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Introduction: Chronic cough is a key symptom that plagues the lives of IPF patients, indeed, it is often the reason the disease is initially diagnosed. The aim of this project was to develop a pre-clinical model of IPF-associated cough and compare the mediator profiles with clinical samples, to aid in the search for effective treatments. Methods: Male guinea pigs (around 325g) were intratracheally dosed with vehicle (saline) or bleomycin (6 USP/kg) and coughs were quantified through manual observation of in-cage video footage, by watching 4.5 hours a day, for 3 weeks. Lung tissue, bronchoalveolar lavage fluid (BALF) and blood was harvested from a parallel group of animals. These were used to assess the extent of fibrosis and measure mediator production (e.g. a marker of oxidative stress: 8-isoprostane). Human BALF was obtained from IPF patients and healthy controls. Results: Spontaneous coughing was recorded in guinea pigs given bleomycin, which was associated with an increase in lung fibrosis and BALF 8-isoprostane (a marker of oxidative stress). We noted similar increases in BALF 8-isoprostane levels in the clinical samples. Conclusions: We have developed a preclinical model to aid in the search for a treatment for IPF-associated coughing. Furthermore, initial data suggests that an increase in oxidative burden (a known trigger of coughing) could be an attractive target.

Rationale: Effective cough treatments are a significant unmet need in patients with lung cancer. Aprepitant is a licensed treatment for nausea and vomiting, which blocks substance P activation of NK-1 (neurokinin 1) receptors, a mechanism also implicated in cough. Objectives: To assess aprepitant in patients with lung cancer with cough and evaluate mechanisms in vagal nerve tissue. Methods: Randomized double-blind crossover trial of patients with lung cancer and bothersome cough. They received 3 days of aprepitant or matched placebo; after a 3-day washout, patients crossed to the alternative treatment. The primary endpoint was awake cough frequency measured at screening and Day 3 of each treatment; secondary endpoints included patient-reported outcomes. In vitro, the depolarization of isolated guinea pig and human vagus nerve sections in grease-gap recording chambers, indicative of sensory nerve activation, was measured to evaluate the mechanism. Measurements and Main Results: Twenty patients with lung cancer enrolled, with a mean age 66 years (67.7); 60% were female and 80% had non–small cell cancer, 50% had advanced stage, and 55% had World Health Organization performance status 1. Cough frequency improved with aprepitant, reducing by 22.2% (95% confidence interval [CI], 2.8–37.7%) over placebo while awake (P = 0.03), 30.3% (95% CI, 12.7–44.3) over 24 hours (P = 0.002), and 59.8% (95% CI, 15.1–86.0) during sleep (P = 0.081). Patient-reported outcomes all significantly improved. Substance P depolarized both guinea pig and human vagus nerve. Aprepitant significantly inhibited substance P–induced depolarization by 78% in guinea pig (P = 0.0145) and 94% in human vagus (P = 0.0145). Conclusions: Substance P activation of NK-1 receptors appears to be an important mechanism driving cough in lung cancer, and NK-1 antagonists show promise as antitussive therapies.

Chronic cough is a burden of many respiratory diseases with no effective & safe treatment. The prostaglandin PGE2 acts on EP receptors (EP1-4) with beneficial & adverse effects in the lungs. We have shown EP4 agonism inhibits activation of airway sensory nerves, blocking cough1. We aimed to determine the signalling downstream of EP4 in airway sensory nerves, to better understand its anti-tussive effect. We isolated guinea pig (GP) vagal ganglia neurons, using retrograde tracer dye to identify airway neurons in vitro. We assessed intracellular calcium & cAMP in these neurons using chemical & virally transfected fluorophores. We also used a grease-gap recording technique to pharmacologically examine EP4 signalling in GP & human vagus nerves in vitro. We have shown that EP4 agonist inhibits capsaicin-induced activation of GP airway neurons. EP4 is known to be a Gαs-coupled GPCR, so we imaged cAMP, observing that EP4 agonism causes cAMP elevation. Dual-fluorophore imaging showed cAMP elevation in neurons where EP4 agonism had inhibitory effects. From previous work we know EP4 agonist inhibits capsaicin-induced GP & human vagus nerve activation. We looked whether the main cAMP effectors (PKA & EPAC) were involved. EPAC (but not PKA) blockers (ESI-09 & KT5720) abolished the inhibitory effect of EP4 agonism. EPAC activator (8-CPT-2Me-cAMP) mimicked the effect of EP4 agonism; this was blocked by EPAC inhibitor. These data reveal a role for cAMP & EPAC in the inhibitory & anti-tussive effects of EP4 agonism on airway sensory nerves. Understanding how EP4 exerts its beneficial effects on airway sensory nerves is important to better inform drug discovery efforts for anti-tussive therapies. 1.Wortley(2019)AJRCCM;199:A2862

BackgroundNavafenterol (AZD8871) belongs to a new class of bronchodilator, the single-molecule muscarinic antagonist and β-agonist, developed for the treatment of COPD. This study aimed to evaluate the efficacy, pharmacokinetics and safety of navafenterol versus placebo and an active comparator treatment for moderate-to-severe COPD.MethodsThis phase 2a, randomised, multicentre (Germany and UK), double-blind, double-dummy, three-way complete crossover study (ClinicalTrials.gov identifier: NCT03645434) compared 2 weeks’ treatment of once-daily navafenterol 600 µg via inhalation with placebo and a fixed-dose combination bronchodilator (umeclidinium/vilanterol (UMEC/VI); 62.5 µg/25 µg) in participants with moderate-to-severe COPD. The primary outcome was change from baseline in trough forced expiratory volume in 1 s (FEV1) on day 15. Secondary end-points included change from baseline in peak FEV1; change from baseline in Breathlessness, Cough and Sputum Scale (BCSS); change from baseline in COPD Assessment Tool (CAT); adverse events; and pharmacokinetics.Results73 participants were randomised. After 14 days, trough FEV1 was significantly improved with navafenterol compared with placebo (least-squares (LS) mean difference 0.202 L; p1 between navafenterol and UMEC/VI (LS mean difference −0.046 L; p=0.075). COPD symptoms (CAT and BCSS) showed significantly greater improvements with both active treatments versus placebo (all pConclusionOnce-daily navafenterol was well tolerated, improved lung function and reduced COPD-related symptoms, similar to an established once-daily fixed-dose combination bronchodilator.

Most airway diseases, including chronic obstructive pulmonary disease (COPD), are associated with excessive coughing. The extent to which this may be a consequence of increased activation of vagal afferents by pathology in the airways (e.g., inflammatory mediators, excessive mucus) or an altered neuronal phenotype is unknown. Understanding whether respiratory diseases are associated with dysfunction of airway sensory nerves has the potential to identify novel therapeutic targets.To assess the changes in cough responses to a range of inhaled irritants in COPD and model these in animals to investigate the underlying mechanisms.Cough responses to inhaled stimuli in patients with COPD, healthy smokers, refractory chronic cough, asthma, and healthy volunteers were assessed and compared with vagus/airway nerve and cough responses in a cigarette smoke (CS) exposure guinea pig model.Patients with COPD had heightened cough responses to capsaicin but reduced responses to prostaglandin E2 compared with healthy volunteers. Furthermore, the different patient groups all exhibited different patterns of modulation of cough responses. Consistent with these findings, capsaicin caused a greater number of coughs in CS-exposed guinea pigs than in control animals; similar increased responses were observed in ex vivo vagus nerve and neuron cell bodies in the vagal ganglia. However, responses to prostaglandin E2 were decreased by CS exposure.CS exposure is capable of inducing responses consistent with phenotypic switching in airway sensory nerves comparable with the cough responses observed in patients with COPD. Moreover, the differing profiles of cough responses support the concept of disease-specific neurophenotypes in airway disease. Clinical trial registered with www.clinicaltrials.gov (NCT 01297790).

Background and Purpose Airway microvascular leak (MVL) involves the extravasation of proteins from post-capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. Prostaglandin E2 (PGE2), a product of cyclooxygenase mediated metabolism of arachidonic acid, binds to 4 receptors, termed EP1-4. PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described. Experimental Approach Evans Blue dye was used as a marker of airway MVL and selective EP receptor agonists and antagonists were used alongside EP receptor deficient mice to define the receptor subtype involved. Key Results PGE2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2-induced MVL was demonstrated in Ptger2-/- and Ptger4-/- mice and in wild type mice pre-treated simultaneously with EP2 (PF-04418948) and the EP4 (ER-819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2-/- and Ptger4-/- mice. Conclusions and Implications PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2. This article is protected by copyright. All rights reserved.

