CB1 Activation Research Articles

The Peptide PnPP-19, a Spider Toxin Derivative, Activates μ-Opioid Receptors and Modulates Calcium Channels.

The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the toxin δ-CNTX-Pn1c (PnTx2-6), isolated from the venom of the spider Phoneutria nigriventer. Behavioural tests suggest that PnPP-19 induces antinociception by activation of CB1, μ and δ opioid receptors. Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation, the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the subtype selectivity for μ-, δ- and/or κ-opioid receptors. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion neurons, and analyzed whether this modulation was opioid-mediated. PnPP-19 selectively activates μ-opioid receptors inducing indirectly inhibition of calcium channels and hereby impairing calcium influx in dorsal root ganglion (DRG) neurons. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce β-arrestin2 recruitment. PnPP-19 is the first spider toxin derivative that, among opioid receptors, selectively activates μ-opioid receptors. The lack of β-arrestin2 recruitment highlights its potential for the design of new improved opioid agonists.

Cannabinoids modulate the affective component of chronic pain

Cannabis sativa preparations have probably been used since millennia for pain relief and are still an important part of our analgesic pharmacopeia. This presentation will summarize our current understanding about the analgesic mechanism of cannabinoids, from molecular mechanisms to the physiological effects. The psychoactive ingredient of C. sativa, 9-tetrahydrocannabinol (THC) activates two different G protein-coupled cannabinoid receptors, CB1 and CB2, both of which mediate different aspects of cannabinoid analgesic effects. CB2 receptors are mostly localized on immune cells and activated microglia. The analgesic effects of CB2 activation are mainly due to anti-inflammatory mechanisms, but probably also through the modulation of algogenic mediators. CB1 is prominently expressed in the central nervous system and is well-known as an important mediator of synaptic plasticity. Activation of CB1 on neurons modulates pain responses at different level, including the processing of nociceptive stimuli and the regulation of descending pain control systems. I will also show evidence that endocannabinoid signaling is involved in the modulation of the affective component of pain.

Secondary metabolites isolated from Pinus roxburghii and interpretation of their cannabinoid and opioid binding properties by virtual screening and in vitro studies

Pinus roxburghii is highly popular as a potent analgesic and anti-inflammatory agent; however its exact mechanism of action was not fully elucidated. We aimed to interpret the analgesic and anti-inflammatory activity of the total ethanol extract of Pinus roxburghii bark (PRE) and its isolated compounds by both in silico molecular modelling and in-vitro cannabinoid and opioid binding activities evaluation for the first time. Comprehensive phytochemical investigation of PRE resulted in the isolation of sixteen compounds that were fully elucidated using 1H NMR and 13C NMR. Four of which namely 1,3,7-trihydroxyxanthone (1), 2,4,7-trihydroxyxanthone (2), isopimaric acid (9) and 3-methoxy-14-serraten-21-one (10) were first to be isolated from PRE. In silico molecular modelling was done using Accelry’s discovery studio 2.5 on the cannabinoid receptor (CB1) and the different opioid receptors (mu, kappa and delta). Results showed that the different isolated constituents exhibited variable degrees of binding with the different examined receptors that undoubtedly explained the observed analgesic and anti-inflammatory activity of PRE. Thus in vitro evaluation of cannabinoid (CB1, CB2) and opioid (μ, κ, δ) binding activities for the isolated compounds was done. PRE and ursolic acid (11) showed a good CB1 receptor binding activity with 66.8 and 48.1% binding, respectively. Isopimaric acid (9) showed good CB2 and mu receptors binding activity estimated by 58.1 and 29.1% binding, respectively. Meanwhile, querectin-3-O-rhamnoside (7) exhibited a moderate κ-opioid receptor activity showing 56.0% binding. Thus, PRE could offer a natural analgesic and anti-inflammatory candidate through the synergistic action of all its components.

