Potent CB1 Research Articles

Phencyclidine-Induced Social Withdrawal Results from Deficient Stimulation of Cannabinoid CB1 Receptors: Implications for Schizophrenia

The neuronal mechanisms underlying social withdrawal, one of the core negative symptoms of schizophrenia, are not well understood. Recent studies suggest an involvement of the endocannabinoid system in the pathophysiology of schizophrenia and, in particular, of negative symptoms. We used biochemical, pharmacological, and behavioral approaches to investigate the role played by the endocannabinoid system in social withdrawal induced by sub-chronic administration of phencyclidine (PCP). Pharmacological enhancement of endocannabinoid levels via systemic administration of URB597, an inhibitor of endocannabinoid degradation, reversed social withdrawal in PCP-treated rats via stimulation of CB1 receptors, but reduced social interaction in control animals through activation of a cannabinoid/vanilloid-sensitive receptor. In addition, the potent CB agonist CP55,940 reversed PCP-induced social withdrawal in a CB₁-dependent manner, whereas pharmacological blockade of CB₁ receptors by either AM251 or SR141716 reduced the time spent in social interaction in control animals. PCP-induced social withdrawal was accompanied by a decrease of anandamide (AEA) levels in the amygdala and prefrontal cortex, and these deficits were reversed by URB597. As CB₁ receptors are predominantly expressed on GABAergic interneurons containing the anxiogenic peptide cholecystokinin (CCK), we also examined whether the PCP-induced social withdrawal resulted from deficient CB₁-mediated modulation of CCK transmission. The selective CCK2 antagonist LY225910 blocked both PCP- and AM251-induced social withdrawal, but not URB597 effect in control rats. Taken together, these findings indicate that AEA-mediated activation of CB₁ receptors is crucial for social interaction, and that PCP-induced social withdrawal results from deficient endocannabinoid transmission.

Selective CB2 agonists with anti-pruritic activity: Discovery of potent and orally available bicyclic 2-pyridones

The CB2 receptor has emerged as a potential target for the treatment of pruritus as well as pain without CB1-mediated side effects. We previously identified 2-pyridone derivatives 1 and 2 as potent CB2 agonists; however, this series of compounds was found to have unacceptable pharmacokinetic profiles with no significant effect in vivo. To improve these profiles, we performed further structural optimization of 1 and 2, which led to the discovery of bicyclic 2-pyridone 18e with improved CB2 affinity and selectivity over CB1. In a mouse pruritus model, 18e inhibited compound 48/80 induced scratching behavior at a dose of 100mg/kg. In addition, the docking model of 18e with an active-state CB2 homology model indicated the structural basis of its high affinity and selectivity over CB1.

Novel Triaryl Sulfonamide Derivatives as Selective Cannabinoid Receptor 2 Inverse Agonists and Osteoclast Inhibitors: Discovery, Optimization, and Biological Evaluation

Cannabinoid receptors have gained increasing attention as drug targets for developing potential therapeutic ligands. Here, we report the discovery and optimization of triaryl sulfonamides as a novel series possessing significant CB2 receptor affinity and selectivity. Four sets of triaryl ligands were designed and synthesized for further structural modifications and led to the identification of eight compounds as potent and selective CB2 inverse agonists with high binding affinity (CB2K(i) < 10 nM). Especially, compound 57 exhibited the strongest binding affinity on the CB2 receptor (CB2K(i) of 0.5 nM) and the best selectivity over the CB1 receptor (selectivity index of 2594). Importantly, 57 also showed potent inhibitory activity on osteoclast formation, and it was confirmed by a cell viability assay that the inhibition effects were not derived from the cytotoxicity. Finally, 3D QSAR studies confirmed our SAR findings that three bulky groups play an important role for CB2 receptor binding affinity.

