Method for preventing or treating neuropathic pain
United States Patent: 7,943,588
Issued: May 17, 2011
Inventors: DeLeo; Joyce A.
(Lebanon, NJ), Tanga; Flobert Y. (Medford, MA)
Assignee: Trustees of
Dartmouth College (Hanover, NH)
Appl. No.: 11/691,783
Filed: March 27, 2007
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The present invention is a method for
preventing or treating neuropathic pain. Using an agent to decrease the
expression or activity of Toll-like receptor 4 (TLR4), behavioral
hypersensitivity is attenuated thereby preventing or treating neuropathic
pain in a subject in need of such treatment.
Description of the
BACKGROUND OF THE INVENTION
Neuropathic pain remains a prevalent, persistent, and debilitating
problem. Attempts to elucidate its mechanisms have focused principally on
peripheral nerves, dorsal root ganglion, and central nervous system (CNS)
neurons. Research efforts have expanded into the burgeoning field of glial/neuronal
transmission and CNS immunologic responses to nerve injury. CNS glia
display immune cell functions in both normal and pathologic conditions,
and there is increasing evidence that neuropathic pain arising from nerve
injury has a CNS neuroimmune component (DeLeo & Yezierski (2001) Pain
90:1-6; DeLeo, et al. (2004) Neuroscientist 10:40-52). Spinal glial
activation triggers rapid, graded CNS expression of proinflammatory
cytokines (including TNF-.alpha., IL-1.beta., and IL-6) that contributes
to the initiation and maintenance of behavioral hypersensitivity after L5
nerve transection (DeLeo & Yezierski (2001) supra; Vuong, et al. (2004)
Cell. Microbiol. 6:269-275; Raghavendra, et al. (2004)
Neuropsychopharmacology 29:327-334). The onset of proinflammatory cytokine
expression correlates with microglial activation and the initiation of
behavioral hypersensitivity (Sommer, et al. (1993) J. Neuropathol. Exp.
Neurol. 52:223-233; Sommer & Myers (1995) Acta Neuropathol. 90:478-485;
Popovich, et al. (1997) J. Comp. Neurol. 377:443-464; Popovich, et al.
(1997) J. Neuropathol. Exp. Neurol. 56:1323-1338), and neuroimmune
activation in painful neuropathy has been established (Vuong, et al.
(2004) supra; Bennett (1999) Proc. Natl. Acad. Sci. USA 96:7737-7738;
Bennett (2000) Clin. J. Pain 16:S139-S143). However, the mechanistic links
between nerve injury, microglial activation, and the genesis of behavioral
hypersensitivity is unclear.
Cells of the innate immune system, including monocytes/macrophages,
natural killer cells, neutrophils, and microglia recognize invariant
molecular structures of pathogens (termed pathogen-associated molecular
patterns, PAMP) by means of stable, genetically conserved,
pattern-recognition receptors on the cell surface. The genes producing
these receptors are homologous to the Toll gene in Drosophila and are
therefore termed Toll-like receptors (Akira & Sato (2003) Scand J. Infect.
Dis. 35:555-562). Toll-like receptor 4 (TLR4) is a transmembrane receptor
protein with extracellular leucine-rich repeat domains and a cytoplasmic
signaling domain. TLR4 expression has been demonstrated in the rodent CNS
(Laflamme & Rivest (2001) FASEB J. 15:155-163; Eklind, et al. (2001) Eur.
J. Neurosci. 13:1101-1106), where in vivo and in vitro studies show that
TLR4 is expressed by microglia (Lehnardt, et al. (2002) J. Neurosci.
22:2478-2486; Lehnardt, et al. (2003) Proc. Natl. Acad. Sci. USA
100:8514-8519). Lipopolysaccharide (LPS, a well known exogenous ligand for
TLR4) and potential endogenous ligands for TLR4 (e.g., members of the heat
shock protein family and proteoglycans) lead to NF-.kappa.B activation and
subsequent induction of proinflammatory cytokines (Vabulas, et al. (2002)
J. Biol. Chem. 277:15107-15112; Tsan & Gao (2004) Am. J. Physiol.
286:C739-C744). TLR4 gene expression is also significantly increased
during all phases of inflammation and is suggested to be related to nerve
injury-induced behavioral hypersensitivity (Raghavendra, et al. 2004. Eur.
J. Neurosci. 20:467-473). Moreover, increased spinal microglial TLR4
activation correlates with the onset of behavioral hypersensitivity in
rats after injury to the L5 spinal nerve, even in the absence of exogenous
TLR4 ligands such as LPS (Tanga, et al. (2004) Neurochem. Int.
