A-317491, a novel potent and selective non-nucleotide antagonist of P2X3 and P2X2/3 receptors, reduces chronic inflammatory and neuropathic pain in the rat.
ABSTRACT P2X3 and P2X2/3 receptors are highly localized on peripheral and central processes of sensory afferent nerves, and activation of these channels contributes to the pronociceptive effects of ATP. A-317491 is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor activation. A-317491 potently blocked recombinant human and rat P2X3 and P2X2/3 receptor-mediated calcium flux (Ki = 22-92 nM) and was highly selective (IC50 >10 microM) over other P2 receptors and other neurotransmitter receptors, ion channels, and enzymes. A-317491 also blocked native P2X3 and P2X2/3 receptors in rat dorsal root ganglion neurons. Blockade of P2X3 containing channels was stereospecific because the R-enantiomer (A-317344) of A-317491 was significantly less active at P2X3 and P2X2/3 receptors. A-317491 dose-dependently (ED50 = 30 micromolkg s.c.) reduced complete Freund's adjuvant-induced thermal hyperalgesia in the rat. A-317491 was most potent (ED50 = 10-15 micromolkg s.c.) in attenuating both thermal hyperalgesia and mechanical allodynia after chronic nerve constriction injury. The R-enantiomer, A-317344, was inactive in these chronic pain models. Although active in chronic pain models, A-317491 was ineffective (ED50 >100 micromolkg s.c.) in reducing nociception in animal models of acute pain, postoperative pain, and visceral pain. The present data indicate that a potent and selective antagonist of P2X3 and P2X2/3 receptors effectively reduces both nerve injury and chronic inflammatory nociception, but P2X3 and P2X2/3 receptor activation may not be a major mediator of acute, acute inflammatory, or visceral pain.
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ABSTRACT: Olfactory ensheathing glial cells (OECs) are a specialized type of glia that form a continuously aligned cellular pathway that actively supports unprecedented regeneration of primary olfactory axons from the periphery into the central nervous system. Implantation of OECs stimulates neural repair in experimental models of spinal cord, brain and peripheral nerve injury and delays disease progression in animal models for neurodegenerative diseases like amyotrophic lateral sclerosis. OECs implanted in the injured spinal cord display a plethora of pro-regenerative effects; they promote axonal regeneration, reorganize the glial scar, remyelinate axons, stimulate blood vessel formation, have phagocytic properties and modulate the immune response. Recently genome wide transcriptional profiling and proteomics analysis combined with classical or larger scale "medium-throughput" bioassays have provided novel insights into the molecular mechanism that endow OECs with their pro-regenerative properties. Here we review these studies and show that the gaps that existed in our understanding of the molecular basis of the reparative properties of OECs are narrowing. OECs express functionally connected sets of genes that can be linked to at least 10 distinct processes directly relevant to neural repair. The data indicate that OECs exhibit a range of synergistic cellular activities, including active and passive stimulation of axon regeneration (by secretion of growth factors, axon guidance molecules and basement membrane components) and critical aspects of tissue repair (by structural remodeling and support, modulation of the immune system, enhancement of neurotrophic and antigenic stimuli and by metabolizing toxic macromolecules). Future experimentation will have to further explore the newly acquired knowledge to enhance the therapeutic potential of OECs.Experimental neurology. 05/2014;
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ABSTRACT: Approximately 50-70% of patients experience incision-induced mechanical nociception after surgery. However, the mechanism underlying incision-induced mechanical nociception is still unclear. Interleukin-10 and brain-derived neurotrophic factor are important pain mediators, but whether interleukin-10 and brain-derived neurotrophic factor are involved in incision-induced mechanical nociception remains uncertain. In this study, forty rats were divided randomly into the incision surgery (n = 32) and sham surgery (n = 8) groups. Plantar incision on the central part of left hind paw was performed under anesthesia in rats from the surgery group. Rats in the sham surgery group received anesthesia, but not an incision. Von Frey test results showed that, compared with the sham surgery group, incision surgery decreased the withdrawal threshold of rats at 0.5, 3, 6 and 24 hours after incision. Immunofluorescence staining in the dorsal root ganglia of the spinal cord (L3-5) showed that interleukin-10 and brain-derived neurotrophic factor were expressed mainly on small- and medium-sized neurons (diameter < 20 μm and 20-40 μm) and satellite cells in the dorsal root ganglia of the spinal cord (L3-5) in the sham surgery group. By contrast, in the surgery group, high expression of interleukin-10 and brain-derived neurotrophic factor appeared in large-sized neurons (diameter > 40 μm) at 6 and 24 hours after incision surgery, which corresponded to the decreased mechanical withdrawal threshold of rats in the surgery group. These experimental findings suggest that expression pattern shift of interleukin-10 and brain-derived neurotrophic factor induced by incision surgery in dorsal root ganglia of rats was closely involved in lowering the threshold to mechanical stimulus in the hind paw following incision surgery. Pain-related mediators induced by incision surgery in dorsal root ganglia of rats possibly underlie mechanical nociception in ipsilateral hind paws.Neural Regeneration Research 12/2013; 8(35):3325-33. · 0.23 Impact Factor