Effects of cannabinoid receptor type 2 in respiratory syncytial virus infection in human subjects and mice

ABSTRACTAn accumulating body of evidence suggests that the endocannabinoid system plays a significant role in pathophysiological processes and impacts disease severity. Here we investigate the possible role of a cannabinoid receptor type 2 (CB2) functional variant in determining disease severity and the potential pharmacological therapeutic effects of CB2 activation in viral respiratory infection. The common missense variant (CAA/CGG; Q63R) of the gene-encoding CB2 receptor (CNR2) was evaluated in 90 inpatient and 90 outpatient children with acute respiratory tract infection (ARTI). The frequency distribution of respiratory syncytial virus (RSV)-the main cause of severe cases of bronchiolitis and pneumonia in children-was studied in all collected samples. The mechanism through which CB2 affects clinical outcomes in case of RSV infection was studied in Balb/c mice model using AM630 as a CB2 antagonist. The potential therapeutic effect of CB2 activation during RSV infection was studied using a selective agonist, JWH133. The CB2 Q63R variation was associated with increased risk of hospitalization in children with ARTI. Children carrying the QQ genotype were more prone to developing severe ARTI (OR = 3.275, 95% CI: 1.221–8.705; p = 0.019). Of all the children enrolled in the study, 83 patients (46.1%) were found positive for RSV infection. The associated risk of developing severe ARTI following RSV infection increased more than two-fold in children carrying the Q allele (OR = 2.148, 95% CI: 1.092–4.224; p = 0.026). In mice, the blockade of CB2 by AM630 during RSV infection enhanced the influx of BAL cells and production of cytokines/chemokines while exaggerating lung pathology. CB2 activation by JWH133 reduces the influx of BAL cells and production of cytokines/chemokines while alleviating lung pathology. Collectively, CB2 is associated with RSV severity during infancy and may serve as a therapeutic target in RSV infection through the alleviation of virus-associated immunopathology.

Cannabinoid‐1 receptor deletion in podocytes mitigates both glomerular and tubular dysfunction in a mouse model of diabetic nephropathy

To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB1 R) in the development of diabetic nephropathy (DN), relative to CB1 R in other renal cell types. We developed a mouse model with a podocyte-specific deletion of CB1 R (pCB1Rko) and challenged this model with streptozotocin (STZ)-induced type-1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1 R activation. High glucose exposure for 48 hours led to an increase in CB1 R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury, reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1-silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycaemia, STZ-treated pCB1Rko mice showed elevated blood glucose similar to that of their wild-type littermates. However, they displayed less albuminuria and less podocyte loss than STZ-treated wild-type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild-type controls, which is mediated, in part, by podocyte-derived endocannabinoids acting via CB1 R on proximal tubular cells. Activation of CB1 R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB1 R blockade.

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.

Finding order in chemical chaos - Continuing characterization of synthetic cannabinoid receptor agonists

Diversion of synthetic cannabinoids from the lab to drugs of abuse has become increasingly prevalent in recent years. Moreover, as earlier synthetic cannabinoids were banned, manufacturers introduced a new supply of novel compounds to serve as replacements. Hence, the chemical diversity of synthetic cannabinoid analogs has also rapidly increased. The present study examined 8 new synthetic cannabinoids: AM-1220, AM-2232, AM-2233, AM-679, EAM-2201, JWH-210, JHW-251, and MAM-2201. Each compound was assessed for binding affinity and functional activation of CB1 and CB2 receptors, and pharmacological equivalence with Δ9-tetrahydrocannabinol (THC) in THC drug discrimination. All compounds bound to and activated CB1 and CB2 receptors, although efficacy at the CB2 receptor was reduced compared to that for the CB1 receptor. Similarly, all compounds stimulated [35S]GTPγS binding through the CB1 receptor, and all compounds except AM-1220 and AM-2233 stimulated [35S]GTPγS binding through the CB2 receptor. Furthermore, these compounds, along with CP55,940, substituted for THC in THC drug discrimination. Rank order of potency in drug discrimination was correlated with CB1 receptor binding affinity. Together, these results suggest that all test compounds share the THC-like subjective effects of marijuana. Interestingly, the most potent compounds in CB1 binding in the present study were also the compounds that have been found recently in the U.S., MAM-2201, EAM-2201, JWH-210, AM-2233, and AM-1220. These results indicate that the evolution of the synthetic cannabinoid drug market may be focused toward compounds with increased potency.This article is part of the Special Issue entitled ‘Designer Drugs and Legal Highs.’

