Non-Canonical Cannabinoid Receptors with Distinct Binding and Signaling Properties in Prostate and Other Cancer Cell Types Mediate Cell Death.

Cannabinoids exert anti-cancer actions; however, the underlying cytotoxic mechanisms and the cannabinoid receptors (CBRs) involved remain unclear. In this study, CBRs were characterized in several cancer cell lines. Radioligand binding screens surprisingly revealed specific binding only for the non-selective cannabinoid [3H]WIN-55,212-2, and not [3H]CP-55,940, indicating that the expressed CBRs exhibit atypical binding properties. Furthermore, [3H]WIN-55,212-2 bound to a single site in all cancer cells with high affinity and varying densities. CBR characteristics were next compared between human prostate cancer cell lines expressing low (PC-3) and high (DU-145) CBR density. Although mRNA for canonical CBRs was detected in both cell lines, only 5 out of 15 compounds with known high affinity for canonical CBRs displaced [3H]WIN-55,212-2 binding. Functional assays further established that CBRs in prostate cancer cells exhibit distinct signaling properties relative to canonical Gi/Go-coupled CBRs. Prostate cancer cells chronically exposed to both CBR agonists and antagonists/inverse agonists produced receptor downregulation, inconsistent with actions at canonical CBRs. Treatment of DU-145 cells with CBR ligands increased LDH-release, decreased ATP-dependent cell viability, and produced mitochondrial membrane potential depolarization. In summary, several cancer cell lines express CBRs with binding and signaling profiles dissimilar to canonical CBRs. Drugs selectively targeting these atypical CBRs might exhibit improved anti-cancer properties.

7-Azaindolequinuclidinones (7-AIQD): A novel class of cannabinoid 1 (CB1) and cannabinoid 2 (CB2) receptor ligands.

A series of N-benzyl-7-azaindolequinuclidinone (7-AIQD) analogs have been synthesized and evaluated for affinity toward CB1 and CB2 cannabinoid receptors and identified as a novel class of cannabinoid receptor ligands. Structure-activity relationship (SAR) studies indicate that 7-AIQD analogs are dual CB1/CB2 receptor ligands exhibiting high potency with somewhat greater selectivity towards CB2 receptors compared to the previously reported indolequinuclidinone (IQD) analogs. Initial binding assays showed that 7-AIQD analogs 8b, 8d, 8f, 8g and 9b (1 µM) produced more that 50% displacement of the CB1/CB2 non-selective agonist CP-55,940 (0.1 nM). Furthermore, Ki values determined from full competition binding curves showed that analogs 8a, 8b and 8g exhibit high affinity (110, 115 and 23.7 nM, respectively) and moderate selectivity (26.3, 6.1 and 9.2-fold, respectively) for CB2 relative to CB1 receptors. Functional studies examining modulation of G-protein activity demonstrated that 8a acts as a neutral antagonist at CB1 and CB2 receptors, while 8b exhibits inverse agonist activity at these receptors. Analogs 8f and 8g exhibit different intrinsic activities, depending on the receptor examined. Molecular docking and binding free energy calculations for the most active compounds (8a, 8b, 8f, and 8g) were performed to better understand the CB2 receptor-selective mechanism at the atomic level. Compound 8g exhibited the highest predicted binding affinity at both CB1 and CB2 receptors, and all four compounds were shown to have higher predicted binding affinities with the CB2 receptor compared to their corresponding binding affinities with the CB1 receptor. Further structural optimization of 7-AIQD analogs may lead to the identification of potential clinical agents.

Significance of Competing Metabolic Pathways for 5F-APINACA Based on Quantitative Kinetics.

In 2020, nearly one-third of new drugs on the global market were synthetic cannabinoids including the drug of abuse N-(1-adamantyl)-1-(5-pentyl)-1H-indazole-3-carboxamide (5F-APINACA, 5F-AKB48). Knowledge of 5F-APINACA metabolism provides a critical mechanistic basis to interpret and predict abuser outcomes. Prior qualitative studies identified which metabolic processes occur but not the order and extent of them and often relied on problematic "semi-quantitative" mass spectroscopic (MS) approaches. We capitalized on 5F-APINACA absorbance for quantitation while leveraging MS to characterize metabolite structures for measuring 5F-APINACA steady-state kinetics. We demonstrated the reliability of absorbance and not MS for inferring metabolite levels. Human liver microsomal reactions yielded eight metabolites by MS but only five by absorbance. Subsequent kinetic studies on primary and secondary metabolites revealed highly efficient mono- and dihydroxylation of the adamantyl group and much less efficient oxidative defluorination at the N-pentyl terminus. Based on regiospecificity and kinetics, we constructed pathways for competing and intersecting steps in 5F-APINACA metabolism. Overall efficiency for adamantyl oxidation was 17-fold higher than that for oxidative defluorination, showing significant bias in metabolic flux and subsequent metabolite profile compositions. Lastly, our analytical approach provides a powerful new strategy to more accurately assess metabolic kinetics for other understudied synthetic cannabinoids possessing the indazole chromophore.

