U.S. patent application number 13/178821 was filed with the patent office on 2012-01-26 for dag-type and indirect protein kinase c activators and anticoagulant for the treatment of stroke.
Invention is credited to Daniel L. Alkon.
Application Number | 20120020948 13/178821 |
Document ID | / |
Family ID | 44359547 |
Filed Date | 2012-01-26 |
United States Patent
Application |
20120020948 |
Kind Code |
A1 |
Alkon; Daniel L. |
January 26, 2012 |
DAG-TYPE AND INDIRECT PROTEIN KINASE C ACTIVATORS AND ANTICOAGULANT
FOR THE TREATMENT OF STROKE
Abstract
The present disclosure provides a method for treating stroke by
administering an anticoagulant, e.g., recombinant tissue
plasminogen activator (rTPA), and a protein kinase C (PKC)
activator, wherein the PKC activator may be administered before,
after, or at the same time as the rTPA. The methods disclosed
herein may limit the size of infarction and/or reduce mortality,
the disruption of the blood-brain barrier, and/or the hemorrhagic
damage due to ischemic stroke compared with rTPA administration
alone; and may also extend the therapeutic time window for
administering rTPA after a stroke. Also disclosed are compositions
and kits comprising rTPA and a PKC activator for treating
stroke.
Inventors: |
Alkon; Daniel L.; (Bethesda,
MD) |
Family ID: |
44359547 |
Appl. No.: |
13/178821 |
Filed: |
July 8, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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61362464 |
Jul 8, 2010 |
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61412753 |
Nov 11, 2010 |
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61412747 |
Nov 11, 2010 |
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Current U.S.
Class: |
424/94.64 ;
514/450 |
Current CPC
Class: |
A61P 9/10 20180101; A61P
25/28 20180101; C12Y 304/21068 20130101; A61P 9/00 20180101; A61K
9/0019 20130101; A61K 31/20 20130101; A61P 25/00 20180101; A61P
43/00 20180101; A61K 38/49 20130101; A61P 7/02 20180101; A61K
31/366 20130101; A61K 31/20 20130101; A61K 2300/00 20130101; A61K
31/366 20130101; A61K 2300/00 20130101; A61K 38/49 20130101; A61K
2300/00 20130101 |
Class at
Publication: |
424/94.64 ;
514/450 |
International
Class: |
A61K 38/49 20060101
A61K038/49; A61P 9/10 20060101 A61P009/10; A61P 7/02 20060101
A61P007/02; A61K 31/365 20060101 A61K031/365 |
Claims
1. A method of treating a subject who has suffered an ischemic
event comprising administering to the subject an anticoagulant and
a protein kinase C (PKC) activator.
2. The method of claim 1, wherein the anticoagulant is tissue
plasminogen activator (TPA).
3. The method of claim 1, wherein the PKC activator binds to the
1,2-diacylglycerol (DAG) site of PKC or indirectly activates
PKC.
4. The method of claim 1, wherein the PKC activator is chosen from
macrocyclic lactones, diacylglycerol derivatives other than phorbol
esters, isoprenoids, daphnane-type diterpenes, bicyclic
triterpenoids, naphthalenesulfonamides, diacylglycerol kinase
inhibitors, and growth factor activators.
5. The method of claim 4, wherein the PKC activator is a
macrocyclic lactone.
6. The method of claim 5, wherein the macrocyclic lactone is chosen
from bryostatin, bryologs, and neristatin.
7. The method of claim 6, wherein the bryostatin is
bryostatin-1.
8. The method of claim 1, wherein the anticoagulant is administered
before the PKC activator.
9. The method of claim 8, wherein the anticoagulant is administered
within 24 hours after the ischemic event.
10. The method of claim 9, wherein the anticoagulant is
administered from about 1 hour to about 12 hours after the ischemic
event.
11. The method of claim 10, wherein the anticoagulant is
administered from about 2 hours to about 6 hours after the ischemic
event.
12. The method of claim 8, wherein the PKC activator is
administered within 24 hours after administration of the
anticoagulant.
13. The method of claim 12, wherein the PKC activator is
administered from about 1 hour to about 12 hours after
administration of the anticoagulant.
14. The method of claim 13, wherein the PKC activator is
administered from about 2 hours to about 6 hours after the
anticoagulant.
15. The method of claim 8, wherein the anticoagulant is
administered within about 6 hours after the ischemic event and the
PKC activator is administered within about 2 hours after the
anticoagulant.
16. The method of claim 15, wherein the anticoagulant is
administered about 3 hours after the ischemic event and the PKC
activator is administered about 2 hours after the
anticoagulant.
17. The method of claim 1, wherein the PKC activator is
administered before the anticoagulant.
18. The method of claim 17, wherein the PKC activator is
administered within 24 hours after the ischemic event.
19. The method of claim 18, wherein the PKC activator is
administered from about 1 hour to about 12 hours after the ischemic
event.
20. The method of claim 19, wherein the PKC activator is
administered from about 2 hours to about 6 hours after the ischemic
event.
21. The method of claim 17, wherein the anticoagulant is
administered within 24 hours after administration of the PKC
activator.
22. The method of claim 21, wherein the anticoagulant is
administered from about 1 hour to about 12 hours after
administration of the PKC activator.
23. The method of claim 22, wherein the anticoagulant is
administered from about 2 hours to about 6 hours after
administration of the PKC activator.
24. The method of claim 17, wherein the PKC activator is
administered within about 6 hours after the ischemic event and the
anticoagulant is administered within about 2 hours after
administration of the PKC activator.
25. The method of claim 24, wherein the PKC activator is
administered about 3 hours after the ischemic event and the
anticoagulant is administered about 2 hours after the PKC
activator.
26. The method of claim 1, wherein mortality is reduced with
respect to administration of the anticoagulant alone.
27. The method of claim 26, wherein mortality 24 hours after the
stroke is reduced by at least 40%.
28. The method of claim 1, wherein hemorrhagic transformation is
reduced compared to administration of the anticoagulant alone.
29. The method of claim 28, wherein the reduction in hemorrhagic
transformation is determined by measuring a reduction in the
subject's hemoglobin level.
30. The method of claim 29, wherein the hemoglobin level is reduced
by about 50%.
31. The method of claim 1, wherein disruption of the blood-brain
barrier is reduced compared to administration of the anticoagulant
alone.
32. A composition comprising a therapeutically effective amount of
a protein kinase C (PKC) activator and a therapeutically effective
amount of an anticoagulant.
33. The composition of claim 32, wherein the anticoagulant is
tissue plasmogin activator (TPA).
34. The composition of claim 32, wherein the PKC activator binds to
the 1,2-diacylglycerol (DAG) site of PKC or indirectly activates
PKC.
35. The composition of claim 32, wherein the PKC activator is
chosen from macrocyclic lactones, diacylglycerol derivatives other
than phorbol esters, isoprenoids, daphnane-type diterpenes,
bicyclic triterpenoids, naphthalenesulfonamides, diacylglycerol
kinase inhibitors, and growth factor activators.
36. The composition of claim 35, wherein the PKC activator is a
macrocyclic lactone.
37. The composition of claim 36, wherein the macrocyclic lactone is
chosen from bryostatin, bryologs, and neristatin.
38. The composition of claim 37, wherein the bryostatin is
bryostatin-1.
39. A kit comprising a composition comprising an anticoagulant and
a composition comprising a protein kinase C (PKC) activator.
40. The kit of claim 39, wherein the anticoagulant is tissue
plasmogin activator (TPA).
41. The kit of claim 39, wherein the PKC activator binds to the
1,2-diacylglycerol (DAG) site of PKC or indirectly activates
PKC.
42. The kit of claim 39, wherein the PKC activator is chosen from
macrocyclic lactones, diacylglycerol derivatives other than phorbol
esters, isoprenoids, daphnane-type diterpenes, bicyclic
triterpenoids, naphthalenesulfonamides, diacylglycerol kinase
inhibitors, and growth factor activators.
43. The kit of claim 42, wherein the PKC activator is a macrocyclic
lactone.
