Role Of CB Research Articles

Fluorescence Sensor for Water in Organic Solvent Using Graphene Oxide- Rhodamine B and Cucurbit[7]uril.

Detection of water in organic solvents gained much importance as these solvents have been used as a medium for conducting organic reactions and water was considered as an inhibitor, when it is present in the reaction medium. There are number of methods available to measure the water content in organic solvents, however, such methods are time consuming and expensive. Here, we developed a facile method for detecting water in organic solvents using an inexpensive fluorescent probe - Rhodamine B decorated Graphene oxide (RBGO). The fluorescent probe, RBGO can be conveniently prepared by mixing the rhodamine B (RB) with graphene oxide (GO) in water. However, the probe will function as a sensor for water in the organic solvents through the release of dye upon interaction with the water present in organic solvents. Surprisingly, combination of cucurbit[7]uril (CB[7]) and RBGO increased the sensitivity of this sensor dramatically for the detection of water. This is the first example of water sensor with best detection limit by the involvement of host molecules such as CB[7]. This sensor displayed the low limit of detection (LOD) for organic solvents (LOD: 0.0015% for DMSO, 0.0025% for DMF), through the two-way process such as decomplexation and encapsulation. We presume that the role of CB [7] can be implemented in other similar sensors to enhance the sensitivity.

Hybrid Macrocyclic Polymers: Self-Assembly Containing Cucurbit[m]uril-pillar[n]arene.

Supramolecular self-assembly by hybrid macrocycles containing both cucurbit[m]uril (CB[m]) and pillar[n]arene was discussed and summarized in this review. Due to different solubility, diverse-sized cavities, and various driving forces in recognizing guests, the role of CB[m] and pillar[n]arene in such hybrid macrocyclic systems could switch between competitor in capturing specialized guests, and cooperator for building advanced hybridized macrocycles, by controlling their characteristics in host–guest inclusions. Furthermore, both CB[m] and pillar[n]arene were employed for fabricating advanced supramolecular self-assemblies such as mechanically interlocked molecules and supramolecular polymers. In those self-assemblies, CB[m] and pillar[n]arene played significant roles in, e.g., microreactor for catalyzing particular reactions to bridge different small pieces together, molecular “joint” to connect different monomers into larger assemblies, and “stabilizer” in accommodating the guest molecules to adopt a favorite structure geometry ready for assembling.

Abstract P2-16-23: Weekly vs. every-3-week (q3wk) carboplatin (Cb) with paclitaxel (P) in neoadjuvant chemotherapy (NACT) for triple-negative breast cancer (TNBC): A retrospective analysis background

Abstract Background: Randomized studies have demonstrated that adding Cb to P as part of NACT for stage II-III TNBC significantly increases pathologic complete response (pCR) rates, but its optimal dose and schedule are uncertain. Since hematologic toxicities associated with higher dose (AUC 5-6) q3wk Cb with P necessitate treatment delays or omissions in some patients (pts), some oncologists favor administering lower dose (AUC 1.5-2) weekly Cb with P instead. While many believe that this approach is better tolerated, there is limited published data on its efficacy in TNBC. Oncologists at University of Vermont and Women and Infants Hospital of Rhode Island decided to analyze results achieved with weekly Cb/P in this setting, particularly pCR rate, and to compare them to those achieved with q3wk Cb/P. Methods: Databases at our institutions were reviewed to identify pts who received P and Cb as initial NACT for TNBC over the past 10 years for whom pathologic results and post-op follow-up were available. pCR was defined as ypT0/isN0, as reported by the institutional pathologists. We also assessed delivery of P and Cb. Disease-free survival was censored as of the patient’s last follow-up visit. Results: Of 76 pts identified (median age 49), 47 received weekly Cb/P and 29 q3wk Cb/P. All but 4 (all in the weekly Cb cohort) subsequently received doxorubicin and cyclophosphamide (AC). In the q3wk Cb cohort, 15 pts also received bevacizumab and 3 each may have received veliparib or atezolizumab on blinded clinical trials. pCR rates are tabulated below: Patient groupNpCR (95% CI)vs. q3week CbAll7654% (42-65%)Stage IIA-IIIA6058%Stage IIIB-IIIC1638%BRCA1/2 mutated989%q3week Cb/P2955% (36-74%)Weekly Cb/P4753% (38-68%)p>0.99 Most pts - 89% of weekly Cb and 90% of q3wk Cb - received ≥11 doses of weekly P; 83% of weekly Cb pts received ≥11 doses of Cb and 90% of q3wk Cb pts received all 4 planned doses of Cb. At median follow-up of 19 months (range <1-118), 93% of pts who achieved pCR and 74% of non-pCRs are free from recurrence; median f/u is much longer (54 vs. 14 months) for the q3wk Cb pts. Conclusions In this retrospective analysis, treatment with weekly Cb/P (typically followed by AC) resulted in a pCR rate similar to that seen with q3wk Cb/P, both in our own pts and as reported from CALGB 40603 (54%) and BrighTNess (58%). Given the ongoing debate on the role of Cb in NACT for TNBC, we believe that the weekly Cb/P regimen should be explored further, as it may be easier to administer without sacrificing efficacy. Citation Format: Kitty E Victoria, Pouyan N Changizzadeh, Bernard Cole, Pulluri Bhargavi, Jessica L Lyon, Marie E Wood, William M Sikov. Weekly vs. every-3-week (q3wk) carboplatin (Cb) with paclitaxel (P) in neoadjuvant chemotherapy (NACT) for triple-negative breast cancer (TNBC): A retrospective analysis background [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P2-16-23.

