Note: Descriptions are shown in the official language in which they were submitted.
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
ZINC IONOPHORES AS ANTI-APOPTOTIC AGENTS
The present invention relates to a method of
preventing or treating apoptosis using a zinc ionophore.
The present invention also relates to a method of
protecting cells against the harmful effects of injurious
agents, for example, oxidants, TNFa, neurotoxins,
ischemia and radiation..
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 cell death normally activated under physiological
conditions, such as involution in tissue remodeling
during morphogenesis, and several immunological
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
processes. The apoptotic process is characterized by
cell shrinkage, chromatin condensation, and
internucleosomal degradation of the cell's DNA
(Verh~aegen et al. (1995) Biochem. Pharmacol. 50(7):1021-
1029 ) .
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. 270:660-670; 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.
130:14595-14665) 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)
-2-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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).
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.
-3-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
Zinc-pyrithione (zinc pyridinethione,
CloHeNz02S2Zn, MW 317.75, commercially available from
Sigma) is the active ingredient in the anti-dandruff
shampoo Head & Shoulders~ (U.S. patents 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 Zn2+ into the target cells. This is
necessitated by the fact that all eukaryotic cells
strictly regulate the membrane transport of Zn2+, making
it very difficult to modulate the intracellular
concentration and distribution of Zn2+. 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
Zn2+ 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). In addition to zinc-pyrithione, another
_4_
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
The ability of zinc-pyrithione and zinc-
diethyldithiocarbamates to protect 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.
Thus, according to the present invention there
is provided a method to protect against apoptosis using
one or more zinc ionophores.
In one embodiment of the present invention
there is provided a method of treating or preventing
apoptosis by administering to a patient in need thereof a
pharmaceutically effective amount of a zinc ionophore.
In another embodiment of the present invention
there is provided a pharmaceutical composition comprising
a zinc ionophore and a pharmaceutically acceptable
carrier.
In a further embodiment of the present
invention there is provided a method of protecting
against the harmful effects of injurious agents selected
from the group consisting of oxidants, TNFa, neurotoxins,
ischemia and radiation by administering to a patient in
-S-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
need of such protection an effective amount of a zinc
ionophore.
These and other features of the invention will
become more apparen t from the following description in
which reference is made to the appended drawings wherein:
FIGURE 1 shows the effect of zinc pyrithione
on
rat cardiac apoptos is.
FIGURE 2 shows the effects of zinc pyrithione
on HSP-70 in heart.
FIGURE 3 shows the anti-apoptotic effect of
zinc pyrithione on Sp1 in brain.
FIGURE 4 shows the effects of zinc pyrithione
on kainic acid indu ced damage in rat brain areas.
FIGURE 5 shows the effects of zinc pyrithione
on the severity of kainic acid induced seizures in rats.
FIGURE 6 shows the protective effects of zinc
pyrithione in PC12 cells subjected to oxidative stress.
FIGURE 7 shows the protective effects of zinc
pyrithione in PC12 cells.
FIGURE 8 shows the anti-apoptotic effect of
zinc-pyrithione in irradiated human primary endothelial
cells.
FIGURE 9 shows the effects of zinc pyrithione
on transcription factor binding activity in human primary
endothelial cells.
FIGURE 10 shows the effects of zinc pyrithione
on the TNFa-induced transcription factor binding activity
in human primary endothelial cells.
-6-
CA 02375852 2001-12-14
wo onoo193 rcTnBOOioloos
FIGURE 11 shows the effects of zinc pyrithione
on cytosolic Ikappa B protein levels in human primary
endothelial cells.
FIGURE 12 shows photomicrographs depicting
histological evidence of protection by zinc-pyrithione in
4 vessel occlusion stroke model in rats.
FIGURE 13 shows the effect of zinc pyrithione
on neuronal survival in 4 vessel occlusion stroke model
in rats.
FIGURE 13A shows the effect of zinc-
diethyldithiocarbamate on neuronal survival in 4 vessel
occlusion stroke model in rats.
FIGURE 14 shows the effect of zinc pyrithione
in rats and a 4x reduction in the number of apoptotic
nuclei .
