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PDF file, 200KB, Role of Sp proteins in regulating level of YY1 protein.

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid–dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor–dependent, thus identifying a new cellular target for this anticancer agent. Mol Cancer Ther; 11(7); 1421–31. ©2012 AACR.

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PDF file, 104KB, BA-mediated downregulation of Sp1, Sp3 and Sp4 partially blocked by CB receptor antagonists.

Buprenorphine (BUP) is the preferred treatment for opioid use disorder during pregnancy but can cause neonatal opioid withdrawal syndrome (NOWS). Norbuprenorphine (NorBUP), an active metabolite of BUP, is implicated in BUP-associated NOWS. We hypothesized that BUP, a low-efficacy agonist of mu opioid receptors, will not antagonize NorBUP, a high-efficacy agonist of mu opioid receptors, in producing NOWS. To test this hypothesis, we treated pregnant Long-Evans rats with BUP (0, 0.01, 0.1 or 1 mg/kg/day) ± NorBUP (1 mg/kg/day) from gestation day 9 until pup delivery, and tested pups for opioid dependence using our established NOWS model. We used LC-MS-MS to quantify brain concentrations of BUP, NorBUP, and their glucuronide conjugates. BUP had little effect on NorBUP-induced NOWS, with the exception of 1 mg/kg/day BUP significantly increasing NorBUP-induced NOWS by 58% in females. BUP and NorBUP brain concentrations predicted NOWS in multiple linear regression models. Interestingly, NorBUP contributed more to NOWS in females (βNorBUP= 51.34, p = 0.0001) than in males (βNorBUP= 19.21, P = 0.093), while BUP was similar for females (βBUP= 10.62, P = 0.0017) and males (βBUP= 11.38, P = 0.009). We are the first to report that NorBUP induces NOWS in the presence of BUP and that there is sexual divergence in the contribution of NorBUP to BUP-associated NOWS. These findings suggest that females are more susceptible to NorBUP-induced NOWS, and that treatment strategies that reduce prenatal NorBUP exposure may be more effective for females than males.

The legal status of kratom in the United States is complex and varies by state. The U.S. Food and Drug Administration (FDA) and the U.S. Drug Enforcement Administration have repeatedly subjected kratom to regulatory review. However, there hasn't been a systematic review of the public's perception of kratom. The present study analyzed open-ended responses from the public to an FDA solicitation for information regarding kratom with the goal of providing a comprehensive assessment of motives for kratom use.To guide decisions regarding kratom regulation, the FDA solicited comments regarding kratom abuse potential, medical usefulness, and impact of scheduling changes from July through August 2021 and posted them to the Federal Register website. We analyzed comments posted during the first 6 weeks of comment solicitation (6,353) using an inductive approach via qualitative content analysis.Respondents reported 106 independent health-related reasons for kratom use, with most categorized as mental health, pain management, substance use disorder, or miscellaneous purposes that included increasing focus, treating insomnia, and decreasing fatigue. Neurological diseases and digestive disorders were also reported. Relatively few (2%) responses reported recreational use, abuse potential, or adverse effects of kratom.Although kratom is not approved as a safe and effective therapy for any indication, individuals use kratom for a broad spectrum of health-related purposes. Limitations of this study include potential bias for respondents with perceived positive experiences using kratom, lack of demographics data, and lack of independent verification of claims made by respondents. Regardless, this study reflects perceptions regarding the therapeutic uses of kratom and provides insight into potential individual-level consequences of regulating kratom in the U.S. It is important to study the public's perception of kratom use, which can aid regulatory purposes and provide clinically important information on individuals' use and valuation of kratom.

