A. Gillissen

Characterization of N-acetylcysteine and ambroxol in anti-oxidant therapy

Reactive free oxygen radicals are known to play an important role in the pathogenesis of various lung diseases such as idiopathic pulmonary fibrosis (IPF), adult respiratory distress syndrome (ARDS) or cystic fibrosis (CF). They can originate from endogenous processes or can be part of exogenous exposures (e.g. ozone, cigarette smoke, asbestos fibres). Consequently, therapeutic enhancement of anti-oxidant defence mechanisms in these lung disorders seems a rational approach. In this regard, N-acetyl-L-cysteine (NAC) and ambroxol have both been frequently investigated. Because of its SH group, NAC scavenges H2O2 (hydrogen peroxide), .OH (hydroxol radical), and HOCl (hypochlorous acid). Furthermore, NAC can easily be deacetylated to cysteine, an important precursor of cellular glutathione synthesis, and thus stimulate the cellular glutathione system. This is most evident in pulmonary diseases characterized by low glutathione levels and high oxidant production by inflammatory cells (e.g. in IPF and ARDS). NAC is an effective drug in the treatment of paracetamol intoxication and may even be protective against side-effects of mutagenic agents. In addition NAC reduces cellular production of pro-inflammatory mediators (e.g. TNF-alpha, IL-1). Also, ambroxol [trans-4-(2-amino-3,5-dibromobenzylamino)-cyclohexane hydrochloride] scavenges oxidants (e.g. .OH, HOCl). Moreover, ambroxol reduces bronchial hyperreactivity, and it is known to stimulate cellular surfactant production. In addition, ambroxol has anti-inflammatory properties owing to its inhibitory effect on the production of cellular cytokines and arachidonic acid metabolites. For both substances effective anti-oxidant and anti-inflammatory function has been validated when used in micromolar concentrations. These levels are attainable in vivo in humans. This paper gives an up-to-date overview about the current knowledge of the hypothesis that oxidant-induced cellular damage underlies the pathogenesis of many human pulmonary diseases, and it discusses the feasibility of anti-oxidant augmentation therapy to the lung by using NAC or ambroxol.

Oxidant scavenger function of ambroxol in vitro: a comparison with N-acetylcysteine.

Highly reactive oxygen metabolites play an important role in inflammatory processes in the lung. Ambroxol (2-amino-3,5-dibromo-N-[trans-4-hydroxycyclohexyl]benzylamine) has been shown to reduce oxidant-mediated cell damage. However, the mechanism of this effect remains unclear. In order to evaluate oxidant scavenger function increasing concentrations of ambroxol (0–10−3 mol/l) were compared with equimolar concentrations ofN-acetylcysteine (NAC) and glutathione (GSH) in vitro to reduce OH• (hydroxyl radical), HOCl (hypochlorous acid), O2− (superoxide anion) and H2O2 (hydrogen peroxide). OH• was measured spectrophotometrically (deoxyribose assay); O2− (xanthine/x-oxidase), H2O2 and HOCl (HOCl/OCl−) were determined by chemiluminescence. Ambroxol, NAC and reduced GSH scavenged OH• significantly at 10−3 mol/l, while HOCl was inhibited at concentrations ≥10−4 mol/l completely (P<0.01). NAC and GSH had no anti-O2− function, while ambroxol (10−4 mol/l) reduced O2− by 14.3±6.7%. In contrast, GSH and NAC scavenged H2O2 at>10−6 mol/l (P<0.01), while ambroxol had no anti-H2O2 effect. Our data demonstrate direct oxidant-reducing capabilities of ambroxol, which may be directly related to the aromatic moiety of the molecule. However, high concentrations (micromolar concentrations) are needed. Due to differences in direct oxidant scavenger function, a combination of ambroxol and NAC could be beneficial in antioxidant therapy.

