U.S. patent application number 12/660088 was filed with the patent office on 2010-06-17 for estrogen beta receptor agonists to prevent or reduce the severity of cardiovascular disease.
Invention is credited to R. Kent Hermsmeyer.
Application Number | 20100152144 12/660088 |
Document ID | / |
Family ID | 32393456 |
Filed Date | 2010-06-17 |
United States Patent
Application |
20100152144 |
Kind Code |
A1 |
Hermsmeyer; R. Kent |
June 17, 2010 |
Estrogen beta receptor agonists to prevent or reduce the severity
of cardiovascular disease
Abstract
A method for preventing or treating vascular hyperreactivity in
which a chemical compound that is an estrogen beta receptor agonist
is administered to a subject suffering from or at risk of vascular
hyperreactivity. Preferably, the administration is topically to the
skin.
Inventors: |
Hermsmeyer; R. Kent;
(Portland, OR) |
Correspondence
Address: |
HOWARD EISENBERG, ESQ.
1220 LIMBERLOST LANE
GLADWYNE
PA
19035
US
|
Family ID: |
32393456 |
Appl. No.: |
12/660088 |
Filed: |
February 19, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10690169 |
Oct 21, 2003 |
7674783 |
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12660088 |
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60428811 |
Nov 22, 2002 |
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Current U.S.
Class: |
514/170 ;
514/182 |
Current CPC
Class: |
A61K 31/57 20130101 |
Class at
Publication: |
514/170 ;
514/182 |
International
Class: |
A61K 31/56 20060101
A61K031/56; A61P 9/00 20060101 A61P009/00 |
Claims
1. A method for reducing the incidence or severity of vascular
hyperreactivity in a patient in need thereof comprising
administering to the patient an effective amount of a selective
estrogen beta receptor agonist that has a higher relative potency
for estrogen receptor beta compared to estrogen receptor alpha than
that of genistein.
2. The method of claim 1 wherein the vascular hyperreactivity of
the patient is manifested by coronary arterial vasospasm.
3. The method of claim 1 wherein the vascular hyperreactivity of
the patient is manifested by hyperreactivity of peripheral
arteries.
4. The method of claim 1 wherein the estrogen beta receptor agonist
is co-administered with a hormone replacement therapy.
5. The method of claim 4 wherein the hormone replacement therapy is
selected from the group consisting of estrogen, androgen, and
progestin therapy.
6. The method of claim 1 wherein the estrogen beta receptor agonist
is administered to the patient by topical application to skin.
7. The method of claim 6 wherein the estrogen beta receptor agonist
is in a topical preparation selected from the group consisting of a
liquid, cream, gel, lotion, ointment, and transdermal patch.
8. The method of claim 1 wherein the estrogen beta receptor agonist
is administered to the patient by other than topical administration
to skin.
9. The method of claim 8 wherein administration is by oral, rectal,
vaginal, topical, sublingual, nasal, intradermal, inhalation, or
sustained implant administration routes.
10. A kit for administering an estrogen beta receptor agonist
compound to a patient suffering from or at risk of vascular
hyperreactivity comprising a container, a topical preparation
containing an estrogen beta receptor agonist in the container, and
instructions for dispensing an amount of the preparation which
provides an amount of the estrogen beta receptor agonist that is
effective to prevent or reduce the severity of vascular
hyperreactivity.
11. The kit of claim 10 wherein the estrogen beta receptor agonist
is 5.alpha.-androstane-3.beta.,17.beta.-diol.
12. The kit of claim 10 wherein the estrogen beta receptor agonist
is a derivative of 5.alpha.-androstane-3.beta.,17.beta.-diol that
has estrogen beta receptor agonist activity.
13. The kit of claim 12 wherein the derivative of
5.alpha.-androstane-3.beta.,17.beta.-diol is selected from the
group consisting of 5.alpha.-androstan-3.beta.,17.beta.-diol-3
hemisuccinate, 5.alpha.-androstan-3.beta.,17.beta.-diol-17-sulphate
sodium salt, 5.alpha.-androstan-3.beta.,17.beta.-diol-3-acetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-17-acetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-diacetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-dibenzoate,
5.alpha.-androstan-3.beta.,17.beta.-diol-dihemisuccinate,
5.alpha.-androstan-3.beta.,17.beta.-diol-diproprionate, and
5.alpha.-androstan-3.beta.,17.beta.-diol-17-hexahydrobenzoate.
14. The kit of claim 10 wherein the estrogen beta receptor agonist
is epiestriol.
15. The kit of claim 10 wherein the estrogen beta receptor agonist
is genistein or diarylpropionitrile.
16. The kit of claim 10 wherein the topical preparation is selected
from the group consisting of a liquid, cream, gel, lotion,
ointment, and transdermal patch.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 10/690,169, filed Oct. 21, 2003, which
application claims the benefit of U.S. Provisional Patent
Application Ser. No. 60/428,811 filed on Nov. 22, 2002, which is
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the use of chemical compounds to
reduce the incidence or severity, and to treat or prevent
cardiovascular disease, including coronary artery disease such as
coronary artery reactivity, cerebrovascular disease such as stroke,
and peripheral vascular disease such as intermittent claudication
or Raynaud's Disease.
BACKGROUND OF THE INVENTION
[0003] Cardiovascular disease, including coronary heart disease,
stroke and other vascular diseases, is the leading cause of death
of men and women in economically-developed countries. The most
common and lethal form of cardiovascular disease is ischemic heart
disease. It has generally been regarded that ischemic heart disease
is caused, primarily, by atherosclerosis of the coronary arteries.
This is a condition where plaques form in the inner lining of the
arteries, causing narrowing of the channel and thereby impairing
blood flow to the heart.
[0004] An increased risk for ischemic heart disease is observed in
women after menopause or ovariectomy and presents a major medical
challenge. It is the leading cause of death in post-menopausal
women, which implicates the importance of the loss of ovarian
steroid hormones.
[0005] Since it is understood that the formation of plaques is
reduced, or even possibly reversed, by a high ratio of high density
lipoproteins (HDL) to low density lipoproteins (LDL), it has been a
strategy to inhibit ischemic heart disease by attempting to
increase this ratio in the blood stream. Estradiol, an ovarian
estrogenic steroid hormone, has been observed to increase the
HDL/LDL ratio, and studies so far suggest that estrogen replacement
therapy for post-menopausal women decreases the incidence of
coronary artery disease, myocardial infarction, and related
cardiovascular events by up to 50%.
