Terpenoids Commonly Found in Cannabis sativa Do Not Modulate the Actions of Phytocannabinoids or Endocannabinoids on TRPA1 and TRPV1 Channels.

Introduction: Cannabis sativa produces hundreds of bioactive compounds, including cannabinoids and terpenoids. It has been proposed that cannabinoids act in synergy with terpenoids to produce the entourage effect, a concept used to explain the therapeutic benefits of medicinal cannabis. One molecular explanation for the entourage effect is that the terpenoids augment the actions of cannabinoids at their molecular drug targets in cells. We recently reported that terpenoids commonly found in cannabis do not influence the functional effects of Δ9-tetrahydrocannabinol (Δ9-THC) on cannabinoid 1 and cannabinoid 2 receptors. The present study aimed to extend on this research by examining whether terpenoids influence the effects of phytocannabinoids and endocannabinoids on human transient receptor potential ankyrin 1 (hTRPA1) and human transient receptor potential vanilloid 1 (hTRPV1) channels heterologously expressed in mammalian cells. Materials and Methods: The activity of terpenoids, phytocannabinoids, and endocannabinoids was assessed in inducible HEK Flp-In T-Rex cells transfected with hTRPA1 and hTRPV1 channels, respectively. Real-time changes in intracellular calcium ([Ca]i) were measured using the Calcium 5 dye and a FlexStation 3 plate reader. Results: α-pinene, ß-pinene, ß-caryophyllene, linalool, limonene, ß-myrcene or α-humulene did not affect [Ca]i in hTRPA1 and hTRPV1 overexpressing cells. Cinnamaldehyde (CA), Δ9-THC, and 2-arachidonoylglycerol (2-AG) activated TRPA1 receptors with high efficacy and similar potency (EC50s of ∼10 µM). Capsaicin and anandamide (AEA) activated TRPV1 receptors with an EC50 of 61 nM and 4.3 µM, respectively, but TRPV1 showed no response to Δ9-THC, cannabidiol, and other minor cannabinoids. Terpenoids did not significantly affect the responses of TRPA1 and TRPV1 receptors to submaximal and maximal concentrations of CA and Δ9-THC or the endocannabinoids AEA and 2-AG. Discussion: We could not find any evidence that the terpenoids tested here activate TRPA1 and TRPV1 channels or modulate their activation by Δ9-THC and other agonists, including endocannabinoids.

Detection of Δ9 THC in oral fluid following vaporized cannabis with varied cannabidiol (CBD) content: An evaluation of two point-of-collection testing devices.

Point-of-collection testing (POCT) for Δ9 -tetrahydrocannabinol (THC) in oral fluid is increasingly used to detect driving under the influence of cannabis (DUIC). However, previous studies have questioned the reliability and accuracy of two commonly used POCT devices, the Securetec DrugWipe® 5 s (DW5s) and Dräger DrugTest® 5000 (DT5000). In the current placebo controlled, double-blind, crossover study we used liquid chromatography-tandem mass spectrometry (LC-MS/MS) to accurately quantify cannabinoid concentrations in the oral fluid of 14 participants at various timepoints (10, 60, 120, and 180 minutes) following vaporization of 125 mg of THC-dominant (11% THC; <1% CBD), THC/CBD equivalent (11% THC; 11% CBD) and placebo (<1% THC; <1% CBD) cannabis. At each timepoint, oral fluid was also screened using the DW5s (10 ng/mL THC cut-off) and DT5000 (10 ng/mL THC cut-off). LC-MS/MS analysis showed peak oral fluid THC concentrations at the 10 minute timepoint with a rapid decline thereafter. This trajectory did not differ with THC dominant and THC/CBD equivalent cannabis. With a 10 ng/mL confirmatory cut-off, 5% of DW5s test results were false positives and 16% false negatives. For the DT5000, 10% of test results were false positives and 9% false negatives. Neither the DW5s nor the DT5000 demonstrated the recommended >80% sensitivity, specificity and accuracy. Accuracy was lowest at 60 minutes, when THC concentrations were often close to the screening cut-off (10 ng/mL). POCT devices can be useful tools in detecting recent cannabis use; however, limitations should be noted, and confirmatory LC-MS/MS quantification of results is strongly advisable.