FIGURE 15 shows middle cerebral artery
occlusion caused infarcts in the left hemisphere of mouse
- brain. Infarcts are visible as white regions in the left
hemi spf~ere . . I
FIGURE 16 shows the reduction in infarct area
upon treatment with zinc-pyrithione in mice with middle
cerebral artery occlusion stroke model.
FIGURE 16A shows the reduction in infarct area
upon treatment with zinc-diethyldithiocarbamate in mice
with middle cerebral artery occlusion stroke model.
FIGURE 17 shows the administration of zinc-
pyrithione decreased infarct volumes in mouse brain.
FIGURE 17A shows the administration of zinc-
diethyldithiocarbamate decreased infarct volumes in mouse
brain.
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
FIGURE 18 shows the effect of zinc-pyrithione
administration on neurological score in mice with middle
cerebral artery occlusion stroke model.
The present invention is directed to a method
of blocking apoptosis. In accordance with the present
invention "blocking" includes treating, preventing,
inhibiting, protecting against and reducing the
occurrence of apoptosis. Likewise and in accordance with
the present invention "treating" includes blocking,
preventing, inhibiting, protecting against and reducing
the occurrence of apoptosis
Previous studies have shown that treatment of
injured cells with zinc can block apoptosis. In these
studies the effectiveness of zinc ionophore was
demonstrated using high concentrations, ranging from 3~.iM
to 5mM. 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
luM, to block apoptosis.
Thus, according to the present invention the
concentration of zinc ionophore used to block apoptosis
ranges from about .005 ug zinc ionophore per kg of body
weight to about 2 mg zinc ionophore per kg of body weight
(i.e. about 600pM zinc ionophore to about 6 ~iM zinc
ionophore). In a further embodiment of the present
invention the concentration of zinc ionophore used to
block apoptosis ranges from about 1.0 ug zinc ionophore
per kg of body weight to about 800 ug zinc ionophore per
_g_
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
kg of body weight. Preferably the concentration of zinc
ionophore used to block apoptosis ranges from about 0.2ug
zinc ionophore per kg of body weight to about 600ug zinc
ionophore per kg of body weight.
In a further embodiment of the present
invention the concentration of zinc ionophore used to
block apoptosis is about 0.9 mg/kg body weight, or about
0.18mg zinc/kg body weight.
According to the present invention, any non-
toxic compound capable of binding zinc with moderate
affinity and having sufficient lipophilic properties to
penetrate cell membranes will be capable of effecting the
protection demonstrated in the present invention with
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.
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-a content. Further, according to the present
invention, the 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 Spl. Still further, according to the
-9-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
present invention the zinc-pyrithione was shown to
upregulate cytoprotective proteins, for example HSP70.
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. With the mouse model
of severe focal ischemia, zinc pyrithione demonstrates
neuroprotective properties, significantly decreasing
brain infarct volume and neurological deficit.
In use, the zinc ionophores, according to the
present invention are administered in a pharmaceutically
effective amount to a patient 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,
-10-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
such as, for example, for about one week to several
months depending upon the condition to be treated.
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.
A decided practical advantage 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. Depending on the route of administration,
the active ingredients which comprise zinc ionophores may
be required to be coated in a material to protect said
zinc ionophores from the action of enzymes, acids and
other natural conditions which may inactivate said zinc
ionophores. In order to administer zinc ionophores by
other than parenteral administration, they should be
-11-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
coated by, or administered with, a material to prevent
inactivation. For example, zinc ionophores 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.
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.
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.
-12-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
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.
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
-13-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
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.
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.
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,
-14-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
Accordingly, in a preferred form of blocking
apoptosis the patient is administered a therapeutically
effective amount of at least one zinc ionophore and a
pharmaceutically acceptable carrier in accordance with
the present invention. A preferred zinc ionophore is
zinc pyrithione. Another preferred zinc ionophore is
zinc diethyldithiocarbamate.
The zinc ionophores of the present invention
are effective against a wide range'of injurious agents,
for example, but not limited to: oxidants, TNFa,
neurotoxins, or radiation.
The zinc ionophores of the present invention
are also effective in treating ischemia. Therefore, in
a preferred form of treating ischemia the patient is
administered a therapeutically effective amount of at
least one zinc ionophore and a pharmaceutically
acceptable carrier. A preferred zinc ionophore is zinc
pyrithione. Another preferred zinc ionophore is zinc
diethyldithiocarbamate.
