Controlling murine and rat chronic pain through A₃ adenosine receptor activation

Abstract

Clinical management of chronic neuropathic pain is limited by marginal effectiveness and unacceptable side effects of current drugs. We demonstrate A₃ adenosine receptor (A₃AR) agonism as a new target-based therapeutic strategy. The development of mechanoallodynia in a well-characterized mouse model of neuropathic pain following chronic constriction injury of the sciatic nerve was rapidly and dose-dependently reversed by the A₃AR agonists: IB-MECA, its 2-chlorinated analog (Cl-IB-MECA), and the structurally distinct MRS1898. These effects were naloxone insensitive and thus are not opioid receptor mediated. IB-MECA was ≥1.6-fold more efficacious than morphine and >5-fold more potent. In addition, IB-MECA was equally efficacious as gabapentin (Neurontin) or amitriptyline, but respectively >350- and >75-fold more potent. Besides its potent standalone ability to reverse established mechanoallodynia, IB-MECA significantly increased the antiallodynic effects of all 3 analgesics. Moreover, neuropathic pain development in rats caused by widely used chemotherapeutics in the taxane (paclitaxel), platinum-complex (oxaliplatin), and proteasome-inhibitor (bortezomib) classes was blocked by IB-MECA without antagonizing their antitumor effect. A₃AR agonist effects were blocked with A₃AR antagonist MRS1523, but not with A₁AR (DPCPX) or A_2AAR (SCH-442416) antagonists. Our findings provide the scientific rationale and pharmacological basis for therapeutic development of A₃AR agonists for chronic pain.—Chen, Z., Janes, K., Chen, C., Doyle, T., Bryant, L., Tosh, D.K., Jacobson, K.A., Salvemini, D. Controlling murine and rat chronic pain through A₃ adenosine receptor activation.

Chronic neuropathic pain is a major unresolved health-care issue with global human and socioeconomic impact (5–10% occurrence in Europe and the United States; ref. 1). Its general incidence is augmented by pain from chemotherapy-induced peripheral neuropathy (CIPN; ref. 2). To this end, patients with malignancies of various etiologies who receive taxane, platinum complex, vinca alkaloids, and/or proteasome-inhibitors risk developing a chronic, distal, and symmetrical sensory peripheral neuropathy often accompanied by a neuropathic pain syndrome (3). This condition may resolve soon after drug termination or last for years (2, 3). Thus, CIPN is one of the most common causes of dose reduction and discontinuation of what is otherwise a life-saving therapy (2, 3).

The causative mechanisms of chronic neuropathic pain are poorly understood, and current pain drugs are only marginally effective and display many side effects (4). Therefore, it is necessary to identify novel therapeutic targets that can effectively resolve this condition. Our study introduces selective A₃ adenosine receptor (A₃AR) agonists as a viable therapeutic strategy in chronic neuropathic pain of distinct etiologies. The purine nucleoside adenosine, as well as its derivatives, generated by metabolically stressed or inflamed cells and tissues exhibits diverse and potent physiological responses in most organs and tissues including the central nervous system (CNS). Rising adenosine concentrations signal a threat to local homeostasis and initiate a myriad of responses in nearby neurons, astrocytes, and microglia cells. The actions of adenosine are mediated through G-protein-coupled receptors, which are classified into 4 subtypes, A₁, A_2A, A_2B, and A₃, each of which has a characteristic profile for agonists and antagonists (5). These receptor subtypes are characterized by their capacity to either increase or decrease intracellular cAMP levels (5). A₁ and A₃ ARs, coupled through G_i proteins, mediate biological effects opposite to A_2A and A_2B ARs, which are coupled to G_s proteins (6). Adenosine acting through one or more of these receptors plays important roles in numerous disease states (7–11). The role of adenosine in pain and the contribution of A₁AR and A_2AAR subtypes have been recognized for several years (12), and A₁AR agonists (10, 12) and A_2AAR agonists (13) have shown efficacy in several preclinical animal models (12). The clinical use of adenosine in pain is underscored by the findings that intrathecal delivery provides long-lasting relief in patients with chronic pain (12). Although selective A₁AR and A_2AAR agonists are in clinical development for neuropathic pain (11, 12, 14), but their use is restricted to local delivery. Systemic administration would risk cardiovascular side effects from activation of the A₁AR expressed in conducting tissues or A_2AAR in vascular smooth muscle (12). In contrast, activation of the A₃AR in humans by potent, selective, and orally bioavailable A₃AR agonists, e.g., IB-MECA [N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide] and its chlorinated counterpart Cl-IB-MECA [2-chloro-N⁶-(3-iodobenzyl)-adenosine-5′-N-methyluronamide] (15, 16), is not associated with cardiac or hemodynamic effects (17). It is important to note that these nucleosides are already in phase 2 and/or 3 clinical trials for autoimmune inflammatory diseases, liver cancer, and hepatitis (17). The A₃AR is highly expressed in many inflammatory cells, including glial cells (18–20), and found in peripheral sensory neurons (21) and neurons (22, 23) in various areas of the CNS (24, 25). However, in contrast to the well-recognized role of the A₁AR and A_2AAR in pain (12), the role of this receptor subtype is not known. One report has examined the effects of A₃AR agonists in a single model: the formalin test in mice (26). In this study the intrathecal delivery of IB-MECA attenuated the inflammatory component, phase 2 but not phase 1, of the formalin test (26). IB-MECA and Cl-IB-MECA provide important pharmacological tools that can be used to explore and dissect the relevance, if any, of A₃AR activation in pain while providing the opportunity for translational application in this setting if warranted by preclinical studies. Focusing initially on chronic neuropathic pain, we demonstrate for the first time that activation of the A₃AR blocks the development of neuropathic pain in models of chronic constriction injury (CCI) following ligation of the sciatic nerve and following the administration of widely used chemotherapeutic agents with distinct antitumor mechanism of action. Our results are anticipated to establish the A₃AR as a scientific foundation for new translational efforts to develop a chronic neuropathic pain treatment, while potentially contributing to a fast-track clinical study of IB-MECA and Cl-IB-MECA.

