Neuropeptide Y fragment capable of releasing hematopoietic stem cells into blood and treating osteoporosis

Abstract

The present invention relates to a novel peptide exhibiting an effect of releasing myelopoiesis stem cells into blood and an osteoporosis therapeutic effect and use thereof and, in particularly, to a novel peptide consisting of an amino acid sequence of SEQ. ID.NO:1 which has effects of releasing hematopoietic stem cells into a bloodstream and decreasing osteoclast cells in bone narrow, and a pharmaceutical composition comprising the novel peptide as an active ingredient for preventing or treating neutropenia, anemia or osteoporosis. Because of side effects, the peptide of the present invention not only increases level of leukocytes, red blood cells and platelets in blood, but also alleviates a decrease in bone density, and therefore, can be very usefully used for the development of a prophylactic or therapeutic agent for neutropenia, anemia or osteoporosis.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein at least one amino acid selected from the group consisting of the fifth and sixth amino acids of the amino acid sequence of SEQ ID NO: 1 is D-type.

2. The peptide according to claim 1, wherein the fifth and sixth amino acids of the amino acid sequence of SEQ ID NO: 1 are D-type.

3. A polynucleotide encoding the peptide of claim 1.

4. A composition comprising the peptide of claim 1 as an active ingredient.

5. The composition of claim 4, wherein the composition is a pharmaceutical composition or a food composition.

6. A method for treating any one disease selected from the group consisting of neutropenia, anemia and osteoporosis in a subject, the method comprising administering an effective amount of a composition to a subject in need thereof, wherein the composition comprises the peptide of claim 1 as an active ingredient.

7. The method of claim 6, wherein the neutropenia may be due to any one or more causes selected from the group consisting of radiation, alcoholism, drugs, allergic diseases, aplastic anemia, autoimmune diseases, T-γ lymphocyte proliferative diseases (T-γ) LPD), myelodysplasia, myeloid fibrosis, dysgammaglobulinemia, paroxysmal nocturnal hemoglobinuria, cancer, vitamin B12 deficiency, folate deficiency, viral infection, bacterial infection, spleen disease, hemodialysis, or transplantation, leukemia, myeloma, lymphoma, metastatic solid tumors that infiltrate and replace the bone marrow, toxins, bone marrow failure, Schwarzmann-Diamond syndrome, cartilage-hair dysfunction, congenital dyskeratosis and type IB glycogen storage disease.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

(2) FIGS. 1A to 1D are a result showing the change in the expression levels of the adhesion factors after treating the three kinds of recombinant peptides, NPY(21-28), NPY(24-31), and NPY(29-36), to osteoblasts that express adhesion factors of myelopoiesis stem cells (1A: summary of the experiment, 1B to 1D: a graph showing the expression levels of adhesion factors with fold change relative to the control group).

(3) FIG. 2A is a summary of the experiment performed to determine the effect of NPY (21-28) on the osteoporosis relief through releasing of myelopoiesis stem cells into blood, and FIG. 2B is the result showing the weight change after administration of each peptide to the animal models of osteoporosis (Sham: control animal models with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(4) FIGS. 3A to 3C are a result showing the expression levels of adhesion factors of hematopoietic stem cell in bone marrow with fold change compared to the control group after administration of each peptide to the animal models of osteoporosis (OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(5) FIG. 4A shows the number of myelopoiesis progenitor cells in the blood after administration of each peptide in the animal models of osteoporosis, and FIG. 4B is a graph showing the number of hematopoietic stem cells in the bone marrow using a marker of myelopoiesis stem cells and quantifying them (OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(6) FIGS. 5A to 5C show the results of micro-CT imaging the overall bone density after administration of each peptide to animal models of osteoporosis (5A), and then graphically quantifying the change of bone density (5B) and the change of bone tissue thickness (5C) (Sham: control animal models with subcutaneous incision only, OVX: osteoporosis with ovarian resection after subcutaneous incision).

(7) FIGS. 6A to 6C show the results of TRAP staining the change in the number of osteoclasts in bone marrow after administration of each peptide to animal models of osteoporosis (6A), and then graphically quantifying the number of osteoclasts (6B) and the surface area occupied by osteoclasts (6C) (Sham: control animal models with subcutaneous incision only, OVX: osteoporosis animal model with ovarian resection after subcutaneous incision).

