Antinociceptive Effect Of DAMGO Research Articles

Spinal interaction between the highly selective μ agonist DAMGO and several δ opioid receptor ligands in naive and morphine-tolerant mice

Since the discovery of opioid receptor dimers their possible roles in opioid actions were intensively investigated. Here we suggest a mechanism that may involve the μ-δ opioid heterodimers. The exact role of δ opioid receptors in antinociception and in the development of opioid tolerance is still unclear. While receptor up-regulation can be observed during the development of opioid tolerance no μ receptor down-regulation could be detected within five days. In our present work we investigated how the selective δ opioid receptor agonists and antagonists influence the antinociceptive effect of the selective μ receptor agonist DAMGO in naïve and morphine-tolerant mice. We treated male NMRI mice with 200 μmol/kg subcutaneous (s.c.) morphine twice daily for three days. On the fourth day we measured the antinociceptive effect of DAMGO alone and combined with delta ligands: DPDPE, deltorphin II (agonists), TIPP and TICPψ (antagonists), respectively, administered intrathecally (i.t.) in mouse tail-flick test. In naive control mice none of the δ ligands caused significant changes in the antinociceptive action of DAMGO. The treatment with s.c. morphine resulted in approximately four-fold tolerance to i.t. DAMGO, i.e. the ED₅₀ value of DAMGO was four times as high as in naive mice. 500 and 1000 pmol/mouse of the δ₁ selective agonist DPDPE enhanced the tolerance to DAMGO while 1000 pmol/mouse of the δ₂ selective agonist deltorphin II did not influence the degree of tolerance. However, both δ antagonists TIPP and TICPψ potentiated the antinociceptive effect of i.t. DAMGO, thus they restored the potency of DAMGO to the control level. The inhibitory action of DPDPE against the antinociceptive effect of DAMGO could be antagonized by TIPP and TICPψ. We hypothesize that during the development of morphine tolerance the formation of μδ heterodimers may contribute to the spinal opioid tolerance. δ ligands may affect the dimer formation differently. Those, like DPDPE may facilitate the dimer formation hence inhibit the antinociceptive effect of DAMGO by causing virtual μ receptor down-regulation. Ligands that do not affect the dimer formation do not influence antinociception either but ligands with the presumed capability of disconnecting the dimers may decrease the spinal tolerance to DAMGO.

Enhanced Brain Delivery of the Opioid Peptide DAMGO in Glutathione PEGylated Liposomes: A Microdialysis Study

Glutathione PEGylated (GSH-PEG) liposomes were evaluated for their ability to enhance and prolong blood-to-brain drug delivery of the opioid peptide DAMGO (H-Tyr-d-Ala-Gly-MePhe-Gly-ol). An intravenous loading dose of DAMGO followed by a 2 h constant rate infusion was administered to rats, and after a washout period of 1 h, GSH-PEG liposomal DAMGO was administered using a similar dosing regimen. DAMGO and GSH-PEG liposomal DAMGO were also administered as a 10 min infusion to compare the disposition of the two formulations. Microdialysis made it possible to determine free DAMGO in brain and plasma, while the GSH-PEG liposomal encapsulated DAMGO was measured with regular plasma sampling. The antinociceptive effect of DAMGO was determined with the tail-flick method. All samples were analyzed using liquid chromatography-tandem mass spectrometry. The short infusion of DAMGO resulted in a fast decline of the peptide concentration in plasma with a half-life of 9.2 ± 2.1 min. Encapsulation in GSH-PEG liposomes prolonged the half-life to 6.9 ± 2.3 h. Free DAMGO entered the brain to a limited extent with a steady state ratio between unbound drug concentrations in brain interstitial fluid and in blood (Kp,uu) of 0.09 ± 0.04. GSH-PEG liposomes significantly increased the brain exposure of DAMGO to a Kp,uu of 0.21 ± 0.17 (p < 0.05). By monitoring the released, active substance in both blood and brain interstitial fluid over time, we were able to demonstrate that GSH-PEG liposomes offer a promising platform for enhancing and prolonging the delivery of drugs to the brain.

