Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS OF DIAGNOSING AND TREATING INFECTED IMPLANTS
FIELD OF THE INVENTION
[0001]The present invention relates to compositions for use in methods for
treating microbial infections on the surfaces of an implant or other surfaces
near an implant in a patient. In particular, the methods are useful for
treating
biofilm infections. The methods include using minimally invasive techniques
such as ultrasound for facilitating the detection of biofilm infections on an
implant or other surface of a patient and destroying the infection by
administering an anthocyanin or an anthocyanidin or metabolite thereof. In
particular, the administration of protocatechuic acid (PCA) or 2,4,6
trihydroxybenzaldehyde (2,4,6 THBA) for use in the treatment of an infected
implant or other surface of a patient is described herein.
BACKGROUND OF THE INVENTION
[0002] Post-operative infections of implants, such as total joint implants
following surgery is frequent, occurring in 1-2% of all cases. The present
diagnosis of infected surgical and medical implants is not always successful.
The clinical presentation is often problematic. Presently, diagnosis depends
zo on both clinical judgment and reliance on standard serologic clinical
tests.
These tests include sedimentation rate (ESR), C-reactive protein (CRP), white
blood cell count (WBC), analysis of aspirated joint fluid, and interpretation
of
intraoperative tissue and fluid test results. Even having evidence of implant
loosening stemming from x-ray identification and pre-operative needle
aspirations can be negative for microbes and cultures of adjacent tissue at
the
time of surgery may be negative for any bacteria. Diagnosis, therefore, can be
complicated and based on a combination of findings, rather than a single one.
Even with these available diagnostic tests, an infected implant often escapes
detection. Clinically, the presentation may not be evident and currently,
there
is no single or combination of tests accurate enough to diagnose an infected
implant.
[0003] The many various methods may fail to identify the source of the
infection in spite of clinical evaluation, laboratory testing, radiological
scans
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and even molecular biology. One of the main reasons for difficulty in
diagnosis
is due to biofilm formation. Common biofilms stem from pathogenic bacteria
and yeast. The biofilm forms a firm attachment to the implant that resists
mobilization. Recently, it has been reported that the only way to mobilize
these biofilms is after surgical removal of the prosthesis and subjection of
the
removed implant to ultrasound (see Trampuz etal., NEJM. 357, pp. 654-663,
2007).
[0004] Once an implant is diagnosed as being infected, there are few good
treatment options. Traditional treatments include antibiotic therapy,
irrigation,
debridement, arthrodesis and even amputation in life threatening situations.
Commonly, the treatment of an infected implant is surgical removal,
placement of a temporary antibiotic impregnated spacer, followed by one or
more surgeries to replace another implant with the attendant risks and
morbidity. Thus, patients having an infection often require complex and
.. expensive treatment that often has low outcome. Each of these methods are
either unsuccessful or are met with complications. The humanitarian and
societal costs are high for the diagnosis and treatment of loose and or
infected total joints. These costs start with the initial diagnostic dilemmas
that
delay definitive treatment or result in erroneous therapies. There is,
therefore,
zo an unmet need for more effective methods of diagnosing an infected
implant
that is in a patient and treating the infected implant. Described herein are
effective methods for detecting an infection of an implant and compounds and
compositions for treating the infected implant that solves this unmet need.
SUMMARY OF THE INVENTION
[0005] In its various embodiments, the present invention relates to
compositions for use in methods for treating infections (e.g., bacterial
infections) on the surfaces of an implant in a patient. In particular, the
methods are useful for treating biofilm infections (e.g., biofilm bacterial
infections). The methods include the use of minimally invasive techniques for
determining the presence of an infection on the implant and destroying the
infection by administering an anthocyanin or an anthocyanidin or metabolites
thereof (e.g., protocatechuic acid or 2,4,6 trihydroxybenzaldehyde).
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[0006] In one embodiment, protocatechuic acid, 2,4,6-
trihydroxybenzaldehyde, or a mixture or combination thereof, for use in a
method of treating a biofilm bacterial infection on a surface of an implant or
a
surface of a patient suspected of having a biofilm bacterial infection is
disclosed. The method includes removing a biofilm-forming bacteria by a
minimally invasive technique comprising needle aspiration or an application of
ultrasound or both to determine a presence of a biofilm around or on the
surface of the implant or surface of the patient, wherein the minimally
invasive
technique dislodges the removed biofilm-forming bacteria from the biofilm
io colony. The method also includes inhibiting or destroying the biofilm
colony
and all biofilm-forming bacteria on or around the surface of the implant in
the
patient or surface of the patient by administering a composition comprising
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof or administering pure crystals or a powder of
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof in an amount effective to inhibit or destroy the biofilm
on
or about the surface of the implant in the patient or the surface of the
patient.
[0007] In some embodiments, the ultrasound is applied at a frequency and
power sufficient to dislodge the biofilm-forming bacteria off the surface of
the
zo implant in a patient or the surface of the patient. In some embodiments,
the
ultrasound is applied at least one of (i) prior to performing a first needle
aspiration and (ii) after performing a first needle aspiration. In some
embodiments, the minimally invasive technique is needle aspiration. In some
embodiments, the minimally invasive technique is needle aspiration and
application of ultrasound. In some embodiments, the ultrasound is applied
prior to performing a first needle aspiration. In some embodiments, the
ultrasound is applied after performing a first needle aspiration. In some
embodiments, the surface of the patient is a closed wound, intact skin, or
skin
having a sinus track.
[0008] In some embodiments, the implant is a medical device, a medical or
surgical implant, total joint prosthesis, a catheter, a dental implant, or a
heart
or vascular graft. In some embodiments, a presence of the biofilm on the
surface of the implant in the patient or the surface of the patient in
previously
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negative tests for a biofilm-forming bacteria following needle aspiration
requires application of ultrasound to dislodge a second portion of biofilm-
form ing bacteria from the biofilm colony for confirmation of the presence of
the
biofilm on the surface of the implant in the patient or surface of the
patient.
[0009] In some embodiments, the ultrasound is applied by a transcutaneous
probe, external stimulation, or lithotripsy. In some embodiments, a presence
of the biofilm on the surface is determined by a needle aspiration prior to
the
application of the composition or the crystals.
[0010] In some embodiments, the biofilm comprises a biofilm-forming bacteria
.. selected from Pseudomonas aeruginosa and Methicillin-resistant
Staphylococcus aureus.
[0011] In some embodiments, the composition is a solution comprising
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof. In some embodiments, the solution comprises about
.. 0.25% to about 50% by weight of protocatechuic acid or 2,4,6-
trihydroxybenzaldehyde or a mixture or combination thereof. In some
embodiments, the solution comprises about 20% to about 30% by weight of
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof.
zo .. [0012] In some embodiments, the composition is a solution comprising
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof and the solution comprises at least one of (i) about 0.25%
to about 50% by weight of protocatechuic acid or 2,4,6-
trihydroxybenzaldehyde or a mixture or combination thereof and (ii) about
20% to about 30% by weight of protocatechuic acid or 2,4,6-
trihydroxybenzaldehyde or a mixture or combination thereof.
[0013] In some embodiments, the composition, powder, or the crystals is
administered once. In some embodiments, the composition, powder, or
crystals is administered continuously, hourly, daily, weekly, or monthly.
.. [0014] In some embodiments, ultrasound is applied to or near the surface of
the implant or surface of the patient having the biofilm bacterial infection
to
dislodge the biofilm colony and facilitate release of a biofilm-forming
bacteria
from the surface. In some embodiments, an aspirate obtained from performing
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the needle aspiration is cultured to determine if any biofilm-forming bacteria
are present, thereby indicating a presence of the biofilm bacterial infection
on
the surface of the implant or the surface of the patient. In some embodiments,
an aspirate obtained from performing the needle aspiration is cultured to
determine if any biofilm forming bacteria are present on the surface of the
implant or the surface of the patient following administration of the
composition or the crystals.
[0015] In some embodiments, an aspirate obtained from performing the
needle aspiration is cultured to determine if any biofilm-forming bacteria are
io present, thereby indicating a presence of the biofilm bacterial
infection on the
surface of the implant or the surface of the patient at least one of (i) prior
to
administration of the composition or the crystals and (ii) following
administration of the composition or the crystals.
[0016] In some embodiments, the ultrasound is applied at a frequency of
about 10 kHz to about 60 kHz. In some embodiments, the ultrasound is
applied at a power density of about 0.1 W/cm2 to about 0.5 W/cm2. In some
embodiments, the ultrasound is applied using at least one of (i) a frequency
of
about 10 kHz to about 60 kHz and (ii) a power density of about 0.1 W/cm2 to
about 0.5 W/cm2. In some embodiments, the ultrasound is applied at a
zo frequency of about 40 kHz and a power density of about 0.22 W/cm2. In
some
embodiments, the ultrasound is applied percutaneously and transcutaneously.
In some embodiments, the surface is further washed with a physiologically
compatible solution to facilitate release of a biofilm-forming bacteria. In
some
embodiments, the surface is washed with a composition comprising an anti-
microbial peptide.
[0017] In some embodiments, a method includes a) performing a needle
aspiration to dislodge and remove a first portion of biofilm-forming bacteria
from the biofilm colony; b) culturing the first portion of biofilm-forming
bacteria
to determine a presence of the biofilm-forming bacteria around or on the
surface of the implant or surface of the patient; c) if the culture is
negative,
applying an ultrasound to dislodge and remove a second portion of biofilm-
form ing bacteria from the biofilm colony; d) if the culture is identified as
positive, administering to a surrounding space in proximity of the surface of
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the implant or the surface of a patient at least one of (i) a solution
comprising
protocatechuic acid in an amount of about 0.25% to about 50% by weight, (ii)
a solution comprising 2,4,6-trihydroxybenzaldehyde in an amount of about
0.25% to about 50% by weight, (iii) a solution comprising a mixture or
combination of protocatechuic acid and 2,4,6-trihydroxybenzaldehyde in an
amount of about 0.25% to about 50% by weight, (iv) crystals of protocatechuic
acid, (v) crystals of 2,4,6-trihydroxybenzaldehyde, (vi) and a mixture or
combination of crystals of protocatechuic acid and 2, 4, 5-
trihydroxybenzaldehyde; wherein the surface of an implant or a surface of a
io patient is at least one of (i) a closed wound, (ii) intact skin, (iii)
skin having
minimal exposure with a fistula, (iv) a medical device, (v) a medical or
surgical
implant, (vi) a dental implant, (vii) total joint prosthesis, (viii) a
catheter, (ix) a
heart graft, and (x) a vascular graft.
[0018] In one aspect, a method includes a) performing a needle aspiration to
determine a presence of a biofilm-forming bacteria around or on the surface of
the implant or surface of the patient; b) culturing the biofilm-forming
bacteria
obtained from the needle aspiration to determine a presence of the biofilm-
form ing bacteria around or on the surface of the implant or surface of the
patient; c) if the culture is negative, then an ultrasound is applied to
dislodge
zo the biofilm colony and biofilm-forming bacteria from the surface of the
implant
or the surface of the patient; d) if the culture is identified as positive,
then
administering to a surrounding space in proximity of the surface of the
implant
or the surface of a patient a solution comprising protocatechuic acid or 2,4,6-
trihydroxybenzaldehyde or a mixture or combination thereof in an amount of
about 0.25% to about 50% by weight or administering crystals of
protocatechuic acid or 2,4,6-trihydroxybenzaldehyde or a mixture or
combination thereof, which is effective to inhibit or destroy the biofilm
colony
and all biofilm-forming bacteria on the surface of the implant or the surface
of
the patient; and e) optionally applying ultrasound prior to performing the
needle aspiration to dislodge the biofilm colony and facilitate release of a
biofilm forming bacteria from the surface; wherein the surface of the patient
is
a closed wound, intact skin or skin having minimal exposure with a fistula;
and
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wherein the implant is a medical device, a medical or surgical implant, a
dental implant, total joint prosthesis, a catheter, or a heart or vascular
graft.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 provides the minimum, maximum and optimum pH for growth
of microorganisms. Acidic environments retard proliferation of various
bacteria. Anthocyanins, anthocyanidins and main metabolites are unstable
relative to basic pH; thus, anthocyanins, anthocyanidins and main metabolites
thereof have the potential to lower the pH of wound tissue as well as any
surfaces and act as bactericidal or bacteriostatic.
io .. [0020] Figure 2 is the metabolic pathway of cyanidin-3-glucoside (C3G)
and
includes the chemical structures of cyanidin-3-glucoside and cyanidin and
their metabolites.
[0021] Figure 3 is the chemical structure of Protocatechuic acid (PCA),
a dihydroxybenzoic acid, a type of phenolic acid. It is a major metabolite of
antioxidant polyphenols found in certain plants, including green tea.
[0022] Figure 4 compares concentrations of C3G and PCA to determine
optimal effective concentrations. Bacterial burdens for P. aeruginosa were
compared after treatment with C3G or PCA at 48 and 96 hours. A
concentration of PCA 25 mM was effective to reduce the bioburden with
zo statistical significance at 48 hours. C3G at 100 and 200 mM
concentrations
were effective at reducing the bioburden at 48 and 96 hours.
[0023] Figure 5 is a chart disclosing potential sources of PCA.
[0024] Figure 6 is a table providing a summary of the effectiveness of certain
anthocyanins, anthocyanidins and a metabolite, including bactericidal or
bacteriostatic activity. During this test, the purity, concentrations and
molecular weight of these test substances (compounds) were known. The
carrier was water and the dose was accurately calculated. Delphinidin limited
growth against C. perfringens, S. aureus, and MRSA. Pelargonidin limited
growth of P. acnes, C. perfingens, S. aureus, MRSA, and S. pyogenes.
Cyanidin Cl was effective against C. difficile, C. perfringens, S. aureus ATCH
6538, S. aureus (MRSA) ATCH 33591, S. mutans, and S. pyogenes. A
proprietary formulation of cyanidin-3-glucoside (approximately 28% C3G by
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weight) had limited effectiveness during this study (18-24 hours for aerobes;
48 hours for anaerobes (C. albicans and L. casei); however, this C3G
formulation, was effective against P. acnes, E. coli, MRSA, K. pneumoniae,
and P. aeruginosa. Protocatechuic acid (PCA), the main metabolite from
anthocyanins and anthocyanidins, was effective against all bacteria tested as
well as C. albicans and K. pneumoniae. Importantly for skin wound treatment,
PCA was effective against S. aureus 6538 and 33591 (MRSA) and
P. aeruginosa. PCA was also effective on C. albicans, which is important
considering its ability to form biofilms and difficulty in treating C.
albicans
when existing with a catheter or implant.
[0025] Figure 7 is a table summarizing in vitro test results of 2, 4, 6
trihydroxybenzaldehyde and demonstrating its ability to act as an
antimicrobial, including as a bactericidal or bacteriostatic. Specifically,
2,4,6
THBA was effective against E. coli, K. pneumoniae, P. aeruginosa, S. aureus
6538 and 33591 (MRSA); further it was effective against a fungi,
Aureobasidium pullulans, ATCC 15233.