Evaluation of tolerability and efficacy of incorporating carboplatin in neoadjuvant anthracycline and taxane based therapy in a BRCA1 enriched triple-negative breast cancer cohort

PurposeThe addition of carboplatin (Cb) to neoadjuvant chemotherapy in triple-negative breast cancer (TNBC) has been demonstrated to improve pathologic complete response (pCR) at the expense of increased toxicity. We aimed to evaluate the effectiveness and tolerability of dose-dense anthracycline & cyclophosphamide (ddAC) followed by weekly paclitaxel (wT) in combination with weekly Cb. MethodsRetrospective data was collected on patients with clinical stage I-III TNBC treated with neoadjuvant ddAC-wTCb (four cycles of ddA 60 mg/m2 and ddC 600 mg/m2 every 2 weeks followed by 12 cycles of wT 80 mg/m2 with Cb AUC 1.5). Indices of tolerability and pCR were evaluated and compared to a historical cohort (n = 76) treated with ddAC-T. A secondary objective was to evaluate the rates of pCR by BRCA status. ResultsFor 43 eligible patients, mean age was 41.5 years, 51% had clinical stage II disease, 81.4% were clinically node positive and 32.6% carried a deleterious BRCA1 mutation. Only 35% completed all scheduled doses of chemotherapy. Grade 3/4 neutropenia was observed in 42.5% of patients. Overall pCR was 51.2%; 44.8% in BRCA wild-type compared to 64.3% in BRCA–associated TNBC (p = 0.232). pCR rates with ddAC-wTCb were similar to historic institutional rates with ddAC-T (51.2% vs. 51.3%, p = 0.987) and were comparable when stratified by BRCA status. In pooled multivariate analysis, only BRCA status (HR 4.00, 95%CI 1.65–9.75, p = 0.002) was significantly associated with pCR. ConclusionNeoadjuvant ddAC-wTCb is less tolerable in clinical practice compared to most clinical trials, with a pCR comparable to historic rates using non-platinum regimen. The role of Cb in neoadjuvant chemotherapy for BRCA mutated TNBC remains uncertain.

Effect of hyperthermia on calbindin-D 28k immunoreactivity in the hippocampal formation following transient global cerebral ischemia in gerbils.

