Cannabinoid CB1 Receptor Binding Research Articles

Cannabidiol improves behavioural and neurochemical deficits in adult female offspring of the maternal immune activation (poly I:C) model of neurodevelopmental disorders

Cognitive impairment is a major source of disability in schizophrenia and current antipsychotic drugs (APDs) have minimal efficacy for this symptom domain. Cannabidiol (CBD), the major non-intoxicating component of Cannabis sativa L., exhibits antipsychotic and neuroprotective properties. We recently reported the effects of CBD on cognition in male offspring of a maternal immune activation (polyinosinic-polycytidilic acid (poly I:C)) model relevant to the aetiology of schizophrenia; however, the effects of CBD treatment in females are unknown. Sex differences are observed in the onset of schizophrenia symptoms and response to APD treatment. Furthermore, the endogenous cannabinoid system, a direct target of CBD, is sexually dimorphic in humans and rodents. Therefore, the present work aimed to assess the therapeutic impact of CBD treatment on behaviour and neurochemical signalling markers in female poly I:C offspring. Time-mated pregnant Sprague-Dawley rats (n = 16) were administered poly I:C (4 mg/kg; i.v.) or saline (control) on gestational day 15. From postnatal day 56, female offspring received CBD (10 mg/kg, i.p.) or vehicle treatment for approximately 3 weeks. Following 2 weeks of CBD treatment, offspring underwent behavioural testing, including the novel object recognition, rewarded alternation T-maze and social interaction tests to assess recognition memory, working memory and sociability, respectively. After 3 weeks of CBD treatment, the prefrontal cortex (PFC) and hippocampus (HPC) were collected to assess effects on endocannabinoid, glutamatergic and gamma-aminobutyric acid (GABA) signalling markers. CBD attenuated poly I:C-induced deficits in recognition memory, social interaction and glutamatergic N-methyl-d-aspartate receptor (NMDAR) binding in the PFC of poly I:C offspring. Working memory performance was similar between treatment groups. CBD also increased glutamate decarboxylase 67, the rate-limiting enzyme that converts glutamate to GABA, and parvalbumin protein levels in the HPC. In contrast to the CBD treatment effects observed in poly I:C offspring, CBD administration to control rats reduced social interaction, cannabinoid CB1 receptor and NMDAR binding density in the PFC, suggesting that CBD administration to healthy rats may have negative consequences on social behaviour and brain maturation in adulthood. Overall, the findings of this study support the therapeutic benefits of CBD on recognition memory and sociability in female poly I:C offspring, and provide insight into the neurochemical changes that may underlie the therapeutic benefits of CBD in the poly I:C model.

Effect of cannabidiol on endocannabinoid, glutamatergic and GABAergic signalling markers in male offspring of a maternal immune activation (poly I:C) model relevant to schizophrenia

The mainstay treatment for schizophrenia is antipsychotic drugs (APDs), which are mostly effective against the positive symptoms (e.g. hallucinations), but provide minimal benefits for the negative symptoms (e.g. social withdrawal) and cognitive deficits. We have recently shown that treatment with the non-intoxicating phytocannabinoid, cannabidiol (CBD), can improve cognition and social interaction deficits in a maternal immune activation (MIA) model relevant to the aetiology of schizophrenia, however, the mechanisms underlying this effect are unknown. An imbalance in the main excitatory (glutamate) and inhibitory (GABA) neurotransmitter systems in the brain plays a role in the pathophysiology of schizophrenia. Therefore, the endocannabinoid system could represent a therapeutic target for schizophrenia as a regulator of glutamate and GABA release via the CB1 receptor (CB1R). This study investigated the effects of chronic CBD treatment on markers of glutamatergic, GABAergic and endocannabinoid signalling in brain regions implicated in social behaviour and cognitive function, including the prefrontal cortex (PFC) and hippocampus (HPC). Time-mated pregnant Sprague-Dawley rats (n = 16) were administered poly I:C (4 mg/kg, i.v.) or saline (control) on gestational day 15. Male offspring were injected with CBD (10 mg/kg, i.p.) or vehicle twice daily from postnatal day 56 for 3 weeks. The prefrontal cortex (PFC) and hippocampus (HPC) were collected for post-mortem receptor binding and Western blot analyses (n = 8 per group). CBD treatment attenuated poly I:C-induced deficits in cannabinoid CB1 receptor binding in the PFC and glutamate decarboxylase 67, the enzyme that converts glutamate to GABA, in the HPC. CBD treatment increased parvalbumin levels in the HPC, regardless of whether offspring were exposed to poly I:C in utero. Conversely, CBD did not affect N-methyl-d-aspartate receptor and gamma-aminobutyric acid (GABA) A receptor binding or protein levels of fatty acid amide hydrolase, the enzyme that degrades the endocannabinoid, anandamide. Overall, these findings show that CBD can restore cannabinoid/GABAergic signalling deficits in regions of the brain implicated in schizophrenia pathophysiology following maternal poly I:C exposure. These findings provide novel evidence for the potential mechanisms underlying the therapeutic effects of CBD treatment in the poly I:C model.