[0026] Figure 8A is a photographic image illustrating in vitro test results of
2,4,6 THBA against P. aeruginosa. Figure 8B is a photographic image
illustrating in vitro test results of 2,4,6 trihydroxybenzaldehyde against
S. aureus 33591 (MRSA). Figure 8C is a photographic image illustrating in
vitro test results of 2,4,6 THBA against P. acnes.
[0027] Figure 9A shows the results of a rodent back skin tape study where
concentrations of PCA and C3G in a vehicle of water were utilized to
determine effectiveness against P. aeruginosa skin infections. Figure 9B
.. shows the results of rodent back skin tape study where concentrations of
PCA
and C3G in a vehicle of water were utilized to determine effectiveness against
P. aeruginosa skin infections.
[0028] Figure 10A shows the results of an additional rodent back skin tape
study to determine effective dosages of PCA and C3G in a vehicle of water
.. that would be bactericidal for P. aeruginosa. Figure 10B shows the results
of
an additional rodent back skin tape study to determine effective dosages of
PCA and C3G in a vehicle of water that would be bactericidal for
P. aeruginosa. Figure 10C shows the results of an additional rodent back skin
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tape study to determine effective dosages of PCA and C3G in a vehicle of
water that would be bactericidal for P. aeruginosa.
[0029] Figure 11A shows the results of a rodent back skin study to determine
the effects of PCA and C3G on the local growth hormones in untreated skin
wounds of rodents. A concentration of 25 mM PCA increased local growth
hormone levels of IGF-1 at the site of the untreated skin wound. Figure 11B
shows the results of a rodent back skin study to determine the effects of PCA
and C3G on the local growth hormones in untreated skin wounds of rodents.
A concentration of 25 mM PCA increased local growth hormone levels of
io TGF-Beta at the site of the untreated skin wound. Figure 11C shows the
results of a rodent back skin study to determine the effects of PCA and C3G
on the local growth hormones in untreated skin wounds of rodents. A
concentration of 25 mM PCA increased local growth hormone levels of EGF
at the site of the untreated skin wound.
[0030] Figure 12A is a photographic image of rodents treated with a topical
solution of C3G (28%); at an acidic pH, this solution maintains a purple or
red
color and quickly metabolized at elevated pH levels, the C3G changes to a
pink or even clear color. In mouse model experiments, however, as observed
in the image, the purple color of C3G remained on the rodent wound surface
zo scar, thus indicating the pH remained acidic on the wound surface. The
C3G
material on the surface was confirmed by subsequent histology. Figure 12B is
a photographic image of tissue from a study utilizing the homogenized wound
tissue method used in this study, whereby the purple color indicates that the
wound probably retained an acidic pH.
[0031] Figure 13A shows the IGF-1 response to 25 mM PCA in various
environments, including tape stripped, tape stripped and infected with P.
aeruginosa, tape stripped and treated with PCA, and tape stripped infected
with P. aeruginosa, and PCA treated. Figure 13B shows the TGF-8 response
to 25 mM PCA in various environments, including tape stripped, tape stripped
and infected with P. aeruginosa, tape stripped and treated with PCA, and tape
stripped infected with P. aeruginosa, and PCA treated. Figure 13C shows the
EGF response to 25 mM PCA in various environments, including tape
stripped, tape stripped and infected with P. aeruginosa, tape stripped and
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treated with PCA, and tape stripped infected with P. aeruginosa, and PCA
treated.
[0032] Figure 14 is a photographic image of a cross section of rodent skin.
[0033] Figure 15 is a photographic image of a cross section of rodent skin.
[0034] Figure 16 is a photographic image of a cross section of rodent skin.
[0035] Figure 17 is a photographic image of a cross section of rodent skin.
[0036] Figure 18 is a photographic image of a cross section of rodent skin.
[0037] Figure 19 is a photographic image of a cross section of rodent skin.
[0038] Figure 20 is a photographic image of a cross section of rodent skin.
io [0039] Figure 21 is a photographic image of a cross section of rodent
skin.
[0040] Figure 22 is a photographic image of a cross section of rodent skin.
[0041] Figure 23 is a photographic image of a cross section of rodent skin.
[0042] Figure 24 is a photographic image of a cross section of rodent skin.
[0043] Figure 25 is a photographic image of a cross section of rodent skin.
[0044] Figure 26 is a photographic image of a cross section of rodent skin.
[0045] Figure 27 is a photographic image of a cross section of rodent skin.
[0046] Figure 28 is a photographic image of a cross section of rodent skin.
[0047] Figure 29 is a photographic image of a cross section of rodent skin.
[0048] Figure 30 is a photographic image of a cross section of rodent skin.
zo [0049] Figure 31 is a photographic image of a cross section of rodent
skin.
[0050] Figure 32 provides a chart studying the effectiveness of anthocyanin
and anthocyanidin metabolites against various microbes, including P. acnes,
C. difficile, E. coli 8739 and 43895, S. aureus 6538, 33591, P. aeruginosa
9027, MRSA 51625 and Legionella 43662, methicillin resistant
staphylococcus epidermis (MRSE), including MRSE ATCC 51625, and others.
[0051] Figure 33 provides a chart summarize results of testing PCA against
Pseudomonas biofilm.
[0052] Figure 34 provides a chart summarize results of testing PCA against
Pseudomonas biofilm.
[0053] Figure 35 provides a chart summarize results of testing PCA against
MRSA biofilm.
[0054] Figure 36 provides a chart summarize results of testing PCA against
MRSA biofilm.
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[0055] Figure 37 provides a chart summarize results of testing PCA against
Pseudomonas biofilm.
[0056] Figure 38 provides a chart summarize results of testing PCA against
MRSA biofilm.
-- [0057] Figure 39 provides a chart of results of testing PCA against P.
acnes
on the skin.
[0058] Figure 40 shows the results for a single spray of 30% PCA in isopropyl
alcohol on 10 million biofilms colonies of Pseudomonas aeruginosa.
[0059] Figure 41 shows the concentration of 30% has lesser effect on MRSA,
but still 90%.
[0060] Figure 42 provides a composite of the results based only for no growth
cultures following treatment by each solution. *Note that the 1% PCA was not
included.
[0061] Figure 43 provides the summation percentages of no growth."
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
[0062] Unless otherwise indicated, all technical and scientific terms used
herein shall have the same meaning as is commonly understood by one of
zo ordinary skill in the art to which the disclosed subject matter belongs.
Unless
otherwise indicated, the following definitions are applicable to this
disclosure.
All publications referred to throughout the disclosure are incorporated by
reference in their entirety. To the extent any definition or usage provided by
any document incorporated herein by reference conflicts with the definition or
-- usage provided herein, the definition or usage provided herein controls.
[0063] As used in the specification and claims, the singular forms "a," "an,"
and "the" include plural referents unless the context clearly dictates
otherwise.
Thus, for example, reference to "a composition" includes mixtures or
combinations of two or more such compositions.
[0064] Throughout the specification and claims, the word "comprise" and
variations of the word, such as "comprising" and "comprises," means
"including but not limited to," and is not intended to exclude, for example,
other components, extracts, additives, or steps. It is also contemplated that
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embodiments described as "comprising" components, the invention also
includes those same inventions as embodiments "consisting of" or "consisting
essentially of."
[0065] Ranges can be expressed herein as "approximately" or from "about"
one particular value, and/or to "about" another particular value. When such a
range is expressed, another embodiment includes from the one particular
value and/or to the other particular value.
[0066] A weight percent of a reagent, component, or compound unless
specifically stated to the contrary, is based on the total weight of the
reagent,
component, composition or formulation in which the reagent, component, or
compound is included, according to its usual definition.
[0067] By "reduce" or other forms of the word, such as "reducing" or
"reduction," is meant decrease or lower a characteristic (e.g., inflammation,
growth or viability of microorganisms, in particular biofilms).
[0068] By "promote" or other forms of the word, such as "promoting," is meant
to induce a particular event or characteristic, or delay the development or
progression of a particular event or characteristic, or to minimize the
chances
that a particular event or characteristic will occur.
[0069] "Treat" or other forms of the word, such as "treating," "treatment" or
zo treated," is used here to mean to administer a composition or to perform
a
method in order to induce, reduce, eliminate, and prevent a characteristic
(e.g., inflammation, growth or viability of microbes). It is generally
understood
that treating involves providing an effective amount of the composition to the
mammal or surface (e.g., near a surface of an implant or patient) for
treatment.
[0070] By the term "effective amount" of a composition or of a compound (e.g.,
PCA or 2,4,6 THBA) as provided herein is meant an amount sufficient to
provide the desired benefit, either a reduction or prevention of microorganism
growth or survival including biofilm colonies. As disclosed herein, the exact
amount required will vary from use to use depending on a variety of
processing parameters, as understood by one of ordinary skill, such as the
type of surface, the type of microorganism to be treated, the surface size,
the
mode of delivery and the like.
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[0071] The term "vehicle" or "vehicle carrier" as used herein refers to the
manner in which the reagents or compositions may be delivered, including as
a liquid, an injectable formulation, a solution, suspension, dispersion, and
oral
compositions and the like.
[0072] The term "growth factors" or "local growth factors" include but are not
limited to, fibroblast growth factor (FGF), FGF-1, FGF-2, FGF-4, platelet-
derived growth factor (PDGF), insulin-binding growth factor (IGF), IGF-1, IGF-
2, epidermal growth factor (EGF), transforming growth factor (TGF), TGF-p,
TGF-a, and collagen growth factors, and/or biologically active derivatives of
these growth factors.
[0073] By "bactericidal" or "antimicrobial" is meant the ability to effect
(e.g.,
eliminate, inhibit decrease, or prevent) microorganism growth, viability,
and/or
survival at any concentration. It also means to kill the microorganism. It is
further meant to eliminate, inhibit, decrease, prevent, destroy, or kill a
biofilm
and/or biofilm-forming microorganisms.
[0074] By "bacteriostatic" is meant the ability to effect (e.g., stabilize or
prevent future growth or prevent new growth) microorganism growth at any
concentration. A bacteriostatic compound, agent or reagent does not
eliminate or kill the bacteria.
zo [0075] By "antiseptic" is meant an antimicrobial reagent or composition
that is
applied to any surface, including skin or tissue, and particulary described
herein on or around an implant or other surface of a patient near the implant
to effect (e.g., eliminate, inhibit, decrease or prevent) microorganism
growth,
viability, and/or survival at any concentration. It is further meant to
eliminate,
inhibit, decrease, prevent, destroy, or kill a biofilm and/or biofilm-forming
microorganisms.
[0076] By "disinfect" or other forms of the word, such as "disinfectant" or
"disinfecting," is meant decrease or lower a characteristic (e.g., eliminate,
reduce, inhibit, decrease, or prevent) microorganism growth, viability or
survival at any concentration.
[0077] By "sanitize" or other forms of the word, such as "sanitizer" or
"sanitizing," is meant decrease or lower a characteristic (e.g., eliminate,
reduce, inhibit, decrease, or prevent) microorganism growth, viability or
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survival at any concentration. It is generally understood that sanitizing
involves providing an effective amount of the composition to any surface
(e.g.,
a surface of an implant or other surface of a patient near the implant).
[0078]By "sterilize" it is meant to kill microbes on the article being
sterilized.
Sterilize and sterilization include cold sterilization methods.
[0079]The term ProC3GTM" (commercially available ChromaDex , Inc.
Irvine, CA product) means a cyanidin 3-glucoside anthocyanin extracted from
black rice and containing approximately 28% cyanidin 3-glucoside by weight
with an additional 5% other anthocyanins.
io [0080]Reference will now be made in detail to specific aspects of the
disclosed materials, compounds, compositions, and methods, examples of
which are illustrated in the following description and examples, and in the
figures and their descriptions.
Methods of Diagnosing and Treating Infected Implants and Surfaces of a
Patient
[0081]The present invention provides methods of diagnosing infected
implants or other surfaces of a patient, compositions and uses for treating
the
infection on the surface of an inserted implant and other surfaces of a
patient.
zo More specifically, the methods and compositions described herein include
the
administration of an anthocyanin or an anthocyanidin or metabolites thereof,
preferably protocatechuic acid (referred to herein as "PCA") and/or 2,4,6
trihydroxybenzaldehyde (referred to herein as "2,4,6 THBA") to inhibit or
completely destroy a biofilm and all biofilm-forming microorganisms on or
around a surface of an implant or of the patient.
[0082] The methods and compositions are used for the treatment of
mammals, including humans. As described above, prior to the invention
described herein there existed a great need for methods and compositions for
diagnosing and treating infected implants. Accordingly, the methods and
compositions described herein are suitable for the diagnosis and treatment of
mammals having an infection on or around the surface of an implant. Such
animals include equine, canine, and feline.
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[0083] Some embodiments of the invention described herein include methods
of diagnosing whether an implant or other surface of a patient is infected
with
an infectious microorganism. In particular, the methods are suitable for
detecting biofilm infections. The methods of diagnosing an infected implant or
other surface of a patient are minimally invasive and may be carried out
completely in an outpatient setting. Exemplary and non-limiting surfaces of
the
patient include a closed wound, intact skin, skin having a sinus track, or any
surface of a patient (internal and external). Exemplary and non-limiting
examples of implants include medical devices, a medical or surgical implant,
total joint prosthesis, dental implants, a catheter, or a heart or vascular
graft.
[0084] In some embodiments, the method for determining whether an implant
is infected or surface of a patient is infected includes performing a needle
aspiration biopsy to detect the presence of a bacterial infection on or around
the surface of an implant. A needle biopsy is performed by aseptically
preparing the skin lying above the area of the biopsy. Next, a needle is
inserted into the skin and into the tissue overlying the implant or other
internal
surface of a patient and interstitial fluid is aspirated from the area
surrounding
the implant or area of infection on the surface of a patient. The biopsy may
be
guided by the use of additional imaging aids including fluoroscopic equipment,
zo .. computed tomography, and or ultrasonography as is known in the art (see
e.g., Yee etal., Journal of Orthopaedic Surgery, 21, pp. 236-240 (2013)).
[0085] In some embodiments, a physiologically compatible solution is utilized
to irrigate the area surrounding the implant. These physiologically compatible
solutions are well known in the art and include saline and Ringer's solution
and the like. Irrigation of the surrounding tissue and implant may increase
the
likelihood of aspirating fluid containing an infectious microorganism. In
addition, movement of the tissue, such as flexion, rotation, and adduction of
joints can be used to facilitate the aspiration of an infectious
microorganism.
