U.S. patent application number 10/759837 was filed with the patent office on 2004-08-26 for zinc ionophores as therapeutic agents.
Invention is credited to Fliss, Henry.
Application Number | 20040167114 10/759837 |
Document ID | / |
Family ID | 27385525 |
Filed Date | 2004-08-26 |
United States Patent
Application |
20040167114 |
Kind Code |
A1 |
Fliss, Henry |
August 26, 2004 |
Zinc ionophores as therapeutic agents
Abstract
The present invention provides methods and compositions
comprising one or more zinc ionophores for protecting tissue from
the harmful effects of apoptosis in patients in need thereof.
Concentrations of zinc-pyrithione and diethyldithiocarbamate in the
picomolar to nanomolar range have a strong protective effect
against apoptosis.
Inventors: |
Fliss, Henry; (Ottawa,
CA) |
Correspondence
Address: |
SCULLY SCOTT MURPHY & PRESSER, PC
400 GARDEN CITY PLAZA
GARDEN CITY
NY
11530
|
Family ID: |
27385525 |
Appl. No.: |
10/759837 |
Filed: |
January 16, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10759837 |
Jan 16, 2004 |
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10205973 |
Jul 26, 2002 |
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6689774 |
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10205973 |
Jul 26, 2002 |
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09759091 |
Jan 12, 2001 |
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6495538 |
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09759091 |
Jan 12, 2001 |
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09602829 |
Jun 23, 2000 |
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6407090 |
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60140632 |
Jun 23, 1999 |
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Current U.S.
Class: |
514/184 ;
514/478; 514/494 |
Current CPC
Class: |
A61K 31/145 20130101;
A61K 31/44 20130101; A61K 31/47 20130101; A61K 31/555 20130101;
A61K 31/40 20130101; A61K 31/27 20130101; A61K 31/07 20130101; A61K
31/405 20130101; A61K 31/355 20130101; A61K 31/315 20130101; A61K
31/325 20130101 |
Class at
Publication: |
514/184 ;
514/478; 514/494 |
International
Class: |
A61K 031/555; A61K
031/315 |
Claims
What is claimed is:
1. A method of treating apoptosis comprising administering to a
subject in need thereof a pharmaceutically effective amount of a
zinc ionophore and a pharmaceutically acceptable carrier.
2. The method of claim 1, wherein the zinc ionophore comprises zinc
pyrithione, heterocyclic amines, dithiocarbamates and Vitamins.
3. The method of claim 2, wherein the zinc ionophore is zinc
pyrithione.
4. The method of claim 2, wherein said heterocyclic amine comprises
5,7-Diiodo-8-hydroxyquinoline and 8-Hydroxyquinoline.
5. The method of claim 2, wherein said dithiocarbamate comprises
pyrrolidine dithiocarbamate, zinc-diethyldithiocarbamate,
disulfiram and zinc-dimethyldithiocarbamate.
6. The method of claim 2, wherein said vitamin is selected from the
group consisting of Vitamin E and Vitamin A.
7. The method of claim 1, wherein the effective amount of a zinc
ionophore ranges from about 0.005 .mu.g per kg of body weight to
about 5.0 mg per kg of body weight.
8. The method of claim 7, wherein the effective amount of a zinc
ionophore ranges from about 0.2 .mu.g per kg of body weight to
about 600 .mu.g per kg of body weight.
9. The method of claim 1, wherein the zinc ionophore is
administered intravenously, intramuscularly, subcutaneously,
intracerebroventricularly- , orally or topically.
10. A method of treating the harmful effects of injurious agents
selected from the group consisting of oxidants, TNF.alpha.,
neurotoxins, and radiation comprising administering to a subject in
need of such protection an effective amount of a zinc ionophore and
a pharmaceutically acceptable carrier.
11. The method of claim 10, wherein the zinc ionophore comprises
zinc pyrithione, heterocyclic amines, dithiocarbamates and
Vitamins.
12. The method of claim 11, wherein the zinc ionophore is zinc
pyrithione.
13. The method of claim 11, wherein said heterocyclic amine
comprises 5,7-Diiodo-8-hydroxyquinoline and 8-Hydroxyquinoline.
14. The method of claim 11, wherein said dithiocarbamate comprises
pyrrolidine dithiocarbamate, zinc-diethyldithiocarbamate,
disulfiram and zinc-dimethyldithiocarbamate.
15. The method of claim 11, wherein said vitamin is selected from
the group consisting of Vitamin E and Vitamin A.
16. The method of claim 10, wherein the effective amount of a zinc
ionophore ranges from about 0.005 .mu.g per kg of body weight to
about 5.0 mg per kg of body weight.
17. The method of claim 16, wherein the effective amount of a zinc
ionophore ranges from about 0.2 .mu.g per kg of body weight to
about 600 .mu.g per kg of body weight.
18. The method of claim 10, wherein the zinc ionophore is
administered intravenously, intramuscularly, subcutaneously,
intracerebroventricularly- , orally or topically.
19. A pharmaceutical composition comprising a zinc ionophore and a
pharmaceutically acceptable carrier.
20. A method of treating ischemia comprising administering to a
subject in need thereof an effective amount of a zinc ionophore and
a pharmaceutically acceptable carrier.
21. The method of claim 20, wherein the zinc ionophore comprises
zinc pyrithione, heterocyclic amines, dithiocarbamates and
Vitamins.
22. The method of claim 21, wherein the zinc ionophore is zinc
pyrithione.
23. The method of claim 21, wherein said heterocyclic amine
comprises of 5,7-Diiodo-8-hydroxyquinoline and
8-Hydroxyquinoline.
24. The method of claim 21, wherein said dithiocarbamate comprises
of pyrrolidine dithiocarbamate, zinc-diethyldithiocarbamate,
disulfiram and zinc-dimethyldithiocarbamate.
25. The method of claim 21, wherein said vitamin is selected from
the group consisting of Vitamin E and Vitamin A.
26. The method of claim 20, wherein the effective amount of a zinc
ionophore ranges from about 0.005 .mu.g per kg of body weight to
about 5.0 mg per kg of body weight.
27. The method of claim 26, wherein the effective amount of a zinc
ionophore ranges from about 0.2 .mu.g per kg of body weight to
about 600 .mu.g per kg of body weight.
28. A method of treating seizures comprising administering to a
subject in need of thereof an effective amount of a zinc ionophore
and a pharmaceutically acceptable carrier.
29. The method of claim 28, wherein the zinc ionophore comprises of
zinc pyrithione, heterocyclic amines, dithiocarbamates and
Vitamins.
30. The method of claim 29, wherein the zinc ionophore is zinc
pyrithione.
31. The method of claim 29, wherein said heterocyclic amine
comprises 5,7-Diiodo-8-hydroxyquinoline and 8-Hydroxyquinoline.
32. The method of claim 29, wherein said dithiocarbamate comprises
pyrrolidine dithiocarbamate, zinc-diethyldithiocarbamate,
disulfiram and zinc-dimethyldithiocarbamate.
33. The method of claim 29, wherein said vitamin is selected from
the group consisting of Vitamin E and Vitamin A.
34. The method of claim 28, wherein the effective amount of a zinc
ionophore ranges from about 0.005 .mu.g per kg of body weight to
about 5.0 mg per kg of body weight.
35. The method of claim 24, wherein the effective amount of a zinc
ionophore ranges from about 0.2 .mu.g per kg of body weight to
about 600 .mu.g per kg of body weight.
36. A method of treating conditions caused by apoptosis comprising
administering to a subject in need thereof an effective amount of a
zinc ionophore and a pharmaceutically acceptable carrier.
37. The method of claim 36, wherein the zinc ionophore comprises
zinc pyrithione, heterocyclic amines, dithiocarbamates and
Vitamins.
38. The method of claim 37, wherein the zinc ionophore is zinc
pyrithione.
39. The method of claim 37, wherein said heterocyclic amine is
selected from the group consisting of 5,7-Diiodo-8-hydroxyquinoline
and 8-Hydroxyquinoline.
40. The method of claim 37, wherein said dithiocarbamate is
selected from the group consisting of pyrrolidine dithiocarbamate,
zinc-diethyldithiocarbamate, disulfiram and
zinc-dimethyldithiocarbamate.
41. The method of claim 37, wherein said vitamin is selected from
the group consisting of Vitamin E and Vitamin A.
42. The method of claim 36, wherein the effective amount of a zinc
ionophore ranges from about 0.005 .mu.g per kg of body weight to
about 5.0 mg per kg of body weight.
43. The method of claim 32, wherein the effective amount of a zinc
ionophore ranges from about 0.2.mu.g per kg of body weight to about
600 .mu.g per kg of body weight.
44. An anti-epileptic composition comprising a zinc ionophore and a
pharmaceutically acceptable carrier.
45. A method of preventing seizures comprising administering to a
subject in need thereof an effective amount of a zinc ionophore and
a pharmaceutically acceptable carrier.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. Ser. No.
09/602,829, filed Jun. 23, 2000, which application claims the
benefit of U.S. Provisional Application No. 60/140,632, filed Jun.
23, 1999.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of protecting
tissue from apoptosis using zinc ionophores. The present invention
also relates to a method of protecting cells against the harmful
effects of injurious agents, for example, oxidants, TNF.alpha.,
neurotoxins, ischemia and radiation.
BACKGROUND OF THE INVENTION
[0003] Zinc plays a critical role in cellular biology, and is
involved in virtually every important cellular process such as
transcription, translation, ion transport, and others (O'Halloran,
T. V. (1993) Science 261:715-725; Cousins, R. J. (1994)
Annu.Rev.Nutr. 14:449-469; Harrison, N. L. et al. (1994)
Neuropharmacology 33:935-952; Berg, J. M. et al. (1996) Science
271:1081-1085). The involvement of cellular zinc in apoptosis has
been recognized for close to 20 years (Sunderman, F. W., Jr. (1995)
Ann.Clin.Lab.Sci. 25:134-142; Fraker, P. J. et al. (1997)
Proc.Soc.Exp.Biol.Med. 215:229-236.). However, the full nature of
this involvement is not fully understood. Apoptosis is a form of
programmed cell death normally activated under physiological
conditions, such as involution in tissue remodelling during
morphogenesis, and several immunological processes. The apoptotic
process is characterized by cell shrinkage, chromatin condensation,
and internucleosomal degradation of the cell's DNA (Verhaegen et
al. (1995) Biochem. Pharmacol. 50(7):1021-1029).
[0004] Numerous in vitro studies have been done recently in an
attempt to elucidate the role of intracellular zinc. Although some
studies have suggested that zinc may actually induce apoptosis (Xu,
J. et al. (1996) Am.J.Physiol. 270:G60-G70; Kim, Y. H. et al.,
(1999) Neuroscience 89:175-182), most have concluded that
increasing the intracellular concentrations of zinc blocks
apoptosis (Sunderman, F. W., Jr. (1995) Ann.Clin.Lab.Sci.
25:134-142; Adebodun, F. et al. (1995) J.Cell.Physiol. 163:80-86;
Zalewski, P. D., et al. (1993) Biochem.J. 296:403-408), and that
decreasing the zinc concentration promotes apoptosis (Jiang, S., et
al. (1995) Lab.Invest. 73:111-117; Treves, S., et al. (1994)
Exp.Cell Res. 211:339-343; Ahn, Y. H., et al. (1998) Exp.Neurol.
154:47-56). The manner in which increased intracellular zinc
affords protection against apoptosis is not clear. (Truong-Tran, A.
Q. et al., (2000) J. Nutr. 130:1459S-1466S) One theory proposes
that zinc inactivates the intracellular endonuclease(s) responsible
for apoptotic DNA fragmentation (Shiokawa, D., et al. (1994)
Eur.J.Biochem. 226:23-30; Yao, M. et al., (1996)
J.Mol.Cell.Cardiol. 28:95-101). Other recent studies have suggested
that zinc-can inhibit caspases (Jiang, S., et al. (1997) Cell Death
Differ. 4:39-50; Perry, D. K., et al. (1997) J.Biol.Chem.
272:18530-18533; Maret, W., et al. (1999) Proc.Natl.Acad.Sci.USA
96:1936-1940), or block the activation of caspases (Aiuchi, T., et
al. (1998) J.Biochem. 124:300-303). However, in view of the large
number of intracellular roles played by zinc, it seems likely that
its anti-apoptotic mechanisms may be more complex, possibly
involving gene expression and cellular signalling pathways. In
fact, recent studies support a role for zinc transients in
intracellular signalling and gene expression (O'Halloran, T. V.
(1993) Science 261:715-725; Berg, J. M., et al., (1996) Science
271:1081-1085).
[0005] In contrast to the large number of in vitro studies, very
few studies have attempted to examine the protective effects of
zinc in vivo. It is important to note that most of the studies that
have explored this possibility have focused on the pretreatment of
tissues with zinc prior to injury. Using this approach, a number of
studies have demonstrated that pretreatment of animals with zinc at
least 24 hours prior to injury provided some measure of protection
against apoptosis (Thomas, D. J. et al., (1991) Toxicology
68:327-337; Matsushita, K., et al., (1996) Brain Res. 743:362-365;
Klosterhalfen, B., et al., (1997) Shock 7:254-262), presumably as a
result of the well established ability of zinc to boost the immune
system (Cunningham-Rundles, S., et al., (1990) Ann.N.Y.Acad.Sci.
587:113-122). Also, one study showed that several days of zinc
dietary supplementation concomitant with i.p. injection of carbon
tetrachloride protected against liver apoptosis (Cabre, M. et al.
(1999) J. Hepatol. 31:228-234). However, no studies have
demonstrated the efficacy of zinc when administered acutely and
post-injury, a much more clinically relevant setting.
