U.S. patent application number 10/817330 was filed with the patent office on 2004-11-04 for formulation of dual cycloxygenase (cox) and lipoxygenase (lox) inhibitors for mammal skin care.
This patent application is currently assigned to Unigen Pharmaceuticals, Inc.. Invention is credited to Jia, Qi.
Application Number | 20040220119 10/817330 |
Document ID | / |
Family ID | 33159803 |
Filed Date | 2004-11-04 |
United States Patent
Application |
20040220119 |
Kind Code |
A1 |
Jia, Qi |
November 4, 2004 |
Formulation of dual cycloxygenase (COX) and lipoxygenase (LOX)
inhibitors for mammal skin care
Abstract
The present invention provides a novel composition of matter
comprised of a mixture of two specific classes of
compounds--Free-B-Ring flavonoids and flavans--for use in the
prevention and treatment of diseases and conditions associated with
the skin. This composition of matter simultaneously inhibits
cyclooxygenase (COX) and lipoxygenase (LOX) enzymatic activity in
normal, aged and damaged dermal cells and tissues. This invention
further provides a method for the prevention and treatment of
diseases and conditions of the skin mediated by cyclooxygenase
(COX) and lipoxygenase (LOX). The method for preventing and
treating COX and LOX mediated diseases and conditions of the skin
is comprised of topically administering to a host in need thereof a
therapeutically effective amount of a composition comprising a
mixture of Free-B-Ring flavonoids and flavans synthesized and/or
isolated from a single plant or multiple plants, preferably in the
Scutellaria and Acacia genus of plants and pharmaceutically and/or
cosmetically acceptable carriers. Finally the present invention
provides a method for the prevention and treatment of COX and LOX
mediated diseases and conditions, including but not limited to sun
burns, thermal burns, acne, topical wounds, minor inflammatory
conditions caused by fungal, microbial and viral infections,
vitilago, systemic lupus erythromatosus, psoriasis, carcinoma,
melanoma, as well as other mammal skin cancers, skin damage
resulting from exposure to ultraviolet (UV) radiation, chemicals,
heat, wind and dry environments, wrinkles, saggy skin, lines and
dark circles around the eyes, dermatitis and other allergy related
conditions of the skin. Use of the composition described herein
also affords the benefit of smooth and youthful skin with improved
elasticity, reduced and delayed aging, enhanced youthful appearance
and texture, and increased flexibility, firmness, smoothness and
suppleness.
Inventors: |
Jia, Qi; (Superior,
CO) |
Correspondence
Address: |
SWANSON & BRATSCHUN L.L.C.
1745 SHEA CENTER DRIVE
SUITE 330
HIGHLANDS RANCH
CO
80129
US
|
Assignee: |
Unigen Pharmaceuticals,
Inc.
Broomfield
CO
80021
|
Family ID: |
33159803 |
Appl. No.: |
10/817330 |
Filed: |
April 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60460736 |
Apr 4, 2003 |
|
|
|
Current U.S.
Class: |
514/27 ;
514/456 |
Current CPC
Class: |
A61K 31/353 20130101;
A61K 36/484 20130101; A61K 36/185 20130101; A61K 36/9062 20130101;
A61K 31/7048 20130101; A61K 8/9789 20170801; A61K 36/11 20130101;
A61Q 19/004 20130101; A61K 36/906 20130101; A61K 36/15 20130101;
A61K 2800/782 20130101; A61K 8/67 20130101; A61K 36/539 20130101;
A61Q 19/08 20130101; A61P 17/00 20180101; A61K 36/60 20130101; A61K
36/486 20130101; A61K 36/47 20130101; A61K 36/48 20130101; A61K
8/498 20130101; A61K 31/352 20130101; A61K 36/53 20130101; A61K
45/06 20130101; A61K 36/28 20130101; A61Q 19/00 20130101; A61K
36/54 20130101; A61K 31/352 20130101; A61K 2300/00 20130101; A61K
31/353 20130101; A61K 2300/00 20130101; A61K 31/7048 20130101; A61K
2300/00 20130101; A61K 36/11 20130101; A61K 2300/00 20130101; A61K
36/15 20130101; A61K 2300/00 20130101; A61K 36/185 20130101; A61K
2300/00 20130101; A61K 36/28 20130101; A61K 2300/00 20130101; A61K
36/47 20130101; A61K 2300/00 20130101; A61K 36/48 20130101; A61K
2300/00 20130101; A61K 36/484 20130101; A61K 2300/00 20130101; A61K
36/486 20130101; A61K 2300/00 20130101; A61K 36/53 20130101; A61K
2300/00 20130101; A61K 36/539 20130101; A61K 2300/00 20130101; A61K
36/54 20130101; A61K 2300/00 20130101; A61K 36/60 20130101; A61K
2300/00 20130101; A61K 36/906 20130101; A61K 2300/00 20130101; A61K
36/9062 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
514/027 ;
514/456 |
International
Class: |
A61K 031/7048; A61K
031/353 |
Claims
1. A method for preventing and treating cyclooxygenase (COX) and
lipoxygenase (LOX) mediated diseases and conditions of the skin,
said method comprising administering to a host in need thereof an
effective amount of a pharmaceutical composition comprising a
mixture of at least one Free-B-ring flavonoid and at least one
flavan.
2. The method of claim 1 wherein the ratio of Free-B-Ring flavonoid
to flavan in said composition is selected from the range of 99:1
Free-B-Ring flavonoid:flavan to 1:99 of Free-B-Ring
flavonoid:flavan.
3. The method of claim 2 wherein the ratio of Free-B-Ring
flavonoid:flavan in the composition of matter is about 20:80.
4. The method of claim 1 wherein said Free-B-Ring flavonoid is
selected from the group of compounds having the following
structure: 9wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5
are independently selected from the group consisting of --H, --OH,
--SH, --OR, --SR, --NH.sub.2, --NHR, --NR.sub.2,
--NR.sub.3.sup.+X.sup.-, a carbon, oxygen, nitrogen or sulfur,
glycoside of a single or a combination of multiple sugars
including, aldopentoses, methyl-aldopentose, aldohexoses,
ketohexose and their chemical derivatives thereof, wherein R is an
alkyl group having between 1-10 carbon atoms; and X is selected
from the group of pharmaceutically acceptable counter anions
including, hydroxyl, chloride, iodide, sulfate, phosphate, acetate,
fluoride and carbonate:
5. The method of claim 1 wherein said flavan is selected from the
group of compounds having the following structure: 10wherein
R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are independently
selected from the group consisting of H, --OH, --SH, --OCH.sub.3,
--SCH.sub.3, --OR, --SR, --NH.sub.2, --NRH, --NR.sub.2,
--NR.sub.3.sup.+X.sup.-, esters of substitution groups,
independently selected from the group consisting of gallate,
acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl
esters and caffeoyl esters; a carbon, oxygen, nitrogen or sulfur
glycoside of a single or a combination of multiple sugars
including, aldopentoses, methyl aldopentose, aldohexoses,
ketohexose and their chemical derivatives thereof; dimer, trimer
and other polymerized flavans; wherein R is an alkyl group having
between 1-10 carbon atoms; and X is selected from the group of
pharmaceutically acceptable counter anions including, but not
limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate,
fluoride, carbonate.
6. The method of claim 1 wherein said Free-B-Ring flavonoid and
said flavan are obtained by organic synthesis or are isolated from
a plant.
7. The method of claim 6 wherein said Free-B-Ring flavonoid and
said flavan are isolated from a plant part selected from the group
consisting of stems, stem barks, trunks, trunk barks, twigs,
tubers, roots, root barks, young shoots, seeds, rhizomes, flowers
and other reproductive organs, leaves and other aerial parts.
8. The method of claim 6 wherein said Free-B-Ring flavonoid is
isolated from a plant family selected from the group consisting of
Annonaceae, Asteraceae, Bignoniaceae, Combretaceae, Compositae,
Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,
Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae and
Zingiberacea.
9. The method of claim 6 wherein said Free-B-Ring flavonoid is
isolated from a plant genus selected from the group consisting of
Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis, Cotula,
Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis, Sapium,
Scutellaria, Molsa, Colebrookea, Stachys, Origanum, Ziziphora,
Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza, Millettia,
Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma, Notholaena,
Pinus, Ulmus and Alpinia.
10. The method claim 6 wherein said flavan is are isolated from a
plant species selected from the group consisting of the Acacia
catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal, Acacia
speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata. A.
mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A.
holoserecia and A. mangium.
11. The method of claim 6 wherein said Free-B-ring flavonoid is
isolated from a plant or plants in the Scutellaria genus of plants
and said flavan is isolated from a plant or plants in the Acacia
genus of plants.
12. The method of claim 1 wherein the composition is administered
in a dosage selected from 0.001 to 200 mg/kg of body weight.
13. The method of claim 1 wherein the composition is administered
in a pharmaceutical, dermatological or cosmetic formulation
comprised of approximately 0.001 weight percent (wt %) to 40.0 wt %
of the mixture of Free-B-Ring flavonoids and flavans in a
pharmaceutically, dermatologically and cosmetically acceptable
carrier.
14. The method of claim 1 wherein the routes of the administration
are selected from the group consisting of topical, aerosol,
suppository, intradermic, intramusclar, and intravenous
administration.
15. The method of claim 14 wherein the route of the administration
is topical.
16. The method of claim 15 wherein the composition is administered
using a nonsticking gauze, a bandage, a swab, a cloth wipe, a
patch, a mask, a protectant, a cleanser, an antiseptic, a solution,
a cream, a lotion, an ointment, a gel or an emulsion, a liquid, a
paste, a soap, or a powder.
17. The method of claim 1 wherein the pharmaceutical composition is
further comprised of a conventional excipient that is
pharmaceutically, dermatologically and cosmetically suitable for
topical application and optionally an adjuvant, and/or a carrier,
and/or a regular or controlled releasing vehicle.
18. The method of claim wherein the COX and LOX mediated diseases
and conditions of the skin are selected from the group consisting
of sun burns, thermal burns, acne, topical wounds, minor
inflammatory conditions caused by fungal, microbial and viral
infections, vitilago, systemic lupus erythromatosus, psoriasis,
carcinoma, melanoma, as well as other mammal skin cancers, skin
damage resulting from exposure to ultraviolet (UV) radiation,
chemicals, heat, wind and dry environments, wrinkles, saggy skin,
lines and dark circles around the eyes, dermatitis and other
allergy related conditions of the skin.
19. A pharmaceutical composition of matter for use in the
prevention and treatment of diseases and conditions related to the
skin comprised of a mixture of at least one Free-B-ring flavonoid
and at least one flavan.
20. The pharmaceutical composition of claim 19 wherein the ratio of
Free-B-Ring flavonoid to flavan in said composition is selected
from the range of 99:1 Free-B-Ring flavonoid:flavan to 1:99 of
Free-B-Ring flavonoid:flavan.
21. The pharmaceutical composition of 20 wherein the ratio of
Free-B-Ring flavonoid:flavan in the composition of matter is about
20:80.
22. The pharmaceutical composition of claim 19 wherein said
Free-B-Ring flavonoid is selected from the group of compounds
having the following structure: 11wherein R.sub.1, R.sub.2,
R.sub.3, R.sub.4, and R.sub.5 are independently selected from the
group consisting of --H, --OH, --SH, --OR, --SR, --NH.sub.2, --NHR,
--NR.sub.2, --NR.sub.3.sup.+X.sup.-, a carbon, oxygen, nitrogen or
sulfur, glycoside of a single or a combination of multiple sugars
including, aldopentoses, methyl-aldopentose, aldohexoses,
ketohexose and their chemical derivatives thereof; wherein R is an
alkyl group having between 1-10 carbon atoms; and X is selected
from the group of pharmaceutically acceptable counter anions
including, hydroxyl, chloride, iodide, sulfate, phosphate, acetate,
fluoride and carbonate.
23. The pharmaceutical composition of claim 19 wherein said flavan
is selected from the group of compounds having the following
structure: 12wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5
are independently selected from the group consisting of H, --OH,
--SH, --OCH.sub.3, --SCH.sub.3, --OR, --SR, --NH.sub.2, --NRH,
--NR.sub.2, --NR.sub.3.sup.+X.sup.-, esters of substitution groups,
independently selected from the group consisting of gallate,
acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl
esters and caffeoyl esters; a carbon, oxygen, nitrogen or sulfur
glycoside of a single or a combination of multiple sugars
including, aldopentoses, methyl aldopentose, aldohexoses,
ketohexose and their chemical derivatives thereof; dimer, trimer
and other polymerized flavans; wherein R is an alkyl group having
between 1-10 carbon atoms; and X is selected from the group of
pharmaceutically acceptable counter anions including, but not
limited to hydroxyl, chloride, iodide, sulfate, phosphate, acetate,
fluoride, carbonate.
24. The pharmaceutical composition of claim 19 wherein said
Free-B-Ring flavonoid and said flavan are obtained by organic
synthesis or are isolated from a plant.
25. The pharmaceutical composition of claim 25 wherein said
Free-B-Ring flavonoid and said flavan are isolated from a plant
part selected from the group consisting of stems, stem barks,
trunks, trunk barks, twigs, tubers, roots, root barks, young
shoots, seeds, rhizomes, flowers and other reproductive organs,
leaves and other aerial parts.
26. The pharmaceutical composition of claim 25 wherein said
Free-B-Ring flavonoid is isolated from a plant family selected from
the group consisting of Annonaceae, Asteraceae, Bignoniaceae,
Combretaceae, Compositae, Euphorbiaceae, Labiatae, Lauranceae,
Leguminosae, Moraceae, Pinaceae, Pteridaceae, Sinopteridaceae,
Ulmaceae and Zingiberacea.
27. The pharmaceutical composition of claim 25 wherein said
Free-B-Ring flavonoid is isolated from a plant genus selected from
the group consisting of Desmos, Achyrocline, Oroxylum, Buchenavia,
Anaphalis, Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium,
Baccharis, Sapium, Scutellaria, Molsa, Colebrookea, Stachys,
Origanum, Ziziphora, Lindera, Actinodaphne, Acacia, Derris,
Glycyrrhiza, Millettia, Pongamia, Tephrosia, Artocarpus, Ficus,
Pityrogramma, Notholaena, Pinus, Ulmus and Alpinia.
28. The pharmaceutical composition of claim 25 wherein said flavan
is are isolated from a plant species selected from the group
consisting of the Acacia catechu, Acacia concinna, Acacia
farnesiana, Acacia Senegal, Acacia speciosa, Acacia arabica, A.
caesia, A. pennata, A. sinuata. A. mearnsii, A. picnantha, A.
dealbata, A. auriculiformis, A. holoserecia and A. mangium.
29. The pharmaceutical composition of claim 25 wherein said
Free-B-ring flavonoid is isolated from a plant or plants in the
Scutellaria genus of plants and said flavan is isolated from a
plant or plants in the Acacia genus of plants.
30. The pharmaceutical composition of claim 19 further comprising a
pharmaceutically acceptable excipient and optionally an adjuvant or
a carrier.
31. The pharmaceutical composition of claim 19, wherein said
composition is formulated for topical application.
32. The pharmaceutical composition of claim 19 where said
composition is formulated in a regular or controlled releasing
vehicle.
33. A method for simultaneously inhibiting the enzymatic activity
of the COX and LOX enzymes in the skin comprised of administering
to a host in need thereof an effective amount of a pharmaceutical
composition comprised of a mixture of at least one Free-B-Ring
flavonoids and one flavan.
34. The method of claim 34 wherein the pharmaceutical composition
is administered in a pharmaceutical, dermatological or cosmetic
formulation comprised of approximately 0.001 weight percent (wt %)
to 40.0 wt % of the mixture of Free-B-Ring flavonoids and flavans
in a pharmaceutically, dermatologically and cosmetically acceptable
carrier.
35. The method of claim 34 wherein the routes of the administration
are selected from the group consisting of topical, aerosol,
suppository, intradermic, intramusclar, and intravenous
administration.
36. The method of claim 36 wherein the route of the administration
is topical.
37. The method of claim 34 wherein the pharmaceutical composition
is administered using a nonsticking gauze, a bandage, a swab, a
cloth wipe, a patch, a mask, a protectant, a cleanser, an
antiseptic, a solution, a cream, a lotion, an ointment, a gel or an
emulsion, a liquid, a paste, a soap, or a powder.
38. The method of claim 34 wherein the pharmaceutical composition
is further comprised of a conventional excipient that is
pharmaceutically, dermatologically and cosmetically suitable for
topical application and optionally an adjuvant, and/or a carrier,
and/or a regular or controlled releasing vehicle.
