U.S. patent application number 11/496151 was filed with the patent office on 2007-06-28 for skin treatment compositions containing copper-pigment complexes.
This patent application is currently assigned to DISCOVERY PARTNERS LLC. Invention is credited to Peter Ladislaus Dorogi, John Patrick McCook, David Bruce Vasily.
Application Number | 20070148224 11/496151 |
Document ID | / |
Family ID | 46325811 |
Filed Date | 2007-06-28 |
United States Patent
Application |
20070148224 |
Kind Code |
A1 |
Dorogi; Peter Ladislaus ; et
al. |
June 28, 2007 |
Skin treatment compositions containing copper-pigment complexes
Abstract
This invention discloses compositions and method for treating
various types of skin disorders, based on topical cutaneous
delivery of copper chemically bound with botanical pigments.
Sodium-copper-chlorophyllin is used as an example, showing benefits
in the treatment of rosacea, acne, oily skin, enlarged pores, and
in relieving skin inflammation. Benefits are also disclosed in
treatment of environmentally caused premature skin aging, via
reductions in fine facial lines and wrinkles, increased tensile
strength of the skin, and increased protection against sunlight via
increased production of melanin. Therapeutic outcomes are improved
when the copper-pigment complex is enclosed within submicron
liposomes.
Inventors: |
Dorogi; Peter Ladislaus;
(Easton, PA) ; Vasily; David Bruce; (Bethlehem,
PA) ; McCook; John Patrick; (Frisco, TX) |
Correspondence
Address: |
THEODORE JAY;SUITE 600
16 N. CHATSWORTH AVE.
LARCHMONT
NY
10538
US
|
Assignee: |
DISCOVERY PARTNERS LLC
|
Family ID: |
46325811 |
Appl. No.: |
11/496151 |
Filed: |
July 31, 2006 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11320280 |
Dec 28, 2005 |
|
|
|
11496151 |
|
|
|
|
Current U.S.
Class: |
424/450 ;
514/185 |
Current CPC
Class: |
A61K 9/06 20130101; A61K
45/06 20130101; A61K 9/0014 20130101; A61P 17/00 20180101; A61Q
19/008 20130101; A61K 47/10 20130101; A61K 9/127 20130101; A61K
8/19 20130101; A61K 47/32 20130101; A61K 8/494 20130101; A61K
31/555 20130101; A61K 2800/58 20130101; A61K 31/555 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/450 ;
514/185 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/555 20060101 A61K031/555 |
Claims
1. A method for beneficially treating one or more human skin
disorders comprising the steps of: [a]producing a composition
containing a non-acidic formulation of copper chemically bound to a
botanical pigment possessing antioxidant qualities; said
composition thus containing a copper-botanical-antioxidant complex;
[b] locating physiological copper-binding sites in the skin that
show signs of chronic skin pathologies or environmental damage; [c]
applying topically to the skin said copper-botanical-antioxidant
complex composition to cause enhanced penetration of the complex to
copper binding sites; and, [d] simultaneously delivering skin
pH-activated copper ions and plant-based antioxidant pigment to
said physiological binding sites to beneficially treat said skin
disorders and to protect against further skin damage.
2. The method of claim 1 wherein said composition is a
pharmaceutical formulation.
3. The method of claim 1 wherein said composition is a
cosmeceutical formulation.
4. The method of claim 1 wherein the human skin disorder consists
of one or more of the following: rosacea, acne, enlarged facial
pores, seborrhea or excessively oily facial skin, acne scars,
post-acne pigmentation, and premature aging of the skin which
includes skin wrinkling, sagging, roughness, blotchiness and uneven
pigmentation caused by sun damage.
5. The method of claim 1, wherein the composition is in the form of
a liquid, gel, spray, lotion, cream, or solid.
6. The method of claim 1, wherein the topical composition is
applied at least once daily for 14 days or more.
7. The method of claim 1, wherein the skin penetration of the
copper-pigment complex is enhanced by encapsulating the
copper-pigment complex within a liposome shell; said liposome shell
preferably containing a linoleic acid-phosphatidyl choline type of
lecithin.
8. The method of claim 1, wherein the copper-pigment antioxidant
complex is sodium-copper-chlorophyllin.
9. The method of claim 1, wherein the composition is applied
topically in conjunction or simultaneously with a secondary
pharmaceutical or cosmeceutical treatment composition for a skin
disorder that includes one or more of the following: rosacea, acne,
enlarged facial pores, seborrhea or excessively oily facial skin,
acne scars, post-acne pigmentation, and premature aging of the skin
which includes skin wrinkling, sagging, roughness, blotchiness and
uneven pigmentation caused by sun damage; said secondary treatment
composition not containing a copper-botanical antioxidant
complex.
10. A composition for topical application for the beneficial
treatment of one or more human skin disorders, said composition
containing: a) an aqueous, non-acidic solution of sodium copper
chlorophyllin within a submicron liposomal structure; b) said
liposomal structure consisting of a linoleic acid/phosphatidyl
choline type lecithin shell; c) said liposomes being dispersed in a
cosmetically, pharmaceutically, or dermatology acceptable carrier
vehicle.
11. The composition of claim 10 wherein the human skin disorder
consists of one or more of the following: rosacea, acne, enlarged
facial pores, seborrhea or excessively oily facial skin, acne
scars, post-acne pigmentation, and premature aging of the skin
which includes skin wrinkling, sagging, roughness, blotchiness and
uneven pigmentation caused by sun damage.
12. The composition of claim 10 where the size of the liposomes is
in the range of 150-350 nanometers in diameter.
13. The composition of claim 10 wherein the concentration of sodium
copper chlorophyllin ranges between 0.0001% and 0.5% by weight of
the total composition.
14. The composition of claim 10 wherein the cosmetically,
pharmaceutically or dermatology acceptable carrier vehicle
comprises a gel, lotion, spray, solid, liquid, emulsion,
microemulsion, suspension, or liposomal dispersion.
15. The composition of claim 10 wherein the pH is adjusted to
between 7.0-8.5 and preferably between 7.2-7.6.
16. A composition for topical application to human skin for
increasing the natural tanning response induced by sunlight or
simulated solar radiation or by UV tanning beds, said composition
containing: a) an aqueous, non-acidic solution of sodium copper
chlorophyllin within a submicron liposomal structure; b) said
liposomal structure consisting of a linoleic acid/phosphatidyl
choline type lecithin shell; c) said liposomes being dispersed in a
cosmetically, pharmaceutically, or dermatology acceptable carrier
vehicle.
17. The composition of claim 16 where the size of the liposomes is
in the range of 150-350 nanometers in diameter.
18. The composition of claim 16 where the concentration of sodium
copper chlorophyllin ranges between 0.0001% and 0.5% by weight of
the total composition.
19. The composition of claim 16 wherein the cosmetically,
pharmaceutically, or dermatology acceptable carrier vehicle
comprises a gel, lotion, liquid or cream emulsion, microemulsion,
suspension, or liposomal dispersion.