[0006] Zinc-pyrithione (zinc pyridinethione,
C.sub.10H.sub.8N.sub.2O.sub.2- S.sub.2Zn, MW 317.75, commercially
available from Sigma) is the active ingredient in the anti-dandruff
shampoo Head & Shoulders.RTM. (U.S. Pat. Nos. 3,236,733, and
3,281,366, both 1966), as well as a number of other topical skin
treatment formulations. It is a fungicide and bactericide at high
concentrations. It is highly lipophilic and therefore penetrates
membranes easily. This permits zinc pyrithione to transport zinc
across cell membranes, thereby conferring on this compound (i.e.
zinc pyrithione) the properties of a zinc ionophore. The
anti-apoptotic effect of zinc pyrithione was first observed in
vitro by Zalewski and coworkers, who showed that micromolar
concentrations of this compound protected lymphocytic leukemia
cells against colchicine-induced apoptosis (Giannakis, C., et al.
(1991) Biochem. Biophys. Res. Commun. 181:915-920). The rationale
for the use of this zinc ionophore was to facilitate the transport
of Zn.sup.2+ into the target cells. This is necessitated by the
fact that all eukaryotic cells strictly regulate the membrane
transport of Zn.sup.2+, making it very difficult to modulate the
intracellular concentration and distribution of Zn.sup.2+.
Zalewski's group has since published a number of other studies, all
of them in vitro, confirming the ability of micromolar
concentrations of zinc-pyrithione to rapidly transport Zn.sup.2+
into cells and to thereby prevent apoptosis (Zalewski, P. D., et
al. (1994) supra; Zalewski, P. D., et al. (1993) Biochem.J.
296:403-408). One confirmatory study, also in vitro, has been
published from another laboratory (Tempel, K.-H. et al., (1993)
Arch.Toxicol. 67:318-324).
[0007] In addition to zinc-pyrithione, another group of zinc
ionophores, the dithiocarbamates, has been shown to affect
apoptosis in vitro. (Orrenius, S. et.al. (1996) Biochem. Soc.
Trans. 24:1032-1038; Stefan, C. et al. (1997) Chem. Res. Toxicol.
10:636-643; Erl, W. et al., (2000) Am. J. Physiol.
278:C1116-C1125). However, no attempts have been made to examine in
vivo the protective effects of zinc-pyrithione, zinc-bound
dithiocarbamates or any other known zinc ionophore at nanomolar or
picomolar concentrations.
SUMMARY OF THE INVENTION
[0008] The ability of zinc-pyrithione and
zinc-diethyldithiocarbamates to protect tissue against apoptosis in
three models of in vivo injury, as well as two in vitro models is
presented. In each case a pronounced anti-apoptotic effect was
achieved. The ability of zinc-pyrithione, zinc
diethyldithiocarbamates and zinc 5,7-diodo-8-hydroxyquinoline to
treat and prevent seizures is also provided.
[0009] Thus, according to the present invention there is provided a
method to protect against apoptosis and treat seizures using one or
more zinc ionophores.
[0010] In one embodiment of the present invention there is provided
a method of treating apoptosis in mammalian tissue such as heart,
brain, and eye tissue by administering to a mammalian subject in
need thereof a pharmaceutically effective amount of at least one
zinc ionophore.
[0011] In another embodiment of the present invention there is
provided a pharmaceutical composition comprising a zinc ionophore
and a pharmaceutically acceptable carrier.
[0012] In a further embodiment of the present invention there is
provided a method of protecting against the harmful effects of
injurious agents such as TNF.alpha., neurotoxins, ischemia and
radiation by administering to a subject in need of such protection
an effective amount of a zinc ionophore.
[0013] In a still further embodiment of the present invention there
is provided a method of treating or preventing seizures by
administering to a subject in need thereof a pharmaceutically
effective amount of at least one zinc ionophore.
BRIEF DESCRIPTION OF THE FIGURES
[0014] These and other features of the invention will become more
apparent from the following description in which reference is made
to the appended drawings wherein:
[0015] FIG. 1 shows the effect of zinc pyrithione on rat cardiac
apoptosis.
[0016] FIG. 2 shows the effects of zinc pyrithione on HSP-70 in
heart.
[0017] FIG. 3 shows the anti-apoptotic effect of zinc pyrithione on
Sp1 in brain.
[0018] FIG. 4 shows the effects of zinc pyrithione on kainic acid
induced damage in rat brain areas.
[0019] FIG. 5 shows the effects of zinc pyrithione on the severity
of kainic acid induced seizures in rats.
[0020] FIG. 6 shows the protective effects of zinc pyrithione in
PC12 cells subjected to oxidative stress.
[0021] FIG. 7 shows the effects of zinc ionophores on the number of
rats with tonic seizures.
[0022] FIG. 8 shows the effects of zinc ionophores on the mortality
in the sc PTZ test. FIG. 9 shows the effect of zinc pyrithione on
latency of audiogenic seizure onset.
[0023] FIG. 10 shows the effect of zinc pyrithione on seizure
severity in the audiogenic epilepsy.
[0024] FIG. 11 shows the protective effects of zinc pyrithione in
PC12 cells.
[0025] FIG. 12 shows the anti-apoptotic effect of zinc-pyrithione
in irradiated human primary endothelial cells.
[0026] FIG. 13 shows the effects of zinc pyrithione on
transcription factor binding activity in human primary endothelial
cells.
[0027] FIG. 14 shows the effects of zinc pyrithione on the
TNF.alpha.-induced transcription factor binding activity in human
primary endothelial cells.
[0028] FIG. 15 shows the effects of zinc pyrithione on cytosolic
Ikappa B protein levels,in human primary endothelial cells.
[0029] FIGS. 16A-16C show photomicrographs depicting histological
evidence of protection by zinc-pyrithione in 4 vessel occlusion
stroke model in rats.
[0030] FIG. 17A shows the effect of zinc pyrithione on neuronal
survival in 4 vessel occlusion stroke model in rats.
[0031] FIG. 17B shows the effect of zinc-pyrithione,
zinc-diethyldithiocarbamate on neuronal survival in 4 vessel
occlusion stroke model in rats.
[0032] FIG. 18 shows the effect of zinc pyrithione in rats and a
4.times. reduction in the number of apoptotic nuclei in a 4 vessel
occlusion stroke model in rats.
[0033] FIG. 19 shows middle cerebral artery occlusion caused
infarcts in the left hemisphere of mouse brain. Infarcts are
visible as white regions in the left hemisphere.
[0034] FIG. 20A shows the reduction in infarct area upon treatment
with zinc-pyrithione in mice with middle cerebral artery occlusion
stroke model.
[0035] FIG. 20B shows the reduction in infarct area upon treatment
with zinc-diethyldithiocarbamate in mice with middle cerebral
artery occlusion stroke model.
[0036] FIG. 21A shows the administration of zinc-pyrithione
decreased infarct volumes in mouse brain.
[0037] FIGS. 21B and 21C show the administration of
zinc-diethyldithiocarbamate decreased infarct volumes in mouse
brain.
[0038] FIG. 22 shows the effect of zinc-pyrithione administration
on a neurological score in mice with middle cerebral artery
occlusion stroke model.
DETAILED DESCRIPTION OF THE INVENTION
[0039] The present invention is directed to a method of protecting
tissue from the harmful effects of apoptosis and treating seizures
by administering an effective amount of one or more zinc ionophores
to a subject in need thereof. By "zinc ionophore" is meant a
therapeutic compound complexed with zinc ions that is capable of
carrying zinc ions across cell membranes. In accordance with the
present invention "treating" includes preventing, blocking,
inhibiting, attenuating, protecting against and reducing the
occurrence of e.g., the harmful effects of apoptosis and
seizures.
[0040] Previous studies have shown in vitro that treatment of
injured cells with zinc can block apoptosis. In these studies the
effectiveness of a zinc ionophore was demonstrated using high
concentrations, ranging from 3 .mu.M to 5 mM. It was found,
according to the present invention that such concentrations are not
suitable for use in any in vivo method. Thus, according to the
present invention, small concentrations of a zinc ionophore in the
nanomolar and picomolar range, such as from about 10 pM to about 1
.mu.m, protect tissue from the harmful effects of apoptosis.
[0041] Thus, according to the present invention the concentration
of zinc ionophore used to protect tissue from the harmful effects
of apoptosis ranges from about 0.005 .mu.g zinc ionophore per kg of
body weight to about 5 mg zinc ionophore per kg of body weight
(i.e. about 600 pM zinc ionophore to about 15 .mu.M zinc
ionophore). In a further embodiment of the present invention the
concentration of zinc ionophore used to protect tissue from the
harmful effects of apoptosis ranges from about 1.0 .mu.g zinc
ionophore per kg of body weight to about 800 .mu.g zinc ionophore
per kg of body weight. Preferably the concentration of zinc
ionophore used to protect tissue from the harmful effects of
apoptosis ranges from about 0.2 .mu.g zinc ionophore per kg of body
weight to about 600 .mu.g zinc ionophore per kg of body weight.
[0042] In a further embodiment of the present invention the
concentration of zinc ionophore used to protect tissue from the
harmful effects of apoptosis is about 0.9 mg/kg body weight, or
about 0.18 mg zinc/kg body weight.
[0043] According to the present invention, any compound capable of
binding zinc with moderate affinity and having sufficient
lipophilic properties to penetrate cell membranes is capable of
effecting the protection demonstrated in the present invention with
e.g., zinc-pyrithione. The following are examples of compounds
which have been shown in accordance with the present invention to
possess zinc-ionophore properties: zinc pyrithione, the
heterocyclic amines including, for example,
5,7-Diiodo-8-hydroxyquinoline, and 8-Hydroxyquinoline; the
dithiocarbamates including, for example, pyrrolidine
dithiocarbamate and diethyldithiocarbamate, disulfiram and
dimethyldithiocarbamate; and Vitamins including, but not limited
to, Vitamin E and Vitamin A. Properties associated with zinc
ionophores include, but are not limited to, an ability to alter
cytosolic PKC-.alpha. content and an ability to alter the nuclear
activity of transcription factors NF-kB, AP-1 and Sp1. According to
the present invention zinc-pyrithione was shown to operate at the
cell signalling level, as demonstrated by its ability to alter
cytosolic PKC-.alpha. content. Further, according to the present
invention, zinc-pyrithione was shown to operate at the
transcriptional level, as demonstrated by its ability to alter the
nuclear activity of transcription factors NF-kB, AP-1 and Sp1.
Still further, according to the present invention zinc-pyrithione
was shown to upregulate cytoprotective proteins, for example
HSP70.
[0044] In accordance with the present invention the zinc ionophores
protect against neuronal cell loss in stroke patients. For example,
zinc pyrithione demonstrates neuroprotective properties, showing
protection against cell loss in the selectively vulnerable zone of
the CA1 region of the hippocampus in a rat model of severe global
ischemia. In the mouse model of severe focal ischemia, zinc
pyrithione demonstrates neuroprotective properties, significantly
decreasing brain infarct volume and neurological deficit.
[0045] The zinc ionophores of the present invention are versatile
in their efficacy and can be used to treat diseases associated with
the human eye including hereditary degenerative retinopathies,
including macular degeneration and retinitis pigmentosa, for
example. Other diseases of the eye treatable with the zinc
ionophores of the present invention include, but are not limited to
cataracts (diabetic and chemically induced, for example), glaucoma,
inflammatory eye diseases, corneal apoptosis associated with
transplantation and Fuchs' dystrophy.
[0046] The zinc ionophores of the present invention can also be
used to treat neurodegenerative diseases such as Alzheimer's
Disease, Parkinson's Disease, Huntington's Disease, Amyotrophic
lateral sclerosis (ALS) and multiple sclerosis, for example.
[0047] The zinc ionophores of the present invention also exhibit
anti-epileptic efficacy. In accordance with the present invention
"anti-epileptic" means "anti-convulsive" and "anti-seizure". For
example, zinc diethyldithiocarbamate demonstrates an anti-seizure
effect in a mouse model of seizure. Using the total elimination of
tonic extension as the principal criterion for anti-seizure
efficacy, zinc diethyldithiocarbamate showed statistically
significant ability to block tonic seizures. Accordingly, the
present invention provides a method for attenuating both the
duration and severity of seizures in mammalian subjects, including
humans.
[0048] In use, at least one zinc ionophore, according to the
present invention is administered in a pharmaceutically effective
amount to a subject in need thereof in a pharmaceutical carrier by
intravenous, intramuscular, subcutaneous, or
intracerebroventricular injection or by oral administration or
topical application. In accordance with the present invention, one
zinc ionophore may be administered, preferably by the intravenous
injection route, alone or in conjunction with a second, different
zinc ionophore. By "in conjunction with" is meant together,
substantially simultaneously or sequentially. In one embodiment,
the zinc ionophores of the present invention, are administered
acutely, such as, for example, substantially immediately following
an injury that results in apoptosis, such as a stroke. The zinc
ionophores may therefore be administered for a short course of
treatment, such as for about 1 day to about 1 week. In another
embodiment, the zinc ionophores of the present invention may be
administered over a longer period of time to ameliorate chronic
apoptotic episodes, such as, for example, for about one week to
several months depending upon the condition to be treated.
[0049] By "pharmaceutically effective amount" as used herein is
meant an amount of zinc ionophore, e.g., zinc-pyrithione, high
enough to significantly positively modify the condition to be
treated but low enough to avoid serious side effects (at a
reasonable benefit/risk ratio), within the scope of sound medical
judgment. A pharmaceutically effective amount of zinc ionophore
will vary with the particular goal to be achieved, the age and
physical condition of the patient being treated, the severity of
the underlying disease, the duration of treatment, the nature of
concurrent therapy and the specific zinc ionophore employed. For
example, a therapeutically effective amount of a zinc ionophore
administered to a child or a neonate will be reduced
proportionately in accordance with sound medical judgment. The
effective amount of zinc ionophore will thus be the minimum amount
which will provide the desired anti-apoptotic effect.