Also defined within the present invention are
compositions suitable for blocking apoptosis which
-15-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
comprise one or more zinc ionophores and a
pharmaceutically acceptable carrier.
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.
The present invention will now be demonstrated
using specific examples that are not to be construed as
limiting.
20
-16-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 1
Screening for ionophores:
Cell Cultures:
Human umbilical vein endothelial cells (HUVEC)
were purchased from Clonetics (San Diego, California) and
passages 2-4 were used for these studies. Cells were
cultured on flame-sterilzed glass coverslips in
Endothelial Basal Medium (Clonetics) supplemented with
l0ng/ml human recombinant epidermal growth factor, 1.0
ug/ml hydrocortisone, 50ug/ml gentamicin, 50ng/ml
amphotetericin B, l2ug/ml bovine brain extract and 2~v/v
fetal bovine serum (all from Clonetics), in a humidified
chamber at 37°C and 5~ COz. To maintain cell populations,
proliferating HUVEC were passaged at 80-90% confluency.
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., (199'7) Am. J.
Physiol. 272:H2095-H2106). The modification consisted of
introducing low concentrations of CaCl2 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
Ca2+- free solution containing ( in mM) : NaCl , 145 ; KC1, 5 ;
MgS09, 1.2; NazHP09, 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 }1M CaCl2 was added to the collagenase
solution. Perfusion with this solution was continued for
-17-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
another 15 to 18 min, followed by a 2 min washout of the
enzyme with fresh perfusate containing 100 ~.iM CaCl2 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
~.un nylon mesh, the cells were allowed to settle and the
supernatant was replaced with a solution containing 2 mM
CaClz. Cells were kept at 37°C in this pre-oxygenated
solution and were studied within 8 hours after isolation,
cellular viability was ensured by regularly replacing the
incubation solution.
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 KC1, 1 mM NaH2 P04 , 1.2
mM MgS04 , 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°C for 18 min. After the addition of 0.082 mg/ml
trypsin inhibitor (Boehringer Mannheim, Indianapolis, I
N) and 0.25 mg/ml DNase I (Boehringer Mannheim), the
tissue was incubated at 25°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°C in solution A containing 2.7
-18-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
mM MgS04 and 0.03 mM CaCl2. After a final centrifugation
the pellet was resuspended in EMEM media (Sigma, St.
Louis, MO) containing 10o dialyzed FBS (Sigma), 25 mM
KC1, 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 ~.un; Falcon).
Cells were plated on glass coverslips coated with poly-D-
lysine (Sigma) in Nunc four-well dishes at a density of
1.5x106 cells per milliliter of medium. Cytosine- [3-
arabinoside (10 u.M; Sigma) was added 24 hr after plating.
Test compounds:
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 Znz+, and
not some other divalent ration 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 ZnCl2 in a 2:1 molar ratio either in water
or DMSO, depending on the solubility of the reactants,
-19-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
and incubating at room temperature for 15 min. The holo-
ionophores were then stored at -20°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 uM of the bolo-ionophore.
When testing the apo-ionophores, an equivalent molar
concentration of the ionophore in the superfusion buffer
(2 uM) was used.
Ionophore screening:
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 uM 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; KC1, 5; MgClz, 1; CaCl2,
2, HEPES, 5; glucose, 10; pH adjusted to 7.4 with NaOH.
The flow rate was maintained at approximately 3 ml/min
-20-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
and the temperature at 37°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-
pyridylmethyl)ethylenediamine (TPEN, 30 uM) was added to
the superfusate at the end of the run. Since TPEN does
not chelate Ca2+, 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 uM) 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.
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.
-21-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
TABLE 1
EXAMPLE OF ZINC IONOPHORES
rrr - excellent
rr - very good
r - good
ZINC PYRITHIONE 1~
DITHIOCARBAMATES
Pyrrolidinedithiocarbamate rr
diethvldithiocarbamate rr
Disulfiram rr
dimethvldithiocarbamate dd
HETEROCYCLIC AMINES
8-Hvdroxycruinoline, Zinc salt r
5,7-Diiodo-8-hydroxyauinoline dr
NSAID
Indomethacin N
VI TAMINS
Vitamin A (all-trans-retinol)r
Vitamin E (alpha-tocopherol) r
-22-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 2
In vivo heart model - ischemic injury.
