U.S. patent application number 13/486035 was filed with the patent office on 2012-12-06 for peptide bfp4 for promoting osteogenesis or vascularization and use thereof.
This patent application is currently assigned to CHONNAM NATIONAL UNIVERSITY HOSPITAL. Invention is credited to Hyung Keun KIM, Ji Hyun KIM, Taek Rim YOON.
Application Number | 20120309675 13/486035 |
Document ID | / |
Family ID | 47260109 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120309675 |
Kind Code |
A1 |
YOON; Taek Rim ; et
al. |
December 6, 2012 |
PEPTIDE BFP4 FOR PROMOTING OSTEOGENESIS OR VASCULARIZATION AND USE
THEREOF
Abstract
Disclosed are a peptide for promoting osteogenesis and
vascularization, and the use thereof. The peptide has a low
molecular weight so that it can be economically produced. In
addition, it promotes osteoblastic differentiation, thus inducing
osteogenesis. Further, the peptide can promote the expression of
VEGF, resulting in vascularization. Accordingly, the peptide is
useful for the prophylaxis or therapy of ischemic diseases.
Inventors: |
YOON; Taek Rim; (Gwangju,
KR) ; KIM; Hyung Keun; (Gwangju, KR) ; KIM; Ji
Hyun; (Gwangju, KR) |
Assignee: |
CHONNAM NATIONAL UNIVERSITY
HOSPITAL
GWANGJU
KR
|
Family ID: |
47260109 |
Appl. No.: |
13/486035 |
Filed: |
June 1, 2012 |
Current U.S.
Class: |
514/1.9 ;
435/320.1; 514/16.7; 514/16.8; 514/16.9; 514/21.5; 530/326;
536/23.5 |
Current CPC
Class: |
C07K 14/51 20130101;
A61P 9/00 20180101; A61P 9/10 20180101; A61P 19/00 20180101; A61P
19/10 20180101; A61P 19/08 20180101; A61P 19/02 20180101; A61P 9/04
20180101; A61K 38/10 20130101 |
Class at
Publication: |
514/1.9 ;
530/326; 536/23.5; 435/320.1; 514/16.7; 514/21.5; 514/16.9;
514/16.8 |
International
Class: |
A61K 38/10 20060101
A61K038/10; C12N 15/12 20060101 C12N015/12; A61P 9/10 20060101
A61P009/10; A61P 19/10 20060101 A61P019/10; A61P 19/02 20060101
A61P019/02; C07K 7/08 20060101 C07K007/08; C12N 15/82 20060101
C12N015/82 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 3, 2011 |
KR |
10-2011-0054179 |
Claims
1. A peptide for promoting osteogenesis or vascularization,
comprising an amino acid sequence of SEQ ID NO: 1.
2. The peptide of claim 1, wherein the amino acid sequence of SEQ
ID NO: 1 is derived from BMP7.
3. The peptide of claim 1, having an activity of promoting
osteoblastic differentiation or Vascular endothelial growth factor
expression.
4. The peptide of claim 1, having an activity of promoting
osteogenesis and vascularization.
5. The peptide of claim 1, wherein the peptide is an isolated
peptide.
6. The peptide of claim 1, wherein the peptide is an isolated and
substantially purified peptide.
7. An isolated polynucleotide, encoding the peptide of claim 1.
8. A recombinant vector, carrying the polynucleotide of claim
7.
9. A pharmaceutical composition for prophylaxis or therapy of a
bone disease or an ischemic disease, comprising as an active
ingredient the peptide of claim 1, a polynucleotide encoding the
peptide, a recombinant vector carrying the polynucleotide, or a
combination thereof.
10. The pharmaceutical composition of claim 9, wherein the bone
disease is selected from the group consisting of osteoporosis,
osteoarthritis, bone fracture, osteoporosis imperfect and a
combination thereof.
11. The pharmaceutical composition of claim 9, wherein the ischemic
disease is selected from the group consisting of ischemic necrosis,
ischemic cerebrovascular disease, ischemic renal diseases, ischemic
lung diseases, ischemic diseases of limbs, ischemic heart diseases,
apoplexy, cerebral infarction, myocardial infarction, ischemic
heart failure, obstructive arteriosclerosis, and a combination
thereof.
