U.S. patent application number 10/071124 was filed with the patent office on 2002-11-21 for pharmaceutical formulations of resveratrol.
Invention is credited to Barillas, Karla Slowing, Jang, Mei-Shiang, Lin, Shengzhao, Moon, Richard C., Ouali, Aomar, Pezzuto, John M..
Application Number | 20020173472 10/071124 |
Document ID | / |
Family ID | 23707110 |
Filed Date | 2002-11-21 |
United States Patent
Application |
20020173472 |
Kind Code |
A1 |
Pezzuto, John M. ; et
al. |
November 21, 2002 |
Pharmaceutical formulations of resveratrol
Abstract
A method is provided for preventing or treating skin conditions,
disorders or diseases, such as may be associated with or caused by
inflammation, sun damage or natural aging. The method involves
administration, preferably topical administration, of an active
agent selected from the group consisting of resveratrol,
pharmacologically acceptable salts, esters, amides, prodrugs and
analogs thereof, and combinations of any of the foregoing.
Pharmaceutical formulations for use in conjunction with the
aforementioned method are provided as well.
Inventors: |
Pezzuto, John M.; (River
Forest, IL) ; Moon, Richard C.; (Plant City, FL)
; Jang, Mei-Shiang; (Chicago, IL) ; Ouali,
Aomar; (Montreal, CA) ; Lin, Shengzhao;
(Montreal, CA) ; Barillas, Karla Slowing; (Madrid,
ES) |
Correspondence
Address: |
REED & ASSOCIATES
800 MENLO AVENUE
SUITE 210
MENLO PARK
CA
94025
US
|
Family ID: |
23707110 |
Appl. No.: |
10/071124 |
Filed: |
February 7, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10071124 |
Feb 7, 2002 |
|
|
|
09430337 |
Oct 29, 1999 |
|
|
|
09430337 |
Oct 29, 1999 |
|
|
|
09005114 |
Jan 9, 1998 |
|
|
|
6008260 |
|
|
|
|
Current U.S.
Class: |
514/25 ;
514/733 |
Current CPC
Class: |
A61K 8/347 20130101;
B82Y 5/00 20130101; A61K 9/0019 20130101; A61K 31/7034 20130101;
A61Q 19/08 20130101; A61P 35/00 20180101; A61K 9/0014 20130101;
A61P 17/16 20180101; A61P 17/12 20180101; A61P 17/00 20180101; A61K
8/60 20130101; A23L 33/105 20160801; A61K 9/1075 20130101; A61K
47/36 20130101; A61P 17/06 20180101; A61K 47/14 20130101; A61K
47/10 20130101; A61K 47/549 20170801; A61K 31/05 20130101 |
Class at
Publication: |
514/25 ;
514/733 |
International
Class: |
A61K 031/7034; A61K
031/05 |
Goverment Interests
[0002] This invention was made with government support under
Contract No. PO 1 CA48112 awarded by the National Cancer Institute.
The United States Government has certain rights in this invention.
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 1998 |
AU |
9888420 |
Claims
1. A method for preventing or treating a skin condition, disorder
or disease that is responsive to resveratrol, comprising
administering to the susceptible or affected area of the
individual's skin a therapeutically effective amount of a topical
pharmaceutical formulation that comprises a topical carrier and an
active agent selected from the group consisting of resveratrol,
pharmacologically acceptable salts, esters, amides, prodrugs and
analogs thereof, and combinations of any of the foregoing.
2. The method of claim 1, wherein the active agent is
cis-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
3. The method of claim 2, wherein the active agent is
cis-resveratrol.
4. The method of claim 2, wherein the active agent is a conjugate
of cis-resveratrol and a mono- or di-saccharide.
5. The method of claim 4, wherein the active agent is
cis-resveratrol glucoside.
6. The method of claim 1, wherein the active agent is
trans-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
7. The method of claim 6, wherein the active agent is
trans-resveratrol.
8. The method of claim 6, wherein the active agent is a conjugate
of trans-resveratrol and a mono- or di-saccharide.
9. The method of claim 8, wherein the active agent is
trans-resveratrol glucoside
10. The method of claim 1, wherein the active agent comprises a
mixture of cis-resveratrol and trans-resveratrol.
11. The method of claim 1, wherein the topical pharmaceutical
formulation comprises an ointment, lotion, cream, emulsion,
microemulsion, gel or solution.
12. The method of claim 1, wherein the topical pharmaceutical
formulation contains approximately 0.25 wt. % to 75 wt. % active
agent.
13. The method of claim 12, wherein the topical pharmaceutical
formulation contains approximately 0.25 wt. % to 30 wt. % active
agent.
14. The method of claim 13, wherein the topical pharmaceutical
formulation contains approximately 0.5 wt. % to 15 wt. % active
agent.
15. The method of claim 14, wherein the topical pharmaceutical
formulation contains approximately 1.0 wt. % to 10 wt. % active
agent.
16. The method of claim 1, wherein the topical pharmaceutical
formulation is administered at least once daily.
17. The method of claim 16, wherein the topical pharmaceutical
formulation is administered one to four times daily.
18. The method of claim 1, wherein the skin condition, disorder or
disease is associated with inflammation.
19. The method of claim 18, wherein the skin condition, disorder or
disease is psoriasis, contact dermatitis, atopic dermatitis,
actinic keratosis, a keratinization disorder, an epidermolysis
bullosa disease, exfoliative dermatitis, seborrheic dermatitis,
erythema multiforme, erythema nodosum, discoid lupus erythematosus,
dermatomyositis or skin cancer.
20. The method of claim 1, wherein the skin condition, disorder or
disease is skin damage caused by the sun or other light
sources.
21. The method of claim 1, wherein the skin condition, disorder or
disease comprises the effects of natural aging on an individual's
skin.
22. A method for preventing or treating a skin condition, disorder
or disease that is responsive to treatment with resveratrol,
comprising administering to a susceptible or affected individual a
pharmaceutical formulation comprised of a microemulsion containing
a therapeutically effective amount of an active agent selected from
the group consisting of resveratrol, pharmacologically acceptable
salts, esters, amides, prodrugs and analogs thereof, and
combinations of any of the foregoing.
23. The method of claim 22, wherein the active agent is
cis-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
24. The method of claim 23, wherein the active agent is
cis-resveratrol.
25. The method of claim 23, wherein the active agent is a conjugate
of cis-resveratrol and a mono- or di-saccharide.
26. The method of claim 25, wherein the active agent is
cis-resveratrol glucoside.
27. The method of claim 22, wherein the active agent is
trans-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
28. The method of claim 27, wherein the active agent is
trans-resveratrol.
29. The method of claim 27, wherein the active agent is a conjugate
of trans-resveratrol and a mono- or di-saccharide.
30. The method of claim 29, wherein the active agent is
trans-resveratrol glucoside.
31. The method of claim 22, wherein the active agent comprises a
mixture of cis-resveratrol and trans-resveratrol.
32. The method of claim 22, wherein the formulation comprises
approximately 0.25 wt. % to 75 wt. % active agent.
33. The method of claim 32, wherein the formulation comprises
approximately 0.25 wt. % to 30 wt. % active agent.
34. The method of claim 33, wherein the formulation comprises
approximately 0.5 wt. % to 15 wt. % active agent.
35. The method of claim 34, wherein formulation comprises
approximately 1.0 wt. % to 10 wt. % active agent.
36. The method of claim 22, wherein the topical pharmaceutical
formulation is administered at least once daily.
37. The method of claim 36, wherein the topical pharmaceutical
formulation is administered one to four times daily.
38. The method of claim 22, wherein the formulation is administered
orally.
39. The method of claim 22, wherein the formulation is administered
parenterally.
40. The method of claim 22, wherein the formulation is administered
at least once daily.
41. The method of claim 40, wherein the formulation is administered
one to four times daily.
42. A topical pharmaceutical formulation for use in preventing or
treating skin conditions, disorders and diseases associated with
inflammation, comprising a topical carrier and a therapeutically
effective concentration of an active agent selected from the group
consisting of resveratrol, pharmacologically acceptable salts,
esters, amides, prodrugs and analogs thereof, and combinations of
any of the foregoing.
43. The formulation of claim 42, wherein the active agent is
cis-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
44. The formulation of claim 43, wherein the active agent is
cis-resveratrol.