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. Previous
studies have demonstrated that this model produces
extensive myocardial apoptosis (Fliss, H. (1996), su ra).
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.
-23-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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 ug/ml proteinase K in 25
mmol/L Tris-HC1 (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 CoCl2, 0.25 mg/ml bovine serum
albumin, 25 mmol/L Tris-HC1, 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°C in a humidified chamber. The reaction was
terminated by washing the sections 3 times (1 min each)
with PBS at RT. The sections were then incubated with 1
ml of a staining solution containing 2.5 mg/ml
fluorescein isothiocyanate-avidin (avidin-FITC), 4X
saline-sodium citrate buffer, 0.1% Triton X-100, and 50
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, 90o 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
-24-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
at 37°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) (Figure 1). Similar
strong protection against apoptosis was observed with
l.2ug/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
15.611.2.
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 (Figure 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 MgClz,
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,OOOxg at 4°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
-25-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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, MA), and was quantified with a densitometer and
Molecular Analyst Software (Bio-Rad Laboratories,
Hercules, California). 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-a (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-a. As PKC-a is a well known
intracelular signalling agent, it was therefore concluded
that Znz+ is capable of modulating intracellular
signalling.
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
-26-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
buffer containing 0.25 M sucrose, 10 mM HEPES, pH 7.6, 25
mM KC1, 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 KC1, 1 mM
EDTA, and 10o glycerol. The homogenate was centrifuged at
100,000 xg at 4°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 MgClz, 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,OOOxg at 4°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 KC1, 0.2 mM EDTA, 20o 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, Wisconsin) were radiolabelled with g[32P]ATP
(Amersham, Arlington Heights, Illinois). 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 KC1, 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,
-27-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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,
California). 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, CA) 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 (Figure 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.
-28-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 3
In Vivo Brain model - kainic acid injury.
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
44:514-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
-29-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
intracellular zinc (Cuajungco, M.P. et al., (1997)
Neurobiol.Dis. 4:137-169).
Experimental model: The experimental approach we 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 (Bong, 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 ~.un) and stained by the Nissl method, using
routine methodology.
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
-30-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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. Figure 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 (Figure 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).
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
Figure 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
-31-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
-32-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 4
In vitro neuronal cell model - oxidative stress
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).
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% COZ at 37°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
-33-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
growth medium (plus serum and NGF) containing 20 uM
hydrogen peroxide (HzOz) . 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°C and were stained with Hoechst 33258 to~
visualize the nuclei.
Results: The data show that zinc-pyrithione (2-500 nM)
provided statistically significant protection against
H202-induced apoptosis, with 10 nM being the optimal
concentration (Figure 6). Compared to control cells
(+NGF), treatment with H202 resulted in the loss of
approximately 50% of the cells through apoptosis (+NGF,
+H202). 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+Hzp2 ; ++,
P<0.02 vs. NGF+Hz02 , Mann-Whitney test, n=4).
In another series of tests (Figure 7), the ability
of zinc-pyrithione to protect against Hz02 was confirmed,
and also demonstrated that this compound can block the
-34-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
apoptosis caused by serum and NGF deprivation. Here too
nM appeared to be the optimally protective
concentration of zinc-pyrithione. Zinc-pyrithione at
concentrations as high as 1000 nM did not show any
5 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 Znz+ alone, at
concentrations equivalent to those of zinc-pyrithione, to
H2O2-treated cells did not block apoptosis (not shown),
10 suggesting that the ionophore pyrithione is necessary for
the transport of Zn2+ into the cytosol, and that it is
this intracellular Znz+ which accounts for the protective
effects of zinc-pyrithione.
-3 5-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 5
In vitro endothelial model - response to ionizing
radiation and TNFa
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. 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 TNFa 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
-3 6-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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 TNFa.
Experimental model:
Cell Culture: Human umbilical vein endothelial cells
(HUVEC) were purchased from Clonetics (San Diego,
California) and used from passages 2-4. Cells were
cultured on gelatin-coated culture dishes in Endothelial
Basal Medium (Clonetics) supplemented with l0ng/ml human
recombinant epidermal growth factor, 1.0 ug/ml
hydrocortisone, 50ug/ml gentamicin, 50ng/ml
amphotetericin B, l2ug/ml bovine brain extract and 2%v/v
fetal bovine serum, in a humidified chamber at 37°C and
5% CO2. To maintain cell populations, proliferating HUVEC
were passaged at 80-90% confluency.
