Selective Cannabinoid Receptor Research Articles

Cross state-dependency of learning between arachidonylcyclopropylamide (ACPA) and muscimol in the mouse dorsal hippocampus

The aim of the present study was to examine cross state-dependent learning between ACPA (a selective cannabinoid CB1 receptor agonist) and muscimol (a selective GABAA receptor agonist) in the step-down inhibitory avoidance learning task. The dorsal hippocampal CA1 regions of adult male NMRI mice were bilaterally cannulated, and all drugs were microinjected into the intended sites of injection.Post-training and/or pre-test administration of ACPA (1 and 2ng/mouse) dose-dependently induced amnesia. Pre-test microinjection of the same doses of ACPA reversed the post-training ACPA-induced amnesia. This event has been named ACPA state-dependent learning (SDL).Post-training and/or pre-test microinjection of muscimol (0.05 and 0.1μg/mouse) dose-dependently induced amnesia. Pre-test administration of the same doses of muscimol reversed the post-training muscimol-induced amnesia, suggesting muscimol SDL.The amnesia induced by post-training administration of ACPA was reversed by pre-test administration of muscimol (0.05 and 0.1μg/mouse). Furthermore, the pre-test microinjection of muscimol (0.025 and 0.05μg/mouse) with an ineffective dose of ACPA (0.5ng/mouse) significantly restored memory retrieval and induced ACPA SDL.In another series of experiments, the amnesia induced by post-training administration of muscimol was reversed by pre-test administration of ACPA (1 and 2ng/mouse). Moreover, pre-test microinjection of ACPA (0.5 and 1ng/mouse) with an ineffective dose of muscimol (0.025μg/mouse) significantly restored memory retrieval and induced muscimol SDL.It is important to note that pre-test intra-CA1 injection of a selective GABAA receptor antagonist, bicuculline (0.125 and 0.25μg/mouse), 5min before the administration of muscimol (0.1μg/mouse) or ACPA (2ng/mouse) dose-dependently inhibited muscimol- and ACPA-induced SDL, respectively. Pre-test intra-CA1 administration of bicuculline (0.0625, 0.125 and 0.25μg/mouse) by itself did not affect memory retention.In conclusion, the data strongly revealed a cross SDL among ACPA and muscimol in the dorsal hippocampal CA1 regions.

Novel Peripherally Restricted Cannabinoid 1 Receptor Selective Antagonist TXX-522 with Prominent Weight-Loss Efficacy in Diet Induced Obese Mice.

The clinical development of the first generation of globally active cannabinoid 1 receptor (CB1R) antagonists was suspended because of their adverse neuropsychiatric effects. Selective blockade of peripheral CB1Rs has the potential to provide a viable strategy for the treatment of severe obesity while avoiding these central nervous system side effects. In the current study, a novel compound (TXX-522) was rationally designed based on the parent nucleus of a classical CB1R-selective antagonist/inverse agonist, rimonabant (SR141716A). Docking assays indicate that TXX-522 was bound with the CB1R in a mode similar to that of SR141716A. TXX-522 showed good binding, CB1R-selectivity (over the CB2R), and functional antagonist activities in a range of in vitro molecular and cellular assays. In vivo analysis of the steady state distribution of TXX-522 in the rat brain and blood tissues and the assay of its functional effects on CB1R activity collectively showed that TXX-522 showed minimal brain penetration. Moreover, the in vivo pharmacodynamic study further revealed that TXX-522 had good oral bioavailability and a potent anti-obesity effect, and ameliorated insulin resistance in high-fat diet-induced obese mice. No impact on food intake was observed in this model, confirming the limited brain penetration of this compound. Thus, the current study indicates that TXX-522 is a novel and potent peripherally acting selective CB1R antagonist with the potential to control obesity and related metabolic disorders.

Effects of arachidonyl-2'-chloroethylamide (ACEA) on the protective action of various antiepileptic drugs in the 6-Hz corneal stimulation model in mice.

