CA2481664A1 - Prevention of breast cancer with novel selective estrogen receptor moldulators - Google Patents
Prevention of breast cancer with novel selective estrogen receptor moldulators Download PDFInfo
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- CA2481664A1 CA2481664A1 CA002481664A CA2481664A CA2481664A1 CA 2481664 A1 CA2481664 A1 CA 2481664A1 CA 002481664 A CA002481664 A CA 002481664A CA 2481664 A CA2481664 A CA 2481664A CA 2481664 A1 CA2481664 A1 CA 2481664A1
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- tamoxifen
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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Description
Prevention of Breast Cancer with Novel Selective Estrogen Receptor Modulators TECHMCAL FIELD
The present invention relates to medical uses of novel selective estrogen receptor modulators (SERM) in prevention and treatment of breast cancer. More particularly, the present invention concerns the use of a class of substituted triterpene sapogenin compounds for the prevention or treatment of breast cancer in a patient in need of such treatment.
BACKGROUND OF THE INVENTION
Breast cancer is one of the most common form of cancer and the most common cause of death in women in the world. For example, in British Columbia of Canada, according to Canadian Cancer Society, for 2004, an estimated 8,900 women will be diagnosed with some form of cancer and there will be 2,600 diagnoses of breast cancer.
A great deal of pharmacological and clinical investigation has been carded out attempting to elucidate the relationship between the hormone estrogen, and the cause, maintenance, and treatment of breast carcinoma.
Although a great deal is known about the relationship of estrogen in the maintenance and treatment of the disease, there is a great deal of controversy associated with the effect of estrogen on the epidemiology of this disease, i.e., whether estrogen is a causative agent (carcinogen) or an obligatory co-factor (permissive) in the initiation of the disease.
The estrogens, which include 17b-estradiol, estrone, and their active metabolites, are major sex-related hormones in women, but additionally, appear to be important homeostatic hormones in women throughout their adult life. Normally, everyone has some level of estrogen.
Hormone Replacement Therapy (HRT); recommended for postmenopausal and peri-menopausal women to alleviate cardiovascular disease, osteoporosis, and other menopausal sequelae, has generated a great deal of debate as to the potential of this therapy to increase the risk of contracting breast carcinoma.
In contrast to the problematic role of estrogen in the initiation of this disease, a great deal (although incomplete) of understanding has been achieved relating estrogen with established breast carcinoma.
Estrogen is a growth factor required by most breast carcinoma cells in the early stages of this disease. Also it has been established, but yet not fully understood why, that during the course of this disease the cancer cells lose their sensitivity to the effects of estrogen. Eventually, a majority of carcinoma cells become no longer dependent on estrogen for growth and are no longer responsive to any hormonally based therapy, which included "anti-estrogens", GNRH agonists, progestins, and androgens.
Endogenous estrogens are thought to play a major role in its development and estrogen receptor Mockers are important drugs in its treatment. It has been shown that longer exposures to estrogens result in an . .".. ," , ,,~_a,wa. ~ anuaam'4~: . . am~.m~vwr-, .v ~~n., rnm,.~,m..R ~
~nur~.~.a~.s ...~ww",..mear~, ~.wa:e ~ .F...y,-~ ..w », M. Ø
,m~,na.w.,..,~,.~, a~....~.~~;.:.;~rC~ax" ..,.. , ;:a,~;;,~,,;"~;as.:..arcm~e.,, ....."..,:,._....._."...._m. "._ ...".
increased risk for breast cancer. Estrogens have effects on many organ systems, beyond the reproductive system, in both females and males. Breast tissue is particularly dependent on circulating estrogens since there is no breast development in aromatase-deficient women and estrogen therapy of these patients leads to normal pre- and postpubertal breast development.
Estrogen effects are exerted through specific receptor: estrogen receptor (ER). The nuclear receptors are ligand-dependent transcription factors that mediate the biological effects of estrogens and anti-estrogens.
Estrogen receptors act mainly by regulating the expression of target genes whose promoters contain specific sequences called estrogen-responsive element (ERE).
After ERE-binding of ligand-bound ER dirners, modulation of transcription occurs via interaction with coactivators or corepressors. All together, these complexes play an important role in the recruitment of transcriptional machinery, the modulation of chromatine structure, and then in the regulation of ER
target-gene expression. The ER conformation differs with the type of ligand, and there is a marked difference in the topology of the ER surface between agonistand antagonist-bound receptors.
Moreover, studies conducted with synthetic anti-estrogens, such as tamoxifen, have shown that the agonist/antagonist profile of a ligand varies with the tissue and the target gene considered. This led to the term of selective estrogen receptor modulators (SERMs) to define this class of drug. ER activity can also be modulated through indirect activation of the ER by growth factors or cytokines independently of the binding of natural or synthetic hormones.
E, f,~'ects of SERMs Most recent drugs targeted to the ER, such as tamoxifen, ICI-164384, and raloxifene act as either ER
antagonists or agonists depending on the species, tissue, and the dose administered. For instance, raloxifene has been reported to act as an antiestrogen in breast tumor tissue and the brain, while it has potentially beneFcial estrogen-like effects in bone and in modulating factors associated with cardio-vascular diseases.
Another example is tamoxifen, which was developed as an antiestrogen for the treatment of breast cancer and was subsequently shown to have estrogen-like effects on bone and the cardiovascular system. However, the potentially beneficial effects of tamoxifen in reducing the risk of osteoporotic fractures and coronary heart disease in postmenopausal women are at least partially offset by its estrogenic effects on the uterus, increasing risk of endometrial cancer development.
A great deal of benefit has been achieved with the use of hormonally based therapeutic interventions. The most widely used therapy is the use of tamoxifen. The five-year survival rate for women with breast carcinoma has been dramatically improved with this therapy; however, the longer-term survival (ten-year+) rate has not improved to the same extent. This lack of improvement in the long-term rate has been attributed to the gradual evolution of the carcinoma cells from estrogen dependence to independence.
Tamoxifen is a nonsteroidal antiestrogen that has been used for the treatment of breast cancer since the early 1970s, successfully helping save the lives of millions of women with breast cancer. Tamoxifen works
The present invention relates to medical uses of novel selective estrogen receptor modulators (SERM) in prevention and treatment of breast cancer. More particularly, the present invention concerns the use of a class of substituted triterpene sapogenin compounds for the prevention or treatment of breast cancer in a patient in need of such treatment.
BACKGROUND OF THE INVENTION
Breast cancer is one of the most common form of cancer and the most common cause of death in women in the world. For example, in British Columbia of Canada, according to Canadian Cancer Society, for 2004, an estimated 8,900 women will be diagnosed with some form of cancer and there will be 2,600 diagnoses of breast cancer.
A great deal of pharmacological and clinical investigation has been carded out attempting to elucidate the relationship between the hormone estrogen, and the cause, maintenance, and treatment of breast carcinoma.
Although a great deal is known about the relationship of estrogen in the maintenance and treatment of the disease, there is a great deal of controversy associated with the effect of estrogen on the epidemiology of this disease, i.e., whether estrogen is a causative agent (carcinogen) or an obligatory co-factor (permissive) in the initiation of the disease.
The estrogens, which include 17b-estradiol, estrone, and their active metabolites, are major sex-related hormones in women, but additionally, appear to be important homeostatic hormones in women throughout their adult life. Normally, everyone has some level of estrogen.
Hormone Replacement Therapy (HRT); recommended for postmenopausal and peri-menopausal women to alleviate cardiovascular disease, osteoporosis, and other menopausal sequelae, has generated a great deal of debate as to the potential of this therapy to increase the risk of contracting breast carcinoma.
In contrast to the problematic role of estrogen in the initiation of this disease, a great deal (although incomplete) of understanding has been achieved relating estrogen with established breast carcinoma.
Estrogen is a growth factor required by most breast carcinoma cells in the early stages of this disease. Also it has been established, but yet not fully understood why, that during the course of this disease the cancer cells lose their sensitivity to the effects of estrogen. Eventually, a majority of carcinoma cells become no longer dependent on estrogen for growth and are no longer responsive to any hormonally based therapy, which included "anti-estrogens", GNRH agonists, progestins, and androgens.
Endogenous estrogens are thought to play a major role in its development and estrogen receptor Mockers are important drugs in its treatment. It has been shown that longer exposures to estrogens result in an . .".. ," , ,,~_a,wa. ~ anuaam'4~: . . am~.m~vwr-, .v ~~n., rnm,.~,m..R ~
~nur~.~.a~.s ...~ww",..mear~, ~.wa:e ~ .F...y,-~ ..w », M. Ø
,m~,na.w.,..,~,.~, a~....~.~~;.:.;~rC~ax" ..,.. , ;:a,~;;,~,,;"~;as.:..arcm~e.,, ....."..,:,._....._."...._m. "._ ...".
increased risk for breast cancer. Estrogens have effects on many organ systems, beyond the reproductive system, in both females and males. Breast tissue is particularly dependent on circulating estrogens since there is no breast development in aromatase-deficient women and estrogen therapy of these patients leads to normal pre- and postpubertal breast development.
Estrogen effects are exerted through specific receptor: estrogen receptor (ER). The nuclear receptors are ligand-dependent transcription factors that mediate the biological effects of estrogens and anti-estrogens.
Estrogen receptors act mainly by regulating the expression of target genes whose promoters contain specific sequences called estrogen-responsive element (ERE).
After ERE-binding of ligand-bound ER dirners, modulation of transcription occurs via interaction with coactivators or corepressors. All together, these complexes play an important role in the recruitment of transcriptional machinery, the modulation of chromatine structure, and then in the regulation of ER
target-gene expression. The ER conformation differs with the type of ligand, and there is a marked difference in the topology of the ER surface between agonistand antagonist-bound receptors.
Moreover, studies conducted with synthetic anti-estrogens, such as tamoxifen, have shown that the agonist/antagonist profile of a ligand varies with the tissue and the target gene considered. This led to the term of selective estrogen receptor modulators (SERMs) to define this class of drug. ER activity can also be modulated through indirect activation of the ER by growth factors or cytokines independently of the binding of natural or synthetic hormones.
E, f,~'ects of SERMs Most recent drugs targeted to the ER, such as tamoxifen, ICI-164384, and raloxifene act as either ER
antagonists or agonists depending on the species, tissue, and the dose administered. For instance, raloxifene has been reported to act as an antiestrogen in breast tumor tissue and the brain, while it has potentially beneFcial estrogen-like effects in bone and in modulating factors associated with cardio-vascular diseases.
Another example is tamoxifen, which was developed as an antiestrogen for the treatment of breast cancer and was subsequently shown to have estrogen-like effects on bone and the cardiovascular system. However, the potentially beneficial effects of tamoxifen in reducing the risk of osteoporotic fractures and coronary heart disease in postmenopausal women are at least partially offset by its estrogenic effects on the uterus, increasing risk of endometrial cancer development.
