Adenosine receptor targets for pain.

The main focus for the development of adenosine targets as analgesics to date has been A1Rs due to its antinociceptive profile in various preclinical pain models. The usefulness of systemic A1R agonists may be limited by other effects (cardiovascular, motor), but enhanced selectivity for pain might occur with partial agonists, potent and highly selective agonists, or allosteric modulators. A2AR agonists exhibit some peripheral pronociceptive effects, but also act on immune cells to suppress inflammation and on spinal glia to suppress pain signaling and may be useful for inflammatory and neuropathic pain. A2BR agonists exhibit peripheral proinflammatory effects on immune cells, but also spinal antinociceptive effects similar to A2AR agonists. A3Rs are now demonstrated to produce antinociception in several preclinical neuropathic pain models, with mechanistic actions on glial cells, and may be useful for neuropathic pain. Endogenous adenosine levels can be augmented by inhibition of metabolism (via adenosine kinase) or increased generation (via nucleotidases), and these approaches have implications for pain. Endogenous adenosine contributes to antinociception by several pharmacological agents, herbal remedies, acupuncture, transcutaneous electrical nerve stimulation, exercise, joint mobilization, and water immersion via spinal and/or peripheral effects, such that this system appears to constitute a major pain regulatory system. Finally, caffeine inhibits A1-, A2A- and A3Rs with similar potency, and dietary caffeine intake will need attention in trials of: (a) agonists and/or modulators acting at these receptors, (b) some pharmacological and herbal analgesics, and (c) manipulations that enhance endogenous adenosine levels, all of which are inhibited by caffeine and/or A1R antagonists in preclinical studies. All adenosine receptors have effects on spinal glial cells in regulating nociception, and gender differences in the involvement of such cells in chronic neuropathic pain indicate gender may also need attention in preclinical and human trials evaluating the efficacy of adenosine-based analgesics.

Topical analgesics for neuropathic pain: preclinical exploration, clinical validation, future development.

Topical analgesics applied locally to skin or to specialized compartments modify pain by actions on sensory nerve endings and/or adjacent cellular elements. With this approach, there are low systemic drug levels, good tolerability and few drug interactions, and combination with oral formulations is feasible. The goal of this review is to provide an overview of the potential for topical analgesics to contribute to improved management of neuropathic pain. Mechanistic and preclinical studies indicate much potential for development of novel topical analgesics for neuropathic pain. In humans, two topical analgesics are approved for use in post-herpetic neuralgia (lidocaine 5% medicated plaster, capsaicin 8% patch), and there is evidence for efficacy in other neuropathic pain conditions. Comparative trials indicate similar efficacy between topical and oral analgesics. Not all individuals respond to topical analgesics, and there is interest in determining factors (patient factors, sensory characteristics) which might predict responsiveness to topical analgesics. There is a growing number of controlled trials and case reports of investigational agents (vasodilators, glutamate receptor antagonists, α2-adrenoreceptor agonists, antidepressants, centrally acting drugs), including combinations of several agents, indicating these produce pain relief in neuropathic pain. There is interest in compounding topical analgesics for neuropathic pain, but several challenges remain for this approach. Topical analgesics have the potential to be a valuable additional approach for the management of neuropathic pain.

Adenosine and ATP receptors.

Adenosine and ATP, via P1 and P2 receptors respectively, can modulate pain transmission under physiological, inflammatory, and neuropathic pain conditions. Such influences reflect peripheral and central actions and effects on neurons as well as other cell types. In general, adenosine A1 receptors produce inhibitory effects on pain in a number of preclinical models and are a focus of attention. In humans, i.v. infusions of adenosine reduce some aspects of neuropathic pain and can reduce postoperative pain. For P2X receptors, there is a significant body of information indicating that inhibition of P2X3 receptors may be useful for relieving inflammatory and neuropathic pain. More recently, data have begun to emerge implicating P2X4, P2X7 and P2Y receptors in aspects of pain transmission. Both P1 and P2 receptors may represent novel targets for pain relief.

Glutamate-evoked release of adenosine and regulation of peripheral nociception.