20. The composition of claim 10 and claim 16 wherein the
pharmaceutical, cosmeceutical, or dermatological vehicle contains
one or more water soluble skin penetration enhancing ingredients
selected from the group that includes propylene glycol, butylene
glycol, pentylene glycol, isopentyl glycol, ethoxydiglycol,
dimethyl isosorbide, acetamide MEA, tetrahydropiperine, various PEG
glyceryl ethers, Levomenol
[(-)-6-Methyl-2-((4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol],
N-Methyl-2-pyrrolidone, dimethyl sulfoxide and methyl sulfone.
Description
[0001] This application is a continuation-in-part of divisional
application Ser. No. 11/320,280 filed Dec. 28, 2005 and titled
"SKIN TREATMENT COMPOSITIONS CONTAINING COPPER-PIGMENT
COMPLEXES"
Peter L. Dorogi, David B. Vasily and John P. McCook
[0002] This Continuation-in-Part differs from the original
application in that the inventors have demonstrated increased
utility for the invention, particularly in the treatment of rosacea
characterized by diffuse facial redness, with currently no
effective topical therapy. The inventors have conducted further
experiments to show that artificial light therapy used to treat
acne, rosacea, and other inflammatory skin conditions is
unnecessary in conjunction with the application of the subject
invention, and, is, in fact, contraindicated. Lastly, this
Continuation-in-Part more fully describes the formulation options
for effective skin penetration of the active copper pigment complex
and the effective pH for formulation stability and dermal and
epidermal efficacy. The details of the testing related to these new
findings and greater utility are described below.
FIELD OF THE INVENTION
[0003] This invention relates to therapeutic substances and methods
for treating, preventing, reversing or inhibiting skin disorders
characterized by redness and broken small blood vessels on the
face, associated with, but not limited to rosacea, as well as skin
disorders with chronic inflammation or sun damage, by topical
application of compositions containing copper bound to a botanical
pigment and delivered in a suitable pharmaceutical or cosmeceutical
vehicle.
BACKGROUND OF THE INVENTION
[0004] Rosacea is typically seen on the face, and is characterized
by persistent erythema and broken small blood vessels
(telangiectasia). In some cases there are red spots (papules) and
sometimes pustules with an acne-like appearance; but in rosacea
they are dome-shaped rather than pointed and there are no
blackheads, whiteheads, deep cysts, or lumps. The broken blood
vessels near the skin surface may leak when flushing or blushing
occurs, creating blotchy red areas on the face. The redness can
come and go, and the skin may eventually become tender, inflamed
and sensitive to the touch. The skin may swell and thicken, and the
redness can become permanent. Rosacea is seen mainly in
fair-skinned people between 30 and 50 years of age. It is estimated
that about 13 million Americans have rosacea.
[0005] In individuals who are genetically predisposed to rosacea,
the triggering cause is believed to be sun damage to small blood
vessels near the skin surface, allowing these vessels to stretch
and become permanently dilated by recurrent flushing or blushing.
The broken blood vessels may thereafter become visible through the
skin surface. Anything that makes such an individual flush or blush
promotes rosacea, including consumption of alcohol or spicy foods,
heating of the skin, and emotional situations such as anxiety or
embarrassment.
[0006] Another suspected cause of rosacea is sensitivity to the
microbial organism Demodex folliculorum (especially in
papulopustular rosacea), a relatively common skin parasite. Diffuse
skin redness may be due to sympathetic (neural) vasomotor
instability, leading to frequent involuntary dilation of the
subcutaneous arteries. Therefore, treatment modalities that
constrict blood vessels and reduce blood flow have been reported to
reduce the diffuse skin redness associated with rosacea, i.e., with
erythematotelangiectatic rosacea. The compositions and methods that
comprise this invention were found to be effective for the
treatment of erythematotelangiectatic rosacea but not
papulopustular rosacea. The term "rosacea" used throughout this
document refers to the erythematotelangiectatic form of the
condition only.
[0007] Treatments that strengthen the connective tissue can help by
preventing break down of blood vessels. Copper is essential for the
maintenance and repair of connective tissue. As a functional
component of the enzyme lysyl oxidase, copper catalyzes the
formation of structural cross-links in collagen. Damage by
environmental factors such as sunlight, pollutants from industrial
combustion reactions, and even second-hand cigarette smoke, is
prevented by the skin's antioxidant enzymes, most notably the
superoxide dismutases (SODs). The predominant form of SOD contains
copper. SOD enzymes also neutralize free radical oxidation
reactions that underlie the signs and symptoms of rosacea, acne,
sunburn, and numerous inflammatory skin pathologies.
[0008] Copper is an essential component of the enzyme tyrosine
hydroxylase, which catalyzes conversion of the amino acid tyrosine
into melanin. With respect to rosacea, tyrosine hydroxylase is also
involved in synthesis of the hormone norepinephrine, the natural
agonist for activating the alpha-adrenergic receptors responsible
for vasoconstriction (reduction of blood flow) in the skin. In
prior art, published U.S. Patent Application #20050020600 describes
topical application of pharmaceutical preparations containing
alpha-adrenergic agonists for the treatment of facial vasomotor
instability. This approach differs fundamentally from the present
invention, wherein we apply copper to the skin in order to increase
the biosynthesis of norepinephrine; but no final agonist is
contained in our compositions.
[0009] Copper provides two routes for treatment of acne. For one,
copper binds to, and activates, certain small peptides that promote
wound healing. An example is the tripeptide known by the acronym
GHK (glycyl-L-histidyl-L-lysine), which promotes the deposition of
new collagen by dermal fibroblasts, stimulating the growth of new
blood vessels, and increasing activity of protease enzymes to
remove previously formed scar tissue. Copper also has
anti-microbial properties and will kill acne-promoting bacteria in
the skin, as demonstrated below.
SUMMARY OF THE INVENTION
[0010] In the present invention, cutaneous topical delivery of
copper is carried out by binding copper with a botanical pigment.
The term "pigment" is meant here and throughout this document to
cover botanical or naturally-derived chromophoric molecules, or
chemically modified botanical chromophores, possessing antioxidant
properties. The copper-pigment complex utilized and demonstrated
here, in a non-limiting way, is sodium-copper-chlorophyllin. The
copper-pigment complex is delivered into the skin with a
penetration-enhancing vehicle.
[0011] To further improve skin penetration, the copper-pigment
complex and its medium may be encapsulated within liposomes. The
lipid wall of the liposomes consists of lecithin and is disclosed
to have additional advantages if it contains linoleic acid,
reported to be therapeutic for acne. The liposomes used are
relatively small, with diameters ranging between 150 and 350
nanometers. Sodium-copper-chlorophyllin loaded liposomes are
mentioned in U.S. Pat. No. 6,663,559: "Method and apparatus for the
photomodulation of living cells". However, this prior art patent is
concerned with the use of sodium-copper-chlorophyllin as a
chromophore in light radiation treatments of the skin. The present
invention does not involve the use of any light sources. In fact,
the resulting increase in skin temperature may worsen rosacea.
