Spinal and peripheral analgesic effects of the CB2 cannabinoid receptor agonist AM1241 in two models of bone cancer-induced pain

Abstract

Background and purpose: The activation of CB(2) receptors induces analgesia in experimental models of chronic pain. The present experiments were designed to study whether the activation of peripheral or spinal CB(2) receptors relieves thermal hyperalgesia and mechanical allodynia in two models of bone cancer pain.

Experimental approach: NCTC 2472 osteosarcoma or B16-F10 melanoma cells were intratibially inoculated to C3H/He and C57BL/6 mice. Thermal hyperalgesia was assessed by the unilateral hot plate test and mechanical allodynia by the von Frey test. AM1241 (CB(2) receptor agonist), AM251 (CB(1) receptor antagonist), SR144528 (CB(2) receptor antagonist) and naloxone were used. CB(2) receptor expression was measured by Western blot.

Key results: AM1241 (0.3-10 mg.kg(-1)) abolished thermal hyperalgesia and mechanical allodynia in both tumour models. The antihyperalgesic effect was antagonized by subcutaneous, intrathecal or peri-tumour administration of SR144528. In contrast, the antiallodynic effect was inhibited by systemic or intrathecal, but not peri-tumour, injection of SR144528. The effects of AM1241 were unchanged by AM251 but were prevented by naloxone. No change in CB(2) receptor expression was found in spinal cord or dorsal root ganglia.

Conclusions and implications: Spinal CB(2) receptors are involved in the antiallodynic effect induced by AM1241 in two neoplastic models while peripheral and spinal receptors participate in the antihyperalgesic effects. Both effects were mediated by endogenous opiates. The use of drugs that activate CB(2) receptors could be a useful strategy to counteract bone cancer-induced pain symptoms.

Figure 1

Antihyperalgesic effect induced by the systemic administration of AM1241 (0.3–3 mg·kg⁻¹, i.p.) or its corresponding solvent (SOL) in mice inoculated with NCTC 2472 osteosarcoma (A) or B16-F10 melanoma cells (B) measured by the unilateral hot plate test. Effect of s.c. AM251 (5 mg·kg⁻¹) or SR144528 (1 mg·kg⁻¹) on the antihyperalgesic effect induced by the i.p. administration of AM1241 (3 mg·kg⁻¹) in mice inoculated with NCTC 2472 osteosarcoma (C) or B16-F10 melanoma cells (D). Each bar represents the mean ± SEM (n = 6–8 mice). **P < 0.01 compared with the right paw of the solvent-treated group, Dunnett's t-test (A, B) or Newman-Keuls test (C, D). ••P < 0.01 compared with the corresponding left paw, Newman-Keuls test. UHP, unilateral hot plate.

Figure 2

(A–B) Effect of the intrathecal administration of SR144528 (5 µg) on the antihyperalgesic effect induced by AM1241 (3 mg·kg⁻¹; i.p.) measured by the unilateral hot plate test in mice inoculated with NCTC 2472 osteosarcoma (A) or B16-F10 melanoma (B) cells. (C–D) Effect of the peri-tumour administration of SR144528 (10 µg) on the antihyperalgesic effect induced by AM1241 (3 mg·kg⁻¹; i.p.) in mice inoculated with NCTC 2472 osteosarcoma (C) or B16-F10 melanoma cells (D). Each bar represents the mean ± SEM (n = 7–9). **P < 0.01 compared with the right paw of the solvent-treated group, ••P < 0.01 compared with the corresponding left paw, Newman-Keuls test. UHP, unilateral hot plate.

Figure 3

Antiallodynic effect induced by the systemic administration of AM1241 (1–10 mg·kg⁻¹, i.p.) or its corresponding solvent (SOL) in mice inoculated with NCTC 2472 osteosarcoma (A) or B16-F10 melanoma cells (B) measured in the von Frey test. Effect of s.c. AM251 (5 mg·kg⁻¹) or SR144528 (1 mg·kg⁻¹) on the antiallodynic effect induced by the i.p. administration of AM1241 (10 mg·kg⁻¹) in mice inoculated with NCTC 2472 osteosarcoma (C) or B16-F10 melanoma cells (D). Each bar represents the mean ± SEM (n = 8–10). *P < 0.05, **P < 0.01 compared with the right or left paw of the solvent-treated group, •P < 0.05, ••P < 0.05 compared with its corresponding left paw, Mann–Whitney U-test.

