COMPOSITION FOR ACCELERATING CELL PROLIFERATION COMPRISING ERYTHROPOIETIN-DERIVED PEPTIDE

Abstract

Provided is a composition for accelerating cell proliferation, including an erythropoietin-derived peptide as an active ingredient. Due to having a simpler structure than that of the existing natural human erythropoietin, the composition easily passes through the tissue-blood barrier, exhibits excellent cell protective activity, does not cause side effects of cell proliferation, and improves a hematopoietic function. Accordingly, the composition is used in the treatment or prevention of an anemic disorder.

Claims

1. A peptide consisting of one or more sequence selected from SEQ ID NOs: 26 to 28.

2. The peptide of claim 1, wherein the peptide is derived from an erythropoietin protein sequence.

3. A composition for accelerating proliferation or differentiation of cells, comprising, as an active ingredient, one or more peptides selected from SEQ ID NOs: 1 to 28.

4. The composition of claim 3, wherein the peptide binds to an erythropoietin receptor.

5. The composition of claim 3, wherein the peptide forms an alpha-helical structure.

6. The composition of claim 3, wherein the peptide has a cell protective activity.

7. The composition of claim 3, wherein the cells are hemocytoblasts, fat cells, pancreatic cells, muscle cells, blood vessel cells, or skin cells.

8. (canceled)

9. A pharmaceutical composition for preventing or treating an anemic disorder, comprising, as an active ingredient, one or more peptides selected from SEQ ID NOs: 1 to 28.

10. The pharmaceutical composition of claim 9, wherein the composition accelerates proliferation of hemocytoblasts.

11. The pharmaceutical composition of claim 9, wherein the anemic disorder is selected from acute or chronic anemia, anemia associated with a kidney disorder, anemia associated with kidney failure, anemia associated with hemopathy, radiation therapy-induced anemia, chemotherapy-induced anemia, anemia associated with a surgical procedure, anemia associated with an infection, anemia associated with nutritional deficiency, abnormal erythropoiesis, initial anemia associated with premature birth, and a combination thereof.

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0035] FIG. 1 is a diagram representing an erythropoietin-derived peptide according to one embodiment and analogues thereof.

[0036] FIGS. 2A-2C are graphs showing binding strength as confirmed by the SPR technique to confirm whether an erythropoietin-derived peptide is able to act on an erythropoietin receptor.

[0037] FIG. 3 is a photograph confirming a secondary alpha helix formation of an erythropoietin-derived peptide (ML1, ML1-C1, ML1-C2, ML1-C3, ML1-H1, ML1-H2, and ML1-H3).

[0038] FIGS. 4A-4D depict graphs showing cell protective effects of erythropoietin-derived peptide treatment of cells in which reactive oxygen species were increased by hydrogen peroxide.

[0039] FIG. 5 depicts graphs showing cell protective effects of a peptide (ML1-L2, ML1-K2, and ML1-R2) prepared by partially modifying sequences of an erythropoietin-derived ML1 peptide.

[0040] FIG. 6 depicts graphs showing cell proliferation rates of a peptide (ML1-L2, ML1-K2, and ML1-R2) prepared by partially modifying sequences of an erythropoietin-derived ML1 peptide.

[0041] FIG. 7 depicts graphs showing cell proliferation rates of an erythropoietin-derive peptide and peptides prepared by partially modifying sequences.

[0042] FIG. 8 shows an illustration of a structure of a complex of erythropoietin receptor (EPOR) and erythropoietin (EPO) of a specific embodiment and binding target sites.

[0043] FIG. 9 depicts a graph showing cell protective and proliferative effects of an erythropoietin-derive peptide and peptides prepared by partially modifying sequences.

[0044] FIG. 10 depicts a graph showing effects of in vivo administration of an erythropoietin-derived peptide (MLP, MLP-C, and MLP-H) on body weight.

[0045] FIGS. 11A-11D depict graphs showing hematopoietic effects of in vivo administration of an erythropoietin-derived peptide (MLP, MLP-C, and MLP-H).

