AU2017237651A1 - Therapeutic compositions and uses thereof - Google Patents

Therapeutic compositions and uses thereof Download PDF

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AU2017237651A1
AU2017237651A1 AU2017237651A AU2017237651A AU2017237651A1 AU 2017237651 A1 AU2017237651 A1 AU 2017237651A1 AU 2017237651 A AU2017237651 A AU 2017237651A AU 2017237651 A AU2017237651 A AU 2017237651A AU 2017237651 A1 AU2017237651 A1 AU 2017237651A1
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dihydroxy
glycerol
propolis
diacetoxy
epithelial
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Stephen John BLOOR
Owen John Catchpole
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Manuka Health New Zealand Ltd
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Manuka Health New Zealand Ltd
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    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/20—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids
    • A61K31/202—Carboxylic acids, e.g. valproic acid having a carboxyl group bound to a chain of seven or more carbon atoms, e.g. stearic, palmitic, arachidic acids having three or more double bonds, e.g. linolenic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/23—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms
    • A61K31/232—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of acids having a carboxyl group bound to a chain of seven or more carbon atoms having three or more double bonds, e.g. etretinate
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

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Abstract

This invention provides pharmaceutical and cosmeceutical compositions, including anti-epithelial cancer compositions, containing glyceride compounds, and compositions comprising one or more of these compounds. Methods of preparing and using such compositions, in particular in the treatment or prevention of epithelial cancers, such as skin cancers, gastrointestinal cancers, and skin disorders, are also provided.

Description

invention provides pharmaceutical and cosmeceutical compositions, including anti-epithelial cancer composi tions, containing glyceride compounds, and compositions comprising one or more of these compounds. Methods of preparing and using such compositions, in particular in the treatment or prevention of epithelial cancers, such as skin cancers, gastrointestinal can cers, and skin disorders, are also provided.
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THERAPEUTIC COMPOSITIONS AND USES THEREOF
FIELD OF INVENTION
This invention relates to compositions for use in skin and gut care and in the treatment and prevention of epithelial cancers, including skin cancers and gastrointestinal cancers. In particular, this invention relates to anti-epithelial cancer compositions comprising one or more glycerides containing dihydroxy fatty acids. Particularly contemplated are skin and gut care and anti-epithelial cancer compositions comprising one or more dihydroxy fatty acid compounds, and the use of such compositions in the treatment or prevention of skin disorders, disorders of the alimentary canal, disorders of mucosal surfaces, and epithelial cancers, including gastrointestinal cancers, such as colorectal, throat, buccal, and gastric cancers, and skin cancers, such as basal cell carcinomas, squamous cell carcinomas, and melanomas.
BACKGROUND OF THE INVENTION
Epithelial cancers include some of the most prevalent cancers in the world. Colorectal cancer is reportedly the second and third most common cancer in women and men, respectively, from developed countries. Colorectal cancer is more prevalent in developed countries - the US, Australia, Europe, and New Zealand having the highest rates - with incidence being as much as 10 times greater than in developing countries. While surgery can be effective, early detection is critical to positive surgical outcomes. Other therapies are largely directed at life extension and palliative care, as the efficacy of current chemotherapies and radiotherapies in treating primary tumours, or metastases outside the lymph nodes is debated.
Throat cancer, also referred to as esophageal cancer, pharyngeal cancer, or laryngeal cancer, encompasses tumours that develop in the tissues of the pharynx, nasopharynx, oropharynx, hypopharynx, larynx (voice box) or tonsils. Therapies for throat cancer include
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Gastric or stomach cancer is the second most common cause of cancer-related death in the world. Diagnosis is often delayed because symptoms may not occur in the early stages of the disease. Surgery to remove the stomach (gastrectomy) is the only treatment that can cure gastric cancers. Chemotherapy and radiation therapy after surgery may improve the chance of a cure.
Skin cancers are also highly prevalent, with more than 3 million diagnosed cases annually in the USA alone. Skin cancer rates are particularly high in Australia and New
Zealand, with incidence being as much as 4 times greater than in the USA. While surgery can be effective, early detection is critical to positive surgical outcomes. Other therapies are largely directed at low risk disease, as the efficacy of current chemotherapies and radiotherapies in treating primary tumours, particularly of malignant melanomas, or metastases outside the lymph nodes, is debated.
Accordingly, there is a need for anti-epithelial cancer compositions, including those suitable for use in the treatment or prevention of skin cancers and/or gastrointestinal cancers, and those which are able to support the maintenance of anti-epithelial cancer activity or augment anti-epithelial cancer activity.
It is an object of the present invention to provide anti-epithelial cancer compositions for use in the treatment or prevention of epithelial cancers, such as skin cancers, including basal cell carcinoma, squamous cell carcinoma, and melanoma, and/or gastrointestinal cancers, such as colorectal, throat, buccal, and gastric cancers, or to at least provide the public with a useful choice.
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SUMMARY OF THE INVENTION
Accordingly, in a first aspect the invention relates to a method of treating or preventing an epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I):
Figure AU2017237651A1_D0001
O OR4 OR5
R-i=OH, OR7or
Figure AU2017237651A1_D0002
OR3 or2 (i) or a pharmaceutically acceptable salt or solvate thereof, wherein R2, R3, R4 and Rs are each independently H or acetyl (CH3CO-),
R6 is H or CH3, R7 is CH3 or C2 to C5 saturated or unsaturated hydrocarbon, and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17.In one embodiment, the invention relates to a method of treating or preventing an epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
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i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In one embodiment, the method comprises administering a composition comprising, consisting essentially or, or consisting of an effective amount of at least one compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
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l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In one embodiment, the invention relates to a method of treating or preventing a skin cancer in a subject, the method comprising administering an effective amount of a composition comprising, consisting essentially or, or consisting of at least one compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
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n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In another aspect the invention relates to a method of inhibiting epithelial tumour formation, inhibiting epithelial tumour growth, or inhibiting epithelial tumour metastasis in a subject, the method comprising administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
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p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In one embodiment the invention relates to a method of inhibiting skin tumour formation, inhibiting skin tumour growth, or inhibiting skin tumour metastasis in a subject, the method comprising administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
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r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
Another aspect relates to a method of inducing apoptosis of one or more neoplastic epithelial cells in a subject, the method comprising administration of an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
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t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In one embodiment, the method is a method of inducing apoptosis of one or more neoplastic skin cells in a subject, the method comprising administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u),
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In one embodiment, the apoptosis is of basal carcinoma cells. In another embodiment the apoptosis is of squamous carcinoma cells. In a further embodiment the apoptosis is of melanoma cells.
In another embodiment, the method is a method of inducing apoptosis of one or more neoplastic gastrointestinal cancer cells in a subject.
For example, the apoptosis is of colorectal cancer cells, throat cancer cells, buccal cancer cells, or gastric cancer cells.
Another aspect relates to a method of modulating proliferation of one or more neoplastic epithelial cells in a subject, the method comprising administration of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
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s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
In one embodiment, the invention relates to a method of modulating proliferation of one or more neoplastic skin cells in a subject.
For example in one embodiment the modulation is reduction. Accordingly the invention relates to a method of reducing proliferation of one or more neoplastic skin cells in a subject, the method comprising administration of an effective amount of a compound selected from the group consisting of 3,8-dihydroxy eicosanoic acid, 1-(3,8-dihydroxy eicosanoyl) glycerol, 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol, 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol, 1-(3,8diacetoxy eicosanoyl) 2,3-diacetoxy glycerol, 3,8-dihydroxy eicosanoic acid methyl ester,
3,8-dihydroxy heneicosanoic acid, 1-(3,8-dihydroxy heneicosanoyl) glycerol, 1-(3,8dihydroxy heneicosanoyl) 2-acetoxy glycerol, 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol, 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol, 3,8-dihydroxy heneicosanoic acid methyl ester, 3,8dihydroxy docosanoic acid, 1-(3,8-dihydroxy docosanoyl) glycerol, 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol, 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol, 1-(3,8dihydroxy docosanoyl) 2,3-diacetoxy glycerol, 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and 3,8-dihydroxy docosanoic acid methyl ester, to a subject in need thereof.
In one embodiment, the proliferation is of basal carcinoma cells. In another embodiment the proliferation is of squamous carcinoma cells. In a further embodiment the proliferation is of melanoma cells.
In one embodiment, the method is a method of modulating proliferation of one or more neoplastic gastrointestinal cancer cells in a subject. For example, the proliferation is of colorectal cancer cells, of throat cancer cells, of buccal cancer cells, or of gastric cancer cells.
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Accordingly the invention relates to a method of reducing proliferation of one or more neoplastic gastrointestinal cancer cells in a subject, the method comprising administration of an effective amount of a compound selected from the group consisting of 3,8-dihydroxy eicosanoic acid, 1-(3,8-dihydroxy eicosanoyl) glycerol, 1-(3,8-dihydroxy eicosanoyl) 2acetoxy glycerol, 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol, 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol, 3,8dihydroxy eicosanoic acid methyl ester, 3,8-dihydroxy heneicosanoic acid, 1-(3,8-dihydroxy heneicosanoyl) glycerol, 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol, 1-(3,8dihydroxy heneicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy heneicosanoyl) 2,3diacetoxy glycerol, 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol, 3,8-dihydroxy heneicosanoic acid methyl ester, 3,8-dihydroxy docosanoic acid, 1-(3,8-dihydroxy docosanoyl) glycerol, 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol, 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol, 1(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and 3,8-dihydroxy docosanoic acid methyl ester, to a subject in need thereof.
Another aspect relates to a method of inducing apoptosis of one or more neoplastic epithelial cells, or of modulating proliferation of one or more neoplastic epithelial cells, for example of one or more neoplastic epithelial cells in vitro or ex vivo, the method comprising contacting the one or more cells with an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
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j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In a further aspect, the invention relates to a composition for use in the treatment or prevention of an epithelial cancer in a subject, the composition comprising, consisting essentially of, or consisting of, a therapeutically effective amount of a compound of formula (I):
Figure AU2017237651A1_D0003
R-i=OH, OR7or
Figure AU2017237651A1_D0004
OR3 or2 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R2, R3, R4 and Rs are each independently H or acetyl (CH3CO-),
R6 is H or CH3, R7 is CH3 or C2 to C6 saturated or unsaturated hydrocarbon and x and y are each independently an integer from 3 to 14,
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PCT/NZ2017/050031 provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17.
In one embodiment, x = 4, R5 = H and y = 9, 10, 11, 12, or 13.
For example, x = 4, R4, Rs, and R6 = H and y = 10, 11 or 12.
Accordingly, in another aspect the invention relates to an anti-epithelial cancer composition comprising, consisting essentially of, or consisting of at least one compound as defined herein, including a compound of formula (I) ora pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester 0) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u),
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In another aspect, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of at least one compound as defined herein, including a compound of formula (I) ora pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u).
In one embodiment, the composition is for use in the treatment or prevention of an epithelial cancer.
In one embodiment, the composition is for maintaining or improving skin health.
In one embodiment, the composition is for maintaining or improving gut health.
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In another aspect the invention relates to a method of inhibiting epithelial tumour formation, preferably skin tumour formation, inhibiting epithelial tumour growth, preferably skin tumour growth, inhibiting epithelial tumour metastasis, preferably skin tumour metastasis or treating or preventing epithelial cancer, preferably skin cancer, in a subject, the method comprising separate, simultaneous or sequential administration of an effective amount of a composition comprising, consisting essentially of, or consisting of a therapeutically effective amount of at least one compound as defined herein, including a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
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u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
Another aspect relates to a method of increasing the responsiveness of a subject to an epithelial cancer therapy comprising administration to the subject of an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
In one embodiment the epithelial cancer therapy is a gastrointestinal cancer therapy. For example, the gastrointestinal cancer therapy is a colorectal cancer therapy, a throat cancer therapy, a buccal cancer therapy, or a gastric cancer therapy.
In one embodiment, the invention relates to a method of increasing the responsiveness of a subject to a skin cancer therapy comprising administration to the subject of a composition as described herein.
In one embodiment the skin cancer therapy is basal cell carcinoma therapy. In another embodiment the skin cancer therapy is squamous cell carcinoma therapy. In a further embodiment the skin cancer therapy is melanoma therapy.
Another aspect relates to a method of increasing the sensitivity of an epithelial tumour in a subject to an epithelial cancer therapy comprising administration to the subject of an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
Another aspect relates to method of resensitising one or more epithelial cancer cells that are resistant to treatment, the method comprising administering an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I), or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof.
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In one embodiment the epithelial tumour is a gastrointestinal tumour. For example, the the gastrointestinal tumour is a colorectal tumour, a throat tumour, a buccal tumour, or a gastric tumour.
In one embodiment, the invention relates to a method of increasing the sensitivity of a skin tumour in a subject to a cancer therapy comprising administration to the subject of a composition as described herein.
In one embodiment the skin tumour is a basal cell carcinoma. In another embodiment the skin tumour is a squamous cell carcinoma. In a further embodiment the skin tumour is a melanoma.
In a further aspect, the invention relates to a method of resensitising to treatment one or more epithelial cancer cells that are resistant to treatment, the method comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein to the one or more epithelial cancer cells.
In one embodiment, the epithelial cancer cells comprise a tumour present in a subject.
In one embodiment, the invention relates to a method of resensitising one or more epithelial cancer cells, preferably skin cancer cells that are resistant to treatment, the method comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein to the one or more epithelial cancer cells, preferably skin cancer cells.
In one embodiment, the skin cancer cells comprise a tumour present in a subject.
The invention also relates to a method of at least partially reversing the resistance of a neoplastic cell in a subject suffering from an epithelial cancer to an epithelial cancer therapy, the method comprising administration to the subject of an effective amount of a a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
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In one embodiment, the method is a method of at least partially reversing the resistance of a neoplastic cell in a subject suffering from skin cancer to a cancer therapy, the method comprising administration to the subject of an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
The present invention further relates to a method of reversing, wholly or in part, the resistance of an epithelial cancer-burdened, preferably a skin cancer-burdened patient to an epithelial cancer therapy, preferably a skin cancer therapy, the method comprising the step of administering to said patient an effective amount of a a composition comprising a a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof, or of a composition as described herein.
In another aspect, the invention provides a method of re-sensitising one or more tumours of an epithelial cancer-burdened patient which are, or are predicted to either be or become, resistant to treatment with an epithelial cancer therapy to treatment with an epithelial cancer therapy, said method comprising the step of administering to said patient an effective amount of a composition comprising a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a therapeutically effective amount of at least one compound selected from any one or more of compounds a) to u) described herein or a pharmaceutically acceptable salt or solvate thereof, of a composition as described herein.
In one embodiment, the method is a method of re-sensitising one or more tumours of a skin cancer-burdened patient which are, or are predicted to either be or become, resistant to treatment with a skin cancer therapy to treatment with a skin cancer therapy, said method comprising the step of administering to said patient an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
In one embodiment, the tumours are resistant to treatment with a chemotherapeutic.
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In one embodiment the subject is a human.In still a further aspect, the present invention relates to a method of improving skin health or gut health, the method comprising administering to a subject in need thereof an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof or of a composition as described herein.
In one embodiment, the composition is a synergistic therapeutic composition. In one embodiment, the composition provides a synergistic therapeutic effect.
In one embodiment the composition comprises a compound described herein and at least one additional therapeutic agent that provide a synergistic therapeutic effect that is greater than the effect of either one alone or greater than the additive effects of either one alone. For example, there is a greater effect on induction of apoptosis, on epithelial cancer cell survival or proliferation such as skin cancer cell survival or proliferation, on resensitisation to therapy, on treatment or prevention of epithelial cancer, such as skin cancer, or the responsiveness of a subject or a tumour to the treatment method. Without wishing to be bound by theory the inventors believe that this enhanced effect may be due to improved bioavailability of the composition, for example through improved water dispersibility. In one embodiment, the compound and the at least one additional therapeutic agent allow the administration of a co-administered or sequentially administered epithelial cancer therapy, such as a skin cancer therapy, to be reduced or increased in dose or in length of administration, as appropriate.
In one embodiment the composition, for example a synergistic therapeutic composition, comprises at least one additional compound or extract derived from poplarderived propolis. For example, the composition additionally comprises at least one compound selected from the group comprising pinocembrin, CAPE, chrysin, pinostrobin chalcone, galangin, benzyl caffeate, benzyl ferulate or caffeic acid.
Another aspect relates to use of at least one compound as defined herein, including a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
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c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), in the manufacture of a medicament or composition for a purpose as herein described.
In one embodiment, the use is use together with at least one additional therapeutic agent in the manufacture of a medicament or composition for a purpose as herein described.
Another aspect relates to use of a composition comprising at least one compound as defined herein, including a compound of formula (I) ora pharmaceutically acceptable salt or solvate thereof, or a compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
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d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), with at least one additional therapeutic agent in the manufacture of a medicament or composition for a purpose as herein described, wherein the medicament or composition is formulated to provide separate, simultaneous or sequential administration of the at least one compound and the at least one additional therapeutic agent.
In one embodiment the medicament or composition comprises cyclodextrin. In one embodiment, the medicament or composition comprises one or more dihydroxy fatty acid compounds as described herein, such as a compound of formula (I), complexed with cyclodextrin.
In another embodiment the compound is provided dissolved in a solvent or mixture of solvents, for example ethanol, ethanol/water, propanol, propanol/water, isopropanol, isopropanol/water, ethyl acetate, hydrocarbon solvents, edible oils or propylene glycol.
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In one embodiment, the medicament or composition is one to which has been added at least one compound selected from any one or more of the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u).
Another aspect relates to a composition for use in inhibiting epithelial tumour formation, inhibiting epithelial tumour growth, inhibiting epithelial tumour metastasis or treating or preventing epithelial cancer in a human subject; inducing apoptosis of one or more neoplastic epithelial cells in a human subject; increasing the responsiveness of a human subject to an epithelial cancer therapy; increasing the sensitivity of an epithelial tumour in a human subject to an epithelial cancer therapy; resensitising one or more
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PCT/NZ2017/050031 epithelial cancer cells in a human subject that are resistant to treatment; at least partially reversing the resistance of a neoplastic cell in a human subject suffering from epithelial cancer to an epithelial cancer therapy; reversing, wholly or in part, the resistance of an epithelial cancer-burdened human patient to an epithelial cancer therapy; or re-sensitising one or more tumours of an epithelial cancer-burdened human patient which are, or are predicted to either be or become, resistant to treatment with an epithelial cancer therapy to treatment with an epithelial cancer therapy.
In one embodiment the composition is for use in inhibiting skin tumour formation, inhibiting skin tumour growth, inhibiting skin tumour metastasis or treating or preventing skin cancer in a human subject; inducing apoptosis of one or more neoplastic skin cells in a human subject; increasing the responsiveness of a human subject to a skin cancer therapy; increasing the sensitivity of a skin tumour in a human subject to a skin cancer therapy; resensitising one or more skin cancer cells in a human subject that are resistant to treatment; at least partially reversing the resistance of a neoplastic cell in a human subject suffering from skin cancer to a skin cancer therapy; reversing, wholly or in part, the resistance of a skin cancer-burdened human patient to a skin cancer therapy; or resensitising one or more tumours of a skin cancer-burdened human patient which are, or are predicted to either be or become, resistant to treatment with a skin cancer therapy to treatment with a skin cancer therapy.
In one example, the invention relates to a composition for use in the treatment or prevention of skin cancer.
In one embodiment, the composition additionally comprises cyclodextrin.
In one embodiment, the composition additionally comprises propolis, for example, poplar-derived propolis.
In one embodiment, the composition additionally comprises an extract derived from poplar, such as a poplar leaf or bud exudate as described herein in the Examples.
In various embodiments, the composition comprises propolis, for example a poplarderived propolis, including a propolis extract or fraction, propolis resin, propolis resin extract and/or an extract derived from poplar, wherein the propolis, propolis resin, propolis resin
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PCT/NZ2017/050031 extract and/or extract from poplar is enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the composition additionally comprises propolis, propolis resin, or propolis resin extract and cyclodextrin.
In one embodiment, the cyclodextrin is alpha-cyclodextrin.
In one embodiment, the cyclodextrin is beta-cyclodextrin.
In one embodiment, the cyclodextrin is gamma-cyclodextrin.
In one embodiment, the cyclodextrin is hydroxypropyl beta-cyclodextrin.
In one embodiment, the cyclodextrin is hydroxypropyl gamma-cyclodextrin.
In one embodiment, the cyclodextrin is a mixture of cyclodextrins.
In one embodiment the anti-skin cancer composition is an anti-basal cell carcinoma composition. In another embodiment the anti-skin cancer composition is an anti-squamous cell carcinoma composition. In a further embodiment the anti-skin cancer composition is an anti-melanoma composition.
In another aspect, the present invention relates to a pharmaceutical composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
In one embodiment, the composition comprises propolis, propolis resin, or propolis resin extract and one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, for example, the composition comprises propolis, propolis resin, or propolis resin extract to which has been added one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In another embodiment, the composition comprises a fraction from propolis and/or poplar bud or leaf exudate, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof.
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In one embodiment, the propolis is poplar-derived propolis.
In one embodiment, the composition additionally comprises cyclodextrin.
In one embodiment, the composition is for maintaining or improving skin health.
Accordingly, in one embodiment the invention relates to a pharmaceutical composition for maintaining or improving skin health, the composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin.
In one embodiment the propolis enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof is propolis resin, for example poplar-derived propolis resin. In one embodiment the resin is propolis resin, for example, poplar-derived propolis resin and the cyclodextrin is alpha, beta or gammacyclodextrin.
In another aspect the invention relates to a method of treating or preventing a skin cancer in a subject, a method of inhibiting skin tumour formation, inhibiting skin tumour growth, or inhibiting skin tumour metastasis, a method of inducing apoptosis of one or more neoplastic skin cells in a subject, a method of modulating proliferation of one or more neoplastic skin cells in a subject, a method of increasing the responsiveness of a subject to a skin cancer therapy, a method of increasing the sensitivity of a skin tumour in a subject to a skin cancer therapy, a method of resensitising one or more skin cancer cells that are resistant to treatment, the method comprising administering an effective amount of a composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin, to a subject in need thereof or to the one or more cells.
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In one embodiment the apoptosis is of skin tumour cells, such as basal carcinoma, squamous carcinoma, or melanoma cells.
In one embodiment the modulation is reduction. Accordingly the invention relates to a method of reducing proliferation of one or more neoplastic skin cells in a subject, the method comprising administration of an effective amount of a composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin, to a subject in need thereof.
In one embodiment the proliferation is of skin tumour cells, such as basal carcinoma, squamous carcinoma, or melanoma cells.
In one embodiment, the skin cancer cells comprise a tumour present in a subject. In one embodiment the skin cancer cells are basal carcinoma cells. In another embodiment the skin cancer cells are squamous carcinoma cells. In a further embodiment the skin cancer cells are melanoma cells.
The invention also relates to a method of at least partially reversing the resistance of a neoplastic cell in a subject suffering from a skin cancer to a skin cancer therapy, the method comprising administration to the subject of a composition comprising, consisting essentially or, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin.
The present invention further relates to a method of reversing, wholly or in part, the resistance of a skin cancer-burdened patient to a skin cancer therapy, the method comprising the step of administering to said patient a composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin.
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In another aspect, the invention provides a method of re-sensitising one or more tumours of a skin cancer-burdened patient which are, or are predicted to either be or become, resistant to treatment with a skin cancer therapy, said method comprising the step of administering to said patient a composition comprising, consisting essentially or, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin.
In one embodiment, the one or more tumours are or are predicted to be or to become resistant to a skin cancer therapy due to increased activation of one or more pro-cancer cell survival signaling pathways within the one or more tumours or within the patient, including increased activation of one or more of the AKT, JNK or JAK/STAT signaling pathways, for example within a sample from the patient, such as a tissue sample, a tumour biopsy, or a blood or plasma sample.