Reduced Tolerance and Asymmetrical Crosstolerance to Effects of the Indole Quinuclidinone Analog PNR-4-20, a G Protein-Biased Cannabinoid 1 Receptor Agonist in Mice: Comparisons with Δ9-Tetrahydrocannabinol and JWH-018.

Most cannabinoid 1 receptor (CB1R) agonists will signal through both G protein-dependent and -independent pathways in an unbiased manner. Recruitment of ß-arrestin 2 desensitizes and internalizes receptors, producing tolerance that limits therapeutic utility of cannabinoids for chronic conditions. We developed the indole quinuclidinone (IQD) analog (Z)-2-((1-(4-fluorobenzyl)-1H-indol-3-yl)methylene)quinuclidin-3-one (PNR-4-20) as a novel G protein-biased agonist at CB1Rs, and the present studies determine if repeated administration of PNR-4-20 produces lesser tolerance to in vivo effects compared with unbiased CB1R agonists Δ9-tetrahydrocannabinol (Δ9-THC) and 1-pentyl-3-(1-naphthoyl)indole (JWH-018). Adult male National Institutes of Health Swiss mice were administered comparable doses of PNR-4-20 (100 mg/kg), Δ9-THC (30 mg/kg), or JWH-018 (3 mg/kg) once per day for five consecutive days to determine tolerance development to hypothermic, antinociceptive, and cataleptic effects. Persistence of tolerance was then determined after a drug abstinence period. We found that unbiased CB1R agonists Δ9-THC and JWH-018 produced similar tolerance to these effects, but lesser tolerance was observed with PNR-4-20 for hypothermic and cataleptic effects. Tolerance to the effects of PNR-4-20 completely recovered after drug abstinence, while residual tolerance was always observed with unbiased CB1R agonists. Repeated treatment with PNR-4-20 and Δ9-THC produced asymmetric crosstolerance to hypothermic effects. Importantly, binding studies suggest PNR-4-20 produced significantly less downregulation of CB1Rs relative to Δ9-THC in hypothalamus and thalamus of chronically treated mice. These studies suggest that the G protein-biased CB1R agonist PNR-4-20 produces significantly less tolerance than unbiased cannabinoid agonists, and that the IQD analogs should be investigated further as a novel molecular scaffold for development of new therapeutics.

Enzymatic analysis of glucuronidation of synthetic cannabinoid 1-naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22).

Recently, there has been a rise in abuse of synthetic cannabinoids (SCBs). The consumption of SCBs results in various effects and can induce toxic reactions, including paranoia, seizures, tachycardia and even death. 1-Naphthyl 1-(4-fluorobenzyl)-1H-indole-3-carboxylate (FDU-PB-22) is a third generation SCB whose metabolic pathway has not been fully characterized. In this study, we conducted in vitro pharmacokinetic analysis of FDU-PB-22 metabolism. Metabolic reactions containing FDU-PB-22 and human liver microsomes (HLMs) were independent of NADPH but not UDP-glucuronic acid (UDPGA), suggesting that UDP-glucuronosyltransferases (UGTs) are the primary enzymes involved in this metabolism. It was further determined that the metabolite extensively formed after incubating FDU-PB-22 with UDPGA in HLMs was the glucuronide of FDU-PB-22 3-carboxyindole (FBI-COOH). Various hepatic UGTs showed enzymatic activity for FBI-COOH. A series of UGT inhibitors showed moderate to strong inhibition of FBI-COOH-glucuronidation in HLMs, suggesting that multiple UGT isoforms are involved in FBI-COOH-glucuronidation in the liver. Interestingly, an extra-hepatic isoform, UGT1A10, exhibited the highest activity with a Km value of 38 µM and a Vmax value of 5.90 nmol/min/mg. Collectively, these results suggest that both genetic mutations of and the co-administration of inhibitors for FDU-PB-22-metabolizing UGTs will likely increase the risk of FDU-PB-22-induced toxicity.