(8) FIGS. 7A to 7C show the results of H & E staining of changes in osteoblasts in bone marrow after administration of each peptide to animal models of osteoporosis (7A), and then graphically quantifying the number of osteoblasts (7B) and the surface area occupied by osteoblasts (7C) (Sham: control animal model with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(9) FIGS. 8A to 8D are a result showing the change in the expression levels of the adhesion factors after treating the three kinds of peptides, NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) recombined by modifying the structure of specific amino acids to increase the receptor affinity of NPY (21-28) to osteoblasts that express adhesion factors of myelopoiesis stem cells (8A: summary of the experiment, 8B to 8D: a graph showing the expression levels of adhesion factors with fold change relative to the control group).

(10) FIG. 9A is a summary of the experiment performed to determine the effect of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) on the osteoporosis relief through releasing myelopoiesis stem cells into blood, and FIG. 9B is the result showing the weight change after administration of each peptide to the animal models of osteoporosis (Sham: control animal model with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(11) FIGS. 10A to 10C are a result showing the expression levels of adhesion factors of hematopoietic stem cell in bone marrow with fold change compared to the control group after administration of each peptide to the animal models of osteoporosis (OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(12) FIG. 11A shows the number of myelopoiesis progenitor cells in the blood after administration of each peptide in the animal models of osteoporosis, and FIG. 11B is a graph showing the number of hematopoietic stem cells in the bone marrow using a marker of myelopoiesis stem cells and quantifying them (OVX: osteoporosis animal model with ovarian resection after subcutaneous incision).

(13) FIGS. 12A to 12C show the results of micro-CT imaging the overall bone density after administration of each peptide to animal models of osteoporosis (12A), and then graphically quantifying the change of bone density (12B) and the change of bone tissue thickness (12C) (Sham: control animal model with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(14) FIGS. 13A to 13C show the results of TRAP staining the change in the number of osteoclasts in bone marrow after administration of each peptide to animal models of osteoporosis (13A), and then graphically quantifying the number of osteoclasts (13B) and the surface area occupied by osteoclasts (13C) (Sham: control animal model with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

(15) FIGS. 14A to 14C show the results of H & E staining of changes in osteoblasts in bone marrow after administration of each peptide to animal models of osteoporosis (14A), and then graphically quantifying the number of osteoblasts (14B) and the surface area occupied by osteoblasts (14C) (Sham: control animal model with subcutaneous incision only, OVX: osteoporosis animal models with ovarian resection after subcutaneous incision).

MODE FOR CARRYING OUT INVENTION

(16) Hereinafter, the present invention will be described in detail.

(17) However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Example 1. Experimental Materials and Experimental Methods

(18) 1-1. Mouse Preparation and Drug Treatment Protocols

(19) All mice used in the experiment were 6 to 8 weeks old mice, C57BL/6 mice, and purchased from Jackson Laboratory (Bar Harbor, Me., USA). NPY (21-28) (SEQ ID NO: 1), NPY (24-31) (SEQ ID NO: 2), NPY (29-36) (SEQ ID NO: 3), NPY D25 (21-28) (SEQ ID NO: 4), NPY D26 (21-28) (SEQ ID NO: 5), NPY D25, 26 (21-28) (SEQ ID NO: 6), NPY (21-36) (Osteopep2) (SEQ ID NO: 7) were prepared from Anygen. Full length NPY (SEQ ID NO: 8) was purchased from Bachem. 10 nM of each peptide was diluted in each medium and injected for in vitro experiments. The NPY D.sup.25(21-28) refers to a peptide in which arginine, the fifth amino acid of the amino acid sequence of NPY (21-28), is modified to D-type, the NPY D.sup.26(21-28) refers to a peptide in which the sixth amino acid histidine is modified to D-form, and the D.sup.25,26(21-28) refers to a peptide in which both the fifth and sixth amino acids are modified to D-form, respectively.