The FAAH inhibitor URB597 suppresses hippocampal maximal dentate afterdischarges and restores seizure-induced impairment of short and long-term synaptic plasticity

Synthetic cannabinoids and phytocannabinoids have been shown to suppress seizures both in humans and experimental models of epilepsy. However, they generally have a detrimental effect on memory and memory-related processes. Here we compared the effect of the inhibition of the endocannabinoid (eCB) degradation versus synthetic CB agonist on limbic seizures induced by maximal dentate activation (MDA) acute kindling. Moreover, we investigated the dentate gyrus (DG) granule cell reactivity and synaptic plasticity in naïve and in MDA-kindled anaesthetised rats. We found that both the fatty acid amide hydrolase (FAAH) inhibitor URB597 and the synthetic cannabinoid agonist WIN55,212-2 displayed AM251-sensitive anti-seizure effects. WIN55,212-2, dose-dependently (0.5–2 mg/kg, i.p.) impaired short-term plasticity (STP) and long-term potentiation (LTP) at perforant path-DG synapses in naïve rats. Strikingly, URB597 (1 mg/kg, i.p.) was devoid of any deleterious effects in normal conditions, while it prevented seizure-induced alterations of both STP and LTP. Our evidence indicates that boosting the eCB tone rather than general CB1 activation might represent a potential strategy for the development of a new class of drugs for treatment of both seizures and comorbid memory impairments associated with epilepsy.

Pharmacology of Cumyl-Carboxamide Synthetic Cannabinoid New Psychoactive Substances (NPS) CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-5F-PINACA, and Their Analogues

Synthetic cannabinoids (SC) are the largest class of new psychoactive substances (NPS), and are increasingly associated with serious adverse effects. The majority of SC NPS are 1,3-disubstituted indoles and indazoles featuring a diversity of subunits at the 1- and 3-positions. Most recently, cumyl-derived indole- and indazole-3-carboxamides have been detected by law enforcement agencies and by emergency departments. Herein we describe the synthesis, characterization, and pharmacology of SCs CUMYL-BICA, CUMYL-PICA, CUMYL-5F-PICA, CUMYL-PINACA, CUMYL-5F-PINACA, and related analogues. All cumyl-derived SCs were potent, efficacious agonists at CB1 (EC50 = 0.43-12.3 nM) and CB2 (EC50 = 11.3-122 nM) receptors in a fluorometric assay of membrane potential, with selectivity for CB1 activation (3.1-53 times over CB2). CUMYL-PICA and CUMYL-5F-PICA were evaluated in rats using biotelemetry, and induced hypothermia and bradycardia at doses of 1 mg/kg. Hypothermia was reversed by pretreatment with a CB1, but not CB2, antagonist, confirming that cumyl-derived SCs are cannabimimetic in vivo.

The role of CB1 in intestinal permeability and inflammation.