Altered metabolism of synthetic cannabinoid JWH-018 by human cytochrome P450 2C9 and variants.

Synthetic cannabinoids (SCBs), synonymous with 'K2', 'Spice' or 'synthetic marijuana', are psychoactive drugs of abuse that frequently result in clinical effects and toxicity more severe than those classically associated with Δ9-tetrahydrocannabinol such as extreme agitation, hallucinations, supraventricular tachycardia, syncope, and seizures. JWH-018 is one of the earliest compounds identified in various SCB products, and our laboratory previously demonstrated that JWH-018 undergoes extensive metabolism by cytochromes P450 (P450), binds to, and activates cannabinoid receptors (CBRs). The major enzyme involved in the metabolism of JWH-018 is CYP2C9, a highly polymorphic enzyme found largely in the intestines and liver, with *1 being designated as the wild type, and *2 and *3 as the two most common variants. Three different major products have been identified in human urine and plasma: JWH-018 (ω)-OH, JWH-018 (ω-1)-OH(R), and JWH-018 (ω-1)-OH(S). The (ω-1)-OH metabolite of JWH-018 is a chiral molecule, and is thus designated as either (ω-1)-OH(R) or (ω-1)-OH(S). Here, in vitro enzyme kinetic assays performed with human recombinant CYP2C9 variants (*1, *2, and *3) revealed that oxidative metabolism by CYP2C9*3 resulted in significantly less formation of (ω)-OH and (ω-1)-OH metabolites. Surprisingly, CYP2C9*2 was roughly 3.6-fold more efficient as the CYP2C9*1 enzyme based on Vmax/Km, increasing the rate of JWH-018 metabolism and allowed for a much more rapid elimination. These results suggest that genetic polymorphisms of P450 enzymes result in the production of varying levels of biologically active JWH-018 metabolites in some individuals, offering a mechanistic explanation for the diverse clinical toxicity often observed following JWH-018 abuse.

The tamoxifen derivative ridaifen-B is a high affinity selective CB2 receptor inverse agonist exhibiting anti-inflammatory and anti-osteoclastogenic effects.

Selective estrogen receptor modulators (SERMs) target estrogen receptors (ERs) to treat breast cancer and osteoporosis. Several SERMs exhibit anti-cancer activity not related to ERs. To discover novel anti-cancer drugs acting via ER-independent mechanisms, derivatives of the SERM tamoxifen, known as the "ridaifen" compounds, have been developed that exhibit reduced or no ER affinity, while maintaining cytotoxicity. Tamoxifen and other SERMs bind to cannabinoid receptors with moderate affinity. Therefore, ER-independent effects of SERMs might be mediated via cannabinoid receptors. This study determined whether RID-B, a first generation ridaifen compound, exhibits affinity and/or activity at CB1 and/or CB2 cannabinoid receptors. RID-B binds with high affinity (Ki = 43.7 nM) and 17-fold selectivity to CB2 over CB1 receptors. RID-B acts as an inverse agonist at CB2 receptors, modulating G-protein and adenylyl cyclase activity with potency values predicted by CB2 affinity. Characteristic of an antagonist, RID-B co-incubation produces a parallel-rightward shift in the concentration-effect curve of CB2 agonist WIN-55,212-2 to inhibit adenylyl cyclase activity. CB2 inverse agonists are reported to exhibit anti-inflammatory and anti-ostoeclastogenic effects. In LPS-activated macrophages, RID-B exhibits anti-inflammatory effects by reducing levels of nitric oxide (NO), IL-6 and IL-1α, but not TNFα. Only reduction of NO concentration by RID-B is mediated by cannabinoid receptors. RID-B also exhibits pronounced anti-osteoclastogenic effects, reducing the number of osteoclasts differentiating from primary bone marrow macrophages in a cannabinoid receptor-dependent manner. In summary, the tamoxifen derivative RID-B, developed with reduced affinity for ERs, is a high affinity selective CB2 inverse agonist with anti-inflammatory and anti-osteoclastogenic properties.

Characterization of structurally novel G protein biased CB1 agonists: Implications for drug development.

