U.S. patent application number 10/274963 was filed with the patent office on 2003-06-12 for cancer treatment composition and method using signal transduction modulators and natural plant essential oils as synergists for conventional treatments.
This patent application is currently assigned to EcoSMART Technologies, Inc.. Invention is credited to Bessette, Steven M., Enan, Essam E..
Application Number | 20030108622 10/274963 |
Document ID | / |
Family ID | 22337523 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030108622 |
Kind Code |
A1 |
Bessette, Steven M. ; et
al. |
June 12, 2003 |
Cancer treatment composition and method using signal transduction
modulators and natural plant essential oils as synergists for
conventional treatments
Abstract
Pharmaceutical compositions containing plant essential oil
compounds, natural or synthetic, or mixtures or derivatives
thereof, with one or more signal transduction modulators, for the
prevention and treatment of cancer.
Inventors: |
Bessette, Steven M.;
(Brentwood, TN) ; Enan, Essam E.; (Franklin,
TN) |
Correspondence
Address: |
William F. Gadiano, Esq.
MCDERMOTT, WILL & EMERY
600 13th Street, N.W.
Washington
DC
20005
US
|
Assignee: |
EcoSMART Technologies, Inc.
|
Family ID: |
22337523 |
Appl. No.: |
10/274963 |
Filed: |
October 22, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10274963 |
Oct 22, 2002 |
|
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09455544 |
Dec 7, 1999 |
|
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|
60111271 |
Dec 7, 1998 |
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Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61K 45/06 20130101;
A61K 36/53 20130101; A61P 43/00 20180101; A61P 15/14 20180101; A61P
35/00 20180101; A61K 36/00 20130101; A61K 36/61 20130101; A61K
31/185 20130101; A61K 36/898 20130101; A61K 31/085 20130101; A61K
31/122 20130101; A61K 31/05 20130101; A61K 31/192 20130101; A61K
31/352 20130101; A61K 38/45 20130101; A61K 31/05 20130101; A61K
2300/00 20130101; A61K 36/53 20130101; A61K 2300/00 20130101; A61K
36/61 20130101; A61K 2300/00 20130101; A61K 36/898 20130101; A61K
2300/00 20130101; A61K 38/45 20130101; A61K 2300/00 20130101; A61K
36/00 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/725 |
International
Class: |
A61K 035/78 |
Claims
What is claimed is:
1. A pharmaceutical composition comprising at least one plant
essential oil compound and at least one signal transduction
modulator.
2. The pharmaceutical composition of claim 1, wherein the plant
essential oil compound is selected from the group consisting of
aldehyde C16 (pure), amyl cinnamic aldehyde, amyl salicylate,
anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde,
cinnamic alcohol, .alpha.-terpineol, carvacrol, carveol, citral,
citronellal, citronellol, p-cymene, diethyl phthalate, dimethyl
salicylate, dipropylene glycol, eucalyptol (cineole), eugenol,
iso-eugenol, galaxolide, geraniol, guaiacol, ionone, d-limonene,
menthol, methyl anthranilate, methyl ionone, methyl salicylate,
a-phellandrene, pennyroyal oil perillaldehyde, 1- or 2-phenyl ethyl
alcohol, 1- or 2-phenyl ethyl propionate, piperonal, piperonyl
acetate, piperonyl alcohol, D-pulegone, terpinen4-ol, terpinyl
acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol,
metabolites of trans-anethole, vanillin, and ethyl vanillin.
3. The pharmaceutical composition of claim 1, wherein the signal
transduction modulator is selected from the group consisting of
cyclic adenosine monophosphate (CAMP)/cAMP-dependent protein
kinase, tyrosine kinase, calcium phospholipid-dependent protein
kinase (PKC), mitogen activated protein kinase family members,
calcium-calmodulin-dependent protein kinase, and growth factor
receptor inhibitors.
4. The pharmaceutical composition of claim 1, further comprising
tamoxifen.
5. The pharmaceutical composition of claim 1, further comprising a
pharmaceutically acceptable carrier.
6. The pharmaceutical composition of claim 1, wherein the soft
tissue cancer is breast cancer.
7. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is eugenol.
8. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is thymol.
9. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is isoeugenol.
10. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is benzyl alcohol.
11. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is carvacrol.
12. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is cinnamic alcohol.
13. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is cinnamic aldehyde.
14. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is citronellal.
15. The pharmaceutical composition of claim 2, wherein the plant
essential oil compound is trans-anethole.
16. A method for treating cancer, comprising administering to a
patient in need thereof a therapeutically effective amount of the
composition of claim 1.
Description
CROSS REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
Patent Application No 60/111,271, filed Dec. 7,1998, the entire
disclosure of which is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The present invention relates, in general, to
therapeutically effective pharmaceutical compositions containing
conventional cancer treatments in combination with signal
transduction modulators and/or natural plant essential oils, and
methods for using same for prophylactically or therapeutically
treating of soft tissue cancer in mammals, including humans, such
as, for example, breast cancer.
