U.S. patent application number 16/297670 was filed with the patent office on 2019-09-19 for bifunctional peptide having capability to reduce inflammation and capability to facilitate differentiation of stem cells into ch.
The applicant listed for this patent is Nano Intelligent Biomedical Engineering Corporation Co. Ltd., Seoul National University R&DB Foundation. Invention is credited to Chong-Pyoung Chung, Jue-Yeon Lee, Yoon Jeong Park.
Application Number | 20190284237 16/297670 |
Document ID | / |
Family ID | 65801964 |
Filed Date | 2019-09-19 |
United States Patent
Application |
20190284237 |
Kind Code |
A1 |
Park; Yoon Jeong ; et
al. |
September 19, 2019 |
BIFUNCTIONAL PEPTIDE HAVING CAPABILITY TO REDUCE INFLAMMATION AND
CAPABILITY TO FACILITATE DIFFERENTIATION OF STEM CELLS INTO
CHONDROCYTES AND USE THEREOF
Abstract
Disclosed are a bifunctional peptide having the capability to
reduce inflammation and the capability to facilitate
differentiation of stem cells into chondrocytes and the use
thereof. Advantageously, the bifunctional peptide is useful for the
prevention or treatment of arthritis accompanied by inflammation
and damage of cartilage tissue due to excellent effects of reducing
inflammation and facilitating differentiation of stem cells into
chondrocytes, can be easily applied to various surgical
regenerative treatments including orthopedic treatments, and can
shorten the treatment period.
Inventors: |
Park; Yoon Jeong; (Seoul,
KR) ; Chung; Chong-Pyoung; (Seoul, KR) ; Lee;
Jue-Yeon; (Gyeonggi-do, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Seoul National University R&DB Foundation
Nano Intelligent Biomedical Engineering Corporation Co.
Ltd. |
Seoul
Chungcheongbuk-do |
|
KR
KR |
|
|
Family ID: |
65801964 |
Appl. No.: |
16/297670 |
Filed: |
March 10, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61L 27/227 20130101;
A61P 19/02 20180101; A61L 2300/41 20130101; A61K 38/00 20130101;
A61L 2300/25 20130101; C07K 7/08 20130101; A61L 27/54 20130101;
A61K 38/10 20130101 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61P 19/02 20060101 A61P019/02 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2018 |
KR |
10-2018-0029912 |
Claims
1. A peptide represented by an amino acid sequence of any one of
SEQ ID NOS: 4 to 8.
2. The peptide according to claim 1, wherein the peptide is a
bifunctional peptide having a capability to reduce inflammation and
a capability to facilitate the differentiation of stem cells into
chondrocytes.
3. A method for preventing or treating arthritis, comprising
administering a peptide represented by an amino acid sequence of
any one of SEQ ID NOS: 1 to 8.
4. The method according to claim 3, wherein the peptide is a
bifunctional peptide having a capability to reduce inflammation and
a capability to facilitate the differentiation of stem cells into
chondrocytes.
5. The method according to claim 3, wherein the peptide is prepared
for injection or topical implantation.
6. The method according to claim 3, wherein the peptide is
administered at a dose of 0.001 to 1,000 mg per 1 kg of a body
weight of a subject in need of treatment.
7. A biomaterial for treating arthritis comprising, as an active
ingredient, a peptide represented by an amino acid sequence of any
one of SEQ ID NOS: 1 to 8.
8. The biomaterial according to claim 7, wherein the peptide is a
bifunctional peptide having a capability to reduce inflammation and
a capability to facilitate the differentiation of stem cells into
chondrocytes.
9. The biomaterial according to claim 7, wherein the biomaterial
comprises any one selected from the group consisting of a barrier
membrane, a porous scaffold, and an implant for bio-implant
produced in the form of a gel.
10. The biomaterial according to claim 9, wherein the barrier
membrane comprises any one selected from the group consisting of
collagen, chitosan, gelatin, polylactide, polylactide glycolide and
polycaprolactone.
11. The biomaterial according to claim 9, wherein the porous
scaffold comprises any one selected from the group consisting of
collagen, chitosan, gelatin, hyaluronic acid, alginic acid,
chondroitin sulfate, polylactide, polylactide glycolide, and
polycaprolactone.
