Tiotropium bromide exerts anti-inflammatory activity in a cigarette smoke mouse model of COPD.

Tiotropium bromide is a long acting muscarinic antagonist (LAMA), marketed under the brand name Spiriva, for the treatment of chronic obstructive pulmonary disease (COPD). Besides its proven direct bronchodilatory activity, recent clinical studies demonstrated that tiotropium is able to reduce the exacerbation rate and impact the clinical course of COPD. One significant pathological feature believed to be causative for the progressive nature of COPD is chronic pulmonary inflammation. The aim of the present study was to investigate the anti-inflammatory activity of tiotropium on cigarette smoke-induced pulmonary inflammation in mice. C57Bl/6 mice were exposed to cigarette smoke (CS) for four days with increasing exposure time for up to 6h per day to elicit pulmonary inflammation and mediator release. One hour before smoke exposure, animals were treated with tiotropium by inhalation (0.01-0.3mg/mL) for 5 min; 18h after the last CS exposure a bronchoalveolar lavage was performed. Tiotropium concentration-dependently inhibited pulmonary neutrophilic inflammation with an IC(50) of 0.058 mg/mL and a maximum inhibition of 60% at 0.3mg/mL. Furthermore, the CS-induced pulmonary release of leukotriene B(4), interleukin-6, keratinocyte-derived chemokine, monocyte chemotactic protein-1, macrophage inflammatory protein-1 alpha and -2, and tumor necrosis factor alpha was dose-dependently reduced. The bronchodilatory activity of tiotropium against acetycholine-induced bronchoconstriction was found to be in the same dose range as the anti-inflammatory activity with an IC(50) of 0.045 mg/mL and a maximum bronchodilation of 90% at 0.3mg/mL. Our data suggest that the beneficial effects of tiotropium on the course of COPD shown in patients may be associated with an anti-inflammatory activity.

Inhibition of airway hyperresponsiveness and pulmonary inflammation by roflumilast and other PDE4 inhibitors.

Roflumilast is an oral, once-daily phosphodiesterase 4 (PDE4) inhibitor with anti-inflammatory activity. We compared the anti-inflammatory effects of roflumilast with those of PDE4 inhibitors rolipram, piclamilast, and cilomilast in ovalbumin (OVA)-sensitized and challenged Brown-Norway rats. Animals were treated orally 1h before OVA challenge with roflumilast (0.3, 1.0, and 3.0mg/kg), rolipram (0.8, 2.8, and 8.3mg/kg), piclamilast (10.0, 20.0, and 30.0mg/kg), or cilomilast (10.3, 34.3, and 103.0mg/kg). Airway hyperresponsiveness (AHR) against adenosine was investigated by measuring airway resistance 200min after OVA challenge. Subsequently, neutrophil influx and tumor necrosis factor-alpha (TNF-alpha) release in the lungs were determined by bronchoalveolar lavage. Direct bronchodilation at the time point of AHR assessment by PDE4 inhibitors was examined in serotonin-challenged animals. Evaluation of neutropenic animals or treatment with anti-TNF-alpha antibody revealed that AHR was independent of neutrophil accumulation or TNF-alpha release. Roflumilast (50% inhibitory dose [ID(50)]=1.5mg/kg) inhibited AHR 3-, 16-, and 27-fold more potently than rolipram, piclamilast, and cilomilast, respectively. Likewise, roflumilast was a more potent inhibitor of neutrophil influx (ID(50)=0.9mg/kg) than rolipram (ID(50)=6.9mg/kg), piclamilast (ID(50)=28.1mg/kg), or cilomilast (ID(50)=37.7mg/kg). Roflumilast, rolipram, and piclamilast-but not cilomilast-suppressed OVA-induced TNF-alpha release in a dose-dependent manner. Roflumilast (ID(50)=0.9mg/kg) exhibited 9- and 23-fold more potent inhibition of TNF-alpha release than rolipram and piclamilast, respectively. Roflumilast did not inhibit serotonin-induced bronchoconstriction 4.5h after administration, suggesting that inhibition of AHR by roflumilast results from anti-inflammatory, not bronchodilatory, effects. This study suggests that roflumilast has anti-inflammatory action and provides rationale for the investigation of roflumilast in asthmatic patients.

In vivo efficacy in airway disease models of roflumilast, a novel orally active PDE4 inhibitor.

