David J Hele

TRPA1 agonists evoke coughing in guinea pig and human volunteers

RATIONALE Cough is the most frequent reason for consultation with a family doctor, or with a general or respiratory physician. Treatment options are limited and one meta-analysis concluded that over-the-counter remedies are ineffective. There is also increasing concern about their use in children. Environmental irritants such as air pollution and cigarette smoke are thought to evoke cough by stimulating airway sensory nerves; however, how this occurs is not fully understood. OBJECTIVES We hypothesized that the TRPA1 (transient receptor potential cation channel, subfamily A, member 1) receptor may have a role as a novel target for tussive agents given that many potential irritants have been shown to activate this channel. METHODS We investigated the effect of TRPA1 ligands on vagal sensory nerve activity in vitro and in guinea pig and human tussive challenge models. MEASUREMENTS AND MAIN RESULTS We demonstrated that TRPA1 agonists such as acrolein activate cloned human TRPA1 channels in HEK293 cells and also vagal sensory nerves in murine, guinea pig, and human tissues. A role for TRPA1 was confirmed, using specific inhibitors and tissue from Trpa1(-/-) gene-deleted animals. Finally, TRPA1 ligands evoked reproducible tussive responses in both a guinea pig model and normal volunteers. CONCLUSIONS This study identifies the TRPA1 receptor as a promiscuous receptor, activated by a wide range of stimuli, making it a perfect target for triggering cough and as such one of the most promising targets currently identified for the development of antitussive drugs.

The discovery of RPR 200765A, a p38 MAP kinase inhibitor displaying a good oral anti-arthritic efficacy

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.

The heterotopic tracheal allograft as an animal model of obliterative bronchiolitis

Heterotopic tracheal allografts in small rodents have been shown to share many characteristics with the development of obliterative bronchiolitis (OB) in the clinic and therefore provide a suitable animal model for the study of OB. The model facilitates the examination of the pathogenesis of the disease and the elucidation of the cellular and molecular mechanisms involved in its development. The model provides a less technically demanding alternative to whole lung transplantation in small rodents and should lead to a speedier identification of new treatments that might prevent the development of post-transplantation OB in the clinic.

Evidence that the anti-spasmogenic effect of the β-adrenoceptor agonist, isoprenaline, on guinea-pig trachealis is not mediated by cyclic AMP-dependent protein kinase

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.

Effect of dopamine receptor agonists on sensory nerve activity: possible therapeutic targets for the treatment of asthma and COPD.

Sensory nerves regulate central and local reflexes such as airway plasma leakage, and cough and their function may be enhanced during inflammation. Evidence suggests that dopamine receptor agonists may inhibit sensory nerve‐mediated responses. In this study dopamine inhibited vagal sensory nerve induced microvascular leakage in the rat. In order to characterize the receptor involved rat vagus preparations were utilized. Quinagolide (D2/3 agonist), ropinirole (D2/3/4 agonist), SKF 38393 (D1/5 agonist), AR‐C68397AA (Viozan™) (dual D2/B2 agonist) and dopamine inhibited hypertonic saline induced depolarization by approximately 50%. Data suggests that AR‐C68397AA and quinagolide also inhibited depolarization of the human vagus. The quinagolide response was blocked by sulpiride (D2/3 antagonist) but not SCH 23390 (D1/5 antagonist); ropinirole was partially blocked by sulpiride, totally blocked by spiperone (at a concentration that blocks all dopamine receptors) but not by SCH 23390. The response to SKF 38393 was not blocked by sulpiride but was by SCH 23390. The inhibition evoked by AR‐C68397AA was only partially blocked by SCH 23390 but not by sulpiride or spiperone whereas dopamine was blocked by spiperone. The effect of dopamine was not stimulus‐specific as it inhibited capsaicin‐induced depolarization of the rat vagus in a spiperone sensitive manner. In conclusion, dopamine receptor ligands inhibit depolarization of the rat and human vagus. These data suggest that dopamine receptor agonists may be of therapeutic benefit in the treatment of symptoms such as cough and mucus secretion which are evident in respiratory diseases such as asthma and chronic obstructive pulmonary disease.

Effect of the p38 kinase inhibitor, SB 203580, on sephadex induced airway inflammation in the rat

SB 203580 is a pyridinyl imidazole compound which inhibits the release of pro-inflammatory cytokines, such as tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), in vitro and in vivo by inhibiting p38 mitogen-activated protein kinase (MAPK). The present study investigated the effects of SB 203580 in a model of airway inflammation induced by the topical administration of Sephadex into the rat airways. This inflammatory response is characterized by the development of lung oedema, airway tissue inflammatory cell recruitment and an increase in lung TNF-alpha and IL-1beta levels. Sephadex-induced lung oedema was accompanied by a significant increase in lung tissue TNF-alpha but not IL-1beta levels. There was also a significant increase in lung tissue macrophages and an increase in eosinophils which did not reach significance. SB 203580 administration significantly inhibited lung oedema (ED50=18 mg x kg(-1)) in a dose-related manner but was without significant effect on lung tissue cell recruitment or cytokine levels. These data suggest that the increase in tumour necrosis factor-alpha and lung oedema are separate processes which both contribute to Sephadex pathology. Furthermore, the inhibitory effect of SB 203580 on Sephadex-induced lung oedema suggests that p38 kinase inhibitors may be of use in pulmonary pathologies in which lung oedema is a feature.