Chronic prenatal exposure to opioids often causes fetal opioid dependence that leads to neonatal opioid withdrawal syndrome (NOWS) shortly after delivery. Rat models of NOWS often require quantifying neonatal withdrawal behaviors using time-consuming, labor-intensive manual scoring methods. The goal of this study was to automate quantification of opioid withdrawal in neonatal rat pups. Accordingly, we used the animal behavior software Ethovision® XT to analyze archived videos of rat pups subjected to precipitated opioid withdrawal testing on postnatal day 0. We compared results obtained from Ethovision® XT with those previously obtained from manual scoring. Two endpoints reported by Ethovision® XT, Distance Moved (cm) and Movement Duration (s), had strong positive linear relationships with manually derived global withdrawal scores (GWS; R2 > 0.73). Sensitivity and specificity of each endpoint to discriminate presence and absence of low-grade withdrawal were assessed by receiver operator characteristic curve analysis, which indicated that Distance Moved and Movement Duration had excellent accuracy (AUC > 0.90). Finally, we analyzed main and interaction effects of prenatal treatment (with vehicle or mu opioid receptor full agonists) and postnatal challenge (with saline or an opioid receptor antagonist) on each endpoint and determined they were similar for the manual and automated methods. These results show that Ethovision® XT software can reliably quantify opioid withdrawal in neonatal rat pups with non-inferiority to manual scoring even in videos that were not originally purposed and optimized for Ethovision® XT analysis. This faster and less labor-intensive method of analysis is expected to accelerate progress in preclinical studies of NOWS.

Childhood maltreatment history is a prevalent risk factor for substance use disorder and has lifelong adverse consequences on psychiatric wellbeing. The role of personality variations in determining childhood maltreatment-associated outcomes is poorly understood. This study sought to test neuroticism and agreeableness as mediator and moderator, respectively, of functional outcomes associated with having a history of childhood maltreatment and presence/absence of cocaine dependence. Ninety-four participants completed the Structured Clinical Interview for DSM-IV (SCID-IV), Childhood Trauma Questionnaire (CTQ), NEO-Five Factor Inventory (NEO-FFI), and the Addiction Severity Index (ASI). The distribution-of-the-product strategy tested if neuroticism mediated the relationship between CTQ and ASI scores. Agreeableness was tested as a moderator using bootstrapped multiple regression analyses with agreeableness*CTQ interaction terms as predictors of ASI scores. Analyses covaried for cocaine dependence to determine its influence. Neuroticism mediated the relationship between severity of childhood maltreatment history and family (ASI-Family) and psychiatric (ASI-Psychiatric) dysfunction in adulthood, independent of cocaine dependence. Agreeableness negatively moderated the effect of childhood maltreatment severity on family dysfunction. Exposure to emotional neglect and abuse selectively drove the mediation and moderation effects. Personality-directed interventions that reduce neuroticism or increase agreeableness may be promising approaches to uncouple childhood maltreatment history from lifelong social and psychiatric dysfunction.

These studies probed the relationship between intrinsic efficacy and tolerance/cross-tolerance between ∆(9)-THC and synthetic cannabinoid drugs of abuse (SCBs) by examining in vivo effects and cellular changes concomitant with their repeated administration in mice. Dose-effect relationships for hypothermic effects were determined in order to confirm that SCBs JWH-018 and JWH-073 are higher efficacy agonists than ∆(9)-THC in mice. Separate groups of mice were treated with saline, sub-maximal hypothermic doses of JWH-018 or JWH-073 (3.0mg/kg or 10.0mg/kg, respectively) or a maximally hypothermic dose of 30.0mg/kg ∆(9)-THC once per day for 5 consecutive days while core temperature and locomotor activity were monitored via biotelemetry. Repeated administration of all drugs resulted in tolerance to hypothermic effects, but not locomotor effects, and this tolerance was still evident 14 days after the last drug administration. Further studies treated mice with 30.0mg/kg ∆(9)-THC once per day for 4 days, then tested with SCBs on day 5. Mice with a ∆(9)-THC history were cross-tolerant to both SCBs, and this cross-tolerance also persisted 14 days after testing. Select brain regions from chronically treated mice were examined for changes in CB1 receptor expression and function. Expression and function of hypothalamic CB1Rs were reduced in mice receiving chronic drugs, but cortical CB1R expression and function were not altered. Collectively, these data demonstrate that repeated ∆(9)-THC, JWH-018 and JWH-073 can induce long-lasting tolerance to some in vivo effects, which is likely mediated by region-specific downregulation and desensitization of CB1Rs.