44. The kit of claim 43, wherein the macrocyclic lactone is chosen
from bryostatin, bryologs, and neristatin.
45. The kit of claim 44, wherein the bryostatin is
bryostatin-1.
46. The kit of claim 39,wherein the PKC activator and the
anticoagulant are formulated together.
47. The kit of claim 39, wherein the PKC activator and the
anticoagulant are formulated separately.
48. The kit of claim 39, wherein the anticoagulant composition is
formulated for intravenous administration.
49. The kit of claim 48, wherein the anticoagulant composition and
the PKC activator composition are both formulated for intravenous
administration.
50. A method of treating stroke in a subject in need thereof
comprising: (a) identifying a subject having suffered a stroke; (b)
administering to the subject a therapeutically-effective amount of
a protein kinase C (PKC) activator; (c) determining whether the
subject suffered an ischemic stroke or hemorrhagic stroke; and (d)
if the subject suffered an ischemic stroke, administering a
therapeutically-effective amount of an anticoagulant.
51. The method of claim 50, wherein step (c) comprises taking a
computed tomography (CT) scan.
Description
[0001] This application claims priority to U.S. Provisional
Application Nos. 61/362,464 filed Jul. 8, 2010, 61/412,753 filed
Nov. 11, 2010, and 61/412,747 filed Nov. 11, 2010, the entire
disclosures of which are incorporated by reference herein.
[0002] The present disclosure relates generally to administration
of an anticoagulant, e.g., recombinant tissue plasminogen activator
(rTPA), and a protein kinase C (PKC) activator to treat a subject
following ischemic stroke. The methods disclosed herein may limit
the size of infarction and/or reduce mortality, the distruption of
the blood-brain barrier, and/or the hemorrhagic damage due to
ischemic stroke compared with rTPA administration alone. The
methods disclosed herein may also extend the therapeutic window in
which rTPA can be administered following a stroke and still be
efficacious. Compositions and kits comprising rTPA and a PKC
activator are also disclosed.
[0003] Stroke
[0004] Stroke, also known as a cerebrovascular accident (CVA), is a
medical emergency and can cause permanent neurologic damage or even
death if not promptly diagnosed and treated. It is the third
leading cause of death and the leading cause of adult disability in
the United States and industrialized European nations. On average,
a stroke occurs every 45 seconds and someone dies every 3 minutes.
Of every 5 deaths from stroke, 2 occur in men and 3 in women.
[0005] A stroke is an acute neurological injury in which the blood
supply to a part of the brain is interrupted, leading to the sudden
loss of neuronal function. The blood supply to the brain may be
interrupted in several ways; the disturbance in perfusion is
commonly arterial, but may be venous.
[0006] Different types of stroke include ischemic stroke and
hemorrhagic stroke. Ischemic stroke or cerebral ischemia is caused
by a temporary or permanent restriction of cerebral blood flow and
oxygen supply caused by, for example, an embolis (embolic stroke)
or blood clot (thrombolyic stroke). In contrast, a hemorrhagic
stroke is caused by the blood vessel rupture (e.g., ruptured
aneurysm), which leads to severe bleeding in the brain.
[0007] In stroke, the part of the brain with disturbed perfusion no
longer receives adequate oxygen (hypoxia). This initiates an
ischemic cascade causing brain cells to die or be seriously
damaged, thereby impairing local brain function. A transient
ischemic attack (TIA) or "mini-stroke" normally lasts less than 24
hours, but is associated with the same symptoms as stroke such as
sudden numbness or weakness of the face, arm, or leg; sudden
confusion, trouble speaking or understanding; sudden trouble seeing
in one or both eyes; and/or sudden trouble walking, dizziness, loss
of balance or coordination. Typically, TIAs do not result in
permanent brain injury through acute infarction (i.e., tissue
death) but they may indicate serious risk of subsequent stroke. An
infarctive stroke typically involves a more severe vessel blockage
that can last longer than 24 hours without intervention. Cerebral
infarctions vary in severity; about one third of the cases result
in death.
[0008] Ischemia may be confined to a specific region of the brain
(focal ischemia), or may affect large areas of brain tissue (global
ischemia). Significant brain injury can occur after the immediate
ischemic event. Neuronal death and injury after cerebral ischemia
involve pathological changes associated with necrosis and delayed
apoptosis. Neurons in the infarction core of focal, severe stroke
are immediately dead and cannot be saved by pharmacologic
intervention. The ischemic penumbra, consisting of the brain tissue
around the core in focal ischemic stroke, and the sensitive
neurons/network in global cerebral ischemia, however, are
maintained by a diminished blood supply. The damage to this
penumbral brain tissue occurs in a "delayed" manner, starting 4-6
hours as the second phase or days and weeks later as the so-called
third phase, after ischemic stroke.
[0009] A consistent consequence of cerebral ischemia/hypoxia in
humans and other mammals is central nervous system dysfunction, the
nature of which depends on the location and extent of injury.
Global cerebral ischemia/hypoxia selectively injures or damages the
pyramidal neurons in the dorsal hippocampal CA1 area, which are
essential for episodic memory, providing a sensitive measure for
monitoring ischemic damage and recovery functionally. After a
cerebral ischemia of about 15 minutes, for example, the hippocampal
CA1 pyramidal cells start to degenerate within 2-3 days, and reach
the maximal extent of cell death a week after the ischemic event.
The sensitive neuronal structures in global cerebral ischemia and
the ischemic penumbra are "at-risk" tissues. They can be salvage
through intervention and further damage limited in the subsequent
days or weeks thereafter, which determine dramatic differences in
long-term disability.
[0010] Following ischemic stroke, there is a transient loss of
blood-brain barrier (BBB) function that happens within minutes or
hours of the event as the interruption in blood flow and lack of
oxygen leads to increased BBB permeability. DiNapoli et al.,
Neurobiology of Aging (2008) vol. 29, pp. 753-764. Disruption of
the BBB, in turn, results in loss of ionic homeostasis and loss of
neurotransmitter homeostasis. Immune cells and toxic compounds can
enter the brain during that period, providing an added neurotoxic
insult. Edema can form during the early stages of ischemia with a
rate related to the rate of sodium transport from blood to brain,
i.e., increased sodium transport across the BBB contributes to
cerebral edema formation. Betz and Coester, Stroke (1990), vol. 21,
pp. 1199-1204. Thus, measurements of both edema and ion uptake in
the brain are indicators of brain pathology following stroke. The
loss of the integrity of the barrier may lead to adverse
hemorrhages as a consequence of thrombolytic therapy, e.g.,
administration of recombinant tissue plasminogen activator (rTPA).
Tanne et al., Nature Reviews Neurology (2008), vol. 4, pp.
644-645.
[0011] Despite the medical emergency presented by stroke, and
preclinical studies suggesting agents that may be effective in
arresting the pathological processes involved, options for treating
stroke remain limited. The main treatment available is rTPA, a
thrombolytic agent and the only drug currently approved by the U.S.
Food and Drug Administration for acute/urgent treatment of ischemic
stroke. The rTPA protein is an enzyme (serine protease) that
initiates local fibrinolysis via fibrin-enhanced conversion of
plasminogen to plasmin. rTPA is used to improve neurologic recovery
and reduce the incidence of disability. Experimental models of
stroke use rTPA, for example, in reperfusion after inducing focal
embolic ischemia via middle cerebral artery occlusion (MCAO).
DiNapoli et al., J. Neurosci Methods (2006), vol. 154, pp.
233-238.
[0012] The effectiveness of rTPA and other potential agents for
arresting infarct development depends on early administration or
even before the ischemic event, if possible. Treatment with rTPA is
designed to achieve early arterial recanalization such that rTPA
must be administered within 3 hours after the event to be
effective. This time dependency limits its clinical usefulness; the
narrow therapeutic time window and exclusion criteria in treating
ischemic stroke leads to about only 5% of candidate patients
receiving effective intravenous thrombolytic therapy. For example,
one study reported 13% mortality at 30 days after an acute ischemic
stroke, with more than two thirds of the deaths related to the
initial stroke. Nedeltcheva et al., Swiss Med. Wkly (2010), vol.