42.4 THE ENDOCANNABINOID SYSTEM IN FIRST-EPISODE PSYCHOSIS

BackgroundThe endocannabinoid (EC) system comprises of fatty acid neurotransmitters such as anandamide and 2-acylglycerol, at least two specific cannabinoid (CB) receptor subtypes and enzyme machinery for synthesis and degradation of ECs. The EC system regulates a wide array of functions in central nervous system and peripheral tissues such as brain development, plasticity, reward and stress sensitivity as well as metabolic functions a such as energy/lipid metabolism including liver function and insulin resistance (1). One of the aims of the METSY project (www.metsy.eu) is to study the central and peripheral EC system in FEPs and their involvement in brain mechanisms in psychosis as well as related metabolic co-morbidities. The cannabinoid CB1 receptor (CB1R) is the most abundant cannabinoid receptor subtype in the human brain and is the predominant mediator of various EC effects. In this first series of METSY experiments we report in vivo brain CB1R binding characteristics in patients with first-episode psychosis (FEP) and controls.MethodsBrain CB1R availability was measured using [18F]-FMPEP-d2 and 3D ECAT HRRT positron emission tomography (PET) using distribution volume (DVt) as a proxy for CB1R availability. Our studies in healthy volunteers revealed a marked sex difference in [18F]-FMPEP DVt with males having higher CB1R availability. The overall CB1R sex difference was widespread but also regionally different with most significant effects in the occipital cortex. Therefore, subsequent studies focused first on male patients with FEP and controls (n=18).ResultsThe results indicate a significant and relatively widespread decrease in CB1R DVt in patients with FEP in fronto-temporal regions, putamen, posterior cingulate as well as parietal regions with large effect sizes. Largely similar results were seen in an independent sample of medication-free male patients with FEP (n=17, the other METSY PET site, King’s College, London, UK) (2). Those studies used another validated CB1R tracer, the [11C]-MePPEP. The reduced CB1R in patients was also state-dependent as [18F]-FMPEP DVt correlated negatively with psychotic symptoms (BPRS) in a highly significant manner.DiscussionEpidemiological studies have convincingly shown that cannabis use is associated with an increased risk of both psychotic symptoms and schizophrenia-like psychoses. We now provide direct in vivo evidence for a robust and partly state-dependent dysregulation of the endocannabinoid system in first-episode psychosis. This is well in line with a recent PET study with CB1R tracer, [11C]-OMAR in patients with schizophrenia (3). Based on previous experimental data the reduced CB1R availability is indicative of an increased endocannabinoid drive in psychosis.AcknowledgementsFunding from the EU’s 7th Framework Programme; METSY - Neuroimaging platform for characterization of metabolic co-morbidities in psychotic disorders (no. 602478).References1) Eriksson O et al The Cannabinoid Receptor-1 Is an Imaging Biomarker of Brown Adipose Tissue. J Nucl Med. 2015 Dec;56(12):1937–41.2) METSY: Endocannabinoid pathways in early psychosis – brain imaging studies (Hietala J, Howes O, Laurikainen H, Borgan F and the METSY PET Imaging group).3) Ranganathan M et al Reduced Brain Cannabinoid Receptor Availability in Schizophrenia. Biol Psychiatry. 2016 Jun 15;79(12):997–1005.