(20) To make an osteoporosis model, 10-18 12 weeks old female mice per group underwent ovarian ablation. A week later, 50 μg/kg of NPY(21-28), NPY D.sup.25(21-28), NPY D.sup.26(21-28), NPY D.sup.25,26(21-28), Osteopep2, NPY and 100 μl of PBS(Gibco) were intraperitoneally administered twice a day for 4 weeks at 12 hour intervals. Alendronate (Sigma), used as a competitive drug, was intraperitoneally administered once a week for 4 weeks at a dose of 50 μg/kg. As a control group, only Sham model of osteoporosis was performed by subcutaneous dissection of female mouse. Mice were placed in experimental groups using the Block randomization method, and all mouse experiments were approved through the Kyungpook National University Institutional Animal Care and Use Committee.

(21) 1-2. Culture of Mesenchymal Stem Cells in Bone Marrow and Induction of Differentiation Into Osteoblasts

(22) Four to six weeks old C57BL/6 mice were sacrificed after anesthesia, and then tibias and femurs were dissected. Bone marrow was harvested from tibias and femurs and single cell suspensions were obtained using a 40 μm cell strainer (Becton-Dickinson LAβware, Franklin Lakes, N.J.). Approximately 107 cells were dispensed into 75-cm 2 flasks containing mesenchymal stem cells Stimulatory Supplements with antibiotics (Stem Cell Technologies, Inc) and MesenCult™ MSCBasal medium. The cells were incubated for 1 week and further incubated for 3 weeks with StemXVivo Osteogenic/Adipogenic Base Media (R & D systems) supplemented with StemXVivo Osteogenic supplement (20×) and penicillin-streptomycin (100×) for differentiation into osteoblasts.

(23) The cultures were replaced every two to three days.

(24) 1-3. Real-Time Quantitative PCR

(25) Real-time quantitative PCR was used to measure expression levels of hematopoietic stem cell adhesion factors (Sdf-1a, KitI, Angpt1) present in osteoblasts. Total RNA was extracted from cell eluate and bone marrow cells using the RNeasy Plus mini kit (Qiagen, Korea, Ltd), and cDNAs were synthesized from 5 μg total RNA using a kit in Clontech (Mountain View, Calif.). In addition, using a Corbett research RG-6000 real-time PCR instrument, Real-time quantitative PCR was performed at 95° C. for 10 minutes; at 95° C., for 10 seconds; at 58° C., for 15 seconds; at 72° C. for 20 seconds as one cycle, and 40 cycles were repeated. Primers used for the real-time quantitative PCR are shown in Table 1.

(26) TABLE-US-00001 TABLE 1 SDF-1 α F 5′-TTCCTATCAGAGCCCATAGAG-3′ SEQ ID  NO: 9 R 5′-CCAGACCATCCTGGATAATG-3′ SEQ ID  NO: 10 Kit ligand F 5′-CCAAAAGCAAAGCCAATTACAAG-3′ SEQ ID  (stem cell NO: 11 factor;  R 5′-AGACTCGGGCCTACAATGGA-3′ SEQ ID  SCF) NO: 12 Angio- F 5′-ACGGGGGTCAATTCTAAG-3′ SEQ ID  poietin-1 NO: 13 (Angpt1) R 5′-GCCATTCCTGACTCCACA-3′ SEQ ID  NO: 14 GAPDH F 5′-TTGCTGTTGAAGTCGCAGGAG-3′ SEQ ID  NO: 15 R 5′-TGTGTCCGTCGTGGATCTGA-3′ SEQ ID  NO: 16

(27) 1-4. Colony-Forming Unit (CFU) Assays

(28) CFU analysis was performed to determine the number of bone marrow hematopoietic progenitor cells in the blood of mice. After anesthetizing the mouse first, 500 μl to 700 μl of blood were collected in a heparin tube by cardiac drawing, and then placed in ammonium chloride solution (Stem Cell Technologies, Inc. 1:10) and placed on ice for 15 minutes to remove red blood cells. The red blood cells were well shaken at intervals of 2 to 3 minutes and centrifuged at 1000 rpm for 7 minutes. The supernatant was removed and washed with IMDM (Gibco) supplied with 2% fetal bovine serum (FBS, Gibco). The washed cells (3×10.sup.5 per mouse) were divided into three 35 mm dishes containing methylcellulose-based media (Methocult, Stem cell), and after the cells were incubated for two weeks, the number of colonies formed in the flask was counted.