MATERIALS AND METHODS

Materials

IB-MECA, Cl-IB-MECA, DPCPX (8-cyclopentyl-1,3-dipropylxanthine), and SCH-442416 [2-(2-furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo-[4,3-e] [1,2,4]triazolo[1,5-C]pyrimidin-5-amine] were purchased from Tocris (Ellisville, MO). MRS1898 ((1′R,2′R,3′S,4′R,5′S)-4-{2-chloro-6-[(3-iodophenylmethyl)amino]purin-9-yl}-1-(methylaminocarbonyl)-bicyclo[3,1,0]hexane-2,3-diol) was synthesized as described previously (27, 28). Morphine was a kind gift from Mallinckrodt (St. Louis, MO, USA). Paclitaxel, oxaliplatin, and bortezomib were purchased commercially from Parenta Pharma (Yardley, PA, USA), Oncology Supply (Dothan, AL, USA), and Selleck Chemicals (Houston, TX, USA), respectively. For cell culture, the media were purchased from Mediatech (Dulbecco's minimal essential medium and McCoy's 5A; Mediatech, Manassas, VA, USA) or Sigma-Aldrich (L12; Sigma Aldrich, St. Louis, MO, USA); fetal bovine serum from Thermo Scientific Hyclone (Waltham, MA, USA); and the penicillin/streptomycin from Invitrogen (Carlsbad, CA, USA). Cell lines were kind gifts from colleagues: SKBR3 (Dr. Joseph Baldassare, St. Louis University), SW480 (Stephanie Knebel, St. Louis University), and RPMI 8226 (Jaki Kornbluth, St. Louis University). MRS1523 [3-propyl-6-ethyl-5[(ethylthio)carbonyl]-2-phenyl-4-propyl-3-pyridine-carboxylate], gabapentin, amitriptyline, and all other chemicals were purchased from Sigma-Aldrich.

Experimental animals

Male Sprague-Dawley rats (200–220 g) or mice (25–30 g) from Harlan (Indianapolis, IN, USA) were housed 3–4/cage (for rats) and 5/cage (for mice) in a controlled environment (12-h light-dark cycles) with food and water available ad libitum. Experiments were performed in accordance with International Association for the Study of Pain, U.S. National Institutes of Health guidelines on laboratory animal welfare, and St. Louis University Institutional Animal Care and Use Committee recommendations. Experimenters were blinded to treatment conditions in all experiments.

CCI model of neuropathic pain

CCI to the sciatic nerve of the left hind leg in mice was performed under general anesthesia using the well-characterized Bennett model (29). Briefly, mice (weighing 25–30 g at the time of surgery) were anesthetized with 3% isoflurane/100% O₂ inhalation and maintained on 2% isoflurane/100% O₂ for the duration of surgery. The left thigh was shaved and scrubbed with Nolvasan, and a small incision (1–1.5 cm in length) was made in the middle of the lateral aspect of the left thigh to expose the sciatic nerve. The nerve was loosely ligated around the entire diameter of the nerve at 3 distinct sites (spaced 1 mm apart) using silk sutures (6.0). The surgical site was closed with a single muscle suture and a skin clip. Pilot studies established that under our experimental conditions peak mechanoallodynia develops by day 5–7 (D5–D7) following CCI. Test substances or their vehicles were given subcutaneously (s.c.), intraperitoneally (i.p.), or orally by gavage (0.1 ml) at peak mechanoallodynia (D7).

Induction of chemotherapy-induced neuropathic pain in rats

Paclitaxel or vehicle (Cremophor EL and 95% anhydrous ethanol in 1:1 ratio) were injected i.p. on 4 alternate days (2 mg/kg on D0, D2, D4, and D6, with a final cumulative dose of 8 mg/kg; ref. 30). Oxaliplatin or vehicle (5% dextrose) was injected i.p. in rats on 5 consecutive days (D0–D4) for a final cumulative dose of 10 mg/kg (31). Bortezomib or vehicle (5% Tween 80, 5% ethanol) was injected i.p. in rats on 5 consecutive days (D0–D4; 0.2 mg/kg) for a final cumulative dose of 1 mg/kg (G. J. Bennett and W. H. Xiao, McGill University, Montreal, QC, Canada; personal communication, November 4, 2011). Test substances or their vehicle were given i.p. (0.2 ml) 30 min before the chemotherapeutic agent or its vehicle on D0 (baseline) and then daily until D15 or D17. Behavioral responses (mechanoallodynia and mechanohyperalgesia) were measured on D0 prior to the first intraperitoneal injection of the chemotherapeutic agent and subsequently at various time points. If testing coincided with a day when rats received test substance, behavioral measurements were taken always before the injection of the test substance. Chemotherapeutic treatments result in bilateral allodynia and hyperalgesia without differences in left and right paw withdrawal threshold (PWT; g) in any group at any time point; thus, values from both paws were averaged. None of the animals exhibited signs of observable toxicity; they exhibited normal posture, grooming, and locomotor behavior, hair coat was normal without signs of piloerection or porphyrin, and body weight gain was normal and comparable to vehicle-treated rats.