Neonatal lipopolysaccharide treatment has long‐term effects on monoaminergic and cannabinoid receptors in the rat

Brain inflammation in early life has been proposed to play important roles in the development of anxiety and psychosis-related behaviors in adulthood, behaviors that rely on the integrity of dopamine and/or serotonin systems. Moreover recent behavioral and anatomical evidence suggests involvement of CB1 receptors in the control of emotion and mood. In this study, we determined the effects of neonatal LPS treatment on dopamine, serotonin, and cannabinoid receptor binding in adulthood. Rats were treated with the bacterial endotoxin lipopolysaccharide (LPS) on postnatal day (PND) 3 and 5. Dopamine D1, D2, serotonin 5HT1A, 5HT2A, and serotonin transporter and cannabinoid CB1 receptor binding across several brain regions were measured autoradiographically in adulthood (PND 85). Neonatal LPS treatment caused a significant increase in dopamine D2 in the nucleus accumbens and olfactory tubercle, a decrease in 5HT1A receptor binding in the hippocampus CA1 and ventromedial hypothalamus. A decrease in CB1 receptor binding after neonatal LPS was observed in the amygdala. Neonatal LPS had no significant impact on dopamine D1, serotonin 5HT2A or serotonin transporter binding in any of the brain regions examined. Our results suggest long lasting, region specific effects and differential impact on dopamine, serotonin and cannabinoid receptor systems following neonatal inflammation, that may form the basis for compromised anxiety and psychosis related behaviors.

Age of stress exposure modulates the immediate and sustained effects of repeated stress on corticolimbic cannabinoid CB1 receptor binding in male rats

Chronic stress is known to modulate cannabinoid CB1 receptor binding densities in corticolimbic structures, in a region-dependent manner; however, the ontogeny of these changes and the degree to which they recover following exposure to stress have yet to be determined. To this extent, we examined both the immediate and sustained effects (following a 40-day recovery period) of a repeated restraint stress paradigm (30-min restraint/day for 10days) on CB1 receptor binding in the prefrontal cortex (PFC), hippocampus and amygdala in both adolescent (stress onset at post-natal day [PND] 35) and adult (stress onset at PND 75) male Sprague–Dawley rats. Consistent with previous reports, we found that repeated stress in adult rats resulted in an increase in CB1 receptor binding in the PFC, a reduction in CB1 receptor binding in the hippocampus and no effect in the amygdala. Interestingly, adolescent rats exposed to repeated restraint stress did not show any change in hippocampal CB1 receptor density, but exhibited an upregulation of CB1 receptor binding in both the PFC and amygdala. In adults, a 40-day recovery period resulted in a normalization of CB1 receptor binding in the PFC, and surprisingly a pronounced upregulation of CB1 receptor binding in the hippocampus, possibly indicative of a rebound effect. Adolescents similarly exhibited this rebound increase in hippocampal CB1 receptor binding, despite a lack in immediate downregulation following repeated restraint. Of particular interest, adolescents exposed to stress were found to have a sustained downregulation of prefrontocortical CB1 receptors in adulthood, which may relate to some of the reported sustained behavioral effects of stress in adolescence. Collectively, these data indicate that the effects of chronic stress on cannabinoid CB1 receptor binding are modulated by the age of stress exposure and period of recovery following the cessation of stress.

