Note: Descriptions are shown in the official language in which they were submitted.
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METHODS FOR TREATING INFLAMMATION AND INFLAMMATORY
DISEASES USING DADPRT INHIBITORS
The present invention relates to methods for treating inflammation and
inflammatory diseases, including arthritis, in animals or manunals. The
invention
also relates to methods for treating animals or mammals having both gram
negative
and gram positive endotoxin symptoms resulting from systemic infections or
resulting
from infestation by Iipopolysaccharides. These methods involve the use of
therapeutically effective amounts of pADPRT inhibitory compounds.
BACKGROUND OF THE INVENTION
The use of pADPRT inhibitory compounds have been reported for treating
cancer and viral infections. Examples of these methods are described in U.S.
Patent
Nos. 5,464,871; 5,473,074; 5,482,975; 5,484,951; 5,516,941; and 5,583,155.
In the published literature, 5-iodo-6-amino-1, 2-benzopyrone (INH,BP), a
novel inhibitor of the nuclear enzyme poly-ADP ribose polymerase (pADPRT) has
recently been shown to inhibit in vivo tumorigencity in a Ha-ras transfected
endothelial cell line; Bauer et al., 1995, "Modification of growth related
enzymatic
pathways and apparent loss of tumorigenicity of a ras-transformed bovine
endothelial
cell line by treatment with 5-iodo-6-amino-1, 2-benzopyrone (INH2BP)," Int. J.
Oncol. 8:239-252; Bauer et aL, 1995, "Reversal of malignant phenotype by 5-
iodo-6-
amino-1, 2-benzopyrone, a non-covalently binding ligand of poly (ADP-ribose)
polymerase," Biochimie 77:347-377. Treatment with INH,BP has also resulted in
changes in topoisomerase I and II and MAP kinase activity; Bauer et aL. 1995,
"Modification of growth related enzymatic pathways and apparent loss of
tumorigencity of a ras-transformed bovine endothelial cell line by treatment
with 5-
iodo-6-amino-1, 2-benzopyrone (INH;BP)," Int. J. Oncol. 8:239-252; Bauer et
a!_.
1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, a
non-
covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77:347-
377.
Based on the effects observed, a hypothesis regarding the potential use of
INH2BP in
the therapy of cancer has been put forward; Bauer et ai., i995, "Modification
of
growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-
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transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1, 2-
benzopyrone (INH2BP)," Int. J. Oncol. 8:239-252; Bauer et al., 1995, "Reversal
of
malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, a non-covalently
binding
ligand of poly (ADP-ribose) polymerase," Biochimie 77:347-377.
Malignant growth and inflammatory processes share the activation of certain
cellular signal transduction pathways, .e.g., MAP kinase; Kyriakis et al.,
1996,
"Sounding the alarm: protein kinase cascades activated by stress and
inflammation,"
J. Biol Chem. 271:24313-24316; Ferrell, JE, 1996, "Tripping the switch
fantastic:
how a protein kinase cascade can convert graded inputs into switch-like
outputs,"
TIBS 21:460-466. Chronic inflammation frequently leads to carcinogenic
transformation, as demonstrated, for example, in the case of the intestinal
epithelium;
Kawai et al., 1993, "Enhancement of rat urinary bladder tumorigenesis by
ipopolysaccharide-induced inflammation," Cancer Res. 53:5172-5; Rosin et al. ,
1994, "Inflammation, chromosomal instability, and cancer: the schistosomiasis
model," Cancer Res. 54 (7 Suppl):1929s-1933s; Choi et al., 1994, "Similarity
of
colorectal cancer in Crohn's disease and ulcerative colitis: implications for
carcinogenesis and prevention," Gut 35:950-4. Based on the connection between
chronic inflammation and carcinogenic transformation, the aim of the present
study
was to investigate whether INl-I,BP affects the course of the inflammatory
process in
vitro and in vivo. In our study, the production of multiple proinflammatory
mediators
was induced by bacterial lipopolysaccharide (endotoxin, LPS). LPS is known to
induce a multitude of cellular reactions and triggers a systemic inflammatory
response. LPS-induced pro-inflammatory mediators include tumor necrosis factor
alpha (TNF), interleukin- 1, interferon-gamma, whereas anti-inflammatory
mediators
include interleukin-10 (IL-10) and interleukin-13; Deltenre et al., 1995,
"Gastric
carcinoma: the Helicobacter pylori trail," Acta Gastroenterol Belg. 58:193-
200;
Beutler, 1995, "TNF, immunity and inflammatory disease: lessons of the past
decade," J. Invest. Med. 42:227-35; Liles et al., 1995, "Review: nomenclature
and
biologic significance of cytokines involved in inflammation and the host
immune
response," J. Infect Dis. 172:1573-80; Giroir, 1993, "Mediators of septic
shock: new
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approaches for interrupting the endogenous inflammatory cascade," Critical
Car. Med.
21:780-9. As a consequence of the production of these inflammatory cytokines,
LPS
initiates the production of inflammatory free radicals (oxygen-centered, such
as
superoxide, and nitrogen-centered radicals, such as nitric oxide [NO]) and of
prostaglandins; Nathan, 1992, "Nitric oxide as a secretory product of
mammalian
cells," FASEB J. 6:3051-3064; Vane, J.R., The Croonian Lecture 1993, "The
endothelium: maestro of the blood circulation," Proc. Roy. Soc. Lond B 343:225-
246;
Szabo, C.; 1995, "Alterations in the production of nitric oxide in various
forms of
circulatory shock," New Horizons 3:3-32. The production of NO in inflammation
is
due to the expression of a distinct isoform of NO synthase (iNOS), while the
production of inflammatory cytokines is explained by the expression of a
distinct
isoform of cyclooxygenase (cyclooxygenase-2, COX-2); Nathan, 1992, "Nitric
oxide
as a secretory product of mammalian cells," FASEB J. 6:3051-3064; Vane, J.R.,
The
Croonian Lecture 1993, "The endothelium: maestro of the blood circulation,"
Proc.
Roy. Soc. Lond B 343:225-246; Szabo, C.; 1995, "Alterations in the production
of
nitric oxide in various forms of circulatory shock," New Horizons 3:3-32.
iNOS,
COX-2, as well as the above mentioned pro-inflammatory cytokines and free
radicals
which play an important role in the LPS-induced inflammatory response; Nathan,
1992, "Nitric oxide as a secretory product of mammalian cells," FASEB J.
6:3051-
3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: maestro of the
blood circulation," Proc. Roy. Soc. Lond B 343:225-246; Szabo, C.; 1995,
"Alterations in the production of nitric oxide in various forms of circulatory
shock,"
New Horizons 3:3-32. Moreover, NO (or its toxic byproduct, peroxynitrite), has
been
implicated as a key mediator leading to the transformation of the inflammatory
response into a carcinogenic process; Bartsch et al., 1994, "Endogenously
formed N-
nitroso compounds and nitrosating agents in human cancer etiology,"
Pharmaco enetics 2:272-7; Liu et at., 1992, "Woodchuck hepatitis virus surface
antigen induces NO synthesis in hepatocytes: possible role in
hepatocarcinogenesis.,"
Carcino enesis 15:2875-7; Ohshima et at., 1994, "Chronic infections and
inflammatory processes as cancer risk factors: possible role of nitric oxide
in
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carcinogenesis," Mutation Res. 305:253-64. In the current studies, we have
first
investigated whether treatment with INH2BP affects the production of the
inflammatory mediators tumor necrosis factor alpha [TNF], interleukin-10,
interleukin-6, NO, and prostaglandin in vivo, in LPS-induced models of
inflammation.
There are a multitude of intracellular processes which precede the production
of proinflammatory mediators. Activation of tyrosine kinases; Levitzki, A.,
1994,
"Signal-transduction therapy. A novel approach to disease management," Eur. J.
Biochem. 226:1-13; Novogrodeky et al. , 1994, "Prevention of
lipopolysaccharide-
induced lethal toxicity by tyrosine kinase inhibitors," Science 264U (Wash):
1319-22;
Marczin et al., 1993, "Tyrosine kinase inhibitors suppress endotoxin-and IL-
lbeta-
induced NO synthesis in aortic smooth muscle cells," Am. J. Physiol. 265:H1014-
1018; mitogen-activated protein kinase (MAP kinase); Matsuda et al., 1994,
"Signaling pathways mediated by the mitogen-activated protein (MAP) kinase
kinase/MAP kinase cascade," J. Leukocyte Biol. 56:548-53; L'Allemain, G.,
1994,
"Deciphering the MAP kinase pathway," Progr. Growth Factor Res. 5:291-334;
Cowley et al., 1994, "Activation of MAP kinase kinase is necessary and
sufficient for
PC12 differentiation and for transformation of NIH 3T3 cells.," Cells 77:841-
52; and
the nuclear factor kappa B (NF-kB) pathway; Baeuerle et al., 1994, "Function
and
activation of NF- B in the immune system," Ann. Rev. Immunol. 12:141-79;
Schreck
et al., 1992, "Nuclear factor kappa B: an oxidative stress-responsive
transcription
factor of eukaryotic cells (a review)," Free Radical Res. Comm. 17:221-37;
Muller et
al., 1993, "Nuclear factor kappa B, a mediator of lipopolysaccharide effects,"
Immunobiol. 187:233-56; are recognized as important factors in the
inflammatory
response and contribute to the expression or production of inflammatory
mediators.
Therefore, we have also investigated whether INHZBP also affects the LPS-
induced
activation of MAP kinase and the NF-kB by LPS. The results of the current
study
demonstrate that INH,BP has potent anti-inflammatory effects by modulating
multiple
components of the LPS-induced inflammatory response.
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SUMMARY OF THE INVENTION
One aspect of the invention is a method for treating inflammation or
inflammatory disease in an animal or mammal, which comprises the steps of
administering an effective amount of an pADPRT inhibitory compound to said
animal
or mammal.
Another aspect of the invention is a method for treating inflammation or
inflammatory disease in an animal or mammal, which comprises the steps of
administering an effective amount of a pADPRT inhibitory compound wherein the
pADPRT inhibitory compound is selected from the group consisting of.
a compound having the formula:
(I) R8
4 i i R1
R3 R2
wherein R,, Rõ R3, Rõ R5 and R are each selected from the group consisting of
hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally
substituted
with alkyl, alkoxy, hydroxy or halo, and only one of R, R2, R3, R4, R5 and, is
amino; a compound having the formula:
tjj)
o, ,NH2
R5 / R1
i
` R2
R4
R3
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wherein R,, Rõ R3, R,, and R5 are each selected from the group consisting of
hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally
substituted
with alkyl, alkoxy, hydroxy or halo, and only one of R,, R2, R3, Rõ and-R5 is
amino;
and a compound having the formula:
(III)
R4 R5
R3 Re
I NH
R2
R1 0
wherein Rõ R,, R3, R,, and R5 are each selected from the group consisting of
hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally
substituted
with alkyl, alkoxy, hydroxy or halo, and only one of Rõ R2, R3, Rõ and R5 is
amino.
Preferred pADPRT compounds include: 6-amino-1, 2-benzopyrone, 3-
nitrosobenzamide, 5-amino- I (2H)-isoquinolinone, 7 -amino- I (2H)-
isoquinolinone,
and 8-amino-1(2H)-isoquinolinone.
Still another aspect of the invention includes a method of treating both gram
negative and gram positive induced symptoms in an animal or mammal, said
method
comprising the step of administering to an animal or mammal a therapeutically
effective amount of a pADPRT inhibitory compound.
Still another aspect of the invention is a method of treating both gram
negative
and gram positive induced endotoxin symptoms in an animal or mammal which
comprises the step of administering to an animal or mammal a therapeutically
effective amount of a pADPRT inhibitory compound wherein the compound is
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selected from the group consisting of compound I, compound II, or compound
III, as described
above.
Still another aspect of the invention is a method of treating both gram
negative and gram
positive induced endotoxin symptoms in an animal or mammal which comprises the
step of
administering to an animal or mammal a therapeutically effective amount of a
pADPRT
inhibitory compound wherein the compound has the structural formula noted
above as
compounds I, II or III.
Still another aspect of the invention is a method of treating arthritis in an
animal or
mammal comprising the step of administering an effective amount of or an
pADPRT inhibitory
compound wherein the compound has the structural formula noted above as
compounds I, II or
III.