140, pp. 254-259. The recommended dose of rTPA is 0.9 mg/kg
(maximum dose 90 mg) where 10% is given by rapid (.about.1 min.) IV
injection and the remainder by constant infusion over 60 min. No
aspirin, heparin, or warfarin should be administered for 24 hours
following rTPA. rTPA is sold under the names alteplase
(Activase.RTM.) and streptokinase (Streptase.RTM.).
[0013] Use of rTPA following stroke is controversial because it
carries an increased risk of intracranial hemorrhage, reperfusion
injury, and diminishing cerebral artery reactivity. Thus, rTPA is
should not be administered to treat hemorrhagic stroke.
Unfortunately, it may not be immediately apparent whether a patient
suffered an ischemic or hemorrhagic stroke, which further limits
the usefulness of rTPA within its limited therapeutic time window.
In addition, hemorrhagic transformation can spontaneously follow
ischemic stroke. For example, one study found that 6.4% of patients
with large strokes developed substantial brain hemorrhage as a
complication from being given rTPA. The National Institute of
Neurological Disorders and Stroke rt-PA Stroke Study Group, N.
Engl. J. Med. (1995), vol. 333, pp. 1581-1587.
[0014] rTPA is contraindicated or advised against in the following
patient populations: [0015] Evidence of intracranial hemorrhage on
pretreatment CT scan [0016] Clinical presentation suggestive of
subarachnoid hemorrhage, even with normal CT scan [0017] Active
internal bleeding [0018] Known bleeding diathesis, including but
not limited to: having a platelet count <100,000/mm; receiving
heparin within 48 hours and having an elevated activated partial
thromboplastin (aPTT) greater than upper limit of normal for
laboratory; and current use of oral anticoagulants (e.g., warfarin
sodium) or recent use with an elevated prothrombin time>15
seconds [0019] Within 3 months any intracranial surgery, serious
head trauma, or previous stroke [0020] History of gastrointestinal
or urinary tract hemorrhage within 21 days [0021] Recent arterial
puncture at a noncompressible site [0022] Recent lumbar puncture
[0023] On repeated measurements, systolic blood pressure greater
than 185 mm Hg or diastolic blood pressure greater than 110 mm Hg
at the time treatment is to begin, and patients requiring
aggressive treatment to reduce blood pressure to within these
limits. [0024] History of intracranial hemorrhage [0025] Abnormal
blood glucose (<50 mg/dL or >400 mg/dL) [0026] Post
myocardial infarction pericarditis [0027] Patient observed to have
seizure at the same time the onset of stroke symptoms were observed
[0028] Known arteriovenous malformation, or aneurysm See, e.g., TPA
Stroke Study Group Guidelines, The Brian Attack Coalition
(available at
http://www.stroke-site.org/guidelines/tpa_guidelines.html).
[0029] Studies suggest an association between hematocrit, reduced
reperfusion and greater infarct size, and between elevated
hemoglobin levels and increased rates of all-cause death. Tanne et
al., BMC Neurology (2010), vol. 10:22, pp. 1-7. Elevated levels of
glycated hemoglobin (HbA1c) increases the risk of heart attacks and
strokes in diabetic patients. Glycated hemoglobin, even at levels
considered in the normal range, can also be an independent
predictor of ischemic stroke in non-diabetic adults. Selvin et al.,
N. Engl. J. Med. (2010), vol. 362, pp. 800-811. Elevated hemoglobin
may also increase the risk of stroke in patients with chronic
kidney disease.
[0030] Low hemoglobin levels (e.g., levels >6.0% or 8.8 g/dL,
anemia) have also been identified as a risk factor for ischemic
stroke, especially following cardiac surgery. In addition, anemia
can worsen brain ischemia following acute ischemic stroke, and is
associated with a poor prognosis and increased mortality after one
year compared with non-anemic stroke patients (hemoglobin <13
g/dL in males, <12 g/dL in women). Tanne et al., BMC Neurology
(2010), 10:22. Studies have also reported that children with sickle
cell anemia have an increased stroke risk.
[0031] Protein Kinase C
[0032] Protein kinase C (PKC) is one of the largest gene families
of non-receptor serine-threonine protein kinases. Since the
discovery of PKC in the early eighties and its identification as a
major receptor for phorbol esters, a multitude of physiological
signaling mechanisms have been ascribed to this enzyme. Kikkawa et
al., J. Biol. Chem. (1982), vol. 257, pp. 13341-13348; Ashendel et
al., Cancer Res. (1983), vol. 43: 4333-4337. The interest in PKC
stems from its unique ability to be activated in vitro by calcium
and diacylglycerol (and phorbol ester mimetics), an effector whose
formation is coupled to phospholipid turnover by the action of
growth and differentiation factors. Activation of PKC involves
binding of 1,2-diacylglycerol (DAG) and/or
1,2-diacyl-sn-glycero-3-phospho-L-serine (phosphatidyl-L-serine,
PS) at different binding sites. An alternative approach to
activating PKC directly is through indirect PKC activation, e.g.,
by activating phospholipases such as phospholipase Cy, by
stimulating the Ser/Thr kinase Akt by way of phosphatidylinositol
3-kinase (P13K), or by increasing the levels of DAG, the endogenous
activator. Nelson et al., Trends in Biochem. Sci. (2009) vol. 34,
pp. 136-145. Diacylglycerol kinase inhibitors, for example, may
enhance the levels of the endogenous ligand diacylglycerol, thereby
producing activation of PKC. Meinhardt et al., Anti-Cancer Drugs
(2002), vol. 13, pp. 725-733. Phorbol esters are not suitable
compounds for eventual drug development because of their tumor
promotion activity. Ibarreta et al. Neuroreport (1999), vol. 10,
pp. 1035-1040).
[0033] The PKC gene family consists of 11 genes, which are divided
into four subgroups: (1) classical PKC .alpha., .beta.1, .beta.2
((.beta.1 and .beta.2 are alternatively spliced forms of the same
gene) and .gamma.; (2) novel PKC .delta., .epsilon., .eta., and
.theta.; (3) atypical PKC .zeta. and /.lamda.; and (4) PKC .mu..
PKC .mu. resembles the novel PKC isoforms but differs by having a
putative transmembrane domain. Blobe et al. Cancer Metastasis Rev.
(1994), vol. 13, pp. 411-431; Hug et al. Biochem. J. (1993) vol.
291, pp. 329-343; Kikkawa et al. Ann. Rev. Biochem. (1989), vol.
58, pp. 31-44. The classical PKC isoforms .alpha., .beta.1,
.beta.2, and .gamma. are Ca.sup.2+, phospholipid, and
diacylglycerol-dependent, whereas the other isoforms are activated
by phospholipid, diacylglycerol but are not dependent on Ca.sup.2+
and no activator may be necessary. All isoforms encompass 5
variable (V1-V5) regions, and the .alpha.,.beta., and .gamma.
isoforms contain four (C1-C4) structural domains which are highly
conserved. All isoforms except PKC .alpha., .beta., and .gamma.
lack the C2 domain, the /.lamda. and .eta. isoforms also lack nine
of two cysteine-rich zinc finger domains in C1 to which
diacylglycerol binds. The C1 domain also contains the
pseudosubstrate sequence which is highly conserved among all
isoforms, and which serves an autoregulatory function by blocking
the substrate-binding site to produce an inactive conformation of
the enzyme. House et al., Science (1987), vol. 238, pp.
1726-1728.
[0034] Because of these structural features, diverse PKC isoforms
are thought to have highly specialized roles in signal transduction
in response to physiological stimuli as well as in neoplastic
transformation and differentiation. Nishizuka, Cancer (1989), vol.
10, pp. 1892-1903; Glazer, pp. 171-198 in Protein Kinase C, I. F.
Kuo, ed., Oxford U. Press, 1994. For a discussion of PKC modulators
see, for example, International Application No. PCT/US97/08141 (WO
97/43268) and U.S. Pat. Nos. 5,652,232; 6,080,784; 5,891,906;
5,962,498; 5,955,501; 5,891,870 and 5,962,504, each incorporated by
reference herein in its entirety.
