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 A(3) adenosine receptor (A(3)AR) 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 A(3)AR 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. A(3)AR agonist effects were blocked with A(3)AR antagonist MRS1523, but not with A(1)AR (DPCPX) or A(2A)AR (SCH-442416) antagonists. Our findings provide the scientific rationale and pharmacological basis for therapeutic development of A(3)AR agonists for chronic pain.

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).

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).

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).

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).

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).

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).

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).