U.S. patent application number 15/398575 was filed with the patent office on 2017-04-27 for novel non-selective cation channel in neuronal cells and methods for treating brain swelling.
The applicant listed for this patent is The United States of America as Represented by the Department of Veterans Affairs, University of Maryland, Baltimore. Invention is credited to Mingkui Chen, J. Marc Simard.
Application Number | 20170112860 15/398575 |
Document ID | / |
Family ID | 38459713 |
Filed Date | 2017-04-27 |
United States Patent
Application |
20170112860 |
Kind Code |
A1 |
Simard; J. Marc ; et
al. |
April 27, 2017 |
NOVEL NON-SELECTIVE CATION CHANNEL IN NEURONAL CELLS AND METHODS
FOR TREATING BRAIN SWELLING
Abstract
The present invention is directed to therapeutic compounds,
treatment methods, and kits affecting the NC.sub.Ca-ATP channel of
neural tissue, including neurons, glia and blood vessels within the
nervous system, and methods of using same. The NC.sub.Ca-ATP
channel is newly expressed in neural tissue following injury such
as ischemia, and is regulated by the sulfonylurea receptor SUR1,
being inhibited by sulfonylurea compounds, e.g., glibenclamide and
tolbutamide, and opened by diazoxide. Antagonists of the
NC.sub.Ca-ATP channel, including SUR1 antagonists, are useful in
the prevention, diminution, and treatment of injured or diseased
neural tissue, including astrocytes, neurons and capillary
endothelial cells, that is due to ischemia, tissue trauma, brain
swelling and increased tissue pressure, or other forms of brain or
spinal cord disease or injury. Agonists of the NC.sub.Ca-ATP
channel may be are useful in the treatment neural tissue where
damage or destruction of the tissue, such as a gliotic capsule, is
desired.
Inventors: |
Simard; J. Marc; (Baltimore,
MD) ; Chen; Mingkui; (Lake Forest, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Maryland, Baltimore
The United States of America as Represented by the Department of
Veterans Affairs |
Baltimore
Washington |
MD
DC |
US
US |
|
|
Family ID: |
38459713 |
Appl. No.: |
15/398575 |
Filed: |
January 4, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14634855 |
Mar 1, 2015 |
|
|
|
15398575 |
|
|
|
|
11359946 |
Feb 22, 2006 |
8980952 |
|
|
14634855 |
|
|
|
|
11229236 |
Sep 16, 2005 |
7872048 |
|
|
11359946 |
|
|
|
|
10391561 |
Mar 20, 2003 |
|
|
|
11359946 |
|
|
|
|
60698272 |
Jul 11, 2005 |
|
|
|
60610758 |
Sep 18, 2004 |
|
|
|
60365933 |
Mar 20, 2002 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/70 20130101;
G01N 27/44791 20130101; A61K 31/565 20130101; A61P 9/10 20180101;
B29L 2031/756 20130101; A61K 31/426 20130101; A61K 31/4439
20130101; A61K 31/7004 20130101; A61K 31/56 20130101; A61K 31/7048
20130101; A61K 9/0019 20130101; B01J 19/0093 20130101; C07K 14/705
20130101; G01N 2500/04 20130101; A61K 31/17 20130101; A61K 31/175
20130101; A61K 31/64 20130101; G01N 33/6872 20130101; A61K 31/00
20130101; Y10S 514/87 20130101; B32B 37/1292 20130101; A61K 31/566
20130101; A61K 31/365 20130101; A61P 43/00 20180101 |
International
Class: |
A61K 31/64 20060101
A61K031/64; A61K 9/00 20060101 A61K009/00; A61K 31/565 20060101
A61K031/565; A61K 31/7004 20060101 A61K031/7004 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
numbers HL082517 and NS048260 awarded by The National Institutes of
Health and a Merit Review grant from the United States Department
of Veterans Affairs. The government has certain rights in the
invention.
Claims
1. A method of ameliorating the effect of a reduction in blood flow
in peri-infarct brain tissue in ischemic disease or injury in a
subject comprising administering to the subject an amount of a
compound effective to inhibit the activity of a NC.sub.Ca-ATP
channel in a neuronal cell, a neuroglia cell, or a neural
endothelial cell.
2. The method of claim 1, where said compound is a compound capable
of effecting hypoglycemia in said subject, further comprising
administration of glucose to said subject effective to at least
partially ameliorate said hypoglycemic effects in said subject.
3. A method of inhibiting neuronal cell swelling in the brain of a
subject, said method comprising administering to the subject a
formulation comprising an effective amount of a compound that
blocks the NC.sub.Ca-ATP channel and a pharmaceutically acceptable
carrier.
4. The method of claim 3, wherein said inhibiting neuronal cell
swelling is further defined as preventing neuronal cell
swelling.
5. The method of claim 3, where said formulation comprises a
compound capable of effecting hypoglycemia in said subject, further
comprising administration of glucose to said subject effective to
at least partially ameliorate said hypoglycemic effects in said
subject.
6. The method of claim 5, wherein said inhibiting neuronal cell
swelling is further defined as preventing neuronal cell
swelling.
7. The method of claim 1, wherein the amelioration of the effect of
a reduction in blood flow comprises a reduction in cytotoxic edema,
ionic edema and/or vasogenic edema.
8. The method of claim 1, wherein the neuroglia cell is selected
from the group consisting of astrocyte, ependymal cell,
oligodentrocyte and microglia.
9. The method of claim 1, wherein the NC.sub.Ca-ATP channel
inhibitor is a type 1 sulfonylurea receptor antagonist selected
from the group consisting of glibenclamide, tolbutamide,
repaglinide, nateglinide, meglitinide, midaglizole, LY397364,
LY389382, glyclazide, glimepiride, estrogen, estradiol, estrone,
estriol, genistein, diethystilbestrol, coumestrol, zearalenone, a
compound that inhibits K.sub.ATP channels.
10. A method of alleviating brain swelling in a subject, comprising
administering to the subject a formulation comprising an effective
amount of a compound that blocks the NC.sub.Ca-ATP channel and a
pharmaceutically acceptable carrier.
11. The method of claim 10, where said formulation is a formulation
capable of effecting hypoglycemia in said subject, further
comprising administration of glucose to said subject effective to
at least partially ameliorate said hypoglycemic effects in said
subject.
12. The method of claim 1, wherein compound effective to inhibit a
NC.sub.Ca-ATP channel is administered alimentarily, parenterally,
topically, mucosally, or by injection into brain parenchema.
13. A pharmaceutical composition comprising a therapeutically
effective amount of a compound that inhibits a NC.sub.Ca-ATP
channel or a pharmaceutically acceptable salt thereof, wherein said
therapeutically effective amount is effective to ameliorate at
least one effect of a reduction in blood flow in peri-infarct brain
tissue in a subject suffering from ischemic disease in the brain or
from brain injury.
14. The pharmaceutical composition of claim 13, wherein the
compound that inhibits a NC.sub.Ca-ATP channel is selected from the
group consisting of glibenclamide, tolbutamide, repaglinide,
nateglinide, meglitinide, midaglizole, LY397364, LY389382,
glyclazide, glimepiride, estrogen, estradiol, estrone, estriol,
genistein, diethystilbestrol, coumestrol, and zearalenone.
15. The pharmaceutical composition of claim 13 wherein the
composition further comprises glucose.
16. The pharmaceutical composition of claim 15, wherein the amount
of said compound that inhibits a NC.sub.Ca-ATP channel or
pharmaceutically acceptable salt thereof is an amount that has a
hypoglycemic effect in a subject to which the pharmaceutical
composition is administered.
17. The pharmaceutical composition of claim 16, wherein the amount
of said glucose is effective to reduce or eliminate a lowering of
the blood glucose concentration by said compound or
pharmaceutically acceptable salt in the subject to which the
pharmaceutical composition is administered.
18. The pharmaceutical composition of claim 13, wherein the
pharmaceutical composition is neuroprotective.
19. The pharmaceutical composition of claim 15, wherein the
pharmaceutical composition is neuroprotective.
20. A method of treating acute cerebral ischemia in a subject
comprising administering to a subject an amount of a compound that
inhibits a NC.sub.Ca-ATP channel or a pharmaceutically acceptable
salt thereof.
21. The method of claim 20, wherein the NC.sub.Ca-ATP channel is
expressed on neuronal cells, neuroglia cells, neural endothelial
cells or a combination thereof.
22. The method of claim 20, wherein said NC.sub.Ca-ATP channel
inhibitor is selected from the group consisting of glibenclamide,
tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,
LY397364, LY389382, glyclazide, glimepiride, estrogen, estradiol,
estrone, estriol, genistein, diethystilbestrol, coumestrol, and
zearalenone.
23. The method of claim 20, wherein the mode of administration of
said NC.sub.Ca-ATP channel inhibitor is selected from the group of
modes of administration consisting of bolus injection, infusion,
and bolus injection in combination with an infusion.
24. The method of claim 20, wherein said NC.sub.Ca-ATP channel
inhibitor is glibenclamide.
25. The method of claim 20, further comprising administering
glucose to said subject.
26. The method of claim 25, wherein said NC.sub.Ca-ATP channel
inhibitor is selected from the group consisting of glibenclamide,
tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,
LY397364, LY389382, glyclazide, glimepiride, estrogen, estradiol,
estrone, estriol, genistein, diethystilbestrol, coumestrol, and
zearalenone.
27. The method of claim 26, wherein the mode of administration of
said NC.sub.Ca-ATP channel inhibitor is selected from the group of
modes of administration consisting of bolus injection, infusion,
and bolus injection in combination with an infusion.
28. The method of claim 20, wherein compound effective to inhibit a
NC.sub.Ca-ATP channel is administered alimentarily, parenterally,
topically, mucosally, or by injection into brain parenchema.
29. A neuroprotective infusion kit comprising a compound that
inhibits a NC.sub.Ca-ATP channel in a neuronal cell, a neuroglia
cell, a neural endothelial cell or a combination thereof and an
intravenous (IV) infusion solution.
30. The neuroprotective infusion kit of claim 29, wherein said IV
infusion solution is an IV infusion solution supplemented with
glucose.
31. The neuroprotective infusion kit of claim 29 further comprising
a neuroprotective bolus kit, wherein the neuroprotective bolus kit
comprises a pre-loaded syringe of a compound that inhibits a
NC.sub.Ca-ATP channel in a neuronal cell, a neuroglia cell, a
neural endothelial cell or a combination thereof within an IV
solution.
32. The kit of claim 31, wherein said IV solution is an IV solution
supplemented with glucose.
33. A method of preventing neural cell swelling in the brain of a
subject, said method comprising administering to the subject a
formulation comprising an effective amount of a compound that
blocks the NC.sub.Ca-ATP channel, glucose, and a pharmaceutically
acceptable carrier.
34. A method of alleviating one or more effects of traumatic brain
injury or cerebral ischemia stemming from neural cell swelling in a
subject, comprising administering to the subject a formulation
comprising an effective amount of a compound that blocks the
NC.sub.Ca-ATP channel, glucose, and a pharmaceutically acceptable
carrier.
35. A method of alleviating one or more effects of traumatic brain
injury or cerebral ischemia in a subject, comprising administering
to the subject a formulation comprising an effective amount of a
sulfonylurea compound and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Non-Provisional
patent application Ser. No. 14/634,855 filed on Mar. 1, 2015, which
is a continuation of U.S. Non-Provisional patent application Ser.
No. 11/359,946 filed on Feb. 22, 2006, issued as U.S. Pat. No.
8,980,952 on Mar. 17, 2015, which is a continuation-in-part of U.S.
Non-Provisional patent application Ser. No. 11/229,236 filed on
Sep. 16, 2005, issued as U.S. Pat. No. 7,872,048 on Jan. 18, 2011,
which claims priority to U.S. Provisional Patent Application No.
60/698,272 filed on Jul. 11, 2005 and to U.S. Provisional
Application No. 60/610,758 filed on Sep. 18, 2004, and U.S.
Non-Provisional patent application Ser. No. 11/359,946 is a
continuation-in part of U.S. Non-Provisional application Ser. No.
10/391,561, now abandoned, filed on Mar. 20, 2003, which claims
priority to U.S. Provisional Patent Application Ser. 60/365,933
filed on Mar. 20, 2002, all of which are hereby incorporated by
reference herein in their entirety.
FIELD OF THE INVENTION
[0003] The present invention generally regards the fields of cell
biology, neurophysiology, and medicine. In particular, the present
invention relates to a novel non-selective monovalent cationic ATP
sensitive ion channel (hereinafter referred to as the NC.sub.Ca-ATP
channel) that is coupled to sulfonylurea receptor type 1 in neural
cells, including astrocytes, neurons and neural endothelial cells,
to compounds and treatments that may modulate NC.sub.Ca-ATP channel
activity, and to kits including compounds useful for treatment of
disease or injury conditions such as stroke or brain trauma.
BACKGROUND OF THE INVENTION
[0004] Injury to the nervous system has serious consequences.
Following traumatic brain injury and stroke, the normal response of
the surrounding brain is to mount a cellular response that includes
formation of reactive astrocytes that are believed to be important
to "contain" and "clean-up" the injury site. Swelling of neural
cells is part of the cytotoxic or cell swelling response that
characterizes brain damage in cerebral ischemia and traumatic brain
injury, and is a major cause of morbidity and mortality. See, Staub
et al., 1993; Kimelberg et al., 1995. A number of mediators have
been identified that initiate swelling of neural cells, including
elevation of extracellular K.sup.+, acidosis, release of
neurotransmitters and free fatty acids. See, Kempski et al., 1991;
Rutledge and Kimelberg, 1996; Mongin et al., 1999. Cytotoxic edema
is a well-recognized phenomenon clinically that causes brain
swelling, which worsens outcome and increases morbidity and
mortality in brain injury and stroke.
[0005] Mechanisms underlying apoptotic death of reactive astrocytes
and other cells have been studied. See, Tanaka et al., 2000; Yu et
al., 2001. The mechanisms responsible for necrotic cell death of
astrocytes, neurons and neural endothelial cells have not been
characterized. Apoptotic cell death is preceded by cell shrinkage
and net loss of K.sup.+. See, Yu et al., 1997; Yu et al., 1999. By
contrast, in necrotic cell death, the plasma membrane is ruptured,
causing cytosolic contents to be released and thereby triggering
tissue inflammation. See, Leist and Nicotera, 1997. Necrotic cell
death may be more deleterious to nearby viable tissues, given the
secondary inflammatory damage that is initiated.
[0006] Necrotic cell death is initiated by osmotic swelling
following influx of Na.sup.+, the major extracellular osmolyte. In
most cell types, accumulation of Na.sup.+ intracellularly is
regarded as a passive process that does not require activation of
specific effectors but that is due instead to defective outward Na'
pumping under conditions of low intracellular adenosine
triphosphate concentration ([ATP]i). See, Leist and Nicotera, 1997;
Trump et al., 1997. Cell blebbing or swelling, an indication of
intracellular Na' overload, is generally regarded as an early sign
of necrotic cell death. See, Leist and Nicotera, 1997; Majno and
Joris, 1995.
[0007] Inhibition of ATP synthesis or ATP depletion also causes
neural cell swelling, blebbing and, if sufficiently severe, plasma
membrane disruption and cell death. See, Jurkowitz-Alexander et
al., 1993. The mechanisms of neural cell swelling associated with
ATP-depletion remained incompletely characterized. See, Lomneth and
Gruenstein, 1989; Juurlink et al., 1992; Rose et al., 1998.
[0008] One potential mechanism would be changes in Na.sup.+ and
K.sup.+ concentration due to inhibition of the
Na.sup.+/K.sup.+-ATPase pump. However, an equivalent degree of
osmotic swelling induced by ouabain-mediated inhibition of the
Na.sup.+/K.sup.+-ATPase pump in neural cells does not produce large
depolarization, blebbing or cell death. See, Jurkowitz-Alexander et
al., 1992; Brismar and Collins, 1993. Failure of the
Na.sup.+/K.sup.+-ATPase pump, therefore, is not the mechanism
critical to swelling of neural cells. None of these studies have
identified the cellular mechanism instrumental in the cell swelling
that is associated with brain damage in cerebral ischemia and
traumatic brain injury and spinal cord injury.
[0009] One subtype of ATP sensitive cation channel is a
non-selective cation channel, that is sensitive to Ca.sup.2+ and
ATP. More specifically, some non-selective cation channels are
activated by intracellular Ca.sup.2+ ([Ca.sup.2+].sub.i) and
inhibited by intracellular ATP ([ATP].sub.i). Although Ca.sup.2+-
and ATP-sensitive cation channels had been identified in a number
of non-neural cell types, they have not been identified in
astrocytes or any other neural cells. See, Sturgess et al., 1987;
Gray and Argent, 1990; Rae et al., 1990; Champigny et al., 1991;
Popp and Gogelein, 1992; Ono et al., 1994, each of which is hereby
incorporated by reference in its entirety. These non-astrocyte
channels comprise a heterogeneous group with incompletely defined
characteristics. They exhibit single-channel conductances in the
range of 25-35 pS, discriminate poorly between Na.sup.+ and
K.sup.+, are impermeable to anions, for the most part impermeable
to divalent cations, and they are blocked by similar concentrations
of the adenine nucleotides ATP, ADP and AMP on the cytoplasmic
side. The function of these non-selective ATP sensitive cation
channels in these non-neural cell types remains enigmatic, in part
because unphysiological concentrations of Ca.sup.2+ are generally
required for channel activation.
[0010] Another subtype of ATP sensitive cation channel is the
ATP-sensitive potassium channel (K.sub.ATP channels) in pancreatic
.beta. cells. One class of insulin secretagogues, the antidiabetic
sulfonylureas, is used to inhibit these K.sub.ATP channels and
stimulate insulin release in diabetes mellitus. See, Lebovitz,
1985. Antidiabetic sulfonylureas mediate their effect on K.sub.ATP
channels via a high affinity sulfonylurea receptor (SUR). See,
Panten et. al., 1989; Aguilar-Bryan et. al., 1995. Several isoforms
of the SUR, termed SUR1, SUR2A, SUR2B, and SUR2C, have been
identified and cloned. See, Aguilar-Bryan et. al., 1995; Inagaki
et. al., 1996; Isomoto et. al., 1996; Lawson, 2000. These receptors
belong to the ATP-binding cassette (ABC) transporter family, of
which the cystic fibrosis transmembrane conductance regulator
(CFTR), another ion channel modulator, is also a member. See,
Higgins, 1992; Aguilar-Bryan et. al., 1995. Notably, the CFTR has
major therapeutic importance, since its genetic absence causes
cystic fibrosis, a fatal disease.
[0011] The sulfonylurea receptor imparts sensitivity to
antidiabetic sulfonylureas such as glibenclamide and tolbutamide.
Also, SUR is responsible for activation of the potassium channel by
a chemically diverse group of agents termed K.sup.+ channel openers
(SUR-activators), such as diazoxide, pinacidil, and cromakalin.
See, Aguilar-Bryan et. al., 1995; Inagaki et. al., 1996; Isomoto
et. al., 1996; Nichols et. al., 1996; Shyng et. al., 1997b. In
various tissues, molecularly distinct SURs are coupled to distinct
channel moieties to form different K.sub.ATP channels with
distinguishable physiological and pharmacological characteristics.
The K.sub.ATP channel in pancreatic .beta. cells is formed from
SUR1 linked with a K.sup.+ channel, whereas the cardiac and smooth
muscle K.sub.ATP channels are formed from SUR2A and SUR2B,
respectively, linked to K.sup.+ channels. See, Fujita and Kurachi,
2000.
Gliotic Capsule
[0012] The gliotic capsule that forms around a "foreign body" in
the brain is an important, albeit neglected, biological system. On
the one hand, the gliotic capsule represents the response of the
brain to an injurious stimulus--an attempt by the brain to wall
off, isolate, dispose of, and otherwise protect itself from the
foreign body. On the other hand, the gliotic capsule forms a
potentially harmful mass of tissue from which originates edema
fluid that contributes to brain swelling, and whose constituent
cells undergo cytotoxic edema, which adds further to brain
swelling. Also, the gliotic capsule protects foreign cells from
immunologic surveillance.
[0013] The essential elements involved in formation of a gliotic
capsule appear to be uniform in many types of CNS pathology, be it
a traumatically implanted foreign body, a metastatic tumor, a brain
abscess, or infarcted necrotic tissue following a stroke. First,
microglia and astrocytes become activated near the site of injury,
with large, stellate-shaped GFAP-positive reactive astrocytes
forming the most prominent cellular component of the response.
Secondly, the foreign nature of the entity is recognized, and the
response is initiated to surround and contain it. Although the
concept of "foreign body" encompasses a large variety of
pathological conditions, the responses in most cases bear a great
deal of similarity to one another.
[0014] The interface between the foreign body and the gliotic
capsule, referred to as the inner zone of the gliotic capsule,
appears to be of great importance in determining the overall
response to injury.
[0015] Thus, a need exists for a physiological target instrumental
in the cell swelling that is associated with brain damage in
cerebral ischemia and traumatic brain injury and in the consequent
morbidity and mortality. There is also a need for specific
treatments for the cytotoxic edema that causes brain swelling,
which worsens outcome and increases morbidity and mortality in
brain injury and stroke. Other and further objects, features, and
advantages will be apparent from the following description of the
presently preferred embodiments of the invention, which are given
for the purpose of disclosure.
SUMMARY OF THE INVENTION
[0016] The present invention is based, in part, on the discovery of
a specific channel, the NC.sub.Ca-ATP channel, which, for example,
is expressed in neurons, glia and neural endothelial cells after
brain trauma. This unique non-selective cation channel is activated
by intracellular calcium and blocked by intracellular ATP
(NC.sub.Ca-ATP channel), and can be expressed in neuronal cells,
neuroglia cells (also termed glia, or glial cells, e.g., astrocyte,
ependymal cell, oligodentrocyte and microglia) or neural
endothelial cells (e.g., capillary endothelial cells) in which the
cells have been or are exposed to a traumatic insult, for example,
an acute neuronal insult (e.g., hypoxia, ischemia, tissue
compression, mechanical distortion, cerebral edema or cell
swelling), toxic compounds or metabolites, an acute injury, cancer,
brain abscess, etc.
[0017] More specifically, the NC.sub.Ca-ATP channel of the present
invention has a single-channel conductance to potassium ion
(K.sup.+) between 20 and 50 pS. The NC.sub.Ca-ATP channel is also
stimulated by Ca.sup.2+ on the cytoplasmic side of the cell
membrane in a physiological concentration range, where
concentration range is from 10.sup.-8 to 10.sup.-5 M. The
NC.sub.Ca-ATP channel is also inhibited by cytoplasmic ATP in a
physiological concentration range, where the concentration range is
from 10.sup.-1 to 10 M. The NC.sub.Ca-ATP channel is also permeable
to the following cations; K.sup.+, Cs.sup.+, Li.sup.+, Na.sup.+; to
the extent that the permeability ratio between any two of the
cations is greater than 0.5 and less than 2.
[0018] More particularly, the present invention relates to the
regulation and/or modulation of this NC.sub.Ca-ATP channel and how
its modulation can be used to treat various diseases and/or
conditions, for example acute neuronal insults (e.g., stroke, an
ischemic/hypoxic insult, a traumatic or mechanical injury) and
diseases or conditions leading to formation of a gliotic capsule.
Yet further, the present invention relates to the regulation and/or
modulation of this NC.sub.Ca-ATP channel and its role in
maintaining or disrupting the integrity of the gliotic capsule. The
modulation and/or regulation of the channel results from
administration of an activator or agonist of the channel or an
antagonist or inhibitor of the channel. Thus, depending upon the
disease, a composition (an antagonist or inhibitor) is administered
to block or inhibit the channel to prevent cell death, for example
to treat cerebral edema that results from ischemia due to tissue
trauma or to increased tissue pressure. In these instances the
channel is blocked to prevent or reduce or modulate depolarization
of the cells. Alternatively, in order to treat or disrupt a gliotic
capsule, it is desirable to open or activate the channel by
administering an agonist or activator compound to cause cell
depolarization resulting in cell death of diseased target
cells.
[0019] In one aspect, the present invention provides novel methods
of treating a patient comprising administering a therapeutic
compound that targets a unique non-selective cation channel
activated by intracellular calcium and blocked by intracellular ATP
(NC.sub.Ca-ATP channel). In specific embodiments, the therapeutic
compound may be an antagonist, and uses thereof in therapies, such
as treatment of cerebral ischemia or edema, benefiting from
blocking and/or inhibiting the NC.sub.Ca-ATP channel. In further
embodiments, where death of cells expressing the NC.sub.Ca-ATP
channel is desired for therapeutic purposes, the therapeutic
compound may be an agonist. Compositions comprising agonists and/or
antagonists of the NC.sub.Ca-ATP channel are also contemplated.
[0020] The invention also encompasses the use of such compounds and
compositions that modulate NC.sub.Ca-ATP channel activity to treat
brain swelling. For example, the present invention relates to
methods for the treatment of brain swelling that results from brain
trauma or cerebral ischemia, resulting in neural cell swelling,
cell death, and an increase in transcapillary formation of ionic
and vasogenic edema. Further provided is a method of preventing
brain swelling and the resulting brain damage through the
therapeutic use of antagonists to the NC.sub.Ca-ATP channel. In one
embodiment, the therapeutic antagonist can be administered to or
into the brain. Such administration to the brain includes injection
directly into the brain, particularly in the case where the brain
has been rendered accessible to injection due to trauma to the
skull, for example. The invention further provides the therapeutic
use of sulfonylurea compounds as antagonists to the NC.sub.Ca-ATP
channel to prevent cell swelling in brain. In one embodiment the
sulfonylurea compound is glibenclamide. In another embodiment, the
sulfonylurea compound is tolbutamide, or any of the other compounds
that have been found to promote insulin secretion by acting on KATP
channels in pancreatic .beta. cells, as listed elsewhere
herein.
[0021] The invention also encompasses agonists and antagonists of
the NC.sub.Ca-ATP channel, including small molecules, large
molecules, and antibodies, as well as nucleotide sequences that can
be used to inhibit NC.sub.Ca-ATP channel gene expression (e.g.,
antisense and ribozyme molecules). An antagonist of the
NC.sub.Ca-ATP channel includes one or more compounds capable of (1)
blocking the channel; (2) preventing channel opening; (3) reducing
the magnitude of membrane current through the channel; (4)
inhibiting transcriptional expression of the channel; and/or (5)
inhibiting post-translational assembly and/or trafficking of
channel subunits.
[0022] The invention relates to assays designed to screen for
compounds or compositions that modulate the NC.sub.Ca-ATP channel,
particularly compounds or compositions that act as antagonists of
the channel, and thereby modulate neural cell swelling and the
concomitant brain swelling. To this end, cell-based assays or
non-cell based assays can be used to detect compounds that interact
with, e.g., bind to, the outside (i.e., extracellular domain) of
the NC.sub.Ca-ATP channel and/or its associated SUR1 regulatory
subunit. The cell-based assays have the advantage in that they can
be used to identify compounds that affect NC.sub.Ca-ATP channel
biological activity (i.e., depolarization). The invention also
provides a method of screening for and identifying antagonists of
the NC.sub.Ca-ATP channel, by contacting neural cells with a test
compound and determining whether the test compound inhibits the
activity of the NC.sub.Ca-ATP channel. In one embodiment, methods
for identifying compounds that are antagonists of the NC.sub.Ca-ATP
are provided. In one embodiment, therapeutic compounds of the
present invention, including NC.sub.Ca-ATP antagonists, are
identified by the compound's ability to block the open channel or
to prevent channel opening, such as by quantifying channel function
using electrophysiological techniques to measure membrane current
through the channel, for example. NC.sub.Ca-ATP antagonists include
compounds that are NC.sub.Ca-ATP channel inhibitors, NC.sub.Ca-ATP
channel blockers, SUR1 antagonists, SUR1 inhibitors, and/or
compounds that reduce the magnitude of membrane current through the
channel, for example. In this embodiment, channel function can be
measured in a preparation of neural cells from a human or animal,
and the test compound can be brought into contact with the cell
preparation by washing it over the cell preparation in solution.
The invention further provides a method of screening for
sulfonylurea compounds that may act as antagonists of the
NC.sub.Ca-ATP channel.
[0023] The present invention relates to drug screening assays to
identify compounds for the treatment of brain swelling, such as the
swelling that occurs after brain injury or cerebral ischemia by
using the NC.sub.Ca-ATP channel as a target. The invention also
relates to compounds that modulate neural cell swelling via the
NC.sub.Ca-ATP channel. The present invention also relates to the
treatment of brain swelling by targeting the NC.sub.Ca-ATP
channel.
[0024] The present invention is also directed to purified
compositions comprising a novel Ca.sup.2+-activated, [ATP].sub.i-
sensitive nonspecific cation channel. In a preferred embodiment of
the present invention, the compositions comprise mammalian neural
cells or membrane preparations expressing the NC.sub.Ca-ATP
channel, most preferably wherein the mammalian neural cells are
freshly isolated reactive astrocytes, neurons or neural endothelial
cells. A preferred example of such a purified composition
comprising the NC.sub.Ca-ATP channel is a membrane preparation
derived from native reactive astrocytes. As demonstrated herein,
when neural cells expressing the NC.sub.Ca-ATP channel are depleted
of intracellular ATP, the NC.sub.Ca-ATP channel opens and the cells
swell and die. However, if the NC.sub.Ca-ATP channel is blocked on
such cells, the cells do not swell and die. The invention is also
based, in part, on the discovery that the NC.sub.Ca-ATP channel is
regulated by a type 1 sulfonylurea receptor, and that antagonists
of this receptor are capable of blocking the NC.sub.Ca-ATP channel
and inhibit neural cell swelling.
[0025] The composition(s) of the present invention may be delivered
alimentarily or parenterally. Examples of alimentary administration
include, but are not limited to orally, buccally, rectally, or
sublingually. Parenteral administration can include, but are not
limited to intramuscularly, subcutaneously, intraperitoneally,
intravenously, intratumorally, intraarterially, intraventricularly,
intracavity, intravesical, intrathecal, or intrapleural. Other
modes of administration may also include topically, mucosally,
transdermally, direct injection into the brain parenchyma.
[0026] An effective amount of an agonist or antagonist of
NC.sub.Ca-ATP channel that may be administered to a cell includes a
dose of about 0.0001 nM to about 2000 .mu.M, for example. More
specifically, doses of an agonist to be administered are from about
0.01 nM to about 2000 .mu.M; about 0.01 .mu.M to about 0.05 .mu.M;
about 0.05 .mu.M to about 1.0 .mu.M; about 1.0 .mu.M to about 1.5
.mu.M; about 1.5 .mu.M to about 2.0 .mu.M; about 2.0 .mu.M to about
3.0 .mu.M; about 3.0 .mu.M to about 4.0 .mu.M; about 4.0 .mu.M to
about 5.0 .mu.M; about 5.0 .mu.M to about 10 .mu.M; about 10 .mu.M
to about 50 .mu.M; about 50 .mu.M to about 100 .mu.M; about 100
.mu.M to about 200 .mu.M; about 200 .mu.M to about 300 .mu.M; about
300 .mu.M to about 500 .mu.M; about 500 .mu.M to about 1000 .mu.M;
about 1000 .mu.M to about 1500 .mu.M and about 1500 .mu.M to about
2000 .mu.M, for example. Of course, all of these amounts are
exemplary, and any amount in-between these points is also expected
to be of use in the invention.
[0027] An effective amount of an agonist or antagonist of the
NC.sub.Ca-ATP channel or related-compounds thereof as a treatment
varies depending upon the host treated and the particular mode of
administration. In one embodiment of the invention the dose range
of the agonist or antagonist of the NC.sub.Ca-ATP channel or
related-compounds thereof will be about 0.01 .mu.g/kg body weight
to about 20,000 .mu.g/kg body weight. The term "body weight" is
applicable when an animal is being treated. When isolated cells are
being treated, "body weight" as used herein should read to mean
"total cell body weight". The term "total body weight" may be used
to apply to both isolated cell and animal treatment. All
concentrations and treatment levels are expressed as "body weight"
or simply "kg" in this application are also considered to cover the
analogous "total cell body weight" and "total body weight"
concentrations. However, those of skill will recognize the utility
of a variety of dosage range, for example, 0.01 .mu.g/kg body
weight to 20,000 .mu.g/kg body weight, 0.02 .mu.g/kg body weight to
15,000 .mu.g/kg body weight, 0.03 .mu.g/kg body weight to 10,000
.mu.g/kg body weight, 0.04 .mu.g/kg body weight to 5,000 .mu.g/kg
body weight, 0.05 .mu.g/kg body weight to 2,500 .mu.g/kg body
weight, 0.06 .mu.g/kg body weight to 1,000 .mu.g/kg body weight,
0.07 .mu.g/kg body weight to 500 .mu.g/kg body weight, 0.08
.mu.g/kg body weight to 400 .mu.g/kg body weight, 0.09 .mu.g/kg
body weight to 200 .mu.g/kg body weight or 0.1 .mu.g/kg body weight
to 100 .mu.g/kg body weight. Further, those of skill will recognize
that a variety of different dosage levels will be of use, for
example, 0.0001 .mu.g/kg, 0.0002 .mu.g/kg, 0.0003 .mu.g/kg, 0.0004
.mu.g/kg, 0.005 .mu.g/kg, 0.0007 .mu.g/kg, 0.001 .mu.g/kg, 0.1
.mu.g/kg, 1.0 .mu.g/kg, 1.5 .mu.g/kg, 2.0 .mu.g/kg, 5.0 .mu.g/kg,
10.0 .mu.g/kg, 15.0 .mu.g/kg, 30.0 .mu.g/kg, 50 .mu.g/kg, 75
.mu.g/kg, 80 .mu.g/kg, 90 .mu.g/kg, 100 .mu.g/kg, 120 .mu.g/kg, 140
.mu.g/kg, 150 .mu.g/kg, 160 .mu.g/kg, 180 .mu.g/kg, 200 .mu.g/kg,
225 .mu.g/kg, 250 .mu.g/kg, 275 .mu.g/kg, 300 .mu.g/kg, 325
.mu.g/kg, 350 .mu.g/kg, 375 .mu.g/kg, 400 .mu.g/kg, 450 .mu.g/kg,
500 .mu.g/kg, 550 .mu.g/kg, 600 .mu.g/kg, 700 .mu.g/kg, 750
.mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg,
12 mg/kg, 15 mg/kg, 20 mg/kg, and/or 30 mg/kg. In particular
embodiments, there may be dosing of from very low ranges (e.g. 1
mg/kg/day or less; 5 mg/kg bolus; or 1 mg/kg/day) to moderate doses
(e.g. 2 mg bolus, 15 mg/day) to high doses (e.g. 5 mg bolus, 30-40
mg/day; and even higher). Of course, all of these dosages are
exemplary, and any dosage in-between these points is also expected
to be of use in the invention. Any of the above dosage ranges or
dosage levels may be employed for an agonist or antagonist, or
both, of NC.sub.Ca-ATP channel or related-compounds thereof.
[0028] The NC.sub.Ca-ATP channel is blocked by antagonists of type
1 sulfonylurea receptor (SUR1) and is opened by SUR1 activators.
More specifically, the antagonists of type 1 sulfonylurea receptor
(SUR1) include blockers of K.sub.ATP channels and the SUR1
activators include activators of K.sub.ATP channels. The channel
can be inhibited by an NC.sub.Ca-ATP channel inhibitor, an
NC.sub.Ca-ATP channel blocker, a type 1 sulfonylurea receptor
(SUR1) antagonist, SUR1 inhibitor, or a compound capable of
reducing the magnitude of membrane current through the channel.
More specifically, the SUR1 antagonist may be selected from the
group consisting of glibenclamide, tolbutamide, repaglinide,
nateglinide, meglitinide, midaglizole, LY397364, LY389382,
gliclazide (also known in the art as glyclazide), glimepiride,
estrogen, estrogen related-compounds (estradiol, estrone, estriol,
genistein, non-steroidal estrogen (e.g., diethystilbestrol),
phytoestrogen (e.g., coumestrol), zearalenone, etc.), and compounds
known to inhibit or block K.sub.ATP channels. MgADP can also be
used to inhibit the channel. Other compounds that can be used to
block or inhibit K.sub.ATP channels include, but are not limited to
tolbutamide, glyburide (1[p-2[5-chloro-O-anisamido)ethyl] phenyl]
sulfonyl]-3-cyclohexyl-3-urea); chlopropamide
(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide
(1-cyclohexyl-3[[p-[2(5-methylpyrazine carboxamido)ethyl] phenyl]
sulfonyl] urea); or
tolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]
carbonyl]-4-methyl). In additional embodiments, non-sulfonyl urea
compounds, such as 2, 3-butanedione and 5-hydroxydecanoic acid,
quinine, and therapeutically equivalent salts and derivatives
thereof, may be employed in the invention.
[0029] The channel is expressed on neuronal cells, neuroglia cells,
neural epithelial cells, neural endothelial cells, or a combination
thereof, for example. The inhibitor blocks the influx of Na.sup.+
into the cells thereby preventing depolarization of the cells.
Inhibition of the influx of Na.sup.+ into the cells thereby at
least prevents or reduces cytotoxic edema and/or ionic edema, and
prevents or reduces hemorrhagic conversion. Thus, this treatment
reduces cell death or necrotic death of neuronal and/or neural
endothelial cells.
[0030] In certain embodiments, the amount of the SUR1 antagonist
administered to the subject is in the range of about 0.0001
.mu.g/kg/day to about 20 mg/kg/day, about 0.01 .mu.g/kg/day to
about 100 .mu.g/kg/day, or about 100 .mu.g/kg/day to about 20
mg/kg/day. Still further, the SUR1 antagonist may be administered
to the subject in the from of a treatment in which the treatment
may comprise the amount of the SUR1 antagonist or the dose of the
SUR1 antagonist that is administered per day (1, 2, 3, 4, etc.),
week (1, 2, 3, 4, 5, etc.), month (1, 2, 3, 4, 5, etc.), etc.
Treatments may be administered such that the amount of SUR1
antagonist administered to the subject is in the range of about
0.0001 .mu.g/kg/treatment to about 20 mg/kg/treatment, about 0.01
.mu.g/kg/treatment to about 100 .mu.g/kg/treatment, or about 100
.mu.g/kg/treatment to about 20 mg/kg/treatment.
[0031] Another embodiment of the present invention comprises a
method of reducing mortality of a subject suffering from a stroke
comprising administering to the subject a compound effective to
inhibit NC.sub.Ca-ATP channels in a neuronal cell, a neuroglia
cell, a neural endothelial cell or a combination thereof. The
compound reduces stroke size and reduces edema located in the
peri-infarct tissue. The compound can be administered alimentary
(e.g., orally, buccally, rectally or sublingually) or parenterally
(e.g., intravenously, intradermally, intramuscularly,
intraarterially, intrathecally, subcutaneously, intraperitoneally,
intraventricularly) and/or topically (e.g., transdermally),
mucosally, or by direct injection into the brain parenchyma.
[0032] Still further, another embodiment comprises a method of
reducing edema in a peri-infarct tissue area of a subject
comprising administering to the subject a compound effective to
inhibit NC.sub.Ca-ATP channels in a neuronal cell, a neuroglial
cell, a neural endothelial cell, or a combination thereof.
[0033] Further embodiments comprises a method of treating a subject
at risk for developing a stroke comprising administering to the
subject a compound effective to inhibit a NC.sub.Ca-ATP channel in
neuronal cell, a neuroglia cell, a neural endothelial cell or a
combination thereof.
[0034] In certain embodiments, the subject is undergoing treatment
for a cardiac condition, thus the condition increases the subjects
risk for developing a stroke. The treatment, for example, may
comprise the use of thrombolytic agents to treat myocardial
infarctions. Still further, the subject may be at risk for
developing a stroke because the subject suffers from atrial
fibrillation or a clotting disorder. Other subjects that are at
risk for developing a stroke include subjects that are at risk of
developing pulmonary emboli, subjects undergoing surgery (e.g.,
vascular surgery or neurological surgery), or subjects undergoing
treatments that increase their risk for developing a stroke, for
example, the treatment may comprise cerebral/endovascular
treatment, angiography or stent placement. In other embodiments,
the subject may be undergoing treatment for vascular disease that
could place the spinal cord at risk for ischemia, such as surgery
requiring aortic cross-clamping, surgery for abdominal aortic
aneurysm, etc. In other embodiments, the patient may be undergoing
surgery for a spinal or spinal cord condition, including
discectomy, fusion, laminectomy, extradural or intradural surgery
for tumor or mass etc., that would place the spinal cord at risk of
injury. In some embodiments of the invention, the subject has a
chronic condition, whereas in other embodiments of the invention,
the subject does not have a chronic condition, such as a short-term
condition.
[0035] Another embodiment of the present invention comprises a
method of treating a subject at risk for developing cerebral edema
comprising administering to the subject a compound effective to
inhibit a NC.sub.Ca-ATP channel in a neuronal cell, a neuroglia
cell, a neural endothelial cell or a combination thereof. The
subject at risk may be suffering from an arterior-venous
malformation, or a mass-occupying lesion (e.g., hematoma) or may be
involved in activities that have an increased risk of brain
trauma.
[0036] Another embodiment of the present invention comprises a
composition comprising a membrane preparation derived from a neural
endothelial cell expressing a NC.sub.Ca-ATP channel, wherein
channel is blocked by antagonists of type 1 sulfonylurea receptor
(SUR1) and opened by SUR1 activators. More specifically, the
channel has the following characteristics: (a) it is a 35 pS type
channel; (b) it is stimulated by cytoplasmic Ca.sup.2+ in the
concentration range from about 10.sup.-8 to about 10.sup.-5 M; (c)
it opens when cytoplasmic ATP is less than about 0.8 .mu.M; and (d)
it is permeable to the monovalent cations K.sup.+, Cs.sup.+,
Li.sup.+ and Na.sup.+.
[0037] In further embodiments, the compound that inhibits the
NC.sub.Ca-ATP channel can be administered in combination with a
thrombolytic agent (e.g., tissue plasminogen activator (tPA),
urokinase, prourokinase, streptokinase, anistreplase, reteplase,
tenecteplase), an anticoagulant or antiplatelet (e.g., aspirin,
warfarin or coumadin), statins, diuretics, vasodilators (e.g.,
nitroglycerin), mannitol, diazoxide or similar compounds that
stimulate or promote ischemic precondition.
[0038] Yet further, another embodiment of the present invention
comprises a pharmaceutical composition comprising a thrombolytic
agent (e.g., tissue plasminogen activator (tPA), urokinase,
prourokinase, streptokinase, anistreplase, reteplase,
tenecteplase), an anticoagulant or antiplatelet (e.g., aspirin,
warfarin or coumadin), statins, diuretics, vasodilators, mannitol,
diazoxide or similar compounds that stimulate or promote ischemic
precondition or a pharmaceutically acceptable salt thereof and a
compound that inhibits a NC.sub.Ca-ATP channel or a
pharmaceutically acceptable salt thereof. This pharmaceutical
composition can be considered neuroprotective, in specific
embodiments. For example, the pharmaceutical composition comprising
a combination of the thrombolytic agent and a compound that
inhibits a NC.sub.Ca-ATP channel is neuroprotective because it
increases the therapeutic window for the administration of the
thrombolytic agent by several hours; for example the therapeutic
window for administration of thrombolytic agents may be increased
by several hours (e.g. about 4-about 8 hrs) by co-administering
antagonist of the NC.sub.Ca-ATP channel.
[0039] Still further, another embodiment comprises a method of
treating acute cerebral ischemia in a subject comprising
administering to a subject an amount of a thrombolytic agent or a
pharmaceutically acceptable salt thereof in combination with an
amount of a compound that inhibits a NC.sub.Ca-ATP channel or a
pharmaceutically acceptable salt thereof. In certain embodiments,
the thrombolytic agent is a tissue plasminogen activator (tPA),
urokinase, prourokinase, streptokinase, anistreplase, reteplase,
tenecteplase or any combination thereof. The SUR1 antagonist can be
administered by any standard parenteral or alimentary route, for
example the SUR1 antagonist may be administered as a bolus
injection or as an infusion or a combination thereof.
[0040] Another embodiment of the present invention comprises a
method of disrupting a gliotic capsule, such as to disrupt the
integrity of the tumor-brain barrier surrounding a tumor in the
brain of a subject comprising administering to the subject a
compound effective to activate a NC.sub.Ca-ATP channel in a
neuronal cell, or a neuroglia cell, a neural endothelial cell or a
combination thereof.
[0041] Where destruction of cells expressing the NC.sub.Ca-ATP
channel is desired, an SUR1 activator or agonist may be
administered, for example, to reduce or remove a gliotic capsule.
The activator compound or agonist can be a type 1 sulfonylurea
receptor agonist. For example, agonists that can be used in the
present invention include, but are not limited to agonist of SUR1,
for example, diazoxide, pinacidil, P1075, cromakalin, or
combinations thereof. Other agonists can include, but are not
limited to diazoxide derivatives, for example
3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide
(NNC 55-9216),
6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide
(BPDZ 154), 7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine
1,1-dioxide (BPDZ 73),
6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine
1,1-dioxide (NNC
55-0118)4,6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-th-
iadiazine 1,1-dioxide (NN414),
3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxide (BPDZ 44),
3-(1',2',2'-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine
1,1-dioxide (BPDZ 62), 3-(1',2',2'-trimethylpropyl)amine-4H-pyrido
(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),
2-alkyl-3-alkylamino-2H-benzo- and
2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxides,
6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine
1,1-dioxide derivatives, 4-N-Substituted and -unsubstituted
3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxides, or combinations thereof. In addition, other
compounds, including 6-chloro-2-methylquinolin-4(1H)-one (HEI 713)
and LN 533021, as well as the class of drugs, arylcyanoguanidines,
are known activators or agonist of SUR1. Other compounds that can
be used include compounds known to activate K.sub.ATP channels.
[0042] Still further, another embodiment of the present invention
comprises a method of inducing cell death of one or more of a
neuronal or a neuroglia cell or a neural endothelial cell
comprising administering to the cell a compound effective to
activate a NC.sub.Ca-ATP channel in the cell. Activation of the
NC.sub.Ca-ATP channel results in an influx of sodium ions
(Na.sup.+) causing depolarization of the cell. The influx of
Na.sup.+ alters the osmotic gradient causing an influx of water
into the cell that leads to cytotoxic edema ultimately resulting in
necrotic cell death.
[0043] Yet further, another embodiment of the present invention
comprises a method of maintaining the integrity of the gliotic
capsule surrounding brain abscess of a subject comprising
administering to the subject a compound effective to inhibit and/or
block at least one NC.sub.Ca-ATP channel in a neuronal cell, a
neuroglia cell, a neural endothelial cell or a combination
thereof.
