Biosynthesis of cannabinoid acids

Malonic acid, mevalonic acid, geraniol and nerol were incorporated into tetrahydrocannabinolic acid and cannabichromenic acid in Cannabis sativa. The pathway from cannabigerolic acid to tetrahydrocannabinolic acid via cannabidiolic acid was established by feeding labelled cannabinoid acids. Cannabichromenic acid was shown to be formed on a side pathway from cannabigerolic acid.     

Targeting the endocannabinoid system with cannabinoid receptor agonists: pharmacological strategies and therapeutic possibilities

Human tissues express cannabinoid CB₁ and CB₂ receptors that can be activated by endogenously released ‘endocannabinoids’ or exogenously administered compounds in a manner that reduces the symptoms or opposes the underlying causes of several disorders in need of effective therapy. Three medicines that activate cannabinoid CB₁/CB₂ receptors are now in the clinic: Cesamet (nabilone), Marinol (dronabinol; Δ⁹-tetrahydrocannabinol (Δ⁹-THC)) and Sativex (Δ⁹-THC with cannabidiol). These can be prescribed for the amelioration of chemotherapy-induced nausea and vomiting (Cesamet and Marinol), stimulation of appetite (Marinol) and symptomatic relief of cancer pain and/or management of neuropathic pain and spasticity in adults with multiple sclerosis (Sativex). This review mentions several possible additional therapeutic targets for cannabinoid receptor agonists. These include other kinds of pain, epilepsy, anxiety, depression, Parkinson''s and Huntington''s diseases, amyotrophic lateral sclerosis, stroke, cancer, drug dependence, glaucoma, autoimmune uveitis, osteoporosis, sepsis, and hepatic, renal, intestinal and cardiovascular disorders. It also describes potential strategies for improving the efficacy and/or benefit-to-risk ratio of these agonists in the clinic. These are strategies that involve (i) targeting cannabinoid receptors located outside the blood-brain barrier, (ii) targeting cannabinoid receptors expressed by a particular tissue, (iii) targeting upregulated cannabinoid receptors, (iv) selectively targeting cannabinoid CB₂ receptors, and/or (v) adjunctive ‘multi-targeting’.     

Eicosanoid mediation of cannabinoid actions

Interactions between cannabinoids and eicosanoids have been observed for the last several decades and account for a variety of cannabinoid actions. These were seen both in vitro and in vivo and may provide a molecular basis for these actions. Some of the topics included in this review are; effects on adenylate cyclase activity, alteration of behavioral responses, reduction of pain sensation, reduction and resolution of inflammation, hypotensive and vasorelaxant responses, anti-cancer and anti-metastatic activities, reduction of intraocular pressure and others. The most widely studied cannabinoids so far are tetrahydrocannabinol and cannabidiol. However, synthetic agents such as CP55,940, ajulemic acid, JWH-133 and WIN-55,212-2 were also investigated for interaction with eicosanoids. The endocannabinoids anandamide and 2-arachidonoylglycerol have been examined as well. Among the eicosanoids mediating cannabinoid actions are PGE₂, 15-deoxy-Δ12, 14-prostaglandin-J₂, lipoxin A₄, lipoxin B₄, and leukotriene B₄. Enzyme activities involved include monoacylglycerylipase, adenylatecyclase, phospholipase A₂, cyclooxygenases-1, 2 and 5, lipoxygenases-12 and 15. Receptors involved include CB1, CB2 and the EP3 and EP3 prostanoid receptors. While not all cannabinoid activities can be accounted for, many are best explained by eicosanoid participation. The recent surge in interest in “medical marijuana” makes understanding mechanisms of cannabinoid actions particularly important.     

The cannabinoid acids,analogs and endogenous counterparts

The cannabinoid acids are a structurally heterogeneous group of compounds some of which are endogenous molecules and others that are metabolites of phytocannabinoids. The prototypic endogenous substance is N-arachidonoyl glycine (NAgly) that is closely related in structure to the cannabinoid agonist anandamide. The most studied phytocannabinoid is Δ⁹-THC-11-oic acid, the principal metabolite of Δ⁹-THC. Both types of acids have in common several biological actions such as low affinity for CB1 anti-inflammatory activity and analgesic properties. This suggests that there may be similarities in their mechanism of action, a point that is discussed in this review. Also presented are reports on analogs of the acids that provide opportunities for the development of novel therapeutic agents, such as ajulemic acid.     

