (FIG. 1a) and SEQ ID NO: 2 (FIG. 1b) prepared by synthetic example 1 of the present invention.

(2) FIG. 2 illustrates graphs showing stability test results of peptides of SEQ ID NO: 1 (FIG. 2a) and SEQ ID NO: 2 (FIG. 2b) prepared by synthetic example 1 of the present invention.

(3) FIG. 3 illustrates osteoclast differentiation inhibitory effects by the treatment with a peptide of SEQ ID NO: 1 (FIG. 3a) and a peptide of SEQ ID NO: 2 (FIG. 3b), prepared by synthetic example of the present invention.

(4) FIG. 4 illustrates the inhibitory effect on the osteoclast differentiation promoting enzyme by the treatment with a peptide of SEQ ID NO: 1 (FIG. 4a) and a peptide of SEQ ID NO: 2 (FIG. 4b), prepared by synthetic example of the present invention.

(5) FIG. 5 illustrates in the inhibitory effects on the mRNA of osteoclast differentiation markers, TRAP and Cathepsin, by a peptide of SEQ ID NO: 1 (FIG. 5a) and a peptide of SEQ ID NO: 2 (FIG. 5b), prepared by synthetic example of the present invention.

(6) FIG. 6 illustrates the inhibitory effects on the mRNA of type 1 and type 2 TNF receptors by the treatment with a peptide of SEQ ID NO: 1 (FIG. 6a) and a peptide of SEQ ID NO: 2 (FIG. 6b), prepared by synthetic example of the present invention.

(7) FIG. 7 illustrates RANKL signal inhibitory effects by the treatment with a peptide of SEQ ID NO: 1 (FIG. 7a) and a peptide of SEQ ID NO: 2 (FIG. 7b), prepared by synthetic example of the present invention.

(8) FIG. 8 illustrates osteoblast differentiation promotion results by the treatment with a peptide of SEQ ID NO: 1 (FIG. 8a) and a peptide of SEQ ID NO: 2 (FIG. 8b), prepared by synthetic example of the present invention.

(9) FIG. 9 illustrates that the treatment with a peptide of SEQ ID NO: 1 (FIG. 9a) and a peptide of SEQ ID NO: 2 (FIG. 9b), prepared by synthetic example of the present invention, increased the expression of p-Smad1/5/8, which is a BMP2 signal.

MODE FOR CARRYING OUT THE INVENTION

(10) Hereinafter, the present invention will be described in detail with reference to examples. These examples are only for illustrating the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

Synthetic Example 1

Synthesis of Asn-Cys-Ser-Asn-Met-Ile-Cys-Glu-Ile-Ile-Thr-His (SEQ ID NO: 1)