Behavioral testing

Mechanoallodynia was measured in CCI and paclitaxel studies after first acclimating the animals to elevated cages with a wire mesh floor for 15 min. The plantar aspect of hindpaws was probed 3 times with calibrated von Frey filaments (Stoelting, Wood Dale, IL, USA; mice: 0.07–2.00 g; rats: 0.407–26 g) according to the “up-and-down” method (32). In oxaliplatin or bortezomib studies, allodynia was assessed with an electronic version of the VF test (dynamic plantar aesthesiometer, model 37450; Ugo Basile, Milan, Italy). Briefly, each rat was placed in a Plexiglas chamber (28×40×35-cm, wire mesh floor), and after its acclimation, a servo-controlled mechanical stimulus was applied to the plantar surface that exerted a progressively increasing punctate pressure up to 50 g within 10 s. Mechanical threshold was assessed 3 times at each time point to yield a mean value, reported as PWT. The development of mechanoallodynia is evidenced by a significant (P<0.05) reduction in mechanical mean PWT at forces that failed to elicit withdrawal responses on D0 before CCI or chemotherapeutic/vehicle treatment. Mechanohyperalgesia was assessed in rats by the Randall and Sellitto paw pressure test (33) using an analgesiometer (Ugo Basile). The nociceptive threshold was defined as the force (g) at which the rat withdrew its paw (cutoff set at 250 g).

Tail-flick and hot plate assay in mice for acute nociception

The tail-flick test, which measures latency of tail withdrawal from a noxious radiant heat source (model 37360; Ugo Basile), was used to measure thermal nociceptive sensitivity in mice with baseline latency of 3–5 s and a cutoff time of 15 s to prevent tissue injury (34). Tail flick latency was taken before and at 30, 60, and 120 min after intraperitoneal injection of IB-MECA, MRS1898 (0.5 μmol/kg), or its vehicle (3% DMSO in saline). For the hot plate test, nociceptive threshold was determined as previously reported by our group by measuring latency (in seconds) of mice placed in a transparent glass cylinder on a hot plate maintained at 52°C (35, 36). Responses indicative of nociception included intermittent lifting and/or licking of the hindpaws or escape behavior. Hot plate latency was taken in mice from all groups before and after drug administration. A cutoff latency of 15 s was employed to prevent tissue damage.

Rotarod test in mice

Mice were trained before experimentation for their ability to remain for 60 s on a revolving Rotarod for mice apparatus (Ugo Basile; accelerating units increasing from 5 to 40 rpm in 60 s). Mice were injected i.p. with IB-MECA, MRS1898 (0.5 μmol/kg), or vehicle (3% DMSO in saline) and then examined at 30, 60, and 120 min after administration for motor impairments on the Rotarod. The latency time to fall off the Rotarod was determined with a cutoff time of 120 s.

Effects of IB-MECA on antitumor activity of paclitaxel, oxaliplatin, and bortezomib

Cells were cultured and assayed in DMEM (SKBR3; human breast cancer cells; 37), L15 (SW480; human colon cancer cells; ref. 38), or RPMI 1620 (RPMI 8226; human multiple myeloma cells; ref. 39) supplemented with heat-denatured 10% FBS and penicillin/streptomycin at 37°C, 0% (SW480) or 5% CO₂ (SKBR3 and RPMI 8226), 95% humidity. The antitumor activities of these agents were measured in cells (6.25×10⁴ cells/well) cultured overnight in 24-well plates (SKBR3 and SW480; BD Biosciences, San Jose, CA, USA) or 12- × 7.5-mm culture tube (RPMI 8226; BD Biosciences) in complete medium. This plating regiment yielded 60% confluent plate cultures for testing. After equilibrating in fresh medium (5 h), cells were treated for 48 h with IB-MECA (10 nM) or its vehicle [0.01% DMSO in phosphate-buffered saline (PBS)] and paclitaxel (1–100 nM) or its vehicle (1% Cremophor E and 0.9% ethanol); oxaliplatin (1–100 μM) or its vehicle (0.01% DMSO in PBS); or bortezomib (1–100 nM) or its vehicle (0.05% dextrose). Two naive control wells were included as a control for 100% survival. Cell survival was determined using a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay adapted from a previously described assay (40, 41). Cells were incubated with MTT (500 μg/ml) for 75 min at 37°C, 0 or 5% CO₂, 95% humidity, removing the medium and dissolving the tetrazolium crystals in isopropanol. The tetrazolium absorption at 560–570 nm (A_560–570nm) was measured from paclitaxel experiments using a Unicam UV1 spectrophotometer (ThermoFisher Scientific) and from oxaliplatin or bortezomib experiments using a Glomax multidetection system (Promega, Madison, WI, USA). The antitumor effects of IB-MECA alone were determined as survivability (%) = (A_560–570nm of chemotherapeutic vehicle + IB-MECA)/(mean A_560–570nm of naive control wells) × 100. The concentration yielding 50% lethality (LC₅₀) of each chemotherapeutic agent + IB-MECA or its vehicle was calculated using 3-parameter nonlinear analysis using survivability (%) = (A_560–570nm of chemotherapeutic + IB-MECA or its vehicle)/(mean A_560–570nm chemotherapeutic vehicle + vehicle of IB-MECA) × 100. The top and bottom plateaus were constrained using GraphPad Prism 5.03 (GraphPad Software, San Deigo, CA, USA).

