Maria G Belvisi

Therapeutic Benefit of a Dissociated Glucocorticoid and the Relevance of In Vitro Separation of Transrepression from Transactivation Activity

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.

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.

Role of p38 MAP kinase in LPS‐induced airway inflammation in the rat

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=15.8u2003mgu2003kg−1) plasma levels of TNF‐α in rats challenged with LPS (1.5u2003mgu2003kg−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 2u2003h time point. Furthermore, the 4u2003h time point was associated with the peak in IL‐1β levels and the initial plateau of neutrophilia observed in the BAL fluid. SB 203580 (100u2003mgu2003kg−1), had no effect on peak TNF‐α levels or the associated neutrophilia in the BAL. Interestingly, the PDE 4 inhibitor RP 73401 (100u2003mgu2003kg−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 18u2003h after LPS treatment (83.6 and 44.5% inhibition, respectively). Alternatively, SB 203580 (100u2003mgu2003kg−1) produced dose‐related inhibition of BAL IL‐1β levels (67.5% inhibition, P<0.01) and BAL neutrophilia (45.9% inhibition, P<0.01) 4u2003h after LPS challenge. P38 protein was present in lung tissue and the level of expression was not affected by LPS treatment. P38 kinase appears to be involved in the release of IL‐1β and the sustained neutrophilic response in the BAL fluid. This data may suggest a role for p38 inhibitors in the treatment of airway inflammatory diseases in which neutrophilia is a feature of the lung pathology.

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.

Differential Effects of Ebselen on Neutrophil Recruitment, Chemokine, and Inflammatory Mediator Expression in a Rat Model of Lipopolysaccharide-Induced Pulmonary Inflammation

We postulated that the seleno-organic compound ebselen would attenuate neutrophil recruitment and activation after aerosolized challenge with endotoxin (LPS) through its effect as an antioxidant and inhibitor of gene activation. Rats were given ebselen (1–100 mg/kg i.p.) followed by aerosolized LPS exposure (0.3 mg/ml for 30 min). Airway inflammatory indices were measured 4 h postchallenge. Bronchoalveolar lavage (BAL) fluid cellularity and myeloperoxidase activity were used as a measure of neutrophil recruitment and activation. RT-PCR analysis was performed in lung tissue to assess gene expression of TNF-α, cytokine-induced neutrophil chemoattractant-1 (CINC-1), macrophage-inflammatory protein-2 (MIP-2), ICAM-1, IL-10, and inducible NO synthase. Protein levels in lung and BAL were also determined by ELISA. Ebselen pretreatment inhibited neutrophil influx and activation as assessed by BAL fluid cellularity and myeloperoxidase activity in cell-free BAL and BAL cell homogenates. This protective effect was accompanied by a significant reduction in lung and BAL fluid TNF-α and IL-1β protein and/or mRNA levels. Ebselen pretreatment also prevented lung ICAM-1 mRNA up-regulation in response to airway challenge with LPS. This was not a global effect of ebselen on LPS-induced gene expression, because the rise in lung and BAL CINC-1 and MIP-2 protein levels were unaffected as were lung mRNA expressions for CINC-1, MIP-2, IL-10, and inducible NO synthase. These data suggest that the anti-inflammatory properties of ebselen are achieved through an inhibition of lung ICAM-1 expression possibly through an inhibition of TNF-α and IL-1β, which are potent neutrophil recruiting mediators and effective inducers of ICAM-1 expression.

Effect of the p38 kinase inhibitor, SB 203580, on allergic airway inflammation in the rat.

