ABT-702 (4-Amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin- 3-yl)pyrido[2,3-d]pyrimidine), a Novel Orally Effective Adenosine Kinase Inhibitor with Analgesic and Anti-Inflammatory Properties. II. In Vivo Characterization in the Rat

Materials and Methods

Subjects.

Male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 200 to 300 g were used for all experiments. These animals were group housed (four per cage) in American Association for the Accreditation of Laboratory Animal Care-approved facilities at Abbott Laboratories in a temperature-regulated environment with lights on between 7:00 AM and 2:00 PM. 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.

Carrageenan-Induced Thermal Hyperalgesia, Edema, and Myeloperoxidase (MPO) Activity.

Paw edema and hyperalgesia were induced by injecting 100 μl of a 1% solution of λ-carrageenan (Sigma Chemical Co., St. Louis, MO) in physiological saline into the plantar surface of the right hindpaw of the rat. The hyperalgesia to thermal stimulation was determined 2 h later using a commercially available paw thermal stimulator (UARDG; Department of Anesthesiology, University of California, San Diego, La Jolla, CA; Dirig and Yaksh, 1995), modeled after that described by Hargreaves et al. (1988). Rats were placed individually in Plexiglas cubicles mounted on a glass surface maintained at 30°C, and allowed a 30-min habituation period. A thermal stimulus, in the form of radiant heat emitted from a focused projection bulb, was then applied to the plantar surface of each hindpaw. The stimulus current was maintained at 4.5 amp and the maximum time of exposure was set at 20 s to limit possible tissue damage. The elapsed time until a brisk withdrawal of the hindpaw from the thermal stimulus was recorded automatically using photodiode motion sensors. In each test session, six rats were randomly selected from ABT-702 or vehicle-treated groups (n = 6 per dose or vehicle group) and placed in the thermal paw stimulator. Each rat was tested in three sequential trials at approximately 5-min intervals. Paw withdrawal latencies were calculated as the mean of the two shortest latencies. In some experiments, a percent vehicle response effect was calculated according to the formula: [latency inflamed paw_{drug group} − mean latency inflamed paw_{vehicle group}]/[mean latency control paw_{vehicle group} − mean latency inflamed paw_{vehicle group}] × 100%.

Paw edema was measured by water displacement plethysmography at selected times after carrageenan injection by placing the paw into the chamber of the plethysmometer (Buxco, Troy, NY) up to the ankle hairline (approximately 1.5 cm). The displacement volume was measured by a transducer and recorded by a computer. ABT-702-treated (n = 6 per dose) and control (ABT-702 vehicle,n = 6) rats were tested in random order. Paw volume displacements were determined for both the carrageenan-treated and control (untreated) paws.

In selected experiments, MPO activity of the carrageenan-injected paw was determined as previously described (Bradley et al., 1982). Rats were euthanized and the plantar surface of the carrageenan-injected hindpaw was dissected. The harvested tissue was then quickly frozen in liquid nitrogen and placed in a −80°C freezer until assayed for MPO activity. At the time of assay, the hindpaw tissue was placed in centrifuge tubes containing 10 ml of 50 mM potassium phosphate buffer, pH 6.0, with 0.5% hexadecyl-trimethylammonium bromide (Sigma Chemical Co.) and homogenized (Brinkman Polytron, setting 6 for 30 s). The homogenate was quickly frozen and thawed for three cycles and then centrifuged for 15 min at 40,000g, 4°C. An aliquot (1 ml) of the supernatant was diluted (1:10, 1:20, and 1:50) and 100 μl of each dilution was added to a 96-well plate. Fifty microliters of a 50 mM potassium phosphate buffer, pH 6.0, solution and 25 μl of the reaction mixture [DMB (O-diansidine dihydrochloride, 95.2 mg/30 ml of H₂O) and H₂O₂ (84 ml 30% H₂O₂/50 ml of H₂O); Sigma Chemical Co.] was added to each well. The assay plate was then immediately inserted into a plate reader (Molecular Devices, Sunnyvale, CA) and the change in absorbance at 460 nm was recorded.