Glutamate (which facilitates peripheral nociception) releases adenosine (which inhibits peripheral nociception via adenosine A(1) receptors) when injected locally into the rat hindpaw. The present study determined whether this locally released adenosine could modulate spontaneous pain behaviors produced by a local injection of 1.5% formalin, by determining the effect of 8-cyclopentyl-theophylline (CPT; selective adenosine A(1) receptor antagonist) on flinching produced by formalin/glutamate combinations. Experiments were performed following a prior conditioning injection of 2.5% formalin into the contralateral hindpaw 3-4 days earlier. CPT augmented flinching behaviors produced by 1.5% formalin/1 micromol glutamate, but had no effect on behaviors produced by formalin or glutamate alone. CPT also augmented flinches generated by formalin/alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and formalin/kainic acid, but not by formalin/N-methyl-D-aspartate (NMDA) combinations. The conditioning leads to a clearer expression of the peripheral inhibitory effect of adenosine (inhibitory effect of an inhibitor of adenosine kinase on flinching also was observed), rather than an increased release of adenosine (no enhanced release observed by microdialysis). Microglia appear to be involved in the conditioning, as microglia are activated in the dorsal spinal cord 3 days following injection of 2.5% formalin, and augmentation of formalin/glutamate-induced flinching by CPT is inhibited by the glial metabolic inhibitor fluorocitrate. The augmentation of flinching by CPT is also eliminated following a spinal pretreatment with MK-801 (NMDA receptor antagonist), cyclohexyladenosine (adenosine A(1) receptor agonist), N(G)-nitro-L-arginine methyl ester HCl (nitric oxide synthetase inhibitor), and chelerythrine (protein kinase C inhibitor). The conditioning pretreatment with 2.5% formalin does not lead to a generalized chemical or thermal hypersensitivity in the contralateral hindpaw. This study demonstrates that prior exposure to 2.5% formalin in the contralateral hindpaw reveals an inhibitory effect of adenosine on peripheral nociception in the presence of glutamate; this conditioning involves microglia and other mechanisms involved in central sensitization.

A pilot study examining topical amitriptyline, ketamine, and a combination of both in the treatment of neuropathic pain.

OBJECTIVE: The involvement of ongoing peripheral activity in the generation of nociceptive input in neuropathic pain suggests that topical drug delivery may be useful as a treatment strategy. This is a pilot study providing initial information regarding the use of novel topical preparations containing amitriptyline (AMI), ketamine (KET), and a combination of both in the treatment of neuropathic pain. METHODS: The study design included a 2 day randomized, double blind, placebo controlled, 4 way cross-over trial of all treatments, followed by an open label treatment phase using the combination cream for 7 days. Twenty volunteers with chronic neuropathic pain were randomly assigned to treatment order and applied 5 mls of each topical treatment (1% AMI, 0.5% KET, combination AMI 1%/KET 0.5%, and placebo) for 2 days. Measures of pain at the end of each block included the short form McGill Pain Questionnaire (MPQ) and visual analog scales (VAS) for present pain intensity and pain relief. Eleven subjects who judged subjective improvement from any treatment in the initial trial entered the open-label trial and used the combination cream for 7 days. Pain levels were recorded daily using the same measures. Blood levels for amitriptyline and ketamine were performed at 7 days to determine whether systemic absorption had occurred. RESULTS: There was no statistically significant difference from placebo after 2 days for any treatment during the double blind component of the trial. In the 11 subjects who used the combination cream, there was a statistically significant effect, with subjects reporting significantly greater analgesia by days 3 to 7 according to measures of pain and pain relief. Blood levels revealed that there was no significant systemic absorption of amitriptyline or ketamine. Only 2 subjects experienced side effects; these were minor and did not lead to discontinuation of the cream. CONCLUSION: This pilot study demonstrated a lack of effect for all treatments in the 2 day double blind placebo controlled trial, followed by analgesia in an open label trial in a subgroup of subjects who chose to use the combination cream for 7 days. Blood analysis revealed no significant systemic absorption of either agent after 7 days of treatment, and creams were well tolerated. A larger scale randomized trial over a longer interval is warranted to examine further effects observed in the open label trial.

Enhanced release of adenosine in rat hind paw following spinal nerve ligation: involvement of capsaicin-sensitive sensory afferents.