[0050] A decided practical advantage of the present invention is
that the zinc ionophore, e.g. zinc-pyrithione, may be administered
in a convenient manner such as by the, intravenous, intramuscular,
subcutaneous, oral or intra-cerebroventricular injection routes or
by topical application, such as in eye drops or eye mist
compositions. Depending on the route of administration, the active
ingredients which comprise zinc ionophores may be required to be
coated in a material to protect the zinc ionophores from the action
of enzymes, acids and other natural conditions which may inactivate
the zinc ionophores. In order to administer zinc ionophores by
other than parenteral administration, the ionophores should be
coated by, or administered with, a material to prevent
inactivation. For example, the zinc ionophores of the present
invention may be co-administered with enzyme inhibitors or in
liposomes. Enzyme inhibitors include pancreatic trypsin inhibitor,
and trasylol. Liposomes include water-in-oil-in-water P40 emulsions
as well as conventional and specifically designed liposomes.
[0051] The zinc ionophores may be administered parenterally or
intraperitoneally. Dispersions can also be prepared, for example,
in glycerol, liquid polyethylene glycols, and mixtures thereof, and
in oils.
[0052] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions (where water soluble) or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases the form must be
sterile and must be fluid to the extent that easy syringability
exists. It must be stable under the conditions of manufacture and
storage. The carrier can be a solvent or dispersion medium
containing, for example, water, DMSO, ethanol, polyol (for example,
glycerol, propylene glycol, liquid polyethylene glycol, and the
like), suitable mixtures thereof and vegetable oils. The proper
fluidity can be maintained, for example, by the use of a coating
such as lecithin, by the maintenance of the required particle size
in the case of dispersion. In many cases it will be preferable to
include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought
about by the use in the compositions of agents delaying absorption,
for example, aluminum monostearate and gelatin.
[0053] Sterile injectable solutions are prepared by incorporating
the zinc ionophore in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
zinc ionophores into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and the freeze-drying technique
which yield a powder of the active ingredient plus any additional
desired ingredient from previously sterile-filtered solution
thereof.
[0054] For oral therapeutic administration, the zinc ionophores may
be incorporated with excipients and used in the form of ingestible
tablets, buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Compositions or preparations
according to the present invention are prepared so that an oral
dosage unit form contains a zinc ionophore concentration sufficient
to treat or block apoptosis in a patient.
[0055] The tablets, troches, pills, capsules, and the like, may
contain the following: a binder such as gum tragacanth, acacia,
corn starch or gelatin; excipients such as dicalcium phosphate; a
disintegrating agent such as corn starch, potato starch, alginic
acid, and the like; a lubricant such as magnesium stearate; and a
sweetening agent such as sucrose, lactose or saccharin may be added
or a flavoring agent such as peppermint, oil or wintergreen or
cherry flavoring. When the dosage unit form is a capsule, it may
contain, in addition to materials of the above type, a liquid
carrier. Various other materials may be present as coatings or to
otherwise modify the physical form of the dosage unit. For
instance, tablets, pills or capsules or zinc ionophore in
suspension may be coated with shellac, sugar or both.
[0056] A syrup or elixir may contain the active compound, sucrose
as a sweetening agent, methyl and propylparabens as preservatives,
a dye and flavoring such as cherry or orange flavor. Of course, any
material used in preparing any dosage unit form should be
pharmaceutically pure and substantially non-toxic in the amounts
employed. In addition, the zinc ionophore may be incorporated into
sustained-release preparations and formulations.
[0057] By "pharmaceutically-acceptable carrier" as used herein is
meant one or more compatible solid or liquid filler diluents or
encapsulating substances. By "compatible" as used herein is meant
that the components of the composition are capable of being
comingled without interacting in a manner which would substantially
decrease the pharmaceutical efficacy of the total composition under
ordinary use situations.
[0058] Some examples of substances which can serve as
pharmaceutical carriers are sugars, such as lactose, glucose and
sucrose; starches such as corn starch and potato starch; cellulose
and its derivatives such as sodium carboxymethycellulose,
ethylcellulose and cellulose acetates; powdered tragancanth; malt;
gelatin; talc; stearic acids; magnesium stearate; calcium sulfate;
vegetable oils, such as peanut oils, cotton seed oil, sesame oil,
olive oil, corn oil and oil of theobroma; polyols such as propylene
glycol, glycerine, sorbitol, manitol, and polyethylene glycol;
agar; alginic acids; pyrogen-free water; isotonic saline; and
phosphate buffer solution; skim milk powder; as well as other
non-toxic compatible substances used in pharmaceutical formulations
such as Vitamin C, estrogen and echinacea, for example. Wetting
agents and lubricants such as sodium lauryl sulfate, as well as
coloring agents, flavoring agents, lubricants, excipients,
tableting agents, stabilizers, anti-oxidants and preservatives, can
also be present.
[0059] Accordingly, in a preferred form of protecting tissue from
the harmful effects of apoptosis and seizures the subject is
administered a therapeutically effective amount of at least one
zinc ionophore and a pharmaceutically acceptable carrier in
accordance with the present invention. The zinc ionophores of the
present invention are also effective in treating ischemia. By
"ischemia" is meant reduced blood flow that results in hypoxia in
one or more organs including brain, heart, muscle and nervous
tissue. For example, ischemia in the brain causes strokes; ischemia
in the heart causes myocardial infarctions. Therefore, in a
preferred form of treating ischemia the subject is administered a
therapeutically effective amount of at least one zinc ionophore and
a pharmaceutically acceptable carrier. A preferred subject is a
human. A preferred zinc ionophore is zinc pyrithione. Another
preferred zinc ionophore is zinc diethyldithiocarbamate.
[0060] The zinc ionophores of the present invention are effective
against a wide range of injurious agents, for example, but not
limited to: oxidants, TNF.alpha., neurotoxins, or radiation.
[0061] Also defined within the present invention are compositions
suitable for protecting tissue from the harmful effects of
apoptosis which comprise one or more zinc ionophores and a
pharmaceutically acceptable carrier.
[0062] Various modifications may be made without departing from the
invention. The disclosure is to be construed as exemplary, rather
than limiting, and such changes within the principles of the
invention as are obvious to one skilled in the art are intended to
be included within the scope of the claims.
[0063] The present invention will now be demonstrated using
specific examples that are not to be construed as limiting.
EXAMPLE 1
[0064] Screening for Ionophores:
[0065] Cell Cultures:
[0066] Human umbilical vein endothelial cells (HUVEC) were
purchased from Clonetics (San Diego, Calif.) and passages 2-4 were
used for these studies. Cells were cultured on flame-sterilzed
glass coverslips in Endothelial Basal Medium (Clonetics)
supplemented with 10 ng/ml human recombinant epidermal growth
factor, 1.0 ug/ml hydrocortisone, 50 ug/ml gentamicin, 50 ng/ml
amphotetericin B, 12 ug/ml bovine brain extract and 2% v/v fetal
bovine serum (all from Clonetics), in a humidified chamber at
37.degree. C. and 5% CO.sub.2. To maintain cell populations,
proliferating HUVEC were passaged at 80-90% confluency.
[0067] Cardiac myocytes were isolated from the ventricular septum
of adult rabbit hearts, following collagenase digestion, in a
manner similar to that described previously (Turan, B. et al.,
(1997) Am. J. Physiol. 272:H2095-H2106). The modification consisted
of introducing low concentrations of CaCl.sub.2 during the
perfusion with collagenase and the dispersion of the myocytes.
Hearts were perfused for about 2 min by gravity under a hydrostatic
pressure of 1 m, with a nominally Ca.sup.2+-free solution
containing (in mM): NaCl, 145; KCl, 5; MgSO.sub.4, 1.2;
Na.sub.2HPO.sub.4, 1.8; HEPES, 5; glucose, 10; pH adjusted to 7.4
with NaOH. Forty ml of this perfusate were then supplemented with
collagenase (1 mg/ml) and perfusion was continued with
recirculation. Within 2-3 min, this treatment resulted in a
complete loss of ventricular pressure. The flow rate was then
adjusted to 15 ml/min and 50 .mu.M CaCl.sub.2 was added to the
collagenase solution. Perfusion with this solution was continued
for another 15 to 18 min, followed by a 2 min washout of the enzyme
with fresh perfusate containing 100 .mu.M CaCl and no collagenase.
The hearts were then removed from the apparatus and the ventricular
septum isolated and minced. Dissociation of the cells was obtained
by gentle agitation of the minced tissue in 50 ml of the same
perfusing solution. Following filtration through a 200 .mu.m nylon
mesh, the cells were allowed to settle and the supernatant was
replaced with a solution containing 2 mM CaCl.sub.2. Cells were
kept at 37.degree. C. in this pre-oxygenated solution and were
studied within 8 hours after isolation, cellular viability was
ensured by regularly replacing the incubation solution.
[0068] Primary cultures of mouse cerebellar granule neurons were
obtained from dissociated cerebella of postnatal day 8 or 9 mice
according to the following protocol (Cregan et al., (1999) J.
Neurosci. 19:7860-7869, incorporated herein by reference). Brains
were removed and placed into separate dishes containing solution A
(124 mM NaCl, 5.37 mM KCl, 1 mM NaH2 PO4 , 1.2 mM MgSO4 , 14.5 mM
D-(1)-glucose, 25 mM HEPES, 3 mg/ml BSA, pH 7.4) in which the
cerebella were dissected, meninges removed, and tissue sliced into
small pieces. The tissue was briefly centrifuged and transferred to
solution A containing 0.25 mg/ml trypsin, then incubated at
37.degree. C. for 18 min. After the addition of 0.082 mg/ml trypsin
inhibitor (Boehringer Mannheim, Indianapolis, Ind.) and 0.25 mg/ml
DNase I (Boehringer Mannheim), the tissue was incubated at
25.degree. C. for 2 min. After a brief centrifugation, the
resulting pellet was gently titrated in solution A yielding
suspension that was further incubated for 10 min at 25.degree. C.
in solution A containing 2.7 mM MgSO4 and 0.03 mM CaCl2. After a
final centrifugation the pellet was resuspended in EMEM media
(Sigma, St. Louis, Mo.) containing 10% dialyzed FBS (Sigma), 25 mM
KCl, 2 mM glutamine (Life Technologies BRL, Gaithersburg, Md.), 25
mM glucose, and 0.1 mg/ml gentamycin (Sigma) and filtered through a
cell strainer (size 70 .mu.m; Falcon). Cells were plated on glass
coverslips coated with poly-D-lysine (Sigma) in Nunc four-well
dishes at a density of 1.5.times.10.sup.6 cells per milliliter of
medium. Cytosine-.beta.-arabinoside (10 .mu.M; Sigma) was added 24
hr after plating.
[0069] Test Compounds:
[0070] Several test compounds with potential zinc-ionophore
activity were screened for their ability to transport zinc into
selected target cells. In order to ascertain that the transported
ion was indeed Zn.sup.2, and not some other divalent cation
contaminant, the test compounds were first complexed with zinc. In
addition to the zinc-complexed ionophores (holo-ionophores), the
zinc-free forms of these compounds (apo-ionophores) were also
tested for the purpose of comparison. Whenever possible, purified
holo-ionophores were purchased commercially (e.g.
zinc-diethyldithiocarbamate, Sigma-Aldrich). However, in most cases
only the apo-ionophores were available commercially. The
holo-ionophores were therefore prepared in our laboratory. Since
zinc ionophores (e.g. pyrithione, diethyldithiocarbamate,
8-hydroxyquinoline) complex with zinc in a 2:1 molar ratio
(ionophore:zinc), stock solutions (generally 15.7 mM) of
holo-ionophores were prepared by combining the apo-ionophore with
ZnCl.sub.2 in a 2:1 molar ratio either in water or DMSO, depending
on the solubility of the reactants, and incubating at room
temperature for 15 min. The holo-ionophores were then stored at
-20.degree. C. Immediately prior to screening, the stock solutions
of these test compounds were thawed and diluted in the superfusion
buffer to give a final concentration of 1 .mu.M of the
holo-ionophore. When testing the apo-ionophores, an equivalent
molar concentration of the ionophore in the superfusion buffer (2
.mu.M) was used.
[0071] Ionophore Screening:
[0072] Screening of the test compounds was performed with cultured
HUVEC, isolated cardiac myocytes, and cultured cerebellar neurons
following an approach described previously (Turan et al., (1997)
Am. J. Physiol. 272:H2095-H2106). Immediately prior to screening,
the cells were loaded with Fura-2, a zinc and calcium-sensitive
indicator, by incubating the cells for 30 min in medium containing
4 .mu.M Fura-2-am (Molecular Probes). Glass coverslips bearing
HUVEC or cerebellar cells were placed directly in a superfusion
chamber on the stage of an epifluorescence inverted microscope
(Nikon Diaphot-DM). With isolated myocytes, an aliquot of Fura-2
loaded cell suspension was placed in the superfusion chamber and
the cells were allowed to adhere to the glass bottom of the chamber
before superfusion was started. The microscope field of view was
adjusted to include one or more individual cells. To establish
baseline fluorescence, the cells were first superfused for a few
minutes with a superfusing solution containing the following (in
mM): NaCl, 140; KCl, 5; MgCl.sub.2, 1; CaCl.sub.2, 2, HEPES, 5;
glucose, 10; pH adjusted to 7.4 with NaOH. The flow rate was
maintained at approximately 3 ml/min and the temperature at
37.degree. C. The cells were then superfused with superfusion
buffer containing a test compound and the fluorescence at 505 nm
was recorded in response to excitation at 340 nm and 380 nm. The
slope of the fluorescence intensity ratio in response to excitation
at 340 and 380 nm was used to determine ionophore activity. In each
test, the membrane-permeant heavy metal chelator
N,N,N',N',-tetrakis(2-pyridylmethy- l)ethylenediamine (TPEN, 30
.mu.M) was added to the superfusate at the end of the run. Since
TPEN does not chelate Ca.sup.2+, loss of fluorescence in response
to TPEN addition confirmed that the fluorescence was attributable
to zinc. In cases where test holo-ionophores did not demonstrate
zinc-ionophore activity, the validity of the negative observations
was confirmed by adding zinc-pyrithione (1 .mu.M) to the
superfusing solution at the end of the test. An increase in
fluorescence in response to the added zinc-pyrithione confirmed
that the cell being tested was viable and responsive.
