Showing papers in "British Journal of Pharmacology in 2010"

Animal research: reporting in vivo experiments: the ARRIVE guidelines

Abstract: The following guidelines are excerpted (as permitted under the Creative Commons Attribution License (CCAL), with the knowledge and approval of PLoS Biology and the authors) from Kilkenny et al (2010) ​ Table

Guidelines for reporting experiments involving animals: the ARRIVE guidelines

Abstract: British Journal of Pharmacology (BJP) is pleased to publish a new set of guidelines for reporting research involving animals, simultaneously with several other journals; the 'ARRIVE' guidelines (Animals in Research: Reporting In Vivo Experiments). This editorial summarizes the background to the guidelines, gives our view of their significance, considers aspects of specific relevance to pharmacology, re-states BJP's guidelines for authors on animal experiments and indicates our commitment to carrying on discussion of this important topic. We also invite feedback via the British Pharmacological Society website.

Ligand binding assays at equilibrium: validation and interpretation.

Abstract: The focus of this review paper is factors affecting data interpretation in ligand binding assays under equilibrium conditions. Protocols for determining Kd (the equilibrium dissociation constant) and KdA (the equilibrium inhibitor constant) for receptor ligands are discussed. The basic theory describing the interaction of a radiotracer and an unlabelled competitor ligand with a receptor is developed. Inappropriate experimental design may result in ligand depletion and non-attainment of equilibrium, distorting the calculation of Kd and KdA. Strategies, both theoretical and practical, will be given to avoid and correct such errors, thus leading to the determination of reliable values for these constants. In determining KdA from competition binding studies, two additional concepts are discussed. First, the necessity to measure an adequate specific binding signal from the bound radiotracer ligand limits the range of affinity constants that can be measured: a particular set of assay conditions may lead to an upper limit on the apparent affinity of unlabelled ligands. Second, an extension of the basic assay methodology can indicate whether the interaction between the tracer and a test ligand is mediated by a competitive or an allosteric mechanism. Finally, the review ends with a discussion of two factors that are often overlooked: buffer composition and the temperature at which the assay is conducted, and the impact these can have on affinity measurements and the understanding of drug interactions. British Journal of Pharmacology (2010) 161, 1219–1237; doi:10.1111/j.1476-5381.2009.00604.x; published online 2 February 2010 This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6

CB2: a cannabinoid receptor with an identity crisis

Abstract: CB2 was first considered to be the ‘peripheral cannabinoid receptor’. This title was bestowed based on its abundant expression in the immune system and presumed absence from the central nervous system. However, multiple recent reports question the absence of CB2 from the central nervous system. For example, it is now well accepted that CB2 is expressed in brain microglia during neuroinflammation. However, the extent of CB2 expression in neurons has remained controversial. There have been studies claiming either extreme-its complete absence to its widespread expression-as well as everything in between. This review will discuss the reported tissue distribution of CB2 with a focus on CB2 in neurons, particularly those in the central nervous system as well as the implications of that presence. As CB2 is an attractive therapeutic target for pain management and immune system modulation without overt psychoactivity, defining the extent of its presence in neurons will have a significant impact on drug discovery. Our recommendation is to encourage cautious interpretation of data that have been presented for and against CB2's presence in neurons and to encourage continued rigorous study. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x

The selectivity of beta-adrenoceptor agonists at human beta1-, beta2- and beta3-adrenoceptors.

Abstract: Beta-adrenoceptor antagonists ("beta-blockers") are one of the most widely used classes of drugs in cardiovascular medicine (hypertension, ischaemic heart disease and increasingly in heart failure) as well as in the management of anxiety, migraine and glaucoma. Where known, the mode of action in cardiovascular disease is from antagonism of endogenous catecholamine responses in the heart (mainly at beta1-adrenoceptors), while the worrisome side effects of bronchospasm result from airway beta2-adrenoceptor blockade. The aim of this study was to determine the selectivity of beta-antagonists for the human beta-adrenoceptor subtypes. (3)H-CGP 12177 whole cell-binding studies were undertaken in CHO cell lines stably expressing either the human beta1-, beta2- or the beta3-adrenoceptor in order to determine the affinity of ligands for each receptor subtype in the same cell background. In this study, the selectivity of well-known subtype-selective ligands was clearly demonstrated: thus, the selective beta1 antagonist CGP 20712A was 501-fold selective over beta2 and 4169-fold selective over beta3; the beta2-selective antagonist ICI 118551 was 550- and 661-fold selective over beta1 and beta3, respectively, and the selective beta3 compound CL 316243 was 10-fold selective over beta2 and more than 129-fold selective over beta1. Those beta2-adrenoceptor agonists used clinically for the treatment of asthma and COPD were beta2 selective: 29-, 61- and 2818-fold for salbutamol, terbutaline and salmeterol over beta1, respectively. There was little difference in the affinity of these ligands between beta1 and beta3 adrenoceptors. The clinically used beta-antagonists studied ranged from bisoprolol (14-fold beta1-selective) to timolol (26-fold beta2-selective). However, the majority showed little selectivity for the beta1- over the beta2-adrenoceptor, with many actually being more beta2-selective. This study shows that the beta1/beta2 selectivity of most clinically used beta-blockers is poor in intact cells, and that some compounds that are traditionally classed as "beta1-selective" actually have higher affinity for the beta2-adrenoceptor. There is therefore considerable potential for developing more selective beta-antagonists for clinical use and thereby reducing the side-effect profile of beta-blockers.

