U.S. patent application number 09/919116 was filed with the patent office on 2001-12-20 for treatment of infarcts.
Invention is credited to Brand, Stephen J., Elliott, Peter J., Goldberg, Alfred L., Plamondon, Louis, Soucy, Francois.
Application Number | 20010053760 09/919116 |
Document ID | / |
Family ID | 27122399 |
Filed Date | 2001-12-20 |
United States Patent
Application |
20010053760 |
Kind Code |
A1 |
Brand, Stephen J. ; et
al. |
December 20, 2001 |
Treatment of infarcts
Abstract
This invention is directed to treating ischemia by administering
proteasome inhibitors, ubiquitin pathway inhibitors, agents that
interfere with the activation of NF-.kappa.B via the ubiquitin
proteasome pathway, or mixtures thereof.
Inventors: |
Brand, Stephen J.; (San
Diego, CA) ; Goldberg, Alfred L.; (Chestnut Hill,
MA) ; Plamondon, Louis; (Watertown, MA) ;
Soucy, Francois; (Arlington, MA) ; Elliott, Peter
J.; (Marlborough, MA) |
Correspondence
Address: |
HALE AND DORR, LLP
60 STATE STREET
BOSTON
MA
02109
|
Family ID: |
27122399 |
Appl. No.: |
09/919116 |
Filed: |
July 30, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09919116 |
Jul 30, 2001 |
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09285170 |
Feb 17, 1998 |
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6271199 |
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09285170 |
Feb 17, 1998 |
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08988339 |
Dec 3, 1997 |
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08988339 |
Dec 3, 1997 |
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08801936 |
Feb 15, 1997 |
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Current U.S.
Class: |
424/94.64 ;
514/15.1; 514/16.4; 514/450 |
Current CPC
Class: |
A61P 9/00 20180101; A61K
38/05 20130101; A61K 31/407 20130101; A61P 9/10 20180101 |
Class at
Publication: |
514/2 ; 514/19;
514/450 |
International
Class: |
A61K 038/05; A61K
038/03; A61K 031/365 |
Claims
What is claimed is:
1. A method of treating ischemic injury in a mammal comprising
administering to said mammal an effective amount of an NF-.kappa.B
activation inhibitor selected from the group consisting of
proteasome inhibitors, ubiquitin pathway inhibitors, agents that
interfere with the activation of NF-.kappa.B via the ubiquitin
proteasome pathway, and mixtures thereof.
2. A method of treating reperfusion injury after ischemia in a
mammal comprising administering to said mammal an effective amount
of an NF-.kappa.B activation inhibitor selected from the group
consisting of proteasome inhibitors, ubiquitin pathway inhibitors,
agents that interfere with the activation of NF-.kappa.B via the
ubiquitin proteasome pathway, and mixtures thereof.
3. A method of preventing, reducing the size or lessening the
severity of infarct resulting from ischemia or reperfusion injury
in a mammal comprising administering to said mammal an effective
amount of an NF-.kappa.B activation inhibitor selected from the
group consisting of proteasome inhibitors, ubiquitin pathway
inhibitors, agents that interfere with the activation of
NF-.kappa.B via the ubiquitin proteasome pathway, and mixtures
thereof.
4. The method of claims 1-3 wherein the ischemia is the result of
vascular occlusion.
5. The method of claim 4 wherein the occlusion is of a cerebral
vessel.
6. The method of claim 4 wherein the occlusion is of a cardiac
vessel.
7. The method of claim 1-3 wherein the method prevents or lessens
the severity of stroke.
8. The method of claim 1-3 wherein the method prevents or lessens
the severity of stroke resulting from the occlusion of the cerebral
vessel.
9. The method of claim 6 wherein the method prevents or lessens the
severity of myocardial infarction resulting from the occlusion of a
cardiac vessel.
10. The method of claim 1-3 wherein the NF-.kappa.B activation
inhibitor is an agent that inhibits phosphorylation of
I.kappa.B-.alpha..
11. The method of claims 1-3 wherein the NF-.kappa.B activation
inhibitor comprises a proteasome inhibitor.
12. The method of claim 11 wherein said proteasome inhibitor is a
peptidyl aldehyde.
13. The method of claim 11 wherein said proteasome inhibitor is a
peptidyl boronic acid or peptidyl boronic ester.
14. The method of claim 11 wherein said proteasome inhibitor is a
lactacystin analog.
15. The method of claim 13 wherein said proteasome inhibitor is
N-(2-pyrazine) carbonyl-L-phenylalanine-L-leucine boronic acid.
16. The method of claim 14 wherein the proteasome inhibitor is
7-n-propyl-clasto-lactacystin .beta.-lactone.
17. The method of claims 1-3 wherein the NF-.kappa.B activation
inhibitor is administered to the mammal less than 12 hours after
the onset of ischemia.
