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Background and purpose Trace amine-associated TA1 receptors play critical roles in regulating dopamine transmission. Previous studies showed that pharmacologically or genetically manipulating the activity of TA1 receptors modulates addiction-like behaviours associated with psychostimulants. However, little is known about whether TA1 receptor modulation would regulate the behavioural effects of opioids. Experimental approach Effects of the selective TA1 receptor partial agonist RO5263397 on the addiction-related and antinociceptive effects of morphine were systematically assessed in male rats and mice. Key results RO5263397 attenuated the expression of morphine-induced behavioural sensitization in wildtype but not TA1 receptor knockout mice. RO5263397 shifted the dose-effect curve of morphine self-administration downward and reduced the breakpoint in a progressive ratio schedule of reinforcement but did not affect food self-administration in rats. RO5263397 decreased the cue- and drug-induced reinstatement of morphine-seeking behaviour in rats. RO5263397 alone did not trigger reinstatement of morphine-seeking behaviour or change locomotor activity in rats with a history of morphine self-administration. However, RO5263397 did not affect the expression of morphine-induced conditioned place preference in mice or rats. RO5263397 did not affect naltrexone-precipitated jumping behaviour or naltrexone-induced conditioned place aversion in morphine-dependent mice. Furthermore, RO5263397 did not affect the analgesic effects of morphine in an acute nociception model in mice and a chronic pain model in rats. Conclusion and implications These results indicated that TA1 receptor activation selectively attenuated the reinforcing, but not withdrawal or antinociceptive effects of morphine, suggesting that selective TA1 receptor agonists might be useful to combat opioid addiction, while sparing the analgesic effects.

Background and purpose Resurgence in the use of chloroquine as a potential treatment for COVID-19 has seen recent cases of fatal toxicity due to unintentional overdoses. Protocols for the management of poisoning recommend diazepam, although there are uncertainties in its pharmacology and efficacy in this context. The aim was to assess the effects of diazepam in experimental models of chloroquine cardiotoxicity. Experimental approach In vitro experiments involved cardiac tissues isolated from rats and incubated with chloroquine alone or in combination with diazepam. In vivo models of toxicity involved chloroquine administered intravenously to pentobarbitone-anaesthetised rats and rabbits. Randomised, controlled treatment studies in rats assessed diazepam, clonazepam and Ro5-4864 administered: (i) prior, (ii) during and (iii) after chloroquine and the effects of diazepam: (iv) at high dose, (v) in urethane-anaesthetised rats and (vi) co-administered with adrenaline. Key results Chloroquine decreased the developed tension of left atria, prolonged the effective refractory period of atria, ventricular tissue and right papillary muscles, and caused dose-dependent impairment of haemodynamic and electrocardiographic parameters. Cardiac arrhythmias indicated impairment of atrioventricular conduction. Studies (i), (ii) and (v) showed no differences between treatments and control. Diazepam increased heart rate in study (iv) and as with clonazepam also prolonged the QTc interval in study (iii). Combined administration of diazepam and adrenaline in study (vi) improved cardiac contractility but caused hypokalaemia. Conclusion and implications Neither diazepam nor other ligands for benzodiazepine binding sites protect against or attenuate chloroquine cardiotoxicity. However, diazepam may augment the effects of positive inotropes in reducing chloroquine cardiotoxicity. Linked articles This article is part of a themed issue on The Pharmacology of COVID-19. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.21/issuetoc.

Background and purpose The concept of opioid ligands biased towards the G protein pathway with minimal recruitment of β-arrestin-2 is a promising approach for the development of novel, efficient, and potentially nonaddictive opioid therapeutics. A recently discovered biased μ-opioid receptor agonist, PZM21, showed analgesic effects with reduced side effects. Here, we aimed to further investigate the behavioural and biochemical properties of PZM21. Experiment approach We evaluated antinociceptive effects of systemic and intrathecal PZM21 administration. Its addiction-like properties were determined using several behavioural approaches: conditioned place preference, locomotor sensitization, precipitated withdrawal, and self-administration. Also, effects of PZM21 on morphine-induced antinociception, tolerance, and reward were assessed. Effects of PZM21 on striatal release of monoamines were evaluated using brain microdialysis. Key results PZM21 caused long-lasting dose-dependent antinociception. It did not induce reward- and reinforcement-related behaviour; however, its repeated administration led to antinociceptive tolerance and naloxone-precipitated withdrawal symptoms. Pretreatment with PZM21 enhanced morphine-induced antinociception and attenuated the expression of morphine reward. In comparison to morphine, PZM21 administration induced a moderate release of dopamine and a robust release of 5-HT in the striatum. Conclusions and implications PZM21 exhibited antinociceptive efficacy, without rewarding or reinforcing properties. However, its clinical application may be restricted, as it induces tolerance and withdrawal symptoms. Notably, its ability to diminish morphine reward implies that PZM21 may be useful in treatment of opioid use disorders.

Background and purpose Aerobic glycolysis is a unique feature of tumour cells that entails several advantages for cancer progression such as resistance to apoptosis. The low MW compound, dichloroacetate, is a pyruvate dehydrogenase kinase inhibitor, which restores oxidative phosphorylation and induces apoptosis in a variety of cancer entities. However, its therapeutic effectiveness is limited by resistance mechanisms. This study aimed to examine the role of the anti-apoptotic hyaluronan (HA) matrix in this context and to identify a potential add-on treatment option to overcome this limitation. Experimental approach The metabolic connection between dichloroacetate treatment and HA matrix augmentation was analysed in vitro by quantitative PCR and affinity cytochemistry. Metabolic pathways were analysed using Seahorse, HPLC, fluorophore-assisted carbohydrate electrophoresis, colourimetry, immunoblots, and immunochemistry. The effects of combining dichloroacetate with the HA synthesis inhibitor 4-methylumbelliferone was evaluated in 2D and 3D cell cultures and in a nude mouse tumour xenograft regression model by immunoblot, immunochemistry, and FACS analysis. Key results Mitochondrial reactivation induced by dichloroacetate metabolically activated HA synthesis by augmenting precursors as well as O-GlcNAcylation. This process was blocked by 4-methylumbelliferone, resulting in enhanced anti-tumour efficacy in 2D and 3D cell culture and in a nude mouse tumour xenograft regression model. Conclusions and implications The HA rich tumour micro-environment represents a metabolic factor contributing to chemotherapy resistance. HA synthesis inhibition exhibited pronounced synergistic actions with dichloroacetate treatment on oesophageal tumour cell proliferation and survival in vitro and in vivo suggesting the combination of these two strategies is an effective anticancer therapy.