45. The formulation of claim 43, wherein the active agent is a
conjugate of cis-resveratrol and a mono- or di-saccharide.
46. The formulation of claim 45, wherein the active agent is
cis-resveratrol glucoside.
47. The formulation of claim 42, wherein the active agent is
trans-resveratrol or a pharmacologically acceptable salt, ester,
amide, prodrug or analog thereof.
48. The formulation of claim 47, wherein the active agent is
trans-resveratrol.
49. The formulation of claim 48, wherein the active agent is a
conjugate of trans-resveratrol and a mono- or di-saccharide.
50. The formulation of claim 49, wherein the active agent is
trans-resveratrol glucoside.
51. The formulation of claim 42, wherein the active agent comprises
a mixture of cis-resveratrol and trans-resveratrol.
52. The formulation of claim 42, wherein the topical carrier
comprises an ointment base and the formulation is an ointment.
53. The formulation of claim 42, wherein the topical carrier
comprises a cream base and the formulation is a cream.
56. The formulation of claim 42, wherein the topical carrier
comprises a lotion base and the formulation is a lotion.
57. The formulation of claim 42, wherein the topical carrier
comprises a gel base and the formulation is a gel.
58. The formulation of claim 42, wherein the topical carrier
comprises an aqueous liquid and the formulation is a solution.
59. The formulation of claim 42, comprising a microemulsion.
60. The formulation of claim 42, comprising approximately 0.25 wt.
% to 75 wt. % active agent.
61. The formulation of claim 60, comprising approximately 0.25 wt.
% to 30 wt. % active agent.
62. The formulation of claim 61, comprising approximately 0.5 wt. %
to 15 wt. % active agent.
63. The formulation of claim 62, comprising approximately 1.0 wt. %
to 10 wt. % active agent.
64. A pharmaceutical formulation comprising: approximately 0.25 wt.
% to 30 wt. % of an active agent selected from the group consisting
of resveratrol, pharmacologically acceptable salts, esters, amides,
prodrugs and analogs thereof, and combinations of any of the
foregoing; approximately 2 wt. % to 20 wt. % emulsifiers;
approximately 2 wt. % to 20 wt. % emollient; approximately 2 wt. %
to 50 wt. % solubilizer; approximately 0.1 wt. % to 0.2 wt. %
preservative; and water.
65. The formulation of claim 64, wherein the emulsifiers are
selected from the group consisting of glyceryl monostearate,
polyoxyethylene stearate, polyethylene glycol, ethylene glycol
palmitostearate, caprilic/capric triglycerides, oleoyl
macrogolglycerides, and combinations thereof.
66. The formulation of claim 64, wherein the emollient is selected
from the group consisting of propylene glycol, glycerol, isopropyl
myristate, PPG-2 ether propionate, and combinations thereof.
67. The formulation of claim 64, wherein the solubilizer is
selected from the group consisting of diethylene glycol monoethyl
ether, diethylene glycol monomethyl ether, diethylene glycol
monoethyl ether oleate, polyethylene glycol, polyethylene castor
oil derivatives, PEG-8 caprylic/capric glycerides, alkyl methyl
sulfoxides, pyrrolidones and dimethyl acetamide.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation-in-part of U.S. patent application
Ser. No. 09/005,114, filed Jan. 9, 1998, the disclosure of which is
incorporated by reference in its entirety.
TECHNICAL FIELD
[0003] The present invention relates generally to pharmaceutical
formulations and methods of use thereof. More particularly, the
invention relates to pharmaceutical formulations of resveratrol
(3,5,4'-trihydroxystilbene) that are useful, for example, in the
prevention and treatment of skin conditions associated with
inflammation, sun damage and aging. The invention finds utility in
the fields of drug delivery, medicine and pharmacology.
BACKGROUND
[0004] It has been noted that there are a number of biologically
active phenolic compounds present in wine, particularly red wine.
Such compounds include, for example, catechin, epicatechin,
quercetin, rutin, trans-resveratrol, cis-resveratrol,
cis-resveratrol glucoside and trans-resveratrol glucoside. See,
e.g., Goldberg et al. (1996) Anal. Chem.68:1688-1694. These
compounds have been shown to protect low-density lipoproteins
against oxidation. The resveratrol isomers, in particular, have
been found to promote vascular relaxation through the generation of
nitric oxide by the endothelium, and to modulate eicosanoid
synthesis in a manner that suggests use in preventing coronary
heart disease. Id. at pp. 1688-89). This discovery appears to
explain the studies demonstrating that moderate consumption of red
wine tends to have a protective effect against heart disease.
Bertelli et al. (1995) Inst. J. Tiss. Reac. XVII(1):1-3. 1
[0005] Resveratrol (3,5,4'-trihydroxystilbene) has been identified
as a constituent not only of grape skins (Soleas et al . (1995) Am.
J. Enol. Vitic. 46(3):346-352) but has also been found to be
present in ground nuts, eucalyptus, and other plant species.
Goldberg et al. (1995), Am. J. Enol. Vitic. 46(2):159-165. A great
deal of interest has been focused on the compound's antifungal
activity and its correlation with resistance to fungal infection.
Id. at 159. Resveratrol may be obtained commercially (typically as
the trans isomer, e.g. from the Sigma Chemical Company, St. Louis,
Mo.), or it may be isolated from wine or grape skins, or it may be
chemically synthesized. Synthesis is typically carried out by a
Wittig reaction linking two substituted phenols through a styrene
double bond, as described by Moreno-Manas et al. (1985) Anal. Quim
81:157-61 and subsequently modified by others (Jeandet et al.
(1991) Am. J. Enol. Vitic. 42:41-46; Goldberg et al. (1994) Anal.
Chem. 66: 3959-63).
[0006] There are more studies concerning trans-resveratrol than the
cis isomer; however, the cis isomer appears to be equally important
from a biological standpoint. Numerous uses have been proposed and
evaluated for the resveratrol isomers. Jang et al. (1997) Science
275:218-220, show that resveratrol has cancer chemopreventive
activity in assays representing three major stages of
carcinogenesis. That is, the authors found that the compound: (1)
acted as an antioxidant and antimutagen and induced phase II
drug-metabolizing enzymes; (2) mediated anti-inflammatory effects
and inhibited cyclooxygenase and hydroperoxidase; and (3) induced
human promyelocytic leukemia cell differentiation. In addition, as
noted above, resveratrol has been extensively studied for its
correlation to the cardiovascular utility of red wine. See, e.g.,
Bertelli et al., supra; Pace-Asciak et al. (1995), Clinica Chimica
Acta 235:207-2191; and Frankel et al. (Apr. 24, 1993), The Lancet
341:1104. Neurologic uses have also been proposed (Lee et al.
(1994), Society for Neuroscience Abstracts 20(1-2):1648).
[0007] To date, however, topical administration of resveratrol is
unknown. The present invention is premised on the unexpected
finding that topical administration of resveratrol is extremely
effective in preventing or treating certain skin conditions,
disorders and diseases, e.g., skin conditions associated with
inflammation, skin damage associated with exposure to the sun, and
the effects of natural aging. It has in addition been discovered
that administration of resveratrol in the form of a microemulsion
is a particularly effective mode of administering the active
agent.
[0008] In addition, resveratrol has found to be useful as a cancer
chemopreventive agent. Known cancer chemopreventive agents include
nonsteroidal antiinflammatory drugs (NSAIDs) such as indomethacin,
aspirin, piroxicam, and sulindac, all of which inhibit
cyclooxygenase, abbreviated hereafter as COX. A COX inhibitory
activity is important in cancer chemoprevention because COX
catalyzes the conversion of arachidonic acid to proinflammatory
substances, such as prostaglandins, which can stimulate tumor cell
growth and suppress immune surveillance. Plescia et al. (1975)
Proc. Natl. Acad. Sci. U.S.A. 72:1848; Goodwin (1984) Am. J. Med.
77:7. In addition, COX can activate carcinogens to forms that
damage genetic material. Zenser et al. (1983), J. Pharmacol. Exp.
Ther. 227:545; Wild et al. (1987) Carcinogenesis 8:541.
Investigators have searched for new cancer chemopreventive agents
by evaluating hundreds of plant extracts for a potential to inhibit
COX. An extract derived from Cassia quinquangulata Rich.
