Synthesis and antinociceptive properties of N-phenyl-N-(1-(2-(thiophen-2-yl)ethyl)azepane-4-yl)propionamide in the mouse tail-flick and hot-plate tests

The goals of this study, were to synthesize N-phenyl-N-(1-(2-(thiophen-2-yl)ethyl)azepane-4-yl)propionamide (1c) and determine its antinociceptive properties. The effect of clonidine on 1c antinociception and the involvement of opioid, α₂-adrenergic, and I₂ imidazoline receptors in 1c antinociception were studied. Also examined was the effect of an endothelin ET_A receptor antagonist on 1c antinociception. Synthesis of 1c was accomplished in two steps using modifications of previously reported methods. Antinociceptive (tail-flick and hot-plate) latencies were measured in male Swiss Webster mice treated with 1c; antagonists + 1c; clonidine + 1c; or antagonists + clonidine + 1c. Mice were pretreated with naloxone (opioid antagonist), yohimbine (α₂-adrenoceptor antagonist), idazoxan (α₂-adrenoceptor/I₂-imidazoline antagonist), BU224 (I₂-imidazoline antagonist) or BQ123 (endothelin ET_A receptor antagonist) to study the involvement of these receptors. Compound 1c produced a dose-dependent increase in antinociceptive latencies; ED₅₀ values were 0.15 mg/kg and 0.16 mg/kg, respectively, in the tail flick and hot plate tests. Naloxone, but not yohimbine, idazoxan or BU224, blocked 1c antinociception. Neither clonidine nor BQ123 potentiated 1c antinociception. Results demonstrate that 1c is 15-times more potent than morphine. The antinociceptive effect of 1c is mediated through opioid receptors. The α₂-adrenergic, I₂-imidazoline and endothelin ET_A receptors are not involved in 1c antinociception.     

In vivo characterization of the effects of ghrelin on the modulation of acute pain at the supraspinal level in mice

Ghrelin, an acylated peptide produced in the stomach, increases food intake and growth hormone secretion, inhibits pro-inflammatory cascade, etc. Ghrelin and its receptor (GHS-R1a) mRNA were found in the area related to the regions for controlling pain transmission, such as the hypothalamus, the midbrain, the spinal cord, etc. Ghrelin has been shown to have antinociceptive activity and also anti-inflammatory properties in inflammatory pain and chronic neuropathic pain. Therefore, the aim of the present study was to investigate the effects of ghrelin for the first time in the acute pain modulation at the supraspinal level, using the tail withdrawal test and hot-plate test in mice. Intracerebroventricular (i.c.v.) administration of ghrelin (mouse, 0.1–3 nmol) produced a dose- and time-related antinociceptive effect in the tail withdrawal test and hot-plate test, respectively. Antinociceptive effect elicited by ghrelin (i.c.v., 1 nmol) was significantly antagonized by opioid receptor antagonist naloxone (i.c.v., 10 nmol co-injection or i.p., 10 mg/kg, 10 min prior to ghrelin) in both tail withdrawal test and hot-plate test. At these doses, naloxone significantly antagonized the antinociceptive effect induced by morphine (i.c.v., 3 nmol). Ghrelin (i.c.v., 1 nmol)-induced antinociception was significantly antagonized by co-injection with 10 nmol [d-Lys3]-GHRP-6, the selective antagonist of GHS-R1a identified more recently, while [d-Lys3]-GHRP-6 (10 nmol) alone induced neither hyperalgesia nor antinociception. Overall this data indicate that ghrelin could produce antinociception through an interaction with GHS-R1a and with the central opioid system. Thus ghrelin may be a promising peptide for developing new analgesic drugs.     