[0046] FIGS. 12A-12B show results of confirming activation regulating effects of an erythropoietin-derived peptide (MLP-C and MLP-H) and erythropoietin (EPO) with respect to an erythropoietin receptor.

MODE OF DISCLOSURE

[0047] Hereinbelow, preferred embodiments are provided to help understanding of the present disclosure. However, as the embodiments disclosed below are provided merely to help understanding of the present disclosure, the scope of the present disclosure is not limited by the embodiments disclosed below.

EXAMPLES

Example 1. Synthesis of Erythropoietin-Derived Peptides

[0048] Erythropoietin-derived peptides of the present disclosure were synthesized as monomers according to a known solid phase peptide synthesis technology (Peptron, Daejeon, Korea).

[0049] In detail, erythropoietin-derived peptides, which are able to bind to crucial amino acid sequences (Arg103, Ser104, Leu105, Leu108, and Arg110) in a sequence of a target site (site 2) of the natural erythropoietin receptor were synthesized, and specific characteristics of the peptides were examined, respectively. To measure concentrations of the synthesized peptides, liquid chromatography/mass-selective detector (HP 1100 series) was used. Purity was measured by high performance liquid chromatography (SHIMADZU prominence HPLC) analysis (>95% purity). The erythropoietin-derived peptides are shown in Table 1 below.

TABLE-US-00001 TABLE 1 SEQ Peptide ID Name Sequence NO ML1 LQLHVDKAVSGLRSLTTLLRALG 1 ML2 LHVDKAVSGLRSLTTLLRAL 2 ML3 TKVNFYAWKR 3 ML4 DKAVSGLRSLTTLLRALGAQKEAI 4 ML5 SGLRSLTTLLRALG 5 ML6 SGLRSLTTLLRALGAQKEAI 6 ML7 WEPLQLHVDKAVSGLRSLTTLLRAL 7 ML8 DKAVSGLRSLTTLLRAL 8 ML1-1 LQLHVLKRVSGLLSHTMLLKALG 9 ML2-1 RHVQKAESGLRSLTKLLREL 10 ML3-1 TRVNYQAWKR 11 ML4-1 KKAVSGLKTLTHILRALGAQKEAI 12 ML5-1 AGLRSRAHLRRALA 13 ML6-1 KGLRSLISLLRALGAQKEAI 14 ML7-1 DEALDLEVDKAATGLRTLTTLIRAL 15 ML8-1 NKAVAGLRSLTVN 16

[0050] Hydrophobicity, charge, and isoelectric point (pl) of the erythropoietin-derived peptides, ML1-1, ML2-1, ML3-1, ML4-1, ML5-1, ML6-1, ML7-1, and ML8-1 were calculated and are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Peptide Charge Target Name Hydrophobicity (pH 7) pI Site ML1-1 8.25 3.4 11.2 2 ML2-1 −4.45 3.2 10.94 2 ML3-1 −10.07 2.9 10.94 1 ML4-1 5 6.1 11.41 2 ML5-1 −4.15 4.1 12.48 2 ML6-1 8.85 2.9 10.94 2 ML7-1 2.05 −2.1 4.59 2 ML8-1 5.7 1.9 11.12 2

Example 2. Erythropoietin-Derived Peptides Using Partial Sequence (1)

[0051] For sequence modification experiments, a binding model of erythropoietin and its receptor was based on a previously known binding structure (Protein Data Bank ID: 1EER). Based on known characteristics of amino acids, amino acids of the erythropoietin-derived peptides were substituted. Amino acids are classified into 4 types ({circle around (1)} non-polar or hydrophobic, {circle around (2)} neutral, {circle around (3)} negatively charged, and {circle around (4)} positively charged) according to polarity of their side chains. Based on information of non-polar (hydrophobic), neutral, negatively charged, or positively charged amino acids, the existing amino acid sequences were substituted to induce modification in respective characteristics.

[0052] Peptides prepared by partially modifying sequences of ML1 peptide and their characteristics are shown in Tables 3 and 4.