(Leguminosae) was identified as a potent COX inhibitor, and on the
basis of bioassay-guided fractionation, trans-resveratrol was
identified as the active compound. See Mannila et al. (19983)
Phytochemistry 33:813, and Jayatilake et al. (1993), J. Nat. Prod.
56:1805.
SUMMARY OF THE INVENTION
[0009] Accordingly, it is a primary object of the invention to
address the above-mentioned needs in the art by providing a method
for preventing or treating certain skin conditions, disorders and
diseases.
[0010] It is another object of the invention to provide such a
method by administering to a patient a formulation comprising an
active agent selected from the group consisting of resveratrol,
pharmacologically acceptable salts, esters, amides, prodrugs and
analogs thereof, and combinations of any of the foregoing.
[0011] It is another object of the invention to provide such a
method wherein the formulation is administered topically.
[0012] It is still another object of the invention to provide such
a method wherein the skin condition, disorder or disease is
associated with inflammation.
[0013] It is yet another object of the invention to provide such a
method in the context of preventing or treating skin-related sun
damage and/or the effects of natural aging on the skin.
[0014] It is an additional object of the invention to provide such
a method in the context of preventing skin cancer.
[0015] It is a further object of the invention to provide such
methods by administering resveratrol in stereoisomerically pure
form, i.e., in either the cis or the trans form.
[0016] It is still a further object of the invention to provide
novel pharmaceutical formulations comprising an active agent
selected from the group consisting of resveratrol,
pharmacologically acceptable salts, esters, amides, prodrugs and
analogs thereof, and combinations of any of the foregoing, in
combination with a pharmaceutically acceptable carrier.
[0017] Additional objects, advantages and novel features of the
invention will be set forth in part in the description which
follows, and in part will become apparent to those skilled in the
art upon examination of the following, or may be learned by
practice of the invention.
[0018] In one embodiment, then, a method is provided for preventing
or treating certain skin conditions, disorders or diseases
comprising administering to the susceptible or affected individual,
preferably by application to the individual's skin, a
pharmaceutical composition comprising a therapeutically effective
amount of an active agent selected from the group consisting of
resveratrol, pharmacologically acceptable salts, esters, amides,
prodrugs and analogs thereof, and combinations of any of the
foregoing. Generally, the active agent will be cis-resveratrol,
trans-resveratrol, or a complex in which one or more of the
compounds' hydroxyl groups are conjugated to a mono- or
di-saccharide, e.g., cis-resveratrol glucoside, trans-resveratrol
glucoside, etc. However, as will be appreciated by those skilled in
the art, and as discussed in detail elsewhere herein, other forms
of the active agents may also be used. The skin condition, disorder
or disease may be, for example, psoriasis, contact dermatitis
(including irritant contact dermatitis and allergic contact
dermatitis), atopic dermatitis (also known as allergic eczema),
actinic keratosis, keratinization disorders (including eczema),
epidermolysis bullosa diseases (including penfigus), exfoliative
dermatitis, seborrheic dermatitis, erythemas (including erythema
multiforme and erythema nodosum), damage caused by the sun or other
light sources, discoid lupus erythematosus, dermatomyositis, skin
cancer, effects of natural aging and the like.
[0019] In another embodiment, a pharmaceutical formulation is
provided comprising an active agent as described above, with
cis-resveratrol, trans-resveratrol, and conjugates of
cis-resveratrol or trans-resveratrol with mono- or di-saccharides
preferred. The formulation is preferably for topical
administration, and thus also comprises a pharmaceutically
acceptable topical carrier selected to correspond to the particular
formulation type, e.g., ointment, lotion, cream, microemulsion,
gel, oil, solution, or the like. The formulation may also contain
one or more additional active agents and/or additives such as
solubilizers, skin permeation enhancers, opacifiers, preservatives
(e.g., anti-oxidants), gelling agents, buffering agents,
surfactants, emulsifiers, emollients, thickening agents,
stabilizers, humectants, colorants, fragrance, and the like.
Inclusion of solubilizers and/or skin permeation enhancers is
particularly preferred.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 contains plots of COX-1 and COX-2 activity vs.
concentration of indomethacin and resveratrol;
[0021] FIG. 2 contains plots of COX-1 and COX-2 hydroperoxidase
activity vs. concentration of indomethacin and resveratrol;
[0022] FIG. 3 is a plot of percent incidence of mouse mammary
lesions vs. resveratrol concentration;
[0023] FIG. 4 contains plots of percent reduction in paw volume vs.
time (hours) for carrageenan-induced inflammation in rats for
various drugs vs. a control group; and
[0024] FIG. 5 contains plots of % tumor incidence (a) and number of
tumors (b) vs. time after resveratrol treatment.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Overview and Definitions:
[0026] Before the present invention is described in detail, it is
to be understood that unless otherwise indicated this invention is
not limited to any particular formulation, carrier, or drug
administration regimen, as such may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only and is not intended to limit
the scope of the present invention.
[0027] It must be noted that as used herein and in the claims, the
singular forms "a," "and" and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "an active agent" in a formulation includes two or
more active agents, reference to "a carrier" includes two or more
carriers, and so forth.
[0028] In this specification and in the claims which follow,
reference will be made to a number of terms which shall be defined
to have the following meanings:
[0029] The term "topical administration" is used in its
conventional sense to mean delivery of a topical drug or
pharmacologically active agent to the skin or mucosa, as in, for
example, the treatment of various skin disorders. In general, when
the term "skin" is used herein, as in administration of a topical
formulation to "the skin," it is to be understood that
administration to mucosal tissue is intended as a possibility as
well.
[0030] The terms "active agent," "drug" and "pharmacologically
active agent" are used interchangeably herein to refer to a
chemical material or compound that is suitable for topical
administration and induces the desired physiological effect. The
terms include derivatives, analogs and prodrugs of such compounds,
as well as pharmaceutically acceptable salts, esters, amides, and
the like.
[0031] The term "resveratrol" is intended to mean either the
cis-isomer of resveratrol, the trans-isomer of resveratrol, or a
mixture of the two isomers. The term is also intended to include
both the naturally occurring active agent and the compound as it
may be chemically synthesized in the laboratory. Further, when the
term "resveratrol" is used herein, it is intended to encompass
pharmacologically acceptable salts, esters, amides, prodrugs and
analogs of resveratrol.
[0032] By the term "effective" or "therapeutically effective"
amount of a drug is meant a nontoxic but sufficient amount of a
compound, to provide the desired effect at a reasonable
benefit/risk ratio attending any medical treatment. The desired
effect and can be alleviation of the signs, symptoms, or causes of
a disease, or any other desired alteration of a biological
system.
[0033] The term "treat" as in "to treat a skin condition" is
intended to include (1) preventing the condition, i.e., avoiding
any clinical symptoms of the condition, (2) inhibiting the
condition, that is, arresting the development or progression of
clinical symptoms, and/or (3) relieving the condition, i.e.,
causing regression of clinical symptoms.
[0034] The terms "condition," "disorder" and "disease" are used
interchangeably herein as referring to a physiological state that
can be prevented or treated by administration of an active agent as
described herein.
[0035] The term "individual" as in treatment of "an individual" is
intended to refer to a mammalian individual afflicted with or prone
to a condition, disorder or disease as specified herein, and
includes both humans and animals.
[0036] By "pharmacologically acceptable" is meant a material that
is not biologically or otherwise undesirable, i.e., the material
may be administered to an individual along with the selected active
agent without causing any undesirable biological effects or
interacting in a deleterious manner with any of the other
components of the pharmaceutical composition in which it is
contained. Similarly, a "pharmacologically acceptable" salt or a
"pharmacologically acceptable" ester of an active agent as provided
herein is a salt or ester which is not biologically or otherwise
undesirable.
[0037] "Penetration enhancer" or "permeation enhancer" as used
herein relates to an increase in the permeability of the skin or
mucosal tissue to the selected pharmacologically active agent,
i.e., so that the rate at which the drug permeates through the skin
or mucosa is increased.
[0038] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not. For example, recitation of an additive
as "optionally present" in a formulation herein encompasses both
the formulation containing the additive and the formulation not
containing the additive.
[0039] Active Agents for Treatment:
[0040] The invention, as noted above, involves the administration
of resveratrol to an individual in order to prevent or treat skin
conditions associated with inflammation.
