U.S. patent application number 10/212847 was filed with the patent office on 2003-03-27 for compositions and method for protecting skin from uv induced immunosuppression and skin damage.
This patent application is currently assigned to Novogen Research Pty. Ltd.. Invention is credited to Husband, Alan James, Kelly, Graham Edmund.
Application Number | 20030059384 10/212847 |
Document ID | / |
Family ID | 3805406 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059384 |
Kind Code |
A1 |
Kelly, Graham Edmund ; et
al. |
March 27, 2003 |
Compositions and method for protecting skin from UV induced
immunosuppression and skin damage
Abstract
A method for protecting skin from either UV-induced
immunosuppression or from UV-induced skin damage comprising the
topical administration of a composition containing an extract of
soy or clover, and/or the isoflavone compounds genistein,
biochanin, dihydro-daidzein, diadzein, formonentin,
dihydro-geneistein, 2-dehydro-O-desmethyl-angolensin,
tetrahydro-daidzein, equol, dehydro-equol, O-desmethyl-angolensin,
or 6-hydroxy-O-desmethyl-angolensin. The invention also comprises
compositions containing these compounds.
Inventors: |
Kelly, Graham Edmund;
(Northbridge, AU) ; Husband, Alan James;
(McMahon's Point, AU) |
Correspondence
Address: |
FINNEGAN, HENDERSON,
FARABOW, GARRETT and DUNNER, L.L.P.
1300 I Street, N.W.
Washington
DC
20005-3315
US
|
Assignee: |
Novogen Research Pty. Ltd.
|
Family ID: |
3805406 |
Appl. No.: |
10/212847 |
Filed: |
August 5, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10212847 |
Aug 5, 2002 |
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09582317 |
Jun 23, 2000 |
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6455032 |
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09582317 |
Jun 23, 2000 |
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PCT/AU98/01054 |
Dec 21, 1998 |
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Current U.S.
Class: |
424/59 |
Current CPC
Class: |
A61Q 19/004 20130101;
A61K 8/492 20130101; A61K 31/12 20130101; A61Q 17/04 20130101; A61K
31/352 20130101; A61P 17/16 20180101; A61K 31/353 20130101; A61Q
19/00 20130101; A61K 2800/92 20130101; A61P 37/06 20180101; A61K
31/404 20130101; A61K 31/4745 20130101; A61K 8/498 20130101 |
Class at
Publication: |
424/59 |
International
Class: |
A61K 007/42 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 24, 1997 |
AU |
PP 1124 |
Claims
1. A composition for application to the skin following UV exposure
or for oral administration prior to or following UV exposure, which
composition comprises a compound of the general Formula (I) 8in
which Z is H, R.sub.1 is H, or R.sub.ACO where R.sub.A is
C.sub.1-10 alkyl or an amino acid, R.sub.2 is H, OH, or OR.sub.B
where R.sub.B is an amino acid or COR.sub.A where R.sub.A is as
previously defined, W is H, A is H or OH, and B is selected from 9W
is H, and A and B taken together form a six membered ring selected
from 10W, A and B taken with the groups with which they are
associated comprise 11W and A taken together with the groups with
which they are associated comprise 12wherein R.sub.3 is H,
COR.sub.A where R.sub.A is as previously defined, CO.sub.2R.sub.c
where R.sub.c is C.sub.1-10 alkyl, or COR.sub.B where R.sub.B is as
previously defined, R.sub.4 is H, COR.sub.D where R.sub.D is H, OH,
C.sub.1-10 alkyl or an amino acid, CO.sub.2R.sub.C where R.sub.C is
as previously defined, COR.sub.E where R.sub.E is H, C.sub.1-10
alkyl or an amino acid, COOH, COR.sub.C where R.sub.C is as
previously defined, or CONHR.sub.E where R.sub.E is as previously
defined, R.sub.5 is H, CO.sub.2R.sub.C where R.sub.C is as
previously defined, or COR.sub.COR.sub.E where R.sub.C and R are as
previously defined, and where the two R.sub.5 groups are attached
to the same group they are the same or different, R.sub.6 is H or
hydroxy C.sub.1-10 alkyl, X is preferably O, but may be N or S, and
Y is 13where R.sub.7 is H, or C.sub.1-10 alkyl, in association with
a dermatologically acceptable or pharmaceutically acceptable
carrier.
