Classical Cannabinoids Research Articles

Abstract 163: Evaluation of ETS2101 and its combination with anti-mCTLA4 monoclonal antibody on CD4 and CD8 TILs in a subcutaneous colorectal cancer model of CT-26

Abstract ETS2101, (HU-211, Dexanabinol) is a synthetic cannabinoid with limited affinity to cannabinoid receptors 1 and 2 (CB1 and CB2), reducing its psychotropic potential in comparison to other molecules in the cannabinoid class. ETS2101 has been found to have effects on immune cell function in vitro. The presence of Tumor Infiltrating Lymphocytes (TILs) has been previously shown to correlate with better patient outcomes during various antitumor therapies in a multitude of cancers. This study utilized immunohistochemistry (IHC) combined with digital image analysis to quantify CD4+ and CD8+ TILs within CT-26 tumors treated with ETS2101 in combination with a checkpoint inhibitor, anti-mCTLA4. A CT-26 colon carcinoma tumor line was implanted s.c. into immune-competent mice. Ten days post implantation groups were treated with ETS2101 administered at 200 mg/kg QD in combination with anti-mCTLA-4 administered as 10 mg/kg i.p. BiW. Thirty seven days post treatment, tumors were collected and IHC was performed for CD4+ and CD8+ TILs on FFPE tumor sections (n=12-15 per group). Whole slide images were generated per IHC section and customized algorithms developed within the Indica Labs HALO platform to classify viable tumor present across each section and to quantify CD4+ or CD8+ stain area within the viable tumor region of interest (ROI). Anti-mCTLA4 monotherapy resulted in regression of 44% tumors (7% CR) and anti-mCTLA4 + ETS2101 combination therapy resulted in regression of 74% tumors (36% CR). There was a significant increase in CD8+ % staining of viable tumor observed following anti-mCTLA4 monotherapy treatment versus control (2.16% v. 1.16%, P<0.0027) and anti-mCTLA4 + ETS2101 combination therapy (2.18% v. 1.10%, P<0.0019) compared to control. A similar trend was observed for CD4+ % staining of viable tumor with a significant increase observed following anti-mCTLA4 monotherapy treatment versus control (1.82% v. 0.49%, P<0.0083) and anti-mCTLA4 + ETS2101 combination therapy (1.93% v. 0.41%, P<0.0028) compared to control. Although, there was no significant difference between anti-mCTLA4 monotherapy treatment and anti-mCTLA4 + ETS2101 combination therapy for either marker alone, following treatment with the combination a statistically significant relationship between best overall response and the ratio of CD8+:CD8+ TILs was observed (P=0.0186). In summary, there was an observed difference in the number of mice exhibiting a partial/complete regression in tumor volume for the combination arm 74% (36% CR) versus the antibody monotherapy arm 44% (7% CR). A statistically significant relationship (p=0.0186) was observed between the best overall tumor response and the ratio of CD8+:CD4+ TILs. IHC combined with image analysis can be utilized to evaluate therapeutic response on TILs within the context of their tumor microenvironment. Citation Format: Lorcan Sherry, Mark Anderson, John Waller, Adam Sardar, Victoria Flores, Daniel Hynes. Evaluation of ETS2101 and its combination with anti-mCTLA4 monoclonal antibody on CD4 and CD8 TILs in a subcutaneous colorectal cancer model of CT-26 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 163. doi:10.1158/1538-7445.AM2017-163

Human Cannabinoid Receptor 2 Ligand-Interaction Motif: Transmembrane Helix 2 Cysteine, C2.59(89), as Determinant of Classical Cannabinoid Agonist Activity and Binding Pose.

