U.S. patent application number 10/140856 was filed with the patent office on 2002-12-19 for compounds and compositions for treating tissue ischemia.
Invention is credited to Al-Abed, Yousef, Bucala, Richard J., Ivanova, Svetlana, Tracey, Kevin J..
Application Number | 20020193412 10/140856 |
Document ID | / |
Family ID | 22378271 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020193412 |
Kind Code |
A1 |
Tracey, Kevin J. ; et
al. |
December 19, 2002 |
Compounds and compositions for treating tissue ischemia
Abstract
There is disclosed a genus of compounds and pharmaceutical
compositions that are protective for mitigating damage associated
with tissue ischemia, particularly stroke (CNS ischemia), and
ischemia of the myocardium. The present invention further provides
a method for treating tissue damage caused by ischemia. Lastly, the
present invention provides a method for treating tissue damage
caused by providing a compound that inhibits the cytotoxic activity
of 3-aminopropanal.
Inventors: |
Tracey, Kevin J.; (Old
Greenwich, CT) ; Al-Abed, Yousef; (New York, NY)
; Ivanova, Svetlana; (Astoria, NY) ; Bucala,
Richard J.; (Cos Cob, CT) |
Correspondence
Address: |
Elie H. Gendloff
Amster, Rothstein & Ebenstein
90 Park Avenue
New York
NY
10016
US
|
Family ID: |
22378271 |
Appl. No.: |
10/140856 |
Filed: |
May 7, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10140856 |
May 7, 2002 |
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09118388 |
Jul 17, 1998 |
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6391899 |
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Current U.S.
Class: |
514/344 ;
514/354; 546/286; 546/314 |
Current CPC
Class: |
A61P 39/00 20180101;
G01N 33/5014 20130101; A61P 43/00 20180101; A61P 9/10 20180101;
G01N 33/502 20130101; G01N 33/5008 20130101; G01N 33/5088 20130101;
A61K 31/4425 20130101; A61K 31/197 20130101; G01N 33/5058 20130101;
A61K 31/426 20130101; G01N 33/5044 20130101 |
Class at
Publication: |
514/344 ;
514/354; 546/314; 546/286 |
International
Class: |
A61K 031/44; C07D
213/46 |
Claims
We claim:
1. An ischemia-damage mitigating compound having a formula I:
6wherein R and R.sub.1 are independently hydrogen, sulfamide,
carboxyamide, cyano, straight or branched C.sub.1-6 alkyl, straight
or branched C.sub.2-6 alkenyl, straight or branched C.sub.1-6
alkoxy, a straight chain C.sub.1-6 alkyl or a straight chain
C.sub.2-6 alkenyl having an ether link or an ester link, toluenyl,
COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R.sub.1
cannot be hydrogen, wherein R.sub.2 and R.sub.3 are independently
hydrogen, sulfamnide, carboxyamide, cyano, straight or branched
C.sub.1-6 alkyl, straight or branched C.sub.2-6 alkenyl, straight
or branched C.sub.1-6 alkoxy, a straight chain C.sub.1-6 alkyl or a
straight chain C.sub.2-6 alkenyl having an ether link or an ester
link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
2. The ischemia-damage mitigating compound of claim 1 wherein R and
R.sub.1 are meta to each other and to the heteroatom.
3. The ischemia-damage mitigating compound of claim 1 wherein R is
COOH.
4. The ischemia-damage mitigating compound of claim 1 wherein
R.sub.1 is COOH.
5. The ischemia-damage mitigating compound of claim 1 wherein
R.sub.2 and R.sub.3 are both hydrogen.
6. The ischemia-damage mitigating compound of claim 1 wherein R and
R.sub.1 are each COOH, and R.sub.2 and R.sub.3 are both
hydrogen.
7. The ischemia-damage mitigating compound of claim 1 wherein the
compound is selected from the group consisting of
1-phenacyl-2,3-dicarboxypyrdiniu- n bromide;
1-phenacyl-2,4-dicarboxypyrdinium bromide;
1-phenacyl-2,5-dicarboxypyrdinium bromide (AP5);
1-phenacyl-2,6-dicarboxy- pyrdinium bromide;
1-phenacyl-2,3-dicarboxyimidepyrdinium bromide;
1-phenacyl-2,4-dicarboxyimidepyrdinium bromide;
1-phenacyl-2,5-dicarboxyi- rnidepyrdinium bromide; and
1-phenacyl-2,6-dicarboxyimidepyrdinium bromide.
8. A pharmaceutical composition comprising a compound from formula
I in a pharmaceutically acceptable carrier, wherein formula I
comprises: 7wherein R and R.sub.1 are independently hydrogen,
sulfamide, carboxyamide, cyano, straight or branched C.sub.1-6
alkyl, straight or branched C.sub.2-6 alkenyl, straight or branched
C.sub.1-6 alkoxy, a straight chain C.sub.1-6 alkyl or a straight
chain C.sub.2-6 alkenyl having an ether link or an ester link,
toluenyl, COOH, nitrate, or halide (Br, Cl, I, F), wherein both R
and R.sub.1 cannot be hydrogen, wherein R.sub.2 and R.sub.3 are
independently hydrogen, sulfamide, carboxyamide, cyano, straight or
branched C.sub.1-6 alkyl, straight or branched C.sub.2-6 alkenyl,
straight or branched C.sub.1-6 alkoxy, a straight chain C.sub.1-6
alkyl or a straight chain C.sub.2-6 alkenyl having an ether link or
an ester link, toluenyl, COOH, nitrate, or halide (Br, Cl, I,
F).
9. The pharmaceutical composition of claim 8 wherein R and R.sub.1
are meta to each other and to the heteroatom.
10. The pharmaceutical composition of claim 8 wherein R is
COOH.
11. The pharmaceutical composition of claim 8 wherein R.sub.1 is
COOH.
12. The pharmaceutical composition of claim 8 wherein R.sub.2 and
R.sub.3 are both hydrogen.
13. The pharmaceutical composition of claim 8 wherein R and R.sub.1
are each COOH, and R.sub.2 and R.sub.3 are both hydrogen.
14. The pharmaceutical composition of claim 8 wherein the compound
is selected from the group consisting of
1-phenacyl-2,3-dicarboxypyrdinium bromide;
1-phenacyl-2,4-dicarboxypyrdinium bromide;
1-phenacyl-2,5-dicarboxypyrdinium bromide (AP5); 1
-phenacyl-2,6-dicarboxypyrdinium bromide;
1-phenacyl-2,3dicarboxyimidepyr- dinium bromide;
1-phenacyl-2,4-dicarboxyimidepyrdinium bromide;
1-phenacyl-2,5-dicarboxyimidepyrdiniun bromide; and
1-phenacyl-2,6-dicarboxyimidepyrdinium bromide.
15. A method for inhibiting tissue damage caused by ischemia,
comprising administering an effective amount of a compound of
formula I, wherein formula I comprises: 8wherein R and R.sub.1 are
independently hydrogen, sulfamide, carboxyamide, cyano, straight or
branched C.sub.1-6alkyl, straight or branched C.sub.2-6. alkenyl,
straight or branched C.sub.1-6 alkoxy, a straight chain C.sub.1-6
alkyl or a straight chain C.sub.2-6 alkenyl having an ether link or
an ester link, toluenyl, COOH, nitrate, or halide (Dr, Cl, I, F),
wherein both R and R.sub.1 cannot be hydrogen, wherein R.sub.2 and
R.sub.3 are independently hydrogen, sulfamide, carboxyamide, cyano,
straight or branched C.sub.1-6 alkyl, straight or branched
C.sub.2-6 alkenyl, straight or branched C.sub.1-6 alkoxy, a
straight chain C.sub.1-6 alkyl or a straight chain C.sub.2-6
alkenyl having an ether link or an ester link, toluenyl, COOH,
nitrate, or halide (Br, Cl, I, F).
16. The method of claim 15 wherein R and R.sub.1 are meta to each
other and to the heteroatom.
17. The method of claim 15 wherein R is COOH.
18. The method of claim 15 wherein R.sub.1 is COOH.
19. The method of claim 15 wherein R.sub.2 and R.sub.3 are both
hydrogen.
20. The method of claim 15 wherein R and R.sub.1 are each COOH, and
R.sub.2 and R.sub.3 are both hydrogen.
21. The method of claim 15 wherein the compound is selected from
the group consisting of 1-phenacyl-2,3-dicarboxypyrdinium bromide;
1-phenacyl-2,4-dicarboxypyrdinium bromide;
1-phenacyl-2,5-dicarboxypyrdin- ium bromide (AP5);
1-phenacyl-2,6-dicarboxypyrdinium bromide;
1-phenacyl-2,3-dicarboxyimidepyrdinium bromide;
1-phenacyl-2,4-dicarboxyi- midepyrdinium bromide;
1-phenacyl-2,5-dicarboxyimidepyrdinium bromide; and
1-phenacyl-2,6-dicarboxyimidepyrdinium bromide.
22. A method for inhibiting tissue damage caused by ischemia,
comprising administering an effective amount of a compound of
formula II, wherein formula II comprises: 9wherein R.sub.1 and
R.sub.2 are independently selected from the group consisting of
hydrogen, hydroxy C.sub.1-6 alkyl, C.sub.1-6 alkoxy C.sub.1-6
alkyl, and R.sub.1 and R.sub.2 together with their ring carbons may
be an aromatic fused ring; wherein Z is hydrogen or an amino group;
wherein Y is hydrogen or a group of the formula --CH.sub.2COR;
wherein R is C.sub.1-6 alkyl, C.sub.1-6 alkoxy, hydroxy, amino,
aryl, or --CH.sub.2R.sub.3 wherein R.sub.3 is H, C.sub.1-6 alkyl,
C.sub.2-6 alkenyl, or C.sub.4-6 aryl.
23. The method of claim 22 wherein the compound of formula II is a
halide (Cl. Br, F or I), tosylate, methanesulfonate or mesitylene
sulfonate salt.
24. A method for treating tissue damage caused by ischemia,
comprising administering an effective amount of a compound that
detoxifies 3-aminopropanal.
25. The method of claim 24 wherein the tissue damage resulting from
ischemia are manifest as myocardial infarction or stroke.
26. An in vivo screening assay comprising administering a polyamine
compound or 3-aminopropanal into the brain parenchyma of a test
animal by microinjection, administering a test compound or control
agent locally or systemically, and measuring cytotoxicity in
stained brain sections from the test animals.
27. An in vitro screening assay comprising exposing cultured glial
cells or neuronal cells related cell lines to 3-aminopropanal at a
concentration of from about 50 to about 1000 .mu.M, adding various
concentrations of test compound or control media to the cell
cultures, incubated under cell culture conditions for a period of
from about 5 minutes to about 20 hours, and determining the
percentage of cell viability.
Description
TECHNICAL FIELD OF THE INVENTION
[0001] The present invention provides a genus of compounds and
pharmaceutical compositions that are protective for mitigating
damage associated with tissue ischemia, particularly stroke (CNS
ischemia), and ischemia of the myocardium. The present invention
further provides a method for treating or preventing tissue damage
precipitated by injury, disease or insult, particularly the tissue
damage caused by ischemia. Lastly, the present invention provides a
method for treating or preventing tissue damage by providing
compounds that and compositions that inhibit or neutralize the
cytotoxic activity of 3-aminopropanal.
BACKGROUND OF THE INVENTION
[0002] Cerebral ischemia, a leading cause of disability and
mortality world-wide, is mediated by a cascade of molecular
cytotoxins that kill potentially viable cells in the brain. The
polyamines, spermine, spermidine, and putrescine, which are among
the most abundant molecules in mammalian brain, have been
implicated in the pathogenesis of ischemic brain damage (Zhang et
al., Proc. Natl. Acad. Sci. U.S.A. 91:10883-10887, 1994; Harman and
Shaw, Br. J. Pharmac. 73:165-174, 1981; Bergeron et al., J. Med.
Chem. 39:5257-5266, 1996; Glantz et al., J. Basic. Clin. Physiol.
Pharmacol. 7:1-10, 1996; Dempsey et al., Neurosurg. 17:635-640,
1985; and Schmitz et al., Neurosurg. 33:882-888, 1993). Polyamine
biosynthesis is increased following the onset of cerebral ischemia,
due to an ischemia-mediated induction of ornithine decarboxylase, a
key synthetic enzyme in the polyamine biosynthetic pathway.
Spermine was linked to development of glutamate-mediated
cytotoxicity, because it can bind to the NR1 subunit of the NMDA
receptor and potentiate glutamate-mediated cell damage (Traynelis
et al., Science 268:873-876, 1995; Traynelis and Cull-Candy. J.
Physiol. (Lond.) 433:727-763, 1991; and Sullivan et al., Neuron
13:929-936, 1994). Administration of experimental therapeutics
which inhibit ornithine decarboxylase limit the development of
ischemic brain damage in experimental animal models of stroke
[ref]. Thus, the accumulation of polyamines in the ischemic brain
occupies an important role in the pathogenesis of stroke (Kindy et
al., J. Cereb. Blood Flow Metab. 14:1040-1045, 1994).
[0003] Brain spermine and spermidine levels are actually decreased
by cerebral ischemia (Paschen, J. Neurochem. 49:35-37, 1987; and
Paschen, Cerebrovasc. Brain Metab. Rev. 4:59-88, 1992). This
observed decline of tissue spernine and spermidine levels is
accompanied by an increase in brain levels of putrescine (Paschen,
Mol. Chem. Neuropathol. 16:241-271, 1992; Paschen, Cerebrovasc.
Brain Metab. Rev. 4:59-88, 1992; Morgan, Bachrach and Heimer, eds.
CRC Publications, 203-229, 1989; and Paschen et al., Acta
Neuropathol. 76:388-394, 1988). Further, intracerebral putrescine
levels correlated significantly with the volume of brain cell
death. Putrescine does not interact with the NMDA receptor, and
does not potentiate its cytotoxic activity. A possible explanation
for these results may reside in the catabolism of polyamines via
the "interconversion pathway" which is dependent upon the activity
of tissue polyamine oxidase (Seiler and Bolkenius, Neurochem. Res.
