Roflumilast N-oxide, a PDE4 inhibitor, improves cilia motility and ciliated human bronchial epithelial cells compromised by cigarette smoke in vitro.

BACKGROUND AND PURPOSE: Mucociliary malfunction occurs in chronic obstructive pulmonary disease (COPD) and compromised functions of ciliated bronchial epithelial cells may contribute to this. Cigarette smoke, a major risk factor for COPD, impairs ciliary beat frequency (CBF). cAMP augments CBF. This in vitro study addressed, in differentiated, primary human bronchial epithelial cells, whether roflumilast N-oxide, a PDE4 inhibitor, (i) augments CBF; (ii) prevents the reduction in CBF induced by cigarette smoke extract (CSE); and (iii) protects against the loss of the ciliated phenotype following long-term CSE exposure. EXPERIMENTAL APPROACH: Air-liquid interface cultured human bronchial epithelial cells were incubated with roflumilast N-oxide and exposed to CSE. CBF was assessed by digital high speed video microscopy (DHSV). Ciliated cells were characterized by ß-tubulin IV staining and analyses of Foxj1 and Dnai2 mRNA and protein (real-time quantitative PCR, Western blotting). KEY RESULTS: Roflumilast N-oxide concentration-dependently triggered a rapid and persistent increase in CBF and reversed the decrease in CBF following CSE. Long-term incubation of bronchial epithelial cells with CSE resulted in a loss in ciliated cells associated with reduced expression of the ciliated cell markers Foxj1 and Dnai2. The PDE4 inhibitor prevented this loss in the ciliated cell phenotype and the compromised Foxj1 and Dnai2 expression. The enhanced release of IL-13 following CSE, a cytokine that diminishes the proportion of ciliated cells and in parallel, reduces Foxj1 and Dnai2, was reversed by roflumilast N-oxide. CONCLUSION AND IMPLICATIONS: Roflumilast N-oxide protected differentiated human bronchial epithelial cells from reduced CBF and loss of ciliated cells following CSE.

Phosphodiesterase inhibitors: history of pharmacology.

The first pharmacological investigations of phosphodiesterase (PDE) inhibitors were developed with the clinical efficacies of drugs isolated from coffee, cacao and tea but only later their relevant ingredients were identified as xanthines that act as PDE. With its diuretic, inotropic and bronchodilating clinical efficacy, use of theophylline anticipated the clinical goals, which were later approached with the first-generation of weakly selective PDE inhibitors in the period from 1980 to 1990. Pharmacological and clinical research with these early compounds provided a vast pool of information regarding desired and adverse actions - although most of these new drugs had to be discontinued due to severe adverse effects. The pharmacological models for cardiac, vascular and respiratory indications were analysed for their PDE isoenzyme profiles, and when biochemical and molecular biological approaches expanded our knowledge of the PDE superfamily, the purified isoenzymes that were now available opened the door for more systematic studies of inhibitors and for generation of highly selective isoenzyme-specific drugs. The development of simple screening models and clinically relevant indication models reflecting the growing knowledge about pathomechanisms of disease are summarised here for today's successful application of highly selective PDE3, PDE4 and PDE5 inhibitors. The interplay of serendipitous discoveries, the establishment of intelligent pharmacological models and the knowledge gain by research results with new substances is reviewed. The broad efficacies of new substances in vitro, the enormous biodiversity of the PDE isoenzyme family and the sophisticated biochemical pharmacology enabled Viagra to be the first success story in the field of PDE inhibitor drug development, but probably more success stories will follow.

Pharmacology, clinical efficacy, and tolerability of phosphodiesterase-4 inhibitors: impact of human pharmacokinetics.

Since more than two decades anti-inflammatory effects of inhibitors of phosphodiesterase-4 have been described in numerous cellular and animal studies and were finally confirmed in clinical trials. The path from an early, pioneering study with Ro20-1724 showing reduction of psoriatric plaque size in 1979 to modern PDE4 inhibitors such as oral apremilast in development for psoriasis, the inhaled PDE4 inhibitor GSK256066 in development for asthma and COPD and finally roflumilast, the first PDE4 inhibitor approved and currently marketed as an oral, once-daily remedy for severe COPD was marked by large progress in chemical optimization based on improved understanding of PDE4 biology and drug-like properties determining the appropriate pharmacokinetic profile. In this chapter aspects of the pharmacology and clinical efficacy of PDE4 inhibitors, which have been in clinical development over the years are summarized with specific emphasis on their clinical pharmacokinetic properties.

