Note: Descriptions are shown in the official language in which they were submitted.
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CHROMOPHORE COMBINATIONS FOR BIOPHOTONIC USES
BACKGROUND OF THE DISCLOSURE
Phototherapy has recently been recognized as having wide range of applications
in both
the medical, cosmetic and dental fields for use in surgeries, therapies and
examinations. For
example, phototherapy has been developed to treat cancers and tumors, to treat
skin conditions,
to disinfect target sites as an antimicrobial treatment, and to promote wound
healing.
Known phototherapy techniques include photodynamic therapy which involves
systemic administration or uptake of a photosensitive agent or chromophore
into the diseased
or injured tissue, followed by site-specific application of activating light.
Other types of
phototherapy include the use of light alone at specific wavelengths to target
tissue using light-
emitting diode (LED) or fluorescent lamps, or lasers.
It is an object of the present disclosure to provide new and improved
compositions and
methods useful in phototherapy.
SUMMARY OF THE DISCLOSURE
The present disclosure provides topical biophotonic compositions and methods
of using
the biophotonic compositions for the biophotonic treatment of living tissue.
Biophotonic
treatment may include skin rejuvenation; tissue repair including wound
healing, scar removal
and scar minimization; treatment of skin conditions such as acne; and
treatment of
periodontitis.
The biophotonic composition of the present disclosure comprises a gelling
agent and at
least two xanthene dyes, wherein a first xanthene dye has an emission spectrum
that overlaps at
least 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70% with an absorption spectrum
of a
second xanthene dye. In some embodiments, the first xanthene dye has an
emission spectrum
that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%,
35-45%, 50-
60%, 55-65% or 60-70% with an absorption spectrum of the second xanthene dye.
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Particularly useful combinations of xanthene dyes include but are not limited
to:
Fluorescein + Eosin Y; Fluorescein + Eosin Y + Rose Bengal; Fluorescein +
Eosin Y +
Phloxine B; Eosin Y + Rose Bengal; Eosin Y + Phloxine B; Fluorescein +
Erythrosine B +
Eosin Y; Eosin Y + Erythrosine; Eosin Y + Erythrosine B + Rose Bengal; Eosin Y
+
Erythrosine B + Phloxine B; Fluorescein + Eosin Y + Erythrosine B + Rose
Bengal; and
Fluorescein + Eosin Y + Erythrosine B + Phloxine B.
The gelling agent may comprise a hygroscopic substance. In addition or in the
alternative, the gelling agent may also be a hydrophilic polymer, a hydrated
polymer or a lipid.
In certain embodiments, the gelling agent comprises one or more of glycerin,
glycols such as
propylene glycol, polyacrylic acid polymers, hyaluronic acid, glucosamine
sulphate or gelatin.
In certain embodiments, the gelling agent is a high molecular weight, cross-
linked
polyacrylic acid polymer having a viscosity in the range of about 20,000-
80,000, 20,000-
100,000, 25,000-90,000, 30,000-80,000, 30,000-70,000, 30,000-60,000, 25,000-
40,000 cP. In
certain embodiments, the cross-linked polyacrylic acid polymer is a carbomer
selected from the
group consisting of, but not limited to, Carbopol 71G NF, 971P NF, 974P NF,
980 NF, 981
NF, 5984 EP, ETD 2020NF, Ultrez 10 NF, 934 NF, 934P NF, 940 NF, 941 NF, or
1342 NF.
In certain embodiments, the biophotonic composition is substantially
translucent and/or
transparent. In certain embodiment, the biophotonic composition has a
translucency of at least
70% at 460 nm. In other embodiments, the composition has a translucency of at
least 20%,
30%, 40%, 50%, 60%, 70%, 75%, 85%, 90%, 95% or 100% at 460 nm.
In certain embodiments, the biophotonic composition is a liquid, a gel, a semi-
solid,
cream, foam, lotion, oil, ointment, paste, suspension, or aerosol spray.
In certain embodiments, the biophotonic composition is encapsulated in a
transparent,
impermeable membrane, or a breathable membrane which allows permeation of
gases but not
liquids. The membrane may comprise a lipid.
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In certain embodiments, the biophotonic composition further comprises an
oxygen-
generating agent. In some embodiments, the oxygen-generating agent comprises
hydrogen
peroxide, carbamide peroxide, benzoyl peroxide, molecular Oxygen or water.
When the
oxygen-releasing agent is a peroxide, it may be present in values less than 6%
H202, from 0.5-
6wt% H202 (or its equivalent), 0.5-5.5%, 0.5-5.0%, 0.5-4.5%, 0.5-4.0%, 0.5-
3.5%, 0.5-3.0%,
0.5-2.5%, 0.5-2%, 0.5-1.5%, or 0.5-1.0%.
In certain embodiments, the biophotonic composition does not generate a
substantial
amount of heat following illumination with light. In some embodiments, the
energy emitted by
the biophotonic composition does not cause tissue damage.
In certain embodiments, the first and second xanthene dyes are present in the
composition in the amount of about 0.001-0.5% per weight of the composition.
In certain embodiments, the biophotonic composition may be applied to or
impregnated
into a material such as a pad, a dressing, a woven or non-woven fabric or the
like. The
impregnated material may be used as a mask (e.g. a face mask) or a dressing.
In certain embodiments, the biophotonic composition further comprises at least
one
waveguide within or adjacent to the composition. The waveguide can be a
particle, a fibre or a
fibrillar network made of a material which can transmit and/or emit light.
In certain embodiments, the composition does not comprise silica, tanning
agents, or
non-fluorescent dyes.
The present disclosure also provides uses of the present composition and
methods for
biophotonic treatment of living tissue.
Accordingly, in some aspects, there is provided a method for providing
biophotonic
therapy to a wound, comprising: applying to a wound a biophotonic composition
comprising at
least a first xanthene dye and a second xanthene dye, wherein the first
xanthene dye has an
emission spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%,
25-35%,
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30-40%, 35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of the
second
xanthene dye; and illuminating the biophotonic composition with light having a
wavelength
that overlaps with an absorption spectrum of the first xanthene dye.
In some embodiments of the method for providing biophotonic therapy to a
wound, the
method promotes wound healing. In certain embodiments of the method, the wound
as
described herein includes for example chronic or acute wounds, such as
diabetic foot ulcers,
pressure ulcers, venous ulcers or amputations. In some embodiments of the
method for
providing biophotonic therapy to a wound, the method promotes reduction of
scar tissue
formation. In certain embodiments, the treatment can be applied in or on the
wound once,
twice, three times, four times, five times or six times a week, daily, or at
any other frequency.
The total treatment time can be one week, two weeks, three weeks, four weeks,
five weeks, six
weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven weeks, twelve
weeks, or any
other length of time deemed appropriate.
In other aspects, there is provided a method for biophotonic treatment of acne
comprising: applying to skin tissue a biophotonic composition comprising at
least a first
xanthene dye and a second xanthene dye, wherein the first xanthene dye has an
emission
spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%,
30-40%,
35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of the second
xanthene dye;
and illuminating the biophotonic composition with light having a wavelength
that overlaps
with an absorption spectrum of the first xanthene dye. In certain embodiments
of the method
for biophotonic treatment acne, the treatment can be applied to the skin
tissue, such as on the
face, once, twice, three times, four times, five times or six times a week,
daily, or at any other
frequency. The total treatment time can be one week, two weeks, three weeks,
four weeks, five
weeks, six weeks, seven weeks, eight weeks, nine weeks, ten weeks, eleven
weeks, twelve
weeks, or any other length of time deemed appropriate. In certain embodiments,
the face may
be split into separate areas (cheeks, forehead), and each area treated
separately. For example,
the composition may be applied topically to a first portion, and that portion
illuminated with
light, and the biophotonic composition then removed. Then the composition is
applied to a
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second portion, illuminated and removed. Finally, the composition is applied
to a third portion,
illuminated and removed.
The disclosed methods for treating acne or wounds may further include, for
example,
administering a systemic or topical drug before, during or after the
biophotonic treatment. The
drug may be an antibiotic, a hormone treatment, or any other pharmaceutical
preparation which
may help to treat acne or wounds. The combination of a systemic treatment
together with a
topical biophotonic treatment can reduce the duration of systemic treatment
time.
In other aspects, there is provided a method for biophotonic treatment of a
skin
disorder, comprising: applying to target skin tissue a biophotonic composition
comprising at
least a first xanthene dye and a second xanthene dye, wherein the first
xanthene dye has an
emission spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%,
25-35%,
30-40%, 35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of the
second
xanthene dye; and illuminating the biophotonic composition with light having a
wavelength
that overlaps with an absorption spectrum of the first xanthene dye.
In other aspects, there is provided a method for promoting skin rejuvenation,
comprising: applying to target skin tissue a biophotonic composition
comprising at least a first
xanthene dye and a second xanthene dye, wherein the first xanthene dye has an
emission
spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%, 25-35%,
30-40%,
35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of the second
xanthene dye;
and illuminating the biophotonic composition with light having a wavelength
that overlaps
with an absorption spectrum of the first xanthene dye.
In other aspects, the present disclosure provides a method for treatment of
periodontal
disease, comprising: applying to a periodontal pocket a biophotonic
composition comprising at
least a first xanthene dye and a second xanthene dye, wherein the first
xanthene dye has an
emission spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%, 20-30%,
25-35%,
30-40%, 35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of the
second
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1
xanthene dye; and illuminating the biophotonic composition with light having a
wavelength
that overlaps with an absorption spectrum of the first xanthene dye.
In other aspects, there is provided a method of using a cascade of energy
transfer
between at least a first and a second fluorescent chromophore to absorb and/or
emit light within
the visible range of the electromagnetic spectrum for treatment of a skin
disorder, treatment of
a wound, skin rejuvenation, treatment of periodontitis. The present methods
and compositions
of the present disclosure may also be used to treat fungal and viral
infections.
In certain embodiments of any method of the present disclosure, the
biophotonic
composition is illuminated for any time period per treatment in which the
biophotonic
composition is activated, for example 1 to 30 minutes. The distance of the
light source from the
biophotonic composition can be any distance which can deliver an appropriate
light power
density to the biophotonic composition and/or the skin tissue, for example 5,
10, 15 or 20 cm.
The biophotonic composition is applied topically at any suitable thickness.
Typically, the
biophotonic composition is applied topically to skin or wounds at a thickness
of at least about
2mm, about 2mm to about 1 Omm.
In certain embodiments, the method of the present disclosure comprises a step
of
illuminating the biophotonic composition for a period of at least 30 seconds,
2 minutes, 3
minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes,
or 30 minutes.
In some embodiments, the biophotonic composition is illuminated for a period
of at least 3
minutes.
In certain embodiments of the methods of the present disclosure, the
biophotonic
composition is removed from the site of a treatment following application of
light.
Accordingly, the biophotonic composition is removed from the site of treatment
within at least
30 seconds, 2 minutes, 3 minutes, 5 minutes, 7 minutes, 10 minutes, 15
minutes, 20 minutes,
25 minutes or 30 minutes after application. In some embodiments, the
biophotonic composition
is illuminated for a period of at least 3 minutes. In some embodiments, the
biophotonic
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composition is removed after a period of at least 3 minutes post application
of the biophotonic
composition to treatment site.
In certain other embodiments, the biophotonic composition is kept in place for
up to
one, two or three weeks, and illuminated with light which may include ambient
light at various
intervals. In this case, the composition may be covered up in between exposure
to light. For
example, the biophotonic composition may be soaked in a dressing and placed
inside or over a
wound and be left in place for an extended period of time (e.g. more than one
day).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts absorption of light in the various layers of the skin (Samson
et al.
Evidence Report/Technology Assessment 2004, 111, pages 1-97).
Figure 2 illustrates the Stokes' shift.
Figure 3 illustrates the absorption and emission spectra of donor and acceptor
chromophores. The spectral overlap between the absorption spectrum of the
acceptor
chromophore and the emission spectrum of the donor chromophore is also shown.
Figure 4 is a schematic of a Jablonski diagram that illustrates the coupled
transitions
involved between a donor emission and acceptor absorbance.
Figures 5A and 5B are absorbance and emission spectra, respectively, of (i)
Fluorescein sodium salt at about 0.09 mg/mL, (ii) Eosin Y at about 0.305
mg/mL, and (iii) a
mixture of Fluorescein sodium salt at about 0.09 mg/mL and Eosin Y at about
0.305 mg/mL,
all in a carbamide gel (Example 1).
Figures 6A and 6B are absorbance and emission spectra, respectively, (i)
Fluorescein
sodium salt at 0.18 mg/mL final concentration, (ii) Eosin Y at about 0.305
mg/mL, and (iii) a
mixture of Fluorescein sodium salt at about 0.18 mg/mL and Eosin Y at about
0.305 mg/mL,
all in an aqueous solution (Example 2).
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Figures 7A and 7B are absorbance and emission spectra, respectively, of (i)
Phloxine B
at 0.25mg/mL final concentration, (ii) Eosin Y at about 0.05 mg/mL, and (iii)
a mixture of
Phloxine B (0.25mg/mL) and Eosin Y (0.05 mg/mL), all in a 12% carbamide gel
(Example 3).
Figures 8A and 8B are absorbance and emission spectra, respectively, of (i)
Phloxine B
at 0.25mg/mL final concentration, (ii) Eosin Y at about 0.08 mg/mL, and (iii)
a mixture of
Phloxine B (0.25mg/mL) and Eosin Y (0.08 mg/mL), all in an aqueous solution
(Example 4).
Figures 9A and 9B are absorbance and emission spectra, respectively, of (i)
Phloxine
B at 1001.1g/g, (ii) Fluorescein at about 100 g/g, and (iii) a mixture of
Phloxine B (100 g/g)
and Fluorescein (100 g/g), all in a 12% carbamide gel (Example 5).
Figures 10A and 10B are absorbance and emission spectra, respectively, of (i)
Phloxine B at 100tig/g, (ii) Fluorescein at about 100 g/g, and (iii) a mixture
of Phloxine B
(100 g/g) and Fluorescein (100 g/g), all in a 12% carbamide gel (Example 6).
Figures nA and 11B are absorbance and emission spectra, respectively, of (i)
Eosin Y
at 0.305 mg/mL final concentration, (ii) Rose Bengal at about 0.085 mg/mL, and
(iii) a mixture
of Eosin Y (0.305mg/mL) and Rose Bengal (0.085 mg/mL), all in a 12% carbamide
gel
(Example 7).
Figure 12 shows that Eosin Y and Rose Bengal act in a synergistic manner
(Example
8).
Figures 13A and 13B show the fluorescence emission (power density) over time
of
compositions comprising (i) Fluorescein + Eosin Y (Figure 11A), and (ii) Eosin
Y + Rose
Bengal (Figure 11B) (Example 9).
Figures 14A and 14B are absorbance and emission spectra, respectively, of (i)
Rose
Bengal at about 0.085 mg/mL, (ii) Fluorescein sodium salt at about 0.44 mg/mL
final
concentration, (ii) Eosin Y at about 0.305 mg/mL, and (iii) a mixture of (i),
(ii) and (iii) in a
carbamide gel (Example 10).
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'
Figures 15A and 15B are absorbance and emission spectra, respectively, of (i)
Rose
Bengal at about 0.085 mg/mL, (ii) Fluorescein sodium salt at about 0.44 mg/mL
final
concentration, (ii) Eosin Y at about 0.305 mg/mL, and (iii) a mixture of (i),
(ii) and (iii) in an
aqueous composition (Example 11).
Figure 16 is an emission spectrum showing the intensity over time of the light
being
emitted from the composition tested in Examples 12 and 13.
Figures 17A and 17B show that the energy density of emitted fluorescence from
Eosin
(top) and Fluorescein (bottom) in a composition increases rapidly with
increasing chromophore
concentration but slows down to a plateau with further concentration increase,
whilst the
activating light decreases with increasing concentration (Example 15).
DETAILED DESCRIPTION
(1) Overview
The present disclosure provides compositions including at least two
photoactive
chromophores which can transfer energy from one to the other and methods
useful for treating
tissue with these compositions for example to promote tissue repair including
wound healing,
for cosmetic treatment of skin such as for skin rejuvenation, for treating
skin disorders such as
acne, and for periodontal treatment.
(2) Definitions
Before continuing to describe the present disclosure in further detail, it is
to be
understood that this disclosure is not limited to specific compositions or
process steps, as such
may vary. It must be noted that, as used in this specification and the
appended claims, the
singular form "a", "an" and "the" include plural referents unless the context
clearly dictates
otherwise.
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As used herein, the term "about" in the context of a given value or range
refers to a
value or range that is within 20%, preferably within 10%, and more preferably
within 5% of
the given value or range.
