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NOTE POUR LE TOME / VOLUME NOTE:
CA 02566721 2006-11-10
WO 2005/110490 PCT/CA2005/000738
Phospholipase C Gamma Modulation and Uses Thereof For Management of Pain and
Nociception
FIELD OF THE INVENTION
The invention relates to the modulation of activity,
function and/or expression of phospholipase C gamma (PLCy) in a
subject and uses thereof, such as for treating and preventing
and pain.
BACKGROUND OF THE INVENTION
The need for new and improved methods and agents for
pain treatment is a significant ongoing concern in medicine.
Acute pain, e.g. related to injury or disease, can be severe
and have critical effects on patient recovery. An even
greater concern is chronic pain, which affects a large
proportion of the population, causing not only significant
discomfort, but can result in low self-esteem, depression,
anger, and can interfere with or completely prevent a sufferer
from typical daily activities.
While a number of studies have been done in this
area, many mechanisms and pathways involved in pain sensation
remain poorly understood. There remains a continued need to
provide new strategies of therapeutic intervention for pain
treatment.
SUMMARY OF THE INVENTION
The invention relates to methods of treating and/or
preventing pain. The invention further relates to methods of
modulating (e.g. inhibiting) activity, expression, and/or
function of a phospholipase C gamma (PLCy) in a subject.
According to one aspect of the present invention,
there is provided a method of treating or preventing pain in a
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2
subject, the method comprising modulating activity, function
and/or expression of a PLCy in the subject.
According to another aspect of the present
invention, there is provided a method of treating or
preventing pain in a subject, the method comprising
administering to the subject an agent capable of modulating
activity, function and/or expression of a PLCY in the subject.
According to still another aspect of the present
invention, there is provided a method of treating or
preventing pain in a subject, comprising administering to the
subject a composition comprising: an agent capable of
modulating activity, function and/or expression of a PLCy in
the subject; and a pharmaceutically acceptable carrier.
According to yet another aspect of the present
invention, there is provided a composition for treatment or
prevention of pain in a subject, comprising: an agent capable
of modulating activity, function and/or expression of a PLCy in
the subject; and a pharmaceutically acceptable carrier.
According to a further aspect of the present
invention, there is provided a package comprising an agent
capable of modulating activity, function and/or expression of
a PLCY in a subject together with instructions for its use in
the treatment or prevention of pain.
According to yet a further aspect of the present
invention, there is provided a package comprising a
composition comprising: an agent capable of modulating
activity, function and/or expression of a PLCy in a subject;
and a pharmaceutically acceptable carrier, together with
instructions for its use in the treatment or prevention of
pain.
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According to still a further aspect of the present
invention, there is provided a use of an agent capable of
modulating activity, function and/or expression of a PLCy in a
subject for the treatment or prevention of pain.
According to another aspect of the present
invention, there is provided a use of an agent capable of
modulating activity, function and/or expression of a PLCy in a
subject for the preparation of a medicament for the treatment
or prevention of pain.
According to yet another aspect of the present
invention, there is provided a use, for the treatment or
prevention of pain, of a composition comprising: an agent
capable of modulating activity, function and/or expression of
a PLCy in a subject; and a pharmaceutically acceptable carrier.
According to another aspect of the present
invention, there is provided a method of identifying or
characterizing an agent for treatment or prevention of pain,
comprising determining PLCy activity, function and/or
expression in the presence of a test agent. In an embodiment,
the method comprises contacting the test agent with a PLCy or a
cell having an activity, function and/or expression of a PLCy;
and determining whether there is modulation (e.g. an
inhibition) of the activity, function and/or expression of a
PLCy in the presence of the agent; wherein the modulation is an
indication that the agent may be used for treatment or
prevention of pain.
The invention further provides a method of
identifying or characterizing a compound for treatment or
prevention of pain, said method comprising: (a) contacting a
test compound with a cell comprising a first nucleic acid
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comprising a transcriptionally regulatory element normally
associated with a PLCr gene, operably linked to a second
nucleic acid comprising a reporter gene capable of encoding a
reporter protein; and (b) determining whether reporter gene
expression or reporter protein activity is decreased in the
presence of said test compound; wherein said decrease in
reporter gene expression or reporter protein activity being is
an indication that said test compound may be used for
treatment or prevention of pain.
The invention further provides a method of
identifying or characterizing an agent for treatment or
prevention of pain, comprising:
contacting the agent and a PLCy ligand with a cell
comprising a PLCy; and
determining whether there is a decrease of the binding of
the ligand to the PLCy in the presence of the agent;
wherein the decrease is an indication that the agent may be
used for treatment or prevention of pain.
The invention further provides a method of
identifying or characterizing an agent for treatment or
prevention of pain, comprising:
contacting the agent and a PLCy ligand with a PLCy; and
determining whether there is decrease of the binding of
the ligand to the PLCr in the presence of the agent;
wherein the decrease is an indication that the agent may be
used for treatment or prevention of pain.
According to still another aspect of the present
invention, there is provided a method for decreasing
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nociception in a subject, comprising modulating activity,
function and/or expression of a PLCti, in the subject.
According to yet another aspect of the present
invention, there is provided a method for decreasing
5 nociception in a subject, comprising administering to the
subject an agent capable of modulating activity, function
and/or expression of a PLCy in the subject.
According to a further aspect of the present
invention, there is provided a method for decreasing
nociception in a subject, comprising administering to the
subject a composition comprising: an agent capable of
modulating activity, function and/or expression of a PLCy in
the subject; and a pharmaceutically acceptable carrier.
In an embodiment, the above-mentioned modulation of
PLCyactivity, function and/or expression is an inhibition of
PLC,activity or expression. In an embodiment the above-
mentioned agent capable of modulating PLCyactivity, function
and/or expression is an agent capable of inhibiting
PLCy,activity, function and/or expression.