2. A composition according to claim 1 wherein compounds of the
Formula (I) are selected from: 14wherein R.sub.7' is H or CH.sub.3,
R.sub.8 is COR where R.sub.D is as previously defined, or H,
R.sub.9 CO.sub.2R.sub.C or COR.sub.E where R.sub.C and R.sub.E are
as previously defined, R.sub.10 is COR.sub.C or COR.sub.COR.sub.E
where R.sub.C and R.sub.E are as previously defined, R.sub.11 is H
or OH, R.sub.12 is H, COOH, CO.sub.2R.sub.C where R.sub.C and is as
previously defined, or CONHR.sub.E where R.sub.E is as previously
defined, R.sub.13 is OH, OR.sub.B where R.sub.B is as previously
defined, or COR.sub.A where R.sub.A is as previously defined,
R.sub.14 is H, or COR.sub.A where R.sub.A is as previously defined,
R.sub.15 is COR.sub.A where R.sub.A is as previously defined,
R.sub.16 is H, COR.sub.B or CO.sub.2R.sub.C where R.sub.B and
R.sub.C are as previously defined, R.sub.17 is H or hydroxy
C.sub.1-10 alkyl, R.sub.18 is H or C.sub.1-10 alkyl, and "---"
represents either a single bond or a double bond.
3. A method for protecting skin from UV induced immunosuppression
and UV induced skin damage which comprises applying to the skin of
a subject after UV exposure a composition which comprises a
compound of the Formula (I) as defined in claim 1 or an extract of
soy or clover, in association with a dermatologically acceptable
carrier.
4. A method for protecting skin from UV induced immunosuppression
and UV induced skin damage which comprises orally administering to
a subject either before and/or after UV exposure a composition
which comprises a compound of the Formula (I) as defined in claim 1
or an extract of soy or clover, in association with a
pharmaceutically acceptable carrier.
5. A method according to claim 3 or 4 wherein the composition
includes .beta.-(1,3)(1,6)-glucan.
6. Use of compounds of the Formula (I) as defined in claim 1 for
the manufacture of a medicament for the treatment, amelioration,
prophylaxis and/or prevention of UV induced immunosuppression in
the skin, and UV induced skin disorders.
7. A composition for application to the skin following UV exposure,
which composition comprises an extract of soy or clover in
association with a dermatologically acceptable carrier.
8. A composition according to claim 1, 2 or 7 which includes
.beta.-(1,3)(1,6)-glucan.
9. A method for protecting skin from UV induced immunosuppression
and UV induced skin damage which comprises applying to the skin of
a subject after UV exposure, or orally before or after UV exposure,
a composition as defined in any of claims 1, 2, 7 or 8.
Description
[0001] This invention relates in one aspect to compositions which
are applied to the skin after ultra violet (UV) radiation exposure,
particularly following exposure to the sun. Such compositions may
be referred to as after-sun compositions. In another aspect the
invention relates to compositions for oral administration which
protect against UV induced skin damage. In another aspect the
invention relates to methods for protecting the skin from UV
induced immunosuppression and UV induced skin damage, such as that
resulting from exposure to the sun.
[0002] Exposure of the skin to ultraviolet radiation causes both
critical damage to the epidermal DNA, which may have long-term
irreversible consequences if remaining unrepaired, and may lead to
a specific impairment of the T lymphocyte immune system.
[0003] In chronically UV-exposed skin, damage to DNA culminates in
carcinogenesis, the most common tumour in man being the basal cell
carcinoma (BCC), followed by squamous cell carcinoma (SCC), and
more rarely malignant melanoma. For example, approximately
two-thirds of the Australian population suffer from non-melanoma
skin cancer at some time in their lives, the proportion increasing
with decreasing latitude, and leading the world. Australia also has
the world's highest incidence of melanoma.
[0004] The immuosuppression caused by UV exposure appears to be a
prerequisite for non-melanoma and melanoma cancer promotion. It is
mediated by a number of mechanisms, such as the formation of
epidermal cis-urocanic acid in UV-irradiated skin, the persistence
of pyrimidine dimers in epidermal DNA, and the upregulation of
inflammatory eicosanoids like PGE2. It is understood that
photoimmunosuppression permits the initiated tumour cell to evade
recognition and rejection by normal immunological mechanisms, to
remain latent for extended periods, and to eventually proliferate
into a tumour.
[0005] Immunocompromised patients, whether genetically (xeroderma
pigmentosum) or pharmacologically (organ transplant recipients)
[see Boyle et al (1984) Lancet, 31 March, 702-705 and Kinlen et al
(1994) Invest Dermatol, Br. J. Med. ii 1461-1466], have a higher
incidence of skin cancer. Also sun-exposed skin areas on humans are
immunologically impaired compared with non-exposed skin [O'Dell et
al (1980) Arch. Dermatol., 116, 559-561], and the level of
immunological responsiveness of skin cancer patients is reduced
compared with non-skin cancer patients [Yoshikawa et al, Invest.
Dermatol., 95:530-536 (1990)]. The immune deficiency following UV
exposure is now known to result from a deficiency in Th1 cell
activity, whereas Th2 responses remain active [Ullrich, S. E.
(1996) Photochem. Photobiol. 64, 254-258].
[0006] The chronic exposure of the skin to solar radiation is well
documented as the cause of the photoageing phenomenon, such as
thickening of skin, drying of skin, increased skin pigmentation,
skin spots and skin lesions.