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ABSTRACT: AbstractATP‐gated P2X3 receptors of sensory ganglion neurons are important transducers of painful stimuli and are modulated by extracellular algogenic substances, via changes in the receptor phosphorylation state. The present study investigated the role of calcium/calmodulin‐dependent serine protein kinase (CASK) in interacting and controlling P2X3 receptor expression and function in mouse trigeminal ganglia. Most ganglion neurons in situ or in culture co‐expressed P2X3 and CASK. CASK was immunoprecipitated with P2X3 receptors from trigeminal ganglia and from P2X3/CASK‐cotransfected human embryonic kidney (HEK) cells. Recombinant P2X3/CASK expression in HEK cells increased serine phosphorylation of P2X3 receptors, typically associated with receptor upregulation. CASK deletion mutants also enhanced P2X3 subunit expression. After silencing CASK, cell surface P2X3 receptor expression was decreased, which is consistent with depressed P2X3 currents. The reduction in P2X3 expression levels was reversed by the proteasomal inhibitor MG‐132. Moreover, neuronal CASK/P2X3 interaction was up‐regulated by nerve growth factor (NGF) signaling and down‐regulated by P2X3 agonist‐induced desensitization. These data suggest a novel interaction between CASK and P2X3 receptors with positive outcome for receptor stability and function. As CASK‐mediated control of P2X3 receptors was dependent on the receptor activation state, CASK represents an intracellular gateway to regulate purinergic nociceptive signaling.Journal of Neurochemistry 01/2013; 126(1). · 4.24 Impact Factor
A-317491, a novel potent and selective non-
nucleotide antagonist of P2X3and P2X2/3receptors,
reduces chronic inflammatory and neuropathic pain
in the rat
Michael F. Jarvis*†, Edward C. Burgard*, Steve McGaraughty*, Prisca Honore*, Kevin Lynch*, Timothy J. Brennan‡,
Alberto Subieta‡, Tim van Biesen*, Jayne Cartmell*, Bruce Bianchi*, Wende Niforatos*, Karen Kage*, Haixia Yu*,
Joe Mikusa*, Carol T. Wismer*, Chang Z. Zhu*, Katharine Chu*, Chih-Hung Lee*, Andrew O. Stewart*,
James Polakowski*, Bryan F. Cox*, Elizabeth Kowaluk*, Michael Williams*, James Sullivan*, and Connie Faltynek*
*Neuroscience Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6123; and‡Department of
Anesthesiology, University of Iowa, Iowa City, IA 52242-1079
Edited by John W. Daly, National Institutes of Health, Bethesda, MD, and approved October 25, 2002 (received for review September 4, 2002)
P2X3 and P2X2/3 receptors are highly localized on peripheral and
central processes of sensory afferent nerves, and activation of these
channels contributes to the pronociceptive effects of ATP. A-317491
is a novel non-nucleotide antagonist of P2X3 and P2X2/3 receptor
activation. A-317491 potently blocked recombinant human and rat
P2X3and P2X2/3receptor-mediated calcium flux (Ki? 22–92 nM) and
was highly selective (IC50>10 ?M) over other P2 receptors and other
neurotransmitter receptors, ion channels, and enzymes. A-317491
also blocked native P2X3 and P2X2/3 receptors in rat dorsal root
ganglion neurons. Blockade of P2X3 containing channels was ste-
reospecific because the R-enantiomer (A-317344) of A-317491 was
significantly less active at P2X3and P2X2/3receptors. A-317491 dose-
dependently (ED50 ? 30 ?mol?kg s.c.) reduced complete Freund’s
potent (ED50 ? 10–15 ?mol?kg s.c.) in attenuating both thermal
hyperalgesia and mechanical allodynia after chronic nerve constric-
pain models. Although active in chronic pain models, A-317491 was
models of acute pain, postoperative pain, and visceral pain. The
present data indicate that a potent and selective antagonist of P2X3
inflammatory nociception, but P2X3 and P2X2/3 receptor activation
calized on peripheral and central processes of sensory afferent
neurons (1–3), has generated much interest in the role of this
receptor in nociceptive signaling (4). The discovery of the P2X3
receptor has provided a putative mechanism for previous reports
that ATP, released from sensory nerves (5), produces fast excita-
tory potentials in dorsal root ganglion (DRG) neurons (6). These
actions appear to be physiologically relevant because iontophoretic
application of ATP to human skin elicits pain (7) and exogenously
applied ATP enhances pain sensations in a human blister base
and as a heteromultimeric combination with the P2X2(P2X2/3)
receptor (1, 2, 9). Both P2X3-containing channels are expressed on
a high proportion of isolectin IB4-positive neurons in DRG (3, 10).