39. A method for improving mammal skin appearance mediated by COX
and LOX pathways comprising administering to a host in need thereof
an effective amount of a composition comprising a mixture of at
least one Free-B-Ring flavonoid and at least one flavan.
40. The method of claim 40 wherein the pharmaceutical composition
is administered in a pharmaceutical, dermatological or cosmetic
formulation comprised of approximately 0.001 weight percent (wt %)
to 40.0 wt % of the mixture of Free-B-Ring flavonoids and flavans
in a pharmaceutically, dermatologically and cosmetically acceptable
carrier.
41. The method of claim 40 wherein the routes of the administration
are selected from the group consisting of topical, aerosol,
suppository, intradermic, intramusclar, and intravenous
administration.
42. The method of claim 42 wherein the route of the administration
is topical.
43. The method of claim 40 wherein the pharmaceutical composition
is administered using a nonsticking gauze, a bandage, a swab, a
cloth wipe, a patch, a mask, a protectant, a cleanser, an
antiseptic, a solution, a cream, a lotion, an ointment, a gel or an
emulsion, a liquid, a paste, a soap, or a powder.
44. The method of claim 40 wherein the pharmaceutical composition
is further comprised of a conventional excipient that is
pharmaceutically, dermatologically and cosmetically suitable for
topical application and optionally an adjuvant, and/or a carrier,
and/or a regular or controlled releasing vehicle.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to a method for the
prevention and treatment of diseases and conditions mediated by
cyclooxygenase (COX) and lipoxygenase (LOX). Specifically, the
present invention relates to a novel composition of matter
comprised of a mixture of a blend of two specific classes of
compounds--Free-B-Ring flavonoids and flavans--for use in the
prevention and treatment of diseases and conditions of the skin
mediated by the COX and LOX pathways. Included in the present
invention is a method for the prevention and treatment of COX and
LOX mediated diseases and conditions, including but not limited to
sun burns, thermal burns, acne, topical wounds, minor inflammatory
conditions caused by fungal, microbial and viral infections,
vitilago, systemic lupus erythromatosus, psoriasis, carcinoma,
melanoma, as well as other mammal skin cancers, skin damage
resulting from exposure to ultraviolet (UV) radiation, chemicals,
heat, wind and dry environments, wrinkles, saggy skin, lines and
dark circles around the eyes, dermatitis and other allergy related
conditions of the skin. Use of the composition described herein
also affords the benefit of smooth and youthful skin with improved
elasticity, reduced and delayed aging, enhanced youthful appearance
and texture, and increased flexibility, firmness, smoothness and
suppleness.
BACKGROUND OF THE INVENTION
[0002] Sunlight has a significant effect on the skin causing
premature aging, skin cancer and a host of other skin changes such
as erythema and tanning. The majority of the damage caused by
sunlight is attributed to ultraviolet (UV) radiation, which has a
wavelength from 200 nm to 400 nm. Ultraviolet radiation is divided
into three categories, UVA, UVB or UVC, depending on wavelength.
UVA, which has a wavelength range from 320-400 nm, can cause
tanning and mild sunburn. UVB, which has a wavelength range from
290-320 nm, can cause sunburn and stimulate pigmentation. UVC,
which has a wavelength range from 100-290 nm, can cause damage but
not tanning. Exposure of the skin to UV radiation induces biphasic
reactions. Thus, upon initial exposure an immediate erythema
reaction occurs, which is a weak reaction that fades within 30
minutes. A delayed erythema reaction occurs after 2-5 hours of
exposure and peaks around 10-24 hours. Enhanced prostaglandin and
leukotriene production are the major mechanisms of action for UV,
sun and chemical/thermal caused erythema. (Wang (2002) Adv.
Dermatol. 18:247).
[0003] The liberation and metabolism of arachidonic acid (AA) from
the cell membrane results in the generation of pro-inflammatory
metabolites by several different pathways. Arguably, two of the
most important pathways to inflammation are mediated by the enzymes
lipoxygenase (LOX) and cyclooxygenase (COX). These are parallel
pathways that result in the generation of leukotrienes and
prostaglandins, respectively, which play important roles in the
initiation and progression of the inflammatory response. These
vasoactive compounds are chemotaxins, which both promote
infiltration of inflammatory cells into tissues and serve to
prolong the inflammatory response. Consequently, the enzymes
responsible for generating these mediators of inflammation have
become the targets in the current invention to develop topically
administered therapeutic agents aimed at the dual inhibition of
inflammation resulting from both pathways which contribute to the
physiological and pathological processes of diseases and conditions
such as sun burn, thermal burns, scald, acne, topical wounds, lupus
erythromatosus, psoriasis, carcinoma, melanoma, and other mammalian
skin cancers.
[0004] Inhibition of the COX enzyme is the mechanism of action
attributed to most non-steroidal anti-inflammatory drugs (NSAIDS).
There are two distinct isoforms of the COX enzyme (COX-1 and
COX-2), which share approximately 60% sequence homology, but differ
in expression profiles and function. COX-1 is a constitutive form
of the enzyme that has been linked to the production of
physiologically important prostaglandins, which help regulate
normal physiological functions, such as platelet aggregation,
protection of cell function in the stomach and maintenance of
normal kidney function. (Dannhardt and Kiefer (2001) Eur. J. Med.
Chem. 36:109-26). The second isoform, COX-2, is a form of the
enzyme that is inducible by pro-inflammatory cytokines, such as
interleukin-1.beta. (IL-1.beta.) and other growth factors.
(Herschmann (1994) Cancer Metastasis Rev. 134:241-56; Xie et al.
(1992) Drugs Dev. Res. 25:249-65). This isoform catalyzes the
production of prostaglandin E.sub.2 (PGE2) from arachidonic acid
(AA). Inhibition of COX is responsible for the anti-inflammatory
activity of conventional NSAIDs.
[0005] Inhibitors that demonstrate dual specificity for COX and LOX
would have the obvious benefit of inhibiting multiple pathways of
arachidonic acid metabolism. Such inhibitors would block the
inflammatory effects of prostaglandins (PG), as well as, those of
multiple leukotrienes (LT) by limiting their production. This
includes the vasodilation, vasopermeability and chemotactic effects
of PGE2, LTB4, LTD4 and LTE4, also known as the slow reacting
substance of anaphalaxis. Of these, LTB4 has the most potent
chemotactic and chemokinetic effects. (Moore (1985) in Prostanoids:
pharmacological, physiological and clinical relevance, Cambridge
University Press, N.Y., pp. 229-230).
[0006] Because the mechanism of action of COX inhibitors overlaps
that of most conventional NSAID's, COX inhibitors are used to treat
many of the same symptoms, including pain and swelling associated
with inflammation in transient conditions and chronic diseases in
which inflammation plays a critical role. Transient conditions
include treatment of inflammation associated with minor abrasions
or contact dermatitis, as well as, skin conditions that are
directly associated with the prostaglandin and leukotriene
pathways, such as skin hyperpigmentation, age spots, vitilago,
systemic lupus erythromatosus, psoriasis, carcinoma, melanoma, and
other mammalian skin cancers. The use of COX inhibitors has been
expanded to include diseases, such as systemic lupus erythromatosus
(SLE) (Goebel et al. (1999) Chem. Res. Toxicol. 12:488-500; Patrono
et al. (1985) J. Clin. Invest. 76:1011-1018), as well as, rheumatic
skin conditions, such as scleroderma. COX inhibitors are also used
for the relief of inflammatory skin conditions that are not of
rheumatic origin, such as psoriasis, in which reducing the
inflammation resulting from the overproduction of prostaglandins
could provide a direct benefit. (Fogh et al. (1993) Acta Derm
Venerologica 73:191-193). Recently over expression of
5-lipoxygenase in the skin of patients with system sclerosis has
been reported. This has led to the suggestion that the LOX pathway
may be of significance in the pathogenesis of system sclerosis and
may represent a valid therapeutic target. (Kowal-Bielecka (2001)
Arthritis Rheum. 44(8):1865). Finally, the increased enzymatic
activity of both the COX-2 and 5-LOX at the site of allergen
injections suggests the potential for using dual COX/LOX inhibitors
to treat the symptoms of both the early and late phases of the skin
allergic response. (Church (2002) Clin. Exp. Allergy.
32(7):1013).
[0007] Topical application of a selective cyclooxygenase inhibitor
has been shown to suppress UVB mediated cutaneous inflammation
following both acute and long-term exposure. Additionally, edema,
dermal neutrophil infiltration and activation, PGE2 levels and the
formation of sunburn cells were reduced by the topical application
of a COX inhibitor. (Wilgus (2000) Prostaglandins Other Lipid
Mediat. 62(4):367). The COX inhibitor Celebrex.TM. has been shown
to reduce the effects of UV induced inflammation when administered
systematically (Wilgus et al. (2002) Adv. Exp. Med. Biol. 507:85),
and topically (Wilgus et al. (2000) Protaglandins Other Lipid
Mediat. 62:367). In animal models, the known COX inhibitor aspirin
and various lipoxygenase inhibitors exhibited vasoprotective
activity against inflammation and vasodepression resulting from UV
irradiation. (Kuhn (1988) Biomed. Biochim. Acta. 47:S320). Acute or
long-term chronic UV exposure causes skin damage and photoageing
that are characterized by degradation of collagen and accumulation
of abnormal elastin in the superficial dermis. A dual COX/LOX
inhibitor can be utilized to prevent and treat collagen degradation
caused by inflammatory infiltration by significantly reducing the
vasodilating, vasopermeability, chemotactic and
chemotaxins-prostaglandins (PG), as well as, those of multiple
leukotrienes (LT). (Bosset (2003) Br. J. Dermatol. 149(4):826; Hase
(2000) Br. J. Dermatol. 142(2):267). Additionally, chemically
induced oxidative stress in mouth skin can be inhibited by
separately administrating COX and LOX inhibitors to reduce
leukocyte adhesion, infiltration and H.sub.2O.sub.2 generation.
(Nakamura (2003) Free Radical Biol. Med. 35(9):997).
[0008] In addition to their use as anti-inflammatory agents,
another potential role for COX inhibitors is in the treatment of
cancer. Over expression of COX has been demonstrated in various
human malignancies and inhibitors of COX have been shown to be
efficacious in the treatment of animals with skin tumors. While the
mechanism of action is not completely understood, the over
expression of COX has been shown to inhibit apoptosis and increase
the invasiveness of tumorgenic cell types. (Dempke et al. (2001) J.
Can. Res. Clin. Oncol. 127:411-17; Moore and Simmons (2000) Current
Med. Chem. 7:1131-1144). Up regulated COX production has been
implicated in the generation of actinic keratosis and squamous cell
carcinoma in skin. Increased amounts of COX were also found in
lesions produced by DNA damage. (Buckman et al. (1998)
Carcinogenesis 19:723). Therefore, control of expression or protein
function of COX would seem to lead to a decrease in the
inflammatory response and the eventual progression to cancer. In
fact, COX inhibitors such as indomethacin and Celebrex.TM. have
been found to be effective in treating UV induced erythema and
tumor formation. (Fischer (1999) Mol. Carcinog. 25:231; Pentland
(1999) Carcinogenesis 20:1939). Recently, the over expression of
lipoxygenase has also been shown to be related to epidermal tumor
development (Muller (2002) Cancer Res. 62(16):4610) and melanoma
carcinogenesis (Winer (2002) Melanoma Res. 12(5):429). The
arachidonic acid (AA) metabolites generated from lipoxygenase
pathways play important roles in tumor growth related signal
transduction suggesting that that the inhibition of lipoxygenase
pathways should be a valid target to prevent cancer progression.
(Cuendet (2000) Drug Metabol Drug Interact 17(4): 109; Steele
(2003) Mutat Res. 523-524:137). Thus, the use of therapeutic agents
having dual COX/LOX inhibitory activity offers significant
advantages in the chemoprevention of cancer.
[0009] Prostaglandins and leukotrienes also play important roles in
the physiological and pathological processes of wounds, burns,
scald, acne, microbial infections, dermatitis, and many other
diseases and conditions of the skin. The activation of a
pro-inflammatory cascade after thermal or chemical burns with
significantly elevated cyclooxygenase and lipoxygenase activities
are well documented and play an important role in the development
of subsequent severe symptoms and immune dysfunction that may lead
to multiple organ failure. (Schwacha (2003) Burns 29(1):1; He
(2001) J. Bum Care Rehabil. 22(1):58).
[0010] Acne is a disease of the pilosebaceous unit with
abnormalities in sebum production, follicular epithelial
desquamation, bacterial proliferation and inflammation. The
inflammatory properties of acne can be detected by polarized light
photography and utilized for clinical diagnosis, including an
evaluation of the extent of the acne and also to determine the
effectiveness of therapy. (Phillips (1997) J. Am. Acad. Dermatol.
37(6):948). Current therapeutic agents for the prevention and
treatment of acne include anti-inflammatory agents, like retinoids,
antimicrobial agents and hormonal drugs. (Leyden (2003) J. Am.
Acad. Dermatol. 49(3 Suppl):S200). Topical application of
anti-inflammatory drugs, such as retinoids (Millikan (2003) J. Am.
Acad. Dermatol. 4(2):75) and the COX inhibitor salicylic acid (Lee
(2003) Dermnatol Surg 29(12):1196) have been clinically
demonstrated as an effective and safe therapy for the treatment of
acne. Additionally, the use of nonsteroidal anti-inflammatory drugs
(NSAIDs) are well documented as therapeutic agents for common and
uncommon dermatoses, including acne, psoriasis, sun burn, erythema
nodosum, cryoglobulinemia, Sweet's syndrome, systemic mastocytosis,
urticarial, liverdoid and nodular vasculitis. (Friedman (2002) J.
Cutan Med. Surg. 6(5):449).
[0011] Flavonoids or bioflavonoids are a widely distributed group
of natural products, which have been reported to have
antibacterial, anti-inflammatory, antiallergic, antimutagenic,
antiviral, antineoplastic, anti-thrombic and vasodilatory activity.
The structural unit common to this group of compounds includes two
benzene rings on either side of a 3-carbon ring as illustrated by
the following general structural formula: 1
[0012] Various combinations of hydroxyl groups, sugars, oxygen and
methyl groups attached to this general three ring structure create
the various classes of flavonoids, which include flavanols,
flavones, flavan-3-ols (catechins), anthocyanins and
isoflavones.
[0013] Free-B-Ring flavones and flavonols are a specific class of
flavonoids, which have no substituent groups on the aromatic B ring
(referred to herein as Free-B-Ring flavonoids), as illustrated by
the following general structure: 2
[0014] wherein
[0015] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of --H, --OH,
--SH, OR, --SR, --NH.sub.2, --NHR, --NR.sub.2,
--NR.sub.3.sup.+X.sup.-, a carbon, oxygen, nitrogen or sulfur,
glycoside of a single or a combination of multiple sugars
including, but not limited to aldopentoses, methyl-aldopentose,
aldohexoses, ketohexose and their chemical derivatives thereof;
[0016] wherein
[0017] R is an alkyl group having between 1-10 carbon atoms;
and
[0018] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, fluoride, sulfate, phosphate, acetate, carbonate, etc.
[0019] Free-B-Ring flavonoids are relatively rare. Out of 9,396
flavonoids synthesized or isolated from natural sources, only 231
Free-B-Ring flavonoids are known (The Combined Chemical Dictionary,
Chapman & Hall/CRC, Version 5:1 June 2001). Free-B-Ring
flavonoids have been reported to have diverse biological activity.
For example, galangin (3,5,7-trihydroxyflavone) acts as antioxidant
and free radical scavenger and is believed to be a promising
candidate for anti-genotoxicity and cancer chemoprevention. (Heo et
al. (2001) Mutat. Res. 488(2):135-150). It is an inhibitor of
tyrosinase monophenolase (Kubo et al. (2000) Bioorg. Med. Chem.
8(7):1749-1755), an inhibitor of rabbit heart carbonyl reductase
(Imamura et al. (2000) J. Biochem. 127(4):653-658), has
antimicrobial activity (Afolayan and Meyer (1997) Ethnopharmacol.
57(3):177-181) and antiviral activity (Meyer et al. (1997) J.
Ethnopharmacol. 56(2):165-169). Baicalein and two other Free-B-Ring
flavonoids, have antiproliferative activity against human breast
cancer cells. (So et al. (1997) Cancer Lett. 112(2):127-133).