TABLE-US-00003 TABLE 3 Peptide Name Sequence SEQ ID NO ML1 LQLHVDKAVSGLRSLTTLLRALG 1 ML1-H1 LQLHVLKAVSGLLTHTTLLKALG 17 ML1-H2 LQLHVLKAVSGLLTLTMIRRALG 18 ML1-H3 LQLHVLKAVAGLRTLAMIRRALA 19

TABLE-US-00004 TABLE 4 Number Net Peptide of Molecular Absorbance Isoelectric Charge Name Residues Weight Coefficient Point (pH 7) Predicted Solubility ML1 23  2461.9 g/mol 0 M.sup.−1cm.sup.−1 pH 11.23 2.1 Low solubility in water ML1-H1 23 2426.94 g/mol 0 M.sup.−1cm.sup.−1 pH 10.73 2.2 Low solubility in water ML1-H2 23 2504.09 g/mol 0 M.sup.−1cm.sup.−1 pH 12.13 3.1 Low solubility in water ML1-H3 23 2515.12 g/mol 0 M.sup.−1cm.sup.−1 pH 12.41 4.1 Low solubility in water

Example 3. Erythropoietin-Derived Peptides Using Partial Sequence (2)

[0053] Partial sequences of the peptides were substituted using the basic sequence of ML1 as in Example 2. In this regard, amino acids were substituted based on the existing binding model of erythropoietin and its receptor without hindering the existing binding structure (a distance between proteins or a protein structure). FIG. 8 illustrates exemplary substitution of amino acid sequences, and since substitution of alanine (Ala) with arginine (Arg) hinders the existing binding, substitution with serine (Ser) may be performed to prevent hindrance of the binding.

[0054] Peptides prepared by modifying the charge of the ML1 peptide and characteristics thereof are shown in Tables 5 and 6.

TABLE-US-00005 TABLE 5 Peptide Name Sequence SEQ ID NO ML1-C1 LDLEVDKAVSGLRSLTTLLRA 20 LG ML1 LQLHVDKAVSGLRSLTTLLRA 1 LG ML1-C2 LQRHVDKRVSGLRSLTTLLR 21 ALG ML1-C3 LQRHVKKRVKGLKSLTTLLRA 22 LG

TABLE-US-00006 TABLE 6 Number Net Peptide of Molecular Absorbance Isoelectric Charge Name Residues Weight Coefficient Point (pH 7) Predicted Solubility ML1-C1 23 2440.83 g/mol 0 M.sup.−1cm.sup.−1 pH 6.96 0 High solubility in water ML1 23  2461.9 g/mol 0 M.sup.−1cm.sup.−1 pH 11.23 2.1 Low solubility in water ML1-C2 23 2590.04 g/mol 0 M.sup.−1cm.sup.−1 pH 12.12 4.1 High solubility in water ML1-C3 23 2616.21 g/mol 0 M.sup.−1cm.sup.−1 pH 12.45 7.1 High solubility in water

Example 4. Erythropoietin-Derived Peptides Using Partial Sequence (3)

[0055] A partial sequence “LHVDKAVSGLRSLTTL” of the ML1 basic sequence was used to prepare peptides having modified amino acids at both ends thereof, as shown in Table 7 below.

TABLE-US-00007 TABLE 7 SEQ ID Peptide Name Sequence NO ML1-L2 L HVDKAVSGLRSLTT L 23 ML1-K2 K HVDKAVSGLRSLTT K 24 ML1-R2 R HVDKAVSGLRSLTT R 25

Example 5. Erythropoietin-Derived Peptides for Use In Vivo (4)

[0056] Using sequences of the ML1, ML1-C3, and ML1-H2, as shown in Table 8 below, peptides, each of which has two amino acids removed from both ends thereof, excluding key regions, to improve efficiency and stability, were additionally prepared.

[0057] Sequences of the additionally prepared MLP, MLP-C, and MLP-H peptides and characteristics thereof are shown in Tables 8 and 9.