[0006] A vasospasm is an abnormally strong and persistent
contraction of the muscles of the coronary arteries which leads to
transmural myocardial ischemia and can result in sudden cardiac
death. The role of coronary vasospasm in cardiovascular disease is
still controversial, and approaches to treatments for
cardiovascular disease have not focused upon methods for reducing
coronary vasospasm. Instead, it is generally believed that coronary
vasospasm is caused by local injury to vessels, such as results
from atherosclerosis and other structural injury, and that
long-term treatment of cardiovascular disease requires prevention
of atherosclerotic plaques, not treatments to prevent
vasospasm.
[0007] Hermsmeyer, U.S. Pat. No. 6,056,972, incorporated herein by
reference and referred to hereafter as the "Hermsmeyer patent",
discloses that low levels of progesterone, either alone or in
combination with estradiol, can inhibit coronary artery reactivity
by a direct effect on coronary arteries and, therefore, can be used
to inhibit certain adverse cardiovascular events and disorders.
Progesterone, administered in conjunction with estradiol,
dramatically reduces the incidence of the adverse effects of
unopposed estrogen. Hermsmeyer discloses that coronary artery
vasospasm can be prevented by administering to a subject
progesterone in an amount to achieve blood levels of progesterone
of between 0.1 nanograms/ml and less than 4 nanograms/ml for at
least 4 hours per day, and wherein said amount results in peak
levels of progesterone of less than 6 nanograms/ml. Hermsmeyer
further discloses that progesterone may be used to treat an
existing vasospasm.
[0008] Hermsmeyer discloses that exaggerated vasoconstrictions of
long (greater than 5 minute) duration are reliably initiated in the
presence of estrogen and progesterone deficiency by the synergistic
combination of two major platelet release products, serotonin and
thromboxane A.sub.2 at concentrations found in platelets.
Hermsmeyer discloses that administration of these two compounds to
menopausal monkeys produces intense, focal constrictions that mimic
arterial vasoconstrictions in humans. These severe induced
vasoconstrictions are prevented or terminated by the administration
of estradiol and/or progesterone.
BRIEF DESCRIPTION OF THE FIGURES
[0009] FIG. 1 is a graph showing the % change in diameter of
coronary arteries following stimulation of constriction in
ovariectomized untreated monkeys and in ovariectomized monkeys
treated with estriol.
[0010] FIG. 2 is a graph showing the persistent protection of
vascular muscle cells in vitro when treated with estriol compared
to vascular muscle cells that were untreated.
[0011] FIG. 3 is a graph showing the estrogen beta receptor
activity of several test compounds in an ER.beta. coactivator
fluorescence polarization assay.
[0012] FIG. 4 is a graph showing the comparative effects of
estriol, 3.beta.Adiol, and epiestriol treatment in vitro on
Ca.sup.2+ responses in rhesus coronary VMC.
[0013] FIG. 5 is a graph showing a comparison of the effects of a
range of 3.beta.Adiol concentrations treatment in vitro on
Ca.sup.2+ responses in rhesus coronary VMC.
[0014] FIG. 6 is a graph showing a comparison of 3.beta.Adiol with
other estrogen receptor beta selective ligands on Ca.sup.2+
responses in rhesus coronary VMC.
[0015] FIG. 7 is a pair of graphs. FIG. 7A shows the effect of a
selective estrogen receptor beta antagonism on the ability of an
estrogen receptor beta agonist to protect VMC from a constrictive
stimulus. FIG. 7B shows the effect of an estrogen receptor beta
antagonist on Ca.sup.2+ responses in coronary VMC.
DESCRIPTION OF THE INVENTION
[0016] The term "prevent" as used herein means to inhibit the
occurrence of a condition, that is to reduce the incidence or the
frequency of occurrence of the condition. The term "prevent" may be
used interchangeably with the term "prophylactically treat". The
term "prevent", as used herein referring to a method, does not mean
or imply that use of the method will provide a guarantee that the
condition will never occur, but rather that the method will inhibit
the occurrence of the condition and that the incidence and/or
frequency of the condition will be reduced.
[0017] It has been surprisingly discovered that estrogen receptor
beta agonists inhibit the development of vascular hyperreactivity,
e.g., coronary vasospasm, in individuals, including those
individuals lacking a significant source of endogenous estrogen,
such as post-menopausal women. According to one embodiment of the
method of the invention, the incidence or severity of vascular
hyperreactivity, including coronary arterial vasospasms, is reduced
by administering to a patient in need thereof an effective amount
of an estrogen receptor beta agonist, thereby inhibiting the severe
long duration vasoconstrictions that define hyperreactivity,
including the formation of coronary arterial vasospasms and thereby
reducing the incidence and/or severity of myocardial ischemia. In a
preferred embodiment, the estrogen receptor beta agonist is
5.alpha.-androstane-3.beta.,17.beta.-diol, also referred to herein
as "3.beta.Adiol". In this application, the invention is
illustrated with 3.beta.Adiol. It is to be understood, however,
that this is merely illustrative and that the invention pertains
not only to 3.beta.Adiol but to derivatives of 3.beta.Adiol that
have estrogen receptor beta agonist activity and other steroids,
such as epiestriol, and non-steroids. Derivative forms of
3.beta.Adiol include but are not limited to
5.alpha.-androstan-3.beta.,17.beta.-diol-3 hemisuccinate,
5.alpha.-androstan-3.beta.,17.beta.-diol-17-sulphate sodium salt,
5.alpha.-androstan-3.beta.,17.beta.-diol-3-acetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-17-acetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-diacetate,
5.alpha.-androstan-3.beta.,17.beta.-diol-dibenzoate,
5.alpha.-androstan-3.beta.,17.beta.-diol-dihemisuccinate,
5.alpha.-androstan-3.beta.,17.beta.-diol-diproprionate, and
5.alpha.-androstan-3.beta.,17.beta.-diol-17-hexahydrobenzoate, and
to any and all known or to-be-discovered estrogen receptor beta
agonists, and pertains particularly to estrogen receptor beta
agonists that are selective over estrogen receptor alpha. Examples
of estrogen receptor beta agonists that are suitable for the
invention include epi-estriol (the .alpha. isomer of estriol),
genistein, and diarylpropionitrile (DPN).
[0018] An effective amount, as described above, is that amount that
is sufficient to prevent or reduce vascular reactivity that leads
to inadequate blood flow to vital organs, as exemplified by
decreasing the severity of coronary arterial vasospasms or to
ameliorate or terminate an existing coronary arterial vasospasm. A
preferred amount of 3.beta.Adiol is that amount which, when
administered to a subject in need thereof, provides a serum
concentration of between 30 to 3000 pg/ml, and most preferably
between 30 to 300 pg/ml. This serum concentration may be higher or
lower than this range, if desired, so long as the amount
administered is sufficient to prevent or reduce the severity of
hyperreactivity such as revealed in ischemia, e.g., coronary
arterial vasospasms, stroke, or intermittent claudication. Of
course, the amount administered of estrogen beta receptor agonists
other than 3.beta.Adiol may differ from the above range depending
upon the precise estrogen beta receptor agonist administered.