Transient Receptor Potential (TRP) A1 channels are expressed on airway sensory nerves and are linked to the initiation of respiratory symptoms and disease pathogenesis. TRPA1 is believed to be an oxidative stress (OS) sensor and is triggered by a range of stimuli e.g. acrolein, PGE2 and bradykinin (BK), hydrogen peroxide (HP), diesel exhaust particles (DEP), hyper-osmolarity (HO) and low temperature (10°C). The aim was to investigate the mechanisms involved in TRPA1 activation on airway sensory nerves and study the role played by OS. We assessed activation of guinea-pig (gp), mice (wild-type versus TRPA1 KO) and human vagus nerve. We used a selective TRPA1 antagonist and three different antioxidants: N-acetyl-L-cysteine (NAC, an extracellular oxidative stress scavenger), Glutathione (GSH, an intracellular anti-oxidant) and VAS2780 (NADPH oxidase inhibitor). The results showed that the TRPA1 antagonist abolished responses to acrolein, HP, DEP, and 10°C and were lost in the tissue from TRPA1 KO mice, whereas responses to PGE2, BK and HO were partially suppressed (approx. 50%). NAC and GSH inhibited acrolein, HP and DEP activation. Whereas, PGE2, BK and HO induced activation appeared to be dependent on NADPH oxidase. Stimulation with 10°C was not sensitive to the anti-oxidants. We have shown a number of different modalities activate TRPA1 on sensory nerves and they, with the exception of 10°C, are mediated via an oxidative stress response. Interestingly, it appears that a number of stimuli are dependent on NADPH oxidase activity to activate TRPA1. This highlights the range of disease relevant mechanisms by which TRPA1 can be activated and implies that modulating the channel is a promising target for future drug discovery.

Heightened cough responses to inhaled capsaicin, a transient receptor potential vanilloid 1 (TRPV1) agonist, are characteristic of patients with chronic cough. However, previously, a TRPV1 antagonist (SB-705498) failed to improve spontaneous cough frequency in these patients, despite small reductions in capsaicin-evoked cough.XEN-D0501 (a potent TRPV1 antagonist) was compared with SB-705498 in preclinical studies to establish whether an improved efficacy profile would support a further clinical trial of XEN-D0501 in refractory chronic cough.XEN-D0501 and SB-705498 were profiled against capsaicin in a sensory nerve activation assay and in vivo potency established against capsaicin-induced cough in the guinea pig. Twenty patients with refractory chronic cough participated in a double-blind, randomized, placebo-controlled crossover study evaluating the effect of 14 days of XEN-D0501 (oral, 4 mg twice daily) versus placebo on awake cough frequency (primary outcome), capsaicin-evoked cough, and patient-reported outcomes.XEN-D0501 was more efficacious and 1,000-fold more potent than SB-705498 at inhibiting capsaicin-induced depolarization of guinea pig and human isolated vagus nerve. In vivo XEN-D0501 completely inhibited capsaicin-induced cough, whereas 100 times more SB-705498 was required to achieve the same effect. In patients, XEN-D0501 substantially reduced maximal cough responses to capsaicin (mean change from baseline, XEN-D0501, -19.3 ± 16.4) coughs; placebo, -1.8 ± 5.8 coughs; P 0.0001), but not spontaneous awake cough frequency (mean change from baseline, XEN-D0501, 6.7 ± 16.9 coughs/h; placebo, 0.4 ± 13.7 coughs/h; P = 0.41).XEN-D0501 demonstrated superior efficacy and potency in preclinical and clinical capsaicin challenge studies; despite this improved pharmacodynamic profile, spontaneous cough frequency did not improve, ruling out TRPV1 as an effective therapeutic target for refractory cough. Clinical trial registered with www.clinicaltrialsregister.eu (2014-000306-36).

Cough is the most common reason to visit a primary care physician, yet it remains an unmet medical need. Fatty acid amide hydrolase (FAAH) is an enzyme that breaks down endocannabinoids, and inhibition of FAAH produces analgesic and anti-inflammatory effects. Cannabinoids inhibit vagal sensory nerve activation and the cough reflex, so it was hypothesised that FAAH inhibition would produce antitussive activity via elevation of endocannabinoids. Primary vagal ganglia neurons, tissue bioassay, in vivo electrophysiology and a conscious guinea pig cough model were utilised to investigate a role for fatty acid amides in modulating sensory nerve activation in vagal afferents. FAAH inhibition produced antitussive activity in guinea pigs with concomitant plasma elevation of the fatty acid amides N-arachidonoylethanolamide (anandamide), palmitoylethanolamide, N-oleoylethanolamide and linoleoylethanolamide. Palmitoylethanolamide inhibited tussive stimulus-induced activation of guinea pig airway innervating vagal ganglia neurons, depolarisation of guinea pig and human vagus, and firing of C-fibre afferents. These effects were mediated via a cannabinoid CB2/Gi/o-coupled pathway and activation of protein phosphatase 2A, resulting in increased calcium sensitivity of calcium-activated potassium channels. These findings identify FAAH inhibition as a target for the development of novel, antitussive agents without the undesirable side-effects of direct cannabinoid receptor agonists.

Transient Receptor Potential Vanilloid 4 (TRPV4) is a broadly expressed, polymodally gated ion channel that plays an important role in many physiological and pathophysiological processes. TRPV4 knockout mice and several synthetic pharmacological compounds that selectively target TRPV4 are now available, which has allowed detailed investigation in to the therapeutic potential of this ion channel. Results from animal studies suggest that TRPV4 antagonism has therapeutic potential in oedema, pain, gastrointestinal disorders, and lung diseases such as cough, bronchoconstriction, pulmonary hypertension, and acute lung injury. A lack of observed side-effects in vivo has prompted a first-in-human trial for a TRPV4 antagonist in healthy participants and stable heart failure patients. If successful, this would open up an exciting new area of research for a multitude of TRPV4-related pathologies. This review will discuss the known roles of TRPV4 in disease, and highlight the possible implications of targeting this important cation channel for therapy.

Background and Purpose Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists are prescribed as bronchodilators for asthma and chronic obstructive pulmonary disease, and it is commonly assumed that they elicit their actions via AC-dependent production of cAMP. However, empirical evidence in support of this is lacking and the exact mechanism by which these drugs acts remains elusive. This is partly due to the existence of at least 10 different isoforms of AC and the absence of any truly selective pharmacological inhibitors. Here, we have used genetically modified mice and model systems to establish the role of AC isoforms in the airway responses to β-adrenoceptor agonists. Experimental Approach Receptors mediating responses to β-adrenoceptor agonists in airway smooth muscle (ASM) and sensory nerve were identified in isolated tissue systems. Expression of mRNA for the AC isoforms in ASM and neurones was determined by qPCR. Functional responses were assessed in AC isoform KO mice and wild-type controls. Key Results Airway and vagal tissue expressed mRNA for various isoforms of AC. AC6 was the most prominent isoform. Responses to β-adrenoceptor agonists in tissues from AC6 KO mice were virtually abolished. Conclusions and Implications AC6 played a critical role in relaxation of ASM to β1-adrenoceptor agonists and in modulation of sensory nerves by β1-3-adrenoceptor agonists. These results further unravel the signalling pathway of this extensively prescribed class of medicine.

Prostaglandin D2 (PGD2) causes cough and levels are increased in asthma suggesting that it may contribute to symptoms. Although the prostaglandin D2 receptor 2 (DP2) is a target for numerous drug discovery programmes little is known about the actions of PGD2 on sensory nerves and cough.We used human and guinea pig bioassays, in vivo electrophysiology and a guinea pig conscious cough model to assess the effect of prostaglandin D2 receptor (DP1), DP2 and thromboxane receptor antagonism on PGD2 responses.PGD2 caused cough in a conscious guinea pig model and an increase in calcium in airway jugular ganglia. Using pharmacology and receptor-deficient mice we showed that the DP1 receptor mediates sensory nerve activation in mouse, guinea pig and human vagal afferents. In vivo, PGD2 and a DP1 receptor agonist, but not a DP2 receptor agonist, activated single airway C-fibres. Interestingly, activation of DP2 inhibited sensory nerve firing to capsaicin in vitro and in vivo.The DP1 receptor could be a therapeutic target for symptoms associated with asthma. Where endogenous PGD2 levels are elevated, loss of DP2 receptor-mediated inhibition of sensory nerves may lead to an increase in vagally associated symptoms and the potential for such adverse effects should be investigated in clinical studies with DP2 antagonists.