These receptors share similar pharmacological profiles (11), but
differ in their acute desensitization kinetics (10, 12). Immunohis-
tochemical studies have shown that P2X3receptor expression is
up-regulated in DRG neurons and ipsilateral spinal cord after
chronic constriction injury (CCI) of the sciatic nerve (13). Addi-
he cloning and characterization of the P2X3receptor, a specific
ATP-sensitive ligand-gated ion channel that is selectively lo-
tionally, CCI results in a specific ectopic sensitivity to ATP that is
not observed on contralateral (uninjured) nerves (14).
Recently, the phenotypic profile of P2X3 receptor gene-
disrupted mice has been reported (15, 16). P2X3(???) mice are
viable and show no overt behavioral perturbations. However,
P2X3(???) mice show reduced pain-related behaviors in re-
sponse to intraplantar ATP or formalin administration, and
ATP-mediated rapidly desensitizing inward currents in DRG
neurons are absent in these mice. Although these observations
reported a transient hyperalgesic response in P2X3(???) mice
after the intraplantar administration of complete Freund’s ad-
juvant (CFA) (16). The effects of P2X3receptor gene disruption
on visceral pain and other models of acute and chronic noci-
ception have not been reported.
There are numerous studies demonstrating that P2 receptor
agonists and antagonists modulate nociceptive behaviors in
rodents (17). However, determination of the specific role of
P2X3 receptor activation in different pain states has been
hampered by the lack of useful receptor ligands for in vivo
studies. Existing P2 receptor agonists nonselectively activate a
variety of P2 receptor subtypes and are metabolically labile (18).
P2 receptor antagonists such as suramin and pyridoxalphos-
phate-6-azophenyl-2?,4?-disulfonic acid lack high affinity and
specificity for individual P2 receptor subtypes (18).
The present studies were undertaken to characterize the antino-
ciceptive effects of A-317491 (Fig. 1A), the first non-nucleotide
antagonist that has high affinity and selectivity for blocking P2X3
homomeric and P2X2/3 heteromeric channels. The chiral pure
S-enantiomer, A-317491, was found to potently block P2X3and
P2X2/3receptor-activated calcium flux in vitro. After s.c. adminis-
tration, A-317491 dose-dependently reduced nociception in neu-
ropathic and inflammatory animal pain models, but generally
lacked acute analgesic efficacy. The R-enantiomer, A-317344,
showed markedly less activity at P2X3and P2X2/3receptors in vitro
and lacked antinociceptive effects in animal models.
Materials and Methods
Ca2?Influx Assay. Stably transfected 1321N1 human astrocytoma
cells expressing rat and human P2X receptors have been de-
scribed (9–11). Activation of human and rat recombinant P2X
receptors was determined on the basis of agonist-induced in-
creases in cytosolic Ca2?concentration as described with minor
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CCI, chronic constriction injury; CFA, complete Freund’s adjuvant; DRG,
dorsal root ganglion.
†To whom correspondence should be addressed. E-mail: email@example.com.
December 24, 2002 ?
vol. 99 ?
no. 26 ?
modifications (11). The fluorescent Ca2?chelating dye fluo-4
was used as an indicator of the relative levels of intracellular
Ca2?in a 96-well format by using a fluorescence imaging plate
reader (Molecular Devices). P2X receptor-expressing cells were
grown to confluence and plated in 96-well black-walled tissue
culture plates ?18 h before the experiment. One to two hours
before the assay, cells were loaded with fluo-4 AM (2.28 ?M;
Molecular Probes) in Dulbecco’s PBS (D-PBS) and maintained
in a dark environment at room temperature. Immediately before
the assay, each plate was washed twice with 250 ?l D-PBS per
well to remove extracellular fluo-4 AM. Two 50-?l additions of
compounds (prepared in D-PBS) were made to the cells during
each experiment. The first test compound (antagonist) addition
was made, and incubation continued for 3 min before the
addition of ?,?-me-ATP (3 ?M final concentration). Measure-
ments continued for 3 min after this final addition. Fluorescence
data were collected at 1- or 5-s intervals throughout the course
of each experiment. Concentration–response data were analyzed
by using GRAPHPAD PRISM (San Diego). Kivalues were estimated
by the equation: Ki? IC50?(1 ? [agonist]?agonist EC50) (19).
Electrophysiology. Whole-cell patch-clamp recordings were ob-
tained as described (10) from stable cell lines or DRG neurons
by using a modified extracellular saline consisting of 155 mM
NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM Hepes, 12
mM glucose, pH 7.4. The patch pipette solution consisted of 140
mM potassium aspartate, 20 mM NaCl, 10 mM EGTA, 5 mM
Hepes. All cells were voltage-clamped at ?60 mV, and series
resistance was compensated 75–90% by using an Axopatch 200B
amplifier (Axon Instruments, Foster City, CA).