The peripheral antinociceptive effect of DAMGO and 6β-glycine-substituted 14-O-methyloxymorphone (HS-731) after systemic administration in a mouse visceral pain model

Inhibition of nociception through activation of peripheral μ opioid receptors (MOR) avoiding the central adverse effects of opioids has added a new possibility to manage pain control. Here we report the antinociceptive effects of the peripherally restricted MOR agonist HS-731, the peptide DAMGO, and morphine in the writhing test in mice. S.c. and i.c.v. HS-731 dose-dependently and completely inhibited writhing, being 24–598 times more potent than the two MOR-selective agonists DAMGO and morphine. However, extremely high s.c./i.c.v. potency ratios were calculated for HS-731 and DAMGO and much lower for morphine. Remarkably, a long duration of action was induced by HS-731 and much shorter by morphine and DAMGO. The antinociceptive effects of systemic opioids were reversed by s.c. naloxone while i.c.v. administration of the MOR selective antagonist CTAP significantly abolished the antinociceptive effect of s.c. morphine but completely failed to antagonize the effects of systemic HS-731 or DAMGO. In addition, in the rat vas deferens HS-731 and DAMGO, but not morphine, showed high intrinsic efficacy and naltrexone-sensitive agonist response at MOR by depressing electrically-evoked contractions of this organ. These data demonstrate that the selectivity and high efficacy of HS-731 and DAMGO at peripheral MOR as well as their inability to cross the blood brain barrier are a cornerstone for producing peripheral antinociception after systemic administration.

In vivo evaluation of (+)-MR200 as a new selective sigma ligand modulating MOP, DOP and KOP supraspinal analgesia

The compound (+)-MR200 [(+)-methyl (1R,2S)-2-{[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl}-1-phenylcyclopropanecarboxylate] is a sigma ligand with increased affinity and selectivity compared to the structurally related ligand haloperidol. From the results of a previous study on the modulation of a systemically injected KOP opioid agonist analgesia by (+)-MR200, we analysed the influence of this sigma ligand on the antinociceptive effect of centrally injected MOP, DOP, and KOP selective agonists using the tail-flick test in rats. The results obtained confirmed that systemic administration of (+)-MR200 (1mg/Kg s.c.) did not modify basal tail-flick latency. Pre-treatment with 1mg/Kg s.c. of (+)-MR200 provided a significant increase in the antinociceptive effect of DAMGO (100ng/rat i.c.v.) and DPDPE (20 μg/rat i.c.v.). Conversely to previous reports, pre-treatment with (+)-MR200 reversed, in these experimental conditions, U-50488H (100μg/rat i.c.v.) analgesia. The mechanism involved in these effects was not clear, but provided additional data on a diverging modulator role of selective sigma-1 antagonists on KOP analgesia.

Effect of MK-801 on the antinociceptive effect of [ d-Ala 2, N-MePhe 4, Gly-ol 5]enkephalin in diabetic mice

The role of N-methyl- d-aspartate (NMDA) receptors in supraspinal and spinal sites on the reduction of supraspinal μ-opioid receptor-induced antinociception in diabetic mice was examined. The antinociceptive effect of i.c.v. [ d-Ala 2, N-MePhe 4, Gly-ol 5]enkephalin (DAMGO, 20 pmol) in diabetic mice was significantly less than that in non-diabetic mice. The antinociceptive effect of i.c.v. DAMGO (20 pmol) was significantly and dose dependently reduced by i.c.v. (+)-5-methyl-10,11-dihydro-5 H-dibenzo[ a, d]cyclohepten-5,10-imine maleate (MK-801) in both non-diabetic (0.03–0.3 nmol) and diabetic mice (0.1–3.0 nmol). While the antinociceptive effect of i.c.v. DAMGO (10 pmol) was significantly enhanced by i.c.v NMDA (0.01–0.1 nmol) in non-diabetic mice, the same doses of i.c.v. NMDA had no significant effect on the antinociceptive effect of i.c.v. DAMGO (20 pmol) in diabetic mice. In non-diabetic mice, the antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose dependently reduced by intrathecal administration of MK-801 (0.1–1.0 nmol). The antinociceptive effect of DAMGO (20 pmol, i.c.v.) was dose-dependently enhanced by MK-801 (0.1–1.0 nmol, i.t.) in diabetic mice. Furthermore, NMDA (0.1 nmol, i.t.) significantly enhanced the antinociceptive effect of DAMGO (10 pmol, i.c.v.) in non-diabetic mice. However, in non-diabetic mice, the antinociceptive effect of DAMGO (40 pmol, i.c.v.) was dose dependently reduced by NMDA (0.03–0.3 nmol, i.t.). These results suggest that NMDA receptor function in supraspinal and spinal sites appear to be modulated differently by the diabetic state, and this functional modulation may play an important role in the reduction of supraspinal μ-opioid receptor-induced antinociception in diabetic animals.

