Note: Descriptions are shown in the official language in which they were submitted.
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, , . .
SELECTIVE ABLATION OF PAIN-SENSING NEURONS BY ADMINISTRATION OF
A VANILLOID RECEPTOR AGONIST
BACKGROUND OF THE INVENTION
Current analgesic therapies often fall short of therapeutic goals and
typically have unacceptable side'effects. In many chronic pain syndromes, such
as those
subsequent to neuropathic injury, pain is not well controlled by any currently
available
method. The sensation of pain is transduced in the periphery by pain-sensing,
i.e.
nociceptive, C- and A-delta primary afferent neurons. These neurons have a
peripheral
nerve ending in the skin or deep tissues and a central'terminal that makes
synaptic contact
with second order neurons in the spinal cord dorsal horn. The impulse is
processed
locally for activation of withdrawal reflexes and relayed to the brain for
conscious
perception and contextually relevant integrated responses. ,
Vanilloid receptor-1 (VR1) is a multimeric cation channel prominently
expressed in nociceptive primary afferent neurons (see, e.g., Cateriua et al.,
Nature
389:8160824, 1997; Tominaga et al., Neuron 531-543, 1998). Activation of the
receptor
typically occurs at the nerve endings via application of painful heat (VR1
transduces heat
pain) or during inflamrnation or exposure to vanilloids. Activation of VR1 by
an agonist,
such as resiniferatoxin or capsaicin, results in the opening of calcium
channels and the
transduction of pain sensation (see, e.g., Szalllasi et al., Mol. Pharmacol.
56:581-587,
1999.) After an initial activation of VR1, VR1 agonists desensitize VR1 to
subsequent
stimuli. This desensitization phenomenon has been exploited in order to
produce
analgesia to subsequent nociceptive challenge. For example, it has been shown
that
topical administration of resinferatoxin (RTX), which is a potent vanilloid
receptor
agonist, at the nerve endings in the skin triggers a long-lasting
insensitivity to chemical
pain stimulation. Furthermore, it has been shown that both subcutaneous and
epidural
administration of the RTX produce thermal analgesia when administered to rats,
with no
restoration of pain sensitivity for over 7 days (see, e.g., Szabo et al.,
Brain Res. 840:92-
98, 1999).
In these studies, however, the VR1 agonist was not administered directly
to the nerve ganglion and the analgesic effect was reversible. Spatially, the
peripheral
Ca2+ toxicity is far removed from the neuronal perikarya in the ganglion so
that
application to the skin does not cause cell death. The present invention is
based on the
discovery that adninistration of VR-1 receptor agonist to the ganglion at the
level of the
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CA 02442049 2008-07-09
neuronal cell body causes neuronal cell death and moreover, that the resulting
selective
ablation of VR1-containing neurons provides a treatment for chronic pain.
The effects of intrathecal capsaiciui on thermal sensitivity in rats has also
been investigated. The results, however, have been conflicting (Nagy et al.,
Braiti Res.
211:497-502, 1981; Palermo et al., Braiti Res. 208:506-510; Yaksh et al.,
Science
206:481-483, 1979; and Russell et al., Pain 25:109-123, 1986). Russell et al.
observed
no thermal analgesia, although in three previous studies, at least some degree
of thermal
analgesia was observed. The conflicting results raised a number of issues such
as the
possibility of complications in data interpretation resulting from spinal cord
damage from
cannula implantation, or solvent toxicity problems. Russell et al. therefore
used a non-
toxic solvent for capsaicin administration to rats and additionally, performed
partial
laminectomies to allow direct visualization of the cauda equina and thus
iuisure drug
delivery to the spinal fluid. No thermal analgesia was demonstrated and the
authors
concluded that intrathecal capsaicin administration is not a reliable method
for producing
thermal analgesia in the rat. The present inventors now resolve this
controversy with the
surprising discovery that intrathecal injection of a vanilloid receptor
agonist such that the
agonist contacts the neuronal cell body in an amount sufficient to cause Ca2+
influx,
results in selective ablation of the neuron, and therefore is an effective
therapy for chronic
pain.
Recent studies have also shown that pain sensing C-fibers appear to
participate in or exacerbate a variety of chronic diseases such as chronic
pancreatitis,
herpes infections, inflammatory or irritable bowel disease and rheumatoid
arthritis.
Generally, these chronic diseases have an inflammatory component in which the
C-fibers
play a role. The present invention therefore also provides a method of
destroying C-fiber
neurons that contribute to chronic disease syndromes, thus providing a method
to
ameliorate or resolve chronic inflammatory conditions. In addition, the
ability to
selectively kill VR- 1 -expressing neurons using the methods of the invention
also provides
a therapy for selectively removing neurons that are reservoirs of virus in
chronic viral
infections such as Herpes vints infection.
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SUMMARY OF THE INVENTION
Various embodiments of this invention provide use of a vanilloid receptor 1
agonist for selectively ablating pain-sensing neurons from a ganglion in a
patient, wherein
said agonist is a resiniferatoxin or a capsaicin; and wherein said agonist is
for
intraganglionic administration by injection to a ganglion selected from the
group consisting
of a dorsal root ganglion, a trigeminal ganglion, and an autonomic ganglion.
The use may
be for preparation of a medicament for such selective ablating.
Various embodiments of this invention provide a composition comprising a
pharmaceutically acceptable carrier and a vanilloid receptor 1 agonist
selected from the
group consisting of a resiniferatoxin or a capsaicin, for use in selectively
ablating pain-
sensing neurons from a ganglion in a patient, wherein said composition is for
intraganglionic administration by injection to a ganglion selected from the
group consisting
of a dorsal root ganglion, a trigeminal ganglion, and an autonomic ganglion.
Various embodiments of this invention provide use of a vanilloid receptor
agonist for preparation of a medicament for selectively ablating pain-sensing
neurons from
a ganglion in a patient, wherein the agonist is a resiniferatoxin and wherein
the medicament
is for intrathecal administration to a dorsal root ganglion. The use may be
for preparation of
a medicament for such selective ablating.
Various embodiments of this invention provide a composition comprising a
pharmaceutically acceptable carrier and a vanilloid receptor agonist for use
in selectively
ablating pain-sensing neurons from a ganglion in a patient, wherein said
agonist is a
resiniferatoxin, and wherein said composition is for intrathecal
administration to a dorsal
root ganglion.