The human cannabinoid subtype 1 receptor (hCB1R) is highly expressed in the CNS and serves as a therapeutic target for endogenous ligands as well as plant-derived and synthetic cannabinoids. Unfortunately, acute use of hCB1R agonists produces unwanted psychotropic effects and chronic administration results in development of tolerance and dependence, limiting the potential clinical use of these ligands. Studies in ß-arrestin knockout mice suggest that interaction of certain GPCRs, including µ-, δ-, κ-opioid and hCB1Rs, with ß-arrestins might be responsible for several adverse effects produced by agonists acting at these receptors. Indeed, agonists that bias opioid receptor activation toward G-protein, relative to ß-arrestin signaling, produce less severe adverse effects. These observations indicate that therapeutic utility of agonists acting at hCB1Rs might be improved by development of G-protein biased hCB1R agonists. Our laboratory recently reported a novel class of indole quinulidinone (IQD) compounds that bind cannabinoid receptors with relatively high affinity and act with varying efficacy. The purpose of this study was to determine whether agonists in this novel cannabinoid class exhibit ligand bias at hCB1 receptors. Our studies found that a novel IQD-derived hCB1 receptor agonist PNR-4-20 elicits robust G protein-dependent signaling, with transduction ratios similar to the non-biased hCB1R agonist CP-55,940. In marked contrast to CP-55,940, PNR-4-20 produces little to no ß-arrestin 2 recruitment. Quantitative calculation of bias factors indicates that PNR-4-20 exhibits from 5.4-fold to 29.5-fold bias for G protein, relative to ß-arrestin 2 signaling (when compared to G protein activation or inhibition of forskolin-stimulated cAMP accumulation, respectively). Importantly, as expected due to reduced ß-arrestin 2 recruitment, chronic exposure of cells to PNR-4-20 results in significantly less desensitization and down-regulation of hCB1Rs compared to similar treatment with CP-55,940. PNR-4-20 (i.p.) is active in the cannabinoid tetrad in mice and chronic treatment results in development of less persistent tolerance and no significant withdrawal signs when compared to animals repeatedly exposed to the non-biased full agoinst JWH-018 or Δ9-THC. Finally, studies of a structurally similar analog PNR- 4-02 show that it is also a G protein biased hCB1R agonist. It is predicted that cannabinoid agonists that bias hCB1R activation toward G protein, relative to ß-arrestin 2 signaling, will produce fewer and less severe adverse effects both acutely and chronically.

Evaluation of Analgesia, Tolerance, and the Mechanism of Action of Morphine-6-O-Sulfate Across Multiple Pain Modalities in Sprague-Dawley Rats.

BACKGROUND: Morphine-6-O-sulfate (M6S) is a mixed µ/δ-opioid receptor (OR) agonist and potential alternative to morphine for treatment of chronic multimodal pain. METHODS: To provide more support for this hypothesis, the antinociceptive effects of M6S and morphine were compared in tests that access a range of pain modalities, including hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold tests. RESULTS: Acutely, M6S was 2- to 3-fold more potent than morphine in HPT and PST tests, specifically, derived from best-fit analysis of dose-response relationships of morphine/M6S half-effective dose (ED50) ratios (lower, upper 95% confidence interval [CI]) were 2.8 (2.0-5.8) in HPT and 2.2 (2.1, 2.4) in PST tests. No differences in analgesic drug potencies were detected in the PPT test (morphine/M6S ED50 ratio 1.2 (95% CI, 0.8-1.4). After 7 to 9 days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all 3 pain tests. Morphine-tolerant rats were not crosstolerant to M6S. The antinociceptive effects of M6S were not sensitive to κ-OR antagonists. However, the δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 55% ± 4% (95% CI, 39-75) in the HPT test, 94% ± 4% (95% CI, 84-105) in the PST test, and 5% ± 17% (95% CI, -47 to 59) or 51% ± 14% (95% CI, 14-84; 6 rats per each group) in the paw pressure threshold test when examined acutely or after 7 days of chronic treatment, respectively. CONCLUSIONS: Activity via δ-ORs thus appears to be an important determinant of M6S action. M6S also exhibited favorable antinociceptive and tolerance profiles compared with morphine in 3 different antinociceptive assays, indicating that M6S may serve as a useful alternative for rotation in morphine-tolerant subjects.

Antinociceptive effects of the 6-O-sulfate ester of morphine in normal and diabetic rats: Comparative role of mu- and delta-opioid receptors.

