Roflumilast inhibits leukocyte-platelet interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes and monocytes.

UNLABELLED: ESSENTIALS: Thrombosis is a major comorbidity in patients with chronic obstructive pulmonary disease (COPD). Roflumilast is a selective phosphodiesterase type-4 (PDE4) inhibitor approved for treatment of severe COPD. PDE4 blockade by roflumilast inhibits prothrombotic functions of neutrophils and monocytes. PDE4 inhibitors may reduce thrombotic risk in COPD as well as in other vascular diseases. BACKGROUND: Roflumilast, an oral selective phosphodiesterase type 4 inhibitor, is approved for the treatment of severe chronic obstructive pulmonary disease (COPD). A recent meta-analysis of trials on COPD revealed that treatment with roflumilast was associated with a significant reduction in the rate of major cardiovascular events. The mechanisms of this effect remain unknown. OBJECTIVES: We tested the hypothesis that roflumilast N-oxide (RNO), the active metabolite of roflumilast, curbs the molecular mechanisms required for leukocyte-platelet (PLT) interactions and prevents the prothrombotic functions of polymorphonuclear leukocytes (PMNs) and monocytes (MNs). METHODS: Using well-characterized in vitro models, we analysed the effects of RNO on: (i) PMN adhesiveness; (ii) the release of neutrophil extracellular traps (NETs); and (iii) tissue factor expression in MNs. Key biochemical events underlying the inhibitory effects of RNO were defined. RESULTS AND CONCLUSIONS: In PMNs, RNO prevented phosphoinositide 3-kinase (PI3K)-dependent phosphorylation of Akt on Ser473, and Src family kinase (SFK)-mediated Pyk2 phosphorylation on Tyr579-580, while inducing protein kinase A-mediated phosphorylation of C-terminal Src kinase, the major negative regulator of SFKs. Modulation of these signaling pathways by RNO resulted in a significant impairment of PMN adhesion to activated PLTs or human umbilical vein endothelial cells, mainly mediated by inhibition of the adhesive function of Mac-1. Moreover RNO curbed SFK/PI3K-mediated NET release by PMNs adherent on fibrinogen-coated surfaces. In MNs interacting with activated PLTs, RNO curbed PI3K-mediated expression of tissue factor. The efficacy of RNO was significantly potentiated by formoterol, a long acting ß-adrenergic receptor agonist. This study reveals novel antithrombotic activities by which roflumilast may exert protective effects against cardiovascular comorbodities in COPD.

The glucose-lowering effects of the PDE4 inhibitors roflumilast and roflumilast-N-oxide in db/db mice.

AIMS/HYPOTHESIS: The cAMP-degrading phosphodiesterase 4 (PDE4) enzyme has recently been implicated in the regulation of glucagon-like peptide-1 (GLP-1), an incretin hormone with glucose-lowering properties. We investigated whether the PDE4 inhibitor roflumilast elevates GLP-1 levels in diabetic db/db mice and whether this elevation is accompanied by glucose-lowering effects. METHODS: Plasma GLP-1 was determined in db/db mice after single oral administration of roflumilast or its active metabolite roflumilast-N-oxide. Diabetes-relevant variables including HbA(1c), blood glucose, serum insulin, body weight, food and water intake, and pancreas morphology were determined in db/db mice treated daily for 28 days with roflumilast or roflumilast-N-oxide. Pharmacokinetic/pharmacodynamic analysis clarified the contribution of roflumilast vs its metabolite. In addition, the effect of roflumilast-N-oxide on insulin release was investigated in primary mouse islets. RESULTS: Single treatment of db/db mice with 10 mg/kg roflumilast or roflumilast-N-oxide enhanced plasma GLP-1 2.5- and fourfold, respectively. Chronic treatment of db/db mice with roflumilast or roflumilast-N-oxide at 3 mg/kg showed prevention of disease progression. Roflumilast-N-oxide abolished the increase in blood glucose, reduced the increment in HbA(1c) by 50% and doubled fasted serum insulin compared with vehicle, concomitant with preservation of pancreatic islet morphology. Furthermore, roflumilast-N-oxide amplified forskolin-induced insulin release in primary islets. Roflumilast-N-oxide showed stronger glucose-lowering effects than its parent compound, consistent with its greater effect on GLP-1 secretion and explainable by pharmacokinetic/pharmacodynamic modelling. CONCLUSIONS/INTERPRETATION: Our results suggest that roflumilast and roflumilast-N-oxide delay the progression of diabetes in db/db mice through protection of pancreatic islet physiology potentially involving GLP-1 and insulin activities.