Cannabinoid receptor 2 (CB2R)-dependent signaling is implicated in neuronal physiology and immune surveillance by brain microglia. Selective CB2R agonists hold therapeutic promise for inflammatory and other neurological disorders. Information on human CB2R (hCB2R) ligand-binding and functional domains is needed to inform the rational design and optimization of candidate druglike hCB2R agonists. Prior demonstration that hCB2R transmembrane helix 2 (TMH2) cysteine C2.59(89) reacts with small-molecule methanethiosulfonates showed that this cysteine residue is accessible to sulfhydryl derivatization reagents. We now report the design and application of two novel, pharmacologically active, high-affinity molecular probes, AM4073 and AM4099, as chemical reporters to interrogate directly the interaction of classical cannabinoid agonists with hCB2R cysteine residues. AM4073 has one electrophilic isothiocyanate (NCS) functionality at the C9 position of its cyclohexenyl C-ring, whereas AM4099 has NCS groups at that position and at the terminus of its aromatic A-ring C3 side chain. Pretreatment of wild-type hCB2R with either probe reduced subsequent [3H]CP55,940 specific binding by ∼60%. Conservative serine substitution of any hCB2R TMH cysteine residue except C2.59(89) did not affect the reduction of [3H]CP55,940 specific binding by either probe, suggesting that AM4073 and AM4099 interact irreversibly with this TMH2 cysteine. In contrast, AM841, an exceptionally potent hCB2R megagonist and direct AM4073/4099 congener bearing a single electrophilic NCS group at the terminus of its C3 side chain, had been demonstrated to bind covalently to TMH6 cysteine C6.47(257) and not C2.59(89). Molecular modeling indicates that the AM4073-hCB2R* interaction at C2.59(89) orients this classical cannabinoid away from TMH6 and toward the TMH2-TMH3 interface in the receptor's hydrophobic binding pocket, whereas the AM841-hCB2R* interaction at C6.47(257) favors agonist orientation toward TMH6/7. These data constitute initial evidence that TMH2 cysteine C2.59(89) is a component of the hCB2R binding pocket for classical cannabinoids. The results further demonstrate how interactions between classical cannabinoids and specific amino acids within the hCB2R* ligand-binding domain act as determinants of agonist pharmacological properties and the architecture of the agonist-hCB2R* conformational ensemble, allowing the receptor to adopt distinct activity states, such that interaction of classical cannabinoids with TMH6 cysteine C6.47(257) favors a binding pose more advantageous for agonist potency than does their interaction with TMH2 cysteine C2.59(89).

Kannabinoide berriak: segurtasun eta eraginkortasun terapeutikoaren bila

Cannabis sativa landarean kannabidiola, kannabigerola edo Δ9-tetrahidrokannabibarina bezalako kannabinoide ez-psikoaktiboak aurki daitezke. Oro har, kannabinoide horiek kannabinoideen hartzaileekiko (CB) afinitate txikia dutenez, ez daukate kannabinoide klasikoek (bereziki Δ9-THCak) duten aktibitate psikoaktiboa. Gizakietan eta animalietan kannabinoide ez-psikoaktiboekin egindako lehenengo ikerketek horien propietate terapeutikoak frogatu dituzte. Efektu horiek azaltzeko itu eta mekanismo desberdinak proposatu dira, besteak beste, hartzaile serotonergikoen aktibazioa edota entzima desberdinen modulazioa.

Regulation of divalent metal transporter-1 by serine phosphorylation.

Divalent metal transporter-1 (DMT1) mediates dietary iron uptake across the intestinal mucosa and facilitates peripheral delivery of iron released by transferrin in the endosome. Here, we report that classical cannabinoids (Δ9-tetrahydrocannabinol, Δ9-THC), nonclassical cannabinoids (CP 55,940), aminoalkylindoles (WIN 55,212-2) and endocannabinoids (anandamide) reduce 55Fe and 54Mn uptake by HEK293T(DMT1) cells stably expressing the transporter. siRNA knockdown of cannabinoid receptor type 2 (CB2) abrogated inhibition. CB2 is a G-protein (GTP-binding protein)-coupled receptor that negatively regulates signal transduction cascades involving serine/threonine kinases. Immunoprecipitation experiments showed that DMT1 is serine-phosphorylated under basal conditions, but that treatment with Δ9-THC reduced phosphorylation. Site-directed mutation of predicted DMT1 phosphosites further showed that substitution of serine with alanine at N-terminal position 43 (S43A) abolished basal phosphorylation. Concordantly, both the rate and extent of 55Fe uptake in cells expressing DMT1(S43A) was reduced compared with those expressing wild-type DMT1. Among kinase inhibitors that affected DMT1-mediated iron uptake, staurosporine also reduced DMT1 phosphorylation confirming a role for serine phosphorylation in iron transport regulation. These combined data indicate that phosphorylation at serine 43 of DMT1 promotes transport activity, whereas dephosphorylation is associated with loss of iron uptake. Since anti-inflammatory actions mediated through CB2 would be associated with reduced DMT1 phosphorylation, we postulate that this pathway provides a means to reduce oxidative stress by limiting iron uptake.