18. The method of claims 1-3 wherein the NF-.kappa.B activation
inhibitor is administered to the mammal less than 6 hours after the
onset of ischemia.
19. The method of claims 1-3 wherein the NF-.kappa.B activation
inhibitor is administered to the mammal before the onset of
ischemia.
20. The method of claims 1-4 further comprising the step of
administering a second agent.
21. The method of claim 21 wherein the second agent is selected
from the group consisting of NF-.kappa.B activation inhibitors,
agents which inhibit the expression or action of proinflammatory
cytokines or cellular adhesion molecules, agents which act to
reperfuse or oxygenate tissues, and agents which assist in
temperature normalization.
22. The method of claim 21 wherein the second agent is selected
from the group consisting of steroids, antiedema drugs,
thrombolytics, clot solubilizing drug, polyanions and
anticoagulants.
23. The method of claim 22 wherein the second agent comprises a
thrombolytic or clot solubilizing drug.
24. The method of claim 24 wherein the thrombolytic clot
solubilizing drug comprises tissue plasminogen activator.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 08/988,339, filed Dec. 3, 1997 which is a
continuation-in-part of U.S. application Ser. No. 08/801,936, filed
Feb. 15, 1997.
FIELD OF THE INVENTION
[0002] This invention relates to treatment of ischemia and
reperfusion injury, including preventing or reducing the size of
infarct after vascular occlusion.
BACKGROUND OF THE INVENTION
[0003] All tissues are sensitive to hypoperfusion and the resulting
lack of oxygen, ischemia. Prolonged ischemia will result in
cellular damage. The magnitude of the injury and the potential for
tissue rescue depends upon the degree and duration of the ischemia.
With long ischemic periods, cellular death occurs (infarction) and
under these conditions the injury is irreversible. On the other
hand, dying cells or cells targeted for cell death may be rescued
by drug treatment, if applied in a timely fashion.
[0004] Major ischemic events of therapeutic concern include, but
are not limited to, heart attacks and stroke. In man, stroke
accounts for 10% of all premature deaths, and of those that survive
the insult, 50% are left severely disabled. Only a small fraction,
10%, of patients actually recover full function.
[0005] Over 1,500,000 Americans suffer from myocardial infarctions
each year. About half of these do not survive to reach the
hospital. However, with the acceptance of thrombolytic therapy such
as streptokinase or tissue plasminogen activator (TPA), the one
month survival rate for patients who do reach the hospital is as
high as 93.6% (Werns, S. W. Textbook of Interventional Cardiology,
ed. Topol, E. J. W B Saunders: 1994, pp142-153). By lysing the clot
early in the course of infarct, ischemic muscle and tissue can be
salvaged. However, reperfusion in and of itself leads to tissue
damage.
[0006] Reperfusion injury may occur as a result of one or more of
the following events: cellular acidosis leading to calcium
overload; increased intracellular osmotic loads of catabolites
leading to cell swelling; free radicals from neutrophils and other
inflammatory cells.
[0007] Neutrophils are seen in reperfused myocardium shortly after
reperfusion. Monocytes/macrophages appear within 24 to 48 hours.
Neutrophil infiltration is three to five fold greater in reperfused
myocardium than in ischemic myocardium, is initiated by adhesion to
endothelial cells, and occurs within 10 minutes of reperfusion.
Neutrophils in and of themselves may become trapped in capillaries
and impede reperfusion. Intravascular neutrophils may block up to
27% of the capillaries, and have been shown to be related to
decreased regional blood flow (Forman et al., Acute Myocardial
Infarction, eds.Gersh et al. Elsevier: 1991, pp 347-370). This can
result in the "no-reflow" phenomenon, where blood flow continues to
decrease after reperfusion.
[0008] It is known that neutrophils must first adhere themselves to
the endothelial cell wall through the interactions with adhesion
molecules. Once attached to the vessel cell wall, the neutrophils
then force themselves between adjacent endothelial cells and move
into the brain tissue, where they release cytotoxic cytokines. The
expression of such adhesion molecules is increased following cell
damage including ischemia. In addition, endothelial cell walls
become more permeable to infiltrating cells due to the release of
nitric oxide (NO). Agents that inhibit the movement (diapedesis) of
neutrophils from surrounding blood vessels into the damaged tissue
may thus be of value in allowing dying cells time to recover from
the ischemic insult.
[0009] There is a need in the art for effective therapies to
prevent or reduce the consequences of ischemia.
SUMMARY OF THE INVENTION
[0010] In a first aspect, the present invention is directed to a
method of treating ischemia in a mammal comprising administering to
said mammal an effective amount of an NF-.kappa.B activation
inhibitor. Preferred NF-.kappa.B activation inhibitors are selected
from the group consisting of proteasome inhibitors, ubiquitin
pathway inhibitors, inhibitors of serine phosphorylation of
I.kappa.B-.alpha., and mixtures thereof. Preferably, the agent is
administered to the mammal after the onset of transient vascular
occlusion and prior to induction of permanent ischemic damage.