Background: Effective cough treatments are a significant unmet need. Aprepitant blocks substance P at NK-1 receptors and is licensed for chemotherapy-induced nausea and vomiting. We assessed Aprepitant in vagal tissue and a proof of concept trial in lung cancer patients with cough. Methods: In vitro, isolated vagus nerve sections were placed in grease gap recording chambers and depolarization to test solutions measured. A randomised double-blind crossover trial enrolled lung cancer patients with cough who received aprepitant (125mg day 1, 80mg day 2&3) or matched placebo; following a 3 day wash out, patients crossed over. The primary endpoint was awake cough frequency (coughs/hr(c/h)) and patients reported cough severity and impact. Results: Substance P depolarised guinea pig (0.083mV±0.0066, n=5) and human vagus (0.0875mV, n=2). Aprepitant (10µM) reduced substance P depolarisation by 78% in guinea pig (0.0180mV±0.0080) and 94% in human vagus (0.0050 mV). Twenty lung cancer patients enrolled: mean age 66yrs(±7.69); 60% female; 70% ex, 25% current and 5% non-smokers; performance status 0=25%, 1=55% and 2=25%. 80% had non-small cell cancer, 50% advanced stage and 20% were on cancer therapy. Awake cough frequency improved with aprepitant [baseline 15.9c/h(95%CI 10.1-28.3), aprepitant 12.8(8.7-18.8), placebo 16.2(11.3-23.0), p=0.03]. Patients reported improved cough severity [VAS baseline 57.0mm (95% CI 47.4-67.2), aprepitant 40.8(34.3-47.3), placebo 49.8(44.2-55.4), p=0.008] and impact [Manchester Cough in Lung Cancer Scale, p Conclusions: This is the first study to show NK-1 antagonism has anti-tussive effects in lung cancer, with only 3 days treatment. Vagal inhibition may mediate this effect.

Adenylyl cyclase (AC) is a key signalling enzyme for many GPCRs and catalyses the conversion of ATP to cAMP which, in turn, is a crucial determinant of many biological responses. β-Adrenoceptor agonists (β 2 ) are commonly prescribed as bronchodilators for respiratory diseases such as asthma and COPD and yet the intracellular signalling mechanisms are still not fully understood. Recently we have shown, using tracheal tissue from WT and GM mice, that, of the 10 different isoforms of AC, AC6 plays a central role in β-agonist, but not PGE 2 , responses (Birrell et al . BJP 2015). The aim of this study was to determine if this mechanism is apparent in translational studies utilising human airway smooth muscle cells (HASMC). We showed that primary HASMCs express AC6 at the mRNA and protein level. We developed a shRNA sequence and vector that targeted AC6 (60+% knockdown of expression). We showed that cAMP production, as measured by FRET, in response to a β 2 agonist was significantly reduced in HASMCs with reduced AC6 expression, whereas responses to forskolin or EP 4 receptor agonist (receptor responsible for PGE 2 -induced relaxation) were not altered. To confirm AC6 functional role of we employed a technique which can detect the contractile status of cultured cells, Ptychography (VL21 Phasefocus UK). Using this system we showed that ACh induced “contraction”, which is reduced by the β 2 agonist in control cells, was not in the cells treated with AC6-shRNA. Responses to a PKA agonist (downstream from AC) however were comparable. We conclude that AC6 plays a critical role in relaxation of HASMCs to β 2 agonists; these results further unravel the signalling pathway of this extensively prescribed class of medicine.

Glucocorticoids are steroid hormones that have pleiotropic effects on development, metabolism, cognitive function and other aspects of physiology. Since the demonstration more than sixty years ago of their capacity to suppress inflammation, synthetic glucocorticoids have been extremely widely used in the treatment of inflammatory diseases. However, their clinical use is limited by numerous, unpredictable and potentially serious side effects. Glucocorticoids regulate gene expression both positively and negatively. Both of these effects are mediated by the glucocorticoid receptor, a ligand-dependent transcription factor. It has become widely accepted that anti-inflammatory effects of glucocorticoids are mostly due to inhibition of transcription, whereas the activation of transcription by the glucocorticoid receptor accounts for the majority of side effects. This dogma (which we refer to as the "transrepression hypothesis") predicts the possibility of uncoupling therapeutic, anti-inflammatory effects from side effects by identifying novel, selective ligands of the glucocorticoid receptor, which preferentially mediate inhibition rather than activation of transcription. It is argued that such "dissociated" glucocorticoid receptor ligands should retain anti-inflammatory potency but cause fewer side effects. Here we critically re-examine the history and foundations of the transrepression hypothesis. We argue that it is incompatible with the complexity of gene regulation by glucocorticoids and poorly supported by experimental evidence; that it no longer aids clear thinking about the actions of the glucocorticoid receptor; and that it will not prove a fruitful basis for continued refinement and improvement of anti-inflammatory drugs that target the glucocorticoid receptor.

Cough is a common symptom of diseases such as asthma and COPD and also presents as a disease in its own right. Treatment options are limited; a recent meta-analysis concluded that over-the-counter remedies are ineffective, and there is increasing concern about their use in children. Transient receptor potential cation channel, subfamily A, member 1 (TRPA1) channels are nonselective cation channels that are activated by a range of natural products (eg, allyl isothiocyanate), a multitude of environmental irritants (eg, acrolein, which is present in air pollution, vehicle exhaust, and cigarette smoke), and inflammatory mediators (eg, cyclopentenone prostaglandins). TRPA1 is primarily expressed in small-diameter, nociceptive neurons where its activation probably contributes to the perception of noxious stimuli. Inhalational exposure to irritating gases, fumes, dusts, vapors, chemicals, and endogenous mediators can lead to the development of cough. The respiratory tract is innervated by primary sensory afferent nerves, which are activated by mechanical and chemical stimuli. Recent data suggest that activation of TRPA1 on these vagal sensory afferents by these irritant substances could lead to central reflexes, including dyspnea, changes in breathing pattern, and cough, which contribute to the symptoms and pathophysiology of respiratory diseases.

Cough is a protective mechanism but can occur excessively in disease. Cough can be modulated by a range of GPCRs which can be either inhibitory or excitatory. Prostaglandin E2 and bradykinin can activate airway sensory nerves via EP3 and B2 receptors receptively and have both been shown to mediate their effects though TRPV1 and TRPA1 receptors. Activation of the β2-adrenoceptor and cannabinoid CB2 receptors can inhibit sensory nerves and prevent cough. It is currently thought that activation of the β2-adrenoceptor causes c-AMP dependent activation of PKA; however, recent research has suggested that the pathway involves PKG-mediated opening of the BKCa channel leading to hyperpolarization.

The nociceptor TRPA1 is thought to be activated through covalent modification of specific cysteine residues on the N terminal of the channel. The precise mechanism of covalent modification with unsaturated carbonyl-containing compounds is unclear, therefore by examining a range of compounds which can undergo both conjugate and/or direct addition reactions we sought to further elucidate the mechanism(s) whereby TRPA1 can be activated by covalent modification. Calcium signalling was used to determine the mechanism of activation of TRPA1 expressed in HEK293 cells with a series of related compounds which were capable of either direct and/or conjugate addition processes. These results were confirmed using physiological recordings with isolated vagus nerve preparations. We found negligible channel activation with chemicals which could only react with cysteine residues via conjugate addition such as acrylamide, acrylic acid, and cinnamic acid. Compounds able to react via either conjugate or direct addition, such as acrolein, methyl vinyl ketone, mesityl oxide, acrylic acid NHS ester, cinnamaldehyde and cinnamic acid NHS ester, activated TRPA1 in a concentration dependent manner as did compounds only capable of direct addition, namely propionic acid NHS ester and hydrocinnamic acid NHS ester. These compounds failed to activate TRPV1 expressed in HEK293 cells or mock transfected HEK293 cells. For molecules capable of direct or conjugate additions, the results suggest for the first time that TRPA1 may be activated preferentially by direct addition of the thiol group of TRPA1 cysteines to the agonist carbonyl carbon of α,β-unsaturated carbonyl-containing compounds.