A great deal of benefit has been achieved with the use of hormonally based therapeutic interventions. The most widely used therapy is the use of tamoxifen. The five-year survival rate for women with breast carcinoma has been dramatically improved with this therapy; however, the longer-term survival (ten-year+) rate has not improved to the same extent. This lack of improvement in the long-term rate has been attributed to the gradual evolution of the carcinoma cells from estrogen dependence to independence.
Tamoxifen is a nonsteroidal antiestrogen that has been used for the treatment of breast cancer since the early 1970s, successfully helping save the lives of millions of women with breast cancer. Tamoxifen works
-2-r , .,. . * a~,.m ~ .. ~*, . ..*,n ~ -~.,~_ ~ < ~,r, a.a.a~ ~ ,. ~~. ~~. . ~
*""~", .-. n v y,,~..~"~ -by binding to the estrogen receptor in the breast and other estrogen-sensitive tissues, thereby preventing the subsequent binding of estrogen. A possible alternative mechanism of tamoxifen's antiestrogenic effect is through the increased production of sex-hormone binding globulin, a protein that binds estrogen and blocks its action. In addition to its antiestrogenic effects, tamoxifen may also inhibit cell division in cancer cells and promote the production of a certain type of tumor-fighting white blood cells called "natural killer cells".
Although tamoxifen acts as an estrogen °'blocker" on the tumor, tamoxifen has an estrogen-like effect on the bone, Liver, and endometrium.
Uses of tamoxifen include the treatment of rnetastatic breast cancer, adjuvant treatment of breast cancer, ductal carcinoma in situ (DCIS), and prevention of breast cancer in women who have a high-risk for getting the disease.
In addition to the benefit of decreasing the risk of developing breast cancer in women who are at high risk, tamoxifen has additional benefits for the bones and cholesterol. Tarnoxifen has been demonstrated to preserve bone in postmenopausal women, which may result in a lessened risk of bone fractures6.
Furthermore, tamoxifen is know to decrease levels of LDL cholesterol, and is associated with increases in HDL cholesterol and triglycerides, typical of the estrogen-like effect on the liver.
For the last decade it has been argued that "antiestrogen" therapy, especially the use of tamoxifen, should be examined for its potential to prevent de novo breast carcinoma. There are a number of side effects and safety concerns with tamoxifen treatment that need to be balanced with the potential benefits. The most troublesome side effects of tamoxifen are worsening of hot flushes, sweats, and vaginal discharge. While the incidence is very low, more serious concerns with tamoxifen treatment include blood clots, liver damage, endometrial cancer, and uterine sarcoma Recently, a "black box"
warning was added to the labeling of tamoxifen that includes a warning of uterine sarcoma in rarE;
instances of 0.17 women per 1000 a year who take tamoxifen. Worldwide, since the initial marketing of tamoxifen, only 159 cases of uterine sarcoma have been reported in women taking the drug. Women who have ever had blood clots should not take tamoxifen for the purpose of prevention of breast cancer. In addition, women who are pregnant, or plan to become pregnant should not take tamoxifen, as it classified as pregnancy category D and may cause birth defects. Due to a drug interaction that could result in bleeding, women who are taking a blood thinner known as Coumadin or warfarin should also avoid taking tamoxifen.
Tamoxifen has been established as a treatment of breast cancer that has benefits far outweighing the risks.
In addition, tamoxifen has demonstrated effectiveness in clinical trials to prevent DCIS from developing into invasive breast cancer, and in reducing the occurrence of breast cancer in women who are at high risk for developing the disease. However, it is in these two clinical scenarios that a woman, along with her healthcare provider, must carefully evaluate the benefits and risks to determine whether tamoxifen is the right treatment choice.
Thus, there is a great need for finding alternative SERM from phytobiocoenose sources which could reduce the side effects and preserve the SERM functions. So far there are about 400 phytoextrogens have been
*""~", .-. n v y,,~..~"~ -by binding to the estrogen receptor in the breast and other estrogen-sensitive tissues, thereby preventing the subsequent binding of estrogen. A possible alternative mechanism of tamoxifen's antiestrogenic effect is through the increased production of sex-hormone binding globulin, a protein that binds estrogen and blocks its action. In addition to its antiestrogenic effects, tamoxifen may also inhibit cell division in cancer cells and promote the production of a certain type of tumor-fighting white blood cells called "natural killer cells".
Although tamoxifen acts as an estrogen °'blocker" on the tumor, tamoxifen has an estrogen-like effect on the bone, Liver, and endometrium.
Uses of tamoxifen include the treatment of rnetastatic breast cancer, adjuvant treatment of breast cancer, ductal carcinoma in situ (DCIS), and prevention of breast cancer in women who have a high-risk for getting the disease.
In addition to the benefit of decreasing the risk of developing breast cancer in women who are at high risk, tamoxifen has additional benefits for the bones and cholesterol. Tarnoxifen has been demonstrated to preserve bone in postmenopausal women, which may result in a lessened risk of bone fractures6.
Furthermore, tamoxifen is know to decrease levels of LDL cholesterol, and is associated with increases in HDL cholesterol and triglycerides, typical of the estrogen-like effect on the liver.
For the last decade it has been argued that "antiestrogen" therapy, especially the use of tamoxifen, should be examined for its potential to prevent de novo breast carcinoma. There are a number of side effects and safety concerns with tamoxifen treatment that need to be balanced with the potential benefits. The most troublesome side effects of tamoxifen are worsening of hot flushes, sweats, and vaginal discharge. While the incidence is very low, more serious concerns with tamoxifen treatment include blood clots, liver damage, endometrial cancer, and uterine sarcoma Recently, a "black box"
warning was added to the labeling of tamoxifen that includes a warning of uterine sarcoma in rarE;
instances of 0.17 women per 1000 a year who take tamoxifen. Worldwide, since the initial marketing of tamoxifen, only 159 cases of uterine sarcoma have been reported in women taking the drug. Women who have ever had blood clots should not take tamoxifen for the purpose of prevention of breast cancer. In addition, women who are pregnant, or plan to become pregnant should not take tamoxifen, as it classified as pregnancy category D and may cause birth defects. Due to a drug interaction that could result in bleeding, women who are taking a blood thinner known as Coumadin or warfarin should also avoid taking tamoxifen.
Tamoxifen has been established as a treatment of breast cancer that has benefits far outweighing the risks.
In addition, tamoxifen has demonstrated effectiveness in clinical trials to prevent DCIS from developing into invasive breast cancer, and in reducing the occurrence of breast cancer in women who are at high risk for developing the disease. However, it is in these two clinical scenarios that a woman, along with her healthcare provider, must carefully evaluate the benefits and risks to determine whether tamoxifen is the right treatment choice.
Thus, there is a great need for finding alternative SERM from phytobiocoenose sources which could reduce the side effects and preserve the SERM functions. So far there are about 400 phytoextrogens have been
-3-_.~,. ~,~~~w..~ .. ate. _ ~.F .__ .~.~_ . _-.........,_ discovered from fruits, vegetables, and corns, etc. These phytoextrogens could be divided into following category from their different chemical structures: Isoflavonoids, Coumestans, Lignans, Stilbenes, Triterpenoids, Sterols, Cycle peptides, Chalcones, Flacanones, and Flavones.
Ginsenosides are group of steroid saponins extracted from Panax ginseng. They possess a four trans-ring rigid steroid skeleton with modified side-chains at C20. Some of the ginsenosides have been shown to bind to the same target proteins as steroidal hormones, including estrogen. It has been reported that ginsenoside Rg-1 had estrogen-like activity, could stimulate breast cancer cell growth and activate ERE
transcription in Hela cells in vitro, which could be blocked by estrogen antagonist ICI 182780. On the other hand, numerous studies demonstrated that some ginsenosides possess anti-cancer activities.
Ginsenoside Rc and Rh2 potently inhibited breast cancer cell proliferation.
Ginsenoside Rg3 could inhibit the growth and angiogenesis of xenotransplanted human breast infiltrating duct carcinoma in nude mice.
Protopanaxadiol type sapogenins are group of sapogenins with no sugar moieties in their chemical structures and without hydroxyl group in C6 position. Our previous studies demonstrated that one of the sapogenins, i.e. 20s-protopanaxadiol (aPPD), induced apoptosis in various tumor cells including glioma cells and breast cancer cells. It is believed that the apoptotic mechanisms involve both caspase-dependent and independent pathways. While the cellular target for aPPD has not been identified, it appears non-toxic for normal tissues, which renders this compound a good anti-cancer drug candidate. There is no report that has been found any sapogenins possesses SERM function.
Clearly, a great need exists for finding alternative SERM from phytobiocoenose sources which preserve the SERM function but reduce the side effect of current chemo-drugs, such as tamoxifen.
BRIEF SUMMARY OF THE INVENTION
This invention relates to a method for the prophylaxis or prevention of breast carcinoma. More particularly, this invention pertains to the use of a group of sapogenins, separately or together, as selective estrogen receptor modulators (SERM). In accordance with the present invention, there is provided in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula I):
20S-Protopanaxadiol
Ginsenosides are group of steroid saponins extracted from Panax ginseng. They possess a four trans-ring rigid steroid skeleton with modified side-chains at C20. Some of the ginsenosides have been shown to bind to the same target proteins as steroidal hormones, including estrogen. It has been reported that ginsenoside Rg-1 had estrogen-like activity, could stimulate breast cancer cell growth and activate ERE
transcription in Hela cells in vitro, which could be blocked by estrogen antagonist ICI 182780. On the other hand, numerous studies demonstrated that some ginsenosides possess anti-cancer activities.
Ginsenoside Rc and Rh2 potently inhibited breast cancer cell proliferation.
Ginsenoside Rg3 could inhibit the growth and angiogenesis of xenotransplanted human breast infiltrating duct carcinoma in nude mice.
Protopanaxadiol type sapogenins are group of sapogenins with no sugar moieties in their chemical structures and without hydroxyl group in C6 position. Our previous studies demonstrated that one of the sapogenins, i.e. 20s-protopanaxadiol (aPPD), induced apoptosis in various tumor cells including glioma cells and breast cancer cells. It is believed that the apoptotic mechanisms involve both caspase-dependent and independent pathways. While the cellular target for aPPD has not been identified, it appears non-toxic for normal tissues, which renders this compound a good anti-cancer drug candidate. There is no report that has been found any sapogenins possesses SERM function.
Clearly, a great need exists for finding alternative SERM from phytobiocoenose sources which preserve the SERM function but reduce the side effect of current chemo-drugs, such as tamoxifen.
BRIEF SUMMARY OF THE INVENTION
This invention relates to a method for the prophylaxis or prevention of breast carcinoma. More particularly, this invention pertains to the use of a group of sapogenins, separately or together, as selective estrogen receptor modulators (SERM). In accordance with the present invention, there is provided in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula I):
20S-Protopanaxadiol
-4-w.~,~v~~~-~ ~ ,x~~a-~.~,~.