[0012] In summary:
[0013] The invention disclosed here is designed, in part, to
deliver copper to binding sites in the skin, where it can be
utilized to form enzymes and wound-healing copper-peptides; for
example, for preventing, reducing and eliminating the signs and
symptoms of rosacea.
[0014] It is another aim of this invention to provide copper to
binding sites where it is utilized to restructure the skin so as to
reduce enlarged facial pores (follicular hypertrophy) and the signs
and symptoms of acne, notably acne-scarring.
[0015] Copper is anti-microbial, and the processes and compositions
disclosed in this invention can be used to deliver copper into
follicles and sebaceous glands, thereby reducing inflammatory acne
by the killing action of copper on the acne-associated bacterium
Propionibacterium acnes.
[0016] The present invention aims to provide copper to
copper-dependent antioxidant enzymes responsible for elimination of
free radicals generated in the skin by ultraviolet light, reactive
oxygen forms, and microbe activity.
[0017] The disclosed method is intended to support copper-binding
enzymes and peptides which are active in repair and replacement of
damaged connective tissue.
[0018] It is a further goal of this invention to improve the skin's
tanning response to ultraviolet light by providing essential copper
to activate the enzyme tyrosine hydroxylase for increased
biosynthesis of melanin.
[0019] It is also the aim of this invention to simultaneously
supply to the skin, besides copper, botanical pigments which are
natural antioxidants, so as to further reduce oxidation damage.
[0020] We disclose that these goals are achievable with therapeutic
units that consist of a copper ion metallically bound to a suitable
botanical pigment. Treatment involves cutaneous application of such
a copper-pigment complex, delivered in a pharmaceutical or
cosmeceutical vehicle that facilitates penetration of the complex
into the skin. In one embodiment of the invention, the
copper-pigment complex is sodium-copper-chlorophyllin. The
copper-pigment complex and its carrier may be encapsulated in
150-350 nanometer diameter liposomes, which are disclosed here to
result in increased skin penetration by the copper-pigment
complex.
[0021] It is understood that in therapeutic applications the
composition containing the copper-pigment complex may be combined
with or applied in conjunction with other pharmaceutical,
cosmeceutical, or nutritional treatments for rosacea, acne, oily
skin, enlarged pores, acne scarring, blotchy skin, facial lines and
wrinkles or other manifestations of photodamage. Topical
application of the copper pigment complex may be sequential or
simultaneous with other topical or even systemic treatments, e.g.,
via injection, inhalation, or ingestion. For example, simultaneous
delivery of the copper pigment complex and another pharmaceutical
preparation may be accomplished by using a co-dispensing
applicator.
[0022] It is disclosed here that combining the compositions and
processes utilizing copper-pigments with currently established
anti-aging, anti-acne, and rosacea treatment technologies will
bring additional benefits to those technologies. For example, the
anti-inflammatory activity and the control of high skin oil
production seen in our studies with sodium-copper-chlorophyllin
could be important additions to established treatments. In the acne
area, copper-pigment technology could be combined with one or more
other treatment compounds that include salicylic acid, retinoids,
benzoyl peroxide, azelaic acid, tetracyclines, clindamycin,
erythromycin, adapalene, tazarotene, resorcinol, colloidal sulfur,
alpha-hydroxy acids or other established anti-acne actives. In the
anti-aging area, combinations with one or more compounds that
include alpha- and beta-hydroxy acids, peptides, alpha- and
beta-keto acids, other antioxidants, sunscreens, isoflavones,
metalloprotease enzyme inhibitors or other established anti-aging
actives may be advantageous. In the treatment of rosacea,
copper-pigment technology may be combined with one or more other
treatment compounds that include topical metronidazole, or oral
therapy with doxycyline, tetracycline, erythromycin, minocycline,
or other established rosacea treatment actives. Co-dispensing the
copper-pigment preparation with other actives formulated at an
acidic pH could also speed the release of the copper, which may
also prove advantageous in some cases.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Damaged collagen and elastin can reduce the tensile strength
of connective tissue. Structural weakness of connective tissue is a
likely cause of telangiectasias, produced by distension and
thinning of blood vessel walls. The increased tissue blood volume
visible through the skin surface is responsible for the diffuse
redness. It is disclosed here that increased cross-linking of
connective tissue due to increased bioavailability of copper can 1)
reduce the signs and symptoms of rosacea, 2) reduce the visibility
of under-eye dark circles, 3) improve tensile strength and
elasticity of skin and thereby diminish the appearance of fine
lines and wrinkles, and 4) reduce the size of enlarged facial
pores.
[0024] It is further disclosed that binding copper with an
antioxidant botanical pigment provides important additional
protection. The skin has its intrinsic mechanisms for repair of
damaged cells and macromolecules reduced after oxidative injury.
Wound healing may also include the body's own production and
secretion of superoxide radicals (.O.sub.2.sup.-) by phagocytic
cells, used to destroy invading microorganisms. Although the
superoxide radical is of itself not very damaging, it can react
with transition metals such as ferrous iron (Fe.sup.+2) and cuprous
copper (Cu.sup.+2) to generate the extremely reactive hydroxyl
radical .OH.sup.-. To offset such potentially harmful side-effects,
we disclose copper-delivery compositions wherein copper is applied
in forms that provide their own antioxidant protection; namely,
copper bound with suitable botanical pigments.
[0025] Elimination of the superoxides is carried out by superoxide
dismutases (SODs). The predominant form of SOD in the skin is the
Cu.sup.+2/Zn.sup.+2-containing dimeric form of the enzyme. It is
disclosed here that increasing the amount of copper in the skin may
result in increased SOD activity, reducing the signs and symptoms
of skin inflammation.
[0026] Seborrheic inflammatory conditions may cause excess oiliness
of the skin, resulting in a cosmetically unattractive shiny
appearance. The anti-inflammatory effect of increased SOD and
introduction of the skin-penetrating botanical antioxidant, may
account for the reduction in facial skin oiliness observed in the
clinical study described below.
[0027] The copper-pigment complex is applied to the skin in a
pharmaceutical or cosmeceutical vehicle, containing
penetration-enhancing ingredients. Non-limiting examples of these
ingredients include propylene glycol, butylene glycol, pentylene
glycol, isopentyl glycol, ethoxydiglycol, dimethyl isosorbide,
acetamide MEA, tetrahydropiperine, various PEG glyceryl ethers,
Levomenol
[(-)-6-Methyl-2-((4-methyl-3-cyclohexen-1-yl)-5-hepten-2-ol],
N-Methyl-2-pyrrolidone, dimethyl sulfoxide and methyl sulfone.