Cough is an essential reflex, but chronic cough can be a problematic symptom of respiratory diseases, for which no effective and safe medications are available. We have shown that inhibition of fatty acid amide hydrolase (FAAH) elevates endocannabinoids (inc. palmitoylethanolamide, anandamide, oleoylethanolamide & linoleoylethanolamide: PEA, AEA, OEA, LEA) and inhibits citric-acid evoked cough in guinea pigs, and PEA inhibits sensory nerve depolarisation1. We investigated the pathway of endocannabinoid inhibition of sensory nerve/neuron activity. PEA, AEA, OEA & LEA inhibited capsaicin-induced depolarisation of isolated guinea pig vagus nerve via the CB2 (but not CB1) receptor, and capsaicin-induced calcium influx in airway-terminating jugular neurons – i.e. cough reflex relevant vagal neurons. Using vagus nerve from PTX dosed guinea pigs we showed PEA inhibition required the Gαi protein. PEA inhibition also required small conductance K+ channel (SKCa) activation, and concurrent with SKCa mediated suppression of nerve activation, PEA inhibited depolarisation by TRPA1 & TRPV4 agonists. Finally, PEA increases the calcium sensitivity of SKCa via protein phosphatase 2A activation. Key experiments paralleled in human vagus nerve indicate this pathway translates to man. In summary, endocannabinoids inhibit sensory nerve activation in guinea pig and human vagus nerve via CB2–Gαi–PP2A–SKCa. This data implies that inhibiting FAAH/elevating endocannabinoids may be beneficial in conditions driven by excessive peripheral sensory nerve activation including chronic cough. 1. Adcock et al. (2011) Proc Br Pharmacol Soc.9: A025p.

Asthma is a debilitating disease of the airways characterised by symptoms such as bronchoconstriction, hyperresponsiveness and cough. Current therapies are ineffective in severe disease highlighting the need for novel treatments. Menthol, a cooling compound found in medicinal products and several OTC medications, is commonly thought to activate the Transient Receptor Potential Melastatin 8 (TRPM8) channel and has been reported to possess beneficial effects such as bronchodilation and suppression of cough, however the mechanism of action is unknown. The aim was to study the beneficial properties of menthol in the airways and investigate the role of TRPM8. TRPM8 was expressed in mouse and guinea pig (GP) vagal ganglia. Using a TRPM8 antagonist (JNJ41876666) and Trpm8 -/- mice in an isolated vagal nerve preparation we showed that both WS3 (TRPM8 agonist) and (-)-menthol caused a small activation of human, GP and mouse vagal nerves which was TRPM8 dependent. Interestingly, pre-incubation of (-)-menthol inhibited vagal nerve activation induced by the tussive stimulus, capsaicin, an effect that was not inhibited by JNJ41876666. WS3 and (-)-menthol caused relaxation of human, mouse and GP trachea, which was not abolished by JNJ41876666 nor in the Trpm8 -/- mouse airway. No expression of TRPM8 was detected in human, GP or mouse airway smooth muscle. (-)-menthol caused a small TRPM8-dependent activation but a robust TRPM8-independent inhibition of vagal sensory nerve activity and relaxation of airway smooth muscle. Elucidating the mechanism behind the beneficial effects of (-)-menthol could lead to the development of promising new therapeutic targets for airway diseases such as asthma.

Hyperpolarized 83 Kr surface quadrupolar relaxation (SQUARE) generates MRI contrast that was previously shown to correlate with surface-to-volume ratios in porous model surface systems. The underlying physics of SQUARE contrast is conceptually different from any other current MRI methodology as the method uses the nuclear electric properties of the spin I = 9/2 isotope 83 Kr. To explore the usage of this non-radioactive isotope for pulmonary pathophysiology, MRI SQUARE contrast was acquired in excised rat lungs obtained from an elastase-induced model of emphysema. A significant 83 Kr T 1 relaxation time increase in the SQUARE contrast was found in the elastase-treated lungs compared with the baseline data from control lungs. The SQUARE contrast suggests a reduction in pulmonary surface-to-volume ratio in the emphysema model that was validated by histology. The finding supports usage of 83 Kr SQUARE as a new biomarker for surface-to-volume ratio changes in emphysema.

BackgroundAsthma and chronic obstructive pulmonary disease (COPD) are chronic inflammatory diseases of the airway. Current treatment options (long acting β-adrenoceptor agonists and glucocorticosteroids) are not optimal as they are only effective in certain patient groups and safety concerns exist regarding both compound classes. Therefore, novel bronchodilator and anti-inflammatory strategies are being pursued. Prostaglandin E2 (PGE2) is an arachidonic acid-derived eicosanoid produced by the lung which acts on four different G-protein coupled receptors (EP1–4) to cause an array of beneficial and deleterious effects. The aim of this study was to identify the EP receptor mediating the anti-inflammatory actions of PGE2 in the lung using a range of cell-based assays and in vivo models.Methods and resultsIt was demonstrated in three distinct model systems (innate stimulus, lipopolysaccharide (LPS); allergic response, ovalbumin (OVA); inhaled pollutant, cigarette smoke) that mice missing functional EP4 (Ptger4−/−) receptors had higher levels of airway inflammation, suggesting that endogenous PGE2 was suppressing inflammation via EP4 receptor activation. Cell-based assay systems (murine and human monocytes/alveolar macrophages) demonstrated that PGE2 inhibited cytokine release from LPS-stimulated cells and that this was mimicked by an EP4 (but not EP1–3) receptor agonist and inhibited by an EP4 receptor antagonist. The anti-inflammatory effect occurred at the transcriptional level and was via the adenylyl cyclase/cAMP/ cAMP-dependent protein kinase (PKA) axis.ConclusionThis study demonstrates that EP4 receptor activation is responsible for the anti-inflammatory activity of PGE2 in a range of disease relevant models and, as such, could represent a novel therapeutic target for chronic airway inflammatory conditions.

Cough is often associated with inflammatory airway disease and is an unmet clinical need. To provide successful future therapies, good disease models are required. Citric acid has been used to provoke cough in humans and to induce cough in guinea pigs and the responses in guinea pig are similar to those in human. Citric acid-induced cough provides a reliable disease model with which to study the mechanisms involved in cough and to evaluate novel therapies.

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor belonging to the nuclear hormone receptor superfamily. PPARgamma regulates several metabolic pathways by binding to sequence-specific PPAR response elements in the promoter region of target genes, including lipid biosynthesis and glucose metabolism. Synthetic PPARgamma agonists have been developed, such as the thiazolidinediones rosiglitazone and pioglitazone. These act as insulin sensitizers and are used in the treatment of type 2 diabetes. Recently however, PPARgamma ligands have been implicated as regulators of cellular inflammatory and immune responses. They are thought to exert anti-inflammatory effects by negatively regulating the expression of pro-inflammatory genes. Several studies have demonstrated that PPARgamma ligands possess anti-inflammatory properties and that these properties may prove helpful in the treatment of inflammatory diseases of the airways. This review will outline the anti-inflammatory effects of synthetic and endogenous PPARgamma ligands and discuss their potential therapeutic effects in animal models of inflammatory airway disease.

The prostanoid receptors on human airway smooth muscle cells (HASMC) that augment the release by IL-1β of granulocyte colony-stimulating factor (G-CSF) have been characterized and the signaling pathway elucidated. PCR of HASM cDNA identified products corresponding to EP2, EP3, and EP4receptor subtypes. These findings were corroborated at the protein level by immunocytochemistry. IL-1β promoted the elaboration of G-CSF, which was augmented by PGE2. Cicaprost (IP receptor agonist) was approximately equiactive with PGE2, whereas PGD2, PGF2α, and U-46619 (TP receptor agonist) were over 10-fold less potent. Neither SQ 29,548 nor BW A868C (TP and DP1receptor antagonists, respectively) attenuated the enhancement of G-CSF release evoking any of the prostanoids studied. With respect to PGE2, the EP receptor agonists 16,16-dimethyl PGE2(nonselective), misoprostol (EP2/EP3selective), 17-phenyl-ω-trinor PGE2(EP1selective), ONO-AE1-259, and butaprost (both EP2selective) were full agonists at enhancing G-CSF release. AH 6809 (10 μM) and L-161,982 (2 μM), which can be used in HASMC as selective EP2and EP4receptor antagonists, respectively, failed to displace to the right the PGE2concentration-response curve that described the augmented G-CSF release. In contrast, AH 6809 and L-161,982 in combination competitively antagonized PGE2-induced G-CSF release. Augmentation of G-CSF release by PGE2was mimicked by 8-BrcAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). These data demonstrate that PGE2facilitates G-CSF secretion from HASMC through a PKA-dependent mechanism by acting through EP2and EP4prostanoid receptors and that effective antagonism is realized only when both subtypes are blocked concurrently.