Rat DRG neurons were prepared as described (10). Lumbar
(L4–6) DRG were dissected and placed in DMEM (HyClone)
containing 0.3% collagenase B (Roche Molecular Biochemicals)
for 60 min at 37°C. The collagenase was replaced with 0.25%
trypsin (GIBCO?BRL) in Ca2??Mg2?-free Dulbecco’s PBS and
further digested for 30 min at 37°C. Ganglia were washed in fresh
DMEM, dissociated by trituration, and plated on polyethyleni-
mine-treated coverslips. Cells were plated in 1 ml DMEM
supplemented with 10% FBS (HyClone), nerve growth factor
(50 ng?ml, Roche Molecular Biochemicals), and 100 units?ml
Drugs were applied to the cells by using a piezoelectric-driven
glass theta tube positioned near the cell. During experiments,
agonists were usually applied every 3 min. A-317491 was both
preapplied and coapplied to cells during agonist application.
Responses were acquired and digitized at 3 kHz, and analyzed by
using PCLAMP software (Axon Instruments). Current amplitudes
were measured at the peak of the response.
Pharmacological Selectivity Studies. The activity of A-317491 (10
selectivity relative to 86 other cell-surface receptors, ion channels,
transport sites, and enzymes including the opioid receptor subtypes
and cycloxygenases 1 and 2, by use of standardized assay protocols
(Cerep, Celle l’Evescault, France) as described (20).
In Vivo Studies: Subjects. In most experiments, male Sprague–
Dawley rats (Charles River Breeding Laboratories) weighing
200–300 g were used. The abdominal constriction assay was
conducted by using male 129J mice weighing 20–25 g (The
Jackson Laboratories). The hotplate assay was conducted by
using male CF-1 mice (Harlan Farms, Portage, MI) weighing
25–30 g. These animals were group-housed in American Asso-
ciation for the Accreditation of Laboratory Animal Care-
approved facilities at Abbott Laboratories in a temperature-
Food and water was available ad libitum except during testing.
All animal handling and experimental protocols were approved
by an institutional animal care and use committee.
Analgesia and Side-Effect Assays. A-317491 and A-317334 were
acute (noxious thermal, mechanical, and chemical stimulation),
inflammatory (intraplantar formalin, carrageenan, and CFA), and
and inflamed colonic distention) and postoperative pain (20–22).
The specific methodologies for these nociceptive assays and the
assessment of rat motor performance, hemodynamics, and general
CNS function are described in detail in the Supporting Text, which
is published as supporting information on the PNAS web site,
www.pnas.org. Unless otherwise noted, all experimental and con-
trol groups contained at least six animals each, and data are
expressed as mean ? SEM. Data analysis was conducted by using
ANOVA and appropriate post hoc comparisons (P ? 0.05) as
described (20, 21). ED50 values were estimated by using least-
squares linear regression.
Compounds. A-317491 and A-317344 were synthesized at Abbott
Laboratories. Compounds were dissolved in sterile water for s.c
administration and administered in a final volume of 1–5 ml?kg,
s.c. Except where noted, compounds were administered 30 min
before nociceptive and side-effect testing.
In Vitro Activities of A-317491. ATP and ?,?-meATP are potent
agonists at both P2X3 and P2X2/3 receptors (11). Because
?,?-meATP is a poor agonist for P2X2receptors (1, 12), it was
used to activate calcium flux in 1321N1 cells expressing human
and rat homomeric P2X3 and heteromeric P2X2/3 receptors.
A-317491 (S-enantiomer) was identified as a potent antagonist
of ?,?-meATP-activated recombinant rat and human P2X3and
P2X2/3 receptors and was significantly more potent than the
R-enantiomer, A-317344 (Table 1). A-317491 showed signifi-
cantly higher affinity in blocking P2X3 receptors (Table 1)
compared with its ability to inhibit functional activation of other
P2X receptors or the P2Y2receptor (Table 2 and Fig. 1C).
to block activation of the human P2X3receptor, as compared with other P2X
receptor subtypes. Representative concentration-effect curves were normalized
to the agonist response (percentage maximal response) in the absence of
A-317491. See Tables 1 and 2 for agonist concentrations and derived Kivalues.
(C) Representative concentration-effect curves for ?,?-meATP in the absence
mean ? SEM from three separate experiments. (D) Schild plot.
www.pnas.org?cgi?doi?10.1073?pnas.252537299Jarvis et al.
