U.S. patent application number 15/720767 was filed with the patent office on 2019-04-04 for peptide for promoting wound healing, its composition and method of using the same.
This patent application is currently assigned to PRO SUNFUN BIOTECH RESEARCH AND DEVELOPMENT CO., LTD.. The applicant listed for this patent is PRO SUNFUN BIOTECH RESEARCH AND DEVELOPMENT CO., LTD.. Invention is credited to Syue-Ting Chen, Min-Chuan Huang, Yu-Chun Liu.
Application Number | 20190099466 15/720767 |
Document ID | / |
Family ID | 65721969 |
Filed Date | 2019-04-04 |
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United States Patent
Application |
20190099466 |
Kind Code |
A1 |
Huang; Min-Chuan ; et
al. |
April 4, 2019 |
PEPTIDE FOR PROMOTING WOUND HEALING, ITS COMPOSITION AND METHOD OF
USING THE SAME
Abstract
Disclosed is a peptide consisting of an amino acid sequence of
HisThrSerThrGluAlaLys (SEQ ID NO: 1). This peptide is effective in
the enhancement of fibroblast cell migration, which promotes wound
healing. Also provided are a pharmaceutical composition for
promoting wound healing comprising the peptide, and a method for
promoting wound healing using the peptide.
Inventors: |
Huang; Min-Chuan; (Taipei,
TW) ; Chen; Syue-Ting; (Taipei, TW) ; Liu;
Yu-Chun; (Taoyuan City, TW) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PRO SUNFUN BIOTECH RESEARCH AND DEVELOPMENT CO., LTD. |
Kaohsiung City |
|
TW |
|
|
Assignee: |
PRO SUNFUN BIOTECH RESEARCH AND
DEVELOPMENT CO., LTD.
Kaohsiung City
TW
|
Family ID: |
65721969 |
Appl. No.: |
15/720767 |
Filed: |
September 29, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/02 20130101;
A61K 9/0014 20130101 |
International
Class: |
A61K 38/02 20060101
A61K038/02; A61K 9/00 20060101 A61K009/00 |
Claims
1-2. (canceled)
3. A pharmaceutical composition for wound healing, comprising a
synthetic peptide consisting of the amino acid sequence of SEQ ID
NO: 1 in an amount effective to enhance fibroblast cell migration
and a pharmaceutically acceptable carrier.
4. The pharmaceutical composition of claim 3, wherein the
composition is for systemic, transdermal or topical
administration.
5. The pharmaceutical composition of claim 4, wherein the
composition is for topical administration.
6. (canceled)
7. The pharmaceutical composition of claim 5, which is in a form of
an ointment, gel, or emulsion.
8. (canceled)
9. The pharmaceutical composition of claim 5, wherein the
composition is for topical administration via a spray device, a
dressing or a paste.
10. A method for promoting wound healing, which comprises
administering to a subject in need thereof the peptide of claim 1
in an amount effective to enhance the fibroblast cell
migration.
11. A method for promoting wound healing, which comprises
administering to a subject in need thereof the composition of claim
3.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to a peptide
effective for promoting wound healing, its composition and method
of using the same.
BACKGROUND OF THE INVENTION
[0002] Wound healing requires the coordination of several cell
types including keratinocytes, fibroblasts, endothelial cells,
macrophages and platelets. The process involves cell proliferation
and migration, collagen deposition and remodeling, wound
contraction and angiogenesis. Fibroblasts are the most important
cells involved in producing and remodeling the extracellular
matrix, and fibroblast cell proliferation and migration play key
roles in the formation of granulation tissue and further wound
repair. Cell migration consisting of a multi-step cyclic process is
necessary for wound repair. The basic migration pattern requires
extension of a protrusion, stable attachment to near the leading
edge of the protrusion, forward movement of the cell body and
release of adhesions and retraction at the cell rear.
(Lauffenburger and Horwitz. Cell migration: a physically integrated
molecular process. Cell 84: 359-369, 1996.) Since fibroblast cell
migration is very important during the wound healing, it may be
used as an in vitro model for investigation of the effects on wound
healing.
[0003] What would be advantageous is a non-toxic, non-antigenic,
inexpensive wound-healing agent having the ability to promote wound
healing and allow non-healing wounds to heal.
BRIEF SUMMARY OF THE INVENTION
[0004] It is unexpectedly found in the present invention that a
peptide having the amino acid sequence of HisThrSerThrGluAlaLys
(SEQ ID NO: 1) is effective in the enhancement of fibroblast cell
migration, which is potential for promotion of wound healing.