Accumulating evidence indicates that cannabinoid CB1 receptor ligands play a pivotal role in seizures, not only in preclinical studies on animals, but also in clinical settings. This study was aimed at characterizing the influence of arachidonyl-2′-chloroethylamide (ACEA–a selective cannabinoid CB1 receptor agonist) co-administered with phenylmethylsulfonyl fluoride (PMSF) on the anticonvulsant potency of various antiepileptic drugs (clobazam, lacosamide, levetiracetam, phenobarbital, tiagabine and valproate) in the 6-Hz corneal stimulation model. Psychomotor seizures in male albino Swiss mice were evoked by a current (32 mA, 6 Hz, 3 s stimulus duration) delivered via corneal electrodes. Potential adverse effects produced by the antiepileptic drugs in combination with ACEA+PMSF were assessed using the chimney test (motor performance), passive avoidance task (remembering and acquisition of learning), and grip-strength test (muscular strength). Brain concentrations of antiepileptic drugs were measured by HPLC to exclude any pharmacokinetic contribution to the observed effect. ACEA (5 mg/kg, i.p.) + PMSF (30 mg/kg, i.p.) significantly potentiated the anticonvulsant potency of levetiracetam (P<0.05), but not that of clobazam, lacosamide, phenobarbital, tiagabine or valproate in the 6-Hz corneal stimulation model. Moreover, ACEA+PMSF did not significantly affect total brain concentrations of levetiracetam in mice. No behavioral side effects were observed in animals receiving combinations of the studied antiepileptic drugs with ACEA+PMSF. In conclusion, the combined administration of ACEA+PMSF with levetiracetam is associated with beneficial anticonvulsant pharmacodynamic interaction in the 6-Hz corneal stimulation model. The selective activation of cannabinoid CB1 receptor-mediated neurotransmission in the brain may enhance levetiracetam-related suppression of seizures in epilepsy patients, contributing to the efficacious treatment of epilepsy in future.

Cannabinoid CB1 receptor inverse agonist MJ08 stimulates glucose production via hepatic sympathetic innervation in rats

As a key insulin target tissue for maintaining systemic glucose homeostasis, the liver plays important roles in improving obesity-associated insulin intolerance via selective cannabinoid CB1 receptor antagonism/inverse agonism. However, it is unclear whether this receptor inverse agonism affects hepatic glucose metabolism. MJ08 is a novel cannabinoid CB1 receptor antagonist/inverse agonist that has superior inverse agonism over the well-known antagonist/inverse agonist, SR141716 (rimonabant). MJ08 remarkably elevates fasting blood glucose independent of inhibition of insulin release in mice. In the current study, MJ08 was used to investigate the mechanism by which liver cannabinoid CB1 receptor inverse activation regulates hepatic glucose metabolism. MJ08 stimulated hepatic glucose production (HGP) in a dose-dependent manner and promoted gluconeogenic gene expression in perfused rat liver. SR141716 exhibited similar but weaker effects. The cannabinoid CB1 receptor agonist (WIN 55,212-2), Gs protein-cyclic AMP (cAMP)-dependent pathway inhibitors (NF449 and H89), β-adrenoceptor antagonist (propranolol), and peripheral sympathetic inhibitor (reserpine) could antagonize MJ08-induced HGP. Furthermore, MJ08 and SR141716 induced monoamine neurotransmitter (noradrenaline) release and increased cAMP content significantly in perfused liver, although only a slight increase was observed in primary cultured hepatocytes. These results indicate that local liver cannabinoid CB1 receptor inverse agonism via hepatic sympathetic innervation is responsible for the HGP induced by MJ08. Thus, high inverse agonistic activity could increase fasting blood glucose levels and should be avoided in the development of peripheral cannabinoid CB1 receptor-targeted weight-loss drugs.