Still another aspect of the invention is a method of treating Chron's Disease
in an animal
or mammal comprising the step of administering an effective amount of an
pADPRT inhibitory
compound wherein the compound has the structural formula noted above as
compounds I, II or
III.
Still another aspect of the invention is a method of treating Barrett's
Disease in an animal
or mammal comprising the step of administering an effective amount of an
pADPRT compound
wherein the compound has the structural formula noted above as compounds I, II
or III.
In one aspect, there is provided the use of a pADPRT inhibitory compound
selected from
the group consisting of:
- a compound having the formula (I):
(I)
R6
O
R5 O
RaI R,
R3 R2
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wherein R,, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, C1-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4,
R5 and R6 is amino, and
- a compound having the formula (III):
(III)
R4 R5
Rg R6
NH
R2
R1 O
wherein R1, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, CI-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4,
R5 and R6 is an amino moiety;
for the treatment of inflammation or inflammatory disease in an animal or
mammal.
In another aspect, there is provided the use of a pADPRT inhibitory compound
for the
treatment of bacterial lipopolysaccharide induced symptoms in an animal or
mammal.
In another aspect, there is provided the use of a pADPRT inhibitory compound
selected from
the group consisting of-
- a compound having the formula (I):
(I)
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R6
O
R5 0
R4 R1
R3 R2
wherein R1, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, C1-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4,
R5 and R6 is amino, and
- a compound having the formula (III):
(III)
R4 R5
R3 R6
I
NH
R2
Ri O
wherein R1, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, C1-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4,
R5 and R6 is an amino moiety;
in the manufacture of a medicament for the treatment of inflammation or
inflammatory
disease in an animal or mammal.
In another aspect, there is provided the use of a pADPRT inhibitory compound
in the
manufacture of a medicament for the treatment of bacterial lipopolysaccharide
induced
symptoms in an animal or mammal.
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In a further aspect, there is provided a pharmaceutical composition for use in
the treatment of
inflammation or inflammatory disease comprising a pADPRT inhibitory compound
selected
from the group:
- a compound having the formula (I):
(I)
R6
O
R5 O
R4 R,
R3 R2
wherein R1, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, C1-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4, R5
and R6 is amino, and
- a compound having the formula (III):
(III)
4 R5
R3 Rs
I
NH
R2
R1 O
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wherein R1, R2, R3, R4, R5 and R6 are each selected from the group consisting
of hydrogen,
hydroxy, amino, C1-C6 alkyl, C1-C5 alkoxy, C3-C8 cycloalkyl or phenol,
unsubstituted or
substituted with C1-C6 alkyl, C1-C5 alkoxy, hydroxy or halo, and only one of
R1, R2, R3, R4,
R5 and R6 is an amino moiety;
and a pharmaceutical excipient.
In still a further aspect, there is provided a pharmaceutical composition for
use in the
treatment of bacterial lipopolysaccharide induced symptoms in an animal or
mammal
comprising a pADPRT inhibitory compound and a pharmaceutical excipient.
The pADPRT inhibitory compounds of the invention may be prepared by the
methods
described in U.S. Patent Nos. 5,464,871; 5,473,074; 5,482,975; 5,484,951;
5,516,941; and
5,583,155.
The preferred compounds for use in the methods of the invention include those
where
the halo group is iodo, and one of the R groups is amino, one of the R groups
may be nitroso
or nitro as described in the aforementioned patents, but preferably the R
group is amino.
Also, it has been found that the pADPRT inhibitory activity is strongly
exhibited when the
iodo moiety is adjacent to the amino moiety. In any event, the compounds to be
used in the
methods of the invention should have pADPRT inhibitory activity.
In one embodiment, there is provided the use of a compound consisting of
compound I,
compound II or compound III, as defined wherein the pADPRT inhibitory compound
is
selected from the group consisting of:
- a compound having the structural formula (I):
(I)
R6
O
R5 O
R4 R1
R3 R2
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R6
O
R5 O
R4 R1
R3 R2
- a compound having the structural formula (II):
(II)
O_~ NH2
R5 R,
I
R4 R2
R3 and
- a compound having the structural formula (III)
(III)
R4 R5
R3 Rs
NH
R2
R, O
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wherein R1, R2, R3, R4, R5 and R6 are, independent of one another, selected
from the group
consisting of hydrogen, hydroxy, amino, nitroso, nitro, halogen, (C1-C6)
alkyl, (C3-C6) alkoxy,
(C3-C7) cycloalkyl, and phenyl and pharmaceutically acceptable salts thereof,
wherein at least
three of the six R1, R2, R3, R4, R5 and R6 substituents are always hydrogen
and at least one of the
six R1, R2, R3, R4, R5 and R6 substituents is an amino moiety.
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The compounds may be used as is, or preferably in combination with a
pharmaceutically acceptable acid addition salt or other suitable
pharmaceutical carrier
known in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Effect of INH,BP on LPS-induced (a) nitrite production, (b) 6-keto
prostaglandin FIa production, (c) TNF production and (d) suppression of
mitochondrial respiration in J774 cells. TNF was measured at 4h, all other
parameters
at 24h after LPS. **represents a significant change in response to LPS when
compared to controls (p<0.01)m; ##represents significant effect of INH,BP in
the
presence of LPS when compared to LPS alone (p<0.01); n=6-12 wells.
FIG. 2. 1NH,BP inhibits iNOS expression in J774 and RAW 264.7 cells.
(a) Representative Northern blots of iNOS and 18s mRNA in J774 cells (A) and
RAW
264.7 macrophages (B) under control conditions (lane 1), at 4h after LPS
treatment
(lane 2) and at 4h after LPS treatment in cells in the presence of INH,BP (100
M)
(lane 3). (b) Effect of INH7BP on iNOS activity in the homogenates of J774
cells
under control conditions (C and C+ INH2BP) and at 12h after LPS treatment (LPS
and
LPS + INH2BP). **represents a significant effect of LPS when compared to
controls
(p<0.01); ##represents significant inhibition by INH2BP (p<0.01); n=4.
(c) Representative iNOS Western blot in control J774 cells and in cells at 12
h after
LPS in the presence or absence of INH2BP.
FIG. 3. (a) Time-dependent loss of the inhibition of nitrite accumulation by
INH2BP (100 M), when given at -1 h prior to LPS together with LPS or at 2, 4
and 6
h after LPS. (b) Effect of INH,BP on nitrite accumulation in J774 cells
stimulated
with the combination of LPS and IFN; n=6-12 wells.
FIG. 4. Effect of INH2BP on the induction of luciferase activity by LPS in
RAW 264.7 cells transiently transfected with either a full length (-1592 bp)
or a
deletional (-367 bp) iNOS promoter-luciferase construct. In cells transfected
with
either the full length or the deletional construct (black bars), treatment
with LPS (10
g/ml), 4 h) led to a 10 to 12-fold induction of luciferase activity, over
control values.
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Co-treatment with INH2BP inhibited LPS-mediated increases in luciferase
activity in
cells transfected with the full length construct, but had no significant
effect in cells
transfected with the -367 bp deletional construct (grey bars). Data are
expressed as
fold increases in luciferase activity over control cells, and are corrected
for respective
beta-galactosidase activity. *represents significant effect of INH2BP in the
presence
of LPS when compared to LPS alone (p<0.05); n = 4 separate transfections.
FIG. 5. INH,BP suppresses the induction of iNOS in conscious rats. iNOS
activity in lung homogenates (a) and plasma nitrite-nitrate concentrations (b)
in
control rats (c), in rats injected with INH,BP (INHBP); in rats injected with
LPS (15
mg/kg i.p. for 6 h); and the effect of treatment with 1NH,BP (10 mg/kg i.p.),
when
given 10 min. prior to LPS (INH,BP + LPS) or at 2 h after LPS (LPS + INH,BP).
**represents a significant effect of LPS when compared to controls (p<0.01);
##represents significant inhibition by the pADPRT inhibitor (p<0.01); n=4-5.
FIG. 6. Effect of INH2BP (10 mg/kg i.p.) on the LPS-induced TNF, IL-10 and
IL-6 response in mice, at 90 min. after LPS administration (4 mg/kg i.p.).
##represents a significant effect of LPS when compared to controls (p<O.OI);
##
represents significant augmentation of the response by INH,BP (p<0.01) ; n=4-
5.
FIG. 7. INH2BP improves survival in mice subjected to endotoxin shock:
effect of INH,BP pretreatment (0.3-10 mg/kg) on endotoxin-induced (120 mg/kg
i.p.)
mortality in mice; n=7-8 animals in each group.
FIG. 8. (a) MAP kinase activity in RAW 264.7 cells treated with vehicle or
LPS (10 g/ml) for 24 h ni presence or absence of 100 M PD 98059 or 150 M
INH,BP. Data represent values obtained in a typical experiment: similar
results were
seen on 3 different experimental days. (b) Representative in gel MAP kinase
assay in
RAW 264.7 cells at 24 h after vehicle or LPS treatment in the presence or
absence of
150 M INH2BP. Lanes 1-4 represent the following groups, respectively: 1:
vehicle-
treated control; 2: LPS treatment; 3: vehicle treatment in the presence of 150
gM
INH2BP; 4: LPS treatment in the presence of 150 M INH2BP.
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FIG. 9. Inhibition of pADPRT with INH,BP does not alter the nuclear
translocation of NF-KB Western blot of nuclear extracts of control J74 cells
and in
cells at 90 min. after LPS treatment in the presence or absence of INH2BP (100
M).
FIG. 10. Describes the effect of INH,BP on the development of carrageenan-
induced paw edema. Data show paw volumes at 1-4h after carrageenan injection
(means + S.E.M., n=6 animals in each group). There was a significant increase
in the
paw volume from hour I (p<0.01), and there was a significant inhibition of the
development of paw edema of INH,BP at 1-4 hours (**p<0.02).
FIG. 11. Describes the effect of INH2BP on the onset of collagen-induced
arthritis. The percentage of arthritic mice (mice showing clinical scores of
arthritis
>1) are represented. The arrow at 21 days represents the time of the second
collagen
immunization, the horizontal bar from day 25 represent the time of the start
of
treatment with INH2BP (N=6) or VEHICLE (N-10).
FIG. 12. Describes the effect of INH2BP on the severity of collagen-induced
arthritis. Median arthritic score during collagen-induced arthritis. The arrow
at 21
days represents the time of the second collagen immunization, the horizontal
bar from
day 25 represent the time of the start of treatment with INH2BP (n-6) or
vehicle
(n=10). There was a significant increase in the arthritic score from day 26
(Ip<0.01),
and there was a significant suppression of the arthritic score by INH,BP
between days
26-35 (#p<0.05).
DE8CRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
Definitions
As used herein:
"Anti-inflammatory" diseases refers to diseases or conditions where there is
an
inflammation of the body tissue. Such disease include for example, Chron's
disease,
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Barrett's disease, arthritis, multiple scelorsis, cardiomyopathic disease,
colitis,
infectious meningitis, encephalitis, and the like.
"Pharmaceutically acceptable acid addition salt" refers to those salts which
retain the biological effectiveness and properties of the free bases and which
are
obtained by reaction with inorganic acids such as hydrochloric acid,
hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid,
salicylic acid
and the like.
"ADPRT" refers to adenosinediphosphoribose transferase and is also known
as poiy(ADP-ribose)polymerase (EC 2.4.99), a specific DNA-binding nuclear
protein
of eucaryotes that catalyzes the polymerization of ADP-ribose. The enzymatic
process is dependent on DNA.
"Alkyl" refers to saturated or unsaturated branched or straight chain
hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl,
isopropyl,
butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
"Akkoxy" refers to the radical -0-alkyl. Typical alkoxy radicals are methoxy,
ethoxy, propoxy, butoxy and pentoxy and the like.
"Cycloalkyl" refers to saturated monocyclic hydrocarbon radical containing 3-
8 carbon atoms such as cycloproply, cyclobutyl, cyclopentyl, cyclohexyl,
cycloheptyl,
cyclooctyl, and the like.
"Substituted phenyl" refers to all possible isomeric phenyl radicals such as
mono or disubstituted with a substituent selected from the group consisting of
alkyl,
alkoxy, hydroxy, or halo.
"Halo" refers to chloro, fluoro, bromo or iodo, and preferably iodo.