The effects of beta-arrestin1 deletion on acute cannabinoid activity,brain cannabinoid receptors and tolerance to cannabinoids in mice

Abstract

Context: Previous studies have indicated a role for beta-arrestin2 in the regulation of brain cannabinoid effects and cannabinoid CB₁ receptors, but whether beta-arrestin1 has a role has not been investigated. Objective: To determine the role of beta-arrestin1 in cannabinoid activity. Materials and methods: Beta-arrestin1 ?/? mice and their wild-type (+/+) counterparts were assayed for antinociceptive and temperature-decreasing effects of two ligands, Δ⁹-tetrahydrocannabinol (THC) and CP55940, after both single and repeated administration. In vitro assays examined the effects of deletion on CB₁ receptor density, agonist-binding and G-protein activation. Results: Deletion of beta-arrestin1 diminished the effects of CP55940 in both antinociception (latency to tail withdrawal) and temperature-depression assays in mice. However, deleting beta-arrestin1 had no effect on the actions of THC in either assay. Antagonist radioligand ([³H]SR141716A) saturation binding indicated no difference between beta-arrestin1 +/+ and ?/? mice in the density or affinity for cannabinoid CB₁ receptors in brain membranes. CP55940 agonist binding in brain membranes from beta-arrestin1 +/+ mice exhibited high- and intermediate-affinity sites, but beta-arrestin1 ?/? membranes exhibited an additional site with low affinity. CP55940 produced greater stimulation of [³⁵S]GTPγS binding to membranes from whole brain of beta-arrestin1 ?/? than +/+ mice. The rates of the development of tolerance to chronic THC or CP55940 administration did not appear to be affected by genotype. Discussion: Beta-arrestin1 appeared to mediate the actions of CP55940, but did not affect the activity of THC. Conclusion: Beta-arrestin1 regulates cannabinoid CB₁ receptor sensitivity in an agonist-selective manner, but may not be the primary mediator of tolerance to cannabinoid agonists.     

Nutritional n-3 polyunsaturated fatty acids deficiency alters cannabinoid receptor signaling pathway in the brain and associated anxiety-like behavior in mice

N-3 polyunsaturated fatty acids (PUFAs) cannot be synthesized de novo in mammals and need to be provided by dietary means. In the brain, the main n-3 PUFA is docosahexaenoic acid (DHA), which is a key component of neuronal membranes. A low dietary level of DHA has been associated with increased risk of developing neuropsychiatric diseases; however, the mechanisms involved remain to be determined. In this study, we found that long-term exposure to an n-3 deficient diet decreases the level of DHA in the brain and impairs the cannabinoid receptor signaling pathway in mood-controlling structures. In n-3 deficient mice, the effect of the cannabinoid agonist WIN55,212-2 in an anxiety-like behavior test was abolished. In addition, the cannabinoid receptor signaling pathways were altered in the prefrontal cortex and the hypothalamus. Consequently, our data suggest that behavioral changes linked to an n-3 dietary deficiency are due to an alteration in the endocannabinoid system in specific brain areas.     

New cannabinoid receptor antagonists as pharmacological tool

Synthesis and pharmacological evaluation of a new series of cannabinoid receptor antagonists of indazole ether derivatives have been performed. Pharmacological evaluation includes radioligand binding assays with [³H]-CP55940 for CB1 and CB2 receptors and functional activity for cannabinoid receptors on isolated tissue. In addition, functional activity of the two synthetic cannabinoids antagonists 18 (PGN36) and 17 (PGN38) were carried out in the osteoblastic cell line MC3T3-E1 that is able to express CB2R upon osteogenic conditions. Both antagonists abolished the increase in collagen type I gene expression by the well-known inducer of bone activity, the HU308 agonist. The results of pharmacological tests have revealed that four of these derivatives behave as CB2R cannabinoid antagonists. In particular, the compounds 17 (PGN38) and 18 (PGN36) highlight as promising candidates as pharmacological tools.     

