1. No category

Brain Advance Access published November 19, 2014
doi:10.1093/brain/awu330
BRAIN 2014: Page 1 of 8
| 1
BRAIN
A JOURNAL OF NEUROLOGY
REPORT
Endogenous adenosine A3 receptor activation
selectively alleviates persistent pain states
Joshua W. Little,1 Amanda Ford,1 Ashley M. Symons-Liguori,2 Zhoumou Chen,1 Kali Janes,1
Timothy Doyle,1 Jennifer Xie,2 Livio Luongo,3 Dillip K. Tosh,4 Sabatino Maione,3
Kirsty Bannister,5 Anthony H. Dickenson,5 Todd W. Vanderah,2 Frank Porreca,2
Kenneth A. Jacobson4 and Daniela Salvemini1
1
2
3
4
5
Saint Louis University School of Medicine, Saint Louis, MO USA
University of Arizona, Department of Pharmacology and Anesthesiology, Tucson, AZ USA
Department of Experimental Medicine, Division of Pharmacology, Second University of Naples, Naples, Italy
National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD USA
University College London, Department of Neuroscience, Physiology and Pharmacology, London, UK
Correspondence to: Daniela Salvemini, Ph.D.,
1402 S. Grand Ave
St. Louis, MO 63104, USA
E-mail: [email protected]
Keywords: adenosine; A3AR; chronic pain; spontaneous pain; rostral ventromedial medulla
Abbreviations: A3AR = adenosine A3 receptor, now known as ADORA3; AR = adenosine receptor; CCI = chronic constriction
injury; ED = effective dose; RVM = rostral ventromedial medulla
Introduction
Chronic pain is an enormous unmet medical need with a
multi-billion dollar impact on society (Pizzo and Clark,
2012). The most successful pharmacological approaches
for the treatment of chronic pain rely on engagement of
endogenous circuits involving opioid, adrenergic, and calcium channel mechanisms (Millan, 2002). However, drugs
exploiting these pathways are often associated with intolerable side effects that result in discontinued use, inadequate
pain relief, and diminished quality of life. The purine nucleoside adenosine functions in extracellular signalling
Received May 21, 2014. Revised September 18, 2014. Accepted October 1, 2014.
Published by Oxford University Press on behalf of the Guarantors of Brain 2014. This work is written by US Government employees and is in the public domain in the US.
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Chronic pain is a global burden that promotes disability and unnecessary suffering. To date, efficacious treatment of chronic pain has
not been achieved. Thus, new therapeutic targets are needed. Here, we demonstrate that increasing endogenous adenosine levels
through selective adenosine kinase inhibition produces powerful analgesic effects in rodent models of experimental neuropathic
pain through the A3 adenosine receptor (A3AR, now known as ADORA3) signalling pathway. Similar results were obtained by the
administration of a novel and highly selective A3AR agonist. These effects were prevented by blockade of spinal and supraspinal A3AR,
lost in A3AR knock-out mice, and independent of opioid and endocannabinoid mechanisms. A3AR activation also relieved non-evoked
spontaneous pain behaviours without promoting analgesic tolerance or inherent reward. Further examination revealed that A3AR
activation reduced spinal cord pain processing by decreasing the excitability of spinal wide dynamic range neurons and producing
supraspinal inhibition of spinal nociception through activation of serotonergic and noradrenergic bulbospinal circuits. Critically,
engaging the A3AR mechanism did not alter nociceptive thresholds in non-neuropathy animals and therefore produced selective
alleviation of persistent neuropathic pain states. These studies reveal A3AR activation by adenosine as an endogenous anti-nociceptive
pathway and support the development of A3AR agonists as novel therapeutics to treat chronic pain.
2
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Materials and methods
Animals
Male Sprague Dawley rats, wild-type C57BL/6 mice, A3AR /
mice (Salvatore et al., 2000), and female BALB/cfC3H mice
were used for all experiments. A total of 301 rats and 63
mice were employed. Procedures for the maintenance and
use of animals were in accordance with the International
Association for the Study of Pain (Seattle, MD), NIH
(Bethesda, MD) guidelines on laboratory animal welfare,
United Kingdom Home Office in compliance with the UK
Animals (Scientific Procedures) Act 1986, and conformed to
the regulations of the SLU, AU, UCL and SUN Committees
on Animal Research. All observations were performed with the
operators blind to the identity of the test substances
administered.