Role of the central amygdala GABA-A receptors in morphine state-dependent memory

AimsIn the present experiments, the effects of bilateral microinjections of the GABA-A receptor agonist and/or antagonist into the central amygdala (CeA) on morphine state-dependent memory were examined.Main methodsIn order to assess memory retrieval, a step-through passive avoidance task was used in adult male Wistar rats.Key findingsSubcutaneous (s.c.) administration of morphine (5 and 7.5 mg/kg) immediately after training (post-training) decreased the memory retrieval. Pre-test administration of the opioid (7.5 mg/kg) also induced amnesia. The response induced by post-training morphine (7.5 mg/kg) was significantly reversed by pre-test administration of the drug (5 and 7.5 mg/kg), indicating morphine state-dependent memory. Pre-test intra-CeA microinjection of muscimol, a GABA-A receptor agonist (0.01, 0.02 and 0.03 µg/rat) reduced morphine state-dependent memory. However, the same doses of muscimol by itself had no effect on memory retrieval. Furthermore, pre-test intra-CeA microinjection of bicuculline, a GABA-A receptor antagonist by itself did not alter memory retrieval. The antagonist also did not change post-training morphine (7.5 mg/kg)-induced amnesia, but in combination with a lower dose of morphine (0.5 mg/kg), improved memory performance. Moreover, muscimol's ability to interfere with morphine state-dependent memory was reversed by co-injection of bicuculline.SignificanceThe results suggest that GABA-A receptor mechanism of the CeA may influence morphine state-dependent memory.     

Design,synthesis, and characterization of 6β-naltrexol analogs,and their selectivity for in vitro opioid receptor subtypes

Since the mu opioid receptor (MOR) is known to be involved in the therapeutically relevant pathways leading to the manifestation of pain and addiction, we are currently studying the specific structural characteristics that promote antagonism at the MOR. The opiates 6β-naltrexol and 6β-naltrexamide function as neutral antagonists in in vitro and in vivo systems previously exposed to morphine, and are under investigation as improved treatments for narcotic dependence. In this research, we synthesized and characterized carbamate and sulfonate ester derivates of 6β-naltrexol that do not contain a protic group at C₆, and evaluated these compounds for opioid receptor affinity. In vitro receptor subtype (μ, κ, and δ opioid receptors) binding data of the carbamate and sulfonate derivatives is reported. All four compounds synthesized exhibited affinity for the MOR better than the standard 6β-naltrexol HCl. Based on K_i data, the order of MOR affinity is as follows: 9 > 13 > 14 > 10 > 6β-naltrexol HCl. Carbamate 9 and tosylate 13 displayed subnanomolar affinity for the MOR, while 10 was the most μ-selective compound synthesized. In conclusion, our data indicate that the absence of a hydrogen-bond donor on the C₆ oxygen enhances rather than impedes the in vitro affinity of naltrexol derivatives for the MOR. Additionally, data also suggest that increasing the bulk around C₆ may allow control of subtype selectivity within these compound series.     

Antagonism by nalmefene of systemic and intrathecal morphine-induced analgesia in mice

Nalmefene is an orally active opiate antagonist structurally related to naloxone and naltrexone. In this study using two different strains of mice (Swiss Cox and ICR), the antagonist activity of nalmefene given subcutaneously (sc) was quantified by determination of the apparent pA2 values against the antinociceptive activity (tail flick and hot plate tests) of morphine given sc or intrathecally (lumbar spinal cord). The apparent pA2 values (constrained to a slope of -1) were 8.06 (7.79-8.33) in Swiss Cox mice and 7.81 (7.62-8.00) in ICR mice in the tail flick test with sc morphine. These values were larger than the corresponding value for naloxone in ICR mice, 7.35 (7.10-7.60). The hot plate test provided similar results: the apparent pA2 values for nalmefene with sc morphine were 8.14 (7.89-8.39) in Swiss Cox mice and 7.81 (7.65-7.97) in ICR mice, values which were different from naloxone 7.33 (7.23-7.42) in ICR mice. Apparent pA2 values for nalmefene with intrathecal morphine were not different from those for naloxone in the tail flick test. Thus, these sets of results suggest that it may be worthwhile to further determine whether systemic nalmefene might possibly possess an advantage over naloxone in antagonizing systemic side effects of morphine arising from local spinal morphine administration.     