Figure 4

(A–B) Effect of the intrathecal administration of SR144528 (5 µg) on the antiallodynic effect induced by AM1241 (10 mg·kg⁻¹; i.p.) measured in the von Frey test in mice inoculated with NCTC 2472 osteosarcoma (A) or B16-F10 melanoma (B) cells. (C–D) Effect of the peri-tumour administration of SR144528 (10 µg) on the antiallodynic effect induced by AM1241 (10 mg·kg⁻¹; i.p.) in mice inoculated with NCTC 2472 osteosarcoma (C) or B16-F10 melanoma cells (D). Each bar represents the mean ± SEM (n = 6–9). **P < 0.01 compared with the right paw of the solvent-treated group, ••P < 0.01 compared with the corresponding left paw, Mann–Whitney U-test.

Figure 5

(A) Antihyperalgesic effect induced by the intrathecal administration of AM1241 (0.03–0.3 µg) or its corresponding solvent (SOL) measured by the unilateral hot plate test in mice intratibially inoculated with NCTC 2472 osteosarcoma cells. Each bar represents the mean ± SEM (n = 6). **P < 0.01 compared with the right paw of the solvent-treated group, P < 0.01 compared with the left paw in the solvent-treated group, Dunnett's t-test. (B) Antiallodynic effect induced by the intrathecal administration of AM1241 (0.1–1 µg) or its corresponding solvent (SOL) measured by the von Frey test in mice intratibially inoculated with NCTC 2472 osteosarcoma cells. Each bar represents the mean ± SEM (n = 7–9). *P < 0.05, **P < 0.01 compared with the right paw of the solvent-treated group, ••P < 0.05 compared with its corresponding left paw, Mann–Whitney U-test. UHP, unilateral hot plate.

Figure 6

(A–B) Effect of the systemic administration of naloxone (3 mg·kg⁻¹; s.c.) on the antihyperalgesic effect induced by AM1241 (3 mg·kg⁻¹; i.p.) measured in the unilateral hot plate test in mice inoculated with NCTC 2472 osteosarcoma (A) or B16-F10 melanoma (B) cells. Each bar represents the mean ± SEM (n = 5). **P < 0.01 compared with the right paw of the solvent-treated group, P < 0.01 compared with the corresponding left paw, Newman-Keuls test. (C–D) Effect of the systemic administration of naloxone (3 mg·kg⁻¹; s.c.) on the antiallodynic effect induced by AM1241 (10 mg·kg⁻¹; i.p.) in mice inoculated with NCTC 2472 osteosarcoma (C) or B16-F10 melanoma cells (D). Each bar represents the mean ± SEM (n = 5). **P < 0.01 compared with the right paw of the solvent-treated group, ••P < 0.01 compared with the corresponding left paw, Mann–Whitney U-test. UHP, unilateral hot plate.

Figure 7

(A) Representative lanes of Western blots for CB₂ receptors, from plantar skin tissue (SKIN), Chinese hamster ovarian cells (CHO), lumbar spinal cord (SC) and lumbar spinal cord incubated with anti-CB₂ receptor and blocking peptide. Molecular weight markers are indicated at the left side of the corresponding lane. (B, C) CB₂ receptor expression measured by Western blot in L2-L6 lumbar segments of the spinal cord or L4-L6 DRG of mice, 2 or 4 weeks after implantation with NCTC 2472 osteosarcoma cells (B) or 1 week after implantation with B16-F10 melanoma cells (C). CB₂ receptor expression was measured in spinal cords of mice inoculated with live or killed tumour cells and in DRG contralateral or ipsilateral to the presence of tumour cells. The fold change in CB₂ receptor protein was estimated by using its corresponding GAPDH as endogenous control. Data are the mean ± SEM (n = 4). DRG, dorsal root ganglia; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.