BACKGROUND OF THE INVENTION
[0003] Breast cancer is a proliferative disease of mammary
epithelial cells and estrogen has been shown to stimulate cell
proliferation of these cells both in culture and in mice (Soto and
Sonnenschein, 1985; Osborne, 1988). Xenoestrogens have been
proposed to stimulate cell proliferation through binding and
activating estrogen receptors (ERs) (Miller et al., 1993; Hoffman,
1992). The incidence of breast cancer has been steadily rising
during the past two or three decades, a trend characterized by
increasing rates among estrogen-responsive tumors, by continuing
increases among older women, and by growing numbers in both
developed and developing countries (Harris et al., 1992). Between
1973-1980, the incidence of breast cancer in the United States
increased a modest 8% among women under 50 years of age, while it
rose 32.1% among women in the age group of 50 years or older (Ries
et al., 1991). This upward shift is consistent with the historical
pattern of accumulation of organochlorine insecticides residues
(xenoestrogens) in the environment (Mussalo-Rauhamaa et al., 1990;
Wolff et al., 1993; Davis et al., 1993). Breast cancer is also the
second leading cause of cancer deaths in women and it is estimated
that in 1998, there will be an additional 43,900 deaths due to
breast cancer. Environmental estrogens or endocrine disruptors have
been suggested to play a role in the etiology or promotion of
breast cancer (Davis et al., 1993; Dewailly et al., 1995).
Experimental evidence reveals that xenoestrogens affect estrogen
production and metabolism and are among the risk factors that cause
breast cancer (Nelson, 1974; Berthois et al., 1986; Henderson et
al., 1993; Jobling et al., 1996; Dees et al., 1997). Most of the
known risk factors for breast cancer, which at least account for
30% of cases (Henderson et al., 1993) are linked with total
life-time exposure to reproductive chemicals such as estrogen and
xenoestrogens.
[0004] It appears evident that soft tissue cancer in mammals is
increasing every year as a result of increased estrogen levels and
increased exposure to environmental xenoestrogens. For example, the
number of prescriptions of estrogen for women in menopause is
rapidly increasing, presently estimated at 50,000,000 prescriptions
annually in the United States alone. This increasing use of
estrogen partially accounts for the higher risk of breast cancer in
both young and middle-aged women. Estrogen is present in all
mammals and is essential in women for reproductive organs such as
ovary, uterus, breast, etc. In men, however, estrogen is required
for sperm production and maturation. The abusive use of estrogen
prescribed for women is at least partially responsible for the
development of soft tissue cancers, especially breast cancer. It is
therefore desirable to antagonize or counteract the adverse effects
of estrogen in women.
[0005] The current FDA-approved treatments, e.g., tamoxifen, in the
United States are effective to some extent in some of the female
population in antagonizing the adverse effects of estrogen.
Unfortunately, these treatments are not totally effective and may
themselves cause additional health related effects, such as uterine
cancer. Thus, if one could identify compounds that would make the
current treatments more effective, or would work in conjunction
with, or in lieu of, the present treatments, it is possible some of
these adverse side effects would be alleviated or even eliminated.
Possible sources of alternative treatments are natural, non-toxic
compounds. It is proposed that these compounds would advantageously
provide for safer and more effective treatments.
[0006] The current literature suggests the use of certain signal
transduction modulators as treatments for breast and colon cancer.
The use of certain monoterpenoid plant essential oils
(alpha-terpineol, linalool, and limonene) is also suggested as a
potential treatment for breast cancer. These monoterpenoids however
are not totally effective and have been proven to be weak
anti-proliferative cancer products. In addition, these data do not
suggest the capability of these compounds to antagonize the action
of estrogen. This may raise the question of how this product may
interact in women with estrogen supplement. None of the data
disclose the synergistic effects of plant essential oils and/or
signal transduction modulators in combination with conventional
treatments to enhance the anti-proliferative and anti-estrogenic
effects of the current treatments without the corresponding
increase in related health risks.
[0007] Accordingly, there is a great need for novel compositions
containing current anti-cancer treatments in combination with novel
ingredients to improve the effective treatment of soft tissue
cancer and to reduce the adverse effects of such current
anti-cancer treatments.
SUMMARY OF THE INVENTION
[0008] An object of the present invention is to provide novel
compositions containing beneficial mixtures of current treatments
for soft tissue cancer with non-toxic plant essential oils, or
derivatives thereof, and/or one or more signal transduction
modulators, for the safe and effective treatment of soft tissue
cancer.
[0009] The above and other objects are accomplished by the present
invention which is directed to novel pharmaceutical compositions
for prophylactically or therapeutically treating soft tissue
cancers. The inventive compositions comprise at least one plant
essential oil compound and/or at least one signal transduction
modulator in admixture with a conventional cancer treatment, e.g.,
tamoxifen.