It is convenient to point out here that "and/or" where used herein is to be
taken as
specific disclosure of each of the two specified features or components with
or without the
other. For example "A and/or B" is to be taken as specific disclosure of each
of (i) A, (ii) B
and (iii) A and B, just as if each is set out individually herein.
"Biophotonic" means the generation, manipulation, detection and application of
photons in a biologically relevant context. In other words, biophotonic
compositions exert
their physiological effects primarily due to the generation and manipulation
of photons.
"Biophotonic composition" is a composition as described herein that may be
activated by light
to produce photons for biologically relevant applications.
"Topical composition" means a composition to be applied to body surfaces, such
as the
skin, mucous membranes, vagina, oral cavity, wounds, and the like. A topical
composition
may be in the form of, including, but not limited to, a cream, gel, ointment,
lotion, levigate,
solution, bioadhesive, salve, milk. The topical composition may impregnate
material such as a
pad, sheet, fabric or fibres, dressings, spray, suspension, foam, or the like.
Terms "chromophore", "photoactivating agent" and "photoactivator" are used
herein
interchangeably. A chromophore means a chemical compound, when contacted by
light
irradiation, is capable of absorbing the light, for example a xanthene dye.
The chromophore
readily undergoes photoexcitation and can then transfer its energy to other
molecules or emit it
as light.
"Oxidant", "oxidizing agent" or "oxygen-releasing agent" which terms are used
interchangeably herein, means a chemical compound that readily transfers
oxygen atoms and
oxidizes other compounds. It includes molecular oxygen as well as oxygen
containing
compounds such as water, peroxide etc..
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"Photobleaching" means the photochemical destruction of a chromophore.
The term "actinic light" is intended to mean light energy emitted from a
specific light
source (e.g., lamp, LED, or laser) and capable of being absorbed by matter
(e.g. the
chromophore or photoactivator defined above). In a preferred embodiment, the
actinic light is
visible light.
"Wound" means an injury to any tissue, including for example, acute, subacute,
delayed
or difficult to heal wounds, and chronic wounds. Examples of wounds may
include both open
and closed wounds. Wounds include, for example, burns, incisions, excisions,
lesions,
lacerations, abrasions, puncture or penetrating wounds, surgical wounds,
contusions,
hematomas, crushing injuries, ulcers (such as for example pressure, venous,
pressure or
diabetic), wounds caused by periodontitis (inflammation of the periodontium),
and gun-shot
wounds.
"Wound healing" means promotion or acceleration of tissue repair including
closure of
a wound, activation of a chronic wound, or minimizing scar formation.
"Skin rejuvenation" means a process of reducing, diminishing, retarding or
reversing
one or more signs of skin aging. For instance, common signs of skin aging
include, but are not
limited to, appearance of fine lines or wrinkles, thin and transparent skin,
loss of underlying fat
(leading to hollowed cheeks and eye sockets as well as noticeable loss of
firmness on the hands
and neck), bone loss (such that bones shrink away from the skin due to bone
loss, which causes
sagging skin), dry skin (which might itch), inability to sweat sufficiently to
cool the skin,
unwanted facial hair, freckles, age spots, spider veins, rough and leathery
skin, fine wrinkles
that disappear when stretched, loose skin, or a blotchy complexion. According
to the present
disclosure, one or more of the above signs of aging may be reduced,
diminished, retarded or
even reversed by the compositions and methods of the present disclosure.
(3) Biophotonic Compositions
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The present disclosure provides biophotonic compositions. Biophotonic
compositions
are compositions that, in a broad sense, comprise chromophore(s) which are
activated by light
and accelerate the dispersion of light energy, which leads to light carrying
on a therapeutic
effect on its own, and/or to the photochemical activation of other agents
contained in the
composition (e.g., the break-down of an oxygen-releasing agent when such agent
is present in
the composition or at the treatment site, leading to the formation of oxygen
radicals, such as
singlet oxygen). The biophotonic compositions of the present disclosure
comprise at least two
xanthene dyes as chromophores.
When a chromophore absorbs a photon of a certain wavelength, it becomes
excited.
This is an unstable condition and the molecule tries to return to the ground
state, giving away
the excess energy. For some chromophores, it is favorable to emit the excess
energy as light
when transforming back to the ground state. This process is called
fluorescence. The peak
wavelength of the emitted fluorescence is shifted towards longer wavelengths
compared to the
absorption wavelengths due to loss of energy in the conversion process. This
is called the
Stokes' shift and is illustrated in Figure 2. In the proper environment (e.g.,
in a biophotonic
composition) much of this energy is transferred to the other components of the
composition or
to the treatment site directly.
Without being bound to theory, it is thought that fluorescent light emitted by
photoactivated chromophores may have therapeutic properties due to its femto-,
pico- or nano-
second emission properties which may be recognized by biological cells and
tissues, leading to
favorable biomodulation. Furthermore, the emitted fluorescent light has a
longer wavelength
and hence a deeper penetration into the tissue than the activating light.
Irradiating tissue with
such a broad range of wavelengths, including in some embodiments the
activating light which
passes through the composition, may have different and complementary effects
on the cells and
tissues. Moreover, the generation of oxygen species (e.g. singlet oxygen) by
photoactivated
chromophores has been observed by the inventors to cause micro-bubbling within
the
composition which can have a physical impact on the tissue to which it is
applied, for example
by dislodging biofilm and debridement of necrotic tissue or providing a
pressure stimulation.
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The biofilm can also be pre-treated with an oxygen-releasing agent to weaken
the biofilm
before treating with the composition of the present disclosure.
Furthermore, it is thought that use of chromophores in a composition to emit
fluorescent light provides the ability to fine-tune the emitted light to a far
greater degree than
using a light source such as an LED or a laser. For example, according to the
therapy or
treatment required, chromophores may be chosen according to their emitted
light wavelength,
and appropriate concentrations used to control the power density of the
emitted light.
The biophotonic compositions of the present disclosure are substantially
transparent/translucent and/or have high light transmittance in order to
permit light dissipation
into and through the composition. In this way, the area of tissue under the
composition can be
treated both with the fluorescent light emitted by the composition and the
light irradiating the
composition to activate it. The % transmittance of the biophotonic composition
can be
measured in the range of wavelengths from 250 nm to 800 nm using, for example,
a Perkin-
Elmer Lambda 9500 series UV-visible spectrophotometer. In some embodiments,
transmittance of the compositions disclosed herein is measured at 460 nm.
As transmittance is dependent upon thickness, the thickness of each sample can
be
measured with calipers prior to loading in the spectrophotometer.
Transmittance values can be
normalized to a thickness of 100 lam (or any thickness) according to:
f2 t2
FT¨corr(A, t2) -= [Cerf (A)t1 ]11 = [FT¨coõ(A, tl )] II )
where, ti=actual specimen thickness, t2=thickness to which transmittance
measurements can be
normalized.
In some embodiments, the biophotonic composition has a transparency or
translucency
that exceeds 15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% at
460 nm.
In some embodiments, the transparency exceeds 70% at 460 nm, 86% at 460 nm,
87% at 460
nm, 88% at 460 nm, 89% at 460 nm, 90% at 460 nm, 91% at 460 nm, 92% at 460 nm,
93% at
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,
460 nm, 94% at 460 nm, 95% at 460 nm, 96% at 460 nm, 97% at 460 nm, 98% at 460
nm or
99% at 460 nm.
The biophotonic compositions of the present disclosure are for topical uses.
These
compositions may be described based on the components making up the
composition.
Additionally or alternatively, the compositions of the present disclosure have
functional and
structural properties and these properties may also be used to define and
describe the
compositions. Individual components of the composition of the present
disclosure are detailed
as below.
(a) Chromophores
The biophotonic topical compositions of the present disclosure comprise at
least two
xanthene dyes as the chromophores. Combining xanthene dyes may increase photo-
absorption
by the combined dye molecules and enhance absorption and photobiomodulation
selectivity.
This creates multiple possibilities of generating new photosensitive, and/or
selective xanthene
dye mixtures.
When such multi-xanthene dye compositions are illuminated with light of an
appropriate wavelength to activate at least one of the xanthene dyes, energy
transfer can occur
between the xanthene dyes. This process, known as resonance energy transfer,
is a
photophysical process through which an excited 'donor' xanthene dye (also
referred to herein
as first xanthene dye) transfers its excitation energy to an 'acceptor'
xanthene dye (also
referred to herein as second xanthene dye). The efficiency and directedness of
resonance
energy transfer depends on the spectral features of donor and acceptor
xanthene dyes. In
particular, the flow of energy between xanthene dyes is dependent on a
spectral overlap
reflecting the relative positioning and shapes of the absorption and emission
spectra. For
energy transfer to occur the emission spectrum of the donor xanthene dye must
preferably
overlap with the absorption spectrum of the acceptor xanthene dye (Figure 3).
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)
Energy transfer manifests itself through decrease or quenching of the donor
emission
and a reduction of excited state lifetime accompanied also by an increase in
acceptor emission
intensity. Figure 4 is a Jablonski diagram that illustrates the coupled
transitions involved
between a donor emission and acceptor absorbance.
To enhance the energy transfer efficiency, the donor xanthene dye should have
good
abilities to absorb photons and emit photons. Furthermore, it is thought that
the more overlap
there is between the donor xanthene dye's emission spectra and the acceptor
xanthene dye's
absorption spectra, the better a donor xanthene dye can transfer energy to the
acceptor xanthene
dye.
In some embodiments, the first xanthene dye has an emission spectrum that
overlaps at
least about 80%, 50%, 40%, 30%, 20%, 10% with an absorption spectrum of the
xanthene dye
chromophore. In one embodiment, the first xanthene dye has an emission
spectrum that
overlaps at least about 20% with an absorption spectrum of the second xanthene
dye. In some
embodiments, the first xanthene dye has an emission spectrum that overlaps at
least 1-10%, 5-
15%, 10-20%, 15-25%, 20-30%, 25-35%, 30-40%, 35-45%, 50-60%, 55-65% or 60-70%
with
an absorption spectrum of the second xanthene dye.
% spectral overlap, as used herein, means the % overlap of a donor xanthene
dye's
emission wavelength range with an acceptor xanthene dye's absorption
wavelength rage,
measured at spectral full width quarter maximum (FWQM). For example, Figure 3
shows the
normalized absorption and emission spectra of donor and acceptor xanthene
dyes. The spectral
FWQM of the acceptor xanthene dye's absorption spectrum is from about 60 nm
(515 nm to
about 575 nm). The overlap of the donor xanthene dye's spectrum with the
absorption
spectrum of the acceptor xanthene dye is about 40 nm (from 515 nm to about 555
nm). Thus,
the % overlap can be calculated as 40nm / 60nm x 100 = 66.6%.
In some embodiments, the second xanthene dye absorbs at a wavelength in the
range of
the visible spectrum. In certain embodiments, the second xanthene dye has an
absorption
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i
wavelength that is relatively longer than that of the first xanthene dye
within the range of about
50-250, 25-150 or 10-100 nm.
As discussed above, the application of light to the compositions of the
present
disclosure can result in a cascade of energy transfer between the xanthene
dyes. In certain
embodiments, such a cascade of energy transfer provides photons that penetrate
the epidermis,
dermis and/or mucosa at the target tissue, including, such as, a site of
wound, or a tissue
afflicted with acne or another skin disorder. In some embodiments, such a
cascade of energy
transfer is not accompanied by concomitant generation of heat. In some other
embodiments,
the cascade of energy transfer does not result in tissue damage.
In some embodiments, the first xanthene dye absorbs at a wavelength in the
range of
the visible spectrum, such as at a wavelength of about 380-800 nm, 380-700, or
380-600 nm.
In other embodiments, the first xanthene dye absorbs at a wavelength of about
200-800 nm,
200-700 nm, 200-600 nm or 200-500 nm. In one embodiment, the first xanthene
dye absorbs
at a wavelength of about 200-600 nm. In some embodiments, the first xanthene
dye absorbs
light at a wavelength of about 200-300 nm, 250-350 nm, 300-400 nm, 350-450 nm,
400-500
nm, 400-600 nm, 450-650 nm, 600-700 nm, 650-750 nm or 700-800 nm.
It will be appreciated by those skilled in the art that optical properties of
a particular
xanthene dye may vary depending on the xanthene dye's surrounding medium.
Therefore, as
used herein, a particular xanthene dye's absorption and/or emission wavelength
(or spectrum)
corresponds to the wavelengths (or spectrum) measured in a biophotonic
composition of the
present disclosure.
Exemplary xanthene dyes include but are not limited to Eosin B (4',5'-
dibromo,21,7'-
dinitr- o-fluorescein, dianion); eosin Y; eosin Y (2',4',5',7'-tetrabromo-
fluoresc- ein, dianion);
eosin (2',4',5',7'-tetrabromo-fluorescein, dianion); eosin (2',4',5',7'-
tetrabromo-fluorescein,
dianion) methyl ester; eosin (2',4',5',7'-tetrabromo-fluorescein, monoanion) p-
isopropylbenzyl
ester; eosin derivative (2',7'-dibromo-fluorescein, dianion); eosin derivative
(4',5'-dibromo-
fluorescein, dianion); eosin derivative (2',7'-dichloro-fluorescein, dianion);
eosin derivative
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,
(4',5'-dichloro-fluorescein, dianion); eosin derivative (2',7'-diiodo-
fluorescein, dianion); eosin
derivative (4',5'-diiodo-fluorescein, dianion); eosin derivative (tribromo-
fluorescein, dianion);
eosin derivative (2',4',5',7'-tetrachlor- o-fluorescein, dianion); eosin;
eosin dicetylpyridinium
chloride ion pair; erythrosin B (2',4',5',7'-tetraiodo-fluorescein, dianion);
erythrosin; erythrosin
dianion; erythiosin B; fluorescein; fluorescein dianion; phloxin B
(2',4',5',7'-tetrabromo-
3,4,5,6-tetrachloro-fluorescein, dianion); phloxin B (tetrachloro-tetrabromo-
fluorescein);
phloxine B; rose bengal (3,4,5,6-tetrachloro-2',4',51,71-tetraiodofluorescein,
dianion); pyronin
G, pyronin J, pyronin Y; Rhodamine dyes such as rhodamines include 4,5-dibromo-
rhodamine
methyl ester; 4,5-dibromo-rhodamine n-butyl ester; rhodamine 101 methyl ester;
rhodamine
123; rhodamine 6G; rhodamine 6G hexyl ester; tetrabromo-rhodamine 123; and
tetramethyl-
rhodamine ethyl ester.
In certain embodiments, the first xanthene dye is present in an amount of
about 0.01-
40% per weight of the composition, and the second xanthene dye is present in
an amount of
about 0.001-40% per weight of the composition. In certain embodiments, the
total weight per
weight of xanthene dyes is in the amount of about 0.01-40.001% per weight of
the
composition. In certain embodiments, the first xanthene dye is present in an
amount of about
0.01-1%, 0.01-2%, 0.05-1%, 0.05-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%,
12.5-
17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%,
32.5-37.5%,
or 35-40% per weight of the composition. In certain embodiments, the second
xanthene dye is
present in an amount of about 0.001-1%, 0.001-2%, 0.001-0.01%, 0.01-0.1%, 0.1-
1.0%, 1-2%,
1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-
25%, 22.5-
27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weight of the
composition.
In certain embodiments, the total weight per weight of xanthene dyes is in the
amount of about
less than 0.5%, less than 0.1%, 0.001-0.1%, 0.01-1%, 0.01-2%, 0.05-2%, 0.001-
0.5%, 0.5-1%,
0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-
22.5%, 20-
25%, 22.5-27.5%, 25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40.05% per
weight of the
composition. All amounts are given as weight percentages per weight of the
total
concentration, and the equivalent weight or volume amounts.
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In certain embodiments, the ratio of the concentrations of the first and
second xanthene
dyes in the composition range from 1:1 to 1:1000. In certain embodiments, the
relative
concentration of Eosin Y: Fluorescein may be such that there is less Eosin Y
than Fluorescein
such as 1000:1 or 100:1 or 10:1 or 60-80%: 20-40%. In certain embodiments, the
ratio of Eosin
Y to Rose Bengal is 1:1 or 70-90%:10-30%. In certain embodiments, the ratio of
Fluorescein to
Eosin Y to Rose Bengal can be 20-40%: 30-60%: 10-20%. The ratio can be
tailored according
to the emitted light spectrum desired for a given treatment or therapy.