We have investigated the bronchodilator and anti-inflammatory properties of roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-dichloropyrid-4-yl]-benzamide), a novel, highly potent, and selective phosphodiesterase 4 (PDE4) inhibitor. Additionally, we compared the effects of roflumilast and its N-oxide, the primary metabolite in vivo, with those of the PDE4 inhibitors piclamilast, rolipram, and cilomilast. Roflumilast inhibited the ovalbumin-evoked contractions of tracheal chains prepared from sensitized guinea pigs (EC(50) = 2 x 10(-7) M) but showed no relaxant effect on tissues contracted spontaneously. In spasmogen-challenged rats and guinea pigs, intravenously administered roflumilast displayed bronchodilatory activity (ED(50) = 4.4 and 7.1 micromol/kg, respectively). Furthermore, roflumilast dose dependently attenuated allergen-induced bronchoconstriction in guinea pigs (ED(50) = 0.1 micromol/kg i.v.). Roflumilast given orally (ED(50) = 1.5 micromol/kg) showed equal potency to its N-oxide (ED(50) = 1.0 micromol/kg) but was superior to piclamilast (ED(50) = 8.3 micromol/kg), rolipram (ED(50) = 32.5 micromol/kg), and cilomilast (ED(50) = 52.2 micromol/kg) in suppressing allergen-induced early airway reactions. To assess the anti-inflammatory potential of orally administered roflumilast, antigen-induced cell infiltration, total protein, and TNFalpha concentration in bronchoalveolar lavage fluid of Brown Norway rats were determined. Roflumilast and its N-oxide equally inhibited eosinophilia (ED(50) = 2.7 and 2.5 micromol/kg, respectively), whereas the reference inhibitors displayed lower potency (ED(50) = 17-106 micromol/kg). Besides, orally administered roflumilast abrogated LPS-induced circulating TNFalpha in the rat (ED(50) = 0.3 micromol/kg), an effect shared by its N-oxide, with both molecules exhibiting 8-, 25-, and 310-fold superiority to piclamilast, rolipram, and cilomilast, respectively. These results, coupled with the in vitro effects of roflumilast on inflammatory cells, suggest that roflumilast represents a potential new drug for the treatment of asthma and chronic obstructive pulmonary disease.

Granulocyte-macrophage colony-stimulating factor amplifies lipopolysaccharide-induced bronchoconstriction by a neutrophil- and cyclooxygenase 2-dependent mechanism.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is used to ameliorate neutropenia in patients after antineoplastic treatment. It has also been suggested as an adjunct treatment in septic patients; however, the recruitment and priming of leukocytes by GM-CSF bears the hazard of a hyperinflammatory response. In particular, the role of GM-CSF in pulmonary functions in septic lungs is still unclear. Therefore, we pretreated rats in vivo with GM-CSF (50 microg/kg, intravenous) and assessed the pulmonary functions of their subsequently prepared isolated perfused lungs when exposed to subtoxic concentrations of lipopolysaccharide (LPS, 2 microg/ml). These lungs showed enhanced expression of cyclooxygenase 2 (COX-2), a significant increase in thromboxane (TX) and tumor necrosis factor (TNF) release into the venous perfusate, and bronchoconstriction. COX-2 inhibition or blocking of the TX receptor abolished the GM-CSF/LPS-induced bronchoconstriction, but not the TNF release. Neutralizing antibodies against TNF did not prevent GM-CSF/LPS-induced bronchoconstriction. After GM-CSF pretreatment, massive neutrophil invasion into the lung occurred. Neutropenic rats were protected against GM-CSF/ LPS-induced lung injury. Similar results were obtained in rats pretreated with G-CSF instead of GM-CSF. We conclude that GM-CSF pretreatment exacerbates pulmonary injury by low-dose LPS via COX-2 expression, TX release, and bronchoconstriction by initiating neutrophil invasion and activation.

Temporal sequence of pulmonary and systemic inflammatory responses to graded polymicrobial peritonitis in mice.

The lungs are the remote organ most commonly affected in human peritonitis. The major goals of this study were to define the dose- and time-dependent relationship between graded septic peritonitis and systemic and pulmonary inflammatory responses in mice. BALB/c mice were treated with intraperitoneal polymicrobial inoculi and sacrificed at 3, 12, and 24 h. The treatment protocol resulted in distinct groups of animals with respect to mortality rate, kinetics, and concentrations of a broad spectrum of pro- and anti-inflammatory endogenous mediators, intrapulmonary bacterial accumulation, and static lung compliance. In sublethally infected mice, pulmonary bacterial proliferation was controlled. Levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-10, interleukin-6, granulocyte colony-stimulating factor (G-CSF), and tumor necrosis factor (TNF) in plasma were elevated 3 h after infection exclusively. At 3 h, MCP-1, gamma interferon, and TNF were detected in extracts of pulmonary tissue or in bronchoalveolar lavage (BAL) fluid. Static lung compliance (C(st)) was transiently decreased at 12 h. In contrast, in lethally infected mice pulmonary bacterial proliferation was not contained. Concentrations of MCP-1, G-CSF, and TNF in plasma were maximal at 24 h, as were pulmonary MCP-1 levels. Lung myeloperoxidase activity was increased at 3, 12, and 24 h. C(st) was reduced after 3 h and did not reach control values at 24 h. Pulmonary cyclooxygenase-2 mRNA and eicosanoids in BAL fluid and plasma were elevated at 3 and 24 h. This study shows that polymicrobial peritonitis in mice leads to dose-dependent systemic and pulmonary inflammation accompanied by a decrease in lung compliance.