[0019] In another embodiment, the invention is a method for
treating a patient experiencing vascular hyperreactivity by
administering an amount of an estrogen receptor beta agonist, such
as 3.beta.Adiol effective to reduce or normalize the
reactivity.
[0020] In another embodiment, the invention is a kit for
administering an estrogen receptor beta agonist, such as
3.beta.Adiol, to a subject in need thereof. The kit includes a
package which houses a container, a topical preparation containing
an estrogen receptor beta agonist in the container, and
instructions. The instructions are for dispensing an amount of the
preparation which amount, when applied topically to the skin, such
as by application of a topical cream, provides an amount of an
estrogen receptor beta agonist effective to prevent or reduce the
severity of vascular hyperreactivity and the tendency for ischemia.
An optional component of the kit is a dispenser for dispensing a
metered amount of the topical preparation.
[0021] In another embodiment, the invention is a kit for
administering an estrogen receptor beta agonist to a patient by
using a transdermal patch. The patch includes a housing, a
reservoir in the housing, a membrane attached to the housing,
adjacent the reservoir, for placement against the epidermis of a
human subject, and an adhesive attached to the housing for holding
the membrane to the epidermis of the subject. The kit preferably
contains instructions for using the patch to treat vascular
hyperreactivity and ischemia. The estrogen receptor beta agonist in
a carrier is contained in the reservoir, and the patch is
constructed and arranged to deliver an amount of the estrogen
receptor beta agonist to the epidermis of the subject to achieve
blood levels of the estrogen receptor beta agonist that are
effective to reduce the incidence or severity of prolonged
vasoconstriction and ischemia.
[0022] In any of the embodiments of the invention, the estrogen
receptor beta agonist may be applied together, either in the same
or separate administration, with a hormone replacement therapy,
such as an estrogen such as estradiol, an androgen such as
testosterone or derivatives thereof, or a progestin such as
progesterone.
[0023] It is further conceived that the estrogen receptor beta
agonists of the invention, including the preferred compound
3.beta.Adiol and derivatives thereof, have utility in the
prevention and treatment of vascular proliferative abnormalities,
such as intimal hyperplasia, restenosis of blood vessels,
post-angioplasty proliferation, Raynaud's Disease, Thromboangiitis
obliterans (Buerger's disease), allograft vasculopathy (such as
occurs in heart and kidney transplant complications), diabetic
angiopathy, and hypertensive vasculopathy.
[0024] These and other aspects of the invention are described in
greater detail below and in the accompanying portions of this
application.
[0025] The disclosure of Hermsmeyer, U.S. Pat. No. 6,056,972, is
incorporated herein in its entirety and particularly for its
disclosure of (1) the kits, as the kits of the present invention
are substantially identical to those of the Hermsmeyer patent
except for the inclusion of an estrogen beta receptor agonist in
place of progesterone, and (2) method of in vivo testing in
Examples 1 and 2 to determine efficacy to prevent or reduce the
severity of vascular hyperreactivity or to reduce the severity or
to terminate prolonged vasoconstrictions and ischemia, as the in
vivo testing applicable to the present invention is substantially
identical to that disclosed in the Hermsmeyer patent except for the
use of an estrogen beta receptor agonist in place of progesterone
and/or estrogen.
[0026] The invention relates to vascular reactivity and, in one
aspect, involves the use of an estrogen beta receptor agonist to
prevent or treat vascular hyperreactivity and ischemia, such as
coronary artery vasospasm, thereby preventing cardiovascular
disorders. A cardiovascular disorder as used herein means
myocardial infarction, ischemic heart disease, heart failure,
stroke, angina pectoris, and peripheral vascular disease. Vascular
reactivity, as used herein, is a measure of the amplitude and
duration of a response of an artery to an applied vasoconstrictor
stimulus. According to the present invention, methods are provided
for reducing the reactivity of arteries, such as coronary arteries
or peripheral arteries, to a vasoconstrictive substance, thereby
preventing or treating ischemia due to prolonged vasoconstriction
including coronary vasospasm. The methods of the invention thus are
adapted to prevent, that is to reduce the incidence, or to relieve
ischemia due to focal or diffuse constriction of abnormally long
duration, e.g. >5 min or as long as 15 min or more) that is
hypothesized to result from local vascular hyperreactivity to
vasoconstrictive substances.
[0027] Artery reactivity can be measured indirectly or directly.
Indirect measures include a cell's response, such as a vascular
smooth muscle cell's response to serotonin (5 HT) and a thromboxane
A.sub.2 mimetic, such as U46619, applied in vitro as described
herein and in the Hermsmeyer patent. Direct measures include in
vivo animal vascular responses to conditions for inducing ischemic
vasoconstriction, e.g. in vivo mechanical injury or in vivo
treatment of a hyperreactive animal with a combination of serotonin
and U46619, also described herein and in the Hermsmeyer patent.
[0028] A vasospasm is an abnormally strong and persistent
contraction of the muscles of the coronary arteries which leads to
transmural myocardial ischemia and often results in sudden cardiac
death. The vasospasm causes the coronary artery to assume a
characteristic "hourglass" shape of prolonged constriction. The
term "vasospasm" is often misused in the literature to refer to a
vasoconstriction which, rather than being abnormal and
life-threatening, is a normal, healthy contraction as a means of
autoregulating blood flow. As used herein, a coronary vasospasm is
defined as epicardial coronary arterial constriction to less than
33%, and preferably less than 25%, of control diameter in focal
areas with adjacent downstream dilation, with the hourglass pattern
thus formed persisting for >5 minutes.
[0029] It is noted that the concept of coronary artery reactivity
has been used in the literature in a sense that is different from
how it is used in this specification. Williams et al., J. American
College of Cardiology, 24 (7):1757-1761 (1994), uses this term to
mean vasodilator capacity in response to acetylcholine, which
causes dilation of normal coronary arteries in direct contrast to
lack of dilation in diseased arteries. According to this usage,
decreases in reactivity are abnormal. Hyperreactivity, as used
herein, refers to an exaggerated amplitude and/or duration of a
constrictor response to a vasoactive substance. Therefore,
hyperreactivity to a vasoconstrictive stimulus underlies coronary
ischemia and vasospasm; decreased vasodilator response to stimuli,
as found in atherosclerotic monkeys by Williams et al., is a
separate and distinct mechanism by which coronary arteries fail to
open, or to remain open in response to acetylcholine, to provide
for increases in coronary blood flow.