The synthetic cannabinoid WIN-55,212 induced-apoptosis in cytotrophoblasts cells by a mechanism dependent on CB1 receptor.

The endocannabinoid system has evolved as a key regulator in several pathological and physiological processes, including placentation, decidualization and implantation. In addition, it is known that Cannabis and cannabinoids negatively affect female reproduction. Although, the biological action of synthetic cannabinoids, such as WIN-55,212, in human fertility and pregnancy outcome remain to be unveiled. A tight balance between proliferation, differentiation and apoptosis of trophoblast cells is required for placental development and pregnancy outcome. Therefore, in this work, the effects of the synthetic cannabinoid WIN-55,212 in placental cytotrophoblast cells were explored. For that, it was used a human choriocarcinoma cell line, BeWo cells, and primary cultures of human cytotrophoblasts isolated from term placentas. Results demonstrate that this synthetic cannabinoid induces cell cycle arrest. We also observed that cell viability loss was associated with a disruption of mitochondrial membrane potential and activation of caspases -9 and -3/-7 independently of reactive oxygen species (ROS) production or recruitment of the endoplasmic reticulum stress marker CHOP. Moreover, these effects were prevented by pre-incubation with a selective cannabinoid receptor 1 (CBR1) antagonist (AM281). Thus, our results provide strong evidences of the apoptotic process induced by WIN-55,212 through the activation of the CBR1, which may reveal the impact of cannabinoids consumption during placental development.

The major neurotransmitter systems in the basolateral amygdala and the ventral tegmental area mediate morphine-induced memory consolidation impairment

In the present study, we investigated the possible participation of the endocannabinoid system in the basolateral amygdala and N-methyl-d-aspartate (NMDA) or GABA-A receptor neurotransmission in the ventral tegmental area in the memory consolidation impairment induced by morphine administration. To measure memory formation, step-through type passive avoidance apparatus was used with adult male Wistar rats. The results showed that intraperitoneal (i.p.) administration of morphine (3 and 6mg/kg) after the successful training phase had an amnestic effect and induced memory consolidation impairment. After training, injection of a selective cannabinoid CB1 receptor agonist, arachydonilcyclopropylamide (ACPA; 0.4–0.6ng/rat) plus systemic injection of an ineffective dose of morphine (0.5mg/kg, i.p.) into the basolateral amygdala impaired memory consolidation suggesting the facilitatory effect of ACPA on morphine response. Also, the results showed that the injection of bicuculline, a GABA-A receptor antagonist (0.3–0.5µg/rat) or NMDA (0.005–0.02µg/rat) into the ventral tegmental area reversed ACPA-induced potentiation of morphine response and improved memory consolidation. It should be considered that the injection of ACPA into the basolateral amygdala and the injection of bicuculline or NMDA into the ventral tegmental area alone could not affect memory consolidation. Taken together, it seems that there is a functional interaction between the basolateral amygdala endocannabinoid system and the ventral tegmental area GABAergic- or glutamatergic neurotransmission in the modulation of morphine-induced memory consolidation impairment.

Rimonabant, a selective cannabinoid1 receptor antagonist, protects against light-induced retinal degeneration in vitro and in vivo

The endocannabinoid system is involved in some neurodegenerative diseases such as Alzheimer's disease. An endogenous constellation of proteins related to cannabinoid1 receptor signaling, including free fatty acids, diacylglycerol lipase, and N-acylethanolamine-hydrolyzing acid amidase, are localized in the murine retina. Moreover, the expression levels of endogenous agonists of cannabinoid receptors are changed in the vitreous fluid. However, the role of the endocannabinoid system in the retina, particularly in the light-induced photoreceptor degeneration, remains unknown. Therefore, we investigated involvement of the cannabinoid1 receptor in light-induced retinal degeneration using in vitro and in vivo models. To evaluate the effect of cannabinoid1 receptors in light irradiation-induced cell death, the mouse retinal cone-cell line (661W) was treated with a cannabinoid1 receptor antagonist, rimonabant. Time-dependent changes of expression and localization of retinal cannabinoid1 receptors were measured using Western blot and immunostaining. Retinal damage was induced in mice by exposure to light, followed by intravitreal injection of rimonabant. Electroretinograms and histologic analyses were performed. Rimonabant suppressed light-induced photoreceptor cell death. Cannabinoid1 receptor expression was upregulated by light exposure. Treatment with rimonabant improved both a- and b-wave amplitudes and the thickness of the outer nuclear layer. These results suggest that the cannabinoid1 receptor is involved in light-induced retinal degeneration and it may represent a therapeutic target in the light-induced photoreceptor degeneration related diseases.