The endocannabinoid system has previously been shown to play a role in the permeability and inflammatory response of the human gut. The goal of our study was to determine the effects of endogenous anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) on the permeability and inflammatory response of intestinal epithelium under normal, inflammatory, and hypoxic conditions. Human intestinal mucosa was modeled using Caco-2 cells. Human tissue was collected from planned colorectal resections. Accumulation of AEA and 2-AG was achieved by inhibiting their metabolizing enzymes URB597 (a fatty acid amide hydrolase inhibitor) and JZL184 (a monoacylglycerol lipase inhibitor). Inflammation and ischemia were simulated with TNF-α and IFN-γ and oxygen deprivation. Permeability changes were measured by transepithelial electrical resistance. The role of the CB1 receptor was explored using CB1-knockdown (CB1Kd) intestinal epithelial cells. Endocannabinoid levels were measured using liquid chromatography-mass spectrometry. Cytokine secretion was measured using multiplex and ELISA. URB597 and JZL184 caused a concentration-dependent increase in permeability via CB1 (P < 0.0001) and decreased cytokine production. Basolateral application of JZL184 decreased permeability via CB1 (P < 0.0001). URB597 and JZL184 increased the enhanced (worsened) permeability caused by inflammation and hypoxia (P < 0.0001 and P < 0.05). CB1Kd cells showed reduced permeability response to inflammation (P < 0.01) but not hypoxia. 2-AG levels were increased in response to inflammation and hypoxia in Caco-2 cells. In human mucosal tissue, inflammation increased the secretion of granulocyte macrophage-colony stimulating factor, IL-12, -13, and -15, which was prevented with ex vivo treatment with URB597 and JZL184, and was inhibited by a CB1 antagonist. The results of this study show that endogenous AEA and 2-AG production and CB1 activation play a key modulatory roles in normal intestinal mucosa permeability and in inflammatory and hypoxic conditions.-Karwad, M. A., Couch, D. G., Theophilidou, E., Sarmad, S., Barrett, D. A., Larvin, M., Wright, K. L., Lund, J. N., O'Sullivan, S. E. The role of CB1 in intestinal permeability and inflammation.

Participation of hypothalamic CB1 receptors in reproductive axis disruption during immune challenge.

Immune challenge inhibits reproductive function and endocannabinoids (eCB) modulate sexual hormones. However, no studies have been performed to assess whether the eCB system mediates the inhibition of hormones that control reproduction as a result of immune system activation during systemic infections. For that reason, we evaluated the participation of the hypothalamic cannabinoid receptor CB1 on the hypothalamic-pituitary-gonadal (HPG) axis activity in rats submitted to immune challenge. Male adult rats were treated i.c.v. administration with a CB1 antagonist/inverse agonist (AM251) (500ng/5 μL), followed by an i.p. injection of lipopolysaccharide (LPS) (5mg/kg) 15minutes later. Plasmatic, hypothalamic and adenohypophyseal pro-inflammatory cytokines, hormones and neuropeptides were assessed 90 or 180minutes post-LPS. The plasma concentration of tumour necrosis factor α and adenohypophyseal mRNA expression of Tnfα and Il1β increased 90 and 180minutes post i.p. administration of LPS. However, cytokine mRNA expression in the hypothalamus increased only 180minutes post-LPS, suggesting an inflammatory delay in this organ. CB1 receptor blockade with AM251 increased LPS inflammatory effects, particularly in the hypothalamus. LPS also inhibited the HPG axis by decreasing gonadotrophin-releasing hormone hypothalamic content and plasma levels of luteinising hormone and testosterone. These disruptor effects were accompanied by decreased hypothalamic Kiss1 mRNA expression and prostaglandin E2 content, as well as by increased gonadotrophin-inhibitory hormone (Rfrp3) mRNA expression. All these disruptive effects were prevented by the presence of AM251. In summary, our results suggest that, in male rats, eCB mediate immune challenge-inhibitory effects on reproductive axis at least partially via hypothalamic CB1 activation. In addition, this receptor also participates in homeostasis recovery by modulating the inflammatory process taking place after LPS administration.