(11) To 700 mg of chloro trityl chloride resin (CTL resin, Nova Biochem Cat No. 01-64-0021) introduced into a reactor was added 10 ml of methylene chloride (MC), followed by stirring for 3 minutes. After removing the solution, 10 ml of dimethylform amide (DMF) was added, followed by stirring for 3 minutes, and then the solvent was again removed. 10 ml of a dichloromethane (DCM) solution was put into the reactor, and 200 mmole Fmoc-L-His(Trt)-OH (Bachem, Swiss) and 400 mmole diisopropyl ethylamine (DIEA) were added, after which the mixture was well dissolved with stirring, and then the reaction was conducted with stirring for 1 hour. After the reaction, the resultant material was washed, and methanol and DIEA (2:1) were dissolved in DCM, followed by a reaction for 10 minutes, and then the resultant material was washed with an excessive amount of DCM/DMF (1:1). After removing the solution, 10 ml of DMF was added, followed by stirring for 3 minutes, and then the solvent was again removed. 10 ml of a deprotection solution (20% piperidine/DMF) was put in the reactor, followed by stirring at room temperature for 10 minutes, and then the solution was removed. The equal amount of a deprotection solution was added, and then the reaction was again maintained for 10 minutes, followed by removal of the solution. The resultant material was washed twice with DMF, once with MC, and once with DMF, for 3 minutes each, thereby preparing His(Trt)-CTL resin. 10 ml of a DMF solution was put in a new reactor, and 200 mmol Fmoc-Thr(tBu)-OH (Bachem, Swiss), 200 mmol HoBt 200 mmole, and 200 mmole Bop were added, and the mixture was dissolved well through stirring. 400 mmole N,N-diisopropylethylamine (DIEA) was divisionally put twice into the reactor, and then the stirring was conducted for at least 5 minutes until all solids were dissolved. The dissolved amino acid mixed solution was put in the reactor containing the deprotected resin, followed by a reaction with stirring at room temperature for 1 hour. After removing the reaction liquid, the stirring was conducted using a DMF solution three times for 5 minutes each, followed by removal. A small amount of the reacted resin was taken to check the reaction degree by Kaiser test (Ninhydrin test). Using the deprotection solution, the deprotection reaction was conducted twice in the same manner as described above, to yield Thr(tBu)-His-(Trt)-CTL resin. After sufficient washing with DMF and MC, Kaiser test was again conducted, and then the following amino acid attachment test was conducted in the same manner as described above. Based on the selected amino acid sequence, the chain reaction was conducted in the order of Fmoc-Ile, Fmoc-Ile, Fmoc-Glu(OtBu), Fmoc-Cys(Trt), Fmoc-Ile, Fmoc-Met, Fmoc-Asn(Trt), Fmoc-Ser(tBu), Fmoc-Cys(Trt), and Fmoc-Asn(Trt). The Fmoc-protective group was removed by reacting twice with the deprotection solution for 10 min each and then conducting washing well. Acetic anhydride, DIEA, and hydroxy benzotriazole (HoBt) were added to conduct acetylation for 1 hour, and then the prepared peptidyl resin was washed three times sequentially with DMF, MC, and methanol, dried under the flow of nitrogen gas, and completely dried by vacuum-drying under phosphorus pentoxide (P.sub.2O.sub.5). 30 ml of a leaving solution [95% trifluoroacetic acid (TFA), 2.5% distilled water, and 2.5% thioanisole] was added, and the reaction was maintained for 2 hours while the mixture was intermittently stirred at room temperature. The resin was filtered, washed with a small amount of a solution, and then mixed with stock solution. The distillation was conducted under reduced pressure to reduce the total volume by half, and then 50 ml of cold ether was added to induce precipitation. Thereafter, the precipitates were collected by centrifugation, followed by washing twice with cold ether. After the stock solution was removed, followed by sufficient drying under nitrogen atmosphere, thereby synthesizing 0.5 g of unpurified peptide 1, NH.sub.2-Asn-Cys-Ser-Asn-Met-Ile-Cys-Glu-Ile-Ile-Thr-His-OH (SEQ ID NO:1) (yield: 89.9%). The molecular weight was determined as 1375.4 Da (theoretical value: 1377.6 Da) by using a molecular weight analysis system (FIG. 1a). The peptide of SEQ ID NO: 2 (NH.sub.2-Arg-Arg-Lys-Leu-Thr-Phe-Tyr-Leu-Lys-Thr-Leu-Glu-OH) was also synthesized by the same method (yield: 92.1%). The molecular weight was determined as 1568.5 Da (theoretical value: 1567.9 Da) by using a molecular weight analysis system (FIG. 1b).

(12) TABLE-US-00001 TABLE 1 Sequences and Molecular Weights  of Synthesized Peptides Analysis value (Mass spectrometer) Analytic Theoretical No. Amino acid sequence value value IL- Asn-Cys-Ser-Asn-Met-Ile- 1375.4 1377.6 3-1 Cys-Giu-Ile-Ile-Thr-His (SEQ ID NO: 1) IL- Arg-Arg-Lys-Leu-Thr-Phe- 1568.5 1567.9 3-2 Tyr-Leu-Lys-Thr-Leu-Giu (SEQ ID NO: 2)

Test Example 1

Thermal Stability of Prepared Peptides

(13) 0.1 mg/ml phosphate buffer solution was prepared from the peptide of SEQ ID NO: 1 or SEQ ID NO: 2, synthesized in synthetic example 1, and standard growth factor (IL-3) purchased from NIBSC (UK). 1 ml of the prepared solution was placed in each glass vial, and allowed to stand at 37° C. The solution standing at 37° C. was sampled on days 0, 1, 3, 5, 10, 20, and 40, and centrifuged for each day to remove denatured peptides or proteins. The supernatant was taken, and quantification using HPLC was conducted (FIGS. 2a and 2b, respectively).

Test Example 2

Verification on Osteoclast Differentiation Inhibitory Effects Using Synthetic Peptides

(14) In order to analyze the IL-3-like action and the inhibitory action of the peptides of SEQ ID NO: 1 and SEQ ID NO: 2, synthesized in synthetic example 1, TRAP staining was conducted using Raw264.7 strain differentiable into osteoclasts while referring to methods, such as tartrate-resistant acid phosphatase staining (Rizzino, et al. Cancer Res. 48:4266(1988)).

