A selective antagonist of histamine H4 receptors prevents antigen-induced airway inflammation and bronchoconstriction in guinea pigs: involvement of lipocortin-1

Background and Purpose

Among the pathogenic mechanisms of asthma, a role for oxidative/nitrosative stress has been well documented. Recent evidence suggests that histamine H₄ receptors play a modulatory role in allergic inflammation. Here we report the effects of compound JNJ 7777120 (JNJ), a selective H₄ receptor antagonist, on antigen-induced airway inflammation, paying special attention to its effects on lipocortin-1 (LC-1/annexin-A1), a 37 kDA anti-inflammatory protein that plays a key role in the production of inflammatory mediators.

Experimental Approach

Ovalbumin (OA)-sensitized guinea pigs placed in a respiratory chamber were challenged with antigen. JNJ (5, 7.5 and 10 mg·kg⁻¹) was given i.p. for 4 days before antigen challenge. Respiratory parameters were recorded. Bronchoalveolar lavage (BAL) fluid was collected and lung specimens taken for further analyses 1 h after antigen challenge. In BAL fluid, levels of LC-1, PGD₂, LTB₄ and TNF-α were measured. In lung tissue samples, myeloperoxidase, caspase-3 and Mn-superoxide dismutase activities and 8-hydroxy-2-deoxyguanosine levels were measured.

Key Results

OA challenge decreased LC-1 levels in BAL fluid, induced cough, dyspnoea and bronchoconstriction and increased PGD₂, LTB₄ and TNF-α levels in lung tissue. Treatment with JNJ dose-dependently increased levels of LC-1, reduced respiratory abnormalities and lowered levels of PGD₂, LTB₄ and TNF-α in BAL fluid.

Conclusions and Implications

Antigen-induced asthma-like reactions in guinea pigs decreased levels of LC-1 and increased TNF-α and eicosanoid production. JNJ pretreatment reduced allergic asthmatic responses and airway inflammation, an effect associated with LC-1 up-regulation.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

In vitro and in vivo characterization of A-940894: a potent histamine H4 receptor antagonist with anti-inflammatory properties

Background and purpose:

The histamine H₄ receptor is widely expressed in cells of immune origin and has been shown to play a role in a variety of inflammatory processes mediated by histamine. In this report, we describe the in vitro and in vivo anti-inflammatory properties of a potent histamine H₄ receptor antagonist, A-940894 (4-piperazin-1-yl-6,7-dihydro-5H-benzo[6,7]cyclohepta[1,2-d]pyrimidin-2-ylamine).

Experimental approach:

We have analysed the pharmacological profile of A-940894 at mouse native, rat recombinant and human recombinant and native, histamine H₄ receptors by radioligand binding, calcium mobilization, mast cell shape change, eosinophil chemotaxis assays and in the mouse model of zymosan-induced peritonitis.

Key results:

A-940894 potently binds to both human and rat histamine H₄ receptors and exhibits considerably lower affinity for the human histamine H₁, H₂ or H₃ receptors. It potently blocked histamine-evoked calcium mobilization in the fluorometric imaging plate reader assays and inhibited histamine-induced shape change of mouse bone marrow-derived mast cells and chemotaxis of human eosinophils in vitro. In a mouse mast cell-dependent model of zymosan-induced peritonitis, A-940894 significantly blocked neutrophil influx and reduced intraperitoneal prostaglandin D₂ levels. Finally, A-940894 has good pharmacokinetic properties, including half-life and oral bioavailability in rats and mice.

Conclusions and Implications:

These data suggest that A-940894 is a potent and selective histamine H₄ receptor antagonist with pharmacokinetic properties suitable for long-term in vivo testing and could serve as a useful tool for the further characterization of histamine H₄ receptor pharmacology.     

Interleukin-22 exacerbates airway inflammation induced by short-term exposure to cigarette smoke in mice

Aim:

Interleukin-22 (IL-22) exhibits both proinflammatory and anti-inflammatory properties in various biological processes. In this study we explored the effects of exogenous recombinant IL-22 (rIL-22) on cigarette smoke (CS)-induced airway inflammation in mice.

Methods:

Male C57BL/6 mice were divided into groups: (1) CS group exposed to tobacco smoke for 3 consecutive days, (2) rIL-22 group received rIL-22 (100 mg/kg, ip), and (3) CS plus rIL-22 group, received rIL-22 (100 mg/kg, ip) before the CS exposure. The airway resistance (Rn), lung morphology, inflammatory cells in the airways, and inflammatory cytokines and CXCR3 ligands in both bronchoalveolar lavage (BAL) fluids and lung tissues were analyzed.