Calbindin D-28K (CB), a Ca2+-binding protein, maintains Ca2+ homeostasis and protects neurons against various insults. Hyperthermia can exacerbate brain damage produced by ischemic insults. However, little is reported about the role of CB in the brain under hyperthermic condition during ischemic insults. We investigated the effects of transient global cerebral ischemia on CB immunoreactivity as well as neuronal damage in the hippocampal formation under hyperthermic condition using immunohistochemistry for neuronal nuclei (NeuN) and CB, and Fluoro-Jade B histofluorescence staining in gerbils. Hyperthermia (39.5 ± 0.2°C) was induced for 30 minutes before and during transient ischemia. Hyperthermic ischemia resulted in neuronal damage/death in the pyramidal layer of CA1–3 area and in the polymorphic layer of the dentate gyrus at 1, 2, 5 days after ischemia. In addition, hyperthermic ischemia significantly decreaced CB immunoreactivity in damaged or dying neurons at 1, 2, 5 days after ischemia. In brief, hyperthermic condition produced more extensive and severer neuronal damage/death, and reduced CB immunoreactivity in the hippocampus following transient global cerebral ischemia. Present findings indicate that the degree of reduced CB immunoreactivity might be related with various neuronal damage/death overtime and corresponding areas after ischemic insults.

Cannabinoid receptor expression in the bladder is altered in detrusor overactivity.

Immunohistochemical (IHC) evidence shows that cannabinoid receptors (CB) are expressed in human bladders and cannabinoid agonists are known to inhibit detrusor contractility. However, the mechanism for this inhibition remains unknown. In addition, the role of CB in detrusor overactivity (DO) is under-investigated. The aim of this study was to compare CB expression in normal and DO human bladders and to further characterise these receptors. Polymer chain reaction (PCR) was used to detect differences in CB transcripts in bladder samples. Differences in CB protein expression was assessed by IHC. Immunofluorescence (IF) was used to evaluate co-localisation of CB with nerve fibres. Receptor density and binding affinity were measured using the cannabinoid radioligand [(3)H]-CP-55,940. There were higher levels of CB1 transcripts in the urothelium of patients with DO and lower levels in the detrusor, compared with normal bladders. Radioligand binding revealed CB density of 421 ± 104 fmol/mg protein in normal human bladders. IHC confirmed these findings at the protein level. IF staining demonstrated co-localisation of CB1 with choline acetyltransferase-(ChAT)-positive nerves in the detrusor and co-localisation with PGP9.5 in both urothelium and detrusor. CB2 was co-localised with both ChAT and PGP9.5 in the urothelium and the detrusor. Cannabinoid receptor expression is reduced in the detrusor of patients with DO, which may play a role in the pathophysiology of the disease. Co-localisation of CB receptors with cholinergic nerves may suggest that CB1, being localised on pre- and postsynaptic terminals, could influence neurotransmitter release. Our findings suggest the potential role of cannabinoid agonists in overactive bladder pharmacotherapy.

Δ9-Tetrahydrocannabinol Impairs the Inflammatory Response to Influenza Infection: Role of Antigen-Presenting Cells and the Cannabinoid Receptors 1 and 2

Δ(9)-tetrahydrocannabinol (Δ(9)-THC) has potent immune modulatory properties and can impair pathogen-induced immune defenses, which in part have been attributed to ligation of the cannabinoid receptors 1 (CB(1)) and 2 (CB(2)). Most recently, dendritic cells (DC) were identified for their potential to enhance influenza-induced immunopathology in mice lacking CB(1) and CB(2) (CB(1) (-/-)CB(2) (-/-)). This study focused on the modulation of the inflammatory immune response to influenza by Δ(9)-THC and the role of CB(1) and/or CB(2) as receptor targets for Δ(9)-THC. C57Bl/6 (wild type) and CB(1) (-/-)CB(2) (-/-) mice were administered Δ(9)-THC (75 mg/kg) surrounding the intranasal instillation of A/PR/8/34 influenza virus. Three days post infection (dpi), Δ(9)-THC broadly decreased expression levels of mRNA induced by the innate immune response to influenza, suppressed the percentage of interferon-gamma (IFN-γ)-producing CD4(+) and interleukin-17-producing NK1.1(+) cells, and reduced the influx of antigen-presenting cells (APC), including inflammatory myeloid cells and monocytes/macrophages, into the lung in a CB(1)- and/or CB(2)-dependent manner. Δ(9)-THC had little effect on the expression of CD86, major histocompatibility complex I (MHC I), and MHC II by APC isolated from the lung. In vitro studies demonstrated that lipopolysaccharide (LPS)-induced maturation was suppressed by Δ(9)-THC in bone marrow-derived DC (bmDC). Furthermore, antigen-specific IFN-γ production by CD8(+) T cells after coculture was reduced by Δ(9)-THC treatment of bmDC in a CB(1)- and/or CB(2)-dependent manner. Collectively, these studies suggest that Δ(9)-THC potently suppresses myeloid cell immune function, in a manner involving CB(1) and/or CB(2), thereby impairing immune responses to influenza infection.

