In vitro and in vivo pharmacology of CP-945,598, a potent and selective cannabinoid CB1 receptor antagonist for the management of obesity

Cannabinoid CB₁ receptor antagonists exhibit pharmacologic properties favorable for the treatment of metabolic disease. CP-945,598 (1-[9-(4-chlorophenyl)-8-(2-chlorophenyl)-9H-purin-6-yl]-4-ethylamino piperidine-4-carboxylic acid amide hydrochloride) is a recently discovered selective, high affinity, competitive CB₁ receptor antagonist that inhibits both basal and cannabinoid agonist-mediated CB₁ receptor signaling in vitro and in vivo. CP-945,598 exhibits sub-nanomolar potency at human CB₁ receptors in both binding (K_i = 0.7 nM) and functional assays (K_i = 0.2 nM). The compound has low affinity (K_i = 7600 nM) for human CB₂ receptors. In vivo, CP-945,598 reverses four cannabinoid agonist-mediated CNS-driven responses (hypo-locomotion, hypothermia, analgesia, and catalepsy) to a synthetic cannabinoid receptor agonist. CP-945,598 exhibits dose and concentration-dependent anorectic activity in two models of acute food intake in rodents, fast-induced re-feeding and spontaneous, nocturnal feeding. CP-945,598 also acutely stimulates energy expenditure in rats and decreases the respiratory quotient indicating a metabolic switch to increased fat oxidation. CP-945,598 at 10 mg/kg promoted a 9%, vehicle adjusted weight loss in a 10 day weight loss study in diet-induced obese mice. Concentration/effect relationships combined with ex vivo brain CB₁ receptor occupancy data were used to evaluate efficacy in behavioral, food intake, and energy expenditure studies. Together, these in vitro, ex vivo, and in vivo data indicate that CP-945,598 is a novel CB₁ receptor competitive antagonist that may further our understanding of the endocannabinoid system.     

3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ-THC and related compounds: synthesis of selective ligands for the CB2 receptor

The synthesis and pharmacology of 15 1-deoxy-Δ⁸-THC analogues, several of which have high affinity for the CB₂ receptor, are described. The deoxy cannabinoids include 1-deoxy-11-hydroxy-Δ⁸-THC (5), 1-deoxy-Δ⁸-THC (6), 1-deoxy-3-butyl-Δ⁸-THC (7), 1-deoxy-3-hexyl-Δ⁸-THC (8) and a series of 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=0–4, 6, 7, where n=the number of carbon atoms in the side chain−2). Three derivatives (17–19) of deoxynabilone (16) were also prepared. The affinities of each compound for the CB₁ and CB₂ receptors were determined employing previously described procedures. Five of the 3-(1′,1′-dimethylalkyl)-1-deoxy-Δ⁸-THC analogues (2, n=1–5) have high affinity (K_i=<20 nM) for the CB₂ receptor. Four of them (2, n=1–4) also have little affinity for the CB₁ receptor (K_i=>295 nM). 3-(1′,1′-Dimethylbutyl)-1-deoxy-Δ⁸-THC (2, n=2) has very high affinity for the CB₂ receptor (K_i=3.4±1.0 nM) and little affinity for the CB₁ receptor (K_i=677±132 nM).

Scheme 3. (a) (C₆H₅)₃PCH₃⁺ Br⁻, n-BuLi/THF, 65°C; (b) LiAlH₄/THF, 25°C; (c) KBH(sec-Bu)₃/THF, −78 to 25°C then H₂O₂/NaOH.     

Role of endocannabinoids and endovanilloids in Ca signalling

Endocannabinoids and endovanilloids are, by definition, endogenous agonists at cannabinoid CB₁ or CB₂ receptors and transient receptor potential vanilloid-type-1 (TRPV1) channels, respectively. Due to the several ways through which cannabinoid receptors influence cytosolic Ca²⁺ concentrations, and to the fact that TRPV1 activation leads to the gating of cations, including Ca²⁺, both endocannabinoids and endovanilloids, taken separately, can strongly influence Ca²⁺ signalling. Moreover, CB₁/CB₂ receptors and TRPV1 channels are often expressed in the same or neighbouring cells, and this can lead to cross-talk between the two receptor types, which is further enriched by the fact that some endocannabinoids, like anandamide and N-arachidonoyldopamine, also activate TRPV1 channels. Finally, both endocannabinoids and endovanilloids also interact with non-cannabinoid, non-TRPV1 receptors and channels, and, although the full physiological relevance of such interactions is yet to be established, the “promiscuity” of these lipophilic molecules can increase even further the potential ways through which they affect Ca²⁺ signalling. Here we discuss the effects of endocannabinoids and endovanilloids on cytosolic Ca²⁺ concentrations and their potential biological consequences.     

