Determination of ajulemic acid and its glucuronide in human plasma by gas chromatography-mass spectrometry

A method using gas chromatography-mass spectrometry (GC-MS) and solid-phase extraction (SPE) was developed for the determination of ajulemic acid (AJA), a non-psychoactive synthetic cannabinoid with interesting therapeutic potential, in human plasma. When using two calibration graphs, the assay linearity ranged from 10 to 750 ng/ml, and 750 to 3000 ng/ml AJA. The intra- and inter-day precision (R.S.D., %), assessed across the linear ranges of the assay, was between 1.5 and 7.0, and 3.6 and 7.9, respectively. The limit of quantitation (LOQ) was 10 ng/ml. The amount of AJA glucuronide was determined by calculating the difference in the AJA concentration before ("free AJA") and after enzymatic hydrolysis ("total AJA"). The present method was used within a clinical study on 21 patients suffering from neuropathic pain with hyperalgesia and allodynia. For example, plasma levels of 599.4+/-37.2 ng/ml (mean+/-R.S.D., n=9) AJA were obtained for samples taken 2 h after the administration of an oral dose of 20 mg AJA. The mean AJA glucuronide concentration at 2h was 63.8+/-127.9 ng/ml.

Do N-arachidonyl-glycine (NA-glycine) and 2-arachidonoyl glycerol (2-AG) share mode of action and the binding site on the β 2 subunit of GABA A receptors?

NA-glycine is an endogenous lipid molecule with analgesic properties, which is structurally similar to the endocannabinoids 2-AG and anandamide but does not interact with cannabinoid receptors. NA-glycine has been suggested to act at the G-protein coupled receptors GPR18 and GPR92. Recently, we have described that NA-glycine can also modulate recombinant α1β2γ2 GABAA receptors. Here we characterize in more detail this modulation and investigate the relationship of its binding site with that of the endocannabinoid 2-AG.

Anticancer activity of anandamide in human cutaneous melanoma cells

Cannabinoids are implicated in the control of cell proliferation, but little is known about the role of the endocannabinoid system in human malignant melanoma. This study was aimed at characterizing the in vitro antitumor activity of anandamide (AEA) in A375 melanoma cells. The mRNA expression of genes that code for proteins involved in the metabolism and in the mechanism of AEA action was assessed by RT-PCR. Cell viability was tested using WST-1 assay and the apoptotic cell death was determined by measuring caspase 3/7 activities. A375 cells express high levels of fatty acid amide hydrolase (FAAH), cyclooxygenase (COX)-2, cannabinoid receptor 1 (CB1), transient receptor potential cation channel subfamily V member 1 (TRPV1) and G-protein-coupled receptor 55 (GPR55) genes. AEA induced a concentration-dependent cytotoxicity with an IC50 of 5.8±0.7 µM and such an effect was associated to a caspase-dependent apoptotic pathway. AEA cytotoxicity was potentiated by FAAH inhibition (2-fold increase, p<0.05) and mitigated by COX-2 or lipoxygenase (LOX) inhibition (5- and 3-fold decrease, respectively; p<0.01). Blocking CB1 receptors partially decreased AEA cytotoxicity, whereas selective antagonism on the TRPV1 barely affected the mechanism of AEA action. Finally, methyl-β-cyclodextrin, a membrane cholesterol depletory, completely reversed the cytotoxicity induced by the selective GPR55 agonist, O-1602, and AEA. Overall, these findings demonstrate that AEA induces cytotoxicity against human melanoma cells in the micromolar range of concentrations through a complex mechanism, which involves COX-2 and LOX-derived product synthesis and CB1 activation. Lipid raft modulation, probably linked to GPR55 activation, might also have a role.

Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50 = 290 nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure–activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Correlating FAAH and anandamide cellular uptake inhibition using N-alkylcarbamate inhibitors: from ultrapotent to hyperpotent.

Besides the suggested role of a putative endocannabinoid membrane transporter mediating the cellular uptake of the endocannabinoid anandamide (AEA), this process is intrinsically coupled to AEA degradation by the fatty acid amide hydrolase (FAAH). Differential blockage of each mechanism is possible using specific small-molecule inhibitors. Starting from the natural product-derived 2E,4E-dodecadiene scaffold previously shown to interact with the endocannabinoid system (ECS), a series of diverse N-alkylcarbamates were prepared with the aim of generating novel ECS modulators. While being inactive at cannabinoid receptors and monoacylglycerol lipase, these N-alkylcarbamates showed potent to ultrapotent picomolar FAAH inhibition in U937 cells. Overall, a highly significant correlation (Spearman's rho=0.91) was found between the inhibition of FAAH and AEA cellular uptake among 54 compounds. Accordingly, in HMC-1 cells lacking FAAH expression the effect on AEA cellular uptake was dramatically reduced. Unexpectedly, 3-(4,5-dihydrothiazol-2-yl)phenyl carbamates and the 3-(1,2,3-thiadiazol-4-yl)phenyl carbamates WOBE490, WOBE491 and WOBE492 showed a potentiation of cellular AEA uptake inhibition in U937 cells, resulting in unprecedented femtomolar (hyperpotent) IC50 values. Potential methodological issues and the role of cellular accumulation of selected probes were investigated. It is shown that albumin impacts the potency of specific N-alkylcarbamates and, more importantly, that accumulation of FAAH inhibitors can significantly increase their effect on cellular AEA uptake. Taken together, this series of N-alkylcarbamates shows a FAAH-dependent inhibition of cellular AEA uptake, which can be strongly potentiated using specific head group modifications. These findings provide a rational basis for the development of hyperpotent AEA uptake inhibitors mediated by ultrapotent FAAH inhibition.