(29) 1-5. Flow Cytometry Analysis (FACs)

(30) To examine the changes in the number of myelopoiesis stem cells in the bone marrow of the mouse, the bone marrows of mice were collected and analyzed by FACs using five antibodies such as Lineage, Sca-1, c-kit, CD150, and CD48 as markers of myelopoiesis stem cells. For the analysis of myelopoiesis stem cell, bone marrow collected from the tibias and the femurs in the animal models of osteoporosis injected with each peptide was removed from red blood cells with ammonium chloride solution (Stem Cell Technologies, Inc. 1:4). After washing with PBS (Gibco) solution containing 10% fetal bovine serum (FBS, Gibco) and 1% sodium azide (Sigma-Aldrich), it was centrifuged at 300×g for 10 minutes. Hematopoietic cells contained in bone marrow were removed with MACs beads (Miltenyi Biotec) using biotinylated lineage antibody (Miltenyi Biotec). The remaining cells were reacted for 30 minutes at 4° C. using Sca-1-PECY7, c-kit-APC, CD150-PE, and CD48-FITC (BD science) antibodies, and then were analyzed by LSRII (BD science) flow cytometry.

(31) 1-6. Micro CT

(32) The femurs were separated from the mouse and refrigerated in 80% ethanol, and in order to measure change of bone density, the bone volume/total volume and trabecular thickness were measured by analyzing the area between 0.7 mm and 2.3 mm from the growth plate baseline with the Quantum FX microCT Imaging System.

(33) 1-7. Immunohistochemistry

(34) The femurs were separated from the mice and fixed in 4% paraformaldehyde for 24 hours, and tissue was decalcified in 10% EDTA for 5 weeks. It was then dehydrated with a series of diluted alcohols, paraffinized, and prepared into 5 mm paraffin sections.

(35) For TRAP staining, sections were deparaffinized, stained with 1.33 mM Fast Red Violet LB Salt (Sigma-Aldrich) in 50 mM sodium acetate (pH 5.0) containing 225 μM Naphthol AS-MX phosphate (Sigma-Aldrich, St Louis, Mo., USA), 0.84% N, N-dimethylformamide (Sigma-Aldrich) and 50 mM sodium tartrate, and incubated for 30 minutes. After incubation, the sections were washed with distilled water and counterstained with 1% methyl green.

(36) For H & E staining, the deparaffinized sections were stained with Harris hematoxyline for 8 minutes, washed with distilled water, and stained with Eosin for 1 minute.

(37) Histomorphometric analysis was performed using the OsteoMeasure program (Extensive interactive Bone Histomorphometry Analysis System).

(38) 1-8. Statistical Analysis

(39) Comparison with each group was performed by one way ANOVA and Tukey's HSD test. All statistical analyzes were performed using SPSS statistical software. It was considered significant for p<0.05.

Example 2 Effects of NPY (21-28), NPY (24-31), NPY (29-36) on the Expression of Myelopolesis Stem Cell Adhesion Factors in Osteoblasts

(40) To investigate the effect of NPY (21-28), NPY (24-31) and NPY (29-36), which are recombinant peptides consisting of eight amino acid sequences, on the expression of hematopoietic stem cell adhesion factors (Sdf-1a, KitI, Angpt1) present in osteoblasts, the following experiments were carried out according to the methods of Examples 1-1 and 1-2, 1-3.

(41) First, bone marrow was harvested from 4 to 6 weeks old C57BL6 mice, and mesenchymal stem cells (BM-MSCs) in bone marrow were collected, and then cultured for 3 weeks in osteoblast differentiation induction medium for the differentiation into osteoblasts. After 28 days, each peptide was treated with 10 nM for 3 days, and bone cells were collected and examined for expression levels of three adhesion factors by real-time quantitative PCR. Osteopep2 and its parent NPY were used as positive controls.

(42) The schematic diagram of the experimental procedure and the result of measuring the expression amount of the adhesion factor are shown in FIGS. 1A to 1D.

