Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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PERIPHERAL BENZODIAZEP1NE RECEPTOR LIGANDS
FIELDS OF THE INVENTION
This invention pertains to the use of compounds which bind with high affinity
to peripheral benzodiazepine receptors as antiinflammatory agents. Such
compounds
:i include isoquinoline and benzodi~~zepine derivatives. These compounds may
be used
in the treatment of human diseases such as rheumatoid arthritis, lupus
erythematosus,
Sjogren's syndrome, osteoarthritis, multiple sclerosis, Behcet's disease,
temporal
aretritis and dementia of the Alzheimer type.
ly BACKGROUND OF THE INVENTION
Historically, two broad classes of benzodiazepine receptors have been
described: central and peripheral. Ligands for central benzodiazepine
receptors, such
as diazepam, flunitrazepan and clonazepam, produce an interaction with GABA"
receptors, enhancing the activity of GABA (gamma-aminobutyric acid). These
15 ligands possess anticonvulsant, muscle relaxant, sedative and anxiolytic
properties.
They are widely used clinically. The receptors are highly concentrated in
brain, but
are also found peripherally.
Ligands that are selective for peripheral benzodiazepine receptors, such as 4'
chlorodiazepam (Ro 5-4864) and 1-(2-chlorophenyl)-N-methyl-N-( 1-methylpropyl)-
3
20 isoquinolinecarboxamide (PK 11.195), do not interact with GABA receptors
and do
not possess the same pharmacological properties as the central benzodiazepine
receptor ligands. Instead they blind to peripheral benzodiazepine receptors
which are
widely distributed throughout the body, including the central nervous system.
The
peripheral benzodiazepine receptor ligands have no well defined
pharmacological
properties, and so far there are no generally aceepted clinical applications
for their
use.
In this application, "peripheral benzodiazepeine receptors" means the class of
peripheral benzodiazepine receptors as distinguished from the class of central
benzodiazepine receptors, and "'peripheral benzodiazepine receptor ligands"
means
:30 ligands that bind with high affuuty to peripheral benzodiazepine
receptors.
PK 11195 has a particularly high affinity for peripheral benzodiazepine
receptors. The equilibrium dissociation constant, or affinity (Kd) of PK 11195
for
SUBSTITUTE SHEET {RULE 26)
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human brain tissue is estimated to be 4.3 nM (Doble et al., 1987). Its
affinity for
mouse peritoneal macrophages is~ reported to be 5 . 6 nM (Zavala and Lenfant,
1987b) .
A compound's ability to displace bound PK 11195 from peripheral
benzodiazepine receptors (Ki) is a convenient measure of the relative strength
of the
compound's binding to these receptors. In brain, this has been determined to
be 3.5
nM for PK / 1195 itself, 44 nlVt for 1-N,N-diethyl-a-methyl-2-phenylquinoline-
4-
propranamide (PK-14067), 178 nM for Ro 5-4864. By contrast, the value for the
central benzodiazepine receptor ligand clonazepam is 46,000 nM (Doble et al.,
1987) .
:l0 The peripheral benzodiazepine receptors are said to be highly associated,
if not
exclusively associated, with the outer mitochondria) membrane. The presumed
association of these receptors with mitochondria (Snyder et al. 1990; Hirsch
et al.
1989) has led to the proposal treat they affect mitochondria) respiration.
However,
this view has been challenged on the basis of a poor correlation between
receptor
:l5 density and the ability of ligands to stimulate respiration (Zisterer et
al. 1992).
The reported effects of peripheral benzodiazepine receptor ligands on immune
functions are diverse and in many respects controversial. PK 11195 has been
shown
to inhibit in vitro mitogen-driven T- and B- cell stimulation, properties
shared by a
number of central benzodiazepine ligands (Ramseir et al., 1993; Bessler et
al., 1992).
20 In contrast, PK 11195 has also been reported to stimulate antibody
production in mice
following immunization with sheep red blood cells (Lenfant and Zavala, 1986;
Zavala
et al. , 1984; Zavala and Lenfa~nt, 1987b) which would enhance the
inflammatory
response. Peripheral benzodiazepine receptor ligands, including Ro 5-4864,
have also
been reported to enhance the rEapiratory burst system of macrophage-like
P388D1
:ZS cells stimulated with arachidonic: acid. PK 11195 in the same situation
reportedly had
little effect (Zavala and lenfant, 1987a).