In an embodiment, the agent as defined herein may be
a PLCYinhibitor, an anti-PLCyantibody, an antisense molecule,
a siRNA, a siRNA-like molecule, or an inhibitor of binding of
a ligand or binding partner to a PLCy. For example, the
PLCyinhibitor may be tricyclodecan-9-yl-xanthogenate, 1-0-
octadecyl-2-O-methyl-rac-glycero-3-phosphorylcholine, neomycin
sulfate, spermine tetrahydrochloride, 1-[6-((17beta-3-
methoxyestra-1,3,5(10)-trien--l7-yl)amino)hexyl]-1H-pyrrole-
2,5-dione, or 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-
yl)amino)hexyl]-2,5-pyrrolidinedione.
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In an embodiment, the antisense molecule is a
nucleic acid that is substantially complementary to a portion
of an mRNA encoding a PLCy. In an embodiment, the antisense
molecule is a nucleic acid that is substantially complementary
to a nucleic acid comprising a nucleotide sequence capable of
encoding a polypeptide having an amino acid sequence selected
from SEQ ID NO:2 and SEQ ID NO:4. In a further embodiment,
the antisense molecule is a nucleic acid that is substantially
complementary to a nucleic acid comprising a nucleotide
sequence selected from SEQ ID NO:1 and SEQ ID NO:3. In an
embodiment, the above-noted portion of an mRNA comprises at
least 5 contiguous bases.
In an embodiment, the siRNA or siRNA-like molecule
is substantially identical to a portion of an mRNA encoding
PLCy. In an embodiment, the s.iRNA or s.iRNA-like molecule is
substantially identical to a portion of an mRNA corresponding
to a DNA sequence capable of encoding a polypeptide having an
amino acid sequence selected from SEQ ID NO:2 and SEQ ID NO:4.
In a further embodiment, the siRNA or siRNA-like molecule is
substantially identical to a portion of an mRNA corresponding
to a DNA sequence selected from SEQ ID NO:1 and SEQ ID NO:3.
In an embodiment, the siRNA or siRNA-like molecule comprises
less than about 30 nucleotides, in a further embodiment about
21 to 23 nucleotides.
In embodiments, the pain is pain associated with
neuropathic pain and/or CNS dysfunction.
In a further embodiment, pain may be neuropathic
pain, somatic or visceral pain. For example, the neuropathic
pain may be associated with a nerve or tract injury. In yet
another embodiment, pain may be chronic inflammatory pain,
pain associated with arthritis, fibromyalgia, back pain,
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cancer-associated pain, pain associated with digestive
disease, pain associated with Crohn's disease, pain associated
with autoimmune disease, pain associated with endocrine
disease, pain associated with diabetic neuropathy, phantom
limb pain, spontaneous pain, chronic post-surgical pain,
chronic temporomandibular pain, causalgia, post-herpetic
neuralgia, AIDS-related pain, complex regional pain syndromes
type I and II, trigeminal neuralgia, chronic back pain, pain
associated with spinal cord injury or recurrent acute pain.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Comparison between the 50% withdrawal threshold for
pain hypersensitivity of rats treated with tricyclodecan-9-yl-
xanthogenate (0) and a vehicle (0).
Figure 2: DNA (SEQ ID N0:1) and polypeptide (SEQ ID NO:2)
sequences of human phospholipase C gamma 1 (PLCG1; accession
nos. NM002660 and NP002651). Coding sequence is defined by
positions 122-3997 of DNA sequence.
Figure 3: DNA (SEQ ID NO:3) and polypeptide (SEQ ID NO:4)
sequences of human phospholipase C gamma 2 (PLCG2; accession
nos. NM002661 and NP002652). Coding sequence defined by
positions 153-3911 of DNA sequence.
DETAILED DESCRIPTION OF THE INVENTION
The invention provides a method for the prevention
and/or treatment of pain in a subject. In embodiments, the
subject may be a vertebrate or a mammal. In an embodiment,
the subject is a mammal, for example a human. The method
comprises inhibiting or decreasing the activity, function
and/or expression of a phospholipase C gamma (PLCY) in a
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subject, e.g. in a cell or tissue of the subject, for example
a central nervous system (CNS) neural cell or tissue. The
method may comprise administering to the subject, systemic or
local, an agent capable of modulating, e.g. inhibiting,
activity, expression and/or function of a PLCY as a means to
attenuate pain. The agent may be administered before, at
about the time of, or subsequent to the onset of pain.
In an embodiment, the CNS neural cell in which the
PLCY activity, function and/or expression is modulated may be
located in the superficial dorsal horn or the spinal cord. In
addition, the cell may also be transsynaptic to a peripheral
nerve cell or sensory fiber from which a signal for pain
originates.
The invention also relates to the treatment of acute
and chronic pain, more specifically to the treatment of
neuropathic pain. "Neuropathic pain", as used herein, refers
to chronic pain associated with nerve injury (e.g. following
crush, transection or compression of nerves or following nerve
degeneration resulting from disease). Neuropathic pain may be
associated with a nerve or tract injury. Moreover, the
neuropathic pain may be associated with visceral and/or
somatic pain.
In an embodiments, the pain may be associated with a
condition chosen from chronic inflammatory pain, pain
associated with arthritis, fibromyalgia, back pain, cancer-
associated pain, pain associated with digestive disease, pain
associated with Crohn's disease, pain associated with
autoimmune disease, pain associated with endocrine disease,
pain associated with diabetic neuropathy, phantom limb pain,
spontaneous pain, chronic post-surgical pain, chronic
temporomandibular pain, causalgia, post-herpetic neuralgia,
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AIDS-related pain, complex regional pain syndromes type I and
II, trigeminal neuralgia, chronic back pain, pain associated
with spinal cord injury, recurrent acute pain, and/or any
combination of the above.
The invention further provides a method for
decreasing nociception in a subject. The method comprises
modulating activity, function and/or expression of a PLCy in a
subject to reduce nociception. "Nociception" as used herein
refers to the sensory component of pain. Pain may be the
result of various stimuli, including but not limited to
pressure, injury, thermal stimuli or chemical (e.g. ionic)
stimuli.