In some embodiments, the xanthene dye combinations are selected such that
their
emitted fluorescent light, on photoactivation, is within one or more of the
green, yellow,
orange, red and infrared portions of the electromagnetic spectrum, for example
having a peak
wavelength within the range of about 490 nm to about 800 nm. In certain
embodiments, the
emitted fluorescent light has a power density of between 0.005 to about 10
mW/cm2, about 0.5
to about 5 mW/cm2, or about 0.05 to about 2 mW/cm2.
Particularly useful combinations of xanthene dyes include but are not limited
to:
Fluorescein + Eosin Y; Fluorescein + Eosin Y + Rose Bengal; Fluorescein +
Eosin Y +
Phloxine B; Eosin Y + Rose Bengal; Eosin Y + Phloxine B; Eosin Y +
Erythrosine;
Fluorescein + Erythrosine B + Eosin Y; Eosin Y + Erythrosine B + Rose Bengal;
Eosin Y +
Erythrosine B + Phloxine B; Fluorescein + Eosin Y + Erythrosine B + Rose
Bengal; and
Fluorescein + Eosin Y + Erythrosine B + Phloxine B.
It is thought that at least some of these combinations have a synergistic
effect at certain
concentration ratios within the composition. For example, at certain
concentration ratios and
with an appropriate activating light, Eosin Y can transfer energy to Rose
Bengal, Erythrosin B
or Phloxine B when activated. This transferred energy is then emitted as
fluorescence and/or by
production of reactive oxygen species (such as singlet oxygen).
The synergistic effect may be apparent by the composition having a light
absorption
spectrum which spans a broader range of wavelengths compared to an individual
light
absorption spectrum of one of the individual chromophores in the composition,
when the
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individual chromophores and the composition are activated by the same
activating light (light
having substantially the same emission spectra). This may confer on the
composition the ability
to be activated by a broader range of activating light wavelengths, for
example by white light
avoiding the need for a precise wavelength of activating light.
The synergistic effect may also be evident through the composition having a
light
emission spectrum which spans a broader range of wavelengths compared to an
individual light
absorption spectrum of one of the individual chromophores in the composition,
when the
individual chromophores and the composition are activated by the same
activating light. This
absorbed and re-emitted light spectrum is thought to be transmitted throughout
the
composition, and also to be transmitted into the site of treatment. This
emitted spectrum will
then illuminate the target tissue with different penetration depths (Figure
1), which may confer
on the target tissue beneficial therapeutic effects. For example green light
has been reported to
have wound healing properties. By emitting a broader range of wavelengths, a
broader range of
therapeutic effects can be achieved. The emitted wavelength can be fine-tuned
using different
chromophore combinations and concentrations.
The synergistic effect may also be evident through the composition having a
higher
light absorption or emission peak compared to an individual light
absorption/emission peak of
one of the individual chromophores in the composition, when the individual
chromophores and
the composition are activated by the same activating light. The ability to
absorb and emit
higher levels of photons may have a therapeutic effect in certain
applications. Furthermore, less
concentration of an individual chromophore may be required to achieve a
certain power
density. Higher power densities can equate to shorter treatment times.
The synergistic effect may also be evident through the composition producing
more
oxygen species, in the presence of an oxygen-releasing agent, compared to
oxygen species
produced by an individual chromophores in the composition, when the individual
chromophores and the composition are activated by the same activating light.
The ability to
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produce higher levels of oxygen species without the need to extend treatment
time or increase
the power density of the activating light may be advantageous in certain
situations.
By means of synergistic effects of the xanthene dye combinations in the
composition,
xanthene dyes which cannot normally be activated by an activating light (such
as a blue light)
can be activated through energy transfer from xanthene dyes which are
activated by the
activating light. In this way, the different properties of photoactivated
xanthene dyes can be
harnessed and tailored according to the cosmetic or the medical therapy
required.
For example, Rose Bengal can generate a high yield of singlet oxygen when
photoactivated in the presence of molecular oxygen, however it has a low
quantum yield in
terms of emitted fluorescent light. Rose Bengal has a peak absorption around
540 nm and so is
normally activated by green light. Eosin Y has a high fluorescence quantum
yield and can be
activated by blue light. By combining Rose Bengal with Eosin Y, one obtains a
composition
which can emit therapeutic fluorescent light and generate singlet oxygen when
activated by
blue light. In this case, the blue light is thought to photoactivate Eosin Y
which transfers some
of its energy to Rose Bengal as well as emitting some energy as fluorescence.
One or more of the chromophores may photobleach during illumination. This can
be a
visible confirmation of 'dose' delivery. As the chromophores photobleach, they
emit less
fluorescence over time. At the same time, they also absorb less of the
activating light over time
and so the tissues receive increasingly higher amounts of the activating
light. In this way, the
chromophores modulate exposure of the tissue to the light which may provide a
somewhat
protective effect.
(b) Additional Chromophores
In addition to the xanthene dye combination, the biophotonic topical
compositions of
the present disclosure may also include, but are not limited to the following:
Chlorophyll dyes
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Exemplary chlorophyll dyes include but are not limited to chlorophyll a;
chlorophyll b;
oil soluble chlorophyll; bacteriochlorophyll a; bacteriochlorophyll b;
bacteriochlorophyll c;
bacteriochlorophyll d; protochlorophyll; protochlorophyll a; amphiphilic
chlorophyll derivative
1; and amphiphilic chlorophyll derivative 2.
Methylene blue dyes
Exemplary methylene blue derivatives include but are not limited to 1-methyl
methylene blue; 1,9-dimethyl methylene blue; methylene blue; methylene blue
(16 µM);
methylene blue (14 µM); methylene violet; bromomethylene violet; 4-
iodomethylene violet;
1,9-dimethy1-3-dimethyl-amino-7-diethyl-a- mino-phenothiazine; and 1,9-
dimethy1-3-
diethylamino-7-dibutyl-amino-phenot- hiazine.
Azo dyes
Exemplary azo (or diazo-) dyes include but are not limited to methyl violet,
neutral red,
para red (pigment red 1), amaranth (Azorubine S), Carmoisine (azorubine, food
red 3, acid red
14), allura red AC (FD&C 40), tartrazine (FD&C Yellow 5), orange G (acid
orange 10),
Ponceau 4R (food red 7), methyl red (acid red 2), and murexide-ammonium
purpurate.
In some aspects of the disclosure, the additional chromophores of the
biophotonic
composition disclosed herein can be independently selected from any of Acid
black 1, Acid
blue 22, Acid blue 93, Acid fuchsin, Acid green, Acid green 1, Acid green 5,
Acid magenta,
Acid orange 10, Acid red 26, Acid red 29, Acid red 44, Acid red 51, Acid red
66, Acid red 87,
Acid red 91, Acid red 92, Acid red 94, Acid red 101, Acid red 103, Acid
roseine, Acid rubin,
Acid violet 19, Acid yellow 1, Acid yellow 9, Acid yellow 23, Acid yellow 24,
Acid yellow 36,
Acid yellow 73, Acid yellow S, Acridine orange, Acriflavine, Alcian blue,
Alcian yellow,
Alcohol soluble eosin, Alizarin, Alizarin blue 2RC, Alizarin carmine, Alizarin
cyanin BBS,
Alizarol cyanin R, Alizarin red S, Alizarin purpurin, Aluminon, Amido black
10B,
Amidoschwarz, Aniline blue WS, Anthracene blue SWR, Auramine 0, Azocannine B,
Azocarmine G, Azoic diazo 5, Azoic diazo 48, Azure A, Azure B, Azure C, Basic
blue 8, Basic
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blue 9, Basic blue 12, Basic blue 15, Basic blue 17, Basic blue 20, Basic blue
26, Basic brown
1, Basic fuchsin, Basic green 4, Basic orange 14, Basic red 2 (Saffranin 0),
Basic red 5, Basic
red 9, Basic violet 2, Basic violet 3, Basic violet 4, Basic violet 10, Basic
violet 14, Basic
yellow 1, Basic yellow 2, Biebrich scarlet, Bismarck brown Y, Brilliant
crystal scarlet 6R,
Calcium red, Carmine, Carminic acid (acid red 4), Celestine blue B, China
blue, Cochineal,
Coelestine blue, Chrome violet CG, Chromotrope 2R, Chromoxane cyanin R, Congo
corinth,
Congo red, Cotton blue, Cotton red, Croceine scarlet, Crocin, Crystal ponceau
6R, Crystal
violet, Dahlia, Diamond green B, Di0C6, Direct blue 14, Direct blue 58, Direct
red, Direct red
10, Direct red 28, Direct red 80, Direct yellow 7, Eosin B, Eosin Bluish,
Eosin, Eosin Y, Eosin
yellowish, Eosinol, Erie garnet B, Eriochrome cyanin R, Erythrosin B, Ethyl
eosin, Ethyl
green, Ethyl violet, Evans blue, Fast blue B, Fast green FCF, Fast red B, Fast
yellow,
Fluorescein, Food green 3, Gallein, Gallamine blue, Gallocyanin, Gentian
violet, Haematein,
Haematine, Haematoxylin, Helio fast rubin BBL, Helvetia blue, Hematein,
Hematine,
Hematoxylin, Hoffman's violet, Imperial red, Indocyanin green, Ingrain blue,
Ingrain blue 1,
Ingrain yellow 1, INT, Kermes, Kermesic acid, Kernechtrot, Lac, Laccaic acid,
Lauth's violet,
Light green, Lissamine green SF, Luxol fast blue, Magenta 0, Magenta I,
Magenta II, Magenta
III, Malachite green, Manchester brown, Martius yellow, Merbromin,
Mercurochrome, Metanil
yellow, Methylene azure A, Methylene azure B, Methylene azure C, Methylene
blue, Methyl
blue, Methyl green, Methyl violet, Methyl violet 2B, Methyl violet 10B,
Mordant blue 3,
Mordant blue 10, Mordant blue 14, Mordant blue 23, Mordant blue 32, Mordant
blue 45,
Mordant red 3, Mordant red 11, Mordant violet 25, Mordant violet 39 Naphthol
blue black,
Naphthol green B, Naphthol yellow S, Natural black 1, Natural red, Natural red
3, Natural red
4, Natural red 8, Natural red 16, Natural red 25, Natural red 28, Natural
yellow 6, NBT,
Neutral red, New fuchsin, Niagara blue 3B, Night blue, Nile blue, Nile blue A,
Nile blue
oxazone, Nile blue sulphate, Nile red, Nitro BT, Nitro blue tetrazolium,
Nuclear fast red, Oil
red 0, Orange G, Orcein, Pararosanilin, Phloxine B, phycobilins, Phycocyanins,
Phycoerythrins. Phycoerythrincyanin (PEC), Phthalocyanines, Picric acid,
Ponceau 2R,
Ponceau 6R, Ponceau B, Ponceau de Xylidine, Ponceau S, Primula, Purpurin,
Pyronin B,
Pyronin G, Pyronin Y, Rhodamine B, Rosanilin, Rose bengal, Saffron, Safranin
0, Scarlet R,
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Scarlet red, Scharlach R, Shellac, Sirius red F3B, Solochrome cyanin R,
Soluble blue, Solvent
black 3, Solvent blue 38, Solvent red 23, Solvent red 24, Solvent red 27,
Solvent red 45,
Solvent yellow 94, Spirit soluble eosin, Sudan III, Sudan IV, Sudan black B,
Sulfur yellow S,
Swiss blue, Tartrazine, Thioflavine S, Thioflavine T, Thionin, Toluidine blue,
Toluyline red,
Tropaeolin G, Trypaflavine, Trypan blue, Uranin, Victoria blue 4R, Victoria
blue B, Victoria
green B, Water blue I, Water soluble eosin, Xylidine ponceau, or Yellowish
eosin.
In certain embodiments, the composition of the present disclosure includes any
of the
additional chromophores listed above in addition to the xanthene dyes, or a
combination
thereof, so as to provide a biophotonic impact at the application site. This
is a distinct
application of these agents and differs from the use of chromophores as simple
stains or as a
catalyst for photo-polymerization.
Chromophores can be selected, for example, on their emission wavelength
properties in
the case of fluorophores, on the basis of their energy transfer potential,
their ability to generate
reactive oxygen species, or their antimicrobial effect. These needs may vary
depending on the
condition requiring treatment. For example, chlorophylls may have an
antimicrobial effect on
bacteria found on the face.
(c) Gelling Agent
The composition may optionally comprise a gelling agent. A gelling agent for
use
according to the present disclosure may comprise any ingredient suitable for
use in a topical
biophotonic formulation as described herein. The gelling agent may be an agent
capable of
forming a cross-linked matrix, including physical and/or chemical cross-links.
The gelling
agent is preferably biocompatible, and may be biodegradable. In some
embodiments, the
gelling agent is able to form a hydrogel or a hydrocolloid. An appropriate
gelling agent is one
that can form a viscous liquid or a semisolid. In preferred embodiments, the
gelling agent
and/or the composition has appropriate light transmission properties. It is
also important to
select a gelling agent which will allow biophotonic activity of the
chromophores. For example,
some chromophores require a hydrated environment in order to fluoresce. The
gelling agent
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may be able to form a gel by itself or in combination with other ingredients
such as water or
another gelling agent, or when applied to a treatment site, or when
illuminated with light.
In some embodiments the composition is in the form of a gel, cream, ointment,
lotion,
paste, spray or foam.
The gelling agent according to various embodiments of the present disclosure
may
comprise polyalkylene oxides, particularly polyethylene glycol and
poly(ethylene oxide)-
poly(propylene oxide) copolymers, including block and random copolymers;
polyols such as
glycerol, polyglycerol (particularly highly branched polyglycerol), propylene
glycol and
trimethylene glycol substituted with one or more polyalkylene oxides, e.g.,
mono-, di- and tri-
polyoxyethylated glycerol, mono- and di-polyoxy-ethylated propylene glycol,
and mono- and
di-polyoxyethylated trimethylene glycol; polyoxyethylated sorbitol,
polyoxyethylated glucose;
acrylic acid polymers and analogs and copolymers thereof, such as polyacrylic
acid per se,
polymethacrylic acid,
poly(hydroxyethylmethacrylate), poly(hydroxyethylacrylate),
poly(methylalkylsulfoxide methacrylate), poly(methylalkylsulfoxide acrylate)
and copolymers
of any of the foregoing, and/or with additional acrylate species such as
aminoethyl acrylate and
mono-2-(acryloxy)-ethyl succinate; polymaleic acid; poly(acrylamides) such as
polyacrylamide
per se, poly(methacrylamide), poly(dimethylacrylamide), and poly(N-isopropyl-
acrylamide);
poly(olefinic alcohol)s such as poly(vinyl alcohol); poly(N-vinyl lactams)
such as poly(vinyl
pyrrolidone), poly(N-vinyl caprolactam), and copolymers thereof,
polyoxazolines, including
poly(methyloxazoline) and poly(ethyloxazoline); and polyvinylamines.
The gelling agent according to certain embodiments of the present disclosure
may
comprise a polymer selected from any of synthetic or semi-synthetic polymeric
materials,
polyacrylate copolymers, cellulose derivatives and polymethyl vinyl
ether/maleic anhydride
copolymers. In some embodiments, the hydrophilic polymer comprises a polymer
that is a high
molecular weight (i.e., molar masses of more than about 5,000, and in some
instances, more
than about 10,000, or 100,000, or 1,000,000) and/or cross-linked polyacrylic
acid polymer. In
some embodiments, the polymer is a polyacrylic acid polymer and has a
viscosity in the range
of about 15,000-100,000, 15,000-90,000, 15,000-80,000, 20,000-80,000, 20,000-
70,000,
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20,000-40,000 cP. In certain embodiment, the polymer is a high molecular
weight, and/or
cross-linked polyacrylic acid polymer, where the polyacrylic acid polymer has
a viscosity in
the range of about 15,000-80,000 cP.
Carbomers may be used. Carbomers are synthetic high molecular weight polymer
of
acrylic acid that are crosslinked with either allylsucrose or allylethers of
pentaerythritol having
a molecular weight of about 3 x 106. The gelation mechanism depends on
neutralization of the
carboxylic acid moiety to form a soluble salt. The polymer is hydrophilic and
produces
sparkling clear gels when neutralized. Carbomer gels possess good thermal
stability in that gel
viscosity and yield value are essentially unaffected by temperature. As a
topical product,
carbomer gels possess optimum rheological properties. The inherent
pseudoplastic flow
permits immediate recovery of viscosity when shear is terminated and the high
yield value and
quick break make it ideal for dispensing. Aqueous solution of Carbopol is
acidic in nature
due to the presence of free carboxylic acid residues. Neutralization of this
solution cross-links
and gelatinizes the polymer to form a viscous integral structure of desired
viscosity.