BACKGROUNDCannabichromene (CBC) is one of the most abundant phytocannabinoids inCannabis spp. It has modest anti-nociceptive and anti-inflammatory effects and potentiates some effects of Δ9-tetrahydrocannabinol (THC)in vivo. How CBC exerts these effects is poorly defined and there is little information about its efficacy at cannabinoid receptors. We sought to determine the functional activity of CBC at CB1 and CB2 receptors.EXPERIMENTAL APPROACHAtT20 cells stably expressing HA-tagged human CB1 and CB2 receptors were used. Assays of cellular membrane potential and loss of cell surface receptors were performed.KEY RESULTSCBC activated CB2 but not CB1 receptors to produce a hyperpolarization of AtT20 cells. Activation of CB2 receptors was antagonised by the CB2 antagonist AM630 and sensitive to pertussis toxin. Co-application of CBC reduced activation of CB2 receptors CP55,940, a potent CB1 and CB2 agonist. Continuous CBC application induced loss of cell surface CB2 receptors and desensitisation of the CB2-induced hyperpolarization.CONCLUSIONS AND IMPLICATIONSCannabichromene is a selective CB2 receptor agonist displaying higher efficacy than THC in hyperpolarising AtT20 cells. CBC may contribute to the potential therapeutic effectiveness of some cannabis preparations, potentially through CB2-mediated modulation of inflammation.

BACKGROUND AND PURPOSE: In patients with pulmonary hypertension (PH) associated with lung disease and/or hypoxia (Group III), decreased pulmonary vascular tone and tissue hypoxia is therapeutically beneficial. PGE(2) and PGI(2) induce potent relaxation of human bronchi from non‐PH (control) patients via EP(4) and IP receptors, respectively. However, the effects of PGE(2)/PGI(2) and their mimetics on human bronchi from PH patients are unknown. Here, we have compared relaxant effects of several PGI(2)–mimetics approved for treating PH Group I with several PGE(2)–mimetics, in bronchial preparations derived from PH Group III and control patients. EXPERIMENTAL APPROACH: Relaxation of bronchial muscle was assessed in samples isolated from control and PH Group III patients. Expression of prostanoid receptors was analysed by western blot and real‐time PCR, and endogenous PGE(2), PGI(2), and cAMP levels were determined by ELISA. KEY RESULTS: Maximal relaxations induced by different EP(4) receptor agonists (PGE(2), L‐902688, and ONO‐AE1‐329) were decreased in human bronchi from PH patients, compared with controls. However, maximal relaxations produced by PGI(2)–mimetics (iloprost, treprostinil, and beraprost) were similar for both groups of patients. Both EP(4) and IP receptor protein and mRNA expressions were significantly lower in human bronchi from PH patients. cAMP levels significantly correlated with PGI(2) but not with PGE(2) levels. CONCLUSION AND IMPLICATIONS: The PGI(2)–mimetics retained maximal bronchodilation in PH Group III patients, whereas bronchodilation induced by EP(4) receptor agonists was decreased. Restoration of EP(4) receptor expression in airways of PH Group III patients with respiratory diseases could bring additional therapeutic benefit.

Background and Purpose The TNF family ligands, B cell activating factor of the TNF family (BAFF, also known as B lymphocyte stimulator, BLyS) and a proliferation‐inducing ligand (APRIL), share the transmembrane activator and calcium‐modulator and cyclophilin ligand (CAML)‐interactor (TACI) as one of their common receptors. Atacicept, a chimeric recombinant TACI/IgG1‐Fc fusion protein, inhibits both ligands. TACI and APRIL also bind to proteoglycans and to heparin that is structurally related to proteoglycans. It is unknown whether the portion of TACI contained in atacicept can bind directly to proteoglycans, or indirectly via APRIL, and whether this could interfere with the anti‐coagulant properties of heparin. Experimental Approach Binding of atacicept and APRIL to proteoglycan‐positive cells was measured by FACS. Activities of heparin and atacicept were measured with activated factor Xa inhibition and cell‐based assays. Effects of heparin on circulating atacicept was monitored in mice. Key Results Atacicept did not bind to proteoglycan‐positive cells, but when complexed to APRIL could do so indirectly via APRIL. Multimers of atacicept obtained after exposure to cysteine or BAFF 60‐mer bound directly to proteoglycans. Atacicept alone, or in complex with APRIL, or in a multimeric form did not interfere with heparin activity in vitro. Conversely, heparin did not influence inhibition of BAFF and APRIL by atacicept and did not change circulating levels of atacicept. Conclusions and Implications Lack of detectable interference of APRIL‐bound or free atacicept on heparin activity makes it unlikely that atacicept at therapeutic doses will interfere with the function of heparin in vivo.

Background and purpose Recent development in drug discovery have shown benzimidazole to be an important pharmacophore,. Benzimidazole derivatives exhibit broad-spectrum pharmacological properties including anti-microbial, anti-diabetic and anti-tumour activity. However, whether benzimidazole derivatives are effective in suppressing angiogenesis and its underlying mechanisms remain incompletely understood. In this study, we aim to characterize the anti-angiogenic mechanisms of a novel 2-aminobenzimidazole-based compound, Jzu 17, in an effort to develop novel angiogenesis inhibitor. Experimental approach Effects of Jzu 17 on endothelial cell proliferation, migration, invasion, and activation of signalling molecules induced by VEGF-A, were analysed by immunoblotting, MTT, BrdU, migration, and invasion assays. We performed tube formation assay, aorta ring sprouting assay, matrigel plug assay, and a mouse model of metastasis to evaluate ex vivo and in vivo anti-angiogenic effects of Jzu 17. Key results Jzu 17 inhibited VEGF-A-induced cell proliferation, migration, invasion, and endothelial tube formation of HUVECs. Jzu 17 suppressed VEGF-A-induced microvessel sprouting ex vivo and attenuated VEGF-A- or tumour cell-induced neovascularization in vivo. Jzu 17 also reduced B16F10 melanoma lung metastasis. In addition, Jzu 17 inhibited the phosphorylation of VEGFR-2 and its downstream signalling molecules in VEGF-A-stimulated HUVECs. Results from computer modelling further showed that Jzu 17 binds to VEGFR-2 with high affinity. Conclusions and implications Jzu 17 may inhibit endothelial remodelling and suppress angiogenesis through targeting VEGF-A-VEGFR-2 signalling. These results also suggest Jzu 17 as a potential lead compound and warrant the clinical development of similar agents in the treatment of cancer and angiogenesis-related diseases.

