Selective CB1 Research Articles

CB2 receptor activation causes an ERK1/2-dependent inflammatory response in human RPE cells

A chronic low-level inflammation contributes to the pathogenesis of age-related macular degeneration (AMD), the most common cause of blindness in the elderly in Western countries. The loss of central vision results from attenuated maintenance of photoreceptors due to the degeneration of retinal pigment epithelium (RPE) cells beneath the photoreceptor layer. It has been proposed that pathologic inflammation initiated in RPE cells could be regulated by the activation of type 2 cannabinoid receptors (CB2). Here, we have analysed the effect of CB2 activation on cellular survival and inflammation in human RPE cells. RPE cells were treated with the selective CB2 agonist JWH-133 in the presence or absence of the oxidative stressor 4-hydroxynonenal. Thereafter, cellular viability as well as the release of pro-inflammatory cytokines and potential underlying signalling pathways were analysed. Our results show that JWH-133 led to increased intracellular Ca2+ levels, suggesting that RPE cells are capable of responding to a CB2 agonist. JWH-133 could not prevent oxidative stress-induced cell death. Instead, 10 µM JWH-133 increased cell death and the release of proinflammatory cytokines in an ERK1/2-dependent manner. In contrast to previous findings, CB2 activation increased, rather than reduced inflammation in RPE cells.

Anti-invasion Effects of Cannabinoids Agonist and Antagonist on Human Breast Cancer Stem Cells.

Studies show that cancer cell invasion or metastasis is the primary cause of death in malignancies including breast cancer. The existence of cancer stem cells (CSCs) in breast cancer may account for tumor initiation, progression, and metastasis. Recent studies have reported different effects of cannabinoids on cancer cells via CB1 and CB2 cannabinoid receptors. In the present study, the effects of ACEA (a selective CB1 receptor agonist) and AM251 (a selective CB1 antagonist) on CSCs and their parental cells were investigated. Breast CSCs derived from MDA-MB-231 cell line were sorted and characterized with CD44+/CD24-/low/ESA+ phenotype. It was observed that ACEA decreased CD44+/CD24-/low/ESA+ cancer stem cell invasiveness. Conversely, AM251 increased the invasion by more than 20% (at the highest concentrations) in both MDA-MB-231 and CSCs. Our results did not show any correlation between reduced invasion and cytotoxic effects of the drug. Since one of the main cancer recurrence factors is anti-cancer drugs fail to inhibit CSC population, this observation would be useful for cancer treatment.

Effects of the cannabinoid CB1 agonist ACEA on salicylate ototoxicity, hyperacusis and tinnitus in guinea pigs

Cannabinoids have been suggested as a therapeutic target for a variety of brain disorders. Despite the presence of their receptors throughout the auditory system, little is known about how cannabinoids affect auditory function. We sought to determine whether administration of arachidonyl-2′-chloroethylamide (ACEA), a highly-selective CB1 agonist, could attenuate a variety of auditory effects caused by prior administration of salicylate, and potentially treat tinnitus. We recorded cortical resting-state activity, auditory-evoked cortical activity and auditory brainstem responses (ABRs), from chronically-implanted awake guinea pigs, before and after salicylate + ACEA. Salicylate-induced reductions in click-evoked ABR amplitudes were smaller in the presence of ACEA, suggesting that the ototoxic effects of salicylate were less severe. ACEA also abolished salicylate-induced changes in cortical alpha band (6–10 Hz) oscillatory activity. However, salicylate-induced increases in cortical evoked activity (suggestive of the presence of hyperacusis) were still present with salicylate + ACEA. ACEA administered alone did not induce significant changes in either ABR amplitudes or oscillatory activity, but did increase cortical evoked potentials. Furthermore, in two separate groups of non-implanted animals, we found no evidence that ACEA could reverse behavioural identification of salicylate- or noise-induced tinnitus. Together, these data suggest that while ACEA may be potentially otoprotective, selective CB1 agonists are not effective in diminishing the presence of tinnitus or hyperacusis.