Apremilast, a cAMP phosphodiesterase-4 inhibitor, demonstrates anti-inflammatory activity in vitro and in a model of psoriasis.

Abstract: Background and purpose: Apremilast is an orally administered phosphodiesterase-4 inhibitor, currently in phase 2 clinical studies of psoriasis and other chronic inflammatory diseases. The inhibitory effects of apremilast on pro-inflammatory responses of human primary peripheral blood mononuclear cells (PBMC), polymorphonuclear cells, natural killer (NK) cells and epidermal keratinocytes were explored in vitro, and in a preclinical model of psoriasis.

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT1A receptors

Abstract: Background and purpose: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT1A receptors. As 5-HT1A receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT1A receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF).

Renin-angiotensin-aldosterone system blockade for cardiovascular diseases: current status.

Abstract: Activation of the renin–angiotensin–aldosterone system (RAAS) results in vasoconstriction, muscular (vascular and cardiac) hypertrophy and fibrosis. Established arterial stiffness and cardiac dysfunction are key factors contributing to subsequent cardiovascular and renal complications. Blockade of RAAS has been shown to be beneficial in patients with hypertension, acute myocardial infarction, chronic systolic heart failure, stroke and diabetic renal disease. An aggressive approach for more extensive RAAS blockade with combination of two commonly used RAAS blockers [ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs)] yielded conflicting results in different patient populations. Combination therapy is also associated with more side effects, in particular hypotension, hyperkalaemia and renal impairment. Recently published ONTARGET study showed ACEI/ARB combination therapy was associated with more adverse effects without any increase in benefit. The Canadian Hypertension Education Program responded with a new warning: ‘Do not use ACEI and ARB in combination’. However, the European Society of Cardiology in their updated heart failure treatment guidelines still recommended ACEI/ARB combo as a viable option. This apparent inconsistency among guidelines generates debate as to which approach of RAAS inhibition is the best. The current paper reviews the latest evidence of isolated ACEI or ARB use and their combination in cardiovascular diseases, and makes recommendations for their prescriptions in specific patient populations.

Signal transduction by protease-activated receptors

Abstract: The family of G protein-coupled receptors (GPCRs) constitutes the largest class of signalling receptors in the human genome, controlling vast physiological responses and are the target of many drugs. After activation, GPCRs are rapidly desensitized by phosphorylation and β-arrestin binding. Most classic GPCRs are internalized through a clathrin, dynamin and β-arrestin-dependent pathway and then recycled back to the cell surface or sorted to lysosomes for degradation. Given the vast number and diversity of GPCRs, different mechanisms are likely to exist to precisely regulate the magnitude, duration and spatial aspects of receptor signalling. The G protein-coupled protease-activated receptors (PARs) provide elegant examples of GPCRs that are regulated by distinct desensitization and endocytic sorting mechanisms, processes that are critically important for the spatial and temporal fidelity of PAR signalling. PARs are irreversibly activated through proteolytic cleavage and transmit cellular responses to extracellular proteases. Activated PAR1 internalizes through a clathrin- and dynamin-dependent pathway independent of β-arrestins. Interestingly, PAR1 is basally ubiquitinated and deubiquitinated after activation and traffics from endosomes to lysosomes independent of ubiquitination. In contrast, β-arrestins mediate activated PAR2 internalization and function as scaffolds that promote signalling from endocytic vesicles. Moreover, activated PAR2 is modified with ubiquitin, which facilitates lysosomal degradation. Activated PARs also adopt distinct active conformations that signal to diverse effectors and are likely regulated by different mechanisms. Thus, the identification of the molecular machinery important for PAR signal regulation will enable the development of new strategies to manipulate receptor signalling and will provide novel targets for the development of drugs.