10:529-544, 1985; Seiler et al., Med. Biol. 59:334-346, 1981;
Bolkenius and Seiler, Int. J. Dev. Neurosci. 4:217-224, 1986; and
Bolkenius et al., Biochim. Biophys. Acta 838:69-76, 1985). This
ubiquitous enzyme, which is present in high levels in brain and
other mammalian tissues, cleaves spermine and spermidine via
oxidative deamination to generate the end products putrescine and
3-aminopropanal (Seiler and Bolkenius. Neurochem. Res. 10:529-544,
1985; Seiler, In Yu et al., eds. Elsevier Science, 333-344, 1995;
Morgan, Essays in Biochemistry 23:82-115, 1987; and Houen et al.,
Acta Chem. Scand. 48:52-60, 1994). 3-. Aminopropanal is known for
its cytotoxicity to primary endothelial cells, fibroblasts, and a
variety of transformed mammalian cell lines (Bouzyk and Rosiek,
Cancer Lett. 39:93-99, 1988; Brunton et al., Toxic. in Vitro
8:337-341, 1994; Gaugas and Dewey, Br. J. Cancer 39:548-557, 1978;
Morgan et al., J. Biochem. 236:97-101, 1986; and Ferrante et al.,
J. Immunol. 133:2157-2162, 1984). 3-Aminopropanal has also been
implicated as a mediator of programmed cell death in murine
embryonic limb buds, and may contribute to the development of
necrosis in some tumors (Parchment and Pierce, Cancer Res
49:6680-6686, 1989; and Kurihara et al., Neurosurg 32:372-375,
1993). Inhibition of polyamine oxidase with aminoguanidine blocked
generation of 3-aminopropanal in cell cultures following the
addition of spermine, and prevented subsequent cytotoxicity
(Ferrante et al., J. Immunol. 133:2157-2162, 1984; Morgan, Essays
in Biochemistry 23:82-115, 1987; and Parchment and Pierce, Cancer
Res. 49:6680-6686, 1989). On a molar basis, the LD.sub.50
concentration of 3-aminopropanal to cells is similar to the
cytotoxicity of glutamate. In contrast, putrescine is not cytotoxic
to cells, even in the millimolar range, but its rate of production
through polyamine oxidation correlates directly with the formation
of a directly cytotoxic aldehyde, 3-aminopropanal.
[0004] In addition, in the data first being reported herein in
glial cells, 3-aminopropanal mediates apoptosis by activation of an
interleukin-1 beta converting enzyme (ICE)-dependent signaling
pathway, whereas in neurons it causes necrotic cell death.
[0005] Cerebral ischemia (stroke) is a debilitating condition
resulting from a sudden cessation of blood flow to an area of the
brain, resulting in a loss of brain tissue. There are no available
therapies to reverse the neurological deficits caused by neuronal
death in the infarct zone. Stroke is a major public health problem
in the U.S. wherein about 550,000 strokes occur each year. Cerebral
ischemia afflicts individuals of all age groups, but the incidence
doubles with each decade over 45 and reaches 1-2% per year in the
population of individuals over 75 years of age. If a patient
survives, major disability can result with loss of ability to
communicate, ambulate, see, coordinate and/or reason. Standard
therapy is often ineffective at preventing brain infarction and is
meant to support cardiovascular and respiratory function, control
intracranial pressure, and prevent recurrent stroke. There is also
a class of protease enzymes that are designed to dissolve blood
clots, only for those strokes caused by blood clots potentially
useful in brain ischemia but (as opposed to bleeding) and these
agents only function to restore some blood flow in limited
situations.
[0006] During the evolution of cerebral infarction (stroke), a core
of densely ischemic tissue becomes rapidly and irreversibly
damaged. Cellular damage in the surrounding area, termed the
"ischemic penumbra," progresses more slowly.
[0007] Following an ischemic insult, the process of tissue
destruction may not be completed for hours or even days (Kirino et
al., Acta Neuropathol. 64:139-147, 1984; and Petito et al.
Neurology 5 37:1281-1286, 1987). There is a temporary window of
opportunity for an intervention to prevent ischemic tissue from
progressing to infarction. In humans, this window is thought to
extend from about 2-4 hours following the onset of ischemia, after
which time the efficacy decreases rapidly (Ginsberg and Pulsinelli,
Ann. Neurol. 36:553-554, 1994). During the therapeutic window, the
target for therapeutic neuroprotection is the ischemic penumbra, a
volume of brain tissue around the ischaemic core, which receives
reduced blood flow and contains compromised, but potentially viable
tissue. Studies have identified important cytotoxic mediators that
cause cell death in the early hours after the onset of
ischemia.
[0008] A number of molecular substrates of normal brain, as well as
extrinsic factors delivered by the circulation, contribute to the
development of cell cytotoxicity during ischemia. These include,
but are not limited to, glutamate, aspartate, nitric oxide,
calcium, free radicals, zinc, cytokines, arachidonic acid
metabolites, and advanced glycation end products (AGEs). Advanced
glycation endproducts are a group of protein modifying adducts that
were implicated in the pathogenesis of diabetic complications. AGEs
were found to be cerebrotoxic in the ischemic penumbra (Zimmerman
et al., Proc. Natl. Acad. Sci. U.S.A. 92:3744-3748, 1995). In
addition, aminoguanidine, a small molecule inhibitor of AGE
cross-linking reactions, effectively abrogated the cerebrotoxicity
of AGEs during focal cerebral ischemia (Zimmerman et al., Proc.
Natl. Acad. Sci. U.S.A. 92:3744-3748, 1995). Aminoguanidine was
also found to be cerebroprotective during focal ischemia in normal,
non-diabetic animals, independent of exogenous AGEs (Zimmerman et
al., Surg. Forum. 45:600-603, 1994). Aminoguanidine further
provided cerebroprotection in a model of focal stroke when
administered within 2 hours after the onset of focal cerebral
ischemia (Cockroft et al., Stroke 27:1393-1398, 1996). It was
considered that the mechanism of action was inhibition of polyamine
oxidase (PAO), an enzyme that produces toxic, reactive aldehyde
metabolites by oxidation of biogenic amines.
[0009] The cascade of cytotoxicity that is initiated by reduced
blood flow is followed by a drop in ATP levels and a reduction of
oxidative phosphorylation. As a result, membrane potentials fall,
leading to release of K.sup.+ and an excessive amount of glutamate
and other excitatory amino acids (EAAs) in a process called
excitotoxicity. This will, in turn, over-activate
N-methyl-D-aspartate (NMDA),
amino-3-hydroxy-5-methyl-4-isoxasole-4-propionate (AMPA), kainate
(KA), and 1S,3R-trans-1-amino-cyclopentyl-1,3-dicarboxylate
(trans-ACPD) receptors (Faroquil and Horrocks, Brain Res.
16:171-191, 1991).
[0010] Elevated glutamate leads to excessive Ca.sup.2+ influx,
primarily by excitatory amino acid receptor channel activation, as
well as swelling and osmotic lysis as a result of depolorization
mediated influx of Na.sup.+, Cl.sup.- and water (Faroquil and
Horrocks, Brain Res. 16:171-191, 1991). This elevation of
intracellular Ca.sup.2+ activates phospholipases, lipases,
proteases and protein kinases, leading to eventual breakdown of
phospholipid membranes, cytoskeletal alterations, arachidonic acid
release, and potentiation of the free radical cascade (Manfred et
al., Biochem. Pharm. 50:1-16, 1995). Other modulators of NMDA
receptors include Zn.sup.2+, histamine, certain neuroactive
steroids, arachidonic acid, polyamines and protons or pH
(Collinridge and Lester, Pharmacol. Rev. 74:143-210, 1989; and
McBain and Mayer, Physiol. Rev. 74:723-760, 1994). Moreover, an
NMDA receptor antagonist, MK-801, can exert a neuroprotective
effect in animal models of cerebral ischemia (Olney et al., J.
Neurosci. 9:1701-1704, 1989).
[0011] Ischemia also leads to formation of reactive oxygen species
PROS), activation of lipid peroxidation, and a reduction in the
endogenous antioxidants ascorbate, glutathione, ubiquinone and
.alpha.-tocopherol in brain tissue. The mitochondrial respiratory
chain and reaction sequences catalyzed by cyclooxygenase and
lipoxygenase are important production sites for superoxide anion
(O.sub.2.sup.-), hydrogen peroxide (H.sub.2O.sub.2) and hydroxy
radical (OH). Activated oxygen species are also formed during
autooxidation of catecholamines and in the xanthine reaction.
[0012] Nitric oxide (NO) is another mediator of tissue injury in
cerebral ischemia NO concentrations increase acutely in the brain
after middle cerebral artery (MCA) occlusion, from approximately 10
nM to 2.2 .mu.M by a porphyrinic microsensor assay (Beckman et al.,
Proc. Natl. Acad. Sci. U.S.A. 87:1620, 1990).
[0013] In addition to these other suspected mediators of ischemic
tissue damage, 3-aminopropanol is an enzymatic by-product of the
oxidative cleavage of the polyamines spermine and spermidine by PAO
in mammalian cells (Holtta, Biochemistry 16:91-100, 1997). The
cytotoxicity resulting from co-incubation of PAO activity with
spermine and spermidine has been abolished by aminoguanidine (Gahl
et al., Chemicobiological Interactins 22:91-98, 1978; and Henle et
al., Cancer Res. 46:175-182, 1986). 3-Aminopropanal has also been
implicated in causing programmed cell death in murine embryonic
limits buds (Parchment et al., Cancer Res. Arch. 49:6680-6686,
1989) and in necrosis of solid tumors.
[0014] These data provides a need in the art to find inhibitors of
PAO activity that are likely to have therapeutic utility in
treating tissue ischemia, particularly mitigating damage to the
ischemic penumbra experienced in stroke, but also in non-neuronal
tissue such as muscle tissue (e.g., smooth muscle and cardiac
muscle).
SUMMARY OF THE INVENTION
[0015] The present invention provides a stroke-damage mitigating
compound having a formula I: 1
[0016] wherein R and R.sub.1 are independently hydrogen, sulfamide,
carboxyamide, cyano, straight or branched C.sub.1-6 alkyl, straight
or branched C.sub.2-6 alkenyl, straight or branched C.sub.1-6
alkoxy, a straight chain C.sub.1-6 alkyl or a straight chain
C.sub.2-6 alkenyl having an ether link or an ester link, toluenyl,
COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R.sub.1
cannot be hydrogen, wherein R.sub.2 and R.sub.3 are independently
hydrogen, sulfamide, carboxyamide, cyano, straight or branched
C.sub.1-6 alky, straight or branched C.sub.2-6 alkenyl, straight or
branched C.sub.1-6 alkoxy, a straight chain C.sub.2-6 alkyl or a
straight chain C.sub.2-6 alkenyl having an ether link or an ester
link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
[0017] Preferably, R and R.sub.1 are meta to each other and to the
heteroatom. Preferably, R is COOH. Preferably, R.sub.1 is COOH.
Preferably, R.sub.2 and R.sub.3 are both hydrogen. Most preferably,
R and R.sub.1 are each COOH, and R.sub.2 and R.sub.3 are both
hydrogen.
[0018] Preferred compounds of formula 1 include, for example,
.sub.1-phenacyl-2,3-dicarboxypyrdinium bromide;
1-phenacyl-2,4-dicarboxyp- yrdinium bromide;
1-phenacyl-2,5-dicarboxypyrdinium bromide (AP5);
1-phenacyl-2,6-dicarboxypyrdinium bromide;
1-phenacyl-2,3-dicarboxyimidep- yrdinium bromide;
1-phenacyl-2,4-dicarboxyimidepyrdinium bromide;
1-phenacyl-2,5-dicarboxyimidepyrdinium bromide; and
1-phenacyl-2,6-dicarboxyimidepyrdinium bromide.
[0019] The present invention provides a pharmaceutical composition
comprising a compound from formula I in a pharmaceutically
acceptable carrier, wherein formula I comprises: 2
[0020] wherein R and R.sub.1 are independently hydrogen,
sulfarnide, carboxyamide, cyano, straight or branched C.sub.1-6
alkyl, straight or branched C.sub.2-6 alkenyl, straight or branched
C.sub.1-6 alkoxy a straight chain C.sub.1-6 alkyl or a straight
chain C.sub.2-6 alkenyl having an ether link or an ester link,
toluenyl, COCH, nitrate, or halide (Br, Cl, I, F), wherein both R
and R.sub.1 cannot be hydrogen, wherein R.sub.2 and R.sub.3 are
independently hydrogen, sulfamide, carboxyarnide, cyano, straight
or branched C.sub.1-6 alkyl, straight or branched C.sub.2-6
alkenyl, straight or branched C.sub.1-6 alkoxy, a straight chain
C.sub.1-6 alkyl or a straight chain C.sub.2-6 alkenyl having an
ether link or an ester link, toluenyl, COOH, nitrate, or halide
(Br, Cl, I, F).
[0021] Preferably, R and R.sub.1 are meta to each other and to the
heteroatom. Preferably, R is COOH. Preferably, R.sub.1 is COOH.
Preferably, R.sub.2 and R.sub.3 are both hydrogen. Most preferably,
R and R.sub.1 are each COOH, and R.sub.2 and R.sub.3 are both
hydrogen.
[0022] The present invention further provides a method for treating
tissue ischernia to mitigate ischemic damage, comprising
administering an effective amount of a compound of formula I,
wherein formula I comprises: 3
[0023] wherein R and R.sub.1 are independently hydrogen, sulfamide,
carboxyamide, cyano, straight or branched C.sub.1-6 alkyl, straight
or branched C.sub.2-6 alkenyl, straight or branched C.sub.1-6
alkoxy, a straight chain C.sub.1-6 alkyl or a straight chain
C.sub.2-6 alkenyl having an ether link or an ester link. toluenyl,
COOH, nitrate, or halide (Br, Cl, I, F), wherein both R and R.sub.1
cannot be hydrogen, wherein R.sub.2 and R.sub.3 are independently
hydrogen, sulfamide, carboxyainide, cyano, straight or branched
C.sub.1-6 alkyl, straight or branched C.sub.2-6 alkenyl, straight
or branched C.sub.1-6 alkoxy, a straight chain C.sub.1-6alkyl or a
straight chain C.sub.2-6 alkenyl having an ether link or an ester
link, toluenyl, COOH, nitrate, or halide (Br, Cl, I, F).