In carrageenan-induced thermal hyperalgesia, paw edema, and MPO activity experiments, ABT-702 was administered orally 1 h before carrageenan, unless otherwise noted. In antagonist experiments, ABT-702 (or vehicle) was administered orally 1 h before carageenan injection, and the antagonists (i.e., theophylline, cyclopentyltheophylline (CPT), 3,7-dimethyl-1-propargylxanthine (DMPX), or vehicle were administered (i.p.) 30 min after ABT-702 treatment.

Formalin Test.

After a 30-min acclimation period to individual observation cages, 50 μl of a 5% formalin solution was injected s.c. into the dorsal aspect of the right hindpaw and the rats were then returned to the clear observation cages, which were suspended above mirrors. Rats were observed for either a continuous period of 60 min or for periods of time corresponding to phase 1 and phase 2 of the formalin test (Abbott et al., 1995). Phase 1 of the formalin test was defined as the period of time immediately after injection of formalin until 10 min after the formalin injection. Effects on phase 2 of the formalin test were determined by monitoring for the 20-min period of time from 30 to 50 min after formalin injection. Nociceptive behaviors were recorded from animals during the session by observing each animal for one 60-s observation period during each 5-min interval. Nociceptive behaviors recorded included flinching, licking, or biting the injected paw. In dose-response studies, ABT-702 (or vehicle) was administered orally 1 h before formalin injection, unless otherwise noted. In antagonist experiments, theophylline (10 mg/kg i.p.) was injected 30 min after ABT-702 treatment.

Spinal Nerve (L5/L6) Ligation Model of Neuropathic Pain.

Rats received unilateral (left side) tight ligation of the L5 and L6 spinal nerves as previously described (Kim and Chung, 1992). For all surgical procedures, rats were maintained under halothane/oxygen anesthesia (4% to induce, 2% to maintain). The L5 and L6 spinal nerves were tightly ligated with 3-0 suture close to the location where they exit the spinal column and care was taken to avoid ligating the L4 spinal nerve. Tactile allodynia was measured using calibrated (force; g) von Frey filaments (Stoelting, Wood Dale, IL) as previously described (Chaplan et al., 1994). Briefly, rats were placed into individual Plexiglas containers (20 × 12.5 × 20 cm) with wire mesh floors, which allowed access to the ventral side of the paw. The von Frey filament was presented perpendicular to the plantar surface of the left hindpaw (ipsilateral to ligation), and held in this position for approximately 6 to 8 s with enough force to cause a slight buckle in the filament. Positive responses included sharp withdrawal in response to stimulus or flinching behavior immediately after removal of the stimulus. The 50% withdrawal threshold was determined using the up-down method of Dixon (1980). A percent maximal protective effect value (% MPE) was calculated for each dose at each pretreatment time according to the following formula: ([postdrug threshold] − [baseline threshold])/([maximum threshold] − [baseline threshold]) × 100%, where maximum threshold was equal to 15 g.

Compound studies began at least 1 week after the nerve ligation surgery. For dose-response experiments, surgically prepared animals were randomly assigned to treatment groups and tested over a 2- to 3-day period. On test days, animals were placed into the individual chambers and allowed to acclimate for 15 to 20 min, after which baseline scores were determined. Only rats with threshold scores ≤4.5 g were considered allodynic and used in further testing. Animals were treated with ABT-702 or vehicle, and threshold scores were determined at intervals thereafter. In repeated dosing experiments, ABT-702 (30 μmol/kg i.p.) or vehicle was administered twice daily for 4 days with treatments being separated by approximately 8 h. On day 5, for the morning treatment, both groups received a challenge dose of ABT-702 (30 μmol/kg i.p.).

Streptozotocin-Induced Diabetes and Tactile Allodynia.

Diabetes was induced using a 75-mg/kg i.p., injection of streptozotocin, and blood glucose levels were assessed 3 to 4 weeks later. Rats with blood glucose levels ≥250 mg/dl (≥14 mM) were considered diabetic and used for further studies. Drug studies began approximately 8 weeks after streptozotocin injection (Lynch et al., 1999). The effects of ABT-702 on tactile allodynia withdrawal thresholds were assessed in these animals in a manner similar to that described above for spinal nerve ligation animals. Only rats with predosing withdrawal thresholds of ≤8.0 g were used in these studies.