[0020] Typically, flavonoids have been tested for biological
activity randomly based upon their availability. Occasionally, the
requirement of substitution on the B-ring has been emphasized for
specific biological activity, such as the B-ring substitution
required for high affinity binding to p-glycoprotein (Boumendjel et
al. (2001) Bioorg. Med. Chem. Lett. 11(1):75-77); cardiotonic
effect (Itoigawa et al. (1999) J. Ethnopharmacol. 65(3): 267-272),
protective effect on endothelial cells against linoleic acid
hydroperoxide-induced toxicity (Kaneko and Baba (1999) Biosci
Biotechnol. Biochem 63(2):323-328), COX-1 inhibitory activity (Wang
(2000) Phytomedicine 7:15-19) and prostaglandin endoperoxide
synthase (Kalkbrenner et al. (1992) Pharmacology 44(1):1-12). Only
a few publications have mentioned the significance of the
unsubstituted B ring of the Free-B-Ring flavonoids. One example, is
the use of 2-phenyl flavones, which inhibit NADPH quinone acceptor
oxidoreductase, as potential anticoagulants. (Chen et al. (2001)
Biochem. Pharmacol. 61(11):1417-1427).
[0021] The mechanism of action with respect to the
anti-inflammatory activity of various Free-B-Ring flavonoids has
been controversial. The anti-inflammatory activity of the
Free-B-Ring flavonoids, chrysin (Liang et al. (2001) FEBS Lett.
496(1):12-18), wogonin (Chi et al. (2001) Biochem. Pharmacol.
61:1195-1203) and halangin (Raso et al. (2001) Life Sci. 68 :921-93
1), has been associated with the suppression of inducible
cyclooxygenase and nitric oxide synthase via activation of
peroxisome proliferator activated receptor gamma (PPAR.gamma.) and
influence on degranulation and AA release. (Tordera et al. (1994)
Z. Naturforsch [C] 49:235-240). It has been reported that oroxylin,
baicalein and wogonin inhibit 12-lipoxygenase activity without
affecting cyclooxygenase. (You et al. (1999) Arch. Pharm. Res.
22(1):18-24). More recently, the anti-inflammatory activity of
wogonin, baicalin and baicalein has been reported as occurring
through inhibition of inducible nitric oxide synthase and cox-2
gene expression induced by nitric oxide inhibitors and
lipopolysaccharide. (Chen et al. (2001) Biochem. Pharmacol.
61(11):1417-1427). It has also been reported that oroxylin acts via
suppression of NF.kappa.B activation. (Chen et al. (2001) Biochem.
Pharmacol. 61(11):1417-1427). Finally, wogonin reportedly inhibits
inducible PGE2 production in macrophages. (Wakabayashi and Yasui
(2000) Eur. J. Pharmacol. 406(3):477-481).
[0022] Inhibition of the phosphorylation of mitrogen-activated
protein kinase and inhibition of Ca.sup.2+ ionophore A23187 induced
PGE.sub.2 release by baicalein has been reported as the mechanism
of anti-inflammatory activity of Scutellariae radix. (Nakahata et
al. (1999) Nippon Yakurigaku Zasshi, 114, Supp. 11:215P-219P;
Nakahata et al. (1998) Am. J. Chin Med. 26:311-323). Baicalin from
Scutellaria baicalensis, reportedly inhibits superantigenic
staphylococcal exotoxins stimulated T-cell proliferation and
production of IL-1.beta., IL-6, tumor necrosis factor-.alpha.
(TNF-.alpha.), and interferon-.gamma. (IFN-.gamma.). (Krakauer et
al. (2001) FEBS Lett. 500:52-55). Thus, the anti-inflammatory
activity of baicalin has been associated with inhibiting the
pro-inflammatory cytokines mediated signaling pathways activated by
superantigens. However, it has also been proposed that the
anti-inflammatory activity of baicalin is due to the binding of a
variety of chemokines, which limits their biological activity. (Li
et al. (2000) Immunopharmacology 49:295-306). Recently, the effects
of baicalin on adhesion molecule expression induced by thrombin and
thrombin receptor agonist peptide (Kimura et al. (2001) Planta Med.
67:331-334), as well as, the inhibition of mitogen-activated
protein kinase cascade (MAPK) (Nakahata et al. (1999) Nippon
Yakurigaku Zasshi, 114, Supp 11:215P-219P; Nakahata et al. (1998)
Am. J. Chin Med. 26:311-323) have been reported.
[0023] The Chinese medicinal plant, Scutellaria baicalensis
contains significant amounts of Free-B-Ring flavonoids, including
baicalein, baicalin, wogonin and baicalenoside. Traditionally, this
plant has been used to treat a number of conditions including
clearing away heat, purging fire, dampness-warm and summer fever
syndromes; polydipsia resulting from high fever; carbuncle, sores
and other pyogenic skin infections; upper respiratory infections,
such as acute tonsillitis, laryngopharyngitis and scarlet fever;
viral hepatitis; nephritis; pelvitis; dysentery; hematemesis and
epistaxis. This plant has also traditionally been used to prevent
miscarriage. (Encyclopedia of Chinese Traditional Medicine,
ShangHai Science and Technology Press, ShangHai, China, 1998).
Clinically Scutellaria is now used to treat conditions such as
pediatric pneumonia, pediatric bacterial diarrhea, viral hepatitis,
acute gallbladder inflammation, hypertension, topical acute
inflammation, resulting from cuts and surgery, bronchial asthma and
upper respiratory infections. (Encyclopedia of Chinese Traditional
Medicine, ShangHai Science and Technology Press, ShangHai, China,
1998). The pharmacological efficacy of Scutellaria roots for
treating bronchial asthma is reportedly related to the presence of
Free-B-Ring flavonoids and their suppression of eotaxin associated
recruitment of eosinophils. (Nakajima et al. (2001) Planta Med.
67(2):132-135).
[0024] To date, a number of naturally occurring Free-B-Ring
flavonoids have been commercialized for varying uses. For example,
liposome formulations of Scutellaria extracts have been utilized
for skin care (U.S. Pat. Nos. 5,643,598; 5,443,983). Baicalin has
been used for preventing cancer, due to its inhibitory effects on
oncogenes (U.S. Pat. No. 6,290,995). Baicalin and other compounds
have been used as antiviral, antibacterial and immunomodulating
agents (U.S. Pat. No. 6,083,921 and WO98/42363) and as natural
anti-oxidants (WO98/49256 and Poland Pub. No. 9,849,256).
Scutellaria baicalensis root extract has been formulated as a
supplemental sun screen agent with additive effects of the
cumulative SPFs of each individual component in a topical
formulation (WO98/19651). Chrysin has been used for its anxiety
reducing properties (U.S. Pat. No. 5,756,538). Anti-inflammatory
flavonoids are used for the control and treatment of anorectal and
colonic diseases (U.S. Pat. No. 5,858,371), and inhibition of
lipoxygenase (U.S. Pat. No. 6,217,875). These compounds are also
formulated with glucosamine collagen and other ingredients for
repair and maintenance of connective tissue (U.S. Pat. No.
6,333,304). Flavonoid esters constitute active ingredients for
cosmetic compositions (U.S. Pat. No. 6,235,294). U.S. application
Ser. No. 10/091,362, filed Mar. 1, 2002, entitled "Identification
of Free-B-Ring Flavonoids as Potent COX-2 Inhibitors," and U.S.
application Ser. No. 10/427,746, filed Jul. 22, 2003, entitled
"Formulation of a Mixture of Free-B-Ring Flavonoids and Flavans as
a Therapeutic Agent" both disclose a method for inhibiting the
cyclooxygenase enzyme COX-2 by administering a composition
comprising a Free-B-Ring flavonoid or a composition containing a
mixture of Free-B-Ring flavonoids to a host in need thereof. This
is the first report of a link between Free-B-Ring flavonoids and
COX-2 inhibitory activity. These applications are specifically
incorporated herein by reference in their entirety.
[0025] Japanese Pat. No. 63027435, describes the extraction, and
enrichment of baicalein and Japanese Pat. No. 61050921 describes
the purification of baicalin.
[0026] Flavans include compounds illustrated by the following
general structure: 3
[0027] wherein
[0028] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from the group consisting of --H, --OH,
--SH, --OCH.sub.3, --SCH.sub.3, --OR, --SR, --NH.sub.2, --NRH,
--NR.sub.2, --NR.sub.3.sup.+X.sup.-, esters of the mentioned
substitution groups, including, but not limited to, gallate,
acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl
esters and caffeoyl esters, and their chemical derivatives thereof;
a carbon, oxygen, nitrogen or sulfur glycoside of a single or a
combination of multiple sugars including, but not limited to,
aldopentoses, methyl aldopentose, aldohexoses, ketohexose and their
chemical derivatives thereof; dimer, trimer and other polymerized
flavans;
[0029] wherein
[0030] R is an alkyl group having between 1-10 carbon atoms;
and
[0031] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, sulfate, phosphate, acetate, fluoride, and carbonate,
etc.
[0032] Catechin is a flavan, found primarily in green tea, having
the following structure: 4
[0033] Catechin works both alone and in conjunction with other
flavonoids found in tea, and has both antiviral and antioxidant
activity. Catechin has been shown to be effective in the treatment
of viral hepatitis. It also appears to prevent oxidative damage to
the heart, kidney, lungs and spleen and has been shown to inhibit
the growth of stomach cancer cells.
[0034] Catechin and its isomer epicatechin inhibit prostaglandin
endoperoxide synthase with an IC.sub.50 value of 40 .mu.M.
(Kalkbrenner et al. (1992) Pharmacol. 44:1-12). Five flavan-3-ol
derivatives, including (+)-catechin and gallocatechin, isolated
from four plant species: Atuna racemosa, Syzygium carynocarpum,
Syzygium malaccense and Vantanea peruviana, exhibit equal to weaker
inhibitory activity against COX-2, relative to COX-1, with
IC.sub.50 values ranging from 3.3 .mu.M to 138 .mu.M (Noreen et al.
(1998) Planta Med. 64:520-524). (+)-Catechin, isolated from the
bark of Ceiba pentandra, inhibits COX-1 with an IC.sub.50 value of
80 .mu.M (Noreen et al. (1998) J. Nat. Prod. 61:8-12). Commercially
available pure (+)-catechin inhibits COX-1 with an IC.sub.50 value
of around 183 to 279 .mu.M depending upon the experimental
conditions, with no selectivity for COX-2. (Noreen et al. (1998) J.
Nat. Prod. 61:1-7).
[0035] Green tea catechin, when supplemented into the diets of
Sprague dawley male rats, lowered the activity level of platelet
PLA.sub.2 and significantly reduced platelet cyclooxygenase levels.
(Yang et al. (1999) J. Nutr. Sci. Vitaminol. 45:337-346). Catechin
and epicatechin reportedly weakly suppress cox-2 gene transcription
in human colon cancer DLD-1 cells (IC.sub.50=415.3 .mu.M). (Mutoh
et al. (2000) Jpn. J. Cancer Res. 91:686-691). The neuroprotective
ability of (+)-catechin from red wine results from the antioxidant
properties of catechin, rather than inhibitory effects on
intracellular enzymes, such as cyclooxygenase, lipoxygenase, or
nitric oxide synthase (Bastianetto et al. (2000) Br. J. Pharmacol.
131:711-720). Catechin derivatives purified from green and black
tea, such as epigallocatechin-3-gallate (EGCG), epigallocatechin
(EGC), epicatechin-3-gallate (ECG), and theaflavins showed
inhibition of cyclooxygenase and lipoxygenase dependent metabolism
of AA in human colon mucosa and colon tumor tissues (Hong et al.
(2001) Biochem. Pharmacol. 62:1175-1183) and induce cox-2
expression and PGE.sub.2 production (Park et al. (2001) Biochem.
Biophys. Res. Commun. 286:721-725). Epiafzelechin isolated from the
aerial parts of Celastrus orbiculatus exhibited dose-dependent
inhibition of COX-1 activity with an IC.sub.50 value of 15 .mu.M
and also demonstrated anti-inflammatory activity against
carrageenin-induced mouse paw edema following oral administration
at a dosage of 100 mg/kg. (Min et al. (1999) Planta Med.
65:460-462).
[0036] Acacia is a genus of leguminous trees and shrubs. The genus
Acacia includes more than 1000 species belonging to the family of
Leguminosae and the subfamily of Mimosoideae. Acacias are
distributed worldwide in tropical and subtropical areas of Central
and South America, Africa, parts of Asia, as well as, Australia,
which has the largest number of endemic species. Acacias are very
important economically, providing a source of tannins, gums,
timber, fuel and fodder. Tannins, which are isolated primarily from
bark, are used extensively for tanning hides and skins. Some Acacia
barks are also used for flavoring local spirits. Some indigenous
species like A. sinuata also yield saponins, which are any of
various plant glucosides that form soapy lathers when mixed and
agitated with water. Saponins are used in detergents, foaming
agents and emulsifiers. The flowers of some Acacia species are
fragrant and used to make perfume. The heartwood of many Acacias is
used for making agricultural implements and also provides a source
of firewood. Acacia gums find extensive use in medicine and
confectionary and as sizing and finishing materials in the textile
industry.
[0037] To date, approximately 330 compounds have been isolated from
various Acacia species. Flavonoids are the major class of compounds
isolated from Acacias. Approximately 180 different flavonoids have
been identified, 111 of which are flavans. Terpenoids are second
largest class of compounds isolated from species of the Acacia
genus, with 48 compounds having been identified. Other classes of
compounds isolated from Acacia include, alkaloids (28), amino
acids/peptides (20), tannins (16), carbohydrates (15), oxygen
heterocycles (15) and aliphatic compounds (10). (Buckingham, The
Combined Chemical Dictionary, Chapman & Hall CRC, version 5:2,
December 2001).
[0038] Phenolic compounds, particularly flavans are found in
moderate to high concentrations in all Acacia species. (Abdulrazak
et al. (2000) Journal of Animal Sciences. 13:935-940).
Historically, most of the plants and extracts of the Acacia genus
have been utilized as astringents to treat gastrointestinal
disorders, diarrhea, indigestion and to stop bleeding. (Vautrin
(1996) Universite Bourgogne (France) European abstract 58-01C:177;
Saleem et al. (1998) Hamdard Midicus. 41:63-67). The bark and pods
of Acacia arabica Willd. contain large quantities of tannins and
have been utilized as astringents and expectorants. (Nadkarni
(1996) India Materia Medica, Bombay Popular Prakashan, pp. 9-17).
Diarylpropanol derivatives, isolated from stem bark of Acacia
tortilis from Somalia, have been reported to have smooth muscle
relaxing effects. (Hagos et al. (1987) Planta Medica. 53:27-31,
1987). It has also been reported that terpenoid saponins isolated
from Acacia victoriae have an inhibitory effect on
dimethylbenz(a)anthracene-induced murine skin carcinogenesis
(Hanausek et al. (2000) Proceedings American Association for Cancer
Research Annual Meeting 41:663) and induce apotosis (Haridas et al.
(2000) Proceedings American Association for Cancer Research Annual
Meeting. 41:600). Plant extracts from Acacia nilotica have been
reported to have spasmogenic, vasoconstrictor and anti-hypertensive
activity (Amos et al. (1999) Phytotherapy Research 13:683-685;
Gilani et al. (1999) Phytotherapy Research. 13:665-669), and
antiplatelet aggregatory activity (Shah et al. (1997) General
Pharmacology. 29:251-255). Anti-inflammatory activity has been
reported for A. nilotica. It was speculated that flavonoids,
polysaccharides and organic acids were potential active components.
(Dafallah and Al-Mustafa (1996) American Journal of Chinese
Medicine. 24:263-269). To date, the only reported 5-lipoxygenase
inhibitor isolated from Acacia is a monoterpenoidal carboxamide
(Seikine et al. (1997) Chemical and Pharmaceutical Bulletin.
45:148-11).
[0039] The extract from the bark of Acacia has been patented in
Japan for external use as a whitening agent (Abe, JP10025238), as a
glucosyl transferase inhibitor for dental applications (Abe,
JP07242555), as a protein synthesis inhibitor (Fukai, JP 07165598),
as an active oxygen scavenging agent for external skin preparations
(Honda, JP 07017847, Bindra U.S. Pat. No. 6,1266,950), and as a
hyaluronidase inhibitor for oral consumption to prevent
inflammation, pollinosis and cough (Ogura, JP 07010768).
[0040] To date, Applicant is unaware of any reports of a
formulation combining only Free-B-Ring-Flavonoids and flavans as
the primary biologically active components for the dual inhibition
of the COX/LOX enzymes that yield significant benefit to mammal
skin conditions.