The chemistry and pharmacology of synthetic cannabinoid SDB-006 and its regioisomeric fluorinated and methoxylated analogs.

Synthetic cannabinoids are the largest and most structurally diverse class of new psychoactive substances, with manufacturers often using isomerism to evade detection and circumvent legal restriction. The regioisomeric methoxy- and fluorine-substituted analogs of SDB-006 (N-benzyl-1-pentyl-1H-indole-3-carboxamide) were synthesized and could not be differentiated by gas chromatography-mass spectrometry (GC-MS), but were distinguishable by liquid chromatography-quadrupole time-of-flight-MS (LC-QTOF-MS). In a fluorescence-based plate reader membrane potential assay, SDB-006 acted as a potent agonist at human cannabinoid receptors (CB1 EC50 = 19 nM). All methoxy- and fluorine-substituted analogs showed reduced potency compared to SDB-006, although the 2-fluorinated analog (EC50 = 166 nM) was comparable to known synthetic cannabinoid RCS-4 (EC50 = 146 nM). Using biotelemetry in rats, SDB-006 and RCS-4 evoked comparable reduction in body temperature (~0.7°C at a dose of 10 mg/kg), suggesting lower potency than the recent synthetic cannabinoid AB-CHMINACA (>2°C, 3 mg/kg).

Intranasal oxytocin in the treatment of anorexia nervosa: Randomized controlled trial during re-feeding.

BACKGROUND: Nutritional rehabilitation in anorexia nervosa (AN) is impeded by fear of food, eating and change leading to treatment resistance. Oxytocin (OT) exerts prosocial effects and modulates trust, fear, anxiety and neuroplasticity. The current placebo-controlled RCT examined the effects of intranasal oxytocin (IN-OT) in AN. The aim was to ascertain whether repeated doses of IN-OT enhance treatment outcomes in AN. METHODS: AN patients self-administered 36 IU IN-OT or placebo daily for 4-6 weeks during hospital treatment. The outcome measures were change in the Eating Disorders Examination (EDE) scale, weight gain, cognitive rigidity, social anxiety, obsessive and autistic symptoms. The effects of the first and last doses of IN-OT were assessed relative to placebo before and after a high-energy afternoon snack, to determine potential dampening of cortisol and anxiety levels by OT. RESULTS: Weight gain was similar in both groups. The EDE eating concern subscale score was significantly lower after IN-OT treatment as was cognitive rigidity. There were no significant differences in social anxiety or any of the other outcomes at follow-up. After four weeks IN-OT, salivary cortisol levels were significantly lowered in anticipation of an afternoon snack compared to placebo. Morning plasma OT levels did not change after chronic IN-OT or with weight restoration. CONCLUSION: IN-OT might enhance nutritional rehabilitation in AN by reducing eating concern and cognitive rigidity. Lower salivary cortisol levels in response to IN-OT suggest diminished neuroendocrine stress responsiveness to food and eating. Such effects require replication with inclusion of more sensitive subjective measures.

Acute and residual effects in adolescent rats resulting from exposure to the novel synthetic cannabinoids AB-PINACA and AB-FUBINACA.

Synthetic cannabinoids (SCs) have rapidly proliferated as recreational drugs, and may present a substantial health risk to vulnerable populations. However, information on possible effects of long-term use is sparse. This study compared acute and residual effects of the popular indazole carboxamide SC compounds AB-PINACA and AB-FUBINACA in adolescent rats with ∆9-tetrahydrocannabinol (THC) and control treatments. Albino Wistar rats were injected (i.p.) with AB-PINACA or AB-FUBINACA every second day (beginning post-natal day (PND) 31), first at a low dose (0.2 mg/kg on 6 days) followed by a higher dose (1 mg/kg on a further 6 days). THC-treated rats received equivalent doses of 6 × 1 mg/kg and 6 × 5 mg/kg. During drug treatment, THC, AB-PINACA, and AB-FUBINACA decreased locomotor activity at high and low doses, increased anxiety-like behaviours and audible vocalisations, and reduced weight gain. Two weeks after dosing was completed, all cannabinoid pre-treated rats exhibited object recognition memory deficits. These were notably more severe in rats pre-treated with AB-FUBINACA. However, social interaction was reduced in the THC pre-treated group only. Six weeks post-dosing, plasma levels of cytokines interleukin (IL)-1α and IL-12 were reduced by AB-FUBINACA pre-treatment, while cerebellar endocannabinoids were reduced by THC and AB-PINACA pre-treatment. The acute effects of AB-PINACA and AB-FUBINACA were broadly similar to those of THC, suggesting that acute SC toxicity in humans may be modulated by dose factors, including inadvertent overdose and product contamination. However, some lasting residual effects of these different cannabinoid receptor agonists were subtly different, hinting at recruitment of different mechanisms of neuroadaptation.