Functional Selectivity of CB2 Cannabinoid Receptor Ligands at a Canonical and Noncanonical Pathway.

The CB2 cannabinoid receptor (CB2) remains a tantalizing, but unrealized therapeutic target. CB2 receptor ligands belong to varied structural classes and display extreme functional selectivity. Here, we have screened diverse CB2 receptor ligands at canonical (inhibition of adenylyl cyclase) and noncanonical (arrestin recruitment) pathways. The nonclassic cannabinoid (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55940) was the most potent agonist for both pathways, while the classic cannabinoid ligand (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran JWH133) was the most efficacious agonist among all the ligands profiled in cyclase assays. In the cyclase assay, other classic cannabinoids showed little [(-)-trans-Δ(9)-tetrahydrocannabinol and (-)-(6aR,7,10,10aR)-tetrahydro-6,6,9-trimethyl-3-(1-methyl-1-phenylethyl)-6H-dibenzo[b,d]pyran-1-ol] (KM233) to no efficacy [(6aR,10aR)-1-methoxy-6,6,9-trimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromene(L759633) and (6aR,10aR)-3-(1,1-dimethylheptyl)-6a,7,8,9,10,10a-hexahydro-1-methoxy-6,6-dimethyl-9-methylene-6H-dibenzo[b,d]pyran]L759656. Most aminoalkylindoles, including [(3R)-​2,​3-​dihydro-​5-​methyl-​3-​(4-​morpholinylmethyl)pyrrolo[1,​2,​3-​de]-​1,​4-​benzoxazin-​6-​yl]-​1-​naphthalenyl-​methanone,​ monomethanesulfonate (WIN55212-2), were moderate efficacy agonists. The cannabilactone 3-(1,1-dimethyl-heptyl)-1-hydroxy-9-methoxy-benzo(c)chromen-6-one (AM1710) was equiefficacious to CP55940 to inhibit adenylyl cyclase, albeit with lower potency. In the arrestin recruitment assays, all classic cannabinoid ligands failed to recruit arrestins, indicating a bias toward G-protein coupling for this class of compound. All aminoalkylindoles tested, except for WIN55212-2 and (1-​pentyl-​1H-​indol-​3-​yl)(2,​2,​3,​3-​tetramethylcyclopropyl)-​methanone (UR144), failed to recruit arrestin. WIN55212-2 was a low efficacy agonist for arrestin recruitment, while UR144 was arrestin biased with no significant inhibition of cyclase. Endocannabinoids were G-protein biased with no arrestin recruitment. The diarylpyrazole antagonist 5-​(4-​chloro-​3-​methylphenyl)-​1-​[(4-​methylphenyl)methyl]-​N-​[(1S,​2S,​4R)-​1,​3,​3-​trimethylbicyclo[2.2.1]hept-​2-​yl]-​1H-​pyrazole-​3-​carboxamide (SR144258) was an inverse agonist in cyclase and arrestin recruitment assays while the aminoalkylindole 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl](4-methoxyphenyl)methanone (AM630) and carboxamide N-(1,3-benzodioxol-5-ylmethyl)-1,2-dihydro-7-methoxy-2-oxo-8-(pentyloxy)-3-quinolinecarboxamide (JTE907) were inverse agonists in cyclase but low efficacy agonists in arrestin recruitment assays. Thus, CB2 receptor ligands display strong and varied functional selectivity at both pathways. Therefore, extreme care must be exercised when using these compounds to infer the role of CB2 receptors in vivo.

Dronabinol for chemotherapy-induced nausea and vomiting unresponsive to antiemetics.

