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Cancer Patent Abstract
Described are skin-care compositions containing non denatured soy
products and optionally other anti-cancer or anti-aging agents.
The compositions can be applied topically to reduce the risk of
UV-induced cutaneous tumors.
Cancer Patent Claims
What is claimed is:
1. A method of inhibiting the progression of a cutaneous tumor
comprising topical application of at least one composition containing
a non-denatured, Kunitz-type soybean trypsin inhibitor in an amount
of from about 0.01-99% by weight.
2. The method of claim 1, wherein said composition is applied at
least once daily on a continuous basis.
3. The method of claim 1, wherein said composition is applied at
least twice daily for at least eight weeks and at least once daily
on a continuous basis thereafter.
4. The method of claim 1, wherein said composition comprises 0.1-10%
by weight of said non denatured soybean trypsin inhibitor and is
applied for at least about four to about ten weeks followed by topical
application of a composition comprising 0.01-10% by weight of said
non denatured soybean trypsin inhibitor on a daily basis thereafter.
5. The method of claim 1, wherein said non-denatured soybean trypsin
inhibitor is contained within liposomes suspended in a cosmetically
acceptable carrier.
6. The method of claim 4, wherein said non-denatured soybean trypsin
inhibitor is contained within liposomes suspended in a cosmetically
acceptable carrier.
7. The method of claim 1, wherein said composition further comprises
a cosmetically acceptable vehicle.
8. The method of claim 1, wherein said composition further comprises
at least one anti-inflammatory agent.
9. The method of claim 1 wherein said composition further comprises
at least one anti-cancer agent.
10. The method of claim 1, wherein said composition further comprises
at least one anti-oxidant.
11. The method of claim 1, wherein said composition further comprises
at least one sunscreen.
12. The method of claim 1, wherein said composition further comprises
from about 0.1 to about 20% emulsifier, and a preservative in an
effective amount.
13. The method of claim 12, wherein said composition further comprises
an anti-oxidant.
14. The method of claim 12, wherein said composition further comprises
an anti-cancer agent.
15. The method of claim 12, wherein said composition further comprises
at least one compound selected from the group consisting of anti-oxidants,
sunscreens, moisturizers, bleaching agents, depigmentation agents,
darkening agents, surfactants, foaming agents, conditioners, humectants,
fragrances, anti-aging agents, anti-inflammatory agents, and anti-cancer
agents.
Cancer Patent Description
FIELD OF THE INVENTION
This invention relates to compositions containing non-denatured
soy products, or soy trypsin inhibitors, and optionally additional
anti-cancer or cosmetically active agents. These compositions can
be applied topically to reduce the risk of UV-induced cutaneous
tumors.
BACKGROUND OF THE INVENTION
Skin, the largest organ of the human body, is continuously exposed
to environmental insults such as smoke, pollution, and ultraviolet
(UV) irradiation. The thinning of the ozone layer, which is expected
to progress for at least several decades, reduces a major barrier
to the passage of ultraviolet-B radiation (UVB) through the atmosphere.
UVB, that is, light whose wavelength is in the range between about
280 and about 320 nm, is the main cause of sunburn, tanning, aging
of the skin, and skin cancer.
The non-melanoma skin cancers (NMSC), including basal-cell and
squamous-cell carcinoma, are the most common types of cancer among
Caucasian populations. The incidence of NMSC has increased worldwide
over the last few decades. Increased recreational and occupational
sunlight exposure is commonly regarded as one of the reasons for
the higher incidence of cutaneous cancers. The increase in UVB exposure
associated with the thinning of the ozone layer is another significant
factor. Mortality from NMSC is low, but the estimated recurrence
rate of about 50% after five years and the local invasiveness of
this type of cancer result in high medical costs. Therefore, NMSC
constitutes a substantial public health concern. (Reviewed in Holick
and Kligman, editors: Biologic effects of light. Walter de Gruyter,
Berlin and New York, 1992).
Photo-carcinogenesis results from a complex interplay of simultaneous
and sequential biochemical events. These events, initiated by irradiation
of an organism with UV light of an appropriate wavelength, include
the formation of DNA photo-products, inaccuracies in DNA repair,
mutation of proto-oncogenes and tumor suppressor genes, and UV-induced
production of radical species which produce subsequent effects on
existing mutations and independently induce further mutations. In
addition, other epigenetic events such as immunological responses,
antioxidant defenses, and dietary factors may influence the course
of carcinogenesis. (Black, H. S., deGruijl, F. R., Forbes P. D.,
Cleaver, J. E., Ananthaswamy, H. N., deFabo, E. C., Ullrich, S.
E., Tyrrell, R. M., Photo-carcinogensis: an overview. J. Photochem.
Photobiol. B 40:1, 29-47, Aug., 1997).
The skin possesses an elaborate antioxidant defense system to deal
with UV-induced oxidative stress. Excessive exposure to UV radiation,
however, can overwhelm the cutaneous antioxidant capacity, leading
to oxidative damage and ultimately to skin cancer and premature
skin aging. Therefore, one strategy for photo-protection is to support
the endogenous antioxidant system by induction or transdermal delivery
of antioxidant enzymes or nonenzymatic antioxidants. Antioxidants
such as glutathione, alpha-tocopherol, ascorbate and beta-carotene
have been found to be very effective in photoprotection. Components
of the antioxidant pathway have also been identified and applied
to the skin of patients. Although skin treatments with single components
of the antioxidant system such as vitamin E were successful against
a wide variety of types of photodamage, they were not shown to affect
the progression of UV-induced tumors. The most promising results
were obtained in studies combining several compounds, which often
resulted in synergy between the protective effects. (Steenvoorden
D. D., van Henegouwen G. M., The use of endogenous antioxidants
to improve photoprotection, J. Photochem. Photobiol., B 41:1-2,
1-10, November, 1997).
Epidemiological studies suggest that components of vegetables,
particularly legumes, are beneficial in lowering the incidence rates
of many types of cancer. For example, the rates of breast, colon
and prostate cancer are significantly lower among the inhabitants
of most countries of the Pacific Basin, but offspring of Pacific
Basin natives who have migrated to the United States develop the
common Western cancers at approximately the same rate as native
Westerners. Such epidemiological studies suggest that dietary and
other environmental factors, rather than genetic differences, contribute
more significantly to the risk of susceptibility to these cancers.
The high consumption of soybean products in Pacific Basin countries,
such as Japan, implicates diet as one factor contributing to the
relatively extremely low rates of cancer mortality in these countries.
(E.g., Wu et al., Soy intake and risk of breast cancer in Asians
and Asian Americans. Am. J. Clin. Nutr. 68: 6 Suppl., 1437S-1443S,
December, 1998).
Soybeans are a rich source of isoflavones, which possess weak estrogenic
activity. Genistein, the main soybean isoflavone, is a specific
inhibitor of protein tyrosine kinases and of other enzymes involved
in signal transduction. Genistein has been shown to suppress the
growth of numerous cancer cells in vitro, and to protect animals
in experimental carcinogenesis models from developing both hormone-
and non-hormone related cancers. (Reviewed in A. R. Kennedy, Chemopreventive
agents: Protease inhibitors, Pharmacology Theories 78 (3), 167-209),
1998 and in Messina et al., Soy intake and cancer risks: A review
of the in vitro and in vivo data, Nutrition and Cancer 21 (2), 113-131,
1994).
Soybeans also contain a number of protease inhibitors such as BBI
and STI. It is important to note that soy foods do not contain high
concentrations of active STI and BBI, because these protease inhibitors
block the action of trypsin and other enzymes needed for protein
digestion. Although STI is denatured by cooking, heat alone does
not inactivate BBI, and consumption of soy products containing high
levels of these protease inhibitors leads to serious digestive problems,
chronic deficiency in amino acid uptake, and cancer. Indeed, the
Chinese did not serve soybeans as food until fermentation techniques
were developed to destroy the anti-digestive properties of the soy
foods (2nd century B.C.E.). During the production of soy foods today,
pureed soybeans are soaked in an alkaline solution and then pressure-heated
to 115.degree. C. in order to denature the protease inhibitors as
much as possible.
Limtrakul et al. attempted to identify a safe level of soy proteins
for nutritional consumption, which would be beneficial in the prevention
of cancer. Skin tumors were chemically induced in mice, which were
fed soy protein isolate (SPI) exclusively, and in mice which were
fed SPI supplemented with soymilk proteins (SMP). It was reported
that "the percentage of tumor-bearing mice and the volume of
tumor tended to be lower in the mice on the SMP diet". Life
Sciences 1993, 53, 1591-1596. When defatted soybeans are treated
first with alkaline, then with acid solution, SPI is the precipitate
and SMP is the supernatant. The Limtrakul study shows the potential
of soy proteins to affect skin cancer progression, when the proteins
are orally consumed. However, it was also emphasized that higher
levels of dietary intake of SMP would result in major health problems.