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The pADPRT inhibitory compounds of the invention (notably compounds
defined above as compounds I, II or III) are potent, specific and non-toxic
anti-
inflammatory compounds, that can be used for conditions and diseases typically
known for inflammation, such as arthritis, Chron's disease, Barrett's disease,
and the
like. Also, these compounds are useful in the treatment of conditions
associated with
gram negative and gram positive induced infections, especially those
associated with
gram negative infections, and including conditions associated with
lipopolysaccharide
condition and sepis. The compounds are especially useful in that they have
very low,
if any toxicity.
In practice, the compounds of the invention or their pharmaceutically
acceptable salts, will be administered in amounts which will be sufficient to
inhibit
inflammatory conditions or disease and/or prevent the development of
inflammation
or inflammatory disease in animals or mammals, and be used in the
pharmaceutical
form most suitable for such purposes.
Administration of the active compounds and salts described herein can be via
any of the accepted modes of administration for therapeutic agents. These
methods
include systemic or local administration such as oral, parenteral,
transdermal,
subcutaneous, or topical administration modes. The preferred method of
administration of these drugs is oral. In some instances it may be necessary
to
administer the composition in other parenteral form.
Depending on the intended mode, the compositions may be in the solid, semi-
solid or liquid dosage form, such as, for example, injectables, tablets,
suppositories,
pills, time-release capsules, powders, liquids, suspensions, or the like,
preferably in
unit dosages. The compositions will include an effective amount of active
pADPRT
inhibitory compound or the pharmaceutically acceptable salt thereof, and in
addition,
it may include any conventional pharmaceutical excipients and other medicinal
or
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pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as
customary in the
pharmaceutical sciences.
For solid compositions such excipients include pharmaceutical grades of
mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,
cellulose,
glucose, sucrose, magnesium carbonate, and the like may be used. The active
pADPRT inhibitory compound defined above, may be also formulated as
suppositories using for example, polyalkylene glycols, for example, propylene
glycol,
as the carrier.
Liquid, particularly injectable compositions can, for example, be prepared by
dissolving, dispersing, etc. the active compound in a pharmaceutical solution
such as,
for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like,
to thereby
form the injectable solution or suspension.
If desired, the pharmaceutical composition to be administered may also
contain minor amounts of nontoxic auxiliary substances such as wetting or
emulsifying agents, pH buffering agents, and other substances such as for
example,
sodium acetate, triethanolamine oleate, etc.
Also, if desired, the pharmaceutical composition to be administered may
contain liposomal formulations comprising a phospholipid, a negatively charged
phopholipid and a compound selected from cholesterol, a fatty acid ester of
cholesterol or an unsaturated fatty acid. Typical neutral phospolipids include
L-a-
phophalidycho line, L-a-phosphatidylinosotol, L-a-phosphatidyl-serine, L-a-
phosphatidylinosotol, L-a-phosphatidic acid, L-a-phosphatidylglycerol, L-a-
lysophosphatidylcholine, sphingomycelin, and cardiolipin.
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Typical negatively charged phospholipids include diacetyl phosphate or
phosphodiglyceride, e.g., dilauroyl, dimyristoyl phosphate. dipalmitoyl
phosphate,
disteroyl phosphate.
Typical cholesterols and cholesterol ethers include cholesterol, 3S-hydroxy-5-
cholestene, polyoxyethanylcholesteryl sebacate, cholesterol-5, 6-epoxide,
cholesteryl
acetate, cholesteryl n-butyl ether, cholesteryl caprate, cholesteryl
dodecanoate,
cholesteryl ethyl ether, cholesteryl heptadecanoate, cholesteryl methyl ester.
Typical unsaturated fatty acids include arachidonic acid, docosahexanoic acid,
.elaidic acid, erucic acid, linoleic acid, linolenic acid, nervonic acid,
oleic acid,
palmitoleic acid, petroselinic acid. The halo nitro compounds may be
encapsulated or
partitioned in a bilayer of liposomes of the liposomal formulation.
In the first embodiment, the liposomes are formed first and then the C-amino,
nitroso or nitro compound is added. Rather than be encapsulated, the C-amino,
nitroso or nitro compound partitions (locates itself) into the lipid bilayer
of the
liposome. To make this composition, typically, the ingredients, e.g.,
phosphatidyl
choline, dicetyl phosphate and cholesterol are blended with a solvent such as
chloroform. After blending the chloroform is driven off. Then water is added
to it.
When the water is added to the liposomes, it makes a multilamellar liposome
(i.e., the
liposomes are similar to an onion skin having many layers). The next step is
to freeze
and thaw them. They are frozen down rapidly in liquid nitrogen. The purpose of
the
rapid freeze and thaw it to make the liposome size more uniform. The liposomes
at
this point are varied in size and you treat and that it one or more,
typically, five, times.
Thawing occurs in a 37 degree water bath. Before the freeze and thaw one
sonicares
the mixture. The combination of sonication and thawing reduces the number of
skins.
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The goal is to produce a unilamellar system. At this point, the C-nitroso
compound is
added to get a 10 millimolar (Mn) concentration. The concentration can be in
excess
of 15 millimolar. For this concentration of lipids, for a 60 milliliter batch,
the total
lipid concentration is 648 mg and 60- milliliters of water is added to that.
The
phosphatidyl choline is 500 mg, the cholesterol is 36 mg; the dicetyl
phosphate is 112
mg.
Increasing the liposome concentration of the mixture permits it to contain
more C-amino, nitroso or nitro compound. For example, it could be twice as
concentrated as it is in the above mixture. For a 60 mil batch, one could
double the
numbers above to have 1000 mg of phosphatidyl choline, 224 mg of dicetyl
phosphate
and 72 mg of cholesterol. Decreasing the concentration decreases the amount of
C-
nitroso compound to get in there. For the hypothetical 60 milliliter batch,
the upper
limit of C-amino compound approaches is 15 millimolar concentration of C-amino
compound. For 3-Nitrosobenzamide this is 135 mg. for a 60 milliliter batch.
The next step is to rehydrate. Then, the next step of the process is extrusion
using an extruder device (Lipex Biomembranes, Inc., Vancouver, British
Columbia,
Canada).
The extrusion process serves two purposes; 1) making the size of the
liposomes uniform; and 2) sterilization.
Extrusion typically involves filtration through a j0.1 micron filter and is
generally followed by freeze drying the mixture to lyophilize the mixture
(takes the
water out of it and makes it a fine powder). This improves solubility so that
one can
put up to about a 40 millimolar solution which is about three times as
concentrated as
prior to free drying. Freeze drying produces a mixture of powdered lipids and
the
powdered C-amino compound. Now one can use the same amount of the C-amino
compound and a smaller amount of liquid making a more concentrated mixture.
For
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example, one may have the same weight of C-amino, nitroso or nitro compound
but
have up to one-third of the original volume.
One could modify steps of the above process by, for example, eliminating
steps such as freeze drying.
This process of the first embodiment does not significantly encapsulate the C-
amino, nitroso or nitro compound. Instead of having the compound in the middle
of
the liposome the compound resides in the membrane itself. The C-amino, nitroso
or
nitro compound partitioned within the membrane of the liposome will migrate to
the
target cells and the lipid will carry the C-amino, nitroso or nitro compound
into the
cell membranes.
Preferably this process makes liposomes having about 0.05-0.45, and more
preferably about 10.1-0.2 micron, diameter. Unilamellar or multilamellar
liposomes
are effective.
The second purpose of extrusion is to sterilize the mixture. To sterilize, the
liposomes are generally made smaller than 45 microns in diameter. Sizes less
than
0.05 microns would theoretically work. The process of the first embodiment has
the
advantage that, for example, in water 3NOBA only has a 0.5 millimolar
concentration.
The present liposomal composition achieves concentrations of 15 millimolar.
Moreover, unlike 3-NOBA merely in aqueous solution, the NOBA-containing
liposomal solution is resistant to ascorbic acid. This makes it useful in
laboratory
mice experiments. The solution may contain the NOBA monomer or NOBA dimer.
In a second embodiment one may start with a film of the lipid components,
hydrate the film with an aqueous solution of drug. This automatically forms
lipids
which entrap (encapsulate) the drug. This occurs with compounds which are
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liposome membrane impermeable. An example of such compounds are those in U.S.
Pat. No. 5,262,564, issued November 16, 1993, e.g., L-cystine sulfinic adducts
of 3-
NOBA.
Parental injectable administration is generally used for subcutaneous,
intramuscular or intravenous injections and infusions. Injectables can be
prepared in
conventional forms, either as liquid solutions or suspensions or solid forms
suitable
for dissolving in liquid prior to injection.
A more recently devised approach for parenteral administration employs the
implantation of a slow-release or sustained-released systems, which assures
that a
constant level of dosage is maintained, according to U.S. Pat. No, 3,710,795.
Any of the above pharmaceutical compositions may contain 0.1-99%,
preferably 1-70% of the active pADPRT inhibitory compounds, especially the
halo-
C-amino, nitroso or nitro compounds of the formulae I, II or III, above as
active
ingredients.
Chronic inflammation is known to facilitate carcinogenic transformation in
various tissues. 5-iodo-6-amino-1,2-benzopyrone (INH,BP), a novel inhibitor of
the
nuclear enzyme poly-ADP ribose polymerase (pADPRT) has recently been shown to
regulate a variety of cellular signal transduction pathways and to abrogate in
vivo
tumorigenicity by a Ha-ras transfected endothelial cell line. As one aspect of
the
present invention demonstrates the effect of pADPRT inhibitory compounds such
as
INH2BP on the activation by endotoxin (bacterial lipopolysaccharide, LPS) on
the
production of the inflammatory mediators tumor necrosis factor alpha (TNF),
interleukin- I O(IL- 10) and interleukin-6 (IL-6), nitric oxide (NO) and
prostaglandins in
vitro and in vivo. In addition, the present invention shows the effect of
pADPRT
inhibitory compounds such as INH_BP on the activation of mitogen-activated
protein
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kinase (MAP kinase) and nuclear factor kB (NF-kB) in vitro. In cultured J774
and
RAW 264.7 macrophages, LPS induced the production of prostaglandin
metabolites,
the release of TNF and the expression of the inducible isoform of NO synthase
(iNOS). The production of prostaglandins and of NO were inhibited by INH,BP in
a
dose-dependent manner, while the short-term release of TNF-alpha was
unaffected.
INH,BP markedly suppressed LPS-mediated luciferase activity in RAW cells
transiently transfected with a full length (-1592 bp) murine macrophage iNOS
promoter-luciferase construct, but not in a deletional construct consisting of
-367 bp.
In vivo, INH,BP pretreatment inhibited :the induction of iNOS by LPS in rats,
did not
affect the LPS-induced TNF and IL-6 response, but enhanced LPS-induced IL-10
production. INH2BP pretreatment markedly improved the survival of mice in a
lethal
model of endotoxin shock. These results demonstrate that pADPRT inhibitory
compounds such as INH7BP have potent anti-inflammatory action in vitro and in
vivo.
Poly-ADP ribose synthetase (PARS) is a nuclear enzyme activated by DNA
single strand breaks. Massive activation of PARS, in response to hydrogen
peroxide-
peroxynitrite- or ionizing radiation-induced extensive DNA single strand
breakage can
initiate an energy-depleting futile cycle culminating in cellular injury. The
production
of peroxynitrite has recently been demonstrated in various forms of
inflammation,
including arthritis and carrageenan-induced paw edema. The present invention
shows
the effect of the novel, potent inhibitor of PARS, pADPRT inhibitory compounds
such as 5-iodo-6-amino-1,2-benzopyrone (INH2BP), in a rat model of carrageenan-
induced paw edema and in a mouse model of collagen-induced paw edema at 1-4h.
Collagen-induced arthritis was induced in male DMA/1J mice, with two
injections of
type II collagen at Day 1 and Day 21. Oral treatment of mice with INH,BP (0.5
g/kg,
daily), starting at the onset of arthritis (Day 25), delayed the development
of the
clinical signs of arthritis at Days 26-35. INH2BP treated animals exhibited a
reduced
arthritic index (arthritic score: 20-50% of the score seen in the vehicle-
treated mice),
and improved histological status, as examined in the knee and paw. These data
demonstrate that the PARS inhibitor INH,BP exhibits anti-inflammatory effects
in
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vivo INH2BP, even with a relatively late start of administration, was able to
delay the
course of the collagen-induced arthritis. The data of the invention support
the view
that PARS activation plays a role in the development of arthritis, and
possibly, other
forms of inflammation and inflammatory diseases.
The following examples serve to illustrate the invention. They should not be
construed as narrowing it, or limiting its scope.
EXAMPLE I
Cell Culture.