12. The biomaterial according to claim 9, wherein the gel comprises
any one selected from the group consisting of collagen, chitosan,
gelatin, hyaluronic acid, alginic acid, poloxamer, propylene
glycol, propylene glycol alginate, chondroitin sulfate and
glycosaminoglycan.
13. The biomaterial according to claim 7, wherein the peptide is
present at a dose of 1 to 50 mg with respect to a unit weight (1 g)
of the biomaterial.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] The priority under 35 USC 119 of Korean Patent Application
No. 10-2018-0029912 filed Mar. 14, 2018 is hereby claimed. The
disclosure of Korean Patent Application No. 10-2018-0029912 is
hereby incorporated herein by reference in its entirety, for all
purposes.
TECHNICAL FIELD
[0002] The present invention relates to a bifunctional peptide
having the capability to reduce inflammation and the capability to
facilitate differentiation from stem cells into chondrocytes and
the use thereof. More particularly, the present invention relates
to a peptide represented by an amino acid sequence of any one of
SEQ ID NOS: 1 to 8, a pharmaceutical composition for preventing or
treating arthritis and a biomaterial for treating arthritis
comprising the same.
BACKGROUND ART
[0003] Damage to cartilage tissue constituting joints may cause
arthritis accompanied by swelling, fever and pain. Arthritis is
classified into about 100 or more types depending on the cause
thereof. The most common type of arthritis is osteoarthritis, which
is a degenerative joint disease mainly caused by aging, and other
types of arthritis include rheumatoid arthritis and psoriatic
arthritis, which are autoimmune diseases, septic arthritis due to
infection and the like.
[0004] Osteoarthritis is a disease in which the joint cartilage
surrounding the joint surface of the bone is worn out, the bone
under the cartilage is exposed, and the synovium around the joint
is inflamed, resulting in pain and deformation. The joints have
soft cartilage serving as a buffer and this results from changes of
cartilage due to aging, improper mechanical pressure, genetic
causes, obesity, trauma to the joints, and cartilage damage due to
inflammation (Blagojevic, M. et al., Osteoarthritis Cartilage 18:
24-33 (2010). Felson, D. T. et al., Osteoarthritis: new insights.
Part 1: The Disease and its risk factors. Ann Intern Med 133:
635-646 (2000).).
[0005] Rheumatoid arthritis is a type of autoimmune disease in
which the body's immune system attacks itself and the synovial
membrane surrounding the joints is inflamed, and may develop in all
age groups from infants to the elderly (Gay S, et al., Ann. Rheum.
Dis. 52: S39-S40 (1993)).
[0006] Cartilage is a tissue including only a cartilage cell, as a
single cell, and an extracellular matrix, and is susceptible to
wear with aging. The cartilage cell is surrounded by the rigid
extracellular matrix and thus is difficult to regenerate once
damaged or degenerated.
[0007] Currently, analgesics, steroids, and nonsteroidal
anti-inflammatory drugs, which are drugs used to treat damaged
cartilage, only nonspecifically relieve pain and inflammation, but
have no fundamental therapeutic effect. Cartilage protectants such
as hyaluronic acid, glucosamine and chondroitin only function to
temporarily protect the joints by alleviating impacts. Bone marrow
stimulation, which is a clinical surgical method used in the
orthopedic surgery, is a method of filling cartilage damage with a
blood clot containing stem cells derived from bone marrow and has
an advantage in that a surgery process is relatively easy and
simple. However, the method is disadvantageous in that, after
surgery, the damaged cartilage is regenerated as fibrous cartilage
rather than hyaline cartilage. Bone-cartilage tissue grafting is a
method of collecting bone-cartilage connective tissue in the site
with a less weight from the patient's own cartilage tissue and then
implanting the bone-cartilage connective tissue into the damaged
cartilage site. Disadvantageously, this method cannot be used to
treat large cartilage damage sites.
[0008] In order to overcome this disadvantage, efforts are being
made to regenerate cartilage using stem cells, scaffolds and growth
factors, which are three major components of tissue engineering.
However, it is difficult for stem cells injected alone into the
damaged cartilage sites to survive and differentiate. In order to
solve this problem, a method of transferring cells using
polymer-based scaffolds is used, but the scaffolds may
disadvantageously cause a spatial limitation, when the cells
proliferate in the scaffolds or when the extracellular matrix is
secreted. Thus, the method of completely regenerating damaged
cartilage remains incomplete.
