U.S. patent application number 16/557132 was filed with the patent office on 2019-12-26 for non-toxic vehicle to solubilize, deliver, and obtain biological activity of steroid hormones at cell, tissue, and organ targets,.
This patent application is currently assigned to Government of the United States as Represented by the Secretary of the Air Force. The applicant listed for this patent is Government of the United States as Represented by the Secretary of the Air Force. Invention is credited to Thomas Shaak, Suizhao Wang.
Application Number | 20190388349 16/557132 |
Document ID | / |
Family ID | 62905385 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190388349 |
Kind Code |
A1 |
Shaak; Thomas ; et
al. |
December 26, 2019 |
NON-TOXIC VEHICLE TO SOLUBILIZE, DELIVER, AND OBTAIN BIOLOGICAL
ACTIVITY OF STEROID HORMONES AT CELL, TISSUE, AND ORGAN TARGETS, IN
VITRO AND IN VIVO
Abstract
A non-toxic vehicle for hormone therapy. The non-toxic vehicle
includes a cationic lipid or a neutral lipid and polyethylene
glycol.
Inventors: |
Shaak; Thomas; (Ocean
Springs, MS) ; Wang; Suizhao; (Biloxi, MS) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Government of the United States as Represented by the Secretary of
the Air Force |
Wright-Patterson AFB |
OH |
US |
|
|
Assignee: |
Government of the United States as
Represented by the Secretary of the Air Force
Wright-Patterson AFB
OH
|
Family ID: |
62905385 |
Appl. No.: |
16/557132 |
Filed: |
August 30, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15875637 |
Jan 19, 2018 |
|
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16557132 |
|
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62449205 |
Jan 23, 2017 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/568 20130101;
A61B 2017/00707 20130101; A61K 2300/00 20130101; G09B 23/34
20130101; A61B 17/32053 20130101; A61M 1/0088 20130101; G09B 23/306
20130101; A61K 31/565 20130101; A61B 10/02 20130101; A61K 47/10
20130101; A61K 47/24 20130101; A61K 31/573 20130101; A61K 9/0019
20130101; A61P 35/00 20180101; A61K 9/1271 20130101; A61K 39/39
20130101 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/573 20060101 A61K031/573; A61K 31/565 20060101
A61K031/565; A61K 9/00 20060101 A61K009/00; A61P 35/00 20060101
A61P035/00; A61K 31/568 20060101 A61K031/568; A61K 47/10 20060101
A61K047/10; A61K 47/24 20060101 A61K047/24; A61K 39/39 20060101
A61K039/39; A61M 1/00 20060101 A61M001/00; G09B 23/30 20060101
G09B023/30; G09B 23/34 20060101 G09B023/34 |
Goverment Interests
RIGHTS OF THE GOVERNMENT
[0002] The invention described herein may be manufactured and used
by or for the Government of the United States for all governmental
purposes without the payment of any royalty.
Claims
1. A method of preparing a hormone treatment with a non-toxic
vehicle, the method comprising: preparing a first solution
comprising an aqueous solution of a hormone and polyethylene glycol
300; preparing a second solution comprising a cationic lipid and
polyethylene glycol 300, wherein a concentration of the cationic
lipid in the polyethylene glycol 300 is 5%; combining the first
solution with the second solution.
2. The method of claim 1, wherein the cationic lipid is selected
from the group consisting of
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride;
2,3-dioleyloxy-N[2-(spermine-carboxaindo)ethyl]-N,N-dimethyl-1--
propanaminium; 5-carboxyspermylglycinedioctadecylaminde;
N,N-dimethyl-N-ethylcarboxamidochloesterol; and
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine.
3. The method of claim 1, wherein the hormone is one or more of
estradiol, testosterone, dihydrotestosterone ("DHT"), and a
glucocorticoid.
4. The method of claim 1, further comprising: introducing a neutral
lipid to the second solution before combining the first and second
solutions.
5. The method of claim 4, wherein the neutral lipid is selected
from the group consisting of dioleoylphosphatidylethanolamine;
palmitoyl-snglycero-phosphoethanolamine; and
1,2-dimyristoyl-snglycero-3-phospho-ethanolamine.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 15/875,637, which claims the benefit of and priority to prior
filed Provisional Application Ser. No. 62/449,205, filed Jan. 23,
2017. The contents of each application is expressly incorporated
herein by reference in its entirety, each in its entirety.
