Phytochemical Characterization and TRPA1/TRPM8 Modulation Profile of the Cannabigerol-Rich Cannabis sativa L. Chemotype IV.

The first detailed phytochemical analysis of the cannabigerol (CBG)-rich chemotype IV of Cannabis sativa L. resulted in the isolation of the expected cannabigerolic acid/cannabigerol (CBGA/CBG) and cannabidiolic acid/cannabidiol (CBDA/CBD) and of nine new phytocannabinoids (5-13), which were fully characterized by HR-ESIMS and 1D and 2D NMR. These included mono- or dihydroxylated CBGA/CBG analogues, a congener with a truncated side chain (10), cyclocannabigerol B (11), and the CBD derivatives named cannabifuranols (12 and 13). Cyclocannabigerol B and cannabifuranols are characterized by a novel phytocannabinoid structural architecture. The isolated phytocannabinoids were assayed on the receptor channels TRPA1 and TRPM8, unveiling a potent dual TRPA1 agonist/TRPM8 antagonist profile for compounds 6, 7, and 14. Chiral separation of the two enantiomers of 5 resulted in the discovery of a synergistic effect of the two enantiomers on TRPA1.

Discovery of a Remarkable Methyl Shift Effect in the Vanilloid Activity of Triterpene Amides.

As part of a study on triterpenoid conjugates, the dietary pentacyclic triterpenoids oleanolic (2a) and ursolic acids (3a) were coupled with vanillamine, and the resulting amides (2b and 3b, respectively) were assayed for activity on the vanilloid receptor TRPV1. Despite a structural difference limited to the location of a methyl group in their conformationally rigid pentacyclic core, oleanoloyl vanillamide dramatically outperformed ursoloyl vanillamide in terms of potency (EC50 = 35 ± 2 nM for 2b and 5.4 ± 2.3 µM for 3b). Using molecular docking and dynamics, this difference was translated into distinct accommodation modes at the TRPV1 vanillyl ligand pocket, suggesting a critical role of a C-H πphenyl interaction between the triterpenoid C-29 methyl and Phe591 of TRPV1. Because the molecular mechanisms underlying the activation process of transient receptor channels (TRPs) remain to be fully elucidated, the observation of spatially restricted structure-activity information is of significant relevance to identify the molecular detail of TRPV1 ligand gating.

Iodine-Promoted Aromatization of p-Menthane-Type Phytocannabinoids.

Treatment with iodine cleanly converts various p-menthane-type phytocannabinoids and their carboxylated precursors into cannabinol (CBN, 1a). The reaction is superior to previously reported protocols in terms of simplicity and substrate range, which includes not only tricyclic tetrahydrocannabinols such as Δ9-THC (2a) but also bicyclic phytocannabinoids such as cannabidiol (CBD, 3a). Lower homologues from the viridin series (2c and 3c, respectively) afforded cannabivarin (CBV), a non-narcotic compound that, when investigated against a series of ionotropic (thermo-TRPs) biological end-points of phytocannabinoids, retained the submicromolar TRPA1-activating and TRPM8-inhibiting properties of CBN, while also potently activating TRPV2. Treatment with iodine provides an easy access to CBN (1a) from crude extracts and side-cuts of the purification of Δ9-THC and CBD from respectively narcotic Cannabis sativa (marijuana) and fiber hemp, substantially expanding the availability of this compound and, in the case of fiber hemp, dissecting it from narcotic phytocannabinoids.

TRPA1 channels as targets for resveratrol and related stilbenoids.

A series of twenty resveratrol analogues was synthesized and tested on TRPA1 and TRPV1 channels. None was able to significantly modulate TRPV1 channels. Conversely, most of them exhibited remarkably higher TRPA1 modulating activity than resveratrol. Optimal potency was observed with ortho monoxygenated stilbenes 6 and 17.

Corrigendum: Oral ultramicronized palmitoylethanolamide: plasma and tissue levels and spinal antihyperalgesic effect.

[This corrects the article DOI: 10.3389/fphar.2018.00249.].

Tetrahydro-ß-carboline derivatives targeting fatty acid amide hydrolase (FAAH) and transient receptor potential (TRP) channels.