[0073] Approximately 50 test compounds were screened for ionophore
activity using this approach (See Table 1). Of those, three groups
of compounds were found to be particularly active zinc ionophores:
pyrithione, dithiocarbamates, and hydroxyquinolines. Several
compounds which do not belong to these groups also showed ionophore
activity but at a lower level. The ionophore activity of pyrithione
appeared to be comparable in all three cell types tested, as were
the activities of diethyldithiocarbamate and
5,7-diiodo-8-hydroxyquinoline.
1TABLE 1 EXAMPLE OF ZINC IONOPHORES .check mark..check mark..check
mark. - excellent .check mark..check mark. - very good .check mark.
- good ZINC-PYRITHIONE .check mark..check mark..check mark.
ZINC-DITHIOCARBAMATES Pyrrolidinedithiocarbamate .check mark..check
mark. diethyldithiocarbamate .check mark..check mark. Disulfiram
.check mark..check mark. dimethyldithiocarbamate .check mark..check
mark. ZINC-HETEROCYCLIC AMINES 8-Hydroxyquinoline, .check mark.
5,7-Diiodo-8-hydroxquinoline .check mark..check mark. ZINC-NSAID
Indomethacin .check mark. ZINC-VITAMINS Vitamin A
(all-trans-retinol) .check mark. Vitamin E (alpha-tocopherol)
.check mark.
EXAMPLE 2
[0074] In vivo Heart Model--Ischemic Injury.
[0075] Experimental model: This model was designed to simulate
myocardial infarcts in rats. The protocol is a modification of one
described in detail previously (Fliss, H. et.al., (1996) Circ.Res.
79:949-956, incorporated herein by reference). Male Sprague-Dawley
rats (250-350 g) were anesthetized, the chest was opened, a
ligature was passed under the coronary artery and was then
fashioned into a snare. The coronary artery was then occluded for a
period of 45 minutes by tightening the snare, at which point the
snare was released and the ischemic myocardium was reperfused.
Following four hours of reperfusion the snare was re-tightened and
the area-at-risk was delineated by intraveneous injection of the
dye Evans blue. The rats were then killed immediately and the
area-at-risk was collected for analysis.
[0076] Previous studies have demonstrated that this model produces
extensive myocardial apoptosis (Fliss, H. (1996), supra). To
examine the protective ability of zinc-pyrithione in this model,
different cumulative doses (from 0.9 mg/kg body weight to 1.2 ug/kg
body weight) of this reagent in a 4% DMSO solution in sterile
saline were injected intravenously through a tail vein in three
equal boluses: at the initiation of reperfusion,-and at 1 and 2 h
after the.initiation of reperfusion. The total volume injected per
rat was 1.5 ml. To examine the protective properties of
zinc-diethyldithiocarbamate (ZnDDC) in this model, one dose of
ZnDDC (0.21 ug/kg body weight) was tested in a manner identical to
that used for zinc pyrithione.
[0077] Results: The data collected to-date show a strong trend
(P<0.1) towards anti-apoptotic effects with very low
concentrations of zinc-pyrithione. TUNEL staining (Fliss, H.
(1996), supra), as described below, was used to identify the
percent of apoptotic myocytes in the affected tissue. Parraffin
sections were deparaffinized and were subsequently permeabilized
with methanol/acetone (1:1) for 10 min at RT, and were washed twice
with PBS. They were then incubated with 20 .mu.g/ml proteinase K in
25 mmol/L Tris-HCl (1 ml/section), pH 6.6, for 15 min at RT, were
washed twice (15 min each) with water, were stained with Hoechst
33258 (0.05 mg/ml) for 30 min at RT, protected from light, and were
washed 3 times (1 min each) with PBS. The sections were then
incubated in 75 ml of a buffer solution containing 200 mmol/L
potassium cacodylate, 2 mmol/L CoCl.sub.2, 0.25 mg/ml bovine serum
albumin, 25 mmol/L Tris-HCl, pH 6.6, 10 mmol/L biotin-16-dUTP
(Boehringer Mannheim Canada, Laval, Quebec), and 25 units of
terminal transferase (Boehringer), for 1 h at 37.degree. C. in a
humidified chamber. The reaction was terminated by washing the
sections 3 times (1 min each) with PBS at RT.
[0078] The sections were then incubated with 1 ml of a staining
solution containing 2.5 mg/ml fluorescein isothiocyanate-avidin
(avidin-FITC), 4.times. saline-sodium citrate buffer, 0.1% Triton
X-100, and 5% powdered milk, for 30 min at RT, protected from
light. The sections were washed 3 times with PBS, were coverslipped
in "anti-fade" solution containing 1 mg/ml p-phenylenediamine, 90%
glycerol, in PBS, and histofluorescence was monitored with a Zeiss
Axiophot microscope. Positive control samples were prepared by
incubating sections with 10 units/ml DNAse I for 20 min at
37.degree. C. prior to treatment with terminal transferase. The
data demonstrate strong inhibition of apoptosis with 2.3 ug
zinc-pyrithione per kg body weight when compared to DMSO carrier
alone (P=0.067) (FIG. 1). Similar strong protection against
apoptosis was observed with 1.2 ug/kg zinc-pyrithione (P=0.053).
Since the zinc constitutes 20% by weight of zinc-pyrithione, the
data suggest that about 0.20 to about 0.50 ug of zinc per kg body
weight provides strong apoptosis protection. Strong protection
(P<0.05) against apoptosis was also observed with the single
dose of ZnDDC tested, i.e. 0.21 ug/kg body weight. Thus, the
percent apoptotic nuclei in the ZnDDC treated hearts was
9.1.+-.3.0, in comparison to the vehicle only group in which the
percent apoptosis was 15.6.+-.1.2.
[0079] Western blot analysis revealed that zinc-pyrithione (0.9
mg/kg.body weight) significantly increased the intracellular
content of Heat Shock Protein 70 (HSP70) in the ischemic myocardium
(FIG. 2). The content of the HSP70 in the ischemic myocardium was
determined using standard immunoblotting techniques. The heart
tissue was homogenized on ice for 45 s using a Polytron homogenizer
at 10,000 rpm in 8 volumes of 10 mM HEPES (pH 7.9), containing 10
mM KCl, 1.5 mM MgCl.sub.2, 0.1% Nonidet P-40, 0.5 mM DTT, 0.5 mM
PMSF, 0.5 mM spermidine, 0.15 mM spermine, 5 mg/ml aprotinin, 5
mg/ml leupeptin, and 5 mg/ml pepstatin. The homogenate was
incubated on ice for 15 minutes and centrifuged at 35,000.times.g
at 4.degree. C. for 15 min. Aliquots (15 mg protein) of the
supernatant were subjected to electrophoresis on 12% polyacrylamide
gels and were transferred to polyvinylidene difluoride (PVDF)
membranes. The membrane was incubated with a polyclonal rabbit
antibody against HSP70, followed by goat anti-rabbit IgG conjugated
to horseradish peroxidase (HRP). Protein band chemiluminescence was
visualized on film according to manufacturer's instructions (NEN
Life Science Products, Boston, Mass.), and was quantified with a
densitometer and Molecular Analyst Software (Bio-Rad Laboratories,
Hercules, Calif.). Since HSP70 has been shown to protect against
apoptosis (Wong, H. R., et al. (1996) Am.J.Respir.Cell Mol.Biol.
15:745-751), and to be induced by zinc (Klosterhalfen, B., et al.,
(1997) Shock 7:254-262), the data suggest that zinc-pyrithione may
exerts its protective effect by upregulating HSP70 synthesis.
Zinc-pyrithione also caused a statistically significant decrease in
the cytosolic concentration of PKC-.alpha. (data not shown), using
the same method as that described for HSP70, with the exception
that instead of using a polyclonal rabbit antibody against HSP70,
we used an antibody against PKC-.alpha.. As PKC-.alpha. is a well
known intracelular signalling agent, it was therefore concluded
that Zn.sup.2+ is capable of modulating intracellular
signalling.
[0080] The limited amount of myocardial tissue available from these
studies has not yet permitted the analysis of transcription factor
activity in this tissue. However, analysis of brain tissue from the
experimental rats by EMSA (electrophoretic mobility shift assays)
showed strong effects on the nuclear content of the two
transcription factors Sp1 and NF-kB. In the Electrophoretic
Mobility Shift Assay (EMSA), brain samples were-homogenized on ice
using six slow strokes of a Teflon pestle homogenizer at 1000 rpm
in 8 volumes of buffer containing 0.25 M sucrose, 10 mM HEPES, pH
7.6, 25 mM KCl, 1 mM EDTA, 10% glycerol, 0.15 mM spermine, and 0.5
mM spermidine. The homogenate was filtered through a 45 mm nylon
sieve and layered over a 10 ml cushion of 2 M sucrose containing 10
mM HEPES, pH 7.6, 25 mM KCl, 1 mM EDTA, and 10% glycerol. The
homogenate was centrifuged at 100,000.times.g at 4.degree. C. for 1
h, the supernatant was discarded, and the pelleted nuclei were
gently resuspended in 40 ml of a lysis buffer containing 20 mM
HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 25%
glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM
spermine, and 5 mg/ml each of aprotinin, leupeptin and pepstatin.
The suspension was incubated on ice for 45 min and centrifuged at
20,000.times.g at 4.degree. C. for 10 min. The supernatant
containing nuclear protein was collected and diluted 1:1 with a
buffer containing 20 mM HEPES, pH 7.9, 50 mM KCl, 0.2 mM EDTA, 20%
glycerol, 0.5 mM DTT, 0.5 mM PMSF, 0.5 mM spermidine, 0.15 mM
spermine, and 5 mg/ml each of aprotinin, leupeptin and pepstatin.
Protein concentrations were determined using the Bio Rad protein.
assay. For EMSA assays, double-stranded consensus oligonucleotides
for NF-kB, AP-1 and Sp1 (Promega, Madison, Wis.) were radiolabelled
with. g[.sup.32P]ATP (Amersham, Arlington Heights, Ill.).
[0081] Five mg of nuclear protein were first incubated for 10 min
at room temperature with 5 mg poly-d[I-C] (Boehringer Manheim,
Montreal, Quebec) in DNA binding buffer (20 mM HEPES, pH 7.9, 0.2
mM EDTA, 0.2 mM EGTA, 100 mM KCl, 5% glycerol, and 2 mM DTT).
Labeled probe (0.2 ng) was then added and the reaction mix
incubated for a an additional 20 min in a final volume of 20 ml.
The reaction mixture was subjected to electrophoresis on 5%
polyacrylamide gel, and the dried gel was exposed to X-ray film.
The intensity of the bands was quantitated with a densitometer and
commercially available software (Molecular Analyst, Bio-Rad
Laboratories, Hercules, Calif.). The subunit composition of NF-kB
was determined with supershift assays. Antibodies (2 mg) to either
p50 or p65 (Santa Cruz Biotechnologies, Inc. Santa Cruz, Calif.)
were added to the incubation mixture and incubated for 20 min prior
to the addition of poly-d[I-C]. No significant changes in the
nuclear content of Sp1 were observed in control rats infused with
zinc-pyrithione without coronary ligation, zinc-pyrithione strongly
protected the brain tissue against the sharp decline in Sp1 caused
by the ischemic episode (FIG. 3). Moreover, zinc-pyrithione
significantly increased the level of NF-kB in the brain of rats
subjected to myocardial ischemia, but had no effect on non-surgical
control brains (data not shown). In view of the well established
role of these transcription factors in apoptosis, our data suggest
that zinc-pyrithione may protect against apoptosis in the heart by
altering cell signalling and gene expression.
EXAMPLE 3
[0082] In vivo Brain Model--Kainic Acid Injury.
[0083] Glutamate is the principal excitatory. neurotransmitter in
the brain, and plays a critical role in the etiology of different
major brain pathologies such as cerebral ischemia,
neurodegeneration, epilepsy, etc (Coyle, J. T. (1993) Science
262:689-695). Compounds which interact with the glutamate receptors
are therefore important tools in the investigation of these
diseases. Kainate is an excitotoxic glutamate analog that produces
excessive neuronal excitation and seizures within hours following
its intraperitoneal injection into adult rats. At 2-3 days after
treatment, neurodegeneration can be observed in the limbic system
in the form of apoptosis (Gillardon, F., et al., (1995)
Neurosci.Lett. 192:85-88). Because the hippocampal subregions in
the rat, particularly the CA3, are enriched in kainate receptors,
they are particularly susceptible to kainate-induced neuronal death
(Meldrum, B. S. (1994) Neurology 44:S14-S23). The neurotoxic effect
of kainate in the rat hippocampal subregions involves a direct
effect on. presynaptic kainate receptors and an indirect effect on
postsynaptic glutamate receptors due to the enhanced. release of
glutamate (Malva, J. O., et al., (1998) Neurochem.Int. 32:1-6). It
appears likely that kainate-induced apoptosis is associated with
the production of reactive oxygen intermediates (Hirata, H. et al.,
(1997) Brain Res.Mol.Brain Res. 48:145-148; Zhang, X., (1997)
Eur.J.Neurosci. 9:760-769; Uz, T., et al., (1996) Neuroscience
73:631-636; Rong, Y. et al., (1996) J.Neurochem. 67:662-668), and
the modulation of intracellular zinc (Cuajungco, M. P. et al.,
(1997) Neurobiol.Dis. 4:137-169).