Locomotor Activity and Rotorod Performance.

Locomotor activity was measured in an open field using photobeam activity monitors (AccuScan Instruments, Columbus, OH). Rats were treated with ABT-702 or vehicle, and placed in activity chambers (42 × 42 × 30 cm) 60 min later. Photobeam breaks were recorded for 30 min and data were collapsed into 10-min intervals for statistical analysis. Rotorod performance was measured using an accelerating rotorod apparatus (Omnitech Electronics, Inc., Columbus, OH). Rats were allowed a 30-min acclimation period in the testing room and then placed on a 9-cm-diameter rod, which increased in speed from 0 to 20 rpm over a 60-s period. The time required for the rat to fall from the rod was recorded, with a maximum score of 60 s. Each rat was given three training sessions before drug treatment. After the training sessions, rats were randomly assigned to treatment groups and injected with either ABT-702 or vehicle. Latencies to fall from the rotorod were determined 30, 60, and 120 min after ABT-702 or vehicle treatment, and these values were used for statistical comparisons.

Heart Rate and Blood Pressure.

This study was carried out using the LabPro telemetry system (Dataquest; Data Sciences International, Minneapolis, MN). Approximately 1.5 weeks before study and under aseptic conditions, male Sprague-Dawley rats were implanted with indwelling telemetry transmitters connected to a small gel-filled catheter that was secured nonocclusively in the abdominal aorta. Signals emitted by the transmitter were detected by individual receivers placed under the animal cages, and translated by the system software into blood pressure waveforms and heart rate values. Arterial pressure and heart rate data were sampled at 5-min intervals and serial 10-min averages were determined. Fifteen rats were instrumented, which allowed the use of a randomized crossover design carried out over two separate days to complete the study. A 3-day washout period was allowed between experiments. ABT-702 or vehicle (n = 6 per dose or vehicle group) was delivered orally via gavage at a dose volume of 5 ml/kg. Mean arterial pressure and heart rate were measured beginning 1 h before, and continuing for 6 h following treatment.

Compounds.

ABT-702 is 4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-d]pyrimidine, and was synthesized as described by C.-H. Lee et al. (C.-H. Lee, M. Jiang, M. Cowart, G. Gfesser, R. Perner, K. H. Kim, Y. G. Gu, M. Williams, M. F. Jarvis, E. A. Kowaluk, A. O. Stewart, and S. S. Bhagwat, submitted). Morphine sulfate was obtained from Mallinckrodt (St. Louis, MO). ADO receptor antagonists and naloxone were obtained from Research Biochemicals International (Natick, MA). ABT-702 was dissolved in a vehicle consisting of 10% dimethyl sulfoxide/34% hydroxypropyl-β-cyclodextrin in sterile water for i.p. administration and 30% polyethylene glycol 400 for p.o. administration. Polyethylene glycol 400 (30%), dimethyl sulfoxide, and hydroxypropyl-β-cyclodextrin were obtained from Sigma (St. Louis, MO). Doses are expressed in micromoles per kilogram of free base, and compounds were administered in a final volume of 1 to 3 ml/kg i.p. or 3 to 5 ml/kg p.o.

Statistics.

Data analysis was carried out using analysis of variance (GB-Stat; Dynamic Microsystems, Inc., Silver Spring, MD) as previously described (Kowaluk et al., 1999). Where appropriate, Fisher's protected least-significant difference was used for post hoc analysis. The level of significance was set at P < .05. ED₅₀ values were estimated using least-squares linear regression. Data are presented as mean ± S.E.M.

Results

Carrageenan-Induced Thermal Hyperalgesia.