[0035] The activation of PKC has been shown to improve learning and
memory. See, e.g., Hongpaisan et al., Proc. Natl. Acad. Sci. (2007)
vol. 104, pp. 19571-19578; International Application Nos.
PCT/US2003/007101 (WO 2003/075850); PCT/US2003/020820 (WO
2004/004641); PCT/US2005/028522 (WO 2006/031337); PCT/US2006/029110
(WO 2007/016202); PCT/US2007/002454 (WO 2008/013573);
PCT/US2008/001755 (WO 2008/100449); PCT/US2008/006158 (WO
2008/143880); PCT/US2009/051927 (WO 2010/014585); and
PCT/US2011/000315; and U.S. application Ser. Nos. 12/068,732;
10/167,491 (now U.S. Pat. No. 6,825,229); 12/851,222; 11/802,723;
12/068,742; and 12/510,681; each incorporated by reference herein
in its entirety. PKC activators have been used to treat memory and
learning deficits induced by stroke upon administration 24 hours or
more after inducing global cerebral ischemia through two-vessel
occlusion combined with a short term (.about.14 minutes) systemic
hypoxia. Sun et al., Proc. Natl. Acad. Sci. (2008) vol. 105, pp.
13620-13625; Sun et al., Proc. Natl. Acad. Sci. (2009) vol. 106,
pp. 14676-14680.
[0036] The present disclosure relates to a method of treating
stroke in a subject who has suffered an ischemic event comprising
administering to the subject an anticoagulant and a protein kinase
C (PKC) activator.
[0037] The present disclosure further provides for a composition
comprising a therapeutically effective amount of a protein kinase C
(PKC) activator and a therapeutically effective amount of an
anticoagulant.
[0038] The present disclosure further provides for a kit comprising
a composition comprising an anticoagulant and a composition
comprising a protein kinase C (PKC) activator.
[0039] Also disclosed herein is a method of treating stroke in a
subject comprising: (a) identifying a subject having suffered a
stroke; (b) administering to the subject a
therapeutically-effective amount of a protein kinase C (PKC)
activator; (c) determining whether the subject suffered an ischemic
stroke or hemorrhagic stroke; and (d) if the subject suffered an
ischemic stroke, administering a therapeutically-effective amount
of an anticoagulant.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 shows a the amount of hemoglobin the ipsilateral and
contralateral cortices following ischemic stroke in rats treated
with either rTPA at 6 hours following stroke, or a combination of
bryostatin-1 administered 2 hours after the stroke, followed 6
hours later by rTPA.
[0041] FIG. 2 shows a the percent of brain edema following ischemic
stroke in rats treated with either rTPA at 6 hours following the
stroke, or a combination of bryostatin-1 administered 2 hours after
the stroke, followed 6 hours later by rTPA.
[0042] FIG. 3 shows the results of uptake of Evans Blue dye in the
ipsilateral and contralateral cortices in rats treated with rTPA 2
hours following the stroke, or a combination of rTPA at 2 hours
followed 6 hours later with bryostatin-1.
[0043] FIG. 4 shows the results of sodium fluoride uptake in the
ipsilateral and contralateral cortices in rats treated with rTPA 2
hours following the stroke, or a combination of rTPA at 2 hours
followed 6 hours later with bryostatin-1.
DETAILED DESCRIPTION
[0044] Particular aspects of the disclosure are described in
greater detail below. The terms and definitions as used in the
present application and as clarified herein are intended to
represent the meaning within the present disclosure. The patent and
scientific literature referred to herein is hereby incorporated by
reference. The terms and definitions provided herein control, if in
conflict with terms and/or definitions incorporated by
reference.
[0045] The singular forms "a," "an," and "the" include plural
reference unless the context dictates otherwise.
[0046] The terms "approximately" and "about" mean to be nearly the
same as a referenced number or value including an acceptable degree
of error for the quantity measured given the nature or precision of
the measurements. As used herein, the terms "approximately" and
"about" should be generally understood to encompass .+-.20% of a
specified amount, frequency or value. Numerical quantities given
herein are approximate unless stated otherwise, meaning that term
"about" or "approximately" can be inferred when not expressly
stated.
[0047] The terms "administer," "administration," or "administering"
as used herein refer to (1) providing, giving, dosing and/or
prescribing by either a health practitioner or his authorized agent
or under his direction a composition according to the disclosure,
and (2) putting into, taking or consuming by the patient or person
himself or herself, a composition according to the disclosure. As
used herein, "administration" of a composition includes any route
of administration, including oral, intravenous, subcutaneous,
intraperitoneal, and intramuscular.
[0048] As used herein, the term "subject" means a mammal, i.e., a
human or a non-human mammal.
[0049] The phrase "a therapeutically effective amount" refers to an
amount of a therapeutic agent that results in a measurable
therapeutic response. A therapeutic response may be any response
that a user (e.g., a clinician) will recognize as an effective
response to the therapy, including improvement of symptoms and
surrogate clinical markers. Thus, a therapeutic response will
generally be an amelioration or inhibition of one or more symptoms
of a disease or condition, e.g., stroke. A measurable therapeutic
response also includes a finding that a symptom or disease is
prevented or has a delayed onset, or is otherwise attenuated by the
therapeutic agent. thus, a "therapeutically effective amount" as
used herein refers to an amount sufficient to reduce one or more
symptom(s) or condition(s) associated with stroke including but not
limited to hemorrhagic transformation, disruption of the
blood-brain barrier, increase in hemoglobin levels, and
mortality.
[0050] As used herein, "protein kinase C activator" or "PKC
activator" means a substance that increases the rate of the
reaction catalyzed by protein kinase C by binding to the protein
kinase C.
[0051] As used herein "macrocyclic lactone" refers to a compound
comprising a macrolide ring, i.e., a large macrocyclic lactone ring
to which one or more deoxy sugars may be attached.
[0052] The term "neurodegeneration" refers to the progressive loss
of structure or function of neurons, including death of
neurons.
[0053] The term "pharmaceutically acceptable" refers to molecular
entities and compositions that are physiologically tolerable and do
not typically produce untoward reactions when administered to a
subject.
[0054] While the present disclosure generally describes use of
rTPA, other anticoagulants and anticoagulant therapies suitable for
the treatment of stroke are also contemplated. Further, it is
understood that the present disclosure is not limited to a specific
manufactured type of TPA (e.g., rTPA), but includes TPA
generally.
[0055] The present disclosure generally relates to compositions,
kits, and methods of use of an anticoagulant, e.g., rTPA and a PKC
activator to treat stroke. In some embodiments, the administration
of a PKC activator may extend the time that rTPA can be
administered after a stroke (e.g., after an ischemic event) while
still retaining efficacy. Further, the combination of rTPA and a
PKC activator may reduce mortality, reduce hemorrhagic
transformation, and/or reduce disruptions to the blood-brain
barrier (BBB) caused by stroke. In addition, the administration of
rTPA and a PKC activator may reduce the level of assayed
hemoglobin, wherein elevated hemoglobin is a risk factor for
reduced reperfusion, greater infarct size, and/or mortality due to
stroke.
[0056] Sliding Temporal Window
[0057] The present disclosure encompasses "sliding temporal
windows" for administration of a PKC activator and rTPA to a stroke
victim. In the methods presently disclosed, a PKC activator may be
administered before, after, and/or in combination with rTPA. In
some embodiments of the present disclosure, rTPA and a PKC
activator are administered at the same time. Thus, the present
disclosure contemplates "sliding temporal windows" for
administration of a PKC activator and rTPA to a subject. The term
"sliding temporal window" refers to the notion that a PKC activator
and rTPA can be administered in any order to a subject that has
suffered a stroke, at any time relative to one another, and at any
time relative to when the stroke occurred.