Phase I hydroxylated metabolites of the K2 synthetic cannabinoid JWH-018 retain in vitro and in vivo cannabinoid 1 receptor affinity and activity

Background

K2 products are synthetic cannabinoid-laced, marijuana-like drugs of abuse, use of which is often associated with clinical symptoms atypical of marijuana use, including hypertension, agitation, hallucinations, psychosis, seizures and panic attacks. JWH-018, a prevalent K2 synthetic cannabinoid, is structurally distinct from Δ⁹-THC, the main psychoactive ingredient in marijuana. Since even subtle structural differences can lead to differential metabolism, formation of novel, biologically active metabolites may be responsible for the distinct effects associated with K2 use. The present study proposes that K2''s high adverse effect occurrence is due, at least in part, to distinct JWH-018 metabolite activity at the cannabinoid 1 receptor (CB1R).

Methods/Principal Findings

JWH-018, five potential monohydroxylated metabolites (M1–M5), and one carboxy metabolite (M6) were examined in mouse brain homogenates containing CB1Rs, first for CB1R affinity using a competition binding assay employing the cannabinoid receptor radioligand [³H]CP-55,940, and then for CB1R intrinsic efficacy using an [³⁵S]GTPγS binding assay. JWH-018 and M1–M5 bound CB1Rs with high affinity, exhibiting K_i values that were lower than or equivalent to Δ⁹-THC. These molecules also stimulated G-proteins with equal or greater efficacy relative to Δ⁹-THC, a CB1R partial agonist. Most importantly, JWH-018, M2, M3, and M5 produced full CB1R agonist levels of activation. CB1R-mediated activation was demonstrated by blockade with O-2050, a CB1R-selective neutral antagonist. Similar to Δ⁹-THC, JWH-018 and M1 produced a marked depression of locomotor activity and core body temperature in mice that were both blocked by the CB1R-preferring antagonist/inverse agonist AM251.

Conclusions/Significance

Unlike metabolites of most drugs, the studied JWH-018 monohydroxylated compounds, but not the carboxy metabolite, retain in vitro and in vivo activity at CB1Rs. These observations, combined with higher CB1R affinity and activity relative to Δ⁹-THC, may contribute to the greater prevalence of adverse effects observed with JWH-018-containing products relative to cannabis.     

Evidence for a cannabinoid receptor in sea urchin sperm and its role in blockade of the acrosome reaction

Delta-9-tetrahydrocannabinol ((?)δ⁹ THC), the primary psychoactive cannabinoid in marihuana, reduces the fertilizing capacity of sea urchin sperm by blocking the acrosome reaction that normally is stimulated by a specific ligand in the egg's jelly coat. The bicyclic synthetic cannabinoid [ H]CP-55,940 has been used as a ligand to demonstrate the presence of a cannabinoid receptor in mammalian brain. We now report that [ H]CP-55,940 binds to live sea urchin (Strongylocentrotus purpuratus) sperm in a concentration, sperm density, and time-dependent manner. Specific binding of [ H]CP-55,940 to sperm, defined as total binding displaced by (?)δ⁹ THC, was saturable: K_D 5.16 ± 1.02 nM; Hill coefficient 0.98 ± 0.004. This suggests a single class of receptor sites and the absence of significant cooperative interactions. Sea urchin sperm contain 712 ± 122 cannabinoid receptors per cell. Binding of [ H]CP-55,940 to sperm was reduced in a dose-dependent manner by increasing concentrations of CP-55,940, (?)δ⁹ THC, and (+)δ⁹ THC. The rank order of potency to inhibit binding of [ H]CP-55,940 to sperm and to block the egg jelly stimulated acrosome reaction was: CP-55,940 > (?)δ⁹THC > (+)δ⁹THC. These findings show that sea urchin sperm contain a stereospecific cannabinoid receptor that may play a role in inhibition of the acrosome reaction. The radioligand binding data obtained with live sea urchin sperm are remarkably similar to those previously published by other investigators using [ H]CP-55,940 on mammalian brain and nonneural tissues. The cannabinoid binding properties of this receptor appear to have been highly conserved during evolution. We postulate that the cannabinoid receptor may modulate cellular responses to stimulation. © 1993 Wiley-Liss, Inc.     