Background and purpose Depression and anxiety are common causes of disability, and innovative tools and potential pharmacological targets are actively sought for prevention and treatment. Therapeutic strategies targeting the relaxin-3 peptide or its primary endogenous receptor, RXFP3, for the treatment of major depression and anxiety disorders have been limited by a lack of compounds with drug-like properties. We proposed that a hydrocarbon-stapled mimetic of relaxin-3, when administered intranasally, might be uniquely applicable to the treatment of these disorders. Experimental approach We designed a series of hydrocarbon-stapled relaxin-3 mimetics and identified the most potent compound using in vitro receptor binding and activation assays. Further, we assessed the effect of intranasal delivery of relaxin-3 and the lead stapled mimetic in rat models of anxiety and depression. Key results We developed an i,i+7 stapled relaxin-3 mimetic that manifested a stabilized α-helical structure, proteolytic resistance, and confirmed agonist activity in receptor binding and activation in vitro assays. The stapled peptide agonist enhanced food intake after intracerebral infusion in rats, confirming in vivo activity. We showed that intranasal delivery of the lead i,i+7 stapled peptide or relaxin-3 had orexigenic effects in rats, indicating a potential clinically translatable route of delivery. Further, intranasal administration of the lead i,i+7 stapled peptide exerted anxiolytic and antidepressant-like activity in anxiety- and depression-related behaviour paradigms. Conclusions and implications Our preclinical findings demonstrate that targeting the relaxin-3/RXFP3 receptor system via intranasal delivery of an i,i+7 stapled relaxin-3 mimetic may represent an effective treatment approach for depression, anxiety, and related neuropsychiatric disorders.

Background and purpose The TRPC1, TRPC4, and TRPC5 proteins form homotetrameric or heterotetrameric, calcium-permeable cation channels that are involved in various disease states. Recent research has yielded specific and potent xanthine-based TRPC1/4/5 inhibitors. Here, we investigated the possibility of xanthine-based activators of these channels. Experimental approach An analogue of the TRPC1/4/5 inhibitor Pico145, AM237, was synthesized and its activity was investigated using HEK cells overexpressing TRPC4, TRPC5, TRPC4-C1, TRPC5-C1, TRPC1:C4 or TRPC1:C5 channels, and in A498 cells expressing native TRPC1:C4 channels. TRPC1/4/5 channel activities were assayed by measuring intracellular concentration of Ca2+ ([Ca2+ ]i ) and by patch-clamp electrophysiology. Selectivity of AM237 was tested against TRPC3, TRPC6, TRPV4, or TRPM2 channels. Key results AM237 potently activated TRPC5:C5 channels (EC50 15-20 nM in [Ca2+ ]i assay) and potentiated their activation by sphingosine-1-phosphate but suppressed activation evoked by (-)-englerin A (EA). In patch-clamp studies, AM237 activated TRPC5:C5 channels, with greater effect at positive voltages, but with lower efficacy than EA. Pico145 competitively inhibited AM237-induced TRPC5:C5 activation. AM237 did not activate TRPC4:C4, TRPC4-C1, TRPC5-C1, TRPC1:C5, and TRPC1:C4 channels, or native TRPC1:C4 channels in A498 cells, but potently inhibited EA-dependent activation of these channels with IC50 values ranging from 0.9 to 7 nM. AM237 (300 nM) did not activate or inhibit TRPC3, TRPC6, TRPV4, or TRPM2 channels. Conclusions and implications This study suggests the possibility for selective activation of TRPC5 channels by xanthine derivatives and supports the general principle that xanthine-based compounds can activate, potentiate, or inhibit these channels depending on subunit composition.

Background and purpose Salinomycin is a well-known inhibitor of human cancer stem cells (CSCs). However, the molecular mechanism(s) by which salinomycin targets colorectal CSCs is poorly understood. Here, we have investigated underlying antitumour mechanisms of salinomycin in colorectal cancer cells and three tumour models. Experimental approach The inhibitory effect of salinomycin on the Wnt/β-catenin pathway was analysed with the SuperTopFlash reporter system. The mRNA expression of Wnt target genes was evaluated with real-time PCR. Effects of salinomycin on β-catenin/TCF4E interaction were examined using co-immunoprecipitation and an in vitro GST pull-down assay. Cell proliferation was determined by BrdU incorporation and soft agar colony formation assay. The stemness of the cells was assessed by sphere formation assay. Antitumour effects of salinomycin on colorectal cancers was evaluated with colorectal CSC xenografts, APCmin/+ transgenic mice, and patient-derived colorectal tumour xenografts. Key results Salinomycin blocked β-catenin/TCF4E complex formation in colorectal cancer cells and in an in vitro GST pull-down assay, thus decreasing expression of Wnt target genes. Salinomycin also suppressed the transcriptional activity mediated by β-catenin/LEF1 or β-catenin/TCF4E complex and exhibited an inhibitory effect on the sphere formation, proliferation, and anchorage-independent growth of colorectal cancer cells. In colorectal tumour xenografts and APCmin/+ transgenic mice, administration of salinomycin significantly reduced tumour growth and the expression of CSC-related Wnt target genes including LGR5. Conclusions and implications Our study suggested that salinomycin could suppress the growth of colorectal cancer by disrupting the β-catenin/TCF complex and thus may be a promising agent for colorectal cancer treatment.

Background and purpose Hydrogen sulfide donors can block the cardiovascular injury of hyperhomocysteinemia. H2 S also lowers serum homocysteine in rats with mild hyperhomocysteinemia, but the pharmacological mechanism is unknown. The present study investigated the mechanism(s) involved. Experimental approach ApoE-knockout mice were fed a Paigen diet and L-methionine in drinking water for 16 weeks to create a mouse model of atherosclerosis with hyperhomocysteinemia. H2 S donors (NaHS and GYY4137) were administered by intraperitoneal injection. We also assayed the H2 S produced (by methylene blue assay and mito-HS [H2 S fluorescence probe]), cystathionine γ lyase (CSE) mRNA and protein expression, and CSE sulfhydration and nitrosylation and its activity. Key results H2 S donor treatment significantly lowered atherosclerotic plaque area, macrophage infiltration, and serum homocysteine level in the mouse model of atherosclerosis with co-existing hyperhomocysteinemia. mRNA and protein levels of CSE, a key enzyme catalyzing homocysteine trans-sulfuration, were down-regulated with hyperhomocysteinemia, and CSE catalytic activity was inhibited. All these effects were reversed with H2 S donor treatment. Hyperhomocysteinemia induced CSE nitrosylation, whereas H2 S sulfhydrated CSE at the same cysteine residues. Nitrosylated CSE decreased and sulfhydrated CSE increased its catalytic and binding activities towards L-homocysteine. Mutation of C252, C255, C307, and C310 residues in CSE abolished CSE nitrosylation or sulfhydration and prevented its binding to L-homocysteine. Conclusions and implications Sulfhydration or nitrosylation of CSE represents a yin/yang regulation of catalysis or binding to L-homocysteine. H2 S donor treatment enhanced CSE sulfhydration, thus lowering serum L-homocysteine, which contributed in part to the anti-atherosclerosis effects in ApoE-knockout mice with hyperhomocysteinemia.