[0044] Still further, another method of the present invention
comprises a method of diagnosing neuronal cell edema and/or
cytotoxic damage in the brain comprising: labeling an antagonist of
SUR1; administering the labeled antagonist of SUR1 to a subject;
measuring the levels of labeled antagonist of SUR1 in the brain of
the subject, wherein the presence of labeled antagonist of SUR1
indicates neuronal cell edema and/or cytotoxic damage in the
brain.
[0045] In further embodiments, the methods can comprise a method of
determining the penumbra following a stroke comprising: labeling an
antagonist of SUR1; administering the labeled antagonist of SUR1 to
a subject; visualizing the labeled antagonist of SUR1 in the brain
of the subject, wherein the presence of labeled antagonist of SUR1
indicates the penumbra.
[0046] Yet further, the present invention comprises a method
monitoring stroke neural disease comprising: labeling an antagonist
of SUR1; administering the labeled antagonist of SUR1 to a subject;
visualizing the labeled antagonist of SUR1 in the brain of the
subject, wherein the presence of labeled antagonist of SUR1
indicates the progression of the disease. In certain embodiments,
the step of visualizing is performed daily to monitor the
progression of the stroke.
[0047] Another embodiment comprises a neuroprotective infusion kit
comprising a compound that inhibits a NC.sub.Ca-ATP channel in a
neuronal cell, a neuroglia cell, a neural endothelial cell or a
combination thereof and an IV solution. The compound and solution
are contained within the same container or within different
containers. More specifically, the compound is contained within the
container of solution.
[0048] The kit may further comprise a neuroprotective bolus kit,
wherein the bolus kit comprises a pre-loaded syringe of a compound
inhibits a NC.sub.Ca-ATP channel in a neuronal cell, a neuroglia
cell, a neural endothelial cell or a combination thereof.
[0049] Still further, another embodiment comprises a
neuroprotective kit comprising a compound that inhibits
NC.sub.Ca-ATP channel in a neuronal cell, a neuroglia cell, an
endothelium cell or a combination thereof and a thrombolytic agent
(e.g., tPA), an anticoagulant (e.g., warfarin or coumadin), an
antiplatelet (e.g., aspirin), a diuretic (e.g., mannitol), a
statin, or a vasodilator (e.g., nitroglycerin).
[0050] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0051] FIGS. 1A-1F. FIG. 1A shows whole cell current clamp
recording before and after exposure to ouabain and before and after
exposure to NaN.sub.3. FIG. 1B shows whole cell voltage-clamp
recordings during ramp pulses (a) before and (b) after exposure to
NaN.sub.3; (c) is the difference current. FIG. 1C shows whole cell
voltage-clamp recordings during step pulses (a) before and (b)
after exposure to NaN.sub.3; (c) is the difference current. FIG. 1D
shows cell-attached patch recording of single ion channel openings
induced by NaN.sub.3 at membrane potentials of (3) -80 mV and (4)
80 mV, compared to control patches at membrane potentials of (1) 80
mV and (2) -80 mV. FIG. 1E shows the cell-attached patch currents
of FIG. 1D, shown at higher time resolution. FIG. 1F shows the
cell-attached patch single-channel current-voltage
relationship.
[0052] FIGS. 2A and 2B. FIG. 2A shows single channel currents
recorded in an inside-out patch at different membrane potentials;
dotted line indicates channel closing. FIG. 2B is a plot of
inside-out patch single channel amplitude vs. membrane
potentials.
[0053] FIGS. 3A-3D. FIG. 3A shows single channel currents recorded
in an inside-out patch with various alkaline ions substituting for
K.sup.+ in the pipette; dotted line indicates channel closing. FIG.
3B is a plot of channel amplitude vs. membrane potential with
various alkaline ions substituting for K.sup.+ in the pipette. FIG.
3C is a plot of channel amplitude measured in inside-out patches
vs. voltage with Ca2.sup.+ and Mg.sup.2+ substituting for K.sup.+
in the pipette. To estimate channel pore size, FIG. 3D is a plot
illustrating the relationship between the permeability (relative to
Cs.sup.+) and the molecular radius of a series of monovalent
organic cations, which included: (a) methanolmine, (b) guanidium,
(c) ethanolamine, (d) diethylamine, (e) piperazine, (f) Tris, and
(g) N-methylglucamine, data indicating an equivalent pore size of
0.67 nm.
[0054] FIGS. 4A and 4B. FIG. 4A shows single channel recordings in
an inside-out patch in the absence and presence of cytoplasmic ATP.
FIG. 4B is a plot of normalized open channel probability (nPo) vs.
concentration of cytoplasmic ATP.
[0055] FIGS. 5A and 5B. FIG. 5A shows current records from an
inside-out patch exposed to different concentrations of
[Ca.sup.2+].sub.i. FIG. 5B the values of nPo measured at the
membrane potentials and [Ca.sup.2+].sub.i indicated.
[0056] FIG. 6 is a plot of mean single channel amplitudes obtained
in an inside-out patch configuration at different potentials
studied and with different [Mg.sup.2+].sub.i; the dotted line
indicates 35 pS conductance.
[0057] FIGS. 7A and 7B show that presence of SUR1 mRNA and absences
of Kir6.1 and Kir 6.2 in reactive astrocytes. Lanes 3 and 5 in FIG.
7A show the presence of SUR1 in insulinoma RIN-m5f cells and NRAs,
respectively. Lanes 4 and 6 in FIG. 7A show that SUR2 is absent in
both cell types. Lanes 3 and 4 in FIG. 7B show that Kir6.1 is
present in insulinoma RIN-m5f cells and Kir6.2 is absent from the
insulinoma cells, respectively. Lanes 5 and 6 in FIG. 7B show that
neither Kir6.1 nor Kir6.2 is present in NRAs, respectively.
[0058] FIG. 8 shows current recordings in an inside-out patch to
illustrate the effects of tryptic digestion on channel sensitivity
to glibenclamide and ATP.
[0059] FIGS. 9A and 9B shows that the channel activator diazoxide
can elicit channel activities under outside-out patch recording
configuration. FIG. 9A shows the outside-out patch recordings with
Na azide and diazoxide applied to the extracellular side of the
membrane. FIG. 9B shows the current records obtained from the
segments marked with the corresponding numbers in FIG. 9A, at
higher temporal resolution.
[0060] FIGS. 10A-10C. FIG. 10A shows outside-out patch recordings
(a) before, (b) during, and (c) after application of glibenclamide
to the extracellular side of the membrane. FIG. 10B shows the
current records of FIG. 10A at higher temporal resolution. FIG. 10C
show a plot of mean single channel amplitudes at the different
potentials studied; the slope of the data indicates 35 pS
conductance of the glibenclamide-sensitive channel.
[0061] FIGS. 11A and 11B show that sulfonylurea compounds inhibit
channel activities. FIG. 11A shows the outside-out patch recordings
with various concentrations of tolbutamide applied to the
extracellular side of the membrane. FIG. 11B shows the
dose-response curves for inhibition of open channel probability by
glibenclamide and tolbutamide to provide a normalized open channel
probability (nPo); data were fit to a standard logistic equation,
with a Hill coefficient of 1 and half-maximum inhibition of 48 nM
and 16.1 .mu.M; values plotted are means (.sup..+-.SE) from 3 and 5
patches for Glibenclamide and Tolbutamide, respectively.
[0062] FIGS. 12A, 12B and 12C show the probability of channel
opening in the presence of 0 .mu.M, 3 .mu.M, and 30 .mu.M
tolbutamide, respectively.
[0063] FIGS. 12D, 12E and 12F show the distribution of open channel
dwell times in the presence of 0 .mu.M, 3 .mu.M, and 30 .mu.M
tolbutamide, respectively.
[0064] FIGS. 12G, 12H and 12I show the distribution of closed
channel dwell times in the presence of 0 .mu.M, 3 .mu.M, and 30
.mu.M tolbutamide, respectively.
[0065] FIG. 13A shows outside-out patch recordings with diazoxide
applied to the extracellular side of the membrane.
[0066] FIG. 13B shows current records at higher temporal resolution
after application of diazoxide and at different membrane
potentials.
[0067] FIG. 13C shows a plot of mean single channel amplitudes at
the different potentials studied; the slope indicates 35 pS
conductance of glibenclamide-sensitive channel.
[0068] FIGS. 14A, 14B and 14C are scanning electron micrographs of
freshly isolated native reactive astrocytes. FIG. 14A shows the
cells when formaldehyde-glutaraldehyde fixation was initiated under
control conditions; FIG. 14B shows the cells fixed 5 min after
exposure to 1 mM NaN.sub.3. FIG. 14C shows the cells fixed 25 min
after exposure to 1 mM NaN.sub.3. Bar, 12 .mu.m.
[0069] FIGS. 15A, 15B and 15C. FIG. 15A has photomicrographs of the
epifluorescence images of cells exposed to different compounds and
labeled with propidium iodide (upper panel a, b and c) or annexin V
(lower panel d, e and f). The compounds were: control (a & d),
1 mM Na azide (b & e), 1 mM Na azide plus 1 .mu.M glibenclamide
(c & f). FIG. 15B has bar graphs showing cell-counts for
propidium iodide labeling; pairwise multiple comparisons indicated
a significant difference (p<0.05) with Na azide treatment; FIG.
15C has bar graphs showing cell-counts for annexin V staining;
pairwise multiple comparisons indicated no significant difference
with any treatment.
[0070] FIG. 16 shows that addition of exogenous
phosphatidylinositol-4,5-bisphosphate (PIP.sub.2) causes activation
of the NC.sub.Ca-ATP channel, despite the presence of ATP in the
bath solution. Initially, channel activity was recorded in an
inside-out patch of membrane from an R1 astrocyte, with a bath
solution containing 1 .mu.M Ca.sup.2+ and 10 .mu.M ATP, which was
sufficient to block channel activity. Addition of 50 .mu.M
PIP.sub.2 resulted in channel activation, reflecting an apparent
decrease in affinity of the channel for ATP.
[0071] FIG. 17 shows that the NC.sub.Ca-ATP channel in an R1
astrocyte is inhibited by estrogen. The initial portion of the
record shows brisk activity from a number of superimposed channels,
recorded in a cell attached patch of membrane from an R1 astrocyte
obtained from a female. Addition of 10 nM estrogen to the bath
promptly resulted in strong inhibition of channel activity. The
mechanism involved is believed to be related to estrogen receptor
mediated activation of phospholipase C (PLC), resulting in
depletion of PIP.sub.2 from the membrane, and reflecting an
apparent increase in affinity for ATP.
[0072] FIGS. 18A and 18B show Western blots demonstrating that R1
astrocytes from both males and females express estrogen receptors
and SUR1, a marker of the NC.sub.Ca-ATP channel. Cell lysates were
obtained from gelatin sponge implants from males (M) and females
(F) and studied at two dilutions (4.times. and 1.times.), with
lysates from uterus used as controls. FIG. 18A was developed using
antibodies directed against estrogen receptors (ER), demonstrating
that both ER.alpha. and ER.beta. are expressed in astrocytes from
both genders. Western blots showed that SUR1 is also expressed by
cells from both genders, with pancreatic tissue used as control as
shown in FIG. 18B.
[0073] FIG. 19 shows that the NC.sub.Ca-ATP channel in an R1
astrocyte from a male is inhibited by estrogen. The initial portion
of the record shows brisk activity from a number of superimposed
channels, recorded in a cell attached patch of membrane from an R1
astrocyte obtained from a male. Addition of 10 nM estrogen to the
bath promptly resulted in strong inhibition of channel
activity.
[0074] FIGS. 20A-20D show the gliotic capsule. FIG. 20A shows a
coronal section of a rat brain sectioned though the site of
implantation of a large gelatin sponge; the sponge (innermost dark
region) is encapsulated by a gliotic capsule (light area), outside
of which is found a region of vasogenic edema (outer dark area),
identified by pre-mortem administration of methylene blue. FIGS.
20B and 20C show low power and high power views, respectively, of
the gliotic capsule immunolabeled for GFAP. FIG. 20D shows a high
power view of GFAP-labeled cells inside of the gelatin sponge
implant.
[0075] FIGS. 21A-21H show immunolabeled astrocytes. FIGS. 21A, 21C,
21E show freshly-isolated large phase-bright R1 astrocytes
immunolabeled for GFAP (FIG. 21C) and vimentin (FIG. 21E). FIGS.
21B, 21D, and 21F show freshly-isolated small phase-dark R2
astrocytes immunolabeled for GFAP (FIG. 21D) and vimentin (FIG.
21F). FIG. 21G shows primary cultures of astrocytes isolated from a
gliotic capsule, with R1 astrocytes developing into large polygonal
cells (b), and R2 astrocytes developing into small bipolar cells
(a). FIG. 21H shows that R2 astrocytes, but not R1 astrocytes, are
labeled with fluorescein tagged chlorotoxin derived from the
scorpion, Leiurus quinquestriatus.
[0076] FIGS. 22A-22D show that the inner zone of the gliotic
capsule expresses SUR1 but not SUR2. Immunolabeling for SUR1 (FIG.
22A) showed prominent expression in cells adjacent to the gelatin
sponge (gf), whereas immunolabeling for SUR2 showed no expression
(FIG. 22B). A single cell enzymatically isolated from a gelatin
sponge implant and immunolabeled for SUR1 is shown (FIG. 22C). FIG.
22D shown RT-PCR for SUR1 in control insulinoma cells (lane 2) and
in isolated R1 astrocytes (lane 3), and for SUR2 in control cardiac
cells (lane 4), but not in isolated R1 astrocytes (lane 5).
[0077] FIGS. 23A-23I show various features of the gliotic capsule.
The gliotic capsule is characterized by GFAP-positive cells that
are several cell-layers thick (FIG. 23A). Only the inner zone of
the gliotic capsule is hypoxic, as demonstrated by pimonidazole
labeling (FIG. 23B) and by immunolabeling for HIF1.alpha. (FIG.
23C). Also, only the inner zone is immunolabeled for SUR1 (FIG.
23D), and for the tight junction proteins, ZO-1 (FIG. 23E) and
occludens (FIG. 23F). FIGS. 23G-23I show that pimonidazole,
HIF1.alpha. and occludens all localize to GFAP-positive astrocytes
that form the inner zone of the gliotic capsule.
[0078] FIGS. 24A and 24B show effects of NC.sub.Ca-ATP channel
inhibition (FIG. 24A) and NC.sub.Ca-ATP channel activation (FIG.
24B) on the gliotic capsule. Animals with gelatin sponge implants
were treated with glibenclamide infusion (FIG. 24A) or diazoxide
infusion (FIG. 24B) via osmotic mini-pumps that delivered the
compounds directly into the area of the gelatin sponge.
Immunolabeling for GFAP showed that channel inhibition with
glibenclamide resulted in formation of a well defined gliotic
capsule (FIG. 24A), whereas channel activation with diazoxide
resulted in formation of a broader, ill-defined capsule (FIG. 24B),
due to diazoxide-induced necrotic death of inner zone cells.
[0079] FIGS. 25A and 25B show that infusion of diazoxide into the
area around the gelatin sponge resulted in a heavy infiltration of
polymorphonuclear leukocytes (PMNs). Nuclear labeling with DAPI
showed densely packed small cells in the vicinity of the gelatin
sponge (FIG. 25A), with immunolabeling using the PMN-specific
marker, MMP-8, demonstrating that these cells were PMNs (FIG. 25B).
It is believed that the strong inflammatory response represented by
the infiltrating PMNs was due to disruption of the barrier between
brain and foreign body (gelatin sponge) normally formed by the
inner zone of the gliotic capsule.
[0080] FIGS. 26A-26L show that R1 astrocytes in the inner zone of
the gliotic capsule typically express SUR1, a marker for the
NC.sub.Ca-ATP channel. The inner zones of the gliotic capsules in
rats with gelatin sponge implants (FIGS. 26A-26C), in rats with
cerebral abscess (FIGS. 26D-26F), and in humans with metastatic
tumor (FIGS. 26J-26L) are shown. Also shown is the area of reactive
gloss adjacent to a stroke in the rat (FIGS. 26G-26I) resulting
from occlusion of the middle cerebral artery. In all cases, a field
of cells is labeled for GFAP and co-labelled for SUR1, as
indicated. Examples of single cells at high power are also shown
for each condition.
[0081] FIGS. 27A-27C shows that stellate astrocytes near the edge
of a stroke up-regulate SUR1 (FIG. 27A), a marker of the
NC.sub.Ca-ATP channel. In the middle of the stroke, cells with
altered morphology including blebbing are also immunolabeled for
SUR1 (FIGS. 27B and 27C).
[0082] FIGS. 28A-28C show that glibenclamide protects from Na
azide-induced channel opening and necrotic cell death. FIG. 28A
shows phase contrast images of 4 different freshly isolated R1
astrocytes observed over the course of 30 min each. The cell
exposed to vehicle solution alone remained phase bright with no
pathological deterioration (control). The cell depleted of ATP by
exposure to Na azide (1 mM) developed progressive blebbing
consistent with cytotoxic edema. Similarly, the cell exposed to the
NC.sub.Ca-ATP channel opener, diazoxide, developed progressive
blebbing consistent with cytotoxic edema. The cell exposed to Na
azide in the presence of glibenclamide remained phase bright with
no pathological deterioration. FIGS. 28B and 28C show cell death of
isolated R1 astrocytes induced by ATP depletion in vitro. Freshly
isolated R1 astrocytes were labeled for necrotic death with
propidium iodide (PI) (FIG. 28B), or for apoptotic death with
annexin V (FIG. 28C), under control conditions, after exposure to
Na azide (1 mM), or after exposure to Na azide in the presence of
glibenclamide (1 .mu.M). Exposure to Na azide resulted mostly in
necrotic death that was largely prevented by glibenclamide.
[0083] FIGS. 29A-29L shows that SUR1 is up-regulated in MCA stroke.
Watershed area between MCA-ACA in 3 different animals 8-16 hr after
MCA stroke, identified by pre-mortem administration of Evans blue
and postmortem perfusion with India ink (FIG. 29A), by TTC staining
(FIG. 29B) and by immunofluorescence imaging for SUR1 (FIG. 29C).
Immunofluorescence images showing SUR1 at 3 hr in the core of the
stroke in cells (FIG. 29D) double-labeled for the neuronal marker,
NeuN (FIG. 29E), and showing SUR1 at 8 hr in the peri-infarct
region in cells (FIGS. 14G and 14J) double-labeled for the
astrocytic marker, GFAP (FIG. 29H), and the endothelial cell
marker, von Willebrand factor (FIG. 29K). Superimposed images of
double-labeled fields are shown (FIGS. 29F, 29I, and 29L).
[0084] FIGS. 30A-30G show that SUR1 but not Kir6.1 or Kir6.2 is
transcriptionally up-regulated in MCA stroke. Western blots for
SUR1 (.apprxeq.180 kDa) at different times (FIG. 30A) and in
different locations (FIG. 15B) after MCA stroke; in (FIG. 30A),
lysates were all from TTC(+) peri-infarct regions of the involved
hemisphere, obtained at the times indicated; in (FIG. 30B), lysates
were all obtained 8 hr after MCA stoke from the regions indicated;
each individual lane in a and b is from a single animal.
Quantification of the data from (FIG. 30A) and (FIG. 30B),
respectively, combined with comparable data for Kir6.1 and Kir6.2;
for each individual blot, data were normalized to values of
.beta.-actin and to the control data for that blot and analyzed
separately; **, p<0.01. In situ hybridization for SUR1, 3 hr
after MCA stroke; paraffin sections showed that large neuron-like
cells (FIG. 30E) and capillaries (FIG. 30F) in the ischemic zone
were labeled, whereas tissues from the same areas on the control
side were not (FIG. 30G).
[0085] FIGS. 31A-31D show patch clamp recordings of NC.sub.CaATP
channel in neuron-like cells in stroke. FIG. 31A shows
phase-contrast image of large neuron-like cells enzymatically
isolated from ischemic region 3 hr following MCAO. FIG. 31B shows
recording of inside-out patch using Cs.sup.+ as the charge carrier;
channel activity was blocked by glibenclamide given as indicated
(arrow); a and b show expanded records of the portions indicated.
FIG. 31C shows recordings at potentials indicated of inside-out
patch using K.sup.+ as the charge carrier; channel activity was
blocked by glibenclamide. FIG. 31D shows a plot of single channel
amplitudes at different voltages showing single channel slope
conductance of 34 pS.
[0086] FIGS. 32A-32E show that glibenclamide reduces mortality,
edema and stroke size in MCA stroke. In FIG. 32A, Mortality was
assessed during 7 days after MCA stroke [double occlusion model
with malignant cerebral edema (MCE)] in two treatment groups, each
comprised of 19 female and 10 male rats, treated with either saline
(empty symbols) or glibenclamide (filled symbols); mortality at 7
days was significantly different. Subgroup analyses for males and
females showed similar results. In FIG. 32B edema was assessed 8 hr
after MCA stroke (MCE model) in two treatment groups, each
comprised of 6 male rats treated with either saline or
glibenclamide; tissues were first processed with TTC to allow
separation into TTC(+) and TTC(-) portions of the involved
hemisphere and contralateral hemisphere, prior to determining
wet/dry weights; values in TTC(+) regions were statistically
different. In FIGS. 32C-32E, stroke size was assessed 48 hr after
MCA stroke [thromboembolic (TE) model] in two treatment groups,
each comprised of 10 male rats, treated with either saline or
glibenclamide; images of TTC-stained coronal sections following MCA
stroke (TE model) in an animal treated with saline (FIG. 32C) and
another treated with glibenclamide (FIG. 32D), showing cortical
sparing often associated with glibenclamide treatment; values of
stroke size, expressed as percent of hemisphere volume (FIG.
32E).
[0087] FIGS. 33A-33D show that tissue distribution of
BODIPY-glibenclamide in MCA stroke. a-c, Fluorescence images of
brain sections in an animal 6 hr after MCA stroke (MCE model) and
administration of BODIPY-glibenclamide; fluorescent labeling was
evident in cells, microvessels (FIG. 33A) and capillaries (FIG.
33C) from ischemic regions, but not in the contralateral hemisphere
(FIG. 18B); the images in (FIGS. 33A and 33B) are from the same
animal, taken with the same exposure time; in (FIG. 33C), the
single layer of nuclei confirms that the structure brightly labeled
by BODIPY-glibenclamide is a capillary. In FIG. 33D,
immunofluorescence image of a brain section from an animal 6 hr
after MCA stroke (MCE model) labeled with anti-SUR1 antibody
showing strong labeling in a capillary and in adjacent neuron-like
cells.
[0088] FIGS. 34A-34H show that glibenclamide reduces hemorrhagic
conversion. FIGS. 34A-34D are from animals co-treated with saline;
FIGS. 34E-34H are from animals co-treated with glibenclamide. The
left column of photographs of coronal sections shows, in rows 1-2
only, intraventricular hemorrhage, plus large areas of hemorrhagic
conversion in ischemic cortical/subcortical regions (red areas on
the right side of pictures; arrows). The right column of
photographs of TTC-processed sections from the same animals show
the areas of infarction.
[0089] FIGS. 35A and 35B show zymography showing gelatinase
activity of matrix metalloproteinases (MMP's) in stroke, and
absence of direct MMP inhibition by glibenclamide. FIG. 35A shows
activation of MMP-9 & MMP-2 in stroke tissue compared to
control; activity of recombinant MMP-9 & MMP-2 shown at left.
FIG. 35B shows gelatinase activity of recombinant enzyme and stroke
tissue under control conditions (CTR), in presence of glibenclamide
(10 .mu.M), and in presence of MMP inhibitor II (300 nM;
Calbiochem).
[0090] FIG. 36 shows phase contrast photomicrograph of cerebral
capillaries freshly isolated from normal brain, after enzymatic
cleaning in preparation for patch clamping.
[0091] FIGS. 37A-37F show that freshly isolated cerebral
endothelial and smooth muscle cells are readily distinguished
electrophysiologically. FIGS. 37A and 37B show superimposed
macroscopic currents recorded during 200 ms depolarizing pulses
from -120 mV to +120 mV in 20 mV steps in an endothelial cell (FIG.
37A) and in an elongated smooth muscle cell (FIG. 37B); holding
potential, -60 mV; nystatin perforated patch technique; bath
solution, standard Krebs with 2 mM Ca.sup.2+; pipette solution, 145
mM K.sup.+. FIGS. 37C and 37D show current-voltage curves computed
from average (mean.+-.SE) currents at the end of 200-ms test pulses
recorded in 9 endothelial cells (FIG. 37C) and 7 smooth muscle
cells (FIG. 37D); same holding potential, technique and solutions
as in FIGS. 37A and 37B. FIGS. 37E and 37F show current voltage
curves recorded during ramp pulses (0.45 mV/ms, holding potential,
-60 mV) in an endothelial cell (FIG. 37E) and in a smooth muscle
cell (FIG. 37F); same holding potential, technique and bath
solution as in FIGS. 37A and 37B, but with pipette solution
containing 145 mM Cs.sup.+ instead of K.sup.+.
[0092] FIG. 38 shows real time RT-PCR showing up-regulation of
SUR1-mRNA in stroke.
[0093] FIGS. 39A-39E show SUR1 knock down (SUR1KD) in R1 astrocytes
protects from ATP-depletion-induced depolarization. FIGS. 39A and
39B show Western blot (FIG. 39A) and quantification of Western
blots (FIG. 39B) of R1 cell lysates confirmed knock down of SUR1
expression by antisense. FIGS. 39C-39E show Na azide caused large
depolarizations in cells exposed to SCR-ODN (FIGS. 39C and 39E) but
little or no depolarization in cells exposed to AS-ODN (FIGS. 39D
and 39E).
[0094] FIGS. 40A-40F show transcription factors in stroke.
Immunofluorescence images of subcortical watershed region between
ACA and MCA territories, from ipsilateral peri-infarct tissues 8 hr
after MCAO (FIGS. 40A-D) and from contralateral control tissues
(FIGS. 40E and 40F). The peri-infarct region showed up-regulation
of both transcription factors, Sp1 (FIGS. 40A and 40C) and
HIF1.alpha. (FIG. 40B) in neuron-like cells and capillaries, as
well as SUR1 in capillaries (FIG. 40D). Control tissues showed
little SP1 and no HIF1.alpha. (FIGS. 40E and 40F).
[0095] FIGS. 41A-41C show an increase in nuclear localization of
the transcription factor, SP1, and SP1 co-localization with SUR1 in
stroke. Immunofluorescence images showing increase of nuclear SP1
labeling in ischemic area 3-hr after MCAO (FIG. 41B), compared to
contralateral side (FIG. 41A). FIG. 41C double labeling of large
neuron-like cell showing nuclear SP1 (green) and
cytoplasmic/plasmalemmal SUR1 (red) in the same cell.
[0096] FIGS. 42A-42D show regulation of SUR1 expression by the
transcription factor, HIF1.alpha.. FIGS. 42A and 42C show Western
blot analysis of HIF1.alpha. protein in R1 astrocytes from gelfoam
implant model of control (CTR) and HIF1.alpha. knock-down (KD).
FIGS. 42B and 42C show SUR1 protein in the same cell lysates.
[0097] FIG. 43 shows relative cerebral blood flow, measured by
Laser Doppler Flowmetry, before (CTR), 1 hr after and 48 hr after
MCAO, in 2 groups, each consisting of 4 male rats, treated with
either saline or glibenclamide; values at 48 hr were statistically
different (by ANOVA; p<0.01).
[0098] FIG. 44 Glibenclamide was just as effective in reducing
edema after stroke with added glucose as without added glucose.
Supplemental glucose (1 gm/kg, i.p.) was administered 4 hr after
MCAO, and animals were sacrificed 8 hr after MCAO for measurements
of edema.
[0099] FIG. 45 Glibenclamide reduces stroke volume even when
administration is delayed up to 2 hours (low dose) or up to 6 hours
(higher dose) following stroke.
[0100] FIG. 46 Glibenclamide reduces hemorrhagic conversion.
Animals treated with intravenous tPA (10 mg/kg over 30 min)
following thromboembolic lesion were also treated with either
saline or glibenclamide. Although 5 of 6 animals co-treated with
saline showed hemorrhagic conversion, only 1 of 6 animals treated
with glibenclamide showed hemorrhagic conversion, demonstrating the
efficacy of glibenclamide treatment to reduce or prevent
hemorrhagic conversion following thromboembolic stroke.
[0101] FIG. 47 shows expression of SUR1 protein in cortical brain
tissues; minimal labeling was observed in control tissues (left
panel), whereas prominent labeling was seen surrounding the site of
the impact ("I", originating from the right side), in brain
contusion (right panel); tissues were harvested 24 hr following
contusion injury.
[0102] FIG. 48 shows high power views of previous image
(above/right panel), showing SUR1 expression following brain
contusion; SUR1 expression was seen in large neuron-like cells
(left panel) and in capillaries co-labeled with SUR1 and von
Willebrand factor (middle and right panels).
DETAILED DESCRIPTION
[0103] The present invention relates to a novel ion channel whose
function underlies the swelling of mammalian neural cells, such as
in response to ATP depletion; treatment methods related to
diseases, trauma, and conditions that lead to the expression of
such channels, including the use of inhibitors of the channel
function to prevent this cell swelling response, which
characterizes brain damage in cerebral ischemia and traumatic brain
injury. The present invention also relates to the use of the
channel to screen for channel inhibitors and activators, and other
uses.
[0104] The NC.sub.Ca-ATP channel of the present invention is
distinguished by certain functional characteristics, the
combination of which distinguishes it from known ion channels. The
characteristics that distinguish the NC.sub.Ca-ATP channel of the
present invention include, but are not necessarily limited to, the
following: 1) it is a non-selective cation channel that readily
allows passage of Na, K and other monovalent cations; 2) it is
activated by an increase in intracellular calcium, and/or by a
decrease in intracellular ATP; 3) it is regulated by sulfonylurea
receptor type 1 (SUR1), which heretofore had been considered to be
associated exclusively with K.sub.ATP channels such as those found
in pancreatic .beta. cells, for example.
[0105] More specifically, the NC.sub.Ca-ATP channel of the present
invention has a single-channel conductance to potassium ion
(K.sup.+) between 20 and 50 pS. The NC.sub.Ca-ATP channel is also
stimulated by Ca.sup.2+ on the cytoplasmic side of the cell
membrane in a physiological concentration range, where said
concentration range is from 10.sup.-8 to iv M. The NC.sub.Ca-ATP
channel is also inhibited by cytoplasmic ATP in a physiological
concentration range, where said concentration range is from about
10.sup.-1 to about 10 .mu.M. The NC.sub.Ca-ATP channel is also
permeable to the following cations; K.sup.+, Cs.sup.+, Li.sup.+,
Na.sup.+; to the extent that the permeability ratio between any two
of said cations is greater than 0.5 and less than 2.
[0106] Some of the preferred embodiments of the present invention
will be described in detail with reference to the attached
drawings.
[0107] This invention may be embodied in many different forms and
should not be construed as being limited to the embodiments set
forth herein.
I. NC.sub.CA-ATP CHANNEL
[0108] A unique non-selective monovalent cationic ATP-sensitive
channel (NC.sub.Ca-ATP channel) was identified first in native
reactive astrocytes (NRAs) and later, as described herein, in
neurons and capillary endothelial cells after stroke or traumatic
brain or spinal cord injury (See at least International application
WO 03/079987 to Simard et al., and Chen and Simard, 2001, each
incorporated by reference herein in its entirety). As with the
K.sub.ATP channel in pancreatic .beta. cells, the NC.sub.CaATP
channel is thought to be a heteromultimer structure comprised of
sulfonylurea receptor type 1 (SUR1) regulatory subunits and
pore-forming subunits (Chen et al., 2003). The pore-forming
subunits have been characterized biophysically, but have yet to be
characterized molecularly.
[0109] The invention is based, in part, on the discovery of a
specific channel, the NC.sub.Ca-ATP channel, defined as a channel
on astrocytes in US Application Publication No. 20030215889, which
is incorporated herein by reference in its entirety. More
specifically, the present invention has further defined that this
channel is not only expressed on astrocytes, it is expressed at
least on neural cells, neuroglial cells, and/or neural endothelial
cells after brain and spinal cord trauma, for example, an hypoxic
event, an ischemic event, or other secondary neuronal injuries
relating to these events.
[0110] The NC.sub.Ca-ATP channel is activated by calcium ions
(Ca.sup.2+) and is sensitive to ATP. Thus, this channel is a
non-selective cation channel activated by intracellular Ca.sup.2+
and blocked by intracellular ATP. When opened by depletion of
intracellular ATP, this channel is responsible for complete
depolarization due to massive Na.sup.+ influx, which creates an
electrical gradient for Cl.sup.- and an osmotic gradient for
H.sub.2O, resulting in cytotoxic edema and cell death. When the
channel is blocked or inhibited, massive Na.sup.+ does not occur,
thereby preventing cytotoxic edema.
[0111] Certain functional characteristics distinguish the
NC.sub.Ca-ATP channel from other known ion channels. These
characteristics can include, but are not limited to, at least some
of the following: 1) it is a non-selective cation channel that
readily allows passage of Na.sup.+, K.sup.+ and other monovalent
cations; 2) it is activated by an increase in intracellular
calcium, and/or by a decrease in intracellular ATP; 3) it is
regulated by sulfonylurea receptor type 1 (SUR1), which heretofore
had been considered to be associated exclusively with K.sub.ATP
channels such as those found in pancreatic .beta. cells.
[0112] More specifically, the NC.sub.Ca-ATP channel of the present
invention has a single-channel conductance to potassium ion
(K.sup.+) between 20 and 50 pS. The NC.sub.Ca-ATP channel is also
stimulated by Ca.sup.2+ on the cytoplasmic side of the cell
membrane in a physiological concentration range, where
concentration range is from 10.sup.-8 to 10.sup.-5 M. The
NC.sub.Ca-ATP channel is also inhibited by cytoplasmic ATP in a
physiological concentration range, where the concentration range is
from 10.sup.-1 to 10 M. The NC.sub.Ca-ATP channel is also permeable
to the following cations; K.sup.+, Cs.sup.+, Li.sup.+, Na.sup.+; to
the extent that the permeability ratio between any two of the
cations is greater than 0.5 and less than 2.
[0113] SUR imparts sensitivity to antidiabetic sulfonylureas such
as glibenclamide and tolbutamide and is responsible for activation
by a chemically diverse group of agents termed "K.sup.+ channel
openers" such as diazoxide, pinacidil and cromakalin (Aguilar-Bryan
et al., 1995; Inagaki et al., 1996; Isomoto et al., 1996; Nichols
et al., 1996; Shyng et al., 1997). In various tissues, molecularly
distinct SURs are coupled to distinct pore-forming subunits to form
different K.sub.ATP channels with distinguishable physiological and
pharmacological characteristics. The K.sub.ATP channel in
pancreatic .beta. cells is formed from SUR1 linked with Kir6.2,
whereas the cardiac and smooth muscle K.sub.ATP channels are formed
from SUR2A and SUR2B linked with Kir6.2 and Kir6.1, respectively
(Fujita et al., 2000). Despite being made up of distinctly
different pore-forming subunits, the NC.sub.Ca-ATP channel is also
sensitive to sulfonylurea compounds.
[0114] Also, unlike the K.sub.ATP channel, the NC.sub.Ca-ATP
channel conducts sodium ions, potassium ions, cesium ions and other
monovalent cations with near equal facility (Chen and Simard, 2001)
suggesting further that the characterization, and consequently the
affinity to certain compounds, of the NC.sub.Ca-ATP channel differs
from the K.sub.ATP channel.
[0115] Other nonselective cation channels that are activated by
intracellular Ca.sup.2+ and inhibited by intracellular ATP have
been identified by others but not in astrocytes or neurons as
disclosed herein. Further, the NC.sub.Ca-ATP channel expressed and
found in astrocytes differs physiologically from the other channels
with respect to calcium sensitivity and adenine nucleotide
sensitivity (Chen et al., 2001).
Summary of NC.sub.Ca-ATP Channel Characteristics
[0116] At least some of the characteristics of cells expressing and
composition comprising the NC.sub.Ca-ATP channel of the present
invention are summarized in Table 1 (taken from experiments with
freshly isolated native reactive astrocytes [NRA]).
TABLE-US-00001 TABLE 1 Properties of cells and membrane
compositions containing the NC.sub.Ca-ATP Channel of the Present
Invention Membrane Preparation derived from freshly Reactive
isolated native Astrocytes reactive astrocytes Monovalent cation
Yes: Yes: permeable? Na.sup.+ Na.sup.+ K.sup.+ K.sup.+ Li.sup.+
Li.sup.+ Rb.sup.+ Rb.sup.+ Cs.sup.+ Cs.sup.+ (Na.sup.+ .apprxeq.
K.sup.+ .apprxeq. (NA.sup.+ .apprxeq. K.sup.+ .apprxeq. Li.sup.+
.apprxeq. Rb.sup.+) Li.sup.+ .apprxeq. Rb.sup.+) Anion permeable?
No No Divalent cation No No permeable? Compounds blocking SUR1
antagonists SUR1 ANTAGONISTS channel activity Channel opening
Intracell. ATP Intracell ATP Requires: depletion depletion
Intracell. Mg.sup.2+ Intracell. Mg.sup.2+ Single Channel ~35 pS ~35
PS Conductance Activation <1.0 .mu.M <1.0 .mu.M [Ca.sup.2+]
[ATP].sub.1 EC.sub.50 (um) 0.79 .mu.M 0.79 .mu.M ADP No channel
effect No channel effect AMP Pore radius 0.41 0.41 (nm)
II. GLIOTIC CAPSULE
[0117] The gliotic capsule forms a potentially harmful mass of
tissue that contributes to brain swelling and mass effect, and that
may shelter foreign cells from surveillance by the immune system.
Applicants are the first to determine that, in a variety
pathological conditions in both rats and humans, reactive
astrocytes (R1 astrocytes) in the inner zone of the gliotic capsule
express a novel SUR1-regulated cation channel, the NC.sub.Ca-ATP
channel, and that this channel directly controls cell viability:
opening the channel is associated with necrotic cell death and
closing the channel is associated with protection from cell death
induced by energy (ATP) depletion.
[0118] As described herein, Applicants are the first to determine
that the inner zone of the gliotic capsule is populated by R1
astrocytes expressing the NC.sub.Ca-ATP channel. Selectively
killing the astrocytes expressing the NC.sub.Ca-ATP channel may aid
in the treatment of conditions that lead to the formation of
gliotic capsules. For example, selectively killing the astrocytes
expressing the NC.sub.Ca-ATP channel disrupts the "tumor brain
barrier" (TBB), causing migration of leukocytes across the TBB and
aiding in treatment of tumors in the brain.
[0119] Also there exists a need for therapeutic compounds capable
of modulating the activity of this target in order to prevent brain
damage. The present invention is directed to a newly characterized
non-selective calcium and ATP sensitive monovalent cation channel,
termed the NC.sub.Ca-ATP channel, which is present in neural cells
and linked to an SUR. The present invention further provides a
method to screen for or identify antagonists to NC.sub.Ca-ATP
channel activity. Further, the present invention provides a method
for the therapeutic use of antagonists, such as sulfonylureas and
other SUR1 blockers, to inhibit this channel's activity and thereby
prevent neural cell swelling and cell death and the concomitant
nervous system damage that includes brain swelling and brain
damage.
[0120] Sodium azide (NaN.sub.3) is a metabolic toxin used to induce
"chemical hypoxia" by depleting intracellular ATP. See, Swanson,
1992. The morphological and electrophysiological responses of
neural cells to NaN.sub.3 are examined in a novel cell preparation.
Freshly isolated native reactive astrocytes (NRAs) from adult rat
brain are used and studied in a native state immediately after
their isolation. Reactive astrocytes are astrocytes that have been
activated or stimulated in vivo, such as those associated with
brain or neural injury. In the post-mortem brains of traumatic
brain injury (TBI) patients, reactive astrocytes are found in
proximity to the injury. The majority of reactive astrocytes
surrounding an injury site in the brain are reactive astrocytes.
Type 1 reactive astrocytes comprise >80% of recoverable reactive
astrocytes, whereas type 2 reactive astrocytes comprise about 5%.
Reactive astrocytes are normally polarized under quiescent
conditions.
[0121] It is readily apparent to one skilled in the art that
various embodiments and modifications can be made to the invention
disclosed in this Application without departing from the scope and
spirit of the invention.
III. DEFINITIONS
[0122] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." Some
embodiments of the invention may consist of or consist essentially
of one or more elements, method steps, and/or methods of the
invention. It is contemplated that any method or composition
described herein can be implemented with respect to any other
method or composition described herein.
[0123] The use of the term "or" in the claims is used to mean
"and/or" unless explicitly indicated to refer to alternatives only
or the alternative are mutually exclusive, although the disclosure
supports a definition that refers to only alternatives and
"and/or."
[0124] As used herein, the term "acute" refers to the onset of a
health effect, usually the effect is a rapid onset that is
considered brief, not prolonged.
[0125] As used herein, the term "acute cerebral ischemia" refers to
a cerebral ischemic event that has a rapid onset and is not
prolonged. The terms "acute cerebral ischemia" and "stroke" can be
used interchangeably."
[0126] As used herein, the term "agonist" refers to a biological or
chemical agent that combines with a receptor on a cell and
initiates the same or equivalent reaction or activity produced by
the binding of an endogenous substance. In the present invention,
the agonist combines, binds, and/or associates with a NC.sub.Ca-ATP
channel of a neuronal cell, a neuroglial cell, or a neural
endothelial cell, such that the NC.sub.Ca-ATP channel is opened
(activated). In certain embodiments, the agonist combines, binds
and/or associates with a regulatory subunit of the NC.sub.Ca-ATP
channel, particularly a SUR1. Alternatively, the agonist combines,
binds, and/or associates with a pore-forming subunit of the
NC.sub.Ca-ATP channel, such that the NC.sub.Ca-ATP channel is
opened (activated). The terms agonist and/or activator can be used
interchangeably.
[0127] As used herein, the term "antagonist" refers to a biological
or chemical agent that acts within the body to reduce the
physiological activity of another chemical or biological substance.
In the present invention, the antagonist blocks, inhibits, reduces
and/or decreases the activity of a NC.sub.Ca-ATP channel of a
neuronal cell, a neuroglia cell or a neural endothelial cell (e.g.,
capillary endothelial cells). In the present invention, the
antagonist combines, binds, associates with a NC.sub.Ca-ATP channel
of neuronal cell, a neuroglia cell or a neural endothelial cell
(e.g., capillary endothelial cells), such that the NC.sub.Ca-ATP
channel is closed (deactivated), meaning reduced biological
activity with respect to the biological activity in the diseased
state. In certain embodiments, the antagonist combines, binds
and/or associates with a regulatory subunit of the NC.sub.Ca-ATP
channel, particularly a SUR1. Alternatively, the antagonist
combines, binds, and/or associates with a pore-forming subunit of
the NC.sub.Ca-ATP channel, such that the NC.sub.Ca-ATP channel is
closed (deactivated). The terms antagonist or inhibitor can be used
interchangeably.
[0128] As used herein, the terms "brain abscess" or "cerebral
abscess" refer to a circumscribed collection of purulent exudate
that is typically associated with swelling.
[0129] As used herein, the terms "blood brain barrier" or "BBB"
refer the barrier between brain blood vessels and brain tissues
whose effect is to restrict what may pass from the blood into the
brain.
[0130] As used herein, the term "cerebral ischemia" refers to a
lack of adequate blood flow to an area, for example a lack of
adequate blood flow to the brain or spinal cord, which may be the
result of a blood clot, blood vessel constriction, a hemorrhage or
tissue compression from an expanding mass.
[0131] As used herein, the term "depolarization" refers to an
increase in the permeability of the cell membrane to sodium ions
wherein the electrical potential difference across the cell
membrane is reduced or eliminated.
[0132] As used herein, the terms "effective amount" or
"therapeutically effective amount" are interchangeable and refer to
an amount that results in an improvement or remediation of the
symptoms of the disease or condition. Those of skill in the art
understand that the effective amount may improve the patient's or
subject's condition, but may not be a complete cure of the disease
and/or condition.
[0133] As used herein, the term "endothelium" refers a layer of
cells that line the inside surfaces of body cavities, blood
vessels, and lymph vessels or that form capillaries.
[0134] As used herein, the term "endothelial cell" refers to a cell
of the endothelium or a cell that lines the surfaces of body
cavities, for example, blood or lymph vessels or capillaries. In
certain embodiments, the term endothelial cell refers to a neural
endothelial cell or an endothelial cell that is part of the nervous
system, for example the central nervous system or the brain or
spinal cord.
[0135] As used herein, the term "gliotic capsule" refers to a
physical barrier surrounding, in whole or in part, a foreign body,
including a metastatic tumor, a cerebral abscess or other mass not
normally found in brain except under pathological conditions. In
certain embodiments, the gliotic capsule comprises an inner zone
comprising neuronal cells, neuroglial cells (e.g., astrocytes)
and/or endothelial cells expressing a NC.sub.Ca-ATP channel.
[0136] As used herein, the term "ionic edema" in brain or nervous
tissue refers to edema arising in tissue in which the blood-brain
barrier remains substantially intact, and is associated with the
movement of electrolytes (e.g. Na.sup.+, Cl.sup.-) plus water into
brain parenchyma.
[0137] As used herein, the term "inhibit" refers to the ability of
the compound to block, partially block, interfere, decrease, reduce
or deactivate a channel such as the NC.sub.Ca-ATP channel. Thus,
one of skill in the art understands that the term inhibit
encompasses a complete and/or partial loss of activity of a
channel, such as the NC.sub.Ca-ATP channel. Channel activity may be
inhibited by channel block (occlusion or closure of the pore
region, preventing ionic current flow through the channel), by
changes in an opening rate or in the mean open time, changes in a
closing rate or in the mean closed time, or by other means. For
example, a complete and/or partial loss of activity of the
NC.sub.Ca-ATP channel as may be indicated by a reduction in cell
depolarization, reduction in sodium ion influx or any other
monovalent ion influx, reduction in an influx of water, reduction
in extravasation of blood, reduction in cell death, as well as an
improvement in cellular survival following an ischemic
challenge.
[0138] The term "morbidity" as used herein is the state of being
diseased. Yet further, morbidity can also refer to the disease rate
or the ratio of sick subjects or cases of disease in to a given
population.
[0139] The term "mortality" as used herein is the state of being
mortal or causing death. Yet further, mortality can also refer to
the death rate or the ratio of number of deaths to a given
population.
[0140] As used herein, the term "neuron" refers to a nerve cell,
also termed a neuronal cell.
[0141] As used herein, the term "neuronal cell" refers to a cell
that is a morphologic and functional unit of the nervous system.
The cell comprises a nerve cell body, the dendrites, and the axon.