This study determined the antinociceptive effects of morphine and morphine-6-O-sulfate (M6S) in both normal and diabetic rats, and evaluated the comparative role of mu-opioid receptors (mu-ORs) and delta-opioid receptors (delta-ORs) in the antinociceptive action of these opioids. In vitro characterization of mu-OR and delta-OR-mediated signaling by M6S and morphine in stably transfected Chinese hamster ovary (CHO-K1) cells showed that M6S exhibited a 6-fold higher affinity for delta-ORs and modulated G-protein and adenylyl cyclase activity via delta-ORs more potently than morphine. Interestingly, while morphine acted as a full agonist at delta-ORs in both functional assays examined, M6S exhibited either partial or full agonist activity for modulation of G-protein or adenylyl cyclase activity, respectively. Molecular docking studies indicated that M6S but not morphine binds equally well at the ligand binding site of both mu- and delta-ORs. In vivo analgesic effects of M6S and morphine in both normal and streptozotocin-induced diabetic Sprague-Dawley rats utilizing the hot water tail flick latency test showed that M6S produced more potent antinociception than morphine in both normal rats and diabetic rats. This difference in potency was abrogated following antagonism of delta- but not mu- or kappa (kappa-ORs) opioid receptors. During 9days of chronic treatment, tolerance developed to morphine-treated but not to M6S-treated rats. Rats that developed tolerance to morphine still remained responsive to M6S. Collectively, this study demonstrates that M6S is a potent and efficacious mu/delta opioid analgesic with a delayed tolerance profile when compared to morphine in both normal and diabetic rats. PERSPECTIVE: This study demonstrates that M6S acts at both mu- and delta-ORs, and adds to the growing evidence that the use of mixed mu/delta opioid agonists in pain treatment may have clinical benefit.

The K2/Spice phenomenon: emergence, identification, legislation and metabolic characterization of synthetic cannabinoids in herbal incense products.

In 2008, the European Monitoring Center for Drugs and Drug Addiction (EMCDDA) detected unregulated, psychoactive synthetic cannabinoids (SCBs) in purportedly all-natural herbal incense products (often known as K2 or Spice) that were being covertly abused as marijuana substitutes. These drugs, which include JWH-018, JWH-073 and CP-47,497, bind and activate the cannabinoid receptors CB1R and CB2R with remarkable potency and efficacy. Serious adverse effects that often require medical attention, including severe cardiovascular, gastrointestinal and psychiatric sequelae, are highly prevalent with SCB abuse. Consequently, progressively restrictive legislation in the US and Europe has banned the distribution, sale and use of prevalent SCBs, initiating cycles in which herbal incense manufacturers replace banned SCBs with newer unregulated SCBs. The contents of the numerous, diverse herbal incense products was unknown when SCB abuse first emerged. Furthermore, the pharmacology of the active components was largely uncharacterized, and confirmation of SCB use was hindered by a lack of known biomarkers. These knowledge gaps prompted scientists across multiple disciplines to rapidly (1) monitor, identify and quantify with chromatography/mass spectrometry the ever-changing contents of herbal incense products, (2) determine the metabolic pathways and major urinary metabolites of several commonly abused SCBs and (3) identify active metabolites that possibly contribute to the severe adverse effect profile of SCBs. This review comprehensively describes the emergence of SCB abuse and provides a historical account of the major case reports, legal decisions and scientific discoveries of the "K2/Spice Phenomenon". Hypotheses concerning potential mechanisms SCB adverse effects are proposed in this review.

Activation of cannabinoid CB2 receptor-mediated AMPK/CREB pathway reduces cerebral ischemic injury.

The type 2 cannabinoid receptor (CB2R) was recently shown to mediate neuroprotection in ischemic injury. However, the role of CB2Rs in the central nervous system, especially neuronal and glial CB2Rs in the cortex, remains unclear. We, therefore, investigated anti-ischemic mechanisms of cortical CB2R activation in various ischemic models. In rat cortical neurons/glia mixed cultures, a CB2R agonist, trans-caryophyllene (TC), decreased neuronal injury and mitochondrial depolarization caused by oxygen-glucose deprivation/re-oxygenation (OGD/R); these effects were reversed by the selective CB2R antagonist, AM630, but not by a type 1 cannabinoid receptor antagonist, AM251. Although it lacked free radical scavenging and antioxidant enzyme induction activities, TC reduced OGD/R-evoked mitochondrial dysfunction and intracellular oxidative stress. Western blot analysis demonstrated that TC enhanced phosphorylation of AMP-activated protein kinase (AMPK) and cAMP responsive element-binding protein (CREB), and increased expression of the CREB target gene product, brain-derived neurotrophic factor. However, TC failed to alter the activity of either Akt or extracellular signal-regulated kinase, two major CB2R signaling pathways. Selective AMPK and CREB inhibitors abolished the neuroprotective effects of TC. In rats, post-ischemic treatment with TC decreased cerebral infarct size and edema, and increased phosphorylated CREB and brain-derived neurotrophic factor expression in neurons. All protective effects of TC were reversed by co-administration with AM630. Collectively, these data demonstrate that cortical CB2R activation by TC ameliorates ischemic injury, potentially through modulation of AMPK/CREB signaling, and suggest that cortical CB2Rs might serve as a putative therapeutic target for cerebral ischemia.

