Human CB1 Receptors Research Articles

Pharmacological Evaluation of a Novel Cannabinoid 2 (CB2) Ligand, PF-03550096, In Vitro and In Vivo by Using a Rat Model of Visceral Hypersensitivity

Previous studies have shown that cannabinoid 2 (CB2)-receptor agonists might have analgesic effects on visceral hypersensitivity. To extend these results, we have determined the pharmacological characteristics of a newly designed CB2ligand, N-[(1S )-1-(aminocarbonyl)-2,2-dimethylpropyl]-3-(3-hydroxy-3-methylbutyl)-2-oxo-2,3-dihydro-1H-benzimidazole-1-carboxamide (PF-03550096), in vitro and in vivo. PF-03550096 showed high affinity to human (Ki= 7.9 ± 1.7 nM) and rat CB2receptors (Ki= 47 ± 5.6 nM). In a cell-based functional assay, PF-03550096 behaved as a full agonist and showed high selectivity for human CB2receptors. Orally administered PF-03550096 (3, 10 mg/kg) inhibited the 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced decrease in colonic pain threshold with statistical significance. The inhibitory effect of PF-03550096 (10 mg/kg) was significantly reversed by a selective CB2antagonist, N-(1S )-endo-1,3,3-trimethylbicyclo[2.2.1]heptan-2-yl-5-(4-chloro-3-methylphenyl)-1(4-methylbenzyl)-pyrazole-3-carboxamide (SR144528), while SR144528 itself did not modify colonic pain threshold. These results indicate that PF-03550096 is a potent CB2agonist and possesses efficacy in a rat model of visceral hypersensitivity.

Analysis of Promoter Regions Regulating Basal and Interleukin-4-Inducible Expression of the Human CB1 Receptor Gene in T Lymphocytes

The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.

A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.

Naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone: A Potent, Orally Bioavailable Human CB1/CB2 Dual Agonist with Antihyperalgesic Properties and Restricted Central Nervous System Penetration

Selective activation of peripheral cannabinoid CB1 receptors has the potential to become a valuable therapy for chronic pain conditions as long as central nervous system effects are attenuated. A new class of cannabinoid ligands was rationally designed from known aminoalkylindole agonists and showed good binding and functional activities at human CB1 and CB2 receptors. This has led to the discovery of a novel CB1/CB2 dual agonist, naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (13), which displays good oral bioavailability, potent antihyperalgesic activity in animal models, and limited brain penetration.

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro

A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells. The [35S]GTPS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor. This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors. This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.

In vitro and in vivo pharmacology of synthetic olivetol‐ or resorcinol‐derived cannabinoid receptor ligands

We have previously reported the development of CB-25 and CB-52, two ligands of CB1 and CB2 cannabinoid receptors. We assessed here their functional activity. The effect of the two compounds on forskolin-induced cAMP formation in intact cells or GTP-gamma-S binding to cell membranes, and their action on nociception in vivo was determined. CB-25 enhanced forskolin-induced cAMP formation in N18TG2 cells (EC50 approximately 20 nM, max. stimulation = 48%), behaving as an inverse CB1 agonist, but it stimulated GTP-gamma-S binding to mouse brain membranes, behaving as a partial CB1 agonist (EC50 =100 nM, max. stimulation = 48%). At human CB1 receptors, CB-25 inhibited cAMP formation in hCB1-CHO cells (EC50 = 1600 nM, max. inhibition = 68% of CP-55,940 effect). CB-52 inhibited forskolin-induced cAMP formation by N18TG2 cells (IC50 = 450 nM, max. inhibition = 40%) and hCB1-CHO cells (EC50 = 2600 nM, max. inhibition = 62% of CP-55,940 effect), and stimulated GTP-gamma-S binding to mouse brain membranes (EC50 = 11 nM, max. stimulation approximately 16%). Both CB-25 and CB-52 showed no activity in all assays of CB2-coupled functional activity and antagonized CP55940-induced stimulation of GTP-gamma-S binding to hCB2-CHO cell membranes. In vivo, both compounds, administered i.p., produced dose-dependent nociception in the plantar test carried out in healthy rats, and antagonised the anti-nociceptive effect of i.p. WIN55,212-2. In the formalin test in mice, however, the compounds counteracted both phases of formalin-induced nociception. CB-25 and CB-52 behave in vitro mostly as CB1 partial agonists and CB2 neutral antagonists, whereas their activity in vivo might depend on the tonic activity of cannabinoid receptors.

In vitro pharmacological characterization of AM1241: a protean agonist at the cannabinoid CB2 receptor?

