The α7 nicotinic receptor dual allosteric agonist and positive allosteric modulator GAT107 reverses nociception in mouse models of inflammatory and neuropathic pain

Abstract

Background and purpose: Orthosteric agonists and positive allosteric modulators (PAMs) of the α7 nicotinic ACh receptor (nAChR) represent novel therapeutic approaches for pain modulation. Moreover, compounds with dual function as allosteric agonists and PAMs, known as ago-PAMs, add further regulation of receptor function.

Experimental approach: Initial studies examined the α7 ago-PAM, GAT107, in the formalin, complete Freund's adjuvant (CFA), LPS inflammatory pain models, the chronic constriction injury neuropathic pain model and the tail flick and hot plate acute thermal nociceptive assays. Additional studies examined the locus of action of GAT107 and immunohistochemical markers in the dorsal horn of the spinal cord in the CFA model.

Key results: Complementary pharmacological and genetic approaches confirmed that the dose-dependent antinociceptive effects of GAT107 were mediated through α7 nAChR. However, GAT107 was inactive in the tail flick and hot plate assays. In addition, GAT107 blocked conditioned place aversion elicited by acetic acid injection. Furthermore, intrathecal, but not intraplantar, injections of GAT107 reversed nociception in the CFA model, suggesting a spinal component of action. Immunohistochemical evaluation revealed an increase in the expression of astrocyte-specific glial fibrillary acidic protein and phosphorylated p38MAPK within the spinal cords of mice treated with CFA, which was attenuated by intrathecal GAT107 treatment. Importantly, GAT107 did not elicit motor impairment and continued to produce antinociceptive effects after subchronic administration in both phases of the formalin test.

Conclusions and implications: Collectively, these results provide the first proof of principle that α7 ago-PAMs represent an effective pharmacological strategy for treating inflammatory and neuropathic pain.

Figure 1

The antinociceptive and anti‐oedema effects of GAT107 in the formalin test. (A) The effect after i.p. administration of GAT107 (0.1, 1, 3 and 10 mg·kg⁻¹) on formalin‐induced pain behaviour in the mouse. Mice were treated with i.p. GAT107 15 min prior to formalin (2.5%, 20 μL) injection into the plantar region of the right hind paw. The cumulative pain response of time of licking was measured during the period of 0–5 (first phase) and 20–45 min (second phase). (B) Anti‐oedema effect of GAT107 (1, 3 and 10 mg·kg⁻¹, i.p.) in the formalin test, as measured by the difference in the ipsilateral paw diameter before and after injection (ΔPD), 1 h after i.pl. injection of 2.5% formalin. (C) Blockade of the antinociceptive effect of GAT107 in the second phase of the formalin test by the α7 antagonist MLA citrate and opioid antagonist naloxone. MLA (10 mg·kg⁻¹, s.c.) and naloxone (2 mg·kg⁻¹, s.c.) were given 15 min before GAT107 (10 mg·kg⁻¹, i.p.) or vehicle (Veh). (D) Antinociceptive effects of GAT107 (10 mg·kg⁻¹, i.p.) in the second phase of the formalin test in the α7 WT and KO mice. Data are given as the mean ± SEM of six animals for each group in A, C and D. In (B) group sizes as follows: Veh (n = 6), GAT107 1 mg·kg⁻¹ (n = 7), 3 mg·kg⁻¹ (n = 7) and 10 mg·kg⁻¹ (n = 7). The differences in experimental numbers within these studies reflect an odd number of animals evenly distributed in the experimental design. *P < 0.05, significantly different from its Veh group; #P < 0.05, significantly different from its corresponding control group (GAT107‐treated group or WT group).

Figure 2

The antinociceptive effects of repeated administration GAT107 in the formalin test. The effect of GAT107 after subchronic i.p. administration of the drug on formalin‐induced pain behaviour in the mouse. Mice were treated with GAT107 (1 and 10 mg·kg⁻¹, i.p.) or vehicle (Veh) for 6 days twice daily with 8 h apart and were challenged with GAT107 (1 and 10 mg·kg⁻¹, i.p.) on day 7 and effects evaluated in formalin (2.5%) test. A Veh control group, in which mice were exposed to 7 days of vehicle, was also included. The cumulative pain response of time of licking was measured during the period of 0–5 min first phase (A) and 20–40 min second phase (B). Data are given as the mean ± SEM of six animals for each group. *P < 0.05, significantly different from its Veh group. #P < 0.05, significantly different from acute group.