The terms neuron, nerve cell, neuronal, neurone, and neurocyte can
be used interchangeably. Neuronal cell types can include, but are
not limited to a typical nerve cell body showing internal
structure, a horizontal cell (of Cajal) from cerebral cortex;
Martinottic cell, biopolar cell, unipolar cell, Pukinje cell, and a
pyramidal cell of motor area of cerebral cortex.
[0142] As used herein, the term "neural" refers to anything
associated with the nervous system. As used herein, the term
"neural cells" includes neurons and glia, including astrocytes. As
used herein, the term "isolated neural cells" means neural cells
isolated from brain.
[0143] As used herein, the terms "neuroglia" or "neuroglial cell"
refers to a cell that is a non-neuronal cellular element of the
nervous system. The terms neuroglia, neurogliacyte, and neuroglial
cell can be used interchangeably. Neuroglial cells can include, but
are not limited to ependymal cells, astrocytes, oligodendrocytes,
or microglia.
[0144] The term "preventing" as used herein refers to minimizing,
reducing or suppressing the risk of developing a disease state or
parameters relating to the disease state or progression or other
abnormal or deleterious conditions.
[0145] The term "reactive astrocytes" means astrocytes found in
brain at the site of a lesion or ischemia. The term "native
reactive astrocytes" or "NRAs" means reactive astrocytes that are
freshly isolated from brain. The term "freshly isolated" as used
herein refers to NRAs that have been purified from brain,
particularly NRAs that were purified from about 0 to about 72 hours
previously. When NRAs are referred to as being "purified from
brain" the word "purified" means that the NRAs are isolated from
other brain tissue and/or implanted gelatin or sponge and does not
refer to a process that simply harvests a population of cells from
brain without further isolation of the cells. As described herein,
the NC.sub.Ca-ATP channel found in reactive astrocytes is present
only in freshly isolated cells; the NC.sub.Ca-ATP channel is lost
shortly after culturing the cells under typical normoxic
conditions. NRAs provide an in vitro model that is more similar to
reactive astrocytes as they exist in vivo in the brain, than
astrocytes grown in culture. The terms "native" and "freshly
isolated" are used synonymously.
[0146] As used herein, the term "reduces" refers to a decrease in
cell death, inflammatory response, hemorrhagic conversion,
extravasation of blood, etc. as compared to no treatment with the
compound of the present invention. Thus, one of skill in the art is
able to determine the scope of the reduction of any of the symptoms
and/or conditions associated with a spinal cord injury in which the
subject has received the treatment of the present invention
compared to no treatment and/or what would otherwise have occurred
without intervention.
[0147] As used herein, the term "stroke" refers to any acute,
clinical event related to the impairment of cerebral circulation.
The terms "acute cerebral ischemia" and "stroke" can be used
interchangeably.
[0148] The terms "treating" and "treatment" as used herein refer to
administering to a subject a therapeutically effective amount of a
composition so that the subject has an improvement in the disease
or condition. The improvement is any observable or measurable
improvement. Thus, one of skill in the art realizes that a
treatment may improve the patient's condition, but may not be a
complete cure of the disease. Treating may also comprise treating
subjects at risk of developing a disease and/or condition.
[0149] As used herein, the term "vasogenic edema" in brain or
nervous tissue refers to edema arising in tissue in which the
blood-brain barrier is not substantially intact, and in which
macromolecules plus water enter into brain parenchyma in addition
to any movement of electrolytes.
[0150] Reactive astrocytes are produced in vivo and harvested from
brain according to a method system similar to that described by
Perillan. See, Chen et al., 2003; Chen et al., 2001, for example.
Harvested cells are then isolated and not cultured; rather, the
freshly isolated reactive astrocytes are studied in a native state
immediately after their isolation from the brain. As described by
Perillan et al. (1999; 2000), cultured astrocytes do not express
the NC.sub.Ca-ATP channel.
[0151] The Examples described herein reveal that NRAs from adult
rat brain express a non-selective cation channel that is activated
by depletion of [ATP], at physiological concentrations of
[Ca.sup.2+].sub.i. This NC.sub.Ca-ATP channel of the present
invention, which is newly identified in NRAs and present in >90%
of membrane patches from such cells, is distinguished from
previously reported non-selective calcium and ATP channels by
exhibiting significantly different properties. These distinguishing
properties of the NC.sub.Ca-ATP of the present invention include:
being activated by submicromolar [Ca.sup.2+] and exhibiting a
different sensitivity to block by various adenine nucleotides.
Opening of the NC.sub.Ca-ATP channel of the present invention by
ATP depletion causes profound membrane depolarization, which
precedes blebbing of the cell membrane. Upon ATP depletion, the
NC.sub.Ca-ATP channel opens to allow Na.sup.+ influx that leads to
cell swelling. This channel is regulated by sulfonylurea receptor
type 1 (SUR1). The channel can be blocked by sulfonylurea, such as
glibenclamide and tolbutamide; treatment with glibenclamide results
in significant reduction in swelling and blebbing and cell death
induced by chemical ATP depletion. This channel participates in the
cation flux involved in cell swelling and cell death. A method of
the present invention includes the use of sulfonylurea compounds to
inhibit the flow of current through the NC.sub.Ca-ATP channel and
inhibit blebbing related to channel opening. Also, use of
sulfonylurea compounds and other compounds that inhibit the flow of
current through the NC.sub.Ca-ATP channel, thus can have a
therapeutic preventative effect on cell swelling and cell death in
the brain and spinal cord.
[0152] In some embodiments, the present invention is directed to
therapeutic compositions and methods of using the same. In one
embodiment, the therapeutic composition is an agonist and/or
antagonist of at least one NC.sub.Ca-ATP channel of a neuronal
cell, a neuroglial cell, or a neural endothelial cell. Further
embodiments of the present invention provide a composition
comprising a membrane preparation expressing the NC.sub.Ca-ATP
channel. For example, the membrane preparation is derived from
neural cells, such as isolated native reactive astrocytes (NRAs),
preferably freshly isolated native reactive astrocytes. The
NC.sub.Ca-ATP channel in the composition has the following
characteristics: (a) it is a 35 pS type channel; (b) it is
stimulated by cytoplasmic Ca.sup.2+; (c) it opens when cytoplasmic
ATP is less than about 0.8 .mu.M; and (d) it is permeable to the
monovalent cations K.sup.+, Cs.sup.+, Li.sup.+ and Na.sup.+ and it
can be blocked by antagonists of the type 1 sulfonylurea
receptor.
[0153] Furthermore, it is an object of the present invention to
provide a method of screening for one or more antagonists of the
NC.sub.Ca-ATP channel, comprising: (a) contacting a test compound
with a composition comprising the NC.sub.Ca-ATP channel; and (b)
identifying test compounds that inhibit an activity of said channel
by measuring said activity in the presence and absence of said test
compound, wherein a test compound that inhibits said activity is
identified as an antagonist of the NC.sub.Ca-ATP channel. For
example, the composition may contain a preparation of neural cells
expressing the NC.sub.Ca-ATP channel or a membrane preparation
expressing the NC.sub.Ca-ATP channel, such as a membrane
preparation derived from isolated native reactive astrocytes (NRAs)
or other cells that express the NC.sub.Ca-ATP channel. The effect
of the compound on this channel may include: (a) blocking the
NC.sub.Ca-ATP channel; (b) closing the NC.sub.Ca-ATP channel; (c)
preventing the NC.sub.Ca-ATP channel from opening; and (d) reducing
the magnitude of membrane current through the NC.sub.Ca-ATP
channel. It is also an object of the present invention to identify
a compound that is an NC.sub.Ca-ATP antagonist, including an
NC.sub.Ca-ATP channel inhibitor, an NC.sub.Ca-ATP channel blocker,
a SUR1 antagonist, SUR1 inhibitor, and/or a compound capable of
reducing the magnitude of membrane current though the channel.
[0154] It is a further object of the invention to provide a method
for identifying compounds that inhibit neural cell swelling,
comprising: (a) contacting a test compound with a composition
comprising the NC.sub.Ca-ATP channel, and (b) determining whether
the test compound blocks the NC.sub.Ca-ATP channel, wherein a test
compound that blocks the NC.sub.Ca-ATP channel is identified as a
compound for inhibiting neural cell swelling.
[0155] It is a further object of the present invention to provide a
method for identifying compounds that inhibit brain swelling,
comprising: (a) contacting a test compound with a composition
comprising the NC.sub.Ca-ATP channel, and (b) determining whether
the test compound blocks the NC.sub.Ca-ATP channel, wherein a test
compound that blocks the NC.sub.Ca-ATP channel is identified as a
compound for inhibiting brain swelling.
[0156] Yet another object of the present invention is to provide a
method for identifying compounds that inhibit brain swelling,
comprising: (a) contacting a test compound with a composition
comprising the NC.sub.Ca-ATP channel, and (b) determining whether
the test compound inhibits neural cell swelling, wherein a test
compound that inhibits neural cell swelling is identified as a
compound for inhibiting brain swelling.
[0157] A further object of the present invention provides a method
for identifying compounds that inhibit neural cell swelling in an
animal, comprising: (a) contacting a test compound with a
composition comprising the NC.sub.Ca-ATP channel and determining
whether the test compound blocks the channel, and (b) administering
the test compound to an animal having a brain injury or cerebral
ischemia, and determining whether the test compound that inhibits
brain swelling of the treated animal, wherein test compounds that
inhibit brain swelling are identified as compounds that inhibit
neural cell swelling in an animal.
[0158] It is a further object of the present invention to provide a
method for identifying compounds that inhibit brain swelling,
comprising: (a) contacting a test compound with a composition
comprising the NC.sub.Ca-ATP channel, and determining whether the
test compound blocks the channel, and (b) administering the test
compound to an animal having a brain injury or cerebral ischemia,
and determining whether the test compound inhibits brain swelling
of the treated animal, wherein test compounds that block the
NC.sub.Ca-ATP channel are identified as compounds that inhibit
brain swelling.
[0159] In each of these objects of the present invention, the
composition preferably comprises a preparation of neural cells
expressing the NC.sub.Ca-ATP channel or a membrane preparation
expressing the NC.sub.Ca-ATP channel, which preferably is derived
from isolated native reactive astrocytes (NRAs). It is a further
object of the present invention to provide the above methods using
a compound that is an antagonist of a type 1 sulfonylurea receptor,
such as a sulfonylurea compound, a benzamido derivative or an
imidazoline derivative.
[0160] It is a further object of the present invention to provide
these methods in which the determining step include, but are not
limited to, detecting or identifying swelling of the native
reactive astrocytes, such as by microscopic observation of cell
appearance (normal, blebbing, swelling); measuring channel
currents; measuring membrane potential; detecting expression of
annexin V; detecting expression of propidium iodide; in vitro
binding assays; and combinations thereof.
[0161] It is a further object of the present invention to provide a
method of preventing neural cell swelling in the brain of a
subject, said method comprising administering to the subject a
formulation containing an effective amount of a compound that
blocks the NC.sub.Ca-ATP channel and a pharmaceutically acceptable
carrier.
[0162] It is a further object of the present invention to provide a
method of alleviating the negative effects of traumatic brain
injury or cerebral ischemia stemming from neural cell swelling in a
subject, comprising administering to the subject a formulation
comprising an effective amount of a compound that blocks the
NC.sub.Ca-ATP channel and a pharmaceutically acceptable carrier.
Such administration may be delivery directly to the brain,
intravenous, subcutaneous, intramuscular, intracutaneous,
intragastric and oral administration. Examples of such compounds
include antagonist of a type 1 sulfonylurea receptor, such as
sulfonylureas like glibenclamide and tolbutamide, as well as other
insulin secretagogues such as repaglinide, nateglinide,
meglitinide, midaglizole, LY397364, LY389382, gliclazide,
glimepiride, MgADP, and combinations thereof.
[0163] It is yet another object of the present invention to provide
a formulation for preventing or inhibiting neural cell swelling in
the brain of a subject, using a formulation that includes a
compound that blocks the NC.sub.Ca-ATP channel and a
pharmaceutically acceptable carrier, wherein the quantity of said
compound is less than the quantity of said compound in formulations
for treating diabetes. It is a further object of the present
invention to provide a formulation for preventing or inhibiting
neural cell swelling in the brain of a subject, using a formulation
that includes a compound that blocks the NC.sub.Ca-ATP channel and
a pharmaceutically acceptable carrier, wherein the quantity of said
compound is at least 2 times less than the quantity of said
compound in formulations for treating diabetes. It is a further
object of the present invention to provide a formulation for
preventing or inhibiting neural cell swelling in the brain of a
subject, using a formulation that includes a compound that blocks
the NC.sub.Ca-ATP channel and a pharmaceutically acceptable
carrier, wherein the quantity of said compound is at least 5 times
less than the quantity of said compound in formulations for
treating diabetes. It is yet another object of the present
invention to provide a formulation for preventing or inhibiting
neural cell swelling in the brain of a subject, using a formulation
that includes a compound that blocks the NC.sub.Ca-ATP channel and
a pharmaceutically acceptable carrier, wherein the quantity of said
compound is at least 10 times less than the quantity of said
compound in formulations for treating diabetes.
[0164] It is therefore another object of the present invention to
provide a method for identifying compounds that inhibit neural cell
swelling, comprising: (a) contacting a test compound with a
composition comprising the Kir2.3 channel, and (b) determining
whether the test compound opens the Kir2.3 channel, wherein a test
compound that opens the Kir2.3 channel is identified as a compound
for inhibiting neural cell swelling.
[0165] It is yet another object of the present invention to provide
a method for a method for identifying compounds that inhibit brain
swelling, comprising: (a) contacting a test compound with a
composition comprising the Kir2.3 channel, and (b) determining
whether the test compound opens the Kir2.3 channel, wherein a test
compound that opens the Kir2.3 channel is identified as a compound
for inhibiting brain swelling.
[0166] It is yet another object of the present invention to provide
a method for a method for identifying compounds that inhibit neural
cell swelling and/or brain swelling in an animal, comprising: (a)
contacting a test compound with a composition comprising the Kir2.3
channel, and (b) determining whether the test compound opens the
Kir2.3 channel, wherein a test compound that opens the Kir2.3
channel is identified as a compound for inhibiting neural cell
swelling and/or brain swelling in an animal.
[0167] It is a further object of the present invention to provide a
method for identifying compounds that prevent, inhibit and/or
alleviate brain swelling in a subject, comprising: (a) contacting a
test compound with a composition comprising the Kir2.3 channel, and
determining whether the test compound opens the Kir2.3 channel, and
(b) administering the test compound to a subject having a brain
injury or cerebral ischemia, and determining whether the test
compound prevents, inhibits and/or alleviates brain swelling in the
subject, wherein test compounds that open the Kir2.3 channel are
identified as compounds that inhibit brain swelling.
[0168] It is a further object of the present invention to provide a
method for identifying compounds that inhibit neural cell swelling
in an animal, comprising: (a) contacting a test compound with a
composition comprising the Kir2.3 channel, and determining whether
the test compound opens the Kir2.3 channel, and (b) administering
the test compound to an animal having a brain injury or cerebral
ischemia, and determining whether the test compound inhibits brain
swelling of the treated animal, wherein test compounds that inhibit
brain swelling are identified as compounds that inhibit neural cell
swelling in an animal.
[0169] It is also an object of the present invention to provide a
method of preventing neural cell swelling in the brain of a
subject, said method comprising administering to the subject a
formulation containing an effective amount of a compound that opens
the Kir2.3 channel and a pharmaceutically acceptable carrier.
[0170] It is a further objection of the present invention to
provide a method of alleviating the negative effects of traumatic
brain injury or cerebral ischemia stemming from neural cell
swelling in a subject, comprising administering to the subject a
formulation comprising an effective amount of a compound that opens
the Kir2.3 channel and a pharmaceutically acceptable carrier. In
the object of the present invention that provide methods assessing
the effect of a compound on the Kir2.3 channel, a preferred
compound is Tenidap
(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-ca-
rboxamide). For example the formulation may provide a daily dose of
Tenidap that is from about 10 mg/day to about 500 mg/day, or, when
administered directly to the brain the daily dose of Tenidap is
from about 500 mg/day to 1.5 gms/day or greater.
IV. EXEMPLARY EMBODIMENTS OF THE PRESENT INVENTION
[0171] In addition to the sulfonylurea receptor 1 (SUR1) being
expressed in R1 astrocytes as part of the NC.sub.Ca-ATP channel,
the present invention further describes that the SUR1 regulatory
subunit of this channel is up-regulated in neurons and capillary
endothelial cells following ischemia, and blocking this receptor
reduces stroke size, cerebral edema and mortality. Thus,
antagonists of the NC.sub.Ca-ATP channel may have an important role
in preventing, alleviating, inhibiting and/or abrogating the
formation of cytotoxic and ionic edema.
[0172] In other embodiments, the therapeutic compound of the
present invention comprises an antagonist of a NC.sub.Ca-ATP
channel of a neuronal cell, a neuroglial cell, a neural endothelial
cell or a combination thereof. Antagonists are contemplated for use
in treating adverse conditions associated with hypoxia and/or
ischemia that result in increased intracranial pressure and/or
cytotoxic edema of the central nervous system. Such conditions
include trauma, ischemic brain injury, namely secondary neuronal
injury, and hemorrhagic infarction. Antagonists protect the cells
expressing the NC.sub.Ca-ATP channel, which is desirable for
clinical treatment in which gliotic capsule integrity is important
and must be maintained to prevent the spread of infection, such as
with a brain abscess. The protection via inhibition of the
NC.sub.Ca-ATP channel is associated with a reduction in cerebral
edema.
[0173] In one aspect, the NC.sub.Ca-ATP channel is blocked,
inhibited, or otherwise is decreased in activity. In such examples,
an antagonist of the NC.sub.Ca-ATP channel is administered and/or
applied. The antagonist modulates the NC.sub.Ca-ATP channel such
that flux through the channel is reduced, ceased, decreased and/or
stopped. The antagonist may have a reversible or an irreversible
activity with respect to the activity of the NC.sub.Ca-ATP channel
of the neuronal cell, neuroglial cell, endothelial cell or a
combination thereof. The antagonist may prevent or lessen the
depolarization of the cells thereby lessening cell swelling due to
osmotic changes that can result from depolarization of the cells.
Thus, inhibition of the NC.sub.Ca-ATP channel can reduce cytotoxic
edema and death of endothelial cells.
[0174] Subjects that can be treated with the therapeutic
composition of the present invention include, but are not limited
subjects suffering from or at risk of developing conditions
associated hypoxia and/or ischemia that result in increased
intracranial pressure and/or with cytotoxic edema of the central
nervous system (CNS). Such conditions include, but are not limited
to trauma (e.g., traumatic brain or spinal cord injury (TBI or
SCI), concussion) ischemic brain injury, hemorrhagic infarction,
stroke, atrial fibrillations, clotting disorders, pulmonary emboli,
arterio-venous malformations, mass-occupying lesions (e.g.,
hematomas), etc. Still further subjects at risk of developing such
conditions can include subjects undergoing treatments that increase
the risk of stroke, for example, surgery (vascular or
neurological), treatment of myocardial infarction with
thrombolytics, cerebral/endovascular treatments, stent placements,
angiography, etc.
[0175] Another aspect of the present invention for the treatment of
ischemia, brain trauma, or other brain injury comprises
administration of an effective amount of a SUR1 antagonist and
administration of glucose. Glucose administration may be at the
time of treatment with an antagonist of the NC.sub.Ca-ATP channel,
such as a SUR1 antagonist, or may follow treatment with an
antagonist of the NC.sub.Ca-ATP channel (e.g., at 15 minutes after
treatment with an antagonist of the NC.sub.Ca-ATP channel, or at
one half hour after treatment with an antagonist of the
NC.sub.Ca-ATP channel, or at one hour after treatment with an
antagonist of the NC.sub.Ca-ATP channel, or at two hours after
treatment with an antagonist of the NC.sub.Ca-ATP channel, or at
three hours after treatment with an antagonist of the NC.sub.Ca-ATP
channel). Glucose administration may be by intravenous, or
intraperitoneal, or other suitable route and means of delivery.
Additional glucose allows administration of higher doses of an
antagonist of the NC.sub.Ca-ATP channel than might otherwise be
possible, so that combined glucose with an antagonist of the
NC.sub.Ca-ATP channel provides greater protection, and may allow
treatment at later times, than with an antagonist of the
NC.sub.Ca-ATP channel alone. Greater amounts of glucose are
administered where larger doses of an antagonist of the
NC.sub.Ca-ATP channel are administered.
[0176] Another aspect of the present invention comprises
co-administration of an antagonist of the NC.sub.Ca-ATP channel
with a thrombolytic agent. Co-administration of these two compound
increases the therapeutic window of the thrombolytic agent by
reducing hemorrhagic conversion. The therapeutic window for
thrombolytic agents may be increased by several (4-8) hours by
co-administering antagonist of the NC.sub.Ca-ATP channel. In
addition to a thrombolytic agent, other agents can be used in
combination with the antagonist of the present invention, for
example, but not limited to antiplatelets, anticoagulants,
vasodilators, statins, diuretics, etc.
[0177] Another aspect of the present invention comprises the use of
labeled SUR1 antagonists to diagnose, determine or monitor stages
of stroke, cerebral edema or visualize the size/boundaries/borders
of a tumor and/or the stroke. For example, the penumbra following
the stroke may be monitored or visualized using labeled SUR1
antagonists.
[0178] Yet further, the compositions of the present invention can
be used to produce neuroprotective kits that are used to treat
subjects at risk or suffering from conditions that are associated
with cytotoxic cerebral edema.
V. EXEMPLARY METHODS OF THE PRESENT INVENTION
[0179] The present invention provides a previously unknown ion
channel found in mammalian neural cells that plays a role in cell
swelling and brain swelling. The present invention further provides
a method of screening for antagonists to the channel and a new use
for antagonists to the channel, including sulfonylurea compounds
such as glibenclamide and tolbutamide, as a treatment for brain
swelling in mammals.
[0180] Methods of the present invention for identifying compounds
that interact with, (e.g., bind to, open, block) the NC.sub.Ca-ATP
channel and employ (i) cell based assays and/or (ii) non-cell based
assay systems. Such compounds may act as antagonists or agonists of
NC.sub.Ca-ATP channel activity. In a preferred embodiment of the
present invention, antagonists that block and/or inhibit the
permeability of the NC.sub.Ca-ATP channel are utilized in methods
for treating neural cell swelling and/or brain swelling.
[0181] The cell based assays use neural cells that express the
NC.sub.Ca-ATP channel, preferably a functional NC.sub.Ca-ATP
channel; the preferred cells are NRAs. The non-cell based assay
systems include membrane preparations that express the
NC.sub.Ca-ATP channel, preferably a functional NC.sub.Ca-ATP
channel. Cell-based assays include, but are not limited to,
compound binding assays, microscopic observation of cell status
(normal, blebbing, swelling, cell death), and measuring channel
currents both before and after exposure to compound. Compositions
comprising membrane preparations expressing the NC.sub.Ca-ATP
channel may be used to identify compounds that interact with, bind
to, block or open the NC.sub.Ca-ATP channel or SUR1. The term
"expressing the NC.sub.Ca-ATP channel" or "expresses the
NC.sub.Ca-ATP channel" means having a functional NC.sub.Ca-ATP
channel. The term "functional NC.sub.Ca-ATP channel" as used herein
means an NC.sub.Ca-ATP channel capable of being detected. One
preferred method of detecting the NC.sub.Ca-ATP channel is by
determining, in vitro or in vivo, whether the channel is open,
closed and/or blocked.
[0182] For example, in a typical experiment using a membrane
preparation, NRAs that express the NC.sub.Ca-ATP channel are used
to produce the membrane preparation. Methods for producing
membranes from whole cells and tissues are well known in the art.
One such method produces purified cell membranes in the form of a
purified microsomal fraction isolated from disrupted cells or a
tissue sample by discontinuous sucrose gradient centrifugation.
Also included are membranes comprised of cell-attached patches,
inside-out patches, or outside-out patches. One example of a tissue
sample expressing NC.sub.Ca-ATP channels is brain tissue adjacent
to brain injury.
[0183] The membrane preparations are used in a number of assays,
including, but not limited to measuring channel currents, both
before and after exposure to compound; and in vitro binding assays.
The experimental conditions for such assays to determine and
quantify the status of the NC.sub.Ca-ATP channel are described
throughout the instant specification, including binding assay
conditions, bath compositions, pipette solutions, concentrations of
ATP and Ca.sup.2+ required, membrane voltage, membrane potentials,
compound quantity ranges, controls, etc.
[0184] Binding assays and competitive binding assays employ a
labeled ligand or antagonist of the NC.sub.Ca-ATP channel. In one
such experiment, labeled Glibenclamide, such as FITC-conjugated
glibenclamide or BODIPY-conjugated glibenclamide or radioactively
labeled glibenclamide is bound to the membranes and assayed for
specific activity; specific binding is determined by comparison
with binding assays performed in the presence of excess unlabelled
antagonist.
[0185] In one method for identifying NC.sub.Ca-ATP channel
blockers, membranes are incubated with a labeled compound shown to
block this channel, in either the presence or absence of test
compound. Compounds that block the NC.sub.Ca-ATP channel and
compete with the labeled compound for binding to the membranes will
have a reduced signal, as compared to the vehicle control samples.
In another aspect of the invention the screens may be designed to
identify compounds that compete with the interaction between
NC.sub.Ca-ATP channel and a known (previously identified herein)
NC.sub.Ca-ATP channel antagonist or SUR1 antagonist, such as
glibenclamide. In such screens, the known NC.sub.Ca-ATP channel
antagonist or SUR1 antagonist is labeled and the test compounds are
then assayed for their ability to compete with or antagonize the
binding of the labeled antagonist.
[0186] The assays described herein can be used to identify
compounds that modulate or affect NC.sub.Ca-ATP channel activity.
For example, compounds that affect NC.sub.Ca-ATP channel activity
include but are not limited to compounds that bind to the
NC.sub.Ca-ATP channel or SUR1, inhibit binding of identified
blockers or ligands (such as glibenclamide), and either
open/activate the channel (agonists) or block/inhibit the channel
(antagonists).
[0187] Assays described can also identify compounds that modulate
neural cell swelling (e.g., compounds which affect other events
involved in neural cell swelling that are activated by ligand
binding to or blocking of the NC.sub.Ca-ATP channel).
VI. COMPOUNDS SCREENED IN ACCORDANCE WITH THE INVENTION
[0188] The compounds for screening in accordance with the invention
include, but are not limited to organic compounds, peptides,
antibodies and fragments thereof, peptidomimetics, that bind to the
NC.sub.Ca-ATP channel and either open the channel (i.e., agonists)
or block the channel (i.e., antagonists). For use in the treatment
of neural cell swelling or brain swelling, compounds that block the
channel are preferred. Agonists that open or maintain the channel
in the open state include peptides, antibodies or fragments
thereof, and other organic compounds that include the SUR1 subunit
of the NC.sub.Ca-ATP channel (or a portion thereof) and bind to and
"neutralize" circulating ligand for SUR1.
[0189] With reference to screening of compounds that affect the
NC.sub.Ca-ATP channel, libraries of known compounds can be
screened, including natural products or synthetic chemicals, and
biologically active materials, including proteins, for compounds
which are inhibitors or activators. Preferably, such a compound is
an NC.sub.Ca-ATP antagonist, which includes an NC.sub.Ca-ATP
channel inhibitor, an NC.sub.Ca-ATP channel blocker, a SUR1
antagonist, SUR1 inhibitor, and/or a compound capable of reducing
the magnitude of membrane current through the channel.
[0190] Compounds may include, but are not limited to, small organic
or inorganic molecules, compounds available in compound libraries,
peptides such as, for example, soluble peptides, including but not
limited to members of random peptide libraries; (see, e.g., Lam, K.
S. et al., 1991, Nature 354: 82-84; Houghten, R. et al., 1991,
Nature 354: 84-86), and combinatorial chemistry-derived molecular
library made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to, members of random
or partially degenerate, directed phosphopeptide libraries; see,
e.g., Songyang, Z. et al., 1993, Cell 72: 767-778), antibodies
(including, but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression library fragments, and
epitope-binding fragments thereof).
[0191] Other compounds which can be screened in accordance with the
invention include but are not limited to small organic molecules
that may or may not be able to cross the blood-brain barrier, gain
entry into an appropriate neural cell and affect the expression of
the NC.sub.Ca-ATP channel gene or some other gene involved in the
NC.sub.Ca-ATP channel activity (e.g., by interacting with the
regulatory region or transcription factors involved in gene
expression); or such compounds that affect the activity of the
NC.sub.Ca-ATP channel or the activity of some other intracellular
factor involved in the NC.sub.Ca-ATP channel activity.
[0192] Computer modeling and searching technologies permit
identification of compounds, or the improvement of already
identified compounds, that can modulate NC.sub.Ca-ATP channel
activity or expression. Having identified such a compound or
composition, the active sites or regions are identified. Such
active sites might typically be ligand binding sites. The active
site can be identified using methods known in the art including,
for example, from study of complexes of the relevant compound or
composition with other ligands, from the amino acid sequences of
peptides, or from the nucleotide sequences of nucleic acids.
Chemical or X-ray crystallographic methods can be used to study
complexes of the relevant compound to find the active site. The
three dimensional geometric structure of the active site is
determined. This can be done by known methods, including X-ray
crystallography, which can determine a complete molecular
structure. On the other hand, solid or liquid phase NMR can be used
to determine certain intra-molecular distances. Any other
experimental method of structure determination can be used to
obtain partial or complete geometric structures. The geometric
structures may be measured with a complexed ligand, natural which
may increase the accuracy of the active site structure
determined.
[0193] If an incomplete or insufficiently accurate structure is
determined, the methods of computer based numerical modeling can be
used to complete the structure or improve its accuracy. Any
recognized modeling method may be used, including parameterized
models specific to particular biopolymers such as proteins or
nucleic acids, molecular dynamics models based on computing
molecular motions, statistical mechanics models based on thermal
ensembles, or combined models. For most types of models, standard
molecular force fields, representing the forces between constituent
atoms and groups, are necessary, and can be selected from force
fields known in physical chemistry. The incomplete or less accurate
experimental structures can serve as constraints on the complete
and more accurate structures computed by these modeling
methods.
[0194] Finally, having determined the structure of the active site,
either experimentally, by modeling, or by a combination, candidate
modulating compounds can be identified by searching databases
containing compounds along with information on their molecular
structure. Such a search seeks compounds having structures that
match the determined active site structure and that interact with
the groups defining the active site. Such a search can be manual,
but is preferably computer assisted. These compounds found from
this search are potential NC.sub.Ca-ATP channel modulating,
preferably blocking, compounds.
[0195] Alternatively, these methods can be used to identify
improved modulating compounds from an already known modulating
compound or ligand. The composition of the known compound can be
modified and the structural effects of modification can be
determined using the experimental and computer modeling methods
described above applied to the new composition. The altered
structure is then compared to the active site structure of the
compound to determine if an improved fit or interaction results. In
this manner systematic variations in composition, such as by
varying side groups, can be quickly evaluated to obtain modified
modulating compounds or ligands of improved specificity or
activity.
[0196] Examples of molecular modeling systems are the CHARMm and
QUANTA programs (Polygen Corporation, Waltham, Mass.). CHARMm
performs the energy minimization and molecular dynamics functions.
QUANTA performs the construction, graphic modeling and analysis of
molecular structure. QUANTA allows interactive construction,
modification, visualization, and analysis of the behavior of
molecules with each other. A number of articles review computer
modeling of drugs interactive with specific proteins, such as
Rotivinen, et al.) 1988, Acta Pharmaceutical Fennica 97: 159-166);
Ripka (1988 New Scientist 54-57); McKinaly and Rossmann (1 989,
Annu. Rev. Pharmacol. Toxicol. 29: 11 1-122); Perry and Davies,
OSAR: Quantitative Structure-Activity Relationships in Drug Design
pp. 189-193 Alan R. Liss, Inc. 1989; Lewis and Dean (1989, Proc. R.
SOC. Lond. 236: 125-140 and 141-162); and, with respect to a model
receptor for nucleic acid components, Askew, et al. (1989, J. Am.
Chem. SOC. 11 1: 1082-1 090). Other computer programs that screen
and graphically depict chemicals are available from companies such
as BioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga,
Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario).
Although these are primarily designed for application to drugs
specific to particular proteins, they can be adapted to design of
drugs specific to regions of DNA or RNA, once that region is
identified.
[0197] Compounds identified via assays such as those described
herein may be useful, for example, in elaborating the biological
function of the NC.sub.Ca-ATP channel and for relief of brain
swelling.
[0198] Assays for testing the efficacy of compounds identified in
the cellular screen can be tested in animal model systems for brain
or spinal cord swelling. Such animal models may be used as test
substrates for the identification of drugs, pharmaceuticals,
therapies and interventions which may be effective in treating
brain or spinal cord swelling. For example, animal models of brain
swelling, such as brain injury, may be exposed to a compound,
suspected of exhibiting an ability to inhibit brain swelling, at a
sufficient concentration and for a time sufficient to elicit such
an inhibition of brain swelling in the exposed animals. The
response of the animals to the exposure may be monitored using
visual means (e.g., radiological, CAT, MRI), measurement of
intracranial pressure, and/or the reversal of symptoms associated
with brain swelling. With regard to intervention, any treatments
which reverse any aspect of brain swelling-associated symptoms
should be considered as candidates for brain swelling therapeutic
intervention. Dosages of test agents may be determined by deriving
dose-response curves, as discussed herein.
[0199] Accordingly, the present invention is useful in the
treatment or alleviation of neural cell swelling and death and
brain swelling, especially those brain insults related to traumatic
brain injury, spinal cord injury, central or peripheral nervous
system damage, cerebral ischemia, such as stroke, or complications
involving and/or stemming from edema, injury, or trauma. Such
damage or complications may be characterized by an apparent brain
damage or aberration, the symptoms of which can be reduced by the
methods of the present invention including the administration of an
effective amount of the active compounds or substances described
herein. According to a specific embodiment of the present invention
the administration of effective amounts of the active compound can
block the channel, which if it remained open would lead to neural
cell swelling and cell death. A variety of antagonists to SUR1 are
suitable for blocking the channel. Examples of suitable SUR1
antagonists include, but are not limited to glibenclamide,
tolbutamide, repaglinide, nateglinide, meglitinide, midaglizole,
LY397364, LY3 89382, gliclazide, glimepiride, MgADP, and
combinations thereof. In a preferred embodiment of the invention
the SUR1 antagonists is selected from the group consisting of
glibenclamide and tolbutamide. Still other therapeutic "strategies"
for preventing neural cell swelling and cell death can be adopted
including, but not limited to methods that maintain the neural cell
in a polarized state and methods that prevent strong
depolarization.
[0200] A. Modulators of the NC.sub.Ca-ATP Channel
[0201] The present invention comprises modulators of the channel,
for example one or more agonists and/or one or more antagonists of
the channel. Examples of antagonists or agonists of the present
invention may encompass respective antagonists and/or agonists
identified in US Application Publication No. 20030215889, which is
incorporated herein by reference in its entirety. One of skill in
the art is aware that the NC.sub.Ca-ATP channel is comprised of at
least two subunits: the regulatory subunit, SUR1, and the pore
forming subunit.
[0202] B. Modulators of SUR1
[0203] In certain embodiments, antagonists to sulfonylurea
receptor-1 (SUR1) are suitable for blocking the channel. Examples
of suitable SUR1 antagonists include, but are not limited to
glibenclamide, tolbutamide, repaglinide, nateglinide, meglitinide,
midaglizole, LY397364, LY389382, glyclazide, glimepiride, estrogen,
estrogen related-compounds estrogen related-compounds (estradiol,
estrone, estriol, genistein, non-steroidal estrogen (e.g.,
diethystilbestrol), phytoestrogen (e.g., coumestrol), zearalenone,
etc.) and combinations thereof. In a preferred embodiment of the
invention the SUR1 antagonists is selected from the group
consisting of glibenclamide and tolbutamide. Yet further, another
antagonist can be MgADP. Other antagonist include blockers of
K.sub.ATP channels, for example, but not limited to tolbutamide,
glyburide (1[p-2[5-chloro-O-anisamido)ethyl] phenyl]
sulfonyl]-3-cyclohexyl-3-urea); chlopropamide
(1-[[(p-chlorophenyl)sulfonyl]-3-propylurea; glipizide
(1-cyclohexyl-3 [[p-[2(5-methylpyrazine carboxamido) ethyl] phenyl]
sulfonyl] urea); or
tolazamide(benzenesulfonamide-N-[[(hexahydro-1H-azepin-1yl)amino]
carbonyl]-4-methyl).
[0204] Agonists that may be used in the present invention include,
but are not limited to, one or more agonists of SUR1, for example,
diazoxide, pinacidil, P1075, cromakalin or activators of K.sub.ATP
channels. Other agonists can include, but are not limited to
diazoixde derivatives, for example
3-isopropylamino-7-methoxy-4H-1,2,4-benzothiadiazine 1,1-dioxide
(NNC 55-9216),
6,7-dichloro-3-isopropylamino-4H-1,2,4-benzothiadiazine 1,1-dioxide
(BPDZ 154), 7-chloro-3-isopropylamino-4H-1,2,4-benzothiadiazine
1,1-dioxide (BPDZ 73),
6-Chloro-3-isopropylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine
1,1-dioxide (NNC 55-0118)4,
6-chloro-3-(1-methylcyclopropyl)amino-4H-thieno[3,2-e]-1,2,4-thiadiazine
1,1-dioxide (NN414),
3-(3-methyl-2-butylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxide (BPDZ 44),
3-(1',2',2'-trimethylpropyl)amino-4H-pyrido(4,3-e)-1,2,4-thiadiazine
1,1-dioxide (BPDZ 62), 3-(1',2',2'-trimethylpropyl)amine-4H-pyrido
(2,3-e)-1,2,4-thiadiazine, 1,1-dioxide (BPDZ 79),
2-alkyl-3-alkylamino-2H-benzo- and
2-alkyl-3-alkylamino-2H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxides,
6-Chloro-3-alkylamino-4H-thieno[3,2-e]-1,2,4-thiadiazine
1,1-dioxide derivatives, 4-N-Substituted and -unsubstituted
3-alkyl- and 3-(alkylamino)-4H-pyrido[4,3-e]-1,2,4-thiadiazine
1,1-dioxides. In addition, other compounds, including
6-chloro-2-methylquinolin-4(1H)-one (HEI 713) and LN 533021, as
well as the class of drugs, arylcyanoguanidines, are known
activators or agonist of SUR1.
[0205] C. Modulators of SUR1 Transcription and/or Translation
[0206] In certain embodiments, the modulator can comprise a
compound (protein, nucleic acid, siRNA, etc.) that modulates
transcription and/or translation of SUR1 (regulatory subunit)
and/or the molecular entities that comprise the pore-forming
subunit.
[0207] D. Transcription Factors
[0208] Transcription factors are regulatory proteins that binds to
a specific DNA sequence (e.g., promoters and enhancers) and
regulate transcription of an encoding DNA region. Thus,
transcription factors can be used to modulate the expression of
SUR1. Typically, a transcription factor comprises a binding domain
that binds to DNA (a DNA-binding domain) and a regulatory domain
that controls transcription. Where a regulatory domain activates
transcription, that regulatory domain is designated an activation
domain. Where that regulatory domain inhibits transcription, that
regulatory domain is designated a repression domain. More
specifically, transcription factors such as Sp1, HIF1.alpha., and
NF.kappa.B can be used to modulate expression of SUR1.
[0209] In particular embodiments of the invention, a transcription
factor may be targeted by a composition of the invention. The
transcription factor may be one that is associated with a pathway
in which SUR1 is involved. The transcription factor may be targeted
with an antagonist of the invention, including siRNA to
downregulate the transcription factor. Such antagonists can be
identified by standard methods in the art, and in particular
embodiments the antagonist is employed for treatment and or
prevention of an individual in need thereof. In an additional
embodiment, the antagonist is employed in conjunction with an
additional compound, such as a composition that modulates the
NC.sub.CA-ATP channel of the invention. For example, the antagonist
may be used in combination with an inhibitor of the channel of the
invention. When employed in combination, the antagonist of a
transcription factor of a SUR1-related pathway may be administered
prior to, during, and/or subsequent to the additional compound.
[0210] E. Antisense and Ribozymes
[0211] An antisense molecule that binds to a translational or
transcriptional start site, or splice junctions, are ideal
inhibitors. Antisense, ribozyme, and double-stranded RNA molecules
target a particular sequence to achieve a reduction or elimination
of a particular polypeptide, such as SUR1. Thus, it is contemplated
that antisense, ribozyme, and double-stranded RNA, and RNA
interference molecules are constructed and used to modulate SUR1
expression.
[0212] F. Antisense Molecules
[0213] Antisense methodology takes advantage of the fact that
nucleic acids tend to pair with complementary sequences. By
complementary, it is meant that polynucleotides are those which are
capable of base-pairing according to the standard Watson-Crick
complementarity rules. That is, the larger purines will base pair
with the smaller pyrimidines to form combinations of guanine paired
with cytosine (G:C) and adenine paired with either thymine (A:T) in
the case of DNA, or adenine paired with uracil (A:U) in the case of
RNA. Inclusion of less common bases such as inosine,
5-methylcytosine, 6-methyladenine, hypoxanthine and others in
hybridizing sequences does not interfere with pairing.
[0214] Targeting double-stranded (ds) DNA with polynucleotides
leads to triple-helix formation; targeting RNA will lead to
double-helix formation. Antisense polynucleotides, when introduced
into a target cell, specifically bind to their target
polynucleotide and interfere with transcription, RNA processing,
transport, translation and/or stability. Antisense RNA constructs,
or DNA encoding such antisense RNAs, are employed to inhibit gene
transcription or translation or both within a host cell, either in
vitro or in vivo, such as within a host animal, including a human
subject.
[0215] Antisense constructs are designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. It is contemplated that the most effective
antisense constructs may include regions complementary to
intron/exon splice junctions. Thus, antisense constructs with
complementarity to regions within 50-200 bases of an intron-exon
splice junction are used. It has been observed that some exon
sequences can be included in the construct without seriously
affecting the target selectivity thereof. The amount of exonic
material included will vary depending on the particular exon and
intron sequences used. One can readily test whether too much exon
DNA is included simply by testing the constructs in vitro to
determine whether normal cellular function is affected or whether
the expression of related genes having complementary sequences is
affected.
[0216] It is advantageous to combine portions of genomic DNA with
cDNA or synthetic sequences to generate specific constructs. For
example, where an intron is desired in the ultimate construct, a
genomic clone will need to be used. The cDNA or a synthesized
polynucleotide may provide more convenient restriction sites for
the remaining portion of the construct and, therefore, would be
used for the rest of the sequence.
[0217] G. RNA Interference
[0218] It is also contemplated in the present invention that
double-stranded RNA is used as an interference molecule, e.g., RNA
interference (RNAi). RNA interference is used to "knock down" or
inhibit a particular gene of interest by simply injecting, bathing
or feeding to the organism of interest the double-stranded RNA
molecule. This technique selectively "knock downs" gene function
without requiring transfection or recombinant techniques (Giet,
2001; Hammond, 2001; Stein P, et al., 2002; Svoboda P, et al.,
2001; Svoboda P, et al., 2000).
[0219] Another type of RNAi is often referred to as small
interfering RNA (siRNA), which may also be utilized to inhibit
SUR1. A siRNA may comprises a double stranded structure or a single
stranded structure, the sequence of which is "substantially
identical" to at least a portion of the target gene (See WO
04/046320, which is incorporated herein by reference in its
entirety). "Identity," as known in the art, is the relationship
between two or more polynucleotide (or polypeptide) sequences, as
determined by comparing the sequences. In the art, identity also
means the degree of sequence relatedness between polynucleotide
sequences, as determined by the match of the order of nucleotides
between such sequences. Identity can be readily calculated. See,
for example: Computational Molecular Biology, Lesk, A. M., ed.
Oxford University Press, New York, 1988; Biocomputing: Informatics
and Genome Projects, Smith, D. W., ea., Academic Press, New York,
1993, and the methods disclosed in WO 99/32619, WO 01/68836, WO
00/44914, and WO 01/36646, specifically incorporated herein by
reference. While a number of methods exist for measuring identity
between two nucleotide sequences, the term is well known in the
art. Methods for determining identity are typically designed to
produce the greatest degree of matching of nucleotide sequence and
are also typically embodied in computer programs. Such programs are
readily available to those in the relevant art. For example, the
GCG program package (Devereux et al.), BLASTP, BLASTN, and FASTA
(Atschul et al.,) and CLUSTAL (Higgins et al., 1992; Thompson, et
al., 1994).
[0220] Thus, siRNA contains a nucleotide sequence that is
essentially identical to at least a portion of the target gene, for
example, SUR1, or any other molecular entity associated with the
NC.sub.Ca-ATP channel such as the pore-forming subunit. One of
skill in the art is aware that the nucleic acid sequences for SUR1
are readily available in GenBank, for example, GenBank accession
L40624, which is incorporated herein by reference in its entirety.
Preferably, the siRNA contains a nucleotide sequence that is
completely identical to at least a portion of the target gene. Of
course, when comparing an RNA sequence to a DNA sequence, an
"identical" RNA sequence will contain ribonucleotides where the DNA
sequence contains deoxyribonucleotides, and further that the RNA
sequence will typically contain a uracil at positions where the DNA
sequence contains thymidine.
[0221] One of skill in the art will appreciate that two
polynucleotides of different lengths may be compared over the
entire length of the longer fragment. Alternatively, small regions
may be compared. Normally sequences of the same length are compared
for a final estimation of their utility in the practice of the
present invention. It is preferred that there be 100% sequence
identity between the dsRNA for use as siRNA and at least 15
contiguous nucleotides of the target gene (e.g., SUR1), although a
dsRNA having 70%, 75%, 80%, 85%, 90%, or 95% or greater may also be
used in the present invention. A siRNA that is essentially
identical to a least a portion of the target gene may also be a
dsRNA wherein one of the two complementary strands (or, in the case
of a self-complementary RNA, one of the two self-complementary
portions) is either identical to the sequence of that portion or
the target gene or contains one or more insertions, deletions or
single point mutations relative to the nucleotide sequence of that
portion of the target gene. siRNA technology thus has the property
of being able to tolerate sequence variations that might be
expected to result from genetic mutation, strain polymorphism, or
evolutionary divergence.
[0222] There are several methods for preparing siRNA, such as
chemical synthesis, in vitro transcription, siRNA expression
vectors, and PCR expression cassettes. Irrespective of which method
one uses, the first step in designing an siRNA molecule is to
choose the siRNA target site, which can be any site in the target
gene. In certain embodiments, one of skill in the art may manually
select the target selecting region of the gene, which may be an ORF
(open reading frame) as the target selecting region and may
preferably be 50-100 nucleotides downstream of the "ATG" start
codon. However, there are several readily available programs
available to assist with the design of siRNA molecules, for example
siRNA Target Designer by Promega, siRNA Target Finder by GenScript
Corp., siRNA Retriever Program by Imgenex Corp., EMBOSS siRNA
algorithm, siRNA program by Qiagen, Ambion siRNA predictor, Ambion
siRNA predictor, Whitehead siRNA prediction, and Sfold. Thus, it is
envisioned that any of the above programs may be utilized to
produce siRNA molecules that can be used in the present
invention.