Animal models
Persistent neuropathic pain models were performed as previously described including: chronic constriction injury (CCI)
(Little et al., 2012), spared nerve injury (Decosterd and
Woolf, 2000), spinal nerve ligation (Kim and Chung, 1992),
chemotherapy-induced peripheral neuropathy (Chen et al.,
2012), and cancer-induced bone pain (Lozano-Ondoua et al.,
2013).
Behaviour for pain models
Mechano-allodynia was assessed using calibrated von Frey filaments. Neurological function and motor coordination were
evaluated by Rotarod motor test. Normal nociception was assessed by tail flick and hot-plate latency tests. Spontaneous and
affective aspects of spinal nerve ligation-induced pain were
assessed using conditioned place preference as described elsewhere (King et al., 2009). In animals with cancer-induced bone
pain, spontaneous flinching and guarding behaviours were
monitored (Lozano-Ondoua et al., 2013).
Drug delivery
Rostral ventromedial medulla (RVM) cannulation, intrathecal
catheterization, and subsequent injections including bilateral
RVM microinjections were performed as previously described
(Little et al., 2012).
In vivo electrophysiology
Extracellular recordings of spinal cord wide dynamic range
neurons (L4-L5 segmental levels) were conducted on postspinal nerve ligation Days 14–18 in isoflurane-anaesthetized
rats.
Statistics
Significant differences were defined as a value of P 5 0.05.
Behavioural data and mechanical and thermal coding were
analysed by mixed-model two-way repeated measures
ANOVA with Bonferroni comparisons. Electrophysiology
data were analysed using a paired Student’s t-test. Sphericity
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within the peripheral nervous system and CNS at four G
protein-coupled adenosine receptor (AR) subtypes: A1,
A2A, A2B, and A3 (Fredholm et al., 2011). With a short
physiological half-life (510 s), adenosine is released to
signal locally following conditions such as tissue trauma
and pain (Fredholm et al., 2011). While adenosine is reported to provide potent and long-lasting pain suppression
in both preclinical animal models and human subject studies, targeting this endogenous pathway for pain management has not yet been achieved (Zylka, 2011). The
analgesic effect of adenosine has been attributed to A1AR
and A2AAR activation (Zylka, 2011); however, these conclusions were made without examining the contribution of
other receptor subtypes. As a consequence, a decade of
preclinical and clinical development efforts to treat chronic
pain have focused on A1AR/A2AAR agonists, but have
failed to yield a viable therapeutic approach due to several
undesirable actions, specifically A1AR/A2AAR-mediated
cardiovascular side effects (Zylka, 2011; Boison, 2013).
Discovery of a way to safely utilize the endogenous adenosine system for analgesia could offer meaningful relief for
pain sufferers. A3AR is highly expressed in many inflammatory cells including glial cells (Abbracchio et al., 1997)
and found in peripheral sensory neurons (Ru et al., 2011)
and neurons in various areas of the CNS (Lopes et al.,
2003). Endogenous adenosine signalling through A3AR
has been demonstrated to be neuroprotective (Boison
et al., 2010; Fishman et al., 2012); however, it is unknown
whether this endogenous pathway affects nociceptive processing. In contrast to the limited therapeutic utility of
A1AR and A2AAR agonists, the A3AR agonist IB-MECA
[N6-(3-iodobenzyl)-adenosine-50 -N-methyluronamide] and
its chlorinated counterpart Cl-IB-MECA have advanced
to clinical trials for non-pain indications and show a
good safety profile (Fishman et al., 2012). Very little is
known about the roles of the A3AR in pain and the
mechanisms and sites of action of A3AR agonists. One
report has examined the effects of IB-MECA in the formalin test in mice (Yoon et al., 2005); intrathecal delivery of
IB-MECA attenuated the inflammatory component, Phase
2 but not Phase 1. We recently reported that IB-MECA is
also effective in models of neuropathic pain induced by
diverse chemotherapeutic agents (paclitaxel, oxaliplatin
and bortezomib) and constriction of the sciatic nerve
(Chen et al., 2012). The effects of IB-MECA were corroborated with the highly selective A3AR agonist MRS5698
(Tosh et al., 2012) in a model of oxaliplatin-induced
neuropathic pain (Janes et al., 2014). In this study, we
extend our previous findings and demonstrate for the
first time that activation of A3AR at spinal and supraspinal
sites is key in the anti-nociceptive effects mediated by
endogenous adenosine and provide pharmacological evidence that identifies a selective A3AR agonist as a potent
non-narcotic agent that produces persistent pain relief
without altering the protective actions of acute physiological pain.