[0058] At least four scenarios are contemplated:
[0059] Scenario 1: In some embodiments of the present disclosure, a
PKC activator may be administered to a subject one or more times
within a given time period after having suffered a stroke, followed
by administration of rTPA one or more times after another time
period. The PKC activator may be administered at any time after the
occurrence of a stroke, generally within about 24 hours. For
example, the PKC activator may be administered to a subject about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours,
about 6 hours, about 7 hours, about 8 hours, about 9 hours, about
10 hours, about 11 hours, about 12 hours, about 13 hours, about 14
hours, about 15 hours, about 16 hours, about 17 hours, about 18
hours, about 19 hours, about 20 hours, about 21 hours, about 22
hours, about 23 hours, or about 24 hours after a stroke. The rTPA
may then be administered to the subject after the PKC activator,
e.g., about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 10 hours, about 11 hours, about 12 hours, about 13
hours, about 14 hours, about 15 hours, about 16 hours, about 17
hours, about 18 hours, about 19 hours, about 20 hours, about 21
hours, about 22 hours, about 23 hours, or about 24 hours after
administration of the PKC activator.
[0060] For example, in some embodiments, the PKC activator is
administered within 24 hours after the ischemic event, such as from
about 1 hour to about 12 hours or from about 2 hours to about 6
hours after the ischemic event. rTPA is then administered within 24
hours after administration of the PKC activator, such as from about
1 hour to about 12 hours or from about 2 hours to about 6 hours
after administration of the PKC activator. In one embodiment, the
PKC activator is administered within about 6 hours after the
ischemic event and the rTPA is administered within about 2 hours
after administration of the PKC activator. In another embodiment,
the PKC activator is administered about 3 hours after the ischemic
event and the rTPA is administered about 2 hours after the PKC
activator.
[0061] Scenario 2: In some embodiments, rTPA may be administered to
a subject one or more times within a given time period after having
suffered a stroke, followed by administration of a PKC activator
one or more times after another time period. The rTPA may be
administered at any time after the occurrence of a stroke,
generally within about 24 hours. For example, the rTPA may be
administered to a subject about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about
12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, or about 24
hours after a stroke. The PKC activator may then be administered to
the subject after the rTPA, e.g., about 1 hour, about 2 hours,
about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7
hours, about 8 hours, about 9 hours, about 10 hours, about 11
hours, about 12 hours, about 13 hours, about 14 hours, about 15
hours, about 16 hours, about 17 hours, about 18 hours, about 19
hours, about 20 hours, about 21 hours, about 22 hours, about 23
hours, or about 24 hours after administration of the rTPA.
[0062] For example, in some embodiments, the rTPA is administered
within 24 hours after the ischemic event, such as from about 1 hour
to about 12 hours or from about 2 hours to about 6 hours after the
ischemic event. The PKC activator is then administered within 24
hours after administration of the rTPA, such as from about 1 hour
to about 12 hours or from about 2 hours to about 6 hours after the
rTPA. In one embodiment, rTPA is administered within about 6 hours
after the ischemic event and the PKC activator is administered
within about 2 hours after the rTPA. In another embodiment, rTPA is
administered about 3 hours after the ischemic event and the PKC
activator is administered about 2 hours after the rTPA.
[0063] Scenario 3: In other embodiments of the present disclosure,
a PKC activator may be administered to a subject one or more times
within a given time period after having suffered a stroke, followed
by rTPA one or more times after another time period, and further
followed by administration of a PKC activator one or more times a
period of time later. For example, the PKC activator may be
administered to a subject about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about
12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, or about 24
hours after a stroke. The rTPA may then be administered to the
subject about 1 hour, about 2 hours, about 3 hours, about 4 hours,
about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9
hours, about 10 hours, about 11 hours, about 12 hours, about 13
hours, about 14 hours, about 15 hours, about 16 hours, about 17
hours, about 18 hours, about 19 hours, about 20 hours, about 21
hours, about 22 hours, about 23 hours, or about 24 hours after
administration of the PKC activator. Thereafter, another PKC
activator may be administered about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours,
about 8 hours, about 9 hours, about 10 hours, about 11 hours, about
12 hours, about 13 hours, about 14 hours, about 15 hours, about 16
hours, about 17 hours, about 18 hours, about 19 hours, about 20
hours, about 21 hours, about 22 hours, about 23 hours, or about 24
hours after administration of the rTPA. The PKC activator
administered before and after the rTPA may be the same or
different.
[0064] Similarly, rTPA may be administered to a subject one or more
times within a given time period after having suffered a stroke,
followed by a PKC activator one or more times after another time
period, and further followed by administration of the same or a
different PKC activator one or more times a period of time
later.
[0065] Scenario 4: In yet other embodiments, a PKC activator and
rTPA may be administered at the same time to a subject after
suffering a stroke. This may be done by directly administering a
composition comprising a PKC activator and rTPA, or administering a
composition comprising a PKC activator and a separate composition
comprising rTPA in succession or rapid succession, one after the
other in either order (i.e., the composition comprising a PKC
activator may be administered first or the composition comprising
rTPA may be administered first).
[0066] In some embodiments, the present disclosure provides a
method for extending the therapeutic window for treating ischemic
stroke with rTPA comprising administering a PKC activator before,
after, or at the same time as rTPA. The recommended time period for
administering rTPA (e.g., Activase.RTM.) is about 3 hours. In one
embodiment of the present disclosure, for example, a PKC activator
is administered to a subject about 2 hours after a stroke followed
by administration of rTPA about 6 hours later (i.e., about 8 hours
after the stroke). In another embodiment, rTPA is administered to a
subject about 6 hours after a stroke followed by administration of
a PKC activator about 2 hours later (i.e., about 8 hours after the
stroke).
[0067] At least one embodiment of the present disclosure provides
for treatment of a subject who has suffered a stroke before it is
known whether the subject suffered an ischemic stroke or a
hemorrhagic stroke. For example, the present disclosure provides
for a method of identifying a subject who has suffered a stroke,
administering a therapeutically-effective amount of a PKC
activator, and determining whether the subject suffered an ischemic
stroke or a hemorrhagic stroke. The determination regarding the
type of stroke suffered may be made by any suitable means known in
the medical arts including, for example, a computed tomography (CT)
scan. If the subject suffered an ischemic stroke, a
therapeutically-effective amount of rTPA may be administered. If
the subject suffered a hemorrhagic stroke, however, rTPA is not
administered. Thus, in some embodiments of the present disclosure,
extending the therapeutic time window for treating stroke with rTPA
allows for a determination of whether a subject suffered an
ischemic stroke or a hemorrhagic stroke.
[0068] PKC Activators
[0069] PKC activators suitable for the methods, compositions, and
kits disclosed herein include, for example, macrocyclic lactones,
e.g., bryostatin and neristatin classes, that act to stimulate PKC.
Of the bryostatin class of compounds, bryostatin-1 has been shown
to activate PKC without tumor promotion. Bryostatin-1 may be
particularly useful as a PKC activator because the dose response
curve is biphasic and bryostatin-1 demonstrates differential
regulation of PKC isozymes including PKC.alpha., PKC.delta. and
PKC.epsilon.. Bryostatin-1 has undergone toxicity and safety
studies in animals and humans, and is actively investigated as an
anti-cancer agent.
[0070] Macrocyclic lactones generally comprise 14-, 15-, or
16-membered lactone rings. Macrolides belong to the polyketide
class of natural products. Macrocyclic lactones and derivatives
thereof are described, for example, in U.S. Pat. Nos. 6,187,568;
6,043,270; 5,393,897; 5,072,004; 5,196,447; 4,833,257; and
4,611,066; and 4,560,774; each incorporated by reference herein in
its entirety. Those patents describe various compounds and various
uses for macrocyclic lactones including their use as an
anti-inflammatory or anti-tumor agent. Szallasi et al. J. Biol.
Chem. (1994), vol. 269, pp. 2118-2124; Zhang et al., Cancer Res.
(1996), vol. 56, pp. 802-808; Hennings et al. Carcinogenesis
(1987), vol. 8, pp. 1343-1346; Varterasian et al. Clin. Cancer Res.
(2000), vol. 6, pp. 825-828; Mutter et al. Bioorganic & Med.
Chem. (2000), vol. 8, pp. 1841-1860; each incorporated by reference
herein in its entirety. The bryostatin and neristatin compounds
were originally isolated from the marine bryozoan Bugula neritina
L.