Immunomodulatory properties of 1,2-dihydro-4-hydroxy-2-oxo-1,8-naphthyridine-3-carboxamide derivative VL15

1,2-Dihydro-4-hydroxy-2-oxo-1,8-naphthyridine-3-carboxamide derivative VL15 has been recently developed as a selective cannabinoid CB2 receptor compound. Given the high selectivity of this compound at the cannabinoid CB2 receptor and the well-known protective function of this receptor in neurological disorders with autoimmune component like multiple sclerosis, we assessed the immunomodulatory properties of VL15. We assessed on activated peripheral blood mononuclear cells), proliferation and viability, cell cycle progression and measured activation markers and the expression of phosphorylated proteins. We found that VL15 reduces PBMC proliferation slightly affecting cell vitality, blocks the cell cycle progression and down-regulates the levels of T cell activation markers as well as the expression of phosphorylated proteins, NF-kB, IKKαβ, IKBα, ERK and Akt. VL15 was also used in drug-permeability assays on Caco-2 cell line to evaluate its oral bioavailability and on MDCKII-hMDR1 cell lines to estimate its propensity to cross the blood-brain barrier by passive diffusion, in order to potentially maintain its efficiency on the infiltrating auto-reactive lymphocytes in the central nervous system. In these models, VL15 showed high intestinal absorption and good blood-brain barrier penetration. Our findings suggest that VL15, by controlling the immune response, might find potential application as orally administered drug in pathologies like multiple sclerosis.

NMDA receptor adjusted co-administration of ecstasy and cannabinoid receptor-1 agonist in the amygdala via stimulation of BDNF/Trk-B/CREB pathway in adult male rats.

Consumption of cannabinoid receptor-1 (CB-1) agonist such as cannabis is widely taken in 3,4- methylenedioxymethamphetamine (MDMA) or ecstasy users; it has been hypothesized that co-consumption of CB-1 agonist might protect neurons against MDMA toxicity. N-methyl-d-aspartate (NMDA) receptors regulate neuronal plasticity and firing rate in the brain through Tyrosine-kinase B (Trk-B) activation. The molecular and electrophysiological association among NMDA and MDMA/Arachidonylcyclopropylamide (ACPA, a selective CB-1 receptor agonist) co-consumption was not well-known. Here, neuronal spontaneous activity, Brain-derived neurotrophic factor (BDNF), Trk-B and cAMP response element binding protein (CREB) phosphorylation levels were recognized in ACPA and MDMA co-injected rats. Besides, we proved the role of NMDA receptor on MDMA and ACPA combination on neuronal spontaneous activity and Trk-B/BDNF pathway in the central amygdala (CeA). Male rats were anesthetized with intra-peritoneal injections of urethane; MDMA, D-2-amino-5-phosphonopentanoate (D-AP5, NMDA receptor antagonist) were injected into CeA. ACPA was administrated by intra-cerebroventricular injection. Thirty minutes following injections, neuronal firing rate was recorded from CeA. Two hours after drug injection, amygdala was collected from brain for molecular evaluations. Single administration of MDMA and/or ACPA reduced firing rates compared with sham group in the CeA dose-dependently. Injection of D-AP5, ACPA and MDMA reduced firing rate compared with sham group (P<0.001). Interestingly, injection of ACPA+MDMA enhanced BDNF, Trk-B and CREB phosphorylation compared with MDMA groups. D-AP5, ACPA and MDMA co-injection decreased BDNF, Trk-B and CREB phosphorylation levels compared with ACPA+MDMA in the amygdala (P<0.01). Probably, NMDA receptors are involved in the protective role of acute MDMA+ACPA co-injection via BDNF/Trk-B/CREB pathways.