TABLE-US-00008 TABLE 8 SEQ ID Peptide Name Sequence NO MLP LHVDKAVSGLRSLTTLLRA 26 MLP-C RHVKKRVKGLKSLTTLLRA 27 MLP-H LHVLKAVSGLLTLTMIRRA 28

TABLE-US-00009 TABLE 9 Number Net Peptide of Molecular Absorbance Isoelectric Charge Name Residues Weight Coefficient Point (pH 7) Predicted Solubility MLP 19 2050.41 g/mol 0 M.sup.−1cm.sup.−1 pH 11.23 2.1 High solubility in water MLP-C 19 2204.71 g/mol 0 M.sup.−1cm.sup.−1 pH 12.45 7.1 High solubility in water MLP-H 19 2590.04 g/mol 0 M.sup.−1cm.sup.−1 pH 12.13 3.1 Low solubility in water

Experimental Example

[0058] Determination of Binding Affinity of Erythropoietin-Derived Peptide to Erythropoietin Receptor (EPOR)

[0059] To determine whether the erythropoietin-derived peptides prepared in Examples 1 to 3 are able to bind to the erythropoietin receptor having the target site to exert their actions, a surface plasmon resonance (SPR) technique was used to determine binding affinity. The SPR technique is to measure interactions between biomolecules in real-time by using an optical principle without specific labeling, and is a system analyzing affinity between two molecules and kinetics, i.e., an association rate (Ka) and a dissociation rate (Kd).

[0060] In detail, real-time SPR analysis was performed using Reichert SPR Biosensor SR 7500C instrument (Reichert Inc., NY, USA). Soluble mouse EPOR chimera proteins (R&D Systems, Minneapolis, Minn., USA) were covalently linked to a carboxymethylated dextran matrix-coated chip (BR-1005-39, Pharmacia Biosensor AB) by an amine coupling procedure using an amine coupling kit (BR-1000-50, GE Healthcare, USA) in accordance with manufacturer's instructions. Each 5 μM, 2.5 μM, and 1.25 μM of the peptide samples of the present disclosure and scrambled peptides were applied at a flow rate of 5 μl/minute, and the experiments were independently performed in duplicate. For signal normalization, DMSO was applied at a flow rate of 5 μl/minute, and after each binding cycle, the sensor chip was regenerated by injecting 25 mM acetic acid at a flow rate of 20 μl/minute.

[0061] As a result, as shown in FIG. 2, the result values were increased according to the concentrations of the erythropoietin-derived peptides of one embodiment, and thus, it was confirmed that the erythropoietin-derived peptides were able to bind to the erythropoietin receptor having the target site to exert their actions. Further, as shown in Tables 10 and 11, it was also confirmed that the erythropoietin-derived peptides of specific embodiments exhibited binding affinities similar to the known binding affinity (˜1 uM).

TABLE-US-00010 TABLE 10 Ka Kd KD ML1 1.311 × 10.sup.3  8.5 × 10.sup.−3 6.46077 μM ML2 .sup. 1.6 × 10 4.4 × 10.sup.−3 273 μM ML3 2.05 × 10.sup.2 .sup. 3 × 10.sup.−3 14.6341 μM ML4  2.2 × 10.sup.2 2.2 × 10.sup.−3 10 μM ML5 3.04 × 10.sup.2 0.1 × 10.sup.−3 0.32894 μM ML6 .sup. 5.0 × 10 4.5 × 10.sup.−2 900 μM ML7 3.00 × 10.sup.2 0.2 × 10.sup.−2 6.666 μM ML8  1.8 × 10.sup.2 0.8 × 10.sup.−1 444.44 μM ML1-1 5.55 × 10.sup.3 5.9 × 10.sup.−3 1.06 μM ML2-1  3.1 × 10.sup.2 4.1 × 10.sup.−3 14.3 μM ML3-1 3.08 × 10.sup.3 1.3 × 10.sup.−2 4.31 μM ML4-1 4.10 × 10.sup.2 1.20 × 10.sup.2   39.34 mM ML5-1 4.42 × 10.sup.2 3.46 × 10.sup.−2  78.28 μM ML6-1  1.9 × 10.sup.2 .sup. 3 × 10.sup.−2 157.8 μM ML7-1 2.26 × 10.sup.2 1.44 × 10.sup.−2  63.70 μM ML8-1 .sup. 6.4 × 10 1.5 × 10.sup.−1 2.37 mM