Inhibition of granulocyte migration by tiotropium bromide

Study objectivesNeutrophil influx into the airways is an important mechanism in the pathophysiology of the inflammatory process in the airways of patients with chronic obstructive pulmonary disease (COPD). Previously it was shown that anticholinergic drugs reduce the release of non-neuronal paracrine mediators, which modulate inflammation in the airways. On this basis, we investigated the ability of the long-acting anticholinergic tiotropium bromide to inhibit a) alveolar macrophage (AM)-mediated chemotaxis of neutrophils, and b) cellular release of reactive oxygen species (ROS).MethodAM and neutrophils were collected from 71 COPD patients. Nanomolar concentrations of tiotropium bromide were tested in AM cultured up to 20 h with LPS (1 μg/ml). AM supernatant was tested for TNFα, IL8, IL6, LTB4, GM-CSF, MIPα/β and ROS. It was further used in a 96-well chemotaxis chamber to stimulate the migration of fluorescence labelled neutrophils. Control stimulants consisted of acetylcholine (ACh), carbachol, muscarine or oxotremorine and in part PMA (phorbol myristate acetate, 0.1 μg/ml). Potential contribution of M1-3-receptors was ascertained by a) analysis of mRNA transcription by RT-PCR, and b) co-incubation with selective M-receptor inhibitors.ResultsSupernatant from AM stimulated with LPS induced neutrophilic migration which could be reduced by tiotropium in a dose dependent manner: 22.1 ± 10.2 (3 nM), 26.5 ± 18,4 (30 nM), and 37.8 ± 24.0 (300 nM, p < 0.001 compared to non-LPS activated AM). Concomitantly TNFα release of stimulated AM dropped by 19.2 ± 7.2% of control (p = 0.001). Tiotropium bromide did not affect cellular IL8, IL6, LTB4, GM-CSF and MIPα/β release in this setting. Tiotropium (30 nM) reduced ROS release of LPS stimulated AM by 36.1 ± 15.2% (p = 0.002) and in carbachol stimulated AM by 46.2 ± 30.2 (p < 0.001). M3R gene expression dominated over M2R and M1R. Chemotaxis inhibitory effect of tiotropium bromide was mainly driven by M3R inhibition.ConclusionOur data confirm that inhibiting muscarinic cholinergic receptors with tiotropium bromide reduces TNFα mediated chemotactic properties and ROS release of human AM, and thus may contribute to lessen cellular inflammation.

Toll-like receptor-9 polymorphisms in sarcoidosis and chronic obstructive pulmonary disease.

The etiology of inflammatory diseases of the lung like sarcoidosis and chronic obstructive pulmonary disease (COPD) is multifactorial. The main trigger for developing a COPD is tobacco smoking while exogenous factors causing sarcoidosis are unclear. In both diseases there is an underlying genetic susceptibility determining both the onset and the course of the diseases. Toll-like receptor (TLR)-9 plays an important role in innate immunity by recognizing bacterial CpG-DNA motifs. It is unclear whether single nucleotide polymorphisms (SNPs) in TLR-9 are able to alter the course of sarcoidosis or COPD, or raise the susceptibility for developing one of the disorders. We examined two SNPs in the promoter region of the TLR-9 gene (T1486C and T1237C) in 175 COPD patients (59% with a stable course of the disease, 41% with an instable course with more than 3 exacerbations over the last 3 years) and 166 sarcoidosis patients (19% with an acute and 81% with a chronic course of the disease lasting >2 years) comparing each group to 233 healthy controls. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis was used for genotyping. The C-allele frequency of T1486C was significantly elevated in COPD patients (p = 0.008). For T1237C there were no significant associations comparing the COPD cohort with the controls. In the sarcoidosis cohort, we could observe a significantly higher prevalence of the C-allele for T1237C in the chronic sarcoidosis cohort in comparison to the control group (p = 0.026). For T1486 there no statistical association was observed. This is the first study showing an association between a SNP (T1486C) in the TLR-9 gene and the onset of COPD. Moreover, we could demonstrate that T1237C is able to alter the course of sarcoidosis as a disease-modifying gene. This study underlines that SNPs in TLR-9 might be involved in acquiring and maintaining lung diseases such as sarcoidosis and COPD.

Inflammatory responses after endothelin B (ETB) receptor activation in human monocytes: new evidence for beneficial anti-inflammatory potency of ETB-receptor antagonism.

