Expression of delta opioid receptors by splenocytes from SEB-treated mice and effects on phosphorylation of MAP kinase

Delta opioid receptors (DORs) are known to modulate multiple T-cell responses. However, little is known about the expression of these receptors. These studies evaluated the expression of DOR mRNA and protein after a single in vivo exposure to staphylococcal enterotoxin B (SEB). SEB (20 microg, ip) significantly enhanced splenocyte DOR mRNA expression 8 and 24 h after injection. SEB also increased the fractions of the total splenocyte (5 to 20%) and T-cell (8 to 50%) populations expressing DOR protein. In saline-treated animals, DOR relative fluorescence intensity per cell was 11.1 +/- 0.62 units (mean +/- SEM), increasing to 16.1 +/- 1.7 after exposure to SEB. DOR fluorescence intensity significantly increased to 33.5 +/- 2.0 units in a subpopulation of T-cells. Thus, SEB significantly increased DOR expression in vivo, affecting both mRNA and protein levels primarily within the T-cell population. To determine whether T-cell DORs modulate the activity of extracellular-regulated kinases (ERKs), the phosphorylation of ERKs 1 and 2 was studied using splenocytes from SEB-treated mice. At concentrations from 10(-8) to 10(-6) M, [d-Ala(2)-d-Leu(5)]-enkephalin, a selective DOR agonist, significantly inhibited anti-CD3-epsilon-induced phosphorylation of the ERKs. Therefore, the DORs expressed by activated T-cells are capable of attenuating T-cell activation that depends on ERK phosphorylation.     

delta-, but not mu-, opioid receptor stabilizes K(+) homeostasis by reducing Ca(2+) influx in the cortex during acute hypoxia

Past work has shown that delta-opioid receptor (DOR) activation by [D-Ala(2),D-Leu(5)]-enkephalin (DADLE) attenuated the disruption of K(+) homeostasis induced by hypoxia or oxygen-glucose deprivation (OGD) in the cortex, while naltrindole, a DOR antagonist blocked this effect, suggesting that DOR activity stabilizes K(+) homeostasis in the cortex during hypoxic/ischemic stress. However, several important issues remain unclear regarding this new observation, especially the difference between DOR and other opioid receptors in the stabilization of K(+) homeostasis and the underlying mechanism. In this study, we asked whether DOR is different from micro-opioid receptors (MOR) in stabilizing K(+) homeostasis and which membrane channel(s) is critically involved in the DOR effect. The main findings are that (1) similar to DADLE (10 microM), H-Dmt-Tic-NH-CH (CH(2)--COOH)-Bid (1-10 microM), a more specific and potent DOR agonist significantly attenuated anoxic K(+) derangement in cortical slice; (2) [D-Ala(2), N-Me-Phe(4), glycinol(5)]-enkephalin (DAGO; 10 microM), a MOR agonist, did not produce any appreciable change in anoxic disruption of K(+) homeostasis; (3) absence of Ca(2+) greatly attenuated anoxic K(+) derangement; (4) inhibition of Ca(2+)-activated K(+) (BK) channels with paxilline (10 microM) reduced anoxic K(+) derangement; (5) DADLE (10 microM) could not further reduce anoxic K(+) derangement in the Ca(2+)-free perfused slices or in the presence of paxilline; and (6) glybenclamide (20 microM), a K(ATP) channel blocker, decreased anoxia-induced K(+) derangement, but DADLE (10 microM) could further attenuate anoxic K(+) derangement in the glybenclamide-perfused slices. These data suggest that DOR, but not MOR, activation is protective against anoxic K(+) derangement in the cortex, at least partially via an inhibition of hypoxia-induced increase in Ca(2+) entry-BK channel activity.     