One-pot heterogeneous synthesis of Δ(3)-tetrahydrocannabinol analogues and xanthenes showing differential binding to CB(1) and CB(2) receptors

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) is the major psychoactive cannabinoid in hemp (Cannabis sativa L.) and responsible for many of the pharmacological effects mediated via cannabinoid receptors. Despite being the major cannabinoid scaffold in nature, Δ(9)-THC double bond isomers remain poorly studied. The chemical scaffold of tetrahydrocannabinol can be assembled from the condensation of distinctly substituted phenols and monoterpenes. Here we explored a microwave-assisted one pot heterogeneous synthesis of Δ(3)-THC from orcinol (1a) and pulegone (2). Four Δ(3)-THC analogues and corresponding Δ(4a)-tetrahydroxanthenes (Δ(4a)-THXs) were synthesized regioselectively and showed differential binding affinities for CB1 and CB2 cannabinoid receptors. Here we report for the first time the CB1 receptor binding of Δ(3)-THC, revealing a more potent receptor binding affinity for the (S)-(-) isomer (hCB1Ki = 5 nM) compared to the (R)-(+) isomer (hCB1Ki = 29 nM). Like Δ(9)-THC, also Δ(3)-THC analogues are partial agonists at CB receptors as indicated by [(35)S]GTPγS binding assays. Interestingly, the THC structural isomers Δ(4a)-THXs showed selective binding and partial agonism at CB2 receptors, revealing a simple non-natural natural product-derived scaffold for novel CB2 ligands.

Identification of endocannabinoid system-modulating N-alkylamides from Heliopsis helianthoides var. scabra and Lepidium meyenii.

The discovery of the interaction of plant-derived N-alkylamides (NAAs) and the mammalian endocannabinoid system (ECS) and the existence of a plant endogenous N-acylethanolamine signaling system have led to the re-evaluation of this group of compounds. Herein, the isolation of seven NAAs and the assessment of their effects on major protein targets in the ECS network are reported. Four NAAs, octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid isobutylamide (1), octadeca-2E,4E,8E,10Z,14Z-pentaene-12-ynoic acid 2'-methylbutylamide (2), hexadeca-2E,4E,9Z-triene-12,14-diynoic acid isobutylamide (3), and hexadeca-2E,4E,9,12-tetraenoic acid 2'-methylbutylamide (4), were identified from Heliopsis helianthoides var. scabra. Compounds 2-4 are new natural products, while 1 was isolated for the first time from this species. The previously described macamides, N-(3-methoxybenzyl)-(9Z,12Z,15Z)-octadecatrienamide (5), N-benzyl-(9Z,12Z,15Z)-octadecatrienamide (6), and N-benzyl-(9Z,12Z)-octadecadienamide (7), were isolated from Lepidium meyenii (Maca). N-Methylbutylamide 4 and N-benzylamide 7 showed submicromolar and selective binding affinities for the cannabinoid CB1 receptor (Ki values of 0.31 and 0.48 μM, respectively). Notably, compound 7 also exhibited weak fatty acid amide hydrolase (FAAH) inhibition (IC50 = 4 μM) and a potent inhibition of anandamide cellular uptake (IC50 = 0.67 μM) that was stronger than the inhibition obtained with the controls OMDM-2 and UCM707. The pronounced ECS polypharmacology of compound 7 highlights the potential involvement of the arachidonoyl-mimicking 9Z,12Z double-bond system in the linoleoyl group for the overall cannabimimetic action of NAAs. This study provides additional strong evidence of the endocannabinoid substrate mimicking of plant-derived NAAs and uncovers a direct and indirect cannabimimetic action of the Peruvian Maca root.

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'.