[0007] UV exposure, such as chronic solar UV radiation causes the
well known effects of reddening of the skin with accompanying
inflammation, known as erythema. This is often referred to as
"sunburn" which is painful, often itchy, and generally results in a
subsequent peeling of the skin which has been subject to chronic
solar irradiation.
[0008] Erythema is particularly prevalent in light skinned
individuals and children. Chronic erythema may predispose
individuals to skin disorders, such as skin cancers in later life.
Depending on an individual's skin colouration, erythema may result
in as little as 20 minutes exposure to the sun.
[0009] With an increasing awareness of the dangers of UV exposure
to skin, "sun blocks" or sunscreens have been available for a
number of years. Sunscreens are applied to the skin prior to sun
exposure. Typically sunscreen compositions contain UVA-type
sunscreen agents and/or UVB-type sunscreen agents. Typical UVA-type
sunscreen agents include certain benzophenones and
dibenzylmethanes. Typical UVB-type sunscreen agents include
substituted para-aminobenzoates, alkyl esters of
para-methoxycinnamate and/or various esters of salicylic acid.
Generally sunscreening agents are used in amounts effective to
provide the desired level of protection from erythema caused by UVA
and/or UVB radiation. Examples of many known sun screening agents
are described in WO 96/14826 which is incorporated herein by
reference.
[0010] Sun screening compositions may contain physical sun
screening agents such as red petrolatum, or titanium dioxide, such
as in amounts from 2-5% by weight of the total composition.
Precipitated silica, kaolin, talc, chalk and the like may also be
used in such compositions.
[0011] A diverse range of compounds have been proposed as UV
absorbers for use in sunscreen compositions. Amongst this enormous
class of sunscreen agents flavonoid compounds, including isoflavone
compounds, have been mentioned. The applicant's investigations
indicate that isoflavone compounds show poor UV absorbing capacity,
contributing as little as 1.5 units sun protection factor (SPF) to
sunscreen compositions.
[0012] Concerns have arisen with regard to the light stability of
various UV absorbers, potential toxic effects of compounds over
long term exposure, complexity and cost of formulations, and
overall effectiveness of sunscreen compositions. Sunscreen
compositions require specific application to the skin prior to UV
exposure. Failure to apply sunscreen, inadequate application to the
skin, and/or loss of sunscreen from the skin all have the potential
to lead to UV damage to the skin.
[0013] Currently available sunscreens are not all efficacious in
protecting skin from UV exposure, such that immune deficiency of
skin and the potentially serious consequences which may ensue
remain unaddressed.
[0014] It has surprisingly been found by the applicant that certain
isoflavone compounds when applied to the skin subsequent to UV
exposure or for oral administration prior to or following UV
exposure, in the form of an after-sun composition, protect the skin
from UV induced immunosuppression and UV induced skin damage. It
has also been found that extracts of soy and clover protect skin
from UV induced immunosuppression and UV induced skin damage.
[0015] In accordance with one aspect of the present invention there
is provided a composition for application to the skin following UV
exposure or for oral administration prior to or following UV
exposure, which composition comprises a compound of the general
Formula (I) 1
[0016] in which
[0017] Z is H,
[0018] R.sub.1 is H, or R.sub.ACO where R.sub.A is C.sub.1-10 alkyl
or an amino acid,
[0019] R.sub.2 is H, OH, or OR.sub.B where R.sub.B is an amino acid
or COR.sub.A where R.sub.A is as previously defined,
[0020] W is H, A is H or OH, and B is selected from 2
[0021] W is H, and A and B taken together form a six membered ring
selected from 3
[0022] W, A and B taken with the groups with which they are
associated comprise 4
[0023] W and A taken together with the groups with which they are
associated comprise 5
[0024] and B is
[0025] wherein
[0026] R.sub.3 is H, COR.sub.A where R.sub.A is as previously
defined, CO.sub.2R.sub.C where R.sub.C is C.sub.1-10 alkyl, or
COR.sub.B where R.sub.B is as previously defined,
[0027] R.sub.4 is H, COR.sub.D where R.sub.D is H, OH, C.sub.1-10
alkyl or an amino acid, CO.sub.2R.sub.C where R.sub.C is as
previously defined, COR.sub.E where R.sub.E is H, C.sub.1-10 alkyl
or an amino acid, COOH, COR.sub.C where R.sub.C is as previously
defined, or CONHR.sub.E where R.sub.E is as previously defined,
[0028] R.sub.5 is H, CO.sub.2R.sub.C where R.sub.C is as previously
defined, or COR.sub.COR.sub.E where R.sub.C and R
[0029] are as previously defined, and where the two R.sub.5 groups
are attached to the same group they are the same or different,
[0030] R.sub.6 is H or hydroxy C.sub.1-10 alkyl,
[0031] X is preferably O, but may be N or S, and
[0032] Y is 6
[0033] where R.sub.7 is H, or C.sub.1-10 alkyl, in association with
a dermatologically acceptable or pharmaceutically acceptable
carrier.
