A novel fluorophosphonate inhibitor of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol with potential anti-obesity effects

Background and Purpose

The development of potent and selective inhibitors of the biosynthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG) via DAG lipases (DAGL) α and β is just starting to be considered as a novel and promising source of pharmaceuticals for the treatment of disorders that might benefit from a reduction in endocannabinoid tone, such as hyperphagia in obese subjects.

Experimental Approach

Three new fluorophosphonate compounds O-7458, O-7459 and O-7460 were synthesized and characterized in various enzymatic assays. The effects of O-7460 on high-fat diet intake were tested in mice.

Key Results

Of the new compounds, O-7460 exhibited the highest potency (IC₅₀ = 690 nM) against the human recombinant DAGLα, and selectivity (IC₅₀ > 10 μM) towards COS-7 cell and human monoacylglycerol lipase (MAGL), and rat brain fatty acid amide hydrolase. Competitive activity-based protein profiling confirmed that O-7460 inhibits mouse brain MAGL only at concentrations ≥10 μM, and showed that this compound has only one major ‘off-target’, that is, the serine hydrolase KIAA1363. O-7460 did not exhibit measurable affinity for human recombinant CB₁ or CB₂ cannabinoid receptors (K_i > 10 μM). In mouse neuroblastoma N18TG2 cells stimulated with ionomycin, O-7460 (10 μM) reduced 2-AG levels. When administered to mice, O-7460 dose-dependently (0–12 mg·kg⁻¹, i.p.) inhibited the intake of a high-fat diet over a 14 h observation period, and, subsequently, slightly but significantly reduced body weight.

Conclusions and Implications

O-7460 might be considered a useful pharmacological tool to investigate further the role played by 2-AG both in vitro and in vivo under physiological as well as pathological conditions.

Linked Articles

This article is part of a themed section on Cannabinoids. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-4 & http://dx.doi.org/10.1111/bph.2012.167.issue-8     

Quantitative assessment of the contribution of sodium‐dependent taurocholate co‐transporting polypeptide (NTCP) to the hepatic uptake of rosuvastatin,pitavastatin and fluvastatin

Hepatic uptake transport is often the rate‐determining step in the systemic clearance of drugs. The ability to predict uptake clearance and to determine the contribution of individual transporters to overall hepatic uptake is therefore critical in assessing the potential pharmacokinetic and pharmacodynamic variability associated with drug–drug interactions and pharmacogenetics. The present study revisited the interaction of statin drugs, including pitavastatin, fluvastatin and rosuvastatin, with the sodium‐dependent taurocholate co‐transporting polypeptide (NTCP) using gene transfected cell models. In addition, the uptake clearance and the contribution of NTCP to the overall hepatic uptake were assessed using in vitro hepatocyte models. Then NTCP protein expression was measured by a targeted proteomics transporter quantification method to confirm the presence and stability of NTCP expression in suspended and cultured hepatocyte models. It was concluded that NTCP‐mediated uptake contributed significantly to active hepatic uptake in hepatocyte models for all three statins. However, the contribution of NTCP‐mediated uptake to the overall active hepatic uptake was compound‐dependent and varied from about 24% to 45%. Understanding the contribution of individual transporter proteins to the overall hepatic uptake and its functional variability when other active hepatic uptake pathways are interrupted could improve the current prediction practice used to assess the pharmacokinetic variability due to drug–drug interactions, pharmacogenetics and physiopathological conditions in humans. Copyright © 2013 John Wiley & Sons, Ltd.     

Evidence for the Putative Cannabinoid Receptor (GPR55)-Mediated Inhibitory Effects on Intestinal Contractility in Mice