The present invention is based on the surprising discovery that direct
application of a vanilloid receptor agonist into the neuron cell body
contained in a ganglion
opens calcium channels in VR1-expressing neuronal perikarya, triggering a
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cascade of events leading to cell death. Accordingly, the present invention
provides
methods of controlling pain and inflammatory disorders that involve activation
of
vanilloid receptor-bearing neurons. In particular, selective deletion of
nociceptive
primary afferent neurons by intraganglionic or intrathecal administration of
the vanilloid
agonist, e.g., resiniferatoxin (RTX) interrupts the signaling pathway and
blocks pain
sensation and neurogenic inflammation. This selective application can be used
for
treatment-resistant trigeminal neuralgia, atypical facial pain, certain types
of neuropathic
pain, for pain management in end-stage disease or palliative care, and for
treatment of
chronic pain that occurs in chronic infections.
Thus, the invention provides a method of selectively ablating pain-sensing
neurons from a ganglion, said method comprising intraganglionic administration
of a
vanilloid receptor agonist to a ganglion selected from the group consisting of
a dorsal root
ganglion, a trigeminal ganglion, or an autonomic ganglion in an amount
sufficient to
ablate the neurons.
In some embodiment, the vanilloid receptor 1 agonist is administered to a
patient suffering from chronic pain. Often, the vanilloid receptor agonist is
selected from
the group consisting of a resiniferatoxin or a capsaicin, such as ovanil.
Preferably, the
VR1 agonist is a resiniferatoxin.
In one embodiment, intraganglionic administration comprises direct
injection into the ganglion.
In an alternative embodiment, intraganglionic administration coinprises
injection into the nerve root.
In one embodiment, the amount that is sufficient to ablate the neurons is
from 50 nanograms to 50 micrograms. Often the amount is from about 500
nanograins to
about 50 micrograms.
In some embodiments, the method further comprises administration of a
local anesthetic, often lidocaine or bupivacaine.
In another aspect, the invention provides a method of selectively ablating
pain-sensing neurons from a ganglion, said method comprising intrathecal
administration
of a vanilloid receptor agonist to a dorsal root ganglion in an amount
sufficient to ablate
the neurons. In some embodiments, intrathecal administration of the VR1
agonist is with
the proviso that the VR1 agonist is not administered to a rat or other rodent.
In a preferred embodiment, the VR1 agonist is a resiniferatoxin.
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In one embodiment, the amount that is sufficient to ablate the neurons is
from 100 nanograms to 500 micrograms. Often, the amount is from about 500
nanograms
to about 500 micrograms.
In some embodiments, the method further comprises administration of a
local anesthetic, often lidocaine or bupivacaine.
In another aspect, the invention provides a kit for selectively ablating pain
sensing neurons from a dorsal root, autonomic, or trigeminal ganglia, said kit
comprising
a compartment containing a vanilloid receptor agonist in an amount sufficient
to ablate
the neurons and instructional materials describing how to use the kit. Such a
kit can also
contain a local anesthetic. Ii1 particular embodiments, the vanilloid receptor
agonist is a
resiniferatoxin.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a micrograph of CGRP immunochemistry in an untreated
trigeminal ganglion.
Figure 2 is a micrograph of CGRP immunochemistry in a treated
trigeminal ganglion.
DETAILED DESCRIPTION
1. Introduction
This invention pertains to the surprising discovery that administration of
vanilloid receptor agonist to a peripheral neuron ganglion is toxic to VR-1
expressing
neurons and is therefore useful to selectively treat acute and chronic pain,
while at the
same time not significantly affecting other somatosensory functions such as
position
sense, light touch, hair movement, pressure or mechanical vibration as well as
mechanical
pinch sensitivity. The neurons that subserve these sensations do not make VR-1
and thus
they are not sensitive to ablation by vanilloid agonist treatment. The
selective ablation of
pain-sensing neurons in these ganglia is useful for the treatment of clhronic
pain,
particularly including, but not limited, to neuropathic pain resulting from
injury to
specific nerves; pain associated with cancer, such as pain resulting from bone
metastases
to the spinal column in prostate cancer; pain associated with inflammatory
diseases such
as acute pancreatitis and pain associated with chronic ganglionic viral
infections
particularly with infection by members of the Herpes virus family such as
Herpes zoster
or Herpes simplex I and II.
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Delivery of VRl agonists to ganglionic neuronal cell bodies is not only an
effective therapy for chronic pain, but can also be used to treat other
chronic
inflammatory conditions where persistent inflammation causes severe
exacerbation of the
underlying disease or may be the cause of the inflammatory condition. For
example,
intraganglionic VR1 agonist can be used to treat herpes virus infections or
pain disorders
caused by herpes virus infection such as post-herpetic neuralgia (shingles).
Herpes
viruses enter a latent state in the dorsal root ganglia, and the C-fiber
neurons are an
important reservoir of the latent virus. Upon stress or reactivation the virus
exits the
latent state and begins replication. Viral particles are transported down the
axon where
they erupt on the skin. In many cases the eruption can be very painful, and
further, can
present additional problems for immunocoinpromised patients or elderly
patients.
Although there has been some progress in antiviral therapy, it remains
difficult to
effectively treat this condition. Because the C-fiber ineuronal cell bodies
express VRl
receptor, the present invention therefore also provides a method of
selectively removing
the neurons that act as a viral reservoir. Upon administration of a VR1
agonist to the
neuronal cell body, death occurs rapidly, and the virus cannot enter a
replicative state.
Thus, the invention also provides a treatment for chronic viral infection,
e.g., Herpes virus
infection, with limited impact on a patient's ability to sense pain (except in
the
dermatomes innervated by the treated ganglia), and provides very effective
pain relief
without compromising other sensory functions.
2. Definitions
The term "VR1 agonist" as used herein refers to a to a compound that
binds to VRl and stimulates calcium uptake. Typically, VR1 agonists comprise a
vanilloid ring that is important for agonist activity.
The term "administering" incorporates the common usage and refers to
any appropriate means to give a pharmaceutical to a patient, taking into
consideration the
phazmaceutical composition and the preferred site of administration (e.g., in
one
embodiment, the pharmaceutical composition of the invention is injected into
the
subarachnoid space as an aqueous solution).
A"patient" as used herein is synonymous with "subject" and refers to any
mammal including, but not limited to, horse, cow, sheep, goat, pig, dog, cat,
rat, mouse,
guinea pig, and primate. In a preferred embodiment, the mammal is a human.
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The term "basal nociceptive responses" incorporates its common usage
and refers to baseline responses to nociceptive, or painf-ul, stimuli.