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

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

Reduced cannabinoid CB1 receptor binding in alcohol dependence measured with positron emission tomography

Brain cannabinoid CB1 receptors contribute to alcohol-related behaviors in experimental animals, but their potential role in humans with alcohol dependence is poorly understood. We measured CB1 receptors in alcohol dependent patients in early and protracted abstinence, and in comparison with control subjects without alcohol use disorders, using positron emission tomography and [(18)F]FMPEP-d2, a radioligand for CB1 receptors. We scanned 18 male in-patients with alcohol dependence twice, within 3-7 days of admission from ongoing drinking, and after 2-4 weeks of supervised abstinence. Imaging data were compared with those from 19 age-matched healthy male control subjects. Data were also analyzed for potential influence of a common functional variation (rs2023239) in the CB1 receptor gene (CNR1) that may moderate CB1 receptor density. On the first scan, CB1 receptor binding was 20-30% lower in patients with alcohol dependence than in control subjects in all brain regions and was negatively correlated with years of alcohol abuse. After 2-4 weeks of abstinence, CB1 receptor binding remained similarly reduced in these patients. Irrespective of the diagnostic status, C allele carriers at rs2023239 had higher CB1 receptor binding compared with non-carriers. Alcohol dependence is associated with a widespread reduction of cannabinoid CB1 receptor binding in the human brain and this reduction persists at least 2-4 weeks into abstinence. The correlation of reduced binding with years of alcohol abuse suggests an involvement of CB1 receptors in alcohol dependence in humans.

Positron emission tomography shows elevated cannabinoid CB1 receptor binding in men with alcohol dependence.

Several lines of evidence link cannabinoid (CB) type 1 (CB (1) ) receptor-mediated endogenous CB (eCB) signaling to the etiology of alcohol dependence (AD). However, to date, only peripheral measures of eCB function have been collected in living humans with AD and no human in vivo data on the potentially critical role of the brain CB (1) receptor in AD have been published. This is an important gap in the literature, because recent therapeutic developments suggest that these receptors could be targeted for the treatment for AD. Medication-free participants were scanned during early abstinence 4weeks after their last drink. Using positron emission tomography (PET) with a high-resolution research tomograph and the CB (1) receptor selective radiotracer [(11) C]OMAR, we determined [(11) C]OMAR volume of distribution ( V (T) ) values, a measure of CB (1) receptor density, in a priori selected brain regions in men with AD (n=8, age 37.4±7.9years; 5 smokers) and healthy control (HC) men (n=8, age 32.5±6.9years; all nonsmokers). PET images reconstructed using the MOLAR algorithm with hardware motion correction were rigidly aligned to the subject-specific magnetic resonance (MR) image, which in turn was warped to an MR template. Time-activity curves (TACs) were extracted from the dynamic PET data using a priori selected regions of interest delineated in the MR template space. In AD relative to HC, [(11) C]OMAR V (T) values were elevated by approximately 20% (p=0.023) in a circuit, including the amygdala, hippocampus, putamen, insula, anterior and posterior cingulate cortices, and orbitofrontal cortex. Age, body mass index, or smoking status did not influence the outcome. These findings agree with preclinical evidence and provide the first, albeit still preliminary in vivo evidence suggesting a role for brain CB (1) receptors in AD. The current study design does not answer the important question of whether elevated CB (1) receptors are a preexisting vulnerability factor for AD or whether elevations develop as a consequence of AD.

Alterations to Melanocortinergic, GABAergic and Cannabinoid Neurotransmission Associated with Olanzapine-Induced Weight Gain

Background/AimSecond generation antipsychotics (SGAs) are used to treat schizophrenia but can cause serious metabolic side-effects, such as obesity and diabetes. This study examined the effects of low to high doses of olanzapine on appetite/metabolic regulatory signals in the hypothalamus and brainstem to elucidate the mechanisms underlying olanzapine-induced obesity.Methodology/ResultsLevels of pro-opiomelanocortin (POMC), neuropeptide Y (NPY) and glutamic acid decarboxylase (GAD65, enzyme for GABA synthesis) mRNA expression, and cannabinoid CB1 receptor (CB1R) binding density (using [3H]SR-141716A) were examined in the arcuate nucleus (Arc) and dorsal vagal complex (DVC) of female Sprague Dawley rats following 0.25, 0.5, 1.0 or 2.0 mg/kg olanzapine or vehicle (3×/day, 14-days). Consistent with its weight gain liability, olanzapine significantly decreased anorexigenic POMC and increased orexigenic NPY mRNA expression in a dose-sensitive manner in the Arc. GAD65 mRNA expression increased and CB1R binding density decreased in the Arc and DVC. Alterations to neurotransmission signals in the brain significantly correlated with body weight and adiposity. The minimum dosage threshold required to induce weight gain in the rat was 0.5 mg/kg olanzapine.ConclusionsOlanzapine-induced weight gain is associated with reduced appetite-inhibiting POMC and increased NPY. This study also supports a role for the CB1R and GABA in the mechanisms underlying weight gain side-effects, possibly by altering POMC transmission. Metabolic dysfunction can be modelled in the female rat using low, clinically-comparable olanzapine doses when administered in-line with the half-life of the drug.