Results:

CS alone significantly elevated IL-22 level in the BAL fluid. Both CS and rIL-22 significantly augmented airway resistance, an influx of inflammatory cells into the airways and lung parenchyma, and significantly elevated levels of pro-inflammatory cytokines (TGFβ1 and IL-17A) and CXCR3 chemokines (particularly CXCL10) at the mRNA and/or protein levels. Furthermore, the effects of rIL-22 on airway resistance and inflammation were synergistic with those of CS, as demonstrated by a further increased Rn value, infiltration of greater numbers of inflammatory cells into the lung, higher levels of inflammatory cytokines and chemokines, and more severe pathological changes in CS plus rIL-22 group as compared to those in CS group.

Conclusion:

Exogenous rIL-22 exacerbates the airway inflammatory responses to CS exposure in part by inducing expression of several proinflammatory cytokines and CXCR3 ligands.     

Biochemical and pharmacological characterization of AZD1981, an orally available selective DP2 antagonist in clinical development for asthma

Background and Purpose

The discovery of DP₂ as a second receptor for PGD₂ has prompted the search for antagonists as potential novel therapies based on the associations between PGD₂ and disease. Here we describe the biochemical and pharmacological properties of 4-(acetylamino)-3-[(4-chlorophenyl)thio]-2-methyl-1H-indole-1-acetic acid (AZD1981), a novel DP₂ receptor antagonist.

Experimental Approach

Binding to DP₂, functional receptor pharmacology and selectivity were studied in both human and animal systems.

Key Results

AZD1981 displaced radio-labelled PGD₂ from human recombinant DP₂ with high potency (pIC₅₀ = 8.4). Binding was reversible, non-competitive and highly selective against a panel of more than 340 other enzymes and receptors, including DP₁ (>1000-fold selective). AZD1981 inhibited DP₂-mediated shape change and CD11b up-regulation in human eosinophils, shape change in basophils and chemotaxis of human eosinophils and Th2 cells with similar potency. AZD1981 exhibited good cross-species binding activity against mouse, rat, guinea pig, rabbit and dog DP₂. Evaluation in mouse, rat or rabbit cell systems was not possible as they did not respond to DP₂ agonists. Agonist responses were seen in guinea pig and dog, and AZD1981 blocked DP₂-mediated eosinophil shape change. Such responses were more robust in the guinea pig, where AZD1981 also blocked DP₂-dependent eosinophil emigration from bone marrow.

Conclusions and Implications

AZD1981 is a DP₂ antagonist that blocks functional responses in eosinophils, Th2 cells and basophils. It exhibited similar potency irrespective of the cell type, DP₂ agonist or species used. This selective orally active agent is currently under clinical evaluation as a potential therapeutic agent in respiratory diseases including asthma.     

Blocking initial infiltration of pioneer CD8+ T-cells into the CNS via inhibition of SHP-2 ameliorates experimental autoimmune encephalomyelitis in mice

Background and Purpose

In contrast to T-cell priming in the periphery, therapeutic strategies targeting the initiation step of T-cell trafficking into the CNS have not been extensively investigated. In this study, we examined the effect of NSC-87877, a potent Src homology 2-containing protein tyrosine phosphatase 2 (SHP-2) inhibitor, on experimental autoimmune encephalomyelitis (EAE) and elucidated its unique mechanism of action.

Experimental Approach

C57BL/6 mice were immunized with myelin oligodendrocyte glycoprotein_35–55 and monitored for clinical severity of disease and histopathological features in the CNS. Levels of cytokines in serum were measured by elisa. Effects of NSC-87877 on expressions of chemokines and cytokines in the CNS were determined by quantitative PCR.

Key Results

NSC-87877-treated mice developed conventional T_H1 and T_H17 responses, but were highly resistant to the induction of EAE. NSC-87877 decreased the accumulation of lymphocytes in the CNS and increased the functional expression of chemokine receptor CXCR7 on CD8⁺ T-cells. Adoptive transfer of T-cells from 2D2-transgenic mice restored EAE susceptibility in NSC-87877-treated mice, indicating that NSC-87877 only targets the initial migration of pioneer T-cells. Furthermore, T-cell-conditioned SHP-2-deficient mice treated with NSC-87877 were no longer resistant to EAE, suggesting that inhibition of SHP-2 contributes to the amelioration of EAE by NSC-87877.