Determining dose yielding 50% effect (ED₅₀) values

Dose response data were curve fitted using the least sum of square method by a normalized 4-parameter, variable slope nonlinear analysis of the reversal (%) of mechanoallodynia in CCI and prevention (%) of mechanoallodynia or mechanohyperalgesia in CIPN using GraphPad Prism 5.03, from which the ED₅₀ was determined and reported with the 95% confidence interval (95% CI). In the CCI model, reversal (%) of mechanoallodynia = (PWT_1h − PWT_D7)/(PWT_D0 − PWT_D7) × 100, where PWT_D0 = PWT (g) at D0; PWT_1h = PWT at 1 h after the administration of IB-MECA, gabapentin, amitriptyline, or combinations of IB-MECA with gabapentin or amitriptyline; and PWT_D7 = PWT at D7 prior to drug administration. In the chemotherapeutic-induced neuropathic pain models, prevention (%) = (PWT_IB-MECA − mean PWT_chemo)/(mean PWT_veh − mean PWT_chemo) × 100, where chemo = paclitaxel or oxaliplatin, and veh = vehicle.

Statistical analysis

Data are expressed as means ± sd for n animals. Behavioral data were analyzed by 2-way repeated measures ANOVA with Bonferroni comparisons (full-time course studies) or 1-way ANOVA Dunnett's comparisons (1 h behavioral data). The dose response curves were compared to a globally fitted curve using the extra sum-of-squares F test comparisons to determine whether the data represented distinct curves between treatments. Significant differences were defined as a value of P < 0.05. All statistical analysis was performed using GraphPad Prism 5.03.

RESULTS

A₃AR agonists block the development of neuropathic pain following CCI via an A₃AR-mediated mechanism

When peak mechanoallodynia develops (D7) following CCI of the mouse sciatic nerve (29), administration of IB-MECA, but not vehicle (3% DMSO in saline, i.p.), rapidly (≤30 min) and dose dependently (0.2–2 μmol/kg, n=5) reversed allodynia, with maximal effect within 1 h (ED₅₀, 0.4 μmol/kg; 95% CI=0.23–0.66; Fig. 1A). IB-MECA lacked effect on PWT in contralateral paws (Fig. 1B). It is noteworthy that when compared to D7, consecutive daily injections (0.5 μmol/kg IB-MECA, n=5) on D8–D15 reversed mechanoallodynia to the same degree as observed on D7 following the first injection (Fig. 1C). This suggests that A₃ARs do not become tolerant to agonist activation, at least over this dosing paradigm. The antiallodynic effect of IB-MECA (0.5 μmol/kg, n=5) was prevented by a 15 min pretreatment with a potent A₃AR antagonist, MRS1523 (5 μmol/kg, n=5; Fig. 2A; ref. 42). MRS1523 is a pyridine derivative that binds to murine and rat A₃ARs with high affinity and has moderate selectivity against A₁AR, but excellent selectivity against A_2AAR and A_2BAR (≥1000-fold; ref. 43). In contrast, the antiallodynic effect of IB-MECA (0.5 μmol/kg, n=5) was not affected by the potent A₁AR antagonist DPCPX (2 μmol/kg, n=5) or by the potent A_2AAR antagonist SCH-442416 (0.2 μmol/kg, n=5; ref. 44 and Fig. 2C). When given alone on D7, MRS1523, DPCPX, or SCH-442416 lacked effect on ipsilateral (Fig. 2A, C) or contralateral (Fig. 2B, D) PWT. Agonist and antagonist doses were chosen from previous studies showing selectivity for their respective receptor subtype (44–46).

Figure 1.

A₃AR agonists reverse mechanoallodynia in the CCI model. A) IB-MECA (i.p.; 0.2, □; 0.5, ●; or 2 μmol/kg; ▵), but not its vehicle (○), on D7 after CCI (arrow) reversed mechanoallodynia in ipsilateral paws. B) IB-MECA did not affect contralateral PWT (g). C) When compared to vehicle (○), daily injections i.p. (D8-D15, arrows) of IB-MECA (0.5 μmol/kg, ●) reversed mechanoallodynia to the same extent as D7. Results are expressed as mean ± sd, n = 5 mice, analyzed by ANOVA with Bonferroni comparisons. *P < 0.001 (D7 or vehicle vs. D0); ^†P < 0.05, ^††P < 0.001 (IB-MECA at each time point posttreatment vs. D7); ^oP < 0.001 (agonist+antagonist vs. agonist alone).

Figure 2.

IB-MECA reverses CCI-induced neuropathic pain through A₃AR-specific mechanisms. Mechanoallodynia developed by D7 after CCI of the sciatic nerve (○) in ipsilateral paws (A, C), but not contralateral paws (B, D), which was reversed by administration of IB-MECA (i.p.; 0.5 μmol/kg; ●; arrow). The A₃AR antagonist, MRS1523 (i.p.; 5 μmol/kg; ♦; A), but not the A₁AR antagonist, DPCPX (2 μmol/kg; ▴; C), or the A_2AAR antagonist, SCH-442416 (i.p.; 0.2 μmol/kg; ▾ (C), prevented the antiallodynic effect of IB-MECA. Neither MRS1523 (◊), DPCPX (▵), nor SCH-442416 (▿), when given alone, had any effect on allodynia on ipsilateral (A, C) or contralateral (B, D) paws. Antagonists were given 15 min before IB-MECA or its vehicle. Results are expressed as means ± sd for n = 5 mice and analyzed by ANOVA with Bonferroni comparisons. *P < 0.001 (D7 vs. D0); ^†P < 0.001 (IB-MECA at t_h vs. D7); °P < 0.001 (IB-MECA+antagonist vs. IB-MECA).