[0010] Additional objects and attendant advantages of the present
invention will be set forth, in part, in the description that
follows, or may be learned from practicing or using the present
invention. The objects and advantages may be realized and attained
by means of the features and combinations particularly recited in
the appended claims. It is to be understood that the foregoing
general description and the following detailed description are
exemplary and explanatory only and are not to be viewed as being
restrictive of the invention, as claimed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] The features and advantages of the present invention will
become more clearly appreciated from the following description
taken in conjunction with the accompanying drawings in which like
elements are denoted by like reference numerals.
[0012] FIG. 1 shows the concentration-response of tamoxifen and
forskolin against estrogen-induced (E.sub.2-induced) proliferation
in breast cancer cells.
[0013] FIG. 2 shows the mixed exposure effect of tamoxifen (TX) and
geldanamycin (GM) on E.sub.2-induced growth in breast cancer
cells.
[0014] FIG. 3 shows the mixed exposure effect of forskolin (FK)
with tamoxifen (TX), and geldanamycin (GM) with TX on
E.sub.2-induced growth in breast cancer cells.
[0015] FIG. 4 shows the time-course effect of tamoxifen (TX) alone
and TX plus forskolin (FK) on E.sub.2-induced growth in breast
cancer cells. Data was calculated as percentage of control
value.
[0016] FIG. 5 shows the time-course effect of tamoxifen (TX) and TX
plus forskolin (FK) on E.sub.2-induced growth in breast cancer
cells. Data is calculated as percentage of E.sub.2-induced cell
growth.
[0017] FIG. 6 shows the mixed exposure effect of tamoxifen (TX, 1
.mu.M) and forskolin (FK, 5 .mu.M) on the growth of breast cancer
cells.
[0018] FIG. 7 shows the mixed exposure effect of tamoxifen (TX, 1
.mu.M) and forskolin (FK, 1 .mu.M) on the growth of breast cancer
cells.
[0019] FIG. 8 shows the effect of different concentrations of
geldanamycin, the c-Src kinase inhibitor and tamoxifen (TX) on the
growth of breast cancer cells.
[0020] FIG. 9 shows the mixed exposure effect of tamoxifen (TX, 5
.mu.M) and mitogen activated protein kinase, MAPK, inhibitor,
PD98059 (2 .mu.M) on the growth of breast cancer cells.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0021] All patents, patent applications and literatures cited in
this description are incorporated herein by reference in their
entirety. In the case of inconsistencies, the present disclosure,
including definitions, will prevail.
[0022] In a preferred embodiment, the present invention provides a
novel pharmaceutical composition comprising at least one plant
essential oil compound derived from either natural or synthetic
sources and at least one signal transduction modulator including
mixtures or derivatives of plant essential oil compound. In a
further preferred embodiment, the present invention is directed to
a novel pharmaceutical composition comprising an anti-cancer
treatment, such as tamoxifen, at least one plant essential oil
compound derived from either natural or synthetic sources or
mixtures or derivatives plant essential oil compounds, and/or at
least one signal transduction modulator.
[0023] The specific plant essential oils disclosed herein or
derivative thereof comprise a monocyclic, carbocyclic ring
structure having six-members and substituted by at least one
oxygenated or hydroxyl functional moiety. Examples of plant
essential oils encompassed within the present invention, include,
but are not limited to, members selected from the group consisting
of aldehyde C16 (pure), amyl cinnamic aldehyde, amyl salicylate,
anisic aldehyde, benzyl alcohol, benzyl acetate, cinnamaldehyde,
cinnamic alcohol, a-terpineol, carvacrol, carveol, citral,
citronellal, citronellol, p-cymene, diethyl phthalate, dimethyl
salicylate, dipropylene glycol, eucalyptol (cineole), eugenol,
iso-eugenol, galaxolide, geraniol, guaiacol, ionone, d-limonene,
menthol, methyl anthranilate, methyl ionone, methyl salicylate,
.alpha.-phellandrene, pennyroyal oil perillaldehyde, 1- or 2-phenyl
ethyl alcohol, 1- or 2-phenyl ethyl propionate, piperonal,
piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen4-ol,
terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil,
thymol, metabolites of trans-anethole, vanillin, ethyl vanillin,
and the like. As these plant essential oil compounds are known and
used for a variety of other purposes, they may be prepared by a
skilled artisan by employing known methods.
[0024] The above plant essential oil compounds can also be
administered in combination with at least one signal transduction
modulator. Non-limiting examples of signal transduction modulators
include members selected from the group consisting of cyclic
adenosine monophosphate (cAMP)/cAMP-dependent protein kinase,
tyrosine kinase, calcium phospholipid-dependent protein kinase
(PKC), mitogen activated protein kinase family members,
calcium-calmodulin-dependent protein kinase, and growth factor
receptor inhibitors. Specific non-limiting examples of such signal
transduction modulators include octopamine, forskolin, PD98059,
geldanamycin and genistein, and staurosporin. When the plant
essential oil compounds of the present invention are administered
in combination therapies with other agents, they may be
administered sequentially or concurrently to a patient in need of
treatment.