It is clear that a need exists for safe, efficacious and economical
agents that prevent or reduce incidence of cancer, particularly
for NMSC, which may be simply and conveniently administered. Further,
economical and prophylactic compositions and methods for the reduction,
prevention or inhibition of the progression of UV-induced cutaneous
tumors are highly desirable. Since topical application is very simple
and convenient, incorporating compositions that reduce skin cancer
incidence into a skin-care product would be extremely advantageous.
While sunscreens are known to reduce the damage resulting from UV
exposure during the period of their application, there is a need
for a skin care product that could also slow the progression of
already-initiated photocarcinogenic processes. It is an object of
the invention to provide such a product.
SUMMARY OF THE INVENTION
The present invention provides a method of reducing the risk of
developing UV-induced tumors of the skin of a mammal by topically
applying a skin-care composition, preferably to an individual who
has already been exposed to or irradiated with UV light. A method
of reducing the growth rate of UV-induced cutaneous tumors by topically
applying the skin-care composition is also provided, as is a method
of preventing the progression of cancer by the same means.
The skin care composition for use in the methods of the invention
is formulated for the topical delivery of a non-denatured soy product
(e.g., to a mammal such as a human) and comprises a soy product
(e.g., a non-denatured soymilk or soybean powder or soybean trypsin
inhibitor) and a vehicle. The composition may optionally comprise
other anti-cancer or cosmetically active agents. Certain skin care
compositions appropriate for use in the present invention have been
described in U.S. patent application Ser. Nos. 09/110,409, 09/621,565
and 09/698,454, filed Jul. 6, 1998, Jul. 21, 2000 and Oct. 27, 2000,
respectively, and in International Application No. WO99/04752. Each
of the foregoing patent documents is incorporated herein by reference.
Other features and advantages of the present invention will be
apparent to those of skill in the art in light of the following
description and claims.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGS. 1A-1C are a series of graphs showing the inhibitory effects
of STI and BBI on the development of skin tumors in SKH-1 mice previously
treated with ultraviolet B light. (BSA--bovine serum albumin; BBI--Bowman-Birk
Inhibitor; STI--Soy Bean Trypsin Inhibitor)
FIGS. 2A-2C are a series of graphs showing the inhibitory effect
of Soymilk on the development of skin tumors in SKH-1 mice previously
treated with ultraviolet B light. The inhibitory effects of heat-denatured
soymilk vs. non-denatured soymilk are compared.
FIGS. 3A-3C are a series of graphs showing the inhibitory effects
caffeine on the development of skin tumors in SKH-1 mice previously
treated with ultraviolet B light.
FIGS. 4A-4C are three photographs showing the reduction in size
and number of UV-induced tumors among mice treated topically with
non-denatured soymilk (FIG. 4A) compared to mice treated with heat-denatured
soymilk (FIG. 4B) or water (FIG. 4C).
FIGS. 5A and 5B are a pair of photographs showing the reduction
in size and number of UV-induced tumors among mice treated topically
with a solution of caffeine in acetone (FIG. 5B) compared to mice
treated with acetone alone (FIG. 5A).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
It is believed that one skilled in the art can, based upon the
description herein, utilize the present invention to its fullest
extent. The following specific embodiments are to be construed as
merely illustrative and not limitative of the remainder of the disclosure.
All publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
The present invention is directed to soy-containing compositions
and methods of use thereof in the prevention and reduction of the
risk of skin cancer. The novel compositions of this invention contain
legume products, and preferably soy products, that may be in the
form of a fluid (e.g., soymilk) or a solid (e.g., a soybean powder
or soymilk powder). What is meant by "soy product" is
a substance derived from the soybean, containing the ingredients
naturally found in soybeans, at the relative concentrations as found
in the beans, excluding water content. In one embodiment, the soy
product is a non denatured soy product.
"Denaturation" is defined in the Bantam Medical Dictionary
(1990 edition) as "the change in the physical and the physiological
properties of a protein, that are brought about by heat, X-rays
or chemicals. These changes include loss of activity (in the case
of enzymes) and loss (or alteration) of antigenicity (in the case
of antigens)".
What is meant by "non-denatured soy product" is a soy
product in which the processing for the derivation of such soy product
(e.g., the temperature, extraction media) did not eliminate its
protease inhibitory activity. In one embodiment, the non-denatured
state of the soy product of this invention is measured by the presence
of an intact soybean trypsin inhibitor (STI) protein.
In another embodiment, the soy product is soymilk. One way to make
soymilk is to soak the soybeans in deionized or purified water for
several hours, and grind them after they were fully hydrated, with
the addition of small quantities of water. (The grinding process
allows the soybean milk to be extracted). After collection, the
soybean milk may be filtered to remove any residual parts of the
bean husk. The soymilk used in this invention can be fresh soymilk
as described above, or may be made from soybean powder and water.
The soybean powder is milled from soybeans and may also be lyophilized,
spray dried, or freeze-dried and the resulting soymilk may or may
not be filtered. Soymilk prepared by these methods may have from
about 1 to about 90% by weight dry soybean powder. Another example
is the use of soymilk powder, made from lyophilized, spray dried
or freeze-dried soymilk, with the addition of water and finished
with or without filtration or homogenization.
Other methods of soybean extraction could also be used to create
the active ingredients used in this invention. In one example, the
active ingredients could be extracted from ground soybeans using
ethanol/water mixtures, followed by the removal of the ethanol from
the extract, in such ways that the protease inhibitory activity
of the soybean will be retained.
The compositions of the present invention may contain from about
1% to about 99%, by weight, of the soy product. For example, when
a liquid soy product (e.g., soymilk) is used, the composition may
contain from about 50% to about 99%, by weight, (e.g., from about
70% to about 99%) of the liquid soy product. For example, when a
solid soy product (e.g., soybean powder or soymilk powder) is used,
the composition may contain from about 1% to about 50%, by weight
(e.g., from about 2% to about 30%, by weight) of the solid soy product.
Compositions comprising solid soy products may also comprise water
(e.g., distilled water or water contained within soymilk) to form
a liquid base for the composition (e.g., to form a cream, lotion,
injectable solution or gel). Such compositions may comprise from
about 50% to about 98%, by weight (e.g., from about 70% to about
98%, by weight) of water. While not limited to these methods of
administration, the compositions of this invention may be delivered
topically, orally, or parenterally, although topical administration
is preferred.
The soy products useful in this invention may be produced from
all soybean species, regardless of their geographic origin, sun
exposure, harvest time and the like. However, specific strains,
geographic origins or growth conditions might be preferred. These
include soybean strains or other legume strains particularly rich
in their trypsin inhibitor (e.g. STI, LTI, BBI) content or strains
in which, under the proper growth conditions trypsin inhibitor enrichment
occurs in the bean. It should be noted that the legume products
useful in the compositions of this invention have a distinctive
odor, which may be tolerable in some cultures, but is undesired
in others. If necessary, the odor of the compositions of this invention
can be reduced by using soybean products derived from specific strains
of soybeans known to be less odiferous, including, but not limited
to, lipoxygenase-2-deficient beans and those having a modified sugar
profile, or the like. A process to reduce oxygen levels in the formulation
may also reduce the odor. Various masking agents or fragrances may
also be used to mask the odor.
In yet another embodiment of the invention, the soy-containing
compositions may optionally comprise additional synthetic or natural
anti-cancer agents. Examples of such agents include, without limitation,
caffeine, Milk Thistle extract, green tea extract, epigallocathechin
gallate, silymarins, glucocorticoids and 5-fluorouracil.
A preferred embodiment of the invention comprises the administration
of soymilk containing compositions before or after the initiation
of UV-induced skin cancer. Especially preferred are embodiments
in which the soymilk is not denatured, leaving STI and BBI intact.
Soymilk also contains genistein and other isoflavones, and anti-oxidants
such as the gamma form of vitamin E, which is essential to the health
of the skin. While not wishing to be held to any particular theory,
it is hypothesized that these different active components also participate
in the prevention of tumor progression. Soymilk also contains lecithins
and other emulsifying molecules that facilitate the transdermal
delivery of the active components.