[0030] Although the present invention has arisen out of studies in
female rhesus monkeys and in vascular muscle cells from rhesus
monkeys, it will be understood by a person of ordinary skill in the
art, that the results of these studies are directly applicable to
human beings, especially to female humans. This is because female
rhesus monkeys have been established as an excellent animal model
for the physiological effects of female sex hormones on women.
These experiments also provide a basis for an estrogen beta
receptor agonist as a prophylactic treatment for myocardial
ischemia in men.
[0031] Thus, the invention is useful in men and women. The
invention is particularly useful in women who have abnormally low
levels of estrogen and progesterone, due to natural circumstances,
surgery, or disease. Typically such women are post-menopausal or
ovariectomized and may suffer from hot flashes and night sweats.
Such women can be otherwise healthy. In particular, such women may
or may not be hypercholesterolemic. In this regard, the invention
has provided the surprising finding that coronary hyperreactivity
can be independent of atherosclerosis, and an estrogen beta
receptor agonist can have its effect directly on artery
vasoconstriction, not only via lipid pathways.
[0032] Animal studies using female rhesus monkeys, described in the
Hermsmeyer patent, have revealed that coronary vasospasm occurs, in
the absence of injury, plaques or other vascular pathology, due to
local regions of vascular muscle hyperreactivity. In particular,
vasospasm occurs in rhesus monkeys in the absence of
atherosclerosis, which demonstrates that pure reactivity is
sufficient to account for practically stopping vital coronary blood
flow. Coronary vasospasm leads to transmural myocardial ischemia
and can result in sudden cardiac death. Moreover, it has been found
that such vascular muscle hyperreactivity can be reproducibly
elicited by the provocation, or challenge, with intracoronary
injection of certain vasoconstricting agents. Thus, it has been
found in these rhesus monkeys that are fed high fiber diets which
prevent atherosclerosis, that life-threatening myocardial ischemia
resulting from coronary hyperreactivity or vasospasm can be
stimulated by drugs in the absence of atherosclerosis or other
vascular pathology.
[0033] The invention is useful in subjects who are otherwise
apparently healthy and in those who are not apparently healthy.
Apparently healthy, as used herein, means individuals who have not
previously had an acute adverse cardiovascular event such as a
myocardial infarction (i.e. individuals who are not at an elevated
risk of a second adverse cardiovascular disorder due to a primary
cardiovascular event). Apparently healthy individuals also do not
otherwise exhibit symptoms of disease. In other words, such
individuals, as examined by a medical professional, would be
characterized as healthy and free of symptoms of disease.
[0034] The invention likewise is useful in
"non-hypercholesterolemic subjects" and in "hypercholesterolemic
subjects". Non-hypercholesterolemic subjects do not fit the current
criteria established for a hypercholesterolemic subject.
Hypercholesterolemic subjects are associated with increased
incidence of a cardiovascular disorder. A hypercholesterolemic
subject has an LDL level of greater than 190 mg/dl, or greater than
160 mg/dl and at least two risk factors selected from the group
consisting of a family history of premature coronary heart disease,
cigarette smoking (more than 10 per day), hypertension, low HDL
(<35 mg/dl), diabetes mellitus, hyperinsulinemia, abnormal
obesity, high lipoprotein (a) and personal history of cerebral
vascular disease or occlusive peripheral vascular disease, or LDL
greater than 130 mg/dl if ischemic heart disease is present.
[0035] The invention thus is useful in connection with treating
populations of patients never before treated with estrogen. Such
patients can be free of symptoms calling for estrogen
treatment.
[0036] It will likewise be understood by those skilled in the art
that, although the invention is illustrated using coronary arteries
and arterial cells, the invention is applicable to other areas of
the body in which vascular muscle cells are present. Thus, the
invention is useful for prevention and treatment of peripheral
vascular disease such as Raynaud's Disease or intermittent
claudication, and in the prevention and treatment of diseases of
vascular proliferative abnormalities such as intimal hyperplasia,
restenosis of blood vessels, post-angioplasty proliferation,
thromboangiitis obliterans, allograft vasculopathy, diabetic
angiopathy, and hypertensive vasculopathy.
[0037] To achieve optimal prophylactic effects, the estrogen beta
receptor agonist may be administered continuously to provide the
desired blood levels. Other forms of administration, such as oral
or intravascular administration, are less preferred because they
are inconvenient and because they do not provide continuous blood
levels.
[0038] The preferred formulation is a topical preparation. As used
herein, "topical" means applied externally to the surface of the
skin. Specifically excluded in the definition of "topical" are
cavities such as the vaginal, rectal or oral cavity. Also excluded
is the corneum. The estrogen beta receptor agonist is preferably
dissolved in a water base in a combination of propylene glycol,
sorbitol, and cetyl and stearyl alcohols. It may be dissolved in a
non-polar oil. Vitamin E (tocopherol) is one such preferred
non-polar oil that may be used for this purpose in the formulation.
Aloe vera and other water-based substances, such as emolients, may
be included as additives to achieve a pleasant skin cream.
[0039] The effectiveness of the estrogen beta receptor agonist in
such a cream as a delivery system is believed to be due, at least
in part, to the combination of direct transdermal absorption into
the subject's bloodstream and the slow rate of transdermal
absorption. As a consequence, the estrogen beta receptor agonist in
the cream formulation applied to the skin is absorbed and released
over many hours, and this provides exposure to a low but relatively
sustained level for a major part of one day.
[0040] Topical preparations, as known in the art, typically are
non-solid, liquid, cream, gel, lotion, or ointment preparations.
They may contain skin penetration enhancers. Skin penetration
enhancers are agents that when co-applied with a drug to the skin
enhance the ability of the drug to penetrate the skin and be
delivered into the blood stream. Skin penetration agents are
discussed in Remington's Pharmaceutical Sciences, Mack Publishing
Co., 18th Edition, Easton, Pa., USA (1990), which is incorporated
herein by reference.
[0041] Patch technologies also may be used as a delivery system for
the estrogen beta receptor agonist. Transdermal patches typically
include a housing, a reservoir in the housing and a membrane
attached to the housing adjacent the reservoir for placement
against the epidermis of the human subject. The patch has also
included an adhesive attached to the housing for holding the
membrane to the epidermis of the subject. Patches suitable for use
in the present invention and capable of delivering an estrogen beta
receptor agonist in the amounts according to the invention may be
found in the following prior art patents: U.S. Pat. No. 3,731,683;
U.S. Pat. No. 3,797,494; and U.S. Pat. No. 4,336,243; U.S. Pat. No.
