Note: Descriptions are shown in the official language in which they were submitted.
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DEXANABINOL AND DEXANABINOL ANALOGS
REGULATE INFLAMMATION RELATED GENES
FIELD OF THE INVENTION
The present invention relates to pharmaceutical compositions comprising as an
active
ingredient non-psychotropic cannabinoid derivatives that modulate the
expression of genes
involved in inflammatory and immune processes. Regulating the transcription of
pro and
anti-inflammatory mediators has useful therapeutic application for prevention
and
treatment of acute and chronic inflammation, autoimmune diseases and related
disorders,
pain, infections, liver diseases, cardiovascular disorders, gastrointestinal
disorders,
disorders of the central and peripheral nervous system including
neurodegenerative
diseases, respiratory diseases, renal diseases, post-operative complications,
tissue rejection
and certain types of cancer.
BACKGROUND OF THE INVENTION
Cannabinoids
Camlabis sativa preparations have long been known as therapeutic agents to
treat
various diseases. The identification of tetrahydrocannabinol (THC) as the
active principle
of marijuana prompted medicinal chemists to develop numerous cannabinoid
analogs.
These novel compounds were designed to exhibit the therapeutically beneficial
properties
of THC without the clinically undesirable psychotropic effects. Potential
therapeutic
applications have classically included known attributes of marijuana itself
such as anti-
emesis, analgesia, anti-glaucoma and appetite stimulation. More recently
recognized roles
for non-psychotropic cannabinoids are as neuroprotective and anti-inflammatory
agents.
The diverse camlabinoid effects are generally attributed to the activation or
inhibition of
various types of receptors. Nevertheless, the mechanisms underlying some
therapeutic
effects of cannabinoid derivatives remain unclear.
Several synthetic cannabinoid derivatives have been prepared since the
discovery of
the natural ligand with an emphasis toward therapeutic non-psychoactive
agents. An
extended family of such compounds were disclosed for example in US Patent Nos.
4,179,517, 4,876,276, 5,538,993, 5,635,530, 6,096,740, and in international
patent
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application WO 01/98289. A pivotal member of this family of compounds is 1,1-
dimethylheptyl-(3S,4S)-7-hydroxy-06-tetrahydrocannabinol, disclosed in US
4,876,276
and denoted therein HU-211. HU-211 was subsequently assigned the trivial
chemical name
dexanabinol. The neuroprotective action of dexanabinol, and later on its
derivatives, was
attributed in part to their ability to bloclc the NMDA receptor. Moreover, the
compounds
seemed to share anti-oxidative and anti-inflammatory properties unrelated to
their capacity
to block the NMDA receptor. This anti-inflammatory activity was associated
with the
ability of those compounds to reduce the secretion of PGE2 produced by the
enzyme
cyclooxygenase-2 (COX-2). COX-2 is one of the cyclooxygenase isoforms involved
in the
metabolism of arachidonic acid (AA) toward prostaglandins (PG) and other
eicosanoids, a
family of compounds known to exhibit inflammatory properties and known to be
involved
in inflammation. Most conventional NSAIDs (non-steroidal anti-inflammatory
drugs)
inhibit COX activity by modifying the enzyme active site thereby preventing
the
transformation of the AA substrate to PGEa (Hint B. et al., J. Pharm. Exp.
Ther. 300: 367-
375, 2002).
Moreover, dexanabinol and later on its analogs were found able to block the
production or action of TNF-a, as disclosed in International Patent
applications WO
97/11668 and WO 01/98289. It was postulated that the inhibition of the
cytokine occurs at
a post-transcriptional stage, since in a model of head injury dexanabinol did
not affect the
levels of TNF-a, mRNA (Shohami E. et al., J. Neuroimmuno. 72: 169-77, 1997).
Human
TNF-a is first translated into a 27 kd transmembrane precursor protein, which
is cleaved
into the secreted 17 kd form by TNF-a converting enzyme (TALE). Based on RT-
PCR
experiments, Shoshany et al. reported that dexanabinol has no significant
effect on TNF-a
mRNA whereas it significantly reduced the levels of TACE mRNA, supporting the
assumption that the drug acts at the level of secretion inhibition
Inflammation
Inflammation is one of the most important processes involved in the defense of
an
organism; however, when it occurs in some organs, such as brain, in response
to an insult,
or if it is inappropriate, such as in autoimmune diseases, inflammation can be
harmful and
therefore requires pharmacological treatment. The inflammatory response
involves many
effector mechanisms that produce a multiplicity of vascular and cellular
reactions.
Vasodilatation, increased microvascular permeability, chemotaxis, cellular
activation, pain
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and finally repair are mediated by the local production and release of several
specific
mediators. Cytokines, chemokines, arachidonic acid derivatives
(prostaglandins,
thromboxanes and leukotrienes), oxygen and possibly nitrogen radicals, play a
regulatory
role in this complex and highly balanced process. Following is a selection of
genes whose
protein product was shown to be involved in a variety of pathways leading to
inflammation: COX-2, IL-2, MCP-1, IL-1 [3, iNOS, TNF-a, IL-6 and IL-10 and
SOCS.
The involvement of these inflammatory mediators in various pathological
conditions
was extensively reviewed (Frangogianis N.G. et al., Cardio. Res. 53: 31-47,
2002; Gerard
C. et al., Nature hnmuno. 2: 108-15, 2001; Nathan C., Nature 420: 846-852,
2002; Quan
N. et al., Histol. Histopathol. 17: 273-88, 2002). Blocking the effect of
these inflammatory
mediators by diverse strategies includes, in addition to the conventional
small molecule
drug approach, the preparation of antibodies, peptidic mimetics or decoys,
oligonucleotides, either antisense or triple-helix forming, directed either
against the ligand
or against its receptor(s). Selective inhibition of COX-2 alone is disclosed
for example in
US patent Nos. 5,783,597, 5,840,746 and 6,025,253 which provide small
molecules for
alleviating, preventing or treating inflammation, neurodegenerative diseases
and
angiogeiuc disorders. US patent 6,344,323 discloses COX-2 antisense
oligonucleotides and
methods of use thereof and US patent 5,776,502 discloses methods of
transcriptional
modulation of various genes including inflammatory mediators using molecules
that bind
to DNA, RNA or protein. The use of antibodies or peptides is exemplified in US
patent
6,277,969 which discloses antibodies to human TNF-a, and peptides thereof for
diagnosis
and treatment of pathologies involving this cytokine.
Despite the progress in this field, there remains an unmet medical need for
effective
therapies for inflammatory diseases. The pro-inflammatory and anti-
inflammatory
mediators have pleiotropic effects, including the property of regulating one
another.
Therefore it is not surprising that the previously described cytokines,
cytokine regulators,
chemokines and "pro-inflammatory" enzymes are involved in numerous diseases
where
they can be either deleterious or beneficial. When appropriately regulated and
balanced,
these agents protect the host by activating defense mechanisms and therefore
their
complete inhibition is not desirable. However, if the inflammation is
inappropriate and the
expression of those mediators is highly dysregulated, then tissue damage may
result.
Compounds that would selectively and simultaneously down regulate pro-
inflammatory
mediators and up-regulate anti-inflammatory ones without totally blocking
their
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physiological beneficial effects would have a clear therapeutic benefit for a
wide range of
disease states.
SUMMARY OF THE INVENTION
The present invention relates to pharmaceutical compositions comprising as an
active
ingredient non-psychotropic cannabinoids and their derivatives that are now
disclosed
unexpectedly to act as direct or indirect regulators of genes involved in
inflammatory
mechanisms. The present invention encompasses any synthetic or natural
cannabinoid
which is essentially devoid of appreciable psychomimetic activity. Currently
preferred are
synthetic non-psychotropic derivatives of dexanabinol, also known as HU-211.
Surprisingly, it is now disclosed that the PGE2 inhibitory activity displayed
by the
preferred compounds does not occur at the level of the COX-2 enzymatic
activity, but
rather at the level of gene regulation. Therefore, some novel non-psychotropic
cannabinoids are useful for the treatment of acute and chronic inflammation,
autoimmune
diseases and related disorders, pain, infections, liver diseases,
cardiovascular disorders,
gastrointestinal disorders, disorders of the central and peripheral nervous
system including
neurodegenerative diseases, respiratory diseases, renal diseases, post-
operative
complications, tissue rejection and certain types of cancer through means not
previously
envisioned. The fact that compounds of the invention act at the level of gene
transcription
by down-regulating pro-inflammatory mediators or by up-regulating anti-
inflammatory
ones or by having both activities simultaneously serve as a basis for treating
a wide range
of conditions with said compounds.
The present invention encompasses pharmaceutical compositions for decreasing
the
transcription of at least one of the pro-inflammatory mediators COX-2, IL-1
Vii, IL-2, iNOS,
TNF-a and MCP-1, comprising as an active ingredient a compound of general
formula (I):
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Formula I
R.
6
R3
having the (3S,4S) configuration and being essentially free of the (3R,4R)
enantiomer,
wherein the dashed line indicates an optional C1-C2 or C6-C1 double bond, and
wherein:
Rl is selected from the group consisting of
a) R' where R' is selected from the group consisting of
A) a linear or branched, saturated or unsaturated, carbon side chain
comprising 1-8
carbon atoms optionally interrupted by 1-3 heteroatoms, and
B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one
or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally
interrupted by 1-4 heteroatoms, and optionally further substituted with one or
more groups selected from
i) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkyl,
ii) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkoxy,
iii) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkylthio,
iv) a halogen,
v) carboxyl,
vi) -COZ-C1-C4 alkyl, wherein the alkyl can be linear, branched or
cyclic, saturated or unsaturated,
vii) keto,
viii) nitro,
ix) a saturated or unsaturated cyclic moiety, an aromatic or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms
comprising one or two-ringed structures, wherein each ring
comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms,
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and optionally further substituted with one or more groups selected
from i)-viii) as defined above,
b) an amine or an amide substituted with at least one substituent as defined
in R'
above,
c) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide
optionally
substituted with one substituent as defined in R' above, and
d) a hydroxyl or an ether -OR' wherein R' is as defined above;
Rz is selected from the group consisting of
a) a halogen,
b) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkyl, and
c) -OR wherein R is selected from the group consisting of
A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or
unsaturated C1-C6 alkyl optionally containing a terminal -OR"' or -OC(O)R"'
moiety wherein R"' is hydrogen or a linear, branched or cyclic, saturated or
unsaturated C1-C6 alkyl, and
B) -C(O)R"' wherein R"' is as previously defined; and
R3 is selected from the group consisting of
a) a linear, branched or cyclic, saturated or unsaturated CI-C12 alkyl,
b) -ORa, in which Ra is a linear, branched or cyclic, saturated or unsaturated
CZ-C9
alkyl which may be substituted at the terminal carbon atom by a phenyl group,
and
c) a linear, branched or cyclic, saturated or unsaturated C1-C7 alkyl-OR"'
wherein R"'
is as previously defined;
and pharmaceutically acceptable salts, esters or solvates thereof.
The present invention also encompasses pharmaceutical composition for
increasing
the transcription of at least one of the anti-inflammatory cytokine IL-10, the
protective
cytokine IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3,
comprising as an active ingredient a compound of general formula (I) as
previously
defined.
Currently more preferred compounds are those wherein R2 is hydroxy or lower
acyloxy and wherein R3 is dimethylheptyl or a dimethylalkyl radical with a
total of at
least 7 carbon atoms.
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According to currently preferred embodiments of the present invention RI is a
heterocyclic moiety selected from the group consisting of an imidazolyl, an
imidazolinyl, a
morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a
pyrrolidinyl, a triazolyl,
and a tetrazolyl, wherein each cyclic moiety may optionally be further
substituted
with at least one substituent selected from the group consisting of C1_6
alkyl, C1_6
alkyloxy, C1_~ alkylthio, keto, carboxy, vitro, saturated or unsaturated
cyclic
moieties or aromatic or heterocyclic moieties wherein each ring comprises 3-8
carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each
independently selected from the group consisting of N, O, and S, wherein each
ring
optionally is further substituted with one or more groups selected from the
group
consisting of C1_6 alkyl, C1_6 alkyloxy, C1_6 alkylthio, keto, carboxy, or
vitro,
wherein C1_6 alkyl, C1_6 alkoxy and C1_6 alkylthio are intended to include
saturated
and unsaturated linear, branched and cyclic structures.
According to more preferred embodiments of the present invention Rl is
selected
from the group consisting of hydroxyl, imidazole, pyrazole, oxazole,
isoxazole,
tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-
imidazole, 2-
methylthio-imidazoline, 4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline,
methyl
sulfide, methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole, 1,3,4-
triazole,
1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl, morpholine,
thiomorpholine,
thiazolidine, glycerol, piperazine, 4-piperidinopiperidine, 4-methylpiperidine
and
tetrahydropyran.
According to additional more preferred embodiments of the present invention Rl
is
selected from the group consisting of mono or di-substituted amines wherein
the
substituent is selected from the group consisting of an C1_g alkyl, C1_6
alkyloxy, C1_6
alkylthio, imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a
piperazinyl, a
pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein
each cyclic
moiety may optionally be further substituted with at least one substituent
selected from the
group consisting of C1_6 alkyl, C1_6 alkyloxy, CI_6 alkylthio, keto, carboxy,
vitro, saturated
or unsaturated cyclic moieties or aromatic or heterocyclic moieties wherein
each ring
comprises 3-~ carbons optionally interrupted by 1-4 heteroatoms, said
heteroatoms each
independently selected from the group consisting of N, O, and S, wherein each
ring
optionally is further substituted with one or more groups selected from the
group
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consisting of C1_6 alkyl, Cl_6 alkyloxy, C1_6 alkylthio, keto, carboxy, or
nitro, wherein C1_6
alkyl, CI_6 alkoxy and C1_6 alkylthio are intended to include saturated and
unsaturated
linear, branched and cyclic structures.
According to another currently preferred embodiment, we disclose a
pharmaceutical
composition which down-regulates gene expression of at least one the pro-
inflammatory
mediators COX-2, IL-1 [3, IL-2, iNOS, TNF-a, and MCP-1, and up-regulates gene
expression of at least one of the anti-inflammatory cytokine IL-10, the
protective cytokine
IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3,
comprising as an
active ingredient a compound of the general formula (I) wherein Rz is OH, R3
is 1,1-
dimethylheptyl, there is a double bond between C6 and C1, and Rl is selected
from the
group consisting of hydroxyl, 2-mercaptoimidazole, imidazole, pyrazole, 4-
methyl-
piperidine, and 4-piperidino-piperidine.
The pharmaceutical compositions may contain in addition to the active
ingredient
conventional pharmaceutically acceptable carriers, diluents and excipients
necessary to
produce a physiologically acceptable and stable formulation.
The pharmaceutical compositions can be administered by any conventional and
appropriate route including oral, parenteral, intravenous, intramuscular,
intralesional,
subcutaneous, transdermal, intrathecal, rectal or intranasal.
Prior to their use as medicaments for preventing, alleviating or treating an
individual
in need thereof, the pharmaceutical compositions will be formulated in unit
dosage. The
selected dosage of active ingredient depends upon the desired therapeutic
effect, the route
of administration and the duration of treatment desired.
A further aspect of the present invention provides a method of preventing,
alleviating
or treating a patient by regulating pro- and anti-inflammatory mediators
selected from
COX-2, IL-1[3, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, by
administering to said patient a therapeutically effective amount of
pharmaceutical
composition containing as an active ingredient a compound of general formula
(I) as
previously defined.
A further aspect of the present invention relates to the use for the
manufacture of a
medicament for preventing, alleviating or treating a disease by regulating pro-
and anti-
inflammatory mediators selected from COX-2, IL-1(3, IL-2, iNOS, TNF-oc, MCP-1,
IL-10,
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IL-6, SOCS-1 and SOCS-3, of a compound of general formula (I) substantially as
shown in
the specification.
BRIEF DESCRIPTION OF THE FIGURES
To assist in the understanding of the invention, and in particular of the data
that are
given in the examples, the following drawing figures are presented herein:
Figure 1 shows the effect of various doses of dexanabinol and its analogs on
IL-2 in Jurkat
cells activated with PMA and Calcium ionophore. In panel A the down-regulatory
effect is
measured at the level of IL-2 gene expression by real-time RT-PCR. In panel B
the down-
regulatory effect is measured at the level of IL-2 secretion.
Fi ure 2 shows the down-regulatory effect of 10 p,M of dexanabinol and its
analogs on
COX-2 gene expression in Jurkat cells activated with PMA and Calcium
ionophore, as
measured by real-time RT-PCR.
Figure 3 shows the effect of dexanabinol and its analogs on gene expression in
the brains
of mice submitted to MCAo 18 hrs before the measurements. In panel A the down-
regulatory effect is measured on the pro-inflammatory mediators COX-2, MCP-1
and IL-2.
In panel B the up-regulatory impact is measured on anti-inflammatory IL-10.
Figure 4 shows the effect of PRS-211,092 ( ~) as compared to vehicle (~) on
expression of
various inflammatory related genes as a function of time from ConA induction
of liver
injury. A: IL-2; B: MCP-1; C: TNF-a; D: IL-1 (3; E: IL-6; F: SOCS-1; and G:
SOCS-3.
Figure 5 shows the effect of various doses of PRS-211,092 and PRS-211,220 on
NF-AT
driven expression of luciferase in activated T cells.
Figuure 6 shows the effect of various doses of dexanabinol, PRS-211,092 and
PRS-211,220,
as well as Celecoxib and Dexamethasone (DXM), as compared to vehicle, on paw
thickness in carrageenan induced paw edema.
Fi,~ure 7 shows the effect of various doses of dexanabinol on tumor growth of
in vivo
implanted LoVo colorectal cancer cells.
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Figure S shows the effect of dexanabinol and PRS-211,220 in vivo in the MPTP
model of
neurodegeneration. In panel A the neurological outcome is measured in a short-
term study.
In panel B the functional outcome is measured using the rotarod test in a long-
term study.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides compositions effective to cause a reduction in
gene
expression of at least one of COX-2, IL-1 (3, IL-2, iNOS, TNF-a and MCP-1.
Moreover,
compositions of the present invention cause an increase in gene expression of
at least one
of the anti-inflammatory cytokine IL-10, the protective cytokine IL-6 and of
the
suppressors of cytokine signaling SOCS-1 and SOCS-3. The mechanism of action
can
either be through direct regulation of gene expression or through indirect
feedback
mechanisms. It will be noted that the compounds of the present invention have
been tested
for their impact on gene expression on a limited set of genes selected for
their known
involvement in the immunomodulatory and/or anti-inflammatory signaling
cascades.