The tenus "chronic pain" and "acute pain" incorporate their coinmon
usages; subjective (e.g., clinical diagnosis) and otlier objective means
(e.g., laboratory
tests, PET) to determine the presence of chronic pain and/or acute pain, and
to distinguish
between these two distinct categories of pain, are described in detail, below.
The term "vanilloid receptor 1" or "VR1" refers to a ligand-gated cation
channel, distantly related to the TRP (transient release potential) proteins,
that can be
activated by vanilloids, heat, and protons. A VRl agonist binds to VR1 and
activates the
VR1 cation channel.
The term "hyperalgesia" refers to an increased response to a stimulus that
is normally painful (see, e.g., Bonica (1990) infra). Its presence is
recognized as a
symptom of chronic pain (i.e., its presence is associated with or is a
sequelae of chronic
pain).
The term "pharmaceutically acceptable excipient" incorporates the
common usage and refers to includes any suitable pharmaceutical excipient,
including,
e.g., water, saline, phosphate buffered saline, Hank's solution, Ringer's
solution,
dextrose/saline, glucose, lactose, or sucrose solutions, magnesium stearate,
sodium
stearate, glycerol monostearate, glycerol, propylene glycol, ethanol, and the
like.
The term "subarachnoid space" or cerebral spinal fluid (CSF) space
incorporates the common usage refers to the anatomic space between the pia
mater and
the arachnoid membrane containing CSF.
The term "intrathecal administration" refers to administration of a
compositions directly into the spinal subarachnoid space.
"Intraganglionic administration" as used herein refers to administration to
a ganglion. Intraganglionic administration can be achieved by direct injection
into the
ganglion and also includes selective nerve root injections, in which the
compound passes
up the connective tissue sleeve around the nerve and enters the ganglion from
the nerve
root just outside the vertebral column. Often, intraganglionic administration
is used in
conjunction with an imaging tecluiique, e.g., employing MRI or x-ray contrast
dyes or
agents, to visualize the targeted ganglion and area of administration.
The term "treating" refers to any indicia of success in the treatment or
amelioration of an injury, pathology, condition, or symptom (e.g., pain),
including any
objective or subjective parameter such as abatement; remission; diminishing of
symptoms
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or making the symptom, injury, pathology or condition more tolerable to the
patient;
decreasing the frequency or duration of the symptom or condition; slowing in
the rate of
degeneration or decline; making the final point of degeneration less
debilitating;
improving a patient's physical or mental well-being; or, in some situations,
preventing the
onset of the symptom or condition, e.g., pain. The treatment or amelioration
of symptoms
can be based on any objective or subjective paratneter; including, e.g., the
results of a
physical examination a.nd/or a psychiatric evaluation, or, simply an
improvement in the
patient's sense of well-being. For example, the methods of the invention
selectively
treats chronic pain by ameliorating the hyperalgesia associated with chronic
pain, while
not significantly affecting non-pain sensory functions such as proprioception,
muscle and
tendon stretch, light touch, vibration sense, motion sensitive
mechanoreceptors that
innervate hair follicles and pressure sense.
3. Distinguishing Chronic from Acute Pain
Pain is always subjective and can have physiologic, pathophysiologic,
psychologic, emotional, and affective dimensions. Pain causation can be
broadly
categorized as organic or psychogenic. Basically, two types of pain exist -
acute pain
and chronic pain. Each possibly is mediated by anatomically different nerves.
Each type
of pain has a different physiologic role. For example, the ability to perceive
and respond
to "acutely" painful stimuli, which usually has the potential to cause tissue
damage,
serves a protective role for the individual. Many treatments for acute pain
cannot
aineliorate chronic pain (this, in fact, is used as one means to objectively
identify
"chronic" versus "acute" pain, as discussed below). There presently exists no
clinically
accepted effective therapy to treat chroiii.c pain without the unwanted side
effect of
significantly dampening protective acute pain responses. The present invention
provides
for targeted removal of neurons, thus limiting the loss of the acute pain
response to those
acute pain sensations transduced by the particular neurons.
Accordingly, in some embodiments, the methods of the invention
comprise selective ablation of neurons in patients or subjects suffering from
chronic pain.
In some embodiments, such as treatment of chronic pain in a young nerve injury
patient,
one or two ganglia, or a particular nerve root are targeted for treatment
using the methods
of the invention, thus providing limited damage to acute pain responses.
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Diagnosing and Assessing Chronic Pain
The invention provides methods of treating chronic pain while at the same
time not significantly affecting the ability to respond to acutely painful,
and potentially
hariuful, stimuli. Thus, proper diagnosis of chronic pain is necessary both to
practice and
to assess the success of the compositions and methods of the invention. Means
to
diagnosis chronic pain include classical clinical and psychological
evaluations, which can
be augmented by various laboratory procedures, as described herein. Such means
are
well-described in the medical/scientific and patent literature; some
illustrative examples
are provided below.
One criteria to diagnose a "chronic" pain is whether the pain persists for a
month beyond the usual course of an acute disease or a reasonable time for an
injury to
heal. This evaluation is made by the clinician on a case by case basis. Acute
diseases or
injuries can heal in 2, 3, or, at most, 6 weeks, depending on the nature of
the condition or
injury, the age and health of the patient, and the like. For example, a
siinple wrist fracture
can remain painful for a week to ten days; however, if pain persists longer
than this
period, a dystropathy could be developing which will be irreversible if not
treated. See,
e.g., Bonica, et al., (1990) "Management of Pain," 2nd Ed., Vol. I, Lea &
Feibiger, Phil.,
PA; Wall and Melzack (1994) "Textbook of Pain," Churchill Livingstone, NY.
Accordingly, a ch.ronic pain is diagnosed by the practitioner based on
clinical and
laboratory results, depending on the particular condition or injury, patient,
and the like
(see also, e.g., Russo (1998) Annu. Rev. Med. 49:123-133).
Another means to identify a "chronic" pain is by diagnosis of a pathologic
process (which is usually also chronic) known to produce or be associated with
chronic
pain. Such conditions are well characterized and include, e.g., chronic pain
syndrome
(see, e.g., Clifford (1993) Can. Fam. Physician 39:549-559), arthralgia,
arthritis (e.g.,
osteoarthritis and rheumatoid arthritis), causalgia, hyperpathia, neuralgia,
neuritis,
radiculagia, fibromyalgia (see, e.g., Simms (1998) Am. J. Med. Sci. 315:346-
350),
orofacial pain and temporomandibular disorders (see, e.g., Binderman (1997)
Curr. Opin.