Background: Cannabinoids inhibit intestinal motility via presynaptic cannabinoid receptor type I (CB1) in enteric neurons while cannabinoid receptor type II (CB2) receptors are located mainly in immune cells. The recently de-orphanized G-protein-coupled receptor, GPR55, has been proposed to be the 'third' cannabinoid receptor. Although gene expression of GPR55 is evident in the gut, functional evidence for GPR55 in the gut is unknown. In this study, we tested the hypothesis that GPR55 activation inhibits neurogenic contractions in the gut. Methods: We assessed the inhibitory effect of the atypical cannabinoid O-1602, a GPR55 agonist, in mouse colon. Isometric tension recordings in colonic tissue strips were used from either wild-type, GPR55(-/-) or CB1(-/-)/CB2(-/-) knockout mice. Results: O-1602 inhibited the electrical field- induced contractions in the colon strips from wild-type and CB1(-/-)/CB2(-/-) in a concentration-dependent manner, suggesting a non-CB1/CB2 receptor-mediated prejunctional effect. The concentration-dependent response of O-1602 was significantly inhibited in GPR55(-/-) mice. O-1602 did not relax colonic strips precontracted with high K(+) (80 mmol/l), indicating no involvement of Ca(2+) channel blockade in O-1602-induced relaxation. However, 10 μmol/l O-1602 partially inhibited the exogenous acetylcholine (10 μmol/l)-induced contractions. Moreover, we also assessed the inhibitory effects of JWH015, a CB2/GPR55 agonist on neurogenic contractions of mouse ileum. Surprisingly, the effects of JWH015 were independent of the known cannabinoid receptors. Conclusion: Taken together, these findings suggest that activation of GPR55 leads to inhibition of neurogenic contractions in the gut and are predominantly prejunctional.     

Evaluation of the endogenous cannabinoid system in mediating the behavioral effects of dipyrone (metamizol) in mice

Dipyrone is a common nonopioid analgesic and antipyretic, which, in many countries, is available over the counter and is more widely used than paracetamol or aspirin. However, the exact mechanisms by which dipyrone acts remain inconclusive. Two novel arachidonoyl-conjugated metabolites are formed in mice following the administration of dipyrone that are dependent on the activity of fatty acid amide hydrolase (FAAH), which also represents the major catabolic enzyme of the endogenous cannabinoid ligand anandamide. These arachidonoyl metabolites not only inhibit cyclooxygenase (COX-1/COX-2) but also bind to cannabinoid receptors at low micromolar concentrations. The relative contributions of cannabinoid receptors and FAAH in the overall behavioral response to dipyrone remain untested. Accordingly, the two primary objectives of the present study were to determine whether the behavioral effects of dipyrone would (a) be blocked by cannabinoid receptor antagonists and (b) occur in FAAH mice. Here, we report that thermal antinociceptive, hypothermic, and locomotor suppressive actions of dipyrone are mediated by a noncannabinoid receptor mechanism of action and occurred after acute or repeated administration irrespective of FAAH. These findings indicate that FAAH-dependent arachidonoyl metabolites and cannabinoid receptors are not requisites by which dipyrone exerts these pharmacological effects under noninflammatory conditions.     

Treatment of advanced lung cancer in the elderly

Lung cancer remains the leading cause of cancer-related mortality in the United States. Almost half of all lung cancer occurs at age > 70 years. The majority of patients with lung cancer present with locally advanced or metastatic disease. Management of advanced lung cancer in the older patient is a commonly encountered clinical scenario. There is a paucity of clinical data guiding the management of lung cancer in the elderly due to underrepresentation of the elderly in clinical trials. The elderly have unique alterations in physiology that put them at a greater risk of toxicity from chemotherapy and biologic therapy. Comorbid conditions, common among the elderly, can further reduce tolerance to therapy. As a consequence, older patients have worse outcomes than younger patients. It is important to look beyond chronologic age to better risk stratify patients when making treatment decisions in older patients with lung cancer. The basic principles of management, especially in the fit elderly, do not differ from those in younger patients. This article provides an overview of management of advanced non-small cell lung cancer. The magnitude of the problem and current treatment guidelines for lung cancer are reviewed with a focus on barriers specific to the elderly. The available clinical trials that have specifically studied the elderly with lung cancer are summarized. The evolving role of palliative care is discussed, as well as the need for integrating geriatric assessment in the care of elderly patients with lung cancer.     