A series of twenty-five derivatives of tetrahydro-ß-carbolines 1-3 was synthesized and assayed on FAAH and TRPV1 and TRPA1 channels. Four carbamates, that is, 5a,c,e, and 9b inhibited FAAH with significant potency and interacted also effectively with TRPV1 and TRPA1 nociceptive receptors, while ureas 7b,d,f, and 8a,b were endowed with specific submicromolar TRPV1 modulating activities.

Fluorescence-Based Assay for TRPV1 Channels.

The transient receptor potential vanilloid 1 ion channel (TRPV1) is a ligand-gated nonselective calcium-permeant cation channel involved in the detection of a wide variety of chemical and physical noxious stimuli, ranging from exogenous and endogenous ligands to noxious heat (>42 °C) and low pH (pH < 5.2). Due to its central role in pain and hyperalgesia, TRPV1 is considered a relevant therapeutic target for the development of analgesic and anti-inflammatory drugs potentially useful to relieve chronic, neuropathic, and inflammatory pain and to treat disorders such as inflammatory bowel disease. In this view, the availability of in vitro assays for the screening of novel TRPV1 modulators is highly desirable. Since TRPV1 activation leads to an increase in the intracellular calcium (Ca2+) levels, the use of Ca2+ fluorescent indicators represent a valuable and sensitive tool for monitoring such intracellular changes. In this chapter, we describe methods for recording and monitoring Ca2+ signals through the fluorescent indicators Fluo-4 acetoxymethyl (AM) and Fura-2 AM in HEK-293 cells transfected with TRPV1 or other thermoTRP channels.

In Silico-Guided Rational Drug Design and Synthesis of Novel 4-(Thiophen-2-yl)butanamides as Potent and Selective TRPV1 Agonists.

We describe an in silico-guided rational drug design and the synthesis of the suggested ligands, aimed at improving the TRPV1-ligand binding properties and the potency of N-(4-hydroxy-3-methoxybenzyl)-4-(thiophen-2-yl) butanamide I, a previously identified TRPV1 agonist. The docking experiments followed by molecular dynamics simulations and thermodynamic analysis led the drug design toward both the introduction of a lipophilic iodine and a flat pyridine/benzene at position 5 of the thiophene nucleus. Most of the synthesized compounds showed high TRPV1 efficacy and potency as well as selectivity. The molecular modeling analysis highlighted crucial hydrophobic interactions between Leu547 and the iodo-thiophene nucleus, as in amide 2a, or between Phe543 and the pyridinyl moiety, as in 3a. In the biological evaluation, both compounds showed protective properties against oxidative stress-induced ROS formation in human keratinocytes. Additionally, while 2a showed neuroprotective effects in both neurons and rat brain slices, 3a exhibited potent antinociceptive effect in vivo..

Synthesis and biological evaluation of [6]-gingerol analogues as transient receptor potential channel TRPV1 and TRPA1 modulators.

In order to explore the structural determinants for the TRPV1 and TRPA1 agonist properties of gingerols, a series of nineteen analogues (1b-5) of racemic [6]-gingerol (1a) was synthesized and tested on TRPV1 and TRPA1 channels. The exploration of the structure-activity relationships, by modulating the three pharmacophoric regions of [6]-gingerol, led to the identification of some selective TRPV1 agonists/desensitizers of TRPV1 channels (3a, 3f, and 4) and of some full TRPA1 antagonists (2c, 2d, 3b, and 3d).

Modulation of thermo-transient receptor potential (thermo-TRP) channels by thymol-based compounds.

A series of thirty-three thymol, p-cymene-3-carboxylic acid, and 3-amino-p-cymene derivatives was synthesized and tested on TRPA1, TRPM8, and TRPV3 channels. Most of them acted as strong modulators of TRPA1, TRPM8, and TRPV3 channels with EC(50) and/or IC(50) values distinctly lower than those of thymol and related monoterpenoids. Some of the compounds examined, that is, 3c, 4e, f, 6b, and 8b exhibited an appreciable subtype-selectivity.