Synthetic cannabinoid AM2201 induces seizures: Involvement of cannabinoid CB1 receptors and glutamatergic transmission

Abuse of synthetic cannabinoids is a serious social problem worldwide. Intentional ingestion of synthetic cannabinoids can cause severe toxicity, including seizures. Here we investigated the effects of acute administration of synthetic cannabinoids on the induction of epileptic seizures by monitoring electroencephalographic activity in freely moving mice. The synthetic cannabinoid, AM2201, induced abnormal, high-amplitude (>2-fold baseline amplitude), sharp-wave activity. The abnormal spike-wave discharges were accompanied by epileptiform behavior: rigid posture, tail extension, rearing with forepaws extended, jumping, and intermittent tonic-clonic jerking movements. The abnormal spike-wave discharges and behavioral changes were suppressed by pretreatment with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630 or the vanilloid receptor antagonist, capsazepine. Furthermore, the group 1 metabotropic glutamate receptor antagonist SIB1757 eliminated AM2201-induced spike-wave discharges and episodes of epileptiform behavior. AM2201 markedly increased the extracellular glutamate concentration in the hippocampus during periods of AM2201-induced abnormal spike-wave discharges and behavioral changes. These findings are the first evidence that AM2201 induces epileptic seizures by enhancing glutamatergic transmission in the hippocampus. Our findings demonstrate that induction of epileptic seizures by synthetic cannabinoids is mediated by CB1 receptors, but not by CB2 receptors, and further suggest that rapid elevation of glutamatergic transmission may play an important role in the induction of seizures following intentional ingestion of synthetic cannabinoids.

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.

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.

Amidoalkylindoles as Potent and Selective Cannabinoid Type 2 Receptor Agonists with in Vivo Efficacy in a Mouse Model of Multiple Sclerosis.

Selective CB2 agonists represent an attractive therapeutic strategy for the treatment of a variety of diseases without psychiatric side effects mediated by the CB1 receptor. We carried out a rational optimization of a black market designer drug SDB-001 that led to the identification of potent and selective CB2 agonists. A 7-methoxy or 7-methylthio substitution at the 3-amidoalkylindoles resulted in potent CB2 antagonists (27 or 28, IC50 = 16-28 nM). Replacement of the amidoalkyls from 3-position to the 2-position of the indole ring dramatically increased the agonist selectivity on the CB2 over CB1 receptor. Particularly, compound 57 displayed a potent agonist activity on the CB2 receptor (EC50 = 114-142 nM) without observable agonist or antagonist activity on the CB1 receptor. Furthermore, 57 significantly alleviated the clinical symptoms and protected the murine central nervous system from immune damage in an experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis.

Antiallodynic effect of β-caryophyllene on paclitaxel-induced peripheral neuropathy in mice

Painful peripheral neuropathy is a common side effect of paclitaxel (PTX). The use of analgesics is an important component for management of PTX-induced peripheral neuropathy (PINP). However, currently employed analgesics have several side effects and are poorly effective. β-caryophyllene (BCP), a dietary selective CB2 agonist, has shown analgesic effect in neuropathic pain models, but its role in chemotherapy-induced neuropathic pain has not yet been investigated. Herein, we used the mouse model of PINP to show the therapeutic effects of BCP in this neuropathy. Male Swiss mice receiving PTX (2 mg kg−1, ip, four alternate days) were treated with BCP (25 mg kg−1, po, twice a day) either during or after PTX administration. Some groups were also pretreated with AM630 (CB2 antagonist, 3 mg kg−1, ip) or AM251 (CB1 antagonist, 1 mg kg−1, ip). Spinal cord samples were collected in different time points to perform immunohistochemical analysis. BCP attenuated the established mechanical allodynia induced by PTX (p < 0.0001) in a CB2-dependent manner. Of note, when given concomitantly with PTX, BCP was able to attenuate the development of PINP (p < 0.0001). Spinal cord immunohistochemistry revealed that preventive treatment with BCP reduced p38 MAPK and NF-κB activation, as well as the increased Iba-1 and IL-1β immunoreactivity promoted by PTX. Our findings show that BCP effectively attenuated PINP, possibly through CB2-activation in the CNS and posterior inhibition of p38 MAPK/NF-κB activation and cytokine release. Taken together, our results suggest that BCP could be used to attenuate the establishment and/or treat PINP.

AM1241 alleviates MPTP-induced Parkinson's disease and promotes the regeneration of DA neurons in PD mice.