Assaying the effect of those non-psychotropic cannabinoid derivatives on a
larger set of
genes, such as found in microarrays, may reveal additional genes that are
involved in the
new gene regulatory action herein disclosed.
The present invention relates to THC-type compounds which are characterized by
an
absolute stereochemistry at the positions 3 and 4 of the molecule (3S,4S),
which is
opposite to the (3R,4R) configuration in the natural series. The natural
compounds of the
(3R,4R) configuration produce undesirable psychotropic "cannabis" type
effects, which
preclude their use for other therapeutically interesting effects. The
compounds of the
invention being of the (3S,4S) configuration are substantially devoid of the
undesired
psychotropic effect and thus can be used for the treatment of various diseases
and
disorders. Thus, in the present specification and claims which follow the term
"essentially
free" qualitatively refer to (3S,4S) compounds of high optical purity
substantially devoid
of the undesired psychotropic effect lying with the (3R,4R) enantiomer. The
quantitative
criterion of the minimum acceptable degree of optical purity of an intended
therapeutic
enantiomer is dictated by the pharmacological potency of the opposite
enantiomer. The
higher the psychotropic activity of the opposite enantiomer, the stricter is
the requirement
for optical purity. Therefore, the quantitative meaning of "essentially free"
depends upon
the nature of each enantiomeric pair. The enantiomeric pair HU-210 and HLT-
211,
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respectively of (3R,4R) and (3S,4S) configuration, is an extreme example of
such a
situation, HCT-210 being hundred times more psychoactive than natural ~9-THC,
the major
active constituent in marijuana. The very highly undesirable psychotropic
effects of HU-
210 require that HU-211 should be of very high enantiomeric purity of at least
99.8%
(Mechoulam R. et al., Tetrahedron Asymmetry 1(5): 315-8, 1990).
In the present specification the term "prodrug" represents compounds which are
rapidly transformed in vivo to the parent compounds of formula (I), for
example by
hydrolysis in blood. Some of the compounds of formula (I) are capable of
further forming
pharmaceutically acceptable salts and esters. "Pharmaceutically acceptable
salts and
esters" means any salt and ester that is pharmaceutically acceptable and has
the desired
pharmacological properties. Such salts include salts that may be derived from
an inorganic
or organic acid, or an inorganic or organic base, including amino acids, which
is not toxic
or undesirable in any way. The present invention also includes within its
scope solvates of
compounds of formula (I) and salts thereof, for example, hydrates. All of
these
pharmaceutical forms are intended to be included within the scope of the
present invention.
In the present specification "inhibiting, reducing, or decreasing effect" is
the ability
to reduce the activity under discussion by at least 20%, preferably 40%, more
preferably
60% and most preferably 80% or greater.
In the present specification "enhancing or increasing effect" is the ability
to increase
the activity under discussion by at least 2 folds, preferably 3 folds, more
preferably 4 folds
and most preferably 5 folds or more.
W the present specification and claims which follow "prophylactically
effective" is
intended to qualify the amount of compound which will achieve the goal of
prevention,
reduction or eradication of the risk of occurrence of the disorder, while
avoiding adverse
side effects. The term "therapeutically effective" is intended to qualify the
amount of
compound that will achieve, with no adverse effects, alleviation, diminished
progression or
treatment of the disorder, once the disorder cannot be further delayed and the
patients are
no longer asymptomatic. The compositions of the present invention are
prophylactic as
well as therapeutic.
The "individual" or "patient" for purposes of treatment includes any human or
mammalian subject affected by any of the diseases where the treatment has
beneficial
therapeutic impact.
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Throughout this specification and the claims which follow, the alkyl
substituents can
be saturated or unsaturated, linear branched or cyclic, the latter only when
the number of
carbon atoms in the alkyl chain is equal or superior to 3.
The genes affected by compounds of the invention are listed below and the
diseases
wherein abnormal regulation of said genes is implicated in the pathological
progression are
briefly reviewed.
COX-2
In addition to their specific physiological functions in several organs, the
kidney and
gastrointestinal (GI) system in particular, prostaglandins have been known for
some time
to play a major role in the inflammatory process. They are involved as
mediators of pain,
edema and vascular permeability in arthritic diseases and they have been
postulated to be
involved in the pathophysiology of colorectal cancer. The biosynthesis of
prostaglandins
depends upon the action of cyclooxygenase (COX), recently found to exist in
the human as
cyclooxygenase type 1 (COX-1) and cyclooxygenase type 2 (COX-2). Both enzymes
are
involved in the synthesis of prostaglandins, COX-1 constitutively and COX-2
following
induction by a number of agents including mitogens, endotoxins, hormones,
cytokines,
stress conditions and growth-factors. As prostaglandins have both
physiological and
pathological roles, it has been assumed that the constitutive COX-1 was
responsible for the
important physiological functions for example in the GI tract, while the
inducible COX-2
was mainly responsible for the pathological effects of prostaglandins in
inflamed tissues.
However, recent pharmacological studies have shown that COX-2 is not
exclusively
expressed in inflamed tissues, but is constitutively present in several organs
where it
synergizes with COX-1 in maintaining homeostasis. Therefore, total inhibition
of COX-2
might not be desirable. Nevertheless, a selective inhibitor of COX-2 is
expected to be
useful in treating the pathophysiological effects of prostaglandins, by virtue
of its anti-
inflammatory, antipyretic and analgesic properties. Indeed COX-2 inhibitors
already exist
on the market and have a wide range of therapeutic benefits. COX-2 inhibitors
were
already found effective in the treatment of osteoarthritis, rheumatoid
arthritis, ocular
inflammation, acute and chronic menstrual pain, gastritis caused by bacterium
helicobacter
pylori. In addition a selective inhibitor would have potential anti-cancer
effects, such as
with breast and colorectal cancer, would be useful in the treatment of polyps
and
angiogenesis and be an attractive candidate for the treatment of neurological
damage either
resulting from spinal cord injury, cerebral ischemia or neurodegenerative
disorders such as
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Alzheimer disease and Parkinson's disease or AIDS associated dementia. A
positive role
for COX-2 inhibition has also been suggested in chronic liver diseases such as
cirrhosis.
Recent fording suggest that COX-2 is a major source of systemic prostacyclin
synthesis,
and its increased production is observed in patients with signs of platelet
activation such as
unstable angina, severe atherosclerosis and during angioplasty.
IL-1 (3
Interleukin-1 (3 (IL-1 [3) is a potent pro-inflammatory cytokine that has been
implicated in a broad spectrum of diseases. Cells known to express Ih-1 [3
include
astrocytes, adrenal cortical cells, NIA cells, macrophages and monocytes,
endothelial cells,
keratinocytes, megakaryocytes and platelets, neurons, neutrophils,
oligodendroglia,
osteoblasts, Schwann cells, trophoblasts, and T cells plus fibroblasts. IL-1
(3 is a key factor
in several inflammatory disorders that accompany for example septic shock,
IBD,
pancreatitis, ulcerative colitis, pulmonary inflammation and wound healing. It
is also
implicated in inflammation leading to connective tissue destruction such as in
rheumatoid
arthritis, osteoarthritis, synovial inflammation and periodontal disease.
Moreover, it is
involved in central nervous system (CNS) pathologies, where it is thought to
exacerbate
neuronal loss. Its level is elevated in brain injury, ALS, AD, PD and
anorexia. However, it
remains unclear how the effects of IL-1 (3 are mediated. Exacerbation may be a
result of
either the direct cytotoxic action of IL-1 (3 on resident cells in the CNS or
it may be a result
of secondary bystander damage by the leukocytes recruited to the brain in
response to IL-
1 ~3 production. Inappropriate production of IL-1 [3 was also observed in
immune disorders
such as allergy, systemic lupus erythematosus (SLE), psoriasis, graft versus
host disease
and MS. IL-1 (3 is up-regulated in patients suffering from Gaucher's disease
(GD), and it is
speculated that this over-expression may relate to the pathophysiology of some
of the
clinical manifestations of GD. The promotion of pancreatic beta-cell
destruction leading to
insulin dependent diabetes mellitus shows dependence on IL-1. The acquired
expression of
IL-1 (3, which might affect the production of other various cytokines as well
as the
regulation of other cellular factors, has been implicated in the progression
of benign
oncologic conditions to severe and often fatal malignancies, in
atherosclerosis and other
cardiovascular disorders, in infectious diseases, in renal and liver
dysfunction, acute
respiratory distress syndrome CARDS), in ischemic and reperfusion injury and
multiple
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organ failure. The long list of diseases wherein IL-1 [3 was found implicated
supports its
central role as a pivotal pro-inflammatory mediator.
IL-2
The induction of the IL-2 gene is a key event in T cell activation that is
required for
the resting cells to become effector cells. Following ligand binding to the T
cell receptor
together with the engagement of a co-stimulatory receptor such as CD28, a
cascade of
cytoplasmic signaling molecules leads to the assembly of several transcription
factors at
their corresponding sites on the IL-2 promoter region in the nucleus. Each one
of these
transcription factors, including AP-1, NF-oB and NEAT, is regulated by
different signaling
pathways, which act in concert to elicit full activation of the IL-2 gene.
Similar mode of
IL-2 gene regulation is obtained by T cell activation with phorbol myristate
acetate (PMA)
and calcium ionophore. hnmunosuppressive drugs such as cyclosporin A strongly
inhibit
the transcription of the IL-2 gene and their contribution to the therapeutic
arsenal is well
known. Compounds able to inhibit the production of the cytokine IL-2 are
potential
immunosuppressive drugs important in the treatment of disorders where T cell
activation
plays a pivotal role, in particular inflammatory conditions with an etiology
including an
autoimmune component such as arthritis, rheumatic diseases, systemic lupus
erythematosus, myasthenia gravis, inflammatory bowel disease, chronic liver
disease, heart
failure, multiple sclerosis, inflammatory demyelinating neuropathies,
psoriasis, diabetes
type 1, parasitic infections, uveitis and other ocular inflammatory
conditions, Sjogren's
syndrome, glomerulonephritis and transplant rej ection.
IL-6
Interleukin-6 (IL-6) is critical to the regulation of the immune and
haematopoietic
systems. The pleiotropic nature of this cytokine family has resulted in a
variety of
pseudonames based on its multiple biological functions. IL-6 elicits B cells
to undergo
proliferation and differentiation into antibody-forming cells; assists in IL-4
dependant IgE
synthesis and T cell activation, growth and differentiation. IL-6 also acts in
conjunction
with IL-3 to induce the proliferation of pluripotent haematopoietic
progenitors. In addition,
this cytokine induces the expression of acute phase proteins. IL-6 is secreted
by T cells, B
cells, mast cells, monocytes, macrophages, hepatocytes, fibroblasts,
endothelial cells,
keratinocytes and many tumor cell lines. Adipocytes, bone marrow stroma cells,
mesangial
cells and some cell types of the central nervous system also produce this
cytokine. IL-6
production is generally correlated with cell activation. IL-6 has been
described as both a
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pro-inflammatory and anti-inflammatory mediator, and its levels are altered in
a number of
diseases: in inflammatory and autoimmune diseases, such as RA and other forms
of
arthritis, SLE, ulcerative colitis, Crohn's disease, pancreatitis, diabetes,
MS, psoriasis; in
infectious diseases, such as HIV, bacterial infections and sepsis, viral and
bacterial
meningitis; in oncologic disorders such as metastatic melanoma, cervical
cancer, myeloid
leukemia, multiple myeloma, Hodgkin's disease, metastatic renal cell
carcinoma, prostate
tumors; in renal insufficiency and dialysis, such as glomerulonephritis,
nephropaties and
hemodialysis; in liver diseases, such as chronic liver diseases, alcoholic
liver cirrhosis,
hepatitis and hepatectomies; in kidney, bone marrow or liver transplantation;
in AD, burns
victims and patients suffering from myocardial infarct. IL-6 is considered to
be an early
marker of injury severity following trauma. The nature of IL-6 action depends
upon time
and site of expression, which ultimately influence if this cytokine will act
as pro- or anti-
inflammatory. However, recent studies suggest that IL-6 should be considered
as a
protective cytokine in the overall balance of cytokine regulation. Proper
regulation of this
important cytokine will have clear beneficial therapeutic impact.
IL-10
Interleukin-10 (IL-10) down-regulates the production of pro-inflammatory
cytokines
and chemokines by activated macrophages, monocytes, polymorphonuclear
leukocytes and
eosinophils. It has been recently suggested that part of this down-regulation
is achieved by
elevation in the levels of SOCS molecules (Suppressors Of Cytokine Signaling).
Therefore
IL-10 is an anti-inflammatory cytokine that plays a role in suppressing immune
and
inflammatory responses. There is evidence that IL-10 can control both T helper
1 (Thl)
type of responses and also Th2 mediated inflammatory processes. IL-10 has a
beneficial
effect on a variety of acute and chronic inflammatory and autoimmune events
including
but not limited to rheumatoid arthritis, ischemia-reperfusion injury,
atherosclerosis,
psoriasis, pemphigus, allergic contact sensitivity reactions, uveitis, organ
transplantation,
injury, infection and sepsis, inflammatory bowel disease, acute pancreatitis,
asthma,
nephrotoxic nephritis and certain malignancies.
iNOS
Nitric oxide (NO) is a short lived molecule required for many physiological
functions in host defence, inflammation and immunity. NO is synthesized by the
enzyme
nitric oxide synthase (NOS) and overproduced during various pathological
inflammatory
states. Three distinct isoforms of NOS have been identified: neuronal (nNOS),
entothelial
CA 02479676 2004-09-17
WO 03/077832 PCT/IL03/00223
(eNOS) and inducible (iNOS). Overproduction of NO by nNOS and iNOS have been
reported in a number of clinical disorders including acute and chronic
neurodegenerative
disorders, convulsions, pain, septic shock, asthma, tissue damage following
inflammation,
Crohn's disease, SLE, osteoarthritis, rheumatoid arthritis, allograft
rejection and in certain
cancers. iNOS is induced by endotoxin or pro-inflammatory cytokines and in
turn
modulate expression of the latter. Selective inhibition of nNOS or iNOS is
expected to
provide a novel therapeutic approach to various diseases.
MCP-1
Monocyte chemoattractant protein-1 (MCP-1) is a pro-inflammatory chemokine of
the C-C family, responsible for the recruitment and activation of mainly
monocytes,
macrophages, basophils, mast cells, T cells, and natural killer cells. The
activated
monocytes, which are recruited to the site of injury, secrete in turn
inflammatory agents
such as TNF-a, IL-1 [3, nitric oxide and prostaglandins. MCP-1 can be involved
in
beneficial processes such as wound healing, but when expressed in excess it
becomes
involved in the pathophysiology of a large number of inflammatory and
autoimmune
diseases. MCP-1 has been implicated in a large number of diseases that affect
various
organs by means of acute or chronic inflammation. Pathological sites of action
of MCP-1
include the skeleton with disorders such as rheumatoid arthritis and various
types of
osseous inflammation, the kidneys with nephrites, nephritic syndromes and
nephrosis
characterized by glomerular nephritides, the eyes with uveitis, vitreoretinal
disorders,
proliferative diabetic retinopathy, and other ocular inflammatory conditions,
the
cardiovascular system with MCP-1 involvement in the early stages of
atherosclerosis, in
restenosis and in the inflammatory response following myocardial infarction,
the
respiratory system with alveolitis, asthma and lung fibrosis or allergic
inflammation, the
digestive tract with inflammatory bowel diseases (IBD) including both
ulcerative colitis
and Crohn's disease. The nervous system is also an important target where MCP-
1 up-
regulation has been observed in various types of pathology. In the central
nervous system,
increases in MCP-1 level have been observed following both head trauma and
brain
ischemia as in stroke, and in immune mediated inflammation as seen in
experimental
autoimmune encephalomyelitis (EAE) or multiple sclerosis (MS), and in the
inflammatory
phenomenon associated with neurodegenerative disorders such as Alzheimer's
disease
(AD). Likewise, MCP-1 is also involved in the development of peripheral
nervous system
(PNS) disorders characterized by mononuclear cell infiltration and related
demyelinating
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disorders. Moreover MCP-1 is involved in vasculitis, angiogenesis, tumor
growth and
metastasis, graft rejection, certain types of bacterial, parasitic or viral
infections, psoriasis,
pemphigus and related disorders, delayed type hypersensitivity reactions of
the skin,
Hodgkin's disease and a number of chronic diseases characterized by a
significant infiltrate
of monocytes, including sarcoidosis, Wegener's granulomatosis and
tuberculosis. MCP-1
is also very important in cases where complications occur following surgical
interventions
such as, for example, angioplasty, atherectomy, circulatory recovery
techniques,
transplants, organ replacement, tissue replacement and prosthetic implants.
Thus, blocking
MCP-1 production and therefore leukocyte recruitment to target tissues in
inflammatory
and autoimmune disease would be a highly effective intervention.
SOCS
Cytokines exert their biological activity through binding to specific cell
surface
receptors, that initiate the appropriate intracellular signal transduction
cascade which in
turn lead to the physiological outcome. Despite their diverse biological
roles, many
common themes have emerged in cytokine signal transduction. Upon receptor
multimerisation, the Janus kinases (JAKs) are activated and phosphorylate,
among other
proteins, the signal transducers and activators of transcription (STATs).
Dimers of
phosphorylated STATs then move into the nucleus where they bind to recognition
sequence in target genes to increase transcription. Such signaling pathway
needs to be also
negatively regulated to ensure the timely switch off of the biological
response. There are at
least three families of proteins that inhibit JAI~ISTAT signaling and the
suppressors of
cytokine signaling (SOCS) belong to one of them. SOCS proteins are an
important element
in a classic negative feedback loop that regulates JAK/STAT signal
transduction initiated
by many cytokines. Following cytokine activation the cell will not only
display increased
transcription of the genes important in mediating the biological effects of
the cytokine but
also genes encoding the SOCS protein which limit the biological effect of the
cytokine. At
least seven SOCS proteins were identified till now and their activity is
presently being
unraveled. SOCS-1 and SOCS-3 are induced by about the same set of cytokines,
including
IL-6, Growth Hormone (GH), IL-10 and GM-CSF, though with different efficiency.