As explained above, the present invention extends to a topical
cosmetic or pharmaceutical composition comprising a non-denatured
soy product (e.g., a non-denatured soymilk or soybean powder) and
a cosmetic or pharmaceutically acceptable vehicle and, optionally,
additional anti-cancer or cosmetically active agents. As used herein,
"topically applying" means directly laying on or spreading
on outer skin, e.g., by use of the hands or an applicator such as
a wipe, roller, or spray.
The phrase "cosmetic or pharmaceutically acceptable"
refers to entities and compositions that are physiologically tolerable
and do not typically produce an allergic or similar untoward reaction
when administered to a human. As used herein, "cosmetically
acceptable" means that the ingredients which the term describes
are suitable for use in contact with tissues (e.g., the skin) without
undue toxicity, incompatibility, instability, irritation, allergic
response, and the like.
The term "vehicle" refers to a diluent, adjuvant, excipient,
or carrier. Such cosmetic or pharmaceutical vehicles can be liquids,
such as water and oils, including those of petroleum, animal, vegetable
or synthetic origin, such as peanut oil, soybean oil, mineral oil,
sesame oil and the like. In the art of formulating skin care compositions,
the vehicle is often an oil-in-water or a water-in-oil emulsion.
Suitable pharmaceutical carriers are described in "Remington's
Pharmaceutical Sciences" by E. W. Martin. Suitable cosmetic
carriers are described below.
The compositions for use in the methods of the present invention
include formulations suitable for topical application to skin. In
one embodiment, the composition comprises a non-denatured soy product
and a cosmetically acceptable topical carrier. In one embodiment,
the cosmetically acceptable topical carrier is from about 50% to
about 99.99%, by weight, of the composition (e.g., from about 80%
to about 99%, by weight, of the composition).
The compositions may be made into a wide variety of product types
that include, but are not limited to, solutions, lotions, creams,
gels, sticks, sprays, ointments, cleansing liquid washes, solid
bars, shampoos, pastes, foams, powders, mousses, shaving creams,
wipes, patches, nail lacquers, wound dressing, adhesive bandages,
hydrogels, and films. Make-up, such as foundations, mascaras, and
lipsticks also form suitable compositions. These product types may
comprise several types of cosmetically acceptable topical carriers
including, but not limited to solutions, emulsions (e.g., microemulsions
and nanoemulsions), gels, solids and liposomes. Certain non-limitative
examples of such carriers are set forth hereinbelow. Other suitable
carriers may be formulated by those of ordinary skill in the art.
Topical compositions useful in the subject invention may be formulated
as a solution comprising an emollient. Such compositions preferably
contain from about 1% to about 50% of an emollient(s). As used herein,
the term "emollient" refers to materials used for the
prevention or relief of dryness, as well as for the protection of
the skin. A wide variety of suitable emollients is known and may
be used in the present invention. Sagarin, Cosmetics, Science and
Technology, 2nd Edition, Vol. 1, pp. 32-43 (1972) and the International
Cosmetic Ingredient Dictionary and Handbook, eds. Wenninger and
McEwen, pp. 1656-61, 1626, and 1654-55 (The Cosmetic, Toiletry,
and Fragrance Assoc., Washington, D.C., 7.sup.th Edition, 1997)
(hereinafter "ICI Handbook") contains numerous examples
of suitable materials.
A lotion can be made from such a solution. Lotions typically comprise
from about 1% to about 20% (e.g., from about 5% to about 10%) of
an emollient(s) and from about 50% to about 90% (e.g., from about
60% to about 80%) of water.
Another type of product that may be formulated from a solution
is a cream. A cream typically comprises from about 5% to about 50%
(e.g., from about 10% to about 20%) of an emollient(s) and from
about 45% to about 85% (e.g., from about 50% to about 75%) of water.
Yet another type of product that may be formulated from a solution
is an ointment. An ointment may comprise a simple base of animal
or vegetable oils or semi-solid hydrocarbons. An ointment may comprise
from about 2% to about 10% of an emollient(s) plus from about 0.1%
to about 2% of a thickening agent(s). A more complete disclosure
of thickening agents or viscosity increasing agents useful herein
can be found in Sagarin, Cosmetics, Science and Technology, 2nd
Edition, Vol. 1, pp. 72-73 (1972) and the ICI Handbook pp. 1693-1697.
The topical compositions useful in the present invention may be
formulated as emulsions. If the carrier is an emulsion, from about
1% to about 10% (e.g., from about 2% to about 5%) of the carrier
comprises an emulsifier(s). Emulsifiers may be nonionic, anionic
or cationic. Suitable emulsifiers are disclosed in, for example,
in McCutcheon's Detergents and Emulsifiers, North American Edition,
pp. 317-324 (1986), and the ICI Handbook, pp.1673-1686.
Lotions and creams can be formulated as emulsions. Typically such
lotions comprise from 0.5% to about 5% of an emulsifier(s). Such
creams would typically comprise from about 1% to about 20% (e.g.,
from about 5% to about 10%) of an emollient(s); from about 20% to
about 80% (e.g., from 30% to about 70%) of water; and from about
1% to about 10% (e.g., from about 2% to about 5%) of an emulsifier(s).
Single emulsion skin care preparations, such as lotions and creams,
of the oil-in-water type and water-in-oil type are well known in
the cosmetic art and are useful in the present invention. Multiphase
emulsion compositions, for example the water-in-oil-in-water type,
as disclosed in U.S. Pat. Nos. 4,254,105 and 4,960,764, may also
be useful in the present invention. In general, such single or multiphase
emulsions contain water, emollients, and emulsifiers as essential
ingredients.
The topical compositions of this invention can also be formulated
as a gel (e.g., an aqueous, alcohol, alcohol/water, or oil gel using
a suitable gelling agent(s)). Suitable gelling agents for aqueous
gels include, but are not limited to, natural gums, acrylic acid
and acrylate polymers and copolymers, and cellulose derivatives
(e.g., hydroxymethyl cellulose and hydroxypropyl cellulose). Suitable
gelling agents for oils (such as mineral oil) include, but are not
limited to, hydrogenated butylene/ethylene/styrene copolymer and
hydrogenated ethylene/propylene/styrene copolymer. Such gels typically
comprise between about 0.1% and 5%, by weight, of such gelling agents.
The topical compositions of the present invention can also be formulated
as a solid formulation (e.g., a wax-based stick, soap bar composition,
powder, or a wipe containing powder).
Liposomal formulations are also useful compositions of the subject
invention. In one embodiment, the soymilk or soybean powder particles
or soy proteins such as STI are contained within the liposome. Examples
of liposomes are unilamellar, multilamellar, and paucilamellar liposomes,
which may or may not contain phospholipids. Such compositions can
be prepared by first combining the non-denatured soy milk product
or the STI with a phospholipid, such as dipalmitoylphosphatidyl
choline, cholesterol and water. An example of a method for producing
liposomes is described in Mezei & Gulasekharam, "Liposomes--A
Selective Drug Delivery System for the Topical Route of Administration;
Gel Dosage Form", Journal of Pharmaceutics and Pharmacology,
Vol. 34 (1982), pp. 473-474. Those of skill in the art may make
suitable modifications of the method described therein.
Epidermal lipids of suitable composition for forming liposomes
may be substituted for the phospholipid. The liposome preparation
may then be incorporated into one of the above carriers (e.g., a
gel or an oil-in-water emulsion) in order to produce the liposomal
formulation. Other compositions and uses of topically applied liposomes
are described in Mezei, M., "Liposomes as a Skin Drug Delivery
System", Topics in Pharmaceutical Sciences (D. Breimer and
P. Speiser, eds.), Elsevier Science Publishers B. V., New York,
N.Y., 1985, pp. 345-358, PCT Patent Application No. WO96/31194,
Niemiec, et al., 12 Pharm. Res. 1184-88 (1995), and U.S. Pat. No.
5,260,065.
In one embodiment, the liposome is nonionic. In one example, the
liposome contains (a) glycerol dilaurate; (b) compounds having the
steroid backbone found in cholesterol; and (c) fatty acid ethers
having from about 12 to about 18 carbon atoms. In a further embodiment,
the liposome comprises glycerol dilaurate, cholesterol, polyoxyethylene-10-stearyl
ether, and polyoxyethylene-9-lauryl ether. In one embodiment, these
ingredients are in a ratio of about 38:12:33:17.
In one embodiment, the liposomes are present in the topical composition
in an amount, based upon the total volume of the composition, of
from about 5 mg/ml to about 100 mg/ml such as from about 10 mg/ml
to about 50 mg/ml.