Conclusions and Implications

NSC-87877 almost completely abolished the development of EAE by blocking the initial infiltration of pioneer CD8⁺ T-cells into the uninflamed CNS. These results reveal a critical role for SHP-2 in regulating EAE pathogenesis and indicate that NSC-87877 is a potential candidate for the treatment of relapsing-remitting multiple sclerosis.     

Histamine is required for H3 receptor-mediated alcohol reward inhibition,but not for alcohol consumption or stimulation

Background and Purpose

Conflicting data have been published on whether histamine is inhibitory to the rewarding effects of abused drugs. The purpose of this study was to clarify the role of neuronal histamine and, in particular, H₃ receptors in alcohol dependence-related behaviours, which represent the addictive effects of alcohol.

Experimental Approach

Alcohol-induced conditioned place preference (alcohol-CPP) was used to measure alcohol reward. Alcohol-induced locomotor stimulation, alcohol consumption and kinetics were also assessed. mRNA levels were quantified using radioactive in situ hybridization.

Key Results

Low doses of H₃ receptor antagonists, JNJ-10181457 and JNJ-39220675, inhibited alcohol reward in wild-type (WT) mice. However, these H₃ receptor antagonists did not inhibit alcohol reward in histidine decarboxylase knock-out (HDC KO) mice and a lack of histamine did not alter alcohol consumption. Thus H₃ receptor antagonists inhibited alcohol reward in a histamine-dependent manner. Furthermore, WT and HDC KO mice were similarly stimulated by alcohol. The expression levels of dopamine D₁ and D₂ receptors, STEP61 and DARPP-32 mRNA in striatal subregions were unaltered in HDC KO mice. No differences were seen in alcohol kinetics in HDC KO compared to WT control animals. In addition, JNJ-39220675 had no effect on alcohol kinetics in WT mice.

Conclusions and Implications

These data suggest that histamine is required for the H₃ receptor-mediated inhibition of alcohol-CPP and support the hypothesis that the brain histaminergic system has an inhibitory role in alcohol reward. Increasing neuronal histamine release via H₃ receptor blockade could therefore be a novel way of treating alcohol dependence.

Linked Articles

This article is part of a themed issue on Histamine Pharmacology Update. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2013.170.issue-1     

Hydrogen sulfide-induced mechanical hyperalgesia and allodynia require activation of both Cav3.2 and TRPA1 channels in mice

BACKGROUND AND PURPOSE

Hydrogen sulfide, a gasotransmitter, facilitates somatic pain signals via activation of Ca_v3.2 T-type calcium channels in rats. Given evidence for the activation of transient receptor potential ankyrin-1 (TRPA1) channels by H₂S, we asked whether TRPA1 channels, in addition to Ca_v3.2 channels, contribute to the H₂S-induced mechanical hyperalgesia and allodynia in mice.

EXPERIMENTAL APPROACH

Mechanical hyperalgesia and allodynia were evaluated by the von Frey test in mice. Ca_v3.2 or TRPA1 channels in the sensory neurons were silenced by repeated intrathecal administration of antisense oligodeoxynucleotides in mice.

KEY RESULTS

Intraplantar administration of NaHS evoked hyperalgesia and allodynia in mice, an effect attenuated or abolished by NNC 55–0396 or mibefradil, T-type calcium channel blockers, and by ascorbic acid or zinc chloride, known to selectively inhibit Ca_v3.2 channels, out of the three isoforms of T-type calcium channels. Silencing of Ca_v3.2 channels in the sensory neurons also prevented the NaHS-induced hyperalgesia and allodynia in mice. The NaHS-induced hyperalgesia and allodynia in mice were significantly suppressed by AP18, a TRPA1 channel blocker, and by silencing of TRPA1 channels in the sensory neurons.

CONCLUSIONS AND IMPLICATIONS

Mechanical hyperalgesia and allodynia induced by NaHS/H₂S required activation of both Ca_v3.2 and TRPA1 channels in mice.     

Decreased production of neuronal NOS-derived hydrogen peroxide contributes to endothelial dysfunction in atherosclerosis

BACKGROUND AND PURPOSE

Reduced NO availability has been described as a key mechanism responsible for endothelial dysfunction in atherosclerosis. We previously reported that neuronal NOS (nNOS)-derived H₂O₂ is an important endothelium-derived relaxant factor in the mouse aorta. The role of H₂O₂ and nNOS in endothelial dysfunction in atherosclerosis remains undetermined. We hypothesized that a decrease in nNOS-derived H₂O₂ contributes to the impaired vasodilatation in apolipoprotein E-deficient mice (ApoE^−/−).