[0034] Preferably the compounds of the Formula (I) are selected
from: 7
[0035] wherein
[0036] R.sub.7' is H or CH.sub.3
[0037] R.sub.8 is COR where R.sub.D is as previously defined, or
H,
[0038] R.sub.9 CO.sub.2R.sub.C or COR.sub.E where R.sub.C and
R.sub.E are as previously defined,
[0039] R.sub.10 is COR.sub.C or COR.sub.COR.sub.E where R.sub.C and
R.sub.E are as previously defined,
[0040] R.sub.11 is H or OH,
[0041] R.sub.12 is H, COOH, CO.sub.2R.sub.C where R.sub.C and is as
previously defined,
[0042] or CONHR.sub.E where R.sub.E is as previously defined,
[0043] R.sub.13 is OH, OR.sub.B where R.sub.B is as previously
defined, or COR.sub.A where R.sub.A is as previously defined,
[0044] R.sub.14 is H, or COR.sub.A where R.sub.A is as previously
defined,
[0045] R.sub.15 is COR.sub.A where R.sub.A is as previously
defined,
[0046] R.sub.16 is H, COR.sub.B or CO.sub.2R.sub.C where R.sub.B
and R.sub.C are as previously defined,
[0047] R.sub.17 is H or hydroxy C.sub.1-10 alkyl,
[0048] R.sub.18 is H or C.sub.1-10 alkyl,
[0049] and "" represents either a single bond or a double bond.
Alkyl groups may be straight or branched chains. C.sub.1-10 alkyl
preferably contains from 1 to 5 carbons, more preferably methyl,
ethyl or propyl.
[0050] Certain of the above compounds may be referred to by the
names genistein (compound 1 where R.sub.7' is H and R.sub.8 is H),
biochanin (compound 1 where R.sub.7' is CH.sub.3 and R.sub.8 is H),
dihy-drodaidzein (compound 2 where is a bond and R.sub.7' is H),
diadzein (compound 2 is a double bond and R.sub.7' is H)
formonentin (compound 2 where is a double bond and R.sub.7' is
CH.sub.3), dihydrogeneistein (compound 5 in the keto form),
2-dehydro-O-desmethyl-angolensin (compound 11), tetrahydrodaidzein
(compound 8), equol (compound 10 when is a single bond),
dehydroequol (compound 10 where is a double bond),
O-desmethyl-angolensin (ODMA--compound 13), and
6-hydroxy-O-desmethylango- lensin (6-hydroxy-ODMA--compound
14).
[0051] Compounds of the Formula (I) include isomers, keto-enol
tautomers, and physiologically acceptable salts. Isomeric forms,
keto-enol forms, and salts can be prepared according to methods
well known in the art, such as described by Brown, W. H.,
Introduction to Organic Chemistry, 4th Edition, Brooks/Cole Pub.
Co., California, (1988).
[0052] In another aspect this invention is concerned with
compositions containing an extract of soy and/or clover in
association with a dermatologically acceptable carrier.
[0053] The compositions of the present invention do not include a
UV absorber or a plurality of UV absorbers added for the purpose of
protecting against UV irradiation. Nor do the compositions of this
invention include any stabilizing compounds which provide UV
stability or other stability to UV absorbing compounds given the
absence of such compounds from the compositions of the present
invention.
[0054] Dermatologically acceptable carriers are those which are
compatible with the skin and can be readily applied to the skin by
standard means. Components of such carriers include, but are not
limited to, one or more of water, glycerol, alcohol, propylene
glycol, fatty alcohols, triglycerides, fatty acid esters, and
mineral oils. Dermatologically acceptable carriers may be in the
form of lotions, creams, gels, mousses, aqueous liquids of varying
viscosity, waxed based sticks, aerosols, alcohol sticks and the
like. Examples of formulations well known in the art may be found
in Balsam, M. S. and Sagrin, E. (editors) Cosmetic Science and
Technology, second edition, Volume 1 and 2, Wiley-Interscience, a
division of John Wiley & Sons Inc, New York, 1972 and Flick, E.
W. Cosmetic and Toiletry Formulations, Noyse Publications 1984,
each of which is incorporated herein by reference.
[0055] Dermatologically acceptable carriers may include one or more
emollients, emulsifiers, surfactants, waxes, thickeners, film
formers, preservatives and perfumes. Such agents are well known in
the art, and are described in Cosmetic Science and Technology and
Cosmetic and Toiletry Formulations referred to above. Further
examples of such additional components are provided for example in
W096/14826.
[0056] An example of the standard lotion for topical application to
the skin contains the following:
1 paraffin oil bees wax triethanolamine olive oil glyceryl
monostearate anhydrous lanolin oleic acid stearic acid water
[0057] The components listed above, other than water, may be
present in an amount from about 0.01% to 10% w/w. Water may be
present in an amount from about 20% to about 90% w/w. Compounds of
the Formula (I) may be present in such a lotion in an amount from
0.05% to 10% w/w.