FIELD OF THE INVENTION
[0003] The present invention relates generally to animal models
and, more particularly, to animal models for educational and
investigational uses.
BACKGROUND OF THE INVENTION
[0004] Steroid hormone therapy is generally considered the
treatment with any steroid hormone, such as estrogen, progesterone,
androgens, and the like. Such treatments have been found useful in
combating the symptoms of menopause, supplementing cancer
treatments, and hormone replacement, to name a few. However, the
use and application of steroid hormones in hormone therapy has been
hampered in conventional methodologies by two difficulties: 1) an
inability to dissolve sufficient amounts of steroid hormones in a
solvent and 2) a lack of delivery vehicle for which a measurable
dose of hormone may be delivered to a target tissue that produces a
biological effect or systemic response. While some conventional
mechanisms have utilized polyethylene glycol ("PEG") for
transmitting the hormone to the target tissue, there are
disadvantages to using PEG alone, such as solubility and
toxicity.
[0005] As a result, there remains a need for non-toxic vehicles
that facilitate the solubility and delivery of small doses of
hormone while effectuating a systemic response.
SUMMARY OF THE INVENTION
[0006] The present invention overcomes the foregoing problems and
other shortcomings, drawbacks, and challenges of conventional
hormone therapy delivery. While the invention will be described in
connection with certain embodiments, it will be understood that the
invention is not limited to these embodiments. To the contrary,
this invention includes all alternatives, modifications, and
equivalents as may be included within the spirit and scope of the
present invention.
[0007] According to embodiments of the present invention, a
non-toxic vehicle for hormone therapy includes a cationic lipid or
a neutral lipid and polyethylene glycol.
[0008] Other embodiments of the present invention are directed to a
method of preparing a hormone treatment with non-toxic vehicle. The
method includes preparing a first solution and a second solution.
The first solution includes an aqueous solution of a hormone and
polyethylene glycol. The second solution includes a cationic lipid,
a neutral lipid, or both, and polyethylene glycol. The prepared
first and second solutions are then combined.
[0009] Additional objects, advantages, and novel features of the
invention will be set forth in part in the description which
follows, and in part will become apparent to those skilled in the
art upon examination of the following or may be learned by practice
of the invention. The objects and advantages of the invention may
be realized and attained by means of the instrumentalities and
combinations particularly pointed out in the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate embodiments of
the present invention and, together with a general description of
the invention given above, and the detailed description of the
embodiments given below, serve to explain the principles of the
present invention.
[0011] FIGS. 1-3 are a flowcharts illustrating a method of
preparing and using an immune system model according to embodiments
of the present invention.
[0012] FIG. 4 is an exemplary photograph of 293T cells of normal,
distinct phenotype.
[0013] FIG. 5 is an exemplary photograph of 293T cells treated with
PEG 300 only.
[0014] FIG. 6 is an exemplary photograph of 293T cells treated with
PEG 300 and .beta.AED
[0015] FIG. 7 is an exemplary photograph of 293T cells treated with
PEG 300 and 17.beta.-estradiol.
[0016] FIG. 8 is an exemplary photograph of 293T cells treated with
PEG 300, .beta.AED, and 17.beta.-estradiol.
[0017] FIG. 9 is an exemplary photograph of 293T cells treated with
PEG 300 and LIPOFECTAMINE.
[0018] FIG. 10 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, and .beta.AED
[0019] FIG. 11 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, and 17.beta.-estradiol.
[0020] FIG. 12 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, .beta.AED, and 17.beta.-estradiol.
[0021] FIG. 13 is a graphical representation of a number of mobile
cells per field of view of in vitro androstene hormone treatments
in 293T cells.
[0022] FIG. 14 is an exemplary photograph of MCF-7 cells treated
with a PEG 300 and LIPOFECTAMINE solution, diluted 1:1000.
[0023] FIG. 15 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AED, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000.
[0024] FIG. 16 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AET, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000.
[0025] FIG. 17 is an exemplary photograph of MCF-7 cells treated
with an E2, PEG 300, and LIPOFECTAMINE solution, diluted
1:5000.