[0084] Experimental model: The experimental approach utilized in
these studies was the well-established model of neurotoxic injury
in rat brains induced by kainic acid (KA). Male Wistar rats
(250-350 g) were anethesized with chloral hydrate (325 mg/kg). KA
(10 mg/kg) was injected intraperitoneally. Zinc pyrithione in 1.2%
DMSO in sterile water was injected intracerebroventricularly
according to the following coordinates: AP-6.8, L+-1.5, DV+3.8 at
the dose of 1 pmol/ventricle. Rats which did not receive zinc
pyrithione were injected with 1.2% DMSO solution alone in water
intracerebroventricularly. zinc pyrithione was injected 15 min
after KA administration. Sham operated rats received 1.2% DMSO in
the lateral ventricles and isotonic saline. intraperitoneally. The
volume of substances injected into ventricles was 1.5 uL. Seizure
activity in the rats was followed during first 4-6 h after KA
administration. Gradation of KA-elicited limbic seizures was
carried out according to accepted protocols (Rong, Y. et al.,
(1996) J.Neurochem. 67:662-668). Sham operated, KA-treated and
KA/zinc treated rats were decapitated after 1-3 days. The brains
were removed, fixed in AFA, paraffin embedded, sectioned (10 .mu.m)
and stained by the Nissl method, using routine methodology.
[0085] Results: Brains of sham-operated rats and control animals
injected icv with zinc-pyrithione did not show any obvious damage
in any brain region. However, administration of KA caused neuronal
degeneration and cell loss in a number of brain regions, with
injury reaching maximal levels within the first day after KA
treatment. Preliminary data suggest that the brain damage is
attributable to apoptosis, as detected by the TUNEL stain (not
shown). Twelve rats were subjected to KA treatment alone and 12
were treated with KA followed by zinc-pyrithione. FIG. 6 shows the
number of rats (out of 12) that displayed detectable signs of
injury in each group. The extent of injury in tissue sections was
determined by thorough histological examination, comparing gross
anatomical features, the number of visible, intact nuclei in each
region, and the presence of other obvious signs of tissue injury.
Reproducible and pronounced damage was seen in the hippocampal
subregions CA1 and CA3, the pyriform cortex (PC), amygdalar region
(AM), and thalamus (TL) in the KA-treated group (FIG. 6). However,
icv administration of zinc-pyrithione provided statistically
significant protection in all regions with the exception of CA3,
where only a protective trend was observed (*, P<0.1 (trend);
**, P<0.05; ***, P<0.01, Fisher's exact test).
[0086] Zinc-pyrithione also changed the pattern of KA-induced
seizures in the rats. The seizure study was performed with 57 rats
in the KA-alone group, and 58 in the KA plus zinc-pyrithione group.
The data presented in FIG. 5 show the number of rats in which a
given seizure severity was the final stage of severity observed.
Notably, zinc-pyrithione caused a statistically significant
3.2-fold decrease in the incidence of the most severe and
irreversible stage of seizures ("jumping", stage 6). In other
words, of the 57 rats treated with KA alone, fully 18 reached the
lethal stage 6, as compared with only 5 out of 58 in the KA plus
zinc-pyrithione group. The data further show that the final stage
of severity tended to be much lower in the zinc-pyrithione group,
as illustrated by the 3.2-fold increase in the number of
zinc-pyrithione treated rats at level 2 ("wet dog shake" stage).
(*, P<0.01, Fisher's exact test). In summary, zinc-pyrithione
significantly decreased KA-induced cell death in a number of brain
regions, and significantly lowered the severity of KA-induced
seizures in rats.
[0087] Effect of Zinc Ionophores on the Subcutaneous PTZ Test in
Rats
[0088] The subcutaneous PTZ (pentylenetetrazole) test in rodents is
commonly used to identify compounds capable of raising seizure
threshold (White at al., Adv. Neurol., 76:29-39, 1998). The ability
of a test compound to inhibit PTZ-induced clonic and tonic
convulsions is currently regarded as predictive of efficacy against
"absence" seizures and/or myoclonic seizures (Suzdak, Jansen, 1995,
Epilepsia, 36(6):612-626). The pharmacological profile of the PTZ
test varies depending on the endpoint selected: PTZ can produce
myoclonic jerks, repeated clonic seizures of forelimbs and
hindlimbs with and without loss of righting reflex, and loss of
righting reflex followed by tonic extension of the forelimbs and
hind limbs. To have the full picture of the PTZ-induced seizures
and of the effects of zinc ionophores, and to make the model more
discriminative (White at al., supra) the behavioral reaction of
each rat was monitored for a period of 2 hours which is much longer
than the usual time period in conventional screening
experiments.
[0089] PTZ (120 mg/kg) was administered subcutaneously to male
Wistar rats (300-450 g). Zinc ionophores were injected i.p. in one
bolus, 30 min before PTZ at a dose of 62.5 nmol/kg and a total
volume of 0.25-0.4 ml. The following groups were studied: vehicle
(2.5% DMSO) n=20;
[0090] zinc pyrithione (ZP, 19.9 .mu.g/kg) n=10; zinc
diethyldithiocarbamate (ZDDC, 22.5 .mu.g/kg) n=19; and zinc
5,7-diiodo-8-hydroxyquinoline (ZDIHQ, 53.7 .mu.g/kg) n=10. Two of
the three zinc ionophores studied, ZDDC and ZP, significantly
decreased mortality in the PTZ model from 80% to 37% and 30%,
respectively (FIG. 7). ZDDC and ZP also significantly decreased the
number of rats displaying the most severe tonic seizures from 90%
to 63% and 60%, respectively (FIG. 8).
[0091] Kruskal-Wallis ANOVA showed a highly significant dependence
of survival time on zinc ionophores administration (Chi
square=16.7; P<0.001).
[0092] For those rats which died within 2 h ("non-survivors"), the
mean time to death increased significantly with all three
ionophores. The survival time increased by 1.5, 1.9, and 1.6 fold
for ZDDC, ZP, and ZDIHQ, respectively. Thus, zinc ionophores not
only decreased the PTZ-induced mortality, but also increased the
survival time of the non-survivors. Since tonic seizures are
believed to be closely related to mortality from seizures, the
decrease in their incidence after ZP or ZDDC injections may be one
of the reason for higher survival in these groups.
[0093] The latency of the onset of clonic and tonic seizures is a
good index of the anti-epileptic potential of the tested
substances. The ionophore data are presented in Table 2. All groups
administered ionophores showed longer mean latencies for all
seizure types and a statistically significant difference was
obtained for the latency of tonus for ZDIHQ. Statistical trends
were observed with ZDDC and ZDIHQ for the increase in latencies for
clonic seizures, and with ZDIHQ for the tonic seizures. It should
be noted that since tonic seizures are the most severe form of
seizure and in most cases lead to death, the observed delay in
tonic seizures is highly significant.
2TABLE 2 Effects of zinc ionophores on latencies of different
seizure types (min, M .+-. S.E.M.). Clonus without Clonus with loss
loss of righting of righting Group reflex reflex Tonic seizure
Vehicle 13.3 .+-. 1.1 15.8 .+-. 1.5 20.3 .+-. 1.7 n = 20 n = 19 n =
18 ZP 14.3 .+-. 1.8 16.8 .+-. 3.2 24.5 .+-. 5.5 n = 10 n = 9 n = 6
ZDDC 17.2 .+-. 1.6 18.5 .+-. 2.0 25.5 .+-. 3.1 n = 19; P < 0.06
n = 16 n = 12; P = 0.1 ZDIHQ 15.6 .+-. 1.7 21.2 .+-. 2.7 28.4 .+-.
4.1 n = 10 n = 10; P < 0.07 n = 9; P = 0.04 P values vs. vehicle
(T-test) are presented.
[0094] The data show that all three zinc ionophores are effective
in blocking the effects of PTZ, although their beneficial effect
varied with respect to different parameters of the PTZ-induced
pathology. ZP and ZDDC significantly decreased mortality and the
incidence of tonic seizures, whereas ZDIHQ delayed tonic seizures.
All ionophores increased survival time.
[0095] Effects of Zinc Pyrithione in Audiogenic Epilepsy Model in
Rats
[0096] Acoustic induction of seizures in rodents is a model of
generalized tonic-clonic limbic seizures induced by sudden exposure
to high intensity sound of up to 100-110 dB. This assay is normally
performed in genetically susceptible strains of rat
(Garcia-Cairasco et al., (1993), Behav. Bran. Res., 58(1-2):57-67;
1998; Zivanovic et al., (1998), Pharmacol. Res., 38(5):347-351), or
in susceptible animals which are identified by screening large
populations of rats. The latter approach was employed by the
present inventor.
[0097] Male Wistar rats (250-350 g) susceptible to audiogenic
epilepsy were identified by screening 200 rats. Animals that
developed seizures in response to a 60 dB sound were selected for
future testing (40 rats, 20% of the overall population). From this
group of susceptible rats, only 21 rats that displayed reproducible
seizures over a span of 3 weeks when stimulated once every 5-7 days
were selected for the study. The reproducibility of the audiogenic
response in these rats meant that each rat could serve as its own
control in these studies.
[0098] The studies were conducted as follows: each of the 21
selected rats was administered in the subsequent weeks either
vehicle alone, or ZP, or no injection, in a random, blinded
fashion, and the response to sound stimulation was recorded. ZP (36
.mu.g/kg) was administered 30 min before the acoustic stimulation
using a bolus intravenous injection of ZP in saline containing 2.5%
DMSO in a total volume of 0.2-0.4 ml. The vehicle-alone group
received only 2.5% DMSO. Control animals did not receive any
injection. Following two weeks of behavioral tests during which
seizure susceptibility was determined, electrodes were implanted in
the motor cortex of selected rats and EEG records were obtained
before and after audiogenic stimulation, as well as before and
after i.v. injection of ZP. The EEG studies aimed to examine the
possible correlation between the anti-epileptic effects of ZP at
the behavioral level and brain activity level. The scale used to
classify decreasing seizure severity was as follows: stage 1--wild
running; stage 2--clonic seizures without loss of righting reflex;
and stage 3--clonic seizures with the loss of righting reflex. The
sound intensity used in these studies (60 dB) was relatively mild,
and did not cause tonic seizures or mortality in the test
animals.
[0099] The data show that ZP delayed the onset of audiogenic
seizures, increasing the latency period by 36% from 42.7.+-.2.2 s
(vehicle) to 58.1.+-.3.9 s (FIG. 9) and decreasing the severity of
seizures by 41% from 1.55.+-.0.15 (vehicle) to 0.91.+-.0.11 (FIG.
10). ANOVA showed a strong dependence of seizure parameters on the
injectate (control, vehicle, ZP): for latency F=9.37, P=0.0003; for
seizure severity F=11.74, P=0.00005. Post-hoc comparisons using the
Duncan test showed a significant difference between the ZP group
and vehicle (P=0.0003for latency, and P=0.002 for seizure
severity). The post hoc test did not show a significant difference
between vehicle (DMSO) and control groups. The data therefore
demonstrate a highly significant protective effect by ZP in the
audiogenic seizure model in Wistar rats, showing both an increase
in latency and a decrease in seizure severity.
[0100] Of the 21 rats used in this study, ZP decreased the seizure
level in 52%. In comparison to vehicle alone, ZP significantly
increased the number of rats Which showed resistance to the
audiogenic stimulus, preventing seizures in fully 19% of the rats
(P<0.05; Chi-square test). ZP also decreased the fraction of
rats displaying clonic seizures from 48% to 14% (P<0.05;
Chi-square test), shifting the seizure profile to the less severe
levels. Thus, the data show a clear delay in the time to seizure
onset, as well as a reduction in seizure severity, as a result of
ZP administration.
[0101] The basal EEG's of the rats displayed a variety of
epi-signs: peaks, sharp waves, and peak-waves. Sound-induced
seizure was preceded by an increase in the incidence of peak groups
and other epi-signs (motor excitation produced some artifacts on
the EEG). Injection of ZP before the sound stimulation decreased
the incidence of peaks and other epi-signs in the basal EEG and
produced a regular theta-rhythm, with only single peaks evident in
the EEG. Audiogenic stimulation 15-30 min after ZP injection
induced the appearance of a regular theta-rhythm, an EEG correlate
of a normal arousal reaction to the sound stimulation. Though some
peaks were still evident, the clear dominance of theta-rhythm
(which was absent when only vehicle was injected) indicated a
normalization of the EEG. Thus, the seizure and EEG data
demonstrates a decrease in sound-induced seizure activity,
confirming an anti-epileptic activity by zinc pyrithione.
EXAMPLE 4
[0102] In vitro Neuronal Cell Model--Oxidative Stress
[0103] Oxidative stress is believed to play an important role in
the apoptotic neuronal cell death associated with many different
neurodegenerative conditions (e.g., Alzheimer's disease,
Parkinson's disease, cerebral ischemia, etc.) (Jenner, P. (1994)
Lancet 344:796-798). The non-differentiated rat pheochromocytoma
PC12 cells are a cell line which differentiates to a neuronal cell
type in the presence of Nerve Growth Factor (NGF), but undergoes
apoptotic cell death when deprived of NGF. These cells also undergo
apoptotic cell death when exposed to oxidants such as hydrogen
peroxide (Satoh, T., et al., (1997) J.Neurosci.Res. 50:413-420;
Maroto, R. et al., (1997) J.Neurochem. 69:514-523; Kubo, T., et
al., (1996) Brain Res. 733:175-183). PC12 cells are therefore a
useful cell model with which to analyze the molecular mechanisms of
apoptosis induced by oxidative stress and other stimuli in neuronal
cells (Kubo, T., et al., (1996) Brain Res. 733:175-183,
incorporated herein by reference).
[0104] Experimental model: PC12 cells were seeded on coverslips
covered with poly-L-lysine in 24-well plates, and were grown in
RPMI-1640 containing 10% FCS, 5% horse-serum, 2 mM glutamine, and
40 mg/kg gentamicin in 5% CO.sub.2 at 37.degree. C. RPMI-1640
medium containing 1% serum (embryonal calf and horse serum, 2:1)
and 50 ng/ml NGF was used to induce differentiation. Differentiated
PC12 monolayers were washed and were induced to undergo apoptosis
in two ways: a) incubation for 4 h with normal growth medium
without serum or NGF, and b) incubation for 4 h with normal growth
medium (plus serum and NGF) containing 20 .mu.M hydrogen peroxide
(H.sub.2O.sub.2). The protective effect of zinc-pyrithione against
both types of apoptosis was tested by preincubating the cells with
this compound for only 5 minutes immediately prior to initiating
apoptosis-inducing treatments a or b. Control cells were pretreated
with the carrier DMSO alone. Pretreatment with zinc-pyrithione,
rather than the more relevant post-treatment approach, was used in
this model because of practical experimental considerations.