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:
Radiation: The cells were washed twice with 37°C D-PBS
and then irradiated in fresh media. Irradiated cells
received a dose of 1000 Rads of gamma-irradiation from a
13'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).
-3 7-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
TNFa: The cells were washed twice with 37°C D-PBS and
then incubated in media containing the TNFa (20ng/ml,
from a stock of l0ug/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).
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,
0.05mg/ml in Hz0), 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.
Preparation of Cytosolic and Nuclear Extracts: Cells
were grown on 100mm2 culture dishes, and following
treatment, were scraped into ice cold PBS and collected
by centrifugation at 200Xg for 5 minutes. The cells were
then resuspended and washed once in 1 ml of ice cold PBS
and centrifuged at 200Xg for 5 minutes at 4°C. The cells
were resuspended in 1 ml of Buffer A (lOmM HEPES, lOmM
KC1, l.5mM MgCl2, pH=7.~9, l.5mM DTT and 0.5mM phenyl
methyl sulphonyl fluoride (PMSF)) and centrifuged at
200Xg for 5 minutes at 4°C. The cells were then
-3 8-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
resuspended and lysed in 300u1 of Buffer A containing
0.1o Nonidet P-40 for 25 minutes on ice. The homogenate
was then spun at 20,OOOXg for 10 minutes at 4°C. The
supernatant containing cytosolic proteins was combined
with an equal volume of Buffer C (20mM HEPES, 50mM KC1,
1.0 mM EDTA, 0.lmM EGTA, 20~ glycerol, pH=7.9, 0.5mM DTT
and 0.5mM PMSF) and was stored at -80°C. The pelleted
nuclei were washed by resuspension in 1m1 of Buffer A and
spun at 20,OOOXg for 1 minute. The supernatant
containing residual cytosolic proteins was discarded and
the pelleted nuclei were resuspended in 35u1 of Buffer B
(20mM HEPES, 420mM NaCl, l.5mM MgClz, 0.2mM EDTA, 25~
glycerol, pH 7.9, 0.5mM DTT, 0.5mM 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,OOOXg for 15 minutes a~
4°C, and was combined with an equal volume of Buffer C
and stored at -80°C.
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.
Electrophoretic Mobility Shift Assay (EMSA): Equal
amounts of nuclear protein (5ug) were incubated with poly
dI-dC (5ug from a stock of 2.5ug/ul in TE buffer) for 10
minutes at RT. This reaction mixture was then incubated
with 0.2ng of 5' end-3zphosphorus-labelled double stranded
oligonucleotide probe for 20 minutes at RT to allow the
binding of nuclear proteins with the labeled probe.
-3 9-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
Loading buffer (5u1 of a mixture containing 20mM HEPES,
100mM KC1, 60% glycerol, 0.5mM EDTA, 0.5mM 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°C
under vacuum. The dried gels were exposed to X-ray film
(Cronex) for up to 2 days at -80°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 2mg 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 (20ng), T4 Polynucleotide kinase and
[g32P]ATP (60uCi) were mixed in kinase buffer (50mM Tris-
HC1, pH 7.6, lOmM MgCl2, 5o glycerol and 5mM DTT) and
incubated at 37°C for 1 hour. Labeled oligonucleotides
were removed by centrifugation through a G-25 Sephadex
Column at 8500rpm for 20 minutes. The labeled
oligonucleotides were then diluted such that 2u1 of the
probe mixture contained approximately 50000-100000cpm.
-40-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
Western Blotting: Equal amounts of cytosolic protein
(3ug) were diluted 1:1 in sample buffer (0.125M Tris-HC1
pH 6.8, 2.6%SDS, 25% glycerol, 0.1m1 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-HC1 pH=6.8, 0.1% SDS, 0.6%
ammonium per sulfate and 0.2% TEMED in H20) and a 10%
running gel (10% acrylamide, 0.3% bis acrylamide, 8%
glycerol, 0.375 Tris-HC1 pH=8.8, 0.1% SDS, 0.04% ammonium
persulfate and 0.05 % TEMED in Hz0) . After
electrophoresis (100V, 50 minutes), the gels were
equilibrated for 15 minutes in ice cold transfer buffer
(25mM Tris HCl, 20% methanol, and 192mM 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°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.