Screening of selected Asian spices for anti obesity-related bioactivities

The potential health effects of 30 spices, commonly used for daily consumption, were submitted to bioactivity screening with several anti-obesity related bioassays: adenosine A1 receptor binding, cannabinoid CB1 receptor binding, TNF-α and 3T3-L1 adipocytes differentiation induction. Sesame seed and red chilli exhibited high binding activity to the adenosine A1 receptor and nutmeg, mace, black pepper and turmeric to the cannabinoid CB1 receptor, while piment and turmeric showed high inhibition of TNF-α accumulation. Black onion seed proved to be the only spice with high 3T3-L1 adipocyte differentiation induction activity. Several well known major compounds found in these active spices were tested with the respective bioassays but did not show activity. Thus, it appears that other minor compounds or the synergistic effects of different constituents are responsible for the observed activity.

Influence of chronic bromocriptine and levodopa administration on cerebral type 1 cannabinoid receptor binding

The endocannabinoid system is an important modulatory system in the brain. Complex interactions with brain dopaminergic circuits have been demonstrated. The aim of this study was to investigate the in vivo effect of the commonly used antiparkinsonian drugs, levodopa (L-DOPA) and bromocriptine, on type 1 cannabinoid (CB1) receptors, using the PET radioligand [(18)F]MK-9470. Seventeen female Wistar rats were studied at baseline and after chronic exposure to either L-DOPA (6 mg/kg/day with 1.5 mg/kg/day carbidopa; n = 6), bromocriptine (4 mg/kg/day; n = 5), or saline (n = 6). [(18)F]MK-9470 binding was assessed in vivo using small animal PET imaging. [(18)F]MK-9470 parametric images were generated, anatomically standardized to Paxinos space and analyzed by voxel-based statistical parametric mapping (SPM2) and a predefined volume-of-interest (VOI) approach. In a 2 x 2 analysis design (condition vs. treatment), no significant changes in absolute or relative [(18)F]MK-9470 binding were present upon chronic exposure to L-DOPA or bromocriptine as compared to saline treatment. The post hoc comparison of chronic scans to baseline within each treatment modality showed regional increases in relative [(18)F]MK-9470 binding in the thalamus (peak average value +6.3%) and in the sensorimotor cortex and hippocampus (peak average value +10.2%) after bromocriptine exposure, while no changes were found for L-DOPA. Chronic administration of L-DOPA and bromocriptine at the applied doses does not produce major cerebral changes in in vivo cannabinoid CB1 receptor binding of [(18)F]MK-9470 in the rat brain. These results also suggest that similar chronic L-DOPA and bromocriptine usage is unlikely to interfere with human PET imaging in healthy conditions using this radioligand.

Role of cannabis and endocannabinoids in the genesis of schizophrenia

Cannabis abuse and endocannabinoids are associated to schizophrenia. It is important to discern the association between schizophrenia and exogenous Cannabis sativa, on one hand, and the endogenous cannabinoid system, on the other hand. On one hand, there is substantial evidence that cannabis abuse is a risk factor for psychosis in genetically predisposed people, may lead to a worse outcome of the disease, or it can affect normal brain development during adolescence, increasing the risk for schizophrenia in adulthood. Regarding genetic predisposition, alterations affecting the cannabinoid CNR1 gene could be related to schizophrenia. On the other hand, the endogenous cannabinoid system is altered in schizophrenia (i.e., increased density of cannabinoid CB1 receptor binding in corticolimbic regions, enhanced cerebrospinal fluid anandamide levels), and dysregulation of this system can interact with neurotransmitter systems in such a way that a "cannabinoid hypothesis" can be integrated in the neurobiological hypotheses of schizophrenia. Finally, there is also evidence that some genetic alterations of the CNR1 gene can act as a protectant factor against schizophrenia or can induce a better pharmacological response to atypical antipsychotics. Cannabis abuse is a risk factor for psychosis in predisposed people, it can affect neurodevelopment during adolescence leading to schizophrenia, and a dysregulation of the endocannabinoid system can participate in schizophrenia. It is also worth noting that some specific cannabinoid alterations can act as neuroprotectant for schizophrenia or can be a psychopharmacogenetic rather than a vulnerability factor.