8-Isoprostanes are bioactive lipid mediators formed via the nonenzymatic peroxidation of arachidonic acid by free radicals and reactive oxygen species. However, their cognate receptors, biological actions, and signaling pathways are poorly studied. Here, we report the effect of a variety of E- and Falpha-ring 8-isoprostanes on the release of granulocyte/macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) from human airway smooth muscle (HASM) cells stimulated with interleukin-1beta (IL-1beta). The elaboration of GM-CSF and G-CSF by IL-1beta was inhibited and augmented, respectively, in a concentration-dependent manner by 8-iso-prostaglandin (PG) E(1) and 8-iso-PGE(2), but not by 8-iso-PGF(1alpha), 8-iso-PGF(2alpha), and 8-iso-PGF(3)alpha. AH 6809 (6-isopropoxy-9-oxoxanthine-2-carboxylic acid), an EP(1)-/EP(2)-/DP-receptor blocking drug, antagonized the inhibitory effect of 8-iso-PGE(1) and 8-iso-PGE(2) on GM-CSF output with an affinity consistent with an interaction at prostanoid receptors of the EP(2)-subtype. In contrast, the facilitation by 8-iso-PGE(1) and 8-iso-PGE(2) of G-CSF release was unaffected by AH 6809 and the selective EP(4)-receptor antagonist L-161,982 [4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide]. However, when used in combination, AH 6809 and L-161,982 displaced 5-fold to the right the 8-iso-PGE and 8-iso-PGE concentration-response curves. The opposing (1)effect of E-ring (2)8-isoprostanes on GM-CSF and G-CSF release was mimicked by 8-bromo-cAMP and abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA). Together, these data demonstrate that E-ring 8-isoprostanes regulate the secretion of GM-CSF and G-CSF from HASM cells by a cAMP- and PKA-dependent mechanism. Moreover, antagonist studies revealed that 8-iso-PGE(1) and 8-iso-PGE(2) act solely via EP(2) -receptors to inhibit GM-CSF release, whereas both EP(2)- and EP(4)-receptor subtypes positively regulate G-CSF output.

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-activated nuclear hormone receptors belonging to the steroid receptor super-family. Previously, the present authors have shown that PPAR-gamma agonists inhibit the release of inflammatory cell survival factors and induce apoptosis in vitro. The aim of this study was to determine the effect of two structurally different PPAR agonists in an in vivo model of lipopolysaccharide (LPS)-induced airway inflammation. Mice were treated with PPAR agonists, rosiglitazone or SB 219994, prior to exposure to aerosolised LPS, and the extent of airway inflammation was assessed 3 h later. In these experiments, the PPAR ligands inhibited LPS-induced airway neutrophilia and associated chemoattractants/survival factors (keratinocyte-derived chemokine and granulocyte-colony stimulating factor) in the mouse lung. The present authors postulate that if a peroxisome proliferator-activated receptor agonist has the same effect in man, and neutrophils are important in the progression of respiratory diseases, such as chronic obstructive pulmonary disease, then this class of compounds could be a potential therapy. Furthermore, several peroxisome proliferator-activated receptor-gamma agonists have been shown to be clinically effective for the treatment of type II diabetes, suggesting that any benefit of peroxisome proliferator-activated receptor-gamma ligands in the progression of respiratory diseases, which may involve airway neutrophilia, could be explored relatively quickly.

Lipopolysaccharide (LPS) is known to generate nitric oxide (NO) in the airway through the activation of nitric-oxide synthase (NOS). The functional consequences of this on the inflammatory response are not clear, with conflicting data published. In the clinic, exhaled NO (ex-NO) is used as a noninvasive biomarker to assess the extent of airway inflammation. It is proposed that monitoring levels of ex-NO could be a useful guide to determining the effectiveness of disease modifying therapies. The aim was, using pharmacological tools, to determine the role of NO in an aerosolized LPS-driven animal model of airway inflammation by assessment of ex-NO, neutrophilia, and inflammatory biomarkers, using a nonselective NOS inhibitor, N G -nitro-l-arginine methyl ester (l-NAME), and a selective inducible NOS (iNOS) inhibitor, N -3 (aminomethyl)benzyl)acetamidine (1400W). Real-time mRNA analysis of the lung tissue indicated an increased gene expression of iNOS following LPS challenge with minimal impact on constitutive NOS isoforms. LPS induced an increase in ex-NO, which appeared to correlate with the increase in iNOS gene expression and airway neutrophilia. Treatment with l-NAME and 1400W resulted in comparable reductions in ex-NO, a reduction in airway neutrophilia, but had little impact on a range of inflammatory biomarkers. This study indicates that the LPS-induced rise in ex-NO is due to enhanced iNOS activity and that NO has a role in airway neutrophilia. Additionally, it appears using ex-NO as a guide to monitoring airway inflammation may have some use, but data should be interpreted with caution when assessing therapies that may directly impact on NO formation.

1. The prostanoid receptor(s) on human airways smooth muscle (HASM) cells that mediates the inhibitory effect of PGE(2) on interleukin (IL)-1 beta-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) release has been classified. 2. IL-1 beta evoked the release of GM-CSF from HASM cells, which was suppressed by PGE(2), 16,16-dimethyl PGE(2) (nonselective), misoprostol (EP(2)/EP(3)-selective), ONO-AE1-259 and butaprost (both EP(2)-selective) with pIC(50) values of 8.61, 7.13, 5.64, 8.79 and 5.43, respectively. EP-receptor agonists that have selectivity for the EP(1)-(17-phenyl-omega-trinor PGE(2)) and EP(3)-receptor (sulprostone) subtypes as well as cicaprost (IP-selective), PGD(2), PGF(2 alpha) and U-46619 (TP-selective) were poorly active or inactive at concentrations up to 10 microM. 3. AH 6809, a drug that can be used to selectively block EP(2)-receptors in HASM cells, antagonised the inhibitory effect of PGE(2), 16,16-dimethyl PGE(2) and ONO-AE1-259 with apparent pA(2) values of 5.85, 6.09 and 6.1 respectively. In contrast, the EP(4)-receptor antagonists, AH 23848B and L-161,982, failed to displace to the right the concentration-response curves that described the inhibition of GM-CSF release evoked by PGE(2) and ONO-AE1-259. 4. Inhibition of GM-CSF release by PGE(2) and 8-Br-cAMP was abolished in cells infected with an adenovirus vector encoding an inhibitor protein of cAMP-dependent protein kinase (PKA) but not by H-89, a purported small molecule inhibitor of PKA. 5. We conclude that prostanoid receptors of the EP(2)-subtype mediate the inhibitory effect of PGE(2) on GM-CSF release from HASM cells by recruiting a PKA-dependent pathway. In addition, the data illustrate that caution should be exercised when using H-89 in studies designed to assess the role of PKA in biological processes.

In the present study, we examined the effect of E-ring 8-isoprostanes on cholinergic neurotransmission in guinea-pig trachea and identified the receptor(s) involved. As isoprostanes are isomeric with prostaglandins, PGE2 and sulprostone (a selective EP3-receptor agonist) were examined in parallel. 8-Iso-PGE1, 8-iso-PGE2 (0.1 nM–1 μM), sulprostone (1 nM–1 μM) and PGE2 (1 μM) suppressed EFS-evoked [3H]ACh release from guinea-pig trachea in a concentration-dependent manner, producing 39.5, 53.9, 61.2 and 59.9% inhibition, respectively, at 1 μM. It should be noted that an established maximum effective concentration was not determined. Neither SQ 29,548 (1 μM; a TP-receptor antagonist) nor AH 6809 (10 μM; an EP1-/EP2-/DP-receptor antagonist) reversed the inhibitory effect of these compounds. L-798,106, a novel and highly selective EP3-receptor antagonist, produced a parallel shift to the right of the concentration–response curves that described the inhibitory action of sulprostone on EFS-evoked contractile responses in guinea-pig vas deferens (an established EP3-receptor-expressing tissue), from which a mean pA2 of 7.48 was derived. On guinea-pig trachea, L-798,106 also antagonised sulprostone-induced inhibition of EFS-induced twitch responses, with similar potency (mean pA2=7.82). The inhibitory effects of 8-iso-PGE1, 8-iso-PGE2, sulprostone and PGE2 on EFS-induced [3H]ACh release was blocked by L-798,106 at a concentration (10 μM) that binds only weakly to human recombinant EP1-, EP2- and EP4-receptor subtypes expressed in HEK 293 cells. These data suggest that E-ring 8-isoprostanes, PGE2 and sulprostone inhibit EFS-evoked [3H]ACh release from cholinergic nerves innervating guinea-pig trachea, by interacting with prejunctional prostanoid receptors of the EP3-subtype. British Journal of Pharmacology (2004) 141, 600–609. doi:10.1038/sj.bjp.0705648