Cannabinoid CB2 receptor ligand profiling reveals biased signalling and off-target activity.

The cannabinoid CB2 receptor (CB2R) represents a promising therapeutic target for various forms of tissue injury and inflammatory diseases. Although numerous compounds have been developed and widely used to target CB2R, their selectivity, molecular mode of action and pharmacokinetic properties have been poorly characterized. Here we report the most extensive characterization of the molecular pharmacology of the most widely used CB2R ligands to date. In a collaborative effort between multiple academic and industry laboratories, we identify marked differences in the ability of certain agonists to activate distinct signalling pathways and to cause off-target effects. We reach a consensus that HU910, HU308 and JWH133 are the recommended selective CB2R agonists to study the role of CB2R in biological and disease processes. We believe that our unique approach would be highly suitable for the characterization of other therapeutic targets in drug discovery research.

Environmental Toxin Acrolein Alters Levels of Endogenous Lipids, Including TRP Agonists: A Potential Mechanism for Headache Driven by TRPA1 Activation.

Exposure to airborne toxins can trigger headaches, but the mechanisms are not well understood. Some environmental toxins, such as acrolein, activate transient receptor potential ankyrin 1 (TRPA1), a receptor involved in pain sensation that is highly expressed in the trigeminovascular system. It has been shown in rat models that repeated exposure to acrolein induces trigeminovascular sensitization to both TRPA1 and TRP vanilloid 1 (TRPV1) agonists, a phenomenon linked to headache. In this study, we test the hypothesis that the sensitization of trigeminovascular responses in rats after acrolein exposure via inhalation is associated with changes in levels of endogenous lipids, including TRPV1 agonists, in the trigeminal ganglia, trigeminal nucleus, and cerebellum. Lipidomics analysis of 80 lipids was performed on each tissue after acute acrolein, chronic acrolein, or room air control. Both acute and chronic acrolein exposure drove widespread alterations in lipid levels. After chronic acrolein exposure, levels of all 6 N-acyl ethanolamines in the screening library, including the endogenous cannabinoid and TRPV1 agonist, N-arachidonoyl ethanolamine, were elevated in trigeminal tissue and in the cerebellum. This increase in TRPV1 ligands by acrolein exposure may indicate further downstream signaling, in that we also show here that a combination of these TRPV1 endogenous agonists increases the potency of the individual ligands in TRPV1-HEK cells. In addition to these TRPV1 agonists, 3 TRPV3 antagonists, 4 TRPV4 agonists, and 25 orphan lipids were up and down regulated after acrolein exposure. These data support the hypothesis that lipid signaling may represent a mechanism by which repeated exposure to the TRPA1 agonist and environmental toxin, acrolein, drives trigeminovascular sensitization.

Endocannabinoid dysregulation in cognitive and stress-related brain regions in the Nrg1 mouse model of schizophrenia.

The endocannabinoid system is dysregulated in schizophrenia. Mice with heterozygous deletion of neuregulin 1 (Nrg1 HET mice) provide a well-characterised animal model of schizophrenia, and display enhanced sensitivity to stress and cannabinoids during adolescence. However, no study has yet determined whether these mice have altered brain endocannabinoid concentrations. Nrg1 application to hippocampal slices decreased 2-arachidonoylglycerol (2-AG) signalling and disrupted long-term depression, a form of synaptic plasticity critical to spatial learning. Therefore we specifically aimed to examine whether Nrg1 HET mice exhibit increased 2-AG concentrations and disruption of spatial learning. As chronic stress influences brain endocannabinoids, we also sought to examine whether Nrg1 deficiency moderates adolescent stress-induced alterations in brain endocannabinoids. Adolescent Nrg1 HET and wild-type (WT) mice were submitted to chronic restraint stress and brain endocannabinoid concentrations were analysed. A separate cohort of WT and Nrg1 HET mice was also assessed for spatial learning performance in the Morris Water Maze. Partial genetic deletion of Nrg1 increased anandamide concentrations in the amygdala and decreased 2-AG concentrations in the hypothalamus. Further, Nrg1 HET mice exhibited increased 2-AG concentrations in the hippocampus and impaired spatial learning performance. Chronic adolescent stress increased anandamide concentrations in the amygdala, however, Nrg1 disruption did not influence this stress-induced change. These results demonstrate for the first time in vivo interplay between Nrg1 and endocannabinoids in the brain. Our results demonstrate that aberrant Nrg1 and endocannabinoid signalling may cooperate in the hippocampus to impair cognition, and that Nrg1 deficiency alters endocannabinoid signalling in brain stress circuitry.