In brain, peripheral benzodiazepine receptors are believed to be associated
with glial cells. However, it is not clear what the physiological function of
these
receptors in brain might be. Various authors have reported increases in
peripheral
30 benzodiazepine receptor levels in rat brain following kainic acid,
ischemic, or
neoplastic lesions. Similar findings have been reported for human brain tissue
in
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patients with brain tumours, neoplasms, multiple sclerosis, cerebrovascular
disorders
and Alzheimer's disease. The teaching is that these increases in peripheral
benzo-
diazepine receptor levels reflect glial proliferation (Diorio et al., 1991;
Leong et al.,
1994).
Following kainic acid lesioning to rat brain, binding of PK 11195 increased
in both time and spatial localization with the appearance of macrophages, but
PK
11195 binding apparently did not correlate with the appearance of astrocytes
(Myers
et al., 1991). In PET studiea of human gliomas, there was a large increase in
binding of PK 11195, but not of Ro 5-4864 (Junck et al., 1989).
It has been suggested that inhibitory agonists of peripheral benzodiazepine
receptors are useful for speeding the recovery of damaged central nervous
system
tissue (Gee, 1993) . The mechanism proposed to account for this effect is
inhibition
of glial cells and macrophages, and inhibition of cytokine production by
macrophages
at the site of acute injury. But Taupin et al. (1991, 1993) find that the
inflammatory
cytokines interleukin-1 and tumor necrosis factor are increased, rather than
decreased
by peripheral benzodiazepine receptor ligands.
In summary, it can be said that the art and teaching in the field is
inconsistent,
Until the United States patent application of McGeer et al. (serial 08/520,
211), there
was no overall concept as to the function of peripheral benzodiazepine
receptors, or
the pharmacological properties of their ligands.
SUMMARY OF THE INVENTION
The invention discloses that ligands which bind with high affinity to
peripheral
benzodiazepine receptors act as antiinflammatory agents. A method of treating
an
inflammatory condition in a mammal is provided. The method comprises
administer-
ing to a mammal requiring such treatment a therapeutically effective amount of
a
compound which binds with high affinity to peripheral benzodiazepine
receptors.
This may require dosages in the range of 0.1-100 mg/kilogram of body mass per
day
or roughly (0.3-320 micromoles/kilogram per day depending on the molecular
weight
of the agent), as determined by a medical practitioner or veterinarian.
Preferably,
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the compound is selected from the group consisting of compounds which bind
with
micromolar or submicromolar affinity to peripheral benzodiazepine receptors,
as, for
example, PK 11195 or pharmacologically acceptable salts thereof.
The invention includes ~pharmaeutical compositions comprising a peripheral
benzodiazepine receptor ligan~d in combination with one or more compatible
pharmaceutically acceptable adjuvents or diluents which may be inert or
physiologi-
cally active. These compositions may be administered by the oral, parenteral
or
rectal route or locally. Compositions of the invention comprising a peripheral
benzodiazepine receptor ligand may be packaged in packaging material that
comprises
l0 a label which indicates that the. composition can be used for treating
inflammatory
conditions.
The present invention provides a method of identifying a compound that is
therapeutically effective for treating an inflammatory condition in a mammal.
The
method comprises selecting a compound:
l5 (1) that binds with micromolar or submicromolar affinity to peripheral
benzodiazepvie receptors; and
{2) that is therapeutically effective in treating inflammatory symptoms
in MRIrlpr rnice.
Additional steps that may be taken in the method of indentifying an
:LO antiinflammatory compound include:
(1) selecting a compound that inhibits respiratory burst in cultured
macrophages;;
(2) selecting a .compound that inhibits the neurotoxic effects of
products secreted by cultured monocytic type cells, such as THP-1
:LS cells, following stimulation by inflammatory agents; and
(3) selecting a compound that exhibits increased binding to human
Alzheimer brain tissue compared to normal human brain tissue.
The compound utilized in the various aspects of the invention may be
selected from the group consistung of: PK 11195, PK-14067, PK 14105, Ro 5-
6993,
:30 Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-
3448,
Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, Ro 5-5122. In each case,
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the compound is preferably 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-
isoquinolinecarboxamide (PK 11195).