Carbomers are available as fine white powders which disperse in water to form
acidic
colloidal suspensions (a 1% dispersion has approx. pH 3) of low viscosity.
Neutralization of
these suspensions using a base, for example sodium, potassium or ammonium
hydroxides, low
molecular weight amines and alkanolamines, results in the formation of
translucent gels.
Nicotine salts such as nicotine chloride form stable water-soluble complexes
with carbomers at
about pH 3.5 and are stabilized at an optimal pH of about 5.6.
In some embodiments of the disclosure, the carbomer is Carbopol. Such polymers
are
commercially available from B.F. Goodrich or Lubrizol under the designation
Carbopol 71G
NF, 420, 430, 475, 488, 493, 910, 934, 934P, 940, 971PNF, 974P NF, 980 NF, 981
NF and the
like. Carbopols are versatile controlled-release polymers, as described by
Brock
(Pharmacotherapy, 14:430-7 (1994)) and Durrani (Pharmaceutical Res. (Supp.)
8:S-135
(1991)), and belong to a family of carbomers which are synthetic, high
molecular weight, non-
linear polymers of acrylic acid, crosslinked with polyalkenyl polyether. In
some embodiments,
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the carbomer is Carbopol0 974P NF, 980 NF, 5984 EP, ETD 2020NF, Ultrez 10 NF,
934 NF,
934P NF or 940 NF. In certain embodiments, the carbomer is Carbopol 980 NF,
ETD 2020
NF, Ultrez 10 NF, Ultrez 21 or 1382 Polymer, 1342 NF, 940 NF.
In certain embodiments, the gelling agent comprises a hygroscopic material. By
hygroscopic material is meant a substance capable of taking up water, for
example, by
absorption or adsorption even at relative humidity as low as 50%, at room
temperature (e.g.
about 25 C). The hygroscopic material may include, but is not limited to,
glucosamine,
glycosaminoglycan, poly(vinyl alcohol), poly(2-hydroxyethylmethylacrylate),
polyethylene
oxide, collagen, chitosan, alginate, a poly(acrylonitrile)-based hydrogel,
poly(ethylene
glycol)/poly(acrylic acid) interpenetrating polymer network hydrogel,
polyethylene oxide-
polybutylene terephthalate, hyaluronic acid, high-molecular-weight polyacrylic
acid,
poly(hydroxy ethylmethacrylate), poly(ethylene glycol), tetraethylene glycol
diacrylate,
polyethylene glycol methacrylate, and poly(methyl acrylate-co-hydroxyethyl
acrylate).
The biophotonic composition of the present disclosure may be further
encapsulated, e.g,
in a membrane. Such a membrane may be transparent, and/or substantially, or
fully
impermeable. The membrane may be impermeable to liquid but permeable to gases
such as air.
In certain embodiments, the composition may form a membrane that encapsulates
the
chromophore(s) of the biophotonic topical composition, where the membrane may
be
substantially impermeable to liquid and/or gas.
The composition may include any other carrier.
(d) Oxygen-releasing agents
According to certain embodiments, the compositions of the present disclosure
may
optionally further comprise an oxygen-releasing agent, for example, as a
source of oxygen.
When a biophotonic composition of the present disclosure comprising an oxygen-
releasing agent is illuminated with light, the xanthene dyes are excited to a
higher energy state.
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When the xanthene dyes' electrons return to a lower energy state, they emit
photons with a
lower energy level, thus causing the emission of light of a longer wavelength
(Stokes' shift). In
the proper environment, some of this energy release is transferred to oxygen
or the reactive
hydrogen peroxide and causes the formation of oxygen radicals, such as singlet
oxygen. The
singlet oxygen and other reactive oxygen species generated by the activation
of the biophotonic
composition are thought to operate in a hormetic fashion. That is, a health
beneficial effect that
is brought about by the low exposure to a normally toxic stimuli (e.g.
reactive oxygen), by
stimulating and modulating stress response pathways in cells of the targeted
tissues.
Endogenous response to exogenous generated free radicals (reactive oxygen
species) is
modulated in increased defense capacity against the exogenous free radicals
and induces
acceleration of healing and regenerative processes. Furthermore, activation of
the composition
can also produce an antibacterial effect. The extreme sensitivity of bacteria
to exposure to free
radicals makes the composition of the present disclosure a de facto
bactericidal composition.
As stated above, the generation of oxygen species by the composition in some
embodiments is accompanied by the micro-bubbling which can contribute to
debridement or
dislodging of biofilm at the site of application. This can allow for the
improved penetration of
the activating and/or fluorescence light to the treatment site for example to
deactivate bacterial
colonies leading to their reduction in number.
Suitable oxygen-releasing agents that may be included in the composition
include, but
are not limited to peroxides such as hydrogen peroxide, urea hydrogen peroxide
and benzoyl
peroxide. Peroxide compounds are oxygen-releasing agents that contain the
peroxy group (R-
0-0-R), which is a chainlike structure containing two oxygen atoms, each of
which is bonded
to the other and a radical or some element.
Hydrogen peroxide (H202) is the starting material to prepare organic
peroxides. H202 is
a powerful oxygen-releasing agent, and the unique property of hydrogen
peroxide is that it
breaks down into water and oxygen and does not form any persistent, toxic
residual compound.
Hydrogen peroxide for use in this composition can be used in a gel, for
example with 6%
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hydrogen peroxide. A suitable range of concentration over which hydrogen
peroxide can be
used in the present composition is from about 0.1% to about 6%.
Urea hydrogen peroxide (also known as urea peroxide, carbamide peroxide or
percarbamide) is soluble in water and contains approximately 35% hydrogen
peroxide.
Carbamide peroxide for use in this composition can be used as a gel, for
example with 16%
carbamide peroxide that represents 5.6 % hydrogen peroxide, or 12 % carbamide
peroxide. A
suitable range of concentration over which urea peroxide can be used in the
present
composition is from about 0.3% to about 16%. Urea peroxide breaks down to urea
and
hydrogen peroxide in a slow-release fashion that can be accelerated with heat
or photochemical
reactions. The released urea [carbamide, (NH2)CO2)l, is highly soluble in
water and is a
powerful protein denaturant. It increases solubility of some proteins and
enhances rehydration
of the skin and/or mucosa.
Benzoyl peroxide consists of two benzoyl groups (benzoic acid with the H of
the
carboxylic acid removed) joined by a peroxide group. It is found in treatments
for acne, in
concentrations varying from 2.5% to 10%. The released peroxide groups are
effective at killing
bacteria. Benzoyl peroxide also promotes skin turnover and clearing of pores,
which further
contributes to decreasing bacterial counts and reduce acne. Benzoyl peroxide
breaks down to
benzoic acid and oxygen upon contact with skin, neither of which is toxic. A
suitable range of
concentration over which benzoyl peroxide can be used in the present
composition is from
about 2.5% to about 5%.
Other oxygen-releasing agents include molecular oxygen, water, perbonates and
carbonates. Oxygen-releasing agents can be provided in powder, liquid or gel
form within the
composition. The composition may include an amount of oxygen-releasing agent,
which is
augmented by the separate application of oxygen-releasing agents to the
treatment site.
Alternatively, oxygen-releasing agents may also be applied to the tissue site
separately
to the composition.
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(e) Healing Factors
The composition of the present disclosure may comprise healing factors.
Healing
factors comprise compounds that promote or enhance the healing or regenerative
process of the
tissues on the application site of the composition. During the photoactivation
of the
composition of the present disclosure, there is an increase of the absorption
of molecules at the
treatment site by the skin, wound or the mucosa. An augmentation in the blood
flow at the site
of treatment is observed for an extent period of time. An increase in the
lymphatic drainage and
a possible change in the osmotic equilibrium due to the dynamic interaction of
the free radical
cascades can be enhanced or even fortified with the inclusion of healing
factors. Suitable
healing factors include, but are not limited to:
Hyaluronic acid (Hyaluronan, hyaluronate): is a non-sulfated
glycosaminoglycan,
distributed widely throughout connective, epithelial and neural tissues. It is
one of the primary
components of the extracellular matrix, and contributes significantly to cell
proliferation and
migration. Hyaluronan is a major component of the skin, where it is involved
in tissue repair.
While it is abundant in extracellular matrices, it contributes to tissues
hydrodynamics,
movement and proliferation of cells and participates in a wide number of cell
surface receptor
interactions, notably those including primary receptor CD44. The
hyaluronidases enzymes
degrade hyaluronan. There are at least seven types of hyaluronidase-like
enzymes in humans,
several of which are tumor suppressors. The degradation products of hyaluronic
acid, the
oligosaccharides and the very-low molecular weight hyaluronic acid, exhibit
pro-angiogenic
properties. In addition, recent studies show that hyaluronan fragments, but
not the native high
molecular mass of hyaluronan, can induce inflammatory responses in macrophages
and
dendritic cells in tissue injury. Hyaluronic acid is well suited to biological
applications
targeting the skin. Due to its high biocompatibility, it is used to stimulate
tissue regeneration.
Studies have shown hyaluronic acid appearing in the early stages of healing to
physically
create room for white blood cells that mediate the immune response. It is used
in the synthesis
of biological scaffolds for wound healing applications and in wrinkle
treatment. A suitable
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range of concentration over which hyaluronic acid can be used in the present
composition is
from about 0.001% to about 3%.
Glucosamine: is one of the most abundant monosaccharides in human tissues and
a
precursor in the biological synthesis of glycosilated proteins and lipids. It
is commonly used in
the treatment of osteoarthritis. The common form of glucosamine used is its
sulfate salt.
Glucosamine shows a number of effects including an anti-inflammatory activity,
stimulation of
the synthesis of proteoglycans and the synthesis of proteolytic enzymes. A
suitable range of
concentration over which glucosamine can be used in the present composition is
from about
0.01% to about 3%.
Allantoin: is a diureide of glyosilic acid. It has keratolytic effect,
increases the water
content of the extracellular matrix, enhances the desquamation of the upper
layers of dead
(apoptotic) skin cells, and promotes skin proliferation and wound healing.
(f) Antimicrobials
The composition of the present disclosure may comprise antimicrobial agents.
Antimicrobials kill microbes or inhibit their growth or accumulation.
Exemplary
antimicrobials (or antimicrobial agent) are recited in U.S. Patent Application
Publications
20040009227 and 20110081530. Suitable antimicrobials for use in the methods of
the present
disclosure include, but not limited to, phenolic and chlorinated phenolic and
chlorinated
phenolic compounds, resorcinol and its derivatives, bisphenolic compounds,
benzoic esters
(parabens), halogenated carbonilides, polymeric antimicrobial agents,
thazolines,
trichloromethylthioimides, natural antimicrobial agents (also referred to as
"natural essential
oils"), metal salts, and broad-spectrum antibiotics.
Specific phenolic and chlorinated phenolic antimicrobial agents that can be
used in the
disclosure include, but are not limited to: phenol; 2-methyl phenol; 3-methyl
phenol; 4-methyl
phenol; 4-ethyl phenol; 2,4-dimethyl phenol; 2,5-dimethyl phenol; 3,4-dimethyl
phenol; 2,6-
dimethyl phenol; 4-n-propyl phenol; 4-n-butyl phenol; 4-n-amyl phenol; 4-tert-
amyl phenol; 4-
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n-hexyl phenol; 4-n-heptyl phenol; mono- and poly-alkyl and aromatic
halophenols; p-
chlorophenyl; methyl p-chlorophenol; ethyl p-chlorophenol; n-propyl p-
chlorophenol; n-butyl
p-chlorophenol; n-amyl p-chlorophenol; sec-amyl p-chlorophenol; n-hexyl p-
chlorophenol;
cyclohexyl p-chlorophenol; n-heptyl p-chlorophenol; n-octyl; p-chlorophenol; o-
chlorophenol;
methyl o-chlorophenol; ethyl o-chlorophenol; n-propyl o-chlorophenol; n-butyl
o-
chlorophenol; n-amyl o-chlorophenol; tert-amyl o-chlorophenol; n-hexyl o-
chlorophenol; n-
heptyl o-chlorophenol; o-benzyl p-chlorophenol; o-benxyl-m-methyl p-
chlorophenol; o-benzyl-
m,m-dimethyl p-chlorophenol; o-phenylethyl p-chlorophenol; o-phenylethyl-m-
methyl p-
chlorophenol; 3-methyl p-chlorophenol 3,5-dimethyl p-chlorophenol, 6-ethyl-3-
methyl p-
chlorophenol, 6-n-propy1-3-methyl p-chlorophenol; 6-iso-propy1-3-methyl p-
chlorophenol; 2-
ethy1-3,5-dimethyl p-chlorophenol; 6-sec-butyl-3-methyl p-chlorophenol; 2-iso-
propy1-3,5-
dimethyl p-chlorophenol; 6-diethylmethy1-3-methyl p-chlorophenol; 6-iso-propy1-
2-ethy1-3-
methyl p-chlorophenol; 2-sec-amyl-3,5-dimethyl p-chlorophenol; 2-diethylmethy1-
3,5-
dimethyl p-chlorophenol; 6-sec-octy1-3-methyl p-chlorophenol; p-chloro-m-
cresol p-
bromophenol; methyl p-bromophenol; ethyl p-bromophenol; n-propyl p-
bromophenol; n-butyl
p-bromophenol; n-amyl p-bromophenol; sec-amyl p-bromophenol; n-hexyl p-
bromophenol;
cyclohexyl p-bromophenol; o-bromophenol; tert-amyl o-bromophenol; n-hexyl o-
bromophenol; n-propyl-m,m-dimethyl o-bromophenol; 2-phenyl phenol; 4-chloro-2-
methyl
phenol; 4-chloro-3-methyl phenol; 4-chloro-3,5-dimethyl phenol; 2,4-dichloro-
3,5-
dimethylphenol; 3,4,5 ,6-tetabromo-2-meth ylphenol- ; 5-methyl-2-pentylphenol;
4-isopropyl-3 -
methylphenol; para-chloro-metaxylenol (PCMX); chlorothymol; phenoxyethanol;
phenoxyisopropanol; and 5-chloro-2-hydroxydiphenylmethane.
Resorcinol and its derivatives can also be used as antimicrobial agents.
Specific
resorcinol derivatives include, but are not limited to: methyl resorcinol;
ethyl resorcinol; n-
propyl resorcinol; n-butyl resorcinol; n-amyl resorcinol; n-hexyl resorcinol;
n-heptyl
resorcinol; n-octyl resorcinol; n-nonyl resorcinol; phenyl resorcinol; benzyl
resorcinol;
phenylethyl resorcinol; phenylpropyl resorcinol; p-chlorobenzyl resorcinol; 5-
chloro-2,4-
dihydroxydiphenyl methane; 4'-chloro-2,4-dihydroxydiphenyl methane; 5-bromo-
2,4-
dihydroxydiphenyl methane; and 4'-bromo-2,4-dihydroxydiphenyl methane.
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Specific bisphenolic antimicrobial agents that can be used in the disclosure
include, but
are not limited to: 2,2'-methylene bis-(4-chlorophenol); 2,4,4'trichloro-2'-
hydroxy-diphenyl
ether, which is sold by Ciba Geigy, Florham Park, N.J. under the tradename
Triclosan0; 2,2'-
methylene bis-(3,4,6-trichlorophenol); 2,2'-methylene bis-(4-chloro-6-
bromophenol); bis-(2-
hydroxy-3,5-dichlorop- henyl) sulphide; and bis-(2-hydroxy-5-
chlorobenzyl)sulphide.
Specific benzoie esters (parabens) that can be used in the disclosure include,
but are not
limited to: methylparaben; propylparaben; butylparaben; ethylparaben;
isopropylparaben;
isobutylparaben; benzylparaben; sodium methylparaben; and sodium
propylparaben.
Specific halogenated carbanilides that can be used in the disclosure include,
but are not
limited to: 3,4,4'-trichlorocarbanilides, such as 3-(4-chloropheny1)-1-(3,4-
dichlorphenyl)urea
sold under the tradename Triclocarban by Ciba-Geigy, Florham Park, N.J.; 3-
trifluoromethy1-
4,4'-dichlorocarbanilide; and 3,3',4-trichlorocarbanilide.
Specific polymeric antimicrobial agents that can be used in the disclosure
include, but
are not limited to: polyhexamethylene biguanide hydrochloride; and
poly(iminoimidocarbonyl
iminoimidocarbonyl iminohexamethylene hydrochloride), which is sold under the
tradename
Vantocil IB.
Specific thazolines that can be used in the disclosure include, but are not
limited to that
sold under the tradename Micro-Check ; and 2-n-octy1-4-isothiazolin-3-one,
which is sold
under the tradename Vinyzene IT-3000 DIDP.