Co-expressions of different opioid receptor types differentially modulate their signaling via G(16)

Combinations of two different types of opioid receptors - delta-, kappa-, mu-opioid receptors (DOR, KOR, and MOR) and opioid receptor-like receptor 1 (ORL(1)) - were co-expressed with the alpha subunit of G(16) in COS-7 cells, and the ability of various selective agonists to induce activation of phospholipase Cbeta was examined. Nociceptin/orphanin FQ-induced response was enhanced when ORL(1) was co-expressed with MOR or KOR but not DOR. The kappa-agonist U50,488H induced a modest inositol phosphate formation when KOR was expressed alone or with MOR, but the response was attenuated when co-expressing with either DOR or ORL(1). It is suggested that the co-expressions of two different opioid receptor types indeed modify their downstream signaling events.     

Cyclic biphalin analogues with a novel linker lead to potent agonist activities at mu,delta, and kappa opioid receptors

In an effort to improve biphalin’s potency and efficacy at the µ-(MOR) and δ-opioid receptors (DOR), a series of cyclic biphalin analogues 1–5 with a cystamine or piperazine linker at the C-terminus were designed and synthesized by solution phase synthesis using Boc-chemistry. Interestingly, all of the analogues showed balanced opioid agonist activities at all opioid receptor subtypes due to enhanced κ-opioid receptor (KOR) activity. Our results indicate that C-terminal flexible linkers play an important role in KOR activity compared to that of the other cyclic biphalin analogues with a hydrazine linker. Among them, analogue 5 is a potent (Ki?=?0.27, 0.46, and 0.87?nM; EC₅₀?=?3.47, 1.45, and 13.5?nM at MOR, DOR, and KOR, respectively) opioid agonist with high efficacy. Based on the high potency and efficacy at the three opioid receptor subtypes, the ligand is expected to have a potential synergistic effect on relieving pain and further studies including in vivo tests are worthwhile.     

mu-Opioid receptors desensitize less rapidly than delta-opioid receptors due to less efficient activation of arrestin

Receptor desensitization by G-protein receptor kinases (GRK) and arrestins is likely to be an important component underlying the development of tolerance to opioid drugs. Reconstitution of this process in Xenopus oocytes revealed distinct differences in the kinetics of GRK and arrestin regulation of the closely related opioid receptors mu (MOR), delta (DOR), and kappa (KOR). We demonstrated that under identical conditions, GRK and arrestin-dependent desensitization of MOR proceeds dramatically slower than that of DOR. Furthermore, GRK3 phosphorylation sites required for opioid receptor desensitization also greatly differ. The determinants for DOR and KOR desensitization reside in the carboxyl-terminal tail, whereas MOR depends on Thr-180 in the second intracellular loop. Although this later finding might indicate an inefficient phosphorylation of MOR Thr-180, increasing the amount of arrestin expressed greatly increased the rate of MOR desensitization to a rate comparable with that of DOR. Similarly, coexpression of a constitutively active arrestin 2(R169E) with MOR and DOR desensitized both receptors in an agonist-dependent, GRK-independent manner at rates that were indistinguishable. Together, these data suggest that it is the activation of arrestin, rather than its binding, that is the rate-limiting step in MOR desensitization. In addition, mutation of Thr-161 in DOR, homologous to MOR Thr-180, significantly inhibited the faster desensitization of DOR. These results suggest that DOR desensitization involves phosphorylation of both the carboxyl-terminal tail and the second intracellular loop that together leads to a more efficient activation of arrestin and thus faster desensitization.     

Isobolographic analysis of the opioid-opioid interactions in a tonic and a phasic mouse model of induced nociceptive pain

Background

Opioids have been used for the management of pain and coadministration of two opioids may induce synergism. In a model of tonic pain, the acetic acid writhing test and in a phasic model, the hot plate, the antinociceptive interaction between fentanyl, methadone, morphine, and tramadol was evaluated.