DETAILED Dl~?SCRIPTION OF THE INVENTION
This invention discloses the use of high affinity peripheral benzodiazepine
receptor ligands as a new class of antiinflammatory compounds. This class of
compounds includes isolquinoliine drivatives such as PK 11195 and
benzodiazepine
derivatives such as Ro 5-4864. The following examples illustrate various
aspects of
this invention, including four assays for defining the antiinflammatory
pharmacologi-
cal profile of such drugs. These assays establish the utility of high affinity
peripheral
benzodiazepine receptor ligands as antiinflammatory agents on the basis of the
following results:
(1) Pronounced therapeutic activity in the MRL-lpr mouse autoimmune
disease. This disorder is an accepted model of a variety of human diseases:
rheumatoid arthritis (Koopma,n and Gay, 1988), systemic lupus erythematosus
(Bartlett et al., 1988), Sjogren"s syndrome (Hayashi et al., 1994), connective
tissue
disease (Rosenberg, 1988), behavioral and neurological disorders (Sakic et
al., 1993)
and CNS inflammation (Vogehweid et al. , 1991) . PK 11195 demonstrated a more
powerful prevention of pathology in this autoimmune disorder than standard
antiinflammatory agents.
(2) Down regulation of respiratory burst activity in cultured macrophages.
PK 11195 was a more potent down regulator than standard antiinflammatory
agents.
(3) Protection from the toxic effects of products secreted from cultured
THP-1 (monocyte type) cells, following stimulation by inflammatory agents,
against
cultured SYSY (neuronal type) cells. PK 11195 and Ro 5-4864 were more
effective
neuroprotective agents than some standard antiinflammatory drugs.
(4) Enhanced high affinity binding to Alzheimer disease brain tissue
compared with neurologically normal brain tissue. Cells involved in the
inflammatory
response in the central nervous system, including T-cells, reactive microglia
(macrophages) and reactive astrocytes (Itagaki et al., 1986), are associated
with
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Alzheimer disease lesions. Therefore, Alzheimer disease tissue is a model for
inflammatory disease of the central nervous system. Both PK 11195 and Ro 5-
4864
showed greater high affinity binding to Alzheimer brain tissue than to
neurologically
normal brain tissue, with the difference being greater for PK 11195.
Together, the foregoing assay results are predictive of the utility of PK
11195 and other peripheral benz;odiazepine receptor ligands in the treatment
of animal
and human disorders of an inflammatory nature. These include, but are not
limited
to, rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome,
osteroarthritis,
multiple sclerosis, inflammatory bowel disease, Behcet's disease, myasthenia
gravis,
temporal arteritis, Hashimoto's disease, dermatitis herpetiformis and other
diseases,
including Alzheimer disease, where chronic inflammation may exacerbate the
fundamental pathology (as discvussed in more detail below).
EXAMPLE 1: Treating Inflammatory Conditions in MRL-lpr Mice
MLR-lpr mice are a widely studied strain which spontaneously develop a
particularly severe autoimmune: disorder. Pathologies that are found in a
variety of
human idiopathic inflammatory and autoimmune diseases are reproduced in these
animals. They are therefore considered to be an outstanding model for such
individual human diseases, although the pathology represents a combination of
several
of them. They are regarded as the best animal model for rheumatoid arthritis
(Koopman and Gay, 1988), systemic lupus erythematosus (Bartlett et al., 1988),
and
Sjogren's syndrome (Hayashi Ert al. , 1994) . They display vascular connective
tissue
disease (Rosenberg, 1988), as seen in several human arthropathies. They
develop
behavioral and neurological disorders (Sakic et al., 1993), as well as
evidence of CNS
inflammation (Vogelweid et al., 1991) which is seen in isolation in such
diseases as
Alzheimer type dementia and multiple sclerosis, but is also part of the
syndrome in
systemic lupus erythematosus and Sjogren's syndrome. The model is considered
to
be a particularly demanding one for screening antiinflammatory agents because
of the
severity and comprehensiveness of the disease pathology. The lesions are
character-
ized by infiltration of B- lymphocytes and T-lymphocytes, indicating that it
is a
typical autoimmune disorder. The lesions include synovial inflammation,
synovial
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cell proliferation, pannus fonnation, and articular cartilage erosion and bone
destruction. Only powerful antiinflammatory agents will inhibit the
devastating
changes that accompany this genetic disorder. Mice of the MRL-lpr strain
spontaneously develop a mild fomn of the disease at 4 to 5 months of age. The
onset
and severity can be accelerated by injection of complete Freund's adjuvant
(CFA),
supplemented to 10 mg/ml with heat-inactivated M. tuberculosis, at 13-14 weeks
of age, with 67-82% of animals becoming affected within 1 month (Ratkay et
al.,
1994; Ratkay et al., 1993).