[0086] Exemplary devices for performing the needle aspiration include a
needle attached to a housing for aspirating fluid from an area surrounding the
implant suspected of having an infection. The aspiration device may include a
connector for attaching a needle to the device and a plunger for extracting a
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sample of the joint fluid from the body of the patient through the needle into
the housing of the device. The length and diameter of the needle depends
upon the location of implant relative to the surface of the skin,
accessibility of
the implant, and ease of obtaining an aspirate. Suitable needles may be of a
.. diameter ranging anywhere from 12 gauge to 30 gauge and from less than an
inch in length to several inches in length as is necessary to reach the
suspected area of infection or surface of an implant that is suspected to have
an infection.
[0087] In some embodiments, the resulting needle aspiration biopsy of tissue
surrounding the surface of the implant is cultured to identify the presence of
any infectious microorganisms. The aspirate may be Gram stained to identify
the presence of any bacteria microorganisms prior to culture.
[0088] In some embodiments, the aspirate is cultured to determine a presence
of an infection. General bacteriological culturing methods are well known in
the art. For example, aspirates can be inoculated on blood agar plates and
grown for 24 to 48 hours under aerobic and anaerobic conditions. The
presence of growth is monitored. Alternatively, aspirates can be inoculated in
a growth broth, which is monitored for turbidity. The microorganism can be
cultured for a day or for up to several weeks to maximize the possibility of
zo detection. Prolonged culture periods can be more effective in
identifying
certain microorganisms including, for example, Propionibacterium, Bacilli, and
Peptostreptococcus species. In addition, blood cultures may be utilized to aid
in the detection of slow growing microorganisms. A positive culture
necessitates treatment of the infected implant with an anthocyanin or
anthocyanidin or metabolite thereof (e.g., PCA or 2,4,6 THBA) as further
described herein.
[0089] In some embodiments, diagnosis of infection of an implant includes
performing additional diagnostic assays, in addition to performing a minimally
invasive needle aspiration biopsy technique. These include serologic tests
including sedimentation rate (ESR) determination, C-reactive protein levels,
white blood cell count, and neutrophil percentage. In addition, genetic
analysis
for the detection of microorganism genetic material may be performed using
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polymerase chain reaction (PCR) detection methods. Other clinical elements
include, loss of function, onset of pain in the implant area, sinus tract,
persistant wound drainage, or fever that may be indicative of an infection.
[0090] In some embodiments, the application of ultrasound is utilized to
facilitate release of an infectious microorganism in situ. The application of
ultrasound is described herein as particularly useful for detecting a biofilm
infection present on or around an implant or other surface of a patient. With
biofilm infections, it is not always possible to obtain a positive detection
for the
presence of pathogenic microorganisms because the microorganisms are in a
quiescent non-growth stage and are enveloped in a thick extracellular matrix.
Without being bound by any theory, it is thought that the application of
ultrasound disrupts the structure of the extracellular matrix of the biofilm
such
that a biofilm-forming microorganism can be detected by any of the
aforementioned methods. Ultrasound techniques for disrupting biofilms from
implant materials in vitro is known in the art (see Granick etal., Eplasty
17(e13), pp. 128-134, 2017 and Trampuz etal., NEJM 357, pp. 654-663,
2007). These techniques have not been contemplated for disrupting a biofilm
infection on an infected implant in situ or other surface of a patient in
vivo. As
first described herein, ultrasound energy can be applied in vivo to facilitate
the
zo release of any biofilm-forming microorganisms present on an infected
implant
or other surface of a patient.
[0091] Conventionally, ultrasound energy has been utilized for moving and
ablating kidney stones through the technique of shock wave lithotripsy (see
e.g., U.S. Patent Nos. 5,496,306, 6,770,039 and 6,123,679). Suitable devices
for performing lithotripsy include electrohydraulic, piezoelectric, and
electromagnetic lithotripters, such as those available from Dornier Med Tech,
Xi Xin Medical Instruments Co. Ltd, and Tissue Regeneration Technologies
(see also e.g., Connors etal., J Urol. 191(1), 2014). The frequency, pressure,
focal width, shock wave rate, and power density of the lithotripter is
selected
such that a biofilm is disrupted, while minimizing potential damage to
surrounding tissues of the patient. Alternatively, the application of
ultrasound
energy can be utilized in a focussed manner to exert a biocidal effect to the
infectious microorganisms and biofilm colonies. In this way, the method of
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applying ultrasound energy through a lithotripter aids in the sanitization of
an
infected implant.
[0092] In some embodiments, the frequency of the oscillating acoustic
pressure wave is from about 1 kHz to about 60 kHz. In some embodiments,
the frequency is about 10 kHz to about 40 kHz. In some embodiments, the
frequency is about 1 kHz, about 5 kHz, about 10 kHz, about 15 kHz, about
20 kHz, about 25 kHz, about 30 kHz, about 35 kHz, about 40 kHz, about
45 kHz, about 50 kHz, about 55 kHz, or about 60 kHz.
[0093] In some embodiments, the pressure generated by the lithotripter is
about 5 MPa to about 160 MPa. In some embodiments, the pressure is about
10 MPa to about 40 MPa. In some embodiments, the pressure is about
1 MPa, about 5 MPa, about 10 MPa, about 15 MPa, about 20 MPa, about
25 MPa, about 30 MPa, about 35 MPa, about 40 MPa, about 50 MPa, about
60 MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about
120 MPa, about 140 MPa, or about 160 MPa.
[0094] In some embodiments, the spatial distribution of acoustic energy or
focal width is from about 1 mm to about 30 mm. In some embodiments, the
focal width is about 5 mm to about 15 mm. In some embodiments, the focal
width about 1 mm, about 5 mm, about 10 mm, about 15 mm, about 20 mm,
zo about 25 mm, or about 30 mm.
[0095] In some embodiments, the shock wave rate is about 10 shock waves
per minute to about 200 shock waves per minute. In some embodiments, the
shock wave rate is about 20 shock waves per minute to about 80 shock
waves per minute. In some embodiments, the shock wave rate is about 10
shock waves per minute, about 20 shock waves per minute, about 30 shock
waves per minute, about 35 shock waves per minute, about 40 shock waves
per minute, about 45 shock waves per minute, about 50 shock waves per
minute, about 55 shock waves per minute, about 60 shock waves per minute,
about 80 shock waves per minute, about 100 shock waves per minute, about
120 shock waves per minute, about 140 shock waves per minute, about
160 shock waves per minute, about 180 shock waves per minute, or about
200 shock waves per minute.
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[0096] In some embodiments, the power density is from about 0.1 W/cm2 to
about 25,000 W/cm2. In some embodiments, the power density is from about
1 W/cm2 to about 10,000 W/cm2. In some embodiments, the power density is
from about 20 W/cm2 to about 5,000 W/cm2. In some embodiments, the power
density is from about 100 W/cm2 to about 3,000 W/cm2.
[0097] The ultrasound may be applied prior to performing a needle aspiration
biopsy. Any microorganisms present in a biofilm can be loosened to increase
the likelihood of detection of the biofilm-forming microorganism.
Alternatively,
the ultrasound may be applied following a first unsuccessful needle aspiration
io biopsy to increase the likelihood that any biofilm-forming bacteria are
detected. Thus, in some embodiments, ultrasound is applied prior to
performing a needle aspiration biopsy. In some other embodiments,
ultrasound is applied after a first needle aspiration biopsy is performed.
[0098] The inventor has further shown that anthocyanins, anthocyanidins and
metabolites thereof, in particular, PCA and 2,4,6 THBA function as broad
spectrum antimicrobial agents. These compounds are useful for treating an
infection on the surface of an implant or other surface of a patient. These
compounds are particularly effective in treating biofilm infections stemming
from yeast and bacteria.
zo [0099] As described above, the methods described herein include
determining
if an infection, including a biofilm infection, is present on or around the
surface
of the implant or other surface of a patient. The methods described herein
further include administering an effective amount of an anthocyanin or an
anthocyanidin or metabolites thereof, preferably PCA and 2,4,6 THBA as an
anti-microbial agent.
[0100] Prior to the invention described herein, there was a need for agents
that can treat infected implants. It was discovered by the inventor that
anthocyanin and anthocyanidin compounds, in particular, PCA and 2,4,6,
THBA are such chemical agents. It has been shown (see figures 6 and 32)
that PCA and 2,4,6 THBA have the ability to kill a wide spectrum of microbes.
For example, figure 6 provides the results of testing showing that PCA, was
effective against all bacteria tested in addition to yeast C. albicans and
highly
virulent K. pneumoniae. PCA was also demonstrated to be effective against
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difficult infectious microorganisms including S. aureus 6538 and 33591
(MRSA) and P. aeruginosa.
[0101] PCA was also effective on C. albicans, which is important considering
its ability to form biofilms and difficulty in treating C. albicans when
existing
within a catheter or an implant. In figure 32, it is shown that PCA is
effective
against C. difficile, P. acnes 6919, E. coli 8739 and 43895, S. aureus 6538,
S. aureus 33591, P. aeruginosa 9027, methicillin resistant staphylococcus
epidermis (MRSE), including MRSE ATCC 51625, and Legionella 43662, and
others. Figure 32 shows that 2,4,6 THBA is effective against E. coli 8739 and
43895, S. aureus 6538, S. aureus 33591, P. aeruginosa 9027, methicillin
resistant staphylococcus epidermis (MRSE), including MRSE ATCC 51625,
and Legionella 43662, and others.
[0102] Implants, such as total joint implants and dental implants, are often
infected with biofilms that are formed from these aforementioned microbes
amongst other types of microbes, which can be difficult to treat. Biofilms are
comprised of bacteria that form colonies and produce a surrounding matrix
film to protect themselves. The biofilm forming bacteria can form colonies
that
attach to foreign bodies, each other and tissues. The bacteria aggregate in
clusters and are surrounded by extracellular polymer matrix. The biofilms are
zo hard to destroy and therefore kill the underlying bacteria and provide
the basis
for much of the antibiotic resistance that has developed. The formation of a
biofilm is a two-step process: 1. adherence of cells to a foreign body
surface;
and 2. accumulation of cells to form multilayered cell clusters.
[0103] The inventor has shown that PCA was able to stop the formation of a
biofilm as well as kill bacterial in already formed biofilms. The biofilms
tested
were Pseudomonas aeruginosa ATCC 700888 and Staphylococcus aureus
ATCC 33591 (MRSA). The tests are described in more details in Examples 7
and 8 and Figures 35 and 36.
[0104] Thus, in some embodiments, following the determination that an
implant or other surface of a patient is infected, the implant or other
surface of
a patient is treated with an effective amount of an anthocyanin or
anthocyanidin or a metabolite thereof in situ. Exemplary and non-limiting
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anthocyanins include cyanidin-3-glucoside or delphinidin-3-glucoside,
cyanidin-3-galactoside, and pelargonidin-3-galactoside. Exemplary and non-
limiting anthocyanidins include cyanidin, delphinidin, pelargonidin, malvidin
and petunidin. In particular, the implant or other surface of a patient is
treated
with a metabolite of anthocyanins and anthocyanidins including PCA, 2,4,6
THBA, and combinations thereof. The anthocyanin or anthocyanidin or
metabolite thereof, including PCA or 2,4,6 THBA, may be administered as
pure crystals or as part of a composition described herein.
[0105] In some embodiments, are methods of prophylactically treating a
preoperative skin incision site, comprising administering an anthocyanin, an
anthocyanidin or a metabolite thereof to a patient in need of such treatment
an effective amount of the anthocyanin or anthocyanidin compound wherein
microbial growth is prevented or reduced prior to any surgery involving the
installation of an implant.
[0106] In some embodiments, a method of disinfecting a surface of an implant
or other surface of a patient includes contacting said surface with an
anthocyanin, an anthocyanidin and/or a metabolite thereof in an effective
amount of the anthocyanin, anthocyanidin and/or metabolite compound
wherein microbial growth is prevented, reduced or eliminated. In some
zo embodiments, the microbial growth that is reduced includes infections
and
biofilm infections on the surface of an implant or other surface of a patient
that
may be an endogenous or exogenous source, including but not limited to
MRSA, P. acnes, S. aureus, P. aeruginosa, E. coli, S. epidermidis,
S. pneumonia, Streptococcus species, C. difficile, and Legionella.
[0107] In some embodiments, the effective amount of the anthocyanin or
anthocyanidin or metabolite thereof, including PCA or 2,4,6 THBA or a
combination thereof is from about 1 pg to about 1000 mg. In some
embodiments, the effective amount is from about 1 pg to about 500 mg. In
some embodiments, the effective amount is from about 1 pg to about 50 mg.
In some embodiments, the effective amount is from about 1 pg to about
10 mg. In some embodiments, the effective amount is from about 1 pg to
about 1000 pg. In some embodiments, the effective amount is from about
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1 pg to about 500 pg. In some embodiments, the effective amount is from
about 1 pg to about 50 pg.
[0108] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is incorporated as pure crystals in an implantable material, such as a
mesh including titanium or stainless steel. The crystals are applied to the
metal where there is surface configuration that provides for housing of the
crystal on the surface. In this way, the crystals remain in place in crystal
form
until activated when subject to fluid common to the mammalian body. The
anthocyanin, anthocyanidin, or metabolite thereof can be present as a
polymorphous, semi-crystalline, hydrate, amorphous or polyamorphous forms.
[0109] The crystals of the anthocyanin or anthocyanidin or metabolite thereof
may be micronized using known micronization techniques known in the art.
The micronized crystals may be useful for embedding upon a surface of to
enhance the dissolution of the compound when provided in a solution or when
administered in vivo to a patient. Thus, in some embodiments, the crystals
have a mean particle distribution of <1000 pm, <500 pm, <400 pm, <300 pm,
<200 pm, <100 pm, <50 pm, or even <10 pm. The particles may have a "d90",
"d50", and "dl 0" distribution of the forgoing sizes, meaning that (90%, 50%,
or
10%, respectively) of particle sizes are less than a specified size or size
zo range. For example, as specified, d90 90 pm means that 90% of the
particle
sizes within a distribution of particles are less than or equal to 90 pm.
[0110] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is substantially pure. In some embodiments, the compounds are at
least 75% to greater than 99% pure. In some embodiments, the compounds
are at least 90%, 95%, 99%, or 99.99% pure as assessed by techniques
routine in the art, such as high performance liquid chromatography (HPLC).
[0111] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is provided as a pharmaceutically acceptable salt form.
Pharmaceutically acceptable salts forms are those formed by, for example,
contacting a free base of the compound with a suitable acid in a suitable
solvent under suitable conditions that will form an acid addition salt. The
salt
form includes alkali metal salts such as, for example, sodium or potassium
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salts, alkaline earth metal salts such as, for example, calcium and magnesium
salts, and salts with organic or inorganic acid such as, for example,
hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric
acid,
citric acid, formic acid, maleic acid, succinic acid, tartaric acid,
methanesulphonic acid, toluenesulphonic acid etc.