12. A method of preventing or treating a disease selected from the
group consisting of a bone disease, an ischemic disease and a
combination thereof, the method comprising: administering a subject
with a therapeutically or pharmaceutically effective amount of a
peptide comprising an amino acid sequence of SEQ ID NO: 1.
13. The method of claim 12, wherein the disease is the bone
disease.
14. The pharmaceutical composition of claim 13, wherein the bone
disease is selected from the group consisting of osteoporosis,
osteoarthritis, bone fracture, osteoporosis imperfect and a
combination thereof.
15. The method of claim 12, wherein the disease is the ischemic
disease.
16. The pharmaceutical composition of claim 15, wherein the
ischemic disease is selected from the group consisting of ischemic
necrosis, ischemic cerebrovascular disease, ischemic renal
diseases, ischemic lung diseases, ischemic diseases of limbs,
ischemic heart diseases, apoplexy, cerebral infarction, myocardial
infarction, ischemic heart failure, obstructive arteriosclerosis,
and a combination thereof.
17. The method of claim 12, wherein the subject is a human.
Description
CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF PRIORITY
[0001] This application claims priority to Korean Patent
Application No. 10-2011-0054179, filed on Jun. 3, 2011, which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] 1. Technical Field
[0003] The present invention relates to a peptide useful for
promoting osteoporosis or vascularization and the use thereof.
[0004] 2. Description of the Related Art
[0005] Bones constitute part of the endoskeleton which physically
supports the body, and play an important role in maintaining the
blood calcium level. In a normal state, bones exhibit dynamic
homeostasis in which both bone resorption and formation actively
proceed in metabolic balance. The disruption of the balance between
bone resorption and formation with a shift towards bone resorption
causes a decrease in bone mineral density or bone mass and bone
strength, leading to osteoporosis.
[0006] Osteoporosis is a bone disease characterized by the high
risk of fracture upon receiving just a slight impact as a result of
weakened bones. Other representative bone diseases are
osteoarthritis and bone defects. Osteoarthritis, also known as
degenerative arthritis, is characterized by a local degenerative
change in the joint as a result of the breakdown and loss of
cartilage. Bone defects may form in many sites of the body,
predominantly because of acute trauma with the accompaniment of
bone matrix loss, acute trauma with the accompaniment of surgical
bone loss, chronic infection with the accompaniment of bone
resection, and chronic nonunion with the accompaniment of segmental
defects.
[0007] The market associated with bone disease around the world
amounts to approximately 130 billion dollars and it is expected to
continuously expand in size. Thus, research institutes and
pharmaceutical companies have made tremendous investments in the
development of medicines for bone diseases.
[0008] Medicines currently used for osteoporosis include
bisphosphonates (alendroate, etidronate, etc.), hormonal agents
(raloxifene), vitamin D agents, calcitonin agents and calcium
agents. Recently, the parathyroid hormone agent FORTEO.TM. has been
commercially available and has an ability to form new bones.
[0009] However, bisphosphonates have a problem associated with the
uptake thereof in that because bisphosphonates are absorbed at a
very low rate and cause erosion of the esophagus, the patients must
take them together with a sufficient amount of water and sit
upright for a time after the uptake of the medication. Hormonal
agents are required to be taken over the entire lifespan of the
patients and the administration thereof over the long term may
cause side effects such as breast cancer, uterine cancer, and
thrombosis. Vitamin D agents are very expensive, and their
pharmaceutical efficacy is not reliable. Calcitonin agents are also
expensive and have a problem associated with the administration
thereof. Calcium agents do not engender significant side effects,
but are limited to prophylactic effects rather than therapeutic
effects.
[0010] The parathyroid hormone agent, FORTEO.TM., which has
recently been commercialized, can induce bone formation and thus
has an advantage over conventional drugs that work to prevent bone
resorption. However, it suffers from the disadvantages of being
administered by daily injection over a long period of time and
being very expensive. There is therefore the need for a novel
medication and method that can increase bone mass and improve bone
quality to substantially reduce the risk of fracture, thus being
applicable to the treatment of osteoporosis.