-41-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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 (Figure
8). 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
(Figure 8). (*, P<0.05 vs. Control; °, P<0.05 vs.
irradiated cells, n=4).
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
(Figure 9). 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; °, P<0.05 vs. irradiated
group, n=4). Zinc-pyrithione had a very similar effect
in TNFa-treated HUVEC (Figure 10), and was particularly
potent at blocking the TNFa-induced increase in NF-kB
content. (*, °, 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
TNFa (Figure 11) .
-42-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 6
EFFECTS OF ZINC PYRITHIONE
ON MODELS OF ISCHEMIC STROKE IN RODENTS
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.
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 (4V0) and a focal ischemia model of middle
cerebral artery occlusion in mice (MCAO). The 4V0
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
-43-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
produced in awake and freely moving rats, and produced
reproducible neuropathology. The 4V0 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
a1.,(1989) supra) the 4V0 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.
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
-44-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
the hemisphere including the cerebral cortex and
subcortical structures, and its severity depended on the
duration of ischemia and the strain of mice used.
Animals
The 4V0 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.
Surgeries
4v0 in rats
Rats under chloral 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 ~ 37-37.5°C, both
before and during the ischemic insult, using a feedback-
-45-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
controlled heating pad and a rectal thermistor
(Homeothermic Blanket System, Harvard Apparatus LTD,
England) . The initial (18t min of ischemia) and final (10t''
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°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).
MCAO in mice
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
20:84-9l, incorporated herein by reference). A 8.0 nylon
microfilament coated with a silicon resin (Xantopren)-
hardener mixture (tiara, 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
-46-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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 ~37°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) .
Zinc ionophore treatments
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 4V0 rat model, four doses of ZP:
3x1.2 ug/kg, 3x6 ug/kg, 3x30 ug/kg, and 3x200 ug/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 ug/kg (3x6 ug/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 4V0 and MCAO models using the dose of 3x7.6 ug/kg, a
regimen which delivers zinc at a dose equivalent to that
delivered with 3x6 ug/kg of zinc pyrithione.
-47-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
Histology
4 vo
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°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
um 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 um-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 dun thick
brain sections from rats sacrificed at 24 h and 96 h
-48-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
after ischemia. Fluorescence was monitored with a Zeiss
Axioplan microscope.
MCAO
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 20 (wt/vol) 2,3,5-
triphenyltetrazolium chloride solution (TTC) in PBS (pH
7.4) for 20 min at 37°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 um sections from the anterior side to
the posterior side at 500 um intervals. ISEL was
performed as described above. Adjacent sections were
stained with cresyl violet (Nissl). The ApopTag~
Peroxidase In situ Apoptosis detection Kit (Intergen)
was also used to detect apoptosis in mouse brain
sections.
-49-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
Statistics
The data were expressed as means tS.E.M. One way-
ANOVA with post hoc Duncan's test or T-test for independent
samples were used for statistical analysis of data.
Zinc ionophore toxicity
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 4V0
(7.5-16.9 ~ at 24 h after ischemia and 4.1-11.2 o at 96 h
after ischemia) did not differ between the vehicle-treated
and ZP-treated groups.
4V0 model (10 min - 1 h - 2 h - injection schedule)
Neuronal cell loss
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 (Figure 12). The ZP-
mediated increase in cell survival varied (1.6-3.5 fold)
and did not show a clear dose-dependence (Figure 13).
Although all doses of ZP tested showed evidence of
protection, the 3x6 ug/kg group reached statistical
significance with approximately 520 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
-50-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
ZP 3x6 ug/kg group significantly different from vehicle at
P<0.01, and the 3x200 ug/kg ZP group showing a trend at
P<0.09. Thus, 3x6 ug/kg appeared to be the optimal
protective dose of ZP in this model. However, the trend for
significant protection shown by the 3x200 ug/kg ZP group,
and the collective evidence of protection at the other
doses suggested that ZP is protective over a broad range of
doses.