The preclinical pharmacology of roflumilast--a selective, oral phosphodiesterase 4 inhibitor in development for chronic obstructive pulmonary disease.

After more than two decades of research into phosphodiesterase 4 (PDE4) inhibitors, roflumilast (3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide) may become the first agent in this class to be approved for patient treatment worldwide. Within the PDE family of 11 known isoenzymes, roflumilast is selective for PDE4, showing balanced selectivity for subtypes A-D, and is of high subnanomolar potency. The active principle of roflumilast in man is its dichloropyridyl N-oxide metabolite, which has similar potency as a PDE4 inhibitor as the parent compound. The long half-life and high potency of this metabolite allows for once-daily, oral administration of a single, 500-microg tablet of roflumilast. The molecular mode of action of roflumilast--PDE4 inhibition and subsequent enhancement of cAMP levels--is well established. To further understand its functional mode of action in chronic obstructive pulmonary disease (COPD), for which roflumilast is being developed, a series of in vitro and in vivo preclinical studies has been performed. COPD is a progressive, devastating condition of the lung associated with an abnormal inflammatory response to noxious particles and gases, particularly tobacco smoke. In addition, according to the Global Initiative for Chronic Obstructive Lung Disease (GOLD), significant extrapulmonary effects, including comorbidities, may add to the severity of the disease in individual patients, and which may be addressed preferentially by orally administered remedies. COPD shows an increasing prevalence and mortality, and its treatment remains a high, unmet medical need. In vivo, roflumilast mitigates key COPD-related disease mechanisms such as tobacco smoke-induced lung inflammation, mucociliary malfunction, lung fibrotic and emphysematous remodelling, oxidative stress, pulmonary vascular remodelling and pulmonary hypertension. In vitro, roflumilast N-oxide has been demonstrated to affect the functions of many cell types, including neutrophils, monocytes/macrophages, CD4+ and CD8+ T-cells, endothelial cells, epithelial cells, smooth muscle cells and fibroblasts. These cellular effects are thought to be responsible for the beneficial effects of roflumilast on the disease mechanisms of COPD, which translate into reduced exacerbations and improved lung function. As a multicomponent disease, COPD requires a broad therapeutic approach that might be achieved by PDE4 inhibition. However, as a PDE4 inhibitor, roflumilast is not a direct bronchodilator. In summary, roflumilast may be the first-in-class PDE4 inhibitor for COPD therapy. In addition to being a non-steroid, anti-inflammatory drug designed to target pulmonary inflammation, the preclinical pharmacology described in this review points to a broad functional mode of action of roflumilast that putatively addresses additional COPD mechanisms. This enables roflumilast to offer effective, oral maintenance treatment for COPD, with an acceptable tolerability profile and the potential to favourably affect the extrapulmonary effects of the disease.

Effect of cyclic AMP-elevating agents on airway ciliary beat frequency in central and lateral airways in rat precision-cut lung slices.

In patients with chronic obstructive pulmonary disease (COPD), mucociliary clearance of the respiratory tract is impaired due to enhanced mucus secretion and deterioration of normal ciliary activity. We investigated the effects of cyclic AMP-elevating agents with a different mode of action on ciliary beat frequency (CBF) in rat large central and small lateral airways by comparing the phosphodiesterase-4 (PDE4) inhibitors rolipram and roflumilast to the beta(2)-adrenoceptor agonist terbutaline and the adenylyl cyclase activator forskolin. Rat precision-cut lung slices were prepared and effects of cyclic AMP-elevating agents on CBF were assessed for up to 4h. In central airways a time- and concentration-dependent increase in CBF was seen for roflumilast (59+/-4%, 1microM, 60min), rolipram (55+/-4%, 1microM, 60min), terbutaline (64+/-8%, 10microM, 60min) and forskolin (55+/-8%, 100microM, 60min). Only roflumilast and rolipram increased CBF in lateral airways, with a similar time course and maximum efficacy (roflumilast 48+/-5%, rolipram 54+/-2%). Incubation of lateral airways with terbutaline (10microM, +11%) or forskolin (100microM, +1%) had negligible effects. As a major novel finding this study reveals that PDE4 inhibitors increased CBF in central as well as in lateral airways, while terbutaline and forskolin affected CBF in proximal airways only.