The mouse macrophage cell lines J774 and RAW 264.7 were cultured in
Dulbecco's modified Eagle's medium (DMEM) as escribed; Szabo et al., 1996,
"DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular
energy
depletion are involved in the cytotoxicity in macrophages and smooth muscle
cells
exposed to peroxynitrite, "Proc. Natl. Acad. Sci. U.S.A. 93: 1753-1758;
Zingarelli et
al., 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP
ribosyl
synthetase and causes cellular energy depletion in macrophages stimulated with
bacterial lipopolysaccaride," J. Immunol. 156: 350-358. In separate studies,
peritoneal macrophages were obtained from male Wistar rats and cultured in
vitro for
24 hours in the absence or presence of LPS and with or without INH,BP. Rats
were
sacrificed and peritoneal macrophages taken and cultured in DMEM. Cells were
treated with E. Coli LPS (10 mg/ml) or LPS and INF (50 /ML) for various
times, in
the presence or absence of various concentrations (1-150 mM) INH2BP or other
pharmacological inhibitors.
MAP kinase related assays.
Raw cells were washed in PBS and collected and lysed using 100 ml of lysis
buffer per million cells. (50 mM Tris-HC I pH 7.4, 1 % NP-40, 0.4 M NaC 1, 0.1
mM
NaVO3, 50 mM KF, 1 mM EGTA, 2 mM PMSF, 25 nM okadaic acid, 1 mg/mL of
each leupeptin, aprotinine, arnastatine and antipaine). Lysis was carried out
for 20
minutes on ice followed by a 14 min. centrifugation at 13000 rpm in an
Eppendorf
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centrifuge. Supernatants were saved and their protein content were assayed
using the
Bio-Rad dye assay.
In gel MAP kinase assay.
Protein samples (50 mg/lane) were electrophoresed in a 100% SDS-PAGE gel
containing immobilized myelin basic protein (MBP, 250 mg/mL gel). After
electrophoresis, the gel was washed once with 50 mM TRIS-HCI pH 7.7 buffer (25
mL, 20 min.), followed by two 30 min. incubations with the same buffer
containing
25% i-propanol. The gel was then washed once with the Tris-Hcl buffer and
soaked
into a solution of 50 mM Tris-HC1 pH 7.7, mM 2-mercaptoethanol, 5 M guanidine
hydrochloride (50 mL) for an hour, changing the incubating solution at 30 min.
The
proteins were then renurtured by incubating the gel in five changes of a
solution of 50
mM TRIS-HCI Ph 7.7, Mm 2-mercaptoethanol, 0.04% NP-40 over a 16 hours period
of time. The gel was then washed twice and preincubated for half an hour in a
solution containing 50 mM TRIS-HC 1 pH 7.7, 5 mM MgC 1, 7 mm 2-
mercaptoethanol. The final incubation was carried out in the same solution
supplemented with 10 mm of'2p-g] ATP (50 mCi/assay) for an hour. At the end of
the incubation, the gel was washed free of unbound radioactivity using 3x25 mL
of
10% TCA and 3x25 ml of 10% acetic acid. dried and autoradiographed; Sasaki el
al.,
1995, "Permissive effect of ceramide on growth factor-induced cell
proliferation,"
Biochem. J. 311:829-34.
MAP kinase Western blotting.
One hundred mg of cell extract proteins were loaded onto a 10% SDS-PAGE
gel, electrophoresed, transblotted onto nitrocellulose membrane and
immunoprobed.
The first antibody (anti-MAP kinase) was from UBI, the second antibody was
alkaline
phosphatase labeled and from NEN Biolabs. Detection was by enhanced
chemiluminesence; Bauer et at., 1995, "Modification of growth related
enzymatic
pathways and apparent loss of tumorigenicity of a ras-transformed bovine
andothelial
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cell line by treatment with 5-iodo-6-amino-l,2-benzopyrone (INH2BP)," Int. J.
Oncol.
8:239-252.
Preparation of nuclear extracts and NF-kB Western blotting.
Cells were treated with LPS in the presence and absence of INH,BP for 90
minutes. Mininuclear extracts were prepared as described; Hassanain et at.,
1993,
"Enhanced gel mobility shift assay for DNA-binding factors," Anal. Biochem.
213:162-7. Briefly, cells were scraped, briefly centrifuged and pellets
resuspended in
400 ml cold Buffer A [Hepes pH 7.9 (10 mM), KCI (10 mM), EDTA (0.1 mM),
EGTA (0.1 mM), DTT (1mM), PMSF (0.5 mM), pepstatin A (1 mg/ml), leupeptin (10
mg/ml), and aprotinin (10 mg/ml)], on ice for 15 minutes, in the presence of
25 ml 1%
NP-40. Then, samples were vortexed, centrifuged for 1 minute at 10,000 g, and
the
pellet resuspended with 100 ml Buffer B {Hepes pH 7.9 (20 mM), NaCl (400 mM),
EDTA (1 mM), EGTA (1mM), DTT (1 mM), PMSF (0.5 mM), pepstatin A (mg/ml),
leupeptin (10 mg/ml) and aprotinin (10 mg/ml)]. After shaking on a rocker
platform
for 15 minutes at 4 C, samples were centrifuged for 15 minutes 15 100,000g at
4 C.
70m1 aliquots were then treated with 150 ml SDS-PAGE sample buffer. Western
blotting was performed as described above, with rabbit anti-mouse NF-kB
primary
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) 1:750 in Tween TBS
(0.02%).
Measurement of nitrite or nitrite/nitrate concentration.
Nitrite in culture supernatants at 24 hours after stimulation was measured as
described; Szabo et al., 1996, "DNA strand breakage, activation of poly-ADP
ribosyl
synthetase, and cellular energy depletion are involved in the cytotoxicity in
macrophages and smooth muscle cells exposed to peroxynitrite, "Proc. Natl.
Acad.
Sci. U.S.A. 93:1753-1758; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA
strand breakage activates poly-ADP ribosyl synthetase and causes cellular
energy
depletion in macrophages stimulated with bacterial lipopolysaccaride," J.
Immunol.
156:350-358; Szabo et al., 1994, "Spermine inhibits the production of nitric
oxide in
immuno-stimulated J774.2 macrophages: requirement of a serum factor," Br. J.
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Pharmacol. 112:355-356; by adding 100 ml of Griess reagent (1% sulfanilamide
and
0.1 % naphthylethylenediamide in 5% phosphoric acid) to 100 ml samples of
medium.
The optical density at 550 nm (OD 550) was measured using a Spectramax 250
microplate reader (molecular Devices, Sunnyvale, CA). For the determination of
total
nitrite/nitrate concentrations in plasma samples, nitrate was reduced to
nitrite by
incubation with nitrate reductase; Zingarelli et al., 1996, "Peroxynitrite-
mediated
DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular
energy depletion in macrophages stimulated with bacterial lipopolysaccaride,"
J. Immunol. 156:350-358.
Measurement of 6-keto prostaglandin F,,.
6-keto prostaglandin F,, production at 4 hours after LPS stimulation was
measured in 100 ml samples of cell culture supernatant using a specific
radloimmunoassay; Szabo et al., 1994, "Spermine inhibits the production of
nitric
oxide in immuno-stimulated J774.2 macrophages: requirement of a serum factor,"
Br. J. Pharmacol. 1 12:355-356.
Cytokine measurements.
Cytokine levels in plasma and cell culture supernatants were determined by
ELISA. Plasma levels of IL-10 and IL-6 were measured using ELISA kits from
Endogen (Endogen Inc., Boston, MA). Concentrations of TNF-a in the plasma and
cell culture supernatants were determined using ELISA kits from Genzyme
(Genzyme
Corp., Boston, MA) as described; Szabo et al., 1997, "Isoproterenal regulates
tumour
necrosis factor, interleukin- 10, interleukin-6 and nitric oxide production
and protects
against the development of vascular hyporeactivity in endotoxemia," Immunology
90:95-100.
Measurement of mitochondrial respiration.
Mitochondrial respiration at 24 hours was assessed by the mitochondrial-
dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
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to formazan; Szabo et al., 1996, "DNA strand breakage, activation of poly-ADP
ribosyl synthetase, and cellular energy depletion are involved in the
cytotoxicity in
macrophages and smooth muscle cells exposed to peroxynitrite, "Proc. Natl.
Acad.
Sci. U.S.A. 93:1753-1758; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA
strand breakage activates poly-ADP ribosyl synthetase and causes cellular
energy
depletion in macrophages stimulated with bacterial lipopolysaccaride," J.
Immunol.
156:350-358.
Northern blotting for iNOS mRNA.
After exposing cells to LPS in the presence or absence of INH,BP for 4 hours,
total RNA was extracted as described using TRIZOL. Aliquots containing 15 mg
total RNA underwent electrophoresis on a 1% agarose gel containing 3%
formaldehyde. RNAs were blot transferred to nylon membrane and UV auto-
crosslinked. Membranes were hybridized as described; Lowenstein et al., 1993,
"Macrophage nitric oxide synthase gene: two upstream regions mediate induction
by
interferon gamma and lipopolysaccharide," Proc. Natl. Acad. Sci. U.S.A.
90:9730-
9734; overnight at 42 C with a murine iNOS cDNA probe (106 .cpm/ml) labeled
with
[32P]dCTP (specific activity, 3,000 Ci/mM; NEN) by random priming (Pharmacia,
Piscataway, NJ). The hybridized filters were serially washed at 53 C using 2X
sodium citrate, sodium chloride, 0.1 % SDS and 25 mM NaHPO4, 1 mM EDTA, 0.1 %
SDS solutions. After probing for iNOS, membranes were stripped with boiling 5
mM
EDTA and rehybridized with a ['`P]-radiolabeled oligonucleotide probe for 18S
ribosomal RNA as a housekeeping gene. After washing, exposure was carried out
overnight using a Phosphor Imager screen.
iNOS Western blotting.
Cells were treated with LPS in the presence and absence of pADPRT inhibitor
for 20 hours. Cells were then scraped in cold PBS and centrifuged at 14000 g
for 30
seconds. The supernatant was removed and lysis buffer containing RIPA (500
mL),
aprotin (10 mg/ml), and PMSF (0.5 mM) was added. DNA was sheered by passing
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samples through a 22 gauge needle. Protein content was determined by the
Bradford
method (BIO-Rad). Cytosolic protein (200 mg/lane) was added to SDS-PAGE
buffer,
boiled for 5 minutes, separated with 7.5% SDS-PAGE, and transferred to
nitrocellulose membranes (0.2 mm) using a Semi-Dry method with an
isotachophoretic buffer system. After 1 hour blocking in 3% gelatin and
subsequent
washing, the samples were immunoblotted in Tween Tris Buffered Saline (TTBS)
and
1% gelatin, with primary rabbit anti-mouse iNOS (upstate Biotechnology, Lake
Placid, NY) 1:1000 in TTBS (0.0%) for 2.5 hours. An alkaline phosphatase-
conjugated goat anti-rabbit iGG antibody was used as secondary antibody.
Antibody
binding was visualized by nitrobule tetrazolium/5-bromo-4-chloro indolyl
phosphate
(NBT/BCIP) in carbonate buffer (BIO-RAD).
Measurement of INOS activity.
Cells were treated with LPS in the presence and absence of pADPRT inhibitor
for 12 hours. The measurement of the calcium- independent conversion of 1-
arginine
to L-citrulline in homogenates of the J774 cells or in lung homogenates was
used as
an indicator of NOS activity as described; Szabo et al., 1994, "Spermine
inhibits the
production of nitric oxide in immuno-stimulated J774.2 macrophages:
requirement of
a serum factor," Br. J. Pharmacol. 112:355-356. Cells were scraped or lungs
were
put into a homogenation buffer composed of: 50 mM Tris HC 1, 0.1 mM EDTA, 0.1
mM EGTA and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) and homogenized in
the buffer on ice using a Tissue Tearor 985-370 homogenizer (Biospec Products,
Racine, WI). Conversion of ['H]-L-arginine (to ['H]-L-citrulline was then
measured
in the homogenates. Homogenates (30 ml) was incubated in the presence of ['H]-
L-
arginine (10 mM, 5 kBq/tube), NADPH (1 mM), calmodulin (30 nM),
tetrahydrobiopterin (5 mM) and EGTA 5 mM) for 20 minutes at 22 C. Reactions
were stopped by dilution with 0.5 ml of ice cold HEPES buffer (pH 5.5)
containing
EGTA 2 mM) and EDTA (2 mM). Reaction mixtures were applied to Dowex 50W
(Na+ form) columns and the eluted ['H]-L-citulline activity was measured by
scintillation counting.