Targeting cannabinoid receptor CB2 in cardiovascular disorders: promises and controversies

Cardiovascular disease is the leading cause of death and disability worldwide, which can be largely attributed to atherosclerosis, a chronic inflammation of the arteries characterized by lesions containing immune and smooth muscle cells, lipids and extracellular matrix. In recent years, the lipid endocannabinoid system has emerged as a new therapeutic target in variety of disorders associated with inflammation and tissue injury, including those of the cardiovascular system. The discovery that Δ-9-tetrahydrocannabinol (Δ9-THC), the main active constituent of marijuana, inhibited atherosclerotic plaque progression via a cannabinoid 2 (CB(2) ) receptor-dependent anti-inflammatory mechanism, and that certain natural and synthetic cannabinoid ligands could modulate the myocardial or cerebral ischaemia-reperfusion-induced tissue damage, have stimulated impetus for a growing number of studies investigating the implication of CB(2) receptors in atherosclerosis, restenosis, stroke, myocardial infarction and heart failure. The aim of this review is to update on recent findings and controversies on the role of CB(2) receptors in cardiovascular disease. Particular emphasis will be placed on novel insights in the potential cellular targets of CB(2) stimulation in cardiovascular system (e.g. endothelial and vascular smooth muscle cells, cardiomyocytes, infiltrating and/or resident monocytes/macrophages and leukocytes, etc.), their interplay and intracellular signalling mechanisms identified, as well as on experimental and clinical studies.

Sativex-like Combination of Phytocannabinoids is Neuroprotective in Malonate-Lesioned Rats, an Inflammatory Model of Huntington’s Disease: Role of CB1 and CB2 Receptors

We have investigated whether a 1:1 combination of botanical extracts enriched in either Δ(9)-tetrahydrocannabinol (Δ(9)-THC) or cannabidiol (CBD), which are the main constituents of the cannabis-based medicine Sativex, is neuroprotective in Huntington's disease (HD), using an experimental model of this disease generated by unilateral lesions of the striatum with the mitochondrial complex II inhibitor malonate. This toxin damages striatal neurons by mechanisms that primarily involve apoptosis and microglial activation. We monitored the extent of this damage and the possible preservation of the striatal parenchyma by treatment with a Sativex-like combination of phytocannabinoids using different histological and biochemical markers. Results were as follows: (i) malonate increased the volume of edema measured by in vivo NMR imaging and the Sativex-like combination of phytocannabinoids partially reduced this increase; (ii) malonate reduced the number of Nissl-stained cells, while enhancing the number of degenerating cells stained with FluoroJade-B, and the Sativex-like combination of phytocannabinoids reversed both effects; (iii) malonate caused a strong glial activation (i.e., reactive microglia labeled with Iba-1, and astrogliosis labeled with GFAP) and the Sativex-like combination of phytocannabinoids attenuated both responses; and (iv) malonate increased the expression of inducible nitric oxide synthase and the neurotrophin IGF-1, and both responses were attenuated after the treatment with the Sativex-like combination of phytocannabinoids. We also wanted to establish whether targets within the endocannabinoid system (i.e., CB(1) and CB(2) receptors) are involved in the beneficial effects induced in this model by the Sativex-like combination of phytocannabinoids. This we did using selective antagonists for both receptor types (i.e., SR141716 and AM630) combined with the Sativex-like phytocannabinoid combination. Our results indicated that the effects of this combination are blocked by these antagonists and hence that they do result from an activation of both CB(1) and CB(2) receptors. In summary, this study provides preclinical evidence in support of a beneficial effect of the cannabis-based medicine Sativex as a neuroprotective agent capable of delaying signs of disease progression in a proinflammatory model of HD, which adds to previous data obtained in models priming oxidative mechanisms of striatal injury. However, the interest here is that, in contrast with these previous data, we have now obtained evidence that both CB(1) and CB(2) receptors appear to be involved in the effects produced by a Sativex-like phytocannabinoid combination, thus stressing the broad-spectrum properties of Sativex that may combine activity at the CB(1) and/or CB(2) receptors with cannabinoid receptor-independent actions.

