Evaluation of cannabimimetic effects of selected minor cannabinoids and Terpenoids in mice.

BACKGROUND: The cannabis plant contains several cannabinoids, and many terpenoids that give cannabis its distinctive flavoring and aroma. Δ9-Tetrahydrocannabinol (Δ9-THC) is the plant's primary psychoactive constituent. Given the abuse liability of Δ9-THC, assessment of the psychoactive effects of minor cannabinoids and other plant constituents is important, especially for compounds that may be used medicinally. This study sought to evaluate select minor cannabinoids and terpenes for Δ9-THC-like psychoactivity in mouse Δ9-THC drug discrimination and determine their binding affinities at CB1 and CB2 receptors. METHODS: Δ9-THC, cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), cannabichromenevarin (CBCV), Δ8-tetrahydrocannabinol (Δ8-THC), (6aR,9R)-Δ10-tetrahydrocannabinol [(6aR,9R)-Δ10-THC], Δ9-tetrahydrocannabinol varin (THCV), ß-caryophyllene (BC), and ß-caryophyllene oxide (BCO) were examined. RESULTS: All minor cannabinoids showed measurable cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor binding, with CBC, CBCV, and CBD, showing the weakest CB1 receptor binding affinity. BC and BCO exhibited negligible affinity for both CB1 and CB2 receptors. In drug discrimination, only Δ8-THC fully substituted for Δ9-THC, while CBN and (6aR,9R)-Δ10-THC partially substituted for Δ9-THC. THCV and BCO did not alter the discriminative stimulus effects of Δ9-THC. CONCLUSION: In summary, only some of myriad cannabinoids and other chemicals found in the cannabis plant bind potently to the identified cannabinoid receptors. Further, only four of the compounds tested herein [Δ9-THC, Δ8-THC, (6aR,9R)-Δ10-THC, and CBN] produced Δ9-THC-like discriminative stimulus effects, suggesting they may possess cannabimimetic subjective effects. Given that the medicinal properties of phytocannabinoids and terpenoids are being investigated scientifically, delineation of their potential adverse effects, including their ability to produce Δ9-THC-like intoxication, is crucial.

In vitro and in vivo pharmacology of nine novel synthetic cannabinoid receptor agonists.

Synthetic cannabinoid receptor agonists (SCRAs) are novel psychoactive substances that bind to and activate CB1 receptors in the brain. The structural manipulations observed in newer SCRAs suggest that manufacturers have incorporated modern drug development techniques into their repertoire, often producing higher CB1 receptor affinity than Δ9-tetrahydrocannabinol (Δ9-THC). This study examined nine SCRAs recently detected by forensic surveillance, some of which caused fatalities: 5F-MDMB-PICA, FUB-144, 5F-MMB-PICA, MMB-4en-PICA, MMB-FUBICA, 5F-EDMB-PINACA, APP-BINACA, MDMB-4en-PINACA, and FUB-AKB48. Compounds were evaluated for CB1 and CB2 receptor binding affinity and functional activation and for their effects on body temperature, time course, and pharmacological equivalence with Δ9-THC in Δ9-THC drug discrimination in mice. All SCRAs bound to and activated CB1 and CB2 receptors with high affinity, with similar or greater affinity for CB2 than CB1 receptors and stimulated [35S]GTPγS binding in CB1 and CB2 expressing cell membranes. All compounds produced hypothermia, with shorter latency to peak effects for SCRAs than Δ9-THC. All SCRAs fully substituted for Δ9-THC in drug discrimination at one or more doses. Rank order potency in producing in vivo effects mostly aligned with rank order CB1 receptor affinities. Potencies for Δ9-THC-like discriminative stimulus effects were similar across sex except Δ9-THC was more potent in females and 5F-MMB-PICA was more potent in males. In summary, 5F-EMDB-PINACA, 5F-MDMB-PICA, MDMB-4en-PINACA, FUB-144, FUB-AKB48, 5F-MMB-PICA, MMB-4en-PICA, and MMB-FUBICA are potent and efficacious SCRAs with pharmacology like that of past SCRAs that have been abused in humans. In contrast, APP-BINACA was efficacious, but had lower potency than most past SCRAs.