In response to a noxious thermal stimulus, the withdrawal latency of the carrageenan-injected, inflamed hindpaw was significantly reduced compared with the contralateral, uninflamed hindpaw (Fig.2A), consistent with the induction of thermal hyperalgesia by carrageenan, as previously described (Hargreaves et al., 1988). ABT-702 dose dependently and fully relieved carrageenan-induced inflammatory hyperalgesia when administered orally (ED₅₀ = 5 μmol/kg, Fig. 2A) or intraperitoneally (ED₅₀ = 0.6 μmol/kg) before carrageenan. Under the same conditions, ABT-702 had no effect on the withdrawal latency of the contralateral, uninflamed paw (Fig. 2A), indicative of a specific antihyperalgesic effect of ABT-702. The antihyperalgesic effects of ABT-702 (30 μmol/kg p.o.) persisted when it was administered up to 6 h before carrageenan, which represents an interval of 8 h before thermal hyperalgesia testing (data not shown). ABT-702 also relieved thermal hyperalgesia when it was administered 1 h after carrageenan injection (ED₅₀ = 10 μmol/kg p.o.). ABT-702 was more potent than morphine (ED₅₀ = 30 μmol/kg p.o.) or the nonsteroidal anti-inflammatory drug ibuprofen (70 μmol/kg p.o.) at relieving thermal hyperalgesia when orally administered before carrageenan (Fig. 2B).

The antihyperalgesic effects of ABT-702 (10 μmol/kg p.o.) were reversed by the nonselective ADO receptor antagonist theophylline (10 mg/kg i.p), the ADO A₁ receptor-selective antagonist CPT (10 mg/kg i.p.), and the ADO A_2Areceptor-selective antagonist DMPX (1 mg/kg i.p.) (Fig.3).

ABT-702 (30 nmol) also fully relieved carrageenan-induced thermal hyperalgesia after intraplantar injection locally at the site of inflammation (Table 1). The effects of intraplantar ABT-702 (30 nmol) were selective for the inflamed paw because ABT-702 injection into the contralateral (control) paw had no effect on the paw withdrawal latency of the inflamed paw (Table 1). This dose of ABT-702 (30 nmol) also had no effect on the paw withdrawal latency of the contralateral (control) paw when injected into either the inflamed or control paw (Table 1).

Carrageenan-Induced Paw Edema.

The acute anti-inflammatory effects of ABT-702 were assessed in the carrageenan-induced paw edema model in the rat. ABT-702 dose dependently reduced paw edema after oral administration (ED₅₀ = 70 μmol/kg, Fig. 4A), exhibiting similar potency and efficacy when it was administered 1 h before (Fig. 4A) or 1 h after (Fig. 4B) carrageenan. ABT-702 also dose dependently reduced neutrophil accumulation at the site of inflammation, as reflected by the reduction of MPO activity in the paw (ED₅₀ = 60 μmol/kg p.o., Fig. 4A). The effects of ABT-702 persisted even when ABT-702 (100 μmol/kg p.o.) was administered up to 9 h before carrageenan (data not shown). ABT-702 showed similar potency and efficacy to ibuprofen and prednisolone when administered orally 1 h after carrageenan (Fig. 4B).

ABT-702 (100 μmol/kg p.o.) mediated edema reduction was completely reversed by the nonselective ADO receptor antagonist theophylline (10 mg/kg i.p.), and partially reversed by either the ADO A₁ selective antagonist CPT (10 mg/kg i.p.), or the ADO A_2A receptor-selective antagonist DMPX (10 mg/kg i.p.) (Fig. 5).

Rat Formalin Test.

In the rat formalin test, 5% formalin was injected into the right hindpaw to induce a characteristic biphasic flinching response. ABT-702 dose dependently reduced both the acute (phase 1) and persistent (phase 2) phases of formalin-evoked flinching (Fig. 6A). ABT-702 was effective after both oral (ED₅₀ = 60 μmol/kg in phase 2, Fig.6) and intraperitoneal (ED₅₀ = 15 μmol/kg in phase 2) administration. ABT-702 was somewhat less potent than morphine (ED₅₀ = 20 μmol/kg p.o.) in blocking phase 2 nociceptive behavior, but markedly more potent and more effective than the NSAID ibuprofen (ED₅₀ >500 μmol/kg p.o.) (Fig. 6B). The antinociceptive effects of ABT-702 (100 μmol/kg p.o.) in phase 2 of the formalin test (27 ± 2 flinches,P < .05 versus vehicle response of 67 ± 7 flinches, P < .05, n = 6) were significantly attenuated by the nonselective ADO receptor antagonist theophylline (10 mg/kg i.p., 44 ± 4 flinches, n = 6, P < .05 versus ABT-702 alone). Theophylline alone (10 mg/kg i.p., 65 ± 3 flinches, n = 6) had no significant effect on nociceptive behaviors compared with vehicle (67 ± 7 flinches, n = 6).