Antinociceptive effects of HUF-101, a fluorinated cannabidiol derivative

Cannabidiol (CBD) is a phytocannabinoid with multiple pharmacological effects and several potential therapeutic properties. Its low oral bioavailability, however, can limit its clinical use. Preliminary results indicate that fluorination of the CBD molecule increases its pharmacological potency. Here, we investigated whether HUF-101 (3, 10, and 30mg/kg), a fluorinated CBD analogue, would induce antinociceptive effects. HUF-101 effects were compared to those induced by CBD (10, 30, and 90mg/kg) and the cannabinoid CB1/2 receptor agonist WIN55,212-2 (1, 3, and 5mg/kg). These drugs were tested in male Swiss mice submitted to the following models predictive to antinociceptive drugs: hot plate, acetic acid-induced writhing, and carrageenan-induced inflammatory hyperalgesia. To evaluate the involvement of CB1 and CB2 receptors in HUF-101 and CBD effects, mice received the CB1 receptor antagonist AM251 (1 or 3mg/kg) or the CB2 receptor antagonist AM630 (1 or 3mg/kg) 30min before HUF-101, CBD, or WIN55,212-2. In the hot plate test, HUF-101 (30mg/kg) and WIN55,212-2 (5mg/kg) induced antinociceptive effects, which were attenuated by the pretreatment with AM251 and AM630. In the abdominal writhing test, CBD (30 and 90mg/kg), HUF-101 (30mg/kg), and WIN55,212-2 (3 and 5mg/kg) induced antinociceptive effects indicated by a reduction in the number of writhing. Whereas the pretreatment with AM630 did not mitigate the effects induced by any drug in this test, the pretreatment with AM251 attenuated the effect caused by WIN55,212-2. In the carrageenan-induced hyperalgesia test, CBD (30 and 90mg/kg), HUF-101 (3, 10 and 30mg/kg) and WIN55,212-2 (1mg/kg) decreased the intensity of mechanical hyperalgesia measured by the electronic von Frey method. The effects of all compounds were attenuated by the pretreatment with AM251 and AM630. Additionally, we evaluated whether HUF-101 would induce the classic cannabinoid CB1 receptor-mediated tetrad (hypolocomotion, catalepsy, hypothermia, and antinociception). Unlike WIN55,212-2, CBD and HUF-101 did not induce the cannabinoid tetrad. These findings show that HUF-101 produced antinociceptive effects at lower doses than CBD, indicating that the addition of fluoride improved its pharmacological profile. Furthermore, some of the antinociceptive effects of CBD and HUF-101 effects seem to involve the activation of CB1 and CB2 receptors.

CB1 and ethanol effects on glutamatergic transmission in the central amygdala of male and female msP and Wistar rats.

The central amygdala (CeA) is involved in the processing of anxiety and stress and plays a role in ethanol consumption. Chronic ethanol recruits stress systems in the CeA, leading to aversive withdrawal symptoms. Although primarily GABAergic, CeA contains glutamatergic afferents, and we have reported inhibitory effects of ethanol on locally evoked glutamatergic responses in CeA of Wistar and Marchigian Sardinian alcohol-preferring (msP) rats. Notably, msP rats display enhanced anxiety, stress and alcohol drinking, simulating the alcohol-dependent phenotype. Endocannabinoids are also involved in regulation of stress, and we previously demonstrated that cannabinoid receptor type 1 (CB1 ) activation decreases CeA GABAergic signaling and blocks ethanol enhancement of GABAergic signaling. Here, we sought to investigate the effects of CB1 activation (WIN 55,212-2; Win) and antagonism (AM251) with and without acute ethanol on glutamatergic synapses in CeA of female and male Wistar and msP rats. Using intracellular sharp pipette electrophysiology, we examined the effects of CB1 compounds on locally evoked excitatory postsynaptic potentials (EPSPs) in CeA and compared effects between strains, gender and estrous cycle. Acute ethanol decreased EPSP amplitudes in Wistars, and in male but not female msPs. Win decreased EPSP amplitudes in msPs, and in male but not female Wistars. Combined application of Win and ethanol resulted in strain-specific effects in female rats. We found no tonic CB1 signaling at glutamatergic synapses in CeA of any groups, and no interaction with ethanol. Collectively, these observations demonstrate sex-strain-specific differences in ethanol and endocannabinoid effects on CeA glutamatergic signaling.

