U.S. patent application number 17/588736 was filed with the patent office on 2022-08-04 for novel peptide derived from pep27 peptide and uses thereof.
The applicant listed for this patent is INDUSTRY ACADEMIC COOPERATION FOUNDATION CHOSUN UNIVERSITY. Invention is credited to Hee Kyoung KANG, Yoon Kyung PARK.
Application Number | 20220242921 17/588736 |
Document ID | / |
Family ID | 1000006169806 |
Filed Date | 2022-08-04 |
United States Patent
Application |
20220242921 |
Kind Code |
A1 |
PARK; Yoon Kyung ; et
al. |
August 4, 2022 |
NOVEL PEPTIDE DERIVED FROM PEP27 PEPTIDE AND USES THEREOF
Abstract
A peptide according to an embodiment of the present in which, in
the amino acid sequence of SEQ ID NO: 1, (i) 2nd and 4th amino
acids are each substituted with tryptophan (W); (ii) 2nd, 4th, 11st
and 13rd amino acids are each substituted with tryptophan; and
(iii) 2nd, 4th, 19th, 20th, 22nd, 23rd, 26th and 27th amino acids
are each substituted with tryptophan. The peptide can be usefully
applied as an active ingredient for antibiotics, cosmetic
compositions, food additives, feed additives, biological pesticides
and quasi-drugs.
Inventors: |
PARK; Yoon Kyung;
(Jeollanam-do, KR) ; KANG; Hee Kyoung; (Gwangju,
KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INDUSTRY ACADEMIC COOPERATION FOUNDATION CHOSUN UNIVERSITY |
Gwangju |
|
KR |
|
|
Family ID: |
1000006169806 |
Appl. No.: |
17/588736 |
Filed: |
January 31, 2022 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A23L 33/18 20160801;
A61Q 19/007 20130101; A61P 31/04 20180101; A61K 38/00 20130101;
A61K 8/64 20130101; A23K 20/147 20160501; A61Q 19/10 20130101; C07K
14/3156 20130101; A23V 2002/00 20130101 |
International
Class: |
C07K 14/315 20060101
C07K014/315; A61P 31/04 20060101 A61P031/04; A61K 8/64 20060101
A61K008/64; A61Q 19/00 20060101 A61Q019/00; A61Q 19/10 20060101
A61Q019/10; A23L 33/18 20060101 A23L033/18; A23K 20/147 20060101
A23K020/147 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 1, 2021 |
KR |
10-2021-0014180 |
Claims
1. A peptide in which, in the amino acid sequence of SEQ ID NO: 1,
(i) 2.sup.nd and 4.sup.th amino acids are each substituted with
tryptophan (W); (ii) 2.sup.nd, 4.sup.th, 11.sup.st and 13.sup.rd
amino acids are each substituted with tryptophan; or (iii)
2.sup.nd, 4.sup.th, 19.sup.th, 20.sup.th, 22.sup.nd, 23.sup.rd,
26.sup.th and 27.sup.th amino acids are each substituted with
tryptophan.
2. The peptide according to claim 1, wherein the peptide consists
of the amino acid sequence of SEQ ID NO: 2.
3. The peptide according to claim 1, wherein the peptide consists
of the amino acid sequence of SEQ ID NO: 3.
4. The peptide according to claim 1, wherein the peptide consists
of the amino acid sequence of SEQ ID NO: 4.
5. An antimicrobial composition comprising the peptide according to
claim 1 as an active ingredient.
6. The antimicrobial composition according to claim 5, wherein the
antimicrobial composition is a pharmaceutical composition.
7. The antimicrobial composition according to claim 5, wherein the
antimicrobial composition is any one selected from the group
consisting of a cosmetic compositions, a food additive, a feed
additive, a biological pesticide, an antiseptic composition and a
quasi-drug compositions.
8. A method of killing or reducing a growth of a microorganism
comprising at least one selected from the group consisting of
gram-positive bacteria, gram-negative bacteria and
antibiotics-tolerant bacteria, the method comprising administering
a composition comprising the peptide of claim 1 to a subject in
need thereof.
9. The method of claim 8, wherein the microorganism comprises the
gram-positive bacteria selected from the group consisting of
Starphylococcus aureus, Bacillus subtilis, Listeria monocytogenes
and a combination thereof.
10. The method of claim 8, wherein the microorganism comprises the
gram-negative bacteria selected from the group consisting of
Escherichia coli, Pseudomonas aeruginosa, Salmonella typhimurium
and a combination thereof.
11. The method of claim 8, wherein the microorganism comprises the
antibiotics-tolerant bacteria selected from the group consisting of
Starphylococcus aureus (S. aureus), Escherichia coli (E. coli),
Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhimurium (S.
typhimurium), and a combination thereof, which have antibiotic
tolerance.
12. The method of claim 8, wherein the peptide consists of the
amino acid sequence of SEQ ID NO: 2.
13. The method of claim 8, wherein the peptide consists of the
amino acid sequence of SEQ ID NO: 3.
14. The method of claim 8, wherein the peptide consists of the
amino acid sequence of SEQ ID NO: 4.
Description
[0001] CROSS-REFERENCE TO RELATED APPLICATION AND CLAIM OF
PRIORITY
[0002] This application claims priority to Korean Patent
Application No. 10-2021-0014180 filed on Feb. 1, 2021 in the Korean
Intellectual Property Office (KIPO), the entire disclosure of which
is incorporated by reference herein.
BACKGROUND
1. Field of the Invention
[0003] The present invention relates to a novel peptide derived
from PEP27 peptide and uses thereof.
2. Description of the Related Art
[0004] Bacterial infections are one of the most common and fatal
causes of human disease. Unfortunately, the abuse of antibiotics
has resulted in antibiotic resistance of bacteria. Indeed, a rate
at which bacteria become resistant to new antibiotics is much
faster than a rate at which analogs of new antibiotics are
developed. For example, bacterial species such as Enterococcus
faecalis, Mycobacterium tuberculosis and Pseudomonas aeruginosa,
which possibly threaten human life, have developed resistance to
all antibiotics known to date.
[0005] Antibiotic tolerance is a distinctive phenomenon from
antibiotic resistance, was firstly discovered in Pneumococcus sp.
in the 1970s, and has provided significant clues to the mechanism
of action of penicillin. Species showing tolerance to antibiotics
stop growing but do not die in the presence of antibiotics at usual
concentrations. Tolerance occurs because, when antibiotics inhibit
cell wall synthetase, bacterial autolytic enzymes such as autolysin
do not activate. Due to this fact, bacteria may be killed by
penicillin that activates endogenous hydrolytic enzymes. On the
other hand, the bacteria may inhibit the activity of the above
enzymes, resulting in survival even during antibiotic therapy. It
is clinically highly significant that bacteria have tolerance to
diverse antibiotics. The reason is that, if it is impossible to
eradicate tolerant bacteria, antibiotic treatment would be less
effective in clinical infections. Further, developing tolerance is
considered to be a pre-requisite for occurrence of resistance to
antibiotics because of survival of strains despite the antibiotic
therapy. Such strains acquire new genetic elements that are
resistant to antibiotics and continue to grow even in the presence
of antibiotics. Indeed, all bacteria resistant to antibiotics are
known to have tolerance too, therefore, development for new
antibiotics capable of killing the antibiotic-resistant bacteria is
required. In aspect of activity mechanism, the antibiotic tolerance
may be classified into two type pathways: first, a phenotypic
tolerance, which occurs when a growth rate is reduced in all
bacteria; and second, a genetic tolerance caused by mutation
occurring in specific bacteria. In both cases, a primary phenomenon
is occurrence of down regulation of autolysin activity, wherein the
down regulation may be transient when it is phenotypically tolerant
to external stimulation. On the other hand, the genetic tolerance
with the resulting mutation, which causes a change in pathway to
regulate cellular hemolysis, may be permanent. The simplest case of
the genetic tolerance is occurrence of defects in the autolysin
enzyme. For various uncertain reasons, strains having tolerance due
to defects of apoptotic enzymes described above have not been
clinically found. On the other hand, clinical tolerance is
exhibited by regulating activity of autolysin. In order to cope
with bacteria that are resistant to antibiotics, development of new
antibiotics is required, and further development of new antibiotics
acting independently of autolysin may also be required.
[0006] Meanwhile, bacteriocin is a natural antimicrobial protein
generated by different types of microorganisms and has bactericidal
activity to bacterial species similar to productive bacteria. The
bacteriocin may be classified into three classes in terms of
structure. The first is lantibiotics, the second is
nonlantibiotics, and the third is proteins secreted by signal
peptides. Animals including insects also produce naturally
occurring peptide antibiotics. Also, the peptide antibiotics may be
divided into three groups in terms of structure. The first is a
cysteine-rich .beta.-sheet peptide, the second is an
.alpha.-helical amphiphilic molecule, and the third is a
proline-rich peptide. These antimicrobial peptides are known to
play an important role in host defense and the innate immune
system. Specifically, these antimicrobial peptides have different
structures depending on the amino acid sequence.
SUMMARY
[0007] An object of the present invention is to provide novel
peptide and uses thereof.
[0008] To achieve the above object, the following technical
solutions are adopted in the present invention.
[0009] 1. A peptide in which, in the amino acid sequence of SEQ ID
NO: 1, (i) 2.sup.nd and 4.sup.th amino acids are each substituted
with tryptophan (W); (ii) 2nd, 4.sup.th, 11.sup.st and 13rd amino
acids are each substituted with tryptophan; or (iii) 2.sup.nd,
4.sup.th, 19.sup.th, 20.sup.th, 22.sup.nd, 23.sup.rd, 26.sup.th and
27.sup.th amino acids are each substituted with tryptophan.
[0010] 2. The peptide according to the above 1, wherein the peptide
consists of the amino acid sequence of SEQ ID NO: 3.
[0011] 3. The peptide according to the above 1, wherein the peptide
has antimicrobial activity against at least one selected from the
group consisting of gram-positive bacteria, gram-negative bacteria
and antibiotics-tolerant bacteria.
[0012] 4. The peptide according to the above 3, wherein the
gram-positive bacteria are at least one selected from the group
consisting of Starphylococcus aureus, Bacillus subtilis and
Listeria monocytogenes.
[0013] 5. The peptide according to the above 3, wherein the
gram-negative bacteria are at least one selected from the group
consisting of Escherichia coli, Pseudomonas aeruginosa and
Salmonella typhimurium.
[0014] 6. The peptide according to the above 3, wherein the
antibiotics-tolerant bacteria are at least one selected from the
group consisting of Starphylococcus aureus (S. aureus), Escherichia
coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and
Salmonella typhimurium (S. typhimurium), which have antibiotic
tolerance.
[0015] 7. An antimicrobial composition including the peptide
according to any one of the above 1 to 6 as an active
ingredient.
[0016] 8. The antimicrobial composition according to the above 7,
wherein the antimicrobial composition is a pharmaceutical
composition.
[0017] 9. The antimicrobial composition according to the above 7,
wherein the antimicrobial composition is any one selected from the
group consisting of cosmetic compositions, food additives, feed
additives, biological pesticides, antiseptic compositions and
quasi-drug compositions.
