U.S. patent application number 09/951036 was filed with the patent office on 2002-06-06 for methods of prevention and treatment of ischemic damage.
Invention is credited to Gordon, Katherine D., Leonard, Robert J., Simpkins, James W..
Application Number | 20020068727 09/951036 |
Document ID | / |
Family ID | 23405633 |
Filed Date | 2002-06-06 |
United States Patent
Application |
20020068727 |
Kind Code |
A1 |
Simpkins, James W. ; et
al. |
June 6, 2002 |
Methods of prevention and treatment of ischemic damage
Abstract
The present invention provides methods of conferring protection
on a population of cells in a subject following an acute
degenerative event, comprising: administering an effective amount
of the compound in an oil formulation over a course that includes
at least one dose within a time that is effectively proximate to
the ischemic event, so as to confer protection on the population of
cells.
Inventors: |
Simpkins, James W.; (Fort
Worth, TX) ; Gordon, Katherine D.; (Winchester,
MA) ; Leonard, Robert J.; (Wellesley, MA) |
Correspondence
Address: |
BROMBERG & SUNSTEIN LLP
125 SUMMER STREET
BOSTON
MA
02110-1618
US
|
Family ID: |
23405633 |
Appl. No.: |
09/951036 |
Filed: |
September 12, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09951036 |
Sep 12, 2001 |
|
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09357446 |
Jul 20, 1999 |
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Current U.S.
Class: |
514/179 ;
514/182 |
Current CPC
Class: |
A61K 31/565
20130101 |
Class at
Publication: |
514/179 ;
514/182 |
International
Class: |
A61K 031/56 |
Claims
We claim:
1. A method for providing a peak plasma concentration of an
estrogen compound to a subject within an effective time for slowing
the progression of an acute degenerative condition, comprising: (a)
providing an estrogen compound formulated in an oil vehicle; (b)
administering the estrogen formulation to the subject; and (c)
causing a peak plasma concentration of estrogen in the subject
within an effective time for slowing the progression of the acute
degenerative condition.
2. A method according to claim 1, wherein the acute degenerative
condition is an ischemic event induced degenerative condition
3. A method according to claim 1, wherein the effective time is 4
hours.
4. A method according to claim 1, wherein the estrogen formulation
is administered subcutaneously.
Description
CROSS REFERENCES
[0001] This application is a continuation of patent application
Ser. No. 09/357,446 filed Jul. 20, 1999 which is herein
incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to the protection of cells
that would otherwise die as a result of an ischemic event.
BACKGROUND ART
[0003] Ischemia is an acute condition associated with an inadequate
flow of oxygenated blood to a part of the body, caused by the
constriction or blockage of the blood vessels supplying it.
Ischemia occurs any time that blood flow to a tissue is reduced
below a critical level. This reduction in blood flow can result
from: (i) the blockage of a vessel by an embolus (blood clot); (ii)
the blockage of a vessel due to atherosclerosis; (iii) the breakage
of a blood vessel (a bleeding stroke); (iv) the blockage of a blood
vessel due to vasoconstriction such as occurs during vasospasms and
possibly, during transient ischemic attacks (TIA) and following
subarachnoid hemorrhage. Conditions in which ischemia occurs
further include (i) myocardial infarction; (ii) trauma; and (iii)
during cardiac and thoracic surgery and neurosurgery (blood flow
needs to be reduced or stopped to achieve the aims of surgery).
During myocardial infarct, stoppage of the heart or damage occurs
which reduces the flow of blood to organs, and ischemia results.
Cardiac tissue itself is also subjected to ischemic damage. During
various surgeries, reduction of blood flow, clots or air bubbles
generated can lead to significant ischemic damage.
[0004] When an ischemic event occurs, there is a gradation of
injury that arises from the ischemic site. The cells at the site of
blood flow restriction, undergo necrosis and form the core of a
lesion. A penumbra is formed around the core where the injury is
not immediately fatal but progresses slowly toward cell death. This
progression to cell death may be reversed upon reestablishment of
blood flow within a short time of the ischemic event.
[0005] Focal ischemia encompasses cerebrovascular disease (stroke),
subarachnoid hemorrhage (SAH) and trauma. Stroke is the third
leading cause of morbidity in the United States, with over 500,000
cases per year, including 150,000 deaths annually. Post-stroke
sequelae are mortality and debilitating chronic neurological
complications which result from neuronal damage for which
prevention or treatment are not currently available.
[0006] Following a stroke, the core area shows signs of cell death,
but cells in the penumbra remain alive for a period of time
although malfunctioning and will, in several days, resemble the
necrotic core. The neurons in the penumbra seem to malfunction in a
graded manner with respect to regional blood flow. As the blood
flow is depleted, neurons fall electrically silent, their ionic
gradients decay, the cells depolarize and then they die.
Endothelial cells of the brain capillaries undergo swelling and the
luminal diameter of the capillaries decrease. Associated with these
events, the blood brain barrier appears to be disrupted, and an
inflammatory response follows which further interrupts blood flow
and the access of cells to oxygen.
[0007] The effects of a stroke on neurons result from the depletion
of energy sources associated with oxygen deprivation which in turn
disrupts the critically important ion pumps responsible for
electrical signaling and neurotransmitter release. The failure of
the ATP-dependant ion specific pumps to maintain ion gradients
through active transport of sodium, chlorine, hydrogen, and calcium
ions out of the cell and potassium ions into the cell results in a
series of adverse biochemical events. For example, increase in
intracellular calcium ion levels results in: (I) the production of
free radicals that extensively damage lipids and proteins; (ii) the
disruption of calcium sensitive receptors such as the N-methyl
D-aspartate (NMDA) and the .alpha.-amino-3-hydroxy-5-methyl--
4-isoxazoleproprionic acid (AMPA) synaptic glutamate receptors;
(iii) the swelling of cells with water as a result of abnormal
accumulation of ions; and (iv) the decrease in intracellular pH.