BACKGROUND AND PURPOSE: (R)‐Ketamine (arketamine) may have utility as a rapidly acting antidepressant. While (R)‐ketamine has lower potency than (R,S)‐ketamine to inhibit NMDA receptors in vitro, the extent to which (R)‐ketamine shares the NMDA receptor‐mediated adverse effects of (R,S)‐ketamine in vivo has not been fully characterised. Furthermore, (R)‐ketamine is metabolised to (2R,6R)‐hydroxynorketamine (HNK), which may contribute to its antidepressant‐relevant actions. EXPERIMENTAL APPROACH: Using mice, we compared (R)‐ketamine with a deuterated form of the drug (6,6‐dideutero‐(R)‐ketamine, (R)‐d(2)‐ketamine), which hinders its metabolism to (2R,6R)‐HNK, in behavioural tests predicting antidepressant responses. We also examined the actions of intracerebroventricularly infused (2R,6R)‐HNK. Further, we quantified putative NMDA receptor inhibition‐mediated adverse effects of (R)‐ketamine. KEY RESULTS: (R)‐d(2)‐Ketamine was identical to (R)‐ketamine in binding to and functionally inhibiting NMDA receptors but hindered (R)‐ketamine's metabolism to (2R,6R)‐HNK. (R)‐Ketamine exerted greater potency than (R)‐d(2)‐ketamine in several antidepressant‐sensitive behavioural measures, consistent with a role of (2R,6R)‐HNK in the actions of (R)‐ketamine. There were dose‐dependent sustained antidepressant‐relevant actions of (2R,6R)‐HNK following intracerebroventricular administration. (R)‐Ketamine exerted NMDA receptor inhibition‐mediated behaviours similar to (R,S)‐ketamine, including locomotor stimulation, conditioned‐place preference, prepulse inhibition deficits, and motor incoordination, with approximately half the potency of the racemic drug. CONCLUSIONS AND IMPLICATIONS: Metabolism of (R)‐ketamine to (2R,6R)‐HNK increases the potency of (R)‐ketamine to exert antidepressant‐relevant actions in mice. Adverse effects of (R)‐ketamine require higher doses than those necessary for antidepressant‐sensitive behavioural changes in mice. However, our data revealing that (R)‐ketamine's adverse effects are elicited at sub‐anaesthetic doses indicate a potential risk for sensory dissociation and abuse liability.

There is evidence for ultra‐sensitive responses to active compounds at concentrations below picomolar levels by proteins and receptors found in species ranging from bacteria to mammals. We have recently shown that such ultra‐sensitivity is also demonstrated by a wide range of prototypical GPCRs, and we have determined the molecular mechanisms behind these responses for three family A GPCRs: the relaxin receptor, RXFP1; the β(2)‐adrenoceptor; and the M(3) muscarinic ACh receptor. Interestingly, there are reports of similar ultra‐sensitivity by more than 15 human GPCR families, in addition to other human receptors and channels. These occur through a diverse range of signalling pathways and produce modulation of important physiological processes, including neuronal transmission, chemotaxis, gene transcription, protein/ion uptake and secretion, muscle contraction and relaxation, and phagocytosis. Here, we summarise the accumulating evidence of ultra‐sensitive receptor signalling to show that this is a common, though currently underappreciated, property of GPCRs. LINKED ARTICLES: This article is part of a themed section on Adrenoceptors—New Roles for Old Players. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.14/issuetoc

Background and Purpose: Triptans are 5-HT1B/1D receptor agonists (that also display 5-HT1F receptor affinity) with antimigraine action, contraindicated in patients with coronary artery disease due to their vasoconstrictor properties. Conversely, lasmiditan was developed as an antimigraine 5-HT1F receptor agonist. To assess the selectivity and cardiovascular effects of lasmiditan, we investigated the binding, functional activity, and in vitro/in vivo vascular effects of lasmiditan and compared it to sumatriptan. Experimental Approach: Binding and second messenger activity assays of lasmiditan and other serotoninergic agonists were performed for human 5-HT1A, 5-HT1B, 5-HT1D, 5-ht1E, 5-HT1F, 5-HT2A, 5-HT2B, and 5-HT7 receptors, and the results were correlated with their potency to constrict isolated human coronary arteries (HCAs). Furthermore, concentration–response curves to lasmiditan and sumatriptan were performed in proximal and distal HCA, internal mammary, and middle meningeal arteries. Finally, anaesthetized female beagle dogs received i.v. infusions of lasmiditan or sumatriptan in escalating cumulative doses, and carotid and coronary artery diameters were measured. Key Results: Lasmiditan showed high selectivity for 5-HT1F receptors. Moreover, the functional potency of the analysed compounds to inhibit cAMP increase through 5-HT1B receptor activation positively correlated with their potency to contract HCA. In isolated human arteries, sumatriptan, but not lasmiditan, induced contractions. Likewise, in vivo, sumatriptan decreased coronary and carotid artery diameters at clinically relevant doses, while lasmiditan was devoid of vasoconstrictor activity at all doses tested. Conclusions and Implications: Lasmiditan is a selective 5-HT1F receptor agonist devoid of vasoconstrictor activity. This may represent a cardiovascular safety advantage when compared to the triptans.

BACKGROUND AND PURPOSE: Despite widespread abuse of cocaine, there are no approved treatments for cocaine use disorder. Chronic cocaine use is associated with up‐regulated dopamine D(3) receptor expression in the brain. Therefore, most D(3)‐based medication development has focused on D(3) antagonists. However, D(3) antagonists do not attenuate cocaine intake under “easy” self‐administration conditions, when response requirements are low. We evaluated a novel, highly selective and metabolically stable D(3) partial agonist, (±)VK4‐40, for its efficacy in reducing cocaine intake and relapse to drug seeking. EXPERIMENTAL APPROACH: The impact of (±)VK4‐40 on cocaine intake and relapse was evaluated using intravenous self‐administration procedures under a fixed‐ratio 2 reinforcement schedule and cocaine‐primed reinstatement conditions in rats. Optogenetic brain‐stimulation reward procedures were used to evaluate the interaction of (±)VK4‐40 and cocaine in the mesolimbic dopamine system in mice. Sucrose self‐administration in rats and a conditioned place preference paradigm in mice were used to evaluate the abuse potential of (±)VK4‐40 alone and other unwanted effects. KEY RESULTS: (±)VK4‐40 dose‐dependently reduced cocaine self‐administration and cocaine‐primed reinstatement of drug‐seeking behaviour. (±)VK4‐40 also inhibited cocaine‐enhanced brain‐stimulation reward caused by optogenetic stimulation of dopamine neurons in the ventral tegmental area. (±)VK4‐40 alone decreased brain‐stimulation reward but produced neither conditioned place preference nor place aversion. This new D(3) partial agonist also failed to alter oral sucrose self‐administration. CONCLUSION AND IMPLICATIONS: The novel D(3) partial agonist, (±)VK4‐40 attenuates cocaine reward and relapse in rodents, without significant unwanted effects. These findings support further investigation of D(3) partial agonists as putative treatments for cocaine use disorder.