4,628,052; U.S. Pat. No. 4,704,282; U.S. Pat. No. 4,788,062; U.S.
Pat. No. 4,906,169; and U.S. Pat. No. 5,164,190, the disclosures of
which are incorporated herein by reference.
[0042] A variety of administration routes besides the topical
route, of course, are acceptable. The methods of the invention
generally speaking, may be practiced using any mode of
administration that is medically-acceptable, meaning any mode that
produces the desired levels of the estrogen beta receptor agonist
without causing clinical unacceptable adverse effects. Such modes
of administration include oral, rectal, vaginal, topical,
sublingual, nasal, intradermal or other parenteral routes such as
inhalation. Intravenous, intramuscular and other injectable routes
are not particularly preferred or suitable for long-term therapy
and prophylaxis.
[0043] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
which constitutes one or more accessory ingredients. In general,
the compositions are prepared by uniformly and intimately bringing
the active compound into association with a lipid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping a
product. As mentioned above, formulations suitable for various
modes of administering can be found in Remington's Pharmaceutical
Sciences.
[0044] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the active compound, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer base systems such as
poly(lactide-glycolate), copolyoxalates, polycaprolactones,
polyester amides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-di- and tri-glycerides; hydrogel
release systems; silastic systems; peptide based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the active compound is contained in a form within a matrix
such as those described in U.S. Pat. Nos. 4,452,775, 4,667,014,
4,748,034 and 5,239,660 and (b) diffusional systems in which an
active component permeates at a controlled rate from a polymer such
as described in U.S. Pat. Nos. 3,832,253, and 3,854,480. In
addition, pump-based hardware delivery systems can be used, some of
which are adapted for implantation.
[0045] Use of a long-term sustained release implant may be
desirable. Long-term release, as used herein, means that the
implant is constructed and arranged to deliver therapeutic levels
of the active ingredient for at least 30 days, and preferably 60
days. Long-term sustained release implants are well-known to those
of ordinary skill in the art and include some of the release
systems described above.
[0046] The invention also includes various kits. Each of the kits
preferably includes instructions for dispensing an amount of an
estrogen beta receptor agonist effective to prevent or treat
vascular hyperreactivity, such as the amounts to achieve the blood
levels as described above. Each of the kits also contains in it a
preparation of an estrogen beta receptor agonist, either
constructed and arranged to deliver the appropriate amounts of the
estrogen beta receptor agonist or with a dispensing means
permitting dispensing of appropriate amounts of the estrogen beta
receptor agonist. Thus, the kit includes a package which preferably
houses instructions as described above. The kit also includes a
topical preparation of an estrogen beta receptor agonist contained
in a container which may be a bottle. A container may be any device
for containing the an estrogen beta receptor agonist, such as a
jar, bottle, vial, tube, packet and the like. This kit also
optionally includes a syringe which may be used to withdraw from
the container the appropriate amount of the topical preparation of
an estrogen beta receptor agonist for use according to the
invention.
[0047] In one preferred embodiment, the optional metering device is
a syringe. The metering device, however, may be any such device
known in the art for dispensing a metered amount of the
preparation, or the kit may lack a metering device. For example,
the metering device may be a calibrated pump that is attached to
the container (i.e., a bottle with a pump dispenser) which is
capable of delivering a metered amount of an estrogen beta receptor
agonist according to the invention. The dispensing device also may
be simply a measuring cup or vial, or pressurized aerosol bottle.
Any means for dispensing a predetermined amount of the estrogen
beta receptor agonist is useful according to the invention.
[0048] A topical cream, gel, lotion, or ointment containing the
estrogen beta receptor agonist also may be contained in individual
packets, each packet containing an appropriate dose for topical
application. The kit may include a plurality of such packets, such
as 30, 60, 100 or more packets, each packet containing a
predetermined amount of cream, such as between about 1.2 to 3.2 ml,
preferably about 2 ml of cream to deliver about 1.0 to 3.0 ml when
squeezed, which in turn contains a predetermined amount of an
estrogen beta receptor agonist. The kits also may include
transdermal patches, which have been described in detail above, as
the means for both containing and the means for dispensing the
estrogen beta receptor agonist.
[0049] The invention is further disclosed in the following
non-limiting examples.
Example 1
Animals
[0050] Six adult ovariectomized (OVX) female rhesus monkeys (Macaca
mulatta) were employed in the studies described below. None of the
monkeys had been exposed to high cholesterol diets and all were
later verified to lack evidence of cardiovascular disease. Each of
the monkeys had a normal physical examination, including blood
chemistry analysis. The monkeys were entered into the study no less
than 3 months after ovariectomy. Monkeys do not endogenously
produce estriol, and therefore all blood or urine levels found must
have resulted from exogenous administration.
Example 2
Catheterization
[0051] Angiography after overnight fasting began with preanesthesia
with ketamine (10 mg/kg body weight intramuscular), intubation with
an endotracheal tube and a light surgical plane of anesthesia with
isoflurane (induced at 1.5% to 3% maintained at 0.75% to 1.25%),
vaporized with 33% nitrous oxide and 67% oxygen, or with 100%
oxygen. Bilateral femoral intraarterial 3F catheters allowed for
coronary catheterization and continuous recording of systemic
arterial blood pressure and heart rate, which were maintained close
to the initial anesthetic value for each monkey. Intravenous
heparin (1,000 U) was injected, and up to 120 ml of pyruvated
Ringer's solution and 0 to 50 ml of dextran solution were used (as
needed) to maintain diastolic blood pressure .gtoreq.60 mm Hg. A
heating pad assisted in maintenance of a body temperature
decrease.ltoreq.2.degree. C. of preanesthesia body temperature
(monitored by rectal thermometer). An electrocardiogram (ECG)
recorded on a Gould eight-channel recorder, cutaneous arterial
oxygen saturation, respiratory rate and end-tidal CO.sub.2 were
also monitored.
[0052] Entire experiments were recorded on videotape (with episodic
fluoroscopic imaging and continuous voice annotation) to permit
computerized quantitative coronary angiography. For reasons
explained in the Hermsmeyer patent, the agent for stimulating the
production of coronary arterial vasospasm in susceptible monkeys
was a combination of serotonin and U46619.
[0053] Placement of the 3F catheter was adjusted to provide
sufficient filling with radio-opaque contrast medium, limited
reflux and isolation of a branch of the coronary arterial tree.