The antinociceptive effects of ABT-702 in the formalin test were studied in combination with morphine. ABT-702 (5 μmol/kg i.p.), administered 30 min before formalin, reduced flinching behaviors by 29 ± 3% (n = 6), whereas morphine (2.6 μmol/kg i.p.), administered 30 min before formalin, reduced flinching by 26 ± 6% (n = 6). The combined administration of ABT-702 with morphine at these minimally effective doses of each compound reduced flinching by 71 ± 9% (P < .05, compared with vehicle-treated rats), suggesting that the effects of ABT-702 were additive with morphine in the formalin test.

Rat Models of Neuropathic Pain.

Tight ligation of the L5/L6 spinal nerves produced tactile allodynia in the ipsilateral hindpaw (mean baseline threshold = 3.2 ± 0.1 g). After oral or intraperitoneal administration, ABT-702 dose dependently increased paw withdrawal latency, restoring paw withdrawal thresholds to levels comparable to those of normal rats (ED₅₀ = 50 μmol/kg p.o., ED₅₀ = 5 μmol/kg i.p., Fig.7A). The effects of ABT-702 (100 μmol/kg p.o.) were near maximal by 1 h after oral administration, and persisted for at least 11 h after dosing (Fig.8). The antiallodynic effects of ABT-702 were similar to those of systemically administered morphine (Fig. 7B). Ibuprofen was inactive in this model at doses up to 500 μmol/kg i.p. (Fig. 7B).

The antiallodynic effects of ABT-702 were completely reversed by the nonselective ADO receptor antagonist theophylline (10 mg/kg i.p.), and by the ADO A₁ receptor-selective antagonist CPT (10 mg/kg i.p.), but not by the ADO A_2Areceptor-selective antagonist DMPX (1 mg/kg i.p.) (Fig.9). The antiallodynic effects of ABT-702 (100 μmol/kg p.o.; 77 ± 1% MPE, P < .05 versus vehicle response of 5 ± 10% MPE, n = 6) were not blocked by the opioid antagonist naloxone (10 mg/kg i.p., 57 ± 15% MPE, n = 6, P > .05 versus ABT-702 alone). At the same dose, naloxone had no effect on tactile allodynia by itself (10 ± 9% MPE versus 5 ± 10% MPE for vehicle, n = 6), but blocked the antiallodynic effect of morphine (96 μmol/kg p.o., 96 ± 4% MPE for morphine versus 24 ± 7% MPE for morphine with naloxone, P< .05, n = 6).

The antinociceptive effects of ABT-702 (30 μmol/kg i.p.) were sustained on the 5th day after repeated twice daily dosing (Fig.10). In contrast, the effects of morphine (21 μmol/kg i.p.) were markedly reduced under similar dosing conditions (Fig. 10), yet both compounds have similar plasma half-lives in the rat.

Tactile Allodynia in Diabetic Rats.

The streptozotocin-induced diabetic rat exhibits a number of anomalies in pain perception, including the development of tactile allodynia (Lynch et al., 1999). ABT-702 relieved the tactile allodynia in these diabetic rats with similar potency and efficacy as was observed in the L5/L6 spinal nerve ligation model (ED₅₀ = 5 μmol/kg i.p., Fig.7A). The effects of ABT-702 were maximal within 30 min of acute administration. The antiallodynic effects of ABT-702 (30 μmol/kg i.p.) in the diabetic rats [88 ± 8% MPE, P < .05 versus vehicle response (19 ± 12% MPE), n = 6] were blocked by the nonselective ADO receptor antagonist theophylline (20 mg/kg i.p., 35 ± 15% MPE, n = 6, P < .05 versus ABT-702 alone). Theophylline alone (20 mg/kg i.p., 17 ± 15% MPE, n = 6) had no significant effect on nociceptive behaviors compared with vehicle (19 ± 12% MPE, n = 6).