[0026] FIG. 18 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AED, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE, diluted
1:5000.
[0027] FIG. 19 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AET, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE, diluted
1:5000.
[0028] FIG. 20 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AET, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000, and a solution of .beta.-AET, PEG 300, and LIPOFECTAMINE,
diluted 1:1000.
[0029] FIG. 21 is an exemplary photograph of MCF-7 cells treated
with a .beta.-AED, PEG 300, and LIPOFECTAMINE solution, diluted
1:1000, a solution of .beta.-AET, PEG 300, and LIPOFECTAMINE,
diluted 1:1000, and a solution of E2, PEG 300, and LIPOFECTAMINE,
diluted 1:5000.
[0030] It should be understood that the appended drawings are not
necessarily to scale, presenting a somewhat simplified
representation of various features illustrative of the basic
principles of the invention. The specific design features of the
sequence of operations as disclosed herein, including, for example,
specific dimensions, orientations, locations, and shapes of various
illustrated components, will be determined in part by the
particular intended application and use environment. Certain
features of the illustrated embodiments have been enlarged or
distorted relative to others to facilitate visualization and clear
understanding. In particular, thin features may be thickened, for
example, for clarity or illustration.
DETAILED DESCRIPTION OF THE INVENTION
[0031] Turning now to the figures, and in particular to FIG. 1, a
flow chart 130 illustrating a method of preparing an immune system
model according to an embodiment of the present invention is shown.
Again, while not necessary, the illustrated embodiments include S.
scrofa; however, other appropriate mammals may be used in the
alternative as the specimen.
[0032] At start, an immune response is stimulated (Block 132) by
injecting a particular antigen into the specimen. Antigens may
include commercially-available bacterial, fungal, or viral specific
antigens configured to stimulate the formation of protective
antibodies (i.e., a thymic dependent response), such as those found
in a vaccine.
[0033] Chronic inflammation conditions include a prolonged and
extended period of inflammation. The chronic inflammatory response
to would related antigens is marked by increased granulocytes, Th1
T cells, M1 macrophages, and a decreased amount of dendritic cells.
More particularly, the thymic immune response includes responses to
T cell dependent antigenic proteins, which may include animal,
bacterial, viral, or fungal proteins. Non-thymic immune responses
include non-specific antigens (non-protein, less specific
particles) that may illicit a non-specific immune reaction, such as
an allergic response or asthma.
[0034] Systemic baseline conditions before and after stimulating
the immune response may extend to the brain, skin, thymus, spleen,
liver, adrenals, gonads, lymph nodes, small intestine, small
intestine mesentery, and circulatory system.
[0035] Continued stimulation generally depends on the system
investigated. Generally, 30 days (or roughly four weeks) are
necessary for a primary and immune response to generate antibodies
from an antigen exposure. A secondary response may be obtained by
reinjecting the antigen after the 30 day primary response.
Accordingly, the desired response may be achieved by a first
stimulation in the first 30 days and, optionally depending on the
phenome to be investigated, at least one subsequent stimulation
every 30 days (or approximately every four weeks) (Block 134).
[0036] With desired response stimulated, modulation of the immune
response may be initiated (Block 136). Immunomodulators include
those chemical agents that modify the immune response by
stimulating antibody formation or inhibiting white blood cell
activity. According to embodiments of the present invention,
immunomodulators may include one or more of delta
5-androsten-3B,17B-diol (.DELTA..sup.5-diol) and delta
5-androsten-3B, 7B,17B-triol (.DELTA..sup.5-triol), and further
optionally with one or more of estradiol, testosterone, and
dihydrotestosterone ("DHT").