Pef=iodontol. 4:144-15), reflex sympathetic dystrophy (see, e.g., Dangel
(1998) Paediatr.
Anaestla. 8:105-112, chronic back pain, certain cancers, and the like.
Chronic pain is also associated with particular injuries to the nerves.
These include, e.g., nerve transection (traumatic or surgical), chronic
abnormal pressure
on a nerve, chemical (e.g., formalin) destruction of nerve tissue, and the
like.
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Chronic pain can also be distinguished from acute pain by its non-
responsiveness to pharmacologic therapies lrnown to significantly ameliorate
or abate
acute pain. When pain is initially diagnosed as acute or of unknown etiology,
the
clinician typically administers one of several analgesics known in the art to
be effective
for acute pain, such as, e.g., a non-steroid anti-inflammatory drug (NSAID),
such as, e.g.,
aspirin, ibuprofen, propoxyphene, tramadol, acetaminophen and the like (see,
e.g., Tramer
(1998) Acta Anaesthesiol. Scand. 42:71-79). If there is no significant
amelioration of
pain, as assessed by the clinician, over an approximately six week period,
then a
provisional diagnosis of chronic pain can be made. Ultimately, as discussed
above, a
diagnosis of chronic pain depends upon determination as to whether pain would
be
expected, given each individual situation.
Other treatments to which chronic pain is also typically incompletely or
totally unresponsive include tricyclic antidepressant administration,
psychotherapy, or
alternative medicines, such as acupuncture, biofeedback, and the like.
Laboratory, radiographic and other types of imaging procedures may also
be used to diagnose chronic pain. In particular, positron emission tomography,
or PET,
now allows the clinician to objectify such otherwise merely subjective
symptoms,
including clironic pain (see, e.g., Reiss (1998) Fortschr. Med. 116:40-43; Di
Piero (1991)
Pain 46:9-12).
4. Vanilloid receptor agonists
VRl agonists as defined herein bind to the VRl receptor and stimulate
calcium activity. VRl agonists are typically characterized by the presence of
a vanilloid
moiety that mediates binding and activation of the receptor. Any number of VR1
receptor agonists are useful for practicing the methods of the invention.
Compounds that
act as VRl receptor agonists include resiniferatoxin and other resiniferatoxin-
like
complex polycyclic compounds such as tinyatoxin, capsaicin and other capsaicin
analogs
such as ovanil, and other compounds that include a vanilloid moiety that
mediates binding
and activation of VRl . In some instances, such as low pH, compounds that lack
a
vanilloid moiety, e.g., anandamide and the eicosinoids prostacyclin and PGE2
can also
functionally activate VRl.
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Resiniferatoxin
In one embodiment, RTX is used as the vanilloid receptor agonist. RTX,
is unlike the structurally related phorbol esters, acts as an ultrapotent
analog of capsaicin,
the pungent principle of the red pepper. RTX is a tricyclic diterpene isolated
from
Eurphorbia resinifera. RTX induces pain, hypothermia, and neurogenic
inflammation;
the acute responses are followed by desensitization to RTX and by cross-
desensitization
to capsaicin. A homovanillyl group is an important structural feature of
capsaicin and the
most prominent feature distinguishing resiniferatoxin from typical phorbol-
related
compounds. Naturally occurring or native RTX has the following structure:
'',o,e n,u,u0 I
\\\\\\~ 11111111110
\\\
H
O OMe
OH
O
OH
RTX and analog compounds such as tinyatoxin as well other compounds,
e.g., 20-homovanillyl esters of diterpenes such as 12-deoxyphorbol 13-
phenylacetate 20-
homovanillate and mezerein 20-homovanillate, are described, for example, in
U.S. Patent
Nos: 4,939,194; 5,021,450; and 5,232,684. Other resiniferatoxin-type phorboid
vanilloids
have also been identified (see, e.g., Szallasi et al., Brit. J. PhNmacol.
128:428-434, 1999).
Often, the C20-homovanillic moiety, the C3-keto group and the ortho-ester
phenyl group
on ring C are important structural elements for activity of RTX and its
analogs. As used
herein, "a resiniferatoxin" or "an RTX" refers to naturally occurring RTX and
analogs of
RTX, including other phorbol vanilloids with VR1 agonist activity.
Capsaicin
Capsaicin is a natural product in capsicum peppers that mediates the "hot"
sensation characteristic of these peppers. As used herein, "a capsaicin" or
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"capsaicinoids" refers to capsaicin and capsaicin-related or analog compounds.
Naturally
occurring or native capsaicin has the structure:
0
""~NH
\ OMe
A number of analogs of capsaicins are known in the art including
vanillylacylamides, homovanillyl acylamides, carbamate derivatives,
sulfonamide
derivatives, urea derivatives, aralkylamides and thioamides, aralkyl
aralkanamides,
phenylacetamides and phenylacetic acid esters are known in the art. In one
embodiment,
the capsaicin analog olvanil (N-vanillyl-9-octadecenamide) is used in the
methods of the
invention. Examples of capsaicin and capsaicin analogs are described, for
example, in the
following patents and patent applications: U.S. Pat. No. 5,962,532; U.S. Pat.
No.
5,762,963; U.S. Pat. No. 5,221,692; U.S. Pat. No. 4,313,958; U.S. Pat. No.
4,532,139;
U.S. Pat. No. 4,544,668; U.S. Pat. No. 4,564,633; U.S. Pat. No. 4,544,669; and
U.S. Pat.
Nos. 4,493,848; 4,532,139; 4,564,633; and 4,544,668.
Other VRI agonists
Other VR1 agonists (see, e.g., WO 00/50387) can also be used to
selectively ablate C-fiber neurons. Such compounds comprise a vanilloid moiety
that
mediates binding and activation of VRl. These compounds include compounds
having
modifications on the C20-homovanillic moiety, the C3-carbonyl, and the ortho-
ester
phenyl moiety.
Useful VR1 agonists for practicing the invention can be readily identified
using standard methodology. The methodology includes such assessments as
measurement of binding to a compound to VR1 and measurement of the ability of
the
compound to stimulate Ca2+ influx. The compound can also be assessed for the
ability to
kill cells that express the vanilloid receptor. These measurements can be
performed using
methods known to those of skill in the art.
The ability of a VR1 agonist to bind VR1 -bearing cells or membranes can
be measured directly or, more typically, in a competition analysis with a
known binding
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compound such as RTX. VRl binding assays are described in a number of
publication,
for example, in various U.S. patents and other publications (e.g., WO 00/503
87, U.S.
