Peptide and use thereof for treatment of disease of brain and nervous system

Abstract

Peptides are easy to pass through a tissue-blood barrier, have excellent physiological activity in the protective activity of cells, and have an economic advantage due to low production costs. In addition, since there is no side effect of cell proliferation, a pharmaceutical composition containing a peptide of an aspect as an active ingredient can be usefully used in the treatment or prevention of neurological disorders and degenerative brain diseases.

Claims

1. A peptide consisting of an amino acid sequence of SEQ ID NO: 3.

2. The peptide of claim 1, wherein the peptide is capable of binding to an erythropoietin (EPO) receptor.

3. The peptide of claim 1, wherein the peptide exhibits a cytoprotective activity.

4. The peptide of claim 3, wherein the peptide has an activity of protecting neurons or brain cells.

5. The peptide of claim 1, wherein the peptide is a peptide that does not have cell-proliferation side effects.

6. A pharmaceutical composition for preventing or treating stroke, the pharmaceutical composition comprising as an active ingredient, the peptide of claim 1.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other aspects, features, and advantages of certain embodiments of the disclosure will be more apparent from the following description taken in conjunction with the accompanying drawings, in which:

(2) FIG. 1A shows an image showing the structure of erythropoietin and FIG. 1B shows a comparison of the peptide of an aspect and the peptide sequence of erythropoietin;

(3) FIG. 2 shows a graph showing the binding affinity of a peptide of an aspect to an erythropoietin receptor compared with a control group;

(4) FIG. 3 is a graph showing cell viability in a stress situation of a peptide of an aspect; con is a negative control without any treatment; none is a cell which is exposed to stress situation only; EPO is an experimental group treated with erythropoietin; and SY-1, SY-2, and SY-3 are experimental groups treated with peptides of SEQ ID NOs 1 to 3, respectively;

(5) FIG. 4A-4C show graphs of cell viability, reactive oxygen species (ROS), and cell death (TUNEL) in a hypoxic environment (H/R condition) of the peptide of an aspect: FIG. 4A shows a graph of cell viability, FIG. 4B shows a graph of ROS, and FIG. 4C shows a graph of cell death;

(6) FIG. 5 shows a schematic diagram of a hypoxia-reoxygenation experiment;

(7) FIG. 6 shows a graph showing cell viability under hypoxia-reoxygenation conditions when treated with the peptide of an aspect;

(8) FIG. 7 shows a graph of cell viability according to concentrations under hypoxia-reoxygenation conditions when treated with the peptide of an aspect;

(9) FIG. 8 shows a graph showing the expression of apoptosis-related proteins under hypoxia-reoxygenation conditions when treated with the peptide of an aspect;

(10) FIG. 9 shows a graph showing Caspase-9 activity under hypoxia-reoxygenation conditions when treated with the peptide of an aspect;

(11) FIG. 10 shows a graph confirming the cell proliferation inhibitory effect of the peptide of an aspect: con is a negative control without any treatment; none is a cell which is exposed to stress situation only; EPO is an experimental group treated with erythropoietin; and SY-1, SY-2, and SY-3 are experimental groups treated with peptides of SEQ ID NOs 1 to 3, respectively; and

(12) FIG. 11A is an image showing that a peptide of an aspect exhibits a cytoprotective effect in a stroke mouse model; and FIG. 11B is a graph showing the effect of stroke treatment in an animal model compared to a control group.

DETAILED DESCRIPTION

(13) Reference will now be made in detail to embodiments, examples of which are illustrated in the accompanying drawings, wherein like reference numerals refer to like elements throughout. In this regard, the present embodiments may have different forms and should not be construed as being limited to the descriptions set forth herein. Accordingly, the embodiments are merely described below, by referring to the figures, to explain aspects of the present description. As used herein, the term “and/or” includes any and all combinations of one or more of the associated listed items. Expressions such as “at least one of,” when preceding a list of elements, modify the entire list of elements and do not modify the individual elements of the list.

(14) Hereinafter, embodiment are presented to help the understanding of the present disclosure. However, the following examples are provided for easier understanding of the present disclosure, and the contents of the present disclosure are not limited by the following examples.

EXAMPLE

Example 1. Synthesis of Erythropoietin-Induced Peptide

(15) The peptide of an aspect was synthesized as a monomer according to a conventionally known solid phase synthesis technology (Peptron, Daejeon, Korea).