Trisubstituted Sulfonamides: A New Chemotype for Development of Potent and Selective CB2 Receptor Inverse Agonists

An extensive exploration of the SAR of a trisubstituted sulfonamides series led to the identification of 39, which is a potent and selective CB2 receptor inverse agonist (CB2Ki = 5.4 nM, and CB1Ki = 500 nM). The functional properties measured by cAMP assays indicated that the selected compounds were CB2 inverse agonists with high potency values (34, EC50 = 8.2 nM, and 39, EC50 = 2.5 nM). Furthermore, an osteoclastogenesis bioassay indicated that trisubstituted sulfonamide compounds showed great inhibition of osteoclast formation.

Effects of a novel CB1 agonist on visual attention in male rats: Role of strategy and expectancy in task accuracy.

The effects of cannabinoid CB1 agonists (including Δ9-tetrahydrocannabinol, the main psychoactive component of marijuana) on attention are uncertain, with reports of impairments, no effects, and occasionally performance enhancements. To better understand these effects, we sought to uncover a role of changing online (within-session) strategy as a possible mediator of the effects of the novel, potent CB1 agonist AM 4054, on a model of sustained attention in male Sprague-Dawley rats. In this operant, two-choice reaction time (RT) task, AM 4054 decreased accuracy in an asymmetric manner; that is, performance was spared on one lever but impaired on the other. Furthermore, this pattern was enhanced by the outcome of the previous trial such that AM 4054 strengthened a win-stay strategy on the "preferred" lever and a lose-shift strategy on the "nonpreferred" lever. This pattern is often found in tests of expectancy; therefore, in a second experiment AM 4054 enhanced expectancy that we engendered by altering the probability of the two stimulus cues. Accuracy was impaired in reporting the less frequent cue, but only after two or more presentations of the more frequent cue. Taking the results of the experiments together, AM 4054 engendered expectancy by increasing the role of previous trial location and outcome on performance of future trials, diminishing stimulus control (and therefore, accuracy). This novel effect of CB1 receptor agonism may contribute to the deleterious effects of cannabinoids on attention.

ChemInform Abstract: Tricyclic Pyrazoles: An Efficient Approach to Cannabinoid Analogues with a Tricyclic Framework Incorporating the Pyrrole and Pyrazole Moieties.

AbstractThe synthesis of cyclic analogues (VIII) and (IX) of the potent and selective CB1 antagonist rimonabant is developed.

New cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA) identified as designer drugs in illegal products

Two new cannabimimetic indazole derivatives, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA, 1) and N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA, 2), have been identified as designer drugs in illegal products. These identifications were based on liquid chromatography–mass spectrometry, gas chromatography–mass spectrometry, high-resolution mass spectrometry, and nuclear magnetic resonance spectroscopy. Because there have been neither chemical nor pharmacological data about compound 1 until now, this is the first report of this compound. Compound 2 was reported as a potent cannabinoid CB1 receptor modulator when synthesized by Pfizer in 2009; but this is the first report of its detection in illegal products.

Scaffold hopping strategy toward original pyrazolines as selective CB2 receptor ligands

In line of a scaffold hopping strategy of pyrazole structures, especially known as potent CB2 receptor antagonists, we exploited an original and convergent synthesis of a new class of C4-benzyl pyrazolines and derivatives from readily available hydrazones and enones (two or three steps). Making use of a mixture of resin supported reagents strategy an efficient domino process allowed the easy construction of various dihydropyrazoles in 63–83% yields. The obtained family of pyrazolines featured significant hCB2/hCB1 selectivity in favor of hCB2 receptors while more than 1000–3000 nM affinity was only measured for hCB1 receptors. This is closely related to pyrazole SR144528 inverse agonist/antagonist, although a partial agonist behavior in the [35S]-GTPγS binding assay was mainly measured in our case pointing out a functional switch in action. Furthermore, this hCB2 selectivity is unique within the pyrazoline CB ligands although the affinity ranging from 251 to 689 nM remains to be improved which give, however, an opportunity for further structure–activity relationship.