High-throughput screening and small animal models, where are we?

Abstract: Current high-throughput screening methods for drug discovery rely on the existence of targets. Moreover, most of the hits generated during screenings turn out to be invalid after further testing in animal models. To by-pass these limitations, efforts are now being made to screen chemical libraries on whole animals. One of the most commonly used animal model in biology is the murine model Mus musculus. However, its cost limit its use in large-scale therapeutic screening. In contrast, the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the fish Danio rerio are gaining momentum as screening tools. These organisms combine genetic amenability, low cost and culture conditions that are compatible with large-scale screens. Their main advantage is to allow high-throughput screening in a whole-animal context. Moreover, their use is not dependent on the prior identification of a target and permits the selection of compounds with an improved safety profile. This review surveys the versatility of these animal models for drug discovery and discuss the options available at this day.

Procalcitonin in sepsis and systemic inflammation: a harmful biomarker and a therapeutic target.

Abstract: The worldwide yearly mortality from sepsis is substantial, greater than that of cancer of the lung and breast combined. Moreover, its incidence is increasing, and its response to therapy has not appreciably improved. In this condition, the secretion of procalcitonin (ProCT), the prohormone of calcitonin, is augmented greatly, attaining levels up to thousands of fold of normal. This hypersecretion emanates from multiple tissues throughout the body that are not traditionally viewed as being endocrine. The serum values of ProCT correlate with the severity of sepsis; they recede with its improvement and worsen with exacerbation. Accordingly, as highlighted in this review, serum ProCT has become useful as a biomarker to assist in the diagnosis of sepsis, as well as related infectious or inflammatory conditions. It is also a useful monitor of the clinical course and prognosis, and sensitive and specific assays have been developed for its measurement. Moreover, it has been demonstrated that the administration of ProCT to septic animals greatly increases mortality, and several toxic effects of ProCT have been elucidated by in vitro experimental studies. Antibodies have been developed that neutralize the harmful effects of ProCT, and their use markedly decreases the symptomatology and mortality of animals that harbour a highly virulent sepsis analogous to that occurring in humans. This therapy is facilitated by the long duration of serum ProCT elevation, which allows for a broad window of therapeutic opportunity. An experimental groundwork has been established that suggests a potential applicability of such therapy in septic humans.

Long‐lasting target binding and rebinding as mechanisms to prolong in vivo drug action

Abstract: An increasing number of examples in the literature suggest that the in vivo duration of drug action not only depends on macroscopic pharmacokinetic properties like plasma half-life and the time needed to equilibrate between the plasma and the effect compartments, but is also influenced by long-lasting target binding and rebinding. The present review combines information from different research areas and simulations to explore the nature of these mechanisms and the conditions in which they are most prevalent. Simulations reveal that these latter phenomena become especially influential when there is no longer sufficient free drug around to maintain high levels of receptor occupancy. There is not always a direct link between slow dissociation and long-lasting in vivo target protection, as the rate of free drug elimination from the effect compartment is also a key influencing factor. Local phenomena that hinder the diffusion of free drug molecules away from their target may allow them to consecutively bind to the same target and/or targets nearby (denoted as 'rebinding') even when their concentration in the bulk phase has already dropped to insignificant levels. The micro-anatomic properties of many effect compartments are likely to intensify this phenomenon. By mimicking the complexity of tissues, intact cells offer the opportunity to investigate both mechanisms under the same, physiologically relevant conditions.

A novel, orally active LPA1 receptor antagonist inhibits lung fibrosis in the mouse bleomycin model

Abstract: Background and purpose: The aim of this study was to assess the potential of an antagonist selective for the lysophosphatidic acid receptor, LPA1, in treating lung fibrosis We evaluated the in vitro and in vivo pharmacological properties of the high affinity, selective, oral LPA1-antagonist (4′-{4-[(R)-1-(2-chloro-phenyl)-ethoxycarbonylamino]-3-methyl-isoxazol-5-yl}-biphenyl-4-yl)-acetic acid (AM966).

Monoclonal antibody TGN1412 trial failure explained by species differences in CD28 expression on CD4+ effector memory T-cells.