[0112] The anthocyanin, anthocyanidin or metabolite thereof may be
administered to treat the infected implant or other surface of a patient by
methods commonly used in the art for the treatment of infected implants with
antimicrobial compounds. These include, but are not limited to, topical
adminstration above the area of infection, transdermal administration above
the area of infection, enteric administration (e.g., oral or rectal),
sublingual
administration, or other parenteral injection, including epidermal injection,
intravenous injection, subcutaneous injection, intra muscular injection, intra-
articular injection, or other injection to administer the anthocyanin or
.. anthocyanidin in proximity to the infected implant or other infected
surface of a
patient.
[0113] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is administered to a patient having an infected implant or other
surface
of a patient at least one time. In some embodiments, once a positive
zo diagnosis of infection of an implant or other surface of a patient is
established,
the anthocyanin, anthocyanidin, or metabolite thereof is administered at a set
frequency. The frequency of administration may be once a week, twice a
week, three times a week, four times a week, five times a week, six times a
week, daily, twice daily, three times daily, four times daily, or hourly.
Alternatively, the anthocyanin, anthocyanidin, or metabolite thereof may be
administered continuously through infusion or irrigation of the infected
surface
of a patient or implant. This type of administration is possible because the
anthocyanin or anthocyanidin or metabolites thereof described herein are safe
and with little to no demonstrated local or systemic toxicity.
[0114] Alternatively, in some other embodiments, the anthocyanin,
anthocyanidin, or metabolite thereof is administered prophylactically to a
patient receiving an implant to prevent or reduce the likelihood of an
infection.
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In some embodiments, the patient is also prophylactically monitored using the
diagnostic methods described herein to identify an infection prior to a
clinical
manifestation of the infection.
[0115] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is administered with a second pharmaceutical agent. Suitable and
non-limiting second pharmaceutical agents include additional anthocyanin or
anthocyanidin compounds or metabolites thereof, antibiotics, analgesics, and
anti-inflammatory agents.
[0116] The therapeutic effective dose may vary depending on a wide variety of
factors. For instance, the dose may vary depending on the formulation,
method of application of the therapeutic reagent or combination with other
reagents, or compositions, compounds or combination of compositions or
compounds to the infected implant or surface of a patient.
Compositions
[0117] Disclosed herein are antimicrobial compositions for treating infected
implants or other infected surfaces of a patient. The disclosed antimicrobial
reagents and compositions can be used to eliminate, reduce, and/or prevent
microorganism growth, viability, or survival. In particular, the compositions
zo including an anthocyanin or anthocyanidin or metabolite thereof in an
effective
amount to eliminate, reduce, and/or prevent microorganism growth, viability,
or survival of a biofilm causing an infection of an implant or other surface
of a
patient.
[0118] The present disclosure provides for pharmaceutical compositions
.. whereby the anthocyanin, anthocyanidin, anthocyanin metabolite,
anthocyanidin metabolite, anthocyanin metabolite, or metabolites thereof, are
isolated reagents. Preferably the present composition includes PCA,
2,4,6 THBA or a combination thereof.
[0119] In some embodiments, the present invention provides for a
pharmaceutical composition for treating an infected implant or other surface
of
a patient including: a) an anthocyanin; b) anthocyanidin; c) a metabolite of
an
anthocyanin or anthocyanidin such as C3G, PCA, 2,4,6 THBA, vanillic and
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hippuric acid. Preferably the present composition includes PCA, 2,4,6 THBA,
or a combination thereof.
[0120] Suitable and non-limiting compositions include those that can be
applied to or near the site of the infected implant or infected surface of a
patient. The composition comprising anthocyanin or anthocyanidin or
metabolite thereof may be in the form of a liquid solution, suspension, a
dispersion, or an emulsion.
[0121] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is about 0.5% to about 90% by weight of the composition. In some
embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about
0.5% to about 70% by weight of the composition. In some embodiments, the
anthocyanin, anthocyanidin, or metabolite thereof is about 0.5% to about 50%
by weight of the composition. In some embodiments, the anthocyanin,
anthocyanidin, or metabolite thereof is about 0.5% to about 30% by weight of
the composition. In some embodiments, the anthocyanin, anthocyanidin, or
metabolite thereof is about 0.5% to about 20% by weight of the composition.
In some embodiments, the anthocyanin, anthocyanidin, or metabolite thereof
is about 5% to about 30% by weight of the composition. In some
embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is about
zo 0.5% to about 20% by weight of the composition. In some embodiments, the
anthocyanin or anthocyanidin or metabolite thereof is about 0.5%, about 1%,
about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about
35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%,
about 70%, about 75%, about 80%, about 85%, or about 90% by weight of the
composition.
[0122] In some embodiments, the anthocyanins or anthocyanidins and
metabolites thereof is provided in concentrations of about 10 to 200 mM. In
other embodiments, anthocyanins, or anthocyanidins, or metabolites thereof
are provided in any recited composition or method of use in a range of
between 20 to 200 mM. In yet other embodiments, the anthocyanin,
anthocyanidin, or metabolites thereof provided in any recited composition or
method of use is provided in a range of between 20 to 100 mM. In yet other
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embodiments, the anthocyanin, anthocyanidin, or metabolites thereof
provided in any recited composition or method of use is provided in a range of
between 50 to 100 mM. In yet other embodiments, the anthocyanin,
anthocyanidin, or metabolites thereof provided in any recited composition or
method of use is provided in a range of between 20 to 50 mM.
[0123] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is in the form of crystals that are embedded into a composition.
Exemplary and non-limiting compositions in which the crystals are embedded
include a cloth or a mesh, such as titanium or stainless steel. In some
embodiments, the anthocyanin, anthocyanidin, or metabolite thereof is in the
form of a powder. A powder form is contemplated for further use in a
composition described herein (e.g., to be dissolved or suspended therein) or
imbedded into a composition.
[0124] In some embodiments, the disclosed composition can be selected from
anthocyanins, anthocyanidins, metabolites of anthocyanin and anthocyanidin
compounds, or a combination thereof. By way of example, the anthocyanin
can be selected from cyanidin-3-glucoside or delphinidin-3-glucoside,
cyanidin-3-galactoside, and pelargonidin-3-galactoside and combinations
thereof. Also by way of example, the anthocyanidins can be selected from
zo cyanidin, delphinidin, pelargonidin, malvidin, petunidin, and
combinations
thereof. In particular, metabolites of anthocyanins and anthocyanidins can be
selected from PCA and 2,4,6 THBA and combinations thereof.
[0125] In some embodiments, the anthocyanin, anthocyanidin, or metabolite
thereof is in a solution. In some embodiments, the metabolite of the
anthocyanin or anthocyanidin is PCA or 2,4,6 THBA. In some embodiments,
the metabolite of the anthocyanin or anthocyanidin is PCA. In some
embodiments, the metabolite of the anthocyanin or anthocyanidin is
2,4,6 THBA.
[0126] In some embodiments, the anthocyanin, anthocyanidin, or a mixture or
combination thereof is provided in a composition including a carrier that
includes 50% to 91% isopropyl alcohol. In some embodiments, PCA,
2,4,6 THBA, or a mixture or combination thereof is provided in a composition
including a carrier that includes 50% to 91 A isopropyl alcohol. In some
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embodiments, PCA is provided in a composition including a carrier that
includes 50% to 91% isopropyl alcohol. In some embodiments, 2,4,6 THBA is
provided in a composition including a carrier that includes 50% to 91 A
isopropyl alcohol.
[0127] The compositions described herein are suitable for routes of
administration including oral, injection, intravenous, subcutaneous,
epidermal,
topical, sublingual, buccal, inhalation, intradermal, subcutaneous, intra
articular, soft tissue, and cutaneous.
[0128] Oral administration of the compositions of this disclosure, including
oral
gavage, may include a liquid or semisolid form, tablet, pill, capsule, powder,
or
gel. Preferably, oral administration will be in a liquid composition.
Compositions including a liquid pharmaceutically inert carrier such as water
may be considered for oral administration. Other pharmaceutically compatible
liquids or semisolids may also be used. The use of such liquids and
semisolids and manufacturing of tablet, pill, powder, or gel compositions is
well known to those of skill in the art. The injectable compositions be in
liquid
or semi-liquid form. Other pharmaceutically compatible liquids or semi-liquids
may also be used. The use of such liquids and semi-liquids is well known to
those of skill in the art.
zo .. [0129] In some embodiments, the composition is formulated as a topical
composition. More preferable, the vehicle of the topical composition delivery
is
in the form of a liquid, salve, soap, spray, foam, cream, emollient, gel,
ointment, balm or transdermal patch.
[0130] In some embodiments, the compositions can be in the form of an
.. aqueous solution. In some embodiments, the compositions disclosed herein
are in the form of a liquid, gel, suspension, dispersion, solid, emulsion,
aerosol, for example, powders, tablets, capsules, pills, liquids, suspensions,
dispersions or emulsions. In addition, the compositions disclosed herein can
be in the form suitable for dilutions. Similarly, the compositions can be in
the
form of a powder, cream, paste, gel or solid that can be reconstituted.
[0131] Other components can be present in the composition, if desired. For
example, the antimicrobial composition can also include at least one additive
selected independently from a carrier, a diluent, an adjuvant, a solubilizing
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agent, a suspending agent, a filler, a surfactant, a secondary antimicrobial
agent, a preservative, a viscosity modifier, a thixotropy modifier, a wetting
agent, an emulsifier, or any combinations thereof. For example, the disclosed
antimicrobial composition can further comprise at least one surfactant
selected from a cationic surfactant, an anionic surfactant, a non-ionic
surfactant, and an amphoteric surfactant. Additionally, the disclosed
antimicrobial and/or pharmaceutical compositions may further comprise
medicament is selected from the group consisting of anesthetic agents,
cleansers, antiseptic agents, scar reducing agents, immunostimulating
agents, antiviral agents, antikeratolytic agents, anti-inflammatory agents,
antifungal agents, antihistamine agents, antibacterial agents, bioadhesive
agents, inhibitors of prostaglandin synthesis, antioxidants, and mixtures
thereof.
[0132] Also, the disclosed antimicrobial compositions can optionally include
one or more additives such as carriers, adjuvants, solubilizing agents,
suspending agents, diluents, surfactants, other antimicrobial agents,
preservatives, fillers, wetting agents, antifoaming agents, emulsifiers, and
additives designed to affect the viscosity or ability of the composition to
adhere to and/or aid in the treatment of an infected implant or other surface
of
zo a patient.
[0133] The disclosed antimicrobial compositions, including the selected active
components, including the anthocyanins or anthocyanidins and metabolites
thereof, are without causing significant undesirable biological effects or
interacting in a deleterious manner with any of the other components of the
composition in which it is contained.
[0134] In other examples, the antimicrobial compositions disclosed herein can
further comprise a carrier. The term "carrier" means a compound,
composition, substance, or structure that, when in combination with a
compound or composition disclosed herein, facilitates preparation,
administration, delivery, effectiveness, or any other feature of the compound
or composition. Examples of carriers include water, isopropyl alcohol ethanol,
polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like),
polyoxyalkylenes, e.g., poly(oxyethylene)-poly(oxypropylene) polymers,
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benzyl alcohol or butanediol, vegetable oils, and suitable mixtures thereof.
"Pharmaceutically acceptable carrier" means a compound, composition,
substance, or structure that is useful in neither preparing a pharmaceutical
composition which is generally safe, non-toxic, and neither biologically nor
otherwise undesirable and includes that which is acceptable for veterinary as
well as human pharmaceutical use. In addition, the carrier maybe aqueous or
oily. The carrier may further include suitable pH modifying agents including
alkali metal salts, such as sodium or potassium hydroxide, or acidic
compounds, such as hydrochloric acid or other weaker organic carboxylic
io acids. In addition, further oxidation stabilizers, e.g., sodium
bisulfite or
preservatives, e.g., benzyl alcohol, may advantageously be incorporated into
the compositions.
[0135] In a further example, the antimicrobial compositions disclosed herein
can also comprise adjuvants such as preserving, wetting, emulsifying,
suspending agents, and dispensing agents. Prevention of the action of other
microorganisms can be accomplished by various antifungal agents, for
example, parabens, chlorobutanol, phenol, and the like.
[0136] Suitable suspending agents can include, for example, ethoxylated
isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
zo microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar
and
tragacanth, or mixtures of these substances, and the like.
[0137] The disclosed antimicrobial compositions can also comprise
solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl
alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl alcohol,
benzyl
benzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, in
particular, cottonseed oil, groundnut oil, corn germ oil, olive oil, castor
oil and
sesame oil, glycerol, tetrahydrofur fury 1 alcohol, polyethyleneglycols and
fatty
acid esters of sorbitan or mixtures of these substances, and the like. The
additives can be present in the disclosed compositions in any amount for the
individual anthocyanin, anthocyanidin or metabolite thereof compound
components.
[0138] The composition may further be in the form of a gel or include an in
situ
gel-forming agent that results in the release of the anthocyanin,
anthocyanidin
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or metabolite thereof over an extended period. Such extended release gel-
form ing compositions are generally known in the art (see e.g.,
WO 1995/035093 and US 20030157178A1).
[0139] Some embodiments are kits for diagnosing and treating an infected
implant or surface of a patient with a therapeutically effective amount of an
anthocyanin or anthocyanidin or metabolite thereof. The kit may include a
minimally invasive sampling device for detecting the presence of an infectious
or pathological microorganism, such as a sterile wrapped needle and syringe.
[0140] The kit further includes an anthocyanin, anthocyanidin or metabolite
thereof in an amount effective for the treatment of an infected implant or
surface of a patient. The anthocyanin, anthocyanidin, or metabolite thereof
can be provided in a liquid or gellable composition described herein.
Alternatively, the anthocyanin, anthocyanidin, or metabolite thereof can be
provided as a crystal present in a vial that can be further prepared as a
liquid
composition or embedded in an implantable material. The amount of the
anthocyanin, anthocyanidin, or metabolite thereof present in the kit may range
from 1 mg to greater than or equal to 10,000 mg. The kit may also include
culturing devices including culture plates, tubes, and growth media as known
in the art. The kit may further include instructions for diagnosing and
treating a
zo patient suspected of having an infection of an implant or other surface
according to the invention.
[0141] It will be readily apparent to one of ordinary skill in the relevant
arts that
suitable modifications and adaptations to the compositions, methods, and
applications described herein can be made without departing from the scope
of any embodiments or aspects thereof. The compositions and methods
provided are exemplary and are not intended to limit the scope of any of the
specified embodiments. All of the various embodiments, aspects, and options
disclosed herein can be combined in any and all variations or iterations. The
scope of the compositions, formulations, methods, and processes described
herein include all actual or potential combinations of embodiments, aspects,
options, examples, and preferences herein described. The examples below
describe non-limiting examples of the inventive embodiments of the invention.
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EXAMPLES
Example 1: Use of in vitro studies for antimicrobial susceptibility testing of
anthocyanins, anthocyanidins, or metabolites and compounds thereof.