The topical compositions useful in the subject invention may contain,
in addition to the aforementioned components, a wide variety of
additional oil-soluble materials and/or water-soluble materials
conventionally used in compositions for use on skin, hair, and nails
at their art-established levels.
In addition to such agents, other emollients and surface active
agents can be incorporated in the emulsions, including glycerol
trioleate, acetylated sucrose distearate, sorbitan trioleate, polyoxyethylene
(1) monostearate, glycerol monooleate, sucrose distearate, polyethylene
glycol (50) monostearate, octylphenoxypoly (ethyleneoxy) ethanol,
decaglycerin penta-isostearate, sorbitan sesquioleate, hydroxylated
lanolin, lanolin, triglyceryl diisostearate, polyoxyethylene (2)
oleyl ether, calcium stearoyl-2-lactylate, methyl glucoside sesquistearate,
sorbitan monopalmitate, methoxy polyethylene glycol-22/dodecyl glycol
copolymer (Elfacos E200), polyethylene glycol-45/dodecyl glycol
copolymer (Elfacos ST9), polyethylene glycol 400 distearate, and
lanolin derived sterol extracts, glycol stearate and glycerol stearate;
alcohols, such as cetyl alcohol and lanolin alcohol; myristates,
such as isopropyl myristate; cetyl palmitate; cholesterol; stearic
acid; propylene glycol; glycerine, sorbitol and the like.
The pharmaceutical or cosmetic composition may be optionally combined
with other ingredients such as moisturizers, cosmetic adjuvants,
anti-oxidants, depigmenting agents, darkening agents, anti-aging
agents, hair removal agents, hair styling agents, nail styling agents,
sunscreens, surfactants, bleaching agents, foaming agents, conditioners,
humectants, fragrances, colorants, viscosifiers, buffering agents,
preservatives, and the like and mixtures thereof. Skin-care compositions
including these components should be formulated so as not to affect
the soy product or soy trypsin inhibitory activity.
Examples of humectants include glycerol, sorbitol, propylene glycol,
ethylene glycol, 1,3-butylene glycol, polypropylene glycol, xylitol,
malitol, lactitol, allantoin, acetamine MEA, oat protein, hyaluronic
acid, and the like. They may be used either singly or in combination.
Because the compositions of this invention are non-denatured, i.e.,
compositions in which the protease inhibitory activity is retained,
they may be more favorable as a medium for microbial growth. Preservatives
are useful for substantially preventing microbial decomposition.
Examples of preservatives include phenoxyethanol and parabens such
as methyl-paraben, ethyl-paraben, and propyl-paraben; salicylic
acid, chlorhexidine hydrochloride, phenoxyethanol, sodium benzoate,
methyl para-hydroxybenzoate, ethyl para-hydroxybenzoate, propyl
para-hydroxybenzoate, butyl para-hydroxybenzoate, isothiazolones
and the like. Other examples of preservatives are listed on pages
1654-55 of the International Cosmetic Ingredient Dictionary and
Handbook, eds. Wenninger and McEwen (CTFA, 7.sup.th ed., 1997),
hereinafter referred to as the "Cosmetic Handbook." The
composition may comprise from about 0.01% to about 20%, by weight
(more preferably, from about 0.5% to about 5%, by weight) of preservative.
Microbial contamination can also be eliminated by gamma irradiation
or microfiltration, or by brief heat treatments that do not result
in the elimination of protease inhibitory activity.
Examples of fragrances and odor masks include menthol, anethole,
carvone, eugenol, limonene, ocimene, n-decylalcohol, citronellol,
a-terpineol, methyl salicylate, methyl acetate, citronellyl acetate,
cineole, linalool, ethyl linalool, vanillin, thymol, spearmint oil,
peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon
oil, pimento oil, cinnamon leaf oil, perilla oil, wintergreen oil,
clove oil, eucalyptus oil and the like.
Examples of surface active agents include sodium alkyl sulfates,
e.g., sodium lauryl sulfate and sodium myristyl sulfate, sodium
N-acyl sarcosinates, e.g., sodium N-lauroyl sarcosinate and sodium
N-myristoyl sarcosinate, sodium dodecylbenzenesulfonate, sodium
hydrogenated coconut fatty acid monoglyceride sulfate, sodium lauryl
sulfoacetate and N-acyl glutamates, e.g., N-palmitoyl glutamate,
N-methylacyltaurin sodium salt, N-methylacylalanine sodium salt,
sodium .alpha.-olefin sulfonate and sodium dioctylsulfosuccinate;
N-alkylaminoglycerols, e.g., N-lauryldiaminoethylglycerol and N-myristyldiaminoethylglycerol,
N-alkyl-N-carboxymethylammonium betaine and sodium 2-alkyl-1-hydroxyethylimidazoline
betaine; polyoxyethylenealkyl ether, polyoxyethylenealkylaryl ether,
polyoxyethylenelanolin alcohol, polyoxyethyleneglyceryl monoaliphatic
acid ester, polyoxyethylenesorbitol aliphatic acid ester, polyoxyethylene
aliphatic acid ester, higher aliphatic acid glycerol ester, sorbitan
aliphatic acid ester, Pluronic.TM. type surface active agent, and
polyoxyethylenesorbitan aliphatic acid esters such as polyoxyethylenesorbitan
monooleate and polyoxyethylenesorbitan monolaurate.
Examples of the binder or thickener include cellulose derivatives
such as alkali metal salts of carboxymethylcellulose, methyl cellulose,
hydroxyethyl cellulose and sodium carboxymethylhydroxyethyl cellulose,
alkali metal alginates such as sodium alginate, propylene glycol
alginate, gums such as carrageenan, xanthan gum, tragacanth gum,
caraya gum and gum arabic, and synthetic binders such as polyvinyl
alcohol, polysodium acrylate and polyvinyl pyrrolidone. Thickening
agents that can be added to the compositions of this invention to
alter viscosity include other polymers such as polyacrylates (e.g.,
polyacrylamide). Other examples of viscosity modifying agents are
listed on pages 1692-97 of the Cosmetic Handbook. To achieve the
appropriate viscosity, compositions of the present invention may
comprise from about 0.01% to about 20%, by weight (e.g., from about
0.1% to about 5%, by weight) of a thickening agent.
Coloring agents and fragrances also are commonly included in such
compositions.
In one embodiment, the topical composition further comprises another
cosmetically active agent in addition to the non-denatured soy product.
A "cosmetically active agent" is a compound (e.g., a synthetic
compound or a compound isolated from a natural source or a natural
extract) that has a cosmetic or therapeutic effect on the skin,
hair, or nails, including, but not limiting to, lightening agents,
darkening agents such as self-tanning agents, anti-acne agents,
shine control agents, anti-microbial agents, anti-inflammatory agents,
anti-mycotic agents, anti-parasite agents, external analgesics,
sunscreens, photoprotectors, antioxidants, keratolytic agents, detergents/surfactants,
moisturizers, nutrients, vitamins, energy enhancers, anti-perspiration
agents, astringents, deodorants, hair removers, firming agents,
anti-callous agents, and agents for hair, nail, and/or skin conditioning.
The compositions of this invention may be applied prior to, concurrently
with or after other active ingredients or compositions to enhance
their effect.
Antioxidants and/or chelating agents may also be used to increase
shelf life and stability of the compositions. Antioxidants may be
added both for formulation stabilization and for biological efficacy.
Antioxidant compounds and their derivatives include, but are not
limited to, water-soluble antioxidants such as sulfhydryl compounds
and their derivatives (e.g., sodium metabisulfite and N-acetyl-cysteine),
lipoic acid and dihydrolipoic acid, resveratrol, acetyl-cysteine
(Iniferine.RTM.) or lactoferrin, and ascorbic acid and ascorbic
acid derivatives (e.g., ascorbyl palmitate and ascorbyl polypeptide).
Oil-soluble antioxidants suitable for use in the compositions of
this invention include, but are not limited to, butylated hydroxytoluene,
retinoids (e.g., retinol and retinyl palmitate), tocopherols (e.g.,
tocopherol acetate), tocotrienols, and ubiquinone. Natural extracts
containing antioxidants suitable for use in the compositions of
this invention, include, but not limited to, extracts containing
flavonoids and isoflavonoids and their derivatives (e.g., genistein
and diadzein), extracts containing resveratrol and the like. Examples
of such natural extracts include grape seed, green tea, pine bark,
propolis, and legume extracts. Other examples of antioxidants may
be found on pages 1612-13 of the Cosmetic Handbook. The compositions
of the present invention may comprises the antioxidant in an amount
of from about 0.001% to about 20%, by weight (e.g., from about 0.01%
to about 10% by weight) of the composition.