Modulation of endogenous adenosine levels by inhibition of adenosine metabolism produces a peripheral antinociceptive effect in a neuropathic pain model. The present study used microdialysis to investigate the neuronal mechanisms modulating extracellular adenosine levels in the rat hind paw following tight ligation of the L5 and L6 spinal nerves. Subcutaneous injection of 50 microl saline into the nerve-injured paw induced a rapid and short-lasting increase in extracellular adenosine levels in the subcutaneous tissues of the rat hind paw ipsilateral to the nerve injury. Saline injection did not increase adenosine levels in sham-operated rats or non-treated rats. The adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine and the adenosine deaminase inhibitor 2'-deoxycoformycin, at doses producing a peripheral antinociceptive effect, did not further enhance subcutaneous adenosine levels in the nerve-injured paw. Systemic pretreatment with capsaicin, a neurotoxin selective for small-diameter sensory afferents, markedly reduced the saline-evoked release of adenosine in rat hind paw following spinal nerve ligation. Systemic pretreatment with 6-hydroxydopamine, a neurotoxin selective for sympathetic afferent nerves, did not affect release. These results suggest that following nerve injury, peripheral capsaicin-sensitive primary sensory afferent nerve terminals are hypersensitive, and are able to release adenosine following a stimulus that does not normally evoke release in sham-operated or intact rats. Sympathetic postganglionic afferents do not appear to be involved in such release. The lack of effect on such release by the inhibitors of adenosine metabolism suggests an altered peripheral adenosine system following spinal nerve ligation.

Chronic administration of amitriptyline and caffeine in a rat model of neuropathic pain: multiple interactions.

This study was designed to determine (1) whether chronic amitriptyline administration was effective in alleviating symptoms of neuropathic pain in a rat model of spinal nerve injury, and (2) whether the effect of amitriptyline involved manipulation of endogenous adenosine, by determining the effect of caffeine, a non-selective adenosine A(1) and A(2) receptor antagonist, on its actions. Nerve injury was produced by unilateral spinal nerve ligation of the fifth and sixth lumbar nerves distal to the dorsal root ganglion, and this resulted in stimulus-evoked thermal hyperalgesia and static tactile mechanical allodynia. Animals received pre- and post-surgical intraperitoneal doses of amitriptyline (10 mg/kg) and caffeine (7.5 mg/kg), alone or in combination, and following surgery, were administered amitriptyline (15-18 mg/kg/day) and caffeine (6-8 mg/kg/day), alone or in combination, in the drinking water. Rats were tested for thermal reaction latencies and static tactile thresholds at 7, 14 and 21 days following surgery. In the paw ipsilateral to the nerve ligation, chronic amitriptyline administration consistently decreased the thermal hyperalgesia produced by spinal nerve ligation over a 3-week period, and this effect was blocked by concomitant caffeine administration at all time intervals. In the contralateral paw, thermal withdrawal latencies were more variable, with the most reproducible finding being a reduction in thermal thresholds in the amitriptyline-caffeine combination group. There was no effect by either drug or the drug combination on the static tactile allodynia produced by spinal nerve ligation in the ipsilateral paw. However, chronic amitriptyline administration induced a tactile hyperaesthesia in the contralateral paw at all time intervals, and this effect was exacerbated by concomitant chronic caffeine administration. The results of this study indicate that chronic administration of amitriptyline is effective in alleviating thermal hyperalgesia, but not static tactile allodynia, in the hindpaw ipsilateral to nerve injury, and the block of this effect by caffeine suggests that this effect is partially achieved through manipulation of endogenous adenosine systems. Additionally, chronic amitriptyline administration induces contralateral hyperaesthetic responses that are augmented by caffeine. Both the symptom-specific effect, and adenosine involvement in amitriptyline action may be important considerations governing its use in neuropathic pain.

Involvement of primary sensory afferents, postganglionic sympathetic nerves and mast cells in the formalin-evoked peripheral release of adenosine.