Effects on Motor Activity.

ABT-702 had no significant effect on motor coordination at 30 to 300 μmol/kg p.o, as measured by the ability of rats to run on an accelerating rotating rod (rotorod assay). ABT-702 also had no effect on spontaneous exploratory activity of rats in a novel open field at 10 μmol/kg p.o., but significantly reduced spontaneous exploratory activity at 30 μmol/kg p.o. (50 ± 6% of vehicle response, P < .05 versus vehicle,n = 6), 100 μmol/kg p.o. (25 ± 3% of vehicle response, P < .05 versus vehicle, n = 6) and 300 μmol/kg p.o. (25 ± 3% of vehicle response,P < .05 versus vehicle, n = 6). Rats were fully awake, responsive to stimuli, and retained the righting reflex, consistent with their ability to perform the rotorod test at all doses tested. While ABT-702 had no significant effects on rotorod performance, its effects on locomotor activity occurred with an ED₅₀ value that was 6-fold higher than that required to reduce thermal hyperalgesia. As such, the potency of ABT-702 to reduce locomotor activity (ED₅₀ = 30 μmol/kg p.o.) was similar to its potency to reduce nociception in the formalin (ED₅₀ = 60 μmol/kg p.o) and L5/L6 spinal nerve ligation (ED₅₀ = 50 μmol/kg p.o) assays.

Cardiovascular Effects.

The cardiovascular effects of ABT-702 were examined using conscious, freely behaving rats instrumented with telemetry transmitters. After oral administration, ABT-702 produced no significant change in mean arterial pressure when administered to conscious rats at 30 or 100 μmol/kg (data not shown). ABT-702 produced a modest reduction in mean arterial pressure at 300 μmol/kg p.o., compared with vehicle; however, mean arterial pressure did not fall below baseline values in ABT-702-treated rats (Fig.11). Heart rate was not significantly altered at any dose tested.

Discussion

The present data demonstrate that the novel AK inhibitor ABT-702 is orally effective to reduce nociception in a variety of rat models of inflammatory and neuropathic pain. ABT-702 exhibited an acute onset (≤1 h) and a prolonged duration of action (8 to 11 h) in these models and was especially potent to fully block inflammatory thermal hyperalgesia (Hargreaves et al., 1988). ABT-702 had a selective antihyperalgesic, rather than analgesic, effect in this model. Although ABT-702, like other AK inhibitors (Kowaluk et al., 1999), has demonstrated analgesic effects in acute thermal threshold tests (e.g., the mouse hot-plate test, Jarvis et al., 2000), it does so at greater than antihyperalgesic doses. ABT-702 also showed equivalent antihyperalgesic activity when administered either before or after carrageenan-evoked tissue injury. The latter effect may be more relevant to the clinical setting where analgesics are administered after injury.

Consistent with the endogenous anti-inflammatory actions of ADO (Firestein, 1996), ABT-702 also dose dependently reduced carrageenan-evoked paw edema and neutrophil accumulation (reflected by MPO activity) at the site of inflammation. ABT-702 equi-effectively reduced paw edema whether it was administered before or after carrageenan, consistent with its antihyperalgesic effects. Interestingly, antihyperalgesia by ABT-702 was evident at doses providing minimal overt reduction of paw edema, suggesting that subtle anti-inflammatory actions that were not detected with the paw edema assay, as well as central mechanisms, may contribute to the antihyperalgesia produced by ABT-702. ABT-702 also significantly reduced paw edema, and decreased radiographic and histological evidence of joint destruction in the rat adjuvant arthritis model of chronic inflammation.2

ABT-702 was also equally effective at relieving tactile allodynia in two rat models of neuropathic pain, the L5/L6 nerve ligation model (Kim and Chung, 1992) and the streptozotocin-induced diabetic neuropathic pain model (Lynch et al., 1999). Full efficacy was observed within 30 to 60 min of intraperitoneal or oral administration of a single, acute dose of ABT-702 in these chronic pain states. ABT-702 also produced antinociceptive effects in the formalin model of persistent pain, dose dependently reducing both the acute (phase I) and persistent (phase 2) phases of the formalin test.