(16) Specifically, erythropoietin-induced peptides capable of binding to amino acid sequences (Arg103, Ser104, Leu105, Leu108, and Arg110) in the sequence of the target region (site 2) of the natural erythropoietin receptor were synthesized, and specific characteristics of each of the peptides were confirmed. A liquid chromatography/mass selective detector (HP 1100 series) was used to measure the concentration of the synthesized peptide. The measurement of purity was carried out by high performance liquid chromatography (SHIMADZU prominence HPLC) analysis (>95% purity).

(17) FIG. 1 shows an image showing the structure of erythropoietin (left) and a comparison of the peptide of an aspect and the peptide sequence of erythropoietin (right).

(18) As a result, as shown in FIG. 1 and Table 1, a novel synthetic peptide was able to be induced.

(19) TABLE-US-00001 TABLE 1 Peptide Sequence name Sequence number SY-1 LQLHVLKAVSGLRTLTTLLRALG 1 SY-2 LQLHVLKAVSGLRTLTMIRRALG 2 SY-3 LQLHVLKAVAGLRTLTMIRRALA 3

(20) For the peptide of an aspect, hydrophobicity, charge, and isoelectric point (pi) were calculated, and are shown in Table 2.

(21) TABLE-US-00002 TABLE 2 Netcharge Extinction Amino (at Hydrophobicity Theoretical Instability Aliphatic GRAVY coefficients Peptide acid pH7.0) (%) MW pl index index index Target (M.sup.−1cm.sup.−1) SY-1 23 3.1 52.17% 2474.03 12.01 36.80 169.57 0.83 91-113 0 (stable) amino acid in human EPO SY-2 23 4.1 52.17% 2547.15 12.3 53.33 152.61 0.613 91 to 113 0 (unstable) amino acid in human EPO SY-3 23 4.1 60.87% 2545.17 12.3 53.33 161.3 0.822 91 to 113 0 (unstable) amino acid in human EPO

Experimental Examples

(22) 1. Confirmation of Binding Affinity of New Peptides

(23) In order to confirm whether the erythropoietin-induced peptide prepared in Example 1 acts by binding to the erythropoietin receptor having a target region, the binding strength was confirmed using a surface plasmon resonance (SPR) technique. The SPR technique uses an optical principle to measure the interaction between biomolecules in real time without a specific label, and is a system that analyzes the affinity and kinetics between two molecules, that is, Ka (association rate) and Kd (dissociation rate).

(24) Specifically, the real-time SPR analysis was performed using Reichert SPR Biosensor SR 7500C equipment (Reichert Inc., NY, USA). Receptor mouse EPOR chimera protein (Soluble mouse EPOR chimera protein)(R&D Systems, Minneapolis, Minn., USA) was covalently bonded to a chip (BR-1005-39, Pharmacia Biosensor AB) coated with carboxy methylated dextran matrix by an amine coupling procedure using an amine coupling kit (BR-1000-50, GE Healthcare, USA) according to the manufacturer's manual. Peptides having concentrations of 5, 2.5 and 1.25 μM according to the present disclosure and scrambled peptides were tested by flowing at a flow rate of 5 μl/minute. In addition, the experiment was independently repeated to increase the accuracy. In addition, DMSO was flowed at 5 μl/minute to normalize the signal. After sufficiently inducing the binding of the peptide of an aspect to the receptor, regeneration was induced by injecting 20 μl/minute of 25 mM acetic acid into a sensor chip.

(25) FIG. 2 shows a graph showing the binding affinity of a peptide of an aspect to an erythropoietin receptor compared with a control group.

(26) As a result, as shown in FIG. 2, it was confirmed that the result value increased in proportion to the concentration of the erythropoietin-induced peptide of an embodiment. This suggests that the peptide of an aspect binds to the target portion of the erythropoietin receptor. In addition, as shown in Table 3, the erythropoietin-induced peptide of an embodiment appears similar to or slightly greater than the previously known binding affinity (up to 1 μM).

(27) TABLE-US-00003 TABLE 3 SY-1 ka kd KD = Kd/Ka 31.5970 0.0446 1.41061 mM SY-2 ka kd KD 32.5770 0.0506 1.55398 mM SY-3 ka kd KD 25.6240 0.0554 2.16266 mM

(28) That is, it can be seen that the binding affinity of the peptide according to an embodiment with respect to the erythropoietin receptor, has a KD value of 0.6 mM to 3.0 mM, for example, 1.41061 mM, 1.55398 mM, and 2.16266 mM.