[0025] The above plant essential oil compounds and signal
transduction modulators of the present invention may be purchased
from conventional sources, may be readily isolated from specific
plants or trees and purified (isolated) or may be synthesized using
conventional techniques. Advantageously, these compounds may be
conveniently synthesized from readily available starting materials.
The relative ease with which the compounds of this invention can be
obtained represents an enormous advantage in the large-scale
production of these compounds.
[0026] It will be appreciated that the therapeutically-active plant
essential oil compounds of the present invention may be modified or
derivatized by appending appropriate functionalities, i.e.,
functional groups, to enhance selective biological properties. Such
modifications are known in the art and include those that increase
biological penetration into a given biological compartment (e.g.,
blood, lymphatic system, central nervous system), increase oral
availability, increase solubility to allow administration by
injection, alter metabolism and alter rate of excretion. In
addition, the plant essential oil compounds may be altered to
pro-drug form such that the desired therapeutically-active form of
the compound is created in the body of the patient as the result of
the action of metabolic or other biochemical processes on the
pro-drug. Some examples of pro-drug forms include ketal, acetal,
oxime, and hydrazone forms of compounds which contain ketone or
aldehyde groups.
[0027] Moreover, the therapeutically-effective plant essential oil
compounds of the present invention may contain one or more
asymmetric carbon atoms and thus may occur as racemates and racemic
mixtures, single enantiomers, diastereomeric mixtures and
individual diastereomers. Each stereogenic carbon may be of the R
or S configuration. All such isomeric forms of these compounds are
expressly included within the purview of the present invention.
[0028] As will be appreciated, the compositions and method of the
present invention include pharmaceutical compositions that comprise
at least one plant essential oil, and pharmaceutically acceptable
salts thereof, in combination with any pharmaceutically acceptable
carrier, adjuvant or vehicle. The term "pharmaceutically acceptable
carrier or adjuvant" refers to a carrier or adjuvant that may be
administered to a patient, together with a plant essential oil
compound of the present invention, and which does not destroy the
pharmacological activity thereof and is nontoxic when administered
in doses sufficient to deliver a therapeutic amount of the
compound.
[0029] Pharmaceutically acceptable salts of the plant essential oil
compounds of this invention include those derived from
pharmaceutically acceptable inorganic and organic acids and bases.
Examples of suitable acid salts include, without limitation,
acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate,
bisulfate, butyrate, citrate, camphorate, camphorsulfonate,
cyclopentanepropionate, digluconate, dodecylsulfate,
ethanesulfonate, formate, fumarate, glucoheptanoate,
glycerophosphate, glycolate, hemisulfate, heptanoate, hexanoate,
hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate,
lactate, maleate, malonate, methanesulfonate,
2-naphthalenesulfonate, nicotinate, nitrate, oxalate, palmoate,
pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,
pivalate, propionate, salicylate, succinate, sulfate, tartrate,
thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,
while not in themselves pharmaceutically acceptable, may be
employed in the preparation of salts useful as intermediates in
obtaining the compounds of the invention and their pharmaceutically
acceptable acid addition salts.
[0030] Salts derived from appropriate bases include alkali metal
(e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium
and N-(C.sub.1-4 alkyl)4+salts. The present invention also
envisions the quatemization of any basic nitrogen-containing groups
of the compounds disclosed herein. Water or oil-soluble or
dispersible products may be obtained by such quaternization.
[0031] Further, pharmaceutically acceptable carriers, adjuvants and
vehicles that may be used in the pharmaceutical compositions of
this invention include, but are not limited to, ion exchangers,
alumina, aluminum stearate, lecithin, self-emulsifying drug
delivery systems (SEDDS) such as d.alpha-tocopherol
polyethyleneglycol 1000 succinate, or other similar polymeric
delivery matrices or systems, serum proteins, such as human serum
albumin, buffer substances such as phosphates, glycine, sorbic
acid, potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, sodium carboxymethylcellulose, polyacrylates,
waxes, polyethylene-polyoxypropylene-block polymers, polyethylene
glycol and wool fat. Cyclodextrins such as alpha-, beta-, and
gamma-cyclodextrin, or chemically modified derivatives such as
hydroxyalkylcyclodextrins, including 2- and
3-hydroxypropyl-.beta.-cyclodextrins, or other solublized
derivatives may also be advantageously used to enhance delivery of
therapeutically-effective plant essential oil compounds of the
present invention.