The agent includes, but is not limited to, that
which directly or indirectly modifies the activity of the
protein and that which modulates the production and/or
stability of the protein (e.g. at the level of transcription,
translation, maturation, post-translational modification,
phosphorylation and degradation). For example, the agent may
be a PLCy inhibitor. In an embodiment, such an inhibitor may
be a peptide or peptide-based compound (e.g. a
peptidomimetic). In further embodiments, the agent may be an
anti-PLCy antibody which is capable of modulating the binding
and/or catalytic activity of a PLCY. Examples of anti-PLC,
antibodies are described in for example Lee et al. (2002, Mol.
Vis., 8: 17-25) and Buckley et al. (2004, J. Biol. Chem., 279:
41807-14). In a further embodiment, the agent may be an
antisense molecule complementary to all or a portion of the
mRNA encoding a PLCy.. In a further embodiment, the agent may
be an siRNA or s.iRNA-Iike molecule.
In a further embodiment, the above-mentioned
inhibition is effected by the inhibition of a binding
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domain(s) of a PLC,. In such a case, the agent is an inhibitor
of binding of a ligand or binding partner to a PLCy. For
example, binding of mitogen-activated protein kinases [e.g.
extracellular signal-related kinase (ERK1 and ERK2)] to a PLCy
5 (e.g. to the D-domain) may be inhibited. As shown in Buckley
et al., 2004, J. Biol. Chem., 279:41807-41814, both ERK and
phospho-ERK (which, like PLCy are activated by BDNF binding of
TrkB) bind the D domain of PLCY and induce phosphorylation of
this isozyme.
10 In one embodiment, the agent capable of modulating
activity and/or expression of a PLC, is a PLCy inhibitor.
Examples of PLCY inhibitors include, but are not limited to,
tricyclodecan-9-yl-xanthogenate, 1-0-octadecyl-2-0-methyl-rac-
glycero-3-phosphorylchol.ine, neomycin sulfate, spermine
tetrahydrochloride, 1-[6-((17beta-3-methoxyestra-1,3,5(10)-
trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione, or 1-[6-
((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-
2,5-pyrrolidinedione. References describing the PLCy inhibiting
action of tricyclodecan-9-yl-xanthogenate (also referred to as
D609) include: Monick, M.M. et al., 1999, J. Immunol. 162,
3005; Maragoudakis, M.E. et al., 1993, Kidney Int. 43, 147;
Schutze, S. et al., 1992, Cell 71, 765; Muller-Decker, K.,
1989, B.iochem. Biophys. Res. Commun. 162, 198; Sauer, G. et
al., 1984, Proc. Nat1. Acad. Sci. USA 81, 3263. The aboved-
2'5 mentioned PLCy, inhibitors are available from EMD Biosciences (a
division of Calbiochem).
In another embodiment, the agent capable of
modulating activity and/or expression of PLCy is an anti-PLCy
antibody. Examples of anti-PLCY antibodies are described in
for example Lee et al. (2002, Mol. Vis., 8: 17-25) and Buckley
et al. (2004, J. Biol. Chem., 279: 41807-14). To prepare such
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an anti-PLCy antibody, a PLCr or fragment, homolog, and/or
variant thereof may be used to immunize a small mammal, e.g.,
a mouse or a rabbit, in order to raise antibodies which
recognize a PLCy. An anti-PLCy antibody may be either
polyclonal or monoclonal. Methods to produce polyclonal or
monoclonal antibodies are well known in the art. For a
review, see Harlow and Lane (1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY and Yelton et al. (1981) Ann. Rev. Biochem. 50:657-
690, both of which are herein incorporated by reference. For
monoclonal antibodies, see Kohler and Milstein (1975) Nature
256:495-497, herein incorporated by reference.
"PLC gamma" or "PLCy" refers to a polypeptide
possessing an activity capable of cleaving a
phosphotidylinositol 4,5 bisphosphate to yield diacylglycerol
and inositol 1,4,5-triphosphate. In embodiments, the PLCyis
selected from the PLCy1 and PLCy2 isoforms. Genes which encode
human PLCy polypeptides are described for example at NCBI
accession Nos. M34667, NM 002660, NM 002661 and NM 182811.
Examples of genes which encode human PLCY polypeptides as well
as the polypeptides they encode are set forth in Figures 2 and
3 and SEQ ID NOs:1-4. For a review of PLCy activity see
Carpenter and Ji (1999, Experimental Cell Research, 253: 15-
24). PLCy activity may be assayed by for example
electrophoretic mobility shift assays (Noh et al., 1998,
Anticancer Research, 18: 2643-8) and myo-inositol 1,4,5-
triphosphate radioceptor assays (Shu et al., 2002, J. Biol.
Chem., 277: 18447-18453).
As noted above, a fragment, homolog and/or variant
of a PLC, which retains activity may also be used in the
methods of the invention. Homologs include protein sequences
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which are substantially identical to the amino acid sequence
of a PLCY, sharing significant structural and functional
homology with a PLCy,. Variants include, but are not limited
to, proteins or peptides which differ from a PLCy by any
modifications, and/or amino acid substitutions, deletions or
additions. Modifications can occur anywhere including the
polypeptide backbone, (i.e. the amino acid sequence), the
amino acid side chains and the amino or carboxy termini. Such
substitutions, deletions or additions may involve one or more
amino acids. Fragments include a fragment or a portion of a
PLC, or a fragment or a portion of a homolog or variant of a
PLCy..
Antibodies may be recombinant, e.g., chimeric (e.g.,
constituted by a variable region of murine origin associated
with a human constant region), humanized (a human
immunoglobulin constant backbone together with hypervariable
region of animal, e.g., murine, origin), and/or single chain.
Both polyclonal and monoclonal antibodies may also be in the
form of immunoglobulin fragments, e. g. , F(ab) '2, Fab or Fab'
fragments. The antibodies may be of any isotype, e.g., IgG or
IgA; and polyclonal antibodies are of a single isotype or a
mixture of isotypes. Anti-PLCy antibodies may be produced and
identified using standard immunological assays, e.g., Western
blot analysis, dot blot assay, or ELISA.
Another class of compounds that can be used to limit
PLCr expression are compounds that lower the level of PLCy
transcripts. By doing so, these compounds limit the number of
PLCr polypeptides that can be produced and can therefore be use
to treat or prevent pain. These compounds include, but are
not limited to, dsRNA, siRNA, siRNA-like molecule, antisense
oligonucleotide or ribozyme.