Usually the left anterior descending coronary artery (LAD) was
chosen, but the left circumflex or right coronary artery may
alternatively be used. After adjustment of the camera angle to
optimally image the coronary vascular tree, warm (35 to 38.degree.
C.) 1- to 2-ml boluses of Hexabrix (Mallinckrodt) radio-opaque
contrast media were injected rapidly by hand to fill optimally.
Fluoroscopic images were recorded on film using an OEC model 9800
digital x-ray system with fluoroscopic C-arm, digitally recorded,
and evaluated in subsequent analysis. All procedures including a
brief 0.2 ml injection of Hexabrix for optimal quantitative
angiography were recorded on videotape for subsequent computer
analysis.
[0054] Every injection of drugs was made by intra-coronary (IC)
route, with a slow, constant flow of 1 ml over 30.+-.1 second. The
time between drug injections was typically 7 to 10 minutes, and no
less than 4 minutes, with pressures and heart rates allowed to
return to .ltoreq.15% change from baseline before the next
injection. All concentrations were syringe concentrations
(uncorrected for dilution by coronary blood flow) to exactly
describe the procedure.
Example 3
Effect of Estriol to Prevent Vasospasm
[0055] A 0.3% estriol topical cream was formulated with the
following components.
Component % Concentration w/w
[0056] estriol0.3 [0057] cetyl alcohol, NF6 [0058] stearyl alcohol,
NF3 [0059] sodium lauryl sulfate, NF0.2 [0060] isopropyl palmitate,
NF1.7 [0061] propylene glycol, USP8 [0062] sorbitol solution,
USP12.9 [0063] lactic acid, USP1.2 [0064] benzyl alcohol, NF2.1
[0065] purified water, USPQS 100
[0066] The six rhesus monkeys of Example 1 were treated daily with
this 0.3% estriol topical cream. The cream was rubbed into the skin
of the monkeys on an area that had been shaven. This dosage of skin
cream is expected to result in an absorption of about 0.3 mg
estriol per ml, based on an assumption of 10% absorption of the 3
mg of estriol in each ml of cream. The estriol was purchased from
Steraloids (Wilton, N.H.).
[0067] After 4 weeks of daily treatment with 0.2 mg/kg of the
estriol topical cream, the monkeys were studied by coronary
catheterization, and reactivity was assessed by injection of
serotonin (100 .mu.M) and U46619 (1 .mu.M). Repeat slow
intracoronary infusions of 1 ml over 30 seconds of the combination
of serotonin and U46619 in this 100:1 molar ratio were used,
adjusted for body weight (0.2 mg/kg) to produce coronary blood
levels of 6.7 .mu.M serotonin and 67 nM U46619. The levels of these
two components were based on calculated coronary flow dilution.
Venous blood samples were collected just prior to beginning the
study and after 2 weeks and 4 weeks of treatment for measurements
of progesterone and estriol. Urine samples were collected just
prior to cardiac catheterization.
[0068] Angiography was carried out under isoflurane (0.75-1.25%)
general anesthesia with 70% O.sub.2 and 30% N.sub.2O. After
ketamine sedation (10 mg/kg) and endotracheal tube placement, both
femoral arteries were cannulated for simultaneous measurement of
systemic blood pressure and coronary catheterization. Intravenous
anticoagulation with 1000 units of heparin and fluid replacement
with 75-150 ml of pyruvated Ringer's solution, and if needed 10-50
ml of dextran, to reach a minimum control diastolic blood pressure
of 60 mm Hg.
[0069] Coronary arteries (left anterior descending or left
circumflex) of the monkeys were catheterized and agents injected as
described above in Example 1.
[0070] Statistical analysis was carried out by Student's t-test
(non-paired) or Chi-square, with the p<0.05 level accepted as
significant.
[0071] Table 1 shows the major finding of this study, which is that
estriol protected against vasospasm. None of the monkeys (6 of 6)
that had been exposed to estriol suffered a vasospasm during the
study. As disclosed in the Hermsmeyer patent, the combination of
serotonin and U46619 predictably produces vasospasm in all
unprotected animals to which it is administered. Therefore, the
results prove decisively that estriol is effective in preventing
coronary vasospasm.
Drug-Induced Coronary Vasospasm in Ovariectomized Animals
Incidence and Steroid Levels
TABLE-US-00001 [0072] TABLE 1 Parameter Estriol No Estriol*
Vasospasm criteria met 0/6 6/6** E.sub.3absorbed, ***2412 +/- 316
(n = 5) ng/ml <1 ng/ml urine ng/ml (total) *Control animals are
historical controls. Such controls are disclosed in the Hermsmeyer
patent **Indicates significant differences between Estriol and No
Estriol at p 0.05 (N = 6 for each group). ***Different from
historical controls at p < 0.05 with unpaired t-test
[0073] The data shows that protection against coronary vasospasm by
administration of estriol was categorical, as none of the E.sub.3
treated monkeys showed a vasospasm in a 12 step intracoronary
serotonin+U46619 injection protocol. For all historical controls,
each of which received only a placebo treatment, all 6 of these
monkeys showed coronary vasospasms that met the criteria of <33%
of control diameter for >5 minutes.
Example 4
Effect of Estriol to on Diameter of Coronary Arteries
[0074] The minimum diameter of coronary arteries of the animals
utilized in the previous examples and treated with the estriol
cream was measured before treatment and following injections of the
provocative stimulus (S+U successive injections) over 30 seconds
and compared with that of control animals before and after the
provocative stimulus. The data, as illustrated in FIG. 1, indicates
that, in OVX monkeys not treated with estriol, the coronary
arteries experienced a vasospasm that reduced the diameter to less
than 33% of control for a period of time greater than 5 minutes.
This contraction occurred in all 6 of 6 of the control OVX animals
studies.
[0075] In contrast, the minimum diameters in all six estriol
treated monkeys were indicative of protection against provoked
vasospasm. The difference in minimum diameter between OVX monkeys
treated and not-treated with estriol was significant at
p<0.05.