Patent No. 5,232,684, supra; Szallasi et al., Molec. Pharmacol. 56:581-587,
1999). In an
exemplary assay, binding activity of a compound containing a vanilloid moiety
can be
assessed be measuring the ability of the compound to displace bound [3H]RTX
from the
VR1 receptor. The analysis can be perfonned using any cell or cell membrane
that has
VR1 receptors. Often, VRl-expressing transfectants or membrane from the spinal
cord
are used. The results are usually expressed in tenns of Ki values that
represent the
concentration of the non-radioactive ligand that displaces half of the bound
labeled RTX.
Preferred VR1 agonists, e.g., RTX, typically have a 10-fold, often a 100-fold,
preferably a
1000-fold higher binding affinity for VRl than native, i.e., the naturally
occurring,
capsaicin.
In order to identify VR1 agonists, binding assays are typically performed
in conjunction with functional assessments that measure the ability of a
compound to
stimulate changes in membrane potential or changes in calcium influx. Changes
in
melnbrane potential or calcium influx can be determined using a variety of
assays well
lcnown to those in the art. For example, VRl-expressing cells such as neurons
from the
dorsal root ganglion or VR1 transfectants can be analyzed by patch clamping
for changes
in whole cell currents that occur upon exposure of the compound being tested
for VR1
activity (see, e.g., the Example section below and Caterina et al., Nature
389:816-824,
1997). Another commonly used method to assess VR1 agonist activity is to
measure the
uptake of calcium using various assays to measure intracellular calcium
concentration.
For example, calcium flux can be measured by assessment of the uptake of
45Ca2+ or by
using fluorescent dyes such as fura-2. For example, a dye such as fura-2,
which
undergoes a change in fluorescence upon binding a single Ca2+ ion, is loaded
into the
cytosol of VR1 -expressing cells. Upon exposure to VR1 agonist, the increase
in cytosolic
calcium is reflected by a change in fluorescence of fura-2 that occurs when
calcium is
bound. Such measurements can also be used to assess the ability of a VR1
agonist to
mobilize intracellular calcium stores from the endoplasmic reticulum (ER). In
preferred
embodiments, VR1 agonists stimulate both a release of Ca2+ from the ER and an
influx of
calcium across the cell membrane.
VRl agonists of this invention are analyzed for the ability to elicit cell
death. In these assays, VRl-expressing cells are exposed to VR1 agonist. VRl-
mediated
cell death is detennined by using morphological assessments and/or staining
with vital
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dyes such as trypan blue (see, e.g., the Examples section and Caterina et al.,
supra).
Preferred VRl agonists for use in the invention typically are 100 times, often
1000 times
more potent than native capsaicin.
Additional compounds e.g., anadamide, and certain eicosanoids such as
prostacyclin and PGE2, can activate VR1, but lack a vanilloid moiety. Such
compounds
can and that are of use in the methods of the invention can also be identified
by
detennining the ability of a compound, , to stimulate calcium uptake andlor
cause cell
death. Such conipounds are typically identified in an assay that compares
activation of
VRl in response to the compound to activation of VRA in response to a known
VR1
agonist, e.g., capsaicin or RTX. comparison to a VRl agonist that comprises a
vanilloid
moiety, often in a competitive functional assay. Preferred compounds are 100-
fold,
preferably 1000-fold, more potent in activating VRl-induced calcium
mobilization in
comparison to native capsaicin.
5. Administration of VR1 agonists
VR1 agonists, such as RTX or olvanil, are formulated as pharmaceuticals
to be used in the methods of the invention to treat chronic pain by selective
ablation of
VRl-expressing neurons. Any VR1 agonist that causes an increase in
intracellular
calcium, preferably by causing both a transmembrane calcium flux and release
of calcium
from the ER, and kills VR1 -expressing cells can be used as a pharmaceutical
in the
invention. Routine means to determine VR1 agonist drug regimens and
formulations to
practice the methods of the invention are well described in the patent and
scientific
literature, and some illustrative examples are set forth below.
Routes of Administnation
The VRl agonists can be administered by any means that delivers the VRl
agonist into the vicinity of the nociceptive primary afferent neuronal cell
body. These
routes of administration include intrathecal administration and
intraganglionic
administration directly into the ganglion or performed by selective nerve root
injections.
in which the compound passes up the connective tissue sleeve around the nerve
and enters
the ganglion from the nerve root just outside the vertebral column (see, e.g.,
TEXTBOOK
OF PAIN, Wall and Melzack, Eds. Harcourt Brace, 4th Ed, 1999).
In one embodiment, the VR1 agonist is injected directly into a ganglion or
at the nerve root using methods standard neurosurgical techniques. Often,
administration
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is perfonned using image analysis using MRI or x-ray contrast dyes, to provide
for direct
delivery to the perikarya. For example, the procedure can be performed in
conjunction
with procedures known in the art, such as CAT scan, fluoroscopy, or open MRI.
In another embodiment, the agonist is administered intrathecally, typically
in a isobaric or hyperbaric pharmaceutically acceptable excipient as further
described
below. Means to administer solutions into the subarachnoid space, i.e.,
intrathecally, into
the CSF, are well known in the art; see, e.g., Oyama, T., U.S. Patent No.
4,313,937.
Determining Dosing Regimens
The pharmaceutical formulations of the invention can be administered in a
variety of unit dosage forms, depending upon the particular condition or
disease, the
degree of chronic pain, the general medical condition of each patient, the
method of
administration, and the like. In one embodiment, the VR1 agonist is
administered in a
pharmaceutically acceptable excipient at a dose of complete with amount.
Details on
dosages are well described in the scientific and patent literature, see, e.g.,
the latest
edition of Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton
PA.
The exact concentration of VRl agonist in a given dose, or the
"therapeutically effective dose" is determined by the medical practitioner, as
discussed
above. The dosage schedule, i.e., the "dosing regimen," will depend upon a
variety of
factors, including the amount of chronic pain present, the duration of the
pain, the stage
and severity of the disease or condition associated witlz the chronic pain (if
any), and the
general state of the patient's health, physical status, age and the like. The
state of the art
allows the clinician to determine the dosage regimen for each individual
patient and, if
appropriate, concurrent disease or condition treated. The illustrative example
provided
below can be used as guidance to determine the dosage regimen, i.e., dose
schedule and
dosage levels administered when practicing the methods of the invention.