In the first set of experiments, mice of the MRL-lpr strain were injected at
:LO 13-14 weeks of age with 0.05 ml of CFA supplemented to 10 mg/ml with heat-
inactivated M. tuberculosis at each of two intradennal thoracic sites
according to the
standard procedure, and, following injection (day 0), were started on a daily
subcutaneous dose of PK 11195 dissolved in alcohol. Daily injections were
continued
until day 14. The animals were sacrificed at day 30 and joint histopathology
:LS assessed. Four dose levels of 1'K 11195 were employed: 0.01 mg/kg (n=15),
0.1
mg/kg (N = 10), 0.5 mg/kg (N=9) and 1 mg/kg (N=7). The results were compared
with those in littennates receiving CFA only (n = 14) or CFA and treatment
with
ethanol alone (n=8). Followuig sacrifice, animals were evaluated for
subsynovial
inflammation, synovial hyperplasia, cartilage destruction and pannus
formation, and
:ZO bone destruction. Using a rating system described by Ratkay et al (1993),
the results
are shown in Table 1. PK 11195 significantly reduced the total
histopathological
score observed in control mice, even at the lowest dose of 0.01 mg/kg. In
comparison
with results obtained by Ratkay et al. (1994), PK 11195, at the 0.1 mg/kg
dose, was
more effective than ten times that dose of indomethacin, forty times that of
:25 cyclosporin, and whole body irradiation at 3 Gy from a 60 cobalt source on
day I of
arthritis injection.
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WO 99/61024 PCT/CA98/00502
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Since alcohol was found to be an irritating vehicle to the mice, dimethyl
sulfoxide (DMSO) was chosen a.s a non-irritating solvent for PK 11195 in
subsequent
experiments. The method for inducing the adjuvant arthritis in these
experiments
remained the same.
The second series of experiments tested the ability of PK 11195 to prevent
MRL-lpr adjuvant arthritis when dissoved in DMSO, using the same criteria as
in the
first experiment. Starting at day 0 (the day of CFA injection), animals
received no
treatment (n = 10), 0.1 ml DrvISO alone (n = 14) or various doses of PK 11195
dissolved in 0.1 ml DMSO subcutaneously until sacrifice at day 30. PK 11195
was
administered at daily doses of 0.01 mg/kg (n = 6), 0.1 mg/kg (n = 6), 0.5
mg/kg
(n = 19) and 1 mg/kg (n = 6;1. About 80 % of the non-txeated animals developed
visibly arthritic joints. There was a dramatically reduced incidence of
visible swelling
in animals receiving higher doses of PK 11195, as well as a delay in the onset
of
disease. This clinical evaluation was confirmed following sacrifice by
histological
analysis of the joints. There was a dose response effect (Table 2), with PK
11195
at 1 mg/kg having the greatest. preventative action. It was interesting to
note that
DMSO alone also seemed to have a mild effect, although it did not reach
significance
at the p < 0.05 statistical leve:f.
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WO 99/61024 PCT/CA98/00502
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These two series of experiments establish that PK 11195 has a preventative
effect against the induction of arthritis in the well established MRL-lpr
animal model.
The third series of experiments tested the ability of PK 11195 to inhibit
MRL-lpr arthritis once it had been established. Mice were primed at day 0 with
CFA, as in the previous two series of experiments. Treatment with DMSO alone,
or PK 11195 in DMSO, was not commenced until day 10, at which time significant
swelling was apparent. PK 111'95 in 0.1 ml DMSO or 0.1 ml DMSO alone was then
administered subcutaneously an a daily basis until sacrifice at day 30.
Erythema and
swelling of the adjuvant injected control group (n = 10) was evident over the
course
of the experiment. DMSO alone (n = 13), and PK 11195 at doses of 0.03 mg/kg (n
= 8). 0.3 mg/kg (n = 16) and 3.0 mg/kg (n = 16) were the treatment groups. PK
11195 at 3.0 mg/kg significantly reduced the swelling (Table 3). This was
confirmed
by histological analyses following sacrifice at day 30. PK 11195 at 3 mg/kg
significantly reduced joint histopatholology (Table 4).