It is preferable to have at least one oil-soluble antioxidant in
the compositions of this invention. The antioxidants should be utilized
in a stabilizing effective amount and may range in total from about
0.001 to 10% based on the weight of the total composition, preferably
from about 0.005 to about 5%. The oil-soluble antioxidants which
are useful in the compositions of the present invention include
butylated hydroxytoluene (BHT), ascorbyl palmitate, butylated hydroxanisole
(BHA), phenyl-.alpha.-naphthylamine, hydroquinone, propyl gallate,
nordihydroguiaretic acid, and mixtures thereof as well as any other
known oil-soluble antioxidant compatible with the other components
of the compositions.
Preferably, a water-soluble antioxidant should also be present
in the water phase of the compositions of this invention. The water-soluble
antioxidants which are useful in the compositions of this invention
include ascorbic acid, sodium metabisulfite, sodium bisulfite, sodium
thiosulfite, sodium formaldehyde sulfoxylate, isoascorbic acid,
thioglyerol, thiosorbitol, thiourea, thioglycolic acid, cysteine
hydrochloride, 1,4-diazobicyclo-(2,2,2)-octane and mixtures thereof
as well as any other known water-soluble antioxidant compatible
with the other components of the compositions.
Chelating agents are also useful in assisting the stabilization
of the compositions of this invention. Examples of chelating agents
include EDTA and derivatives thereof (e.g., disodium EDTA and dipotassium
EDTA), Iniferine .RTM., lactoferrin, and citric acid. Other examples
of chelating agents are listed on page 1626 of the Cosmetic Handbook.
The compositions of the present invention may comprise the chelating
agent in an amount of from about 0.001% to about 20%, by weight
(e.g., from about 0.01% to about 10% by weight) of the composition.
Other active ingredients such as sunscreen materials may be utilized
in the compositions of the present invention provided that they
are physically and chemically compatible with the other components
of the compositions. Sunscreens may include organic or inorganic
sunscreens, such as methoxyoctylcinnamate and other cinnamate compounds,
titanium dioxide and zinc oxide and the like.
Various irritancy mitigants may be added to the compositions of
this invention. Irritancy mitigants such as .alpha.-bisabolol, panthenol,
allantoin, ginkgo biloba, stearoyl glycerrhetinic acid (licorice
extract), tea tree oil, butchers' broom, calendula, ginseng and
the like may be added.
Other ingredients may include agents that assist in protecting
the skin from aging, such as sunscreens, anti-oxidant vitamins such
as ascorbic acid, vitamin B, biotin, pantothenic acid, vitamin D,
vitamin E and vitamin C, and sodium bisulfite. Yeast extract, ginkgo
biloba, bisabolol, panthenol, alpha hydroxy acids and oligosaccharides
such as melibiose are among other ingredients which assist in preventing
aging of the skin by such means as irritation mitigation, oxidation
mitigation, healing, affecting retinoid metabolism and inhibiting
the production of elastase.
The compositions of this invention may also contain other depigmenting
agents in addition to the soy product. What is meant by depigmentation
is the lightening of the color of an area of skin, including but
not limited to, the global lightening of the user's skin tone/complexion
(e.g., the face, hands, or whole body, which is uneven as a result
of aging skin, or darker than desired because of ethnicity or pathology,
and the like), the evening of skin color tone, or the specific lightening
of age spots, freckles, or darker pigmented areas such as, but not
limited to, post-inflammatory hyper-pigmentary lesions.
Examples of such depigmenting agents include, but are not limited
to, lipoic acid, dihydrolipoic acid, resveratrol, ascorbic acid,
kojic acid, hydroquinone, isoflavones, retinoids (e.g., retinol,
retinoic acid, and retinyl palmitate), tyrosinase inhibitors, melanosome
transfer inhibitors, and selective cytotoxic agents for melanocytes,
or natural extracts, e.g., licorice extract, gatuline A (pilewort
extract), and micromerol (butylene glycol and apple extract), providing
these activities. The amount of the depigmenting agent used will
depend on the activity of the compound, and will typically range
from about 0.001% to about 20%, by weight (e.g., from about 0.01%
to about 10%, by weight) of the composition.
Other skin color evening ingredients, such as skin darkening or
sunless tanning agents, may also be effective in the skin care compositions
for use in this invention.
The composition of the present invention may also contain compounds
that enhance the feel of the composition on the skin of the user.
Examples of such compounds include, but are not limited to, oils,
silicones (e.g., siloxane polymers such as dimethicone) and skin-conditioning
agents such as emollients, and humectants. Examples of such skin
conditioning agents may be found of pages 1656-1670 of the Cosmetic
Handbook.
Compositions which assist in the reduction of lines and wrinkles
may also be added to the compositions of this invention. For example,
alpha hydroxy acids, hyaluronic acid, Gatuline R (fagus silvitica
extract), pigments and scattering aids such as zinc oxide and titanium
dioxide may be used in the compositions of this invention in this
capacity.
Anti-inflammatory agents may also be used in the compositions of
this invention. Not only should these agents assist in mitigating
irritation, they may assist in treating wrinkles and lines in the
skin. Steroidal anti-inflammatory agents, including but not limited
to, corticosteroids such as hydrocortisone, hydroxyltriamcinolone,
alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasone
dipropionate, clobetasol valerate, desonide, desoxycorticosterone
acetate, dexamethoasone, dichlorisone, deflorasonediacetate, diflucortolone
valerate, fluadronolone, fluclarolone acetonide, fludrocortisone,
flumethasone pivalate, fluosinolone acetonide, fluocionide, flucortine
butylester, fluocortolone, flupredidene (flupredylidene) acetate,
flurandronolone, halcinonide, hydrocortisone acetate, hydrocortisone
butyrate, methylprednisolone, triamcinolone acetonide, cortisone,
cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,
fluradrenalone acetonide, medrysone, amciafel, amcinafide, betamethasone
and its esters, chlorprednisone acetate, clocortelone, clescinolone,
dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone,
fluperolone, fluprednisolone, hydrocortisone valerate, hydrocortisone
cyclopentylpropionate, hydrocortamate, meprednisone, paramethasone,
prednisolone, prednisone, beclomethasone dipropionate, triamcinolone
and mixtures thereof may be used. Preferably, hydrocortisone or
natural extracts with similar activity may be used.
Nonsteroidal anti-inflammatory agents may also be employed in the
compositions of this invention, such as salicylates, acetic acid
derivatives, fenamates, propionic acid derivatives and pyrazoles
or mixtures thereof. Other synthetic and natural anti-inflammatory
agents may also be used.
Additional active ingredients having topical activity may be utilized
in the compositions of this invention. Azole-type anti-fungal and
anti-bacterial agents may be employed in the compositions of this
invention in their base form. For example, ketoconazole, miconazole,
itraconazole, elubiol, and like related imidazole antifungals and
antibacterials are useful in the topical formulations of this invention.
It can be readily appreciated that a transdermal route of administration
may be enhanced by use of a dermal penetration enhancer, e.g., such
as enhancers described in U.S. Pat. No. 5,164,189, U.S. Pat. No.
5,008,110, and U.S. Pat. No. 4,879,119, issued Nov. 7, 1989 to Aruga
et al. In one embodiment, a composition of the present invention
can be delivered in a controlled release system, such as using a
transdermal patch, liposomes, or other modes of administration.
In another embodiment, polymeric materials can be used [see Medical
Applications of Controlled Release, Langer and Wise (eds.), CRC
Press: Boca Raton, Fla. (1974); Controlled Drug Bioavailability,
Drug Product Design and Performance, Smolen and Ball (eds.), Wiley:
New York (1984); Ranger and Peppas, J. Macromol. Sci. Rev. Macromol.
Chem. 23:61 (1983); see also Levy et al., Science 228:190 (1985);
During et al., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg.
71:105 (1989)].
In another embodiment, a controlled release system can be placed
in proximity of the target tissues of the mammal, thus requiring
only a fraction of the systemic dose [see, e.g., Goodson, in Medical
Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)].
In particular, a controlled release system can be introduced into
an animal in proximity of the site of inappropriate immune activation
or a tumor. Other controlled release systems are discussed in a
review by Langer [Science 249:1527-1533 (1990)].
In yet another embodiment of the invention, the soybean trypsin
inhibitor may be produced by recombinant means. The nucleotide and
protein sequences of STI are known. See GenBank Accession No. AF314823.