Typically, VR1 agonists to a particular ganglion are administered to create
a temporary environment from about 1 to 5 minutes achieved by injection of the
agonist.
Based on objective and subjective criteria, as discussed herein, any dosage
can be used as
required and tolerated by the patient. Multiple administrations can also be
performed as
required. For intraganglionic administration to a dorsal root or autonomic
ganglion, a
typical volume injected is from 50 to 300 microliters delivering a total
amount of VRl
agonist that ranges from about 50 nanograms to about 50 micrograms. Often the
amount
administered is from 200 ng to 1 ug. The VR1 can be administered as a bolus or
infused
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over a period of time, typically from 1 to 5 minutes. For intraganglionic
administration
to a trigeminal ganglion, a volume of from about 100 microliters to about 500
microliters
is typically used to delivered from about 50 nanograms to about 50 micrograms
of VRl
agonist. The VR1 agonist can be infused over a length of time from about 1 to
5 minutes,
or can be delivered as one or more boluses. Dosages in the ranges of 100
nanograms to
500 micrograms are often used. For intrathecal administration, an amount from
about 0.5
to 5 ccs, often 3 ccs are injected into the subarachnoid space. The total
amount of VRl
agonist in the injected volume is usually from about 500 nanograms to about
500
micrograms.
VRl agonist can be prepared as pharmaceutical compositions by
combination with appropriate medical carriers or diluents. Examples of aqueous
solutions that can be used in VRl formulations include, e.g., water, saline,
phosphate
buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose
solutions and
the like. The formulations can contain pharmaceutically acceptable auxiliary
substances
to enhance stability, deliverability or solubility, such as buffering agents,
tonicity
adjusting agents, wetting agents, detergents and the like. Additives can also
include
additional active ingredients such as bactericidal agents, or stabilizers. For
example, the
solution can contain sodium acetate, sodium lactate, sodium chloride,
potassium chloride,
calcium chloride, sorbitan monolaurate or triethanolamine oleate. These
compositions
can be sterilized by conventional, well-known sterilization techniques, or can
be sterile
filtered. The resulting aqueous solutions can be packaged for use as is, or
lyophilized, the
lyophilized preparation being combined with a sterile aqueous solution prior
to
administration.
The VR1 agonists are often administered in specific formulations such as
isobaric or hyperbaric solutions that may additionally contain other agents
such as a long
acting local anesthetic. The density,of the solution can be controlled using
methods
known to those of skill in the art. For example, a solution can be made more
hyperbaric
by the addition of iohexol, iodixanol, metrizamide, sucrose, trehalose,
glucose, or other
biocompatible molecules with high specific gravity.
In some embodiments, the VRl agonist is administered in conjunction
with a local anesthetic. A local anesthetic refers to a drug that provides
temporary
numbness and pain relief in a specific region. Local anesthetics are well
known to those
of skill in the art. These includes, dibucaine, bupivacaine, ropivacaine,
etidocaine,
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tetracaine, ropivicaine, procaine, chlorocaine, prilocaine, mepivacaine,
lidocaine,
xylocaine, 2-chloroprocaine, and acid addition salts or mixtures thereof.
The VRl agonists can also be administered in conjunction with other
agents. For example, the VR1 agonist can be administered with a dye or tracer
compound when image-guided administration procedures are performed. Common
agents include a radio-opaque dye or manetic resonance contrast agent such as
gadlinium.
The VR1 agonists for use to selectively ablate VR1 -expressing neurons are
administered to a subject such as a mammal, preferably, a primate or a human,
but can
also be used for other mammals such as horses, cows, sheep, pigs, dogs, cats,
rabbits, or
other animals.
5. Kits
After a pharmaceutical comprising a VRl agonist for use in the methods of
the invention has been formulated in a acceptable carrier, it can be placed in
an
appropriate container and labeled for treatment of an indicated condition,
such as chronic
pain. For administration of VR1 agonists, such labeling would include, e.g.,
instructions
concerning the amount, frequency and method of administration. In one
embodiment, the
invention provides for a kit for the treatment of chronic pain in a human
which includes a
the VRl agonist and instructional material teaching the indications, dosage
and schedule
of administration of the agonist. Often, such kits also include a local
anesthetic.
It is understood that the examples and embodiments described herein are
for illustrative purposes only and that various modifications or changes in
light thereof
will be suggested to persons skilled in the art and are to be included within
the spirit and
purview of this application and scope of the appended claims.
Although the foregoing invention has been described in some detail by
way of illustration and example for purposes of clarity of understanding, it
will be readily
apparent to one of ordinary skill in the art in light of the teachings of this
invention that
certain changes and modifications may be made thereto without departing from
the spirit
or scope of the appended claims.
EXAMPLES
The following examples are provided by way of illustration only and not
by way of limitation. Those of skill in the art will readily recognize a
variety of
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noncritical parameters that could be changed or modified to yield essentially
similar
results.
Example 1. Administration of VR-1 agonist to cells expressing VR-1.
The effect of VR-1 agonist administration to cells expression VR-1 was
measured using the following methodology.
A VR1 expression vector encoding a VR-1/Green Fluorescent Protein
construct was expressed in Cos7 and HEK293 cells using transient transfection.
Western
blot analysis showed that VR1 eGFP protein exhibited GFP-specific
immunoreactivity
and was not cleaved. The cells in the population that fluoresced green were
voltage
clamped and the holding potential adjusted to -60 mV. The first application of
10 M
capsaicin (CAP) to the cells induced a large inward current (N=5). Multiple
exposures
resulted in a gradual decrease, indicating receptor desensitization. The
VRleGFP-
mediated current was attenuated by co-incubation of an antagonist, 10 M
capsazepine
(CPZ). Current versus voltage relationships demonstrated that the VR1eGFP-
mediated
current was not voltage sensitive. The reversal potential was calculated to be
78.3 mV,
suggesting mixed cation selectivity for the channel.
Resiniferatoxin (RTX), in much lower concentration, induced a current
similar to that of CAP; however, a single application of 125 pM RTX (N=12
cells)
resulted in complete desensitization. Coincidentally, the membrane capacitance
of.
VR1 eGFP-transfected cells dropped dramatically (6 12.5 pF), indicating an
about 600
ma loss of plasma membrane due to RTX treatment. This calculation assumes a
capacitance of 1 F/cm2 of inenlbrane. However, the time constant remained the
same.