[0018] The peptide of the present invention has excellent
antimicrobial activity and low cytotoxicity, whereby it can be
usefully used as an active ingredient of antibiotics, cosmetic
compositions, food additives, feed additives, biological
pesticides, quasi-drugs and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] 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:
[0020] FIG. 1 illustrates results of confirming the secondary
structure of each of the antimicrobial PEP27 peptide (control) and
a new peptide, that is, PEP27-2 as a PEP27 analog in which an amino
acid residue is substituted (experimental group), respectively, in
a membrane-like environment, wherein (a) of FIG. 1 shows an aqueous
solution (10 mM PBS solution), (b) of FIG. 1 shows a bacterial
membrane-mimic artificial membrane composed of PE/PG(7/3), and (c)
of FIG. 1 shows an eukaryotic cell-mimic artificial membrane
composed of PC/CH/SM (1/1/1);
[0021] FIG. 2 illustrates results of confirming the action of PEP27
peptide as the control and PEP27-2 as the new peptide,
respectively, on Escherichia coli membrane by flow cytometry
(FACS);
[0022] FIG. 3 illustrates results of confirming the binding ability
of PEP27 peptide as the control and PEP27-2 as the new peptide,
respectively, to DNA, which is an internal substance of bacteria,
wherein the ratio of peptide:DNA is DNA alone (0), 0.25:1, 0.5:1,
1:1, 1.5:1, 2:1, 3:1 and 4:1, respectively;
[0023] FIG. 4 illustrates results of confirming the action of PEP27
peptide as the control and PEP27-2 as the new peptide,
respectively, on Staphylococcus aureus membrane by a scanning
electron microscope (SEM);
[0024] FIG. 5 illustrates results of confirming internalization of
PEP27-2 peptide labeled with FITC on human keratinocyte line (HaCaT
cell line, Dr. N E. Fusenig, Heidelberg, Germany) (FITC-PEP27-2) by
a fluorescence microscope;
[0025] FIG. 6 illustrates results of confirming the healing effects
of PEP27 peptide and antibiotics, that is, ceftriaxone, when used
alone or in combination with the same for treatment of abscess
formed after infection with Starphylococcus aureus MW2 which is a
multidrug-tolerant strain;
[0026] FIG. 7 illustrates results of determining a total number of
bacteria (CFU/g) recovered by gathering abscess portions formed
after infection with Starphylococcus aureus MW2 which is a
multidrug-tolerant strain;
[0027] FIG. 8 illustrates results of confirming strain distribution
as well as the healing effects of PEP27 peptide and antibiotics,
ceftriaxone, when used alone or in combination with the same for
treatment of abscess formed after infection with the strain formed
by inserting green fluorescent protein (GFP) gene into
Starphylococcus aureus MW2 which is a multidrug-tolerant strain
(Starphylococcus aureus MW2-GFP);
[0028] FIG. 9 illustrates results of confirming a change in abscess
tissues when PEP27 peptide and antibiotics, that is, ceftriaxone,
respectively, are used alone or in combination with the same for
treatment of abscess formed after infection with Starphylococcus
aureus MW2 which is a multidrug-tolerant strain; and
[0029] FIG. 10 illustrates results of confirming the relative
expression rate of genes related to inflammatory response in
abscess tissues formed after infection with Starphylococcus aureus
MW2 which is a multidrug-tolerant strain (TNF-.alpha.: tumor
necrosis factor-alpha, IL-1.beta.: interleukin-1 beta, IL-6:
interleukin-6, iNOS: nitrogen oxide synthetase, COX-2:
cyclooxygenase-2).
[0030] FIG. 11 and FIG. 12 illustrate time kill kinetics of PEP27
and its analogs. E. coli ATCC 25922 (A of FIG. 11) and S. aureus
ATCC 25923 (B of FIG. 11) were incubated with PEP27 or PEP27-2 at
their MICs for 0-80 min. Symbols represent the mean.+-.SD of pooled
data from three independent experiments (***P<0.001). Total
bacterial population was stained with the QUANTOM total cell
staining dye (FIG. 12). The intracellular fluorescence was imaged
using the QUANTOM Tx microbial cell counter, which captures and
counts cells automatically. Data in FIG. 11 were analyzed by
one-way ANOVA.
[0031] FIG. 13 illustrates the dose-response curve for hemolytic
activity against mRBCs (n=3 per condition). Data in FIG. 13 were
analyzed by one-way ANOVA. **P<0.01.
[0032] FIG. 14 illustrates the dose-response curve for cytotoxic
activity against HaCaT (A of FIG. 14) (n=4 per condition) and MCF-7
(B of FIG. 14) (n=3 per condition) cells. Symbols represent
mean.+-.SD from triplicate determinations. Data in FIG. 14 were
analyzed by two-way ANOVA. **P<0.01.
[0033] FIG. 15 illustrates the effect of PEP27 and PEP27-2 on
bacterial membrane permeability. A of FIG. 15 illustrates inner
membrane permeability. Hydrolysis of ONPG (n=3 per condition) due
to the release of cytoplasmic .beta.-galactosidase in E. coli ATCC
25922 cells treated with the indicated peptides at 1.times.MIC was
measured for 60 min at 420 nm. ***P<0.001 vs PEP27. B of FIG. 15
to E of FIG. 15 illustrate the changes in cytoplasmic membrane
potential indicated by the membrane potential-sensitive dye
DiSC.sub.3-5 (n=4 per condition) in E. coli ATCC 25922 cells
treated with PEP27 (B of FIG. 15), PEP27-2 (C of FIG. 15), buforin
2 (D of FIG. 15), or melittin (E of FIG. 15).
[0034] FIG. 16 illustrates the Calcein release from PE/PG (7/3,
w/w) (A of FIG. 16) and PC/CH/SM (1/1/1, w/w/w) (B of FIG. 16) LUVs
induced by PEP27 and PEP27-2 (n=3 per condition).
[0035] FIG. 17 illustrates inhibition of biofilm formation. A of
FIG. 17 illustrates Fluorescence microscopy analysis of biofilms
formed by MDR bacteria after treatment with PEP27-2 at
1.times.MBIC. Live cells were stained with SYTO9. B of FIG. 17 and
C of FIG. 17 illustrate the effect of PEP27-2 on biofilm formation
by standard bacteria (B of FIG. 17) and MDR bacteria (C of FIG.
17).
[0036] FIG. 18 illustrates the synergistic action of PEP27-2. A of
FIG. 18 and B of FIG. 18 illustrate that the synergistic action of
PEP27-2 and antibiotics killed S. aureus MW2. Logarithmically grown
(5.times.10.sup.5) S. aureus MW2 was incubated with indicated
combinations of PEP27-2 and antibiotics in PBS containing 0.5% TSB
at 37.degree. C. shaking. After 3 h of incubation, several
dilutions of the bacterial suspensions were plated onto TSB agar
plates. The next day S. aureus MW2 CFUs were counted. Each
experiment was performed in triplicate. The data represent the
average of three independent experiments.+-.SD: *P<0.05,
**P<0.01, ***P<0.001 vs untreated control. Data were analyzed
by one-way ANOVA. C of FIG. 18 illustrates the combination index of
PEP27-2 and antibiotics. Combination index was calculated using
CompuSyn (ComboSyn Inc.) and indicated in medium effect plots as a
function of the bacteria antagonistic effects as a function of the
bacteria fractions affected by the combinatorial antibiotic
treatment. Combination index value of 1 indicated additive effects,
whereas values <1 and >1 indicated synergistic and
antagonistic effects, respectively.
DETAILED DESCRIPTION
[0037] Hereinafter, the present invention will be described in
detail.
[0038] The present invention provides a peptide in which, in the
amino acid sequence of SEQ ID NO: 1, (i) 2.sup.nd and 4.sup.th
amino acids are each substituted with tryptophan (W); (ii)
2.sup.nd, 4.sup.th, 11.sup.st and 13.sup.rd amino acids are each
substituted with tryptophan; or (iii) 2.sup.nd, 4.sup.th,
19.sup.th, 20.sup.th, 22.sup.nd, 23.sup.rd, 26.sup.th and 27.sup.th
amino acids are each substituted with tryptophan.
[0039] PEP27, a parental peptide consisting of the amino acid
sequence of SEQ ID NO: 1, is secreted from Streptococcus
pneumoniae, and is known as a "death signal peptide" existing at
Vex123-Pep27-VncRS gene site.
TABLE-US-00001 (SEQ ID NO: 1)
MRKEFHNVLSSGQLLADKRPARDYNRK-NH.sub.2
[0040] PEP27 peptide is possibly prepared by any conventional
peptide synthesis method known in the art, and the preparation
method thereof is not particularly limited. For example, the method
for synthesis of PEP27 peptide used herein may include the typical
chemical synthesis method of peptide in the art, specifically, the
solution-phase peptide synthesis method, the solid-phase peptide
synthesis method, the fragment condensation method, and the F-moc
or T-BOC chemical method.
[0041] The peptide of the present invention may be peptides
consisting of any one amino acid sequence selected from the group
consisting of SEQ ID NOS: 2 to 4.
TABLE-US-00002 (SEQ ID NO: 2) MWKWFHNVLSSGQLLADKRPARDYNRK-NH.sub.2
(SEQ ID NO: 3) MWKWFHNVLSWGWLLADKRPARDYNRK-NH.sub.2 (SEQ ID NO: 4)
MWKWFHNVLSSGQLLADKWWAWWYNWW-NH.sub.2
[0042] The peptide consisting of the amino acid sequence of SEQ ID
NO: 2 is a peptide, in which 2.sup.nd and 4.sup.th amino acids in
PEP27 as the parental peptide are each substituted with tryptophan
(W), and refers to as PEP27-1.
[0043] The peptide consisting of the amino acid sequence of SEQ ID
NO: 3 is a peptide, in which 2.sup.nd, 4.sup.th, 11.sup.st and
13.sup.rd amino acids in PEP27 as the parental peptide are each
substituted with tryptophan (W), and refers to as PEP27-2.
[0044] The peptide consisting of the amino acid sequence of SEQ ID
NO: 4 is a peptide, in which 2.sup.nd, 4.sup.th, 19.sup.th,
20.sup.th, 22.sup.nd, 23.sup.rd, 26.sup.th and 27.sup.th amino
acids in PEP27 as the parental peptide are each substituted with
tryptophan (W), and refers to as PEP27-5.
[0045] "--NH.sub.2" in each of the amino acid sequences of SEQ ID
NOS: 1 to 4 indicates that a carboxyl group at C terminal was
changed by amidation.
[0046] Specifically, the peptide of the present invention may be a
peptide consisting of the amino acid sequence of SEQ ID NO: 3.
[0047] The amino acid substitution as described above may induce
increase/decrease of electrode and reduce cytotoxicity of the
peptide. Further, the peptide may represent antimicrobial activity
against gram-positive bacteria, gram-negative bacteria and
antibiotics-tolerant bacteria by the amino acid substitution
described above.
[0048] The peptide of the present invention may exhibit
antimicrobial activity against gram-positive bacteria,
gram-negative bacteria and antibiotics-tolerant bacteria.
[0049] The gram-positive bacteria may be at least one selected from
the group consisting of Staphylococcus, Listeria, Corynebacterium,
Lactobacillus and Bacillus, but they are not limited thereto.
Specifically, the peptide of the present may represent excellent
antimicrobial activity against at least one selected from
Staphylococcus, Bacillus or Listeria.
[0050] The gram-negative bacteria may be at least one selected from
the group consisting of Pseudomonas, Escherichia, Salmonella,
Leptospira and Rickettsia, but they are not limited thereto.
Specifically, the peptide of the present invention may exhibit
excellent antimicrobial activity to at least one selected from the
group consisting of Pseudomonas, Escherichia or Salmonella.
[0051] The antibiotics-tolerant bacteria may be at least one
selected from the group consisting of Pseudomonas aeruginosa,
Escherichia coli, Salmonella typhimurium and Staphylococcus aureus,
which have antibiotic tolerance, but they are not limited thereto.