The broad spectrum of efficacy across pain types (nociceptive, inflammatory, neuropathic) demonstrated for ABT-702 is consistent with the spectrum of effectiveness that has been described for prototypical AK inhibitors, P1 receptor agonists, as well as ADO itself (Sawynok, 1999; Kowaluk and Jarvis, 2000). Prototypical AK inhibitors (5′-amino-deoxyadenosine, 5-iodotubercidin and 5′-deoxy-5-iodotubercidin) have demonstrated efficacy in acute thermal threshold tests (Keil and DeLander, 1992; Kowaluk et al., 1999), in carrageenan-induced thermal hyperalgesia (Poon and Sawynok, 1998), in the formalin test (Poon and Sawynok, 1998), and in diabetic neuropathic pain (Lynch et al., 1999). However, the majority of previous studies with commercially available prototypical AK inhibitors have used the spinal route of administration due, in particular for 5′amino,5′-deopxyadenosine, to relatively weak cellular penetration and poor central nervous system access of these compounds. The present studies with ABT-702 have established the antinociceptive potential of AK inhibition after the more clinically useful systemic, and in particular oral, route of administration. The demonstration that ABT-702 exhibits anti-inflammatory effects also adds to the body of evidence supporting the anti-inflammatory efficacy of systemically administered AK inhibitors, including GP515, in animal models (Firestein et al., 1994; Firestein, 1996).

The antinociceptive and acute anti-inflammatory effects of ABT-702 were blocked by ADO receptor-selective antagonists, and/or by the nonselective receptor antagonist theophylline. These data are consistent with potentiation of endogenous ADO and subsequent ADO receptor activation as a mechanism of action for ABT-702. Studies with the highly selective ADO A₁ receptor antagonist CPT (Sawynok and Reid, 1996) and ADO A_2Areceptor-selective antagonist doses of DMPX (Seale et al., 1988), suggest that both A₁ and A_2A ADO receptor interactions contribute to the antihyperalgesic and anti-inflammatory effects of ABT-702. In contrast, the effects of ABT-702 in the L5/L6 nerve ligation model of neuropathic pain appear to be mediated primarily via activation of the ADO A₁, but not the A_2A, receptor by endogenous ADO. Similarly, the effects of ABT-702 to relieve acute thermal pain in the mouse hot-plate test involved A₁, but not A_2A, receptor activation (Jarvis et al., 2000). These data suggest differing control mechanisms for the relief of inflammatory pain compared with neuropathic and acute physiological pain by ABT-702.

The observed role of ADO A₁ receptor activation in the antinociceptive effects of ABT-702 across pain models is consistent with evidence implicating the activation of spinal ADO A₁ receptors in antinociception (Sawynok, 1999). Studies with ADO receptor-selective agonists and antagonists have demonstrated a pharmacology for spinal antincociception that is primarily ADO A₁ receptor mediated (Keil and DeLander, 1992; Lee and Yaksh, 1996; Poon and Sawynok, 1998; Sawynok, 1999). ADO A₁ receptors are localized to the dorsal horn of the spinal cord (Choca et al., 1987). In addition, electrophysiological studies in intact rats indicate that ADO A₁ receptor agonists modulate acutely evoked and inflammation-evoked responses of spinal cord dorsal horn neurons (Reeve and Dickenson, 1995). In the central nervous system, ADO activates A₁ receptors to inhibit cAMP production, increase K⁺ currents, and decrease Ca²⁺ currents (Kowaluk and Jarvis, 2000). Presynaptically, ADO inhibits neurotransmitter and neuropeptide release, including glutamate, substance P, and calcitonin-gene related peptide (Santicioli et al., 1992). Postsynaptically, ADO suppresses sensory transmission as a result of the activation of K⁺ conductances and membrane hyperpolarization (Salter et al., 1993; Li and Perl, 1994). All of these actions of ADO have been implicated as key mediators of the central sensitization characterizing inflammatory and neuropathic pain states.