Antihyperalgesic effect of CB1 receptor activation involves the modulation of P2X3 receptor in the primary afferent neuron

Cannabinoid system is a potential target for pain control. Cannabinoid receptor 1 (CB1) activation play a role in the analgesic effect of cannabinoids once it is expressed in primary afferent neurons. This study investigates whether the anti-hyperalgesic effect of CB1 receptor activation involves P2X3 receptor in primary afferent neurons. Mechanical hyperalgesia was evaluated by electronic von Frey test. Cannabinoid effect was evaluated using anandamide or ACEA, a non-selective or a selective CB1 receptor agonists, respectively; AM251, a CB1 receptor antagonist, and antisense ODN for CB1 receptor. Calcium imaging assay was performed to evaluated α,β-meATP-responsive cultured DRG neurons pretreated with ACEA. Anandamide or ACEA administered in peripheral tissue reduced the carrageenan-induced mechanical hyperalgesia. The reduction in the carrageenan-induced hyperalgesia induced by ACEA was completely reversed by administration of AM251 as well as by the intrathecal treatment with antisense ODN for CB1 receptor. Also, ACEA reduced the mechanical hyperalgesia induced by bradykinin and by α,β-meATP, a P2X3 receptor non-selective agonist, but not by tumor necrosis factor alpha (TNF-α), interleukin-1 beta (IL-1β) and chemokine-induced chemoattractant-1 (CINC-1). Finally, CB1 receptors are co-localized with P2X3 receptors in DRG small-diameter neurons and the treatment with ACEA reduced the number of α,β-meATP-responsive cultured DRG neurons. Our data suggest that the analgesic effect of CB1 receptor activation is mediated by a negative modulation of the P2X3 receptor in the primary afferent neurons.

Activation of cannabinoid receptor 2 attenuates mechanical allodynia and neuroinflammatory responses in a chronic post-ischemic pain model of complex regional pain syndrome type I in rats.

Complex regional pain syndrome type 1 (CRPS-I) remains one of the most clinically challenging neuropathic pain syndromes and its mechanism has not been fully characterized. Cannabinoid receptor 2 (CB2) has emerged as a promising target for treating different neuropathic pain syndromes. In neuropathic pain models, activated microglia expressing CB2 receptors are seen in the spinal cord. Chemokine fractalkine receptor (CX3CR1) plays a substantial role in microglial activation and neuroinflammation. We hypothesized that a CB2 agonist could modulate neuroinflammation and neuropathic pain in an ischemia model of CRPS by regulating CB2 and CX3CR1 signaling. We used chronic post-ischemia pain (CPIP) as a model of CRPS-I. Rats in the CPIP group exhibited significant hyperemia and edema of the ischemic hindpaw and spontaneous pain behaviors (hindpaw shaking and licking). Intraperitoneal administration of MDA7 (a selective CB2 agonist) attenuated mechanical allodynia induced by CPIP. MDA7 treatment was found to interfere with early events in the CRPS-I neuroinflammatory response by suppressing peripheral edema, spinal microglial activation and expression of CX3CR1 and CB2 receptors on the microglia in the spinal cord. MDA7 also mitigated the loss of intraepidermal nerve fibers induced by CPIP. Neuroprotective effects of MDA7 were blocked by a CB2 antagonist, AM630. Our findings suggest that MDA7, a novel CB2 agonist, may offer an innovative therapeutic approach for treating neuropathic symptoms and neuroinflammatory responses induced by CRPS-I in the setting of ischemia and reperfusion injury.