In addition, with ZnDDC at 3x7.6 ug/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 3x6 ug/kg of ZP, a dose which
delivered the same amount of zinc (Figure 13A).
ISEL staining
As described above, ZP at 3x6 ug/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.
At 24 h after ischemia no apoptotic cells were
observed in the sham, vehicle-treated, or ZP (3x6 ug/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 (131.4 114.6 in CA1 region, n=5).
However, in rats treated with 3x6 ug/kg ZP, the number of
apoptotic nuclei was approximately 4 times lower
(33.617.8, n=9, P<0.0001, t-test) (Figure 14). No
-51-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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.
Neurological deficit
The neurological deficit data in 4V0 rats are
presented in Table 2. 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 4V0 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 4V0 rats
despite clear evidence of histological protection.
4 YO model (3 h, 4 h, 6 h injection schedule)
Administration of ZP in boluses of 6 ug/kg at 3,
4, and 6 h after the ischemic episode (3x6 ug/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 3x6 ug/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 (103.5115.2 vs.
73.2f15.0). The neurological deficit data for this ZP
administration schedule are presented in Table 2. The
-52-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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
S did not significantly change its neuroprotective effect
at the dose of 3x6 ug/kg, indicating a possible wide
therapeutic window for ZP in the 4V0 stroke model.
MCAO
MCAO for 1 h produced significant infarcts in the
left hemisphere of mouse brain (Figure 15). Three doses
of ZP were used (3x1.2 ug/kg, 3x6 ug/kg, and 3x30 }lg/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 (Figure 16). 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 3x7.6 ug/kg also
significantly decreased the infarct area in this model
(Figure 16A).
Table 3 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
-53-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
previously for this strain of mouse (tiara, 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.
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
(Figure 17). 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 3x7.6
ug/kg also significantly decreased both the absolute and
relative infarct volumes in this model (Figure 17A).
Clear evidence of protection by ZP was also observed
with the neurological score (Figure 18). 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
-54-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
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 3x1.2 ug/kg group, 61.5 % in
the 3x6 ug/kg group, and 63.6% in the 3x30 ug/kg group.
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 3x30 ug/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 3x1.2 ug/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) (Figure 20).
-5 S-
CA 02375852 2001-12-14
i~VO 01/00193 PCT/IB00/01005
TABLE 2. Neurological deficit in 4V0 rats (neurological
score, meantS.E.M.)
Grou N 24 h 96 h
Vehicle 8 2.111.0 1.410.9
ZP 3x1.2 ug/kg 9 2.10.9 0.410.2
ZP 3x6 ug/kg 10 1.410.4 1.410.6
ZP 3x30 k 5 2.81.0 1.610.7
ZP 3x200 ug/kg 5 2.41.3 2.011.1
ZP 3x6 k * 6 1.30.7 0.80.4
* ZP administered 3, 4, and 6 h after the ischemic episode.
TABLE 3. Infarct volumes and neurological deficit in mice
following 60 min MCAO (meantS.E.M.)
Group N Infarct Infarct volume, Neurologica
volume, mm' ~ of 1 deficit
contralateral
hemis here
Control (no 9 61.213.6 33.612.5 2.610.5
in'ections)
Vehicle-treated 10 71.313.9 41.514.6 1.710.5
ZP 3x1.2 k 13 44.012.6*** 24.911.5*** 0.310.2**
ZP 3x6 k 13 50.614.7*** 28.712.5*** 0.810.3*""
ZP 3x30 k 11 45.511.5***"24.910.8***" 0.7t0.3*
"
*, 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
-56-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
EXAMPLE 7
In vivo heart model - ischemic injury in pigs.