The antibiotics described above may include at least one selected
from the group consisting of aminoglycoside series (aminoglycoside,
gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.),
sulfonamide series, beta-lactam series (beta-lactam,
amoxicillin/clavulanic acid), chloramphenicol series, erythromycin
series, fluorphenicol series, phosphomycin series, kanamycin
series, lincomycin series, methicillin series, quinolone series,
streptomycin series, tetracycline series, trimethoprim series and
vancomycin series of antibiotics, but they are not limited
thereto.
[0052] The peptide of the present invention may represent low
cytotoxicity within antimicrobial concentration in regard to
human-derived cells while having excellent antimicrobial activity
to the gram-positive bacteria, gram-negative bacteria and
antibiotics-tolerant bacteria.
[0053] Further, the present invention provides an antimicrobial
composition including the peptide described above as an active
ingredient.
[0054] The antimicrobial composition may be a pharmaceutical
composition. That is, the present invention provides a
pharmaceutical composition for antimicrobial use, which includes
the peptide described above as an active ingredient.
[0055] Peptide analogs derived from PEP27 antimicrobial peptide of
the present invention, that is, amino acid sequences of SEQ ID NOS:
2 to 4 may show strong antimicrobial activity and have low
cytotoxicity to human-derived cells, whereby the peptide of the
present invention can be usefully used as an active ingredient of
an antimicrobial pharmaceutical composition. For example, the
peptide of the present invention may be used as one of antibiotics.
For example, the peptide of the present invention may be used in
combination with other antibiotics and, when used in combination
with other antibiotics, may represent synergistic antimicrobial
activity compared to using the same alone.
[0056] The pharmaceutical composition may further include any
appropriate carrier, excipient and diluents, which are commonly
used in manufacturing a pharmaceutical composition, in addition to
the peptide described above as an active ingredient.
[0057] The carrier, excipient and diluents possibly included in the
pharmaceutical composition may include, for example, lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, acacia rubber, alginate, gelatin, calcium
phosphate, calcium silicate, cellulose, methyl cellulose,
microcrystalline cellulose, polyvinyl pyrrolidone, water,
methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium
stearate and mineral oil.
[0058] The pharmaceutical composition may be administered orally or
parenterally during clinical administration, and may be used in the
form of any typical pharmaceutical formulation. Parenteral
administration may refer to administration through a route other
than oral administration, such as rectal, intravenous, peritoneal,
muscle, arterial, transdermal, nasal, inhalation, ocular and
subcutaneous administration.
[0059] The pharmaceutical composition may further contain one or
more active ingredients exhibiting the same or similar functions as
the peptide which is the active ingredient described above.
[0060] For example, the pharmaceutical composition of the present
invention may further include any antibiotics known in the art.
[0061] The known antibiotics may be at least one selected from the
group consisting of aminoglycoside series (aminoglycoside,
gentamicin, neomycin, etc.), penicillin series (ampicillin, etc.),
sulfonamide series, beta-lactam series (beta-lactam,
amoxicillin/clavulanic acid), chloramphenicol series, erythromycin
series, fluorphenicol series, phosphomycin series, kanamycin
series, lincomycin series, methicillin series, quinolone series,
streptomycin series, tetracycline series, trimethoprim series and
vancomycin series of antibiotics. Specifically, the antibiotics may
be at least one selected from the group consisting of meropenem,
ceftazidime, ceftriaxone, doripenem, ertapenem, imipenem,
cilastatin, cefadroxil, cefazolin, cephalexin, cefaclor, cefotetan,
cefoxitin, cefprozil, cefuroxime, cefdinir, cefditoren, cefixime,
cefotaxime, cefpodoxime, ceftibuten, cefepime and ceftaroline. More
specifically, the antibiotics may be at least one selected from the
group consisting of meropenem, ceftazidime and ceftriaxone.
[0062] An effective dose of the peptide of the present invention
may range from 0.1 to 2 mg/kg, and the peptide may be administered
once to 3 times a day.
[0063] With regard to the peptide of the present invention, a total
effective amount of the peptide may be administered to a patient in
the form of a bolus or in a single dose by infusion or the like for
a relatively short period of time. Alternatively, the peptide may
be administered according to fractionated treatment protocol in
which multiple doses are administered over a long period of time.
With regard to a concentration of the peptide described above,
considering that an effective dosage to a patient is determined by
taking into account different factors such as the patient's age and
health condition, as well as administration routes and the number
of treatments of a drug, the effective dosage of the peptide of the
present invention may be appropriately determined for specific uses
of the peptide as an antibiotic by those skilled in the art.
[0064] The antimicrobial composition may be any one selected from
the group consisting of a cosmetic composition, food additives,
feed additives, biological pesticides, an antiseptic composition
and a quasi-drug composition.
[0065] The present invention provides an antimicrobial cosmetic
composition which includes the peptide described above as an active
ingredient.
[0066] The amino acid sequences of SEQ ID NOS: 2 to 4 as peptide
analogs derived from PEP27 antimicrobial peptide according to the
present invention may show strong antimicrobial activity and have
low cytotoxicity to human-derived cells, whereby the peptide of the
present invention can be usefully used as an active ingredient of
an antimicrobial cosmetic composition.
[0067] The cosmetic composition may further include components
generally used in a cosmetic composition, in addition to the
peptide described above as an active ingredient. For example, the
cosmetic composition may include at least one among conventional
auxiliary agents such as antioxidants, stabilizers, solubilizers,
vitamins, pigments and fragrances, and carriers.
[0068] The cosmetic composition may include the peptide as an
active ingredient in an amount of 0.1 to 50% by weight ("wt. %"),
and preferably 1 to 10 wt. %.
[0069] The cosmetic composition may be prepared in any type of
formulations commonly manufactured in the art, for example, may be
formulated in the form of solution, suspension, emulsion, paste,
gel, cream, lotion, powder, soap, surfactant-containing cleansing,
oil, powder foundation, emulsion foundation, wax foundation, spray,
etc., but it is not limited thereto.
[0070] If the formulation of the cosmetic composition is a paste,
cream or gel, animal oil, vegetable oil, wax, paraffin, starch,
tragacanth, cellulose derivatives, polyethylene glycol, silicone,
bentonite, silica, talc, or zinc oxide may be used as a carrier
component.
[0071] If the formulation of the cosmetic composition is a powder
or spray, lactose, talc, silica, aluminum hydroxide, calcium
silicate or polyamide powder may be used as a carrier component. In
particular, for a spray formulation, a propellant such as
chlorofluorohydrocarbon, propane/butane or dimethylether may be
further included.
[0072] If the formulation of the cosmetic composition is a solution
or emulsion, a solvent, solubilizer or emulsifier may be used as a
carrier component. For example, water, ethanol, isopropanol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,
propyleneglycol, 1,3-butylglycol oil, glycerol aliphatic ester,
polyethylene glycol or fatty acid ester of sorbitan may be
used.
[0073] If the formulation of the cosmetic composition is a
suspension, a liquid diluent such as water, ethanol or
propyleneglycol, a suspending agent such as ethoxylated isostearyl
alcohol, polyoxyethylene sorbitol ester and polyoxyethylene
sorbitan ester, microcrystalline cellulose, aluminum metahydroxide,
bentonite, agar, tragacanth, or the like may be used as a carrier
component.
[0074] If the formulation of the cosmetic composition is a
surfactant-containing cleansing agent, aliphatic alcohol sulfate,
aliphatic alcohol ether sulfate, sulfosuccinic acid monoester,
isethionate, imidazolinium derivatives, methyl taurate,
sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine,
fatty alcohols, fatty acid glycerides, fatty acid diethanolamides,
vegetable oils, lanolin derivatives or ethoxylated glycerol fatty
acid esters may be used as a carrier component.
[0075] The present invention provides an antimicrobial food
additive which includes the peptide described above as an active
ingredient.
[0076] The amino acid sequences of SEQ ID NOS: 2 to 4 as peptide
analogs derived from PEP27 antimicrobial peptide of the present
invention may show strong antimicrobial activity and have low
cytotoxicity to human-derived cells, whereby the peptide of the
present invention can be usefully used as an active ingredient of
an antimicrobial food additive.
[0077] When the peptide of the present invention is used as a food
additive, the peptide may be added at it is or may be used together
with other food ingredients. A mixing amount of the active
ingredient may be appropriately determined depending on the purpose
of use. For example, the peptide of the present invention may be
added in an amount of 15 parts by weight ("wt.parts") or less, and
preferably 10 wt.parts or less based on the raw material. However,
in the case of long-term intake, the above amount may be the above
range or less, and since there is no problem in terms of stability,
the active ingredient may be used even in an amount of the above
range or more.
[0078] The food to which the peptide of the present invention is
able to be added may include, for example, meat, sausage, bread,
chocolate, candies, snacks, confectionery, pizza, ramen, other
noodles, gums, dairy products including ice cream, various soups,
beverages, tea, drinks, alcoholic beverages and vitamin complexes,
and all foods in the usual sense are included.
[0079] The present invention provides an antimicrobial feed
additive which includes the peptide described above as an active
ingredient.
[0080] The amino acid sequences of SEQ ID NOS: 2 to 4 as peptide
analogs derived from PEP27 antimicrobial peptide of the present
invention may show strong antimicrobial activity and have low
cytotoxicity to human-derived cells, whereby the peptide of the
present invention can be usefully used as an active ingredient of
an antimicrobial feed additive.
[0081] When the peptide of the present invention is used as the
feed additive, effects of replacing existing antibiotics,
suppressing the growth of harmful food pathogens thus to enhance
the health condition of the animal body, improving the weight gain
and meat quality of livestock, and increasing milk production and
immunity may be acquired. When the peptide of the present invention
is used as a feed additive, the feed may be prepared in the form of
fermented feed, blended feed, pellet form, and silage.
[0082] The fermented feed may be produced by adding a variety of
microorganism groups or enzymes as well as the peptide of the
present invention to the feed and then fermenting organic
substances therein, while the blended feed may be produced by
admixing various types of general feeds as well as the peptide of
the present invention. Further, the pellet-type feed may be
produced by applying heat and pressure to the blended feed in a
pellet machine, while the silage may be produced by fermenting
fresh-cut forage with microorganisms. In addition, wet fermented
feed may be produced by collecting and transporting organic
materials such as food waste, sterilizing the same, blending the
same with excipients for adjusting water content at a predetermined
ratio, followed by fermenting the mixture at a temperature suitable
for fermentation for 24 hours or more and adjusting the water
content to reach about 70%. Fermented dry feed may be produced by
adjusting the water content through a drying process to reach 30 to
40%.
[0083] The present invention also provides an antimicrobial
antiseptic composition, an antimicrobial biological pesticide and
an antimicrobial quasi-drug, each of which includes the peptide of
the present invention as an active ingredient.
[0084] The amino acid sequences of SEQ ID NOS: 2 to 4 as peptide
analogs derived from PEP27 antimicrobial peptide of the present
invention may show strong antimicrobial activity and have low
cytotoxicity to human-derived cells, whereby the peptide of the
present invention can be usefully used as an active ingredient of
an antimicrobial antiseptic composition, an antimicrobial
biological pesticide and an antimicrobial quasi-drug,
respectively.
[0085] The antimicrobial antiseptic composition may include, for
example, food preservatives, cosmetic preservatives and
pharmaceutical preservatives. The food preservatives, cosmetic
preservatives and pharmaceutical preservatives are additives used
to prevent deterioration, spoilage, discoloration and chemical
modification. For example, fungicides and antioxidants may be
included, and functional antibiotics for suppressing the growth of
microorganisms such as bacteria, mold, yeast, etc. thus to inhibit
the growth of spoilage microorganisms or sterilize the same in food
and medicines may be further included. Desirable conditions for
such antiseptic composition as described above may include
non-toxicity and effectiveness even with trace amounts.