Tetrahydropyrazolo[4,3-c]pyridine derivatives as potent and peripherally selective cannabinoid-1 (CB1) receptor inverse agonists

Peripherally restricted CB1 receptor inverse agonists hold potential as useful therapeutics to treat obesity and related metabolic diseases without causing undesired CNS-mediated adverse effects. We identified a series of tetrahydropyrazolo[4,3-c]pyridine derivatives as potent and highly peripherally selective CB1 receptor inverse agonists. This discovery was achieved by introducing polar functional groups into the molecule, which increase the topological polar surface area and reduce its brain-penetrating ability.

Tetrahydroindazole derivatives as potent and peripherally selective cannabinoid-1 (CB1) receptor inverse agonists

A series of potent and receptor-selective cannabinoid-1 (CB1) receptor inverse agonists has been discovered. Peripheral selectivity of the compounds was assessed by a mouse tissue distribution study, in which the concentrations of a test compound in both plasma and brain were measured. A number of peripherally selective compounds have been identified through this process. Compound 2p was further evaluated in a 3-week efficacy study in the diet-induced obesity (DIO) mouse model. Beneficial effects on plasma glucose were observed from the compound-treated mice.

Current drugs for weight loss

Current drugs for weight loss

Peripherally Selective Cannabinoid 1 Receptor (CB1R) Agonists for the Treatment of Neuropathic Pain.

Alleviation of neuropathic pain by cannabinoids is limited by their central nervous system (CNS) side effects. Indole and indene compounds were engineered for high hCB1R affinity, peripheral selectivity, metabolic stability, and in vivo efficacy. An epithelial cell line assay identified candidates with <1% blood-brain barrier penetration for testing in a rat neuropathy induced by unilateral sciatic nerve entrapment (SNE). The SNE-induced mechanical allodynia was reversibly suppressed, partially or completely, after intraperitoneal or oral administration of several indenes. At doses that relieve neuropathy symptoms, the indenes completely lacked, while the brain-permeant CB1R agonist HU-210 (1) exhibited strong CNS side effects, in catalepsy, hypothermia, and motor incoordination assays. Pharmacokinetic findings of ∼0.001 cerebrospinal fluid:plasma ratio further supported limited CNS penetration. Pretreatment with selective CB1R or CB2R blockers suggested mainly CB1R contribution to an indene's antiallodynic effects. Therefore, this class of CB1R agonists holds promise as a viable treatment for neuropathic pain.

Anti-nociceptive and desensitizing effects of olvanil on capsaicin-induced thermal hyperalgesia in the rat.

BackgroundOlvanil (NE 19550) is a non-pungent synthetic analogue of capsaicin, the natural pungent ingredient of capsicum which activates the transient receptor potential vanilloid type-1 (TRPV1) channel and was developed as a potential analgesic compound. Olvanil has potent anti-hyperalgesic effects in several experimental models of chronic pain. Here we report the inhibitory effects of olvanil on nociceptive processing using cultured dorsal root ganglion (DRG) neurons and compare the effects of capsaicin and olvanil on thermal nociceptive processing in vivo; potential contributions of the cannabinoid CB1 receptor to olvanil’s anti-hyperalgesic effects were also investigated.MethodsA hot plate analgesia meter was used to evaluate the anti-nociceptive effects of olvanil on capsaicin-induced thermal hyperalgesia and the role played by CB1 receptors in mediating these effects. Single cell calcium imaging studies of DRG neurons were employed to determine the desensitizing effects of olvanil on capsaicin-evoked calcium responses. Statistical analysis used Student’s t test or one way ANOVA followed by Dunnett’s post-hoc test as appropriate.ResultsBoth olvanil (100 nM) and capsaicin (100 nM) produced significant increases in intracellular calcium concentrations [Ca2+]i in cultured DRG neurons. Olvanil was able to desensitise TRPV1 responses to further capsaicin exposure more effectively than capsaicin. Intraplantar injection of capsaicin (0.1, 0.3 and 1 μg) produced a robust TRPV1-dependant thermal hyperalgesia in rats, whilst olvanil (0.1, 0.3 and 1 μg) produced no hyperalgesia, emphasizing its lack of pungency. The highest dose of olvanil significantly reduced the hyperalgesic effects of capsaicin in vivo. Intraplantar injection of the selective cannabinoid CB1 receptor antagonist rimonabant (1 μg) altered neither capsaicin-induced thermal hyperalgesia nor the desensitizing properties of olvanil, indicating a lack of involvement of CB1 receptors.ConclusionsOlvanil is effective in reducing capsaicin-induced thermal hyperalgesia, probably via directly desensitizing TRPV1 channels in a CB1 receptor-independent fashion. The results presented clearly support the potential for olvanil in the development of new topical analgesic preparations for treating chronic pain conditions while avoiding the unwanted side effects of capsaicin treatments.