[0024] Preferably, R and R.sub.1 are meta to each other and to the
heteroatom. Preferably, R is COOH. Preferably, R.sub.1 is COOH.
Preferably, R.sub.2 and R.sub.3 are both hydrogen. Most preferably,
R and R.sub.1 are each COOH, and R.sub.2 and R.sub.3 are both
hydrogen.
[0025] The invention further provides a method for treating tissue
ischemia to mitigate ischemic damage, comprising administering an
effective amount of a compound of formula II, wherein formula II
comprises: 4
[0026] wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of hydrogen, hydroxy C.sub.1-6 alkyl,
C.sub.1-6 alkoxy C.sub.1-6 alkyl, and R.sub.1 and R.sub.2 together
with their ring carbons may be an aromatic fused ring; wherein Z is
hydrogen or an amino group; wherein Y is hydrogen or a group of the
formula --CH.sub.2COR; wherein R is C.sub.1-6 alkyl, C.sub.1-6
alkoxy, hydroxy, amino, aryl, or --CH.sub.2R.sub.3 wherein R.sub.3
is H, C.sub.1-6 alkyl, C.sub.2-6 alkenyl, or C.sub.4-6 aryl.
Preferably, the compound of formula II is a halide (Cl, E Br, F or
I), tosylate, methanesulfonate or mesitylene sulfonate salt.
[0027] The present invention further provides a method for
inhibiting tissue damage, comprising administering an effective
amount of a compound that inhibits or neutralizes the cytotoxic
activity of 3-aminopropanal. Preferably, the diseases resulting
from tissue ischemia are myocardial infarction or stroke.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1 shows that polyamine oxidase activity increased
during cerebral ischemia, and was inhibited by aminoguanidine and
chloroquine. Polyamine oxidase activity was measured in brain
homogenates. Data shown are mean.+-.s.d.; n=3. "Normal"=sham
operated control brain homogenate; "Ischemia Vehicle"=homogenate
prepared 2 hr after the onset of middle cerebral artery occlusion;
"Ischemia AG"=addition of aminoguanidine (1 mM) at time=-5 minutes
prior to spermine; and "Ischemia CHLQ"=addition of chloroquine (1
mM) at time=-5 minutes prior to spermine. * P<0.05 vs Normal; #
P<0.05 vs Ischemia Vehicle.
[0029] FIG. 2 shows that brain 3-aminopropanal levels increase
during cerebral ischemia. Brain 3-aminopropanal levels were
measured by derivatization and HPLC assay (as described infra) in
rats subjected to permanent focal cerebral ischemia.
3-Aminopropanal was not detected in sham-operated controls. It
should be noted that 3-aminopropanal tissue levels increased
markedly within two hours after middle cerebral artery occlusion,
and continued to increase further for at least 25 hr. Data shown
are mean.+-.s.d., n=3 animals/group. *P<0.05 vs t=0 hours by
ANOVA.
[0030] FIG. 3 shows the brain-damaging effects of intracortically
administered polyamines and a metabolite thereof, 3-aminopropanal.
Brain damage (infarct or cytotoxic volume) was measured after
intracortical microinjection of spermine, spermidine,
3-aminopropanal and putrescine. Data shown are volume of brain
damage (mm.sup.3) as measured by integrating the area of negative
TTC staining over the entire brain hemisphere in animals injected
with the polyamines shown; mean.+-.s.e.m., n=6-8 group. * P<0.05
vs vehicle. Of note, putrescine did not cause tissue damage;
polyamine substrates metabolized by PAO to yield 3-aminopropanal,
or 3-aminopropanal itself, were brain damaging.
[0031] FIG. 4 shows that aminoguanidine and chloroquine protected
against intracortical spermine toxicity. All animals received
intracortical spermine (25 .mu.g in 2 .mu.l) by
stereotactically-guided microinjection as described infra.
Experimental animals were treated with aminoguanidine or
chloroquine in connection with the intracortical spermine according
to the following dose schedules: systemic aminoguanidine was 320
mg/kg, i.p. 30 minute pretreatment followed by subsequent doses of
110 mg/kg i.p. each 8 hr after intracortical spermine;
intracortical aminoguanidine was a single dose (320 mg/kg)
simultaneously with intracortical spermine; chloroquine was a
single intraperitoneal dose (25 mg/kg) 30 minutes prior to the
spermine injection. Data shown are infarct volume (mm.sub.3)
assessed quantitatively 48 hr after the intracortical spermine
injection (mean.+-.s.e., n=6-8 group). *P<0.05 vs
spermine/vehicle.
[0032] FIG. 5 shows that inhibition of ICE (caspase-1), but not of
CPP-32 (caspase-3) blocked 3-aminopropanal-induced glial apoptosis
in vitro. Cells were pretreated with the ICE (IL-1.beta. converting
enzyme) inhibitor (Ac-YVAD-CMK) at concentrations of 0.4 .mu.M
(triangles) or 40 .mu.M (circles) for 3 hours, followed by
treatment with 3-aminopropanal for an additional 5 hours, and
analyzed for cell viability by the MTT assay. Controls consisted of
DMSO-treated cells (squares) to assess for non-specific solvent
effects. Data are mean.+-.s.d., n=3 wells/experiment.
[0033] FIG. 6 shows cerebroprotection by three different phenylacyl
pyridinium derivatives (PAPDs). Animals received either PBS vehicle
(n=13) or PAPD treatment by i.p. injection beginning 15 minutes
after the onset of ischemia. PICVA-25 (n=9; total dose 200 mg/kg),
PICVA-27 (n=12; total dose 200 mg/kg), PICVA-13 (n=8; total dose
400 mg/kg). Data shown are mean stroke (infarct) volumes expressed
as mm.sup.3 s.e.m. *P<0.05 by one way ANOVA.
[0034] FIG. 7 shows a dose:response relationship for compound
PICVA-13 in an in vivo screening assay for cerebroprotection
following experimentally induced focal brain ischemia. Animals
received either PBS vehicle (n=13) or PICVA-13 i.p. beginning at 15
minutes after the onset of ischemia. PICVA-13 was administered at
60 mg/kg (n=6), 200 mg/kg (n=6), 400 mg/kg (n=11) or 600 mg/kg
(n=13). Data shown are infarct volumes expressed as
mm.sub.3.+-.s.e.m. *P<0.05 by one way ANOVA.
[0035] FIG. 8 shows that the beneficial, cerebroprotective effects
of PICVA- 13 treatment are available for at least two hours after
ischemia begins. Animals received either PBS vehicle (n=10) or
PICVA- 13 beginning two hours after the onset of ischemia, at dose
of 400 mg/kg (n=8) or 600 mgAg (n=9). Data shown are infarct
volumes expressed as mm.sub.3.+-.s.e.m. *P<0.05 by one way
ANOVA.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention arose out of a series of experiments
wherein cerebral damage subsequent to induced focal ischemia was
found to be mediated by the induction of brain polyamine oxidase
activity. Moreover, the cytotoxic end product 3-aminopropanal
accumulated in the ischemic brain at levels that are lethal to
neurons and glial cells. These data further demonstrated that
inhibition of polyamine oxidase activity with structurally distinct
pharmaceutical compounds prevented the formation of
3-aminopropanal, and provided significant protection against the
development of cerebral damage following permanent cerebral artery
occlusion in rats. Finally, data is presented that certain
compounds with the potential to react with 3-aminopropanal are
efficacious in limiting the extent of tissue damage following
ischemia
[0037] 3-Aminopropanal
[0038] Four closely related lines of evidence support the role of
3-aminopropanal as a cytotoxic mediator of cellular and tissue
damage in cerebral ischemia. First, cerebral ischemia mediates an
early induction of polyamine oxidase activity. Second, the
cytotoxic enzyme product 3-aminopropanal accumulates during the
early response to cerebral ischemia (FIG. 2), but is not produced
in normally perfused controls. Third, 3-aminopropanal production in
the ischemic brain increases prior to the onset of significant
cellular degeneration, with tissue 3-aminopropanal levels rising
further during the period of progressive cell death. Fourth,
3-aminopropanal is a potent cytotoxin which activates apoptosis via
an ICE-dependent mechanism in glial cells, and necrosis in neurons.
Considered together, these data offer an explanation for the
correlation between brain levels of putrescine, a stable end
product of terminal polyamine oxidation, and infarct volume
(Dempsey et al., Neurosurg. 17:635-640, 1985; Traynelis et al., J.
Physiol. (London) 433:727-763, 1991; Gilad et al., Mol. Chem.
Neuropathol. 18:197-210, 1993), since catabolism of spermine and
spermidine by polyamine oxidase produces both a stable, non-toxic
end product (putrescine) and a potent cytotoxin (3-aminopropanal).
The latter product mediates cell death, and the former accumulates
in correlation to the extent of damage.
[0039] Previous observations suggest that polyamines can prevent
apoptosis in neuronal cultures (Harada and Sugimoto, Brain Res
753:251-259,1997; and Xie et al., Exp. Cell Res 230:386-392, 1997),
or amplify glutamate-mediated cell cytotoxicity (Pegg et al.,
Biochem. Soc. Trans. 22:846-35 852, 1994). Cell survival in the
ischemic zone is likely to be critically dependent upon the balance
between the direct effects of polyamines, and the cytotoxic effects
of their metabolite, 3-aminopropanal. There has been some
controversy as to whether both 3-aminopropanal and
3-acetamidopropanal can produce acrolein in vivo, a known mediator
of cytotoxicity and apoptosis (Li et al., Toxicol. Appl. Pharmacol.
145:331-339, 1997; and Fernandez et al., Br. J. Cancer
72:1194-1199, 1995). Thus, it is likely that several products of
polyamine oxidation could further augment the cytotoxicity of
3-aminopropanal. When considered together and without being bound
by theory, these observations add further credence to the
hypothesis that enhanced polyamine oxidation during ischemia is
deleterious.
[0040] Without being bound by theory, these data suggest the
following mechanism of brain cell death during cerebral ischemia:
dead and doing cells in the densely hypoxic core release stores of
intracellular spermine and spermidine, which is catabolized by
polyamine oxidase. The resultant production of 3-aminopropanal
causes apoptosis in surrounding glial cells, and necrosis of
neurons, which in turn release more spermine and sperrnidine as
substrate for polyamine oxidase. This cytotoxic mechanism spreads
to involve a larger volume of potentially viable cells surrounding
the ischemic core. It is likely that in concert with the excitatory
amino acids, activated oxygen species, nitric oxide, TNF, IL-1,
IL-6, and platelet-activating factor (Meistrell III et al., SHOCK
8:341-348, 1997; Zhang et al., Stroke 27:317-323, 1996; Coyle and
Puttfarcken, Science 262:689-695,193; Rothwell and Strijbos, Int J
Dev. Neurosci 13:179-185, 1995; Irikura et al., Proc. Natl. Acad.
Sci. U.S.A. 92:6823-6827, 1995; Rothwell and Relton, Cerebrovasc
Brain Metab Rev 5:178-198, 1993;Taupin et al., J Neuroimmunol.
42:177-185, 1993; Saito et al., Neurosci Lett 206:149-152, 1996;
Choi, J Neurobiol 23:1261-1276, 1992; and Montague et al., Science
263:973-76, 1994), 3-aminopropanal is positioned as a proximal
mediator in the cytoxicity cascade of cerebral ischemia. Previous
observations have noted that inhibition of ICE (IL- 1 .beta.
converting enzyme) protects against the development of apoptosis
during cerebral ischemia (Friedlander et al., J Exp. Med.
185:933-940, 1997; Gillardon et al., Brain Res. Mol. Brain Res.
50:16-22, 1997; and Hara et al., J. Cereb. Blood Flow Metab.
17:370-375, 1997). Since the stimulus to apoptosis during cerebral
ischemia is unknown, it is reasonable to suggest a proximal role
for 3-aminopropanal. Further, it was previously reported that TNF
synthesis is upregulated during the first 12 hours of brain
ischemia, which cytokine participates in the subsequent development
of brain damage (Meistrell III et al., SHOCK 8:341-348, 1997).
Spermine is a direct inhibitor of TNF synthesis in human peripheral
blood mononuclear cells (Zhang et al., J. Exp. Med. 185:1-10,
1997). Moreover, the role of decreasing spermine levels during
cerebral ischemia is unexplained and not reported.
[0041] Centrally administered 3-aminopropanal directly stimulated
intracerebral TNF synthesis (data not illustrated in examples).
These data provide additional evidence for a proximal role of
3-aminopropanal in the pathogenesis of stroke.
[0042] Early production and potent cytotoxicity of 3-aminopropanal
define a proximal role of this aldehyde in the ischemic mediator
cascade. Thus, polyamine oxidation contributes to cell cytotoxicity
and these data do not contradict the potential role of other
cytotoxic factors. Polyamines and polyamine oxidase are ubiquitous
in all mammalian tissues (Seiler, Biochimica et Biophysica Acta.
615:480-488, 1980).
[0043] Table 1 provides a time course study of cell viability after
exposure to 3-aminopropanal. Glial or neuronal cells were exposed
to an LD.sub.100 concentration of 3-aminopropanal (750 .mu.M) for
the times indicated, followed by replacing the media with fresh
OPTI-MEM (medium) for a total incubation time of 20 hr. Cell
viability was determined by MTT assay (data shown are
mean.+-.s.e).
1 Cell Viability (% alive) Time (min) Glial cells (HTB14) Neurons
(HTB11) 5 96 .+-. 3 29 .+-. 6 60 92 .+-. 1 13 .+-. 4 120 78 .+-. 7
6 .+-. 2 1200 5 .+-. 5 3 .+-. 1
[0044] These data support the notion that polyamine oxidase-derived
3-aminopropanal is a mediator of the brain damaging sequelae of
cerebral ischemia, which can be therapeutically modulated. Brain
polyamine oxidase activity was increased significantly within two
hours after the onset of ischemia in brain homogenates (15.8.+-.0.9
nmol/mg protein) as compared to homogenates prepared from the
normally perfused contralateral side (7.4.+-.0.5 nmol/h/mg protein)
(P<0.05). The major catabolic products of polyamine oxidase are
putrescine and 3-aminopropanal. Although 3-aminopropanal is a
potent cytotoxin, essential information was previously lacking on
whether 3-aminopropanal is produced during cerebral ischemia.