[0028] The copper-antioxidant complex and its aqueous vehicle may
be delivered within a liposomal dispersion, wherein the lipid shell
of the liposome consists of lecithin. Although lecithin has been
used for similar applications, the role of lecithin has been viewed
strictly as an encapsulating material that facilitates the
penetration of hydrophilic substances into the skin. The present
invention discloses that if the type of lecithin used is a
structural combination of linoleic acid and phosphatidyl choline
(high linoleic acid content lecithin), then the liposome material
itself may reduce acne symptoms. Liposomes composed of this lipid
and loaded with sodium-copper-chlorophyllin were manufactured for
us by Rovi Cosmetics (ROVI GmbH & Co., Kosmetische Rohstoffe
KG, Schluchtern, Germany). Liposomes of this type can also be made
in situ in a suitable cosmetic or pharmaceutical vehicle by
dissolving the copper-pigment complex in water and encapsulating
the aqueous copper complex with Phospholipon 80.RTM. or
Phospholipon 85.RTM.. Lecithin of this type was obtained from the
American Lecithin Company, originally manufactured by Phospholipid
GmbH (Cologne, Germany) and typically contains more than 50%
linoleic acid. The copper-complex loaded liposomes are disclosed as
novel treatments of rosacea, acne, and other inflammatory skin
pathologies. The invention combines four types of ingredients:
copper ions, antioxidant botanical pigments, skin penetration
enhancing vehicles, and therapeutic lipids in the form of
liposomes.
[0029] Another aspect of the invention is a method for controlled
release of copper in the skin, based on suitable formulation of the
pH of the therapeutic unit compared with the internal pH of the
skin. In the case of sodium-copper-chlorophyllin, copper is bound
to the chlorophyllin by a metallic bond and can be replaced by two
protons: a more acidic environment releases the copper, increasing
the concentration of the free form, Cu.sup.+2. It is thereby
another aspect of the invention that the therapeutic unit is
formulated in a vehicle at a slightly alkaline pH: typically, a pH
range of 7.2 to 7.6 gives good results. Aqueous solutions of
sodium-copper-chlorophyllin complex are typically alkaline, in the
pH-range 8.0-9.5 prior to any pH adjustment via acidifying agents
or buffering agents. When liposomal dispersants are used to enhance
skin penetration, the pH of the liposomal dispersion is adjusted to
a slightly alkaline pH of 7.2-7.6 to stabilize
sodium-copper-chlorophyllin.
[0030] The more acidic environment of the skin's outer mantle
releases copper from the pigment. Encapsulation by liposomes acts
as a shield against this "acid shock", resulting in deeper skin
penetration, slower release, and potentially enhanced utilization
of copper. In a "proof-of-principle" study with one human subject,
increased utilization of copper was demonstrated for the liposomal
system; the experiment is described in detail in Study 6 below.
This shows that 1) the aqueous composition containing
sodium-copper-chlorophyllin and penetration-enhancing agents
delivers copper to the enzyme tyrosine hydroxylase situated at the
base of the epidermis and 2) encapsulation of the copper-pigment
complex within a liposome increases the amount of copper
delivered.
[0031] Another aspect of the present invention is that controlled
dissociation of the sodium-copper-chlorophyllin complex is
possible, in part because of the high stability of this complex.
Normally, inorganic and organic copper salts, including
copper-peptides, are not very stable with regard to binding copper.
The inherent stability and lack of reactivity of
sodium-copper-chlorophyllin is essential for this process to work
successfully. We demonstrated the stability of this copper-pigment
complex by exposing a sample of the material to intense pulsed
light (IPL) flashes in the visible spectral range absorbed by this
dye. Stability was monitored by measurement of the dye's full
absorbance spectrum, since degradation of the dye would be
detectable as changes in the absorbance spectrum. Even after
application of ten pulses at the maximum fluence of the flash lamp,
the absorbance spectrum of sodium-copper-chlorphyllin remained
unchanged, demonstrating high stability for this material.
[0032] In nature, many types of botanical pigments protect plants
against the free radicals generated from molecular oxygen.
Antioxidant pigments are essential for the survival of plants, and
the pigments usually contain a metal-ion such as magnesium, zinc,
or copper. Although our experiments utilized
sodium-copper-chlorophyllin, other examples of botanical pigments
containing metals and possessing free radical scavenging properties
are considered to be part of this invention. A non-limiting list of
examples of such pigments includes carotenoids, chlorophylls,
anthocyanins, betalains and phycobilins.
[0033] We summarize the invention in greater detail as follows:
[0034] In one preferred embodiment of this invention, the carrier
vehicle of the copper-pigment complex is a liposomal dispersion,
containing lecithin liposomes of very small size, typically between
150-350 nanometers. Bound Cu.sup.+2 ions are loaded into the
liposomes as an aqueous solution of sodium-copper-chlorophyllin.
Chlorophyllin itself is a highly effective antioxidant and is
expected to neutralize any free radical load put on the skin by
reactions between Cu.sup.+2, hydrogen peroxide, and ultraviolet
light.
[0035] Copper ions are favored to dissociate from chlorophyllin due
to the acidity of the skin compared with the formulated higher pH
of the composition. This release process is expectedly slowed by
liposome encapsulation. Alternatively, dissociation of the
copper-pigment complex can be increased by increasing the skin
temperature, for example by optical or sonic heating; or, the
release can be slowed by cooling the skin.
[0036] Because copper ions bind with many skin peptides and
proteins, benefits of this type of topical treatment depend on the
applied copper reaching the critical copper-binding sites.
Combination of these particular liposomes, characterized by their
high stability and small size, loaded with copper bound to a
suitable, stable botanical pigment, and delivered within a
penetration-enhancing vehicle, are all essential parts of this
invention.
[0037] In turn, the aims of the present invention are to provide
compositions and processes for enhancing cutaneous physiological
functions that are, at least in part, dependent on the presence of
copper. These copper-dependent functions include repair and growth
of connective tissue, regulation of mitochondrial energy
metabolism, formation of melanin, and reduction of active oxygen
species. The invention discloses the process consisting of:
[0038] 1) producing a composition containing a non-acidic
formulation of copper chemically bound to a botanical pigment
possessing antioxidant qualities, the composition thus containing a
copper-botanical-antioxidant complex;
[0039] 2) identification of carrier vehicle containing penetration
enhancing agents and encapsulation of the copper-pigment
antioxidant complex in stable, submicron liposomes containing
relatively high linoleic acid content;
[0040] 3) locating physiological copper-binding sites in the skin
that show signs of chronic skin pathologies or environmental
damage;
[0041] 4) applying topically to the skin the encapsulated
copper-botanical-antioxidant complex composition to cause enhanced
penetration of the complex to copper binding sites; and,
[0042] 5) simultaneously delivering skin pH-activated copper ions
and plant-based antioxidant pigment to said physiological binding
sites to beneficially treat said skin disorders and to protect
against further skin damage.
[0043] It is disclosed that copper ion (Cu.sup.+2) that is
metallically bound to a botanical pigment, chlorophyllin, can
function as a biologically effective copper-delivery system when
suitably formulated for topical application. The pigment part of
the complex can function as an antioxidant in the skin, thereby
reducing inflammatory oxidizing radicals and skin inflammation. The
list of copper-dependent enzymes that are normally active in the
skin, and thereby affected, includes lysyl oxidase (cross-links
collagen fibrils to form the structurally supportive collagen
fibers), superoxide dismutase (active in the reduction of damaging
oxygen species), tyrosine hydroxylase (important in the synthesis
of melanin and the neurotransmitter norepinephrine). Other
copper-containing enzymes found in the skin are cytochrome C
oxidase (involved in production of the skin's high-energy metabolic
substrates) and dopamine beta-hydroxylase (involved in regulation
of dopamine and norepinephrine). By explicitly targeting donation
of copper to enzymes and copper-dependent wound-healing peptides,
via the compositions and processes disclosed in this invention, we
imply to cover all copper-dependent needs of the skin.