The CB2 receptor has been proposed as a novel target for the treatment of pain, and CB2 receptor agonists defined in in vitro assays have demonstrated analgesic activity in animal models. Based on its in vivo analgesic efficacy, AM1241 has been classified as a CB2-selective agonist. However, in vitro characterization of AM1241 in functional assays has not been reported. In this study, AM1241 was characterized across multiple in vitro assays employing heterologous recombinant receptor expression systems to assess its binding potencies at the human CB2 and CB1 receptors and its functional efficacies at the human CB2 receptor. AM1241 exhibited distinct functional properties depending on the assay conditions employed, a unique profile in contrast to those of the agonist CP 55,940 and the inverse agonist SR144528. AM1241 displayed neutral antagonist activities in FLIPR and cyclase assays. However, when cyclase assays were performed using lower forskolin concentrations for stimulation, AM1241 exhibited partial agonist efficacy. In addition, it behaved as a partial agonist in ERK (or MAP) kinase assays. The unusual phenomenon of inconsistent functional efficacies suggests that AM1241 is a protean agonist at the CB2 receptor. We postulate that functional efficacies displayed by protean agonists in various assay systems may depend on the levels of receptor constitutive activities exhibited in the assay systems, and therefore, efficacies observed in in vitro assays may not predict in vivo activities.

Exploration of natural alkylamides and synthetic analogs as source for new ligands for the cannabinoid type-2 receptor

The cannabinoid type-2 (CB2) receptor is an attractive target for the development of drugs against inflammatory disease, atherosclerosis, and osteoporosis. Based on the discovery that alkylamides (alkamides) from Echinacea constitute a new class of CB2-specific cannabinomimetics1, we have screened a series of synthetic analogs of Echinacea alkylamides as well as other plant-derived natural N-alkyl amides for competitive binding to the CB2-receptor. Because dodeca-2E,4E-dienoic acid isobutylamide from Echinacea has a high affinity to the human CB2-receptor we synthesized analogs with modified head groups, as reported for the endogenous cannabinoid anandamide2. The resulting preliminary structure-activity relationship clearly shows that the CB2 receptor binding mode of Echinacea alkylamides is different from anandamide. To further explore the potential of natural N-alkyl amides as a new general class of CB2 ligands we have initiated a screening of plant extracts. The hexane extracts of the medicinal plants Spilanthes oleracea L. and Lepidium meyenii Walper. which are known to contain distinct types of alkylamides, were tested in receptor binding assays. The alkylamide-fraction of the L. meyenii extract showed significant receptor binding and five isolated benzylated alkylamides (macamides) were assessed for both their CB2-receptor affinity as well as CB2-mediated functional effects. Our data show that natural alkylamides from Echinacea and Lepidium are promising candidates for the development of novel CB2-receptor ligands.

Synthesis, cannabinoid receptor activity, and enzymatic stability of reversed amide derivatives of arachidonoyl ethanolamide

Retroanandamide ( 2f) and its 10 analogues ( 1a– e, 2a– e) were synthesized and evaluated for the cannabinoid receptor activation by a [ 35S]GTPγS binding assay using rat cerebellar membranes, and Chinese hamster ovary cell membranes expressing human CB2 receptors. The primary goal of the study was to develop cannabinoid receptor agonists having improved enzymatic stability compared to endogenous N-arachidonoyl ethanolamide (AEA). Furthermore, by reversing the amide bond of AEA, the formation of arachidonic acid would be prevented. Finally, an effect of the carbonyl carbon position on the cannabinoid receptor activity was explored by synthesizing retroanandamide analogues having different chain lengths ( 1a– e, C 19; 2a– f, C 20). All the synthesized compounds, except 2c, behaved as partial agonists for the both cannabinoid receptors. In rat brain homogenate, the reversed amides possessed significantly higher stability against FAAH induced degradation than AEA. Therefore, the reversed amide analogues of AEA may serve as enzymatically stable structural basis for the drug design based on the endogenous cannabinoids.

(609): Species dependent pharmacology of human, mouse and rat CB2 receptors

(609): Species dependent pharmacology of human, mouse and rat CB2 receptors

Synthesis and SAR Studies of 2-Oxoquinoline Derivatives as CB2 Receptor Inverse Agonists

The highly CB2 selective cannabinoid receptor inverse agonist, 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid N-benzo[1,3]dioxol-5-ylmethyl)amide (JTE-907; 9b), served as the lead compound for investigating the structure-activity relationships of its analogues and in the search for more potent and effective CB2 receptor inverse agonists. A series of aromatic amides of 7-methoxy-2-oxo-8-pentyloxy-1,2-dihydroquinoline-3-carboxylic acid 6 was synthesized, and the CB2 receptor activities of the compounds were determined by a [35S]GTPgammaS-binding assay using membranes of CHO cells stably transfected with the human CB2 receptor. As a result, all the compounds were defined as full CB2 receptor inverse agonists, and additionally, except for two 3,4-dihydroxyphenylalkylamides, they were found to be equally potent as SR144528.