[0037] If desired, delivery of the immunomodulator may be assisted,
according to one embodiment of the present invention, by preparing
a non-toxic vehicle (Block 138). Briefly, embodiments of the
present invention overcome previous difficulties of steroid hormone
solubility and delivery of measureable doses to produce biological
effects. Accordingly, and as illustrated in the flow chart 138 of
FIG. 2, the non-toxic vehicle includes preparation of a composition
comprising a cationic or neutral lipid and polyethylene glycol
("PEG") (Block 140). Suitable cationic and neutral lipids may
include, for example,
N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride
("DOTMA"); dioleoylphosphatidylethanolamine ("DOPE");
2,3-dioleyloxy-N-[2-(spermine-carboxaindo)ethyl]-N,N-dimethyl-1-propanami-
nium ("DOSPA"); 5-carboxyspermylglycinedioctadecylaminde ("DOGS");
N,N-dimethyl-N-ethylcarboxamidochloesterol ("DC-Chol");
1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine ("DPPE");
palmitoyl-sn-glycero-phosphoethanolamine ("PPE"); and
1,2-dimyristoyl-snglycero-3-phospho-ethanolamine ("DMPE").
[0038] Cellular membranes generally comprise a phospholipid bilayer
such that outer surfaces of the cellular membrane comprise the
hydrophilic head groups of the phospholipids with the hydrophobic
tails directed centrally between the outer surfaces. Cationic and
neutral lipids may assist in trafficking hormone to and across the
cellular membrane. Suitable cationic and neutral lipid solutions
may include, for example, the commercially-available LIPOFECTAMINE
(ThermoFisher Scientific Inc., Waltham, Mass.) or any appropriate
PEG formulation in any weight may be used, including but not
limited to, PEG 200, PEG 300, PEG 1500, and so forth.
[0039] The liposomal formulation may be prepared in an aqueous
solution comprising two solutions. In a first solution, the
immunomodulator (.beta.-AED (Steraloids, Inc., Newport, R.I.) or
(.beta.-AET (Steraloids, Inc.)) is dissolved in a polyester (for
example, PEG), the latter of which may be pre-warmed. In a second
solution, a dilution of the cationic and neutral lipid, for
example, a 3:1 mixture of cationic to neutral lipid (DOSPA:DOPE) as
in LIPOFECTAMINE, may be introduced into a polyester, mixed, and
incubated (incubation may be at 42.degree. C. for 30 min). The
first and second solutions may then be combined.
[0040] A solution comprising the immunomodulator may then be
introduced to the liposomal formulation (Block 142). Evidence of
undissolved hormone may include the presence of crystals in in
vitro cultures. If crystals are present, then the solution may be
warned to above 42.degree. C.
[0041] While not wishing to be bound by theory, it is thought that
the cationic liposome formulation provides a solvation and delivery
base. While PEG has the ability to, alone, dissolve and deliver
hormones, when cationic liposome formulations are used, the PEG
solution is diluted, improving solubility, and decreasing
toxicity.
[0042] Returning to FIG. 1, the immunomodulator, with or without
the treatment vehicle of Block 138, may administered directly or
indirectly through the mandibular gland, the thoracic duct, or
through the lymphatic drainage system. Alternatively, the
immunomodulator may be subcutaneously injected in, or delivered by
a transdermal patch applied to, an anterior portion of the patient
where lymph vessels of the thoracic limb drain to various lymph
nodes (mandibular, parotid, accessory mandibular, lateral
retropharyngical, ventral superficial cervical, dorsal superficial
cervical, and axillares primae costae (costo-axillary)).
Alternatively still, the immunomodulator may be subcutaneously
injected in, or delivered by a transdermal patch applied to, a
posterior portion of the patient where lymph vessels of the
abdominal wall, pelvic wall, and pelvic limbs drain to various
lymph nodes (subiliac, superficial inguinal, accessory superficial
inguinal, superficial popliteal, deep popliteal, external sacral,
anterior sacral, medial iliacs, and lateral iliac).
[0043] Generally, only one application (whether subcutaneous
injection or transdermal patch) is required for immunomodulation
("Yes" branch of Decision Block 144). However, it would be
appreciated by those having ordinary skill in the art and the
benefit of the disclosure made herein that other embodiment of the
invention of immunomodulatory schemes or other combinations of
immunomodulators may be induced by one subcutaneous injection or
transdermal application.
[0044] The initial dosage of immunomodulatory (Block 136), or any
subsequent dosage ("No" branch of Decision Block 144) may require
sequential hormone treatments (Block 146) with a repeat dose of the
immunomodulatory. For example, hydrocortisone followed by an
application of 37 ng/kg or higher of
androst-5-ene-3.beta.,17.beta.-diol (".beta.-AED") or
5-androstene-3.beta.,7.beta.,17.beta.-triol (".beta.-AET"). Still
other combinations with additional hormones, such as testosterone
or estrogen, may be used to achieve a desired method of
immunomodulation.