Altered patterns of filopodia production in CHO cells heterologously expressing zebra finch CB1 cannabinoid receptors

Recent findings indicate that cannabinoid-altered vocal development involves elevated densities of dendritic spines in a subset of brain regions involved in zebra finch song learning and production suggesting that cannabinoid receptor activation may regulate cell structure. Here we report that activation of zebra finch CB₁ receptors (zfCB₁, delivered by a lentivector to CHO cells) produces dose-dependent biphasic effects on the mean length of filopodia expressed: Low agonist concentrations (3 nM WIN55212-2) increase lengths while higher concentrations reduce them. In contrast, treatment of zfCB₁-expressing cells with the antagonist/inverse agonist SR141716A causes increases in both mean filopodia length and number at 30 and 100 nM. These results demonstrate that CB₁ receptor activation can differentially influence filiopodia elongation depending on dose, and demonstrate that manipulation of cannabinoid receptor activity is capable of modulating cell morphology.     

Human orexin/hypocretin receptors form constitutive homo- and heteromeric complexes with each other and with human CB1 cannabinoid receptors

Human OX₁ orexin receptors have been shown to homodimerize and they have also been suggested to heterodimerize with CB₁ cannabinoid receptors. The latter has been suggested to be important for orexin receptor responses and trafficking. In this study, we wanted to assess the ability of the other combinations of receptors to also form similar complexes. Vectors for expression of human OX₁, OX₂ and CB₁ receptors, C-terminally fused with either Renilla luciferase or GFP² green fluorescent protein variant, were generated. The constructs were transiently expressed in Chinese hamster ovary cells, and constitutive dimerization between the receptors was assessed by bioluminescence energy transfer (BRET). Orexin receptor subtypes readily formed homo- and hetero(di)mers, as suggested by significant BRET signals. CB₁ receptors formed homodimers, and they also heterodimerized with both orexin receptors. Interestingly, BRET efficiency was higher for homodimers than for almost all heterodimers. This is likely to be due to the geometry of the interaction; the putatively symmetric dimers may place the C-termini in a more suitable orientation in homomers. Fusion of luciferase to an orexin receptor and GFP² to CB₁ produced more effective BRET than the opposite fusions, also suggesting differences in geometry. Similar was seen for the OX₁–OX₂ interaction. In conclusion, orexin receptors have a significant propensity to make homo- and heterodi-/oligomeric complexes. However, it is unclear whether this affects their signaling. As orexin receptors efficiently signal via endocannabinoid production to CB₁ receptors, dimerization could be an effective way of forming signal complexes with optimal cannabinoid concentrations available for cannabinoid receptors.     

Type 1 Cannabinoid Receptor Ligands Display Functional Selectivity in a Cell Culture Model of Striatal Medium Spiny Projection Neurons

Modulation of type 1 cannabinoid receptor (CB₁) activity has been touted as a potential means of treating addiction, anxiety, depression, and neurodegeneration. Different agonists of CB₁ are known to evoke varied responses in vivo. Functional selectivity is the ligand-specific activation of certain signal transduction pathways at a receptor that can signal through multiple pathways. To understand cannabinoid-specific functional selectivity, different groups have examined the effect of individual cannabinoids on various signaling pathways in heterologous expression systems. In the current study, we compared the functional selectivity of six cannabinoids, including two endocannabinoids (2-arachidonyl glycerol (2-AG) and anandamide (AEA)), two synthetic cannabinoids (WIN55,212-2 and CP55,940), and two phytocannabinoids (cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (THC)) on arrestin2-, Gα_i/o-, Gβγ-, Gα_s-, and Gα_q-mediated intracellular signaling in the mouse STHdh^Q7/Q7 cell culture model of striatal medium spiny projection neurons that endogenously express CB₁. In this system, 2-AG, THC, and CP55,940 were more potent mediators of arrestin2 recruitment than other cannabinoids tested. 2-AG, AEA, and WIN55,212-2, enhanced Gα_i/o and Gβγ signaling, with 2-AG and AEA treatment leading to increased total CB₁ levels. 2-AG, AEA, THC, and WIN55,212-2 also activated Gα_q-dependent pathways. CP55,940 and CBD both signaled through Gα_s. CP55,940, but not CBD, activated downstream Gα_s pathways via CB₁ targets. THC and CP55,940 promoted CB₁ internalization and decreased CB₁ protein levels over an 18-h period. These data demonstrate that individual cannabinoids display functional selectivity at CB₁ leading to activation of distinct signaling pathways. To effectively match cannabinoids with therapeutic goals, these compounds must be screened for their signaling bias.     