Rudofsky, et al. ((2004) Diabetes Care 27:179-183) further teach that type
II diabetics that are carriers of polymorphic forms of two TLR4 genes have
a lower prevalence of diabetic neuropathy, wherein WO 2005/068442
discloses the use of certain compounds to treat pain in patients without
SUMMARY OF THE INVENTION
The present invention relates to a method of preventing or treating
individuals suffering from neuropathic pain. The method involves
administering to a subject in need of treatment an effective amount of an
agent that decreases the expression or activity of TLR4 thereby preventing
or treating neuropathic pain in the subject.
DETAILED DESCRIPTION OF THE INVENTION
It has now been found that modulators of TLR4 can be used to attenuate
behavioral hypersensitivity associated with neuropathic pain. Functional
links between TLR4, microglial activation, and the initiation of
behavioral hypersensitivity were assessed by analyzing glial activation
and behavioral hypersensitivity after spinal L5 nerve transection in
wild-type mice and in genetically manipulated mice lacking normal TLR4
expression. Two mouse strains with distinct genetic defects at the TLR4
loci were used, namely a TLR4 knockout (KO) mouse (C57BL/10ScNJ), which
has a complete deletion of the TLR4 gene and thus cannot synthesize TLR4
mRNA or protein, and the TLR4 point-mutant mouse (C3H/HeJ), which
expresses only a mutated (nonfunctional) TLR4 protein because of an amino
acid substitution at position 712 in the third exon of the TLR4 gene (Poltorak,
et al. (1998) Science 282:2085-2088; Poltorak, et al. (2000) Proc. Natl.
Acad. Sci. USA 97:2163-2167). Glial activation and behavioral
hypersensitivity after L5 nerve transection were also assessed in normal
rats and in rats injected intrathecally with antisense
oligodeoxynucleotide (ODN) to decrease CNS expression of TLR4. Both series
of experiments established a role for TLR4 and CNS innate neuroimmune
activation in the onset of behavioral hypersensitivity.
To demonstrate that TLR4 was critical for pain induction, TLR4 KO mice
(C57BL/10SCNJ; n=21) and point-mutant mice were analyzed. TLR4 KO mice
(C57BL/10SCNJ; n=21) and point-mutant mice (C3H/HeJ; n=8) displayed
significantly attenuated mechanical allodynia compared with their
respective wild-type controls (C57BL10/ScSnJ; n=15 and C3H/HeN; n=7) for
0.008-g (P<0.001) and 0.015-g (P<0.001) von Frey filament stimulations
beginning at day 1 after surgery (FIGS. 1A and 1B (see Original Patent)).
FIG. 1C (see Original Patent) shows that TLR4 KO and point-mutant mice
also displayed a significantly attenuated response to heat for days 1-7
(P<0.05) and 10-14 after surgery (P<0.001). FIG. 1D (see Original Patent)
shows that the tail-flick response to immersion in 49.degree. C. water was
also significantly attenuated for these mice for days 1-7 (P<0.05) and
10-14 after surgery (P<0.001).
To determine whether knockout of TLR4 could attenuate spinal glial
activation, expression of CD11b and CD14 was analyzed. A 3- to 5-fold
decrease in expression of mRNA for CD11b and CD14 was observed in TLR4 KO
mice compared with the wild-type control group beginning at day 3 after
surgery (P<0.001). A graded, significant up-regulation of TLR4 mRNA was
observed in the wild-type control group (P<0.001). No TLR4 mRNA was
detected in the TLR4 KO mice. Messenger RNA for the astrocytic activation
marker glial fibrillary acid protein (GFAP) also decreased in TLR4 KO mice
relative to the wild-type control group, starting at day 7 after surgery
A link between innate immunity and spinal proinflammatory cytokine
expression was also established. Significantly (3- to 5-fold) lower spinal
expression of mRNA for IFN-.gamma., IL-1.beta., and TNF-.alpha. was
observed after injury in the TLR4 KO mice as compared with the wild-type
control group (P<0.001).
In injured wild-type mice, a robust immunoreactive staining of CD11b/CR3
throughout the dorsal horn ipsilateral to the L5 nerve transection (laminae
I-IV) was observed on days 3, 7, and 14 after surgery. By contrast,
CD11b/CR3 immunoreactivity was reduced almost to baseline levels in the
ipsilateral dorsal horn of injured TLR4 KO mice. These immunohistochemical
protein data support the mRNA findings for CD11b and establish a link
between microglial activation and TLR4.
To demonstrate that behavioral hypersensitivity could be attenuated using
a small molecule inhibitor, a TLR4 antisense oligodeoxynucleotide (ODN)
was injected into the CNS of rats. It was initially confirmed that FITC-labeled
random control ODN was incorporated into the spinal cord. Co-labeling with
DAPI demonstrated the proximity of the FITC-labeled ODN to the nucleus,
showing that the modified ODN was incorporated into the CNS parenchyma.