Abstract: Background and purpose In 2006, a life-threatening 'cytokine storm', not predicted by pre-clinical safety testing, rapidly occurred in all six healthy volunteers during the phase I clinical trial of the CD28 superagonist monoclonal antibody (mAb) TGN1412 To date, no unequivocal explanation for the failure of TGN1412 to stimulate profound cytokine release in vitro or in vivo in species used for pre-clinical safety testing has been established Here, we have identified a species difference almost certainly responsible for this disparate immunopharmacology Experimental approach Polychromatic flow cytometry and intracellular cytokine staining were employed to dissect the in vitro immunopharmacology of TGN1412 and other therapeutic mAbs at the cellular level to identify differences between humans and species used for pre-clinical safety testing Key results In vitro IL-2 and IFN-γ release from CD4+ effector memory T-cells were key indicators of a TGN1412-type response This mechanism of cytokine release differed from that of other therapeutic mAbs, which can cause adverse reactions, because these other mAbs stimulate cytokine release primarily from natural killer cells In contrast to humans, CD28 is not expressed on the CD4+ effector memory T-cells of all species used for pre-clinical safety testing, so cannot be stimulated by TGN1412 Conclusions and implications It is likely that activation of CD4+ effector memory T-cells by TGN1412 was responsible for the cytokine storm Lack of CD28 expression on the CD4+ effector memory T-cells of species used for pre-clinical safety testing of TGN1412 offers an explanation for the failure to predict a 'cytokine storm' in humans

JWH018, a common constituent of ‘Spice’ herbal blends, is a potent and efficacious cannabinoid CB1 receptor agonist

Abstract: Background and purpose: ‘Spice’ is an herbal blend primarily marketed in Europe as a mild hallucinogen with prominent cannabis-like effects and as a legal alternative to cannabis. However, a recent report identified a number of synthetic additives in samples of ‘Spice’. One of these, the indole derivative JWH018, is a ligand for the cannabinoid receptor 1 (CB1) cannabinoid receptor and inhibits cAMP production in CB1 receptor-expressing CHO cells. Other effects of JWH018 on CB1 receptor-mediated signalling are not known, particularly in neurons. Here we have evaluated the signalling pathways activated by JWH018 at CB1 receptors. Experimental approach: We investigated the effects of JWH018 on neurotransmission in cultured autaptic hippocampal neurons. We further analysed its activation of ERK1/2 mitogen activated protein kinase (MAPK) and internalization of CB1 receptors in HEK293 cells stably expressing this receptor. Key results: In cultured autaptic hippocampal neurons, JWH018 potently inhibited excitatory postsynaptic currents (IC50= 14.9 nM) in a concentration- and CB1 receptor-dependent manner. Furthermore, it increased ERK1/2 MAPK phosphorylation (EC50= 4.4 nM). We also found that JWH018 potently induced rapid and robust CB1 receptor internalization (EC50= 2.8 nM; t1/2= 17.3 min). Conclusions and implications: JWH018, a prominent component of several herbal preparations marketed for their psychoactivity, is a potent and effective CB1 receptor agonist that activates multiple CB1 receptor signalling pathways. Thus, it is likely that the subjective effects of ‘Spice’ are due to activation of cannabinoid CB1 receptors by JWH018, added to this herbal preparation.

Comparative pharmacology of chemically distinct NADPH oxidase inhibitors.

Abstract: BACKGROUND AND PURPOSE Oxidative stress [i.e. increased levels of reactive oxygen species (ROS)] has been suggested as a pathomechanism of different diseases, although the disease-relevant sources of ROS remain to be identified. One of these sources may be NADPH oxidases. However, due to increasing concerns about the specificity of the compounds commonly used as NADPH oxidase inhibitors, data obtained with these compounds may have to be re-interpreted. EXPERIMENTAL APPROACH We compared the pharmacological profiles of the commonly used NADPH oxidase inhibitors, diphenylene iodonium (DPI), apocynin and 4-(2-amino-ethyl)-benzolsulphonyl-fluoride (AEBSF), as well as the novel triazolo pyrimidine VAS3947. We used several assays for detecting cellular and tissue ROS, as none of them is specific and artefact free. KEY RESULTS DPI abolished NADPH oxidase-mediated ROS formation, but also inhibited other flavo-enzymes such as NO synthase (NOS) and xanthine oxidase (XOD). Apocynin interfered with ROS detection and varied considerably in efficacy and potency, as did AEBSF. Conversely, the novel NADPH oxidase inhibitor, VAS3947, consistently inhibited NADPH oxidase activity in low micromolar concentrations, and interfered neither with ROS detection nor with XOD or eNOS activities. VAS3947 attenuated ROS formation in aortas of spontaneously hypertensive rats (SHRs), where NOS or XOD inhibitors were without effect. CONCLUSIONS AND IMPLICATIONS Our data suggest that triazolo pyrimidines such as VAS3947 are specific NADPH oxidase inhibitors, while DPI and apocynin can no longer be recommended. Based on the effects of VAS3947, NADPH oxidases appear to be a major source of ROS in aortas of SHRs.