Chemotherapy-induced nausea and vomiting (CINV) is one of the most common symptoms feared by patients, but may be prevented or lessened with appropriate medications. Several antiemetic options exist to manage CINV. Corticosteroids, serotonin receptor antagonists, and neurokinin receptor antagonists are the classes most commonly used in the prevention of CINV. There are many alternative drug classes utilized for the prevention and management of CINV such as antihistamines, benzodiazepines, anticonvulsants, cannabinoids, and dopamine receptor antagonists. Medications belonging to these classes generally have lower efficacy and are associated with more adverse effects. They are also not as well studied compared to the aforementioned agents. This review will focus on dronabinol, a member of the cannabinoid class, and its role in CINV. Cannabis sativa L. (also known as marijuana) contains naturally occurring delta-9-tetrahydrocannibinol (delta-9-THC). The synthetic version of delta-9-THC is the active ingredient in dronabinol that makes dronabinol an orally active cannabinoid. Evidence for clinical efficacy of dronabinol will be analyzed in this review as monotherapy, in combination with ondansetron, and in combination with prochlorperazine.

How Present Synthetic Cannabinoids Can Help Predict Symptoms in the Future

New synthetic cannabinoids appear regularly in the illicit drug market, often in response to legal restrictions. These compounds are characterized by increasing binding affinities (Ki) to CB1 and CB2 receptors. Increasing affinity to CB receptors can occur by substitution of a halogen on the terminal position of the pentyl chain of the classical synthetic cannabinoids. Fluorination of the aliphatic side chain of established cannabinoid agonists is a popular pathway of modifying existing active drugs and synthesizing novel drugs to increase potency. Biological impacts of these compounds that have been reported include seizures, body temperature losses that may lead to cardiac distress, as well as cases reporting delirium and severe neural incapacities. These symptoms have been reported in case reports with evidence that like their cannabinoid counterparts, these compounds distribute post-mortem into a variety of tissues, especially adipose tissue. Researchers have detected and identified certain synthetic cannabinoid compounds on botanicals using a variety of separation and detection systems such as GC-FID and GC-MS, as well as LC-MS/MS and NMR. Researchers have utilized a variety of chromatographic methods including LC MS/MS, to identify the parent compounds, their metabolites and structural analogs in biological samples such as urine and blood. The aim of this review is to spotlight the newer generations of synthetic cannabinoids with a more powerful affinity towards the cannabinoid receptors to continue to help researchers and clinicians further predict symptoms as well as future compounds and treatments.

The Structure-Function Relationships of Classical Cannabinoids: CB1/CB2 Modulation.

The cannabinoids are members of a deceptively simple class of terpenophenolic secondary metabolites isolated from Cannabis sativa highlighted by (−)-Δ9-tetrahydrocannabinol (THC), eliciting distinct pharmacological effects mediated largely by cannabinoid receptor (CB1 or CB2) signaling. Since the initial discovery of THC and related cannabinoids, synthetic and semisynthetic classical cannabinoid analogs have been evaluated to help define receptor binding modes and structure–CB1/CB2 functional activity relationships. This perspective will examine the classical cannabinoids, with particular emphasis on the structure–activity relationship of five regions: C3 side chain, phenolic hydroxyl, aromatic A-ring, pyran B-ring, and cyclohexenyl C-ring. Cumulative structure–activity relationship studies to date have helped define the critical structural elements required for potency and selectivity toward CB1 and CB2 and, more importantly, ushered the discovery and development of contemporary nonclassical cannabinoid modulators with enhanced physicochemical and pharmacological profiles.

Structure–activity relationships of synthetic cannabinoid designer drug RCS-4 and its regioisomers and C4 homologues

RCS-4 [(4-methoxyphenyl)-1-yl-(1-pentyl-1H-indol-3-yl)methanone] represents the first of several N-alkyl-3-(methoxybenzoyl)indoles identified by forensic scientists as synthetic cannabinoid (SC) designer drugs. Despite the detection of RCS-4 and several analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4, and RCS-4-C4) in products intended for human consumption, relatively little is known about this class of cannabinoids. The synthesis of all regioisomers of RCS-4 and their C4 homologues is described. This study also systematically explored the structure–activity relationships of this class of SCs at human CB1 and CB2 receptors using an in vitro fluorometric imaging plate reader membrane potential assay. All compounds demonstrated agonist activity at CB1 (EC50 = 54–574 nM) and CB2 (EC50 = 4.5–46 nM) receptors, with the C4 homologues showing a preference for CB2 receptors over CB1 receptors (31–42 times). Since most of the analogues (RCS-2, RCS-3, RCS-2-C4, RCS-3-C4 and RCS-4-C4) are not subject to regulation in much of the world, despite their activities towards CB1 and CB2 receptors, there is a possibility that these analogues will emerge on the black market.