(15) Raw264.7 cell lines (ATCC) were cultured in Duibecco's modified Eagle's medium (DMEM, Gibco, U.S.A.) supplemented with 10% fetal bovine serum (FBS, Sigma) using each 250 ml-flask for tissue culture. The cultured cell lines were carefully detached from the bottom of the culture container using a pipette, followed by centrifugation, to obtain only cell precipitates. The cell precipitates were re-suspended in DMEM culture medium supplemented with 10% FBS, and then added to a 46-well plate for tissue culture plate at 1×10.sup.4 cells per each well. For the induction of differentiation, the blank sample and RAW264.7 cells were treated with 10 ng/ml RANKL, 50 ng/ml TNF-α, and 1 μg/ml or 10 μg/ml synthesized peptides dissolved in 10% distilled water in a sterile state, and then cultured under 5% CO.sub.2 at 37° C. for 72 hours. After 72 hours, the medium was changed with the same culture liquid, and then the blank sample and the cells were treated with 10 ng/ml RANKL, 50 ng/ml TNF-α, and 1 μg/ml or 10 μg/ml synthesized peptides, and then cultured under the same conditions for 48 hours. After the culturing was completed for a total of five days, the upper layer was removed. For cell fixation, a fixation buffer containing 25 ml of a citrate solution, 65 ml of acetone, and 8 ml of 37% formaldehyde was prepared. The cells were fixed with the fixation buffer for 30 seconds, and then washed three times with phosphate buffer saline (PBS). The washing solution was removed, and the cells were stained with the leukocyte alkaline phosphatase kit (Sigma, U.S.A.).

(16) FIG. 3 illustrates the inhibitory results of the differentiation of Raw264.7 cells, of which the differentiation can be induced by RANKL and TNF-α, by IL-3-1 (FIG. 3a) and IL-3-2 (FIG. 3b). As shown in FIGS. 3a and 3b, the peptides of the present invention can inhibit the RANKL- and TNF-α-induced differentiation of Raw264.7 into osteoclasts.

Test Example 3

Verification on Osteoclast Differentiation Inhibitory Effects Using Synthetic Peptides

(17) Raw264.7 cells were treated in combination with 10 ng/ml RANKL, 50 ng/ml TNF-α, or the 1 or 10 μg/ml peptide synthesized in synthetic example 1 to induce differentiation for five days, and then the inhibition degree of the activity of TRAP, an osteoclast differentiation marker, was tested. After culture, the culture liquid was removed, and then 100 μl of a lysis buffer (20 mM tris buffer, 3% triton X-100) was added to disrupt cell walls. A reaction solution is prepared from 500 μl of a citrate solution (18 mM citric acid, 9 mM sodium chloride, 12 mM surfactant; pH 3.5), 50 μl of a tartrate solution, and 500 μl of a 20 mM phosphate substrate. 100 μl of the lysate and 100 μl of the reaction solution were added in the same amount, and then the reaction was conducted at 37° C. for 30 minutes. For colorimetric analysis, the absorbance was determined at 405 nm using a spectrophotometer. It was validated that, in cases where the treatment with the peptide of SEQ ID NO: 1 or SEQ ID NO: 2 together with RANKL and TNF-α, the peptides inhibited the activity of TRAP, which is an osteoclast differentiation marker, in a concentration-dependent manner (FIGS. 4a and 4b).

Test Example 4

Verification on Inhibitory Effect on Osteoclast Differentiation Marker mRAN Using Synthetic Peptides