Quercetin potentiates insulin secretion and protects INS-1 pancreatic β-cells against oxidative damage via the ERK1/2 pathway

Abstract: BACKGROUND AND PURPOSE Quercetin lowers plasma glucose, normalizes glucose tolerance tests and preserves pancreatic β-cell integrity in diabetic rats. However, its mechanism of action has never been explored in insulin-secreting β-cells. Using the INS-1 β-cell line, the effects of quercetin were determined on glucose- or glibenclamide-induced insulin secretion and on β-cell dysfunctions induced by hydrogen peroxide (H2O2). These effects were analysed along with the activation of the extracellular signal-regulated kinase (ERK)1/2 pathway. N-acetyl-L-cysteine (NAC) and resveratrol, two antioxidants also known to exhibit some anti-diabetic properties, were used for comparison.

Adolescent cannabis use and psychosis: epidemiology and neurodevelopmental models

Abstract: Cannabis is one of the most widely used illicit drugs among adolescents, and most users first experiment with it in adolescence. Adolescence is a critical phase for brain development, characterized by neuronal maturation and rearrangement processes, such as myelination, synaptic pruning and dendritic plasticity. The endocannabinoid system plays an important role in fundamental brain developmental processes such as neuronal cell proliferation, migration and differentiation. Therefore changes in endocannabinoid activity during this specific developmental phase, induced by the psychoactive component of marijuana, Δ9-tetrahydrocannabinol, might lead to subtle but lasting neurobiological changes that can affect brain functions and behaviour. In this review, we outline recent research into the endocannabinoid system focusing on the relationships between adolescent exposure to cannabinoids and increased risk for certain neuropsychiatric diseases such as schizophrenia, as highlighted by both human and animal studies. Particular emphasis will be given to the possible mechanisms by which adolescent cannabis consumption could render a person more susceptible to developing psychoses such as schizophrenia. This article is part of a themed issue on Cannabinoids. To view the editorial for this themed issue visit http://dx.doi.org/10.1111/j.1476-5381.2010.00831.x

Markedly reduced toxicity of a hydrogen sulphide-releasing derivative of naproxen (ATB-346)

Abstract: Background and purpose: Hydrogen sulphide is an important mediator of gastric mucosal defence. The use of non-steroidal anti-inflammatory drugs continues to be limited by their toxicity, particularly in the upper gastrointestinal tract. We evaluated the gastrointestinal safety and anti-inflammatory efficacy of a novel hydrogen sulphide-releasing derivative of naproxen, ATB-346 [2-(6-methoxy-napthalen-2-yl)-propionic acid 4-thiocarbamoyl-phenyl ester]. Experimental approach: The ability of ATB-346 versus naproxen to cause gastric damage was evaluated in healthy rats and in rats with compromised gastric mucosal defence. Effects on the small intestine and on the healing of ulcers were also assessed. The ability of ATB-346 to inhibit cyclooxygenase-1 and 2 and to reduce inflammation in vivo was also evaluated. Key results: ATB-346 suppressed gastric prostaglandin E2 synthesis as effectively as naproxen, but produced negligible damage in the stomach and intestine. In situations in which the gastric mucosa was rendered significantly more susceptible to naproxen-induced damage (e.g. ablation of sensory afferent nerves, inhibition of endogenous nitric oxide or hydrogen sulphide synthesis, co-administration with aspirin, antagonism of KIR6.x channels), ATB-346 did not cause significant damage. Unlike naproxen and celecoxib, ATB-346 accelerated healing of pre-existing gastric ulcers. In a mouse airpouch model, ATB-346 suppressed cyclooxygenase-2 activity and inhibited leukocyte infiltration more effectively than naproxen. ATB-346 was as effective as naproxen in adjuvant-induced arthritis in rats, with a more rapid onset of activity. Unlike naproxen, ATB-346 did not elevate blood pressure in hypertensive rats. Conclusions and implications: ATB-346 exhibits anti-inflammatory properties similar to naproxen, but with substantially reduced gastrointestinal toxicity.

A monobromobimane-based assay to measure the pharmacokinetic profile of reactive sulphide species in blood

Abstract: Background and purpose: Hydrogen sulphide (H2S) is a labile, endogenous metabolite of cysteine, with multiple biological roles. The development of sulphide-based therapies for human diseases will benefit from a reliable method of quantifying H2S in blood and tissues.