[0086] When the composition of the present invention is used as a
quasi-drug composition, the peptide as an active ingredient may be
added at it is or used together with other quasi-drugs or
quasi-drug components.
[0087] The quasi-drug composition may include, for example,
disinfectant cleaner, shower foam, mouthwash, wet tissue, a
detergent soap, hand wash, humidifier filler, mask, ointment, patch
or filter filler.
[0088] Further, the present invention provides an antimicrobial
treatment method which includes administering a pharmaceutically
effective amount of the antimicrobial peptide to a subject.
[0089] The subject may be a mammal other than a human, but it is
not limited thereto.
[0090] Hereinafter, the present invention will be concretely
described by means of the following examples. However, these
examples are only illustrative of the present invention, and the
scope and range of the present invention are duly not limited
thereto.
EXAMPLE
[0091] 1. Synthesis and Purification of Peptides
[0092] According to the method of synthesizing a liquid peptide
(Merrifield, R B., J. Am. Chem. Soc., 85, 2149, 196) of Merrifield,
a peptide was synthesized (SEQ ID NO: 2) by substituting 2.sup.nd
and 4.sup.th amino acid residues, which are present in the amino
acid sequence of PEP27 as the parental peptide defined with the
amino acid sequence of SEQ ID NO: 1, with tryptophan (W). Further,
another peptide was synthesized by substituting 11.sup.st,
13.sup.rd or 19.sup.th, 20.sup.th, 22.sup.nd, 23.sup.rd, 26.sup.th
and 27.sup.th amino acid residues, which are present in the amino
acid sequence of SEQ ID NO: 2, with tryptophan (W) (Table 1).
[0093] Specifically, the peptide having a carboxyl terminal in the
form of NH.sub.2 designed in the present invention was obtained
using a link amide MBHA-resin as a starting material, and the
peptide having a carboxyl terminal in the form of OH was obtained
using Fmoc (9-fluorenyl methoxycarbonyl)-amino acid-Wang resin as a
starting material. Extension of peptide chains by Fmoc-amino acid
coupling was implemented by DCC (N-hydroxy benzotriazole
(HOBt)-dicyclo-hexycarbodiimide) method. After coupling the
Fmoc-amino acid at the amino terminal of each peptide, Fmoc group
was removed with NMP (20% piperidine/N-methyl pyrrolidone)
solution, followed by washing several times with NMP and DCM
(dichloromethane) and then drying the same with nitrogen gas. To
the prepared peptide, a mixed solution of trifluoroacetic acid
(TFA), phenol, thioanisole, H.sub.2O and triisopropylsilane at a
ratio of 85:5:5:2.5:2.5 (v/v), respectively, was added, followed by
reaction for 2 to 3 hours to remove the protective group and to
separate the peptide from the resin. Thereafter, the peptide was
obtained by precipitation with diethylether. The obtained crude
peptide was purified in reverse phase (RP)-HPLC column (Delta Pak,
C18300 .ANG., 15, 19.0 mm.times.30 cm, Waters, USA) in an
acetonitrile gradient containing 0.05% TFA. After hydrolyzing the
synthetic peptide at 110.degree. C. with 6N hydrochloric acid, the
residue was concentrated under reduced pressure and dissolved in
0.02N hydrochloric acid, followed by measuring the constitutional
composition of amino acid using an amino acid analyzer (Hitachi
8500 A). Then, in order to determine purity and a molecular weight
of the peptide, MALDI mass spectrometry (Hill, et al., Rapid
Commun. Mass Spectrometry, 5: 395, 1991) was performed.
[0094] As a result, as shown in Table 1 below, the peptides defined
with the amino acid sequences of SEQ ID NOS: 1 to 4 were
synthesized with the purity of 95% or more, and the molecular
weights thereof were confirmed to be substantially the same as
expected molecular weights.
TABLE-US-00003 TABLE 1 Name SEQ Molec- of ID ular peptide Amino
acid sequence NO. weight PEP27 MRKEFHNVLSSGQLLADKR 1 3228.7
PARDYNRK-NH.sub.2 PEP27-1 MWKWFHNVLSSGQLLADKR 2 3316.9
PARDYNRK-NH.sub.2 PEP27-2 MWKWFHNVLSWGWLLADKR 3 3474.1
PARDYNRK-NH.sub.2 PEP27-5 MWKWFHNVLSSGQLLADKW 4 3625.2
WAWWYNWW-NH.sub.2
[0095] With reference to the sequence of PEP27 (SEQ ID NO: 1),
types of substituted amino acids of novel peptides (SEQ ID NOS: 2
to 4) and substitution sites thereof are shown in Table 2
below.
TABLE-US-00004 TABLE 2 Number of amino acid PEP27 PEP27-1 PEP27-2
PEP27-5 1 M 2 R W W W 3 K 4 E W W W 5 F 6 H 7 N 8 V 9 L 10 S 11 S W
12 G 13 Q W 14 L 15 L 16 A 17 D 18 K 19 R W 20 P W 21 A 22 R W 23 D
W 24 Y 25 N 26 R W 27 K W (Blank: no substitution of amino
acid)
[0096] 2. Measurement of Antimicrobial Activity
[0097] In order to evaluate the antimicrobial activity of the
peptides prepared by the method of above 1, a minimal inhibitory
concentration (MIC) value as the minimum concentration of a
peptide, in which cells are not divided, was measured.
[0098] Specifically, the strains shown in Table 3 below were
purchased, cultured in a medium having a constitutional composition
suitable for each strain to a mid-log phase, and then diluted to a
concentration of 2.times.10.sup.4 cells/100 .mu.l to prepare the
product on a micro-titration plate (Nunc, USA). Then, PEP27,
PEP27-1, PEP27-2 or PEP27-5 peptide synthesized in the above 1 was
added to each well by serial dilution 1/2 times, followed by
incubation at 37.degree. C. for 18 hours. Thereafter, using a
micro-titration plate reader (Merck Elisa reader, Germany),
absorbance was measured at a wavelength of 600 nm to determine the
MIC value for each strain. PEP27 as the parental peptide was used
as a control, while buforin 2 and melittin were used as comparison
peptides.
TABLE-US-00005 TABLE 3 Name of strain Division Origin Deposit No.
Staphylococcus Gram-positive American Type ATCC25923 aureus
bacteria Culture Collection (ATCC) ATCC ATCC29213 Antibiotics-
Isolated cell line tolerant gram- USA 300 positive bacteria
Isolated cell line MW2 (USA 400) Culture Collection CCARM3089 of
Antimicrobial Resistant Microbes (CCARM) CCARM CCARM3090 CCARM
CCARM3518 Transformed strain MW2-GFP Pseudomonas Gram-negative ATCC
ATCC15692 aeruginosa bacteria ATCC ATCC27853 Antibiotics- Isolated
cell line 4007 tolerant gram- Isolated cell line 4891 negative
bacteria Escherichia coli Gram-negative ATCC ATCC25922 bacteria
ATCC ATCC27853 Antibiotics- CCARM CCARM1229 tolerant gram CCARM
CCARM1238 negative bacteria Salmonella Gram-negative Korean Culture
Type KCTC1926 typhimurium bacteria Collection (KCTC) Antibiotics-
CCARM CCARM8007 tolerant gram- CCARM CCARM8008 negative bacteria
Bacillus subtilis Gram-positive KCTC KCTC1998 bacteria Listeria
Gram-positive KCTC KCTC3710 monocytogenes bacteria
[0099] As a result, as shown in Table 4 below, it was confirmed
that PEP27-2 peptide showed noticeably excellent antimicrobial
activity to gram-positive bacteria, gram-negative bacteria and
antibiotics-tolerant bacteria, as compared to the control, that is,
the parental PEP27 peptide and other novel peptides.
TABLE-US-00006 TABLE 4 Minimal inhibitory concentration (.mu.M)
Peptide Strain PEP27 PEP27-1 PEP27-2 PEP27-5 Buforin Melittin
Gram-positive bacteria S. aureus 32 32 2 >64 16 2 ATCC25923 S.
aureus 32 32 2 >64 16 2 ATCC29213 B. subtilis 64 32 4 >64 32
4 KCTC1998 L. monocytogens 32 64 8 >64 64 4 KCTC3710
Gram-negative bacteria E. coli 64 64 4 >64 64 2 ATCC25922 E.
coli 64 32 4 >64 64 2 ATCC27325 P. aeruginosa 64 32 4 >64 32
2 ATCC15692 P. aeruginosa 64 32 4 >64 32 2 ATCC27853 Sa.
typhimurium 64 32 8 >64 32 4 KCTC1926 Antibiotics-tolerant
gram-positive bacteria S. aureus 32 32 4 >64 32 2 CCARM3089 S.
aureus 32 16 4 >64 32 4 CCARM3090 S. aureus 32 32 4 >64 32 2
CCARM3518 S. aureus 32 32 2 >64 32 2 USA300 S. aureus MW2 32 32
3 >64 64 4 Antibiotics-tolerant gram-negative bacteria E. coli
64 32 4 >64 64 2 CCARM1229 E. coli 64 32 4 >64 64 4 CCARM1238
P. aeruginosa 64 32 4 >64 32 2 4007 P. aeruginosa 64 32 4 >64
64 2 4891 Sa. typhimurium 64 32 4 >64 32 4 CCARM8007 Sa.
typhimurium 64 32 4 >64 32 4 CCARM8009
[0100] The results of time-kill analysis of PEP27 and PEP27-2
against E. coli ATCC 25922 and S. aureus ATCC 25923 are shown in
FIGS. 11 and 12.
[0101] Time-killing kinetics were analyzed for E. coli ATCC 25922
and S. aureus ATCC 25923 in the presence of PEP27-2 at their
respective MICs. The bacterial cells were cultured overnight until
they reached the exponential growth phase; they were then incubated
at 37.degree. C. for an additional 10 min from 0 min to 1 h. The
total bacterial population was then stained with QUANTOM total cell
staining dye, after which the stained cells were mixed with QUANTOM
cell loading buffer and loaded into QUANTOM M50 Slides. The slides
were centrifuged using the QUANTOM centrifuge to evenly distribute
them throughout the counting chamber and ensure accurate cell
counts. Intracellular fluorescence was detected using the QUANTOM
Tx microbial cell counter, which captures and counts cells
automatically (Logos Biosystems, Gyeonggi-do, South Korea).
[0102] PEP27-2 induced a decrease in E. coli cell viability of
1.86.times.10.sup.9 and in S. aureus of 1.60.times.10.sup.9 (98.63%
and 97.48%, respectively) after 20 min of treatment, with complete
killing at 30 and 40 min, respectively (A of FIG. 11 and B of FIG.
11).
[0103] To monitor the changes in bacterial viability caused by the
peptides, we monitored the fluorescence spectra of bacterial
samples stained with QUANTOM total cell staining dye. The results
showed that PEP27-2 killed both strains within 60 min in the
time-kill kinetics (FIG. 12).
[0104] 3. Measurement of Hemolytic Activity
[0105] In order to compare the cytotoxicity of the peptides
prepared by the method of above 1, erythrocyte hemolytic activity
of each of the synthesized peptides was measured.
[0106] Specifically, red blood cells of a rat (Balb/c, 6 weeks old,
female) were diluted with PBS (pH 7.0) to a concentration of 8%,
and PEP27, PEP27-1, PEP27-2 and PEP27-5 peptides were used for
treatment of the cells at concentrations of 3.13, 6.25, 12.5, 25.01
50.0 and 100.0 .mu.M/well, respectively, followed by reaction at
37.degree. C. for 1 hour. Then, an amount of hemoglobin contained
in the supernatant obtained by centrifugation at 1,000.times.g was
determined by measuring the absorbance at a wavelength of 414 nm.