Functionalization of β-caryophyllene generates novel polypharmacology in the endocannabinoid system

The widespread dietary plant sesquiterpene hydrocarbon β-caryophyllene (1) is a CB2 cannabinoid receptor-specific agonist showing anti-inflammatory and analgesic effects in vivo. Structural insights into the pharmacophore of this hydrocarbon, which lacks functional groups other than double bonds, are missing. A structure-activity study provided evidence for the existence of a well-defined sesquiterpene hydrocarbon binding site in CB2 receptors, highlighting its exquisite sensitivity to modifications of the strained endocyclic double bond of 1. While most changes on this element were detrimental for activity, ring-opening cross metathesis of 1 with ethyl acrylate followed by amide functionalization generated a series of new monocyclic amides (11a, 11b, 11c) that not only retained the CB2 receptor functional agonism of 1 but also reversibly inhibited fatty acid amide hydrolase (FAAH), the major endocannabinoid degrading enzyme, without affecting monoacylglycerol lipase (MAGL) and α,β hydrolases 6 and 12. Intriguingly, further modification of this monocyclic scaffold generated the FAAH- and endocannabinoid substrate-specific cyclooxygenase-2 (COX-2) dual inhibitors 11e and 11f, which are probes with a novel pharmacological profile. Our study shows that by removing the conformational constraints induced by the medium-sized ring and by introducing functional groups in the sesquiterpene hydrocarbon 1, a new scaffold with pronounced polypharmacological features within the endocannabinoid system could be generated. The structural and functional repertoire of cannabimimetics and their yet poorly understood intrinsic promiscuity may be exploited to generate novel probes and ultimately more effective drugs.

Quantification of anandamide and 2-arachidonoylglycerol plasma levels to examine potential influences of tetrahydrocannabinol application on the endocannabinoid system in humans

The effects of tetrahydrocannabinol (THC) and endogenous cannabinoids (endocannabinoids, ECs) are both mediated by activation of the cannabinoid receptors CB1 and CB2. Exogenous activation of these receptors by THC could therefore alter EC levels. We tested this hypothesis in healthy volunteers (n = 25) who received a large intravenous dose of THC (0.10 mg/kg). Effects on the EC system were quantified by serial measurements of plasma ECs after THC administration. Eleven blood samples were drawn during the first 5 h after THC administration and two more samples after 24 and 48 h. THC, its metabolites THC-OH (biologically active) and THC-COOH (non-active), and the ECs anandamide and 2-arachidonoylglycerol (2-AG) were quantified by liquid chromatography-mass spectrometry. EC-plasma levels showed a biphasic response after THC injection reaching maximal values at 30 min. Anandamide increased slightly from 0.58 ± 0.21 ng/ml at baseline to 0.64 ± 0.24 ng/ml (p < 0.05) and 2-AG from 7.60 ± 4.30 ng/ml to 9.50 ± 5.90 ng/ml (p < 0.05). After reaching maximal concentrations, EC plasma levels decreased markedly to a nadir of 300 min after THC administration (to 0.32 ± 0.15 ng/ml for anandamide and to 5.50 ± 3.01 ng/ml for 2-AG, p < 0.05). EC plasma concentrations returned to near baseline levels until 48 h after the experiment. THC (0.76 ± 0.16 ng/ml) and THC-OH (0.36 ± 0.17 ng/ml) were still measurable at 24 h and remained detectible until 48 h after THC administration. Although the underlying mechanism is not clear, high doses of intravenous THC appear to influence endogenous cannabinoid concentrations and presumably EC-signalling.

Quantitative analysis of arachidonic acid, endocannabinoids, N-acylethanolamines and steroids in biological samples by LCMS/MS: Fit to purpose.

Arachidonic acid (5Z,8Z,11Z,14Z-eicosatetraenoic acid; C20:4) (arachidonate, AA) is a vital polyunsaturated omega-6 fatty acid (PUFA) without its presence the mammalian brain, muscles, and possibly other organs cannot develop or function [1] and [2]. AA fulfils numerous known and possibly yet unknown functions as integral part of mammalian phospholipid membranes and as free AA which also acts as a precursor of a variety of biologically active lipid mediators generally referred to as eicosanoids (e.g., prostaglandins, leukotrienes). A more recent class of eicosanoids is composed of the endogenous cannabinoids (endocannabinoids) 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide (anandamide, AEA), which act on cannabinoid CB1 and CB2 receptors but also modulate ion channels and nuclear receptors [3] and [4]. In recent years, the role of endocannabinoids as prominent anti-inflammatory and neuromodulatory eicosanoids has been shown by numerous studies [5].