Because the fast-desensitizing properties of the homomeric
P2X3receptors limit analysis of antagonist competitiveness (23),
by using P2X2/3receptors. A-317491 was found to be a compet-
itive antagonist of rat P2X2/3receptors, with increasing concen-
trations of A-317491 producing rightward parallel shifts in the
?,?-meATP dose–response curve (Fig. 1B). A Schild analysis
(Fig. 1D) of these data yielded a pA2value of 232 nM, which is
in agreement with the estimated Kivalue of 92 nM at rat P2X2/3
receptors (Table 1).
Fig. 2 summarizes electrophysiological studies of the effects of
A-317491 on ?,?-meATP-induced currents in both stably trans-
fected cells and DRG neurons. Application of ?,?-meATP
evoked rapidly desensitizing currents in cells expressing human
P2X3receptors. A-317491 produced a concentration-dependent
block of human P2X3currents with an IC50value of 97 nM (n ?
3, Ki? 17 nM, Fig. 2 A and B). Similar results were obtained with
recombinant human P2X3receptors when ATP (3 ?M) was used
as the agonist (IC50? 99 nM, n ? 3, Ki? 4 nM) and with human
P2X2/3receptors when ?,??meATP (10 ?M) was used as the
agonist (IC50? 169 nM, n ? 2–6, Ki? 20 nM). A-317491 was
equally effective at blocking rapidly desensitizing P2X3-
mediated currents in rat DRG neurons (Fig. 2 C and D).
A-317491 produced a concentration-dependent block of DRG
currents with an IC50value of 15 nM (n ? 3). At all receptor
subtypes, the effects of A-317491 were reversible, and essentially
complete block of current was observed at a concentration of 10
?M. At this concentration, no nonspecific effects of the com-
pound were observed on cellular input resistance or voltage-
clamp holding current.
A-317491 was also evaluated by Cerep for activity at 86 other
receptors, enzymes, and ion channels (17). A-317491 was inac-
tive (IC50? 10 ?M) at most of these other proteins. The only
interaction with an IC50? 10 ?M was at the ? opioid receptor
(IC50 ? 5 ?M); in contrast, 10 ?M A-317491 caused ?10%
inhibition of binding to the ? opioid receptor and only 15%
inhibition of binding to the ? opioid receptor. Preliminary
pharmacokinetic studies in rats indicated that 10 ?mol?kg
A-317491 had high (?80%) systemic bioavailability after s.c.
dosing (estimated plasma concentration ? 15 ?g?ml, ?99%
protein bound) and a half-life in plasma of 11 h. A-317491 did
not undergo any detectable metabolism (oxidation or glucu-
ronidation) in in vitro assays using human and rat liver micro-
somes (unpublished observations).
Antinociceptive Activity of A-317491. To further characterize the
nociceptive role of P2X3 receptor activation, A-317491 was
evaluated in a variety of animal pain models after s.c. adminis-
tration. A-317491 was most potent in reducing mechanical
allodynia and thermal hyperalgesia in the CCI model (ED50?
10 and 15 ?mol?kg s.c., Fig. 3 A and B) as compared with the
other animal models of nociception tested (Table 3). A-317491
was fully effective in blocking nociception in this model, whereas
the R-enantiomer, A-317344, was inactive (Fig. 3C). The antino-
ciceptive effects of A-317491 in the CCI model were rapid in
onset and persisted for 5 h after s.c. administration (Fig. 3D). In
contrast to its full efficacy in the CCI model, A-317491 was only
partially effective (50% reduction at 100 ?mol?kg s.c.) in
reducing tactile allodynia thresholds in the L5?L6 nerve ligation
model (Table 3).
Table 1. In vitro activity of A-317491 and A-317344 at human
and rat P2X3and P2X2/3receptors
22 ? 8
22 ? 3
92 ? 11
9 ? 2
1,300 ? 30
1,100 ? 200
Kicalculated by the method of Cheng–Prusoff (19) using the IC50values
determined in the presence of 3 ?M agonist (?,?-meATP); mean ? SEM (n ?
3–10). pEC50values for ?,?-meATP at rat and human P2X3receptors averaged
6.21 ? 0.05; pEC50values at rat and human P2X2/3receptors averaged 5.65 ?
0.08 (ref. 11 and unpublished observations).
Table 2. Activity of A-317491 at other P2X and the P2Y2receptors
7.73 ? 0.27
6.11 ? 0.20
6.30 ? 0.17
5.86 ? 0.15
7.99 ? 0.31
4.97 ? 0.17
4.33 ? 0.13
Data represent means ? SEM from at least three separate experiments.
3 ?M ?,?-meATP (control), and the percentage control response was mea-
sured in the presence of increasing concentrations of A-317491 (n ? 3). (B)
Representative ?,?-meATP-induced current traces recorded from a P2X3-
expressing cell before (control) and during application of 300 nM A-317491.
(C) A-317491 concentration–response curve measured in rat DRG neurons.