Experimental model: Adult male pigs weighing 27~2 kg will
be used for this study. The pigs will be premedicated
with subcutaneous diazepam 5 mg and azaperon
intramuscularly 5 mg/kg. Anesthesia will be initiated
with intravenous thiopental 300-500 mg, and will be
maintained with intravenous thiopental. After intubation,
the pigs will be mechanically ventilated with a Drager
respirator AV-1 (OZ in room air: FiOz, 0.3; PCO2,
controlled), and central venous and arterial lines will
be introduced. Before a midline thoracotomy, a 7.5-mg
bolus of the analgeticum piritramid will be given
intravenously. The pericardium will be opened and fixed
to the border of the sternum. A left atrial pressure
(LAP) catheter will be introduced. Coronary occlusion by
a snare will be applied by tunneling the left anterior
descending (LAD) coronary artery with a monofil suture
between the proximal and medial third behind the first
diagonal branch. At the same level, the vena cordis magna
will be cannulated for blood analysis with a small
catheter. A myocardial POz probe will then be implanted
into the expected center of the area at risk. A
temperature probe will be positioned next to the POZ
sensor. Baseline values will be acquired during a 1-hour
preoperative period. Coronary occlusion will be achieved
by tightening the snare around the LAD for 60 min. The
snare will then be loosened to initiate reperfusion. Two
models will be tested: I. An acute non-survival model
with a total of 6 h reperfusion, and II. A recovery model
with 7 days of reperfusion. Each of the two models will
-5 7-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
consist of 5 groups: Sham operated (Sham),
Ischemia/reperfusion alone (I/R), I/R plus vehicle alone
(I/R + DMSO), I/R plus zinc pyrithione (I/R + ZP), and
I/R plus Zinc-diethyldithiocarbamate (I/R + ZD). The zinc
ionophores will be infused into the LAD in three boluses
of 5m1 saline, at 0, 1, and 2h after the initiation of
reperfusion to give a cumulative dose of 1 ug/kg body
weight. Vehicle will be administered in a similar and
blinded fasion. Hemodynamic and POz measurements and
blood samples will be obtained at 5, 10, 20, 30, and 60
minutes after coronary occlusion; and after 5, 10, 20,
30, 60, 90, and 120 minutes of coronary reperfusion.
Global and regional contractility will be recorded by
regional 2Dultrasound before, after 15 and 60 minutes of
coronary occlusion, and after 15, 60, and 120 minutes of
reperfusion. At the end of reperfusion, the pigs will be
killed, and their hearts will be recovered for further
analysis.
ECG, right atrial, pulmonary artery, and
arterial pressure and LAP will be recorded on a Siemens
Sirecust 404-1 at different time points. Cardiac output
will be determined by thermodilution (5 mL NaCl 0.9%,
room temperature) and by continuous measurement with a
Baxter Vigilance monitor. The Licox catheter probe
measurement system will be used in which the flexible
Licox catheter POz microprobe is in direct contact with
the myocardium. One milliliter of heparinized venous and
arterial blood will be drawn with polypropylene syringes
from the femoral artery and the vena cordis magna.
Lactate and arteriovenous OZ differences will be
determined before, after 60 minutes of coronary
occlusion, and after 10 and 120 minutes of reperfusion.
-S 8-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
Blood gas analysis will be performed with the Radiometer
Copenhagen Arterial Bloodgas Laboratory 3. For lactate
determination, samples will be centrifuged at 20008 (10
minutes at 4°C), plasma will be decanted, and lactate
will be measured with the Lactate Analyzer model 23L from
Yellow Springs Instrument Co Inc. Venous blood samples (3
ml) will be collected in polypropylene tubes containing
citrate and will be centrifuged at 20008 for 15 minutes
at 4°C. Plasma creatine kinase activity will be
determined and expressed as international units per
milliliter. Troponin-T will be measured.
With model I (non-survival), after 6 h of
reperfusion, the LAD will be reoccluded. Then, 40 ml of
Evan's blue (2% wt/vol solution) will be injected into
the pulmonary artery to stain perfused myocardium.
Unstained myocardium will be defined as the area at risk.
After cardioplegia with 20 mL potassium chloride IV
(20~), 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 4-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 1a
triphenyltetrazolium chloride (TTC, Sigma) and 8~ dextran
(molecular weight, 77.800) for 20 minutes at 37°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
-59-
CA 02375852 2001-12-14
WO 01/00193 PCT/IB00/01005
tissue. The three tissue sections-nonischemic (area not
at risk), ischemic non-necrotic, and ischemic necrotic
tissue will be weighed. The arterial pressure-rate
product (mean arterial pressure times heart rate) will be
taken as a global parameter of myocardial contractility.
TUNEL staining (Fliss et al. (1996) supra), as described
above, will be used to identify the percent of apoptotic
myocytes in the myocardial tissue.
-60-