[0009] In the case of cartilage damage accompanied by inflammation
such as osteoarthritis and rheumatoid arthritis, it is difficult to
regenerate cartilage without treating the inflammation, although
various techniques for cartilage regeneration are attempted.
Therefore, two functions of inflammation treatment and cartilage
regeneration are needed.
[0010] Histone deacetylase (HDAC) is an enzyme that deacetylates
histones to condense chromatin and inhibit gene transcription. A
histone protein is an element that constitutes the nucleosome with
DNA. The structure of the histone protein can be variously
modified. Histone acetylation and methylation, along with DNA
methylation, are important acquired mechanisms to regulate the
expression of certain genes. In general, when the expression of the
certain genes is activated, the histone tail is acetylated by
histone acetyltransferase (HAT), whereas, when the histone tail is
deacetylated by histone deacetylase, i.e., HDAC, the expression of
certain genes is inactivated, that is, gene expression is
inhibited.
[0011] The roles of HDACs in treating a variety of diseases,
including inflammatory diseases and degenerative diseases including
neuropathy, and modulators thereof have been studied. Previous
studies have shown that HDAC inhibitors are effective not only in
treating inflammation, but also in facilitating specific
differentiation of stem cells into nerve, bone and liver tissues.
However, there are no peptide-based HDAC inhibitors other than low
molecular-weight synthetic substances and hereditary substances
such as siRNA.
[0012] Accordingly, as a result of intensive studies to solve the
problems of the prior art as described above, the present inventors
have found a specific peptide, which is capable of facilitating
differentiation of stem cells into chondrocytes while reducing
inflammation of joint by inhibiting the expression of HDAC and the
production of cytokine interleukin 6, and have completed the
present invention on the basis thereof.
DISCLOSURE
Technical Problem
[0013] Therefore, it is one object of the present invention to
provide a bifunctional peptide having the capability to reduce
inflammation and the capability to facilitate differentiation of
stem cells into chondrocytes.
[0014] It is another object of the present invention to provide a
pharmaceutical composition for preventing or treating arthritis
comprising the bifunctional peptide.
[0015] It is another object of the present invention to provide a
biomaterial for treating arthritis comprising the bifunctional
peptide.
Technical Solution
[0016] In accordance with the present invention, the above and
other objects can be accomplished by the provision of a
bifunctional peptide represented by an amino acid sequence of any
one of SEQ ID NOS: 1 to 8.
[0017] In accordance with another aspect of the present invention,
provided is a pharmaceutical composition for preventing or treating
arthritis comprising the bifunctional peptide as an active
ingredient.
[0018] In accordance with yet another aspect of the present
invention, provided is a biomaterial for treating arthritis
comprising the bifunctional peptide.
[0019] In accordance with another aspect of the present invention,
provided is a method for preventing or treating arthritis
comprising administering to the subject the bifunctional
peptide.
[0020] In accordance with another aspect of the present invention,
provided is the bifunctional peptide for use in a method of
preventing or treating arthritis.
[0021] In accordance with another aspect of the present invention,
provided is a pharmaceutical composition comprising the
bifunctional peptide for use in a method of preventing or treating
arthritis.
[0022] In accordance with another aspect of the present invention,
provided is a use of the bifunctional peptide for the manufacture
of a medicament for preventing or treating arthritis.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] The above and other objects, features and other advantages
of the present invention will be more clearly understood from the
following detailed description taken in conjunction with the
accompanying drawings, in which:
[0024] FIG. 1 show the results of Western blotting (A) and band
density (B) to identify the degree of protein expression of HDAC1
and HDAC5 after treatment of RAW 264.7 cells with the peptides of
SEQ ID NOS: 1 to 8 and then induction of inflammation by LPS
(lipopolysaccharide).
[0025] FIG. 2 show the results of measurement of the amount of
produced cytokine interleukin 6 after treatment of RAW 264.7 cells
with different concentrations of the peptides of SEQ ID NOS: 1 to 8
and induction of inflammation by LPS.
[0026] FIG. 3 show the expression levels of collagen type II and
aggrecan genes measured using RT-PCR after treatment of mesenchymal
stem cells (MSCs) with peptides of SEQ ID NOS: 1 and 8 and culture
of the MSCs in a cartilage differentiation medium for 14 days.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as appreciated by those skilled
in the field to which the present invention pertains. In general,
the nomenclature used herein is well-known in the art and is
ordinarily used.