[0011] Ischemic diseases are caused by locally restricting the
blood supply as a result of various pathological abnormalities in
the blood vessels, with resultant damage or malfunction of tissues.
Blood supply through vessels is essential for wound healing or
tissue regeneration. Vascular diseases, such as arteriosclerosis,
myocardial infarction, and angina pectoris are caused by restricted
blood flow.
[0012] Vascular therapy is used to treat diseases by generating
vessels. Vascular endothelial growth factor (VEGF) is already used
as a therapeutic for severe ischemia. Other vasculogenic or
angiogenic factors such as fibroblast growth factor (FGF),
epidermal growth factor (EGF) and platelet-derived endothelial
growth factor (PDEGF) have also been studied for purposes of
clinical use. However, these factors are proteins and so are
difficult to separate and purify. In addition, they are too
expensive for clinical application.
[0013] Leading to the present invention, intensive and thorough
research into the treatment of bone diseases and ischemic diseases,
conducted by the present inventors under the background, resulted
in the finding that BFP 4 (bone forming peptide 4) can be
economically synthesized and has the activity of promoting
osteogenesis and vascularization.
SUMMARY
[0014] It is an aspect of the present invention to provide an
isolated peptide useful for the promotion of osteogenesis or
vascularization, including an amino acid sequence of SEQ ID NO:
1.
[0015] The peptide may be an isolated and/or substantially purified
peptide.
[0016] It is another aspect of the present invention to provide an
isolated polynucleotide encoding the peptide.
[0017] It is a further aspect of the present invention to provide a
recombinant vector carrying the polynucleotide.
[0018] It is still a further aspect of the present invention to
provide a pharmaceutical composition for the prevention or
treatment of a bone disease or an ischemic disease, including the
peptide, a polynucleotide encoding the peptide, or a recombinant
vector carrying the polynucleotide as an active ingredient.
[0019] It is still yet another aspect of the present invention to
provide a method of preventing or treating a disease selected from
the group consisting of a bone disease, an ischemic disease and a
combination thereof, the method including administering a subject
with a therapeutically or pharmaceutically effective amount of a
peptide comprising an amino acid sequence of SEQ ID NO: 1.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A and 1B are a diagram showing the amino acid
sequence, and structure (FIG. 1A) and net charge of BFP 4 (FIG.
1B).
[0021] FIG. 2 is of photographs showing the ability of BFP 4 to
promote osteoblastic differentiation, as analyzed by Alizarin red
staining.
[0022] FIG. 3 is a graph showing the non-cytotoxicity of BFP 4.
[0023] FIGS. 4A and 4B are of graphs showing BFP 4-induced
increases in ALP activity and calcium level, respectively.
[0024] FIG. 5 is a photograph showing the BFP 4-induced expression
of runx2, osteocalcin and ALP genes by western blot.
[0025] FIG. 6 is of graphs showing the BFP 4-induced expression of
cell surface markers (CD44 and CD51) associated with osteoblastic
differentiation, as analyzed by FACS.
[0026] FIG. 7 is of fluorescence microphotographs showing the BFP
4-induced expression of CD44 and CD51.
[0027] FIG. 8 is of X-ray photographs showing the BFP 4-induced
osteogenesis in transplanted regions of mice.
[0028] FIG. 9 is a photograph showing the BFP 4-induced expression
of VEGF.
[0029] FIG. 10 is a photograph showing the BFP 4-induced
vascularization in Matrigel transplanted into an animal.
DETAILED DESCRIPTION
[0030] Exemplary embodiments of the present invention will be
described in detail below with reference to the accompanying
drawings. However, the exemplary embodiments of the present
invention described herein are merely examples, and the scope of
the present invention is not limited thereto.
[0031] In accordance with one aspect thereof, the present invention
provides a peptide for promoting osteogenesis or vascularization,
including the amino acid sequence of SEQ ID NO: 1.