[0041] Resveratrol may be administered in natural form, i.e., as
isolated from grape skins, wine or other plant-derived
compositions, or it may be administered as chemically synthesized
in the laboratory (e.g., using the methods of Moreno-Manas et al.,
Jeandet et al., or Goldberg et al. (1994), cited earlier herein),
or as obtained commercially, e.g., from the Sigma Chemical Company
(St. Louis, Mo.). Preferred methods for obtaining resveratrol from
a natural source is to extract the compound from P. capsudatum or
from the dried roots of C. quinquangulata which may be harvested in
Peru. The dried ground plant material may be extracted with a
suitable solvent, e.g., methanol, preferably followed by
concentration and dilution with water. After washing with hexane or
an equally suitable nonpolar organic solvent, the aqueous layer may
be partitioned with, for example, ethyl acetate. The ethyl acetate
extract is then separated into fractions using, for example,
chloroform-methanol (0 to 30% methanol) as eluent over a silica gel
chromatographic column. Fractions with higher concentrations of
resveratrol may be combined and subjected to further column
chromatography until the product is obtained in sufficiently high
yield.
[0042] The active agent may be administered in the form of a
pharmacologically acceptable salt, ester, amide, prodrug or analog
or as a combination thereof. Salts, esters, amides, prodrugs and
analogs of resveratrol may be prepared using standard procedures
known to those skilled in the art of synthetic organic chemistry
and pharmaceutical formulation, described, for example, by J.
March, "Advanced Organic Chemistry: Reactions, Mechanisms and
Structure," 4th Ed. (New York: Wiley-Interscience, 1992), and in
Remington's Pharmaceutical Sciences, 19th Ed. (Easton, Pa.: Mack
Publishing Company, 1995). For example, basic addition salts are
prepared from the neutral drug using conventional means, involving
reaction of one or more of the active agent's free hydroxyl groups
with a suitable base. Generally, the neutral form of the drug is
dissolved in a polar organic solvent such as methanol or ethanol
and the base is added thereto. The resulting salt either
precipitates or may be brought out of solution by addition of a
less polar solvent. Suitable bases for forming basic addition salts
include, but are not limited to, inorganic bases such as sodium
hydroxide, potassium hydroxide, ammonium hydroxide, calcium
hydroxide, trimethylamine, or the like. Preparation of esters
involves functionalization of hydroxyl groups which may be present
within the molecular structure of the drug. The esters are
typically acyl-substituted derivatives of free alcohol groups,
i.e., moieties which are derived from carboxylic acids of the
formula RCOOH where R is alkyl, and preferably is lower alkyl.
Esters can be reconverted to the free acids, if desired, by using
conventional hydrogenolysis or hydrolysis procedures. Preparation
of amides and prodrugs can be carried out in an analogous manner.
Other derivatives and analogs of the active agents may be prepared
using standard techniques known to those skilled in the art of
synthetic organic chemistry, or may be deduced by reference to the
pertinent literature.
[0043] Preferred derivatives of cis- and trans-resveratrol are
those in which one or more of the compounds' hydroxyl groups,
typically the 3-hydroxyl group, is conjugated to a mono- or
di-saccharide, generally the 1-position of a monosaccharide.
Examples of saccharides which may be conjugated to the resveratrol
molecule include, but are not limited to, glucose, galactose,
maltose, lactose and sucrose. Cis-resveratrol glucoside and
trans-resveratrol glucoside are particularly preferred.
[0044] Pharmaceutical Formulations:
[0045] In a preferred embodiment, the active agent is incorporated
into a topical formulation containing a topical carrier that is
generally suited to topical drug administration and comprising any
such material known in the art. The topical carrier is selected so
as to provide the composition in the desired form, e.g., as an
ointment, lotion, cream, microemulsion, gel, oil, solution, or the
like, and may be comprised of a material of either naturally
occurring or synthetic origin. It is essential, clearly, that the
selected carrier not adversely affect the active agent or other
components of the topical formulation. Examples of suitable topical
carriers for use herein include water, alcohols and other nontoxic
organic solvents, glycerin, mineral oil, silicone, petroleum jelly,
lanolin, fatty acids, vegetable oils, parabens, waxes, and the
like.
[0046] Particularly preferred formulations herein are colorless,
odorless ointments, lotions, creams, microemulsions and gels.
[0047] Ointments are semisolid preparations which are typically
based on petrolatum or other petroleum derivatives. The specific
ointment base to be used, as will be appreciated by those skilled
in the art, is one that will provide for optimum drug delivery,
and, preferably, will provide for other desired characteristics as
well, e.g., emolliency or the like. As with other carriers or
vehicles, an ointment base should be inert, stable, nonirritating
and nonsensitizing. As explained in Remington's, cited in the
preceding section, ointment bases may be grouped in four classes:
oleaginous bases; emulsifiable bases; emulsion bases; and
water-soluble bases. Oleaginous ointment bases include, for
example, vegetable oils, fats obtained from animals, and semisolid
hydrocarbons obtained from petroleum. Emulsifiable ointment bases,
also known as absorbent ointment bases, contain little or no water
and include, for example, hydroxystearin sulfate, anhydrous lanolin
and hydrophilic petrolatum. Emulsion ointment bases are either
water-in-oil (W/O) emulsions or oil-in-water (O/W) emulsions, and
include, for example, cetyl alcohol, glyceryl monostearate, lanolin
and stearic acid. Preferred water-soluble ointment bases are
prepared from polyethylene glycols (PEGs) of varying molecular
weight; again, reference may be had to Remington's, supra, for
further information.
[0048] Lotions are preparations to be applied to the skin surface
without friction, and are typically liquid or semiliquid
preparations in which solid particles, including the active agent,
are present in a water or alcohol base. Lotions are usually
suspensions of solids, and preferably, for the present purpose,
comprise a liquid oily emulsion of the oil-in-water type. Lotions
are preferred formulations herein for treating large body areas,
because of the ease of applying a more fluid composition. It is
generally necessary that the insoluble matter in a lotion be finely
divided. Lotions will typically contain suspending agents to
produce better dispersions as well as compounds useful for
localizing and holding the active agent in contact with the skin,
e.g., methylcellulose, sodium carboxymethylcellulose, or the like.
A particularly preferred lotion formulation for use in conjunction
with the present invention contains propylene glycol mixed with a
hydrophilic petrolatum such as that which may be obtained under the
trademark Aquaphor.RTM. from Beiersdorf, Inc. (Norwalk, Conn.).
[0049] Creams containing the active agent are, as known in the art,
viscous liquid or semisolid emulsions, either oil-in-water or
water-in-oil. Cream bases are water-washable, and contain an oil
phase, an emulsifier and an aqueous phase. The oil phase is
generally comprised of petrolatum and a fatty alcohol such as cetyl
or stearyl alcohol; the aqueous phase usually, although not
necessarily, exceeds the oil phase in volume, and generally
contains a humectant. The emulsifier in a cream formulation, as
explained in Remington 's, supra, is generally a nonionic, anionic,
cationic or amphoteric surfactant.
[0050] Microemulsions are thermodynamically stable, isotropically
clear dispersions of two immiscible liquids, such as oil and water,
stabilized by an interfacial film of surfactant molecules
(Encyclopedia of Pharmaceutical Technology (New York: Marcel
Dekker, 1992), volume 9). For the preparation of microemulsions,
surfactant (emulsifier), co-surfactant (co-emulsifier), an oil
phase and a water phase are necessary. Suitable surfactants include
any surfactants that are useful in the preparation of emulsions,
e.g., emulsifiers that are typically used in the preparation of
creams. The co-surfactant (or "co-emulsifer") is generally selected
from the group of polyglycerol derivatives, glycerol derivatives
and fatty alcohols. Preferred emulsifier/co-emulsifier combinations
are generally although not necessarily selected from the group
consisting of: glyceryl monostearate and polyoxyethylene stearate;
polyethylene glycol and ethylene glycol palmitostearate; and
caprilic and capric triglycerides and oleoyl macrogolglycerides.
The water phase includes not only water but also, typically,
buffers, glucose, propylene glycol, polyethylene glycols,
preferably lower molecular weight polyethylene glycols (e.g., PEG
300 and PEG 400), and/or glycerol, and the like, while the oil
phase will generally comprise, for example, fatty acid esters,
modified vegetable oils, silicone oils, mixtures of mono- di- and
triglycerides, mono- and di-esters of PEG (e.g., oleoyl macrogol
glycerides), etc.