TABLE-US-00011 TABLE 11 Km Ka Kd KD ML1 1.26E+05 1310.8 8.47E−03 6.46077 μM ML1-H1 4.79E+05 1.01E+03 7.94E−03 7.84542 μM ML1-H2 1.00E+10 3434.3 1.05E−03 306.977 nM ML1-H3 1.00E+10 4157.6 4.77E−03 1.14651 μM ML1-C1 9.23E+05 1745.7 0.2617  149.921 μM ML1-C2 4.58E+05 1.59E+03 0.01876 11.7609 μM ML1-C3 1.46E+05 1104.9 0.01086 9.82836 μM

[0062] In other words, it could be confirmed that the peptides according to specific embodiments are those derived from the erythropoietin binding site, and thus have binding affinity to the erythropoietin receptor.

[0063] Determination of Secondary Alpha-Helix Formation of Erythropoietin-Derived Peptide

[0064] It was determined whether the erythropoietin-derived peptides synthesized in Examples 2 and 3 are able to form a stable alpha-helix, like natural erythropoietin.

[0065] As a result, as shown in FIG. 3, it was confirmed that the erythropoietin-derived peptides synthesized in Examples 2 and 3 formed a stable secondary alpha-helix, like natural erythropoietin.

[0066] Determination of Cell Protective Effect of Erythropoietin-Derived Peptide (1)

[0067] To determine whether the erythropoietin-derived peptides prepared in Examples 1 to 3 exhibit cell protective effects, cell viability was determined under stress conditions where an increase in reactive oxygen species was induced by hydrogen peroxide (H.sub.2O.sub.2).

[0068] In detail, to evaluate cell viability, an MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, Wis., USA) was performed. PC12 cells were seeded in a 96-well plate (5×10.sup.4 cells per well), and an increase in reactive oxygen species was induced using 150 μM of hydrogen peroxide (H.sub.2O.sub.2). Thereafter, 25 ng/ml of nerve growth factor (NGF) was added as a positive control group, and 1 IU/ml of the erythropoietin compound, 0.25 pM, 1 pM, 2 pM, or 4 pM of the peptide of Example 1, each 0.25 pM, 1 pM, 2 pM, 10 pM, or 100 pM of the peptides of Examples 2 and 3, or 0.1 pM, 1 pM, 50 pM, or 0.5 nM of the peptide of Example 4 was added, and 20 μl of an MTS solution was added to each well, and left for 3 hours. The initial number of cells (0 hour) and the number of cells after 48 hours were counted. Intracellular soluble formazan produced by cell reduction was determined by recording absorbance of each 96-well plate at a wavelength of 490 nm using a VERSA MAX.

[0069] As a result, as shown in FIG. 4, it was confirmed that the erythropoietin-derived peptides protected cells from cell death caused by the increase in reactive oxygen species. It could be confirmed that this result was similar to the cell protective effect by treatment with the natural erythropoietin compound.

[0070] Determination of Cell Protective Effect of Erythropoietin-Derived Peptide (2)

[0071] To determine whether the erythropoietin-derived peptide prepared in Example 4 exhibits the cell protective effect, mitochondrial activity was determined under stress conditions where an increase in reactive oxygen species was induced by hydrogen peroxide (H.sub.2O.sub.2).

[0072] In detail, PC12 cells or human SH-SY5Y cells were seeded in a 96-well plate (5×10.sup.4 cells per well), and an increase in reactive oxygen species was induced using 150 μM of hydrogen peroxide (H.sub.2O.sub.2). Thereafter, 25 ng/ml of NGF was added as a positive control group, and 1 IU/ml of the erythropoietin compound, or 0.1 pM, 1 pM, 50 pM, or 0.5 nM of the peptide of Example 4 was added.