[0223] H. Ribozymes
[0224] Ribozymes are RNA-protein complexes that cleave nucleic
acids in a site-specific fashion. Ribozymes have specific catalytic
domains that possess endonuclease activity (Kim and Cech, 1987;
Forster and Symons, 1987). For example, a large number of ribozymes
accelerate phosphoester transfer reactions with a high degree of
specificity, often cleaving only one of several phosphoesters in an
oligonucleotide substrate (Cech et al., 1981; Reinhold-Hurek and
Shub, 1992). This specificity has been attributed to the
requirement that the substrate bind via specific base-pairing
interactions to the internal guide sequence ("IGS") of the ribozyme
prior to chemical reaction.
[0225] Ribozyme catalysis has primarily been observed as part of
sequence specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cech et al., 1981). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known
ribonucleases and approaching that of the DNA restriction enzymes.
Thus, sequence-specific ribozyme-mediated inhibition of gene
expression is particularly suited to therapeutic applications
(Scanlon et al., 1991; Sarver et al., 1990; Sioud et al., 1992).
Most of this work involved the modification of a target mRNA, based
on a specific mutant codon that is cleaved by a specific ribozyme.
In light of the information included herein and the knowledge of
one of ordinary skill in the art, the preparation and use of
additional ribozymes that are specifically targeted to a given gene
will now be straightforward.
[0226] Other suitable ribozymes include sequences from RNase P with
RNA cleavage activity (Yuan et al., 1992; Yuan and Altman, 1994),
hairpin ribozyme structures (Berzal-Herranz et al., 1992; Chowrira
et al., 1993) and hepatitis .delta. virus based ribozymes (Perrotta
and Been, 1992). The general design and optimization of ribozyme
directed RNA cleavage activity has been discussed in detail
(Haseloff and Gerlach, 1988; Symons, 1992; Chowrira, et al., 1994;
and Thompson, et al., 1995).
[0227] The other variable on ribozyme design is the selection of a
cleavage site on a given target RNA. Ribozymes are targeted to a
given sequence by virtue of annealing to a site by complimentary
base pair interactions. Two stretches of homology are required for
this targeting. These stretches of homologous sequences flank the
catalytic ribozyme structure defined above. Each stretch of
homologous sequence can vary in length from 7 to 15 nucleotides.
The only requirement for defining the homologous sequences is that,
on the target RNA, they are separated by a specific sequence which
is the cleavage site. For hammerhead ribozymes, the cleavage site
is a dinucleotide sequence on the target RNA, uracil (U) followed
by either an adenine, cytosine or uracil (A,C or U; Perriman, et
al., 1992; Thompson, et al., 1995). The frequency of this
dinucleotide occurring in any given RNA is statistically 3 out of
16.
[0228] Designing and testing ribozymes for efficient cleavage of a
target RNA is a process well known to those skilled in the art.
Examples of scientific methods for designing and testing ribozymes
are described by Chowrira et al. (1994) and Lieber and Strauss
(1995), each incorporated by reference. The identification of
operative and preferred sequences for use in SUR1 targeted
ribozymes is simply a matter of preparing and testing a given
sequence, and is a routinely practiced screening method known to
those of skill in the art.
[0229] I. Inhibition of Post-Translational Assembly and
Trafficking
[0230] Following expression of individual regulatory and
pore-forming subunit proteins of the channel, and in particular
aspects of the invention, these proteins are modified by
glycosylation in the Golgi apparatus of the cell, assembled into
functional heteromultimers that comprise the channel, and then
transported to the plasmalemmal membrane where they are inserted to
form functional channels. The last of these processes is referred
to as "trafficking".
[0231] In specific embodiments of the invention, molecules that
bind to any of the constituent proteins interfere with
post-translational assembly and trafficking, and thereby interfere
with expression of functional channels. One such example is with
glibenclamide binding to SUR1 subunits. In additional embodiments,
glibenclamide, which binds with femtomolar affinity to SUR1,
interferes with post-translational assembly and trafficking
required for functional channel expression.
VII. EXEMPLARY METHODS OF SCREENING FOR MODULATORS
[0232] Further embodiments of the present invention can include
methods for identifying modulators of the NC.sub.Ca-ATP channel,
for example, agonist or antagonist, that modify the activity and/or
expression. These assays may comprise random screening of large
libraries of candidate substances; alternatively, the assays may be
used to focus on particular classes of compounds selected with an
eye towards structural attributes that are believed to make them
more likely to modulate the function or activity or expression of
the NC.sub.Ca-ATP channel.
[0233] By function, it is meant that one may assay for mRNA
expression, protein expression, protein activity, or channel
activity, more specifically, the ability of the modulator to open
or inhibit or block the NC.sub.Ca-ATP channel. Thus, the compounds
for screening in accordance with the invention include, but are not
limited to natural or synthetic organic compounds, peptides,
antibodies and fragments thereof, peptidomimetics, that bind to the
NC.sub.Ca-ATP channel and either open the channel (e.g., agonists)
or block the channel (e.g., antagonists). For use in the treatment
of neural cell swelling or brain swelling, compounds that block the
channel are preferred. Agonists that open or maintain the channel
in the open state include peptides, antibodies or fragments
thereof, and other organic compounds that include the SUR1 subunit
of the NC.sub.Ca-ATP channel (or a portion thereof) and bind to and
"neutralize" circulating ligand for SUR1.
[0234] With reference to screening of compounds that affect the
NC.sub.Ca-ATP channel, libraries of known compounds can be
screened, including natural products or synthetic chemicals, and
biologically active materials, including proteins, for compounds
which are inhibitors or activators. Preferably, such a compound is
an NC.sub.Ca-ATP antagonist, which includes an NC.sub.Ca-ATP
channel inhibitor, an NC.sub.Ca-ATP channel blocker, a SUR1
antagonist, SUR1 inhibitor, and/or a compound capable of reducing
the magnitude of membrane current through the channel.
[0235] Compounds may include, but are not limited to, small organic
or inorganic molecules, compounds available in compound libraries,
peptides such as, for example, soluble peptides, including but not
limited to members of random peptide libraries; (see, e.g., Lam, K.
S. et al., 1991, Nature 354: 82-84; Houghten, R. et al., 1991,
Nature 354: 84-86), and combinatorial chemistry-derived molecular
library made of D- and/or L-configuration amino acids,
phosphopeptides (including, but not limited to, members of random
or partially degenerate, directed phosphopeptide libraries; see,
e.g., Songyang, Z. et al., 1993, Cell 72: 767-778), antibodies
(including, but not limited to, polyclonal, monoclonal, humanized,
anti-idiotypic, chimeric or single chain antibodies, and FAb,
F(ab').sub.2 and FAb expression library fragments, and
epitope-binding fragments thereof).
[0236] Other compounds that can be screened in accordance with the
invention include but are not limited to small organic molecules
that may or may not cross the blood-brain barrier, gain entry into
an appropriate neural or endothelial cell and affect the expression
of the NC.sub.Ca-ATP channel gene or some other gene involved in
the NC.sub.Ca-ATP channel activity (e.g., by interacting with the
regulatory region or transcription factors involved in gene
expression, or by interfering with post-translational channel
assembly or trafficking); or such compounds that affect the
activity of the NC.sub.Ca-ATP channel or the activity of some other
intracellular factor involved in the NC.sub.Ca-ATP channel
activity.
[0237] To identify, make, generate, provide, manufacture or obtain
modulator, one generally will determine the activity of the
NC.sub.Ca-ATP channel in the presence, absence, or both of the
candidate substance, wherein an inhibitor or antagonist is defined
as any substance that down-regulates, reduces, inhibits, blocks or
decreases the NC.sub.Ca-ATP channel expression or activity, and
wherein an activator or agonist is defined as any substance that
up-regulates, enhances, activates, increases or opens the
NC.sub.Ca-ATP channel. For example, a method may generally
comprise:
[0238] (a) providing a candidate substance suspected of activating
or inhibiting the NC.sub.Ca-ATP channel expression or activity in
vitro or in vivo;
[0239] (b) assessing the ability of the candidate substance to
activate or inhibit the NC.sub.Ca-ATP channel expression or
activity in vitro or in vivo;
[0240] (c) selecting a modulator; and
[0241] (d) manufacturing the modulator.
[0242] In certain embodiments, an alternative assessing step can be
assessing the ability of the candidate substance to bind
specifically to the NC.sub.Ca-ATP channel in vitro or in vivo;
[0243] In further embodiments, the NC.sub.Ca-ATP channel may be
provided in a cell or a cell free system and the NC.sub.Ca-ATP
channel may be contacted with the candidate substance. Next, the
modulator is selected by assessing the effect of the candidate
substance on the NC.sub.Ca-ATP channel activity or expression. Upon
identification of the modulator, the method may further provide
manufacturing of the modulator.
[0244] An effective amount of modulator of an NC.sub.Ca-ATP channel
(which may be an agonist or antagonist, and is preferably an
antagonist) that may be administered to a cell includes a dose of
about 0.0001 nM to about 2000 .mu.M. More specifically, doses of an
agonist to be administered are from about 0.01 nM to about 2000
.mu.M; about 0.01 .mu.M to about 0.05 .mu.M; about 0.05 .mu.M to
about 1.0 .mu.M; about 1.0 .mu.M to about 1.5 .mu.M; about 1.5
.mu.M to about 2.0 .mu.M; about 2.0 .mu.M to about 3.0 .mu.M; about
3.0 .mu.M to about 4.0 .mu.M; about 4.0 .mu.M to about 5.0 .mu.M;
about 5.0 .mu.M to about 10 .mu.M; about 10 .mu.M to about 50
.mu.M; about 50 .mu.M to about 100 .mu.M; about 100 .mu.M to about
200 .mu.M; about 200 .mu.M to about 300 .mu.M; about 300 .mu.M to
about 500 .mu.M; about 500 .mu.M to about 1000 .mu.M; about 1000
.mu.M to about 1500 .mu.M and about 1500 .mu.M to about 2000 .mu.M.
Of course, all of these amounts are exemplary, and any amount
in-between these points is also expected to be of use in the
invention.
[0245] The NC.sub.Ca-ATP channel modulator or related-compound
thereof can be administered parenterally or alimentarily.
Parenteral administrations include, but are not limited to
intravenously, intradermally, intramuscularly, intraarterially,
intrathecally, intraventricularly, intratumorally, subcutaneous, or
intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158;
5,641,515; and 5,399,363 (each specifically incorporated herein by
reference in its entirety). Alimentary administrations include, but
are not limited to orally, buccally, rectally, or sublingually.
[0246] The administration of the therapeutic compounds and/or the
therapies of the present invention may include systemic, local
and/or regional and may oral, intravenous, and intramuscular.
Alternatively, other routes of administration are also contemplated
such as, for example, arterial perfusion, intracavitary,
intraperitoneal, intrapleural, intraventricular, intratumoral,
intraparenchyma and/or intrathecal. If desired the therapeutic
compound may be administered by the same route as the
chemotherapeutic agent, even if the therapeutic compound and the
chemotherapeutic agent are not administered simultaneously. The
skilled artisan is aware of determining the appropriate
administration route using standard methods and procedures. In one
example, where assessment of a response to chemotherapy, both
peripherally and centrally is desired, the health care professional
may use a systemic administration.
[0247] Treatment methods will involve treating an individual with
an effective amount of a composition containing an agonist of
NC.sub.Ca-ATP channel or related-compound thereof. An effective
amount is described, generally, as that amount sufficient to
detectably and repeatedly to ameliorate, reduce, minimize or limit
the extent of a disease or its symptoms. More specifically, it is
envisioned that the treatment with the an antagonist of
NC.sub.Ca-ATP channel or related-compounds thereof will reduce cell
swelling and brain swelling following stroke, brain trauma, or
other brain injury, and will reduce brain damage following stroke,
brain trauma or other brain injury or spinal cord injury.
[0248] The effective amount of "therapeutically effective amounts"
of the an antagonist of NC.sub.Ca-ATP channel or related-compounds
thereof to be used are those amounts effective to produce
beneficial results, particularly with respect to stroke or brain
trauma treatment, in the recipient animal or patient. Such amounts
may be initially determined by reviewing the published literature,
by conducting in vitro tests or by conducting metabolic studies in
healthy experimental animals. Before use in a clinical setting, it
may be beneficial to conduct confirmatory studies in an animal
model, preferably a widely accepted animal model of the particular
disease to be treated. Preferred animal models for use in certain
embodiments are rodent models, which are preferred because they are
economical to use and, particularly, because the results gained are
widely accepted as predictive of clinical value.
[0249] As is well known in the art, a specific dose level of active
compounds such as an antagonist of NC.sub.Ca-ATP channel or
related-compounds thereof for any particular patient depends upon a
variety of factors including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination, and the severity of the particular disease undergoing
therapy. The person responsible for administration will determine
the appropriate dose for the individual subject. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biologics standards.
[0250] An effective amount of an antagonist of NC.sub.Ca-ATP
channel or related-compounds thereof as a treatment varies
depending upon the host treated and the particular mode of
administration. In one embodiment of the invention the dose range
of the agonist of NC.sub.Ca-ATP channel or related-compounds
thereof will be about 0.0001 .mu.g/kg body weight to about 500
mg/kg body weight. The term "body weight" is applicable when an
animal is being treated. When isolated cells are being treated,
"body weight" as used herein should read to mean "total cell body
weight". The term "total body weight" may be used to apply to both
isolated cell and animal treatment. All concentrations and
treatment levels are expressed as "body weight" or simply "kg" in
this application are also considered to cover the analogous "total
cell body weight" and "total body weight" concentrations. However,
those of skill will recognize the utility of a variety of dosage
range, for example, 0.0001 .mu.g/kg body weight to 450 mg/kg body
weight, 0.0002 .mu.g/kg body weight to 400 mg/kg body weight,
0.0003 .mu.g/kg body weight to 350 mg/kg body weight, 0.0004
.mu.g/kg body weight to 300 mg/kg body weight, 0.0005 .mu.g/kg body
weight to 250 mg/kg body weight, 5.0 .mu.g/kg body weight to 200
mg/kg body weight, 10.0 .mu.g/kg body weight to 150 mg/kg body
weight, 100.0 .mu.g/kg body weight to 100 mg/kg body weight, or
1000 .mu.g/kg body weight to 50 mg/kg body weight. Further, those
of skill will recognize that a variety of different dosage levels
will be of use, for example, up to about 0.0001 .mu.g/kg, up to
about 0.0002 .mu.g/kg, up to about 0.0003 .mu.g/kg, less than about
0.0004 .mu.g/kg, less than about 0.005 .mu.g/kg, less than about
0.0007 .mu.g/kg, less than about 0.001 .mu.g/kg, less than about
0.1 .mu.g/kg, less than about 1.0 .mu.g/kg, less than about 1.5
.mu.g/kg, less than about 2.0 .mu.g/kg, less than about 5.0
.mu.g/kg, less than about 10.0 .mu.g/kg, less than about 15.0
.mu.g/kg, less than about 30.0 .mu.g/kg, less than about 50
.mu.g/kg, less than about 75 .mu.g/kg, less than about 80 .mu.g/kg,
less than about 90 .mu.g/kg, less than about 100 .mu.g/kg, less
than about 200 .mu.g/kg, less than about 300 .mu.g/kg, less than
about 400 .mu.g/kg, less than about 500 .mu.g/kg, less than about 1
mg/kg, less than about 2 mg/kg, less than about 3 mg/kg, less than
about 5 mg/kg, less than about 10 mg/kg, less than about 100 mg/kg.
Further, those of skill will recognize that a variety of different
dosage levels will be of use, for example, 0.0001 .mu.g/kg, 0.0002
.mu.g/kg, 0.0003 .mu.g/kg, 0.0004 .mu.g/kg, 0.005 .mu.g/kg, 0.0007
.mu.g/kg, 0.001 .mu.g/kg, 0.1 .mu.g/kg, 1.0 .mu.g/kg, 1.5 .mu.g/kg,
2.0 .mu.g/kg, 5.0 .mu.g/kg, 10.0 .mu.g/kg, 15.0 .mu.g/kg, 30.0
.mu.g/kg, 50 .mu.g/kg, 75 .mu.g/kg, 80 .mu.g/kg, 90 .mu.g/kg, 100
.mu.g/kg, 120 .mu.g/kg, 140 .mu.g/kg, 150 .mu.g/kg, 160 .mu.g/kg,
180 .mu.g/kg, 200 .mu.g/kg, 225 .mu.g/kg, 250 .mu.g/kg, 275
.mu.g/kg, 300 .mu.g/kg, 325 .mu.g/kg, 350 .mu.g/kg, 375 .mu.g/kg,
400 .mu.g/kg, 450 .mu.g/kg, 500 .mu.g/kg, 550 .mu.g/kg, 600
.mu.g/kg, 700 .mu.g/kg, 750 .mu.g/kg, 800 .mu.g/kg, 900 .mu.g/kg, 1
mg/kg, 5 mg/kg, 10 mg/kg, 12 mg/kg, 15 mg/kg, 20 mg/kg, and/or 30
mg/kg. Of course, all of these dosages are exemplary, and any
dosage in-between these points is also expected to be of use in the
invention. Any of the above dosage ranges or dosage levels may be
employed for an agonist of NC.sub.Ca-ATP channel or
related-compounds thereof.
[0251] Administration of the therapeutic agonist of NC.sub.Ca-ATP
channel composition of the present invention to a patient or
subject will follow general protocols for the administration of
chemotherapeutics, taking into account the toxicity, if any, of the
agonist of NC.sub.Ca-ATP channel. It is expected that the treatment
cycles would be repeated as necessary. It also is contemplated that
various standard therapies, as well as surgical intervention, may
be applied in combination with the described therapy.
[0252] The treatments may include various "unit doses." Unit dose
is defined as containing a predetermined quantity of the
therapeutic composition (an agonist of NC.sub.Ca-ATP channel or its
related-compounds thereof) calculated to produce the desired
responses in association with its administration, e.g., the
appropriate route and treatment regimen. The quantity to be
administered, and the particular route and formulation, are within
the skill of those in the clinical arts. Also of import is the
subject to be treated, in particular, the state of the subject and
the protection desired. A unit dose need not be administered as a
single injection but may comprise continuous infusion over a set
period of time.
[0253] According to the present invention, one may treat stroke,
brain trauma, or other brain or spinal cord injury by systemic
administration, such as intravenous, intra-arterial, peritoneal, by
administration via pump, or by direct injection into the brain or
ventricles with an antagonist of NC.sub.Ca-ATP channel or
related-compound composition. Alternatively, the brain or spinal
cord may be infused or perfused with the composition using any
suitable delivery vehicle. Systemic administration or oral
administration may be performed, and, in embodiments of the present
invention, local or regional administration may be performed.
Continuous administration also may be applied where appropriate,
for example, where a patient may be monitored on an on-going basis.
Delivery via syringe or catheterization is one effective method.
Continuous perfusion may take place for a period from about 1-2
hours, to about 2-6 hours, to about 6-12 hours, to about 12-24
hours, to about 1-2 days, to about 1-2 wk or longer following the
initiation of treatment. Generally, the dose of the therapeutic
composition via continuous perfusion will be equivalent to that
given by a single or multiple injections, adjusted over a period of
time during which the perfusion occurs. Multiple injections
delivered as single dose comprise about 0.1 to about 1 ml volumes.
In embodiments, the volume to be administered may be about 4-10 ml
(preferably 10 ml), while in further embodiments a volume of about
1-3 ml will be used (preferably 3 ml).
VIII. METHODS OF CEREBRAL ISCHEMIA TREATMENT
[0254] Treatment with an Antagonist
[0255] In other embodiments, the therapeutic compound of the
present invention comprises an antagonist of a NC.sub.Ca-ATP
channel of a neuronal cell, a neuroglial cell, a neural endothelial
cell or a combination thereof. Antagonists are contemplated for use
in treating adverse conditions associated with intracranial
pressure and/or ionic or cytotoxic edema of the central nervous
system. Such conditions include trauma (e.g., traumatic brain or
spinal cord injury (TBI or SCI, respectively)), ischemic brain or
spinal cord injury, primary and secondary neuronal injury, stroke,
arteriovenous malformations (AVM), mass-occupying lesion (e.g.,
hematoma), and hemorrhagic infarction. Antagonists protect the
cells expressing the NC.sub.CA-ATP channel, which is desirable for
clinical treatment in which ionic or cytotoxic edema is formed, in
which capillary integrity is lost following ischemia, and in which
gliotic capsule integrity is important and must be maintained to
prevent the spread of infection, such as with a brain abscess.
Those of skill in the art realize that a brain abscess is a
completely enclosed and results in cerebral swelling. The
protection via inhibition of the NC.sub.Ca-ATP channel is
associated with a reduction in cerebral ionic and cytotoxic edema.
Thus, the compound that inhibits the NC.sub.Ca-ATP channel is
neuroprotective.
[0256] In one aspect, the NC.sub.Ca-ATP channel is blocked,
inhibited, or otherwise is decreased in activity. In such examples,
an antagonist of the NC.sub.Ca-ATP channel is administered and/or
applied. The antagonist modulates the NC.sub.Ca-ATP channel such
that flux (ion and/or water) through the channel is reduced,
ceased, decreased and/or stopped. The antagonist may have a
reversible or an irreversible activity with respect to the activity
of the NC.sub.Ca-ATP channel of the neuronal cell, neuroglial cell,
a neural endothelial cell or a combination thereof. Thus,
inhibition of the NC.sub.Ca-ATP channel can reduce cytotoxic edema
and death of endothelial cells which are associated with formation
of ionic edema and with hemorrhagic conversion.
[0257] Accordingly, the present invention is useful in the
treatment or alleviation of acute cerebral ischemia. According to a
specific embodiment of the present invention the administration of
effective amounts of the active compound can block the channel,
which if remained open leads to neuronal cell swelling and cell
death. A variety of antagonists to SUR1 are suitable for blocking
the channel. Examples of suitable SUR1 antagonists include, but are
not limited to glibenclamide, tolbutamide, repaglinide,
nateglinide, meglitinide, midaglizole, LY397364, LY389382,
glyclazide, glimepiride, estrogen, estrogen related-compounds and
combinations thereof. In a preferred embodiment of the invention
the SUR1 antagonists is selected from the group consisting of
glibenclamide and tolbutamide. Another antagonist that can be used
is MgADP. Still other therapeutic "strategies" for preventing
neural cell swelling and cell death can be adopted including, but
not limited to methods that maintain the neural cell in a polarized
state and methods that prevent strong depolarization.
[0258] In further embodiments, inhibitors or antagonist of the
NC.sub.Ca-ATP channel can be used to reduce or alleviate or
abrogate hemorrhagic conversion. The pathological sequence that
takes place in capillaries after ischemia can be divided into 3
stages, based on the principal constituents that move from the
intravascular compartment into brain parenchyma (Ayata 2002; Betz,
1996; Betz 1989). The first stage is characterized by formation of
"ionic" edema, during which the BBB remains intact, with movement
of electrolytes (Na.sup.+, Cl.sup.-) plus water into brain
parenchyma. The second stage is characterized by formation of
"vasogenic" edema, due to breakdown of the BBB, during which
macromolecules plus water enter into brain parenchyma. The third
stage is characterized by hemorrhagic conversion, due to
catastrophic failure of capillaries, during which all constituents
of blood extravasate into brain parenchyma. In accordance with
Starling's law, understanding these phases requires that 2 things
be identified: (i) the driving force that "pushes" things into
parenchyma; and (ii) the permeability pore that allows passage of
these things into parenchyma.
[0259] Thus, the use of the antagonist or related-compounds thereof
can reduce the mortality of a subject suffering from a stroke
and/or rescue the penumbra area or prevent damage in the penumbra
area which comprises areas of tissue that are at risk of becoming
irreversibly damaged.
[0260] With the administration of an antagonist of the
NC.sub.Ca-ATP channel, endothelial cell depolarization is
abrogated, slowed, reduced or inhibited due to the opening of the
NC.sub.Ca-ATP channel. Thus, abrogation of cell depolarization
results in abrogation or inhibition of Na.sup.+ influx, which
prevents a change in osmotic gradient thereby preventing an influx
of water into the endothelial cell and stopping cell swelling,
blebbing and cytotoxic edema. Thus, preventing or inhibiting or
attenuating endothelial cell depolarization can prevent or reduce
hemorrhagic conversion.
[0261] Neuronal cells in which the antagonist of the NC.sub.Ca-ATP
channel may be administered may include any cell that expresses
SUR1, for example any neuronal cell, neuroglial cell or a neural
endothelia cell.
[0262] Subjects that may be treated with the antagonist or
related-compound thereof include those that are suffering from or
at risk of developing trauma (e.g., traumatic brain or spinal cord
injury (TBI or SCI)), ischemic brain or spinal cord injury, primary
and secondary neuronal injury, stroke, arteriovenous malformations
(AVM), brain abscess, mass-occupying lesion, hemorrhagic
infarction, or any other condition associated with cerebral hypoxia
or cerebral ischemia resulting in cerebral edema and/or increased
intracranial pressure, for example, but not limited to brain mass,
brain edema, hematoma, end stage cerebral edema, encephalopathies,
etc. Thus, the antagonist can be a therapeutic treatment in which
the therapeutic treatment includes prophylaxis or a prophylactic
treatment. The antagonist or related-compounds thereof are
neuroprotective.
[0263] Other subjects that may be treated with the antagonist of
the present invention include those subjects that are at risk or
predisposed to developing a stroke. Such subjects can include, but
are not limited to subjects that suffer from atrial fibrillations,
clotting disorders, and/or risk of pulmonary emboli.
[0264] In certain embodiments, a subject at risk for developing a
stroke may include subjects undergoing treatments, for example, but
not limited to cerebral/endovascular treatments, surgery (e.g.,
craniotomy, cranial surgery, removal of brain tumors (e.g.,
hematoma), coronary artery bypass grafting (CABG), angiography,
stent replacement, other vascular surgeries, and/or other CNS or
neurological surgeries), and treatment of myocardial infarction
(MI) with thrombolytics, as well as surgeries on aortic abdominal
aneurysms and major vessels that provide blood supply to the spinal
cord. In such cases, the subject may be treated with the antagonist
or related-compound of the present invention prior to the actual
treatment. Pretreatment can include administration of the
antagonist and/or related-compound months (1, 2, 3, etc.), weeks
(1, 2, 3, etc.), days (1, 2, 3, etc.), hours (1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12), or minutes (15, 30, 60, 90, etc.) prior to the
actual treatment or surgery. Treatment of the antagonist and/or
related-compound can continue during the treatment and/or surgery
and after the treatment and/or surgery until the risk of developing
a stroke in the subject is decreased, lessened or alleviated.
[0265] In further embodiments, the antagonist of the present
invention can be given to a subject at risk of developing head/neck
trauma, such as a subject involved in sports or other activities
that have an increased risk of head/neck trauma.
[0266] An effective amount of an antagonist of the NC.sub.Ca-ATP
channel that may be administered to a cell includes a dose of about
0.0001 nM to about 2000 .mu.M. More specifically, doses of an
agonist to be administered are from about 0.01 nM to about 2000
.mu.M; about 0.01 .mu.M to about 0.05 .mu.M; about 0.05 .mu.M to
about 1.0 .mu.M; about 1.0 .mu.M to about 1.5 .mu.M; about 1.5
.mu.M to about 2.0 .mu.M; about 2.0 .mu.M to about 3.0 .mu.M; about
3.0 .mu.M to about 4.0 .mu.M; about 4.0 .mu.M to about 5.0 .mu.M;
about 5.0 .mu.M to about 10 .mu.M; about 10 .mu.M to about 50
.mu.M; about 50 .mu.M to about 100 .mu.M; about 100 .mu.M to about
200 .mu.M; about 200 .mu.M to about 300 .mu.M; about 300 .mu.M to
about 500 .mu.M; about 500 .mu.M to about 1000 .mu.M; about 1000
.mu.M to about 1500 .mu.M and about 1500 .mu.M to about 2000 .mu.M.
Of course, all of these amounts are exemplary, and any amount
in-between these points is also expected to be of use in the
invention.
[0267] The antagonist or related-compound thereof can be
administered parenterally or alimentary. Parenteral administrations
include, but are not limited to intravenously, intradermally,
intramuscularly, intraarterially, intrathecally, subcutaneous, or
intraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158;
5,641,515; and 5,399,363 (each specifically incorporated herein by
reference in its entirety). Alimentary administrations include, but
are not limited to orally, buccally, rectally, or sublingually.
[0268] The administration of the therapeutic compounds and/or the
therapies of the present invention may include systemic, local
and/or regional administrations, for example, topically (dermally,
transdermally), via catheters, implantable pumps, etc.
Alternatively, other routes of administration are also contemplated
such as, for example, arterial perfusion, intracavitary,
intraperitoneal, intrapleural, intraventricular and/or intrathecal.
The skilled artisan is aware of determining the appropriate
administration route using standard methods and procedures. Other
routes of administration are discussed elsewhere in the
specification and are incorporated herein by reference.
[0269] Treatment methods will involve treating an individual with
an effective amount of a composition containing an antagonist of
NC.sub.Ca-ATP channel or related-compound thereof. An effective
amount is described, generally, as that amount sufficient to
detectably and repeatedly to ameliorate, reduce, minimize or limit
the extent of a disease or its symptoms. More specifically, it is
envisioned that the treatment with the an antagonist of
NC.sub.Ca-ATP channel or related-compounds thereof will inhibit
cell depolarization, inhibit Na.sup.+ influx, inhibit an osmotic
gradient change, inhibit water influx into the cell, inhibit
cytotoxic cell edema, decrease stroke size, inhibit hemorrhagic
conversion, and decrease mortality of the subject.
[0270] The effective amount of an antagonist of NC.sub.Ca-ATP
channel or related-compounds thereof to be used are those amounts
effective to produce beneficial results, particularly with respect
to stroke treatment, in the recipient animal or patient. Such
amounts may be initially determined by reviewing the published
literature, by conducting in vitro tests or by conducting metabolic
studies in healthy experimental animals. Before use in a clinical
setting, it may be beneficial to conduct confirmatory studies in an
animal model, preferably a widely accepted animal model of the
particular disease to be treated. Preferred animal models for use
in certain embodiments are rodent models, which are preferred
because they are economical to use and, particularly, because the
results gained are widely accepted as predictive of clinical
value.
[0271] As is well known in the art, a specific dose level of active
compounds such as an antagonist of the NC.sub.Ca-ATP channel or
related-compounds thereof for any particular patient depends upon a
variety of factors including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, route of administration, rate of excretion, drug
combination, and the severity of the particular disease undergoing
therapy. The person responsible for administration will determine
the appropriate dose for the individual subject. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biologics standards.
[0272] One of skill in the art realizes that the effective amount
of the antagonist or related-compound thereof can be the amount
that is required to achieve the desired result: reduction in the
risk of stroke, reduction in intracranial pressure, reduction in
cell death, reduction in stroke size, reduction in spinal cord
injury, etc. This amount also is an amount that maintains a
reasonable level of blood glucose in the patient, for example, the
amount of the antagonist maintains a blood glucose level of at
least 60 mmol/1, more preferably, the blood glucose level is
maintain in the range of about 60 mmol/1 to about 150 mmol/1. Thus,
the amounts prevents the subject from becoming hypoglycemic. If
glucose levels are not normal, then one of skill in the art would
administer either insulin or glucose, depending upon if the patient
is hypoglycemic or hyperglycemic.
[0273] Administration of the therapeutic antagonist of
NC.sub.Ca-ATP channel composition of the present invention to a
patient or subject will follow general protocols for the
administration of therapies used in stroke treatment, such as
thrombolytics, taking into account the toxicity, if any, of the
antagonist of the NC.sub.Ca-ATP channel. It is expected that the
treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described
therapy.
IX. PHARMACEUTICAL FORMULATIONS AND METHODS OF TREATING NEURAL CELL
SWELLING AND BRAIN SWELLING
[0274] A. Compositions of the Present Invention
[0275] The present invention also contemplates therapeutic methods
employing compositions comprising the active substances disclosed
herein. Preferably, these compositions include pharmaceutical
compositions comprising a therapeutically effective amount of one
or more of the active compounds or substances along with a
pharmaceutically acceptable carrier.
[0276] As used herein, the term "pharmaceutically acceptable"
carrier means a non-toxic, inert solid, semi-solid liquid filler,
diluent, encapsulating material, formulation auxiliary of any type,
or simply a sterile aqueous medium, such as saline. Some examples
of the materials that can serve as pharmaceutically acceptable
carriers are sugars, such as lactose, glucose and sucrose, starches
such as corn starch and potato starch, cellulose and its
derivatives such as sodium carboxymethyl cellulose, ethyl cellulose
and cellulose acetate; powdered tragacanth; malt, gelatin, talc;
excipients such as cocoa butter and suppository waxes; oils such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,
corn oil and soybean oil; glycols, such as propylene glycol,
polyols such as glycerin, sorbitol, mannitol and polyethylene
glycol; esters such as ethyl oleate and ethyl laurate, agar;
buffering agents such as magnesium hydroxide and aluminum
hydroxide; alginic acid; pyrogen-free water; isotonic saline,
Ringer's solution; ethyl alcohol and phosphate buffer solutions, as
well as other non-toxic compatible substances used in
pharmaceutical formulations.
[0277] Wetting agents, emulsifiers and lubricants such as sodium
lauryl sulfate and magnesium stearate, as well as coloring agents,
releasing agents, coating agents, sweetening, flavoring and
perfuming agents, preservatives and antioxidants can also be
present in the composition, according to the judgment of the
formulator. Examples of pharmaceutically acceptable antioxidants
include, but are not limited to, water soluble antioxidants such as
ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium
metabisulfite, sodium sulfite, and the like; oil soluble
antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole
(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,
aloha-tocopherol and the like; and the metal chelating agents such
as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,
tartaric acid, phosphoric acid and the like.
[0278] B. Dose Determinations
[0279] By a "therapeutically effective amount" or simply "effective
amount" of an active compound, such as glibenclamide or
tolbutamide, is meant a sufficient amount of the compound to treat
or alleviate the brain swelling at a reasonable benefit/risk ratio
applicable to any medical treatment. It will be understood,
however, that the total daily usage of the active compounds and
compositions of the present invention will be decided by the
attending physician within the scope of sound medical judgment. The
specific therapeutically effective dose level for any particular
patient will depend upon a variety of factors including the
disorder being treated and the severity of the brain injury or
ischemia; activity of the specific compound employed; the specific
composition employed; the age, body weight, general health, sex and
diet of the patient; the time of administration, route of
administration, and rate of excretion of the specific compound
employed; the duration of the treatment; drugs used in combination
or coinciding with the specific compound employed; and like factors
well known in the medical arts.
[0280] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell assays or
experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds which exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects may be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0281] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED50 with little or
no toxicity. The dosage may vary within this range depending upon
the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
based assays. A dose may be formulated in animal models to achieve
a circulating plasma concentration range that includes the IC50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0282] The total daily dose of the active compounds of the present
invention administered to a subject in single or in divided doses
can be in amounts, for example, from 0.01 to 25 mg/kg body weight
or more usually from 0.1 to 15 mg/kg body weight. Single dose
compositions may contain such amounts or submultiples thereof to
make up the daily dose. In general, treatment regimens according to
the present invention comprise administration to a human or other
mammal in need of such treatment from about 1 mg to about 1000 mg
of the active substance(s) of this invention per day in multiple
doses or in a single dose of from 1 mg, 5 mg, 10 mg, 100 mg, 500 mg
or 1000 mg.
[0283] In certain situations, it may be important to maintain a
fairly high dose of the active agent in the blood stream of the
patient, particularly early in the treatment. Such a fairly high
dose may include a dose that is several times greater than its use
in other indications. For example, the typical anti-diabetic dose
of oral or IV glibenclamide is about 2.5 mg/kg to about 15 mg/kg
per day; the typical anti-diabetic dose of oral or IV tolbutamide
is about to 0.5 gm/kg to about 2.0 gm/kg per day; the typical
anti-diabetic dose for oral gliclazide is about 30 mg/kg to about
120 mg/kg per day; however, much larger doses may be required to
block neural cell swelling and brain swelling.
[0284] For example, in one embodiment of the present invention
directed to a method of preventing neuronal cell swelling in the
brain of a subject by administering to the subject a formulation
containing an effective amount of a compound that blocks the
NC.sub.Ca-ATP channel and a pharmaceutically acceptable carrier;
such formulations may contain from about 0.1 to about 100 grams of
tolbutamide or from about 0.5 to about 150 milligrams of
glibenclamide. In another embodiment of the present invention
directed to a method of alleviating the negative effects of
traumatic brain injury or cerebral ischemia stemming from neural
cell swelling in a subject by administering to the subject a
formulation containing an effective amount of a compound that
blocks the NC.sub.Ca-ATP channel and a pharmaceutically acceptable
carrier.
[0285] In situations of traumatic brain injury or cerebral ischemia
(such as stroke), or cerebral hypoxia, it may be important to
maintain a fairly high dose of the active agent to ensure delivery
to the brain of the patient, particularly early in the treatment.
Hence, at least initially, it may be important to keep the dose
relatively high and/or at a substantially constant level for a
given period of time, preferably, at least about six or more hours,
more preferably, at least about twelve or more hours and, most
preferably, at least about twenty-four or more hours. In situations
of traumatic brain injury or cerebral ischemia (such as stroke), it
may be important to maintain a fairly high dose of the active agent
to ensure delivery to the brain of the patient, particularly early
in the treatment.
[0286] When the method of the present invention is employed to
treat conditions involving bleeding in the brain, such as traumatic
brain injury or cerebral ischemia (such as stroke), delivery via
the vascular system is available and the compound is not
necessarily required to readily cross the blood-brain barrier.
[0287] C. Formulations and Administration
[0288] The compounds of the present invention may be administered
alone or in combination or in concurrent therapy with other agents
which affect the central or peripheral nervous system, particularly
selected areas of the brain.
[0289] Liquid dosage forms for oral administration may include
pharmaceutically acceptable emulsions, microemulsions, solutions,
suspensions, syrups and elixirs containing inert diluents commonly
used in the art, such as water, isotonic solutions, or saline. Such
compositions may also comprise adjuvants, such as wetting agents;
emulsifying and suspending agents; sweetening, flavoring and
perfuming agents.
[0290] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions may be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution, suspension or emulsion in a nontoxic
parenterally acceptable diluent or solvent, for example, as a
solution in 1,3-butanediol. Among the acceptable vehicles and
solvents that may be employed are water, Ringer's solution, U.S.P.
and isotonic sodium chloride solution. In addition, sterile, fixed
oils are conventionally employed as a solvent or suspending medium.
For this purpose any bland fixed oil can be employed including
synthetic mono- or diglycerides. In addition, fatty acids such as
oleic acid are used in the preparation of injectables.
[0291] The injectable formulation can be sterilized, for example,
by filtration through a bacteria-retaining filter, or by
incorporating sterilizing agents in the form of sterile solid
compositions, which can be dissolved or dispersed in sterile water
or other sterile injectable medium just prior to use.
[0292] In order to prolong the effect of a drug, it is often
desirable to slow the absorption of a drug from subcutaneous or
intramuscular injection. The most common way to accomplish this is
to inject a suspension of crystalline or amorphous material with
poor water solubility. The rate of absorption of the drug becomes
dependent on the rate of dissolution of the drug, which is, in
turn, dependent on the physical state of the drug, for example, the
crystal size and the crystalline form. Another approach to delaying
absorption of a drug is to administer the drug as a solution or
suspension in oil. Injectable depot forms can also be made by
forming microcapsule matrices of drugs and biodegradable polymers,
such as polylactide-polyglycoside. Depending on the ratio of drug
to polymer and the composition of the polymer, the rate of drug
release can be controlled. Examples of other biodegradable polymers
include polyorthoesters and polyanhydrides. The depot injectables
can also be made by entrapping the drug in liposomes or
microemulsions, which are compatible with body tissues.
[0293] Suppositories for rectal administration of the drug can be
prepared by mixing the drug with a suitable non-irritating
excipient, such as cocoa butter and polyethylene glycol which are
solid at ordinary temperature but liquid at the rectal temperature
and will, therefore, melt in the rectum and release the drug.
[0294] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, gelcaps and granules. In such
solid dosage forms the active compound may be admixed with at least
one inert diluent such as sucrose, lactose or starch. Such dosage
forms may also comprise, as is normal practice, additional
substances other than inert diluents, e.g., tableting lubricants
and other tableting aids such as magnesium stearate and
microcrystalline cellulose. In the case of capsules, tablets and
pills, the dosage forms may also comprise buffering agents. Tablets
and pills can additionally be prepared with enteric coatings and
other release-controlling coatings.
[0295] Solid compositions of a similar type may also be employed as
fillers in soft and hard-filled gelatin capsules using such
excipients as lactose or milk sugar as well as high molecular
weight polyethylene glycols and the like.
[0296] The active compounds can also be in micro-encapsulated form
with one or more excipients as noted above. The solid dosage forms
of tablets, capsules, pills, and granules can be prepared with
coatings and shells such as enteric coatings and other coatings
well known in the pharmaceutical formulating art. They may
optionally contain opacifying agents and can also be of a
composition that they release the active ingredient(s) only, or
preferably, in a certain part of the intestinal tract, optionally
in a delayed manner. Examples of embedding compositions which can
be used include polymeric substances and waxes.
[0297] Dosage forms for topical or transdermal administration of a
compound of this invention further include ointments, pastes,
creams, lotions, gels, powders, solutions, sprays, inhalants or
patches. Transdermal patches have the added advantage of providing
controlled delivery of active compound to the body. Such dosage
forms can be made by dissolving or dispersing the compound in the
proper medium. Absorption enhancers can also be used to increase
the flux of the compound across the skin. The rate can be
controlled by either providing a rate controlling membrane or by
dispersing the compound in a polymer matrix or gel. The ointments,
pastes, creams and gels may contain, in addition to an active
compound of this invention, excipients such as animal and vegetable
fats, oils, waxes, paraffins, starch, tragacanth, cellulose
derivatives, polyethylene glycols, silicones, bentonites, silicic
acid, talc and zinc oxide, or mixtures thereof.
[0298] The method of the present invention employs the compounds
identified herein for both in vitro and in vivo applications. For
in vivo applications, the invention compounds can be incorporated
into a pharmaceutically acceptable formulation for administration.
Those of skill in the art can readily determine suitable dosage
levels when the invention compounds are so used.
[0299] As employed herein, the phrase "suitable dosage levels"
refers to levels of compound sufficient to provide circulating
concentrations high enough to effectively block the NC.sub.Ca-ATP
channel and prevent or reduce neural cell swelling in vivo.
[0300] In accordance with a particular embodiment of the present
invention, compositions comprising at least one SUR1 antagonist
compound (as described above), and a pharmaceutically acceptable
carrier are contemplated.
[0301] Exemplary pharmaceutically acceptable carriers include
carriers suitable for oral, intravenous, subcutaneous,
intramuscular, intracutaneous, and the like administration.
Administration in the form of creams, lotions, tablets, dispersible
powders, granules, syrups, elixirs, sterile aqueous or non-aqueous
solutions, suspensions or emulsions, and the like, is
contemplated.
[0302] For the preparation of oral liquids, suitable carriers
include emulsions, solutions, suspensions, syrups, and the like,
optionally containing additives such as wetting agents, emulsifying
and suspending agents, sweetening, flavoring and perfuming agents,
and the like.
[0303] For the preparation of fluids for parenteral administration,
suitable carriers include sterile aqueous or non-aqueous solutions,
suspensions, or emulsions. Examples of non-aqueous solvents or
vehicles are propylene glycol, polyethylene glycol, vegetable oils,
such as olive oil and corn oil, gelatin, and injectable organic
esters such as ethyl oleate. Such dosage forms may also contain
adjuvants such as preserving, wetting, emulsifying, and dispersing
agents. They may be sterilized, for example, by filtration through
a bacteria-retaining filter, by incorporating sterilizing agents
into the compositions, by irradiating the compositions, or by
heating the compositions. They can also be manufactured in the form
of sterile water, or some other sterile injectable medium
immediately before use. The active compound is admixed under
sterile conditions with a pharmaceutically acceptable carrier and
any needed preservatives or buffers as may be required.
[0304] The treatments may include various "unit doses." Unit dose
is defined as containing a predetermined quantity of the
therapeutic composition (an antagonist of the NC.sub.Ca-ATP channel
or its related-compounds thereof) calculated to produce the desired
responses in association with its administration, e.g., the
appropriate route and treatment regimen. The quantity to be
administered, and the particular route and formulation, are within
the skill of those in the clinical arts. Also of import is the
subject to be treated, in particular, the state of the subject and
the protection desired. A unit dose need not be administered as a
single injection but may comprise continuous infusion over a set
period of time.
X. COMBINATION TREATMENTS
[0305] In the context of the present invention, it is contemplated
that an antagonist of the NC.sub.Ca-ATP channel or
related-compounds thereof may be used in combination with an
additional therapeutic agent to more effectively treat a cerebral
ischemic event, and/or decrease intracranial pressure. In some
embodiments, it is contemplated that a conventional therapy or
agent, including but not limited to, a pharmacological therapeutic
agent may be combined with the antagonist or related-compound of
the present invention.
[0306] Pharmacological therapeutic agents and methods of
administration, dosages, etc. are well known to those of skill in
the art (see for example, the "Physicians Desk Reference", Goodman
& Gilman's "The Pharmacological Basis of Therapeutics",
"Remington's Pharmaceutical Sciences", and "The Merck Index,
Eleventh Edition", incorporated herein by reference in relevant
parts), and may be combined with the invention in light of the
disclosures herein. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject, and such individual
determinations are within the skill of those of ordinary skill in
the art.