[0044] We have described the importance of copper ions in skin
biology, given the necessary presence of copper at catalytic sites
of various enzymes, noting also its association with small
peptides, such as GHK, which modulate recovery from injury. Whereas
botanically-derived pigments such as chlorophylls and carotenoids
have been used previously in skin care formulations for their
antioxidant potential, binding copper ions with such botanical
pigments, specifically to deliver copper, is new. Chlorophyll and
chlorophyllin have been used to sanitize and deodorize wounds, to
treat burns, blisters, ulcerations, psoriasis, and as additives to
hair growth preparations. Copper ions, in bound forms such as
copper sulfate, copper glycosides, copper sucralphate and copper
gluconate, have had prior use as topical anti-inflammatory agents
and in the treatment of spider veins, cellulite, poison ivy, as
well as in antiviral compositions. Sodium-copper-chlorophyllin has
been previously used as a photomodulation agent: whereby light
energy absorbed by this compound, for example from a laser, is
transferred to a neighboring, endogenous skin-cell pigment, thereby
"energizing" the cell. The copper-pigment may thereby function as a
skin or hair growth stimulation agent. However, copper of this form
has not been viewed to have an active role, other than stabilizing
the molecular structure of chlorophyllin.
[0045] Copper-chlorophyllin has been used previously as an internal
deodorant in tablet form, in combination with proteolytic enzymes
for the debridement and healing of ulcerative wounds and as a
colorant in dentifrice, bone cement, and certain dry foods.
Oil-soluble copper-chlorophyll and water soluble
sodium-copper-chlorophyllin have not been used commercially in
pharmaceutical or cosmeceutical skin care products, except for
limited use as a deodorant and wound healing additive to products
used to treat deep, open wounds such as decubitus ulcers and
colostomy openings. The world patent literature does show the
potential use of sodium-copper-chlorophyllin as deodorant or
disinfectant additive to cosmetic bath cleansers, facial masques,
and general purpose moisturizing creams. However, neither
copper-chlorophyll nor sodium-copper-chlorophyllin have ever been
used commercially in products as topical treatments for chronic
dermatoses or any skin disease, for the treatment of photodamaged
skin or for the treatment of specific facial cosmetic issues such
as enlarged pores, acne scars, or under eye circles, as disclosed
in this invention.
[0046] It may be assumed that sodium-copper-chlorophyllin has not
been used in cosmetic products in any significant way simply
because the material is a dark-green pigment, even at low
concentrations. For example, sodium-copper-chlorophyllin exhibits a
dark-green color in water at 0.1% by weight. Topical use products
are typically uncolored or lightly colored with dyes to avoid
staining of the skin or with insoluble and non-staining inorganic
pigments. Green is, of course, not a natural skin tone. Our studies
show that sodium-copper-chlorophyllin within liposomes, or in an
aqueous gel vehicle with water soluble penetration enhancers, will
penetrate the skin. We disclose here that concentrations up to 0.1%
by weight for very light skin and up to 0.5% by weight for very
dark skin can be used topically; that is, the pigment is absorbed
and is not visibly evident.
[0047] The novelty of the present invention is supported by the
following facts: [0048] 1. Based on history of use, one would not
expect a priori that the oil-soluble copper-chlorophyll or the
water-soluble copper-chlorophyllin would readily penetrate intact
skin or even skin compromised by acne or rosacea; [0049] 2. Based
on history of use, one would not expect the low level of
copper-chlorophyllin, for example, 0.1% by weight--a level used as
a colorant in dentifrice and foods, to produce visible improvements
in skin condition; [0050] 3. Based on history of use, one would not
expect visible reductions in pore size, uneven skin coloring, and
increased tensile strength of skin after twice daily use of
sodium-copper-chlorophyllin for only 2 to 3 weeks; [0051] 4. Based
on history of use, one would not expect significant antimicrobial
activity against the bacterium involved in acne (P. acnes) and
hence significant anti-acne activity.
[0052] These findings are detailed in discussion of experimental
studies presented in the next section.
[0053] The potential of sodium-copper-chlorophyllin to be a
copper-delivery agent, transferring chlorophyllin-bound copper to
copper-dependent enzymes in the skin, is new, as is the concept of
using the pigment to reduce free radicals produced by the
additional free copper load.
[0054] We next outline the preparation of one such "therapeutic
unit", in an embodiment of the invention that utilizes
sodium-copper-chlorophyllin as the copper-pigment complex.
Botanical pigments invariably possess a metal-ligand binding site:
in natural chlorophyll this binding site is occupied by a magnesium
atom. Copper is substituted for magnesium by treating the
chlorophyll with an acid, thereby replacing the magnesium with two
hydrogen ions (protons). Thereafter, the protons are replaced by a
cuprous copper ion (Cu.sup.+2) by alkaline hydrolysis with a copper
salt solution. This process yields oil-soluble copper-chlorophyll.
Subsequent alkaline hydrolysis with a sodium salt opens up the
cyclopentone ring of chlorophyll and replaces the ester groups with
sodium, forming water-soluble sodium-copper-chlorophyllin.
[0055] Liposomes loaded with sodium-copper-chlorophyllin were
prepared at our request by Rovi Cosmetics (Schluchtem, Germany).
Composition (by weight) consisted typically of lecithin (10.00%),
sodium-copper-chlorophyllin (5.00%), ethyl alcohol (3.33%),
Phenonip (0.50%), and water buffered with potassium dihydrogen
phosphate. The pH of the "raw" aqueous liposome dispersion ranged
from 7.5 to 8.5. The material was stored in a dark, cool (5.degree.
C.) area until used in a treatment composition as detailed
below.
[0056] Liposomes containing sodium-copper-chlorophyllin can also be
prepared by using high linoleic acid lecithin (Phospholipon 80.RTM.
or Phospholipon 85.RTM.) supplied by American Lecithin Company
(Oxford, Conn.) or by Phospholipid GmbH (Cologne, Germany). Typical
liposome formulations using Phospholipon lecithin and sodium copper
chlorophyllin are detailed below in Exhibit 1 (Formula number
JPM-01-03-B; all ingredients percentages are by weight)
Exhibit 1
Sodium Copper Chlorophyllin Liposome; Formula # JPM-01-03-B
TABLE-US-00001 [0057] Ingredient % w/w Phospholipon 85G
(Phospholipid GmbH) 10.00 Sodium Copper Chlorophyllin, USP* 5.00
Hydrolite-5 (Symrise, Inc.) 3.00 Butylene Glycol 4.00
Phenoxyethanol 0.30 Deionized Water 77.70 *Supplied by Seltzer
Chemical Inc., Carlsbad, CA
[0058] The above liposome formula (JPM-01-03-B) is made by first
combining and dissolving the butylene glycol, Hydrolite-5
(1,2-pentylene glycol), and phenoxyethanol in the deionized water.