Injection of formalin into the rat hind paw produces a dose-dependent local peripheral release of adenosine. Low doses of formalin (0.5-2.5%) evoke release during the first 10 min following injection, while a high dose of formalin (5%) evokes release lasting for 60 min. The current study was designed to determine the possible origin of release produced by two doses of formalin (1.5% and 5%). Microdialysis probes were implanted into the subcutaneous tissue under the glabrous skin of the hind paw of anaesthetized rats, and adenosine was determined by high performance liquid chromatography. Pretreatment with capsaicin, a neurotoxin selective for unmyelinated small diameter primary afferent nerves, markedly reduced the adenosine released by 1.5% formalin and the early phase of release by 5% formalin. Acute injection of 1% capsaicin to the hind paw of untreated rats also induced adenosine release. Pretreatment with 6-hydroxydopamine, a neurotoxin selective for sympathetic postganglionic nerve terminals, had no effect on release evoked by 1.5% formalin, but significantly reduced adenosine release during the late phase of release induced by 5% formalin. Pretreatment with compound 48/80, which degranulates mast cells, had no effect on adenosine release evoked by either concentration of formalin. We conclude that the origin of the adenosine released peripherally by formalin depends on the formalin concentration. At the lower concentration (1.5%), release is predominantly from unmyelinated sensory afferent nerve terminals, while at the higher concentration (5%), unmyelinated afferent nerve terminals are involved in the early phase, while sympathetic postganglionic nerve terminals are involved in the later phase. Mast cells do not contribute to release of adenosine evoked by either concentration of formalin.

Antidepressants as analgesics: an overview of central and peripheral mechanisms of action.

Antidepressants, given systemically, are widely used for the treatment of various chronic and neuropathic pain conditions in humans. In animal studies, antidepressants exhibit analgesic properties in nociceptive, inflammatory and neuropathic test systems, with outcomes depending on the specific agent, the particular test, the route of administration and the treatment method used. Although early studies focused on central (i.e., supraspinal, spinal) actions, more recent studies have demonstrated a local peripheral analgesic effect of antidepressants. These peripheral actions raise the possibility that topical formulations of antidepressants may be a useful alternative drug delivery system for analgesia. Antidepressants exhibit a number of pharmacological actions: they block reuptake of noradrenaline and 5-hydroxytryptamine, have direct and indirect actions on opioid receptors, inhibit histamine, cholinergic, 5-hydroxytryptamine and N-methyl-D-aspartate receptors, inhibit ion channel activity, and block adenosine uptake. The involvement of these mechanisms in both central and peripheral analgesia produced by antidepressants is considered. Data illustrating the preclinical peripheral analgesic actions of antidepressants are presented, as are some aspects of the mechanisms by which these actions occur.

Potentiation of formalin-evoked adenosine release by an adenosine kinase inhibitor and an adenosine deaminase inhibitor in the rat hind paw: a microdialysis study.

The present study examined the effects of local subcutaneous administration of formalin on adenosine release from the rat hind paw, and the effects of inhibitors of adenosine metabolism on such release. Microdialysis probes were inserted into the subcutaneous tissue of the plantar surface of rat hind paws. Samples were collected every 10 min at a perfusion rate of 2 microl/min and high performance liquid chromatography was used to measure adenosine levels. At lower concentrations of formalin (0.5-2.5%), a significant increase in adenosine levels was observed in the first 10 min after formalin injection, while at the highest concentration of formalin (5%), the increase in adenosine release was observed over 60 min. Co-administration of the adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine (100 nmol) with formalin, significantly increased adenosine release evoked by 0.5-1.5% formalin, but did not produce a further enhancement of release evoked by 5% formalin. The adenosine deaminase inhibitor 2'-deoxycoformycin (100 nmol) significantly increased adenosine levels at 5% formalin but had no effect at lower concentrations of formalin. In confirmation of previous studies, subcutaneous injection of formalin (0.5-5%) produced a characteristic biphasic concentration-related expression of nociceptive behaviours and an increase in paw volume. This study directly demonstrates that formalin can evoke a concentration-dependent local release of adenosine from the rat hind paw. The ability of an adenosine kinase inhibitor and an adenosine deaminase inhibitor to modulate this release is dependent on substrate adenosine concentrations.

Caffeine blockade of the thermal antihyperalgesic effect of acute amitriptyline in a rat model of neuropathic pain.