Cannabinoid receptor subtypes 1 and 2 mediate long-lasting neuroprotection and improve motor behavior deficits after transient focal cerebral ischemia

The endocannabinoid system is crucially involved in the regulation of brain activity and inflammation. We have investigated the localization of cannabinoid CB1 and CB2 receptors in adult rat brains before and after focal cerebral ischemia due to endothelin-induced transient occlusion of the middle cerebral artery (eMCAO). Using immunohistochemistry, both receptor subtypes were identified in cortical neurons. After eMCAO, neuronal cell death was accompanied by reduced neuronal CB1 and CB2 receptor-linked immunofluorescence. In parallel, CB1 receptor was found in activated microglia/macrophages 3days post eMCAO and in astroglia cells at days 3 and 7. CB2 receptor labeling was identified in activated microglia/macrophages or astroglia 3 and 7days post ischemia, respectively. In addition, immune competent CD45-positive cells were characterized by pronounced CB2 receptor staining 3 and 7days post eMCAO. KN38-72717, a potent and selective CB1 and CB2 receptor agonist, revealed a significant, dose-dependent and long-lasting reduction of cortical lesion sizes due to eMCAO, when applied consecutively before, during and after eMCAO. In addition, severe motor deficits of animals suffering from eMCAO were significantly improved by KN38-7271. KN38-7271 remained effective, even if its application was delayed up to 6h post eMCAO. Finally, we show that the endocannabinoid system assembles a comprehensive machinery to defend the brain against the devastating consequences of cerebral ischemia. In summary, this study underlines the therapeutic potential of CB1 and/or CB2 receptor agonists against neurodegenerative diseases or injuries involving acute or chronic imbalances of cerebral blood flow and energy consumption.

4-Oxo-1,4-dihydropyridines as Selective CB2 Cannabinoid Receptor Ligands Part 2: Discovery of New Agonists Endowed with Protective Effect Against Experimental Colitis

Further on to our earlier work on the 4-oxo-1,4-dihydropyridine, we describe herein our strategy to get access to potent selective CB₂ receptor agonists. Thus, we designed and synthesized 29 compounds, evaluated on both hCB₁ and hCB₂ cannabinoid receptors, and assessed 11 of them in the TNBS-induced colitis model in mice. Compound 48 was found to be the most efficient of our series, exhibiting an exquisite protection against experimental colitis, superior to the one observed after treatment with Pentasa.

The anti‐nausea effects of CB1 agonists are mediated by an action at the visceral insular cortex

Conditioned gaping reactions reflect nausea-induced behaviour in rats. Cannabinoid 1 receptor (CB(1) ) agonists interfere with the establishment of nausea-induced conditioned gaping; however, it is not known if their effects are mediated by an action at peripheral or central CB(1) receptors. We utilized the conditioned gaping model of nausea to evaluate the effect of peripheral and central administration of the peripherally restricted CB(1) agonist, CB13, on the establishment of LiCl-induced gaping in rats. We further evaluated the ability of HU-210 administered to the gustatory insular cortex (GIC) or visceral insular cortex (VIC) to interfere with LiCl-induced conditioned gaping and determined if this effect was mediated by CB(1) receptors. Central, but not peripheral, CB13 suppressed LiCl-induced conditioned gaping. Central administration of the potent CB(1) agonist, HU-210, delivered to the VIC, but not the GIC, suppressed the establishment of LiCl-induced gaping reactions, but not LiCl-induced suppression of hedonic reactions or conditioned taste avoidance. This pattern of results suggests that HU-210 delivered to the VIC prevented LiCl-induced nausea, but not learning per se. The suppression of LiCl-induced conditioned gaping by HU-210 was mediated by CB(1) receptors because it was prevented by co-administration of CB(1) antagonist/inverse agonist, AM-251, into the VIC. A high dose of AM-251 (20 µg) administered alone into the VIC did not produce conditioned gaping reactions. The nausea-relieving effects of CB(1) agonists, but not the nausea-inducing effects of CB(1) inverse agonists, are mediated, at least in part, by their action at the VIC in rats.