[0073] When mitochondrial activity is suppressed, mitochondrial swelling due to abnormalities of the mitochondrial membrane potential, dysfunction due to oxidative stress such as reactive oxygen species or free radicals, dysfunction due to genetic factors, and dysfunction due to defects in oxidative phosphorylation for mitochondrial energy production occur. Thus, mitochondrial activity may be determined by measuring the mitochondrial membrane potential. Tetramethylrhodamine methyl ester (TMRM) staining of mitochondria was performed, and since TMRM staining intensity is increased in proportion to the mitochondrial membrane potential, the intracellular mitochondrial membrane potential was determined by measuring the TMRM staining intensity using a microplate reader (excitation, 485 nm; emission, 535 nm).

[0074] As a result, as shown in FIG. 5, it was confirmed that the erythropoietin-derived peptides suppressed inhibition of mitochondrial activity caused by increased reactive oxygen species. It could be confirmed that this result was similar to the effect by treatment with the natural erythropoietin compound.

[0075] Determination of Cell Proliferation-Inhibitory Effect of Erythropoietin-Derived Peptide

[0076] Side effects such as cell proliferation were determined for the three peptides (ML1-L2, ML1-K2, and ML1-R2) prepared in Example 4.

[0077] In detail, to determine cell proliferation degree, an MTS assay (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, Wis., USA) was performed. PC12 cells were seeded in a 96-well plate (5×10.sup.4 cells per well), and 1 pM of scrambled peptide (Scr) as a negative control group, 0.5 IU/ml, 1 IU/ml, or 10 IU/ml of the erythropoietin compound, or 1 pM, 10 pM, or 0.5 nM of the peptide of Example 4 was added, and 20 μl of an MTS solution was added to each well, and left for 3 hours. The initial number of cells (0 hour) and the number of cells after 48 hours were counted. Intracellular soluble formazan produced by cell reduction was determined by recording absorbance of each 96-well plate at a wavelength of 490 nm using a VERSA MAX.

[0078] As a result, as shown in FIG. 6, it was confirmed that all the peptides showed cell proliferation rates similar to that of the control group, and showed no side effect of cell proliferation.

[0079] Determination of Cell Proliferation-Inhibitory and -Enhancing Effects of Erythropoietin-Derived Peptide

[0080] To determine the side effect, such as cell proliferation, of the peptides prepared in Examples 1 to 3, cell viability was evaluated by an MTT assay.

[0081] In detail, PC12 cells were cultured in a DMEM (Dulbecco's Modified Eagle's Medium) medium (Hyclone, USA) and an RPM11640 medium (Hyclone, Utah, USA), each supplemented with 10% fetal bovine serum (FBS, Hyclone, Utah, USA), 100 unit/ml penicillin, and 100 μg/ml streptomycin (Hyclone, Utah, USA) in an incubator supplied with 5% CO.sub.2 and under a condition of 37° C. PC12 cell lines were seeded in a 96-well plate at a density of 5×10.sup.4 cells/ml, and cultured under conditions of 37° C. and 5% CO.sub.2 for 24 hours. Thereafter, the cells were treated with each of the peptides of Examples 1 to 3, which were prepared at a concentration of 10 ng/ml, followed by incubation for 24 hours. Thereafter, 20 μl of 5 mg/ml 3-[4,5-dimethyl-thiazol]-2,5-diphenyl-tetrazolium bromide (MTT) reagent was added thereto, and allowed to react for 2 hours. After reaction, 200 μl of dimethyl sulfoxide (DMSO, Duksan, Gyeonggi-do, Korea) was added thereto to completely dissolve formed formazan, and absorbance at 570 nm was measured using a microplate reader (Molecular Devices, CA, USA).