[0028] In the present invention, the experiment was carried out by
preparing a bifunctional peptide having the capability to
facilitate the differentiation of stem cells into chondrocytes and
the capability to reduce inflammation, and then treating RAW 264.7
cells and mesenchymal stem cells (MSCs) having inflammation induced
by LPS (lipopolysaccharide) with the bifunctional peptide. As a
result, it was found that the expression of HDAC1 and HDAC5 and the
production of inflammatory cytokine interleukin 6 (IL-6) were
decreased and that the expression of cartilage-cell-specific genes
was increased.
[0029] Accordingly, in one aspect, the present invention is
directed to a peptide represented by an amino acid sequence of any
one of SEQ ID NOS: 1 to 8.
TABLE-US-00001 SEQ ID NO: 1: GKCSTRGRKCCRRKK SEQ ID NO: 2:
GKCSTRGRKCMRRKK SEQ ID NO: 3: GKCSTRGRKMCRRKK SEQ ID NO: 4:
GKCSTRGRKMMRRKK SEQ ID NO: 5: GKMSTRGRKCCRRKK SEQ ID NO: 6:
GKMSTRGRKMCRRKK SEQ ID NO: 7: GKMSTRGRKCMRRKK SEQ ID NO: 8:
GKMSTRGRKMMRRKK
[0030] In the present invention, SEQ ID NO: 2 is obtained by
substituting cysteine of SEQ ID NO: 1 with methionine, wherein such
substitution aims at preventing oxidation in the SS bond present in
adjacent cysteine-cysteine or distant cysteine-cysteine structures.
The total number of cysteines in SEQ ID NO: 1 is 3, and SEQ ID NOS:
2 to 8 are obtained by substituting these cysteines with
methionine.
[0031] In the present invention, the peptide may have dual
functions including the capability of reducing inflammation and the
capability of facilitating differentiation of stem cells into
chondrocytes.
[0032] In the present invention, the peptide is preferably produced
through a solid-phase peptide synthesis, when taking into
consideration the fact that the peptide can be produced in a large
amount compared to a protein produced by a recombinant DNA
technique, but the present invention is not limited thereto.
[0033] Meanwhile, the present inventors have found through in-vitro
experiments that the peptide can reduce the inflammation of cells
and the capability to facilitate differentiation of stem cells into
chondrocytes.
[0034] In another aspect, the present invention is directed to a
pharmaceutical composition for preventing or treating arthritis
comprising the bifunctional peptide as an active ingredient.
[0035] As used herein, the term "composition" is considered to
include any product containing a certain ingredient as well as any
product that is prepared directly or indirectly by a combination of
certain ingredients.
[0036] In the present invention, the composition may further
contain a pharmaceutically acceptable carrier, wherein the carrier
is selected from the group consisting of saline, sterilized water,
Ringer's solution, buffered saline, dextrose solution, maltodextrin
solution, glycerol, and ethanol, but the present invention is not
limited thereto.
[0037] In the present invention, the composition may further
contain at least one adjuvant selected from the group consisting of
an excipient, a buffer, an antimicrobial antiseptic, a surfactant,
an antioxidant, a tonicity adjuster, a preservative, a thickener
and a viscosity modifier, but the present invention is not limited
thereto. Each ingredient can be selected from a raw material
commonly used in the art and suitably modified and used within the
range acceptable for the art.
[0038] In the present invention, the composition may be formulated
into any one selected from the group consisting of injections, oral
mucosa agents, capsules, transdermal agents and gel-type agents,
but the present invention is not limited thereto, and can be
prepared into a suitable formulation using a method well-known in
the art (Joseph Price Remington, Remington's Pharmaceutical
Science, 17th edition, Mack Publishing Company, Easton. Pa.).
[0039] In the present invention, the composition may be
administered through a route commonly used in the pharmaceutical
field, preferably parenteral administration. For example, the
composition may be administered through an intravenous,
intraperitoneal, intramuscular, intraarterial, oral, intracardiac,
intramedullary, intrathecal, transdermal, intestinal, subcutaneous,
sublingual or topical administration route. In general, the
composition contains, as an active ingredient, a therapeutically
effective amount of the bifunctional peptide having the capability
to reduce inflammation activity and the capability to facilitate
differentiation of stem cells into chondrocytes according to the
present invention.