However, the close temporal proximity of the pretreatment to the
initiation of the injurious treatment is more representative of a
concomitant exposure of the cells to both zinc-pyrithione and the
injurious agent, rather than an authentic pretreatment. At the end
of the 4 h incubation, the cells were fixed with methanol:acetone
(1:1) at -20.degree. C. and were stained with Hoechst 33258 to
visualize the nuclei.
[0105] Results: The data show that zinc-pyrithione (2-500 nM)
provided statistically significant protection against
H.sub.2O.sub.2-induced apoptosis, with 10 nM being the optimal
concentration (FIG. 6). Compared to control cells (+NGF), treatment
with H.sub.2O.sub.2 resulted in the loss of approximately 50% of
the cells through apoptosis (+NGF, +H.sub.2O.sub.2). However,
treatment with as little as 2 nM zinc-pyrithione for 5 min
significantly attenuated cell death. (*, P<0.005 vs. +NGF; +,
P<0.05 vs. NGF+H.sub.2O.sub.2; ++, P<0.02 vs.
NGF+H.sub.2O.sub.2, Mann-Whitney test, n=4).
[0106] In another series of tests (FIG. 11), the ability of
zinc-pyrithione to protect against H.sub.2O.sub.2 was confirmed,
and also demonstrated that this compound can block the apoptosis
caused by serum and NGF deprivation. Here too 10 nM appeared to be
the optimally protective concentration of zinc-pyrithione.
Zinc-pyrithione at concentrations as high as 1000 nM did not show
any effects on cell growth in control cultures. (*, P<0.05 vs.
+NGF; +, P<0.05 vs. respective "without ZP" group, Mann-Whitney
test, n=3). The addition of Zn.sup.2+ alone, at concentrations
equivalent to those of zinc-pyrithione, to H.sub.2O.sub.2-treated
cells did not block apoptosis (not shown), suggesting that the
ionophore pyrithione is necessary for the transport of Zn.sup.2+
into the cytosol, and that it is this intracellular Zn.sup.2+ which
accounts for the protective effects of zinc-pyrithione.
EXAMPLE 5
[0107] In vitro Endothelial Model--Response to Ionizing Radiation
and TNF.alpha.
[0108] Ionizing radiation has been shown to induce apoptosis in a
variety of cell and tissue types (Van Antwerp, D. J., et al. (1996)
Science 274:787-789; Findik, D., et al., (1995) J.Cell.Biochem.
57:12-21) including endothelial cells (Langley, R. E., et al.
(1997) Br.J.Cancer 75:666-672). Although the mechanisms by which
ionizing radiation induces apoptosis have yet to be resolved, it
has been shown to directly induce DNA damage, to generate the
formation of reactive oxygen species and to alter membrane
structure (Datta, R., et al. (1997) J.Biol.Chem. 272:1965-1969),
all of which can contribute to apoptotic cell death.
[0109] One well established effect of ionizing radiation is its
alteration of gene transcription by means of the modulation of
transcription factors. For example, radiation has been shown to
induce the activation of the transcription factor NF-kB in several
cell types (Valerie, K., et al., (1995) Biochemistry
34:15768-15776), including endothelial cells (Hallahan, D. et al.,
(1995) Biochem.Biophys.Res.Commun. 217:784-795). The cytokine
TNF.alpha. is also capable of causing apoptosis in endothelial
cells either alone (Slowik, M. R., et al., (1997) Lab.Invest.
77:257-267; Spyridopoulos, I., et al., (1998) Circulation
98:2883-2890) or synergistically with other agents (Eissner, G., et
al., (1995) Blood 86:4184-4193). Here too, the apoptotic events are
heavily regulated by alterations in transcription factor activity
(Hu, X. L., (1998) Blood 92:2759-2765). The studies performed with
this model were designed to examine the ability of zinc-pyrithione
to block radiation-induced apoptosis, and to elucidate the effect
of zinc-pyrithione at the transcriptional level in response to
either ionizing radiation or TNF.alpha..
[0110] Experimental model:
[0111] Cell Culture: Human umbilical vein endothelial cells (HUVEC)
were purchased from Clonetics (San Diego, Calif.) and used from
passages 2-4. Cells were cultured on gelatin-coated culture dishes
in Endothelial. Basal Medium (Clonetics) supplemented with 10 ng/ml
human recombinant epidermal growth factor, 1.0 ug/ml
hydrocortisone, 50 ug/ml gentamicin, 50 ng/ml amphotetericin B, 12
ug/ml bovine brain extract and 2% v/v fetal bovine serum, in a
humidified chamber at 37.degree. C. and 5% CO.sub.2. To maintain
cell populations, proliferating HUVEC were passaged at 80-90%
confluency.
[0112] Experimental Treatments: HUVEC were grown to confluency, and
then given an additional 24 hours to achieve quiescence prior to
experimental treatment. The following treatments were
performed:
[0113] Radiation: The cells were washed twice with 37.degree. C.
D-PBS and then irradiated in fresh media. Irradiated cells received
a dose of 1000 Rads of gamma-irradiation from a Cesium source
(Atomic Energy of Canada). The cells were then incubated for 2
hours (cytosolic and nuclear protein extraction) or 8 hours
(Hoechst staining and DNA electrophoresis).
[0114] TNF.alpha.: The cells were washed twice with 37.degree. C.
D-PBS and then incubated in media containing the TNF.alpha. (20
ng/ml, from a stock of 10 .mu.g/ml prepared in phosphate buffered
saline (PBS)-1% bovine serum albumin. Control cells received fresh
media alone. The cells were then incubated for 2 hours (cytosolic
and nuclear protein extraction) or 8 hours (Hoechst staining and
DNA electrophoresis).
[0115] Hoechst Staining: Cells were grown on round, gelatin coated
12 mm glass coverslips, and following treatment, were fixed
with-0.5 ml of 1% glutaraldehyde in PBS for 10 minutes at room
temperature (RT). The cells were then. washed twice with PBS for 5
minutes, and permeabilized with 0.5 ml of 1:1 methanol/acetone for
10 minutes at RT, followed by two five minute PBS washes. The cells
were then incubated with Hoechst 33258 (bis-benzimide, 0.05 mg/ml
in H.sub.2O), a fluorescent DNA binding dye, for 30 minutes at room
temperature, in the dark. The nuclear morphology of the cells was
then visualized under a Zeiss Axiophot fluorescence microscope.
[0116] Preparation of Cytosolic and Nuclear Extracts: Cells were
grown on 100 mm.sup.2 culture dishes, and following treatment, were
scraped into ice cold PBS and collected by centrifugation at
200.times.g for 5 minutes. The cells were then resuspended and
washed once in 1 ml of ice cold PBS and centrifuged at 200.times.g
for 5 minutes at 4.degree. C. The cells were resuspended in 1 ml of
Buffer A (10 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, pH=7.9, 1.5 mM DTT
and 0.5 mM phenyl methyl sulphonyl fluoride (PMSF)) and centrifuged
at 200.times.g for 5 minutes at 4.degree. C. The cells were then
resuspended and lysed in 300 ul of Buffer A containing 0.1% Nonidet
P-40 for 25 minutes on ice. The homogenate was then spun at
20,000.times.g for 10 minutes at 4.degree. C. The supernatant
containing cytosolic proteins was combined with an equal volume of
Buffer C (20 mM HEPES, 50 mM KCl, 1.0 mM EDTA, 0.1 mM EGTA, 20%
glycerol, pH=7.9, 0.5 mM DTT and 0.5 mM PMSF) and was stored at
-80.degree. C. The pelleted nuclei were washed by resuspension in 1
ml of Buffer A and spun at 20,000.times.g for 1 minute. The
supernatant containing residual cytosolic proteins was discarded
and the pelleted nuclei were resuspended in 35 ul of Buffer B (20
mM HEPES, 420 mM NaCl, 1.5 mM MgCl.sub.2, 0.2 mM EDTA, 25%
glycerol, pH 7.9, 0.5 mM DTT, 0.5 mM PMSF, and the protease
inhibitors spermidine, spermine, aprotinin, leupeptin and
pepstatin) for 45 minutes on ice in order to extract the nuclear
proteins. The nuclear extract was then obtained following
centrifugation at 20,000.times.g for 15 minutes at 4.degree. C.,
and was combined with an equal volume of Buffer C and stored at
-80.degree. C.
[0117] Determination of Protein Concentration: The protein
concentration in the nuclear and cytosolic extracts was determined
using the Bradford Assay (Biorad) using bovine serum albumin as the
standard.
[0118] Electrophoretic mobility Shift Assay (EMSA): Equal amounts
of nuclear protein (5 ug) were incubated with poly dI-dC (5 ug from
a stock of 2.5 ug/ul in TE buffer) for 10 minutes at RT. This
reaction mixture was then incubated with 0.2 ng of 5'
end-.sup.32phosphorus-labelled double stranded oligonucleotide
probe for 20 minutes at RT to allow the binding of nuclear proteins
with the labeled probe. Loading buffer (5 ul of a mixture
containing 20 mM HEPES, 100 mM KCl, 60% glycerol, 0.5 mM EDTA, 0.5
mM EGTA and 0.125% bromophenol blue) was added to the reaction
mixture prior to the electrophoresis on a 5% native polyacrylamide
gel. The gels were run in Tris-Glycine solution for 1.5 hours at
200V and were then dried between filter paper and cellophane for
1.5 hours at 80.degree. C. under vacuum. The dried gels were
exposed to X-ray film (Cronex) for up to 2 days at -80.degree. C.
For competition assays, the reaction mixture was incubated with a
125-fold excess of unlabeled probe for 20 minutes at RT prior to
the addition of the labeled probe. For supershift assays, the
reaction mixture was incubated with 2 mg of rabbit polyclonal
anti-NFkB p50 or p65 antibody (Santa Cruz Biotechnology) for 20
minutes at RT immediately subsequent to the addition of the labeled
probe. The bound antibody retards the mobility of the protein-DNA
complex, resulting in a shifted band. The consensus
oligonucleotides for the transcription factors NFkB (5'-ACT TGA GGG
GAC TTT CCC AGG C-3'), AP-1 (5'-CGC TTG ATG AGT CAG CCG GAA-3') and
Sp1 (5'-ATT CGA TCG GGG CGG GGC GAG C-3') (Promega) and were
labeled as suggested by Promega with minor modifications. Briefly,
oligonucleotides (20 ng), T4 Polynucleotide kinase and [g32P]ATP
(60 uCi) were mixed in kinase buffer (50 mM Tris-HCl, pH 7.6; 10 mM
MgCl.sub.2, 5% glycerol and 5 mM DTT) and incubated at 37.degree.
C. for 1 hour. Labeled oligonucleotides were removed by
centrifugation through a G-25 Sephadex Column at 8500 rpm for 20
minutes. The labeled oligonucleotides were then diluted such that 2
ul of the probe mixture contained approximately 50000-100000
cpm.
[0119] Western Blotting: Equal amounts of cytosolic protein (3 ug)
were diluted 1:1 in sample buffer (0.125M Tris-HCl pH 6.8, 2.6%
SDS, 25% glycerol, 0.1 ml beta-mercaptoethanol and bromo phenol
blue). The mixture was placed in boiling water for 5 minutes to
denature the proteins and was then subjected to SDS-PAGE for 2.5
hours at 100V in running buffer. The gels consisted of a stacking
gel (4.5% acrylamide, 0.125M Tris-HCl pH=6.8, 0.1% SDS, 0.6%
ammonium persulfate and 0.2% TEMED in H.sub.2O) and a 10% running
gel (10% acrylamide, 0.3% bis acrylamide, 8% glycerol, 0.375
Tris-HCl pH=8.8, 0.1% SDS, 0.04% ammonium persulfate and 0.05%
TEMED in H.sub.2O). After electrophoresis (100V, 50 minutes), the
gels were equilibrated for 15 minutes in ice cold transfer buffer
(25 mM Tris HCl, 20% methanol, and 192 mM glycine), then
transferred onto a polyvinyllidene difluoride membrane for 1 hour
at 100V. The blots were then blocked overnight in 5% skimmed milk
in Tris-Buffered Saline containing 0.1% Tween-20 (TBS-T) at
4.degree. C. with constant shaking. The blots were then washed with
TBS-T and incubated for 1.5 hours in primary antibody (anti-IkBa,
Santa Cruz Biotechnology) diluted 1:1000 in 2% skimmed milk in
TBS-T and sodium azide at RT with constant shaking. The blots were
then washed with TBS-T and incubated for 30 minutes in horseradish
peroxidase labeled goat anti-rabbit IgG diluted 1:10000 in 2%
skimmed milk in TBS-T at RT with constant shaking. Following
treatment with the secondary antibody, the blots were extensively
washed with TBS-T and incubated for 1 minute with chemiluminescent
substrate. The blots were then exposed to X-ray film for 1-5
minutes;
[0120] Results: The data show protection by zinc-pyrithione against
endothelial apoptosis, and also show that this protective effect is
associated with transcriptional modulation. Treatment of irradiated
HUVEC (IR) with zinc-pyrithione significantly blocked apoptosis
(FIG. 12). The data also show that zinc is required for this effect
since the sodium salt of pyrithione was not effective in preventing
apoptosis. DMSO alone was also ineffective. No apoptosis was caused
in control cultures by zinc-pyrithione, sodium-pyrithione, or DMSO
alone (FIG. 12). (*, P<0.05 vs. Control; .degree., P<0.05 vs.
irradiated cells, n=4).
