Comparative in Vivo Investigation of Intrathecal and Intracerebroventricular Administration with Melanocortin Ligands MTII and AGRP into Mice

Central administration of melanocortin ligands has been used as a critical technique to study energy homeostasis. While intracerebroventricular (ICV) injection is the most commonly used method during these investigations, intrathecal (IT) injection can be equally efficacious for the central delivery of ligands. Importantly, intrathecal administration can optimize exploration of melanocortin receptors in the spinal cord. Herein, we investigate comparative IT and ICV administration of two melanocortin ligands, the synthetic MTII (Ac-Nle-c­[Asp-His-DPhe-Arg-Trp-Lys]-NH₂) MC4R agonist and agouti-related peptide [AGRP­(87-132)] MC4R inverse agonist/antagonist, on the same batch of age-matched mice in TSE metabolic cages undergoing a nocturnal satiated paradigm. To our knowledge, this is the first study to test how central administration of these ligands directly to the spinal cord affects energy homeostasis. Results showed, as expected, that MTII IT administration caused a decrease in food and water intake and an overall negative energy balance without affecting activity. As anticipated, IT administration of AGRP caused weight gain, increase of food/water intake, and increase respiratory exchange ratio (RER). Unexpectantly, the prolonged activity of AGRP was notably shorter (2 days) compared to mice given ICV injections of the same concentrations in previous studies (7 days or more).− It appears that IT administration results in a more sensitive response that may be a good approach for testing synthetic compound potency values ranging in nanomolar to high micromolar in vitro EC₅₀ values. Indeed, our investigation reveals that the spine influences a different melanocortin response compared to the brain for the AGRP ligand. This study indicates that IT administration can be a useful technique for future metabolic studies using melanocortin ligands and highlights the importance of exploring the role of melanocortin receptors in the spinal cord

Clinically Employed Opioid Analgesics Produce Antinociception via μ‑δ Opioid Receptor Heteromers in Rhesus Monkeys

Morphine and related drugs are widely employed as analgesics despite the side effects associated with their use. Although morphine is thought to mediate analgesia through mu opioid receptors, delta opioid receptors have been implicated in mediating some side effects such as tolerance and dependence. Here we present evidence in rhesus monkeys that morphine, fentanyl, and possibly methadone selectively activate mu-delta heteromers to produce antinociception that is potently antagonized by the delta opioid receptor antagonist, naltrindole (NTI). Studies with HEK293 cells expressing mu-delta heteromeric opioid receptors exhibit a similar antagonism profile of receptor activation in the presence of NTI. In mice, morphine was potently inhibited by naltrindole when administered intrathecally, but not intracerebroventricularly, suggesting the possible involvement of mu-delta heteromers in the spinal cord of rodents. Taken together, these results strongly suggest that, in primates, mu-delta heteromers are allosterically coupled and mediate the antinociceptive effects of three clinically employed opioid analgesics that have been traditionally viewed as mu-selective. Given the known involvement of delta receptors in morphine tolerance and dependence, our results implicate mu-delta heteromers in mediating both antinociception and these side effects in primates. These results open the door for further investigation in humans

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR₅)

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR₅ heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR₅ antagonist pharmacophores linked through homologous spacers (14–24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (<b>MCC22</b>; <b>3e</b>) with a 22-atom spacer exhibited profound antinociception (i.t. ED₅₀ = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, <b>MCC22</b> was ∼3500× more potent than a mixture of mu agonist and CCR₅ antagonist monovalent ligands. These data strongly suggest that <b>MCC22</b> acts by bridging the protomers of a MOR-CCR₅ heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR₅ heteromer as a novel target for the treatment of chronic pain

Inhibition of Inflammatory and Neuropathic Pain by Targeting a Mu Opioid Receptor/Chemokine Receptor5 Heteromer (MOR-CCR₅)

Chemokine release promotes cross-talk between opioid and chemokine receptors that in part leads to reduced efficacy of morphine in the treatment of chronic pain. On the basis of the possibility that a MOR-CCR₅ heteromer is involved in such cross-talk, we have synthesized bivalent ligands (MCC series) that contain mu opioid agonist and CCR₅ antagonist pharmacophores linked through homologous spacers (14–24 atoms). When tested on lipopolysaccharide-inflamed mice, a member of the series (<b>MCC22</b>; <b>3e</b>) with a 22-atom spacer exhibited profound antinociception (i.t. ED₅₀ = 0.0146 pmol/mouse) that was 2000× greater than morphine. Moreover, <b>MCC22</b> was ∼3500× more potent than a mixture of mu agonist and CCR₅ antagonist monovalent ligands. These data strongly suggest that <b>MCC22</b> acts by bridging the protomers of a MOR-CCR₅ heteromer having a TM5,6 interface. Molecular simulation studies are consistent with such bridging. This study supports the MOR-CCR₅ heteromer as a novel target for the treatment of chronic pain

Putative Kappa Opioid Heteromers As Targets for Developing Analgesics Free of Adverse Effects

It is now generally recognized that upon activation by an agonist, β-arrestin associates with G protein-coupled receptors and acts as a scaffold in creating a diverse signaling network that could lead to adverse effects. As an approach to reducing side effects associated with κ opioid agonists, a series of β-naltrexamides <b>3</b>–<b>10</b> was synthesized in an effort to selectively target putative κ opioid heteromers without recruiting β-arrestin upon activation. The most potent derivative <b>3</b> (INTA) strongly activated KOR-DOR and KOR-MOR heteromers in HEK293 cells. In vivo studies revealed <b>3</b> to produce potent antinociception, which, when taken together with antagonism data, was consistent with the activation of both heteromers. <b>3</b> was devoid of tolerance, dependence, and showed no aversive effect in the conditioned place preference assay. As immunofluorescence studies indicated no recruitment of β-arrestin2 to membranes in coexpressed KOR-DOR cells, this study suggests that targeting of specific putative heteromers has the potential to identify leads for analgesics devoid of adverse effects