[0040] In the present invention, the preparation for topical
administration may be in the form of a gel or film. The gel base
preferably includes one or more selected from the group consisting
of collagen, chitosan, hyaluronic acid, alginic acid, propylene
glycol, propylene glycol alginate, poloxamer and chondroitin
sulfate, but the present invention is not limited thereto.
[0041] In the present invention, the composition is preferably
administered at a dose of 0.001 to 1,000 mg, more preferably 0.1 to
100 mg, per 1 kg of a body weight of the subject in need of
treatment, but is not limited thereto, and the range of the dose
may vary according to body weight, age, gender, a state of health
and diet of a patient, administration time, administration method,
excretion rate and severity of disease. The dose can be determined
in consideration of these factors by an ordinary expert in the
art.
[0042] In another aspect, the present invention is directed to a
method for preventing or treating arthritis comprising
administering to the subject the bifunctional peptide.
[0043] In another aspect, the present invention is directed to the
bifunctional peptide for use in a method of preventing or treating
arthritis.
[0044] In another aspect, the present invention is directed to a
pharmaceutical composition comprising the bifunctional peptide for
use in a method of preventing or treating arthritis.
[0045] In another aspect, the present invention is directed to a
use of the bifunctional peptide for preventing or treating
arthritis.
[0046] In another aspect, the present invention is directed to a
use of the bifunctional peptide for the manufacture of a medicament
for preventing or treating arthritis.
[0047] In another aspect, the present invention is directed to a
biomaterial for treating arthritis comprising the bifunctional
peptide.
[0048] In the present invention, in order to bind the bifunctional
peptide sequence to the biomaterial, a chemical bond can be induced
using a cross-linking agent. At this time, treatment is carried out
to introduce a functional group, for example, a thiol group (--SH),
which is capable of binding to the cysteine at the end of the
peptide, or to form amine (NH.sub.2), so that the bifunctional
peptide can be smoothly applied to a cross-linking reaction using a
cross-linking agent.
[0049] Examples of the crosslinking agent that can be used in the
present invention include, but are not limited to,
1,4-bismaleimidobutane (BMB), 1,11-bismaleimidotetraethyleneglycol
(BM[PEO]4), 1-ethyl-3-[3-dimethyl aminopropyl]carbodiimide
hydrochloride (EDC),
succinimidyl-4-[N-maleimidomethylcyclohexanel-carboxy-[6-amidocaproate]]
(SMCC) and sulfonates thereof (sulfo-SMCC), succinimidyl
6-[3-(2-pyridyldithio)-propionamido] hexanoate (SPDP) and
sulfonates thereof (sulfo-SPDP),
m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and sulfonates
thereof (sulfo-MBS), and succinimidyl [4-(p-maleimidophenyl)
butyrate] (SMPB) and sulfonates thereof (sulfo-SMPB).
[0050] In the present invention, the biomaterial may include any
one selected from the group consisting of a barrier membrane, a
porous scaffold, and an implant for bio-implant produced in the
form of a gel, but the present invention is not limited thereto.
Any kind and form of barrier membrane, porous scaffold and gel used
in the art can be used.
[0051] In the present invention, the barrier membrane is preferably
selected from the group consisting of collagen, chitosan, gelatin,
polylactide, polylactide glycolide and polycaprolactone, but the
present invention is not limited thereto.
[0052] In addition, the porous scaffold may be any one selected
from the group consisting of collagen, chitosan, gelatin,
hyaluronic acid, alginic acid, chondroitin sulfate, polylactide,
polylactide glycolide, and polycaprolactone, but the present
invention is not limited thereto.
[0053] The gel is preferably selected from the group consisting of
collagen, chitosan, gelatin, hyaluronic acid, alginic acid,
poloxamer, propylene glycol, propylene glycol alginate, chondroitin
sulfate and glycosaminoglycan, but the present invention is not
limited thereto.
[0054] Hereinafter, the present invention will be described in more
detail with reference to the following examples. However, it will
be obvious to those skilled in the art that these examples are
provided only for illustration of the present invention and should
not be construed as limiting the scope of the present
invention.