In one embodiment, the invention provides a method of inactivating or suppressing one or more pro-cancer cell survival signaling pathways within the one or more tumours or within the patient. For example, the invention relates to a method of inactivating or suppressing one or more of the AKT, JNK or JAK/STAT signaling pathways within the one or more tumours.
In one embodiment, the one or more tumours are or are predicted to be or to become resistant to a skin cancer therapy due to increased activation of one or more of the AKT,
JNK or JAK/STAT signaling pathways within the tumour(s).
In one embodiment, the invention provides a method of preventing tumours becoming resistant to a primary skin cancer therapy, wherein the resistance is at least in part mediated by increased activation of one or more of the AKT, JNK or JAK/STAT signaling pathways, for example within the tumour(s).
In one embodiment, the tumours are resistant to treatment with a chemotherapeutic.
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In one embodiment the skin cancer is basal cell carcinoma. In another embodiment the skin cancer is squamous cell carcinoma. In a further embodiment the skin cancer is melanoma.
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and alphacyclodextrin, for example poplar-derived propolis, propolis resin, or propolis resin extract and cyclodextrin, such as poplar-derived propolis and alpha-cyclodextrin.
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and betacyclodextrin, for example poplar-derived propolis, propolis resin, or propolis resin extract and cyclodextrin, such as poplar-derived propolis, propolis resin, or propolis resin extract and beta-cyclodextrin.
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and gamma-cyclodextrin, for example poplar-derived propolis, propolis resin, or propolis resin extract and cyclodextrin, such as poplar-derived propolis, propolis resin, or propolis resin extract and gamma-cyclodextrin.
In a further aspect, the invention provides a synergistic composition comprising propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and cyclodextrin. In one embodiment, the composition is a synergistic therapeutic composition.
In one embodiment, the composition provides a synergistic therapeutic effect.
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In one embodiment the propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin provide a synergistic therapeutic effect that is greater than the effect of either one alone or greater than the additive effects of either one alone.
For example, there is a greater effect on induction of apoptosis, on skin cancer cell survival or proliferation, on resensitisation to therapy, on treatment or prevention of skin cancer, or the responsiveness of a subject or a tumour to the treatment method. In one embodiment, the propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin allow the administration of a co-administered or sequentially administered skin cancer therapy to be reduced or increased in dose or in length of administration, as appropriate.
Another aspect relates to use of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin in the manufacture of a composition for a purpose as herein described.
Another aspect relates to use of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin with at least one additional therapeutic agent in the manufacture of a composition for a purpose as herein described. In one embodiment, the use is of enriched propolis, propolis resin, or propolis resin extract and alpha-cyclodextrin. In one embodiment, the use is of enriched propolis, propolis resin, or propolis resin extract and beta-cyclodextrin. In one embodiment, the use is of enriched propolis and gamma-cyclodextrin.
Another aspect of the invention relates to use of a complex comprising propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or
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Another aspect relates to a composition comprising, consisting essentially of or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin for use in improving skin health. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and alpha-cyclodextrin. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and gamma-cyclodextrin.
In another aspect, the invention relates to a composition comprising, consisting essentially of or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin for use in treating or preventing a skin cancer in a subject.
In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and alphacyclodextrin. In one embodiment, the composition comprises, consists essentially of, or consists of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and gamma-cyclodextrin.
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Another aspect relates to a product comprising, consisting essentially of or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin, optionally with one or more, two or more or three or more additional therapeutic agents as a combined preparation for simultaneous, separate or sequential use for a purpose as described herein.
Another aspect relates to a composition for use in the treatment or prevention of a skin cancer.
One aspect relates to a method of isolating or purifying a compound or mixture of compounds of formula (I) from propolis or poplar, propolis resin or an extract or exudate thereof comprising the steps of providing poplar, propolis, propolis resin or an extract or exudate thereof, and isolating or purifying the compound or mixture of compounds from the poplar, propolis, propolis resin or an extract or exudate thereof.
In various embodiments the method comprises one or more of the following nonlimiting steps:
a) fractionating the poplar, propolis, propolis resin or extract or exudate thereof by chromatography, for example, column chromatography, reverse phase chromatography, normal phase chromatography, or supercritical fluid chromatography, and/or solvent partitioning and/or supercritical extraction to produce one or more fractions comprising one or more of the compounds of formula (I),
b) fractionating the poplar, propolis, propolis resin or extract or exudate thereof or one or more fractions, by preparative HPLC and/or polymeric resin fractionation to produce one or more fractions comprising one or more of the compounds of formula (I), and, optionally,
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c) further purifying the compound or compounds of formula (I) from the one or more fractions of step b) and/or step c).
In a further embodiment, the poplar, propolis, propolis resin or extract or exudate could be subjected to a hydrolysis, methylation or acetylation reaction, and then one or more of steps a), b) or c) performed to isolate and purify one or more compounds of formula (I).
A further aspect relates to a marker of anti-epithelial cancer efficacy comprising a compound of formula (I).
Another aspect relates to a method of evaluating the anti-epithelial cancer efficacy of a composition or product comprising
a) providing a sample of the composition or product, and
b) either
i) determining the presence of one or more compounds of formula (I) in the sample, or ii) measuring the amount and/or concentration of one or more compounds of formula (I) in the sample and determining whether the amount and/or concentration of one or more of the one or more compounds in the sample is equal to, or greater than, a reference amount of that compound known to provide anti-epithelial cancer efficacy, and optionally, iii) determining the biological activity of compound(s) and/or fractions containing the compounds using an in-vitro or in-vivo bioassay.
A further aspect relates to a method of evaluating the anti-epithelial cancer efficacy of a composition or product comprising
a) receiving information about the presence and/or amount of one or more compounds of formula (I) in a sample of the composition or product, and
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b) determining whether the amount of each compound in the sample is equal to, or greater than, a reference amount of that compound known to provide anti-epithelial cancer efficacy.
A further aspect relates to a method of evaluating the anti-epithelial cancer efficacy of a composition or product comprising
a) confirming the presence and/or amount of one or more compounds of formula (I) in a sample of the composition or product using gas, liquid or supercritical fluid chromatography, and
b) determining whether the amount of each compound in the sample is equal to, or greater than, a reference amount of that compound known to provide anti-epithelial cancer efficacy.
In various embodiments the method of evaluating the anti-epithelial cancer efficacy of a composition or product comprises any one or more of the following steps:
a) fractionating the sample by solvent partitioning and/or supercritical extraction and/or chromatography, for example, reverse phase chromatography to produce one or more fractions enriched in one or more of the compounds of formula (I),
b) further fractionating the one or more fractions enriched in one or more of the compounds of formula (I) by one or more of solvent or supercritical extraction or chromatography to produce one or more fractions highly enriched in one or more of the compounds of formula (I),
c) analysing the highly enriched fraction(s), for example by one or more of gas, liquid or supercritical chromatography, to determine the presence and/or amount of one or more of the compound(s) of formula (I) in the sample.
In various embodiments the method of evaluating the anti-epithelial cancer efficacy of a composition or product comprises any one or more of the following steps:
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a. fractionating the sample by chromatography, for example, reverse phase chromatography to produce one or more chromatography fractions,
b. fractionating the sample or chromatography fraction by preparative HPLC to produce one or more HPLC fractions,
c. analysing the trace produced by preparative HPLC to determine the presence and/or amount of the compound in the sample, and/or
d. subjecting the sample, one or more reverse chromatography fractions or one or more HPLC fractions to mass spectrometry to determine the presence and/or amount of the one or more compounds of formula (I) in the sample.
In one embodiment, the determination of the presence and/or amount of one or more of the compound(s) of formula (I) in the sample is by mass spectrometry, for example, liquid chromatography mass spectrometry (LC-MS).
In one aspect the invention relates to a composition or product comprising one or more compounds of formula (I) wherein the amount and/or concentration of one or more compounds of formula (I) is specified on an indicator, such as but not limited to a product label, certificate of analysis, website, promotional material associated with the composition or product.
In various embodiments the compound of formula (I) is selected from the group consisting of
a) 3,8-dihydroxy eicosanoic acid,
b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
g) 3,8-dihydroxy eicosanoic acid methyl ester
h) 3,8-dihydroxy heneicosanoic acid,
i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
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j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
n) 3,8-dihydroxy heneicosanoic acid methyl ester
o) 3,8-dihydroxy docosanoic acid,
p) 1-(3,8-dihydroxy docosanoyl) glycerol,
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
u) 3,8-dihydroxy docosanoic acid methyl ester.
In one embodiment the composition or product is, or comprises, propolis, propolis resin, or propolis resin extract, for example, propolis, propolis resin, or propolis resin extract comprising or enriched in one or more compounds of formula (I).
In one embodiment, the indicator associated with the composition or product is a label or packaging insert.
In one embodiment, the indicator associated with the composition or product is a
Certificate Of Analysis (COA).
In one embodiment, the indicator associated with the composition or product is a website promoting the product.
In one embodiment, the indicator associated with the composition or product is a brochure or pamphlet promoting the product.
The following embodiments may relate to any of the above aspects.
In various embodiments, the epithelial cancer is an epidermal cancer or an epidermoid cancer.
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In various embodiments, the epithelial cancer is a gastrointestinal cancer, such as a colorectal cancer, throat cancer, oesophageal cancer, buccal cancer, or gastric cancer. For example, in one embodiment the colorectal cancer is a colon adenocarcinoma. In another embodiment, the oesophageal cancer is an oesophageal squamous carcinoma. In another embodiment, the gastric cancer is a gastric carcinoma.
In other embodiments, the epithelial cancer is a skin cancer, such as a basal cell carcinoma, a squamous cell carcinoma, or a melanoma.
In various embodiments, the composition additionally comprises one or more of caffeic acid phenylether ester (CAPE), caffeic acid, pinocembrin, benzyl caffeate, benzyl ferulate, benzyl isoferulate, chrysin, cinnamyl caffeate, pinostrobin chalcone, galangin, and pinobanksin.
In exemplary embodiments, the composition is one to which has been added one or more of CAPE, caffeic acid, pinocembrin, benzyl caffeate, benzyl ferulate, benzyl isoferulate, cinnamyl caffeate, pinostrobin chalcone, chrysin, galangin, and pinobanksin-3-acetate.
In one embodiment, the composition has a CAPE concentration of greater than about
1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175, 200,
250, 300, 350, 400, 450, 500, or 600 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
In one embodiment, the composition has a pinocembrin concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 75, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, or 600 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 600 mg/g).
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In one embodiment, the composition has a galangin concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a chrysin concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a pinobanksin concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a caffeic acid concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150, 175,
200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a benzyl caffeate concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150,
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175, 200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a benzyl ferulate concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150,
175, 200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a cinnamyl caffeate concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150,
175, 200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In one embodiment, the composition has a cinnamyl ferulate concentration of greater than about 1, 2, 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 70, 75, 80, 90, 100, 125, 150,
175, 200, 250, 300, 350, 400, 450, 500, or 500 mg/g and useful ranges may be selected between any of these values (for example, about 1 to about 5, about 1 to about 10, about 2 to about 20, about 5 to about 20, about 5 to about 25, about 10 to about 25, about 10 to about 40, about 15 to about 100, or about 20 to about 500 mg/g).
In various embodiments, the cyclodextrin is alpha-cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins comprising alpha-cyclodextrin.
In various embodiments, the cyclodextrin is beta-cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins comprising beta-cyclodextrin.
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In various embodiments, the cyclodextrin is gamma-cyclodextrin, or the cyclodextrin is present as a combination of cyclodextrins comprising gamma-cyclodextrin.
In one embodiment, the cyclodextrin is chemically-modified cyclodextrin, examples of which are described in Stella and He, Cyclodextrins, Toxicologic Pathology, 36, 2008, 30-42.
In one embodiment, the chemically-modified cyclodextrin is hydroxy propyl beta cyclodextrin or hydroxy propyl gamma cyclodextrin
In one embodiment, the propolis is present in the anti-epithelial cancer composition as a propolis extract or fraction.
In one embodiment, the propolis present in the anti-epithelial cancer composition is free of wax. For example, the propolis has been dewaxed using extraction processes known in the art. Dewaxed propolis is often known or referred to as 'propolis resin'.
In one embodiment, the propolis is poplar-derived or Poplar propolis. For example, the poplar-derived propolis is at least in part derived from the bud and leaf exudates of one or more species of poplars, birches, larches or willows.
In one embodiment, the composition comprises from about 1.0%wt to about 99 %wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I). In one embodiment the composition comprises from about 1.0%wt to about %wt propolis resin enriched in one or more compounds of formula (I).
In various embodiments, the composition comprises from about l%wt to about %wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about l%wtto about 25%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about l%wt to about 30%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about l%wtto about 40%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about l%wt to about 50%wt propolis, propolis resin, or propolis resin extract enriched in one or more
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15%wt to about 99%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about 20%wt to about 25%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about
20%wt to about 30%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about 20%wt to about 40%wt propolis, propolis resin,
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PCT/NZ2017/050031 or propolis resin extract enriched in one or more compounds of formula (I), from about
20%wt to about 50%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), from about 20%wt to about 99%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), or about 25%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I), or about 30%wt propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I).
In various embodiments, the composition comprises from about l%wt to about %wt propolis resin enriched in one or more compounds of formula (I), from about l%wt to about 25%wt propolis resin enriched in one or more compounds of formula (I), from about l%wt to about 30%wt propolis resin enriched in one or more compounds of formula (I), from about l%wt to about 40%wt propolis resin enriched in one or more compounds of formula (I), from about l%wt to about 50%wt propolis resin enriched in one or more compounds of formula (I), from about 5%wt to about 25%wt propolis resin enriched in one or more compounds of formula (I), from about 5%wt to about 30%wt propolis resin enriched in one or more compounds of formula (I), from about 5%wt to about 40%wt propolis resin enriched in one or more compounds of formula (I), from about 5%wt to about
50%wt propolis resin enriched in one or more compounds of formula (I), from about 5%wt to about 99%wt propolis resin enriched in one or more compounds of formula (I), from about 10%wt to about 25%wt propolis resin enriched in one or more compounds of formula (I), from about 10%wt to about 30%wt propolis resin enriched in one or more compounds of formula (I), from about 10%wtto about 40%wt propolis resin enriched in one or more compounds of formula (I), from about 10%wtto about 50%wt propolis resin enriched in one or more compounds of formula (I), from about 10%wt to about 99%wt propolis resin enriched in one or more compounds of formula (I), from about 15%wt to about 25%wt propolis resin enriched in one or more compounds of formula (I), from about 15%wt to
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PCT/NZ2017/050031 about 30%wt propolis resin enriched in one or more compounds of formula (I), from about
15%wt to about 40%wt propolis resin enriched in one or more compounds of formula (I), from about 15%wt to about 50%wt propolis resin enriched in one or more compounds of formula (I), from about 15%wt to about 99%wt propolis resin enriched in one or more compounds of formula (I), from about 20%wt to about 25%wt propolis resin enriched in one or more compounds of formula (I), from about 20%wt to about 30%wt propolis resin enriched in one or more compounds of formula (I), from about 20%wt to about 40%wt propolis resin enriched in one or more compounds of formula (I), from about 20%wt to about 50%wt propolis resin enriched in one or more compounds of formula (I), from about
20%wt to about 99%wt propolis resin enriched in one or more compounds of formula (I), or about 25%wt propolis resin enriched in one or more compounds of formula (I), or about
30%wt propolis resin propolis resin enriched in one or more compounds of formula (I).
In one embodiment the propolis, propolis resin, or propolis resin extract in the composition is entirely encapsulated within the cyclodextrin.
In one embodiment the molar ratio of propolis, propolis resin, or propolis resin extract to cyclodextrin in the composition is no greater than about 1:1.
In one embodiment, the propolis, propolis resin, or propolis resin extract is poplarderived propolis, propolis resin, or propolis resin extract.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 3,8-dihydroxy eicosanoic acid. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy eicosanoyl) glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8dihydroxy eicosanoyl) 2-acetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis
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PCT/NZ2017/050031 resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy eicosanoyl)
2,3-diacetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-diacetoxy eicosanoyl) 2,3diacetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 3,8-dihydroxy eicosanoic acid methyl ester. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 3,8-dihydroxy heneicosanoic acid. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy heneicosanoyl) glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol. In one embodiment, the poplarderived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 3,8dihydroxy heneicosanoic acid methyl ester. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 3,8-dihydroxy docosanoic acid. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy docosanoyl) glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol. In one embodiment, the
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PCT/NZ2017/050031 poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol. In one embodiment, the poplarderived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in 1(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol. In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract comprises and/or is enriched in and 3,8dihydroxy docosanoic acid methyl ester.
In one embodiment, the poplar-derived propolis has any one or more of compounds
a) to u) described herein at a concentration of greater than about lmg/kg, about 1.5mg/kg, about 2mg/kg, about 2.5mg/kg, about 3mg/kg, about 3.5mg/kg, about 4mg/kg, about
4.5mg/kg, about 5mg/kg, about 5.5mg/kg, about 5mg/kg, about 7.5mg/kg, about lOmg/kg, about 15mg/kg, about 20mg/kgabout 25mg/kg, about 30mg/kg, about 40mg/kg, about 50mg/kg, about 75mg/kg, about lOOmg/kg, about 125mg/kg, about 150mg/kg, about 175mg/kg, about 200mg/kg, 250mg/kg, about 300mg/kg, about 350mg/kg, about
400mg/kg, about 450mg/kg, about 500 mg/kg, about 550mg/kg, or about 500mg/kg.
In some embodiments the poplar-derived propolis, propolis resin, or propolis resin extract has from at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 5, 7.5, 10, 15,
20, 25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 500,
550, 700, 750, 800, 850, 900, 950, or lOOOmg/g of any two or more, three or more, or four or more, of compounds a) to u) described herein, and useful ranges may be selected from any of these values (for example from about 1 to about 400, about 1 to about 100, about 5 to about 1000, about 5 to about 500, about 5 to about 300, or about 5 to about
100). For example, in some embodiments the poplar-derived propolis has 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol and 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol, or 1(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol and 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol, or 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol, 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol and 1WO 2017/164750
PCT/NZ2017/050031 (3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol and any one or more of compounds a) to
c), e) to j), or I) to u) at a concentration of about lmg/g to lOOOmg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 3,8-dihydroxy eicosanoic acid concentration of at least about 0.lmg/g, about
0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about
3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 5mg/g, about
7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about
40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about
400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy eicosanoyl) glycerol concentration of at least about 0.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 5mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about
150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol concentration of at least about 0.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 5mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
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300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g,
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PCT/NZ2017/050031 about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 3,8-dihydroxy eicosanoic acid methyl ester concentration of at least about
O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 5mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about
125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 3,8-dihydroxy heneicosanoic acid concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 5mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about
150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy heneicosanoyl) glycerol concentration of at least about
O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 5mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about
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125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
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25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 3,8-dihydroxy heneicosanoic acid methyl ester concentration of at least about
O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about
125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 3,8-dihydroxy docosanoic acid concentration of at least about O.lmg/g, about
0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about
3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about
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7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about
40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about
400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy docosanoyl) glycerol concentration of at least about
O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about 2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about 5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout 25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about
125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about 300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about 550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 500mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
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5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a 1-(3,8-dihacetoxy docosanoyl) 2,3-diacetoxy glycerol concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In one embodiment, the poplar-derived propolis, propolis resin, or propolis resin extract has a and 3,8-dihydroxy docosanoic acid methyl ester concentration of at least about O.lmg/g, about 0.5mg/g, about lmg/g, about 1.5mg/g, about 2mg/g, about
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2.5mg/g, about 3mg/g, about 3.5mg/g, about 4mg/g, about 4.5mg/g, about 5mg/g, about
5.5mg/g, about 6mg/g, about 7.5mg/g, about lOmg/g, about 15mg/g, about 20mg/gabout
25mg/g, about 30mg/g, about 40mg/g, about 50mg/g, about 75mg/g, about lOOmg/g, about 125mg/g, about 150mg/g, about 175mg/g, about 200mg/g, 250mg/g, about
300mg/g, about 350mg/g, about 400mg/g, about 450mg/g, about 500 mg/g, about
550mg/g, or about 600mg/g.
In various embodiments, the poplar-derived propolis, propolis resin, or propolis resin extract additionally comprises any combination of two or more of CAPE, chrysin, galangin, pinocembrin, pinobanksin, benzyl caffeate, benzyl ferulate, benzyl isoferulate, cinnamyl caffeate, cinnamyl ferulate, pinostrobin chalcone, and caffeic acid.
In various embodiments, the composition comprises or is administered separately, simultaneously or sequentially with at least one additional therapeutic agent, preferably the at least one additional therapeutic agent is an anti-tumour agent, preferably the antitumour agent is selected from an anti-tumour food factor, a chemotherapeutic agent, or an immunotherapeutic agent.
In various embodiments, the skin cancer therapy, the therapeutic agent, or the antitumour agent is effective to induce apoptosis, for example, induce apoptosis in one or more skin cancer cells or in one or more neoplastic cells.
In one embodiment, the composition is a consumer good.
In one embodiment, the composition is a composition for topical administration.
In one embodiment, the topical composition comprises one or more penetrants, photo-protectants, UV-protectants, vitamins, moisturizers, oils, hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, pigments, odor absorbers or dyestuffs.
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In one embodiment the composition is a food, drink, food additive, drink additive, dietary supplement, nutritional product, medical food, nutraceutical, medicament or pharmaceutical.
In various embodiments, the composition is formulated for oral, topical, or parenteral administration.
In one embodiment, a composition formulated for oral administration comprises gamma-cyclodextrin.
In one embodiment, a composition formulated for topical administration comprises alpha-cyclodextrin.
In one embodiment, a composition formulated for topical administration comprises beta-cyclodextrin.
In one embodiment, the composition comprises one or more additional anti-epithelial cancer agents.
In one embodiment, the composition is a pharmaceutical composition.
In various embodiments, the chemotherapeutic agent is selected from the group comprising mitotic inhibitors, such as vinca alkaloids, including vincristine, vinblastine, vinorelbine, vindesine, vinflunine, podophyllotoxin, taxanes, including docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel, and epothilones, such as ixabepilone; topoisomerase I inhibitors, such as topotecan, irinotecan, camptothecin, rubitecan, and belotecan, topoisomerase type II inhibitors, including amsacrine, etoposide, etoposide phosphate, and teniposide, anthracyclines, such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, amrubicin, pirarubicin, valrubicin, and zorubicin, and anthracenediones, such mitoxantrone and pixantrone; anti metabolites, including dihydrofolate reductase inhibitors, such as aminopterin, methotrexate, pemetrexed, thymidylate synthase inhibitors, such as raltitrexed and pemetrexed, adenosine deaminase inhibitors, including pentostatin, halogenated or ribonucleotide reductase inhibitors, such as cladribine, clofarabine, and
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PCT/NZ2017/050031 fludarabine, thiopurines, including thioguanine and mercaptopurine, thymidylate synthase inhibitors, including fluorouracil, capecitabine, tegafur, carmofur, and floxuridine, DNA polymerase inhibitors, such as cytarabine, ribonucleotide reductase inhibitors, such as gemcitabine, hypomethylating agents, including azacitidine, and decitabine, and ribonucleotide reductase inhibitors, such as hydroxyurea; cell-cycle nonspecific antineoplastic agents, including alkylating agents such as nitrogen mustards, including mechlorethamine, cyclophosphamide, ifosfamide, trofosfamide, chlorambucil, melphalan, prednimustine, bendamustine, uramustine, estramustine, nitrosoureas, including carmustine, lomustine, semustine, fotemustine, nimustine, ranimustine, and streptozocin, alkyl sulfonates, including busulfan, mannosulfan, and treosulfan, aziridines, including carboquone, thioTEPA, triaziquone, and triethylenemelamine, alkylating-like agents, including platinum agents such as cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, satraplatin, hydrazines, such as procarbazine, triazenes, such as dacarbazine, temozolomide, altretamine, and mitobronitol, and streptomycins, such as actinomycin, bleomycin, daunomycin,mitomycin, and plicamycin; photosensitizers, including aminolevulinic acid, methyl aminolevulinate, efaproxiral, and porphyrin derivatives, such as porfimer sodium, talaporfin, temoporfin, and verteporfin; enzyme inhibitors, including farnesyltransferase inhibitors such as tipifarnib, cyclin-dependent kinase inhibitors, such as alvocidib and seliciclib, proteasome inhibitors, such as bortezomib, phosphodiesterase inhibitors, such as anagrelide, IMP dehydrogenase inhibitors, such as tiazofurine, lipoxygenase inhibitors, such as masoprocol, and PARP inhibitors, such as olaparib; receptor antagonists, such as endothelin receptor antagonists including atrasentan, retinoid X receptor antagonists, such as bexarotene, and testolactone; and other chemotherapeutics, including amsacrine, trabectedin, retinoids such as alitretinoin and tretinoin, arsenic trioxide, asparagine depleters such as asparaginase or pegaspargase, celecoxib, demecolcine, elesclomol, elsamitrucin, etoglucid, and lonidamine.