2‐arachidonyl glycerol activates platelets via conversion to arachidonic acid and not by direct activation of cannabinoid receptors

There are conflicting views in the literature as to whether cannabinoids have an impact on platelet activity and to what extent cannabinoid receptors are involved. This is an important issue to resolve because platelet effects of putative therapeutic cannabinoid inhibitors and stimulators will have an impact on their potential benefits and safety. The data presented in this manuscript clearly show that the endocannabinoid 2-arrachidonyl glycerol can activate platelet activity, but that the effects are mediated through an aspirin-sensitive pathway that is not affected by cannabinoid receptor antagonists or FAAH inhibition, but is abolished by MAGL inhibition. The findings question the role of cannabinoid receptors in platelet function and suggest that platelet function is unlikely to be directly affected by cannabinoid receptor antagonists, at least in the acute phase. Cannabinoid receptor-1 (CB(1)) antagonists suppress appetite and induce weight loss. Direct antagonism of CB(1) receptors on platelets might be an additional benefit for CB(1) antagonists, but the role of CB(1) receptors in platelets is controversial. We tested the hypothesis that the endocannabinoid, 2-arachidonyl glycerol (2-AG), induces platelet aggregation by a COX-mediated mechanism rather than through CB(1) receptor activation, in blood obtained from healthy volunteers and patients with coronary artery disease receiving low dose aspirin. Aggregatory responses to the cannabinoids 2-AG and Delta(9)-THC were examined in blood sampled from healthy volunteers (n= 8) and patients (n= 12) with coronary artery disease receiving aspirin using whole blood aggregometry. The effects of CB(1) (AM251) and CB(2) (AM630) antagonists, as well as fatty acid amide hydrolase (FAAH) and monoacyl glycerol lipase (MAGL) inhibitors and aspirin on 2-AG-induced aggregation were also assessed. AM251 (100 nm-30 microm) had no effect on platelet aggregation induced by either ADP (P= 0.90) or thrombin (P= 0.86). 2-AG, but not Delta(9)-THC, induced aggregation. 2-AG-induced aggregation was unaffected by AM251 and AM630 but was abolished by aspirin (P < 0.001) and by the MAGL inhibitor, URB602 (P < 0.001). Moreover, the aggregatory response to 2-AG was depressed (by >75%, P < 0.001) in blood from patients with coronary artery disease receiving aspirin compared with that from healthy volunteers. 2-AG-mediated activation of platelets is via metabolism to arachidonic acid by MAGL, and not through direct action on CB(1) or CB(2) receptors, at least in the acute phase.

CB 2 cannabinoid receptor antagonist SR144528 decreases mu-opioid receptor expression and activation in mouse brainstem: Role of CB 2 receptor in pain

