Antinociceptive effects of MSVIII-19, a functional antagonist of the GluK1 kainate receptor

Abstract

The ionotropic glutamate receptor subunit, GluK1 (GluR5), is expressed in many regions of the nervous system related to sensory transmission. Recently, a selective ligand for the GluK1 receptor, MSVIII-19 (8,9-dideoxy-neodysiherbaine), was synthesized as a derivative of dysiherbaine, a toxin isolated from the marine sponge Lendenfeldia chondrodes. MSVIII-19 potently desensitizes GluK1 receptors without channel activation, rendering it useful as a functional antagonist. Given the high selectivity for GluK1 and the proposed role for this glutamate receptor in nociception, we sought to test the analgesic potential of MSVIII-19 in a series of models of inflammatory, neuropathic, and visceral pain in mice. MSVIII-19 delivered intrathecally dose-dependently reduced formalin-induced spontaneous behaviors and reduced thermal hypersensitivity 3 hours after formalin injection and 24 hours after complete Freund's adjuvant-induced inflammation, but had no effect on mechanical sensitivity in the same models. Intrathecal MSVIII-19 significantly reduced both thermal hyperalgesia and mechanical hypersensitivity in the chronic constriction injury model of neuropathic pain, but had no effect in the acetic acid model of visceral pain. Peripheral administration of MSVIII-19 had no analgesic efficacy in any of these models. Finally, intrathecal MSVIII-19 did not alter responses in Tail-flick tests or performance on the accelerating RotaRod. These data suggest that spinal administration of MSVIII-19 reverses hypersensitivity in several models of pain in mice, supporting the clinical potential of GluK1 antagonists for the management of pain.

Figure 1

Representative neuronal kainate receptor currents evoked by glutamate in the absence and presence of 10 µM MSVIII-19 are shown in the top panel. Also shown is a representative trace showing the lack of any detectable currents induced by 30 µM MSVIII-19 alone. Glutamate (10 mM) was applied for 100 ms to a small-diameter, acutely isolated mouse DRG neuron held in voltage clamp at −70 mV. MSVIII-19 was applied for at least one minute prior to the test glutamate application. Data in the inhibition-response curve were fit to a single site logistic equation with a variable slope to derive an IC₅₀ of 179 nM. The Hill slope was relatively shallow (0.55), as was the case with inhibition of recombinant GluK1 and GluK1/GluK5 receptors. Data and logistic fits for the latter are shown for comparative purposes and were reported in our previous publication[22].

Figure 2. Spinally but not peripherally administered MSVIII-19 is analgesic in the formalin model

A, Intrathecal injection of MSVIII-19 dose-dependently reduced spontaneous pain-related behaviors in both the first and second phases of the formalin test. (0.5 nmol, n=10). Vehicle n=9; 0.1 nmol, n=11; 0.5 nmol, n=7; 1 nmol, n=11; 2 nmol, n=9. At the 5 minute time point, MSVIII-19 significantly reduced responses at the 0.5 (P<0.05), 1.0 (P<0.01), and 2.0 nmol (P<0.001) doses (ANOVA with Bonferroni post-hoc test). At the 20 and 25 minute timepoints, all dose of MSVIII-19 significantly reduced responses (P<0.001). B). Summed data for the first phase (0–10min) and second phase (15–60min) showing significant dose-dependent inhibition of both the first (p<0.01) and second (p<0.001) phases by intrathecally administered MSVIII-19. C) Peripheral (intraplantar) administration of MSVIII-19 (0.5nmol) had no effect on spontaneous pain-related behaviors in the formalin test.

Figure 3. Spinally administered MSVIII-19 selectively reduces thermal hypersensitivity in the formalin model

Thermal or mechanical baselines were taken, followed by paw injection with 5% formalin. Three hours after formalin injection, mice were tested for thermal (A) or mechanical (B) sensitivity. The mice then received an intrathecal injection of either vehicle or MSVIII-19 (0.5 nmol), and were then tested for post-treatment thermal or mechanical sensitivity. Formalin induced significant thermal and mechanical hypersensitivity relative to pre-formalin baselines (P < 0.01; ANOVA with Tukey’s post-hoc test). Thermal hypersensitivity was significantly reduced by MSVIII-19 relative to vehicle, whereas mechanical hypersensitivity was not affected. *** p< 0.001

Figure 4. Spinally but not peripherally administered MSVIII-19 selectively reduces thermal hypersensitivity in the CFA model of inflammatory pain

After measurement of thermal and mechanical baselines, mice received a paw injection of CFA. Twenty four hours after CFA injection, mice were tested for thermal (A) or mechanical (B) sensitivity. The mice then received an intrathecal injection of either vehicle or MSVIII-19 (0.5 nmol), and were then tested for posttreatment thermal or mechanical sensitivity. CFA induced significant thermal and mechanical hypersensitivity relative to pre-CFA baselines (P < 0.01; ANOVA with Tukey’s post-hoc test). Thermal hypersensitivity was significantly reduced by MSVIII-19 relative to vehicle, whereas mechanical hypersensitivity was not affected. *** p< 0.001. C) and D) show that intraplantar administration of MSVIII-19 (2 nmol) has no effect on thermal or mechanical sensitivity in the CFA model.

Figure 5. Spinally but not peripherally administered MSVIII-19 reduces thermal and mechanical hypersensitivity in the CCI model of neuropathic pain

After measurement of thermal and mechanical baselines, mice underwent surgery to induce chronic constriction injury of the sciatic nerve. Two weeks after CCI surgery, mice were tested for thermal (A) or mechanical (B) sensitivity. CCI induced both thermal and mechanical hypersensitivity (P < 0.01; ANOVA with Tukey’s post-hoc test). The mice then received an intrathecal injection of either vehicle or MSVIII-19 (0.5 nmol), and were then tested for post-treatment thermal or mechanical sensitivity. Both thermal and mechanical hypersensitivity were significantly reduced by MSVIII-19 relative to vehicle. ** p < 0.01; *** p< 0.001. C) and D) show that intraplantar administration of MSVIII-19 (2 nmol) has no effect on thermal or mechanical sensitivity in the CCI model of neuropathic pain.

Figure 6. MSVIII-19 has no effect in the acetic acid model of visceral pain

Following intrathecal injection of either vehicle or MSVIII-19 (0.5 nmol), mice received an intraperitoneal injection of 0.6% acetic acid, and abdominal writhes were counted for a period of 20 min, and are reported in 5 min bins. There were no significant differences between MSVIII-19 and vehicle-injected groups.

Figure 7. MSVIII-19 has no effect in the tail-flick assay of thermal pain and has no effect on locomotor behavior in the accelerating rotarod

A) Mice were first tested for baseline tailflick latency. (Pre-inj). They then received an intrathecal injection of MSVIII-19 (0.5nmol), and tailflick latency was then measured again (Post-inj). There was no difference between MSVIII-19 (0.5 nmol) and vehicle-injected groups. B) The effects of intrathecial MSVIII-19 (0.5–2.0nmol) were compared to vehicle-treated mice in locomotor performance on the accelerating rotarod. Mice were pretreated with an intrathecal injection of vehicle (n=23) or MSVIII-19 (0.5 nmol, n=9; 1.0 nmol, n=9; 2.0 nmol, n=14). There were no significant effects of MSVIII-19 as compared to vehicle.