[0142] This example describes the method for testing the antimicrobial
susceptibility of anthocyanins, anthocyanidins, or metabolites and compounds
thereof. The Kirby-Bauer method of disc diffusion was used for testing,
following a standard set of procedures recommended by the NCCLS. In this
methodology, a set of discs saturated with either testing compounds or a
control was placed on inoculated agar plates. The plates were inoculated with
organisms listed in the tables provided in FIG.6, including C. difficile,
P. acnes, C. perfringens, L. casei, C. albicans, E. coli, ATTC 8739 and ATCC
43895, S. aureus, S. mutans, S. pyogenes, P. aeruginosa, and K.
pneumoniae. The control sample was amoxicillin, an antimicrobial with very
.. effective broad-spectrum antibiotic properties. Samples included
delphinidin,
pelargonidin, cyanidin Cl, 28% cyanindin-3-glucoside (C3G), protocatechuic
acid (PCA) and 2,4,6 trihydroxybenzaldehyde (2,4,6 THBA).
[0143] After 18, 24, or 48 hours of incubation, depending upon the
microorganism, each plate was examined. The diameters of the zones of
zo complete inhibition were measured, including the diameter of the disc.
Zones
were measured to the nearest millimeter, using sliding calipers. The size of
the zones of inhibition was interpreted by referring to NCCLS standard.
Results were interpreted as follows: NI was no inhibition of growth under the
test sample, I was inhibition of growth under the test sample, NZ indicated no
zone of inhibition surrounding the test sample, and CZ indicated a clear zone
of inhibition surrounding the sample and zone width in millimeters. See
figure 6 for complete results.
Results
[0144] Referring to figure 6 and figure 32, the testing samples had
bactericidal
and bacteriostatic activity against many of the organisms. Of note, P. acnes,
an organism that is very difficult to treat, often requiring multiple current
antibiotics for effective treatment, was susceptible to both C3G and PCA.
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Indeed, both of these test samples were bactericidal against P. acnes.
Additionally, PCA was also effective against Staphlococcus aureus ATCC
33591, known as Methicillin Resistant Staph aureus (MRSA), Staphlococcus
epidermidis ATCC 51625, known as Methicillin Resistant Staph epidermidis
.. (MRSE), E. coli 8739 and 43895, and Legionella 43662.
[0145] PCA was also shown to have some effectiveness against
Pseudomonas aeruginosa, a common pathogen in wounds, especially burns,
as well as chronic lung infections. Amoxicillin, the control sample, had no
effect on P. aeruginosa. Similarly, Candida albicans, frequently a co pathogen
in wounds, was susceptible to PCA.
[0146] PCA was also shown to have some effectiveness against
Pseudomonas aeruginosa, a common pathogen in wounds, especially burns.
Amoxicillin, the control sample, had no effect on P. aeruginosa. Similarly,
Candida albicans, frequently a co pathogen in wounds, was susceptible to
PCA.
[0147] In summary, the present invention provides advantages over the prior
art, including providing anthocyanin, anthocyanidin, their metabolites or
combinations thereof to a wound to provide a reduction or elimination of
bacteria. It is contemplated that the invention will also find use in the
zo treatment of surfaces, including medical devices and medical implants,
to
reduce or eliminate bacteria.
Example 2: Use of mouse model to determine dose levels and intervals of test
samples.
Methods:
[0148] Mice had back skin tape stripped and the stripped site (wound) was
infected with P. aeruginosa (ACTA 9027). The test reagents were applied
topically in an aqueous solution on the stripped site at two hours and daily
for
four days.
[0149] Cyanidin 3-glucoside (C3G), an anthocyanin, and its main metabolite
PCA were formulated and tested at several doses. The aqueous carrier was
water. The C3G formulation included 50 mM, 100 mM and 200 mM dose
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concentrations. Similarly, the PCA formulation included at 50, 100 and
200 mM dose concentrations.
Results
[0150] Results were collected from the mice at day five. Both C3G and PCA
decreased the bacterial burden; however, none were statistically significant.
See figure 9A. There was a trend towards a decreasing concentration of PCA,
with 50 mM being the most effective. The most effective dose of C3G was 100
mM. It is contemplated that because C3G degrades to PCA in this
environment, the test results may indicate that C3G was not being tested
alone, but rather was a combination of C3G and its metabolites, including a
combination of C3G and PCA as the effective agents.
Example 3: Use of mouse model to further determine effective dose levels
and dose intervals of test samples.
Methods:
[0151] Mice had back skin tape stripped and the stripped site (wound) was
infected with P. aeruginosa (ACTA 27853). The test reagents were applied
topically in an aqueous solution on the stripped site at two hours and daily
on
zo day 1,2 and 3.
[0152] C3G, an anthocyanin and its main metabolite PCA were formulated
and tested at several doses. The aqueous carrier was water. The C3G
formulation included 100 mM and 200 mM dose concentrations and the PCA
formulation included 25 and 50 mM dose concentrations.
Results
[0153] Results were collected from the mice at day two and four. Both C3G
and PCA decreased the bacterial burden at 48 and 96 hours. (See Figure 9B).
The most significant decrease of bacteria was observed at 25 mM of and 100
and 200 mM of C3G. Although PCA at 25 mM reduced the bacterial burden at
both time periods, its activity was statistically significant at 48 hours. C3G
at
both 100 mM and 200 mM significantly reduced the bacterial burden at 48 and
96 hours.
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Example 4: Use of a mouse model for wound healing.
Methods:
[0154] Mice were shaved but unstrapped and uninfected (normal rodent skin).
The test reagents were applied topically in an aqueous solution on the
unstripped site at two hours and daily on day 1, 2 and 3.
[0155] Testing reagents consisted of C3G and PCA formulated at one dose,
100 pM in an aqueous solution.
Results:
[0156] Referring to figure 10, there was little or no stimulation of IGF-1 and
TGF-8 at local levels observed at the 100 pM concentration of testing
reagents. In fact, levels of EGF actually decreased below normal levels. There
was observed a decrease of all three local growth hormones at 100 uM of
C3G. These results suggest that mice skin differs in response to a dose that
has been shown to stimulate human synovium to produce IGF-1. Thus, this
low of a dose is not useful for rodents for this purpose.
Example 5: Use of mouse model to determine isolated effect of 25 mM
solution of PCA in various environments.
Methods:
[0157] Four different conditions were used: mice had back skin tape stripped
and the stripped site (wound) was infected with P. aeruginosa; mice had back
skin stripped and were not infected, mice had taped stripped, infected and
treated with PCA, mice were tape stripped, uninfected, and treated with PCA.
When used, the PCA test reagent was applied topically in an aqueous
solution on the stripped site at two hours and 24 hours.
[0158] The testing reagents consisted of and PCA formulated at one dose,
25mM, in an aqueous solution. Levels of IGF-1, TGF-8, and EGF levels in the
skin tissue at 48 hours were measured by ELISA. There were two control
groups; the stripped skin and the stripped skin and infected.
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Results:
[0159] Referring to figure 13, the infected stripped skin showed the highest
level with IGF-1 (statistically significant) and TGF-p. This is representative
of
tissue response to injury and infection; similarly, the EGF response was very
inconsistent compared to the other two growth hormones.
[0160] The EGF response levels were different than either IGF-1or TGF-p.
They were highest in the stripped and uninfected wound and lowest in the
stripped, infected and treated wound. Therefore, the treatment optimized the
amount of hormone production compared to the untreated infection. This is
beneficial to limit scarring while promoting healing over the controls.
Overall,
PCA at 25 mM acts on stripped and infected mice skin and optimizes the IGF-
1 production and optimizes the local growth hormones.
Example 6: Use of mice to establish wound promoting effect of compositions.
Method:
[0161] Fifteen rodents were used to establish the histological findings of
stripped skin, stripped and infected skin, and stripped, infected and treated
wound. There were two control groups and four experimental groups
according to the following:
zo [0162] Control Group 1: three mice with only tape stripped wounds on the
back. These mice were not infected or treated. The skin was harvested at
time zero, 2 and 48 hours for histology examination.
[0163] Control Group 2: three had tape stripped wounds and infection. Tissue
submitted at 2 and 48 hours for histological examination.
[0164] Experimental Groups: There were 4 experimental groups. In these
groups, mice had skin stripped wounds and infection. Treatment varied by
reagent and dosage. Testing reagents included PCA at 25 and 50 mM and
C3G at 100 and 200 mM.
[0165] Pseudomonas aeruginosa (ATCC 27853) procured from American
Type Culture Collection, Manassas, VA was used to infect the experimental
groups of mice. The organism was grown overnight at 37 C at ambient
atmosphere trypticase soy agar plates supplemented with 5% sheep blood
cells. The culture will be aseptically swabbed and transferred to tubes of
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trypticase soy broth. The optical density will be determined at 600 nm. The
cultures will be diluted to provide an inoculum of approximately 9.0 logio CFU
per mouse in a volume of 100 pL. lnoculum count was estimated before
inoculation by optical density and confirmed after inoculation by dilution and
back count.
[0166] The testing reagents were topically applied at 2 and 24 hours with
100 uL of fluid spread over the wound.
[0167] The following histological assessments were conducted:
[0168] Surface Cellularity: The histological assessment included the presence
or absence of the surface cellularity and the depth of the cells.
[0169] Dermis: vascularity and inflammation.
[0170] Thickness: The thickness of the dermal layer was observed.
[0171] Hair Follicles: The hair follicles and the layer of surrounding cells
were
observed. Hair follicles presence is critically important to skin wound
healing.
(Gharzi A, Reynolds AJ, Jahoda CA. Plasticity of hair follicle dermal cells in
wound healing and induction. Exp Dermatol. 2003 Apr; 12 (2):126-36). The
dermal sheath surrounding the hair follicle has the progenitor cells for
contributing fibroblasts for wound healing. (Johada CA, Reynolds AJ. Hair
follicle dermal sheath cells: unsung participants in wound healing. Lancet.
zo 2001 Oct 27; 358(9291):1445-8).
[0172] Vascularity: Vascularity was observed, but an assessment of
angiogenesis was not performed on the 48 hour material since new
vascularity takes three to twelve days to develop. (Busuioc CJ, et al. Phases
of cutaneous angiogenesis process in experimental third-degree skin burns:
histological and immunohistochemical study. Rom J Morphol Embryo!. 2013;
54(1):163-710.)
[0173] Inflammation: The presence of cellular infiltration was observed and
its
location.
[0174] Skin Thickness: The thickness of the skin was estimated related to the
uninfected, untreated wound. This depth was estimated on the uniform
histology photomicrographs from the surface to the muscle layer.
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Results:
[0175] The following results were observed in each group:
CONTROL GROUP 1: Uninfected and untreated.
[0176] Time Zero: (See figures 14-15) At time zero following the wound
stripping there was cellular covering of the surface. The dermal layer was not
thickened. The hair follicles have a single cellular lining. There was minimal
vascularity and no inflammation. The depth of the tissue was considered zero
for future bench mark 0+.
[0177] 2 hours: (See figures 16-17) At 2 hours following the wound stripping
the surface remained covered with cellularity. The dermal layer was minimally
thickened. The follicles and cellular lining was the same. There was minimal
increase in vascularity and inflammation. The increase in the depth of the
tissue was considered 0.5+.
[0178] 48 hours: (See figures 18-19) At 48 hours the wound stripped,
uninfected, untreated specimens showed natural history response of surface
cellular proliferation and thickness. The dermal layer was thickened. The hair
follicles were present with single layer cellular lining. The vascularity was
increased in amount compared to the 2 hour specimens. The inflammation
was present throughout the derm is and muscle layer. The thickness was
zo considered 0.5+.
CONTROL GROUP 2: Infected and untreated.
[0179] 2 hours: (See figures 20-21) The histological assessment showed the
wound stripped, infected, but untreated controls at 2 hours to have multiple
cellular covering on surface. There was minimal thickening of the dermal
layer. The hair follicles were abundant and had double layer cellular lining.
There was minimal vascularity and no inflammation in the specimens. The
thickness was assigned 0.5+.
[0180] 48 hours: (See figures 22-23) At 48 hours the surface cellular covering
was gone. The dermal layer had minimal thickening. The hair follicles were
present, with minimal cellularity lining. There was marked increase in
vascularity and minimal inflammation in dermis layer. The depth was
considered 0.5+ compared to time zero.
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EXPERIMENTAL GROUP PCA 25 mM
[0181]48 hours: (See figures 24-25) The cellular covering of the surface was
abundant and multiple cell layers. The dermal layer was thickened. The hair
follicles were prominent with multiple cellular lining. There was collagen
proliferation between the epidermis and dermis. Additionally, there was
moderate vascularity, but less than that seen in infected untreated group.
There was abundant inflammation and it was greater than was seen in the
PCA 50 dose. Thickness was assigned 2+.
EXPERIMENTAL GROUP PCA 50 mM
[0182]48 hours: (See figures 26-27) The surface was covered with multiple
layers of cells. The dermal layer was thicker. The hair follicles had double
layer of cells. There was increased vascularity. Inflammation also increased
in
the dermis and below the muscle layer. The tissue thickness was assigned
2+.
EXPERIMENTAL GROUP C3G 100 mM
[0183]48 Hours: (See figures 28-29) There was multiple cellular covering of
the surface. The dye of the C3G was apparent on the skin surface indicating it
zo had not changed color due to pH nor completely degraded. The dermal
layer
was thicker. The hair follicle had single and double cellular lining. The
vascularity was prominent. There was inflammation in the dermis and
muscular layer and below. The thickness of the tissue was assigned 2+.
EXPERIMENTAL GROUP C3G 200 mM
[0184]48 Hours: (See figures 30-31) There was evidence of the C3G material
remaining on the skin surface. The surface cellular layer was multiple cells
thick. The dermal layer was thickened. The hair follicles had single and
double
cellular lining. The vascularity was increased. There was inflammation in the
dermis and muscular layer. The thickness was assigned 2+.
[0185]These results confirm that an anthocyanin (-38% C-3-G as the source)
and the main metabolite of anthocyanins and anthocyanidins, protocatechuic
acid (PCA) when applied topically at various calculated doses to the stripped
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skin wound of a rodent were bactericidal in 48 to 96 hours. There was a
10,000 fold kill of Pseudomonas aeruginosa in 48 hours with both reagents
and dose.
[0186] The results also show by histology a simultaneous healing of the
.. experimentally created wound in the same time frame. C-3-G and PCA in two
different doses stimulated tissue repair as evidence by histology.
[0187] Specifically, the experimental model provided evidence of a
histological
contrast between the control and experimental groups. At 48 hours, Control
Group 2 that was wound stripped and infected showed a clear contrast to the
uninfected Control Group 1. In the skin stripped infected group there was loss
of the epithelial cellular covering, no follicular cellular proliferation,
marked
increase in vascularity and little inflammatory response. This histological
condition provided clear contrast to the treatment groups. All treatment
groups
by comparison showed healing response with multiple layer cellular
.. proliferation on the surface, multiple layer cellular proliferation along
the hair
follicles, less vascularity, but an inflammatory cellular response in the
dermis
and muscular levels. See figures 14-31. PCA at a concentration of 25 mM
also showed collagen layer formation between the epidermis and derm is.