[0011] In a second aspect, the present invention is directed to a
method of preventing or lessening the severity reperfusion injury
in a mammal comprising administering to said mammal an effective
amount of an NF-.kappa.B activation inhibitor. Preferred
NF-.kappa.B activation inhibitors are selected from the group
consisting of proteasome inhibitors, ubiquitin pathway inhibitors,
inhibitors of serine phosphorylation of I.kappa.B-.alpha., and
mixtures thereof.
[0012] In a third aspect, the present invention is directed to a
method of preventing, reducing the size of, or lessening the
severity of infarction in a mammal comprising administering to said
mammal an effective amount of an NF-.kappa.B activation inhibitor.
Preferred NF-.kappa.B activation inhibitors are selected from the
group consisting of proteasome inhibitors, ubiquitin pathway
inhibitors, inhibitors of serine phosphorylation of
I.kappa.B-.alpha., and mixtures thereof. In preferred embodiments,
the method according to this aspect of the invention prevents or
lessens severity of infarction after occlusion of a cerebral vessel
or a cardiac vessel. In certain preferred embodiments, the method
prevents the occlusion from resulting in stroke, or lessens the
severity of a stroke resulting from cerebral vessel occlusion.
[0013] In a fourth aspect, the present invention is directed to a
method of treating ischemia or reperfusion injury, including
preventing or lessening the severity of infarction in a mammal
comprising administering to the mammal an adjunct therapeutic, in
addition to administering an NF-.kappa.B activation inhibitor.
Preferred NF-.kappa.B activation inhibitors are selected from the
group consisting of proteasome inhibitors, ubiquitin pathway
inhibitors, inhibitors of serine phosphorylation of
I.kappa.B-.alpha., and mixtures thereof. Certain preferred adjunct
therapeutics include without limitation, agents such as steroids
which further inhibit NF-.kappa.B activation or inhibit the
expression or action of proinflammatory cytokines or cellular
adhesion molecules; agents which act to either reperfuse or
oxygenate tissues, such antiedema drugs, thrombolytics such as TPA,
streptokinase and urokinase, polyanions such as heparin,
anticoagulants; and agents that assist in temperature
normalization.
[0014] Preferred NF-.kappa.B activation inhibitors inhibit
NF-.kappa.B activation by the ubiquitin-proteasome pathway. In
certain preferred embodiments, the NF-.kappa.B activation inhibitor
inhibits phosphorylation of I.kappa.B-.alpha.. In certain preferred
embodiments, the NF-.kappa.B activation inhibitor is a proteasome
inhibitor. Preferably, the proteasome inhibitor is selected from
the group consisting of peptidyl aldehydes, boronic acids, boronic
esters, lactacystin, and lactacystin analogs. In certain preferred
embodiments, NF-.kappa.B activation inhibitor is a ubiquitin
pathway inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 shows the cascade of NF-.kappa.B activation leading
to reperfusion injury, including infarct. Points of intervention by
the methods according to the invention are indicated.
[0016] FIG. 2 shows the ubiquitin-proteasome pathway.
[0017] FIG. 3 is directed to reduction of infarct volume following
middle cerebral artery (MCA) occlusion by administration of the
proteasome inhibitor
N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid.
[0018] FIG. 4 is directed to reduction of infarct size, expressed
as a percentage of the contralateral hemisphere, by administration
of the proteasome inhibitor
N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid.
[0019] FIG. 5 is directed to reduction of infarct volume by
administration of the proteasome inhibitor
7-n-propyl-clasto-lactacystin .beta.-lactone following middle
cerebral artery (MCA) occlusion.
[0020] FIG. 6 is directed to reduction of neurological score by
administration of the proteasome inhibitor
7-n-propyl-clasto-lactacystin .beta.-lactone following middle
cerebral artery (MCA) occlusion.
[0021] FIG. 7 is directed to reduction of infarct volume by
administration of the proteasome inhibitor
7-n-propyl-clasto-lactacystin .beta.-lactone.
[0022] FIG. 8 is directed to reduction of neurological score by
administration of the proteasome inhibitor
7-n-propyl-clasto-lactacystin .beta.-lactone.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The invention relates to treatment of ischemia and
reperfusion injury, including preventing, reducing the size or
lessening the severity of infarct after vascular occlusion. The
patent applications, patents and literature references cited herein
indicate the knowledge in this field and are hereby incorporated by
reference in their entirety. In the case of inconsistencies the
present disclosure will prevail.
[0024] It has now been unexpectedly discovered that the
ubiquitin-proteasome pathway is a target for treating ischemia and
reperfusion injury, including preventing, reducing the size, or
lessening the severity of infarcts following vascular occlusions
such as occur during heart attack or stroke, and that inhibitors of
NF-.kappa.B activation via the ubiquitin-proteasome pathway can
provide effective therapy for these conditions. The invention
provides surprisingly effective methods for treating ischemia or
reperfusion injury.