The main pathological feature of Parkinson's disease (PD) is the loss of dopaminergic neurons in the substantia nigra. In this study, we investigated the role of cannabinoid receptor 2 (CB2R) agonist AM1241 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in a mouse model of PD. Upon treatment with AM1241, the decreased CB2R level in the PD mouse brain was reversed and the behavior score markedly elevated, accompanied with a dose-dependent increase of dopamine and serotonin. In addition, western blot assay and immunostaining results suggested that AM1241 significantly activated PI3K/Akt/MEK phosphorylation and increased the expression of Parkin and PINK1, both in the substantia nigra and hippocampus. The mRNA expression analysis further demonstrated that AM1241 increased expression of the CB2R and activated Parkin/PINK1 signaling pathways. Furthermore, the increased number of TH-positive cells in the substantia nigra indicated that AM1241 regenerated DA neurons in PD mice, and could therefore be a potential candidate for PD treatment. The clear co-localization of CB2R and DA neurons suggested that AM1241 targeted CB2R, thus also identifying a novel target for PD treatment. In conclusion, the selective CB2 agonist AM1241 has a significant therapeutic effect on PD mice and resulted in regeneration of DA neurons following MPTP-induced neurotoxicity. The possible mechanisms underlying the neurogenesis effect of AM1241 might be the induction of CB2R expression and an increase in phosphorylation of the PI3K/AKT signaling pathway.

Involvement of Gaba and Cannabinoid Receptors in Central Food Intake Regulation in Neonatal Layer Chicks: Role of CB1 and Gabaa Receptors

ABSTRACT Feeding behavior is regulated via a complex network which interacts via diverse signals from central and peripheral tissues. Endocannabinoids modulate release of GABA in a variety of regions of the central nervous system. Endocannabinoids and GABAergic system have an important role in the central regulation of appetite. Thus, the present study examines the possible interaction of central canabinoidergic and GABAergic systems on food intake in 3-h food-deprived (FD3) neonatal layer-type chicks. The results of this study showed that intracerebroventricular (ICV) injection of 2-AG (2-Arachidonoylglycerol, selective CB1 receptors agonist, 2µg) significantly increased food intake and this effect of 2-AG was attenuated by Picrotoxin (GABAA antagonist, 0.5µg) (P 0.05). Also, hyperphagic effect of CB65 (CB2 receptors agonist, 1.25µg) was not affected by Picrotoxin or CGP54626 (p>0.05). Moreover, the food intake of chicks was significantly increased by ICV injection of GABAA agonist (Gaboxadol, 0.2 µg) and SR141716A (CB1 receptors antagonist, 6.25µg) significantly decreased Gaboxadol-induced hyperphagia (P 0.05). These data showed there might be an interaction between central cannabinoidergic and GABAergic systems via CB1 and GABAA receptors in control of food intake in neonatal layer chicks.

Development of Highly Affine and Selective Fluorinated Cannabinoid Type 2 Receptor Ligands.

Cannabinoid type 2 receptors (CB2 receptors) are involved in various pathological processes, and the visualization of their in vivo availability with positron emission tomography (PET) is of high interest. The study focuses on the introduction of fluorine into the structure of the highly affine and selective CB2 receptor ligand N-(adamantan-1-yl)-5-ethyl-2-methyl-1-phenyl-1H-imidazole-4-carboxamide (5). A novel series of compounds was developed by modifying (i) the adamantane-3-position, (ii) the imidazole-N-phenyl ring, and (iii) the imidazole-2-position, and the impact on the CB2 binding affinity and selectivity toward cannabinoid type 1 receptors (CB1) was evaluated. This study identified compound 15 as one of the most potent (Ki(CB2) = 0.29 nM) and selective (CB1/CB2 > 10000) CB2 receptor ligands discovered so far, eligible for the development of an 18F-labeled PET radiotracer.

Development of Quinazoline/Pyrimidine-2,4(1H,3H)-diones as Agonists of Cannabinoid Receptor Type 2.

Starting from a prototypical structure 1, we describe our efforts to design and obtain novel quinazoline/pyrimidine-2,4(1H,3H)-diones with high CB2 agonist potency and selectivity as well as improved physicochemical characteristics, mainly hydrophilicity. The most potent and selective CB2 agonists, 8 and 36, in this series were also endowed with lower logP values than that of GW842166X and lead compound 1. These derivatives appear to be promising lead compounds for the development of future CB2 agonists.

Pharmaco-toxicological effects of the novel third-generation fluorinate synthetic cannabinoids, 5F-ADBINACA, AB-FUBINACA, and STS-135 in mice. In vitro and in vivo studies.