Figure 3

The anti‐allodynic and antihyperalgesic effects of systemic GAT107 in the CFA‐induced chronic inflammatory pain model. The (A) anti‐allodynic and (B) antihyperalgesic effects of after i.p. administration of various doses of GAT107 (1, 3 and 10 mg·kg⁻¹). The mechanical paw withdrawal thresholds and differences in paw withdrawal latencies (ΔPWL = contralateral − ipsilateral hind paw latencies) were determined 3 days after i.pl. injection of CFA (50%). To determine the blockade of the anti‐allodynic effect of GAT107 by the α7 antagonist MLA citrate, MLA was administered (C) systemically (10 mg·kg⁻¹, s.c.) and (D) spinal (10 μg in 5 μL, i.t.) 15 min before GAT107 (10 mg·kg⁻¹, i.p.) injection. (E) Blockade of the antihyperalgesic effects of GAT107 by MLA (10 mg·kg⁻¹, s.c.) in the Hargreaves test. Data are given as the mean ± SEM of eight animals for each group in A and B and six animals for each group in C, D and E. *P < 0.05, significantly different from its vehicle (Veh) group. #P < 0.05, significantly different from its corresponding control group (GAT107‐treated group). BL, baseline.

Figure 4

The anti‐allodynic and anti‐inflammatory effects of spinal and peripheral GAT107 in the CFA‐induced chronic inflammatory pain model. A head‐to‐head comparison of anti‐allodynic effects of (A) spinal (0.3 and 3 μg per mouse, i.t.) and (C) peripheral (3 and 9 μg per mouse, i.pl.) GAT107 in CFA‐induced inflammatory pain. The anti‐inflammatory effect of (B) spinal and (D) peripheral injection of GAT107, measured by the difference in the ipsilateral paw diameter before and after CFA injection (ΔPD), was assessed 1 h after GAT107 injection. Data are given as the mean ± SEM of six animals for each group in A and B. (C and D) Group sizes as follows: vehicle (n = 5), GAT107 3 μg (n = 6) and 9 μg (n = 6). The differences in experimental numbers within these studies reflect an odd number of animals evenly distributed in the experimental design. *P < 0.05, significantly different from its vehicle group. BL, baseline.

Figure 5

Immunoreactivity of GFAP and p‐p38MAPK following GAT107‐induced reversal of allodynia. (A) Prior to CFA injection, all groups exhibited similar baseline (BL) thresholds. CFA produced significant allodynia. Behavioural responses following i.t. GAT107 (3 μg) produced maximal reversal of allodynia. At peak reversal, animals were killed, and spinal tissue was collected. (B) Compared with vehicle (Veh)‐injected mice given i.t. GAT107 or equivolume of Veh, CFA mice demonstrated a robust increase in dorsal horn GFAP immunoreactivity given i.t. Veh. Representative images at 20× magnification of GFAP fluorescent staining (green). (C) Compared with Veh‐injected mice given i.t. GAT107 or equivolume Veh, CFA mice produced a robust p‐p38MAPK increase in dorsal horn spinal cord tissues following i.t. Veh injection. GAT107 administered i.t. reversed CFA‐induced increases in p‐p38MAPK immunoreactivity. Representative images at 20× magnification of p‐p38MAPK fluorescent staining (red). In all images, the scale bar is equal to 50 μm. Data are given as the mean ± SEM of four animals for each group.

Figure 6

The anti‐allodynic effects of GAT107 in LPS‐induced inflammatory pain model. The anti‐allodynic effects after i.p. administration of various doses of GAT107 (1, 3 and 10 mg·kg⁻¹). The mechanical paw withdrawal thresholds were determined 24 h after i.pl. injection of LPS (2.5 μg in 20 μL). Data are given as the mean ± SEM of seven animals for each group. *P < 0.05, significantly different from its vehicle group. BL, baseline.

Figure 7

The anti‐allodynic effects of GAT107 in CCI‐induced neuropathic pain. The anti‐allodynic effects after i.p. injection of GAT107 (1, 3 and 10 mg·kg⁻¹) in (A) CCI and (B) sham mice were determined using the von Frey test. The antihyperalgesic effects of GAT107 (1, 3 and 10 mg·kg⁻¹) were tested 30 min after its injection in (C) CCI and (D) sham mice using the Hargreaves test. Data are given as the mean ± SEM of 10 animals for each group in (A) and six animals for each group in (B, C and D). *P < 0.05, significantly different from its vehicle group. BL, baseline.

Figure 8

Effects of GAT107 on acetic acid (AA)‐induced stretching and CPA. (A) i.p. injection of GAT107 (1, 3 and 10 mg·kg⁻¹) attenuated AA‐induced writhing behaviour. (B) GAT107 (1, 3 and 10 mg·kg⁻¹, i.p.) attenuated AA‐induced CPA. Data are given as the mean ± SEM of six animals for each group in (A). (B) Group sizes as follows: vehicle (Veh) and AA controls (n = 7) and others (n = 6). The differences in experimental numbers within these studies reflect an odd number of animals evenly distributed in the experimental design. *P < 0.05, compared with the Veh‐injected mice; #P < 0.05, compared with the AA‐injected mice.

Figure 9

Effects of GAT107 in (A) the tail flick and (B) hot plate tests. The antinociceptive effects of GAT107 (10 mg·kg⁻¹, i.p.) were measured at multiple time points (in min) after injection. Data are presented as %MPE ± SEM of six animals for each group.