Functional and fine structural changes in isolated rat lungs challenged with endotoxin ex vivo and in vitro.

The aim of this study was to relate changes in rat lung functions caused by the endotoxin lipopolysaccharide (LPS) to alterations in structure. The following four experimental groups were used: 1), control in vitro, perfusion for 150 minutes; 2), LPS in vitro, perfusion for 150 minutes and infusion of 5 mg of LPS after 40 minutes; 3), control ex vivo, perfusion for 10 minutes; and 4), LPS ex vivo, lungs perfused for 10 minutes from rats treated for 110 minutes with 20 mg/kg LPS intraperitoneally. Histologically, blood-derived leukocytes were detectable only in lungs from group 4, where neutrophils were found in capillaries, interstitium, and endothelial pouches. LPS treatment increased pulmonary resistance and decreased pulmonary compliance in group 4 (ex vivo), and, to a greater extent, in group 2 (in vitro). In these two groups, formation of giant lamellar bodies in the type II pneumocytes was observed. By histological examination, the bronchoconstriction induced by LPS in vitro was localized to the terminal bronchioles. At 2 hours after LPS treatment, no edema and no change in precapillary and postcapillary resistance, capillary pressure, vascular compliance, capillary permeability, and the wet/dry ratio was observed. Thus, our major findings are that LPS induced constriction of the terminal bronchioles in vitro, formation of giant lamellar bodies in type II pneumocytes ex vivo and in vitro, and trapping of neutrophils in endothelial pouches in vivo.

Contributions of thromboxane and leukotrienes to PAF-induced impairment of lung function in the rat.

This study was carried out to further clarify the role of eicosanoids in platelet-activating factor (PAF)-induced pulmonary vasoconstriction, bronchoconstriction, and edema formation in the isolated perfused rat lung. Infusion of PAF into the isolated perfused rat lung caused vasoconstriction [mean effective concn (EC50) = 0.88 nmol], caused bronchoconstriction (EC50 = 0.71 nmol), and increased the capillary filtration coefficient (EC50 = 1.4 nmol). Two minutes after injection of 50 nmol PAF, a high thromboxane concentration (3,000 pg/ml) and a low peptidoleukotriene concentration (150 pg/ml) were found in the effluent perfusate. PAF-induced vaso- and bronchoconstriction were unaffected by the non-redox 5-lipoxygenase inhibitor ZM-230,487 but were markedly attenuated by inhibition of cyclooxygenase with acetylsalicylic acid or thromboxane-receptor antagonism with BM-13177. Dual inhibition of cyclooxygenase and 5-lipoxygenase had the most pronounced inhibitory effect on PAF-induced vaso- and bronchoconstriction. None of the inhibitors tested prevented the increase in vascular permeability. We conclude that thromboxane is the major vasoconstrictor and bronchoconstrictor induced by PAF, whereas leukotrienes contribute a significant but minor part in this system. PAF-induced microvascular permeability was not inhibited by blockade of arachidonate metabolism and therefore seems to be mediated by a mechanism independent of eicosanoids.

An improved setup for the isolated perfused rat lung.

This article will describe a method for perfusing and ventilating rat lungs which allows assessment of lung mechanics, segmental vascular resistance, gas exchange, and vascular permeability. Isolated perfused rat lungs (IPL) were ventilated by negative pressure ventilation and were perfused at constant pressure with recirculating, albumin-containing Krebs-Henseleit buffer. A newly constructed respirator pump facilitated well-defined ventilation of the lungs and permitted switching between negative and positive pressure ventilation. Using this setup lungs were ventilated with positive pressure during both surgery and measurement of lung weight. Once the lungs were prepared, electronic control circuits ensured stability of the perfusate pH and perfusate pressure. Such preparations were stable for at least 3 hr. Breathing mechanics, for example, tidal volume, pulmonary compliance, and pulmonary resistance, perfusate characteristics, such as pulmonary vascular resistance, pulmonary pre- and postcapillary resistance, perfusate pH, PO2, and PCO2, and the capillary filtration coefficient were determined. All measured parameters were within normal physiological range, and the lungs were biochemically active, and responded to various stimuli.