As a control which is a standard for the degree of cell
destruction, the absorbance of the obtained supernatant was
measured after treatment using 1% Triton X-100 (Sigma, USA) and
reaction at 37.degree. C. for 1 hour. With reference to 100%
erythrocytes hemolysis activity, hemolysis of each peptide was
calculated from the measured absorbance using Equation 1 below.
Erythrocyte destruction ability (%)=(Absorbance A-Absorbance
B)/(Absorbance C-Absorbance B).times.100 [Equation 1]
[0107] (wherein, absorbance A represents the absorbance of a
reaction solution treated with each peptide measured at a
wavelength of 414 nm; absorbance B represents the absorbance of a
reaction solution treated with PBS measured at a wavelength of 414
nm; and absorbance C represents the absorbance of a reaction
solution treated with 1% Triton X-100 measured at a wavelength of
414 nm).
[0108] As a result, as shown in Table 5 below and FIG. 13, although
PEP27 peptide as the parental peptide showed only 3.58% of
hemolytic action to rat erythrocytes when treated at a
concentration of 100 .mu.M, it was confirmed that PEP27-2 peptide
appeared occurrence of 75.8% hemolysis on erythrocytes even at the
concentration of 100 .mu.M, thereby increasing toxicity. However,
toxicity was increased due to increase in activity, and it was
confirmed that the toxicity is not found within the range of
antimicrobial activity.
TABLE-US-00007 TABLE 5 Erythrocyte destruction ability (%) Peptide
Peptide Peptide Peptide Peptide Peptide concentration concentration
concentration concentration concentration concentration 100 .mu.M
50 .mu.M 25 .mu.M 12.5 .mu.M 6.25 .mu.M 3.13 .mu.M PEP27 3.58 3.64
2.76 1.79 1.26 0 (SEQ ID NO: 1) PEP27-1 50.65 44.51 25.75 1.97 1.05
0 (SEQ ID NO: 2) PEP27-2 75.84 62.47 40.51 14.96 2.30 0 (SEQ ID NO:
3) PEP27-5 3.25 2.34 0 0 0 0 (SEQ ID NO: 4) Buforin 2 5.77 3.10
2.05 0.92 0.29 0 Melittin 99.17 97.76 83.99 69.75 49.41 27.33
[0109] 4. Confirmation of Cytotoxicity in Normal Cell Lines
[0110] In order to confirm the cytotoxicity of the peptides
prepared by the method of above 1 in normal cell lines, toxicity
was tested in HaCaT (human keratinocyte fibroblasts cell, A of FIG.
14) and MCF-7(human breast adenocarcinoma cell, B of FIG. 14)
cells.
[0111] Specifically, Thiazolyl blue tetrazolium bromide (MTT, Merck
KGaA) was used to determine the cytotoxicity of PEP27 and its
analogs against HaCaT (human keratinocyte fibroblasts, n=4 per
condition) and MCF-7 (human breast adenocarcinoma, n=3 per
condition) cells. The cells were cultured in Dulbecco's modified
Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS, Gibco, Grand Island, N.Y., USA) and 1%
penicillin-streptomycin (Gibco) at a density of 2.times.10.sup.4
cells/well (96-well plate). Peptides were then added at various
concentrations and incubated for 24 h at 37.degree. C., after which
MTT (final concentration, 0.5 mg/mL) was added to each well and
incubated for 4 h. The formazan product was solubilized in DMSO,
and the absorbance at 570 nm was measured to induce cytotoxicity.
Cells treated with 0.1% Triton X-100 were used as the 100%
cytotoxic control sample.
[0112] As a result, as shown in FIG. 14, at a high concentration of
100 .mu.M, the cell viabilities following treatment with PEP27,
PEP27-1, PEP27-2, and PEP27-5 were 100%, 41%-42%, 19%-23%, and
100%, respectively. Melittin strongly reduced the viability of all
mammalian cells tested, with the lowest cell viability of 0.76%.
PEP27-2 showed more potent antimicrobial activity than the parental
peptide. PEP27-2 showed cytotoxic effects when high concentrations
of the peptide were used to treat mouse erythrocytes and human
cells. These results agree with those obtained for the control
peptide melittin. However, unlike melittin, PEP27-2 was not toxic
within the range of antimicrobial activity or 4.times.MIC
concentration. Melittin begins to show toxicity within the range of
antibacterial activity that increases rapidly from 2.times.MIC.
These results indicate that PEP27-2 is a viable candidate,
particularly for human pathogenic bacteria, including MDR bacteria
if developed as a novel antimicrobial drug usable at low
concentrations.
[0113] 5. Measurement of Circular Dichroism Spectrum
[0114] In order to confirm whether the peptide prepared by the
method of above 1 form the .alpha.-helical structure, which is a
secondary structure, a spectrum was measured by a circular
dichroism method.
[0115] Specifically, PEP27, PEP27-1, PEP27-2 or PEP27-5 peptide in
a suspension of 1 mM large unilamellar vesicles (LUV) that consists
of 10 mM PBS (pH 7.4), PE/PG
(L-.alpha.-phosphatidylethanolamine/L-.alpha.-phosphatidyl-DL-glycerol;
7/3, w/w) or PC/CH/SM
(L-.alpha.-phosphatidylcholine/cholesterol/sphingomyelin; 1/1/1,
w/w/w) was added at 40 .mu.M to a cell of 0.1 cm length
(path-length) and fixed, followed by measuring a circular dichroism
spectrum on a Jasco 810 spectrophotometer at a temperature of
25.degree. C. The .alpha.-helical structure calculation formula for
the circular dichroic spectrum was used in Equation 2 below.
[ .theta. ] = .theta. obs 10 l c [ Equation .times. .times. 2 ]
##EQU00001##
[0116] (wherein .theta..sub.obs represents millidegrees of a
signal, l represents an optical path-length of a cell size (cm),
and c represents a concentration (mol/L) of added peptide).
[0117] As a result, when the peptide was added to the 10 mM PBS
solution, no structure was formed, whereas, when the peptide was
added to the LUV solution consisting of PE/PG (7/3) or PC/CH/SM
(1/1/1), it could be seen that the .alpha.-helix structure, which
is a secondary structure, was formed in all peptides even with
difference in structures therebetween. From the results, it was
confirmed that the antimicrobial peptide of the present invention
could form the .alpha.-helical structure on a PE/PG (7/3) solution
similar to a membrane of bacteria, which is a microorganism and a
PC/CH/SM (1/1/1) solution similar to an eukaryotic membrane (FIG.
1).
[0118] 6. Cell Membrane Disruption
[0119] To investigate the interactions with the peptide-bacterial
cell membrane, we estimated the permeability of PEP27-2 in E. coli
cytoplasmic membranes producing (3-galactosidase.
[0120] Changes in the permeability of the inner membrane of E. coli
caused by the peptides were estimated by measuring (3-galactosidase
activity using o-nitrophenyl-.beta.-galactosidase (ONPG, Merck
KGaA, n=3 per condition) as a substrate, as described previously.
Mid-log-phase E. coli ATCC 25922 cells were washed with 10 mM PBS
(pH 7.4) containing 100 mM NaCl and resuspended in 10 mM PBS (pH
7.4) to an OD.sub.600 of 1.2. The cell suspension was incubated
with 1 mM ONPG and 1.times.MIC of the peptides. Fluorescence was
measured at excitation and emission wavelengths of 622 and 670 nm,
respectively. Time-dependent changes in the relative fluorescence
intensity at 420 nm were measured with a Versa-Max microplate
reader (Molecular Devices, Sunnyvale, Calif., USA).
[0121] As shown in A of FIG. 15, PEP27 did not permeate the inner
membrane at the MIC. PEP27-2 slightly induced inner membrane
permeabilization compared to PEP27 at its MIC. However, the
membrane-permeable activity of PEP27-2 was weak compared to that of
melittin peptides. Melittin at the MIC rapidly increases the
permeabilization of the bacterial inner membrane. This finding
indicates that PEP27-2 did not completely destroy the cytoplasmic
membranes when killing E. coli.
[0122] We also used E. coli cells and the membrane
potential-sensitive cyanine dye 3,3'-dipropylthiadicarbocyanine
iodide (DiSC.sub.3-5) to test the ability of PEP27 and PEP27-2 to
depolarize Gram-negative bacterial membranes (B of FIG. 15 to E of
FIG. 15).
[0123] Depolarization of the cytoplasmic membrane by the peptides
was measured using the membrane potential-sensitive dye
DiSC.sub.3-5 (Merck KGaA, n=4 per condition) with intact E. coli
cells as previously described.66 Mid-log-phase E. coli ATCC 25922
cells were washed with 5 mM HEPES buffer containing 20 mM glucose
and resuspended to an OD.sub.600 of 0.05 in 5 mM HEPES buffer
containing 20 mM glucose and 0.1 M KCl. The cell suspension was
incubated with 1 .mu.M DiSC.sub.3-5 for 1 h. Next, 1.times.,
2.times., and 4.times.MIC of PEP27-2 was added to the mixture of E.
coli cells and DiSC.sub.3-5. Fluorescence was measured at
excitation and emission wavelengths of 622 and 670 nm,
respectively.
[0124] When the bacterial cytoplasmic membrane is disrupted, the
membrane potential dissipates E. coli, and DiSC.sub.3-5 is released
into the culture medium, resulting in a fluorescence increase.
After stabilization for 10 min by treatment with DiSC.sub.3-5, AMPs
were added, and the plasma membrane potential of E. coli was
confirmed by monitoring the change in fluorescence intensity for
another 30 min.
[0125] When cells were treated with PEP27-2 and melittin, the
fluorescence intensity increased dose-dependently. This suggests a
significant membrane depolarization effect in E. coli. Melittin at
4.times.MIC showed an increase in fluorescence intensity to nearly
140, whereas 4.times.MIC of PEP27 and PEP27-2 increased the
fluorescence intensity only up to 80. PEP27 and PEP27-2 showed very
similar results as the control (buffer), suggesting that minimal
membrane depolarization is involved in membrane interactions.
[0126] 7. AMP-Induced Calcein Release in LUVs
[0127] To examine the mechanism of action of PEP27 and PEP27-2, we
evaluated the changes in bacterial cell membrane permeability by
monitoring the release of calcein, a fluorescent marker, from
calcein entrapped LUVs with different lipid bilayer
compositions.
[0128] Peptide-induced membrane permeabilization was quantified by
monitoring calcein release from LUVs (n=3 per condition). Different
calcein-entrapped LUVs [PE/PG (7/3) or PC/CH/SM (1/1/1)] were
prepared as described above, except that the dry lipid films were
resuspended in 1 mL of PBS (pH 7.2) containing 70 mM calcein. Gel
filtration chromatography on a Sephadex G-50 column was performed
to separate free calcein from that entrapped in LUVs. Thereafter,
the LUVs with entrapped calcein were mixed with peptides at various
ratios, and the fluorescence of the released calcein was measured
with a SpectraMax M3 Multi-Mode Microplate Reader (Molecular
Devices, Sunnyvale, Calif., USA) at excitation and emission
wavelengths of 480 and 520 nm, respectively. The results obtained
for a sample treated with 0.1% Triton X-100 were considered
complete (100%) release.
[0129] The results are shown in A and B of FIG. 16. PEP27, PEP27-2,
and CPP buforin 2 induced little or no calcein release from the
PE/PG and PC/CH/SM LUVs. Even at high peptide-to-lipid (P/L) ratios
of 0.2, PEP27, PEP27-2, and buforin 2 only caused leakage (12%,
20%, and 20%, respectively) in the PE/PG and PC/CH/SM liposomes.