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically ei~'ective amount of a compound having the structure (Formula III):
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula III):
Comuound III
Common Name: 3,12,20,26-tetrahydroxy-dammar-24z-ene;
Structural Formula:
OH
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula VI):
Compound VI
Common Name-. 3,12,20,25-tetrahydroxy-dammarane;
Structural Formula:
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically ei~'ective amount of a compound having the structure (Formula III):
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula III):
Comuound III
Common Name: 3,12,20,26-tetrahydroxy-dammar-24z-ene;
Structural Formula:
OH
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula VI):
Compound VI
Common Name-. 3,12,20,25-tetrahydroxy-dammarane;
Structural Formula:
-5-r . a.x., ... w ."~~, sw~a,."", ~roMu.~,.e.." ~yys s..,avSLms~..
a,a~usys..~~.rem~nm~x.,,....~x~;xMda,..~a"~w~s.-z"p~"~r."~,..~wm.;~.:~.avua~.~wr.~aar~...,.. ~ ~w .. »..~-u.-~,~.,.,_.,..-....__.~._.....,..-_.,.......
~~,iF°~Pro~0.C~xryrp~wwra.
HO ,r H
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula V):
Compound V
Common Name: 3,12,22-trihydroxy-22-de-isopentenyl-dammarane;
Structural Formula:
r a HO
or a pharmaceutically acceptable salt or pro-drug thereof, The invention further relates to a method for preventing breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for treatment of breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for prevention and treatment of breast cancer by administrating to human for a sufficient term an effective dose of tamoxifen and one or more compounds selected from formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has
a,a~usys..~~.rem~nm~x.,,....~x~;xMda,..~a"~w~s.-z"p~"~r."~,..~wm.;~.:~.avua~.~wr.~aar~...,.. ~ ~w .. »..~-u.-~,~.,.,_.,..-....__.~._.....,..-_.,.......
~~,iF°~Pro~0.C~xryrp~wwra.
HO ,r H
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula V):
Compound V
Common Name: 3,12,22-trihydroxy-22-de-isopentenyl-dammarane;
Structural Formula:
r a HO
or a pharmaceutically acceptable salt or pro-drug thereof, The invention further relates to a method for preventing breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for treatment of breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for prevention and treatment of breast cancer by administrating to human for a sufficient term an effective dose of tamoxifen and one or more compounds selected from formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has
-6-not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The compounds of the present invention are selective estrogen receptor modulators (SERM), that is, compounds which produce estrogen agonism in one or more desired target tissues while producing estrogen antagonism and/or minimal (i.e. clinically insignificant) agonism in reproductive tissue such as the breast or uterus.
DRAWINGS
In drawings which illustrate specific embodiments of the invention, but which should not be construed as restricting the spirit or scope of the invention in any way:
1. Figure 1 shows the binding assay to estrogen receptors.
2. Figure 2a shows the effect of aPPD on the activation of ERE-luc gene transcription in the txansiently ERE-luc transfected MCF-7 cells. This indicate aPPD had weak estrogenic effect 3. Figure 2b shows the inhibition effect of aPPD and Tamoxifen on the ER
activation.
4. Figure 2c shows the synergetic inhibitory effects of aPPD and Tamoxifen on the estrogen-induced ERE-luc transcriptional activation.
5. Figure 3 shows the aniproliferative effect of aPPD and Tamoxifen.
6. Figure 4a shows aPPD inhibits tumor initiation and progression of MCF-7 cells in mice xenografts.
The compounds of the present invention are selective estrogen receptor modulators (SERM), that is, compounds which produce estrogen agonism in one or more desired target tissues while producing estrogen antagonism and/or minimal (i.e. clinically insignificant) agonism in reproductive tissue such as the breast or uterus.
DRAWINGS
In drawings which illustrate specific embodiments of the invention, but which should not be construed as restricting the spirit or scope of the invention in any way:
1. Figure 1 shows the binding assay to estrogen receptors.
2. Figure 2a shows the effect of aPPD on the activation of ERE-luc gene transcription in the txansiently ERE-luc transfected MCF-7 cells. This indicate aPPD had weak estrogenic effect 3. Figure 2b shows the inhibition effect of aPPD and Tamoxifen on the ER
activation.
4. Figure 2c shows the synergetic inhibitory effects of aPPD and Tamoxifen on the estrogen-induced ERE-luc transcriptional activation.
5. Figure 3 shows the aniproliferative effect of aPPD and Tamoxifen.
6. Figure 4a shows aPPD inhibits tumor initiation and progression of MCF-7 cells in mice xenografts.
7. Figure 4b shows the comparison of inhibitory effects between aPPD and Tamoxifen on breast tumor growth in SCID Mice xenocraft DETAILED DESCRIPTION
DEFINITION
The terms "prevention of', "prophylaxis" and "prevent" includes reducing the likelihood of a patient incurring or developing breast cancer.
The term "de novo", as used in the current invention, means the lack of transformation or metamorphosis of normal breast cells to cancerous or malignant cells in the first instance.
Such a transformation may occur in stages in the same or daughter cells via an evolutionary process or may occur in a single, pivotal event.
This de novo process is in contrast to the metastasis, colonization, or spreading of already transformed or malignant cells from the primary tumor site to new locations. 'this invention also relates to the administration of a compound of formula I and Formula II to a patient who is at risk of developing de novo breast cancer.
The term "pro-drug," as used herein means a compound of the present invention bearing a group which is metabolically cleaved in a human to produce a therapeutically active compound of the present invention. In ...~,a.w ,"~.~.....~. .~.:~s:.~ ,~~:ae.:s ,.,ar,.e~~, :~:=fix:-rxx: ~-:..~:~.~v~.~.~--.~.. ~,<
... ~~r,-,.>._... . . ~, particular, such prodrug compounds include those in which either or both of the substituent groups R1, R2, R3, and R4 of the structure shown above or groups which are metabolically cleaved in the body to yield a corresponding compound of the present invention.
COMPOUNDS OF THE PRESENT INVENTION
Through the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
This invention relates to compounds of formula I are used as SERM for preventing breast cancer with following the structure (Formula I):
20S-ProtopartaxadPo!
or a pharmaceutically acceptable salt or pro-drug thereof.
The methods provided by this invention are practiced by administering to a patient in need thereof a dose of a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof, which is effective to prevent breast cancer.
Preferred compounds of the present invention include PAM-120 (Formula II):
_g_ ,_",rtm nmr,.v w' '6:..'d'U4 ..A'~~u:".."&~'"".
9N~~~;a~drYn',Yak'~P.yas5u.YAY~~a:.4YWrte:.amMmro-....wm-~.-..-...."_....."......
.G..yn...,.c~: ..sl.~wWA n .,_. y, ~ .. ~ rv-..,...~LY/~i~rs 4'~~~ - wrnxs.
.Ana..rer~..mw...-,-s...n,~.wm..~...,«
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula III):
Compound III
Common Name: 3,12,20,26-tetrahydroxy-dammar-24z-ene;
Structural Formula:
OH
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula Vd):
Compound VI
Common Name: 3,12,20,25-tetrahydroxy-dammarane;
Structural Formula:
HO
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula V):
Compound V
Common Name: 3,12,22-trihydroxy-22-de-isopentenyl-dammarane;
Structural Formula:
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
The invention further relates to a method for preventing breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for treatment of breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for prevention and treatment of breast cancer by administrating to human for a sufficient term an effective dose of tamoxifen and one or more compounds selected from formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The compounds of the present invention are selective estrogen receptor modulators (SERM), that is, compounds which produce estrogen agonism in one or more desired target tissues while producing estrogen antagonism andlor minimal (i.e. clinically insignificant) agonism in reproductive tissue such as the breast or uterus.
The present invention provided that aPPD is a partial agonist and antagonist for estrogen receptors, i.e.
SERM, Compounds with similar biocharactors have been known as selective estrogen receptor modulators (SEItMs). Like aPPD, the SERMs bind to estrogen receptors and have tissue-specific effects that allow them to function as estrogen agonists in some tissues and estrogen antagonists in other tissues. These compounds include clomiphene citrate, tamoxifen, toremifene and raloxifene.
Molecular mechanisms of SERM is believed to attribute to conformational modification of estrogen receptor protein after bound with different ligands, which affects the affinity of the receptor to other proteins that normally associate with the ... ,. ~",~...~ ~ ,.., ~ ra~. . r,~.l, :.;~A,~ ~ ~.-m,F~_.._...~~ . ~.,.u.. .-._~ ___...~___ . .~~~ o- "."M, ,~~.~,~...-,~~.-..
receptor for its physiological activity. Tamoxifen has been widely used in prevention and of breast cancer for the past decades due to its estrogen antagonistic effect on breast tissue.
Meanwhile, it also has an estrogen-like effect on lipids, bone, and the endornetrium. The agonistic effect on endometrium rnay relate to its reported contribution to the initiation and progression of endomel;rial cancer. It is not clear whether aPPD is a true SERM at the present time. The estrogen-related activity of aPPD
remains to be investigated in cells of other tissue origins and its conformational effect on estrogen receptor protein after bound needs to be understood.
The present invention provided that aglycone protopanaxadiol(aPPD) which possess the SERM functions by showing 1) aglycone protopanaxadiol can bind to estrogen receptors with a low affinity and activates the receptor to certain degree, indicated by its ability of stimulating ERE
mediated report gene expression; 2) aPPD can compete with estradiol for the E2 receptors and block its activity in stimulating ERE mediated gene expression; 3) aPPD can block E2 stimulated breast cancer cell proliferation both in vitro and in vivo;
4) aPPD can synergistically enhance the effect of Tarnoxifen for both antagonistic effect on E2 stimulated gene expression and the cytotoxicity on MCF7 cells.
The present invention provided a method of using aPPD together with tamoxifen can synergistically act with Tamoxifen to either block estrogen activity on gene regulation ar abolish estrogen stimulated cell proliferation. Tamoxifen has been known for its cytotoxicity(Petinari, 2004), mechanisms of which may relate to activation of caspases, elevation of intracellular calcium level and inhibition of insulin-like growth factor pathway(Jan, 2000; Kanter-Lewensohn, 2000; Salami, 2003). Tamoxifen is also known for its chemosensitizing effect when used in combination with other cytotoxic agents (Koh, 2003; Ramachandran, 2004). The synergistic cytotoxicity of Tamoxifen and aPPD occurred at the concentrations of 2uM and lOuM for each compounds, respectively. The synergy in cytotoxicity resulted in massive cell death that might be independent of their estrogen related activity. On the other hand, the synergy between aPPD and Tamoxifen were also demonstrated in their inhibition on estrogen stimulated activity and the synergy between aPPD and Tamoxifen occurred in both antiestrogenic and cytatoxic effects on the breast cancer cells. The synergetic effects between aPPD and tamoxifen could lead to reduce the side effect of tamoxifen by applying less dosage of tamoxifen. The synergistic effects may contribute to the powerful inhibition in estrogen-dependent breast cancer growth in the animal model. This was especially demonstrated in initial stage of tumor growth in E2-supported breast cancer model.