[0058] Compositions according to the present invention may be
readily prepared according to standard procedures known in the art
for the preparation of compositions for topical application to the
skin, for example as described by Balsam, M. S. and Sagrin, E.
(editors) Cosmetic Science and Technology, second edition, Volume 1
and 2, Wiley-Interscience, a division of John Wiley & Sons Inc,
New York, 1972 and Flick, E. W. Cosmetic and Toiletry Formulations,
Noyse Publications, 1984. By way of example, compositions may be
prepared by blending together the compounds of the Formula (I)
(optionally dissolved in a solvent such as DMSO, ethanol, paraffin
oil, olive oil or other suitable solvents) and one or more
dermatological acceptable carriers, such as first by
dissolving.
[0059] Pharmaceutically acceptable carriers and dosage forms for
oral administration are well known in the art, and are described
for example in Remingtons Pharmaceutical Sciences, Mack Publishing
Co., Ed. Osol, 10th Edition. The carrier may be a solid or a
liquid, or both and is preferably formulated with compounds of the
formula (I) or extracts of soy or clover as a unit dose, for
example, a tablet, which may contain from 0.5% to 60% by weight of
the active component. One or more active compounds may be
formulated with one or more carriers by well known techniques of
pharmacy. Formulations may be prepared by uniformly and intimately
admixing the active compound with a liquid or finely divided solid
carrier or both, and then, if necessary, shaping the resulting
mixture to form a unit dosage. Moulded tablets may, for example, be
made by moulding together powdered components moistened with an
inert liquid binder. Compositions may be administered before and/or
after UV exposure. Conveniently compositions are administered
daily, for example giving a daily administration of 0.05 mg to 500
mg per day of active components.
[0060] Compounds of the Formula (I) may be prepared according to
procedures disclosed in PCT/AU97/00563 which is incorporated herein
by reference. Compounds of the Formula (I) may be purified from
human urine according to the procedures of Jaonnou et al (1995) J.
Steroid. Biochem. Molec. Biol., 54, 167-184 which is incorporated
herein by reference.
[0061] Extracts of soy or clover may be prepared according to
WO93/23069, the teachings of which are incorporated by reference.
As described in WO93/23069 soy or clover may be extracted with a
mixture of organic solvent (such as ethanol, chloroform, acetone,
ethyl acetate and the like) and water. The ratio of solvent in
water may be from 0.1% to 99.9%, preferably 40% to 60%.
[0062] Red clover (T. pratense) and subterranean clover (T.
subterranean) are preferred clovers. Raw plant material may be
dried, chaffed or otherwise comminuted and then subject to
extraction. The resultant organic solvent layer following
extraction is removed such as by distillation, and the aqueous
layer and residual material from the organic layer concentrated as
desired, such as by distillation. In respect of soy, beans may be
treated to remove the hull (such as by using a tumble mill which
splits the beans into two cotyledons and a hypocotyl which may be
separated from one another). Cotyledons, and optionally hypocotyls,
may be comminuted and then subject to extraction as described
above. Extracts may include one or more compounds of the Formula
(I).
[0063] Extracts are formulated with a dermatologically acceptable
carrier as herein described to give a composition for application
to the skin, or formulated for oral administration. Compositions
may contain from 0.01% to 10% w/w of extract.
[0064] The composition of the present invention may include
.beta.-(1-3)(1,6)-glucan (such as prepared according to
PCT/AU96/00138, the teachings of which are incorporated herein by
reference), which may be present in an amount from 0.1% to 30% w/w.
If particulate, the glucan may be dissolved in 0.1% DMSO or other
suitable dissolving solvent. Such compositions may exhibit
synergism between the respective components resulting in highly
potent protection from UV induced skin damage. Accordingly, this
invention in another aspect related to compositions and methods of
treatment, as described herein, which include
.beta.-(1-3)(1,6)-glucan.
[0065] UV induced skin damage refers to any sunlight or other UV
damage which effects skin, whether of a human or animal. Such
damage includes erythema (reddening and swelling of the skin, often
referred to as sunburn) photoaging of skin such as
hyperkeratinization and elastosis and skin lesions such as
precancerous and cancerous lesions, for example actinic keratoses
and pre-malignant and malignant skin cancers.
[0066] According to another aspect of this invention there is
provided a method for protecting skin from UV induced
immunosuppression and UV induced skin damage which comprises
applying to the skin of a subject after UV exposure a composition
which comprises a compound of the Formula (I) as described in
association with a dermatologically acceptable carrier. The
composition may include .beta.-(1,3)(1,6)-glucan.
[0067] In another aspect of this invention there is provided a
method for protecting skin against UV induced immunosuppression and
UV induced skin damage which comprises orally administering to a
subject either before and/or after UV exposure a compound of the
Formula (I) or an extract of soy or clover.
