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Cancer Patent Abstract
Pharmaceutical compositions containing plant essential oils, natural
or synthetic, or mixtures or derivatives thereof, for the prevention
and treatment of soft tissue cancer in mammals.
Cancer Patent Claims
What is claimed is:
1. A method for inhibiting the growth of breast cancer cells, the
method comprising applying a therapeutically effective amount of
eugenol to a population of breast cancer cells for a sufficient
time to observe inhibition of growth in said breast cancer cells.
2. The method of claim 1, wherein the breast cancer cells are MCF-7
cells.
Cancer Patent Description
FIELD OF THE INVENTION
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, 1981). 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 (Reese et al., 1991). This upward shift is consistent with
the historical pattern of accumulation of organochlorine insecticide
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., 1994).
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.
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. A possible source
of alternative treatments are natural, non-toxic compounds. It is
proposed that these compounds would advantageously provide for safer
and more effective treatments.
The use of certain monoterpenoid plant essential oils (alpha-terpineol,
linalool, and limonene) is 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 of action of estrogen. This may raise the question
of how this product may interact in women with estrogen supplement.
Accordingly, there is a great need for novel pharmaceutical compositions
containing non-toxic ingredients that may be effectively used in
the prevention or treatment of soft tissue cancer in mammals.
SUMMARY OF THE INVENTION
A primary object of the present invention is to provide novel compositions
that contain certain plant essential oils, natural or synthetic
in source, or mixtures or derivatives thereof, as a prophylactic
for, or a treatment of, soft tissue cancer.
The above and other objects are accomplished by the present invention
which is directed to novel pharmaceutical compositions containing
at least one plant essential oil compound, including mixtures or
derivatives thereof, which are synthetically made or obtained from
natural sources. The present invention is also directed to methods
for using such novel pharmaceutical compositions for prophylactically
or therapeutically treating soft tissue cancers.
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 instrumentalities 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.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
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.
In a preferred embodiment, the present invention provides a novel
pharmaceutical composition for preventing or treating cancer, the
composition comprising at least one plant essential oil compound
including mixtures or derivatives of plant essential oil compounds
derived from either natural or synthetic sources.
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, .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, .alpha.-phellandrene, pennyroyal oil perillaldehyde,
1- or 2-phenyl ethyl alcohol, 1- or 2-phenyl ethyl propionate, piperonal,
piperonyl acetate, piperonyl alcohol, D-pulegone, terpinen-4-ol,
terpinyl acetate, 4-tert butylcyclohexyl acetate, thyme oil, thymol,
metabolites of trans-anethole, vanillin, ethyl vanillin, and the
like.
As plant essential oil compounds are known and used for other purposes,
they may be prepared by a skilled artisan by employing known methods.
In addition, they 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 compositions
of the present invention can be synthesized represents an enormous
advantage in the large-scale production of these compounds.
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.
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.
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.
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.
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 quaternization
of any basic nitrogen-containing groups of the compounds disclosed
herein. Water or oil-soluble or dispersible products may be obtained
by such quaternization.
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.
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.
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.
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.
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
glycols.
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-octyldodecanol,
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.
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.
Another acceptable pharmaceutical preparation would be an encapsulated
form of the plant essential oils, 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 polymers acceptable to the US FDA or composed
of lipids or other suitable materials. 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.
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.
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.
The prophylactic use of the present invention may require the daily
intake of a prophylactically-effective amount.
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.
The compositions and methods of the present invention will be further
illustrated in the following, non-limiting Examples. The 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. 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.
EXAMPLE 1
Antiproliferative Effect on Human Epithelial Breast Cancer Cells
(MCF-7)
MCF-7 cells were cultured in growth 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. After 24 hours of treatment
cell proliferation was measured using .sup.3H-thymidine incorporation.
These test chemicals were tested in the presence and absence of
estrogen to address if they have anti-estrogenic activity in addition
to their antiproliferative effect. The study was done in triplicate
and a control was used with solvent only. Control received solvent
only at <0.1% ethanol. Estrogen was tested at 1 nM (=0.27 ng
E.sub.2/ml). Exemplary plant essential oil compounds were tested
at 50 ug/ml. Results are shown in Table 1.
TABLE-US-00001 TABLE 1 .sup.3H-thymidine incorporation % of % of
dpm/ug control estrogen Test Chemical protein value value Control
414.2 100 Estrogen 658.6 158 100 .alpha.-terpineol 480.5 116 .alpha.-terpineol
+ estrogen 679.1 164 103 Trans-anethole 434.9 105 Trans-anethole
+ estrogen 589.8 142 89 Carvacrol 207.2 50 Carvacrol + estrogen
181.7 43 28 Cinnamic alcohol 18.5 4 Cinnamic alcohol + estrogen
220.8 53 34 Eugenol 215.6 52 Eugenol + estrogen 289.1 70 44 Iso-eugenol
119 29 Iso-eugenol + estrogen 164 40 25 Thymol 97.3 23 Thymol +
estrogen 108.2 26 16 Citronellal 85.2 20 Citronellal + estrogen
284.0 69 43 p-cymene 419.0 101 p-cymene + estrogen 632.0 152 96
Eucalyptol 398.0 96 Eucalyptol + estrogen 568.0 137 86 Methyl salicylate
444.0 107 Methyl salicylate + estrogen 693.9 168 106
EXAMPLE 2
Antiproliferative Effect
MCF-7 cells were cultured in growth 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. After 24 hours of treatment
cell proliferation was measured using .sup.3H-thymidine incorporation.