SUMMARY OF THE INVENTION
[0041] The present invention includes methods that are effective in
simultaneously inhibiting both the cyclooxygenase (COX) and
lipoxygenase (LOX) enzymes, for use in the prevention and treatment
of diseases and conditions related to the skin. The method for the
simultaneous dual inhibition of the COX and LOX enzymes is
comprised administering, preferably topically, a composition
comprised of a mixture of Free-B-Ring flavonoids and flavans
synthesized and/or isolated from a single plant or multiple plants
to a host in need thereof. This composition of matter is referred
to herein as Soliprin.TM.. The efficacy of this method was
demonstrated with purified enzymes, in different cell lines, in
multiple animal models and eventually in a human clinical study.
The ratio of the Free-B-Ring flavonoids to flavans in the
composition can be in the range of 99.9:0.1 of Free-B-Ring
flavonoids:flavans to 0.1:99.9 Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-Ring flavonoids to flavans is selected from the group
consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of this
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is 80:20. In a preferred embodiment, the
Free-B-Ring flavonoids are isolated from a plant or plants in the
Scutellaria genus of plants and the flavans are isolated from a
plant or plants in the Acacia genus of plants.
[0042] The present invention also includes methods for the
prevention and treatment of COX and LOX mediated diseases and
conditions of the skin. The method for preventing and treating COX
and LOX mediated diseases and conditions of the skin is comprised
of administering, preferably topically, to a host in need thereof
an effective amount of a composition comprising a mixture of
Free-B-Ring flavonoids and flavans synthesized and/or isolated from
a single plant or multiple plants and a pharmaceutically acceptable
carrier. The ratio of the Free-B-Ring flavonoids to flavans in the
composition can be in the range of 99.9:0.1 of Free-B-Ring
flavonoids:flavans to 0.1:99.9 Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-Ring flavonoids to flavans is selected from the group
consisting of approximately 90: 10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of this
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is 80:20. In a preferred embodiment, the
Free-B-Ring flavonoids are isolated from a plant or plants in the
Scutellaria genus of plants and the flavans are isolated from a
plant or plants in the Acacia genus of plants.
[0043] The Free-B-Ring flavonoids, also referred to herein as
Free-B-Ring flavones and flavonols, that can be used in accordance
with the following invention include compounds illustrated by the
following general structure: 5
[0044] wherein
[0045] R.sub.1, R.sub.2, R.sub.3, R.sub.4, and R.sub.5 are
independently selected from the group consisting of --H, --OH,
--SH, OR, --SR, --NH.sub.2, --NHR, --NR.sub.2,
--NR.sub.3.sup.+X.sup.-, a carbon, oxygen, nitrogen or sulfur,
glycoside of a single or a combination of multiple sugars
including, but not limited to aldopentoses, methyl-aldopentose,
aldohexoses, ketohexose and their chemical derivatives thereof;
[0046] wherein
[0047] R is selected from an alkyl group having between 1-10 carbon
atoms; and
[0048] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.
[0049] The Free-B-Ring flavonoids of this invention may be obtained
by synthetic methods or extracted from the family of plants
including, but not limited to Annonaceae, Asteraceae, Bignoniaceae,
Combretaceae, Compositae, Euphorbiaceae, Labiatae, Lauranceae,
Leguminosae, Moraceae, Pinaceae, Pteridaceae, Sinopteridaceae,
Ulmaceae and Zingiberacea. The Free-B-Ring flavonoids can be
extracted, concentrated, and purified from the following genus of
high plants, including but not limited to Desmos, Achyrocline,
Oroxylum, Buchenavia, Anaphalis, Cotula, Gnaphalium, Helichrysum,
Centaurea, Eupatorium, Baccharis, Sapium, Scutellaria, Molsa,
Colebrookea, Stachys, Origanum, Ziziphora, Lindera, Actinodaphne,
Acacia, Derris, Glycyrrhiza, Millettia, Pongamia, Tephrosia,
Artocarpus, Ficus, Pityrogramma, Notholaena, Pinus, Ulmus and
Alpinia.
[0050] The flavans that can be used in accordance with the
following invention include compounds illustrated by the following
general structure: 6
[0051] wherein
[0052] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from the group consisting of H, --OH, --SH,
--OCH.sub.3, --SCH.sub.3, --OR, --SR, --NH.sub.2, --NRH,
--NR.sub.2, --NR.sub.3.sup.+X.sup.-, esters of the mentioned
substitution groups, including, but not limited to, gallate,
acetate, cinnamoyl and hydroxyl-cinnamoyl esters, trihydroxybenzoyl
esters and caffeoyl esters; thereof carbon, oxygen, nitrogen or
sulfur glycoside of a single or a combination of multiple sugars
including, but not limited to, aldopentoses, methyl aldopentose,
aldohexoses, ketohexose and their chemical derivatives thereof;
dimer, trimer and other polymerized flavans;
[0053] wherein
[0054] R is selected from an alkyl group having between 1-10 carbon
atoms; and
[0055] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.
[0056] The flavans of this invention may be obtained from a plant
or plants selected from the genus of Acacia. In a preferred
embodiment, the plant is selected from the group consisting of
Acacia catechu, Acacia concinna, Acacia farnesiana, Acacia Senegal,
Acacia speciosa, Acacia arabica, A. caesia, A. pennata, A. sinuata.
A. mearnsii, A. picnantha, A. dealbata, A. auriculiformis, A.
holoserecia and A. mangium.
[0057] In one embodiment, the present invention includes a method
for preventing and treating a number of COX and LOX mediated
diseases and conditions of the skin including, but not limited to
sun burns, thermal burns, acne, topical wounds, minor inflammatory
conditions caused by fungal, microbial and viral infections,
vitilago, systemic lupus erythromatosus, psoriasis, carcinoma,
melanoma, as well as other mammal skin cancers. In another
embodiment the present invention includes a method for preventing
and treating skin damage resulting from exposure to ultraviolet
(UV) radiation, chemicals, heat, wind and dry environments. In yet
another embodiment the present invention includes a method for
preventing and treating wrinkles, saggy skin, lines and dark
circles around the eyes, dermatitis and other allergy related
conditions of the skin.
[0058] The present invention further includes therapeutic
compositions comprising the therapeutic agents of the present
invention. In addition to their use for the prevention and
treatment of the above described diseases and conditions of the
skin, the therapeutic compositions described herein can also be
used to sooth sensitive skin and to provide smooth and youthful
skin with improved elasticity, reduced and delayed aging, enhanced
youthful appearance and texture, and increased flexibility,
firmness, smoothness and suppleness.
[0059] The method of prevention and treatment according to this
invention comprises administering topically to a host in need
thereof a therapeutically effective amount of the formulated
Free-B-Ring flavonoids and flavans isolated from a single source or
multiple sources. The purity of the individual and/or a mixture of
multiple Free-B-Ring flavonoids and flavans includes, but is not
limited to 0.01% to 100%, depending on the methodology used to
obtain the compound(s). In a preferred embodiment, doses of the
mixture of Free-B=Ring flavonoids and flavans containing the same
are an efficacious, nontoxic quantity generally selected from the
range of 0.001% to 100% based on total weight of the topical
formulation. Persons skilled in the art using routine clinical
testing are able to determine optimum doses for the particular
ailment being treated.
[0060] The present invention includes an evaluation of different
compositions of Free-B-Ring flavonoids and flavans using enzymatic
and in vivo models to optimize the formulation and obtain the
desired physiological activity. The efficacy and safety of this
formulation is also demonstrated in human clinical studies. The
compositions of this invention can be administered by any method
known to one of ordinary skill in the art. The modes of
administration include, but are not limited to, enteral (oral)
administration, parenteral (intravenous, subcutaneous, and
intramuscular) administration and topical application. In the
preferred embodiment the method of treatment according to this
invention comprises administering topically to a host in need
thereof a therapeutically effective amount of a mixture of
Free-B-Ring flavonoids and flavans synthesized and/or isolated from
a single plant or multiple plants.
[0061] It is to be understood that both the foregoing general
description and the following detailed description are exemplary
and explanatory only and are not restrictive of the invention as
claimed.
BRIEF DESCRIPTION OF THE FIGURES
[0062] FIG. 1 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by a standardized Free-B-Ring flavonoid extract
(83% baicalin based on HPLC) which was isolated from Scutellaria
baicalensis. The extract was examined for its inhibition of the
peroxidase activity of recombinant ovine COX-1 (.diamond-solid.)
and ovine COX-2 (.box-solid.). The data is presented as percent
inhibition vs. inhibitor concentration (.mu.g/mL). The IC.sub.50
for COX-1 was calculated as 0.24 .mu.g/mL/unit of enzyme while the
IC.sub.50 for COX-2 was calculated as 0.48 .mu.g/mL/unit.
[0063] FIG. 2 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by the purified component baicalin which was
isolated from Scutellaria baicalensis. The compound was examined
for its inhibition of the peroxidase activity of recombinant ovine
COX-1 (.diamond-solid.) and ovine COX-2 (.box-solid.). The data is
presented as percent inhibition vs. inhibitor concentration
(.mu.g/mL). The IC.sub.50 for COX-1 was determined to be 0.44
.mu.g/mL/unit of enzyme and the IC.sub.50 for COX-2 was determined
to be 0.28 .mu.g/mL/unit.
[0064] FIG. 3 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by the purified component baicalein isolated from
Scutellaria baicalensis. The compound was examined for its
inhibition of the peroxidase activity of recombinant ovine COX-1
(.diamond-solid.) and ovine COX-2 (.box-solid.). The data is
presented as percent inhibition vs. inhibitor concentration
(.mu.g/mL). The IC.sub.50 for COX-1 was determined to be 0.18
.mu.g/mL/unit of enzyme and the IC.sub.50 for COX-2 was determined
to be 0.28 .mu.g/mL/unit.
[0065] FIG. 4 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by a standardized flavan extract containing 50%
total flavans which was isolated from Acacia catechu. The extract
was examined for its inhibition of the peroxidase activity of
recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2
(.box-solid.). The data is presented as percent inhibition vs.
inhibitor concentration (.mu.g/mL). The IC.sub.50 for COX-1 was
calculated as 0.17 .mu.g/mL/unit of enzyme and the IC.sub.50 for
COX-2 was calculated as 0.41 .mu.g/mL/unit.
[0066] FIG. 5 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by the a composition of matter comprised of greater
than 90% flavans isolated from Acacia catechu. The composition was
examined for its inhibition of the peroxidase activity of
recombinant ovine COX-1 (.diamond-solid.) and ovine COX-2
(.box-solid.). The data is presented as percent inhibition vs.
inhibitor concentration (.mu.g/mL). The IC.sub.50 for COX-1 was
calculated as 0.11 .mu.g/mL/unit of enzyme and the IC.sub.50 for
COX-2 was calculated as 0.42 .mu.g/mL/unit.
[0067] FIG. 6 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by a formulation produced by combining an extract
of Free-B-Ring flavonoids isolated from the roots of Scutellaria
baicalensis and an extract of flavans isolated from the bark of
Acacia catechu in a ratio of 80:20. This composition of matter,
referred to hereinafter as Soliprin.TM., was examined for its
inhibition of the peroxidase activity of recombinant ovine COX-1
(.diamond-solid.) and ovine COX-2 (.box-solid.). The data is
presented as percent inhibition vs. inhibitor concentration
(.mu.g/mL). The IC.sub.50 for COX-1 was calculated as 0.76
.mu.g/mL/unit of enzyme and the IC.sub.50 for COX-2 was calculated
as 0.80 .mu.g/mL/unit.
[0068] FIG. 7 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by a formulation produced by combining an extract
of Free-B-Ring flavonoids isolated from the roots of Scutellaria
baicalensis and an extract of flavans isolated from the bark of
Acacia catechu in a ratio of about 50:50. The composition,
Soliprin.TM., was examined for its inhibition of the peroxidase
activity of recombinant ovine COX-1 (.diamond-solid.) and ovine
COX-2 (.box-solid.). The data is presented as percent inhibition
vs. inhibitor concentration (.mu.g/mL). The IC.sub.50 for COX-1 was
calculated as 0.38 .mu.g/mL/unit of enzyme and the IC.sub.50 for
COX-2 was determined to be 0.84 .mu.g/mL/unit.
[0069] FIG. 8 depicts graphically a profile of the inhibition of
COX-1 and COX-2 by a formulation produced by combining an extract
of Free-B-Ring flavonoids isolated from the roots of Scutellaria
baicalensis and an extract of flavans isolated from the bark of
Acacia catechu in a ratio of about 20:80. The composition,
Soliprin.TM., was examined for its inhibition of the peroxidase
activity of recombinant ovine COX-1 (.diamond-solid.) and ovine
COX-2 (.box-solid.). The data is presented as percent inhibition
vs. inhibitor concentration (.mu.g/mL). The IC.sub.50 of this
composition for COX-1 was 0.18 .mu.g/mL/unit of enzyme and the
IC.sub.50 for COX-2 was 0.41 .mu.g/mL/unit.
[0070] FIG. 9 depicts graphically a profile of the inhibition of
5-LO by the flavan extract from Acacia catechu. The composition was
examined for its inhibition of recombinant potato 5-lipoxygenase
activity (.diamond-solid.) as described in Example 4. The data is
presented as percent inhibition of assays without inhibitor. The
IC.sub.50 for 5-LO was 1.38 .mu.g/mL/unit of enzyme.
[0071] FIG. 10 illustrates the High Pressure Liquid Chromatography
(HPLC) chromatogram of a typical formulation comprised of a mixture
of Free-B-Ring flavonoids isolated from the roots of Scutellaria
baicalensis and flavans isolated from the bark of Acacia catechu in
a ratio of 80:20 carried out under the conditions as described in
Example 9.
[0072] FIG. 11 depicts graphically the effect of increasing
concentrations of Soliprin.TM. on the amount of LPS-induced newly
synthesized LTB.sub.4 (.diamond-solid.) as determined by ELISA in
THP-1 or HT-29 cells (ATCC) as described in Example 10. The
Soliprin.TM. was produced through the combination of standardized
extracts of Free-B-Ring flavonoids isolated from the roots of
Scutellaria baicalensis and flavans isolated from the bark of
Acacia catechu in a ratio of 80:20. The activity of the
Soliprin.TM. formulation is expressed as % inhibition of induced
LTB.sub.4 synthesis.
[0073] FIG. 12 compares the LTB.sub.4 levels as determined by ELISA
that remain in HT-29 cells after treatment with 3 .mu.g/mL
Soliprin.TM. in non-induced cells to treatment with 3 .mu.g/mL
ibuprofen as described in Example 10. The Soliprin.TM. formulation
demonstrated 80% inhibition of LTB4 production in the HT-29 cells
after two days of treatment.
[0074] FIG. 13 illustrates graphically ear-swelling data as a
measure of inhibition of inflammation as described in Example 11.
Soliprin.TM. produced through the combination of standardized
extracts of Free-B-Ring flavonoids isolated from the roots of
Scutellaria baicalensis and flavans isolated from the bark of
Acacia catechu in a ratio of 80:20 was compared to untreated mice
and mice given indomethacin (1.5 .mu.g/kg) via oral gavage. The
data is presented as the difference in micron measurement of the
untreated vs. the treated ear lobe for each mouse.
[0075] FIG. 14 illustrates graphically the effect of 100 .mu.g/kg
of Soliprin.TM., produced through the combination of standardized
extracts of Free-B-Ring flavonoids isolated from the roots of
Scutellaria baicalensis and flavans isolated from the bark of
Acacia catechu in a ratio of 80:20 on the AA injected ankles of
mice (Soliprin.TM.+arachidoni- c acid) compared to non-treated mice
(no treatment+arachidonic acid), mice without AA injections
(negative control) or mice that were injected with the liquid
carrier (vehicle control).
[0076] FIG. 15 depicts graphically the changes in hairless mice
skin erythema scores in different treatment groups as a function of
time following irradiation of the mice with UV light as described
in Example 12. The mice in Groups B-1, A-1, B-2 and A-2 were
treated with Soliprin.TM. either before (Groups B-1 and B-2) or
after (A-1 and A-2) irradiation. The Soliprin.TM. was produced
through the combination of standardized extracts of Free-B-Ring
flavonoids isolated from the roots of Scutellaria baicalensis and
flavans isolated from the bark of Acacia catechu in a ratio of
80:20. With reference to FIG. 15, it can be seen that topical
applications of Soliprin.TM., both before and after UV radiation,
significantly reduced erythema scores as compared with the control
group and the group that was administered the standard treatment
agent-Sooth-a-caine.