Neuropeptide B/W receptor 1 peptidomimetic agonists: Structure-activity relationships and plasma stability.

Neuropeptides B and W (NPB and NPW) are endogenous ligands of the Neuropeptide B/W Receptor 1 (NPBWR1) which has been implicated in a wide range of functions including regulation of pain and energy homeostasis. There is currently little information on the structure-activity relationships (SAR) of these two neuropeptides. In a quest to develop stable and potent NPBWR1 peptidomimetic agonists, we performed systematic SAR by truncation, Alanine/Glycine and d-amino acid scans, and replacement with unnatural amino acids. Evaluation in the NPBWR1 calcium assay revealed that the C-terminal GRAAGLL and N-terminal WYK regions constitute the two-epitope pharmacophore for NPBWR1 agonism. Replacement of the N-terminal Trp with its desaminoTrp residue resulted in compound 30 which exhibited nanomolar potency comparable to the endogenous NPB at NPBWR1 (Calcium assay: EC50 = 8 nM vs. 13 nM, cAMP assay: 2.7 nM vs 3.5 nM) and enhanced metabolic stability against rat plasma (39.1 min vs. 11.9 min).

Exploring determinants of agonist efficacy at the CB1 cannabinoid receptor: Analogues of the synthetic cannabinoid receptor agonist EG-018.

Neutral antagonists of GPCRs remain relatively rare-indeed, a large majority of GPCR antagonists are actually inverse agonists. The synthetic cannabinoid receptor agonist (SCRA) EG-018 was recently reported as a low efficacy cannabinoid receptor agonist. Here we report a comparative characterization of EG-018 and 13 analogues along with extant putative neutral antagonists of CB1 . In HEK cells stably expressing human CB1 , assays for inhibition of cAMP were performed by real-time BRET biosensor (CAMYEL), G protein cycling was quantified by [35 S]GTPγS binding, and stimulation of pERK was characterized by AlphaLISA (PerkinElmer). Signaling outcomes for the EG-018 analogues were highly variable, ranging from moderate efficacy agonism with high potency, to marginal agonism at lower potency. As predicted by differing pathway sensitivities to differences in ligand efficacy, most EG-018-based compounds were completely inactive in pERK alone. The lowest efficacy analogue in cAMP assays, 157, had utility in antagonism assay paradigms. Developing neutral antagonists of the CB1 receptor has been a long-standing research goal, and such compounds would have utility both as research tools and in therapeutics. Although these results emphasize again the importance of system factors in determining signaling outcomes, some compounds characterized in this study appear among the lowest efficacy agonists described to date and therefore suggest that development of neutral antagonists is an achievable goal for CB1 .

Development of 3-(4-Chlorophenyl)-1-(phenethyl)urea Analogues as Allosteric Modulators of the Cannabinoid Type-1 Receptor: RTICBM-189 is Brain Penetrant and Attenuates Reinstatement of Cocaine-Seeking Behavior.

We have shown that CB1 receptor negative allosteric modulators (NAMs) attenuated the reinstatement of cocaine-seeking behaviors in rats. In an effort to further define the structure-activity relationships and assess the druglike properties of the 3-(4-chlorophenyl)-1-(phenethyl)urea-based CB1 NAMs that we recently reported, we introduced substituents of different electronic properties and sizes to the phenethyl group and evaluated their potency in CB1 calcium mobilization, cAMP, and GTPγS assays. We found that 3-position substitutions such as Cl, F, and Me afforded enhanced CB1 potency, whereas 4-position analogues were generally less potent. The 3-chloro analogue (31, RTICBM-189) showed no activity at >50 protein targets and excellent brain permeation but relatively low metabolic stability in rat liver microsomes. Pharmacokinetic studies in rats confirmed the excellent brain exposure of 31 with a brain/plasma ratio Kp of 2.0. Importantly, intraperitoneal administration of 31 significantly and selectively attenuated the reinstatement of the cocaine-seeking behavior in rats without affecting locomotion.