To generalize the benefit of A₃AR agonism in neuropathic pain, 2 additional selective A₃AR agonists were tested: a 2-chlorinated analog (Cl-IB-MECA) and the structurally distinct MRS1898. MRS1898 is a well-characterized, potent A₃AR agonist containing a rigid bicyclic ring substitution of ribose that maintains a receptor-preferred conformation (27). Cl-IB-MECA (0.6 μmol/kg, n=5; Fig. 3A) and MRS1898 (0.5 μmol/kg, n=5; Fig. 3C) rapidly and maximally (≤1 h) reversed mechanoallodynia, effects blocked by MRS1523 (5 μmol/kg, n=5; Fig. 3A) but not by DPCPX (2 μmol/kg, n=5) or SCH-442416 (0.2 μmol/kg, n=5; Fig. 3C). Doses of Cl-IB-MECA and MRS1898 were selected from previous studies (27). Cl-IB-MECA and MRS1898 had no effect on PWT in contralateral paws (Fig. 3B, D). Collectively, our results establish that A₃AR agonists of 2 distinct classes reverse mechanoallodynia through an A₃AR mechanism and without involving other ARs.

Figure 3.

Cl-IB-MECA and MRS1898 reverse CCI-induced neuropathic pain through an A₃AR-specific mechanism. When given i.p. on D7 and compared to vehicle (○), administration (arrow) of Cl-IB-MECA (0.6 μmol/kg; ●; A, B) or MRS1898 (0.5 μmol/kg; ●; C, D) reversed mechanoallodynia in ipsilateral (A, C), with no effects on contralateral paws (B, D). The A₃R antagonist, MRS1523 (5 μmol/kg; ♦), blocked the ability of Cl-IB-MECA (A) or MRS1898 (C) to reverse mechanoallodynia. The A₁AR antagonist, DPCPX (2 μmol/kg; ▴) or the A_2AAR antagonist, SCH-442416 (i.p.; 0.2 μmol/kg;▾) did not prevent the antiallodynic effects of MRS1898 (C). Neither MRS1523 (◊), DPCPX (▵), nor SCH-442416 (▿), when given alone, had any effect on allodynia on ipsilateral (A, C) or contralateral (B, D) paws. Antagonists were given 15 min before Cl-IB-MECA and MRS1898 or its vehicle. Results are expressed as means ± sd for n = 5 mice and analyzed by ANOVA with Bonferroni comparisons. *P < 0.001 (D7 vs. D0); ^†P < 0.001 (A₃AR agonists± antagonists at t_h vs. D7); °P < 0.001 (A₃AR agonists+antagonist vs. agonists).

Antiallodynic effects of A₃AR agonists are naloxone independent

A high dose (i.p.) of nonselective opioid receptor antagonist naloxone (25 μmol/kg, n=5) was administered 15 min before IB-MECA (0.5 μmol/kg, n=5) or MRS1898 (0.5 μmol/kg, n=5). Naloxone did not interfere with the ability of the A₃AR agonists to reverse established mechanoallodynia in the CCI model (Fig. 4A), thus excluding an opioid-dependent mechanism. Given alone on D7, naloxone did not affect ipsilateral (Fig. 4A) or contralateral (Fig. 4B) PWT.

Figure 4.

Naloxone does not block antiallodynic effects of A₃AR agonists. A) In ipsilateral paws, the reversal of mechanoallodynia by IB-MECA or MRS1898 (0.5 μmol/kg) was not prevented by naloxone (25 μmol/kg). B) No differences in PWT (g) were observed in contralateral paws. Results are expressed as means ± sd, n = 5 mice, analyzed by ANOVA with Dunnett's comparisons. *P < 0.001 (D7 or vehicle vs. D0); ^†P < 0.001 (IB-MECA at 1 h post-treatment vs. D7).

A₃AR agonists have no effect on acute nociception

IB-MECA or MRS1898 (0.5 μmol/kg, n=5) tested at 30, 60, and 120 min lacked effect on acute nociception in the mouse tail flick (Fig. 5A). On the other hand, morphine injected s.c. (35 μmol/kg; n=5) and used as a positive control elicited potent acute antinociceptive effects with a significant (P<0.001) increase in tail-flick latency (Fig. 5A). Similarly, IB-MECA or MRS1898 (0.5 μmol/kg, n=5) had no effect when tested on the hot plate (not shown), supporting the lack of a role for A₃AR agonists in modulating normal nociception.

Figure 5.

A₃AR agonists have no effect on acute nociception and Rotarod test. A) Unlike morphine (35 μmol/kg, s.c., ▴), IB-MECA (0.5 μmol/kg, ○) and MRS1898 (0.5 μmol/kg, □) lacked effect on mouse tail flick latency. B) Mouse Rotarod latency was similar with IB-MECA (0.5 μmol/kg, solid bar), MRS1898 (0.5 μmol/kg, shaded bar), or vehicle (open bar). Results are expressed as means ± sd, n = 5 mice, analyzed by ANOVA with Bonferroni comparisons. ^†P < 0.001 (morphine vs. t_0h).

A₃AR agonists have no effect on the Rotarod test

IB-MECA or MRS1898 (0.5 μmol/kg, n=5) did not induce Rotarod deficits, ruling out potential motor function impairment in mice (Fig. 5B). In addition, A₃AR agonists when tested at the highest dose lacked observable signs of lethargy or sedation; i.e., they exhibited normal posture, no loss of normal activity such as grooming, no effect on alert and exploratory behavior, no effect on spontaneous locomotor activity, and no loss of motor coordination, paw dragging, or their ability to remain upright.