The alteration in metabolism within the cell further results in the
accumulation of ions in the cells as energy sources are depleted.
For example, anaerobic glycolysis that forms lactic acid, replaces
the normal aerobic glycolysis pathways in the mitochondria. This
results in acidosis that results in further accumulation of calcium
ions in the cell.
[0008] Despite the frequency of occurrence of ischemia (including
stroke) and despite the serious nature of the outcome for the
patient, treatments for these conditions have proven to be elusive.
There are two basic approaches that have been undertaken to rescue
degenerating cells in the penumbra. The first and most effective
approach to date has been the identification of blood clot
dissolvers that bring about rapid removal of the vascular blockage
that restricts blood flow to the cells. Recombinant tissue
plasminogen activator (TPA) has been approved by the Federal Drug
Administration for use in dissolving clots that cause ischemia in
thrombotic stroke. Nevertheless, adverse side effects are
associated with the use of TPA. For example, a consequence of the
breakdown of blood clots by TPA treatment is cerebral hemorrhaging
that results from blood vessel damage caused by the ischemia. A
second basic approach to treating degenerating cells deprived of
oxygen is to protect the cells from damage that accumulates from
the associated energy deficit. To this end, glutamate antagonists
and calcium channel antagonists have been most thoroughly
investigated. None of these have proven to be substantially
efficacious but they are still in early clinical development. The
athophysiology and treatment of focal cerebral ischemia has been
reviewed by B. K. Seisjo, J. Neurosurgery, 1992, vol. 77, p.
169-184 and 337-354.
[0009] In addition to the targets of drug development described by
Seisjo (1992), epidemiological studies have shown that women
undergoing hormone replacement therapy with estrogen and
progesterone experienced a reduction in the incidence and severity
of heart disease. This correlation was further investigated for
stroke with mixed results. A 10-year epidemiological study on
48,000 women reported by Stampfer et al. (New England Journal of
Medicine, 1991, vol. 325, p. 756) concluded that there was a
correlation between use of estrogen and decrease in incidence of
coronary heart disease, but no decrease in the incidence of stroke
was observed. In contrast, a report by Wren (The Medical Journal of
Australia, 1992, vol. 157, p. 204) who reviewed 100 articles
directed to the question as to whether estrogens reduce the risk of
atherosclerosis and myocardial infarction, concluded that estrogens
in hormone replacement therapies significantly reduce the incidence
of myocardial infarction and stroke and may accomplish this at the
site of the blood vessel wall. This conclusion was further
supported by Falkeborn et al. Arch Intern. Med., 1993, vol. 153, p.
1201. The above correlation between estrogen replacement therapy
and reduced incidence of stroke relies on epidemiological data
only. No biochemical data were analyzed to interpret or support
these conclusions, nor is there any information as to reduction in
ischemic lesion or morbidity with hormone use. Furthermore, these
studies were restricted to the patients receiving long-term hormone
replacement treatment. No studies were performed on patients who
might be administered estrogen therapeutically shortly before,
during, or after a stroke for the first time. Furthermore, the
studies were limited to estrogens utilized in estrogen replacement
therapy. No studies were performed on any non-sex related estrogens
that might be used in treating males or females.
[0010] Studies have been conducted on the neuroprotective effects
of steroids in which glucocorticosteroid for example was found to
have a positive effect in reducing spinal cord injury but had a
negative effect on hippocampal neurodegeneration. For example, Hall
(J. Neurosurg vol. 76, 13-22 (1992)) noted that the glucocorticoid
steroid, methylprednisolone, believed to involve the inhibition of
oxygen free radical-induced lipid peroxidation, could improve the
6-month recovery of patients with spinal cord injury when
administered in an intensive 24-hour intravenous regimen beginning
within 8 hours after injury. However, when the steroid was examined
for selective protection of neuronal necrosis of hippocampal
neurons, it was found that the hippocampal neuronal loss was
significantly worsened by glucocorticoid steroid dosing suggesting
that this hormone is unsuitable for treating acute cerebral
ischemic. Hall reported that substitution of a complex amine on a
non-glucocorticoid steroid in place of the 21'-hydroxyl
functionality results in an enhancement of lipid anti-oxidant
activity. No data were provided concerning the behavior of this
molecule in treating ischemic events or in neuroprotection of
neurons in the brain. Additionally, free radical scavenging
activity has been reported for a lazaroid, another
non-glucocorticoid steroid having a substituted 21'-hydroxyl
functionality, but there is no evidence that this compound is
significantly efficacious for treating stroke or other forms of
ischemia.
[0011] There is a need for effective treatments for stroke and
other forms of ischemia that are safe, and may be administered
preventatively to men and women who are susceptible to such
conditions, and may further be used after the ischemia has occurred
so as to protect cells from progressive degeneration that is
initiated by the ischemic event. There is further a need for
therapeutic strategies, to treat victims of stroke or other forms
of ischemic events such as myocardial infarction, in which the
active drug could enter the bloodstream very rapidly, reach peak
levels within minutes, and sustain lower, therapeutic drug dosage
levels for a significant period of time (e.g., hours)
thereafter.
SUMMARY OF THE INVENTION
[0012] The invention satisfies the above need. Novel methods are
provided for prevention and treatment of ischemic damage using
estrogen compounds.
[0013] A preferred embodiment of the invention provides a method
for conferring protection on a population of cells associated with
an ischemic focus, in a subject following an ischemic event that
includes the steps of providing subcutaneously an estrogen compound
in a drug delivery system in which the estrogen compound is
dissolved in oil with or without additional excipients such as
solvents, stabilizers or preservatives, so as to confer protection
on the population of cells. Further embodiments include selecting a
proximate time for administering the effective dose of the estrogen
compound that is prior to the ischemic event. Alternatively, the
estrogen compound may be administered within an effective proximate
time after the ischemic event. The method of the invention may be
applied to any of a cerebrovascular disease, subarachnoid
hemorrhage, myocardial infarct, surgery, and trauma. In particular,
when the ischemic event is a stroke, the protected cells include at
least one of neurons and endothelial cells.