[0025] The present inventors have discovered that blocking
proteasome function reduces the effects of ischemia, such as
reducing infarct size following vascular occlusion. This can be
done by direct proteasome inhibition (shown with
N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic acid and
7-n-propyl-clasto-lactacystin .beta.-lactone) or by blocking
ubiquitination of proteasome-targeted proteins such as
I.kappa.B-.alpha.. Any inhibitors which affect activation of
NF-.kappa.B via the ubiquitin/proteasome pathway in eukaryotic
cells are expected to be effective in preventing or treating
infarction, including infarction following vascular occlusion and
are thus in the scope of the present invention.
[0026] In accordance with the present invention, treatment of
ischemia, including reperfusion injury, prevention of infarction
and reduction in size or lessening of severity of infarct is
achieved by administering to a mammal an effective amount of an
NF-.kappa.B activation inhibitor. Preferred NF-.kappa.B activation
inhibitors are selected from the group consisting of proteasome
inhibitors, ubiquitin pathway inhibitors, inhibitors of serine
phosphorylation of I.kappa.B-.alpha., and mixtures thereof.
[0027] In a first aspect, the present invention is directed to a
method of treating ischemia in a mammal comprising administering to
said mammal an effective amount of an NF-.kappa.B activation
inhibitor. All tissues are sensitive to lack of oxygen (ischemia)
resulting from hypoperfusion. Major ischemic events of therapeutic
concern include, but are not limited to, heart attacks and stroke.
Ischemia also can affect other tissues, including retinal, liver,
kidney, bone, placental and spinal tissue. Prolonged ischemia
results in cellular damage, manifest, in the case of cerebral
ischemia, as neurological dysfunction. Agents currently used in
treatment of stroke are targeted at 1) reversing excessive
excitotoxic phenomena associated with an ischemic episode; or 2)
increasing blood flow to ischemic tissue.
[0028] Ischemic injury may commonly result from e.g., vascular
occlusion such as by an embolus or thrombus, hemorrhage, near
drowning and near suffocation. Without wishing to be bound by
theory, it is believed that ischemia causes a massive release of
the excitotoxic amino acid, glutamate, from presynaptic nerve
endings in the brain which act on N-methyl-D-aspartate (NMDA)
receptors on adjacent cells. Once activated, NMDA receptors allow
excessive calcium to enter the cell, which in turn activates a
number of secondary pathways that ultimately lead to cellular
protein degradation and cell death. In the search for effective
therapies, efforts have been made to target either the release of
presynaptic glutamate (via .kappa.-opiate receptor stimulation), or
blockade of NMDA receptor activation (either directly, with NMDA
antagonists, or indirectly, with glycine antagonists). Calcium
channel blockers and calpain inhibitors have also been
investigated. While individual drugs have shown activity in both
preclinical and clinical situations, the benefit is limited due to
the speed at which the cascade occurs, the time taken for the drugs
to be given, and the effectiveness of the therapy. The only drug
used clinically to increase blood flow is tissue plasminogen
activator (TPA), which aids in the rapid solubilization of clots
that are responsible for the vessel blockade. While effective to a
limited extent in stroke patients, by actually promoting bleeding,
drugs like TPA can be lethal to those patients that have cerebral
hemorrhage. As such, TPA cannot be given until the patient has been
confirmed as having a stroke rather than hemorrhage. For a stroke
patient, the time taken for this analysis to occur obviously
increases the length of time of the ischemia and hence the amount
of salvageable tissue is reduced. Agents such as NF-.kappa.B
activation inhibitors that can act on the ischemic cascade system
itself are not limited by such a prolonged diagnosis period, as
they are not detrimental in cerebral hemorrhage patients.
[0029] Preferred NF-.kappa.B activation inhibitors inhibit
NF-.kappa.B activation by the ubiquitin-proteasome pathway. In
certain preferred embodiments, the NF-.kappa.B activation inhibitor
inhibits phosphorylation of I.kappa.B-.alpha.. In certain preferred
embodiments, the NF-.kappa.B activation inhibitor is a proteasome
inhibitor. In such embodiments the inhibition of the proteasome is
preferably less than complete inhibition. Preferably, the
proteasome inhibitor is selected from the group consisting of
peptidyl aldehydes, boronic acids, boronic esters, lactacystin, and
lactacystin analogs. In certain preferred embodiments, the
NF-.kappa.B activation inhibitor is a ubiquitin pathway
inhibitor.