DETAILED DESCRIPTION OF THE INVENTION
[0077] Various terms are used herein to refer to aspects of the
present invention. To aid in the clarification of the description
of the components of this invention, the following definitions are
provided.
[0078] It is to be noted that the term "a" or "an" entity refers to
one or more of that entity; for example, a flavonoid refers to one
or more flavonoids. As such, the terms "a" or "an", "one or more"
and "at least one" are used interchangeably herein.
[0079] "Free-B-Ring Flavonoids" as used herein are a specific class
of flavonoids, which have no substitute groups on the aromatic
B-ring, as illustrated by the following general structure: 7
[0080] wherein
[0081] R.sub.1, R.sub.2, R.sub.3, R4, and R.sub.5 are independently
selected from the group consisting of --H, --OH, --SH, OR, --SR,
--NH.sub.2, --NHR, --NR.sub.2, --NR.sub.3.sup.+X.sup.-, a carbon,
oxygen, nitrogen or sulfur, glycoside of a single or a combination
of multiple sugars including, but not limited to aldopentoses,
methyl-aldopentose, aldohexoses, ketohexose and their chemical
derivatives thereof;
[0082] wherein
[0083] R is an alkyl group having between 1-10 carbon atoms;
and
[0084] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.
[0085] "Flavans" as used herein refer to a specific class of
flavonoids, which can be generally represented by the following
general structure: 8
[0086] wherein
[0087] R.sub.1, R.sub.2, R.sub.3, R.sub.4 and R.sub.5 are
independently selected from the group consisting of H, --OH,
[0088] --SH, --OCH.sub.3, --SCH.sub.3, --OR, --SR, --NH.sub.2,
--NRH, --NR.sub.2, --NR.sub.3.sup.+X.sup.-, esters of substitution
groups, including, but not limited to, gallate, acetate, cinnamoyl
and hydroxyl-cinnamoyl esters, trihydroxybenzoyl esters and
caffeoyl esters and their chemical derivatives thereof; carbon,
oxygen, nitrogen or sulfur glycoside of a single or a combination
of multiple sugars including, but not limited to, aldopentoses,
methyl aldopentose, aldohexoses, ketohexose and their chemical
derivatives thereof; dimer, trimer and other polymerized
flavans;
[0089] wherein
[0090] R is an alkyl group having between 1-10 carbon atoms;
and
[0091] X is selected from the group of pharmaceutically acceptable
counter anions including, but not limited to hydroxyl, chloride,
iodide, sulfate, phosphate, acetate, fluoride, carbonate, etc.
[0092] "Therapeutic" as used herein, includes treatment and/or
prophylaxis. When used, therapeutic refers to humans as well as
other animals.
[0093] "Pharmaceutically or therapeutically effective dose or
amount" refers to a dosage level sufficient to induce a desired
biological result. That result may be the alleviation of the signs,
symptoms or causes of a disease or any other alteration of a
biological system that is desired.
[0094] "Placebo" refers to the substitution of the pharmaceutically
or therapeutically effective dose or amount dose sufficient to
induce a desired biological that may alleviate the signs, symptoms
or causes of a disease with a non-active substance.
[0095] A "host" or "patient" is a living subject, human or animal,
into which the compositions described herein are administered.
Thus, the invention described herein may be used for veterinary as
well as human applications and the terms "patient" or "host" should
not be construed in a limiting manner. In the case of veterinary
applications, the dosage ranges can be determined as described
below, taking into account the body weight of the animal.
[0096] Note that throughout this application various citations are
provided. Each citation is specifically incorporated herein in its
entirety by reference.
[0097] The current invention provides methods for the extraction
(Example 1, Table 1) of plants that contain Free-B-Ring flavonoids
and flavans with organic and aqueous solvents. The crude extracts
were assayed for cyclooxygenase inhibitory activity (Example 2,
Tables 2 and 3). Purified Free-B-Ring flavonoids and flavans
demonstrated inhibitory activity against cyclooxygenase (COX) and
lipoxygenase (LOX), respectively, as shown in Examples 3 and 4.
Methods for analyzing and quantifying the extracts are described in
Examples 5 and 6 and the procedures to generate standardized
Free-B-Ring flavonoids and flavans from botanical origins are
provided in Examples 7 and 8.
[0098] In one embodiment of the present invention, the standardized
Free-B-Ring flavonoid extract is comprised of the active compounds
having a purity of between 1-99% (by weight) of total Free-B-Ring
flavonoids as defined in Examples 1, 2, 5 and 8. Baicalin is the
major active component in the extract, which accounts for
approximately 50-90% (by weight) of the total Free-B-Ring
flavonoids. In a preferred embodiment, the standardized extract
contains >70% total Free-B-Ring flavonoids in which >75% of
the Free-B-Ring flavonoids is baicalin.
[0099] In one embodiment, the standardized flavan extract is
comprised of the active compounds having a purity of between 1-99%
(by weight) total flavans as defined in Examples 1, 4, 6 and 7.
Catechin is the major active component in the extract and accounts
for 50-95% (by weight) of the total flavans. In a preferred
embodiment, the standardized flavan extract contains >80% total
flavans in which >70% of flavans is catechin.
[0100] In one embodiment, Soliprin.TM. is produced by mixing the
above two extracts or synthetic compounds in a ratio from 99:1 to
1:99. The preferred ratios of Free-B-Ring flavonoids to flavans are
80:20 as defined in Example 9 and Table 10 and 15:85 as defined in
Example 9.
[0101] The concentration of Free-B-Ring flavonoids in Soliprin.TM.
can be from about 1% to 99% and the concentration of flavans in
Soliprin.TM. can be from 99% to 1%. In a preferred embodiment of
the invention, the concentration of total Free-B-Ring flavonoids in
Soliprin.TM. is approximately 20% with a baicalin content of
approximately 15% of total weight of the Soliprin.TM.; and the
concentration of total flavans in Soliprin.TM. is approximately 75%
with a catechin content of approximately 70%. In this embodiment,
the total active components (Free-B-Ring flavonoids plus flavans)
in Soliprin.TM. are >90% of the total weight.
[0102] The present invention includes methods that are effective in
simultaneously inhibiting both the cyclooxygenase (COX) and
lipoxygenase (LOX) enzymes, for use in the prevention and treatment
of diseases and conditions related to the skin. The method for the
simultaneous dual inhibition of the COX and LOX enzymes is
comprised of administering, preferably topically a composition
comprised of a mixture of Free-B-Ring flavonoids and flavans
synthesized and/or isolated from a single plant or multiple plants
to a host in need thereof. This composition of matter is referred
to herein as Soliprin.TM.. The efficacy of this method was
demonstrated with purified enzymes, in different cell lines, in
multiple animal models and eventually in a human clinical study.
The ratio of the Free-B-Ring flavonoids to flavans in the
composition can be in the range of 99.9:0.1 of Free-B-Ring
flavonoids:flavans to 0.1:99.9 Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-Ring flavonoids to flavans is selected from the group
consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of this
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is 20:80. In a preferred embodiment, the
Free-B-Ring flavonoids are isolated from a plant or plants in the
Scutellaria genus of plants and the flavans are isolated from a
plant or plants in the Acacia genus of plants.
[0103] The present invention also includes methods for the
prevention and treatment of COX and LOX mediated diseases and
conditions of the skin. The method for preventing and treating COX
and LOX mediated diseases and conditions of the skin is comprised
of administering, preferably topically, to a host in need thereof
an effective amount of a composition comprised of a mixture of
Free-B-Ring flavonoids and flavans synthesized and/or isolated from
a single plant or multiple plants and a pharmaceutically acceptable
carrier. The ratio of the Free-B-Ring flavonoids to flavans in the
composition can be in the range of 99.9:0.1 of Free-B-Ring
flavonoids:flavans to 0.1:99.9 Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-ring flavonoids to flavans is selected from the group
consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of this
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is 20:80. In a preferred embodiment, the
Free-B-Ring flavonoids are isolated from a plant or plants in the
Scutellaria genus of plants and the flavans are isolated from a
plant or plants in the Acacia genus of plants.
[0104] In one embodiment, the present invention includes a method
for preventing and treating a number of COX and LOX mediated
diseases and conditions of the skin including, but not limited to
sun burns, thermal burns, acne, topical wounds, minor inflammatory
conditions caused by fungal, microbial and viral infections,
vitilago, systemic lupus erythromatosus, psoriasis, carcinoma,
melanoma, as well as other mammal skin cancers. In another
embodiment the present invention includes a method for preventing
and treating skin damage resulting from exposure to UV radiation,
chemicals, heat, wind and dry environments. In yet another
embodiment the present invention includes a method for preventing
and treating wrinkles, saggy skin, lines and dark circles around
the eyes, dermatitis and other allergy related conditions of the
skin.
[0105] The present invention further includes therapeutic
compositions comprising the therapeutic agents of the present
invention. In addition to their use for the prevention and
treatment of the above described diseases and conditions of the
skin, the therapeutic compositions described herein can be used to
sooth sensitive skin and to provide smooth and youthful skin with
improved elasticity, reduced and delayed aging, enhanced youthful
appearance and texture, and increased flexibility, firmness,
smoothness and suppleness.
[0106] The Free-B-Ring flavonoids that can be used in accordance
with the instant invention include compounds illustrated by the
general structure set forth above. The Free-B-Ring flavonoids of
this invention may be obtained by synthetic methods or may be
isolated from the family of plants including, but not limited to
Annonaceae, Asteraceae, Bignoniaceae, Combretaceae, Compositae,
Euphorbiaceae, Labiatae, Lauranceae, Leguminosae, Moraceae,
Pinaceae, Pteridaceae, Sinopteridaceae, Ulmaceae, and Zingiberacea.
The Free-B-Ring flavonoids can be extracted, concentrated, and
purified from the following genus of high plants, including but not
limited to Desmos, Achyrocline, Oroxylum, Buchenavia, Anaphalis,
Cotula, Gnaphalium, Helichrysum, Centaurea, Eupatorium, Baccharis,
Sapium, Scutellaria, Molsa, Colebrookea, Stachys, Origanum,
Ziziphora, Lindera, Actinodaphne, Acacia, Derris, Glycyrrhiza,
Millettia, Pongamia, Tephrosia, Artocarpus, Ficus, Pityrogramma,
Notholaena, Pinus, Ulmus and Alpinia.
[0107] The flavonoids can be found in different parts of plants,
including but not limited to stems, stem barks, twigs, tubers,
roots, root barks, young shoots, seeds, rhizomes, flowers and other
reproductive organs, leaves and other aerial parts. Methods for the
isolation and purification of Free-B-Ring flavonoids are described
in U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002,
entitled "Identification of Free-B-Ring Flavonoids as Potent Cox-2
Inhibitors," which is incorporated herein by reference in its
entirety.
[0108] The flavans that can be used in accordance with the method
of this invention include compounds illustrated by the general
structure set forth above. The flavans of this invention are
isolated from a plant or plants selected from the Acacia genus of
plants. In a preferred embodiment, the plant is selected from the
group consisting of Acacia catechu (A. catechu), A. concinna, A.
farnesiana, A. Senegal, A. speciosa, A. arabica, A. caesia, A.
pennata, A. sinuata. A. mearnsii, A. picnantha, A. dealbata, A.
auriculiformis, A. holoserecia and A. mangium.
[0109] The flavans can be found in different parts of plants,
including but not limited to stems, stem barks, trunks, trunk
barks, twigs, tubers, roots, root barks, young shoots, seeds,
rhizomes, flowers and other reproductive organs, leaves and other
aerial parts. Methods for the isolation and purification of flavans
are described in U.S. application Ser. No. 10/104,477, filed Mar.
22, 2002, entitled "Isolation of a Dual Cox-2 and 5-Lipoxygenase
Inhibitor from Acacia," which is incorporated herein by reference
in its entirety.
[0110] The present invention implements a strategy that combines a
series of in vivo inflammation and toxicity studies as well as in
vitro biochemical, cellular, and gene expression screens to
identify active plant extracts that specifically inhibit COX and
LOX enzymatic activity, impact mRNA gene expression and reduce
inflammation. The methods used herein to identify active plant
extracts that specifically inhibit COX and LOX are described in
Examples 1 and 2, as well as in U.S. application Ser. No.
10/091,362, filed Mar. 1, 2002, entitled "Identification of
Free-B-Ring Flavonoids as Potent Cox-2 Inhibitors;" U.S.
application Ser. No. 10/104,477, filed Mar. 22, 2002, entitled
"Isolation of a Dual Cox-2 and 5-Lipoxygenase Inhibitor from
Acacia," and U.S. application Ser. No. 10/427,746, filed Apr. 30,
2003, entitled "Formulation With Dual Cox-2 And 5-Lipoxygenase
Inhibitory Activity," each of which is incorporated herein by
reference in its entirety.
[0111] The biochemical assay, used to measure inhibition of COX,
relies on the protein's peroxidase activity in the presence of heme
and arachidonic acid. This study which is described in Example 3,
showed that the purified Free-B-Ring flavonoids, baicalin and
baicalein isolated from Scutellaria baicalensis and the flavan
extract isolated from Acacia catechu, and each individual
standardized extract containing high concentrations of Free-B-Ring
flavonoids and flavans inhibited COX activity (FIGS. 1-5).
Additionally, compositions having different ratios of each of the
individual standardized extracts (i.e., 80:20, 50:50 and 20:80
Free-B-Ring flavonoids:flavans), prepared as illustrated in Example
9, were all highly effective at inhibiting the COX activity in
vitro (FIGS. 6-8). The inhibition of LOX activity by a flavan
extract isolated from Acacia catechu, was assessed using a
lipoxygenase screening assay in vitro as described in Example 4.
The results are illustrated in FIG. 9. In addition, cell assays
that targeted inhibition of compounds in the breakdown of
arachidonic acid in the LOX pathway, namely leukotriene B4 were
performed using a Soliprin.TM. sample as described in Example 10.
The LTB.sub.4 inhibition results by Soliprin.TM. are illustrated in
FIGS. 11 and 12.
[0112] In vivo efficacy was demonstrated by the application of skin
irritating substances, such as AA, to the ears and ankle joint of
mice and measuring the reduction of swelling in mice treated with
Soliprin.TM. as described in Example 11. The results are set forth
in FIGS. 13 and 14. Finally, the efficacy of topical application of
Soliprin.TM. formulation in preventing and treating UV induced skin
erythema is illustrated in Example 12 and FIG. 15. In the study
described in Example 12, Soliprin.TM. in a blend ratio of 80:20 as
of Free-B-Ring flavonoids:flavans was dissolved in water and
applied topically at two concentration to the skin of hairless mice
both before and after UV exposure, respectively. The erythema
scores of the hairless mice from four Soliprin.TM. groups, in both
concentrations and regardless the applications time as before or
after UV exposure, all showed much less redness in smaller skin
areas as compared to severe and extended erythema in both the
control group and the group that was treated with Sooth-A Cain.
[0113] Example 13 (Tables 11 and 12) describes a general method for
the preparation of a Soliprin.TM. cream using pharmacologically,
dermatologically and cosmetic acceptable excipients. For purposes
of illustration this Example provides a detailed procedure for the
preparation of both a 0.5 wt % and 1.5 wt % Soliprin.TM. cream.
Finally, both of the Soliprin.TM. creams prepared as described in
Example 13 were evaluated on human skin for potential irritation
and induction of contact sensitization. A total of 97 and 101
subjects completed induction and challenge with the 0.5% and 1.5%
Soliprin.TM. creams, respectively. Test results show that
Soliprin.TM. creams at 0.5% and 1.5% concentration produced minimal
irritation and did not elicit evidence of induced contact
sensitization.
[0114] In summary, the present invention includes methods that are
effective in simultaneously inhibiting both the COX and LOX
enzymes. The method for the simultaneous dual inhibition of the COX
and LOX pathways is comprised of administering a composition
comprising a mixture of Free-B-Ring flavonoids and flavans
synthesized and/or isolated from a single plant or multiple plants
to a host in need thereof. The ratio of Free-B-Ring flavonoids to
flavans in the composition can be in the range of 99:1 Free-B-Ring
flavonoids:flavans to 1:99 of Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-Ring flavonoids to flavans is selected from the group
consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70,-20:80 and 10:90. In a preferred embodiment of the
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is approximately 20:80. In a preferred
embodiment, the Free-B-Ring flavonoids are isolated from a plant or
plants in the Scutellaria genus of plants and flavans are isolated
from a plant or plants in the Acacia genus of plants.