EXAMPLE 1
Synthesis of Bifunctional Peptide Having Capability to Reduce
Inflammation and Capability to Facilitate Differentiation of Stem
Cells into Chondrocytes
[0055] Bifunctional peptides (SEQ ID NOS: 1 to 8) having
anti-inflammatory activity and the capability to facilitate
differentiation of stem cells into chondrocytes were synthesized in
a predetermined order from the N terminal through F-moc solid-phase
peptide synthesis. The synthesized peptide sequences were cleaved
from the resin, washed, lyophilized and then separated and purified
by liquid chromatography. The molecular weights of the purified
peptides were identified through a MALDI-TOF assay.
TABLE-US-00002 SEQ ID NO: 1: GKCSTRGRKCCRRKK SEQ ID NO: 2:
GKCSTRGRKCMRRKK SEQ ID NO: 3: GKCSTRGRKMCRRKK SEQ ID NO: 4:
GKCSTRGRKMMRRKK SEQ ID NO: 5: GKMSTRGRKCCRRKK SEQ ID NO: 6:
GKMSTRGRKMCRRKK SEQ ID NO: 7: GKMSTRGRKCMRRKK SEQ ID NO: 8:
GKMSTRGRKMMRRKK
EXAMPLE 2
Preparation of Pharmaceutical Composition for Preventing or
Treating Arthritis Comprising Bifunctional Peptide Having
Capability to Reduce Inflammation and Capability to Facilitate
Differentiation of Stem Cells into Chondrocytes
[0056] A pharmaceutical composition for preventing or treating
arthritis comprising the bifunctional peptide having the capability
to reduce inflammation and the capability to facilitate
differentiation of stem cells into chondrocytes, as an active
ingredient, was prepared (Table 1).
TABLE-US-00003 TABLE 1 Ingredients Weight (mg) Bifunctional
peptides (SEQ ID NOS: 1 to 8) 1 Sodium chloride, USP 8.18 Sodium
succinate 1.62 WFI 987.5 Sodium hydroxide, NF and/or acetic acid,
NF Total 1 g, pH 6
EXAMPLE 3
Production of Biomaterial for Treating Arthritis Including
Bifunctional Peptide Having Capability to Reduce Inflammation and
Having Capability to Facilitate Differentiation of Stem Cells into
Chondrocytes
[0057] The collagen biomaterial produced in the form of a barrier
membrane or a porous scaffold was added to 2 ml of phosphate buffer
solution (pH 7.4) to hydrate the surface thereof, sulfo-SMCC was
added thereto as a crosslinking agent at a concentration of 5
mg/ml, and stirring was conducted for 2 hours to introduce a
functional group into the surface of the biomaterial. After
reaction at room temperature for 2 hours, the collagen biomaterial
was washed, a solution of 10 mg of the peptide of SEQ ID NO: 4 in
100 .mu.l of phosphate buffer was added thereto, reaction was
conducted for 24 hours and the resulting product was then washed to
produce a collagen biomaterial including an immobilized
peptide.
Test Example 1
Evaluation of Inhibition of HDAC Expression by Bifunctional Peptide
Having Capability to Reduce Inflammation and Capability to
Facilitate Differentiation of Stem Cells into Chondrocytes
[0058] In order to identify the change of HDAC expression by the
bifunctional peptide at a molecular level, RAW 264.7 cells were
plated at a density of 70% in a 6-well plate, and after hours, the
RAW 264.7 cells were subjected to cell starvation in DMEM medium
containing 0.5% FBS for 2 hours. After the cells were treated with
medium containing 100 .mu.M of each of the peptides of SEQ ID NOS:
1 to 8 for 1 hour, the cells were treated with LPS at a
concentration of 1 .mu.g/ml to induce an inflammatory reaction for
30 minutes. Proteins were lysed using RIPA lysis buffer (25 mM
TrisHCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1%
SDS) containing a protease inhibitor and a phosphatase inhibitor.
Proteins were assayed by BCA protein assay, and expression levels
of HDAC1 and HDAC5 proteins were identified through Western
blotting (FIG. 1). For the Western blotting, the sample was loaded
at a constant amount on an 8% SDS PAGE gel along with a size
marker, centrifuged for about 2 hours and then transferred to a
nitrocellulose membrane. The transferred membrane was blocked with
5% skim milk for 1 hour and was reacted with the first antibody at
a ratio of 1:1000 overnight. Then, the membrane was washed with
TBST containing 0.1% tween-20 and reacted with a HRP-coated
secondary antibody for 1 hour and then chemiluminescence was
identified with an ECL substrate.