[0071] In one embodiment of the present disclosure, the PKC
activator is a macrocyclic lactone such as a bryostatin or
neristatin. Bryostatins include, for example, bryostatin-1,
bryostatin-2, bryostatin-3, bryostatin-4, bryostatin-5,
bryostatin-6, bryostatin-7, bryostatin-8, bryostatin-9,
bryostatin-10, bryostatin-11, bryostatin-12, bryostatin-13,
bryostatin-14, bryostatin-15, bryostatin-16, bryostatin-17, and
bryostatin-18. In at least one embodiment, the bryostatin is
bryostatin-1. Neristatins suitable for the present disclosure
include, for example, neristatin-1.
[0072] Analogs of bryostatin, commonly referred to as bryologs, are
one particular class of PKC activators that are suitable for use in
the present disclosure. Table 1 summarizes structural
characteristics of several bryologs and demonstrates variability in
their affinity for PKC (ranging from 0.25 nM to 10 .mu.M).
Structurally, they are all similar. While bryostatin-1 has two
pyran rings and one 6-membered cyclic acetal, in most bryologs one
of the pyrans of bryostatin-1 is replaced with a second 6-membered
acetal ring. This modification reduces the stability of bryologs,
relative to bryostatin-1, for example, in both strong acid or base,
but has little significance at physiological pH. Bryologs also have
a lower molecular weight (ranging from about 600 g/mol to 755
g/mol), as compared to bryostatin-1 (988), a property which
facilitates transport across the blood-brain barrier.
TABLE-US-00001 TABLE 1 Bryologs. PKC Affin Name (nM) MW Description
Bryostatin-1 1.35 988 2 pyran + 1 cyclic acetal + macrocycle Analog
1 0.25 737 1 pyran + 2 cyclic acetal + macrocycle Analog 2 6.50 723
1 pyran + 2 cyclic acetal + macrocycle Analog 7a -- 642 1 pyran + 2
cyclic acetals + macrocycle Analog 7b 297 711 1 pyran + 2 cyclic
acetals + macrocycle Analog 7c 3.4 726 1 pyran + 2 cyclic acetals +
macrocycle Analog 7d 10000 745 1 pyran + 2 cyclic acetals +
macrocycle, acetylated Analog 8 8.3 754 2 cyclic acetals +
macrocycle Analog 9 10000 599 2 cyclic acetals
[0073] Analog 1 exhibits the highest affinity for PKC. Wender et
al., Curr. Drug Discov. Technol. (2004), vol. 1, pp. 1-11; Wender
et al. Proc. Natl. Acad. Sci. (1998), vol. 95, pp. 6624-6629;
Wender et al., J. Am. Chem. Soc. (2002), vol. 124, pp. 13648-13649,
each incorporated by reference herein in their entireties. Only
Analog 1 exhibits a higher affinity for PKC than bryostatin. Analog
2, which lacks the A ring of bryostatin-1, is the simplest analog
that maintains high affinity for PKC. In addition to the active
bryologs, Analog 7d, which is acetylated at position 26, has
virtually no affinity for PKC.
##STR00001##
[0074] B-ring bryologs may also be used in the present disclosure.
These synthetic bryologs have affinities in the low nanomolar
range. Wender et al., Org Lett. (2006), vol. 8, pp. 5299-5302,
incorporated by reference herein in its entirety. B-ring bryologs
have the advantage of being completely synthetic, and do not
require purification from a natural source.
##STR00002##
[0075] A third class of suitable bryostatin analogs is the A-ring
bryologs. These bryologs have slightly lower affinity for PKC than
bryostatin-1 (6.5 nM, 2.3 nM, and 1.9 nM for bryologs 3, 4, and 5,
respectively) and a lower molecular weight.
[0076] Bryostatin analogs are described, for example, in U.S. Pat.
Nos. 6,624,189 and 7,256,286.
[0077] A number of derivatives of diacylglycerol (DAG) bind to and
activate PKC. Niedel et al., Proc. Natl. Acad. Sci. (1983), vol.
80, pp. 36-40; Mori et al., J. Biochem. (1982), vol. 91, pp.
427-431; Kaibuchi et al., J. Biol. Chem. (1983), vol. 258, pp.
6701-6704. However, DAG and DAG derivatives are of limited value as
drugs. Activation of PKC by diacylglycerols is transient, because
they are rapidly metabolized by diacylglycerol kinase and lipase.
Bishop et al. J. Biol. Chem. (1986), vol. 261, pp. 6993-7000;
Chuang et al. Am. J. Physiol. (1993), vol. 265, pp. C927-C933;
incorporated by reference herein in their entireties. The fatty
acid substitution determines the strength of activation.
Diacylglycerols having an unsaturated fatty acid are most active.
The stereoisomeric configuration is important; fatty acids with a
1,2-sn configuration are active while 2,3-sn-diacylglycerols and
1,3-diacylglycerols do not bind to PKC. Cis-unsaturated fatty acids
may be synergistic with diacylglycerols. In at least one
embodiment, the term "PKC activator" expressly excludes DAG or DAG
derivatives.
[0078] Isoprenoids are PKC activators also suitable for the present
disclosure. Farnesyl thiotriazole, for example, is a synthetic
isoprenoid that activates PKC with a K.sub.d of 2.5 .mu.M. Farnesyl
thiotriazole, for example, is equipotent with dioleoylglycerol, but
does not possess hydrolyzable esters of fatty acids. Gilbert et
al., Biochemistry (1995), vol. 34, pp. 3916-3920; incorporated by
reference herein in its entirety. Farnesyl thiotriazole and related
compounds represent a stable, persistent PKC activator. Because of
its low molecular weight (305.5 g/mol) and absence of charged
groups, farnesyl thiotriazole would be expected to readily cross
the blood-brain barrier.
##STR00003##
[0079] Octylindolactam V is a non-phorbol protein kinase C
activator related to teleocidin. The advantages of octylindolactam
V (specifically the (-)-enantiomer) include greater metabolic
stability, high potency (EC.sub.50=29 nM) and low molecular weight
that facilitates transport across the blood brain barrier. Fujiki
et al. Adv. Cancer Res. (1987), vol. 49 pp. 223-264; Collins et al.
Biochem. Biophys. Res. Commun. (1982), vol. 104, pp. 1159-4166,
each incorporated by reference herein in its entirety.
##STR00004##
[0080] Gnidimacrin is a daphnane-type diterpene that displays
potent antitumor activity at concentrations of 0.1 nM-1 nM against
murine leukemias and solid tumors. It acts as a PKC activator at a
concentration of 0.3 nM in K562 cells, and regulates cell cycle
progression at the G1/S phase through the suppression of Cdc25A and
subsequent inhibition of cyclin dependent kinase 2 (Cdk2) (100%
inhibition achieved at 5 ng/ml). Gnidimacrin is a heterocyclic
natural product similar to bryostatin, but somewhat smaller
(MW=774.9 g/mol).
[0081] Iripallidal is a bicyclic triterpenoid isolated from Iris
pallida. Iripallidal displays anti-proliferative activity in a NCI
60 cell line screen with GI.sub.50 (concentration required to
inhibit growth by 50%) values from micromolar to nanomolar range.
It binds to PKCa with high affinity (K.sub.i=75.6 nM). It induces
phosphorylation of Erk1/2 in a RasGRP3-dependent manner. Its
molecular weight is 486.7 g/mol. Iripallidal is about half the size
of bryostatin and lacks charged groups.
##STR00005##
[0082] Ingenol is a diterpenoid related to phorbol but less toxic.
It is derived from the milkweed plant Euphorbia peplus. Ingenol
3,20-dibenzoate, for example, competes with [3H] phorbol dibutyrate
for binding to PKC (K.sub.i=240 nM). Winkler et al., J. Org. Chem.
(1995), vol. 60, pp. 1381-1390, incorporated by reference herein.
Ingenol-3-angelate exhibits antitumor activity against squamous
cell carcinoma and melanoma when used topically. Ogbourne et al.