In the present study, we sought to determine whether administration of caffeine, a non-selective adenosine receptor antagonist, would affect the thermal antihyperalgesic efficacy of acute amitriptyline in a rat model of neuropathic pain. Rats were rendered neuropathic by unilateral tight ligation of the fifth and sixth lumbar spinal nerves, and tested for thermal hyperalgesia using a focused beam of light. Systemic administration of caffeine (1.5-7.5 mg/kg), at the same time as amitriptyline, blocked the thermal antihyperalgesic effect of 10 mg/kg amitriptyline. The greatest degree of block exerted by caffeine was observed with 3.75 mg/kg (100% block), a dose that had no observable intrinsic effect. Spinal administration of amitriptyline (60 microg) exhibited a mild antihyperalgesic effect that was unaffected by pretreatment with intrathecal caffeine (100 microg). Peripheral administration of amitriptyline into the neuropathic paw (under brief anesthesia) produced an antihyperalgesic effect at both 30 and 100 nmol, with a greater effect being observed at 100 nmol. Coadministration of caffeine (1500 nmol) partially antagonized the effects of both doses of amitriptyline. The results of this study suggest that the thermal antihyperalgesic effect of acute amitriptyline in this model may involve enhancement of an endogenous adenosine tone. This involvement is important in light of the widespread consumption of caffeine, which may potentially act to reduce the benefits of amitriptyline in the treatment of neuropathic pain.

Involvement of mast cells, sensory afferents and sympathetic mechanisms in paw oedema induced by adenosine A(1) and A(2B/3) receptor agonists.

Both the adenosine A(1) receptor agonist N(6)-cyclopentyladenosine and the adenosine A(2B/3) receptor agonist N(6)-benzyl-5'-N-ethylcarboxamido adenosine (N(6)-B-NECA) produce an acute paw oedema response following local s.c. injection into the rat hindpaw. This study characterized aspects of the mechanisms by which these responses occur by determining the effect of compound 48/80 (mast cell depleting agent), capsaicin (sensory neurotoxin) and 6-hydroxydopamine (sympathetic nervous system neurotoxin) on the paw oedema response produced by these agents. Compound 48/80 markedly reduced the increase in paw volume produced by both N(6)-cyclopentyladenosine and N(6)-B-NECA. Capsaicin significantly reduced paw oedema induced by N(6)-cyclopentyladenosine but not N(6)-B-NECA. In contrast, 6-hydroxydopamine reduced paw oedema induced by N(6)-B-NECA but not N(6)-cyclopentyladenosine. These results indicate an involvement of mast cells in paw oedema produced by both adenosine A(1) and A(2B/3) receptor agonists. For N(6)-cyclopentyladenosine, this involvement may be a secondary involvement due to activation of a neurogenic mechanism, but for N(6)-B-NECA, it may be a direct effect on mast cells. The nature of the involvement of the sympathetic nervous system in the action of N(6)-B-NECA is not entirely clear.

Antinociceptive and anti-inflammatory properties of an adenosine kinase inhibitor and an adenosine deaminase inhibitor.

Spinal administration of an adenosine kinase inhibitor, alone or in combination with an adenosine deaminase inhibitor, produces antinociception in inflammatory pain tests. In the present study, we examined the antinociceptive and anti-inflammatory effects produced by the peripheral (intraplantar) administration of 5'-amino-5'-deoxyadenosine (an adenosine kinase inhibitor), 2'-deoxycoformycin (an adenosine deaminase inhibitor), and combinations of both agents in the carrageenan-induced thermal hyperalgesia and paw oedema model in the rat. When injected in the ipsilateral paw immediately prior to carrageenan injection, both agents produced antinociception only at the highest dose (1 micromol), whereas a reduction in paw swelling was evident at a lower dose (300 nmol). Significant augmentation in both the antinociceptive and anti-inflammatory effects was seen when 5'-amino-5'-deoxyadenosine and 2'-deoxycoformycin were co-administered in equimolar doses at all dose levels. Both effects were mediated via activation of adenosine receptors, as indicated by blockade by an adenosine receptor antagonist. When administered into the contralateral paw, 1 micromol 5'-amino-5'-deoxyadenosine+1 micromol 2'-deoxycoformycin produced prominent antinociception, indicating a systemic drug activity. There was only a modest reduction in paw oedema in the carrageenan-injected (ipsilateral) paw, suggesting that much of this activity was locally mediated. Reversal of systemic effects on thermal thresholds by an intrathecal adenosine receptor antagonist implicates a spinal site of action in this instance. An ipsilateral administration of 1 micromol 5'-amino-5'-deoxyadenosine, but not 1 micromol 2'-deoxycoformycin, reduced carrageenan-induced c-Fos expression in the spinal dorsal horn, and this was further reduced by the peripheral co-injection of the two agents. These results provide evidence for a predominantly spinal antinociceptive effect and a predominantly peripheral anti-inflammatory effect produced by inhibitors of adenosine kinase and adenosine deaminase.