Results

The potency of opioids in the writhing test compared to the hot plate assay was from 2.5 (fentanyl) to 15.5 (morphine) times, respectively. The ED₅₀ was used in a fixed ratio for each of the six pairs of opioid combinations, which, resulted in a synergistic antinociception except for methadone/tramadol and fentanyl/tramadol which were additive, in the hot plate. The opioid antagonists naltrexone, naltrindole and nor-binaltorphimine, suggests that the synergism of morphine combinations are due to the activation of MOR subtypes with partially contribution of DOR and KOR, however fentanyl and methadone combinations are partially due to the activation of MOR and DOR subtypes and KOR lack of participation. The antinociceptive effects of tramadol combinations, are partially due to the activation of MOR, DOR and KOR opioid subtypes.

Conclusion

These results suggets that effectiveness and magnitude of the interactions between opioids are dependent on pain stimulus intensity.     

Opioid-receptor gene expression and localization in cancer cells

This study examined the presence and cellular localization of three types of opioid receptors (MOR, DOR and KOR) in five human cancer cell lines: MCF-7, MDA-MB-231, HT-29, MGH-U1 and SH-SY5Y. Expression levels of opioid receptors were measured quantitatively using real-time PCR, and the localizations of the receptors in the cells were determined by immunocytochemistry. All three types of opioid receptors were present in each of the five cell lines examined. However, three of the cell lines (MCF-7, HT-29 and SH-SY5Y) showed significantly higher levels of MOR mRNA and protein than the other two types of receptors (DOR and KOR). Immunocytochemistry revealed that MOR, DOR and KOR receptors were predominantly present on the surface of cell membranes, although these receptors were also occasionally present in the cell cytoplasm.     

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.     

Delta-Opioid Receptor Analgesia Is Independent of Microglial Activation in a Rat Model of Neuropathic Pain

The analgesic effect of delta-opioid receptor (DOR) ligands in neuropathic pain is not diminished in contrast to other opioid receptor ligands, which lose their effectiveness as analgesics. In this study, we examine whether this effect is related to nerve injury-induced microglial activation. We therefore investigated the influence of minocycline-induced inhibition of microglial activation on the analgesic effects of opioid receptor agonists: morphine, DAMGO, U50,488H, DPDPE, Deltorphin II and SNC80 after chronic constriction injury (CCI) to the sciatic nerve in rats. Pre-emptive and repeated administration of minocycline (30 mg/kg, i.p.) over 7 days significantly reduced allodynia and hyperalgesia as measured on day 7 after CCI. The antiallodynic and antihyperalgesic effects of intrathecally (i.t.) administered morphine (10–20 µg), DAMGO (1–2 µg) and U50,488H (25–50 µg) were significantly potentiated in rats after minocycline, but no such changes were observed after DPDPE (10–20 µg), deltorphin II (1.5–15 µg) and SNC80 (10–20 µg) administration. Additionally, nerve injury-induced down-regulation of all types of opioid receptors in the spinal cord and dorsal root ganglia was not influenced by minocycline, which indicates that the effects of opioid ligands are dependent on other changes, presumably neuroimmune interactions. Our study of rat primary microglial cell culture using qRT-PCR, Western blotting and immunocytochemistry confirmed the presence of mu-opioid receptors (MOR) and kappa-opioid receptors (KOR), further we provide the first evidence for the lack of DOR on microglial cells. In summary, DOR analgesia is different from analgesia induced by MOR and KOR receptors because it does not dependent on injury-induced microglial activation. DOR agonists appear to be the best candidates for new drugs to treat neuropathic pain.     

Role of Opioid Receptors Signaling in Remote Electrostimulation - Induced Protection against Ischemia/Reperfusion Injury in Rat Hearts

Aims

Our previous studies demonstrated that remote electro-stimulation (RES) increased myocardial GSK3 phosphorylation and attenuated ischemia/ reperfusion (I/R) injury in rat hearts. However, the role of various opioid receptors (OR) subtypes in preconditioned RES-induced myocardial protection remains unknown. We investigated the role of OR subtype signaling in RES-induced cardioprotection against I/R injury of the rat heart.