This series of experunents established that PK 11195 has a pronounced
therapeutic effect on already established arthritis in the MRL-lpr model.
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WO 99/61024 PCT/CA98100502
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WO 99/61024 PCT/CA98/00502
- 13 -
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EXAMPLE 2: Inhibiting~Resniiratory Burst in Cultured Macrophages
The respiratory burst system is an attack mechanism possessed by
professional phagocytes such as peritoneal macrophages. Its main function is
to
protect the body from hostile: invaders by generating superoxide radicals, but
inappropriate activation can damage host tissue. Oxidative stress is believed
to be
one of the more harmful concomitants of inflammation. The respiratory burst
system
can be activated in cultured peritoneal macrophages in multiple ways,
including
exposure to zymosan particles opsonized by complement proteins (Klegeris and
McGeer, 1994). The ability of PK 11195 to inhibit respiratory burst by
administra-
tion before or after exposure of peritoneal macrophages to opsonized zymosan
is
shown in Table 5. At 10-', PK 11195 inhibited respiratory burst by more than
50 %
whether given before or after o;psonized zymosan. This indicates that the
effect was
downstream from, or independent of, the opsonized zymosan receptors. Table 5
also
shows the comparative effects of three agents effective in inflammatory
disorders:
indomethacin, prednisone and dapsone. Each of these agents was substantially
less
effective than PK 11195, and tended to show less inhibition when administered
after
opsonized zymosan than before. These data illustrate that PK 11195 inhibits
respiratory burst more powerfully and by different mechanisms than these well
known
antiinflammatory agents. It its also more effective than 4'-chlorodiazepam,
the
prototype benzodiazepine ligand for peripheral benzodiazepine receptors.
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Table 5: Inhibition of Respiratory Burst in Cultured Rat Peritoneal
Macrophages by
Various Substances
Percent of Control
( t S. E. )
when Substance was ivg en
Substance ConcentrationBefore OZ# After OZ#
PK 11195 10-~M 99.0 t 5.6 97.9 f 0.2
10-sM 87. 9 t 5 . 5 83 . 5 t
8 . 7
10~M 48 .9 t 10. 8 47.1 t 13
.6
Ro 5-4864 10~M 94. 6 t 12.2 not done
10-'M 85 .4 t 2.5 not done
10~'M 74.7 t 1. 9 not done
Dapsone 10-SM 94.7 t 1. 3 96.4 t 1.
6
10~M 73.6 t4.6 90.3 t6.2
Indomethacin10-sM 101. 3 t 3 . 5 not done
10~M 86. 3 t 10. 9 100. 9 t
2
10-3M 36 t 12. 5 64. 8 t
9.4
Prednisone 10~'M 88 . 8 t 7 . 2 101. 8 t
4. 9
10-'M 62.5 t 8.1 67 t 11.6
OZ# = opsonized zymosan
For methods see Klegeris and McGeer, 1994
EXAMPLE 3: Reduction of Cvtotoxicitv to Neuronal SYSY Cells Caused by
Secretorv Products of Monocvtic THP-1 Cells Stimulated by Inflammatory
Mediators
Cultured cells of the monocytic type, such as THP-1 cells, when stimulated
by inflammatory agents, secrete products which in their aggregate are toxic to
cultured neurons.
Human monocytic THP-1 cells were plated in 24-well plates at a concentra
tion of Sx105 cells per well in 1 ml of Dulbecco's Modified Eagle Medium
Nutrient
Mixture-F12 containing 5% festal bovine serum (FBS). THP-1 cells were
incubated
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in the presence or absence of drugs for 30 minutes prior to the addition of an
inflammatory stimulus consisting; of 1 Jug/ml lipopolysaccharide (LPS) with
333 U/ml
of interferon-~y (IFN-~y) . After :24 hours incubation in a humidified 5 %
C02/95 % air
atmosphere at 37°C, 0.5 ml aliquots of cell-free supernatants were
transferred to the
wells containing SYSY cells which had been plated 24 hours earlier. After 72
hours
of culture, cell death was evaluated by the amount of lactate dehydrogenase
(LDH)
which had been released into the medium from lysed cells. Cell survival was
measured by the ability of the cell culture to reduce the formazan dye 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).