(See figures/photos 24 and 25). This response is beneficial in the use of
zo anthocyanin and anthocyanidins and metabolites thereof as a cosmetic
agent
to promote wound healing and improve skin health, including wrinkle
reduction or removal. This method of use of anthocyanin and anthocyanidin
metabolites, and particularly PCA, is based upon the two fold response; the
collagen layer increase and the skin swelling that increased the depth of the
skin.
Example 7: PCA 's effect on Pseudomonas aeruginosa ATCC 700888 and
Staphylococcus aureus ATCC 33591 (MRSA) biofilms
[0188] The inventor has shown that a composition comprising PCA was able
to stop the growth of a biofilm formation as well as destroy already formed
biofilms. The biofilms tested were Pseudomonas aeruginosa ATCC 700888
and Staphylococcus aureus ATCC 33591 (MRSA).
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[0189]The following amounts were tested on a polyester cloth and sintered
316 stainless steel mesh: The cloth was a piece cut from a polyester pillow
case. The cloth was soaked in the PCA solution and air dried for 24 hours.
The cloth was dried when tested.
1: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: cloth material,
1-1
2: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: cloth material,
1-2
3: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: 3-ply sintered
mesh, 1-1
4: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: 3-ply sintered
mesh, 1-2
5: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: 5-ply sintered
mesh, 1-1
6: 20 grams of PCA in 100m1 of 70% isopropyl alcohol, vehicle: 5-ply sintered
mesh, 1-2
7: Glass Slide (to serve as control article), 1-1
8: Glass Slide (to serve as control article), 1-2
9: PCA crystals imbedded, vehicle: 3-ply sintered 40 micron mesh, 1-1
10: PCA crystals imbedded, vehicle: 3-ply sintered 40 micron mesh, 1-2
11: PCA crystals imbedded, vehicle: 5-ply sintered 40 micron mesh, 1-1
12: PCA crystals imbedded, vehicle: 5-ply sintered 40 micron mesh, 1-2
[0190]The bacteria (Pseudomonas aeruginosa ATCC 700888 and
Staphylococcus aureus ATCC 33591 (MRSA)) were placed in reactors and
allowed to grow and form biofilms. Then cloths and metal were treated by
coating with PCA solutions and then were left to dry. Two sets of the
stainless
steel mesh had crystals imbedded into the mesh to replicate placement into a
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mesh or coated joint implant. A standard ASTM E-2647 drip flow biofilm
reactor was used to grow a biofilm and the treated surfaces (as well as the
control) were placed into the reactors and the biofilm was allowed to grow for
about 6 hours. The samples received a continuous nutrient flow for an
additional time period for about 48 hours to promote a steady growth rate of
the biofilm. Then the biofilm was removed, analyzed and a microbial count
and log density measurements were taken for each sample. Colony forming
unites ("CFU") were counted (which is an estimate of the number of viable
bacterial. Log density is the calculation of the biofilm present.
[0191] It was found that a 10% concentration as not as effective against
Pseudomonas, but a 20% concentration of PCA was very effective. See
figures 33 and 34. Figure 34 shows that the materials that were treated had a
very much smaller log density and CFUs than the control material (glass
slide).
[0192] It was found that a 30% concentration of PCA against MRSA was very
effective. See figures 35 and 36.
Example 8: Spray on Solution of PCA and Time Study of PCA 's effect on
Pseudomonas aeruginosa ATCC 700888 and Staphylococcus aureus ATCC
zo 33591 (MRSA) bio films
[0193] Next the time necessary to destroy biofilms and kill bacteria was
tested. A biofilm consisting of over 10 million organisms were formed on a
glass slide. A single spray of 30% PCA mixed with isopropyl alcohol was
applied to the glass slide. The colony forming units (CFU) were examined at
30 minutes and 60 minutes after the single spray. Incidentally, typical tests
for
testing the ability of an antibiotic to work are done for 48 hours. The tests
performed were as follows:
[0194] Glass slides were inoculated at Time 0. Batch phase was performed for
6 hours to allow for biofilm formation on the glass slides. The drip flow
mechanism was then turned on to provide a continuous flow of nutrients to the
glass slides over 48 hours. After 48 hours, 2 sets of glass slides were
sprayed
with a 30% PCA solution. One set was removed and analyzed for biofilm
reduction after 30 minutes. The other set was removed after 60 minutes.
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[0195] 2 sets of control slides were also removed and analyzed after 30
minutes and 60 minutes. The control slides were not treated with 30% PCA
and were used for comparative purposes.
[0196]The Log Reduction Calculations were performed as follows: The mean
30% PCA treated samples were compared to the mean positive control
samples, per time point evaluated.
[0197]For Pseudomonas aeruginosa, after 30 minutes of the application of
the 30% PCA spray, there was a 3.3 log reduction. After 60 minutes, there
was 2.2 log reduction, which amounts to a 99.9% reduction in the number
io colony forming units. See figure 37.
[0198]For Staphylococcus aureus, after 30 minutes of the application of the
30% PCA spray, there was a reduction of 60 million CFUs to 3 million and
after 60 minutes, there was reduction, of 25 million to 2 million CFUs. See
figure 38.
[0199] Thus, the results show that a 30% PCA spray killed 90% of the biofilms
in 30 minutes. The FDA only requires a 90% reduction.
[0200] It is noted that this above experiment was chosen to replicate a
clinical
condition, involving a metal or linen implant even to the extreme because
there would never be that concentration of biofilms nor bacteria in practice
zo flowing over the implants. Accordingly, the invention also provides a
method
of blocking initial attachment of the bacteria to the implant and therefor
preventing growth/development of a biofilm on an implant.
[0201] It was determined that the effective amount of an anthocyanin,
anthocyanidin or metabolite thereof (e.g., PCA or 2,4,6 THBA) depends upon
the species of bacterial to be eradicated. It was discovered that a 10%
concentration of PCA was not effective on Pseudomonas aeruginosa (see
figure 33), whereas a 20% concentration was effective (see figure 34).
[0202] The biofilms destroying properties of coating metal and linen for
Methicillin resistant Stapylococcus aureus required higher dose than
.. Pseudomonas aeruginosa (see figure 35). At a concentration of 30%, it was
effective (see figure 36). The application on an implant allowed to dry had
the
above results. However, when a glass surface is covered with 10 million
biofilms colonies the results differ with the concentration and the bacteria
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biofilms to be eradicated. The glass surface experiments are described in
example 8. Generally, Glass slides were inoculated at Time 0. Batch phase
was performed for 6 hours to allow for biofilm formation on the glass slides.
The drip flow mechanism was then turned on to provide a continuous flow of
nutrients to the glass slides over 48 Hours. After 48 hours, 2 sets of glass
slides were sprayed with a 30% PCA solution. One set was removed and
analyzed for biofilm reduction after 30 minutes. The other set was removed
after 60 minutes. 2 sets of control slides were also removed and analyzed
after 30 minutes and 60 minutes. The control slides were not treated with 30%
.. PCA and were used for comparative purposes. The Log Reduction
Calculations were performed as follows: The mean 30% PCA treated samples
were compared to the mean positive control samples, per time point
evaluated. The results were as follows for a single spray of 30% PCA in
isopropyl alcohol on 10 million biofilms colonies of Pseudomonas aeruginosa.
See figure 40. The concentration of 30% has lesser effect on MRSA, but still
90%. See figure 41. These experimental concentrations of biofilms covered
pathogens that far exceed the concentrations and numbers found in practice.
Example 9: Testing against Pro pionibacterium acnes
zo [0203] BALB/c mice were infected with Propionibacterium acnes via
intradermal injection and treated topically with varying concentrations of a
novel test compound, PCA, at 2, 24, 48, and 72 hours following challenge.
Efficacy was evaluated by CFU analysis from skin samples harvested at 96
hours post challenge.
[0204] These data demonstrate that P. acnes establishes a steady intradermal
colonization in the skin of BALB/c mice. When administered topically, PCA at
60 mg/kg, demonstrated a bacteriostatic effect and reduced P. acnes CFU
burden in mouse skin by a statistically significant amount. All lower amounts
of PCA showed no such effect.
[0205] Female BALB/c mice, ordered from Harlan and weighing 17-19 g, were
acclimated to housing conditions and handled in accordance with AUP
number TP-18-13. The animals were acclimated for 4 days prior to bacterial
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challenge. Only animals deemed healthy and fully immunocompetent were
included in this study. Cages were prepared with 2 mice per cage.
[0206] The animals were fed Teklad Global Rodent Diet (Harlan) and water ad
libitum. Mice were housed in static cages with Teklad 1/8" corn cob bedding
inside bioBubble Clean Rooms that provide H.E.P.A filtered air into the
bubble environment at 100 complete air changes per hour. All treatments and
infectious challenges were carried out in the bioBubble environment. The
environment was controlled to a temperature range of 74 4 F and a humidity
range of 30-70%. Treatment groups were identified by cage card. All
procedures carried out in this experiment were conducted in compliance with
all the laws, regulations and guidelines of the National Institutes of Health
and
with the approval of the TransPharm Animal Care and Use Committee.
Bacterial Cultures
[0207] Propionibacterium acnes (1100; ATCC 6919), procured directly from
the American Type Culture Collection.
Skin Preparation
[0208] On Day -1, each mouse was anesthetized in an lsoflurane induction
zo chamber and the lesion site was cleared of hair. An area of
approximately
2.0 cm x 2.0 cm of skin on the dorsal area of each mouse was cleared
through use of the depilatory agent Nair .
Challenge
[0209] Cultures were grown for 96 hrs at 37 C in an anaerobic atmosphere on
TS agar plates supplemented with 5% sheep blood cells. The culture was
aseptically swabbed and transferred to tubes of TS broth and allowed to grow
for 72 hours. The cultures were diluted to provide challenge inoculum of
approximately 6.0-7.0 10g10 CFU per 50 pL in PBS. On Day 0 each mouse
was anesthetized using lsoflurane. Each animal on the study was
administered 50 pL of the bacterial suspension via intradermal injection in
the
dorsal area that was previously denuded of hair. The final CFU count from the
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challenge suspension determined that 6.0 log10 CFU per mouse were
delivered.
Formulation and Dosing
[0210] The test treatment, PCA, was provided by the study Sponsor and
formulated using sterile water. Treatments were administered topically in a
dose volume of 0.1 mL. Treatments were given at 2, 24, 48, and 72 hours
post challenge at 60 mg/kg (78 mM; Group 3), 30 mg/kg (39 mM; Group 4) or
mg/kg (19.5 mM; Group 5).
Table 1: Animal Challenge, Treatment and Harvest Schedule
Group n Intradermal Treatment ROA Schedule CFU
P. acnes harvest*
1 2 6.0 log untreated NA NA 2 hr
2 2 6.0 log untreated NA NA 96 hr
3 2 6.0 log PCA 60 mg/kg Topical 2, 24, 48, 72 96 hr
hrs
4 2 6.0 log PCA 30 mg/kg Topical 2, 24, 48, 72 96 hr
hrs
5 2 6.0 log PCA 15 mg/kg Topical 2, 24, 48, 72 96 hr
hrs
*Relative to Challenge at 0 hr
RESULTS AND DISCUSSION
Infection/Treatment/General Observations
[0211] None of the study subjects displayed any acute adverse events
associated with the treatments. None of the test subjects succumbed to the
infection or showed signs of morbidity, which could be attributed to
penetration of the infection into the circulatory system or deep tissue. No
zo treatment group displayed adverse signs beyond those expected for mice
which have received a superficial bacterial infection.
[0212] The test article preparations were administered topically at 2, 24, 48,
and 72 hours following the bacterial challenge. While untreated mice were
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harvested at 2 hours post infection, CFU burden was not detected. However
at 96 hours post infection, the CFU burden rose from 6.0 log10 to 6.65 log10,
indicating a successful inoculation.
[0213]At 96 hours following challenge, mice were humanely euthanized and
skin was aseptically removed from the infection site. Skin samples were
placed in homogenation vials with 2.0 mL PBS, weighed and homogenized
using a mini-bead beater. Homogenate was serially diluted and plated
anaerobically on TSA agar plates for enumeration of colony forming units per
gram of skin tissue.
[0214]The mean bacterial burden of the untreated group at 96 hours was
6.65 10g10. CFU levels in all treated groups were compared to the untreated
group to determine statistical significance. Only the high dose of PCA
(60 mg/kg) showed significant reduction of CFU burden when compared to
the untreated control (P=0.0285). That an approximately 1 10g10 reduction
was observed indicates that at this concentration PCA, is bacteriostatic and
not bactericidal. All other treatments were statistically non-different than
the
untreated control (Figure 39).
[0215]These data demonstrate that P. acnes establishes a steady intradermal
colonization in the skin of BALB/c mice. When administered topically, PCA at
zo 60 mg/kg (78 mM solution), demonstrated a bacteriostatic effect and
reduced
P. acnes CFU burden in mouse skin by a statistically significant amount. All
lower amounts of PCA showed no such effect.
Example 10: Antibiotic testing with PCA or 2,4,6 THBA using propylene glycol
[0216]PCA or 2,4,6 THBA were combined with propylene glycol (PPG). The
PPG was placed on a paper disc and then either PCA or 2,4,6 THBA was
applied. The paper disc was then placed on colonies of various bacterial in a
Petri dish. At a certain uniform time they were inspected and classified in
the
following categories:
NI: no inhibition of bacteria growth under the sample
I: inhibition of bacterial growth under the sample
NZ: no accompanying zone of inhibition
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CZ: clear zone of inhibition surrounding the sample and zone
measured in millimeters (mm).
[0217] The results were as follows:
.. Water/PCA/Klebssilla pneumonia ATCC4352: I/CZ/2mm
PPG/PCA/Kiebssilla pneumonia ATCC4352: I/CZ/3mm
Water/PCA/Pseudomonas aeruginosa ATCC9027: I/CZ/10 mm
PPG/PCA/Pseudomonas aeruginosa ATCC9027: I/CZ/4 mm
Water/2,4,6 THBA/Pseudomonas aeruginosa ATCC9027: I/CZ/2 and 3 mm
PPG/2,4,6 THBA /Pseudomonas aeruginosa ATCC9027: I/CZ/3 mm
Water/2,4,6 THBA/Staphylococcus aureus ATCC33591 (MRSA): I/CZ/ 14 and
15 mm
PPG/2,4,6 THBA /Staphylococcus aureus ATCC33591 (MRSA): I/CZ/12 mm
Example 11: PCA to sterilize/disinfect human skin study
[0218] A randomized double blinded study was performed at Loma Linda
zo .. Medical School. It involved 4 phases over two years' time. The methods
were
as follows:
[0219] Phase 1: The active test reagent was topically applied 1.54% PCA in
sterile water to the anterior shoulder region. This 1.54% solution of PCA in
.. water was used effectively in our prior animal wound studies. The controls
were Chloraprep (2% Chlorhexidine in 70% isopropyl alcohol) and Betadine
(9.0% to 12.0% available iodine in water). Cultures were taken before
application and 20 minutes after application. The initial harvest was by a
surface swab. Application was by soaked sponge, without force or scrubbing.