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Functional assay of NOS promotor.
Since under our experimental conditions, J774 cells were resistant to our
attempts to transiently transfect them using the calcium phosphate,
lipofectin, and
lipofectamin methods, transfection studies were performed in RAW 264.7 cells.
iNOS promoter activity was evaluated by transient transfection of AW 264.7
cells
with reporter gene constructs incorporating the 5' murine macrophage iNOS
promoter
region upstream from the reporter gene luciferase; Lowenstein et al., 1993,
"Macrophage nitric oxide synthase gene: two upstream regions mediate induction
by
interferon gamma and lipopolysaccharide," Proc. Natl. Acad. Sci. U.S.A.
90:9730-
9734; (kindly provided by Dr. Charles J. Lowenstein, Johns Hopkins
University).
Two constructs were used: a full length promoter construct (-1592 bp) and a
deletional construct consisting of -367 bp. Cells were plated into 6-well
culture plates
at -50% confluence and transfected with the respective iNOS promoter-
luciferase
construct in equimolar amounts using cationic liposomes (Lipofectin, Gibco).
In
order to control for differences in transfection efficiencies, cells were co-
transfected
with pSV40-b-galactosidase. After transfection, cells were allowed to recover
overnight, then subsequently treated with media alone (control), LPS (10
mg/ml), or
LPS plus INH,BP (100 mM). After 4 hours of treatment, cells were washed once
in
PBS, lysed in reporter lysis buffer (Promega), and analyzed for luciferase
activity was
corrected for respective raw-galactosidase activity and is expressed as fold
increase
over control cells (transfected and treated with media alone).
In vivo experiments.
Male Wistar rats and Male BALB/c mice were obtained from Charles River
Laboratories (Wilmington, MA or Budapest, Hungary). Animals received food and
water ad libitum, and lighting was maintained on 12 hour cycle. Rats were
injected
i.p. with E. coli LPS (15 mg/kg) and sacrificed at 6 hours. Plasma samples
were taken
for nitrite/nitrate determinations and lung samples for iNOS measurements.
Separate
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groups of rats were treated with INH1BP (10 mg/kg i.p.) 10 minutes prior to
LPS or 2
hours after LPS injection.
In studies for the measurement of LPS-induced cytokine response, mice were
injected i.p. with either drug vehicle, or with INH,BP (10 mg/kg) in a volume
of 0.1
ml/10 g body weight. Half an hour later they were challenged with 4 mg/kg of
i.p.
LPS. The animals were killed at 90 minutes after LPS treatment, blood was
collected
in ice-cold Eppendorf tubes containing EDTA, and centrifuged for 10 minutes at
4 C.
The plasma was stored at -7 C until assayed.
In survival studies with mice, animals were subjected to i.p. injection of LPS
(120 mg/kg) at time 0 and survival was monitored for 42 hours after LPS.
Separate
groups of mice received vehicle or INH2BP treatment (0.1-10 mg/kg i.p.) at
times -18
hours, -4 hours, 0 hours, 6 hours, 24 hours and 30 hours relative to LPS.
Materials.
DMEM, RPM 1, TRIZOL and fetal calf serum were from Gibco (Grand Island,
NY). ['H]NAD+ and [32P]NAD+ were obtained from DuPont NEN (Boston, MA).
Alcohol dehydrogenase and ND+ were obtained from Boehringer Mannheim
(Indianapolis, IN). PD 98059 was obtained from Cal biochem (La Jolla, CA). All
other drugs were obtained from Sigma (St. Louis, MO).
Statistical evaluation.
All values in the figures and text are expressed as mean + standard error of
the
mean (S.E.M.) of n observations (n > 4). Student's unpaired t-test was used to
compare means between groups. A p-value less than 0.05 was considered
statistically
significant.
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Results
INH2BP suppresses LPS-induced nitric oxide and prostaglandin but no
TNF-a production in J774 macrophages
INH,BP treatment caused a dose-dependent inhibition of LPS-induced nitrite
formation in J774 macrophages (Fig. la). Similarly, INH,BP suppressed LPS-
induced
production of 6-keto prostaglandin F,3 (Fig. lb), but not the production of
TNF (Fig.
lc), and restored the LPS-induced suppression of mitochondrial respiration
(Fig. ld).
INH,BP caused a marked inhibition of iNOS mRNA and protein expression (Fig. 2a-
c). The inhibition of nitrite production by INH,BP was greatly diminished when
the
agent was given several hours LPS, as opposed to prior to the stimulus of iNOS
induction (Fig. 3a). Moreover, the inhibitory effect of INH,BP on iNOS was
greatly
reduced when LPS was used in combination was interferon-gamma (INF-g 50 u/mL)
for immunostimulation (Fig. 3b).
Selective regulation of the induction of the iNOS promoter by INH2BP
In order to further study the regulation of iNOS promoter by IN112BP we
performed transient assays using murine macrophage NOS promoter- luciferase
constructs. Consistent with previous data; Lowenstein et al., 1993,
"Macrophage
nitric oxide synthase gene: two upstream regions mediate induction by
interferon
gamma and lipopolysaccharide," Proc. Natl. Acad. Sci. U.S.A. 90:9730-9734, we
found an important role for LPS-mediated transcriptional regulation of murine
macrophage iNOS, as evidence by an -10- to 12-fold induction of luciferase
activity
by LPS (Fig. 4). Co-treatment of cells transfected with the full length (-1592
bp)
promoter construct with INH2BP, completely inhibited LPS-mediated luciferase
activity (Fig. 4). However, similar co-treatment of cells transfected with the
-367 bp
deletional construct did not significantly affect LPS-mediated luciferase
activity
(Fig. 4).
In vivo anti-inflammatory effects of INH2BP
INH2BP pretreatment significantly reduced the LPS-induced increase in
plasma nitrite-nitrate and the increase in pulmonary iNOS activity in
conscious rats
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(Fig. 5). The inhibitory effect of INH,BP on NO production was reduced when
the
agent was added to the cells or to the animals several hours after LPS
stimulation
(Fig. 5). Similarly to the transformed cell lines, treatment with 100 mM
INH,BP
significantly reduced (by 56 + 7%, p<0.01) nitrite production in primary cells
(peritonea] macrophages obtained from rats) stimulated with LPS (10 mg/ml) in
vitro
(n=4).
Similarly to the in vitro results (Fig. lc), INH,BP did not significantly
affect
the LPS-induced increase in plasma TNF levels in mice (Fig. 6a). Nor did
INH,BP
affect LPS-induced IL-6 production (Fig. 6C). However, INH2BP caused an
augmentation of the LPS-induced IL-10 plasma response (Fig. 6b)
Pretreatment of mice by INH,BP caused a significant and dose-dependent
improvement in the survival rate subjected to lethal doses of LPS (Fig. 7).
INHZBP activity abolishes LPS-induced activation of MAP kinase but
does not alter activation and nuclear translocation of NF-kB.
There are a multitude of intracellular processes which precede the induction
if
iNOS and the production of other inflammatory mediators. Activation of
tyrosine
kinases; Levitzki, A., 1994, "Signal-transduction therapy. A novel approach to
disease management," Eur. J. Biochem. 226:1-13; Novogrodsky et al., 1994,
"Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase
inhibitors," Science 264 (Wash):1319-22; Marczin et al., 1993, "Tyrosine
kinase
inhibitors suppress endotoxin-and IL-lbeta-induced NO synthesis in aortic
smooth
muscle cells," Am. J. Physiol. 265:H1014-1018, mitogen-activated protein
kinase
(MAP kinase); Matsuda et al., 1994, "Signaling pathways mediated by the
mitogen-
activated protein (MAP) kinase kinase/MAP kinase cascade," J. Leukocyte Biol.
56:548-53; L'Allemain, G., 1994, "Deciphering the MAP kinase pathway," Pro r.
Growth Factor Res. 5:291-334; Cowley et al., 1994, "Activation of MAP kinase
kinase is necessary and sufficient for PC 12 differentiation and for
transformation of
NIH 3T3 cells," Cells 77:841-52; and the NF-kB pathway; Baeuerle et al., 1994,
"Function and activation of NF- kB in the immune system," Ann. Rev. Immunol.
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12:141-79; Schreck et al., 1992, "Nuclear factor kappa B: an oxidative stress-
responsive transcription factor of eukaryotic cells (a review)," Free Radical
Res.
Comm. 17:221-37; Muller et al., 1993, "Nuclear factor kappa B, a mediator of
lipopolysaccharide effects," Immunobiol. 187:233-56; are recognized as
important
factors in the inflammatory mediators. We investigated, therefore, whether
INH2BP
affects the activation of MPA kinase and the MF-kB in response to LPS
stimulation in
order to elucidate the potential involvement of these pathways in the
inhibitory effect
by INH2BP of the inflammatory process.
There was a significant basal MAP kinase activity in unstimulated RAW 264.7
macrophages. LPS treatment (10 mg/ml, 24 hours) induced an approximately 2.5-
fold
increase in the MAP kinase activity (Fig. 8), without affecting the amount of
immunoreactive MAP kinase content, as demonstrated by Western blotting (not
shown). Pretreatment of the cells for 3 days with INH2BP (150 mM) suppressed
basal
MAP kinase activity by approximately 50% and abolished the LPS-induced
increase
in MAP kinase (not shown). Basal MAP kinase activity was slightly suppressed
by
the MAP kinase kinase inhibitor; Pang et at., 1995, "Inhibition of MAP kinase
kinase
blocks the differentiation of PC-12 cells induced by nerve growth factor," J.
Biol.
Chem. 270:13585-8; PD 98059 (100 mM), and LPS-induced MAP kinase activation
was also inhibited (Fig. 8). In agreement with recent data in cardiac
myocytes; Singh
et at., 1996, "Regulation of cytoline-inducible nitric oxide synthesis in
cardiac
myocytes and microvascular endothelial cells.," J. Biol Chem. 271:1111-1117;
LPS-
induced nitrite production was also suppressed by PD 98059 (by 53%, at 100 mM,
n=3).
Similar to recent observations in a range of monocytic cell lines; Baeuerle et
al., 1994, "Function and activation of NF- B in the immune system," Ann. Rev.
Immunol. 12:141-79; we found basal (constitutive) nuclear NF-kB in the J774
cells
and RAW 264.7 cells. LPS stimulation caused an increase in nuclear
translocation of
NF-kB, and inhibition of INH2BP did not affect nuclear translocation of NF-kB
in
response to LPS (Fig. 9).
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DISCUSSION
Poly (ADP-ribose) synthetase (pADPRT) is a protein-modifying and ADP-
polymerizing enzyme which is present abundantly in the nucleus; Ueda et at.,
1985,
"ADP-ribosylation," Ann. Rev. Biochem. 54:73-100. The physiological function
of
pADPRT has been the subject of much debate. In contrast to the original
proposal,
which claimed that pADPRT is a DNA repair enzyme, now it is clear that pADPRT
is
not directly involved in DNA repair; Lindahl et al., 1995, "Post-translational
modification of poly(ADP-ribose) polymerase induced by DNA strand breaks,"
Trends Biochem. Sci. 20:405-411; and cells from transgenic mice in which the
pADPRT gene has been ablated have normal DNA repair characteristics; Buki et
al.,
1995, "Identification of domains of poly(ADP-ribose) polymerase for protein
binding
and self association "J. Biol. Chem. 270:3370-3377. Under physiologic
conditions
pADPRT can bind to numerous cellular protein and DNA site and can exert
pleitropic
cellular regulatory functions; Bauer et at., 1995, "Modification of growth
related
enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed
bovine
endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone
(INH,BP),"
Int. J. Oncol. 8:239-252; Bauer et al., 1995, "Reversal of malignant phenotype
by 5-
iodo-6-amino- 1,2-benzopyrone, a non-covalently binding ligand of poly (ADP-
ribose)
polymerase," Biochimie 77:347-377; Buki et at., 1995, "Identification of
domains of
poly (ADP-ribose) polymerase for protein binding and self association, "J.
Biol.
Chem. 270:3370-3377. pADPRT activation has also been proposed to serve as a
mechanism to induce cell death, in particular after radiation injury, and
oxidant stress;
Cochrane, 1991, "Mechanisms of oxidant injury of cells," Molec. Aspects Med.