Rational design of cannabinoid type-1 receptor allosteric modulators: Org27569 and PSNCBAM-1 hybrids.

Allosteric modulation offers an alternate approach to target the cannabinoid type-1 receptor (CB1) for therapeutic benefits. Examination of the two widely studied prototypic CB1 negative allosteric modulators (NAMs) Org27569 and PSNCBAM-1 revealed structural resemblance and similar structure-activity relationships (SARs). In silico docking and dynamics simulation studies using the crystal structure of CB1 co-bound with CP55,940 and Org27569 suggested that Org27569 and PSNCBAM-1 occupied the same binding pocket and several common interactions were present in both series with the CB1 receptor. A new scaffold was therefore designed by merging the key structural features from the two series and the hybrids retained these binding features in the in silico docking studies. In addition, one such hybrid displayed similar functions to Org27569 in dynamic simulations by preserving a key R2143.50-D3386.30 salt bridge and maintaining an antagonist-like Helix3-Helix6 interhelical distance. Based on these results, a series of hybrids were synthesized and assessed in calcium mobilization, [35S]GTPγS binding and cAMP assays. Several compounds displayed comparable potencies to Org27569 and PSNCBAM-1 in these assays. This work offers new insight of the SAR requirement at the allosteric site of the CB1 receptor and provides a new scaffold that can be optimized for the development of future CB1 allosteric modulators.

Sex, species and age: Effects of rodent demographics on the pharmacology of ∆9-tetrahydrocanabinol.

Cannabis edibles are becoming more common in an increasingly diverse population of users, and the impact of first pass metabolism on cannabis's pharmacological profile across age and sex is not well understood. The present study examined the impact of age, sex and rodent species on the effects of intraperitoneal (i.p.) delta-9-tetrahydrocannabinol (THC) and its primary psychoactive metabolite, 11-OH-THC, in rodent models of psychoactivity and molecular assays of cannabinoid receptor type-1 (CB1) pharmacology. Like oral THC, i.p. THC also undergoes first pass metabolism. In both species and sexes, 11-OH-THC exhibited marginally higher affinity (~1.5 fold) than THC and both served as partial agonists in [35S]GTPγS binding with equivalent potency; 11-OH-THC exhibited slightly greater efficacy in rat brain tissue. In ICR mice, 11-OH-THC exhibited greater potency than THC in assays of catalepsy (7- to 15-fold) and hypothermia (7- to 31-fold). Further, 11-OH-THC was more potent in THC drug discrimination (7- to 9-fold) in C57Bl/6 J mice, with THC-like discriminative stimulus effects being CB1-, but not CB2-, mediated. THC's discriminative stimulus also was stable across age in mice, as its potency did not change over the course of the experiment (~17 months). While sex differences in THC's effects were not revealed in mice, THC was significantly more potent in females Sprague-Dawley rats than in males trained to discriminate THC from vehicle. This study demonstrates a cross-species in the psychoactive effects of i.p. THC across sex that may be related to differential metabolism of THC into its psychoactive metabolite 11-OH-THC, suggesting that species is a crucial design consideration in the preclinical study of phytocannabinoids.

In vitro and in vivo pharmacological evaluation of the synthetic cannabinoid receptor agonist EG-018.