[0082] As a result, as shown in FIG. 7, it was confirmed that all the peptides showed cell proliferation rates similar to that of the control group, while ML1-C2 and ML1-C3 had cell proliferative effects to a degree similar to that of the natural erythropoietin. In other words, the peptides have cell proliferative effects similar to that of the natural erythropoietin and thus may be utilized as an alternative material therefor.

[0083] Comparison of Cell Protective and Proliferative Effects of Erythropoietin-Derived Peptide

[0084] Cell protective and proliferative effects of the seven peptides (ML1, ML1-C1, ML1-C2, ML1-C3, ML1-H1, ML1-H2, and ML1-H3) prepared in Example 3 were statistically compared to that of EPO.

[0085] In detail, the effects shown in FIG. 4 and FIG. 7 were adjusted with respect to the effects by EPO and quantified, and clustering was performed by a K-means clustering algorithm.

[0086] As a result, as shown in FIG. 9, ML1-C1, ML1-C2 and ML1-C3 were clustered as having similar effects as EPO in terms of cell protection and effects. However, it was analyzed that ML1, ML1-H1, ML1-H2, and ML1-H3 showed a large difference in terms of cell proliferative effects compared to EPO groups, while showing no large difference thereto in terms of cell protective effects.

[0087] Determination of In Vivo Hematopoietic Effects of Erythropoietin-Derived Peptide

[0088] In vivo hematopoietic activities in a mouse were measured for the peptides prepared in Example 5, MLP, MLP-C, and MLP-H.

[0089] In detail, the prepared peptide or scrambled (Scr) peptide as a negative control group was administered by intraperitoneal injection to mice at a dose of 0 μg/kg, 1 μg/kg, or 100 μg/kg repeatedly, everyday over 14 days. Thereafter, a body weight test and a blood test were performed.

[0090] As a result, as shown in FIG. 10, it could be confirmed that all the peptides gave rise to no significant changes in body weight of the mice. Further, it could be confirmed that as shown in FIG. 11A, hematocrit, which is the volume of red blood cells in the blood expressed as a volume percentage, was significantly increased according to an administration dose only in the mice treated with MLP-C, and as shown in FIG. 11B, in case of the mice administered with 100 μg/kg, there were significant increases in red blood cell count, hemoglobin content, mean corpuscular hemoglobin concentration, and mean corpuscular hemoglobin. However, as shown in FIG. 11D, it could be confirmed that there was no large change in terms of platelet concentration. In other words, the peptide according to an aspect was able to induce hematopoiesis in vivo, and thus, the peptide is not only able to improve the hematopoietic function, but also may be used in the prevention or treatment of a hematopoietic disorder.

[0091] Determination of Erythropoietin Receptor (EPOR) Activation Regulation by Erythropoietin-Derived Peptide

[0092] Erythropoietin receptor activation levels by MLP-C and MLP-H, among the peptides prepared in Example 5, were compared to that of the natural erythropoietin.

[0093] In detail, a DNA plasmid capable of expressing an erythropoietin receptor fused to green fluorescence protein was transfected into HEK 293 cell lines, and stable cell lines stably expressing the erythropoietin receptor fused to green fluorescence protein were prepared. After treatment with EPO as a positive control group, and MLP-C or MLP-H, a western blot was performed by sampling at time 0, 30, 60, and 90 minutes, and activation levels of the erythropoietin receptor were determined through ERK1/2 phosphorylation levels.

[0094] As a result, as shown in FIG. 12, it could be confirmed that, while EPO and MLP-C exhibit maximum activity at 30 minutes and decline thereafter, MLP-H maintains activation even after 30 minutes. In other words, while MLP-C exhibits a brief but potent activation with respect to the erythropoietin receptor, MLP-H exhibits an activation that is weak yet lasts for a prolonged period of time, and therefore, the erythropoietin-derived peptide according to an aspect is capable of regulating activation of the erythropoietin receptor. Accordingly, the erythropoietin-derived peptide may be used as a therapeutic agent for conditions that require cell proliferative effects, such as anemia.