LDH activity in cell culture supernatants was measured by the enzymatic
assay described by Decker and Lohmann-Matthes (1988) in which formation of the
formazan product of the iodonit~rotetrazolium dye INT was followed
colorimetxically.
Optical densities were measured, and the amount of LDH was expressed as
specific
cell lysis (in percent) calculated) by the following formula:
%iysed cells = 100 x [D,A(treated)-DA(untreated)]/DA(lysed)
where DA is absorbance of supernatants at 490 nm from which background
absorbance has been subtracted.. ~A(treated) was the measurement on
supernatant
from drug-treated cells, AA(u:ntreated) that from cells incubated only with
fresh
medium, and DA(lysed) that from cells where complete lysis was achieved with 1
Triton X-100.
The MTT assay was perfumed as described by Mosmann (1983) and by
Hansen et al. (1989) . This method is based on the conversion of MTT to
colored
formazan by viable but not lby dead cells. The viability of SYSY cells was
determined by adding MT'T to the SYSY cell cultures to reach the final
concentration
of 1 mg/ml. Plates were placf;d overnight at 37°C and optical densities
at 570 nm
were measured by transferring 100 ~cl aliquots to 96-well plates and using the
plate
reader with a corresponding filter. The percentage of viable cells was
calculated by
the following formula:
viable cells = 100 x [OD (treated)-OD(lysed)]/[OD(untreated)-OD(lysed)]
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WO 99/61024 PCT/CA98/00502
where OD is the optical density. As in the LDH experiments, treated refers to
cells
treated with drug, untreated to those incubated only with fresh medium, and
lysed to
those where complete lysis was achieved with 1 % Triton X-100.
The results are shown in Table 6. They demonstrate that both PK 11195
and Ro 5-4864 significantly prevented toxicity to neuronal cultures at doses
ranging
from 2x10~M to SxlO-SM. Agents which failed to have any effect in this assay
at
equivalent concentrations included such well known antiinflammatory agents as
prednisone, dexamethasone and propentofylline.
Table 6: Inhibition of THP-1. Human Monocytic Cell Toxicity Towards SYSY
Human Neuroblastoma Cells
A. LDH assa y after
72 hours
incubation
~ Dead cellsSignificance Level
Substance Concentration( + S E (randamized blocks
) ANOVA)
PK 11195 SxlO-s 33.411.9 F=11.6, p = 0.0007
2x10-5 27.413.4
2x10 35.313.3
0 45.112.6
Ro 5-4864 5x10-5 28.714.9 F=6.1, p = 0.009
2x10-5 30.616.1
2x10- 38.512.2
0 43.112.9
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WO 99/61024 PCT/CA9g/00502
- ig -
B. MTT assay after 72 hours uicubation
%~ Live cells Significance Level
Substance Concentration (+ S E.) (randomized blocks ANOVA)
PK 11195 5x10-5 ti1.7t15.6 F=9.7, p = 0.002
2xlo-s ti5.7 t 14.4
2x10- 47.9 t 8.0
0 35.4 t5.6
Ro 5-4864 5x10-5 Ei0.6f 1.2 F=7.8, p = 0.004
2x10-' '.>8.4t7.9
:l0 2x10- 44.5 t6.9
0 39.1 t6.4
These experiments establish that PK 11195 and Ro 5-4864 have a protective
effect against neurotoxic products secreted by monocyte type cells following
inflammatory induction by the well known inflammatory mediators
lipopolysaccharide
and gamma-interferon.
EXAMPLE 4~ Bindins to Human Alzheimer Brain Tissue
To date, the only method by which the levels of putative peripheral
benzodiazepine receptors in tis sue can be determined is by high affinity
binding of
specific ligands such as PK 11195. To compare the levels in Alzheimer and
normal
brain tissue, we assessed the high affinity binding of PK 11195 to Alzheimer
and
normal brain tissue. The standard method of Schoemaker et al. (1983) was
employed.
The results are shown in Table 7 for Alzheimer and control brain tissue.
As shown in the table, Alzheimer cases had 2.7 fold higher binding of PK 11195
than
control brain tissue. This is greater than the difference observed with Ro 5-
4864
(McGeer et al., 1988).