[0121] EMSA tests showed that irradiation-induced apoptosis is
associated with a significant increase in nuclear NF-kB content,
and that zinc-pyrithione, but not sodium-pyrithione or DMSO alone,
blocked this increase (FIG. 13). Zinc-pyrithione also lowered the
nuclear content of AP-1, but did not appear to affect Sp1 in this
model. (*, P<0.05 vs. Control; .degree., P<0.05 vs.
irradiated group, n=4). Zinc-pyrithione had a very similar effect
in TNF.alpha.-treated HUVEC (FIG. 14, and was particularly potent
at blocking the TNF.alpha.-induced increase in NF-kB content. (*,
.degree., same as above, n=3). Since NF-kB is associated with the
cytosolic inhibitor IkB which governs its activity in the cell, the
effect of zinc-pyrithione on the cytosolic level of this protein
was examined. The data show that zinc-pyrithione lowered the
cytosolic content of IkB in cells treated with either radiation or
TNF.alpha. (FIG. 15).
EXAMPLE 6
Effects of Zinc Pyrithione on Models of Ischemic Stroke in
Rodents
[0122] Stroke is an extremely variable clinical condition which
reflects the variability of the underlying disease process. The
vascular occlusion can occur at many different sites in the brain
and the cause of the occlusion, the severity of the problem, and
the degree of reversibility can all contribute to the variability
of outcome. In contrast, in experimental animal models most of
these variables can be controlled or eliminated, enabling a
meaningful interpretation of the results. Generally, stroke models
are grouped into those producing either global or focal ischemia
(Ginsberg, M. D. & Busto, R. (1989) Stroke, 20:1627-1642,
incoporated herein by reference; Ginsberg, M. D. & Busto, R.
(1998) Small-Animal Models of global and focal cerebral ischemia.
In Cerebrovascular Disease: Pathophysiology, Diagnosis, and
Management (ed. Malden, M. A.), pp. 14-35, Blackwell Science,
incoporated herein by reference). It is generally understood that
global models are more relevant to cardiac arrest, while focal
models are of greater relevance to acute ischemic stroke.
[0123] To study the possible neuroprotective effects of zinc
pyrithione (ZP) two models of stroke were used: a global
ischemia-reperfusion model of 4 vessel occlusion in rats (4VO) and
a focal ischemia model of middle cerebral artery occlusion in mice
(MCAO). The 4VO approach sealed off the two carotid and two
vertebral arteries which carry all the blood to the brain. This
approach permitted severe forebrain ischemia to be produced in
awake and freely moving rats, and produced reproducible
neuropathology. The 4VO is a two-stage operative procedure
(Pulsinelli, et al., (1979) Stroke, 10: 267-272, incorporated
herein by reference). In the first stage, the vertebral arteries
were exposed and permanently sealed by electrocauterization
(Pulsinelli, et al., (1988) Stroke, 19:913-914, incorporated herein
by reference). This occlusion of the vertebral arteries does not in
itself cause serious injury in the rat. It is the second-stage
which initiated the injurious ischemic episode. It was performed 24
h later and involved the brief occlusion of the carotid arteries,
shutting off all blood flow to the brain. Although there was a
marked mortality during both stages even in laboratories which are
highly experienced in this procedure (Ginsberg, et al., (1989)
supra) the 4VO is a favorite stroke model because it results in
highly reproducible damage in the CA1 region of the hippocampus, as
well as in some other brain regions.
[0124] MCAO in mice is one of the most clinically relevant stroke
models. It shuts off blood flow to only a portion of the brain,
producing a focal injury which closely resembles the clinical
situation with stroke patients. This procedure was performed with
an intraluminal thread. A nylon suture was introduced into the
external carotid artery and was gently advanced into the internal
carotid artery. The diameter of the suture was such that it lodged
in the anterior cerebral artery, occluding the medial cerebral
artery at its origin. Brain damage in this model was observed as
early as several hours after the ischemic episode, with optimal
injury occurring at 24 h. The injury occupied a large part of the
hemisphere including the cerebral cortex and subcortical
structures, and its severity depended on the duration of ischemia
and the strain of mice used.
[0125] Animals
[0126] The 4VO procedure was performed on male Wistar rats weighing
240-300 g. The rats were housed in groups of 5 in plastic cages
with free access to food and water. The MCAO protocol was performed
on male C57BL/6 mice weighing 20-28 g which were housed in groups
of 10 in plastic cages with free access to food and water. All
experiments were performed in accordance with the National
Institutes of Health Guidelines for the Care and Use of Laboratory
Animals.
[0127] Surgeries
[0128] 4VO in Rats
[0129] Rats under chloraI hydrate anesthesia (325 mg/kg) were
positioned in a stereotaxic frame. The vertebral arteries were
exposed and permanently occluded by electrocautery at the first
cervical vertebra. Snares (surgical silk strings) were then placed
loosely around each common carotid artery without interrupting the
carotid blood flow. The animals were then allowed to recover for 24
hours with free access to water. On the following day, the rats
were lightly anesthetized with ether, were secured to surgical
boards, ventral side up, and their common carotid arteries were
exposed. Forebrain ischemia was initiated by tightening the snares
around the carotid arteries for 10 min. The body temperature of the
rats was carefully maintained at .about.37-37.5.degree. C., both
before and during the ischemic insult, using a feedback-controlled
heating pad and a rectal thermistor (Homeothermic Blanket System,
Harvard Apparatus LTD, England). The initial (1.sup.st min of
ischemia) and final (10.sup.th minute of ischemia) temperature did
not differ among all groups of rats studied. After the ischemic
episode, the temperature was maintained in similar fashion at
37.degree. C. for at least 4 h. Only rats that showed signs of
severe neurological injury, such as a loss of the righting reflex,
pupil dilation, etc., were included in this study. In the
sham-operated controls, the vertebral and carotid arteries were
exposed, but were not occluded. Evaluations of neurological deficit
were performed at 24 and 96 h after ischemia, and were based on a
scoring system which recorded activity level, motility, pain
reflex, grabbing reflex, and the ability to see and hear
(Miljkovic, L. M., et al., (1997) Ann. Emerg. Med. 29, 758-765,
incorporated herein by reference).
[0130] MCAO in Mice
[0131] Mice (C57BL/6) were anesthetized with an intraperitoneal
injection of chloral hydrate (350 mg/kg) and xylasine (4 mg/kg).
Focal cerebral ischemia was produced by occlusion of the MCA using
the intraluminal filament technique (Longa, Z. E., et al., (1989)
Stroke 20:84-91, incorporated herein by reference). A 8.0 nylon
microfilament coated with a silicon resin (Xantopren)-hardener
mixture (Hara, H., et al., (1996) J. Cereb. Blood Flow Metab.
16:605-611, incorporated herein by reference) was inserted into the
left common carotid-artery, and was advanced 10-11 mm distal to the
carotid bifurcation so as to occlude the MCA and posterior
communicating artery. The filament was left in this position for 1
h. For reperfusion, the animals were re-anesthetized briefly and
the filament was withdrawn to restore the blood flow. Core
temperature was maintained at .about.37.degree. C. with a
homeothermic blanket for a period of 2 h following reperfusion. The
neurological deficit caused by the ischemic insult was scored after
2 h of reperfusion according to the scheme of Bederson, J. B., et
al. (1986) Stroke 17:472-476, incorporated herein by reference: 0,
no observable neurological deficit (normal); 1, failure to extend
the right forepaw (mild); 2, circling to the contralateral side
(moderate); 3, falling to the right (severe); 4, inability to walk
spontaneously (most severe).
[0132] Zinc Ionophore Treatments
[0133] The zinc pyrithione (ZP) data presented were derived with a
treatment protocol in which ZP was injected in three boluses, at 10
min, 1 h, and 2 h after the termination of the ischemic episode,
through a tail vein catheter. With the 4VO rat model, four doses of
ZP: 3.times.2 .mu.g/kg, 3.times.6 .mu.g/kg, 3.times.30 .mu.g/kg,
and 3.times.200 .mu.g/kg were tested. With the mouse MCAO model
only the three lowest concentrations of ZP were tested. Some data
were also obtained with a second ZP treatment protocol in which
three boluses of 6 .mu.g/kg (3.times.6 .mu.g/kg) were injected at
3, 4, and 6 h after the termination of the ischemic episode. In
addition, a treatment protocol with zinc-diethyldithiocarbamate
(ZnDDC) was performed with both the 4VO and MCAO models using the
dose of 3.times.7.6 .mu.g/kg, a regimen which delivers zinc at a
dose equivalent to that. delivered with 3.times.6 .mu.g/kg of zinc
pyrithione.
[0134] The zinc ionophore solutions were prepared by diluting a
stock solution of ZP and ZnDDC in DMSO with saline. The final
concentration of DMSO in the injectate was 2.5%. Control animals
receiving vehicle-alone were injected with 2.5% DMSO in saline.
[0135] Histology
[0136] 4VO
[0137] Four days post surgery, control and experimental animals
were deeply anaesthetized with sodium thiopental (60 mg/kg,
intraperitoneally) and were perfused transcardially with 250 ml of
AFA fixative (96% alcohol, 39% formalin, glacial acetic acid,
7:2:1). After the AFA perfusion the heads were collected intact and
were kept at 4.degree. C. for 4-5 h. The brains were then removed
and immersed in the same fixative for 1 h, and were then stored in
70% alcohol. Each forebrain was cut into three frontal blocks and
imbedded in paraffin. Ten .mu.m thick sections were cut from a
region 3.0-4.0 mm posterior to the bregma. The sections were
stained with cresyl violet (Nissl). Computer images of the stained
sections were prepared, and the total number of viable pyramidal
neurons was counted in a 500 .mu.m-long section of the CA1 region
in the hippocampus. The person doing the cell counts was blinded as
to the identity of the experimental groups. In Situ End Labeling
(ISEL), a protocol for identifying apoptotic cells by staining
fragmented DNA, was performed according to a protocol developed in
the laboratory of Dr. Fliss (Schmidt-Kastner, et al., (1997) Stroke
28: 163-170, incorporated herein by reference) using deparaffinized
10 .mu.M thick brain sections from rats sacrificed at 24 h and 96 h
after ischemia. Fluorescence was monitored with a Zeiss Axioplan
microscope.
[0138] MCAO
[0139] Mice were killed 24 h after reperfusion with an overdose of
sodium thiopental (60 mg/kg, intraperitoneally), and the brain was
rapidly removed and sectioned coronally into five 1.7 mm slices.
The slices were then placed in 2% (wt/vol)
2,3,5-triphenyltetrazolium chloride solution (TTC) in PBS (pH 7.4)
for 20 min at 37.degree. C. This procedure, which tests
mitochondrial activity, stains viable tissue a bright red, while
the infarcted regions remain white. Following TTC staining the
sections were fixed in 10% formalin overnight. The area of infarct
in each section was determined using an image-analysis system. The
infarct volume was subsequently calculated by summing the infarct
areas in the sequential 1.7 mm-thick sections with correction for
edema. The person measuring infarct volumes was blinded as to the
identity of the experimental groups. For ISEL staining, the brains
were removed, were frozen rapidly, and were sectioned with a
cryostat into 20 .mu.m sections from the anterior side to the
posterior side at 500 .mu.m intervals. ISEL was performed as
described above. Adjacent sections were stained with cresyl violet
(Nissl). The ApopTag.RTM. Peroxidase In situ Apoptosis detection
Kit (Intergen) was also used to detect apoptosis in mouse brain
sections.
[0140] Statistics
[0141] The data were expressed as means .+-.S.E.M. One way-ANOVA
with post hoc Duncan's test or T-test for independent samples were
used for statistical analysis of data.
[0142] Zinc Ionophore Toxicity
[0143] No noticeable changes in behavior or appearance were
detected in animals injected with lower doses of ZP, or the single
dose of ZnDDC, when compared to those receiving vehicle alone. The
weight loss in rats after 4VO (7.5-16.9% at 24 h after ischemia and
4.1-11.2% at 96 h after ischemia) did not differ between the
vehicle-treated and ZP-treated groups.
[0144] 4VO Model (10 min-1 h-2 h-injection Schedule) Neuronal Cell
Loss
[0145] Approximately 15% of the neurons in the CA1 region survived
the ischemic insult in the vehicle-treated group when compared to
the sham-operated animals. However, treatment with ZP showed
pronounced protection and increased the number of viable cells
(FIG. 16). The ZP-mediated increase in cell survival varied
(1.6-3.5 fold) and did not show a clear dose-dependence (FIG. 17A).
Although all doses of ZP tested showed evidence of protection, the
3.times.6 .mu.g/kg group reached statistical significance with
approximately 52% of the pyramidal cells surviving the ischemic
episode. One-way ANOVA (sham, vehicle, and ZP-treated groups)
showed a significant dependence of the viable cell number in the
CA1 on the experimental treatment conditions (F=12.9,
P<0.000001). The post hoc Duncan's test showed the difference of
all ZP and vehicle-treated groups from shams at P<0.01, with the
ZP 3.times.6 .mu.g/kg group significantly different from vehicle at
P<0.01, and the 3.times.200 .mu.g/kg ZP group showing a trend at
P<0.09. Thus, 3.times.6 .mu.g/kg appeared to be the optimal
protective dose of ZP in this model. However, the trend for
significant protection shown by the 3.times.200 .mu.g/kg ZP group,
and the collective evidence of protection at the other doses
suggested that ZP is protective over a broad range of doses.
[0146] In addition, with ZnDDC at 3.times.7.6 .mu.g/kg, the number
of viable neurons in the CA1 region was 99.5.+-.19.9, showing that
the protection with this dose of ZnDDC was similar to that achieved
with 3.times.6 .mu.g/kg of ZP, a dose which delivered the same
amount of zinc (FIG. 17B).