These test chemicals were tested in the presence and absence of
estrogen to address if they have anti-estrogenic activity in addition
to their antiproliferative effect. The study was done in triplicate
and a control was used with solvent only. Control received solvent
only at <0.1% ethanol. Estrogen was tested at 1 nM (0.27 ng E.sub.2/ml).
Plant essential oil compounds were tested at 50 ug/ml. Results are
shown Table 2.
TABLE-US-00002 TABLE 2 .sup.3H-thymidine incorporation dpm/.mu.g
% of control % of estrogen Test Chemical protein value value Control
438.1 100 Estrogen 664.2 151.6 100 .alpha.-terpineol 490.7 112.0
.alpha.-terpineol + estrogen 636.7 145.3 96 Guaiacol 712.3 162.6
Guaiacol + estrogen 788.8 180.1 119 R-(+)-Limonene 393.1 89.7 R-(+)-Limonene
+ estrogen 522.6 119.3 79 .alpha.-phellandrene 348.4 79.5 .alpha.-phellandrene
+ estrogen 635.3 145.0 96
EXAMPLE 3
Dose-Response Effect on E.sub.2-Induced Cell Growth
MCF-7 cells were cultured in growth medium supplemented with 10%
fetal bovine serum (FBS). At 85% confluence, cells were sub-cultured
in 6 well petri-dishes and supplemented with 5% FBS serum stripped
medium, phenol red free for 24 hours prior to the treatment of different
concentrations of the test chemicals. After 5 days of treatment
cells were trypsinized, collected using Eppendorf microcentrifuge.
The cell pellets were resuspended in 1% trypan blue and three aliquots
(10 ul each) of the viable cell suspension were counted using a
haemocytometer assay. Each sample was then counted three times and
the data shown is the average of three counts. These test chemicals
were tested in the presence of 10 nM estrogen (=2.7 ng estrogen/ml).
Two wells per test concentration were used. This experiment was
repeated two times. Control received solvent only at <0.1% ethanol.
Results are shown in Table 3.
TABLE-US-00003 TABLE 3 Cell count .times. (10.sup.4)/ml Treatment
well #1 well #2 average Day 5. % of control Control 22 22 22.0 100
E.sub.2 (10 nM) 36 28 32.0 145 % anti-E.sub.2 E.sub.2/thymol (20
.mu.g/ml) 10 14 12.0 62.50 E.sub.2/thymol (10 .mu.g/ml) 12 12 12.0
62.50 E.sub.2/thymol (5 .mu.g/ml) 20 18 19.0 40.60 E.sub.2/thymol
(1 .mu.g/ml) 32 32 32.0 00.00 E.sub.2/isoeugenol (20 .mu.g/ml) 10
10 10.0 69.00 E.sub.2/isoeugenol (10 .mu.g/ml) 6 10 8.0 75.00 E.sub.2/isoeugenol
(5 .mu.g/ml) 18 23 21.0 35.60 E.sub.2/isoeugenol (1 .mu.g/ml) 21
21 21.0 35.60 E.sub.2/eugenol (20 .mu.g/ml) 10 16 13.0 60.40 E.sub.2/eugenol
(10 .mu.g/ml) 18 14 16.0 50.00 E.sub.2/eugenol (5 .mu.g/ml) 19 19
19.0 41.70 E.sub.2/eugenol (1 .mu.g/ml) 19 29 24.0 25.00 E.sub.2/benzyl
alcohol (50 .mu.g/ml) 24 18 21.0 35.40 E.sub.2/cinnamic aldehyde
00 02 01.0 96.90 (50 .mu.g/ml)
Our data show a dose-response relationship of plant essential oil
compounds, and their antiestrogenicity against E.sub.2-induced abnormal
cell growth and proliferation in human epithelial breast cancer
cells (MCF-7). These data demonstrated that thymol at low dose (5
ug/ml) provided 40% protection against the E.sub.2-induced abnormal
growth in cancer breast cells. In addition, these data also showed
that eugenol (1 ug/ml) and isoeugenol (1 ug/ml) expressed 35% and
25% protection, respectively, against the E.sub.2-induced abnormal
growth in cancer breast cells (see example 3). Further, cinnamic
aldehyde provided approximately 96% control against the E.sub.2-induced
abnormal growth in cancer breast cells at 50 ug/ml (see example
3).
The above Examples show, inter alia, that certain plant essential
oils and combinations thereof are anti-proliferative, anti-estrogenic
and/or anti-mitogenic compounds that are useful for prophylactically
or therapeutically treating soft tissue cancers.
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.
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