Leucettamols, bifunctionalized marine sphingoids, act as modulators of TRPA1 and TRPM8 channels.

Leucettamols, bifunctionalized sphingoid-like compounds obtained from a marine sponge Leucetta sp., act as non-electrophilic activators of the TRPA1 channel and potent inhibitors of the icilin-mediated activation of the TRPM8 channel, while they are inactive on CB1, CB2 and TRPV1 receptors. Leucettamols represent the first compounds of marine origin to target TRPA1 and the first class of natural products to inhibit TRPM8 channels. The preparation of a small series of semi-synthetic derivatives revealed interesting details on the structure-activity relationships within this new chemotype of simple acyclic TRP modulators.

N-palmitoyl-vanillamide (palvanil) is a non-pungent analogue of capsaicin with stronger desensitizing capability against the TRPV1 receptor and anti-hyperalgesic activity.

N-acyl-vanillamide (NAVAM) analogues of the natural pungent principle of capsicum, capsaicin, were developed several years ago as potential non-pungent analgesic compounds. N-oleoyl-vanillamide (olvanil) and N-arachidonoy-vanillamide (arvanil), in particular, were described in several publications and patents to behave as potent anti-hyperalgesic compounds in experimental models of chronic and inflammatory pain, and to activate both "capsaicin receptors", i.e. the transient receptor potential of vanilloid type-1 (TRPV1) channel, and, either directly or indirectly, cannabinoid receptors of type-1. Here we report the biochemical and pharmacological characterization of a so far neglected NAVAM, N-palmitoyl-vanillamide (palvanil), and propose its possible use instead of capsaicin, as a possible topical analgesic. Palvanil exhibited a kinetics of activation of human recombinant TRPV1-mediated intracellular calcium elevation significantly slower than that of capsaicin (t(1/2)=21s and 8s, respectively at 1µM). Slow kinetics of TRPV1 agonists were previously found to be associated with stronger potencies as TRPV1 desensitizing agents, which in turn are usually associated with lower pungency and stronger anti-hyperalgesic activity. Accordingly, palvanil desensitized the human recombinant TRPV1 to the effect of capsaicin (10nM) with significantly higher potency than capsaicin (IC(50)=0.8nM and 3.8nM, respectively), this effect reaching its maximum more rapidly (50 and 250min, respectively). Palvanil was also more potent than capsaicin at desensitizing the stimulatory effect of TRPV1 by low pH together with anandamide, which mimics conditions occurring during inflammation. In the eye-wiping assay carried out in mice, palvanil was not pungent and instead caused a strong and long-lasting inhibition of capsaicin-induced eye-wiping. Finally, intraplantar palvanil inhibited the second phase of the nociceptive response to formalin in mice. In conclusion, palvanil appears to be a non-pungent analogue of capsaicin with stronger desensitizing effects on TRPV1 and hence potentially higher anti-hyperalgesic activity.

Cannabinoid CB1 receptor elevation of intracellular calcium in neuroblastoma SH-SY5Y cells: interactions with muscarinic and delta-opioid receptors.

Although coupled to G(i/o) proteins, cannabinoid CB(1) receptors can also activate intracellular Ca(2+) ([Ca(2+)](i)) accumulation through not fully understood mechanisms. We report that in, human neuroblastoma SH-SY5Y cells, CB(1) activation with the specific agonist arachidonoylchloroethanolamide (ACEA), weakly elevates [Ca(2+)](i) and that this effect, when using low (1-100 nM) concentrations of ACEA, is enhanced by the previous activation of G(q/11)-coupled M(3) muscarinic receptors with carbachol, dose-dependently and up to approximately 8-fold. A similar behaviour was also observed with carbachol and the G(i/o)-coupled delta-opioid receptor. Furthermore, stimulation of CB(1) receptors produced a concentration-dependent leftward shift of the elevation of [Ca(2+)](i) by delta-opioid receptors. These stimulatory effects were variedly attenuated by selective antagonists of each receptor, pertussis toxin, inhibitors of phospholipase C (U73122 and D609), and, when assessed in the presence of extracellular Ca(2+), by the block of voltage-activated calcium channels. Cholera toxin only slightly inhibited the G(q/11)-G(i/o)-mediated cross-talk, but induced a stronger inhibition of the G(i/o)-G(i/o)-mediated interaction. These findings suggest that activation of M(3) muscarinic receptors might produce a qualitative alteration of the signaling associated with G(i/o)-coupled receptors, and that sequential activation of CB(1) and delta-opioid receptors, both coupled to G(i/o), produces instead synergistic effects on [Ca(2+)](i) elevation.

