Antinociceptive properties of losmapimod in two acute pain models in rats: behavioural analysis, immunohistochemistry, dose response, and comparison with usual analgesic drugs

Abstract

Background: The p38 protein is a ubiquitous mitogen-activated protein kinase involved in the proinflammatory signalling pathway and in the pain response after various noxious stimuli. Many p38 inhibitors have been developed and shown to provide effective analgesia in animal models. They are, however, mainly administered intrathecally or intravenously. Our study aimed to evaluate losmapimod, a novel oral p38 inhibitor, in two murine acute pain models.

Methods: Losmapimod (12 mg kg^-1) was compared with paracetamol, ketamine, and morphine using thermal and mechanical stimulation after carrageenan injection. A dose-effect study was also performed with this model. Behavioural testing was also performed in a plantar incision model to confirm the analgesic effect of losmapimod. Expression of activated p38 in neurones, microglia, and astrocytes was also investigated at 2, 15, and 24 h after carrageenan injection.

Results: Losmapimod was both antiallodynic and antihyperalgesic in the carrageenan pain model and provided an antinociceptive effect similar to that of morphine. The dose of 12 mg kg^-1 was shown to be the ED₇₈ and ED₆₄ after thermal and mechanical stimulation, respectively. After plantar incision, losmapimod provided a significant antinociceptive effect. No life-threatening side-effect was observed in the behavioural study. Losmapimod prevented neurone and microglial activation at 2 and 15 h after carrageenan injection, respectively, but no effect was found on astrocytic activation.

Conclusion: Losmapimod appears to be a promising drug in severe acute pain conditions. Losmapimod could also be helpful for postoperative pain control, as suggested by its effect after plantar incision.

Keywords: acute pain; antinociceptive drug; losmapimod; p38 inhibition; rodent.

Fig 1

Behavioural results after carrageenan injection. Nine rats were included in each group. Paw withdrawal threshold after von Frey hair stimulation (upper panel) and Hargreaves heat lamp application (lower panel) as a function of time. Data are expressed as median and inter-quartile range. To improve the clarity of the figure after von Frey hair application, values in ‘losmapimod+carrageenan’ and ‘paracetamol+carrageenan’ groups were slightly modified (from 245 to 235 kPa on D2 and D3, and from 245 to 220 kPa on D1 and D2, respectively). Briefly, carrageenan 3% 100 μl was administered subcutaneously in the left hind paw. Ketamine 20 mg kg⁻¹ and morphine 3 mg kg⁻¹ were also administered subcutaneously. Paracetamol 500 mg kg⁻¹ and losmapimod 12 mg kg⁻¹ were administered orally. P<0.01 for comparison between ‘losmapimod+carrageenan’ group and all other groups after Hargreaves test. After von Frey hair stimulation; P<0.01 for comparison between the ‘losmapimod+carrageenan’ group and all others except the ‘morphine+carrageenan’ group where the difference did not reach significance.

Fig 2

Dose–response curves of losmapimod-induced antinociceptive effect using the Hargreaves (left panel) and von Frey hair (right panel) tests. C represents the losmapimod concentration. Each point represents one dose (0, 2, 4, 12, and 50 mg kg⁻¹).

Fig 3

Behavioural results after plantar incision. Paw withdrawal threshold after von Frey hair stimulation (upper panel) and Hargreaves heat lamp application (lower panel) as a function of time. Data are expressed as median and inter-quartile range. Losmapimod was administered orally at 30 mg kg⁻¹. The difference was statistically significant (P<0.01) between the two groups, both after mechanical and thermal stimulation.

Fig 4

Phosphorylated p38 (p-p38) expression in left DRG neurones at 2 h in ‘carrageenan’ (a to c) and ‘losmapimod+carrageenan’ group (d to f). Neurones are stained in green (Neu-N marker) (a and d), phosphorylated p38 is stained in red (b and e), and blue corresponds to DAPI staining (unspecific marker of cell nucleus). Triple-stained cells were counted and appear in yellow (c and f). Examples of triple-stained cells are pointed with white arrows (c and f). The white scale bar corresponds to 50 μm. DAPI, 4,6-diamidino-2-phenylindol dihydrochloride.

Fig 5

Ipsilateral (left side of spinal cord) phosphorylated p38 expression in microglia (a to f) and astrocytes (g to l) at 15 h in ‘carrageenan’ (a to c and g to i) and ‘losmapimod+carrageenan’ (d to f and j to l) groups. Microglial and astrocytic cells are stained in green (CD11b and GFAP, respectively) (a, d, j, and l), phosphorylated p38 is stained in red (b, e, h, and k) and blue corresponds to DAPI staining (unspecific marker of cell nucleus). Triple-stained cells were counted and appear in yellow (c, f, i, and l). Examples of triple-stained cells are pointed with white arrows (c, f, i, and l). The white scale bar corresponds to 50 μm. DAPI, 4,6-diamidino-2-phenylindol dihydrochloride; GFAP, glial fibrillary acidic protein.