[0059] FIG. 1A shows the results of Western blotting to identify
the expression levels of HDAC1 and HDAC5 proteins by the peptides
of SEQ ID NOS: 1 to 8. FIG. 1B shows the results of normalization
of HDAC1 and HDAC5 bands by actin bands. The expression of HDAC 1
and HDAC5 was increased upon treatment with LPS alone, but the
expression levels of HDAC1 and HDCA5 proteins were inhibited by the
peptides when simultaneously treated with LPS and peptides of SEQ
ID NOS: 1 to 8. It can be seen from these results that the peptides
of SEQ ID NOS: 1 to 8 were effective as HDAC inhibitors.
Test Example 2
Evaluation of Inhibitory Effect on Production of Inflammatory
Cytokine by Bifunctional Peptide Having Capability to Reduce
Inflammation and Capability to Facilitate Differentiation of Stem
Cells into Chondrocytes
[0060] RAW 264.7 cells were seeded at a density of 5.times.10.sup.5
cells/ml in a 24-well plate in order to identify the production of
IL-6 by the bifunctional peptide. Each well was treated with each
of the peptides of SEQ ID NOS: 1 to 8 at a concentration of 100
.mu.M. After 1 hour, the well was treated with LPS at a
concentration of 1 .mu.g/ml and cultured for 17 hours. The
concentration of IL-6 released from the cell culture medium was
measured using an ELISA kit (R&D, Minneapolis, Minn. USA).
[0061] FIG. 2 is a graph showing a change in the amount of
interleukin 6 produced by the peptides of SEQ ID NOS: 1 to 8. The
concentration of IL-6 was increased in RAW 264.7 macrophages
supplemented with LPS, but the production of interleukin 6 was
decreased in a concentration-dependent manner in a medium treated
with LPS and the peptides of SEQ ID NOs: 1 to 8. This means that
the peptides of SEQ ID NOS: 1 to 8 were effective in reducing
inflammation.
Test Example 3
Evaluation of Differentiation Capability from Mesenchymal Stem
Cells to Chondrocytes by Bifunctional Peptide Having Capability to
Reduce Inflammation and Capability to Facilitate Differentiation of
Stem Cells into Chondrocytes
[0062] In order to identify the ability of the bifunctional peptide
to differentiate into chondrocytes from stem cells, the peptides of
SEQ ID NOS: 1 to 8 were each dissolved in PBS at a concentration of
1 mM. 1.times.10.sup.6 cells/mL of the cultured mesoderm-derived
stem cells were seeded in a 24-well plate, treated with 100 .mu.m
of each of the peptides of SEQ ID NOS: 1 to 8, and further treated
therewith whenever the medium was replaced. The cells were cultured
for 21 days in a cartilage differentiation medium containing
DMEM-HG, sodium pyruvate, 100 nM dexamethasone, 20 ug/mL proline,
37.5 .mu.g/mL ascorbic 2-phosphate, 1% PS, 10 ng/mL TGF-.beta.1, 1%
FBS, and 1.times. insulin-transferrin-selenium (ITS+), and then the
capability to differentiate into chondrocytes was evaluated. Each
of the cells was disrupted using TRIzol reagent, and total RNA was
extracted. The extracted RNA was reverse-transcribed into cDNA and
amplified through a polymerase chain reaction. Collagen type II and
Aggrecan were used to identify cartilage differentiation. 1 .mu.l
of each of 10 pM type collagen II, aggrecan, and GAPDH gene sense
and antisense primers, 25 .mu.l of Power CYBR Green PCR master mix
(Invitrogen) and 22 .mu.l of DW were added to 1 .mu.l of the cDNA
template to produce a sample, and the sample was measured using a
StepOnePlus (Invitrogen). The nucleic acid sequences of the PCR
primers used for the amplification of respective genes and the
sizes of the PCR products are as follows.