Methods for recombinant expression of STI are well known to those
of ordinary skill in the art. In an alternative embodiment, the
STI so produced may be modified at the genetic level (e.g. replacing
amino acids to change local charges, to enhance skin penetration
without compromising activity, or to enhance activity without compromising
skin penetration) or chemically post synthesis (e.g. additional
lipid or sugar groups) to enhance uptake of the STI into the skin
of the patient.
Various and numerous methods are known in the art for transdermal
administration of a drug, e.g., via a transdermal patch. Transdermal
patches are described in for example, U.S. Pat. No. 5,407,713, issued
Apr. 18, 1995 to Rolando et al.; U.S. Pat. No. 5,352,456, issued
Oct. 4, 1004 to Fallon et al.; U.S. Pat. No. 5,332,213 issued Aug.
9, 1994 to D'Angelo et al.; U.S. Pat. No. 5,336,168, issued Aug.
9, 1994 to Sibalis; U.S. Pat. No. 5,290,561, issued Mar. 1, 1994
to Farhadieh et al.; U.S. Pat. No. 5,254,346, issued Oct. 19, 1993
to Tucker et al.; U.S. Pat. No. 5,164,189, issued Nov. 17, 1992
to Berger et al.; U.S. Pat. No. 5,163,899, issued Nov. 17, 1992
to Sibalis; U.S. Pat. Nos. 5,088,977 and 5,087,240, both issued
Feb. 18, 1992 to Sibalis; U.S. Pat. No. 5,008,110, issued Apr. 16,
1991 to Benecke et al.; and U.S. Pat. No. 4,921,475, issued May
1, 1990 to Sibalis.
Compositions of the present invention may be prepared by mixing
the desired ingredients. For example, soymilk is mixed with the
chelating agent, preservative, and/or antioxidant. A thickener is
then added to the system, and the mixture is further mixed until
it reaches homogeneity at the desired viscosity. The compositions
of the present invention may be prepared under an argon, nitrogen,
or other inert gaseous blanket in order to enhance formulation stability
and/or to reduce soybean odor. The compositions of this invention
may be packaged in a tube, a sealed packet, a jar, a pump, a bottle,
a can, a pledget, a towelet, a wipe or the like. An airtight package
such as an aluminum tube, aluminum pocket, pump, laminate tube,
or the like, can also be used to further enhance product stability.
The skin-care compositions for use in the methods of this invention
may be applied daily for at least four weeks, and more preferably
at least eight weeks, by which an effect upon the appearance of
skin should be observed. Application may be continued as long as
desired to maintain the condition of the skin and to reduce skin
cancer risk.
The topically active pharmaceutical or cosmetic composition should
be applied in an amount effective to effect the desired changes
in the skin. As used herein, "amount effective" shall
mean an amount sufficient to cover the region of skin surface where
preventing cancer, inhibiting the growth rate of a cutaneous tumor,
or reducing the risk of cancer is desired. Preferably, the composition
is applied to the skin surface such that, based upon a cm.sup.2
of skin surface, from about 2 .mu.l/cm.sup.2 to about 500 .mu.l/cm.sup.2
of topically active agent is present when preventing cancer, inhibiting
the growth rate of a cutaneous tumor, or reducing the risk of cancer
is desired.
The following examples are provided to describe the invention in
further detail. These examples are provided for illustrative purposes
only, and are not to be construed as limiting the invention.
EXAMPLE 1
Soymilk Reduces UVB-Induced Tumorigenesis
Female SKH-1 mice, 6-7 weeks old, were purchased from Charles River
Laboratories (Kingston, N.Y.) and were housed for at least one week
before use. Mice were given water and Purina Laboratory Chow 5001
diet (Ralston-Purina, St. Louis, Mo.) ad libitum . Animal care was
based on the "Guide for the Care and Use of Laboratory Animals",
NIH Publication No. 85-23. Ultraviolet B light (UVB) lamps (FS72T12-UVB-HO)
were from National Biological Corp. (Twinsburg, Ohio). Mice were
irradiated with UV lamps that emit both UVB (280-320 nm, 75-80%
of total energy) and UVA (320-375 nm, 20-25% of total energy). The
UVB dose was quantified with a Spectra 305 dosimeter from DaevIin
Co. (Byran, Ohio). The radiation was further calibrated with a Research
radiometer/photometer model IL1700 from International Light Inc.
(Newburgport, Mass.).
Mice were irradiated with UVB light (30 mJ/cm.sup.2) for 25 to
30 seconds twice a week for 20 weeks. UVB irradiation was stopped
when the first tumor was visible on the back of one mouse. This
mouse was removed, and the population was classified as "at
high risk for skin tumor development." Three weeks later, mice
with no tumors were randomized into groups of 30 mice. One group
(the control) was not treated, and other groups were topically treated,
once a day, five days a week, with 100 .mu.l of test material. Agents
tested in this example included STI and BBI, and either freshly
prepared soymilk or freshly prepared soymilk that was heat denatured
at 90.degree. C. for 20 min. Controls include vehicle alone and
an unrelated protein (BSA).
The number of skin tumors and tumor sizes were measured every three
weeks, using the methods described in Lou et al., (Effects of oral
administration of tea, decaffeinated tea, and caffeine on the formation
and growth of tumors in high risk SKH-1 mice previously treated
with ultraviolet B light. Nutrition and Cancer 33, p. 146-153, 1999).
The results of these measurements are summarized in Table 1 and
shown in FIGS. 1A-1C and FIGS. 2A and 2B. An increase in tumor volume
per mouse during the 21 weeks of the treatment phase was observed
in each treatment regimen. The daily treatment with heated soymilk
did not affect the rate of increase in tumor volume per mouse relative
to control. Surprisingly, daily treatment with fresh soymilk had
a dramatic effect on tumor volume. Tumor growth was significantly
slower, and final tumor volumes were reduced by a factor of two
to three in the mice treated with fresh soymilk, relative to the
untreated or heated soymilk treated mice. See FIG. 2C. Table 1 shows
the effect of fresh soymilk on tumor progression as measured by
several additional parameters. The percent of tumor-bearing mice
was reduced following treatment with fresh soymilk, as were the
number of tumors per mouse and the tumor volume per tumor.
FIGS. 1A-1C show the effects of treatment of BBI and STI on mice
irradiated with UVB. Surprisingly, using STI alone had an unexpected
and significant effect on tumor progression. See FIGS. 1A-1C. The
dramatic difference in results produced by treatment with fresh
soymilk versus heat denatured soymilk, see FIGS. 2A-2C, also points
to STI as an active anti-tumor ingredient, since it is well known
that STI is denatured by cooking, but heat alone will not denature
BBI, nor does it affect phytoestrogens. FIGS. 4A-4C are three photographs
showing a random group of mice from each treatment group. This figure
clearly demonstrates the reduced tumorigenicity in the mice treated
with fresh soymilk vs heated treated soymilk or water. Compare FIG.
4A with 4B or 4C. These data indicate that topical treatment with
fresh soymilk reduces UVB-induced tumor growth and progression in
a high risk, pre-exposed population. These data also suggest that
some of the active(s) of fresh soymilk that are involved in the
tumor growth reduction are heat sensitive.
TABLE-US-00001 TABLE I Effects of Topical Applications of Soymilk,
STI, BBI, and Caffeine on the Development of Skin Tumors in SKH-1
Mice Previously Treated with Ultraviolet B Light Tumor Tumor Number
of Percent of volume per volume per mouse per Weight per mice with
Tumors per tumor mouse Week Treatment group mouse(g) tumors mouse
(mm.sup.3) (mm.sup.3) 0 No treatment 30/30 29.9 .+-. 0.5 0 0 0 0
Water 29/29 30.0 .+-. 0.3 0 0 0 0 Soymilk 30/30 29.9 .+-. 0.5 0
0 0 0 Heated Soymilk 30/30 30.0 .+-. 0.5 0 0 0 0 Liposomes 30/30
30.0 .+-. 0.4 0 0 0 0 STI 30/30 30.2 .+-. 0.4 0 0 0 0 BBI 30/30
30.1 .+-. 0.4 0 0 0 0 BSA in 30/30 30.1 .+-. 0.2 0 0 0 0 liposomes
Acetone 30/30 30.1 .+-. 0.2 0 0 0 0 Caffeine 30/30 30.0 .+-. 0.2
0 0 0 0 (1.2 mg) 6 No treatment 30/30 30.3 .+-. 0.4 47.7% 0.7 .+-.