Virtual Screening of Novel CB2 Ligands Using a Comparative Model of the Human Cannabinoid CB2 Receptor

To identify novel selective CB2 lead compounds, a comparative model of the CB2 receptor was constructed using the high-resolution bovine rhodopsin X-ray structure as a template. The CB2 model was utilized both in building the database queries and in filtering the hit compounds by a docking and scoring method. In G-protein activation assays, 1-isoquinolyl[3-(trifluoromethyl)phenyl]methanone (40, NRB 04079) was found to act as a selective agonist at the human CB2 receptor.

Synthesis and Activity of 4,5‐Diarylimidazoles as Human CB1 Receptor Inverse Agonists.

AbstractFor Abstract see ChemInform Abstract in Full Text.

The Endocannabinoid Noladin Ether Acts as a Full Agonist at Human CB2 Cannabinoid Receptors

Noladin ether (NE) is a putative endogenously occurring cannabinoid demonstrating agonist activity at CB1 receptors. Because of reported selective affinity for CB1 receptors, the pharmacological actions of NE at CB2 receptors have not been examined. Therefore, the purpose of this study was to characterize the binding and functional properties of NE at human CB2 receptors stably expressed in Chinese hamster ovary (CHO) cells as well as in HL-60 cells, which express CB2 receptors endogenously. Surprisingly, in transfected CHO cells, NE exhibits a relatively high nanomolar affinity for CB2 receptors (K(i) = 480 nM), comparable to that observed for the endocannabinoid 2-arachidonoyl glycerol (2-AG) (K(i) = 1016 nM). Furthermore, NE activates G proteins and inhibits the intracellular effector adenylyl cyclase with equivalent efficacy relative to the full cannabinoid agonists 2-AG and CP 55,940 (CP) [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl) cyclohexanol]. The rank order of potency for G protein activation and effector regulation by the three agonists is similar to their apparent affinity for CB2 receptors; CP > NE > or = 2-AG. Regulation of adenylyl cyclase activity by all agonists is inhibited by pertussis toxin pretreatment or by coincubation with AM630 [6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)-methanone], a CB2 antagonist. Chronic treatment with NE or CP results in CB2 receptor desensitization and down-regulation. All agonists also inhibit adenylyl cyclase activity in HL-60 cells. Together, these data indicate that NE acts as a full agonist at human CB2 receptors and thus might have important physiological functions at peripheral cannabinoid receptors.

Cysteine 2.59(89) in the Second Transmembrane Domain of Human CB2 Receptor Is Accessible within the Ligand Binding Crevice: Evidence for Possible CB2 Deviation from a Rhodopsin Template

In this study, the sensitivity of the CB2 receptor to methanethiosulfonate (MTS) derivatives was tested, and a native cysteine residue conferring the sensitivity was identified. By incubating human embryonic kidney 293 cells stably transfected with CB2 receptors and MTS derivatives such as MTS ethylammonium (MTSEA), [(3)H]HU-243 binding was inhibited. Pretreatment of the CB2 receptor with cannabinoid ligands prevented this inhibition, suggesting that MTSEA modification occurred within the binding crevice. To identify the cysteine(s) responsible for the MTSEA sensitivity, 10 CB2 mutants were prepared in which the eight cysteines in transmembrane domains or extracellular loop 2 were mutated to serine or alanine, one at a time or in combination. Five mutants exhibited specific [(3)H]HU-243 binding, with K(d) and B(max) values similar to those of wild-type CB2. However, five other mutants had no detectable ligand binding and were not detected on cell membranes by Western blot analysis. Among the five mutants with normal binding, only the sensitivity to MTSEA of the C2.59(89)S mutant was reduced significantly. These data demonstrate that C2.59(89) is the residue that mainly confers the inhibitory effect of MTSEA on ligand binding. Furthermore, the magnitude of the second-order rate constant (1.14 +/- 0.28 M(-1)s(-1)) for the MTSEA reaction with wild-type CB2 suggests that C2.59(89) resides at the margin of the CB2 binding site crevice. The accessibility of C2.59(89) to MTSEA provides experimental evidence for a possible conformational difference between TMH2 of CB2 versus Rho. Modeling studies undertaken to explore the origin of such differences suggest it is possibly caused by the conformational influence of S2.54(84).