[0051] Gel formulations are semisolid systems consisting of either
suspensions made up of small inorganic particles (two-phase
systems) or large organic molecules distributed substantially
uniformly throughout a carrier liquid (single phase gels). Single
phase gels can be made, for example, by combining the active agent,
a carrier liquid and a suitable gelling agent such as tragacanth
(at 2 to 5%), sodium alginate (at 2-10%), gelatin (at 2-15%),
methylcellulose (at 3-5%), sodium carboxymethylcellulose (at 2-5%),
carbomer (at 0.3-5%) or polyvinyl alcohol (at 10-20%) together and
mixing until a characteristic semisolid product is produced. Other
suitable gelling agents include methylhydroxycellulose,
polyoxyethylene-polyoxypropylene, hydroxyethylcellulose and
gelatin. Although gels commonly employ aqueous carrier liquid,
alcohols and oils can be used as the carrier liquid as well.
[0052] Various additives, known to those skilled in the art, may be
included in the topical formulations of the invention. Examples of
additives include, but are not limited to, solubilizers, skin
permeation enhancers, opacifiers, preservatives (e.g.,
anti-oxidants), gelling agents, buffering agents, surfactants
(particularly nonionic and amphoteric surfactants), emulsifiers,
emollients, thickening agents, stabilizers, humectants, colorants,
fragrance, and the like. Inclusion of solubilizers and/or skin
permeation enhancers is particularly preferred, along with
emulsifiers, emollients and preservatives. An optimum topical
formulation comprises approximately: 2 wt. % to 60 wt. %,
preferably 2 wt. % to 50 wt. %, solubilizer and/or skin permeation
enhancer; 2 wt. % to 50 wt. %, preferably 2 wt. % to 20 wt. %,
emulsifiers; 2 wt. % to 20 wt. % emollient; and 0.01 to 0.2 wt. %
preservative, with the active agent and carrier (e.g., water)
making of the remainder of the formulation.
[0053] A skin permeation enhancer serves to facilitate passage of
therapeutic levels of active agent to pass through a reasonably
sized area of unbroken skin. Suitable enhancers are well known in
the art and include, for example: lower alkanols such as methanol
ethanol and 2-propanol; alkyl methyl sulfoxides such as
dimethylsulfoxide (DMSO), decylmethylsulfoxide (C.sub.10MSO) and
tetradecylmethyl sulfoxide; pyrrolidones such as 2-pyrrolidone,
N-methyl-2-pyrrolidone and N-(-hydroxyethyl)pyrrolidone; urea;
N,N-diethyl-m-toluamide; C.sub.2-C.sub.6 alkanediols; miscellaneous
solvents such as dimethyl formamide (DMF), N,N-dimethylacetamide
(DMA) and tetrahydrofurfuryl alcohol; and the 1-substituted
azacycloheptan-2-ones, particularly
1-n-dodecylcyclazacycloheptan-2-one (laurocapram; available under
the trademark Azone.RTM. from Whitby Research Incorporated,
Richmond, Va.).
[0054] Examples of solubilizers include, but are not limited to,
the following: hydrophilic ethers such as diethylene glycol
monoethyl ether (ethoxydiglycol, available commercially as
Transcutol.RTM.) and diethylene glycol monoethyl ether oleate
(available commercially as Softcutol.RTM.); polyethylene castor oil
derivatives such as polyoxy 35 castor oil, polyoxy 40 hydrogenated
castor oil, etc.; polyethylene glycol, particularly lower molecular
weight polyethylene glycols such as PEG 300 and PEG 400, and
polyethylene glycol derivatives such as PEG-8 caprylic/capric
glycerides (available commercially as Labrasol.RTM.); alkyl methyl
sulfoxides such as DMSO; pyrrolidones such as 2-pyrrolidone and
N-methyl-2-pyrrolidone, and DMA. Many solubilizers can also act as
absorption enhancers. A single solubilizer may be incorporated into
the formulation, or a mixture of solubilizers may be incorporated
therein.
[0055] Suitable emulsifiers and co-emulsifiers include, without
limitation, those emulsifiers and co-emulsifiers described with
respect to microemulsion formulations. Emollients include, for
example, propylene glycol, glycerol, isopropyl myristate,
polypropylene glycol-2 (PPG-2) myristyl ether propionate, and the
like.
[0056] Other active agents may also be included in the formulation,
e.g., other anti-inflammatory agents, analgesics, antimicrobial
agents, antifungal agents, antibiotics, vitamins, antioxidants, and
sunblock agents commonly found in sunscreen formulations including,
but not limited to, anthranilates, benzophenones (particularly
benzophenone-3), camphor derivatives, cinnamates (e.g., octyl
methoxycinnamate), dibenzoyl methanes (e.g., butyl methoxydibenzoyl
methane), p-aminobenzoic acid (PABA) and derivatives thereof, and
salicylates (e.g., octyl salicylate).
[0057] In the preferred topical formulations of the invention, the
active agent is present in an amount in the range of approximately
0.25 wt. % to 75 wt. % of the formulation, preferably in the range
of approximately 0.25 wt. % to 30 wt. % of the formulation, more
preferably in the range of approximately 0.5 wt. % to 15 wt. % of
the formulation, and most preferably in the range of approximately
1.0 wt. % to 10 wt. % of the formulation.
[0058] In an alternative embodiment, a pharmaceutical formulation
is provided for oral or parenteral administration, in which case
the formulation comprises a resveratrol-containing microemulsion as
described above, but may contain alternative pharmaceutically
acceptable carriers, vehicles, additives, etc. particularly suited
to oral or parenteral drug administration. Alternatively, a
resveratrol-containing microemulsion may be administered orally or
parenterally substantially as described above, without
modification.
[0059] Utility:
[0060] The pharmaceutical formulations of the invention are useful
for treating humans and animals suffering from or prone to certain
skin conditions, disorders or diseases associated with or caused by
inflammation, sun damage or natural aging. For example, the
compositions find utility in the prevention or treatment of
psoriasis, contact dermatitis (including irritant contact
dermatitis and allergic contact dermatitis), atopic dermatitis
(also known as allergic eczema), actinic keratosis, keratinization
disorders (including eczema), epidermolysis bullosa diseases
(including penfigus), exfoliative dermatitis, seborrheic
dermatitis, erythemas (including erythema multiforme and erythema
nodosum), damage caused by the sun or other light sources, discoid
lupus erythematosus, dermatomyositis, skin cancer and the effects
of natural aging. The formulations are administered topically, to
the skin or mucosal tissue, as an ointment, lotion, cream,
microemulsion, gel, solution or the like, as described in the
preceding section, within the context of a dosing regimen effective
to bring about the desired result. The preferred dose of active
agent is in the range of about 0.005 to about 1 micromoles per kg
per day, preferably about 0.05 to about 0.75 micromoles per kg per
day, more typically about 0.075 to about 0.5 micromoles per kg per
day. It will be recognized by those skilled in the art that the
optimal quantity and spacing of individual dosages will be
determined by the nature and extent of the condition being treated,
the site of administration, and the particular individual
undergoing treatment, and that such optimums can be determined by
conventional techniques. That is, an optimal dosing regimen for any
particular patient, i.e., the number and frequency of doses, can be
ascertained using conventional course of treatment determination
tests. Generally, a dosing regimen herein involves administration
of the topical formulation at least once daily, and preferably one
to four times daily, until symptoms have subsided.
[0061] The topical formulations of the invention can also be used
as chemopreventive compositions. When used in a chemopreventive
method, susceptible skin is treated prior to any visible condition
in a particular individual.
[0062] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description, as well as the examples which
follow, are intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modifications will be
apparent to those skilled in the art to which the invention
pertains.
[0063] All patents, patent documents, and publications cited herein
are hereby incorporated by reference in their entireties.
[0064] Experimental
[0065] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the formulations of the
invention, and are not intended to limit the scope of what the
inventors regard as their invention. Efforts have been made to
ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.) but some errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. and pressure is at or near
atmospheric. All solvents, reagents and formulation components were
of Pharmaceutical Grade.