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It is intended that reference to a range of numbers disclosed herein (for example, 1 to
10) also incorporates reference to all rational numbers within that range (for example, 1,
1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example, 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are hereby expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.
In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.
The invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, in any or all combinations of two or more of said parts, elements or features, and where specific integers are mentioned herein that have known equivalents in the art to which the invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
DETAILED DESCRIPTION
The present invention relates to fatty acid compounds and compositions comprising these dihydroxy fatty acid glycerides having anti-epithelial cancer activity. Pharmaceutical
Compositions, for example anti-epithelial cancer compositions, provide and/or enhance antiepithelial cancer efficacy, and in certain embodiments enhance the activity and
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PCT/NZ2017/050031 physicochemical properties of compositions comprising these compounds, such as propolis and poplar extracts. Nutraceutical compositions, for example skin and gut health compositions provide health benefits. In certain embodiments, for example where propolis or propolis extracts or fractions or poplar extracts or fractions are present, the activity and physicochemical properties of the compositions are enhanced.
In one aspect, the invention relates to a method of treating or preventing an epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a a composition comprising a therapeutically effective amount of a compound of formula (I):
Figure AU2017237651A1_D0005
OR4 0R5
R-i=OH, OR7or °‘ or2 (I) or a pharmaceutically acceptable salt or solvate thereof, wherein R2, R3, R4 and Rs are each independently H or acetyl (CH3CO-),
R6 is H or CH3, R7 is CH3 or C2 to C5 saturated or unsaturated hydrocarbon, and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17.
In one embodiment, x is greater than or equal to 4.In one embodiment, the invention relates to a method of treating or preventing an epithelial cancer in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a therapeutically effective amount of at least one compound selected from any one or more of the group consisting of
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a) 3,8-dihydroxy eicosanoic acid,
OH
Figure AU2017237651A1_D0006
OH OH
b) 1-(3,8-dihydroxy eicosanoyl) glycerol
OH
Figure AU2017237651A1_D0007
c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
Figure AU2017237651A1_D0008
Figure AU2017237651A1_D0009
Figure AU2017237651A1_D0010
f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
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Figure AU2017237651A1_D0011
g) 3,8-dihydroxy eicosanoic acid methyl ester
OH
Figure AU2017237651A1_D0012
OCH3 OH
h) 3,8-dihydroxy heneicosanoic acid,
OH
Figure AU2017237651A1_D0013
OH OH
i) 1-(3,8-dihydroxy heneiconsanoyl) glycerol
OH
Figure AU2017237651A1_D0014
j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
OH
Figure AU2017237651A1_D0015
k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol, .0 OH
HO'
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Figure AU2017237651A1_D0016
m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
Figure AU2017237651A1_D0017
OH
Figure AU2017237651A1_D0018
OCH3 OH
o) 3,8-dihydroxy docosanoic acid,
OH
Figure AU2017237651A1_D0019
OH OH
p) 1-(3,8-dihydroxy docosanoyl) glycerol
OH
Figure AU2017237651A1_D0020
q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
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Figure AU2017237651A1_D0021
r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
Figure AU2017237651A1_D0022
s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
Figure AU2017237651A1_D0023
t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol,
Figure AU2017237651A1_D0024
OH
Figure AU2017237651A1_D0025
pharmaceutically acceptable salt or solvate of any one of a) to u), to a subject in need thereof.
The compounds and pharmaceutical compositions, for example anti-cancer compositions, are in some embodiments used to treat or prevent epithelial cancers, or in other embodiments, for example in which propolis, propolis extracts or fractions, propolis resin, or propolis resin extract or poplar extracts or fractions are present, are used to
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PCT/NZ2017/050031 improve skin health, and/or enhance the activity and physicochemical properties of propolis, propolis extracts or fractions, propolis resin, or propolis resin extract or materials with propolis contained.
Accordingly, provided that the anti-epithelial cancer compositions are formulated so as to be suitable for administration to a mammalian subject, for example they consist of materials that are safe to the human body, they can be used for manufacturing antiepithelial cancer pharmaceutical compositions and drugs, as well as nutraceutical compositions, consumer goods, such as beverages, foods, lotions, skin creams and the like.
Furthermore, as the anti-epithelial cancer activity of embodiments of the compositions described herein is maintained for a sustained period, the dosage or frequency of administration of the composition can be reduced, or higher efficacy is provided, or both.
The phrases anti-epithelial cancer compositions or compositions having antiepithelial cancer activity (used interchangeably herein) of this invention contemplate any kind of compositions. Examples include anti-epithelial cancer compositions containing propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin or anti-epithelial cancer compositions containing materials with propolis, propolis resin, or propolis resin extract and cyclodextrin. Synergistic compositions, including those which enhance any anti-epithelial cancer activity observed in either propolis or in cyclodextrin alone are particularly contemplated. In certain embodiments the anti-epithelial cancer compositions may be anti-basal cell carcinoma, anti-squamous cell carcinoma or anti-melanoma compositions. In other embodiments, the anti-epithelial cancer compositions are anti-gastrointestinal cancer compositions, such as anti-colorectal cancer, anti-gastric cancer or anti-throat cancer compositions.
The term and/or can mean and or or.
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The terms cancer and cancerous refer to a physiological condition in mammals that is typically characterized by abnormal or unregulated cell proliferation, cell survival, cell motility, neoplasticity, and/or oncogenicity. Cancer and cancer pathology can be associated, for example, with metastasis, interference with the normal functioning of neighbouring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.
Specifically included are basal cell carcinomas, squamous cell carcinomas, melanomas, and precancerous conditions, which can include dermal tumours, epithelial tumours, tumours of the buccal cavity, for example, squamous cell cancers of the buccal cavity, carcinomas in situ, as well as invasive basal cell, squamous cell, or melanoma cancers, and secondary tumours derived therefrom. Also specifically included are cancers of the gastrointestinal tract, such as colorectal cancers and precancerous conditions, which can include epithelial tumours, nonepitheliaI tumours, carcinomas, for example, carcinomas in situ, as well as invasive colorectal cancers. Also included are gastric cancers and precancerous conditions, which can include epithelial tumours, adenocarcinomas, gastric lymphomas, carcinoid tumours, stromal tumours. Also included are throat cancers and precancerous conditions, which can include epithelial tumours, squamous cell carcinomas, adenocarcinomas. Cancers of mucosal tissues are similarly specifically contemplated. Cancers may be, for example, carcinomas in situ, as well as invasive cancers.
The term comprising as used in this specification means consisting at least in part of. When interpreting statements in this specification that include that term, the features, prefaced by that term in each statement, all need to be present but other features can also be present. Related terms such as comprise and comprised are to be interpreted in the same manner.
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An effective amount is the amount required to confer therapeutic effect. The interrelationship of dosages for animals and humans (based on milligrams per meter squared of body surface) is described by Freireich, et al. (1955). Body surface area can be approximately determined from height and weight of the subject. See, e.g., Scientific
Tables, Geigy Pharmaceuticals, Ardley, New York, 1970, 537. Effective doses also vary, as recognized by those skilled in the art, dependent on route of administration, excipient usage, and the like.
As used herein, an extract or a fraction of poplar is suitable for use in the present invention provided it at least retains one or more anti-epithelial cancer activities, or comprises one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Exemplary extracts or fractions of poplar for use herein are those enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Examples of such functional extracts include the anti-epithelial cancer preparation described herein in the Examples. It will be appreciated that poplar extracts or fractions enriched in one of more of the compounds of formula (I) can be prepared additively or subtractively, whereby at least partially isolated or purified compounds of formula (I) may be added to a composition, such as poplar or a poplar extract or fraction, or by removal from poplar or a poplar extract or fraction of compounds other than one or more compounds of formula (I).
As used herein, an extract or a fraction of propolis is suitable for use in the present invention provided it at least retains one or more anti-epithelial cancer activities or efficacies exhibited by propolis, and/or comprises one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Exemplary extracts or fractions of propolis for use herein are those enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. Examples of such functional extracts include the anti-epithelial cancer tincture described herein in the
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Examples. It will be appreciated that propolis extracts or fractions enriched in one of more of the compounds of formula (I) can be prepared additively or subtractively, whereby at least partially isolated or purified compounds of formula (I) may be added to a composition, such as propolis or a propolis extract or fraction, or by removal from propolis or a propolis extract or fraction of compounds other than one or more compounds of formula (I).
As used herein, chromatography refers to a separation process in which compounds dissolved or dispersed or otherwise transported in a solvent phase are contacted with a solid phase whereby the solid phase selectively retards one or more of the compounds relative to the other compounds present in the solvent, thus enabling separation to occur. Examples of types of chromatographic processes include column chromatography, liquid chromatography, gas chromatography, supercritical chromatography, size exclusion chromatography, preparative chromatography, solid phase extraction. Chromatography processes can be used to determine the concentration of a compound or compounds in a mixture, identify unknown compounds, and/or isolate one or more compounds to, for example obtain detailed structural analysis data, use as an analytical standard and/or for bioassay.
Methods and assays to determine one or more biological effects elicited by one or more of the compounds or compositions described herein, for example, compositions comprising one or more compounds of formula (I), optionally together with propolis or poplar extracts, are well known in the art and examples are described herein, and such methods and assays can be used to identify or verify efficacy, for example, efficacy of one or more one or more of the compounds or compositions described herein, including a composition comprising one or more functional extracts or functional fractions of propolis, such as propolis, a propolis extract, a poplar extract, or a propolis or poplar fraction enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. For example, an assay of the ability of one or more of
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PCT/NZ2017/050031 the compounds or compositions described herein to increase one or more oncogenic traits in a cell, such as those described herein in the Examples, is amenable to verify efficacy of one or more of the compounds or compositions described herein, or to identify one or more functional extracts or functional fractions of propolis or poplar.
As used herein, propolis contemplates propolis produced by bees from any botantical source. In one embodiment, the propolis is poplar-derived propolis. Poplar-derived propolis is also known under different names, such as Poplar propolis. For example, the propolis is derived principally from the bud and leaf exudates of one or more species of poplars, and to a lesser extent birches, larches or willows. Propolis has been classified into seven major classes based on plant source which gives rise to characteristic compounds being present (Sforcin and Bankova, 2011. Propolis: is there a potential for the development of new drugs? J. Ethnopharmacology, 133: 253-250.). These classes are
Poplar from Europe, North America, Southern South America, New Zealand which have high levels of aglycone flavonoids such as chrysin and galangin; Brazilian green, which contains prenylated p-coumaric acids such as artepillan C; Birch from Russia also rich in aglycone flavonoids such as apigenin, rhamnocitrin and kaemferide; Red propolis sourced from Clusia species from Cuba, Brazil, Mexico, Venezuala which contains polyprenylated benzophenones including nemorosone and xanthochymol; Mediterranean from Greece,
Sicily, Crete, Malta rich in diterpenes that are sourced from conifers; and Pacific from
Okinawa, Taiwan, Indonesia, which contain 'propolins'.
Identification and verification of the anti-cancer constituent(s) present in propolis resin has been challenging because of the complex and multicomponent nature of the propolis resin. Poplar propolis resin has also been shown to contain a number of types of compounds, including sugars, glycerol, simple fatty acids, volatile oils and glycerol esters of phenolic acids. A group of compounds not previously reported in propolis having hitherto unknown anti cancer activities are described herein. These compounds are long chain fatty
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PCT/NZ2017/050031 acids and their equivalent glycerides. Furthermore, these dihydroxy free fatty acids and glycerides may be partially to fully acetylated. These compounds have the structure of formula (I):
OR,
OR.
R-i=OH, OR7or
Figure AU2017237651A1_D0026
OR3 or2 (I) wherein R2, R3, FU and Rs are each independently H or acetyl (CH3CO-),
R6 is H or CH3, R7 is CH3 or C2 to C6 saturated or unsaturated hydrocarbon and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and 10 when R6 is CH3, x+y is greater than 9 and less than or equal to 17.
As will be appreciated by those skilled in the art, the identity, and in certain cases the suitability for particular uses, including use in the present invention, of propolis may be determined by analysis of the composition of the propolis. The presence and amount of specific compounds, (including the compounds discussed herein) - frequently referred to as marker compounds, allows a determination of the suitability of a particular source of propolis, for example poplar-derived propolis compared to Brazilian propolis, for a particular use. In certain embodiments of the present invention, the presence or amount of one or more marker compounds is determined or assayed, for example as a preliminary grading step, prior to formulation of the composition. In other embodiments, the presence or amount of one or more marker compounds is determined and reported, for example, on an
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The indicator may be in any form, including, but not limited to, labelling incorporated into the product, for example, etched or printed on the surface of the product or composition, for example, an indicator printed or etched onto a capsule or tablet, packaging or labelling associated with the product such as a label attached to, or incorporated on, a package containing the product or composition, materials provided with, but separate to, the product or composition, for example a Certificate of Analysis; a website, a brochure, a pamphlet, or a display associated with the sale or marketing of the product or composition.
The compounds of formula (I) may be isolated and/or purified from poplar-derived propolis using methods known in the art. Exemplary methods are presented herein.
Similarly, compounds of formula (I) may be isolated and/or purified from botanical sources, such as poplar and poplar extracts or exudates, again using methods known in the art.
Exemplary methods are also presented herein. These methods are also amenable to use in determining the concentration or amount of one or more compounds of formula (I) or pharmaceutically acceptable salt or solvate thereof present in a composition as herein described, for example for the purposes of reporting said concentration or amount.
Alternatively, the compounds of formula (I) may be prepared synthetically using methods well known in the art. For example, synthesis via the Grignard reaction (Smith,
Michael B.; March, Jerry (2007), Advanced Organic Chemistry: Reactions, Mechanisms, and
Structure (6th ed.), New York: Wiley-Interscience, ISBN 0-471-72091-7) using known starting materials is specifically contemplated in the synthesis of a compound of formula (I).
For example, it is contemplated that compounds of formula (I) could be prepared from the starting materials 3,8-dihydroxyoctanoic acid; and a Grignard reagent selected from dodecyl magnesium bromide, tridecyl magnesium bromide or tetradecyl magnesium bromide. These
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Grignard reagents may be prepared from the corresponding long chain fatty alcohol dodecan-l-ol (lauryl alcohol), 1-tridecanol or 1-tetradecanol (myristol alcohol). It is further contemplated that the primary alcohol of the 3,8-dihydroxyoctanoic acid would be selectively oxidized to the aldehyde to enable Grignard coupling of the starting materials.
The resultant 3,8-dihydroxy fatty acid can then be esterified using suitable lipases, or via standard chemical esterification with glycerol, glycerol-2-acetate or glycerol 2,3-acetate to give the main compounds of formula (I).
When used in respect of a composition described herein or a component of such a composition, the phrases enhanced activity or enhanced anti-epithelial cancer activity and grammatical equivalents and derivatives thereof is intended to mean that when present in the composition, an equivalent amount or concentration of the anti-epithelial cancer agent has increased anti-epithelial cancer activity compared to that of the agent in the absence of the composition (such as the isolated agent), and/or the stability of the composition is improved relative to the single component and/or the bioavailability of the composition is improved relative to the single component. For example, the enhanced activity is at least about 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,
170, 175, 180, 185, 190, 195, 200%, or more of the original activity, and useful ranges may be selected between any of these values (for example, from about 105 to about 200%, from about 120 to about 200%, from about 140 to about 200%, from about 150 to about
200%, from about 180 to about 200%, and from about 190 to about 200%). In certain embodiments, compositions may exhibit enhanced anti-epithelial cancer activity, that is, exhibit at least about 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165,
170, 175, 180, 185, 190, 195, 200%, or more of the anti-epithelial cancer activity of propolis alone, or of cyclodextrin alone. Similarly, preferred compositions are capable of supporting the maintenance of enhanced anti-epithelial cancer activity, and can be said to retain enhanced anti-epithelial cancer activity, ideally until utilized using the methods
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PCT/NZ2017/050031 contemplated herein. The enhanced activity (including enhanced maintenance of activity) is believed, without wishing to be bound by any theory, to result from synergy amongst the various components of the compositions, or from improved stability of the composition or from improved bioavailability of the composition due to, for example, improved water dispersibility.
The term oral administration includes oral, buccal, enteral and intra-gastric administration.
The term parenteral administration includes but is not limited to topical (including administration to any dermal, epithelial or mucosal surface), subcutaneous, intravenous, intraperitoneal, intramuscular and intratumoural (including any direct administration to a tumour) administration.
The term pharmaceutically acceptable carrier is intended to refer to a carrier including but not limited to an excipient, diluent or auxiliary that can be administered to a subject as a component of a composition. Preferred carriers do not reduce the activity of the composition and are not toxic when administered in doses sufficient to deliver an effective amount of propolis or extracts thereof, or, when administered, of another antiepithelial cancer agent.
The term (s) following a noun contemplates the singular or plural form, or both.
The term subject is intended to refer to an animal, preferably a mammal, more preferably a mammalian companion animal or human. Preferred companion animals include cats, dogs and horses. Other mammalian subjects include an agricultural animal, including a horse, a pig, a sheep, a goat, a cow, a deer, or a fowl, or a laboratory animal, including a monkey, a rat, or a mouse.
The term treat and its derivatives should be interpreted in their broadest possible context. The term should not be taken to imply that a subject is treated until total recovery. Accordingly, treat broadly includes maintaining a subject's disease progression
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PCT/NZ2017/050031 or symptoms at a substantially static level, increasing a subject's rate of recovery, amelioration and/or prevention of the onset of the symptoms or severity of a particular condition, or extending a patient's quality of life. The term treat also broadly includes the maintenance of good health for sensitive individuals and building stamina for disease prevention.
Exemplary uses of the invention
The methods and compositions described herein may be used in the treatment or prevention of epithelial cancers, neoplastic disorders associated with epithelial cells, and the symptoms of such cancers, including the symptoms of cancer treatment, and associated disorders.
In one example, the methods and compositions described herein may be used in the treatment or prevention of skin cancers, such as melanomas, neoplastic disorders associated with melanomas, and the symptoms of melanoma, melanoma treatment, and associated disorders. Melanoma (also referred to as malignant melanoma) is a neoplastic condition affecting melanocytes.
Melanomas are the least common, but most aggressive and threatening of the skin cancers. Where possible, the preferred treatment is complete surgical removal which can be curative if metastasis has not occurred.
The methods and compositions described herein may be used in the treatment or prevention of basal cell carcinomas, neoplastic disorders associated with basal carcinoma cells, and the symptoms of basal cell carcinoma, basal cell carcinoma treatment, and associated disorders. Basal cell carcinoma is a neoplastic condition affecting the basal cells of the dermis.
Basal cell carcinoma originates from lowest layer of the epidermis. Where possible, the preferred treatment is complete surgical removal which can be curative.
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In certain embodiments, the methods and compositions are used in the treatment or prevention of squamous cell carcinomas, neoplastic disorders associated with squamous carcinoma cells, and the symptoms of squamous cell carcinoma, squamous cell carcinoma treatment, and associated disorders.
Squamous cell carcinoma is a neoplastic condition arising in cells of the middle layer of epidermis. Although squamous cell carcinoma is less common than basal cell carcinoma, metastases are more likely and can be fatal if not treated.
The invention provides methods and compositions for inhibiting skin tumour formation, inhibiting skin tumour growth, inhibiting skin tumour metastasis, or treating or preventing a skin cancer in a subject in need thereof. Without wishing to be bound by any theory, applicants believe that inhibition occurs at least in part, for example, by prevention of UV-initiated damage to DNA or the prevention or reduction in formation of reactive oxygen species.
In certain embodiments, the invention also relates to methods of at least partially reversing the resistance of a neoplastic cell in a subject suffering from a skin cancer to a skin cancer therapy, or to a method of reversing, wholly or in part, the resistance of a skin cancer-burdened patient to a skin cancer therapy, or to a method of re-sensitising one or more tumours of a skin cancer-burdened patient which are, or are predicted to either be or become, resistant to treatment with a skin cancer therapy, said methods comprising the step of administering to said patient a composition comprising, consisting essentially of, or consisting of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) and cyclodextrin.
In one embodiment, the one or more tumours are or are predicted to be or to become resistant to a skin cancer therapy due increased activation of one or more pro-cancer cell survival signaling pathways within the one or more tumours or within the patient, including increased activation of one or more of the AKT, JNK or JAK/STAT signaling pathways, for
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PCT/NZ2017/050031 example within a sample from the patient, such as a tissue sample, a tumour biopsy, or a blood or plasma sample.
Pro-cancer cell survival signaling pathways implicated in the onset and development of skin cancers are known in the art.
The methods and compositions may be used in the treatment or prevention of melanomas, neoplastic disorders associated with melanoma cells, and the symptoms of melanoma, melanoma treatment, and associated disorders.
The most common therapies are surgical excision of tumours and radiation therapy.
Chemotherapeutic agents may be used in combination surgery and/or radiation.
The methods and compositions may also be used for maintaining or improving skin health.
This includes the treatment or prevention of a condition associated with poor skin health, low immunity and skin inflammation. For example, the methods and compositions are useful for or in the treatment or prevention of skin aging, sun burn, dermatitis, eczema, psoriasis, ichthyosis and related inflammatory conditions, and in the treatment or prevention of red, irritated, dry, cracked or itchy skin.
Propolis and materials comprising propolis
Propolis is available in New Zealand and elsewhere, commonly as a resinous sticky solid. Propolis may be obtained from bee-hives with the resulting propolis held in storage, for example to assess the propolis content. Typically the propolis, or an extract thereof, is processed to a fine particulate form or a concentrated tincture. Various methods of preparing active propolis, or an extract thereof, to a particulate form or concentrated tincture are known. Most commonly, crude propolis is extracted using ethanol or ethanol/water mixtures to produce a dilute tincture. Wax associated with the crude propolis is at best poorly soluble in the solvent and so is mostly not extracted. Any extracted wax can be removed by cooling the dilute tincture and then settling, filtration, or centrifugation.
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The tincture can then be concentrated by partial to complete evaporation of the solvent to give a concentrated tincture, optionally followed by freeze drying to give a powder.
Alternatively, the tincture can be spray dried to give a powder. Fractions can be prepared by using methods known in the art such as chromatography (such as HPLC) using, for example, a size exclusion matrix or a reverse phase matrix, or supercritical fractionation. A typical solvent for use in such a chromatographic process is ethanol or another water miscible alcohol.