[0115] The present further includes methods for the prevention and
treatment of COX and LOX mediated skin diseases and conditions. The
method for preventing and treating COX and LOX mediated skin
diseases and conditions is comprised of administering to a host in
need thereof an effective amount of a composition comprising a
mixture of Free-B-Ring flavonoids and flavans synthesized and/or
isolated from a single plant or multiple plants together with a
pharmaceutically acceptable carrier. The ratio of Free-B-Ring
flavonoids to flavans can be in the range of 99:1 Free-B-Ring
flavonoids:flavans to 1:99 of Free-B-Ring flavonoids:flavans. In
specific embodiments of the present invention, the ratio of
Free-B-Ring flavonoids to flavans is selected from the group
consisting of approximately 90:10, 80:20, 70:30, 60:40, 50:50,
40:60, 30:70, 20:80 and 10:90. In a preferred embodiment of the
invention, the ratio of Free-B-Ring flavonoids:flavans in the
composition of matter is approximately 20:80. In a preferred
embodiment, the Free-B-ring flavonoids are isolated from a plant or
plants in the Scutellaria genus of plants and flavans are isolated
from a plant or plants in the Acacia genus of plants.
[0116] Applicant believes that U.S. application Ser. No.
10/104,477, filed Mar. 22, 2002, entitled "Isolation of a Dual
COX-2 and 5-Lipoxygenase Inhibitor from Acacia," is the first
report of a composition of matter isolated from the Acacia genus of
plants that demonstrates dual specificity for COX and LOX and that
U.S. application Ser. No. 10/091,362, filed Mar. 1, 2002, entitled
"Identification of Free-B-Ring Flavonoids as Potent COX-2
Inhibitors," is the first report of a correlation between
Free-B-Ring flavonoid structure and COX inhibitory activity. These
discoveries led to a novel blending of two classes of specific
compounds--Free-B-Ring Flavonoids and flavans--to form a
composition of matter, referred to herein as Soliprin.TM., which
can be used for the prevention and treatment of COX and LOX
mediated diseases and conditions, as described in U.S. application
Ser. No. 10/427,746, filed Apr. 30, 2003, entitled "Formulation
With Dual Cox-2 And 5-Lipoxygenase Inhibitory Activity." COX and
LOX mediated diseases and conditions include, but are not limited
to diseases and conditions of the skin including, but are not
limited to sun burns, thermal burns, acne, topical wounds, minor
inflammatory conditions caused by fungal, microbial and viral
infections, vitilago, systemic lupus erythromatosus, psoriasis,
carcinoma, melanoma, as well as other mammal skin cancers, skin
damage resulting from exposure to UV radiation, chemicals, heat,
wind and dry environments, wrinkles, saggy skin, lines and dark
circles around the eyes, dermatitis and other allergy related
conditions of the skin. Although not limited by theory, it is
believed that the mechanism of action of this class of compounds is
the direct dual inhibition of both COX and LOX enzymatic
activity.
[0117] The present invention further includes therapeutic
compositions comprising the therapeutic agents of the present
invention including various formulations thereof. Methods for the
preparation of these compositions, together with methods for the
determination of their purity and specific composition are
described in Examples 5-9 and FIG. 10.
[0118] In a preferred embodiment, the method of prevention and
treatment of COX and LOX mediated skin related diseases and
conditions according to this invention comprises administering
topically to a host in need thereof a therapeutically effective
amount of the formulated Free-B-Ring flavonoids and/or flavans
isolated from a single source or multiple sources. The purity of
the individual and/or a mixture of Free-B-Ring flavonoids and
flavans includes, but is not limited to 0.01% to 100%, depending on
the methodology used to obtain the compound(s). In a preferred
embodiment, doses of the mixture of Free-B-Ring flavonoids and/or
flavans containing that same are an efficacious, nontoxic quantity
generally selected from the range of 0.001% to 100% based on total
weight of the topical formulation. Persons skilled in the art using
routine clinical testing are able to determine optimum doses for
the particular ailment being treated.
[0119] The present invention includes evaluation of the different
composition of Free-B-Ring flavonoids and flavan using enzymatic
and in vivo anti-inflammation models to optimize the formulation
and obtain the greatest potency as described below. The present
invention provides a commercially viable process for the isolation,
purification and combination of Acacia flavans with Free-B-Ring
flavonoids to yield a composition of matter having desirable
physiological activity. In addition to their use for the prevention
and treatment of the above described diseases and conditions of the
skin, the therapeutic compositions described herein can also be
used to sooth sensitive skin and to provide smooth and youthful
skin with improved elasticity, reduced and delayed aging, enhanced
youthful appearance and texture, and increased flexibility,
firmness, smoothness and suppleness.
[0120] The compositions of the present invention can be formulated
as pharmaceutical compositions which include other components such
as a pharmaceutically and/or cosmetically acceptable excipient, an
adjuvant, and/or a carrier. For example, compositions of the
present invention can be formulated in an excipient that the host
to be treated can tolerate. An excipient is an inert substance used
as a diluent or vehicle for a drug. Examples of such excipients
include, but are not limited to water, buffers, saline, Ringer's
solution, dextrose solution, mannitol, Hank's solution,
preservatives and other aqueous physiologically balanced salt
solutions. Nonaqueous vehicles, such as fixed oils, sesame oil,
ethyl oleate, or triglycerides may also be used. Other useful
formulations include suspensions containing viscosity enhancing
agents, such as sodium carboxymethylcellulose, sorbitol, or
dextran. Excipients can also contain minor amounts of additives,
such as substances that enhance isotonicity and chemical stability.
Examples of buffers include phosphate buffer, bicarbonate buffer,
tris buffer, histidine, citrate, and glycine, or mixtures thereof,
while examples of preservatives include, but are not limited to
thimerosal, m- or o-cresol, formalin and benzyl alcohol. Standard
formulations can either be liquid or solids, which can be taken up
in a suitable liquid as a suspension or solution for
administration. Thus, in a non-liquid formulation, the excipient
can comprise dextrose, human serum albumin, preservatives, etc., to
which sterile water or saline can be added prior to
administration.
[0121] In one embodiment of the present invention, the composition
can also include an adjuvant or a carrier. Adjuvants are typically
substances that generally enhance the biological response of a
mammal to a specific bioactive agent. Suitable adjuvants include,
but are not limited to, Freund's adjuvant; other bacterial cell
wall components; aluminum-based salts; calcium-based salts; silica;
polynucleotides; toxoids; serum proteins; viral coat proteins;
other bacterial-derived preparations; gamma interferon; block
copolymer adjuvants, such as Hunter's Titermax adjuvant
(Vaxcel.TM., Inc. Norcross, Ga.); Ribi adjuvants (available from
Ribi ImmunoChem Research, Inc., Hamilton, Mont.); and saponins and
their derivatives, such as Quil A (available from Superfos
Biosector A/S, Denmark). Carriers are typically compounds that
increase the half-life of a therapeutic composition in the treated
host. Suitable carriers include, but are not limited to, polymeric
controlled release formulations, biodegradable implants, liposomes,
bacteria, viruses, oils, esters, and glycols.
[0122] In one embodiment, the composition is prepared as a
controlled release formulation, which slowly releases the
composition of the present invention into the host. As used herein,
a controlled release formulation comprises a composition of the
present invention in a controlled release vehicle. Suitable
controlled release vehicles will be known to those skilled in the
art. Preferred controlled release formulations are biodegradable
(i.e., bioerodible).
[0123] The therapeutic agents of the instant invention are
preferably administered topically by any suitable means, known to
those of skill in the art for topically administering therapeutic
compositions including, but not limited to as an ointment, gel,
lotion, or cream base or as an emulsion, as a patch, dressing or
mask, a nonsticking gauze, a bandage, a swab or a cloth wipe. Such
topical application can be locally administered to any affected
area, using any standard means known for topical administration. A
therapeutic composition can be administered in a variety of unit
dosage forms depending upon the method of administration. For
particular modes of delivery, a therapeutic composition of the
present invention can be formulated in an excipient of the present
invention. A therapeutic reagent of the present invention can be
administered to any host, preferably to mammals, and more
preferably to humans. The particular mode of administration will
depend on the condition to be treated.
[0124] In one embodiment, a suitable ointment is comprised of the
desired concentration of the mixture of Free-B-Ring flavonoids and
flavans, that is an efficacious, nontoxic quantity generally
selected from the range of 0.001% to 100% based on total weight of
the topical formulation, from 65 to 100% (preferably 75 to 96%) of
white soft paraffin, from 0 to 15% of liquid paraffin, and from 0
to 7% (preferably 3 to 7%) of lanolin or a derivative of synthetic
equivalent thereof. In another embodiment the ointment may comprise
a polyethylene--liquid paraffin matrix.
[0125] In one embodiment, a suitable cream is comprised of an
emulsifying system together with the desired concentration of the
mixture of Free-B-Ring flavonoids and flavans as provided above.
The emulsifying system is preferably comprised of from 2 to 10% of
polyoxyethylene alcohols (e.g. the mixture available under the
trademark Cetomacrogol.TM. 1000), from 10 to 25% of stearyl
alcohol, from 20 to 60% of liquid paraffin, and from 10 to 65% of
water; together with one or more preservatives, for example from
0.1 to 1% of N,N"-methylenebis[N'-[3-(hyd-
roxymethyl)-2,5-dioxo-4-imidazolidinyl]urea] (available under the
name Imidurea USNF), from 0.1 to 1% of alkyl 4-hydroxybenzoates
(for example the mixture available from Nipa Laboratories under the
trade mark Nipastat), from 0.01 to 0.1% of sodium butyl
4-hydroxybenzoate (available from Nipa Laboratories under the trade
mark Nipabutyl sodium), and from 0.1 to 2% of phenoxyethanol.
Example 13 describes the formulation of two different
concentrations of the composition of this invention as a cream and
Example 14 describes a study undertaken to evaluate the cream for
irritation and sensitization of the skin. From this study it was
determined that Soliprin.TM. is a safe composition that can be
applied topically at an efficacious concentration without causing
irritation or sensitization of the skin.
[0126] In one embodiment, a suitable gel is comprised of a
semi-solid system in which a liquid phase is constrained within a
three dimensional polymeric matrix with a high degree of
cross-linking. The liquid phase may be comprised of water, together
with the desired amount of the mixture of Free-B-Ring flavonoids
and flavans, from 0 to 20% of water-miscible additives, for example
glycerol, polyethylene glycol, or propylene glycol, and from 0.1 to
10%, preferably from 0.5 to 2%, of a thickening agent, which may be
a natural product, for example tragacanth, pectin, carrageen, agar
and alginic acid, or a synthetic or semi-synthetic compound, for
example methylcellulose and carboxypolymethylene (carbopol);
together with one or more preservatives, for example from 0.1 to 2%
of methyl 4-hydroxybenzoate (methyl paraben) or
phenoxyethanol-differential. Another suitable base, is comprised of
the desired amount of the mixture of Free-B-Ring flavonoids and
flavans, together with from 70 to 90% of polyethylene glycol (for
example, polyethylene glycol ointment containing 40% of
polyethylene glycol 3350 and 60% of polyethylene glycol 400,
prepared in accordance with the U.S. National Formulary (USNF)),
from 5 to 20% of water, from 0.02 to 0.25% of an anti-oxidant (for
example butylated hydroxytoluene), and from 0.005 to 0.1% of a
chelating agent (for example ethylenediamine tetraacetic acid
(EDTA)).
[0127] The term soft paraffin as used above encompasses the cream
or ointment bases white soft paraffin and yellow soft paraffin. The
term lanolin encompasses native wool fat and purified wool fat.
Derivatives of lanolin include in particular lanolins which have
been chemically modified in order to alter their physical or
chemical properties and synthetic equivalents of lanolin include in
particular synthetic or semisynthetic compounds and mixtures which
are known and used in the pharmaceutical and cosmetic arts as
alternatives to lanolin and may, for example, be referred to as
lanolin substitutes.
[0128] One suitable synthetic equivalent of lanolin that may be
used is the material available under the trademark Softisan.TM.
known as Softisan 649. Softisan 649, available from Dynamit Nobel
Aktiengesellschaft, is a glycerine ester of natural vegetable fatty
acids, of isostearic acid and of adipic acid; its properties are
discussed by H. Hermsdorf in Fette, Seifen, Anstrichmittel, Issue
No. 84, No.3 (1982), pp. 3-6.
[0129] The other substances mentioned hereinabove as constituents
of suitable ointment or cream bases and their properties are
discussed in standard reference works, for example pharmacopoeia.
Cetomacrogol 1000 has the formula
CH.sub.3(CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.nOH, wherein m may
be 15 or 17 and n may be 20 to 24. Butylated hydroxytoluene is
2,6-di-tert-butyl-p-cresol. Nipastat is a mixture of methyl, ethyl,
propyl and butyl 4-hydroxybenzoates.
[0130] The compositions of the invention may be produced by
conventional pharmaceutical techniques. Thus the aforementioned
compositions, for example, may conveniently be prepared by mixing
together at an elevated temperature, preferably 60-70.degree. C.,
the soft paraffin, liquid paraffin if present, and lanolin or
derivative or synthetic equivalent thereof. The mixture may then be
cooled to room temperature, and, after addition of the hydrated
crystalline calcium salt of mupirocin, together with the
corticosteroid and any other ingredients, stirred to ensure
adequate dispersion.
[0131] Regardless of the manner of administration, the specific
dose is calculated according to the approximate body weight of the
host. Further refinement of the calculations necessary to determine
the appropriate dosage for treatment involving each of the above
mentioned formulations is routinely made by those of ordinary skill
in the art and is within the scope of tasks routinely performed by
them without undue experimentation, especially in light of the
dosage information and assays disclosed herein. These dosages may
be ascertained through use of the established assays for
determining dosages utilized in conjunction with appropriate
dose-response data.
[0132] It should be noted that the invention described herein may
be used for veterinary as well as human applications and that the
term "host" should not be construed in a limiting manner. In the
case of veterinary applications, the dosage ranges can be
determined as described above, taking into account the body weight
of the animal.
[0133] The compositions of this invention can be administered by
any method known to one of ordinary skill in the art. The modes of
administration include, but are not limited to, enteral (oral)
administration, parenteral (intravenous, subcutaneous, and
intramuscular) administration and topical application. The method
of treatment according to this invention comprises administering
internally or topically to a patient in need thereof a
therapeutically effective amount of a mixture of Free-B-Ring
flavonoids and flavans synthesized and/or isolated from a single
plant or multiple plants. In a preferred embodiment the composition
is administered topically.
[0134] The following examples are provided for illustrative
purposes only and are not intended to limit the scope of the
invention.
EXAMPLES
Example 1
Preparation of Organic and Aqueous Extracts from Acacia and
Scutellaria Plants
[0135] Plant material from Acacia catechu (L) Willd. barks,
Scutellaria orthocalyx roots, Scutellaria baicalensis roots or
Scutellaria lateriflora whole plant was ground to a particle size
of no larger than 2 mm. Dried ground plant material (60 g) was then
transferred to an Erlenmeyer flask and methanol:dichloromethane
(1:1) (600 mL) was added. The mixture was shaken for one hour,
filtered and the biomass was extracted again with methanol:
dichloromethane (1:1) (600 mL). The organic extracts were combined
and evaporated under vacuum to provide the organic extract (see
Table 1 below). After organic extraction, the biomass was air dried
and extracted once with ultra pure water (600 mL). The aqueous
solution was filtered and freeze-dried to provide the aqueous
extract (see Table 1 below).
1TABLE 1 Yield of Organic and Aqueous Extracts of Acacia and
Scutellaria Species Plant Source Amount Organic Extract Aqueous
Extract Acacia catechu barks 60 g 27.2 g 10.8 g Scutellaria
orthocalyx roots 60 g 4.04 g 8.95 g Scutellaria baicalensis 60 g
9.18 g 7.18 g roots Scutellaria lateriflora 60 g 6.54 g 4.08 g
(whole plant)
Example 2
Inhibition of COX-2 and COX-1 Peroxidase Activity by Plant Extracts
from Acacia catechu, Various Scutellaria Species and Other
Plants
[0136] The bioassay directed screening process for the
identification of specific COX-2 inhibitors was designed to assay
the peroxidase activity of the enzyme as described below.
[0137] Peroxidase Assay. The assay to detect inhibitors of COX-2
was modified for a high throughput platform (Raz). Briefly,
recombinant ovine COX-2 (Cayman) in peroxidase buffer (100 mM TBS,
5 mM EDTA, 1 .mu.M Heme, 1 mg epinephrine, 0.094% phenol) was
incubated with extract (1:500 dilution) for 15 minutes. Quantablu
(Pierce) substrate was added and allowed to develop for 45 minutes
at 25.degree. C. Luminescence was then read using a Wallac Victor 2
plate reader. The results are presented in Table 2.