Synthetic cannabinoid receptor agonists (SCRAs) possess high abuse liability and complex toxicological profiles, making them serious threats to public health. EG-018 is a SCRA that has been detected in both illicit products and human samples, but it has received little attention to date. The current studies investigated EG-018 at human CB1 and CB2 receptors expressed in HEK293 cells in [3H]CP55,940 competition binding, [35S]GTPγS binding and forskolin-stimulated cAMP production. EG-018 was also tested in vivo for its ability to produce cannabimimetic and abuse-related effects in the cannabinoid tetrad and THC drug discrimination, respectively. EG-018 exhibited high affinity at CB1 (21 nM) and at CB2 (7 nM), but in contrast to typical SCRAs, behaved as a weak partial agonist in [35S]GTPγS binding, exhibiting lower efficacy but greater potency, than that of THC at CB1 and similar potency and efficacy at CB2. EG-018 inhibited forskolin-stimulated cAMP with similar efficacy but lower potency, compared to THC, which was likely due to high receptor density facilitating saturation of this signaling pathway. In mice, EG-018 (100 mg/kg, 30 min) administered intraperitoneally (i.p.) did not produce effects in the tetrad or drug discrimination nor did it shift THC's ED50 value in drug discrimination when administered before THC, suggesting EG-018 has negligible occupancy of brain CB1 receptors following i.p. administration. Following intravenous (i.v.) administration, EG-018 (56 mg/kg) produced hypomotility, catalepsy, and hypothermia, but only catalepsy was blocked by the selective CB1 antagonist rimonabant (3 mg/kg, i.v.). Additional studies of EG-018 and its structural analogues could provide further insight into how cannabinoids exert efficacy through the cannabinoid receptors.

Synthesis and Pharmacological Evaluation of 1-Phenyl-3-Thiophenylurea Derivatives as Cannabinoid Type-1 Receptor Allosteric Modulators.

We previously reported diarylurea derivatives as cannabinoid type-1 receptor (CB1) allosteric modulators, which were effective in attenuating cocaine-seeking behavior. Herein, we extended the structure-activity relationships of PSNCBAM-1 (2) at the central phenyl ring directly connected to the urea moiety. Replacement with a thiophene ring led to 11 with improved or comparable potencies in calcium mobilization, [35S]GTPγS binding, and cAMP assays, whereas substitution with nonaromatic rings led to significant attenuation of the modulatory activity. These compounds had no inverse agonism in [35S]GTPγS binding, a characteristic that is often thought to contribute to adverse psychiatric effects. While 11 had good metabolic stability in rat liver microsomes, it showed modest solubility and blood-brain barrier permeability. Compound 11 showed an insignificant attenuation of cocaine seeking behavior in rats, most likely due to its limited CNS penetration, suggesting that pharmacokinetics and distribution play a role in translating the in vitro efficacy to in vivo behavior.

Toxic by design? Formation of thermal degradants and cyanide from carboxamide-type synthetic cannabinoids CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, NNEI, and MN-18 during exposure to high temperatures.

PURPOSE: The use of novel synthetic cannabinoids as intoxicants continues in spite of associated health risks. These compounds are typically smoked or vaporized, but many synthetic cannabinoids contain thermally labile chemical moieties. This study investigated the thermal stability six carboxamide-type synthetic cannabinoids (CUMYL-PICA, 5F-CUMYL-PICA, AMB-FUBINACA, MDMB-FUBINACA, NNEI, and MN-18) in order to characterise potential user exposure to thermolysis products. METHODS: Compounds were heated sequentially to 200, 400, 600 and 800 °C using a thermolysis probe, and the resultant thermolysis products were analysed via GC-MS. A secondary analysis quantified thermolytically generated cyanide via LC-MS/MS. RESULTS: All six synthetic cannabinoids underwent thermal degradation when heated above 400 °C, and released a variety of potentially toxic products, including toluene, naphthalene, and 1-naphthalamine. Compound-specific degradants were tentatively identified together with a general degradative pathway for carboxamide-type synthetic cannabinoids, which proceeds via indole- or indazole-amide formation and subsequent dehydration to an indole- or indazole-carbonitrile. This degradative pathway culminated in the thermolytic liberation of cyanide, in amounts up to 27 µg per mg of starting material. CONCLUSIONS: People who smoke carboxamide-type synthetic cannabinoids are likely to be exposed to range of potentially toxic thermal degradants, including cyanide. These degradants could have significant health impacts in human users.