Tumour necrosis factor‐α (TNF‐α) and interleukin 1β (IL‐1β) have been implicated in the pathogenesis of asthma. The p38 kinase inhibitor, SB 203580 inhibits TNF‐α and IL‐1β production in vitro and in vivo. In this study the effect of SB 203580 on allergen‐induced airway TNF‐α production and inflammatory cell recruitment was investigated in sensitized Brown Norway rats. The allergen‐induced increase in bronchoalveolar lavage (BAL) TNF‐α was inhibited by SB 203580 at every dose tested (10–100u2003mgu2003kg−1, p.o.). In contrast, neither ovalbumin‐induced eosinophilia or neutrophilia were inhibited by SB 203580 (10–100u2003mgu2003kg−1, p.o.). In conclusion, SB 203580 inhibits BAL TNF‐α production by 95% without inhibiting either antigen‐induced airway eosinophilia or neutrophilia. This data suggests that either the residual TNF‐α is sufficent to drive allergen‐induced inflammatory cell recruitment into the lung or that TNF‐α is not involved in allergen‐induced inflammatory cell recruitment.

Mediator involvement in antigen-induced bronchospasm and microvascular leakage in the airways of ovalbumin sensitized Brown Norway rats

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 H1 receptor antagonist, mepyramine, a 5‐HT receptor antagonist, methysergide, and a cys‐leukotriene‐1 receptor antagonist, montelukast. Ovalbumin at 1u2003mgu2003kg−1 i.v. caused a significant increase in microvascular leakage in the airways and at 3u2003mgu2003kg−1 i.v. caused a significant increase in airways resistance. Histamine (1u2003mgu2003kg−1 i.v.), 5‐HT (0.1u2003mgu2003kg−1 i.v.) and leukotriene D4 (LTD4, 50u2003μgu2003kg−1u2003i.v.) caused a significant increase in microvascular leakage in the airways. Mepyramine (1u2003mgu2003kg−1 i.v.), methysergide (0.1u2003mgu2003kg−1 i.v.), or montelukast (30u2003mgu2003kg−1 i.v.) inhibited histamine, 5‐HT or LTD4 ‐induced microvascular leakage respectively. Methysergide (0.1u2003mgu2003kg−1 i.v.) reduced ovalbumin‐induced microvascular leakage in the trachea and at 0.3u2003mgu2003kg−1 i.v. inhibited bronchospasm (38 and 58%, respectively). Montelukast (30u2003mgu2003kg−1 p.o.) reduced ovalbumin‐induced microvascular leakage in airway tissue to basal levels (78%) and inhibited ovalbumin‐induced bronchospasm (50%). Mepyramine (3u2003mgu2003kg−1 i.v.) had no effect on ovalbumin‐induced leakage or bronchospasm. A combination of all three compounds (mepyramine, methysergide and montelukast) reduced ovalbumin‐induced microvascular leakage in airway tissue to basal levels (70u2003–u200378%) and almost completely inhibited bronchospasm (92%). 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.

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.

Characterization of the effects of cannabinoids on guinea-pig tracheal smooth muscle tone: role in the modulation of acetylcholine release from parasympathetic nerves

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 1u2003μM, P<0.05, respectively). The CB1 receptor antagonist SR 141716A (1u2003μM), had no effect on ACh release and failed to reverse the inhibitory effect of CP55,940 (1u2003μM). Paradoxically, CP55,940 had no significant effect on EFS‐evoked cholinergic contractile responses. Furthermore, CP55,940 did not relax pre‐contracted tracheal strips or affect contractile responses to exogenous ACh. This lack of activity on smooth muscle tone is consistent with the fact that no detectable specific binding of [3H] CP55,940 was found in tracheal homogenates. These data suggest that cannabinoid agonists inhibit ACh release from cholinergic nerve terminals via activation of CB2 receptors but that this inhibitory action does not impact on functional responses such as cholinergic contraction.

Effect of endothelin antagonists, including the novel ETA receptor antagonist LBL 031, on endothelin‐1 and lipopolysaccharide‐induced microvascular leakage in rat airways

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 (1u2003nmoleu2003kg−1) or LPS (30u2003mgu2003kg−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 30u2003mgu2003kg−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 (3u2003mgu2003kg−1). Pre‐treatment with the selective ETA receptor antagonist, LBL 031 (0.1u2003mgu2003kg−1) or PD 156707 (10u2003mgu2003kg−1), or the mixed ETA/B receptor antagonist, bosentan (30u2003mgu2003kg−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 (3u2003mgu2003kg−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.