Methods & Results

Male Spraque-Dawley rats were used. RES was performed on median nerves area with/without pretreatment with various receptors antagonists such as opioid receptor (OR) subtype receptors (KOR, DOR, and MOR). The expressions of Akt, GSK3, and PKCε expression were analyzed by Western blotting. When RES was preconditioned before the I/R model, the rat''s hemodynamic index, infarction size, mortality and serum CK-MB were evaluated. Our results showed that Akt, GSK3 and PKCε expression levels were significantly increased in the RES group compared to the sham group, which were blocked by pretreatment with specific antagonists targeting KOR and DOR, but not MOR subtype. Using the I/R model, the duration of arrhythmia and infarct size were both significantly attenuated in RES group. The mortality rates of the sham RES group, the RES group, RES group + KOR antagonist, RES group + DOR/MOR antagonists (KOR left), RES group + DOR antagonist, and RES group + KOR/MOR antagonists (DOR left) were 50%, 20%, 67%, 13%, 50% and 55%, respectively.

Conclusion

The mechanism of RES-induced myocardial protection against I/R injury seems to involve multiple target pathways such as Akt, KOR and/or DOR signaling.     

Mitochondrial ERK plays a key role in delta-opioid receptor neuroprotection against acute mitochondrial dysfunction

It is well established that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effects of DOR activation on mitochondrial dysfunction in neurons are poorly elucidated. In this investigation, we studied the effects of [d-Ala2, d-Leu5] enkephalin (DADLE), a potent DOR agonist, on acute mitochondrial dysfunction and ensuing cell damage induced by sodium azide in primary rat cortical neuronal cultures, and explored possible mechanisms underlying. Here, we show that DADLE reverses NaN₃-induced acute mitochondrial dysfunction by selectively activating DOR, mainly including mitochondrial membrane depolarization, mitochondrial Ca²⁺ overload and reactive oxygen species generation. DOR stimulation also inhibits cytochrome c release and caspase-3 activation, and attenuates neuronal death caused by acute NaN₃ insults. Furthermore, DOR activation with DADLE protects neurons from acute NaN₃ insults mainly through PKC-ERK pathway, and mitochondrial ERK activation is especially required for DOR neuroprotection against acute mitochondrial dysfunction.     

Peripheral opioidergic regulation of the tracheobronchial mucociliary transport system.

We hypothesized that, in the airway mucosa, opioids are inhibitory neural modulators that cause an increase in net water absorption in the airway mucosa (as in the gut). Changes in bidirectional water fluxes across ovine tracheal mucosa in response to basolateral application of the opioid peptides beta-endorphin, dynorphin A-(1-8), and [d-Ala(2), d-Leu(5)]-enkephalin (DADLE) were measured. beta-Endorphin and dynorphin A-(1-8) decreased luminal-to-basolateral water fluxes, and dynorphin A-(1-8) and DADLE increased basolateral-to-luminal water flux. These responses were electroneutral. In seven beagle dogs, administration of aerosolized beta-endorphin (1 mg) to the tracheobronchial airways decreased the clearance of radiotagged particles from the bronchi in 1 h from 34.7 to 22.0% (P < 0.001). Naloxone abrogated the beta-endorphin-induced changes in vitro and in vivo. Contrary to our hypothesis, the opioid-induced changes in water fluxes would all lead to a predictable increase in airway surface fluid. The beta-endorphin-induced increases in airway fluid together with reduced bronchial mucociliary clearance may produce procongestive responses when opioids are administered as antitussives.     