[0032] The pharmaceutical compositions of this invention may be
administered orally, parenterally, by inhalation spray, topically,
rectally, nasally, buccally, vaginally or via an implanted
reservoir, however, oral administration or administration by
injection is preferred. The-pharmaceutical compositions of this
invention may contain any conventional non-toxic
pharmaceutically-acceptable carriers, adjuvants or vehicles. In
some cases, the pH of the formulation may be adjusted with
pharmaceutically acceptable acids, bases or buffers to enhance the
stability of the formulated compound or its delivery form. The term
parenteral as used herein includes subcutaneous, intracutaneous,
intravenous, intramuscular, intraarticular, intrasynovial,
intrasternal, intrathecal, intralesional and intracranial injection
or infusion techniques.
[0033] The pharmaceutical compositions may be in the form of a
sterile injectable preparation, for example, as a sterile
injectable aqueous or oleaginous suspension. This suspension may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents (such as, for example, Tween 80) and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a non-toxic
parenterally-acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are mannitol, water, Ringer's
solution and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose, any bland fixed oil may be
employed including synthetic mono- or diglycerides. Fatty acids,
such as oleic acid and its glyceride derivatives are useful in the
preparation of injectables, as are natural
pharmaceutically-acceptable oils, such as olive oil or castor oil,
especially in their polyoxyethylated versions. These oil solutions
or suspensions may also contain a long-chain alcohol diluent or
dispersant such as Ph. Helv or a similar alcohol.
[0034] The pharmaceutical compositions of the present invention may
be orally administered in any orally acceptable dosage form
including, but not limited to, capsules, tablets, and aqueous
suspensions and solutions. In the case of tablets for oral use,
carriers which are commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, are also typically
added. For oral administration in a capsule form, useful diluents
include lactose and dried corn starch. When aqueous suspensions are
administered orally, the active ingredient is combined with
emulsifying and suspending agents. If desired, certain sweetening
and/or flavoring and/or coloring agents may be added.
[0035] The pharmaceutical compositions of the present invention may
also be administered in the form of suppositories for rectal
administration. These compositions can be prepared by mixing a
compound of this invention with a suitable non-irritating excipient
which is solid at room temperature but liquid at the rectal
temperature and therefore will melt in the rectum to release the
active components. Such materials include, but are not limited to,
cocoa butter, beeswax and polyethylene glycol.
[0036] Although rare, topical administration of the pharmaceutical
compositions of the present invention is especially useful when the
desired treatment involves areas or organs readily accessible by
topical application. For application topically to the skin, the
pharmaceutical composition should be formulated with a suitable
ointment containing the active components suspended or dissolved in
a carrier. Carriers for topical administration of the compounds of
this invention include, but are not limited to, mineral oil, liquid
petroleum, white petroleum, propylene glycol, polyoxyethylene
polyoxypropylene compound, emulsifying wax and water.
Alternatively, the pharmaceutical composition can be formulated
with a suitable lotion or cream containing the active compound
suspended or dissolved in a carrier. Suitable carriers include, but
are not limited to, mineral oil, sorbitan monostearate, polysorbate
60, cetyl esters wax, cetearyl alcohol, 2-octyidodecanol, benzyl
alcohol and water. The pharmaceutical compositions of this
invention may also be topically applied to the lower intestinal
tract by rectal suppository formulation or in a suitable enema
formulation. Topically-transdermal patches are also included in
this invention.
[0037] The pharmaceutical compositions of this invention may be
administered by nasal aerosol or inhalation. Such compositions are
prepared according to techniques well-known in the art of
pharmaceutical formulation and may be prepared as solutions in
saline, employing benzyl alcohol or other suitable preservatives,
absorption promoters to enhance bioavailability, fluorocarbons,
and/or other solubilizing or dispersing agents known in the
art.
[0038] Another acceptable pharmaceutical preparation would be an
encapsulated form of the plant essential oil compounds, as is, or
modified as per the prior descriptions. The walls of the capsules
could be designed to release the plant essential oils rapidly, i.e.
one minute, hour or day, or it could be designed to release over
some designated period of time, i.e. days, weeks or months. The
wall materials could be natural or synthetic is polymers acceptable
to the US FDA or composed of lipids or other suitable
materials.
[0039] These capsules could be delivered either orally or by
injection and could be either water or oil based depending upon the
desired method of use or required rate of release.
[0040] Dosage levels of between about 0.001 and about 100 mg/kg
body weight per day, preferably between about 0.5 and about 75
mg/kg body weight per day of the active ingredient compound are
useful in the prevention and treatment of soft tissue cancers.
Typically, the pharmaceutical compositions of this invention will
be administered from about 1 to about 5 times per day or
alternatively, as a continuous infusion. Such administration can be
used as a chronic or acute therapy. The amount of active ingredient
that may be combined with the carrier materials to produce a single
dosage form will vary depending upon the host treated and the
particular mode of administration. A typical preparation will
contain from about 5% to about 95% active compound (w/w).
Preferably, such preparations contain from about 20% to about 80%
active compound.