Endothelin (ET) stimulates potent ETA/ETB receptors important in the pathogenesis of pulmonary arterial hypertension (PAH) and fibrosis. Though therapy with ET-receptor antagonists is well established uncertainty exists whether selective ETA or dual ETA/ETB-receptor antagonism is superior in PAH. The objective of this study was to further elucidate the pro-inflammatory effects of ET-1 on ETB receptors in cultured human monocytes (10(5)/20 h) compared with non-specific stimulation with LPS in vitro and to define the antagonizing effects of bosentan, a dual ETA/ETB-receptor antagonist, on inflammatory mediator production. We further hypothesized that ETB-receptor antagonism reduces the requirement of PGE2 to control inflammatory mediator production. Activation of the monocyte ETB subtype by ET (1 ng/ml) concentration-dependently stimulated TNF-alpha (744%) >PGE2 (570%) > IL-1 beta (112%) and had no effect on 5-lipoxygenase metabolism. Compared with ET a different profile of IL-1 beta >TNF-alpha >PGE2 was induced by LPS. ETB-receptor antagonism attenuated ET- and LPS-responses in monocytes, in particular of TNF-alpha and PGE2 to a similar extend (40%) that were only demonstrable following LPS at therapeutic plasma concentrations of bosentan and had no effect on IL-1 beta. Inhibition of ETB receptors in LPS-stimulated monocytes by bosentan was responded with suppression of PGE2 and increased production of leukotrienes indicating strong effects in the cyclooxygenase pathway that is known to control cellular ET transcription. These data suggest an important signaling pathway between ET-induced cytokine production following ETB-receptor activation with no further control of ET transcription by PGE2 required following ETB receptor antagonism. Therefore, in states of inflammation increased ETB-receptor expression and activation mediated by elevated ET concentrations may be an underestimated mechanism, which warrants the application of combined ETA/ETB-receptor antagonists.

Beta-2-Agonists Have Antioxidant Function in vitro. 2. The Effect of Beta-2-Agonists on Oxidant-Mediated Cytotoxicity and on Superoxide Anion Generated by Human Polymorphonuclear Leukocytes

Therapeutic agents which may be able to enhance the antioxidant screen of the epithelial surface of the lung have the potential to influence the progression of lung inflammation. This study evaluates the efficacy of a variety of antiasthma drugs to reduce oxidant-mediated cytotoxicity and to inhibit superoxide anion generated by human polymorphonuclear leukocytes. We quantified in vitro the prevention of H2O2-mediated cytotoxicity (lactate dehydrogenase release assay) using the antiasthma drugs as follows: ipratropium bromide, salbutamol (salbutamol base), fenoterol (fenoterol hydrobromide), terbutaline terbutaline sulfate), isoproterenol, prednisolone (prednisolone hydrogensuccinate), beclomethasone (17,21-beclomethasone dipropionate) and reduced glutathione. Furthermore, fenoterol and isoproterenol were evaluated ex vivo to reduce superoxide anion (O2-) generated by freshly isolated polymorphonuclear cells (PMN) from smokers with chronic obstructive lung disease (n = 10). Using a concentration of 10(-4) M, reduction of cytotoxicity was quite different among beta(2)-agonists: fenoterol (97.8%) > isoproterenol (67.6% > salbutamol (41.8%) > terbutaline (30.5%) > ipratropium bromide (18.1%). Corticosteroids and theophylline had no antioxidant effect. The cellular O2- production of freshly isolated PMN was significantly (p < 0.05, comparisons 0 vs. > or = 10(-7) M) reduced with fenoterol and isoproterenol at concentrations > or = 10(-7) M. Propranolol had no inhibitory effect on antioxidant properties of beta(2)-agonists. We hypothesize that the antioxidant function of beta(2)-agonists is related to the number and formation of hydroxyl groups of the phenol rings within their molecular structure. These results demonstrate that beta(2)-agonists have in part a good intrinsic scavenger function on reactive oxygen species when used in micromolar concentrations. However, to achieve this effect supratherapeutic concentrations were necessary. Thus, the conceivable benefit of beta(2)-agonists in the treatment of high oxidant burden in vivo seems doubtful.

Leitlinie der Deutschen Gesellschaft für Pneumologie und Beatmungsmedizin zur Diagnostik und Therapie von erwachsenen Patienten mit akutem und chronischem Husten

The first set of German guidelines for diagnosis and treatment of patients suffering from acute or chronic cough was published in 2004. [1]. Scientific developments over the past five years necessitate an update. The guidelines evaluate and establish required diagnostic and therapeutic measures. The purpose of this document is to assist in ascertaining underlying causes and treating cough, in order to eliminate or minimize impairments of patients’ health. The guidelines aim to introduce scientifically founded, evidence-based steps for diagnosis and treatment of cough and optimize cost-effectiveness. Recommendations are assessed through the GRADE system (The Grades of Recommendation, Assessment, Development and Evaluation) [2]. Nevertheless, each patient is entitled to individual diagnosis and treatment. A specific case can justify divergence from these guidelines. Anatomy and physiology of cough !