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In an embodiment, expression of a nucleic acid
encoding a polypeptide of interest, or a fragment thereof, may
be inhibited or prevented using RNA interference (RNAi)
technology, a type of post-transcriptional gene silencing.
RNAi may be used to create a pseudo "knockout", i.e. a system
in which the expression of the product encoded by a gene or
coding region of interest is reduced, resulting in an overall
reduction of the activity of the encoded product in a system.
As such, RNAi may be performed to target a nucleic acid of
interest or fragment or variant thereof, to in turn reduce its
expression and the level of activity of the product which it
encodes. Such a system may be used for functional studies of
the product, as well as to treat disorders related to the
activity of such a product. RNAi is described in for example
Hammond et al. (2001), Sharp (2001), Caplen et al. (2001),
Sedlak (2000) and published US patent applications 20020173478
(Gewirtz; published November 21, 2002) and 20020132788 (Lewis
et al.; published November 7, 2002). Reagents and kits for
performing RNAi are available commercially from for example
Ambion Inc. (Austin, TX, USA) and New England Biolabs Inc.
(Beverly, MA, USA).
The initial agent for RNAi in some systems is
thought to be dsRNA molecule corresponding to a target nucleic
acid. The dsRNA is then thought to be cleaved into short
interfering RNAs (siRNAs) which are 21-23 nucleotides in
length (19-21 bp duplexes, each with 2 nucleotide 3'
overhangs). The enzyme thought to effect this first cleavage
step has been referred to as "Dicer" and is categorized as a
member of the RNase III family of dsRNA-specific
ribonucleases. Alternatively, RNAi may be effected via
directly introducing into the cell, or generating within the
cell by introducing into the cell a suitable precursor (e.g.
vector encoding precursor(s), etc.) of such an siRNA or siRNA-
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like molecule. A siRNA may then associate with other
intracellular components to form an RNA-induced silencing
complex (RISC). The RISC thus formed may subsequently target
a transcript of interest via base-pairing interactions between
its siRNA component and the target transcript by virtue of
homology, resulting in the cleavage of the target transcript
approximately 12 nucleotides from the 3' end of the siRNA.
Thus the target mRNA is cleaved and the level of protein
product it encodes is reduced.
RNA.i may be effected by the introduction of suitable
in vitro synthesized siRNA or siRNA-like molecules into cells.
RNAi may for example be performed using chemically-synthesized
RNA (Brown et al., 2002). Alternatively, suitable expression
vectors may be used to transcribe such RNA either in vitro or
in vivo. In vitro transcription of sense and antisense
strands (encoded by sequences present on the same vector or on
separate vectors) may be effected using for example T7 RNA
polymerase, in which case the vector may comprise a suitable
coding sequence operably-linked to a T7 promoter. The in
vitro-transcribed RNA may in embodiments be processed (e.g.
using E. coli RNase III) in vitro to a size conducive to RNAi.
The sense and antisense transcripts are combined to form an
RNA duplex which is introduced into a target cell of interest.
Other vectors may be used, which express small hairpin RNAs
(shRNAs) which can be processed into siRNA-like molecules.
Various vector-based methods are described in for example
Brummelkamp et al. (2002), Lee et al. (2002), Miyagashi and
Taira (2002), Paddison et al. (2002) Paul et al. (2002) Sui et
al. (2002) and Yu et al. (2002). Various methods for
introducing such vectors into cells, either in vitro or in
vivo (e.g. gene therapy) are known in the art.
Accordingly, in an embodiment expression of a
nucleic acid encoding a PLCY, or a fragment thereof, may be
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inhibited by introducing into or generating within a cell an
siRNA or siRNA-like molecule corresponding to a nucleic acid
encoding a polypeptide of interest, or a fragment thereof, or
to an nucleic acid homologous thereto. "siRNA-like molecule"
5 refers to a nucleic acid molecule similar to an siRNA (e.g. in
size and structure) and capable of eliciting siRNA activity,
i.e. to effect the RNAi.-mediated inhibition of expression. In
various embodiments such a method may entail the direct
administration of the siRNA or siRNA-like molecule into a
10 cell, or use of the vector-based methods described above. In
an embodiment, the siRNA or siRNA-like molecule is less than
about 30 nucleotides in length. In a further embodiment, the
siRNA or siRNA-like molecule is about 21-23 nucleotides in
length. In an embodiment, siRNA or siRNA-like molecule
15 comprises a 19-21 bp duplex portion, each strand having a 2
nucleotide 3' overhang. In embodiments, the siRNA or siRNA-
like molecule is substantially identical to a nucleic acid
encoding a PLCy, or a fragment or variant (or a fragment of a
variant) thereof. Such a variant is capable of encoding a
protein having activity similar to a PLCy. In embodiments, the
sense strand of the siRNA or siRNA-like molecule is
substantially identical to SEQ ID NOs: 1 or 3, or a fragment
thereof (RNA having U in place of T residues of the DNA
sequence).
In yet another embodiment, transformation of cells
with antisense constructs may be used to inhibit expression of
a PLCy. Antisense constructs are nucleic acid molecules that
may be transcribed to provide an antisense molecule that is
substantially complementary to all or a portion of the mRNA
encoding a PLCr, so that expression of the antisense construct
interferes with the expression of the PLCr. In an embodiment,
the just noted antisense molecule is antisense to a DNA
sequence coding a PLCY, in an embodiment, a human PLCy. In
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some embodiments, antisense constructs of the invention may
therefore encode five or more contiguous nucleic acid-residues
substantially complimentary to a contiguous portion of a
nucleic acid sequence encoding a PLCy.
In further embodiments, polypeptides and nucleic acids
which are substantially identical to those noted herein may be
utilized in the context of the present invention.
"Homology" and "homologous" refers to sequence
similarity between two peptides or two nucleic acid molecules.