3-aminopropanal accumulated in the ischemic brain within 2 hours
after permanent forebrain ischemia in rats, but not in normally
perfused brain.
[0045] Cytotoxic levels of 3-aminopropanal (750 .mu.M) were
achieved prior to the onset of significant cerebral cell damage,
and increased in a time-dependent manner. Elevated 3-aminopropanal
levels occurred in association with the development of progressive
neuronal and glial cell death. Glial cell cultures that were
exposed to 3-aminopropanal underwent apoptosis (L.D..sub.50=275
.mu.M) whereas neurons were killed by necrotic mechanisms
(L.D..sub.50=90 .mu.M). The tetrapeptide ICE inhibitor
(Ac-YVAD-CMK) prevented 3-aminopropanal-mediated apoptosis in glial
cells. Finally, treatment of rats with two structurally distinct
inhibitors of polyamine oxidase (aminoguanidine and chloroquine)
attenuated brain polyamine oxidase activity, prevented the
production of 3-aminopropanal, and significantly protected against
the development of ischemic brain damage in vivo. Considered
together, these results indicate that polyamine oxidase-derived
3-aminopropanal is a mediator of the brain damaging sequelae of
cerebral ischemia, which can be therapeutically modulated.
[0046] Synthesis Compounds and Formulations
[0047] The compounds of Formula I may have an asymmetric carbon
atom which may occur in the R, R.sub.1 and R.sub.3 side-chains;
optical isomers may likewise occur in the R, R.sub.1 and R.sub.3
side-chains of the compounds of Formula II. The isomers of a
racemic mixture of any of these compounds can be separated by
methods known to those skilled in the art and the isomeric
preparations thus purified and isolated can be tested, by methods
known to those skilled in the art or taught herein, to determine if
such an isomeric preparation (as opposed to the corresponding
racemic mixture) is more desirable, e.g., has less toxicity or
greater potency, for use in the methods of this invention.
[0048] The compounds, compositions and treatment methods of this
invention can be used to prevent or inhibit tissue necrosis and
cell death in a variety of settings, particularly as the
circumstances may be understood to involve the evolution of
3-aminopropanal and its role as a cytotoxin that propagates
cellular damage. Such additional indications have in common the
need to inhibit "innocent bystander" cell death. In other words,
the compounds, compositions and methods of this invention find
particular utility in preventing the spread of cell death outward
from a fatally damaged, non-viable core into a potentially viable
surrounding or penumbral zone. The initiating damage at the core
may be due to any of a variety of causes (e.g., ischemia, embolism,
thrombosis, hypoperfusion, injury, trauma, disease, heat, cold,
chemical exposure, surgery, exposure to endogenous, environmental
or therapeutic cytotoxins, radiation, necrosis, apoptosis, etc.);
the compounds, compositions and methods of this invention will then
find utility in preventing or limiting cellular damage and cell
death in tissue adjacent to the non-viable core. Without accepting
any limitation implied by mechanistic explanation, the compounds,
compositions and methods of this invention are thought to operate
in such diverse settings by molecular interference with and
neutralization of the cytotoxic activity of 3-aminopropanal, which
cytotoxin arises naturally from metabolism of polyamines released
in the core and in the penumbra as cellular damage propagates
outward from the initiating site. In each such instance, the
compounds, compositions and methods of this invention may be
usefully employed to limit tissue damage ancillary or adjacent to
the site of initiating damage.
[0049] Compounds of formula I can be synthesized according to the
following procedure. A general procedure for the synthesis of
N-Phenyacyl Pyridinum Bromide derivatives: A solution of Pyridine
(1.58 g, 20.0 rnmol) and Bromoacetophenone (4.00 g, 20.0 mmol) in
50 ml of ethanol was refluxed for 6 h. The reaction progress was
monitored by TLC using a mixture of ethyl acetate:methanol:water
(4:2:1 and 1.0% of ammonia) as developing solvent. Upon cooling the
reaction mixture, the product came out of solution as white
precipitate which was filtered and recrystallized from ethanol as
white needle crystal (MP. 216.4-220.degree. C.) in 97% yield.
Further characterizations of the new products were carried out
using NMR and mass spectrometer.
[0050] Compounds according to formula 2 have been described, for
example, in U.S. Pat. No. 5,656,261, the disclosure of which is
incorporated by reference herein. Such compounds include, for
example, 3-amino-thiazolium mesitylenesulfonate;
2,3-diamino-thiazolium mesitylenesulfonate;
3-(2-methoxy-2-oxoethyl) -4,5-dimethylthiazolium bromide;
3-(2-methoxy-2-oxoethyl)-4-methylthiazolium bromide;
3-(2-phenyl-2-oxoethyl)-4-methylthiazolium bromide;
3-(2-phenyl-2-oxoethyl) -4,5-dimethylthiazolium bromide;
3-amino-4-methyl-thiazolium mesitylene sulfonate;
3-(2-methoxy-2-oxoethyl- )-5-methyl-thiazolium bromide;
3-(2-phenyl-2-oxoethyl)-5-methyl-thiazolium bromide; 3-(2-
[4.sup.1-bromophenyl]-2-oxoethyl) -thiazolium bromide;
3-(2-[4.sup.1-bromophenyl]-2-oxoethyl) -4-methyl-thiazolium
bromide; 3-(2-[4.sup.1- bromophenyl]-2-oxoethyl)
-5-methyl-thiazolium bromide; 3-(2-
[4.sup.1-bromophenyl]-2-oxoethyl) -4,5-dimethylthiazolium bromide;
3-(2-methoxy-2-oxoethyl) -4-methyl-5-(2-hydroxyethyl) -thiazolium
bromide; 3-(2-phenyl-2-oxoethyl)
-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide; 3-(2-
[4.sup.1-bromophenyl]-2-oxoethyl) -4-methyl-5-(2-hydroxyet- hyl)
thiazolium bromide; 3,4-dimethyl-5-(2-hydroxyethyl)-thiazolium
iodide; 3-ethyl-5-(2-hydroxyethyl) -4-methyl thiazolium bromide;
3-benzyl-5-(2-hydroxyethyl))-4-methyl thiazolium chloride;
3-(2-methoxy-2-oxoethyl) -benzothiazolium bromide;
3-(2-phenyl-2-oxoethyl) -benzothiazolium bromide; 3-(2-
[4.sup.1-bromophenyl]-2-oxoethyl) -benzo-thiazolium bromide;
3-(carboxymethyl) -benzothiazolium bromide;
2,3-diamino-benzothiazolium mesitylenesulfonate;
3-(2-amino-2-oxoethyl) -thiazolium bromide;
3-(2-amino-2-oxoethyl)-4-methyl-thiazolium bromide;
3-(2-amino-2-oxoethyl)-5-methyl-thiazolium bromide;
3-(2-amino-2-oxoethyl)-4,5-dimethyl-thiazolium bromide;
3-(2-amino-2-oxoethyl)-benzothiazolium bromide;
3-(2-amino-2-oxoethyl) -4-methyl-5-(2-hydroxyethyl) thiazolium
bromide; 3-amino-5-(2-hydroxyethy- l)-4-methyl-thiazolium
mesitylenesulfonate; 3-(2-methyl-2-oxoethyl) thiazolium chloride;
3-amino-4-methyl-5-(2-acetoxyethyl) thiazolium mesitylenesulfonate;
3-(2-phenyl-2-oxoethyl) thiazolium bromide;
3-(2-methoxy-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl) thiazolium
bromide; 3-(2-amino-2-oxoethyl)-4-methyl-5-(2-acetoxyethyl)
thiazolium bromide; 2-amino-3-(2-methoxy-2-oxoethyl) thiazolium
bromide; 2-amino-3-(2-methoxy-2-oxoethyl) benzothiazolium bromide;
2-amino-3-(2-amino-2-oxoethyl) thiazolium bromide;
2-amino-3-(2-amino-2-oxoethyl) benzothiazolium bromide;
3-[2-(4'-methoxyphenyl)-2-oxoethyl]-thiazolinium bromide; 3-[2-(2
',4'-dimethoxyphenyl)-2-oxoethyl]-thiaxolinium bromide;
3-[2-(4'-fluorophenyl-2-oxoethyl]-thiazolinium bromide;
3-[2-(2',4'-difluorophenyl)-2-oxoethyl]-thiazolinium bromide;
3-[2-(4'-diethylaminophenyl)-2-oxoethyl]-thiazolinium bromide;
3-propargyl-thiazolium bromide; 3-propargyl-4-methyl thiazolium
bromide; 3-propargyl-5-methyl thiazolium bromide; 3-propargyl-4,
5-dimethyl thiazolium bromide;
3-propargyl-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide,
3-(2-[3'-methoxyphenyl]-2-oxoethyl)-thiazolium bromide;
3-(2-[3'-methoxy phenyl]-2-oxoethyl)-4 methyl-5-
(2'-hydroxyethyl)-thiazo- lium bromide;
3-(2-[3'-methoxyphenyl]-2-oxoethyl)-benzothiazolium bromide;
2,3-diamino-4-chlorobenzothiazolium mesitylenesulfonate;
2,3-diamino-4-methyl-thiazolium mesitylene sulfonate;
3-amino4-methyl-5-vinyl-thiazolium 5 mesitylene sulfonate;
2,3-diamino-6-chlorobenzothiazolium mesitylenesulfonate;
2,6-diamino-benzothiazole dihydrochloride;
2,6-diamino-3[2-(4'-methoxyphe- nyl)-2-oxoethyl]benzothiazolium
bromide; 2,6-diamino-3[2-(3'-methoxyphenyl-
)-2-oxoethyl]benzothiazolium bromide;
2,6-diamino-3[2-(4'-diethylaminophen-
yl)-2-oxoethyl]benzothiazolium bromide;
2,6-diamino-3[2-(4'-bromophenyl)-2- -oxoethyl]benzothiazolium
bromide; 2,6-diamino-3 (2-(2-phenyl-2-oxoethyl) benzothiazolium
bromide; 2,6-diamino-3[2-(4'-fluorophenyl-2-oxoethyl]benz-
othiazolium bromide; 3-acetamido-4-methyl-5-thiazolyl-ethyl acetate
mesitylenesulfonate; 2,3-diamino-5-methylthiazolium
mesitylenesulfonate;
3-[2-(2'-naphthyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide; 3-[2-(3',5'-Di-tert-butyl-4'-hydroxyphenyl)
-2-oxoethyl]4-methyl-5-(2'-hydroxyethyl) -thiazolium bromide;
3-[2-(2',6'-Dichlorophenethylamino)-2-oxoethyl]-4-methyl-5-
(2'-hydroxyethyl)-thiazolium-bromide;
3'[2-Dibutylamino-2-oxoethyl]-4-met- hyl-5- (2'-hydroxyethyl)
-thiazolium bromide; 3-[2-4'carbethoxyanilino)-2--
oxoethyl]-4-methyl-5-(2'-hydroxyethyl) -thiazolium bromide;
3-[2-(2',6'-Diisopropylanilino)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)--
thiazolium bromide; 3-amino 4-methyl-5-[2-(2',6'-dichlorobenzyloxy)
ethyl]-thioazolium mesitylenesulfonate;
3-[2-(4'-carbmethoxy-3'-hydroxyan- ilino)
-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)-thiazolium bromide;
2,3-Diamino-4,5-dimethyl thiazolium mesitylene sulfonate;
2,3-Diamino-4-methyl-5-hydroxyethyl-thiazolium mesitylene
sulfonate; 2,3-Diamino-5-(3',4'-trimethylenedioxy phenyl)
thiazolium mesitylene sulfonate;
3[2-(1',4'-benzodioxan-6-yl)-2-oxoethyl]-4-methyl-5-(2'-hydrox-
yethyl)-thiazolium bromide; 3'[2-(3',4'-trimethylenedioxyphenyl)
-2-oxoethyl]-4-methyl-5-(2-hydroxyethyl)-thiazolium bromide; 3-
(2-[1',4-benzodioxan-6-yl]-2-oxoethyl)-thiazolium bromide;
3-[2-(3',4'-trimethylenedioxyphenyl)-2-oxoethyl]-thiazolium
bromide; 3-[2-
(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-thiazolium
bromide;
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethyl]-4-methyl-
-thiazolium bromide;
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl)-2-oxoethy-
l]-5-methyl-thiazolium bromide;
3-[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl- )
-2-oxoethyl]-4,5-dimethyl-thiazolium bromide;
3-[2-(3',5'-di-tert-butyl4- '-hydroxyphenyl)
-2-oxoethyl-]benzothiazolium bromide; 1-methyl-3-
[2-(3',5'-di-tert-butyl-4'-hydroxyphenyl) -2-oxoethyl]-imidazolium
bromide; 3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-thiazolinium bromide;
3-[2-(4'-n-pentylphenyl)-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl)
-thiazolinium bromide; 3-[2- 4'-diethylaminophenyl)
-2-oxoethyl]-4-methyl-5-(2'-hydroxyethyl) -thiazolinium bromide;
3-(2-phenyl-2-oxoethyl) -4-methyl-5-vinyl-thiazolium bromide;
3-[2-(3',5'-tert-butyl-4'-hydroxyphenyl)
-2-oxoethyl]-4-methyl-5-vinyl-th- iazolium bromide;
3-(2-tert-butyl-2-oxoethyl)-thiazolium bromide;
3-(2-tert-butyl-2-oxoethyl) 4-methyl-5-(2'-hydroxyethyl)
-thiazolium bromide; 3-(3'-methoxybenzyl)
4-methyl-5-(2'-hydroxyethyl)-thiazolium chloride;
3-(2',6'-dichlorobenzyl) 4-methyl-5-(2'-hydroxyethyl)-thiazoliu- m
chloride; 3-(2'-nitrobenzyl) 4-methyl-5-
(2'-hydroxyethyl)-thiazolium bromide; 3[2-(4'-chlorophenyl)
-2-oxoethyl]-thiazolium bromide;
3[2-(4'-chlorophenyl)-2-oxoethyl]4-methyl-5-(2'-hydroxyethyl)-thiazolium
bromide; and
3[2-(4'-methoxyphenyl)-2-oxoethyl]4-methyl-5-(2'hydroxyethyl- )
-thiazolium bromide.