The role of mu- and delta-opiate receptors in the realization of the autonomic effects of opioid peptides

In the first series of experiments on the isolated mouse vas deferens and guinea-pig ileum the capacity of 10 opioid peptides to activate mu- and delta-receptors was evaluated. [DAla2, DLeu5]-enkephalin (DADLE) and [DAla2, MePhe4, Gly5-ol]-enkephalin (DAMPGE) were the most selective agonists of delta- and mu-opiate receptors, respectively. In the second series of experiments on urethan-anesthetized rats it was shown, that intravenous administration of DADLE or DAMPGE (10(-7) M/kg each) elicited hypotension, bradycardia and expiratory apnoe. These effects disappeared both after naloxone injection and bilateral cervical vagotomy. A reflex nature of the vegetative effects of opioid peptides and the role of both mu- and delta-receptors in their realization are suggested.     

Effects of antisense oligodeoxynucleotides against opioid receptors on the receptor mRNA contents

It was demonstrated in the previous study that the microinjection of antisense oligodeoxynucleotide (AS ODN) against mu-opioid receptor (MOR) into periaqueductal gray (PAG) of rat brain selectively decreased the MOR mRNA content in PAG, and the decrease in MOR mRNA content was enhanced by pretreatment of the PAG with MOR AS ODN. In the present investigation, effects of the pretreatment of PAG with AS ODN against kappa- or delta-opioid receptor (KOR or DOR) on the decrease in the MOR mRNA content induced by MOR AS ODN were examined. Both KOR and DOR AS ODNs significantly decreased the target mRNA contents, while they did not significantly change MOR mRNA content. The decrease in MOR mRNA content induced by MOR AS ODN, however, was significantly enhanced by the pretreatment of PAG with either KOR or DOR AS ODNs. Results show that the AS ODN has both the specific target mRNA decreasing action and the nonspecific enhancing action on the AS-induced decrease in the mRNA content.     

Opioid-induced down-regulation of RGS4: role of ubiquitination and implications for receptor cross-talk

Regulator of G protein signaling protein 4 (RGS4) acts as a GTPase accelerating protein to modulate μ- and δ- opioid receptor (MOR and DOR, respectively) signaling. In turn, exposure to MOR agonists leads to changes in RGS4 at the mRNA and/or protein level. Here we have used human neuroblastoma SH-SY5Y cells that endogenously express MOR, DOR, and RGS4 to study opioid-mediated down-regulation of RGS4. Overnight treatment of SH-SY5Y cells with the MOR agonist DAMGO or the DOR agonist DPDPE decreased RGS4 protein by ～60% accompanied by a profound loss of opioid receptors but with no change in RGS4 mRNA. The decrease in RGS4 protein was prevented by the pretreatment with pertussis toxin or the opioid antagonist naloxone. The agonist-induced down-regulation of RGS4 proteins was completely blocked by treatment with the proteasome inhibitors MG132 or lactacystin or high concentrations of leupeptin, indicating involvement of ubiquitin-proteasome and lysosomal degradation. Polyubiquitinated RGS4 protein was observed in the presence of MG132 or the specific proteasome inhibitor lactacystin and promoted by opioid agonist. The loss of opioid receptors was not prevented by MG132, demonstrating a different degradation pathway. RGS4 is a GTPase accelerating protein for both Gα(i/o) and Gα(q) proteins. After overnight treatment with DAMGO to reduce RGS4 protein, signaling at the Gα(i/o)-coupled DOR and the Gα(q)-coupled M(3) muscarinic receptor (M(3)R) was increased but not signaling of the α(2) adrenergic receptor or bradykinin BK(2) receptor, suggesting the development of cross-talk between the DOR and M(3)R involving RGS4.     