METHOD OF THE PRSENT dNVENTlON
Material 17 I3-Estradiol and Tamoxifen purchased from Sigma, Careseng and aPPD supplied by Pegasus Pharmaceuticals Inc., [3H] 17- 13 estadiol (89 Ci/mmol), purchased from Perken Elmer, were dissolved in absolute ethanol as stock solutions. Before applied to the cultured cells, each of the compounds was diluted into 1000 times solution with ethanol and then further diluted with fresh culture media into working -II-solutions. MTT, from Sigma, was dissolved in RPMI-phenol free media as Smg/ml stock solution. DNA
purification Kit was bought from Qiagen, USA. lipofectamin 2000 were purchased from Invitrogen Life Technology. Luciferase assay system, and 13-glycosiladase assay kit were purchased from Promega, USA.
The rest chemicals were purchased from Sigma.
Breast adenoma cells MCF-7 are gift from BC Cancer agency. Fetal calf serum and DMEM were bought from Invitrogen Canada.
Methods Whole cell binding assay: MCF-7 cells were cultured in phenol red-free DMEM
containing 5%
charcoal-stripped Fetal bovine Serum in IOcm culture dish for fve days and then 80,000 cells/well were seeded in a 24-well plate. After 24 hours the cells were changed into serum free medium with lOnM
[3H]-17- 13 estradiol in the absence (total binding, TB) or the presence (nonspecific binding, NSB) of 200-fold excess 17- f3 estradiol or in the presence (competitive binding) of increasing concentration of 17-13 estradiol or aPPD. The same medium with 0.2% ethanol was used a.s vehicle control. The whole cell binding assay was carried out as described by Lee(19), briefly the cells were incubated in 5% C02 incubator at 37oC for 60 minutes. Then the cells were washed with ice-cold phosphate buffer (PBS) for three times to remove free ligands and the bound and intracellular [3H]-estradiol was extracted with cold absolute ethanol and quantified in a (3-scintillation counter at 30% e~ciency.
Binding data were analyzed with Scatchard. A 50% inhibition of binding was defined as 50% of [3H]-17- f3 estradiol displacement in the presence of unlabeled 17- f3 estradiol or aPPD. Data were the average (+SD) of triplicate measurements.
Co-transfection and lueiferase assays: MCF-7 cells were cultured in phenol red-free DMEM
supplemented with charcoal-stripped 5% FCS for five days before transfection.
40,000 cells/well were plated in 24-well plates in the above medium. Cells were transfected with various constructs using lipofectamin 2000 as described by the manufacturer. Briefly each transfection was performed in triplicate, with the ratio of 2_lof lipofectamin 2000 _ total DNA. Each transfection contained O.lug of EREII-Luc (provided by Dr Peter J. Kushner, UGSF) and 0.2 ug of HSV LacZ. After four hours the transfection medium was replaced with lml of DMEM medium with charcoal-stripped 5% FCS, IOnM E2, and/or 5-20uM aPPD, and/or lOuM Tamoxifen. The final concentration of ethanol was 0.2%. After the cells were treated for 24 hours, the media was removed the wells. The cells were washed with Mg2+- and Ca2+-free PBS twice, and were lysed with lysis buffer. The cells were quick frozen to -80 and thawed. The lysate was centrifuged at 14,000 for one minute, the supernatant was combined with Iuciferase assay buffer and the chemiluminescence was measured for one second. Simultaneously 50 ul of supernatant was combined with 50 ul of Assay 2X buffer incubate the reaction at 37°C for 30 minutes or until a faint yellow color has developed. Stop the reactions by adding SOOpI of 1M Sodium Carbonate. Read the absorbance at 420nm.
Luciferase activity was normalized for transfection efficiency by the concomitant assay of -glycosidase activity.
:~R~.,~ ,~, Proliferation Assay: MCF-7 cells were grew in phenol red-free DMEM
supplemented with charcoal-stripped 5% FCS for five days. Five thousand cells per well were plated in 96-well plates in the above medium. The next day, the culture medium was removed by aspiration and 200u1 of fresh medium containing IOnM estradiol, andlor various concentrations of aPPD wil;h /without Tamoxifen was added.
0.2% of ethanol was as vehicle control. After three days treatment, the cell number was determined by MTT assay according to the manufacturer instruction. Briefly, the medium was removed and replaced with SOuI of O.Smg/ml MTT. The cells were incubated in C02 incubator for 4 hours, and the purple crystal was solubilized with 100u1 of lysis buffer overnight. The optical density (O.D.) of each well was measured on spectrophotometer at 570nm.
1VICF-7 Xenograft model in SLID mice: A total of 34 female SCID mice were randomly assigned into seven groups with four to seven mice in each group. One day before the xenografts, each mouse, was received a 4mm subcutaneous silastic E2 pellet, which constantly released E2 to support the estrogen dependent breast cancer cells growth. Mice in estrogen minus control ;8roup were received a 4mm pellet containing no E2. MCF-7 cells were cultured in phenol red-frze DMEM
supplemented with charcoal-stripped 5% Fetal bovine serum. When the cells were in exponent phase, the cells were harvested and resuspend in 100 million cells per ml in the same culture :medium. Ten million cells were subcutaneously inoculated with 27g needles in each mouse. All the procedures were in accordance with the guidelines set by the animal care committee, at University of British Columbia. The mice were treated with aPPD and Tamoxifen daily from the second day of tumor inoculation for 31 days. The treatment groups were as the follows. (a). E2 + vehicle (n=4); (b) E2 + aPPD lOmg/kg/day (n=6); (c) E2 +aPPDIOmg/kg/day + Tamoxifen l.Sg/kg/day (n=7); (d) E2 + tamoa:ifen 3mg/kg/day (n=7); (f) E2 +
Caraseng 20mg/kg/day (n=6); (g) E2 minus control (n=4). The tumor size was measured from the tenth days post implantation.
Sera Estradiol level determination: Samples of plasma were collected by cardiopuncture from each mouse before sacrifice. Serum E2 level was determined by Commercial ELISA kit (Biocheck Inc, American), according the manufacture instruction.
Whole Cell BinddngAssays Whole cell binding assay has been carried out to test whether aPPD could bind to Estrogen receptors (ERs).
Because aPPD was very difficult to be radiolabeled, we used cold aPl?D to compete with [3H]-17-beta-estradiol for estrogen receptors. ER positive MCF-7 breast cancer cell tine, of which estrogen receptor alpha was predominant, was incubated in the presence of 10 nM [3H]-17-beta-estradiol and increasing concentrations of unlabelled E2 or aPPD as competitors for 60 minutes. As shown in Fig.2, aPPD weakly inhibited binding of [3H]-17-beta- estradiol to its receptors with an estimated IC50 of 26.3+/-1.08 uM. As a comparison, unlabeled 17-beta- estradiol has the IC50 of 70+/-2.34nM, suggesting that the affinity of aPPD
to estrogen receptors was 370-fold less than the estrogen itself.
Effects of aPPD on estrogen regulated gene expression To investigate whether the binding of aPPD to ER could induce the expression of ER regulated genes, a construct with a luciferase gene downstream to a promoter containing ER
regulatory element (ERE) was transfected into ER positive MCF-7 cells. As illustrated in Fig.3a, aPPD (2.5-lOuM) activated the ERE
luciferase gene transcription. Comparing to background, aPPD induced luciferase expression by
DEFINITION
The terms "prevention of', "prophylaxis" and "prevent" includes reducing the likelihood of a patient incurring or developing breast cancer.
The term "de novo", as used in the current invention, means the lack of transformation or metamorphosis of normal breast cells to cancerous or malignant cells in the first instance.
Such a transformation may occur in stages in the same or daughter cells via an evolutionary process or may occur in a single, pivotal event.
This de novo process is in contrast to the metastasis, colonization, or spreading of already transformed or malignant cells from the primary tumor site to new locations. 'this invention also relates to the administration of a compound of formula I and Formula II to a patient who is at risk of developing de novo breast cancer.
The term "pro-drug," as used herein means a compound of the present invention bearing a group which is metabolically cleaved in a human to produce a therapeutically active compound of the present invention. In ...~,a.w ,"~.~.....~. .~.:~s:.~ ,~~:ae.:s ,.,ar,.e~~, :~:=fix:-rxx: ~-:..~:~.~v~.~.~--.~.. ~,<
... ~~r,-,.>._... . . ~, particular, such prodrug compounds include those in which either or both of the substituent groups R1, R2, R3, and R4 of the structure shown above or groups which are metabolically cleaved in the body to yield a corresponding compound of the present invention.
COMPOUNDS OF THE PRESENT INVENTION
Through the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
This invention relates to compounds of formula I are used as SERM for preventing breast cancer with following the structure (Formula I):
20S-ProtopartaxadPo!
or a pharmaceutically acceptable salt or pro-drug thereof.
The methods provided by this invention are practiced by administering to a patient in need thereof a dose of a compound of the present invention or a pharmaceutically acceptable salt or solvate thereof, which is effective to prevent breast cancer.
Preferred compounds of the present invention include PAM-120 (Formula II):
_g_ ,_",rtm nmr,.v w' '6:..'d'U4 ..A'~~u:".."&~'"".
9N~~~;a~drYn',Yak'~P.yas5u.YAY~~a:.4YWrte:.amMmro-....wm-~.-..-...."_....."......
.G..yn...,.c~: ..sl.~wWA n .,_. y, ~ .. ~ rv-..,...~LY/~i~rs 4'~~~ - wrnxs.
.Ana..rer~..mw...-,-s...n,~.wm..~...,«
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula III):
Compound III
Common Name: 3,12,20,26-tetrahydroxy-dammar-24z-ene;
Structural Formula:
OH
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula Vd):
Compound VI
Common Name: 3,12,20,25-tetrahydroxy-dammarane;
Structural Formula:
HO
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
In accordance with the present invention, there is provided a method for the prophylaxis or prevention of breast carcinoma in a patient in need of such treatment comprising administering a therapeutically effective amount of a compound having the structure (Formula V):
Compound V
Common Name: 3,12,22-trihydroxy-22-de-isopentenyl-dammarane;
Structural Formula:
HO
or a pharmaceutically acceptable salt or pro-drug thereof.
The invention further relates to a method for preventing breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for treatment of breast cancer by administrating to human for a sufficient term an effective dose of a compound of formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The invention further relates to a method for prevention and treatment of breast cancer by administrating to human for a sufficient term an effective dose of tamoxifen and one or more compounds selected from formula I, II, III, VI, and V or pharmaceutically acceptable salt or pro-drug thereof, where the human has not been diagnosed with, but is determined to be at risk for, developing breast cancer.