[0307] Non-limiting examples of a pharmacological therapeutic agent
that may be used in the present invention include an
antihyperlipoproteinemic agent, an antiarteriosclerotic agent, an
anticholesterol agent, an antiinflammatory agent, an
antithrombotic/fibrinolytic agent, anticoagulant, antiplatelet,
vasodilator, and/or diuretics. Thromoblytics that are used can
include, but are not limited to prourokinase, streptokinase, and
tissue plasminogen activator (tPA) Anticholesterol agents include
but are not limited to HMG-CoA Reductase inhibitors, cholesterol
absorption inhibitors, bile acid sequestrants, nicotinic acid and
derivatives thereof, fibric acid and derivatives thereof. HMG-CoA
Reductase inhibitors include statins, for example, but not limited
to atorvastatin calcium (Lipitor.RTM.), cerivastatin sodium
(Baycol.RTM.), fluvastatin sodium (Lescol.RTM.), lovastatin
(Advicor.RTM.), pravastatin sodium (Pravachol.RTM.), and
simvastatin (Zocor.RTM.). Agents known to reduce the absorption of
ingested cholesterol include, for example, Zetia.RTM.. Bile acid
sequestrants include, but are not limited to cholestryramine,
cholestipol and colesevalam. Other anticholesterol agents include
fibric acids and derivatives thereof (e.g., gemfibrozil,
fenofibrate and clofibrate); nicotinic acids and derivatives
thereof (e.g., nician, lovastatin) and agents that extend the
release of nicotinic acid, for example niaspan. Antiinflammatory
agents include, but are not limited to non-sterodial
anti-inflammatory agents (e.g., naproxen, ibuprofen, celeoxib) and
sterodial anti-inflammatory agents (e.g., glucocorticoids).
Anticoagulants include, but are not limited to heparin, warfarin,
and coumadin. Antiplatelets include, but are not limited to
aspirin, and aspirin related-compounds, for example acetaminophen.
Diuretics include, but are not limited to such as furosemide
(Lasix.RTM.), bumetanide (Bumex.RTM.), torsemide (Demadex.RTM.),
thiazide & thiazide-like diuretics (e.g., chlorothiazide
(Diuril.RTM.) and hydrochlorothiazide (Esidrix.RTM.), benzthiazide,
cyclothiazide, indapamide, chlorthalidone, bendroflumethizide,
metolazone), amiloride, triamterene, and spironolacton.
Vasodilators include, but are not limited to nitroglycerin.
[0308] Thus, in certain embodiments, the present invention
comprises co-administration of an antagonist of the NC.sub.Ca-ATP
channel with a thrombolytic agent. Co-administration of these two
compounds will increase the therapeutic window of the thrombolytic
agent. Examples of suitable thrombolytic agents that can be
employed in the methods and pharmaceutical compositions of this
invention are prourokinase, streptokinase, and tissue plasminogen
activator (tPA).
[0309] In certain embodiments, the present invention comprises
co-administration of an antagonist of the NC.sub.Ca-ATP channel
with glucose or related carbohydrate to maintain appropriate levels
of serum glucose. Appropriate levels of blood glucose are within
the range of about 60 mmol/1 to about 150 mmol/liter. Thus, glucose
or a related carbohydrate is administered in combination to
maintain the serum glucose within this range.
[0310] When an additional therapeutic agent, as long as the dose of
the additional therapeutic agent does not exceed previously quoted
toxicity levels, the effective amounts of the additional
therapeutic agent may simply be defined as that amount effective to
reduce cerebral edema when administered to an animal in combination
with an agonist of NC.sub.Ca-ATP channel or related-compounds
thereof. This may be easily determined by monitoring the animal or
patient and measuring those physical and biochemical parameters of
health and disease that are indicative of the success of a given
treatment. Such methods are routine in animal testing and clinical
practice.
[0311] To inhibit hemorrhagic conversion, reduce cell swelling,
etc., using the methods and compositions of the present invention,
one would generally contact a cell with antagonist of NC.sub.Ca-ATP
channel or related-compounds thereof in combination with an
additional therapeutic agent, such as tPA, aspirin, statins,
diuretics, warfarin, coumadin, mannitol, etc. These compositions
would be provided in a combined amount effective to inhibit
hemorrhagic conversion, cell swelling and edema. This process may
involve contacting the cells with agonist of NC.sub.Ca-ATP channel
or related-compounds thereof in combination with an additional
therapeutic agent or factor(s) at the same time. This may be
achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes an antagonist of
the NC.sub.Ca-ATP channel or derivatives thereof and the other
includes the additional agent.
[0312] Alternatively, treatment with an antagonist of NC.sub.Ca-ATP
channel or related-compounds thereof may precede or follow the
additional agent treatment by intervals ranging from minutes to
hours to weeks to months. In embodiments where the additional agent
is applied separately to the cell, one would generally ensure that
a significant period of time did not expire between the time of
each delivery, such that the agent would still be able to exert an
advantageously combined effect on the cell. In such instances, it
is contemplated that one would contact the cell with both
modalities within about 1-24 hr of each other and, more preferably,
within about 6-12 hr of each other.
[0313] Typically, for maximum benefit of the thrombolytic agent, or
therapy must be started within three hours of the onset of stroke
symptoms, making rapid diagnosis and differentiation of stroke and
stroke type critical. However, in the present invention,
administration of the NC.sub.Ca-ATP channel with a thrombolytic
agent increases this therapeutic window. The therapeutic window for
thrombolytic agents may be increased by several (4-8) hours by
co-administering antagonist of the NC.sub.Ca-ATP channel.
[0314] Further embodiments include treatment with SUR1 antagonist,
thrombolytic agent, and glucose. Glucose administration may be at
the time of treatment with SUR1 antagonist, or may follow treatment
with SUR1 antagonist (e.g., at 15 minutes after treatment with SUR1
antagonist, or at one half hour after treatment with SUR1
antagonist, or at one hour after treatment with SUR1 antagonist, or
at two hours after treatment with SUR1 antagonist, or at three
hours after treatment with SUR1 antagonist). Glucose administration
may be by intravenous, or intraperitoneal, or other suitable route
and means of delivery. Additional glucose allows administration of
higher doses of SUR1 antagonist than might otherwise be possible.
Treatment with glucose in conjunction with treatment with SUR1
antagonist (at the same time as treatment with SUR1 antagonist, or
at a later time after treatment with SUR1 antagonist) may further
enlarge the time window after stroke, trauma, or other brain injury
when thrombolytic treatment may be initiated.
[0315] Yet further, the combination of the antagonist and tPA
results in a decrease or prevention of hemorrhagic conversion
following reperfusion. Hemorrhagic conversion is the transformation
of a bland infarct into a hemorrhagic infarct after restoration of
circulation. It is generally accepted that these complications of
stroke and of reperfusion are attributable to capillary endothelial
cell dysfunction that worsens as ischemia progresses. Thus, the
present invention is protective of the endothelial cell dysfunction
that occurs as a result of an ischemic event.
[0316] Endothelial cell dysfunction comprises three phases. Phase
one is characterized by formation of ionic edema with the blood
brain barrier still intact. The second phase is characterized by
formation of vasogenic edema in which the blood brain barrier is no
longer intact. Phase three is characterized by hemorrhagic
conversion due to failure of capillary integrity during which all
constituents of blood, including erythrocytes, extravasate into
brain parenchyma. Disruption of BBB involves ischemia-induced
activation of endothelial cells that results in expression and
release of MMPs, specifically, MMP-2 (gelatinase A) and MMP-9
(gelatinase B).
[0317] Since hemorrhagic conversion increases mortality of the
patient, it is essential that these patients receive treatment in
an urgent manner. For example, it is known that hemorrhagic
conversion typically results in patients if reperfusion and tPA
treatment is delayed beyond 3 hr or more after thrombotic stroke.
Thus, the administration of the antagonist of the present invention
will reduce necrotic death of ischemic endothelial cells, and will
thereby prolong the therapeutic window for tPA, thereby decreasing
mortality of the patient.
XI. DIAGNOSTICS
[0318] The antagonist or related-compound can be used for
diagnosing, monitoring, or prognosis of ischemia or damage to
neurons, glial cells or in monitoring neuronal cells in zones of
cerebral edema, metastatic tumors, etc.
[0319] A. Genetic Diagnosis
[0320] One embodiment of the instant invention comprises a method
for detecting expression of any portion of a Na.sub.Ca-ATP channel,
for example, expression of the regulatory unit, SUR1, and/or
expression of the pore-forming subunit. This may comprise
determining the level of SUR1 expressed and/or the level of the
pore-forming subunit expressed. It is understood by the present
invention that the up-regulation or increased expression of the
Na.sub.Ca-ATP channel relates to increased levels of SUR1, which
correlates to increased neuronal damage, such as cerebral
edema.
[0321] Firstly, a biological sample is obtained from a subject. The
biological sample may be tissue or fluid. In certain embodiments,
the biological sample includes cells from the brain and/or cerebral
endothelial cells or microvessels and/or gliotic capsule. For
example, in metastatic tumors, glial cells are activated and form a
capsule around the tumor.
[0322] Nucleic acids used are isolated from cells contained in the
biological sample, according to standard methodologies (Sambrook et
al., 1989). The nucleic acid may be genomic DNA or fractionated or
whole cell RNA. Where RNA is used, it may be desired to convert the
RNA to a complementary DNA (cDNA). In one embodiment, the RNA is
whole cell RNA; in another, it is poly-A RNA. Normally, the nucleic
acid is amplified.
[0323] Depending on the format, the specific nucleic acid of
interest is identified in the sample directly using amplification
or with a second, known nucleic acid following amplification. Next,
the identified product is detected. In certain applications, the
detection may be performed by visual means (e.g., ethidium bromide
staining of a gel). Alternatively, the detection may involve
indirect identification of the product via chemiluminescence,
radioactive scintigraphy of radiolabel or fluorescent label or even
via a system using electrical or thermal impulse signals (Affymax
Technology; Bellus, 1994).
[0324] Following detection, one may compare the results seen in a
given subject with a statistically significant reference group of
normal subjects and subjects that have been diagnosed with a
stroke, cancer, cerebral edema, etc.
[0325] Yet further, it is contemplated that chip-based DNA
technologies such as those described by Hacia et al., (1996) and
Shoemaker et al., (1996) can be used for diagnosis. Briefly, these
techniques involve quantitative methods for analyzing large numbers
of genes rapidly and accurately. By tagging genes with
oligonucleotides or using fixed probe arrays, one can employ chip
technology to segregate target molecules as high density arrays and
screen these molecules on the basis of hybridization. See also
Pease et al., (1994); Fodor et al., (1991).
[0326] B. Other Types of Diagnosis
[0327] In order to increase the efficacy of molecules, for example,
compounds and/or proteins and/or antibodies, as diagnostic agents,
it is conventional to link or covalently bind or complex at least
one desired molecule or moiety.
[0328] Certain examples of conjugates are those conjugates in which
the molecule (for example, protein, antibody, and/or compound) is
linked to a detectable label. "Detectable labels" are compounds
and/or elements that can be detected due to their specific
functional properties, and/or chemical characteristics, the use of
which allows the antibody to which they are attached to be
detected, and/or further quantified if desired.
[0329] Conjugates are generally preferred for use as diagnostic
agents. Diagnostics generally fall within two classes, those for
use in in vitro diagnostics, such as in a variety of immunoassays,
and/or those for use in vivo diagnostic protocols, generally known
as "molecule-directed imaging".
[0330] Many appropriate imaging agents are known in the art, as are
methods for their attachment to molecules, for example, antibodies
(see, for e.g., U.S. Pat. Nos. 5,021,236; 4,938,948; and 4,472,509,
each incorporated herein by reference). The imaging moieties used
can be paramagnetic ions; radioactive isotopes; fluorochromes;
NMR-detectable substances; X-ray imaging.
[0331] In the case of paramagnetic ions, one might mention by way
of example ions such as chromium (III), manganese (II), iron (III),
iron (II), cobalt (II), nickel (II), copper (II), neodymium (III),
samarium (III), ytterbium (III), gadolinium (III), vanadium (II),
terbium (III), dysprosium (III), holmium (III) and/or erbium (III),
with gadolinium being particularly preferred. Ions useful in other
contexts, such as X-ray imaging, include but are not limited to
lanthanum (III), gold (III), lead (II), and especially bismuth
(III).
[0332] In the case of radioactive isotopes for therapeutic and/or
diagnostic application, one might mention .sup.211astatine,
.sup.11carbon, .sup.14carbon, .sup.51chromium, .sup.36chlorine,
.sup.57cobalt, .sup.58cobalt, .sup.67copper, .sup.152Eu,
.sup.67gallium, .sup.3hydrogen, .sup.123iodine, .sup.125iodine,
.sup.131iodine, .sup.59 iron, .sup.32phosphorus, .sup.186rhenium,
.sup.188rhenium, .sup.75selenium, .sup.35sulphur,
.sup.99mtechnicium and/or .sup.90yttrium. .sup.125I is often being
preferred for use in certain embodiments, and .sup.99m technicium
and/or .sup.111indium are also often preferred due to their low
energy and suitability for long range detection.
[0333] Among the fluorescent labels contemplated for use as
conjugates include Alexa 350, Alexa 430, AMCA, BODIPY 630/650,
BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX,
Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX,
6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514,
Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin,
ROX, TAMRA, TET, Tetramethylrhodamine, and/or Texas Red.
[0334] Another type of conjugates contemplated in the present
invention are those intended primarily for use in vitro, where the
molecule is linked to a secondary binding ligand and/or to an
enzyme (an enzyme tag) that will generate a colored product upon
contact with a chromogenic substrate. Examples of suitable enzymes
include urease, alkaline phosphatase, (horseradish) hydrogen
peroxidase or glucose oxidase. Preferred secondary binding ligands
are biotin and/or avidin and streptavidin compounds. The use of
such labels is well known to those of skill in the art and are
described, for example, in U.S. Pat. Nos. 3,817,837; 3,850,752;
3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each
incorporated herein by reference.
[0335] The steps of various other useful immunodetection methods
have been described in the scientific literature, such as, e.g.,
Nakamura et al., (1987). Immunoassays, in their most simple and
direct sense, are binding assays. Certain preferred immunoassays
are the various types of radioimmunoas says (RIA) and immunobead
capture assay. Immunohistochemical detection using tissue sections
also is particularly useful. However, it will be readily
appreciated that detection is not limited to such techniques, and
Western blotting, dot blotting, FACS analyses, and the like also
may be used in connection with the present invention.
[0336] Immunologically-based detection methods for use in
conjunction with Western blotting include enzymatically-,
radiolabel-, or fluorescently-tagged secondary molecules/antibodies
against the SUR1 or regulatory subunit of the NC.sub.Ca-ATP channel
are considered to be of particular use in this regard. U.S. patents
concerning the use of such labels include U.S. Pat. Nos. 3,817,837;
3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and
4,366,241, each incorporated herein by reference. Of course, one
may find additional advantages through the use of a secondary
binding ligand such as a second antibody or a biotin/avidin ligand
binding arrangement, as is known in the art.
[0337] In addition to the above imaging techniques, one of skill in
the art is also aware that positron emission tomography, PET
imaging or a PET scan, can also be used as a diagnostic
examination. PET scans involve the acquisition of physiologic
images based on the detection of radiation from the emission of
positrons. Positrons are tiny particles emitted from a radioactive
substance administered to the subject.
[0338] Thus, in certain embodiments of the present invention, the
antagonist or related-compound thereof is enzymatically-,
radiolabel-, or fluorescently-tagged, as described above and used
to diagnosis, monitor, and/or stage neuronal damage by cerebral
edema. For example, the enzymatically-, radiolabel-, or
fluorescently-tagged antagonist or related-compound thereof can be
used to determine the size, limits and/or boundaries of tumors. It
is difficult to determine the boundaries of certain tumors, for
example, metastatic tumors. In metastatic tumors, glial cells are
activated and form a capsule or gliotic capsule around the tumor.
Thus, the labeled antagonist or related-compound thereof can be
used to determine the border of tumor, which can enhance the
efficiency of its removal by the surgeon. Still further, the
labeled antagonist or related-compound thereof may be used to
determine or define the penumbra or the areas at risk for later
infarction or damage after a stroke.
[0339] C. Formulations and Routes for Administration of
Compounds
[0340] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more modulators of
NC.sub.Ca-ATP channel (antagonist and/or agonist) or
related-compounds or additional agent dissolved or dispersed in a
pharmaceutically acceptable carrier. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of a
pharmaceutical composition that contains at least one modulators of
NC.sub.Ca-ATP channel (antagonist and/or agonist) or
related-compounds or additional active ingredient will be known to
those of skill in the art in light of the present disclosure, as
exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, incorporated herein by reference. Moreover,
for animal (e.g., human) administration, it will be understood that
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biological
Standards.
[0341] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, gels,
binders, excipients, disintegration agents, lubricants, sweetening
agents, flavoring agents, dyes, such like materials and
combinations thereof, as would be known to one of ordinary skill in
the art (see, for example, Remington's Pharmaceutical Sciences,
18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated
herein by reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the
pharmaceutical compositions is contemplated.
[0342] The modulators of NC.sub.Ca-ATP channel (antagonist and/or
agonist) or related-compounds may comprise different types of
carriers depending on whether it is to be administered in solid,
liquid or aerosol form, and whether it need to be sterile for such
routes of administration as injection. The present invention can be
administered intravenously, intradermally, transdermally,
intrathecally, intraventricularly, intraarterially,
intraperitoneally, intranasally, intravaginally, intrarectally,
topically, intramuscularly, subcutaneously, mucosally, orally,
topically, locally, inhalation (e.g., aerosol inhalation),
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), or by other method
or any combination of the forgoing as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference).
[0343] The modulators of NC.sub.Ca-ATP channel (antagonist and/or
agonist) or related-compounds may be formulated into a composition
in a free base, neutral or salt form. Pharmaceutically acceptable
salts, include the acid addition salts, e.g., those formed with the
free amino groups of a proteinaceous composition, or which are
formed with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. Salts formed with the free carboxyl groups can
also be derived from inorganic bases such as for example, sodium,
potassium, ammonium, calcium or ferric hydroxides; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as formulated for parenteral
administrations such as injectable solutions, or aerosols for
delivery to the lungs, or formulated for alimentary administrations
such as drug release capsules and the like.
[0344] Further in accordance with the present invention, the
composition of the present invention suitable for administration is
provided in a pharmaceutically acceptable carrier with or without
an inert diluent. The carrier should be assimilable and includes
liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar
as any conventional media, agent, diluent or carrier is detrimental
to the recipient or to the therapeutic effectiveness of the
composition contained therein, its use in administrable composition
for use in practicing the methods of the present invention is
appropriate. Examples of carriers or diluents include fats, oils,
water, saline solutions, lipids, liposomes, resins, binders,
fillers and the like, or combinations thereof. The composition may
also comprise various antioxidants to retard oxidation of one or
more component. Additionally, the prevention of the action of
microorganisms can be brought about by preservatives such as
various antibacterial and antifungal agents, including but not
limited to parabens (e.g., methylparabens, propylparabens),
chlorobutanol, phenol, sorbic acid, thimerosal or combinations
thereof.
[0345] In accordance with the present invention, the composition is
combined with the carrier in any convenient and practical manner,
i.e., by solution, suspension, emulsification, admixture,
encapsulation, absorption and the like. Such procedures are routine
for those skilled in the art.
[0346] In a specific embodiment of the present invention, the
composition is combined or mixed thoroughly with a semi-solid or
solid carrier. The mixing can be carried out in any convenient
manner such as grinding. Stabilizing agents can be also added in
the mixing process in order to protect the composition from loss of
therapeutic activity, i.e., denaturation in the stomach. Examples
of stabilizers for use in an the composition include buffers, amino
acids such as glycine and lysine, carbohydrates such as dextrose,
mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol,
mannitol, etc.
[0347] In further embodiments, the present invention may concern
the use of a pharmaceutical lipid vehicle compositions that include
modulators of NC.sub.Ca-ATP channel (antagonist and/or agonist) or
related-compounds, one or more lipids, and an aqueous solvent. As
used herein, the term "lipid" will be defined to include any of a
broad range of substances that is characteristically insoluble in
water and extractable with an organic solvent. This broad class of
compounds is well known to those of skill in the art, and as the
term "lipid" is used herein, it is not limited to any particular
structure. Examples include compounds which contain long-chain
aliphatic hydrocarbons and their derivatives. A lipid may be
naturally occurring or synthetic (i.e., designed or produced by
man). However, a lipid is usually a biological substance.
Biological lipids are well known in the art, and include for
example, neutral fats, phospholipids, phosphoglycerides, steroids,
terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,
lipids with ether and ester-linked fatty acids and polymerizable
lipids, and combinations thereof. Of course, compounds other than
those specifically described herein that are understood by one of
skill in the art as lipids are also encompassed by the compositions
and methods of the present invention.
[0348] One of ordinary skill in the art would be familiar with the
range of techniques that can be employed for dispersing a
composition in a lipid vehicle. For example, the modulators of
NC.sub.Ca-ATP channel (antagonist and/or agonist) or
related-compounds may be dispersed in a solution containing a
lipid, dissolved with a lipid, emulsified with a lipid, mixed with
a lipid, combined with a lipid, covalently bonded to a lipid,
contained as a suspension in a lipid, contained or complexed with a
micelle or liposome, or otherwise associated with a lipid or lipid
structure by any means known to those of ordinary skill in the art.
The dispersion may or may not result in the formation of
liposomes.
[0349] The actual dosage amount of a composition of the present
invention administered to an animal patient can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic and/or prophylatic interventions, idiopathy
of the patient and on the route of administration. Depending upon
the dosage and the route of administration, the number of
administrations of a preferred dosage and/or an effective amount
may vary according to the response of the subject. The practitioner
responsible for administration will, in any event, determine the
concentration of active ingredient(s) in a composition and
appropriate dose(s) for the individual subject.
[0350] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein.
Naturally, the amount of active compound(s) in each therapeutically
useful composition may be prepared is such a way that a suitable
dosage will be obtained in any given unit dose of the compound.
Factors such as solubility, bioavailability, biological half-life,
route of administration, product shelf life, as well as other
pharmacological considerations will be contemplated by one skilled
in the art of preparing such pharmaceutical formulations, and as
such, a variety of dosages and treatment regimens may be
desirable.
[0351] Pharmaceutical formulations may be administered by any
suitable route or means, including alimentary, parenteral, topical,
mucosal or other route or means of administration. Alimentary
routes of administration include administration oral, buccal,
rectal and sublingual routes. Parenteral routes of administration
include administration include injection into the brain parenchyma,
and intravenous, intradermal, intramuscular, intraarterial,
intrathecal, subcutaneous, intraperitoneal, and intraventricular
routes of administration. Topical routes of administration include
transdermal administration.
[0352] D. Alimentary Compositions and Formulations
[0353] In preferred embodiments of the present invention, the
modulators of NC.sub.Ca-ATP channel (antagonist and/or agonist) or
related-compounds are formulated to be administered via an
alimentary route. Alimentary routes include all possible routes of
administration in which the composition is in direct contact with
the alimentary tract. Specifically, the pharmaceutical compositions
disclosed herein may be administered orally, buccally, rectally, or
sublingually. As such, these compositions may be formulated with an
inert diluent or with an assimilable edible carrier, or they may be
enclosed in hard- or soft-shell gelatin capsule, or they may be
compressed into tablets, or they may be incorporated directly with
the food of the diet.
[0354] In certain embodiments, the active compounds may be
incorporated with excipients and used in the form of ingestible
tablets, buccal tables, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like (Mathiowitz et al., 1997; Hwang et
al., 1998; U.S. Pat. Nos. 5,641,515; 5,580,579 and 5,792, 451, each
specifically incorporated herein by reference in its entirety). The
tablets, troches, pills, capsules and the like may also contain the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc. When the dosage unit form is a capsule, it
may contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills, or capsules may be coated with shellac,
sugar, or both. When the dosage form is a capsule, it may contain,
in addition to materials of the above type, carriers such as a
liquid carrier. Gelatin capsules, tablets, or pills may be
enterically coated. Enteric coatings prevent denaturation of the
composition in the stomach or upper bowel where the pH is acidic.
See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small
intestines, the basic pH therein dissolves the coating and permits
the composition to be released and absorbed by specialized cells,
e.g., epithelial enterocytes and Peyer's patch M cells. A syrup of
elixir may contain the active compound sucrose as a sweetening
agent methyl and propylparabens as preservatives, a dye and
flavoring, such as cherry or orange flavor. Of course, any material
used in preparing any dosage unit form should be pharmaceutically
pure and substantially non-toxic in the amounts employed. In
addition, the active compounds may be incorporated into
sustained-release preparation and formulations.
[0355] For oral administration the compositions of the present
invention may alternatively be incorporated with one or more
excipients in the form of a mouthwash, dentifrice, buccal tablet,
oral spray, or sublingual orally-administered formulation. For
example, a mouthwash may be prepared incorporating the active
ingredient in the required amount in an appropriate solvent, such
as a sodium borate solution (Dobell's Solution). Alternatively, the
active ingredient may be incorporated into an oral solution such as
one containing sodium borate, glycerin and potassium bicarbonate,
or dispersed in a dentifrice, or added in a
therapeutically-effective amount to a composition that may include
water, binders, abrasives, flavoring agents, foaming agents, and
humectants. Alternatively the compositions may be fashioned into a
tablet or solution form that may be placed under the tongue or
otherwise dissolved in the mouth.
[0356] Additional formulations which are suitable for other modes
of alimentary administration include suppositories. Suppositories
are solid dosage forms of various weights and shapes, usually
medicated, for insertion into the rectum. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0357] E. Parenteral Compositions and Formulations
[0358] In further embodiments, modulators of NC.sub.Ca-ATP channel
(antagonist and/or agonist) or related-compounds may be
administered via a parenteral route. As used herein, the term
"parenteral" includes routes that bypass the alimentary tract.
Specifically, the pharmaceutical compositions disclosed herein may
be administered for example, but not limited to intravenously,
intradermally, intramuscularly, intraarterially,
intraventricularly, intrathecally, subcutaneous, or
intraperitoneally U.S. Pat. Nos. 6,7537,514, 6,613,308, 5,466,468,
5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated
herein by reference in its entirety).
[0359] Solutions of the active compounds as free base or
pharmacologically acceptable salts may be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions may also be prepared in glycerol, liquid polyethylene
glycols, and mixtures thereof and in oils. Under ordinary
conditions of storage and use, these preparations contain a
preservative to prevent the growth of microorganisms. The
pharmaceutical forms suitable for injectable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation of sterile injectable solutions or
dispersions (U.S. Pat. No. 5,466,468, specifically incorporated
herein by reference in its entirety). In all cases the form must be
sterile and must be fluid to the extent that easy injectability
exists. It must be stable under the conditions of manufacture and
storage and must be preserved against the contaminating action of
microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, DMSO, polyol (i.e., glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), suitable mixtures thereof,
and/or vegetable oils. Proper fluidity may be maintained, for
example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial and
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride. Prolonged absorption of the injectable
compositions can be brought about by the use in the compositions of
agents delaying absorption, for example, aluminum monostearate and
gelatin.
[0360] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous, and
intraperitoneal administration. In this connection, sterile aqueous
media that can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage may
be dissolved in 1 ml of isotonic NaCl solution and either added to
1000 ml of hypodermoclysis fluid or injected at the proposed site
of infusion, (see for example, "Remington's Pharmaceutical
Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some
variation in dosage will necessarily occur depending on the
condition of the subject being treated. The person responsible for
administration will, in any event, determine the appropriate dose
for the individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0361] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof. A
powdered composition is combined with a liquid carrier such as,
e.g., water or a saline solution, with or without a stabilizing
agent.
[0362] F. Miscellaneous Pharmaceutical Compositions and
Formulations
[0363] In other preferred embodiments of the invention, the active
compound modulators of NC.sub.CaATP channel (antagonist and/or
agonist) or related-compounds may be formulated for administration
via various miscellaneous routes, for example, topical (i.e.,
transdermal) administration, mucosal administration (intranasal,
vaginal, etc.) and/or inhalation.
[0364] Pharmaceutical compositions for topical administration may
include the active compound formulated for a medicated application
such as an ointment, paste, cream or powder. Ointments include all
oleaginous, adsorption, emulsion and water-solubly based
compositions for topical application, while creams and lotions are
those compositions that include an emulsion base only. Topically
administered medications may contain a penetration enhancer to
facilitate adsorption of the active ingredients through the skin.
Suitable penetration enhancers include glycerin, alcohols, alkyl
methyl sulfoxides, pyrrolidones and laurocapram. Possible bases for
compositions for topical application include polyethylene glycol,
lanolin, cold cream and petrolatum as well as any other suitable
absorption, emulsion or water-soluble ointment base. Topical
preparations may also include emulsifiers, gelling agents, and
antimicrobial preservatives as necessary to preserve the active
ingredient and provide for a homogenous mixture. Transdermal
administration of the present invention may also comprise the use
of a "patch". For example, the patch may supply one or more active
substances at a predetermined rate and in a continuous manner over
a fixed period of time.
[0365] In certain embodiments, the pharmaceutical compositions may
be delivered by eye drops, intranasal sprays, inhalation, and/or
other aerosol delivery vehicles. Methods for delivering
compositions directly to the lungs via nasal aerosol sprays has
been described e.g., in U.S. Pat. Nos. 5,756,353 and 5,804,212
(each specifically incorporated herein by reference in its
entirety). Likewise, the delivery of drugs using intranasal
microparticle resins (Takenaga et al., 1998) and
lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,
specifically incorporated herein by reference in its entirety) are
also well-known in the pharmaceutical arts. Likewise, transmucosal
drug delivery in the form of a polytetrafluoroetheylene support
matrix is described in U.S. Pat. No. 5,780,045 (specifically
incorporated herein by reference in its entirety).
[0366] The term aerosol refers to a colloidal system of finely
divided solid of liquid particles dispersed in a liquefied or
pressurized gas propellant. The typical aerosol of the present
invention for inhalation will consist of a suspension of active
ingredients in liquid propellant or a mixture of liquid propellant
and a suitable solvent. Suitable propellants include hydrocarbons
and hydrocarbon ethers. Suitable containers will vary according to
the pressure requirements of the propellant. Administration of the
aerosol will vary according to subject's age, weight and the
severity and response of the symptoms.
XII. DIAGNOSTIC OR THERAPEUTIC KITS
[0367] Any of the compositions described herein may be comprised in
a kit. In a non-limiting example, it is envisioned that a compound
that selectively binds to or identifies SUR1 may be comprised in a
diagnostic kit. Such compounds can be referred to as an "SUR1
marker", which may include, but are not limited to antibodies
(monoclonal or polyclonal), SUR1 oligonucleotides, SUR1
polypeptides, small molecule or combinations thereof, antagonist,
agonist, etc. It is envisioned that any of these SUR1 markers may
be linked to a radioactive substance and/or a fluorescent marker
and/or a enzymatic tag for quick determination. The kits may also
comprise, in suitable container means a lipid, and/or an additional
agent, for example a radioactive or enzymatic or florescent
marker.
[0368] The kits may comprise a suitably aliquoted SUR1 marker,
lipid and/or additional agent compositions of the present
invention, whether labeled or unlabeled, as may be used to prepare
a standard curve for a detection assay. The components of the kits
may be packaged either in aqueous media or in lyophilized form. The
container means of the kits will generally include at least one
vial, test tube, flask, bottle, syringe or other container means,
into which a component may be placed, and preferably, suitably
aliquoted. Where there are more than one component in the kit, the
kit also will generally contain a second, third or other additional
container into which the additional components may be separately
placed. However, various combinations of components may be
comprised in a vial. The kits of the present invention also will
typically include a means for containing the SUR1 marker, lipid,
additional agent, and any other reagent containers in close
confinement for commercial sale. Such containers may include
injection or blow molded plastic containers into which the desired
vials are retained.
[0369] Therapeutic kits of the present invention are kits
comprising an antagonist, agonist or an related-compound thereof.
Depending upon the condition and/or disease that is being treated,
the kit may comprise an SUR1 antagonist or related-compound thereof
to block and/or inhibit the NC.sub.Ca-ATP channel or the kit may
comprise an SUR1 agonist or related-compound thereof to open the
NC.sub.Ca-ATP channel. Such kits will generally contain, in
suitable container means, a pharmaceutically acceptable formulation
of SUR1 antagonist, agonist or related-compound thereof. The kit
may have a single container means, and/or it may have distinct
container means for each compound. For example, the therapeutic
compound and solution may be contained within the same container;
alternatively, the therapeutic compound and solution may each be
contained within different containers. A kit may include a
container with the therapeutic compound that is contained within a
container of solution.
[0370] When the components of the kit are provided in one and/or
more liquid solutions, the liquid solution is an aqueous solution,
with a sterile aqueous solution being particularly preferred. The
SUR1 antagonist, agonist or related-compounds thereof may also be
formulated into a syringeable composition. In which case, the
container means may itself be a syringe, pipette, and/or other such
like apparatus, from which the formulation may be applied to an
infected area of the body, injected into an animal, and/or even
applied to and/or mixed with the other components of the kit.
[0371] Examples of aqueous solutions include, but are not limited
to ethanol, DMSO and/or Ringer's solution. In certain embodiments,
the concentration of DMSO or ethanol that is used is no greater
than 0.1% or (1 ml/1000 L).
[0372] However, the components of the kit may be provided as dried
powder(s). When reagents and/or components are provided as a dry
powder, the powder can be reconstituted by the addition of a
suitable solvent. It is envisioned that the solvent may also be
provided in another container means.
[0373] The container means will generally include at least one
vial, test tube, flask, bottle, syringe and/or other container
means, into which the SUR1 antagonist, agonist or related-compounds
thereof is suitably allocated. The kits may also comprise a second
container means for containing a sterile, pharmaceutically
acceptable buffer and/or other diluent.
[0374] The kits of the present invention will also typically
include a means for containing the vials in close confinement for
commercial sale, such as, e.g., injection and/or blow-molded
plastic containers into which the desired vials are retained.
[0375] Irrespective of the number and/or type of containers, the
kits of the invention may also comprise, and/or be packaged with,
an instrument for assisting with the injection/administration
and/or placement of the SUR1 antagonist, agonist or
related-compounds thereof within the body of an animal. Such an
instrument may be a syringe, pipette, forceps, and/or any such
medically approved delivery vehicle.
[0376] In addition to the SUR1 antagonist, agonist or
related-compounds thereof, the kits may also include a second
active ingredient. Examples of the second active ingredient include
substances to prevent hypoglycemia (e.g., glucose, D5W, glucagon,
etc.), thrombolytic agents, anticoagulants, antiplatelets, statins,
diuretics, vasodilators, etc. These second active ingredients may
be combined in the same vial as the SUR1 antagonist, agonist or
related-compounds thereof or they may be contained in a separate
vial.
[0377] Still further, the kits of the present invention can also
include glucose testing kits. Thus, the blood glucose of the
patient is measured using the glucose testing kit, then the SUR1
antagonist, agonist or related-compounds thereof can be
administered to the subject followed by measuring the blood glucose
of the patient.
[0378] In addition to the above kits, the therapeutic kits of the
present invention can be assembled such that an IV bag comprises a
septum or chamber which can be opened or broken to release the
compound into the IV bag. Another type of kit may include a bolus
kit in which the bolus kit comprises a pre-loaded syringe or
similar easy to use, rapidly administrable device. An infusion kit
may comprise the vials or ampoules and an IV solution (e.g.,
Ringer's solution) for the vials or ampoules to be added prior to
infusion. The infusion kit may also comprise a bolus kit for a
bolus/loading dose to be administered to the subject prior, during
or after the infusion.
EXAMPLES
[0379] The following examples are provided for further illustration
of the present invention, and do not limit the invention. The
examples provided herein are for illustrative purposes only, and
are in no way intended to limit the scope of the present invention.
While the invention has been described in detail, and with
reference to specific embodiments thereof, it will be apparent to
one with ordinary skill in the art that various changes and
modifications can be made in the specific embodiments which are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention. Experiments
and exemplary procedures are described below which provide
additional enabling support for the present invention. In
particular, in vitro studies using freshly isolated reactive
astrocytes and in vivo studies using appropriate animal models are
disclosed.
Cell Preparation
[0380] Reactive astrocytes are produced in vivo and harvested from
adult brain in the following manner: gelatin sponges (Gelfoam.RTM.,
Upjohn Co., Kalamazoo Mich.) are implanted into a stab wound in the
parietal lobe of 8 week old Wistar rats as described herein. Sponge
pieces are harvested at 8 days and washed three times in
phosphate-buffered saline (PBS, pH 7.4) to remove adherent tissue.
Depending on the number of NRAs required for a particular study,
the sponge pieces may be harvested earlier or later after
implantation into a stab wound, with the preferred harvest being
conducted from about 2 days to about 30 days after implantation,
and the most preferred range being conducted from about 2 days to
about 3 days after implantation.
[0381] NRAs are freshly isolated from the sponge pieces in the
following manner: washed pieces are placed in an Eppendorf tube
containing artificial cerebrospinal fluid (aCSF) composed of (mM):
124 mM NaCl, 5.0 mM, 1.3 mM MgCl.sub.2, 2.0 mM CaCl.sub.2, 26 mM
NaHCO.sub.3, and 10 mM D-glucose; at pH 7.4, .apprxeq.290 mOsm,
wherein the aCSF contains papain 20 U/ml, trypsin inhibitor 10
mg/ml and DNase 0.01% (Worthington, Lakewood, N.J.), the entirety
of which is referred to as a "digestion system."
[0382] This digestion system is transferred to an incubator
(humidified 90%/10% air/C02, 37.degree. C.) for 20 minutes, and is
gently triturated every 5 minutes. The cell suspension is
centrifuged at 3,000 rpm for 1 minute. The pelleted cells are
resuspended in aCSF and stored at 4.degree. C. until studied.
[0383] For some studies, prior to resuspension in aCSF, the
pelleted cells can be further purified by removing red blood cells
(RBCs) using density gradient centrifugation in Histopaque-1 077
(Sigma Diagnostics, St. Louis, Mo.). This further purification
process can produce a population of cells in which <<1% are
RBCs, as determined by phase contrast microscopy.
Scanning Electron Microscopy (SEM)
[0384] To study cell blebbing and swelling, freshly isolated cells
are exposed at room temperature to NaN.sub.3 then, after various
time intervals, cells are fixed using iced 4% formaldehyde+1%
glutaraldehyde for 24 hours then dehydrated using serial
concentrations (35, 50, 75, 95, 100%) of ethanol. Specimens are
critical point dried (Tousimis), gold coated (Technics), and viewed
using an AMR 1000 scanning electron microscope.
Electrophysiology
[0385] Experiments are carried out at room temperature,
22-25.degree. C., using NRAs within 24 hour of cell isolation. An
aliquot of these freshly isolated NRAs is placed in the recording
chamber filled with extracellular bath solution containing (a):
NaCl 130, KCl 10, CaCl.sub.2 1, MgCl.sub.2 1, HEPES 32.5, glucose
12.5, pH 7.4. After viable cells adhere to the surface, flushing
with excess solution washes away residual debris not previously
removed by centrifugation. Membrane currents are amplified
(Axopatch 200A, Axon Instruments, Foster City, Calif.) and sampled
on-line at 5 kHz using a microcomputer equipped with a digitizing
board (Digidata 1200A, Axon Instruments) and running Clampex
software (version 8.0, Axon Instruments). Membrane currents are
recorded in intact cells using both the cell-attached and the
nystatin-perforated whole-cell configurations, according to methods
described in Horn and Marty, 1988. Membrane currents are recorded
in cell-free isolated membrane patches, using both the inside-out
and outside-out configurations, such as those described in Hamill
et al., 1981. Patch clamp pipettes, pulled from borosilicate glass
(Kimax, Fisher Scientific, Pittsburgh, Pa.), have resistances of
6-8 M.OMEGA. a for single channel recordings and 2-4 M.OMEGA. a for
experiments using the nystatin-perforated whole-cell technique. The
bath electrode is a Ag/AgCl pellet (Clark Electromedical, Reading,
England) that is placed directly in the bath except when the bath
[Cl--] is altered, in which case an agar bridge made with 3 M KC 1
is used to connect to the bath.
[0386] The terms "intracellular" and "cytoplasmic" are
interchangeable, as are the terms "extracellular" and "external".
The terms "voltage" and "potential" are interchangeable when
referring to membrane voltage or membrane potential. "Clamping" a
cell membrane refers to holding the voltage across the cell
membrane constant and measuring changes in membrane current as
membrane resistance changes due to ion channel opening and closing
("voltage clamp") or holding the current across the cell membrane
constant and measuring changes in membrane voltage as membrane
resistance changes due to ion channel opening and closing ("current
clamp"). When a membrane voltage is imposed on the cell, for
example with a "ramp" or "pulse", it is understood that the cell
membrane has been voltage-clamped and membrane current is being
measured. When membrane "resting potential" is measured, it is
understood that the cell membrane has been current-clamped and
membrane voltage is being measured.
[0387] The "whole-cell" experimental configuration refers to a
situation in which a recording pipette penetrates the cell membrane
so that the pipette solution is continuous with the cytoplasm or
the membrane under the pipette is perforated using nystatin, the
external solution is in contact with the extracellular membrane,
and current or voltage recordings represent measurements from the
entire cell membrane. The "cell-attached patch" experimental
configuration refers to a situation in which the pipette contacts
the cell so that the patch is still forming part of the intact cell
membrane and channels in the patch are recorded. The "outside-out
patch" experimental configuration refers to a situation in which an
excised patch of cell membrane is sealed to the tip of a recording
pipette so that the pipette solution is in contact with the
extracellular side of the membrane, the external solution is in
contact with the cytoplasmic side of the membrane, and current or
voltage recordings represent measurements from the excised patch of
membrane. The "inside-out patch" experimental configuration refers
to a situation in which an excised patch of cell membrane is sealed
to the tip of a recording pipette so that the pipette solution is
in contact with the cytoplasmic side of the membrane, the external
solution is in contact with the extracellular side of the membrane,
and current or voltage recordings represent measurements from the
excised patch of membrane.
[0388] The term "patches" includes, but is not limited to:
inside-out patches, outside-out patches, an excised patch of a cell
membrane, or a cell-attached patch. The term "membrane preparation"
includes patches as well as cell membranes isolated from mammalian
cells or tissues. Isolated mammalian cell membranes are produced by
methods well known in the art. One example of such a membrane
preparation is a microsomal fraction purified from disrupted cells
or a tissue sample by discontinuous sucrose gradient
centrifugation.
[0389] Patches with seal resistance of <3 G.OMEGA. and access
resistance of >50 M.OMEGA. are discarded. Macroscopic membrane
currents are measured during step pulses (600 ms) or during ramp
pulses (-140 to +50 mV at 0.32 mV/ms) from a holding potential of
-67 mV.
Recording Solutions
[0390] For whole cell macroscopic recordings, a nystatin perforated
patch technique is used, with a bath solution containing (mM): NaCl
130, KCl 10, CaCl.sub.2 1, MgCl.sub.2 1, HEPES 32.5, glucose 12.5,
pH 7.4. The pipette solution contains (mM): KCl 55, K.sub.2SO4 75,
MgCl.sub.2 8, and HEPES 10, pH 7.2. Nystatin, 50 mg (Calbiochem) is
dissolved in dimethylsulfoxide (DMSO), 1 ml. Working solutions are
made before the experiment by adding 16.5 .mu.l nystatin stock
solution to 5 ml of the base pipette solution to yield a final
concentration of nystatin of 165 .mu.g/ml and DMSO 3.3 .mu.l/ml.
This composition of the pipette solution includes K.sub.2SO.sub.4
instead of a portion of the KCl that would otherwise be included.
The SO.sub.4.sup.2- anion, unlike Cl--, is not permeable through
the nystatin pore. Reducing the pipette [Cl.sup.-] reduces the
driving force for Cl-- into the cell, thereby minimizing osmotic
swelling of the cell that might otherwise occur during
electrophysiological recording (Horn and Marty, 1988).
[0391] For cell-attached patch recording, a bath solution is used
containing (mM): NaCl 130, KCl 10, CaCl.sub.2 1, MgCl.sub.2 1,
HEPES 32.5, glucose 12.5, pH 7.4. The pipette contains (mM): KCl
145, MgCl.sub.2 1, CaCl.sub.2 0.2, EGTA 5, HEPES 10, pH 7.28. The
measured osmolarity of the extracellular solution is .apprxeq.300
mOsm (Precision Systems, Natick, Mass.).
[0392] For most inside-out patch recording, a bath solution is used
containing (mM): CsCl 145, CaCl.sub.2 4.5, MgCl.sub.2 1, EGTA 5,
HEPES 32.5, glucose 12.5, pH 7.4. The pipette contains (a): CsCl
145, MgCl.sub.2 1, CaCl.sub.2 0.2, EGTA 5, HEPES 10, pH 7.28. For
other inside-out patch recordings, Cs.sup.+ in the above solutions
is replaced with equimolar K.sup.+.
[0393] For the inorganic cation substitution experiments, Cs.sup.+
in the pipette is typically replaced by equimolar concentrations of
individual test ions (Cook et al., 1990).
[0394] For outside-out patch recording, the pipette solution
contains (mM): CsCl 145, MgCl.sub.2 1, CaCl.sub.2 0.2, EGTA 5,
HEPES 10, pH 7.28. The standard bath solution contains (mM): CsCl
145, CaCl24.5, MgCl.sub.2 1, EGTA 5, HEPES 32.5, glucose 12.5, pH
7.4. For the organic cation substitution experiments, Cs+ in the
bath is replaced with equimolar concentrations of test cation.
[0395] For experiments requiring low concentration of free
Ca.sup.2+ in bath solution, Ca.sup.2+-EGTA buffered solution is
employed, and free [Ca.sup.2+] is calculated using the program
WEBMAXC v2.10 available through the Stanford University World Wide
Web Site. For [Ca.sup.2+]=1 .mu.M, 5 mM EGTA is used and 4.5 mM
Ca.sup.2+ salt. [Ca.sup.2+]=1 .mu.M is also used in solutions to
test intracellular ATP and Mg.sup.2+ activities.
[0396] Single-channel amplitudes used to calculate slope
conductance are obtained by fitting a Gaussian function to an
all-points amplitude histogram of records obtained at various
potentials. To calculate open channel probability (nPo) at various
potentials and with different test agents, the all-points histogram
is fit to a Gaussian function and the area under the fitted curve
for the open channel is divided by the area under the fitted curve
for the closed plus open channel. Values of nPo at different
concentration of test agents are fit to a standard logistic
equation using a least-squares method.
[0397] For estimating ionic permeabilities of various cations
relative to that for K.sup.+, each permeability (Px/PK) is obtained
from its reversal potential (Erev) by fitting to the
Goldman-Hodgkin-Katz (GHK) equation well known in the art. See
Goldman 1943; Hodgkin and Katz, 1949. Current-voltage data are fit
to the GHK equation, assuming that both K+ and the test ion are
permeant.