Palmitoylethanolamide regulates development of intestinal radiation injury in a mast cell-dependent manner.

BACKGROUND: Mast cells and neuroimmune interactions regulate the severity of intestinal radiation mucositis, a dose-limiting toxicity during radiation therapy of abdominal malignancies. AIM: Because endocannabinoids (eCB) regulate intestinal inflammation, we investigated the effect of the cannabimimetic, palmitoylethanolamide (PEA), in a mast competent (+/+) and mast cell-deficient (Ws/Ws) rat model. METHODS: Rats underwent localized, fractionated intestinal irradiation, and received daily injections with vehicle or PEA from 1 day before until 2 weeks after radiation. Intestinal injury was assessed noninvasively by luminol bioluminescence, and, at 2 weeks, by histology, morphometry, and immunohistochemical analysis, gene expression analysis, and pathway analysis. RESULTS: Compared with +/+ rats, Ws/Ws rats sustained more intestinal structural injury (p = 0.01), mucosal damage (p = 0.02), neutrophil infiltration (p = 0.0003), and collagen deposition (p = 0.004). PEA reduced structural radiation injury (p = 0.02), intestinal wall thickness (p = 0.03), collagen deposition (p = 0.03), and intestinal inflammation (p = 0.02) in Ws/Ws rats, but not in +/+ rats. PEA inhibited mast cell-derived cellular immune response and anti-inflammatory IL-6 and IL-10 signaling and activated the prothrombin pathway in +/+ rats. In contrast, while PEA suppressed nonmast cell-derived immune responses, it increased anti-inflammatory IL-10 and IL-6 signaling and decreased activation of the prothrombin pathway in Ws/Ws rats. CONCLUSIONS: These data demonstrate that the absence of mast cells exacerbate radiation enteropathy by mechanisms that likely involve the coagulation system, anti-inflammatory cytokine signaling, and the innate immune system; and that these mechanisms are regulated by PEA in a mast cell-dependent manner. The eCB system should be explored as target for mitigating intestinal radiation injury.

CB1 and CB2 receptors are novel molecular targets for Tamoxifen and 4OH-Tamoxifen.

Tamoxifen (Tam) is classified as a selective estrogen receptor modulator (SERM) and is used for treatment of patients with ER-positive breast cancer. However, it has been shown that Tam and its cytochrome P450-generated metabolite 4-hydroxy-Tam (4OH-Tam) also exhibit cytotoxic effects in ER-negative breast cancer cells. These observations suggest that Tam and 4OH-Tam can produce cytotoxicity via estrogen receptor (ER)-independent mechanism(s) of action. The molecular targets responsible for the ER-independent effects of Tam and its derivatives are poorly understood. Interestingly, similar to Tam and 4OH-Tam, cannabinoids have also been shown to exhibit anti-proliferative and apoptotic effects in ER-negative breast cancer cells, and estrogen can regulate expression levels of cannabinoid receptors (CBRs). Therefore, this study investigated whether CBRs might serve as novel molecular targets for Tam and 4OH-Tam. We report that both compounds bind to CB1 and CB2Rs with moderate affinity (0.9-3 µM). Furthermore, Tam and 4OH-Tam exhibit inverse activity at CB1 and CB2Rs in membrane preparations, reducing basal G-protein activity. Tam and 4OH-Tam also act as CB1/CB2R-inverse agonists to regulate the downstream intracellular effector adenylyl cyclase in intact cells, producing concentration-dependent increases in intracellular cAMP. These results suggest that CBRs are molecular targets for Tam and 4OH-Tam and may contribute to the ER-independent cytotoxic effects reported for these drugs. Importantly, these findings also indicate that Tam and 4OH-Tam might be used as structural scaffolds for development of novel, efficacious, non-toxic cancer drugs acting via CB1 and/or CB2Rs.

Differential drug-drug interactions of the synthetic Cannabinoids JWH-018 and JWH-073: implications for drug abuse liability and pain therapy.