The compounds of the present invention are selective estrogen receptor modulators (SERM), that is, compounds which produce estrogen agonism in one or more desired target tissues while producing estrogen antagonism andlor minimal (i.e. clinically insignificant) agonism in reproductive tissue such as the breast or uterus.
The present invention provided that aPPD is a partial agonist and antagonist for estrogen receptors, i.e.
SERM, Compounds with similar biocharactors have been known as selective estrogen receptor modulators (SEItMs). Like aPPD, the SERMs bind to estrogen receptors and have tissue-specific effects that allow them to function as estrogen agonists in some tissues and estrogen antagonists in other tissues. These compounds include clomiphene citrate, tamoxifen, toremifene and raloxifene.
Molecular mechanisms of SERM is believed to attribute to conformational modification of estrogen receptor protein after bound with different ligands, which affects the affinity of the receptor to other proteins that normally associate with the ... ,. ~",~...~ ~ ,.., ~ ra~. . r,~.l, :.;~A,~ ~ ~.-m,F~_.._...~~ . ~.,.u.. .-._~ ___...~___ . .~~~ o- "."M, ,~~.~,~...-,~~.-..
receptor for its physiological activity. Tamoxifen has been widely used in prevention and of breast cancer for the past decades due to its estrogen antagonistic effect on breast tissue.
Meanwhile, it also has an estrogen-like effect on lipids, bone, and the endornetrium. The agonistic effect on endometrium rnay relate to its reported contribution to the initiation and progression of endomel;rial cancer. It is not clear whether aPPD is a true SERM at the present time. The estrogen-related activity of aPPD
remains to be investigated in cells of other tissue origins and its conformational effect on estrogen receptor protein after bound needs to be understood.
The present invention provided that aglycone protopanaxadiol(aPPD) which possess the SERM functions by showing 1) aglycone protopanaxadiol can bind to estrogen receptors with a low affinity and activates the receptor to certain degree, indicated by its ability of stimulating ERE
mediated report gene expression; 2) aPPD can compete with estradiol for the E2 receptors and block its activity in stimulating ERE mediated gene expression; 3) aPPD can block E2 stimulated breast cancer cell proliferation both in vitro and in vivo;
4) aPPD can synergistically enhance the effect of Tarnoxifen for both antagonistic effect on E2 stimulated gene expression and the cytotoxicity on MCF7 cells.
The present invention provided a method of using aPPD together with tamoxifen can synergistically act with Tamoxifen to either block estrogen activity on gene regulation ar abolish estrogen stimulated cell proliferation. Tamoxifen has been known for its cytotoxicity(Petinari, 2004), mechanisms of which may relate to activation of caspases, elevation of intracellular calcium level and inhibition of insulin-like growth factor pathway(Jan, 2000; Kanter-Lewensohn, 2000; Salami, 2003). Tamoxifen is also known for its chemosensitizing effect when used in combination with other cytotoxic agents (Koh, 2003; Ramachandran, 2004). The synergistic cytotoxicity of Tamoxifen and aPPD occurred at the concentrations of 2uM and lOuM for each compounds, respectively. The synergy in cytotoxicity resulted in massive cell death that might be independent of their estrogen related activity. On the other hand, the synergy between aPPD and Tamoxifen were also demonstrated in their inhibition on estrogen stimulated activity and the synergy between aPPD and Tamoxifen occurred in both antiestrogenic and cytatoxic effects on the breast cancer cells. The synergetic effects between aPPD and tamoxifen could lead to reduce the side effect of tamoxifen by applying less dosage of tamoxifen. The synergistic effects may contribute to the powerful inhibition in estrogen-dependent breast cancer growth in the animal model. This was especially demonstrated in initial stage of tumor growth in E2-supported breast cancer model.
METHOD OF THE PRSENT dNVENTlON
Material 17 I3-Estradiol and Tamoxifen purchased from Sigma, Careseng and aPPD supplied by Pegasus Pharmaceuticals Inc., [3H] 17- 13 estadiol (89 Ci/mmol), purchased from Perken Elmer, were dissolved in absolute ethanol as stock solutions. Before applied to the cultured cells, each of the compounds was diluted into 1000 times solution with ethanol and then further diluted with fresh culture media into working -II-solutions. MTT, from Sigma, was dissolved in RPMI-phenol free media as Smg/ml stock solution. DNA
purification Kit was bought from Qiagen, USA. lipofectamin 2000 were purchased from Invitrogen Life Technology. Luciferase assay system, and 13-glycosiladase assay kit were purchased from Promega, USA.
The rest chemicals were purchased from Sigma.
Breast adenoma cells MCF-7 are gift from BC Cancer agency. Fetal calf serum and DMEM were bought from Invitrogen Canada.
Methods Whole cell binding assay: MCF-7 cells were cultured in phenol red-free DMEM
containing 5%
charcoal-stripped Fetal bovine Serum in IOcm culture dish for fve days and then 80,000 cells/well were seeded in a 24-well plate. After 24 hours the cells were changed into serum free medium with lOnM
[3H]-17- 13 estradiol in the absence (total binding, TB) or the presence (nonspecific binding, NSB) of 200-fold excess 17- f3 estradiol or in the presence (competitive binding) of increasing concentration of 17-13 estradiol or aPPD. The same medium with 0.2% ethanol was used a.s vehicle control. The whole cell binding assay was carried out as described by Lee(19), briefly the cells were incubated in 5% C02 incubator at 37oC for 60 minutes. Then the cells were washed with ice-cold phosphate buffer (PBS) for three times to remove free ligands and the bound and intracellular [3H]-estradiol was extracted with cold absolute ethanol and quantified in a (3-scintillation counter at 30% e~ciency.
Binding data were analyzed with Scatchard. A 50% inhibition of binding was defined as 50% of [3H]-17- f3 estradiol displacement in the presence of unlabeled 17- f3 estradiol or aPPD. Data were the average (+SD) of triplicate measurements.
Co-transfection and lueiferase assays: MCF-7 cells were cultured in phenol red-free DMEM
supplemented with charcoal-stripped 5% FCS for five days before transfection.
40,000 cells/well were plated in 24-well plates in the above medium. Cells were transfected with various constructs using lipofectamin 2000 as described by the manufacturer. Briefly each transfection was performed in triplicate, with the ratio of 2_lof lipofectamin 2000 _ total DNA. Each transfection contained O.lug of EREII-Luc (provided by Dr Peter J. Kushner, UGSF) and 0.2 ug of HSV LacZ. After four hours the transfection medium was replaced with lml of DMEM medium with charcoal-stripped 5% FCS, IOnM E2, and/or 5-20uM aPPD, and/or lOuM Tamoxifen. The final concentration of ethanol was 0.2%. After the cells were treated for 24 hours, the media was removed the wells. The cells were washed with Mg2+- and Ca2+-free PBS twice, and were lysed with lysis buffer. The cells were quick frozen to -80 and thawed. The lysate was centrifuged at 14,000 for one minute, the supernatant was combined with Iuciferase assay buffer and the chemiluminescence was measured for one second. Simultaneously 50 ul of supernatant was combined with 50 ul of Assay 2X buffer incubate the reaction at 37°C for 30 minutes or until a faint yellow color has developed. Stop the reactions by adding SOOpI of 1M Sodium Carbonate. Read the absorbance at 420nm.
Luciferase activity was normalized for transfection efficiency by the concomitant assay of -glycosidase activity.
:~R~.,~ ,~, Proliferation Assay: MCF-7 cells were grew in phenol red-free DMEM
supplemented with charcoal-stripped 5% FCS for five days. Five thousand cells per well were plated in 96-well plates in the above medium. The next day, the culture medium was removed by aspiration and 200u1 of fresh medium containing IOnM estradiol, andlor various concentrations of aPPD wil;h /without Tamoxifen was added.
0.2% of ethanol was as vehicle control. After three days treatment, the cell number was determined by MTT assay according to the manufacturer instruction. Briefly, the medium was removed and replaced with SOuI of O.Smg/ml MTT. The cells were incubated in C02 incubator for 4 hours, and the purple crystal was solubilized with 100u1 of lysis buffer overnight. The optical density (O.D.) of each well was measured on spectrophotometer at 570nm.
1VICF-7 Xenograft model in SLID mice: A total of 34 female SCID mice were randomly assigned into seven groups with four to seven mice in each group. One day before the xenografts, each mouse, was received a 4mm subcutaneous silastic E2 pellet, which constantly released E2 to support the estrogen dependent breast cancer cells growth. Mice in estrogen minus control ;8roup were received a 4mm pellet containing no E2. MCF-7 cells were cultured in phenol red-frze DMEM
supplemented with charcoal-stripped 5% Fetal bovine serum. When the cells were in exponent phase, the cells were harvested and resuspend in 100 million cells per ml in the same culture :medium. Ten million cells were subcutaneously inoculated with 27g needles in each mouse. All the procedures were in accordance with the guidelines set by the animal care committee, at University of British Columbia. The mice were treated with aPPD and Tamoxifen daily from the second day of tumor inoculation for 31 days. The treatment groups were as the follows. (a). E2 + vehicle (n=4); (b) E2 + aPPD lOmg/kg/day (n=6); (c) E2 +aPPDIOmg/kg/day + Tamoxifen l.Sg/kg/day (n=7); (d) E2 + tamoa:ifen 3mg/kg/day (n=7); (f) E2 +
Caraseng 20mg/kg/day (n=6); (g) E2 minus control (n=4). The tumor size was measured from the tenth days post implantation.
Sera Estradiol level determination: Samples of plasma were collected by cardiopuncture from each mouse before sacrifice. Serum E2 level was determined by Commercial ELISA kit (Biocheck Inc, American), according the manufacture instruction.
Whole Cell BinddngAssays Whole cell binding assay has been carried out to test whether aPPD could bind to Estrogen receptors (ERs).
Because aPPD was very difficult to be radiolabeled, we used cold aPl?D to compete with [3H]-17-beta-estradiol for estrogen receptors. ER positive MCF-7 breast cancer cell tine, of which estrogen receptor alpha was predominant, was incubated in the presence of 10 nM [3H]-17-beta-estradiol and increasing concentrations of unlabelled E2 or aPPD as competitors for 60 minutes. As shown in Fig.2, aPPD weakly inhibited binding of [3H]-17-beta- estradiol to its receptors with an estimated IC50 of 26.3+/-1.08 uM. As a comparison, unlabeled 17-beta- estradiol has the IC50 of 70+/-2.34nM, suggesting that the affinity of aPPD
to estrogen receptors was 370-fold less than the estrogen itself.
Effects of aPPD on estrogen regulated gene expression To investigate whether the binding of aPPD to ER could induce the expression of ER regulated genes, a construct with a luciferase gene downstream to a promoter containing ER
regulatory element (ERE) was transfected into ER positive MCF-7 cells. As illustrated in Fig.3a, aPPD (2.5-lOuM) activated the ERE
luciferase gene transcription. Comparing to background, aPPD induced luciferase expression by
8.48+/-1.72 fold (P<0.01). Interestingly, the maximum activation of ERE-mediated luciferase gene expression (10.32+/-1.14 fold) was reached with 5 uM aPPD but not lOuM. To confirm the specificity of aPPD on ER-mediated transactivation, we examined whether Tamoxifen, a specific ER antagonist, could inhibit the effect of aPPD. As shown in Fig.3a, 5uM of tamoxifen completely blocked aPPD induced reporter gene expression, which suggested that the aPPD-stimulated luciferase transcription was specifically mediated by ERs.