Chronic stress differentially regulates cannabinoid CB1 receptor binding in distinct hippocampal subfields

Exposure to chronic stress has been found to decrease cannabinoid CB1 receptor expression in the hippocampus; however, the specificity of this phenomenon to specific subfields of the hippocampus has not been characterized. To this extent, male Sprague-Dawley rats were exposed to 21 days of restraint stress (6 h/day), after which autoradiographical analysis of cannabinoid CB1 receptor binding site densities were examined in the CA1, CA3 and dentate gyrus subfields of the hippocampus. Chronic stress was found to produce a significant reduction in cannabinoid CB1 receptor binding in the dentate gyrus, while increasing cannabinoid CB1 receptor binding in the CA3. There was no effect of chronic stress on cannabinoid CB1 receptor binding in the CA1, or two other proximal regions, the retrosplenial cortical gyrus and the laterodorsal thalamus. Given the role of hippocampal cannabinoid CB1 receptor activity in the maintenance of synaptic integrity and neuroplasticity in the hippocampus, these data suggest that changes in cannabinoid CB1 receptor activity following stress may contribute to stress-induced modulation of these processes.

Adaptations of Striatal Endocannabinoid System During Stress

The endocannabinoid system (ECS) plays a fundamental role in the regulation of synaptic transmission. Exposure to stressful events triggers synaptic adaptations in many brain areas. The activity of the ECS in stress-responsive neural circuits suggests that it may be involved in the behavioral responses and synaptic effects typical of stress. In this review, we discuss evidence demonstrating that striatal ECS is modulated by stress. Chronic stress exposure alters endocannabinoid levels, cannabinoid CB1 receptor binding and cannabinoid CB1 receptor-mediated control of inhibitory synaptic transmission in the striatum. Recent studies have shown that impairment of endocannabinoid signalling is associated with inability to adapt to chronic stress and to the development of maladaptive behaviors. The ECS represents a novel potential pharmacological target to treat stress-associated neuropsychiatric conditions.

HU210-Induced Downregulation in Cannabinoid CB1 Receptor Binding Strongly Correlates with Body Weight Loss in the Adult Rat

In vitro autoradiography was used to examine changes in cannabinoid CB1 receptors (targeted with [(3)H] CP55,940) in rats treated with the potent cannabinoid agonist HU210. Animals were administered with HU210 (25, 50, 100 microg/kg) for 4 or 14 days or received a single 100 microg/kg injection of HU210 and sacrificed 24 h later. The acute dose resulted in a decrease in binding in the caudate putamen and hippocampus. A dose dependent, region-specific reduction (P < 0.0001) in [(3)H] CP55,940 binding was seen in all brain regions examined after 4 and 14 days treatment. A decrease in body weight was recorded during the first 4 days of treatment but after this animals began to gain weight. Correlations (0.865 < r < 0.659, P < 0.0001) between body weight on day four and CB1 receptor binding were found in all brain regions examined suggesting that downregulation of CB1 receptors may contribute to the induction of tolerance to body weight loss induced by HU210.