In turn
SOCS-1 inhibits IL-6, GH signaling, IL-2, IL-4, Interferons (IFN) a,, (3 and
y, LIF,
oncostatin M and trombopoeitin. SOCS-3 down-regulates leptin, GH, IFN-a, IFN-
[3 and
IFN-y. Thus, induction of SOCS proteins by one cytokine also attenuates the
response for
additional cytokines different from the original inducer, by a phenomenon of
cross
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WO 03/077832 PCT/IL03/00223
inhibition. These proteins are potentially important regulators of
inflammatory and
immune responses of hematopoiesis and hormone response.
TNF-a
Tumor necrosis factor (TNF) has been attributed' a central role in
inflammatory
processes. In all disorders with an inflammatory component the connnon process
is that the
initial injury, whether initiated by an infectious, chemical, or environmental
agent,
produces focal tissue necrosis in the target organ. As a result of this
damage, tissue-fixed
macrophages and circulating monocytes migrate to the damaged site, become
activated and
secrete products that cause additional cell damage or induction of
inflammatory products
thus amplifying the response. TNF-a is a cytokine produced primarily by
monocytes and
macrophages. Beside its physiologic effect, TNF-a is cytotoxic and regulates
inflammatory processes through induction for instance of IL-1, IL-6, IL-8,
macrophage
inflammatory protein (MIP)-2, granulocyte-macrophage colony stimulating factor
(GM-
CSF) and adhesion molecules. At elevated levels TNF-a is involved in septic
shock
syndrome, autoimmune and inflammatory processes including Crohn's disease,
brain
injury, venous thromboses, arteriosclerosis, vasculitis, IBD, MS, EAE, SLE,
AD, PD,
AIDS dementia, contact dermatitis, mixed connective tissue disease, arthritis,
organ
specific toxic response, hepatic injury during sepsis and reperfusion, chronic
inflammatory
lung diseases, muscle wasting and cachexia. The therapeutic importance of TNF-
a
blockade is tremendous, nevertheless it should be kept in mind that TNF-a is
also involved
in normal physiological and repair processes and totally eliminating this
cytokine would be
detrimental.
By virtue of their anti-inflammatory and immunomodulatory properties,
resulting
from the gene regulation of the pro- and anti-inflammatory mediators selected
COX-2, IL-
1[3, IL-2, iNOS, TNF-a, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, it will be
recognized
that the compositions according to the present invention will be useful for
treating
indications having an inflammatory or autoimmune mechanism involved in their
etiology
or pathogenesis. Such diseases or disorders are exemplified by multiple
sclerosis,
amyotrophic lateral sclerosis, systemic lupus erythematosis, myasthenia
gravis, Sjogren's
syndrome, diabetes mellitus type I, late onset diabetes type 2, sarcoidosis;
skeletal and
connective tissue disorders including arthritis, rheumatoid arthritis,
osteoarthritis and
rheumatoid diseases; ocular inflammation related disorders including uveitis,
vitreoretinal
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WO 03/077832 PCT/IL03/00223
disorders, proliferative diabetic retinopathy, allergic conjunctivitis;
metabolic diseases that
involve abnormalities in lipid peroxisomes and lipid peroxidation and/or
oxidative stress;
skin related disorders including psoriasis, pemphigus and related syndromes,
delayed-type
hypersensitivity and contact dermatitis; respiratory diseases including cystic
fibrosis,
chronic bronchitis, emphysema, chronic obstructive pulmonary disease, asthma,
allergic
rlunitis or lung inflammation, idiopathic lung fibrosis, tuberculosis, and
alveolitis; kidney
diseases including autoimmune nephritis, renal ischemia, nephrites, nephritic
syndromes
and nephrosis characterized by glomerular nephritides; liver diseases both
acute and
chronic such as autoimmune hepatitis, cirrhosis, hepatitis and fulininant
hepatic failure;
gastrointestinal diseases including inflammatory bowel diseases, ulcerative
colitis, Crohn's
disease and gastritis, polyposis and cancer of the bowel, especially the
colon; infectious
diseases generated by certain bacterial, viral and parasitic invasion and
sepsis that might
result from injury; and post-operative complications following angioplasty,
circulatory
recovery techniques, prosthetic implants and tissue or organ transplants,
including graft
rej ection.
When the site of action is the central or the peripheral nervous system, the
pharmaceutical compositions comprising as an active ingredient a compound
regulating the
gene expression of the pro- and anti-inflammatory mediators selected from COX-
2, IL-1 (3,
IL-2, iNOS, TNF-a,, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, act as
neuroprotectors. By
virtue of their neuroprotective properties, it will be recognized that the
compositions
according to the present invention will be useful in treating acute
neurological disorders,
resulting either from ischemic or traumatic damage, including but not limited
to stroke,
head trauma and spinal cord injury. The composition of the present invention
may also be
effective in treating demyelinating disorders and certain chronic degenerative
diseases that
are characterized by gradual selective neuronal loss such as Parkinson's
disease,
Alzheimer's disease, AIDS dementia, Huntington's chorea, amyotrophic lateral
sclerosis,
Kennedy's syndrome, motor neuron disease and prion-associated
neurodegeneration.
By virtue of their analgesic properties it will be recognized that the
compositions
according to the present invention will be useful in treating pain including
peripheral,
visceral, neuropathic, inflammatory and referred pain.
The compositions of the present invention may also be effective in
cardiovascular
protection and/or treatment of atheroma, atherosclerosis, and consequences
thereof,
restenosis, angioplasty, myocardial ischemia and myocardial infarction.
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Another feature of the present invention is the ability of the disclosed
compounds in
chemoprevention in oncological processes including polyps, tumor growth,
angiogenesis
and metastasis of certain types of cancer, including breast and colon cancer.
The pharmaceutical compositions of the present invention, which down-regulate
gene expression of at least one the pro-inflammatory mediators COX-2, IL-1 (3,
IL-2, iNOS,
TNF-a,, and MCP-1, comprise as an active ingredient a compound of the general
formula
(I):
Formula I
6
R.
R3
having the (3S,4S) configuration and being essentially free of the (3R,4R)
enantiomer,
wherein the dashed line indicates an optional C1-C2 or C6-C1 double bond, and
wherein:
Rl is selected from the group consisting of
a) R' where R' is selected from the group consisting of
A) a linear or branched, saturated or unsaturated, carbon side chain
comprising 1-8
carbon atoms optionally interrupted by 1-3 heteroatoms, and
B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one
or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally
interrupted by 1-4 heteroatoms, and optionally further substituted with one or
more groups selected from
i) a linear, branched saturated or unsaturated
or cyclic, C1-C6 alkyl,
ii) a linear, branched saturated or unsaturated
or cyclic, C1-C6 alkoxy,
iii)a linear, branched saturated or mlsaturated
or cyclic, Cl-C6 alkylthio,
iv) a halogen,
v) carboxyl,
vi) -C02-C1-C4 alkyl, wherein the alkyl can be linear, branched or
cyclic, saturated or unsaturated,
CA 02479676 2004-09-17
WO 03/077832 PCT/IL03/00223
vii) keto,
viii) nitro,
ix) a saturated or unsaturated cyclic moiety, an aromatic or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms
comprising one or two-ringed structures, wherein each ring
comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms,
and optionally further substituted with one or more groups selected
from i)-viii) as defined above,
b) an amine or an amide substituted with at least one substituent as defined
in R'
above,
c) a tluol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide
optionally
substituted with one substituent as defined in R' above, and
d) a hydroxyl or an ether -OR' wherein R' is as defined above;
Ra is selected from the group consisting of
a) a halogen,
b) a linear, branched or cyclic, saturated or unsaturated Cl-C6 alkyl, and
c) -OR wherein R is selected from the group consisting of
A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or
unsaturated C1-C6 alkyl optionally containing a terminal -OR"' or -OC(O)R"'
~ moiety wherein R"' is hydrogen or a linear, branched or cyclic, saturated or
unsaturated C1-C6 alkyl, and
B) -C(O)R"' wherein R"' is as previously defined; and
R3 is selected from the group consisting of
a) a linear, branched or cyclic, saturated or unsaturated Cl-Clz alkyl,
b) -ORa, in which Ra is a linear, branched or cyclic, saturated or unsaturated
Cz-C9
alkyl which may be substituted at the terminal carbon atom by a phenyl group,
and
c) a linear, branched or cyclic, saturated or unsaturated C1-C7 alkyl-OR"'
wherein R"'
is as previously defined;
and pharmaceutically acceptable salts, esters or solvates thereof.
The pharmaceutical compositions of the present invention, which up-regulate
gene
expression of at least one of the anti-inflammatory cytokine IL-10, the
protective cytokine
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WO 03/077832 PCT/IL03/00223
IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3, comprise
as an
active ingredient a compound of the general formula (I):
Formula I
6
having the (3S,4S) configuration and being essentially free of the (3R,4R)
enantiomer,
wherein the dashed line indicates an optional C1-C2 or C6-Cl double bond, and
wherein:
Rl is selected from the group consisting of
a) R' where R' is selected from the group consisting of
A) a linear or branched, saturated or unsaturated, carbon side chain
comprising 1-8
carbon atoms optionally interrupted by 1-3 heteroatoms, and
B) a saturated or unsaturated cyclic moiety, an aromatic moiety or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms comprising one
or two-ringed structures, wherein each ring comprises 3-8 carbons, optionally
interrupted by 1-4 heteroatoms, and optionally further substituted with one or
more groups selected from ,
i) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkyl,
ii) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkoxy,
iii) a linear, branched or cyclic, saturated or unsaturated CI-C6 alkylthio,
iv) a halogen,
v) carboxyl,
vi) -C02-C1-C4 alkyl, wherein the alkyl can be linear, branched or
cyclic, saturated or unsaturated,
vii) keto,
viii) vitro,
ix) a saturated or unsaturated cyclic moiety, an aromatic or a
heterocyclic moiety; the cyclic moiety having from 5-20 atoms
comprising one or two-ringed structures, wherein each ring
22
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WO 03/077832 PCT/IL03/00223
comprises 3-8 carbons, optionally interrupted by 1-4 heteroatoms,
and optionally further substituted with one or more groups selected
from i)-viii) as defined above,
b) an amine or an amide substituted with at least one substituent as defined
in R'
above,
c) a thiol, a sulfide, a sulfoxide, a sulfone, a thioester or a thioamide
optionally
substituted with one substituent as defined in R' above, and
d) a hydroxyl or an ether -OR' wherein R' is as defined above;
R2 is selected from the group consisting of
a) a halogen,
b) a linear, branched or cyclic, saturated or unsaturated C1-C6 alkyl, and
c) -OR wherein R is selected from the group consisting of
A) -R", wherein R" is hydrogen or a linear, branched or cyclic, saturated or
unsaturated C1-C6 alkyl optionally containing a terminal -OR"' or -OC(O)R"'
moiety wherein R"' is hydrogen or a linear, branched or cyclic, saturated or
unsaturated Cl-C6 alkyl, and
B) -C(O)R"' wherein R"' is as previously defined; and
R3 is selected from the group consisting of
a) a linear, branched or cyclic, saturated or unsaturated C1-Cla alkyl,
b) -ORa, in which Ra is a linear, branched or cyclic, saturated or unsaturated
CZ-C9
allcyl which may be substituted at the terminal carbon atom by a phenyl group,
and
c) a linear, branched or cyclic, saturated or unsaturated C1-C7 alkyl-OR"'
wherein R"'
is as previously defined;
and pharmaceutically acceptable salts, esters or solvates thereof.
Currently more preferred compounds are those wherein R2 is hydroxy or lower
acyloxy and wherein R3 is dimethylheptyl or a dimethylalkyl radical with a
total of at
least 7 carbon atoms.
According to currently preferred embodiments of the present invention Rl is a
heterocyclic moiety selected from the group consisting of an imidazolyl, an
imidazolinyl, a
morpholino, a piperidyl, a piperazinyl, a pyrazolyl, a pyrrolyl, a
pyrrolidinyl, a tria,zolyl,
and a tetrazolyl, wherein each cyclic moiety may optionally be further
substituted
with at least one substituent selected from the group consisting of C1_6
alkyl, C1_s
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WO 03/077832 PCT/IL03/00223
alkyloxy, C1_6 alkylthio, keto, carboxy, vitro, saturated or unsaturated
cyclic
moieties or aromatic or heterocyclic moieties wherein each ring comprises 3-8
carbons optionally interrupted by 1-4 heteroatoms, said heteroatoms each
independently selected from the group consisting of N, O, and S, wherein each
ring
optionally is further substituted with one or more groups selected from the
group
consisting of C1_6 alkyl, C1_6 alkyloxy, C1_6 alkylthio, keto, carboxy, or
vitro,
wherein C1_6 alkyl, C1_6 alkoxy and C1_6 alkylthio are intended to include
saturated
and unsaturated linear, branched and cyclic structures.
According to more preferred embodiments of the present invention Rl is
selected
from the group consisting of hydroxyl, imidazole, pyrazole, oxazole,
isoxazole,
tetrahydropyridine, pyrazoline, oxazoline, pyrrolidine, imidazoline, 2-thio-
imidazole, 2-
methylthio-imidazoline, 4-methyl-2-imidazoline, 4,4-dimethyl-2-imidazoline,
methyl
sulfide, methylsulfoxide, acetamido, benzamide, cyano, 1,2,4-triazole, 1,3,4-
triazole,
1,2,3,4-tetrazole, 1,2,3,5-tetrazole, thiophene, phenyl, morpholine,
thiomorpholine,
thiazolidine, glycerol, piperazine, 4-piperidinopiperidine, 4-methylpiperidine
and
tetrahydropyran.
According to additional more preferred embodiments of the present invention Rl
is
selected from the group consisting of mono or di-substituted amines wherein
the
substituent is selected from the group consisting of an C1_6 alkyl, C1_6
alkyloxy, C1_6
alkylthio, imidazolyl, an imidazolinyl, a morpholino, a piperidyl, a
piperazinyl, a
pyrazolyl, a pyrrolyl, a pyrrolidinyl, a triazolyl, and a tetrazolyl, wherein
each cyclic
moiety may optionally be further substituted with at least one substituent
selected from the
group consisting of C1_6 alkyl, C1_6 alkyloxy, Ci_6 alkylthio, keto, carboxy,
vitro, saturated
or unsaturated cyclic moieties or aromatic or heterocyclic moieties wherein
each ring
comprises 3-8 carbons optionally interrupted by 1-4 heteroatoms, said
heteroatoms each
independently selected from the group consisting of N, O, and S, wherein each
ring
optionally is further substituted with one or more groups selected from the
group
consisting of C1_6 alkyl, C1_6 alkyloxy, C1_6 alkylthio, keto, carboxy, or
vitro, wherein Cl_6
alkyl, C1_6 alkoxy and C1_6 alkylthio are intended to include saturated and
unsaturated
linear, branched and cyclic structures.
According to another currently preferred embodiment, we disclose a
pharmaceutical
composition which down-regulates gene expression of at least one the pro-
inflammatory
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WO 03/077832 PCT/IL03/00223
mediators COX-2, IL-1 (3, IL-2, iNOS, TNF-a and MCP-l, and up-regulates gene
expression of at least one of the anti-inflannnatory cytokine IL-10, the
protective cytokine
IL-6 and of the suppressors of cytokine signaling SOCS-1 and SOCS-3,
comprising as an
active ingredient a compound of the general formula (I) wherein RZ is OH, R3
is 1,1-
dimethylheptyl, there is a double bond between C6 and C1, and Rl is selected
from the
group consisting of hydroxyl, 2-mercaptoimidazole, imidazole, pyrazole, 4-
methyl-
piperidine, and 4-piperidino-piperidine.
Specific pharmaceutical compositions of particular interest comprise as an
active
ingredient compounds within formula (I) previously disclosed as HU-211, also
known as
dexanabinol, PRS-211,092, PRS-211,095, PRS-211,220, PRS-211,251 and PRS-
211,257
in International Patent application WO 01/98289.
The novel compositions contain in addition to the active ingredient
conventional
pharmaceutically acceptable Garners, diluents and excipients necessary to
produce a
physiologically acceptable and stable formulation. Some compounds of the
present
invention are characteristically hydrophobic and practically insoluble in
water with high
lipophilicity, as expressed by their high octanol/water partition coefficient
expressed as log
P values, and formulation strategies to prepare acceptable dosage forms will
be applied.
Enabling therapeutically effective and convenient administration of the
compounds of the
present invention is an integral part of this invention.
For water soluble compounds standard formulations will be utilized. Solid
compositions for oral administration such as tablets, pills, capsules,
softgels or the like may
be prepared by mixing the active ingredient with conventional,
pharmaceutically
acceptable ingredients such as corn starch, lactose, sucrose, mannitol,
sorbitol, talc,
polyvinylpyrrolidone, polyethyleneglycol, cyclodextrins, dextrans, glycerol,
polyglycolized glycerides, tocopheryl polyethyleneglycol succinate, sodium
lauryl sulfate,
polyethoxylated castor oils, non-ionic surfactants, stearic acid, magnesium
stearate,
dicalcium phosphate and gums as pharmaceutically acceptable diluents. The
tablets or pills
can be coated or otherwise compounded with pharmaceutically acceptable
materials known
in the art, such as microcrystalline cellulose and cellulose derivatives such
as
hydroxypropylmethylcellulose (HPMC), to provide a dosage form affording
prolonged
action or sustained release. Other solid compositions can be prepared as
suppositories, for
rectal administration. Liquid forms may be prepared for oral administration or
for
injection, the term including but not limited to subcutaneous, transdermal,
intravenous,
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intrathecal, intralesional, adjacent to or into tumors, and other parenteral
routes of
administration. The liquid compositions include aqueous solutions, with or
without organic
cosolvents, aqueous or oil suspensions including but not limited to
cyclodextrins as
suspending agent, flavored emulsions with edible oils, triglycerides and
phospholipids, as
well as elixirs and similar pharmaceutical vehicles. In addition, the
compositions of the
present invention may be formed as aerosols, for intranasal and like
administration.
Topical pharmaceutical compositions of the present invention may be formulated
as
solution, lotion, gel, cream, ointment, emulsion or adhesive film with
pharmaceutically
acceptable excipients including but not limited to propylene glycol,
phospholipids,
monoglycerides, diglycerides, triglycerides, polysorbates, surfactants,
hydrogels,
petrolatum or other such excipients as are known in the art.
Prior to their use as medicaments, the pharmaceutical compositions will
generally be
formulated in unit dosage. The active dose for humans is generally in the
range of from
0.05 mg to about 50 mg per kg body weight, in a regimen of 1-4 times a day.