The capacitance changes suggests either shedding or internalization of
VR1eGFP membranes (Zimmerberg et al. Proc. Natl. Acad. Sci USA 84: 1585-1589,
1987). To verify that the decrease in capacitance was mediated by VR1eGFP, the
initial
slope of the current evoked by RTX was plotted versus the change in
capacitance for each
cell. The slope of the evoked current correlated with the change in
capacitance. In non-
transfected cells, RTX neither evoked currents nor induced a change in
capacitance. In
accordance with the electrophysiological data, exposure to RTX induced Ca2+
uptake in
VR1eGFP-expressing HEK293 and Cos7 cells. This demonstrated that VR1eGFP can
mediate ligand-induced Caz+-influx. RTX induced Ca2+ uptake with an ED5o = 0.1
0.05
nM (N=3) while capsaicin induced Ca2+ uptake wit11 an ED50 = 0.5 0.15 M,
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A VR1-tagged with a 12 amino acid s-epitope was also tested in an
NIH3T3 cell line expressing "VRls", a C-terminal epsilon epitope-tagged
vanilloid 1
receptor.
Preparation of the plasmid vector expressing C-terminally epsilon epitope
tagged vanilloid receptor was performed as follows. Plasmid expressing the rat
vanilloid
receptor (VR1) extended with the short, 12 amino acid s-tag (KGFSYFGEDLMP) C-
terminally was constructed in a vector driven by the metallothionine (pMTH)
promoter.
The backbone vector has been previously described (Olah et al., Anal Biochem
221:94-
102; 1994). Briefly, Sal I and Mlu I restriction endonuclease sites were
incorporated into
a VR1 PCR fragment. After digestion of the PCR fragment with these enzynles,
the size-
separated cDNA insert was ligated in the pEMTH plasmid vector at the
compatible Xho I
and Mlu I sites (Olah et al., 1994). The chimeric constructs were verified by
sequencing
and transiently transfected into NIH 3T3 cells employing the protocol provided
for the
lipofectamine reagent (Life Sciences, Gaithersburg, MD).
Preparation of VRls expressing cell line - To prepare cell lines
permanently expressing the recombinant VR1 with the C-terminal E-tag NIH 3T3
cells
were transiently transfected with the pMTH-VR1E plasmid. GenePorter (GP)
purchased
from Gene Therapy Systems was used as transfection reagent. NIH 3T3 cells were
seeded
in 24 well plates a day before transfection, then 2 g pMTH-VRls plasmid
DNA/well
was used together with 25 and 10 l of GP reagent, respectively. The DNA and
the GP
were mixed in serum free Opti-MEM for 15 minutes at room temperature then
placed on
the cultured cells. After 3 hrs at 34 C the incubation medium was supplemented
with
equal volume of complete DMEM containing 10% FBS, 1% streptomycin, and 1%
glutamine. To prevent acidification of the culture medium the pH was buffered
to 7.5
with 20 mM HEPES. To diminish heat induced activation of VR1, cells were
cultured in
incubator adjusted to 34 C. After 24 hrs cells were transferred into selection
medium
prepared in complete DMEM buffered with 20 mM HEPES to pH = 7.5, containing
0.8
g/ml geneticin (G418). The selection medium was changed every second days.
After one
month G418 resistant colonies were tested with vanilloid-induced Ca2+-
transport assays.
A colony (A5) exhibiting RTX-induced 45Ca2+-uptake > 50 fold above the base
line
determined with non-transfected NIH3T3 cells was chosen for further studies.
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Similar results were obtained for the VRl tagged with the 12 anlino acid s-
epitope, compared to the VR1 eGFP, indicating that a C-terminal tag per se,
does not
significantly change the calcium uptalce parameters.
Assessment of the quantitative characteristic of [3H]RTX binding to eGFP-
and e-tagged VR1 expressed in Cos7 cells showed that progressive [3H]RTX was
almost
completely inhibited by co-incubation with 10 uMCPZ. Both tagged recombinants
exhibited a high affinity, dose-dependent interaction and cooperativity among
the
receptors. No significant [3H]RTX binding was detect in cells transfected with
the eGFP
plasmid alone.
Confocal microscopy showed that VR1eGFP was prominently found in the
ER and to a lesser degree, in the plasma membrane. The physiological evidence,
however, demonstrated the presence of functional receptor protein in the
plasma
membrane. Incubation of VR1eGFP-expressing cells with 1 nM RTX for 20 sec
induced
a dramatic fragmentation of the ER, appearance of microvilli at the plasma
membrane,
and rounding up of filamentous mitochondria. Although mitochondria reacted
rapidly to
1 nM RTX or 1 uM CAP, no nlixing between the VR1 eGFP vesicles and the
mitochondrial membranes was observed. Without external Ca2+, the vanilloid-
induced
membrane alterations were delayed 5-10 min (vs. msec) in VR1eGFP-expressing
cells.
In cells expressing only eGFP, the mitochondria and ER did not change in
response to
vanilloid treatment.
Both electrophysiology and fluorescent microscopy demonstrated dramatic
membrane remodeling in response to vanilloids in cells expressing VRl eGFP.
VRl eGFP
and VRls expression in transiently transfected cells conferred vanilloid-
induced plasma
membrane 45 Ca2+ flux. The effect of RTX on the cytosolic Ca2+ was then
examined by
microfluorometry in transfected cells loaded with the Ca2+ monitoring dye,
Fura-2 AM.
The resting [Ca2+]i was similar in Cos 7 cells transfect with either VR1 eGFP
or eGFP
plasmid. Addition of 1 nM RTX induced a rapid (within 10 sec) elevation of
[Ca2+]; in
VR1 eGFP-expressing cells that peaked at 500 nM at about 1 min and, consistent
with the
ER and mitochondrial damage, did not return to resting levels. In the absence
of external
Ca2+, vanilloids were less effective. Cells expressing eGFP only showed no
increase in
[Ca2+]i.
Time lapse confocal microscopy demonstrated the in vivo dynamics of
vanilloid action on live VRl eGFP-expressing cells. Prior to RTX treatment,
VRl eGFP -
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decorated ER showed a similar morphology. Within 30 seconds of RTX treatment,
the
ER condensed and the nuclear envelope.was outlined by the VRl eGFP
fluorescence.
These changes coincided with the increase in [Ca2+]i. VR1eGFP-expressing cells
were
visualized with 1 second scans at 1 minute intervals for one hour using
confocal
microscopy. Three minutes after addition of 1 nM RTX, the cells showed
extensive
accumulation of VR1eGFP at the nuclear membrane and in membrane vesicles
around the
nucleus, similar to that observed at 90 seconds. With time, progressively
growing blebs
were noted in the nuclear membrane. Cells showed membrane degradation
concluding
with bursting of the plasma membrane within an hour, often at about 45
minutes. Lower
doses of RTX (<_ 0.1 nM) evoked slower nuclear membrane fragmentation.