(18) Raw264.7 cells were treated in combination with 10 ng/ml RANKL, 50 ng/ml TNF-α, or the 1 or 10 pg/ml peptide synthesized in synthetic example 1 to induce differentiation for five days, and then the inhibition degree of the cathepsin K mRNA expression was analyzed. FIGS. 5a and 5b show that the mRNA levels of cathepsin K were reduced by the treatment with the peptides of the present invention, respectively. In addition, FIGS. 6a and 6b verifies that the mRNA expression levels of type 1 and type 2 TNF receptors, which have been increased by the treatment with RANKL or TNF-α, were reduced by the treatment with all the peptides of the present invention. Target-specific primer sequences used in PCR were as follows: TRAP forward primer sequence, 5′-AAATCACTCTTTAAGAACAG-3′ (SEQ ID NO:3) and TRAP reverse primer sequence, 5′-TTATTGAATAGCAGTGACAG-3′ (SEQ ID NO:4) (annealing temperature, 45° C.); cathepsin K forward primer sequence, 5′-CCTCTCTTGGTGTCCATACA-3′ (SEQ ID NO:5) and cathepsin K reverse primer sequence, 5′-ATCTCTCTGTACCCTCTGCA-3′ (SEQ ID NO:6) (annealing temperature, 53° C.); GAPDH forward primer sequence, 5′-GGTGTGAACGGATTTGGCCGTATTG-3′ (SEQ ID NO:7) and GAPDH reverse primer sequence, 5′-CCGTTGAATTTGCCGTGAGTGGAGT-3′ (SEQ ID NO:8) (annealing temperature, 55° C.); type 1 TNF receptor forward primer sequence, 5′-acctttacggcttcccagaa-3′ (SEQ ID NO:9) and type 1 TNF receptor reverse primer sequence, 5′-tccttacagccacacaccgt-3′ (SEQ ID NO:10) (annealing temperature, 55° C.); type 2 TNF receptor forward primer sequence, 5′-aggctggaaagcccctaact-3′ (SEQ ID NO:11) and type 2 TNF receptor reverse primer sequence, 5′-atgggggtactggagacagg-3′ (SEQ ID NO:12) (annealing temperature, 55° C.); and actin forward primer sequence, 5′-CGTGGGCCGCCCTAGGCA-3′ (SEQ ID NO:13) and actin reverse primer sequence, 5′-TTGGCTTAGGGTTCAGGGGG-3′ (SEQ ID NO:14) (annealing temperature, 55° C.).

(19) Therefore, the peptides of the present invention can inhibit all of TRAP, cathepsin K, and type 1 and type 2 TNF receptors, which are increased by RANKL and TNF-α (FIGS. 5a and 5b and FIGS. 6a and 6b).

Test Example 5

Verification on RANKL Signal Inhibition by Synthetic Peptides

(20) Raw264.7 cells were treated with the peptides synthesized in synthetic example 1, and after 30 minutes, the nuclear translocation of NF-κB, which is a representative signal of RANKL protein, was checked. The effect of each peptide was verified through western blotting using polyclonal antibody to NF-κB (Cat. No sc-372, SantaCruz, USA). The treatment with the peptides of the present invention confirmed the activation and nuclear translocation of NE-κB (FIGS. 7a and 7b). The phosphorylation of c-Jun, which is another representative signal of RANKL protein, and the nuclear translocation of the phosphorylated c-Jun were confirmed. The effect of each peptide was verified through western blotting using polyclonal antibodies to phospho-c-Jun (Cat. No sc-1694, SantaCruz, USA) (FIGS. 7a and 7b).

(21) Considering the test results of test examples 1 to 5, the peptides of the present invention inhibit osteoclast differentiation very effectively through the inhibition of RANKL-RANK signaling activation.

Test Example 6

Verification on Promotion of Osteogenic Differentiation Marker Gene by Synthetic Peptides

(22) MC3T3-E1 cells were treated with 10 or 50 pg/ml of the peptides synthesized in synthetic example 1 to induce the differentiation for two days, and tests for verifying the mRNA expression of osteoblast differentiation markers, such as osteocalcin (OCN), osteoprotegerin (OPG), bone sialoprotein (BSP), and osteopontin (OPN), were conducted. Target-specific primer sequences used in PCR were as follows: OCN forward primer sequence, 5′-gcgctctgtctctctgacct-3′ (SEQ ID NO:15) and OCN reverse primer sequence, 5′-tttgtaggcggtcttcaagc-3′ (SEQ ID NO:16) (annealing temperature, 60° C.); OPG forward primer sequence, 5′-ctgcctgggaagaagatcag-3′ (SEQ ID NO:17) and OPG reverse primer sequence, 5′-ttgtgaagctgtgcaggaac-3′ (SEQ ID NO:18) (annealing temperature, 60° C.); BSP forward primer sequence, 5′-aaagtgaaggaaagcgacga-3′ (SEQ ID NO:19) and BSP reverse primer sequence, 5′-gttccttctgcacctgcttc-3′ (SEQ ID NO:20) (annealing temperature, 60° C.); OPN forward primer sequence, 5′-GATGAATCTGACGAATCTCAC-3′ (SEQ ID NO:21) and OPN reverse primer sequence, 5′-CTGCTTAATCCTCACTAACAC-3′ (SEQ ID NO:22) (annealing temperature, 50° C.). FIG. 8 shows that, when the cells were treated with the peptides of the present invention, the mRNA levels of the genes, which are the osteoblast differentiation markers, were increased by the peptides of SEQ ID NO: 1 or SEQ ID NO: 2. Therefore, the peptides of the present invention can promote osteoblast differentiation by increasing the genetic expressions of OCN, OPG, BSP, and OPN, which are osteoblast differentiation markers.