Modulation of delta opioid agonist-induced antinociception by repeated morphine pretreatment in rhesus monkeys

AimsRepeated treatment with morphine increases antinociceptive effects of delta opioid agonists in rodents by a mechanism that may involve increased cell-surface expression of delta receptors. The present study evaluated effects of repeated morphine treatment on behavioral effects of the delta agonist SNC80 and the mu agonist fentanyl in rhesus monkeys.Main methodsIn an assay of schedule-controlled responding, three monkeys responded for food reinforcement under a fixed-ratio 30 schedule. In an assay of thermal nociception, tail-withdrawal latencies were evaluated in three monkeys using thermal stimulus intensities of 48 and 54 °C. In both assays, the effects of SNC80 (0.032–3.2 mg/kg) and fentanyl (0.001–0.056 mg/kg) were evaluated after repeated treatment with saline or a regimen of morphine doses modeled on the regimen that enhanced delta agonist antinociception and apparent delta receptor availability in previous rodent studies.Key findingsBoth SNC80 and fentanyl dose-dependently decreased rates of schedule-controlled responding, and repeated morphine treatment did not significantly alter these effects. In the assay of thermal nociception, SNC80 had little effect on tail-withdrawal latencies from water heated to 48 or 54 °C, whereas fentanyl increased tail-withdrawal latencies at both temperatures. Repeated morphine tended to increase the antinociceptive effects of SNC80 and to decrease the antinociceptive effects of fentanyl, but these effects of repeated morphine were small and were significant only at the higher stimulus intensity (54 °C).SignificanceThese results provide limited support for the proposition that prior stimulation of mu receptors selectively increases the antinociceptive effects of delta agonists in rhesus monkeys.     

Attenuation of systemic morphine-induced analgesia by central administration of ghrelin and related peptides in mice

Ghrelin, an acylated 28-amino peptide secreted in the gastric endocrine cells, has been demonstrated to stimulate the release of growth hormone, increase food intake, and inhibit pro-inflammatory cascade, etc. Ghrelin mainly combines with its receptor (GHS-R1α) to play the role in physiological and pathological functions. It has been reported that ghrelin plays important roles in the control of pain through interaction with the opioid system in inflammatory pain and acute pain. However, very few studies show the effect of supraspinal ghrelin system on antinociception induced by intraperitoneal (i.p.) administration of morphine. In the present study, intracerebroventricular (i.c.v.) injection of ghrelin (0.1, 1, 10 and 100 nmol/L) produced inhibition of systemic morphine (6 mg/kg, i.p.) analgesia in the tail withdrawal test. Similarly, i.c.v. injection GHRP-6 and GHRP-2 which are the agonists of GHS-R1α, also decreased analgesia effect induced by morphine injected intraperitoneally in mice. Furthermore, these anti-opioid activities of ghrelin and related peptides were not blocked by pretreatment with the GHS-R1α selective antagonist [d-Lys³]-GHRP-6 (100 nmol/L, i.c.v.). These results demonstrated that central ghrelin and related peptides could inhibit the analgesia effect induced by intraperitoneal (i.p.) administration of morphine. The anti-opioid effects of ghrelin and related peptides do not interact with GHS-R1a. These findings may pave the way for a new strategy on investigating the interaction between ghrelin system and opioids on pain modulation.     

Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine.In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid (μ, δ, k), cannabinoid (CB₁, CB₂), glutamatergic (NMDA), GABAergic (GABA_A, GABA_B), serotoninergic (5HT_2A) and adrenergic (α₂) receptors.The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABA_A and moderate affinity for GABA_B, NMDA and δ opioid receptors.WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABA_A, GABA_B, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.     

Kososan,a standardized traditional Japanese herbal medicine,reverses sleep disturbance in socially isolated mice via GABAA-benzodiazepine receptor complex activation

PurposeKososan (KSS), a traditional Japanese medicine with a distinct aroma, is clinically used to treat affective disorders but its antidepressant-like effect has not been thoroughly investigated. In this study, we investigated the effects of inhaled and orally administered KSS on sleep disturbances in socially isolated mice.MethodsFour-weeks-old male ddy mice were housed either in social isolation or in groups for 4–6 weeks before the experiment. KSS was orally administered (0.5 or 1.0 g/kg) or inhaled (0.5, 1.0, or 2.5 g/0.125 m³) 60 min before pentobarbital administration. Stress levels in mice were evaluated by the duration of pentobarbital-induced sleeping time.ResultsSleeping time was shorter in socially-isolated mice than in group-housed mice. Oral and inhaled KSS prolonged sleeping time in stressed mice, but had no effect on sleeping time of group-housed mice. Prolonged sleeping time after oral KSS was significantly inhibited (p < 0.05) by bicuculline (3 mg/kg, i.p.), a GABA_A antagonist, but not by flumazenil (3 mg/kg, i.p.), a selective benzodiazepine antagonist. Prolonged sleeping time after KSS inhalation was significantly inhibited (p < 0.05) by flumazenil but not by bicuculline.ConclusionsOur findings suggest that KSS activates GABA_A-benzodiazepine receptor complex and reverses shortened pentobarbital-induced sleep caused by social isolation.     