[0051] Therapeutic Uses, Routes of Administration and
Formulations
[0052] The present invention provides novel compounds,
pharmaceutical formulations and methods of treatment to mitigate
ischemic damage. Effective doses of the therapy, as described
below, may be formulated in suitable pharmacological carriers and
may be administered by any appropriate means, including, but not
limited to injection (intravenous, intraperitoneal, intramuscular,
intracranial, intramyocardial, subcutaneous), by absorption through
epithelial or mucocutaneous linings (oral mucosa, rectal and
vaginal epithelial linings, nasopharyngeal mucosa, intestinal
mucosa); orally, transdermally or any other means available within
the pharmaceutical arts.
[0053] A compound can be administered to a human patient by itself
or in pharmaceutical compositions where it is mixed with suitable
carriers or excipients at doses to treat or mitigate tissue
ischemia or to mitigate ischemic damage. Preferably, the organ
sites of treatment are the CNS and the myocardium. A
therapeutically effective dose further refers to that amount of the
compound sufficient to treat or mitigate tissue ischemia or to
mitigate ischemic damage. Therapeutically effective doses may be
administered alone or as adjunctive therapy in combination with
other treatments for tissue ischemia or to mitigate tissue ischemic
damage. Techniques for the formulation and administration of the
compounds of the instant application may be found in "Remington's
Pharmaceutical Sciences" Mack Publishing Co., Easton, Pa., latest
addition.
[0054] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intracranial, intrascerebroventricular, intramedullary injections,
as well as intrathecal, direct intraventricular, intravenous,
intraperitoneal, intranasal, or intraocular injections, and
optionally in a depot or sustained release formulation.
[0055] Furthermore, one may administer the agent of the present
invention in a targeted drug delivery system, for example in a
liposome coated with a tissue-targeting antibody. The liposomes
will be targeted to and taken up selectively by target tissue
cells.
[0056] The pharmaceutical compositions may be manufactured by means
of conventional mixing, dissolving, dragee-making, levitating,
emulsifying, encapsulating, entrapping, or lyophilizing processes.
Pharmaceutical compositions for use in accordance with the
invention may be formulated in conventional manner using one or
more physiologically acceptable carriers comprising excipients and
auxiliaries which facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Proper
formulation is dependent upon the route of administration
chosen.
[0057] For injection, the agents of the invention may be formulated
in aqueous solutions, preferably in physiologically compatible
buffers, such as Hank's solution, Ringer's solution, or
physiological saline buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are usually known in the art.
[0058] For oral administration, the compounds can be formulated by
combining the active compounds with pharmaceutically acceptable
carriers. Such carriers enable the compounds to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. Pharmaceutical preparations for oral use can be obtained
solid excipient, optionally grinding a resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries, if desired, to obtain tablets or dragee cores.
Suitable excipients are, in particular, fillers such as sugars,
including lactose, sucrose, mannitol, or sorbitol; cellulose
preparations such as, for example, maize starch, wheat starch, rice
starch, potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethylcellulose, sodium carboxymethylcellulose, and/or
polyvinylpyrrolidone (PVP). If desired, disintegrating agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar, or
alginic acid or a salt thereof such as sodium alginate.
[0059] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0060] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for such administration.
[0061] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner. For
administration by inhalation, the compounds for use according to
the invention are delivered in the form of an aerosol spray
presentation from pressurized packs or a nebulizer, with the use of
a suitable propellant, such as, dichlorodifluoromethane,
trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide
or other suitable gas. In the case of a pressurized aerosol the
dosage unit may be determined by providing a valve to deliver a
metered amount. Capsules and cartridges of gelatin for use in an
inhaler or insufflator may be formulated containing a powder mix of
the compound and a suitable powder base such as lactose or
starch.
[0062] The compounds may be formulated for parenteral
administration by injection, such as, by bolus injection or
continuous infusion. Formulations for injection may be presented in
unit dosage form, e.g., in ampoules or in multi-dose containers,
with an added preservative. The compositions may take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulary agents such as suspending, stabilizing
and/or dispersing agents.
[0063] Pharmaceutical formulations for parenteral administration
include, for instance, aqueous solutions of the active compounds in
water-soluble form. Additionally, suspensions of the active
compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Aqueous injection
suspensions may contain substances which increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers or agents which increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Alternatively, the active ingredient may be in powder
form for constitution with a suitable vehicle before injection,
such as, sterile pyrogen-free water.
[0064] Liposomes and emulsions are known examples of delivery
vehicles or carriers for hydrophobic drugs. Certain organic
solvents such as dimethylsulfoxide also may be employed, although
usually at the cost of greater toxicity. Additionally, the
compounds may be delivered using a sustained-release system, such
as semipermeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Various of sustained-release materials have
been established and are well known by those skilled in the art.
Sustained-release capsules may, depending on their chemical nature,
release the compounds for a few weeks up to over 100 days.
Depending on the chemical nature and the biological stability of
the therapeutic reagent, additional strategies for stabilization
may be employed.
[0065] The pharmaceutical compositions also may comprise suitable
solid or gel phase carriers or excipients. Examples of such
carriers or excipients include but are not limited to calcium
carbonate, calcium phosphate, various sugars, starches, cellulose
derivatives, gelatin, and polymers such as polyethylene
glycols.
[0066] Many of the compounds described herein and identified as
useful for treating or mitigating tissue ischemia or mitigating
ischemic damage may be provided as salts with pharmaceutically
compatible counterions. Pharmaceutically compatible salts may be
formed with many acids. including but not limited to hydrochloric,
sulfuric, acetic, lactic, tartaric, malic, succinic, etc.; or
bases. Salts tend to be more soluble in aqueous or other protonic
solvents that are the corresponding free base forms. Examples of
pharmaceutically acceptable salts, carriers or excipients are well
known to those skilled in the art and can be found, for example, in
Remington's Pharmaceutical Sciences, 18th Edition, A. R. Gennaro,
Ed., Mack Publishing Co., Easton, Pa., 1990. Such salts include,
but are not limited to, sodium, potassium, lithium, calcium,
magnesium, iron, zinc, hydrochloride, hydrobromide, hydroiodide,
acetate, citrate, tartrate, malate salts, and the like.
[0067] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve their intended purpose.
More specifically, a therapeutically effective amount means an
amount effective to prevent development of or to alleviate the
existing symptoms of the subject being treated. Determination of
the effective amounts is well within the capability of those
skilled in the art, especially in light of the detailed disclosure
provided herein.
[0068] For any compound used in the method of the invention, the
therapeutically effective dose can be estimated initially from cell
culture assays. In addition, therapeutically useful doses from in
vivo experiments are provided with the in vivo data of preferred
embodiments illustrated herein. Such information can be used to
more accurately determine useful doses in humans.
[0069] A therapeutically effective dose refers to that amount of
the compound that results in a reduction in the otherwise expected
severity of stroke of damage in a prolongation of survival in a
patient. Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical, pharmacological, and
toxicological procedures in cell cultures or experimental animals
for determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio between LD.sub.50 and ED.sub.50. Compounds
which exhibit high therapeutic indices are preferred. The data
obtained from cell culture assays or animal studies can be used in
formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized.
[0070] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active moiety which are sufficient to
maintain the desired modulating effects, or minimal effective
concentration (MEC). The MEC will vary for each compound but can be
estimated from in vitro data; such as, the concentration necessary
to achieve a 50-90% inhibition of polyarnine-derived or
3-aminopropanal-mediated cellular damage (in vitro) or achieve a
reduction in the otherwise expected severity of stroke or ischemic
damage in vivo using the assays described herein. Dosages necessary
to achieve the MEC will depend on individual characteristics and
route of administration. However, HPLC assays, bioassays or
immunoassays can be used to determine plasma concentrations.
[0071] The amount of composition administered will be dependent on
the subject being treated, on the subject's weight, the severity of
the affliction, the manner of administration and the judgment of
the prescribing physician.
[0072] Screening Assays
[0073] The present invention further provides an in vivo screening
assay comprising administering a polyamine compound or
3-aminopropanal into the brain parenchyma of a test animal by
microinjection, administering a test compound or control agent
locally or systemicly, and measuring cortical cytotoxicity in
stained brain sections from the test animals generated by the
location of microinjection. Preferably, the polyamine compound is
spermine or spermidine or metabolites thereof. Preferably, the test
animal is a rat. Preferably, cytotoxicity of stained brain sections
is measured by planimetric analysis. Example 2 below provides an
experiment using this in vivo assay procedure and demonstrates
cytotoxic activity of 3-aminopropanal, spermine and spermidine but
not putrescine. The model was verified by systemic administration
of polyamine oxidase inhibitors chloroquine and aminoguanidine.
[0074] The present invention further provides an in vitro screening
assay comprising exposing cultured glial cells or neuronal cells
related cell lines to 3-aminopropanal at a concentration of a from
about 50 to about 1000 .mu.M, adding various concentrations of test
compound or control Amedia to the cell cultures, incubated under
cell culture conditions for a period of from about 5 minutes to
about 20 hours, and determining the percentage of cell viability.
Preferably, the concentration of 3-aminopropanal in the cell
culture medium is approximately 80 .mu.M. Preferably, the method
for determining cell viability is by measuring
metabolically-dependent reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-dip- henyltetrazolium (MTT).
Another method for determining cell death is by looking for
evidence of apoptosis, for example by a terminal doxynucleotidyl
transferase mediated dUTP nick end labeling (TUNEL) method
(Gavrieli et al., J. Cell. Bio. 119:493-501, 1992). Example 3 below
provides additional working details for the in vitro screening
assay, and Example 5 below shows exemplary assay results with a
variety of test compounds.
EXAMPLE 1
3-Aminopropanal Mediates Tissue Damage Resulting from Ischemia
[0075] Polyamine Oxidase Activity is Increased following
Ischemia
[0076] To determine the role of polyamine oxidase activity and the
metabolites it generates from polyamines in the tissue damage
attendant to ischemia, an animal model of stroke was conducted.
Lewis rats were subjected to focal cerebral ischemia by
microsurgical occlusion of the middle cerebral artery in a
standardized model as described previously (Zimmerman et al., Proc.
Natl. Acad. Sci. U.S.A. 92:3744-3748, 1995; Cockroft et al., Stroke
27:1393-1398, 1996; and Meistrell III et al., SHOCK 8:341-348,
1997). Briefly, the ipsilateral common carotid artery was ligated
and divided, the middle cerebral artery coagulated and divided
distal to the lenticulostriate branch, and the contralateral common
carotid temporarily occluded for 1 hr. The onset of ischemia in
these experiments was defined as the time the middle cerebral
artery was cut. For measurement of infarct volume, the animals were
euthanized at the time indicated and fresh brain sections prepared
(1 mm), immersed in 2,3,5-triphenyltetrazolium chloride (TTC) in
154 mM NaCl for 30 min at 37.degree. C., and total cerebral infarct
volume measured by computerized quantitative planimetry as
described elsewhere (Zimmnerman et al., Proc. Natl. Acad. Sci.
U.S.A. 92:3744-3748, 1995; Cockroft et al., Stroke 27:1393-11398,
1996; and Bederson et al., Experientia 41:1209-1211, 1986). Similar
measurements of stroke volume (infarct size) were obtained in
separate experiments using planimetric analysis of brain sections
stained with hematoxylin and eosin. All procedures involving
animals in this example and in the following examples were
conducted in conformity with institutional guidelines and under the
approval of the Animal Care and Use Committee of North Shore
University Hospital--New York University Medical School.
[0077] To measure tissue polyamine oxidase activity, brain
homogenates were prepared from the anatomic region perfused by the
middle cerebral artery, and total polyamine oxidase activity
determined using a method described previously (Seiler and
Bolkenius, Neurochem. Res. 10:529-544, 1985; Seiler et al., Med.
Biol. 59:334-346, 1981; and Milani et al., J. Neurosci. Res.
28:434-441, 1991), relying on the metabolic conversion of added
spermine. Briefly, two hours after permanent occlusion of the
middle cerebral artery, a 4 mm thick coronal section of the
ipsilateral hemisphere encompassing the zone of ischemia (beginning
3 mm caudal from the frontal pole) was manually homogenized on ice
in 1.5 ml of Hanks media containing 1 mM phenylmethylsulfonyl
fluoride (PMSF) and centrifuged at 43,000 X g for 30 min. Brain
polyamine oxidase activity in the resultant homogenate supernatant
was determined by addition of spermine at time zero (50 .mu.l of a
1 mM stock solution added per 1 ml of homogenate supernatant). For
experiments using enzyme inhibitors, aminoguanidine or chloroquine
in the pharmacological concentration range (50 .mu.M - 5 mM) was
added 5 minutes prior to spermine. The spermine-containing
supernatants were maintained at 37.degree. C., and at time points
up to 60 min after the addition of spermine, duplicate 200 .mu.l
samples were removed and enzyme activity was stopped by addition of
10 .mu.l of 60% perchloric acid (PCA). Samples for HPLC analysis to
detect spermine were prepared with dansyl chloride (200 .mu.l of 10
mg/ml dansyl chloride solution in acetone and 200 .mu.l saturated
aqueous Na.sub.2CO.sub.3 per 50 .mu.l sample homogenate
supernatant; 10 min incubation at 65.degree. C.; brief
centrifugation to clear) and dansylated spermine was detected by
fluoresence after fractionation by HPLC (20 .mu.l injectate; Vydac
analytical C4 column eluted in a linear gradient from 0-100%
methanol (in H.sub.2O) over 35 min). Enzyme activity was corrected
for the protein content of the homogenate supernatants, and data
expressed as mean.+-.s.d.; n=3 brain homogenates per condition.