Currents were activated by 10 ?M ?,?-meATP, and the percentage control
response was measured in the presence of increasing concentrations of
A-317491 (n ? 3). (D) Representative ?,?-meATP-induced current traces
recorded from a rat DRG neuron before (control) and during application of
300 nM A-317491.
(A) A-317491 concentration–response curve measured in stable cells
Jarvis et al.PNAS ?
December 24, 2002 ?
vol. 99 ?
no. 26 ?
After 48 h of inflammation induced by the intraplantar
administration of CFA, A-317491 fully blocked thermal hyper-
algesia (Fig. 4A). The antinociceptive effects of A-317491 were
specific to the injured paw, as the paw withdrawal latencies for
the uninjured paw were not significantly altered by A-37491 at
the doses tested. The antinociceptive effects of A-317491 in this
model were also stereospecific because the R-enantiomer,
A-317344, was inactive in this model (Fig. 4B). As was observed
in the CCI model, the analgesic effects of A-317491 were rapid
in onset and lasted for 8 h after s.c. administration (Fig. 4C).
A-317491 produced an equivalent amount of antinociception
after twice-daily administration for 4 days as was observed after
acute administration (Fig. 4D).
A-317491 also dose-dependently reduced nociceptive re-
sponses in chemically induced pain models including the persis-
tent phase of the formalin test and a murine model of abdominal
pain, the acetic acid-induced abdominal constriction assay
(ACA) (Table 3; Fig. 5). The R-enantiomer, A-317344, was
completely inactive in the ACA assay. As shown in Table 3,
A-317491 was significantly more effective in reducing nocicep-
tion in the persistent phase of the formalin assay compared with
its activity in the acute phase immediately after formalin ad-
ministration. A-317491 was also generally ineffective at doses up
to 100 ?mol?kg s.c. in reducing nociception elicited by a variety
of other acute noxious stimuli including thermal, mechanical,
capsaicin, and acute inflammatory hyperalgesia (Table 3).
The effects of A-317491 were also evaluated in a model of
postoperative pain induced by the incision of the skin, fascia, and
plantaris muscle in the rat (22). In this model, A-317491 (100
?mol?kg s.c.) administered 30 min before or after the surgery, or
locally (300 nmol) around the incision site 5 min before or after the
surgery, did not decrease the mechanical allodynia observed 2 h
modify the development of mechanical allodynia (von Frey hair
sensitivity) 1 and 2 days after surgery (Table 3). A-317491 (100
?mol?kg s.c.) also had no effect on visceral pain in the rat as shown
by its inability to attenuate either the visceromotor response
observed after acute noxious colonic distension or inflammation-
induced visceral hyperalgesia (Table 3).
Effects on Motor Activity, CNS, and Cardiovascular Function.
A-317491 had no significant effect (P ? 0.05) on motor coor-
dination at doses up to 300 ?mol?kg s.c., as measured by the
ability of rats to run on an accelerating rotating rod (rotorod
assay, control latency ? 59 ? 1 s, A-317491 300 ?mol?kg
latency ? 53 ? 2 s). A-317491 also had no effect on spontaneous
exploratory activity of rats in a novel open field at 100 ?mol?kg
s.c., but a statistically significant (32 ? 7%, P ? 0.05) reduction
hair) thresholds (A) and thermal paw withdrawal latencies (B) in the CCI
model. I, Paw responses ipsilateral to the nerve injury; and E, paw responses
of the contralateral paw (mean ? SEM). (C) A-317491 (100 ?mol?kg s.c.), but
not A-317344 (100 ?mol?kg s.c.) attenuates mechanical allodynia (open bars)
contralateral paw. (D) Time course for the onset and duration of A-317491
ical allodynia thresholds of CCI animals treated with vehicle.*, P ? 0.05 as
compared with vehicle-treated animals (n ? 6 per group).
Table 3. Analgesic profile of A-317491
ModelED50, ?mol?kg s.c.% Effect, 100 ?mol?kg s.c.
L5?L6 nerve ligation
Formalin test (persistent phase)
Chronic thermal hyperalgesia (CFA)
Acute thermal hyperalgesia (Carrageenan)
Mouse abdominal constriction
Rat colonic distention
Rat visceral hyperalgesia
Postoperative somatic pain
Rat acute thermal
Rat acute mechanical
Rat intraplantar capsaicin
Formalin (acute phase)
100 ? 8*
110 ? 7*
50 ? 8*
60 ? 3*
74 ? 11*
15 ? 5
2778 ? 2*
0 ? 10
0 ? 4
?10 ? 10
7 ? 5
25 ? 7
*Significantly different (P ? 0.05) from vehicle-treated control animal responses (n ? 6 per group).
www.pnas.org?cgi?doi?10.1073?pnas.252537299Jarvis et al.
of spontaneous exploratory activity was observed at 300
?mol?kg s.c. Rats were fully awake, were responsive to stimuli,
and retained the righting reflex, consistent with their ability to
perform the rotorod test at all doses tested.