Cannabis is the illicit drug that is most abused during pregnancy worldwide, resulting in prenatal exposure rates of ∼ 5–15% of the total population. Many challenges exist in detecting prenatal cannabis exposure (PCE) in the obstetric patient population, in understanding the motivation to use cannabis during pregnancy, and in determining the effects of PCE on offspring. This chapter summarizes the scientific literature regarding the prevalence of PCE, and the detection methods used to estimate exposure prevalence. The characteristics of pregnant women who are most vulnerable to using cannabis are also described. Finally, the effects of PCE on the childhood course of physical, neurological, cognitive, behavioral, and emotional development are discussed. The purpose of the current chapter is to inform readers about the potentially negative impact of PCE at both the individual and societal level, by providing reliable, scientifically valid, peer-reviewed information on this controversial subject.

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.

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.

Marijuana use among women is highly prevalent, but the societal conversation on marijuana rarely focuses on how marijuana affects female reproduction and endocrinology. This article reviews the current scientific literature regarding marijuana use and hypothalamic-pituitary-ovarian (HPO) axis regulation, ovarian hormone production, the menstrual cycle, and fertility. Evidence suggests that marijuana can reduce female fertility by disrupting hypothalamic release of gonadotropin releasing hormone (GnRH), leading to reduced estrogen and progesterone production and anovulatory menstrual cycles. Tolerance to these effects has been shown in rhesus monkeys, but the effects of chronic marijuana use on human female reproduction are largely unknown. Marijuana-induced analgesia, drug reinforcement properties, tolerance, and dependence are influenced by ovarian hormones, with estrogen generally increasing and progesterone decreasing sensitivity to marijuana. Carefully controlled regulation of the Endocannabinoid System (ECS) is required for successful reproduction, and the exogenous cannabinoids in marijuana may disrupt the delicate balance of the ECS in the female reproductive system.

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) =

K2 and several similar purported “incense products” spiked with synthetic cannabinoids are abused as cannabis substitutes. We hypothesized that metabolism of JWH-073, a prevalent cannabinoid found in K2, contributes to toxicity associated with K2 use. Competition receptor binding studies and G-protein activation assays, both performed by employing mouse brain homogenates, were used to determine the affinity and intrinsic activity, respectively, of potential monohydroxylated (M1, M3–M5) and monocarboxylated (M6) metabolites at cannabinoid 1 receptors (CB1Rs). Surprisingly, M1, M4 and M5 retain nanomolar affinity for CB1Rs, while M3 displays micromolar affinity and M6 does not bind to CB1Rs. JWH-073 displays equivalent efficacy to that of the CB1R full agonist CP-55,940, while M1, M3, and M5 act as CB1R partial agonists, and M4 shows little or no intrinsic activity. Further in vitro investigation by Schild analysis revealed that M4 acts as a competitive neutral CB1R antagonist ( K b ∼ 40 nM). In agreement with in vitro studies, M4 also demonstrates CB1R antagonism in vivo by blunting cannabinoid-induced hypothermia in mice. Interestingly, M4 does not block agonist-mediated responses of other measures in the cannabinoid tetrad ( e.g. , locomotor suppression, catalepsy or analgesia). Finally, also as predicted by in vitro results, M1 exhibits agonist activity in vivo by inducing significant hypothermia and suppression of locomotor activity in mice. In conclusion, the present study indicates that further work examining the physiological effects of synthetic cannabinoid metabolism is warranted. Such a complex mix of metabolically produced CB1R ligands may contribute to the adverse effect profile of JWH-073-containing products.