Metabolism of classical cannabinoids and the synthetic cannabinoid JWH-018.

Although the putative pharmacological targets of synthetic cannabinoids (SCBs) abused in "K2" and "Spice" are similar to Δ(9) -tetrahydrocannabinol (Δ(9) -THC), it remains unclear why SCB toxicity is similar yet different from marijuana. There are obvious potency and efficacy differences, but also important metabolic differences that help explain the unique adverse reactions associated with SCBs. This brief review discusses the limited research on the metabolism of the SCB JWH-018 and contrasts that with the metabolism of Δ(9) -THC.

3'-functionalized adamantyl cannabinoid receptor probes.

The aliphatic side chain plays a pivotal role in determining the cannabinergic potency of tricyclic classical cannabinoids, and we have previously shown that this chain could be substituted successfully by adamantyl or other polycyclic groups. In an effort to explore the pharmacophoric features of these conformationally fixed groups, we have synthesized a series of analogues in which the C3 position is substituted directly with an adamantyl group bearing functionality at one of the tertiary carbon atoms. These substituents included the electrophilic isothiocyanate and photoactivatable azido groups, both of which are capable of covalent attachment with the target protein. Our results show that substitution at the 3'-adamantyl position can lead to ligands with improved affinities and CB1/CB2 selectivities. Our work has also led to the development of two successful covalent probes with high affinities for both cannabinoid receptors, namely, the electrophilic isothiocyanate AM994 and the photoactivatable aliphatic azido AM993 analogues.

A structure–reactivity relationship driven approach to the identification of a color test protocol for the presumptive indication of synthetic cannabimimetic drugs of abuse

The number of analyses of synthetic cannabimimetic drugs of abuse by forensic laboratories in the United States grew rapidly from 2010 to 2012 and then declined somewhat in 2013. In 2010, according to the National Forensic Laboratory Information System (NFLIS), 3,287 reports by federal, state and local forensic laboratories were identified as containing synthetic cannabinoids. In 2011 and 2012, the numbers increased to 23,693 and 42,503, respectively. 27,119 reports were identified in 2013. Several commonly encountered structural sub-classes of these synthetic designer drugs, namely the naphthoylindoles, benzoylindoles, phenylacetylindoles, and cyclopropoylindoles contain a ketone functional group. The Duquenois-Levine color test for the presumptive identification of classical cannabinoids such as Δ9-tetrahydrocannabinol is negative for the synthetic cannabimimetics. The van Urk color test for the presumptive identification of indole containing drugs of abuse is also negative for these compounds. The use of 2,4-dinitrophenylhydrazine as an alternative color test reagent (targeting the keto moiety rather than the indole) for presumptive identification of these classes of drugs was investigated.

Cross-substitution of Δ9-tetrahydrocannabinol and JWH-018 in drug discrimination in rats

Synthetic indole-derived cannabinoids, originally developed to probe cannabinoid CB1 and CB2 receptors, have become widely abused for their marijuana-like intoxicating properties. The present study examined the effects of indole-derived cannabinoids in rats trained to discriminate Δ9-tetrahydrocannabinol (Δ9-THC) from vehicle. In addition, the effects of Δ9-THC in rats trained to discriminate JWH-018 from vehicle were assessed. Adult male Sprague–Dawley rats were trained to discriminate 3mg/kg Δ9-THC or 0.3mg/kg JWH-018 from vehicle. JWH-018, JWH-073, and JWH-210 fully substituted in Δ9-THC-trained rats and Δ9-THC substituted in JWH-018-trained rats. In contrast, JWH-320, an indole-derived cannabinoid without affinity for CB1 receptors, failed to substitute for Δ9-THC. Pre-treatment with 1mg/kg rimonabant significantly reduced responding on the JWH-018-associated lever in JWH-018-trained rats. These results support the conclusion that the interoceptive effects of Δ9-THC and synthetic indole-derived cannabinoids show a large degree of overlap, which is predictive of their use for their marijuana-like intoxicating properties. Characterization of the extent of pharmacological differences among structural classes of cannabinoids, and determination of their mechanisms remain important goals.