[0005] Accordingly, the present invention provides in one aspect a
synthestic peptide consisting of the amino acid sequence of SEQ ID
NO: 1, which is also named as Peptide No. 9. The peptide provides
an efficacy t in promotion of wound healing.
[0006] In another aspect, the present invention provides a cosmetic
or pharmaceutical composition for promoting wound healing,
comprising an effective amount of Peptide No. 9 and a cosmetically
or pharmaceutically acceptable carrier.
[0007] In one further aspect, the present invention provides a
method for promoting wound healing, which comprises administering
to a subject in need thereof the peptide having the amino acid
sequence of SEQ ID NO: 1 in an amount effective to enhance the
migration of fibroblast cells in the subject.
[0008] In one embodiment of the method according to the invention,
the peptide is topically administered to the subject.
[0009] It is to be understood that both the foregoing general
description and the following description are exemplary and
explanatory only and are not restrictive of the invention.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0010] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. For the purpose of
illustrating the invention, there are shown in the drawings
embodiments which are presently preferred.
[0011] In the drawings:
[0012] FIG. 1A provides the representative images of HaCaT
keratinocyte cell migration after 24-hour culture with 50 .mu.g/ml
Peptide NO.9 in transwell migration assay (Scale bar, 1 mm),
showing that Peptide NO.9 enhanced HaCaT keratinocyte cell
migration.
[0013] FIG. 1B provides the result of the statistic analysis of
HaCaT keratinocyte cell migration after 24-hour culture with 50
.mu.g/ml Peptide NO. 9 in transwell migration assay (*P<0.05),
showing that Peptide NO.9 provided a significant efficacy in
enhancing HaCaT keratinocyte cell migration.
[0014] FIG. 2A provides representative images of CCD966SK
fibroblast cell migration after 24-hour culture treated with 50
.mu.g/ml Peptide NO. 9 in transwell migration assay, showing that
Peptide NO.9 enhanced CCD966SK fibroblast cell migration.
[0015] FIG. 2B provides the results of the statistic analysis of
migration after 24-hour culture treated with 50 .mu.g/ml Peptide
NO.9 in transwell migration assay (**P<0.01), showing that
Peptide NO.9 provided a significant efficacy in enhancing HaCaT
CCD966SK fibroblast cell migration.
[0016] FIG. 3A provides the viability of HaCaT keratinocyte cells
after 24-hour culture treated with 50 .mu.g/ml Peptide NO. 9 in MTT
assay, indicating that Peptide NO. 9 did not significantly affect
viability of HaCaT cells.
[0017] FIG. 3B provides the viability of HaCaT keratinocyte cells
after 48-hour culture treated with 50 .mu.g/ml Peptide NO. 9 in MTT
assay, indicating that Peptide NO. 9 did not significantly affect
viability of HaCaT cells.
[0018] FIG. 3C provides the viability of HaCaT keratinocyte cells
after 72-hour culture treated with 50 .mu.g/ml Peptide NO. 9 in MTT
assay, indicating that Peptide NO. 9 did not significantly affect
viability of HaCaT cells.
[0019] FIG. 4A shows the viability of CCD966SK cells which were
treated with 50 .mu.g/ml Peptide NO.9 for 24 hours, indicating that
Peptide NO. 9 did not significantly affect viability of CCD966SK
cells.
[0020] FIG. 4B shows the viability of CCD966SK cells which were
treated with 50 .mu.g/ml Peptide NO.9 for 48 hours, indicating that
Peptide NO. 9 did not significantly affect viability of CCD966SK
cells.
[0021] FIG. 4C shows the viability of CCD966SK cells which were
treated with 50 .mu.g/ml Peptide NO.9 for 72 hours, indicating that
Peptide NO. 9 did not significantly affect viability of CCD966SK
cells.
DETAILED DESCRIPTION OF THE INVENTION
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
[0023] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one."
[0024] The term "peptide" is used herein in its conventional sense,
i.e., a polymer in which the monomers are amino acids and are
joined together through amide bonds, alternatively referred to as a
polypeptide. When the amino acids are .alpha.-amino acids, either
the L-optical isomer or the D-optical isomer may be used.
Additionally, unnatural amino acids, for example. .beta.-alanine,
phenylglycine and homoarginine are also meant to be included.
Standard abbreviations for amino acids are used.