In one embodiment propolis or concentrated propolis tincture is combined with other compounds that enhance the properties of propolis, for example a compound that enhances the ease of formulation or administration, or that enhances anti-epithelial cancer activity, or that enhances the stability of one or more anti-epithelial cancer activities present in propolis. Examples of additional compounds are those that improve the therapeutic benefits of the propolis. Exemplary compositions in which one or more compounds present in propolis, and in poplar-derived propolis in particular, including biologically active compounds such as CAPE, caffeic acid, pinocembrin, benzyl caffeate, cinnamyl caffeate, benzyl ferulate, cinnamyl ferulate, chrysin, tectochrysin, galangin, pinobanksin, pinostrobin chalcone, and pinobanksin-3-acetate are added are specifically contemplated. In other examples, additional compounds are included to improve or maintain the physiological benefits of the composition, for example mannitol can be added to enhance the diuretic properties of the resulting composition. Alternatively or additionally other compounds such as excipients, and/or propellants could be added to improve the dosing, manufacturability or delivery properties of the composition.
In particularly contemplated embodiments, dewaxed propolis resin, optionally with one or more additional compounds added, is encapsulated with cyclodextrin and the admixture dried. Further processing of the admixture, for example, to obtain a particle size distribution
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PCT/NZ2017/050031 that enables ready admixture with the other components of the composition, ease of tableting, or ease of administration to a subject, is conducted.
In typical embodiments, the propolis or propolis resin is sterilized, for example by heating to kill bacteria, protozoa, yeast, fungi and other organisms that naturally may be present in the propolis.
Poplar extracts
As described in the Examples herein, exemplary compounds of formula (I) have been identified in extracts prepared from poplar (Populus spp.), and particularly from exudates prepared from buds, leaf material, and twigs. Without wishing to be bound by any theory, applicant believes the compounds described herein are present in or associated with poplar derived propolis and propolis resin. Those skilled in the art will recognise that for use in the present invention, poplar or propolis extracts may be processed to a form suitable for further processing and encapsulation, for example with cyclodextrin or extracted with a solvent (e.g., alcohol) while maintaining the bioactive ingredients. Typically the propolis or poplar extract is processed to a fine particulate form or a concentrated tincture.
Cyclodextrins and materials comprising cyclodextrin
Cyclodextrins are cyclic molecules composed of glucopyranose ring units which form toroidal structures. The interior of the cyclodextrin molecule is hydrophobic and the exterior is hydrophilic, making the cyclodextrin molecule water soluble. The degree of solubility can be altered through substitution of the hydroxyl groups on the exterior of the cyclodextrin.
Similarly, the hydrophobicity of the interior can be altered through substitution, though generally the hydrophobic nature of the interior allows accommodation of relatively hydrophobic guests within the cavity. Accommodation of one molecule within another is known as complexation and the resulting product is referred to as an inclusion complex.
Cyclodextrins are typically identified with reference to the number of monomeric units that comprise the molecule, wherein alpha-cyclodextrin (a -cyclodextrin) comprises six
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PCT/NZ2017/050031 monomeric units, beta-cyclodextrin (β -cyclodextrin) comprises seven monomeric units, and gamma-cyclodextrin (γ -cyclodextrin) comprises eight monomeric units. Larger cyclodextrin molecules have been described, including a well-characterised cyclodextrin containing 32 1,4-anhydroglucopyranoside units.
Cyclodextrin molecules may conveniently be derivatised, by for example chemical modification, for example to alter one or more of the physicochemical properties thereof.
Examples of cyclodextrin derivatives include methylated cyclodextrins, sulfobutylcyclodextrin, maltosylcyclodextrin, hydroxypropylcyclodextrin, for example betahydroxylpropylcyclodextrin and gamma-hydroxypropylcyclodextrin, and salts thereof. Those skilled in the art will recognise that various derivates of cyclodextrin may be suitable for particular purposes, for example, certain derivatives of cyclodextrin are not be acceptable for administration to human subjects, but are suitable for industrial uses.
Cyclodextrins comprising the anti-epithelial cancer compositions described herein may be commercially available, or may be prepared independently by methods well known to those skilled in the art. It will be apparent to those skilled in the art that cyclodextrins used in the anti-epithelial cancer compositions for administration to a subject, for example a cyclodextrin for manufacturing a beverage, food, or pharmaceutical should be safe to human body, and preferably is a pharmaceutically acceptable cyclodextrin.
In particularly contemplated embodiments, alpha-cyclodextrin, gamma-cyclodextrin or combinations comprising alphan-cyclodextrin, gamma-cyclodextrin, or both, are used. In such embodiments, anti-epithelial cancer activity is substantially enhanced, as presented herein in the examples. Such compositions comprising alpha-cyclodextrin and/or gammacyclodextrin can be formulated to provide, for example, enhanced mouth feel or palatability, for example compositions comprising alpha-cyclodextrin and/or gamma-cyclodextrin and propolis exhibit a strong tendency to mask any distasteful flavours present in the propolis.
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Cyclodextrins suitable for use in the present invention can be obtained from commercial sources, or can be prepared independently by methods well known in the art, such as from starch by enzymatic conversion. In certain embodiments, CAVAMAX W6, W7 or W8 FOOD, an alpha, beta or gamma-cyclodextrin commercially supplied by Wacker AG, is used.
In certain embodiments, such as those directed to topical application of compositions, non-food grade cyclodextrins are used, for example, cyclodextrins including substituted cyclodextrins from the CAVASOL range of cyclodextrins are contemplated.
Epithelial cancers
Epithelial cancers amenable to treatment using the compositions described herein include carcinomas, such as squamous cell carcinomas, basal cell carcinomas, adenocarcinomas, large cell carcinoms, small cell carcinomas, and transitional cell carcinomas. It will be understood that carcinoma is the term generally used for a malignant epithelial tumour. There are two main types of carcinomas, categorised by the type of epithelium from which they are derived. These are squamous cell carcinomas, which are derived from squamous epithelium, and adenocarcinomas, which are derived from glandular epithelium.
In various embodiments, the epithelial cancer is selected from the group comprising gastric carcinoma (intestinal type), gastric carcinoma (difuse type/mucinous), moderately differentiated adenocarcinoma of the colon, poorly differentiated adenocarcinoma (mucinous) of the colon, hepatocellular carcinoma including poorly differentiated hepatocellular carcinoma, renal cell carcinoma (Grawitz tumor), endometrioid carcinoma, carcinoma of the breast including invasive carcinoma of the breast, metastatic carcinoma (lymph node), colorectal carcinoma, oral carcinomas including esophageal carcinoma, pharyngeal cancer, or laryngeal cancer, skin cancer including basal cell skin cancer, squamous cell skin cancer, and melanoma, and ovarian carcinoma.
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The treatment of oral or throat cancer, also referred to as esophageal cancer, pharyngeal cancer, or laryngeal cancer, and encompassing tumours that develop in the tissues of the pharynx, nasopharynx, oropharynx, hypopharynx, larynx (voice box) or tonsils, using the methods and compositions described herein is particularly contemplated.
Similarly, the treatment of gastric or stomach cancer, and of basal cell skin cancer, squamous cell skin cancer, and melanoma, are particularly contemplated.
Compositions
Exemplary anti-epithelial cancer compositions comprise one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof together with a pharmaceutically acceptable carrier.
Compositions suitable for administration to a subject may be formulated as a food, drink, food additive, drink additive, dietary supplement, nutritional product, cosmeceutical, medical food, nutraceutical, medical supply, medical device, medicament or pharmaceutical.
Appropriate formulations may be prepared by an art skilled worker with regard to that skill and the teaching of this specification.
The compositions useful herein may be formulated to allow for administration to a subject by any chosen route, including but not limited to oral or parenteral (including topical, subcutaneous, intramuscular and intravenous) administration. Those skilled in the art will appreciate that the route of administration to a subject will typically take into account the purpose for which the composition is being administered - for example, where a pharmaceutical composition is being administered to improve skin health or treat or prevent a skin cancer, the route of administration will typically be chosen taking into account the nature of the health aspect or skin cancer being targeted.
In general, for oral administration a dietary (a food, food additive or food supplement for example), nutraceutical or pharmaceutical composition useful herein may be formulated by a skilled worker according to known formulation techniques. In certain embodiments,
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PCT/NZ2017/050031 compositions formulated for oral administration comprise gamma-cyclodextrin. In certain embodiments, compositions formulated for oral administration comprise alpha-cyclodextrin.
Thus, a pharmaceutical composition useful according to the invention may be formulated with an appropriate pharmaceutically acceptable carrier (including excipients, diluents, auxiliaries, and combinations thereof) selected with regard to the intended route of administration and standard pharmaceutical practice. See for example, Remington's
Pharmaceutical Sciences, 16th edition, Osol, A. Ed., Mack Publishing Co., 1980.
While one suitable route of administration of certain embodiments, such as those comprising one or more compounds of formula (I), is oral, it should be understood that any mode of administration may be suitable for any composition, including administration by multiple routes, including different routes for different agents. Therefore, inhalation (nasal or buccal inhalation) and vaginal and rectal administration of any composition is also contemplated. Intramedullar, epidural, intra-articular, and intra-pleural administration of any composition is also contemplated. Administration of a composition, optionally with at least one additional anti-epithelial cancer factor, by a first administration route accompanied by separate, simultaneous or sequential administration of one or more other agents, including one or more other anti-epithelial cancer agents, by a second administration route is also contemplated; for example, oral administration of a composition accompanied by topical administration of the at least one additional anti-epithelial cancer agent.
The compositions may also be formulated as a dosage form. A dosage form useful herein may be administered orally as a powder, liquid, tablet or capsule. Suitable dosage forms may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents, or have an enteric coating. Suitable enteric coatings are known. Enteric coatings surrounding the active ingredients and prevent the release of the active ingredients in the stomach but allow release after the dosage form has left the stomach. Dosage forms useful herein may be adapted for immediate, delayed, modified,
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PCT/NZ2017/050031 sustained, pulsed or controlled release of the active components. Suitable formulations may contain additional agents as required, including emulsifying, antioxidant, flavouring or colouring agents.
Capsules can contain any standard pharmaceutically acceptable materials such as gelatin or cellulose. Tablets can be formulated in accordance with conventional procedures by compressing mixtures of the active ingredients with a solid carrier and a lubricant.
Examples of solid carriers include starch and sugar bentonite. Active ingredients can also be administered in a form of a hard shell tablet or a capsule containing a binder, e.g., lactose or mannitol, a conventional filler, and a tabletting agent. Pharmaceutical compositions can also be administered via the parenteral route. Examples of parenteral dosage forms include aqueous solutions, isotonic saline or 5% glucose of the active agent, or other well-known pharmaceutically acceptable excipient. Solubilising agents well-known to those familiar with the art, can be utilized as pharmaceutical excipients for delivery of the anti-epithelial cancer agent.
Injectable dosage forms may be formulated as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. The dosage form may also be emulsified. The one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof may be mixed with carriers such as, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
Sustained-release preparations may be prepared. Suitable examples of sustainedrelease preparations include semi-permeable matrices of solid hydrophobic polymers containing the one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, optionally together with propolis, and/or a poplar extract, and or cyclodextrin, and when present the at least one additional anti-epithelial cancer agent. The matrices may be in the form of shaped articles, e.g., films, or
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PCT/NZ2017/050031 microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (see US
3,773,919), copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, and degradable lactic acid-glycolic acid copolymers such as the
LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate).
Topical formulations comprising compositions, such as those comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, or enriched propolis, propolis resin, or propolis resin extact and cyclodextrin, and/or when present the at least one additional anti-epithelial cancer agent, are particularly contemplated. Topical formulations may be prepared as lotions, creams, ointments, pastes or salves using known carriers for such applications. In certain embodiments, compositions formulated for topical administration comprise alpha, beta or gamma-cyclodextrin.
In certain embodiments, topical formulations comprise one or more penetrants, such as one or more alkyl lactates, one or more antioxidants, such as Vitamin E (alphatocopherol) or another naturally-occurring antioxidant including polyphenolic antioxidants such as proanthocyanidins and chlorogenic, quinic, and ferulic acids, one or more photoprotectants or UV-protectants, such as TiO2 or carnosic acid, one or more lipids, collagen, keratin or other proteins.
In certain embodiments, the topical compositions comprise one or more carriers that are common in cosmetics, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents, preserving agents, antioxidants, solvents, fragrances, fillers, UVprotectants, pigments, odor absorbers and dyestuffs. Typically, the composition will contain an amount conventionally used in the art, for example, from 0.01% to 20% relative to the total weight of the composition. Depending on their nature and the specific embodiment, these carriers are introduced into a lipid phase, into an aqueous phase, or into one or more
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In certain embodiments, and particularly when the composition is an emulsion, the proportion of the lipid/fatty phase may range from 5% to 80% by weight, for example from
5% to 50% by weight relative to the total weight of the composition. The oils, emulsifiers and co-emulsifiers contemplated for use in the composition in emulsion form are chosen from those conventionally used in the art. When present, the emulsifier and co-emulsifier are present in the composition in a proportion ranging from 0.3% to 30% by weight, for example from 0.5% to 20% by weight, relative to the total weight of the composition.
In certain embodiments, the composition comprises one or more oils such as one or more mineral oils, such as liquid petroleum jelly, oils of plant origin, such as avocado oil or soybean oil, oils of animal origin, for example lanolin, synthetic oils, for example perhydrosqualene, silicone oils, such as cyclomethicone, and fluoro oils, including perfluoropolyethers. Fatty alcohols, such as cetyl alcohol, fatty acids and waxes, for example carnauba wax or ozokerite, are also used as fatty substances in certain embodiments.
In certain embodiments, the emulsifiers and co-emulsifiers are fatty acid esters of polyethylene glycol, such as PEG stearate, or fatty acid esters of glycerol, such as glyceryl stearate, or mixtures thereof.
The use of hydrophilic gelling agents is contemplated in certain formulations, where such agents include carboxyvinyl polymers, such as carbomer, acrylic copolymers such as acrylate/alkylacrylate copolymers, polyacrylamides, polysaccharides, natural gums and clays, and lipophilic gelling agents including modified clays, for instance bentonites, metal salts of fatty acids, hydrophobic silica and polyethylenes.
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Dermabase cream, Unibase cream, and Vanicream are representative examples of commercially available base creams for use as a pharmaceutically acceptable carrier in certain embodiments.
The topical formulations will in certain embodiments also contain moisturizers, depigmenting or pigmenting agents, antimicrobial agents, or free-radical scavengers.
In certain embodiments, topical compositions are formulated as aqueous formulations, such as an aqueous skin cream, for example a water-in-oil or oil-in-water emulsion.
Such formulations may be administered directly, for example, applied directly on to a wound, sprayed onto a surgical site, etc, or may be applied indirectly, such as by impregnation into a bandage or dressing or sprayed onto surgical equipment, dressings and the like.
Parenteral unit dosage forms comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, optionally with at least one additional therapeutic agent, are also provided.
In one example, the anti-epithelial cancer composition is a powder that is obtained after mixing a propolis tincture, for example a propolis tincture enriched in one or more of the compounds of formula (I) or a pharmaceutically-acceptable salt or solvate thereof, with cyclodextrin and one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, then adding water and homogenizing the composition, and then spray-drying or freeze-drying. Other exemplary anti-epithelial cancer compositions of the present invention include solutions, including for example, those in which propolis tincture or fraction thereof and cyclodextrin are mixed and then dispersed in water, those in which propolis or materials with propolis contained and cyclodextrin are independently dissolved or dispersed in water, and then admixed, for example by kneading, and further those in which propolis powder or resin is firstly dissolved in another organic solvent or organic solvent-water mixture in which it is soluble, such as for example ethanol,
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PCT/NZ2017/050031 propylene glycol, ethyl acetate, isopropyl alcohol, and mixtures thereof with water, and the resultant solution is admixed with cyclodextrin, then added to water, and further mixed, for example by kneading and then dried by means known in the art, such as spray or freezedrying. Alternatively, water can be admixed with cyclodextrin at or below the solubility of cyclodextrin in water, to which propolis tincture is then admixed, further water is added and mixed, and then the resultant dispersion is dried by means known in the art, such as spray or freeze-drying. In certain embodiments, anti-epithelial cancer compositions prepared as powders as described above may be preferred, for example because they may maintain stronger anti-epithelial cancer activity or may maintain anti-epithelial cancer activity for a longer period than that of solutions of anti-epithelial cancer compositions prepared as described above.
The content of propolis, propolis resin or propolis resin extract enriched in at least one compound of formula (I) and cyclodextrin of the present invention can be at any level as long as the expected anti-epithelial cancer activity is realized. Similarly, the content of compound(s) of formula (I), propolis, propolis resin or propolis resin extract and cyclodextrin of the present invention can be at any level as long as the expected antiepithelial cancer activity is realized.
Without wishing to be bound by any theory, the applicants believe that the propolis, propolis resin, or propolis resin extract enriched in at least one compound of formula (I) in the composition will be entirely encapsulated when the molar ratio of said propolis, propolis resin, or propolis resin extract to cyclodextrin is no greater than 1:1.
In some embodiments the molar ratio of propolis, propolis resin, or propolis resin extract enriched in at least one compound of formula (I) to cyclodextrin may exceed 1:1 in the compositions. In such compositions the excess propolis, propolis resin, or propolis resin extract will not be encapsulated by the cyclodextrin.
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Other anti-epithelial cancer substances generally known can be combined with the anti-epithelial cancer compositions, depending upon the application to which the composition is to be put.
Without wishing to be bound by any theory, the applicants believe that the enhanced anti-epithelial cancer activity observed in exemplary compositions of the present invention comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin may be due at least in part to a synergy between the one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, particularly when present as propolis resin or concentrated fractions of propolis resin, and cyclodextrin. In certain embodiments, the exemplary composition exhibiting a synergy is a composition comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, such as a propolis extract or fraction, propolis resin extract or a poplar extract or exudate enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and alpha-cyclodextrin. In another contemplated embodiment, the composition exhibiting a synergy is a composition comprising one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, such as a propolis extract or fraction, propolis resin extract or a poplar extract or exudate enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, and gamma-cyclodextrin.
In one embodiment the present invention relates to use of propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and cyclodextrin, such as propolis and alpha-cyclodextrin and/or gamma-cyclodextrin, optionally with at least one anti-epithelial cancer agent, in the manufacture of a food, drink, food additive, drink additive, dietary
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In one embodiment, exemplary compositions comprising propolis, propolis resin, propolis resin extract, poplar extracts, or cyclodextrin is formulated for oral administration.
In another embodiment, the composition is formulated for parenteral, including topical, administration. In certain embodiments, the composition is for inducing apoptosis, treating or preventing a skin cancer, maintaining or improving skin health or one or more other uses as described above.
In one embodiment the composition is in the form of a powder, a tablet, a caplet, a pill, a hard or soft capsule or a lozenge.
In one embodiment the composition is in the form of a sachet, a dispensable powder, granules, a suspension, an elixir, a liquid, a drink, or any other form that can be added to food or drink, including for example water or fruit juice. In one embodiment the composition is an enteral product, a solid enteral product or a liquid enteral product.
In one embodiment, the composition is in the form of a cream, ointment, a paste, a drop solution including eye drops or ear drops, an inhaler or as an inhalable composition, a dressing, a pad, or a spray.
In one embodiment the composition further comprises one or more constituents (such as antioxidants) which prevent or reduce degradation of the composition during storage or after administration.
In one embodiment, compositions useful herein include any edible consumer product, particularly one which is able to carry one or more cyclodextrins. When the composition comprises a proteinaceous factor as the at least one additional anti-epithelial cancer agent, the edible consumer product is one able to carry protein. Examples of suitable edible consumer products include baked goods, powders, liquids, confectionary products, reconstituted fruit products, snack bars, food bars, muesli bars, spreads, sauces, dips, dairy
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PCT/NZ2017/050031 products including ice creams, yoghurts and cheeses, drinks including dairy and non-dairy based drinks (such as milk drinks including milk shakes, and yogurt drinks), milk powders, sports or nutritional supplements including dairy and non-dairy based sports or nutritional supplements, food additives such as protein sprinkles and dietary supplement products including daily supplement tablets. Within this embodiment, a composition useful herein may also be an infant formula, in powder or liquid form. Suitable nutraceutical compositions useful herein may be provided in similar forms. Particularly contemplated are compositions additionally comprising milk or one or more milk products or components of milk, such as milk protein, whey protein, colostrums, milk fat, or any fractions of milk or one or more milk products or components of milk, such as a milk fat fraction, a milk protein fraction, a whey protein fraction, a colostrums fraction, or the like.
Compositions useful herein may further include other factors such as calcium, zinc, magnesium, selenium, vitamin C, vitamin D, vitamin E, vitamin K2, complex carbohydrates, edible or cooking oils including palm, olive, soybean, canola, corn, sunflower, safflower, peanut, grape seed, sesame, nut, almond, cashew, hazelnut, macadamia, pecan, pistachio, and walnut, and other edibles include acai, amaranth, apricot, argan, artichoke, avocado, babassu, bean, blackcurrant seed, borage seed, borneo tallow nut, bottle gourd, buffalo gourd, carob pod (algaroba), cohune, coriander seed, evening primrose, false flax, hemp, kapok seed, lallemantia, meadowfoam seed, mustard, okra seed (hibiscus seed), perilla seed, pequi, pine nut, poppyseed, prune kernel, pumpkin seed, quinoa, ramtil, rice bran, tea (camellia), thistle, watermelon seed, or wheat germ oil, or a combination thereof.
In various embodiments, compositions may comprise at least one additional therapeutic agent, wherein the at least one additional therapeutic agent is an antibiotic, such as an aminoglycoside, such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin, tobramicin, or paromomycin; an ansamycin, such as geldanamycin, or herbimycin; a carbacephem, such as loracarbef; carbapenems, such as, ertapenem,
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PCT/NZ2017/050031 doripenem, imipenem/cilastatin, or meropenem; cephalosporins (first generation), such as cefadroxil, cefazolin, cefalotin or cefalothin, orcefalexin; cephalosporins (second generation), such as cefaclor, cefamandole, cefoxitin, cefprozil, or cefuroxime;
cephalosporins (third generation), such as cefixime, cefdinir, cefditoren, cefoperazone, cefotaxime, cefpodoxime, ceftazidime, ceftibuten, ceftizoxime, or ceftriaxone;
cephalosporins (fourth generation), such as cefepime; cephalosporins (fifth generation), such as ceftobiprole; glycopeptides, such as teicoplanin, or vancomycin; macrolides, such as azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, troleandomycin, telithromycin, or spectinomycin; monobactams, such as aztreonam; penicillins, such as amoxicillin, ampicillin, azlocillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, meticillin, nafcillin, oxacillin, penicillin, piperacillin, or ticarcillin; polypeptides, such as bacitracin, colistin, or polymyxin b; quinolones, such as ciprofloxacin, enoxacin, gatifloxacin, levofloxacin, lomefloxacin, moxifloxacin, norfloxacin, or ofloxacin;
sulfonamides, such as mafenide, sulfonamidochrysoidine (archaic), sulfacetamide, sulfadiazine, sulfamethizole, sulfanilimide (archaic), sulfasalazine, sulfisoxazole, trimethoprim, or trimethoprim-sulfamethoxazole (co-trimoxazole) (tmp-smx); tetracyclines, such as demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline; others such as arsphenamine, chloramphenicol, clindamycin, lincomycin, ethambutol, fosfomycin, fusidic acid, furazolidone, isoniazid, linezolid, metronidazole, mupirocin, nitrofurantoin, platensimycin, pyrazinamide, quinupristin/dalfopristin, rifampicin (rifampin in US), thiamphenicol, tinidazole, dapsone, clofazimine; or a cyclic lipopeptides, such as daptomycin, a glycylcycline, such as tigecycline, or an oxazolidinones, such as linezolid.