[0045] Referring now to FIG. 3, a flowchart illustrating a method
150 for causing cellular death of triple-negative breast cancer
cells by hormone modulation is described according to an embodiment
of the present invention is shown. Triple-negative breast cancer
comprises a heterogeneous subset of breast cancer-types. While
accounting for 15% to 25% of all breast cancer cases, the triple
negative cancers are not supported by estrogen and progesterone
because the cells are ER and PR receptor negative and have an
increased number of HER2 receptors.
[0046] At start, a combination of adrenal hormones, including
.DELTA.5-androsten-3.beta.,17.beta.-diol and
.DELTA.5-androsten-3.beta.,7.beta.,17.beta.-triol (both at a
concentration of about 25 .mu.M) with or without estradiol ("E2")
is prepared (at a concentration of about 5 nM) (Block 152). The
adrenal hormone solutions may be prepared and administered
separately or together.
[0047] Optionally, the preparation may include a suitable vehicle
(Block 154), such as PEG, ethanol, dimethylsulfoxide ("DMSO"), or
the PEG-LIPOFECTAMINE vehicle according to other embodiments of the
present invention, for example as described with reference to FIG.
2. In such embodiments, the respective adrenal hormone solution may
be mixed with the cationic or neutral lipid and
polyethyleneglycol.
[0048] In vitro treatment may include applying the adrenal hormones
with vehicle to a cell growth medium. In vivo treatment may be
injected directly into a tumor, applied to an area from which a
tumor was surgically removed, or injected subcutaneously.
[0049] Only one application (whether subcutaneous injection or
transdermal patch) is required for immunomodulation ("Yes" branch
of Decision Block 156). However, it would be appreciated by those
having ordinary skill in the art and the benefit of the disclosure
made herein that other embodiment of the invention of
immunomodulatory schemes or other combinations of immunomodulators
may be induced by one subcutaneous injection or transdermal
application.
[0050] The initial dosage of immunomodulatory (Block 154), or any
subsequent dosage ("No" branch of Decision Block 156) may require
sequential hormone treatments (Block 158) with a repeat dose of the
immunomodulatory. For example, hydrocortisone, testosterone, or
estrogen, may be used to achieve a desired method of
immunomodulation.
[0051] Although not specifically illustrated herein, it would be
understood that the various embodiments of the treatment vehicle
described herein need not be limited to delivery of an
immunomodulator or in the modeling of an immune response.
Accordingly, the treatment vehicle may be used, when appropriate or
desired, to achieve delivery of hormone, steroid, or the like to
achieve a desired physiological effect that would otherwise
difficult to achieve.
[0052] Preparation and delivery of hormone in according to
embodiments as describe herein provide the benefit in that the
treated cultures are not morphologically or phenotypically changed
as compared to control cells. Full dissolution of the hormone
reduces toxicity and reduces interference with physiological
effects of the hormones. Moreover, use of the non-toxic vehicle
reduces component residue at the point of application, allowing for
more precise measurement of dosage administration.
[0053] The following examples illustrate particular properties and
advantages of some of the embodiments of the present invention.
Furthermore, these are examples of reduction to practice of the
present invention and confirmation that the principles described in
the present invention are therefore valid but should not be
construed as in any way limiting the scope of the invention.
EXAMPLE 1
[0054] A non-toxic vehicle for administration of androstene
hormones according to embodiments of the present invention was
prepared from a first solution and a second solution. The first
solution, comprising liposomal formulation, was placed in PEG 300
to a concentration of 5% and heated in a sonicating water bath to
about 45.degree. C. The second solution, comprising a selected
hormone (one or more of 5.DELTA.-androsten-3.beta., 17.alpha.-diol,
5.DELTA.-androsten-3.beta., 17.beta.-diol, or
5.DELTA.-androsten-3.beta.,7.beta.,17.beta.-triol), was placed in
PEG 300 and heated in a sonicating water bath to about 45.degree.