Pharmacology of Valinate and tert-Leucinate Synthetic Cannabinoids 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and Their Analogues.

Indole and indazole synthetic cannabinoids (SCs) featuring l-valinate or l-tert-leucinate pendant group have recently emerged as prevalent recreational drugs, and their use has been associated with serious adverse health effects. Due to the limited pharmacological data available for these compounds, 5F-AMBICA, 5F-AMB, 5F-ADB, AMB-FUBINACA, MDMB-FUBINACA, MDMB-CHMICA, and their analogues were synthesized and assessed for cannabimimetic activity in vitro and in vivo. All SCs acted as potent, highly efficacious agonists at CB1 (EC50 = 0.45-36 nM) and CB2 (EC50 = 4.6-128 nM) receptors in a fluorometric assay of membrane potential, with a general preference for CB1 activation. The cannabimimetic properties of two prevalent compounds with confirmed toxicity in humans, 5F-AMB and MDMB-FUBINACA, were demonstrated in vivo using biotelemetry in rats. Bradycardia and hypothermia were induced by 5F-AMB and MDMB-FUBINACA doses of 0.1-1 mg/kg (and 3 mg/kg for 5F-AMB), with MDMB-FUBINACA showing the most dramatic hypothermic response recorded in our laboratory for any SC (>3 °C at 0.3 mg/kg). Reversal of hypothermia by pretreatment with a CB1, but not CB2, antagonist was demonstrated for 5F-AMB and MDMB-FUBINACA, consistent with CB1-mediated effects in vivo. The in vitro and in vivo data indicate that these SCs act as highly efficacious CB receptor agonists with greater potency than Δ(9)-THC and earlier generations of SCs.

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

Identification of N-arachidonoyl dopamine as a highly biased ligand at cannabinoid CB1 receptors.

BACKGROUND AND PURPOSE: N-arachidonyl dopamine (NADA) has been identified as a putative endocannabinoid, but there is little information about which signalling pathways it activates. The purpose of this study was to identify the signalling pathways activated by NADA in vitro. EXPERIMENTAL APPROACH: Human or rat cannabinoid CB1 receptors were expressed in AtT20, CHO or HEK 293 cells. NADA displacement of radiolabelled cannabinoids, and CB1 receptor mediated activation of K channels or ERK phosphorylation, release of intracellular calcium ([Ca]i ) and modulation of adenylyl cyclase were measured in addition to NADA effects on CB1 receptor trafficking. KEY RESULTS: At concentrations up to 30 µM, NADA failed to activate any signalling pathways via CB1 receptors, with the exception of mobilization of [Ca]i . The elevations of [Ca]i were insensitive to pertussis toxin, and reduced or abolished by blockers of Gq /11 -dependent processes including U73122, thapsigargin and a peptide antagonist of Gq /11 activation. Prolonged NADA incubation produced modest loss of cell surface CB1 receptors. The prototypical cannabinoid agonist CP55940 signalled as expected in all assays. CONCLUSIONS AND IMPLICATIONS: NADA is an ineffective agonist at most canonical cannabinoid receptor signalling pathways, but did promote mobilization of [Ca]i via Gq -dependent processes and some CB1 receptor trafficking. This signalling profile is distinct from that of any known cannabinoid, and suggests that NADA may have a unique spectrum of effects in vivo. Our results also indicate that it may be possible to identify highly biased CB1 receptor ligands displaying a subset of the pharmacological or therapeutic effects usually attributed to CB1 ligands.