EXAMPLE 1
Preparation of a Resveratrol Cream
[0066] A topical pharmaceutical composition containing
trans-resveratrol was prepared in the form of a cream, as
follows:
1 Phase Component Wt. % 1 Polyethylene Glycol and Ethyleneglycol
Palmitostearate 5% Caprilic/Capric Triglycerides 5% Oleoyl
Macrogolglycerides (Labrafil M 1944CS) 4% Cetyl Alcohol 5.5% PPG-2
Myristyl Ether Propionate (Crodamol PMP) 6% 2 Xanthan Gum 0.3%
Purified Water 48% 3 Propylene Glycol 1% Methylparaben 0.18%
Propylparaben 0.02% 4 Trans-Resveratrol 10% Diethylene Glycol
Monoethyl Ether (Transcutol) 15%
[0067] The composition was prepared as follows. The xanthan gum was
dispersed in water, and allowed to stand. Phase 1 (the oil phase)
was heated to 75.degree. C. Phase 2 was then heated to 75.degree.
C. Under high speed agitation, phase 1 was mixed into phase 2. The
temperature was maintained at 75.degree. C., and rapid stirring was
continued for 10 min. The mixture was cooled slowly while stirring
was continued at low speed. At 40.degree. C., Phase 3 was added.
The trans-resveratrol was then dispersed into diethylene glycol
monoethyl ether, heated to 40.degree. C., and then cooled down to
30.degree. C. while stirring slowly (Phase 4). At 30.degree. C.,
Phase 4 was added to the cream, mixed well, and cooled down to room
temperature with slow mixing. An off-white, stable cream was
obtained.
EXAMPLE 2
Microemulsion of Resveratrol
[0068] A microemulsion of resveratrol was prepared having the
following components:
2 Component Wt. % Trans-Resveratrol 10% Diethylene Glycol Monoethyl
Ether (Transcutol) 47.4% PEG-8 Caprylic/Capric Triglycerides
(Labrasol) 23.7% Oleoyl Macrogolglycerides (Labrafil M 1944 CS)
7.9% PEG 400 4.7% Water 0.3%
[0069] Trans-resveratrol was dispersed into diethylene glycol
monoethyl ether. PEG-8 caprylic/capric glycerides and oleoyl
macrogolglycerides were added, with agitation. PEG 400 was then
slowly added, again, with agitation, followed by addition of water.
A stable, transparent, slightly yellow microemulsion was
obtained.
EXAMPLE 3
Inhibition of TPA-Induced Inflammation Test
[0070] Inhibition of inflammation induced by repeated application
of 12-O-tetradecanoylphorbol-13-acetate (TPA) can be used to
indicate the efficacy of various formulations used to treat
inflammation. The test method is modified from that described by
Marks et al. (1976) Cancer Res. 36:2636. TPA is known to induce
changes in murine skin which mimic many of the inflammatory and
epithelial changes which in occur in human skin diseases such as
psoriasis. Thus, this mouse-based model is believed to be a good
indicator of human response as well.
[0071] The composition of Example 1 (1 g, removing any excess) is
initially applied to both ears of ten mice. The inflammatory
stimulus (i.e., TPA) is then applied to the right ear of each
mouse. Four hours later, edema is measured by weighing discs
removed from the ear using a biopsy punch. A control group receives
TPA in the right ear and the composition without the resveratrol to
the left ear. The ratio of weight between discs receiving
composition and TPA and TPA alone is calculated to give the percent
inhibition of control swelling. The same process is carried out for
the microemulsion of Example 2. The composition of Example 1
provides a 72% inhibition of swelling while the microemulsion of
Example 2 provides a 74% inhibition of swelling as compared to the
control. Thus, both formulations are shown to provide effective
inhibition of inflammation.
EXAMPLE 4
Inhibition of Sunburn Cell Formation
[0072] The composition of Example 1 (1 g applied to 1 cm.sup.2
area, removing any excess) is applied to the skin of mice. UV-B
(100 mJ/cm.sup.2) is irradiated onto the skin (health lamp
SE20/SLE, Toshiba). Twenty four hours later, skin is biopsied at
the treatment area and at a treatment-free area to serve as a
control. The biopsied samples are stained using hematoxylin and
eosin, after which the number of sunburn cells is counted under a
microscope. The ratio of sunburn cells in areas receiving the
treatment and in treatment-free areas is calculated to give the
percent inhibition of sunburn cell formation. The same process is
repeated using the microemulsion of Example 2. The composition of
Example 1 is shown to provide 76% less sunburned cells and the
microemulsion is shown to provide 79% less sunburned cells. Thus,
both formulations are shown to provide effective inhibition of
sunburn cell formation.
EXAMPLE 5
Inhibition of Wrinkle Formation in Hairless Mice
[0073] To each hairless mouse (HR/ICR, 6 weeks old), the
composition of Example 1 (1 g applied to 1 cm.sup.2 area, removing
any excess) is applied. Approximately 10 minutes later, UV-B is
irradiated unto the mouse using 6 bulbs of health lamps (SL20-SLE,
Toshiba) so that the dose per irradiation is not greater than 1 MED
(minimal erythema dose). Application of the formulation followed by
irradiation is repeated five times per week over sixteen weeks.
While the irradiation energy is measured using a UV-radiometer
(UVR-305/S65D, Tokyo Optical K.K.) so that the dose per irradiation
is not greater than 1 MED, energy is irradiated at an intensity of
0.28 mW/cm.sup.2 with the total dose, 100 mJ/cm.sup.2. As a
control, a composition the same in all respects as the composition
of Example 1 except lacking resveratrol is applied and the mouse is
similarly irradiated following the protocol provided above. The
same process is carried out for the microemulsion of Example 2.
[0074] The level of wrinkle formation is then visually determined
using the following standards (wrinkle indices): (1) no wrinkle are
formed; (2) small amounts of wrinkles are formed; (3) moderate
amounts of wrinkles are formed; and (4) considerable amounts of
wrinkles are formed.
[0075] In order to analyze the wrinkles, replicas of different skin
areas (3 replicas per mouse) each having a round shape with a
diameter of 1 cm are obtained using a hydrophilic exaflex,
hydrophilic vinyl silicone imaging agent. Each replica is placed
horizontally and lightened from an angle of 30.degree.. The area of
shadows formed by the wrinkles are analyzed as the area ratio using
an image analyzing apparatus.
[0076] The results are shown below:
3 Example Wrinkle Index Area ratio of image analysis (%) 1 2.06
3.01 2 1.97 2.85 control 3.78 6.46
[0077] Thus, both formulations are shown to inhibit wrinkle
formation.
EXAMPLE 6
Prevention of Sun Damage in Humans
[0078] Once an MED (minimal erythema dose) is established for each
of 10 patients, the composition of Example 1 is applied (under
occlusion) to each patient 2 hours before UVB irradiation. Two
areas of skin per patient are exposed to UVB irradiation: an area
receiving the composition of Example 1 and an area receiving the
same composition but without resveratrol. One hour after UVB
irradiation, each patient has the same composition reapplied to the
appropriate area of skin. For the next three days, each patient is
evaluated and the irradiation sites are photographed. The results
of the 10 patients are compiled and evaluated. The same process is
followed for the microemulsion of Example 2. It is shown that
resveratrol prevents sun damage in humans.
EXAMPLE 7
Topically Administered Resveratrol in Chemoprevention
[0079] Resveratrol was found to inhibit cellular events associated
with tumor initiation, promotion, and progression. As discussed
hereafter, the activity of resveratrol was demonstrated on the
basis of ability of resveratrol to inhibit the cyclooxygenase
activity of COX-1 (i.e., median effective dose ED.sub.50 of 15
.mu.M). This activity correlates with antitumor promotion. Although
the inhibitory activity of resveratrol was less than that of some
NSAIDs, such as indomethacin (ED.sub.50=2.3 .mu.M), the resveratrol
activity was much greater than the activity of compounds such as
aspirin (ED.sub.50=880 .mu.M). Also, unlike indomethacin and most
other NSAIDs, resveratrol inhibited the hydroperoxidase activity of
COX-1 (ED.sub.50=3.7 .mu.M).
[0080] Resveratrol-mediated inhibition was specific for the
cyclooxygenase activity of COX-1 because there was no discernable
activity when oxygen uptake was assessed with COX-2, an inducible
form of the enzyme associated with responses such as inflammation,
and inhibition of the hydroperoxidase activity of COX-2
(ED.sub.50=85 .mu.M) was greatly reduced relative to the activity
observed with COX-1.