SR 141716 (Rimonabant) precipitates withdrawal in marijuana-dependent mice

Repeated marijuana use is known to lead to physical dependence in humans; however, its dependence liability has yet to be adequately assessed in laboratory animals. The goals of the present study were to: assess whether the CB(1) antagonist SR 141716 (rimonabant) precipitates withdrawal in mice that had been repeatedly exposed to marijuana smoke, and to compare these precipitated withdrawal effects to those elicited following intravenous administration of its chief psychoactive component Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716 elicited a significant increase in paw tremors in mice that were repeatedly dosed with either marijuana or Delta(9)-THC. Unexpectedly, the blood and brain concentrations of Delta(9)-THC following marijuana exposure were considerably lower than those found following Delta(9)-THC injection when comparing an equivalent magnitude of paw tremors in both conditions. Finally, Delta(9)-THC dose-dependently alleviated SR 141716-induced paw tremors in marijuana-dependent mice, but marijuana itself failed to reverse the precipitated withdrawal effect. It is likely that marijuana exposure generated insufficient Delta(9)-THC brain levels (i.e., 203+/-19 ng/g) to reverse the withdrawal signs compared with the brain levels following intravenous injection (i.e., 1862+/-82 ng/g). These findings taken together indicate that mice exposed repeatedly to marijuana smoke exhibit similar precipitated withdrawal effects as Delta(9)-THC-injected mice.     

Adjuvant therapy of stage II breast cancer treated with CMFVP, radiation therapy and VATH following lumpectomy. A pilot trial

A pilot study was undertaken to assess the feasibility, toxicity, and efficacy of combined radiation therapy and chemotherapy in the adjuvant treatment of node-positive. Stage II patients with breast carcinoma who had undergone lumpectomy. Therapy consisted of three phases, starting with a six-week CMFVP (cyclophosphamide, methotrexate, 5-fluorouracil, vincristine, prednisone) induction, followed by radiation therapy to the breast, and concluding with four cycles of VATH (vinblastine, Adriamycin, thiotepa, Halotesin). Twenty-seven patients were entered with an average age of 51.5 years (median 50 yrs) and a mean follow-up of 46.2 months. Twenty-three patients (85.2%) are alive and 19 (70.3%) disease free. There were no ipsilateral local recurrences. Cosmetic results were good to excellent in 26/27 patients. The doses of VATH were not compromised by the prior therapy. The regimen was found to be tolerable and is a reasonable approach in the adjuvant treatment of this particular patient population.     

Visualization of neuromuscular junctions over periods of several months in living mice

Identified neuromuscular junctions were followed in the sternomastoid muscle of living mice for several months by repeated staining with the fluorescent dye 4-(4-diethylaminostyryl)-N-methylpyridinium iodide (4-Di-2-ASP; Magrassi et al., 1987). Overall terminal growth occurred at many endplates; however, the branching pattern of presynaptic arbors was largely unchanged, even after several months. The absence of significant remodeling over time was not a result of dye-staining, since sprouting was readily induced at residual motor endings by partial denervation. We conclude that--apart from overall growth--most neuromuscular junctions in the adult mouse are stable over intervals that represent a significant fraction of the animal's lifetime.     

CT findings in complications of acquired renal cystic disease

A 42-year-old man with end-stage renal disease developed acquired renal cystic disease. The left kidney underwent tumorous degeneration necessitating nephrectomy. Eight months later acute hemorrhagic renal cyst rupture culminated in right nephrectomy.     

Relation of animal size to convergence, divergence, and neuronal number in peripheral sympathetic pathways

The enormous range of animal size raises a fundamental problem: How do larger animals maintain adequate control of peripheral structures that are many times more massive and extensive than the homologous structures in smaller animals? To explore this question, we have determined neuronal number, the number of axons that innervate each neuron (convergence) and the number of neurons innervated by each axon (divergence), in a peripheral sympathetic pathway of several mammals (mouse, hamster, rat, guinea pig, and rabbit). The average adult weights of these species vary over approximately a 65-fold range. However, the number of superior cervical ganglion cells increases by only a factor of 4 between the smallest of these animals (mice; about 25 gm) and the largest (rabbits; about 1700 gm); the number of spinal preganglionic neurons that innervate the ganglion increases by only a factor of 2. Thus, the number of nerve cells in the sympathetic system does not increase in proportion to animal size. On the other hand, our results indicate that there are systematic differences across these species in the number of axons that innervate each ganglion cell and in the number of ganglion cells innervated by each axon. We suggest that modulation of convergence and divergence in sympathetic ganglia allows this part of the nervous system to effectively activate homologous peripheral targets over a wide range of animal size.