Background and purpose Liquorice is the root of Glycyrrhiza glabra, which is a popular food in Europe and China that has previously shown benefits for skeletal fatigue and nutrient metabolism. However, the mechanism and active ingredients remain largely unclear. The aim of this study was to investigate the active ingredients of liquorice for muscle wasting and elucidate the underlying mechanisms. Experimental approach RNA-Seq and bioinformatics analysis were applied to predict the main target of liquorice. A machine learning model and a docking tool were used to predict active ingredients. Isotope labelling experiments, immunostaining, Western blots, qRT-PCR, ChIP-PCR and luciferase reporters were utilized to test the pharmacological effects in vitro and in vivo. The reverse effects were verified through recombination-based overexpression. Key results The liposoluble constituents of liquorice improved muscle wasting by inhibiting protein catabolism and fibre atrophy. We further identified FoxO1 as the target of liposoluble constituents of liquorice. In addition, hispaglabridin B (HB) was predicted as an inhibitor of FoxO1. Further studies determined that HB improved muscle wasting by inhibiting catabolism in vivo and in vitro. HB also markedly suppressed the transcriptional activity of FoxO1, with decreased expression of the muscle-specific E3 ubiquitin ligases MuRF1 and Atrogin-1. Conclusions and implications HB can serve as a novel natural food extract for preventing muscle wasting in chronic kidney disease and possibly other catabolic conditions.

BACKGROUND AND PURPOSE: Abdominal aortic aneurysm (AAA) is a degenerative disease affecting human health, but there are no safe and effective medications for AAA therapy. Cycloastragenol (CAG), derived from Astragali Radix, has various pharmacological effects. However, whether CAG can protect against AAA remains elusive. In this study, we investigated whether CAG has an inhibitory effect on AAA and its related mechanism. EXPERIMENTAL APPROACH: The AAA mouse model was induced by incubating the abdominal aorta with elastase. CAG was administered by gavage at different doses beginning on the same day or 14 days after inducing AAA to explore its preventive or therapeutic effects respectively. The preventive effects of CAG on AAA were verified in another AAA mouse model induced by angiotensin II in ApoE(−/−) mouse. In vitro experiments were implemented on rat vascular smooth muscle cells (VSMCs) stimulated by TNF‐α. KEY RESULTS: Compared to the control AAA model group, CAG (125 mg·kg(−1) body weight day(−1)) reduced the incidence of AAA, the dilatation of aorta and elastin degradation in media in both mouse models of AAA. CAG suppressed the inflammation, oxidation, phenotype switch and apoptosis in TNF‐α‐stimulated VSMCs, ameliorated the expression and activity of MMPs and decreased the activation of the ERK/JNK signalling pathway. CAG also inhibited the degradation of elastin in TNF‐α‐stimulated VSMCs. CONCLUSION AND IMPLICATIONS: CAG presents protective effects against AAA through down‐regulation of the MAPK signalling pathways and thus attenuates inflammation, oxidation, VSMC phenotype switch and apoptosis and the expression of MMPs as well as increasing elastin biosynthesis.

Background and Purpose Emerging evidence indicates that hypertension is mediated by immune mechanisms. We hypothesized that exposure to Porphyromonas gingivalis antigens, commonly encountered in periodontal disease, can enhance immune activation in hypertension and exacerbate the elevation in BP, vascular inflammation and vascular dysfunction. Experimental Approach Th1 immune responses were elicited through immunizations using P. gingivalis lysate antigens (10 μg) conjugated with aluminium oxide (50 μg) and IL‐12 (1 μg). The hypertension and vascular endothelial dysfunction evoked by subpressor doses of angiotensin II (0.25 mg·kg−1·day−1) were studied, and vascular inflammation was quantified by flow cytometry and real‐time PCR. Key Results Systemic T‐cell activation, a characteristic of hypertension, was exacerbated by P. gingivalis antigen stimulation. This translated into increased aortic vascular inflammation with enhanced leukocyte, in particular, T‐cell and macrophage infiltration. The expression of the Th1 cytokines, IFN‐γ and TNF‐α, and the transcription factor, TBX21, was increased in aortas of P. gingivalis/IL‐12/aluminium oxide‐immunized mice, while IL‐4 and TGF‐β were unchanged. These immune changes in mice with induced T‐helper‐type 1 immune responses were associated with an enhanced elevation of BP and endothelial dysfunction compared with control mice in response to 2 week infusion of a subpressor dose of angiotensin II. Conclusions and Implications These results support the concept that Th1 immune responses induced by bacterial antigens such as P. gingivalis can increase sensitivity to subpressor pro‐hypertensive insults such as low‐dose angiotensin II, thus providing a mechanistic link between chronic infection, such as periodontitis, and hypertension. Linked Articles This article is part of a themed section on Immune Targets in Hypertension. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.12/issuetoc

BACKGROUND AND PURPOSE: Altered function or expression of GABA(A) receptors contributes to anxiety disorders. Benzodiazepines are widely prescribed for the treatment of anxiety. However, the long‐term use of benzodiazepines increases the risk of developing drug dependence and tolerance. Thus, it is urgent to explore new therapeutic approaches. Metformin is widely used to treat Type 2 diabetes and other metabolic syndromes. However, the role of metformin in psychiatric disorders, especially anxiety, remains largely unknown. EXPERIMENTAL APPROACH: We examined the effects of metformin on anxiety‐like behaviour of rats in open field test and elevated plus maze test. We also observed the effect of metformin (10 μM, in vitro; 100 mg·kg(−1), in vivo) on the trafficking of GABA(A) receptors, as mechanisms underlying the anxiolytic effects of metformin. KEY RESULTS: Metformin (100 mg·kg(−1), i.p. 30 min) displayed a robust and rapid anxiolytic effect, without tolerance. Metformin up‐regulated the surface expression of GABA(A) receptors and increased miniature inhibitory postsynaptic currents (mIPSCs). AMP‐activated protein kinase (AMPK) activated by metformin‐induced stimulation of forkhead box O3a (FoxO3a) transcriptional activity, followed by increased expression of GABA(A) receptor‐associated protein (GABARAP) and its binding to GABA(A) receptors finally resulted in the membrane insertion of GABA(A) receptors. CONCLUSIONS AND IMPLICATIONS: Metformin increased mIPSCs by up‐regulating the membrane insertion of GABA(A) receptors, via a pathway involving AMPK, FoxO3a, and the GABA(A) receptor‐associated protein. Thus metformin has a potential new use in the treatment of anxiety disorders.