Cannabinoid CB<Subscript>2</Subscript> Receptor-Mediated Anti-nociception in Models of Acute and Chronic Pain

The endocannabinoid system consists of cannabinoid CB₁ and CB₂ receptors, endogenous ligands and their synthesising/metabolising enzymes. Cannabinoid receptors are present at key sites involved in the relay and modulation of nociceptive information. The analgesic effects of cannabinoids have been well documented. The usefulness of nonselective cannabinoid agonists can, however, be limited by psychoactive side effects associated with activation of CB₁ receptors. Following the recent evidence for CB₂ receptors existing in the nervous system and reports of their up-regulation in chronic pain states and neurodegenerative diseases, much research is now aimed at shedding light on the role of the CB₂ receptor in human disease. Recent studies have demonstrated anti-nociceptive effects of selective CB₂ receptor agonists in animal models of pain in the absence of CNS side effects. This review focuses on the analgesic potential of CB₂ receptor agonists for inflammatory, post-operative and neuropathic pain states and discusses their possible sites and mechanisms of action. Jhaveri and Sagar joint first author.     

Synthesis and binding study of certain 6-arylalkanamides as molecular probes for cannabinoid receptor subtypes

Tetrahydrocannabinol and other mixed cannabinoid (CB) receptors CB₁/CB₂ receptor agonists are well established to elicit antinociceptive effects and psychomimetic actions, however, their potential for abuse have dampened enthusiasm for their therapeutic development. In an effort to refine a semi-rigid structural framework for CB₂ receptors binding, we designed novel compounds based on aromatic moiety and flexible linker with various amides mimicking the outlook of the endogenous anandamide which could provide as CB₂ receptor ligand. In this direction, we developed and synthesized new aryl or arylidene hexanoic acid amides and aryl alkanoic acid diamide carrying different head groups. These new compounds were tested for their affinities for human recombinant CB receptors CB₁ and CB₂ and fatty acid amide hydrolase. Although, the preliminary screening of these compounds demonstrated weak binding activity towards CB receptor subtypes at 10 µmole, yet this template still could serve up as probes for further optimization and development of affinity ligand for CB receptors.     

Altered patterns of filopodia production in CHO cells heterologously expressing zebra finch CB1 cannabinoid receptors

Recent findings indicate that cannabinoid-altered vocal development involves elevated densities of dendritic spines in a subset of brain regions involved in zebra finch song learning and production suggesting that cannabinoid receptor activation may regulate cell structure. Here we report that activation of zebra finch CB₁ receptors (zfCB₁, delivered by a lentivector to CHO cells) produces dose-dependent biphasic effects on the mean length of filopodia expressed: Low agonist concentrations (3 nM WIN55212-2) increase lengths while higher concentrations reduce them. In contrast, treatment of zfCB₁-expressing cells with the antagonist/inverse agonist SR141716A causes increases in both mean filopodia length and number at 30 and 100 nM. These results demonstrate that CB₁ receptor activation can differentially influence filiopodia elongation depending on dose, and demonstrate that manipulation of cannabinoid receptor activity is capable of modulating cell morphology.     

Δ-Tetrahydrocannabinol regulates the p53 post-translational modifiers Murine double minute 2 and the Small Ubiquitin MOdifier protein in the rat brain

The phytocannabinoid Δ⁹-Tetrahydrocannabinol (Δ⁹-THC), the main psychoactive cannabinoid in cannabis, activates a number of signalling cascades including p53. This study examines the role of Δ⁹-THC in regulating the p53 post-translational modifier proteins, Murine double minute (Mdm2) and Small Ubquitin-like MOdifier protein 1 (SUMO-1) in cortical neurons. Δ⁹-THC increased both Mdm2 and SUMO-1 protein expression and induced the deSUMOylation of p53 in a cannabinoid receptor type 1 (CB₁)-receptor dependent manner. We demonstrate that Δ⁹-THC decreased the SUMOylation of the CB₁ receptor. The data reveal a novel role for cannabinoid receptor activation in modulating the SUMO regulatory system.