Formerly considered as an exclusively peripheral receptor, it is now accepted that CB 2 cannabinoid receptor is also present in limited amounts and distinct locations in the brain of several animal species, including mice. However, the possible roles of CB 2 receptors in the brain need to be clarified. The aim of our work was to study the μ-opioid receptor (MOR) mRNA expression level and functional activity after acute in vivo and in vitro treatments with the endocannabinoid noladin ether (NE) and with the CB 2 receptor antagonist SR144528 in brainstem of mice deficient in either CB 1 or CB 2 receptors. This study is based on our previous observations that noladin ether (NE) produces decrease in the activity of MOR in forebrain and this attenuation can be antagonized by the CB 2 cannabinoid antagonist SR144528, suggesting a CB 2 receptor mediated effect. We used quantitative real-time PCR to examine the changes of MOR mRNA levels, [ 35S]GTPγS binding assay to analyze the capability of μ-opioid agonist DAMGO to activate G-proteins and competition binding assays to directly measure the ligand binding to MOR in mice brainstem. After acute NE administration no significant changes were observed on MOR signaling. Nevertheless pretreatment of mice with SR144528 prior to the administration of NE significantly decreased MOR signaling suggesting the involvement of SR144528 in mediating the effect of MOR. mRNA expression of MORs significantly decreased both in CB 1 wild-type and CB 1 knockout mice after a single injection of SR144528 at 0.1 mg/kg when compared to the vehicle treated controls. Consequently, MOR-mediated signaling was attenuated after acute in vivo treatment with SR144528 in both CB 1 wild-type and CB 1 knockout mice. In vitro addition of 1 μM SR144528 caused a decrease in the maximal stimulation of DAMGO in [ 35S]GTPγS binding assays in CB 2 wild-type brainstem membranes whereas no significant changes were observed in CB 2 receptor knockouts. Radioligand binding competition studies showed that the noticed effect of SR144528 on MOR signaling is not mediated through MORs. Our data demonstrate that the SR144528 caused pronounced decrease in the activity of MOR is mediated via CB 2 cannabinoid receptors.

The cannabinoid antagonist AM251 attenuates nicotine self-administration and nicotine-seeking behaviour in rats

The cannabinoid receptor subtype (CB 1) antagonist rimonabant (SR141716) has been shown to decrease nicotine self-administration and attenuate nicotine-evoked dopamine release in the nucleus accumbens; effects that support recent findings on its clinical efficacy as a smoking cessation aid. The present experiments aim to advance our understanding on the role of CB 1 receptors in rodent models of nicotine dependence. AM251, a selective antagonist at CB 1 receptors dose-dependently (1, 3 and 10 mg/kg IP) suppressed intravenous nicotine (0.03 mg/kg per infusion) self-administration in rats during three successive days of pre-treatment. This reduction was short lasting since behaviour was reinstated by suspending AM251 pre-treatment. This was relatively specific to nicotine self-administration since the profile of these reductions produced by AM251 was significantly different from the responses maintained by food pellets. In a model of nicotine-seeking behaviour, rats that had been extinguished by removal of nicotine and associated cues, and presented with a priming dose of nicotine (0.2 mg/kg SC) with the cues, showed robustly reinstated responses to nicotine-seeking behaviour. Acute pre-treatment with AM251 (1–10 mg/kg IP) dose-dependently attenuated the reinstatement effects produced by nicotine and the contingently presented cues. These preclinical findings support the use of rimonabant as a smoking cessation aid and highlight the CB 1 receptor as a viable target to control intake of nicotine and prevent relapse.

Anti-dyskinetic effects of cannabinoids in a rat model of Parkinson's disease: Role of CB 1 and TRPV1 receptors

Levodopa is the most commonly prescribed drug for Parkinson's disease (PD). Although levodopa improves PD symptoms in the initial stages of the disease, its long-term use is limited by the development of side effects, including abnormal involuntary movements (dyskinesias) and psychiatric complications. The endocannabinoid system is emerging as an important modulator of basal ganglia functions and its pharmacologic manipulation represents a promising therapy to alleviate levodopa-induced dyskinesias. Rats with 6-OHDA lesions that are chronically treated with levodopa develop increasingly severe axial, limb, locomotor and oro-facial abnormal involuntary movements (AIMs). Administration of the cannabinoid agonist WIN 55,212-2 attenuated levodopa-induced axial, limb and oral AIMs dose-dependently via a CB 1-mediated mechanism, whereas it had no effect on locomotive AIMs. By contrast, systemic administration of URB597, a potent FAAH inhibitor, did not affect AIMs scoring despite its ability to increase anandamide concentration throughout the basal ganglia. Unlike WIN, anandamide can also bind and activate transient receptor potential vanilloid type-1 (TRPV1) receptors, which have been implicated in the modulation of dopamine transmission in the basal ganglia. Interestingly, URB597 significantly decreased all AIMs subtypes only if co-administered with the TRPV1 antagonist capsazepine. Our data indicate that pharmacological blockade of TRPV1 receptors unmasks the anti-dyskinetic effects of FAAH inhibitors and that CB 1 and TRPV1 receptors play opposite roles in levodopa-induced dyskinesias.