Pharmacology of Indole and Indazole Synthetic Cannabinoid Designer Drugs AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, and 5F-ADBICA.

Synthetic cannabinoid (SC) designer drugs based on indole and indazole scaffolds and featuring l-valinamide or l-tert-leucinamide side chains are encountered with increasing frequency by forensic researchers and law enforcement agencies and are associated with serious adverse health effects. However, many of these novel SCs are unprecedented in the scientific literature at the time of their discovery, and little is known of their pharmacology. Here, we report the synthesis and pharmacological characterization of AB-FUBINACA, ADB-FUBINACA, AB-PINACA, ADB-PINACA, 5F-AB-PINACA, 5F-ADB-PINACA, ADBICA, 5F-ADBICA, and several analogues. All synthesized SCs acted as high potency agonists of CB1 (EC50 = 0.24-21 nM) and CB2 (EC50 = 0.88-15 nM) receptors in a fluorometric assay of membrane potential, with 5F-ADB-PINACA showing the greatest potency at CB1 receptors. The cannabimimetic activities of AB-FUBINACA and AB-PINACA in vivo were evaluated in rats using biotelemetry. AB-FUBINACA and AB-PINACA dose-dependently induced hypothermia and bradycardia at doses of 0.3-3 mg/kg, and hypothermia was reversed by pretreatment with a CB1 (but not CB2) antagonist, indicating that these SCs are cannabimimetic in vivo, consistent with anecdotal reports of psychoactivity in humans.

Effects of bioisosteric fluorine in synthetic cannabinoid designer drugs JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135.

Synthetic cannabinoid (SC) designer drugs featuring bioisosteric fluorine substitution are identified by forensic chemists and toxicologists with increasing frequency. Although terminal fluorination of N-pentyl indole SCs is sometimes known to improve cannabinoid type 1 (CB1) receptor binding affinity, little is known of the effects of fluorination on functional activity of SCs. This study explores the in vitro functional activities of SC designer drugs JWH-018, UR-144, PB-22, and APICA, and their respective terminally fluorinated analogues AM-2201, XLR-11, 5F-PB-22, and STS-135 at human CB1 and CB2 receptors using a FLIPR membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 2.8-1959 nM) and CB2 (EC50 = 6.5-206 nM) receptors, with the fluorinated analogues generally showing increased CB1 receptor potency (∼2-5 times). Additionally, the cannabimimetic activities and relative potencies of JWH-018, AM-2201, UR-144, XLR-11, PB-22, 5F-PB-22, APICA, and STS-135 in vivo were evaluated in rats using biotelemetry. All SCs dose-dependently induced hypothermia and reduced heart rate at doses of 0.3-10 mg/kg. There was no consistent trend for increased potency of fluorinated SCs over the corresponding des-fluoro SCs in vivo. Based on magnitude and duration of hypothermia, the SCs were ranked for potency (PB-22 > 5F-PB-22 = JWH-018 > AM-2201 > APICA = STS-135 = XLR-11 > UR-144).

Novel endogenous N-acyl amides activate TRPV1-4 receptors, BV-2 microglia, and are regulated in brain in an acute model of inflammation.

A family of endogenous lipids, structurally analogous to the endogenous cannabinoid, N-arachidonoyl ethanolamine (Anandamide), and called N-acyl amides have emerged as a family of biologically active compounds at TRP receptors. N-acyl amides are constructed from an acyl group and an amine via an amide bond. This same structure can be modified by changing either the fatty acid or the amide to form potentially hundreds of lipids. More than 70 N-acyl amides have been identified in nature. We have ongoing studies aimed at isolating and characterizing additional members of the family of N-acyl amides in both central and peripheral tissues in mammalian systems. Here, using a unique in-house library of over 70 N-acyl amides we tested the following three hypotheses: (1) Additional N-acyl amides will have activity at TRPV1-4, (2) Acute peripheral injury will drive changes in CNS levels of N-acyl amides, and (3) N-acyl amides will regulate calcium in CNS-derived microglia. Through these studies, we have identified 20 novel N-acyl amides that collectively activate (stimulating or inhibiting) TRPV1-4. Using lipid extraction and HPLC coupled to tandem mass spectrometry we showed that levels of at least 10 of these N-acyl amides that activate TRPVs are regulated in brain after intraplantar carrageenan injection. We then screened the BV2 microglial cell line for activity with this N-acyl amide library and found overlap with TRPV receptor activity as well as additional activators of calcium mobilization from these lipids. Together these data provide new insight into the family of N-acyl amides and their roles as signaling molecules at ion channels, in microglia, and in the brain in the context of inflammation.