OBJECTIVES/SPECIFIC AIMS: Rodent models can be used to study neonatal abstinence syndrome (NAS), but the applicability of findings from the models to NAS in humans is not well understood. The objective of this study was to develop a rat model of norbuprenorphine-induced NAS and validate its translational value by comparing blood concentrations in the norbuprenorphine-treated pregnant rat to those previously reported in pregnant women undergoing buprenorphine treatment. METHODS/STUDY POPULATION: Pregnant Long-Evans rats were implanted with 14-day osmotic minipumps containing vehicle, morphine (positive control), or norbuprenorphine (0.3–3 mg/kg/d) on gestation day 9. Within 12 hours of delivery, pups were tested for spontaneous or precipitated opioid withdrawal by injecting them with saline (10 mL/kg, i.p.) or naltrexone (1 or 10 mg/kg, i.p), respectively, and observing them for well-validated neonatal withdrawal signs. Blood was sampled via indwelling jugular catheters from a subset of norbuprenorphine-treated dams on gestation day 8, 10, 13, 17, and 20. Norbuprenorphine concentrations in whole blood samples were quantified using LC/MS/MS. RESULTS/ANTICIPATED RESULTS: Blood concentrations of norbuprenorphine in rats exposed to 1–3 mg/kg/d of norbuprenorphine were similar to levels previously reported in pregnant women undergoing buprenorphine treatment. Pups born to dams treated with these doses exhibited robust withdrawal signs. Blood concentrations of norbuprenorphine decreased across gestation, which is similar to previous reports in humans. DISCUSSION/SIGNIFICANCE OF IMPACT: These results suggest that dosing dams with 1–3 mg/kg/day norbuprenorphine produces maternal blood concentrations and withdrawal severity similar to those previously reported in humans. This provides evidence that, at these doses, this model is useful for testing hypotheses about norbuprenorphine that are applicable to NAS in humans.

Drug addictions are debilitating disorders that are highly associated with personality abnormalities. Early life stress (ELS) is a common risk factor for addiction and personality disturbances, but the relationships between ELS, addiction, and personality are poorly understood.Ninety-five research participants were assessed for and grouped by ELS history and cocaine dependence. NEO-FFI personality measures were compared between the groups to define ELS- and addiction-related differences in personality traits. ELS and cocaine dependence were then examined as predictors of personality trait scores. Finally, k-means clustering was used to uncover clusters of personality trait configurations within the sample. Odds of cluster membership across subject groups was then determined.Trait expression differed significantly across subject groups. Cocaine-dependent subjects with a history of ELS (cocaine+/ELS+) displayed the greatest deviations in normative personality. Cocaine dependence significantly predicted four traits, while ELS predicted neuroticism and agreeableness; there was no interaction effect between ELS and cocaine dependence. The cluster analysis identified four distinct personality profiles: Open, Gregarious, Dysphoric, and Closed. Distribution of these profiles across subject groups differed significantly. Inclusion in cocaine+/ELS+, cocaine-/ELS+, and cocaine-/ELS- groups significantly increased the odds of expressing the Dysphoric, Open and Gregarious profiles, respectively.Cocaine dependence and early life stress were significantly and differentially associated with altered expression of individual personality traits and their aggregation as personality profiles, suggesting that individuals who are at-risk for developing addictions due to ELS exposure may benefit from personality centered approaches as an early intervention and prevention.

Recently, hydroxylated metabolites of JWH-018, a synthetic cannabinoid found in many K2/Spice preparations, have been shown to retain affinity and activity for cannabinoid type 1 receptors (CB1Rs). The activity of glucuronidated metabolites of JWH-018 is not known; hence, this study investigated the affinity and activity of a major metabolite, JWH-018-N-(5-hydroxypentyl) β-D-glucuronide (018-gluc), for CB1Rs. The 018-gluc binds CB1Rs (K(i) = 922 nM), has no effect on G-protein activity, but antagonizes JWH-018 activity at CB1Rs. The data suggests that hydroxylation by cytochrome P450s and subsequent glucuronidation by UDP-glucuronosyltransferases produces a metabolite, 018-gluc, which possesses antagonistic activity at CB1Rs.

Human users of synthetic cannabinoids (SCBs) JWH-018 and JWH-073 typically smoke these drugs, but preclinical studies usually rely on injection for drug delivery. We used the cannabinoid tetrad and drug discrimination to compare in vivo effects of inhaled drugs with injected doses of these two SCBs, as well as with the phytocannabinoid Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Mice inhaled various doses of Δ(9)-THC, JWH-018 or JWH-073, or were injected intraperitoneally (IP) with these same compounds. Rectal temperature, tail flick latency in response to radiant heat, horizontal bar catalepsy, and suppression of locomotor activity were assessed in each animal. In separate studies, mice were trained to discriminate Δ(9)-THC (IP) from saline, and tests were performed with inhaled or injected doses of the SCBs. Both SCBs elicited Δ(9)-THC-like effects across both routes of administration, and effects following inhalation were attenuated by pretreatment with the CB1 antagonist/inverse agonist rimonabant. No cataleptic effects were observed following inhalation, but all compounds induced catalepsy following injection. Injected JWH-018 and JWH-073 fully substituted for Δ(9)-THC, but substitution was partial (JWH-073) or required relatively higher doses (JWH-018) when drugs were inhaled. These studies demonstrate that the SCBs JWH-018 and JWH-073 elicit dose-dependent, CB1 receptor-mediated Δ(9)-THC-like effects in mice when delivered via inhalation or via injection. Across these routes of administration, differences in cataleptic effects and, perhaps, discriminative stimulus effects, may implicate the involvement of active metabolites of these compounds.