Since levels of aPPD stimulated luciferase gene expression was much lower than that of E2, which was 8.79 times more potent than aPPD, and since aPPD stimulated ERE activity was not dose-dependent anymore when concentration of aPPD above SuM, we speculated that aPPD might inhibit E2 induced ERE
activity. Thus, E2 stimulated luciferase activity was measured in ER positive MCF-7 cells in the presence of aPPD or Tamoxifen. As shown in Fig.3b, both aPPD and Tamoxifen inhibited IOnM E2-induced ERE-luciferase transcription dose-dependently, with the IC50 of 12.5 uM and 2.8uM, respectively. To explore possible interactions between Tamoxifen and aPPD, 5 or 10 uM aPPD was used in combination with various concentrations of Tamoxifen to measure ERE mediated luciferase activity in the presence of IOnM of E2. The results are demonstrated in Fig.3c. The inhibition of ERE-luciferase transcription by 5uM and lOuM of aPPD alone (without Tamoxifen) was 20.1+/-4,9% and 38.4+/-2.8%, respectively (p<0.01). The lowest effective inhibitory dose of tamoxifen was 2uM(21.2+/-4.7%, P<0.05) in the present study. However, in the presence of 5 or IOuM of aPPD, 0.5 and 1 uM of Tamoxifen showed significant inhibitory effect on E2 stimulated ERE-luciferase acitivity. The combiined treatment with 5uM aPPD and Tamoxifen had synergistic effect when concentration of Tamoxifen was below 4uM
(p<0.01).
aPPD inhibited E2 stimulated MCF-7 proliferation and synergistically enhanced tamoxifen cytotoxicity:
To evaluate effects of aPPD on ER stimulated cell growth, the proliferation assay was performed on the MCF-7 human breast cancer cells. MCF-7 cells were treated for 48 hours with lOnM E2 plus 0-lSuM
Tamoxifen in the presence or absence of IOuM aPPD. As shown in Fig.4, lOnM of E2 increased the MCF-7 cell growth by17.0+/-0.08 %, p<0.05) compared to vehicle treated control, lOuM
aPPD alone antagonized E2-stimulated MCF-7 proliferation by 15.2+/-0.03% (P<0.05). Tamoxifen alone also abolished E2-stimulated MCF-7 proliferation at the concentration below 5 uM (p<0.05).
Cytotoxicity of Tamoxifen appeared at concentration of IOuM and lSuM, resulting 36%+/-0.05% and 99.8+/-0.02% of cell death in the presence of IOnM E2. The cytotoxic effect of Tamoxifen was significantly increased by aPPD in a synergistic fashion. As shown in FigSa, co-treatment with lOuM aPPD and 5uM
Tamoxifen caused 53.2+/-5.3% of inhibition in cell growth. (P<0.01), while aPPD and Tamoxifen alone had only 15.2+/-0.03% and 12.8% +/-0.02% respectively. Even greater synergistic effect in Tamoxifen and aPPD
(lOuM) caused cytotoxicity was seen at lOuM Tamoxifen, which resulted in almost 100% cell death in 48 hours, while Tamoxifen alone only caused 36%+/-0.05% of growth inhibition in the presence of E2 compared to untreated cells.
aPPD inhibited in vivo ER-dependent tumor Growth To confirm the inhibitory effect of aPPD on ER-dependent tumor growth, aPPD
was orally administrated in animals bearing MCF-7 tumors with subcutaneously implanted silastic E2 pellet.
The tumor became measurable one week after implantation, and the measurement started on day 10 post implantation. The tumor growth rates were dependent on the size of the E2 pellets implanted. As shown in Fig.4, the tumor volume was more than twenty folds of its initial size in animals of control group by day 31 Post-implantation. In the contrast, there was no measurable tumor growth in the non-E2 supported control group. The Tumor volume eventually regressed from the average volume of 0.416+/-0.25mm3 to 0.13+/-0.08mm3 over two weeks, thus indicating that the MCF-7 breast cancer growing was totally E2-dependent, and the intrinsic E2 level was not sufficient for supporting progression of the xenografts.
In animals with estradiol support but daily given aPPD lOmg/lcg, the initial tumor volume was 0.052+/-0.000 mm3, and decreased to immeasurable size by day 15. Compared with the tumor volume of Non-E2 supported group on the tenth day, the mean tumor volume of the aPPD
group was 13% of that of Non-E2 supported group, suggesting that aPPD efficiently blocked ER stimulated tumor growth.
Tamoxifen (3mg/kg, i.p.) also effectively inhibited tumor growth in E2 implanted animals (21 of 21 mice, P<0.01.). The same as the group with lOmg/kg aPPD plus l.5mg/kg Tamoxifen. In another group, E2-supported animals were given a commercial product Careseng that contains 50% of aPPD and other aglycone ginsenosides. By day 31, the end of experiment, there was no significant difference between the 4 treatment groups and the non-E2 supported group. It is worthwhile to notice that there was a difference in tumor sizes on the first day of measurement (day 10 post implantation) among the treatment groups. Mean volume of Tamoxifen only treated group is significantly larger than aPPD and Tamoxifen combined treated group or caraseng group (0.10 +/-0.Ol lmm3, p<0.01), which may reflect the difference in potency of these treatments Fig Sb. The above evidence data indicated that aPPD could also effectively inhibit tumor formation and progression. aPPD had synergetic effect with tamoxifen.
Plasma E2 level:
To ensure that there was sufficient levels of estrogen in the system of all treated animals, the plasma E2 concentration was measured with ELISA. The results were show in Table 1. The plasma E2 concentration in the non-E2 supported group was only at the pg level. While the E2 plasma levels in animals of treated groups varied, there was no significant difference among 4 treatment groups and the E2 supported control group. In addition, correlation coefficient analysis using samples from E2 control group indicated no correlation between the E2 level and tumor within the variation range (data not shown), suggesting that ,~v~~W.-there was sufficient E2 to support tumor growth even with lowest E2 level.
PHARMACEUTICAL FORMULATIONS' The compounds of this invention are administered by a variety of routes including oral, rectal, transdermal, subucutaneus, intravenous, intramuscular, and intranasal.
These compounds preferably are formulated prior to administration, the selection of which will be decided by the attending physician. Thus, another aspect of the present invention is a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, optionally containing an effective amount of estrogen or progestin, and a pharmaceutically acceptable carrier, diluent, or excipient.
The total active ingredients in such formulations comprises from ~.1% to 99.9%
by weight of the formulation.
By "pharmaceutically acceptable" it is meant the earner, diluent, excipients and salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
Pharmaceutical formulations of the present invention are prepared by procedures known in the art using well known and readily available ingredients. For example, the compounds of Formula I, either alone, or in combination with an estrogen or progestin compound, are formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, solutions, injectables, aerosols, powders, and the like.
The total active ingredients in such formulations comprises from 0.1% to 99.9%
by weight of the formulation.
By "pharmaceutically acceptable" it is meant the carrier, diluent, excipients and salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
The formulations may be specially formulated for oral administration, in solid or liquid form, for parenteral injection, topical or aerosol administration, or for rectal or vaginal administration by means of a suppository.
The pharmaceutical compositions of this invention can be administered to humans and other mammals orally, rectally, intravaginally, parenterally, topically (by means of powders, ointments, creams, or drops), bucally or sublingually, or as an oral or nasal spray. The term "parenteral administration" refers herein to modes of administration which include intravenous, intramuscular, intraperitoneal, instrasternal, subcutaneous, or intraarticular injection or infusion.
Pharmaceutical compositions of this invention for parenteral administration comprise sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders which are reconstituted immediately prior to use into sterile solutions or suspensions.
Examples of suitable sterile aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, physiological saline solution, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), and thelike), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of coating materials such as lecithin, by the maintenance of proper particle size in the case of dispersions and suspensions, and by the use of surfactants.
Parenteral compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms is ensured by the inclusion of antibacterial and antifungal agents, for example, paxaben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of injectable formulations may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug following subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension or crystalline or amorphous material of low water solubility or by dissolving or suspending the drug in an oil vehicle. In the case of the subcutaneous or intramuseular injection of a suspension containing a form of the drug with low water solubility, the rate of absorption of the drug depends upon its rate of dissolution.
Injectable "depot" formulations of the compounds of this invention are made by forming microencapsulated matrices of the drug in biodegradable polymers such as poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acid, poly (orthoesters), and poly (anhydrides) these materials which are described in the art.
Depending upon the ratio of drug to polymer and the characteristics of the particular polymer employed, the rate of drug release can be controlled.
Injectable formulations are sterilized, for example, by filtration through bacterial-retaining filters, or by presterilization of the components of the mixture prior to their admixture, either at the time of manufacture or just prior to administration (as in the example of a dual chamber syringe package).
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active component is mixed with at least one inert, pharmaceutically acceptable carrier such as sodium citrate, or dicalcium phosphate, and/or (a) fillers or extenders such as starches, lactose, glucose, mannitol, and silicic acid, (b) binding agents such as carboxymethyl cellulose, alginates, gelatin, poly(vinylpyrrolidine), sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates and sodium carbonate, (e) solution retarding agents such as pazafFn, (f) absorption accelerating agents such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerin monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium - 1'7 -stearate, solid polyethylene glycols), sodium Iauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills and granules can also be prepared with coatings or shells such as enteric coatings or other coatings well known in the pharmaceutical formulating art. The coatings may contain opacifying agents or agents which release the active ingredients) in a particular part of the digestive tract, as for example, acid soluble coatings for release of the active ingredients) in the stomach, or base soluble coatings for release of the active ingredients) in the intestinal tract.
The active ingredients) may also be microencapsulated in a sustained-release coating, with the microcapsules being made part of a pill of capsule formulation.
Liquid dosage forms for oral administration of the compounds of this invention include solution, emulsions, suspensions, syrups and elixirs. In addition to the active components, liquid formulations may include inert diluents commonly used in the art such as water or other pharmaceutically acceptable solvents, solubilizing agents and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols), fatty acid esters of sorbitol, and mixtures thereof.
Besides inert diluents, the liquid oral formulations may also include adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Liquid compositions of a similar type may also comprise the fill in soft or hard gelatin capsules using excipients such as lactose as well as high molecular weight polyethylene glycols) and the like.
Liquid suspension, in addition to the active ingredients) may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite clay, agar-agar, and tragacanth, and mixtures thereof.
The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.