Marijuana substitutes often contain blends of multiple psychoactive synthetic cannabinoids (SCBs), including the prevalent SCBs (1-pentyl-1H-indole-3-yl)-1-naphthalenyl-methanone (JWH-018) and (1-butyl-1H-indole-3-yl)-1-naphthalenyl-methanone (JWH-073). Because SCBs are frequently used in combinations, we hypothesized that coadministering multiple SCBs induces synergistic drug-drug interactions. Drug-drug interactions between JWH-018 and JWH-073 were investigated in vivo for Δ(9)-tetrahydrocannabinol (Δ(9)-THC)-like discriminative stimulus effects, analgesia, task disruption, and hypothermia. Combinations (JWH-018:JWH-073) of these drugs were administered to mice in assays of Δ(9)-THC discrimination, tail-immersion, and food-maintained responding, and rectal temperatures were measured. Synergism occurred in the Δ(9)-THC discrimination assay for two constant dose ratio combinations (1:3 and 1:1). A 1:1 and 2:3 dose ratio induced additivity and synergy, respectively, in the tail-immersion assay. Both 1:1 and 2:3 dose ratios were additive for hypothermia, whereas a 1:3 dose ratio induced subadditive suppression of food-maintained responding. In vitro drug-drug interactions were assessed using competition receptor-binding assays employing mouse brain homogenates and cannabinoid 1 receptor (CB1R)-mediated inhibition of adenylyl cyclase activity in Neuro2A wild-type cells. Interestingly, synergy occurred in the competition receptor-binding assay for two dose ratios (1:5 and 1:10), but not in the adenylyl cyclase activity assay (1:5). Altogether, these data indicate that drug-drug interactions between JWH-018 and JWH-073 are effect- and ratio-dependent and may increase the relative potency of marijuana substitutes for subjective Δ(9)-THC-like effects. Combinations may improve the therapeutic profile of cannabinoids, considering that analgesia but not hypothermia or task disruption was potentiated. Importantly, synergy in the competition receptor-binding assay suggests multiple CB1R-SCB binding sites.

Human metabolites of synthetic cannabinoids JWH-018 and JWH-073 bind with high affinity and act as potent agonists at cannabinoid type-2 receptors.

K2 or Spice is an emerging drug of abuse that contains synthetic cannabinoids, including JWH-018 and JWH-073. Recent reports indicate that monohydroxylated metabolites of JWH-018 and JWH-073 retain high affinity and activity at cannabinoid type-1 receptors (CB1Rs), potentially contributing to the enhanced toxicity of K2 compared to marijuana. Since the parent compounds also bind to cannabinoid type-2 receptors (CB2Rs), this study investigated the affinity and intrinsic activity of JWH-018, JWH-073 and several monohydroxylated metabolites at human CB2Rs (hCB2Rs). The affinity of cannabinoids for hCB2Rs was determined by competition binding studies employing CHO-hCB2 membranes. Intrinsic activity of compounds was assessed by G-protein activation and adenylyl cyclase (AC)-inhibition in CHO-hCB2 cells. JWH-073, JWH-018 and several of their human metabolites exhibit nanomolar affinity and act as potent agonists at hCB2Rs. Furthermore, a major omega hydroxyl metabolite of JWH-073 (JWH-073-M5) binds to CB2Rs with 10-fold less affinity than the parent molecule, but unexpectedly, is equipotent in regulating AC-activity when compared to the parent molecule. Finally, when compared to CP-55,940 and Δ(9)-tetrahydrocannabinol (Δ(9)-THC), JWH-018, JWH-018-M5 and JWH-073-M5 require significantly less CB2R occupancy to produce similar levels of AC-inhibition, indicating that these compounds may more efficiently couple CB2Rs to AC than the well characterized cannabinoid agonists examined. These results indicate that JWH-018, JWH-073 and several major human metabolites of these compounds exhibit high affinity and demonstrate distinctive signaling properties at CB2Rs. Therefore, future studies examining pharmacological and toxicological properties of synthetic cannabinoids present in K2 products should consider potential actions of these drugs at both CB1 and CB2Rs.

Evaluation of (Z)-2-((1-benzyl-1H-indol-3-yl)methylene)-quinuclidin-3-one analogues as novel, high affinity ligands for CB1 and CB2 cannabinoid receptors.