J. W. Little et al.
A3AR activation suppresses persistent pain
BRAIN 2014: Page 3 of 8
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Figure 1 Endogenous A3AR activation is anti-nociceptive. ABT702 (5 mg/kg, intraperitoneal; filled square) but not vehicle (open square)
increased paw withdrawal thresholds (PWTs) in rats at 7 days (D7) post-CCI. Pretreatment (15 min before) with MRS1523 (2 mg/kg, intraperitoneal; open circle), but not vehicle (open square), attenuated the effects of ABT702 (A). When compared to Day 7 wild-type (WT; white bar)
mice, ABT702 (5 mg/kg, intraperitoneal) treated A3AR / CCI-mice (hatched bar) had significantly lower paw withdrawal thresholds (B). The
beneficial effects of ABT702 (5 mg/kg, intraperitoneal; filled square) in CCI-rats were transiently reversed by MRS1523 (2 mg/kg, intraperitoneal;
open circle) but not its vehicle (Veh) administered at peak efficacy (C). Data are mean SD for n = 7 (A) or n = 5 (B and C) animals/group and
analysed by two-way ANOVA with Bonferroni comparisons. #P 5 0.05 versus Day 0; *P 5 0.05 versus Day 7; †P 5 0.05 versus ABT702.
Results
The adenosine kinase inhibitor
ABT-702 reverses neuropathic pain
We first explored the contribution of A3AR signalling to
anti-nociception during persistent pain using a rodent
model of CCI (Bennett and Xie, 1988). Adenosine kinase
is a key intracellular enzyme regulating intra- and extracellular concentrations of adenosine and its inhibition effectively potentiates extracellular adenosine concentration and
signalling (Boison, 2013). Systemic administration of the
selective non-nucleoside adenosine kinase inhibitor ABT702 (Jarvis et al., 2000) at peak CCI-induced pain (postinjury Day 7) reversed established hypersensitivity to mechanical stimuli (mechano-allodynia) as measured by changes
in paw withdrawal threshold (g) (Fig. 1A). The antiallodynic effects of ABT-702 were partially attenuated by
pretreatment or post-treatment with the selective A3AR antagonist MRS1523 (Li et al., 1998) and in A3AR / mice
(Salvatore et al., 2000) with CCI (Fig. 1A–C). MRS1523
alone had no effect on paw withdrawal threshold. Partial
attenuation is consistent with the reported contribution of
A1AR to the effects of ABT-702 (Kowaluk et al., 2000).
Additionally, the long-lasting effects of ABT-702 also agree
with previous reports where its anti-allodynic action in a
neuropathic pain model was sustained up to 11 h (Kowaluk
et al., 2000). ABT-702 did not alter paw withdrawal
thresholds
in
unaffected
contralateral
paws
(Supplementary Fig. 1A). To examine the importance of
A3AR signalling in persistent neuropathic pain of a different aetiology, we tested the effects of ABT-702 in chemotherapy (i.e. paclitaxel)-induced peripheral neuropathy
(Chen et al., 2012), a common dose-limiting toxicity and
cause of dose reduction for many chemotherapeutic agents
(Farquhar-Smith, 2011). ABT-702 reversed peak (Day 25)
chemotherapy-induced mechano-allodynia and mechanohyperalgesia in an A3AR-dependent manner as anti-nociception was partially blocked or reversed by MRS1523
(Supplementary Fig. 1B–E).