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.

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.

Triterpenoids such as betulinic acid (BA) and synthetic analogs of oleanolic acid [2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO)] and glycyrrhetinic acid [2-cyano-3,11-dioxo-18β-oleana-1,12-dien-30-oc acid (CDODA)] are potent anticancer agents that exhibit antiproliferative, antiangiogenic, anti-inflammatory and pro-apoptotic activities. Although their effects on multiple pathways have been reported, unifying mechanisms of action have not been reported. Studies in this laboratory have now demonstrated that several triterpenoids including BA and some derivatives, celastrol, methyl ursolatee, β-boswellic acid derivatives, and the synthetic analogs CDDO, CDODA and their esters decreased expression of specificity protein (Sp) transcription factors and several pro-oncogenic Sp-regulated genes in multiple cancer cell lines. The mechanisms of this response are both compound- and cell context-dependent and include activation of both proteasome-dependent and -independent pathways. Triterpenoid-mediated induction of reactive oxygen species (ROS) has now been characterized as an important proteasome-independent pathway for downregulation of Sp transcription factors. ROS decreases expression of microRNA-27a (miR-27a) and miR-20a/miR-17-5p and this results in the induction of the transcriptional “Sp-repressors” ZBTB10 and ZBTB4, respectively, which in turn downregulate Sp and Sp-regulated genes. Triterpenoids also activate or deactive nuclear receptors and G-protein coupled receptors, and these pathways contribute to their antitumorigenic activity and may also play a role in targeting Sp1, Sp3 and Sp4 which are highly overexpressed in multiple cancers and appear to be important for maintaining the cancer phenotype.

The β2-AR (β2-adrenergic receptor) is an important target for respiratory and CVD (cardiovascular disease) medications. Clinical studies suggest that N-terminal polymorphisms of β2-AR may act as disease modifiers. We hypothesized that polymorphisms at amino acids 16 and 27 result in differential trafficking and down-regulation of β2-AR variants following β-agonist exposure. The functional consequences of the four possible combinations of these polymorphisms in the human β2-AR (designated β2-AR-RE, β2-AR-GE, β2-AR-RQ and β2-AR-GQ) were studied using site-directed mutagenesis and recombinant expression in HEK-293 cells (human embryonic kidney cells). Ligand-binding assays demonstrated that after 24 h exposure to 1 μM isoprenaline, isoforms with Arg16 (β2-AR-RE and β2-AR-RQ) underwent increased down-regulation compared with isoforms with Gly16 (β2-AR-GE and β2-AR-GQ). Consistent with these differences in down-regulation between isoforms, prolonged isoprenaline treatment resulted in diminished cAMP response to subsequent isoprenaline challenge in β2-AR-RE relative to β2-AR-GE. Confocal microscopy revealed that the receptor isoforms had similar co-localization with the early endosomal marker EEA1 following isoprenaline treatment, suggesting that they had similar patterns of internalization. None of the isoforms exhibited significant co-localization with the recycling endosome marker Rab11 in response to isoprenaline treatment. Furthermore, we found that prolonged isoprenaline treatment led to a higher degree of co-localization of β2-AR-RE with the lysosomal marker LAMP1 (lysosome-associated membrane protein 1) compared with that of β2-AR-GE. Taken together, these results indicate that a mechanism responsible for differential responses of these receptor isoforms to the β-agonist involves differences in the efficiency with which agonist-activated receptors are trafficked to the lysosomes for degradation, or differences in degradation in the lysosomes.