Homology can be determined by comparing each position in the
aligned sequences. A degree of homology between nucleic acid
or between amino acid sequences is a function of the number of
identical or matching nucleotides or amino acids at positions
shared by the sequences. As the term is used herein, a
nucleic acid sequence is "homologous" to another sequence if
the two sequences are substantially identical and the
functional activity of the sequences is conserved (as used
herein, the term 'homologous' does not infer evolutionary
relatedness). Two nucleic acid sequences are considered
substantially identical if, when optimally aligned (with gaps
permitted), they share at least about 50% sequence similarity
or identity, or if the sequences share defined functional
motifs. In alternative embodiments, sequence similarity in
optimally aligned substantially identical sequences may be at
least 60%, 70%, 75%, 80%, 85%, 90% or 95%. As used herein, a
given percentage of homology between sequences denotes the
degree of sequence identity in optimally aligned sequences.
An "unrelated" or "non-homologous"- sequence shares less than
40% identity, though preferably less than about 25 % identity,
with any of SEQ ID NOs 1-4.
Substantially complementary nucleic acids are
nucleic acids in which the "complement" of one molecule is
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substantially identical to the other molecule. Qptimal
alignment of sequences for comparisons of identity may be
conducted using a variety of algorithms, such as the local
homology algorithm of Smith and Waterman, 1981, Adv. App1.
Math 2: 482, the homology alignment algorithm of Needleman and
Wunsch, 1970, J. Mol. Biol. 48:443, the search for similarity
method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA
85: 2444, and the computerised implementations of these
algorithms (such as GAP, BESTFIT, FASTA and TFASTA in the
Wisconsin Genetics Software Package, Genetics Computer Group,
Madison, WI, U.S.A.). Sequence identity may also be determined
using the BLAST algorithm, described in Altschul et al., 1990,
J. Mol. Biol. 215:403-10 (using the published default
settings). Software for performing BLAST analysis may be
available through the National Center for Biotechnology
Information (through the internet at
http://www.ncbi.nlm.nih.gov/). The BLAST algorithm involves
first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence that
either match or satisfy some positive-valued threshold score T
when aligned with a word of the same length in a database
sequence. T is referred to as the neighbourhood word score
threshold. Initial neighbourhood word hits act as seeds for
initiating searches to find longer HSPs. The word hits are
extended in both directions along each sequence for as far as
the cumulative alignment score can be increased. Extension of
the word hits in each direction is halted when the following
parameters are met: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the
cumulative score goes to zero or below, due to the
accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The
BLAST algorithm parameters W, T and X determine the
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sensitivity and speed of the alignment. The BLAST program may
use as defaults a word length (W) of 11, the BLOSUM62 scoring
matrix (Henikoff and Henikoff, 1992, Proc. Natl. Acad. Sci. ,
USA 89: 10915-10919) alignments (B) of 50, expectation (E) of
10 (or 1 or 0.1 or 0.01 or 0.001 or 0.0001), M=5, N=4, and a
comparison of both strands. One measure of the statistical
similarity between two sequences using the BLAST algorithm is
the smallest sum probability (P(N)), which provides an
indication of the probability by which a match between two
nucleotide or amino acid sequences would occur by chance. In
alternative embodiments of the invention, nucleotide or amino
acid sequences are considered substantially identical if the
smallest sum probability in a comparison of the test sequences
is less than about 1, preferably less than about 0.1, more
preferably less than about 0.01, and most preferably less than
about 0.001.
Ari alternative indication that two nucleic acid
sequences are substantially complementary is that the two
sequences hybridize to each other under moderately stringent,
or preferably stringent, conditions. Hybridization to filter-
bound sequences under moderately stringent conditions may, for
example, be performed in 0.5 M NaHPO4r 7% sodium dodecyl
sulfate (SDS), 1 mM EDTA at 65 C, and washing in 0.2 x
SSC/0.1% SDS at 42 C (see Ausubel, et al. (eds), 1989, Current
Protocols in Molecular Biology, Vol. 1, Green Publishing
Associates, Inc., and John Wiley & Sons, Inc., New York, at p.
2.10.3). Alternatively, hybridization to filter-bound
sequences under stringent conditions may, for example, be
performed in 0.5 M NaHPO9r 7% SDS, 1 mM EDTA at 65 C, and
washing in 0.1 x SSC/0.1% SDS at 68 C (see Ausubel, et al.
(eds), 1989, supra). Hybridization conditions may be modified
in accordance with known methods depending on the sequence of
interest (see Tijssen, 1993, Laboratory Techniques in
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Biochemistry and Molecular Biology -- Hybridization with
Nucleic Acid Probes, Part I, Chapter 2 "Overview of principles
of hybridization and the strategy of nucleic acid probe
assays", Elsevier, New York). Generally, stringent conditions
are selected to be about 5 C lower than the thermal melting
point for the specific sequence at a defined ionic strength
and pH.
In alternative embodiments, the invention provides
antisense molecules and ribozymes for exogenous administration
to bind to, degrade and/or inhibit the translation of PLCy
mRNA. Examples of therapeutic antisense oligonucleotide
applications, incorporated herein by reference, include: U.S.
Pat. No. 5,135,917, issued Aug. 4, 1992; U.S. Pat. No.
5,098,890, issued Mar. 24, 1992; U.S. Pat. No. 5,087,617,
issued Feb. 11, 1992; U.S. Pat. No. 5,166,195 issued Nov. 24,
1992; U.S. Pat. No. 5,004,810, issued Apr. 2, 1991; U.S. Pat.
No. 5,194,428, issued Mar. 16, 1993; U.S. Pat. No. 4,806,463,
issued Feb. 21, 1989; U.S. Pat. No. 5,286,717 issued Feb. 15,
1994; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423;
BioWorld Today, Apr. 29, 1994, p. 3.
Preferably, in antisense molecules, there is a
sufficient degree of complementarity to the PLCr mRNA to avoid
non-specific binding of the antisense molecule to non-target
sequences under conditions in which specific binding is
desired, such as under physiological conditions in the case of
in vivo assays or therapeutic treatment or, in the case of in
vitro assays, under conditions in which the assays are
conducted. The target mRNA for antisense binding may include
not only the information to encode a protein, but also
associated ribonucleotides, which for example form the 5'-
untranslated region, the 3'-untranslated region, the 5' cap
region and intron/exon junction ribonucleotides. A method of
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screening for antisense and ribozyme nucleic acids that may be
used to provide such molecules as inhibitors of the invention
is disclosed in U.S. Patent No. 5,932,435 (which is
incorporated herein by reference).