Opiate antagonist prevents μ- and δ-opiate receptor dimerization to facilitate ability of agonist to control ethanol-altered natural killer cell functions and mammary tumor growth

In the natural killer (NK) cells, δ-opiate receptor (DOR) and μ-opioid receptor (MOR) interact in a feedback manner to regulate cytolytic function with an unknown mechanism. Using RNK16 cells, a rat NK cell line, we show that MOR and DOR monomer and dimer proteins existed in these cells and that chronic treatment with a receptor antagonist reduced protein levels of the targeted receptor but increased levels of opposing receptor monomer and homodimer. The opposing receptor-enhancing effects of MOR and DOR antagonists were abolished following receptor gene knockdown by siRNA. Ethanol treatment increased MOR and DOR heterodimers while it decreased the cellular levels of MOR and DOR monomers and homodimers. The opioid receptor homodimerization was associated with an increased receptor binding, and heterodimerization was associated with a decreased receptor binding and the production of cytotoxic factors. Similarly, in vivo, opioid receptor dimerization, ligand binding of receptors, and cell function in immune cells were promoted by chronic treatment with an opiate antagonist but suppressed by chronic ethanol feeding. Additionally, a combined treatment of an MOR antagonist and a DOR agonist was able to reverse the immune suppressive effect of ethanol and reduce the growth and progression of mammary tumors in rats. These data identify a role of receptor dimerization in the mechanism of DOR and MOR feedback interaction in NK cells, and they further elucidate the potential for the use of a combined opioid antagonist and agonist therapy for the treatment of immune incompetence and cancer and alcohol-related diseases.     

GABA(A) and GABA(B) receptors differentially modulate volume and frequency in ventilatory compensation in obese Zucker rats.

The aim of this study was to investigate whether GABA(A) and/or GABA(B) receptor-mediated mechanisms contribute to the impaired ventilatory response and reduced maximal aerobic exercise capacity in obese Zucker rats. Ten lean and 10 obese Zucker rats were studied at 12 wk of age. Minute ventilation (Ve), tidal volume (Vt), and breathing frequency (f) during room air breathing and in response to 10 min of hypercapnia (8% CO(2)) and 30 min of hypoxia (10% O(2)) were measured by the barometric method, and peak oxygen consumption (Vo(2 peak)) was measured by an enclosed metabolic treadmill following the randomized blinded subcutaneous administration of equal volumes of DMSO (vehicle), bicuculline (selective GABA(A) receptor antagonist, 1 mg/kg), and phaclofen (selective GABA(B) receptor antagonist, 1 mg/kg). Administration of bicuculline and phaclofen to lean animals had no effect on Ve and Vo(2 peak). Similarly, phaclofen failed to alter Ve and Vo(2 peak) in obese rats, although it did significantly increase f after 5-20 min of hypoxia. In contrast, bicuculline increased Ve and Vt relative to DMSO during room air breathing and after 10-30 min of hypoxic exposure in obese rats, but it did not increase Ve at 5 min of hypoxemia. Bicuculline increased Vo(2 peak) relative to DMSO in obese Zucker rats. We conclude that endogenous GABA acting on GABA(A) receptors can modulate Ve and Vo(2 peak) in obese but not in lean Zucker rats, whereas endogenous GABA acting on GABA(B) receptors modulates f during hypoxia (5-20 min) in obese rats in a very different manner from that when acting on GABA(A) receptors.     

Noladin ether, a putative endocannabinoid, inhibits mu-opioid receptor activation via CB2 cannabinoid receptors