[0032] SEQ ID NO: 1:
Phe-Phe-Lys-Ala-Thr-Glu-Val-His-Phe-Arg-Ser-Ile-Arg-Ser-Thr
[0033] As used herein, the term "osteogenesis" is intended to refer
to the process of laying down new bone material, including the
formation of bone matrix by osteoblasts and the mineralization
thereof.
[0034] The term "vascularization," as used herein, is intended to
refer to the physical process of blood vessel formation, that is,
the generation of new blood vessels into cells, tissues or organs,
including both the de novo production of endothelial cells
(vasculogenesis) and the growth of new blood vessels from
pre-existing ones (angiogenesis).
[0035] As used herein, the term "promoting osteogenesis or
vascularization" or grammatical variations thereof means bringing
about osteogenesis or vascularization within a short time,
including the induction of osteogenesis and vascularization.
[0036] By the term "peptide," as used herein, is meant a short
polymer of amino acids linked by peptide bonds. All of the amino
acids may have an L- or D-configuration. The peptide of the present
invention includes the amino acid sequence of SEQ ID NO: 1.
Variations in the peptides including fusion with other amino acids
or peptides are within the scope of the present invention so long
as it has the activity of promoting osteogenesis or
vascularization.
[0037] The amino acid useful in the present invention may include a
naturally occurring amino acid, a synthetic amino acid, and an
amino acid analog and an amino acid mimic, which act like naturally
occurring amino acids. The term "naturally occurring amino acid"
means an amino acid encoded by genetic code. The amino acid analog
is a compound which has hydrogen atoms, a carboxyl group, an amino
group and an .alpha. carbon atom linked to an R group, with a
modification given to the R group (e.g., norleucine) or the peptide
backbone. The term "amino acid mimic" means a chemical compound
which differs from amino acids in chemical structure, but acts like
a naturally occurring amino acid.
[0038] Also, homologues of the peptide of the present invention
fall within the scope of the present invention. The term
"homologues", as used herein, means peptides which exhibit
substantially identical physiological activity to that of the
peptide of SEQ ID NO: 1. The homologues exhibiting "identical
physiological activity" may be peptides that share an amino acid
sequence homology of at least 60%, for example, at least 70% and
for another example, at least 90% with the peptide of SEQ ID NO:
1.
[0039] The amino acid sequence of SEQ ID NO: 1 may be derived from
BMP7, and for example, from the prodomain of Bone morphogenetic
protein 7 (BMP7). Bone morphogenetic proteins (BMPs) are known as
factors capable of inducing ossification, but exist in a trace
amount in nature. BMPs have high-molecular weights and the
recombinant preparation thereof is very expensive. In addition,
many restrictions are put on the use of BMPs as medications in
terms of physical properties and administration due to the
proteinous properties thereof. In contrast, the peptide of the
present invention is a low-molecular weight compound including 15
amino acids and can be economically produced. Also in terms of
function, the peptide of the present invention exhibits an
osteogenic effect identical to or higher than that of BMP7.
[0040] The peptide of the present invention may promote osteoblast
differentiation or VEGF expression.
[0041] In an embodiment, the peptide of the present invention may
be a peptide having the activity of promoting osteogenesis and
vascularization.
[0042] In an experiment in which the differentiation of osteoblast
cells was examined, the peptide of the present invention was found
to promote osteoblastic differentiation at higher levels than did
BMP7, as measured by Alizarin Red staining (FIG. 2). Also, an
animal test with mice showed that even a greater extent of bone
formation was achieved at sites treated with the peptide of the
present invention than with BMP7 (FIG. 8). Therefore, the peptide
of the present invention can be useful for the prevention and
treatment of bone diseases.
[0043] The expression of Vascular endothelial growth factor (VEGF),
known as a potent factor involved in vascularization, stimulates
the formation of new blood vessels. In an example of the present
invention, a higher level of VEGF was expressed by the peptide of
the present invention than by BMP7 (FIG. 9). An animal test of mice
showed that the peptide of the present invention induced the
formation of new blood vessels in matrigel (FIG. 10). Accordingly,
the peptide of the present invention can be useful for the
prevention and treatment of ischemic diseases.