[0030] The transcription factor NF-.kappa.B is a member of the Rel
protein family. The Rel family of transcriptional activator
proteins can be divided into two groups. The first group requires
proteolytic processing, and includes p105 and p100, which are
processed to p50 and p52, respectively. The second group does not
require proteolytic processing and includes p65 (Rel A), Rel
(c-Rel), and Rel B. NF-.kappa.B comprises two subunits, p50 and an
additional member of the Rel gene family, e.g., p 65. Unprocessed
p105 can also associate with p65 and other members of the Rel
family. In most cells, the p50-p65 heterodimer is present in an
inactive form in the cytoplasm, bound to I.kappa.B-.alpha.. The
ternary complex can be activated by the dissociation and
destruction of I.kappa.B-.alpha., while the p65/p105 heterodimer
can be activated by processing of p105.
[0031] The ubiquitin-proteasome pathway plays an essential role in
the regulation of NF-.kappa.B activity, being responsible both for
processing of p105 to p50 and for the degradation of the inhibitor
protein I.kappa.B-.alpha.. (Palombella et al., WO95/25533) In order
to be targeted for degradation by the proteasome, I.kappa.B-.alpha.
must first undergo selective phosphorylation at serine residues 32
and 36, followed by ubiquitination. (Alkalay et al., Proc. Natl.
Acad. Sci. USA 92: 10599 (1995); Chen, WO97/35014)
[0032] Once activated, NF-.kappa.B translocates to the nucleus,
where it plays a central role in the regulation of a remarkably
diverse set of genes involved in the immune and inflammatory
responses (Grilli et al., International Review of Cytology 143:1-62
(1993)). For example, NF-.kappa.B is required for the expression of
a number of genes involved in the inflammatory response, such as
TNF-.alpha. gene and genes encoding the cell adhesion molecules
E-selectin, P-selectin, ICAM, and VCAM (Collins, T., Lab. Invest.
(1993) 68:499. NF-.kappa.B is also required for the expression of a
large number of cytokine genes such as IL-2, IL-6, granulocyte
colony stimulating factor, and IFN-.beta.. Inducible nitric oxide
synthetase is also under regulatory control of NF-.kappa.B.
[0033] Proteasome inhibitors block I.kappa.B-.alpha. degradation
and activation of NF-.kappa.B (Palombella et al. WO 95/25533
published Sep. 28, 1995; Traenckner, et al., EMBO J. (1994)
13:5433). Proteasome inhibitors also block TNF-.alpha. induced
expression of the leukocyte adhesion molecules E-selectin, VCAM-1,
and ICAM-1 (Read, et al., Immunity (1995) 2:493). These cell
adhesion molecules play a critical role in supporting the
emigration of leukocytes from the bloodstream to extravascular
sites of injury such as ischemic tissue. Although intended to serve
a repair function, the resultant influx of cells, particularly
neutrophils, can promote damage by release of cytokines that speed
cell death and signal additional cells (e.g., macrophages) to
invade the area.
[0034] In a second aspect, the present invention is directed to a
method of preventing or lessening the severity of reperfusion
injury in a mammal comprising administering to said mammal an
effective amount of an NF-.kappa.B activation inhibitor.
Reperfusion injury may occur as a result of one or more of the
following events: cellular acidosis leading to calcium overload;
increased intracellular osmotic loads of catabolites leading to
cell swelling; free radicals from neutrophils and other
inflammatory cells. Preferred NF-.kappa.B activation inhibitors are
selected from the group consisting of proteasome inhibitors,
ubiquitin pathway inhibitors, inhibitors of serine phosphorylation
of I.kappa.B-.alpha., and mixtures thereof.
[0035] In a third aspect, the present invention is directed to a
method of preventing, reducing the size of, or lessening the
severity of infarction in a mammal comprising administering to said
mammal an effective amount of an NF-.kappa.B activation inhibitor.
Preferred NF-.kappa.B activation inhibitors are selected from the
group consisting of proteasome inhibitors, ubiquitin pathway
inhibitors, inhibitors of serine phosphorylation of
I.kappa.B-.alpha., and mixtures thereof. In preferred embodiments,
the method according to this aspect of the invention prevents,
reduces the size or lessens the severity of infarction after
occlusion of a cerebral vessel or a cardiac vessel. In certain
preferred embodiments, the method prevents the occlusion from
resulting in stroke, or lessens the severity of a stroke resulting
from cerebral vessel occlusion.
[0036] The most common form of stroke is thrombotic stroke, where
occlusion of cerebral blood vessels is believed to be caused by a
plug of aggregated platelets. Often, these platelet plugs are
released as emboli from platelet thrombi on atherosclerotic plaques
in major carotid or cerebral vessels. Thrombotic strokes often have
a characteristic "stuttering" onset in which an initial modest,
often reversible, neurological deficit is followed by a more
severe, irreversible stroke. The initial event often reflects
transient cerebrovascular obstruction by platelet thrombi, which is
potentially reversible. Indeed, clinically, a mild stroke can be
viewed as the extreme end of the spectrum of transient ischemic
attacks (TIA)--a reversible neurological deficit in which a
cerebral vessel is transiently occluded by an embolic platelet
thrombus, which subsequently disaggregates, thus allowing flow to
be reestablished. Therefore, the administration of an agent as
disclosed herein after the onset of transient vascular occlusion is
contemplated by the present invention. Another important form of
stroke is stroke after cerebral hemmorhage, as discussed above. It
is believed that the agents disclosed herein will have broad range
efficacy in preventing, reducing the size, or lessening the
severity of infarcts resulting from a variety of causes, including
thrombotic stroke and stroke following cerebral hemmorhage. As a
practical matter, the reduction of infarct size or lessening of
infarct severity will be inferred from a reduction in symptoms
associated with the infarct, including without limitation
neurological symptoms and cardiac performance symptoms.