The complex G protein-coupled receptor kinase 2 (GRK2) interactome unveils new physiopathological targets

Abstract: GRK2 is a ubiquitous member of the G protein-coupled receptor kinase (GRK) family that appears to play a central, integrative role in signal transduction cascades. GRKs participate together with arrestins in the regulation of G protein-coupled receptors (GPCR), a family of hundreds of membrane proteins of key physiological and pharmacological importance, by triggering receptor desensitization from G proteins and GPCR internalization, and also by helping assemble macromolecular signalosomes in the receptor environment acting as agonist-regulated adaptor scaffolds, thus contributing to signal propagation. In addition, emerging evidence indicates that GRK2 can phosphorylate a growing number of non-GPCR substrates and associate with a variety of proteins related to signal transduction, thus suggesting that this kinase could also have diverse ‘effector’ functions. We discuss herein the increasing complexity of such GRK2 ‘interactome’, with emphasis on the recently reported roles of this kinase in cell migration and cell cycle progression and on the functional impact of the altered GRK2 levels observed in several relevant cardiovascular, inflammatory or tumour pathologies. Deciphering how the different networks of potential GRK2 functional interactions are orchestrated in a stimulus, cell type or context-specific way is critical to unveil the contribution of GRK2 to basic cellular processes, to understand how alterations in GRK2 levels or functionality may participate in the onset or development of several cardiovascular, tumour or inflammatory diseases, and to assess the feasibility of new therapeutic strategies based on the modulation of the activity, levels or specific interactions of GRK2.

GPR55 ligands promote receptor coupling to multiple signalling pathways

Abstract: Background and purpose: Although GPR55 is potently activated by the endogenous lysophospholipid, L-α-lysophosphatidylinositol (LPI), it is also thought to be sensitive to a number of cannabinoid ligands, including the prototypic CB1 receptor antagonists AM251 and SR141716A (Rimonabant®). In this study we have used a range of functional assays to compare the pharmacological activity of selected cannabinoid ligands, AM251, AM281 and SR141716A with LPI in a HEK293 cell line engineered to stably express recombinant, human GPR55.

Evidence that the plant cannabinoid cannabigerol is a highly potent α2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist.

Abstract: Background and purpose: Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor.

Depression-resistant endophenotype in mice overexpressing cannabinoid CB2 receptors

Abstract: Background and purpose: The present study evaluated the role of CB2 receptors in the regulation of depressive-like behaviours. Transgenic mice overexpressing the CB2 receptor (CB2xP) were challenged with different types of acute and chronic experimental paradigms to evaluate their response in terms of depressive-like behaviours. Experimental approach: Tail suspension test (TST), novelty-suppressed feeding test (NSFT) and unpredictable chronic mild stress tests (CMS) were carried out in CB2xP mice. Furthermore, acute and chronic antidepressant-like effects of the CB2 receptor-antagonist AM630 were evaluated by means of the forced swimming test (FST) and CMS, respectively, in wild-type (WT) and CB2xP mice. CB2 gene expression, brain-derived neurotrophic factor (BDNF) gene and protein expressions were studied in mice exposed to CMS by real-time PCR and immunohistochemistry, respectively. Key results: Overexpression of CB2 receptors resulted in decreased depressive-like behaviours in the TST and NSFT. CMS failed to alter the TST and sucrose consumption in CB2xP mice. In addition, no changes in BDNF gene and protein expression were observed in stressed CB2xP mice. Interestingly, acute administration of AM630 (1 and 3 mg·kg−1, i.p.) exerted antidepressant-like effects on the FST in WT, but not in CB2xP mice. Chronic administration of AM630 for 4 weeks (1 mg·kg−1; twice daily, i.p.) blocked the effects of CMS on TST, sucrose intake, CB2 receptor gene, BDNF gene and protein expression in WT mice. Conclusion and implications: Taken together, these results suggest that increased CB2 receptor expression significantly reduced depressive-related behaviours and that the CB2 receptor could be a new potential therapeutic target for depressive-related disorders.