EXPERIMENTAL APPROACH

Changes in isometric tension were recorded on a myograph; simultaneously, NO and H₂O₂ were measured using carbon microsensors. Antisense oligodeoxynucleotides were used to knockdown eNOS and nNOS in vivo. Western blot and confocal microscopy were used to analyse the expression and localization of NOS isoforms.

KEY RESULTS

Aortas from ApoE^−/− mice showed impaired vasodilatation paralleled by decreased NO and H₂O₂ production. Inhibition of nNOS with L-Arg^NO2-L-Dbu, knockdown of nNOS and catalase, which decomposes H₂O₂ into oxygen and water, decreased ACh-induced relaxation by half, produced a small diminution of NO production and abolished H₂O₂ in wild-type animals, but had no effect in ApoE^−/− mice. Confocal microscopy showed increased nNOS immunostaining in endothelial cells of ApoE^−/− mice. However, ACh stimulation of vessels resulted in less phosphorylation on Ser852 in ApoE^−/− mice.

CONCLUSIONS AND IMPLICATIONS

Our data show that endothelial nNOS-derived H₂O₂ production is impaired and contributes to endothelial dysfunction in ApoE^−/− aorta. The present study provides a new mechanism for endothelial dysfunction in atherosclerosis and may represent a novel target to elaborate the therapeutic strategy for vascular atherosclerosis.     

Liposome-encapsulated dexamethasone attenuates ventilator-induced lung inflammation

BACKGROUND AND PURPOSE

Systemic glucocorticoid therapy may effectively attenuate lung inflammation but also induce severe side-effects. Delivery of glucocorticoids by liposomes could therefore be beneficial. We investigated if liposome-encapsulated dexamethasone inhibited ventilator-induced lung inflammation. Furthermore, we evaluated whether targeting of cellular Fcγ-receptors (FcγRs) by conjugating immunoglobulin G (IgG) to liposomes, would improve the efficacy of dexamethasone-liposomes in attenuating granulocyte infiltration, one of the hallmarks of lung inflammation.

EXPERIMENTAL APPROACH

Mice were anaesthetized, tracheotomized and mechanically ventilated for 5 h with either ‘low’ tidal volumes ∼7.5 mL·kg⁻¹ (LV_T) or ‘high’ tidal volumes ∼15 mL·kg⁻¹ (HV_T). At initiation of ventilation, we intravenously administered dexamethasone encapsulated in liposomes (Dex-liposomes), dexamethasone encapsulated in IgG-modified liposomes (IgG-Dex-liposomes) or free dexamethasone. Non-ventilated mice served as controls.

KEY RESULTS

Dex-liposomes attenuated granulocyte infiltration and IL-6 mRNA expression after LV_T-ventilation, but not after HV_T-ventilation. Dex-liposomes also down-regulated mRNA expression of IL-1β and KC, but not of CCL2 (MCP-1) in lungs of LV_T and HV_T-ventilated mice. Importantly, IgG-Dex-liposomes inhibited granulocyte influx caused by either LV_T or HV_T-ventilation. IgG-Dex-liposomes diminished IL-1β and KC mRNA expression in both ventilation groups, and IL-6 and CCL2 mRNA expression in the LV_T-ventilated group. Free dexamethasone prevented granulocyte influx and inflammatory mediator expression induced by LV_T or HV_T-ventilation.

CONCLUSIONS AND IMPLICATIONS

FcγR-targeted IgG-Dex-liposomes are pharmacologically more effective than Dex-liposomes particularly in inhibiting pulmonary granulocyte infiltration. IgG-Dex-liposomes inhibited most parameters of ventilator-induced lung inflammation as effectively as free dexamethasone, with the advantage that liposome-encapsulated dexamethasone will be released locally in the lung thereby preventing systemic side-effects.     

Pharmacological characterization of GSK1004723, a novel, long-acting antagonist at histamine H(1) and H(3) receptors

BACKGROUND AND PURPOSE

Preclinical pharmacological characterization of GSK1004723, a novel, dual histamine H₁ and H₃ receptor antagonist.