Effects of the phosphodiesterase type 4 inhibitor roflumilast on early and late allergic response and airway hyperresponsiveness in Aspergillus-fumigatus-sensitized mice.

BACKGROUND: Inhibitory effects of roflumilast on responses characteristic of allergic asthma were investigated in a fungal asthma model in BALB/c mice. METHODS: Mice were sensitized with Aspergillus antigen (Afu) and exposed to Afu or vehicle, and given roflumilast 1 or 5 mg/kg. Early airway response (EAR) and late airway hyperresponsiveness (AHR) to methacholine were measured via plethysmography. Bronchoalveolar lavage (BAL) was used to assess inflammatory cell count. RESULTS: In Afu-exposed mice, roflumilast dose-dependently reduced the EAR [26% at 1 mg/kg (NS) and 94% at 5 mg/kg (p < 0.01)] and AHR [46% at 1 mg/kg (NS) and 128% at 5 mg/kg (p < 0.05)]. Roflumilast 5 mg/kg reduced neutrophil, eosinophil and lymphocyte counts [87% (p < 0.01), 40% (NS) and 67% (p < 0.01), respectively] in BAL fluid versus controls. CONCLUSIONS: In this model, roflumilast inhibited the EAR, suppressed AHR and reduced inflammatory cell infiltration.

Effect of roflumilast on inflammatory cells in the lungs of cigarette smoke-exposed mice.

BACKGROUND: We reported that roflumilast, a phosphodiesterase 4 inhibitor, given orally at 5 mg/kg to mice prevented the development of emphysema in a chronic model of cigarette smoke exposure, while at 1 mg/kg was ineffective. Here we investigated the effects of roflumilast on the volume density (VV) of the inflammatory cells present in the lungs after chronic cigarette smoke exposure. METHODS: Slides were obtained from blocks of the previous study and VV was assessed immunohistochemically and by point counting using a grid with 48 points, a 20x objective and a computer screen for a final magnification of 580x. Neutrophils were marked with myeloperoxidase antibody, macrophages with Mac-3, dendritic cells with fascin, B-lymphocytes with B220, CD4+ T-cells with CD4+ antibody, and CD8+T-cells with CD8-alpha. The significance of the differences was calculated using one-way analysis of variance. RESULTS: Chronic smoke exposure increased neutrophil VV by 97%, macrophage by 107%, dendritic cell by 217%, B-lymphocyte by 436%, CD4+ by 524%, and CD8+ by 417%. The higher dose of roflumilast prevented the increase in neutrophil VV by 78%, macrophage by 82%, dendritic cell by 48%, B-lymphocyte by 100%, CD4+ by 98% and CD8+ VV by 88%. The lower dose of roflumilast did not prevent the increase in neutrophil, macrophage and B-cell VV but prevented dendritic cells by 42%, CD4+ by 55%, and CD8+ by 91%. CONCLUSION: These results indicate (i) chronic exposure to cigarette smoke in mice results in a significant recruitment into the lung of inflammatory cells of both the innate and adaptive immune system; (ii) roflumilast at the higher dose exerts a protective effect against the recruitment of all these cells and at the lower dose against the recruitment of dendritic cells and T-lymphocytes; (iii) these findings underline the role of innate immunity in the development of pulmonary emphysema and (iiii) support previous results indicating that the inflammatory cells of the adaptive immune system do not play a central role in the development of cigarette smoke induced emphysema in mice.

Roflumilast fully prevents emphysema in mice chronically exposed to cigarette smoke.