[0037] In a fourth aspect, the present invention is directed to a
method to treating ischemia or reperfusion injury, including
without limitation reducing the size or lessening the severity of
infarction in a mammal comprising administering to the mammal an
adjunct therapeutic, in addition to administering an NF-.kappa.B
activation inhibitor. Preferred NF-.kappa.B activation inhibitors
are selected from the group consisting of proteasome inhibitors,
ubiquitin pathway inhibitors, inhibitors of serine phosphorylation
of I.kappa.B-.alpha., and mixtures thereof. Certain preferred
adjunct therapeutics include without limitation, agents which such
as steroids which further inhibit NF-.kappa.B activation or inhibit
the expression or action of proinflammatory cytokines or cellular
adhesion molecules; agents which act to either reperfuse or
oxygenate tissues, antiedema drugs, thrombolytics such as TPA,
streptokinase and urokinase, polyanions such as heparin,
anticoagulants; and agents that assist in temperature
normalization. Agents that inhibit the action of cytokines or
cellular adhesion molecules include, without limitation,
antibodies, or an antibody derivative, which may more preferably be
a monoclonal antibody, a human antibody, a humanized antibody, a
single-chain antibody, a chimeric antibody, or an antigen-binding
antibody fragment. The use of any of the agents discussed or
disclosed herein in combination with any other agent or agents used
in the treatment of stroke or myocardial infarction is further
contemplated within the scope of the present invention.
[0038] In the present description, the following definitions will
be used.
[0039] "Treatment" shall mean preventing or lessening ischemic
injury or reperfusion injury, including the prevention of
infarction or reduction in size or lessening in severity of
infarct, including without limitation infarct after vascular
occlusion. Any amelioration of any symptom of the infarct pursuant
to treatment using any proteasome inhibitor, ubiquitin pathway
inhibitor, or agent that interferes with activation of NF-.kappa.B
via the ubiquitin proteasome pathway is within the scope of the
invention.
[0040] The term "mammals" is intended to include humans.
[0041] "Inhibitors of NF-.kappa.B activation" or "NF-.kappa.B
activation inhibitors" shall mean any substance which inhibits of
NF-.kappa.B activation via the ubiquitin proteasome pathway, and
shall include any substance that 1) inhibits the proteasome or the
activity thereof; 2) inhibits ubiquitination of I.kappa.B-.alpha.
or p105; or 3) inhibits phosphorylation of I.kappa.B-.alpha. or
p105.
[0042] "Ubiquitin pathway inhibitor" shall mean any substance which
directly or indirectly inhibits ubiquitination or the transfer of
ubiquitin to proteins. Non-limiting examples of ubiquitin pathway
inhibitors include those disclosed in Berleth et al, Biochem.
35(5):1664-1671, (1996). Inhibitors of I.kappa.B-.alpha.
phosphorylation are also known (Chen, Cell 84:853 (1996)).
[0043] "Proteasome inhibitor" shall mean any substance which
directly or indirectly inhibits the proteasome or the activity
thereof. Non-limiting examples of proteasome inhibitors for use in
the present invention include peptide aldehydes (Stein et al. WO
95/24914 published Sep. 21, 1995; Siman et al. WO 91/13904
published Sep. 19, 1991; lqbal et al. J. Med. Chem. 38:2276-2277
(1995)), peptide boronic acids (Adams et al. WO 96/13266 published
May 9, 1996; Siman et al. WO 91/13904), lactacystin, and
lactacystin analogs (Fenteany et al. Proc. Natl. Acad. Sci. USA
(1994) 91:3358; Fenteany et al. WO 96/32105, published Oct. 17,
1996).
[0044] Peptide aldehyde proteasome inhibitors for use in the
present invention preferably are those disclosed in Stein et al. WO
95/24914 published Sep. 21, 1995 or Siman et al. WO 91/13904
published Sep. 19, 1991, both hereby incorporated by reference in
their entirety.
[0045] Boronic acid or ester compounds for use in the present
invention preferably are those disclosed in Adams et al. WO
96/13266 published May 9, 1996, or Siman et al. WO 91/13904, both
of which are hereby incorporated by reference in their
entirety.