[0044] The peptide of the present invention may be prepared using a
well-known peptide synthesis method or by culturing transformed
host cells. In the latter case, typically, a polynucleotide
encoding the peptide is introduced into an expression vector which
is then transformed into a host cell, and the tansformant is
cultured to produce the peptide. To culture the transformant, any
appropriate method known in the art may be used.
[0045] In accordance with another aspect thereof, the present
invention provides a polynucleotide encoding the peptide of the
present invention, and a recombinant vector carrying the same.
[0046] As used herein, the term "polynucleotide" refers to a
polymer composed of many nucleotide monomers covalently bonded in a
chain, as exemplified by DNA (deoxyribonucleic acid) or RNA
(ribonucleic acid). The polynucleotide of the present invention
includes a polynucleotide encoding the peptide of the present
invention.
[0047] In the polynucleotide of the present invention, various
modifications may be made in the encoding region provided that they
do not change the amino acid sequence of the polypeptide expressed
from the coding region, due to codon degeneracy or in consideration
of the codons preferred by the organism in which they are to be
expressed, and various modifications or alterations may be
introduced even in regions other than the coding region so long as
they have no influence on the expression of the gene. That is to
say, the polynucleotide of the present invention may be modified at
one or more nucleic acid bases by substitution, deletion, insertion
or a combination thereof as long as the resulting polynucleotides
encode functionally equivalent polypeptides, and they are also
within the scope of the present invention.
[0048] The recombinant vector of the present invention is a means
for expressing the peptide of the present invention within a host
cell. Any expression vector such as a plasmid vector, a cosmid
vector, a bacteriophage vector, and so on may be used so long as it
is known for an expression vector in the art. The vector may be
readily constructed by the artisan of ordinary skill using a
well-known DNA recombination technique.
[0049] In accordance with a further aspect thereof, the present
invention provides a pharmaceutical composition for the prophylaxis
or therapy of a bone disease or an ischemic disease, including as
an active ingredient the polypeptide of the present invention, a
polynucleotide encoding the polypeptide, or a recombinant vector
carrying the polynucleotide.
[0050] In accordance with still a further aspect thereof, the
present invention provides a method for the prophylaxis or therapy
of a bone disease or an ischemic disease, including administering
the pharmaceutical composition to a subject which has been attacked
with or is likely to be attacked with the bone disease or the
ischemic disease.
[0051] As used herein, the term "prevention" refers to any measure
taken in order to suppress or delay the onset of bone diseases or
ischemic diseases by administering the pharmaceutical composition
of an embodiment of the present invention. The term "treatment," as
used herein, refers to any measure taken in order to improve or
beneficially alter symptoms of bone diseases or ischemic diseases
by administering the pharmaceutical composition of an embodiment of
the present invention.
[0052] The term "subject," as used herein, refers to any animal
including humans, which has been attacked with or is likely to be
attacked with the bone disease or ischemic disease. The bone
disease may be selected from the group consisting of osteoporosis,
osteoarthritis, bone fracture, osteogenesis imperfect and a
combination thereof. The ischemic disease may be one selected from
the group consisting of ischemic necrosis, ischemic cerebrovascular
diseases, ischemic renal diseases, ischemic lung diseases, ischemic
diseases of the limbs, ischemic heart diseases, apoplexy, cerebral
infarction, myocardial infarction, ischemic heart failure, angina
pectoris, obstructive arteriosclerosis and a combination
thereof.
[0053] So long as it allows the pharmaceutical composition of the
embodiment of the present invention to reach tissues or cells of
interest (e.g. bone-defected site), any administration route may be
taken. Depending on the purposes of administration, the
pharmaceutical composition of the embodiment of the present
invention may be administered directly or indirectly via
intraperitoneal, intravenous, intramuscular, subcutaneous,
interdermal, oral, intrapulmonary, intrarectal, or intracellular
routes. In this context, the pharmaceutical composition of the
embodiment of the present invention may be administered by means of
any device capable of transporting an active ingredient into target
cells.