(-)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels.

A series of twenty-two (-)-menthylamine derivatives was synthesized and tested on TRPM8, TRPV1, and TRPA1 channels. Five of the novel compounds, that is, 1d, 1f, 2b, 2c, and 2e behaved as potent TRPM8 antagonists with IC(50) values versus icilin and (-)-menthol between 20 nM and 0.7 microM, and were between 4- and approximately 150-fold selective versus TRPV1 and TRPA1 activation. Compound 1d also induced caspase 3/7 release in TRPM8-expressing LNCaP prostate carcinoma cells, but not in non-TRPM8 expressing DU-145 cells. Five other derivatives, that is, 1a, 1g, 1h, 2f, and 2h were slightly less potent than previous compounds but still relatively selective versus TRPV1 and TRPA1.

Synthesis and biological evaluation of piperazinyl carbamates and ureas as fatty acid amide hydrolase (FAAH) and transient receptor potential (TRP) channel dual ligands.

The evaluation of a series of piperazinyl carbamates and ureas, designed on the basis of previously reported TRPV1 antagonists and FAAH inhibitors, led to the identification of some 'dual-action' compounds targeting both FAAH and TRPV1 or TRPA1 receptors.

Taste-guided identification of high potency TRPA1 agonists from Perilla frutescens.

Perilla frutescens is a food plant widely used in Asian cuisine. This plant was investigated for its interesting taste and somatosensory properties. Perillaldehyde and perillaketone are among the components of the aromatic extracts from P. Frutescens. These compounds were shown here to activate the cloned TRPA1 channel when expressed in an heterologous cell system and are therefore suggested to be responsible for the chemesthetic properties of this plant.

Evaluation of the production of exopolysaccharides by newly isolated Halomonas strains from Tunisian hypersaline environments.

Ten Halomonas strains were screened from different Tunisian hypersaline environments for the production of exopolysaccharides (EPS), characterized and identified basing on 16S rRNA gene sequencing. EPS production was therefore studied using two different culture media M1 (complex medium) and M2 (semi-complex medium). Selected isolates produced different EPS amounts ranging from 86 to 170 mg L-1 and 26 to 105 mg L-1 when grown on M1 and M2, respectively. The use of M1 encouraged stronger bacterial growth associated with greater EPS production compared to M2. Nevertheless, the highest EPS yield (YEPS/X) was observed for strains grown on M2. When cultivated on M1, all isolates produced EPS exhibiting almost the same monosaccharide profile with mannose, glucose and arabinose being the main monomers. However, the produced EPS on M2 were characterized by heterogeneous monosaccharide profiles among the different species, mostly consisting of glucomannan that could be a versatile material used for many further applications.

Effects of non-euphoric plant cannabinoids on muscle quality and performance of dystrophic mdx mice.

BACKGROUND AND PURPOSE: Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, results in chronic inflammation and irreversible skeletal muscle degeneration. Moreover, the associated impairment of autophagy greatly contributes to the aggravation of muscle damage. We explored the possibility of using non-euphoric compounds present in Cannabis sativa, cannabidiol (CBD), cannabidivarin (CBDV) and tetrahydrocannabidivarin (THCV), to reduce inflammation, restore functional autophagy and positively enhance muscle function in vivo. EXPERIMENTAL APPROACH: Using quantitative PCR, western blots and [Ca2+ ]i measurements, we explored the effects of CBD and CBDV on the differentiation of both murine and human skeletal muscle cells as well as their potential interaction with TRP channels. Male dystrophic mdx mice were injected i.p. with CBD or CBDV at different stages of the disease. After treatment, locomotor tests and biochemical analyses were used to evaluate their effects on inflammation and autophagy. KEY RESULTS: CBD and CBDV promoted the differentiation of murine C2C12 myoblast cells into myotubes by increasing [Ca2+ ]i mostly via TRPV1 activation, an effect that undergoes rapid desensitization. In primary satellite cells and myoblasts isolated from healthy and/or DMD donors, not only CBD and CBDV but also THCV promoted myotube formation, in this case, mostly via TRPA1 activation. In mdx mice, CBD (60 mg·kg-1 ) and CBDV (60 mg·kg-1 ) prevented the loss of locomotor activity, reduced inflammation and restored autophagy. CONCLUSION AND IMPLICATIONS: We provide new insights into plant cannabinoid interactions with TRP channels in skeletal muscle, highlighting a potential opportunity for novel co-adjuvant therapies to prevent muscle degeneration in DMD patients. LINKED ARTICLES: This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc.