Effects of acute and chronic morphine and narcotic antagonists on brain energy metabolism

Morphine sulphate (4 mg/kg to 32 mg/kg) produced a dose-dependent decrease in brain malate as antinociception increased. Decreased brain malate persisted 72 hours after implantation of morphine pellets by which time mice had become tolerant to antinociception. This finding suggests that malate decrease, unlike changes of other metabolites in other studies, might not be simply a result of general metabolic changes. Malate change as well as antinociception was prevented by prior injection of naloxone (3.0 mg/kg) or naltrexone (0.6 mg/kg) in acute experiments. Malate decrease in pelleted mice was no longer present if withdrawal was produced by naloxone or naltrexone in mice implanted with morphine pellets for 72 hours. Brain P-creatine was elevated in all mice implanted with morphine pellets even after withdrawal, thus, apparently, representing a more generalized effect than malate change.     

Effects of methylnaltrexone on morphine-induced cough suppression in guinea pigs

We administered methylnaltrexone, a peripheral opioid receptor antagonist, to guinea pigs previously injected with morphine sulfate to determine whether the compound could block opioid-induced cough suppression without blocking antinociception. The effects of methylnaltrexone (2.0, 1.6, 0.8 mg/kg) and of naltrexone (0.32, 0.16, 0.02 and 0.01 mg/kg) were compared in animals who had been injected with morphine sulfate (8.1 mg/kg). At 2.0 mg/kg methylnaltrexone, number of coughs returned to baseline value and nociception remained unaffected. At the two higher doses of naltrexone (0.32 and 0.16 mg/kg), morphine-induced antitussive effect was blocked, but antinociception was reversed. Our results suggested that methylnaltrexone possesses opioid antagonist activity in receptors peripheral to the blood-brain barrier. Its peripheral activity makes methylnaltrexone a clinically interesting agent for maintaining the cough reflex in those who must take opioids for analgesia.     

Effect of Bacopa monniera on liver and kidney toxicity in chronic use of opioids

In the present study, we investigated the protective effect of Bacopa monniera, an indigenous Ayurvedic medicinal plant in India, against morphine-induced liver and kidney toxicity in rats. Morphine intoxicated rats received 10-160 mg/kg body weight of morphine hydrochloride intraperitoneally for 21 days. Bacopa monniera Extract (BME) pretreated rats were administered with BME (40 mg/kg) orally once a day 2 h before the injection of morphine for 21 days. Pretreatment with BME has shown to possess a significant protective effect against morphine-induced liver and kidney functions in terms of serum glutamate oxaloacetate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, lactate dehydrogenases and gamma-glutamyl transferase activities and urea, creatinine and uric acid level respectively. Histopathological changes of liver and kidney were also in accordance with the biochemical findings. The results of this study indicate that Bacopa monniera extract exerted a protection against morphine-induced liver and kidney toxicity.     

The putative somatostatin antagonist cyclo-somatostatin has opioid agonist effects in gastrointestinal preparations