Selective inhibition of FAAH produces antidiarrheal and antinociceptive effect mediated by endocannabinoids and cannabinoid‐like fatty acid amides

The endogenous cannabinoid system (ECS) plays a crucial role in multiple physiological processes in the central nervous system and in the periphery. The discovery that selective cannabinoid (CB) receptor agonists exert a potent inhibitory action on gastrointestinal (GI) motility and pain has placed the ECS in the center of attention as a possible target for the treatment of functional GI diseases. However, side effects of CB agonists prompted the search for novel therapeutic targets. Here, the effect of PF-3845, a potent and selective fatty acid amide hydrolase (FAAH) inhibitor in the GI tract was investigated. The effect of PF-3845 on GI motility was characterized in vitro and in vivo, using mouse models that mimic physiological and pathophysiological conditions. The antinociceptive action of PF-3845 was evaluated on the basis of behavioral pain models. Endocannabinoid degradation product levels after inhibition of FAAH were quantified using HPLC-MS/MS. PF-3845 significantly inhibited mouse colonic motility in vitro and in vivo. Selective inhibition of FAAH reversed hypermotility and reduced pain in mouse models mimicking functional GI disorders. The effects of PF-3845 were mediated by endogenous CBs and non-CB lipophilic compounds via classical (CB1) and atypical CB receptors. These data expand our understanding of the ECS function and provide a novel framework for the development of future potential treatments of functional GI disorders.

Legal controls on cannabimimetics: An international dilemma?

Since early 2009, over 80 illicitly produced synthetic cannabinoid receptor agonists have been notified to the European Monitoring Centre for Drugs and Drug Addiction (EMCDDA). Yet more have been reported in other countries or offered for sale on websites. These cannabinoids typically act as agonists at CB1 receptors, and mimic the effects of Δ(9)-tetrahydrocannabinol (THC), the principal psychoactive constituent of Cannabis L. As with other 'new psychoactive substances', they have shown the limitations of current drug control procedures. First, the regular appearance of new compounds makes specific listing impractical and overwhelms any attempt to create risk assessments on a substance-by-substance basis. Secondly, the lack of human pharmacological and toxicological data hinders any objective attempt to show that synthetic cannabinoids are harmful. The UK has had a long experience of using generic legislation to control groups of compounds. However, cannabimimetic activity arises in a large number of distinct chemical families, and it is clear that generic control can no longer cope with this diversity whilst remaining intelligible to non-chemists. Analogue control presents further difficulties, and does not appear to offer an acceptable solution. For these reasons, legislatures in many countries are creating novel forms of regulation separate from domestic drug laws. The generic definition of classical cannabinoids, introduced into the UK Misuse of Drugs Act in 1971, also shows signs of weakness. Thus the growing interest in the clinical potential of tetrahydrocannabivarin (THCV) is inhibited, at least in the UK, by its unintended status as a Schedule 1 substance.

Structural analogs of pyrazole and sulfonamide cannabinoids: Effects on acute food intake in mice

Obesity contributes to a multitude of serious health problems. Given the demonstrated role of the endogenous cannabinoid system in appetite regulation, the purpose of the present study was to evaluate structural analogs of two cannabinoids, rimonabant (cannabinoid CB1 receptor antagonist) and O-2050 (sulfonamide analog of Δ8-tetrahydrocannabinol), that showed appetite suppressant effects in previous studies. Structure–activity relationships of these two lead compounds were examined in several assays, including cannabinoid CB1 and CB2 receptor binding, food intake, and an in vivo test battery (locomotor activity, antinociception, ring immobility, and body temperature) in mice. Rimonabant and O-2050 reliably decreased feeding in mice; however, their analogs decreased feeding only at higher doses, even though some compounds had quite good cannabinoid CB1 binding affinity. Results of the in vivo test battery were inconsistent, with some of the compounds producing effects characteristic of cannabinoid agonists while other compounds were inactive or were antagonists against an active dose of Δ9-tetrahydrocannabinol. These results demonstrate that reduction of food intake is not a characteristic effect of pyrazole and sulfonamide cannabinoid analogs with favorable cannabinoid CB1 binding affinity, suggesting that development of these classes of cannabinoids for the treatment of obesity will require evaluation of their effects in a broad spectrum of pharmacological assays.