5 Antisense molecules (oligonucleotides) of the
invention may include those which contain intersugar backbone
linkages such as phosphotriesters, methyl phosphonates, short
chain alkyl or cycloalkyl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages,
10 phosphorothioates and those with CH2--NH--O--CH2r CH2--N (CH3) --
0--CH2 (known as methylene(methylimino) or MMI backbone), CH2-
-0--N ( CH3 ) --CHZ, CH2--N ( CH3 ) --N ( CH3 ) --CH2 and 0--N ( CH3 ) --CH2 --
CH2 backbones (where phosphodiester is 0--P--O--CH2).
Oligonucleotides having morpholino backbone structures may
15 also be used (U.S. Pat. No. 5,034,506). In alternative
embodiments, antisense oligonucleotides may have a peptide
nucleic acid (PNA, sometimes referred to as "protein nucleic
acid") backbone, in which the phosphodiester backbone of the
oligonucleotide may be replaced with a polyamide backbone
20 wherein nucleosidic bases are bound directly or indirectly to
aza nitrogen atoms or methylene groups in the polyamide
backbone (Nielsen et al., 1991, Science 254:1497 and U.S. Pat.
No. 5,539,082). The phosphodiester bonds may be substituted
with structures that are chiral and enantiomerically specific.
Persons of ordinary skill in the art will be able to select
other linkages for use in practice of the invention.
Oligonucleotides may also include species which
include at least one modified nucleotide base. Thus, purines
and pyrimidines other than those normally found in nature may
be used. Similarly, modifications on the pentofuranosyl
portion of the nucleotide subunits may also be effected.
Examples of such modifications are 2'-O-alkyl- and 2'-halogen-
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substituted nucleotides. Some specific examples of
modifications at the 2' position of sugar moieties which are
useful in the present invention are OH, SH, SCH3, F, OCN,
O(CH2) n NH2 or O(CH2) n CH3 where n is from 1 to about 10; C1 to
Clo lower alkyl, substituted lower alkyl, alkaryl or aralkyl;
Cl; Br; CN; CF3 ; OCF3 ; 0-, S-, or N'-alkyl; 0-, S-, or N-
alkenyl; SOCH3 ; SOZ CH3; ON02 ; NO2 ; N3; NH2; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino;
substituted silyl; an RNA cleaving group; a reporter group; an
intercalator; a group for improving the pharmacokinetic
properties of an oligonucleotide; or a group for improving the
pharmacodynamic properties of an oligonucleotide and other
substituents having similar properties. One or more
pentofuranosyl groups may be replaced by another sugar, by a
sugar mimic such as cyclobutyl or by another moiety which
takes the place of the sugar.
In some embodiments, the antisense oligonucleotides
in accordance with this invention may comprise from about 5 to
about 100 nucleotide units. As will be appreciated, a nucleotide
unit is a base-sugar combination (or a combination of analogous
structures) suitably bound to an adjacent nucleotide unit
through phosphodiester or other bonds forming a backbone
structure.
The invention also provides pharmaceutical
compositions (medicaments) comprising an agent capable of
modulating activity and/or expression of a PLCY, and a
pharmaceutically acceptable carrier. In an embodiment, such
compositions include the agent, in a therapeutically or
prophylactically effective amount sufficient to treat or
attenuate pain, and a pharmaceutically acceptable carrier.
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A "therapeutically effective amount" refers to an
amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic result, such as
reduction of pain. A therapeutically effective amount of an
agent capable of modulating activity and/or expression of a
PLCy, may vary according to factors such as the disease state,
age, sex, and weight of the individual, and the ability of the
agent to elicit a desired response in the individual. Dosage
regimens may be adjusted to provide the optimum therapeutic
response. A therapeutically effective amount is also one in
which any toxic or detrimental effects of the agent are
outweighed by the therapeutically beneficial effects. A
"prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to
achieve the desired prophylactic result, such as preventing or
inhibiting onset of pain or increases in the severity of pain.
A prophylactically effective amount can be determined as
described above for the therapeutically effective amount. For
any particular subject, specific dosage regimens may be
adjusted over time according to the individual need and the
professional judgement of the person administering or
supervising the administration of the compositions.
As used herein "pharmaceutically acceptable carrier",
or "excipient" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like that are
physiologically compatible. In one embodiment, the carrier is
suitable for parenteral administration. Alternatively, the
carrier can be suitable for intravenous, intraperitoneal,
intramuscular, intracranial, intrathecal, sublingual or oral
administration. Pharmaceutically acceptable carriers include
sterile aqueous solutions or dispersions and sterile powders
for the extemporaneous preparation of sterile injectable
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solutions or dispersion. The use of such media and agents for
pharmaceutically active substances is well known in the art.
Except insofar as any conventional media or agent is
incompatible with the active agent, use thereof in the
pharmaceutical compositions of the invention is contemplated.
Supplementary active compounds can also be incorporated into
the compositions.
Therapeutic compositions typically must be sterile
and stable under the conditions of manufacture and storage.
The composition can be formulated as a solution,
microemulsion, liposome, or other ordered structure suitable
to high drug concentration. The carrier can be a solvent or
dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example,
by the use of a coating such as lecithin, by the maintenance
of the required particle size in the case of dispersion and by
the use of surfactants. In many cases, it will be preferable
to include isotonic agents, for example, sugars, polyalcohols
such as mannitol, sorbitol, or sodium chloride in the
composition. Prolonged absorption of the injectable
compositions can be brought about by including in the
composition an agent which delays absorption, for example,
monostearate salts and gelatin. Moreover, the agent capable
of modulating activity and/or expression of a PLCY, can be
administered in a time release formulation, for example in a
composition which includes a slow release polymer. The active
compounds can be prepared with carriers that will protect the
compound against rapid release, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used,
such as ethylene vinyl acetate, polyanhydrides, polyglycolic
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acid, collagen, polyorthoesters, polylactic acid and
polylactic, polyglycolic copolymers (PLG). Many methods for
the preparation of such formulations are patented or generally
known to those skilled in the art.