Example 5
Culture of Rhesus Monkey Coronary Arteries
[0076] Hearts or major blood vessels are removed at necropsy from
rhesus monkeys sacrificed for other purposes and are immediately
immersed in cold ionic solution for mammals (ISM2). ISM2 contains,
in mM, 100 NaCl, 16 NaHCO3, 0.5 NaH.sub.2PO.sub.4, 4.7 KCl, 1.8
CaCl.sub.2, 0.4 MgCl.sub.2, 0.4 HgSO.sub.4, 50 HEPES, and 5.5
dextrose, at pH 7-3 to 7.4. Single cells are enzymatically
dissociated in a multi-step process for hearts and coronary
arteries are dissected from the anterior and posterior walls of the
ventricles and the atrioventricular sulcus. The coronary artery
segments are cleaned and then cut into small pieces and rinsed in
culture solution for mammals--5th generation (CV5M) for 10 min,
followed by potassium glutamate (KG) solution for 10 min, both at
room temperature. CV5M contains 4.0 mM L-glutamine, 100 .mu.g/mL
ciprofloxacin, 50 mM HEPES (pH 7.3), and 16 mM NaHCO.sub.3
dissolved in 90% MEM-Earles's salts and 10% horse serum. KG
solution contains, in mM, 140 K-glutamate, 50 HEPES (pH 7.3), 16
NaH.sub.2PO.sub.4, and 16.5 dextrose.
[0077] The coronary artery tissue is then incubated at 37.degree.
C. for 60 min in an enzyme solution (430 U/ml collagenase and 2
U/ml elastase with 2 mg/ml bovine serum albumin and 100 .mu.M
Ca.sup.2+ in 10 ml of 37.degree. C. KG) with slow stirring. The
supernatant is transferred to 25 ml of 4.degree. C. KG holding
solution with 10% horse serum (KG-H), and 10 ml of enzyme solution
is added to the remaining un-dispersed tissue fragments. This
enzymatic treatment is repeated three times.
[0078] Enzyme exposure is minimized by cold and added serum.
Holding solutions (with 10% serum) containing the dispersed cells
are centrifuged at 200 G and 4.degree. C. for 5 min. After the
supernatant is removed, the cells are re-suspended in 5 ml of
37.degree. C. CV5M and incubated for 1 hr at 37.degree. C. to
permit preferential attachment of fibroblasts. The remaining cells
in suspension are counted and concentrated to .about.100,000 living
cells/ml by centrifugation, and re-suspended in CV5M at 37.degree.
C. VMC are then seeded on clean glass coverslips in culture plates,
and maintained undisturbed in 95% air-5% CO2 and 95% humidity at
37.degree. C. for attachment for 12 hr, and then flooded with CV5M.
Cells from the suspension are also similarly seeded in growth
optimized culture flasks and flooded with CV5M after 12 hr.
[0079] Media changes to a 1% medium (CV5MM, identical to CV5M
except the concentration of horse serum is 1%) are used to reduce
growth factors after 2-4 days. Strict pH control (7.3-7.4) at each
temperature (4.degree. and 37.degree. C.) is maintained by
recognition and allowance for the temperature coefficient of the
HEPES buffer. Subculture of cells is performed by brief
trypsinization, followed by neutralization of trypsin, wash in
media, reconstitution of cell suspension in medium with 1% serum,
and plating onto culture plates and coverslips. For Ca.sup.2+
imaging experiments, VMC from primary culture are used. Under these
culture conditions with low serum (1%) in the media, VMC can retain
their functional properties of contraction and relaxation,
receptors, and ion channels for many weeks and for at least 2
splits and replating of cells.
Example 6
Intracellular Ca.sup.2+ by Fluo3 and PKC (Protein Kinase C) by
Hypericin Fluorescence (2 Wavelength Fluorescence) in VMC
[0080] Imaging of intracellular Ca.sup.2+ changes is performed
using the fluorescent indicator, Fluo3. Glass coverslips with
attached VMC are placed into 300 .mu.L LF 922 laminar flow chambers
on the stage of a Zeiss Axiovert 200M microscope, and suffused with
ISM2 continuously at 1 mL/min. Live cell fluorescent data
acquisition is performed with a photon counting Hamamatsu VIM
camera in conjunction with image processing software. Briefly, VMC
are loaded with 3 .mu.M Fluo3 for 10 min (stopped flow), then
washed (continuously suffused) with ISM2 for 5 min. VMC are exposed
for stimulation to a combination of serotonin and U46619, a stabile
thromboxane A2 mimetic. This combination, containing serotonin at a
concentration of 10 .mu.M and U46619 at a concentration of 100 nM,
is referred to as S+U. The VMC are exposed to the S+U combination
for 15 sec (stopped flow), and then continuously suffused with
ISM2. The S+U combination, even though present for only seconds,
reliably induces long-lasting, characteristically ischemia
producing vasoconstrictions in ovariectomized rhesus monkeys.
Ca.sup.2+ images are recorded according to a timed protocol
(Ca.sup.2+ as fluo3 500-520 nm fluorescence intensity and
distribution at 1, 2, 5, 10, 15, 20 and 30 min after stimulation
and PKC as hypericin 600-660 nm fluorescence intensity at 3, 4, 9,
16, 21 and 31 min) for 31 min after stimulation. Images are
collected and intensities are measured using Image J image
analysis.
Example 7
Persistent Protection of VMC In Vitro by Estriol
[0081] Surgically menopausal (OVX) monkeys were placed into two
groups, a control, untreated group, and a second group treated with
estriol cream as described above. VMC from monkeys in the two
groups were obtained and cultured as described above in Example 5
and imaged for changes in intracellular Ca.sup.2+ levels as
described in Example 6. The data is shown in FIG. 2.
[0082] VMC isolated from OVX monkeys treated for 4 weeks with daily
transdermal estriol and then cultured for 2 weeks before the time 0
challenge (15 sec 100 nM serotonin and 100 nM U46619 (S+U)
stimulus) showed a protected state brief Ca.sup.2+ spike of 10-30
sec., declining after 2 min to nearly baseline level. In contrast,
VMC from untreated OVX cultured in steroid-free media for 1-2 weeks
responded hyperactively to the same 15 sec duration of S+U stimulus
challenge at time 0 with an initial Ca.sup.2+ spike followed 5 to
10 minutes later with a second phase of Ca.sup.2+ signal elevation
indicating a hyperreactive state. The difference between the two
groups was significant, p<0.05.
Example 8
Estrogen Receptor Beta Activity of Test Compounds
[0083] An estrogen receptor beta coactivator fluorescence
polarization assay was performed by PanVera/Invitrogen discovery
screening applying the company's ER.beta. Coactivator Assay Kit
(Invitrogen Corp., Carlsbad, Calif.) using fluorescence
polarization with known estrogen receptor ligands (estradiol,
ICI182,780, tamoxifen, and OH-tamoxifen) and test compounds
(estriol, 3.beta.Adiol, and epiestriol). Data is shown in FIG.
3.