IB-MECA increases the potency of the analgesic effects of morphine, gabapentin, and amitriptyline in CCI

Morphine (0.11–35 μmol/kg, n=5), but not its vehicle (saline), given s.c. on D7 led to a rapid peak (≤0.5 h) and dose-dependent reversal of mechanoallodynia in ipsilateral paws (Fig. 6A), but not in contralateral paws (Fig. 6B). Morphine at the time of maximal reversal (0.5 h) displayed an ED₅₀ of 2.1 μmol/kg (95% CI=1.5–3.0), which was 5-fold less potent than IB-MECA (Fig. 7). Moreover, IB-MECA (E_max=100%) was more efficacious than morphine alone (E_max=62±3%, Fig. 7). Gabapentin (18–584 μmol/kg, i.p., n=5; Fig. 6C) or amitriptyline (3–191 μmol/kg, oral, n=5; Fig. 6E), but not their vehicle (saline), on D7 led to a rapid (≤0.5 h) and dose-dependent reversal of mechanoallodynia in ipsilateral paws that peaked at 1 h. The ED₅₀ of gabapentin and amitriptyline at maximal reversal (1 h) was, respectively, 140 μmol/kg (95% CI=122–162) and 31 μmol/kg (95% CI=22–43; Fig. 7). Therefore, gabapentin and amitriptyline were > 350- and 75-fold less potent than IB-MECA. Gabapentin and amitriptyline had no effect on contralateral PWT (Fig. 6D, F).

Figure 6.

Morphine, gabapentin, or amitriptyline reverse mechanoallodynia in CCI-induced neuropathic pain. The development of mechanoallodynia observed on D7 after CCI in the ipsilateral paw (□, n=6) was reversed in a dose- and time-dependent manner by morphine (0.11, ○; 0.35,●; 1.05, ■; 3.5, ▴; 11, ▾; or 35 μmol/kg, ♦; A), gabapentin (18, ■; 58, ▴; 175, ▾; or 584 μmol/kg, ♦; C), or amitriptyline (3.2,●; 9.6, ■; 32, ▴; 96, ▾; or 191 μmol/kg, ♦; E) in ipsilateral paws. These agents had no effect in contralateral paws (B, D, F). Results are expressed as means ± sd for n = 5 mice and analyzed by ANOVA with Bonferroni comparisons. *P < 0.001 (D7 vs. D0); ^†P < 0.05, ^††P < 0.001 (morphine, gabapentin, or amitriptyline at t_h vs. D7).

Figure 7.

Relative potencies of IB-MECA, morphine, gabapentin, and amitriptyline in CCI. As tested on D7 and at time of peak reversal, IB-MECA (●) was > 5-, >350-, and > 75-fold, respectively, more potent in reversing established mechanoallodynia when compared to morphine (▾), gabapentin (■), or amitriptyline (▴). In addition, IB-MECA was more efficacious than morphine but equiefficacious with gabapentin or amitriptyline. Results expressed as means ± sd, n = 5 mice, difference between curves were analyzed by extra sum-of-squares F test comparisons. *P < 0.001 (morphine, gabapentin or amitriptyline vs. IB-MECA); ^†P < 0.001 (morphine, gabapentin or amitriptyline vs. gabapentin, amitriptyline, or morphine+IB-MECA).

It is noteworthy that a low IB-MECA dose devoid of antiallodynic effects in CCI (0.2 μmol/kg, n=5) augmented the ability of morphine (0.11–35 μmol/kg, n=5; Fig. 8A), gabapentin (1.8–175 μmol/kg, n=5; Fig. 8B), or amitriptyline (1–96 μmol/kg, n=5; Fig. 8C) to reverse established mechanoallodynia, as evidenced by a significant (P<0.001) leftward dose response shift. To this end, the ED₅₀ at peak reversal for morphine decreased from 2.1 μmol/kg (95% CI=1.5–3.0) to 0.98 μmol/kg (95% CI=0.66–1.5) when combined with IB-MECA, whereas the ED₅₀ at peak reversal for gabapentin or amitriptyline decreased from 140 μmol/kg (95% CI=122–162) and 31 μmol/kg (95% CI=22–43) to 27 μmol/kg (95% CI=21–34) and 13 μmol/kg (95% CI=11–16), respectively, when combined with IB-MECA. This combined treatment lacked effect on contralateral PWT (not shown). Collectively, IB-MECA increased the potency of morphine by > 2-fold and that of gabapentin and amitriptyline by > 5- and 2-fold, respectively. Moreover, IB-MECA also enhanced the efficacy of morphine by 1.6-fold (Fig. 8A).

Figure 8.

IB-MECA augments the antiallodynic effects of morphine, gabapentin, or amitriptyline in CCI. When compared to morphine (0.11–35 μmol/kg, s.c., ▾; A), gabapentin (18–584 μmol/kg, i.p., ■; B), or amitriptyline (3–191 μmol/kg, oral, ▴; C) alone on D7, coadministration of a low dose of IB-MECA (0.2 μmol/kg) significantly increased their antiallodynic effects as revealed by a shift to the left in the dose-response of morphine (▿; A), gabapentin (□; B), and amitriptyline (▵; C). Moreover, IB-MECA (0.2 μmol/kg) increased the efficacy of morphine (A). Results expressed as means ± sd, n = 5 mice, difference between curves were analyzed by extra sum-of-squares F test comparisons. *P < 0.001 (morphine, gabapentin, or amitriptyline vs. IB-MECA); ^†P < 0.001 (morphine, gabapentin, or amitriptyline vs. gabapentin, amitriptyline or morphine+IB-MECA).