5F-ADBINACA, AB-FUBINACA, and STS-135 are 3 novel third-generation fluorinate synthetic cannabinoids that are illegally marketed as incense, herbal preparations, or research chemicals for their psychoactive cannabis-like effects. The present study aims at investigating the in vitro and in vivo pharmacological activity of 5F-ADBINACA, AB-FUBINACA, and STS-135 in male CD-1 mice, comparing their in vivo effects with those caused by the administration of Δ9 -THC and JWH-018. In vitro competition binding experiments revealed a nanomolar affinity and potency of the 5F-ADBINACA, AB-FUBINACA, and STS-135 on mouse and human CB1 and CB2 receptors. Moreover, these synthetic cannabinoids induced neurotoxicity in murine neuro-2a cells. In vivo studies showed that 5F-ADBINACA, AB-FUBINACA, and STS-135 induced hypothermia; increased pain threshold to both noxious mechanical and thermal stimuli; caused catalepsy; reduced motor activity; impaired sensorimotor responses (visual, acoustic, and tactile); caused seizures, myoclonia, and hyperreflexia; and promoted aggressiveness in mice. Behavioral and neurological effects were fully prevented by the selective CB1 receptor antagonist/inverse agonist AM 251. Differently, the visual sensory response induced by STS-135 was only partly prevented by the AM 251, suggesting a CB1 -independent mechanism. For the first time, the present study demonstrates the pharmaco-toxicological effects induced by the administration of 5F-ADBINACA, AB-FUBINACA, and STS-135 in mice and suggests their possible detrimental effects on human health.

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.

Role of the amygdala GABA-A receptors in ACPA-induced deficits during conditioned fear learning.

The basolateral amygdala (BLA) is a key structure for the emotional processing and storage of memories associated with emotional events, especially fear. On the other hand, endocannabinoids and CB1 receptors play a key role in learning and memory partly through long-term synaptic depression of GABAergic synapses in the BLA. The aim of this study was to explore the effects of GABA-A receptor agonist and antagonist in the fear-related memory acquisition deficits induced by ACPA (a selective CB1 cannabinoid receptor agonist). This study used context and tone fear conditioning paradigms to assess fear-related memory in male NMRI mice. Our results showed that the pre-training intraperitoneal administration of ACPA (0.5mg/kg) or (0.1 and 0.5mg/kg) decreased the percentage of freezing time in the contextual and tone fear conditioning, respectively. This indicated an impaired context- or tone-dependent fear memory acquisition. Moreover, the pre-training intra-BLA microinjection of GABA-A receptor agonist, muscimol, at 0.05 and 0.5μg/mouse impaired context-dependent fear memory, while the same doses of GABA-A antagonist, bicuculline, impaired tone-dependent fear memory. However, a subthreshold dose of muscimol or bicuculline increased the effect of ACPA at 0.1 and 0.5 or 0.05mg/kg on context- or tone-dependent fear memory, respectively. In addition, bicuculline at the lower dose increased the ACPA response on locomotor activity compared to its respective group. Such findings highlighted an interaction between BLA GABAergic and cannabinoidergic systems during the acquisition phase of conditioned fear memories.

Selective cannabinoid 2 receptor stimulation reduces tubular epithelial cell injury following renal ischemia‐reperfusion injury

Acute kidney injury (AKI) is an increasingly common clinical problem and has been studied for its association with chronic kidney disease progression. Currently, therapies to treat AKI are not available, so further insight into new therapeutic targets are essential in the treatment of this disease. In this study, a novel cannabinoid receptor type 2 (CB2) agonist, SMM‐295, was designed and synthesized for testing as a potential therapeutic to treat acute kidney injury. Molecular docking of SMM‐295 into a CB2 active state model showed that SMM‐295 interacts well with key amino acids to stabilize the active state. In cell culture, SMM‐295 was shown to exhibit ~100‐fold selectivity for CB2‐ versus CB1‐expressing 293 cells. Moreover, the CLogP values were consistent with a more aqueous solubility for SMM‐295 (CLogP=4.96) compared with a commonly used CB2 agonist, JWH‐133 (CLogP=5.72). Subsequent in vivo testing of the CB2 agonist was performed using a mouse model of AKI. Bilateral ischemia‐reperfusion injury was performed in mice, which is a common model to mimic human AKI, and SMM‐295 (6 mg/kg) was immediately administered IP to the mice upon reperfusion of the kidneys following the ischemic episode and every 24 hours until the end of the experiment. Histological damage assessment after 48 hours following reperfusion demonstrated around a 50% reduction (P&lt;0.05) in tubular damage in the presence of SMM‐295 (n=5) compared to vehicle‐treated mice (n=4). This was consistent with a significant reduction (P&lt;0.05) in both plasma creatinine and NGAL by approximately 60% with SMM‐295 administration (n=5) versus vehicle alone (n=7). SMM‐295 also showed better therapeutic results compared to the commercially available CB2 agonist JWH‐133 which also led to a drop in both serum creatinine levels and tubular injury, but did not reach statistical significance (P&gt;0.05). Conversely, CB2 inverse agonist, SMM‐189, showed significant increases (P&lt;0.05) in both serum creatinine (152%; n=4) at 24 hours and tubular injury (47%; n=3) at 48 hours following IRI. SMM‐295 treated mice (n=5) also showed significant reduction (32%; P&lt;0.05) in PCNA‐positive cells in the outer medulla compared to the vehicle treated group (n=4) 48 hours following IRI. Western blot analysis of kidney lysates in SMM‐295 treated mice (n=5) at 48 hours following IRI demonstrate increased levels of cell survival proteins including total and active forms of Akt as well as Bcl‐2 compared to vehicle treated mice (n=3). These data would suggest that selective CB2 activation can play a beneficial role in protecting tubular epithelial cells following transient injury stimulus application to the kidneys, and may open the door to new small molecule activators of the cannabinoid systems for renal‐directed therapies.Support or Funding InformationThis study was partially funded by NIH RO1 DK090123 (F.P.) and institutional funds (F.P.).