Background The IL-1 family of cytokines is known to play an important role in inflammation therefore understanding the mechanism by which they are produced is paramount. Despite the recent plethora of publications dedicated to the study of these cytokines, the mechanism by which they are produced in the airway following endotoxin, Lipopolysaccharide (LPS), exposure is currently unclear. The aim was to determine the mechanism by which the IL-1 cytokines are produced after LPS inhaled challenge. Methods Mice were challenged with aerosolised LPS, and lung tissue and bronchiolar lavage fluid (BALF) collected. Targets were measured at the mRNA and protein level; caspase activity was determined using specific assays. Results BALF IL-1b/IL-18, but not IL-1a, was dependent on Ice Protease-Activating Factor (IPAF), and to a lesser extent Apoptosis-associated Speck-like protein containing a CARD (ASC). Interestingly, although we measured an increase in mRNA expression for caspase 1 and 11, we could not detect an increase in lung enzyme activity or a role for them in IL-1a/b production. Further investigations showed that whilst we could detect an increase in caspase 8 activity at later points in the time course (during resolution of inflammation), it appeared to play no role in the production of IL-1 cytokines in this model system. Conclusions TLR4 activation increases levels of BALF IL-1b/IL-18 via an IPAF dependent and caspase 1/11/8 independent pathway. Furthermore, it would appear that the presence of IL-1a in the BALF is independent of these pathways. This novel data sheds light on innate signalling pathways in the lung that control the production of these key inflammatory cytokines.

In human airway smooth muscle cells, the levels of RANTES were increased upon stimulation with interleukin-1beta together with tumour necrosis factor-alpha (TNF-alpha) (10 ng ml(-1) for each). In this study, we have assessed the effects of prostaglandin E(2) and the beta(2)-adrenoceptor agonist, fenoterol on RANTES (regulated upon activation, normal T cell expressed and secreted) release by these cells. The levels of RANTES released by human airway smooth muscle cells were measured after 24 h of treatment. Prostaglandin E(2) and fenoterol, only in presence of a cyclo-oxygenase inhibitor indomethacin (10(-6) M), provoked a concentration-dependent reduction in RANTES release. These data suggest that, in settings where cyclo-oxygenase activity is low, both drugs may relieve the symptoms of airway diseases by reducing RANTES production.

The spasmolytic and anti-spasmogenic activity of β-adrenoceptor agonists on airways smooth muscle is thought to involve activation of the cyclic AMP/cyclic AMP-dependent protein kinase (PKA) cascade. Here we have tested the hypothesis that PKA mediates the anti-spasmogenic activity of isoprenaline and other cyclic AMP-elevating agents in guinea-pig isolated trachea by utilizing a number of cell permeant cyclic AMP analogues that act as competitive ‘antagonists’ of PKA. Anion-exchange chromatography of guinea-pig tracheae resolved two peaks of PKA activity that corresponded to the type I (∼5%) and type II (∼93%) isoenzymes. Pre-treatment of tracheae with zardaverine (30 μM), vasoactive intestinal peptide (VIP) (1 μM) and the non-selective activator of PKA, Sp-8-CPT-cAMPS (10 μM), produced a non-parallel rightwards shift in the concentration-response curves that described acetylcholine (ACh)-induced tension generation. The type II-selective PKA inhibitor, Rp-8-CPT-cAMPS (300 μM), abolished this effect. Pre-treatment of tracheae with Sp-8-Br-PET-cGMPS (30 μM) produced a non-parallel rightwards shift of the concentration-response curves that described ACh-induced tension generation. The selective cyclic GMP-dependent protein kinase (PKG) inhibitor, Rp-8-pCPT-cGMPS (300 μM), abolished this effect. Pre-treatment of tracheae with isoprenaline (1 μM) produced a 10 fold shift to the right of the ACh concentration-response curve by a mechanism that was unaffected by Rp-8-Br-cAMPS (300 μM, selective inhibitor of type I PKA), Rp-8-CPT-cAMPS (300 μM) and Rp-8-pCPT-cGMPS (300 μM). We conclude that the anti-spasmogenic activity of Sp-8-CPT-cAMPS, zardaverine and VIP in guinea-pig trachea is attributable to activation of the cyclic AMP/PKA cascade whereas isoprenaline suppresses ACh-induced contractions by a mechanism(s) that is independent of PKA and PKG. British Journal of Pharmacology (2001) 133, 1201–1212; doi:10.1038/sj.bjp.0704213

We investigated the effect of the p38 kinase inhibitor SB 203580 on airway inflammation induced by aerosolized lipopolysaccharide (LPS) in male Wistar rats. SB 203580 significantly inhibited (ED50=15.8 mg kg−1) plasma levels of TNF-α in rats challenged with LPS (1.5 mg kg−1, i.p.). Aerosolized LPS induced a peak in TNF-α levels and the initiation of a neutrophilic response in bronchoalveolar lavage (BAL) fluid at the 2 h time point. Furthermore, the 4 h time point was associated with the peak in IL-1β levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100 mg kg−1), had no effect on peak TNF-α levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100 mg kg−1) significantly reduced both TNF-α levels and neutrophilic inflammation. However, the BAL fluid from rats pre-treated with either compound significantly inhibited TNF-α release from cultured human monocytes 18 h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100 mg kg−1) produced dose-related inhibition of BAL IL-1β levels (67.5% inhibition, P

8-Iso-prostaglandin F(2 alpha) is present in increased amounts in airway inflammation. 8-Iso-prostaglandin F(2 alpha) constricts the airways via the activation of thromboxane A(2) receptors. However, thromboxane A(2) receptors are also present pre-junctionally on cholinergic nerve terminals innervating guinea pig trachea. We have demonstrated that 8-iso-prostaglandin F(2 alpha) inhibited electrical field stimulation-evoked [3H]acetylcholine release in a concentration-dependent manner, an effect that was not inhibited by the selective thromboxane A(2) receptor antagonist 4(Z)-6-[(2,4,5 cis)2-(2-chlorophenyl)-4-(2-hydroxyphenyl)1,3-dioxan-5-yl]hexenoic acid (ICI 192,605). These data suggest that 8-iso-prostaglandin F(2 alpha) inhibits acetylcholine release through a receptor distinct from the thromboxane A(2) receptor and provides evidence that isoprostanes may have a 'dual' role as both beneficial and deleterious mediators in airway disease.

RPR132331, a 2-(2-dioxanyl)imidazole, was identified as an inhibitor of tumour necrosis factor (TNF)alpha release from lipopolysaccharide (LPS)-stimulated human monocytes. An intensive programme of work exploring the biology, toxicity and physical chemistry of a novel series of inhibitors, derived from RPR132331, has led to the identification of RPR200765A, a development candidate for the treatment of rheumatoid arthritis (RA). RPR200765A is a potent and selective inhibitor of p38 MAP kinase (IC50 = 50 nM). It inhibits LPS-stimulated TNFalpha release both in vitro, from human monocytes (EC50 = 110 nM), and in vivo in Balb/c mice (ED50 = 6 mg/kg). At oral doses between 10 and 30 mg/kg/day it reduces the incidence and progression in the rat streptococcal cell wall (SCW) arthritis model when administered in either prophylactic or therapeutic dosing regimens. The compound, which is a mesylate salt and exists as a stable monohydrate, shows good oral bioavailabiltiy (F = 50% in the rat) and excellent chemical stability. The data from the SCW disease model suggests that RPR200765A could exhibit a profile of disease modifying activity in rheumatoid arthritis (RA) patients which is not observed with current drug therapies.

Glucocorticoids (GCs) are the mainstay of asthma therapy; however, major side effects limit their therapeutic use. GCs influence the expression of genes either by transactivation or transrepression. The antiinflammatory effects of steroids are thought to be due to transrepression and the side effects, transactivation. Recently, a compound, RU 24858, has been identified that demonstrated dissociation between transactivation and transrepression in vitro. RU 24858 exerts strong AP-1 inhibition (transrepression), but little or no transactivation. We investigated whether this improved in vitro profile results in the maintenance of antiinflammatory activity (evaluated in the Sephadex model of lung edema) with reduced systemic toxicity (evaluated by loss in body weight, thymus involution, and bone turnover) compared with standard GCs. RU 24858 exhibits comparable antiinflammatory activity to the standard steroid, budesonide. However, the systemic changes observed indicate that transactivation events do occur with this GC with similar potency to the standard steroids. In addition, the GCs profiled showed no differentiation on quantitative osteopenia of the femur. These results suggest that in vitro separation of transrepression from transactivation activity does not translate to an increased therapeutic ratio for GCs in vivo or that adverse effects are a consequence of transrepression.