[0014] The method utilizes an estrogen compound which may include
alpha isomers or beta isomers of estrogen compounds. Examples of
different isomers are provided wherein the estrogen compound is
selected from the group consisting of 17.alpha.-estradiol and
17.beta.-estradiol.
[0015] In a preferred embodiment of the invention, a method is
provided for protecting cells in a subject from degeneration during
or after an ischemic event. The steps of the method include
identifying a susceptible subject, providing an effective dose of
an estrogen compound prior to or after the ischemic event, and
protecting cells from degeneration otherwise occurring in the
absence of the estrogen compound.
[0016] In a further embodiment of the invention, a method is
provided for treating stroke in a subject, including the steps of
providing an effective dose of an estrogen compound in a
pharmaceutical formulation and administering the formulation to the
subject so as to reduce the adverse effects of the stroke.
[0017] The invention in another embodiment provides a method for
conferring protection on a population of cells associated with
ischemia, in a subject following an ischemic event, comprising: (a)
providing an estrogen compound formulated in an oil vehicle; and
(b) administering an effective amount of the compound over a course
that includes at least one dose within a time that is effectively
proximate to the ischemic event, so as to confer protection on the
population of cells. Further in this embodiment in (b) the estrogen
compound is administered by subcutaneous injection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] These and other features, aspects, and advantages of the
present invention will be better understood with reference to the
following description, appended claims, and accompanying drawings,
where:
[0019] FIG. 1 is a bar graph that shows the effects of pretreatment
of ovariectomized rats, with 17.beta.-estradiol, initiated 24 hours
prior to ischemia induced by middle cerebral artery occlusion
(MCAO); where the 17.beta.-estradiol is administered as a
subcutaneous 5 mm Silastic.RTM. implant (E2) or via the
estradiol-chemical delivery system (E2-CDS) (1 mg/kg body weight)
and a control is provided (a sham pellet). Values are given as the
mean plus and minus the standard error of the mean (.+-.SEM) for
the percent ischemic area in 3 brain slices. The asterisk indicates
that the observed p value was less than 0.05 (*=p<0.05) vs. sham
group. The number of samples for sham=6, for 17.beta.-estradiol=8,
and for E2-CDS groups=10.
[0020] FIG. 2 is a bar graph that shows the effects of treatment of
ovariectomized (OVX) rats with 17.beta.-estradiol, at 2 hours prior
to ischemia induced by MCAO, where the 17.beta.-estradiol (10
.mu.g/kg) is injected subcutaneously in an oil vehicle. Rats were
decapitated 24 hours after the MCAO. Rat brains were dissected
coronally as region A-E, 24 hours after MCAO. Values were given as
the mean.+-.SEM where n=8 for OVX+E.sub.2 group and n=6 for OVX
group(control). * p<0.05 vs. corresponding vehicle control
groups.
[0021] FIG. 3 is a bar graph that shows the effects of pretreatment
of ovariectomized rats with 17.alpha.-estradiol, initiated 24 hours
prior to ischemia induced by MCAO, where the 17.alpha.-estradiol is
administered in a 5 mm Silastic.RTM. tube, and the negative control
is a 5 mm Silastic.RTM. tube without estrogen (sham). Rats were
decapitated 24 hours after the MCAO. Values are given as the
mean.+-.SEM for the percent ischemic area in 5 brain slices. A to E
designate the distance caudal to the olfactory bulb A=5 mm, B=7 mm,
C=9 mm, D=11 mm, and E=13 mm. *=p<0.05 vs. sham group for the
equivalent brain slice; for sham n=10 and for 17.alpha.-estradiol
groups, n=13.
[0022] FIG. 4 is a bar graph that shows the effects of
post-treatment of ovariectomized rats with 17.beta.-estradiol or an
hydroxypropyl cyclodextrin (HPCD) control at 40 minutes (a) and 90
minutes (b) post onset of MCAO. The 17.beta.-estradiol was
formulated in an estradiol chemical delivery system (E2-CDS) at a
concentration of 1 mg/kg body weight and injected intravenously.
Rats were decapitated 24 hours after the MCAO. Values are given as
the mean.+-.SEM for the percent ischemic area in 5 brain slices. A
to E designate the distance caudal to the olfactory bulb A=5 mm,
B=7 mm, C=9 mm, D=11 mm and E=13 mm. Where *=p<0.05 vs HPCD
group for the same brain slice, N=9 for vehicle, and 13 for E2-CDS
groups.
[0023] FIG. 5 is a bar graph that shows the effects of
17.beta.-estradiol (2 nM) on brain capillary endothelial cell
(BCEC) mortality following 24 hours of hypoglycemia. The control
consists of the ethanol vehicle only. The glucose concentrations in
the cell media were adjusted from 20 mg % to 200 mg % by adding
appropriate amount of D-(+)-glucose to the glucose-free media. BCEC
were incubated for 24 hours (a) and 48 hours (b). Trypan blue
staining was used to distinguish live cells from dead cells. Two
cell countings at two different hemacytometer squares were
averaged. Mean.+-.SEM are depicted (n=8-12). *p<0.05 vs.
corresponding vehicle control.
[0024] FIG. 6 is a bar graph that shows the effects of
17.beta.-estradiol (2 nm) on BCEC mortality following anoxia. The
control consists of the ethanol vehicle without estrogen. Cell
media contained 200 mg % glucose. Culture dishes containing BCEC
were placed in nitrogen filled chamber for 4 hours. Trypan blue
staining was used to distinguish live cells from dead cells. Two
cell countings at two different hemacytometer squares were
averaged. Mean.+-.SEM are depicted (n=8-12). *p<0.05 vs.
corresponding vehicle control.
