Hermann Tenor

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.

PDE isoenzymes as targets for anti-asthma drugs

Phophodiesterase (PDE) isoenzyme profiles of human cell preparations and tissues have been analysed by a semiquantitative method using selective PDE inhibitors and activators. Neutrophils, eosinophils and monocytes contain PDE IV exclusively. Lymphocytes, alveolar macrophages and endothelial cells contain PDE IV and PDE III, and in addition, PDE I is measured in macrophages and PDE II in endothelial cells. These basal cell-specific PDE isoenzyme profiles appear to be modified by: 1) substrate concentration; 2) kinase-dependent phosphorylation; and 3) regulated rate of synthesis. Therefore, PDE isoenzyme profiles represent dynamic patterns, which apparently adapt to pathological and environmental conditions. In parallel functional studies, the influence of mono-selective (rolipram, PDE IV; motapizone, PDE III), dual-selective (zardaverine) and non-selective (theophylline) PDE inhibitors were compared. Corresponding to isoenzyme analysis, it was demonstrated that both PDE III and PDE IV have to be inhibited for complete suppression of either tumour necrosis factor-alpha (TNF-alpha) release from macrophages, or lymphocyte proliferation (PDE III/IV cells). In eosinophils (PDE IV cells) platelet-activating factor (PAF)-induced chemotaxis or C5a-stimulated degranulation are only weakly inhibited by rolipram alone. After addition of a beta 2-agonist, however, the efficacy of rolipram is enhanced due to concomitant influence of synthesis and breakdown of cyclic adenosine monophosphate (cAMP). Theophylline inhibits PDE isoenzyme activities and functions of inflammatory cells with similar potency, and exhibits higher functional efficacy as compared to rolipram.

Phosphodiesterase profiles of highly purified human peripheral blood leukocyte populations from normal and atopic individuals: A comparative study

BACKGROUND Several previous reports have suggested an increased activity of cAMP phosphodiesterases (PDEs) and a higher sensitivity of these enzymes toward PDE inhibitors in leukocytes of patients suffering from atopic dermatitis. OBJECTIVE The purpose of the present study was to comprehensively analyze and compare the PDE expression and activity profile of highly purified populations of leukocytes from normal and atopic blood donors. In addition, the influence of PDE inhibitors on function of leukocytes from normal and atopic individuals was investigated. METHODS Density gradient centrifugation, elutriation, and magnetic cell sorting techniques were used to purify eosinophils, monocytes, and B and T lymphocytes from peripheral human blood. Complementary DNA-polymerase chain reaction was used to analyze PDE4 subtype messenger RNA (mRNA) expression levels in addition to PDE isoenzyme activities. PDE4 inhibitor sensitivity was determined in monocyte homogenates from both groups. Functionally, suppression of lipopolysaccharide-induced synthesis of tumor necrosis factor-alpha in monocytes as well as phytohemagglutinin-induced T cell proliferation in peripheral blood mononuclear cell fractions by PDE4 and PDE3/4 inhibitors was compared. RESULTS Identical PDE activities and mRNA expression profiles were found in all cells from normal and atopic donors except that there was an increase in the mRNA levels of PDE4A and PDE4B2 in atopic T cells, which was, however, not reflected in overall PDE4A activity. In addition, no differences in sensitivity of the functional responses to PDE inhibitors were noted. The mixed PDE3/4 inhibitor zardaverine was a more potent inhibitor of T cell proliferation than rolipram, a selective PDE4 inhibitor. CONCLUSION No evidence for alterations of PDE activities in atopy is provided by our findings.

Roflumilast, a phosphodiesterase 4 inhibitor, alleviates bleomycin-induced lung injury

Background and purpose:  The effects of a phosphodiesterase 4 (PDE4) inhibitor, roflumilast, on bleomycin‐induced lung injury were explored in ‘preventive’ and ‘therapeutic’ protocols and compared with glucocorticoids.

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug targets.

Roflumilast inhibits leukocyte-endothelial cell interactions, expression of adhesion molecules and microvascular permeability

The present study addressed the effects of the investigational PDE4 inhibitor roflumilast on leukocyte‐endothelial cell interactions and endothelial permeability in vivo and in vitro.