Structured summary

MINT-7266621: Cb1 (uniprotkb:P20272) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation(MI:0006)MINT-7266633: SUMO-1 (uniprotkb:Q5I0H3) and Cb1 (uniprotkb:P20272) colocalize (MI:0403) by fluorescence microscopy (MI:0416)MINT-7266611: p53 (uniprotkb:P10361) physically interacts (MI:0915) with SUMO-1 (uniprotkb:Q5I0H3) by anti bait coimmunoprecipitation (MI:0006)     

Functional importance of Ψ38 and Ψ39 in distinct tRNAs,amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast

The numerous modifications of tRNA play central roles in controlling tRNA structure and translation. Modifications in and around the anticodon loop often have critical roles in decoding mRNA and in maintaining its reading frame. Residues U₃₈ and U₃₉ in the anticodon stem–loop are frequently modified to pseudouridine (Ψ) by members of the widely conserved TruA/Pus3 family of pseudouridylases. We investigate here the cause of the temperature sensitivity of pus3Δ mutants of the yeast Saccharomyces cerevisiae and find that, although Ψ₃₈ or Ψ₃₉ is found on at least 19 characterized cytoplasmic tRNA species, the temperature sensitivity is primarily due to poor function of tRNA^Gln(UUG), which normally has Ψ₃₈. Further investigation reveals that at elevated temperatures there are substantially reduced levels of the s²U moiety of mcm⁵s²U₃₄ of tRNA^Gln(UUG) and the other two cytoplasmic species with mcm⁵s²U₃₄, that the reduced s²U levels occur in the parent strain BY4741 and in the widely used strain W303, and that reduced levels of the s²U moiety are detectable in BY4741 at temperatures as low as 33°C. Additional examination of the role of Ψ_38,39 provides evidence that Ψ₃₈ is important for function of tRNA^Gln(UUG) at permissive temperature, and indicates that Ψ₃₉ is important for the function of tRNA^Trp(CCA) in trm10Δ pus3Δ mutants and of tRNA^Leu(CAA) as a UAG nonsense suppressor. These results provide evidence for important roles of both Ψ₃₈ and Ψ₃₉ in specific tRNAs, and establish that modification of the wobble position is subject to change under relatively mild growth conditions.     

CB₁ receptor activation inhibits neuronal and astrocytic intermediary metabolism in the rat hippocampus

Cannabinoid CB₁ receptor (CB₁R) activation decreases synaptic GABAergic and glutamatergic transmission and it also controls peripheral metabolism. Here we aimed at testing with ¹³C NMR isotopomer analysis whether CB₁Rs could have a local metabolic role in brain areas having high CB₁R density, such as the hippocampus. We labelled hippocampal slices with the tracers [2-¹³C]acetate, which is oxidized in glial cells, and [U-¹³C]glucose, which is metabolized both in glia and neurons, to evaluate metabolic compartmentation between glia and neurons. The synthetic CB₁R agonist WIN55212-2 (1 μM) significantly decreased the metabolism of both [2-¹³C]acetate (−11.6 ± 2.0%) and [U-¹³C]glucose (−11.2 ± 3.4%) in the tricarboxylic acid cycle that contributes to the glutamate pool. WIN55212-2 also significantly decreased the metabolism of [U-¹³C]glucose (−11.7 ± 4.0%) but not that of [2-¹³C]acetate contributing to the pool of GABA. These effects of WIN55212-2 were prevented by the CB₁R antagonist AM251 (500 nM). These results thus suggest that CB₁Rs might be present also in hippocampal astrocytes besides their well-known neuronal localization. Indeed, confocal microscopy analysis revealed the presence of specific CB₁R immunoreactivity in astrocytes and pericytes throughout the hippocampus.In conclusion, CB₁Rs are able to control hippocampal intermediary metabolism in both neuronal and glial compartments, which suggests new alternative mechanisms by which CB₁Rs control cell physiology and afford neuroprotection.     