Background and Purpose Atopic dermatitis (AD) is a multifactorial skin condition with complex interactions of innate and adaptive immune responses. There are several existing therapies for AD, including topical glucocorticosteroids, emollients, phototherapies, calcineurin inhibitors and immunosuppressants, such as cyclosporine A. Although these therapies reduce inflammation, they also cause serious side effects. Therefore, it is necessary to develop new therapeutic approaches for AD treatment without side effects. There are several studies on natural materials or toxins, such as herbs, ginseng extract and snake venom, for AD treatment. However, treatment of AD with bee venom and its major component, melittin has rarely been studied. Experimental Approach Effects of bee venom and melittin were studied in a model of AD in vivo induced by 1‐chloro‐2,4‐dinitrobenzene (DNCB) in female Balb/c mice and in cultures of human keratinocytes, stimulated by TNF‐α/IFN‐γ. The potential pharmacological effects of bee venom and melittin on these in vivo and in vitro AD‐like skin disease models were studied. Key Results Bee venom and melittin exhibited potent anti‐atopic activities, shown by decreased AD‐like skin lesions, induced by DNCB in mice. In vitro studies using TNF‐α/IFN‐γ‐stimulated human keratinocytes showed that bee venom and melittin inhibited the increased expression of chemokines, such as CCL17 and CCL22, and pro‐inflammatory cytokines, including IL‐1β, IL‐6 and IFN‐γ, through the blockade of the NF‐κB and STAT signalling pathways. Conclusions and Implications Our results suggest that bee venom and melittin would be suitable for epicutaneous application, as topical administration is often appropriate for the treatment of AD.

Background and Purpose Numerous claims are made for cannabis' therapeutic utility upon human seizures, but concerns persist about risks. A potential confounder is the presence of both Δ9‐tetrahydrocannabinol (THC), variously reported to be pro‐ and anticonvulsant, and cannabidiol (CBD), widely confirmed as anticonvulsant. Therefore, we investigated effects of prolonged exposure to different THC/CBD cannabis extracts on seizure activity and associated measures of endocannabinoid (eCB) system signalling. Experimental Approach Cannabis extract effects on in vivo neurological and behavioural responses, and on bioanalyte levels, were measured in rats and dogs. Extract effects on seizure activity were measured using electroencephalography telemetry in rats. eCB signalling was also investigated using radioligand binding in cannabis extract‐treated rats and treatment‐naïve rat, mouse, chicken, dog and human tissue. Key Results Prolonged exposure to cannabis extracts caused spontaneous, generalized seizures, subserved by epileptiform discharges in rats, but not dogs, and produced higher THC, but lower 11‐hydroxy‐THC (11‐OH‐THC) and CBD, plasma concentrations in rats versus dogs. In the same rats, prolonged exposure to cannabis also impaired cannabinoid type 1 receptor (CB1 receptor)‐mediated signalling. Profiling CB1 receptor expression, basal activity, extent of activation and sensitivity to THC suggested interspecies differences in eCB signalling, being more pronounced in a species that exhibited cannabis extract‐induced seizures (rat) than one that did not (dog). Conclusions and Implications Sustained cannabis extract treatment caused differential seizure, behavioural and bioanalyte levels between rats and dogs. Supporting radioligand binding data suggest species differences in eCB signalling. Interspecies variations may have important implications for predicting cannabis‐induced convulsions from animal models. Linked Articles This article is part of a themed section on 8th European Workshop on Cannabinoid Research. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.10/issuetoc

BACKGROUND AND PURPOSE: Enhanced late sodium current (late INa ) in heart failure and long QT syndrome type 3 is proarrhythmic. This study investigated the antiarrhythmic effect and mode of action of the selective and potent late INa inhibitor GS-458967 (GS967) against Torsades de Pointes arrhythmias (TdP) in the chronic atrioventricular block (CAVB) dog. EXPERIMENTAL APPROACH: Electrophysiological and antiarrhythmic effects of GS967 were evaluated in isolated canine ventricular cardiomyocytes and CAVB dogs with dofetilide-induced early afterdepolarizations (EADs) and TdP, respectively. Mapping of intramural cardiac electrical activity in vivo was conducted to study effects of GS967 on spatial dispersion of repolarization. KEY RESULTS: GS967 (IC50 ~200nM) significantly shortened repolarization in canine ventricular cardiomyocytes and sinus rhythm (SR) dogs, in a concentration and dose-dependent manner. In vitro, despite addition of 1μM GS967, dofetilide-induced EADs remained present in 42% and 35% of cardiomyocytes from SR and CAVB dogs, respectively. Nonetheless, GS967 (787±265nM) completely abolished dofetilide-induced TdP in CAVB dogs (10/14 after dofetilide to 0/14 dogs after GS967), while single ectopic beats (sEB) persisted in 9 animals. In vivo mapping experiments showed that GS967 significantly reduced spatial dispersion of repolarization: cubic dispersion was significantly decreased from 237±54ms after dofetilide to 123±34ms after GS967. CONCLUSION AND IMPLICATIONS: GS967 terminated all dofetilide-induced TdP without completely suppressing EADs and sEB in vitro and in vivo, respectively. The antiarrhythmic mode of action of GS967, through the reduction of spatial dispersion of repolarization, seems to predominantly impede the perpetuation of arrhythmic events into TdP rather than their initiating trigger.

Background and Purpose Non‐alcoholic steatohepatitis (NASH) is the hepatic manifestation of metabolic syndrome and is characterized by steatosis, inflammation and fibrosis. Soluble guanylate cyclase (sGC) stimulation reduces inflammation and fibrosis in experimental models of lung, kidney and heart disease. Here, we tested whether sGC stimulation is also effective in experimental NASH. Experimental Approach NASH was induced in mice by feeding a choline‐deficient, l‐amino acid‐defined, high‐fat diet. These mice received either placebo or the sGC stimulator IW‐1973 at two different doses (1 and 3 mg·kg−1·day−1) for 9 weeks. IW‐1973 was also tested in high‐fat diet (HFD)‐induced obese mice. Steatosis, inflammation and fibrosis were assessed by Oil Red O, haematoxylin–eosin, Masson's trichrome, Sirius Red, F4/80 and α‐smooth muscle actin staining. mRNA expression was assessed by quantitative PCR. Levels of IW‐1973, cytokines and cGMP were determined by LC‐MS/MS, Luminex and enzyme immunoassay respectively. Key Results Mice with NASH showed reduced cGMP levels and sGC expression, increased steatosis, inflammation, fibrosis, TNF‐α and MCP‐1 levels and up‐regulated collagen types I α1 and α2, MMP2, TGF‐β1 and tissue metallopeptidase inhibitor 1 expression. IW‐1973 restored hepatic cGMP levels and sGC expression resulting in a dose‐dependent reduction of hepatic inflammation and fibrosis. IW‐1973 levels were ≈40‐fold higher in liver tissue than in plasma. IW‐1973 also reduced hepatic steatosis and adipocyte hypertrophy secondary to enhanced autophagy in HFD‐induced obese mice. Conclusions and Implications Our data indicate that sGC stimulation prevents hepatic steatosis, inflammation and fibrosis in experimental NASH. These findings warrant further evaluation of IW‐1973 in the clinical setting.