AimsSpecificity of receptor antagonists used is crucial for clarifying physiological/pathophysiological roles of the respective endogenous agonist. We studied the effects (somatostatin antagonist and possibly other actions) of cyclo-somatostatin (CSST), a putative somatostatin receptor antagonist on the guinea-pig small intestine, a preparation where somatostatin causes inhibition of nerve-mediated contractions.Main methodsIn isolated organ experiments, half-maximal cholinergic “twitch” contractions of the guinea-pig small intestine were evoked or tonic contractions of the rat stomach fundus strip (in the presence of physostigmine) were elicited by electrical field stimulation. The effects of somatostatin (somatostatin-14), CSST, naloxone, as well as of direct smooth muscle stimulants were examined.Key findingsSomatostatin (10 nM–1 μM) caused transient inhibition of the twitch contraction, in a naloxone-insensitive manner. Surprisingly, CSST (0.3–1 μM) also inhibited twitch contractions (more than 50% reduction at 1 μM). This effect was prevented by the opioid receptor antagonist naloxone. Responses to acetylcholine or histamine were not or only minimally inhibited by CSST (up to 3 μM). CSST (0.3 μM in the absence or 1–10 μM in the presence of naloxone) failed to inhibit the effect of somatostatin. The SST₂ receptor antagonist CYN-154806 (3 μM) attenuated the effect of somatostatin and failed to evoke naloxone-sensitive inhibition of the twitch response. The naloxone-sensitive inhibitory effect of CSST on cholinergic contractions was also confirmed in the rat stomach fundus preparation.SignificanceCyclo-somatostatin exerts opioid agonist activity in the two preparations tested, while it does not behave as a somatostatin-receptor antagonist in the guinea-pig intestine.     

Blockade of Nitric Oxide Overproduction and Oxidative Stress by <Emphasis Type="Italic">Nigella sativa</Emphasis> Oil Attenuates Morphine-Induced Tolerance and Dependence in Mice

The effects of Nigella sativa oil on morphine-induced tolerance and dependence in mice and possible mechanism(s) of these effects were investigated, for the first time, in this study. Repeated administration of Nigella sativa oil (4 ml/kg, p.o.) along with morphine (5 mg/kg, s.c.) attenuated the development of tolerance, as measured by the hot plate test, and dependence, as assessed by naloxone (5 mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of morphine to mice or by administration of naloxone to morphine-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments were inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of the oil on morphine-induced tolerance and dependence and on naloxone-induced biochemical alterations in morphine-dependent mice was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25 mg/kg). Similarly, concurrent i.p. administration of the NO synthase inhibitors; L-N (G)-nitroarginine methyl ester (10 mg/kg), aminoguanidine (20 mg/kg) and 7-nitroindazole (25 mg/kg) or the antioxidant, N-acetylcysteine (50 mg/kg) enhanced this inhibitory effect of the oil. On the other hand, this effect was antagonized by concurrent i.p. administration of the nitric oxide precursor, L-arginine (300 mg/kg). These results provide evidence that Nigella sativa oil, through inhibition of morphine-induced NO overproduction and oxidative stress, appears to have a therapeutic potential in opioid tolerance and dependence.     

Probable involvement of Ca2 +-activated Cl− channels (CaCCs) in the activation of CB1 cannabinoid receptors

AimsRecently, we demonstrated that peripheral antinociception induced by δ opioid receptor is dependent of Ca^2 +-activated Cl^? channels (CaCCs). Because opioid and cannabinoid receptors share some common mechanisms of action, our objective was to identify a possible relationship between CaCCs and the endocannabinoid system.Main methodsTo induce hyperalgesia, rat paws were treated with intraplantar prostaglandin E₂ (PGE₂, 2 μg). Nociceptive thresholds to pressure (grams) were measured using an algesimetric apparatus 3 h following injection. Probabilities were calculated using ANOVA/Bonferroni's test, and values that were less than 5% were considered to be statistically significant.Key findingsAdministration of the cannabinoid agonist CB₁ anandamide (12.5, 25 and 50 μg/paw) and the cannabinoid agonist CB₂ PEA (5, 10 and 20 μg/paw) decreased the PGE₂-induced hyperalgesia in a dose-dependent manner. The possibility of the higher doses of anandamide (50 μg) and PEA (20 μg) having a central or systemic effect was excluded because the administration of the drug into the contralateral paw did not elicit antinociception in the right paw. As expected, the antinociceptive effects induced by anandamide and PEA were blocked by the CB₁ and CB₂ receptor antagonists AM251 and AM630, respectively. The peripheral antinociception was induced by anandamide but not PEA and was dose-dependently inhibited by the CaCC blocker niflumic acid (8, 16 and 32 μg).SignificanceThese results provide the first evidence for the involvement of CaCCs in the peripheral antinociception induced by activation of the CB₁ cannabinoid receptor.     