EFFECTIVEAMOUNTAND DOSAGE
As used herein, the teen "effective amounf° means an amount of compound of the present invention which is capable of inhibiting or preventing breast tumor formation. The specific dose of a compound administered according to this invention is determined by the particular circumstances surrounding each situation including, for example, the potency of the compound administered, the route of administration, the prior medical history of the patient, and the pathological condition being treated. A typical daily dose will contain a nontoxic dosage level of from about O.S mg to about SOg/day/70kg body weight of a compound of the present invention. Preferred daily doses generally will be from about 50 rng to about I00 mg/day/70kg body weight.
The exact dose is determined, in accordance with the standard practice in the medical arts of dose titrating the patient; that is, initially administering a low dose of the compound, and gradually increasing the does until the desired therapeutic effect is observed.
PREPARATION OF COMPOUNDS OF THE PRESENT IlV6'ENTION
The compounds used in the methods of the current invention can be available commercially from Pegasus Pharmaceuticals Group Ine, Richmond, British Columbia, Canada, or can be made according to established procedures, such as those detailed in U.S. Patent published applications (No.
09/910,887, 09/982,018) The following examples are presented to further illustrate the preparation of compounds of the present invention. The Examples are not to be read as limiting the scope of the invention as it is defined by the appended claims.
Example 1 Preparation of compounds of Formula I\II1III1VI\V
[001] Ginseng crude extract 10 g was dissolved in 40 mL of 95% ethanol [002} Add 40 mL of 5 N NaOH
[003] Pour into the reaction tank, and set temperature to 240C, and pressure to 3.5 Mpa, for L5 hours [004] Reduce temperature to room temperature, and take the products out the tank [005] Add HCI to neutralize pH to about 7, and expend the volume to 800 mL
using water [006J Extract 3 times with acetic ester, 100 mL each time [007] Combine all the extracts, and reduce the pressure to dry. Thus, obtain 3.8 g of dried extracts [008] Grind and dissolved the extract in 20 mL of methanol, and rnix the methanol solution with silica gel
Since levels of aPPD stimulated luciferase gene expression was much lower than that of E2, which was 8.79 times more potent than aPPD, and since aPPD stimulated ERE activity was not dose-dependent anymore when concentration of aPPD above SuM, we speculated that aPPD might inhibit E2 induced ERE
activity. Thus, E2 stimulated luciferase activity was measured in ER positive MCF-7 cells in the presence of aPPD or Tamoxifen. As shown in Fig.3b, both aPPD and Tamoxifen inhibited IOnM E2-induced ERE-luciferase transcription dose-dependently, with the IC50 of 12.5 uM and 2.8uM, respectively. To explore possible interactions between Tamoxifen and aPPD, 5 or 10 uM aPPD was used in combination with various concentrations of Tamoxifen to measure ERE mediated luciferase activity in the presence of IOnM of E2. The results are demonstrated in Fig.3c. The inhibition of ERE-luciferase transcription by 5uM and lOuM of aPPD alone (without Tamoxifen) was 20.1+/-4,9% and 38.4+/-2.8%, respectively (p<0.01). The lowest effective inhibitory dose of tamoxifen was 2uM(21.2+/-4.7%, P<0.05) in the present study. However, in the presence of 5 or IOuM of aPPD, 0.5 and 1 uM of Tamoxifen showed significant inhibitory effect on E2 stimulated ERE-luciferase acitivity. The combiined treatment with 5uM aPPD and Tamoxifen had synergistic effect when concentration of Tamoxifen was below 4uM
(p<0.01).
aPPD inhibited E2 stimulated MCF-7 proliferation and synergistically enhanced tamoxifen cytotoxicity:
To evaluate effects of aPPD on ER stimulated cell growth, the proliferation assay was performed on the MCF-7 human breast cancer cells. MCF-7 cells were treated for 48 hours with lOnM E2 plus 0-lSuM
Tamoxifen in the presence or absence of IOuM aPPD. As shown in Fig.4, lOnM of E2 increased the MCF-7 cell growth by17.0+/-0.08 %, p<0.05) compared to vehicle treated control, lOuM
aPPD alone antagonized E2-stimulated MCF-7 proliferation by 15.2+/-0.03% (P<0.05). Tamoxifen alone also abolished E2-stimulated MCF-7 proliferation at the concentration below 5 uM (p<0.05).
Cytotoxicity of Tamoxifen appeared at concentration of IOuM and lSuM, resulting 36%+/-0.05% and 99.8+/-0.02% of cell death in the presence of IOnM E2. The cytotoxic effect of Tamoxifen was significantly increased by aPPD in a synergistic fashion. As shown in FigSa, co-treatment with lOuM aPPD and 5uM
Tamoxifen caused 53.2+/-5.3% of inhibition in cell growth. (P<0.01), while aPPD and Tamoxifen alone had only 15.2+/-0.03% and 12.8% +/-0.02% respectively. Even greater synergistic effect in Tamoxifen and aPPD
(lOuM) caused cytotoxicity was seen at lOuM Tamoxifen, which resulted in almost 100% cell death in 48 hours, while Tamoxifen alone only caused 36%+/-0.05% of growth inhibition in the presence of E2 compared to untreated cells.
aPPD inhibited in vivo ER-dependent tumor Growth To confirm the inhibitory effect of aPPD on ER-dependent tumor growth, aPPD
was orally administrated in animals bearing MCF-7 tumors with subcutaneously implanted silastic E2 pellet.
The tumor became measurable one week after implantation, and the measurement started on day 10 post implantation. The tumor growth rates were dependent on the size of the E2 pellets implanted. As shown in Fig.4, the tumor volume was more than twenty folds of its initial size in animals of control group by day 31 Post-implantation. In the contrast, there was no measurable tumor growth in the non-E2 supported control group. The Tumor volume eventually regressed from the average volume of 0.416+/-0.25mm3 to 0.13+/-0.08mm3 over two weeks, thus indicating that the MCF-7 breast cancer growing was totally E2-dependent, and the intrinsic E2 level was not sufficient for supporting progression of the xenografts.
In animals with estradiol support but daily given aPPD lOmg/lcg, the initial tumor volume was 0.052+/-0.000 mm3, and decreased to immeasurable size by day 15. Compared with the tumor volume of Non-E2 supported group on the tenth day, the mean tumor volume of the aPPD
group was 13% of that of Non-E2 supported group, suggesting that aPPD efficiently blocked ER stimulated tumor growth.
Tamoxifen (3mg/kg, i.p.) also effectively inhibited tumor growth in E2 implanted animals (21 of 21 mice, P<0.01.). The same as the group with lOmg/kg aPPD plus l.5mg/kg Tamoxifen. In another group, E2-supported animals were given a commercial product Careseng that contains 50% of aPPD and other aglycone ginsenosides. By day 31, the end of experiment, there was no significant difference between the 4 treatment groups and the non-E2 supported group. It is worthwhile to notice that there was a difference in tumor sizes on the first day of measurement (day 10 post implantation) among the treatment groups. Mean volume of Tamoxifen only treated group is significantly larger than aPPD and Tamoxifen combined treated group or caraseng group (0.10 +/-0.Ol lmm3, p<0.01), which may reflect the difference in potency of these treatments Fig Sb. The above evidence data indicated that aPPD could also effectively inhibit tumor formation and progression. aPPD had synergetic effect with tamoxifen.
Plasma E2 level:
To ensure that there was sufficient levels of estrogen in the system of all treated animals, the plasma E2 concentration was measured with ELISA. The results were show in Table 1. The plasma E2 concentration in the non-E2 supported group was only at the pg level. While the E2 plasma levels in animals of treated groups varied, there was no significant difference among 4 treatment groups and the E2 supported control group. In addition, correlation coefficient analysis using samples from E2 control group indicated no correlation between the E2 level and tumor within the variation range (data not shown), suggesting that ,~v~~W.-there was sufficient E2 to support tumor growth even with lowest E2 level.
PHARMACEUTICAL FORMULATIONS' The compounds of this invention are administered by a variety of routes including oral, rectal, transdermal, subucutaneus, intravenous, intramuscular, and intranasal.
These compounds preferably are formulated prior to administration, the selection of which will be decided by the attending physician. Thus, another aspect of the present invention is a pharmaceutical composition comprising an effective amount of a compound of Formula I, or a pharmaceutically acceptable salt thereof, optionally containing an effective amount of estrogen or progestin, and a pharmaceutically acceptable carrier, diluent, or excipient.
The total active ingredients in such formulations comprises from ~.1% to 99.9%
by weight of the formulation.
By "pharmaceutically acceptable" it is meant the earner, diluent, excipients and salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
Pharmaceutical formulations of the present invention are prepared by procedures known in the art using well known and readily available ingredients. For example, the compounds of Formula I, either alone, or in combination with an estrogen or progestin compound, are formulated with common excipients, diluents, or carriers, and formed into tablets, capsules, suspensions, solutions, injectables, aerosols, powders, and the like.
The total active ingredients in such formulations comprises from 0.1% to 99.9%
by weight of the formulation.
By "pharmaceutically acceptable" it is meant the carrier, diluent, excipients and salt must be compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.
The formulations may be specially formulated for oral administration, in solid or liquid form, for parenteral injection, topical or aerosol administration, or for rectal or vaginal administration by means of a suppository.
The pharmaceutical compositions of this invention can be administered to humans and other mammals orally, rectally, intravaginally, parenterally, topically (by means of powders, ointments, creams, or drops), bucally or sublingually, or as an oral or nasal spray. The term "parenteral administration" refers herein to modes of administration which include intravenous, intramuscular, intraperitoneal, instrasternal, subcutaneous, or intraarticular injection or infusion.
Pharmaceutical compositions of this invention for parenteral administration comprise sterile aqueous or nonaqueous solutions, dispersions, suspensions, or emulsions, as well as sterile powders which are reconstituted immediately prior to use into sterile solutions or suspensions.
Examples of suitable sterile aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, physiological saline solution, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol), and thelike), and suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Proper fluidity is maintained, for example, by the use of coating materials such as lecithin, by the maintenance of proper particle size in the case of dispersions and suspensions, and by the use of surfactants.
Parenteral compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the action of microorganisms is ensured by the inclusion of antibacterial and antifungal agents, for example, paxaben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents such as sugars, sodium chloride, and the like. Prolonged absorption of injectable formulations may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
In some cases, in order to prolong the effect of the drug, it is desirable to slow the absorption of the drug following subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension or crystalline or amorphous material of low water solubility or by dissolving or suspending the drug in an oil vehicle. In the case of the subcutaneous or intramuseular injection of a suspension containing a form of the drug with low water solubility, the rate of absorption of the drug depends upon its rate of dissolution.
Injectable "depot" formulations of the compounds of this invention are made by forming microencapsulated matrices of the drug in biodegradable polymers such as poly(lactic acid), poly(glycolic acid), copolymers of lactic and glycolic acid, poly (orthoesters), and poly (anhydrides) these materials which are described in the art.