7-Alkyl-3-benzylcoumarins: A Versatile Scaffold for the Development of Potent and Selective Cannabinoid Receptor Agonists and Antagonists

A series of 7-alkyl-3-benzylcoumarins was designed, synthesized, and tested at cannabinoid CB(1) and CB(2) receptors in radioligand binding and cAMP accumulation studies. 7-Alkyl-3-benzylcoumarins were found to constitute a versatile scaffold for obtaining potent CB receptor ligands with high potency at either CB(1) or CB(2) and a broad spectrum of efficacies. Fine-tuning of compound properties was achieved by small modifications of the substitution pattern. The most potent compounds of the present series include 5-methoxy-3-(2-methylbenzyl)-7-pentyl-2H-chromen-2-one (19a, PSB-SB-1201), a selective CB(1)antagonist (K(i) CB(1) 0.022 μM), 5-methoxy-3-(2-methoxybenzyl)-7-pentyl-2H-chromen-2-one (21a, PSB-SB-1202), a dual CB(1)/CB(2)agonist (CB(1)K(i) 0.032 μM, EC(50) 0.056 μM; CB(2)K(i) 0.049 μM, EC(50) 0.014 μM), 5-hydroxy-3-(2-hydroxybenzyl)-7-(2-methyloct-2-yl)-2H-chromen-2-one (25b, PSB-SB-1203), a dual CB(1)/CB(2) ligand that blocks CB(1) but activates CB(2) receptors (CB(1)K(i) 0.244 μM; CB(2)K(i) 0.210 μM, EC(50) 0.054 μM), and 7-(1-butylcyclopentyl)-5-hydroxy-3-(2-hydroxybenzyl)-2H-chromen-2-one (27b, PSB-SB-1204), a selective CB(2) receptor agonist (CB(1)K(i) 1.59 μM; CB(2)K(i) 0.068 μM, EC(50) 0.048 μM).

Contrasting effects of different cannabinoid receptor ligands on mouse ingestive behaviour

This study characterized the effects of seven diverse cannabinoid receptor agonists (and one antagonist) on ingestive behaviour in nondeprived adult, male CD1 mice. Microstructural analysis of licking for a range of concentrations of condensed milk (10, 15 and 20%) was carried out following administration of vehicle or: Δ⁹-tetrahydrocannabinol (Δ⁹-THC) at 1, 3 or 6 mg/kg; CP55,940 at 10, 30 or 50 µg/kg; Win 55,212-2 at 0.5, 1 or 3 mg/kg; HU-210 at 0.01, 0.03 or 0.1 mg/kg; methanandamide at 1, 3 or 6 mg/kg; arachidonyl-2'-chloroethylamide at 1, 3 or 6 mg/kg and JWH133 at 1, 3 or 6 mg/kg. The cannabinoid receptor antagonist/inverse agonist rimonabant was also tested at 0.3, 1 or 3 mg/kg. Test sessions comprised three 30 s presentations of the milk concentrations separated by 10 s interpresentation intervals. The nonselective CB1 receptor agonists Δ⁹-THC, CP55,940 and Win 55,212-2 increased the number of licks for condensed milk, primarily by a significant increase in bout number. The potent and nonselective CB1 receptor agonist HU-210 and the selective CB1 receptor agonists methanandamide and arachidonyl-2'-chloroethylamide did not significantly affect licking behaviour but did significantly increase the latency to start licking. The CB1 receptor antagonist rimonabant produced effects that were opposite in direction to those produced by Δ⁹-THC, CP55,940 and Win 55,212-2. Finally, the selective CB2 receptor agonist JWH133 had no significant effects on behaviour. These data add to reports that cannabinoid agonists can enhance the appetitive aspects of feeding, but they also demonstrate that not all CB1 receptor agonists do this, and therefore the relationship between action at CB1 receptors and appetitive feeding effects is not straightforward.