The second harvest was performed with the back edge of a sterile knife blade
scraping with pressure in attempt to maximize the harvest from the deeper
sebaceous glands and hair follicles. The specimens were placed in culture
media. Bacteriology was performed at WuXiAppTec in Marrietta, GA.
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[0220] Phase 2 included eleven medical students and was same method as
Phase 1. However the PCA vehicle was changed to 70% isopropyl alcohol.
This allowed a higher concentration of PCA than possible in sterile water,
10%. Phase 2a: The 70% isopropyl alcohol vehicle was tested for its
bactericidal properties. All cultures that were negative or markedly reduced
with PCA topical solution were examined for exact nature of the index bacteria
and the post treatment cultures that showed no or minimal growth. In this way
it was learned what specific bacterial strains PCA could eliminate or reduce.
Results:
[0221] Phase 1 showed the aqueous solution of 1.54% PCA to be partially
effective as compared to the controls. In phase 1 of the Loma Linda Studies,
1`)/0 PCA in water showed no growth in 7 of 22 subjects on aerobic culture and
10 cultures showed reduced growth. By anaerobic culture, 6 of 22 cultures
showed no growth and 15 showed reduced colony growth. Six heavy growth
pre-treatment cultures prior to the 1.24% PCA treatment were chosen to
examine the isolates to learn what pathogens were killed or not killed. Most
of
the bacteria eradicated were non pathogens. Sample culture #29 showed 5
zo unique colonies by aerobic culture and 2 unique colonies by anaerobic
culture. After 1.24% PCA treatment there was no growth on either culture.
Therefore pre-treatment cultures were examined for the species. The chart
below is the result of the specific species colony identified and the method
of
identification used. The organisms were predominately non pathogens except
for P. acnes. However all of which were removed by the treatment.
GRAM STAIN & ORGANISM METHOD OF
SAMPLE ID CELL IDENTIFICATION
IDENTIFICATION
MORPHOLOGY
1(A) Gram positive cocci Micrococcus luteus / Biochemical
Analysis
lylae
1(B) Gram positive cocci Micrococcus luteus / Biochemical
Analysis
lylae
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1(C) Gram positive cocci
Micro coccus luteus Biochemical Analysis
1(D) Gram positive cocci
Micro coccus luteus Biochemical Analysis
1(E) Gram positive cocci
Staphylococcus DNA Sequencing
epidermidis
2(A) Gram positive cocci
Staphylococcus capitis Biochemical Analysis
2(B) Gram positive rods
Propionibacterium DNA Sequencing
acnes
[0222] Two other index pre-treatment cultures showed heavy growth of
Propionibacterium acnes, a potential pathogen. The post treatment cultures
showed the colonies of P. acnes was decreased to 5 colonies in one and 1
colony in another, but not eliminated. This suggested that PCA may be
effective against P. acnes if a higher concentration was applied in subsequent
Phases of this study. Testing with 1.24% in water killed bacteria of normal
flora as shown above chart. To increase efficacy and to improve skin
penetration to the depth of the hair follicles that can harbor bacteria, Phase
II
io of the study used 70% isopropyl alcohol (30% water) 85 ml so as to
increase
the concentration of PCA.
[0223] Phase II: This study involved 11 human subjects. There were 5 males
and 6 females. The ages were 23-33 years. There were two reagents. The
control was 70% isopropyl alcohol. The PCA source was a phytochemical
extract from Nanjing Zelang Medical Technology Co. LTD. This source was
chosen due to markedly reduced cost of goods compared to that which is
biochemically manufactured. The experimental dose was 9 +/- % PCA in 70%
isopropyl alcohol. 10 grams of PCA was placed in 100 ml of isopropyl alcohol.
zo The isopropyl alcohol allowed for a greater dose of PCA than water.
(allowed
more PCA to be dissolved). The initial harvest was by a surface swab.
Application was by soaked sponge, without force or scrubbing. The second
harvest was performed with the back edge of a sterile knife blade scraping
with pressure in attempt to maximize the harvest from the deeper sebaceous
glands and hair follicles.
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[0224] Cultures were stored in ice and shipped in FedEx ice box to
WuXiapptec in Marietta, GA for aerobic and anaerobic cultures and held for
21 days. Method used for bacterial species identification was Vitek MS-
MALDI-TOF MS. It was determined that the dose or concentration of PCA
needed to be increased, which required changing the water to 70% isopropyl
alcohol. This is the same as used with Chloraprep which is 2% w/v
chlorhexidine gluconate in 70% isopropyl alcohol. 10% PCA in 70% isopropyl
alcohol was compared to the control 70% isopropyl alcohol in Phase II. The
results of Phase II showed the following summary of the no growth culture
results after treatment.
Aerobic Anaerobic
PCA/IP alcohol 10/11 no growth 9/11 no growth
Control IP alcohol 6/11 no growth* 4/11 no growth
*Note that 3/11 of the 70% IPA groups showed increased colony growth after
IPA treatment alone.
[0225] Phase 2 results with 9+% solution of PCA were compared to similar
test Phase 1 and this showed this to comparable to Betadine in effectiveness,
but not with Chloraprep, which killed all the bacterial colonies. This PCA/70%
isopropyl alcohol solution was effective against 10/11 index cultured aerobic
bacteria and reducing on anaerobic culture in 9 of 11 subjects. The two
zo showing residual colonies were one (n=1) colony of Propionibacterium
acnes
in each of the two cultures. Phase 2a showed that 70% isopropyl alcohol
(IPA) alone had few antibacterial properties. Of course sterile water used in
Phase 1 had no antibacterial properties, therefore the testing was on the
effectiveness of the PCA.
[0226] The Phase 2 study showed the importance of PCA reagent
concentration or dose. A different application necessitated an increase dose.
PCA at 1.54 % although effective in an animal open skin wound was not a
very effective antibacterial reagent on intact human skin. Therefore the
concentration of PCA needed to be increased and could not be increased with
water, but only be accomplished with the vehicle of 70% isopropyl alcohol.
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[0227] Phase 2 showed that PCA was more effective in the higher
concentration eliminating all bacteria and reducing Propionibacterium acnes
to one colony in two separate instances. It should be noted that the 70%
isopropyl alcohol vehicle had little anti-bacterial properties. The water was
sterile and the alcohol tested negative in vitro. Therefore, they did not
contribute other than as a vehicle for topical application in this
environment.
[0228] The interval of 20 minutes was chosen for testing was similar to what
would be expected if used as a surgical preparation.
[0229] The first and 2nd method of harvesting differed. The 2nd was
accomplished with pressure wiping with the back end of a sterile scalpel so as
to maximize the harvest even from sub surface sweat gland and hair follicles.
[0230] The strengths of this study were in the careful attention given to the
method to disadvantage the topical applications effectiveness, yet PCA was
effective and comparable to Betadine tested similarly as Phase 1. The PCA
used was a single molecule of 99% pure biochemically synthesized PCA. The
water an and the isopropyl alcohol could be discounted as a contributor to the
effect as it contributed nothing to the bactericidal effect; sterile water and
null
effect of IPA in vitro testing.
[0231] The method used placed a burden on effectiveness of the reagents by
zo the short time (20 minutes) to act and the 2nd harvest maximizing the
potential yield due to the pressure scraping with back edge of a scalpel.
[0232] This study showed the effectiveness of a single reagent, PCA, the
common phytochemical metabolite. It showed that a single concentrated
metabolite working alone is bactericidal on human skin. It showed the
importance of dose variation depending upon the intended topical application;
wound versus intact skin. It showed the effectiveness absent any potential
antibacterial boost from the isopropyl alcohol vehicle.
[0233] It established the criteria necessary for consideration as a drug;
single
reagent, known dose effective in two different host environments, topical
application route, frequency of application (once), duration (20 min) time to
gain the intended result.
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[0234]Phase 3: Another test was conducted where the PCA was dissolved in
15 ml propylene glycol. The PPH is a skin penetration enhancer and assists in
dissolving PCA.
[0235] In another test, 5 ml of essential oil of Peppermint was added. This
has
.. skin penetration enhancer properties and anti-microbial properties.
[0236]Based upon Phase I and II results, it was determined that the dose or
concentration of PCA should be increased. It also was determined that it
would be ideal to have something in the composition that possessed skin
penetration properties. Therefore propylene glycol (PPG) was added. Fasano
WJ, ten Berge WF, Banton MI, Heneweer M, Moore NP. Dermal penetration
of propylene glycols: measured absorption across human abdominal skin in
vitro and comparison with a QSAR model. Toxicol In Vitro. 2011
Dec;25(8):1664-70. Then to further the skin penetration an essential oil was
added; i.e. an essence of peppermint oil (EOPO) (Nature oil, 1800 Miller
Parkway, Streetsburo, OH 44241 100% pure [Japan]. Chen J, Jiang Q-D, Wu
Y-M, Liu P, Yao J-H, Lu Q, Zhang H, Duan J-A. Potential of Essential Oils as
Penetration Enhancers for Transdermal Administration of Ibuprofen to Treat
Dysmenorrhoea Molecules 2015, 20, 18219-18236. Note that both PPG and
EOPO are skin penetration enhancers but also have anti- microbial
zo .. properties. P. acnes normally reside deep in the skin's hair follicles
and or
sebaceous glands. Therefore non-penetration common commercial
disinfectants are not effective as reported in the literature and noted in the
Background section above.
[0237]The composition of matter was created when 20 grams of PCA was
placed in 85 ml 70% isopropyl alcohol making a concentration of PCA
17% (+/-). Then 15 ml propylene glycol and 5 ml of essential oil of peppermint
were added. All reagents had skin penetration properties. The opposite
shoulder had only the control 70% isopropyl alcohol vehicle applied. The
subjects volunteered they liked the peppermint smell. The following summary
is the no growth culture results after treatment.
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Reagent Aerobic Anaerobic
17%+/-PCA 10/12 11/12
composition
70% IPA 10/12 8/12
alone
The analysis of the 2 pre PCA treatment subjects that grew subsequent
positive cultures were analyzed as follows:
[0238] PCA Pre Treatment Aerobic Species Analysis: The index subject #18
aerobic growth was too numerous to count. There were 2 different species;
Staphylococcus epidermidis / hominis and Micrococcus luteus. The Subject
#18's post PCA treatment aerobic culture, #20 showed 2 colonies; S. capitis
and S. epidermidis. For reasons unknown, neither of these species was
identified in the index culture, and both are considered non-pathogens. The
pre-PCA treatment index culture on subject #37 had colonies too numerous to
count (TNTC) with heavy growth. The colony species were reported as one;
Staphylococcus capitus. The Subject #37's post PCA treatment aerobic
culture #39, showed one (1) colony growth, but not the former S. capitus, but
was identified Staphylococcus epidermidis as the residual. As in prior phases,
the residual growth was most often a non-pathogen on aerobic culture.
[0239] Anaerobic Species analysis: The 17% PCA composite solution resulted
in 11 of 12 subject's subsequent cultures having no growth. Pre PCA
treatment subject #18 had bacteria colonies too numerous to count (TNTC) on
anaerobic culture. There were 4 separate colonies with the following species;
zo S. epidermidis (n=3) and Propionibacterium avidium. After this PCA
treatment
there was no anaerobic growth. Subject #37 index anaerobic culture showed
79 colonies. There were 4 prominent colony species; Staphylococcus
epidermidis, Staphylococcus capitis /caprae, Staphylococcus capitis and
Propionibacterium acnes. The one post PCA culture that showed a bacterial
colony culture was subject #37 with post PCA treatment anaerobic culture
being #39. There were 2 two colonies of same species; Staphylococcus
epidermidis. There were no Propionibacterium acnes colonies. Thus, the
results of Phase 3 showed the effectiveness of 17%+/- PCA in a composition
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of matter that had skin penetration properties; propylene glycol and essence
of peppermint oil.
[0240] In Phase IV the method was different from the previous phases:
[0241] Method: The variations in materials and methods in this phase were
based upon results of the previous phases. They included changes in the
solution, the method of dissolving the PCA, the timing of skin application,
plus
using two applications to simulate the present day recommendations of
surgical skin preparation.
io .. [0242] The solution used was 20 mg of ground PCA (Nanjing Zelang Medical
Technology Co. Ltd.) in 95 ml 91% isopropyl alcohol and 5 ml of essential oil
of peppermint 100% pure. The method placed the 20 grams of ground PCA in
sterile container with volume markers. Then added 85 ml of 91% isopropyl
alcohol; warm and shake. 5 ml of essential oil of peppermint was added. A
repeat of warming and shaking was instituted. The container was filled to
100 ml, warm and repeated the shaking. It took perhaps 15 to 20 minutes for
the ground crystals to dissolve.
[0243] An area was marked at 4 corners on the anterior axilla area of both
shoulders. There was a topical reagent application. After 5 minutes a second
zo application was made. The control group was again Chloraprep . The
experimental side was applied with manual motion and pressure on a sterile
sponge soaked with the PCA solution. The subjects put hands on head for 10
minutes and then 10 minutes of moving arms around. Culture #1 was
harvested with scraping at 20 minutes post 2nd application. Subjects rested
their arms at side for 40 minutes which is 60 minutes after the first
application
and 40 minutes after culture #1. A second culture is taken with scraping. The
post application cultures were taken of each shoulder skin area with scraping
with back side of a sterile surgical knife blade to maximize the yield.
Results of Phase IV:
[0244] The index pre-treatment culture's average colony counts of the 4
groups were similar.
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Aerobic Anaerobic
PCA 34.4 average 44.4 average
Chloraprep 41.75 average 49.83 average
[0245] The only group showing no growth in all 12 treated subjects was the
PCA/peppermint solution treated aerobic group at 20 minutes. Other than that
group, neither PCA nor Chloraprep sterilized the skin 100% at 20 or 60
minutes. However, Chloraprep had more no growth" cultures in total. The
following summary is the no growth cultures results after treatment.
Aerobic anaerobic
PCA 20 minutes 12/12 4/12
PCA 60 minutes 7/12 6/12
Chloraprep 20 minutes 10/12 8/12
Chloraprep 60m mutes 10/12 10/12
[0246] The 20% PCA in 91% IPA and PPO was most effective at 20 minutes
on aerobic culture, but not otherwise. Chloraprep was partially effective at
minutes and maintained similar effectiveness on aerobic culture and same
effectiveness on anaerobic cultures at 60 minutes. The results were different
on Chloraprep at 20 minutes from Phase 1 when there was no growth
following this reagent application.