Compensatory activation of cannabinoid CB2 receptor inhibition of GABA release in the rostral ventromedial medulla (RVM) in inflammatory pain

The rostral ventromedial medulla (RVM) is a relay in the descending pain modulatory system and an important site of endocannabinoid modulation of pain. Endocannabinoids inhibit GABA release in the RVM but it is not known if this effect persists in chronic pain states. In the present studies, persistent inflammation induced by complete Freund's adjuvant (CFA) increased GABAergic miniature inhibitory postsynaptic currents (mIPSCs). Endocannabinoid activation of cannabinoid (CB1) receptors known to inhibit presynaptic GABA release was significantly reduced in the RVM of CFA‐treated rats compared to naïve rats. The reduction in CFA‐treated rats correlated with decreased CB1 receptor protein expression and function in the RVM. Paradoxically, the non‐selective CB1/CB2 receptor agonist WIN55,212 inhibited GABAergic mIPSCs in both naïve and CFA‐treated rats. However, WIN55,212 inhibition was reversed by the CB1 receptor antagonist rimonabant in naïve rats but not in CFA‐treated rats. WIN55,212‐mediated inhibition in CFA‐treated rats was blocked by the CB2 receptor‐selective antagonist SR144528 indicating that CB2 receptor function in the RVM is increased during persistent inflammation. Consistent with these results, CB2 receptor agonists, AM1241 and GW405833 inhibited GABAergic mIPSC frequency only in CFA‐treated rats and the inhibition was reversed with SR144258. When administered alone, SR144528 and another CB2 receptor‐selective antagonist AM630 increased mIPSC frequency in the RVM of CFA‐treated rats indicating that CB2 receptors are tonically activated by endocannabinoids. Our data provide evidence that CB2 receptor function emerges in the RVM in persistent inflammation and that selective CB2 receptor agonists may be useful for treatment of persistent inflammatory pain.Support or Funding InformationThese studies were supported by the grants from NIH (DA035316 and R56NS093894), and America Heart Association (13SDG14590005, MH.L.).

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.

Role of orexin-2 and CB1 receptors within the periaqueductal gray matter in lateral hypothalamic-induced antinociception in rats.

Orexin plays an important role in pain modulation. Orexin-1 and orexin-2 receptors (Ox1r and Ox2r) are found at high density in the ventrolateral periaqueductal gray matter (vlPAG). Our previous study showed that chemical stimulation of the lateral hypothalamus with carbachol induces antinociception in the tail-flick test, a model of acute pain, and Ox1r-mediated antinociception in the vlPAG is modulated by the activity of vlPAG CB1 receptors. In the current study, TCS OX2 29, an Ox2r antagonist (5, 15, 50, 150, and 500 nmol/l), was microinjected into the vlPAG 5 min before the administration of carbachol (125 nmol/l). TCS OX2 29 dose dependently reduced carbachol-induced antinociception. In a second set of experiments, animals were treated with carbachol 5 min after intra-vlPAG administration of 15 nmol/l TCS OX2 29 and 1 nmol/l AM251 (a selective CB1 receptor antagonist), or 150 nmol/l TCS OX2 29 and 10 nmol/l AM251. The findings showed that the antinociceptive effect of orexin is partially mediated by activation of vlPAG Ox2 receptors. Furthermore, the administration of ineffective doses of Ox2 and CB1 receptor antagonists reduced the lateral hypothalamus-induced antinociception. It seems that Ox2 and CB1 receptors act through different pathways and Ox2r-mediated antinociception is not dependent on CB1 receptor activity.