Cannabinoid Receptor Signaling in Central Regulation of Feeding Behavior: A Mini-Review.

Cannabinoids are lipid messengers that modulate a variety of physiological processes and modify the generation of specific behaviors. In this regard, the cannabinoid receptor type 1 (CB1) represents the most relevant target molecule of cannabinoids so far. One main function of central CB1 signaling is to maintain whole body energy homeostasis. Thus, cannabinoids functionally interact with classical neurotransmitters in neural networks that control energy metabolism and feeding behavior. The promotion of CB1 signaling can increase appetite and stimulate feeding, while blockade of CB1 suppresses hunger and induces hypophagia. However, in order to treat overeating, pharmacological blockade of CB1 by the inverse agonist rimonabant not only suppressed feeding but also resulted in psychiatric side effects. Therefore, research within the last decade focused on deciphering the underlying cellular and molecular mechanisms of central cannabinoid signaling that control feeding and other behaviors, with the overall aim still being the identification of specific targets to develop safe pharmacological interventions for the treatment of obesity. Today, many studies unraveled the subcellular localization of CB1 and the function of cannabinoids in neurons and glial cells within circumscribed brain regions that represent integral parts of neural circuitries controlling feeding behavior. Here, these novel experimental findings will be summarized and recent advances in understanding the mechanisms of CB1-dependent cannabinoid signaling being relevant for central regulation of feeding behavior will be highlighted. Finally, presumed alternative pathways of cannabinoids that are not driven by CB1 activation but also contributing to control of feeding behavior will be introduced.

Activation of cannabinoid receptor type II by AM1241 protects adipose-derived mesenchymal stem cells from oxidative damage and enhances their therapeutic efficacy in myocardial infarction mice via Stat3 activation.

The poor survival of cells in ischemic sites diminishes the therapeutic efficacy of stem cell therapy. Previously we and others have reported that Cannabinoid receptor type II (CB2) is protective during heart ischemic injury for its anti-oxidative activity. However, whether CB2 activation could improve the survival and therapeutic efficacy of stem cells in ischemic myocardium and the underlying mechanisms remain elusive. Here, we showed evidence that CB2 agonist AM1241 treatment could improve the functional survival of adipose-derived mesenchymal stem cells (AD-MSCs) in vitro as well as in vivo. Moreover, AD-MSCs adjuvant with AM1241 improved cardiac function, and inhibited cardiac oxidative stress, apoptosis and fibrosis. To unveil possible mechanisms, AD-MSCs were exposed to hydrogen peroxide/serum deprivation to simulate the ischemic environment in myocardium. Results delineated that AM1241 blocked the apoptosis, oxidative damage and promoted the paracrine effects of AD-MSCs. Mechanistically, AM1241 activated signal transducers and activators of transcription 3 (Stat3) through the phosphorylation of Akt and ERK1/2. Moreover, the administration of AM630, LY294002, U0126 and AG490 (inhibitors for CB2, Akt, ERK1/2 and Stat3, respectively) could abolish the beneficial actions of AM1241. Our result support the promise of CB2 activation as an effective strategy to optimize stem cell-based therapy possibly through Stat3 activation.

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.).

Effect of Fatty Acid Amide Hydrolase (FAAH) Inhibitors Isolated from Nutmeg on Anxiety