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WO 99/61024 PCT/CA98/00502
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Table 7: Specific High Affinity Binding of PK 11195 (at 3 nM) to Alzheimer and
Control Cortical Tissue (in fentamoles/mg protein)
Alzheimer Control
4g0 201
585 94
400 265
450 118
395 141
408 111
422 222
Means+ S.E. 449~~25 165+25
(difference significant at p < 0.001 )
A criterion by which the potency of other isoquinoline or benzodiazepine
derivatives can be compared with PK 11195 is their ability to displace PK
11195 in
a competitive binding assay (Doble et al., 198.
EXAMPLE 5: A Method of Identifying_Novel Antiinflammatory Compounds
The invention provides a method of identifying a compound that is
therapeutically effective for treating an inflammatory condition in a mammal.
The
method comprises selecting a compound:
(1) that binds with micromolar or submicromolar affinity to peripheral
benzodiazepine receptors; and
(2) that is therapeutically effective in treating inflammatory symptoms
in MRL-lpr mice.
Additional steps may be taken in the method of indentifying anti-
inflammatory compounds;
(1) selecting the compound that inhibits respiratory burst in cultured
macrophages;
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WO 99/61024 PCT/CA98/00502
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(2) selecting the compound which inhibits the secretion of neurotoxic
products by cultured monocyte type cells following exposure to an
inflammatory stimulus; or
(3) selecting the compound that exhibits increased binding to human
Alzheimer brain tissue compared to normal human brain tissue.
In the method of identifying antiinflammatory compounds, the compound
utilized may be selected from vthe group consisting of: PK 11195, PK-14067, PK
14105, Ro 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro
5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-4608, Ro 5-6524, Ro
5-5122, therapeutically acceptable salts of these compounds or mixtures of
these
compounds or their salts.
EXAMPLE 6: Methods of Treatment
A method of treating an inflammatory condition in a mammal is provided.
The method comprises administering to a mammal requiring such treatment a
therapeutically effective amount of a compound, or a pharmaceutically
acceptable salt
of the compound, the compound being selected from the group consisting of
compounds which bind with micromolar or submicromolar affinity to peripheral
benzodiazepine receptors. The: method may be practiced where the mammal is a
human being and the compounf. binds with micromoiar or submicromolar affinity
to
human peripheral benzodiazepine receptors. The inflammatory condition may be
rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome, osteoarthritis,
multiple sclerosis, Behcet's disease, temporal arteritis, and, without being
limited by
the foregoing, any inflammatory disorder which calls for the use of
antiinflammatory
agents. Alternatively, the inflammatory condition may be dementia of the
Alzheimer
type and the peripheral benzodiazepine receptors may be those that are found
in brain.
Dementia of the Alzheimer type is included in this category since it has been
shown
to be characterized by chronuc inflammation of the brain and to respond to
antiinflammatory therapy (U. S. Patent 5,192, 753; European patent 0 642 336
BI) .
A preferred compound is PK 11195. However, the compound may be
selected from the group consisting of: PK 11195, PK-14067, PK 14105, Ro 5-
6993,
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WO 99/61024 PCT/CA9t3/00502
-21 -
Ro 5-4864, Ro S-6900, Ro 5-6945, Ro 5-6669, Ro 5-6902, Ro 5-6531, Ro 5-3448,
Diazepam, Ro 7-5520, Ro S-5115, Ro 5-4608, Ro S-6524, Ro 5-5122,
therapeutically
acceptable salts of these compounds or mixtures of these compounds or their
salts,
or other compounds that bind with as high or higher affinity to peripheral
benzo-
diazepine receptors.
EXAMPLE 7: Formulations
The invention includes phanmaeutical compositions comprising a peripheral
benzodiazepine receptor ligand in combination with one or more compatible
:l0 pharmaceutically acceptable adjuvents or diluents which may be inert or
physiologi-
cally active. These compositions may be administered by the oral, parenteral
or
rectal route or locally. The peripheral benzodiazepine receptor ligand may be
PK
11195. However, the ligand may be selected from the group consisting of: PK
11195, PK-14067, PK 14105, F;o 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-
:15 6669, Ro 5-6902, Ro 5-6531, Ro 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro
5-
4608, Ro 5-6524, Ro S-5122, therapeutically acceptable salts of these
compounds or
mixtures of these compounds or their salts, or other compounds that bind with
as high
or higher affinity to peripheral benzodiazepine receptors.