EXPERIMENTAL APPROACH

GSK1004723 was characterized in vitro and in vivo using methods that included radioligand binding, intracellular calcium mobilization, cAMP production, GTPγS binding, superfused human bronchus and guinea pig whole body plethysmography.

KEY RESULTS

In cell membranes over-expressing human recombinant H₁ and H₃ receptors, GSK1004723 displayed high affinity, competitive binding (H₁ pKi = 10.2; H₃ pKi = 10.6). In addition, GSK1004723 demonstrated slow dissociation from both receptors with a t_1/2 of 1.2 and 1.5 h for H₁ and H₃ respectively. GSK1004723 specifically antagonized H₁ receptor mediated increases in intracellular calcium and H₃ receptor mediated increases in GTPγS binding. The antagonism exerted was retained after cell washing, consistent with slow dissociation from H₁ and H₃ receptors. Duration of action was further evaluated using superfused human bronchus preparations. GSK1004723 (100 nmol·L⁻¹) reversed an established contractile response to histamine. When GSK1004723 was removed from the perfusate, only 20% recovery of the histamine response was observed over 10 h. Moreover, 21 h post-exposure to GSK1004723 there remained almost complete antagonism of responses to histamine. In vivo pharmacology was studied in conscious guinea pigs in which nasal congestion induced by intranasal histamine was measured indirectly (plethysmography). GSK1004723 (0.1 and 1 mg·mL⁻¹ intranasal) antagonized the histamine-induced response with a duration of up to 72 h.

CONCLUSIONS AND IMPLICATIONS

GSK1004723 is a potent and selective histamine H₁ and H₃ receptor antagonist with a long duration of action and represents a potential novel therapy for allergic rhinitis.     

α-Galactosylceramide suppresses murine eosinophil production through interferon-γ-dependent induction of NO synthase and CD95

Background and Purpose

α-Galactosylceramide (α-GalCer), a pleiotropic immunomodulator with therapeutic potential in neoplastic, autoimmune and allergic diseases, activates invariant natural killer T-cells throughCD1-restricted receptors for α-GalCer on antigen-presenting cells, inducing cytokine secretion. However the haemopoietic effects of α-GalCer remain little explored.

Experimental Approach

α-GalCer-induced modulation of eosinophil production in IL-5-stimulated bone marrow cultures was examined in wild-type (BALB/c, C57BL/6) mice and their mutants lacking CD1, inducible NOS (iNOS), CD95 and IFN-γ, along with the effects of lymphocytes; IFN-γ; caspase and iNOS inhibitors; non-steroidal anti-inflammatory drugs (NSAIDs) and LTD₄; and dexamethasone.

Key Results

α-GalCer (10⁻⁶–10⁻⁸M) suppressed IL-5-stimulated eosinopoiesis by inducing apoptosis. α-GalCer pretreatment in vivo (100 μg·kg⁻¹, i.v.) suppressed colony formation by GM-CSF-stimulated bone marrow progenitors in semi-solid cultures. α-GalCer and dexamethasone synergistically promoted eosinophil maturation. Suppression of eosinophil production by α-GalCer was prevented by aminoguanidine and was undetectable in bone marrow lacking iNOS, CD95, CD28; or CD1d. Separation on Percoll gradients and depletion of CD3+ cells made bone marrow precursors unresponsive to α-GalCer. Responsiveness was restored with splenic lymphocytes. Experiments with (i) IFN-γ-deficient bone marrow, alone or co-cultured with spleen T-cells from wild-type, but not from CD1d-deficient, donors; (ii) IFN-γ neutralization; and (iii) recombinant IFN-γ, showed that these effects of α-GalCer were mediated by IFN-γ. Effects of α-GalCer on eosinophil production were blocked by LTD₄ and NSAIDs.

Conclusions and Implications

α-GalCer activation of IFN-γ-secreting, CD1d-restricted lymphocytes induced iNOS-CD95-dependent apoptosis in developing eosinophils. This pathway is initiated by endogenous regulatory lymphocytes, antagonised by LTD₄, NSAIDs and aminoguanidine, and modified by dexamethasone.     

Functional pharmacology of H1 histamine receptors expressed in mouse preoptic/anterior hypothalamic neurons

BACKGROUND AND PURPOSE

Histamine H₁ receptors are highly expressed in hypothalamic neurons and mediate histaminergic modulation of several brain-controlled physiological functions, such as sleep, feeding and thermoregulation. In spite of the fact that the mouse is used as an experimental model for studying histaminergic signalling, the pharmacological characteristics of mouse H₁ receptors have not been studied. In particular, selective and potent H₁ receptor agonists have not been identified.