Diarylureas Containing 5-Membered Heterocycles as CB1 Receptor Allosteric Modulators: Design, Synthesis, and Pharmacological Evaluation.

Allosteric modulators have attracted significant interest as an alternate strategy to modulate CB1 receptor signaling for therapeutic benefits that may avoid the adverse effects associated with orthosteric ligands. Here we extended our previous structure-activity relationship studies on the diarylurea-based CB1 negative allosteric modulators (NAMs) by introducing five-membered heterocycles to replace the 5-pyrrolidinylpyridinyl group in PSNCBAM-1 (1), one of the first generation CB1 allosteric modulators. Many of these compounds had comparable potency to 1 in blocking the CB1 agonist CP55,940 stimulated calcium mobilization and [35S]GTP-γ-S binding. Similar to 1, most compounds showed positive cooperativity by increasing [3H]CP55,940 binding, consistent with the positive allosteric modulator (PAM)-antagonist mechanism. Interestingly, these compounds exhibited differences in ability to increase specific binding of [3H]CP55,940 and decrease binding of the antagonist [3H]SR141716. In saturation binding studies, only increases in [3H]CP55,940 Bmax, but not Kd, were observed, suggesting that these compounds stabilize low affinity receptors into a high affinity state. Among the series, the 2-pyrrolyl analogue (13) exhibited greater potency than 1 in the [35S]GTP-γ-S binding assay and significantly enhanced the maximum binding level in the [3H]CP5,5940 binding assay, indicating greater CB1 receptor affinity and/or cooperativity.

Sex, THC, and hormones: Effects on density and sensitivity of CB1 cannabinoid receptors in rats.

BACKGROUND: The recent NIH mandate to consider sex as a biological variable in preclinical research has focused attention on delineation of sex differences in behavior. To investigate mechanisms underlying sex differences in Δ9-tetrahydrocannabinol (THC) effects, we examined the effects of sex and gonadal hormones on CB1 receptors in cerebellum, hippocampus, prefrontal cortex, and striatum. METHODS: Adult Sprague-Dawley rats underwent gonadectomy (GDX) or sham-GDX. Half of the GDX females and males received estradiol or testosterone replacement (GDX+H), respectively. All rats were injected with vehicle or 30 mg/kg THC twice daily for 1 week before brain collection. CP55,940-stimulated [35S]GTPγS and [3H]SR141716A saturation binding assays were performed. RESULTS: With exception of enhanced receptor activation in the hippocampi of female rats compared to males, vehicle-treated rats exhibited minimal sex differences in CB1 receptor densities or G-protein coupling. Repeated treatment with THC resulted in pronounced CB1 receptor desensitization and downregulation in both sexes in all brain regions with a greater magnitude of change in females. CONCLUSIONS: These results suggest that sex differences in the density and G-protein coupling of brain CB1 receptors may play a limited role in sex differences in acute THC effects not mediated by the hippocampus. In contrast, sex differences after repeated THC were common, with females (intact, GDX, and GDX+H) showing greater downregulation or desensitization in all four brain regions compared to the respective male groups. This result is consistent with a finding that women tend to progress to tolerance and dependence quicker than men after initiation of cannabis use.

Synthetic Cannabinoid Hydroxypentyl Metabolites Retain Efficacy at Human Cannabinoid Receptors.