[0025] As used herein, the term "subject" refers to a vertebrate or
vertebrates, preferably mammals, including, for example, humans,
laboratory animals such as rats and mice, and farm animals, such as
horses and cows; particularly humans. Hereinafter, a human serving
as a subject is specifically referred to as a "human subject."
[0026] As used herein, the term "carrier" or "cosmetically or
pharmaceutically acceptable carrier" refers to any material
commonly used on the formulations of cosmetic or pharmaceutical
compositions used to enhance stability, sterility and
deliverability. When the peptide delivery system is formulated as a
solution or suspension, the delivery system is in an acceptable
carrier, preferably an aqueous carrier. A variety of aqueous
carriers may be used, e.g., water, buffered water, 0.8% saline,
0.3% glycine, hyaluronic acid and the like. The compositions may
contain physiologically acceptable auxiliary substances as required
to approximate physiological conditions, such as pH adjusting and
buffering agents, tonicity adjusting agents, wetting agents and the
like, for example, sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate, etc.
[0027] The term "systemic" or "systemically" as used herein refers
to a route of administration of medication or other substance into
the circulatory system so that the entire body of a subject to be
administered is affected. The administration may take place via
enteral administration (through which the absorption of the
medication or other substance through gastrointestinal tracts) or
parenteral administration such as injection, infusion or
implantation.
[0028] The term "topical" or "topically" is used herein its
conventional sense as referring to a spot which can be in or on any
part of the body, including but not limited to the epidermis, any
other dermis, or any other body tissue. Topical administration or
application means the direct contact of the peptide with tissue,
such as skin or membrane which contains melanin-producing
cells.
[0029] The term "effective amount" as used herein refers to a
sufficient amount of the peptide according to the invention to
provide desired therapeutic or cosmetic effects, or the induction
of a particular type of response. The effective amount required
varies from subject to subject, depending on the disease state,
physical conditions, age, sex, species and weight of the subject,
etc. However, an appropriate effective amount can be determined by
one of ordinary skill in the art using only routine
experimentation. For example, the peptide according to the
invention may be administered systemically, transdermally or
topically.
[0030] The term "pharmaceutically acceptable carrier" as used
herein encompasses any of the standard pharmaceutical carriers.
Such carriers may include, but are not limited to: saline, buffered
saline, dextrose, water, glycerol, ethanol, propylene glycol,
cremophor, nanoparticles, liposome, polymer, and combinations
thereof. In addition to standard carriers, a pharmaceutical
composition of the present invention may be supplemented with one
or more excipients that are normally employed in common standard
formulations, such as surfactants, solubilizers, stabilizers,
emulsifiers, thickeners, and preservatives. Such excipients are
well known to those skilled in the art.
[0031] As shown in the examples, the peptide having the amino acid
sequence of SEQ ID NO: 1, which may be artificially synthesized by
a standard method or in any manner commonly used or known to one of
ordinary skill. It was confirmed to have an effect in enhancing the
expression of keratinocyte cells, and the migration of the
fibroblast cells. Therefore, the invention also provides a method
for promoting wound healing, which comprises administering to a
subject in need thereof the peptide having the amino acid sequence
of SEQ ID NO: 1 in an amount effective to enhance the expression of
collagen or elastin in fibroblast cells, and the migration of the
fibroblast cells.
[0032] The pharmaceutical composition of the present invention may
be constituted with one or more pharmaceutically acceptable
carriers into any form suitable for the mode of administration
selected, including systemic and topical administrations via
enteral or parenteral administration such as injection, infusion or
implantation, oral, transdermal or topical administration. In
certain embodiments of the invention, the composition may be
formulated with a pharmaceutically or cosmetically acceptable
carrier as a topical formulation in a solution, ointment, gel,
serum, cream, lotion, powder, emulsion or any form for
administration. In some particular examples, the formulation may be
administered via a spray device, a dressing, or a paste.
[0033] The present invention is further illustrated by the
following examples, which are provided for the purpose of
demonstration rather than limitation.
EXAMPLES
Example 1: Preparation of the Peptide of SEQ ID NO: 1
[0034] The peptide consisting of the sequence of SEQ ID NO: 1
(HisThrSerThrGluAlaLys) was synthesized by MDBio, Inc. (Taipei,
Taiwan) and the purity and composition of peptide was confirmed by
high performance liquid chromatography (HPLC) and mass
spectrometry. Peptide stock was stored at -20.degree. C. after
dissolving 10 mg of lyophilized peptide powder in 250 .mu.l of
double deionized water (dd H.sub.2O).