The preferred
range of dosage is from 0.1 mg to about 20 mg per kg body weight. However, it
is evident
to the man skilled in the art that dosages would be determined by the
attending physician,
according to the disease to be treated, its severity, the method and frequency
of
administration, the patient's age, weight, gender and medical condition,
contraindications
and the like. The dosage will generally be lower if the compounds are
administered locally
rather than systematically, and for prevention or chronic treatment rather
than for acute
therapy.
A further aspect of the present invention provides a method of preventing,
alleviating
or treating a patient by regulating pro- and anti-inflammatory mediators
selected from
COX-2, IL-1[3, IL-2, iNOS, TNF-a,, MCP-1, IL-10, IL-6, SOCS-1 and SOCS-3, by
administering to said patient a therapeutically effective amount of
pharmaceutical
composition containing as an active ingredient a compound of general formula
(I) as
previously defined.
A further aspect of the present invention relates to the use for the
manufacture of a
medicament for preventing, alleviating or treating a disease by regulating pro-
and anti-
inflammatory mediators selected from COX-2, IL-1 (3, IL-2, iNOS, TNF-a,, MCP-
1, IL-10,
IL-6, SOCS-1 and SOCS-3, of a compound of general formula (I) substantially as
shown in
the specification.
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The principles of the present invention will be more fully understood in the
following
examples, which are to be construed in a non-limitative manner.
PHYSIOLOGICAL EXAMPLES
Unless otherwise indicated the test compounds are prepared as follows: for in
vitro
assays the compounds are first dissolved in DMSO and then stepwise diluted in
the assay
buffer, generally tissue culture medium, down to a final concentration of 0.1%
DMSO. For
in vivo assays the test compounds are first diluted in cremophor:ethanol (70%
and 30%
w/w respectively) and further diluted 1:20 in physiological buffer, generally
saline, to
reach the appropriate dose. Alternatively, compounds can be first disolved in
PEG:ethanol
(1:1) and then diluted in Intralipid. Thus, the vehicle is the original
"solvent" diluted in the
appropriate buffer.
During this study, we concentrated our attention toward genes involved in
inflammatory processes either directly, e.g. cytokines and chemokines, or
indirectly, e.g.
regulators of cytokine signaling pathways or transcription. These genes are
either encoding
pro-inflammatory mediators, such as the cytokines IL-1 (3, IL-2, TNF-oc, the
chemokine
MCP-1 and the enzyme COX-2, or anti-inflammatory mediators such as the anti-
inflammatory cytokine IL-10, the protective cytokine IL-6 or upstream
regulators of
cytokine such as the suppressors of cytokine signaling SOCS-1 and SOCS-3. Gene
expression was assessed by RNA level quantitation and when possible by direct
protein
quantitation. Whatever the experimental system or the gene to be tested the
following
procedure were followed for quantitation.
A- Impact of tricyclic dextrocannabinoids on gene expression.
Example 1.
RNA preparation and real-time RT-PCR.
Total RNA was prepared using SV total RNA isolation system (Promega). The
cells
or tissues were homogenized in lysis buffer. The lysates were transferred to
an RNA
isolation column, treated with DNAse, washed and eluted according to kit
instructions.
RNA concentrations were determined using GeneQuant II (Phamacia-Amersham).
Complementary DNA (cDNA) was synthesized from total RNA using SUPERSCRIPT II
reverse transcriptase (Life Technologies). 2 ~g of total RNA were combined
with an oligo
(dT)15 primer, 0.5 mM dNTP mix, 8 units of reverse transcriptase and other
reaction
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WO 03/077832 PCT/IL03/00223
components up to a final volume of 20 ~,1, according to the kit instructions.
The reaction
mixture was incubated at 42°C for 45 min and inactivated at 70°C
for 15 minutes.
Quantitative real-time RT-PCR includes 1 ~,l of the cDNA, 300 nM of the
appropriate
forward and reverse primers (see below) and 7.5 ~,1 of the reaction mix
containing buffer,
nucleotides, Taq polymerase and syber green (Syber Green master mix, Applied
Biosystems), in a total reaction volume of 15 ~.1. Gene amplification was
obtained using
the GeneAmp 5700 sequence detection system (Applied Biosystems). Amplification
included one stage of 10 minutes at 95°C followed by 40 cycles of a 2-
steps loop: 20
seconds at 95°C, and 1 minute at 60°C. During each annealing
step, the amount of the
amplified product is measured by the fluorescence of the double strand DNA
binding dye,
syber Green. The cycle of threshold (CT), representing the PCR cycle at which
an increase
in fluorescence above a baseline signal can be first detected, is determined
for each
product. A delay of one PCR cycle in the CT is translated into a two-fold
decrease in
starting template molecules and vice versa. The changes in the CT of the
specific gene
product are normalized to the changes in the CT of a reference gene
cyclophilin A or
GAPDH. Results are expressed as fold increase of gene expression in the test
system above
the appropriate control, such as inactivated cell lines or vehicle "treated"
animals. In all
cases, results are also normalized to either one of the reference house-
keeping genes.
Primer sequences used:
Mouse COX-2 forward 5'-TTCCGTTTCTCGTGGTCACTT-3'
Mouse COX-2 reverse S'- AGCGCTGAGGTTTTCCTGAA-3'
Mouse haptoglobin forward 5'-GCTGGGATCCTGAGCTTTGA-3'
Mouse haptoglobin reverse 5'- TTGGCCATGGTTTCCTGAAC-3'
Mouse IL-1 (3 forward 5'-ACACTCCTTAGTCCTCGGCCA-3'
Mouse IL-1 (3 reverse 5'-CCATCAGAGGCAAGGAGGAA-3'
Mouse IL-2 forward 5'-GAAACTCCCCAGGATGCTCAC-3'
Mouse IL-2 reverse 5'-GCCGCAGAGGTCCAAGTTC-3'
Mouse IL-6 forward 5'-AGAAGGAGTGGCTAAGGACCAA-3'
Mouse IL-6 reverse 5'-GGCATAACGCACTAGGTTTGC-3'
Mouse IL-10 forward 5'-GCCCTTTGCTATGGTGTCCTT-3'
Mouse IL-10 reverse 5'-TCCCTGGTTTCTCTTCCCAA-3'
Mouse iNOS forward 5'-TTCACCTCACTGTGGCCGT-3'
Mouse iNOS reverse 5'- GCACTCTCTTGCGGACCATC-3'
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Mouse MCP-1 forward 5'-TCACAGTTGCCGGCTGG-3'
Mouse MCP-1 reverse 5'-TCTTTGGGACACCTGCTGCT-3'
Mouse SAA-3 forward 5'-CAGAAGTTCACGGGACATGGA-3'
Mouse SAA-3 reverse 5'- CCAGCAGGTCGGAAGTGGT-3'
Mouse SOCS-1 forward 5'-GCATCCCTCTTAACCCGGTACT-3'
Mouse SOCS-1 reverse 5'-AATAAGGCGCCCCCACTTA-3'
Mouse SOCS-3 forward 5'-AGGCACTCCCCGGGAGTAC-3'
Mouse SOCS-3 reverse 5'-GGCCACGTTGGAGGAGAGA-3'
Mouse TNF-a forward 5'-AAGGACTCAAATGGGCTTTCC-3'
Mouse TNF-a reverse 5'-CCTCATTCTGAGACAGAGGCAAC-3'
Mouse cyclophilin A forward 5'-TCGCCATTGCCAAGGAGTAG-3'
Mouse cyclophilin A reverse 5'-GGTCACCCCATCAGATGGAA-3'
Mouse GAPDH forward 5'-GGTTGTCTCCTGCGACTTCAA-3'
Mouse GAPDH reverse 5'-GTAGGCCATGAGGTCCACCA-3'
Human COX-2 forward 5'- TCCTGCCTACTGGAAGCCAA-3'
Human COX-2 reverse 5'- AGCCCTTCACGTTATTGCAGAT-3'
Human IL-2 forward 5'- GGGACTTAATCAGCAATATCAACGT-3'
Human IL-2 reverse 5'- TTCTACAATGGTTGCTGTCTCATCT-3'
Human cyclophilin A forward 5'-GCATACGGGTCCTGGCATC-3'
Human cyclophilin A reverse 5'-TGCCATCCAACCACTCAGTCT-3'
Human GAPDH forward 5'-ACCCACTCCTCCACCTTTGA-3'
Human GAPDH reverse 5'-CTGTTGCTGTAGCCAAATTCGT-3'
Example 2.
Quantitation of protein using ELISA.
The technique used to quantify the amount of a given protein in a liquid
sample, either
tissue culture supernatant or body fluid, is based on Enzyme Linked
ImmunoSorbent
Assay (ELISA) methodology. Either commercially available or established in
house, the
assay is based on the capture of the protein of interest by specific
antibodies bound to the
bottom of an ELISA plate well. Unbound material is washed away, the captured
protein is
then exposed to a secondary antibody generally labeled with horseradish
peroxidase (HRP)
or alkaline phosphatase (ALP). Again the unbound material is washed away, the
samples
are then incubated with the appropriate substrate yielding a colorimetric
reaction. The
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reaction is stopped and reading is carried out in a spectrophotometer at the
appropriate
wavelength. Samples are tested at least in duplicate and the appropriate
standard curve,
consisting of serial dilutions of the recombinant target protein, is
incorporated on each
plate. Concentration of the protein in the sample is calculated from the
standard curve.
Example 3.
Quantitation of COX-2 gene expression in LPS activated macrophages and LPS
injected mice brains.
We previously showed that dexanabinol and its analogs reduce the levels of
secreted
PGEZ in LPS activated mouse macrophages (RAW 264.7) cells in vitro (WO
01/98289).
The ICSO for inhibition of PGEz secretion were determined and found to be 10
~.M, 10 p,M,
4 pM, and 8 ~,M, for dexanabinol, PRS-211,092, PRS-211,095 and PRS-211,220,
respectively. For comparison, the ICSO for inhibition of PGE2 secretion for
the known anti-
inflammatory drugs Celecoxib, Rofecoxib and NS-398 were respectively 5 nM, 100
nM
and 100 nM in the same experimental setup. We assumed then that this
phenomenon
observed at the level of PGE2 secretion was due to direct inhibition of COX-2
enzymatic
activity. To check this hypothesis we have now tested in vitro the enzymatic
activity of
recombinant COX-2 on its substrate in the presence of dexanabinol and its
analogs PRS-
211,092, PRS-211,095 and PRS-211,220. The assay reproduces in vitro the true
enzymatic
activity, the test compounds, up to 10 ~.M, were preincubated for 5 minutes
with purified
COX-2, then the natural substrate AA was added and the reaction was allowed to
proceed
for 2 minutes. HCl was used to stop the enzyme catalysis and saturated
stannous chloride
was added to stabilize the PGH2 product into PGF2a. The concentration of PGF2a
was
measured using an Enzyme Immuno Assay (EIA), a standard curve was prepared and
results were derived from this standard curve. Whereas classical COX-2
inhibitors such as
VIOXX~, Indomethacin and NS-398 strongly inhibited COX-2 enzymatic activity in
this
assay, no inhibitory effect was observed for dexanabinol and its analogs.
Therefore, we
decided to test whether dexanabinol and its analogs have an unexpected impact
on COX-2
gene expression and for this purpose we utilized LPS stimulated RAW 264.7
cells using
real-time RT-PCR.
RAW 264.7 macrophages, a mouse cell line (ATCC # TIB 71), were grown in
Dulbecco's modified Eagle's medium (DMEM) with 4 mM L-glutamine adjusted to
contain
1.5 g/L sodium bicarbonate, 4.5 g/L glucose, and 10% heat inactivated fetal
bovine serum.
Cells were grown in tissue culture flasks and seeded at appropriate density
into 24 wells
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tissue culture plates. 0.5 x 106 Raw cells in one milliliter were stimulated
with 2 ~g/ml
Lipopolysaccharide E.coli OSS:BS (DIFCO Laboratories). The mouse macrophages
were
pre-treated for one hour with controls or 10 ~M of dexanabinol and its
analogs, unless
indicated otherwise, and later on activated with LPS. RNA samples were
extracted from
the cells 2.5 hrs after activation and COX-2 gene expression levels were
analyzed by real-
time RT-PCR as previously described.
The results of this experiment are expressed as fold activation of COX-2 over
non-
activated macrophages, after normalization to cyclophilin A expression. When
cells are
treated with vehicle only we observe a maximal 6-fold increase in COX-2
expression. A
decrease of 67% in RNA levels of COX-2 is observed when the activation is
carned out in
presence of 10 ~M dexanabinol. At this concentration all three analogs (PRS-
211,092,
PRS-211,095 and PRS-211,220) cause an inhibition of at least 50%. Together
with the fact
that COX-2 enzyme activity is not directly affected by these compounds, these
results
indicate that PGE2 concentrations, that were originally monitored, were
reduced as a result
of decrease in COX-2 gene expression and not as previously thought by
inhibition of
enzymatic activity. However, at this stage we do not know if COX-2 gene
regulation is
directly achieved by our compounds, or results indirectly from the regulation
of other
genes with secondary impact on COX-2 through feedback mechanisms.
To test if the gene transcription regulatory effect of compounds of the
invention was
of physiological consequence in the whole animal, we injected either PBS or
100 ng LPS
in vivo into mice brains intra cerebral ventricular (i.c.v.). Each treatment
group was
composed of at least five C57/BL male mice (6-8 weeks old, 25 g average body
weight,
Harlan, Israel). The mice were anesthetized with a mixture of 35 mg/kg pental
and 8 mg/kg
xylazine. LPS was dissolved in saline at 20 ng/~.1 and 5 ~1 were injected in
each ventricule
at a rate of 1 ~.1/min with the help of a syringe pump and a brain infusion
canula. After
each injection, the cannula is left iu situ for one more minute to avoid
reflux. The various
treatment groups, controls including the cremophor:ethanol vehicle,
dexanabinol and its
analogs (20 mg/kg), were injected i.p. (0.1 ml/10 g body weight)
simultaneously with the
i.c.v injection of LPS. Six hours following LPS injection, the animals were
sacrificed by
i.p. injection of 100 mg/kg pentobarbitone sodium and their brains were
removed and kept
at -80°C until next step. RNA was extracted from each whole brain and
COX-2 gene
expression levels were analyzed by real-time RT-PCR as previously described.
The results
of this experiment are expressed as fold activation of COX-2 in LPS versus PBS
injected
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brains. LPS injected brains treated with vehicle only show a 5.3 fold increase
in COX-2
RNA level, mice treated with 20 mg/kg dexanabinol display a reduction of 53%
in the
level of COX-2 activation.
From these experimental results, it is evident that the active ingredients of
the present
invention are effective in reducing COX-2 RNA level both in vitro and in vivo.
Consequently, the compounds may be therapeutically effective in the wide
variety of
COX-2 related disorders.
Example 4.
Quantitation of secretion and gene expression in LPS activated macrophages.
To test if COX-2 is the sole gene whose transcription is affected by compounds
of
the invention, additional genes were further analyzed by real-time RT-PCR in
the above-
described model of activated macrophages. This part of the study was performed
with
PRS-211,092 at doses up to 20 p,M. The level of TNF-a and of MCP-1 was
elevated by
19-fold and 55-fold respectively in activated as compared to resting
macrophages, but
PRS-211,092 did not affect the level of expression of any of these genes at
the doses
tested. The levels of iNOS and IL-1(3 were significantly elevated in activated
macrophages
by 266-fold and 1 x,960-fold respectively. PRS-211,092 inhibited gene
expression in a dose
dependent manner with an ICSO of 4 ~M for iNOS and of 17.5 ~M for IL-1 (3. In
this
experiment COX-2 gene expression was elevated 70-fold by activation and under
these
conditions the dose related effect of PRS-211,092 yielded an ICSO of 21.5 ~,M.
Moreover, it was confirmed that the reduction in IL-1 (3 gene expression
correlates to
a reduction in IL-1(3 secretion. The ICso of dexanabinol, PRS-211,092 and PRS-
211,220
for inhibition of IL-1 [3 secretion are 6 ~M, 3 ~,M and 4 ~,M, respectively.
PRS-211,095
was tested at the single concentration of 10 ~.M and found to inhibit IL-1(3
secretion by
70%, similarly to PRS-211,092 and PRS-211,220 that both yielded 76% inhibition
at that
same concentration.
From these experimental results, it is evident that the active ingredients of
the present
invention are effective not only in affecting COX-2 RNA level but also in
specifically
reducing RNA levels of additional inflammatory related genes such as IL-1 ~i
and iNOS. As
expected the impact on gene expression correlates with a decrease in secretion
of the
relevant inflammatory mediators. The fact that COX-2 is not the sole gene
whose
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WO 03/077832 PCT/IL03/00223
transcription is affected provide a significant advantage to compound of the
invention over
classical anti-inflammatory therapies which are designed to block one mediator
at a time.
A compound that can reduce the expression and secretion of multiple
inflammatory
mediators simultaneously harbors in a single molecule the approach of combined
therapy
now used to replace the single target classical anti-inflammatory strategies.
Example 5.
Quantitation of IL-1(3 gene expression and activated microglia in LPS injected
mice
brains.
The local i.c.v. injection of LPS in mice brain stimulates a wide range of
inflammatory responses and cascades where various cytokines and chemokines are
implicated. Following the results obtained in examples 3 and 4, we decided to
assess the
effect of additional active compounds of the invention on other genes involved
in the
inflammatory response. Specifically, we tested the impact of dexanabinol and
PRS-
211,092, both at 20 mg/kg, on the expression of pro-inflammatory IL-1(3
cytokine. The
protocol was basically as previously described with minor modifications. The
amount of
LPS injected in the mice brain was raised to 250 ng and the animals were
sacrificed 24
hours after LPS and treatment injection for mRNA analysis, carried out as
previously
described. Moreover, some animals were kept alive and sacrificed 72 hours
after LPS and
treatment inj ection for immunohistochemical analysis. For this purpose, after
euthanizing
the animals by injecting i.p. 100 mg/lcg of sodium pentobarbitone, brains were
removed
and fixed in 4% formaldehyde for at least 72 hours. Brains were then washed
with PBS
and transferred for cryoprotection in a solution of 30% sucrose in PBS, until
they sank.
After the brains sank in the sucrose they were frozen using the cryostat
special fast-
freezing technique (-60°C). The brains were cryosectioned (18 Vim) at
the level of the
entire hippocampus. Immunohistochemistry staining was carned out using
polyclonal rat
anti-mouse F4/80 (Serotec, USA) and goat anti-rat-IgG-peroxidase (Jackson,
USA), for the
detection of activated microglia cells. The slides were stained using the 3,3-
diaminobenzidine tetrahydrochloride (DAB) chromagen detection kit of the
automated
immunostaining system (Vantana, France). Quantitative analysis was carned out
by
counting the number of immunoreactive cells/mm2 at the level of the
hippocampus.