12:
137-147; Berger, 1991, "Oxidant-induced cytotoxicity: a challenge for
metabolic
modulation," Am. J. Respir. Cell. Biol. Biol. 4:1-3. One of the important
physiological functions of pADPRT may be the regulation of enzyme induction,
gene
expression and cell differentiation; Bauer et at., 1995, "Modification of
growth related
enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed
bovine
endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone
(INH,BP),"
Int. J. Oncol. 8:239-252.; Bauer et at., 1995, "Reversal of malignant
phenotype by 5-
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iodo-6-amino- 1,2-benzopyrone, a non-covalently binding ligand of poly (ADP-
ribose)
polymerase," Biochimie 77:347-377; Minaga et al., 1978, "Induction of cardiac
L-
ornithine decarboxylase by nicotinamide and its regulation by putrescine,"
Eur. J.
Biochem. 91:577-85; Griffin et al., 1984, "The in vivo effect of benzamide and
phenobarbital on liver enzymes: poly(ADP-ribose) polymerase, cytochrome P-450,
styrene oxide hydrolase, cholesterol oxide hydrolase, cholesterol oxide
hydrolase,
glutathione S-transferase and UDP-glucuronyl transferase," Biochem. Biophys.
Res.
Comm. 122:770-5. The induction of alkaline phosphatase by INH2BP; Bauer et
al.,
1995, "Modification of growth related enzymatic pathways and apparent loss of
tumorigenicity of a ras-transformed bovine endothelial cell line by treatment
with 5-
iodo-6-amino-l,2-benzopyrone (INH,BP)," Int. J. Oncol. 8:239-252; is a
probable
cause of inactivation of certain phosphorylation dependent enzymes, e.g., MAP
kinase
topoisomerase I and topoisomerase II. INH,BP in bovine endothelial cells
transfected
with Ha-ras abrogates tumorigenicity, arrests cell multiplication, increases
toposomerase I, toposomerase II, and MAP kinase activity, down-regulates DNA-
methyl-transferase and protein kinase C, and ODC increases the
hypophosphorylation
of Rb protein, and inhibits the expression of the ras gene without the loss of
the
oncogene, Bauer et al., 1995, "Modification of growth related enzymatic
pathways
and apparent loss of tumorigenicity of a ras-transformed bovine endothelial
cell line
by treatment with 5-iodo-6-amino-1,2-benzopyrone INH,BP)," Int. J. Oncoi.
8:239-
252; Bauer et al., 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-
1,2-
benzopyrone, a non-covalently binding ligand of poly (ADP-ribose) polymerase,"
Biochimie 77:347- 377.
Based on the recently described anticancer actions of INH,BP; Bauer et al.,
1995, "Modification of growth related enzymatic pathways and apparent loss of
tumorigenicity of a ras-transformed bovine endothelial cell line by treatment
with 5-
iodo-6-amino-1,2- benzopyrone (INH1BP)," Int. J. Oncol. 8:239-252; Bauer et
al.,
1995, "Reversal of malignant phenotype by 5-iodo-6- amino-l,2-benzopyrone, a
non-
covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77:347-
377;
and the link between chronic inflammation and cancer, with special reference
to NO
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production (see: Introduction), here we investigated whether INH2BP modulates
the
LPS-induced inflammatory response in vitro and in vivo. We found that several
of the
pathways and mediators studied (MAP kinase, prostaglandins, NO) were
suppressed
by INH2BP, whereas others (TNF, IL-6, NF kB) were unaffected, or augmented (IL-
10). Generally, the present data of the invention shows that pADPRT inhibitory
compounds such as 1NHZBP exert anti- inflammatory actions, and the combination
of
these effects may underlie the improvement in survival rate in the animals or
mammals pretreated with this inhibitor of pADPRT.
EXAMPLE 2
INH2BP suppresses the LPS-induced induction of iNOS.
By way of background, the inducible isoform of nitric oxide (NO) synthease
(iNOS) is expressed in response to pro-inflammatory stimuli in a variety of
cells.
Overproduction of NO by iNOS plays an important role in shock and
inflammation;
Nathan, 1992, "Nitric oxide as a secretory product of mammalian cells," FASEB
J.
6:3051-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: maestro
of
the blood circulation," Proc. Roy. Soc. Lond B 343:225-246; Szabo, C.; 1995,
"Alterations in the production of nitric oxide in various forms of circulatory
shock,"
New Horizons 3:3-32; and may predispose to carcinogenic transformation;
Bartsch et
al., 1994, "Endogenously formed N-nitroso compounds and nitrosating agents in
human cancer etiology," Pharmacogenetics 2:272-7; Liu et al., 1992, "Woodchuck
hepatitis virus surface antigen induces NO synthesis in hepatocytes: possible
role in
hepatocarcinogenesis.," Carcinogenesis 15:2875-7; Ohshima et al., 1994,
"Chronic
infections and inflammatory processes as cancer risk factors: possible role of
nitric
oxide in carcinogenesis," Mutation Res. 305:253-64. The promoter region of the
murine iNOS gene has been cloned, and separate regions responsible for
inducibility
in response to LPS and to IFN have been identified. LPS-mediated induction if
iNOS
appears to involve the mobilization and nuclear translocation of NF-kB, with
subsequent binding to the iNOS promoter. The induction of iNOS can also be
inhibited by pharmacological inhibitors of tyrosine kinase and NF-kB
activation;
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Szabo, C.; 1995, "Alterations in the production of nitric oxide in various-
forms of
circulatory shock," New Horizons 3:3-32.
The inhibitory effect of INH,BP on iNOS expression was indicated by the
inhibition on nitrite production., iNOS mRNA expression and iNOS protein
expression. The regulation occurs in the early stage of iNOS induction, since
INH2BP
gradually loses its effectiveness when applied at increasing times after the
stimulus
for iNOS induction. The regulation of INH,BP of iNOS induction occurs both in
vitro and in whole animals. In addition, our data show that the LPS-induced
production of cyclooxygenase metabolites, similar to the induction of iNOS, is
modulated by 1NH2BP. The production of cyclooxygenase metabolites by pro-
inflammatory cytokines is due to novel mRNA and protein synthesis, and
expression
of COX-2, by a process which shares similarities with the process of iNOS
induction;
Vane et al., 1995, "New insights into the mode of action of anti- inflammatory
drugs,"
Inflamm. Res. 44:1-10. The inhibition of the LPS-induced expression of
inflammatory mediators, however, is not a non-specific "response to INH2BP,
since
the induction of TNF by LPS was not affected by this agent in the J774 cells.
Interestingly, the inhibitory effect of INH2BP on iNOS was greatly reduced
when LPS was used in combination with INF for immunostimulation. This effect
may be due to the fact that IFN-induced transcription factors such as
interferonregulatory factor; Martin et al., 1994, "Role of interferon
regulatory factor I
in induction of nitric oxide synthase," J. Exp. Med. 180:977-84; bypass the
inhibition
of the iNOS induction by the above mentioned agents.
Previous in vitro studies have suggested that induction of iNOS is modulated
by pharmacological inhibitors of pADPRT in macrophages in vitro; Hauschildt et
al.,
1992, "Induction of nitric oxide synthase in L929 cells by tumour-necrosis
factor
alpha is prevented by inhibitors of poly (ADP-ribose) polymerase," Biochem. J.
288:255-260; Pellat-Seceunyk et al., 1994, "Nicotinamide inhibits nitric oxide
synthase mRNA induction in activated macrophages," Biochem. J. 297:53-58.
However, in these studies, the pADPRT inhibitors 30 aminobenzamide and
nicotinamide were used at high concentrations (10-30 mM), which inhibited
total
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protein and RNA synthesis, and may have had additional, pharmacological
actions,
such as free radical scavenging; Hauschildt et al., 1992, "Induction of nitric
oxide
synthase in L929 cells by tumour-necrosis factor alpha is prevented by
inhibitors of
poly (ADP-ribose) polymerase," Biochem. J. 288:255- 260. The present
experiments,
using INH,BP, further suggest the pleiotropic involvement of pADPRT in the
process
of iNOS mRNA transcription. In order to study the regulation of the iNOS
promoter
by INH,BP, transient transfection assays were performed using murine
macrophage
iNOS promoter luciferase constructs. These data with the deletional constructs
indirectly suggest that INH,BP regulates a transcription event which involves
the
murine iNOS promoter region between -1592 and -367 bp. ADP-ribosylation of
histones and nucleases may be involved in the maintenance of a relaxed
chromatin
structure; Bauer et al., 1995, "Modification of growth related enzymatic
pathways and
apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell
line by
treatment with 5-iodo-6-amino-l,2-benzopyrone (INH,BP)," Int. J. Oncol. 8:239-
252;
Bauer et al., 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-l,2-
benzopyrone, a non-covalently binding ligand of poly (ADP-ribose) polymerase,"
Biochimie 77:347-377; Ueda et al., 1985, "ADP-ribosylation," Ann. Rev.
Biochem.
54:73-100. Based on previous experimental data; Bauer et al., 1995,
"Modification of
growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-
transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-
benzopyrone (INH,BP)," Int. J. Oncol. 8:239-252; Bauer et al., 1995, "Reversal
of
malignant phenotype by 5-iodo-6-amino- 1,2-benzopyrone, a non-covalently
binding
ligand of poly (ADP-ribose) polymerase," Biochimie 77:347-377; is reasonable
to
suggest that in these experimental systems pADPRT inhibitory compounds, e.g.,
INH,BP, pretreatment inhibits auto-poly-ADP-ribosylation of pADPRT and
histones.
Such action is known to trigger the conversion of relaxed to condensed
chromatin,
and, by way of upregulation of nucleases and other DNA structure regulatory
enzymes; Bauer et al., 1995, "Modification of growth related enzymatic
pathways and
apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell
line by
treatment with 5-iodo-6-amino-1,2-benzopyrone (INH,BP)," Int. J. Oncol. 8:239-
252;
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Bauer et al., 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1,2-
benzopyrone, a non-covalently binding ligand of poly (ADP-ribose) polymerase,"
Biochimie 77:347-377; may affect promoter functions.
EXAMPLE 3
Effect of inhibition of INH2BP on MAP kinase and NF-kB activation
These results have demonstrated that 1NH2BP treatment inhibits LPS-induced
activation of MAP kinase. In respect, these data are similar to findings with
transformed endothelial cells; Bauer et a!., 1995, "Modification of growth
related
enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed
bovine
endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone
(INH,BP),"
Int. J. Oncol. 8:239-252. It is probable that the inhibition of MAP kinase
activation
occurs by a pleiotropic cellular response trigger by INH2BP. MAP kinase has
been
shown to be activated in various cell types treated with LPS or various pro-
inflammatory cytokines (TNF-alpha, interleukin-1, nerve growth factor);
Kyriakis et
al., 1996, "Sounding the alarm: protein kinase cascades activated by stress
and
inflammation," J. Biol Chem. 271:24313-24316; Matsuda et al. , 1994,
"Signaling
pathways mediated by the mitogen-activated protein (MAP) kinase kinase/MAP
kinase ascade," J. Leukocyte Biol. 56:548-53; Cowley et al., 1994, "Activation
of
MAP kinase kinase is necessary and sufficient for PC12 differentiation and for
transformation of NIH 3T3 cells.," Cells 77:841-52; Pang et al., 1995,
"Inhibition of
MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve
growth
factor," J. Biol. Chem. 22Q:13585-8; Willis et al., 1996, "Differential
induction of the
mitigen activated protein kinase pathway by bacterial lipopolysaccharide in
cultured
monocytes and astrocytes," Biochem. J. 313:519-524; Saklatvala et al., 1993,
"Interleukin 1 and tumour necrosis factor-alpha activate the mitogen-activated
protein
(MAP) kinase kinase in cultured cells," FEBS Lett. 334:189-92. A variety of
extracellular signals converge at the MAP kinase kinase/MAP kinase cascade
through
different MAP kinase kinase - kinases and elicit a vice spectrum of cellular
responses;
Kyriakis et at., 1996, "Sounding the alarm: protein kinase cascades activated
by
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stress and inflammation," J. Biol Chem. 271:24313-24316; Ferrell, JE, 1996,
"Tripping the switch fantastic: how a protein kinase cascade can convert
graded inputs
into switchlike outputs," TIBS 21:460-466. Blockade of MAP kinase or MAP
kinase
kinase modifies a multitude of intracellular pathways and inhibits cellular
differentiation and proliferation; Kyriakis et al., 1996, "Sounding the alarm:
protein
kinase cascades activated by stress and inflammation," J. Biol Chem. 271:24313-
24316; Matsuda et al., 1994, "Signaling pathways mediated by the mitogen-
activated
protein (MAP) kinase kinase/MAP kinase cascade," J. Leukocyte Biol. 56:548-53;
Cowley et al., 1994, "Activation of MAP kinase kinase is necessary and
sufficient for
PC12 differentiation and for transformation of NIH 3T3 cells.," Cells 77:841-
52; Pang
et al., 1995, "Inhibition of MAP kinase kinase blocks the differentiation of
PC-12
cells induced by nerve growth factor," J. Biol. Chem. 270:13585-8; Willis et
al.,
1996, "Differential induction of the mitigen-activated protein kinase pathway
by
bacterial lipopolysaccharide in cultured monocytes and astrocytes," Biochem.