Example 2: Cell Culture
[0035] Human keratinocyte HaCaT and skin fibroblast CCD-966SK cells
were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing
10% (v/v) FBS and 1% (v/v) antibiotics in 5% CO.sub.2 at 37.degree.
C.
Example 3: Transwell Migration Assay
[0036] HaCaT cells (5.times.10.sup.4) or CCD-966SK cells
(5.times.10.sup.3) in 0.25 ml serum-free DMEM were seeded into the
upper chamber with an 8-.mu.m pore size membrane (Corning, USA) and
0.5 ml serum free DMEM with or without 50 .mu.g/ml Peptide NO.9
were loaded to the lower chamber in 24-well culture plate. After 24
hours incubation, cells were fixed and stained with 0.5% (w/v)
crystal violet (Sigma) containing 20% (v/v) methanol. The number of
migrated cells from 5 random fields was counted under the
microscope. Results obtained were analyzed by student's t-test and
graphed as mean.+-.SD.
[0037] The results were shown in FIG. 1A and FIG. 1B. As shown in
FIG. 1A providing representative images of HaCaT keratinocyte cell
migration after 24-hour culture with 50 .mu.g/ml Peptide NO. 9, and
FIG. 1B providing the result of the statistic analysis, Peptide NO.
9 provided an effect in enhancement of keratinocyte cell migration
of HaCaT cells (P<0.05).
[0038] It is also illustrated in FIGS. 2A and 2B that Peptide NO.9
enhanced CCD966SK fibroblast cell migration. As shown in FIG. 2A
providing representative images of CCD966SK fibroblast cell
migration after 24-hour culture with 50 .mu.g/ml Peptide NO. 9 in
transwell migration assay, and FIG. 2B showing that the Peptide NO.
9 provided an effect in enhancement of keratinocyte cell migration
of CCD966SK cells (P<0.01).
Example 4: MTT Assay
[0039] HaCaT keratinocyte cells (3.times.10.sup.3, each cell line)
in 100 .mu.I complete DMEM were seeded in 96-well plates with or
without 50 .mu.g/ml Peptide NO. 9, respectively. Ten microliters of
5 mg/ml 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide solution (MTT; Sigma) was added to each well for the
indicated times and incubated at 37.degree. C. for 3 hours. after
which 100 .mu.l 10% SDS in 0.01 N HCl was added to dissolve the MTT
formazan crystals. The resultant optical density was measured
spectrophotometrically at dual wavelengths, 550 and 630 nm.
[0040] The viability of HaCaT keratinocyte cells after 24-hour,
48-hour or 72-hour culture treated with 50 .mu.g/ml Peptide NO. 9
were tested by MTT assay. As shown in FIG. 3A (for 24 hour), FIG.
3B (for 48 hour) and FIG. 3C (for 74 hour), Peptide NO. 9 did not
significantly affect viability of HaCaT cells.
[0041] In the same manner, the viability of CCD966SK keratinocyte
cells after 24-hour culture FIG. 4A shows the viability of CCD966SK
cells which were treated with 50 .mu.g/ml Peptide NO. 9 for 24
hours, indicating that Peptide NO. 9 did not significantly affect
viability of CCD966SK cells.
[0042] FIG. 4B shows the viability of CCD966SK cells which were
treated with 50 .mu.g/ml Peptide NO. 9 for 48 hours, indicating
that Peptide NO.9 did not significantly affect viability of
CCD966SK cells.
[0043] FIG. 4C shows the viability of CCD966SK cells which were
treated with 50 .mu.g/ml Peptide NO.9 for 72 hours, indicating that
Peptide NO.9 did not significantly affect viability of CCD966SK
cells.
[0044] Given the above, it is concluded that the peptide according
to the invention (i.e., Peptide No. 9) provides an unexpected
efficacy in promotion of wound healing through the enhancement of
fibroblast cell migration, instead of fibroblast cell
viability.
[0045] It will be appreciated by those skilled in the art that
changes could be made to the embodiments described above without
departing from the broad inventive concept thereof. It is
understood, therefore, that this invention is not limited to the
particular embodiments disclosed, but it is intended to cover
modifications within the spirit and scope of the present invention
as defined by the appended claims.
Sequence CWU 1
1
217PRTArtificial SequenceSynthetic 1His Thr Ser Thr Glu Ala Lys1
527PRTArtificial SequenceSynthetic 2Pro Asp Ser Thr Glu Ala Lys1
5
* * * * *