EXPERIMENTAL APPROACH

Ca²⁺ imaging using fura-2 fluorescence signals and whole-cell patch-clamp recordings were carried out in mouse preoptic/anterior hypothalamic neurons in culture.

KEY RESULTS

The H₁ receptor antagonists mepyramine and trans-triprolidine potently antagonized the activation by histamine of these receptors with IC₅₀ values of 0.02 and 0.2 μM respectively. All H₁ receptor agonists studied had relatively low potency at the H₁ receptors expressed by these neurons. Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine had full-agonist activity with potencies similar to that of histamine. In contrast, 2-pyridylethylamine and betahistine showed only partial agonist activity and lower potency than histamine. The histamine receptor agonist, 6-[2-(4-imidazolyl)ethylamino]-N-(4-trifluoromethylphenyl)heptanecarboxamide (HTMT) had no agonist activity at the H₁ receptors H1 receptors expressed by mouse preoptic/anterior hypothalamic neurons but displayed antagonist activity.

CONCLUSIONS AND IMPLICATIONS

Methylhistaprodifen and 2-(3-trifluoromethylphenyl)histamine were identified as full agonists of mouse H₁ receptors. These results also indicated that histamine H₁ receptors in mice exhibited a pharmacological profile in terms of agonism, significantly different from those of H₁ receptors expressed in other species.     

Structure-activity relationships of pentamidine-affected ion channel trafficking and dofetilide mediated rescue

Background and Purpose

Drug interference with normal hERG protein trafficking substantially reduces the channel density in the plasma membrane and thereby poses an arrhythmic threat. The chemical substructures important for hERG trafficking inhibition were investigated using pentamidine as a model drug. Furthermore, the relationship between acute ion channel block and correction of trafficking by dofetilide was studied.

Experimental Approach

hERG and K_IR2.1 trafficking in HEK293 cells was evaluated by Western blot and immunofluorescence microscopy after treatment with pentamidine and six pentamidine analogues, and correction with dofetilide and four dofetilide analogues that displayed different abilities to inhibit I_Kr. Molecular dynamics simulations were used to address mode, number and type of interactions between hERG and dofetilide analogues.

Key Results

Structural modifications of pentamidine differentially affected plasma membrane levels of hERG and K_IR2.1. Modification of the phenyl ring or substituents directly attached to it had the largest effect, affirming the importance of these chemical residues in ion channel binding. PA-4 had the mildest effects on both ion channels. Dofetilide corrected pentamidine-induced hERG, but not K_IR2.1 trafficking defects. Dofetilide analogues that displayed high channel affinity, mediated by pi-pi stacks and hydrophobic interactions, also restored hERG protein levels, whereas analogues with low affinity were ineffective.

Conclusions and Implications

Drug-induced trafficking defects can be minimized if certain chemical features are avoided or ‘synthesized out’; this could influence the design and development of future drugs. Further analysis of such features in hERG trafficking correctors may facilitate the design of a non-blocking corrector for trafficking defective hERG proteins in both congenital and acquired LQTS.     

Prostanoid receptor antagonists: development strategies and therapeutic applications

Background and purpose:

Histamine H₃ receptor antagonists are currently being evaluated in clinical trials for a number of central nervous system disorders including narcolepsy. These agents can increase wakefulness (W) in cats and rodents following acute administration, but their effects after repeat dosing have not been reported previously.

Experimental approach:

EEG and EMG recordings were used to investigate the effects of acute and repeat administration of the novel H₃ antagonist GSK189254 on the sleep–wake cycle in wild-type (Ox+/+) and orexin knockout (Ox−/−) mice, the latter being genetically susceptible to narcoleptic episodes. In addition, we investigated H₃ and H₁ receptor expression in this model using radioligand binding and autoradiography.

Key results:

In Ox+/+ and Ox−/− mice, acute administration of GSK189254 (3 and 10 mg·kg⁻¹ p.o.) increased W and decreased slow wave and paradoxical sleep to a similar degree to modafinil (64 mg·kg⁻¹), while it reduced narcoleptic episodes in Ox−/− mice. After twice daily dosing for 8 days, the effect of GSK189254 (10 mg·kg⁻¹) on W in both Ox+/+ and Ox−/− mice was significantly reduced, while the effect on narcoleptic episodes in Ox−/− mice was significantly increased. Binding studies revealed no significant differences in H₃ or H₁ receptor expression between Ox+/+ and Ox−/− mice.