Peripheral antinociceptive actions of desipramine and fluoxetine in an inflammatory and neuropathic pain test in the rat.

Amitriptyline, a non-selective noradrenaline (NA) and 5-hydroxytryptamine (5-HT) reuptake inhibitor, has recently been demonstrated to produce a peripheral antinociceptive action in an inflammatory (formalin test) and a neuropathic pain model (spinal nerve ligation). In the present study, we determined whether desipramine, a selective NA reuptake inhibitor, and fluoxetine, a selective 5-HT reuptake inhibitor, could produce peripheral antinociceptive actions in these same tests. Effects on paw volume also were determined. In the 2.5% formalin test, desipramine and fluoxetine 10-300 nmol produced a dose-related reduction in phase 2 (16-60 min) flinching and biting/licking behaviours when coadministered with the formalin. Phase 1 flinch behaviours (0-12 min) were significantly reduced at the highest dose. These actions are peripherally mediated, as they were not seen when desipramine or fluoxetine (100, 300 nmol) were injected into the contralateral hindpaw. The peripheral action of desipramine and fluoxetine was not altered by coadministration of caffeine 1500 nmol. In the spinal nerve ligation model, desipramine 100 nmol, but not fluoxetine 100 nmol, produced a peripheral anti-hyperalgesic action in the hindpaw corresponding to the ligated side when thresholds were determined using a thermal paw stimulator. In paw volume experiments, desipramine, at doses which are maximally effective in behavioural tests, produced only a slight increase in paw volume, but fluoxetine (10-300 nmol) produced a robust and sustained dose-related increase in paw volume. Amitriptyline also produced minimal effects on paw volume. When coinjected with formalin, no agent significantly altered the degree of paw swelling produced by formalin. The increase in paw volume produced by fluoxetine was inhibited by ketanserin (5-HT2 receptor antagonist), mepyramine (histamine H1 receptor antagonist) and phentolamine (alpha-adrenergic receptor antagonist), but not by the other selective 5-HT receptor antagonists tested or caffeine. The pronounced peripheral pain alleviating actions in the absence of marked changes in paw volume produced by desipramine and amitriptyline, but not fluoxetine, in the formalin test and the spinal nerve ligation model suggest that these agents could be developed as cream or gel formulations to recruit a peripheral antinociceptive action in inflammatory and neuropathic pain states. Such a formulation might permit the attainment of higher and more efficacious concentrations in the region of the sensory nerve terminal, with limited systemic side effects.

Peripheral antinociceptive action of amitriptyline in the rat formalin test: involvement of adenosine.

The present study determined (1) whether amitriptyline could produce a local antinociceptive action in the formalin test, (2) whether endogenous adenosine was involved in this action, and (3) which other systems might contribute to such an action. Coadministration of amitriptyline 10-100 nmol with 2.5% formalin produced a dose-related reduction in phase 1 (0-12 min) and phase 2 (16-60 min) flinching behaviours, as well as in phase 2 biting/licking time (no phase 1 expression). This action was not seen or only partially expressed at low concentrations of formalin (0.5%, 0.75%). Coadministration of caffeine with amitriptyline partially reversed the antinociceptive action of amitriptyline against both behaviours at 2.5% formalin. At 1.5% formalin, caffeine still produced only a partial reversal of effect; this appeared to be due to a block of adenosine A1 receptors, as it was also seen with the selective adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine. Using antagonists for a number of other systems, no evidence for an involvement of alpha-adrenergic, histamine, excitatory amino acid or opioid receptors in the action of amitriptyline was observed or inferred. A local anaesthetic action for amitriptyline remains a possibility for the residual action. These results indicate that amitriptyline can produce a local peripheral antinociceptive action which is mediated, in part, by an interaction with endogenous adenosine, most likely an inhibition of the cellular uptake of adenosine with a consequent activation of adenosine A1 receptors on sensory nerve terminals. Local application of amitriptyline by cream or gel might prove to be a useful method of drug delivery in inflammatory pain states.