Involvement of Spinal CB1 Cannabinoid Receptors on the Antinociceptive Effect of Celecoxib in Rat Formalin Test

Background: The pivotal role of cyclooxygenase-2 (COX-2) inhibitors in inflammatory pain is well documented, but its mechanism is not entirely clear. Nonsteroidal anti-inflammatory drugs (NSAIDs) as inhibitors of COX could inhibit fatty acid amide hydrolase, the enzyme responsible for the metabolism of endocannabinoids. Therefore, it is expected that celecoxib administration (selective COX-2 inhibitor) preserve the increased level of endocannabinoids after noxious stimuli, which leads to more pain suppression. Objectives: This study was designed to evaluate the interaction between the intrathecally administered celecoxib in combination with rimonabant, a selective cannabinoid CB1 receptor antagonist/reverse agonist on the pain behavior induced by formalin test in rat. Materials and Methods: Male Wistar rats with inserted lumbar intrathecal catheters were randomly grouped and tested in blinded manner by the same experimenter. Antinociceptive effects of different intrathecally doses of celecoxib (0.5, 5 and 10 µg/rat) in absence and with pretreatment of rimonabant at doses of 100 and 200 µg/rat were examined on the formalin test. Results: Celecoxib reduced the pain behavior in both phases of the formalin test, but this antinociceptive effect was a dose-dependent manner in the late phase. Rimonabant alone induced hyperalgesia as compared with the control group and pretreatment of rats with rimonabant reversed the analgesic activity of celecoxib. Conclusions: Antinociceptive effect of celecoxib may be mediated partly through the cannabinoid system. These effects are possibly attributed to inhibition of endocannabinoid degradation and consequently enhancement of endocannabinoids concentration at the spinal cord level. However, the hyperalgesic effects of rimonabant are not fully responsible for the reversal of celecoxib analgesia. Accordingly, NSAIDs intensify their effects through maintenance of the endocannabinoid tone. Therefore, combination of NSAIDs and cannabinoid agents could be used for synergistic effects.

PnPP-19, a spider toxin peptide, induces peripheral antinociception through opioid and cannabinoid receptors and inhibition of neutral endopeptidase.