Specific trichloromethylthioimides that can be used in the disclosure include,
but are
not limited to: N-(trichloromethylthio)phthalimide, which is sold under the
tradename
Fungitrol ; and N-trichloromethylthio-4-cyclohexene-1,2-dicarboximide, which
is sold under
the tradename Vancide .
Specific natural antimicrobial agents that can be used in the disclosure
include, but are
not limited to, oils of: anise; lemon; orange; rosemary; wintergreen; thyme;
lavender; cloves;
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hops; tea tree; citronella; wheat; barley; lemongrass; cedar leaf; cedarwood;
cinnamon;
fleagrass; geranium; sandalwood; violet; cranberry; eucalyptus; vervain;
peppermint; gum
benzoin; basil; fennel; fir; balsam; menthol; ocmea origanuin; hydastis;
carradensis;
Berberidaceac daceae; Ratanhiae longa; and Curcuma longa. Also included in
this class of
natural antimicrobial agents are the key chemical components of the plant oils
which have been
found to provide antimicrobial benefit. These chemicals include, but are not
limited to: anethol;
catechole; camphene; thymol; eugenol; eucalyptol; ferulic acid; farnesol;
hinokitiol; tropolone;
limonene; menthol; methyl salicylate; carvacol; terpineol; verbenone;
berberine; ratanhiae
extract; caryophellene oxide; citronellic acid; curcumin; nerolidol; and
geraniol.
Specific metal salts that can be used in the disclosure include, but are not
limited to,
salts of metals in groups 3a-5a, 3b-7b, and 8 of the periodic table. Specific
examples of metal
salts include, but are not limited to, salts of: aluminum; zirconium; zinc;
silver; gold; copper;
lanthanum; tin; mercury; bismuth; selenium; strontium; scandium; yttrium;
cerium;
praseodymiun; neodymium; promethum; samarium; europium; gadolinium; terbium;
dysprosium; holmium; erbium; thalium; ytterbium; lutetium; and mixtures
thereof. An example
of the metal-ion based antimicrobial agent is sold under the tradename
HealthShield , and is
manufactured by HealthShield Technology, Wakefield, Mass. [give other examples
here e.g.
smith and nephew]
Specific broad-spectrum antimicrobial agents that can be used in the
disclosure include,
but are not limited to, those that are recited in other categories of
antimicrobial agents herein.
Additional antimicrobial agents that can be used in the methods of the
disclosure
include, but are not limited to: pyrithiones, and in particular pyrithione-
including zinc
complexes such as that sold under the tradename Octopirox,O;
dimethyidimethylol hydantoin,
which is sold under the tradename Glydant ; methylchloroisothiazolinone/
methylisothiazolinone, which is sold under the tradename Kathon CG ; sodium
sulfite;
sodium bisulfite; imidazolidinyl urea, which is sold under the tradename
Germall 115 ;
diazolidinyl urea, which is sold under the tradename Germall 11C); benzyl
alcohol v2-bromo-2-
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nitropropane-1,3-diol, which is sold under the tradename BronopolO; formalin
or
formaldehyde; iodopropenyl butylcarbamate, which is sold under the tradename
Polyphase
P100 ; chloroacetamide; methanamine; methyldibromonitrile glutaronitrile (1,2-
dibromo-2,4-
dicyanobutane), which is sold under the tradename Tektamer0; glutaraldehyde; 5-
bromo-5-
nitro-1,3-dioxane, which is sold under the tradename Bronidoxia; phenethyl
alcohol; o-
phenylphenol/sodium o-phenylphenol sodium hydroxymethylglycinate, which is
sold under the
tradename Suttocide AC); polymethoxy bicyclic oxazolidine; which is sold under
the tradename
Nuosept CC); dimethoxane; thimersal; dichlorobenzyl alcohol; captan;
chlorphenenesin;
dichlorophene; chlorbutanol; glyceryl laurate; halogenated diphenyl ethers;
2,4,4'-trichloro-2'-
hydroxy-diphenyl ether, which is sold under the tradename Triclosan and is
available from
Ciba-Geigy, Florham Park, N.J.; and 2,2'-dihydroxy-5,5'-dibromo-diphenyl
ether.
Additional antimicrobial agents that can be used in the methods of the
disclosure
include those disclosed by U.S. Pat. Nos. 3,141,321; 4,402,959; 4,430,381;
4,533,435;
4,625,026; 4,736,467; 4,855,139; 5,069,907; 5,091,102; 5,639,464; 5,853,883;
5,854,147;
5,894,042; and 5,919,554, and U.S. Pat. Appl. Publ. Nos. 20040009227 and
20110081530.
(g) Collagens and Agents that Promote Collagen Synthesis
The compositions of the present disclosure may include collagens and agents
that
promote collagen synthesis. Collagen is a fibrous protein produced in dermal
fibroblast cells
and forming 70% of the dermis. Collagen is responsible for the smoothing and
firming of the
skin. Therefore, when the synthesis of collagen is reduced, skin aging will
occur, and so the
firming and smoothing of the skin will be rapidly reduced. As a result, the
skin will be flaccid
and wrinkled. On the other hand, when metabolism of collagen is activated by
the stimulation
of collagen synthesis in the skin, the components of dermal matrices will be
increased, leading
to effects, such as wrinkle improvement, firmness improvement and skin
strengthening. Thus,
collagens and agents that promote collagen synthesis may also be useful in the
present
disclosure. Agents that promote collagen synthesis (i.e., pro-collagen
synthesis agents) include
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amino acids, peptides, proteins, lipids, small chemical molecules, natural
products and extracts
from natural products.
For instance, it was discovered that intake of vitamin C, iron, and collagen
can
effectively increase the amount of collagen in skin or bone. See, e.g., U.S.
Patent Application
Publication 20090069217. Examples of the vitamin C include an ascorbic acid
derivative such
as L-ascorbic acid or sodium L-ascorbate, an ascorbic acid preparation
obtained by coating
ascorbic acid with an emulsifier or the like, and a mixture containing two or
more of those
vitamin Cs at an arbitrary rate. In addition, natural products containing
vitamin C such as
acerola and lemon may also be used. Examples of the iron preparation include:
an inorganic
iron such as ferrous sulfate, sodium ferrous citrate, or ferric pyrophosphate;
an organic iron
such as heme iron, ferritin iron, or lactoferrin iron; and a mixture
containing two or more of
those irons at an arbitrary rate. In addition, natural products containing
iron such as spinach or
liver may also be used. Moreover, examples of the collagen include: an extract
obtained by
treating bone, skin, or the like of a mammal such as bovine or swine with an
acid or alkaline; a
peptide obtained by hydrolyzing the extract with a protease such as pepsine,
trypsin, or
chymotrypsin; and a mixture containing two or more of those collagens at an
arbitrary rate.
Collagens extracted from plant sources may also be used.
Additional pro-collagen synthesis agents are described, for example, in U.S.
Patent
Patents 7598291, 7722904, 6203805 , 5529769, etc, and U.S. Patent Application
Publications
20060247313, 20080108681, 20110130459, 20090325885, 20110086060, etc.
(4) Methods of Use
The biophotonic compositions of the present disclosure have numerous uses.
Without
being bound by theory, the biophotonic compositions of the present disclosure
may promote
wound healing or tissue repair. The biophotonic compositions of the present
disclosure may
also be used to treat a skin disorder. The biophotonic compositions of the
present disclosure
may also be used to treat acne. The biophotonic compositions of the present
disclosure may
also be used for skin rejuvenation. The biophotonic compositions of the
present disclosure may
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also be used for treating acute inflammation. Therefore, it is an objective of
the present
disclosure to provide a method for providing biophotonic therapy to a wound,
where the
method promotes wound healing. It is also an objective of the present
disclosure to provide a
method for providing biophotonic therapy to a skin tissue afflicted with acne,
wherein the
method is used to treat acne. It is also an objective of the present
disclosure to provide a
method for providing biophotonic therapy to a skin tissue afflicted with a
skin disorder,
wherein the method is used to treat the skin disorder. It is also an objective
of the present
disclosure to provide a method for providing biophotonic therapy to skin
tissue, wherein the
method is used for promoting skin rejuvenation.
In certain embodiments, the present disclosure provides a method for providing
a
biophotonic therapy to a wound, the method comprising: applying (e.g., by
topical application)
a biophotonic composition of the present disclosure to a site of a wound, and
illuminating the
biophotonic composition with light having a wavelength that overlaps with an
absorption
spectrum of the first xanthene dye (e.g., donor xanthene dye) of the
biophotonic composition.
In yet another aspect, the present disclosure provides a method for promoting
skin
rejuvenation. In certain embodiments, the present disclosure provides a method
for providing
skin rejuvenation, the method comprising: applying (e.g., by topical
application) a biophotonic
composition of the present disclosure to the skin, and illuminating the
biophotonic composition
with light having a wavelength that overlaps with an absorption spectrum of
the first xanthene
dye (e.g., donor xanthene dye) of the biophotonic composition.
In yet another aspect, the present disclosure provides a method for providing
biophotonic therapy to a target skin tissue afflicted with a skin disorder. In
certain
embodiments, the present disclosure provides a method for providing a
biophotonic therapy to
a target skin tissue, the method comprising: applying (e.g., by topical
application) a
biophotonic composition of the present disclosure to a target skin tissue, and
illuminating the
biophotonic composition with light having a wavelength that overlaps with an
absorption
spectrum of the first xanthene dye (e.g., donor xanthene dye) of the
biophotonic composition.
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In yet another aspect, the present disclosure provides a method for providing
biophotonic therapy to a target skin tissue afflicted with acne. In certain
embodiments, the
present disclosure provides a method for providing a biophotonic therapy to a
target skin tissue
afflicted with acne, the method comprising: applying (e.g., by topical
application) a
biophotonic composition of the present disclosure to a target skin tissue, and
illuminating the
biophotonic composition with light having a wavelength that overlaps with an
absorption
spectrum of the first xanthene dye (e.g., donor xanthene dye) of the
biophotonic composition.
In other embodiments, the present disclosure provides a method for treating
acute
inflammation, the method comprising: topically applying a biophotonic
composition of the
present disclosure to a target skin tissue with acute inflammation, and
illuminating the
biophotonic composition with light having a wavelength that overlaps with an
absorption
spectrum of the first xanthene dye (e.g., donor xanthene dye) of the
biophotonic composition.
The biophotonic compositions suitable for use in the methods of the present
disclosure
may be selected from any of the embodiments of the biophotonic compositions
described
above. For instance, the biophotonic compositions useful in the method of the
present
disclosure may comprise a first xanthene dye that undergoes at least partial
photobleaching
upon application of light. The first xanthene dye may absorb at a wavelength
of about 200-800
nm, 200-700 nm, 200-600 nm or 200-500 nm. In one embodiment, the first
xanthene dye
absorbs at a wavelength of about 200-600 nm. In some embodiments, the first
xanthene dye
absorbs light at a wavelength of about 200-300 nm, 250-350 nm, 300-400 nm, 350-
450 nm,
400-500 nm, 450-650 nm, 600-700 nm, 650-750 nm or 700-800 nm. The absorption
spectrum
of the second xanthene dye should overlap at least about 80%, 50%, 40%, 30%,
or 20% with
the emission spectrum of the first xanthene dye. In some embodiments, the
first xanthene dye
has an emission spectrum that overlaps at least 1-10%, 5-15%, 10-20%, 15-25%,
20-30%, 25-
35%, 30-40%, 35-45%, 50-60%, 55-65% or 60-70% with an absorption spectrum of
the second
xanthene dye.
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Illumination of the biophotonic composition with light may cause a transfer of
energy
from the first xanthene dye to the second xanthene dye. Subsequently, the
second xanthene dye
may emit energy as fluorescence and/or generate reactive oxygen species. In
certain
embodiments of the methods the present disclosure, energy transfer caused by
the application
of light is not accompanied by concomitant generation of heat, or does not
result in tissue
damage.
The biophotonic compositions useful for the present methods can be formulated
with
any carrier. In certain embodiments, the carrier is a gelling agent. The
gelling agent may
include, but is not limited to, lipids such as glycerin, glycols such as
propylene glycol,
hyaluronic acid, glucosamine sulfate, cellulose derivatives (hydroxypropyl
methylcellulose,
hydroxyethyl cellulose, hydroxypropyl cellulose, methylcellulose and the
like), noncellulose
polysaccharides (galactomannans, guar gum, carob gum, gum arabic, sterculia
gum, agar,
alginates and the like) and acrylic acid polymers.
In the methods of the present disclosure, any source of actinic light can be
used. Any
type of halogen, LED or plasma arc lamp or laser may be suitable. The primary
characteristic
of suitable sources of actinic light will be that they emit light in a
wavelength (or wavelengths)
appropriate for activating the one or more chromophores present in the
composition. In one
embodiment, an argon laser is used. In another embodiment, a potassium-titanyl
phosphate
(KTP) laser (e.g. a GreenLightTM laser) is used. In another embodiment,
sunlight may be used.
In yet another embodiment, a LED photocuring device is the source of the
actinic light. In yet
another embodiment, the source of the actinic light is a source of light
having a wavelength
between about 200 to 800 nm. In another embodiment, the source of the actinic
light is a source
of visible light having a wavelength between about 400 and 600 nm.
Furthermore, the source of
actinic light should have a suitable power density. Suitable power density for
non-collimated
light sources (LED, halogen or plasma lamps) are in the range from about 1
mW/cm2 to about
200 mW/cm2. Suitable power density for laser light sources are in the range
from about 0.5
mW/cm2 to about 0.8 mW/cm2.
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In some embodiments of the methods of the present disclosure, the light has an
energy
at the subject's skin, wound or mucosa surface of between about 1 mW/cm2 and
about 500
mW/cm2, 1-300 mW/cm2, or 1-200 mW/cm2, wherein the energy applied depends at
least on
the condition being treated, the wavelength of the light, the distance of the
subject's skin from
the light source, and the thickness of the biophotonic compsoition. In certain
embodiments, the
light at the subject's skin is between about 1-40 mW/cm2, or 20-60 mW/cm2, or
40-80
mW/cm2, or 60-100 mW/cm2, or 80-120 mW/cm2, or 100-140 mW/cm2, or 120-160
mW/cm2,
or 140-180 mW/cm2, or 160-200 mW/cm2, or 110-240 mW/cm2, or 110-150 mW/cm2, or
190-
240 mW/cm2.
In some embodiments, a mobile device can be used to activate embodiments of
the
biophotonic composition of the present disclosure, wherein the mobile device
can emit light
having an emission spectra which overlaps an absorption spectra of the donor
xanthene dye in
the biophotonic composition. The mobile device can have a display screen
through which the
light is emitted and/or the mobile device can emit light from a flashlight
which can
photoactivate the biophotonic composition.
In some embodiments, a display screen on a television or a computer monitor
can be
used to activate the biophotonic composition, wherein the display screen can
emit light having
an emission spectra which overlaps an absorption spectra of the donor xanthene
dye in the
biophotonic composition.
In certain embodiments, the first and/or the second xanthene dye can be
photoactivated
by ambient light which may originate from the sun or other light sources.
Ambient light can be
considered to be a general illumination that comes from all directions in a
room that has no
visible source. In certain embodiments, the first and/or the second xanthene
dye can be
photoactivated by light in the visible range of the electromagnetic spectrum.
Exposure times to
ambient light may be longer than that to direct light.
In certain embodiments, different sources of light can be used to activate the
biophotonic compositions, such as a combination of ambient light and direct
LED light.
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The duration of the exposure to actinic light required will be dependent on
the surface
of the treated area, the type of lesion, trauma or injury that is being
treated, the power density,
wavelength and bandwidth of the light source, the thickness of the biophotonic
composition,
and the treatment distance from the light source. The illumination of the
treated area by
fluorescence may take place within seconds or even fragment of seconds, but a
prolonged
exposure period is beneficial to exploit the synergistic effects of the
absorbed, reflected and
reemitted light on the composition of the present disclosure and its
interaction with the tissue
being treated. In one embodiment, the time of exposure to actinic light of the
tissue, skin or
wound on which the biophotonic composition has been applied is a period
between 1 minute
and 5 minutes. In another embodiment, the time of exposure to actinic light of
the tissue, skin
or wound on which the biophotonic composition has been applied is a period
between 1 minute
and 5 minutes. In some other embodiments, the biophotonic composition is
illuminated for a
period between 1 minute and 3 minutes. In certain embodiments, light is
applied for a period
of 1-30 seconds, 15-45 seconds, 30-60 seconds, 0.75-1.5 minutes, 1-2 minutes,
1.5-2.5
minutes, 2-3 minutes, 2.5-3.5 minutes, 3-4 minutes, 3.5-4.5 minutes, 4-5
minutes, 5-10
minutes, 10-15 minutes, 15-20 minutes, 20-25 minutes, or 20-30 minutes. In yet
another
embodiment, the source of actinic light is in continuous motion over the
treated area for the
appropriate time of exposure. In yet another embodiment, multiple applications
of the
biophotonic composition and actinic light are performed. In some embodiments,
the tissue, skin
or wound is exposed to actinic light at least two, three, four, five or six
times. In some
embodiments, a fresh application of the biophotonic composition is applied
before exposure to
actinic light.