A phosphodiesterase 4 inhibitor, roflumilast N-oxide, inhibits human lung fibroblast functions in vitro.

The PDE4 inhibitor roflumilast mitigates bleomycin-induced lung fibrotic remodeling in rodents. In the current study it was explored whether roflumilast N-oxide, the active metabolite of roflumilast influences functions of cultured lung fibroblasts. Cells of the human foetal lung fibroblast strain GM06114 were stimulated with TNF-alpha (5 ng ml(-1)) and cell surface ICAM-1 and eotaxin release were assessed. [methyl-(3)H] thymidine incorporation was measured following stimulation with bFGF (10 ng ml(-1)). alpha-Smooth muscle actin (protein), CTGF (mRNA) and fibronectin (mRNA) were determined secondary to TGFbeta1 (1 ng ml(-1)). In the presence of PGE(2) (1 nM), roflumilast N-oxide reduced TNF-alpha-induced ICAM-1 and eotaxin by about 70% and >90% with half-maximum inhibition at 0.9 nM and 0.5 nM, respectively. Roflumilast N-oxide also attenuated [methyl-(3)H] thymidine incorporation secondary to bFGF by about 75% with half-maximum inhibition at 0.7 nM when cells were co-incubated with IL-1beta (10 pg ml(-1)). In the presence of this cytokine roflumilast N-oxide (1 microM) diminished TGFbeta1-induced expression of alpha-smooth muscle actin and transcripts of CTGF and fibronectin. In addition, IL-1beta up-regulated PDE4 activity in the lung fibroblasts. Taken together, these findings indicate that roflumilast N-oxide directly targets human lung fibroblasts, which may at least partially explain the efficacy of roflumilast to mitigate a pulmonary fibrotic response in vivo.

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.

Methotrexate treatment suppresses local cytokine and chemokine production in rat adjuvant arthritis.

Rheumatoid arthritis (RA) is a disease that is still insufficiently controlled by current treatments. Methotrexate (M7X), a small molecular weight compound, has been the gold standard in the treatment of RA. It has several anti-inflammatory activities, but their contribution to its antiarthritic effects has not yet been established. We conducted a rat adjuvant arthritis study, in which we investigated the effect of MTX on local cytokine and chemokine production in arthritic paws. Our data demonstrate that MTX was able to significantly suppress cytokine and chemokine release in the inflamed joints in a dose-dependent fashion, accompanied by amelioration of the disease as indicated by reduced paw swelling and arthritic scores. These findings prompt further studies to clarify whether these suppressive effects of MTX on local cytokine and chemokine release are direct or whether they are a result of other preceding anti-inflammatory activities of the compound.

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.

Automatic leukocyte differentiation in bronchoalveolar lavage fluids of guinea pigs and brown-Norway rats.

Asthma is considered to be a chronic inflammatory response of the airways characterized by a leukocyte infiltration into the lungs. Whereas lymphocytes and macrophages are involved in the initiation and propagation of inflammation, both neutrophils and in particular eosinophils are considered to play major effector roles. Therefore, allergic animal models in various species have been established to assess leukocyte infiltration by bronchoalveolar lavage (BAL) of antigen-sensitized and antigen-challenged animals as an inflammatory parameter in asthma pharmacology. Differential leukocyte counts in BAL fluids are routinely assessed by visual microscopic analysis of stained slides after cytocentrifugation. This procedure is very time-consuming, and the underlying standard morphological criteria may vary between different observers. In the present paper, we propose an alternative automatic method for leukocyte differentiation in BAL fluids from ovalbumin-treated guinea pigs and Brown-Norway rats using Cobas Helios 5Diff from Hoffmann-La Roche. BAL samples are directly applied to the analyzer and are automatically mixed with "Eosinofix," which stabilizes leukocyte membranes and specifically stains eosinophils. By a combination of electric (resistance) and optical (light scatter) analysis, the lymphocytes, monocytes/macrophages, neutrophils, and eosinophils are discriminated and the total leukocyte numbers are obtained. For both animal species we found high correlations for all leukocyte populations by comparing the results obtained with Cobas Helios 5Diff and conventional microscopic analysis. The major advantage of the automatic method is the much lower (about one-third) time requirement.

Dose-response comparisons of five lung surfactant factor (LSF) preparations in an animal model of adult respiratory distress syndrome (ARDS).