Conference Report (42nd Annual Meeting of the Italian Society of Neurology, 22-25 October 2011, Turin, Italy)

Conference Report (42nd Annual Meeting of the Italian Society of Neurology, 22-25 October 2011, Turin, Italy)

Chemiluminescence detection of cannabinoids and related compounds with acidic potassium permanganate

This is the first report of chemiluminescence from the reaction of cannabinoids with acidic potassium permanganate, which we have applied to the high performance liquid chromatography (HPLC) determination of cannabidiol (CBD) in industrial-grade hemp. The intensities of the light-producing reactions with two commercially available cannabinoid standards were compared to that of seven model phenolic analytes. Resorcinol, representing the parent phenolic moiety of the cannabinoid class, was shown to react with the permanganate reagents in a manner more similar to phenol than to its hydroxyphenol positional isomers, pyrocatechol and hydroquinone. Alkyl substituents on the phenolic ring, however, have a considerable impact on emission intensity that is dependent upon the position of the groups and the composition of the permanganate reagent. This analytical approach has potential for the determination of other cannabinoids including Δ(9) -tetrahydrocannabinol in drug-grade cannabis.

Tentative identification of phase I metabolites of HU-210, a classical synthetic cannabinoid, by LC–MS/MS

(6aR,10aR)-9-(Hydroxymethyl)-6,6-dimethyl-3-(2-methyloctan-2-yl)-6a,7,10,10a-tetrahydrobenzo[c]chromen-1-ol (HU-210) is a synthetic cannabinoid, with a classical cannabinoid structure similar to Δ(9)-tetrahydrocannabinol (Δ(9)-THC). In this study, the in vitro metabolism of HU-210 was investigated in human liver microsomes to characterize associated phase I metabolites. HU-210 was incubated with human liver microsomes, and the reaction mixture was analyzed using LC-MS/MS. HU-210 was metabolized in human liver microsomes, yielding about 24 metabolites. These metabolites were structurally characterized on the basis of accurate mass analyses and MS/MS fragmentation patterns. The major metabolic route for HU-210 was oxygenation. Metabolites M1-M7 were identified as mono-oxygenated metabolites; M8-M15, mono-hydroxylated metabolites; M16-M20, di-oxygenated metabolites; and M21-M24, di-hydroxylated metabolites. These results provide evidence for in vivo HU-210 metabolism, and they may be applied to the analysis of HU-210 and its relevant metabolites in biological samples.

Update on the Role of Cannabinoid Receptors after Ischemic Stroke

Cannabinoids are considered as key mediators in the pathophysiology of inflammatory diseases, including atherosclerosis. In particular, they have been shown to reduce the ischemic injury after acute cardiovascular events, such as acute myocardial infarction and ischemic stroke. These protective and anti-inflammatory properties on peripheral tissues and circulating inflammatory have been demonstrated to involve their binding with both selective cannabinoid type 1 (CB1) and type 2 (CB2) transmembrane receptors. On the other hands, the recent discoveries of novel different classes of cannabinoids and receptors have increased the complexity of this system in atherosclerosis. Although only preliminary data have been reported on the activities of novel cannabinoid receptors, several studies have already investigated the role of CB1 and CB2 receptors in ischemic stroke. While CB1 receptor activation has been shown to directly reduce atherosclerotic plaque inflammation, controversial data have been shown on neurotransmission and neuroprotection after stroke. Given its potent anti-inflammatory activities on circulating leukocytes, the CB2 activation has been proven to produce protective effects against acute poststroke inflammation. In this paper, we will update evidence on different cannabinoid-triggered avenues to reduce inflammation and neuronal injury in acute ischemic stroke.