The involvement of both ADO A₁ and A_2A receptors in the anti-inflammatory effects of ABT-702 is consistent with the role of these ADO receptor subtypes in the inhibition of neutrophil function and inflammatory mediator-evoked vascular leakage by endogenous ADO (Firestein, 1996). The anti-inflammatory effects of locally released endogenous ADO may also account, in part, for the ADO A_2Areceptor-mediated component underlying ABT-702 antihyperalgesia in the carrageenan model. Consistent with such a peripheral mechanism, ABT-702 relieved carrageenan-induced thermal hyperalgesia when administered locally at the site of carrageenan inflammation. Similar local, peripheral antinociceptive effects have been observed with the prototypical AK inhibitor 5′amino,5′-deopxyadenosine in the rat formalin test (Sawynok et al., 1998). The possibility that spinal A_2A receptors also contribute to ABT-702-mediated antihyperalgesia cannot be ruled out, given the presence of ADO A_2A receptors in the dorsal spinal cord (Choca et al., 1987), as well as pharmacological evidence suggesting involvement of ADO A_2A receptors in spinal antinociception (Sawynok, 1999).

Previous efforts to develop direct-acting ADO agonists as therapeutic entities have been hampered by their mechanism-based cardiovascular and sedative side effects (Williams, 1996). ABT-702 had no effects on motor coordination, as reflected by rotorod performance, at doses providing maximal antinociception in all animal models studied, including models of nociceptive, inflammatory, and neuropathic pain. ABT-702 also had no significant effect on exploratory locomotor activity at doses providing relief of carrageenan-induced thermal hyperalgesia (≤10 μmol/kg p.o.), although effects were observed at higher doses. ABT-702 had no effect on mean arterial pressure in rats at oral doses providing maximal antinociception in rat carrageenan-induced thermal hyperalgesia, and at doses providing near-maximal antinociception in the formalin and neuropathic pain models after oral administration. A modest reduction in mean arterial pressure was observed at the maximally effective oral dose in the latter two models. No effects were seen on heart rate at any dose tested. Thus, ABT-702 provided antihyperalgesic effects at doses devoid of classical ADO-mediated side effects.

Consistent with its in vitro pharmacology (Jarvis et al., 2000), the antinociceptive effects of ABT-702 can be differentiated from classical analgesic and anti-inflammatory agents, morphine and ibuprophen. ABT-702 was markedly more potent than ibuprofen to relieve carrageenan-induced thermal hyperalgesia, and was more potent and effective than ibuprofen in the formalin test. ABT-702 fully relieved tactile allodynia in the Chung model, whereas ibuprofen was completely ineffective in this model. The antinociceptive effects of ABT-702 do not involve opioid systems because the antinociceptive effects of ABT-702 in both a chronic neuropathic pain model and in an acute thermal nociceptive test (Jarvis et al., 2000) were not attenuated by the opioid receptor antagonist naloxone. The observation that the antinociceptive effects of ABT-702 and morphine in the formalin test are additive is also consistent with distinct mechanisms of action for ABT-702 and morphine. Importantly, ABT-702 was also shown to have less potential to develop tolerance to its antinociceptive effects than morphine.

In summary, the novel, potent, and selective non-nucleoside AK inhibitor ABT-702 is orally effective to ameliorate nociceptive behaviors in a spectrum of well characterized animal models of pain, including inflammatory pain, chemically induced persistent pain, and neuropathic pain. It is especially potent to relieve the hyperalgesia of inflammatory pain, at doses devoid of effects on blood pressure, heart rate, and motor activity. Thus, ABT-702 is a novel non-nucleoside AK inhibitor that shows antinociceptive and anti-inflammatory activity in vivo.