Carbamoyl tetrazoles as inhibitors of endocannabinoid inactivation: a critical revisitation.

We have synthesized a series of 18 1,5- and 2,5-disubstituted carbamoyl tetrazoles, including LY2183240 (1) and LY2318912 (7), two compounds previously described as potent inhibitors of the cellular uptake of the endocannabinoid anandamide, and their regioisomers 2 and 8. We confirm that compound 1 is a potent inhibitor of both the cellular uptake and, like the other new compounds synthesized here, the enzymatic hydrolysis of anandamide. With the exception of 9, 12, 15, and the 2,5-regioisomer of LY2183240 2, the other compounds were all found to be weakly active or inactive on anandamide uptake. Several compounds also inhibited the enzymatic hydrolysis of the other main endocannabinoid, 2-arachidonoylglycerol, as well as its enzymatic release from sn-1-oleoyl-2-arachidonoyl-glycerol, at submicromolar concentrations. Four of the novel compounds, i.e. 3, 4, 17, and 18, inhibited anandamide hydrolysis potently (IC50=2.1-5.4nM) and selectively over all the other targets tested (IC50 >or= 10microM), thus representing new potentially useful tools for the inhibition of fatty acid amide hydrolase.

First "hybrid" ligands of vanilloid TRPV1 and cannabinoid CB2 receptors and non-polyunsaturated fatty acid-derived CB2-selective ligands.

12-Phenylacetyl-ricinoleoyl-vanillamide (phenylacetylrinvanil, PhAR, IDN5890), is an ultra-potent agonist of human vanilloid TRPV1 receptors also endowed with moderate affinity for human cannabinoid CB(2) receptors. To improve its CB(2) affinity and temper its potency at TRPV1, the modification of the polar headgroup and the lipophilic 12-acylgroup of PhAR was pursued. Replacement of the vanillyl headgroup of PhAR with various aromatic or alkyl amino groups decreased activity at TRPV1 receptors, although the dopamine, cyclopropylamine, 1'-(R)- and 1'-(S)-methyl-ethanolamine, and ethanolamine derivatives retained significant potency (EC(50) 31-126 nM). Within these compounds, the 12-phenylacetylricinoleyl cyclopropylamide and ethanolamide were the strongest ligands at CB(2) receptors, with K(i) of 22 and 44 nM, and 14- and >20-fold selectivity over cannabinoid CB(1) receptors, respectively. The propyl- and allyl-derivatives also exhibited high affinity at CB(2) receptors (K(i)=40 and 22 nM, with 40 and >80-fold selectivity over CB(1) receptors, respectively), but no activity at TRPV1 receptors. The cyclopropyl- and allyl-derivatives behaved as CB(2) inverse agonists in the GTP-gamma-S binding assay. Addition of para-methoxy, -tert-butyl or -chlorine groups to the 12-phenylacetyl moiety of PhAR produced compounds that retained full potency at TRPV1 receptors, but with improved selectivity over CB(2) or CB(1) receptors. Thus, the manipulation of PhAR led to the development of the first CB(2)/TRPV1 dual ligands and of an entirely new class of inverse agonists at CB(2) receptors. Both types of compounds might find application in the treatment of inflammation, and represent new molecular probes to investigate the endocannabinoid-endovanilloid signalling system.