Conclusions and implications:

These studies provide further evidence to support the potential use of H₃ antagonists in the treatment of narcolepsy and excessive daytime sleepiness. Moreover, the differential effects observed on W and narcoleptic episodes following repeat dosing could have important implications in clinical studies.     

AVE 0991, a non-peptide mimic of angiotensin-(1–7) effects,attenuates pulmonary remodelling in a model of chronic asthma

BACKGROUND AND PURPOSE

AVE 0991 (AVE) is a non-peptide compound, mimic of the angiotensin (Ang)-(1–7) actions in many tissues and pathophysiological states. Here, we have investigated the effect of AVE on pulmonary remodelling in a murine model of ovalbumin (OVA)-induced chronic allergic lung inflammation.

EXPERIMENTAL APPROACH

We used BALB/c mice (6–8 weeks old) and induced chronic allergic lung inflammation by OVA sensitization (20 μg·mouse⁻¹, i.p., four times, 14 days apart) and OVA challenge (1%, nebulised during 30 min, three times per·week, for 4 weeks). Control and AVE groups were given saline i.p and challenged with saline. AVE treatment (1 mg·kg⁻¹·per day, s.c.) or saline (100 μL·kg⁻¹·per day, s.c.) was given during the challenge period. Mice were anaesthetized 72 h after the last challenge and blood and lungs collected. In some animals, primary bronchi were isolated to test contractile responses. Cytokines were evaluated in bronchoalveolar lavage (BAL) and lung homogenates.

KEY RESULTS

Treatment with AVE of OVA sensitised and challenged mice attenuated the altered contractile response to carbachol in bronchial rings and reversed the increased airway wall and pulmonary vasculature thickness and right ventricular hypertrophy. Furthermore, AVE reduced IL-5 and increased IL-10 levels in the BAL, accompanied by decreased Ang II levels in lungs.

CONCLUSIONS AND IMPLICATIONS

AVE treatment prevented pulmonary remodelling, inflammation and right ventricular hypertrophy in OVA mice, suggesting that Ang-(1–7) receptor agonists are a new possibility for the treatment of pulmonary remodelling induced by chronic asthma.     

Potential therapeutic effect of Allium cepa L. and quercetin in a murine model of Blomia tropicalis induced asthma

Background

Asthma is an inflammatory condition characterized by airway hyperresponsiveness and chronic inflammation. The resolution of inflammation is an essential process to treat this condition. In this study we investigated the effect of Allium cepa L. extract (AcE) and quercetin (Qt) on cytokine and on smooth muscle contraction in vitro and its therapeutic potential in a murine model of asthma.

Methods

AcE was obtained by maceration of Allium cepa L. and it was standardized in terms of quercetin concentration using high performance liquid chromatography (HPLC). In vitro, using AcE 10, 100 or 1000 μg/ml or Qt 3.5, 7.5, 15 μg/ml, we measured the concentration of cytokines in spleen cell culture supernatants, and the ability to relax tracheal smooth muscle from A/J mice. In vivo, Blomia tropicalis (BT)-sensitized A/J mice were treated with AcE 100, 1000 mg/kg or 30 mg/kg Qt. We measured cell influx in bronchoalveolar lavage (BAL), eosinophil peroxidase (EPO) in lungs, serum levels of Bt-specific IgE, cytokines levels in BAL, and lung histology.

Results

We observed a reduction in the production of inflammatory cytokines, a relaxation of tracheal rings, and a reduction in total number of cells in BAL and EPO in lungs by treatment with AcE or Qt.

Conclusion

AcE and Qt have potential as antiasthmatic drugs, as they possess both immunomodulatory and bronchodilatory properties.     

Bitter tasting compounds dilate airways by inhibiting airway smooth muscle calcium oscillations and calcium sensitivity

Background and Purpose

While selective, bitter tasting, TAS2R agonists can relax agonist-contracted airway smooth muscle (ASM), their mechanism of action is unclear. However, ASM contraction is regulated by Ca²⁺ signalling and Ca²⁺ sensitivity. We have therefore investigated how the TAS2R10 agonists chloroquine, quinine and denotonium regulate contractile agonist-induced Ca²⁺ signalling and sensitivity.