Background and purpose Naturally occurring dysfunction in NaV channels results in complex disorders such as chronic pain, making these channels an attractive target for new therapies. In the pursuit of novel NaV modulators, we investigated spider venoms for new inhibitors of NaV channels. Experimental Approach We used high-throughput screens to identify a NaV modulator in venom of the spider Davus fasciatus. Further characterization of this venom peptide was undertaken using fluorescent and electrophysiological assays, molecular modeling and a rodent pain model. Key Results We identified a potent NaV inhibitor named μ-TRTX-Df1a. This 34-residue peptide fully inhibited responses mediated by NaV1.7 endogenously expressed in SH-SY5Y cells. Df1a also inhibited CaV3 currents but had no activity against KV2. The modelled structure of Df1a, which contains an inhibitor cystine knot motif, is reminiscent of the NaV channel toxin ProTx-I. Electrophysiology revealed that Df1a inhibits all NaV subtypes tested (hNaV1.1–1.7). Df1a also slowed fast inactivation of NaV1.1, NaV1.3 and NaV1.5, and modified the voltage-dependence of activation and inactivation of most NaV subtypes. Df1a preferentially binds to the domain II voltage-sensor and has additional interactions with the voltage sensors domains III and IV, which likely explains its modulatory features. Df1a was analgesic in vivo, reversing the spontaneous pain behaviors induced by the NaV activator OD1. Conclusion and Implication μ-TRTX-Df1a shows potential as a new molecule for the development of therapies to treat voltage-gated ion channels mediated pain disorders.

Background and Purpose Results regarding protective effects of dipeptidyl peptidase 4 (DPP4) inhibitors in renal ischaemia–reperfusion injury (IRI) are conflicting. Here we have compared structurally unrelated DPP4 inhibitors in a model of renal IRI. Experimental Approach IRI was induced in uninephrectomized male rats by renal artery clamping for 30 min. The sham group was uninephrectomized but not subjected to IRI. DPP4 inhibitors or vehicle were given p.o. once daily on three consecutive days prior to IRI: linagliptin (1.5 mg·kg−1·day−1), vildagliptin (8 mg·kg−1·day−1) and sitagliptin (30 mg·kg−1·day−1). An additional group received sitagliptin until study end (before IRI: 30 mg·kg−1·day−1; after IRI: 15 mg·kg−1·day−1). Key Results Plasma‐active glucagon‐like peptide type 1 (GLP‐1) increased threefold to fourfold in all DPP4 inhibitor groups 24 h after IRI. Plasma cystatin C, a marker of GFR, peaked 48 h after IRI. Compared with the placebo group, DPP4 inhibition did not reduce increased plasma cystatin C levels. DPP4 inhibitors ameliorated histopathologically assessed tubular damage with varying degrees of drug‐specific efficacies. Renal osteopontin expression was uniformly reduced by all DPP4 inhibitors. IRI‐related increased renal cytokine expression was not decreased by DPP4 inhibition. Renal DPP4 activity at study end was significantly inhibited in the linagliptin group, but only numerically reduced in the prolonged/dose‐adjusted sitagliptin group. Active GLP‐1 plasma levels at study end were increased only in the prolonged/dose‐adjusted sitagliptin treatment group. Conclusions and Implications In rats with renal IRI, DPP4 inhibition did not alter plasma cystatin C, a marker of glomerular function, but may protect against tubular damage.

Background and purpose The κ receptor has a central role in modulating neurotransmission in central and peripheral neuronal circuits that subserve pain and other behavioural responses. Although κ receptor agonists do not produce euphoria or lead to respiratory suppression, they induce dysphoria and sedation. We hypothesized that brain‐penetrant κ receptor ligands possessing biased agonism towards G protein signalling over β‐arrestin2 recruitment would produce robust antinociception with fewer associated liabilities. Experimental approach Two new diphenethylamines with high κ receptor selectivity, HS665 and HS666, were assessed following i.c.v. administration in mouse assays of antinociception with the 55°C warm‐water tail withdrawal test, locomotor activity in the rotorod and conditioned place preference. The [35S]‐GTPγS binding and β‐arrestin2 recruitment in vitro assays were used to characterize biased agonism. Key results HS665 (κ receptor agonist) and HS666 (κ receptor partial agonist) demonstrated dose‐dependent antinociception after i.c.v. administration mediated by the κ receptor. These highly selective κ receptor ligands displayed varying biased signalling towards G protein coupling in vitro, consistent with a reduced liability profile, reflected by reduced sedation and absence of conditioned place aversion for HS666. Conclusions and implications HS665 and HS666 activate central κ receptors to produce potent antinociception, with HS666 displaying pharmacological characteristics of a κ receptor analgesic with reduced liability for aversive effects correlating with its low efficacy in the β‐arrestin2 signalling pathway. Our data provide further understanding of the contribution of central κ receptors in pain suppression, and the prospect of dissociating the antinociceptive effects of HS665 and HS666 from κ receptor‐mediated adverse effects.

BACKGROUND AND PURPOSE Intracranial aneurysm (IA), common in general public, causes lethal subarachnoid hemorrhage on rupture. Prevention of rupture of IA is therefore socially important. However, there is currently no medical treatment. Recent studies suggest that IA is a disease of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrated macrophages. Sphingosine-1-phosphate receptor type 1 (S1P1) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, in prevention of IA in animal model. EXPERIMENTAL APPROACH A selective S1P1 agonist, ASP4058, was used. Its effects on endothelial permeability and migration of macrophages across endothelial cell monolayer were tested in vitro using a Transwell system, and effects of ASP4058 on size of IAs induced in a rat model was evaluated in rat IA model. KEY RESULTS S1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC-dextran leakage through endothelial monolayer and suppressed migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 suppressed vascular permeability, macrophage infiltration and size of IAs by acting as an S1P1 agonist in rat model. This effect was mimicked by two other structurally-unrelated S1P1 agonists. CONCLUSION AND IMPLICATIONS A selective S1P1 agonist is a strong drug candidate for IA treatment through promoting endothelial cell barrier and suppressing trans-endothelial migration of macrophages in IA lesions. Non-approved abbreviations anterior cerebral artery, ACA; 3-aminopropionitrile, BAPN; Chinese hamster ovary, CHO; diastolic blood pressures, DBP; geometric mean fluorescence intensity, geoMFI; human carotid artery endothelial cells, HCtAEC; Intracranial aneurysm, IA; mean blood pressures, MBP; methylcellulose, MC; olfactory artery, OA; α-smooth muscle actin, SMA; systolic blood pressures, SBP; Sphigosine-1-phosphate, S1P; Transmission Electron Microscopy, TEM; wall shear stress, WSS