In the methods of the present disclosure, the biophotonic composition may be
optionally removed from the site of treatment following application of light.
In certain
embodiments, the biophotonic composition is left on the treatment site for
more than 30
minutes, more than one hour, more than 2 hours, more than 3 hours. It can be
illuminated with
ambient light. To prevent drying, the composition can be covered with a
transparent or
translucent cover such as a polymer film, or an opaque cover which can be
removed before
illumination.
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(5) Wounds and Wound Healing
The biophotonic compositions and methods of the present disclosure may be used
to
treat wounds and promote wound healing. Wounds that may be treated by the
biophotonic
compositions and methods of the present disclosure include, for example,
injuries to the skin
and subcutaneous tissue initiated in different ways (e.g., pressure ulcers
from extended bed
rest, wounds induced by trauma, wounds induced by conditions such as
periodontitis) and with
varying characteristics. In certain embodiments, the present disclosure
provides biophotonic
compositions and methods for treating and/or promoting the healing of, for
example, burns,
incisions, excisions, lacerations, abrasions, puncture or penetrating wounds,
surgical wounds,
contusions, hematomas, crushing injuries, gun shots, sores and ulcers.
Biophotonic compositions and methods of the present disclosure may be used to
treat
and/or promote the healing of chronic cutaneous ulcers or wounds, which are
wounds that have
failed to proceed through an orderly and timely series of events to produce a
durable structural,
functional, and cosmetic closure. The vast majority of chronic wounds can be
classified into
three categories based on their etiology: pressure ulcers, neuropathic
(diabetic foot) ulcers and
vascular (venous or arterial) ulcers.
In certain other embodiments, the present disclosure provides biophotonic
compositions
and methods for treating and/or promoting healing, Grade I-IV ulcers. In
certain embodiments,
the application provides compositions suitable for use with Grade II ulcers in
particular.
Ulcers may be classified into one of four grades depending on the depth of the
wound: i) Grade
I: wounds limited to the epithelium; ii) Grade II: wounds extending into the
dermis; iii) Grade
III: wounds extending into the subcutaneous tissue; and iv) Grade IV (or full-
thickness
wounds): wounds wherein bones are exposed (e.g., a bony pressure point such as
the greater
trochanter or the sacrum).
For example, the present disclosure provides biophotonic compositions and
methods for
treating and/or promoting healing of a diabetic ulcer. Diabetic patients are
prone to foot and
other ulcerations due to both neurologic and vascular complications.
Peripheral neuropathy can
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cause altered or complete loss of sensation in the foot and/or leg. Diabetic
patients with
advanced neuropathy lose all ability for sharp-dull discrimination. Any cuts
or trauma to the
foot may go completely unnoticed for days or weeks in a patient with
neuropathy. A patient
with advanced neuropathy loses the ability to sense a sustained pressure
insult, as a result,
tissue ischemia and necrosis may occur leading to for example, plantar
ulcerations.
Microvascular disease is one of the significant complications for diabetics
which may also lead
to ulcerations. In certain embodiments, compositions and methods of treating a
chronic wound
are provided here in, where the chronic wound is characterized by diabetic
foot ulcers and/or
ulcerations due to neurologic and/or vascular complications of diabetes.
In other examples, the present disclosure provides biophotonic compositions
and
methods for treating and/or promoting healing of a pressure ulcer. Pressure
ulcer includes bed
sores, decubitus ulcers and ischial tuberosity ulcers and can cause
considerable pain and
discomfort to a patient. A pressure ulcer can occur as a result of a prolonged
pressure applied
to the skin. Thus, pressure can be exerted on the skin of a patient due to the
weight or mass of
an individual. A pressure ulcer can develop when blood supply to an area of
the skin is
obstructed or cut off for more than two or three hours. The affected skin area
can turns red,
becomes painful and can become necrotic. If untreated, the skin breaks open
and can become
infected. An ulcer sore is therefore a skin ulcer that occurs in an area of
the skin that is under
pressure from e.g. lying in bed, sitting in a wheelchair, and/or wearing a
cast for a prolonged
period of time. Pressure ulcer can occur when a person is bedridden,
unconscious, unable to
sense pain, or immobile. Pressure ulcer often occur in boney prominences of
the body such as
the buttocks area (on the sacrum or iliac crest), or on the heels of a foot.
In other examples, the present disclosure provides biophotonic compositions
and
methods for treating and/or promoting healing of acute wounds.
Additional types of wound that can be treated by the biophotonic compositions
and
methods of the present disclosure include those disclosed by U.S. Pat. Appl.
Publ. No.
20090220450, which is incorporated herein by reference.
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Wound healing in adult tissues is a complicated reparative process. For
example, the
healing process for skin involves the recruitment of a variety of specialized
cells to the site of
the wound, extracellular matrix and basement membrane deposition,
angiogenesis, selective
protease activity and re-epithelialization.
There are three distinct phases in the wound healing process. First, in the
inflammatory
phase, which typically occurs from the moment a wound occurs until the first
two to five days,
platelets aggregate to deposit granules, promoting the deposit of fibrin and
stimulating the
release of growth factors. Leukocytes migrate to the wound site and begin to
digest and
transport debris away from the wound. During this inflammatory phase,
monocytes are also
converted to macrophages, which release growth factors for stimulating
angiogenesis and the
production of fibroblasts.
Second, in the proliferative phase, which typically occurs from two days to
three weeks,
granulation tissue forms, and epithelialization and contraction begin.
Fibroblasts, which are key
cell types in this phase, proliferate and synthesize collagen to fill the
wound and provide a
strong matrix on which epithelial cells grow. As fibroblasts produce collagen,
vascularization
extends from nearby vessels, resulting in granulation tissue. Granulation
tissue typically grows
from the base of the wound. Epithelialization involves the migration of
epithelial cells from the
wound surfaces to seal the wound. Epithelial cells are driven by the need to
contact cells of like
type and are guided by a network of fibrin strands that function as a grid
over which these cells
migrate. Contractile cells called myofibroblasts appear in wounds, and aid in
wound closure.
These cells exhibit collagen synthesis and contractility, and are common in
granulating
wounds.
Third, in the remodeling phase, the final phase of wound healing which can
take place
from three weeks up to several years, collagen in the scar undergoes repeated
degradation and
re-synthesis. During this phase, the tensile strength of the newly formed skin
increases.
However, as the rate of wound healing increases, there is often an associated
increase in
scar formation. Scarring is a consequence of the healing process in most adult
animal and
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human tissues. Scar tissue is not identical to the tissue which it replaces,
as it is usually of
inferior functional quality. The types of scars include, but are not limited
to, atrophic,
hypertrophic and keloidal scars, as well as scar contractures. Atrophic scars
are flat and
depressed below the surrounding skin as a valley or hole. Hypertrophic scars
are elevated scars
that remain within the boundaries of the original lesion, and often contain
excessive collagen
arranged in an abnormal pattern. Keloidal scars are elevated scars that spread
beyond the
margins of the original wound and invade the surrounding normal skin in a way
that is site
specific, and often contain whorls of collagen arranged in an abnormal
fashion.
In contrast, normal skin consists of collagen fibers arranged in a basket-
weave pattern,
which contributes to both the strength and elasticity of the dermis. Thus, to
achieve a smoother
wound healing process, an approach is needed that not only stimulates collagen
production, but
also does so in a way that reduces scar formation.
The biophotonic compositions and methods of the present disclosure promote the
wound healing by promoting the formation of substantially uniform
epithelialization;
promoting collagen synthesis; promoting controlled contraction; and/or by
reducing the
formation of scar tissue. In certain embodiments, the biophotonic compositions
and methods of
the present disclosure may promote wound healing by promoting the formation of
substantially
uniform epithelialization. In some embodiments, the biophotonic compositions
and methods of
the present disclosure promote collagen synthesis. In some other embodiments,
the biophotonic
compositions and methods of the present disclosure promote controlled
contraction. In certain
embodiments, the biophotonic compositions and methods of the present
disclosure promote
wound healing, for example, by reducing the formation of scar tissue or by
speeding up the
wound closure process. In certain embodiments, the biophotonic compositions
and methods of
the present disclosure promote wound healing, for example, by reducing
inflammation. In
certain embodiments, the biophotonic composition can be used following wound
closure to
optimize scar revision. In this case, the biophotonic composition may be
applied at regular
intervals such as once a week, or at an interval deemed appropriate by the
physician.
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The biophotonic composition may be soaked into a woven or non-woven material
or a
sponge and applied as a wound dressing. A light source, such as LEDs or
waveguides, may be
provided within or adjacent the wound dressing or the composition to
illuminate the
composition. The waveguides can be optical fibres which can transmit light,
not only from
their ends, but also from their body. For example, made of polycarbonate or
polymeth ylmethacryl ate.
Adjunct therapies which may be topical or systemic such as antibiotic
treatment may
also be used. Negative pressure assisted wound closure can also be used to
assist wound
closure and/or to remove the composition.
(6) Acne and Acne Scars
The biophotonic compositions and methods of the present disclosure may be used
to
treat acne. As used herein, "acne" means a disorder of the skin caused by
inflammation of skin
glands or hair follicles. The biophotonic compositions and methods of the
disclosure can be
used to treat acne at early pre-emergent stages or later stages where lesions
from acne are
visible. Mild, moderate and severe acne can be treated with embodiments of the
biophotonic
compositions and methods. Early pre-emergent stages of acne usually begin with
an excessive
secretion of sebum or dermal oil from the sebaceous glands located in the
pilosebaceous
apparatus. Sebum reaches the skin surface through the duct of the hair
follicle. The presence of
excessive amounts of sebum in the duct and on the skin tends to obstruct or
stagnate the normal
flow of sebum from the follicular duct, thus producing a thickening and
solidification of the
sebum to create a solid plug known as a comedone. In the normal sequence of
developing acne,
hyperkeratinazation of the follicular opening is stimulated, thus completing
blocking of the
duct. The usual results are papules, pustules, or cysts, often contaminated
with bacteria, which
cause secondary infections. Acne is characterized particularly by the presence
of comedones,
inflammatory papules, or cysts. The appearance of acne may range from slight
skin irritation to
pitting and even the development of disfiguring scars. Accordingly, the
biophotonic
compositions and methods of the present disclosure can be used to treat one or
more of skin
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irritation, pitting, development of scars, comedones, inflammatory papules,
cysts,
hyperkeratinazation, and thickening and hardening of sebum associated with
acne.
The composition may be soaked into or applied to a woven or non-woven material
or a
sponge and applied as a mask to body parts such as the face, body, arms, legs
etc. A light
source, such as LEDs or waveguides, may be provided within or adjacent the
mask or the
composition to illuminate the composition. The waveguides can be optical
fibres which can
transmit light, not only from their ends, but also from their body. For
example, made of
polycarbonate or polymethylmethacrylate.
The biophotonic compositions and methods of the present disclosure may be used
to
treat various types of acne. Some types of acne include, for example, acne
vulgaris, cystic acne,
acne atrophica, bromide acne, chlorine acne, acne conglobata, acne cosmetica,
acne
detergicans, epidemic acne, acne estivalis, acne fulminans, halogen acne, acne
indurata, iodide
acne, acne keloid, acne mechanica, acne papulosa, pomade acne, premenstral
acne, acne
pustulosa, acne scorbutica, acne scrofulosorum, acne urticata, acne
varioliformis, acne
venenata, propionic acne, acne excoriee, gram negative acne, steroid acne, and
nodulocystic
acne.
(7) Skin Aging and Rejuvenation
The dermis is the second layer of skin, containing the structural elements of
the skin,
the connective tissue. There are various types of connective tissue with
different functions.
Elastin fibers give the skin its elasticity, and collagen gives the skin its
strength.
The junction between the dermis and the epidermis is an important structure.
The
dermal-epidermal junction interlocks forming finger-like epidermal ridges. The
cells of the
epidermis receive their nutrients from the blood vessels in the dermis. The
epidermal ridges
increase the surface area of the epidermis that is exposed to these blood
vessels and the needed
nutrients.
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The aging of skin comes with significant physiological changes to the skin.
The
generation of new skin cells slows down, and the epidermal ridges of the
dermal-epidermal
junction flatten out. While the number of elastin fibers increases, their
structure and coherence
decrease. Also the amount of collagen and the thickness of the dermis decrease
with the ageing
of the skin.
Collagen is a major component of the skin's extracellular matrix, providing a
structural
framework. During the aging process, the decrease of collagen synthesis and
insolubilization of
collagen fibers contribute to a thinning of the dermis and loss of the skin's
biomechanical
properties.
The physiological changes to the skin result in noticeable aging symptoms
often
referred to as chronological-, intrinsic- and photo-ageing. The skin becomes
drier, roughness
and scaling increase, the appearance becomes duller, and most obviously fine
lines and
wrinkles appear. Other symptoms or signs of skin aging include, but are not
limited to, thinning
and transparent skin, loss of underlying fat (leading to hollowed cheeks and
eye sockets as well
as noticeable loss of firmness on the hands and neck), bone loss (such that
bones shrink away
from the skin due to bone loss, which causes sagging skin), dry skin (which
might itch),
inability to sweat sufficiently to cool the skin, unwanted facial hair,
freckles, age spots, spider
veins, rough and leathery skin, fine wrinkles that disappear when stretched,
loose skin, a
blotchy complexion.
The dermal-epidermal junction is a basement membrane that separates the
keratinocytes
in the epidermis from the extracellular matrix, which lies below in the
dermis. This membrane
consists of two layers: the basal lamina in contact with the keratinocytes,
and the underlying
reticular lamina in contact with the extracellular matrix. The basal lamina is
rich in collagen
type IV and laminin, molecules that play a role in providing a structural
network and
bioadhesive properties for cell attachment.
Laminin is a glycoprotein that only exists in basement membranes. It is
composed of
three polypeptide chains (alpha, beta and gamma) arranged in the shape of an
asymmetric cross
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and held together by disulfide bonds. The three chains exist as different
subtypes which result
in twelve different isoforms for laminin, including Laminin-1 and Laminin-5.
The dermis is anchored to hemidesmosomes, specific junction points located on
the
keratinocytes, which consist of a-integrins and other proteins, at the basal
membrane
keratinocytes by type VII collagen fibrils. Laminins, and particularly Laminin-
5, constitute the
real anchor point between hemidesmosomal transmembrane proteins in basal
keratinocytes and
type VII collagen.
Laminin-5 synthesis and type VII collagen expression have been proven to
decrease in
aged skin. This causes a loss of contact between dermis and epidermis, and
results in the skin
losing elasticity and becoming saggy.
Recently another type of wrinkles generally referred to as expression
wrinkles, got
general recognition. These wrinkles require loss of resilience, particularly
in the dermis,
because of which the skin is no longer able to resume its original state when
facial muscles
which produce facial expressions exert stress on the skin, resulting in
expression wrinkles.
The compositions and methods of the present disclosure promote skin
rejuvenation. In
certain embodiments, the compositions and methods of the present disclosure
promote collagen
synthesis. In certain other embodiments, the compositions and methods of the
present
disclosure may reduce, diminish, retard or even reverse one or more signs of
skin aging
including, but not limited to, appearance of fine lines or wrinkles, thin and
transparent skin,
loss of underlying fat (leading to hollowed cheeks and eye sockets as well as
noticeable loss of
firmness on the hands and neck), bone loss (such that bones shrink away from
the skin due to
bone loss, which causes sagging skin), dry skin (which might itch), inability
to sweat
sufficiently to cool the skin, unwanted facial hair, freckles, age spots,
spider veins, rough and
leathery skin, fine wrinkles that disappear when stretched, loose skin, or a
blotchy complexion.
In certain embodiments, the compositions and methods of the present disclosure
may induce a
reduction in pore size, enhance sculpturing of skin subsections, and/or
enhance skin
translucence.