In other embodiments, the at least one additional therapeutic agent is an antifungal, such as a polyene antifungal, such as natamycin, rimocidin, filipin, nystatin, amphotericin B, candicin; imidazoles, such as miconazole, ketoconazole, clotrimazole, econazole, bifonazole, butoconazole, fenticonazole, isoconazole, oxiconazole, sertaconazole, sulconazole, or
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PCT/NZ2017/050031 tioconazole; triazoles, such as fluconazole, itraconazole, isavuconazole, ravuconazole, posaconazole, voriconazole, or terconazole; thiazoles such as abafungin; allylamines, such as terbinafine, amorolfine, naftifine, or butenafine; echinocandins, such as anidulafungin, caspofungin, or micafungin; others such as benzoic acid, ciclopirox, tolnaftate, undecylenic acid, flucytosine or 5-fluorocytosine, griseofulvin, haloprogin, and sodium bicarbonate; or alternatives such as allicin, tea tree oil, citronella oil, iodine, lemon grass, olive leaf, orange oil, palmarosa oil, patchouli, lemon myrtle, neem seed oil, coconut oil, zinc, or selenium.
Alternatively the agent is selected from any of those described herein.
The efficacy of a composition useful according to the invention can be evaluated both in vitro and in vivo. See, e.g., the examples below. Briefly, in one embodiment the composition can be tested for its ability, to for example, inhibit neoplastic cell proliferation in vitro. For in vivo studies, the composition can be fed to, injected into, or topically applied to an animal (e.g., a mouse) and its effects on skin cancer cell survival, proliferation, metastasis, or one or more symptoms of a skin cancer or associated disease or disorder are then assessed. Based on the results, an appropriate dosage range, frequency, and administration route can be determined.
The compositions useful herein may be used alone or in combination with one or more other anti-epithelial cancer agents, or one or more additional therapeutic agents. The antiepithelial cancer agent or additional therapeutic agent may be or comprise a food, drink, food additive, drink additive, food component, drink component, dietary supplement, nutritional product, medical food, nutraceutical, cosmeceutical, medical device, medical supply, medicament or pharmaceutical. The anti-epithelial cancer agent or additional therapeutic agent is preferably effective to attenuate one or more neoplastic diseases or disorders or one or more of the symptoms of one or more neoplastic diseases or disorders, or otherwise confer a benefit on the subject to whom it is administered. Preferred
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PCT/NZ2017/050031 therapeutic agents include therapeutic food factors, immunogenic or immunostimulatory agents, wound healing agents, and the like.
It should be understood that the additional anti-epithelial cancer or therapeutic agents listed above (both food based and pharmaceutical agents) may also be employed in a method according to the invention where they are administered separately, simultaneously or sequentially with a composition useful herein.
As will be appreciated, the dose of the composition administered, the period of administration, and the general administration regime may differ between subjects depending on such variables as the severity of symptoms of a subject, the type of disorder to be treated, the mode of administration chosen, and the age, sex and/or general health of a subject. However, by way of general example, from about 1 mg to about 5000 mg per kg body weight of a composition useful herein is administered, 1 mg to about 4000 mg per kg body weight of a composition useful herein is administered, 1 mg to about 3000 mg per kg body weight of a composition useful herein is administered, 1 mg to about 2000 mg per kg body weight of a composition useful herein is administered, 1 mg to about 1000 mg per kg body weight of a composition useful herein is administered, per administration or per day, preferably about 5 to about 100 mg per kg, preferably per day. In one embodiment, the administration is of from about 0.05 mg to about 250 mg per kg body weight of a composition useful herein.
In various embodiments, sufficient composition is administered to deliver from about
0.001 mg to about 50 mg of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof per kg body weight, from about 0.001 mg to about 40 mg per kg body weight, from about 0.001 mg to about 30 mg per kg body weight, from about 0.001 mg to about 20 mg per kg body weight, from about 0.001 mg to about lOmg per kg body weight, from about 0.001 mg to about 5 mg per kg body weight, from about 0.001 mg to about 1 mg per kg body weight, from about 0.001 mg to about 0.5 mg per kg body weight,
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PCT/NZ2017/050031 from about 0.001 mg to about 0.1 mg per kg body weight, or from about 0.001 mg to about
0.05 mg of a compound of formula (I) or a pharmaceutically acceptable salt or solvate thereof per kg body weight, per administration or per day.
It should be appreciated that administration may include a single dose, such as a single daily dose, or administration of a number of discrete divided doses as may be appropriate. It should be understood that a person of ordinary skill in the art will be able without undue experimentation, having regard to that skill and this disclosure, to determine an effective dosage regime (including dose and timing of administration) for a given condition.
The present invention also relates to a dietary, nutraceutical, cosmeceutical or oral pharmaceutical composition comprising, consisting essentially of or consisting of one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof, optionally together with propolis or a material comprising propolis in combination with cyclodextrin. In certain embodiments the composition consists essentially of about 1 to 99 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about 1 to 80 wt % cyclodextrin. For example, the composition consists essentially of about 20 to 80 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about 20 to 80 wt % cyclodextrin. In another example, the composition consists essentially of about 20 to 40 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about 60 to 80 wt % cyclodextrin.
Dietary, nutraceutical, cosmeceutical or oral pharmaceutical compositions comprising, consisting essentially of or consisting of propolis, propolis resin, or propolis resin extract
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PCT/NZ2017/050031 enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof or a material comprising propolis, propolis resin, or propolis resin extract that is encapsulated by cyclodextrin are provided. In certain embodiments the composition consists essentially of about 1 to 30 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about 70 to 99 wt % cyclodextrin. For example, the composition consists essentially of about 10 to 25 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about to 90 wt % cyclodextrin. In another example, the composition consists essentially of about 20 to 30 wt % propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof and about 70 to 80 wt % cyclodextrin.
In one embodiment a composition comprises propolis, propolis resin, or propolis resin extract enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof or a propolis fraction enriched in one or more compounds of formula (I) or one or more pharmaceutically acceptable salts or solvates thereof. In one embodiment the composition comprises at least about 1, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 50, 70, 80, 90, or 99% by weight propolis, propolis resin, propolis resin extract or a propolis fraction, and useful ranges may be selected from any of these values (for example, from about 1 to about 25% by weight, from about 1 to about 30% by weight, from about 5 to about 30% by weight, from about 15 to about 30% by weight, from about to about 30% by weight, from about 25 to about 30% by weight, from about 10 to about
50% by weight, from about 15 to about 50% by weight, from about 40 to about 99% by weight, from about 45 to about 99% by weight, from about 50 to about 99% by weight, from about 55 to about 99% by weight, from about 50 to about 99% by weight, from about
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PCT/NZ2017/050031 to about 99% by weight, from about 70 to about 99% by weight, from about 75 to about
99% by weight, from about 80 to about 99% by weight, from about 85 to about 99% by weight, from about 90 to about 99% by weight, or from about 95 to about 99% by weight).
In one embodiment a composition comprises cyclodextrin, for example alphacyclodextrin and/or gamma-cyclodextrin. In one embodiment the composition comprises at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or at least about 90% by weight cyclodextrin, and useful ranges may be selected from any of these values (for example, from about 1 to about 99% by weight, from about 5 to about 99% by weight, from about 10 to about 99% by weight, from about 15 to about 99% by weight, from about 20 to about 99% by weight, from about 25 to about 99% by weight, from about to about 99% by weight, from about 35 to about 99% by weight, from about 40 to about
99% by weight, from about 45 to about 99% by weight, from about 50 to about 99% by weight, from about 55 to about 99% by weight, from about 60 to about 99% by weight, from about 65 to about 99% by weight, from about 70 to about 99% by weight, from about to about 99% by weight, from about 80 to about 99% by weight, from about 85 to about
99% by weight, from about 90 to about 99% by weight, or from about 95 to about 99% by weight).
When used in combination with another anti-epithelial cancer agent or therapeutic agent, the administration of a composition useful herein and the other anti-epithelial cancer agent or therapeutic agent may be simultaneous or sequential. Simultaneous administration includes the administration of a single dosage form that comprises all components or the administration of separate dosage forms at substantially the same time. Sequential administration includes administration according to different schedules, preferably so that there is an overlap in the periods during which the composition useful herein and other therapeutic agent are provided.
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Additionally, it is contemplated that a composition in accordance with the invention may be formulated with additional active ingredients which may be of benefit to a subject in particular instances. For example, therapeutic agents that target the same or different facets of the disease process may be used.
Accordingly, foods and beverages comprising anti-epithelial cancer compositions of this invention can be used for general foods and health food. Since the anti-epithelial cancer compositions of the present invention mask the taste of propolis, they can be eaten as they are or in the form of powder. They can be used as an ingredient or raw material for cake, biscuit, cookie, chocolate, sweets and other confectionary, including drops or chewing gum.
The compositions may be added to water as a drink, can be used as a taste modifier for beverages such as milk, tea, coffee, hot chocolate, etc., and as an ingredient or raw material for fruit juice beverages, sports drink, etc.
Exemplary anti-epithelial cancer compositions and methods for preparing such compositions will now be described with reference to the following examples.
EXAMPLES
Example 1: Initial bioassay guided fractionation of propolis tincture
This example describes an assessment of the anti-gastrointestinal cancer activity of fractions of propolis produced by preparative chromatography. This study was performed using proliferation assays in the human colon cancer adenocarcinoma cell line, DLD-1.
Production of propolis tincture fractions by column chromatography
Fractionation was carried out using a glass column packed with Merck Lichroprep CI8 reversed phase stationary phase (15 x 4 cm) which had been washed with methanol (MeOH) (200 ml) and equilibrated with 20% aqueous ethanol (EtOH) (500 ml). Propolis tincture dry solids (5.445 g) dissolved in EtOH (5 ml) was loaded onto the top of the column using a piston pump. Elution was carried out as a stepped gradient (250 ml) consisting of
20%, 30%, 40%, 50%, 50%, 70%, 80%, and 90% aqueous EtOH, followed by two 100%
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EtOH steps, then elution with 2-propanol (IPA),ethyl acetate (EtOAc), acetone, and chloroform (CHCI3). Solvent from the various fractions was removed under vacuum on a rotary evaporator followed by freeze drying overnight. The two 100% EtOH fractions were pooled as were the remaining four non-polar fractions (IPA, EtOAc, acetone, and CHCI3) for biological assay work due to their relatively low masses. The fractions are shown in Table 1 according to the percentage of ethanol used in the elution step from the column, e.g. 20%,
30%, 40%, 50%, 60%, 70%, 80%, and 90% aqueous EtOH, and 100% EtOH.
Table 1. Propolis fractionation mass and test sample numbers
Sample # Test Sample ID Elution solvent Fraction mass (g) % of recovered mass
1 Propolis fraction 1 20% EtOH 0.1127 2.07
2 Propolis fraction 2 30% EtOH 0.0658 1.21
3 Propolis fraction 3 40% EtOH 0.2408 4.42
4 Propolis fraction 4 50% EtOH 0.5022 9.22
5 Propolis fraction 5 60% EtOH 2.2333 41.01
6 Propolis fraction 6 70% EtOH 0.9843 18.07
7 Propolis fraction 7 80% EtOH 0.4697 8.62
8 Propolis fraction 8 90% EtOH 0.2413 4.43
9 Propolis fraction 9 100% EtOH (2) 0.3018 5.54
10 Propolis fraction 10 IPA, EtOAc, acetone, and CHCI3 0.042 0.77
11 Propolis tincture dry solids
Materials and methods for the anti-gastrointestinal cancer anti-proliferative assay for DLD-1 human colon adenocarcinoma cells, KYSE-30 human oesophageal squamous cell carcinoma and NCI-N87 human gastric carcinoma
Test samples shown above in Table 1 obtained by the fractionation of propolis tincture dry solids were assessed for their ability to modulate the viability and proliferation of human colorectal adenocarcinoma cells (DLD-1) as assessed by the MTT assay. The propolis tincture dry solids starting material was also included in the bioassay. A positive control, 5fluorouracil (5-FU) was included in addition to an unsupplemented cell control (negative
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PCT/NZ2017/050031 control) in the study. In later examples, test samples were also assessed for the ability to modulate the viability and proliferation of human oesophageal cells (KYSE-30) and human gastric carcinoma cells (NCI-N87) as assessed by the MTT assay.
Description of test materials and test methods
Human gastro-intestinal cancer cell lines DLD-1, KYSE-30 and NCI-N87 were revived from cryostorage and cultured in the presence of the test and reference samples. The culture conditions for the cells were those described by the supplier of the cells (ATCC). An
MTT assay was then performed on the cultures to determine the effect of the samples on the cell proliferation.
The methodology was based on the procedures reported by:
Smolka, AJ, Goldenring, JR, Gupta, S and Charles E Hammond, CE. Inhibition of gastric H,KATPase activity and gastric epithelial cell IL-8 secretion by the pyrrolizine derivative
ML 3000. (2004). BMC Gastroenterology. 4: 4.
Chailler, P and Menard, D (2005). Establishment of Human Gastric Epithelial (HGE) Cell
Lines Exhibiting Barrier Function, Progenitor, and Prezymogenic Characteristics.
Journal of Cellular Physiology 202: 253-274.
Trainer, D.L., Kline, T., McCabe, F.L., Faucette, L.F., Field, J., Chaikin, M., Anzano, M.,
Rieman, D., Hoffstein, S., Li, D-J., Gennaro, D., Buscarino, C., Lynch, M., Poste, G.
And Greig, R. (1988). Biological characterization and oncogene expression in human colorectal carcinoma cells lines. International Journal of Cancer 41: 287295.
Shimada, Y, Imamura, M, Wagata, T, Yamaguchi, N, Tobe, T. (1992) Characterization of 21 newly established Esophageal Cancer Cell Lines. Cancer 59: 277 - 284.
Sutter, AP, Hopfner, M, Huether, A, Maaser, K, Scherubi, H. (2005) Targeting the epithelial growth factor receptor by erlotinib (Tarceva™) for the treatment of esophageal cancer. Inter, J Cancer. 118: 1814 - 1822
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Palyi, I. (1989) Heterogeneity of the Response to Inducers of Differentiation and to
Cytostatics of Tumor Cell Populations. Pathology Research & Practice. 184: 11-17.
Fritzsche, C, Zeller, G, Knaup, KX, Roemer, K. (2004) No anti-apoptotic effects of single copies of mutant p33 genes in drug-treated tumor cells. Anti-Cancer Drugs. 15: 679688.
Yang, Y, Zhou, Z, He, S, Fan, T, et al. (2012) Treatment of prostate carcinoma with (Galectin-3)-targeted HPMA copolymer-(G3-C12)-5-Fluorouracil conjugates.
Biomaterials. 33: 2260-2271.
Nakagawa, Y., Iinuma, M., Naoe, T., Nozawa, Y. And Akao, Y. (2007). Characterized mechanism of α-mangostin-induce cell death: Caspase-independent apoptosis with release of endonuclease-G from mitochondria and increased miR-143 expression in human colorectal cancer DLD-1 cells. Bioorganic & Medicinal Chemistry 15: 56205628.
Minegaki, T., Takara, K., Hamaguchi, R., Tsujimoto, M. And Nishiguchi, K. (2013). Factors affecting the sensitivity of human-derived esophageal carcinoma cell lines to 5fluorouracil and cisplatin. Oncology Letters 5: 427-434.
Nakamura, A., Nakajima, G., Okuyama, R., Kuramochi, H., Kondoh, Y., Kanemura, T.,
Takechi, T., Yamamoto, M. And Hayashi, K. (2014). Enhancement of 5-fluorouracilinduced cytotoxicity by leucovorin in 5-fluorouracil-resistant gastric cancer cells with upregulated expression of thymidylate synthase. Gastric Cancer 17: 188-195.
Tankiewicz-Kwedlo, A., Pawlak, D., Domaniewski, T. And Buczko, W. (2010). Effect of erythropoietin, 5-fluorouracil and SN-38 on the growth of DLD-1 cells.
Pharmacological Reports 62: 926-937.
Sample Preparation
Working solutions were prepared by dissolving the test fractions in 15% ethanol (ETOH)/HBSS to a concentration of 2 mg/ml solids.
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Experimental Procedures
Characterisation of the Test System
1. Human colorectal adenocarcinoma cells were obtained from ATCC (CCI-221, DLD-1).
2. Human gastric carcinoma cells were obtained from ATCC (CRL-5822, NCI-N87).
3. Human oesophageal squamous cell carcinoma cells were obtained from Sigma Aldrich (ECACC, KYSE-30)
4. The DLD-1 cell medium obtained from GIBCO was Dulbecco's Modified Eagle's Medium (DMEM) had 10% Foetal Bovine Serum (FBS), lOOU/ml Penicillin and lOOmg/ml Streptomycin added. The medium was stored at 4°C
5. The NCI-N87 cell medium obtained from Sigma was RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate. The medium was stored at 4°C
6. The KYSE-30 cell medium obtained from Sigma was Ham's F12:RPMI-1640 (50:50) medium modified to contain 2 mM L-glutamine. For the complete medium 10% FBS, lOOU/ml Penicillin and 2.5pg/ml Gentamicin was added. The medium was stored at 4°C.
7. Penicillin-streptomycin solution consisting of lOOOOunits/mL penicillin, 10 mg/ml streptomycin in 0.9% NaCI, stored at -20°C was obtained from Sigma (#P-0781).
8. Trypsin-EDTA solution consisting of 0.25% Trypsin/EDTA was obtained from Invitrogen (15400054).
9. Phosphate buffered saline (PBS) was prepared in-house.
10. Hanks Balanced Salt Solution (HBSS), stored at 4°C was obtained from GIBCO (14185-052).
11. Foetal Bovine Serum, stored at -20°C was obtained from GIBCO (10091-148).
12. MTT Reagent, 100 mg/vial, obtained from SIGMA (M-2128) was dissolved in PBS at 10 mg/ml and stored at -20 °C. 5 mg/ml MTT solution was prepared in PBS and stored at 4 °C as working solution.
13. MTT lysis buffer consisting of 10 % sodium dodecyl sulphate (SDS)/45% Dimethyl Formamide was prepared by dissolving 20 g SDS in 100 mL of double-distilled water (DDW), adding 90 mL of Dimethyl Formamide, adjusting the pH to 4.7 using glacial acetic acid, and then diluting with DDW to a final volume of 200 ml.
14. 5-Fluorouracil (5-FU) was obtained from Sigma (F-6627). Working solutions in 15 % ETOH/HBSS were prepared as required and then diluted to the required concentration just prior to bioassay.
Medium preparation
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The medium for the propagation of each of the cells lines is given above. Each medium was prepared following the instructions of the manufacturer and supplemented with penicillin-streptomycin solution (10ml per litre). FBS (at 10%) was added just before use.
Culturing of cells
1. Each of the cell lines were revived from cryostorage.
2. Following initial propagation using the media described above (see Medium Preparation), the culture was sub-cultured using the trypsin-EDTA as follows. The media was removed and 5ml of the trypsin-EDTA solution added and incubated at 37°C for 5 min or until all the cells had detached. The trypsin was neutralised by adding an equal volume of DMEM medium and the culture centrifuged at 300g (1200rpm) for 5 min at 4°C.
3. The supernatant was decanted and the cell pellets re-suspended in medium DMEM, FBS (10%), penicillin (lOOunits/ml), streptomycin (100pg/ml). The cells were cultured at 37°C in 5% CC>2/95% air.
4. After reaching confluence, the cells were detached using trypsin-EDTA and centrifuged as described in 2 above.
5. The supernatants were discarded and the cells re-suspended in DMEM and supplements as described in 3 above at 1.0 x 104 cells per ml.
6. Into each well of three 96 well plates, 180μI of the cells (1,800 cells/well) or medium was added. The plates were incubated at 5% (302/95% air at 37°C for 48 h which was sufficient to allow the cells to adhere.
7. To each well, 20μI of each of the test samples or positive control was added. For the 'medium' or 'cells only' controls, 20μI of 15%ETOH/HBSS was added to each well. Each sample was assessed in replicates of 3 - 6, while the controls on each of three micro-titer plates were assessed in replicates of 3 - 9 (combined triplicates). The final concentration of the sample in each well was 200 pg/ml unless noted.
8. The total volume in each well was 200pl.
9. The plates were incubated at 37°C in 5% CC>2/95% air for 19 h.
Cell Proliferation Assay
1. On completion of the incubation, 20 pi of MTT working solution (5 mg/ml) was added to all wells and incubated for 3-4 hr at 37° C in 5% 002/95% air.
2. During this incubation period it was noted that a strong colour unexpectedly appeared in some of the wells of a number of samples, in both the Cells plus Sample wells and the Sample plus Medium Blank wells. At the end of this period the supernatants were removed from each well and each well was gently washed with HBSS twice, to remove the purple colour, before adding the MTT lysis buffer as described in Step 3 below.
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100
3. 100 μΙ of MTT lysis buffer was then added and the plates incubated overnight at 37°C in 5% CO2/95% air. The plates were centrifuged at 1200rpm for 10 minutes to pellet any remaining insoluble material. From each well 200μΙ aliquots were transferred to fresh 95 well plates. The plates were read by a VersaMax microplate reader at 570 nm.
4. Results were expressed as the percentage proliferation of cells cultured in the presence of the sample in comparison to the cells only control. The blank reading was subtracted from all wells as a background reading.
Results
A summary of the effects of controls and test samples on the proliferation of the DLD1 cells for propolis fractions S#1 - S#10 and comparative propolis dry solids S#ll is presented in Table 2 below, where NC = Negative Control (cells only), PC = Positive Control (5-fluorouracil, tested at 7.50 ng/ml). In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical
Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 2. Effects of the propolis fraction samples on the proliferation of DLD-1 cells.
Sample ID & number of replicates, n Mean (OD 570nm) SEM p Values (<0.05) % Inhibition
NC-l Cells Only, n=9 0.7047 0.0252 1.00 0.00
PC-1 Cells - 5-FU, 7.50ng/ml, n=9 0.6575 0.0172 NS 6.69
Cells - S#l. Propolis Fraction #1, 200 pg/ml, n=6 0.2246 0.0297 2E-08 68.13
Cells - S#2. Propolis Fraction #2, 200 pg/ml, n=5 0.1919 0.0250 2E-08 72.77
Cells - S#3. Propolis Fraction #3, 200 pg/ml, n=6 0.5359 0.0153 2.3E-04 23.95
Cells - S#4. Propolis Fraction #4, 200 pg/ml, n=6 0.5779 0.0203 3.2E-03 17.99
Cells - S#5. Propolis Fraction #5, 200 0.4159 0.0172 1.2E-06 40.97
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pg/ml, n=6
Cells - S#6. Propolis Fraction #6, 200 0.6488 0.0197 NS 7.93
pg/ml, n=6
Cells - S#7. Propolis Fraction #7, 200 pg/ml, n=6 0.6037 0.0135 9.2E-03 14.32
Cells - S#8. Propolis Fraction #8, 200 pg/ml, n=6 0.1970 0.0152 IE-08 72.04
Cells - S#9. Propolis Fraction #9, 200 pg/ml, n=6 0.5932 0.0194 7.0E-03 15.82
Cells - S#10. Propolis Fraction #10, 200 pg/ml, n=6 0.6375 0.0309 NS 9.54
Cells - S#ll. Propolis tincture dry solids, 200 pg/ml, n=6 0.4750 0.0207 2E-05 32.59
The most active propolis fractions were found to be fractions S#l, S#2, S#5 and S#8.
Propolis fractions S#l, S#2 and S#5 contained known propolis phenolics and flavonoids and so were not investigated further in this work. Further studies were conducted to determine the compounds present in propolis fraction S#8, which was shown by HPLC to not contain any previously identified phenolic compounds. These studies are described in Example 2.