[0041] Upon improvement of a patient's condition, a maintenance
dose of a compound, composition or combination of this invention
may be administered, if necessary. Subsequently, the dosage or
frequency of administration, or both, may be reduced, as a function
of the symptoms, to a level at which the improved condition is
retained when the symptoms have been alleviated to the desired
level, treatment should cease. Patients may, however, require
intermittent treatment on a long-term basis upon any recurrence of
disease symptoms.
[0042] The prophylactic use of the present invention may require
the daily intake of a prophylactically-effective amount.
[0043] As the skilled artisan will appreciate, lower or higher
doses than those recited above may be required. Specific dosage and
treatment regimens for any particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health status, sex, diet,
time of administration, rate of excretion, drug combination, the
severity and course of a cancer, the patient's disposition to
cancer and the judgment of the treating physician.
[0044] The compositions and methods of the present invention will
be further illustrated in the following non-limiting Examples that
show the synergistic effects of signal transduction modulators with
tamoxifen. When reading the following examples, it will be
appreciated that the growth and proliferation of MCF-7 cells are
strictly estrogen-dependent. In the presence of estrogen, the cells
grow, confluent and form foci, the landmark of tumor diagnosis. In
the absence of estrogen, the growth of these cells is slow and the
formation of foci is rare. The following Examples are illustrative
of various embodiments only and do- not limit the claimed invention
regarding the materials, conditions, weight ratios, process
parameters and the like recited herein. Examples 14 exemplify,
inter alia, the present invention against breast cancer in
menopausal women who are taking estrogen supplement. Examples 5-7
exemplify, inter alia, the present invention against breast cancer
in pre-menopausal women and/or elderly women who are not taking
estrogen supplement, but have potential risk factors to develop
breast cancer.
Example 1
[0045] Dose-Response of Tamoxifen on Estrogen (E.sub.2)-Induced
Cell Proliferation MCF-7 cells were cultured in growth DMEM medium
supplemented with 10% fetal bovine serum (FBS). At 85% confluence,
cells were sub-cultured in 5% FBS serum stripped medium, phenol red
free for 24 hours prior to the treatment of the test chemical.
Cells were treated with 10 nM E.sub.2 (=2.7 ng E.sub.2/ml) with and
without different concentrations of tamoxifen (TX) 24 hours prior
to cell proliferation assessment. Cell proliferation was measured
using .sup.3H-thymidine incorporation. The study was done in
triplicate and a control was used with solvent only. Control
received solvent only at <0.1% ethanol.
[0046] Results are shown in Table 1. In Table 1, percentage of
control is calculated either based on the control (solvent only,
ethanol 0.1%) or based on the E.sub.2 values which is between
parenthesis.
1TABLE 1 Dose-Response Effect of TX .sup.3H-thymidine incorporation
Test dpm/.mu.g % of % Treatment Conc. dpm protein control
protection Control 1993 306.6 100 (solvent only) E.sub.2 10 nM
16388 483.4 157 proliferation TX 1 .mu.M 2586 129.3 42 58 TX +
E.sub.2 12336 379.6 124 (79) proliferation (21) TX 5 .mu.M 1848
112.0 36 64 TX + E.sub.2 4007 260.2 85 (53) 15 (47) TX 10 .mu.M
1545 99.8 33 67 TX + E.sub.2 3239 118.5 39 (25) 61 (75)
[0047] Table 1 demonstrates that the antagonistic action of TX
against the E.sub.2-induced cell proliferation is dose-dependent.
TX at low concentrations between 0.56 ug/ml to 2.8 ug/ml (=1 - 5
.mu.M) is potent against the E.sub.2-induced cell
proliferation.
Example 2
[0048] Dose-response of Signal Transduction Modulators on
E2-Induced Cell Proliferation To determine the impact of two key
signaling proteins, cAMP/PKA system and tyrosine kinase family
member, c-Src kinase (p.sup.src60) on estrogen-induced cell
proliferation in human epithelial breast cancer cells, MCF-7 cells
were cultured in growth DMEM medium supplemented with 10% fetal
bovine serum (FBS). At 85% confluence, cells were sub-cultured in
5% FBS serum stripped medium, phenol red free for 24 hours prior to
the treatment of the test chemical. Cells were treated with 10 nM
E.sub.2 (=2.7 ng E.sub.2/ml) with and without different
concentrations (1-10 .mu.M=0.41-4.1 .mu.g/ml) of CAMP inducer,
forskolin (FK) and c-Src kinase inhibitor geldanamycin (GM) 24
hours prior to cell proliferation assessment. Tamoxifen (TX) was
used in parallel as positive control. Cell proliferation was
measured using .sup.3H-thymidine incorporation. The study was done
in triplicate and a control was used with solvent only. Control
received solvent only at <0.1% ethanol. Results are shown in
Table 2 and FIG. 1.