C. The first and second solutions were then combine and stirred
until dissolved.
[0055] Biological function was tested in 293T cells, which is a
human embryonic cell line transformed with Large T antigen or SV40.
This hypotriploid (polyploid) cell line is particularly useful in
such transformation studies and has very distinct phenotypes when
grown. All androstene hormone treatment applications were at
concentrations of 25 .mu.M.
[0056] FIG. 4 is an exemplary photograph of 293T cells of normal,
distinct phenotype. There are small cell islands, large cell
islands, and mobile cells/cell processes.
[0057] FIG. 5 is an exemplary photograph of 293T cells treated with
PEG 300 only. The cells demonstrate the same characteristics as the
normal cells of FIG. 3.
[0058] FIG. 6 is an exemplary photograph of 293T cells treated with
PEG 300 and .beta.AED. Resultant cell islands are larger and there
is a significant reduction in mobile cells.
[0059] FIG. 7 is an exemplary photograph of 293T cells treated with
PEG 300 and 17.beta.-estradiol. The culture includes many more cell
processes as compared to estradiol alone.
[0060] FIG. 8 is an exemplary photograph of 293T cells treated with
PEG 300, .beta.AED, and 17.beta.-estradiol. There is a visible
decrease in mobile cells over the treatment with .beta.AED
alone.
[0061] FIG. 9 is an exemplary photograph of 293T cells treated with
PEG 300 and LIPOFECTAMINE, the latter of which increased
granularity. Otherwise, cells were similar to the Blanks, despite
presence of PEG 300.
[0062] FIG. 10 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, and .beta.AED. The treatment
combination demonstrated a drastic decrease in, or morphological
change, in mobile elements.
[0063] FIG. 11 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, and 17.beta.-estradiol. The
combination produced many large islands with mobile processes,
small islands with mobile process, and mobile cells forming
interconnections with other elements.
[0064] FIG. 12 is an exemplary photograph of 293T cells treated
with PEG 300, LIPOFECTAMINE, .beta.AED, and 17.beta.-estradiol. As
compared to cells of FIG. 11, mobile cells and processes are
largely non-existent.
[0065] FIG. 13 is a graphical representation of a number of mobile
cells per field of view of in vitro androstene hormone treatments
in 293T cells, described above, wherein column A is a blank, column
B is PEG 300 with LIPOFECTAMINE, column C is PEG 300 with
LIPOFECTAMINE with 25 .mu.M .beta.-AED, column D is PEG 300 with
LIPOFECTAMINE with 5 nM E2, column E is PEG 300 with LIPOFECTAMINE
with 25 .mu.M .beta.-AED and 5 nM E2, column F is PEG 300 with
LIPOFECTAMINE, column G is PEG 300 with LIPOFECTAMINE and 5 nM E2,
column H is PEG 300 with LIPOFECTAMINE and 25 .mu.M .beta.-AED, and
column I is PEG 300 with LIPOFECTAMINE, 25 .mu.M .beta.-AED, and 5
nM E2.
EXAMPLE 2
[0066] Hormone dependent, noninvasive, epithelial phenotype (ER/PR
positive MCF-7 breast cancer cells) and hormone independent,
invasive, mesenchymal phenotype (ER/PR negative MDA-MB-231 breast
cancer cell) cell suspensions were acquired and prepared in
complete medium (DMEM/F12, 10% FBS, 1% penicillin/streptomycin
(Gibco)). Cellular concentration was adjusted to 2.times.10.sup.5
cells/mL.
[0067] Androstene hormone solutions according to embodiments of the
present invention were prepared from a first solution and a second
solution. For the first solution, 3.0 mg of .beta.-AED or
.beta.-AET was added to warm PEG 300 to prepare 50 mM stock. For
the second solution, 10 .mu.L of LIPOFECTAMINE (3:1 DOSPA: DOPE)
was introduced to 100 .mu.L of PEG 300, mixed, and incubated in a
water bath at 42.degree. C. for 30 min. The first and second
solutions were then mixed in a sonicator water bath at 42.degree.
C. for 1 hr. The final concentration was 50 mM .beta.-AED (or AET,
as used).