The positive control melittin (a pore-forming AMP) induced maximum
leakage of 100% at a P/L ratio of 0.05 (A and B of FIG. 16). These
results confirm that PEP27-2 does not destroy the bacterial cell
membrane and killed bacteria via other mechanisms.
[0130] 8. Flow Cytometry Measurement
[0131] In order to confirm whether the peptide prepared by the
method of above 1 acts on the bacterial membrane, PEP27-2 peptide
having better antimicrobial activity than the parental peptide was
analyzed through flow cytometry.
[0132] Specifically, the minimal inhibitory concentration (MIC)
values of the PEP27 parental peptide or the PEP27-2 peptide were
used for treatment of E. coli, respectively, followed by reaction
at 37.degree. C. for 1 hour. Thereafter, the supernatant was
removed using a centrifuge (10,000 rpm) and stained with propidium
iodide (PI) at a concentration of 10 .mu.g/ml at 4.degree. C. for
30 minutes. Then, the unbound propidium iodide (PI) was removed
using the centrifuge, and 1 ml of physiological saline (PBS) was
added to remove agglomeration of the cells. Thereafter, effects of
the peptide on the bacterial membrane were determined using a
Bechman flow cytometer.
[0133] As a result, as shown in FIG. 2, it was confirmed that the
PEP27-2 peptide (21.84%) was a little superior to the PEP27
parental peptide (19.9%) in terms of membrane destruction ability
in Escherichia coli cell. For the positive control without
peptides, the percentage of PI-positive E. coli cells was very low
(4.48%), indicating that the bacterial cell membranes were intact.
However, like buforin 2 as a cell permeable peptide, the above
peptides were found not to have an ability of damaging a bacterial
membrane. It was confirmed that PEP27 and PEP27-2 could extinct
Escherichia coli without destruction of the E. coli membrane. Among
control peptides, melittin destructing a cell membrane was found to
have cell membrane destruction ability in Escherichia coli,
therefore, the peptide of the present invention was demonstrated to
have a mechanism different from that of melittin.
[0134] 9. Analysis of Binding of Antimicrobial Peptide and DNA
[0135] In order to specifically determine what mechanism the
synthetic peptide of the present invention prepared by the method
of above 1 exhibits antimicrobial activity, it was investigated
through electrophoresis whether PEP27, PEP27-1, PEP27-2 or PEP27-5
synthetic peptide binds to DNA, which is an internal material of
bacteria.
[0136] Specifically, 300 ng of plasmid DNA (pRSETB) was reacted
with the peptide by a relative ratio (reactions were performed at
peptide/DNA ratios of DNA alone, 0.25:1, 0.5:1, 1:1, 1.5:1, 2:1,
3:1, 4:1, respectively) at 37.degree. C. for 10 minutes, followed
by conducting electrophoresis on a 1% agarose gel, staining the
same with ethidium bromide (EtBr) and monitoring through UV.
[0137] As a result, as shown in FIG. 3, melittin as a comparison
peptide was not bound to any DNA, while all the PEP27, PEP27-2 and
the control peptide were bound to DNA even with difference in
binding levels (FIG. 3). Herein, PEP272-2 peptide showed DNA
binding ability higher than PEP27. From the above results, it was
confirmed that, due to substitution of some amino acid residues,
PEP27-2 peptide has higher binding ability to DNA, which is an
internal material of bacteria, as compared to the parental peptide,
thereby exhibiting higher antimicrobial activity.
[0138] 10. Scanning Electron Microscope (SEM) Analysis
[0139] In order to confirm whether the synthetic peptides of the
present invention prepared by the method of above 1 would damage
the bacterial cell membrane, it was investigated through a scanning
electron microscope.
[0140] Specifically, after suspending E. coli and Staphylococcus
aureus cells in PBS at a concentration of 0.2 in terms of
OD.sub.600, the cells were treated with PEP27-2 peptide at MIC and
then reacted at 37.degree. C. for 1 hour. As a control, a strain
not treated with peptide was used. After incubation, the cells were
recovered, treated with 2.5% glutaraldehyde at 4.degree. C. for 18
hours, and washed twice with PBS buffer solution. The fixed cells
were dehydrated for 10 minutes using 100% ethanol and diluted
ethanol (50%, 70%, 90% and 100%) in sequential order, and then the
samples were dried and coated with platinum, followed by
observation through a low vacuum scanning electron microscope.
[0141] As a result, as shown in FIG. 4, it was confirmed that the
peptide-untreated control cells had a bright and smooth surface
whereas the cells treated with melittin, which is a membrane
destruction peptide used as a comparison control, showed
significant membrane damage. The cell surface exposed to the
peptide was observed to have blister-like damage due to formation
of pores and, in some cases, leakage of contents in the cytoplasm
was observed. However, the cells treated with PEP27-2 peptide
appeared very little cell damage, which is almost the same as the
control. As a result, it was confirmed that bacteria became extinct
without destruction of the cell membrane.
[0142] 11. Cell Absorption of Peptide
[0143] With regard to the synthetic peptide prepared by the method
of above 1, an ability of penetrating cells was determined through
a phase-contrast microscope. Specifically, human keratinocyte line
(HaCaT cells) was dispensed on a 6-well plate provided with a
polylysine coating cover slip (SPL Life Sciences, Gyeonggi-do,
Korea) at 2.times.10.sup.5 cells/well and cultured for 24 hours,
and then treated with fluorescein isothiocyanate (FITC)-labeled
PEP27-2 (FITC-PEP27-2) at a concentration of 5 .mu.M, followed by
incubation in a 5% CO.sub.2 incubator for 1 hour. After 1 hour, a
nucleus of the cell was treated and stained with Hoechst 33342
nucleic acid dye solution (Invitrogen, Carlsbad, Calif., USA; 8
.mu.M) for 10 minutes. After washing the stained product with PBS
three times, fluorescence in the cells was imaged using a
phase-contrast microscope (EVOS.TM. FL Auto 2 Imaging System,
Thermo Fisher Scientific).
[0144] As a result, as shown in FIG. 5, FITC-PEP27-2 was obviously
absorbed in HaCaT cells and uniformly distributed as green
fluorescence in the cells, thus appearing in both of the cytoplasm
and the nucleus. In order to confirm localization of PEP27-2 in the
nucleus, contrast staining of the nucleus of the cell treated with
FITC-labeled peptide was performed with a nucleus staining dye
Hoechst 33342. From the stained cell, it was confirmed that PEP27-2
is distributed in both of the cytoplasm and the nucleus of HaCaT
cells. And in HaCaT cells, cellular absorption of FITC-PEP27-2 into
the cytoplasm and the nucleus was confirmed. On the other hand, the
control peptide, that is, TAT (GRKKRRQRRRPQ(SEQ ID NO:5)) which is
a cell permeable peptide having 11 amino acid residues and is
highly positive-charged, was found to be absorbed into the
cytoplasm and the nucleus of HaCaT cell like the PEP27-2
peptide.
[0145] 12. Measurement of Anti-Biofilm Activity
[0146] Biofilms may form on a wide variety of surfaces, including
living or dead surfaces, and biofilm-associated bacteria are more
tolerant to antibiotics than planktonic bacterial cells, making
them antibiotic-resistant. To assess the efficacy of PEP27-2
against biofilms, we observed the anti-biofilm activity of PEP27-2
in S. aureus (ATCC 25923 and CCARM 3090), E. coli (ATCC 25922 and
CCARM 1238), and P. aeruginosa (ATCC 27853 and 4891).
[0147] All tested bacterial strains formed widespread biofilms in
the absence of PEP27-2 (A of FIG. 17). The maximum percent biofilm
inhibition by PEP27-2 was 83.4% against S. aureus ATCC 25923, 91.8%
against E. coli ATCC 25922, and 81.2% against P. aeruginosa ATCC
27853 (B of FIG. 17). In addition, we confirmed that PEP27-2
efficiently inhibits biofilm formation by MDR bacterial strains
(73.3% against S. aureus CCARM 3090, 84.3% against E. coli CCARM
1238, and 81.1% against P. aeruginosa 4891) (C of FIG. 17).
[0148] We also observed minimal biofilm inhibitory concentration
(MBIC) against MDR S. aureus, E. coli, and P. aeruginosa.
[0149] Specifically, among the strains listed in Table 3 above,
Staphylococcus aureus, E. coli and Pseudomonas erujinosa,
respectively, were cultured in each medium to a mid-log phase,
diluted to a cell concentration of 5.times.10.sup.4 cells/100
.mu.l, and then, inoculated into a micro-plate (SPL). Then, PEP27-2
peptide was diluted 1/10 times with 10 mM PBS solution (pH 7.2) and
10 .mu.l of the solution was added to each well, followed by
incubation at 37.degree. C. for 24 hours. After completely removing
the supernatant, the solution was fixed with 100% methanol for 15
minutes, stained with a crystal violet dye solution for 1 hour,
washed 3 times, and then dissolved in 95% ethanol, followed by
measuring the absorbance at a wavelength of 595 nm using a micro
titration plate reader, thereby determining a biofilm minimal
inhibitory concentration value for each strain.
[0150] As a result, as shown in Table 6 below, it was confirmed
that PEP27-2 peptide exhibited strong biofilm inhibitory activity
in all strains, which is substantially similar to that of melittin
as the control peptide.
TABLE-US-00008 TABLE 6 Biofilm minimal inhibitory concentration
(.mu.M) PEP27-2 Melittin Gram-positive bacteria S. aureus ATCC25923
4 4 S. aureus ATCC29213 4 4 Gram-negative bacteria E. coli
ATCC25922 8 4 E. coli ATCC27325 8 4 P. aeruginosa ATCC15692 8 4 P.
aeruginosa ATCC27853 8 4 Antibiotics-tolerant gram-positive
bacteria S. aureus CCARM3089 4 4 S. aureus CCARM3090 4 4 S. aureus
CCARM3518 4 8 Antibiotics-tolerant gram-negative bacteria E. coli
CCARM1229 8 4 E. coli CCARM1238 8 4 P. aeruginosa 4007 8 4 P.
aeruginosa 4891 8 8
[0151] 13. Confirmation of Antimicrobial Activity of Antimicrobial
Peptide and Antibiotics
[0152] In order to compare the antimicrobial activity of PEP27-2,
which is the peptide having the most excellent antimicrobial
activity among the peptides prepared by the method of above 1, with
existing antibiotics, MIC concentrations of specific antibiotics
were investigated with regard to the same gram-positive bacteria
(Staphylococcus aureus) and the same gram-negative bacteria
(Pseudomonas erujinosa) stains. Specifically, subject strains were
prepared by the method of above 2, and were treated with the
antibiotics, that is, meropenem, ceftazidime, ceftriaxone or
vancomycin, respectively, followed by determining MICs of the
antibiotics.
[0153] As a result, as shown in Table 7 below, it was confirmed
that PEP27-2 express antimicrobial activity and MIC of PEP27-2 to
11 types of strains has ranged from 2 to 4 .mu.M. Specifically, it
was confirmed that meropenem, ceftazidime, ceftriaxone and
vancomycin express antimicrobial activity and MICs of these
antibiotics have ranged from 0.25 to 512 .mu.M (Table 7).