Evaluation of N-substituent structural variations in opioid receptor profile of LP1

The benzomorphan scaffold has great potential as lead structure and the nature of the N-substituent is able to influence affinity, potency, and efficacy at all three opioid receptors. Building upon these considerations, we synthesized a new series of LP1 analogues by introducing naphthyl or heteroaromatic rings in propanamide side chain of its N-substituent (9–15). In vitro competition-binding assays in HEK293 cells stably expressing MOR, DOR or KOR showed that in compound 9 the 1-naphthyl ring led to the retention of MOR affinity (K_i^MOR = 38 ± 4 nM) displaying good selectivity versus DOR and KOR. In the electrically stimulated GPI, compound 9 was inactive as agonist but produced an antagonist potency value (pA₂) of 8.6 in presence of MOR agonist DAMGO. Moreover, subcutaneously administered it antagonized the antinociceptive effects of morphine with an AD₅₀ = 2.0 mg/kg in mouse-tail flick test. Modeling studies on MOR revealed that compound 9 fit very well in the binding pocket but in a different way in respect to the agonist LP1. Probably the replacement of its N-substituent on the III, IV and V TM domains reflects an antagonist behavior. Therefore, compound 9 could represent a potential lead to further develop antagonists as valid therapeutic agents and useful pharmacological tools to study opioid receptor function.     

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.     

Inverse agonists and neutral antagonists at µ opioid receptor (MOR): possible role of basal receptor signaling in narcotic dependence

The mu opioid receptor, MOR, displays spontaneous agonist-independent (basal) G protein coupling in vitro. To determine whether basal MOR signaling contributes to narcotic dependence, antagonists were tested for intrinsic effects on basal MOR signaling in vitro and in vivo, before and after morphine pretreatment. Intrinsic effects of MOR ligands were tested by measuring GTPgammaS binding to cell membranes and cAMP levels in intact cells. beta-CNA, C-CAM, BNTX, and nalmefene were identified as inverse agonists (suppressing basal MOR signaling). Naloxone and naltrexone were neutral antagonists (not affecting basal signaling) in untreated cells, whereas inverse agonistic effects became apparent only after morphine pretreatment. In contrast, 6alpha- and 6beta-naltrexol and -naloxol, and 6beta-naltrexamine were neutral antagonists regardless of morphine pretreatment. In an acute and chronic mouse model of morphine-induced dependence, 6beta-naltrexol caused significantly reduced withdrawal jumping compared to naloxone and naltrexone, at doses effective in blocking morphine antinociception. This supports the hypothesis that naloxone-induced withdrawal symptoms result at least in part from suppression of basal signaling activity of MOR in morphine-dependent animals. Neutral antagonists have promise in treatment of narcotic addiction.     

Acute effects of cocaine,morphine and their combination on bioenergetic function and susceptibility to oxidative stress of rat liver mitochondria

AimsCocaine and heroin are frequently co-abused in a combination known as speedball. Despite the relevance of the liver in the metabolism and detoxification of these drugs, little is known about the impact of speedball on liver function.Main methodsIn this work, we evaluated the effects of cocaine, morphine and morphine + cocaine (Mor + Coc) combination (1:1) in isolated rat liver mitochondria, upon glutamate/malate or succinate energization, on bioenergetics and oxidative stress-related parameters by using Clark O₂, Ca^2 +, TPP⁺ and pH electrodes and by measuring thiobarbituric acid reactive substances (TBARS) and H₂O₂ production.Key findingsCocaine and Mor + Coc at the higher concentrations (1 mM) similarly increased O₂ consumption at state 2, state 4 and state oligomycin. In these conditions, maximum respiration was decreased only upon glutamate/malate energization, suggesting an involvement of complex I. Morphine (1 mM) only increased state 2 respiration. Cocaine and Mor + Coc induced a similar decrease in maximum mitochondrial membrane potential and in ADP-induced depolarization, whereas morphine had no effect. The drugs and their combination similarly decreased mitochondrial ATPase activity and had no effect on Ca^2 +-induced permeability transition. Morphine and Mor + Coc prevented lipid peroxidation, since in these conditions there was a decrease in O₂ consumption and in TBARS upon ADP/Fe^2 + stimulus, and a decrease in H₂O₂ formation, suggesting an antioxidant effect. Interestingly, heroin did not share morphine antioxidant properties.SignificanceOur results show that the sequential direct exposure of liver mitochondria to morphine and cocaine does not alter the effects observed in the presence of each drug alone.