[0025] FIG. 7 is a bar graph that shows the effects of
17.beta.-estradiol (2 nm) on BCEC mortality compared with a control
(ethanol vehicle) following a combination treatment of both anoxia
and hypoglycemia. Cell media contained 200 mg % or 100 mg %
glucose. Culture dishes containing BCEC were placed in either an
incubator or a nitrogen filled chamber for two hours. Trypan blue
staining was used to distinguish live cells from dead cells. Two
cell countings at two different hemacytometer squares were
averaged. Mean.+-.SEM are depicted (n=8.12). *<0.05 vs.
corresponding vehicle control.
[0026] FIG. 8 is a bar graph that shows the effects of
post-treatment of ovariectomized (OVX) rats with 17.beta.-estradiol
at 0.5 hour, 1 hour, 2 hours, 3 hours or 4 hours following ischemic
induced by MCAO. The estrogen compound was administered by a
combination of an intravenous preparation (100 .mu.g/kg) of
HPCK-complexed 17.beta.-estradiol and Silastic.RTM. pellet at the
times post-occlusion indicated. Ovariectomized, non-treated
animals(OVX) and non-ovariectomized, non-treated animals (INT) were
used as controls (n=12 and n=6, respectively). At 48 hours
following MCAO, ischemic lesion volume was determined using 2,3,5
-triphenyltetrazolium (TTC) staining.
[0027] FIG. 9 is a graph that shows the effects on drug kinetics of
administering an estrogen compound in single subcutaneous bolus
injection in oil on the ordinate, as a function of time on the
abscissa.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0028] The invention provides an effective treatment for stroke and
other forms of ischemia that may safely be administered to men and
women so as to protect cells from progressive degeneration that is
initiated by the ischemic event.
[0029] Estrogen compounds are defined here and in the claims as any
of the structures described in the 11th edition of "Steroids" from
Steraloid Inc., Wilton, N.H., incorporated herein by reference.
Included in this definition are non-steroidal estrogens described
in the aforementioned reference. Other estrogens included in this
definition are estrogen derivatives, estrogen metabolites, estrogen
precursors, and modifications of the foregoing as well as molecules
capable of binding cell associated estrogen receptor as well as
other molecules where the result of binding triggers a
characteristic estrogen effect. Any diastereomer or enantiomer of
compounds described herein is included in the definitions herein.
Also included are mixtures of more then one estrogen. The term
"estradiol" or "estrogen" is included in the meaning of estrogen
compound.
[0030] .beta.-estrogen and .alpha.-estrogen are isomers of
estrogen.
[0031] The term "E2" is synonymous with
.beta.-estradiol,17.beta.-estradio- l, E.sub.2, and
.beta.-E.sub.2.
[0032] An "animal subject" is defined here and in the claims as a
higher organism including humans.
[0033] The term "non-sex hormone" is defined here and in the claims
as an estrogen compound having diminished, minimal or no
sex-related effect on the subject.
[0034] Estrogen compounds are here shown to protect cells from
degeneration in the penumbra of the ischemic lesion. (Examples 1
and 2) Estrogen compounds are further shown to be protective of a
plurality of cell types, including neuronal cells and endothelial
cells (Examples 1-3). According to the invention, estrogen
compounds may be used to protect cells from the effects of oxygen
deprivation and glucose deprivation and consequently from energy
deprivation associated with ischemia.
[0035] In an embodiment of the invention, a method of treatment is
provided that is suitable for human male and female subjects and
involves administering an effective dose of estrogen either before
or after a stroke has occurred.
[0036] In certain circumstances according to the invention, it is
desirable to administer estrogen prior to a predicted ischemic
event. Such circumstances arise when, for example, a subject has
already experienced a stroke. In this case, the subject will have
an increased probability of experiencing a second stroke. Subjects
who are susceptible to transient ischemic attacks also have an
increased risk of a stroke. Subjects who suffer a subarachnoid
hemorrhage may experience further ischemic events induced by
vasospasms that constrict the blood vessels. Subjects who
experience trauma to organs such as the brain are also susceptible
to an ischemic event. The above situations exemplify circumstances
when a subject would benefit from pretreatment with an estrogen
compound. Such pretreatment may be beneficial in reducing the
adverse effects of a future ischemic event when administered in the
short term, such as within 24 hours before the event (Example 1) or
in the long term, where administration begins immediately after an
event such as a stroke and continues prophylactically for an
extended period of time. An example of time of administration for
prophylactic use may extend from days to months depending of the
particular susceptibility profile of the individual. In these
circumstances, a course of at least one dose of estrogen may be
administered over time so that an effective dose is maintained in
the subject. For short term treatments, parenteral administration
may be used as an alternative to the delivery of a dose by any of
the routes specified below. The optimal dose of estrogen compound
for prophylactic use should provide a plasma concentration of
10-500 pg/ml of estrogen compound, however higher doses are also
acceptable. In these circumstances, the use of non-sex estrogen
compounds such as the .alpha.-estrogen isomers are of particular
utility in men and women because the sex-related functions of the
hormone are avoided.
[0037] According to embodiments of the invention, estrogen
compounds are effective in reducing the adverse effects of an
ischemic event such as cerebrovascular disease, subarachnoid
hemorrhage, or trauma. Accordingly, the compound is administered as
soon as possible after initiation of the event and preferably
within 12 hours, more particularly, within 5 hours following the
event. It is desirable that an increased concentration of estrogen
compound be maintained in the plasma for at least several hours to
several days following the ischemic event. The increased
concentration of estrogen compound in the plasma should be in the
range of 10-12,000 pg/ml of estrogen compound.
[0038] The present invention demonstrates for the first time that
pretreatment with estrogens or early post-treatment of an estrogen
compound can significantly reduce the size of the necrotic area
following an ischemic event. This effect of pretreatment with an
estrogen compound is independent of the isomeric form and the route
of administration of the estrogen compound. .alpha.-isomers of
estrogen have been shown to be as effective as .beta.-isomers of
estrogen in protecting cells from the effects of ischemia. The
method as exemplified in Example 1 and FIGS. 1, 2 and 3 confirm
that the protective activity of estrogen compounds is not dependent
on the sex-related activity of the hormone (estrogenicity).