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(8) Skin Disorders
The biophotonic compositions and methods of the present disclosure may be used
to
treat skin disorders that include, but are not limited to, erythema,
telangiectasia, actinic
telangiectasia, psoriasis, skin cancer, pemphigus, sunburn, dermatitis,
eczema, rashes,
impetigo, lichen simplex chronicus, rhinophyma, perioral dermatitis,
pseudofolliculitis barbae,
drug eruptions, erythema multiforme, erythema nodosum, granuloma annulare,
actinic
keratosis, purpura, alopecia areata, aphthous stomatitis, drug eruptions, dry
skin, chapping,
xerosis, ichthyosis vulgaris, fungal infections, parasitic infection, viral
infections, herpes
simplex, intertrigo, keloids, keratoses, milia, moluscum contagiosum,
pityriasis rosea, pruritus,
urticaria, and vascular tumors and malformations. Dermatitis includes contact
dermatitis, atopic
dermatitis, seborrheic dermatitis, nummular dermatitis, generalized
exfoliative dermatitis, and
statis dermatitis. Skin cancers include melanoma, basal cell carcinoma, and
squamous cell
carcinoma.
Some skin disorders present various symptoms including redness, flushing,
burning,
scaling, pimples, papules, pustules, comedones, macules, nodules, vesicles,
blisters,
telangiectasia, spider veins, sores, surface irritations or pain, itching,
inflammation, red, purple,
or blue patches or discolorations, moles, and/or tumors. Accordingly, the
biophotonic
compositions and methods of the present disclosure can be used to treat
redness, flushing,
burning, scaling, pimples, papules, pustules, comedones, macules, nodules,
vesicles, blisters,
telangiectasia, spider veins, sores, surface irritations or pain, itching,
acute inflammation, red,
purple, or blue patches or discolorations, moles, and/or tumors. Acute
inflammation can
present itself as pain, heat, redness, swelling and loss of function. It
includes those seen in
allergic reactions such as insect bites e.g.; mosquito, bees, wasps, poison
ivy, post-ablative
treatment.
The composition may be soaked into or applied to a woven or non-woven material
or a
sponge and applied as a mask to body parts to treat skin disorders. A light
source, such as
LEDs or waveguides, may be provided within or adjacent the mask or the
composition to
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illuminate the composition. The waveguides can be optical fibres which can
transmit light, not
only from their ends, but also from their body. For example, made of
polycarbonate or
polymethylmethacrylate.
(9) Kits
The present disclosure also provides kits for preparing and/or applying any of
the
compositions of the present disclosure. The kit may include a biophotonic
topical composition
of the present disclosure. The composition may include an oxygen-releasing
agent present in
amount about 0.01% - 40%, 0.01% - 1.0%, 0.5% - 10.0%, 5% - 15%, 10% - 20%, 15%
- 25%,
20% - 30%, 15.0% - 25%, 20% - 30%, 25% - 35%, or 30% - 40% by weight to weight
of the
composition. The first xanthene dye may be present in an amount of about 0.01-
40% per
weight of the composition, and a second xanthene dye may be present in an
amount of about
0.01-40% per weight of the composition. In certain embodiments, the first
xanthene dye is
present in an amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-
10%, 7.5-
12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%,
27.5-32.5%,
30-35%, 32.5-37.5%, or 35-40% per weight of the composition. In certain
embodiments, the
second xanthene dye is present in an amount of about 0.001-0.1%, 0.05-1%, 0.5-
2%, 1-5%,
2.5-7.5%, 5-10%, 7.5-12.5%, 10-15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%,
22.5-27.5%,
25-30%, 27.5-32.5%, 30-35%, 32.5-37.5%, or 35-40% per weight of the
composition. In
certain embodiments, the amount of xanthene dyes may be in the amount of about
0.05-40.05%
per weight of the composition. In certain embodiments, the amount of xanthene
dyes may be in
the amount of about 0.001-0.1%, 0.05-1%, 0.5-2%, 1-5%, 2.5-7.5%, 5-10%, 7.5-
12.5%, 10-
15%, 12.5-17.5%, 15-20%, 17.5-22.5%, 20-25%, 22.5-27.5%, 25-30%, 27.5-32.5%,
30-35%,
32.5-37.5%, or 35-40.05% per weight of the composition.
In some embodiments, the kit includes more than one composition, for example,
a first
and a second composition. The first composition may include the oxygen-
releasing agent and
the second composition may include the xanthene dyes in a liquid or as a
powder. In some
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embodiments, the kit includes containers comprising the compositions of the
present
disclosure.
The composition (s) may be contained in containers. The containers may be
light
impermeable, air-tight and/or leak resistant. Exemplary containers include,
but are not limited
to, syringes, vials, or pouches. For example, the container may be a dual-
chamber syringe
where the contents of the chambers mix on expulsion of the compositions from
the chambers.
In another example, the pouch may include two chambers separated by a
frangible membrane.
In another example, one component may be contained in a syringe and injectable
into a
container comprising the second component. The container may be a spray can
which may or
may not be pressurized. The composition may be in liquid and/or gaseous form.
The biophotonic composition may also be provided in a container comprising one
or
more chambers for holding one or more components of the biophotonic
composition, and an
outlet in communication with the one or more chambers for discharging the
biophotonic
composition from the container.
In other embodiments, the kit comprises a systemic or topical drug for
augmenting the
treatment of the composition. For example, the kit may include a systemic or
topical antibiotic
or hormone treatment for acne treatment or wound healing.
Written instructions on how to use the biophotonic composition in accordance
with the
present disclosure may be included in the kit, or may be included on or
associated with the
containers comprising the compositions of the present disclosure.
In certain embodiments, the kit may comprise a further component which is a
dressing.
The dressing may be a porous or semi-porous structure for receiving the
biophotonic
composition. The dressing may comprise woven or non-woven fibrous materials.
In certain embodiments of the kit, the kit may further comprise a light source
such as a
portable light with a wavelength appropriate to activate the chromophore in
the biophotonic
composition. The portable light may be battery operated or re-chargeable.
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In certain embodiments, the kit may further comprise one or more waveguides.
Identification of equivalent compositions, methods and kits are well within
the skill of
the ordinary practitioner and would require no more than routine
experimentation, in light of
the teachings of the present disclosure. Practice of the disclosure will be
still more fully
understood from the following examples, which are presented herein for
illustration only and
should not be construed as limiting the disclosure in any way.
EXAMPLES
The examples below are given so as to illustrate the practice of various
embodiments of
the present disclosure. They are not intended to limit or define the entire
scope of this
disclosure.
Example 1 ¨ Absorption/emission spectra of Fluorescein and Eosin Y in a gel
The photodynamic properties of (i) Fluorescein sodium salt at about 0.09
mg/mL, (ii) Eosin Y
at about 0.305 mg/mL, and (iii) a mixture of Fluorescein sodium salt at about
0.09 mg/mL and
Eosin Y at about 0.305 mg/mL, all in a gel (comprising about 12% carbamide
peroxide), were
evaluated. A flexstation 384 II spectrophotometer was used to measure emitted
fluorescence
with the following parameters: mode fluorescence, excitation 460 nm, emission
spectra 465-
750 nm. The absorbance was read using a synergy HT microplate reader: mode
absorbance;
spectra between 300-650nm.
The absorption and emission spectra are shown in Figures 5A and 5B which
indicate
an energy transfer between the chromophores in the combination. In particular
a broader
absorption and emission spectra was achieved with the Eosin Y and chromophore
combination,
compared with the individual chromophores. This means that the multiple
chromophore
composition can be activated with a broader bandwidth of light, and that the
multiple
chromophore light can emit a broader bandwidth of light after illumination. In
other words,
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emission from the multi-chromophore composition occured in a broader range of
wavelengths
compared to the individual chromophores. In this example, the composition
emitted light in the
green, yellow and orange wavelengths of the visible spectra. Photobleaching of
Eosin Y was
observed during illumination. Furthermore, results (not shown) indicate that
the presence of
peroxide in the gel does not affect the absorbance and emission spectra.
Peroxide is optional in
compositions and methods of the present disclosure.
Example 2 - Absorption/emission spectra of a Fluorescein and Eosin Y aqueous
solution
The photodynamic properties of (i) Fluorescein sodium salt at 0.18 mg/mL final
concentration,
(ii) Eosin Y at about 0.305 mg/mL, and (iii) a mixture of Fluorescein sodium
salt at about 0.18
mg/mL and Eosin Y at about 0.305 mg/mL, all in an aqueous solution were
evaluated. A
flexstation 384 II spectrophotometer was used to measure emitted fluorescence
with the
following parameters: mode fluorescence, excitation 460 nm, emission spectra
465-750 nm.
The absorbance was read using a synergy HT microplate reader: mode absorbance;
spectra
between 300-650nm.
The absorption and emission spectra are shown in Figures 6A and 6B which
indicate
an energy transfer between the chromophores in the combination. Also, as with
Figures 5A
and 5B, a broader emission spectra was achieved with the Eosin Y and
chromophore
combination, compared with the individual chromophores. The composition
emitted light in the
green, yellow and orange wavelengths of the visible spectra. The difference in
the absorption
and emission spectra between Examples 1 and 2 may be explained by the optical
difference in
the media (gel in Example 1 and aqueous solution in this example) as well as
possibly the
effect of doubling the fluorescein concentration. It can be seen that adding
Fluorescein to Eosin
Y, broadens the bandwidth of the absorption and emission peaks of Eosin Y.
This confers on
the multiple chromophore combination, the ability to absorb a broader range of
wavelengths
for photoactivation and to emit a wider range of wavelengths which may confer
different
therapeutic effects at the same time. Photobleaching of Eosin Y was observed
during
illumination.
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Example 3 - Absorption/emission spectra of Phloxine B and Eosin Y in a gel
The photodynamic properties of (i) Phloxine B at 0.25mg/mL final
concentration, (ii) Eosin Y
at about 0.05 mg/mL, and (iii) a mixture of Phloxine B (0.25mg/mL) and Eosin Y
(0.05
mg/mL), all in a 12% carbamide gel were evaluated. A flexstation 384 II
spectrophotometer
was used to measure emitted fluorescence with the following parameters: mode
fluorescence,
excitation 460 nm, emission spectra 465-750 nm. The absorbance was read using
a synergy HT
microplate reader: mode absorbance; spectra between 300-650nm.
The absorption and emission spectra are shown in Figures 7A and 7B which
indicate
an energy transfer between the chromophores in the combination. As before,
broader
absorption and emission spectra were achieved with the Phloxine B and Eosin Y
chromophore
combination, compared with the individual chromophores. The composition
emitted light in the
green, yellow, orange and red wavelengths of the visible spectra.
Example 4 - Absorption/emission spectra of an aqueous solution of Phloxine B
and Eosin 17
The photodynamic properties of (i) Phloxine B at 0.25mg/mL final
concentration, (ii) Eosin Y
at about 0.08 mg/mL, and (iii) a mixture of Phloxine B (0.25mg/mL) and Eosin Y
(0.08
mg/mL), all in an aqueous solution were evaluated. A flexstation 384 II
spectrophotometer was
used to measure emitted fluorescence with the following parameters: mode
fluorescence,
excitation 460 nm, emission spectra 465-750 nm. The absorbance was read using
a synergy HT
microplate reader: mode absorbance; spectra between 300-650nm.
The absorption and emission spectra are shown in Figures 8A and 8B which
indicate
an energy transfer between the chromophores in the combination. Broader
absorption and
emission spectra were achieved with the Phloxine B and Eosin Y chromophore
combination,
compared with the individual chromophores. The composition emitted light in
the green,
yellow, orange and red wavelengths of the visible spectra.
Example 5 - Absorption/emission spectra of Phloxine B and Fluorescein in a gel
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The photodynamic properties of (i) Fluorescein at about 100 g/g final
concentration, (ii)
Phloxine B at about 100 g/g, and (iii) a mixture of Fluorescein (100 g/g) and
Phloxine B
(100pg/g), all in a 12% carbamide gel were evaluated. A flexstation 384 II
spectrophotometer
was used to measure emitted fluorescence with the following parameters: mode
fluorescence,
excitation 460 nm, emission spectra 465-750 nm. The absorbance was read using
a synergy HT
microplate reader: mode absorbance; spectra between 300-650nm.
The absorption and emission spectra are shown in Figures 9A and 9B which
indicate
an energy transfer between the chromophores in the combination. For this
particular
combination of chromophores and at this concentration, for the chromophore
combination two
peaks corresponding to fluorescein and phloxine B emission was absorved, with
a higher peak
at around 577 nm absorption, compared with the individual chromophores.
Example 6 - Absorption/emission spectra of Fluorescein and Rose Bengal in a
gel
The photodynamic properties of (i) Fluorescein at about 100 g/g final
concentration, (ii) Rose
Bengal at about 100 g/g, and (iii) a mixture of Fluorescein (100 g/g) and
Phloxine B
(100 g/g), all in a 12% carbamide gel were evaluated. A flexstation 384 II
spectrophotometer
was used to measure emitted fluorescence with the following parameters: mode
fluorescence,
excitation 460 nm, emission spectra 465-750 nm. The absorbance was read using
a synergy HT
microplate reader: mode absorbance; spectra between 300-650nm.
The absorption and emission spectra are shown in Figures 10A and 10B which
indicate
an energy transfer between the chromophores in the combination. For this
particular
combination of chromophores and at this concentration, two emission peaks were
observed in
the combined chromophore composition with the combined composition having a
higher peak
at around 580 nm, compared with the individual chromophores.
Example 7 - Absorption/emission spectra of Rose Bengal and Eosin Y in a gel
The photodynamic properties of (i) Eosin Y at 0.305 mg/mL final concentration,
(ii) Rose
Bengal at about 0.085 mg/mL, and (iii) a mixture of Eosin Y (0.305mg/mL) and
Rose Bengal
(0.085 mg/mL), all in a 12% carbamide gel were evaluated. A flexstation 384 II
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spectrophotometer was used to measure emitted fluorescence with the following
parameters:
mode fluorescence, excitation 460 nm, emission spectra 465-750 nm. The
absorbance was read
using a synergy HT microplate reader: mode absorbance; spectra between 300-
650nm.
The absorption and emission spectra are shown in Figures 11A and 118 which
indicate
an energy transfer between the chromophores in the combination. For this
particular
combination of chromophores and at this concentration, a higher absorption was
achieved with
the chromophore combination, compared with the individual chromophores. The
emission
spectra of this specific combination had a lower power density than for Eosin
Y alone. In the
absence of a temperature rise in the composition during or after illumination,
this apparent loss
of energy may be attributed to reactive Oxygen species generation (see Example
8 below).
Example 8 - Eosin and Rose Bengal generate oxygen species
The synergy between two chromophores according to various embodiments of the
present
disclosure was investigated by preparing the following:
1 ¨ Eosin Y (0.035%) + Rose Bengal (0.085%) in a 12% carbamide gel.
2 ¨ Rose Bengal (0.085%) in a 12% carbamide gel.
Rose Bengal is known to have a high quantum yield in terms of singlet oxygen
production in the presence of oxygen-releasing agents when photoactivated by
green light (a
singlet oxygen quantum yield of approximately 75% in water [Murasecco-Suardi
et al,
Helvetica Chimica Acta, Vol. 70, pp.1760-73, 1987]). Eosin Y is known to have
a high
quantum yield in terms of emitted fluorescent light when photoactivated and
can be at least
partially activated by blue light when in a gel. Photoactivated Eosin Y has a
much lower
quantum yield in terms of singlet oxygen production in the presence of oxygen-
releasing agents
(a singlet oxygen quantum yield when fully activated of approximately 4%
[Gandin et al,
Photochemistry and Photobiology, Vol.37, pp.27I-8, 1983]).
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When Eosin Y and Rose Bengal are combined, it appears that both chromophores
are
activated by the same blue light as evidenced by Figure 12.
Figure 12, left panel, shows a photograph of the composition when viewed under
a
light microscope (x250) before exposure to an activating blue light. Very few
bubbles were
seen in both compositions. Following illumination with blue light a dramatic
increase in
bubbles was seen with the composition comprising a combination of Eosin Y and
Rose Bengal,
but not with the composition comprising Rose Bengal alone or Eosin Y alone
(not shown).
This suggests that there is a transfer of energy from Eosin Y to Rose Bengal
leading to the
formation oxygen species. Eosin Y alone in a carbamide gel presented similar
properties to
Rose Bengal. A similar effect was observed with Fluorescein and Rose Bengal.
Example 9¨ Variation of the chromophore concentration ratios
The effect of varying the concentrations of the individual chromophores in
multiple
chromophore compositions, according to embodiments of the present disclosure,
were
investigated. The fluorescence emission over time of compositions comprising
(i) Fluorescein
¨ Eosin Y, and (ii) Eosin Y ¨ Rose Bengal, are presented in Figures 13A and
13B respectively.