(43) As shown in FIGS. 1A to 1D, when compared to osteoblasts (control) not treated with peptides, the expression levels of Sdf-1a, KitI, and Angpt1 were decreased only in osteoblasts treated with NPY (21-28) among NPY (21-28), NPY (24-31), and NPY (29-36) peptides (p<0.05, n=3 per group). From these results, it was found that only NPY (21-28) of the three peptides recombined with eight amino acids decreased the expression levels of myelopoiesis stem cell adhesion factors present in osteoblasts, which may induce the release of myelopoiesis stem cell from bone marrow to blood. In other words, NPY (21-28) was found to be a peptide comprising an active site capable of inducing the release of myelopoiesis stem cell into blood.

Example 3. Effect of NPY (21-28) of the Present Invention on Releasing Myelopolesis Stem Cells into Blood in Animal Models of Osteoporosis

(44) In order to investigate whether the NPY (21-28) of the present invention affects the blood release of myelopoiesis stem cells in the animal models of osteoporosis, the following experiments were carried out according to the methods of Examples 1-1, 1-3, 1-4, and 1-5. In order to make animal models of osteoporosis, 12 weeks old C57BL/6 female mice were subjected to ovarian ablation for 10 to 18 animals in each group. One week later, 50 μg/kg of NPY (21-28) and 100 μl of PBS (Gibco) were intraperitoneally administered twice a day for 4 weeks at 12 hour intervals. As a control group, mice that underwent only subcutaneous incisions in normal mice were used. Weight change was measured weekly, and bone marrow and blood were collected one hour after the last dose at 4 weeks, and the release of myelopoiesis progenitor and stem cells into blood was analyzed. The positive control group, NPY (21-36), Osteopep2 and full length NPY (50 μg/kg, intraperitoneally administered twice a day for 4 weeks) were used. Alendronate used as a competitive drug for osteoporosis treatment was intraperitoneally administered at a dose of 50 μg/kg for 4 weeks once a week. The schematic diagram of an experimental procedure is shown in FIG. 2A.

(45) 3-1. Effect of NPY (21-28) on Weight Gain by Osteoporosis in Animal Models of Osteoporosis

(46) In order to investigate the effect of NPY (21-28) of the present invention on weight gain induced by osteoporosis, body weight was measured once a week for a total of 4 weeks of NPY (21-28) injection. The results are shown in FIG. 2B. As shown in FIG. 2B, the group injected with PBS in the animal models of osteoporosis gained weight according to time, while the group injected with NPY (21-28) did not change in weight (p<0.05, n=10-18 per group). From the above results, it can be seen that NPY (21-28) can suppress the weight gain caused by osteoporosis.

(47) 3-2. Effect of NPY (21-28) on the Expression of Myeloid Stem Cell Adhesion Factors in Animal Models of Osteoporosis

(48) To investigate the effect of NPY (21-28) of the present invention on the expression level of adhesion factor involved in the maintenance of myelopoiesis stem cells in bone marrow of the animal models of osteoporosis, bone marrow was harvested from tibias and femurs of mice 1 hour after the last administration of NPY (21-28) at 4 weeks. The expression levels of the adhesion factors were examined by real-time quantitative PCR, which is the method described in the Example 1-3.

(49) The results are shown in FIGS. 3A to 3C. As shown in FIGS. 3A to 3C, expression levels of major adhesion factors of Sdf-1a, KitI, and Angpt1 were decreased (p<0.05, n=3-4 per group).

(50) 3-3. Effect of NPY (21-28) on Releasing Myelopolesis Progenitor Cells and Myelopoiesis Stem Cells into Blood in Animal Models of Osteoporosis

(51) To investigate the effect of NPY (21-28) of the present invention on myelopoiesis progenitor cells and myelopoiesis stem cells released into the blood in osteoporosis animal model, it was confirmed through the CFU assay and FACs, which are the methods described in Examples 1-4 and 1-5.

(52) The results are shown in FIGS. 4A and 4B. As shown in FIGS. 4A and 4B, (a) CFU assay showed that NPY (21-28) increases releasing myelopoiesis progenitor cells into blood in the animal model of osteoporosis (p<0.05, n=4-5 per group). As a result, it was confirmed through the FACs that (b) the number of myelopoiesis stem cells remaining in the bone marrow was reduced (p<0.05, n=4-5 per group).