1. To determine which mediators are involved in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitised Brown Norway rats we investigated the effect of a histamine H(1) receptor antagonist, mepyramine, a 5-HT receptor antagonist, methysergide, and a cys-leukotriene-1 receptor antagonist, montelukast. 2. Ovalbumin at 1 mg kg(-1) i.v. caused a significant increase in microvascular leakage in the airways and at 3 mg kg(-1) i.v. caused a significant increase in airways resistance. 3. Histamine (1 mg kg(-1) i.v.), 5-HT (0.1 mg kg(-1) i.v.) and leukotriene D(4) (LTD(4), 50 microg kg(-1) i.v.) caused a significant increase in microvascular leakage in the airways. 4. Mepyramine (1 mg kg(-1) i.v.), methysergide (0.1 mg kg(-1) i.v.), or montelukast (30 mg kg(-1) i.v.) inhibited histamine, 5-HT or LTD(4) -induced microvascular leakage respectively. 5. Methysergide (0.1 mg kg(-1) i.v.) reduced ovalbumin-induced microvascular leakage in the trachea and at 0.3 mg kg(-1) i.v. inhibited bronchospasm (38 and 58%, respectively). Montelukast (30 mg kg(-1) p.o.) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (78%) and inhibited ovalbumin-induced bronchospasm (50%). Mepyramine (3 mg kg(-1) i.v.) had no effect on ovalbumin-induced leakage or bronchospasm. 6. A combination of all three compounds (mepyramine, methysergide and montelukast) reduced ovalbumin-induced microvascular leakage in airway tissue to basal levels (70 - 78%) and almost completely inhibited bronchospasm (92%). 7. Antigen-induced bronchospasm appears to equally involve the activation of 5-HT and cys-leukotriene-1 receptors whereas ovalbumin-induced microvascular leakage appears to be predominantly mediated by cys-leukotriene-1 receptors.

The effect of the novel ETA receptor antagonist LBL 031 and other selective and mixed endothelin receptor antagonists on endothelin-1 (ET-1)-induced and lipopolysaccharide (LPS)-induced microvascular leakage was assessed in rat airways Intravenously administered ET-1 (1 nmole kg−1) or LPS (30 mg kg−1) caused a significant increase in microvascular leakage in rat airways when compared to vehicle treated animals. Pre-treatment with the selective ETA receptor antagonists, LBL 031 or PD 156707, or the mixed ETA/B receptor antagonist, bosentan (each at 30 mg kg−1), reduced ET-1-induced leakage to baseline levels. ET-1-induced leakage was not reduced by pre-treatment with the ETB selective antagonist BQ 788 (3 mg kg−1). Pre-treatment with the selective ETA receptor antagonist, LBL 031 (0.1 mg kg−1) or PD 156707 (10 mg kg−1), or the mixed ETA/B receptor antagonist, bosentan (30 mg kg−1), reduced LPS-induced leakage by 54, 48 and 59% respectively. LPS-induced leakage was not affected by pre-treatment with the ETB selective antagonist BQ 788 (3 mg kg−1). The data suggests that ET-1-induced microvascular leakage in the rat airway is ETA receptor mediated and that part of the increase induced by LPS may be due to the actions of ET-1. Therefore, a potent ETA receptor selective antagonist, such as LBL 031, may provide a suitable treatment for inflammatory diseases of the airways, especially those involving LPS and having an exudative phase, such as the septic shock-induced adult respiratory distress syndrome. British Journal of Pharmacology (2000) 131, 1129–1134; doi:10.1038/sj.bjp.0703691

We investigated the ability of the cannabinoid agonists CP55,940 (CB1/CB2) and anandamide (endogenous cannabinoid) to modulate electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. We assessed whether modulation of transmitter release translated to an impact on functional responses by investigating the effect of these agents on contractile responses evoked by EFS and ACh. Furthermore, we evaluated the ability of these compounds to elicit bronchodilation in pre-contracted guinea-pig tracheal strips. CP55,940 and anandamide significantly inhibited EFS-evoked ACh release (maximal inhibition of 35.1±2.9% and 33.4±6.4% at 1 μM, P

The aim of the present study was to develop a chronic in vivo model of pulmonary beta(2)-adrenoceptor desensitization and to elucidate the nature and molecular basis of this state. Subcutaneous infusion of rats with albuterol for 7 days compromised the ability of albuterol, given acutely, to protect against acetylcholine-induced bronchoconstriction. The bronchoprotective effect of prostaglandin E(2), but not forskolin, was also impaired, indicating that the desensitization was heterologous and that the primary defect in signaling was upstream of adenylyl cyclase. beta(2)-Adrenoceptor density was reduced in lung membranes harvested from albuterol-treated animals, and this was associated with impaired albuterol-induced cyclic adenosine monophosphate (cAMP) accumulation and activation of cAMP-dependent protein kinase ex vivo. Gsalpha expression was reduced in the lung and tracheae of albuterol-treated rats, and cholera toxin-induced cAMP accumulation was blunted. Chronic treatment of rats with albuterol also increased cAMP phosphodiesterase activity and G protein-coupled receptor kinase-2, but the extent to which these events contributed to beta(2)-adrenoceptor desensitization was unclear given that forskolin was active in both groups of animals and that desensitization was heterologous. Collectively, these results indicate that albuterol effects heterologous desensitization of pulmonary Gs-coupled receptors in this model, with downregulation of Gsalpha representing a primary molecular etiology.

The anti-spasmogenic potential of SK&F 94120 (PDE3-selective), rolipram (PDE4-selective), zaprinast (PDE5-selective), zardaverine (dual PDE3/4 inhibitor) and theophylline (non-selective) was evaluated in guinea-pig trachealis. SK&F 94120 or rolipram (10 and 100 μM) antagonized histamine-induced tension generation in a concentration-dependent and non-competitive manner whereas ACh-induced contractions were unaffected. Similarly, SK&F 94120 and rolipram in combination were anti-spasmogenic with respect to both contractile agonists to an extent that was greater than the effect of either drug alone. Identical results were obtained with zardaverine (1, 10 and 100 μM) and theophylline (100 μM and 1 mM). Zaprinast protected guinea-pig trachealis against histamine-, but not ACh-induced contractile responses in a manner that was indistinguishable from the results obtained with SK&F 94120. However, in contrast to the interaction between SK&F 94120 and rolipram, no further antagonism was seen when zaprinast and rolipram were used in combination. Pre-treatment of tissues with SNP (10 and 100 μM) antagonized histamine-induced tension generation in a concentration-dependent and non-competitive manner. However, no further antagonism was produced when SNP and rolipram were used concurrently. Likewise, the protection afforded by a combination SNP and SK&F 94120 was no greater than that produced by SNP alone. These results demonstrate that an inhibitor of PDE3 enhances the anti-spasmogenic activity of rolipram but not drugs that elevate cyclic GMP mass. Moreover, the ability of SNP and zaprinast to protect guinea-pig trachealis against histamine-induced contractions apparently is not due to the inhibition of PDE3. British Journal of Pharmacology (1999) 128, 327–336; doi:10.1038/sj.bjp.0702779

In this study we have evaluated the pharmacological profile of the muscarinic antagonist glycopyrrolate in guinea-pig and human airways in comparison with the commonly used antagonist ipratropium bromide. Glycopyrrolate and ipratropium bromide inhibited EFS-induced contraction of guinea-pig trachea and human airways in a concentration-dependent manner. Glycopyrrolate was more potent than ipratropium bromide. The onset of action (time to attainment of 50% of maximum response) of glycopyrrolate was similar to that obtained with ipratropium bromide in both preparations. In guinea-pig trachea, the offset of action (time taken for response to return to 50% recovery after wash out of the test antagonist) for glycopyrrolate (t1/2 [offset]=26.4±0.5 min) was less than that obtained with ipratropium bromide (81.2±3.7 min). In human airways, however, the duration of action of glycopyrrolate (t1/2 [offset]>96 min) was significantly more prolonged compared to ipratropium bromide (t1/2 [offset]=59.2±17.8 min). In competition studies, glycopyrrolate and ipratropium bromide bind human peripheral lung and human airway smooth muscle (HASM) muscarinic receptors with affinities in the nanomolar range (Ki values 0.5–3.6 nM). Similar to ipratropium bromide, glycopyrrolate showed no selectivity in its binding to the M1–M3 receptors. Kinetics studies, however, showed that glycopyrrolate dissociates slowly from HASM muscarinic receptors (60% protection against [3H]-NMS binding at 30 nM) compared to ipratropium bromide. These results suggest that glycopyrrolate bind human and guinea-pig airway muscarinic receptors with high affinity. Furthermore, we suggest that the slow dissociation profile of glycopyrrolate might be the underlying mechanism by which this drug accomplishes its long duration of action. British Journal of Pharmacology (1999) 127, 413–420; doi:10.1038/sj.bjp.0702573