Synthetic cannabinoids (SCs) are novel psychoactive substances that are easily acquired, widely abused as a substitute for cannabis, and associated with cardiotoxicity and seizures. Although the structural bases of these compounds are scaffolds with known affinity and efficacy at the human cannabinoid type-1 receptor (hCB1), upon ingestion or inhalation they can be metabolized to multiple chemical entities of unknown pharmacological activity. A large proportion of these metabolites are hydroxylated on the pentyl chain, a key substituent that determines receptor affinity and selectivity. Thus, the pharmacology of SC metabolites may be an important component in understanding the in vivo effects of SCs. We examined nine SCs (AB-PINACA, 5F-AB-PINACA, ADB/MDMB-PINACA, 5F-ADB, 5F-CUMYL-PINACA, AMB-PINACA, 5F-AMB, APINACA, and 5F-APINACA) and their hydroxypentyl (either 4-OH or 5-OH) metabolites in [3H]CP55,940 receptor binding and the [35S]GTPγS functional assay to determine the extent to which these metabolites retain activity at cannabinoid receptors. All of the SCs tested exhibited high affinity (<10 nM) and efficacy for hCB1 and hCB2 The majority of the hydroxypentyl metabolites retained full efficacy at hCB1 and hCB2, albeit with reduced affinity and potency, and exhibited greater binding selectivity for hCB2 These data suggest that phase I metabolites may be contributing to the in vivo pharmacology and toxicology of abused SCs. Considering this and previous reports demonstrating that metabolites retain efficacy at the hCB1 receptor, the full pharmacokinetic profiles of the parent compounds and their metabolites need to be considered in terms of the pharmacological effects and time course associated with these drugs.

Molecular and Behavioral Pharmacological Characterization of Abused Synthetic Cannabinoids MMB- and MDMB-FUBINACA, MN-18, NNEI, CUMYL-PICA, and 5-Fluoro-CUMYL-PICA.

Synthetic cannabinoids are a class of novel psychoactive substances that exhibit high affinity at the cannabinoid type-1 (CB1) receptor and produce effects similar to those of Δ-9-tetrahydrocannabinol (THC), the primary psychoactive constituent of cannabis. Illicit drug manufacturers are continually circumventing laws banning the sale of synthetic cannabinoids by synthesizing novel structures and doing so with little regard for the potential impact on pharmacological and toxicological effects. Synthetic cannabinoids produce a wide range of effects that include cardiotoxicity, seizure activity, and kidney damage, and they can cause death. Six synthetic cannabinoids, recently detected in illicit preparations, MMB-FUBINACA, MDMB-FUBINACA, CUMYL-PICA, 5F-CUMYL-PICA, NNEI, and MN-18 were assessed for: 1) receptor binding affinity at the human CB1 and human CB2 receptors, 2) function in [35S]GTPγS and cAMP signaling, and 3) THC-like effects in a mouse drug discrimination assay. All six synthetic cannabinoids exhibited high affinity for human cannabinoid receptors type-1 and type-2 and produced greater maximal effects than THC in [35S]GTPγS and cAMP signaling. Additionally, all six synthetic cannabinoids substituted for THC in drug discrimination, suggesting they probably possess subjective effects similar to those of cannabis. Notably, MDMB-FUBINACA, a methylated analog of MMB-FUBINACA, had higher affinity for CB1 than the parent, showing that minor structural modifications being introduced can have a large impact on the pharmacological properties of these drugs. This study demonstrates that novel structures being sold and used illicitly as substitutes for cannabis are retaining high affinity at the CB1 receptor, exhibiting greater efficacy than THC, and producing THC-like effects in models relevant to subjective effects in humans.

Kinetic and metabolic profiles of synthetic cannabinoids NNEI and MN-18.