A3AR activation by MRS5698
reverses persistent neuropathic
pain without tolerance
We next investigated the potential utility of a highly selective, orally bioavailable A3AR agonist MRS5698 to modulate persistent pain; MRS5698 (Fig. 2A inset) has a high
affinity for A3AR (43 nM) and excellent selectivity (5104fold over human, rat, and mouse A1AR or A2AAR) (Tosh
et al., 2012). Subcutaneous MRS5698 administration
during peak CCI-induced neuropathic pain in rats reversed
mechano-allodynia in a dose-dependent manner (ED50 =
0.35 mg/kg or 0.6 mmol/kg at 1 h; Fig. 2A) comparable to
morphine at peak effect (ED50 = 0.5 mg/kg or 1.8 mmol/kg)
and to IB-MECA (ED50 = 0.2 mg/kg or 0.4 mmol/kg; n = 6)
with a fast onset of action (530 min) and full efficacy
within 1 h post-dosing. MRS5698 lacked effect on paw
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was tested with Mauchly’s test, Greenhouse-Geisser corrections were used where required. Conditioned place preference
difference scores were analysed using the paired t-test.
Statistical analyses were performed using SPSS v21 (IBM)
and Graphpad Prism v6.02.
Details of all procedures and reagents are described fully in
the Supplementary material.
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J. W. Little et al.
withdrawal threshold in contralateral paws (Supplementary
Fig. 2A). Further characterization of MRS5698 revealed a
wide therapeutic index, inferred from a lack of sedation or
motor deficits in animals tested on a Rotarod at 30 mg/kg
(i.e. 100 the anti-allodynic ED50; Supplementary Fig. 2B)
and the non-lethal toleration of oral doses up to 150 mg/kg
in rats (i.e. 400 the anti-allodynic ED50). MRS5698 was
effective when given by multiple systemic routes of administration (i.e. intraperitoneal, subcutaneous, and intravenous) and protective in other well-established rat
neuropathic pain models including spared nerve injury
(Decosterd and Woolf, 2000) and spinal nerve ligation
(Kim and Chung, 1992). Indeed, therapeutic administration
of MRS5698 (1 mg/kg) reversed maximal mechano-allodynia within 1 h by 82 15% (mean SD, n = 5) and by
98 3.3% (n = 7) in spared nerve injury and spinal nerve
ligation, respectively. MRS5698 was similarly efficacious in
a more complex model of persistent pain with multiple
aetiologies: cancer-induced bone pain (Lozano-Ondoua
et al., 2013). Noteworthy, MRS5698 administered on
Day 10 post-cancer-induced bone pain induction provided
dose-dependent relief of spontaneous pain behaviours
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Figure 2 A3AR activation reverses CCI-induced mechano-allodynia without analgesic tolerance or altering normal nociception. MRS5698 but not vehicle (Veh, open circle) increased paw withdrawal thresholds (PWTs) Day 7 post-CCI in rats (A) and decreased
flinching and guarding behaviours resultant from cancer-induced bone pain in mice (B). Pretreatment with MRS1523 (2 mg/kg, intraperitoneal;
black bar), but not vehicle blocked the effects of MRS5698 (1 mg/kg; grey bar) in CCI-rats (C). MRS5698 (1 mg/kg) reversed paw withdrawal
threshold in wild-type (WT; grey bar) but not A3AR / CCI-mice (hatched bar) (D). Pretreatment with naloxone (8 mg/kg, intraperitoneal; light
grey bar), SR144528 (3 mg/kg, intraperitoneal; grey hatched bar), or rimonabant (1 mg/kg, intraperitoneal; dark grey bar) did not attenuate
MRS5698’s effects (1 mg/kg; black bar) in CCI-mice (E). Repeated daily injections of morphine (3 mg/kg, subcutaneous; grey circle) but not
MRS5698 (1 mg/kg; filled square) led to a loss of anti-allodynic effect (peak reversal, 1 h post-dosing) by the fourth day in CCI-rats (F). MRS5698
(10 mg/kg/d; filled square) but not vehicle (open circle) delivered via a 7-day (D7) subcutaneous minipump reversed mechano-allodynia in CCImice that was transiently reversed by a single injection of MRS1523 (2 mg/kg, intraperitoneal) on day 14 (D14), 6 days after minipump implantation
(G). MRS5698 (1 mg/kg) had no effect on tail flick latency (TFL, H) or in the hot-plate test (I) in naı¨ve rats. When compared to sham (hatched
bars), MRS5698 (1 mg/kg) produced robust chamber-paired place preference at Day 14 post-spinal nerve ligation (SNL, black bars) in rats (J). Data
are mean SD (A–I) or SEM (J) for n = 4–7 (A and C–I), n = 8 (B), or n = 10–12 (J) animals/group and analysed by two-way ANOVA with
Bonferroni comparisons or paired Student’s t-test. #P 5 0.05 versus Day 0; *P 5 0.05 versus Day 7; †P 5 0.05 versus MRS5698 or Day 14;
§
P 5 0.05 versus Day 7 at 1 h or Veh/Sham. BL = Baseline.