[0078] Polyamine oxidase activity was significantly higher in
homogenates prepared from ischemic hemispheres as compared to the
contralateral (control) hemispheres (PAO activity after
ischemia=15.8.+-.0.9 /h/mg protein vs. PAO activity in
contralateral controls=7.4.+-.0.5 nmol/h/mg protein, P<0.05; see
FIG. 1). This increase in brain polyamine oxidase activity was
detected as early as 2 hours after the onset of cerebral ischemia,
suggesting that enhanced PAO activity occurred as part of the
earliest response to ischemia.
[0079] Two known, structurally distinct inhibitors of polyamine
oxidase activity, aminoguanidine and chloroquine (Seiler et al.,
Med. Biol. 59:334-346, 1981; Holtta, Biochem. 16:91-100, 1977;
Flayeh, Clin. Chem. 34:401-403, 1988; and Gahl and Pitot, Biochem.
J. 202:603-611, 1982), were selected to determine the specificity
of this spermine-metabolizing activity. Addition of either of these
agents to homogenates of ischemic brain dose-dependently inhibited
polyamine oxidase activity (see FIG. 1); chloroquine I.C..sub.50=40
.mu.M; aminoguanidine I.C..sub.50=400 .mu.M. These data indicate
that within 2 hours after the onset of cerebral ischemia there is a
specific induction of brain polyamine oxidase activity, and that
this activity is susceptible to inhibition by known inhibitors of
polyamine oxidase.
[0080] 3-Aminooronanal Levels are Increased Following Ischemia
[0081] To determine whether the increase in brain PAO activity
associated with focal cerebral ischemia causes enhanced
3-aminopropanal production, a method to detect 3-aminopropanal in
tissue samples was developed. To develop the method,
3-aminopropanal was prepared by hydrolysis of the diethyl acetal
and then derivatized using 2,4-dinitrophenylhydrazine.
Specifically, 3-aminopropanal was prepared by hydrolysis of 145 mM
3-aminopropanal diethyl acetal (TCI America) in 1.5 M HCl for 5
hours at room temperature. The reaction mixture was applied to a
column (3 cm.times.6 cm) of Dowex-50 (H+-form) ion exchange resin
and eluted with a step gradient of 0-3 M HCl (160 ml; flow rate 0.7
ml/min). Fractions containing aldehyde, as determined by the method
of Bachrach and Reches (Bachrach and Reches, Anal. Biochem.
17:38-30 48, 1966), were concentrated in a centrifugal evaporator
at room temperature. The concentration of aldehyde (i.e.,
3-aminopropanal) was determined spectrophotometrically at 531 nm,
based on a reaction of aldehydes with
4-amino-3-hydrazino-5-mercapto-1,2,4-triazole (`Purpald`; Aldrich)
(Dickinson and Jacobsen, Chemical Communications 1719-720, 1970)
with reference to a standard curve using propionaldehyde (Sigma).
Acidic solutions of the aldehyde were neutralized with NaOH to
physiological pH immediately before use. Vehicle control solutions
were prepared, consisting of the same stoichiometric amounts of HCl
and NaOH.
[0082] To derivatize 3-aminopropanal, 2,4-dinitrophenylhydrazine
(2,4-DNPH; 0.5 g of 2,4-DNPH dissolved in 11 ml of concentrated
HCl/ethanol (1: 10, v/v)) was refluxed for 10 seconds with aqueous
3-aminopropanal. The resulting
3-aminopropionaldehyde-2,4-dinitrophenylhy- drazone derivative
(3-AP-2,4-DNPH) was precipitated at room temperature, and collected
by filtration. .sup.1H-NMR spectrometry (DMSO-d.sub.6 and
CDCl.sub.3, 270 MHz) of purified 2,4-dinitrophenylhydrazone
derivative was employed to confirm its structure. The NMR spectrum
revealed the presence of syn and anti isomers (1:1) with resonance
at .delta.8.83 and .delta.11.35. A standard curve was generated by
an HPLC assay of the dansylated derivative of the compound (see
below).
[0083] An additional standard curve was constructed to quantify
recovery of derivatized 3-aminopropanal from tissue homogenates.
Briefly, a 4 mm thick brain slice obtained from the region perfused
by the middle cerebral artery was homogenized manually, followed by
addition of 3-aminopropanal (at 100, 150, 200, 300, 1000 nmol/ml)
and 1.5 ml of 2,4-DNPH reagent. The samples were refluxed in the
presence of the 2,4-DNPH reagent for 10 seconds, then 20 .mu.l of
60% perchloric acid (PCA) was added to stop the reaction, followed
by 200 .mu.l of water. The samples were vigorously vortexed,
centrifuged at 14,000 rpm for 30 min, and the supernatant
concentrated to near dryness in a centrifugal evaporator. Samples
were redissolved in 100 .mu.l of water, centrifuged for 10 min at
14, 000 rpm to clear precipitates, then prepared for assay by
HPLC.
[0084] An HPLC detection system was used for detection of the
derivatization products of 3-aminopropanal and
2,4-dinitrophenylhydrazine as follows. A Hewlett-Packard Model 1090
liquid chromatograph (Wilmington, Del., U.S.A.) equipped with an
autosampler, photo diode-array and fluorescence detectors, and
Chemstation operating software was used for all analyses. Detection
by fluorescence was used, based on the reaction of
5-dimethyl-aminonapthalene sulfonyl-chloride (Dansyl chloride;
Molecular Probes; relative fluorescence at 340 nm with excitation
at 430 nm) with primary and secondary amines. Dansylation was
performed by reacting 50 .mu.l of the sample with 200 .mu.l of 10
mg/ml dansyl chloride solution in acetone, 200 .mu.l of saturated
Na.sub.2CO.sub.3 solution, 3 .mu.l 60% PCA and 3 .mu.l 1 mM
1,7-diaminoheptane (Sigma), followed by incubation at 65.degree. C.
for 10 min. 20 .mu.l of the resulting derivatized sample was
injected onto a Vydac C-4 250.times.4.6 mm column with 5 mn
particle size. Using a flow rate of 1.0 ml/min, runs were initiated
at 100% A (dH.sub.2O ) and a linear gradient to 100% B (methanol)
was performed over 45 min., followed by 5 min. of 100% B and a
return to 100% A over 5 min.
[0085] HPLC analysis of the 2,4-DNPH-derivatized, dansylated
products revealed two peaks (24 min and 27 min) in equal ratio,
attributable to the geometric isomers shown below (without
dansylation): 5
[0086] Electrospray ionization mass spectroscopy (EIMS) of the
HPLC-purified products conformed to the expected mass ion, m/z 251,
for the non-dansylated isomers.
[0087] Brain homogenates were prepared as above after 2 hours or 25
hours of ischemia from rats subjected to permanent focal cerebral
ischemia, and the homogenates were derivatized with
2,4-dinitrophenyihydrazine as above. HPLC analysis of the
dansylated derivatized homogenates revealed the appearance of two
peaks, and EIMS confirmed their identities as the isomeric
3-aminopropanal-2,4-dinitrophenylhydrazine reaction products. The
3-aminopropanal derivatization products were not detected by this
HPLC-based assay in brain homogenates prepared from sham-operated,
normally perfused control animals, indicating that ischernia
mediated the appearance of 3-aminopropanal.
[0088] Thus, use of the quantitative HPLC-based assay for
derivatized 3-AP (described above), which can reproducibly detect
1-2 .mu.M 3-aminopropanal in brain tissue, confirmed that
3-aminopropanal is not present or produced in homogenates prepared
from normally perfused brain. However, within 2 hours after the
onset of cerebral ischemia, it was observed that there were
significantly increased 3-aminopropanal levels, which increased
further in a time-dependent manner for a least 25 hr after the
onset of ischemia (see FIG. 2; results are expressed following
normalization for protein content as measured by the Bradford
method (Biorad), and after correction for HPLC injection volume
using an internal standard of 1,7-diaminoheptane. Data are
mean.+-.s.d.; n=3-4 animals per group). When considered with the
foregoing observation that cerebral ischemia mediates an early
induction of brain polyamine oxidase activity, these findings
indicate that ischemia induces PAO activity which produces 3-AP as
a metabolite, and that this enzyme pathway continues to generate
3-aminopropanal during at least the first day after the onset of
cerebral ischemia.
[0089] The HPLC assay employed may well have underestimated the
amount of 3-aminopropanal produced in the ischemic brain, because
3-aminopropanal is a reactive molecule which can bind to the amino
and sulfhydryl groups of proteins (Brunton et al., Toxic. In Vitro
8:337-341, 1994; and Seiler, Digestion 46:319-330, 1990), thereby
decreasing its availability for derivatization and detection.
Nonetheless, after correcting the measured levels for total brain
protein (213 g/kg), brain 3-aminopropanal concentrations after
ischemia reached a highly cytotoxic range (0.75 to 2.0 mM).
[0090] A further set of experiments was conducted to examine the
time course of 3-aminopropanal production relative to the
development of brain cell death. These experiments were necessary
in order to determine whether the enzymatic formation of
3-aminopropanal was temporally upstream of the onset of brain cell
death. Accordingly, the volume of cell death was measured by
preparing sections from ischemic brain, staining with the vital
dye, 2,3,5-triphenyltetrazolium chloride (TTC), and integrating the
total area of dead tissue across multiple sections. For the first
three hours of ischemia, cells in the region of the occluded middle
cerebral artery were observed to be largely viable (total volume of
cell death=2.+-.2 mm.sup.3). Histological examination of
hematoxylin and eosin-stained brain sections confirmed that cells
were morphologically intact and had not yet developed degenerative
changes at a time when 3-aminopropanal levels were already
significantly increased. Over the subsequent 25 hours, it was
observed that the volume of brain cell death "spread" to involve a
significantly larger region (infarct volume at 25 hours=71.+-.24
mm.sup.3; vs infarct volume at 3 hours=2.+-.2 mn.sup.3; P<0.05),
and this "spreading" cell death developed in association with
increasing 3-aminopropanal levels. These findings give evidence
that significant accumulation of 3-aminopropanal occurs during the
early response to cerebral ischemia, and precedes the development
of overt, progressive brain cell death.
Example 2
In Vivo Screening Assay for Agents that Inhibit
3-Aminopropanal-Mediated Tissue Damage.
[0091] Exogenous 3-Aminopropanal, or Polyamines Metabolizable to
Yield 3-Aminopropanal, Cause Brain Damage In Vivo
[0092] Since polyamine oxidase activity is present in normal
mammalian brain (Paschen, Cerebrovasc. Brain Metab. Rev. 4:59-88,
1992; and Seiler and Bolkenius, Neurochem. Res. 10:529-544, 1985),
the next experiment investigated whether increased extracellular
levels of substrate (e.g., spermine or spermidine) would stimulate
the production of 3-aminopropanal and induce local cell death. To
investigate this possibility, spermine, spermidine, and metabolites
thereof were administered into the cerebral cortex of rats by
direct stereotactic microinjection, and the brain volume of
resulting cell death measured by TTC staining brain sections.
Polyamines or 3-aminopropanal were administered into rat brain
cortex in vivo by stereotactically-guided microinjection into a
location selected to correspond to the stereotactic/anatomic
co-ordinates perfused by the middle cerebral artery. Briefly, male
Lewis rats (270-300 g) were anesthetized and placed in a
stereotactic head frame (Stoelting Co., Wood Dale, Ill., U.S.A.).
The incisor bar was adjusted until the plane defined by the lambda
and bregma was parallel to the base plate. A microsurgical
craniotomy was performed 1.7 mm anterior to bregma, and 5 mm right
of the midline, and the tip of a 29-gauge needle advanced 2 mm deep
to the dural opening. Polyamine-containing solution (25 .mu.g/2
.mu.l) prepared in sterile saline (NaCl, 154 mM) was injected over
3 minutes, the needle then left undisturbed for 5 minutes, then
removed. Animals were euthanized 48 hr later, the brains excised
and sectioned in 1 mm thick slices in the coronal plane, and the
sections immersed for 30 minutes at 37.degree. C. in a solution
containing 2,3,5-triphenyl-2H-tetrazolium chloride (2% in NaCl, 154
mM). Brain infarction was visualized as areas of unstained (white)
tissue which were easily contrasted against viable tissue, which
stained red. Slices were placed in buffered 10% formalin and
infarct size quantitatively assessed by planimetric analysis. In
separate studies, histopathological analysis of brain sections
verified the location of the injectate and the correlation of
tissue damage revealed by TTC staining. Groups of 3-4 animals were
used for each of the experimental conditions as noted.
[0093] There was brain tissue damage in animals given intracortical
spermine or spermidine, but cell death was not observed after
intracortically administered putrescine, a polyamine which cannot
be degraded by polyamine oxidase to generate 3-aminopropanal (see
FIG. 3). The quantities of 3-aminopropanal administered (25 .mu.g
per injection) were similar to the amounts produced endogenously
during ischemia (approximately 350 .mu.M assuming a volume of
distribution of a typical middle cerebral artery infarction).
Intracortical administration of these amounts of 3-aminopropanal
caused significant cytotoxicity in the cerebral cortex. Systemic
administration of the polyamine oxidase inhibitors chloroquine and
aminoguanidine conferred significant protection against the
development of spermine-mediated intracortical damage (see FIG. 4),
suggesting that polyamine oxidase activity is necessary to mediate
the cytotoxicity of extracellular spermine. Intracortical
administration of aminoguanidine also conferred significant
protection against intracortical spermine-mediated cell death (see
FIG. 4), indicating that the cerebroprotective effects of the
enzyme inhibitor occur locally in the brain cortex, and not via
some unanticipated peripheral drug action. Of note, aminoguanidine
failed to significantly attenuate the direct cytotoxicity of
intracortical 3-aminopropanal (see FIG. 4), suggesting that the
protective mechanism of aminoguanidine against spermine
cytotoxicity is through inhibition of polyamine oxidase, and not by
directly inhibiting the cytotoxicity of 3-aminopropanal.
[0094] Thus, increased extracellular levels of spermine,
spermidine, or 3-aminopropanal (but not putrescine) are cytotoxic
in vivo, in this case to cerebral cortical cells. Furthermore, this
model provides a convenient assay for the activity of test
compounds and compositions to inhibit 3-aminopropanal-mediated
tissue damage. Test compounds or compositions may be administered
in any of a variety of dosage formats (e.g., intravenous,
intraperitoneal, oral, intramuscular, intracranial), either before
or at various time periods after experimental introduction of
3-aminopropanal into tissue, such as brain. The activity of test
compounds and compositions to antagonize the in vivo cytotoxic
effects of the administered 3-aminopropanal may then be
conveniently evaluated, for instance by vital staining to estimate
the extent of induced cell death or tissue infarction.