This mixture is heated to approximately 50.degree. C. before adding
the sodium copper chlorophyllin. This mixture is mixed until the
chlorophyllin salt is fully dissolved. The mixture is then cooled
to 25-30.degree. C. and the Phospholipon lecithin is added. The
aqueous solution of sodium copper chlorophyllin and the lecithin
are homogenized with a Waring.RTM. type blender, Osterizer.RTM.,
Eppenbach.RTM. or Silverson.RTM. homogenizer or a similar mixer
capable of high shear mixing. High shear mixing is continued at
3000-5000 rpm for approximately 15 minutes to create a uniform
liposome dispersion with the average liposome particle measuring
between 150-350 nanometers.
[0059] The resulting liposomal dispersion of sodium copper
chlorophyllin is a dark green, syrup-like liquid with a pH of
between 8.5-9.5. Buffer solution may be added to maintain the pH of
the final liposome dispersion between 7.5-8.5.
[0060] Liposome dispersions of the sodium copper chlorophyllin can
also be made by changing the ratio of Phospholipon from the 2:1
ratio of lecithin: sodium copper chlorophyllin used in Exhibit 1 to
higher or lower ratios of Phospholipon to chlorophyllin salt.
[0061] In another embodiment of the invention, the raw liposomal
dispersion supplied by Rovi Cosmetics GmbH was formulated into a
cosmeceutically acceptable gel of the following composition shown
below in Exhibit 2 (all components are listed in percentage by
weight):
Exhibit 2
Chlorophyllin Treatment Gel; Formula #28-145
TABLE-US-00002 [0062] Ingredient % w/w Carbopol 940; 2% dispersion
55.00 1,3-Butylene Glycol 4.00 Ethanol SD 40, 190 Proof 3.50 Sodium
Lactate, 60% (Patlac NAL; RITA) 1.60 Pentylene Glycol (Hydrolite-5)
4.00 Phenoxyethanol 0.75 Sodium Hydroxide solution, 25% 1.50
Rovisome I chlorophyllin* (Rovi) 2.00 Deionized Water (add a
sufficient amount to make) 100.00 *Rovisome I chlorophyllin is a
custom liposomal dispersion containing 5% w/w
sodium-copper-chlorophyllin complex in a high linoleic acid
lecithin shell.
[0063] The final concentration of sodium-copper-chlorophyllin in
the above treatment gel is 0.1% w/w. The pH of the gel was
typically adjusted to between 7.2-7.6 with NaOH solution and the
formula amount shown is increased until a minimum pH of 7.2 is
achieved. The formula shown in Exhibit 1 above would be made by
first creating a Carbopol.RTM. (Noveon Corporation) gel adjusted to
a pH of between 7.2-7.6 with sodium hydroxide solution, adding all
ingredients other than the Rovisome chlorophyllin liposome and, as
a last step, uniformly dispersing the Rovisome chlorophyllin
liposome throughout the neutralized gel. Formulas typified by
Exhibit 2 have shown to maintain viscosity, pH and uniform
dispersion of the sodium copper chlorophyllin liposome through
accelerated stability tests that would project to several years of
shelf life without significant change.
[0064] The topical gel shown in Exhibit 2 has also been formulated
by creating submicron (150-350 nanometer) liposomes of
sodium-copper-chlorophyllin complex with the use of Phospholipon
85G supplied by American Lecthin Company as previously shown in
Exhibit 1. This liposome formula (JPM-01-03-B) was then
incorporated in a topical gel with the final formula depicted in
Exhibit 3 below (all ingredient percentages are by weight):
Exhibit 3
Chlorophyllin Treatment Gel; Formula # JPM-01-35
TABLE-US-00003 [0065] Ingredient % w/w Carbopol 940; 2% dispersion
55.00 1,3-Butylene Glycol 4.00 Sodium Lactate, 60% (Patlac NAL;
RITA) 1.60 Pentylene Glycol (Hydrolite-5) 4.00 Phenoxyethanol 0.75
Sodium Hydroxide solution, 25% 1.50 Sod. Copper Chlorophyllin
liposome # JPM-01-03-B* 2.00 Deionized Water (add a sufficient
amount to make) 100.00 *Formula JPM-01-03-B is a liposomal
dispersion of 5% w/w sodium-copper-chlorophyllin complex in a high
linoleic acid lecithin shell (Phospholipon 85G).
[0066] The final concentration of sodium-copper-chlorophyllin in
the above treatment gel (Exhibit 3) is 0.1% w/w. The pH of the gel
was typically adjusted to between 7.2-7.6 with NaOH solution and
the formula amount shown of NaOH solution is increased until a
minimum pH of 7.2 is achieved. The formula shown in Exhibit 3 is
made by first creating a Carbopol gel adjusted to a pH of between
7.2-7.6 with sodium hydroxide solution, adding all ingredients
other than the chlorophyllin liposome and, as a last step,
uniformly dispersing the chlorophyllin liposome (JPM-01-03-B)
throughout the neutralized gel.
[0067] The following studies were conducted to evaluate the
effectiveness of such compositions.
Study 1
[0068] This study looked at the effects of 0.10% by weight
sodium-copper-chlorophyllin, encapsulated within lecithin liposomes
and dispersed in an aqueous gel base, on facial redness in rosacea.
Digital photos were taken, and spectrophotometric measurements
quantified erythema. Color was expressed by the spectrophotometer
in "tristimulus" parameters L*, a*, b*, where L* expresses the
"lightness" or brightness of the skin color, a* expresses the
relative redness of the color using a standard green-to-red color
space axis, and b* expresses the "yellowness" of the skin on a
standard blue-to-yellow color space axis. The value of a* is a
highly sensitive expression of the degree of erythema. Normal
facial skin is measured to have a*-values around 10, whereas
erythemic conditions typical of rosacea elevate a* values to 15,
20, or even higher.
[0069] The gel containing 0.10% sodium-copper-chlorophyllin was
applied by the patients to their own face twice daily for six
weeks. Photographs and calorimetric readings were done at the
pre-treatment visit and repeated at 2, 4 and 6 weeks thereafter.
The patients did not use any other treatments that would reduce
redness. Color measurements were made at each of the four office
visits. The measurement sites selected were the forehead, the left
and right malar regions (cheeks), the nose, and the chin.
Examination of the collected data indicated that colorimeter
readings on the chin are characterized by large sampling error,
because the contour of the chin can not be reproducibly interfaced
with the optical window of the calorimeter. Therefore, readings of
chin color were omitted from the analysis. The four remaining
facial sites (forehead, both cheeks, and the nose) were not
erythemic in all patients, and non-involved sites were also
ignored. With these necessary qualifying conditions, a grand total
of 24 facial regions, distributed among 7 patients, were identified
as significantly elevated in skin redness at the outset and were
followed throughout the study.