15 [0247] There were 9 cultures that warranted selection for species
determination. #8 pre PCA treatment anaerobic culture had 8 colony count but
zero at 20 and 60 minutes. The interest was to learn what bacterial were
eliminated. There were two species; S. capitis and S. epidermidis. #12 post
PCA at 60 minutes showed 1 spreader colony on the plate (SPR) 50 colonies.
zo This contrasted with the pre-treatment of 28 colonies. The species found
in
#12 aerobic were Klebsiella pneumonia/oxytoca and Micrococcus leuteus.
These are not common pathogens. #14 pre PCA anaerobic showed 115
colonies. The species were Propionibacterium acnes, S. lugdunensis, and
Kocuria varians. There was no growth at 20 minutes showing that potential
pathogen P. acnes was eliminated. However, there were 4 aerobic colonies at
60 minutes in #19. They were Gram Negative Rods: Stenotrophomonas
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maltophilia: rare pathogen and Stenotrophomonas maltophilia. The Gram
Positive cocci were Kytococcus sedentarius: rare opportunistic pathogen and
Micrococcus luteus / lylae: opportunistic pathogen, particularly in hosts
with compromised immune systems, such as HIV patients. #19 was a pre
PCA anaerobic culture with 39 count showing species of P. acnes and
S. epidermidis. #21 was the post PCA at 20 minutes and showed 1 anaerobic
colony of P. acnes, a reduction from 39 colonies.
[0248]There was culture of Gram Positive Rods with spores identified as:
Lysinibacillus sphaericus / fusiformis: rare pathogen. #23 showed no growth
at 60 minutes for aerobic or anaerobic. Therefore all aerobic bacteria
including the one anaerobic colony of P. acnes was gone at 60 minutes. #31
aerobic was pre PCA showing 134 colonies. The species were Micrococcus
luteus and Micrococcus luteus/lylae. There was no growth at 20 or 60 minutes
of these normal flora species. #31 anaerobic was a pre PCA showing 28
colonies of Staphylococcus lugdunensis and Bacillus cereus/thuringiensis.
There was no growth of either at 20 and 60 minutes. #32 was pre Chloraprep
with aerobic colony count of 59 with the following species: Micrococcus
luteus/lylae, Micrococcus luteus, and Kocuria kristinae. There was no colony
growth at 20 or 60 minutes. #32 was pre Chloraprep anaerobic with 17
zo .. colonies with the following species: S. epidermidis and Gemella
bergeri/sanguinis. The post treatment showed no growth at 20 or 60 minutes.
It should be noted in any of the literature or these experiments that re-
colonization occurs with skin bacteria, likely in 30 minutes after treatment.
[0249]Species Analysis: The pre-treatment species were predominately
saprotrophic or commensal organisms. Propionibacterium acnes was
identified in two subjects. After PCA treatment the specific species analysis
showed predominately saprotrophic or commensal organisms. The potential
pathogen Propionibacterium acnes was identified with 19 colonies pre-PCA
treatment in #14 but showed no growth at 20 minutes after PCA treatment.
P. acnes was identified pre-PCA treatment in #19 with 19 colonies and post
treatment at 20 minutes there was one remaining colony of P. acnes.
However, there was no growth at 60 minutes.
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Discussion of Phase IV:
[0250] This solution of 20 grams of PCA, 75 ml of 91 % isopropyl alcohol and
ml of essence of peppermint oil was effective in removing potential
pathogens, including Propionibacterium acnes. Only commensal bacteria with
5 rare pathogenicity remained. The control Chloraprep did not removed most
of the bacterial colonies, but not all. Many saprotrophic or commensal
organisms were left intact. The PCA composition was not as effective as
Chloraprep against anaerobes, but where there was post PCA growth the
species-specific analysis showed that Propionibacterium was controlled.
[0251] A composite of the results based only no growth cultures following
treatment by each solution are shown in figure 42. *Note that the 1`)/0 PCA
was not included. The summation percentages of no growth" are shown in
figure 43.
Summary of the 4 phase testing at Loma Linda:
[0252] The questions proposed in the purpose of this proof of principle pilot
study were answered in the affirmative. The optimal dose of PCA to act as a
human skin disinfectant is greater than 10%. There is a facilitating delivery
vehicle, which is at least 70% isopropyl alcohol, which allows higher
zo concentrations of PCA to go into solution than water, propylene glycol,
and/or
essential oils alone. The results were optimized by the addition of known skin
penetration enhancers; propylene glycol and essence of peppermint oil.
P. acnes normally resides deep in the skin's hair follicles and or sebaceous
glands. Therefore, non-penetration common commercial disinfectants are not
effective.
[0253] PCA in one or more of these vehicles provided a broad-spectrum
disinfectant effect comparable to existing commercial products; isopropyl
alcohol, Chloraprep and Betadine .
[0254] One of two result assessment approaches was considered in this
study. One is related to government regulations and is called non-inferiority
status, or the showing the test article is essentially the same as a marketed
product. The reason to choose this method is that the existing marketed
products in the study had side effects, complications, and or higher cost.
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D'Agostino Sr RB, Massarol JM, Sullivan LM. Non-inferiority trials: design
concepts and issues ¨ the encounters of academic consultants in statistics
Statist. Med. 2003; 22:169-186.
[0255] The other method is to establish clear superiority to an existing
product. Both were considered in this study. The various PCA solutions
showed a non-inferiority status to 70% Isopropyl alcohol, Betadine and
Chloraprep . The PCA solutions in this study showed superiority to all but
Chloraprep . After the first Phase and each subsequent Phase results, the
materials and methods were modified in attempt to improve the results of the
test reagent PCA. The vehicle was changed from water to isopropyl alcohol to
facilitate an increase dose of PCA. In addition, the vehicle was change to
include reagents known to have skin penetration properties as well as
antibiotic properties; i.e. propylene glycol and essence of peppermint oil. In
Phase III, the best PCA results in both aerobic and anaerobic cultures were
obtained with a mixture of 17% PCA, propylene glycol and essence of
peppermint oil. The anaerobic cultured bacteria are those likely to be below
the skin surface and perhaps were affected by the addition of the skin
penetrator enhancing reagents. PCA effectiveness was demonstrated in
Phase I, even with the low dose of a 1.24 % aqueous solution. This aqueous
zo solution of PCA decreased the colonies of Propionibacterium acnes that
were
too numerous to count down to about to 1 and 5 colonies on same subject
after treatment. Solutions of PCA at 10% or higher removed all pathogens,
including P. acnes, but not all bacteria. Based upon the literature and the
results of this study, the goal of complete sterilization of the human skin
may
not be possible, predictable or desirable. In fact, sterilization may not be
desirable as it may eliminate bacteria that are beneficial to skin health and
homeostasis. It does seem reasonable to remove all bacteria species that
have pathologic potential. The irony of this goal is that it is reported that
Propionibacterium acnes although in some instances has become a
pathogen, it also has beneficial properties secreting a novel antioxidant.
PCA,
which decreased and or eliminated P. acnes in these studies, is an anti-
oxidant so its presence as such may substitute for the biological anti-oxidant
benefit of P. acnes. To further confuse any such study of this nature, it has
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been reported that the amount of bacteria on the skin prior to surgery are
not directly related to surgical site infection. (Cronquist AB, Jokob K, Lai
L,
Latta PD, Larson EL. Relationship between Skin Microbial Counts and
Surgical Site Infection after Neurosurgery. Clin Infect Dis. (2001) 33
(8):1302-
1308).
[0256] In Phase I, Chloraprep showed sterilization of the skin in all
subjects.
However, this was not replicated in Phase IV at 20 minutes or at the extended
time of 60 minutes. It should be noted that Saltzman, et al. reported in the
literature that Chloraprep had a 7% incidence of residual bacteria;
Propionibacterium acnes and Staphylococcus aureus persisted after
treatment and his harvest was at a short time in which the reagent was still
moist and there was not enough time perhaps for recolonization. (Saltzman
MD, Nuber GW, Gryzlo SM, Mareck GS, Koh JL. Efficacy of Surgical
Preparation Solutions in Shoulder Surgery. J Bone Joint Surg Am, 2009 Aug
01; 91(8): 1949 -1953). There is no clear reason for the difference in
Chloraprep results in the literature since there were many variables in
methods and timing of harvesting. There is not an apparent explanation for
the differing results at 20 minutes in Phase I and IV in our study. Yet in
Phase
IV Chloraprep was better than 20% PCA in 91% isopropyl alcohol and 5 ml
zo of essence of peppermint oil except at 20-minute aerobic cultures, which
were
sterile with the PCA solution. Note that isopropyl alcohol was not in Phase IV
solution.
[0257]Another measure of a treatment method would be the effectiveness to
reduce bacteria colony counts. This was common with PCA solutions in all
phases. A reduction in bacteria is a well-known principle in surgery in order
to
reduce the chance of infection. (Anglen J. Perspectives on Modern
Orthopedics. Wound Irrigation in Musculoskeletal Injury. J Am Acad Ortho
Surg. July/August 2001. 9 (4). 219-226).
[0258]The identification of anthocyanins and anthocyanidins or combinations
of anthocyanins, anthocyanidins or their metabolites that are bactericidal or
antimicrobial was determined by conducting in vitro testing described above.
Anthocyanidins that were tested at 100 mM (44.938 grams per liter) with less
than one milliliter per dose included delphinidin, pelargonidin, and cyanidin
Cl
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and cyanidin-3-glucoside. Protocatechuic acid and 2, 4, 6
trihydroxybenzaldehyde, the anthocyanidin metabolites, were also tested at
the same concentrations. Referring to FIGS. 6-8, delphinidin limited growth
against C. perfringens, S. aureus, and MRSA. Pelargonidin limited growth of
P. acnes, C. perfringens, S. aureus, MRSA, and S. pyogenes. Cyanidin Cl
was effective against C. difficile, C. perfringens, S. aureus ATCH 6538,
S. aureus (MRSA) ATCH 33591, S. mutans, and S. pyogenes. C3G
(approximately 28% by weight) had limited effectiveness during this study (18-
24 hours for aerobes; 48 hours for anaerobes (C. albicans and L. casei). This
proprietary C3G formulation, however, was effective against P. acnes, E. coli,
MRSA, K. pneumoniae, and P. aeruginosa. Protocatechuic acid (PCA), the
main metabolite from anthocyanins and anthocyanidins, was effective against
all bacteria tested as well as C. albicans and K. pneumoniae. Importantly for
skin wound treatment, PCA was effective against S. aureus 6538 and 33591
(MRSA) and P. aeruginosa. PCA was also effective on C. albicans, which is
important considering its ability to form biofilms and difficulty in treating
C. albicans when existing with a catheter or implant. 2, 4, 6
trihydroxybenzaldehyde was effective against E. coli, K. pneumoniae,
P. aeruginosa, S. aureus 6538 and 33591 (MRSA); it also was effective
zo against A. pullulans, ATCC 15233, a fungi.
[0259] While specific dosages of certain anthocyanins and anthocyanidins
were determined to have the above mentioned effects against certain
bacteria, in vivo testing were conducted to determine optimal dosages and to
confirm the ability of a topical application of these compounds to have
antimicrobial effect while prompting healing of a wound. It was hypothesized
that certain dose and interval topical application of a water soluble solution
of
PCA and/or C3G (28% of C3G by weight) at certain concentrations based
upon molecular weight would kill or reduce the bioburden of Pseudomonas
aeruginosa while healing the wound as evidence by optimization of the local
growth hormones and confirmed by histological evidence. Referring to FIG.9,
a decrease in bacterial burden in the skin at 96 hours days was noted (CFU
means colony forming units). A concentration of 50 mM of PCA was found to
be most effective; higher concentrations of PCA were not as effective at
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decreasing bacterial burdens. The most effective concentration of C3G was
100 mM. Importantly, histological evaluations of skin samples from the study
confirmed healing at 48 and 96 hours with proliferation of parafollicular
cells
and migration to cover the skin surface. There was minimal inflammation in
the dermis. There was collagen proliferation in the derm is.
[0260] As provided in figure 1, bacteria have a range of pH at which growth is
optimized, and most bacteria are more viable at basic pH ranges. Generally,
anthocyanins, anthocyanidins and their metabolites also have an acidic pH
and have the potential to have bactericidal or bacteriostatic modes of action.
Because C3G and PCA reagents have an acidic pH, their bactericidal or
bacteriostatic mode of action is by direct contact with the bacteria.
[0261] Anthocyanins and anthocyanidins were further studied to determine
effects on wound healing, including whether they had any effect on the
optimization of local growth hormone activity at the wound site along with
other supporting histological evidence of promoting healing.
[0262] Local growth hormones are important substances in the control of
wound healing. Equally as important, however, is to optimize the amount of
these hormones desirable for promoting wound healing while avoiding scar
formation and keloids.
zo [0263] Examples of common local growth hormones related to skin wound
healing include Epidermal growth factor (EGF), Insulin-like growth factor-1
(IGF-1) and Transforming Growth Factor ¨Beta (TGF-8). Epidermal growth
factor or EGF is a growth factor that stimulates cell growth, proliferation,
and differentiation by binding to its receptor EGFR. IGF-1 is important in
skin
repair by stimulating keratinocyte proliferation and migration as well as
collagen production by fibroblasts. Its expression is important during wound
healing such that retarded healing has been correlated with reduced IGF-1
levels. While local administration of IGF-1 to wound sites enhanced wound
closure and stimulated granulation tissue formation, increased IGF-1 receptor
expression was reported in chronic wounds and in hypertrophic scars.
Additionally, IGF-1 stimulation was associated with increased invasive
capacity of keloid fibroblasts. Systemic delivery of IGF-1 also caused
hyperglycemia, electrolyte imbalance, and edema. Therefore, it is desirable to
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have slightly elevated but not over elevated IGF-1 by a treatment modality.
TGF-6 also is important in skin would healing; however, it is considered a pro-
fibrotic growth factor and increased levels of TGF-6 or prolonged presence
has been identified as causing hypertrophic scaring.
[0264] Referring to figures 11-13, tests were performed on rodent skin to
explore the effects of PCA on the local growth hormones in rodent skin. A
concentration of 25 mM PCA increased local growth hormone levels at the
site of the untreated skin wound. In particular, figure 11 demonstrates that a
single reagent or compound would optimize local growth hormones to
io promote healing without scarring. Approximately 25 mM PCA was the
optimal
reagent and dose. As demonstrated in figure 13, optimization is possible
using the compositions of the present invention. In figure 13, all three local
growth hormones were lowered in the simulated clinical pathological
environment (stripped and infected); however, the lowering of these hormones
.. was not to the extent of absences. Hence, the necessary IGF-1 is still
above
the controls in this environment; however, the scar forming properties of the
other two hormones have been markedly reduced. Therefore, optimization of
local growth hormones is achieved. In figure 13, the optional concentration of
PCA was confirmed as 25 mM PCA in this situation and environment,
zo meaning local growth hormone growth levels were optimized at this dosage
such that IGF-1 as moderately elevated while TGF-6 and EGF levels were
decreased. This is important to promote wound healing while preventing
potential scarring.
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