Morphine-induced mechanical hypersensitivity in mice requires δ receptors, ß-arrestin2, and c-Src activity.

Morphine diminishes pain, but its long-term use is compromised by tolerance and hyperalgesia. Studies implicate δ receptors, ß-arrestin2 and Src kinase in tolerance. We examined whether these proteins are also involved in morphine-induced hypersensitivity (MIH). A common pathway for tolerance and hypersensitivity may provide a single target to guide improved analgesic approaches. We examined mechanical sensitivity using automated von Frey in wild-type (WT) and transgenic male and female C57Bl/6 mice before and after hind paw inflammation by complete Freund's adjuvant (CFA). CFA-evoked hypersensitivity ceased on day 7 in WT but persisted for the 15-day testing period in µ-/- . Recovery was delayed until day 13 in δ-/- . We explored the expression of opioid genes in the spinal cord using quantitative RT-PCR. Restoration to basal sensitivity in WT occurred with increased δ expression. By contrast, κ expression was reduced, while µ remained unchanged. Daily morphine reduced hypersensitivity in WT on day 3 compared to controls; however, hypersensitivity recurred on day 9 and beyond. By contrast, WT had no recurrence of hypersensitivity in the absence of daily morphine. We used ß-arrestin2-/- , δ-/- and Src inhibition by dasatinib in WT to establish whether these approaches, which diminish tolerance, also attenuate MIH. While none of these approaches affected CFA-evoked inflammation or acute hypersensitivity, all caused sustained morphine anti-hypersensitivity, abolishing MIH. Like morphine tolerance, MIH in this model requires δ receptors, ß-arrestin2 and Src activity. Our findings suggest that MIH is caused by a tolerance-induced reduction in endogenous opioid signalling. KEY POINTS: Morphine is effective for treating severe acute pain, but tolerance and hypersensitivity often develop during its use in treating chronic pain. It is unclear whether these detrimental effects share similar mechanisms; if so, it might be possible to develop a single approach to minimise both phenomena. Mice deficient in µ receptors, δ receptors or ß-arrestin2 and wild type mice treated with the Src inhibitor dasatinib exhibit negligible morphine tolerance. We show that these same approaches also prevent the development of morphine-induced hypersensitivity during persistent inflammation. This knowledge identifies strategies, such as the use of Src inhibitors, which may mitigate tolerance and morphine induced hyperalgesia.

TRV130 partial agonism and capacity to induce anti-nociceptive tolerance revealed through reducing available µ-opioid receptor number.

BACKGROUND AND PURPOSE: ß-Arrestin2 recruitment to µ-receptors may contribute to the development of opioid side effects. This possibility led to the development of TRV130 and PZM21, opioids reportedly biased against ß-arrestin2 recruitment in favour of G-protein signalling. However, low efficacy ß-arrestin2 recruitment by TRV130 and PZM21 may simply reflect partial agonism overlooked due to overexpression of µ-receptors. EXPERIMENTAL APPROACH: Efficacies and apparent potencies of DAMGO, morphine, PZM21 and TRV130 as stimulators of ß-arrestin2 recruitment and inhibitors of cAMP accumulation were assessed in CHO cells stably expressing µ-receptors. Receptor availability was depleted through prior exposure of cells to the irreversible antagonist, ß-FNA. We also examined whether µ-receptor availability influences TRV130 anti-nociception and/or tolerance using the tail withdrawal assay in wild-type C57BL/6 and µ+/- mice. KEY RESULTS: Morphine, PZM21 and TRV130 were partial agonists in the ß-arrestin2 recruitment assay. Only TRV130 exhibited partial agonism in the cAMP assay. Exposure to ß-FNA to reduce µ-receptor availability further limited the efficacy of TRV130 and revealed morphine and PZM21 to be partial agonists. Despite having partial efficacy in vitro, TRV130 caused potent anti-nociception (ED50 : 0.33 mg·kg-1 ) in wild-type mice, without tolerance after daily administration for 10 days. TRV130 caused similar anti-nociception in µ+/- mice, with marked tolerance on day 4 of injections. CONCLUSION AND IMPLICATIONS: Our findings emphasise the importance of receptor reserve when characterising µ-receptor agonists. Reduced receptor availability reveals that TRV130 is a partial agonist capable of tolerance, despite having limited efficacy for ß-arrestin2 recruitment to the µ-receptor.

Activation of µ-opioid receptors by MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) and its fluorinated derivatives.

BACKGROUND AND PURPOSE: A fluorinated derivative (2F-MT-45) of the synthetic µ-opioid receptor agonist MT-45 (1-cyclohexyl-4-(1,2-diphenylethyl)piperazine) was recently identified in a seized illicit tablet. While MT-45 is a Class A drug, banned in a number of countries, nothing is known about the pharmacology of 2F-MT-45. This study compares the pharmacology of MT-45, its fluorinated derivatives and two of its metabolites. EXPERIMENTAL APPROACH: We used a ß-arrestin2 recruitment assay in CHO cells stably expressing µ receptors to quantify the apparent potencies and efficacies of known (MT-45, morphine, fentanyl and DAMGO) and potential agonists. In addition, the GloSensor protein was transiently expressed to quantify changes in cAMP levels. We measured Ca2+ to investigate whether MT-45 and its metabolites have effects on GluN1/N2A NMDA receptors stably expressed in Ltk- cells. KEY RESULTS: The fluorinated MT-45 derivatives have higher apparent potencies (2F-MT-45: 42 nM) than MT-45 (1.3 µM) for inhibition of cAMP accumulation and ß-arrestin2 recruitment (2F-MT-45: 196 nM; MT-45: 23.1 µM). While MT-45 and 2F-MT-45 are poor recruiters of ß-arrestin2, they have similar efficacies for reducing cAMP levels as DAMGO. Two MT-45 metabolites displayed negligible potencies as µ receptor agonists, but one, 1,2-diphenylethylpiperazine, inhibited the NMDA receptor with an IC50 of 29 µM. CONCLUSION AND IMPLICATIONS: Fluorinated derivatives of MT-45 are potent µ receptor agonists and this may pose a danger to illicit opioid users. Inhibition of NMDA receptors by a metabolite of MT-45 may contribute to the reported dissociative effects.