[0070] The redness of these 24 facial regions, measured at weeks 0,
2, 4, and 6 of twice-daily treatment, were fit to a statistical
regression line:
y=-0.017x+0.969+Error,
where "Error" refers to the calculated experimental estimation of
a*, y is the fractional skin redness parameter defined as the value
of a* at week x divided by the value a* measured at the
pre-treatment visit (x=0). Consequently, y has the value 1.0 for
the pre-treatment visit, and subsequent values of y represent the
fractional value of a* relative to its value at x=0. This
regression analysis had a two-tailed probability value 0.0063,
which is statistically highly significant, and therefore the -1.7%
slope of the linear regression line is a meaningful conclusion from
the data. This drop rate amounts roughly to a 10% drop in a* over
the six-week duration of the study, corresponding to a drop in a*
of 1-2 units for these patients: which is a visible
improvement.
Study 2
[0071] In prior art, U.S. Pat. No. 6,663,559 "Method and apparatus
for the photomodulation of living cells",
sodium-copper-chlorophyllin is mentioned as a suitable ingredient
for light-based therapy of various skin disorders, specifically
treatment of the skin with LED arrays. Sodium-copper-chlorophyllin
was disclosed therein to enhance the transfer of light energy into
the skin, due to the chromophoric nature of chlorophyllin.
Physiological benefits obtained from release and subsequent
biological uptake of copper and the antioxidant action of
chlorophyllin has heretofore not been recognized.
[0072] We first investigated the impact of LED-treatment on
rosacea, without any copper-pigment complex, and compared the
outcome against the improvements found for the copper-pigment
complex, described in Study 1. Four rosacea patients were treated
twice weekly for two weeks with an LED light source (Gentle Waves
light-emitting-diode system using yellow light at a wavelength of
588 nanometers). Linear regression analysis of normalized a*
values, as in Study 1, measured at two time points, week 0
(pre-treatment) and week 2, gave the equation
y=0.05x+1+Error,
with a 2-tailed probability of 0.20: not statistically significant
(Although not significant, the regression analysis suggests an
average rise, not a decrease in a* over the two-week LED
treatment). For comparison, limiting the analysis of Study 1 data
to only the first two weeks of sodium-copper-chlorophyllin
treatment, gives the regression line
y=-0.054x+1+Error,
with a 2-tailed probability of 0.0022, showing a statistically
significant drop in a* values of 5.4% on average per week for the
first two weeks of treatment with the sodium-copper-chlorophyllin
liposome gel composition.
[0073] It is therefore arguable that the reduction in skin redness
seen in Study 1 is due to biochemical, not optical, properties of
sodium-copper-chlorophyllin. Also, these results are not a foregone
conclusion from prior art that utilized sodium-copper-chlorophyllin
in support of light-based treatment modalities. In fact, our study
suggests that the erythemic component of rosacea may at first be
aggravated by light exposure, whereas sodium-copper-chlorophyllin
without light therapy quickly provides substantial results.
Study 3
[0074] A similar study was carried out on skin redness in two
rosacea patients, utilizing an intense pulsed light (IPL) source
and results were compared against the outcome obtained on the same
patients using the same 0.10% sodium-copper-chlorophyllin gel.
Regression analysis of changes in the redness parameter a* of nine
IPL-treated facial sites (cheeks), receiving light-treatment at the
6 and 10 week time points, gave the statistical result
y=1-0.002x+Error,
predicting essentially a flat response over the 10-week study, with
a two-tailed probability of 0.81: which is not statistically
significant. Subsequent treatment of these patients with the
sodium-copper-chlorophyllin gel gave the linear regression
model
y=-0.020x+0.989+Error,
with a 2-tailed probability of 0.0016, which is highly
statistically significant. These results essentially corroborate
those found in Study 1 and in Study 2: during the first few weeks
of light treatment, the copper-pigment complex reduces the measured
value of the skin redness parameter a* by about 2% per week,
whereas the LED and IPL treatments do not yet appear to elicit a
significant response.
Study 4
[0075] In this clinical trial, an aqueous gel base containing the
dispersion of lecithin liposomes and 0.10% by weight
sodium-copper-chlorophyllin was evaluated for its effectiveness in
treating large pores on the nose and/or cheeks, acne, oiliness of
skin, and blotchiness (uneven reddish skin color) of ten subjects
with mild to moderate acne. The gel was applied to the nose and
cheeks twice daily for four weeks. Skin condition was evaluated by
both the patients themselves and by an expert clinical grader.
Methods of clinical evaluation included visual examination
(counting of acne lesions and enlarged pores, visible skin oiliness
and smoothness of skin texture) and measurements of oiliness using
Sebutape.RTM., and digital photography. Evaluations were carried
out at the start of the study and after four weeks of treatment.
Results: After four weeks of treatment most of the ten patients had
a decrease in skin oiliness (8/10), most had fewer enlarged pores
(9/10), a few had less acne (3/10), less sebaceous thickening of
the skin (4/10), and smoother skin (3/10). In their
self-assessment, all ten patients felt that their skin condition
improved, especially with regard to reduced oiliness, pore size and
overall appearance. Sebutape measurements were made at four facial
sites: the right side and left side of the forehead, plus the nose
and the chin. A global parameter of overall skin oiliness was
calculated by summing these four Sebutape measurement values for
each patient at the start of the study, and again at two weeks and
at four weeks into the treatment. The Sebutape results showed an
average 9% reduction in the amount of skin-surface oil after two
weeks and an average reduction of 13% at four weeks; the latter is
statistically highly significant. It was also noticed that most of
the patients had reduced inflammation (redness), particularly one
patient with blotchy facial redness. Overall, the study indicated
that the treatment results in dramatic reduction of
inflammation.
Study 5
[0076] This study was conducted at a different clinical research
site, and again examined the benefits of a twice-daily facial
application of the 0.10% sodium-copper-chlorophyllin gel. Ten
subjects, men and women 18-30 years of age, were enrolled in the
study. Each subject had mild to moderate acne, with large, visible
pores on the nose and/or cheeks, oily skin and blotchy skin
coloration. Clinical grading of enlarged facial pores, oiliness and
blotchiness were performed during the panelists' initial visit and
repeated after three weeks of treatment. Acne was evaluated by
counting inflammatory lesions (papules, pustules and nodules) and
non-inflammatory lesions (open and closed comedones) for the full
face (forehead, left and right cheeks and chin) at the initial
visit and after three weeks. The subjects also provided
self-assessment diary data of their skin condition throughout the
study. Results: The following table summarizes the percentage of
statistically significant improvements determined at the three-week
time point compared with pre-treatment values, as determined by
clinical grading.
TABLE-US-00004 Attribute Percentage Improvement Oiliness -36.1%
Enlarged pores -21.9% Blotchiness -26.8% Closed Comedones -28.3%
Global Acne Score -13.0% Face Overall -18.6%
[0077] The majority of subjects (8/10) noted various degrees of
improvement in their own skin condition, mainly with respect to
reduced oiliness, visibility of pores and evenness of color and
texture. Digital photographic analysis utilizing the VISIA.RTM.
clinical grading system calculated significant reduction in pores,
acne-related porphyrins, and improvement in overall evenness of
skin.