[0046] More preferably, the boronic acid compound for use in the
present invention is selected from the group consisting of:
[0047]
N-(4-morpholine)carbonyl-.beta.-(1-naphthyl)-L-alanine-L-leucine
boronic acid
[0048] N-(8-quinoline)sulfonyl-.beta.-(1-naphthyl)-L-alanine
-L-alanine-L-leucine boronic acid,
[0049] N-(2-pyrazine)carbonyl-L-phenylalanine-L-leucine boronic
acid, and
[0050]
N-(4-morpholine)carbonyl-[O-(2-pyridylmethyl)]-L-tyrosine-L-leucine
boronic acid.
[0051] Lactacystin and lactacystin analog compounds for use in the
present invention preferably are those disclosed in Fenteany et al.
WO 96/32105, published Oct. 17, 1996, hereby incorporated by
reference in its entirety. More preferably, the lactacystin analog
is selected from lactacystin, clasto-lactacystin .beta.-lactone,
7-ethyl-clasto-lactacysti- n .beta.-lactone and
7-n-propyl-clasto-lactacystin .beta.-lactone are used for the
methods of the invention. Most preferably the lactacystin analog is
7-n-propyl-clasto-lactacystin .beta.-lactone.
[0052] The agents disclosed herein may be administered by any
route, including intradermally, subcutaneously, orally,
intraarterially or intravenously. Preferably, administration will
be by the intravenous route. Preferably parenteral administration
may be provided in a bolus or by infusion.
[0053] The concentration of a disclosed compound in a
pharmaceutically acceptable mixture will vary depending on several
factors, including the dosage of the compound to be administered,
the pharmacokinetic characteristics of the compound(s) employed,
and the route of administration. Effective amounts of agents for
treating ischemia or reperfusion injury would broadly range between
about 10 .mu.g and about 50 mg per Kg of body weight of a recipient
mammal. The agent may be administered in a single dose or in repeat
doses. Treatments may be administered daily or more frequently
depending upon a number of factors, including the overall health of
a patient, and the formulation and route of administration of the
selected compound(s).
[0054] The disclosed compound may be administered at any time
before, during, or after the onset of ischemia. In certain
preferred embodiments, the agent is administered after the onset of
ischemia, but at a time early enough to reverse damage to some or
all of the affected tissue. Preferably, the agent is administered
less than 12 hours, more preferably less than 6 hours and still
more preferably less than about 3 hours after onset of the ischemic
event. Treatment may be initiated before, during or after
reperfusion of the ischemic tissue. In many instances, the time of
reperfusion cannot be accurately determined, but it is preferred
that treatment begin before, during or soon after reperfusion to
prevent or lessen additional damaging consequences which may result
from reperfusion.
[0055] In the event of a clot inducing an ischemic episode, drugs
such as TPA which break up the clot can be administered to reduce
the potential tissue damage. Once dosed, the drug acts quickly to
remove the vascular blockade and, therefore, the time at which the
ischemic event ends can be determined. In one preferred embodiment,
the inhibitor of NF-.kappa.B activation is administered at the same
time or immediately following the clot dissolving drug.
[0056] In certain other preferred embodiments, the inhibitor of
NF-.kappa.B activation is administered prior to the onset of
ischemia. The onset of ischemia can be predicted in the case of
certain medical procedures, such as surgical procedures. In another
preferred embodiment, the disclosed compound is administered just
prior to or immediately following the release of ischemia and onset
of reperfusion during such a medical procedure (e.g., angioplasty
procedures).
[0057] The following examples are intended to further illustrate
certain preferred embodiments of the invention and are not limiting
in nature.
EXAMPLES
Example 1
[0058] Methods
[0059] Six male Sprague Dawley rats (300 g) were anesthetized with
haloethane and subjected to middle cerebral artery (MCA) occlusion
using a nylon filament for 2 h. Subsequently, the filament was
removed and reperfusion of the infarcted tissue occurred for 24
hours before the rat was sacrificed.
[0060] Staining of coronal sections (2.0 mm.times.7-8) with
triphenyltetrazolium chloride (TTC) taken throughout the brain were
evaluated under blinded conditions using image analysis to
determine infarct size.
[0061] The infarct was also expressed as a percentage of the
contralateral (non-infarcted) hemisphere to provide an indication
of how much of the ipsilateral (infarcted) hemisphere was actually
damaged by the procedure. Because edema is present in the infarcted
hemisphere, it is often impossible to directly ascertain the
percentage of the ipsilateral hemisphere that has been damaged.
[0062] Dosing Regimen
[0063] Rats were given iv bolus injections (1.0 mL/kg) of either
vehicle (10% PEG 200/saline; n=3) or
N-(2-pyrazine)carbonyl-L-phenylalanine-L-leu- cine boronic acid
(0.03 mg/kg; n=3) at 30 minutes, 2 hours, and 6 hours after the
start of the occlusion.
[0064] Results
[0065] Infarct volume was decreased by 62% on the ipsilateral
hemisphere in treated animals (FIG. 1). This reflects a decrease in
total damage of the hemisphere from 19% to 2% (FIG. 2).