Sterile injectable solutions can be prepared by
incorporating the active agent (e.g. an agent capable of
modulating activity and/or expression of a PLCY) in the
required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally, dispersions
are prepared by incorporating the active agent into a sterile
vehicle which contains a basic dispersion medium and the
required other ingredients from those enumerated above. In
the case of sterile powders for the preparation of sterile
injectable solutions, the preferred methods of preparation are
vacuum drying and freeze-drying which yields a powder of the
active ingredient plus any additional desired ingredient from
a previously sterile-filtered solution thereof. In accordance
with an alternative aspect of the invention, an agent capable
of mQdulating activity and/or expression of a PLCY in a
subject, may be formulated with one or more additional
compounds that enhance its solubility.
In another embodiment, an agent of the invention is
administered such that it comes into contact with a CNS tissue
or a CNS neuron. As used herein, the "central nervous system"
or CNS is the portion of the nervous system comprising the
brain and the spinal cord (e.g. in the lumbar region). By
contrast, the "peripheral nervous system" or PNS is the
portion of the nervous system other than the brain and the
spirial cord. In a further embodiment, the CNS tissue is the
superficial dorsal horn, in a further embodiment, a lamina I
neuron. As such, in embodiments an agent of the invention can
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be administered to treat CNS cells in vivo via direct
intracranial or intrathecal injection or injection into the
cerebrospinal fluid. Alternatively, the agent can be
administered systemically (e.g. intravenously, or orally) in a
5 form capable of crossing the blood brain barrier and ente-ring
the CNS. "Neural" and "neuronal" are used herein
interchangeably and both relate to neurons and the nervous
system.
In a further embodiment of the present invention,
10 therapeutic compositions of the present invention, comprising
an agent capable of decreasing/inhibiting activity and/or
expression of a PLCy in a CNS cell may be provided in
kits/containers or packages (e.g. commercial packages) which
further comprise instructions for their use for the treatment
15 of pain. Similarly, the invention provides a package
comprising an agent capable of decreasing/inhibiting activity
and/or expression of a PLCy together with instructions for the
prevention and/or treatment of pain.
The invention further provides a use of an agent
20 capable of decreasing/inhibiting activity and/or expression of
a PLCy or a composition comprising an agent capable of
decreasing/inhibiting activity and/or expression of a PLCY for
the prevention and/or treatment pain, or for the preparation
of a medicament for the prevention and/or treatment of pain.
25 In another aspect, the invention relates to the use
of PLCy as a target in screening methods that may be used for
the identification and characterization of agents capable of
decreasing/inhibiting activity and/.or expression of a PLCy.
Therefore, the invention further provides a method of
determining whether a candidate agent is capable of modulating
activity and/or expression of a PLCy in a subject, and in turn
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is useful for the prevention and treatment of pain. In an
embodiment, the method comprises contacting a cell (e.g. a
CNS-derived cell) with the candidate agent and determining
whether PLCy activity has been decreased or inhibited in the
presence of the test agent. In a further embodiment, the
assay may be carried out in a cell-free system, by providing a
suitable preparation or sample of active PLCY, contacting the
PLCr with the candidate agent, and similarly determining
whether its activity is inhibited or decreased in the presence
of the test compound. In a further embodiment, the assay may
comprise assessing whether a candidate compound inhibits the
binding of a binding partner or ligand to a PLCy. In such a
case, the method comprises contacting a PLC, or a cell
comprising a PLCy with a candidate agent and the binding
partner or ligand, and determining whether the binding of the
binding partner or ligand to the PLCy is decreased in the
presence of the candidate or test compound. In embodiments,
the binding partner or ligand is a mitogen-activated protein
kinase or homolog or fragment thereof (such as ERK1 or ERK2,
or a homolog or fragment thereof). In an embodiment, binding
to the D-domain of PLCy is determined.
Inhibition or a decrease of PLCr activity or of the
above-mentioned binding is indicative that the test agent may
be used for the treatment or the prevention of pain.
As used herein, a "CNS-derived cell" is a cell
isolated or derived from a CNS tissue, and in embodiments
includes both primary neuronal cultures, immortalized neuronal
cell lines, as well as accepted in vitro neuronal model
systems (e.g. cells differentiated into neurons in vitro). In
an embodiment, the above-mentioned cell possesses a PLCY
activity.
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The above-mentioned method may be employed either
with a single test agent or a plurality or library (e.g. a
combinatorial library) of test agents. In the latter case,
synergistic effects provided by combinations of agents may
also be identified and characterized. The above-mentioned
agents may be used for prevention and/or treatment of pain, or
may be used as lead agents for the development and testing of
additional agents having improved specificity, efficacy and/or
pharmacological (e.g. pharmacokinetic) properties. In an
embodiment the agent may be a prodrug which is altered into
its active form at the appropriate site of action, e.g. in CNS
tissue (e.g. in the spinal cord). In certain embodiments, one
or a plurality of the steps of the screening/testing methods
of the invention may be automated.
The invention further relates to methods for the
identification and characterization of agents capable of
decreasing/inhibiting PLC, gene expression. Such a method may
comprise determining PLC7 gene expression in the presence
versus the absence of a test agent. Sizch gene expression may
be determined by detection of the corresponding RNA or
protein, or via the use of a suitable reporter construct
comprising a transcriptional regulatory element(s) normally
associated with such PLCy gene, operably-linked to a reporter
gene. A first nucleic acid sequence may "operably-linked"
with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is
operably-linked to a coding sequence if the promoter affects
the transcription or expression of the coding sequences.