2TABLE 2 Dose-Response Effect on E.sub.2 Induced MCF-7 Cell
Proliferation .sup.3H-Thymidine incorporation/.mu.g protein
Treatment (% of control) % protection Control 100 E.sub.2 (10 nM)
290 proliferation In the presence of 10 nM E.sub.2 FK 1 .mu.M 34 66
FK 5 .mu.M 22 78 FK 10 .mu.M 14 86 TX 1 .mu.M 79 21 TX 5 .mu.M 54
46 TX 10 .mu.M 25 75 GM 1 ng/ml no effect none GM 5 ng/ml no effect
none GM 10 ng/ml 94 6
[0049] Table 2 illustrates that a) cAMP/PKA cascade, and estrogen
receptor pathway are involved in the cancer cell proliferation, b)
the antiestrogenic action of TX and FK is dose-dependent, and c)
GM, the c-Src kinase inhibitor, at 10 ng/ml did not inhibit the
E.sub.2-induced cell proliferation.
Example 3
[0050] Effect of FK or GM on TX Efficacy Against E.sub.2-Induced
Cell Proliferation
[0051] MCF-7 cells were cultured in growth DMEM medium supplemented
with 10% fetal 10 bovine serum (FBS). At 85% confluence, cells were
sub-cultured in 5% FBS serum stripped medium, phenol red free for
24 hours prior to the treatment of the test chemical. Cells were
treated with 10 nM E.sub.2 (=2.7 ng E.sub.2/ml) with and without
two concentrations of FK (1 and 5 .mu.M =0.41 and 2.05 pg FK/ml).
In another experiment cells were treated with 10 nM E.sub.2 (=2.7
ng E.sub.2/ml) with three concentraion of GM (1, 5 and 10 ng/ml) 24
hours prior to cell proliferation assessment. Tamoxifen (TX) was
used in parallel as positive control. Cell proliferation was
measured using .sup.3H-thymidine incorporation. The study was done
in triplicate and a control was used with solvent only. Control
received solvent only at <0.1% ethanol. Results are shown in
Tables 3 and 4 and FIGS. 2 and 3.
[0052] The data in Table 3 shows that the synergistic action
between TX and FK depends on the ratio between the two chemicals
when they are applied as a mixture. The ratio recommended is 1 part
TX: 3.7 parts FK, based on .mu.g of active ingredient per ml. In
addition, although GM alone did not antagonize the estrogenic
activity of E2, it was able to increase the efficacy of TX
significantly (Table 4). Our data showed that when 10 ng GM/ml was
mixed with 5 .mu.M TX (=2.8 ug TX/ml), a 75% protection (vs 46%
protection by 5 .mu.M TX alone) was revealed (Table 4). Another
confirmation for the synergistic action of GM on TX against
E.sub.2-induced breast cancer cell growth emerged from the data in
FIG. 2. These data illustrate that 1 .mu.M of TX provides an
approximate 25% protection against the biological activity of
E.sub.2. In the presence of 10 ng GM/ml, 45% increase in the
antiestrogenicity of 1 .mu.M TX (=0.56 ug TX/ml) was found.
Interestingly, 5 .mu.M TX alone induced 45% protection against the
E.sub.2-induced cancer cell growth. Thus, the concentration of TX
can be reduced by 20 percent to minimize (if not abolish)
detrimental side effects without adversely affecting efficacy, as
shown in FIG. 3 which illustrates the synergistic relationship
between TX and FK or GM.
3TABLE 3 Dose-Response Effect on E.sub.2 Induced MCF-7 Cell
Proliferation .sup.3H-Thymidine incorporation/.mu.g protein
Treatment % of control % protection Control 100 E.sub.2 (10 nM) 290
proliferation In the presence of 10 nM E.sub.2 FK 1 .mu.M 34 66 FK
5 .mu.M 22 78 FK 10 .mu.M 14 86 TX 1 .mu.M 79 21 TX 5 .mu.M 54 46
TX 10 .mu.M 25 75 GM 1 ng/ml no effect none GM 5 ng/ml no effect
none GM 10 ng/ml 94 6 With 1 .mu.M FK: FK + TX 1 .mu.M + E.sub.2 43
57 FK + TX 5 .mu.M + E.sub.2 21 79 FK + TX 10 .mu.M + E.sub.2 13 87
With 5 .mu.M FK: FK + TX 1 .mu.M + E.sub.2 21 79 FK + TX 5 .mu.M +
E.sub.2 12 88 FK + TX 10 .mu.M + E.sub.2 9 91
[0053]
4TABLE 4 Interaction Between TX and GM on MCF-7 Cell Proliferation
.sup.3H-Thymidine incorporation/.mu.g protein Treatment (% of
control) % protection E.sub.2 10 Nm 100 proliferation GM 1 ng/ml
100 0 GM 5 ng/ml 100 0 GM 10 ng/ml 90 10 With 5 .mu.M TX: + E.sub.2
54 46 + E.sub.2 + GM 1 ng/ml 54 46 + E.sub.2 + GM 5 ng/ml 50 50 +
E.sub.2 + GM 10 ng/ml 25 75 E.sub.2 + FK 5 .mu.M + GM 10 ng/ml + TX
31 69 5 .mu.M
Example 4
[0054] Effect of cAMP Inducer on Efficacy and Stability of
Tamoxifen MCF-7 cells were cultured in growth DMEM medium
supplemented with 10% fetal bovine serum (FBS). At 85% confluence,
cells were sub-cultured in 5% FBS serum stripped medium, phenol red
free for 24 hours prior to the treatment of the test chemical.