Brain levels of prostaglandins, endocannabinoids, and related lipids are affected by mating strategies.

Background. Endogenous cannabinoids (eCBs) are involved in the development and regulation of reproductive behaviors. Likewise, prostaglandins (PGs) drive sexual differentiation and initiation of ovulation. Here, we use lipidomics strategies to test the hypotheses that mating immediately activates the biosynthesis and/or metabolism of eCBs and PGs and that specific mating strategies differentially regulate these lipids in the brain. Methods. Lipid extractions and tandem mass spectrometric analysis were performed on brains from proestrous rats that had experienced one of two mating strategies (paced or standard mating) and two nonmated groups (chamber exposed and home cage controls). Levels of PGs (PGE2 and PGF2alpha), eCBs (AEA and 2-AG, N-arachidonoyl glycine), and 4 related lipids (4 N-acylethanolamides) were measured in olfactory bulb, hypothalamus, hippocampus, thalamus, striatum, midbrain, cerebellum, and brainstem. Results. Overall, levels of these lipids were significantly lower among paced compared to standard mated rats with the most dramatic decreases observed in brainstem, hippocampus, midbrain, and striatum. However, chamber exposed rats had significantly higher levels of these lipids compared to home cage controls and paced mated wherein the hippocampus showed the largest increases. Conclusions. These data demonstrate that mating strategies and exposure to mating arenas influence lipid signaling in the brain.

Targeted lipidomics in Drosophila melanogaster identifies novel 2-monoacylglycerols and N-acyl amides.

Lipid metabolism is critical to coordinate organ development and physiology in response to tissue-autonomous signals and environmental cues. Changes to the availability and signaling of lipid mediators can limit competitiveness, adaptation to environmental stressors, and augment pathological processes. Two classes of lipids, the N-acyl amides and the 2-acyl glycerols, have emerged as important signaling molecules in a wide range of species with important signaling properties, though most of what is known about their cellular functions is from mammalian models. Therefore, expanding available knowledge on the repertoire of these lipids in invertebrates will provide additional avenues of research aimed at elucidating biosynthetic, metabolic, and signaling properties of these molecules. Drosophila melanogaster is a commonly used organism to study intercellular communication, including the functions of bioactive lipids. However, limited information is available on the molecular identity of lipids with putative biological activities in Drosophila. Here, we used a targeted lipidomics approach to identify putative signaling lipids in third instar Drosophila larvae, possessing particularly large lipid mass in their fat body. We identified 2-linoleoyl glycerol, 2-oleoyl glycerol, and 45 N-acyl amides in larval tissues, and validated our findings by the comparative analysis of Oregon-RS, Canton-S and w1118 strains. Data here suggest that Drosophila represent another model system to use for the study of 2-acyl glycerol and N-acyl amide signaling.

The synthesis and pharmacological evaluation of adamantane-derived indoles: cannabimimetic drugs of abuse.

Two novel adamantane derivatives, adamantan-1-yl(1-pentyl-1H-indol-3-yl)methanone (AB-001) and N-(adamtan-1-yl)-1-pentyl-1H-indole-3-carboxamide (SDB-001), were recently identified as cannabimimetic indoles of abuse. Conflicting anecdotal reports of the psychoactivity of AB-001 in humans, and a complete dearth of information about the bioactivity of SDB-001, prompted the preparation of AB-001, SDB-001, and several analogues intended to explore preliminary structure-activity relationships within this class. This study sought to elucidate which structural features of AB-001, SDB-001, and their analogues govern the cannabimimetic potency of these chemotypes in vitro and in vivo. All compounds showed similar full agonist profiles at CB1 (EC50 = 16-43 nM) and CB2 (EC50 = 29-216 nM) receptors in vitro using a FLIPR membrane potential assay, with the exception of SDB-002, which demonstrated partial agonist activity at CB2 receptors. The activity of AB-001, AB-002, and SDB-001 in rats was compared to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC) and cannabimimetic indole JWH-018 using biotelemetry. SDB-001 dose-dependently induced hypothermia and reduced heart rate (maximal dose 10 mg/kg) with potency comparable to that of Δ(9)-tetrahydrocannabinol (Δ(9)-THC, maximal dose 10 mg/kg), and lower than that of JWH-018 (maximal dose 3 mg/kg). Additionally, the changes in body temperature and heart rate affected by SDB-001 are of longer duration than those of Δ(9)-THC or JWH-018, suggesting a different pharmacokinetic profile. In contrast, AB-001, and its homologue, AB-002, did not produce significant hypothermic and bradycardic effects, even at relatively higher doses (up to 30 mg/kg), indicating greatly reduced potency compared to Δ(9)-THC, JWH-018, and SDB-001.