[0054] The pharmaceutical composition of an embodiment of the
present invention may further include a pharmaceutically acceptable
vehicle. When containing a pharmaceutically acceptable vehicle, the
pharmaceutical composition of an embodiment of the present
invention may exist in various oral or non-oral dosage forms. In
this regard, the pharmaceutical composition of an embodiment of the
present invention may be formulated in combination with diluents or
an excipient such as a filler, a thickener, a binder, a wetting
agent, a disintegrant, a surfactant, etc. Solid preparations
intended for oral administration may take the form of tablets,
pills, powders, granules, capsules, and the like. In regards to
these solid agents, the peptide of an embodiment of the present
invention may be formulated in combination with at least one
excipient such as starch, calcium carbonate, sucrose, lactose, or
gelatin. Liquid preparations intended for oral administration
include suspensions, internal use solutions, emulsions, syrups, and
the like. In addition to a simple diluent such as water or liquid
paraffin, various excipients, such as wetting agents, sweetening
agents, aromatics, preservatives, and the like may be contained in
the liquid preparations. Also, the pharmaceutical composition of
the embodiment of the present invention may be administered via a
non-oral route. For this, sterile aqueous solutions, non-aqueous
solvents, suspensions, emulsions, lyophilizates, suppositories, and
the like may be used. Injectable propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and esters such as ethyl
oleate may be suitable for non-aqueous solvents and suspensions.
The basic materials of suppositories include Witepsol, macrogol,
Tween 61, cacao butter, laurin butter, and glycerogelatin.
[0055] The dosage form of the pharmaceutical composition of the
present invention may be selected from the group consisting of a
tablet, a pill, a powder, a granule, a capsule, a suspension, an
internal use solution, an emulsion, a syrup, a sterile aqueous
solution, a non-aqueous solution, a suspension, a lyophilizate, and
a suppository.
[0056] The pharmaceutical composition of the present invention is
administered in a therapeutically or pharmaceutically effective
amount. The term "therapeutically or pharmaceutically effective
amount," as used herein, is intended to refer to a sufficient
amount of the pharmaceutical composition to treat a disease, at a
reasonable benefit/risk ratio applicable to any medical treatment.
The effective amount may vary depending on various factors
including the severity of the disease being treated, the patient's
age and sex, drug activity, sensitivity to drugs, the time of
administration, the route of administration, the rate of excretion,
the period of time of treatment, the co-administration of drugs,
and other parameters well known in the art.
[0057] For treating bone diseases or ischemic diseases, the
pharmaceutical composition of the present invention may be used in
combination with surgery, hormones, drugs, and/or biological
reaction regulators.
[0058] A better understanding of the present invention may be
obtained through the following examples which are set forth to
illustrate, but are not to be construed as limiting the present
invention.
Example 1
Synthesis of BFP 4
[0059] The peptide having the amino acid sequence of SEQ ID NO: 1
can be artificially synthesized using a well-known method. The
synthesis of the peptide was entrusted to Peptron Co., Ltd (Korea).
The peptide having the amino acid sequence of SEQ ID NO: 1 was
named BFP 4 (bone forming peptide 4).
[0060] Phe-Phe-Lys-Ala-Thr-Glu-Val-His-Phe-Arg-Ser-Ile-Arg-Ser-Thr
(SEQ ID NO: 1)
[0061] Analysis with a commercial program showed that BFP 4 is
composed of 15 amino acid residues with a molecular weight of
1826.1 and has an alpha-helix structure as illustrated in FIG.
1A.
Example 2
Preparation of Osteoblast Cells and Osteogenic Differentiation
Medium
[0062] For use as osteoblast cells in this example, mesenchymal
stem cells isolated from Balb/c mouse bone marrow stromal cells
were seeded at a density of 1.times.10.sup.4 cells in a DMEM
(Dulbecco's Modified Eagle Medium) supplemented with 10% FBS and
incubated at 37.degree. C. for about 3 days in a 5% CO.sub.2
atmosphere.
[0063] An osteogenic differentiation medium (ODM) was prepared by
supplementing DMEM with 50 .mu.g/mL ascorbic acid, 10.sup.-8 M
dexamethasone and 10 mM beta-glycophosphate.