In 2014 and 2015, synthetic cannabinoid receptor agonists NNEI (N-1-naphthalenyl-1-pentyl-1H-indole-3-carboxamide) and MN-18 (N-1-naphthalenyl-1-pentyl-1H-indazole-3-carboxamide) were detected in recreationally used and abused products in multiple countries, and were implicated in episodes of poisoning and toxicity. Despite this, the pharmacokinetic profiles of NNEI and MN-18 have not been characterized. In the present study NNEI and MN-18 were incubated in rat and human liver microsomes and hepatocytes, to estimate kinetic parameters and to identify potential metabolic pathways, respectively. These parameters and pathways were then examined in vivo, via analysis of blood and urine samples from catheterized male rats following intraperitoneal (3 mg/kg) administration of NNEI and MN-18. Both NNEI and MN-18 were rapidly cleared by rat and human liver microsomes, and underwent a range of oxidative transformations during incubation with rat and human hepatocytes. Several unique metabolites were identified for the forensic identification of NNEI and MN-18 intake. Interestingly, NNEI underwent a greater number of biotransformations (20 NNEI metabolites versus 10 MN-18 metabolites), yet parent MN-18 was eliminated at a faster rate than NNEI in vivo. Additionally, in vivo elimination was more rapid than in vitro estimates. These data highlight that even closely related synthetic cannabinoids can possess markedly distinct pharmacokinetic profiles, which can vary substantially between in vitro and in vivo models.

Novel Diarylurea Based Allosteric Modulators of the Cannabinoid CB1 Receptor: Evaluation of Importance of 6-Pyrrolidinylpyridinyl Substitution.

Allosteric modulators of the cannabinoid CB1 receptor have recently been reported as an alternative approach to modulate the CB1 receptor for therapeutic benefits. In this study, we report the design and synthesis of a series of diarylureas derived from PSNCBAM-1 (2). Similar to 2, these diarylureas dose-dependently inhibited CP55,940-induced intracellular calcium mobilization and [35S]GTP-γ-S binding while enhancing [3H]CP55,940 binding to the CB1 receptor. Structure-activity relationship studies revealed that the pyridinyl ring of 2 could be replaced by other aromatic rings and the pyrrolidinyl ring is not required for CB1 allosteric modulation. 34 (RTICBM-74) had similar potencies as 2 in all in vitro assays but showed significantly improved metabolic stability to rat liver microsomes. More importantly, 34 was more effective than 2 in attenuating the reinstatement of extinguished cocaine-seeking behavior in rats, demonstrating the potential of this diarylurea series as promising candidates for the development of relapse treatment of cocaine addiction.

The great divide: Separation between in vitro and in vivo effects of PSNCBAM-based CB1 receptor allosteric modulators.

While allosteric modulators of the cannabinoid type-1 receptor (CB1) continue to be developed and characterized, the gap between the in vitro and in vivo data is widening, raising questions regarding translatability of their effects and biological relevance. Among the CB1 allosteric modulators, PSNCBAM-1 has received little attention regarding its effects in vivo. Recently, pregnenolone was reported to act as an allosteric modulator of CB1, blocking THC's effects in vitro and in vivo, highlighting the potential of CB1 allosteric modulators for treatment of cannabis intoxication. We investigated the pharmacological effects of PSNCBAM-1 and two structural analogs, RTICBM-15 and -28, as well as pregnenolone, in both signaling and behavioral assays including [35S]GTPγS binding, the cannabinoid tetrad and drug discrimination. While the CB1 allosteric modulator PSNCBAM-1 attenuated THC-induced anti-nociception and its structural analog RTICBM-28 reduced THC's potency in drug discrimination, most cannabinoid effects in mice were unaffected. In contrast to the mouse studies, PSNCBAM-1 and analogs insurmountably antagonized CP55,940- and THC-stimulated [35S]GTPγS binding and exhibited negative binding cooperativity with [3H]SR141716 with similar apparent affinities. Notably, RTICBM-28, which contains a cyano substitution at the 4-chlorophenyl position of PSNCBAM-1, exhibited enhanced binding cooperativity with CP55,940. In contrast to previous findings, pregnenolone did not block THC's effects in drug discrimination or [35S]GTPγS. These data further highlight the difficulty in translating pharmacological effects of CB1 allosteric modulators in vivo but confirm the established pharmacology of PSNCBAM-1 and analogs in molecular assays of CB1 receptor function.

Thermolytic Degradation of Synthetic Cannabinoids: Chemical Exposures and Pharmacological Consequences.