RATIONALE: There is a need for new agents capable of suppressing the inflammatory response in chronic obstructive pulmonary disease. OBJECTIVES: This study evaluated the effects of roflumilast, a phosphodiesterase 4 (PDE4) inhibitor on acute lung inflammation and chronic lung changes in models of cigarette exposure in mice. METHODS: Roflumilast was given orally either at 1 mg/kg (R1) or at 5 mg/kg (R5). In the acute model (five cigarettes for 20 minutes), bronchoalveolar lavage fluid (BALF) changes were investigated at 4 and 24 hours. In the chronic model (three cigarettes/day for 7 months), morphometric and biochemical parameters were assessed at 7 months. MEASUREMENTS AND MAIN RESULTS: Acute exposure caused a fivefold increase in BALF neutrophils. Both doses of roflumilast partially prevented (by 30%) this increase. In addition, after smoke exposure, R1 increased BALF interleukin-10 by 79% and R5 by 129%. Chronic smoke exposure caused a 1.8-fold increase in lung macrophage density, emphysema, an increase of the mean linear intercept (+21%), a decrease of the internal surface area (-13%), and a drop (-13%) in lung desmosine content. R1 did not have any effect, whereas R5 prevented the increase in lung macrophage density by 70% and fully prevented the other changes. In addition, in the smoke-exposure group, 63% of the mice showed goblet cell metaplasia, and neither of the doses of roflumilast had any effect. CONCLUSIONS: This study shows for the first time that a PDE4 inhibitor partially ameliorates lung inflammation and fully prevents parenchymal destruction induced by cigarette smoke.

Roflumilast inhibition of pulmonary leukotriene production and bronchoconstriction in ovalbumin-sensitized and -challenged Guinea pigs.

This study investigated the effects of roflumilast, a PDE4 inhibitor, on slow-reacting substance of anaphylaxis (SRS-A)-mediated bronchoconstriction and pulmonary leukotriene (LT) release in ovalbumin (OVA)-sensitized and -challenged guinea pigs. Animals were treated with roflumilast orally (0.04, 0.12, 0.4, or 4 mg/kg) or placebo 1 hour before OVA challenge. Bronchoconstriction was quantified by measuring airway conductance (Gaw) and dynamic lung compliance (Cdyn). Roflumilast significantly attenuated the decrease in Gaw (50% inhibitory dose [ID50] = 0.33 mg/kg) and Cdyn (ID50 = 0.25 mg/kg) in a dose-dependent manner and significantly inhibited Cys-LT (ID50 = 0.06 mg/kg) and LTB4 (ID50 = 0.05 mg/kg) release versus placebo-treated animals. Roflumilast did not affect LTD4-induced bronchoconstriction. These findings support the role of roflumilast as an anti-inflammatory treatment for asthma.

Inhibition of inflammation and remodeling by roflumilast and dexamethasone in murine chronic asthma.

Phosphodiesterase (PDE) inhibitors have potential as alternatives or adjuncts to glucocorticoid therapy in asthma. We compared roflumilast (a selective PDE4 inhibitor) with pentoxifylline (a nonselective inhibitor) and dexamethasone in ameliorating the lesions of chronic asthma in a mouse model. BALB/c mice sensitized to ovalbumin were chronically challenged with aerosolized antigen for 6 weeks. During weeks 5 and 6, groups of animals were treated with roflumilast or dexamethasone by daily gavage or with pentoxifylline by daily intraperitoneal injection. Airway hyper-reactivity (AHR) was evaluated by whole-body plethysmography and airway lesions by histomorphometry and immunohistochemistry. Compared with vehicle alone, treatment with roflumilast or dexamethasone significantly reduced accumulation of eosinophils and chronic inflammatory cells, subepithelial collagenization, and thickening of the airway epithelium. Dexamethasone also reduced goblet cell hyperplasia/metaplasia, subepithelial accumulation of transforming growth factor-beta1, and epithelial cytoplasmic immunoreactivity for nuclear factor-kappaB. Treatment with pentoxifylline inhibited only eosinophil recruitment and epithelial thickening. Roflumilast and dexamethasone slightly decreased AHR, whereas this was significantly reduced by pentoxifylline. Thus, in this model of chronic asthma, both roflumilast and dexamethasone were potent inhibitors of airway inflammation and remodeling. Roflumilast did not diminish accumulation of transforming growth factor-beta1, suggesting that it might affect remodeling by mechanisms distinct from glucocorticoids.