J.
313:519-524; Saklatvala et al., 1993, "Interleukin 1 and tumour necrosis
factor-alpha
activate the mitogen-activated protein (MAP) kinase kinase in cultured cells,"
FEBS
Lett. 334:189-92. Recently, inhibition of MAP kinase kinase with PD 98059 has
been
shown to suppress the expression of iNOS mRNA in cultured endothelial cells
and a
cardiac myocytes; Singh et al., 1996, "Regulation of cytoline-inducible nitric
oxide
synthesis in cardiac myocytes and microvascular endothelial cells.," J. Biol.
Chem.
271:1111-1117. This finding is in line with our observation that PD 98059
causes a
marked suppression of nitrite production by LPS in the RAW macrophages.
Since activation of NF-kB is a major pathway in the inflammatory response,
and it is involved in the induction of iNOS by LPS, but not by INF; Szabo, C.;
1995,
"Alterations in the production of nitric oxide in various forms of circulatory
shock,"
New Horizons 3:3-32; Martin et at., 1994, "Role of interferon regulatory
factor 1 in
induction of nitric oxide synthase," J. Exp. Med. 180:977-84, we sought to
investigate
potential effect of INH,BP on NF-kB. Our results demonstrate that INH2BP does
not
alter the nuclear translocation of NF-kB activation, or the modulation of NF-
kB-
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mediated cellular events by INH,BP, if any, may occur at a cellular event
distal to
nuclear translocation of NF-kB.
EXAMPLE 4
Pathophysiological and therapeutic implications; INHZBP modulates the
inflammatory process at multiple levels
Reduction by pADPRT inhibitors of the expression of pro-inflammatory genes
iNOS and COX-2, and the subsequent reduced formation of NO and prostaglandins
may be beneficial in various forms of inflammation; Nathan, 1992, "Nitric
oxide as a
secretory product of mammalian cells," FASEB J. 6:3051-3064; Vane, J.R., The
Croonian Lecture 1993, "The endothelium: maestro of the blood circulation,"
Proc.
Roy. Soc. Lond B 343:225-246; Szabo, C.; 1995, "Alterations in the production
of
nitric oxide in various forms of circulatory shock," New Horizons 3:3-32; Vane
et al.,
1995, "New insights into the mode of action of anti-inflammatory drugs,"
Inflamm.
B. 44:1-10. In addition, enhanced release of IL-10 may have additional anti-
inflammatory actions; Liles et al., 1995, "Review: nomenclature and biologic
significance of cytokines involved in inflammation and the host immune
response," J.
Infect Dis. 172:1573-80; Giroir, 1993, "Mediators of septic shock: new
approaches for
interrupting the endogenous inflammatory cascade," Critical Car. Med. 21:780-
9;
Szabo et al., 1997, "Isoproterenal regulates tumour necrosis factor,
interleukin-10,
interleukin-6 and nitric oxide production and protects against the development
of
vascular hyporeactivity in endotoxemia," Immunology 90:95-100. It is
conceivable
that such effects significantly contribute to the improvement by pADPRT
inhibitory
compounds, e.g., INH,BP pretreatment and the survival rate of mice challenged
with
lethal doses of endotoxin. However, the delineation of the exact mechanisms by
which INH2BP exerts effects on the LPS-induced expression of the various
inflammatory mediators requires further detailed investigations. On one hand,
it is
conceivable that pADPRT activity or the binding of pADPRT protein is involved
in
the regulation of the production of inflammatory mediators and/or the
expression of
genes that code for components of the inflammatory process. On the other hand,
it is
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probable that indirect down-regulation of MAP kinase activity by INH,BP; Bauer
et
al., 1995, "Modification of growth related enzymatic pathways and apparent
loss of
tumorigenicity of a ras transformed bovine endothelial cell line by treatment
with 5-
iodo-6-amino-1,2-benzopyrone (INH,BP)," Int. J. Oncol. 8:239-252; may also
contribute to the observed effects, as predicted by other studies; Kyriakis et
al., 1996,
"Sounding the alarm: protein kinase cascades activated by stress and
inflammation,"
J. Biol Chem. 271:24313-24316; Ferrell, JE, 1996, "Tripping the switch
fantastic:
how a protein kinase cascade can convert graded inputs into switch-like
outputs,"
TIBS 21:460-466. The present results demonstrate the therapeutic potential of
pADPRT inhibitory compounds such as INH,BP in various inflammatory diseases.
EXAMPLE 5
Some of the cytotoxic effects of nitric oxide (NO) are related to the
production
of peroxynitrite, a reactive oxidant formed by the rapid reaction of NO and
superoxide; Crow et at., 1995, "The role of peroxynitrite in nitric oxide-
mediated
toxicity", Current Top Microbiol. Immunol. 196:57-73; Pryor et al., 1995, "The
chemistry of peroxynitrite: a product from the reaction of nitric oxide with
superoxide", Am. J. Physiol. L699- L772. The formation of peroxynitrite has
been
demonstrated in a variety of inflammatory conditions, including systemic
inflammation induced by endotoxin; Szabo el al., 1995, "Alterations in nitric
oxide
production in various forms of circulatory shock" New Horizons 3:2-32;
arthritis;
Kaur et al., "Evidence for nitric oxide-mediated oxidative damage in chronic
inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid
patients",
FEBS Lett. 1359:9-12; and carageenan induced paw edema; *Salvemini et at.,
1996.
In fact, from pharmacological studies, utilizing NO synthase (NOS) inhibitors
and
superoxide dismutase mimetics, it was concluded that peroxynitrite plays an
important
pathogenetic role in the development of in the inflammatory process; Szabo C,
1996,
"The role of peroxynitrite in the pathophysiology of shock, inflammation and
schemia-reperfusion injury", Shock 6:79-88; *Salvemini et at., 1996;
*Zingarelli et
al., 1997. Moreover, it has been demonstrated that some of the agents
currently used
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in the treatment of arthritis are, in fact, scavengers of peroxynitrite;
Whiteman et al.,
1996 "Protection against peroxynitrite dependent tyrosine nitration and alpha
1-
antiproteinase inactivation by some anti-inflammatory drugs and by the
antibiotic
tetracycline" Annals. of the Rheumatic Diseases 55:383-7. The realization that
a
significant part of the NO-related cytotoxicity is due to the formation of
peroxynitrite
has necessitated the development of novel therapeutic approaches based around
the
formation and action of peroxynitrite.
One of the intracellular pathways triggered by peroxynitrite is related to DNA
single strand breakage and activation of poly (ADP-ribose) synthetase (PARS);
Szabo
et al., 1996, "The role of peroxynitrite in the pathophysiology of shock,
inflammation
and schemia-reperfusion injury", Shock 6: 79-88; *Szabo, 1996b). Pronounced
activation of PARS can rapidly deplete the intracellular concentration if its
substrate,
NAD+, slowing the rate of glycolysis, electron transport, and, therefore, ATP
formation, resulting in cell dysfunction; *Berger, 1991; *Cochrane, 1991.
Accordingly, inhibitors of PARS protect against cellular injury under these
conditions. This mechanism, known as the "PARS suicide hypothesis", has
previously been characterized in relation to H,O,-induced oxidant damage and
radiation injury; *Berger, 1991; *Cochrane, 1991; and has recently been
implicated in
the NO- and peroxynitrite-related cellular injury in endotoxic shock, stroke,
ischemia-
reperfusion injury, and diabetes mellitus; Szabo et al., 1996, "The role of
peroxynitrite
in the pathophysiology of shock, inflammation and schemia-reperfusion injury",
Shock 6:79-88; *Zhang et al., 1994, *Heller, et al., 1995.
The potential role of PARS in arthritis has recently been put forward by
Kroger and colleagues. In a potassium peroxochromate-induced model,
nicotinamide
treatment caused 1 25-35% reduction in the mean arthritic score; *Miesel et
al., 1996.
However, from that study, the mechanism of the inhibition remained undefined,
since
no clear distinction could be drawn between the free radical scavenging
activity and
the PARS inhibitory effect of nicotinamide; *Miesel et al., 1995. In the
present study,
with the aid of 5-iodo-6-amino-1,2- benzopyrone (INH2BP), a novel, potent
inhibitor
of PARS activity; *Bauer et al., 1995a, *Bauer et al., 1995b; we investigated
the
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effect of pharmacological inhibition of PARS on the course of carrageenan-
induced
paw edema and collageninduced arthritis. The results of our study support the
view
that inhibition of PARS is of anti-inflammatory potential.
EXAMPLE 6
Induction and evaluation of carrageenan- induced paw edema
Male Wistar rats (250-300 g, Charles River Laboratories, Wilmington, MA)
were used in these studies. Animals received a subplantar injection 0.1 ml
saline
containing I% 1-carrageenan into the right hind paw. This phiogogenic agent
was
given to either INH,BP-treated animals or to animals treated with vehicle.
Animals
were treated with INH,BP (0.5 g/kg p.o) -24h and -2h before the injection of
carrageenan. The volume of the paw was measured by phlethysmometry immediately
after the injection as previously described; *Sautebin, et al., 1995.
Subsequent
readings of the volume of the same paw were carried out at 60 minute intervals
and
compared to the initial readings. For these experiments, n=6 vehicle-treated
and n=6
INH,BP treated animals were used.
EXAMPLE 7
Induction and evaluation of collagen-induced arthritis
Male DBA/I J mice (9 weeks, Jackson Laboratory, Bar Harbor, ME) were used
for these studies. Chick type II collagen (CII) was dissolved in 0.01 M acetic
acid at a
concentration of 2 mg/ml by stirring overnight at 4 C. Dissolved CII was
frozen at
-70 C until use. Complete Freund's adjuvant (CFA) was prepared by the addition
of
Mycobacterium tuberculosis H37ra at a concentration of 2 mg/ml. Before
injection,
CII was emulsified with an equal volume of CFA. Collagen-induced arthritis was
induced as previously described; Hughes et al., 1994, "Induction of T helper
cell
hyporesponsiveness in an experimental model of autoimmunity by using
nonmitogenic anti-CD3 monoclonal antibody", J. Immunol. 153:3319-3325. On day
1, mice were injected intradermally at the base of the tail with 100 ml CII).
On day
21, a second injection of CII in CFA was administered. Animals were treated
with
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either vehicle (n=10) or with INH,BP (n=60 (0.5 g/kg p.o.) every 24 hours,
starting
from Day 25. Mice were evaluated daily for arthritis by using a macroscopic
scoring
system ranging from 0 to 4 (1 - swelling and/or redness of the paw or one
digit; 2 -
two joints involved; 3 - more than two joints involved; 3 - more than two
joints
involved; and 4 = severe arthritis of the entire paw and digits). The
arthritic index for
each mouse was calculated by adding the four scores of the individual paws. At
the
end of the experiments (Day 35), animals were sacrificed under anesthesia and
paws
and knees were removed and fixed for histological examination. Histological
examination was done by an investigator blinding for the treatment regime.
Data analysis and presentation
For the studies with carrageenan-induced paw edema, paw volumes in the
treated and untreated groups of animals were compared with unpaired Student's
test.
For the arthritis studies, Mann-Whitney U-test (2-tailed, independent) was
used to test
the statistical differences in the arthritic indices. This nonparametric
statistic was
used to compare medians, rather than means, because the scale of measurement
was
ordinal, and the distribution values were typically nonnormally distributed;
Hughes et
al., 1994, "Induction of T helper cell hyporesponsiveness in an experimental
model of
auto-immunity by using nonmitogenic anti-CD3 monoclonal antibody", J. Immunol.