Phase III randomized trial of postoperative radiotherapy (RT) with or without carboplatin (Cb) in patients (pts) with head and neck squamous cell carcinoma (HNSCC)

6078 Background: Postoperative RT plus cisplatin is a standard treatment in high-risk resected HNSCC. However, the role of Cb in the same setting has not been established. Methods: Eligible were pts with macroscopically resected stage III/IV HNSCC with high-risk pathologic features (=3 nodes, extracapsular extension, perineural or angiolymphatic invasion, or involved margins), ECOG performance status 0–2, and adequate laboratory parameters. Pts were randomized to receive RT 59.4 Gy (1.8 Gy/day) versus the same RT plus Cb 100 mg/m2 intravenously once weekly during RT. The primary endpoint was the 2-year disease-free survival (DFS); with a projected sample size 100 patients/arm, the power was 0.81 with alpha 0.1 to detect a 15% improvement (50% to 65%). Results: From 4/94 to 4/02, 76 pts were randomized, of whom 72 pts were eligible and analyzable (36/arm). The study was prematurely closed due to slow accrual. Median age 54 years (24–83); male/female: 60 pts/12 pts; primary sites: oral cavity (n=26), oropharynx (n=23), hypopharynx (n=8), larynx (n=15); stage III/IV: 6 pts/66 pts; ECOG performance status 0/1/2: 40 pts/30 pts/2 pts. Median follow-up was 5.3 years. The 2-, 3- and 5-year DFS with RT/Cb vs. RT was 70%, 60%, and 46% vs. 55% (p=0.21), 51% (p=0.48), and 43% (p=0.79) and the 2-, 3- and 5-year overall survival (OS) was 74%, 71%, and 47% vs. 51% (p=0.04), 51% (p=0.07) and 40% (p=0.59). There were no statistically significant differences in median DFS or OS. In the subset of pts with primary sites other than the oropharynx (n=49), RT/Cb resulted in better OS vs. RT (p=0.05, Wilcoxon; p=0.27, log-rank). RT/Cb was associated with infrequent grade III/IV toxicities, including leukopenia (RT/Cb 8% vs. RT 0%), stomatitis (11% vs. 3%), and dermatitis (6% vs. 3%). Gastrostomy tube was placed in 15 pts (RT/Cb 8 vs. RT 7). Conclusions: Our data do not show a statistically significant improvement in DFS with RT/Cb vs. RT but this may reflect insufficient sample size. The potential benefit of RT/Cb for a subset of pts merits further study. No significant financial relationships to disclose.

Effects of inhibition of fatty acid amide hydrolase vs. the anandamide membrane transporter on TRPV1‐mediated calcium responses in adult DRG neurons; the role of CB1 receptors

The aim of the present study was to investigate the relationship between TRPV1 stimulation and endocannabinoid-driven CB(1) receptor-mediated inhibition of activity in adult rat dorsal root ganglion (DRG) neurons, a model of primary afferent nociceptors. Calcium-imaging studies were performed to compare the effects of the fatty acid amide hydrolase (FAAH) inhibitor URB597 (1 microm) vs. the anandamide (AEA) uptake inhibitor UCM707 (1 microm) on capsaicin (100 nm) and N-arachidonoyl dopamine (NADA; 1 microm)-evoked changes in intracellular calcium [Ca(2+)](i) in DRG neurons. The ability of the CB(1) receptor antagonist AM251 (1 microm) to modulate the effects of URB597 and UCM707 was also determined. Suprafusion of NADA and capsaicin evoked robust increases in [Ca(2+)](i) in DRG neurons (89 +/- 4% and 132 +/- 6% of the depolarizing KCl response, respectively). Co-incubation with URB597 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (39 +/- 3% and 79 +/- 4% of KCl response, respectively). Similarly, co-incubation with UCM707 significantly attenuated both NADA and capsaicin-evoked increases in [Ca(2+)](i) (59 +/- 7% and 72 +/- 4% of KCl response, respectively). The CB(1) receptor antagonist AM251 significantly attenuated the effects of URB597 on NADA-evoked increases in [Ca(2+)](i) but not the effects of URB597 on capsaicin-evoked increases in [Ca(2+)](i). By contrast, AM251 significantly attenuated the inhibitory effects of UCM707 on both NADA and capsaicin-evoked increases in [Ca(2+)](i.) These data suggest that transport of both NADA and capsaicin into DRG neurons and the subsequent activation of TRPV1 is partly governed by FAAH-dependent mechanisms as well as via the putative AEA membrane transporter.