Long-term consequences of URB597 administration during adolescence on cannabinoid CB1 receptor binding in brain areas

Despite the alarming increment in the use and abuse of cannabis preparations among young people, little is known about possible long-term consequences of targeting the endocannabinoid system during the critical developmental period of adolescence. Therefore, we aimed to analyze possible long-lasting neurobiological consequences of enhancing endocannabinoid signalling during adolescence, by means of blocking anandamide (AEA) hydrolysis. Adolescent Wistar male rats were administered an inhibitor of AEA hydrolysis, i.e. URB597 (0, 0.1 or 0.5 mg/kg/day from postnatal days 38 to 43). The expression of brain cannabinoid receptor type 1 (CB1R) was then analyzed by [ 3H]CP-55,940 auto-radiographic binding at adulthood. Repeated URB597 administration during adolescence persistently modified CB1R binding in a region-dependent manner. A long-lasting decrease of CB1R binding levels was found in caudate-putamen, nucleus accumbens, ventral tegmental area and hippocampus, while an opposite increment was observed in the locus coeruleus. Present results provide evidence for long-lasting effects of adolescent URB597 administration. Activation of endocannabinoid transmission during the still plastic phase of adolescence may have implications for the maturational end-point of the endocannabinoid system itself, which could lead to permanent alterations in neuronal brain circuits and behavioural responses. Insights into the developmental trajectories of this neuromodulatory system may help us to better understand and prevent outcomes of neonatal and adolescent cannabis exposure.

Design, chemical synthesis, and biological evaluation of novel triazolyl analogues of taranabant (MK-0364), a cannabinoid-1 receptor inverse agonist

Being obese has various health problems that are related to type 2 diabetes mellitus, cardiovascular disease, hypertension, hyperlipidemia, and fibrinolytic abnormalities. Merck's taranabant (MK-0364), a CB1R inverse agonist, is currently in Phase 3 clinical trials, and is being actively pursued by Merck toward obesity market. Merck intends to file for FDA approval of taranabant in 2008. In order to increase solubility and potency of taranabant, or even possibly improve safety, novel triazole analogues of taranabant have been designed and synthesized. We introduced a pivotal asymmetric center via the Evans chiral auxiliary methodology and set up 1,2,4-triazole via substitution of α-bromoketone. Subsequently, diastereoselective reduction was accomplished to install adjacent essential asymmetric center. This method allowed us to prepare readily sub-gram scale of the target compounds in a convenient way. The synthesized analogues were subjected to biological evaluation involving cannabinoid CB1 receptor binding affinity. While the parent taranabant bears highly potent binding affinity to cannabinoid CB1 receptor, neither of the analog structures improved CB1 receptor binding affinities.

The design, synthesis and testing of desoxy-CBD: Further evidence for a region of steric interference at the cannabinoid receptor

Cannabidiol CBD, a non-psychoactive constituent of marihuana, has been reported to possess essentially no affinity for cannabinoid CB1 receptor binding sites in the brain. Our hypothesis concerning CBD's lack of affinity for the cannabinoid CB1 receptor is that CBD is not capable of clearing a region of steric interference at the CB1 receptor and thereby not able to bind to this receptor. We have previously characterized this region of steric interference at the CB1 receptor [P.H. Reggio, A.M. Panu, S. Miles J. Med. Chem. 36, 1761–1771 (1993)] in three dimensions using the Active Analog Approach. We report here a conformational analysis of CBD which, in turn, led to the design of a new analog, desoxy-CBD. Modeling results for desoxy-CBD predict that this compound is capable of clearing the region of steric interference by expending 3.64 kcal/mol of energy in contrast to the 12.39 kcal/mol expenditure required by CBD. Desoxy-CBD was synthesized by condensation of 3-pentylphenol with p-mentha-2,8-dien-l-ol mediated by DMF-dineopental acetal. Desoxy-CBD was found to behave as a partial agonist in the mouse vas deferens assay, an assay which is reported to detect the presence of cannabinoid receptors. The compound produced a concentration related inhibition of electrically-evoked contractions of the mouse vas deferens, possessing an ic 50 of 30.9 nM in this assay. Taken together, these results support the hypothesis of the existence of a region of steric interference at the CB1 receptor. While the energy expenditure to clear this region was too high for the parent compound, CBD, the removal of the C6' hydroxyl of CBD produced a molecule (desoxy-CBD) able to clear this region and produce activity, albeit at a reduced level.