C-terminal hydrazide modification changes the spinal antinociceptive profiles of endomorphins in mice

Previously, we have demonstrated that endomorphins (EMs) analogs with C-terminal hydrazide modification retained the μ-opioid receptor affinity and selectivity, and exhibited potent antinociception after intracerebroventricular (i.c.v.) administration. In the present study, we extended our studies to evaluate the antinociceptive profiles of EMs and their analogs EM-1-NHNH₂, EM-2-NHNH₂ given spinally in the radiant heat paw withdrawal test. Following intrathecal (i.t.) administration, EM-1, EM-2, EM-1-NHNH₂ and EM-2-NHNH₂ dose-dependently increased the latency for paw withdrawal response. EM-1-NHNH₂ displayed the highest antinociceptive effects, with the ED₅₀ values being 1.63 nmol, more potent than the parent EM-1 (1.96 nmol), but with no significant difference. By contrast, the analgesic activities of EM-2 and its analog EM-2-NHNH₂ were almost equivalent (P>0.05). Naloxone and β-funaltrexamine (β-FNA) almost completely attenuated the antinociceptive effects of EMs and their analogs EM-1-NHNH₂, EM-2-NHNH₂ (10 nmol, i.t.), indicating the involvement of μ-opioid receptors. Notably, the antinociception of EM-1 was not significantly antagonized by naloxonazine, a selective μ₁-opioid receptor antagonist, but partially reversed the effects of EM-2, suggesting that EM-1 and EM-2 may produce antinociception through distinct μ₁- and μ₂-opioid receptor subtypes. Moreover, naloxonazine didn't significantly block the antinociceptive effects of EM-1-NHNH₂ and EM-2-NHNH₂, and nor-BNI, the κ-opioid receptor antagonist, attenuated the analgesic effects of EM-2, but not EM-1, EM-1-NHNH₂ or EM-2-NHNH₂. These results indicated that C-terminal amide to hydrazide conversion changed the antinociceptive opioid mechanisms of EM-2 but not EM-1 at the spinal level. Herein, the acute antinociceptive tolerance were further determined and compared. EM-1-NHNH₂ and EM-2-NHNH₂ shifted the dose-response curve rightward by only 2.8 and 1.5-fold as determined by tolerance ratio, whereas EM-1 and EM-2 by 3.4 and 4.6-fold, respectively, indicating substantially reduced antinociceptive tolerance. The present study demonstrated that C-terminal hydrazide modification changes the spinal antinociceptive profiles of EMs.