Phosphodiesterase isoenzyme families in human osteoarthritis chondrocytes – functional importance of phosphodiesterase 4

We studied whether selective inhibitors of cyclic nucleotide hydrolysing phosphodiesterase (PDE) isoenzymes influence IL‐1β‐induced nitric oxide (NO) release from human articular chondrocytes. In addition, the pattern of PDE isoenzymes contributing to cyclic nucleotide hydrolysis in human chondrocytes was characterized. Chondrocytes were isolated from human osteoarthritic cartilage and cultured in alginate beads. IL‐1β‐induced chondrocyte products (nitric oxide and prostaglandin E2) were measured in culture supernatants after 48 h incubation time. PDE activities were assessed in chondrocyte lysates. Inducible nitric oxide synthase (iNOS) and PDE4A‐D proteins were detected by immunoblotting. The selective PDE4 inhibitors Piclamilast and Roflumilast partially attenuated IL‐1β‐induced NO production whereas selective inhibitors of PDE2 (EHNA), PDE3 (Motapizone) or PDE5 (Sildenafil) were inactive. Indomethacin reversed the reduction of IL‐1β‐induced NO by PDE4 inhibitors. It was shown that autocrine prostaglandin E2 (PGE2) enabled PDE4 inhibitors to reduce IL‐1β‐induced NO in this experimental setting. Major PDE4 and PDE1 activities were identified in chondrocyte lysates whereas only minor activities of PDE2, 3 and 5 were found. IL‐1β and cyclic AMP‐mimetics upregulated PDE4 activity and this was associated with an augmentation of PDE4B2 protein. Based on the view that nitric oxide contributes to cartilage degradation in osteoarthritis our study suggests that PDE4 inhibitors may have chondroprotective effects.

Roflumilast inhibits the release of chemokines and TNF-α from human lung macrophages stimulated with lipopolysaccharide

BACKGROUND AND PURPOSE Lung macrophages are critically involved in respiratory diseases. This study assessed the effects of the PDE4 inhibitor roflumilast and its active metabolite, roflumilast N‐oxide on the release of a range of chemokines (CCL2, 3, 4, CXCL1, 8, 10) and of TNF‐α, from human lung macrophages, stimulated with bacterial lipopolysaccharide LPS.

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.

Effects of Roflumilast, a Phosphodiesterase-4 Inhibitor, on Hypoxia- and Monocrotaline-Induced Pulmonary Hypertension in Rats

Phosphodiesterase type 4 (PDE4) is involved in the hydrolysis of cAMP in pulmonary vascular smooth muscle (PA-SMC) and immune inflammatory cells. Given that intracellular cAMP accumulation inhibits contraction and growth of PA-SMCs as well as inflammatory cell functions, we investigated the effects of the PDE4 inhibitor 3-cyclopropylmethoxy-4-difluoromethoxy-N-[3,5-di-chloropyrid-4-yl]-benzamide (roflumilast), on pulmonary hypertension (PH) in rats. Treatment with roflumilast (0.5 or 1.5 mg · kg-1 day-1) from day 1 to day 21 after monocrotaline (MCT) injection (60 mg · kg-1 s.c.) attenuated PH development: pulmonary artery pressure, right ventricular hypertrophy, and muscularization of distal vessels on day 21 were decreased compared to control MCT-treated rats. Roflumilast (1.5 mg · kg-1 day-1) also reduced the increases in interleukin-6 and monocyte chemotactic protein-1 mRNAs observed in lung tissue on day 21 without affecting the rise in interleukin-1β mRNA on days 1 and 21. Roflumilast (1.5 mg · kg-1 day-1) from day 21 to day 42 after MCT reversed established PH, almost normalizing pulmonary artery pressure and structure, and suppressing proliferating cell nuclear antigen-positive cells in pulmonary vascular walls. Treatment with roflumilast similarly attenuated PH development due to chronic hypoxia. Treatment of human PA-SMCs with roflumilast N-oxide, the active metabolite of roflumilast, at concentrations up to 10-6 M, potentiated PA-SMC growth inhibition induced by prostacyclin (10-6 M) or interleukin-1β (10 ng · ml-1) but was inactive on its own. In conclusion, the PDE4 inhibitor roflumilast significantly attenuates pulmonary vascular remodeling and hypertension induced by chronic hypoxia or MCT and reverses established PH after MCT administration.