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Statistical analysis.
Results are expressed as mean~SD. Data were analyzed using analysis of
variance
(ANOVA) followed by post-hoc Fisher test and t-test. A value of p<0.05 is
considered to
be statistically significant.
Twenty-four hours following LPS injection, we observed a rise in the level of
IL-1(3
mRNA, normalized to cyclophilin. LPS injected animals have 18 fold higher IL-
1(3 mRNA
levels than saline injected animals. Treatment with 20 mg/kg of PRS-211,092,
administered simultaneously with the LPS stimulation, reduced the amount of IL-
1 (3
mRNA fold activation by 56% as compared to its vehicle.
Immunohistochemical analysis for the glial cell marker revealed that i.c.v.
injection
of LPS caused massive gliosis, restricted to the hippocampus. Sham operated
animals
displayed a baseline of zero activated microglia while LPS i.c.v. injected
animals had on
average as many as 488 immunoreactive cells/mm2. Treatment with 20 mg/kg of
PRS-
211,092 significantly reduced the amount of activated microglia by 64%, while
20 mg/kg
of dexanabinol caused a reduction of 40%, both as compared to animals treated
with
vehicle alone.
Altogether, these results show that active compounds of the invention act
through
modulation of inflammatory mediators in in vivo models of neuroinflammation as
well as
in in vitro models of activated cells of the immune system, such as the
macrophages. Their
effect is not only expressed by modulation of gene expression of soluble
inflammatory
mediators, but also by a significant decrease in the amount of activated cells
involved in
the inflammatory process.
Example 6.
Quantitation of gene expression and protein secretion in activated T cells.
IL-2 gene expression.
The induction of the IL-2 gene is the hallmark event of T cell activation that
is
required for the resting cells to become effector cells. Similar mode of IL-2
gene regulation
is obtained by T cell activation with PMA (phorbol-12-myristate-13-acetate)
and calcium
ionophore.
The human acute lymphoma T cell line Jurkat (ATCC # TIB 152) was used to test
the possible immunosuppressive effect of dexanabinol and its analogs on T cell
activation.
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The Jurkat cells were grown in RPMI 1640 medium with 2 mM L-glutamine adjusted
to
contain 1.5 g/L sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM
sodium
pyruvate, and 10% heat inactivated fetal bovine serum. Cells were grown in
tissue culture
flasks and seeded at appropriate density into 24 wells tissue culture plates.
2 x 106 cells in
one milliliter were stimulated using 10 ng/ml of PMA (Sigma) and 1 ~,M A23187
calcium
ionophore (Sigma). Cyclosporin A (Sandoz), a known immunosuppressive drug, was
used
as positive control. The controls and test compounds were added at indicated
concentrations one hour before stimulation. RNA samples were extracted from
the cells 6
hrs after activation and IL-2 gene expression levels were analyzed by real-
time RT-PCR as
previously described.
Figure lA depicts the result of this experiment as fold activation of IL-2
over non-
activated T cells. The results are plotted after normalization to cyclophilin
A expression.
Dexanabinol and its analogs inhibited IL-2 gene expression in a dose related
manner with
maximal inhibitory activity at the highest dose tested, 10 ~,M. At this
concentration
dexanabinol, also known as PRS-211,007, reduced IL-2 fold of activation by
41%, PRS-
211,092 by 69%, PRS-211,095 by 70% and PRS-211,220 by 84% (the latter not
shown).
Cyclosporin A completely blocked IL-2 transcription at 10 nM and inhibited it
to 50% at 1
nlVI.
IL-2 secretion.
In parallel, the supernatant from each well was collected 24 hours after the
activation
and analyzed for the presence of secreted IL-2 by ELISA. The principles of the
assay are
as previously described. The secondary antibodies were conjugated with HRP,
following
substrate addition the peroxidase catalyzed color change is stopped by
acidification. The
absorbance measured at 450 nm is proportional to the concentration of IL-2 in
the sample
or standard. A standard curve is obtained by plotting the concentrations of
recombinant IL-
2 standards versus their absorbances. The IL-2 concentrations in experimental
samples axe
then determined using the standard curve.
Results are shown in Figure 1B, where the amount of secreted IL-2 (ng/ml) is
plotted
for each treatment group. When the activated cells are treated with vehicle
only the
maximal level of IL-2 secretion is 8.73 ng/ml. In correlation with gene
expression
inhibition, dexanabinol and its analogs PRS-211,092, PRS-211,095 and PRS-
211,220
inhibited the secretion of IL-2 in a dose dependent manner at ICSO of 8 ~.M,
0.4 ~,M, 1 ~.M
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WO 03/077832 PCT/IL03/00223
and 2 ~,M respectively. For comparison, the positive control Cyclosporin A
inhibited IL-2
concentrations in the growth medium with an ICso of 0.06 nM in the same
experimental
setup.
Taken together, these results indicate that dexanabinol and its analogs down-
regulate
IL-2, either directly or indirectly, both at the level of its RNA and at the
level of its
secretion, as was shown in activated Jurkat T cells. This implies that
compounds of the
invention may have a beneficial therapeutic impact on T cell mediated
disorders.
COX-2 gene expression.
In addition, we used this experimental system to test the impact of
dexanabinol and
its analogs on COX-2 gene expression. Following activation of Jurkat cells
with PMA and
calcium ionophore we obtained elevated COX-2 gene expression levels. The
impact of the
test compounds on COX-2 was assessed as previously described for IL-2. Figure
2 depicts
the results of this experiment as fold activation of COX-2 over non-activated
T cells. The
results are plotted after normalization to GAPDH expression. We observe that
COX-2 gene
expression was inhibited by dexanabinol, also known as PRS-211,007, and PRS-
211,092,
PRS-211,095, and PRS-211,220, by 88%, 87%, 85% and 92% respectively. The
positive
control cyclosporin A was also very potent in this assay. COX-1 RNA levels
were not
changed throughout the experiment (data not shown) supporting the specificity
of action of
dexanabinol and its analogs.
From these experimental results, we confirm that the active ingredient of the
present
invention is effective in reducing COX-2 RNA level in another in vitro system,
composed
of T cells instead of macrophages. Moreover, these results prove that the
effect of the
compounds of the present invention is not limited to down-regulation at the
level of gene
expression, since the decrease in RNA is correlated to a decrease in protein
secretion, as
shown for IL-2. Consequently, the compounds may be therapeutically effective
in the wide
variety of COX-2 and IL-2 related disorders.
Example 7.
Quantitation of gene expression and protein secretion in activated Mast cells.
Mast cells are multifunctional bone marrow derived cells that upon activation
release
many potent inflammatory mediators. Release is done either from preformed
granules,
trough the process of degranulation, or following stimulation-induced de novo
synthesis.
The molecules released by Mast cells include biogenic amines such as
histamine,
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chemokines, cytokines, enzymes, growth factors, peptides, arachidonic acid
products and
proteoglycans. It should be noted that mast cells are also known to play a key
role in
generating pain signal.
RBL-2H3 cells (ATCC # CRL-2256) are grown in EMEM medium with Earle's
BSS, 2 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM
non
essential amino acids, 1.0 mM sodium pyruvate, and 15% heat inactivated fetal
calf serum.
Cells are grown in tissue culture flasks and seeded at appropriate density
into 24 wells
tissue culture plates. 2 x 105 cells in one milliliter are seeded. Following
overnight
incubation, the plated cells are preincubated for half an hour with test
compounds and
controls and then stimulated with 10 ng/ml of PMA (Sigma) and 1 ~,M A23187
calcium
ionophore (Sigma). The degranulation process is allowed to proceed at
37°C for various
periods of time, depending on the level of analysis and the molecule
monitored. Cells are
collected after half an hour for RNA preparation while supernatants are
collected two and
an half hours after stimulation, for the analysis of hexoaminidase and PGE2
secretion, and
IL-4 and TNF-a, gene expression and secretion. For COX-2 gene expression RNA
is
collected one hour after stimulation. The Src family inhibitor PP2 or the PKC
inhibitor
GF109203X (both from Calbiochem) are used as positive control. The controls
and test
compounds are added at indicated concentrations before stimulation. The
concentrations of
the agent under study are measured in commercially available EIA, ELISA or
enzymatic
assays. Inhibition is calculated versus vehicle treated cells. In parallel,
RNA samples are
extracted from the cells at the appropriate predetermined time points after
activation and
gene expression levels are analyzed by real-time RT-PCR as previously
described wherein
the constitutively expressed COX-1 is used to normalize gene expression.
Example 8.
Quantitation of gene expression in brain tissue following Middle Cerebral
Artery
Occlusion.
Transient MCAo in mice
This model corresponds to cerebral ischemia as observed in stroke. Mice
(C57BL,
male, 25 gr average body weight, Harlan, Israel) were anaesthetized with
halothane in 30%
oxygen and 70% nitrogen (4% for induction in an anesthesia chamber, and 1-2%
in a
facemask for maintenance). A midline incision was made in the skin of the
neck, and the
tissue underneath was bluntly dissected. The right common carotid artery (CCA)
and its
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junction with the external carotid artery (ECA) and internal carotid artery
(ICA) were
explored by blunt dissection. The branches of the ECA, the occipital and the
superior
thyroid artery, were then cauterized. The CCA was then transiently closed by
positioning
around it a 5-0 silk suture material (Assut, Switzerland). Two cm pieces of
the nylon suture
material were cut and placed in a solution of 1% Poly-L-Lysine and then dried
in an oven
(60°C) for 60 minutes. The tip of each piece was rounded under a flame.
The ECA is
permanently occluded with the same type of suture material. A third closure,
transient this
time, was done in the ICA with 5-0 silk suture material. A small hole is cut
in the ECA and
the nylon thread is inserted into the ICA while avoiding entrance into the
pterygopalatine
artery. The thread is inserted 11 mm until a slight resistance is felt. Then a
5-0 silk suture
knot secures the thread. One cm of the thread left outside are then cut. The
skin wound is
closed by 5-0 silk suture material.
Following the operation, the animals were allowed to wake up in the cage. One-
hour
after insult initiation animals were clinically tested to verify the success
of MCA occlusion.
The evaluating system was based on works by Belayev et al., (Stroke 27: 1616-
23, 1996;
Brain Res. 833: 181-90, 1999). It consisted of two tests: the postural reflex
test and the fore
limb-placing test. The postural reflex was evaluated while the animal was
suspended by the
tail, whereas the fore limb-placing test was performed while the animal was
held by the
stomach. Table 1 summarizes the tests and their scoring system.
Table 1: Neurological evaluation of mice with MCAo.
Item Normal Deficit
Score
Postural reflex test (hang test) * 0 2
Placing test (performed on each side) #
Visual placing
Forward 0 2
Sideways 0 2
Tactile placing
Dorsal surface of paw 0 2
Lateral surface of paw 0 2
Proprioceptive placing 0 2
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* Scores are as follows: 0 no observable deficit, 1 limb flexion during
hang test, 2 deficit on lateral push.
# Scores are as follows: 0 complete immediate placing, 1 incomplete or
delayed placing (>2 seconds), 2 absence of placing.
Only animals with total scores between 8 tol2 were included in the study.
Ninety
minutes after initiation of the insult, the selected animals are resedated
using the same
method, the neck wound is then re-opened and the nylon thread is pulled out of
the ICA.
The skin wound is then closed with 5-0 silk suture material. The controls and
test
compounds, (dexanabinol also known as PRS-211,007, PRS-211,092, PRS-211,095,
and
PRS-211,220) are administered 1 minute before the end of the insult. All
treatments are
delivered i.v. 5 mg/kg (except PRS-211,220 0.5 mg/kg). Vehicle is administered
5 ml/kg.
Each treatment group comprised 6 to 8 animals. The drugs were dissolved in PEG-
Ethanol
and diluted in Intralipid (Pharmacia Upjohn). Eighteen hours later, animals
were sacrificed
by i.p. injection of pentobarbitone sodium 100 mg/kg. Brains were then
removed, and total
RNA was prepared from the ipsilateral half of the brains. Gene expression
levels were
analyzed by real-time RT-PCR as previously described. Results are expressed as
fold
activation over sham operated animals. Gene expression was normalized to house-
keeping
gene cyclophilin.
COX-2 gene expression in MCAo brains.
Dexanabinol and its analogs were already shown to be effective in reducing
brain
damage after stroke and improving outcome in the middle artery cerebral
occlusion
(MCAo) model in rats and mice. The purpose of this experiment was to check if
these
functional improvements were achieved by the newly identified mechanism of
action.
Therefore, COX-2 RNA levels were assessed 18 hrs following MCAo. The results
obtained with all genes tested are displayed in Figure 3. Vehicle only treated
animals
displayed a 5-fold activation of COX-2 gene expression versus sham operated
animals.
Treatment with dexanabinol and the PRS-211,092 clearly reduced this outcome by
38%
and 48% respectively, in comparison with the vehicle treated group (Figure
3A).
MCP-1 gene expression in MCAo brains.
Chemokines are low molecular weight, secreted proteins that chemoattractant
and
activate specific subpopulations of leukocytes. Monocyte chemoattractant
protein-1 (MCP-
1) is highly specific for monocytes, which are recruited to the site of
injury, become
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activated and secrete inflammatory. Increased MCP-1 RNA levels following MCAo
were
previously reported (Che et al., Brain Research 902: 171-7, 2001).
Itnrnunohistochemistry
studies showed that both ischemic neurons (after 12 hours of ischemic insult)
and
astrocytes (two days after insult) expressed MCP-1. We tested the effect of
dexanabinol
and PRS-211,092 treatments on MCP-1 RNA levels in mice brains after 18 hours
of
MCAo. Vehicle treated animals displayed a 16 fold increase in MCP-1 gene
expression
versus sham operated animals. Treatment with dexanabinol and PRS-211,092
reduced this
outcome by 41% and 63% respectively, in comparison to the vehicle treated and
the
untreated groups (Figure 3A).
IL-2 gene expression in MCAo brains.
Dexanabinol and its analogs were already shown to be effective in reducing IL-
2
both at the level of gene expression and at the level of secretion in
activated T cells.
Therefore, we wished to verify their impact in vivo and we assessed IL-2 RNA
levels in
mice brains 18 hrs following MCAo. Vehicle only treated animals displayed a 4-
fold
activation of IL-2 gene expression versus sham operated animals. Treatment
with
dexanabinol and the PRS-211,092 clearly reduced this outcome by 177% and 130%
respectively, in comparison with the vehicle treated group (Figure 3A).
IL-10 gene expression in MCAo brains.
IL-10 is a potent anti-inflammatory cytokine strongly related to the
previously
described pro-inflammatory genes. Moreover, it has already been reported that
IL-10 gene
expression levels increase in rat brain following MCAo (Zhai et al, J. Neurol.
Sci. 152:
119-24, 1997) and that IL-10 administration reduces rat brain injury following
focal stroke
(Sperat et al, Neurosci. Lett. 251: 189-92, 1998). We wish to check that this
phenomenon
can be repeated in the mice model of MCAo and that our compounds have a
further
positive impact on the expression of this anti-inflammatory cytokine. We
tested the effect
of dexanabinol and its analogs on IL-10 RNA levels in mice brains after 18
hours of
MCAo. Vehicle treated animals displayed a 35 fold increase in IL-10 gene
expression
versus sham operated animals. Treatment with dexanabinol and PRS-211,092
further
increased this outcome by 4.4-fold and 2.3-fold respectively, in comparison to
the vehicle
treated and the untreated groups (Figure 3B).
Moreover, many more genes encoding for instance cytokines, adhesion molecules,
or
transcription factors, were reported to display abnormal levels of expression
in cerebral
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WO 03/077832 PCT/IL03/00223
ischemia. Out of a total of twenty four such agents screened in this study
only the four
genes previously described were affected by compounds of the invention.
Surprisingly, in
this model IL-1 (3, IL-6 and iNOS gene expression were not modulated by
dexanabinol or
PRS-211,092, at least not at the time point selected for analysis.
From these experimental results, we confirm that the active ingredient of the
present
invention is effective in reducing in vivo the RNA level of 3 important pro-
inflammatory
mediators, COX-2, IL-2 and MCP-l, while it is increasing the RNA level of the
anti-
inflammatory cytokine IL-10. Consequently, the compounds may be
therapeutically
effective in the wide variety of immune/inflammatory related disorders.
Moreover, it
should be noted that compounds of the invention are specific both in terms of
the
inflammatory related genes they regulate and in terms of the cellular targets
or tissues in
which they act.
Example 9.
Quantitation of gene expression in liver and spleen following ConA injection
in mice.
The ConA model for T cell mediated injury.
The most common causes of life threatening T cell mediated liver damage in
humans
are infections with hepatitis B or C viruses and autoimmune hepatitis.
Different animal
models of autoimmune liver injury have been developed, including acute liver
failure in
mice induced by intravenous inj ection of the T cell stimulatory plant lectin
concanavalin A
(ConA). ConA has high affinity for the hepatic sinus. Treatment of mice with
ConA
activates T cells that accumulate in the liver and release cytokines (IL-6, IL-
10, TNF-a,
INF-y, IL-2) that regulate liver damage. Pretreatment with the
immunosuppressor drugs
such as cyclosporin A or FK506 completely prevents liver injury caused by ConA
injection, demonstrating the major role of T cell activation in this model. We
have shown
that human T-lymphocytes (Jurkat cell line), activated by PMA and calcium
ionophore, are
inhibited by dexanabinol and its analogs (PRS-211,092, PRS-211,095 and PRS-
211,220).
Therefore, we decided to test in vivo the potency of these compounds to
regulate T cell
induced cytokine expression levels and reduce liver inflammatory damage using
the ConA
model.
Each experimental group contained at least twenty BALB/c inbred female mice
(25 g
average weight, Harlan, Israel). The negative control group was composed of
mice inj ected
with saline instead of ConA. The injection of ConA (Sigma) was done i.v. at
the base of
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the tail at the dose of 15 mg/kg in saline. The treatments were injected i.v.
at S mg/kg , 30
minutes before ConA injection, unless indicated otherwise. Compounds were
dissolved in
cremophor:ethanol, further diluted with saline before injection, and vehicle
only was
included as an internal control. Analysis was performed at predetermined time
points
following ConA injection.