A3AR activation suppresses persistent pain
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| 5
Figure 3 Spinal and RVM A3AR contribute to the reversal of mechano-allodynia. Quantitative reverse transcription PCR (A) and
western blot (B) revealed detectable levels of A3AR in lower lumbar spinal cord (SC) and RVM of rats. Bands are from representative gels are
shown above. Intrathecal (filed triangle, i.th.; 2 h post-ABT702) or RVM (inverted filled triangle, 6 h post-ABT702) administration of MRS1523
(1 nmol) but not vehicle in rats on Day 7 post-CCI transiently reversed the effects of ABT702 (5 mg/kg, intraperitoneal) (C). Data are mean SD
for n = 6 animals/group and analysed by two-way ANOVA with Bonferroni comparisons. #P 5 0.05 versus Day 0; *P 5 0.05 versus Day 7;
§
P 5 0.05 versus ABT702 at 2 h; ’P 5 0.05 versus ABT702 at 6 h.
MRS5698 does not alter normal
nociception
MRS5698 tested at the highest effective dose had no effect
in tests that measure the acute thermal nociceptive component of physiological pain: tail flick and hot-plate (Fig. 2H
and I).
MRS5698 produces conditioned place
preference in nerve-injured rats
In addition to evoked pain behaviours, patients with neuropathic conditions commonly experience ongoing (spontaneous) pain that corresponds to the affective/motivational
aspects of neuropathic pain. Relief of ongoing pain
behaviours can engage the mesolimbic reward circuit (King
et al., 2009; Navratilova et al., 2012) and be unmasked in
animals using conditioned place preference. During ongoing
pain, pairing an anti-nociceptive treatment that is not inherently rewarding, such as peripheral nerve block, with a context (chamber) can elicit conditioned place preference (King
et al., 2009). Ideally, a pain therapeutic offers pain relief without abuse potential from inherent reward. Preconditioning
baseline for total time spent in the chambers was not significantly different between vehicle and MRS5698 paired chambers for all animals (Supplementary Fig. 2C). We found that
systemic MRS5698 (1 mg/kg) was associated with enhanced
chamber preference at Day 14 post-spinal nerve ligation [difference score in seconds (i.e. post-conditioning minus preconditioning): 132.2 42.8 s], but not sham surgery (difference
score: 24.6 46.7 s) (Fig. 2J). These results suggest that
A3AR activation reverses spontaneous pain behaviours without inherent reward.
Spinal and supraspinal A3AR
contribute to the reversal of
mechano-allodynia
To explore sites and neurobiological mechanisms underlying the anti-nociceptive effects of A3AR activation, we
examined A3AR in CNS regions related to nociceptive processing. We found A3AR/Adora3 mRNA transcript and
protein in the spinal cord and RVM (Fig. 3A and B), key
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(flinching, guarding) (ED50 = 0.1 mg/kg or 0.2 mmol/kg at
1 h; Fig. 2B). As A3AR agonists are in clinical trials as
anticancer agents (Fishman et al., 2012), the potential
dual pharmacological properties (anticancer effects and
pain-relieving properties) of an A3AR agonist may offer a
significant therapeutic advantage.
The specificity of MRS5698 at A3AR was confirmed with
pharmacological and genetic studies in the CCI model: its
anti-nociceptive effects were blocked by MRS1523 and lost
in A3AR / mice (Fig. 2C and D). MRS5698 anti-nociception was independent of endogenous opioid and cannabinoid pathways. Naloxone (non-selective m-opioid antagonist)
(Jurna, 1988), rimonabant (cannabinoid receptor 1 antagonist) (Rinaldi-Carmona et al., 1995), or SR144528 (cannabinoid receptor 2 antagonist) (Rinaldi-Carmona et al.,
1998) pretreatment did not attenuate the effects of
MRS5698 (Fig. 2E). Repeated injections of MRS5698 in
rats did not lead to tolerance or a loss of efficacy since
consecutive daily injections of MRS5698 on Days 8–15
reversed mechano-allodynia as effectively as on Day 7
(Fig. 2F). Continuous anti-nociception was observed with
a 6-day subcutaneous mini-pump infusion of MRS5698
started on Day 7 post-CCI; acute exposure to MRS1523
reduced these effects, confirming A3AR signalling (Fig. 2G).