Example 3
Assay of BFP 4 for Ability to Promote Osteoblastic
Differentiation
[0064] Because calcium is deposited when the mesenchymal stem cells
differentiate into osteoblasts, the degree of osteoblastic
differentiation was analyzed by measuring mineralization with
Alizarin red staining. That is, the greater advance in osteoblastic
differentiation produced a wider region positive to Alizarin red.
After the cells in the osteogenic differentiation medium were
treated with BFP 4 in the presence of Alizarin Red, the staining of
Alizarin red was monitored to determine the degree of osteoblastic
differentiation. In this regard, the mesenchymal cells were
transferred into an osteogenic differentiation medium and incubated
for three days. Thereafter, BFP 4 or BMP7 was added in
concentrations of 0.001 .mu.g/mL, 0.01 .mu.g/mL, 0.05 .mu.g/mL, and
0.1 .mu.g/mL to the medium, followed by incubation for an
additional 2 days. Subsequently, the mesenchymal stem cells were
fixed for one hour with ice-cooled 70% ethanol and stained for
about 10 min with Alizarin red-S to analyze the degree of
deposition of calcium. The results are shown in FIG. 2.
[0065] As can be seen in FIG. 2, the most intensive staining was
detected in the cells treated with BFP 4 at a concentration of 0.01
.mu.g/mL. Particularly, BFP 4 according to the present invention
was found to produce more intensive colors than did BMP7 (FIG. 2).
These data indicate that activity for osteoblastic differentiation
of BFP 4 is more potent than that of BMP7.
Example 4
Assay for Cytotoxicity of BFP 4
[0066] At concentrations effective for calcium deposition, BFP 4
was analyzed for cytotoxicity. The results are shown in FIG. 3. In
this context, MTT
((3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a
yellow tetrazole) assay was performed using an MTT Cell
Proliferation Assay Kit (Cayman Chemical) according to the
instructions of the manufacturer.
[0067] As seen in FIG. 3, no cytotoxicity was detected at
concentrations of BFP 4 which are effective for promoting
osteogenic differentiation.
Example 5
Assay of BFP 4 for Ability to Increase ALP Activity and Calcium
Level
[0068] During osteoblastic differentiation, alkaline phosphatase
(ALP), a marker enzyme characteristic of osteoblasts, and calcium
were quantitatively analyzed using a DALP-250 QuantiChrom Alkaline
Phosphatase Assay Kit (Gentaur) and a DICA-500 QuantiChrom Calcium
Assay Kit (Gentaur), respectively. The results are depicted in
FIGS. 4A and 4B.
[0069] As can be seen in FIGS. 4A and 4B, both the activity of ALP
and the concentration of calcium were significantly increased at
BFP 4 concentrations of 0.01 .mu.g/mL and 0.1 .mu.g/mL.
Example 6
Assay for BFP 4-Induced Osteoblast-Specific Gene Expression
[0070] The ability of BFP 4 to induce the expression of runx2,
osteocalcin and alkaline phosphatase genes, which are marker genes
characteristic of osteoblasts, was assayed. The results are shown
in FIG. 5. In this regard, RNA was isolated from the cells treated
with the peptide and reverse transcribed into DNA which was then
used as a template for PCR. Electrophoresis of the PCR products
determined the expression levels of the marker genes.
[0071] As shown in FIG. 5, BFP 4 induced expression of the
osteoblast-specific genes.
Example 7
Assay of BFP 4-Induced Cell Surface Marker Expression During
Osteoblastic Differentiation
[0072] The effect of BFP 4 on the expression of CD44 and CD51,
surface proteins characteristically emerging during differentiation
from mesenchymal stem cells into osteoblasts, was measured using
FACS. The results are shown in FIG. 6.
[0073] As seen in FIG. 6, when BFP 4 was added to the osteogenic
differentiation medium, the cell surface marker CD44 was expressed
at a high level in the mesenchymal cells which were undergoing
osteoblastic differentiation. Particularly, a peak in the
expression level of CD44 was detected at a BFP 4 concentration of
0.1 .mu.g/mL. Also, CD51 was expressed at higher levels in the
presence of BFP 4 during the osteoblastic differentiation (FIG.