Anticancer Drugs (2007), vol. 18, pp. 357-362, incorporated by
reference herein.
##STR00006##
[0083] Napthalenesulfonamides, including
N-(n-heptyl)-5-chloro-1-naphthalenesulfonamide (SC-10) and
N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, are members of
another class of PKC activators. SC-10 activates PKC in a
calcium-dependent manner, using a mechanism similar to that of
phosphatidylserine. Ito et al., Biochemistry (1986), vol. 25, pp.
4179-4184, incorporated by reference herein.
Naphthalenesulfonamides act by a different mechanism than
bryostatin and may show a synergistic effect with bryostatin or
member of another class of PKC activators. Structurally,
naphthalenesulfonamides are similar to the calmodulin (CaM)
antagonist W-7, but are reported to have no effect on CaM
kinase.
[0084] Diacylglycerol kinase inhibitors may also be suitable as PKC
activators in the present disclosure by indirectly activating PKC.
Examples of diacylglycerol kinase inhibitors include, but are not
limited to,
6-(2-(4-[(4-fluorophenyl)phenylmethylene]-1-piperidinyl)ethyl)-7-meth-
yl-5H-thiazolo[3,2-a]pyrimidin-5-one (R59022) and
[34244-(bis-(4-fluorophenyl)methylene]piperidin-1-yl)ethyl]-2,3-dihydro-2-
-thioxo-4(1H)-quinazolinone (R59949).
[0085] A variety of growth factors, such as fibroblast growth
factor 18 (FGF-18) and insulin growth factor, function through the
PKC pathway. FGF-18 expression is up-regulated in learning, and
receptors for insulin growth factor have been implicated in
learning. Activation of the PKC signaling pathway by these or other
growth factors offers an additional potential means of activating
PKC.
[0086] Growth factor activators, including 4-methyl catechol
derivatives like 4-methylcatechol acetic acid (MCBA) that stimulate
the synthesis and/or activation of growth factors such as NGF and
BDNF, also activate PKC as well as convergent pathways responsible
for synaptogenesis and/or neuritic branching.
[0087] In some embodiments of the present disclosure, administering
a PKC activator and rTPA may reduce mortality in a subject 24 hours
after stroke. For example, mortality after 24 hours may be reduced
by at least 20%, at least 30%, at least 40%, or at least 50%. In at
least one embodiment, administering a PKC activator and rTPA
reduces mortality 24 hours after stroke by at least 40%.
[0088] In some embodiments, the combination of a PKC activator and
rTPA may reduce disruption of the blood-brain barrier after stroke.
The combination of a PKC activator and rTPA may also reduce
hemorrhagic transformation. In some embodiments, for example,
administering a PKC activator and rTPA after a stroke reduces
hemoglobin levels, wherein a reduction in hemoglobin indicates a
reduction in hemorrhagic transformation and/or a reduction in
disruption of the blood-brain barrier. In some embodiments, the
hemoglovin level is reduced by about 30%, about 35%, about 40%,
about 45%, about 50%, about 55%, or about 60%. In at least one
embodiment, for example, administering a PKC activator and rTPA
after stroke reduces hemoglobin levels by about 50%. Reduced
disruption of the blood-brain barrier may also be assessed by
measuring extravasation of albumin. DiNapoli et al., Neurobiology
of Aging (2008), vol. 29, pp. 753-764.
[0089] In some embodiments, administering a PKC activator and rTPA
may limit the size of the infarction due to stroke, e.g., limit the
tissue damage caused by an ischemic event.
[0090] Formulation and Administration
[0091] The formulations of the pharmaceutical compositions
described herein may be prepared by any suitable method known in
the art of pharmacology. In general, such preparatory methods
include bringing the active ingredient into association with a
carrier or one or more other accessory ingredients, then, if
necessary or desirable, shaping or packaging the product into a
desired single- or multi-dose unit.
[0092] Although the descriptions of pharmaceutical compositions
provided herein are principally directed to pharmaceutical
compositions suitable for ethical administration to humans, it will
be understood by skilled artisan that such compositions are
generally suitable for administration to animals of all sorts.
Modification of pharmaceutical compositions suitable for
administration to humans order to render the compositions suitable
for administration to various animals is well understood, and the
ordinarily skilled veterinary pharmacologist can design and perform
such modification with merely ordinary, if any, experimentation.
Subjects to which administration of the pharmaceutical compositions
of the invention is contemplated include, but are not limited to,
humans and other primates, and other mammals.
[0093] In some embodiments, the PKC activator and anticoagulant,
e.g., rTPA, are formulated together. In other embodiments, the PKC
activator and rTPA are formulated separately.
[0094] The compositions disclosed herein may be administrated by
any suitable route including oral, parenteral, transmucosal,
intranasal, inhalation, or transdermal routes. Parenteral routes
include intravenous, intra-arteriolar, intramuscular, intradermal,
subcutaneous, intraperitoneal, intraventricular, intrathecal, and
intracranial administration. A suitable route of administration may
be chosen to permit crossing the blood-brain barrier. Rapoport et
al., J. Lipid Res. (2001) vol. 42, pp. 678-685.
[0095] The compositions disclosed herein may be formulated
according to conventional methods, and may include any
pharmaceutically acceptable additives, such as excipients,
lubricants, diluents, flavorants, colorants, buffers, and
disintegrants. See e.g., Remington's Pharmaceutical Sciences,
20.sup.th Ed., Mack Publishing Co. 2000.
[0096] In some embodiments, the PKC activator is formulated in a
solid oral dosage form. For oral administration, the composition
may take the form of a tablet or capsule prepared by conventional
means with pharmaceutically acceptable excipients such as binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose); fillers (e.g., lactose,
microcrystalline cellulose or calcium hydrogen phosphate);
lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycolate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets may be
coated by methods generally known in the art. Liquid preparations
for oral administration may take the form of, for example,
solutions, syrups or suspensions, or they may be presented as a dry
product for constitution with water or other suitable vehicle
before use. Such liquid preparations may be prepared by
conventional means with pharmaceutically acceptable additives such
as suspending agents (e.g., sorbitol syrup, cellulose derivatives
or hydrogenated edible fats); emulsifying agents (e.g., lecithin or
acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl
alcohol or fractionated vegetable oils); and preservatives (e.g.,
methyl or propyl-phydroxybenzoates or sorbic acid). The
preparations may also comprise buffer salts, flavoring, coloring
and sweetening agents as appropriate.
[0097] In other embodiments of the present disclosure, the PKC
activator may be formulated for parenteral administration such as
bolus injection or continuous infusion. Formulations for injection
may be presented in unit dosage form, e.g., in ampoules or in
multi-dose containers, with an added preservative. The compositions
may take such forms as suspensions, solutions, dispersions, or
emulsions in oily or aqueous vehicles, and may contain formulatory
agents such as suspending, stabilizing and/or dispersing
agents.
[0098] In some embodiments, the PKC activator may be formulated
with a pharmaceutically-acceptable carrier for administration.
Pharmaceutically acceptable carriers include, but are not limited
to, one or more of the following: excipients; surface active
agents; dispersing agents; inert diluents; granulating and
disintegrating agents; binding agents; lubricating agents;
sweetening agents; flavoring agents; coloring agents;
preservatives; physiologically degradable compositions such as
gelatin; aqueous vehicles and solvents; oily vehicles and solvents;
suspending agents; dispersing or wetting agents; emulsifying
agents, demulcents; buffers; salts; thickening agents; fillers;
emulsifying agents; antioxidants; antibiotics; antifungal agents;
stabilizing agents; and pharmaceutically acceptable polymeric or
hydrophobic materials. Other "additional ingredients" which may be
included in the pharmaceutical compositions of the invention are
generally known in the art and may be described, for example, in
Remington's Pharmaceutical Sciences, Genaro, ed., Mack Publishing
Co., Easton, Pa., 1985, incorporated by reference herein.