[0138] Table 2 sets forth the inhibition of enzyme by the organic
and aqueous extracts obtained from five plant species, including
the bark of Acacia catechu, roots of two Scutellaria species and
extracts from three other plant species, which are comprised of
structurally similar Free-B-Ring flavonoids. Data is presented as
the percent of peroxidase activity relative to the recombinant
ovine COX-2 enzyme and substrate alone. The percent inhibition by
the organic extract ranged from 30% to 90%.
2TABLE 2 Inhibition of COX-2 Peroxidase Activity by Various Species
Inhibition of COX-2 Inhibition of COX-2 Plant Source by organic
extract by aqueous extract Acacia catechu (bark) 75% 30%
Scutellaria orthocalyx (root) 55% 77% Scutellaria baicalensis
(root) 75% 0% Desmodium sambuense 55% 39% (whole plant) Eucaluptus
globulus (leaf) 30% 10% Murica nana (leaf) 90% 0%
[0139] Comparison of the relative inhibition of the COX-1 and COX-2
isoforms requires the generation of IC.sub.50 values for each of
these enzymes. The IC.sub.50 is defined as the concentration at
which 50% inhibition of enzyme activity in relation to the control
is achieved by a particular inhibitor. In these experiments,
IC.sub.50 values were found to range from 6 to 50 .mu.g/mL and 7 to
80 .mu.g/mL for the COX-2 and COX-1 enzymes, respectively, as set
forth in Table 3. Comparison of the IC.sub.50 values of COX-2 and
COX-1 demonstrates the specificity of the organic extracts from
various plants for each of these enzymes. The organic extract of
Scutellaria lateriflora for example, shows preferential inhibition
of COX-2 over COX-1 with IC.sub.50 values of 30 and 80 .mu.g/mL,
respectively. While some extracts demonstrate preferential
inhibition of COX-2, others do not. Examination of the HTP
fractions and purified compounds from these fractions is necessary
to determine the true specificity of inhibition for these extracts
and compounds.
3TABLE 3 IC.sub.50 Values of Organic Extracts for Human and Ovine
COX-2 and COX-1 IC.sub.50 Human IC.sub.50 Ovine IC.sub.50 Ovine
COX-2 COX-2 COX-1 Plant Source (.mu.g/mL) (.mu.g/mL) (.mu.g/mL)
Acacia catechu (bark) 3 6.25 2.5 Scutellaria orthocalyx (root) Not
done 10 10 Scutellaria baicalensis (root) 30 20 20 Scutellaria
lateriflora 20 30 80 (whole plant) Eucaluptus globulus (leaf) Not
done 50 50 Murica nana (leaf) 5 6 7
Example 3
Inhibition of COX-1 and COX-2 Peroxidase Activity
[0140] In order to screen for compounds that inhibited the COX-1
and COX-2 activities, a high throughput, in vitro assay was
developed that utilized the inhibition of the peroxidase activity
of both enzymes. (Needleman et al. (1986) Annu Rev Biochem. 55:69).
Briefly, the composition or compound being examined was titrated
against a fixed amount of COX-1 and COX-2 enzymes. A cleavable,
peroxide chromophore was included in the assay to visualize the
peroxidase activity of each enzyme in presence of arachidonic acid
as a cofactor. Typically, assays were performed in a 96-well
format. Each inhibitor, taken from a 10 mg/mL stock solution in
100% DMSO, was tested in triplicate at room temperature using the
following range of concentrations: 0, 0.1, 1, 5, 10, 20, 50, 100,
and 500 .mu.g/mL. To each well, 150 .mu.L of 100 mM Tris-HCl, pH
7.5 was added along with 10 .mu.L of 22 .mu.M Hematin diluted in
tris buffer, 10 .mu.L of inhibitor diluted in DMSO and 25 units of
either the COX-1 or COX-2 enzyme. The components were mixed for 10
seconds on a rotating platform, followed by the addition of 20
.mu.L of 2 mM N,N,N'N'-tetramethyl-p-pheny- lenediamine
dihydrochloride (TMPD) and 20 .mu.L of 1.1 mM arachidonic acid to
initiate the reaction. The plate was shaken for 10 seconds and then
incubated 5 minutes before reading the absorbance at 570 nm. The
inhibitor concentration vs. % inhibition was plotted and the
IC.sub.50 determined by taking the half-maximal point along the
isotherm and intersecting the concentration on the X-axis. The
IC.sub.50 was then normalized to the number of enzyme units in the
assay. The results are summarized in Table 4.
4TABLE 4 Inhibition of COX Enzyme Activity by Purified Free-B-Ring
Flavonoids Free-B-Ring Flavonoids Inhibition of COX-1 Inhibition of
COX-2 Baicalein 107% 109% 5,6-Dihydroxy-7- 75% 59% methoxyflavone
7,8-Dihydroxyflavone 74% 63% Baicalin 95% 97% Wogonin 16% 12%
[0141] The dose responses and IC.sub.50 values for a standardized
Free-B-Ring flavonoid extract, baicalin, and baicalein isolated
from the roots of Scutellaria baicalensis are provided in FIGS. 1,
2 and 3, respectively. The dose responses and IC.sub.50 values for
two standardized flavan extract (50% and >90% flavans,
respectively) isolated from the heartwood of Acacia catechu are
provided in FIGS. 4 and 5, respectively. The dose responses and
IC.sub.50 values for three formulations of Free-B-Ring flavonoids
and flavans of varying composition are provided in FIG. 6 (80:20
blending), FIG. 7 (50:50 blending) and FIG. 8 (20:80 blending),
respectively.
Example 4
Inhibition of 5-Lipoxygenase by Catechin isolated from Acacia
catechu
[0142] One of the most important pathways involved in the
inflammatory response is produced by non-heme, iron-containing
lipoxygenases (5-LO, 12-LO, and 15-LO), which catalyze the addition
of molecular oxygen onto fatty acids such as AA (AA) to produce the
hydroperoxides 5-, 12- and 15-HPETE, which are then converted to
leukotrienes. There were early indications that the flavan extract
from A. catechu may provide some degree of LOX inhibition, thereby
preventing the formation of 5-HPETE. A Lipoxygenase Inhibitor
Screening Assay Kit (Cayman Chemical, Inc., Cat #760700) was used
to assess whether an extract isolated from A. catechu containing
>90% flavans directly inhibited LOX in vitro. The 15-LO from
soybeans normally used in the kit was replaced with potato LOX,
after a buffer change from phosphate to a tris -based buffer using
microfiltration was performed. This assay detects the formation of
hydroperoxides through an oxygen sensing chromagen. Briefly, the
assay was performed in triplicate by adding 90 .mu.L of 0.17
units/.mu.L potato 5-LO, 20 .mu.L of 1.1 mM AA, 100 .mu.L of
oxygen-sensing chromagen and 10 .mu.L of purified flavan inhibitor
to final concentrations ranging from 0 to 500 .mu.g/mL. The
IC.sub.50 for 5-LO inhibition from this composition was determined
to be 1.38 .mu.g/mL/unit of enzyme. The results are set forth in
FIG. 9.
Example 5
HPLC Quantification of Free-B-Ring Flavonoids in Active Extracts
Isolated from Scutellaria orthocalyx (Roots) Scutellaria
baicalensis (Roots) and Oroxylum indicum (Seeds)
[0143] The presence and quantity of Free-B-Ring flavonoids in five
active extracts isolated from three different plant species as
described in Examples 1 and 2 were determined by HPLC and the
results are set forth in the Table 5, below. The Free-B-Ring
flavonoids were quantitatively analyzed by HPLC on a Luna C-18
column (250.times.4.5 mm, 5 .mu.m) using a 1% phosphoric acid and
acetonitrile gradient from 80% to 20% in 22 minutes. The
Free-B-Ring flavonoids were detected using a UV detector at 254 nm
and identified based on retention time by comparison with baicalin,
baicalein and other Free-B-Ring flavonoid standards.
5TABLE 5 Free-B-Ring Flavonoid Content in Active Plant Extracts %
Total % Extractible amount of Free-B-Ring Weight from Free-B-Ring
Flavonoids Active Extracts of Extract BioMass Flavonoids in Extract
S. orthocalyx 8.95 g 14.9% 0.2 mg 0.6% (aqueous extract) S.
orthocalyx 3.43 g 5.7% 1.95 mg 6.4% (organic extract) S.
baicalensis 7.18 g 12.0% 0.03 mg 0.07% (aqueous extract) S.
baicalensis 9.18 g 15.3% 20.3 mg 35.5% (organic extract) Oroxylum
indicum 6.58 g 11.0% 0.4 mg 2.2% (organic extract)
Example 6
HPLC Quantification of Active Extracts from Acacia catechu
[0144] The flavans in the organic and aqueous extracts isolated
from Acacia catechu as illustrated in Examples 1 and 2 were
quantified by HPLC using a PhotoDiode Array detector (HPLC/PDA) and
a Luna C18 column (250 mm.times.4.6 mm). The flavans were eluted
from the column using an acetonitrile gradient from 10% to 30% ACN
over a period of 20 minutes, followed by 60% ACN for five minutes.
The results are set forth in Table 6. The flavans were quantified
based on retention time and PDA data using catechin and epicatechin
as standards. The retention times for the two major flavans were
12.73 minutes and 15.76 minutes, respectively.
6TABLE 6 Free-B-Ring Flavonoid Content in Active Plant Extracts
Active Extracts from Weight of % Extractible % Flavans bark of A.
catechu Extract from BioMass in Extract Aqueous Extract 10.8 g
18.0% 0.998% Organic Extract 27.2 g 45.3% 30.37%
Example 7
Preparation of a Standardized Extract from Acacia catechu
[0145] Acacia catechu (500 mg of ground root) was extracted twice
with 25 mL (2.times.25 mL) of the following solvent systems. (1)
100% water, (2) 80:20 water:methanol, (3) 60:40 water:methanol, (4)
40:60 water:methanol, (5) 20:80 water:methanol, (6) 100% methanol,
(7) 80:20 methanol:THF, (8) 60:40 methanol:THF. The two extracts
from each individual extraction were combined concentrated and
dried under low vacuum. The identification of the chemical
components in each extract was achieved by HPLC using a PhotoDiode
Array detector (HPLC/PDA) and a 250 mm.times.4.6 mm C18 column. The
chemical components were quantified based on retention time and PDA
data using catechin and epicatechin as standards. The results are
set forth in Table 7. As shown in Table 7, the flavan extract
generated from solvent extraction with 80% methanol/water provided
the highest concentration of flavan components.
7TABLE 7 Solvents for Generating Standardized Flavan Extracts from
Acacia catechu Total Extraction Weight of % Extractible amount of %
Catechins Solvent Extract from BioMass Catechins in Extract 100%
water 292.8 mg 58.56% 13 mg 12.02% water:methanol 282.9 mg 56.58%
13 mg 11.19% (80:20) water:methanol 287.6 mg 57.52% 15 mg 13.54%
(60:40) water:methanol 264.8 mg 52.96% 19 mg 13.70% (40:60)
water:methanol 222.8 mg 44.56% 15 mg 14.83% (20:80) 100% methanol
215.0 mg 43.00% 15 mg 12.73% methanol:THF 264.4 mg 52.88% 11 mg
8.81% (80:20) methanol:THF 259.9 mg 51.98% 15 mg 9.05% (60:40)
[0146] Higher purity material can be obtained by recrystallization
of extracts having a catechin content of between 8%-15% using an
alcohol/water and/or aqueous solvents as the recrystallization
solvent. It may be necessary to decolorize prior to
recrystallization by adding active charcoal or other decolorization
agent to a heated saturated solution of the extract. The high
purity catechins then crystallized upon cooling of the heated
saturated solution. The crystals were then filtered to remove
solvent, dried and ground into a fine powder. Recrystallization can
be repeated as necessary to achieve a the desired level of purity
(60%-100% of catechin flavans).
Example 8
Preparation of Standardized Free-B-Ring Flavonoid Extracts from
Various Scutellaria species
[0147] Scutellaria orthocalyx (500 mg of ground root) was extracted
twice with 25 mL of the following solvent systems. (1) 100% water,
(2) 80:20 water:methanol, (3) 60:40 water:methanol, (4) 40:60
water:methanol, (5) 20:80 water:methanol, (6) 100% methanol, (7)
80:20 methanol:THF, (8) 60:40 methanol:THF. The extracts were
combined, concentrated and dried under low vacuum. Identification
of chemical components in each extract was performed by HPLC using
a PhotoDiode Array detector (HPLC/PDA) and a 250 mm.times.4.6 mm
C18 column. The chemical components were quantified based on
retention time and PDA data using baicalein, baicalin,
scutellarein, and wogonin as standards. The results are set forth
in Table 8.
8TABLE 8 Quantification of Free-B-Ring Flavonoids Extracted from
Scutellaria orthocalyx % Extractible Total % Extraction Weight of
from amount of Flavonoids in Solvent Extract BioMass Flavonoids
Extract 100% water 96 mg 19.2% 0.02 mg 0.20% Water:methanol 138.3
mg 27.7% 0.38 mg 0.38% (80:20) Water:methanol 169.5 mg 33.9% 0.78
mg 8.39% (60:40) Water:methanol 142.2 mg 28.4% 1.14 mg 11.26%
(40:60) Water:methanol 104.5 mg 20.9% 0.94 mg 7.99% (20:80) 100%
methanol 57.5 mg 11.5% 0.99 mg 10.42% methanol:THF 59.6 mg 11.9%
0.89 mg 8.76% (80:20) methanol:THF 58.8 mg 11.8% 1.10 mg 10.71%
(60:40)
[0148] Scutellaria baicalensis (1000 mg of ground root) was
extracted twice using 50 mL of a mixture of methanol and water as
follows: (1) 100% water, (2) 70:30 water:methanol, (3) 50:50
water:methanol, (4) 30:70 water:methanol, (5) 100% methanol. The
extracts were combined, concentrated and dried under low vacuum.
Identification of the chemical components was performed by HPLC
using a PhotoDiode Array detector (HPLC/PDA), and a 250
mm.times.4.6 mm C18 column. The chemical components in each extract
were quantified based on retention time and PDA data using
baicalein, baicalin, scutellarein, and wogonin standards. The
results are set forth in Table 9.
9TABLE 9 Quantification of Free-B-Ring Flavonoids Extracted from
Scutellaria baicalensis % Extractible Total % Extraction Weight of
from amount of Flavonoids in Solvent Extract BioMass Flavonoids
Extract 100% water 277.5 mg 27.8% 1 mg 0.09% Water:methanol 338.6
mg 33.9% 1.19 mg 11.48% (70:30) Water:methanol 304.3 mg 30.4% 1.99
mg 18.93% (50:50) Water:methanol 293.9 mg 29.4% 2.29 mg 19.61%
(30:70) 100% methanol 204.2 mg 20.4% 2.73 mg 24.51%
[0149] Higher purity Free-B-Ring flavonoids can be obtained by
recrystallization of extracts having a Free-B-Ring flavonoid
content of between 8-15% using alcohol/water as a recrystallization
solvent. It may be necessary to decolorize prior to
recrystallization by adding active charcoal or other decolorization
agent to a heated saturated solution of the extract. The
Free-B-Ring flavonoids crystallized upon cooling. The crystals were
filtered, dried and ground into a fine powder. Recrystallization
can be repeated as necessary to achieve a the desired level of
purity (60%-100% of Free-B-Ring flavonoids).
Example 9
Preparation of a Formulation with a Standardized Free-B-Ring
Flavonoid Extract from the Roots of Scutellaria baicalensis and a
Standardized Flavan Extract from the Bark of Acacia catechu
[0150] A novel composition of matter, referred to herein as
Soliprin.TM. was formulated using two standardized extracts
isolated from Acacia and Scutellaria, respectively, together with
one or more excipients. A general example for preparing such a
composition is set forth below. The Acacia extract used in this
example contained >80% total flavans, as catechin and
epicatechin, and the Scutellaria extract contained >80%
Free-B-Ring flavonoids, which was primarily baicalin. The
Scutellaria extract also contained other minor amounts of
Free-B-Ring flavonoids as set forth in Table 11. One or more
excipients/preservatives was also added to the composition of
matter. The ratio of flavans and Free-B-Ring flavonoids can be
adjusted based on the indications and the specific requirements
with respect to inhibition of COX vs. LO, requirements of skin
penetration, and potency requirements of the product, such as
duration of potency required, etc. The quantity of the excipients
can be adjusted based on the actual active content of each
ingredient. A blending table for each individual batch of product
must be generated based on the product specification and QC results
for individual batch of ingredients. Additional amounts of active
ingredients in the range of 2-5% are recommended to meet the
product specification.