[0147] ISEL Staining
[0148] As described above, ZP at 3.times.6 .mu.g/kg showed
significant protection against neuronal cell death. However, the
data do not indicate if this cell death was apoptotic or necrotic
in nature. To determine if the ZP-dependent increase in cell
survival was attributable to a lower incidence of apoptosis we
performed ISEL on brain sections.
[0149] At 24 h after ischemia no apoptotic cells were observed in
the sham, vehicle-treated, or ZP (3.times.6 .mu.g/kg)-treated
groups (n=5 for each group), suggesting that apoptosis required a
longer post-ischemic period to manifest itself in this model.
Apoptosis was more commonly observed at 96 h after ischemia.
Numerous ISEL-positive nuclei were observed in the hippocampi of
vehicle-treated rats (131.4.+-.14.6 in CA1 region, n=5). However,
in rats treated with 3.times.6 .mu.g/kg ZP, the number of apoptotic
nuclei was approximately 4 times lower (33.6.+-.7.8, n=9,
P<0.0001, t-test) (FIG. 18). No apoptosis was detected in any
brain region of the sham-operated rats. The data therefore
indicated that ZP has a potent anti-apoptotic effect in this model,
and that it is this effect which accounted for the observed
neuronal protection.
[0150] Neurological Deficit
[0151] The neurological deficit data in 4VO rats are presented in
Table 3. A method of scoring in which increasing neurological
scores are indicative of decreasing neurological function compared
to a perfect score of 0 for the shams was employed. For example, a
large number of the 4VO rats did not show any neurological deficit
(score 0), despite the fact that they sustained an almost complete
loss of cells in the CA1 region. Therefore, the administration of
ZP did not influence the neurological deficit in the 4VO rats
despite clear evidence of histological protection.
[0152] 4VO-model (3 h, 4 h, 6 h Injection Schedule)
[0153] Administration of ZP in boluses of 6 .mu.g/kg at 3, 4, and 6
h after the ischemic episode (3.times.6 .mu.g/kg) significantly
increased neuronal viability in the CA1 region, with the number of
viable neurons increasing 2.5 fold vs. the vehicle-treated group.
The cell count was 73.2.+-.15.0 in the ZP-treated group vs.
29.2.+-.7.8 in the vehicle-treated group (P<0.03, t-test). The
viable cell counts in the 3.times.6 .mu.g/kg groups of both
injection regimens (10 min, 1 h, 2 h or 3, 4, and 6 h after
ischemic episode) did not differ significantly (103.5.+-.15.2 vs.
73.2.+-.15.0). The neurological deficit data for this ZP
administration schedule are presented in Table 3. The neurological
score in "3, 4, 6 h schedule" rats did not differ from that in the
other regimen (10 min, 1 h, 2 h after ischemic episode). These data
therefore indicated that delaying the first administration of ZP by
3 hours did not significantly change its neuroprotective effect at
the dose of 3.times.6 .mu.g/kg, indicating a possible wide
therapeutic window for ZP in the 4VO stroke model.
[0154] MCAO
[0155] MCAO for 1 h produced significant infarcts in the left
hemisphere of mouse brain (FIG. 19). Three doses of ZP were used
(3.times.1.2 .mu.g/kg, 3.times.6 .mu.g/kg, and 3.times.30 .mu.g/kg)
and infarct areas, infarct volumes and neurological scores were
measured. ZP at all three doses significantly decreased the infarct
area at a distance of 3.4-5.8 mm from the frontal pole (FIG. 20A).
One-way ANOVA (vehicle and ZP-treated groups) showed a dependence
of infarct size in sections 3, 4, and 5 (3.4, 4.1, and 5.8 mm from
the frontal pole) on the experimental treatments with F=7.8
(P=0.0003), 13.0 (P=0.000003), and 11.7 (P=0.00001), respectively.
The post hoc Duncan's test showed a significant difference between
infarct size and vehicle at P<0.01 in sections 3-5 for each ZP
dose. The infarct size did not differ significantly between the ZP
groups. In addition, ZnDDC at 3.times.7.6 .mu.g/kg also
significantly decreased the infarct area in this model (FIG.
20B).
[0156] Table 4 presents data on the absolute and relative (% of the
contralateral hemisphere) infarct volumes in the mouse MCAO model
in response to zinc pyrithione treatment. The calculated infarct
volumes in the ischemic control mice were similar to those reported
previously for this strain of mouse (Hara, H., et al. (1996) J.
Cereb. Blood Flow Metab. 16:605-611; MacManus, J. P., et al.,
(1999) NeuroReport 10:2711-2714; Nagayama, M., et al., (1999) J
Cereb. Blood Flow Metab. 11:1213-1219; Nogawa, S., et al., (1998)
Proc. Natl. Acad. Sci. U S A. 95: 10966-10971; Takagi, Y., et al.,
(1999) Proc. Natl. Acad. Sci. U S A. 96:4131-4136). Vehicle alone
(2.5% DMSO) produced apparently contradictory effects. It tended to
increase the infarct volume but concomitantly decreased the
neurological score. However, these effects were not statistically
significant.
[0157] The administration of ZP at all three doses statistically
significantly decreased both the absolute (data not shown) and
relative infarct volumes by 29.0-38.2% and 30.8-40.0%, depending on
the dose, respectively) compared with the vehicle-treated group
(FIG. 21A). Comparison of control, vehicle-, and ZP-treated groups
using one-way ANOVA showed a significant dependence of both
absolute and relative infarct volumes on the experimental
treatments (F=10.1, P<0.00005; F=8.2, P<0.00005,
respectively). Highly significant differences (Duncan's test)
between the control and each of the ZP groups (p<0.05), as well
as between the vehicle and the ZP groups (P<0.001) was shown for
both the absolute and relative infarct values. In addition, ZnDDC
at 3.times.7.6 .mu.g/kg also significantly decreased both the
absolute and relative infarct volumes in this model (FIGS. 21B and
21C).
[0158] Clear evidence of protection by ZP was also observed with
the neurological score (FIG. 22). In the vehicle treated groups 4
out of the 10 mice (40%) showed. no apparent neurological deficits
(zero neurological score) even though they had significant
infarctions. Such animals were not common in the control group
(12.5%). In contrast, in the ZP-treated groups the number of mice
developing infarct but having.no neurological deficit increased to
84.6% in the 3.times.1.2 .mu.g/kg group, 61.5% in the 3.times.6
.mu.g/kg group, and 63.6% in the 3.times.30 .mu.g/kg group.
[0159] Moreover, no severe deficits (neurological score of 3 or 4)
were observed in the ZP-treated groups (the only exception was one
mouse with the score of 3 in the 3.times.30 .mu.g/kg group).
One-way ANOVA showed a statistically significant dependence of
neurological score on ZP (F=2.9, P<0.05). The difference between
the vehicle and the 3.times.1.2 .mu.g/kg group was significant
(P<0.01, post hoc Duncan's test), while the difference between
the vehicle and the two other ZP doses tended to be significant
(P<0.06, trend).
3TABLE 3 Neurological deficit in 4VO rats (neurological score, mean
.+-. S.E.M.) Group N 24 h 96 h Vehicle 8 2.1 .+-. 1.0 1.4 .+-. 0.9
ZP 3 .times. 1.2 .mu.g/kg 9 2.1 .+-. 0.9 0.4 .+-. 0.2 ZP 3 .times.
6 .mu.g/kg 10 1.4 .+-. 0.4 1.4 .+-. 0.6 ZP 3 .times. 30 .mu.g/kg 5
2.8 .+-. 1.0 1.6 .+-. 0.7 ZP 3 .times. 200 .mu.g/kg 5 2.4 .+-. 1.3
2.0 .+-. 1.1 ZP 3 .times. 6 .mu.g/kg* 6 1.3 .+-. 0.7 0.8 .+-. 0.4
*ZP administered 3, 4, and 6 h after the ischemic episode.
[0160]
4TABLE 4 Infarct volumes and neurological deficit in mice following
60 min MCAO (mean .+-. S.E.M.) Infarct volume, % Infarct of
contralateral Neurological Group N volume, mm.sup.3 hemisphere
deficit Control (no 9 61.2 .+-. 3.6 33.6 .+-. 2.5 2.6 .+-. 0.5
injections) Vehicle-treated 10 71.3 .+-. 3.9 41.5 .+-. 4.6 1.7 .+-.
0.5 ZP 3 .times. 1.2 .mu.g/kg 13 44.0 .+-. 2.6***' 24.9 .+-.
1.5***' 0.3 .+-. 0.2**" ZP 3 .times. 6 .mu.g/kg 13 50.6 .+-. 4.7***
28.7 .+-. 2.5*** 0.8 .+-. 0.3*" ZP 3 .times. 30 .mu.g/kg 11 45.5
.+-. 1.5***' 24.9 .+-. 0.8***' 0.7 .+-. 0.3*" *P < 0.06 vs.
vehicle-treated group; **P < 0.01 vs. vehicle-treated group;
***P < 0.001 vs. vehicle-treated group. 'P < 0.05 vs. control
group; "P < 0.01 vs. control group
EXAMPLE 7
[0161] In vivo Heart Model--Ischemic Injury in Pigs.
[0162] Experimental Model:
[0163] Adult pigs of either sex weighing 20-30 kg will be used for
this study. The pigs will be premedicated with an intramuscular
injection of Ketamine at a dose of 11 mg/kg, and Midazolam at a
dose of 0.2 mg/kg, 30 min prior to induction of anesthesia.
Anesthesia will be initiated with Isoflurane with oxygen to a
surgical plane before the animals will be intubated with an
endotracheal tube. Anesthesia will be maintained with Isoflurane
(generally 2%) and oxygen (1-2 L/min). Assisted ventilation (10-15
ml/kg tidal volume; rate 20-40 breathes per minute) will be used
for the surgical procedure. Fluid will be administered through an
ear vein using saline 0.9% at a rate of 10 ml/kg/hour. The rate
will be increased accordingly in the presence of hemorrhage.
Temperature will be maintained with a circulating warm water
blanket. Central venous and arterial lines will be introduced in
the carotid artery and the internal jugular veins for a Swan Ganz
sheath and a catheter for drug administration. Local anesthesia
will be provided by means of epidural injection of morphine (1 mg)
diluted in 5 ml 0.9% NaCl. Local nerve blocks will also be achieved
by infiltrating Marcaine along the proximal and lateral borders of
the sternum. Local blocks will be repeated every 4 hours. A
Lidocaine drip will be administered via one of the jugular
catheters at a rate of 20-40 ml/hr. A midline sternotomy will be
performed. A lidocaine bolus at 1-2 mg/kg will be administered
before any manipulation of the heart. The pericardium will be
opened and fixed to the border of the sternum to form a pericardial
cradle. A snare will be applied by tunneling the LAD with a monofil
suture approximately half way down the vessel. Coronary occlusion
will be achieved by tightening the snare around the LAD for 45 min.
The snare will then be loosened to initiate reperfusion.
[0164] ECG, central venous pressure, pulmonary artery, and arterial
pressure will be monitored continuously on a Siemens Sirecust
404-1. Cardiac output will be determined by thermodilution (5 ml
NaCl 0.9%, room temperature). Arteriovenous O.sub.2 differences
will be determined before, 20, and 45 minutes after coronary
occlusion, and after 10 and 120 minutes of reperfusion. Blood gas
analysis will be performed with the Radiometer Copenhagen ABL 77.
Venous blood samples (3 ml) will be collected in polypropylene
tubes containing citrate and will be centrifuged at 2000 g for 15
minutes at 4.degree. C. Plasma creatine kinase activity will be
determined and expressed as international units per milliliter.
Troponin-T will be measured.
[0165] Two models will be tested: I. An acute non-survival model
with a total of 4 h reperfusion, and II. A recovery model with 7
days of reperfusion. Each of the two models will consist of 5
groups: 1) Sham operated, 2) Ischemia/reperfusion alone, 3) I/R
plus vehicle alone, 4) I/R plus zinc pyrithione, and 5) I/R plus
Zinc-diethyldithiocarbamate. The zinc ionophores , or vehicle, will
be infused into the jugular vein at a dose of 8.3 ng/kg body weight
at 0, 1, and 2 h after the initiation of reperfusion in a total of
30 ml of saline in a blinded fashion under maintenance of constant
flow and pressure. Hemodynamic and PO.sub.2 measurements, as well
as blood samples will be obtained before; 5, 10, 30, and 45 minutes
after coronary occlusion; and after 5, 10, 30, 60, 120, and 240
minutes of coronary reperfusion. At the end of reperfusion, the
pigs will be sacrificed with 2M KCl (1 ml/kg), and their hearts
will be recovered for further analysis.
[0166] With model I (non-survival), after 4 h of reperfusion, the
left anterior descending coronary artery (LAD) will be reoccluded.
Then, 60 ml of Evan's blue (2% wt/vol solution) will be injected
into the left atrium to stain perfused myocardium. Unstained
myocardium will be defined as the area at risk. After injection of
2M KCl, the heart will be excised. The right ventricle, the large
vessels, and fat tissue will be removed. The left ventricle will
then be sliced perpendicular to the axis of the left side of the
heart from the apex to the AV groove in 6-7 mm slices. The
unstained part of the left ventricular myocardium will be separated
from the Evan's blue-stained portion and immersed in a 0.09-mol/L
sodium phosphate buffer, pH 7.4, containing 1% triphenyltetrazolium
chloride (TTC, Sigma) and 8% dextran (molecular weight, 77.800) for
20 minutes at 37.degree. C. The TTC dye will form a dark-red
formazan complex in the presence of viable myocardial cells that
contain active dehydrogenases and cofactors. Dead cells will remain
unstained. The ischemic but non-necrotic, red-stained tissue will
be separated from the unstained, infarcted tissue. The three tissue
sections-nonischemic (area not at risk), ischemic non-necrotic, and
ischemic necrotic tissue will be weighed. TUNEL staining, as
described above (Fliss, et al. (1996), incorporated herein by
reference), will be used to identify the percent of apoptotic
myocytes in the myocardial tissue.
* * * * *