Test Example 7

Verification on Promotion of Osteogenic Differentiation Signal by Synthetic Peptides

(23) In order to verify whether pSmad1/5/8, which is a signal involved in osteogenic differentiation, was activated by the present peptides, MC3T3-E1 cells were dispensed into 6-well plates at 2×10.sup.5 cells, and then cultured for 24 hours under the conditions of 37° C. and 5% CO.sub.2. After 24 hours, the medium was changed with a serum-free medium, and then the cells were starved for 24 hours, treated with the 10 μg/ml peptides or 50 ng/ml BMP2, used as a positive control, for 30 minutes, washed with PBS, and dissolved in a lysis buffer, thereby obtaining proteins, which were then subjected to western blotting.

(24) In accordance with the foregoing results, it was confirmed that the treatment with the peptides of SEQ ID NO: 1 or SEQ ID NO: 2 of the present invention led to the phosphorylation of Smad1/5/8, and this means that the bone formation promoting signal is transmitted due to the treatment with the peptides of the present invention, so that the osteoblast differentiation is maintained (FIGS. 9a and 9b, respectively).

Formulation Example 1

Emollient Lotion

(25) Emollient lotion, which includes nanosomes containing peptide 1 or 2, prepared in synthetic example 1, and has the following composition, was prepared by a general skin lotion preparation method.

(26) TABLE-US-00002 TABLE 2 Emollient Lotion Composition Component Content (wt %) peptide nanosome 0.001 1,3-butylene glycol 6.0 glycerin 4.0 PEG 1500 1.0 sodium hyaluronate 1.0 polysolvate 20 0.5 ethanol 8.0 preservative, coloring Suitable benzophenone-9 0.05 aroma Small purified water balanced Total 100

Formulation Example 2

Moisturizing Cream

(27) Nutritional cream, which includes nanosomes containing peptide 1 or 2, prepared in synthetic example 1, and has the following composition, was prepared by a general moisturizing cream preparation method.

(28) TABLE-US-00003 TABLE 3 Moisturizing Cream Composition Component Content (wt %) peptide nanosome 0.001 meadowfoam oil 3.0 cetearyl alcohol 1.5 stearic acid 1.5 glyceryl stearate 1.5 Liquid paraffin 10.0 wax 2.0 Polysolvate 60 0.6 sorbitan sesquioleate 2.5 squalane 3.0 1,3-butylene glycol 3.0 glycerin 5.0 triethanolamine 0.5 tocopheryl acetate 0.5 preservative, coloring suitable aroma suitable purified water blanced Total 100

Formulation Example 3

Moisturizing Skin Lotion

(29) Nutritional skin lotion, which includes nanosomes containing peptide 1 or 2, prepared in synthetic example 1, and has the following composition, was prepared by a general skin lotion preparation method.

(30) TABLE-US-00004 TABLE 4 Moisturizing Skin Lotion Composition Component Content (wt %) peptide nanosome 0.002 1,3-butylene glycol 4.0 glycerin 4.0 stearyl alcohol 0.8 glyceryl stearate 1.0 Triethanolamine 0.13 tocopheryl acetate 0.3 liquid paraffin 5.0 squalane 3.0 macadamia nut oil 2.0 Polysolvate 60 1.5 sorbitan sesquioleate 0.5 carboxy vinly polymer 1.0 preservative, coloring suitable aroma suitable purified water blanced Total 100

Formulation Example 4

Essence

(31) Essence, which includes nanosomes containing peptide 1 or 2, prepared in synthetic example 1, and has the following composition, was prepared by a general essence preparation method.

(32) TABLE-US-00005 TABLE 5 Essence Composition Component Content (wt %) peptide nanosome 0.005 glycerin 10.0 1,3-butylene glycol 5.0 PEG 1500 2.0 allantoin 0.1 DL-panthenol 0.3 EDTA-2Na 0.02 hydroxyethyl cellulose 0.1 sodium hyaluronate 8.0 carboxy vinyl polymer 0.2 triethanolamine 0.18 octyldodeces-16 0.4 ethanol 6.0 aroma, preservative, coloring purified water Total 100

(33) Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for one embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.