A-317491 (10–300 ?mol?kg s.c.) was also evaluated in a
number of assays to assess general CNS function. No significant
differences (P ? 0.05) from vehicle-treated animals were noted
for A-317491-treated mice in the Irwin test, the ethanol and
barbital interaction assays, and the pentylenetetrazol-induced
seizure assay (data not shown). Statistically significant, but
dose-independent, anticonvulsant effects were found for
A-317491 (100 ?mol?kg s.c., 10% protection, P ? 0.05) in the
mouse electroconvulsive shock model, and a 0.4–0.6°C increase
in rectal temperature was observed at 10 and 30 ?mol?kg s.c.
General CNS depression (respiration, sensory-motor deficits)
and lethality were observed at a dose of 1,000 ?mol?kg s.c. in
mice. The cardiovascular effects of A-317491 were examined by
using conscious, freely behaving rats instrumented with telem-
etry transmitters. After s.c. administration, A-317491 produced
no statistically significant changes in mean arterial pressure or
heart rate when administered to conscious rats at 300 ?mol?kg.
These data demonstrate that A-317491 is a potent and selective
antagonist of P2X3and P2X2?3receptors. Like the nucleotide-
based antagonist 2?,3?-O-2,4,6-trinitrophenyl (TNP)-ATP (22),
A-317491 is a competitive antagonist of P2X2/3receptors. How-
ever, unlike TNP-ATP, which also has high affinity for P2X1
receptors (24), A-317491 exhibits ?100-fold selectivity for P2X3
and P2X2/3receptors compared with its activity at other P2X
receptor subtypes. A-317491 shows only very weak or no affinity
for a large selection of other cell surface receptors, ion channels,
not inhibit ectonucleotidase activity as measured by [32P]ATP
degradation (unpublished observations). The specificity of the
antagonist actions of A-317491 for P2X3and P2X2/3receptor
blockade is further supported by the significantly weaker activity
of the R-enantiomer, A-317344, as a P2X3receptor antagonist.
Electrophysiological data from both recombinant and native
P2X3 receptor-mediated responses demonstrate that receptor
block is rapid in onset, reversible, and devoid of nonspecific
effects. A-317491 is also not susceptible to metabolic dephos-
phorylation like TNP-ATP (25). Thus, A-317491 represents the
first non-nucleotide, potent and selective, antagonist of P2X3-
A-317491 effectively reduced nociception in the CFA-induced
model of chronic inflammatory pain and was particularly potent
CCI neuropathic pain model. The enhanced antinociceptive
efficacy of A-317491 in the CCI model is consistent with the
previously documented up-regulation of P2X3-containing chan-
nels in rat DRG and spinal dorsal horn in this model (13).
Although less active, A-317491 also significantly reduced tactile
allodynia thresholds in the L5?L6 nerve injury model. After
L5?L6 nerve ligation, there is a significant decrease in the
density of IB4-positive small diameter neurons in the L5?L6
DRG and a corresponding reduction in P2X3immunoreactivity
(26). However, a subpopulation of IB4 negative larger diameter
neurons in the L5?L6 DRG remain intact, show P2X3immu-
noreactivity, and demonstrate both fast (P2X3-like) and slowly
(P2X2/3-like) desensitizing responses to ATP (26). Taken to-
gether, these data provide neurochemical and functional evi-
dence that activation of P2X3and P2X2/3receptors is modulated
during chronic pain and blockade of these receptors can reduce
nociception mediated by both small and larger diameter sensory
neurons in chronic pain states.
The antinociceptive effects of A-317491 in both the CFA and
CCI models were rapid in onset and pharmacologically specific
because similar antinociceptive effects were not observed after
systemic administration of A-317344, the less active R-
enantiomer. The antinociceptive effects of A-317491 in the CFA
model were also maintained after repeated administration twice
daily for 4 days. Thus, A-317491 shows reduced potential to
produce tolerance compared with morphine, which has signifi-
cantly reduced antinociceptive activity after repeated dosing
(21). The antinociceptive effects of A-317491 were not accom-
panied by side effects commonly associated with other analgesic
48 h after intraplantar administration of CFA. F, Responses of CFA-injected
paw. E, Paw withdrawal latencies of uninjected contralateral paw (mean ?
SEM). (B) A-317491 (100 ?mol?kg s.c.), but not A-317344 (100 ?mol?kg s.c.),
attenuates CFA-induced thermal hyperalgesia (open bars) in the rat. Hatched
bars represent responses of the contralateral paw. (C) Time course for the
(F) in the rat. E, Paw withdrawal latencies of CFA-injected paws from animals
antinociceptive effects of A-317491 (100 ?mol?kg s.c., open bars) after re-
peated dosing in the CFA model were not significantly different as compared
with vehicle-pretreated animals (chronic) or animals that received a single
(acute) administration of A-317491. Hatched bars represent responses of the
contralateral uninjected paw.*, P ? 0.05 as compared with vehicle-treated
animals (n ? 6 per group). ?, P ? 0.05 as compared with CFA-treated paw.