[0095] 3-Aminopropanal Mediates Both Cellular Necrosis and
Apoptosis In Vivo
[0096] A series of experiments were undertaken to elucidate the
endogenous mechanisms underlying tissue damage during ischemia.
Specifically, the fate of brain cells in animals subject to
intracortical administration of polyamines or 3-aminopropanal was
studied. Histopathologic examinations performed on brain tissue
harvested 24 hr after intracortical 3-aminopropanal microinjection
localized degenerative changes to the tissue surrounding the
injection zone. Stereotactically guided microinjections of 3-AP or
various polyamines were made into the cerebral cortex. Tissues were
prepared for histology by anesthetizing the animals and then
perfusing transcardially in sequence with saline (30 ml, pH=7.4),
formaldehyde (100 ml, 4% in PBS, pH=6.5), then formaldehyde (100
ml, 4%, pH=8.5). Tissues were allowed to postfix for 30 minutes.
then perfused with sucrose (50 ml, 20% in 0.1M PBS, pH=7.4). Brains
were then removed, stored in sucrose overnight at 4.degree. C., and
sectioned into 10 mm thick sections. Deoxynucleotidyltransferase
(TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) staining was
performed using a kit according to the manufacturers instructions
(ApopTag, Oncor). Cells near the 3-aminopropanal injection site
were necrotic, as evidenced by eosin-positive staining. Moreover,
in the same region, cells were undergoing programmed cell death, as
evidenced by TUNEL positive staining. These changes were not
observed in the injection zone when either vehicle or putrescine
was administered. Direct intracortical administration of spermine
also caused cell necrosis and apoptosis, and this degeneration was
significantly inhibited by administration of aminoguanidine. Thus,
the accumulation of brain cell damage in vivo, in response to
intracortical extracellular 3-aminopropanal administration, occurs
through both cellular necrosis and programmed cell death.
Example 3
In Vitro Screening Assays for Agents to Prevent or Minimize Tissue
Damage and Cell Death Following Ischemia
[0097] 3-Aminopronanal Induces Apoptosis in Glial Cells, but
Cellular Necrosis in Neuronal Cells
[0098] To directly investigate the cytotoxic mechanisms and
corresponding signaling cascades induced by 3-aminopropanal,
cultured human glial (HTB14) and neuronal (HTB 11) cell lines were
exposed to 3-aminopropanal. The glial cell line (HTB14) (Ponten and
Macintyre, Acta Pathol. Microbiol. Scand. 74:465-486, 1968) and
neuronal cell line (HTB11) (Bluestein, Proc. Natl. Acad. Sci.
U.S.A. 75:3965-3969, 1978) were obtained from the American Type
Culture Collection (ATCC) and cultured in minimal Eagle's medium
(MEM; Gibco), supplemented to 10% fetal bovine serum (FBS;
Hyclone), 1 mM Na pyruyate (Sigma), 0.5% penicillin/streptomycin
(Sigma), in a humidified atmosphere of 5% CO.sub.2 in air at
37.degree. C. For all experiments involving exposure to
3-aminopropanal, cells were grown in 96-well microtiter plates to
90-95% confluence. Prior to all experiments the medium was replaced
with fresh serum-free medium (Opti-MEM I), in order to minimize
interaction of 3-aminopropanal with serum proteins. For all
experiments utilizing a short duration of 3-aminopropanal exposure
(5 minutes to 2 hours in 96-well plates), the cells were washed at
the times indicated, and then incubated in Opti-MEM I for up to 20
hours before assessment of cell viability. Assessment of
involvement of caspase-1 and caspase-3 in cell death following 3-AP
exposure was by pretreating cells with the caspase-l inhibitor,
Ac-YVAD-CMK (BACHEM), or the caspase-3 inhibitor, Ac-DEVD-CHO
(Peptides International), for 3 hours followed by addition of
3-aminopropanal for 5 additional hours. DMSO controls were
performed to assess for effects of solvent. Cell viability was
measured by the metabolically-dependent reduction of
3-(4,5-dimethylthiazol-2-yl)-2,5-dip- henyltetrazolium bromide
(MTT) (Sigma) (Sieuwerts et al., Eur. J Clin. Chem. Clin. Biochem.
33:813-823, 1995). Data are expressed as mean.+-.s.d.; n=3-6 wells
per condition, and experiments performed in triplicate.
[0099] Following 20 hr of incubation, the cytotoxic LD.sub.50 for
3-aminopropanal was estimated as 275.+-.10 .mu.M for the glial cell
line, HTB-14, and 90.+-.20 .mu.M for the neuronal cell line, HTB
11. 3-Aminopropanal was somewhat more cytotoxic in primary rat
astroglial cell cultures (LD.sub.50=80.+-.9 .mu.M). A time course
study revealed that 3-aminopropanal exposure for as little as 5
minutes was significantly cytotoxic to neuronal cells, but a longer
exposure was required to mediate significant cytotoxicity in glial
cells (see Table I below).
2TABLE I Time course study of cell viability after exposure to
3-aminopropanal. Gial or neruonal cell line cultures were exposed
to an LD.sub.100 concentration of 3-aminopropanal (750 or 350
.mu.M, respectively) for the indicated time, then medium was
replaced with serum-free (no 3-AP) for a total incubation time of
20 hrs. Cell viability was then determined by MIT assay (data shown
are mean .+-. s.e.). Cell viability was 100 .+-. 4% for all times
in vehicle-treated controls. Cell Viability (% alive) Time (min)
Glial cells (HTB14) Neurons (HTB11) 5 96 .+-. 3 29 .+-. 6 60 92
.+-. 1 13 .+-. 4 120 78 .+-. 7 6 .+-. 2 1200 5 .+-. 5 3 .+-. 1
[0100] This latent onset suggested that glial cell death might be
dependent upon apoptosis-mediated pathways. In agreement with this
possibility, an apoptosis-specific DNA fragmentation pattern was
observed following exposure of HTB-14 glial cells to
3-aminopropanal, but not after corresponding exposure of cultured
neuronal cells. HTB- 11.
[0101] Additional evidence of apoptosis by flow cytometric
detection of DNA strand breaks was obtained using the terminal
deoxynucleotidyl transferase mediated dUTP nick end labeling
(TUNEL) method (Gavrieli et al., J. Cell. Biol. 119:493-501, 1992).
Terminal deoxynucleotidyl transferase mediated dUTP nick end
labeling (TUNEL) staining was performed on cell cultures treated
with 3-aminopropanal, as indicated. Cells were harvested and
collected by centrifugation at 1,500 rpm for 5 min. The pellets
were fixed with 1X ORTHO Permeafix (Orthodiagnostics) at room
temperature for 40 min. After washing with Dulbecco's
phosphate-buffered saline containing 1% bovine serum albumin
(PBS-BSA), cells were stained by the TUNEL method using the Oncor
Apop Tag Direct Fluorescein kit (Oncor), in accordance with the
manufacturer's instructions. A negative control was performed by
preparing a reaction solution that was devoid of TdT. A Becton
Dickinson flow cytometer was used for all analyses; five to ten
thousand events (ungated) were collected according to a single
color procedure.
[0102] In these experiments, 76% of the glial cells stained
TUNEL-positive following 13 hours of exposure to 275 .mu.M
3-aminopropanal, whereas vehicle-treated control cells were
uniformly TUNEL-negative. Multi-parameter flow cytometry revealed
that glial cell populations exposed to 3-aminopropanal exhibited a
decrease in cellular forward light scatter and an increase in side
scatter, consistent with the typical cell shrinkage, chromatin
condensation, and nuclear fragmentation of apoptosis. Apoptosis of
glial cells was also confirmed by subdiploid staining with
propidium iodide and with Annexin V/PI.
[0103] In contrast to the results in the glial cell line (HTB-14),
3-aminopropanal did not induce apoptosis in neuronal cell cultures
(HTB 11) using similar experimental methods. DNA samples from HTB11
or HTB14 cells (20-30.times.10.sup.6 cells) were assayed for strand
breakage by resuspending harvested cells (treated with 3-AP or
control) in a reaction buffer containing proteinase K, and
incubating overnight at 55.degree. C. RNase was added to a final
concentration of 50 mg/ml and the samples were incubated at
37.degree. C. for one hour. DNA was extracted three times with
phenol/chloroform and twice with chloroform and precipitated in two
volumes of chilled 100% ethanol and 0.3M sodium acetate (pH 5.2).
The DNA was resuspended in 50 .mu.l of dH.sub.2O, fractionated by
1.5% agarose gel electrophoresis, and stained with SYBR Green I
nucleic acid stain (Molecular Probes).
[0104] Electrophoresis of DNA prepared from 3-aminopropanal-treated
neurons revealed no evidence of chromosomal DNA degradation. In
addition, no increase in TUNEL-positivity under these conditions
was observed, although a forward scatter/side scatter analysis
revealed significant cell death after 3-aminopropanal treatment
(55.7%), but not in vehicle-treated controls (9.1%).
[0105] There was also no evidence of apoptosis as measured with
Annexin V, a method used to detect loss of cell membrane
phospholipid asymmetry that has be associated with apoptosis.
Annexin V/propidium iodide (PI) staining was performed using a kit
in accordance to the manufacturer's instructions (The Apoptosis
Detection Kit, R&D Systems, Minneapolis). Cells were analyzed
by flow cytometry within one hour of completion of staining. The
AnnexinV-FITC and PI signals were quantitated independently
according to a two-color flow cytometric procedure.
[0106] Apoptosis was induced in neuronal cells by exposure to
camptothecin (Furuya et al., Anticancer Res 17:2089-2093, 1997) (15
.mu.g/ml for 20 hr) as assessed by TUNEL and Annexin V methods,
indicating that the absence of apoptosis after 3-aminopropanal
exposure was not due to some unanticipated generalized cellular
defect in these neuron-like cells. Thus, in contrast to glial
cells, exposure of neuronal cells to 3-aminopropanal causes
primarily necrotic cell death.
[0107] The in vitro cell culture experiments described in this
example provide convenient assays for the activity of test
compounds and compositions to inhibit 3-aminopropanal-mediated
cytotoxicity, predictive of like benefits in mitigating tissue
damage in vivo. Test compounds or compositions may be introduced
simultaneously with, before or at various time periods after
experimental introduction of 3-aminopropanal into the cell
cultures, which may be glia-like (e.g., HTB-14), neuron-like (e.g.,
HTB-11) or typical of other cells or tissues subject to ischemic
damage in vivo. The activity of test compounds and compositions to
antagonize the in vitro cytotoxic effects of the administered
3-aminopropanal may then be conveniently evaluated, for instance by
staining to estimate the extent of induced cell death and further
to characterize said cytotoxicity as cellular necrosis vs.
apoptosis.
[0108] Caspase-1 plays a role in the 3-aminopropanal-induced
apoptosis of glial cell cultures.
[0109] The cysteine proteases ICE (Interleukin-1 beta Converting
Enzyme; caspase-1) and cysteine protease P-32 (CPP-32; caspase-3)
have been implicated in the cellular signaling pathways mediating
apoptosis during cerebral ischemia (Bhat et al., J. Neurosci.
16:4146-4154, 1996; Loddick et al., Neuroreport. 7:1465-1468, 1996;
Hara et al., Proc. Natl. Acad. Sci. U.S.A. 94:2007-2012, 1997; and
Friedlander et al., J. Exp. Med. 185:933-940, 1997). To investigate
whether these proteases were required for the induction of
apoptosis by 3-aminopropanal in glial cells, HTB14 cells were
treated for three hours with a tetrapeptide ICE inhibitor
(Ac-YVAD-CMK), or with a CPP-32 inhibitor (Ac-DEVD-CHO), followed
by a five hour treatment with 3-aminopropanal. Treatment with the
ICE inhibitor, but not the CPP-32 inhibitor, conferred
dose-dependent inhibition of 3-aminopropanal-induced cell death
(see FIG. 5), indicating that ICE proteases are required for
3-aminopropanal-induced apoptosis. These data give evidence for a
specific role of ICE in the 3-aminopropanal-induced signaling that
mediates apoptosis in glial cells, and further exemplify the
utility of these in vitro screening assays for identifying
compounds and compositions with activity in mitigating
3-aminopropanal-induced tissue damage.
Example 4
In Vivo Screening Assay for Compounds and Compositions with
Activity in Mitigating Tissue Damage Following Ischemia
[0110] Administration of Polyamine Oxidase Inhibitors In Vivo
Attenuates 3-Aminopropanal Production and Protects Against
Ischemia-Induced Tissue Damage
[0111] The mechanism of ischemia-induced cell death and tissue
damage introduced herein predicts that administration of polyamine
oxidase inhibitors during cerebral ischemia in vivo will reduce
both the accumulation of 3-aminopropanal and the volume of cerebral
infarction. Accordingly, these end points were measured after
administering two structurally distinct polyamine oxidase
inhibitors to rats in the standardized model of permanent middle
cerebral artery occlusion described above. Aminoguanidine,
administered after the onset of cerebral ischemia (320 mg/kg i.p.
15 minutes post-ischemia, then 110 mg/kg i.p. every 8 hrs),
significantly reduced the volume of cerebral damage (Cockroft et
al., Stroke 27:1393-1398, 1996). Aminoguanidine administered by
this established treatment protocol efficaciously prevented the
increase of brain 3-aminopropanal levels (see Table II). Rats were
subjected to permanent middle cerebral artery occlusion
(n=4/group), and infarct volume measured 25 hours after the onset
of ischemia as described above. Treated animals received either
aminoguanidine (AG; 320 mg/kg i.p. 15 minutes post-ischemia,
followed by 100 mg/kg i.p. every 8 hr), chloroquine (CQ; 25 mg/kg
i.p. 15 minutes post-ischemia), or vehicle (saline, i.p.) given 15
minutes post-ischemia. Table II shows that two structurally
distinct inhibitors of polyamine oxidase (AG and CQ) attenuate
cerebral infarction and that AG can prevent the ischemia-associated
local increase in 3-aminopropanal.