[0081] These results are illustrated in the plots of FIGS. 1-3.
FIG. 1 shows the inhibitory effects of indomethacin on COX-1 and of
resveratrol on COX-1 and COX-2. FIG. 1 contains plots of percent
activity vs. concentration of indomethacin or resveratrol (in
.mu.M). COX activity was measured by measuring oxygen consumption
at 37.degree. C. The procedure is set forth in F. J. G. Van der
Ouderaa et al., Methods Enzymol., 86, 60 (1982), and R. J. Kulmacz
et al. in Prostaglandins and Related Substances. A Practical
Approach, C. Benedetto et al., Eds. (IRL Press, Oxford, 1987), pp.
209-227, each incorporated herein by reference. The reactions were
initiated by adding 0.6 mM arachidonic acid to a mixture containing
0.1 M (molar) sodium phosphate (pH 7.4), 1.0 mM (millimolar)
phenol, and 0.01 mM hemin; microsomes (0.2 mg of protein) derived
from sheep seminal vesicles were used as a crude source of COX-1 or
recombinant human COX-2 (0.1 mg of protein); and the test compound.
FIG. 1 shows the dramatic inhibitory effect of resveratrol on
COX-1.
[0082] FIG. 2 shows the inhibitory effect of indomethacin on COX-1
hydroperoxidase activity, and resveratrol on COX-1 or COX-2
hydroperoxidase activity. Hydroperoxidase activity was determined
by spectrophotometry. The reaction mixtures contained 0.1 M
tris-NCl (pH 8.5), 1.2 .mu.M (micromolar) hemin, 0.24 mM,
N,N,N',N'-tetramethyl-p-phen- ylenediamine (TMPD), COX-1 (36 .mu.g
of protein) or COX-2 (45 .mu.g of protein), and a test compound.
Hydrogen peroxide (250 .mu.M) was used to initiate the reaction,
and changes in absorbence at 595 nm (nanometers) were measured
Inhibitory activity was calculated by comparing the initial rate of
change in absorbance in the presence of a test compound with that
observed with dimethyl sulfoxide (DMSO) solvent only. Each point
represents the mean of two replicate determinations. FIG. 2 shows
that resveratrol strongly inhibits COX-1 hydroperoxidase
activity.
[0083] FIG. 3 shows the inhibition of DMBA
(7,12-dimethylbenz(a)anthracene- )-induced preneoplastic lesions in
mouse mammary gland culture by treatment with resveratrol. Mammary
glands were incubated with resveratrol for 10 days and DMBA for 24
hours on day three. The procedure is set forth in Moon et al. in
Chemistry and Biology of Retinoids, Eds. Dawson et al. (Boca Raton,
Fla.: CRC Press, 1990), at pp. 501-518. Percent incidence of
mammary lesions was determined after an additional 14 days of
incubation. The data from resveratrol-treated groups was compared
to control groups and the results expressed as a percentage.
Resveratrol demonstrated an excellent ability to inhibit
DMBA-induced preneoplastic lesions in mouse mammary gland
cultures.
[0084] Based on the results illustrated in FIGS. 1-3, the
antiinflammatory activity of resveratrol was investigated. In a
carrageenan-induced model of inflammation in rats, resveratrol
significantly reduced pedal edema both in the acute phase (3 to 7
hours) and in the chronic phase (24 to 144 hours). The
edema-suppressing activity of resveratrol was greater than that of
phenylbutazone, and was similar to that of indomethacin. These data
are set forth in FIG. 4. Overall, the data in FIG. 4 demonstrates
the ability of resveratrol to inhibit tumor promotion.
[0085] In particular, FIG. 4 shows the effects of resveratrol (at 3
mg/kg and 8 mg/kg of body weight), phenylbutazone, and indomethacin
on carrageenan-induced inflammation in rats. The procedure is set
forth in Slowing et al. (1994) J. Ethnopharmacol. 43:9 (1994),
incorporated herein by reference. In these tests, female Wistar
rats (150 to 200 g body weight) were divided into groups of seven
animals each. All rats received 0.1 ml of Freund's complete
adjuvant, available from Sigma Chemical Co., St. Louis, Mo., by
intradermal injection into the tail. Animals were used 7 days after
injection of adjuvant. One hour after oral administration of
resveratrol (3 or 8 mg per kilogram of body weight) or reference
drugs including phenylbutazone (80 mg/kg) and indomethacin (5
mg/kg), the rats were injected with 0.1 ml of a 2% (w/v) suspension
of carrageenan in saline solution into the left hind paw. For the
control group, a 1:1 mixture of Tween 80 (i.e., polysorbate 80,
available from ICI Americas, Inc., Wilmington, Del.) and water
(0.2/3.3, v/v) and 1% (w/v) methylcellulose was used as a vehicle.
The left hind paw volume of each rat was measured by water
plethysmography on a Letica, Model L17500, before the adjuvant
injection, and, again, 6 days later, before the injection of
carrageenan. Paw volumes were determined within 3 to 144 hours
after injection of carrageenan. Inhibition of edema was calculated
relative to the mean edema of the vehicle-treated control
group.
[0086] Percent reduction was determined by comparing the paw volume
of rats in the control group (treated with carrageenan only) to paw
volume of rats in the drug-treated group. Dosing was repeated daily
for 7 days. Hours refers to hours after carrageenan injection. The
data for the indomethacin group at 120 hours and 140 hours were not
reliable because of the induction of secondary lesions. The plots
in FIG. 4 show that resveratrol was effective in reducing
carrageenan-induced inflammation in rats.
[0087] Resveratrol also was found to inhibit events associated with
tumor initiation. For example, resveratrol inhibited free-radical
formation (ED.sub.50=27 .mu.M), in a dose-dependent manner, when
human promyelocytic leukemia (HL-60) cells were treated with TPA.
The procedure is set forth in Sharma et al. (1994) Cancer Res.
54:5848. In these tests, HL-60 cells were maintained in RPMI 1640
medium (available from Gibco BRL, Grand Island, N.Y.) supplemented
with 5% heat-inactivated calf serum, penicillin G sodium (100
U/ml), and streptomycin sulphate (100 .mu.g/ml) (Gibco BRL) at
37.degree. C. in a humidified atmosphere at 5% carbon dioxide in
air. Differentiation was induced by a 7-day treatment with 1.3%
DMSO, and the cells were cultured in 96-well plates
(1.times.10.sup.6 cells per well) in Hanks' balanced salt solution
containing 30 mM Hepes, pH 7.6. After the addition of TPA (8 .mu.M)
to induce free radical formation, cytochrome c (160 .mu.M) and
resveratrol were added. The cells were incubated for 1 hour at
37.degree. C., and antioxidant activity was determined by
monitoring absorbance at 550 nm. The same reaction mixture, but
lacking the HL-60 cells, was used as a blank control.
[0088] Resveratrol also functioned as an antimutagen, as
illustrated by a dose-dependent inhibition of the mutagenic
response induced by treatment of Salmonella typhimitrium strain
TM677 with 7,12-dimethyl-benz(a)anthrac- ene (DMBA) (ED.sub.50=4
.mu.M). This procedure is set forth in Shamon et al. (1995)
Anticancer Res. 14:1775 (1995), incorporated herein by reference.
In these tests, a reaction mixture was prepared containing
Salmonella typhimurium strain TM677, S9 liver homogenate derived
from Aroclor 1254-pretreated rats, a NADPH-generating system (NADPH
is the reduced form of nicotinamide adenine dinucleotide
phosphate), and various concentrations of resveratrol, which were
added one minute before the addition of 80 .mu.M DMBA. After
incubation for 2 hours at 37.degree. C., the bacteria were
recovered by centrifugation, resuspended, and plated (in
triplicate) on minimal agar in the presence or absence of
8-azaguanine. The plates then were incubated for 48 hours at
37.degree. C., and the results were expressed as mutant fractions,
i.e., the average number of colonies capable of growing in the
presence of 8-azaguanine divided by the average number of colonies
capable of growing in the absence of 8-azaguanine, after correcting
for dilution factors. The percent inhibition was calculated
relative to control plates that were treated with DMSO only.