The endocannabinoid system (ECS) is a complex neuromodulatory network involved in the regulation of numerous physiological functions. These effects have been substantiated by the use of known exogenous cannabinoid, Δ9‐THC, the active component of marijuana. Thus, the ECS has emerged as an appealing pharmacologic target in drug discovery. Anecdotal reports of Myristica fragrans, commonly known as nutmeg, being used as a substitute for marijuana, suggest that one or more of its components may interact with the ECS. Previous studies in our laboratory demonstrated that administration of nutmeg extracts produced behavioral tetrad effects similar to Δ9‐THC. However, the mechanism of such action requires further exploration. Receptor binding assays revealed that nutmeg extracts did not have direct CB1 or CB2 receptor binding activity. Additional mechanistic studies showed that indirect interaction through the inhibition of fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), key endocannabinoid degradation enzymes, was observed. The objective of the current study was to isolate the active components of nutmeg extract responsible for the FAAH inhibition activity and to evaluate them in an animal model of anxiety. Using bioassay guided fractionation and various chromatographic techniques, three compounds were isolated, spectroscopically characterized, and identified (MF 109‐5, MF30‐7, and MF117‐3). Concentration and time inhibition studies were conducted. The IC50 values calculated for the three compounds were 4.57 ± 0.66, 7.50 ± 2.02, and 38.29 ± 6.18 mM, respectively. The compounds were further evaluated in the elevated plus maze animal model of anxiety in comparison to diazepam as the positive control. The three nutmeg compounds were tested at doses ranging from 5 to 120 mg/kg, i.p. injection. While diazepam demonstrated a significant dose‐response increase in open arm entry and permanence time, indicative of anxiolytic action, none of the isolated compounds had a significant effect. In conclusion, the study resulted in the identification of three nutmeg compounds that significantly inhibit FAAH enzyme and hence shed light on potential mechanism of cannabis‐like action of nutmeg. While none of the compounds showed anxiolytic activity in the elevated plus maze model, additional pharmacokinetic studies are needed to optimize the dosing regimen required for significant increase in endocannabinoids that might be needed for elaboration of the anxiolytic effect.Support or Funding InformationResearch reported in this publication was supported by the National Institute on Drug Abuse of the National Institutes of Health under Award Number R24DA036410. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Activation of CB1 receptors by 2-arachidonoylglycerol attenuates vasoconstriction induced by U46619 and angiotensin II in human and rat pulmonary arteries.

Recent evidence suggests that endocannabinoids acting via cannabinoid CB1 receptors may modulate vascular responses of various vasoconstrictors in the rodent systemic vasculature. The aim of the study was to investigate whether endocannabinoids modulate the contractile responses evoked by a thromboxane A2 analog (U46619), angiotensin II (ANG II), serotonin (5-HT), and phenylephrine, which stimulate distinct Gq/11 protein-coupled receptors (thromboxane, ANG II type 1, 5-HT2, and α1-adrenergic receptors) in isolated endothelium-intact human and rat pulmonary arteries (hPAs and rPAs, respectively). The CB1 receptor antagonist AM251 (1 μM) and diacylglycerol lipase (2-arachidonoylglycerol synthesis enzyme) inhibitor RHC80267 (40 μM) enhanced contractions induced by U46619 in hPAs and rPAs and by ANG II in rPAs in an endothelium-dependent manner. AM251 did not influence vasoconstrictions induced by 5-HT or phenylephrine in rPAs. The monoacylglycerol lipase (2-arachidonoylglycerol degradation enzyme) inhibitor JZL184 (1 μM), but not the fatty acid amide hydrolase (anandamide degradation enzyme) inhibitor URB597 (1 μM), attenuated contractions evoked by U46619 in hPAs and rPAs and ANG II in rPAs. 2-Arachidonoylglycerol concentration-dependently induced relaxation of hPAs, which was inhibited by endothelium denudation or AM251 and enhanced by JZL184. Expression of CB1 receptors was confirmed in hPAs and rPAs using Western blotting and immunohistochemistry. The present study shows the protective interaction between the endocannabinoid system and vasoconstriction in response to U46619 and ANG II in the human and rat pulmonary circulation. U46619 and ANG II may stimulate rapid endothelial release of endocannabinoids (mainly 2-arachidonoylglycerol), leading to CB1 receptor-dependent and/or CB1 receptor-independent vasorelaxation, which in the negative feedback mechanism reduces later agonist-induced vasoconstriction.