Impact of treatment was monitored at three levels. First, blood samples (200-
400 ~.l)
were collected at predetermined time points after ConA injection, using retro-
orbital
puncture. After short centrifugation (5000 rpm for 2 min) serum was recovered
and stored
at -80°C until further use for determination of cytokines
concentrations by ELISA and
aminotransferase release from the liver as a marker for liver injury.
In parallel, the concentration of cytokines was also determined in the 'organs
of
interests. For this purpose, the mice were killed by dislocation of the
cervical vertebrae, at
predetermined time points following ConA inj ection. The spleen and the liver
were
removed. Part of the liver was fixed in 4% formaldehyde for histology and the
other part
was kept at -80°C for protein or RNA extraction. The spleens were
weighted and a small
part of the spleen was fixed in 4% formaldehyde, while most of the organ is
cultured
according to the following procedure. Each spleen is squeezed through a cell
strainer with
the rough end of a 5 ml syringe into 4 ml of RPMI medium. Large tissue
fragments are
removed by gravity sedimentation and the supernatants are collected. Cells are
washed 3
times with 5 ml of erythrocyte lysis buffer (Boehringer), resuspended in 4 ml
RPMI
medium supplemented with 2 mM L-glutamine adjusted to contain 1.5 g/L sodium
bicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1.0 mM sodium pyruvate, and 10%
heat
inactivated fetal bovine serum, and plated in a 6 wells culture dish. Cells
are incubated for
24 hours and cytokine concentrations in the supernatant were determined by
ELISA as
previously described.
ALT release from ConA injured liver.
Alanine aminotransferase (ALT) is an enzyme found mainly in the liver and it
is
measured to determine whether the liver is damaged or diseased in a variety of
human
conditions, especially hepatitis and cirrhosis. The level of its release into
the bloodstream
linearly correlates with the severity of the liver injury and thus it can be
used both for
diagnosis purposes and to monitor the efficiency of a treatment. The time
course of plasma
ALT release after ConA injection is known from the literature to peak at eight
hours. At
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this time point the ALT concentration measured was about 1700 units/1 and 5
mg/kg of
PRS-211,092 when administered 30 minutes before ConA injection significantly
reduced
ALT concentrations by 57%. We used this assay to preliminarily assess the
temporal
window of PRS-211,092 efficiency and the compound was administered either 30
or 60
minutes after induction of injury. At both time points PRS-211,092 still
reduced
significantly ALT concentrations by 61% and 51% respectively. FK-506 was used
as
positive control and at the dose of 1 mg/kg, it reduced ALT concentrations by
92%, 93%
and 86% when injected at the three previously described time points. Thus, the
test
compound has an important beneficial impact on liver injury as assessed by the
reduction
of ALT, a robust marker of the liver injury. This therapeutic effect is
maintained even
when the treatment is administered up to at least one hour after injury.
IL-2 gene expression in liver and spleen, and secretion, in the ConA model.
The induction of IL-2 expression in the liver of ConA injected mice and the
effect of
immunosuppressors in such a model have already been reported (Okamoto T. and
Kobayashi T., Jpn. J. Pharmacol. 77: 261-3, 1998). The level of pro-
inflammatory IL-2
expression was assessed in the liver at four time points: 15 minutes, 1, 4 and
8 hours
following ConA injection. The effect of the compound is compared to its
vehicle, which
has no effect of its own on gene expression as assessed by the fact that
vehicle treated
animals yield results similar to saline treated animals (data not shown).
Increase in IL-2
gene expression is detected only from one hour on (Figure 4A). At this time
point, vehicle
treated animals displayed a 428-fold increase in IL-2 RNA levels versus saline
injected
animals. Treatment with 5 mg/kg i.v. of PRS-211,092 reduced this outcome by
37%. When
the analysis is carned out 4 and 8 hours following ConA injection, the
increase in IL-2
expression is correspondingly 817 and 549-fold and the test compound reduce
these effects
by 56% and 53% respectively. The effect of PRS-211,092 is statistically
significant at all
time points from one hour on.
Similarly, the level of IL-2 gene expression was assessed in the spleen of the
ConA
injected mice, one and four hours following injury. In this organ, vehicle
treated animals
displayed at each time point respectively a 309-fold and a 997-fold increase
in IL-2 RNA
levels versus saline injected animals. Treatment with 5 mg/kg i.v. of PRS-
211,092 reduced
this outcome by 46% when tested one hour after injury and by 32% when tested
at four
hours after injury.
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In parallel, the spleen was removed and splenocytes were cultured for 24 hours
to
allow the assessment of secretion levels. Saline injected animals yielded
undetectable
background concentrations of IL-2, ConA injected animals treated with vehicle
only
yielded 1 ng/ml IL-2 while ConA injected animals treated with PRS-211,092
produced
only 0.6 ng/ml, a 40% reduction in secretion from splenocytes. Moreover, the
concentration of IL-2 was assessed in the plasma, PRS-211,092 succeeded to
reduce the
vehicle treated concentrations from 0.88 ng/ml down to 0.52 ng/ml, i.e. a 41%
reduction.
In this in vivo model we show that the reduction in IL-2 RNA levels is
supported and
correlated with a reduction in protein secretion. This was previously shown in
the in vitro
model of activated T cells.
MCP-1 gene expression in liver in the ConA model.
Animals were sacrificed at the four time points following ConA injection
previously
described and the level of pro-inflammatory MCP-1 expression was assessed in
the liver.
Zilcrease in MCP-1 gene expression is detected only from one hour on (40-fold
increase
over saline injected animals), but at this time point treatment with 5 mg/kg
PRS-211,092
has no effect (Figure 4B). Four and eight hours following ConA inj ection, the
increase in
MCP-1 expression is correspondingly 111 and 287-fold and the test compound
reduces
these effects by 29% and 55% respectively. The effect of PRS-211,092 is
statistically
significant at both time points.
TNF-oc gene expression in liver in the ConA model.
TNF-oc, a cytokine produced mainly by activated macrophages, has pleiotropic
effects both beneficial, as in liver regeneration, and deleterious, when it
has direct
cytotoxic role in human hepatocytes. TNF-a, has been shown to be a crucial
factor in
immune mediated hepatitis and is also a mediator of hepatotoxicity in patients
with
alcoholic liver disease, fulminant hepatic failure and viral hepatitis. In
most of these
disorders, the concentration of TNF-a, correlate inversely with patients
survival.
Animals were sacrificed at the four time points previously described and the
level of
pro-inflammatory TNF-a expression was assessed in the liver. Increase in TNF-a
gene
expression is detected immediately, as early as 15 minutes following ConA
injection (21-
fold increase over saline injected animals), and already at this time point
treatment with 5
mg/kg PRS-211,092 has an inhibitory effect of 32%. One, four and eight hours
following
ConA injection, the increase in TNF-a, expression is correspondingly 162, 81
and 38-fold.
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The test compound reduces these effects by 27% and 17%, and then maintains the
same
level of TNF-a expression (Figure 4C).
Moreover, it was confirmed in vitro that the reduction in TNF-a gene
expression
observed in vivo is indeed in correlation with a reduction in TNF-a secretion.
For this
purpose, the previously described experimental system of LPS activated
macrophages was
used. The ICso of dexanabinol, PRS-211,092, PRS-211,095 and PRS-211,220 for
inhibition
of TNF-a secretion are all between 10 to 20 ~M.
IL-1(3 gene expression in liver in the ConA model.
Animals were sacrificed at the four time points previously described and the
level of
pro-inflammatory IL-1 [3 expression was assessed in the liver. Increase in IL-
1 (3 gene
expression is detected immediately, as early as 15 minutes following ConA
injection (14-
fold increase over saline injected animals), but at this time point treatment
with 5 mg/kg
PRS-211,092 has an enhancing effect of 64% (Figure 4D). One, four and eight
hours
following ConA injection, the increase in IL-1(3 expression is correspondingly
35, 20 and
15-fold. The test compound reduces these effects by 52%, 46% and 27%,
respectively. The
effect of PRS-211,092 is statistically significant at all time points. The
effect of cytokine
modulation might be in some cases a time dependent issue. At certain period,
under given
circumstances, the presence of a cytokine might be beneficial while it will
become
deleterious in another time window. The fact that IL-1(3 is first up-regulated
may have an
initial positive effect, such as induction of SOCS-1, further sustained by its
later inhibition,
as suggested by the decrease in ALT at the end of the study that supports the
overall
hepatoprotective activity of the test compound.
IL-6 gene expression in liver and spleen in the ConA model.
A bimodal role has been suggested for IL-6 in the ConA induced model of
hepatitis.
Some studies supported the claim that IL-6 production favors the development
of hepatic
injury, while in other studies IL-6 predominantly displayed a hepatoprotective
activity.
These contradictory observations have been reconciled when a thorough time
course
experiment was carried out with neutralizing antibodies. The results of this
study indicate
that IL-6 presence in the early phase of the disease is critical for evoking a
strong
hepatoprotective effect, while continued high concentrations of the cytokine
are harmful
for the liver (Tagawa Y-I et al., J. Leukoc. Biol. 67: 90-6, 2000).
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Animals were sacrificed at the four time points previously described and the
level of
expression of the protective IL-6 cytokine was assessed in the liver. Increase
in IL-6 gene
expression is detected 15 minutes following ConA injection (4-fold increase
over saline
injected animals), and at this time point treatment with 5 mg/kg PRS-211,092
has an
enhancing effect of 470% (Figure 4E). One, four and eight hours following ConA
injection, the increase in IL-6 expression is correspondingly 90, 38 and 10-
fold. The test
compound continues to have an enhancing effect on IL-6 gene expression but
reduced to
38% at one hour after injury. At four hours PRS-211,092 reduces IL-6 ConA
induced
overexpression by 31%, and looses efficacy when the analysis is carned out
eight hours
after liver injury induction. The effect of PRS-211,092 is statistically
significant at all time
points. As observed in the case of IL-1[3, PRS-211,092 has a bimodal effect on
IL-6
expression. The fact that IL-6 is first up-regulated when it acts as a
protective mediator and
then inhibited when its hepatotoxic role predominates, supports the overall
hepatoprotective activity of the test compound.
Preliminary studies carned out with the spleens of the ConA inj ected mice
suggest
that similarly to the situation observed in the liver, IL-6 gene expression is
during the first
hour after injury initially up-regulated by treatment with 5 mg/kg i.v. of PRS-
211,092.
Again, this up-regulation of IL-6 when the cytokine acts predominantly as a
protective
mediator is in line with the overall anti-inflammatory and liver protective
activity of the
test compound.
IL-10 gene expression in liver and spleen, and secretion, in the ConA model.
The roles of the anti-inflammatory cytokine IL-10 in ConA model of murine
liver
injury have already been reported and it has been shown that administration of
anti-IL-10
antibodies results in aggravated liver injury (Kato et al, Hepatology Research
20: 232-43,
2001). Animals were treated with 5 mg/kg i.v. of PRS-211,092 and euthanized 15
minutes,
1, 4 or 8 hours following ConA induced liver injury. Vehicle treated animals
displayed on
average a 9-fold increase in IL-10 liver or spleen RNA levels versus saline
injected
animals at all time points tested. Treated animals displayed further increased
IL-10 gene
expression by 1.5 fold in the spleen as early as 15 minutes after injury
followed by a 3.2
fold increase 1 hr after injury. A similar increase of about 1.4 fold is
observed in the liver
only 8 hours after injury, suggesting that IL-10 is first up-regulated in the
spleen then in
the liver. In parallel to RNA quantitation in the whole organ, the spleen was
removed at
one hour after injury and splenocytes were cultured for 24 hours to allow the
assessment of
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secretion levels. Saline injected animals yielded undetectable background
levels of IL-10,
ConA injected animals treated with vehicle only yielded 331 pg/ml IL-10 while
ConA
injected animals treated with PRS-211,092 produced 549 pg/ml, a 1.7 fold
increase in
secretion from splenocytes, which correlated with the increased RNA expression
in the
spleen. Last, the level of IL-10 was determined in plasma 1 hour following
ConA injection
and 5 mg/kg i.v. of PRS-211,092 significantly (p=0.03) increased the level of
this anti-
inflammatory cytokine from 493 pg/ml in vehicle treated animals to 726 pg/ml
in PRS-
211,092 treated animals, a 1.5 fold increase which also correlates with the
increase in gene
expression observed in the spleen at this early time point. In this in vivo
model we show
that the increase in IL-10 RNA levels is supported and correlated with an
increase in
protein secretion.
From these experimental results, we confirm in a second in vivo model that the
active ingredient of the present invention is effective in reducing the RNA
level of pro-
inflammatory mediators while it is increasing the RNA level of the anti-
inflammatory
cytokine IL-10. Consequently, it further supports the fact that compounds of
the invention
may be therapeutically effective in the wide variety of immune/inflammatory
related
disorders.
Expression of genes involved in cytokine signal transduction, in the ConA
model.
The purpose of this study was to determine if a common mechanism lye beyond
the
observations that active compounds of the invention are efficient modulators
of anti- and
pro-inflammatory mediators. In recent years, progress was made in unraveling
cytokine
signal transduction. This work led to the recognition of Janus kinases (JAKs)
and signal
transducers and activators of transcription (STATs) as positive regulators;
and of
suppressers of cytokine signaling (SOCS), protein inhibitors of activated
STATs (PIAS)
and the SH-2 containing phosphatase, as negative regulators of this signaling
pathway. In
this study we checked the effect of ConA induced liver injury on the levels of
SOCS-1 and
SOCS-3, as previously described. Both SOCS-1 and SOCS-3 have important
recognized
roles in the liver. SOCS-1 deficient mice suffer from three major
abnormalities:
lymphopoenia, macrophage infiltration of several organs including the liver,
heart, lung
and skin, and severe fatty degeneration of the liver. On the other hand, SOCS-
3 is the main
SOCS gene induced in the liver by GH. Thus, we tested if PRS-211,092 had an
effect on
the expression of these negative regulators of gene expression.
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As can be seen from Figures 4F and 4G, the pattern of impact on gene
expression is a
mirror image of the impact of PRS-211,092 on the cytokine themselves. While
PRS-
211,092 lowered the almost linear slope of MCP-1 expression over time from
liver injury,
it symmetrically increased the slope of SOCS-1 gene expression. Similarly the
pattern of
increase in SOCS-3 due to PRS-211,092 treatment is the mirror image of the
decrease in
IL-2, TNF-a,, IL-1 ~i and IL-6. These results, showing activity of the test
compound on the
modulation of regulators of cytokine signal transduction, support the fact
that the active
compounds of the invention act through regulation of transcription of genes
involved in
inflammatory processes.
Moreover, when taken together with the results obtained with the downstream
regulated cytokines and chemokines, we can make the following observation.
From the
graphs displayed in Figure 4 we see that generally speaking PRS-211,092 has a
clear
moderating effect on the expression of inflammatory related genes, or their
regulators, that
are activated during the course of liver injury in the ConA model. PRS-211,092
down-
regulates the expression of positive inflammatory mediators and up-regulates
the
expression of negative mediators. The cytokines and chemokine tested often act
in concert
and cross regulate one another to yield the final physiological outcome. When
the ultimate
effect of the above-described gene modulation is tested by a biological marker
of liver
injury, such as ALT, SAA-3 and haptoglobin, we conclude that the overall end
result is
indeed hepatoprotection.
Expression of genes involved in acute phase response, in the ConA model.
The acute phase response is an innate body defense seen during acute
inflammation,
infection and trauma, which involves the altered production of certain blood
proteins
termed acute phase proteins (APPS). As previously stated activated macrophages
and other
leukocytes release pro-inflammatory cytokines such as TNF-a, IL-1 and IL-6.
These
cytokines in turn stimulate hepatocytes to synthesize and secrete acute phase
proteins such
as C-reactive protein (CRP), mannose-binding lectin (MBL), haptoglobin and
serum
amyloid A (SAA). Measurement of acute phase protein level is used to monitor
the
severity of the innate response and thus predict the prognosis of the disease.
The purpose
of this study was to determine if indeed the modulation of anti- and pro-
inflammatory
mediators in the liver lead to the appropriate down-regulation of positive
APPS, both at the
level of gene expression and secretion. In this study we checked the effect of
ConA
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induced liver injury on the levels of SAA-3 and haptoglobin, and we tested if
PRS-211,092
had an effect on the expression of these acute phase proteins, as previously
described.
Administration of 5 mg/kg PRS-211,092, 1 mg/kg FK-506 or vehicle thirty
minutes
before ConA had the following effects on the APPS tested four and eight hours
after
induction of liver injury. PRS-211,092 significantly reduced the levels of
liver gene
expression of SAA-3 by 55% and 48%, and of haptoglobin by 56% and 66%, at the
respective time points. The positive control FK-506 yielded on average at both
time points
of four and eight hours after injury 97% and 78% reduction in gene expression
of SAA-3
and haptoglobin respectively. These results further strengthen the fact that
the modulatory
effect of active compound of the invention on gene expression of the cytokine
network of
pro- and anti-inflammatory mediators has an overall therapeutic benefit, as
monitored by
the significant decrease in the APPS, SAA-3 and haptoglobin.
Example 10.
Hepatoprotection through inhibition of apoptosis in HepG2 cell lines.
The purpose of this study is to assess whether the hepatoprotective effect of
the
active compounds of the invention is achieved through inhibition of apoptotic
events in
cells of hepatic lineage. The proteolytic cleavage of caspases and poly-ADP-
ribose
polymerase (PARP) is a known marker of apoptosis, and is investigated by
immunoblotting or immunohistochemistry in HepG2 cells. Apoptosis is induced in
these
cells either by TNF-oc, anti-CD95 or ethanol.
HepG2 (ATCC # HB-8065) are human hepatocellular carcinoma cells and are grown
in Eagle's Minimum Essential medium, supplemented with 10% FCS, 2 mM L-
glutamine,
1 mM sodium pyruvate, 0.1 mM nonessential amino acids, and 1.5 g/1 sodium
bicarbonate.
A total of 1 x 106 cells/well are seeded in 6-well plates and are treated with
either anti-
CD95 (0.5 mg/ml) or with TNF-a, (10 ng/ml), and cycloheximide (10 mg/ml). For
ethanol
induced apoptosis, the range of the inducer is 100 to 400 ~,M and cells must
be cultured for
24 hours under those conditions. Test compounds and controls are added to the
cells one
hour before the induction of apoptosis, unless otherwise stated. After 6 hours
for TNF-a or
anti-CD95 or 24 hours for ethanol induction, cells are washed in cold PBS and
lysed in 1%
Triton X-100, 50 mM Tris-HCI, pH 7.6, and 150 mM NaCl containing 3 mg/ml
leupeptin,
3 mg/ml aprotinin, 3 mg/ml pepstatin A and 2 mM phenylmethylsulfonyl .uoride.
