Possible mechanism of bronchoprotection by SIN-1 in anaesthetized guinea pigs: roles of nitric oxide and peroxynitrite.

BACKGROUND: S-morpholinosydnonimine (SIN-1) is thought to generate peroxynitrite. Recent reports suggested that peroxynitrite possessed a potent vascular relaxant activity via guanylate cyclase activation. However, no previous studies have examined the relaxant effect of peroxynitrite on airway smooth muscle. OBJECTIVE: To determine the mechanism of bronchoprotection by SIN-1, considering in particular the involvement of nitric oxide (NO) and peroxynitrite. METHODS: Peroxynitrite formation was assayed by monitoring the oxidizing activity of dihydrorhodamine 123, and NO was measured polarographically as a redox current in vitro. We examined the effect of SIN-1 delivered to the airway by ultrasonic nebulization against bronchoconstriction induced by acetylcholine in anaesthetized guinea pigs. RESULTS: SIN-1 produced peroxynitrite in a time- and concentration-dependent manner, but did not produce NO in vitro. However, when mixed with glutathione (GSH) and bronchoalveolar lavage fluid (BALF), peroxynitrite formation by SIN-1 was inhibited and SIN-1 induced the release of NO. SNAP (S-nitroso-N-acetyl-penicillamine) and SIN-1 each inhibited acetylcholine-induced bronchoconstriction in a dose-dependent manner in vivo. Though GSH alone did not have any effect on baseline airway resistance and acetylcholine-induced bronchoconstriction, pretreatment with GSH significantly enhanced SNAP- and SIN-1-induced bronchoprotection. In addition, pretreatment with carboxy-PTIO, a NO scavenger, completely inhibited bronchoprotective effect of SNAP on acetylcholine-induced bronchoconstriction, but partially inhibited SIN-1-induced bronchoprotection. CONCLUSION: These findings demonstrated that SIN-1 is a potent peroxynitrite-releasing compound and caused significant bronchoprotection against acetylcholine. The mechanism of bronchoprotection by SIN-1 appears to be mediated by peroxynitrite but also at least in part through NO regeneration, which may involve GSH and airway thiols as a consequence of exposure to peroxynitrite.     

The bronchoprotective effect of inhaling methacholine by using total lung capacity inspirations has a marked influence on the interpretation of the test result

Methacholine tests are widely used as a diagnostic aid for asthma. Their strength has been reputed to be the high sensitivity and very infrequent occurrence of false-negative test results (ie, high negative predictive value). There are 2 commonly used methods that have been outlined by the American Thoracic Society. These methods were thought to give equivalent results. However, in 3 investigations in which we have compared the 2 methods, we have demonstrated a marked lack of comparability. In subjects with borderline to mild airway responsiveness (tidal breathing, methacholine PC20 >2 mg/mL), the 5 deep inhalations required of the dosimeter method produce marked bronchoprotection in some subjects with asthma. The result of this bronchoprotection is that in 55 subjects with asthma, 50% of those whose tidal breathing PC20 value was greater than 2 mg/mL and 25% of the total had negative methacholine challenge results. This indicates that the standardized dosimeter method has an unacceptable loss of diagnostic sensitivity. We recommend that the dosimeter method not be performed as outlined by the American Thoracic Society and that methacholine should be administered by means of submaximal inhalations or tidal breathing.     

Deep inspiration and airway smooth muscle adaptation to length change

In normal subjects a deep inspiration (DI) taken during bronchoconstriction substantially reduces airway narrowing (bronchodilation) and a DI taken prior to bronchoconstriction attenuates subsequent airway narrowing (bronchoprotection). Although the exact mechanism(s) for these phenomena are unclear the time course of these effects supports the hypothesis that they are mediated through actions of airway smooth muscle (ASM). There is convincing evidence that both the bronchodilation and bronchoprotection actions of DI are deficient or absent in asthmatic subjects. Various theories have been proposed such as a failure of transmission of stress and strain to the ASM in asthma, stretch-induced contraction of smooth muscle in asthmatics, a failure to release bronchodilating substances and differential effects on cross-bridge dynamics or contractile element rearrangement. In this brief review we focus on the mechanical consequences of DI on the ASM. We suggest that a failure of plastic rearrangement of the contractile apparatus following DI is at the basis of the abnormal response to DI in asthma.     

Pharmacological evaluation for anti-asthmatic and anti-inflammatory potential of Woodfordia fruticosa flower extracts

Context: Woodfordia fruticosa Kurz. (Lythraceae) flowers are ethnopharmacologically acclaimed in the Indian medicinal system to treat asthma.

Objective: To evaluate W. fruticosa flower extracts for anti-asthmatic effect.

Materials and methods: Ethyl acetate, acetone, methanol, and hydro-alcohol extracts of W. fruticosa flowers were obtained successively and standardized. Ability of extracts to stabilize free radicals and compound-48/80-induced mast cell degranulation was evaluated. In vitro anti-inflammatory potential of extracts at 100 and 200?µg/ml by membrane stabilization and in vivo inhibition of rat paw edema up to 5?h (100 and 200?mg/ml; p.o.) was evaluated. In vitro bronchorelaxant effect was examined against histamine- and acetylcholine (1?µg/ml; independently)-induced guinea pig tracheal contraction. Extracts were evaluated for bronchoprotection (in vivo) ability against 0.1% histamine- and 2% acetylcholine-induced bronchospasm in guinea pigs at 100 and 200?mg/ml; p.o.