(29) 2. Confirmation of the Cytoprotective Effect of New Peptides

(30) 2.1 Confirmation of Cell Viability in Stressful Situations

(31) To confirm whether the peptide of Example 1 exhibits a cytoprotective effect, cell viability was confirmed in a stress situation in which an increase in reactive oxygen was induced with hydrogen peroxide (H.sub.2O.sub.2).

(32) Specifically, cell viability was evaluated using MTS analysis (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, Wis., USA). PC12 cells were seeded into a 96 well plate (5×10.sup.4 cells per well/), and an increase in reactive oxygen was induced with 300 μM hydrogen peroxide (H.sub.2O.sub.2). Next, 0.5 IU/ml of an erythropoietin compound, and a peptide of an aspect were added, separately, and then 20 μl of MTS solution was added to each well, followed by waiting for 3 hours. Next, the absorbance of each well of the 96-well plate was recorded using VERSA MAX at a wavelength of 490 nm to compare the initial cell number (0 hour) and the cell number after 48 hours.

(33) FIG. 3 is a graph showing cell viability in a stress situation of a peptide of an aspect; con is a negative control without any treatment; none is a cell which is exposed to stress situation only; EPO is an experimental group treated with erythropoietin; and SY-1, SY-2, and SY-3 are experimental groups treated with peptides of SEQ ID NOs 1 to 3, respectively.

(34) As a result, as shown in FIG. 3, it was confirmed that the peptide of an aspect protects cells from apoptosis due to an increase in active oxygen. This shows that the peptide of an aspect exhibits a remarkable cell protective effect compared to when treated with a natural erythropoietin compound.

(35) 2.2 Confirmation of Cell Viability in Hypoxic Environment

(36) Cell viability, reactive oxygen species (ROS), and cell death (TUNEL) were evaluated to confirm whether the produced peptide exhibits a protective effect on cells in a hypoxic environment (H/R condition).

(37) First, the degree of cell viability was confirmed. Specifically, HT-22 cells cultured on a 96-well plate or a 6-well plate for 24 hours were maintained under hypoxic condition for 18 hours in a sealed airtight container of Anaeropack (Mitsubishi Gas Company, Tokyo, Japan). The Anaeropack absorbs oxygen and generates carbon dioxide, thereby inducing a hypoxic environment. Next, reoxygenation was performed at 5% CO.sub.2 and 37° C. for 3 hours. Next, after 19 hours of treatment with EPO or a peptide of an aspect in the hypoxic environment, cell viability assays were performed.

(38) FIG. 4 shows graphs of cell viability, reactive oxygen species (ROS), and cell death (TUNEL) in a hypoxic environment (H/R condition) of the peptide of an aspect: A shows a graph of cell viability, B shows a graph of ROS, and C shows a graph of cell death.

(39) As a result, as shown in FIG. 4, it was confirmed that the peptide of an aspect protects cells from apoptosis due to H/R condition. In addition, it can be seen that this effect is remarkable compared to the cell protective effect obtained by treatment with a natural erythropoietin compound.

(40) Next, intracellular ROS was measured.

(41) Specifically, HT22 cells were seeded into each well of a 96-well plat and cultured for 24 hours. After treatment with erythropoietin and the peptide of an aspect according to the conditions of each group, 5 μM of CM-H2DCFDA was added and an additional culture was performed for 30 minutes. Next, after removing residual H2DCFDA that did not bind to ROS by using PBS, the fluorescence intensity of DCF was measured at 488 nm/525 nm (ex/em) by using a fluorescent plate reader.

(42) As a result, as shown in FIG. 4, it was confirmed that the prepared peptide protects cells from ROS caused by H/R conditions. In addition, it can be seen that this effect is remarkable compared to the cell protective effect obtained by treatment with a natural erythropoietin compound.

(43) Next, cell death (TUNEL) was measured.