[0398] To estimate the pore size of the NC.sub.Ca-ATP channel of
the present invention, the relative permeabilities of organic
cations are evaluated. The Stokes-Einstein radius (rse) is
calculated from the limiting conductivities () of the ions with the
formula: r.sub.SE.lamda.=constant. The constant is determined from
the behavior of TEA at 25.degree. C., for which .lamda.=44.9
cm.sup.2.OMEGA.-1, r.sub.SE=0.204 nm. The Stoke-Einstein radius is
then converted to the molecular radius using correction factors
read off from FIG. 6.1 in Robinson and Stokes, 1970. The equivalent
limiting conductance for ethanolamine is given (ibid.) and those of
other ions are calculated from their molecular weight by the
formula, MW 0.5.lamda.=constant. The constant is determined by the
value for ethanolamine at 25.degree. C.: MW=62.1 and .lamda.=4.42
cm.sup.2.OMEGA.-1 equi. Relative permeabilities (Px/PCs) are then
plotted against the calculated ionic radii. The effect of solute
size on the rate of penetration (permeability) through pores is
expressed by the Renkin equation (Renkin, 1955):
a/a.sub.0=[1-(r/R)].sup.2[1-2.104(r/R)+2.09(rR).sup.3-0.95(r/R).sup.5]
(1)
in which a, a.sub.0, r, and R are the effective area of the pore,
the total cross sectional area of the pore, radius of the solute,
and radius of the pore, respectively.
[0399] Junction potentials are determined with an electrometer by
measuring the diffusion potential established across a dialysis
membrane and are subtracted when appropriate. Holding currents are
not subtracted from any of the recordings. Difference currents are
obtained by simply subtracting current records before and after
perfusing NaN.sub.3, with no other processing being employed.
Example 1
Morphological Changes with ATP Depletion Using NaN.sub.3
[0400] Cultured neural cells have been shown to swell upon ATP
depletion. See, Jurkowitz-Alexander et al., 1992;
Jurkowitz-Alexander et al., 1993. Freshly isolated NRAs depleted of
ATP also results in cell swelling. Ischemia or traumatic injury in
brain also causes depletion of ATP in brain neural cells.
[0401] The surfaces of freshly isolated NRAs are highly complex,
exhibiting small membrane evaginations and fine processes that
decorate the entire cell surface, as shown in the scanning electron
micrograph in FIG. 14A. Exposure of NRAs to NaN.sub.3 (1 mM) causes
changes in the surface appearance, characterized early-on by loss
of complex structure and development of surface blebs (FIG. 14B),
followed later by a grossly swollen appearance with complete loss
of fine structure and formation of multiple large blebs (FIG. 14C).
Therefore, NRAs undergo blebbing and swelling after NaN.sub.3--
induced ATP depletion.
[0402] Phase contrast microscopy is also useful for assessing this
process, although fine structure cannot be resolved. Blebbing is
visibly apparent 10-15 minutes after exposure to NaN.sub.3.
Morphological changes of this sort are attributable to loss of
cytoskeletal integrity, combined with action of an osmotic force
that causes swelling of the cell.
[0403] To assess the contribution of the osmotic gradient to cell
swelling, the experiment is repeated in the presence of mannitol,
an impermeant oncotic agent. Mannitol (50 mM), at a concentration
sufficient to increase osmolarity of the extracellular solution
from 300 to 350 mOsm, delays bleb formation >30 minutes after
exposure to NaN.sub.3. Cellular ATP also can be depleted using
exposure to NaCN (2.5 mM) plus 2-deoxyglucose (10 mM). See, Johnson
et al., 1994. Similar morphologic changes, including cell membrane
blebbing and delay of blebbing by mannitol are obtained following
exposure to NaCN and 2-deoxyglucose. This demonstrates that the
effect of NaN.sub.3 is due in fact to ATP depletion and not to any
other non-specific effect of NaN.sub.3.
Example 2
General Electrophysiological Properties of NRAs
[0404] The macroscopic currents of whole cell preparations of N u s
are characterized by small inward currents at negative potentials,
large outward currents at positive potentials, and a flat "plateau"
region at intermediate potentials. NRAs exhibit macroscopic
currents that are consistent with observations in primary cultured
cells of the same origin. See, Chen et al., 2003; Chen et al.,
2001. The NRAs exhibited inward currents negative to the K.sup.+
equilibrium potential (E.sub.K) are usually <100 pA, much
smaller than values reported in cultured neonatal astrocytes
(Ransom and Sontheimer, 1995), but consistent with findings in
astrocytes freshly isolated from injured brain (Bordey and
Sontheimer, 1998; Schroder et al., 1999). The large outward
currents in NRAs are partially blocked by charybdotoxin (100 nM),
iberiotoxin (1 00 nM) and tetraethylammonium chloride (5 mM),
consistent with the presence of a large conductance
Ca.sup.2+-activated K.sup.+ channel. See, Perillan et al., 1999.
The outward current that remains in the presence of charybdotoxin
can be further blocked by 4-aminopyridine (5 mM), and exhibits
kinetic properties typical of a delayed rectifier K.sup.+ channel.
Consistent with a previous report (Perillan et al., 1999), fast
inward voltage dependent currents attributable to Na.sup.+ channels
are observed in less that 1% of NRAS.
NaN.sub.3 Elicits Depolarizing Inward Current Due to 35 DS
Channel
[0405] Current clamp recordings are used to investigate the effect
of ATP depletion by NaN.sub.3 in NRAs. For these experiments, a
nystatin-perforated patch method is used to assure that the
metabolic disruption comes from drug application and not cell
dialysis. Extracellular application of NaN.sub.3 (1 mM; room
temperature) results in a large and swift depolarization of the
cells (FIG. 1A). NaN.sub.3 rapidly depolarizes the cells to
E.sub.m.apprxeq.0 mV (-4.3.+-.0.9 mV). Depolarization usually
starts .about.1 minute after addition of NaN.sub.3, is complete in
<3 minutes, and is irreversible on washout of drug. Ouabain is a
known Na.sup.+/K.sup.+-ATPase blocker. See, Brismar and Collins,
1993. The magnitude of the depolarization observed with NaN.sub.3
far exceeds the small reversible depolarization induced by ouabain
(1 mM). This indicates that the large depolarization observed after
exposure to NaN.sub.3 is not caused by Na.sup.+/K.sup.+-ATPase pump
failure.
[0406] The time course of depolarization with NaN.sub.3 is
appreciably more rapid than the time course for development of cell
membrane blebbing observed with the same treatment. Also, neither
the time course nor the magnitude of the depolarization is affected
by raising the extracellular osmolarity with 50 mM mannitol, a
treatment that substantially delays bleb formation. Thus,
depolarization is a primary event, not secondary to cell swelling
or stretch.
[0407] Voltage-clamp recordings show that exposure to NaN.sub.3
results in a net increase of inward current in NRAs. Recordings
obtained using both ramp (FIG. 1B) and step pulses (FIG. 1C) show
significantly larger currents after NaN.sub.3 treatment, as shown
by comparing the recordings before (a) and after (b) NaN.sub.3
treatment. A plot of the "difference currents", obtained by
subtracting the current-voltage curve before drug from that after
drug (line c in FIG. 1B), indicates that the new current turned on
by NaN.sub.3 reverses near 0 mV. A reversal potential near 0 mV is
indicative that the NaN.sub.3-induced current results from a
non-specific cation conductance.
[0408] To further characterize the NaN.sub.3-induced current,
cell-attached patch recordings are used. Exposure to NaN.sub.3
elicits single channel currents in patches that exhibit no single
channel currents prior to addition of drug (FIG. 1D). After
addition of NaN.sub.3, recordings at low temporal resolution reveal
a large increase in current variance that, after increasing
temporal resolution, is revealed to be due to single channel events
(FIG. 1E at 3 and 4). The amplitudes of single-channel events
recorded at different membrane potentials are plotted in FIG. 1F,
which shows that NaN.sub.3 activates a single channel conductance
of .apprxeq.35 pS that exhibits weak inward rectification when
measured in the cell-attached configuration.
[0409] Additional experiments are carried out in the cell-attached
configuration with the pipette solution supplemented with various
drugs. The NaN.sub.3-induced single channel currents are not
blocked by 10 mM TEA, 5 mM 4-AP, 100 nM iberiotoxin, 100 nM
charybdotoxin, or 1 .mu.M tetrodotoxin (4-6 patches for each
compound). These experiments indicating that a typical K.sup.+ or
Na.sup.+ channel is not involved. Also, because 0.2 mM Ca.sup.2+ is
included in the pipette solution, these single channel openings are
unlikely to be due to monovalent cation influx via an L-type
Ca.sup.2+ channel.
[0410] Similar depolarization and activation of a 35 pS channel are
obtained when cellular ATP is depleted using exposure to NaCN (2.5
mM) plus 2-deoxyglucose (10 mM). This demonstrates that the effect
of NaN.sub.3 is caused by ATP depletion and not by any other
non-specific effect of NaN.sub.3.
[0411] Apart from ATP depletion, patch excision is also a highly
reliable method for channel activation. Of the more than 120 cells
studied in the cell-attached configuration, spontaneous channel
activity attributable to a .apprxeq.35 pS conductance is detected
in only 2 cells. Thus, the NC.sub.Ca-ATP channel of the present
invention is typically silent in metabolically healthy cells. By
contrast, a .apprxeq.35-pS channel is present in >90% of
inside-out patches formed from NRAs not exposed to NaN.sub.3 or
other metabolic toxins, thus demonstrating that an intracellular
element lost on patch excision normally prevents channel
activation.
[0412] Another potential mechanism of channel activation other than
patch excision is regulatory volume decrease (RVD). Cell swelling
is widely recognized as a stimulus that initiates RVD, a phenomenon
accompanied by activation of various currents, including a
non-selective cation channel in some systems. See, Ono et al.,
1994. When membrane patches are studied in a cell-attached
configuration, hyposmotic stimulation (210 mosmo/kgH.sub.2O)
activated single channel events, but none exhibit a .apprxeq.35 pS
conductance. This finding indicates that the depolarization and
channel activation observed with NaN.sup.3 are not part of an RVD
response secondary to NaN.sub.3-- induced cell swelling, and
accords with the previously noted observation that
NaN.sub.3-induced depolarization preceded cell swelling. This fact
is supported by the observation that the NC.sub.Ca-ATP channel is
seldom observed in cell attached patches from healthy cells, but
becomes evident in >90% of patches after conversion to an
inside-out configuration. Also, the NC.sub.Ca-ATP channel is lost
shortly after culturing reactive astrocytes.
Example 3
Relative Permeabilities and Pore-Size
[0413] The channel is further characterized using membrane patches
in the inside-out configuration. Records obtained during test
pulses to various potentials with equal [K.sup.+] on both sides of
the membrane are shown in FIG. 2A. Amplitude histograms are
constructed of events observed at potentials from -140 mV to +100
mV, and values (mean.+-.SE) for 4 patches are plotted and show in
FIG. 2B. Fit of the data to a linear equation indicates a slope
conductance of 35 pS, with an extrapolated reversal potential
(E.sub.rev) of +0.1 mV, close to the expected K.sup.+ reversal
potential (E.sub.K) of 0 mV.
[0414] In addition to conducting K.sup.+, the channel transports a
variety of alkaline ions (FIG. 3A), indicating that it is a
non-selective cation channel. In inside-out patches, the
conductance of the channel is measured with various alkaline ions
in the pipette solution, including Cs.sup.+, Na.sup.+, Rb.sup.+,
K.sup.+, and Li.sup.+, always with equimolar K.sup.+ in the bath
solution. Current-voltage data are fit to the GHK equation.
Na.sup.+ is shown to have a nearly equal slope conductance (32.6
pS) compared to K.sup.+ (35.2 pS), but the slope conductance is
reduced with other cations (FIG. 3B). Measurements of E.sub.rev are
used to estimate relative permeabilities for the series of alkaline
ions. Values for relative permeabilities derived from the GHK
equation are P.sub.Cs.sup.+/P.sub.K.sup.+=1.06,
P.sub.Na.sup.+/P.sub.K.sup.+=1.04,
P.sub.Kb.sup.+/P.sub.K.sup.+=1.02, and
P.sub.Li.sup.+/PK.sup.+=0.96, indicating that this channel is
nearly equally permeable to all monovalent cations.
[0415] The permeability of the NC.sub.Ca-ATP channel of the present
invention to anions, such as Cl.sup.-, is also assessed. After
measuring single channel current amplitudes at different potentials
with 145 mM KCl, the bath solution is changed to equimolar K.sup.+
gluconate. When an agar bridge is used, the solution change
resulted in a change in E.sub.rev<0.5 mV, indicating that the
NC.sub.Ca-ATP channel of the present invention is essentially
impermeable to anions.
[0416] The permeability of the instant channel to divalent cations,
Ca.sup.2+ and Mg.sup.2+, is also investigated (FIG. 3C). When
potassium ion in the pipette solution is replaced with 75 mM
Ca.sup.2+ or Mg.sup.2+, inward currents are not detected. Fit to
the GHK equation gives best fit values for E.sub.rev,<<-65 mV
for Ca.sup.2+ and Mg.sup.2+ respectively, giving relative
permeabilities with respect to K.sup.+ of <<0.001, indicating
that this channel is essentially impermeable to divalent
cations.
[0417] Because the NC.sub.Ca-ATP channel of the present invention
discriminates very poorly among monovalent inorganic cations (FIGS.
3A and B), experiments are performed to determine the equivalent
pore size of the channel by measuring channel permeability,
relative to Cs.sup.+, for a wide range of organic cations. Using an
outside-out patch configuration, single-channel current-voltage
relations are plotted to obtain E.sub.rev for a number of organic
cations. Permeability ratios are then derived from fits to the GHK
equation. For each of the organic cations (a) nethanolamine, (b)
guanidium, (c) ethanolamine, (d) diethylamine, (e) piperazine, (f)
Tris, and (g) N-methylglucamine, the mean value of relative
permeability measured is plotted against its hydrated molecular
radius (FIG. 3D, empty circles). The permeability ratios define a
smoothly declining series of values that are well fit by the Renkin
equation. The Renkin equation describes the permeation of a rigid
sphere through a cylindrical pore. Renkin, 1955. Least-squares, fit
to the equation, indicates an equivalent pore radius of 0.67 nm for
the NC.sub.Ca-ATP channel of the present invention. A 0.67 nm pore
radius is similar to pore sizes of 6 A, found for the Ca.sup.2+
channel (McCleskey and Almers, 1985) and 7.4 A, found for the nAChR
channel (Adams et al., 1980). Junction potentials determined
according to the methods described herein generally did not exceed
5 mV.
Example 4
Inhibition by [ATP].sub.i
[0418] The NC.sub.Ca-ATP channel is inhibited by intracellular ATP,
based on the finding that this channel is turned on after depleting
intracellular ATP by exposure to NaN.sub.3 (See FIGS. 1B, 1C, 1D
and 1E) or to NaCN plus 2-deoxyglucose. This fact is supported by
the observation that the NC.sub.Ca-ATP channel of the present
invention is seldom observed in cell-attached patches from healthy
cells, but becomes evident in >90% of patches after conversion
to an inside-out configuration.
[0419] Inside-out patches are used to demonstrate that the channel
is sensitive to block by ATP on the cytoplasmic side of the
membrane. Patches are studied using Cs.sup.+ as the charge carrier,
to assure that no K.sup.+ channel, such as Kir2.3 or K.sub.ATP, is
contributing to patch activity. With no ATP and 1 .mu.M Ca.sup.2+
in the bath, the NC.sub.Ca-ATP channel exhibits vigorous openings.
1 mM ATP causes profound diminution in channel activity, an effect
that is readily reversed on washout (FIG. 4A); however, channel
availability is unaffected by 1 mM AMP or ADP. The open channel
probability (nPo) is measured at different [ATP].sub.i, and these
values are normalized to that obtained at [ATP].sub.i=0 mM, and
fitted to a standard logistic equation. As shown in FIG. 4B, the
NC.sub.Ca-ATP channel is blocked by [ATP], in a dose-dependent
manner. Half maximum inhibition (IC.sub.50) is observed at
[ATP].sub.i, =0.79 .mu.M with a Hill coefficient of 1, and channel
activity is completely abolished at [ATP]i>30 .mu.M. ADP and
AMP, have no effect on the NC.sub.Ca-ATP channel activity in
inside-out patches.
[0420] This in vitro assay for determining the concentration of the
test compound which achieves a half-maximal inhibition of channel
activity (ICSO) may be used to formulate dose in animal models to
achieve a circulating plasma concentration range that includes the
IC.sub.50.
Example 5
Activation by [Ca.sup.2+].sub.i
[0421] The Ca.sup.2+ concentration on the cytoplasmic side of the
membrane is also found to regulate activity of the NC.sub.Ca-ATP
channel of the present invention. The relationship between
NC.sub.Ca-ATP channel activity and [Ca.sup.2+].sub.i is examined
using inside-out patches studied at membrane potential (Em)=-80 mV.
Changing [Ca.sup.2+].sub.i clearly affects activity of the
NC.sub.Ca-ATP channel (FIG. 5A). When free [Ca.sup.2+].sub.i is
<30 nM, no channel activity is apparent. With
[Ca.sup.2+].sub.i>30 nM, the open probability (nPo) increases in
accordance with the [Ca.sup.2+].sub.i, up to .mu.M of
[Ca.sup.2+].sub.i at which activity is near maximum. The effect of
Ca.sup.2+ on channel availability is found to depend on membrane
voltage. Values of nPo from 4-9 patches obtained at three different
potentials, Em=-40 mV, -80 mV and -120 mV, are normalized to values
observed with 3 .mu.M [Ca.sup.2+].sub.i. These data are fit to a
standard logistic equation using a Hill coefficient of 1.5 and
half-maximum values of 0.12 .mu.M, 0.31 .mu.M and 1.5 .mu.M at -40
mV, -80 mV and -120 mV, respectively (FIG. 5B). These data indicate
that channel activity is strongly dependent on [Ca.sup.2+].sub.i at
physiologically relevant concentrations, and that the effect of
Ca.sup.2+ is voltage dependent, consistent with a Ca.sup.2+ binding
site inside the electric field of the membrane.
Example 6
Internal Mg.sup.2+ Causes Rectification
[0422] Because certain channels are sensitive to intracellular
Mg.sup.2+ (Chuang et al., 1997; Perillan et al., 2000), experiments
are carried out to determine whether the channel rectification
observed in cell-attached patch recordings (see FIG. 1F) might be
due to intracellular Mg.sup.2+. Using inside-out patches studied
with equimolar K.sup.+ on both sides of the membrane, [Mg.sup.2+]
is varied on the cytoplasmic side. Single channel records and
channel amplitudes observed with different [Mg.sup.2+].sub.i are
shown (FIG. 6). No rectification is evident with [Mg.sup.2+].sub.i
30 .mu.M, but at [Mg.sup.2+].sub.i.apprxeq.100 .mu.M, increasingly
strong rectification is present. At 100 .mu.M, Mg.sup.2+ appears to
produce a flickery block.
Example 7
Identifying the Presence of SUR in NRAs
[0423] To determine if SUR receptors are present in NRAs, the
binding of glibenclamide to these cells is assessed by fluorescence
microscopy. Eight week old Wistar rats are injured by a stab wound
into the subcortical white matter and implantation of a gelatin
sponge as previously described herein. Eight days later, tissue
sections of formaldehyde-fixed brains from injured animals are
incubated for 60 minutes at room temperature with 20 nM
FITC-conjugated glibenclamide. A fluorescence image of the gelatin
sponge shows labeled cells lining the cavities of the sponge. In
brain adjacent to the injury, essentially no glibenclamide binding
is apparent. These data indicate that SUR, which are not normally
present in subcortical white matter, are expressed in neural cells
following traumatic injury.
RT-PCR
[0424] Total RNA is extracted from cells and used to synthesize
cDNA, which is amplified from reactive astrocytes is analyzed by
RT-PCR on an agarose gel stained with ethidium bromide. FIG. 7A is
a photograph of the gel showing the RT-PCR for SUR1 and SUR2. FIG.
7B is a photograph of a gel showing the RT-PCR for Kir6.1 and
Kir6.2. Lanes 3 and 4 in FIGS. 7A and 7B show the RT-PCR for
insulinoma cells. Lanes 5 and 6 show the RT-PCR for reactive
astrocytes. Lane 1 in FIGS. 7A and 7B represents ladder size
markers; Lane 2 in FIGS. 7A and 7B is a blank control. In FIG. 7A,
lanes 3 and 4 show the SUR1 and SUR2 experiments, respectively, in
insulinoma cells. Insulinoma cells are known to express SUR1, but
not SUR2. Lanes 5 and 6 in FIG. 7A show the SUR1 and SUR2
experiments in reactive astrocytes, respectively. FIG. 7A shows
that SUR1 mRNA is present in reactive astrocytes, as well as in the
control insulinoma cells. SUR2 is absent in both cell types. In
FIG. 7B, lanes 3 and 4 show the Kir6.1 and Kir6.2 experiments in
insulinoma cells, respectively. Kir6.1 is present in insulinoma
cells, but Kir6.2 is not. Kir6 is the potassium channel associated
with SUR1 in insulinoma cells. Lane 5 and 6 in FIG. 7B show that
neither Kir6.1 nor Kir6.2 is present in reactive astrocytes.
Therefore, reactive astrocytes express SUR1 mRNA, but Kir6.1 and
Kir6.2 mRNA is absent from the cells.
[0425] The presence of SUR1 in reactive astrocytes combined with
the regulation of the NC.sub.Ca-ATP channel in astrocytes by SUR
antagonists indicates that SUR regulates the NC.sub.Ca-ATP channel
of the present invention.
Example 8
Tryptic Digests
[0426] A characteristic feature of SUR-regulated K.sub.ATP function
is that tryptic digestion of the cytoplasmic face of the channel,
but not its extracellular face causes loss of inhibition by
sulfonylureas, without altering sensitivity to ATP and without
changing the biophysical properties of the channel. The effect of
trypsin on NC.sub.Ca-ATP function is shown in FIG. 8. Under control
conditions, channel activity in the inside-out patch configuration
is strongly inhibited by 1 .mu.M glibenclamide. Exposure to 100
pg/ml trypsin on the cytoplasmic side of the membrane for 3 minutes
yields a patch that still exhibits strong channel activity, but
that channel activity is completely unaffected by glibenclamide.
After such trypsin treatment of the cytoplasmic side, the
biophysical properties of the channel, including open channel
conductance, open channel times, Ca.sup.2+-mediated activation are
unchanged, and the channel still maintains its typical sensitivity
to ATP. By contrast, exposure of the extracellular side of the
membrane has no effect on glibenclamide inhibition. These trypsin
digest data on the NC.sub.Ca-ATP channel of the present invention
provide additional supporting evidence that SUR1 is involved in
regulation of the NC.sub.Ca-ATP channel, because the results
compare to previous findings from SUR1-regulated K.sub.ATP
channels. Linkage of a SUR to a non-selective ATP sensitive cation
channel, has not been shown previously.
Assays for Compounds or Compositions that Block NC.sub.Ca-ATP
Channel and Inhibit Neural Cell Swelling
Example 9
Effects of Sulfonylurea Compounds
[0427] Sulfonylurea compounds are known to modulate the
sulfonylurea receptor. A sulfonylurea receptor is generally
associated with K.sub.ATP channels as a regulatory component, and
is found in various tissues, including rat NRAs. Notably, the
K.sub.ATP channels Kir6.1 and Kir6.2 are not present in rat NRAs
(FIG. 7B). It is possible to activate the NC.sub.Ca-ATP channel
with SUR ligand diazoxide in outside-out patches (FIGS. 9A and 9B).
NaN.sub.3 does not elicit channel activity in isolated membrane
patches, indicating that it works via ATP depletion rather than any
direct effect on the channel.
Example 10
In Vitro Assays for Determining Dose-Dependent Blockage of the
Nc.sub.ca-Atp Channel
[0428] SUR1 blocking compounds, such as glibenclamide and
tolbutamide, are known to have an inhibitory effect on K.sub.ATP
channels. In one embodiment, the present invention arrives at the
objects of the invention by providing a method in which the direct
inhibitory effect of glibenclamide and tolbutamide on NC.sub.Ca-ATP
channels is determined (FIGS. 10 and 11). Inside-out patches are
used to show the inhibitory effect of sulfonylureas. To ensure that
no K.sup.+ channel, particularly K.sub.ATP is contributing to patch
current, Cs.sup.+ is used as the charge carrier. Channel activity
is profoundly diminished by the addition of 10 .mu.M glibenclamide
(FIG. 10A at b), and the activity is shown to be due to a 35 pS
cation channel, which is consistent with the NC.sub.Ca-ATP channel
of the present invention (FIG. 10C). Another sulfonylurea,
tolbutamide, is also shown to inhibit NC.sub.Ca-ATP channel
activity (FIGS. 11A and 11B). As shown in FIG. 11B, the
NC.sub.Ca-ATP channel is blocked by the sulfonylureas in a
dose-dependent manner. With tolbutamide, half maximum inhibition
(EC.sub.50) is observed at 16.1 .mu.M with a Hill coefficient of
1.3, and channel activity is completely lost at concentrations
>300 .mu.M. With glibenclamide, EC.sub.50 is observed at 48
.mu.M with a Hill coefficient of 1.2. The sensitivity of the
NC.sub.Ca-ATP channel of the present invention to blocking in NRAs
with both of these sulfonylurea compounds corresponds closely to
that reported in pancreatic .beta. cells and in expression systems
with SUR1, but not SUR2.
[0429] This in vitro assay for determining the concentration of the
test compound which achieves a half-maximal inhibition of channel
activity may be used to formulate dose in animal models to achieve.
a circulating plasma concentration range.
Example 11
Mechanism of Channel Regulation by Sulfonylureas
[0430] The NC.sub.Ca-ATP channel of the present invention exhibits
two open states, with a shorter and a longer dwell time, each less
than 10 ms. FIG. 12 shows data from a patch exhibiting an open
channel probability (nPo) of 0.63, with open dwell time values
.tau..sub.0-1 .tau..sub.0-2 and of 1.9 and 8.2 ms. After successive
application of 3 .mu.M tolbutamide (FIGS. 12B and 12E) and 30 .mu.M
tolbutamide (FIGS. 12C and 12F), nPo decreased to 0.44 and 0.09,
respectively, but the open dwell time values are not appreciably
affected by the drug. Closed channel dwell times are increased in
duration and frequency by tolbutamide (FIGS. 12H and 12I). Thus,
the channel of the present inventions exhibits a form of channel
inhibition in which the blocking compound had no effect on open
channel dwell times and a progressive increase in long closures.
This form of channel inhibition is similar to that produced by
sulfonylureas acting on the K.sub.ATP channel in pancreatic .beta.
cells. See, Gillis et al., 1989; Babeenko et al., 1999).
Example 12
[0431] Application of 100 .mu.M of the SUR-activator diazoxide
activates the 35 pS channel of the present invention, causing weak
inward rectification in cell-attached patches (FIGS. 13A, 13B and
13C). To determine the type of SUR affecting activation of the
NC.sub.Ca-ATP channel of the present invention, experiments are
conducted using sulfonylurea compounds that preferentially activate
SUR2 over SUR1, namely cromakalin, and pinacidil. Both cromakalin
and pinacidil had no effect on the NC.sub.Ca-ATP channel of the
present invention, which is consistent with other data described
herein indicating that SUR1 is associated with the NC.sub.Ca-ATP
channel of the present invention, and activation of the channel is
not mediated by SUR2.
Example 13
Sur-Mediated Cell Swelling
[0432] After addition of NaN.sub.3 to deplete ATP in cells, cell
blebbing typically becomes apparent in 7-10 minutes. Diazoxide is
an SUR1 agonist or SUR1 activator. When diazoxide alone is added to
the cells, blebbing occurs even without ATP depletion, Diazoxide,
therefore, opens the channel directly without ATP depletion by
activating SUR1. However, when cells are pretreated with
glibenclamide, addition of NaN.sub.3 does not cause blebbing, even
after 30 minutes. Thus, activation of NC.sub.Ca-ATP channel by ATP
depletion or by the channel opener, diazoxide, can result in
blebbing and swelling of NRAs, and that swelling can be prevented
by blocking the channel with glibenclamide. ATP depletion by Na
azide can result in necrotic cell death of NRAs. These findings
accord with the data described herein that glibenclamide protects
from the opening of the NC.sub.Ca-ATP channel following ATP
depletion, and that opening of this channel is responsible for cell
blebbing.
[0433] The antagonist used in the methods of the present invention
includes a compound that interferes with NC.sub.Ca-ATP function.
Typically, the effect of an antagonist is observed as a blocking of
NC.sub.Ca-ATP current in conditions under which the channel has
been activated and current can be measured in the absence of the
antagonist.
[0434] In addition to SUR1 specific sulfonylurea compounds, agents
that block SUR1, also include compounds that are structurally
unrelated to sulfonylureas. Such SUR1 blockers include a class of
insulin secretagogues compounds that bind to the SUR, which were
identified and developed for the treatment of type 2 diabetes. The
benzarnido derivatives: repaglinide, nateglinide, and meglitinide
represent one such class of insulin secretagogues, that bind to the
SUR. Nateglinide is an amino acid derivative. Also, imidazoline
derivatives have been identified that interact with the
sulfonylurea receptor (SUR) 1 subunit such as midaglizole
(KAD-1229), LY397364 and LY389382.
[0435] In one preferred embodiment of the present invention,
compounds that preferentially block SUR1, but not SUR2, are used in
the method of the present invention. Such compounds include
tolbutamide and gliclazide. The following compounds block both SUR1
and SUR2: glibenclamide, glimepiride, repaglinide, and meglitinide.
In yet another embodiment of the method of the present invention,
administration is combined with MgADP, which has been show to
produce an apparent increase of sulfonylurea efficacy on channels
containing SUR1, but not SUR2.
Example 14
[0436] To determine whether NC.sub.Ca-ATP activation by ATP
depletion initiates necrosis of reactive astrocytes that express
this channel, studies are conducted to determine if glibenclamide
is capable of protecting reactive astrocytes from cell death by
inhibiting NC.sub.Ca-ATP channel activity via its action on SUR1.
Two types of cell death, apoptosis and necrosis, are assessed
following ATP depletion.
[0437] Thus, activation of NC.sub.Ca-ATP channel is responsible for
necrotic death of NRAs following ATP depletion, and that
glibenclamide can prevent this form of cell death.
[0438] In this Example, the preparation of freshly isolated NRAs
was further purified by removal of RBCs, as described herein to
provide a cell population having <1% RBCs. Over 95% of cells had
resting potentials near E.sub.K, suggesting that the enzymatic
dissociation method had not appreciably harmed the cells. Over 95%
of cells are positive for the astrocyte marker, glial fibrillary
acidic protein (GFAP) as determined by immunofluorescence. When
examined by phase microscopy, the NRAs are of various sizes,
ranging from 11-45 .mu.m in diameter, some of which are phase
bright and others are phase dark. A subgroup of phase bright cells
had multiple short but distinct cell processes that are shorter
than the cell soma. In this Example, only larger (.apprxeq.30 .mu.m
diameter), phase bright cells with short processes (<1 cell
length) are studied. This population of NRAs reliably express
NC.sub.Ca-ATP channels.
[0439] Experiments are conducted at room temperature (22-25.degree.
C.) within 24 hr of cell isolation. An aliquot of cells is placed
on a chamber slide (LAB-TEK, Naperville, Ill.) filled with
extracellular bath solution containing (a): NaCl 130, KCl 10,
CaCl.sub.2 1, MgCl.sub.2 1, HEPES 32.5, glucose 12.5, pH 7.4. After
viable cells adhered to the surface, any residual debris not
previously removed by centrifugation is washed away by flushing
with excess solution. Cells are subjected to ATP depletion by 1 mM
Na azide to activate (open) the NC.sub.Ca-ATP channels, and then
incubated with glibenclamide (1 .mu.M).
[0440] Thereafter, the cells are examined by propidium iodide (PI)
staining for evidence of cellular membrane permeabilization, an
indication of early oncotic or necrotic cell death. See, Barros et
al., 2001. The cells are also examined by fluorescein-tagged
annexin V binding for evidence of externalization of the
phosphoaminolipid phosphotidylserine from the inner face of the
plasma membrane to the outer surface, an early indication of
apoptosis. See, Clodi et al., 2000; Rucker-Martin et al., 1999.
Staining procedure are conducting according to manufacture
directions (Vybrant Apoptosis Assay Kit 2, Molecular Probes).
Slides are mounted using ProLong antifade mounting medium
(Molecular Probes). Signals are visualized using a Nikon Diaphot
epifluorescent microscope (Leitz Wetzlar). Images are captured and
stored using a SenSys digital camera (Roper Scientific Inc.) and
IPLab software (version 3.0; Scanalytics Inc.). Annexin V-positive
cells or PI-positive cells are counted in 20 individual fields
using a 20.times. objective lens. Mean values of positive cells in
20 fields for various treatment groups are compared using ANOVA
Pairwise multiple comparisons, with p<0.05 being considered as
indicating a significant difference.
[0441] The fluorescence microscopy photos shown in FIG. 15A show
that under baseline (control) conditions, both annexin V-positive
and PI-positive cells (photos a and d, respectively) are rare in
the cell isolates. After a 10-min incubation with Na azide (1 mM),
the number of PI-positive cells increased substantially (p<0.05)
(FIG. 15A at photo b and FIG. 15B). This indicates that ATP
depletion triggers necrotic death in these cells. By contrast, Na
azide treatment caused the number of annexin V-positive cells to
increase slightly; the increase not being statically significant
(p>0.05) (FIG. 15A at photo e and FIG. 15C). This indicates that
apoptotic death was not a major endpoint of ATP depletion in these
cells.
[0442] Pretreatment of cells with glibenclamide (1 .mu.M) at the
time of administration of Na aide dramatically decreased the number
of PI-positive cells (p<0.05; FIG. 15A at photo c and FIG. 15B),
indicating significant protection from necrotic death following ATP
depletion. The number of NRAs undergoing apoptotic death also
decreased with glibenclamide, as indicated by annexin V labeling
(FIG. 15A at photo f and FIG. 15C), but values for this group were
not significantly different.
[0443] This data indicate that the NC.sub.Ca-ATP channel is
involved in the mechanism of the necrotic cell death of reactive
astrocytes. This Example shows that necrotic, rather than
apoptotic, cell death is the principal endpoint of ATP depletion in
these cells. Therefore, ATP depletion by Na azide initiates cell
death by removal of the ATP block of the NC.sub.Ca-ATP channel,
thus initiating oncotic cell swelling. Involvement of this channel
in oncotic cell swelling is confirmed by showing that necrotic
death can also be induced by diazoxide, the channel opener that
activates the NC.sub.Ca-ATP channel in these cells, and could be
blocked by glibenclamide, which prevents opening of the
NC.sub.Ca-ATP channel. The involvement of the NC.sub.Ca-ATP channel
in cell death of reactive astrocytes provides a mechanism and
target of death in these cells, as well as the importance of
blocking the NC.sub.Ca-ATP channel to prevent the death of reactive
astrocytes, which occurs in traumatic brain injury.
Example 15
In Vitro Assays for Determining the Ability of a Test Compound to
Provide Dose-Dependent Blockage of the Nc.sub.ca-Atp Channel
[0444] NC.sub.Ca-ATP channels blocking compounds can be identified
by a method in which the direct inhibitory effect of the test
compound on NC.sub.Ca-ATP channels is determined. Inside-out
patches are used to show the inhibitory effect of the compound. To
ensure that no K.sup.+ channel, particularly K.sub.ATP is
contributing to patch current, Cs.sup.+ is used as the charge
carrier. Compounds that profoundly diminish channel activity, and
the activity is shown to be due to a 35 pS cation channel, such a
compound is identified as a compound that blocks the NC.sub.Ca-ATP
channels and is capable of inhibiting neuronal cell swelling and
brain swelling. Varying concentrations of the compound are used to
determine whether the NC.sub.Ca-ATP channel is blocked by the
compound in a dose-dependent manner. The concentration at which
half maximum inhibition (EC.sub.50) is observed and the
concentration at which channel activity is completely lost are
determined. The sensitivity of the NC.sub.Ca-ATP channel of the
present invention to blocking in NRAs with the test compound can be
compared. This in vitro assay for determining the concentration of
the test compound which achieves a half-maximal inhibition of
channel activity may be used to formulate dose in animal models to
achieve a circulating plasma concentration range.
Example 16
In Vivo Assays for Determining Dose-Dependent Blockage of the
Nc.sub.ca-Atp Channel
[0445] The concentration of the test compound which achieves a
half-maximal inhibition of channel activity is used to formulate
dose in animal models to achieve a circulating plasma concentration
range. The dose of test compound that achieves a circulating plasma
concentration range calculated by methods known in the art is
administered to an animal having brain injury or cerebral ischemia.
To determine whether the test compound prevents, inhibits or
diminishes brain swelling, the epidural pressure and/or
intracranial pressure of the animal is measured, such as by using a
microballoon, to quantitatively monitor brain swelling. Also, the
swelling can be monitored by magnetic resonance (MR) imaging. Three
different studies start administration prior to, at the time of, or
after the brain injury. A compound that provided diminishes brain
swelling, as compared to controls, is identified as a compound
capable of inhibiting neuronal cell swelling and brain swelling.
Varying concentrations of the compound are used to determine
whether the compound delivers efficacy in a dose-dependent manner.
The dose at which half maximum inhibition is observed and the
concentration at which brain swelling is most quickly alleviated
are determined. Formulations are produced comprising the optimal
effective dose of the test compound for preventing, inhibiting, or
diminishing brain swelling, along with a pharmaceutically
acceptable carrier.
Example 17
Additional Mechanisms for Maintaining NRAs in a Polarized State
[0446] When reactive astrocytes are strongly depolarized due to
opening of the NC.sub.Ca-ATP channel, they undergo blebbing and
swelling and eventually sustain necrotic cell death. As stated
above, when reactive astrocytes are strongly depolarized due to
opening of a non-selective channel that is sensitive to Ca.sup.2+
and ATP (NC.sub.Ca-ATP channel), they undergo blebbing and swelling
and eventually sustain necrotic cell death. The death of these
reactive astrocytes can be prevented if strong depolarization can
be prevented, in other words, if the cells can be maintained in a
polarized state.
[0447] One potential way of maintaining the NRAs in a polarized
state is to open the Kir2.3 channel. NRAs are exposed to the Kir2.3
channel opener, Tenidap
(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-ca-
rboxamide), to maintain Kir2.3 channels open. Native reactive
astrocytes freshly harvested from adult rat brains after injury are
exposed to Tenidap to evaluate the drug's ability to open the
Kir2.3 channel in these cells. Preferably, type 1 reactive (R1)
astrocytes are harvested and used in this assay. One of the
subtypes of reactive astrocytes is the type R1 astrocyte. Type R1
astrocytes comprise the largest population of recoverable
astrocytes at the site of brain injury. They are characteristically
located in the region of tissue surrounding the injury site, many
of which are found to have migrated into the injury site itself.
See, Perillan, et al., 1999.
[0448] The reactive astrocytes that are part of the cellular
response to TBI and stroke are comprised of at least two subtypes.
One of the subtypes of reactive astrocytes is the type R1
astrocyte. Type R1 astrocytes comprise the largest population of
recoverable astrocytes at the site of brain injury. They are
characteristically located in the region of tissue surrounding the
injury site, with many of these cells also being found to have
migrated into the injury site itself. See, Perillan, et al.
1999.
[0449] Type R1 astrocytes are the predominant type of reactive
astrocyte in the NRA preparations. Type R1 astrocytes express two
critically important ion channels in their cell membrane: (a) the
Kir2.3 channel, which is present in cultured as well as freshly
isolated cells; and (b) the NC.sub.Ca-ATP channel, which is present
only in freshly isolated reactive astrocytes and lost shortly after
culturing. The Kir2.3 is an inward rectifier channel that is
critically important for maintaining the cell polarized to a normal
resting potential near the potassium reversal potential
(.apprxeq.-75 mV). When this channel is inactivated or inhibited,
the cell depolarizes to a potential near the chloride reversal
potential (.apprxeq.-25 mV). Characteristic features of the
NC.sub.Ca-ATP channel are: 1) it is a non-selective cation channels
that allows passage of Na.sup.+, K.sup.+, and other monovalent
cations quite readily; 2) it is activated by an increase in
intracellular calcium, and/or by a decrease in intracellular ATP;
and 3) it is regulated by sulfonylurea receptor type 1 (SUR1). SUR1
had been considered to be associated exclusively with K.sub.ATP
channels, such as those found in pancreatic .beta. cells.
[0450] Opening of the NC.sub.Ca-ATP channel following ATP
depletion, as with ischemia or hypoxia, causes depolarization of
the cell due to influx of Na.sup.+. This influx of Na.sup.+
increases the osmotic load within the cell, and as a result,
H.sub.2O enters the cell to equilibrate the osmotic load. The
result is an excess of Na.sup.+ and H.sub.2O intracellularly, a
pathological response that produces cell blebbing and cell swelling
and that is known as cytotoxic edema. Left unchecked, this
pathological response eventually leads to cell death. As disclosed
herein, this cell death is mostly necrotic cell death but to a
lesser extent, apoptotic cell death as well.
[0451] A number of approaches may be used to meliorate brain
swelling due to cytotoxic edema. One currently used treatment for
treating patients in relevant clinical situations is based on
increasing extracellular osmolarity to reduce the driving force for
influx of H.sub.2O. This strategy also reduces blebbing in isolated
cells.
[0452] A more specific strategy to reduce cytotoxic edema is
inactivating or blocking the NC.sub.Ca-ATP channel that is
primarily responsible for the influx of Na.sup.+ that draws
H.sub.2O into the cell and that actually causes cytotoxic edema.
One highly selective approach to inactivating this channel is to
exploit the unique relationship between the channel and the
controlling regulatory subunit, SUR1. A variety of drugs have been
developed that interact with SUR1 in pancreatic .beta. cells to
block the K.sub.ATP channel in those cells and thereby treat
diabetes. Some of these drugs belong to the class of agents called
sulfonylureas. As described herein, drugs that block the K.sub.ATP
channel, such as glibenclamide and tolbutamide, are highly
effective at blocking the NC.sub.Ca-ATP channel in type R1
astrocytes. Drugs capable NC.sub.Ca-ATP channel blocking in NRAs
(a) prevents cell blebbing in response to ATP depletion, (b)
significantly reduces cell death following ATP depletion. Also, the
use of glibenclamide to treat brain swelling in an animal suffering
from stroke or brain injury is described herein.
[0453] Yet another strategy to oppose the effect of the
NC.sub.Ca-ATP channel and reduce cytotoxic edema would be to
counteract depolarization of the cell that accompanies opening of
the NC.sub.Ca-ATP channel. One way to accomplish this is to enhance
opening of the Kir2.3 channels that are also present in these
cells. An anti-inflammatory compound, Tenidap
(5-chloro-2,3-dihydro-3-(hydroxy-2-thienylmethylene)-2-oxo-1H-indole-1-ca-
rboxamide), is an opener of Kir2.3 channels. See, Popp et al.,
1992; Liu et al., 2002. Tenidap is evaluated for its ability to
reduce cell blebbing and swelling and necrotic cell death in
response to ATP depletion in the isolated cells as well as in situ
in injured rat brain. To assess whether Tenidap opens the Kir2.3
channels in type R1 astrocytes, using methods similar to those
described herein for evaluating the status of the NC.sub.Ca-ATP
channel. Results from such experiments that show Tenidap to open
Kir2.3 channels in type R1 astrocytes, and reduce cell blebbing and
cell death in response to ATP depletion would indicate the
usefulness of Tenidap in treating brain swelling and cytotoxic
edema resulting from TBI or cerebral ischemia. The effective amount
of Tenidap is that amount capable of reducing brain swelling or
cerebral ischemia due to the drug's ability to inhibit neural cell
swelling and necrotic cell death.
[0454] SUR1 blockers are believed to be the most specific, reliable
blockers and to provide the fewest untoward side effects. Further,
a combination of treatments including use of two to more of osmotic
diuretics, NC.sub.Ca-ATP channel blockers such as glibenclamide and
Kir2.3 channel openers such as Tenidap may provide excellent
efficacy in ameliorating cytotoxic edema and reducing morbidity and
mortality in brain injury and stroke. Thus, concomitant or
successive administration of an NC.sub.Ca-ATP channel blocker and a
Kir2.3 channel opener is expected to provide excellent efficacy in
ameliorating cytotoxic edema and reducing morbidity and mortality
in brain injury and stroke. For example, administration of
glibenclamide and Tenidap would be useful for ameliorating
cytotoxic edema and reducing morbidity and mortality in brain
injury and stroke.
Example 18
Modulation by Estrogen
[0455] A characteristic feature of K.sub.ATP channels (Kir6.1,
Kir6.2) is that channel affinity for ATP is modulated by the
presence of the membrane lipid, PIP.sub.2. The open-state stability
of K.sub.ATP channels is increased by application of PIP.sub.2 to
the cytoplasmic side of the membrane (Ashcroft, 1998; Baukrowitz et
al., 1998; Rohacs et al., 1999). An increase in the open-state
stability is manifested as an increase in the channel open
probability in the absence of ATP, and in a corresponding decrease
in sensitivity to inhibition by ATP (Enkvetchakul et al., 2000;
Haruna et al., 2000; Koster et al., 1999; and Larsson et al.,
2000).
[0456] Given the numerous similarities between the K.sub.ATP
channel and the NC.sub.Ca-ATP channel, the inventors postulated
that ATP-sensitivity of the NC.sub.Ca-ATP channel would respond to
PIP.sub.2 in the same way. This was tested by studying
NC.sub.Ca-ATP channels in inside out patches with Cs.sup.+ as the
charge carrier, and with 1 .mu.M Ca.sup.2+ and 10 .mu.M ATP in the
bath, with the latter expected to fully block the channel. Under
these conditions, only the NC.sub.Ca-ATP channel was recorded in R1
astrocytes. When PIP.sub.2 (50 .mu.M) was added to the bath,
channel activity became prominent (FIG. 16), as predicted by
analogy to the effect of PIP.sub.2 on K.sub.ATP channels. This
channel activity was blocked by glibenclamide, confirming identity
of the channel.
[0457] To determine if a receptor-mediated mechanism was involved
in the modulation of NC.sub.Ca-ATP channel activity, a well known
phospholipase C (PLC) was used to study if PLC activation would
cause degradation and consumption of PIP.sub.2 and thereby increase
affinity for ATP, e.g., reduce channel opening. Estrogen is a well
known PLC activator in brain as well as elsewhere (Beyer et al.,
2002; Le Mellay et al., 1999; Qui et al., 2003). For this
experiment, cell attached patches were studied to prevent
alteration of intracellular signaling machinery. NC.sub.Ca-ATP
channel activity was produced by exposure to Na azide to cause
depletion of cellular ATP (FIG. 2, initial part of the record).
[0458] When estrogen (E2; 10 nM) was applied to the bath, activity
due to the NC.sub.Ca-ATP channel was soon terminated (FIG. 17).
This suggested that estrogen exerted regulatory control over the
NC.sub.Ca-ATP channel, and suggested that an estrogen receptor
capable of rapid (non-genomic) activation of signaling cascades was
present on these cells.