The synthetic peptide PnPP-19 has been studied as a new drug candidate to treat erectile dysfunction. However, PnTx2-6, the spider toxin from which the peptide was designed, induces hyperalgesia. Therefore, we intended to investigate the role of PnPP-19 in the nociceptive pathway. Nociceptive thresholds were measured by paw pressure test. PnPP-19 was administered intraplantarly alone or with selective cannabinoid or opioid receptor antagonists. The hydrolysis of PnPP-19 by neutral endopeptidase (NEP) (EC 3.4.24.11), an enzyme that cleaves enkephalin, was monitored by HPLC and the cleavage sites were deduced by LC-MS. Inhibition by PnPP-19 and Leu-enkephalin of NEP enzyme activity was determined spectrofluorimetrically. PnPP-19 (5, 10 and 20 μg per paw) induced peripheral antinociception in rats. Specific antagonists of μ opioid receptors (clocinnamox), δ opioid receptors (naltrindole) and CB1 receptors (AM251) partly inhibited the antinociceptive effect of PnPP-19. Inhibition of fatty acid amide hydrolase by MAFP or of anandamide uptake by VDM11 enhanced PnPP-19-induced antinociception. NEP cleaved PnPP-19 only after a long incubation, and Ki values of 35.6 ± 1.4 and 14.6 ± 0.44 μmol·L(-1) were determined for PnPP-19 and Leu-enkephalin respectively as inhibitors of NEP activity. Antinociception induced by PnPP-19 appears to involve the inhibition of NEP and activation of CB1, μ and δ opioid receptors. Our data provide a greater understanding of the antinociceptive effects of PnPP-19. This peptide could be useful as a new antinociceptive drug candidate.

Effect of nucleus accumbens shell 5-HT4 receptors on the impairment of ACPA-induced emotional memory consolidation in male Wistar rats.

The present study investigates the effects of 5-HT4 receptors of the nucleus accumbens (NAc) shell on the impairment of emotional memory consolidation induced by cannabinoid CB1 receptor stimulation. The elevated plus maze test-retest paradigm was used to assess memory in adult male Wistar rats. Intra-NAc shell administration of ACPA (selective cannabinoid CB1 receptor agonist 0.006 µg/rat) and RS23597 (5-HT4 receptor antagonist 0.01 µg/rat), immediately after training, decreased emotional memory consolidation, suggesting a drug-induced amnesia, whereas post-training intra-NAc shell microinjections of RS67333 (5-HT4 receptor agonist 0.016 µg/rat) increased emotional memory consolidation. Interestingly, RS67333 exerted a dual effect on ACPA-induced behaviors, potentiating and restoring amnesia caused by the subthreshold and effective doses of ACPA, respectively. However, neither RS23597 nor AM251 (CB1 receptor antagonist 30, 60 and 120 ng/rat) affected emotional memory consolidation. Nonetheless, a subthreshold dose of AM251 (120 ng/rat) reversed the amnesia induced by ACPA (0.006 µg/rat) and RS23597 (0.01 µg/rat). None of the above doses altered the locomotor activity. In conclusion, our results suggest that the NAc-shell 5-HT4 receptors are involved in the modulation of ACPA-induced amnesia.

Investigations on the 4-Quinolone-3-carboxylic Acid Motif. 7. Synthesis and Pharmacological Evaluation of 4-Quinolone-3-carboxamides and 4-Hydroxy-2-quinolone-3-carboxamides as High Affinity Cannabinoid Receptor 2 (CB2R) Ligands with Improved Aqueous Solubility.

4-Quinolone-3-carboxamide derivatives have long been recognized as potent and selective cannabinoid type-2 receptor (CB2R) ligands. With the aim to improve their physicochemical properties, basically aqueous solubility, two different approaches were followed, entailing the substitution of the alkyl chain with a basic replacement or scaffold modification to 4-hydroxy-2-quinolone structure. According to the first approach, compound 6d was obtained, showing slightly reduced receptor affinity (K(i) = 60 nM) compared to the lead compound 4 (0.8 nM) but greatly enhanced solubility (400-3400 times depending on the pH of the medium). On the other hand, shifting from 4-quinolone to 4-hydroxy-2-quinolone structure enabled the discovery of a novel class of CB2R ligands, such as 7b and 7c, characterized by K(i) < 1 nM and selectivity index [SI = K(i)(CB1R)/K(i)(CB2R)] > 1300. At pH 7.4, compound 7c resulted by 100-fold more soluble than 4.