In vivo TSPO and cannabinoid receptor type 2 availability early in post-stroke neuroinflammation in rats: a positron emission tomography study

BackgroundUpregulated levels of 18-kDa translocator proteins (TSPO) and type 2 endocannabinoid receptors (CB2) are considered to reflect different aspects of microglia-related neuroinflammatory responses in the brain. Relative to the increase in the TSPO expression that occurs slightly later during neuroinflammation in a proinflammatory fashion, CB2 activation is considered to relate to the neuroprotective responses that occurs predominantly at an early stage of brain disorders. These findings, however, were deduced from studies with different animal samples under different experimental settings. Here, we aimed to examined the differences in TSPO binding and CB2 availability at an early stage of stroke in the same animal using positron emission tomography (PET).MethodsWe used a total of eight Sprague-Dawley rats that underwent photothrombotic stroke surgery. The binding levels of a TSPO tracer [11C](R)PK11195 and a CB2 tracer [11C]NE40 were measured at 24 h after the surgery in the same animal using PET in combination with immunohistochemistry for CB2 and several other markers. A morphological inspection was also performed with X-ray computed tomography for small animals.ResultsThe levels of [11C]NE40 binding potential (BPND) were significantly higher in the cerebral cortical region on the lesion side than those on the non-lesion side, whereas no difference was found in the levels of [11C](R)PK11195 BPND between hemispheres. The tracer influx index (R1) data were all reduced on the lesion side irrespective of tracers. This increase in [11C]NE40 BPND was concomitant with an elevation in CB2 expression mainly within the microglia in the peri-infarct area, as shown by immunohistochemical examinations with Iba-1, CD11b/c+, and NG2+ staining.ConclusionsThe present results provide in vivo evidence of different responses of microglia occurring in the acute state of stroke. The use of the CB2 tracer [11C]NE40 allows us to evaluate the roles played by the neuroprotective aspect of microglia in acute neuroinflammatory processes.

The Ratio of 2-AG to Its Isomer 1-AG as an Intrinsic Fine Tuning Mechanism of CB1 Receptor Activation.

Endocannabinoids are pleiotropic lipid messengers that play pro-homeostatic role in cellular physiology by strongly influencing intracellular Ca2+ concentration through the activation of cannabinoid receptors. One of the best-known endocannabinoid ‘2-AG’ is chemically unstable in aqueous solutions, thus its molecular rearrangement, resulting in the formation of 1-AG, may influence 2-AG-mediated signaling depending on the relative concentration and potency of the two isomers. To predict whether this molecular rearrangement may be relevant in physiological processes and in experiments with 2-AG, here we studied if isomerization of 2-AG has an impact on 2-AG-induced, CB1-mediated Ca2+ signaling in vitro. We found that the isomerization-dependent drop in effective 2-AG concentration caused only a weak diminution of Ca2+ signaling in CB1 transfected COS7 cells. We also found that 1-AG induces Ca2+ transients through the activation of CB1, but its working concentration is threefold higher than that of 2-AG. Decreasing the concentration of 2-AG in parallel to the prevention of 1-AG formation by rapid preparation of 2-AG solutions, caused a significant diminution of Ca2+ signals. However, various mixtures of the two isomers in a fix total concentration – mimicking the process of isomerization over time – attenuated the drop in 2-AG potency, resulting in a minor decrease in CB1 mediated Ca2+ transients. Our results indicate that release of 2-AG into aqueous medium is accompanied by its isomerization, resulting in a drop of 2-AG concentration and simultaneous formation of the similarly bioactive isomer 1-AG. Thus, the relative concentration of the two isomers with different potency and efficacy may influence CB1 activation and the consequent biological responses. In addition, our results suggest that 1-AG may play role in stabilizing the strength of cannabinoid signal in case of prolonged 2-AG dependent cannabinoid mechanisms.