[0459] Next, to determine whether estrogen receptors could be
detected in R1 astrocytes from males and females. Gelatin sponge
implants were harvested 7 days after implantation in a group of 3
female rats (F) and another group of 3 male rats (M). Pooled
protein from each group was analyzed at 2 dilutions (4.times.=50
.mu.g total protein; 1.times.=12.5 .mu.g total protein) by Western
blotting, with protein from uterus being used as a control (FIG.
18A). Membranes were blotted with an antibody that recognized both
.alpha. and .beta. estrogen receptors. Both males and females
showed prominent bands at the appropriate molecular weights for the
.alpha. (66 kDa) and .beta. (55 kDa) receptors (FIG. 18) (Hiroi et
al., 1999). The same samples of protein from males and females were
also used to confirm presence of SUR1, with protein from pancreas
used as a positive control (FIG. 18B). Notably, estrogen receptors
have previously been reported in astrocytes from males and females
(Choi et al., 2001). In cerebral cortex, the .beta. isoform is
reportedly more abundant (Guo et al., 2001) as suggested by the
Western blot.
[0460] Next, the electrophysiological experiment of FIG. 17 was
repeated using R1 astrocytes harvested from male rats. As above,
cell attached patches were studied in which NC.sub.Ca-ATP channel
activity was activated by depletion of intracellular ATP following
exposure to Na azide (FIG. 4A). Examination of the record at higher
temporal resolution confirmed activity of a well defined channel of
the appropriate conductance for the NC.sub.Ca-ATP channel (FIG.
4B). When estrogen was applied to the bath (FIG. 4, E2, 10 nM,
arrow), activity due to the NC.sub.Ca-ATP channel was quickly
terminated (FIG. 19). These data provided further evidence that
estrogen exerted regulatory control over the NC.sub.Ca-ATP channel,
and suggested, in addition, that this response was equally robust
in R1 astrocytes from males and females.
[0461] By analogy to the effects of estrogen, other mechanisms that
deplete PIP.sub.2, including other receptor-mediated mechanism as
well as more direct activators of PLC such as G-proteins etc.,
would be expected to have a similar inhibitory effect on activity
of the NC.sub.Ca-ATP channel and thereby exert a protective
effect.
Example 19
The Gliotic Capsule
[0462] The standard model involved placing a stab injury into the
parietal lobe of an anesthetized rat and implanting a sterile
foreign body (gelatin sponge; Gelfoam.RTM.) into the stab wound.
Variants of the standard model included impregnating the sponge
with a substance (e.g., lipopolysaccharide, LPS) or infusing a
substance continuously in vivo using an osmotic mini-pump with the
delivery catheter placed directly into the sponge. The injury
procedure was well tolerated by the animals, with virtually no
morbidity or mortality and minimal pain. After an appropriate time
in vivo, the whole brain was harvested for histological or
immunohistochemical study of tissue sections. Alternatively, if the
sponge itself was gently removed from the brain, the inner zone of
the gliotic capsule adheres to the sponge and was excised along
with it. Thus, the sponge was assayed for protein (e.g., Western)
or mRNA (RT-PCR), or it was enzymatically dissociated to yield
constituent cells for electrophysiological or other single-cell
measurements.
[0463] The gliotic capsule was well developed 7-10 days after
injury. The gliotic capsule was visualized in coronal sections by
perfusing the animal with Evans Blue prior to perfusion-fixation of
the brain (FIG. 20A). A region of edema (dark) was seen to outline
the avascular gliotic capsule (light) that surrounded the gelatin
sponge (dark). Immunohistochemical examination with anti-GFAP
antibodies showed that the brain parenchyma in the vicinity of the
sponge harbors many GFAP-positive reactive astrocytes (FIG. 20B;
arrow showed where the gelatin sponge was). At higher power, these
intraparenchymal GFAP-positive cells were shown to be large and to
bear many prominent cell processes (FIG. 20C, arrow). Examining the
gelatin sponge itself showed GFAP-positive reactive astrocytes that
migrated into the interstices of the sponge (FIG. 20D, arrow).
Example 20
Isolation of Cells from the Gliotic Capsule
[0464] Phase contrast microscopy of cells freshly isolated by
papain digestion of the inner zone of the gliotic capsule and
gelatin sponge revealed three types of cells. Most of the cells
(>90%) were large, round, have no cell processes and were
phase-bright (FIG. 21A). A number of cells (3-5%) were small,
round, have no cell processes and were phase-dark (FIG. 21B).
Occasionally, a cell was found that was intermediate in size, was
phase-bright and had multiple processes that were more than one
cell diameter in length (Chen et al., 2003). Immunofluorescence
study showed that all of these cells were strongly positive for
typical astrocyte markers, including GFAP (FIG. 21C,D) and vimentin
(FIG. 21E,F). Microglia were not prominent in the inner zone of the
gliotic capsule itself, as indicated by sparse labeling for OX-42.
Cells of the inner zone of the gliotic capsule were negative for
the 02A progenitor marker, A2B5, and the fibroblast marker, prolyl
4-hydroxylase (Dalton et al., 2003).
[0465] As with freshly isolated cells, three morphologically
distinct types of cells were observed in primary culture. Most
cells (>90%) were large polygonal cells (FIG. 21Gb), a few
(3-5%) were small bipolar cells (FIG. 21Ga), and only occasionally
were process-bearing stellate-shaped cells observed (Perillan et
al., 2000). All of these cells were strongly labeled with anti-GFAP
antibodies (FIG. 21H). Experiments in which cells obtained by
enzymatic digestion were followed individually in primary culture
showed that the large phase-bright cells develop into large
polygonal cells (FIG. 21Gb), and the small phase-dark cells
developed into small bipolar cells (FIG. 21Ga) (Dalton et al.,
2003).
[0466] The three morphologically distinguishable types of
GFAP-positive astrocytes from the inner zone of the gliotic capsule
exhibited very different macroscopic whole cell
electrophysiological profiles:
[0467] (i) Electrophysiological studies on stellate astrocytes
showed that they expressed Kir2.3 and Kir4.1 inward rectifier
channels, and immunolabeling experiments suggested that they also
expressed K.sub.ATP channels comprised of SUR1 and Kir6.1 subunits
(Chen et al., 2003; Perillan et al., 2000);
[0468] (ii) Electrophysiological studies on R2 astrocytes showed
that they expressed a novel Ca.sup.2+-activated Cl-- channel that
was sensitive to the polypeptide toxin from the scorpion, Leiurus
quinquestriatus (Dalton et al., 2003). Only the R2 astrocyte
expressed this channel.
[0469] (iii) Electrophysiological studies on R1 astrocytes showed
that they express Kir2.3 inward rectifier channels that are
regulated by TGF.beta.1 via PKC.delta. (Perillan et al., 2002;
Perillan et al., 2000). When freshly isolated but not after
culturing, R1 astrocytes also expressed a novel SUR1-regulated
NC.sub.Ca-ATP channel (Chen et al., 2003; Chen et al., 2001).
Example 21
Expression of SUR1
[0470] Glibenclamide binds to sulfonylurea receptors, SUR1 and
SUR2, with higher affinity for SUR1. Immunofluorescence studies
were performed using anti-SURx antibodies. The inner zone of the
gliotic capsule immediately outside of the gelatin sponge (gf in
FIG. 22) was strongly labeled with anti-SUR1 antibody (FIG. 22A)
but not with anti-SUR2 antibody (FIG. 22B). Although individual
cells were not discerned at low magnification, higher magnification
showed that SUR1 label was uniformly distributed in individual
cells after isolation (FIG. 22C).
[0471] Evidence for transcription of SUR1, but not SUR2 was also
found in RT-PCR experiments run on mRNA from gelatin sponges
isolated 7 days after implantation. The signal observed in
astrocytes (FIG. 22D, lane 3) was present at the appropriate
position on the gel, similar to that from control insulinoma
RIN-m5f cells (FIG. 22D, lane 2). By contrast, mRNA for SUR2 is not
transcribed in reactive astrocytes (FIG. 22D, lane 5) although it
is in cardiomyocytes used as control (FIG. 22D, lane 4).
Example 22
Characterization of the Inner Zone of the Gliotic Capsule
[0472] To examine whether or not all GFAP-positive reactive
astrocytes in the gliotic capsule are SUR1 positive, brains from
rats that had been implanted 1 week earlier with a gelatin sponge,
then perfusion-fixed and equilibrated in 40% sucrose in PBS
.times.2 days were studied. Cryostat sections were double labeled
with anti-GFAP and anti-SUR1 antibodies and studied with
immunofluorescence. For this and other immunolabeling experiments,
standard control protocol included use of the appropriate
immunogenic peptide when available or omission of primary
antibody.
[0473] Five animals were sectioned and imaged with low power
images. The images invariably showed that the depth (thickness) of
the GFAP response from the edge of the gelatin sponge was
several-fold greater than the depth of the SUR1 response.
Measurements of the depth of the GFAP response yielded values of
about 400-500 .mu.m (FIG. 23A; in FIGS. 23A-23I, the location of
the gelatin sponge implant was always to the left; bar in FIG. 23F
equals 100 .mu.m). By contrast, the prominent portion of the SUR1
response extended for a depth of only 25-50 .mu.m (FIG. 23D).
Outside of the SUR1-positive zone was a wide region of
GFAP-positive reactive astrocytes that were mostly SUR1 negative.
The SUR1 response was always located precisely at the interface
with the foreign body, in the innermost zone of the gliotic
capsule. Cells that were SUR1 positive were always GFAP positive.
It was evident from this experiment that cells clinging to the
gelatin sponge and that were harvested with it were likeliest to
express SUR1. Also, it was clear that R1 astrocytes in this
innermost region comprised a unique subpopulation of reactive
astrocytes. From this observation emerged the concept of the "inner
zone" of the gliotic capsule as being a unique entity, distinct
from the remainder of the gliotic capsule.
Example 23
Other Characteristics of the Inner Zone of the Gliotic Capsule
[0474] Other studies were performed to further evaluate the inner
zone of the gliotic capsule. In previous experiments, it was found
that primary culture of R1 astrocytes under normoxic culture
conditions resulted in loss of the SUR1-regulated NC.sub.Ca-ATP
channel after 3 days, whereas cultured under hypoxic conditions
resulted in continued expression of the channel (Chen et al.,
2003). Thus, it was determined that expression of the channel
required hypoxic conditions, and thus the inner zone of the gliotic
capsule where SUR1 expressing R1 astrocytes were found might also
be hypoxic. To evaluate this, the histochemical marker,
pimonidazole, was used which at pO.sub.2<10 mm Hg, forms
irreversible covalent adducts with cellular proteins that can be
detected immunohistochemically (Arteel et al, 1998; Hale et al.,
2002; Kennedy et al., 1997).
[0475] Briefly, rats were prepared with a stab injury and
implantation of a gelatin sponge. Rats were allowed to survive 1
week. Pimonidazole was administered prior to death, and
cryosections were processed for immunofluorescence study using the
appropriate antibody to detect pimonidazole adducts. Cryosections
were double labeled for GFAP. This experiment confirmed the
presence of hypoxic conditions restricted to the SUR1-positive
inner zone of the gliotic capsule, with the most prominent
pimonidazole labeling extending only 20-50 .mu.m deep (FIG. 23B;
GFAP not shown but the depth of the GFAP response resembled that in
FIG. 23A). High resolution imaging showed that pimonidazole
labeling (FIG. 23G, upper right) was present in large GFAP-positive
astrocytes (FIG. 23G, lower left).
[0476] It was reasoned that hypoxia of the inner zone might lead to
up-regulation/activation of the hypoxia-responsive transcription
factor, HIF-1. To examine this, immunolabeling was performed of
sections with anti-HIF-1.alpha. antibodies with co-labeling for
GFAP. This experiment confirmed that HIF-1.alpha. labeling was
mostly restricted to the SUR1-positive inner zone of the gliotic
capsule, with labeling extending only 20-50 .mu.m deep (FIG. 23C;
GFAP not shown but the depth of the GFAP response resembled that in
FIG. 23A). High resolution imaging showed that HIF-1.alpha.
labeling (FIG. 23H, upper right) was present in large GFAP-positive
astrocytes (FIG. 23H, lower left).
[0477] Expression of tight junction proteins was also examined. Two
tight junction proteins, ZO-1 and occludin-5, were studied,
labeling alternate cryosections with antibodies directed against
these proteins. Sections were double labeled for GFAP. Again, only
the innermost layer 20-50 .mu.m deep was labeled for either ZO-1 or
occludin-5 (FIGS. 23E and 23F; GFAP not shown but the depth of the
GFAP response resembled that in FIG. 23A). High resolution imaging
showed that occludin-5 labeling (FIG. 23I, upper right) was present
in large GFAP-positive astrocytes (FIG. 23I, lower left).
[0478] Thus, the inner zone of the gliotic capsule, with its R1
astrocytes that express SUR1-regulated NC.sub.Ca-ATP channels and
tight junction proteins, may be acting as an important barrier
between the foreign body and the brain, e.g., a foreign body-brain
barrier (FbBB). If true, one would expect that breaching the
barrier might significantly affect the overall response to
injury.
Example 24
Manipulation of the Inner Zone
[0479] Rats were prepared with a stab injury and implantation of a
gelatin sponge according to our usual protocol and were allowed to
survive 1 week. At time of surgery, rats were also implanted with
osmotic mini-pumps subcutaneously with the delivery catheter placed
in the brain at the site of injury. Animals received pumps with
either glibenclamide (1 .mu.M at 0.5 .mu.l/hr.times.7 days) or
diazoxide (10 .mu.M at 0.5 .mu.l/hr.times.7 days). No systemic
toxicity was observed, neurological behavior was not impaired, and
animals appeared healthy and were not febrile.
[0480] Cryosections of injured brains were examined for GFAP. In
animals receiving glibenclamide, a well defined gliotic capsule was
visualized that was sharply demarcated from surrounding brain, with
the inner zone appearing to be densely populated by GFAP-positive
cells (FIG. 24A; gelatin sponge to the right). By contrast, animals
receiving diazoxide showed an expanded GFAP-positive response that
extended farther from the foreign body, with an outer region that
was poorly demarcated, and an inner zone that was loose and not
compact (FIG. 24B; gelatin sponge to the right).
[0481] Cryosections were also examined with the nuclear label,
DAPI. In sections from glibenclamide-treated animals, most of the
labeling was attributable to GFAP-positive astrocytes. However, in
sections from diazoxide-treated animals, DAPI labeling showed
"sheets" of small nucleated cells (dull spots in FIG. 25A). On
inspection, these sheets of cells appeared to be polymorphonuclear
leukocytes (PMNs, neutrophils). This was confirmed by labeling with
MMP-8, a PMN-specific marker (FIG. 25B). It is important to note
that no evidence of infection was present, and microbiological
cultures of explanted materials showed no bacterial growth,
including aerobic and anaerobic cultures, indicating that the
inflammatory response was not due to infection.
[0482] Thus, protecting inner zone R1 astrocytes with glibenclamide
appeared to have restrained the overall GFAP-response to injury,
whereas killing inner zone R1 astrocytes with diazoxide appeared to
have caused an expansion of the overall GFAP-response and
recruitment of tremendous numbers of neutrophils. These
observations strongly reinforced the concept of the "inner zone" of
the gliotic capsule as being a unique entity, with a critical
function in determining the overall response to injury.
Example 25
SUR1 in Multiple Brain Pathologies
[0483] Tissues were obtained from the 3 rat models (trauma, abscess
and stroke) and from the gliotic capsule surrounding human
metastatic tumor, and double immunolabeling was performed with
antibodies directed against GFAP and SUR1. Low power views showed a
layer of tissue adjacent to the gelatin sponge implant with
positive immunolabeling for GFAP that coincided with positive
immunolabeling for SUR1 (FIG. 26A,B). Examination of individual
cells at high power showed that the SUR1 immunolabel was present in
large stellate-shaped astrocytes, confirming the presence of
SUR1-positive R1 astrocytes in the inner zone of the gliotic
capsule surrounding a foreign body implant (FIG. 26C).
[0484] A brain abscess model in the rat was studied. The abscess
was produced by implanting an autologous fecal pellet subcortically
under general anesthesia. These animals survived quite well,
although they showed evidence of mild weight loss. When sacrificed
1 week after surgery, a purulent cavity was found surrounded by a
gliotic capsule. Low power views of the gliotic capsule adjacent to
the area of puss showed cells with positive immunolabeling for GFAP
that coincided with positive immunolabeling for SUR1 (FIG. 26D,E).
Examination of individual cells at high power showed that the SUR1
immunolabel was present in large stellate-shaped astrocytes,
confirming the presence of SUR1-positive R1 astrocytes in the inner
zone of the gliotic capsule surrounding brain abscess (FIG.
26F).
[0485] A standard stoke model in the rat was studied. The stroke
was produced by intra-carotid insertion of a thread up to the
bifurcation of the internal carotid artery, placed under general
anesthesia. Animals surviving the stroke were sacrificed at 1 week
and the brain was examined. Low power views of tissues adjacent to
the area of stroke showed cells with positive immunolabeling for
GFAP that coincided with positive immunolabeling for SUR1 (FIG.
26G,H). Examination of individual cells at high power showed that
the SUR1 immunolabel was present in large stellate-shaped
astrocytes, confirming the presence of SUR1-positive R1 astrocytes
in the gliotic capsule surrounding stroke (FIG. 26I).
[0486] Tissue was obtained from humans undergoing surgery for
resection of metastatic brain tumors. At surgery, the gliotic
capsule that surrounds the metastasis is readily distinguished from
the tumor itself and from edematous white matter. Low power views
of the gliotic capsule adjacent to the metastasis showed cells with
positive immunolabeling for GFAP that coincided with positive
immunolabeling for SUR1 (FIG. 26J,K). Examination of individual
cells at high power showed that the SUR1 immunolabel was present in
large stellate-shaped astrocytes with multiple well-developed
processes, confirming the presence of SUR1-positive R1 astrocytes
in the gliotic capsule surrounding metastatic brain tumor in humans
(FIG. 26L).
[0487] These data show for the first time SUR1 up-regulation in
reactive astrocytes at the site of formation of a gliotic capsule
consistent with expression of SUR1-regulated NC.sub.Ca-ATP channels
in R1 astrocytes. The data indicate that SUR1 expression in R1
astrocytes in the gliotic capsule was a common phenomenon in
numerous pathological conditions that affect the brain. These data
highlight a unique opportunity to manipulate R1 astrocytes of the
inner zone selectively by exploiting pharmacological agents that
act at SUR1 and that can therefore determine death or survival of
these cells.
[0488] Overall, these observations strongly reinforced the concept
of the "inner zone" of the gliotic capsule as being a unique
entity, distinct from the remainder of the gliotic capsule.
Example 26
The Nc.sub.ca-Atp Channel and Necrotic Death
[0489] NC.sub.Ca-ATP channels were studied in a rodent model of
stroke. In the penumbra, SUR1 labeling was found in stellate-shaped
cells (FIG. 27A) that were also GFAP-positive. In the middle of the
stroke, stellate cells were absent, but SUR1 labeling was found in
round cells exhibiting a bleb-like appearance (FIG. 27B,C) that
were also GFAP-positive (not shown). The round cells with blebbing
in situ resembled reactive astrocytes in vitro undergoing necrotic
death after exposure to Na azide. The effect of glibenclamide vs.
saline was determined. Glibenclamide or saline was administered via
subcutaneously-implanted osmotic mini-pump (1 .mu.M at 0.5
.mu.l/hr). In saline treated rats, 3-day mortality after stroke was
68%, whereas in glibenclamide-treated rats, 3-day mortality was
reduced to 28% (n=29 in each group; p<0.001, by .chi..sup.2). In
separate animals, the stroke hemisphere in glibenclamide-treated
rats contained only half as much excess water as in saline-treated
rats (n=5 in each group; p<0.01, by t-test), confirming an
important role of the NC.sub.Ca-ATP channel in edema formation.
[0490] SUR1 was also studied in a rodent model of trauma. The
effect of direct infusion of drugs into the site of trauma was
examined using an implanted osmotic mini-pump. The channel
inhibitor, glibenclamide, was used to reduce death of reactive
astrocytes, and the channel activator, diazoxide, to promote
astrocyte death. Glibenclamide infusion reduced the overall injury
response, stabilized the gliotic capsule around the foreign body
implant, and minimized the inflammatory response compared to
control.
[0491] Conversely, diazoxide essentially destroyed the gliotic
capsule and incited a huge inflammatory response, characterized by
massive influx of polymorphonuclear cells (PMNs) (FIG. 25A, B).
These data suggested that NC.sub.Ca-ATP channel plays a critical
role in the injury response, and they strongly support the
hypothesis that inflammation is closely linked to activity of the
NC.sub.Ca-ATP channel and necrotic death of reactive
astrocytes.
Example 27
Permanent MCA Models
[0492] Adult male or female Wistar rats (275-350 gm) were fasted
overnight then anesthetized (Ketamine, 60 mg/kg plus Xylazine, 7.5
mg/kg, i.p.). The right femoral artery was cannulated, and
physiological parameters, including temperature, pH, pO.sub.2,
pCO.sub.2 and glucose were monitored. Using a ventral cervical
incision, the right external carotid and pterygopalatine arteries
were ligated. The common carotid artery was ligated proximally and
catheterized to allow embolization of the internal carotid
artery.
[0493] For thromboembolic (TE) stroke, 7-8 allogeneic clots, 1.5 mm
long, were embolized. Allogeneic, thrombin-induced, fibrin-rich
blood clots were prepared (Toomy et al., 2002).
[0494] For large MCA strokes with malignant cerebral edema (MCE),
the inventors first embolized microparticles (Nakabayashi et al.,
1997) [polyvinyl alcohol (PVA) particles; Target Therapeutics,
Fremont Calif.; 150-250 .mu.m diameter, 600 .mu.g in 1.5 ml
heparinized-saline], followed by standard permanent intraluminal
suture occlusion (Kawamura et al., 1991) using a monofilament
suture (4-0 nylon, rounded at the tip and coated with
poly-L-lysine) advanced up to the ICA bifurcation and secured in
place with a ligature.
[0495] After stroke, animals are given 10 ml glucose-free normal
saline by dermoclysis. Rectal temperature was maintained at
.apprxeq.37.degree. C. using a servo-controlled warming blanket
until animals awoke from anesthesia. Blood gases and serum glucose
at the time of stroke were: pO.sub.2, 94.+-.5 mm Hg; pCO.sub.2,
36.+-.5 mm Hg; pH, 7.33.+-.0.01; glucose 142.+-.6 mg/dl in controls
and pO.sub.2, 93.+-.3 mm Hg; pCO.sub.2, 38.+-.2 mm Hg; pH,
7.34.+-.0.01; glucose 152.+-.7 mg/dl in glibenclamide-treated
animals.
[0496] With both models, animals awoke promptly from anesthesia and
moved about, generally exhibited abnormal neurological function,
typically circling behavior and hemiparesis. Mortality with the
thromboembolic (TE) model was minimal, whereas with the malignant
cerebral edema (MCE) model, animals exhibited delayed
deterioration, often leading to death. Most deaths occurred 12-24
hr after MCA occlusion, with necropsies confirming that death was
due to bland infarcts. Rarely, an animal died <6 hr after stroke
and was found at necropsy to have a subarachnoid hemorrhage, in
which case it was excluded from the study. Mortality in untreated
animals with MCE and bland infarcts was 65%, similar to that in
humans with large MCA strokes (Ayata & Ropper, 2002).
Example 28
Studies on Stroke Size, Mortality, Tissue-Water, and Drug
Localization
[0497] After MCA occlusion (both TE and MCE models), mini-osmotic
pumps (Alzet 2002, Durect Corporation, Cupertino, Calif.) were
implanted subcutaneously that delivered either saline or
glibenclamide (Sigma, St. Louis, Mo.; 300 .mu.M or 148 .mu.g/ml,
0.5 .mu.l/hr subcutaneously, no loading dose). Stroke size (TE
model), measured as the volume of TTC(-) tissue in consecutive 2 mm
thick slices and expressed as the percent of hemisphere volume, was
compared 48 after stroke in 2 treatment groups, each comprised of
10 male rats, treated with either saline or glibenclamide.
Mortality (MCE model) was compared during the first week after
stroke in 2 treatment groups, each comprised of 29 rats (19 female
plus 10 male), treated with either saline or glibenclamide. Edema
(MCE model) was compared at 8 hr after stroke in 2 treatment
groups, each comprised of 11 male rats, treated with either saline
or glibenclamide; rats in each of these 2 treatment groups were
subdivided into 2 subgroups, with the first of these being used to
analyze water in the entire involved hemisphere (no TTC
processing), and the second being used to analyze water in the
TTC(+) vs. TTC(-) portions of the involved hemisphere. For
localization of fluorescent-tagged drug, 20 male rats were
subjected to MCA stroke (MCE model) and were implanted with
mini-osmotic pumps that delivered BODIPY-conjugated glibenclamide
(BODIPY-FL-glyburide, Molecular Probes, Eugene, Oreg.; 300 .mu.M or
235 .mu.g/ml, 0.5 .mu.l/hr subcutaneously, no loading dose). Of
these, 15 rats were used for validation of drug action (mortality,
tissue water and glucose) and 5 rats were used for determination of
drug distribution.
Example 29
Immunolabeling
[0498] Brains were perfusion-fixed (4% paraformaldehyde) and
cryoprotected (30% sucrose). Cryosections (10 .mu.m) were prepared
and immunolabeled using standard techniques (Chen et al., 2003).
After permeabilizing (0.3% Triton X-100 for 10 min), sections were
blocked (2% donkey serum for 1 hr; Sigma D-9663), then incubated
with primary antibody directed against SUR1 (1:300; 1 hr at room
temperature then 48 h at 4.degree. C.; SC-5789; Santa Cruz
Biotechnology). After washing, sections were incubated with
fluorescent secondary antibody (1:400; donkey anti-goat Alexa Fluor
555; Molecular Probes, OR). For co-labeling, primary antibodies
directed against NeuN (1:100; MAB377; Chemicon, CA); GFAP (1:500;
CY3 conjugated; C-9205; Sigma, St. Louis, Mo.) and vWf (1:200;
F3520, Sigma) were used and tissues were processed according to
manufacturers' recommendations. Species-appropriate fluorescent
secondary antibodies were used as needed. Fluorescent signals were
visualized using epifluorescence microscopy (Nikon Eclipse
E1000).
Example 30
TTC Staining, Stroke Size
[0499] Freshly harvested brains were cut into 2-mm thick coronal
sections, and slices were exposed to TTC (0.125% w/v in 62.5 mM
Tris-HCl, 13 mM MgCl.sub.2, 1.5% dimethylformamide) for 30 min at
37.degree. C. For stroke size, stained sections were photographed
and images were analyzed (Scion Image) to determine the percent of
the involved hemisphere occupied by TTC(-) tissue; no correction
for edema was performed. For some determinations of water or SUR1
protein content, individual coronal sections were divided under
magnification into 3 parts: (i) the non-involved, control
hemisphere; (ii) the TTC(+) portion of the involved hemisphere;
(iii) the TTC(-) portion of the involved hemisphere. For each
animal, pooled tissues from the 3 parts were then processed for
tissue water measurements or for Western blots.
Example 31
Tissue Water Content
[0500] Tissue water was quantified by the wet/dry weight method
(Hua et al., 2003). Tissue samples were blotted to remove small
quantities of adsorbed fluid. Samples were weighed with a precision
scale to obtain the wet weight (WW), dried to constant weight at
80.degree. C. and low vacuum, and then reweighed to obtain the dry
weight (WD). The percent H.sub.2O of each tissue sample was then
calculated as (WW-WD).times.100/WW.
Example 32
Immunoblots
[0501] Tissues lysates and gels were prepared (Perillan et al.,
2002). Membranes were developed for SUR1 (SC-5789; Santa Cruz
Biotechnology), Kir6.1 (Santa Cruz) or Kir6.2 (Santa Cruz).
Membranes were stripped and re-blotted for .beta.-actin (1:5000;
Sigma, St. Louis, Mo.), which was used to normalize the primary
data. Detection was carried out using the ECL system (Amersham
Biosciences, Inc.) with routine imaging and quantification (Fuji
LAS-3000).
Example 33
In Situ Hybridization
[0502] Non-radioactive digoxigenin-labeled probes were made
according to the manufacturer's protocol (Roche) using SP6 or T7
RNA polymerase. RNA dig-labeled probes (sense and anti-sense) were
generated from pGEM-T easy plasmids (Promega) with the SUR1 insert
(613 bp) flanked by the primers: 5' AAGCACGTCAACGCCCT 3' (forward;
SEQ ID NO: 1); 5' GAAGCTTTTCCGGCTTGTC 3' (reverse; SEQ ID NO: 2).
Fresh-frozen (10 .mu.m) or paraffin-embedded (4 .mu.m) sections of
rat brain (3, 6, 8 hours after MCA stroke) were used for in situ
hybridization (Anisimov et al., 2002).
Example 34
Inner Zone of the Gliotic Capsule
[0503] To assess if other causes of hypoxia, for example arterial
occlusion, resulted in up-regulation of SUR1, two rodent models of
permanent focal cerebral ischemia as described in the examples were
used.
[0504] The MCE model was used to evaluate SUR1 protein and mRNA,
and to assess effects of SUR1 inhibition on edema and survival,
while the TE model was used to measure effects of SUR1 inhibition
on stroke size. Absence of perfusion (FIG. 29A), TTC staining
(Mathews et al., 2000) (FIG. 29B) and GFAP immunolabeling were used
to distinguish infarct from peri-infarct regions.
[0505] SUR1 expression increased transiently in the core of the
infarct. Here, an increase in SUR1 became evident as early as 2-3
hr after MCA occlusion (FIG. 29D), well before onset of necrosis,
and later disappeared as necrosis set in (FIG. 29C, right side of
figure). At these early times before necrosis, SUR1 was very
prominent in neurons that co-labeled with NeuN (FIG. 29D-F).
[0506] In peri-infarct regions, including the classical ischemic
"watershed" zone between anterior cerebral artery (ACA) and MCA
territories, SUR1 expression increased later than in the core but
was sustained. By 6-12 hr, SUR1 expression sharply demarcated
infarct and peri-infarct areas (FIG. 29C). Here, SUR1 expression
was found in neurons, astrocytes and capillary endothelial cells,
as shown by co-labeling with NeuN, GFAP (FIGS. 29G-I) and von
Willebrand factor (FIG. 29J-L), respectively. SUR1 is not normally
expressed in such abundance in these cortical and subcortical areas
(Treherne & Ashford, 1991; Karschin et al., 1997) as is evident
in contralateral tissues (FIG. 29C, left side of figure).
[0507] Western blots showed an increase in expression of SUR1
protein, most prominently in peri-infarct regions (FIG. 30A-D).
However, the pore-forming subunits of K.sub.ATP channels, Kir6.1 or
Kir6.2, were not up-regulated (FIG. 30C-D). In situ hybridization
showed SUR1 transcripts in neurons and capillaries from regions of
ischemia that were not present in control tissues (FIG. 30E-G),
suggesting that SUR1, but not K.sub.ATP channels, was
transcriptionally up-regulated in cerebral ischemia.
[0508] Thus, these data suggest that SUR1, but not Kir6.1 or
Kir6.2, is transcriptionally up-regulated in cerebral ischemia,
first in regions that are destined to undergo necrosis, and later
in peri-infarct regions.
Example 35
SUR1 Up-Regulation
[0509] FIG. 30A-G discussed in Example 34 showed that SUR1 was
significantly up-regulated in stroke. It also showed that the
pore-forming subunits, Kir6.1 and Kir6.2, were not up-regulated in
stroke, suggesting that K.sub.ATP channels were not involved. To
prove that SUR1 up-regulation is due to NC.sub.Ca-ATP channels and
not to K.sub.ATP channels, patch clamp recordings of neurons and
endothelial cells from ischemic regions were performed. Large
neuron-like cells were enxymatically isolated 3-hr (FIG. 31A) and
6-hr after stroke. Patch clamp study was carried out using Cs.sup.+
in the bath and pipette, to block all K.sup.+ channels including
K.sub.ATP channels. These experiments showed robust cation channel
activity that was blocked by glibenclamide, as predicted for the
NC.sub.Ca-ATP channel (FIG. 31B). In addition, when channel
activity was recorded with K.sup.+, the slope conductance was 34 pS
(FIG. 31C,D), as previously reported in freshly isolated R1
astrocytes, and much less than the 70-75 pS reported for KATP
channels.
Example 36
Function of SUR1 in Cerebral Ischemia
[0510] To determine the function of SUR1 that was newly expressed
in cerebral ischemia, the effects of glibenclamide, a highly
selective inhibitor of SUR1 was studied. The effect of
glibenclamide on mortality (MCE model) was studied. In a large
group of animals, both male and female, treatment with
glibenclamide resulted in a dramatic reduction in mortality
compared to saline, from 65% to 24% (p<0.002; FIG. 32A).
[0511] Since glibenclamide had been shown to ameliorate cytotoxic
edema of astrocytes induced by energy depletion (Chen et al.,
2003), it was reasoned that the beneficial effect on mortality was
related to edema. The effect of glibenclamide on the formation of
edema 8 hr after induction of stroke (MCE model) was examined. This
is a time that preceded death of any animal in the mortality study.
In the first of two experiments, water content in the involved and
uninvolved hemispheres was measured using the methods described
above. For the control hemisphere, water was 77.9.+-.0.2%. For the
involved hemisphere, water rose by 3.4%, to 81.3.+-.0.5% for the
group treated with saline, whereas it rose by only 2.0%, to
79.9.+-.0.3%, for the group treated with glibenclamide. These
values were significantly different (p<0.05), consistent with an
important role of SUR1 in formation of edema.
[0512] Next, to better characterize the location of edema, the
water content after dividing coronal brain sections into viable
TTC(+) and non-viable TTC(-) parts was examined. Water in the
uninvolved hemisphere was 78.0.+-.0.1% (FIG. 32B), similar to the
previous value of 77.9.+-.0.2%, indicating that TTC processing had
not altered water content. For the involved hemisphere, water in
the TTC(+) tissue rose by 5.4%, to 83.4.+-.1.1% for the group
treated with saline, whereas it rose by only 2.5%, to 80.5.+-.0.3%,
for the group treated with glibenclamide (FIG. 32B). These values
were significantly different (p<0.05). By contrast, values for
water in TTC(-) tissues, 78.7.+-.1.0% and 78.6.+-.0.4% with saline
and with glibenclamide, respectively, were not different (p=0.97),
and were only slightly higher than the value for the uninvolved
hemisphere (78.0%), reflecting a need for ongoing blood flow to
increase tissue water (FIG. 32B) (Ayata & Ropper, 2002).
[0513] In these animals, serum glucose at 8 hr when edema was
measured remained in a range unlikely to have an effect on
ischemia-induced damage (Li et al., 1994; Wass & Lanier, 1996)
(122.+-.4 vs. 93.+-.3 mg/dl for saline and glibenclamide-treated
animals, respectively; 11 rats/group). Together, these data
indicated that the edema was located almost entirely in viable
peri-infarct (penumbral) tissue adjacent to the early core of the
stroke, and that glibenclamide was highly effective in reducing it,
consistent with an important role for SUR1 in formation of
edema.
[0514] Thus, the data with low-dose glibenclamide, which is highly
selective for SUR1 (Gribble & Reimann, 2003; Meyer et al.,
1999) provided compelling evidence of a critical role for SUR1 in
formation of cerebral edema.
Example 37
The Effect of Stroke Size
[0515] A non-lethal thromboembolic (TE) model was used to assess
stroke size 48 hr after induction of stroke.
[0516] With the TE model, glibenclamide treatment resulted in a
highly significant reduction in stroke volume, compared to saline
controls (32.5.+-.4.9% vs. 15.5.+-.2.3%; p<0.01) (FIG. 32C-E).
Essentially all animals, regardless of treatment group, suffered
infarctions involving the basal ganglia, which were supplied by
terminal lenticulostriate arterioles. However, reduced stroke
volumes in the glibenclamide group were often associated with
marked sparing of the cerebral cortex (FIG. 32C-D), a phenomenon
previously reported with decompressive craniectomy (Doerfler et
al., 2001). With glibenclamide, cortical sparing may reflect
improved leptomeningeal collateral blood flow due to reduced
cerebral edema and reduced intracranial pressure.
Example 38
MCE Model Following Stroke
[0517] The fluorescent derivative, BODIPY-glibenclamide, was used
to label tissues in vivo following stroke (MCE model).
[0518] When delivered in the same manner as the parent compound,
the fluorescent derivative exhibited similar protective effects,
but was less potent [7-day mortality, 40% (n=10); water in the
TTC(+) portion of the involved hemisphere at 8 hr, 82.7.+-.1.4%
(n=5); serum glucose, 109.+-.4 mg/dl], consistent with reduced
efficacy of the labeled drug (Zunkler et al., 2004). The low
systemic dose of drug used yielded minimal labeling in the
uninvolved hemisphere (FIG. 33B) and pancreas, and none in the
unperfused core of the stroke. However, cells in peri-infarct
regions were clearly labeled, with well-defined labeling of large
neuron-like cells and of microvessels (FIG. 33A), including
capillaries (FIG. 33C), that showed prominent expression of SUR1
(FIG. 33D). Preferential cellular labeling in ischemic brain likely
reflected not only an increase in glibenclamide binding sites, but
also an increase in uptake, possibly due to alteration of the blood
brain barrier.
[0519] Thus, the data indicated the presence of NC.sub.Ca-ATP
channels in capillary endothelium and neurons in addition to their
previously described presence in astrocytes (Chen et al., 2001;
Chen et al., 2003). Additional patch clamp experiments on neurons
and microvessels isolated from ischemic cortex 1-6 hr after MCA
occlusion (MCE model) confirmed the presence of NC.sub.Ca-ATP
channels, showing a non-selective cation channel of around 30-35 pS
conductance, that was easily recorded with Cs.sup.+ as the charge
carrier, and that was blocked by glibenclamide. This channel was
not present in cells from non-ischemic cerebral tissues.
[0520] In view of the above, it is suggested that SUR1-regulated
NC.sub.Ca-ATP channels that are opened by ATP depletion and that
are newly expressed in ischemic neurons, astrocytes and endothelial
cells constitute an important, heretofore unidentified pathway for
Na.sup.+ flux required for formation of cytotoxic and ionic edema.
Together, these findings suggest a critical involvement of SUR1 in
a new pathway that determines formation of edema following cerebral
ischemia. Molecular therapies directed at SUR1 may provide
important new avenues for treatment of many types of CNS injuries
associated with ischemia.
Example 39
Co-Administration of Glibenclamide and tPA
[0521] A rodent model of thromboembolic stroke was used (Aoki et
al., 2002; Kijkhuizen et al., 2001; Kano et al., 2000; Sumii et
al., 2002; Tejima et al., 2001). Briefly, male spontaneously
hypertensive rats that have been fasted overnight are anesthetized
using halothane (1-1.5% in a 70/30 mixture of N.sub.2O/O.sub.2)
with spontaneous respiration (Lee et al., 2004; Sumii et al.,
2002). Rectal temperature was maintained at .apprxeq.37.degree. C.
with a thermostat-controlled heating pad. The right femoral artery
was cannulated, and physiological parameters, including
temperature, mean blood pressure, pH, pO.sub.2, and pCO.sub.2,
glucose were monitored. Temporary focal ischemia was obtained with
an embolic model that used allogeneic clots to occlude the MCA.
Allogeneic, thrombin-induced, fibrin-rich blood clots were prepared
using methods adapted from Niessen et al. (Asahi et al., 2000;
Niessen et al., 2003; Sumii et al., 2002). Seven clots, 1.5 mm
long, were used for embolizing.
[0522] Using a ventral cervical incision, the internal and external
carotid arteries were exposed. The external carotid artery and
pterygopalatine arteries were ligated. Removable surgical clips
were applied to the common and internal carotid arteries. The
modified PE-50 catheter containing the clots was inserted
retrograde into the external carotid artery and advanced up to the
internal carotid artery. The temporary clips were removed, and the
clots were injected. Incisions were closed.
[0523] After stroke, animals were given glucose-free normal saline,
10 ml total, by dermoclysis. Temperature was maintained until
animals were awake and were moving about.
[0524] Just prior to the time designated for treatment
(reperfusion), animals were re-anesthetized and the femoral vein
was cannulated. At the time designated for treatment, saline, or a
loading dose of glibenclamide (1.5 .mu.g/kg, i.v., Sigma, St.
Louis) was first administered. Then, reperfusion was achieved with
i.v. administration of rtPA (10 mg/kg, Alteplase, Genetech;
dissolved in 2 ml distilled water, given over 30 min) (Buesseb et
al., 2002). Then, using a dorsal thoracic incision, a mini-osmotic
pump (Alzet 2002, Durect Corporation, Cupertino, Calif.) was
implanted subcutaneously that delivered either saline or
glibenclamide (300 .mu.M or 148 .mu.g/ml, 0.5 .mu.l/hr s.q.).
Physiological parameters, including temperature, mean blood
pressure (tail cuff plethysmography), blood gases and glucose were
monitored.
[0525] At the same time of 6 hr, animals were co-treated with
either saline or glibenclamide (loading dose of 1.5 .mu.g/kg i.v.
plus implantation of a mini-osmotic pump containing 148
.mu.g/ml=300 .mu.M delivered at 1/2 .mu.l/hr). Animals were
euthanized 24 hr following stroke and brains were perfused to
remove blood from the intravascular compartment. Coronal sections
of the fresh brains were prepared and photographed, following which
sections were processed for TTC staining to identify areas of
infarction.
[0526] All animals (5/5) co-treated with saline showed large
regions of hemorrhagic conversion in cortical and subcortical
parenchymal areas of infarction, along with evidence of
intraventricular hemorrhage (FIG. 34A-D). In contrast, only 1/5
animals co-treated with glibenclamide had hemorrhagic conversion,
with 4/5 showing no evidence of hemorrhage (FIG. 34E-H).
[0527] These data suggest that there was protection from
hemorrhagic conversion with the administration of glibenclamide, as
well as reduction in stroke size, ionic edema, and vasogenic
edema.
Example 40
Isolation of Brain Capillaries and Endothelial Cells
[0528] The method was adapted in part from Harder et al. (1994)
with modifications as previously reported (Seidel, 1991). Briefly,
a rat was deeply anesthetized, the descending aorta was ligated,
the right atrium was opened and the left ventricle was cannulated
to allow perfusion of 50 ml of a physiological solution containing
a 1% suspension of iron oxide particles (particle size, 10 .mu.m;
Aldrich Chemical Co.). The brain was removed, the pia and pial
vessels were stripped away and the cortical mantel is minced into
pieces 1-2 mm.sup.3 with razor blades. The tissue pieces were
incubated with trypsin plus DNAse and then sieved through nylon
mesh (210 .mu.m). Retained microvessels were resuspended in
collagenase, agitated and incubated at 37.degree. C. for an
additional 10 min. To terminate the digestion, microvessels were
adhered to the side of the container with a magnet and washed
repeatedly to remove enzyme and cellular debris.
[0529] Using these methods yielded healthy-appearing microvascular
structures that were suitable for further digestion to obtain
single cells (FIG. 36) for further experiments.
[0530] Isolated endothelial cells were studied using freshly
isolated endothelial cells using a nystatin-perforated patch
technique. With physiological solutions, the cells exhibited a
prominent, strongly rectifying inward current at negative
potentials, and a modest outward current at positive potentials
(FIG. 37A), yielding a characteristic current-voltage curve with
near-zero current at intermediate potentials (FIG. 37C), similar to
previous observations in freshly isolated endothelial cells (Hogg
et al., 2002). When K.sup.+ in the pipette solution was replaced
with Cs.sup.+, K.sup.+ channel currents were completely blocked. In
endothelial cells, this yielded a current-voltage curve that was
linear (FIG. 37E). These data demonstrated that voltage dependent
channels in freshly isolated endothelial cells are exclusively
K.sup.+ channels that do not carry Na.sup.+.
Example 41
Isolation of Neurons
[0531] Neurons were isolated from vibratome sections.
Immunolabeling experiments indicated that ischemic NeuN-positive
neurons expressed SUR1 within 2-3 hr after MCAO, before necrosis
was evident. Therefore, tissues were prepared at 2-3 hr after MCAO.
The brain was divided coronally at the level of the bregma, and
cryosections were prepared from one half and vibratome sections
were prepared from the other half. Cryosections (10 .mu.m) were
used for TTC staining (Mathews et al., 2000) or alternatively,
high-contrast silver infarct staining (SIS), (Vogel et al., 1999)
to identify the region of ischemia, and for immunolabeling, to
verify SUR1 up-regulation in neurons double labeled for NeuN.
Vibratome sections (300 .mu.m) were processed (Hainsworth et al.,
2000; Kay et al., 1986; Moyer et al., 1998) to obtain single
neurons for patch clamping. Selected portions of coronal slices
were incubated at 35.degree. C. in HBSS bubbled with air. After at
least 30 min, the pieces were transferred to HBSS containing 1.5
mg/ml protease XIV (Sigma). After 30-40 min of protease treatment,
the pieces were rinsed in enzyme-free HBSS and mechanically
triturated. For controls, cells from mirror-image cortical areas in
the uninvolved hemisphere were used. Cells were allowed to settle
in HBSS for 10-12 min in a plastic Petri dish mounted on the stage
of an inverted microscope. Large and medium-sized pyramidal-shaped
neurons were selected for recordings. At this early time of 2-3 hr,
only neurons and capillaries, not astrocytes, show up-regulation of
SUR1.
[0532] Once the cells were isolated patch clamp experiments using
well known methods including whole-cell, inside-out, outside-out
and perforated patch were used (Chen et al., 2003; Chen et al.,
2001; Perillan et al., 2002; Perillan et al., 2000; Perillan et
al., 1999)
Example 42
MMP Inhibition by Glibenclamide
[0533] Activation of MMP-9 & MMP-2 in stroke tissue was
compared to controls. Briefly, gelatinase activity of recombinant
enzyme and stroke tissue under control conditions (CTR), in
presence of glibenclamide (10 .mu.M), and in presence of
MMP-inhibitor II (300 nM; Calbiochem).
[0534] Next, the supernatants underwent a gelatinase purification
process with gelatin-Sepharose 4B (Pharmacia), and Zymography was
performed on the purified supernatants in sodium dodecyl sulfate
gels containing gelatin (Rosenberg, 1994). Dried gels were scanned
with a transparency scanner, and images were analyzed by
densitometry. The relative lysis of an individual sample was
expressed as the integrated density value of its band and divided
by the protein content of the sample.
[0535] Zymography confirmed that gelatinase activity was increased
after stroke (FIG. 35A), and showed that gelatinase activity
assayed in the presence of glibenclamide (FIG. 35B, Glibenclamide)
was the same as that assayed without (FIG. 35B, CTR), although
gelatinase activity was strongly inhibited by commercially
available MMP inhibitor II (FIG. 35B, MMP-2/MMP-9 inhibitor). These
data demonstrated that glibenclamide did not directly inhibit
gelatinase activity, and suggested that the reduction of
hemorrhagic conversion observed with glibenclamide likely came
about due to a beneficial, protective effect of glibenclamide on
ischemic endothelial cells.