(44) Specifically, the culture solution was first removed, washing was performed three times with phosphate-buffered saline (PBS), and then, the cells were immobilized with 4% PFA for 10 minutes at room temperature. Next, after washing twice with PBS for 5 minutes, proteinase K (20 μg/ml) was instilled and the cells were incubated for 10 minutes at 37° C. After washing, 1% H.sub.2O.sub.2 (in PBS) was reacted at room temperature for 5 minutes, washing was performed using PBS, 75 μl of equilibration buffer was instilled, and then, the reaction was carried out for 10 seconds, and then the equilibration buffer was removed. Next, after instilling 55 μl/5 cm.sup.2 of working strength TdT (terminal deoxynucleotidyl transferase) enzyme, the reaction was caused for 1 hour while humidity was maintained at 37° C., and a working strength stop/wash buffer was added thereto and a reaction was used at room temperature for 10 minutes. After washing with PBS, 65 μl/5 cm.sup.2 of anti-digoxigenin peroxidase was instilled and a reaction was caused at room temperature for 30 minutes while humidity was maintained constant. Finally, after washing with PBS, a reaction was caused with a mixed solution including 0.02% 3,3-diaminobenzidine tetrahydrochloride (DAB), which is a coloring agent, and 0.003% H.sub.2O.sub.2 at room temperature for 10 minutes, and then the result was observed with an optical microscope.

(45) As a result, as shown in FIG. 4, it was confirmed that the peptide of an aspect protects cells from apoptosis due to H/R condition.

(46) 2.3 Comparison of Cell Viability of Peptides in Hypoxic Environment

(47) Cell viability was compared when treated with SY-1, SY-2 and SY-3 of H/R cells cultured under low oxygen conditions (Hypoxia) and later cultured under oxygen conditions (Reoxygenation).

(48) FIG. 5 shows a schematic diagram of a hypoxia-reoxygenation experiment.

(49) As shown in FIG. 5, the hypoxic step was performed on the cells for 18 hours, and then reoxygenation was performed for 3 hours. Thereafter, 19 hours after the treatments with peptides of an aspect SY-1, SY-2, and SY-3 and control groups EPO, ML1, and ML1-1, a reaction was identified. At this time, the concentration of EPO was 0.4 IU.

(50) FIG. 6 shows a graph showing cell viability under hypoxia-reoxygenation conditions when treated with the peptide of an aspect.

(51) FIG. 7 shows a graph of cell viability according to concentrations under hypoxia-reoxygenation conditions when treated with the peptide of an aspect.

(52) Table 4 show cell viability under hypoxia-reoxygenation conditions according to concentrations.

(53) TABLE-US-00004 TABLE 4 ML1 (#1) cont H/R EPO 1 uM 2.5 uM 5 uM 1.123 0.628 0.745 0.705 0.662 0.698 1.106 0.611 0.761 0.673 0.693 0.671 1.109 0.614 0.755 0.696 0.696 0.682 Average 1.113 0.618 0.754 0.691 0.684 0.684 STDEV 0.009 0.009 0.008 0.017 0.019 0.014 ML1-1 (#9) cont H/R EPO 1 uM 2.5 uM 5 uM 1.114 0.638 0.747 0.694 0.673 0.681 1.109 0.622 0.759 0.668 0.696 0.688 1.008 0.611 0.764 0.689 0.675 0.665 Average 1.077 0.624 0.757 0.684 0.681 0.678 STDEV 0.060 0.014 0.009 0.014 0.013 0.012 SY-1 cont H/R EPO 1 uM 2.5 uM 5 uM 1.135 0.627 0.745 0.662 0.649 0.648 1.112 0.616 0.761 0.657 0.663 0.653 1.126 0.619 0.765 0.644 0.652 0.641 Average 1.124 0.621 0.757 0.654 0.655 0.647 STDEV 0.012 0.006 0.011 0.009 0.007 0.006 SY-2 cont H/R EPO 1 uM 2.5 uM 5 uM 1.125 0.631 0.758 0.658 0.671 0.652 1.109 0.604 0.763 0.673 0.667 0.677 1.122 0.614 0.761 0.682 0.689 0.655 Average 1.119 0.616 0.761 0.671 0.676 0.661 STDEV 0.009 0.014 0.003 0.012 0.012 0.014 SY-3 cont H/R EPO 1 uM 2.5 uM 5 uM 1.113 0.628 0.747 0.722 0.739 0.707 1.101 0.611 0.776 0.736 0.741 0.732 1.092 0.614 0.763 0.719 0.724 0.741 average 1.102 0.618 0.762 0.726 0.735 0.727 STDEV 0.011 0.009 0.015 0.009 0.009 0.018

(54) As a result, as shown in FIGS. 5 to 7 and Table 4, it was confirmed that the cell viability was higher when treated with the peptide of an aspect than treated with the control groups, and when treated with SY-3, the cell viability was the highest.

(55) Next, the expression levels of the apoptosis-related proteins Caspase-9, Caspase-3, Bax, and β-actin of the cells (H/R cells) were measured using Western blot and compared.