The G protein-coupled cannabinoid-1 (CB1) receptor of mammalian brain: Inhibition by phthalate esters in vitro

This research examines the in vitro interaction of phthalate diesters and monoesters with the G protein-coupled cannabinoid 1 (CB₁) receptor, a presynaptic complex involved in the regulation of synaptic activity in mammalian brain. The diesters, n-butylbenzylphthalate (nBBP), di-n-hexylphthalate (DnHP), di-n-butylphthalate (DnBP), di-2-ethylhexylphthalate (DEHP), di-isooctylphthalate (DiOP) and di-n-octylphthalate (DnOP) inhibited the specific binding of the CB₁ receptor agonist [³H]CP-55940 to mouse whole brain membranes at micromolar concentrations (IC₅₀s: nBBP 27.4 μM; DnHP 33.9 μM; DnBP 45.9 μM; DEHP 47.4 μM; DiOP 55.4 μM; DnOP 75.2 μM). DnHP, DnBP and nBBP achieved full (or close to full) blockade of [³H]CP-55940 binding, whereas DEHP, DiOP and DnOP produced partial (55-70%) inhibition. Binding experiments with phenylmethane-sulfonylfluoride (PMSF) indicated that the ester linkages of nBBP and DnBP remain intact during assay. The monoesters mono-2-ethylhexylphthalate (M2EHP) and mono-isohexylphthalate (MiHP) failed to reach IC₅₀ at 150 μM and mono-n-butylphthalate (MnBP) was inactive. Inhibitory potencies in the [³H]CP-55940 binding assay were positively correlated with inhibition of CB₁ receptor agonist-stimulated binding of [³⁵S]GTPγS to the G protein, demonstrating that phthalates cause functional impairment of this complex. DnBP, nBBP and DEHP also inhibited binding of [³H]SR141716A, whereas inhibition with MiHP was comparatively weak and MnBP had no effect. Equilibrium binding experiments with [³H]SR141716A showed that phthalates reduce the B_max of radioligand without changing its K_d. DnBP and nBBP also rapidly enhanced the dissociation of [³H]SR141716A. Our data are consistent with an allosteric mechanism for inhibition, with phthalates acting as relatively low affinity antagonists of CB₁ receptors and cannabinoid agonist-dependent activation of the G-protein. Further studies are warranted, since some phthalate esters may have potential to modify CB₁ receptor-dependent behavioral and physiological outcomes in the whole animal.     

Active endocannabinoids are secreted on extracellular membrane vesicles

Endocannabinoids primarily influence neuronal synaptic communication within the nervous system. To exert their function, endocannabinoids need to travel across the intercellular space. However, how hydrophobic endocannabinoids cross cell membranes and move extracellularly remains an unresolved problem. Here, we show that endocannabinoids are secreted through extracellular membrane vesicles produced by microglial cells. We demonstrate that microglial extracellular vesicles carry on their surface N-arachidonoylethanolamine (AEA), which is able to stimulate type-1 cannabinoid receptors (CB₁), and inhibit presynaptic transmission, in target GABAergic neurons. This is the first demonstration of a functional role of extracellular vesicular transport of endocannabinoids.     

Expression,surface immobilization,and characterization of functional recombinant cannabinoid receptor CB2

Human peripheral cannabinoid receptor CB₂, a G protein-coupled receptor (GPCR) involved in regulation of immune response has become an important target for pharmaceutical drug development. Structural and functional studies on CB₂ may benefit from immobilization of the purified and functional receptor onto a suitable surface at a controlled density and, preferably in a uniform orientation. The goal of this project was to develop a generic strategy for preparation of functional recombinant CB₂ and immobilization at solid interfaces. Expression of CB₂ as a fusion with Rho-tag (peptide composed of the last nine amino acids of rhodopsin) in E. coli was evaluated in terms of protein levels, accessibility of the tag, and activity of the receptor. The structural integrity of CB₂ was tested by ligand binding to the receptor solubilized in detergent micelles, captured on tag-specific monoclonal 1D4 antibody-coated resin. Highly pure and functional CB₂ was obtained by sequential chromatography on a 1D4- and Ni-NTA-resin and its affinity to the 1D4 antibody characterized by surface plasmon resonance (SPR). Either the purified receptor or fusion CB₂ from the crude cell extract was captured onto a 1D4-coated CM4 chip (Biacore) in a quantitative fashion at uniform orientation as demonstrated by the SPR signal. Furthermore, the accessibility of the extracellular surface of immobilized CB₂ and the affinity of interaction with a novel monoclonal antibody NAA-1 was studied by SPR. In summary, we present an integral strategy for purification, surface immobilization, ligand- and antibody binding studies of functional cannabinoid receptor CB₂.     

Is lipid signaling through cannabinoid 2 receptors part of a protective system?