Mu-opioid and CB1-cannabinoid agonists produce analgesia; however, adverse effects limit use of drugs in both classes. Additive or synergistic effects resulting from concurrent administration of low doses of mu- and CB1-agonists may produce analgesia with fewer side effects. Synergism potentially results from interaction between mu-opioid receptors (MORs) and CB1 receptors (CB1Rs). AM-251 and rimonabant are CB1R antagonist/inverse agonists employed to validate opioid–cannabinoid interactions, presumed to act selectively at CB1Rs. Therefore, the potential for direct action of these antagonists at MORs is rarely considered. This study determined if AM-251 and/or rimonabant directly bind and modulate the function of MORs. Surprisingly, AM-251 and rimonabant, but not a third CB1R inverse agonist AM-281, bind with mid-nanomolar affinity to human MORs with a rank order of affinity ( K i ) of AM-251 (251 nM) > rimonabant (652 nM) > AM281 (2135 nM). AM-251 and rimonabant, but not AM-281, also competitively antagonize morphine induced G-protein activation in CHO-hMOR cell homogenates ( K b = 719 or 1310 nM, respectively). AM-251 and rimonabant block morphine inhibition of cAMP production, while only AM-251 elicits cAMP rebound in CHO-hMOR cells chronically exposed to morphine. AM-251 and rimonabant (10 mg/kg) attenuate morphine analgesia, whereas the same dose of AM-281 produces little effect. Therefore, in addition to high CB1R affinity, AM-251 and rimonabant bind to MORs with mid-nanomolar affinity and at higher doses may affect morphine analgesia via direct antagonism at MORs. Such CB1-independent actions of these antagonists may contribute to reported inconsistencies when CB1/MOR interactions are examined via pharmacological methods in CB1-knockout versus wild-type mice.

Treatment of ErbB2-overexpressing BT474 and MDA-MB-453 breast cancer cells with 1 to 10 μmol/L betulinic acid inhibited cell growth, induced apoptosis, downregulated specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and decreased expression of ErbB2. Individual or combined knockdown of Sp1, Sp3, Sp4 by RNA interference also decreased expression of ErbB2 and this response was because of repression of YY1, an Sp-regulated gene. Betulinic acid–dependent repression of Sp1, Sp3, Sp4, and Sp-regulated genes was due, in part, to induction of the Sp repressor ZBTB10 and downregulation of microRNA-27a (miR-27a), which constitutively inhibits ZBTB10 expression, and we show for the first time that the effects of betulinic acid on the miR-27a:ZBTB10-Sp transcription factor axis were cannabinoid 1 (CB1) and CB2 receptor–dependent, thus identifying a new cellular target for this anticancer agent. Mol Cancer Ther; 11(7); 1421–31. ©2012 AACR.

The therapeutic promise of trans-resveratrol (tRes) is limited by poor bioavailability following rapid metabolism. We hypothesise that trans-arachidin-1 (tA1) and trans-arachidin-3 (tA3), peanut hairy root-derived isoprenylated analogs of tRes, will exhibit slower metabolism/enhanced bioavailability and retain biological activity via cannabinoid receptor (CBR) binding relative to their non-prenylated parent compounds trans-piceatannol (tPice) and tRes, respectively.The activities of eight human UDP-glucuronosyltransferases (UGTs) toward these compounds were evaluated. The greatest activity was observed for extrahepatic UGTs 1A10 and 1A7, followed by hepatic UGTs 1A1 and 1A9. Importantly, an additional isoprenyl and/or hydroxyl group in tA1 and tA3 slowed overall glucuronidation. CBR binding studies demonstrated that all analogs bound to CB1Rs with similar affinities (5-18 µM); however, only tA1 and tA3 bound appreciably to CB2Rs. Molecular modelling studies confirmed that the isoprenyl moiety of tA1 and tA3 improved binding affinity to CB2Rs. Finally, although tA3 acted as a competitive CB1R antagonist, tA1 antagonised CB1R agonists by both competitive and non-competitive mechanisms.Prenylated stilbenoids may be preferable alternatives to tRes due to increased bioavailability via slowed metabolism. Similar structural analogs might be developed as novel CB therapeutics for obesity and/or drug dependency.