0.2 1.5 .+-. 0.7 1.1 .+-. 0.5 Water 29/29 30.4 .+-. 0.5 27.6% 0.4
.+-. 0.2 0.5 .+-. 0.0 0.2 .+-. 0.1 Soymilk 30/30 29.9 .+-. 0.4 6.7%
0.1 .+-. 0.0 0.5 .+-. 0.0 0 Heated Soymilk 30/30 30.4 .+-. 0.6 36.7%
0.4 .+-. 0.1 1.1 .+-. 0.4 0.5 .+-. 0.2 Liposomes 30/30 30.0 .+-.
0.4 40.0% 0.7 .+-. 0.2 2.0 .+-. 0.7 1.4 .+-. 0.6 STI 30/30 29.7
.+-. 0.5 16.7% 0.2 .+-. 0.1 3.5 .+-. 1.9 0.8 .+-. 0.6 BBI 30/30
30.0 .+-. 0.5 30.0% 0.4 .+-. 0.1 0.9 .+-. 0.3 0.3 .+-. 0.1 BSA in
30/30 30.3 .+-. 0.4 20.0% 2.0 .+-. 0.1 2.5 .+-. 1.9 0.6 .+-. 0.5
liposomes Acetone 30/30 29.6 .+-. 0.4 36.7% 0.7 .+-. 0.2 2.6 .+-.
0.9 1.9 .+-. 0.8 30/30 29.3 .+-. 0.4 16.7% 0.2 .+-. 0.1 1.3 .+-.
0.7 0.2 .+-. 0.1 9 No treatment 30/30 30.5 .+-. 0.3 43.3% 0.8 .+-.
0.2 3.3 .+-. 1.5 2.7 .+-. 1.3 Water 29/29 30.6 .+-. 0.5 48.3% 1.0
.+-. 0.3 1.3 .+-. 0.3 1.3 .+-. 0.6 Soymilk 30/30 30.1 .+-. 0.4 20.0%
0.2 .+-. 0.1 0.5 .+-. 0.0 0.1 .+-. 0.0 Heated Soymilk 30/30 30.3
.+-. 0.5 46.7% 0.7 .+-. 0.1 4.8 .+-. 1.9 3.2 .+-. 1.3 Liposomes
30/30 30.2 .+-. 0.4 46.7% 1.9 .+-. 0.5 1.7 .+-. 0.6 3.2 .+-. 1.2
STI 29/30 30.0 .+-. 0.4 41.4% 0.9 .+-. 0.2 3.0 .+-. 1.4 2.6 .+-.
1.4 BBI 30/30 29.9 .+-. 0.5 27.6% 0.6 .+-. 0.2 1.7 .+-. 0.8 1.0
.+-. 0.7 BSA in 30/30 30.5 .+-. 0.4 50.0% 1.1 .+-. 0.3 2.7 .+-.
1.2 2.9 .+-. 1.4 liposomes Acetone 30/30 30.4 .+-. 0.5 60.0% 1.4
.+-. 0.5 2.8 .+-. 0.9 4.0 .+-. 1.5 Caffeine 30/30 29.4 .+-. 0.4
13.3% 0.2 .+-. 0.1 3.4 .+-. 2.2 0.7 .+-. 0.5 (1.2 mg) 12 No treatment
30/30 30.4 .+-. 0.4 70.0% 1.9 .+-. 0.3 2.8 .+-. 0.7 5.4 .+-. 1.7
Water 29/29 31.1 .+-. 0.5 58.6% 1.9 .+-. 0.5 2.3 .+-. 0.5 4.4 .+-.
1.2 Soymilk 30/30 30.4 .+-. 0.4 40.0% 0.7 .+-. 0.2 1.8 .+-. 0.4
1.2 .+-. 0.4 Heated Soymilk 30/30 30.2 .+-. 0.6 53.3% 1.0 .+-. 0.2
14.4 .+-. 6.0 14.9 .+-. 6.3 Liposomes 30/30 30.0 .+-. 0.4 70.0%
4.0 .+-. 0.9 4.4 .+-. 1.0 17.8 .+-. 5.2 STI 29/30 29.8 .+-. 0.5
46.7% 1.9 .+-. 0.5 4.8 .+-. 1.3 9.4 .+-. 3.9 BBI 30/30 30.6 .+-.
0.4 50.0% 1.5 .+-. 0.4 2.4 .+-. 0.8 3.7 .+-. 1.8 BSA in 30/30 30.4
.+-. 0.5 80.0% 2.6 .+-. 0.6 3.5 .+-. 1.0 9.1 .+-. 3.1 liposomes
Acetone 30/30 30.0 .+-. 0.4 80.0% 4.5 .+-. 0.9 5.2 .+-. 1.0 23.4
.+-. 7.1 Caffeine 30/30 29.0 .+-. 0.3 50.0% 1.3 .+-. 0.4 3.2 .+-.
0.8 4.0 .+-. 1.9 15 No treatment 30/30 30.6 .+-. 0.4 76.7% 2.9 .+-.
0.5 5.8 .+-. 1.4 16.8 .+-. 5.7 Water 29/29 31.2 .+-. 0.4 82.8% 3.5
.+-. 0.6 2.8 .+-. 0.6 9.7 .+-. 3.4 Soymilk 30/30 30.7 .+-. 0.3 46.7%
1.1 .+-. 0.3 3.0 .+-. 0.8 3.4 .+-. 1.0 Heated Soymilk 30/30 30.6
.+-. 0.5 70.0% 2.3 .+-. 0.4 10.1 .+-. 3.6 22.9 .+-. 7.9 Liposomes
30/30 29.6 .+-. 0.9 90.0% 6.2 .+-. 1.1 7.5 .+-. 1.3 46.2 .+-. 13.2
STI 29/30 30.1 .+-. 0.4 55.2% 3.1 .+-. 0.7 5.8 .+-. 2.1 17.8 .+-.
7.9 BBI 30/30 30.6 .+-. 0.5 66.7% 2.2 .+-. 0.4 4.1 .+-. 0.8 9.0
.+-. 2.4 BSA in 30/30 30.5 .+-. 0.4 90.0% 5.3 .+-. 0.8 4.0 .+-.
0.7 21.4 .+-. 6.6 liposomes Acetone 30/30 30.0 .+-. 0.3 83.3% 6.6
.+-. 1.0 7.4 .+-. 1.3 48.3 .+-. 16.7 Caffeine 30/30 29.4 .+-. 0.3
56.7% 2.1 .+-. 0.5 3.2 .+-. 0.6 6.7 .+-. 2.2 (1.2 mg) 18 No treatment
30/30 31.6 .+-. 0.4 83.3% 4.2 .+-. 0.6 9.0 .+-. 2.0 38.0 .+-. 12.8
Water 28/29 32.0 .+-. 0.5 96.6% 5.4 .+-. 0.6 4.0 .+-. 1.3 21.5 .+-.
7.5 Soymilk 30/30 30.7 .+-. 0.4 66.7% 1.9 .+-. 0.5 6.5 .+-. 1.8
12.3 .+-. 4.6 Heated Soymilk 30/30 31.0 .+-. 0.6 83.3% 4.1 .+-.
0.7 6.9 .+-. 2.4 28.7 .+-. 9.8 Liposomes 30/30 30.9 .+-. 0.5 90.0%
7.1 .+-. 1.0 8.7 .+-. 1.1 61.7 .+-. 15.0 STI 28/30 30.0 .+-. 0.4
63.3% 4.2 .+-. 0.9 7.3 .+-. 2.2 30.7 .+-. 11.9 BBI 30/30 30.7 .+-.
0.5 76.7% 3.2 .+-. 0.6 5.9 .+-. 1.3 19.0 .+-. 4.6 BSA in 30/30 30.9
.+-. 0.5 90.0% 6.5 .+-. 0.9 6.3 .+-. 1.0 41.1 .+-. 9.7 liposomes
Acetone 29/30 30.7 .+-. 0.5 86.7% 6.9 .+-. 1.0 8.4 .+-. 1.3 57.4
.+-. 13.7 Caffeine 30/30 29.6 .+-. 0.4 80.0% 3.6 .+-. 0.6 6.2 .+-.
1.3 22.1 .+-. 6.2 21 No treatment 30/30 31.5 .+-. 0.3 100.0% 5.9
.+-. 0.7 8.8 .+-. 2.1 51.9 .+-. 16.3 Water 28/29 32.0 .+-. 0.4 96.6%
6.3 .+-. 0.6 6.4 .+-. 2.4 40.6 .+-. 15.6 Soymilk 30/30 31.1 .+-.