Experimental Approach

Airways in mouse lung slices were contracted with either methacholine (MCh) or 5HT and bronchodilation assessed using phase-contrast microscopy. Ca²⁺ signalling was measured with 2-photon fluorescence microscopy of ASM cells loaded with Oregon Green, a Ca²⁺-sensitive indicator (with or without caged-IP₃). Effects on Ca²⁺ sensitivity were assessed on lung slices treated with caffeine and ryanodine to permeabilize ASM cells to Ca²⁺.

Key Results

The TAS2R10 agonists dilated airways constricted by either MCh or 5HT, accompanied by inhibition of agonist-induced Ca²⁺ oscillations. However, in non-contracted airways, TAS2R10 agonists, at concentrations that maximally dilated constricted airways, did not evoke Ca²⁺ signals in ASM cells. Ca²⁺ increases mediated by the photolysis of caged-IP₃ were also attenuated by chloroquine, quinine and denotonium. In Ca²⁺-permeabilized ASM cells, the TAS2R10 agonists dilated MCh- and 5HT-constricted airways.

Conclusions and Implications

TAS2R10 agonists reversed bronchoconstriction by inhibiting agonist-induced Ca²⁺ oscillations while simultaneously reducing the Ca²⁺ sensitivity of ASM cells. Reduction of Ca²⁺ oscillations may be due to inhibition of Ca²⁺ release through IP₃ receptors. Further characterization of bronchodilatory TAS2R agonists may lead to the development of novel therapies for the treatment of bronchoconstrictive conditions.     

Characterization of histamine H3 receptors in Alzheimer's Disease brain and amyloid over-expressing TASTPM mice

Background and purpose:

Histamine H₃ receptor antagonists are currently being evaluated for their potential use in a number of central nervous system disorders including Alzheimer''s Disease (AD). To date, little is known about the state of H₃ receptors in AD.

Experimental approach:

In the present study we used the radiolabelled H₃ receptor antagonist [³H]GSK189254 to investigate H₃ receptor binding in the amyloid over-expressing double mutant APPswe × PSI.MI46V (TASTPM) transgenic mouse model of AD and in post-mortem human AD brain samples.

Key results:

No significant differences in specific H₃ receptor binding were observed between wild type and TASTPM mice in the cortex, hippocampus or hypothalamus. Specific [³H]GSK189254 binding was detected in sections of human medial frontal cortex from AD brains of varying disease severity (Braak stages I–VI). With more quantitative analysis in a larger cohort, we observed that H₃ receptor densities were not significantly different between AD and age-matched control brains in both frontal and temporal cortical regions. However, within the AD group, [³H]GSK189254 binding density in frontal cortex was higher in individuals with more severe dementia prior to death.

Conclusions and implications:

The maintenance of H₃ receptor integrity observed in the various stages of AD in this study is important, given the potential use of H₃ antagonists as a novel therapeutic approach for the symptomatic treatment of AD.     

Altered histamine H3 receptor radioligand binding in post-mortem brain samples from subjects with psychiatric diseases

Background and purpose:

Histamine is a modulatory neurotransmitter in the brain. Auto- and hetero-histamine H₃ receptors are present in human brain and are potential targets of antipsychotics. These receptors may also display disease-related abnormalities in psychiatric disorders. Here we have assessed how histamine H₃ receptors in human brain may be affected in schizophrenia, bipolar disorder, major depression.

Experimental approach:

Histamine H₃ receptor radioligand binding assays were applied to frozen post-mortem prefrontal and temporal cortical sections and anterior hippocampal sections from subjects with schizophrenia, bipolar disorder, major depression and matched controls.

Key results:

Compared with the controls, increased H₃ receptor radioligand binding was found in dorsolateral prefrontal cortex of schizophrenic subjects (especially the ones who were treated with atypical antipsychotics), and bipolar subjects with psychotic symptoms. No differences in H₃ receptor radioligand binding were found in the temporal cortex. In hippocampal formation of control subjects, H₃ receptor radioligand binding was prominent in dentate gyrus, subiculum, entorhinal cortex and parasubiculum. Decreased H₃ binding was found in the CA4 area of bipolar subjects. Decreased H₃ binding in CA2 and presubiculum of medication-free bipolar subjects was also seen.

Conclusions and implications:

The results suggest that histamine H₃ receptors in the prefrontal cortex take part in the modulation of cognition, which is impaired in schizophrenic subjects and bipolar subjects with psychotic symptoms. Histamine H₃ receptors probably regulate connections between hippocampus and various cortical and subcortical regions and could also be involved in the neuropathology of schizophrenia and bipolar disorder.