Genetic polymorphisms and drug interactions modulating CYP2D6 and CYP3A activities have a major effect on oxycodone analgesic efficacy and safety

Abstract: BACKGROUND AND PURPOSE: The major drug-metabolizing enzymes for the oxidation of oxycodone are CYP2D6 and CYP3A. A high interindividual variability in the activity of these enzymes because of genetic polymorphisms and/or drug-drug interactions is well established. The possible role of an active metabolite in the pharmacodynamics of oxycodone has been questioned and the importance of CYP3A-mediated effects on the pharmacokinetics and pharmacodynamics of oxycodone has been poorly explored. EXPERIMENTAL APPROACH: We conducted a randomized crossover (five arms) double-blind placebo-controlled study in 10 healthy volunteers genotyped for CYP2D6. Oral oxycodone (0.2 mg x kg(-1)) was given alone or after inhibition of CYP2D6 (with quinidine) and/or of CYP3A (with ketoconazole). Experimental pain (cold pressor test, electrical stimulation, thermode), pupil size, psychomotor effects and toxicity were assessed. KEY RESULTS: CYP2D6 activity was correlated with oxycodone experimental pain assessment. CYP2D6 ultra-rapid metabolizers experienced increased pharmacodynamic effects, whereas cold pressor test and pupil size were unchanged in CYP2D6 poor metabolizers, relative to extensive metabolizers. CYP2D6 blockade reduced subjective pain threshold (SPT) for oxycodone by 30% and the response was similar to placebo. CYP3A4 blockade had a major effect on all pharmacodynamic assessments and SPT increased by 15%. Oxymorphone C(max) was correlated with SPT assessment (rho(S)= 0.7) and the only independent positive predictor of SPT. Side-effects were observed after CYP3A4 blockade and/or in CYP2D6 ultra-rapid metabolizers. CONCLUSIONS AND IMPLICATIONS: The modulation of CYP2D6 and CYP3A activities had clear effects on oxycodone pharmacodynamics and these effects were dependent on CYP2D6 genetic polymorphism.

Non-synaptic receptors and transporters involved in brain functions and targets of drug treatment

Abstract: Beyond direct synaptic communication, neurons are able to talk to each other without making synapses. They are able to send chemical messages by means of diffusion to target cells via the extracellular space, provided that the target neurons are equipped with high-affinity receptors. While synaptic transmission is responsible for the ‘what’ of brain function, the ‘how’ of brain function (mood, attention, level of arousal, general excitability, etc.) is mainly controlled non-synaptically using the extracellular space as communication channel. It is principally the ‘how’ that can be modulated by medicine. In this paper, we discuss different forms of non-synaptic transmission, localized spillover of synaptic transmitters, local presynaptic modulation and tonic influence of ambient transmitter levels on the activity of vast neuronal populations. We consider different aspects of non-synaptic transmission, such as synaptic–extrasynaptic receptor trafficking, neuron–glia communication and retrograde signalling. We review structural and functional aspects of non-synaptic transmission, including (i) anatomical arrangement of non-synaptic release sites, receptors and transporters, (ii) intravesicular, intra- and extracellular concentrations of neurotransmitters, as well as the spatiotemporal pattern of transmitter diffusion. We propose that an effective general strategy for efficient pharmacological intervention could include the identification of specific non-synaptic targets and the subsequent development of selective pharmacological tools to influence them.

A novel peripherally restricted cannabinoid receptor antagonist, AM6545, reduces food intake and body weight, but does not cause malaise, in rodents.

Abstract: BACKGROUND AND PURPOSE Cannabinoid CB1 receptor antagonists reduce food intake and body weight, but clinical use in humans is limited by effects on the CNS. We have evaluated a novel cannabinoid antagonist (AM6545) designed to have limited CNS penetration, to see if it would inhibit food intake in rodents, without aversive effects. EXPERIMENTAL APPROACH Cannabinoid receptor binding studies, cAMP assays, brain penetration studies and gastrointestinal motility studies were carried out to assess the activity profile of AM6545. The potential for AM6545 to induce malaise in rats and the actions of AM6545 on food intake and body weight were also investigated. KEY RESULTS AM6545 binds to CB1 receptors with a Ki of 1.7 nM and CB2 receptors with a Ki of 523 nM. AM6545 is a neutral antagonist, having no effect on cAMP levels in transfected cells and was less centrally penetrant than AM4113, a comparable CB1 receptor antagonist. AM6545 reversed the effects of WIN55212-2 in an assay of colonic motility. In contrast to AM251, AM6545 did not produce conditioned gaping or conditioned taste avoidance in rats. In rats and mice, AM6545 dose-dependently reduced food intake and induced a sustained reduction in body weight. The effect on food intake was maintained in rats with a complete subdiaphragmatic vagotomy. AM6545 inhibited food intake in CB1 receptor gene-deficient mice, but not in CB1/CB2 receptor double knockout mice. CONCLUSIONS AND IMPLICATIONS Peripherally active, cannabinoid receptor antagonists with limited brain penetration may be useful agents for the treatment of obesity and its complications.

Aiming drug discovery at lysophosphatidic acid targets.