Effects of a μ-opioid receptor agonist on G-protein activation in streptozotocin-induced diabetic mice

Many clinical and experimental studies have suggested that diabetes or hyperglycemia alter pain sensitivity, and sensitivity to several drugs. It has been reported that the antinociceptive potency of morphine is decreased in several rodent models of hyperglycemia, including streptozotocin-induced diabetes, an animal models of type I diabetes. The present study was designed to investigate in streptozotocin-induced diabetic mice the effect of the selective μ-opioid agonist [ d-Ala 2, NMePhe 4, Gly-ol 5]enkephalin (DAMGO) on G-protein activation by monitoring guanosine-5′- O-(3-[ 35S]thio)triphosphate ([ 35S]GTPγS) binding to pons/medulla membranes, which contain the key areas for opioid antinociception. In the tail-flick test, DAMGO (1–10 ng, intracerebroventricularly) produced a marked dose-dependent antinociception in non-diabetic mice. In streptozotocin-induced diabetic mice, the effect of DAMGO was significantly attenuated as compared to that in non-diabetic mice. In the [ 35S]GTPγS binding assay, DAMGO (0.1–10 μM) increased the binding of [ 35S]GTPγS to pons/medulla membranes from non-diabetic mice in a concentration-dependent manner, affording approximately 100% maximal stimulation at 10 μM. The maximal stimulation of [ 35S]GTPγS binding by DAMGO (10 μM) in streptozotocin-induced diabetic mice (100.55±3.12%), was similar to non-diabetic mice. The present results indicated that the antinociceptive effect of DAMGO given supraspinally was less potent in streptozotocin-induced diabetic mice than that in non-diabetic mice, whereas the μ-opioid receptor-mediated G-protein activation in pons/medulla was unaltered in streptozotocin-induced diabetic mice. Thus, the attenuation of DAMGO-induced antinociception in streptozotocin-induced diabetic mice is probably caused by dysfunction in cellular pathways after the activation of G-proteins.

Effects of mu and delta opioid agonists and antagonists on affective vocal and reflexive pain responses during social stress in rats.

The present experiments evaluated the influence of intraventricular mu and delta opioid receptors on affective vocal and reflexive responses to aversive stimuli in socially inexperienced, as well as defensive and submissive responses in defeated, adult male Long-Evans rats. Defeat stress consisted of: (1) an aggressive confrontation in which the experimental intruder rat exhibited escape, defensive and submissive behaviors [i.e., upright, supine postures and ultrasonic vocalizations (USV)], and subsequently, (2) protection from the resident stimulus rat with a wire mesh screen for 10-20 min. Defeat stress was immediately followed by an experimental session with tactile startle (20 psi). The mu opioid receptor agonists morphine (0.1-0.6 microg i.c.v.) and [D-Ala2-N-Me-Phe4-Gly5-ol]-enkephalin (DAMGO; 0.01-0.3 microg i.c.v.), and the delta opioid receptor agonist [D-Pen2,5]-enkephalin (DPDPE; 10-100 microg i.c.v.) dose-dependently decreased startle-induced USV and increased tail-flick latencies in socially inexperienced and defeated rats. Of greater interest, morphine, DAMGO and DPDPE increased the occurrence of the submissive crouch posture, and defeated rats were more sensitive than socially inexperienced rats to the startle-induced USV-suppressive and antinociceptive effects of morphine and DPDPE. The antinociceptive effects of DAMGO were likewise obtained at lower doses in defeated rats. Finally, the USV-suppressive effects of morphine and DAMGO were reversed with the mu receptor antagonist naltrexone (0.1 mg/kg i.p.), but the USV-suppressive effects produced by DPDPE were not reversed with the delta receptor antagonist naltrindole (1 mg/kg i.p.). These results confirm mu, but not delta opioid receptor activation as significant in affective vocal, passive-submissive behavior, as well as reflexive antinociception. Furthermore, similar to previous studies with restraint and electric shock stress, the facilitation of mu opioid effects on vocal responses and antinociception is consistent with the proposal that defeat stress activated endogenous opioid mechanisms.