[0151] Scutellaria baicalensis root extract (38.5 kg) (lot
#RM052302-01) having a Free-B-Ring flavonoid content of 82.2%
(baicalin); Acacia catechu bark extract (6.9 kg) (lot #RM052902-01)
with total flavan content of 80.4%; and excipient (5.0 kg of
Candex) were combined to provide a Soliprin.TM. formulation (50.4
kg) having a blending ratio of 85:15 by weight of the active
Free-B-Ring flavonoids and flavans. Table 10 provides the
quantification of the active Free-B-Ring flavonoids and flavans of
this specific batch of Soliprin.TM. (Lot #G1702-COX-2), determined
using the methods provided in Examples 6 and 8. With reference to
Table 10, this specific batch of Soliprin.TM. contains 86% total
active ingredients, including 75.7% Free-B-Ring flavonoids and
10.3% flavans. FIG. 10 illustrates the HPLC chromatogram of a
representative Soliprin.TM. sample which had a blending ratio of
80:20 by weight of the active Free-B-Ring flavonoids and
flavans.
10TABLE 10 Free-B-Ring Flavonoid and Flavan Content of a Soliprin
.TM. Formulation Active Components % Content 1. Flavonoids a.
Baicalin 62.5% b. Minor Flavonoids i. Wogonin-7-glucuronide 6.7%
ii. Oroxylin A 7-glucuronide 2.0% iii. Baicalein 1.5% iv. Wogonin
1.1% v. Chrysin-7-glucuronide 0.8% vi.
5-Methyl-wogonin-7-glucuronide 0.5% vii. Scutellarin 0.3% viii.
Norwogonin 0.3% ix. Chrysin <0.2% x. Oroxylin A <0.2% c.
Total Free-B-ring Flavonoids 75.7% 2. Flavans a. Catechin 9.9% b.
Epicatechin 0.4% c. Subtotal Flavans 10.3% 3. Total Active
Ingredients 86%
[0152] Using the same approach, the following batches of
Soliprin.TM. were prepared using a combination of a standardized
Free-B-Ring flavonoid extract from Scutellaria baicalensis roots
and a standardized flavan extract from Acacia catechu bark having a
blending ratio of 12:88 and 15:85, respectively.
[0153] Scutellaria baicalensis root extract (58.0 g) (lot
#RM021203-01) having a Free-B-Ring flavonoid content of 87.9% (as
baicalin) and Acacia catechu bark extract (442.0 g) (lot
#RM050603-01) with total flavan content of 84.9% were blended to
provide a Soliprin.TM. composition (500 g, lot #QJ205-19) having a
blending ratio of 12:88 by weight. Utilizing the methods provided
in Examples 6 and 8, the Free-B-Ring flavonoid content of
(baicalin) was 9.65% and flavan content (total catechin and
epicatechin) was 73.2% in this specific batch of Soliprin.TM. (lot
#QJ205-19).
[0154] Scutellaria baicalensis root extract (300 g) (lot
#RM060403-01) having a Free-B-Ring flavonoid content of 82.9% (as
baicalin) and Acacia catechu bark extract (1700 g) (lot
#RM050603-01) with total flavan content of 90.8% were blended to
provide a Soliprin.TM. composition (2000 g, lot #A1904) having a
blending ratio of 15:85 by weight. Utilizing the methods provided
in Examples 6 and 8, the Free-B-Ring flavonoid content (baicalin)
was 15.6% and flavan content (total catechin and epicatechin) was
75.0% in this specific batch of Soliprin.TM. (lot #A1904).
Example 10
Measurements of Dose Response and IC.sub.50 Values of 5-LO Enzyme
Inhibition from a Formulation of Soliprin.TM.
[0155] A Soliprin.TM. formulation (80:20) was prepared as described
in Example 9. (See also Example 14 of U.S. patent application Ser.
No. 10/427,746, filed Apr. 30, 2003, entitled "Formulation With
Dual COX-2 And 5-Lipoxygenase Inhibitory Activity," which is
incorporated herein by reference in its entirety) using a
combination of a standardized Free-B-Ring flavonoid extract from
Scutellaria baicalensis roots and a standardized flavan extract
from Acacia catechu bark with a blending ratio of 80:20. The sample
was titrated in tissue culture media containing THP-1 or HT-29
cells; monocyte cell lines that express COX-1, COX-2 and 5-LOX. A
competitive ELISA for Leukotriene B4 (LTB4; Neogen, Inc.,
Cat#406110) was used to assess the effect of this Soliprin.TM.
formulation on newly synthesized levels of LTB4 present in each
cell line as a measure Soliprin.TM.'s inhibitory effect on the
5-LOX pathway. The assay was performed in duplicate by adding
160,000 to 180,000 cells per well in 6-well plates. The
Soliprin.TM. formulation was added to the THP-1 cultures at 3, 10,
30 and 100 .mu.g/mL and incubated overnight (.about.12-15 hrs) at
37.degree. C. with 5% CO.sub.2 in a humidified environment. The
results are set forth in FIG. 11, which shows that the production
of newly LPS-induced LTB4 was almost completely inhibited by the
addition of Soliprin.TM. to the THP-1 cultures between 3 and 10
.mu.g/mL.
[0156] Soliprin.TM. and ibuprofen, another known 5-LOX inhibitor,
were added to the HT-29 cells at 3 .mu.g/mL and incubated 48 hrs at
37.degree. C. with 5% CO.sub.2 in a humidified environment. Each
treated cell line was then harvested by centrifugation and
disrupted by gentle dounce homogenization lysis in physiological
buffers. As shown in FIG. 12, Soliprin.TM. inhibited generation of
80% of the newly synthesized LTB4 in HT-29 cells. Ibuprofen only
showed a 20% reduction in the amount of LTB4 over the same time
period.
Example 11
Evaluation of the Efficacy of Soliprin.TM. with in vivo Mouse Ear
Swelling Model
[0157] A Soliprin.TM. formulation was prepared using a combination
of a standardized Free-B-Ring flavonoid extract from Scutellaria
baicalensis roots and a standardized flavan extract from Acacia
catechu bark with a blending ratio of 80:20 as described in Example
9. To test whether this composition could be used to treat
inflammation in vivo, the composition was administered by oral
gavage to 4-5 week old ICR mice (Harlan Labs) one day before
treatment of their ears with arachidonic acid (AA). Test mice were
fed dose equivalents of 50, 100 and 200 mg/kg of Soliprin.TM.
suspended in olive oil while control mice were fed only olive oil.
The following day, 20 .mu.L of 330 mM AA in 95% alcohol was applied
to one ear of each mouse, while alcohol was applied to the other
ear as a control. Mice treated with Soliprin.TM. showed a
measurable dose response that tracked with increasing doses of
Soliprin.TM., as demonstrated in FIG. 13. With reference to FIG.
13, the 200 mg/kg dose reduces swelling by over 50% as compared to
the "No treatment" control. The 50 mg/kg dose of Soliprin.TM. was
as effective as the 50 mg/kg dose of another strong
anti-inflammatory, indomethacin.
[0158] In another animal model designed to demonstrate the
anti-inflammatory activity of Soliprin.TM. the 80:20 formulation
described above was orally administrated to mice in a dose of 100
mg/kg suspended in olive oil .about.12 hours before injection of 20
.mu.L of 100 mM AA in 95% ethanol into the hind ankle joints of 4-5
week old ICR mice (Harlan Labs). The test group was fed the
Soliprin.TM. formulation, while another group was not given the
formulation. Control groups included mice that had not received
arachidonic acid injections (negative control) and a group that had
95% ethanol without AA injected (vehicle control). These groups
were also not given Soliprin.TM.. The results are set forth in FIG.
14. With reference to FIG. 14, the mice given Soliprin.TM. that
were injected with AA showed background levels of swelling as
compared to the controls and the untreated arachidonic injected
group. These results demonstrate the effectiveness of Soliprin.TM.
for reducing swelling in joints, the site of action.
Example 12
Evaluation of the Efficacy of Soliprin.TM. in Preventing and
Treating Damage Resulting from Exposure of Skin to UV Radiation
[0159] Six groups of hairless female mice (five mice per group)
(Strain SKH-1, Harlan Labs) were irradiated, while anesthetized,
for three minutes on three consecutive days with 0.626 mW/cm.sup.2
to test the effectiveness of the Soliprin.TM. formulation in
preventing and treating damage resulting from exposure of skin to
UV radiation. The Soliprin.TM. formulation was prepared using a
combination of a standardized Free-B-Ring flavonoid extract from
Scutellaria baicalensis roots and a standardized flavan extract
from Acacia catechu bark with a blending ratio of 80:20 as
described in Example 9. The six treatment groups were as
follows:
11 Group # 1 Control group: no treatment before or after UV
irradiation 2 Positive control: treated with a topical application
of Sooth-A-Caine (Banana Boat) after UV irradiation 3 Soliprin .TM.
Treatment B-1: treated with topical application of 1 mg/mL Soliprin
.TM. in water before UV irradiation 4 Soliprin .TM. Treatment A-1:
treated with topical application of 1 mg/mL Soliprin .TM. in water
after UV irradiation 5 Soliprin .TM. Treatment B-2: treated with
topical application of 5 mg/mL Soliprin .TM. in water before UV
irradiation 6 Soliprin .TM. Treatment A-2: treated with topical
application of 5 mg/mL Soliprin .TM. in water after UV
irradiation
[0160] After three days of UV exposure and treatment, the mice were
scored on level of erythema (redness) using the following scale:
0--no visible erythema; 1--very slight erythema; 2--well defined
erythema; 3--severe erythema; and 4--tumor formation. Erythema was
scored by eye for each group. The results are set forth in FIG. 15.
With reference to FIG. 15 it can be seen that the control group
(Group 1) had severe redness on day 3 (72 hours after the three day
exposure to UV radiation). The Sooth-a-caine group also had maximum
redness on day 3 (Group 2). The redness for the Soliprin.TM.
treated groups (Groups 3-6) never exceeded a score of 2. These
scores, though subjective, show that Soliprin.TM. is effective in
both preventing and treating UV caused skin erythema.
[0161] Photographs of representative mice on day four clearly
demonstrate differences between the control group, the
Sooth-a-cain.TM. treated groups and the Soliprin.TM. treated groups
(data not shown). The control group and Sooth-a-cain.TM. treated
animals exhibited very extensive patterns and redness of erythema
compared to the animals treated with the Soliprin.TM. formulation
both before and after UV exposure. The animals treated before UV
irradiation with 5 mg/mL Soliprin.TM. exhibited the least amount of
erythema as compared to all of the other animals.
Example 13
Formulation of the Soliprin.TM. Composition into a Cream
[0162] Two different concentrations of Soliprin.TM. (0.5% and 1.5%
by weight of Soliprin.TM.) (lot #A1904 as described in Example 9)
were formulated as creams as illustrated in the following
procedures and in Tables 11 and 12.
[0163] Soliprin.TM. (Lot #A1904) was dissolved in water at room
temperature and homogenized with a blender until it was fully
dispersed in solution (approximately 5 minutes). At room
temperature and without stirring or agitating the solution,
Ultrez-21 carbomer was added by sprinkling onto the surface of the
solution and allowing it to fully wet (no white areas visible) and
fall into the solution. With gentle stirring, the solution was then
heated to 40.degree. C. and glycerin was added (Part A). The
mixture was then stirred for an additional 5 minutes. The remaining
components (Part B) were weighed and heated to 40.degree. C. while
mixing. At 40.degree. C., the remaining components (Part B) were
added to Part A and the resulting composition was mixed well until
homogenous (approximately 5 minutes). The emulsion was cooled to
30.degree. C. and the pH was adjusted to approximately 5.5 (5.3 to
5.7) by titrating with neutralizer while stirring with a stir bar
and/or spatula. The emulsion became highly viscous due to
neutralization-induced conformational change of the carbomer. The
emulsion eventually achieved a suitable viscosity for an emulsion
cream. The emulsion cream was then mixed until uniform after which
it was poured into a clean storage vessel and stored at 2.degree.
to 8.degree. C. for one month.
12TABLE 11 Ingredient list for a 0.5% Soliprin Cream Phase
Ingredient % (w/w) Weight (g) Aqueous Water, Purified 85.00 1275.0
Soliprin (Lot#A1904) 0.50 7.5 Ultrez 21 Carbomer 0.50 7.5 Glycerin
8.00 120.0 Oil PEG-7 Glyceryl Cocoate 3.00 45.0 Caprylic/Capric
Triglyceride 2.67 40.0 PH Sodium Hydroxide (18% w/v), 0.00 0.0
Neutralizer Molecular Biology Grade SUM 7 Ingredients 99.7
1495.0
[0164]
13TABLE 12 Ingredient list in a 1.5% Soliprin Cream Phase
Ingredient % (w/w) Weight (g) Aqueous Water, Purified 84.00 1260.0
Soliprin (Lot#A1904) 1.50 22.5 Ultrez 21 Carbomer 0.50 7.5 Glycerin
8.00 120.0 Oil PEG-7 Glyceryl Cocoate 3.00 45.0 Caprylic/Capric
Triglyceride 2.67 40.0 pH Sodium Hydroxide (18% w/v), Neutralizer
Molecular Biology Grade SUM 7 Ingredients 99.7 1495.0
Example 14
Evaluation of a Soliprin.TM. Cream for Irritation and Induction of
Contact Sensitization by Repetitive Application to Human Skin
[0165] The Soliprin.TM. was tested on human skin using an
adaptation of the Draize Patch Test (Marzulli and Maibach (1977)
Contact Allergy: Predictive Testing in Humans. In Advances in Modem
Toxicology, Dermatotoxicology and Pharmacology. Eds. Marzulli, F. N
and Maibach, H. I. 4, 353-372). The test sites were located on the
upper arm or the paraspinal region of the back. Each test article
had an induction site and a challenge site. The induction site was
comprised of two sub-sites: an original-site and a move-site.
Patches, which contains 0.2 ml of Soliprin cream on each patch,
were applied repeatedly to the original-site unless a sufficiently
strong irritation reaction developed, requiring the patch to be
applied to the move-site. Patches were applied by a clinical
research institute and were removed and discarded by the subjects
approximately 24 or 48/72 hours later. In the induction phase,
repetitive application of the test article to the same site on the
skin and a total of 9 induction patches were applied within a
4-week period. The rest period was 10 to 21 days between
application of the last induction patch and application of the
challenge patch. During this time no test article or any other
material was applied to the test area. At the challenge phase, the
test article was applied to a naive site on the opposite side of
the body and discarded by the subjects approximately 24 or 48 hours
later.
[0166] Skin responses to each patch application were examined and
graded under light supplied by a 100-watt incandescent blue bulb
according to the designated scoring scale. In instances where a
strong irritation reaction warranted application of the test
article to the move-site, residual scores were be recorded through
the end of induction (or until resolved if reactions persist after
induction is completed) for all previously exposed sites. All skin
reactions were recorded. During the challenge phase, skin responses
were evaluated approximately 48 and 72 or 96 hours after patch
application. Conclusions, with regard to induced sensitivity, were
derived primarily from the challenge evaluations.
[0167] The two Soliprin.TM. creams prepared in the Example 13 at
0.5% and 1.5% Soliprin.TM. concentrations were evaluated according
to the above protocol. A total of 120 subjects were recruited for
each group. Ninety-seven subjects completed the study for the 0.5%
Soliprin.TM. group and 101 subjects completed the study for 1.5%
Soliprin.TM. group. There was no evidence of sensitization reaction
for either the 0.5% and 1.5% Soliprin.TM. creams. For the 0.5%
Soliprin.TM., during induction, sixteen subjects exhibited
occasional occurrences of slight to mild erythema (scores of +
and/or 1). At challenge, four subjects exhibited slight to mild
erythema at 48 hours that cleared by 96 hours. For 1.5%
Soliprin.TM., during induction, twenty-six subjects exhibited
occasional occurrences of slight to mild erythema (scores of +
and/or 1). At challenge, one subjects exhibited slight to mild
erythema at 48 hours that cleared by 96 hours.
[0168] This study demonstrates that Soliprin.TM. is a safe
ingredient that can be applied topically to human skin at an
efficacious concentration without causing irritation or
sensitization.
* * * * *