Tricyclic Pyrazoles: An Efficient Approach to Cannabinoid Analogues with a Tricyclic Framework Incorporating the Pyrrole and Pyrazole Moieties

In this paper we report the synthesis of some new cannabinoid analogues that share with rimonabant, a potent and selective CB1 antagonist, the 2,4-dichlorophenyl and piperidin-1-yl substituents on the pyrazole and amide nitrogens, respectively. The general synthesis involves the preparation of a cyclic ketone condensed with a pyrrole, followed by Claisen condensation with diethyl oxalate; from here, an aza-annulation reaction with a substituted hydrazine followed by an amidation step complete the synthesis.

Discovery and optimization of novel purines as potent and selective CB2 agonists

A focused screening strategy identified thienopyrimidine 1 as a hCB2 cannabinoid receptor agonist with moderate selectivity over the hCB1 receptor. This initial hit suffered from poor in vitro metabolic stability and high in vivo clearance. Structure–activity relationships describe the optimization and modification to a less lipophilic purine core. Examples from this novel series were found to be highly potent and fully efficacious agonists of the human CB2 receptor with excellent selectivity against CB1. Compound 10 possesses good biopharmaceutical properties, is highly water soluble and demonstrates robust oral activity in rodent models of joint pain.

Detection and Quantification of New Designer Drugs in Human Blood: Part 1 - Synthetic Cannabinoids

Synthetic cannabinoids sprayed on herbal mixtures have been abused as a new designer drug all over the world since 2004. In 2008, the first compounds, CP 47,497 and JWH-018, were identified as active ingredients in these mixtures. Most of the compounds have been synthesized for research purposes and are potent CB1 and/or CB2 receptor agonists. To investigate the presence of synthetic cannabinoids in blood samples, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed using only 100 µL of blood. After the addition of 0.2 mL of trizma buffer, the blood was extracted using liquid-liquid extraction with 1 mL of 1-chlorobutane containing 10% of isopropanol for 5 min on a shaker at 1,500 rpm. After centrifugation at 12,000 rpm for 1 min, the separated solvent layer was transferred to an autosampler vial and evaporated to dryness under N₂. The residue was reconstituted in methanol and injected into a Shimadzu 8030 LC-MS-MS system to separate and detect 25 synthetic cannabinoids. The method has been validated according to international guidelines and was found to be selective for all tested compounds. Calibration was satisfactory from 0.5-100 ng/mL, and from 5.0-500 ng/mL. for HU-210, CP 47,497 and the CP 47,497 C-8 homolog, respectively. The extraction efficiencies ranged from 30-101% and the matrix effects from 67-112%. Accuracy data were within the acceptance interval of ±15% (±20% at the lower limit of quantification) of the nominal values for all drugs.

Design, Synthesis, and Pharmacological Characterization of Indol-3-ylacetamides, Indol-3-yloxoacetamides, and Indol-3-ylcarboxamides: Potent and Selective CB2 Cannabinoid Receptor Inverse Agonists

In our search for new cannabinoid receptor modulators, we describe herein the design and synthesis of three sets of indole-based ligands characterized by an acetamide, oxalylamide, or carboxamide chain, respectively. Most of the compounds showed affinity for CB2 receptors in the nanomolar range, with K(i) values spanning 3 orders of magnitude (377-0.37 nM), and moderate to good selectivity over CB1 receptors. Their in vitro functional activity as inverse agonists was confirmed in vivo in the formalin test of acute peripheral and inflammatory pain in mice, in which compounds 10a and 11e proved to be able to reverse the effect of the CB2 selective agonist COR167.

Synergistic interaction of pregabalin with the synthetic cannabinoid WIN 55,212-2 mesylate in the hot-plate test in mice: an isobolographic analysis