.alpha.-isomers of estrogen compounds are non-sex hormones, yet
these compounds are as effective at protecting the brain against
ischemic damage as the .beta.-isomers. Example 1 further
demonstrates that the observed reduction in mortality of
ovariectomized rats when treated with 17.beta.-estradiol is not
dependent on the route of administration, since the protective
effect was similar when the same estrogen compound was administered
as a subcutaneous implant or as an intravenous injection.
Regardless of the route of administration or the formulation, the
estrogen compounds have a remarkable effect on the ability of
animals to survive an ischemic event.
[0039] The demonstration that estrogen is efficacious in protection
of cells in an ischemic area is demonstrated in the examples below
using rat models in which the middle cerebral artery (MCA) is
experimentally occluded, the middle cerebral artery occlusion
(MCAO) model. This animal model is well known in the art to
simulate an in vivo ischemic event such as may occur in a human
subject. The experimental occlusion of the MCA causes a large
unilateral ischemic area that typically involves the basal ganglion
and frontal, parietal, and temporal cortical areas (Menzies et al.
Neurosurgery 31, 100-106 (1992)). The ischemic lesion begins with a
smaller core at the site perfused by the MCA and grows with time.
This penumbral area around the core infarct is believed to result
from a propagation of the lesion from the core outward to tissue
that remains perfused by collateral circulation during the
occlusion. The effect of a therapeutic agent on the penumbra
surrounding the core of the ischemic event may be examined when
brain slices are obtained from the animal. The MCA supplies blood
to the cortical surfaces of frontal, parietal, and temporal lobes
as well as basal ganglia and internal capsule. Slices of the brain
are taken around the region where the greatest ischemic effect
occurs. These regions have been identified as region B, C, and D in
Examples 2 and 3. These regions are not as readily compensated by
alternative sources of blood flow as are regions A and E. This is
because the MCA is the terminal artery on which the lace of
collateral arteries supplying the MCA-distributed area relies,
thereby making the MCA-occlusion induced ischemia uncompensatible.
On the other hand, anastomoses between MCA and the anterior carotid
artery (ACA) in region A and between MCA and the posterior carotid
artery (PCA) in region E (Examples 1 and 2), may compensate for the
MCA occlusion-induced ischemia as observed in the present
study.
[0040] In order to study the effect of estrogen on the propagation
of the lesion following an ischemic event, rats were ovariectomized
and two weeks later were exposed to various estrogen preparations
prior to or following MCAO. (Examples 1 and 2). Untreated,
ovariectomized rats had a mortality of 65%. Pretreatment with
E2-CDS or 17.beta.-estradiol itself decreased mortality from 16%
and 22%, respectively. This marked reduction in mortality was
accompanied by a reduction in the ischemic area of the brain from
25.6.+-.5.7% in the untreated, ovariectomized rats to 9.1.+-.4.2%
and 9.8.+-.4.0 in the E2-CDS or 17.beta.-estradiol treated rats,
respectively. Similarly, pretreatment with non-sex hormones,
exemplified by 17.alpha.-estradiol, reduced ischemic area by 55 to
81% (Example 1). When administered 40 or 90 minutes after MCAO,
17.beta.-estradiol reduced ischemic area by 45-90% or 31%,
respectively (Example 2). Non-sex hormones were also highly
protective when administered following induction of ischemia. These
results demonstrate the neuroprotective effect of estrogen
compounds in the brain following an ischemic event.
[0041] Reduction in available oxygen and glucose for energy
metabolism is a feature of an ischemic event. This has a negative
impact on the blood vessels that may be required to supply
nutrients once the occlusion is reversed. The negative effect on
blood vessels following ischemia further increases the long-term
damage associated with the event. This effect can be reproduced in
vitro as described in Example 3. In these circumstances, it has
been shown here, estrogen compounds are capable of protecting brain
capillary endothelial cells from cell death that would otherwise
occur during hypoglycemia and anoxia during an ischemic event
(FIGS. 5-7). As a consequence of this protection, the integrity of
the vascular supply and the blood brain barrier is preserved by
estrogen compounds such that following reperfusion of the brain
after the ischemic event, blood flow and transport functions can
once again occur.
[0042] Estrogen compounds are shown here to be effectively
delivered subcutaneously in an oil vehicle (Example 1 and FIG. 9).
This mode of delivery was successful at achieving blood levels of
4,610 pg/ml of the estrogen compound within 30 minutes. Sustained
delivery was achieved also, as animal blood levels of 2,004 pg/ml
was at the four hour time point (FIG. 9).
EXAMPLES
Example 1
Measurement of the effect of estrogen compound administered prior
to ischemic events
[0043] Rats were used as experimental models to test the effects of
estrogen compounds in protecting against ischemic damage. To remove
the naturally occurring source of estrogen, ovariectomies were
performed prior to induction of ischemia.
[0044] Subsequent to the ovariectomy, rats were treated with an
estrogen compound either by subcutaneous delivery with
Silastic.RTM. tubes 24 hours prior to the MCA occlusion or by
intravenous delivery as follows:
[0045] Subcutaneous sustained delivery: 17.beta.- or
17.alpha.-estradiol was packed into 5 mm long Silastic.RTM. tubes
(Dow-Corning, Midland, Mich.) according to the method of Mohammed
et al. 1985 Ann. Neurol 18, 705-711. Sham (empty) tubes were
similarly prepared as estrogen negative controls. The pellets were
implanted subcutaneously (sc) into ovariectomized rats 24 hours
prior to MCAO. 5 mm of Silastic.RTM. tubing containing estrogen
resulted in plasma levels of about 100-200 pg/ml.