Background and purpose Dihydrotanshinone I (DHI), a lipophilic component of traditional Chinese medicine Salvia miltiorrhiza Bunge, has various therapeutic effects. We investigated the anti-fibrotic effect of DHI and its underlying mechanisms in vitro and in vivo. Experimental approach Rats subjected to bile duct ligation (BDL) were treated with DHI (25 mg·kg-1 ·day-1 , i.p.) for 14 days. Serum biochemical and liver tissue morphological analyses were performed. The human hepatic stellate cell line LX-2 served as a liver fibrosis model in vitro. Liver fibrogenic genes, yes-associated protein (YAP) downstream genes and autophagy markers were examined using western blot and real-time PCR analyses. Similar analyses were done in rat primary hepatic stellate cells (pHSCs). Autophagy flux was assessed by immunofluorescence. Key results In BDL rats, DHI administration attenuated liver necrosis, bile duct proliferation and collagen accumulation and reduced the expression of genes associated with fibrogenesis, including Tgfb1, Mmp-2, Acta2 and Col1a1. DHI (1, 5, 10 μmol·L-1 ) time- and dose-dependently suppressed the protein level of COL1A1, TGFβ1 and α-SMA in LX-2 cells and rat pHSCs. Furthermore, DHI blocked the nuclear translocation of YAP, which inhibited the YAP/TEAD2 interaction and its downstream fibrogenic genes, connective tissue growth factor, SOX4 and survivin. This stimulated autophagic flux and accelerated the degradation of liver collagen. Conclusions and implications DHI exerts anti-fibrotic effects in BDL rats, LX-2 cells and rat pHSCs by inhibiting the YAP and TEAD2 complex and stimulating autophagy. These findings indicate that DHI may be a potential therapeutic for the treatment of liver fibrosis.

Background and Purpose Tuberculosis (TB) remains a major global health threat and is now the leading cause of death from a single infectious agent worldwide. The current TB drug regimen is inadequate, and new anti‐tubercular agents are urgently required to be able to successfully combat the increasing prevalence of drug‐resistant TB. The purpose of this study was to investigate a piperidinol compound derivative that is highly active against the Mycobacterium tuberculosis bacillus. Experimental Approach The antibacterial properties of the piperidinol compound and its corresponding bis‐Mannich base analogue were evaluated against M. smegmatis and Gram‐negative organisms. Cytotoxicity studies were undertaken in order to determine the selectivity index for these compounds. Spontaneous resistant mutants of M. smegmatis were generated against the piperidinol and corresponding bis‐Mannich base lead derivatives and whole genome sequencing employed to determine the genetic modifications that lead to selection pressure in the presence of these compounds. Key Results The piperidinol and the bis‐Mannich base analogue were found to be selective for mycobacteria and rapidly kill this organism with a cytotoxicity selectivity index for mycobacteria of >30‐fold. Whole genome sequencing of M. smegmatis strains resistant to the lead compounds led to the identification of a number of single nucleotide polymorphisms indicating multiple targets. Conclusion and Implications Our results indicate that the piperidinol moiety represents an attractive compound class in the pursuit of novel anti‐tubercular agents. Linked Articles This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc

Background and Purpose Reducing glucocorticoid exposure in the brain via intracellular inhibition of the cortisol‐regenerating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) has emerged as a therapeutic strategy to treat cognitive impairment in early Alzheimer's disease (AD). We sought to discover novel, brain‐penetrant 11β‐HSD1 inhibitors as potential medicines for the treatment of AD. Experimental Approach Medicinal chemistry optimization of a series of amido‐thiophene analogues was performed to identify potent and selective 11β‐HSD1 inhibitors with optimized oral pharmacokinetics able to access the brain. Single and multiple ascending dose studies were conducted in healthy human subjects to determine the safety, pharmacokinetic and pharmacodynamic characteristics of the candidate compound. Results UE2343 was identified as a potent, orally bioavailable, brain‐penetrant 11β‐HSD1 inhibitor and selected for clinical studies. No major safety issues occurred in human subjects. Plasma adrenocorticotropic hormone was elevated (a marker of systemic enzyme inhibition) at doses of 10 mg and above, but plasma cortisol levels were unchanged. Following multiple doses of UE2343, plasma levels were approximately dose proportional and the terminal t 1/2 ranged from 10 to 14 h. The urinary tetrahydrocortisols/tetrahydrocortisone ratio was reduced at doses of 10 mg and above, indicating maximal 11β‐HSD1 inhibition in the liver. Concentrations of UE2343 in the CSF were 33% of free plasma levels, and the peak concentration in CSF was ninefold greater than the UE2343 IC50. Conclusions and Implications UE2343 is safe, well tolerated and reaches the brain at concentrations predicted to inhibit 11β‐HSD1. UE2343 is therefore a suitable candidate to test the hypothesis that 11β‐HSD1 inhibition in brain improves memory in patients with AD.

Background and purpose Cerebral vasospasm and neuronal apoptosis after subarachnoid haemorrhage (SAH) is the major cause of morbidity and mortality in SAH patients. So far, single-target agents have not prevented its occurrence. Memantine, a non-competitive NMDA re3ceptor antagonist, is known to alleviate brain injury and vasospasm in experimental models of SAH. Impairment of NO availability also contributes to vasospasm. Recently, we designed and synthesized a memantine nitrate MN-08, which has potent dual functions: neuroprotection and vasodilation. Here, we have tested the therapeutic effects of MN-08 in animal models of SAH. Experimental approach Binding to NMDA receptors (expressed in HEK293 cells), NO release and vasodilator effects of MN-08 were assessed in vitro. Therapeutic effects of MN-08 were investigated in vivo, using rat and rabbit SAH models. Key results MN-08 bound to the NMDA receptor, slowly releasing NO in vitro and in vivo. Consequently, MN-08 relaxed the pre-contracted middle cerebral artery ex vivo and increased blood flow velocity in small vessels of the mouse cerebral cortex. It did not, however, lower systemic blood pressure. In an endovascular perforation rat model of SAH, MN-08 improved the neurological scores and ameliorated cerebral vasospasm. Moreover, MN-08 also alleviated cerebral vasospasm in a cisterna magna single-injection model in rabbits. MN-08 attenuated neural cell apoptosis in both rat and rabbit models of SAH. Importantly, the therapeutic benefit of MN-08 was greater than that of memantine. Conclusion and implications MN-08 has neuroprotective potential and can ameliorate vasospasm in experimental SAH models.

Background and purpose Like chili peppers, gingers produce pungent stimuli by a group of vanilloid compounds that activate the nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel. How these compounds interact with TRPV1 remains unclear. Experimental approach We used computational structural modelling, functional tests (electrophysiology and calcium imaging), and mutagenesis to investigate the structural mechanisms underlying ligand-channel interactions. Key results The potency of three principal pungent compounds from ginger -shogaol, gingerol, and zingerone-depends on the same two residues in the TRPV1 channel that form a hydrogen bond with the chili pepper pungent compound, capsaicin. Computational modelling revealed binding poses of these ginger compounds similar to those of capsaicin, including a "head-down tail-up" orientation, two specific hydrogen bonds, and important contributions of van der Waals interactions by the aliphatic tail. Our study also identified a novel horizontal binding pose of zingerone that allows it to directly interact with the channel pore when bound inside the ligand-binding pocket. These observations offer a molecular level explanation for how unique structures in the ginger compounds affect their channel activation potency. Conclusions and implications Mechanistic insights into the interactions of ginger compounds and the TRPV1 cation channel should help guide drug discovery efforts to modulate nociception.