[0068] Compositions are applied to the skin following sun exposure,
and are generally applied after each exposure to the skin, or
alternatively at the end of a day following a series of exposure of
the skin to sun.
[0069] Compositions may be applied to the skin by any convenient
means known in the art, such as by way of being rubbed on, rolled
on, sprayed on, wiped on or the like. The mode of application would
generally depend upon the nature of the formulation, whether a
cream, foam, lotion, roll-on "stick", liquid of varying viscosity,
or the like.
[0070] Particularly preferred compounds of the Formula (I) for use
in compositions and methods of the present invention are genistein
(compound 1 where R.sub.7' is, and R.sub.8 is H), equol (compound
10 where is a bond) and dehydroequol (compound 10 where is a double
bond), tetrahydrodaidzein (compound 8) and O-desmethyl-angolensin
(ODMA--compound 13).
[0071] In another aspect of this invention there is provided use of
compounds of the Formula (I) or an isoflavone extract of soy or
clover for the manufacture of a medicament for the treatment,
amelioration, prophylaxis and/or prevention of UV induced
immunosuppression in the skin, and UV induced skin damage.
[0072] In another aspect this invention is concerned with an
article which includes a composition as herein defined. Examples of
such articles include wipes having applied thereto the
aforementioned compositions, emollient sticks, spray devices,
containers or the like. O'Dell et al (1980 Arch. Dermatol, 116,
559-561) have shown that sun-exposed skin areas on humans are
immunologically impaired according to non-exposed skin. A standard
model for testing immunosuppression, that is a model with reference
to human immunosuppression, is the hairless mouse. A standard test
in the hairless mouse model is the contact hypersensitivity (CHS)
reaction to an immune irritant such as oxazolone (Asherson and
Ptak, Immunology, (1968) 15:405-416). The compound oxazolone
induces a vigorous immune response in the hairless mouse which may
be measured by ear swelling. Immune suppression by UV light or
other agents can be readily tested in the model. See for example
Kondo, S, McKenzie, R. C., Sauder, D. N., J. Invest. Dermatol.,
103:811-814; Reeve et al, Cancer Letts., 108:271-279 (1996); and
Reeve et al, J. Invest. Dermatol., 103:801-806 (1994). As will be
shown in the non-limiting examples which follow, compositions
comprising compounds of the Formula (I) were active in the
treatment/prevention/amelioration of UV induced immunosuppression
in the hairless mouse model, which as discussed above is directly
referable to the situation in human skin, for example, Yoshikawa et
al, J. Invest. Dermatol., 95:530-536, (1990).
[0073] Hairless mouse strains such as the HRA-Skh-1 mice are a
standard mouse model used to study solar damage to human skin
[Canfield et al, Pathology, 17:613-616, (1985)]. Exposure of the
hairless mouse to UV light mimics "sunburn" in humans. With
continued irradiation treatment, this on-going damage is reflected
in progressive thickening of the skin which histologically mimics
hyperkeratinization and elastosis associated with photoaging and
chronically sun-exposed skin in humans. Pre-malignant tumours begin
to appear with several weeks of completion of the ultra violet
light regimen. Over an ensuing time period there is a progressive
development of pre-malignant and malignant tumours, the histology
and behaviour of which closely mimic actinic keratoses and
pre-malignant and malignant skin cancers that develop in humans in
response to sunlight.
[0074] It has been found by the inventors that compositions
containing compounds of the Formula (I), or extracts as herein
described, with or without .beta.-(1,3)(1,6)-glucan when applied to
the skin in a dermatologically acceptable carrier following ultra
violet irradiation, or by oral administration before and/or after
ultra violet irradiation, provides protection from UV induced skin
damage such as erythema, photoaging effects (as evidenced by
progressive thickening of the skin) and a development of other UV
induced lesions of the skin including pre-malignant and malignant
skin cancers.
[0075] This invention will now be described with reference to the
following non-limiting examples which illustrate various
embodiments of the invention.
EXAMPLE 1
Preparation of Lotions Containing Compositions of the Formula
(I)
[0076] A lotion ("base or test lotion") was prepared by mixing
together the following components:
2 ml ml paraffin oil 80 glyceryl monostearate 60 olive oil 60 oleic
acid 25 anhydrous lanolin 60 water 1200 stearic acid 58
triethanolamine 27 beeswax 10
[0077] Compounds of the Formula (I) were dissolved in 0.1%
dimethylsulfoxide (DMSO) and incorporated into the base lotion in
an amount from 0.01% to 10% w/w.
[0078] Test lotion "A" was prepared containing 20 .mu.M of various
compounds of the Formula (I) dissolved in 0.1% DMSO (a primary
solvent for the compounds). Test lotion `B` was prepared containing
20 .mu.M of various compounds of the Formula (I) dissolved in 0.1%
DMSO in combination with a solution of .beta.-(1,3)(1,6)-glucan
(10% w/w).