153:3319-3325.
Values in Figure 10 are expressed as mean + standard error of the man of n
observations, where n represents the number of rats (6 animals for each
group).
Values in Fig. 11 represent incidences (%), whereas values in Fig. 12
represent
medians. A p-value less than 0.05 was considered statistically significant
(I'<0.05;
**p<0.02).
Materials
5-iodo-6-amino-l,2-benzopyrone (INH2BP) was prepared as described
previously (*Bauer et al., 1995a; * Bauer et al., 1995b). Chick type 11
collagen was
from Elastin Products Company, Inc. (Owensville, MO). Mycobacterium
tuberculosis
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H37Ra was from Difco (Detroit, MI). All other chemicals were from Sigma
Chemical Co. (St. Louis, MO). Subplantar injection of carrageenan into the rat
paw
led to a time-dependent increase in paw volume with a maximal response at 3h
(Fig.
10). This carrageenan induced paw edema was significantly reduced by treatment
with INH,BP (Fig. 10).
In the collagen-induced arthritis model in mice, between Days 26-35 after the
first collagen immunization, animals progressively developed arthritis, as
evidenced
by an increase in the arthritis incidence and an increase in the arthritic
score (Figs. 11-
12). Treatment with INH,BP reduced the incidence of arthritis until Day 33 and
reduced the severity of the disease throughout the experimental period. By Day
30,
arthritic score increased to 10, whereas median arthritic scores in the INH2BP
treated
animals remained around 5 (Fig. 12). By Day 35, all vehicle-treated animals,
and
most of the INH-,BP treated animals had some degree of arthritis (Fig. 11).
However,
even at Day 35, the median arthritic scores were significantly decreased by
INH,BP
treatment (Fig 12).
At Day 35, histological evaluation of the paws in the vehicle-treated
arthritic
animals revealed signs of severe suppurative arthritis, with massive mixed
(neutrophil,
macrophages and lymphocyte) infiltration into the larger ankle joints and the
terminal
digits. In addition, a severe or moderate necrosis, hyperplasia and sloughing
of the
synovium could be seen, together with the extension of the inflammation into
the
adjacent musculature with fibrosis and increased mucous production. In the
INH2BP
animals, the degree of arthritis was significantly reduced. Nevertheless,
there was still
a significant degree of arthritis in these animals, with a moderate, primarily
neutrophil
infiltration into several of the larger joints, coupled with mild to moderate
necrosis
and hyperplasia of the synovium. Similar to these findings in the paw, signs
of severe
suppurative arthritis were found in the knee, which was reduced by treatment
with
INH2BP (not shown).
Discussion
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No, peroxynitrite, oxyradicals and products of the inducible cyclooxygenase
have independently been proposed as important factors in the pathogenesis of
various
forms of inflammation, including arthritis (see Introduction and also: Brahn,
1991,
"Animal models of rheumatoid arthritis. Clues to etiology and treatment" Clin.
Orthop. Rel. Res. 265:42-53; Kaur et al., 1994, "Evidence for nitric oxide-
mediated
oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial
fluid
from rheumatoid patients. FEBS Lett. 1359:9-12; Oyanagui Y, 1994, "Nitric
oxide
and superoxide radical are involved in both initiation and envelopment of
adjuvant
arthritis in rats" Life Sci. 54:PL285-9; Miesel et al., 1994, "Effects of
allupurinol on
in vivo suppression of arthritis in mice and ex vivo modulation of phagocytic
production of oxygen radicals in whole human blood", Inflammation 6:597-612;
Whiteman et al., 1996 "Protection against peroxynitrite dependent tyrosine
nitration
and alpha 1-antiproteinase inactivation by some anti-inflammatory drugs and by
the
antibiotic tetracycline" Annals. of the Rheumatic Diseases 55:383-7; Anderson
et al.,
1996, "Selective inhibition of cyclooxygenase (COX)-2-reverses inflammation
and
expression of COX-2 and interleukin 6 in rat adjuvant arthritis", J. Clin.
Invest.
97:2672-2679. The present study, demonstrating anti-inflammatory effects of
INH,BP in the carrageenan-induced paw edema model and in the collagen induced
arthritis model supports the view that PARS is involved in the progression of
the
inflammatory process and the pharmacological inhibition of PARS is of anti-
inflammatory potential.
The primary mode of action of INHZBP is likely to be related to interruption
of
the futile intracellular cascade characterized by DNA injury. PARS activation,
ADP
ribosylation and NAD+ and ATP depletion in various cell types of the inflamed
joints.
Inhibition of this pathway with various inhibitors of PARS, such as 3-
aminobenzamide, nicotinamide and INH,BP has been shown to protect multiple
cell
types from injury; Berger, 1991; *Cochrane, 1991; Szabo et al., 1996, "The
role of
peroxynitrite in the pathophysiology of shock, inflammation and
schemiareperfusion
injury", Shock 6:79-88; *Szabo, 1996b.
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The overproduction of NO in inflammatory conditions is due to the
suppression of the inducible isoform of NOS (iNOS); Nathan, 1992, "Nitric
oxide as a
secretory product of mammalian cells", FASEB J. 6:3051-3064; Szabo, 1995,
"Alterations in nitric oxide production in various forms of circulatory
shock", New
Horizons 3:2-32; Southan, et al., 1996, "Spontaneous rearrangement of
aminoaklylguanidines into mercaptoalkylguadidines - a novel class of nitric
oxide
synthase inhibitors with selectivity toward the inducible isoform" Br. J.
Pharmacol.
117:619-632. Several lines of evidence suggest a role for iNOS and NO
overproduction in the pathogenesis of arthritis (see for reviews: Stenovic-
Racic, et al.,
1993, "Nitric oxide and arthritis", Arthr. Rhemat. 36:1036- 1044; *Evans et
al., 1995.
First, the expression of iNOS and the production of large amounts of NO has
been
demonstrated in chondrocytes from experimental animals and humans (Haeselmann
et
al., 1994, "Nitric oxide and proteoglycan synthesis by human articular
chondrocytes
in alginate culture", FEBS Lett. 352:361- 364; Sakurai et al., 1995, "Nitric
oxide
production and inducible nitric oxide synthase expression in inflammatory
arthritis",
J. Clin. Invest. 96:2357-63; Grabowski et al., 1996, "Nitric oxide production
in cells
derived from the human joint", Br. J. Rheumatol. 35:207-12; Murrell et al.,
1996,
"Nitric oxide: an important articular free radical", J. Bone Joint Sur. - Am.
78:265-74.
Second, an increase in the circulating levels of nitrite/nitrate (the
breakdown products
of NO) has been demonstrated in patients with arthritis; (Farrell et at.,
1992),
"Increased concentrations of nitrite in synovial fluid and serum samples
suggest
increased nitric oxide synthesis in rheumatic diseases" Ann. Rhem. Dis.
51:1219-22;
Stichtenoth et at, 1995, "Urinary nitrate excretion is increased in patients
with
rheumatoid arthritis and reduced by predisolone", Ann. Rhem. Dis. 54:820-4.
Third,
the development of arthritis has been shown to be reduced by non-isoform-
selective
inhibitors of NOS (*Ialential et al, 1993; McCartney-Francis et a., 1993,
"Suppression
of arthritis by an inhibitor of nitric oxide synthase", J. Exp. Med. 178:749-
753;
Weinberg et al., 1994, "The role of nitric oxide in the pathogenesis of
spontaneous
murine autoimmune disease, increased nitric oxide production and nitric oxide
synthase expression in MRL-lpr/ipr mice, and reduction of spontaneous
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glomerulonephritis and arthritis by orally administered NG-monomethyl-L-
arginine",
J. Exp. Med. 1979:651-60; Stefanovic-Racic et al., 1994, "N-monomethyl
arginine, an
inhibitor of nitric oxide synthase, suppresses the development of adjuvant
arthritis in
rats" Arthr. Rheumat. 37:1062-9; and, more recently, by inhibitors with
selectivity for
iNOS; (Connor et a!., 1995, "Suppression of adjuvant-induced arthritis by
selective
inhibition of inducible nitric oxide synthase", Eur. J. Pharmacol. 273:15-24.
In this
respect it is noteworthy that pretreatment of multiple cell types with PARS
inhibitors
(including 3-aminobenzaminde, nicotinamide as well as INH,BP) prior to
immunostimulation has been shown to suppress the expression of mRNA for iNOS
and reduce NO production; (*Hauschildt et al., 1992, *Pellat- Seceunyk et al.,
1994;
Zingarelli et at., 1996, "Peroxynitrite-mediated DNA strand breakage activates
poly-
ADP ribosyi synthetase and causes cellular energy depletion in macrophages
stimulated with bacterial lipopolysaccharide" J. Immunol. 156:350-358; *Szabo
et al
1997. From these experimental data it may be concluded that PARS via a not yet
characterized mechanism, also regulates the process of iNOS expression, and
that this
effect may represent an additional mode of beneficial action of PARS
inhibition in
various forms of inflammation. However, caution should be exercised when
interpreting the above findings. For instance, in the in vitro studies quoted
above,
extremely high concentrations of the PARS inhibitors 3-amiobenzamide and
nicotinamide were required (10-30 mM) in order to demonstrate suppression of
iNOS
induction. These high concentrations of these agents may have additional
pharmacological actions, such as inhibition of total protein and RNA
synthesis, and/or
free radical scavenging actions; *Hauschildt et al., 1992, *Pellat-Seceunyk et
a!.,
1994; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA strand breakage
activates
poly-ADP ribosyl synthetase and causes cellular energy depletion in
macrophages
stimulated with bacterial lipopolysaccharide" J. Immunol. 156:350- 358.
INH2BP, on
the other hand, effectively suppressed the expression of iNOS even at lower,
non-
cytotoxic concentrations (100-300 mM). However, in the case of INH2BP, several
modes of action should be considered, since this agent is an inducer of
alkaline
phosphatases, with secondary, pleiotropic modulation of cellular responses;
*Bauer et
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al., 1996; *Szabo et al., 1997). Experiments in cells or animals with ablation
of the
PARS gene are required to definitely address the question as to whether
inhibition of
PARS per se suppresses the process of NOS induction.
In a recent study of Ehrlich and colleagues, it was shown that in cultured
rabbit synovial fibroblasts, the cytokine-induced expression of collagenase
activity
was suppressed by 3-aminobenzamide; *Ehrlich et al., 1995. It is now possible
to
determine the pharmacological action (which nevertheless, would be expected to
suppress the course of the arthritis process), the property of the particular
inhibitor
used, or whether it is, indeed, related to a reduction of the catalytic
activity of PARS.
In this respect, it is noteworthy that, based on studies with pharmacological
inhibitors,
PARS has been implicated in the regulation of a variety of genes, including
the major
histocompatibility complex class II gene (*Hiromatsu et a!., 1992; Taniguchi
et al.,
1993), ras c-myc (*Bauer et al., 1996, *Nagao et a!. 1991), DNA
methyltransferase
gene (*Bauer et al, 1996) and protein kinase C (*Bauer et a!., 1996).
Taken together, the present work demonstrated the amelioration of the
development of local inflammatory response and the inhibition of the
progression of
collagen-induced arthritis by INH,BP . Although, for the last decade, a role
for PARS
has been proposed in DNA repair, recent observations demonstrate that the
ablation of
the gene for PARS does not compromise DNA repair: PARS knockout animals
appear normal and viable (*Wang et a!., 1995). This observation strengthens
the anti-
inflammatory potential of pharmacological inhibitors of PARS. PARS inhibition
(as
opposed to iNOS inhibition) is unlikely to interfere with the important
antimicrobial
effects of NO, since invading microorganisms do not contain PARS. On the other
hand, PARS inhibition is not only expected to inhibit part of the oxidant-
induced
cytotoxicity, and thus may be more effective when applied in combination with
other
free radical scavengers or other immunosuppressive agents. The results of the
present
studies support he view that PARS inhibition, alone, or in combination with
other
anti-inflammatory agents, represents a promising novel anti-inflammatory
approach.
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In a similar manner as shown in the above examples, compounds of formulae
II, and III are used to treat inflammation or inflammatory diseases, as well
as treating
gram negative and gram positive infections.
The foregoing written specification is considered to be sufficient to enable
one
skilled in the art to practice the invention. Indeed, various modifications of
the above-
described modes for carrying out the invention which are obvious to those
skilled in
the field of pharmaceutical cormulation or related fields are intended to be
within the
scope of the following claims.
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