Example 2: Bioassay guided fractionation of the non-phenolic fraction
This example describes the further bioassay-guided fractionation of the propolis fraction S#8 prior to compound identification. Four further sub-fractions numbered S#28 to
S#31 were produced. This study was performed using the proliferation of the colon cancer adenocarcinoma cell line DLD-1 by MTT assays as described in Example 1.
Preparative HPLC to produce sub-fractions S#28 to S#31
The 90% aqueous EtOH elution fraction (propolis fraction S#8) produced for
Example 1 was further fractionated by preparative HPLC. The propolis fraction S#8 was dissolved in neat EtOH, and then chromatographed by preparative HPLC on a Phenomenex
Synergi C-12 column (4p,-RP Max 80A 250 x 30 mm) using a Gilson 321 preparative pump and Agilent 1100 series diode array detector. Injection volumes of between 0.5 and 1.5 ml
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102 were employed. A flow rate of 20 ml/min. was employed. Solvents were 80% aqueous
MeOH (containing 0.1% TFA) and EtOAc/MeOH (4:1 vol/vol). The initial eluent composition consisted of the 80% aqueous methanol. The solvent composition was held for 5 minutes at the initial conditions before the EtOAc/MeOH solvent concentration was increased linearly to
100% over 35 minutes. The chromatography was carried out at room temperature (18 20°C). Fractions were collected manually with online detection carried out at 210 nm, 268 nm and 327 nm and also by evaporative light scattering detection (ELSD). The main components of this fraction were quite non-polar, eluting late in the chromatogram, and showed minimal UV absorption at the wavelengths typically used for analysis on other propolis fractions containing phenolics, i.e., 268 and 327 nm. However, ELSD revealed a complex mix of components. Due to the destructive nature of ELSD preparative HPLC fractions were collected manually with online detection carried out at 210 nm. As the chromatography did not produce distinct isolated peaks but rather broad rises and falls in the baseline four fractions were collected during the run and all were prepared for biological assay.
Materials and methods for the DLD-1 colon cancer anti-proliferative assay
Test samples shown below in Table 3 were assessed for their ability to modulate the viability and proliferation of human colorectal adenocarcinoma cells (DLD-1) as assessed by the MTT assay. A positive control, 5-fluorouracil (5-FU) was included in addition to an unsupplemented cell control (NC, negative control) in the study. The concentration of 5-FU was increased from Example 1 to induce a stronger antiproliferative response. The DLD-1 anti-proliferation assay was performed as described in Example 1.
Table 3. Test Samples
Sample No Test Sample ID and concentration Sample No Test Sample and concentration
S#28 90% FI, 200 pg/ml NC-1 Cells only
S#29 90% F2, 200 pg/ml PC-1 Cells + 5-FU 0.65 pg/ml
S#30 90% F3, 200 pg/ml PC-2 Cells + 5-FU 1.95 pg/ml
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S#31 90% F4, 200 pg/ml
The samples were dissolved as working solutions at 2 mg/ml in 15% EtOH in HBSS.
In the assay the final concentration of the samples was 200 pg/ml, with a final EtOH concentration of 1.5%.
Results and Discussion
A summary of the effects of the positive control and test samples on the proliferation of the cells after 24 hours incubation is presented in Table 4, where NC = Negative Control (cells only), PC = Positive Control (5-fluorouracil, tested at 0.55 and 1.95 pg/ml). In the
Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the
Measurement associated with the Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 4. Effect of the propolis sub-fraction samples on DLD-1 cell proliferation.
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n = 12 0.3239 0.0135 NS 0.00
PC-1 Cells + 5-FU, 0.65 pg/ml, n = 12 0.3023 0.0154 NS 6.66
PC-2 Cells + 5-FU, 1.95 pg/ml, n = 12 0.2689 0.0116 5.3E-03 16.96
Cells + S#28. 90% FI, n=6 0.1909 0.0137 1.3E-05 41.06
Cells + S#29. 90% F2, n = 6 0.2528 0.0106 3.3E-03 21.96
Cells + S#30. 90% F3, n=5 0.0000 N/A N/A 100
Cells + S#31. 90% F4, n=6 0.2776 0.0118 4.2E-02 14.30
All of the propolis sub-fraction samples tested inhibited the proliferation of the DLD-1 colon cancer cells. The most active fraction was S#30 90 % F3, which completely inhibited proliferation and was cytotoxic at the concentration tested. Further work was carried out in
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Example 3 to identify the nature of the bioactive compounds present in the test samples used in Examples 1 (propolis fraction S#8) and 2 (propolis sub-fraction S#30).
Example 3: Identification of glycerides in propolis as active compounds
This example describes the isolation and identification of novel dihydroxy fatty acid glycerides from NZ propolis resin fractions equivalent to Example 1 (propolis fraction S#8) and 2 (propolis sub-fraction S#30). 1 gram of 40 % propolis tincture containing 40 % propolis resin (400 mg dry solids) was fractionated using a silica gel column using a stepwise solvent gradient to produce 14 fractions with weight distribution given in Table 5.
Each fraction was analysed by UHPLC using both a UV (258 nm for detection of phenolics) and ELSD detector (for detection of all compounds). Propolis resin and selected fractions were also analyzed by LC-MS to enable structural elucidation. Purified subfractions were also analysed by NMR. Washing the column sequentially with hexane, hexane/diethyl ether and diethyl ether eluted most of the phenolic material from the silica (fractions 1-2; 3-7; 815 9 respectively) including flavonoids, caffeate and ferulate esters. A brown coloured fraction remained on the silica. Elution of the brown colour began when ethyl acetate was employed (fractions 11-12). Most of the remaining dark colour was then eluted with acetone and methanol (fractions 13, 14). The mass distribution shows that most of the propolis mass is in the diethyl ether fractions which had a light yellow colour, however a significant percentage of the total mass was eluted in the fractions eluted with ethyl acetate (fractions
11, 12). Water soluble material was recovered in fractions 13, 14 or retained on the column.
Table 5. Weight and solvent gradient for silica gel fractionation of propolis.
Fraction Eluant Volume (ml) Weight (mg) % of total eluted
1 Hexane 10 1 0.3
2 Hexane 10 <1 < 0.3
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3 20% ether /hexane 10 1 0.3
4 40% ether /hexane 10 2 0.6
5 40% ether/hexane 10 8 2.3
6 60% ether /hexane 10 10 2.8
7 60% ether /hexane 10 62 17.5
8 Diethyl ether 10 70 19.8
9 Diethyl ether 10 51 14.4
10 Dichloromethane 20 53 15.0
11 Ethyl acetate 20 47 13.3
12 Ethyl acetate 20 38 10.7
13 Acetone/ methanol 11 3.1
14 Methanol 3 0.8
Results and discussion
The novel compounds appeared as sharp peaks in the 34-44 minutes region of an
ELSD chromatogram of fractions 12, 13. The late peaks observed in the ELSD which were sharp and well resolved were able to be linked to a set of peaks seen in LC-MS runs of crude propolis and enriched fractions 12 and 13. No UV absorption was associated with these peaks. Mass spectrometry (MS) shows good peaks in positive ion mode where M + l and
M + Na peaks were observed. The main components of this set of compounds have molecular weights of 450, 474, 488 and 502, equating to a homologous series where the components differed by a methylene group. All of the mass spectra had common peaks at
159 and 117 atomic mass units (amu). In the negative ion mode the compounds had quasi-molecular ions at M+45 due to formate adducts. High resolution MS gave a molecular formula of C25H48O7 for the 450 molecular weight compound and extra methylenes for the others in the series. Sub-fractions of crude fractions 12, 13 were then fully acetylated to aid the separation of the compounds of interest and to provide more definitive spectroscopic data. The mass spectra of the peracetylated mixture showed in general that the
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The initial chromatographic fractionation described above was repeated several times to generate more of the enriched fractions 11 - 13. The compounds of interest were also readily separated from these enriched fractions by silica gel chromatography to yield sufficient material for NMR analysis. The spectra showed the presence of four acetate groups, including a single acetate which was present in some of the natural compounds. A substituted glycerol was present and the acid attached to the glycerol was also hydroxylated at the 3 position. The rest of the molecule was comprised of a fatty acid chain which also has a second hydroxyl group. The fatty acid is a dihydroxy C20, C21 C22 or C23 for the main compounds. The compounds are thus glycerides.
GC-MS analysis of a TMS treated glyceride rich fraction produced by reversed phase column chromatography showed the natural mixture of hydroxylated fatty acid lipids present in propolis was quite complex. This fraction had the common fatty acids already known to be present in propolis (palmitic, oleic and stearic acid) along with the monoglycerides. The complexity was reduced by base hydrolysis to remove both the glycerol and acetyl moieties to give four major and a number of minor dihydroxy fatty acids.
This fitted with the structural type described above, but did not identify where the acetyl group resides. This combined with the range of fatty acids and positional/stereo isomers for the hydroxyl groups gives rise to the large number of analogues. The main compounds identified by chromatographic isolation followed by structural elucidation using MS and NMR were monoacylglycerides of dihydroxy C20, C21 C22 or C23 fatty acids (esterification at position Cl of glycerol). The fatty acids in the monoglycerides were predominantly straight chain C20-C22. The fatty acid was hydroxylated at position C3, and a second hydroxyl group was separated from the first by at least one methylene group; and was not on the final methyl group of the fatty acid. The glycerol moiety may be acetylated at one or both of
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Example 4: Production of a glyceride concentrate and isolation and purification of glycerides
In this example, the position of the second hydroxyl group on the fatty acids was determined, as was the placement of the acetyl group. Individual glyceride compounds were also isolated. A large-scale extract was prepared from 1.1 kg of dewaxed propolis resin using ethyl acetate as the solvent. The propolis resin was coarsely chopped and soaked in ethyl acetate at room temperature with mechanical stirring for 2 hrs. The solvent with dissolved extract was filtered but not concentrated. Around 20 % of this extract solution was chromatographed using silica gel, after first absorbing the extract onto a portion of silica gel and evaporating the ethyl acetate using a rotary evaporator. The pre-absorbed solid was then applied to the top of a large silica gel column and the column eluted with 1:1 hexane/ether and 8 x 150 ml fractions were collected, then the column was eluted with diethyl ether for 4 further 150 ml fractions before beginning elution with ethyl acetate. The ethyl acetate fractions (fractions #13 - #15) collected were much darker in colour than the earlier fractions. A final fraction (#17) was collected after elution with acetone. Silica
TLC (visualisation using phosphomolybdic acid in ethanol followed by heat) and LC-MS were used to track progression of the purification process. The glyceride rich fractions from silica gel chromatography were then combined and subjected to reversed phase C18 column chromatography using mixtures of water and ethanol without acid to yield a final glyceride rich fraction (GLY-conc). This process was repeated with more of the ethyl acetate extract as required.
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Analysis of all the fractions by LC-MS showed that the ethyl acetate fractions #13 #16 and acetone fraction #17 contained the main glyceride compounds. These fractions were also rich in the brown coloured material, consistent with Example 3. Fractions #13 and #14 were combined; and #15 and #16 were also combined. The #13/#14 combined fraction was used for structural identification as follows. Approximately 100 mg of the #13/#14 mixture was mixed with 4 ml THF:MeOH:H2O and 55 mg LiOH. This was stirred at ice temperature for 30 minutes and then at room temperature for 48 hrs. The reaction mixture was acidified with acetic acid and extracted with chloroform. The dried extract was then esterified with diazomethane and subjected to silica gel chromatography (ethyl acetate/hexane). A fraction comprising one main spot (Rf approximately 0.3 with 1:1
EtOAc:hexane, visualisation by phosphomolybdic acid in ethanol with heat) was collected.
This purified sample was then analysed by GC-MS after treatment with BSTFA. The 9 free fatty acid peaks numbered 4-12 resulting from hydrolysis of the glycerides in the MS spectrum were a homologous series. Major peak 4 had a characteristic peak at 383 (corresponding to loss of methyl and TMSOH) while the subsequent major peaks 5 (397), 7 (411), 10 (425) 11 (439) and 12 (453) were the C19 through to C24 dihydroxy fatty acids (methyl ester TMS ethers). Another feature was fragment ions at m/z 301 and 247 seen for all the peaks. These fragments, especially the 301 fragment, are characteristic of 3,8dihydroxy acid TMS ethers that were obtained by the saponification, methylation and sialylation of 3-acetoxy, 8-hydroxy fatty acid glycerols esterified at position C2 on the glycerol [Asai, T., Hara, N., Kobayashi, S., Kohshima, S., Fujimoto, Y. (2009). Acylglycerols (=glycerides) from the glandular trichome exudate on the leaves of Paulownia tomentosa.
Helvetica Chimica Acta. 92: 1473-1494], The other possible dihydroxy acids 3,6; 3,7; and
3,9 have different fragmentation patterns. Thus the dihydroxy acids in the glyceride fraction are the C19 to C24 3,8-dihydroxy fatty acids. The relative peak size indicates that the C20 and C22 chain lengths were the dominant compounds. Minor peaks 6, 8, 9, 13 with almost
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C24 acids i.e. a branched methyl at the terminal end of the fatty acid. This fits with evidence from NMR on the per-acetylated compound which suggested minor amounts of methyl branching.
The #15/16 combined fraction was then used for preparative HPLC followed by
Sephadex LH20 size exclusion chromatography to obtain two mono-acetate compounds
GLY-lAc-1, and GLY-lAc-2 (C20 and C21 dihydroxy fatty acid monoacetates respectively).
Fraction #17 was used for preparative HPLC followed by Sephadex LH20 size exclusion chromatography to obtain three no-acetate compounds GLY-0Ac-l, GLY-OAc-2 and GLYOAc-3 (C20, C21 and C22 dihydroxy fatty acid monoglycerides respectively). The separation progress was tracked using HPLC with ELSD and the purity of the individual compounds isolated was assessed to be >95% by HPLC, LC-MS and NMR.
Example 5: Production of glyceride concentrate, free fatty acids, methyl esters, diacetates, peracetates and cyclodextrin complexes
Hydrolysis of a sample of the glyceride concentrate obtained in Example 4 to produce free fatty acids (FFA-conc) was performed as described in Reis et al (Reis, M.G., De Faria,
A.D., Do Amaral, M.D.C.E., Marsaioli, A.J. (2003). Oncidinol - A novel diacylglycerol from
Ornithophora radicans Barb. Rodr. (Orchidaceae) floral oil. Tetrahedron Letters 44: 85198523). Approx. 100 mg of the glyceride concentrate was mixed with 4 ml THF:MeOH:H2O and 55 mg LiOH. This was stirred at ice temperature for 30 minutes and then at room temperature (RT) for 48 hrs. The reaction mixture was acidified with acetic acid and extracted with chloroform. The free fatty acid mixture was purified using silica gel chromatography (ethyl acetate/hexane).
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A sample of the purified free fatty acid mixture was also methylated to produce dihydroxy fatty acid methyl esters (ME-conc). Methylation was performed by addition of diazomethane solution (Aldrich, trimethylsilyldiazomethane, 2.0M solution in hexanes) to 10 mg of free fatty acid mixture. After reacting for 1 hr at room temperature the reaction was quenched with acetic acid and the solution evaporated to dryness under N2. The methyl ester mixture was purified using silica gel chromatography (ethyl acetate/hexane). A fraction comprising one main spot (Rf approximately 0.3 with 1:1 EtOAc:Hex, visualisation phosphomolybdic acid in ethanol followed by heat) was collected.
Di hydroxy fatty acid glyceride diacetates (GLY-2AC-1 mix) were prepared by partial acetylation of the glyceride concentrate (15 mg stirred at room temperature with 1 equivalent of acetic anhydride overnight in THF, containing a catalytic amount of dimethylaminopyridine). The reaction mixture was purified to give one spot on TLC.
Per-acetylation of the glyceride rich fraction (PerAc-conc)-was carried out by dissolving the partially purified glyceride fraction (approx. 150 mg) in acetic anhydride (20 mL) and adding a small amount of dimethyl amino pyridine. After stirring at room temperature overnight the reaction was worked up by adding methanol and toluene (approx. 20 mL of each) and evaporating to dryness using a rotary evaporator. The residue was the chromatographed on silica gel using hexane/ethyl acetate mixtures to obtain a purified peracetate mixture (approx. 80 mg).
Cyclodextrin complexes of the glyceride concentrate obtained in Example 4 were produced as follows. Approximately 400 mg of glyceride concentrate was accurately weighed into a flask. 1.2 g of ethanol was then added to the vial, and the contents stirred to dissolve the solids to make a 25 % by mass solution of glycerides in ethanol. The glycerides rapidly dissolved in the ethanol. Approximately 300 mg of alpha, beta and gamma cyclodextrins were separately accurately weighed into mortars. Approximately 400 mg of the 25 % glycerides tincture was then added by calibrated syringe to each mortar
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The contents of each mortar were added to a weighed round bottom flask. The flask contents were frozen by rotating the flask in a dry ice/acetone mixture. The flask contents were then freeze-dried. 370 mg of gamma-cyclodextrin glyceride complex (g-CD GLY cone);
390 mg of beta-cyclodextrin glyceride complex (β-CD GLY cone); and 350 mg of alphacyclodextrin glyceride complex (α-CD GLY cone) were recovered from the flasks.
Example 6: Antiproliferative activity of dihydroxy fatty acid glycerides against human skin cancer cell lines: melanoma cell line A-2058
This example shows that a number of glyceride compounds present in NZ propolis have novel anti-skin cancer activity against the human melanoma cell line A-2508. This example also describes the general in-vitro bioassay method for determining the antiproliferative activity of purified fractions and highly purified glycerides against human skin cancer cell lines; and the activity of these test compounds against human melanoma cell line A-2058. The three human skin cancer cell lines were:
1) Human Melanoma cell line (A-2058).
2) Human Epidermoid (Squamous) Carcinoma cell line (A-431).
3) Human Basal Cell Carcinoma cell line (TE 354.T).
The cell lines were revived from cryostorage and cultured in the presence of the test and reference samples. An MTT assay was then performed on the cultures to determine the effect of the samples on the cell viability and proliferation. The percentage standard error of the mean was assessed and extreme outliers were removed if the SEM% >15. Preliminary statistical significance was assessed with an independent Student t-test at a < 0.05 (with and without outliers). The methodology was based on the procedures reported by:
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Ahn, NG, Campbell JS (1993). Metabolic labeling of mitogen-activated protein kinase in A431 cells demonstrates phosphorylation on serine and threonine residues. Proc Natl
Acad Sci. 90: 5143 - 5147.
Galan-Cobo, A et al (2014). Functional inhibition of aquaporin-3 with a gold-based compound induces blockage of cell proliferation. J Cellul. Physiol. 229: 1787 - 1801.
Roomi, MW, Kalinovsky, J et al (2013) Effect of a nutrient mixture on matrix metalloproteinase-9 dimers in various human cancer cell lines. Inter J Oncol. 44: 936 - 942.
Huang, HC, Lin, MK et al (2013). Cardenolides and bufadienolide glycosides from
Kalanchoe tubiflora and evaluation of cytotoxicity. Planta Medica. 79: 1362 - 1369.
Tilli, CMLJ, Stavast-Kooy, AJW et al (2003). The garlic-derived organosulfur component ajoene decreases basal cell carcinoma tumor size by inducing apoptosis. Arch
Dermatol Res. 295: 117 - 123.
Sample Preparation
The test samples were dissolved in 15% Ethanol (EtOH)/HBSS to make a working solution, which was then diluted 10 times to give a final concentration in the cells of 50 pg/ml unless noted.
Experimental Procedures
Characterisation of the Test System
1. Human melanoma cell line (A-2058, ATCC CRL11147) was obtained from ATCC, Bethesda, MD, USA.
2. Human squamous (epidermoid) carcinoma cell line (A-431, ATCC CRL1555) was obtained from ATCC, Bethesda, MD, USA.
3. Human basal cell carcinoma cell line (TE 354.T, ATCC CRL7762) was obtained from ATCC, Bethesda, MD, USA.
4. Dulbecco's Modified Eagle's Medium (DMEM) was obtained from GIBCO (12100-038). 10% Foetal Bovine Serum (FBS), lOOU/ml Penicillin and lOOmg/ml Streptomycin were added before use.
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5. Penicillin-streptomycin solution, stored at -20°C consisting of 10000 units/ml penicillin and lOmg/ml streptomycin in 0.9% NaCI was obtained from Sigma (#P0781).
5. Trypsin-EDTA solution consisting of 0.25% Trypsin/EDTA was obtained from Invitrogen (#15400054).
7. Phosphate buffered saline (PBS) was prepared in-house.
8. Hanks Balanced Salt Solution (HBSS), stored at 4°C was obtained from GIBCO (# 14185-052).
9. Foetal Bovine Serum (FBS) stored at -20° C was obtained from GIBCO (# 10091148).
10. MTT Reagent, 100 mg/vial, was obtained from Sigma (M-2128) and then dissolved in PBS at 10 mg/ml and stored at -20 °C. A 5mg/ml MTT solution was prepared in PBS and stored at 4 °C as the working solution.
11. MTT lysis buffer: 10 % sodium dodecyl sulphate (SDS)/45% Dimethyl Formamide was prepared by dissolving 20 g SDS in 100 ml of double-distilled water (DDW), and then adding 90 ml of Dimethyl Formamide to the solution. The pH was adjusted to 4.7 by glacial acetic acid, and DDW added up to a final volume of 200 ml.
12. 5-Fluorouracil (5-FU) was supplied by Sigma (F-5527). Three working solutions were prepared at 19.5pg/ml, 5.5pg/ml and 1.95pg/ml dissolved in 15% Ethanol/HBSS. Final concentrations were 1.95pg/ml (15pM), 0.55pg/ml (5pM) and 0.195pg/ml (1.5pM).
Medium Preparation
The culture conditions for the cells were those suggested by the supplier of the cells (ATCC). The medium for the propagation of each of the cells lines was DMEM as given above. The medium was prepared following the ATCC instructions and supplemented with penicillin-streptomycin solution (10ml per litre). FBS (at 10%) was also added just before use.
Culturing of Cells:
1. Each of the cell lines obtained from the American Type Culture Collection USA was revived from cryostorage.
Following initial propagation using the DMEM media described above the cultures were sub-cultured using trypsin-EDTA. The media was removed and 5ml of the
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3. The supernatant was decanted and the cell pellets re-suspended in DMEM that contained FBS (10%), penicillin (100 units/ml), streptomycin (100pg/ml).
4. After reaching confluence, the cells were detached using trypsin-EDTA as described in 2 above and centrifuged.
5. The supernatants were discarded and the cells re-suspended in DMEM and supplements as described in 3 above at varying concentrations of cells per ml. The cell concentration of the A-431 culture was increased from 1 x 104 to 5 x 104 cells/ml, whereas the concentrations of the other two cell lines (A2058 and TE 354.T), were increased to 2 x 104 cells/ml.
5. For the three cell lines, into each well of 95 well plates, 180μI of the cells or medium was added. The plates were incubated using 5% CO2/95% air at 37°C for varying time periods to allow the cells to adhere. For the A-431 culture, the pre-incubation time was 3hrs, whereas the pre-incubation times for the other two cell lines (A2058 and TE 354.T) were 24hrs.
7. 20pl of each of the test compounds or 5-FU was added to each well. To the wells labelled 'medium' or 'cells only', 20pl of 15%ETOH/HBSS was added. The number of times each test sample or control was assessed is noted in the Examples tables.
8. The total volume in each well was 200pl.
9. The plates were further incubated at 37°C in 5% CC>2/95% air for 24hr.
Cell Proliferation Assay
1. On completion of the incubation, 20 pi of MTT working solution (5mg/ml) was added to all wells and the plates incubated for 3-4 hr at 37° C in 5% 002/95% air. The plates were monitored every 30-50 minutes and if a few cells showed the presence of crystals then the lysis buffer was added as in Step 2.