Abstract: Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is the prototype member of a family of lipid mediators and second messengers. LPA and its naturally occurring analogues interact with G protein-coupled receptors on the cell surface and a nuclear hormone receptor within the cell. In addition, there are several enzymes that utilize LPA as a substrate or generate it as a product and are under its regulatory control. LPA is present in biological fluids, and attempts have been made to link changes in its concentration and molecular composition to specific disease conditions. Through their many targets, members of the LPA family regulate cell survival, apoptosis, motility, shape, differentiation, gene transcription, malignant transformation and more. The present review depicts arbitrary aspects of the physiological and pathophysiological actions of LPA and attempts to link them with select targets. Many of us are now convinced that therapies targeting LPA biosynthesis and signalling are feasible for the treatment of devastating human diseases such as cancer, fibrosis and degenerative conditions. However, successful targeting of the pathways associated with this pleiotropic lipid will depend on the future development of as yet undeveloped pharmacons.

6‐Shogaol, an active constituent of ginger, inhibits breast cancer cell invasion by reducing matrix metalloproteinase‐9 expression via blockade of nuclear factor‐κB activation

Abstract: BACKGROUND AND PURPOSE Shogaols are reported to possess anti-inflammatory and anticancer activities. However, the antimetastatic potential of shogaols remains unexplored. This study was performed to assess the effects of shogaols against breast cancer cell invasion and to investigate the underlying mechanisms. EXPERIMENTAL APPROACH The anti-invasive effect of a series of shogaols was initially evaluated on MDA-MB-231 breast cancer cells using the matrigel invasion assay. The suppressive effects of 6-shogaol on phorbol 12-myristate 13-acetate (PMA)-induced matrix metalloproteinase-9 (MMP-9) gelatinolytic activity and nuclear factor-κB (NF-κB) activation were further determined. KEY RESULTS Shogaols (6-, 8- and 10-shogaol) inhibited PMA-stimulated MDA-MB-231 cell invasion with an accompanying decrease in MMP-9 secretion. 6-Shogaol was identified to display the greatest anti-invasive effect in association with a dose-dependent reduction in MMP-9 gene activation, protein expression and secretion. The NF-κB transcriptional activity was decreased by 6-shogaol; an effect mediated by inhibition of IκB phosphorylation and degradation that subsequently led to suppression of NF-κB p65 phosphorylation and nuclear translocation. In addition, 6-shogaol was found to inhibit JNK activation with no resulting reduction in activator protein-1 transcriptional activity. By using specific inhibitors, it was demonstrated that ERK and NF-κB signalling, but not JNK and p38 signalling, were involved in PMA-stimulated MMP-9 activation. CONCLUSIONS AND IMPLICATIONS 6-Shogaol is a potent inhibitor of MDA-MB-231 cell invasion, and the molecular mechanism involves at least in part the down-regulation of MMP-9 transcription by targeting the NF-κB activation cascade. This class of naturally occurring small molecules thus have potential for clinical use as antimetastatic treatments.

Thymoquinone inhibits proliferation, induces apoptosis and chemosensitizes human multiple myeloma cells through suppression of signal transducer and activator of transcription 3 activation pathway

Abstract: BACKGROUND AND PURPOSE Constitutive activation of the signal transducer and activator of transcription 3 (STAT3) pathway is frequently encountered in several human cancers including multiple myeloma (MM). Thus, agents that suppress STAT3 phosphorylation have a potential for treatment of MM. In the present report, we investigated whether thymoquinone (TQ), the main component isolated from the medicinal plant Nigella sativa, modulated the STAT3 signalling pathway in MM cells. EXPERIMENTAL APPROACH The effect of TQ on both constitutive and IL-6-induced STAT3 activation, associated protein kinases, STAT3-regulated gene products involved in proliferation, survival and angiogenesis, cellular proliferation and apoptosis in MM cells, was investigated. KEY RESULTS We found that TQ inhibited both constitutive and IL-6-inducible STAT3 phosphorylation which correlated with the inhibition of c-Src and JAK2 activation. Vanadate reversed the TQ-induced down-regulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that TQ can induce the expression of Src homology-2 phosphatase 2 that correlated with suppression of STAT3 activation. TQ also down-regulated the expression of STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin, Mcl-1 and vascular endothelial growth factor. Finally, TQ induced the accumulation of cells in sub-G1 phase, inhibited proliferation and induced apoptosis, as indicated by poly ADP ribose polymerase cleavage. TQ also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. CONCLUSIONS AND IMPLICATIONS Our study has identified STAT3 signalling as a target of TQ and has thus raised its potential application in the prevention and treatment of MM and other cancers.