TABLE-US-00004 Type II collagen SEQ ID NO: 9: forward primer:
5'-GGCAATAGCAGGTTCACGTACA-3' SEQ ID NO: 10: reverse primer:
5'-CGATAACAGTCTTGCCCCACTT-3' Product size: 79 bp Aggrecan SEQ ID
NO: 11: forward primer: 5'-TCGAGGACAGCGAGGCC-3' 24-16 SEQ ID NO:
12: reverse primer: 5'-TCGAGGGTGTAGCGTGTAGAGA-3' Product size: 85
bp GAPDH (glyceraldehyde-3-phosphate dehydrogenase) SEQ ID NO: 13:
forward primer: 5'-ATGGGGAAGGTGAAGGTCG-3' SEQ ID NO: 14: reverse
primer: 5'-TAAAAGCAGCCCTGGTGACC-3' Product size: 119 bp
[0063] The RT-PCR results are shown in FIG. 3. The expression of
collagen type II and Aggrecan genes was increased when treated with
each of the peptides of SEQ ID NOS: 1 to 8 in a cartilage
differentiation medium. This means that stem cells differentiated
into chondrocytes. It could be seen from this that the peptides of
SEQ ID NOS: 1 to 8 were effective in facilitating the
differentiation of stem cells into chondrocytes.
[0064] Although specific configurations of the present invention
have been described in detail, those skilled in the art will
appreciate that preferred embodiments of this description are given
for illustrative purposes and should not be construed as limiting
the scope of the present invention. Therefore, the substantial
scope of the present invention is defined by the accompanying
claims and equivalents thereto.
Advantageous effects
[0065] The bifunctional peptide according to the present invention
has a function of reducing inflammation by inhibiting the
expression of HDAC1 and HDAC5 and the production of cytokine
interleukin 6, and exerts an effect of facilitating differentiation
of stem cells into chondrocytes by improving the expression of
collagen type II and aggrecan, which are cartilage-cell-specific
genes, when mesenchymal stem cells (MSCs) are treated with the
bifunctional peptide. Thus, advantageously, the bifunctional
peptide can be effectively used as a pharmaceutical composition for
preventing or treating arthritis accompanied by inflammation and
damage of cartilage tissue and a biomaterial, and can be easily
applied to various surgical regenerative treatments including
orthopedic treatments and can shorten the treatment period.
Sequence CWU 1
1
14115PRTArtificial Sequencebifunctional peptide 1Gly Lys Cys Ser
Thr Arg Gly Arg Lys Cys Cys Arg Arg Lys Lys1 5 10
15215PRTArtificial Sequencebifunctional peptide 2Gly Lys Cys Ser
Thr Arg Gly Arg Lys Cys Met Arg Arg Lys Lys1 5 10
15315PRTArtificial Sequencebifunctional peptide 3Gly Lys Cys Ser
Thr Arg Gly Arg Lys Met Cys Arg Arg Lys Lys1 5 10
15415PRTArtificial Sequencebifunctional peptide 4Gly Lys Cys Ser
Thr Arg Gly Arg Lys Met Met Arg Arg Lys Lys1 5 10
15515PRTArtificial Sequencebifunctional peptide 5Gly Lys Met Ser
Thr Arg Gly Arg Lys Cys Cys Arg Arg Lys Lys1 5 10
15615PRTArtificial Sequencebifunctional peptide 6Gly Lys Met Ser
Thr Arg Gly Arg Lys Met Cys Arg Arg Lys Lys1 5 10
15715PRTArtificial Sequencebifunctional peptide 7Gly Lys Met Ser
Thr Arg Gly Arg Lys Cys Met Arg Arg Lys Lys1 5 10
15815PRTArtificial Sequencebifunctional peptide 8Gly Lys Met Ser
Thr Arg Gly Arg Lys Met Met Arg Arg Lys Lys1 5 10
15922DNAArtificial Sequencebifunctional peptide 9ggcaatagca
ggttcacgta ca 221022DNAArtificial Sequencebifunctional peptide
10cgataacagt cttgccccac tt 221117DNAArtificial Sequencebifunctional
peptide 11tcgaggacag cgaggcc 171222DNAArtificial
Sequencebifunctional peptide 12tcgagggtgt agcgtgtaga ga
221319DNAArtificial Sequencebifunctional peptide 13atggggaagg
tgaaggtcg 191419DNAArtificial Sequencebifunctional peptide
14atggggaagg tgaaggtcg 19
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