This example demonstrates that application of VR1 anagonist to VRl-
expressing cells triggers a cascade of events leading to cell death. These
data showed that
VRl activation produces a transmembrane calcium flux which fragments the ER
and
mitochondria, causes propidium iodide uptake.
Example 2. VR1 agonist injection into the trigeminal ganglion
Methods
Trigeminal microinjections: Male Sprague Dawley rats (300g) were
anesthetized with a combination of ketamine/xylazine and placed in a
stereotaxic frame.
A 26 gauge stainless steel catheter, sharpened with a bevel of -0.5mm, was
positioned at
2.5 mm posterior and 1.5 min lateral to bregma. The needle was advanced till
it touched
the base of the skull. At this point the tip has penetrated through the
trigeminal ganglion,
which is -1.2 mm in depth. The needle was retracted 0.5 mm and RTX (200 ng)
was
injected in a volume of 2 microliters over 1.5 to 2 min. The RTX was diluted
with 0.9%
saline from a stock solution which contained lmg/ml of RTX, 10% ethanol, 10%
Tween
80 and 80% normal.saline. The vehicle that was injected was a 1:10 dilution of
the RTX
stock vehicle using 0.9% saline as the diluent. The needle is left in place
for an additional
minute, withdrawn, and the scalp incision is closed with stainless steel
clips.
Behavioral/physiological assessmeiits: Nociceptive behavior was assessed
by the capsaicin eye-wipe test. In this test a 0.01% solution of capsaicin is
instilled into
the eye. This causes the rat to squint, blink and wipe the eye with the
forepaw. At the
concentration used the wiping behavior lasts approximately one minute and the
quantitative end point is the total number of wipes in one minute. Loss of
nociceptive
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primary afferents also affects peripheral inflammation. This can be assessed
by
measuring peripheral plasma extravasation using intravenous administration of
Evans
blue dye during inflammation or activation of primary afferent endings by
capsaicin. In
the presence of nociceptive nerves topical capsaicin activates the primary
afferent nerve
ending and releases transmitters, these dilate the blood vessels and allow
plasma proteins
to extravasate into the tissue. Evans blue binds strongly to albumin and the
skin turns
blue due to the leakage of the blue albumin into the skin. There was a
dramatic blockade
of plasma extravasation by intraganglionic RTX.
Anatomical assessments: Rats were perfused with 4% paraformaldehyde
via an intracardiac puncture and aortic cannulation. The ganglion and the
brain
stem/spinal cord region centered on the obex were removed. Tissues were
stained for
CGRP and Substance P, both peptides are made by primary afferent neurons that
co-
express the VR1 receptor. The nociceptive inputs from one ganglion are
lateralized and
project only to one side of the trigeminal nucleus caudalis. Thus, one side
can be
compared to the other in the same animal.
Results: In the eye wipe test, the unilateral control showed 20 2 wipes in
the first minute. Treatment with 0.2 ug of RTX injected into the trigeminal
ganglion
resulted in no eye wipes within the first minutes (n=8 rats). In animals
receiving 0.02 ug
of RTX (n=4), the control values were 28 2 wipes in the first minute. RTX
treatment
resulted in a decrease in the number of wipes to 7 3 in the first minute.
Control animals
(n=10) receiving an injection of the vehicle alone showed 24 wipes in the
first minute on
the unaffected side compared to 23 wipes in the first minute on the injected
side.
Staining of the ganglion for CGRP demonstrate that cells in the ganglion
that received the RTX were killed by direct injection. RTX selectively removes
C-fiber
neurons from the ganglion. See Figures 1 and 2 where some of the large neurons
remain
intact after RTX injection. Their cell bodies are lightly toned rather than
dark black. The
loss of plasma extravasation on the ganglion-injected side of the rat's head
showed that
loss of cells in the ganglion is manifested by a corresponding loss of pain-
sensing nerves
in the skin.
The experiments demonstrated that direct administration of a vanilloid
receptor agonist to the trigeminal ganglion results in killing of VR- 1 -
expressing cells and
a concordant decrease in sensitivity to pain.
Example 3. Intrathecal administration of RTX.
21
CA 02442049 2008-07-09
` , .
Pain sensitivity following intrathecal administration of RTX to rats was
measured using a test for thennal sensitivity well known to those in the art,
the paw
withdrawal latency test (e.g., Hargreaves (1988) Pain 32:77-88). Sprague
Dawley rats
received 6 ug of RTX in a 5 microliter volume administered by lumbar puncture.
The
baseline sensitivity was 8.4 1.3 second. Following administration of RTX,
the paw
withdrawal latency was 18:1:1.2 seconds. No attenuation of mechanical pinch
sensitivity
was observed. Thus, RTX administration resulted in attenuation of thermal
sensitivity.
Example 4. Administration of a VRl agonist to a patient suffering from chronic
pain
Selective ablation of VRl-expressing neurons is often used to treat patients
suffering from chronic pain. For example, the method can be used to treat a
patient with
chronic pain resulting from injury to a single nerve. In this example, because
the injury is
to a single nerve, one dorsal root ganglion is treated. The ganglion is
visualized, often by
CAT scan or fluoroscopy. Prior to injection of the VRl agonist, e.g., RTX, a
local
anesthetic is administered. The RTX (for example, 400 ng) is administered in a
volume
of, e.g., 100 ul as a single injection directly into the dorsal root ganglion
over one minute.
Following administration the needle is removed and the patient undergoes
observation.
Administration of RTX results in the reduction of the symptoms of chronic pain
the
treated patient.
22
i i
CA 02442049 2003-09-22
, = ,
SEQUENCE LISTING
<110> THE GOVERNMENT OF THE UNITED STATES, AS REPRESENTED BY THE
SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SERVICES
<120> SELECTIVE ABLATION OF PAIN-SENSING NEURONS BY ADMINISTRATION OF A
VANILLOID RECEPTOR AGONIST
<130> 40330-2072
<140> WO PCT/USO1/09425
<141> 2001-03-22
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 12
<212> PRT
<213> Artificial
<220>
<223> Epsilon-tag
<400> 1
Lys Gly Phe Ser Tyr Phe Gly Glu Asp Leu Met Pro
1 5 10
22a