The mammalian body has a highly developed immune system which guards against continuous invading protein attacks and aims at preventing, attenuating or repairing the inflicted damage. It is conceivable that through evolution analogous biological protective systems have been evolved against non-protein attacks. There is emerging evidence that lipid endocannabinoid signaling through cannabinoid 2 (CB₂) receptors may represent an example/part of such a protective system/armamentarium. Inflammation/tissue injury triggers rapid elevations in local endocannabinoid levels, which in turn regulate signaling responses in immune and other cells modulating their critical functions. Changes in endocannabinoid levels and/or CB₂ receptor expressions have been reported in almost all diseases affecting humans, ranging from cardiovascular, gastrointestinal, liver, kidney, neurodegenerative, psychiatric, bone, skin, autoimmune, lung disorders to pain and cancer, and modulating CB₂ receptor activity holds tremendous therapeutic potential in these pathologies. While CB₂ receptor activation in general mediates immunosuppressive effects, which limit inflammation and associated tissue injury in large number of pathological conditions, in some disease states activation of the CB₂ receptor may enhance or even trigger tissue damage, which will also be discussed alongside the protective actions of the CB₂ receptor stimulation with endocannabinoids or synthetic agonists, and the possible biological mechanisms involved in these effects.     

The evolution and comparative neurobiology of endocannabinoid signalling

CB₁- and CB₂-type cannabinoid receptors mediate effects of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide in mammals. In canonical endocannabinoid-mediated synaptic plasticity, 2-AG is generated postsynaptically by diacylglycerol lipase alpha and acts via presynaptic CB₁-type cannabinoid receptors to inhibit neurotransmitter release. Electrophysiological studies on lampreys indicate that this retrograde signalling mechanism occurs throughout the vertebrates, whereas system-level studies point to conserved roles for endocannabinoid signalling in neural mechanisms of learning and control of locomotor activity and feeding. CB₁/CB₂-type receptors originated in a common ancestor of extant chordates, and in the sea squirt Ciona intestinalis a CB₁/CB₂-type receptor is targeted to axons, indicative of an ancient role for cannabinoid receptors as axonal regulators of neuronal signalling. Although CB₁/CB₂-type receptors are unique to chordates, enzymes involved in biosynthesis/inactivation of endocannabinoids occur throughout the animal kingdom. Accordingly, non-CB₁/CB₂-mediated mechanisms of endocannabinoid signalling have been postulated. For example, there is evidence that 2-AG mediates retrograde signalling at synapses in the nervous system of the leech Hirudo medicinalis by activating presynaptic transient receptor potential vanilloid-type ion channels. Thus, postsynaptic synthesis of 2-AG or anandamide may be a phylogenetically widespread phenomenon, and a variety of proteins may have evolved as presynaptic (or postsynaptic) receptors for endocannabinoids.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

The influence of beta-arrestin2 on cannabinoid CB1 receptor coupling to G-proteins and subcellular localization and relative levels of beta-arrestin1 and 2 in mouse brain

Abstract

Context: Beta-arrestins are known to couple to some G-protein-coupled receptors (GPCRs) to regulate receptor internalization, G-protein coupling and signal transduction, but have not been investigated for most receptors, and for very few receptors in vivo. Previous studies have shown that beta-arrestin2 deletion enhances the efficacy of specific cannabinoid agonists. Objective: The present study hypothesized that brain cannabinoid CB₁ receptors are regulated by beta-arrestin2. Methods: Beta-arrestin2+/+ and ?/? mice were used. Western blotting was used to determine the relative levels of each beta-arrestin subtype in mouse brain. Receptor binding was measured to determine whether deletion of beta-arrestin2 influences agonist binding to brain CB₁ receptors, or the subcellular localization of CB₁ in brain membranes subjected to differential centrifugation. A variety of cannabinoid agonists from different chemical classes were investigated for their ability to activate G-proteins in the presence and absence of beta-arrestin2 in cerebellum, hippocampus and cortex. Results: No differences were found in the density of beta-arrestin1 or cannabinoid CB₁ receptors in several brains of beta-arrestin2+/+ versus ?/? mice. Differences between genotypes were found in the proportion of high- and low-affinity agonist binding sites in brain areas that naturally express higher levels of beta-arrestin2. Cortex from beta-arrestin2?/? mice contained less CB₁ in the P1 fraction and more CB₁ in the P2 fraction compared to beta-arrestin2+/+. Of the agonists assayed for activity, only Δ⁹-tetrahydrocannabinol (THC) exhibited a difference between genotypes, in that it was less efficacious in beta-arrestin2?/? than +/+ mouse membranes. Conclusion: Beta-arrestin2 regulates cannabinoid CB₁ receptors in brain.