After
centrifugation (10 minutes, 13,000 rpm, 4°C), the cell lysates are
separated by sodium
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dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a
polyvinylidene
di.uoride membrane (Amersham, Germany). Membranes are blocked with 5% milk
powder in Tris-buffered saline and then incubated for 1 hour with 1 mg/ml of
either anti-
cleaved caspase-3, anti-cleaved caspase-7, or antibodies recognizing the full-
length forms
of the proteins. Membranes axe then washed 4 times with Tris-buffered
saline/0.05%
Tween-20 and incubated with the respective peroxidase-conjugated secondary
antibodies
for 1 hour. After extensive washing, bound antibodies are detected by enhanced
chemiluminescent staining.
Example 11.
Inhibition of IL-2 related transcription factors in activated T cells.
We have established that IL-2 gene expression and secretion are inhibited by
the
active compounds of the invention both in vitro, in PMA/Calcium ionophore
activated T
cells, and in vivo in the MCAo model for determination of neuroprotection and
in the
ConA induced model of liver injury for determination of hepatoprotection. IL-2
is tightly
regulated at the level of transcription. The purpose of this study was to
check if the test
compounds have an effect on some of the transcription factors involved in the
regulation of
the IL-2 promoter.
Two different lines were established, in each case Jurkat cells were
cotransfected
with a plasmid containing a luciferase reporter gene controlled by either NF-
AT (Clontech
cat# 52088) or AP-1 (Clontech cat# 52087) transcription elements and with a
plasmid
containing the neomycin resistance gene. Transfection was carned out using the
cationic
lipid reagent method (Dimeri-C, Invitrogen Life Technologies). In order to
isolate stable
clones, the transfected human T cells were submitted to gentamicin selection
for four
weeks, and positive clones were identified. The stable clones, transfected
with the various
transcription elements controlling luciferase, were grown as previously
described for
Jurkat cells, with the addition of 300 ~.g/ml gentamicin. On the experimental
day, the cells
containing each reporter were plated in a 24 well plate at 106 cells/ml/well.
Test
compounds were resuspended in DMSO, and added for one hour before cell
activation, at
predetermined doses wherein DMSO final concentration is 0.1%. Cyclosporin A
was used
as positive control at the dose of 100 nM. Cells were then stimulated with 10
ng/ml PMA
and 2 ~.M calcium ionophore for 6 hours. At the end of the experiment, cells
were
collected, rinsed in PBS and lyzed for 15 minutes on ice in 50 ~1 luciferase
lysis buffer
(Promega). Cell debris were removed by spinning down the cell lysate for 5
minutes at
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high speed at 4°C. Luciferase activity was measured in a black 96 well
plate, where 10 p.l
of cleared cell lysate were combined with 90 ~,1 of luciferin substrate
(Promega).
Luminescence was immediately measured in the appropriate reader (X-flour,
Tecan).
Results are shown in Figure 5, where we can see that activation of the cells
yield a
significant increase in NF-AT driven luciferase expression, from 60
luminescence units
(LU) to 2449 LU. Treatment of the cells with test compounds caused a dose
dependent
reduction in NF-AT driven luciferase expression. PRS-211,092 has an ICso of
1.5 ~,M and
PRS-211,220 has an ICSO of 3.9 ~.M. In this experimental setup, 100 nM of
cyclosporin A
caused a total inhibition. Similarly, we tested the activity of 10 p,M PRS-
211,092 on cells
stably transfected with the AP-1 reporter. Cell activation increased the level
of AP-1 driven
luciferase expression from 182 LU to 19391 LU, however neither PRS-211,092 nor
cyclosporin A affected this outcome. These results further support that the
active
compounds of the invention act through regulation of transcription of genes
involved in
inflammatory processes.
Example 12.
DNA arrays.
DNA-array based technologies are widely used in gene regulation research, most
commonly to measure differential gene expression, that is comparing the
relative level of
RNA transcripts in different samples. The purpose of this study is to allow a
preliminary
screen of the impact of dexanabinol and its analogs on the regulation of a
large amount of
genes. The technology is based on hundreds (macro-arrays) to thousands (micro-
arrays) of
sequence-specific DNA fragments spotted on a solid matrix such as glass slides
or
membranes. RNA samples from the examined tissue or cells are reverse-
transcribed into
cDNA, labeled and hybridized with the array. The number of labeled transcripts
hybridized
to a single spot is turned to a radioactive, fluorescence or chemiluminescence
signal and
detected by the appropriate instrument. The quantification of the signal on
each spot
measures the level of expression of the specific gene. We have used membrane-
based
focused macroarrays each consisting of gene families representing a biological
regulatory
pathway such as cytokines or chemokine arrays, commercially made by SuperArray
Inc.
cDNA samples from mice brains after MCAo treated with either vehicle or test
compound
were labeled with biotin, hybridized to the array-membranes and detected using
a
chemiluminescence detector, according to SuperArray instructions. The genes
that are
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expressed differentially between treatment asld control are subj ected to
confirmation
analysis using real-time quantitative PCR, as previously described.
B- Impact of tricyclic dextrocannabinoids on animal models for various
diseases.
Example 13.
Effect of the compounds in carrageenan induced paw edema.
The purpose of this study is to test in vivo the anti-inflammatory activity of
the
compounds in paw edema induced by injection of 1% carrageenan in the animal
hind paw.
Female Balb/c mice (20 gr average body weight, Harlan, Israel) are
anesthetized with a
combination of xylazine and pentobarbitone diluted in sterile saline, 15 and 6
mg/kg i.p.
respectively. Anesthetized mice are injected subcutaneously, in the subplantar
region of
one (right) paw with 0.05 ml of 1% w/v Carrageenan in sterile water. The
contralateral
(left) paw is not injected as data from the literature, confirmed by our own
experience,
showed that injection of 0.05 ml of normal saline did not affect later
thickness or volume
measurements. The test compounds, including known anti-inflammatory controls,
are
dissolved in cremophor:ethanol and further diluted 1:20 or 1:50 in sterile
saline before i.p.
injection that takes place immediately before the carrageenan injection. Three
hours after
injection the animals are resedated following the previously described
procedure. Paw
thickness is measured using a dial thickness gauge (Spring-dial, constant low
pressure
gauge, Mitutoyo, TG/L-1, O.Olmm) and paw volume is measured using a
plethysmometer
(model #7150, Ugo Basile, Italy). Paw Edema is expressed as the difference
between the
right treated and the left untreated paws of the same animal, either as D Paw
Volume
(OPV) in millimeters cube or as O Paw Thickness (APT) in millimeters. Each
group
comprises at least 10 animals. Results can be further normalized to the ~PV
and APT
values of each treatment group at 0 mg/kg (vehicle only). At the end of the
study, animals
are euthanized with an i.p. injection of 100 mg/kg pentobarbitone.
The differences between ~PV and OPT among various treatment groups are
analyzed
by analysis of variance (ANOVA) followed by post-hoc Fisher test. A value of
p<0.05 is
considered to be statistically significant.
Results are depicted in Figure 6 where the % inhibition of paw thickness,
normalized
to vehicle, is plotted against the dose of the test compound. Dexanabinol
yields a reduction
of about 29% in paw thickness at doses ranging from 0.2 to 0.5 mg/kg. PRS-
211,092 yields
a reduction of about 22% in paw thickness at the very low dose of 0.25 mg/kg.
PRS-
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211,220 reduces even more paw thickness by 31% at the even lower dose of 0.1
mg/kg.
These results are statistically significant as compared to vehicle treated
animals. At these
doses, known anti-inflammatory drugs such as Celecoxib and Dexamethasone (DXM)
yield respectively 24% and 26% reduction in paw thickness at 0.1 mg/kg, 28%
and 31% at
0.25 mg/kg and both 33% at 0.5 mg/kg. It should be kept in mind that these
commercially
available drugs display serious side effects that prevent chronic uses without
complementary protective medication. The fact that compounds of the invention
have anti-
inflammatory activity comparable to these drugs is very encouraging since
compounds of
this family have the advantage of being devoid of side effects, thus making
them
interesting candidates for the replacement of existing anti-inflammatory
drugs. These
results support that the active compounds of this invention, which act through
modulation
of pro/anti-inflammatory mediators, have an anti-inflammatory effect that may
be relevant
to a wide range of human conditions with inflammatory components.
Example 14.
Effect of compounds in cancer chemoprotection.
In vitro.
Cells from several tumor-derived cell lines are tested for their proliferation
capacity
in presence of our test compounds. Pancreatic tumor cell lines were obtained
from ATCC.
Panc-1 (ATCC # CRL-1469) were cultured in DMEM supplemented with 4 mM
Glutamine, 4.5 g/L glucose, 1.5 g/L bicarbonate, antibiotics (penicillin,
nystatin and
streptomycin) and 10% heat inactivated fetal calf serum. Aspc-1 (ATCC # CRL-
1682)
were cultured in RPMI supplemented with 2 mM Glutamine, 10 mM HEPES, 1 mM
sodium pyruvate, 4.5 g/L glucose, 1.5 g/L bicarbonate, antibiotics
(penicillin, nystatin and
streptomycin) and 20% heat inactivated fetal calf serum. Cells were seeded in
a 24 well
plate (105 cells/ml/well) and grown overnight. The cells are incubated with
the test
compounds (1-100 ~M) or vehicle (0.1% DMSO final concentration). Cell
viability was
determined 24 hours later using standard crystal violet staining. The culture
medium was
removed from the wells and the cells were fixed by adding 1 ml/well of 2%
formaldehyde
in PBS for 10 minutes. Following fixation the cells are washed three times
with PBS and
250 ~,1 of 0.5% (w/v) crystal violet is added to each well and the plates were
incubated for
30 minutes at room temperature with gentle agitation. The stained cells were
then washed
three times with double distilled water and the color was extracted by adding
to each well
250 p,l of 10% acetic acid. The plates were agitated for 15 minutes at room
temperature
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and 100 ~,1 were transferred in duplicate to a 96 well plate for reading.
Optical density
(OD) of the cells was measured at 620 nm in an ELISA reader and results are
expressed as
viable cells. Absorbance of untreated cells is recorded as 100%. The
experimental
results show that Aspc-1 proliferation was not affected by the presence of
dexanabinol up
to 15 ~,M whereas Panc-1 cells proliferation was inhibited by 26% at this same
concentration. The ICSO (dose inhibiting cell growth by 50%) is determined.
Moreover, the cells are stained for activated caspase 3 to determine whether
they
died through an apoptotic mechanism. The medium from the wells is discarded
and cells
are fixed by adding 1 ml of 4% formaldehyde in PBS, for 10 min. Cells are
washed twice
with PBS-0.1% Tween20 (PBS-T) and permeabilized with cold methanol for 20 min.
The
cells are washed twice with PBS-T and incubated with 1 ml blocking solution
(3% BSA,
PBS-T) for 30 min. The primary antibody (rabbit anti- cleaved caspase 3
(asp175) Cell
Signaling Technology, diluted 1:50 with blocking solution) is added and the
cells
incubated for 60 min. at 37°C. The cells axe washed twice with PBS-T.
The secondary
antibody (HRP conjugated anti-rabbit IgG diluted 1:200 with blocking solution)
is added to
the wells and incubated for 60 min. at RT. Cells are washed twice with PBS-T
and
incubated for 10 min with a fluoresceine tyramide reagent (NEN, diluted 1:50
with
amplification diluent). Cells are washed twice with PBS-T and the signal
visualized by
fluorescence or confocal microscope. Beside monitoring activated caspase-3,
the
expression of apoptosis-related genes in cells treated with dexanabinol and
its analogs is
compared to that in untreated cells. The procedure for real-time RT-PCR is as
previously
described. For each gene, a pair of specific PCR primers is designed and the
reaction is
done according to the ABI protocols. The quantification of each gene
expression level is
normalized to a housekeeping gene and compared to RNA samples from non-treated
cells.
In vivo.
Once we have shown that dexanabinol and its analogs were inhibitors of tumor
cell
proliferation in vitro, we wished to test if our compounds were as efficient
in vivo. For this
purpose the tumor cells LoVo (originating from a colorectal tumor; ATCC # CCL-
229)
were grown in DMEM containing 4.5 g/L glucose, 2 mM Glutamine, 1% Pen/Strep
and
10% heat inactivated fetal calf serum. The cells were harvested using Trypsin-
EDTA, the
detached cells were rinsed in PBS, and counted. Predetermined amounts (1x106
cells iii
constant volume of 0.12 ml/animal) were injected s.c. above the right femoral
joint in nude
CD-1 male mice (average weight 20-25 gr, Harlan, Israel). Each treatment group
was
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composed of at least 7 animals. Each animal was clinically monitored daily.
The growth of
the tumor was also monitored during the daily visits but actual measurements
were
recorded once a week. When tumors reach the appropriate size, animals are
treated with
either vehicle, 5 ml/kg/day, or with our test compounds, in the range of 2.5
to 10
mg/kg/day.
Thirty-six out of the 40 implanted mice developed a visible tumor within 5 to
6
weeks from tumor implantation. All animals were treated first on the 36th-42"d
day of
tumor implantation and the treatment lasted 8 weeks. One animal died in each
treatment
group between the 5th and the 7th week of treatment. The results are expressed
as percent of
tumor growth at the various days of the treatment as compared to baseline day
1. The
results are depicted in Figure 7 where we can observe that the efficacy of
dexanabinol is
inversely proportional to its dose in the range tested. 2.5 mg/kg (~) seems
more efficient
than 5 mg/kg (~), itself better than 10 mg/kg (x), which has very little
effect, if at all, as
compared to vehicle (--~--). By the end of the treatment, 2.5 mg/kg
dexanabinol reduced
the tumor growth by 89%, 5 mg/kg by 61 % and 10 mg/kg by 21 %. These
experimental
results strongly suggest that the active compound of this invention, which
acts through
modulation of pro/anti-inflammatory mediators, may be therapeutically
effective against
certain types of tumors.
Example 15.
Treatment of neurodegenerative disorders: the MPTP model
Parkinson's disease (PD) is a neurodegenerative disorder characterized by
tremor,
slowness of movements, stiffiiess and poor balance. Most, if not all, of these
disabilities are
due to a profound reduction in striatal dopamine content caused by loss of
dopaminergic
neurons in the Substantia Nigra pays compacta (SNpc) and of their projecting
nerve fibers
in the striatum. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a well
known
neurotoxin that can cause depletion of dopamine content in the striatum and a
reduction in
the number of nigrostriatal dopaminergic neurons in several species including
humans
(Turski L. et al, Nature 349: 573, 1991). Neuroinflammatory phenomena are
observed in
this model. The aim of the present study is to examine the effect of compounds
involved in
gene regulation of pro- and anti-inflammatory mediators on the progression of
MPTP-
induced dopaminergic toxicity. The study is carned out in two time windows. In
the short-
term model, the neurological outcome is assessed, namely by measuring the
effect of the
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test compounds on the number of immunoreactive cells. In the long-term model,
the
functional outcome is assessed in the rotarod system following various
treatments.
Animal treatment and~rocedure~ the short-term model for neurological outcome.
The mice (C57/BL male mice, average weight 30 g, Harlan, Israel) were
administered i.p. with 4 injections of MPTP (Sigma, USA) (20 mg/kg, 5 ml/kg)
in saline
(Teva Medical Israel) at 2 hours interval on day 1. The test compounds and
vehicle control
(cremophor:ethanol diluted in saline) were injected i.p. once just before the
first MPTP
administration at a volume dose of 5 ml/kg. Dexanabinol was tested at 10, 20,
and 30
mg/kg and PRS-211,220 was tested at 0.5, 1, and 5 mg/kg. Each treatment group
was
composed of at least seven animals. Seven days following the MPTP treatment
the animals
are euthanized by i.p. administration of 100 mg/kg pentobarbitone sodium (CTS,
Israel)
and their brains are removed for tyrosine hydroxylase (TH) detection using
immunohistochemistry.
TH inununoreactivity at the level of the SNpc
Brains were fixed by immersion in 4% formaldehyde for at least 72 hours. The
brains
were then washed with PBS and transferred to 30% sucrose in PBS until they
sank. After
the brains sank in the sucrose they were frozen using the cryostat special
fast-freezing
technique (-60°C). The brains were cryosectioned (20 ~,m) at the level
of the striatum and
at the level of the substantia nigra (SN). Immunohistochemistry staining was
carned out
using Rabbit anti-tyrosine hydroxylase (1:250, Calbiochem, USA). The slides
were stained
using the 3,3-diaminobenzidine tetrahydrochloride (DAB) chromagen detection
kit of the
automated immtmostaining system (Vantana, France). Quantitative analysis was
carried
out by counting the number of TH-immunoreactive cells/mma at the widest area
of the
SNpc.
Statistical analysis.
Results are expressed as mean~SD. Data were analyzed using analysis of
variance
(ANOVA) followed by post-hoc Fisher test. A value of p<0.05 is considered to
be
statistically significant.
The results are depicted in Figure 8A, where we can see that MPTP injections
resulted in a reduction of about 65% in the number of TH-IR cells at the SNpc
level as
compared to the saline injected animals. Treatment of the animals with the
test compounds
results in a dose-dependent preservation of the TH-IR cells. Percent
preservation is
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calculated by dividing the number of rescued cells (treatment-MPTP) by the
maximal
possible number of rescued cells (saline-MPTP). Best rescue were observed with
20 mg/kg
of dexanabinol, which preserved about 25% of the TH-IR cells, increasing the
dose to 30
mg/kg having no significant effect, and 5 mg/lcg of PRS-211,220, which
preserved
approximately 50% of the TH-IR cells. Vehicle alone had no effect on TH-IR
preservation.
These two doses of test compounds were ftirther used to determine the
functional outcome.
Animal treatment and~rocedure~ the long-term model for functional outcome.
The mice (C57BL male mice, average weight 30 g, Harlan, Israel) were
administered i.p. with MPTP (Sigma, USA) (40 mg/kg, 5 ml/kg) in saline (Teva
Medical,
Israel) at 2 weeks interval for four times. The test compounds and controls
were injected
i.p. once just before the first MPTP administration at a volume dose of 5
ml/kg.
Dexanabinol was tested at 20 mg/kg and PRS-211,220 was tested at 5 mg/kg. Each
treatment group was composed of at least fourteen animals.