0.3 83.3% 2.7 .+-. 0.5 4.8 .+-. 1.3 12.9 .+-. 4.4 Heated Soymilk
29/30 31.3 .+-. 0.6 90.0% 6.7 .+-. 0.9 8.9 .+-. 3.2 59.9 .+-. 20.9
Liposomes 30/30 30.9 .+-. 0.4 96.7% 8.2 .+-. 1.1 9.0 .+-. 1.5 73.7
.+-. 15.0 STI 28/30 30.6 .+-. 0.4 80.0% 5.6 .+-. 0.9 8.6 .+-. 3.2
48.7 .+-. 23.3 BBI 30/30 30.8 .+-. 0.4 90.0% 4.5 .+-. 0.6 7.4 .+-.
2.0 33.2 .+-. 9.6 BSA in 30/30 30.7 .+-. 0.4 90.0% 7.1 .+-. 0.8
6.3 .+-. 1.0 44.7 .+-. 11.6 liposomes Acetone 28/30 30.5 .+-. 0.4
90.0% 6.9 .+-. 0.9 9.6 .+-. 2.0 65.7 .+-. 0.9 Caffeine 30/30 29.9
.+-. 0.3 76.7% 4.0 .+-. 0.7 5.3 .+-. 1.0 21.5 .+-. 6.0 SKH-1 mice
(7-8 weeks old) were treated with ultraviolet B light (UVB; 30 mJ/cm.sup.2)
twice weekly for 20 weeks and UVB treatment was stopped. Three weeks
later, the mice (with no visible tumors) were randomized into 10
groups (30 mice per group) and the mice were treated topically:
group 2, 100 ul water; group 3, 100 ul Soymilk; group 4, 100 ul
heated Soymilk; group 5, 100 ul liposomes; group 6, trypsin inhibitor
(STI; 0.8 mg; T9003, Sigma) in 100 ul liposomes; group 7, Bowman-Birk
protease inhibitor (BBI; 0.8 mg; T97770, Sigma) in 100 ul liposomes;
group 8, bovine serum albumin (BSA; 0.8 mg) in 100 ul liposomes;
group 9, 100 ul acetone; group 10, caffeine, CAF; 1.2 mg) in 100
ul acetone once a day 5 days per week for 21 weeks.
EXAMPLE 2
Caffeine Reduces UVB-Induced Tumorigenesis
The experiment described in Example 1 was also performed using
a topical caffeine treatment. Caffeine was purchased from Sigma
(St. Louis, Mo.). Acetone was from Fisher Scientific (Springfield,
N.J.). Mice were treated daily, five days a week with caffeine (1.2
mg) in 100 .mu.l acetone or with 100 .mu.l of acetone only. Experimental
procedures and measurements were identical to those described in
Example 1, and the two experiments were performed at the same time,
using the same UVB-irradiated population.
FIGS. 3A-3C show that tumor progression following acetone treatment
was very similar to that of the untreated group graphed in FIG.
2. The caffeine treated mice showed a marked delay in tumor progression,
as evident from the tumor volume per mouse data in FIG. 3C. The
additional parameters reported in Table 1 further support this observation
of the beneficial effect of caffeine on tumor progression. The percentage
of tumor-bearing bearing mice was reduced following caffeine treatment,
as were the number of tumors per mouse and the tumor volume per
tumor. FIGS. 5A and 5B are two pictures, each depicting a random
group of mice from acetone treated (FIG. 5A) vs. caffeine treated
(FIG. 5B mice). This figure clearly demonstrates the reduced tumorigenicity
in the caffeine-treated mice.
EXAMPLE 3
Preparation of Soymilk from Soybean Powder
160 g of soybean powder (Sunlight Foods, Taipei, Taiwan) was added
to about 1440 g of deionized water. The mixture was stirred at room
temperature for about 1 hour. The mixture was then filtered through
a sieve having holes of 75 .mu.m diameter. The filtrate resulted
in about 1.1 kg of soymilk.
EXAMPLE 4
Preparation of Soymilk Gel from Soymilk
The following compositions of this invention were prepared as follows.
The weight percentages of each ingredient in the compositions are
indicated below in Table 2 and Table 3. First, the soymilk, as prepared
in example 3, was placed into a first beaker. The preservative Phenonip.RTM.
(a mixture of the preservatives methyl-paraben, propyl-paraben,
ethyl-paraben, and phenoxy-ethanol sold by NIPA, Wilmington, Del.)
or the preservative phenoxyethanol were added to the soymilk. Next,
the chelating agent Disodium EDTA and in some examples the humectant
glycerin were added to the first beaker and mixed with the soymilk.
It is also possible to further add cyclomethicone, or dimethicone
(tradename Dow Corning 200 Fluid .RTM.), or PolySorbate 20, or Aluminum
Starch Octyl Succinate, or Sucrose Cocoate, or PEG-6 Capric/Caprylic
Triglycerides to the soymilk mixture at this step as required in
some examples in Table 2 and Table 3. A mixture of the thickener
polyacrylamide, laureth-7, and C13-14 isoparaffins (sold by Seppic,
Paris, France under the Tradename Sepigel.RTM.) was added to a second
beaker along with the anti-oxidant BHT. The ingredients in the second
beaker were then added to the ingredients of the first beaker and
mixed until homogenous. The anti-oxidants ascorbic acid, sodium
ascorbyl phosphate, lactoferrin, or tocopherol were then added to
the beaker and homogeneously mixed to form the resulting gel.
EXAMPLE 5
Preparation of Soymilk Gel from Soybean Powder, Soymilk Powder
or Soybean Extract
The following compositions of this invention were prepared as follows.
The weight percentage of each ingredient in each of the preparations
is indicated below in Table 3. First, the soymilk powder (Devansoy
Farms, Carroll, Iowa) or the soybean powder (Sunlight Foods, Taipei,
Taiwan) or the Soybean Extract and deionized water were placed into
a first beaker and mixed to reconstitute the soy powder. The preservative
Phenonip.RTM. and the chelating agent Disodium EDTA were then added
to the first beaker and mixed with the soymilk. A mixture of polyacrylamide,
laureth-7, and C13-14 isoparaffins was added to a second beaker
along with the anti-oxidant BHT. The ingredients in the second beaker
were then added to the ingredients of the first beaker and mixed
until homogenous.
TABLE-US-00002 TABLE 2 Soybean Essence formulations 24 26 27 28
33 34 35 Soymilk 94.40% 92.40% 90.70% 94.70% Phenoxyethanol and
Parabens 1.00% 1.00% 1.00% 1.00% 1.00% 1.00% 1.00% Glycerin 5.00%
Disodium EDTA 0.05% 0.05% 0.05% 0.05% 0.05% 0.05% 0.05% Polyacrylamide/Laureth-7/C.sub.13-14
Isoparrafin 3.50% 3.50% 3.20% 3.20% 3.20% 3.20% 3.20% Ascorbic Acid
1.00% Butylated Hydroxytoluene 0.05% 0.05% 0.05% 0.05% 0.05% 0.05%
0.05% Deionized Water 90.70% 90.70% 85.70% Lactoferrin 1.00% 1.00%
Tocopherol 1.00% Dow Corning 200 Fluid 1.00% Soymilk Powder 5.00%
Soybean Extract using Ethanol/Water Mixture 5% 10% TOTAL 100% 100%
100% 100% 100% 100% 100%
TABLE-US-00003 TABLE 3 Soybean Essence formulations Soybean Essences
1 6 8 21 23 Soymilk 87.42% 89.04% 96.09% 96.05% 95.70% Phenoxyethanol
0.73% Phenoxyethanol and Parabens 1.00% 1.00% 1.00% 1.00% Glycerin
2.50% 2.50% Cyclomethicone 2.00% Aluminum Starch Ocetyl Succinate
0.75% Sucrose Cocoate 1.00% 1.00% PEG-6 Capric/Caprylic Triglycerides
3.00% 3.00% Disodium EDTA 0.10% 0.10% 0.05% Polyacrylamide/Laureth-7/C.sub.13-14
Isoparrafin 2.50% 2.75% 2.90% 2.90% 3.20% Ascorbic Acid 0.01% Butylated
Hydroxytoluene 0.10% 0.01% 0.05% 0.05% Polysorbate 20 0.50% TOTAL
100% 100% 100% 100% 100%
While certain preferred embodiments of the present invention have
been described and specifically exemplified above, it is not intended
that the invention be limited to such embodiments. Various modifications
may be made thereto without departing from the scope and spirit
of the present invention, as set forth in the following claims. |