As can be seen in Figure 13A, the emission properties of the following were
investigated: (i) 109 pg/g of Eosin Y + 10pg/g of fluorescein, (ii) 109 pg/g
of Eosin Y +
100pg/g of fluorescein, (iii) 109 pg/g of Eosin Y, (iv) 10pg/g of fluorescein,
(v) 100p g/g of
fluorescein, all in a carbamide peroxide gel. An SP-100 spectroradiometer was
used to measure
the power density spectra (mW/cm2 versus wavelength) of a photonic signal
detected from the
various compositions when illuminated with blue light (wavelength of about 440
to 480 nm at a
power density of less than 150 mW/cm2 for about 5 minutes). Fluorescence is
measured as
light within the 519-700nm range.
As can be seen, the emitted fluorescence of all concentrations decay over
time. This
decay is often accompanied by a photobleaching of one or more of the
chromophores in the
composition. A higher concentration of fluorescein in a multiple chromophore
composition
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provides a higher initial emitted fluorescence which also lasts longer, i.e.
has a longer lifetime.
For the Eosin Y (109 gig) and Fluorescein (100 g/g) composition, the initial
emitted
fluorescence is slightly lower than that of a composition comprising 100 g/g
fluorescein alone.
This may be attributed to use of energy to form oxygen species (as described
in Example 6
above). Therefore, the relative concentrations of the chromophores within a
multiple
chromophore composition can be varied to tailor the resultant fluorescence and
oxygen species
properties.
In Figure 13B, the following compositions were evaluated (i) 109 gig of Eosin
Y +
1 g/g of rose bengal (ratio of about 10:1), (ii) 109 gig of Eosin Y + 100 g/g
of rose bengal
(ratio of about 1:1), (iii) 109 mg/g of Eosin Y, (iv) lpg/g of rose bengal,
(v) 100 g/g of rose
bengal, all in a carbamide peroxide gel. The same decay trend observed in
Figure 13A was
also observed for eosin Y alone, eosin Y- 1 pg/g rose bengal, as well as eosin
Y-10 g/g rose
Bengal (not shown). The very low fluorescence levels for both concentrations
of rose bengal
alone when activated by blue light can also be observed. Surprisingly, for the
composition of
109jug/g of Eosin Y + 100 g/g of rose bengal a sustained fluorescence was
observed, albeit at
a lower level than that of Eosin Y alone, and Eosin Y + li_tg/g of rose
bengal. In this
composition, no photobleaching of Eosin Y was observed. Without wishing to be
limited by
theory, it is believed that Eosin Y is not photobleaching as at this ratio of
Eosin Y/rose Bengal,
Eosin Y is able to transfer all of its absorbed energy to rose bengal which
then emits the energy
and thus prevents the photodegradation of the eosin Y molecules. The peak
emission
wavelength of the 109 pg/g Eosin Y + 100 g/g rose bengal composition is closer
to that of rose
bengal's peak emission wavelength than that of eosin y.
A similar sustained fluorescence effect was observed for a composition
comprising
fluorescein, eosin Y and rose bengal at relative concentration ratios of about
1:10:10 (not
shown).
Example 10 - Absorption/emission spectra of Fluorescein, Eosin Y and Rose
Bengal in a gel
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The photodynamic properties of (i) Rose Bengal at about 0.085 mg/mL, (ii)
Fluorescein
sodium salt at about 0.44 mg/mL final concentration, (ii) Eosin Y at about
0.305 mg/mL, and
(iii) a mixture of (i), (ii) and (iii) according to an embodiment of the
present disclosure in a gel
comprising about 12% carbamide peroxide were evaluated. A flexstation 384 II
spectrophotometer was used to measure emitted fluorescence with the following
parameters:
mode fluorescence, excitation 460 nm, emission spectra 465-750 nm. The
absorbance was read
using a synergy HT microplate reader: mode absorbance; spectra between 300-
650nm.
The absorbance and emission spectra are shown in Figures 14A and 14B which
indicate an energy transfer between the chromophores in the chromophore
combination. As is
clear from Figure 14B, the bandwidth of the Fluorescein, Eosin Y and Rose
Bengal
combination is wider than that of Eosin Y alone.
Example 11 - Absorption/emission spectra of Fluorescein, Eosin Y and Rose
Bengal in an
aqueous solution
The photodynamic properties of (i) Rose Bengal at about 0.085 mg/mL, (ii)
Fluorescein
sodium salt at about 0.44 mg/mL final concentration, (ii) Eosin Y at about
0.305 mg/mL, and
(iii) a mixture of (i), (ii) and (iii) in an aqueous solution according to an
embodiment of the
present disclosure were evaluated. A flexstation 384 II spectrophotometer was
used to measure
emitted fluorescence with the following parameters: mode fluorescence,
excitation 460 nm,
emission spectra 465-750 nm. The absorbance was read using a synergy HT
microplate reader:
mode absorbance; spectra between 300-650nm.
The absorbance and emission spectra are shown in Figures 15A and 15B which
indicate an energy transfer between the chromophores in the chromophore
combination, in the
absence of a peroxide but in the presence of other oxygen-releasing agents
(e.g. water).
In reference to the absorption and emission spectra of the compositions of the
present
disclosure within a carbamide peroxide gel, the same spectra was obtained for
the same
chromophores in a gel without the peroxide.
Example 12 - Angiogenic potential of a composition of the disclosure
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A human skin model was developed to assess the angiogenic potential of
compositions of the
present disclosure. Briefly, a composition comprising Eosin Y and Erythrosine
was placed on
top of a human skin model containing fibroblasts and keratinocytes. The skin
model and the
composition were separated by a nylon mesh of 20 micron pore size. The
composition was then
irradiated with blue light ('activating light') for 5 minutes at a distance of
5 cm from the light
source. The activating light consisted of light emitted from an LED lamp
having an average
peak wavelength of about 400-470 nm, and a power intensity measured at 10 cm
of 7.7 J/cm2
to 11.5 J/cm2. Upon illumination with the activating light, the composition
emitted fluorescent
light. Since the composition was in limited contact with the cells, the
fibroblasts and
keratinocytes were exposed mainly to the activating light and the fluorescent
light emitted from
the composition. Conditioned media from the treated human 3D skin model were
then applied
to human aortic endothelial cells previously plated in Matrigela The formation
of tubes by
endothelial cells was observed and monitored by microscopy and image analysis
after 24
hours. The conditioned media from 3D skin models treated with light
illumination induced
endothelial tube formation in vitro, suggesting an indirect effect of the
light treatment (blue
light and fluorescence) on angiogenesis via the production of factors by
fibroblasts and
keratinocytes. Plain media and conditioned media from untreated skin samples
were used as a
control, and did not induce endothelial tube formation.
Figure 16 is an emission spectrum showing the intensity over time of the light
being
emitted from the biophotonic composition as measured using the
spectroradiometer of Example
9. It can be reasonably inferred that other chromophore combinations
exhibiting a comparable
emission spectra would also induce angiogenesis. As can be seen from Figure
16, the emitted
fluorescence light had a wavelength of about 520-620 nm with a peak at around
560 nm.
Similar emission spectra were observed using Eosin Y and Fluorescein (Figure
5B); Eosin Y
and Phloxine B (Figure 7B, Figure 8B); Eosin Y and Rose Bengal (Figure 11B);
Fluorescein,
Eosin Y and Rose bengal (Figure 14B, Figure 15B). Other chromophore
combinations with
similar emission spectra are also possible, which can be reasonably expected
to have
angiogenic properties.
Example 13 - Protein secretion and gene expression profiles
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Wounded and unwounded 3D human skin models (EpiDermFT, MatTek Corporation)
were
used to assess the potential of a composition of the present disclosure to
trigger distinct protein
secretion and gene expression profiles. Briefly, a composition comprising
Eosin and
Erythrosine were placed on top of wounded and unwounded 3D human skin models
cultured
under different conditions (with growth factors (1X), 50% growth factors
(0.5X) and no growth
factors (OX)). The different conditions mimicked non-compromised healing, semi-
starvation
conditions and starvations conditions, respectively. The skin models and the
composition were
separated by a nylon mesh of 20 micron pore size. Each skin model-composition
combination
was then irradiated with blue light ('activating light') for 5 minutes at a
distance of 5 cm from
the light source. The activating light consisted of light emitted from an LED
lamp having an
average peak wavelength of about 440-470 nm, a power density of 60-150mW/cm2
at 5 cm,
and a total energy density after 5 minutes of about 18-39 J/cm2. The controls
consisted of 3D
skin models not illuminated with light.
Gene expression and protein secretion profiles were measured 24 hours post-
light
exposure. Cytokine secretion was analyzed by antibody arrays (RayBio Human
Cytokine
antibody array), gene expression was analyzed by PCR array (PAHS-013A,
SABioscience) and
cytotoxicity was determined by GAPDH and LDH release. Results (Tables 1 and 2)
showed
that the light treatment is capable of increasing the level of protein
secreted and gene
expression involved in the early inflammatory phase of wound healing in
wounded skin inserts
and in non-starvation conditions. In starvation conditions mimicking chronic
wounds, there
was no increase in the level of inflammatory protein secreted when compared to
the control.
Interestingly, the effect of the light treatment on unwounded skin models has
a much lower
impact at the cellular level than on wounded skin insert, which suggests an
effect at the cellular
effect level of the light treatment. It seems to accelerate the inflammatory
phase of the wound
healing process. Due to the lack of other cell types such as macrophages in
the 3D skin model,
the anti-inflammatory feed-back is absent and may explain the delay in wound
closure.
Cytotoxicity was not observed in the light treatments. The eosin y and
erythrosine b
composition had the same emission properties as illustrated in Figure 16. As
stated above, it
can be reasonably inferred that other chromophore combinations exhibiting a
comparable
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emission spectra would also induce secretion of proteins or gene expression as
seen in this
Example.
Table 1 ¨ List of proteins with statistically significant difference secretion
ratio between treated
and untreated control at day 3. Two arrows mean that the ratio was over 2
folds.
Medium 1X Medium 0.5X Medium OX
Increase ENA78 p=0.04 TT Angiogenin p=0.03 I
I1-1R4/ST2 p=0.02 l't CXCL16 p=0.04
I
MMP3 p=0.01 TT
MCP-2 p=0.04 TT
Decrease BMP6 p=0.01 .1, BMP6 p=0.02 1
TNFa p=0.005 1
Table 2 ¨ List of genes with statistically significant difference expression
ratio between treated
and untreated control during the first 24 hours. Two arrows mean that the
ratio was over 2
folds.
Medium 1X Medium 0.5X Medium OX
Increase CTGF p=0.02 i CTGF P=0.04 i MMP3 p=0.007 IT
ITGB3 p=0.03 i ITGB3 p=0.05 i LAMA1 p=0.03 I
MMP1 p=0.03 i MMP1 p=0.02 Ti ITGA2 p=0.03 I
MMP3 p=0.01 i MMP10 p=0.003 IT
THBS1 P=0.02 i MMP3 p=0.007 IT
MMP8 p=0.02 it
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THBS1 p=0.03 't
Decrease HAS1 p=0.009 4_4 NCAM1 p=0.02 14.
NCAM1 p=0.05 4,4, VCAN p=0.02 4,
VCAM1 p=0.03 14 LAMC1 p=0.002 4.
COL7A1 p=0.04 I. COL6A1 p=0.007 4,
CTNNA1 p=0.03 4. MMP7 p=0.003 4,
Example 14 ¨ Eosin Y and Fluorescein induce collagen formation
A composition according to an embodiment of the present invention, comprising
0.01% Eosin
Y and 0.01% Fluorescein in a carrier matrix (1.8% carbopol gel) was evaluated
for its potential
to induce collagen formation. Dermal human fibroblasts were plated in glass-
bottomed dishes
with wells (MatTek ). There were approximately 4000 cells per well. After 48
hours, the
glass-bottomed dishes were inverted and the cells were treated through the
glass bottom with
(i) a no light (control), (ii) sunlight exposure for about 13 minutes at noon
(control), (iii) the
composition applied to the glass well bottom on the other side of the cells
(no light exposure),
(iv) the composition applied to the glass well bottom on the other side of the
cells (sun light
exposure for about 13 minutes at noon), and (v) the composition applied to the
glass well
bottom on the other side of the cells (blue light exposure for about 5
minutes). In the case of
(iii), (iv) and (v), there was no direct contact between the cells and the
composition. In the case
of (iv) and (v), the cells were exposed to emitted light from and through the
Eosin Y and
Fluorescein composition when exposed to sunlight and blue light respectively.
An at least
partial photobleaching was observed in (iv) and (v). After the treatment, the
cells were washed
and incubated in regular medium for 48 hours. A collagen assay was then
performed on the
supernatant using the Picro-Sirius red method. This involved adding Sirius red
dye solution in
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picric acid to the supernatant, incubating with gentle agitation for 30
minutes followed by
centrifugation to form a pellet. The pellet was washed first with 0.1N HC1 and
then 0.5 N
NaOH to remove free dye. After centrifugation, the suspension was read at 540
nm for collagen
type I. The results are shown in Table 1.
Table 1 ¨ A qualitative comparison of collagen type I concentration in a
dermal human
fibroblast supernatant exposed to (i) a no light (control), (ii) sunlight
exposure for about 13
minutes at noon (control), (iii) any light emitted from a Eosin Y and
Fluorescein composition
through a glass separation (no light exposure), (iv) any light emitted from a
Eosin Y and
Fluorescein composition through a glass separation (sun light exposure for
about 13 minutes at
noon), and (v) the composition applied to the glass well bottom on the other
side of the cells
(blue light exposure for about 5 minutes). ++ indicates collagen levels about
twice as high as +,
and +++ indicates collagen levels about three times as high as +.
No light Sunlight Eosin Y + Eosin and Eosin and
(control) (control) Fluorescein ¨ Fluorescein ¨ Fluorescein ¨
no light sunlight blue light
Collagen + + ++ +++ +++
concentration
There was a statistical difference between the collagen levels induced by the
Eosin Y
and Fluorescein composition exposed to sunlight and blue light compared to the
no light and
sunlight alone controls.
Collagen generation is indicative of a potential for tissue repair including
stabilization of
granulation tissue and decreasing of wound size. It is also linked to
reduction of fine lines, a
decrease in pore size, improvement of texture and improvement of tensile
strength of intact
skin. The emission spectra of the Eosin Y and Fluorescein composition of this
example had a
single peak emission with a wavelength that ranged from about 480-620 nm.
Following
illumination with sunlight, the power density of the peak was reduced
indicating an at least
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WO 2014/040176 PCT/CA2013/000786
partial photobleaching in 13 minutes, which was also observed by a change in
colour of the
composition. The rate of fluorescence emission/photobleaching was slower when
illuminated
by sunlight (white light) compared to Eosin Y and Fluorescein compositions
(e.g. compositions
of Examples 5 and 6) when activated by blue light.
Example 15 ¨ Selecting the concentration of chromophore in the biophotonic
composition
The fluorescence spectra of compositions with different concentrations of
chromophores were
investigated using a spectroradiometer and an activating blue light (as in
Example 9).
Exemplary fluorescence spectra of Eosin Y and Fluorescein are presented in
Figures 17A and
17B. It was found that emitted fluorescence from the chromophore increases
rapidly with
increasing concentration but slows down to a plateau with further
concentration increase.
Activating light passing through the composition decreases with increasing
chromophore
composition as more is absorbed by the chromophores. Therefore, the
concentration of
chromophores in compositions of the present disclosure can be selected
according to a required
ratio and level of activating light and fluorescence treating the tissue based
on this example. In
some embodiments, it will be after the zone of rapid increase, i.e. between
0.5 and 1 mg/mL for
Eosin Y (Figure 17A).
Therefore, concentration can be selected according to required activating
light and
fluorescence. In some embodiments, it will be after zone of rapid increase,
i.e. between 0.5 and
1 mg/mL for Eosin Y (Figure 17A).
Compositions with rose bengal behave slightly differently and become more
opaque
with increasing concentration which may be due to bubble formation.
Similarly, the relationship between the power density of light received by the
tissues
with illuminating time was investigated. It was found that the power density
of the activating
light was low initially and increased with time. This correlates with the
light absorbing
chromophores photobleaching and more of the activating light passing through
the composition
to reach tissues. In parallel, the fluorescent light emitted by the
composition decreased with
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WO 2014/040176 PCT/CA2013/000786
time as one or more of the chromophores photobleached. Overall, the total
power density of the
light treating the tissues increased gradually over illumination time.
It should be appreciated that the invention is not limited to the particular
embodiments
described and illustrated herein but includes all modifications and variations
falling within the
scope of the invention as defined in the appended claims.
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