Seven days following each MPTP injection, the animals were submitted to a
functional test using the rotarod apparatus as described by Rozas et al.
(Rozas G. et al., J.
of Neuroscience Methods 83: 165-75, 1998). The performance of the animals in
the
rotarod system, which measures overall locomotor ability, reflects their
ability to achieve
functions generally affected in PD. The animals were trained for 4 days before
beginning
the experiment. Their task was to stay on the accelerating rod without falling
for 12
minutes (3 minutes at each speed). The tested speeds were: 15, 19, 23 and 27
rpm. Animal
performance on the rod was scored as follows: each animal could obtain a
maximum of 3
points (1 for each minute) for full walking on the rod at each speed.
Therefore, an animal
could get a maximum score of 12 points (3 for each speed). Catching the
circling beam of
the rod without walking subtracted 0.5 points for every 3 circles circled by
the animal. The
first 3 circles did not affect the score. Fifty-two days following initiation
of study animals
were euthanized by i.p. administration of 100 mglkg pentobarbitone sodium
(CTS, Israel).
The results are shown in Figure 8B, where we can see that each additional MPTP
injection generate a further decrease in the animals ability to perform the
rotarod test.
Though this trend can be observed as early as in the first session following
the first MPTP
injection, the effect of MPTP on animal performance becomes statistically
significant only
from the third session and on. Indeed in the two first sessions we did not
observe a
significant effect of any of the treatment group on the rotarod score of the
animals (data not
shown). In the following sessions, we observe that the vehicle has no effect
of its own, that
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dexanabinol at 20 mg/kg shows a positive trend at the fourth session and that
PRS-211,220
at 5 mg/kg yields a statistically significant improvement in functional
outcome at both later
sessions when compared to vehicle.
Another parameter that was measured in this experiment is mortality, which was
relatively high in this model. Animals that received only saline, and no MPTP,
displayed,
as expected, 0% mortality (0/14 animals). However, MPTP injected animals
displayed
69% mortality (11/16 animals) over the period of the study, reflecting the
severity of the
model. Treatment with 20 mg/kg dexanabinol dramatically lowered this figure
down to
11% mortality (1/9 animals), while treatment with 5 mg/kg PRS-211,220 was even
more
effective with 0% mortality (0/7 animals).
Altogether the results of this study show that the active compounds of this
invention,
which act through modulation of pro/anti-inflammatory mediators, may be
therapeutically
effective against the neuroinflammatory component of neurodegenerative
diseases. The
effect of these compounds is not only major on the mortality rate but above a
certain level
of TH-IR cells rescue, also significant on the functional abilities of the
survivors.
Example 16.
Peripheral noxious pain: the formalin test.
Pain mediated by the peripheral nervous system, is tested in the 'formalin
test' for
cutaneous (peripheral) pain (Tjolson A. et al, Pain 51: 5-17, 1992). First the
test
compounds are injected i.p. Then formalin is injected s.c. in the plantar
surface of the hind
paw of a mouse 90 min after the test compound. Immediately after formalin
administration
pain is assessed (every 5 min for 1 hr) by the number of times the animal
licks the
formalin-injected paw.
Example 17.
Neuropathic pain: attenuation of mechanical allodynia.
The aim of this study is to assess the potential analgesic effects of our
compounds in
an animal model of neuropathic pain. A peripheral monopathy was induced in the
right
hind limb of rats following a chronic constriction of the sciatic nerve
(Bennet G.J. and Xie
Y-K. Pain 33: 87-107, 1988). The development of mechanical allodyna was
monitored
using an established behavioral test (Von Frey filaments).
Pre-surgery baseline values are ascertained as the mean of two pre-surgery
values.
Once the baseline values had been established, the animals are surgically
prepared by
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constricting the right sciatic nerve with 4 chromic cat gut loose ligatures.
On day 11 post-
operation, the animals that have developed mechanical allodyna are arbitrarily
allocated to
the various treatment groups based on the pre-surgery values.
The design is randomized, performed in a masked fashion as to whether drug or
vehicle is being given. The animals, male Sprague-Dawley rats, are allowed to
acclimatize
to the behavioral testing equipment before testing. On the testing day, the
animals are
given a single dose of one of the test compounds in a volume of 2.5 ml/kg.
Following 15
and 180 minutes a series of Von Frey filaments (pre-calibrated before testing)
are applied
to the plantar surface of the hind paw, from below. The filaments are applied
in ascending
order starting with the weakest force; 0.37 g or filament handle no. 3.61),
and the
withdrawal threshold for both the ipsilateral and contralateral hind paws is
evaluated. Each
filament is indented on the mid-plantar surface of the foot to the point where
it just starts to
bend; this is repeated approximately 8-10 times per filament at a frequency of
approximately 1 Hz. The withdrawal threshold is defined as being the lowest
force of two
or more consecutive Von Frey's filaments to elicit a reflex withdrawal
response (i.e. a brief
paw flick) and is measured in grams.
Example 18.
Visceral pain.
The ability of the tricyclic dextrocannabinoid to decrease visceral pain is
tested using the acetic acid induced model. ICR male mice (average body weight
30
g, Harlan, Israel) are pre-treated with i.v. inj ection of either vehicle
(Cremophor:Ethanol 70:30 w/w further diluted 1:20 into saline; 5 ml/kg), or
test
compounds. Each treatment group comprises at least five animals. Fifteen
minutes
later, the mice are injected i.p. with 10 ml/kg of 0.6% acetic acid in water
and the
number of writhes are counted during a 5 minutes period, starting 5 minutes
after
the acetic acid administration. A writhing is considered as contraction of the
abdominal muscles accompanied by an elongation of the body and extension of
the
hind limb. Results are expressed as mean number of writhes ~ SEM. Data was
analyzed using analysis of variance (ANOVA) followed by Tukey's post hoc test.
A
value of p<0.05 was considered statistically significant.
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Example 19.
Diabetes type I: the NOD mice model.
The purpose of the present study is to establish a model in non-obese diabetic
(NOD)
mice to test the protective activity of dexanabinol and its analogs in an
experimental setup
relevant to human insulin-dependent diabetes mellitus.
NOD/It female mice (70-80 days old at study onset, Harlan, Israel) are
weighted at
day 1. Their baseline glucose level is established using a drop of blood
obtained by
sectioning the tip of the tail and a glucometer with the appropriate
glucosticks (Elite,
Bayer). Mice are then injected i.p. with cyclophosphamide (Sigma) diluted in
saline at a
dose of 300 mg/kg. The appearance of glucose in the urine of the animals is
monitored
every two days using a urine multistick (Bayer). When this test indicates that
the animals
reach glucourea, then the level of glucose in the blood is reassessed during
two consecutive
days after overnight starvation. Animals are defined as diabetic if their
glucose blood
levels are above 300 mg/dl. Three days following the diagnostic of diabetes,
the animals
are sacrificed by i.p. injection of 100 mg/kg pentobarbitone. Their spleen and
pancreas are
removed for further study including FAGS analysis of the T cells
subpopulations in the
spleen and histo- and immuno-pathological evaluation of the pancreas.
The histopathological evaluation is carried out on ten Langerhans islands for
each
animal and the scoring is according to the following method (Sempe P. et al,
Eur. J.
Immunol. 21: 1163-9, 1991). The severity of the damage is scored according to
the level of
mononuclear infiltrate: 0- no infiltration, 1- periductular infiltrate, 2-
peri-islet infiltrate, 3-
intra-islet infiltrate, 4- intra-islet infiltrate associated with (3-cell
destruction. The mean
score for the pancreas of each animal is calculated by dividing the total
score by the
number of islets examined.
Example 20.
Renal ischemia.
The purpose of the present study is to test the nephro-protective activity of
dexanabinol and its analogs in an acute renal ischemia model in rats.
Male Sprague Dawley rats (250 gr average body weight, Harlan, Israel) are
anesthetized with a combination of xylazine and pentobaxbitone 8 and 35 mg/kg
i.p.
respectively. Then a 45-minutes ischemia is induced bilaterally on both
kidneys. The
sedated animals are positioned on their backs. The abdomen skin is shaved and
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with 70% ethanol. A midline skin incision is performed (2-3 cm long) and the
abdomen is
opened through an incision in the linea Alba. The kidneys are explored after
gentle
removal of the intestines to the opposite direction. While this is done, the
intestines are
covered with wet (warm saline 37°C) sterile sponges. The renal arteries
are isolated by
blunt dissection from the surrounding fat, and occluded together with the
renal veins in the
kidney hilus by arterial micro clips (FST Canada). Kidneys that become pale
immediately
after artery occlusion are considered ischemic. Only animals showing that both
kidneys are
ischemic are included in the study. During the ischemic insult the intestines
axe returned
into the abdominal cavity. The wound is covered with wet sponges (they were
kept wet by
rinsing warm saline).In addition, rectal temperature is monitored to remain
between 37°C-
38°C. Rectal temperature is measured using a thermistor (YSI USA model
400) and a
measuring unit (Cole Parmer model 8402-00).
Forty-five minutes after the ischemia initiation, the artery clips are
removed.
Reperfusion is verified by the return of the pink color of the kidney. The
wound is then
closed with 3-0 silk suture material (Assut, Switzerland) in two layers
(abdomen wall and
skin). At 1, 3 and 7 days post ischemic insult animals are lightly
anesthetized in an
anesthesia chamber with ether and blood samples are collected after an infra
orbital sinus
puncture. Blood is collected into eppendorf tubes, and centrifuged (4000 rpm
for 5
minutes). Serum is then separated and kept at -20°C before evaluation
of blood levels of
creatinine and blood urea nitrogen (BUN). At the end of the study, animals are
euthanized
with pentobarbitone sodium 100 mg/kg i.p. Kidneys are removed, weighted and
kept in 4%
formaldehyde solution for possible fizrther usage.
Treatments are administered i.v. into the femoral vein at 5 ml/kg to 10
animals per
group, immediately after the end of the ischemic insult. Results are compared
to ischemic
(vehicle treated) and sham (the same procedure, without renal artery
occlusion).
Statistical analysis.
The blood levels of BUN and creatinine are compared using ANOVA followed by
Duncan's post-hoc test.
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Example 21.
Inflammatory bowel disease: the acetic acid-induced model.
The purpose of this study is to evaluate the activity of test compounds in a
masked
study of acetic acid-induced inflarmnatory bowel disease in rats.
Male Sprague Dawley rats (10 weeks old, 200-250 gr, Harlan, Israel) are
lightly
anaesthetized by i.p. injection of a ketamine:rompun combination (100:10 mg/kg
respectively). A polyethylene catheter (outer diameter 1.7 mm) is inserted
through the
rectum 5 cm into the colon. And 2 ml of 5% acetic acid are then slowly
administered into
the colon. Fifteen seconds later the colon is washed with 3 ml saline and 15
seconds later
with additional 3 ml of saline. linmediately after, each group of animals are
treated with
either one of the appropriate treatments. All treatments are administered once
daily for 7
days. Animals are clinically followed for 1 week. During this period, the
following
parameters are daily monitored and recorded: body weight, presence of blood in
the stool
and stool consistency. These findings are scored according to table 1 (Murthy
S.N. et al
Dig. Dis. Sci. 38: 1722-34, 1993).
Table 1: Criteria for Scoring Disease Activity Index (DAI#) of IBD.
Score Weight Loss Stool ConsistencyOccult Blood or
* Gross Bleeding
0 None Normal Negative
1 l-5 Loose Stool Negative
2 5-10 Loose Stool Hemoccult Positive
3 10-15 Diarrhea Hemoccult Positive
4 >15 Diarrhea Gross Bleeding
DAI- (combined score of weight loss, stool consistency, and bleeding)l3.
* Normal stool - well formed pellets; loose stools - pasty stool that does not
stick to
the anus; and diarrhea - liquid stools that sticks to the anus.
Seven days post disease induction animals are sacrificed with pentobarbital
100
mg/kg i.p. The whole colon is excised, slit longitudinally and examined under
a
magnifying glass, and any visible damage is recorded and scored according to
table 2
(along et al, J. Phann. Exp. Ther. 274: 475-80, 1995).
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Table 2: Gross Pathology Scoring Method for Evaluating the Severity of IBD.
Score Pathology
0 No damage
1 Localized hyperemia and/or edema
2 Two or sites of hyperemia and/or edema
3 Localized erosion
4 Localized ulcer
More then 1 site of erosion/or ulcer, or 1 erosion
site or ulcer
extending > 2 cm along the length of the colon
Statistical analysis.
The clinical outcome is analyzed using analysis of variance (ANOVA) followed
by
Duncan's post-hoc test. A non-parametric test (Wilcoxon Rank Sum Test) is used
for
5 evaluating the gross pathology findings.
Example 22.
Experimental autoimmune diseases: CIA, EAE and DTH.
Autoimmune diseases are associated with elevated levels of inflammatory
cytokines.
The rodent models most commonly studied are experimental allergic
encephalomyelitis
(EAE), a model for multiple sclerosis in the human, experimental autoimmune
arthritis, a
model for rheumatoid arthritis in the human and delayed type hypersensitivity
(DTH), a
model for allergic reactions in the human. EAE is an autoimmune neurological
disease
elicited by sensitization of the animals to myelin basic protein from the
central nervous
system, which is also lazown as basic encephalitogeuc protein. Experimental
autoimmune
arthritis is induced in animals by immunization with collagen in complete
Freund's adjuvant:
the model is therefore named collagen induced arthritis (CIA). Delayed type
hypersensitivity
is induced by the application of dinitrofluorobenzene according to a strict
time-schedule,
therefore the model generated correspond to allergic contact dermatitis in the
human. The
purpose of the present study is to test the ability of our compounds to
prevent or attenuate the
clinical signs of these three autoirmnune disease models.
Collagen Induced Arthritis.
Adult DBA/1 male mice (20 g average body weight, Harlan, Israel), at least
eight per
treatment group are used in this study. Bovine collagen type 2 is dissolved in
0.05 M acetic
acid at a concentration of 2 mg/ml by stirnng ON at 4°C. The collagen
solution is further
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emulsified in an equal volume of Complete Freund's Adjuvant (CFA). Each animal
is
administered with 100 ~g collagen type 2 in 0.1 ml CFA emulsion. The collagen
is
administered s.c. at the base of the tail. Twenty-one day after priming, the
mice receive an
intradermal booster injection of 100 ~.g collagen in Incomplete Freund's
adjuvant.
The volume of each hind paw is measured using a plethysmometer (Hugo Basill,
Italy), and the thickness using a dial, constant pressure gauge, (Mitutoyo,
Japan).
Measurements are performed before collagen administration and every second day
throughout the designated follow-up period. All treatments are administered
intraperitoneally. At the end of the treatment period the animals are
sacrificed with
pentobarbital 100 mg/kg i.p.
Statistical analysis.
The differences between the severity of the paw swelling among various
treatment
groups are compared using analysis of variance ANOVA followed by post-hoc t-
Test. A
value of p<0.05 is considered to be statistically significant.
Experimental Autoimmune Encephalomyelitis.
Various animal models of autoimmune encephalomyelitis are known in the art,
depending on the method of induction, the strain of the animal and the antigen
employed to
induce the disease. The impact of the test compounds is tested in EAE using
Lewis rats in
which the onset of disease is observed by the appearance of clinical symptoms
about 10 days
after induction. The disease progresses and the clinical score increases and
peaks around day
15 and spontaneous recovery is observed around day 18 after induction of the
disease. The
animals (at least 10 per test group at initiation of study) are maintained on
a 12 hours light/12
hours dark regimen, at a constant temperature of 22°C, with food and
water ad libitum. EAE
is induced in these animals by immunization with purified guinea pig myelin
basic protein
emulsified in Complete Freund's Adjuvant. Guinea pig myelin basic protein
(MBP) is
prepared from spinal cord homogenates defatted with chloroform/ethanol and the
isolated
protein is purified using ion exchange chromatography. Each animal receives 50
~.g of the
purified protein. A solution of MBP (0.5 mg/ml) is emulsified with an equal
volume of
Complete Freund's Adjuvant containing 4 mg/ml of mycobacterium tuberculosis,
and each
animal receives 100 p,l (50 ~,1 in each hind foot pad).
Animals are treated with test compounds or vehicle control, administered
intravenously in a volume of 5 ml/kg, for three consecutive days starting from
the onset of
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the disease (~ at day 10 following disease induction). Methyl prednisolone is
used as
positive control and it is administered daily for 5 consecutive days i.v. at
20 mg/kg starting
from day of disease induction by MBP inj ection. The results are recorded as
clinical score;
score of 0 indicates a normal animal with no clinical signs, 1 indicates tail
paralysis, 2
indicates paraplegia, 3 indicates quadriplegia, 4 indicates complete body
paralysis and 5
indicates death.
Statistical anal
The differences between the severity of the clinical outcomes among various
treatment groups was analyzed by analysis of variance (ANOVA) followed by post-
hoc
Fisher test. A value of p<0.05 is considered to be statistically significant.
Delayed Type Hyt~ersensitivity in mice model.
Adult female BALB/c mice (20 g average body weight, Harlan, Israel) were
sensitized on day 0 and day 1 by application of 30 ~,1 of 0.15%
Dinitrofluorobenzene
(DNFB) diluted in acetone on the shaved skin of the abdomen. On day 6 the
animals were
challenged by application of 10 ~,l of DNFB in acetone on one ear. The
contralateral ear
was not challenged but received the application of 10 ~1 acetone. Test
compounds were
administered at increasing doses from 0 to 15 mg/kg i.p. twice, the first
injection was
immediately after DNFB challenge (on day 6) and the second injection was 16
hours after
challenge (on day 7). Each treatment group comprised at least 7 animals.
Dexamethasone
(DXM) was used as positive control. Ear thickness was determined (in 0.01 mm
units) 24
hours after challenge (and 6 hours after second treatment on day 7) using a
dial thickness
gauge (Mitutoyo, Japan).
Results are analyzed as ear thickness of DNFB treated over DNFB untreated
contralateral ear. The impact of the test compound is further assessed by
comparing its
mean impact on the animals of the treatment group to the response generated by
the
appropriate vehicle only.
Although the present invention has been described with respect to various
specific
embodiments presented thereof for the sake of illustration only, such
specifically disclosed
embodiments should not be considered limiting. Many other such embodiments
will occur
to those skilled in the art based upon applicants' disclosure herein, and
applicants propose
to be bound only by the spirit and scope of their invention as defined in the
appended
claims.