A small library of N-benzyl indolequinuclidinone (IQD) analogs has been identified as a novel class of cannabinoid ligands. The affinity and selectivity of these IQDs for the two established cannabinoid receptor subtypes, CB1 and CB2, was evaluated. Compounds 8 (R=R(2)=H, R(1)=F) and 13 (R=COOCH3, R(1)=R(2)=H) exhibited high affinity for CB2 receptors with Ki values of 1.33 and 2.50 nM, respectively, and had lower affinities for the CB1 receptor (Ki values of 9.23 and 85.7 nM, respectively). Compound 13 had the highest selectivity of all the compounds examined, and represents a potent cannabinoid ligand with 34-times greater selectivity for CB2R over CB1R. These findings are significant for future drug development, given recent reports demonstrating beneficial use of cannabinoid ligands in a wide variety of human disease states including drug abuse, depression, schizophrenia, inflammation, chronic pain, obesity, osteoporosis and cancer.

Deuterated buprenorphine retains pharmacodynamic properties of buprenorphine and resists metabolism to the active metabolite norbuprenorphine in rats.

Introduction: An active metabolite of buprenorphine (BUP), called norbuprenorphine (NorBUP), is implicated in neonatal opioid withdrawal syndrome when BUP is taken during pregnancy. Therefore, reducing or eliminating metabolism of BUP to NorBUP is a novel strategy that will likely lower total fetal exposure to opioids and thus improve offspring outcomes. Precision deuteration alters pharmacokinetics of drugs without altering pharmacodynamics. Here, we report the synthesis and testing of deuterated buprenorphine (BUP-D2). Methods: We determined opioid receptor affinities of BUP-D2 relative to BUP with radioligand competition receptor binding assays, and the potency and efficacy of BUP-D2 relative to BUP to activate G-proteins via opioid receptors with [35S]GTPγS binding assays in homogenates containing the human mu, delta, or kappa opioid receptors. The antinociceptive effects of BUP-D2 and BUP were compared using the warm-water tail withdrawal assay in rats. Blood concentration versus time profiles of BUP, BUP-D2, and NorBUP were measured in rats following intravenous BUP-D2 or BUP injection. Results: The synthesis provided a 48% yield and the product was ≥99% deuterated. Like BUP, BUP-D2 had sub-nanomolar affinity for opioid receptors. BUP-D2 also activated opioid receptors and induced antinociception with equal potency and efficacy as BUP. The maximum concentration and the area under the curve of NorBUP in the blood of rats that received BUP-D2 were over 19- and 10-fold lower, respectively, than in rats that received BUP. Discussion: These results indicate that BUP-D2 retains key pharmacodynamic properties of BUP and resists metabolism to NorBUP and therefore holds promise as an alternative to BUP.

Cytochrome P450-mediated oxidative metabolism of abused synthetic cannabinoids found in K2/Spice: identification of novel cannabinoid receptor ligands.

Abuse of synthetic cannabinoids (SCs), such as [1-naphthalenyl-(1-pentyl-1H-indol-3-yl]-methanone (JWH-018) and [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM2201), is increasing at an alarming rate. Although very little is known about the metabolism and toxicology of these popular designer drugs, mass spectrometric analysis of human urine specimens after JWH-018 and AM2201 exposure identified monohydroxylated and carboxylated derivatives as major metabolites. The present study extends these initial findings by testing the hypothesis that JWH-018 and its fluorinated counterpart AM2201 are subject to cytochrome P450 (P450)-mediated oxidation, forming potent hydroxylated metabolites that retain significant affinity and activity at the cannabinoid 1 (CB(1)) receptor. Kinetic analysis using human liver microsomes and recombinant human protein identified CYP2C9 and CYP1A2 as major P450s involved in the oxidation of the JWH-018 and AM2201. In vitro metabolite formation mirrored human urinary metabolic profiles, and each of the primary enzymes exhibited high affinity (K(m) = 0.81-7.3 µM) and low to high reaction velocities (V(max) = 0.0053-2.7 nmol of product · min(-1) · nmol protein(-1)). The contribution of CYP2C19, 2D6, 2E1, and 3A4 in the hepatic metabolic clearance of these synthetic cannabinoids was minimal (f(m) = <0.2). In vitro studies demonstrated that the primary metabolites produced in humans display high affinity and intrinsic activity at the CB(1) receptor, which was attenuated by the CB(1) receptor antagonist (6aR,10aR)-3-(1-methanesulfonylamino-4-hexyn-6-yl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran (O-2050). Results from the present study provide critical, missing data related to potential toxicological properties of "K2" parent compounds and their human metabolites, including mechanism(s) of action at cannabinoid receptors.