Study 6
[0078] A small clinical study was carried out using 5 panelists, to
investigate whether sodium-copper-chlorophyllin enhances skin
tanning following UV-light irradiation. We were in effect testing
whether the gel preparation increases tyrosinase activity by
increasing the availability of copper in the skin. The net effect
of sodium-copper-chlorophyllin on tanning was somewhat
unpredictable, because even if copper stimulates melanogenesis by
increasing the activity of tyrosinase, the impact of UV may be
reduced, because chlorophyllin is a strong UV absorber (sunscreen)
and chlorophyllin is also a strong antioxidant, expected to reduce
erythema. We therefore compared the degree of tanning produced by
the sodium-copper-chlorophyllin (0.10% by weight) against the
tanning produced by an identical preparation of
sodium-magnesium-chlorophyllin (0.10% by weight). The
magnesium-complex of chlorophyllin corresponds to the actual
botanical-derived form of chlorophyll, and has approximately the
same UV-absorbance and sunscreen attributes as the copper form.
Therefore, any increase in melanin formation by the copper-complex
over that for the magnesium-complex would be due to copper.
[0079] Five people, 18-65 years of age, participated in the study.
All were Fitzpatrick skin type III or IV; that is, all five
subjects had substantial tanning capability. The minimal erythemal
dose (MED) was determined on the lower back of each subject over
the first two days of the study. Thereafter 200 microliters of a
treatment gel containing 0.10% by weight of either
sodium-copper-chlorophyllin or sodium-magnesium chlorophyllin in a
liposomal dispersion as described in Studies 1 and 2 was applied to
1 cm.sup.2 sites on the lower back and covered with a
semi-occlusive skin patch. The gels were re-applied and patched
daily for 5 consecutive days to randomly assigned and coded sites
on either side of the lower back. After 5 days, the final patches
were removed and the test areas were irradiated with simulated
solar light, at dosages of either 1.5 or 2.0 MED. Two untreated
sites were also irradiated at 1.5 and 2.0 MED for comparison. The
irradiated sites, treated and control, were visually graded for
"Darkness" and "Degree of Tanning" at 4 days and 7 days
post-irradiation. Darkness scores refer to total skin pigment
(hemoglobin and melanin), whereas Tanning scores refer more to
melanin. Treated areas of each subject were photographed at 7 days
after irradiation using macrophotographic techniques, with and
without polarized light. Results: The Darkness ranking (in which
the sites are ranked on a scale of 1-6, with 1 being the darkest),
resulted in the following average scores:
TABLE-US-00005 Treatment Average Darkness Score Std. Dev. Untreated
3.95 2.03 Mg-Chlorophyllin 3.55 1.60 Cu-Chlorophyllin 2.80 1.47
[0080] Given the small sample size, the differences amongst the
three types of sites are not statistically significant, but
directionally the scores show the copper-chlorophyllin treated
sites to be the darkest.
[0081] The Degree of Tanning scores proved to be more
interesting:
TABLE-US-00006 Treatment Average Tanning Score Std. Dev. Untreated
5.55 1.48 Mg-Chlorophyllin 5.25 0.98 Cu-Chlorophyllin 6.15 1.84
(Degree of Tanning is ranked on a scale: 0 = very light tan and 10
= very deep tan).
[0082] This study, although involving only 5 subjects, showed the
expected directional differences in the tanning response, in that
1) the magnesium-chlorophyllin treated sites had less tanning than
the untreated sites, presumably because of the UV-light absorption
and antioxidant protection afforded by chlorophyllin, and 2) the
presence of the copper atom gave a substantial boost in the tanning
response compared to that seen with Mg-chlorophyllin, presumably
showing the copper effect.
[0083] A small study, on one subject, was conducted to see whether
encapsulation of the sodium-copper-chlorophyllin within the
liposome enhanced skin tanning, i.e., increased penetration and
bioavailability of copper. The study compared treatment with 0.10%
by weight sodium-copper-chlorophyllin in a treatment gel
composition similar to Exhibit 2, with and without liposome. Three
areas on the back received 1.0 MED simulated solar radiation: one
area had received no treatment, one area had been pre-treated with
the gel without liposome and the third area was treated with the
liposome gel. An expert blinded grader scored the resulting skin
tanning at 10 days after irradiation, using the Degree of Tanning
scale 0 to 10 (darkest). The control, untreated area showed very
little tanning and was given a tanning score of 1-2; the area
treated using the gel without liposome was scored as 6.0-6.5; the
area treated with the liposome gel was scored as 8. These results
suggest copper from the copper-pigment complex applied in a vehicle
with penetration-enhancing agents reaches and is picked up by
melanocytes, with and without the liposome, but that the liposome
enhances penetration beyond that achievable with the penetration
enhancers used.
Study 7
[0084] The unexpected result that the sodium-copper-chlorophyllin
gel used in the above described clinical studies reduced
acne-associated inflammation and porphyrins led us to propose that
we were observing a copper-mediated antimicrobial effect on
Propionibacterium acnes. To test this hypothesis, a "kill rate"
test against P. acnes was conducted at a microbiological testing
laboratory. The study compared the antimicrobial properties of the
treatment gel containing 0.1% sodium-copper-chlorophyllin against
the same gel composition with 0.1% sodium-magnesium-chlorophyllin.
Antimicrobial activity of the two gels was determined using the
standard methodology of counting the number of organisms on test
plates covered with the respective gels after 1 hour and 24 hours
of incubation. The kill rate is calculated as the logarithmic
reduction in the concentration of organisms when compared with the
concentration of organisms in the original inoculation material.
Results are summarized in the following table:
Treatment Gel with 0.1% w/w Sodium Copper Chlorophyllin
TABLE-US-00007 [0085] Organism Inoculum Level Average Log Reduction
P. acnes 2.95 .times. 10.sup.5 No Growth 5.47 1 hour P. acnes 2.95
.times. 10.sup.5 No Growth 5.47 24 hours
Treatment Gel with 0.1% w/w Sodium Magnesium Chlorophyllin
TABLE-US-00008 [0086] Organism Inoculum Level Average Log Reduction
P. acnes 2.95 .times. 10.sup.5 6.500 1.66 1 hour P. acnes 2.95
.times. 10.sup.5 2.000 2.17 24 hours
[0087] A log reduction of 1.66 or 2.17 obtained with the
sodium-magnesium-chlorophyllin gel at one hour and 24 hours,
respectively, is considered ineffective antimicrobial activity, and
may in fact be due to just the gel base itself. On the other hand,
the total kill of P. acnes seen with sodium-copper-chlorophyllin
gel at both incubation times suggests very strongly that the copper
exerts significant antimicrobial activity, and we propose it is the
copper that dissociates from chlorophyllin that is responsible for
the reduction of acne symptoms seen in the clinical studies. The
treatment gel vehicle used in the above studies was identical to
the formula shown in Exhibit 2 except for the use of Rovisome I
chlorophyllin (sodium copper chlorophyllin liposome; Rovi) in one
treatment gel and Rovisome II chlorophyllin (sodium magnesium
chlorophyllin liposome; Rovi) in the other treatment gel.
* * * * *