enkephalin--ala(2)-mephe(4)-gly(5)- and Nociceptive-Pain

The pivotal role of the stereocenter at the N-substituent of the 6,7-benzomorphan scaffold was investigated combining synthetic and pharmacological approaches. 2R- and 2S-diastereoisomers of the multitarget MOR/DOR antinociceptive ligand LP2 (1) were synthesized and their pharmacological profile was evaluated in in vitro and vivo assays. From our results, 2S-LP2 (5) showed an improved pharmacological profile in comparison to LP2 (1) and 2R-LP2 (4). 2S-LP2 (5) elicited an antinociceptive effect with a 1.5- and 3-times higher potency than LP2 (1) and R-antipode (4), respectively. In vivo effect of 2S-LP2 (5) was consistent with the improved MOR/DOR efficacy profile assessed by radioligand binding assay, to evaluate the opioid receptor affinity, and BRET assay, to evaluate the capability to promote receptor/G-protein and receptor/β-arrestin 2 interaction. 2S-LP2 (5) was able to activate, with different efficacy, G-protein pathway over β-arrestin 2, behaving as biased agonist at MOR and mainly at DOR. Considering the therapeutic potential of both multitarget MOR/DOR agonism and functional selectivity over G-protein, the 2S-LP2 (5) biased multitarget MOR/DOR agonist could provide a safer treatment opportunity.

Topics: Analgesics, Opioid; Animals; Benzomorphans; Dose-Response Relationship, Drug; Drug Discovery; Humans; Mice; Molecular Structure; Nociceptive Pain; Pain Measurement; Receptors, Opioid, delta; Receptors, Opioid, mu; Structure-Activity Relationship

Opioids produce antinociception by activation of G protein signaling linked to the mu-opioid receptor (MOPr). However, opioid binding to the MOPr also activates β-arrestin signaling. Opioids such as DAMGO and fentanyl differ in their relative efficacy for activation of these signaling cascades, but the behavioral consequences of this differential signaling are not known. The purpose of this study was to evaluate the behavioral significance of G protein and internalization dependent signaling within ventrolateral periaqueductal gray (vlPAG). Antinociception induced by microinjecting DAMGO into the vlPAG was attenuated by blocking Gαi/o protein signaling with administration of pertussis toxin (PTX), preventing internalization with administration of dynamin dominant-negative inhibitory peptide (dyn-DN) or direct inhibition of ERK1/2 with administration of the MEK inhibitor, U0126. In contrast, the antinociceptive effect of microinjecting fentanyl into the vlPAG was not altered by administration of PTX or U0126, and was enhanced by administration of dyn-DN. Microinjection of DAMGO, but not fentanyl, into the vlPAG induced phosphorylation of ERK1/2, which was blocked by inhibiting receptor internalization with administration of dyn-DN, but not by inhibition of Gαi/o proteins. ERK1/2 inhibition also prevented the development and expression of tolerance to repeated DAMGO microinjections, but had no effect on fentanyl tolerance. These data reveal that ERK1/2 activation following MOPr internalization contributes to the antinociceptive effect of some (e.g., DAMGO), but not all opioids (e.g., fentanyl) despite the known similarities for these agonists to induce β-arrestin recruitment and internalization.

Topics: Analgesics, Opioid; Animals; Butadienes; Drug Tolerance; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Enzyme Inhibitors; Fentanyl; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Nociceptive Pain; Periaqueductal Gray; Rats, Sprague-Dawley