Results: Standardization studies revealed that the methanol extract exhibited highest polyphenolic (62.66 GAE), and flavonoid (6.32 RE) content and HPLC fingerprinting confirmed the presence of gallic acid (R_t 1.383). IC₅₀ values for DPPH scavenging and metal chelation by the methanol extract were 40.42 and 31.50?µg/ml. Methanol and ethyl acetate extracts at 100?µg/ml exhibited 06.52 and 07.12% of histamine release. Methanol, ethyl acetate, and hydro alcohol extracts at 200?mg/kg demonstrated 32.73, 29.83, 26.75% and 32.46, 9.38, 26.75% inhibition of egg albumin and carrageenan-induced inflammation, respectively. Methanol extract exhibited 100% bronchorelaxation and 48.83% bronchoprotection.

Conclusion: Woodfordia fruticosa flower (WFF) extracts exhibited anti-asthmatic effect by demonstrating bronchoprotection, bronchorelaxation, anti-inflammatory, antioxidant, and mast cell stabilization ability.     

Comparison of bronchoprotective and bronchodilator effects of a single dose of formoterol delivered by hydrofluoroalkane and chlorofluorocarbon aerosols and dry powder in a double blind, placebo-controlled, crossover study

BACKGROUND: In response to the phasing out of chlorofluorocarbon (CFC) inhalers, a metered dose hydrofluoroalkane (HFA) formulation, Modulite (Chiesi Farmaceutici S.p.A, Parma, Italy), to be delivered with a pressurized metered dose inhaler (pMDI), has been developed. Modulite is a HFA formulation technology that has been designed to provide stable and uniform dose delivery of HFA-based formulations to enable an easy transition from CFC to HFA inhalers. OBJECTIVES: The aim of this study was to compare the bronchoprotective and bronchodilator effects of a single dose of 12 microg of formoterol from the HFA Modulite inhaler with the Foradil Aerolizer (dry powder inhaler, DPI) and the Foradil CFC inhalers (Novartis Health Consumer, Basel, Switzerland). METHODS: This was a double blind, double dummy, randomized, placebo-controlled, crossover study conducted in 38 subjects with mild to moderate asthma (mean forced expiratory volume in 1 s [FEV1] 87.5% predicted). The primary endpoint was methacholine challenge provocative dose required for 20% fall in the FEV1 (PD20) 90 min post dose. Bronchodilation was assessed with spirometry (FEV1, FVC, FEF25-75) and impulse oscillometry (resistance at 5 and 20 Hz, reactance at 5 Hz and resonant frequency) over the 90 min post dose. In a subset of 12 subjects formoterol plasma levels, serum potassium and glucose were determined up to 480 min post dose. RESULTS: The three formoterol formulations demonstrated significant (P < or = 0.05) improvements in bronchoprotection compared to placebo and non-inferiority of the HFA preparation compared to the CFC and DPI preparations was demonstrated. Geometric mean PD20 values were 0.51 mg with HFA, 0.62 mg with DPI, 0.62 mg with CFC and 0.2 mg with placebo. The log transformed mean differences in PD20 doubling dose between HFA and (a) DPI was -0.28 (95% CI -0.84-0.29, P = 0.57) (b) CFC was -0.28 (95% CI -0.84-0.28, P = 0.57) and (c) placebo was 1.38 (95% CI 0.82-1.94, P < 0.001). Serum potassium, glucose and formoterol plasma profiles were comparable for the CFC, HFA and DPI devices. CONCLUSION: Our findings of similar efficacy, pharmacokinetics and systemic effects of the HFA formoterol inhaler compared to the CFC and DPI preparations supports the potential use of this novel formulation in the treatment of asthma.     

Chronic treatment in vivo with β-adrenoceptor agonists induces dysfunction of airway β(2) -adrenoceptors and exacerbates lung inflammation in mice

BACKGROUND AND PURPOSE

Inhalation of a β-adrenoceptor agonist (β-agonist) is first-line asthma therapy, used for both prophylaxis against, and acute relief of, bronchoconstriction. However, repeated clinical use of β-agonists leads to impaired bronchoprotection and, in some cases, adverse patient outcomes. Mechanisms underlying this β₂-adrenoceptor dysfunction are not well understood, due largely to the lack of a comprehensive animal model and the uncertainty as to whether or not bronchorelaxation in mice is mediated by β₂-adrenoceptors. Thus, we aimed to develop a mouse model that demonstrated functional β-agonist-induced β₂-adrenoceptor desensitization in the context of allergic inflammatory airway disease.

EXPERIMENTAL APPROACH

We combined chronic allergen exposure with repeated β-agonist inhalation in allergen-treated BALB/C mice and examined the contribution of β₂-adrenoceptors to albuterol-induced bronchoprotection using FVB/NJ mice with genetic deletion of β₂-adrenoceptors (KO). Associated inflammatory changes – cytokines (ELISA), cells in bronchoalevolar lavage and airway remodelling (histology) and β₂-adrenoceptor density (radioligand binding) – were also measured.

KEY RESULTS

β₂-Adrenoceptors mediated albuterol-induced bronchoprotection in mice. Chronic treatment with albuterol induced loss of bronchoprotection, associated with exacerbation of the inflammatory components of the asthma phenotype.

CONCLUSIONS AND IMPLICATIONS

This animal model reproduced salient features of human asthma and linked loss of bronchoprotection with airway pathobiology. Accordingly, the model offers an advanced tool for understanding the mechanisms of the effects of chronic β- agonist treatment on β-adrenoceptor function in asthma. Such information may guide the clinical use of β-agonists and provide insight into development of novel β-adrenoceptor ligands for the treatment of asthma.