[0079] An extract of red clover was prepared according to
WO93/23069. The aqueous extract, or a particulate or dried extract
dissolved in 0.1% DMSO is incorporated into the base lotion in an
amount of 0.5% w/w.
EXAMPLE 2
UV Induced Immunosuppression
[0080] The CHS reaction in mice is a standard model for
immunosuppression and is a representative model for human
immunosuppression (Yoshikawa et al, Invest. Dermatol., 95:530-536
(1990)).
[0081] The CHS reaction was carried out according to the protocol
of Reeve et al, Immunology, 78:99-104 (1993). Hairless albino
Sh:HR-1 mice were divided into two treatment groups. The first
treatment group designated group 1 was not subject to UV
irradiation, and received either 0.2 ml of test lotion "A" or test
lotion `B` painted on to the dorsal skin on four consecutive days
(that is, 4 .mu.mol/mouse, or approximately 0.1 .mu.mol/cm.sup.2).
The second treatment group, group 2, were UV irradiated with a
minimal erythemal dose of UV light which simulates the toxic effect
of sunlight on the skin. After each daily UV treatment 0.2 ml of
treatment solution was applied to the dorsal skin as for treatment
group 1. Mid-dorsal skinfold thickness was measured every twenty
four hours to indicate erythema oedema. CHS to oxazolone was
induced on abdominal skin seven days after the first treatment
followed by challenge on the ears on day 15, and average ear
swelling was determined as a measure of immune response.
[0082] Genistein, daidzein, equol, tetrahydrodaidzein, ODMA,
dehydroequol and clover extract were tested in this experiment in
test lotion "A". Equol, genistein and clover extract were also
tested in tes lotion `B`. The results expressed below are given as
a percentage of suppression of CHS measured with reference to the
non-UV exposed treatment group 1, which is induced by UV exposure.
Results are as follows:
3 % Suppression of CHS Hours of ear challenge 18 21 Base lotion 24
37 Genistein 14 13 Daidzein 27 37 Equol 12 5 tetrahydrodaidzein 33
31 dehydroequol 10 4 Clover extract 8 9 Genistein and glucan 11 9
Equol and glucan 8 3 Clover extract and glucan 5 5
[0083] The above results clearly show that genistein, dehydroequol
and equol have significant activity in prevention/amelioration of
UV induced immunosuppression as does the clover extract. In
contrast, the compounds daidzein and tetrahydrodaidzein did not
ameliorate the effects of UV induced immunosuppression.
[0084] Erythema/oedema was measured at day four and day seven of
the treatment groups. These results show that genistein and equol
suppressed erythema/oedema. For example, at seven days after
treatment for base lotion "A" average skin fold thickness was six
units (that is 0.006 in), for the daidzein containing lotion
average skin fold increase was seven units, and for
tetrahydrodaidzein average skin fold thickness was five units. In
contrast, for equol, genistein, dehydroequol and ODMA there was a
negative average skin fold increase of approximately 0.2, which
corresponds to substantially no increase in skin fold thickness.
This result shows that compositions of the Formula (I) are
effective in protecting against UV induced skin damage,
particularly erythema, oedema, and thickening of the skin.
EXAMPLE 3
[0085] Tests were performed at the Australian Photobiology Testing
Facility at the University of Sydney. Human subjects were tested in
a controlled solarium. A grid was established using reference
coordinate points on the back of each subject, thereby providing a
series of treatment sites on the back of each patient where the
erythema-reducing potential of compositions according to the
invention could be tested.
[0086] Compounds of the Formula (I) were dissolved in DMSO and
incorporated into base lotion "A" to contain 100 .mu.M of active
ingredient, and 0.5% DMSO.
[0087] Topical applications were made to the back of the patient
within the treatment grid which was established to provide a series
of sites for UV irradiation, and subsequent application of
compositions. Topical application consisted of 2 mg/cm.sup.2 lotion
applied firmly, followed by a second 2 mg/cm.sup.2 applied but not
rubbed in. Each topical treatment was therefore 0.4 nmols/cm.sup.2
compound in the Formula (I).
[0088] The treatment procedure was as follows. On the first day of
treatment a minimum erythemal dose of UV (MED) was established for
each subject, this being the minimum degree of UV light which on
visual determination produces a reddening of the skin. From this
data, which is specific for each patient, a 0.75 level of minimum
erythema dose was calculated (0.75.times.MED). The first treatment
day was a Monday. Subsequent treatments were as follows:
[0089] Tuesday
[0090] Expose 3.times.3 skin sites to 0.75 MED UV.
[0091] Immediately, at four hours and at six hours post-UV,
[0092] 1 apply nil to three sites
[0093] 2 apply topical base lotion to three sites
[0094] 3 apply topical equol lotion to three sites according to
technique above.