A₃AR agonists block the development of chemotherapy-induced neuropathic pain without interfering with antitumor effects

In order to test whether beneficial effects of A₃AR agonists could be extended to another form of neuropathic pain, we investigated their pharmacological activity in models of neuropathic pain induced by widely used chemotherapeutics in distinct classes and with well-known, distinct antitumor mechanisms of action: paclitaxel, oxaliplatin, and bortezomib. Although chemotherapeutic dosing in each model is completed within several days, we continued dosing agonists until the time when pain typically occurs (between D15 and D17). The delay to symptom onset (also noted in patients) introduces uncertainty in the time of onset of relevant pathological process. When compared to vehicle, paclitaxel administration led to neuropathic pain (mechanoallodynia and mechanohyperalgesia) that peaked by D16, plateaued through D25, and was dose-dependently attenuated by daily (D0–D15) administration of IB-MECA (0.02–0.2 μmol/kg/d, i.p., n=6; Fig. 9A, B) but not vehicle (3% DMSO in saline). The D16 ED₅₀ values for prevention of paclitaxel-induced mechanoallodynia and mechanohyperalgesia were 0.02 and 0.03 μmol/kg/d (95% CI=0.018–0.024 and 0.024–0.033). It is noteworthy that following discontinuation of IB-MECA treatment on D15, neuropathic pain did not emerge through D25 (Fig. 9A, B). The effects of IB-MECA (0.2 μmol/kg/d, n=6) were prevented by coadministration with MRS1523 (5 μmol/kg/d, n=6; Fig. 9C, D). A₃AR agonism is clearly necessary, since Cl-IB-MECA (0.2 μmol/kg/d, n=5) or MRS1898 (0.2 μmol/kg/d, n=3) completely blocked paclitaxel-induced neuropathic pain (not shown), eliminating the possibility that a pharmacological action particular to IB-MECA causes the protective effects.

Figure 9.

Repeated dosing with IB-MECA blocks chemotherapy-induced neuropathic pain. When compared to the vehicle group (○), paclitaxel alone (●), or oxaliplatin alone (●) led to a time-dependent development of mechanoallodynia (A, E) and mechanohyperalgesia (B, F), which was blocked by daily injections (D0-D15/D17) with IB-MECA (i.p.; 0.02, ■; 0.05, ▴; or 0.2 μmol/kg/d, ▾). Effects of IB-MECA (0.2 μmol/kg/d) in paclitaxel-induced neuropathic pain were antagonized by coadministration of MRS1523 (5 μmol/kg/d; ♦; C, D). At the highest dose, IB-MECA alone (0.2 μmol/kg, ▿; A–F) or MRS1523 alone (5 μmol/kg/d,◊; C, D) lacked effect in comparision to vehicle groups. Results expressed as means ± sd, n = 6 rats, analyzed by ANOVA with Bonferroni comparisons. *P < 0.001 (chemotherapeutic agent vs. vehicle); ^†P < 0.01 or ^††P < 0.001 (chemotherapeutic agent+IB-MECA vs. chemotherapeutic agent); ^oP < 0.05, ^ooP < 0.01, ^oooP < 0.001 (paclitaxel+IB-MECA+MRS1523 vs. paclitaxel+IB-MECA).

The beneficial effects of IB-MECA were not restricted to paclitaxel. The development of oxaliplatin-induced neuropathic pain (peaking by D17 and plateauing through D25) was attenuated dose dependently by daily (D0–D17) administration of IB-MECA (0.02–0.2 μmol/kg/d, n=6; Fig. 9E, F) and did not emerge on drug termination. IB-MECA prevented mechanoallodynia and mechanohyperalgesia with D17 ED₅₀ values of 0.05 and 0.06 μmol/kg/d, respectively (95% CI=0.039–0.053 and 0.048–0.07). Finally, IB-MECA (0.2 μmol/kg/d, n=5) blocked the development of bortezomib-induced mechanoallodynia and mechanohyperalgesia, which did not emerge when treatment was discontinued on D17 (not shown). IB-MECA administered alone (0.2 μmol/kg/d, n=6) did not affect PWT in any of the chemotherapy models used (Fig. 9). Additional experiments confirming an A₃AR-dependent mechanism in oxaliplatin- or bortezomib-induced neuropathic pain were unnecessary as the IB-MECA doses matched those used with paclitaxel. None of the drugs tested affected body weight, and all animals gained weight to the same extent over the course of the experiment (not shown). The potent antitumor effects of A₃AR agonists are well documented (47). The effects of IB-MECA on the antitumor activity of paclitaxel in human breast cancer cells (SKBR3; ref. 37), oxaliplatin in human colon cancer cells (SW480; ref. 38), or bortezomib in human multiple myeloma cells (RPMI 8226 ref. 39) were assessed using an MTT assay adapted from a previously described assay (40, 41). At a dose yielding <20% decrease in cell survival when used alone, IB-MECA (10 nM) did not diminish the antitumor effects of paclitaxel, oxaliplatin, or bortezomib on human breast, colon, and multiple myeloma cancer cells (Table 1). Higher doses of IB-MECA were not used because these doses had direct antitumor effects on all 3 cell lines tested and so could interact positively with antitumor effects of the chemotherapeutic to provide benefit.

Table 1.