2. 100 μΙ of MTT lysis buffer was then added and the plates incubated overnight at 37°C in 5% 002/95% air. The plates were centrifuged at 300g (1200rpm) for 10 minutes to pellet any remaining insoluble material. From each well a 200μΙ aliquot was transferred to fresh 95 well plates. The plates were read in a VersaMax microplate reader at 550 nm.
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3. Results were expressed as the percentage absorbance of cells cultured in the presence of the sample in comparison to that of the cells only control. The blank reading was subtracted from all wells as a background reading.
The glyceride test samples GLY-lAc-1 and GLY-lAc-2 (dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol, C20 and C21 fatty acid respectively) and GLY-OAc1, GLY-OAc-2 and GLY-OAc-3 (dihydroxy fatty acid glyceride containing no acetates, C20,
C21, and C22 fatty acid respectively) were tested at a final concentration of 50 pg/ml, and compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 0.195, 0.65 and 1.95 pg/ml respectively; and
PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml.
Results and discussion
This example shows that a number of glyceride compounds present in NZ propolis have novel anti-skin cancer activity against the human melanoma cell line A-2508.The bioassay results are shown in Table 6. In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical
Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05; % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 6: Antiproliferative activity of dihydroxy fatty acid glycerides against human melanoma cell line A-2508
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.1260 0.0046 1 0.00
PC-1 Cells + 5-FU, 0.195 pg/ml, n=6 0.1339 0.0067 NS 0
PC-2 Cells + 5-FU, 0.65 pg/ml, n=6 0.1187 0.0043 NS 5.78
PC-3 Cells + 5-FU, 1.95 pg/ml, n=6 0.1383 0.0045 NS 0
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PC-4 Cells + MGP, 50 pg/ml, n=3 0.0548 0.0016 1.7E-05 56.5
S#12 Cells + GLY-lAc-1, 50 pg/ml, n=2 0.0249 0.0037 2.4E-05 80.2
S#13 Cells + GLY-lAc-2, 50 pg/ml, n=2 0.0236 0.0006 1.4E-06 81.3
S#14 Cells + GLY-OAc-1, 50 pg/ml, n=2 0.0157 0.0001 1.3E-05 87.5
S#15 Cells + GLY-OAc-2, 50 pg/ml, n=2 0.0106 0.0001 9.7E-06 91.6
S#16 Cells + GLY-OAc-3, 50 pg/ml, n=2 0.0358 0.0055 5.2E-05 71.6
All of the highly purified compounds inhibited proliferation of human melanoma skin cancer cell lines by 72 - 92 % when tested at 50 pg/ml. The most potent of these compounds was the non-acetylated dihydroxy fatty acid glyceride GLY-OAc-2 with a fatty acid chain length of 22 carbons. The antiproliferative activity of these dihydroxy fatty acid glycerides was superior to the positive control monoglycerol palmitate (MGP), also tested at pg/ml. The 5-fluorouracil had low and non-significant activity at the three concentrations tested of 0.195, 0.55 and 1.95 pg/ml.
Example 7: Antiproliferative activity of dihydroxy fatty acid glycerides against human epidermoid carcinoma A-431
This example shows that a number of glyceride compounds present in NZ propolis have novel anti-skin cancer activity against the human epidermoid cancer cell line A-431.
The glyceride test samples GLY-lAc-1 (C20 dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol) and GLY-0Ac-l (C20 dihydroxy fatty acid glyceride containing no acetates on the glycerol) were tested for their antiproliferative activity against human epidermoid carcinoma cell line A-431 at a final concentration of 50 pg/ml. The cell proliferation assay was performed as described in Example 5. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3
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PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 Mg/ml.
The bioassay results are shown in Table 7. In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the
Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 7: Antiproliferative activity of dihydroxy fatty acid glycerides against human epidermoid cancer cell line A-431
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.2621 0.0158 NS 0.00
PC-1 Cells + 5-FU, 0.195 pg/ml, n=6 0.2364 0.0064 NS 9.8
PC-2 Cells + 5-FU, 0.65 pg/ml, n=6 0.2416 0.0111 NS 7.8
PC-3 Cells + 5-FU, 1.95 pg/ml, n=6 0.2561 0.0086 NS 2.3
PC-4 Cells + MGP, 50 pg/ml, n=3 0.0360 0.0034 2.5E-05 86.3
S#12 Cells + GLY-lAc-1, 50 gg/ml, n=3 0.0507 0.0037 3.3E-04 80.6
S#14 Cells + GLY-OAc-1, 50 gg/ml, n=3 0.0484 0.0025 3.6E-05 81.5
The two monoglyceride compounds tested, GLY-lAc-1 and GLY-0Ac-l, have almost identical levels of inhibition of proliferation at 80.6 and 81.5 % respectively when tested at
Mg/ml- This example shows that the number of acetates on the glycerol is not significant with respect to activity. The antiproliferative activity of these dihydroxy fatty acid glycerides was similar to the positive control monoglycerol palmitate (MGP), also tested at 50 Mg/ml.
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The 5 fluorouracil had low and non-significant activity at the three concentrations tested of
0.195, 0.65 and 1.95 pg/ml.
Example 8: Antiproliferative activity of dihydroxy fatty acid glycerides against 5 human basal cell carcinoma TE 354.T
The glyceride test samples GLY-lAc-1 (C20 dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol) and GLY-OAc-1 (C20 dihydroxy fatty acid glyceride containing no acetates on the glycerol) were tested for their antiproliferative activity against human basal cell carcinoma cell line TE 345.T at a final concentration of 50 pg/ml. The cell proliferation assay was performed as described in Example 6. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 0.195, 0.65 and 1.95 pg/ml respectively; and
PC-4 consisting of glycerol monopalmitate tested at 50 pg/ml.
This example shows that a number of glyceride compounds present in NZ propolis have novel anti-skin cancer activity against the human basal cancer cell line TE 354.T. The bioassay results are shown in Table 8. In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical
Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 8: Antiproliferative activity of dihydroxy fatty acid glycerides against human basal cell carcinoma cell line TE 354.T
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.2696 0.0096 NS 0.00
PC-1 Cells + 5-FU, 0.195 pg/ml, n=6 0.2548 0.0051 NS 5.49
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PC-2 Cells + 5-FU, 0.65 pg/ml, n=6 0.2656 0.0061 NS 1.49
PC-3 Cells + 5-FU, 1.95 pg/ml, n=6 0.2553 0.0070 NS 5.31
PC-4 Cells + MGP, 50 pg/ml, n=3 0.2277 0.0065 0.0247 15.6
S#12 Cells + GLY-lAc-1, 50 pg/ml, n=3 0.1959 0.0037 1.3E-03 27.3
S#14 Cells + GLY-OAc-1, 50 pg/ml, n=3 0.1672 0.0236 1.7E-03 38.0
The two monoglyceride compounds tested, GLY-lAc-1 and GLY-OAc-1, have similar levels of inhibition of proliferation at 27.3 and 38.0 % respectively when tested at 50 pg/ml.
This example shows that having zero acetates on the glycerol gives an improvement in bioactivity for this skin cancer cell line. The antiproliferative activity of these dihydroxy fatty acid glycerides was superior to the positive control monoglycerol palmitate (MGP), also tested at 50 pg/ml. The 5 fluorouracil had low and non-significant activity at the three concentrations tested of 0.195, 0.65 and 1.95 pg/ml.
Example 9: Antiproliferative activity of dihydroxy fatty acid glycerides and derivatives against human colon adenocarcinoma cell line DLD-1
This example shows the antiproliferative activity of individual glycerides GLY-lAc-1 and GLY-lAc-2 (dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol, C20 and C21 fatty acid chain length) and GLY-OAc-1 and GLY-OAc-2 (non-acetylated dihydroxy fatty acid glycerides, C20 and C21 fatty acid chain length) were tested for anti-proliferative activity at a final concentration of 50 pg/ml against colon adenocarcinoma cell line DLD-1.
GLY-lAc-1 was also retested in a second assay at 50 and 25 pg/ml to provide an indicative dose response. The cell proliferation assay was performed as described in Examples 1 and
2. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2 consisting of 5-FU at a test concentration of 0.65 and 1.95 pg/ml respectively; and PC-3 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml. The
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120 cells only data NC-1 for the first and NC-2 second bioassay trials are also included in the table.
This example shows that a number of glyceride compounds present in NZ propolis have novel anti-gastrointestinal cancer activity against the human colon adenocarcinoma cell line DLD-1. The bioassay results are shown in Table 9. In the Table, OD is Optical
Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 9: Antiproliferative activity of dihydroxy fatty acid glycerides against human adenocarcinoma cell line DLD-1
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=3 0.4035 0.0131 1 0.00
PC-1 Cells + 5-FU, 0.65 pg/ml, n=3 0.3626 0.0392 NS 10.1
PC-2 Cells + 5-FU, 1.95 pg/ml, n=3 0.3173 0.0267 NS 21.3
PC-3 Cells + MGP, 50 pg/ml, n=3 0.1133 0.0111 1.6E-04 71.9
S#12 Cells + GLY-lAc-1, 50 gg/ml, n=3 0.1277 0.0008 1.1E-04 68.3
S#13 Cells + GLY-lAc-2, 50 gg/ml, n=2 0.0347 0.0104 6.4E-04 91.4
S#14 Cells + GLY-OAc-1, 50 gg/ml, n=2 0.0810 0.0036 8.3E-04 79.9
S#15 Cells + GLY-OAc-2, 50 gg/ml, n=3 0.0979 0.0141 1.8E-04 75.7
NC-2 Cells only (trial 2), n=6 0.3269 0.0379 1 0.00
S#4a Cells + GLY-lAc-1, 50 pg/ml (trial 2), n=6 0.0191 0.0193 1.5E-05 94.2
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S#4b Cells + GLY-lAc-1, 25 pg/ml 0.2832 0.0134 NS 13.3
(trial 2), n=6
The inhibition of proliferation for all monoglycerides with one or zero acetates attached to glycerol was in the range 58 - 91 % when tested at 50 pg/ml. The most potent of these compounds in the first assay was the mono-acetylated dihydroxy fatty acid glyceride GLYlAc-2. The antiproliferative activity of these dihydroxy fatty acid glycerides was similar to or superior to the positive control monoglycerol palmitate (MGP), also tested at 50 pg/ml. GLYlAc-1 was retested at 50 and 25 pg/ml. The antiproliferative activity was 94 % at 50 pg/ml but only 13 % at 25 pg/ml, and so the LD50 for this compound is somewhere between 25 and 50 pg/ml. The 5-fluorouracil had low and non-significant activity at the two concentrations tested of 0.55 and 1.95 pg/ml.
Example 10: Antiproliferative activity of dihydroxy fatty acid glycerides mixtures, derivates and cyclodextrin complexes against human adenocarcinoma cell line DLD-1
This example shows the antiproliferative activity against the human colon cancer cell line DLD-1 of a purified glyceride fraction GLY-conc; derivatives of the purified glycerides including free fatty acids (FFA-conc), methyl esters (ME-conc) and peracetylated glycerides (PerAc-conc); and cyclodextrin complexes of the glycerides g-CD GLY-conc (gammacyclodextrin encapsulated glyceride concentrate), and α-CD GLY-conc (alpha-cyclodextrin encapsulated glyceride concentrate). These fractions and samples were prepared as described in Examples 4 and 5, and were tested for anti-proliferative activity at a final effective concentration of 50 pg/ml glyceride concentrate against colon adenocarcinoma cell line DLD-1. The cyclodextrin-encapsulated glycerides samples containing 25 % by mass glyceride concentrate were tested at a concentration of 200 pg/ml, which is equivalent to 50 pg/ml glycerides. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 5.5,
19.5 and 58.5 pg/ml; and PC-4 consisting of glycerol monopalmitate tested at 50 pg/ml.
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The bioassay results are shown in Table 10. In the Table, OD is Optical Density measured at
570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical
Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 10: Antiproliferative activity of dihydroxy fatty acid glyceride concentrates, derivatives and complexes against human colon adenocarcinoma cell line DLD-1
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.3269 0.0379 1 0.00
PC-1 Cells + 5-FU, 6.5 pg/ml, n=6 0.3222 0.0337 NS 1.44
PC-2 Cells + 5-FU, 19.5 pg/ml, n=6 0.3301 0.0202 NS 0.00
PC-3 Cells + 5-FU, 58.5 pg/ml, n=6 0.2883 0.0054 NS 11.8
PC-4 Cells + MGP, 50 pg/ml, n=5 0.0052 0.0022 3.1E-05 98.4
S#9 Cells + GLY-conc, 50 pg/ml, n = 5 0.0600 0.0126 1.6E-04 81.6
S#10 Cells + FFA-conc, 50 gg/ml, n=6 0.2935 0.0074 NS 10.2
S#ll Cells + ME-conc, 50 pg/ml, n=6 0.1720 0.0061 2.4E-03 47.4
S#12 Cells + PerAc-conc, 50 gg/ml, n=6 0.2648 0.0152 NS 19.0
S#13 Cells + g-CD GLY-conc, 200 gg/ml, n=6 0.2356 0.0104 4.2E-02 27.9
S#15 Cells + α-CD GLY-conc, 200 gg/ml, n=6 0.2205 0.0121 2.3E-02 32.6
This example shows that a glyceride mixture isolated from NZ propolis; derivatives of the glycerides including free fatty acids, alkyl esters and peracetates; and cyclodextrinencapsulated glycerides have novel anti-gastrointestinal cancer activity against the human colon adenocarcinoma cell line DLD-1. The most potent of the test substances, with
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123 inhibition of proliferation of 82 % is the monoglyceride concentrate. The most potent of the derivatives and complexes were the methylesters at 47 % inhibition followed by the alpha and gamma cyclodextrin complexes at 33 and 28 % respectively. The peracetates and free fatty acids had the lowest antiproliferative activity at 19 and 10 % respectively. It is likely that the test concentration of these derivatives was too low. The 5-fluorouracil had low and non-significant activity at the concentrations tested of 5.5, 19.5 and 58.5 pg/ml.
Example 11: Antiproliferative activity of dihydroxy fatty acid glycerides against human gastric carcinoma cell line NCI-N87
This example shows the antiproliferative activity of individual glycerides GLY-lAc-1 and GLY-lAc-2 (dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol, C20,
C21, C22 fatty acid chain lengths), GLY-0Ac-l, GLY-OAc-2, GLY-OAc-3 (non-acetylated dihydroxy fatty acid glycerides, C20, C21, C22 fatty acid chain lengths) and GLY-2AC-1 mix (dihydroxy fatty acid glyceride acetylated at positions C2 and C3 on the glycerol, mixture of dihydroxy fatty acid chain lengths) were tested for anti-proliferative activity at a final concentration of 50 pg/ml against human gastric carcinoma cell line NCI-N87. GLY-lAc-1 was also tested at 25 and 10 pg/ml to provide an indicative dose response. The cell proliferation assay was performed as described in Examples 1 and 2. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 5.5, 19.5 and 58.5 pg/ml; and PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml. The bioassay results are shown in
Table 11. In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard
Error of the Measurement associated with the Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05; % inhibition is the percentage reduction of proliferation compared
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Table 11: Antiproliferative activity of dihydroxy fatty acid glycerides against human gastric cancer cell line NCI-N87
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-l Cells only, n=6 0.0864 0.0128 1.00 0.00
PC-1 Cells + 5-FU, 6.5 pg/ml, n=6 0.0693 0.0062 NS 19.83
PC-1 Cells + 5-FU, 19.5 pg/ml, n=6 0.0792 0.0067 NS 8.32
PC-1 Cells + 5-FU, 58.5 pg/ml, n=6 0.0793 0.0023 NS 8.22
PC-4 Cells + MGP, 50 pg/ml, n=6 0.0000 0.0000 N/A 100
S#1 Cells + GLY-OAc-1, 50 pg/ml, n=6 0.0000 0.0000 N/A 100.00
S#2 Cells + GLY-OAc-2, 50 pg/ml, n = 6 0.0000 0.0000 N/A 100.00
S#3 Cells + GLY-OAc-3, 50 pg/ml, n=6 0.0000 0.0000 5.0E-05 99.97
S#4a Cells + GLY-lAc-1, 50 pg/ml, n=6 0.0000 0.0000 N/A 100.00
S#4b Cells + GLY-lAc-1, 25 pg/ml, n=2 0.0118 0.0016 1.9E-02 86.38
S#4c Cells + GLY-lAc-1, 10 pg/ml, n=6 0.0530 0.0041 3.2E-02 38.61
S#7 Cells + GLY-lAc-2, 50 pg/ml, n = 6 0.0000 0.0000 N/A 100.00
S#8 Cells + GLY-2AC-1 mix, 50 pg/ml, n=4 0.0414 0.0042 2.5E-02 52.08
This example shows that a large number of glyceride compounds present in NZ propolis have novel and very potent anti-gastrointestinal cancer activity against the human gastric cancer cell line NCI-N87. All the isolated no-acetate and monoacetate compounds were cytotoxic (100 % inhibition of proliferation) when tested at 50 pg/ml. The mixture of diacetate glyceride compounds, GLY-2AC-1 mix were strongly antiproliferative but not
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125 cytotoxic at 52 % inhibition. Reducing the test concentration of the mono-acetate compound GLY-lAc-1 from 50 to 25 pg/ml reduced the anti-proliferative activity to 86 % (still very strongly active). Reducing the test concentration still further from 25 to 10 pg/ml reduced the anti-proliferative activity to 39 %. The LD50 is therefore somewhere between and 10 pg/ml. The 5-fluorouracil had low and non-significant activity at the concentrations tested of 6.5, 19.5 and 58.5 pg/ml, with the highest activity obtained at 6.5 pg/ml. The glycerol monopalmitate (MGP) was also cytotoxic at the test concentration of 50 pg/ml.
Example 12: Antiproliferative activity of dihydroxy fatty acid glycerides mixtures, derivatives and cyclodextrin complexes against human gastric carcinoma cell line NCI-N87
This example shows the antiproliferative activity against the human gastric carcinoma cell line NCI-N87 of a purified glyceride fraction GLY-conc; derivatives of the purified glycerides including free fatty acids (FFA-conc), methyl esters (ME-conc) and peracetylated glycerides (PerAc-conc); and cyclodextrin complexes of the glycerides g-CD GLY-conc (gamma-cyclodextrin encapsulated glyceride concentrate), and α-CD GLY-conc (alphacyclodextrin encapsulated glyceride concentrate). These fractions and samples were prepared as described in Examples 4 and 5, and were tested for anti-proliferative activity at a final effective concentration of 50 pg/ml glyceride concentrate against gastric carcinoma cell line NCI-N87. The cyclodextrin-encapsulated glycerides samples containing 25 % by mass glyceride concentrate were tested at a concentration of 200 pg/ml, equivalent to 50 pg/ml glycerides. The test samples were compared against the negative control NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 6.5,
19.5 and 58.5 pg/ml; and PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml. The bioassay results are shown in Table 12. In the Table, OD is Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the
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Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 12: Antiproliferative activity of dihydroxy fatty acid glyceride concentrates, derivatives and complexes against human gastric carcinoma cell line NCI-N87
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.0864 0.0128 1.000 0.00
PC-1 Cells + 5-FU, 6.5 pg/ml, n=6 0.0693 0.0062 NS 19.83
PC-1 Cells + 5-FU, 19.5 pg/ml, n=6 0.0792 0.0067 NS 8.32
PC-3 Cells + 5-FU, 58.5 pg/ml, n=6 0.0793 0.0023 NS 8.22
PC-4 Cells + MGP, 50 pg/ml, n=6 0.0000 0.0000 N/A 100
S#9 Cells + GLY-conc, 50 pg/ml, n = 2 0.0150 0.0039 2.2E-02 82.65
S#10 Cells + FFA-conc, 50 pg/ml, n=6 0.0388 0.0056 6.6E-03 55.14
S#ll Cells + ME-conc, 50 pg/ml, n = 5 0.0260 0.0039 2.5E-03 69.86
S#12 Cells + PerAc-conc, 50 pg/ml, n=4 0.0347 0.0058 1.4E-02 59.89
S#13 Cells + g-CD GLY-conc, 200 pg/ml, n=5 0.0299 0.0042 3.8E-03 65.41
S#14 Cells + β-CD GLY-conc, 200 pg/ml, n=4 0.0193 0.0033 3.2E-03 77.67
S#15 Cells + α-CD GLY-conc, 200 pg/ml, n=6 0.0274 0.0022 1.0E-03 68.34
This example shows that a glyceride mixture isolated from NZ propolis; derivatives including free fatty acids, alkyl esters and peracetates; and cyclodextrin-encapsulated glycerides have novel anti-gastrointestinal cancer activity against the human gastric carcinoma cell line NCI-N87. The most potent of the test substances, with inhibition of
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127 proliferation of 82 % is the monoglyceride concentrate. The most potent of the derivatives and complexes were the methylesters at 70 % inhibition, beta-cyclodextrin complex at 78 % alpha cyclodextrin complex at 68 % and gamma cyclodextrin complex at 65 % respectively. The peracetates and free fatty acids had lower but still strong antiproliferative activity at 60 and 55 % respectively. The 5-fluorouracil had low and non-significant activity at the concentrations tested of 6.5, 19.5 and 58.5 pg/ml, with the highest activity obtained at 6.5 pg/ml. The glycerol monopalmitate (MGP) was cytotoxic at the test concentration of pg/ml.
Example 13: Antiproliferative activity of dihydroxy fatty acid glycerides against human oesophageal carcinoma cell line KYSE-30
This example shows the antiproliferative activity of individual glycerides GLY-lAc-1 and GLY-lAc-2 (dihydroxy fatty acid glyceride acetylated at position C3 of the glycerol, C20,
C21 fatty acid chain length respectively), GLY-0Ac-l, GLY-OAc-2, GLY-OAc-3 (nonacetylated dihydroxy fatty acid glycerides, C20, C21, C22 fatty acid chain length respectively) and GLY-2AC-1 mix (dihydroxy fatty acid glyceride acetylated at positions C2 and C3 of the glycerol, mixture of fatty acid chain lengths) were tested for anti-proliferative activity at a final concentration of 50 pg/ml against human oesophageal carcinoma cell line
KYSE-30. GLY-lAc-1 was also tested at 25 and 10 pg/ml to provide an indicative dose response. The cell proliferation assay was performed as described in Examples 1 and 2. The test samples were compared against the negative control NC-1 (cells only); positive controls
PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 6.5, 19.5 and 58.5 pg/ml;
and PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml. The bioassay results are shown in Table 13. In the Table, OD is Optical Density measured at 570 nm;
SEM is the Standard Error of the Measurement associated with the Mean Optical Density value measured (NS = not significant); p is the probability value that the measurement is
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128 statistically significant via the Student t-test, here taken to be < 0.05; % inhibition is the percentage reduction of proliferation compared to the negative control, with a large number indicating the test compound has anticancer proliferation potential.