(53) From the above results, the administration of NPY (21-28) of the present invention can induce releasing myelopoiesis progenitors and its stem cells into blood by reducing the expression levels of adhesion factors of myelopoiesis stem cells present in osteoblasts. The effect of NPY (21-28) was confirmed to be superior to Osteopep2 and NPY used as a positive control.

Example 4. Effect of NPY (21-28) of the Present Invention on the Prevention and Treatment of Osteoporosis

(54) In order to investigate whether releasing myelopoiesis stem cells into blood by administration of NPY (21-28) of the present invention has the effect of alleviating a decrease of bone density of osteoporosis, the following experiments were carried out according to the methods of Examples 1-6 and 1-7.

(55) 4-1. Changes in Bone Density by NPY (21-28) Administration in Animal Models of Osteoporosis

(56) Femurs were separated from mice after the last administration of NPY (21-28) at 4 weeks. To determine the change of bone density, bone density (bone volume/total volume) and bone tissue thickness (trabecular thickness) were measured with the Quantum FX microCT Imaging System.

(57) The results are shown in FIGS. 5A to 5C. As shown in FIGS. 5A to 5C, the microCT photographs showed that the percentage of bone density (BV/TV, %) and bone tissue thickness (trabecular thickness, mm) of the mice injecting NPY in the animal models of osteoporosis were increased compared to the mice injected with PBS. In addition, the effect of NPY (21-28) was better than that of the Osteopep2 and NPY as positive control group. It showed similar effects as the mice injected with the competitive drug Alendronate (p<0.05, n=4-5 per group).

(58) 4-2. Osteoclast Changes in Bone Marrow by NPY (21-28) Administration in Animal Models of Osteoporosis

(59) After the last administration at 4 weeks of NPY (21-28), the femurs were separated from the mice and the number of osteoclasts in the bone marrow was measured by TRAP staining.

(60) The results are shown in FIGS. 6A to 6C. As shown in FIGS. 6A to 6C, the number of TRAP positive osteoclasts in bone marrow of mice injecting NPY (Number of osteoclast/Bone surface, mm) and the osteoclast surface (Osteoclast surface/Bone surface, %) were reduced compared to mice injected with PBS (p<0.05, n=3-4 per group). In addition, the effect of NPY (21-28) was not only superior to Osteopep2 and NPY as the positive control group, but it was also found to be superior to the competitive drug Alendronate.

(61) 4-3. Changes of Osteoblast in Bone Marrow by NPY (21-28) Administration in Animal Models of Osteoporosis

(62) After the last administration of 4 weeks of NPY (21-28), the femurs were separated from the mice, and the number of osteoblasts in bone marrow was measured by H & E staining.

(63) The results are shown in FIGS. 7A to 7C. As shown in FIGS. 7A to 7C, the number of osteoblast (Number of osteoblast/Bone surface, mm) and the osteoblast surface (Osteoblast surface/Bone surface, %) adjacent to the bone in the bone marrow of the mice to which NPY (21-28) was administered were increased compared to mice injected with PBS, and this effect was better than that of Osteopep2 as the positive control group (p<0.05, n=4 per group).

(64) From these results, it was found that NPY (21-28) inhibits the reduction in bone density and bone tissue thickness of osteoporosis mice by reducing the number of osteoclasts that differentiate from hematopoietic stem cells in the bone marrow and by simultaneously increasing the number of osteoblasts after inducing the release of myelopoiesis stem cells into blood in animal models of osteoporosis. In addition, NPY (21-28) is a short sequence peptide comprising the active site of Osteopep2 and/or NPY used as a positive control and showed a better effect than these. Thus, NPY (21-28) was found that it has an effect of preventing and treating osteoporosis.

Example 5. Effects of NPY D.SUP.25.(21-28), NPY D.SUP.26.(21-28), NPY D.SUP.25,26.(21-28)), in which the Structure of Specific Amino Acids were Modified to Increase Receptor Affinity of NPY (21-28) on the Expression of Myelopolesis Stem Cell Adhesion Factors in Osteoblasts

(65) To investigate effects of NPY D.sup.25(21-28), NPY D.sup.26(21-28), NPY D.sup.25,26(21-28)), in which the structure of specific amino acids were modified to increase receptor affinity of NPY (21-28) on the expression of myelopoiesis stem cell adhesion factors (Sdf-1a, KitI, Angpt1) in osteoblasts, the following experiments were carried out according to the methods of Examples 1-1 and 1-2, 1-3.