[0099] In some embodiments, the PKC activator may be formulated
with a hydrophobic carrier for administration. Hydrophobic carriers
include inclusion complexes, dispersions (such as micelles,
microemulsions, and emulsions), and liposomes. Exemplary
hydrophobic carriers include inclusion complexes, micelles, and
liposomes. See, e.g., Remington's: The Science and Practice of
Pharmacy 20th ed., ed. Gennaro, Lippincott: Philadelphia, Pa. 2003.
The PKC activators presently disclosed may be incorporated into
hydrophobic carriers, for example as at least 1%, at least 5%, at
least 10%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, or at least 90% of the
total carrier by weight. In addition, other compounds may be
included either in the hydrophobic carrier or the solution, e.g.,
to stabilize the formulation.
[0100] In some embodiments, the PKC activator may also be
formulated as a depot preparation. Such long acting formulations
may be administered by implantation (for example subcutaneously or
intramuscularly) or by intramuscular injection. Thus, for example,
the PKC activator may be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salt.
[0101] In another embodiment, the PKC activator may be delivered in
a vesicle, such as a micelle, liposome, or an artificial
low-density lipoprotein (LDL) particle. See, e.g., U.S. Pat. No.
7,682,627.
[0102] The doses for administration may suitably be prepared so as
to deliver from about 1 mg to about 10 g, such as from about 10 mg
to about 1 g, or for example, from about 250 mg to about 500 mg of
the PKC activator per day. When prepared for topical administration
or parenteral formulations they may be made in formulae containing
from about 0.01% to about 60% by weight of the final formulation,
such as from about 0.1% to about 30% by weight, such as from about
1% to about 10% by weight. A suitable dose can be determined by
methods known in the art and according to clinically relevant
factors such as the age of the patient.
[0103] In at least one embodiment, the PKC activator is formulated
for intravenous administration. The PKC activator may be formulated
for intravenous administration of a dose ranging from about 25
.mu.g/m.sup.2 to about 50 .mu.g/m.sup.2. In some embodiments, the
PKC activator and rTPA are both formulated for intravenous
administration. The rTPA may be formulated for intravenous
administration of a dose of about 0.9 mg/kg. The PKC and rTPA may
be formulated together for intravenous administration, or they may
be formulated separately for intravenous administration.
[0104] Kits
[0105] The present disclosure further relates to kits that may be
utilized for preparing and administering pharmaceutical
compositions of an anticoagulant, e.g., rTPA, and a PKC activator
disclosed herein to a subject in need thereof. The kits may also
comprise devices such as syringes for administration of the
pharmaceutical compositions described herein.
[0106] In some embodiments, the kits may comprise one or more
vials, syringes, needles, ampules, cartridges, bottles or other
such vessels for storing and/or subsequently mixing compositions of
rTPA and PKC activator disclosed herein. In certain embodiments,
the devices, syringes, ampules, cartridges, bottles or other such
vessels for storing and/or subsequently mixing the compositions of
rTPA and a PKC activator disclosed herein may, or may not have more
than one chamber.
[0107] In still further embodiments, the compositions of rTPA and a
PKC activator disclosed herein may be stored in one or more
graduated vessels (such as a syringe or syringes or other device
useful for measuring volumes).
[0108] In certain embodiments, the kits may comprise pharmaceutical
compositions of rTPA and a PKC activator stored within the same or
separate ampules, vials, syringes, cartridges, bottles or other
such vessels.
[0109] The kits may also comprise one or more anesthetics,
preferably local anesthetics. In certain embodiments, the
anesthetics are in a ready-to-use formulation, such as, for example
an injectable formulation (optionally in one or more pre-loaded
syringes) or a formulation that may be applied topically to an area
where the compositions of rTPA and PKC activator disclosed herein
are to be administered.
[0110] Topical formulations of anesthetics may be in form an
anesthetic applied to a pad, swab, towelette, disposable napkin,
cloth, patch, bandage, gauze, cotton ball, Q-tip.TM., ointment,
cream, gel, paste, liquid, or any other topically applied
formulation. Anesthetics for use with the present invention may
include, but are not limited to lidocaine, marcaine, cocaine and
xylocaine, for example.
[0111] The kits may also contain instructions relating to the use
of the pharmaceutical compositions of rTPA and a PKC activator and
procedures for mixing, diluting or combining formulations of rTPA
and a PKC activator. The instructions may also contain directions
for properly diluting formulations of rTPA and/or a PKC activator
to obtain a desired pH or range of pHs and/or a desired specific
activity and/or protein concentration after mixing but prior to
administration. The instructions may also contain dosing
information. The instructions may also contain material directed to
methods for selecting subjects for treatment with the disclosed
pharmaceutical compositions of rTPA and a PKC activator. The kits
may also include additional buffers, syringes, needles, needle-less
injection devices, sterile pads or swabs.
[0112] The following examples are intended to illustrate the
present disclosure without, however, being limiting in nature. It
is understood that the skilled artisan will envision additional
embodiments consistent with the disclosure provided herein.
EXAMPLES
Example 1
Focal Ischemia Model of Stroke
[0113] A transient animal model of focal ischemia was used for
these experiments. The middle cerebral artery (MCA) was surgically
dissected and occluded in anesthetized rats by ligature, followed
by reperfusion after a defined period (about 2 hours). Animal
models transient ischemia via occlusion of the MCA (MCAO) are
described in, e.g., Sicard and Fisher, Exp. & Transl. Stroke
Med. (2009), vol. 1, pp. 1-7.
Example 2
Drug Administration
[0114] In a first experiment, rTPA was administered intravenously
(.about.0.9 mg/kg) 6 hours after the ischemic event, followed 2
hours later with a single intravenous administration of
bryostatin-1 in a dosage range of from about 25 .mu.g/m.sup.2 to 50
.mu.g/m.sup.2.
[0115] In a second experiment, bryostatin-1 was administered
intravenously (about 25 .mu.g/m.sup.2 to 50 .mu.g/m.sup.2) 2 hours
after the ischemic event, followed by intravenous administration of
rTPA (.about.0.9 mg/kg) about 6 hours later.
[0116] In a third experiment, rTPA was administered intravenously
(.about.0.9 mg/kg) 2 hours after the ischemic event, followed by
intravenous administration of bryostatin-1 in a dosage range of
from about 25 .mu.g/m.sup.2 to 50 .mu.g/m.sup.2 about 6 hours
later.
Example 3
Results
[0117] 1. Mortality
[0118] rTPA given 6 hours after the stroke, followed 2 hours later
with bryostatin-1 led to 0% mortality 24 hours later (N=9 animals).
In contrast, if rTPA was given 6 hours after the stroke, in the
absence of subsequent treatment with bryostatin, 44% mortality was
observed (N=6 animals).
[0119] 2. Hemorrhage, Edema, and Blood-Brain Barrier
Disruptions
[0120] Bryostatin-1 administered 2 hours after the stroke, followed
6 hours later by rTPA, resulted in a 50% reduction of assayed
hemoglobin in the cortex and striatum, as compared to rTPA given 6
hours after the stroke without prior bryostatin-1 treatment (FIG.
1). Brain edema was also significantly reduced with this
combination of rTPA and bryostatin-1 (FIG. 2).
[0121] The BBB permeability typically increase prior to the
occurrence of edema following focal ischemia, such that edema can
be used to measure BBB disruptions at the site of the ischemic
lesion. In addition, the hemorrhage process is involved in the BBB
disruption and edema. In one experiment, uptake of Evans Blue dye
was used to measure BBB permeability, i.e., disruption, and
hemorrhaging in ischemic animal models of stroke. FIG. 3 shows that
combinations of bryostatin and administered according to the
methods of the present disclosure significantly reduced uptake of
Evans Blue dye in the ipsilateral and contralateral cortices.
[0122] Lastly, increased transport of sodium across the (BBB)
contributes to cerebral edema formation in ischemic stroke. FIG. 4
shows that uptake of NaF in the ipsilateral and contralateral
cortices is also reduced with the disclosed administration regimens
of bryostatin-1 and rTPA.
[0123] The foregoing results demonstrate that the combination of
bryostatin-1 with rTPA following ischemic stroke unexpectedly and
significantly reduces mortality and brain injury following ischemic
stroke.
* * * * *
References