Example 43
Up-Regulation of SUR1-mRNA in Stroke
[0536] Additional molecular evidence for involvement of SUR1 in
stroke was obtained using quantitative RT-PCR.
[0537] Total RNA was extracted and purified from samples of
homogenized brain tissues contralateral (CTR) and ipsilateral to
MCAO (STROKE) using guanidine isothyocyonatye. cDNA was synthesized
with 4 .mu.g of total RNA per 50 .mu.l of reaction mixture using
TaqMan RT kit (Applied Biosystems). Relative values of SUR1-mRNA
were obtained by normalizing to H1f0 (histone 1 member 0). The
following probes were used SUR1 forward: GAGTCGGACTTCTCGCCCT (SEQ
ID NO: 3); SUR1 reverse: CCTTGACAGTGGCCGAACC (SEQ ID NO: 4); SUR1
TaqMan Probe: 6-FAM-TTCCACATCCTGGTCACACCGCTGTTAMRA (SEQ ID NO: 5);
H1f0 forward: CGGACCACCCCAAGTATTCA (SEQ ID NO: 6); H1f0 reverse:
GCCGGCACGGTTCTTCT (SEQ ID NO: 7); H1f0 TaqMan Probe:
6-FAM-CATGATCGTGGCTGCTA TCCAGGCA-TAMRA (SEQ ID NO: 8).
[0538] These data showed that mRNA for SUR1 was significantly
increased in the core region, 3 hr after MCAO (FIG. 38).
Example 44
SUR1 Knockdown (SUR1KD) is Protective
[0539] To further test involvement of SUR1, SUR1 expression was
"knocked down" in situ by infusing oligodeoxynucleotide (ODN) for
14 days using a mini-osmotic pump, with the delivery catheter
placed in the gelfoam implantation site in the brain, in the
otherwise standard model that the inventors use for R1 astrocyte
isolation (Perillan et al., 1980, Perillan et al., 2002, Perillan
et al., 2000, Perillan et al., 1999). Knockdown of SUR1 expression
(SUR1KD) was achieved using antisense (AS; 5'-GGCCGAGTGGTTCTCGGT-3'
(SEQ ID NO: 9)) (Yokoshiki et al., 1999) oligodeoxynucleotide
(ODN), with scrambled (SCR; 5'-TGCCTGAGGCGTGGCTGT-3' (SEQ ID NO:
10)) ODN being used as control.
[0540] Immunoblots of gliotic capsule showed significant reduction
in SUR1 expression in SUR1 knockdown (SUR1KD) tissues compared to
controls receiving scrambled sequence ODN (FIGS. 39A and 39B).
[0541] The inventors enzymatically isolated single cells from
SUR1KD and controls using a standard cell isolation protocols
described above (Chen et al., 2003) to assess functional responses
to ATP depletion induced by Na azide. In R1 astrocytes from control
tissues, Na azide (1 mM) caused rapid depolarization due to
Na.sup.+ influx attributable to activation of NC.sub.Ca-ATP
channels (FIG. 39C). Notably, this depolarizing response was
opposite the hyperpolarizing response observed when K.sub.ATP
channels were activated. In R1 astrocytes from SUR1KD, however, Na
azide had little effect on resting membrane potential (FIG. 39D).
In controls, application of Na azide resulted in depolarization of
64.+-.3.7 mV, whereas in cells for SUR1KD, depolarization was only
8.7.+-.1.7 mV (FIG. 39E).
[0542] In addition, membrane blebbing that typically follows
exposure to Na azide was not observed in cells from SUR1KD,
confirming the role for SUR1 in cytotoxic edema of R1
astrocytes.
Example 45
Molecular Factors that Regulate SUR1 Expression
[0543] Based on work in pancreatic .beta. cells, a number of SP1
transcription factor binding sites have been identified in the
proximal SUR1 promoter region that are considered to be important
for activation of SUR1 transcriptional activity (Ashfield et al.,
1998; Hilali et al., 2004). Notably, SP1 has essentially not been
studied in stroke (Salminen et al., 1995).
[0544] Briefly, the ischemic peri-infarct tissues was immunolabeled
for SP1, which is important for SUR1 expression, for HIF1.alpha.,
which is widely recognized to be up-regulated in cerebral ischemia
(Semenza 2001; Sharp et al., 2000) and for SUR1 itself. SP1 was
prominently expressed in large neuron-like cells and in capillaries
(FIG. 40A, 40C) in regions confirmed to be ischemic by virtue of
expression of HIF1.alpha. (FIG. 40B). Notably, capillaries that
expressed SP1 also showed prominent expression of SUR1 (FIG. 40C,
40D). Contralateral control tissues showed little immunolabeling
for SP1 and none for HIF1.alpha. (FIG. 40E, 40F).
[0545] Nuclear SP1 localization was significantly augmented
early-on in stroke (FIG. 41A, 41B), and nuclear SP1 was found in
large neuron-like cells that express SUR1 following MCAO (FIG.
41C).
[0546] HIF1.alpha. knock-down animals were obtained by infusion of
antisense oligodeoxynucleotide at the site of gelfoam implant. FIG.
42 confirms the HIF1.alpha. knock-down animals results in a
significant decrease in SUR1 expression (FIG. 42B, 42D), providing
strong evidence that not only SP1 but also HIF1.alpha. is likely to
be an important regulator of SUR1 expression.
Example 46
Blood Flow in Peri-Infarct Tissue is Protected by Treatment with
Nc.sub.ca-Atp Channel Antagonist
[0547] Block of SUR1 by systemic administration of low-dose
glibenclamide reduces cerebral edema, infarct volume and mortality,
with the reduction in infarct volume being associated with cortical
sparing. A thromboembolic (TE) stroke model associated with
non-lethal infarctions from MCAO was used to study effects on
infarct volume in rats. Given the striking effects on mortality and
edema, the inventors sought to determine whether glibenclamide
would have a favorable effect on infarct volume. This was not
feasible with the MCE model because of the high incidence of early
mortality. We therefore utilized a non-lethal thromboembolic (TE)
model that would allow assessment of infarct volume at 2 and 7 days
after MCAO. At 2 days, glibenclamide treatment resulted in a highly
significant reduction in infarct volume, compared to saline
controls (35.5.+-.4.4% vs. 16.7.+-.2.2%; p<0.01). A similar
observation was made at 7 days (15.2.+-.1.2%; p<0.01),
indicating again that the effect of treatment was durable.
[0548] All animals, regardless of treatment group, suffered
infarctions involving the basal ganglia, which are supplied by
terminal arterioles. However, reduced infarct volumes in the
glibenclamide groups were often associated with marked sparing of
the cerebral cortex, a phenomenon previously reported with
decompressive craniectomy. (Doerfler, et al., (2001)). We
hypothesized that cortical sparing with glibenclamide might reflect
improved leptomeningeal collateral blood flow, which could be due
to reduced cerebral edema. The effective dose of glibenclamide was
75 ng/hr. Direct vasodilation was not expected, since glibenclamide
is normally vasoconstrictive due to block of KATP channels.
(Lindauer, et al., (2003), and Tomiyama, et al., (1999)).
[0549] Blood flow was measured using laser Doppler flowmetry in
order to determine the effects of glibenclamide treatment on
cerebral blood flow. Using the same TE model, measurements of
relative cerebral blood flow were obtained for somatosensory cortex
supplied by the middle cerebral artery (MCA). Laser Doppler
flowmetry showed values in the involved hemisphere that were
significantly reduced 1 hr after middle cerebral artery occlusion
(MCAO) in both saline- and glibenclamide-treated groups (FIG. 43).
However, flow measurements recovered completely by 48 hr in
glibenclamide-treated animals but not in saline-treated animals
(FIG. 43), consistent with the cortical sparing observed.
Methods
[0550] Relative cerebral blood flow (TE model) was measured using
laser Doppler flowmetry (LDF) in 2 groups, each consisting of 4
male rats, treated with either saline or glibenclamide. Prior to
MCAO, two 1.5-mm pits were carefully drilled halfway through the
skull over the left and right somatosensory cortex (MCA territory),
3 mm posterior and 3 mm lateral to the bregma. A two-channel LDF
instrument (DRT4; Moor Instruments, Axminster, UK) was used to
simultaneously measure blood flow in both hemispheres. LDF readings
were normalized by adjusting the depth of the pits to obtain a
ratio of blood flow of .about.1.0 between sides. Once this ratio
had been obtained, five sets of LDF measurements were taken at 1
min intervals, values for each location were averaged and the ratio
of ipsilateral to contralateral LDF values was calculated. This
technique minimized effects of intra-measurement differences in
probe position, angle, lighting condition, etc. Once baseline CBF
had been determined, skin incisions over the pits were closed and
the procedure for MCAO was initiated. Relative CBF measurements
were later repeated at 1 hr and 48 hours after MCAO, using the same
pits and the same method of averaging 5 bilateral measurements
obtained at 1-min intervals.
[0551] Edema (MCE model) was analyzed 8 hr after MCAO in 2 series
of animals. In the first series, tissue water was analyzed in the
uninvolved vs. involved hemisphere of 2 groups of 11 male rats,
treated with either saline or glibenclamide (no TTC
processing).
[0552] In the second series, tissue water was analyzed in the
uninvolved hemisphere and in the TTC(+) vs. TTC(-) portions of the
involved hemisphere in 3 groups of 6 male rats treated with either
saline alone, vehicle (saline plus DMSO) or glibenclamide. Tissue
water was quantified by the wet/dry weight method. Tissue samples
were blotted to remove small quantities of adsorbed fluid. Samples
were weighed with a precision scale to obtain the wet weight
(W.sub.W), dried to constant weight at 80.degree. C. and low
vacuum, and then reweighed to obtain the dry weight (W.sub.D). The
percent H.sub.2O of each tissue sample was then calculated as
(W.sub.W-W.sub.D).times.100/W.sub.W.
[0553] Infarct volume (TE model), measured as the volume of TTC(-)
tissue in consecutive 2 mm thick slices and expressed as the
percent of hemisphere volume, was compared in 3 treatment groups,
consisting of 9, 9 and 7 male rats, treated with saline and
assessed at 2 days, or treated with glibenclamide and assessed at 2
days or 7 days after MCAO.
[0554] Permanent MCA occlusion (MCAO) models. This study was
performed in accordance with the guidelines of the Institutional
Animal Care and Use Committee. Adult male or female Wistar rats
(275-350 gm) were fasted overnight then anesthetized (Ketamine, 60
mg/kg plus Xylazine, 7.5 mg/kg, i.p.). The right femoral artery was
cannulated, and physiological parameters, including temperature,
pH, pO.sub.2, pCO.sub.2 and glucose were monitored. Using a ventral
cervical incision, the right external carotid and pterygopalatine
arteries were ligated. The common carotid artery was ligated
proximally and catheterized to allow embolization of the internal
carotid artery. For the thromboembolic (TE) stroke model, 7-8
allogeneic clots, 1.5 mm long, were embolized. Allogeneic,
thrombin-induced, fibrin-rich blood clots were prepared as
described. For large MCA infarcts with malignant cerebral edema
(MCE), the inventors first embolized microparticles [polyvinyl
alcohol (PVA) particles; Target Therapeutics, Fremont Calif.;
150-250 .mu.m diameter, 600 .mu.g in 1.5 ml heparinized-saline],
followed by standard permanent intraluminal suture occlusion using
a monofilament suture (4-0 nylon, rounded at the tip and coated
with poly-L-lysine) advanced up to the ICA bifurcation and secured
in place with a ligature. After MCAO, animals were given 10 ml of
glucose-free normal saline by dermoclysis. Rectal temperature was
maintained at about 37.degree. C. using a servo-controlled warming
blanket until animals awoke from anesthesia. Blood gases and serum
glucose at the time of MCAO were: pO.sub.2, 94.+-.5 mm Hg;
pCO.sub.2, 36.+-.5 mm Hg; pH, 7.33.+-.0.01; glucose 142.+-.6 mg/dl
in controls and pO.sub.2, 93.+-.3 mm Hg; pCO.sub.2, 38.+-.2 mm Hg;
pH, 7.34.+-.0.01; glucose 152.+-.7 mg/dl in glibenclamide-treated
animals. With both models, animals awoke promptly from anesthesia
and moved about, generally exhibited abnormal neurological
function, typically circling behavior and hemiparesis. Mortality
with the TE model was minimal, whereas with the MCE model, animals
exhibited delayed deterioration, often leading to death. Most
deaths occurred 12-24 hr after MCAO, with necropsies confirming
that death was due to bland infarcts. Rarely, an animal died <6
hr after MCAO and was found at necropsy to have a subarachnoid
hemorrhage, in which case it was excluded from the study. Mortality
in untreated animals with MCE and bland infarcts was 65%, similar
to that in humans with large MCA strokes.
[0555] Within 2-3 min after MCAO (both TE and MCE models),
mini-osmotic pumps (Alzet 2002, 14 day pump, 0.5 .mu.l/hr; Durect
Corporation, Cupertino, Calif.) were implanted subcutaneously that
delivered either saline (0.9% NaCl), vehicle (saline plus DMSO) or
glibenclamide in vehicle, subcutaneously (no loading dose).
Glibenclamide (Sigma, St. Louis, Mo.) was prepared as a 10 mM stock
solution in DMSO, with 15 .mu.l stock solution diluted into 500
.mu.l saline to give a final concentration of 148 .mu.g/ml or 300
.mu.M in the pump. The effective dose of glibenclamide was 75
ng/hr. The effective dose of DMSO was 15 nl/hr, which is what was
delivered in vehicle-treated animals.
[0556] TTC (triphenyltetrazolium chloride) staining was measured to
determine infarct volume. Freshly harvested brains were cut into
2-mm thick coronal sections, and slices were exposed to TTC (0.125%
w/v in 62.5 mM Tris-HCl, 13 mM MgCl.sub.2, 1.5% dimethylformamide)
for 30 min at 37.degree. C. For infarct volume, stained sections
were photographed and images were analyzed (Scion Image) to
determine the percent of the involved hemisphere occupied by TTC(-)
tissue; no correction for edema was performed. For some
determinations of water content or SUR1 protein content, individual
coronal sections were divided under magnification into 3 parts: (i)
the uninvolved, control hemisphere; (ii) the TTC(+) portion of the
involved hemisphere; (iii) the TTC(-) portion of the involved
hemisphere. For each animal, tissues from the 3 parts were then
processed for tissue water measurements, or Western blots.
[0557] These findings indicate that the SUR1-regulated
NC.sub.Ca-ATP channel is critically involved in development of
cerebral edema, that modulation of the SUR1-regulated NC.sub.Ca-ATP
channel can lead to improved blood flow in peri-infarct tissue, and
that targeting SUR1 provides an important new therapeutic approach
to stroke.
Example 47
NC.sub.ca-Atp Channel Antagonist Treatment Reduces Edema Even with
Added Glucose Treatment
[0558] Although the dose of glibenclamide was low, a drop in serum
glucose concentration in glibenclamide-treated animals was noted in
the experiments described above. The drop in glucose by
glibenclamide raised the question whether the beneficial effect of
glibenclamide on edema was mediated directly via NC.sub.Ca-ATP
channels, or indirectly via reduction in serum glucose.
[0559] Tissue water as a measure of edema was measured in rats in a
middle cerebral artery occlusion (MCAO) model of stroke. As in
Example 46, the effective dose of glibenclamide was 75 ng/hr
delivered by subcutaneously implanted Alzet mini-osmotic pump.
Animals treated with glibenclamide (GLIB) alone experienced reduced
serum glucose. For example, serum glucose concentration at 8 hr,
when edema was measured, was 122.+-.4 for saline-treated animals
(SALINE) vs. 93.+-.3 mg/dl for glibenclamide-treated animals (GLIB)
(see FIG. 44). Administration of glucose 4 hours after occlusion
resulted in serum glucose concentrations of 141.+-.4 mg/dl at 8
hours after occlusion.
[0560] Edema measurements in the same brain areas and at the same
time in animals treated with GLIB indicated that GLIB reduced edema
irrespective of the glucose concentration. In these animals,
supplemental glucose (1 gm/kg, i.p.) was administered 4 hr after
MCAO. This dose of glucose is reported to produce levels of
hyperglycemia of 300 mg/dl, when measured shortly after
administration. Animals were sacrificed 8 hr after MCAO for
measurements of edema (FIG. 44, GLIB+GLUCOSE). Serum glucose 4 hr
after glucose administration (i.e., at time of sacrifice, 8 hr
after MCAO) was still elevated (141.+-.4 mg/dl). However, in these
animals, GLIB was just as effective in reducing edema, even in the
face of hyperglycemia.
[0561] These results indicate that adding glucose does not impair
the protective effect of SUR1 antagonist treatment, and may enhance
the protective effect of SUR1 antagonist treatment.
Example 48
Delayed Treatment with Glibenclamide Reduces Stroke Volume in Rats
Following Middle Cerebral Artery Occlusion (MCAO)
[0562] Stroke volume in rats was measured as discussed above.
Glibenclamide (3.3 .mu.g/kg or 33.0 .mu.g/kg) was given as
indicated in FIG. 45. Animals treated with the higher dose of
glibenclamide were also given 1 gm/kg glucose in order to
counteract hypoglycemia caused by the glibenclamide.
[0563] Stroke Model:
[0564] thromboembolic embolization of allogeneic clots via internal
carotid artery in male Wistar rats, 275-325 gm. Treatment: within
2-3 min after MCAO, animals were implanted with mini-osmotic pumps
fitted with catheters of a length calibrated to delay onset of drug
delivery by the amount of time indicated; the pumps were filled
with glibenclamide, 300 .mu.M, that was delivered at a rate of 0.5
.mu.l/hr, giving an effective infusion rate of 75 ng/hr for
glibenclamide, and an effective delivery rate of 15 nl/hr for DMSO
(used as vehicle solvent); at the designated time, animals were
also injected intraperitoneally with a loading dose of
glibenclamide, either 3.3 or 33 .mu.g/kg, and in the case of the
higher dose of glibenclamide, with a supplemental dose of glucose
of 1 gm/kg. Stroke volume was determined at 48 after MCAO from the
volume of TTC(-) tissue and is expressed as the percent of
hemisphere volume in FIG. 45. Values of "n" indicate the number of
rats per group; asterisks (*) indicates a statistically significant
(P<0.05) difference in volume compared to saline (SAL) control
as illustrated in FIG. 45.
[0565] A significant reduction in stroke volume was observed when
glibenclamide infusion was begun: (i) immediately after stroke,
with no loading dose; (ii) 2 hr after stroke, with a loading dose
of 3.3 .mu.g/kg; (iii) and up to 6 hr after stroke with a loading
dose of 33 .mu.g/kg. Thus the lower dose of glibenclamide (3.3
.mu.g/kg) was effective at reducing stroke volume in experimental
animals subjected to middle cerebral artery occlusion (MCAO) when
the glibenclamide was given at 0 or 2 hours after MCAO. Although
some reduction in stroke volume was seen at 4 hours after MCAO with
the lower dose of glibenclamide, the difference was not
statistically significant with this number of animals. However,
statistically significant reductions in stroke volume (as compared
to control) were observed in animals treated with the higher dose
of glibenclamide (33.0 .mu.g/kg, with co-administered glucose)
given at 4 and at 6 hours after MCAO, as shown in FIG. 45. Thus, a
larger dose of 33.0 .mu.g/kg was effective up to three times as
long after MCAO as was the smaller dose of glibenclamide.
[0566] These data indicate that the beneficial effect of
glibenclamide can be obtained even with substantial delay in
treatment, consistent with the beneficial effect being due to a
reduction in edema that permits leptomeningeal collateral flow that
helps salvage cortical structures. These data also demonstrate that
co-administration of glibenclamide with glucose is effective in
reducing stroke volume, that such co-administration with glucose
allows treatment with higher doses of glibenclamide without the
possibly deleterious effects of lowered blood glucose, and allows
for effective sulfonylurea treatment with greater delay before
initiating treatment after stroke than appeared possible with lower
sulfonylurea doses.
Example 49
Glibenclamide Reduces Hemorrhagic Conversion
[0567] Hemorrhagic conversion is a serious condition that often
follows stroke or ischemic insult, in which reperfusion to ischemic
tissue causes further damage to compromised tissue as anoxic and
acidic fluids which had accumulated in non-perfused tissues flows
to other tissues as blood flow is restored to the region. Further,
damage can come from leaky endothelial cells and blood vessels
distal to the ischemic damage. Accordingly, an outcome study was
designed as indicated to determine the effect of glibenclamide on
hemorrhagic conversion.
[0568] In this study, male rats of the spontaneously hypertensive
(SHR) strain were subjected to a thromboembolic stroke and then
treated with tissueplasminogen activator (tPA) to dissolve the clot
and restore perfusion to non-perfused brain tissue. In particular,
thromboembolic stroke was performed six hours after initiation of
the experimental stroke, tPA was administered intravenously (10
mg/kg over 30 min), along with either saline (control) or
glibenclamide. Glibenclamide-treated animals were given a loading
dose of 1.5 .mu.g/kg intravenously (i.v.) and a sub-cutaneous
(s.c.) mini osmotic pump was implanted that delivered 148 .mu.g/ml
(equivalent to 300 .mu.M at 1/2 .mu.l/hr) to the animals.
[0569] The internal carotid artery (ICA) of male SHR rats were
embolized with allogeneic thrombi to produced MCAO. Six hours
later, animals were treated with tPA (10 mg/kg i.v over 30 min) and
co-treated with either saline or glibenclamide (1.5 mg/kg i.v.
bolus plus implantation of a s.c. pump that delivered a 300 mM
solution at 0.5 ml/hr). At 24 hr after stroke, brains were perfused
to remove intravascular blood, sectioned coronally, photographed,
and processed for TTC staining. Results are shown in FIG. 46. Rows
1-2 (A-D) are from animals co-treated with saline; rows 3-4 (E-H)
are from animals co-treated with glibenclamide. The left column of
photographs of coronal sections shows, in rows 1-2 only,
intraventricular hemorrhage, plus large areas of hemorrhagic
conversion in ischemic cortical/subcortical regions (red areas on
the right side of pictures; arrows). The right column of
photographs of TTC-processed sections from the same animals show
the areas of infarction.
[0570] As shown in FIG. 46, the incidence of hemorrhage within the
stroke region (measured at 24 hours) was reduced by glibenclamide
treatment as compared with control. Although 5 of 6 animals
co-treated with saline showed hemorrhagic conversion, only 1 of 6
animals treated with glibenclamide showed hemorrhagic conversion,
demonstrating the efficacy of glibenclamide treatment to reduce or
prevent hemorrhagic conversion following thromboembolic stroke.
FIG. 46 thus demonstrates that glibenclamide treatment reduces
hemorrhagic conversion in tPA-treated animals, and extends the time
window after ischemic insult within which tPA may be administered
without deleterious effects.
[0571] The foregoing disclosure of the preferred embodiments of the
present invention has been presented for purposes of illustration
and description. It is not intended to be exhaustive or to limit
the invention to the precise forms disclosed. Many variations and
modifications of the embodiments described herein will be apparent
to one of ordinary skill in the art in light of the above
disclosure. The scope of the invention is to be defined only by the
claims appended hereto, and by their equivalents.
[0572] Further, in describing representative embodiments of the
present invention, the specification may have presented the method
and/or process of the present invention as a particular sequence of
steps. However, to the extent that the method or process does not
rely on the particular order of steps set forth herein, the method
or process should not be limited to the particular sequence of
steps described. As one of ordinary skill in the art would
appreciate, other sequences of steps may be possible. Therefore,
the particular order of the steps set forth in the specification
should not be construed as limitations on the claims. In addition,
the claims directed to the method and/or process of the present
invention should not be limited to the performance of their steps
in the order written, and one skilled in the art can readily
appreciate that the sequences may be varied and still remain within
the spirit and scope of the present invention. Further, in
describing representative embodiments of the present invention, the
specification may have presented the method and/or process of the
present invention as a particular sequence of steps. However, to
the extent that the method or process does not rely on the
particular order of steps set forth herein, the method or process
should not be limited to the particular sequence of steps
described. As one of ordinary skill in the art would appreciate,
other sequences of steps may be possible. Therefore, the particular
order of the steps set forth in the specification should not be
construed as limitations on the claims. In addition, the claims
directed to the method and/or process of the present invention
should not be limited to the performance of their steps in the
order written, and one skilled in the art can readily appreciate
that the sequences may be varied and still remain within the spirit
and scope of the present invention.
Example 50
Brain Contusion Results in Up-Regulation of SUR1
[0573] Contusion Model: Adult Wistar rats were anesthetized
(Ketamine and Zylazine) and underwent aseptic surgery to create a
right parietal craniectomy that exposed the dura. A contusion
injury was obtained using a weight-drop device, consisting of an
impactor (a thin light rod with a 5-mm polypropylene ball at the
tip, guided within a glass cylinder) that was gently placed on the
exposed dura and that was activated by weight drop (10-gm weight
dropped from 2.5 cm). Controls underwent sham surgery that included
craniectomy but no weight drop. Brains were harvested 24 hours
later and cryosectioned to assess for SUR1 expression using
immunohistochemistry. The antibody used for immunohistochemistry
had previously been shown to be highly specific for SUR1 and to
label only a single band (180 kDa) in the range between 116-290 kDa
in peri-infarct brain tissues (see Simard et al., Nature Medicine,
2006). Immunolabeling showed prominent up-regulation of SUR1 in the
region of contusion (see FIGS. 47 and 48), consistent with
contusion-induced up-regulation of NCCa-ATP channels.
REFERENCES
[0574] All patents and publications cited herein are hereby
incorporated by reference in their entirety herein. Full citations
for the references cited herein are provided in the following
list.
PATENTS AND PATENT APPLICATIONS
[0575] WO 03/079987 [0576] U.S. Pat. No. 5,637,085 [0577] U.S. Pat.
No. 6,391,911
PUBLICATIONS
[0577] [0578] Adams et al. (1980) J Gen Physiol 75: 493-510. [0579]
Aguilar-Bryan et al. (1995) Science 268: 423-426. [0580]
Aguilar-Bryan L, et al., Science. 1995; 268:423-426. [0581] Ammala
C, et al., Nature. 1996; 379:545-548. [0582] Anisimov, S. V., et
al., Mech. Dev. 117, 25-74 (2002). [0583] Aoki K, et al., Acta
Neuropathol (Berl). 2003; 106:121-124. [0584] Arteel G E, et al.,
Eur J Biochem. 1998; 253:743-750. [0585] Ashcroft F M. Science.
1998; 282:1059-1060. [0586] Ayata, C. & Ropper, A. H. J. Clin.
Neurosci. 9, 113-124 (2002). [0587] Babenko A P, et al., Annu Rev
Physiol. 1998; 60:667-687. [0588] Ballanyi, K. J. Exp. Biol. 207,
3201-3212 (2004). [0589] Barclay J, et al., J Neurosci. 2002;
22:8139-8147. [0590] Barros et al. (2001) Hepatology 33: 114-122.
[0591] Baukrowitz T, et al., Science. 1998; 282:1141-1144. [0592]
Becker J B, et al., Ann N Y Acad Sci. 2001; 937:172-187. [0593]
Beyer C, et al., J Steroid Biochem Mol Biol. 2002; 81:319-325.
[0594] Blurton-Jones M, et al., J Comp Neurol. 2001; 433:115-123.
[0595] Bordey and Sontheimer (1998) Epilepsy Res 32: 286-303.
[0596] Brismar and Collins (1993) J Physiol (Lond) 460: 365-383.
[0597] Bussink J, et al., Radiat Res. 2000; 154:547-555. [0598]
Cevolani D, et al., Brain Res Bull. 2001; 54:353-361. [0599]
Champigny et al. (1991) Biochem Biophys Res Commun 176: 1 196-1
203. [0600] Chen H., et al., J. Neurol. Sci. 118, 109-6 (1993).
[0601] Chen M, et al., J Neurosci. 2003; 23:8568-8577. [0602] Chen
M, Simard J M. J Neurosci. 2001; 21:6512-6521 [0603] Choi I, et
al., Mol Cell Endocrinol. 2001; 181:139-150. [0604] Christensen and
Hofbann (1992) J Membr Biol 129: 13-36. [0605] Chuang et al. (1997)
Cell 89: 1 121-1 132. [0606] Cook et al. (1990) J Membr Biol 114:
37-52. [0607] Cress A E. Biotechniques. 2000; 29:776-781. [0608]
Dalton S, et al., Glia. 2003; 42:325-339. [0609] Dhandapani K, et
al., Endocrine. 2003; 21:59-66. [0610] Dhandapani K M, et al., Biol
Reprod. 2002; 67:1379-1385. [0611] Dhandapani K M, et al., BMC
Neurosci. 2002; 3:6. [0612] Diab A, et al., Infect Immun. 1999;
67:2590-2601. [0613] Doerfler et al. (2001) Stroke 32, 2675-2681.
[0614] Doerfler, A., et al., Stroke 32, 2675-2681 (2001). [0615]
Drain P, et al., Proc Natl Acad Sci USA. 1998; 95:13953-13958.
[0616] Dubik D et al., Oncogene. 1992; 7:1587-1594. [0617] El Ashry
D, et al., J Steroid Biochem Mol Biol. 1996; 59:261-269. [0618]
Enkvetchakul D, et al., Biophys J. 2000; 78:2334-2348. [0619] Falk
E M, et al., Pharmacol Biochem Behav. 2002; 72:617-622. [0620]
Fischer S, et al., J Cell Physiol. 2004; 198:359-369. [0621] Foy M
R, et al., Brain Res. 1984; 321:311-314. [0622] Fujita A, et al.,
Pharmacol Ther. 2000; 85:39-53. [0623] Fujita and Kurachi (2000)
Pharmacol Ther 2000 January: 85 (1):39-53. [0624] Garcia-Estrada J,
et al., Brain Res. 1993; 628:271-278. [0625] Garcia-Ovejero D, et
al., J Comp Neurol. 2002; 450:256-271. [0626] Garcia-Segura L M, et
al., Prog Neurobiol. 2001; 63:29-60. [0627] Garlid K D, et al.,
Circ Res. 1997; 81:1072-1082. [0628] Giaccia A J, et al., Int J
Radiat Oncol Biol Phys. 1992; 23:891-897. [0629] Gray and Argent
(1990) Biochim Biophys Acta 1029: 33-42. [0630] Gribble, F. M.
& Reimann, F. Diabetologia 46, 875-891 (2003). [0631] Grover G
J. Can J Physiol Pharmacol. 1997; 75:309-315. [0632] Guo X Z, et
al., Cell Res. 2001; 11:321-324. [0633] Hainsworth et al.,
Neuropharmacology. 2001; 40:784-791. [0634] Hale L P, et al., Am J
Physiol Heart Circ Physiol. 2002; 282:H1467-H1477. [0635] Halstead
J, et al., J Biol Chem. 1995; 270:13600-13603. [0636] Hamill et al.
(1981) Pflugers Arch 391: 85-100. [0637] Harder et al., Am J
Physiol. 1994; 266:H2098-H2107. Haruna T, et al., Pflugers Arch.
2000; 441:200-207. [0638] Harvey et al. (1999) Br J Pharmacol 126:
51-60. [0639] Haug A, et al., Arch Toxicol. 1994; 68:1-7. [0640]
Higashijima T, et al., J Biol Chem. 1990; 265:14176-14186. [0641]
Higgins (1992) Annu Rev Cell Biol 8: 67-1 13. [0642] Higgins C F.
Annu Rev Cell Biol. 1992; 8:67-113. [0643] Hiroi H, et al., J Mol
Endocrinol. 1999; 22:37-44. [0644] Hobbs M V, et al., J Immunol.
1993; 150:3602-3614. [0645] Hogg et al., FEBS Lett. 2002;
522:125-129. [0646] Hogg et al., Lung. 2002; 180:203-214. [0647]
Hohenegger M, et al., Proc Natl Acad Sci USA. 1998; 95:346-351.
[0648] Honda K, et al., J Neurosci Res. 2000; 60:321-327. [0649]
Horn and Marty (1988) J Gen Physiol 92:145-159. [0650] Hossain M A,
et al., J Biol Chem. 2000; 275:27874-27882. [0651] Hua Y, et al., J
Cereb Blood Flow Metab. 2003; 23:1448-1454. [0652] Hunt R A, et
al., Hypertension. 1999; 34:603-608. [0653] Huovinen R, et al., Int
J Cancer. 1993; 55:685-691. [0654] Ignotz R A, et al., J Cell
Biochem. 2000; 78:588-594. [0655] Inagaki et al. (1996) Neuron
16:1011-1017. [0656] Inagaki N, et al., Neuron. 1996; 16:1011-1017.
[0657] Isomoto et al. (1996) J Biol Chem 271: 24321-24324. [0658]
Isomoto S, et al., J Biol Chem. 1996; 271:24321-24324. [0659] Jain,
Sci. Amer. 271: 58-65, 1994. [0660] Johnson et al. (1994) J
Neurosci 14: 4040-4049. [0661] Jorgensen M B, et al., Exp Neurol.
1993; 120:70-88. [0662] Jovanovic A, et al., Lab Invest. 1998;
78:1101-1107. [0663] Jurkowitz-Alexander et al. (1992) J Neurochem
59: 344-352. [0664] Jurkowitz-Alexander et al. (1993) J Neurochem
61:1581-1584. [0665] Juurlink B H, Chen Y, Hertz L (1992) Can J
Physiol Pharmacol 70 Suppl: S344-S349. [0666] Kakinuma Y, et al.,
Clin Sci (Lond). 2002; 103 Suppl 48:210S-214S. [0667] Kangas L.
Cancer Chemother Pharmacol. 1990; 27:8-12. [0668] Kangas L. J
Steroid Biochem. 1990; 36:191-195. [0669] Kanthasamy A, et al.,
Neuroscience. 2002; 114:917-924. [0670] Karschin, C., et al., FEBS
Lett. 401, 59-64 (1997). [0671] Kawamura, S., et al., Acta
Neurochir. (Wien.) 109, 126-132 (1991). [0672] Kay et al., J
Neurosci Methods. 1986; 16:227-238. [0673] Ke C, et al., Neurosci
Lett. 2001; 301:21-24. [0674] Kelly M J, et al., Steroids. 1999;
64:64-75. [0675] Kempski et al. (1991). Ann N Y Acad Sci 633:
306-317. [0676] Kennedy A S, et al., Int J Radiat Oncol Biol Phys.
1997; 37:897-905. [0677] Kielian T, et al., J Immunol. 2001;
166:4634-4643. [0678] Kim and Fu (1993) J Membr Biol 135: 27-37.
[0679] Kimelberg et al. (1989) Mol Chem Neuropathol 11(1): 1-31.
[0680] Kimelberg et al. (1995) J Cereb Blood Flow Metab 15: 409-4
16. [0681] Kimura D. Sci Am. 1992; 267:118-125. [0682] Kohshi K, J
Neurol Sci. 2003; 209:115-117. [0683] Kom et al. (1991) Perforated
patch recording. In: Methods in Neuroscience. Electrophysiology and
Microinjection. (Conn P M, ed), pp 364-373. San Diego: Academic
Press. [0684] Korbmacher et al. (1995) J Membr Biol 146: 29-45.
[0685] Koster J C, J Gen Physiol. 1999; 114:203-213. [0686] Kucich
U, et al., Arch Biochem Biophys. 2000; 374:313-324. [0687] Kuiper G
G, et al., Endocrinology. 1997; 138:863-870. [0688] Kuiper G G, et
al., Proc Natl Acad Sci USA. 1996; 93:5925-5930. [0689] Larsson O,
et al., Diabetes. 2000; 49:1409-1412. [0690] Lawson (2000) Kidney
Int 2000 March: 57 (3): 838-845. [0691] Lawson K. Kidney Int. 2000;
57:838-845. [0692] Le Mellay V, et al., J Cell Biochem. 1999;
75:138-146. [0693] Leaney J L, Tinker A. Proc Natl Acad Sci USA.
2000; 97:5651-5656. [0694] Lebovitz (1985) Oral hypoglycemic
agents. Amsterdam: Elsevier. [0695] Li, P. A., et al., Neurosci.
Lett. 177, 63-65 (1994). [0696] Lieberherr M, et al., J Cell
Biochem. 1999; 74:50-60. [0697] Lindauer et al. (2003) J. Cereb.
Blood Flow Metab 23, 1227-1238. [0698] Liss B, Roeper J. Mol Membr
Biol. 2001; 18:117-127. [0699] Liu et al. (2002) Eur. J. Pharmacol.
435: 153-160. [0700] Liu Y, et al., Circulation. 1998;
97:2463-2469. [0701] Lomneth and Gruenstein (1989) Am J Physiol
257: C817-C824. [0702] Majno and Joris (1995) Am J Path 01 146:
3-15. [0703] Maruyama and Petersen (1984) J Membr Biol 81: 83-87.
[0704] Mateo J, et al., Biochem J. 2003; 376:537-544. [0705]
Mathews et al., J Neurosci Methods. 2000; 102:43-51. [0706] McNally
J G, et al., Methods. 1999; 19:373-385. [0707] Meyer, M., et al.,
Br. J. Pharmacol. 128, 27-34 (1999). [0708] Mongin et al. (1999) Am
J Physiol 277: C823-C832. [0709] Moon R C, Constantinou A I. Breast
Cancer Res Treat. 1997; 46:181-189. [0710] Moyer et al., J Neurosci
Methods. 1998; 86:35-54. [0711] Munoz A, et al., Stroke. 2003;
34:164-170. [0712] Murayama T, et al., J Cell Physiol. 1996;
169:448-454. [0713] Murphy K, et al., Mol Pharmacol. 2003; in
press. [0714] Nakabayashi, K. et al. AJNR Am. J. Neuroradiol. 18,
485-491 (1997). [0715] Nichols C G, et al., Science. 1996;
272:1785-1787. [0716] Nichols et al. (1996) Science 272: 1785-1787.
[0717] Oehmichen M, et al., Exp Toxicol Pathol. 2000; 52:348-352.
[0718] Oehmichen M, et al., Neurotoxicology. 2001; 22:99-107.
[0719] Olive P L, et al., Br J Cancer. 2000; 83:1525-1531. [0720]
Ono et al. (1994) Am J Physiol 267: F558-F565. [0721] Paczynski R
P, et al., Stroke. 2000; 31:1702-1708. [0722] Paech K, et al.,
Science. 1997; 277:1508-1510. [0723] Panten et al. (1989) Biochem
Pharmacol 38: 1217-1229. [0724] Panten U, et al., Biochem
Pharmacol. 1989; 38:1217-1229. [0725] Papadopoulos M C, et al., Mt
Sinai J Med. 2002; 69:242-248. Perillan et al. (1999) Glia 27:
213-225. [0726] Perillan et al. (2000) Glia 3 1: 181-192. [0727]
Perillan et al. (2002) J. Biol. Chem. 277: 1974-1980. [0728]
Perillan P R, et al., J Biol Chem. 2002; 277:1974-1980. [0729]
Perillan P R, et al., Glia. 1999; 27:213-225. [0730] Perillan P R,
et al., Glia. 2000; 31:181-192. [0731] Phillips M I, Zhang Y C.
Methods Enzymol. 2000; 313:46-56. [0732] Piiper A, et al., Am J
Physiol. 1997; 272:G135-G140. [0733] Pogue B W, et al., Radiat Res.
2001; 155:15-25. [0734] Popp and Gogelein (1992) Biochim Biophys
Acta 1108: 59-66. [0735] Proks P, et al., J Physiol. 1999; 514 (Pt
1):19-25. [0736] Qiu J, et al., J Neurosci. 2003; 23:9529-9540.
[0737] Rae et al. (1990) Exp Eye Res 50: 373-384. [0738] Rama Rao K
V, et al., J Neurosci Res. 2003; 74:891-897. [0739] Rama Rao K V,
et al., Neuroreport. 2003; 14:2379-2382. [0740] Ramirez V D, Zheng
J. Front Neuroendocrinol. 1996; 17:402-439. [0741] Ransom and
Sontheimer (1995) J Neurophysiol 73: 333-346. [0742] Raucher D, et
al., Cell. 2000; 100:221-228. [0743] Renkin (1955) J Gen Physiol
38: 225-243. [0744] Robinson and Stokes (1970) Electrolyte
Solutions. London: Buttenvorths. [0745] Robinson A P, et al.,
Immunology. 1986; 57:239-247. [0746] Robinson S P, et al., Eur J
Cancer Clin Oncol. 1988; 24:1817-1821. [0747] Rohacs T, et al., J
Biol Chem. 1999; 274:36065-36072. [0748] Rose et al. (1998) J
Neurosci 18: 3554-3562. [0749] Rossignol F, et al., Gene. 2002;
299:135-140. [0750] Rucker-Martin et al. (1999) Basic Res Cardiol
94: 171-179. [0751] Ruknudin A, et al., J Biol Chem. 1998;
273:14165-14171. [0752] Ruscher K, et al., J Neurosci. 2002;
22:10291-10301. [0753] Russo J, et al., IARC Sci Publ. 1990; 47-78.
[0754] Russo J, Russo I H. Lab Invest. 1987; 57:112-137. [0755]
Rutledge and Kimelberg (1996) J Neurosci 16: 7803-78 1 1. [0756]
Saadoun S, et al., Br J Cancer. 2002; 87:621-623. [0757] Schroder
et al. (1999) Glia 28: 166-1 74. [0758] Schubert P, et al., Ann N Y
Acad Sci. 2000; 903:24-33. [0759] Seidel et al., Cell Tissue Res.
1991; 265:579-587. [0760] Seino, S. Annu. Rev. Physiol 61, 337-362
(1999). [0761] Semenza G L. Biochem Pharmacol. 2000; 59:47-53.
[0762] Shaywitz B A, et al., Nature. 1995; 373:607-609. [0763]
Shyng et al. (1997) J Gen Physiol 110: 141-153. [0764] Shyng S, et
al., J Gen Physiol. 1997; 110:643-654. [0765] Sigworth and Sine
(1987) Biophys J 52: 1047-1 054. [0766] Singer C A, et al., J
Neurosci. 1999; 19:2455-2463. [0767] Singh M, et al., J Neurosci.
1999; 19:1179-1188. [0768] Smith S S, et al., Brain Res. 1987;
422:40-51. [0769] Smith S S, et al., Brain Res. 1988; 475:272-282.
[0770] Sohrabji F, et al., Proc Natl Acad Sci USA. 1995;
92:11110-11114. [0771] Staub et al. (1993) Brain Res 610: 69-74.
[0772] Stone D J, et al., J Neurosci. 1998; 18:3180-3185. [0773]
Streit W J, et al., Prog Neurobiol. 1999; 57:563-581. [0774]
Sturgess et al. (1987) Pflugers Arch 409: 607-6 1 5. [0775] Sun M
C, et al., J Neurosurg. 2003; 98:565-569. [0776] Swanson R A (1992)
Neurosci Lett 147: 143-146. [0777] Sylvia V L, et al, J Steroid
Biochem Mol Biol. 2000; 73:211-224. [0778] Tanaka et al. (2000) J
Biol Chem 275: 10388-10393. [0779] Teixeira C, et al., Cancer Res.
1995; 55:3902-3907. [0780] Thrash-Bingham C A, et al., J Natl
Cancer Inst. 1999; 91:143-151. [0781] Toker A. Curr Opin Cell Biol.
1998; 10:254-261. [0782] Tomiyama, et al. (1999) Stroke 30,
1942-1947. [0783] Toomey, J. R. et al. Stroke 33, 578-585 (2002).
[0784] Toran-Allerand C D. J Steroid Biochem Mol Biol. 1996;
56:169-178. [0785] Torner L, et al., J Neurosci. 2001;
21:3207-3214. [0786] Treherne, J. M. & Ashford, M. L.
Neuroscience 40, 523-531 (1991). [0787] Tucker S J, et al., EMBO J.
1998; 17:3290-3296. [0788] Tucker S J, et al., Nature. 1997;
387:179-183. [0789] Ubl et al. (1988) J Membr Biol 104: 223-232.
[0790] Vogel et al., Stroke. 1999; 30:1134-1141. [0791] Wallace W,
et al., Biotechniques. 2001; 31:1076-8, 1080, 1082. [0792] Walz et
al. (1994) J Neurosci Res 38: 12-18. [0793] Wang J Y, et al., Glia.
2000; 32:155-164. [0794] Wang Y L. Methods Cell Biol. 1998;
56:305-315. [0795] Wass, C. T. & Lanier, W. L. Mayo Clin. Proc.
71, 801-812 (1996). [0796] Wiesener M S, et al., FASEB J. 2003;
17:271-273. [0797] Woolley C S. Curr Opin Neurobiol. 1999;
9:349-354. [0798] Xie L H, et al., Proc Natl Acad Sci USA. 1999;
96:15292-15297. [0799] Yajima Y, et al., Endocrinology. 1997;
138:1949-1958. [0800] Young, W. & Constantini, S. The
Neurobiology of Central Nervous System Trauma. Salzman, S. K. &
Faden, A. I. (eds.), pp. 123-130 (Oxford University Press, New
York, 1994). [0801] Yu et al. (2001) Glia 35: 121-130. [0802] Zhang
L, et al., Brain Res Mol Brain Res. 2002; 103:1-11. [0803] Zhang Y,
et al., J Neurosci. 2001; 21:RC176. [0804] Zheng J, Ramirez V D. J
Steroid Biochem Mol Biol. 1997; 62:327-336. [0805] Zunkler, B. J.,
et al., Biochem. Pharmacol. 67, 1437-1444 (2004).
[0806] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
Sequence CWU 1
1
10117DNAArtificial SequenceSynthetic Oligonucleotide Primer
1aagcacgtca acgccct 17219DNAArtificial SequenceSynthetic
Oligonucleotide Primer 2gaagcttttc cggcttgtc 19319DNAArtificial
SequenceSynthetic Oligonucleotide Primer 3gagtcggact tctcgccct
19419DNAArtificial SequenceSynthetic Oligonucleotide Primer
4ccttgacagt ggccgaacc 19525DNAArtificial SequenceSynthetic
Oligonucleotide Probe 5ttccacatcc tggtcacacc gctgt
25620DNAArtificial SequenceSynthetic Oligonucleotide Primer
6cggaccaccc caagtattca 20717DNAArtificial SequenceSynthetic
Oligonucleotide Primer 7gccggcacgg ttcttct 17825DNAArtificial
SequenceSynthetic Oligonucleotide Probe 8catgatcgtg gctgctatcc
aggca 25918DNAArtificial SequenceSynthetic Oligonucleotide Primer
9ggccgagtgg ttctcggt 181018DNAArtificial SequenceSynthetic
Oligonucleotide Primer 10tgcctgaggc gtggctgt 18
* * * * *