Oleoyl serine, an endogenous N-acyl amide, modulates bone remodeling and mass.

Bone mass is determined by a continuous remodeling process, whereby the mineralized matrix is being removed by osteoclasts and subsequently replaced with newly formed bone tissue produced by osteoblasts. Here we report the presence of endogenous amides of long-chain fatty acids with amino acids or with ethanolamine (N-acyl amides) in mouse bone. Of these compounds, N-oleoyl-l-serine (OS) had the highest activity in an osteoblast proliferation assay. In these cells, OS triggers a Gi-protein-coupled receptor and Erk1/2. It also mitigates osteoclast number by promoting osteoclast apoptosis through the inhibition of Erk1/2 phosphorylation and receptor activator of nuclear-κB ligand (RANKL) expression in bone marrow stromal cells and osteoblasts. In intact mice, OS moderately increases bone volume density mainly by inhibiting bone resorption. However, in a mouse ovariectomy (OVX) model for osteoporosis, OS effectively rescues bone loss by increasing bone formation and markedly restraining bone resorption. The differential effect of exogenous OS in the OVX vs. intact animals is apparently a result of an OVX-induced decrease in skeletal OS levels. These data show that OS is a previously unexplored lipid regulator of bone remodeling. It represents a lead to antiosteoporotic drug discovery, advantageous to currently available therapies, which are essentially either proformative or antiresorptive.

The endocannabinoid anandamide is a precursor for the signaling lipid N-arachidonoyl glycine by two distinct pathways.

BACKGROUND: N-arachidonoyl glycine (NAGly) is an endogenous signaling lipid with a wide variety of biological activity whose biosynthesis is poorly understood. Two primary biosynthetic pathways have been proposed. One suggests that NAGly is formed via an enzymatically regulated conjugation of arachidonic acid (AA) and glycine. The other suggests that NAGly is an oxidative metabolite of the endogenous cannabinoid, anandamide (AEA), through an alcohol dehydrogenase. Here using both in vitro and in vivo assays measuring metabolites with LC/MS/MS we test the hypothesis that both pathways are present in mammalian cells. RESULTS: The metabolic products of deuterium-labeled AEA, D4AEA (deuterium on ethanolamine), indicated that NAGly is formed by the oxidation of the ethanolamine creating a D2NAGly product in both RAW 264.7 and C6 glioma cells. Significantly, D4AEA produced a D0NAGly product only in C6 glioma cells suggesting that the hydrolysis of AEA yielded AA that was used preferentially in a conjugation reaction. Addition of the fatty acid amide (FAAH) inhibitor URB 597 blocked the production of D0NAGly in these cells. Incubation with D8AA in C6 glioma cells likewise produced D8NAGly; however, with significantly less efficacy leading to the hypothesis that FAAH-initiated AEA-released AA conjugation with glycine predominates in these cells. Furthermore, the levels of AEA in the brain were significantly increased, whereas those of NAGly were significantly decreased after systemic injection of URB 597 in rats and in FAAH KO mice further supporting a role for FAAH in endogenous NAGly biosynthesis. Incubations of NAGly and recombinant FAAH demonstrated that NAGly is a significantly less efficacious substrate for FAAH with only ~50% hydrolysis at 30 minutes compared to 100% hydrolysis of AEA. Co-incubations of AEA and glycine with recombinant FAAH did not, however, produce NAGly. CONCLUSION: These data support the hypothesis that the signaling lipid NAGly is a metabolic product of AEA by both oxidative metabolism of the AEA ethanolamine moiety and through the conjugation of glycine to AA that is released during AEA hydrolysis by FAAH.