(66) The schematic diagram of the experimental procedure and the result of measuring the expression amount of the adhesion factors are shown in FIGS. 8A to 8D. As shown in FIGS. 8a to 8d, it was confirmed that the expression levels of Sdf-1a, KitI, and Angpt1 decreased in osteoblasts treated with NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28), compared to the osteoblasts (control) not treated with peptides. In particular, NPY D.sup.25,26 (21-28) was confirmed to further reduce the amount of Angpt1 expression than NPY (21-28).

(67) From the above results, it was found that NPY D.sup.25(21-28), NPY D.sup.25(21-28), and NPY D.sup.25,26(21-28) peptides which transformed the structure of a specific amino acid into D-form decreased the expression level of myelopoiesis stem cell adhesion factor present in osteoblasts, which may induce releasing of myelopoiesis stem cell from bone marrow into blood.

Example 6. Effects of NPY D.SUP.25.(21-28), NPY D.SUP.26.(21-28), NPY D.SUP.25,26.(21-28) on Releasing Myelopolesis Stem Cells into Blood in Animal Models of Osteoporosis

(68) In order to investigate whether the NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) affects releasing myelopoiesis stem cells into blood in the animal models of osteoporosis, the following experiments were carried out according to the methods of Examples 1-1, 1-3, 1-4, and 1-5.

(69) 6-1. Effects of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) on Weight Gain by Osteoporosis in Animal Models of Osteoporosis

(70) In order to investigate the effects of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) of the present invention on weight gain induced by osteoporosis, body weight was measured once a week for a total of 4 weeks in which each peptide was injected.

(71) The results are shown in FIGS. 9A and 9B. As shown in FIGS. 9A and 9B, the group injected with PBS in the animal models of osteoporosis gained weight according to time, while the group injected with NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) did not change in weight (p<0.05, n=10-18 per group). From the above results, it can be seen that NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) can suppress the weight gain caused by osteoporosis.

(72) 6-2. Effects of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) on the Expression of Myeloid Stem Cell Adhesion Factors in Animal Models of Osteoporosis

(73) To investigate the effect of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) of the present invention on the expression level of adhesion factor involved in the maintenance of myelopoiesis stem cells in bone marrow of the animal models of osteoporosis, bone marrow was harvested from tibias and femurs of mice 1 hour after the last administration of each peptide at 4 weeks. The expression levels of the adhesion factors were examined by real-time quantitative PCR, which is the method described in the Example 1-3.

(74) The results are shown in FIGS. 10A to 10C. As shown in FIGS. 10A to 10C, expression levels of major adhesion factors of Sdf-1a, KitI, and Angpt1 were decreased, and the effects were generally superior to those of NPY (p<0.05, n=3 per group).

(75) 6-3. Effects of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) on Releasing Myelopolesis Progenitor Cells and Myelopolesis Stem Cells into Blood in Animal Models of Osteoporosis

(76) To investigate the effect of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) on myelopoiesis progenitor cells and its stem cells released into the blood in osteoporosis animal model, it was confirmed through the CFU assay and FACs which are the methods described in Examples 1-4 and 1-5.

(77) The results are shown in FIGS. 11A and 11B. As shown in FIGS. 11A and 11B, (a) CFU assay showed that NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) increases releasing myelopoiesis progenitor cells into blood in the animal model of osteoporosis (p<0.05, n=3 per group). As a result, it was confirmed through the FACs that (b) the number of myelopoiesis stem cells remaining in the bone marrow was reduced (p<0.05, n=4-5 per group). On the other hand, the effects of these D-type peptides were confirmed to be superior to those of NPY (21-28).

(78) From the above results, the administration of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) of the present invention can induce releasing myelopoiesis progenitors and its stem cells into blood by reducing the expression levels of adhesion factors of myelopoiesis stem cells present in osteoblasts. In particular, among these peptides, NPY D.sup.25,26 (21-28) was found to induce releasing myelopoiesis stem cells into blood with the highest efficiency, and the effect was even better than that of NPY (21-28).