BackgroundThe aim of the study was to determine the type of interaction between pregabalin (a 3rd-generation antiepileptic drug) and WIN 55,212-2 mesylate (WIN – a highly potent non-selective cannabinoid CB1 and CB2 receptor agonist) administered in combination at a fixed ratio of 1:1, in the acute thermal pain model (hot-plate test) in mice. MethodsLinear regression analysis was used to evaluate the dose-response relationships between logarithms of drug doses and their resultant maximum possible antinociceptive effects in the mouse hot-plate test. From linear equations, doses were calculated that increased the antinociceptive effect by 30% (ED30 values) for pregabalin, WIN, and their combination. The type of interaction between pregabalin and WIN was assessed using the isobolographic analysis. ResultsResults indicated that both compounds produced a definite antinociceptive effect, and the experimentally-derived ED30 values for pregabalin and WIN, when applied alone, were 29.4mg/kg and 10.5mg/kg, respectively. With isobolography, the experimentally derived ED30 mix value for the fixed ratio combination of 1:1 was 5.7mg/kg, and differed significantly from the theoretically calculated ED30 add value of 19.95mg/kg (p<0.01), indicating synergistic interaction between pregabalin and WIN in the hot-plate test in mice. ConclusionsIsobolographic analysis demonstrated that the combination of WIN with pregabalin at a fixed ratio of 1:1 exerted synergistic interaction in the mouse model of acute thermal pain. If the results from this study could be adapted to clinical settings, the combination of WIN with pregabalin might be beneficial for pain relief in humans.

A new cannabinoid CB2 receptor agonist HU‐910 attenuates oxidative stress, inflammation and cell death associated with hepatic ischaemia/reperfusion injury

Cannabinoid CB(2) receptor activation has been reported to attenuate myocardial, cerebral and hepatic ischaemia-reperfusion (I/R) injury. We have investigated the effects of a novel CB(2) receptor agonist ((1S,4R)-2-(2,6-dimethoxy-4-(2-methyloctan-2-yl)phenyl)-7,7-dimethylbicyclo[2.2.1]hept-2-en-1-yl)methanol (HU-910) on liver injury induced by 1 h of ischaemia followed by 2, 6 or 24 h of reperfusion, using a well-established mouse model of segmental hepatic I/R. Displacement of [(3) H]CP55940 by HU-910 from specific binding sites in CHO cell membranes transfected with human CB(2) or CB(1) receptors (hCB(1/2) ) yielded K(i) values of 6 nM and 1.4 µM respectively. HU-910 inhibited forskolin-stimulated cyclic AMP production by hCB(2) CHO cells (EC(50) = 162 nM) and yielded EC(50) of 26.4 nM in [(35) S]GTPγS binding assays using hCB(2) expressing CHO membranes. HU-910 given before ischaemia significantly attenuated levels of I/R-induced hepatic pro-inflammatory chemokines (CCL3 and CXCL2), TNF-α, inter-cellular adhesion molecule-1, neutrophil infiltration, oxidative stress and cell death. Some of the beneficial effect of HU-910 also persisted when given at the beginning of the reperfusion or 1 h after the ischaemic episode. Furthermore, HU-910 attenuated the bacterial endotoxin-triggered TNF-α production in isolated Kupffer cells and expression of adhesion molecules in primary human liver sinusoidal endothelial cells stimulated with TNF-α. Pretreatment with a CB(2) receptor antagonist attenuated the protective effects of HU-910, while pretreatment with a CB(1) antagonist tended to enhance them. HU-910 is a potent CB(2) receptor agonist which may exert protective effects in various diseases associated with inflammation and tissue injury. This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

Design of a Potent CB1 Receptor Antagonist Series: Potential Scaffold for Peripherally-Targeted Agents.

Antagonism of cannabinoid-1 (CB1) receptor signaling has been demonstrated to inhibit feeding behaviors in humans, but CB1-mediated central nervous system (CNS) side effects have halted the marketing and further development of the lead drugs against this target. However, peripherally restricted CB1 receptor antagonists may hold potential for providing the desired efficacy with reduced CNS side effect profiles. In this report we detail the discovery and structure-activity-relationship analysis of a novel bicyclic scaffold (3) that exhibits potent CB1 receptor antagonism and oral activity in preclinical feeding models. Optimization of physical properties has led to the identification of analogues which are predicted to have reduced CNS exposure and could serve as a starting point for the design of peripherally targeted CB1 receptor antagonists.