Table 13: Antiproliferative activity of dihydroxy fatty acid glycerides against 5 human oesophageal carcinoma cell line KYSE-30
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.1519 0.0072 1.000 0.00
PC-1 Cells + 5-FU, 6.5 pg/ml, n=6 0.1336 0.0083 NS 12.02
PC-2 Cells + 5-FU, 19.5 pg/ml, n=6 0.1407 0.0053 NS 7.34
PC-3 Cells + 5-FU, 58.5 pg/ml, n=6 0.1263 0.0064 2.4E-02 16.85
PC-4 Cells + MGP, 50 pg/ml, n=6 0.0385 0.0047 1.2E-07 74.68
S#1 Cells + GLY-OAc-1, 50 pg/ml, n = 5 0.0732 0.0131 3.7E-04 51.78
S#2 Cells + GLY-OAc-2, 50 pg/ml, n = 6 0.1076 0.0087 2.9E-03 29.13
S#3 Cells + GLY-OAc-3, 50 pg/ml, n=4 0.0269 0.0045 1.2E-06 82.28
S#4a Cells + GLY-lAc-1, 50 pg/ml, n=4 0.0206 0.0062 2.1E-07 86.46
S#4b Cells + GLY-lAc-1, 25 pg/ml, n=5 0.0766 0.0097 1.3E-04 49.55
S#4c Cells + GLY-lAc-1, 10 pg/ml, n=6 0.1378 0.0083 NS 9.23
S#7 Cells + GLY-lAc-2, 50 pg/ml, n = 5 0.1006 0.0163 1.2E-02 33.76
S#8 Cells + GLY-2AC-1 mix, 50 pg/ml, n=6 0.1137 0.0033 7.3E-04 25.13
This example shows that a number of glyceride compounds present in NZ propolis have novel and potent anti-gastrointestinal cancer activity against the human oesophageal carcinoma cell line KYSE-30. All the isolated no-acetate and monoacetate compounds were moderately to strongly antiproliferative (34 - 86 %) when tested at 50 pg/ml, with the most
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LD50 is therefore around 25 pg/ml. The 5-fluorouracil had low but significant activity of 17 % when tested at 58.5 pg/ml, but non-significant activity at the lower concentrations of 6.5 and 19.5 pg/ml. The glycerol monopalmitate (MGP) had strong antiproliferative activity of % when tested at 50 pg/ml.
Example 14: Antiproliferative activity of dihydroxy fatty acid glycerides mixtures, and derivatives against human oesophageal squamous carcinoma cell line KYSE30
This example shows the antiproliferative activity against the human oesophageal squamous carcinoma cell line KYSE-30 of a purified glyceride fraction GLY-conc; derivatives of the purified glycerides including free fatty acids (FFA-conc), methyl esters (ME-conc) and peracetylated glycerides (PerAc-conc). These fractions and samples were prepared as described in Examples 4 and 5, and were tested for anti-proliferative activity at a final effective concentration of 50 pg/ml glyceride concentrate against oesophageal squamous carcinoma cell line KYSE-30. The test samples were compared against the negative control
NC-1 (cells only); positive controls PC-1, PC-2, PC-3 consisting of 5-FU at a test concentration of 6.5, 19.5 and 58.5 pg/ml; and PC-4 consisting of glycerol monopalmitate (MGP) tested at 50 pg/ml. The bioassay results are shown in Table 14. In the Table, OD is
Optical Density measured at 570 nm; SEM is the Standard Error of the Measurement associated with the Mean Optical Density value measured; p is the probability value that the measurement is statistically significant via the Student t-test, here taken to be < 0.05 (NS = not significant); % inhibition is the percentage reduction of proliferation compared to the
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Table 14: Antiproliferative activity of dihydroxy fatty acid glyceride concentrates and derivatives against human oesophageal squamous carcinoma cell line KYSE5 30
Sample ID & number of replicates, n Mean (OD 570nm) SEM P Values (<0.05) % Inhibition
NC-1 Cells only, n=6 0.1519 0.0072 1.000 0.00
PC-1 Cells + 5-FU, 6.5 pg/ml, n=6 0.1336 0.0083 NS 12.02
PC-2 Cells + 5-FU, 19.5 pg/ml, n=6 0.1407 0.0053 NS 7.34
PC-3 Cells + 5-FU, 58.5 pg/ml, n=6 0.1263 0.0064 2.4E-02 16.85
PC-4 Cells + MGP, 50 pg/ml, n=6 0.0385 0.0047 1.2E-07 74.68
S#9 Cells + GLY-conc, 50 pg/ml, n=6 0.1137 0.0033 7.2E-04 44.03
S#10 Cells + FFA-conc, 50 pg/ml, n=6 0.0850 0.0039 1.0E-05 21.47
S#ll Cells + ME-conc, 50 pg/ml, n=6 0.1193 0.0028 1.8E-03 27.59
S#12 Cells + PerAc-conc, 50 pg/ml, n=6 0.1100 0.0028 2.9E-04 32.03
This example shows that a glyceride mixture isolated from NZ propolis; derivatives including free fatty acids, alkyl esters and peracetates have novel anti-gastrointestinal cancer activity against the human oesophageal squamous carcinoma cell line KYSE-30. The most potent of the test substances, with inhibition of proliferation of 44 % is the monoglyceride concentrate. The most potent of the derivatives were the peracetates at 32 % inhibition. The methyl esters and free fatty acids had slightly lower antiproliferative activity at 28 and 21 % respectively. The 5-fluorouracil had low but significant activity of
16.8 % inhibition at the concentration tested of 58.5 pg/ml, but non-significant activity at
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Example 15: Isolation of dihydroxy fatty acid glycerides from Poplar trees.
This example shows that certain species or hybrids of poplar are a source for di hydroxy fatty acid glycerides found in New Zealand propolis, and are thus an alternative botanical source of these glycerides to propolis or to chemical synthesis.
Samples of poplar buds, twigs and leaf material were collected from the Plant and
Food Research poplar collection at Aokautere (Manawatu, NewZealand) and from the
Wellington Regional Council nursery at the Akura Conservation Centre (Masterton, New
Zealand) from September to February. Most samples were from coppiced trees as these were easily accessed.
Samples were weighed and extracted with absolute ethanol at room temperature for approximately 2 hrs. The samples were extracted as is without maceration or slicing. The extracts were dried and weighed and samples of each prepared for LC-MS analysis by making up solutions in ethanol at 5 mg/ml. Samples of leaf and flower material were collected from two Paulownia tomentosa trees, and from mature willow tree leaf and buds in the Hutt Valley, for comparative analysis. The material was extracted by dipping the plant material into ethyl acetate to extract the waxy outer layers of the material only.
All samples were analysed using the LC-MS method developed for the glycerides present in propolis as described in Example 4. This analysis method was able to detect the novel dihydroxy fatty acid glycerides at very low levels, along with other glycerides (if present).
Results
Qualitative levels of dihydroxy fatty acid glycerides in twig, leaf or buds of a variety of poplar species are shown in Table 15.
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Table 15: Qualitative glyceride content in New Zealand poplar cultivars.
Cultivar Populus hybrid/clone Plant part
Twig Leaf Bud
Kulu ciliata Wall, ex Royle np np ND
Oxford maximowiczii Henry x berolinensis Dipp. np np ND
Pecam maximowiczii x nigra np np ND
Selwyn deltoides x nigra + + + + + + ND
Tasman X euramericana Guinier (= X canadensis Moench) + + + + + + +
Luisa Avanzo X euramericana Guinier (= X canadensis Moench) + + + + + + ND
Fraser X euramericana Guinier (= X canadensis Moench) + + + + + ND
Yunnan yunnanensis Dode np np ND
Geyles maximowiczii x nigra np np ND
Italica nigra L. np np ND
np = glycerides not present, ++ moderate levels of glycerides, + + + high levels of glycerides, ND = not determined.
The analysis showed that the same dihydroxy fatty acid glycerides seen in propolis were present in many of the more commonly planted New Zealand poplar cultivars analysed. The glycerides were found on leaves, twigs and buds of many poplars and appeared to be generally associated with the resin on these parts.
The fatty acid profile for the glycerides was mostly the same as that seen in the propolis, suggesting poplar is the source of the propolis glycerides. It is anticipated that the individual glycerides could be isolated from poplar exudates in a similar manner to that used in Example 4. Poplar cultivars such as 'Tasman' and 'Fraser' are typical examples of the widely planted Populus x Euramerica (also known as Canadian poplar and Carolina poplar) while Populus deltoides x nigra hybrids such as Selwyn are also common in New Zealand.
The dihydroxy fatty acid glycerides were generally found to be absent in some of the other cultivars such as the P. maximowiczii x nigra clones and the Chinese poplar species 'Yunan'.
Glycerides were absent in hybrids not containing a cross with deltoides. These types of
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133 glycerides were also absent in samples of willow leaf and buds, while in Paulownia low levels of different types of glycerides were present, showing that these plant species are not the source of the glyceride in propolis, and are not suitable plant species for isolating the glyceride compounds described herein.
Example 16: Determination of the chirality of the novel glycerides
This example shows that the naturally occurring novel dihydroxy fatty acid glycerides have 3R, 8R stereochemistry. Chiral analysis of alcohol groups of organic molecules is generally performed by using chiral derivatisation reagents. These reagents are available in
R and S forms and usually contain an aromatic group which when attached to the alcohol group is able to affect, by shielding, the 1H NMR chemical shifts of neighbouring groups.
The variation in the shift of neighbouring groups for the separately prepared R and S derivatives indicates the original alcohol chirality. For the novel glycerides the stereochemistry of the alcoholic groups of the 3, 8-dihydroxy fatty acids is of most interest.
As the length of the fatty acid chain has no effect of the chemical shift of the protons at C2,-3 and -8 positions, a crude glyceride mixture was used for this work.
Preparation of R, S diesters and 1H-NMR analysis
The crude glyceride mixture was hydrolysed using LiOH as described in Example 4.
The free fatty acid methyl ester was then prepared from the hydrolysed glyceride mixture as described in Example 4 by using diazomethane. R- and S- α-methoxyphenylacetic acid (MPA), N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and 4dimethylaminopyridine (DMAP) were purchased from Sigma. The chiral ester derivatisation method used was that described by Freire et al 2005. 10 mg of the dihydroxy free fatty acid methyl ester mixture was dissolved in approximately 0.8 mL CH2CI2 (dry) in a 5 mL reaction vial. The solution was stirred at room temperature using a magnetic stirrer and 23 mg of Rα-methoxyphenylacetic acid, 10 pL of EDC and a catalytic amount of DMAP were added.
The reaction was worked up by washing the reaction solution with water, 1M HCI, sodium
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134 bicarbonate and water. The organic layer was dried under vacuum and purified using a small silica gel column to obtain the diester product (approximately 8 mg). The S-diester was obtained in a similar fashion. 1H NMR samples were dissolved in CDCI3 and run at
500MHz. The products were then examined by 1H NMR.
Results and discussion
The signals for H-2, H-3 and H-8 were sufficiently well separated for detailed analysis.
The first observation was that the two products were single species by NMR. This would only be the case if the original alcohols were chiral, i.e. the C-3 and C-8 hydroxyls are stereospecific, each either R or S. The changes in the shifts of these derivatives, Ad(S-R), are shown in Table 16. Based on comparison with similar compounds in the literature (Tables 17 and 18), the results show that the stereochemical configuration is 3R, 8R for all the glycerides.
Table 16: 1H NMR chemical shift differences between R and S MPA esters for
3,8 dihydroxy fatty acid methyl esters.
Hydrogen δ δ Di R δ Di S Δ 6(S-R)
Di hydroxy fatty fatty acid fatty acid
acid methyl methyl ester methyl ester
ester
Ha-C(2) 2.5 2.6 2.43 -0.17
Hb-C(2) 2.41 2.5 2.35 -0.15
H-3 4.0 5.22 5.15 -0.07
H-8 3.6 4.8 4.9 + 0.1
Confirmation of stereochemistry by comparison with the literature
The changes in the shifts of Δδ(5-Κ) can then be compared with relevant examples from the literature to confirm the stereochemistry. There are two examples in the literature
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135 where direct comparisons are valid and a similar derivatisation agent has been used, these are 3,7-diacetoxy docosahexanoic acid, otherwise known as byrsonic acid (Reis et al 2007), and l-acetyl-2-(3,7-diacetoxyeiconsaonyl)-glycerol, otherwise known as oncidinol (Reis et al 2003). Byrsonic acid was hydrolysed to remove the acetoxy groups to produce a 3,7 dihydroxy fatty acid. 3,7 diesters were then prepared using the chiral derivatisation agent
Mosher's acid (methoxy{trifluoromethyl}phenylacetic acid), and were then examined by 1H
NMR. The changes in Δ 5(S-R) were as shown in Table 17 which allowed Reis et al 2007 to assign the acid as (3R,7/?)-3,7-dihydroxy docosanoic acid.
Table 17: 1H NMR chemical shift differences between R and S Mosher esters for 3,7 dihydroxy fatty acid methyl esters derived from byrsonic acid.
Hydrogen Δ 0(S-R)
Ha-C(2) -0.11
Hb-C(2) -0.13
H-3 -0.08
H-7 + 0.07
Similarly, Reis et al 2003 also hydrolyzed oncidinol to remove the glycerol and acetoxy groups to give a 3,7 dihydroxy fatty acid, the acid was derivatized to a diester using Mosher acid and the 3,7 diester was examined by 1H NMR. The changes in Ad(S-R) as shown in
Table 18 enabled the acid to be assigned as a 3R,7R dihydroxy acid.
Table 18: 1H NMR chemical shift differences between R and S Mosher esters for 3,7 dihydroxy fatty acid methyl esters derived from oncidinol.
Hydrogen Δ 0(S-R)
Ha-C(2) -0.11
Hb-C(2) -0.12
H-3 -0.06
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H-7 + 0.08
For both byrsonic acid and oncinidol, the results are very similar to those seen for the novel glycerides in Table 15. The Ad(S-R) values are negative for H-2 and H-3 and positive for the remote hydroxyl position H8, confirming that the stereochemical configuration is 3R,
8R for all the glycerides.
Freire F, Seco JM, Quinoa E, Riguera R. The prediction of the absolute stereochemistry of primary and secondary 1,2-diols by 1H NMR spectroscopy: principles and applications.
Chemistry 2005 19;ll(19):5509-22).
Reis, M.G., De Faria, A.D., Dos Santos, I.A., Amaral, M.D.C.E., Marsaioli, A.J. Byrsonic acid
- The clue to floral mimicry involving oil-producing flowers and oil-collecting bees.
Journal of Chemical Ecology. 2007, 33 (7), pp. 1421-1429
Reis, M.G., De Faria, A.D., Do Amaral, M.D.C.E., Marsaioli, A.J. Oncidinol - A novel diacylglycerol from Ornithophora radicans Barb. Rodr. (Orchidaceae) floral oil. 2003.
Tetrahedron Letters. 44 (45), pp. 8519-8523
INDUSTRIAL APPLICABILITY
Anti-epithelial cancer compositions of this invention, including those comprising propolis or an extract or fraction thereof enriched in compounds of formula (I) and cyclodextrin, and those comprising isolated or purified compounds derived from propolis or fraction thereof can be used in the pharmaceutical and medical fields, for example in medical devices, medical supplies, and pharmaceuticals and in consumer goods including foods and beverages, compositions, nutraceuticals, cosmeceuticals, and functional foods.
Methods of using such compositions, for example in the treatment of epithelial cancers, such as skin cancers and symptoms thereof have application in the medical field.
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WHAT

Claims (16)

  1. WE CLAIM IS:
    1. A method of treating or preventing an epithelial cancer in a subject or of inducing apoptosis of one or more neoplastic epithelial cells in a subject, the method comprising administering an effective amount of one or more compounds of formula (I):
    OR4 or5
    R-i=OH, OR7or
    OR3 or2 (I) wherein R2, R3, FU and Rs are each independently H or acetyl (CH3CO-),
    R6 is H or CH3, R7 is CH3 or C2 to C6 saturated or unsaturated hydrocarbon and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17, or one or more pharmaceutically acceptable salts or solvates thereof, to a subject in need thereof.
  2. 2. A pharmaceutical composition comprising a therapeutically effective amount of a compound of a compound of formula (I):
    O OR4 OR5
    R-i=OH, OR7or
    O- or2
    WO 2017/164750
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    138 (I) wherein R2, R3, R4 and R5 are each independently H or acetyl (CH3CO-),
    R6 is H or CH3, R7 is CH3 or C2 to C6 saturated or unsaturated hydrocarbon and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17.
  3. 3. The composition of claim 2, wherein R6 is H.
  4. 4. The composition of claim 2, wherein R6 is CH3.
  5. 5. The pharmaceutical composition according to claim 2 or 3 comprising a therapeutically effective amount of at least one compound selected from any one or more of
    a) 3,8-dihydroxy eicosanoic acid,
    b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
    c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
    d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
    e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
    f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
    g) 3,8-dihydroxy eicosanoic acid methyl ester
    h) 3,8-dihydroxy heneicosanoic acid,
    i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
    j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
    k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
    l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
    m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
    n) 3,8-dihydroxy heneicosanoic acid methyl ester 0) 3,8-dihydroxy docosanoic acid,
    p) 1-(3,8-dihydroxy docosanoyl) glycerol,
    q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
    r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
    s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
    WO 2017/164750
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    139
    t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
    u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u), for use in treating or preventing an epithelial cancer.
    5. The use of a compound of formula (I):
    O OR4 OR5
    R-i=OH, OR7 or
    OR3 or2 (I) wherein R2, R3, R4 and Rs are each independently H or acetyl (CH3CO-),
    R
  6. 6 is H or CH3, R7 is CH3 or C2 to C5 saturated or unsaturated hydrocarbon and x and y are each independently an integer from 3 to 14, provided that when R6 is H, x+ y is greater than 10 and less than or equal to 18, and when R6 is CH3, x+y is greater than 9 and less than or equal to 17, in the manufacture of a composition for use in inhibiting epithelial tumour formation, inhibiting epithelial tumour growth, inhibiting epithelial tumour metastasis or treating or preventing epithelial cancer in a subject; inducing apoptosis of one or more neoplastic epithelial cells in a subject; increasing the responsiveness of a subject to an epithelial cancer therapy; increasing the sensitivity of an epithelial tumour in a subject to an epithelial cancer therapy; resensitising one or more epithelial cancer cells in a subject that are resistant to treatment; at least partially reversing the resistance of a neoplastic cell in a subject suffering from epithelial cancer to an epithelial cancer therapy; reversing, wholly or in part, the resistance of an epithelial cancer-burdened patient to an epithelial cancer therapy; or re-sensitising one or more tumours of an epithelial cancer-burdened patient which are, or are predicted to either be or become, resistant to
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    140 treatment with an epithelial cancer therapy to treatment with an epithelial cancer therapy.
  7. 7. The use of claim 5, wherein the subject is a human subject.
  8. 8. The use of claim 5 or 7, wherein the compound is a compound as defined in any one of claims 2, 3 or 4.
  9. 9. The use of at least one compound selected from any one or more of the group consisting of
    a) 3,8-dihydroxy eicosanoic acid,
    b) 1-(3,8-dihydroxy eicosanoyl) glycerol,
    c) 1-(3,8-dihydroxy eicosanoyl) 2-acetoxy glycerol,
    d) 1-(3,8-dihydroxy eicosanoyl) 3-acetoxy glycerol,
    e) 1-(3,8-dihydroxy eicosanoyl) 2,3-diacetoxy glycerol,
    f) 1-(3,8-diacetoxy eicosanoyl) 2,3-diacetoxy glycerol,
    g) 3,8-dihydroxy eicosanoic acid methyl ester
    h) 3,8-dihydroxy heneicosanoic acid,
    i) 1-(3,8-dihydroxy heneicosanoyl) glycerol,
    j) 1-(3,8-dihydroxy heneicosanoyl) 2-acetoxy glycerol,
    k) 1-(3,8-dihydroxy heneicosanoyl) 3-acetoxy glycerol,
    l) 1-(3,8-dihydroxy heneicosanoyl) 2,3-diacetoxy glycerol,
    m) 1-(3,8-diacetoxy heneicosanoyl) 2,3-diacetoxy glycerol,
    n) 3,8-dihydroxy heneicosanoic acid methyl ester
    o) 3,8-dihydroxy docosanoic acid,
    p) 1-(3,8-dihydroxy docosanoyl) glycerol,
    q) 1-(3,8-dihydroxy docosanoyl) 2-acetoxy glycerol,
    r) 1-(3,8-dihydroxy docosanoyl) 3-acetoxy glycerol,
    s) 1-(3,8-dihydroxy docosanoyl) 2,3-diacetoxy glycerol,
    t) 1-(3,8-diacetoxy docosanoyl) 2,3-diacetoxy glycerol, and
    u) 3,8-dihydroxy docosanoic acid methyl ester, or a pharmaceutically acceptable salt or solvate of any one of a) to u),
    WO 2017/164750
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    141 optionally with at least one additional therapeutic agent, in the manufacture of a composition for use in inhibiting epithelial tumour formation, inhibiting epithelial tumour growth, inhibiting epithelial tumour metastasis or treating or preventing epithelial cancer in a subject; inducing apoptosis of one or more neoplastic epithelial cells in a subject; increasing the responsiveness of a subject to an epithelial cancer therapy; increasing the sensitivity of an epithelial tumour in a subject to an epithelial cancer therapy; resensitising one or more epithelial cancer cells in a subject that are resistant to treatment; at least partially reversing the resistance of a neoplastic cell in a subject suffering from epithelial cancer to an epithelial cancer therapy; reversing, wholly or in part, the resistance of an epithelial cancer-burdened patient to an epithelial cancer therapy; or re-sensitising one or more tumours of an epithelial cancer-burdened patient which are, or are predicted to either be or become, resistant to treatment with an epithelial cancer therapy to treatment with an epithelial cancer therapy.
  10. 10. A composition of any one of claims 2 to 5 for use in inhibiting epithelial tumour formation, inhibiting epithelial tumour growth, inhibiting epithelial tumour metastasis or treating or preventing epithelial cancer in a human subject; inducing apoptosis of one or more neoplastic epithelial cells in a human subject; increasing the responsiveness of a human subject to an epithelial cancer therapy; increasing the sensitivity of an epithelial tumour in a human subject to an epithelial cancer therapy; resensitising one or more epithelial cancer cells in a human subject that are resistant to treatment; at least partially reversing the resistance of a neoplastic cell in a human subject suffering from epithelial cancer to an epithelial cancer therapy; reversing, wholly or in part, the resistance of an epithelial cancer-burdened human patient to an epithelial cancer therapy; or re-sensitising one or more tumours of an epithelial cancer-burdened human patient which are, or are predicted to either be or become, resistant to treatment with an epithelial cancer therapy to treatment with an epithelial cancer therapy.
  11. 11. The composition of claim 10 for use in the treatment or prevention of epithelial cancer.
  12. 12. A method of isolating or purifying a compound or mixture of compounds of formula (I) from propolis or poplar, or an extract or exudate thereof comprising the steps of
    WO 2017/164750
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    142
    a. providing a poplar extract, poplar-derived propolis, or an extract or exudate thereof, and
    b. isolating or purifying the compound from the poplar, propolis, or an extract or exudate thereof.
  13. 13. The method of claim 12 wherein the poplar extract, poplar-derived propolis, or extract or exudate thereof is from Populus deltoides, hybrids of Populus deltoids including Populus deltoides x nigra or Populus euramericana Guinier including cultivars, crosses and hybrids thereof.
  14. 14. The method of claim 13 wherein the Populus euramericana Guinier is a cultivar selected from the following group: Selwyn, Tasman, Luisa Avanzo, and Fraser.
  15. 15. The method of any one of claims 12 to 14 comprising one or more of the following steps:
    a. fractionating the poplar, propolis, or extract or exudate thereof by chromatography, for example, column chromatography, reverse phase chromatography, normal phase chromatography, or supercritical fluid chromatography, and/or solvent partitioning and/or supercritical extraction to produce one or more fractions comprising one or more of the compounds of formula (I),
    b. fractionating the poplar, propolis, or extract or exudate thereof or one or more fractions, by preparative HPLC and/or polymeric resin fractionation to produce one or more fractions comprising one or more of the compounds of formula (I), and, optionally,
    c. further purifying the one or more compounds of formula (I) from the one or more fractions of step b) and/or step c).
  16. 16. A composition or product comprising one or more compounds of formula (I) wherein the amount and/or concentration of one or more compounds of formula (I) is specified on an indicator associated with the composition or product.
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