[0046] Intravenous (iv) delivery: 17.beta.-estradiol was prepared
for iv delivery using an estrogen-chemical delivery system (E2-CDS)
as described in Brewster et al., Reviews in the Neurosciences 2,
241-285 (1990) and Estes et al., Life Sciences 40: 1327-1334
(1987). E2-CDS was complexed with hydroxypropyl-.beta.-cyclodextrin
(HPCD) (Brewster et al. J. Parenteral Science and Technology 43:
231-240, (1989)). The complexation achieved was 32 mg of E2-CDS per
gram HPCD. In the first study, a single intravenous (iv) injection
of E2-CDS (1 mg/kg body weight) was administered at 24 hours prior
to MCAO. The control was administered HPCD only. The chemical
delivery system is formulated so that the estrogen is slowly
released from the carrier. This delivery system has been shown to
effectively deliver estrogen in a sustained manner to the brain.
Indeed, the dose of E2-CDS used in Examples 1 and 2 (1 mg/kg) is
sufficient to provide 1000 pg/gm brain tissue at 24 hours post
administration.
[0047] At 7 to 8 days after ovariectomy, a method for occluding the
middle carotid artery was applied to the rat using modifications of
the methods of Longa et al. (1989) Stroke, vol. 20, 84-91; and
Nagasawa et al. (1989) Stroke, vol. 20, 1037-1043, with certain
modifications, as described herein.
[0048] Animals were anesthetized by intraperitoneal (ip) injection
with ketamine (60 mg/kg) and xylazine (10 mg/kg). Rectal
temperature was monitored and maintained between 36.5 and
37.0.degree. C. with a heat lamp throughout the entire procedure.
The left carotid artery was exposed through a midline cervical
incision. The left stemohyloid, sternomastoid, digastric (posterior
belly) and the omohyloid muscles were divided and retracted. Part
of the greater horn of the hyloid bone was cut to facilitate
exposure of the distal external carotid artery (ECA). The common
carotid artery (CCA), ECA, and internal carotid artery (ICA) were
dissected away from adjacent nerves. The distal ECA and its
branches, the CCA, and the pterygopalatine arteries were coagulated
completely. A microvascular clip was placed on the ICA near skull
base. A 2.5 cm length of 3-0 monofilament nylon suture was heated
to create a globule for easy movement and blocking of the lumen of
the vessel. This was introduced into the ECA lumen through the
puncture. The suture was gently advanced to the distal ICA until it
reached the clipped position. The microvascular clip was then
removed and the suture was inserted until resistance was felt. The
distance between the CCA bifurcation and the resistive point was
about 1.8 cm. This operative procedure was completed within 10
minutes without bleeding. After the prescribed occlusion time (40
minutes), the suture was withdrawn from the ICA and the distal ICA
was immediately cauterized.
[0049] Animals that survived until the scheduled sacrifice time
were sacrificed by decapitation. Scheduled post-ischemic sacrifices
occurred at 6 hours, 24 hours and 1 week post MCAO (Table 1). For
the 6-hour sample, animals were monitored continuously. For the
24-hour sample, animals were observed for about 4 hours and were
then returned to their cages. Similarly, animals scheduled for the
1 week post-ischemic sacrifice were monitored for the first 4 hours
after surgery and then daily thereafter.
[0050] The brains were isolated from the decapitated heads, sliced
into 3 or 5 coronal tissue slices as described below and then
stained with hematoxylin and eosin to determine the extent of the
ischemic area. Stained slices were photographed and subsequently
imaged using a Macintosh Cadre 800 computer, equipped with an Image
1.47 software program for the assessment of the cross-sectional
area of the ischemic lesion. These images and the calculated area
of ischemic damage were stored in the program for later retrieval
and data reduction. The significance of differences in mortality
among the different treatment groups was determined using
Chi-Square analysis.
[0051] The results obtained using different routes of
administration and different isomeric forms of estrogen compounds
are provided below.
[0052] The administration of an estrogen compound by subcutaneously
using Silastic.RTM. tubes or by controlled intravenous delivery, at
24 hours prior to the ischemic event, caused brain lesion size and
mortality to be reduced.
[0053] Three coronal slices were made at 1, 5, and 7 mm posterior
to the olfactory bulb. Only 35% of the control (sham) animals
survived until the scheduled post-ischemic sacrifice time (Table
1). In contrast, 78% and 84% of animals, treated 24 hours prior to
MCAO with either 17.beta.-estradiol in a Silastic.RTM. tube (E2
implant) or with E2-CDS at 1 mg/kg administered by an intravenous
injection survived until the scheduled post-ischemic sacrifice time
at 6 hours, 1 day, and 1 week. Elevated levels of
17.beta.-estradiol were detected in all samples at the time of
sacrifice. The reduction in mortality in the estrogen compound
pretreatment group was most notable at 1 day and 1 week after MCAO
(Table 1). Furthermore, the reduced mortality in the estrogen
compound treated rats was correlated with the reduction of ischemic
area in animals that survived to the scheduled 1 day or 1 week
post-ischemic sacrifice time (FIG. 1). Control (sham) rats had
ischemic lesions that occupied 25.6.+-.5.7% of the cross-sectional
area of brain sections evaluated (FIG. 1). By contrast, rats
treated with 17.beta.-estradiol in Silastic.RTM. tubes or E2-CDS
had ischemic lesions that occupied only 9.8.+-.4.0 and 9.1.+-.4.2%,
respectively, of the brain area evaluated. The significance of
differences among groups was determined by analysis of variance
(ANOVA) and the Fischer's test was used for the post hoc
comparison. Determination of areas under the curves were not done
here as only three brain slices were taken.
[0054] The results shown in FIG. 2 illustrate the significant
protective effect of estrogen compounds in tissue slices A-D in
animals treated with subcutaneous injection of 17.beta.-estradiol
(10 .mu.g/ml) two hours prior to an ischemic event.