1 We have demonstrated recently that exogenous prostaglandin E2 (PGE2) inhibits electrical field stimulation (EFS)-induced acetylcholine (ACh) release from parasympathetic nerve terminals innervating guinea-pig trachea. In the present study, we have attempted to characterize the pre-junctional prostanoid receptor(s) responsible for the inhibitory action of PGE2 and to assess whether other prostanoids modulate, at a prejunctional level, cholinergic neurotransmission in guinea-pig trachea. To this end, we have investigated the effect of a range of both natural and synthetic prostanoid agonists and antagonists on EFS-evoked [3H]-ACh release. 2 In epithelium-denuded tracheal strips pretreated with indomethacin (10 μM), PGE2 (0.1 nM–1 μM) inhibited EFS-evoked [3H]-ACh release in a concentration-dependent manner with an EC50 and maximal effect of 7.62 nM and 74% inhibition, respectively. Cicaprost, an IP-receptor agonist, PGF2α and the stable thromboxane mimetic, U46619 (each at 1 μM), also inhibited [3H]-ACh release by 48%, 41% and 35%, respectively. PGD2 (1 μM) had no significant effect on [3H]-ACh release. 3 The selective TP-receptor antagonist, ICI 192,605 (0.1 μM), completely reversed the inhibition of cholinergic neurotransmission induced by U-46619, but had no significant effect on similar responses effected by PGE2 and PGF2α. 4 A number of EP-receptor agonists mimicked the ability of PGE2 to inhibit [3H]-ACh release with a rank order of potency: GR63799X (EP3-selective)>PGE2>M&B 28,767 (EP3 selective)>17-phenyl-ω-trinor PGE2 (EP1-selective). The EP2-selective agonist, AH 13205 (1 μM), did not affect EFS-induced [3H]-ACh release. 5 AH6809 (10 μM), at a concentration 10 to 100 times greater than its pA2 at DP-, EP1- and EP2-receptors, failed to reverse the inhibitory effect of PGE2 or 17-phenyl-ω-trinor PGE2 on [3H]-ACh release. 6 These results suggest that PGE2 inhibits [3H]-ACh release from parasympathetic nerves supplying guinea-pig trachea via an interaction with prejunctional prostanoid receptors of the EP3-receptor subtype. Evidence for inhibitory prejunctional TP- and, possibly, IP-receptors was also obtained although these receptors may play only a minor role in suppressing [3H]-ACh release when compared to receptors of the EP3-subtype. However, the relative importance of the different receptors will depend not only on the sensitivity of guinea-pig trachea to prostanoids but on the nature of the endogenous ligands released locally that have activity on parasympathetic nerves. British Journal of Pharmacology (1998) 123, 1246–1252; doi:10.1038/sj.bjp.0701720

We have examined whether 4-tert-butyl-N-[6-(2-hydroxy-ethoxy)-5-(2-methoxy-phenoxy)-2,2′-bipyrimidin-4-yl]-benzenesulfonamide (bosentan; endothelin ETA/B receptor antagonist) and (R)2-[(R)-2-[(S)-2-[[1-(hexahydro-1H-azepinyl)]carbonyl] amino-4-methylpentanoil]amino-3-[3-(1-methyl-1H-indoyl)]propionyl]amino-3-(2-pyridyl) propionic acid (FR 139317; endothelin ETA receptor antagonist) inhibit contractions of human airway smooth muscle induced by endothelin-1 or Suc-[Glu9,Ala11,15]enthothelin-1-(8–21) (IRL 1620; endothelin ETB receptor agonist). Endothelin-1 and IRL 1620 were equipotent. Bosentan and FR 139317 (each 10 μM) produced a small shift in response curves to endothelin-1 (1.6- and 1.5-fold, respectively). However, bosentan was more potent against contractions elicited by IRL 1620 (10 μM, 11.2-fold shift) suggesting that these agonists exhibit different kinetic interactions with endothelin receptors or implying an interaction with a novel endothelin ETB receptor subtype in human airways.

Cyclo-oxygenase (COX) is the enzyme that converts arachidonic acid to prostaglandin H2 (PGH2) which can then be further metabolized to prostanoids which modulate various airway functions. COX exists in at least two isoforms. COX-1 is expressed constitutively, whereas COX-2 is expressed in response to pro-inflammatory stimuli. Prostanoids are produced under physiological and pathophysiological conditions by many cell types in the lung. However, the regulation of the different COX isoforms in human airway smooth muscle (HASM) cells has not yet been determined. COX-1 and COX-2 protein were measured by Western blot analysis with specific antibodies for COX-1 and COX-2. COX-2 mRNA levels were assessed by Northern blot analysis by use of a COX-2 cDNA probe. COX activity was determined by measuring conversion of either endogenous or exogenous arachidonic acid to three metabolites, PGE2, thromboxane B2 or 6-ketoPGF1α by radioimmunoassay. Under control culture conditions HASM cells expressed COX-1, but not COX-2, protein. However, a mixture of cytokines (interleukin-1β (IL-1β), tumour necrosis factor α (TNFα) and interferon γ (IFNγ) each at 10 ng ml−1) induced COX-2 mRNA expression, which was maximal at 12 h and inhibited by dexamethasone (1 μm; added 30 min before the cytokines). Furthermore, COX-2 protein was detected 24 h after the cytokine treatment and the expression of this protein was also inhibited by dexamethasone (1 μm) and cyclohexamide (10 μg ml−1; added 30 min before the cytokines). Untreated HASM cells released low or undetectable amounts of all COX metabolites measured over a 24 h period. Incubation of the cells with the cytokine mixture (IL-1β, TNFα, IFNγ each at 10 ng ml−1 for 24 h) caused the accumulation of PGE2 and 6-keto-PGF1α. In experiments where COX-2 metabolized endogenous stores of arachidonic acid, treatment of HASM cells with IL-1β in combination with TNFα caused a similar release of PGE2 to that when the three cytokines were given in combination. In other experiments designed to measure COX-2 activity directly, cells were treated with cytokines for 24 h before fresh culture medium was added containing exogenous arachidonic acid (30 μm for 15 min) after which PGE2 was measured. IL-1β and TNFα increased COX-2 activity and an additional small increase was produced by the three cytokines in combination. These findings suggest that the increased expression of COX-2 is intimately involved in the exaggerated release of prostanoids from HASM cells exposed to pro-inflammatory cytokines. These data indicate a role for airway smooth muscle cells, in addition to their contractile function, as inflammatory cells involved in the production of mediators which may contribute to the inflammatory response seen in diseases such as asthma. British Journal of Pharmacology (1997) 120, 910–916; doi:10.1038/sj.bjp.0700963

Sensory nerves play an important role in airway disease by mediating central reflexes such as cough, and local axon reflexes resulting in the peripheral release of neuropeptides. We have tested whether the benzimidazolone compound, NS1619, an opener of large conductance calcium-activated potassium (BK Ca) channels, inhibits the activity of sensory fibers, and central and local airway reflexes in guinea pig airways. In in vitro single fiber recording experiments, NS1619 applied to identified receptive fields in the trachea inhibited the firing of A(delta)-fibers evoked by hypertonic saline and distilled water, and bradykinin-evoked firing of C-fibers. Electrically evoked nonadrenergic noncholinergic contractions of isolated bronchi mediated by the release of neurokinin A (NKA) from C-fibers, but not those elicited by exogenous NKA, were inhibited by NS1619. These effects of NS1619 were prevented by iberiotoxin, a selective blocker of BK Ca channels. In conscious guinea pigs, cough evoked by aerosolized citric acid was also inhibited by NS1619. These data show that BK Ca channel activation inhibits sensory nerve activity, resulting in a reduction of both afferent and efferent function. BK Ca channel openers may therefore be of potential benefit in reducing neurogenic inflammation and central reflexes seen during inflammatory conditions of the airways, and may represent a new class of antitussive drug.