GABA A receptors modulate cannabinoid-evoked hypothermia

Cannabinoids evoke hypothermia by stimulating central CB 1 receptors. GABA induces hypothermia via GABA A or GABA B receptor activation. CB 1 receptor activation increases GABA release in the hypothalamus, a central locus for thermoregulation, suggesting that cannabinoid and GABA systems may be functionally linked in body temperature regulation. We investigated whether GABA receptors modulate the hypothermic actions of [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1ij]quinolin-6-one] (WIN 55212-2), a selective cannabinoid agonist, in male Sprague–Dawley rats. WIN 55212-2 (2.5 mg/kg im) produced a rapid hypothermia that peaked 45–90 min postinjection. The hypothermia was attenuated by bicuculline (2 mg/kg ip), a GABA A antagonist. However, SCH 50911 (1–10 mg/kg ip), a GABA B blocker, did not antagonize the hypothermia. Neither bicuculline (2 mg/kg) nor SCH 50911 (10 mg/kg) by itself altered body temperature. We also investigated a possible role for CB 1 receptors in GABA-generated hypothermia. Muscimol (2.5 mg/kg ip), a GABA A agonist, or baclofen (5 mg/kg ip), a GABA B agonist, evoked a significant hypothermia. Blockade of CB 1 receptors with SR141716A (2.5 mg/kg im) did not antagonize muscimol- or baclofen-induced hypothermia, indicating that GABA-evoked hypothermia does not contain a CB 1-sensitive component. Our results implicate GABA A receptors in the hypothermic actions of cannabinoids and provide further evidence of a functional link between cannabinoid and GABA systems.

Role of CB1 and CB2 receptors in the inhibitory effects of cannabinoids on lipopolysaccharide-induced nitric oxide release in astrocyte cultures.

The purpose of this study was to investigate the role of the central cannabinoid receptor (CB(1)) in mediating the actions of the endogenous cannabinoid agonist anandamide and the synthetic cannabinoid CP-55940. Activation of primary mouse astrocyte cultures by exposure to bacterial lipopolysaccharide (LPS) caused a marked (approximately tenfold) increase in nitric oxide (NO) release. Coincubation with the cannabinoid agonists anandamide or CP-55940 markedly inhibited release of NO (-12% to -55%). This effect was abolished by SR-141716A (1 microM), a CB1 receptor antagonist. SR-141716A alone also significantly increased NO release in response to LPS, suggesting that endogenous cannabinoids modify inflammatory responses. In contrast, coincubation with the CB2 receptor antagonist SR-144528 (1 microM) abolished the inhibitory effects of the endogenous cannabinoid anandamide on LPS-induced NO release, although this may reflect nonspecific effects of this ligand or cannabinoid actions through atypical receptors of anandamide. We also showed that endogenous or synthetic cannabinoids inhibit LPS-induced inducible NO synthase expression (mRNA and protein) in astrocyte cultures. These results indicate that CB1 receptors may promote antiinflammatory responses in astrocytes.

Endogenous cannabinoid anandamide increases heart resistance to arrhythmogenic effects of epinephrine: role of CB(1) and CB(2) receptors.

Intravenous injection of 10 mg/kg anandamide reduces the incidence and duration of epinephrine-induced arrhythmias in rats. SR141716A and SR144528, antagonists of cannabinoid receptor I and II did not abolish the antiarrhythmic effect of anandamide. These data suggest that the antiarrhythmic effect of anandamide is nonspecific or mediated via unknown cannabinoid receptors, but not associated with activation of cannabinoid receptors I and II.

Role of CB 1 cannabinoid receptors in the regulation of rat gastric acid secretion

Role of CB 1 cannabinoid receptors in the regulation of rat gastric acid secretion