(56) FIG. 8 shows a graph showing the expression of apoptosis-related proteins under hypoxia-reoxygenation conditions when treated with the peptide of an aspect.

(57) FIG. 9 shows a graph showing Caspase-9 activity under hypoxia-reoxygenation conditions when treated with the peptide of an aspect.

(58) As a result, as shown in FIGS. 8 and 9, the expression of the apoptosis-related proteins Caspase-9, Caspase-3, Bax and β-actin was lower when treated with the peptide of an aspect than when treated with the control groups, and in particular, when treated with SY-3, the cell viability was the highest.

(59) 4. Confirmation of Cell Proliferation Inhibitory Effect of New Peptide

(60) Side effects such as cell proliferation of the three peptides SY-1, SY-2 and SY-3 prepared in Example 1 were confirmed.

(61) Specifically, MTS analysis (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, Wis., USA) method was used. PC12 cells were seeded into a 96 well plate (5×10.sup.4 cells per well), and then an erythropoietin compound and a peptide of an aspect were added thereto. Next, 20 μl of the MTS solution was added to each well and the cells were allowed to stand for 3 hours. Initial cell count (0 hours) and cell count after 48 hours were measured. Intracellular soluble formazan was determined by recording the absorbance of each well of the 96 well plate by using VERSA MAX at a wavelength of 490 nm.

(62) As a result, as shown in FIG. 10, the cell proliferation rate of all of the peptides of an aspect was similar to those of the control groups, and as a result, it was confirmed that there were no cell-proliferation side effects.

(63) 4. Confirmation of Stroke Treatment and Brain Protection Effects of Novel Peptides in Animal Models

(64) 4.1 Preparation of Animal Models

(65) An MCAO animal model was prepared for use as the animal model used in this experiment.

(66) First, a mouse was anesthetized with a mixture of 70% N.sub.2O and 30% O.sub.2 and 3% isoflurane, and the anesthesia was maintained with 1.5 to 2.0% isofluorene. The neck skin of the experimental animal stably anesthetized was incised, and then, the bifurcation of the common carotid artery was exposed. The external carotid artery of the exposed part was tied and the internal carotid artery was carefully separated from the vagus nerve. Through the external carotid stump, the silicone coated 7-0 surgical nylon monofilament was pushed to the end of the middle cerebral artery (MCA) through the right internal carotid artery. Laser doppler flowmetry (perimed 5000 system, sweden) was used to determine whether focal cerebral ischemia was induced. During the operation, a heating pad (CMA 150, Stockholm, Sweden) was used to monitor the temperature of the rectum and the body temperature was maintained at 37±0.5° C. The nylon monofilament was maintained for 90 minutes, and then the monofilament structure was removed therefrom and re-perfusion was performed. Recovery of the clogged blood flow was confirmed using a laser Doppler flow meter. Next, for recovery after surgery, the mouse were recovered for 3 hours in a warm box at room temperature (30° C.). After the end of the re-perfusion time (24 hr. after reperfusion following 90 min MCAO), the brain was isolated by a humane method to obtain a brain tissue sample.

(67) 4.2 Confirmation of the Brain Protective Effect of the Novel Peptide

(68) In order to confirm whether the peptide according to an aspect was effective in animal experiments as in cell experiments, 24 hours after damage to the brain of the animal model of 4.1, the animal model was cut off, and the brain was excised and washed with refrigerated physiological saline. Next, the washed brain was placed in a brain matrix (Stoelting Co, USA) and cut into 2 mm thickness by using a cutting knife to prepare a brain section. The prepared brain section was put in 15 ml of 0.125% TTC solution (62.5 mM Tris-HCl, 13 mM MgCl.sub.2, 1.5% DMF), stained for 90 minutes at 37° C., washed with physiological saline, and imaged using a scanner. The obtained data was stored and analyzed on a computer.

(69) As a result, as shown in FIG. 11, it was confirmed that the peptide of an aspect has an effect of protecting the brain and treating a stroke in the mouse stroke model. In addition, in the case of the animal experiment, the effect of the new peptide compared to erythropoietin was much clearer than that of the cell experiment.

(70) Peptides according to an aspect are easy to pass through a tissue-blood barrier, have excellent physiological activity in the protective activity of cells, and have an economic advantage due to low production costs. In addition, since there is no side effect of cell proliferation, a pharmaceutical composition containing a peptide of an aspect as an active ingredient can be usefully used in the treatment or prevention of cranial nervous system diseases.

(71) It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments. While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.