Example 2
[0066] Methods
[0067] Male Sprague Dawley rats (250-400 g) were anesthetized with
haloethane and subjected to middle cerebral artery (MCA) occlusion
using a nylon filament for 2 h. Subsequently, the filament was
removed and reperfusion of the infarcted tissue occurred for 24
hours before the rat was sacrificed.
[0068] Immediately after the filament was withdrawn, the animals
were evaluated using a neurological scoring system. Neurological
scores were expressed on a scale from 0 to 10, with 0 representing
no neurological deficit and 10 representing severe neurological
deficit. After 24 hours and before sacrifice, animals were
evaluated a second time using the same neurological scoring
system.
[0069] Staining of coronal sections (2.0 mm.times.7-8) with
triphenyltetrazolium chloride (TTC) taken throughout the brain were
evaluated under blinded conditions using image analysis to
determine infarct size.
[0070] Dosing Regimen
[0071] Rats were given iv bolus injections (1.0 mL/kg) of either
vehicle (50% propylene glycol/saline; n=8) or
7-n-propyl-clasto-lactacystin .beta.-lactone (0.1 mg/kg; n=6) at 2
hours after the start of the occlusion.
[0072] Results
[0073] In animals treated with 7-n-propyl-clasto-lactacystin
.beta.-lactone, infarct volume was decreased by 70% (FIG. 5).
[0074] All animals had a neurological score of 10.+-.0 immediately
after the 2 hour ischemic episode. At 24 hours, the vehicle-treated
rats had a mean score of 8.7.+-.0.6, whereas rats treated with a
single 0.1 mg/kg dose of 7-n-propyl-clasto-lactacystin
.beta.-lactone had a mean score of 5.5.+-.1 (FIG. 6). These data
represent a 40% neurological improvement for the drug-treated
animals.
[0075] Conclusion
[0076] 7-n-propyl-clasto-lactacystin .beta.-lactone provides
significant protection in both the degree of neurological deficit
and infarcted brain damage.
Example 3
[0077] Methods
[0078] Male Sprague Dawley rats (250-400 g) were anesthetized with
haloethane and subjected to middle cerebral artery (MCA) occlusion
using a nylon filament for 2 h. Subsequently, the filament was
removed and reperfusion of the infarcted tissue occurred for 24
hours before the rat was sacrificed.
[0079] Immediately after the filament was withdrawn, the animals
were evaluated using a neurological scoring system. Neurological
scores were expressed on a scale from 0 to 10, with 0 representing
no neurological deficit and 10 representing severe neurological
deficit. After 24 hours and before sacrifice, animals were
evaluated a second time using the same neurological scoring
system.
[0080] Staining of coronal sections (2.0 mm.times.7-8) with
triphenyltetrazolium chloride (TTC) taken throughout the brain were
evaluated under blinded conditions using image analysis to
determine infarct size.
[0081] Dosing Regimen
[0082] Rats were given iv bolus injections (1.0 mL/kg) of either
vehicle (50% propylene glycol/saline; n=8) or
7-n-propyl-clasto-lactacystin .beta.-lactone (0.3 mg/kg; n=7) at 2
hours after the start of the occlusion. Two additional groups of
rats were given iv bolus injections (1.0 mL/kg) of
7-n-propyl-clasto-lactacystin .beta.-lactone at 0 minutes, 2 hours,
and 6 hours after the start of the occlusion. One group (0.1
mg/kg.times.3; n=6) received 0.1 mg/kg at each of these times,
while another group (0.3 mg/kg.times.3; n=7) received 0.3 mg/kg at
each of the three timepoints.
[0083] Results
[0084] In animals treated with a single dose of
7-n-propyl-clasto-lactacys- tin .beta.-lactone, infarct volume was
decreased by 50% (FIG. 3). Infarct volume was not significantly
decreased in either the 0.1 mg/kg.times.3 dosage group or the 0.3
mg/kg.times.3 dosage group (FIG. 7).
[0085] All animals had a neurological score of 10.+-.0 immediately
after the 2 hour ischemic episode. At 24 hours, the vehicle-treated
rats had a mean score of 8.7.+-.0.6, whereas rats treated with a
single 0.3 mg/kg dose of 7-n-propyl-clasto-lactacystin
.beta.-lactone had a mean score of 4.+-.1 (FIG. 8). These data
represent a 60% neurological improvement for the drug-treated
animals. No significant improvement in neurological score was
observed in either the 0.1 mg/kg.times.3 dosage group or the 0.3
mg/kg.times.3 dosage group (FIG. 8).
[0086] Conclusion
[0087] 7-n-propyl-clasto-lactacystin .beta.-lactone provides
significant protection in both the degree of neurological deficit
and infarcted brain damage.
[0088] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention, which is defined by the following
claims.
* * * * *