TABLE-US-00009 Minimal inhibitory concentration (.mu.M)
Microorganism PEP27-2 Meropenem Ceftazidime Ceftriaxone Vancomycin
S. aureus 2 2 16 8 0.25 ATCC25923 S. aureus 2 2 3 4 0.25 ATCC29213
S. aureus 4 64 512 512 0.25 CCARM3089 S. aureus 4 128 512 512 0.5
CCARM3090 S. aureus 4 64 64 256 0.25 CCARM3518 S. aureus 2 2 32 16
0.25 USA300 S. aureus MW2 4 2 32 32 0.25 P. aeruginosa 4 2 4 16 512
ATCC15692 P. aeruginosa 4 2 2 8 256 ATCC27853 P. aeruginosa 4 4 16
16 512 4007 P. aeruginosa 4 8 16 64 512 4891
[0154] 14. Confirmation of Synergistic Effects of Combination
Treatment of Antimicrobial Peptide and Antibiotics
[0155] Based on MIC values determined with regard to respective
mixtures, synergistic effects of the peptide and the antibiotics in
relation to antimicrobial activity to strains were assessed. For
assessment, fractional inhibitory concentration (FIC) analysis was
implemented by checkerboard assay.
[0156] Specifically, after inoculating 100 .mu.l of bacteria
(5.times.10.sup.5 CFU/mL) to each well of a 96-well plate, 50 .mu.l
of peptide solution diluted from the MIC value (solution 2:1
diluted by stages) was added to each well. Thereafter, an
antibiotic solution was diluted and added to each well by 50 .mu.l.
Further, the above procedures were implemented under opposite
conditions, so as to determine MIC value of each mixed solution.
FIC values were calculated as follows: FIC index=FIC (A)+FIC
(B)=[A]/MIC(A)+[B]/MIC(B), wherein [A] represents the MIC of
PEP27-2 when used in combination with antibiotics, MIC (A)
represents the MIC of PEP27-2 alone, FIC (A) represents FIC of
PEP27-2, and [B], MIC (B) and FIC (B) represent values
corresponding to the antibiotics. With the calculated FIC values,
it was assessed as follows: <0.5--synergy effect; 0.5 to
4--indifference; >4.0--antagonism.
[0157] As a result, it was confirmed that any combination of the
peptide and the antibiotics did not appear antagonism. Accordingly,
as shown in Table 8 below and FIG. 18, with regard to
Staphylococcus aureus CCARM 3089 and CCARM 3090, synergistic
effects were demonstrated when meropenem, ceftazidime or
ceftriaxone was combined with 0.25.times.MIC of PEP27-2. Further,
with regard to Pseudomonas erujinosa 4891 and Staphylococcus aureus
MW2, synergistic effects were demonstrated only when either
meropenem or ceftriaxone was combined with 0.25.times.MIC of
PEP27-2 (Tables 8 and 9). As shown in Tables 9 and 10 below,
synergistic effects were expressed in most of antibiotics-tolerant
strains as compared to a few of standard strains.
TABLE-US-00010 TABLE 8 Fractional inhibitory Treated concentration
Effects of Strain peptide/antibiotics (FIC) combination S. aureus
PEP27-2/Meropenem 1 Indifferent ATCC2523 PEP27-2/Ceftazidime 0.75
Indifferent PEP27-2/Ceftriaxone 0.75 Indifferent S. aureus
PEP27-2/Meropenem 1 Indifferent ATCC29213 PEP27-2/Ceftazidime 0.75
Indifferent PEP27-2/Ceftriaxone 0.75 Synergy S. aureus
PEP27-2/Meropenem 0.75 Indifferent USA300 PEP27-2/Ceftazidime 0.75
Indifferent PEP27-2/Ceftriaxone 0.75 Indifferent S. aureus
PEP27-2/Meropenem 0.5 Synergy MW2 PEP27-2/Ceftazidime 0.75
Indifferent PEP27-2/Ceftriaxone 0.5 Synergy S. aureus
PEP27-2/Meropenem 0.5 Synergy CCARM3089 PEP27-2/Ceftazidime 0.5
Synergy PEP27-2/Ceftriaxone 0.5 Synergy S. aureus PEP27-2/Meropenem
0.38 Synergy CCARM3090 PEP27-2/Ceftazidime 0.5 Synergy
PEP27-2/Ceftriaxone 0.5 Synergy S. aureus PEP27-2/Meropenem 0.75
Indifferent CCARM3518 PEP27-2/Ceftazidime 0.75 Indifferent
PEP27-2/Ceftriaxone 0.75 Indifferent
TABLE-US-00011 TABLE 9 Fractional inhibitory Treated concentration
Effects of Strain peptide/antibiotics (FIC) combination P.
aeruginosa PEP27-2/Meropenem 0.63 Indifferent ATCC15692
PEP27-2/Ceftazidime 0.75 Indifferent PEP27-2/Ceftriaxone 0.5
Synergy P. aeruginosa PEP27-2/Meropenem 0.75 Indifferent ATCC27853
PEP27-2/Ceftazidime 1 Indifferent PEP27-2/Ceftriaxone 0.75
Indifferent P. aeruginosa PEP27-2/Meropenem 0.75 Indifferent 4007
PEP27-2/Ceftazidime 0.5 Synergy PEP27-2/Ceftriaxone 0.5 Synergy P.
aeruginosa PEP27-2/Meropenem 0.5 Synergy 4891 PEP27-2/Ceftazidime
0.75 Indifferent PEP27-2/Ceftriaxone 0.5 Synergy
[0158] 15. Measurement of Healing Ability of Abscess Infected with
Multidrug-Tolerant Staphylococcus Aureus
[0159] The efficacy of PEP27-2, which is the peptide having the
most excellent antimicrobial activity among the peptides prepared
by the method of above 1 was evaluated in vivo using a mouse
model.
[0160] Specifically, the epidermis on the back side (dorsal side)
of 6-7 week old BALB/c mouse was infected with Staphylococcus
aureus MW2 having GFP gene inserted therein (S. aureus MW2-GFP,
1.times.10.sup.9 CFU/10 .mu.l PBS). After 2 hours form infection,
PEP27-2 alone (0.2 mg/kg, 50 .mu.l), ceftriaxone alone (0.2 mg/kg,
50 .mu.l) or a combination of the peptide and the antibiotic (0.05
mg/kg PEP27-2+0.1 mg/kg ceftriaxone, 50 .mu.l) was injected.
Further, the control mice were injected with the same amount of PBS
(20 .mu.l) without peptide. On 1.sup.st, 2.sup.nd, 3.sup.rd,
4.sup.th, 5.sup.th and 7.sup.th days after treatment, the shape of
abscess was photographed, and surrounding tissues as well as the
abscess were gathered. The abscess tissue was homogenized in 500
.mu.l of sterile PBS using a tissue grinder. A serial diluted
solution of the homogenate was spread on an agar plate and cultured
for 24 hours in order to quantify the Staphylococcus aureus MW2
strain.
[0161] As a result, it was confirmed that abscess is not formed in
the experimental group to which only PBS was injected without
infection of Staphylococcus aureus MW2 strain (FIG. 6). On the
other hand, for the experimental group to which only PBS was
injected after infection of Staphylococcus aureus MW2, it could be
seen that abscess was formed on day 1 after infection, a size of
the abscess did not decrease till 7.sup.th day, which in turn
caused severe inflammation. However, in all of the experimental
groups in which only PEP27-2 was injected, only ceftriaxone was
injected, and a combination of PEP27-2+ceftriaxone was injected,
respectively, after infection of Staphylococcus aureus MW2, it
could be seen that the size of formed abscess was smaller than the
experimental group in which only PBS was injected. On the other
hand, it was confirmed visually that abscess healing effects when
treated with the combination described above were significantly
similar to or slightly better than treatment using PEP27-2 alone,
after infection of Staphylococcus aureus MW2 (FIG. 6). In order to
confirm the abscess healing effects more obviously, a homogenate of
abscess tissue was cultured followed by counting the number of
bacteria. Even from these results, it was demonstrated that, when
treated using PEP27-2 or ceftriaxone after bacterial infection, the
number of bacteria was reduced by 80.0% or 46.0% on day 3,
respectively, and was further reduced by 89.3% or 61.5% on day 7,
respectively. In addition, it was confirmed that the number of
colonies after combination treatment was about 91.1%, and thus the
number of Staphylococcus aureus MW2 strains was lower than that
after single treatment (FIG. 7). Therefore, it could be understood
that the combination treatment has synergistic effects of improving
the removal of bacteria on infected site, as compared to the single
treatment using PEP27-2 or ceftriaxone alone. Furthermore, it was
confirmed that strains in the abscess portion could be effectively
removed even with treatment using PEP27-2 alone.
[0162] 16. Live Fluorescence Imaging of Abscess Mouse Model
[0163] For real-time tracking the progress of infection with regard
to the peptide that has the most excellent antimicrobial activity
among the peptides prepared by the method of above 1, fluorescence
(GFP)-labeled Staphylococcus aureus MW2 (Staphylococcus aureus
MW2-GFP) was used. Fluorescence images were taken from the
beginning of infection to 6 hours, 1 day, 2 days and 3 days by
means of an FOBI fluorescence imaging system (Neo Science, Suwon,
Korea), followed by analysis thereof using live image software.
[0164] As a result, on 0 hour after infection of Staphylococcus
aureus MW2-GFP at a high dose of (1.times.10.sup.9 CFU), there was
no difference in fluorescence signals between the experimental
groups (FIG. 8). Further, on the skin surface above the abscess
treated only with Staphylococcus aureus MW2-GFP, the fluorescence
signal was continuously increased for 3 days. On the other hand,
the fluorescence signal after treatment using PEP27-2 alone was
substantially similar to that obtained after combination treatment,
whereas the fluorescence signal after treatment using ceftriaxone
alone was not reduced (FIG. 8). On the basis of the fluorescent
abscess portion, it was confirmed that the combination treatment of
PEP27-2 and ceftriaxone showed synergistic effects.
[0165] 17. Staining of Abscess Tissue Fragment Infected with
Multidrug-Tolerant Staphylococcus Aureus
[0166] Among tissues on the abscess portion and infected portion
gathered in the above 13, the tissue on day 7 was fixed with 4%
buffered formalin. The infection portion or abscess portion fixed
with formalin was embedded in paraffin, while staining a fragment
of the cross-section with hematoxylin and eosin.
[0167] As a result, the infected portion of a mouse infected with
Staphylococcus aureus MW2 alone was extended from the skin tissue
to the deep skeletal muscle tissue, and neutrophils and macrophages
were observed around the infected portion. Due to deep inflammation
in the upper dermis, damage and necrosis of skeletal muscles, a
large abscess with shells/scabs was formed on the top of the skin
(FIG. 9). By counting the number of bacteria and live fluorescence
imaging, it was confirmed that Staphylococcus aureus MW2 with
increased scabs was contained in the skin surface. On 2 hours after
infection of Staphylococcus aureus MW2, the mice were treated with
PEP27-2, ceftriaxone or a combination thereof, respectively. From
the treated mice, it was confirmed that the number of visible scabs
formed on the skin surface and the number of bacteria were markedly
reduced. However, it was confirmed that inflammatory response
occurred most noticeably in the tissue fragment during the
combination treatment (FIG. 9).
[0168] 18. Analysis of Pro-Inflammatory Cytokine Gene Expression
Using Quantitative Real-Time PCR
[0169] mRNA expression of inflammatory mediators such as tumor
necrosis factor-alpha (TNF-.alpha.), interleukin-1 beta
(IL-1.beta.), interleukin-6 (IL-6), nitrogen oxide synthetase
(iNOS) and cyclooxygenase-2 (COX-2) is changed during skin
infection and wound healing. For mice not infected with bacteria,
infected mice, and mice subjected to treatment using PEP27-2 alone,
ceftriaxone alone or a combination thereof after infection, the
expression of inflammatory genes in the mice were measured thus to
verify the efficacy of each of PEP27-2 single treatment and
combination treatment with antibiotics.