4'-O-methylhonokiol increases levels of 2-arachidonoyl glycerol in mouse brain via selective inhibition of its COX-2-mediated oxygenation

BACKGROUND AND PURPOSE 4'-O-methylhonokiol (MH) is a natural product showing anti-inflammatory, anti-osteoclastogenic, and neuroprotective effects. MH was reported to modulate cannabinoid CB2 receptors as an inverse agonist for cAMP production and an agonist for intracellular [Ca2+]. It was recently shown that MH inhibits cAMP formation via CB2 receptors. In this study, the exact modulation of MH on CB2 receptor activity was elucidated and its endocannabinoid substrate-specific inhibition (SSI) of cyclooxygenase-2 (COX-2) and CNS bioavailability are described for the first time. METHODS CB2 receptor modulation ([35S]GTPγS, cAMP, and β-arrestin) by MH was measured in hCB2-transfected CHO-K1 cells and native conditions (HL60 cells and mouse spleen). The COX-2 SSI was investigated in RAW264.7 cells and in Swiss albino mice by targeted metabolomics using LC-MS/MS. RESULTS MH is a CB2 receptor agonist and a potent COX-2 SSI. It induced partial agonism in both the [35S]GTPγS binding and β-arrestin recruitment assays while being a full agonist in the cAMP pathway. MH selectively inhibited PGE2 glycerol ester formation (over PGE2) in RAW264.7 cells and significantly increased the levels of 2-AG in mouse brain in a dose-dependent manner (3 to 20 mg kg(-1)) without affecting other metabolites. After 7 h from intraperitoneal (i.p.) injection, MH was quantified in significant amounts in the brain (corresponding to 200 to 300 nM). CONCLUSIONS LC-MS/MS quantification shows that MH is bioavailable to the brain and under condition of inflammation exerts significant indirect effects on 2-AG levels. The biphenyl scaffold might serve as valuable source of dual CB2 receptor modulators and COX-2 SSIs as demonstrated by additional MH analogs that show similar effects. The combination of CB2 agonism and COX-2 SSI offers a yet unexplored polypharmacology with expected synergistic effects in neuroinflammatory diseases, thus providing a rationale for the diverse neuroprotective effects reported for MH in animal models.

AM 6545: A Novel Peripheral CB1 Antagonist

Obesity and other related metabolic disorders are a common problem in the United States. Consequently, several drug therapies have been developed in an attempt to address this problem. Many older appetite suppressants, such as amphetamines, were dangerous and potentially addictive. For the last few years, the endocannabinoid system was investigated as a potential target for appetite suppression. Unfortunately, early cannabinoid CB1 antagonists came with an unacceptable side effect profile of their own, which is largely due to central actions of these drugs. In an attempt to reduce the side effect profile, researchers are investigating peripherally acting cannabinoid antagonists, which do not penetrate the blood brain barrier. This study investigated AM 6545, a novel peripheral cannabinoid antagonist, for its effects on food reinforced instrumental behavior. In the end, the results indicated that AM6545 produced a dose-related suppression of lever pressing for food reinforcement.

Advanced Protein Modeling Method: Benchmarking and Applications in Computer-Aided Drug Discovery

Development of homology modeling methods will remain an area of active research. These methods aim to develop and model increasingly accurate three-dimensional structures of yet uncrystallized therapeutically relevant proteins e.g. Class A G-Protein Coupled Receptors. Incorporating protein flexibility is one way to achieve this goal. Here, I will discuss the enhancement and validation of the ligand-steered modeling, originally developed by Dr. Claudio Cavasotto, via cross modeling of the newly crystallized GPCR structures. This method uses known ligands and known experimental information to optimize relevant protein binding sites by incorporating protein flexibility. The ligand-steered models were able to model, reasonably reproduce binding sites and the co-crystallized native ligand poses of the β2 adrenergic and Adenosine 2A receptors using a single template structure. They also performed better than the choice of template, and crude models in a small scale high-throughput docking experiments and compound selectivity studies. Next, the application of this method to develop high-quality homology models of Cannabinoid Receptor 2, an emerging non-psychotic pain management target, is discussed. These models were validated by their ability to rationalize structure activity relationship data of two, inverse agonist and agonist, series of compounds. The method was also applied to improve the virtual screening performance of the β2 adrenergic crystal structure by optimizing the binding site using β2 specific compounds. These results show the feasibility of optimizing only the pharmacologically relevant protein binding sites and applicability to structure-based drug design projects.

Exceptionally potent inhibitors of fatty acid amide hydrolase: The enzyme responsible for degradation of endogenous oleamide and anandamide

The development of exceptionally potent inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the degradation of oleamide (an endogenous sleep-inducing lipid), and anandamide (an endogenous ligand for cannabinoid receptors) is detailed. The inhibitors may serve as useful tools to clarify the role of endogenous oleamide and anandamide and may prove to be useful therapeutic agents for the treatment of sleep disorders or pain. The combination of several features—an optimal C12–C8 chain length, π-unsaturation introduction at the corresponding arachidonoyl Δ8,9/Δ11,12 and oleoyl Δ9,10 location, and an α-keto N4 oxazolopyridine with incorporation of a second weakly basic nitrogen provided FAAH inhibitors with Kis that drop below 200 pM and are 102–103 times more potent than the corresponding trifluoromethyl ketones.