Possible involvement of protein kinase C in the attenuation of [d-Ala2, NMePhe4, Gly-ol5]enkephalin-induced antinociception in diabetic mice

The effects of pretreatment with a highly selective protein kinase C inhibitor, calphostin C, on the antinociception induced by the intracerebroventricular (i.c.v.) administration of the mu-opioid receptor agonist [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAMGO) were studied in diabetic and non-diabetic mice. The antinociceptive potency of i.c.v. DAMGO in diabetic mice was lower than that in non-diabetic mice. I.c.v. pretreatment with phorbol 12,13-dibutyrate (50 pmol), a protein kinase C activator, for 60 min attenuated the antinociception induced by i.c.v. DAMGO in non-diabetic mice, but not in diabetic mice. I.c.v. pretreatment with calphostin C (3 pmol) for 60, 120 and 240 min, but not 10 min, increased the antinociceptive effect of DAMGO (10 ng) in diabetic mice, but not in non-diabetic mice. The dose-response curve for DAMGO-induced antinociception in diabetic mice, but not in non-diabetic mice, was shifted to the left by i.c.v. pretreatment with calphostin C (3 pmol) for 60 min. In non-diabetic mice, i.c.v. pretreatment with a high dose of calphostin C (10 pmol) for 60 and 120 min potentiated DAMGO-induced antinociception. These results indicate that protein kinase C may be involved in DAMGO-induced antinociception in mice. Furthermore, these results suggest that the attenuation of DAMGO-induced antinociception in diabetic mice may be due in part to increased protein kinase C activity.

Selective attenuation of μ-opioid receptor-mediated effects in rat sensory neurons by intrathecal administration of antisense oligodeoxynucleotides

To test the hypothesis that the expression of specific proteins on peripheral terminals of primary afferents can be attenuated by intrathecal administration of antisense oligodeoxynucleotides (ODNs), we administered ODNs antisense to the μ-opioid receptor to male Sprague-Dawley rats via chronically implanted intrathecal cannulae. Antisense but not mismatch ODN treatment significantly decreased peripheral ( d-Ala 2, N-Me-Phe 4, Gly 5-ol)-enkephalin (DAMGO) inhibition of prostaglandin E 2 (PGE 2) hyperalgesia. Antisense treatment affected neither the magnitude of PGE 2 hyperalgesia nor the antinociception produced by a peripherally administered adenosine A 1-agonist. The antinociceptive effects of DAMGO was fully recovered 8 days after cessation of ODN treatment. DAMGO-induced inhibition of voltage-gated Ca 2+ currents (VGCC), in cultured dorsal root ganglion (DRG) neurons from rats treated with ODNs, was lso significantly reduced by antisense but not mismatch ODNs. Taken together, these observations suggest that intrathecal administration of antisense ODNs can be used to study the function of proteins present in the peripheral terminals of primary afferent neurons.

Evidence for differential modulation of μ-opioid receptor-mediated antinociceptive and antitussive activities by spleen-derived factor(s) from diabetic mice

The effects of spleen-derived factor(s) from diabetic mice on the antinociceptive and antitussive effects of μ-opioid receptor agonists were examined in mice. The antinociceptive effects were examined 1 week after adoptive transfer of the supernatant of spleen cell homogenate (SSCH) from diabetic mice (SSCH-D). Naive mice which had been injected with SSCH-D were less sensitive to the antinociceptive effects of μ-opioid receptor agonists, such as morphine and [ d-Ala 2, N-MePhe 4, Gly-ol 5]enkephalin (DAMGO), than mice which had been injected with SSCH from non-diabetic mice. The antinociceptive effects of DAMGO was also significantly lower in naive mice injected with SSCH-D than in SSCH from non-diabetic mice (SSCH-ND)-treated naive mice, when assessed 2 weeks after adoptive transfer of SSCH. The sensitivity to the antinociceptive effect of [ d]-Pen 2,5]enkephalin (DPDPE), a δ-opioid receptor agonist, was significantly enhanced 2 weeks, but not 1 week, after adoptive transfer of SSCH-D. On the other hand, adoptive transfer of SSCH-D to naive mice had no significant effect on the recipients' antitussive sensitivities to morphine and DAMGO when assessed 1 week after transfer of SSCH-D. However, when the antitussive effect was assessed 2 weeks after adoptive transfer of SSCH, the antitussive effect of DAMGO was significantly lower in naive mice injected with SSCH-D than in SSCH-ND-treated naive mice. The reduction in the antitussive effect of DAMGO in naive mice had been injected with SSCH-D 2 weeks before testing was abolished when they were pretreated with naltrindole, a selective δ-opioid receptor antagonist. These findings suggest that some factor(s) derived from spleen cells may primarily contribute to the reduction in the antinociceptive effects, but not the antitussive effects, of supraspinal μ-opioid receptors. Furthermore, the dysfunction of μ-opioid receptors may up-regulate δ-opioid receptors, which would reduce μ-opioid receptor-mediated antitussive effects.