[0170] Specifically, total RNA was isolated from wound tissues
using Trizol reagent. From the prepared total RNA, cDNA was
synthesized using a cDNA synthesis kit. qPCR was implemented using
qPCR 2.times.premix (SYBR Green), followed by determining
expression amounts of TNF-.alpha., IL-1.beta., IL-6, iNOS and COX-2
among inflammatory genes. Expression levels were quantified based
on .beta.-actin, and the PCR process was conducted as followed: at
50.degree. C. for 2 minutes and at 95.degree. C. for 10 minutes;
95.degree. C. for 15 seconds, 60.degree. C. for 1 minute, 40
cycles; 95.degree. C. for 15 seconds, 60.degree. C. for 1 minute,
95.degree. C. for 30 seconds, and 60.degree. C. for 15 seconds. The
gene expression was calculated after standardization to
.beta.-actin level through .DELTA..DELTA.Ct method. Transcription
of the control cell was set to 1 and other experimental groups have
multiple of the set value.
[0171] As a result of confirming the expression of TNF-.alpha.,
IL-1.beta., IL-6, iNOS and COX-2, it was observed that the
expression of each of the inflammatory cytokines was increased in
the abscess portion of the skin infected with Staphylococcus aureus
MW2 strain. Further, in the conditions in which the skin wounds
infected with bacteria were subjected to PEP27-2 single treatment,
ceftriaxone single treatment or combination treatment thereof, it
was confirmed that the expression of inflammatory cytokines was
suppressed (FIG. 10). Further, the combination treatment of PEP27-2
and ceftriaxone exhibited stronger suppression of the inflammatory
cytokines than the single treatment using either thereof except for
TNF-.alpha.. On the other hand, in the case of TNF-.alpha., PEP27-2
single treatment and combination treatment of PEP27-2 and
ceftriaxone, the expression was similarly suppressed. This result
was demonstrated that the combination treatment had synergistic
effects in suppressing the expression of some inflammatory
mediators derived from the mouse infected with Staphylococcus
aureus MW2. Therefore, it was confirmed that, when PEP27-2 is
combined with ceftriaxone injection, antimicrobial effects against
Staphylococcus aureus MW2 would be exhibited in vivo, thereby
inhibiting inflammation occurring in response to infection.
[0172] From the above experimental results, it could be confirmed
that three types of PEP27 analogs (SEQ ID NOS: 2 to 4) according to
the present invention had low cytotoxicity to normal cells, and
exhibited antimicrobial activity to gram-positive bacteria,
gram-negative bacteria and antibiotics-tolerant bacteria, which is
substantially excellent than or equal to that of PEP27 as the
parental peptide. Specifically, among the synthesized peptides,
PEP27-2 peptide (SEQ ID NO: 3) was demonstrated to exhibit
remarkably excellent antimicrobial activity compared to the PEP27
parental peptide. In addition, when the synthetic peptide analog is
used for treatment in combination with antibiotics, it was
confirmed that synergistic effects could be obtained for
antimicrobial activity to the gram-positive bacteria, gram-negative
bacteria and antibiotics-tolerant bacteria.
Preparative Example 1: Preparation of Pharmaceutical
Formulation
[0173] 1-1. Preparation of Powder
TABLE-US-00012 TABLE 10 Component Weight (mg) Peptide of the
present 20 mg invention Lactose 20 mg
[0174] After admixing the above components, the mixture was filled
in an airtight cloth bag to prepare powder.
[0175] 1-2. Preparation of Tablets
TABLE-US-00013 Component Weight (mg) Peptide of the present 10 mg
invention Corn starch 100 mg Lactose 100 mg Magnesium stearate 2
mg
[0176] After admixing the above components, the mixture was punched
to form a tablet formulation according to the conventional tablet
formation method.
[0177] 1-3. Preparation of Capsules
TABLE-US-00014 TABLE 12 Component Weight (mg) Peptide of the
present 10 mg invention Crystalline cellulose 3 mg Lactose 14.8 mg
Magnesium stearate 0.2 mg
[0178] After admixing the above components, the mixture was charged
in a gelatin capsule to form a capsule formulation according to the
conventional capsule preparation method.
[0179] 1-4. Preparation of Liquid Formulation
TABLE-US-00015 TABLE 13 Component Weight (mg) Peptide of the
present 20 mg invention Isomerized sugar 10 g Mannitol 5 g Purified
water Appropriate amount
[0180] According to the conventional preparation method of liquid
preparation, each of the components was added to purified water to
dissolve it, followed by adding lemon zest in an appropriate amount
and blending the above components, then, purified water was added
to adjust a total amount to 100 ml. Thereafter, the mixture was
charged in a brown bottle, followed by sterilization thus to
prepare the liquid formulation.
[0181] 1-5. Preparation of Injection Formulation
TABLE-US-00016 TABLE 14 Component Addition amount Peptide of the
present 10 mg/ml invention Diluted hydrochloric acid BP Till
reaching pH 7.6 Main sodium chloride BP Max. 1 ml
[0182] The peptide of the present invention was dissolved in an
appropriate volume of the main sodium chloride BP for primary use,
the pH of the resulting solution was adjusted to pH 7.6 with
diluted hydrochloric acid BP, and the volume was adjusted with the
main sodium chloride BP and then sufficiently blended. The solution
was charged in a 5 ml type I ampoule made of transparent glass, the
glass was dissolved and air-sealed under an upper grid, followed by
sterilizing the same in an autoclave at 120.degree. C. for 15
minutes or longer, thereby preparing an injection formulation.
Preparative Example 2: Preparation of Cosmetics
[0183] 2-1. Emollient Lotion (Skin)
[0184] In order to prepare an antimicrobial emollient skin lotion
containing the peptide of the present invention, the following
components could be admixed as listed in Table 15 below to prepare
the emollient skin lotion according to the conventional
manufacturing method in the field of cosmetics.
TABLE-US-00017 TABLE 15 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention 1,3-butyleneglycol 3.0 Glycerin 5.0
Polyoxyethylene (60) 0.2 hydrogenated castor oil Ethanol 8.0 Citric
acid 0.02 Sodium citrate 0.06 Preservative Trace amount Flavor
Trace amount Purified water To 100
[0185] 2-2. Nutritional Lotion (Lotion)
[0186] In order to prepare an antimicrobial nutritional lotion
containing the peptide of the present invention, the following
components could be admixed as listed in Table 16 below to prepare
the nutritional lotion according to the conventional manufacturing
method in the field of cosmetics.
TABLE-US-00018 TABLE 16 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Squalane 10.0 Polyoxyethylene sorbitan
2.0 monooleate Guaiacum oil 0.1 to 30 1,3-butyleneglycol 8.0
Glycerin 5.0 Polyoxyethylene (60) 0.2 hydrogenated castor oil
Ethanol 8.0 Citric acid 0.02 Sodium citrate 0.06 Preservative Trace
amount Flavor Trace amount Purified water To 100
[0187] 2-3. Essence
[0188] In order to prepare an antimicrobial essence containing the
peptide of the present invention, the following components could be
admixed as listed in Table 17 below to prepare the essence
according to the conventional manufacturing method in the field of
cosmetics.
TABLE-US-00019 TABLE 17 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Sitosterol 1.7 Polyglyceryl 2-oleate
1.5 Ceramide 0.7 Ceteares-4 1.2 Cholesterol 1.5 Dicetyl phosphate
0.4 Concentrated glycerin 5.0 Carboxylvinyl polymer 0.2 Xanthan gum
0.2 Preservative Trace amount Flavor Trace amount Purified water To
100
[0189] 2-4. Face Wash (Cleansing Foam)
[0190] In order to prepare an antimicrobial face wash (cleansing
foam) containing the peptide of the present invention, the
following components could be admixed as listed in Table 18 below
to prepare the cleansing foam according to the conventional
manufacturing method in the field of cosmetics.
TABLE-US-00020 TABLE 18 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Sodium N-acylglutamate 20.0 Glycerin
10.0 PEG-400 15.0 Propyleneglycol 10.0 POE(15) oleylalcohol ether
3.0 Laurin derivative 2.0 Methyl paraben 0.2 EDTA-4Na 0.03 Flavor
0.2 Purified water To 100
[0191] 2-5. Nourishing Cream
[0192] In order to prepare an antimicrobial nutrient cream
containing the peptide of the present invention, the following
components could be admixed as listed in Table 19 below to prepare
the nourishing cream according to the conventional manufacturing
method in the field of cosmetics.
TABLE-US-00021 TABLE 19 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Vaseline 7.0 Liquid paraffin 10.0
Beewax 2.0 Polysorbate 60 2.5 Sorbitan sesquioleate 1.5 Squalane
3.0 Propyleneglycol 6.0 Glycerin 4.0 Triethanolamine 0.5 Xanthan
gum 0.5 Tocopherol acetate 0.1 Flavor, preservative Trace amount
Purified water To 100
[0193] 2-6. Massage Cream
[0194] In order to prepare an antimicrobial massage cream
containing the peptide of the present invention, the following
components could be admixed as listed in Table 20 below to prepare
the massage cream according to the conventional manufacturing
method in the field of cosmetics.
TABLE-US-00022 TABLE 20 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Propyleneglycol 6.0 Glycerin 4.0
Triethanolamine 0.5 Beewax 2.0 Tocophenyl acetate 0.1 Polysorbate
60 3.0 Sorbitan sesquioleate 2.5 Cetearyl alcohol 2.0 Liquid
paraffin 30.0 Xanthan gum 0.5 Flavor, preservative Trace amount
Purified water To 100
[0195] 2-7. Pack
[0196] In order to prepare an antimicrobial pack containing peptide
of the present invention, the following components could be admixed
as listed in Table 21 below to prepare the pack according to the
conventional manufacturing method in the field of cosmetics.
TABLE-US-00023 TABLE 21 Component Content (wt. %) Peptide of the
present 0.1 to 30 invention Propyleneglycol 2.0 Glycerin 4.0
Polyvinyl alcohol 10.0 Ethanol 7.0 PiG-40 hydrogenated 0.8 castor
oil Triethanolamine 0.3 Flavor, preservative Trace amount Purified
water To 100
[0197] A sequence listing electronically submitted with the present
application on Jan. 31, 2022 as an ASCII text file named
20220131_Q73822LC09_TU_SEQ, created on Jan. 31, 2022 and having a
size of 2,000 bytes, is incorporated herein by reference in its
entirety.
Sequence CWU 1
1
5127PRTArtificial SequencePEP27MOD_RES(27)..(27)AMIDATION 1Met Arg
Lys Glu Phe His Asn Val Leu Ser Ser Gly Gln Leu Leu Ala1 5 10 15Asp
Lys Arg Pro Ala Arg Asp Tyr Asn Arg Lys 20 25227PRTArtificial
SequencePEP27-1MOD_RES(27)..(27)AMIDATION 2Met Trp Lys Trp Phe His
Asn Val Leu Ser Ser Gly Gln Leu Leu Ala1 5 10 15Asp Lys Arg Pro Ala
Arg Asp Tyr Asn Arg Lys 20 25327PRTArtificial
SequencePEP27-2MOD_RES(27)..(27)AMIDATION 3Met Trp Lys Trp Phe His
Asn Val Leu Ser Trp Gly Trp Leu Leu Ala1 5 10 15Asp Lys Arg Pro Ala
Arg Asp Tyr Asn Arg Lys 20 25427PRTArtificial
SequencePEP27-5MOD_RES(27)..(27)AMIDATION 4Met Trp Lys Trp Phe His
Asn Val Leu Ser Ser Gly Gln Leu Leu Ala1 5 10 15Asp Lys Trp Trp Ala
Trp Trp Tyr Asn Trp Trp 20 25512PRTArtificial SequenceTAT 5Gly Arg
Lys Lys Arg Arg Gln Arg Arg Arg Pro Gln1 5 10
* * * * *