Daily intrathecal injection of 10 .mu.g of TLR4 antisense ODN resulted in
moderate but significant attenuation of both mechanical allodynia (FIG. 2A (see Original Patent))
and thermal hyperalgesia (FIG. 2B); more significant attenuation was
observed with 20-.mu.g daily injections (P<0.001). Daily injections of
saline or of 10 or 20 .mu.g of mismatch ODN did not alter mechanical or
Because of the potential for TLR4 antisense ODN backbone toxicity, the
maximum daily intrathecal administration of TLR4 antisense ODN was 20 .mu.g.
This dosage provided a 56% decrease in spinal TLR4 mRNA expression
(P<0.001). Decreased expression of TLR4 paralleled the decreased
expression of the microglial activation markers CD11b (51%) and CD14 (44%)
(P<0.001). The decrease in TLR4 also led to a significant decrease in
proinflammatory cytokines, i.e., TNF-.alpha. (17%), IL-6 (52%), and
IL-1.beta. (53%); P<0.001 in all cases in TLR4 antisense ODN-treated rats
as compared with rats treated with saline or mismatch ODN.
The data presented herein demonstrate a key role for microglial TLR4 in
the induction of behavioral hypersensitivity in rodent models of
neuropathy. These data also show that the mRNA expression of the
microglial activation markers, TLR4, CD11b/CR3, and CD14, were only
elevated at the initiation phase of behavioral hypersensitivity (days 3
and 7). Conversely, the astrocytic activation marker GFAP showed an
opposite pattern with a graded increase in the mRNA expression level at
later time points (days 7 and 14). These results are consistent with
findings suggesting that microglia are involved in the initiating phase of
behavioral hypersensitivity, whereas astrocytes are involved in the
maintenance phase (Tanga, et al. (2004) Neurochem. Int. 45:397-407;
Raghavendra, et al. (2003) J. Pharmacol. Exp. Ther. 306:624-630). Many
factors have been postulated to activate microglia after injury, but the
finding that microglial TLR4 plays a crucial part as a receptor in the
induction phase of behavioral hypersensitivity in rodent models of
neuropathy has not been suggested in the art.
After L5 nerve transection injury, mediators are released or activated,
causing secondary swelling and damage to neurons. Such mediators include
ATP, glutamate, acidosis, free saturated or unsaturated fatty acids, high-extracellular
potassium, serotonin, bradykinin, substance P, histamine, and products
from the cyclooygenase and lipoxygenase pathway of the arachidonic acid
metabolism (Kempski & Volk (1994) Acta Neurochir. Suppl. 60:7-11). At the
spinal cord level, these alterations lead to a hyperexcitable state with
intense nociceptive input to the dorsal horn and the chemical
sensitization of high-threshold nociceptors to transmit low-intensity
The findings presented herein indicate that TLR4 is a microglial sensor
that triggers glial activation and the dynamic CNS immune response that
ensues in response to nerve damage. Inhibition of TLR4 expression was
found to produce a significant and dose-related attenuation of L5 nerve
transection-induced tactile and thermal hypersensitivity, reduce
expression of mRNA for microglial markers, and reduce expression of mRNA
for spinal proinflammatory cytokine. These findings indicate a CNS role
for TLR4 and innate immunity in the etiology of neuropathic pain. The
ability of TLR4 to activate a pathway leading to central sensitization and
nerve injury-induced behavioral hypersensitivity provides a means for
regulating glial activation, and thus, alleviating chronic pain due to
nerve damage. Accordingly, the present invention relates to methods for
attenuating behavioral hypersensitivity associated with neuropathic pain
and for preventing or treating neuropathic pain using an effective amount
of an agent that decreases the expression or activity of TLR4. As used in
the context of the present invention, neuropathic pain is pain that
originates from a damaged nerve or nervous system.
Generally, an agent of the present invention is administered to an
individual or patient identified as being in need of such prevention or
treatment using routes (e.g., injection, infusion, or inhalation) and
dosages that are determined to be appropriate by those of skill in this
art. As used in the context of the present invention, an individual in
need of treatment can include a human, zoo animal, companion animal,
laboratory animal or livestock.
An effective amount of agent administered is defined as an amount which
prevents, attenuates, or reduces behavioral hypersensitivity associated
with neuropathic pain. Behavioral hypersensitivity of pain may include
sensations that are sharp, aching, throbbing, gnawing, deep, squeezing, or
colicky in nature and can be measured by, for example, exposure to thermal
hyperalgesia or mechanical hyperalgesia.