[0095] Wednesday
[0096] Assess erythema (none visible as expected).
[0097] Re-expose same 3.times.3 skin sites to 0.75 MED UV.
[0098] Immediately, at four hours and at six hours post-UV, apply
topical lotions as above.
[0099] Thursday
[0100] Assess erythema visually by ranking the colour intensity of
the 3.times.3 skin sites.
[0101] Re-expose same 3.times.3 skin sites to one MED UV.
[0102] Topical applications as before
[0103] Friday
[0104] Assess erythema.
[0105] The results showed a progressively more intense erythema
development peaking at fifty five and a half hours which gradually
faded. There was no statistical difference between erythema
intensity at triplicate sites receiving UV alone, compared with
sites receiving UV plus base lotion. There was a significant
reduction in erythema intensity at sites receiving UV plus a lotion
containing compounds of the Formula (I), compared with UV alone or
base lotion plus UV. A representative result is presented below
where the compound of the Formula (I) was equol.
4 Average erythema intensity (n = 3) +/-SEM Time UV + SEM SEM +
Point UV UV + compound of UV SEM + compound of Hours alone vehicle
Formula (I) alone vehicle Formula (I) 48 0.3 0.33 0.21 0.08 0.07
0.04 52 0.58 0.67 0.21 0.14 0.18 0.04 55.5 1.5 1.25 0.67 0.29 0.31
0.18 71 1 1.17 0.67 0 0.36 0.27 75 1 1.04 0.38 0 0.44 1 77 0.67
1.17 0.54 0.14 0.36 0.21
[0106] The above results are significant when tested by student's
t-test at individual inspection times. These results demonstrate
that compositions containing compounds of the Formula (I) can be
used in therapeutic treatment to prevent UV damage after sun
exposure in humans.
EXAMPLE 4
[0107] The CHS reaction was carried out as in Example 2. Hairless
albino Sh:HR-1 mice were divided into eight treatment groups as
shown:
[0108] 1. base lotion+MED (UVB) for 3 days
[0109] 2. glucan (20%) in base lotion+1 MED (UVB) for 3 days
[0110] 3. Equol (2.5 uM) in base lotion+1 MED (UVB) for 3 days
[0111] 4. Equol (2.5uM)+glucan (20%) in base lotion+1 MED (UVB) for
3 days
[0112] 5. base lotion and no irradiation for three days
(Control)
[0113] 6. glucan (20%) in base lotion and no irradiation for three
days (Control)
[0114] 7. Equol (2.5 uM) in base lotion and no irradiation for
three days (Control)
[0115] 8. Equol (2.5 uM)+glucan (20%) in base lotion and no
irradiation for three days (Control)
[0116] On days 7 and 8 the mice were sensitised with 2% oxazolone
in alcohol (100 ul/mouse). On day 14, the ears were challenged with
2% oxazolone (20 ul/mouse). On day 15, the ears were measured for
skin fold thickness and suppression of chronic hypersensitivity.
The results indicated that both glucan and equol were effective in
decreasing the percentage suppression of chronic hypersensitivity
and skin fold thickness, and that in combination they provided
potentiated protection when compared to the control groups.
5 Difference in skin fold Lotion % Suppression of CHS thickness
from day 1 (mm) Base lotion only 41% 47.7 Glucan only 4.5% 25 Equol
only 6% 29.8 Glucan + equol 2.5% 16.8
EXAMPLE 5
[0117] Hairless albino Sh:HR-1 mice were divided into two groups
each containing 20 animals. The first group receiving red clover
extract diet (prepared as described in WO 93/23069), and the second
control group receiving a phytoestrogen free diet. Both groups
received 1 minimal erythemal does of UVB light to stimulate the
toxic effect of sunlight on the skin daily for 10 weeks. At weeks
11, 13, 14, 15, 16 and 17 the number of skin tumours on each animal
were measured and calculated as tumour multiplicity, i.e. average
number of tumours per mouse. The results showed a significant
protection (p<0.05) against UVB induced carcinogenesis in mice
fed a red clover diet after 17 weeks.
6 Time (weeks) From Tumour Multiplicity: Tumour Multiplicity:
Initial UVB Irradiation Control Red Clover 11 0 0 13 0.13 0.25 14
0.37 0.46 15 0.82 0.76 16 1.65 1.01 17 2.62 1.51
[0118] The same model is used with compositions containing
compounds of the formula (I). Protection against UVB induced
carcinogenesis is observed.
[0119] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising" or the term "includes" or variations thereof, will
be understood to imply the inclusion of a stated element or integer
or group of elements or integers but not the exclusion of any other
element or integer or group of elements or integers. In this
regard, in construing the claim scope, an embodiment where one or
more features is added to any of claims is to be regarded as within
the scope of the invention given that the essential features of the
invention as claimed are included in such an embodiment.
[0120] All publications referred to herein are incorporated by
reference.
* * * * *