[0055] Rats were ovariectomized, treated with a single dose of
17.beta.-estradiol (10 .mu.g/kg ) by a sc injection, 14 days after
the ovariectomy and two hours prior to the ischemic event as
described above. This injection was sufficient to achieve a plasma
concentration of 250 pg/ml at the time of occlusion. The animals
were sacrificed at 24 hours and the brains extracted. Estrogen
compound replacement of ovariectomized rats reduced by 46.3% and
44.1% (p<0.05) ischemic lesion size of the whole coronal section
at region C and D, respectively (FIG. 2). These regions correspond
to sections taken at 9 and 11 mm caudal to the olfactory bulb.
[0056] The results shown in FIG. 3 illustrate the significant
protective effect of 17.alpha.-estradiol in tissue slices A-E in
animals treated with a sustained subcutaneous delivery of
17.alpha.-estradiol initiated 24 hours prior to the ischemic
event.
[0057] Ovariectomized rats were treated with 5 mm Silastic.RTM.
tubes containing 17.alpha.-estradiol at 24 hours prior to MCAO. At
24 hours after the MCAO, the animals were sacrificed and the brains
extracted. Five, 2 mm thick coronal sections were made at 5, 7, 9,
11, and 13 mm posterior of the olfactory bulb. The slices were then
incubated for 30 minutes in a 2% solution of 2,3,5-triphenyl
tetrazolium (TTC; Sigma Chemical Corp., St. Louis, Mo.) in
physiological saline at 37.degree. C. Sham-treated rats showed the
expected ischemic lesion, with the maximum ischemic area
(24.1.+-.2.4%) occurring in slice C (9 mm posterior to the
olfactory bulb ) and smaller lesion areas occurring in more rostral
and caudal slices (FIG. 3). The significance of differences between
sham and steroid-treated groups, were thus determined and data from
two groups were compared for each experiment. To determine the area
under the lesion curve for a given treatment, the trapezoidal
method was used. Areas calculated for each animal were grouped and
the differences between groups were determined by the student t
test.
[0058] Animals pretreated with 17.alpha.-estradiol exhibited
smaller ischemic areas compared with the sham treated animals in
all slices evaluated (FIG. 3, A-E). Specifically, slices C, D and E
(sections taken at 7, 9, and 11 mm posterior to the olfactory
bulb), ischemic area was reduced significantly by 55%, 66%, and
81%, respectively (FIG. 3). The area under the ischemic lesion
curve for the sham-treated, and the 17.alpha.-estradiol groups was
8.1.+-.0.8 and 3.7.+-.1.3, respectively (Table 2).
Example 2
Measurement of the effect of estrogen compounds administered after
the ischemic event
[0059] To test the extent to which estrogen treatment was effective
after the onset of the occlusion, ovariectomized rats were treated
iv with a sustained release of either E2-CDS or with a control
(HPCD vehicle), the positive sample causing a brain tissue
concentration of estrogen of 1000 pg estrogen/gm brain tissue, 24
hours after administration. The estrogen compound was administered
at 40 minutes and 90 minutes after the onset of the MCAO (FIGS. 4a
and b, Table 2) and the animals sacrificed at 24 hours after the
MCAO. Five 2 mm thick coronal sections were made at 5, 7, 9, 11,
and 13 mm posterior of the olfactory bulb as described in Example
1.
[0060] Post-treatment at 40 minutes: As shown in FIG. 4a, the
control rats (HPCD treated) had large ischemic areas in all slices
sampled, with the maximum ischemic area of 25.6.+-.2.7% observed in
slice C. E2-CDS treatment reduced ischemic area in all slices
sampled (FIG. 4). The extent of reduction in ischemic area ranged
from 90% in slice A (5 mm posterior of the olfactory bulb) to 45%
in slice C (9 m posterior to the olfactory bulb) (FIG. 4a). The
integrated area under the ischemic lesion curve was 10.1.+-.1.6 for
the vehicle treated rats and 4.5.+-.0.9 for the E2-CDS animals
(Table 2).
[0061] Post-treatment at 90 minutes: Rats were treated with E2-CDS
or HPCD vehicle at 90 minutes after the onset of the occlusion
(FIG. 4b and Table 2). Again, HPCD treated animals showed a large
lesion in all slices sampled, with the maximum ischemic area seen
in slice C (20.5.+-.3.1% of the slice area). Treatment with E2-CDS
reduced the mean ischemic area in all slices examined, however, the
differences were not statistically significant. An evaluation of
the area under the ischemic curve for the two groups revealed that
treatment with E2-CDS reduced the ischemic area by 37.1%, from
8.2.+-.1.7 (HPCD treated animals) to 5.2.+-.1.7 (E2-CDS treated
animals).
Example 3
Estrogen compounds protect brain capillary endothelial cells under
conditions associated with focal ischemic
[0062] Primary rat brain capillary endothelial cells (BCEC)
cultures were prepared following the method of Goldstein, J.
Neurochemistry vol. 25, 715-717, 1975, incorporated herein by
reference.
[0063] Hypoglycemia experiments were undertaken. 17.beta.-estradiol
(2 nm) or control (ethanol vehicle) were added to BCEC cultures.
The glucose concentration of the culture media was then adjusted
from 20 mg % to 200 mg % by adding appropriate amount of
D-(+)-glucose to the glucose-free media and monitored by Glucose
and L-Lactate Analyzer (YSI model 2300 STAT plus, YSI, Inc., Yellow
Springs, Ohio). The hypoglycemic cultures were maintained for 24
hours or 48 hours prior to staining with Trypan blue.
[0064] Anoxia environment was created by placing culture dishes
containing BCEC with or without 2 mn 17.beta.-estradiol in the
Modular Incubator Chamber (Billups-Rothenberg, Inc., Delmar,
Calif.). Nitrogen gas was influxed to replace the oxygen inside the
chamber. The chamber was sealed and placed in the incubator for
four hours for nonhypoglycemic cultures and 2 hours for
hypoglycemic cultures.