IB-MECA does not interfere with the chemotherapeutic antitumor activity of 3 anticancer agents

Cell line and treatment	LC50	n	P
SKBR3 breast cancer cells
Paclitaxel (10 nM)	7.0 nM	5	1.0
Paclitaxel + IB-MECA (10 nM)	7.1 nM	5
SW480 colon cancer cells
Oxaliplatin (10 nM)	3.8 μM		0.89
Oxaliplatin + IB-MECA (10 nM)	3.2 μM	6
RPMI 8226 multiple myeloma cells
Bortezomib (10 nM)	27 nM	5	0.25
Bortezomib + IB-MECA (10 nM)	29 nM	5

DISCUSSION

Despite extensive research efforts, chronic neuropathic remains a large unmet medical need, and novel therapies are needed. Our results demonstrate for the first time that A₃AR agonists are viable as standalone therapeutics in the management of chronic neuropathic pain of at least 2 distinct etiologies. These results are anticipated to provide the pharmacological rationale for a “proof-of-concept” for using selective A₃AR agonists as a new approach in chronic neuropathic pain. From a translational perspective, this could conceivably lead to a fast-track investigation of A₃AR agonists (IB-MECA or Cl-IB-MECA) that are already in clinical trials for other indications (8, 17).

In a well-characterized model of chronic neuropathic pain resulting from constriction of the sciatic nerve in mice (29), A₃AR agonists were found to be potent and efficacious standalone agents able to reverse established neuropathic pain. Moreover, when used at a dose that alone had no effect, an A₃AR agonist significantly enhanced the analgesic effects of currently used analgesics: morphine, gabapentin, and amitriptyline. Opiate/narcotic analgesics, typified by morphine, are the most effective treatments for acute and chronic severe pain. However, their clinical utility is often hampered by the development of analgesic tolerance, which requires escalating doses to achieve equivalent pain relief (48). This complex pathophysiological cycle represents a critical barrier to the quality of life of these patients due to the resulting oversedation, reduced physical activity, constipation, respiratory depression, high potential for addiction, and other side effects (48). Gabapentin, an anticonvulsant that attenuates glutamatergic neurotransmission by binding to α2δ subunits of voltage-dependent Ca²⁺ channels, and amitriptyline, a dual serotonin and norepinephrine reuptake inhibitor, are used in chronic neuropathic pain states but display limited efficacy and side effects (4). If translated clinically, these results may provide an opportunity to increase the efficacy of current analgesics at lower doses, thereby reducing side-effect profiles.

Extending this concept to other neuropathic pain forms, we examined A₃AR agonists in models of chemotherapy-induced neuropathic pain. Chronic neuropathic pain accompanying CIPN greatly reduces the success of chemotherapeutics by limiting doses and decreasing quality of life by imparting psychological distress, fatigue, sleep disorders, cognitive deficit, and diminished activity (2). We now demonstrate that A₃AR agonism is a viable strategy to block neuropathic pain in rats from widely used chemotherapeutic agents having distinct antineoplastic mechanisms of action, namely, paclitaxel for treatment of breast and ovarian cancer, oxaliplatin for metastatic colon cancer and other gastrointestinal tumors, and bortezomib for multiple myelomas (2) without reducing antitumoral effects. The findings that neuropathic pain caused by distinct chemotherapeutics did not emerge for several days after discontinuing the A₃AR agonist treatment suggest that A₃AR agonists may prevent underlying causative mechanisms necessary for neuropathic pain development rather than simply providing a transient decrease in enhanced nociceptive processing, as would occur with other analgesics (e.g., gabapentin, opiates). Thus, A₃AR-targeted approaches could potentially be disease-modifying in nature; in-depth studies to better define this possibility are warranted.

Clinically, patients undergoing chemotherapy could potentially benefit from A₃AR agonist-facilitated use of significantly increased doses of each chemotherapeutic agent for maximal tumor regression. The effect on quality of life would be substantial, and more lives may be saved using chemotherapy. Patients currently not qualifying for treatment (or continued treatment) with drugs like paclitaxel due to impending (or worsening) neuropathy would instead benefit from full-strength antitumor dosages, now tolerable with a coadministered “neuroprotective” agent. Additional clinically relevant observations with A₃AR agonists include direct antitumor effects against various tumors (both in vitro and in vivo), a reduction of chemotherapy-induced myelotoxicity and encouraging preliminary clinical results (47). Taken together with their ability to prevent chemotherapy-induced neuropathic pain, these distinct properties of A₃AR agonists may combine to produce especially useful therapy.

The biochemical basis underlying the protective and beneficial effects of A₃AR agonists in chronic neuropathic pain is unknown. Mitotoxicity in peripheral sensory afferents and neuroinflammation in spinal cord contribute to the development of chronic neuropathic pain of distinct etiologies (49–51). It is noteworthy that mitoprotection is important in the cardioprotective role of A₃AR agonists, which are also potent anti-inflammatory agents (47). We are currently investigating the hypothesis that mitoprotective effects and/or attenuation of neuroinflammatory processes in spinal cord mediate the underlying protective A₃AR role. In addition, activation of the A₃AR can limit neuronal excitation in some situations, and A₃AR agonists are neuroprotective (5, 47). For example, adenosine acting at a cerebral A₃AR is neuroprotective in stroke models (52). A₃AR agonists have been shown to limit ischemic damage (53) and confer prolonged protection from the effects of middle cerebral artery ligation (54), while A₃ receptor-knockout mice are more susceptible to neurodegeneration after hypoxic challenge (55). In addition, activation of the A₃AR contributes to the inhibition of cortical synaptic potentials during hypoxia (56) and inhibits synaptic transmission in the enteric nervous system (57). More recently, several A₃AR agonists were reported to also attenuate glutamate and NMDA-mediated Ca²⁺ rise in neurons in vitro and thus neuronal excitability (22, 58), suggesting that the A₃AR impacts glutamatergic signaling. Clearly, modulation of neuronal excitability is a likely mechanism whereby A₃AR agonists might exert beneficial effects in chronic neuropathic pain settings. Clinical trials with A₃AR agonists should be considered and explored to advance the treatment of chronic pain, thus alleviating human suffering and impacting the quality of life of patients.