(A) A-317491 dose-dependently increases paw withdrawal latencies
dose-dependently reduces nociception in the mouse acetic acid-induced ab-
dominal constriction assay. Data represent mean ? SEM.*, P ? 0.05 as
compared with vehicle-treated animals (n ? 6 per group).
(Left) Antinociceptive effects of A-317491 in the persistent phase of
Jarvis et al.PNAS ?
December 24, 2002 ?
vol. 99 ?
no. 26 ?
agents. A-317491 produced no significant effects on cardiovas-
cular function and did not significantly alter motor performance
at doses up to 100 ?mol?kg s.c. in the rat. Additional studies
in the mouse indicated that A-317491 was generally devoid
of dose-dependent effects on CNS function up to doses of
300 ?mol?kg s.c.
The antinociceptive effects of A-317491 in the CFA and CCI
models are in agreement with other recent data demonstrating
that intrathecal P2X3 antisense (27) and P2X3 receptor gene
disruption (15, 16) reduce nociceptive sensitivity. Specifically,
systemic administration of A-317491 was found to produce a
similar reduction (?50%) of spontaneous formalin-induced
persistent nociception as was produced by P2X3 antisense or
observed in P2X3gene-disrupted animals. However, in contrast
to the reported hyperalgesic effects of P2X3 receptor gene
disruption (16), A-317491 produced significant antihyperalgesia
in models of both chronic inflammatory hyperalgesia and neu-
Unlike its analgesic effects in the chronic inflammatory and
the neuropathic pain assays, A-317491 was ineffective in models
of acute nociception involving a variety of noxious stimuli
including heat, mechanical, and chemical (capsaicin, carra-
geenan, and formalin) stimulation. Although A-317491 was
effective in reducing nociception in the acetic acid-induced
mouse abdominal constriction assay, it was ineffective in reduc-
ing visceromotor responses after acute noxious colonic disten-
sion or inflammation-induced visceral hyperalgesia in the rat.
A-317491 was also ineffective in reducing pain-related behaviors
in a plantar incision model of postoperative somatic pain (22).
In comparison to its analgesic efficacy in the CFA and neuro-
pathic chronic pain models, these data suggest that activation of
P2X3 and P2X2/3 receptors may be more involved in specific
aspects of chronic inflammatory hyperalgesia and nerve injury-
induced allodynia than in acute or visceral pain states.
Although the reasons for the differential analgesic efficacy of
A-317491 in various pain states remain unclear, this pattern of
activity may reflect different relative contributions of glutama-
tergic neurotransmission in various pain states. For example,
recent studies have shown that the pharmacology of the plantar
incision model of postoperative pain is different from the
pharmacology of more classical models of inflammatory and
spinal N-methyl-D-aspartate (NMDA) receptor and metabo-
tropic glutamatergic receptor transmission have little antinoci-
ceptive effect in normal animals, but have antinociceptive prop-
erties in several models of chronic pain such as thermal
hyperalgesia and mechanical allodynia observed in inflamma-
tory (carrageenan, CFA) or neuropathic (sciatic nerve constric-
tion, spinal nerve ligation) pain states (29, 30). Interestingly, it
has recently been shown that spinal NMDA receptor and
metabotropic glutamatergic receptor antagonists are ineffective
at reducing incision-induced mechanical allodynia, suggesting a
(28). Because previous data has shown that activation of P2X
receptors facilitates the release of glutamate in spinal dorsal
horn neurons (31), the analgesic effects of A-317491 may depend
on glutamaterigically mediated nociceptive processes.
Previous pharmacological studies of the nociceptive role of
P2X3-containing channels have been limited to the evaluation of
low-affinity nonselective antagonists like suramin and pyridox-
alphosphate-6-azophenyl-2?,4?-disulfonic acid (17) or more
recently to the potent, but unstable antagonist, 2?,3?-O-2,4,6-
trinitrophenyl-ATP, after local (32) or intrathecal administra-
tion (33, 34). In the absence of selective ligands, molecular
approaches like gene disruption (15, 16) and gene knockdown
(antisense) (27) studies have provided some evidence that
P2X3-containing channels contribute to nociception. A-317491
represents an important advance in P2X3receptor pharmacol-
ogy because of its high degree of P2X receptor selectivity and its
and the P2X3 gene disruption data (15, 16, 27) indicate that
processes of peripheral and central sensitization states associ-
ated with some forms of chronic inflammatory pain and to
nociceptive states arising from nerve injury.
We thank Dr. Gerald F. Gebhart and Elizabeth Kamp (University of
Iowa) for contributing visceral nociception data.
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