Cells were treated with 10 nM E.sub.2 (=2.7 ng E.sub.2/ml) with and
without a mixture of 1 .mu.M FK and 1 .mu.M TX. Cell growth was
measured 1, 2 and 5 days following treatment. Cell growth was
determined by cell count using trypan blue exclusion. The study was
done in duplicate and a control was used with solvent only. Control
received solvent only at <. 1% ethanol. Results are shown in
FIGS. 4 and 5.
[0055] FIG. 4 illustrates that when the efficacy of TX and FK
against E2 action was calculated based on the control value the
following was found: (a) TX (1 .mu.M =0.56 ug TX/ml) alone was able
to antagonize the E.sub.2 action up to 48 hr after treatment and
(b) this action of TX was sustained up to 4-5 days in the presence
of 1 .mu.M FK (FIG. 4).
[0056] FIG. 5 shows that when the efficacy of TX and FK was
calculated as percentage of the E.sub.2-induced cell growth value
the same conclusion was found except more stability and protection
by TX was found in the presence of FK.
Example 5
[0057] Effect of Forskolin on Tamoxifen efficacy
[0058] MCF-7 cells were cultured in growth DMEM medium supplemented
with 10% fetal bovine serum (FBS). At 85% confluence, cells were
sub-cultured in 5% FBS serum stripped medium, phenol red free for
24 hours prior to the treatment of the test chemical. Cells were
treated with and without a mixture of either 5 .mu.M FK (2.05 .mu.g
FK/ml) and 1 .mu.M TX (=0.56 ug TX/ml) or a mixture of 1 .mu.M TX
(=0.56 ug TX/ml) and 1 .mu.M FK. Cell growth was measured 5 days
following treatment. Cell growth was determined by cell count using
trypan blue exclusion. The study was done in duplicate and a
control was used with solvent only. Control received solvent only
at <0.1% ethanol. Results are shown in FIGS. 6 and 7.
Example 6
[0059] Effect of c-Src Kinase Inhibitor GM on Tamoxifen
Efficacy
[0060] MCF-7 cells were cultured in growth DMEM medium supplemented
with 10% fetal bovine serum (FBS). At 85% confluence, cells were
sub-cultured in 5% FBS serum stripped medium, phenol red free for
24 hours prior to the treatment of the test chemical. Cells were
treated with and without a mixture of either 5 ng GM/ml and 5 .mu.M
TX (=2.8 ug TX/ml) or a mixture of 10 ng GM/ml and 5 .mu.M TX (=2.8
ug TX/ml). Cell growth was measured 5 days following treatment.
Cell growth was determined by cell count using trypan blue
exclusion. The study was done in duplicate and a control was used
with solvent only. Control received solvent only at <0.1%
ethanol. Results are shown in FIG. 8.
Example 7
[0061] Effect of mitogen activated protein kinases inhibitor
PD98059 on TX efficacy
[0062] MCF-7 cells were cultured in growth DMEM medium supplemented
with 10% fetal bovine serum (FBS). At 85% confluence, cells were
sub-cultured in 5% FBS serum stripped medium, phenol red free for
24 hours prior to the treatment of the test chemical. Cells were
treated with and without a mixture of 5 .mu.M TX (=2.8 ug TX/ml)
and 2 .mu.M PD98059. Cell growth was measured 5 days following
treatment. Cell growth was determined by cell count using trypan
blue exclusion. The study was done in duplicate and a control was
used with solvent only. Control received solvent only at <0.1%
ethanol. Results are shown in FIG. 9. p The above Examples show,
inter alia, that (a) signal transduction cascades are vital in the
cancer cell growth and cell proliferation and (b) CAMP inducers
(e.g., FK), c-Src kinase inhibitors (e.g., GM) and MAPK inhibitors
(e.g., PD98059) synergize the anticancer activity of tamoxifen in
breast cancer cells. In general, one or more signal transduction
modulators may be used in combination with plant essential oil
compounds and derivatives thereof to provide anti-proliferative,
anti-estrogenic and/or anti-mitogenic compositions for
prophylactically and/or therapeutically treating soft tissue
cancers.
[0063] Although illustrative embodiments of the present invention
have been described in detail, it is to be understood that the
present invention is not limited to those precise embodiments, and
that various changes and modifications can be effected therein by
one skilled in the art without departing from the scope and spirit
of the invention as defined by the appended claims.
* * * * *