Polymorphisms of the ß2 adrenoreceptor gene in chronic obstructive pulmonary disease

Background: The ß2-adrenergic receptors are cell surface receptors playing a central role in the pharmacological targeting asthma and chronic obstructive pulmonary disease (COPD). Recent studies suggest that patients who are homozygous for one of the two important polymorphisms of the ß2-adrenergic receptor (ADRB2) gene at codon 16 (arginine to glycine) and 27 (glutamine to glutamate) may have a reduced response to ß2-agonists. Since smoking patients who are Gly16 homozygotes have an increased risk of airway obstruction we hypothesized that ß2-adrenoreceptor gene polymorphisms may be also a cofounder for COPD development and disease severity. Methods: We investigated 190 COPD patients and 172 healthy volunteers in a case-control study. DNA was isolated from whole blood and ß2-AR gene polymorphisms Arg/Gly16 and Gln/Glu27 were determined using allele-specific polymerase chain reaction (PCR). Results: In COPD patients with Gly/Gly16 was found more frequently than in healthy smokers (29.47% COPD versus 18.18% controls, p = 0.026). All other gene polymorphisms of the ADRB2 gene at codon 16 were equally distributed between groups. ß2-adrenoreceptor gene polymorphisms were neither a cofounder for COPD exacerbations (≥ 3 hospitalizations within the last 3 years) nor for disease severity (FEV1 ≤ 30% predicted). Conclusion: Our study suggests that the Gly16 allele of the ß2-AR gene predisposes to COPD development but not for exacerbation rates and disease severity. In contrast, Gln/Glu27 polymorphism was irrelevant in our COPD cohort.

Beta-2-Agonists Have Antioxidant Function in vitro. 1. Inhibition of Superoxide Anion, Hydrogen Peroxide, Hypochlorous Acid and Hydroxyl Radical

beta(2)-Agonists are known to have anti-inflammatory efficacy. In this context, beta(2)-agonists are also capable of inhibiting oxidant production of cultured inflammatory cells. As the mechanisms of this function still remain speculative, the purpose of this study was to quantify the efficacy of beta(2)-agonists in vitro to inhibit superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH.) and hypochlorous acid (HOCl). We tested the following antiasthma drugs: ipratropium bromide, salbutamol (salbutamol base), fenoterol (fenoterol hydrobromide), terbutaline (terbutaline sulfate), isoproterenol, prednisolone (prednisolone hydrogensuccinate), beclomethasone (beclomethasone dipropionate) and theophylline (theophylline sulfate). Antioxidant function was quantified by using the following assay systems: O2- (ferricytochrome c + xanthine/xanthine oxidase), H2O2 (phenol red + 5.10(-6) M H2O2), OH. (deoxyribose assay) and HOCI (HOCl/OCl- in luminol-dependent chemiluminescence). At 10(-4) M, the anti-H2O2 and anti-O2- capacity was as follows: salbutamol/terbutaline < fenoterol < isoproterenol. All beta(2)-agonists (10(-4) M) tested reduced HOCl activity by > 50% (p < 0.01). In contrast, moderate OH. reduction (10-30%) by the beta(2)-agonists is regarded as an nonspecific effect, due to the high concentrations needed (10(-3) M). Corticosteroids and theophylline had no antioxidant effect. These results demonstrate the different redox potentials of different phenol types within the molecular structure of the beta(2)-agonists. The good antioxidative function of isoproterenol is related to ortho formation of the phenol ring, whereas fenoterol has tow phenol rings which can be oxidized. A direct oxidant scavenger function may explain the ability of beta(2)-agonists to reduce the oxidant production of inflammatory cells in vitro.

Smoking cessation in patients with COPD

Chronic obstructive pulmonary disease (COPD) is a leading cause of death worldwide. Cigarette smoking is the main cause of COPD. Quitting smoking is thus the most effective treatment strategy and central in COPD prevention. A number of guidelines on prevention, diagnosis, therapy and rehabilitation of COPD have been published. To help implementing and standardizing smoking cessation in COPD a guideline was published 2008 in Germany focusing on this complex issue. The present guideline is an update of the 2008 guideline and has a high grade of evidence (S3 according to the AWMF; Arbeitsgemeinschaft wissenschaftlicher medizinischer Fachgesellschaften). The guideline gives comprehensive and practical information on how to integrate smoking cessation as an central part of COPD therapy.