3 TABLE II Infarct Brain 3-aminopropanal Volume (mm.sup.3) level
(.mu.mol/g protein) Vehicle 71 .+-. 24 13 .+-. 8 Aminoguanidine 12
.+-. 2*# not detectable Chloroquine 27 .+-. 8* not tested *P <
0.05 vs vehicle. #from (Cockroft et al., Stroke 27:1393-1398,
1996)
[0112] In agreement with the above asserted mechanisms of tissue
damage in ischemia, administration of chloroquine (a known
inhibitor of polyamine oxidase) also protected against
ischemia-induced tissue damage, even when such administration was
delayed 15 minutes after occlusion of the middle cerebral artery
(Table II). It was previously reported that the protective effects
of aminoguanidine were not attributable to altering peripheral
cardiovascular parameters that influence the volume of brain damage
(Cockroft et al., Stroke 27:1393-1398, 1996). Similarly,
chloroquine did not significantly alter systemic homeostatic
responses to cerebral ischemia; physiological parameters measured
before and during ischemia (blood pressure, heart rate, body
temperature, arterial blood gases) did not differ between groups of
subjects treated with vehicle or chloroquine. Thus, the
cerebroprotective effects of chloroquine cannot be attributed to
alterations in the peripheral cardiovascular response to cerebral
ischemia, and seem instead to relate to the activity of chloroquine
to inhibit PAO activity.
[0113] Previously, ladecola and colleagues reported that iNOS is
upregulated 24-48 hr after cerebral ischemia, and that delayed
administration of aminoguanidine can prevent secondary NO- mediated
brain damage in a delayed therapeutic window (Zhang et al.. Stroke
27:317-323, 1996). The evidence of the present Examples, on the
other hand, indicates that polyamine oxidase activity is
upregulated much earlier after cerebral ischemia (within 2 hours),
and that early administration of aminoguanidine inhibits the
generation of 3-aminopropanal. Although the most direct
interpretation of these data is that two structurally distinct
inhibitors of polyamine oxidase prevented ischemic damage by
preventing the formation of 3-aminopropanal, a series of additional
experiments was performed to exclude other possibilities.
[0114] Addition of even suprapharmacological amounts of chloroquine
(1 mM) failed to inhibit iNOS activity measured in RAW 264.7 cell
lysates (INOS activity in control cultures=13300.+-.250 DPM/.mu.g
protein vs. INOS activity in chloroquine-treated
cultures=11800.+-.900 DPM/mg protein; P>0.05). Thus, these data
exclude the unlikely possibility that chloroquine protection
occurred through an unanticipated inhibition of iNOS.
[0115] When cell viability was measured in the presence of PAO
inhibitors, it was observed that the LD.sub.50 for 3-aminopropanal
after overnight incubation in HTB11 cells was similar whether or
not aminoguanidine or chloroquine were added. Thus, these data
exclude the unlikely possibility that aminoguanidine or chloroquine
might protect cells by directly interfering with the cytotoxic
activity of 3-aminopropanal.
[0116] When aminoguanidine was added to primary neuronal cultures
treated with N-methy-D-aspartic acid (NMDA), no attenuation of
cytotoxicity was observed (Table III). Thus, these data exclude the
unlikely possibility that the mechanism of aminoguanidine
protection is mediated via altering the sensitivity of cells to the
cytotoxicity of glutamate. Cultured hippocampal neurons (12-15 days
in vitro) were exposed to NMDA (500 .mu.M) for 5 min in MEM
(without serum) supplemented with glutamine (100 .mu.M) and glycine
(10 .mu.M), rinsed in Earle's Basic Salt Solution, and incubated
under standard conditions for 24 hr (37.degree. C.). Neuronal
survival was assessed microscopically, by counting a sample of cell
somas for uptake vs. exclusion of trypan blue. Aminoguanidine or
the non-competitive excitatory amino acid receptor antagonist,
MK-801, was added just prior to NMDA and in the final rinse at the
concentrations shown. Data are mean.+-.s.e. of nine wells from
three replicate experiments. Table III shows that aminoguanidine
was not protective against NMDA neurotoxicity.
4TABLE III Cell viability (% dead) [Aminoguanidine] NMDA MK-801
Neurotoxicity (.mu.M) (500 .mu.M) (20 .mu.M) (% dead) 0 - - 3 .+-.
1 0 + - 88 .+-. 4 0 + + 8 .+-. 3 1000 + - 79 .+-. 4 1000 - - 7 .+-.
2
[0117] A further experiment was conducted to determine whether
3-aminopropanal mediated cell death through induction of iNOS
activity. Addition of iNOS inhibitors (L-N.sup.G-monomethylarginine
(L-NMMA) or aminoguanidine) to 3-aminopropanal-treated glial cells
failed to attenuate the development of TUNEL-positivity as measured
by cytofluorography. Although these data have excluded a number of
plausible alternative mechanisms through which aminoguanidine or
chloroquine might protect against cerebral ischemia, it remains
theoretically possible that other activities of chloroquine might
additionally contribute to the observed protection against
infarction (i.e., inhibition of free radical formation,
phospholipase activity or protein synthesis). However, such
alternative mechanisms do not account for the present chain of
evidence showing that: 1) administration of inhibitors of polyamine
oxidase activity limits the formation of 3-aminopropanal in the
setting of ischemia; 2) 3-aminopropanal cytotoxicity cannot be
blocked with chloroquine; and 3) either chloroquine or
aminoguanidine prevented the brain damaging effects of either
intracortical spernine or ischemia.
Example 5
In Vivo and In Vitro Screening Assay Results: Active Compounds
[0118] Compounds Protective against 3-Aminoprolanal-Induced
Cytotoxicity In Vitro
[0119] Use of the in vitro screening assays described in Example 3
enables the identification of compounds with predicted clinical
utility in ameliorating the extent of tissue damage following
ischemia, particularly as therapeutic agents against the brain
damage associated with stroke. Replicate glial cell cultures (HTB
14) or neuronal cell cultures (HTB 11) are cultured under
conditions well-known in the art, and exposed to a concentration
series of 3-aminopropanal (e.g., a 2X dilution series from 3200
.mu.M to 25 .mu.M). Test compounds are added simultaneously with,
or at various times before or after addition of 3-aminopropanal,
according to the detailed procedures of Example 3, allowing the
test compounds to be evaluated for beneficial, protective effects
(or for toxic effects to be noted) in an in vitro assay that is
predictive of like ameliorative effects on the tissue damage
attendant to focal ischemia in vivo. Use of glia-like and
neuron-like cells in this in vitro assay is particularly adapted to
modeling the cytotoxicity and tissue damage of cerebral ischemia or
stroke. A variety of test compounds were evaluated by these methods
at a concentration of 1.0 mM, with results summarized in Table IV
below:
5TABLE IV Effect of test compounds on 3-AP cytotoxicity No effect
or weakly protective Toxic Protective Glial cell assay (HTB14)
putrescine 3-(2-amino-2-oxo- p25 aminoguanidine ethyl)-4-methyl-5-
p27b penicillamine (hydroxyethyl)- 3-(2-methoxy-2-
2,3-diamino-thiazolium thiazolium bromide oxoethyl)-benzothia-
O-mesitylenesulfonate 2-mercaptoimidazole zolium bromide
3-(2-amino-2-oxoethyl)-4- p117a 2-mercapto-1- methyl-thiazolium
bromide p213a methyl-imidazole thiamine HCl cysteine
2-mercaptopyridine N-acetylcysteine 6-mercaptopurine riboside
2-mercaptoethyl- p213b amine glutathione 1-(carboxymethyl)-
pyridinium chloride hydrazide p27a Neuronal cell assay (HTB11)
N-acetylcysteine p25 glutathione p27a p27b cysteine wherein: p25 is
3-(2-phenyl-2-oxoethyl)-thiazolium bromide, and this compound also
has been coded as PICVA-25; p27b is
3-(2-phenyl-2-oxoethyl)-4-methyl-5-(hydroxyethyl)-thiazolium
bromide p27a is N-(2-phenyl-2-oxoethyl)-pyridinium bromide, and
this compound has also been coded as AP1 and as PICVA-27; p213a is
2-(2-phenyl-2-oxoethyl)-2-mercaptoimidazole ether p213b is
N,N'-bis-(2-phenyl-2-oxoethyl)-imidazolium bromide p117a is
N-(2-phenyl-2-oxoethyl)-2-mercaptopyridinium bromide.
[0120] In an alternative embodiment of the screening assay of
Example 3, various concentrations of the test compound (e.g.,
10-1000 .mu.M) are incubated with the indicator cells in presence
of a fixed concentration of 3-AP (e.g., 200 .mu.M). The toxicity of
the test compounds may be evaluated in parallel cultures incubated
without 3-AP; generally, the desired test compound will show
cellular toxicity at much higher doses than those that confer
protection against 3-AP (e.g., 10-10,000-fold). The results of such
tests are summarized in Table V, below:
6TABLE V Effect of test compounds on 3-AP cytotoxicity No effect or
Toxic or Protective (50% Effective weakly protective no effect
dose; 50% Toxic dose) Glial cell assay (HTB14) AP6 AP9 AP5 (150
.mu.M; 7 mM) AP2 AP12 AP7 AP19 p27a (425 .mu.M; 5 mM) YA1 AP20 AP21
(100 .mu.M; not tested) YA2 AP23 AP22 (100 .mu.M; 1 mM) AP18 AP28
AP24 3,5-di-tert.- ascorbic acid butyl-4-hydroxy- 34p toluene
wherein: AP6 is N-(2-phenyl-2-oxoethyl)-2-(2'-pyridine)-pyridinium
bromide. AP2 is N-(2-phenyl-2-oxoethyl)-quinolinium bromide. AP7 is
N-(2-phenyl-2-oxoethyl)-pyrazinium bromide. YA1 is
2-phenyl-2-oxoethyl-dimethylphosphonate. YA2 is
N-(2-phenyl-2-oxoethyl)-triethylammonium bromide. AP18 is
N-(2-phenyl-2-oxoethyl)-4-tert.-butylpyridinium bromide. AP24 is
N-(2-phenyl-2-oxoethyl)-3-n-butylpyridinium bromide. 34P is
pyridine-3,5-dicarboxylic acid. AP9 is N-(2-phenyl-2-oxoethyl)-4--
N,N-dimethylamino-pyridinium bromide. AP12 is
N-(2-phenyl-2-oxoethyl)-pyrazinium bromide. AP19 is
N-(2-phenyl-2-oxoethyl)-3-fluoropyridinium bromide. AP20 is
N-(2-phenyl-2-oxoethyl)-4-ethylpyridinium bromide. AP23 is
N-(2-phenyl-2-oxoethyl)-2,6-dihydroxymethylpyridinium bromide AP28
is N-(2-phenyl-2-oxoethyl)-3 ,5-diiodo-4-pyridinone. AP5 is
N-(2-phenyl-2-oxoethyl)-2,5-dicarboxypyridinium bromide; and this
compound has also been coded PICVA-13. AP21 is
N-(2-phenyl-2-oxoethyl)-3,4-dicarboxyamide-pyridinium bromide. AP22
is N-(2-phenyl-2-oxoethyl)-3-bromo-5-carboxypyridinium bromide.
[0121] Compounds Protective Against 3-Aminopropanal-Induced
Cytotoxicity In Vivo
[0122] Use of the in vivo screening assays described in Examples 2
and 4 enables the identification of compounds with predicted
clinical utility in ameliorating the extent of tissue damage
following ischemia, particularly as therapeutic agents against the
brain damage associated with stroke. Use of these in vivo screening
assays is particularly valuable to further validate the potential
beneficial effects of compounds and compositions identified as
pharmacologic inhibitors of 3-AP toxicity in the companion in vitro
assays of Example 3. Three phenylacyl pyridinium derivatives
identified in in vitro as inhibitors of 3-AP cytotoxicity were
evaluated by the in vivo assay methods described above. As shown in
FIG. 6, when either of three different phenylacyl pyridinium
derivatives (designated PICVA- 13, PICVA-25 and PICVA-27) were
administered i.p. beginning 15 minutes after the onset of ischemia
(operationally defined as the time of division of the MCA), the
volume of brain tissue eventually infarcted was significantly
reduced as compared to vehicle-treated controls. With compound
PICVA-13, this protective effect was dose-dependent (see FIG. 7).
The protective effect of compound PICVA-13 also was obtained at a
dose of 600 mg/kg when PICVA-13 was administered as late as two
hours after the onset of ischemia (see FIG. 8; 70% reduction in
infarct size; P<0.05). This dose-dependent protection by
PICVA-13 treatment was independent of systemic parameters known to
influence the extent of brain damage in stroke (e.g., blood
pressure, heart rate, temperature, serum glucose, and arterial pH,
PO.sub.2 and PCO.sub.2.; which parameters sampled at 60 and 120
minutes after onset of ischemia did not differ significantly from
baseline). Also, an index of brain edema did not differ between
PICVA-13-treated and control treated subjects. This example
provides evidence that pharmacologic inhibitors of 3-aminopropanal
cytotoxicity can significantly limit the volume of brain damaged in
experimental stroke, predictive of the utility of such agents as
therapeutic agents for human clinical use.
[0123] Whole Animal Toxicity of Phenylacyl Pyridinium Compounds
[0124] In the in vivo screening tests reported above, appreciable
mortality (25% and 38%, respectively) was associated with treatment
with compounds PICVA-27 and PICVA-25; no deaths occurred in
association with PICVA-13 treatment. Further testing showed that no
otherwise untreated mice died at the seven-day timepoint after
administration of doses of PICVA-13 ranging from 1.0 mg/kg to 1000
mg/kg (two mice treated per dosage condition; 1.0, 10, 100, 500 and
1000 mg/kg). Male Balb/c mice weighing between 20 and 25 grams were
treated with PICVA-13 by intraperitoneal injection, and the mice
were observed for survival over the ensuing seven days. All mice
survived and no mice exhibited overt signs of toxicity,
demonstrating normal grooming and feeding over the observation
period. This lack of toxicity established compound PICVA-13 as the
preferred compound.
* * * * *