As will be understood by those of skill in this art, the specific dose
level for any particular patient will depend on a variety of factors,
including the activity of the specific compound employed; the age, body
weight, general health, and sex of the individual being treated; the time
and route of administration; the rate of excretion; other drugs that have
previously been administered; and the severity of the particular disease
The method of the present invention is particularly useful for preventing
and/or treating pain associated with neuropathies, polyneuropathies (e.g.,
as in diabetes and trauma), neuralgias (e.g., post-zosterian neuralgia,
postherpetic neuralgia, trigeminal neuralgia, algodystrophy, and
HIV-related pain); musculo-skeletal pain such as osteo-traumatic pain,
arthritis, osteoarthritis, spondylarthritis as well as phantom limb pain,
back pain, vertebral pain, post-surgery pain; cancer-related pain;
vascular pain such as pain resulting from Raynaud's syndrome, Horton's
disease, arteritis, and varicose ulcers; as well as pain associated with
multiple sclerosis, Crohn's Disease, and endometriosis.
Agents for decreasing the expression or activity of TLR4 can be selected
from a variety of compound classes including small organic molecules,
antisense oligonucleotides (including siRNA and the like), ribozymes,
anti-TLR4 antibodies or fragments thereof. In particular embodiments, the
instant agent decreases the expression or activity of TLR4 receptor by 10%
to 100% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%)
relative to a control level of TLR4 expression or activity such as that
found in a subject with neuropathic pain and has not received treatment.
Agents known in the art to antagonize the activity of TLR4 include, but
are not limited to, E5564 (also known as compound 1287 or SGEA) (Mullarkey
et al. (2003) J. Pharmacol. Exp. Ther. 304(3):1093-1102, 2003). This agent
is also described in U.S. Pat. No. 5,681,824. A cell-permeable TLR4
inhibitory peptide is also known in the art for blocking TLR4 activity
(see Huang, et al. (2005) Cancer Research 65:5009-5014). Antagonistic
antibodies such as monoclonal antibody MTS510 to mouse TLR4 (commercially
available from eBioscience, San Diego, Calif.) can also be used. See,
e.g., Qi & Shelhamer (2005) J. Biol. Chem. 280:38969-38975. For
therapeutic use in species such as humans, such an antibody can be
humanized or fragmented to improve efficacy.
Agents which inhibit the expression of TLR4 include, e.g., TLR4 antisense
ODN as well as TLR4 short interfering RNAs (siRNAs). Antisense ODN
molecules are commercially available to the skilled artisan from sources
such as Biognostik (Gottingen, Germany), whereas suitable siRNA molecules
are disclosed by Huang, et al. (2005) supra or Qi & Shelhamer (2005) supra
(e.g., sense, gat ccG TTC CAT TGC TTG GCG AAT TTC AAG AGA ATT CGC CAA GCA
ATG GAA CTT TTT Tg (SEQ ID NO:1) and antisense, aat tcA AAA AAG TTC CAT
TGC TTG GCG AAT TCT CTT GAA ATT CGC CAA GCA ATG GAA Cg (SEQ ID NO:2)). As
will be appreciated by the skilled artisan, antisense ODN, siRNA, or
ribozymes can be readily generated based on the known nucleic acid
sequence encoding TLR4. Examples of known TLR4 sequences are found in
GENBANK, including those having the following Accession Nos. (listed by
species): cow (Bos taurus; NM.sub.--174198, AB056444, and AF310952); pig (Sus
scrofa; AY289532); chicken (Gallus gallus; AY064697); horse (Equus
caballus; AY005808); dog (Canis farniliaris; AB080363); cat (Felis catus;
BAB43947); human (Homo sapiens; NM.sub.--138557, NM.sub.--138556, NM.sub.--138554,
NM.sub.--003266, AF177765, AF172171, AF172170, AF172169, AH009665,
AF177766, and U88880); mouse (Mus musculus; NM021297, AL805946, AF177767,
AF222309, AF185285, AF110133, and AF095353); rat (Rattus norvegicus;
NM.sub.--019178); Chinese hamster (Cricetulus griseus; AF153676); gorilla
(Gorilla gorilla; AF497565, AF497564, AF497563, and AH011592); orangutan (Pongo
pygmaeus; AF497562, AF497561, AF497560, and AH011591); olive baboon (Papio
hamadryas anubis; AH008378, AF180964, AF180963, and AF180962); pygmy
chimpanzee (Pan paniscus; AH008351, AF179220, AF179219, and AF179218); and
rhesus monkey (Macaca rnulatta; AF162474).
It is contemplated that the agents of the present invention can be used
alone or in combination with other treatments known to alleviate pain.
Claim 1 of 1 Claim
1. A method of preventing or treating
neuropathic pain comprising administering intrathecally to a subject in
need of treatment an effective amount of an agent that decreases the
expression or activity of TLR4, wherein said agent comprises a TLR4
antisense oligonucleotide, MTS510, or E5564, thereby preventing or
treating neuropathic pain in the subject.
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