[0084] Data on the X-axis of the graph of FIG. 3 are represented as
log of concentrations from 0.038 nM to 1240 nM tested for each of
the perspective ligands. The ligand concentration that produces a
half-maximal increase or decrease in polarization equals the ligand
EC50 for the ER.beta./D22 interaction. The ordinate Y-axis, mP,
indicates fluorescence polarization units. Increase in polarization
indicates agonist activity. Conversely, a decrease in polarization
indicates antagonist activity. The date represent mean+/-standard
deviation of the polarization values from the assay performed in
triplicate.
[0085] As shown in FIG. 3, estriol and estradiol are estrogen beta
agonists, even though these compounds have significant estrogen
alpha agonistic activity as well. Also, epiestriol and
3.beta.Adiol, compounds that are selective for estrogen beta over
estrogen alpha receptors, have beta receptor activity similar to
that of estriol and estradiol. The other test ligands showed no
estrogen beta receptor agonist activity and, at higher
concentrations, function as estrogen beta receptor antagonists.
Example 9
Comparison of Effects of Estriol, 3.beta.Adiol, and Epiestriol
[0086] FIG. 4 shows the comparative effects of estriol,
3.beta.Adiol, and epiestriol treatment in vitro on Ca.sup.2+
responses in rhesus coronary VMC. Following treatment for 72 hours
with either 300 pM 3.beta.Adiol, 1 nM estriol, or 1 nM epiestriol,
the time course of Ca.sup.2+ signal in response to a 15 sec
stimulation with S+U as described above was determined. Compared to
controls, estriol was found to be protective. However, protection
with estriol was found to be weaker than the estrogen beta receptor
selective agonists 3.beta.Adiol and epiestriol. The data was found
to be significant at p<0.05.
Example 10
Effect of Concentration of 3.beta.Adiol
[0087] The study of Example 9 was repeated using only 3.beta.Adiol
at 4 different concentrations ranging from 30 pM to 3 nM. Data is
shown graphically in FIG. 5.
[0088] As shown, a 3.beta.Adiol concentration of 30 pM resulted in
a Ca+2 response similar to that of untreated control VMC. At
concentrations greater than 30 pM, a protected state was
established. The data was found to be significant at p<0.05.
Example 11
Comparison of Different Estrogen Beta Receptor Agonists
[0089] The studies of Examples 8 to 10 were repeated but using 3
different estrogen beta receptor agonists, 3.beta.Adiol (300 pM),
genistein (10 nM), and DPN (1 nM). Data is shown graphically in
FIG. 6.
[0090] As shown, each of the estrogen beta receptor agonists
produced a protected state in the VMC as compared to control
(p<0.05). Protection with 3.beta.Adiol was superior to that
obtained with either genistein or DPN.
Example 12
Effect of Selective ER.beta. Antagonism by RR-Tetrahydrochrysene
(RRTHC)
[0091] The studies of Examples 8 to 11 were repeated with VMC that
were pretreated with the estrogen beta receptor antagonist RRTHC.
Data is shown graphically in FIGS. 7 A and B.
[0092] As shown in FIG. 7A, the time course of Ca2+ signal in
response to a 15 second stimulation, at time 0, with S+U showed
that protection of 72 hour treatment in vitro with 300 pM
3.beta.Adiol is blocked in VMC that are pre-treated with RRTHC (30
.mu.M, 3 hours prior to and during 3.beta.Adiol). The data was
found to be significant at p<0.05.
[0093] As shown in FIG. 7B, the time course of Ca2+ signal in
response to a 15 sec stimulation with S+U showed an exaggerated
hyperreactive state beyond control after 72 hours in RRTHC in
vitro, but not alter 1 to 6 hour treatment with RRTHC. The data was
found to be significant at p<0.05.
Example 13
Solutions and Drugs Utilized in Examples
[0094] Table 2 shows the cell culture solutions and drugs utilized
in the Examples. Phenol red, known to have estrogenic activity, is
omitted from all solutions. Maintenance medium (used after the VMC
are well attached) is added after 2-4 days and for subsequent media
changes is CV5M1, identical to CV5M except that horse serum
concentration is reduced to 1%. Estrogen and progesterone are
undetectable (<0.1 ng/ml) in CV5M culture medium.
TABLE-US-00002 TABLE 2 SOLUTION COMPOSITION NOTES KG 140 mM
K-glutamate, 50 pH measured at mM HEPES, 16 mM temperature of use
NaH.sub.2PO.sub.4, 16.5 mM glucose, (4.degree. C., 22.degree. C.,
37.degree. C.) pH 7.35 KGH KG + 10% horse serum HyClone, Logan, UT
KGHN KGH + 1 .mu.M nifedipine Collagenase 400 .mu./ml 37.degree. KG
Worthington Type 2 Biochemical Corp., Lakewood, NJ Elastase 0.5
.mu./ml 37.degree. KG Worthington Biochemical Corp., Lakewood NJ
CV5M MEM-Earle salts, 4.0 mM L- pH measured at glutamine, 50 mM
HEPES, temperature of use 16 mM NaHCO.sub.3, 100 .mu.g/ml
(4.degree. C., 22.degree. C., 37.degree. C.) ciprofloxacin, pH
7.35, + 10% horse serum CV5M5 CV5M + horse serum (5%) CV5M1 CV5M +
horse serum (1%) ISM2 100 mM NaCl, 16 mM NaHCO.sub.3, 0.5 mM
NaH.sub.2PO.sub.4, 4.7 mM KCl, 1.8 mM CaCl.sub.2, 0.4 mM
MgCl.sub.2, 0.4 mM MgSO.sub.4, 50 mM HEPES, 5.5 mM glucose, ph 7.35
Fluo3 3 .mu.M/2% DMSO/ISM2 Molecular Probes, Eugene, OR Hypericin
100 nM/4.5% Tocris, Ellsville, MO hydroxypropylcyclodextrin/
sonicated just before ISM2 use S = serotonin 10 .mu.M/0.9% NaCl
Sigma, St. Louis, MO U = U46619 100 nM/0.9% NaCl Sigma, St. Louis,
MO
[0095] All solutions are titrated for pH with HEPES acid and
Na+-HEPES base and measured to be accurate at the intended use
temperatures (4.degree. C., 22.degree. C., and 37.degree. C. for
ice, benchtop, and incubator solutions, respectively). Stock
solutions of 10 mM fluo3 in DMSO are diluted 1000.times. in ISM2.
Hypericin 200 nM stock in 0.1% DMSO are mixed 1:1 with 20 .mu.M
.beta.-hydroxypropylcyclodextrin in ISM2. Solutions of serotonin
are diluted in ISM2 from freshly made 10 mM stock.
[0096] Further modifications, uses, and applications of the
invention described herein will be apparent to those skilled in the
art. It is intended that such modifications be encompassed in the
following claims.
* * * * *