Acute paw oedema induced by local injection of adenosine A(1), A(2) and A(3) receptor agonists.

The present study used plethysmometry to examine oedema following local injection of selective adenosine A(1), A(2) and A(3) receptor agonists and inhibitors of adenosine metabolism into the hindpaw of the rat. N(6)-Cyclopentyladenosine and L-N(6)-phenylisopropyladenosine (A(1)), 2-[p(2-carboxyethyl) phenethylamino]-5'-N-ethylcarboxamidoadenosine hydrochloride (CGS21680) (A(2A)) and N(6)-benzyl-5'-N-ethylcarboxamido adenosine (N(6)-B-NECA) (A(3)) all produced an increase in paw volume (N(6)N(6)-cyclopentyladenosine, L-N(6)CGS21680). At the highest dose, each agent also produced a systemically mediated suppression of oedema. Oedema by N(6)-cyclopentyladenosine was blocked by caffeine, 8-cyclopentyl-1,3-dimethylxanthine and enprofylline. Oedema by CGS21680 was blocked by caffeine and 8-cyclopentyl-1, 3-dimethylxanthine. Oedema by N(6)-B-NECA was blocked by enprofylline, but not by caffeine or 8-cyclopentyl-1, 3-dimethylxanthine, or by systemic administration of MRS 1191. Oedema by both N(6)-cyclopentyladenosine and N(6)-B-NECA was blocked by mepyramine, ketanserin and phentolamine, but that by CGS21680 was not. The adenosine kinase inhibitor 5'-amino-5'-deoxyadenosine and the adenosine deaminase inhibitor 2'-deoxycoformycin produced only a limited increase in paw volume, and this was blocked by caffeine. This study demonstrates an acute paw oedema response following local administration of adenosine A(1), A(2) and A(3) receptor agonists, which likely results from different mechanisms of action in each case.

Adenosine receptor activation and nociception.

Adenosine and ATP exert multiple influences on pain transmission at peripheral and spinal sites. At peripheral nerve terminals in rodents, adenosine A1 receptor activation produces antinociception by decreasing, while adenosine A1 receptor activation produces pronociceptive or pain enhancing properties by increasing, cyclic AMP levels in the sensory nerve terminal. Adenosine A3 receptor activation produces pain behaviours due to the release of histamine and 5-hydroxytryptamine from mast cells and subsequent actions on the sensory nerve terminal. In humans, the peripheral administration of adenosine produces pain responses resembling that generated under ischemic conditions and the local release of adenosine may contribute to ischemic pain. In the spinal cord, adenosine A receptor activation produces antinociceptive properties in acute nociceptive, inflammatory and neuropathic pain tests. This is seen at doses lower than those which produce motor effects. Antinociception results from the inhibition of intrinsic neurons by an increase in K+ conductance and presynaptic inhibition of sensory nerve terminals to inhibit the release of substance P and perhaps glutamate. There are observations suggesting some involvement of spinal adenosine A2 receptors in pain processing, but no data on any adenosine A3 receptor involvement. Endogenous adenosine systems contribute to antinociceptive properties of caffeine, opioids, noradrenaline, 5-hydroxytryptamine, tricyclic antidepressants and transcutaneous electrical nerve stimulation. Purinergic systems exhibit a significant potential for development as therapeutic agents. An understanding of the contribution of adenosine to pain processing is important for understanding how caffeine produces adjuvant analgesic properties in some situations, but might interfere with the optimal benefit to be derived from others.

Antinociception by adenosine analogs and inhibitors of adenosine metabolism in an inflammatory thermal hyperalgesia model in the rat.