Synthetic cannabinoids are manufactured clandestinely with little quality control and are distributed as herbal "spice" for smoking or as bulk compound for mixing with a solvent and inhalation via electronic vaporizers. Intoxication with synthetic cannabinoids has been associated with seizure, excited delirium, coma, kidney damage, and other disorders. The chemical alterations produced by heating these structurally novel compounds for consumption are largely unknown. Here, we show that heating synthetic cannabinoids containing tetramethylcyclopropyl-ring substituents produced thermal degradants with pharmacological activity that varied considerably from their parent compounds. Moreover, these degradants were formed under conditions simulating smoking. Some products of combustion retained high affinity at the cannabinoid 1 (CB1) and CB2 receptors, were more efficacious than (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3-hydroxypropyl)cyclohexanol (CP55,940) in stimulating CB1 receptor-mediated guanosine 5'-O-(3-thiotriphosphate) (GTPγS) binding, and were potent in producing Δ9-tetrahydrocannabinol-like effects in laboratory animals, whereas other compounds had low affinity and efficacy and were devoid of cannabimimetic activity. Degradants that retained affinity and efficacy also substituted in drug discrimination tests for the prototypical synthetic cannabinoid 1-pentyl-3-(1-naphthoyl)indole (JWH-018), and are likely to produce psychotropic effects in humans. Hence, it is important to take into consideration the actual chemical exposures that occur during use of synthetic cannabinoid formulations to better comprehend the relationships between dose and effect.

Design, synthesis, and analysis of antagonists of GPR55: Piperidine-substituted 1,3,4-oxadiazol-2-ones.

A series of 1,3,4-oxadiazol-2-ones was synthesized and tested for activity as antagonists at GPR55 in cellular beta-arrestin redistribution assays. The synthesis was designed to be modular in nature so that a sufficient number of analogues could be rapidly accessed to explore initial structure-activity relationships. The design of analogues was guided by the docking of potential compounds into a model of the inactive form of GPR55. The results of the assays were used to learn more about the binding pocket of GPR55. With this oxadiazolone scaffold, it was determined that modification of the aryl group adjacent to the oxadiazolone ring was often detrimental and that the distal cyclopropane was beneficial for activity. These results will guide further exploration of this receptor.

CB1 allosteric modulator Org27569 is an antagonist/inverse agonist of ERK1/2 signaling.

Allosteric modulation of cannabinoid type-1 receptors (CB1) is a novel means through which signaling bias may be exerted. Org27569 remains the most-characterized CB1 allosteric modulator yet there are conflicting reports regarding its effects on extracellular signal-regulated kinase (ERK) signaling. We conducted a systematic evaluation of Org27569 signaling through ERK. We have found that Org27569 is an antagonist of human CB1 (hCB1) mediated ERK signaling in HEK293 cells where it fully blocks CP55,940- but does not completely inhibit THC- and 2-AG-stimulated ERK1/2 activation. In hCB1 HEK293 cells, CP55,940 (1 µM) treatment produced a significant increase in puncta at 20, 40, 60, and 120 min, consistent with receptor internalization. Org27569 (10 µM) co-treatment prevented internalization at each time point and alone had no effect. These data demonstrate that Org27569 can block the CP55,940- induced internalization of CB1 receptors. Org27569 reduced basal ERK phosphorylation in hCB1 HEK293 cells but not in untransfected cells, demonstrating that Org27569 acts via the CB1 receptor to produce this effect. Furthermore, inverse agonism was through inhibition of Gi/o as overnight treatment with pertussis toxin abated this response. Finally, to delineate Org27569's effects on ERK1/2 in subcellular compartments, subcellular fractionation was performed; Org27569 produced a significant decrease in ERK phosphorylation in the nuclear-enriched and cytosolic fractions. Altogether, these data are consistent with previous studies demonstrating that CB1-mediated ERK1/2 activation is Gi/o-dependent and that Org27569 is an inverse agonist of CB1 receptors. To our knowledge this is the first reported demonstration of inverse agonism of ERK signaling by Org27569.