Example 7. Effects of NPY D.SUP.25.(21-28), NPY D.SUP.26.(21-28), and NPY D.SUP.25,26.(21-28) on Preventing and Treating Osteoporosis

(79) In order to investigate whether releasing myelopoiesis stem cells into blood by administration of NPY (21-28) has the effect of alleviating a decrease of bone density of osteoporosis, the following experiments were carried out according to the methods of Examples 1-6 and 1-7.

(80) 7-1. Changes in Bone Density by NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) Administration in Animal Models of Osteoporosis

(81) Femurs were separated from mice after the last administration of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) at 4 weeks. To determine the change of bone density, bone density (bone volume/total volume) and bone tissue thickness (trabecular thickness) were measured with the Quantum FX microCT Imaging System.

(82) The results are shown in FIGS. 12A to 12C. As shown in FIGS. 12A to 12C, the microCT photographs showed that the percentage of bone density (BV/TV, %) and bone tissue thickness (trabecular thickness, mm) of the mice injected with NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) in the animal models of osteoporosis were increased compared to the mice injected with PBS. In addition, this effect was equal to or higher than that of NPY (21-28), and the effect was similar to that of mice injected with the competitive drug Alendronate. In particular, animals treated with NPY D.sup.25,26(21-28) showed the best effect (p<0.05, n=4-5 per group).

(83) 7-2. Osteoclast Changes in Bone Marrow by Administration of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) in Animal Models of Osteoporosis

(84) After the last administration at 4 weeks of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28), the femurs were separated from the mice and the number of osteoclasts in the bone marrow was measured by TRAP staining.

(85) The results are shown in FIGS. 13A to 13C. As shown in FIGS. 13A to 13C, the number of TRAP positive osteoclasts in bone marrow of mice injected with NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) (Number of osteoclast/Bone surface, mm) and the osteoclast surface (Osteoclast surface/Bone surface, %) were reduced compared to mice injected with PBS (p<0.05, n=4 per group). As with bone density, NPY D.sup.25,26(21-28) was found to reduce osteoclasts in bone marrow with the highest efficiency.

(86) 7-3. Changes of Osteoblast in Bone Marrow by Administration of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) in Animal Models of Osteoporosis

(87) After the last administration of 4 weeks of NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28), the femurs were separated from the mice and the number of osteoblasts in bone marrow was measured by H & E staining.

(88) The results are shown in FIGS. 14A to 14C. As shown in FIGS. 14A to 14C, the number of osteoblast (Number of osteoblast/Bone surface, mm) and the osteoblast surface (Osteoblast surface/Bone surface, %) adjacent to the bone in the bone marrow of the mice to which NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) were administered were increased compared to mice injected with PBS (p<0.05, n=4 per group).

(89) From these results, it was found that NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28), which modified the structure of specific amino acids of NPY (21-28), inhibit the reduction in bone density and bone tissue thickness of osteoporosis mice by reducing the number of osteoclasts that differentiate from hematopoietic stem cells in the bone marrow and by simultaneously increasing the number of osteoblasts after inducing the release of myelopoiesis stem cells into blood in animal models of osteoporosis.

(90) Therefore, it can be shown that NPY(21-28) and NPY D.sup.25(21-28), NPY D.sup.26(21-28), and NPY D.sup.25,26(21-28) recombined through structural modifications therefrom are effective for preventing and treating osteoporosis. These short fragment peptides may not only have a better effect compared to the previously reported long peptides, but may also represent additional advantages such as improved stability, high absorption of tissue, and ease of manufacture.

INDUSTRIAL APPLICABILITY

(91) The peptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention effectively induces releasing hematopoietic stem cells into blood by reducing the expression level of hematopoietic stem cell adhesion factors in bone marrow. This induces a decrease in the number of osteoclasts in bone marrow and an increase in the number of osteoblasts, and has the effect of alleviating the decrease in bone density. Therefore, the peptide consisting of the amino acid sequence of SEQ ID NO: 1 of the present invention can be very usefully used for the development of a prophylactic or therapeutic agent for osteoporosis, which is highly likely to be used industrially.