The present study examined the spinal antinociceptive effects of adenosine analogs and inhibitors of adenosine kinase and adenosine deaminase in the carrageenan-induced thermal hyperalgesia model in the rat. The possible enhancement of the antinociceptive effects of adenosine kinase inhibitors by an adenosine deaminase inhibitor also was investigated. Unilateral hindpaw inflammation was induced by an intraplantar injection of lambda carrageenan (2 mg/100 microl), which consistently produced significant paw swelling and thermal hyperalgesia. Drugs were administered intrathecally, either by acute percutaneous lumbar puncture (individual agents and combinations) or via an intrathecal catheter surgically implanted 7-10 days prior to drug testing (antagonist experiments). N6-cyclohexyladenosine (CHA; adenosine A1 receptor agonist; 0.01-1 nmol), 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680; adenosine A2A receptor agonist; 0.1-10 nmol), 5'-amino-5'-deoxyadenosine (NH2dAdo; adenosine kinase inhibitor: 10-300 nmol), and 5-iodotubercidin (ITU; adenosine kinase inhibitor; 0.1-100 nmol) produced, to varying extents, dose-dependent antinociception. No analgesia was seen following injection of 2'-deoxycoformycin (dCF; an adenosine deaminase inhibitor; 100-300 nmol). Reversal of drug effects by caffeine (non-selective adenosine A1/A2 receptor antagonist; 515 nmol) confirmed the involvement of the adenosine receptor, while antagonism by 8-cyclopentyl-1,3-dimethylxanthine (CPT; adenosine A1 receptor antagonist; 242 nmol), but not 3,7-dimethyl-1-propargylxanthine (DMPX; adenosine A2A receptor antagonist; 242 nmol), evidenced an adenosine A1 receptor mediated spinal antinociception by NH2dAdo. dCF (100 nmol), which was inactive by itself, enhanced the effects of 10 nmol and 30 nmol NH2dAdo. Enhancement of the antinociceptive effect of ITU by dCF was less pronounced. None of the antinociceptive drug regimens had any effect on paw swelling. These results demonstrate that both directly and indirectly acting adenosine agents, when administered spinally, produce antinociception through activation of spinal adenosine A1 receptors in an inflammatory model of thermal hyperalgesia. The spinal antinociceptive effects of selected adenosine kinase inhibitors can be significantly augmented when administered simultaneously with an adenosine deaminase inhibitor.

Formalin-induced nociceptive behavior and edema: involvement of multiple peripheral 5-hydroxytryptamine receptor subtypes.

The role of 5-hydroxytryptamine and its receptor subtypes in the development of acute inflammation was investigated using the rat paw formalin test as a model for pain (measured by flinching behavior) and edema formation (measured by plethysmometry). The role of endogenously released 5-hydroxytryptamine was assessed using 5-hydroxytryptamine receptor subtype-selective antagonists co-injected with 2.5% formalin, while the receptor subtypes involved in the inflammatory process were further defined by co-injection of 5-hydroxytryptamine or 5-hydroxytryptamine receptor subtype-selective agonists with 0.5% formalin in anticipation of an augmented response. When co-administered with 2.5% formalin, propranolol, tropisetron or GR113808A, but not ketanserin, effectively blocked nociceptive behavior. In the presence of 0.5% formalin, 5-carboxamidotryptamine, 1-(m-chlorophenyl) biguanide or 5-methoxytryptamine, but not (+/-)-1-4-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane, augmented the flinching response. These data suggest involvement of 5-hydroxytryptamine1, 5-hydroxytryptamine3 and 5-hydroxytryptamine4 receptors in peripheral nociception. There may be some dissociation of nociception and edema formation, since no single 5-hydroxytryptamine receptor antagonist inhibited edema formation with 2.5% formalin; however, with 0.5% formalin, edema formation was enhanced by co-administration of 5-hydroxytryptamine, 5-carboxamidotryptamine, (+/-)-1-4-(4-iodo-2,5-dimethoxyphenyl)-2-aminopropane or 5-methoxytryptamine, but not 1-(m-chlorophenyl) biguanide. These data suggest involvement of 5-hydroxytryptamine1, 5-hydroxytryptamine2 and possibly 5-hydroxytryptamine4 receptors in edema formation. These results confirm the involvement of 5-hydroxytryptamine1 and 5-hydroxytryptamine3 receptor subtypes in peripheral nociception associated with acute inflammation and further suggest an involvement of the more recently characterized 5-hydroxytryptamine4 receptor in this process. There appears to be a dissociation in 5-hydroxytryptamine receptors involved in peripheral nociception and edema formation.