1. We have examined the effects of five different lung surfactant factor (LSF) preparations in the rat lung lavage model. In this model repetitive lung lavage leads to lung injury with some similarities to adult respiratory distress syndrome with poor gas exchange and protein leakage into the alveolar spaces. These pathological sequelae can be reversed by LSF instillation soon after lavage. 2. The tested LSF preparations were: two bovine: Survanta and Alveofact: two synthetic: Exosurf and a protein-free phospholipid based LSF (PL-LSF) and one Recombinant LSF at doses of 25, 50 and 100 mg kg-1 body weight and an untreated control group. 3. Tracheotomized rats (10-12 per dose) were pressure-controlled ventilated (Siemens Servo Ventilator 900C) with 100% oxygen at a respiratory rate of 30 breaths min-1, inspiration expiration ratio of 1:2, peak inspiratory pressure (PIP) of 28 cmH2O at positive end-expiratory pressure (PEEP) of 8 cmH2O. Two hours after LSF administration, PEEP and in parallel PIP was reduced from 8 to 6 (1st reduction), from 6 to 3 (2nd reduction) and from 3 to 0 cmH2O (3rd reduction). 4. Partial arterial oxygen pressure (PaO2, mmHg) at 5 min and 120 min after LSF administration and during the 2nd PEEP reduction (PaO2(PEEP23/3)) were used for statistical comparison. All LSF preparations caused a dose-dependent increase for the PaO2(120'), whereas during the 2nd PEEP reduction only bovine and recombinant LSF exhibited dose-dependency. Exosurf did not increase PaO2 after administration of the highest dose. At the highest dose Exosurf exerted no further improvement but rather a tendency to relapse. The bovine and the Recombinant LSF are superior to both synthetic LSFpreparations.5. In this animal model and under the described specific ventilatory settings, even between bovine LSFpreparations there are detectable differences that are pronounced when compared to synthetic LSFwithout any surfactant proteins. We conclude that the difference between bovine and synthetic LSFpreparations can be overcome by addition of the surfactant protein C.

Comparative investigation of the effects of zardaverine and theophylline on pulmonary function in rats.

Zardaverine (Byk Gulden, Konstanz, Germany) is a new selective phosphodiesterase (PDE) III/IV inhibitor. The bronchodilating and bronchoprotective potency of zardaverine and the nonselective PDE inhibitor theophylline was compared by measuring typical spontaneous and forced respiratory function parameters in anesthetized female Wistar rats using whole-body plethysmography. Zardaverine (3, 10, 30 mumol/kg) and theophylline (30, 100, 300 mumol/kg), respectively, were given orally in 4% Methocel/0.9% saline solution 20 min before measurement. One week before treatment, control measurements were performed in the same animals after administration of the vehicle. When spontaneously breathing, the 30 mumol/kg zardaverine- (300 mumol/kg theophylline-) treated animals showed significant changes: a 23% (14% ns) decrease in lung resistance and a 43% (25%) increase in dynamic compliance. These changes can be interpreted as an indication of bronchodilation, since quasistatic lung compliance was unchanged. In the acetylcholine challenge test, treatment with only 10 mumol/kg zardaverine (but 300 mumol/kg theophylline) revealed significant changes compared to control measurement: a 37% (28%) lower resistance and 85% (44%) higher compliance. It has been determined that zardaverine is more than 30 times as potent as theophylline in inhibiting acetylcholine-induced bronchospasms, which is also supported by forced expiratory flow data.

Randomized placebo-controlled study on the characteristics and duration of action of surfactant treatment in premature lambs.

In a placebo-controlled study, the characteristics and duration of action of a commercially available bovine lung surfactant factor (LSF; 50 mg/kg body weight) were investigated in premature lambs at 124-127 days gestational age. Exact mating of ewes was controlled by progesterone hormone analysis. To minimize unwanted effects of narcotics through transplacental transfer to the fetus, spinal anaesthesia of the ewes was performed. The physiological status 'in utero' was demonstrated by arterial blood gas analysis (partial arterial oxygen pressure (PaO2), partial arterial carbon dioxide pressure (PaCO2) and arterial blood pH) of the fetus before umbilical cord ligature. The values for blood gases, blood pressure and lung mechanics measured after onset of ventilation and before LSF treatment were indicative of fetal maturity. Immediately after LSF treatment, PaO2 increased significantly, then decreased for about 2 h and finally stabilized on a plateau until the end of the experiment. In contrast, arterial blood pH and PaCO2 values normalized more slowly and remained normal for a longer period in the LSF group, whereas both parameters did not normalize in the placebo group. Total dynamic lung compliance also showed a slow improvement and remained on a higher level for a longer period than PaO2. Using a low respiratory rate it was possible to ventilate at lower peak inspiratory pressures and this resulted in higher compliance values. In conclusion, the presented data suggest that artificial ventilation greatly influences lung mechanics. Due to a relatively short substance effect concerning PaO2 we assume that higher or repeated doses of LSF may be necessary to produce sustained improvements in clinical trials.