In contrast, morphine lost its anti-nociceptive effects when
given over a similar time course (Fig. 2F).
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| BRAIN 2014: Page 6 of 8
J. W. Little et al.
administration of MRS1523 (1 nmol, open square), but not vehicle (Veh, filled square), blocked the anti-allodynic effects of MRS5698 (1 mg/kg,
subcutaneous) in CCI-rats (Day 7). MRS1523 (open triangle) or vehicle (open circle) alone had no effect (A and B). In spinal nerve ligation (SNL)
rats (Day 14), MRS5698 (1 mg/kg, subcutaneous; black bars) significantly reduced the excitability of spinal neurons to peripherally applied natural
and electrical stimuli (C). RVM microinjection of MRS5698 (0.3, filled triangle; 1, inverted filled triangle; or 3 nmol, filled square), but not vehicle
(open circle) dose-dependently reversed CCI-induced mechano-allodynia in rats; these effects were blocked by RVM-injection of MRS1523
(1 nmol, open square) (D). The RVM MRS5698 (3 nmol, filled square) effects were transiently reversed by intrathecal delivery of methysergide
(30 mg; filled diamond) or yohimbine (30 mg; filled circle). Methysergide (open diamond) or yohimbine (open circle) alone had no effect (E). (F)
Schematic representation of mechanisms underlying A3AR-induced anti-nociception revealed in our study. Data are mean SD (A, B, D and E)
or SEM (C) for n = 4–5 (A, B, D and E) or n = 8 (C) animals/group and analysed by two-way ANOVA with Bonferroni or paired Student’s t-test.
#
P 5 0.05 versus Day 0; *P 5 0.05 versus Day 7 or baseline; †P 5 0.05 versus MRS5698. PD = post-discharge; IP = input; WU = wind up;
PAC = periaqueductal grey; LC = locus coeruleus; PWT = paw withdrawal threshold.
regions for nociceptive processing and modulation (Ossipov
et al., 2010). A3AR/Adora3 mRNA was expressed in the
lower lumbar (L4–6) spinal cord (mean Ct = 27.8 0.3;
n = 6) and micropunched RVM (mean Ct = 28.8 0.2;
n = 6) tissues (Fig. 3A); for comparison, Hprt1 was used
as an endogenous control gene in both the spinal cord
and
RVM
(mean
Ct = 20.8 0.4
and
mean
Ct = 21.5 0.2, respectively). Similarly, western blot revealed A3AR/ADORA3 protein was expressed in these
same tissues (Fig. 3B). Consistent with these A3AR expression patterns, spinal or bilateral intra-RVM microinjections
of MRS1523 transiently abrogated ABT-702 anti-nociception, suggesting A3AR activation by endogenous adenosine
at spinal and supraspinal sites (Fig. 3C). Furthermore,
intrathecal or intra-RVM administration of MRS1523
blocked the ability of systemically administered MRS5698
to reverse mechano-allodynia (Fig. 4A and B). These findings confirm A3AR expression in pain-related CNS areas
and demonstrate the functional relevance of A3AR to adenosine-mediated anti-nociception.