beta 2 -agonists block tussive responses in guinea pigs via an atypical cAMP-dependent pathway

β2-Adrenoceptor agonists are the most effective bronchodilators currently available, and are used for symptom management in asthmatics. However, whether β2-agonists are also antitussive is controversial. Identifying an antitussive role for β2-agonists and dissecting the possible mechanism of action may help to explain the inconsistencies in the clinical literature and lead to the development of novel therapeutic agents. The aim of the present study was to determine whether or not β2-agonists attenuate the tussive response in guinea pig and human models, and, if so, to identify the mechanism(s) involved. Depolarisation of vagal sensory nerves (human and guinea pig) was assessed as an indicator of sensory nerve activity. Cough was measured in a conscious guinea pig model. A β2-agonist, terbutaline, dose-dependently inhibited the cough response to tussive agents in conscious guinea pigs. Terbutaline and another β2-agonist, fenoterol, blocked sensory nerve activation in vitro. Using these mechanistic models, it was established that β2-agonists suppress the tussive response via a nonclassical cyclic adenosine monosphosphate-dependent pathway that involves the activation of protein kinase G and, subsequently, the opening of large-conductance calcium-activated potassium channels. In conclusion, β2-adrenoceptor agonists are antitussive, and this property occurs due to a direct inhibition of sensory nerve activation. These findings may help to explain the confusion that exists in the clinical literature, and could be exploited to identify novel therapies for the treatment of cough, which is a significant unmet medical need.

New Advances and Potential Therapies for the Treatment of Asthma

Asthma is a disease of the airways with an underlying inflammatory component. The prevalence and healthcare burden of asthma is still rising and is predicted to continue to rise in the current century. Inhaled β2-adrenoceptor agonists and corticosteroids form the basis of the treatments available to alleviate the symptoms of asthma. There is a need for novel, safe treatments to tackle the underlying inflammation that characterizes asthma pathology. Furthermore, there is a requirement for new treatments to be developed as oral therapy in order to alleviate patient compliance issues, especially in children. A multitude of new approaches and new targets are being investigated, which may provide opportunities for novel therapeutic interventions in this debilitating disease. For simplicity, these approaches can be divided into two categories. The first comprises therapies directed against specific components or steps seen in allergic asthma. By ‘components’ we mean the key inflammatory cells (T cells [in particular Th2], B cells, eosinophils, mast cells, basophils and antigen presenting cells [APC]) and mediators (immunoglobulin E [IgE], cytokines, histamines, leukotrienes and prostanoids) believed to be involved in the chronic inflammation seen in asthma. By ‘steps’ we mean the allergic response, such as antigen processing and presentation, Th2-cell activation, B-cell isotype switching, mast cell involvement and airway remodeling. The other category of novel approaches to disease modification in asthma encompasses general anti-inflammatory therapies including phosphodiesterase 4 (PDE4) inhibitors, p38 mitogen-activated protein kinase (MAPK) inhibitors, peroxisome proliferator-activated receptor-γ (PPARγ) agonists, and lipoxins.

New approaches to the modulation of inflammatory processes in airway disease models: ATS 2001, May 18-23, San Francisco

The 97th American Thoracic Society meeting proved to be an excellent meeting, providing a wealth of new information on inflammatory diseases of the airways. Once again there appeared to be an increased emphasis on chronic obstructive pulmonary disease (COPD) with most of the major drug companies concentrating a large part of their efforts in this field. An assessment of the new British Thoracic Society guidelines, which are designed to promote better management of COPD, was also presented at the meeting. Potential new treatments for inflammatory diseases of the airways including COPD were described, ranging from phase III trial data with GlaxoSmithKlines PDE4 inhibitor, Cilomilast (Ariflo®) to the development of AstraZenecas novel dual dopamine D2-receptor/β2-adrenoreceptor agonist, Viozan™. Of particular interest was Byk Guldens Ciclesonide, a new corticosteroid with equivalent efficacy to the market leaders but with an improved safety profile. The same company also presented data on their PDE4 inhibitor, Roflumilast, which is now in phase II/III. Bayer presented data on their PDE4 inhibitor, BAY 19-8004, in a smoking animal model and claimed greater anti-inflammatory efficacy than with a steroid. Asta Medica (now known as Elbion) also described a new potent PDE4 inhibitor, AWD 12-281, with anti-inflammatory activity. In the bronchodilator field, an analysis of data from a one-year trial with Boehringer Ingelheims Tiotropium revealed a possible improvement in lung function in COPD patients; this needs to be confirmed in a specifically designed study. Inhibitors of p38 (c-Jun NH2-terminal kinase and syk kinase) were also discussed as anti-inflammatory agents with potential in the treatment of COPD and asthma. GlaxoSmithKlines p38 kinase inhibitor, SB 239063, appeared to be the most advanced of these with clinical data expected in two to three years. Lyn kinase was also discussed as a novel target for inflammatory airway diseases.