Comparison of a phospholipid-based protein-free surfactant and a natural bovine surfactant (SURVANTA) during pressure and volume-controlled ventilation in an improved rabbit fetus model.

During pressure- or volume-controlled ventilation different surfactant preparations were compared in an improved rabbit fetus model. Based on a self-designed software program, this model enables on-line registration of lung mechanics and heart rate in up to ten fetuses. Using a commercially available bovine lung surfactant (SURVANTA) as standard, we compared animals treated with a protein-free surfactant preparation containing only phospholipids, PL (dipalmitoylphosphatidylcholine:palmitoyloleoylphosphatidylglycerol++ +, DPPC:POPG 70:30) plus palmitic acid (PA) with an untreated ventilated control group. During pressure-controlled ventilation the insufflation pressure (IP) was decreased and increased stepwise with and without positive end-expiratory pressure (PEEP). SURVANTA was significantly more potent than PL plus PA and both differed significantly from the untreated controls. With additional PEEP the differences between SURVANTA and PL+PA disappeared but the differences to the controls were still present. We found that, with additional PEEP, active natural surfactants lead to ECG-irregularities, which indicates that PEEP influences pulmonary and cardiovascular function and compromises the benefits of surfactant therapy. Also during volume-controlled ventilation SURVANTA was superior to PL+PA and the untreated controls. In order to raise the level of activity of pure PL mixtures to that of natural bovine surfactants, we suggest that a surface active protein (probably SP-C) must be added to such mixtures.

Zardaverine: a cyclic AMP specific PDE III/IV inhibitor.

Zardaverine is shown to inhibit selectively two out of five isoenzyme classes of phosphodiesterases, namely PDE III from human platelets and PDE IV from human polymorphonuclear leucocytes (PMN) with IC50 values of 0.58 and 0.17 microM, respectively. Motapizone or rolipram, for comparison, are more selective recognizing either PDE III or PDE IV. On the cellular level, agonist induced aggregation of platelets or activated secretions of PMN and several proinflammatory cells are synergistically inhibited by zardaverine and adenylate cyclase activators such as prostaglandins or forskolin. Application of zardaverine and selective drugs show that the effects of PDE inhibitors in platelets are mediated by a PDE III isoenzyme, whereas by a PDE IV isoenzyme in neutrophils, eosinophils, basophils and mast cells. An antiinflammatory potential in vivo of zardaverine is demonstrated by the inhibition of bronchial cell infiltration following inhalative allergen challenge in sensitized guinea pigs. Zardaverine and dexamethasone prevent bronchial eosinophilia and neutrophilia with similar dosage of 30 microM/kg orally, suggesting that this PDE III/IV inhibitor may be useful for both, bronchorelaxation and reduction of inflammation in asthma therapy.

Dual effect of external calcium on the frequency of miniature synaptic potentials in frog sympathetic ganglion cells.

The effect of calcium on spontaneous transmitter release and on the release induced by tetanic stimulation and by raising the external potassium concentration ([K]0) was studied in sympathetic ganglion cells of Rana esculenta. 1. In standard Ringer's solution the frequency of miniature excitatory postsynaptic potentials (mepsp) ranged from 0.05--2.0 s-1 (0.05 +/- 0.09 s-1, n = 37) at room temperature. 2. At a [K]0 of 2.5 mM mepsp frequency was approximately linearly related to the logarithm of the external calcium concentration (log [Ca]0) (0.1 mM less than or equal to [Ca]0 less than or equal to 20 mM). 3. Duration and amplitude of the potentiation of transmitter release after tetanic preganglionic stimulation increased depending on [Ca]0. 4. Mepsp frequency was strongly dependent on [K]0 between 10 and 20 mM; the frequency being increased to about 40 times control level at a [K]0 of 20 mM. 5. Raising [Ca]0 up to 1.8 mM in high K solutions resulted in an increase in mepsp frequency followed by a decrease at higher [Ca]0. 6. These results are consistent with the idea that the effect of calcium on mepsp frequency depends on: (a) the driving force for calcium entry, (b) the effect of Ca ions on the potential gradient within the nerve membrane.