[0065] Cell mortality was counted using Trypan blue staining
method. Cell death percentage was calculated as dead cell/alive
cell.times.100%.
[0066] Statistical methods used included two-way analysis of
variance, applied to determine the significance of the difference
among the experimental groups. Kruskal-Wallis nonparametric
analysis was used for data presented as percentage. The
Mann-Whitney U tests were used when Kruskal-Wallis showed
significance among groups. P<0.05 was considered
significant.
[0067] The results are shown in FIGS. 5a and 5b for cells deprived
of glucose. The normal glucose concentration in the media is 200 mg
% and there is little difference in % cell death between cultures
with and without estrogen supplement. However, reduction in medium
glucose content to 100 mg %, 40 mg %, and 20 mg % caused cell
death, and 17.beta.-estradiol saved cell loss by 35.9%, 28.4% and
23.% (p<0.05), respectively, compared with corresponding control
groups not exposed to the estrogen compound. It was further noted
that there were floating cells, which meant more dead cells, in the
control groups than in the estradiol-treated groups. Since these
cells were excluded when counting cell mortality, the protective
effects of estradiol may be underestimated. A similar beneficial
effect was observed over a 24 hour and 48 hour hypoglycemic
treatment (FIGS. 5a and b, respectively).
[0068] Anoxia had a more dramatic effect in cell viability as shown
in FIG. 6 for cells in media containing 200 mg % glucose. Anoxia
induced cell death as much as 48.8% and 39.8% in the control and E2
reduced cell death by 28.4% (p<0.05) at 1 hour and 18.4%
(p<0.05) at 4 hour anoxic insults.
[0069] When cells were exposed to both hypoglycemia (100 mg %
hypoglycemia) and anoxia conditions (2 hours), 17.beta.-estradiol
was effective in protecting cultured BCEC from the cumulative
effect of both conditions (FIG. 7).
[0070] The in vitro assay is representative of events that follow
ischemia such as that induced by MCAO where oxygen and glucose
supplies to the of the blood brain barrier endothelial cells are
reduced.
Example 4
Comparison of post-treatment at 0.5, 1, 2, 3 and 4 hour time
points
[0071] Ovariectomized rats were treated with both an intravenous
injection (100 .mu.g/kg) of HPCD-complexed 17.beta.-estradiol and a
17.beta.-estradiol containing Silastic.RTM. pellet at the times
indicated after the onset of occlusion (FIG. 8). HPCD and
HPCD-encapsulated 17.beta.-estradiol were purchased from Sigma (St.
Louis, Mo.). Ovariectomized, non-treated animals (OVX) and
non-ovariectomized, non-treated animals (INT) were used as controls
(n=12 and n=6, respectively). At 48 hours following MCAO, animals
were sacrificed and ischemic lesion volume was determined by
obtaining brain sections as previously described and staining with
TTC. FIG. 8 shows that significant protection was observed when
drugs were administered at 0.5, 1, 2, or 3 hours
post-occlusion.
Example 5
Delivery of an estrogen compound using an oil vehicle
[0072] To test the kinetics of uptake of an estrogen compound in an
oil vehicle, male Sprague-Dawley rats (Taconic) were given
17.beta.-estradiol in a subcutaneous bolus injection, and drug
levels in the blood were determined over a 25 hour period. The drug
was dissolved in corn oil at 100 .mu.g/ml and the final dosage
delivered was 100 .mu.g/kg. Blood samples were drawn at 30 minutes
prior to drug administration, 30 minutes after drug administration,
4 hours after drug administration and 24 hours after drug
administration. Venous blood was collected into heparinized tubes,
centrifuged and the plasma was collected and frozen. Levels of
17.beta.-estradiol were determined using a commercially supplied
radioimmunoassay kit.
[0073] As shown in FIG. 9, there was a significant, very rapid
uptake of the 17.beta.-estradiol into the bloodstream, peaking in
this experiment at the 30 minute time point (at 4,610 pg/ml). At 4
hours, the levels of circulating 17.beta.-estradiol was 2,004
pg/ml. By 25 hours, 17.beta.-estradiol blood levels had fallen off
to near zero.
[0074] These delivery kinetics indicate that the delivery vehicle
described here in which the estrogen compound was dissolved in oil
and delivered by a single subcutaneous injection into animals
serves the dual purpose of initiating rapid uptake of the compound
into the blood, and providing for sustained delivery of the
compound for hours thereafter.
1TABLE 1 Effects of Pretreatment with 17 .beta.-Estradiol or an
Estradiol-Chemical Delivery System (E2-CDS) on Mortality Following
Middle Cerebral Artery Occlusion. Time of Number of Number of
Number of Planned Animals Animals Animals Treatment Sacrifice
Tested Alive Dead Survival Sham 6 hrs 12 5 7 42 1 Day 18 6 12 33 1
Week 5 1 4 20 Total 35 12 23 35 E2 Implant 6 hrs 6 3 3 50 1 Day 8 8
0 100* 1 Week 4 3 1 75* Total 18 14 4 78* E2-CDS 6 hrs 7 5 2 71 1
Day 8 7 1 88* 1 Week 4 4 0 100 Total 19 16 3 84* *p <0.05 versus
sham control group at each of the time points, as determined by Chi
Squares analysis.
[0075]
2TABLE 2 Effects of Estrogens on the Area Under the Ischemic Lesion
Curve in Ovariectomized Rats. Steroid Treatment Area Under Curve
Sham 24 hour pretreatment 8.1 .+-. 0.8 17.alpha.-estradiol 24 hour
pretreatment 3.7 .+-. 1.3* HPCD Vehicle 40 min post-treatment 10.1
.+-. 1.6 E2-CDS 40 min post-treatment 4.5 .+-. 0.9* HPCD Vehicle 90
min post-treatment 8.2 .+-. 1.7 E2-CDS 90 min post-treatment 5.21
.+-. 1.7 *p <0.02 versus sham control by Students t test
* * * * *