Tablets, pills, powders (gelatin capsules or cachets) or granules, may be
:ZO used as solid compositions for oral administration. In these compositions,
the active
ingredient according to the invention may be mixed with one or more inert
diluents
such as starch, cellulose, sucrose, lactose or silica. These compositions may
also
contain substances other than diluents, for example one or more lubricants
such as
magnesium stearate or talcum, a colorant, a coating (dragees) or a lacquer.
25 Pharmaceutically acceptable solutions, suspensions, emulsions, syrups and
elixirs containing inert diluents ouch as water, ethanol, glycerol, benzoic
acid, benzyl
alcohol, sodium benzoate, dimethyl sulfoxide, vegetable oils or liquid
paraffin may
be used as liquid composition> for oral administration. These compositions may
contain substances other than diluents, for example wetting agents,
sweeteners,
30 thickeners, flavoring agents or stabilizers.
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WO 99/61024 PCT/CA9$/00502
-22-
Sterile compositions for parenteral administration may preferably be nan-
aqueous solutions, suspensions or emulsions. Water, ethanol, propylene glycol,
polyethylene glycol, benzoic acid, benzyl alcohol, sodium benzoate, dimethyl
sulfoxide, vegetable oils, especially olive oil, injectable organic acids
esters, for
example ethyl oleate or other suitable organic solvents may be used as the
solvent or
the carrier.
These compositions may also contain adjuvants, especially wetting agents,
tonicity regulating agents, emulsifiers, dispersants and stabilizers. The
sterilization
may be carried out in several ways, for example by aseptic filtration,
incorporating
DLO a sterilizing agent, by irradiation or by heating. They may also be
prepared in the
form of sterile solid compositions which may be dissolved at the time of use
in a
sterile medium suitable for injection.
Compositions for rectal administration are suppositories or rectal capsules,
which contain, in addition to the active product, excipients such as cocoa
butter,
:l5 semi-synthetic glycerides or polyethylene glycols.
Compositions for local administration may be for example creams,
ointments, lotions, eye lotions, mouth-washes, nasal drops or aerosols.
The dosage depends on the effect sought, the length of treatment and the
administration route employed. In general, the medical practitioner {or
veterinarian)
a0 will determine the appropriate dosage depending on the age, weight and all
other
factors specific to the subject to be treated. The approximate dosage range
may be
chosen from the dosage range shown to be effective in resisting damage in the
MRL-
lpr murine rheumatoid arthritis model, i.e. 0.1 mg/kg to 100 mg/kg, with the
most
probable range being 1-10 mg/kg body weight per day.
:25
EXAMPLE 8: Articles of Manufacture
Compositions of the invention comprising a peripheral benzodiazepine ligand
may be packaged in packaging material that comprises a label which indicates
that the
composition can be used for treating inflammatory conditions. Such conditions
30 include rheumatoid arthritis, lupus erythematosus, Sjogren's syndrome,
osteoarthritis,
multiple sclerosis,Behcet's disease, temporal arteritis and dementia of the
Alzheimer
CA 02332825 2000-11-20
WO 99/61024 PCT/CA98/00502
- 23 -
type. The peripheral benzodiazepine receptor ligand of the composition
preferably
exhibits micromolar or submicromolar affinity to peripheral benzodiazepine
receptors,
for example PK 11195. The ligand may be selected from the group consisting of:
PK
11195, PK-14067, PK 14105, R.o 5-6993, Ro 5-4864, Ro 5-6900, Ro 5-6945, Ro 5-
S 6669, Ro 5-6902, Ro 5-6531, R.o 5-3448, Diazepam, Ro 7-5520, Ro 5-5115, Ro 5-
4608, Ro S-6524, Ro 5-5122, W erapeutically acceptable salts of these
compounds or
mixtures of these compounds or their salts, or other compounds that bind with
as high
or higher affinity to peripheral benzodiazepine receptors.
As will be apparent to those skilled in the art of the invention in the light
of
l0 the foregoing disclosure, many alterations and modifications are possible
in the
practice of this invention without departing from the spirit and scope
thereof.
Accordingly, the scope of the invention is to be considered in accordance with
the
substance defined by the claims.
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WO 99/61024 PCT/CA98/00502
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