Pharmacological and pharmacokinetic characterization of the cannabinoid receptor 2 agonist, GW405833, utilizing rodent models of acute and chronic pain, anxiety, ataxia and catalepsy

To date, two cannabinoid receptors have been identified, CB1 and CB2. Activation of these receptors with non-selective cannabinoid receptor agonists reduces pain sensitivity in animals and humans. However, activation of CB1 receptors is also associated with central side effects, including ataxia and catalepsy. More recently, a role for selective CB2 agonists in pain modification has been demonstrated. GW405833, a selective CB2 agonist, was recently reported to partially reverse the inflammation and hyperalgesia in a rat model of acute inflammation. In the current report, we extend the characterization and therapeutic potential of this compound. For the first time, we show that GW405833 selectively binds both rat and human CB2 receptors with high affinity, where it acts as a partial agonist (∼50% reduction of forskolin-mediated cAMP production compared to the full cannabinoid agonist, CP55,940). We also report for the first time that intraperitoneal administration of GW405833 (0.3–100 mg/kg) to rats shows linear, dose-dependent increases in plasma levels and substantial penetration into the central nervous system. In addition, GW405833 (up to 30 mg/kg) elicits potent and efficacious antihyperalgesic effects in rodent models of neuropathic, incisional and chronic inflammatory pain, the first description of this compound in these models. In contrast, analgesia, sedation and catalepsy were not observed in this dose range, but were apparent at 100 mg/kg. Additionally, GW405833 was not antihyperalgesic against chronic inflammatory pain in CB2 knockout mice. These data support the tenet that selective CB2 receptor agonists have the potential to treat pain without eliciting the centrally-mediated side effects associated with non-selective cannabinoid agonists, and highlight the utility of GW405833 for the investigation of CB2 physiology.

Synthesis and activity of 4,5-diarylimidazoles as human CB1 receptor inverse agonists

Structure–activity relationship studies directed toward the optimization of 4,5-diarylimidazole-2-carboxamide analogs as human CB1 receptor inverse agonists resulted in the discovery of the two amide derivatives 24a and b (hCB1 IC 50 = 6.1 and 4.0 nM) which also demonstrated efficacy in overnight feeding studies in the rat for reduction in both food intake and overall body weight.

Identification and characterisation of a novel splice variant of the human CB1 receptor

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA’s are expressed at low levels in a variety of tissues.

SR147778 [5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide], a new potent and selective antagonist of the CB1 cannabinoid receptor: biochemical and pharmacological characterization.

Based on binding, functional, and pharmacological data, this study introduces SR147778 [5-(4-bromophenyl)-1-(2,4-dichloro-phenyl)-4-ethyl-N-(1-piperidinyl)-1H-pyrazole-3-carboxamide] as a highly potent, selective, and orally active antagonist for the CB1 receptor. This compound displays nanomolar affinity (Ki = 0.56 and 3.5 nM) for both the rat brain and human CB1 recombinant receptors, respectively. It has low affinity (Ki = 400 nM) for both the rat spleen and human CB2 receptors. Furthermore, it shows no affinity for any of the over 100 targets investigated (IC50 > 1 microM). In vitro, SR147778 antagonizes the inhibitory effects of CP 55,940 [(1R,3R,4R)-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-4-(3-hydroxypropyl)cyclohexan-1-ol] on both the mouse vas deferens contractions (pA2 value = 8.1) and on forskolin-stimulated adenylyl cyclase activity in the U373 MG cell lines (pA2 value = 8.2) but not in Chinese hamster ovary (CHO) cells permanently expressing the human peripheral cannabinoid receptor (hCB2). SR147778 is able to block the mitogen-activated protein kinase activity induced by CP 55,940 in the CHO cell line expressing human brain cannabinoid receptor (IC50 = 9.6 nM) but was inactive in cells expressing hCB2. After oral administration, SR147778 displaced the ex vivo [3H]-CP 55,940 binding to mouse brain membranes (ED50 = 3.8 mg/kg) with a long duration of action, whereas it did not interact with the CB2 receptor expressed in the mouse spleen. Using different routes of administration, SR147778 (0.3-3 mg/kg) is shown to antagonize pharmacological effects (hypothermia, analgesia, and gastrointestinal transit) induced by R-(+)-(2,3-dihydro-5-methyl-3-[[4-morpholinyl]methyl] pyrol [1,2,3-de]-1,4-benzoxazin-6-yl)(1-naphthalenyl) methanone in mice. Finally, per se, SR147778 (0.3-10 mg/kg) is able to reduce ethanol or sucrose consumption in mice and rats and food intake in fasted and nondeprived rats.

Potent imidazole and triazole CB1 receptor antagonists related to SR141716

Diarylimidazolecarboxamides and diaryltriazolecarboxamides related to SR141716 were synthesized and tested for binding to the human CB1 receptor. Suitably substituted imidazoles are comparably potent to the clinical candidate, whereas the analogous triazoles are less so due to the absence of an additional substituent on the azole ring.