[0068] A .beta.-estradiol solution was also prepared by adding 2.7
mg of .beta.-estradiol to 200 .mu.L warmed PEG 300 with
LIPOFECTAMINE and then further diluted 1:1000 with PEG-300. The
final concentration was 50 .mu.M.
[0069] A diluted .beta.-AED (or .beta.-AET) solution was prepared
with complete medium at a final concentration of 50 .mu.M (1:1000
dilute of 50 mM). Likewise, a diluted .beta.-E2 solution was
prepared with complete medium at a final concentration of 10 nM
(1:5000 dilute of 50 .mu.M).
[0070] Using a 24-well plate, mixtures of 0.5 mL of cells per well
with 0.5 mL of medium diluted hormone vehicle. Cell cultures were
maintained at 90% humidity, 7% CO.sub.2, and 37.degree. C.
[0071] Results are provided in tabular format below:
TABLE-US-00001 TABLE 1 MDA-MB-231 MCF7 Cell Death Cell Death
Additional Treatment Amount Amount Observation .beta.-AED Not
observed Not observed Cell particles observed .beta.-AED + E2 70%
Not observed Cell particles observed .beta.-AED + .beta.-AET 80%
Not observed Cell particles observed .beta.-AED + .beta.-AET + E2
90% Not observed Cell particles observed .beta.-AET Not observed
Not observed N/A E2 Not observed Not observed N/A
[0072] The MDA-MB-231 cell cultures treated with (1) 25 .mu.M
.beta.-AED and 5 nM E2, (2) 25 .mu.M .beta.-AED and 25 .mu.M AET,
or (3) 25 .mu.M .beta.-AED, 25 .mu.M AET and 5 nM E2 yielded cell
death rates of 70%, 80%, and 90%, respectively. Cell death was not
observed in MCF7 cell cultures treated with the same doses.
[0073] FIG. 14 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising PEG 300 with 5% LIPOFECTAMINE
solution diluted 1:1000.
[0074] FIG. 15 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M. As compared to FIG. 14, the cell
density of FIG. 15 is not as heath and there are areas of decreased
density. FIG. 15 also presents small particles (identified by
arrows) that were produced by cells in response to the treatment
with .beta.-AED
[0075] FIG. 16 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AET in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M. The cell growth is thick and
similar to the cell growth of FIG. 14.
[0076] FIG. 17 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 .mu.M solution of E2 in PEG
300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a
concentration of 5 nM. The cell growth is thick and similar to the
cell growth of FIGS. 14 and 16.
[0077] FIG. 18 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M and 25 .mu.M stock solution of E2 in
PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a
concentration of 5 nM. The cell growth is thick and similar to the
cell growth of FIGS. 14, 16, and 17.
[0078] FIG. 19 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AET in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M and 25 .mu.M stock solution of E2 in
PEG 300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a
concentration of 5 nM. The cell growth is thick and similar to the
cell growth of FIGS. 14 and 16-18.
[0079] FIG. 20 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M and 25 mM stock solution of
.beta.-AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M. The cell growth is thick and
similar to the cell growth of FIGS. 14 and 16-19.
[0080] FIG. 21 is a microscopic photograph of MCF-7 cells grown for
5 days in a cell medium comprising 25 mM stock solution of
.beta.-AED in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000
to a concentration of 25 .mu.M, 25 mM stock solution of .beta.-AED
in PEG 300 with 5% LIPOFECTAMINE solution diluted 1:1000 to a
concentration of 25 .mu.M, and 25 .mu.M stock solution of E2 in PEG
300 with 5% LIPOFECTAMINE solution diluted 1:5000 to a
concentration of 5 nM. While the figure presents a full growth of
cells, the cell layer is not as thick as was presented in FIGS. 14
and 16-19. The treatment caused the cells to produce small cell
bodies.
[0081] While the present invention has been illustrated by a
description of one or more embodiments thereof and while these
embodiments have been described in considerable detail, they are
not intended to restrict or in any way limit the scope of the
appended claims to such detail. Additional advantages and
modifications will readily appear to those skilled in the art. The
invention in its broader aspects is therefore not limited to the
specific details, representative apparatus and method, and
illustrative examples shown and described. Accordingly, departures
may be made from such details without departing from the scope of
the general inventive concept.
* * * * *