Structural biology of human cannabinoid receptor-2 helix 6 in membrane-mimetic environments

We detail the structure and dynamics of a synthetic peptide corresponding to transmembrane helix 6 (TMH6) of human cannabinoid receptor-2 (hCB2) in biomembrane-mimetic environments. The peptide’s NMR structural biology is characterized by two α-helical domains bridged by a flexible, nonhelical hinge region containing a highly-conserved CWFP motif with an environmentally sensitive, Pro-based conformational switch. Buried within the peptide’s flexible region, W²⁵⁸ may hydrogen-bond with L²⁵⁵ to help stabilize the Pro-kinked hCB2 TMH6 structure and position C²⁵⁷ advantageously for interaction with agonist ligands. These characteristics of hCB2 TMH6 are potential structural features of ligand-induced hCB2 activation in vivo.     

Alkamides and a neolignan from Echinacea purpurea roots and the interaction of alkamides with G-protein-coupled cannabinoid receptors

Multiple chromatographic separations of the CHCl₃-soluble extract of the roots of Echinacea purpurea led to the isolation of 19 compounds. Four natural products, three alkamides and nitidanin diisovalerianate, were identified, and five further compounds were detected for the first time in this species. Additionally, 10 known E. purpurea metabolites were isolated. The structures were determined by mass spectrometry and advanced 1D and 2D NMR techniques. The bioactivity of the isolated compounds was studied in [³⁵S]GTPγS-binding experiments performed on rat brain membrane preparations. Both partial and inverse agonist compounds for cannabinoid (CB1) receptors were identified among the metabolites, characterized by weak to moderate interactions with the G-protein signaling mechanisms. The G-protein-modulating activities of the Echinacea compounds are rather far from the full agonist effects seen with the CB1 receptor agonist reference compound arachidonyl-2′-chloroethylamide (ACEA). However, upon coadministration with ACEA, a number of them proved capable of inhibiting the stimulation of the pure agonist, thereby demonstrating cannabinoid receptor antagonist properties.     

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.     

Inhibition of neuroblastoma adenylate cyclase by cannabinoid and nantradol compounds

This study was undertaken to ascertain the effects of cannabinoid drugs on prostanoid-stimulated adenylate cyclase in neuroblastoma cells. This report demonstrates that Δ⁹-tetrahydrocannabinol (THC) and levonantradol could decrease initial rate cyclic AMP accumulation in response to prostacyclin in intact cells. Basal accumulation was also diminished. Prostanoid-stimulated adenylate cyclase in a membrane preparation from these cells was inhibited by cannabinoid and nantradol compounds. However, this inhibition was not competitive with prostaglandin E₁ or prostacyclin. Further, inhibition was also observed when the enzyme was stimulated by peptide hormones at the secretin receptor. In contrast, enzyme activated by NaF was not inhibited by cannabinoid compounds. Cyclic AMP phosphodiesterase activity in subcellular fractions was unaltered by these agents. These data demonstrate that cannabinoid and nantradol compounds decrease cyclic AMP accumulation in neuronally derived cells, and that this results from an inhibition of basal and hormone-stimulated adenylate cyclase activity.     

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel,high affinity ligands for CB1 and CB2 cannabinoid receptors

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R = R² = H, R¹ = F) and 13 (R = COOCH₃, R¹ = R² = H) exhibited high affinity for CB2 receptors with K_i values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (K_i values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.     

AM630 is a competitive cannabinoid receptor antagonist in the guinea pig brain

AM 630 has been demonstrated to be a cannabinoid receptor antagonist in the mouse brain and vas deferens. Conversely, it was recently reported that AM630 acts as a cannabinoid agonist in the guinea pig ileum. This research was designed to determine whether the difference in the action of AM630 is species specific. Studies conducted in guinea pig brain reveal that AM630 antagonizes the stimulatory effect of the cannabinoid agonist WIN 55,212-2 on [³⁵S]GTPγS binding suggesting that difference in AM630 activity in different tissues is not due to species Variation.     

Hit-to-lead optimization of pyrrolo[1,2-a]quinoxalines as novel cannabinoid type 1 receptor antagonists

Hit-to-lead optimization of a novel series of N-alkyl-N-[2-oxo-2-(4-aryl-4H-pyrrolo[1,2-a]quinoxaline-5-yl)-ethyl]-carboxylic acid amides, derived from a high throughput screening (HTS) hit, are described. Subsequent optimization led to identification of in vitro potent cannabinoid 1 receptor (CB1R) antagonists representing a new class of compounds in this area.     