Depending upon the ratio of drug to polymer and the characteristics of the particular polymer employed, the rate of drug release can be controlled.
Injectable formulations are sterilized, for example, by filtration through bacterial-retaining filters, or by presterilization of the components of the mixture prior to their admixture, either at the time of manufacture or just prior to administration (as in the example of a dual chamber syringe package).
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active component is mixed with at least one inert, pharmaceutically acceptable carrier such as sodium citrate, or dicalcium phosphate, and/or (a) fillers or extenders such as starches, lactose, glucose, mannitol, and silicic acid, (b) binding agents such as carboxymethyl cellulose, alginates, gelatin, poly(vinylpyrrolidine), sucrose and acacia, (c) humectants such as glycerol, (d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, silicates and sodium carbonate, (e) solution retarding agents such as pazafFn, (f) absorption accelerating agents such as quaternary ammonium compounds, (g) wetting agents such as cetyl alcohol and glycerin monostearate, (h) absorbents such as kaolin and bentonite clay, and (i) lubricants such as talc, calcium stearate, magnesium - 1'7 -stearate, solid polyethylene glycols), sodium Iauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also contain buffering agents.
Solid dosage forms such as tablets, dragees, capsules, pills and granules can also be prepared with coatings or shells such as enteric coatings or other coatings well known in the pharmaceutical formulating art. The coatings may contain opacifying agents or agents which release the active ingredients) in a particular part of the digestive tract, as for example, acid soluble coatings for release of the active ingredients) in the stomach, or base soluble coatings for release of the active ingredients) in the intestinal tract.
The active ingredients) may also be microencapsulated in a sustained-release coating, with the microcapsules being made part of a pill of capsule formulation.
Liquid dosage forms for oral administration of the compounds of this invention include solution, emulsions, suspensions, syrups and elixirs. In addition to the active components, liquid formulations may include inert diluents commonly used in the art such as water or other pharmaceutically acceptable solvents, solubilizing agents and emulsifiers such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethyl formamide, oils (in particular, cottonseed, ground nut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols), fatty acid esters of sorbitol, and mixtures thereof.
Besides inert diluents, the liquid oral formulations may also include adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
Liquid compositions of a similar type may also comprise the fill in soft or hard gelatin capsules using excipients such as lactose as well as high molecular weight polyethylene glycols) and the like.
Liquid suspension, in addition to the active ingredients) may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite clay, agar-agar, and tragacanth, and mixtures thereof.
The pharmaceutically acceptable salts generally have enhanced solubility characteristics compared to the compound from which they are derived, and thus are often more amenable to formulation as liquids or emulsions.
EFFECTIVEAMOUNTAND DOSAGE
As used herein, the teen "effective amounf° means an amount of compound of the present invention which is capable of inhibiting or preventing breast tumor formation. The specific dose of a compound administered according to this invention is determined by the particular circumstances surrounding each situation including, for example, the potency of the compound administered, the route of administration, the prior medical history of the patient, and the pathological condition being treated. A typical daily dose will contain a nontoxic dosage level of from about O.S mg to about SOg/day/70kg body weight of a compound of the present invention. Preferred daily doses generally will be from about 50 rng to about I00 mg/day/70kg body weight.
The exact dose is determined, in accordance with the standard practice in the medical arts of dose titrating the patient; that is, initially administering a low dose of the compound, and gradually increasing the does until the desired therapeutic effect is observed.
PREPARATION OF COMPOUNDS OF THE PRESENT IlV6'ENTION
The compounds used in the methods of the current invention can be available commercially from Pegasus Pharmaceuticals Group Ine, Richmond, British Columbia, Canada, or can be made according to established procedures, such as those detailed in U.S. Patent published applications (No.
09/910,887, 09/982,018) The following examples are presented to further illustrate the preparation of compounds of the present invention. The Examples are not to be read as limiting the scope of the invention as it is defined by the appended claims.
Example 1 Preparation of compounds of Formula I\II1III1VI\V
[001] Ginseng crude extract 10 g was dissolved in 40 mL of 95% ethanol [002} Add 40 mL of 5 N NaOH
[003] Pour into the reaction tank, and set temperature to 240C, and pressure to 3.5 Mpa, for L5 hours [004] Reduce temperature to room temperature, and take the products out the tank [005] Add HCI to neutralize pH to about 7, and expend the volume to 800 mL
using water [006J Extract 3 times with acetic ester, 100 mL each time [007] Combine all the extracts, and reduce the pressure to dry. Thus, obtain 3.8 g of dried extracts [008] Grind and dissolved the extract in 20 mL of methanol, and rnix the methanol solution with silica gel
[009] Dry up the mixture, and then grind to fine powder
[010] Load the Silica gel column [011 ] Wash the column with 60 mI, of petroleum ether :benzene (1:3), and thus, 50 mg of Formula II
compound and 45 mg of Formula V compound were obtained. Eluted with petroleum ether :benzene (1:2) to obtain Formula VI compound (23 mg) [012J Wash the column with 90 mL of chloroform:methanol (95:3), and thus 250 mg Formula I compound was obtained, and Formula III compound (26 mg) were yielded by elutf;d with 100 ml chloroform:
methanol (100:5).
Example 2 Preparation of compounds of Formula I\II\III\VI\V
[001] 10 g of Ginseng crude extract was added into reaction tank [002] Add to the reaction tank 100 mL of S N NaOH
., . "," ,...~;~u,~ø~,..~,.,;:~::.~.a~.~:~~ ,~.~y~~.~.:.m.=,,,-~~.,_ ....
,:<..,,- .,.~,,~,~y.~__..~d..
[003) Set temperature to 270C and pressure to 4.5 Mpa for 1 hour [004] Reduce temperature to room temperature, then take out the products [005] Neutralize the pH to 7 using HCl [006) Filter and keep the solids [007] Dissolve the solids in 10 mL of 95% Ethanol [008) Add water to make ethanol content less than 5%
[009] Sit still overnight [010] Filter and keep the solids
compound and 45 mg of Formula V compound were obtained. Eluted with petroleum ether :benzene (1:2) to obtain Formula VI compound (23 mg) [012J Wash the column with 90 mL of chloroform:methanol (95:3), and thus 250 mg Formula I compound was obtained, and Formula III compound (26 mg) were yielded by elutf;d with 100 ml chloroform:
methanol (100:5).
Example 2 Preparation of compounds of Formula I\II\III\VI\V
[001] 10 g of Ginseng crude extract was added into reaction tank [002] Add to the reaction tank 100 mL of S N NaOH
., . "," ,...~;~u,~ø~,..~,.,;:~::.~.a~.~:~~ ,~.~y~~.~.:.m.=,,,-~~.,_ ....
,:<..,,- .,.~,,~,~y.~__..~d..
[003) Set temperature to 270C and pressure to 4.5 Mpa for 1 hour [004] Reduce temperature to room temperature, then take out the products [005] Neutralize the pH to 7 using HCl [006) Filter and keep the solids [007] Dissolve the solids in 10 mL of 95% Ethanol [008) Add water to make ethanol content less than 5%
[009] Sit still overnight [010] Filter and keep the solids
[011] Dry the solids
[012] Dissolved the solids in 10 mL of methanol
(013] Filter and keep the solution
[014] Dry the solution to obtain 3.6 g of products
[015) Mix the products with 11 g of silica gel
[016] Grind and then load the silica gel column [017[Ol l) Wash the column with 60 mL of petroleum ether :benzene (1:3), and thus, 43 mg of Formula II
compound and 51 mg of Formula V compound were obtained. Eluted with petroleum ether :benzene (1:2) to obtain Formula VI compound (21 mg) [OI2] Wash the column with 90 mL of chloroform:methanol (95:3), and thus 261 mg Formula I compound was obtained, and Formula III compound (27 mg) were yielded by eluted with 100 ml chloroform:
methanol (100:5).
FORMULATION EXAMPLES
In the formulations which follow, "active ingredient" means a compound or combination of compounds of Formula I, II, III, VI and V, or a salt or solvate thereof.
Formulation Example 1 Gelatin Capsules Ingredient (mglcapsule) m/m Quantity Active ingredient40mg 27%
Absolute Ethanol120mg 32.4%
Cremophor Rh40lSOmg 40.6%
Formulation Example 2 Oral Solution Ingredient (mglbottle) m/m Quantity Active ingredient100mg 28.75%
Absolute Ethanol75mg 21.43%
Cremophor Rh40175mg 50%
compound and 51 mg of Formula V compound were obtained. Eluted with petroleum ether :benzene (1:2) to obtain Formula VI compound (21 mg) [OI2] Wash the column with 90 mL of chloroform:methanol (95:3), and thus 261 mg Formula I compound was obtained, and Formula III compound (27 mg) were yielded by eluted with 100 ml chloroform:
methanol (100:5).
FORMULATION EXAMPLES
In the formulations which follow, "active ingredient" means a compound or combination of compounds of Formula I, II, III, VI and V, or a salt or solvate thereof.
Formulation Example 1 Gelatin Capsules Ingredient (mglcapsule) m/m Quantity Active ingredient40mg 27%
Absolute Ethanol120mg 32.4%
Cremophor Rh40lSOmg 40.6%
Formulation Example 2 Oral Solution Ingredient (mglbottle) m/m Quantity Active ingredient100mg 28.75%
Absolute Ethanol75mg 21.43%
Cremophor Rh40175mg 50%
Claims
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002481664A CA2481664A1 (en) | 2004-09-30 | 2004-09-30 | Prevention of breast cancer with novel selective estrogen receptor moldulators |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA002481664A CA2481664A1 (en) | 2004-09-30 | 2004-09-30 | Prevention of breast cancer with novel selective estrogen receptor moldulators |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2481664A1 true CA2481664A1 (en) | 2006-03-30 |
Family
ID=36121727
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002481664A Abandoned CA2481664A1 (en) | 2004-09-30 | 2004-09-30 | Prevention of breast cancer with novel selective estrogen receptor moldulators |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2481664A1 (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2446641A (en) * | 2007-02-14 | 2008-08-20 | Tcp Innovations Ltd | Triphenylethylene TGF-beta stimulating agents |
| CN105640968A (en) * | 2016-01-14 | 2016-06-08 | 孙妙囡 | Ginseng saponin combination and application thereof |
-
2004
- 2004-09-30 CA CA002481664A patent/CA2481664A1/en not_active Abandoned
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB2446641A (en) * | 2007-02-14 | 2008-08-20 | Tcp Innovations Ltd | Triphenylethylene TGF-beta stimulating agents |
| GB2446641B (en) * | 2007-02-14 | 2010-10-13 | Tcp Innovations Ltd | Improved compositions and combinations 2 |
| CN105640968A (en) * | 2016-01-14 | 2016-06-08 | 孙妙囡 | Ginseng saponin combination and application thereof |
| CN105640968B (en) * | 2016-01-14 | 2019-03-19 | 孙妙囡 | A kind of Panaxsaponin composition and its application |
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