6).
Example 8
Assay for BFP 4-Induced Expression of Cell Surface Markers CD44 and
CD51
[0074] The BFP 4-induced expression of the cell surface markers
CD44 and CD51 was observed using a fluorescence microscope, and the
results are given in FIG. 7. In this regard, DAPI
(4',6-diamidino-2-phenylindole) staining was performed to ascertain
the viability of the cells during osteoblastic differentiation, and
anti-CD44 or CD51 antibodies which appear when they bind to the
antigens, were used for fluorescence analysis.
[0075] As can be seen in FIG. 7, both CD44 and CD51 were expressed
in the cells. Higher expression levels of CD44 and CD51 were
observed in the osteogenic differentiation medium in the presence
of BFP 4.
Example 9
In Vivo Assay of BFP 4 for Ability to Promote Osteogenesis In
Vivo
[0076] An animal test with mice (n=6) was performed to examine the
ability of BFP 4 to promote osteogenesis in vivo. First,
mesenchymal stem cells were treated twice with ODM over two days to
induce osteoblastic differentiation. Upon the second treatment with
ODM, BMP7 or BFP 4 was added to the cells, followed by incubation
for 24 hours. Cells were harvested and transplanted in the same
number into the back of the mice, with collagen serving as a
scaffold. Using X-rays, bone formation was examined 4 and 8 weeks
after the transplantation. The results are given in FIG. 8.
[0077] As can be seen in the X-ray photographs of FIG. 8, four
weeks after transplantation, new bones were formed in the region
treated with BFP 4. In addition, as seen in the photographs taken 8
weeks after transplantation, a significantly higher extent of bone
formation was achieved in the region treated with BFP 4 than that
treated with BMP7. These experimental results imply that BFP 4 has
higher activity of promoting osteoblastic differentiation compared
to the conventional factor BMP7, and can be useful for the
prevention and treatment of bone diseases.
Example 10
Assay for BFP 4-Induced VEGF Expression
[0078] To examine the effect of BFP 4 on vascularization, BFP 4 was
assayed for its ability to induce VEGF expression. In this regard,
proteins were isolated from the cells treated with the peptide and
subjected to electrophoresis before measurement using an anti-VEGF
antibody.
[0079] As seen in FIG. 9, the expression of VEGF was induced by BFP
4 and to a higher level than by BMP7.
Example 11
In Vivo Assay for Vascularization of BFP 4
[0080] To examine the ability of BFP 4 to form new blood vessels in
vivo, a mouse animal test was performed using Matrigel. In this
context, cells were treated with the peptide and mixed well with
Matrigel before transplantation into the back of the mice. Eight
weeks after the transplantation, the Matrigel was detached from the
mouse back which was then observed with regard to the formation of
new blood vessels.
[0081] As shown in FIG. 10, blood vessels were newly formed in the
Matrigel 8 weeks after transplantation. Particularly, more
vascularization was induced by BFP4 than by BMP7
[0082] As shown above, the peptide for promoting osteogenesis in
accordance with the present invention has a low molecular weight so
that it can be economically synthesized. In addition, the peptide
of the present invention can promote osteoblastic differentiation,
thus inducing osteogenesis. Hence, the peptide of the present
invention is useful in the prevention or treatment of bone
diseases.
[0083] Also, the peptide for promoting vascularization in
accordance with the present invention can induce the expression of
VEGF, resulting in vascularization. Therefore, the peptide of the
present invention is applicable to the prevention or treatment of
ischemic diseases.
[0084] It will be apparent to those skilled in the art that various
modifications can be made to the above-described exemplary
embodiments of the present invention without departing from the
spirit or scope of the invention. Thus, it is intended that the
present invention covers all such modifications provided they come
within the scope of the appended claims and their equivalents.
Sequence CWU 1
1
1115PRTArtificial Sequencepeptide for promoting osteoporosis or
vascularization 1Phe Phe Lys Ala Thr Glu Val His Phe Arg Ser Ile
Arg Ser Thr1 5 10 15
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