Synthesis and in vitro autoradiographic evaluation of a novel high-affinity radioiodinated ligand for imaging brain cannabinoid subtype-1 receptors

There is strong interest to study the involvement of brain cannabinoid subtype-1 (CB₁) receptors in neuropsychiatric disorders with single photon emission computed tomography (SPECT) and a suitable radioligand. Here we report the synthesis of a novel high-affinity radioiodinated CB₁ receptor ligand ([¹²⁵I]8, [¹²⁵I]1-(2-iodophenyl)-4-cyano-5-(4-methoxyphenyl)-N-(piperidin-1-yl)-1H-pyrazole-3-carboxylate, [¹²⁵I]SD7015). By autoradiography in vitro, [¹²⁵I]8 showed selective binding to CB₁ receptors on human brain postmortem cryosections and now merits labeling with iodine-123 for further evaluation as a SPECT radioligand in non-human primate.     

Oxidative stress and cannabinoid receptor expression in type‐2 diabetic rat pancreas following treatment with Δ9‐THC

The objectives of study were (a) to determine alteration of feeding, glucose level and oxidative stress and (b) to investigate expression and localization of cannabinoid receptors in type‐2 diabetic rat pancreas treated with Δ⁹‐tetrahydrocannabinol (Δ⁹‐THC). Rats were randomly divided into four groups: control, Δ⁹‐THC, diabetes and diabetes + Δ⁹‐THC groups. Diabetic rats were treated with a single dose of nicotinamide (85 mg/kg) 15 min before injection of streptozotocin (65 mg/kg). Δ⁹‐THC was administered intraperitoneally at 3 mg/kg/day for 7 days. Body weights and blood glucose level of rats in all groups were measured on days 0, 7, 14 and 21. On day 15 after the Δ⁹‐THC injections, pancreatic tissues were removed. Blood glucose levels and body weights of diabetic rats treated with Δ⁹‐THC did not show statistically significant changes when compared with the diabetic animals on days 7, 14 and 21. Treatment with Δ⁹‐THC significantly increased pancreas glutathione levels, enzyme activities of superoxide dismutase and catalase in diabetes compared with non‐treatment diabetes group. The cannabinoid 1 receptor was found in islets, whereas the cannabinoid 2 receptor was found in pancreatic ducts. Their localization in cells was both nuclear and cytoplasmic. We can suggest that Δ⁹‐THC may be an important agent for the treatment of oxidative damages induced by diabetes. However, it must be supported with anti‐hyperglycaemic agents. Furthermore, the present study for the first time emphasizes that Δ⁹‐THC may improve pancreatic cells via cannabinoid receptors in diabetes. The aim of present study was to elucidate the effects of Δ⁹‐THC, a natural cannabinoid receptor agonist, on the expression and localization of cannabinoid receptors, and oxidative stress statue in type‐2 diabetic rat pancreas. Results demonstrate that the cannabinoid receptors are presented in both Langerhans islets and duct regions. The curative effects of Δ⁹‐THC can be occurred via activation of cannabinoid receptors in diabetic rat pancreas. Moreover, it may provide a protective effect against oxidative damage induced by diabetes. Thus, it is suggested that Δ⁹‐THC can be a candidate for therapeutic alternatives of diabetes symptoms. Copyright © 2014 John Wiley & Sons, Ltd.     

CB2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB₂ cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB₂ receptors in the brain need to be clarified. The aim of our work was to study the μ-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB₂ receptor antagonist SR144528 in brainstem of mice deficient in either CB₁ or CB₂ receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB₂ cannabinoid antagonist SR144528, suggesting a CB₂ receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [³⁵S]GTPγS binding assay to analyze the capability of μ-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB₁ wild-type and CB₁ knockout mice after a single injection of SR144528 at 0.1 mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB₁ wild-type and CB₁ knockout mice. In vitro addition of 1 μM SR144528 caused a decrease in the maximal stimulation of DAMGO in [³⁵S]GTPγS binding assays in CB₂ wild-type brainstem membranes whereas no significant changes were observed in CB₂ receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB₂ cannabinoid receptors.