[0089] In addition, resveratrol induced quinone reductase activity
with cultured mouse hepatoma (Hepa 1c1c7) cells (concentration
required to double activity, 21 .mu.M). See Prochaska et al. (1988)
Anal. Biochem. 169:328. This result is important because phase II
enzymes, such as quinone reductase, are capable of metabolically
detoxifying carcinogens. See Zhang et al. (1994), Proc. Natl. Acad.
Sci., U.S.A. 91: 3247. An identical response profile was observed
with cultured BP.sup.1Cl hepatoma cells (a derivative of Hepa 1c1c7
cells that is incapable of phase I enzyme induction), indicating
that resveratrol is a monofunctional inducer.
[0090] In addition, the ability of resveratrol to inhibit the
progression stage of carcinogenesis was tested by treating cultured
HL-60 cells with resveratrol. See Suh et al. (995)Anticancer
Res.15:233. Under normal culture conditions, these cells have
unlimited proliferative capacity. In a dose-dependent manner,
resveratrol-induced expression of nitroblue tetrazolium reduction
activity, a marker of granulocyte formation (ED.sub.50=11 .mu.M),
and nonspecific acid esterase activity, a marker of macrophase
(monocyte) formation (ED.sub.5019 .mu.M). Concurrently,
incorporation of [.sup.3H]thymidine was inhibited (ED.sub.50=18
.mu.M), which is indicative of terminal differentiation to a
nonproliferative phenotype.
[0091] In order to more directly assess the cancer chemopreventive
activity of resveratrol, the effects of resveratrol in a mouse
mammary gland culture model of carcinogenesis was investigated.
Resveratrol inhibited, in a dose-dependent manner, the development
of DMBA-induced preneoplastic lesions (ED.sub.50=3.1 .mu.M) (see
FIG. 3). No signs of toxicity were observed, as judged by
morphological examination of the glands.
[0092] In addition, tumorigenesis in the two-stage mouse skin
cancer model in which DMBA was used as initiator and TPA as
promoter was studied. During an 18-week study, mice treated with
DMBA-plus TPA developed an average of two tumors per mouse with 40%
tumor incidence, as illustrated in FIG. 5(a).
[0093] In particular, FIG. 5 shows the effect of resveratrol on
tumorigenesis in the two-stage mouse skin model. Six groups of 20
female CD-1 mice (4 to 5 weeks old) were treated topically with 200
.mu.mol of DMBA in 0.2 ml of acetone on the shaved dorsal region.
See Gerhuser et al. (1995) Nature Med. 1:260 (1995). One week
later, the mice were treated with 5 .mu.mol of TPA in 0.2 ml of
acetone alone or together with 1, 5, 10, or 25 .mu.mol of
resveratrol in 0.2 ml of acetone, twice a week for 18 weeks.
Animals were weighed weekly and observed for tumor development once
every week. FIG. 5A shows percent incidence of observable skin
tumors. FIG. 5B shows total number of observable skin tumors.
Overall, FIG. 5 shows that resveratrol greatly reduced the
incidence of tumors in the mouse skin model.
[0094] Application of 1, 5, 10, or 25 .mu.mol of resveratrol,
together with TPA twice a week for 18 weeks, reduced the number of
skin tumors per mouse by 68, 81, 76, and 98%, respectively, and the
percentage of mice with tumors was lowered by 50, 63, 63, and 88%,
respectively (FIG. 5(b)). No overt signs of resveratrol-induced
toxicity were observed, as judged by visual inspection of the skin,
gross morphological examination of major organ systems, or change
in body weights, relative to controls.
[0095] To further demonstrate the ability of resveratrol to act as
a cancer chemopreventive agent, tests were performed to determine
whether resveratrol possesses activity against chemically induced
neoplastic transformation in the C3H/10T1/2 clone 8 cells. This
10T1/2 cell line of mouse embryo fibroblasts has been widely used
as an experimental system to study neoplastic transformation
induced by a variety of agents, such as polycyclic hydrocarbons,
methylating agents, and X-rays. Two-stage transformation assays,
with 3-methylcholanthrene (MCA) or DMBA as inducer and TPA as
promoter, were performed in these tests.
[0096] C3H10T1/2 C1 8 cells were purchased from the American Type
Culture Collection (ATCC CCL 226, Rockville, Md.) and maintained in
Eagle's basal medium with Earle's salts (EBME) containing 10% (v/v)
fetal bovine serum (FBS, Gibco Laboratories, Chagrin Falls, Ohio)
without antibiotics, and sodium bicarbonate (2.2 g/1). Cells were
seeded at 5.times.10.sup.4 per 75 cm.sup.2 flask and grown in a
humidified incubator containing 5% carbon dioxide in air at
37.degree. C. Cells used in all these experiments were passage
between 10-13.
[0097] Two-stage transformation assays in C3H10T1/2 cells were
performed according to a procedure modified from that described in
Mondal et al. (1976) Cancer Res. 36:2254-2260. One thousand cells
obtained from freshly confluent flasks were seeded per well in
12-well plate containing 2 ml of medium (12 wells per experimental
point). After incubation at 37.degree. C. for three days, MCA or
DMBA (2 mg/ml stock solution in DMSO) was added to give final
concentrations of 0.25 .mu.g/ml. Carcinogen-containing medium was
removed from the growing cultures after 24 hours and cells were
rinsed once with PBS before addition of fresh EBME supplemented
with 10% FBS and gentamycin (25 .mu.g/ml). Following five days of
further incubation without carcinogen, medium was changed to fresh
medium containing TPA (0.1 .mu.g/ml) or TPA and different
concentrations of resveratrol. TPA and test compounds were added to
the medium each time the medium was changed. Medium was changed on
all plates twice weekly. After confluency was reached, the FBS
concentration was reduced to 5% and amphotericin B (fungizone, 1.5
.mu.g/ml) was added to the medium (see Reznikoff et al. (1973)
Cancer Res. 33:3239-3249 and Bertram (1979) Cancer Lett.
7:289-298). Subsequent medium changes were performed weekly.
Cultures were fixed with methanol and stained with Giemsa at the
seventh week. Type II and III transformed foci were scored
according to the teachings of Landolph "Transformation Assay of
Established Cell Lines: Mechanism and Application," Eds. Kakunaga
et al. (Toronto: Oxford University Press, 1985), at pp.
185-199.
[0098] Table 1 summarizes the effects of various cancer
chemopreventive agents, including resveratrol, on chemical-induced
neoplastic transformation.
4TABLE 1 Effects of various chemopreventive agents on
chemical-induced neoplastic transformation Total No. of No. of
wells Total Total foci/no. foci/total Wells No. No. wells no. of
with foci Treatment type II type III scored wells (%) 0.5% DMSO 0 0
0/12 0/12 0 control MCA (5.0)* 9 2 11/12 8/12 67 MCA (0.25) 0 0
0/12 0/12 0 MCA (0.25); 8 0 8/12 7/12 58 TPA (0.1) MCA (0.25); 3 0
3/12 2/12 17 TPA (0.1); resveratrol 2.5 .mu.M MCA (0.25; 3 1 4/12
2/12 17 TPA (0.1); resveratrol 5.0 .mu.M 0.5% DMSO 0 0 0/12 0/12 0
MCA (0.25); 1 0 1/12 1/12 8.3 TPA (0.1); resveratrol 10 0.5% DMSO 0
0 0/12 0/12 0 control DMBA (5.0) 4 0 4/12 2/12 17 DMBA (0.25) 1 0
1/12 1/12 8.3 DMBA (0.25); 4 0 4/12 4/12 33 TPA (0.1) DMBA (0.25);
3 0 3/12 3/12 25 TPA (0.1); resveratrol 2.5 DMBA (0.25); 1 1 2/12
2/12 17 TPA (0.1); resveratrol 5.0 0.5% DMSO 0 0 0/12 0/12 0
control DMBA (0.25); 1 1 2/12 2/12 17 TPA (0.1); resveratrol 10
.mu.M DMBA (0.25); 0 0 0/12 0/12 0 TPA (0.1); aspirin 200 .mu.M
DMBA (0.25); 0 0 0/12 0/12 0 TPA (0.1); all-trans-retinyl acetate
(0.3) DMBA (0.25); 4 0 4/12 4/12 33 TPA (0.1); Vit E. 100 .mu.M
*Numbers in parenthesis, concentration (.mu.g/ml).
[0099] The above tests and data show that resveratrol can be
administered to mammals as a prophylactic against chemically
induced cancers.
* * * * *