We previously reported that a multifunctional opioid/neuropeptide FF receptor agonist, DN-9, achieved peripherally restricted analgesia with reduced side effects. To develop stable and orally bioavailable analogues of DN-9, eight lactam-bridged cyclic analogues of DN-9 between positions 2 and 5 were designed, synthesized, and biologically evaluated. In vitro cAMP assays revealed that these analogues, except 7, were multifunctional ligands that activated opioid and neuropeptide FF receptors. Analogue 1 exhibited improved potency for κ-opioid and NPFF 2 receptors. All analogues exhibited potent, long-lasting, and peripherally restricted antinociception in the tail-flick test without tolerance development after subcutaneous administration and produced oral analgesia. Oral administration of the optimized compound analogue 1 exhibited powerful, peripherally restricted antinociceptive effects in mouse models of acute, inflammatory, and neuropathic pain. Remarkably, orally administered analogue 1 had no significant side effects, such as tolerance, dependence, constipation, or respiratory depression, at effective analgesic doses.
Background
The nonapeptide DN‐9 functions as a multifunctional agonist to opioid and neuropeptide FF (NPFF) receptors and exhibits antinociceptive effects at the central and peripheral levels.
Methods
The effects of DN‐9 on small and colonic intestinal transit were evaluated using the upper gastrointestinal (GI) transit test and colonic bead expulsion assay, respectively. Opioid and NPFF receptor antagonists were used to investigate the mechanisms of DN‐9‐induced GI inhibition. Furthermore, the agonism of the DN‐9 analog [Phg9]‐DN‐9 to opioid and NPFF receptors was tested by the cAMP assay.
Key results
Intracerebroventricular administration of DN‐9 dose‐dependently slowed upper GI transit and colonic expulsion via mu‐ and kappa‐opioid receptors in the brain, independent of the delta‐opioid receptor. Similarly, intraperitoneal injection of DN‐9 dose‐dependently inhibited GI propulsion via the peripheral opioid receptors. DN‐9‐induced GI transit inhibitions were significantly aggravated by the NPFF receptor antagonist RF9. Moreover, the DN‐9 analog [Phg9]‐DN‐9, an agonist at mu‐, delta‐, and kappa‐opioid receptors but not NPFF receptors, inhibited GI more potently than DN‐9. In addition, intracerebroventricular NPFF significantly attenuated the central inhibitory effects induced by [Phg9]‐DN‐9 and morphine. However, central and peripheral injections of NPFF or RF9 almost had no significant effects on GI transit by itself.
Conclusion and Inferences
Intracerebroventricular and intraperitoneal administrations of DN‐9 inhibit GI transit via opioid receptors in mice by central and peripheral mechanisms, respectively. In addition, the NPFF agonism of DN‐9 possesses antiopioid effects on GI transit, which might explain the reduced constipation at the antinociceptive doses.
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