The regulator of G-protein signaling protein type 4 (RGS4) accelerates the guanosine triphosphatase activity of G(αi) and G(αo), resulting in the inactivation of G-protein-coupled receptor signaling. An opioid receptor (OR), a G(αi)-coupled receptor, plays an important role in pain modulation in the central nervous system. In this study, we examined whether (1) spinal RGS4 affected nociceptive responses in the formalin pain test, (2) this RGS4-mediated effect was involved in OR activation, and (3) the µ-OR agonist-induced antinociceptive effect was modified by RGS4 modulation.. Formalin (1%, 20 µL) was injected subcutaneously into the right hindpaws of male 129S4/SvJae×C57BL/6J (RGS4(+/+) or RGS4(-/-)) mice, and the licking responses were counted for 40 minutes. The time periods (seconds) spent licking the injected paw during 0 to 10 minutes (early phase) and 10 to 40 minutes (late phase) were measured as indicators of acute nociception and inflammatory pain response, respectively. An RGS4 inhibitor, CCG50014, and/or a µ-OR agonist, [D-Ala², N-MePhe⁴, Gly-ol]-enkephalin (DAMGO), were intrathecally injected 5 minutes before the formalin injection. A nonselective OR antagonist, naloxone, was intraperitoneally injected 30 minutes before the CCG50014 injection.. Mice that received the formalin injection exhibited typical biphasic nociceptive behaviors. The nociceptive responses in RGS4-knockout mice were significantly decreased during the late phase but not during the early phase. Similarly, intrathecally administered CCG50014 (10, 30, or 100 nmol) attenuated the nociceptive responses during the late phase in a dose-dependent manner. The antinociceptive effect of the RGS4 inhibitor was totally blocked by naloxone (5 mg/kg). In contrast, intrathecal injection of DAMGO achieved a dose-dependent reduction of the nociceptive responses at the early and late phases. This analgesic effect of DAMGO was significantly enhanced by the genetic depletion of RGS4 or by coadministration of CCG50014 (10 nmol).. These findings demonstrated that spinal RGS4 inhibited the endogenous or exogenous OR-mediated antinociceptive effect in the formalin pain test. Thus, the inhibition of RGS4 activity can enhance OR agonist-induced analgesia. The enhancement of OR agonist-induced analgesia by coadministration of the RGS4 inhibitor suggests a new therapeutic strategy for the management of inflammatory pain.

Topics: Analgesics; Analgesics, Opioid; Animals; Behavior, Animal; Disease Models, Animal; Dose-Response Relationship, Drug; Drug Synergism; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Formaldehyde; Injections, Spinal; Male; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Knockout; Narcotic Antagonists; Nociception; Nociceptive Pain; Pain Measurement; Receptors, Opioid, mu; RGS Proteins; Spinal Cord; Thiazolidinediones; Time Factors

Respiratory depression is a therapy-limiting side effect of opioid analgesics, yet our understanding of the brain circuits mediating this potentially lethal outcome remains incomplete. Here we studied the contribution of the rostral ventromedial medulla (RVM), a region long implicated in pain modulation and homeostatic regulation, to opioid-induced respiratory depression. Microinjection of the μ-opioid agonist DAMGO in the RVM of lightly anesthetized rats produced both analgesia and respiratory depression, showing that neurons in this region can modulate breathing. Blocking opioid action in the RVM by microinjecting the opioid antagonist naltrexone reversed the analgesic and respiratory effects of systemically administered morphine, showing that this region plays a role in both the analgesic and respiratory-depressant properties of systemically administered morphine. The distribution of neurons directly inhibited by RVM opioid microinjection was determined with a fluorescent opioid peptide, dermorphin-Alexa 594, and found to be concentrated in and around the RVM. The non-opioid analgesic improgan, like DAMGO, produced antinociception but, unlike DAMGO, stimulated breathing when microinjected into the RVM. Concurrent recording of RVM neurons during improgan microinjection showed that this agent activated RVM ON-cells, OFF-cells, and NEUTRAL-cells. Since opioids are known to activate OFF-cells but suppress ON-cell firing, the differential respiratory response to these two analgesic drugs is best explained by their opposing effects on the activity of RVM ON-cells. These findings show that pain relief can be separated pharmacologically from respiratory depression and identify RVM OFF-cells as important central targets for continued development of potent analgesics with fewer side effects.

Topics: Analgesics, Opioid; Animals; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Evoked Potentials; Male; Medulla Oblongata; Morphine; Naltrexone; Narcotic Antagonists; Neurons; Nociception; Nociceptive Pain; Rats; Rats, Sprague-Dawley; Respiratory Insufficiency