During neuropathic pain, spinal nociceptive processing is
enhanced by the increased spontaneous activity of wide
dynamic range pain projection neurons, which are modulated by both spinal and supraspinal (e.g. RVM) mechanisms (Ossipov et al., 2010). Thus, the integrated effect of
systemic MRS5698 was tested to determine if A3AR
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Figure 4 Mechanisms of A3AR-induced anti-nociception during persistent neuropathic pain. Intrathecal (i.th, A) or intra-RVM (B)
A3AR activation suppresses persistent pain
| 7
limitation of therapeutic approaches that enhance adenosine signalling (Fredholm et al., 2011; Zylka, 2011;
Boison, 2013); however, A3AR agonists in clinical trials,
IB-MECA and Cl-IBMECA, have no reported serious side
effects (Fishman et al., 2012). Yet, while these prototypical
A3AR agonists were anti-nociceptive in our preclinical studies (Chen et al., 2012), there may be therapeutic advantages using highly selective A3AR agonists such as
MRS5698. These are anticipated to potentially allow aggressive dose escalation in difficult or complicated clinical
settings; their high degree of selectivity should confer an
advantage as spill over effect on A1AR and A2AAR is significantly reduced. It has long been appreciated that harnessing the potent analgesic effects of adenosine could
provide a breakthrough step towards an effective treatment
for chronic pain. Our findings suggest that this goal may be
achieved by focusing future work on the A3AR pathway as
its activation provides robust anti-nociception across several models of neuropathic pain, and in the more complex
pain state of cancer-induced bone pain, which includes inflammatory and neuropathic features.
Acknowledgements
We are grateful to Dr Gary Bennett for his invaluable
advice throughout the course of these studies and for critically reviewing our work.
Funding
This work was supported by grants from the National
Cancer Institute (RO1CA169519), NIH predoctoral fellowship (5T32GM008306), NIDDK Intramural Research
Program, FIRB “Futuro in Ricerca” MIUR, Italy
(RBFR126IGO_001) and with additional support from
the Saint Louis Cancer Centre.
Supplementary material
Supplementary material is available at Brain online.
Discussion
Despite extensive research efforts, chronic pain remains a
large unmet medical need and novel therapies are required
as alternative options when the standards of care are not
sufficient or effective. Our results substantially extend our
previous findings exploring A3AR in pain (Chen et al.,
2012) by identifying an endogenous analgesic A3AR pathway within key regions of the CNS that is distinguished by
its potent efficacy and state-dependent nature, functioning
to suppress only pathological pain without altering the
normal pain threshold or activating reward centres in
normal rats. Cardiovascular side effects are the major
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activation inhibits nociceptive processing by reducing the
responsiveness of spinal wide dynamic range neurons. As
previously shown, baseline neuronal responses to a variety
of stimuli were similar in sham and neuropathic animals
since the model involves a loss of two-thirds of the afferent
input to the spinal cord; yet the wide dynamic range neurons in neuropathic animals maintain higher than predicted
levels of activity (Chapman et al., 1998). Subcutaneous
MRS5698 administered to nerve-injured rats during maximal mechano-allodynia significantly reduced evoked wide
dynamic range neuron responses to non-noxious and noxious mechanical, thermal, and electrical stimulation with
peak effects at 1 h post-dosing (Fig. 4C), but had no
effect in sham-operated rats (Supplementary Fig. 2D and
E), further supporting that A3AR activation produces
state-dependent anti-nociception. The effects were clear
and prolonged and tended to be larger for the mechanical
stimuli (Fig. 4C). The drug effects were apparent at 30 min
and maximal at 1 h. There was an incomplete recovery by
3 h. For comparable levels of neuronal firing, MRS5698
reduced the low intensity thermal response (42 C) by
65% compared to 84% for the 8 g mechanical stimulus.
The corresponding values for the medium stimuli were
48% and 66%, and 32% and 54% for the highest intensities. Even with the highest mechanical and thermal stimuli
the neuronal responses were still significantly reduced.
Next, we evaluated the contribution of the RVM to the
anti-allodynic effects of A3AR activation. The RVM is a
primary source of descending inhibition of spinal nociception by engaging serotonergic and noradrenergic bulbospinal circuits (Ossipov et al., 2010); therefore, we
investigated whether A3AR activation in the RVM
employs these circuits. Intra-RVM MRS5698 dose-dependently reversed CCI mechano-allodynia in an A3AR-dependent manner as intra-RVM pretreatment of MRS1523
prevented anti-nociception (Fig. 4D). Intrathecal delivery
of the serotonin receptor antagonist, methysergide (Hoyer
et al., 1994) or the 2 noradrenergic receptor antagonist,
yohimbine (Goldberg and Robertson, 1983) (Fig. 4E), also
attenuated MRS5698’s anti-nociceptive effects; these data
imply that A3AR activation in the RVM engages bulbospinal inhibitory circuits to suppress spinal nociception.
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