Generally, operably-linked DNA sequences are contiguous and,
where necessary to join two protein coding regions, in reading
frame. However, since, for example, enhancers generally
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function when separated from the promoters by several
kilobases and intronic sequences may be of variable lengths,
some polynucleotide elements may be operably-linked but not
contiguous. "Transcriptional regulatory element" is a generic
term that refers to DNA sequences, such as initiation and
termination signals, enhancers, and promoters, splicing
signals, polyadenylation signals which induce or control
transcription of protein coding sequences with which they are
operably-linked. The expression of such a reporter gene may
be measured on the transcriptional or translational level,
e.g. by the amount of RNA or protein produced. RNA may be
detected by for example Northern analysis or by the reverse
transcriptase-polymerase chain reaction (RT-PCR) method (see
for example Sambrook et al (1989) Molecular Cloning: A
Laboratory Manual (second edition), Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, USA). Protein
levels may be detected either directly using affinity reagents
(e.g. an antibody or fragment thereof [for methods, see for
example Harlow, E. and Lane, D(1988) Antibodies: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, NY]; a ligand which binds the protein) or by other
properties (e.g. fluorescence in the case of green fluorescent
protein) or by measurement of the protein's activity, which
may entail enzymatic activity to produce a detectable product
(e.g. with altered spectroscopic properties) or a detectable
phenotype (e.g. alterations in cell growth). Suitable
reporter genes include but are not limited to chloramphenicol
acetyltransferase, beta-D galactosidase, luciferase, or green
fluorescent protein.
The invention further provides a method for
diagnosing or prognosticating pain associated with CNS
dysfunction. In an embodiment, the pain associated with such
CNS dysfunction is neuropathic pain. In an embodiment, the
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method comprises determining whether there is modulation of
activity and/or expression of a PLCy in a CNS neural cell
relative to a corresponding control activity and/or expression
of PLCr. In this particular method, a difference in the test
level relative to a control level is an indication that the
subject is experiencing pain associated with CNS dysfunction.
In an embodiment, the method may comprise determining whether
PLC7 activity and/or expression is modulated relative to a
control activity and/or expression. In yet another
embodiment, the PLC7 activity and/or expression can be selected
from an established standard, such as a corresponding PLCy
activity and/or expression level determined in the subject at
an earlier time; a corresponding PLCy activity and/or
expression level determined in said subject when the subject
is experiencing less pain (relative to the current sensation
of pain noted above) or substantially no pain; or a
corresponding PLCy activity and/or expression level determined
in a control subject experiencing less pain (relative to the
current sensation of pain in the test subject noted above) or
substantially no pain. In an embodiment, a subject or control
subject experiencing less pain or substantially no pain
Tresents no evident lesions to his central or peripheral
nervous system (e.g. neuropathic pain) or persistent pain.
For example, PLCr activity and/or expression may be
determined by administering, to a subject, an indicator
compound (such as a compound indicative of activity and/or
expression of PLCy) that is capable of contacting a CNS neural
cell of that subject. Following the administration of the
indicator compound, assessment of the in vivo signal
associated with such indicator compound may be performed. In
an embodiment, an indicator compound, such as an
immunodetection-based reagent (e.g. antibody, single chain
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antibody or Fab fragment directed against the PLC, polypeptide)
or a suitable substrate which yields detectable products as a
result of PLCy activity, may be employed. Following injection
of the indicator compound, an imaging technique may be
5 performed to assess the in vivo signal associated with the
indicator compound. The imaging technique may enable the
assessment of the in vivo signal of the indicator compound.
In an embodiment, the methods of
diagnosis/prognostication noted above may be performed in
10 conjunction with the therapeutic/prophylactic methods noted
above, for preventing or treating pain in a subject. Such a
method thus comprises the diagnosis or prognostication of pain
and modulation of activity and/or expression of a PCLy in the
subject, thereby to prevent or treat pain.
15 Although various embodiments of the invention are
disclosed herein, many adaptations and modifications may be
made within the scope of the invention in accordance with the
common general knowledge of those skilled in this art. Such
modifications include the substitution of known equivalents
20 for any aspect of the invention in order to achieve the same
result in substantially the same way. Numeric ranges are
inclusive of the numbers defining the range. In the claims,
the word "comprising" is used as an open-ended term,
substantially equivalent to the phrase "including, but not
25 limited to". The following examples are illustrative of
various aspects of the invention, and do not limit the broad
aspects of the invention as disclosed herein.
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EXAMPLES
Example 1:
Eleven adult, Sprague-Dawley rats received an
experimental chronic constriction injury to the sciatic nerve
(Mosconi & Kruger, 1996, Pain 64:37-57). Pain
hypersensitivity, indicative of neuropathic pain, increased
progressively over a two week interval and was monitored using
the Von Frey technique (Chaplan et a1., 1994, J. Neurosci.
Methods 53:55-63). Following the development of allodynia
(characterized by a 50% withdrawal threshold (WD50) of -< 2.0
g), rats were administered 500 ng tricyclodecan-9-yl-
xanthogenate by intrathecal catheter, the tricyclodecan-9-yl-
xanthogenate was prepared in a solution of saline (0.9% NaCl)
with 10% v/v dimethyl sulfoxide. Tricyclodecan-9-yl-
xanthogenate is available from EMD Biosciences (a division of
Calbiochem). A solution of saline (0.9% NaCl) with 10% v/v
dimethyl sulfoxide was injected into control rats as a
vehicle. The volume of the vehicle administered was the same
as for the PLCy inhibitor solution.
Seven allodynic rats received single intrathecal
injections of a vehicle, whereas 4 rats received single
injections of tricyclodecan-9-yl-xanthogenate; the mean WD50
for both groups was 1.9 0.2 g prior to injections. In as
little as 4 hours post-injection, the WD50 of rats administered
tricyclodecan-9-yl-xanthogenate was significantly different
from those that received the vehicle (tricyclodecan-9-yl-
xanthogenate, 8.8 2.1 g vs. vehicle, 4.1 0.7 g; p < 0.05),
and significantly different from the initial, pre-injection
WD50 (p < 0.05, mixed design ANOVA). This significant
reduction of pain hypersensitivity persisted for greater than
6 hours.
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Throughout this application, various references are
referred to describe more fully the state of the art to which
this invention pertains. The disclosures of these references
are hereby incorporated by reference into the present
disclosure.
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