We examined the occurrence of possible changes in mRNA expression and the functional activity of opioid receptors after acute in vivo and in vitro treatment with the putative endogenous cannabinoid noladin ether. While noladin ether (NE) demonstrates agonist activity at CB1 cannabinoid receptors, recent data indicate that NE acts as a full agonist at CB2 cannabinoid receptors too. Considering the functional interactions between opioids and cannabinoids, it is of interest to examine whether NE affects the opioid system. To that end, we studied the influence of NE on mu-opioid receptor (MOR) mRNA expression and MOR mediated G-protein signaling. We used real-time PCR and [35S]GTPgammaS binding assays to examine the changes of MOR mRNA levels and the capability of the mu-opioid agonist peptide ([D-Ala2,(NMe)Phe4,Gly5-ol]enkephalin (DAMGO) in activating regulatory G-proteins via MORs in forebrain membrane fractions of wild-type (w.t., CB1+/+) and CB1 receptor deficient transgenic mice (knockout, CB1-/-). We found, that the expression of MOR mRNAs significantly decreased both in CB1+/+ and CB1-/- forebrain after a single injection of NE at 1 mg/kg when compared to control. Consequently, MOR-mediated signaling is attenuated after acute in vivo treatment with NE in both CB1+/+ and CB1-/- mice. Inhibition on MOR mediated activation is observed after in vitro NE administration as well. Radioligand binding competition studies showed that the noticed effect of NE on MOR signaling is not mediated through MORs. Both in vivo and in vitro attenuations of NE can be antagonized by the CB2 selective antagonist SR144528. Taken together, our data suggest that the NE caused pronounced decrease in the activity of MOR is mediated via CB2 cannabinoid receptors.     

Opioid receptor selectivity profile change via isosterism for 14-O-substituted naltrexone derivatives

Isosterism is commonly used in drug discovery and development to address stability, selectivity, toxicity, pharmacokinetics, and efficacy issues. A series of 14-O-substituted naltrexone derivatives were identified as potent mu opioid receptor (MOR) antagonists with improved selectivity over the kappa opioid receptor (KOR) and the delta opioid receptor (DOR), compared to naltrexone. Since esters are not metabolically very stable under typical physiological conditions, their corresponding amide analogs were thus synthesized and biologically evaluated. Unlike their isosteres, most of these novel ligands seem to be dually selective for the MOR and the KOR over the DOR. The restricted flexibility of the amide bond linkage might be responsible for their altered selectivity profile. However, the majority of the 14-N-substituted naltrexone derivatives produced marginal or no MOR stimulation in the ³⁵S-GTP[γS] assay, which resembled their ester analogs. The current study thus indicated that the 14-substituted naltrexone isosteres are not bioisosteres since they have distinctive pharmacological profile with the regard to their opioid receptor binding affinity and selectivity.     

Antinociceptive profile of LP1, a non-peptide multitarget opioid ligand

AimsOpioid drugs are the principal treatment option for moderate to severe pain and exert their biological effects through interactions with opioid receptors that are widely distributed throughout the CNS and peripheral tissues. Ligands capable of simultaneously targeting different receptors could be successful candidates for the treatment of chronic pain. Enhanced antinociception coupled with a low incidence of side effects has been demonstrated for ligands possessing mixed mu-opioid receptor (MOR) and delta-opioid receptor (DOR) activity. We previously reported that 3-[(2R,6R,11R)-8-hydroxy-6,11-dimethyl-1,4,5,6-tetrahydro-2,6-methano-3-benzazocin-3(2 H)-yl]-N-phenylpropanamide (LP1) acted as a MOR-DOR ligand in in vitro functional assays and moreover this drug produced a valid antinociception that was longer lasting than that of morphine. The aim of this work was to determine whether the antinociceptive effect produced by LP1 was central or peripheral and to assess which opioid receptor subtypes are involved in its effects.Main methodsWe explored the effects of naloxone methiodide (NX-M), a quaternary opioid antagonist, administered either intracerebroventricularly (i.c.v.) or subcutaneously (s.c.), on LP1-mediated antinociception in male Sprague–Dawley rats. In addition, we administered s.c. selective antagonists for MOR, DOR and kappa-opioid receptor (KOR) to investigate the effects of LP1. To characterise this drug's DOR profile better, we also investigated the effects of LP1 on DPDPE, a selective DOR agonist.Key findingsData obtained by tail flick test showed that LP1 induced predominantly MOR-mediated supraspinal antinociception and was able to counteract DPDPE analgesia.SignificanceLP1, a multitarget opioid ligand, is a supraspinal acting antinociceptive agent that is useful for the treatment of chronic pain.